NOVEL METHODS FOR DETECTION OF BIOACTIVE SUBSTANCES AND THEIR EFFECTS IN ORGANISMS AND IN THE ENVIRONMENT. Tatjana Radovanović Vukajlović

Transcription

1 UNIVERSITY OF NOVA GORICA GRADUATE SCHOOL NOVEL METHODS FOR DETECTION OF BIOACTIVE SUBSTANCES AND THEIR EFFECTS IN ORGANISMS AND IN THE ENVIRONMENT DISSERTATION Tatjana Radovanović Vukajlović Mentor: Prof. dr. Mladen Franko Nova Gorica, 2017

2 Learn from yesterday, live for today, hope for tomorrow. The important thing is not to stop questioning. Albert Einstein

3 To my husband Nemanja & son Filip. I II

4 ABSTRACT Since the concentration of bioactive substances and infectious agents in organisms and in the environment are low highly sensitive techniques such as: chromatography technology coupled with mass spectrometry (GC/MS, LC/MS and LC MS/MS) and transmission electron microscopy (TEM) are needed for their detection. These techniques are highly sensitive, but time consuming, requiring use of expensive apparatus and large quantities of reagents and organic solvents which are harmful for the environment. Because there is a growing need for analysis of a large number of environmental samples it is necessary to develop new, so called vanguard methods that enable rapid and reliable screening of large numbers of samples in the shortest possible time. Analysis with such screening methods are often less accurate or even semi-quantitative, but nevertheless allow reliable identification of nonproblematic samples and in practice they limit the use of demanding classical analytical methods to only a few percent of all the samples. Therefore, general objectives of the thesis were development of novel methods for sensitive, fast and cost effective detection of pharmaceuticals, viruses and viral particles in waters and biological fluids and for detection of their effects in organisms. Novel methods were based on the combination of TLS (Thermal Lens Spectrometry), microfluidics and immunological methods such as ELISA. TLS as highly sensitive technique (allowing detection of absorbances of less than 10-6 ) coupled with microfluidic technology allows detection of very low analyte concentration, shorter time for analysis, higher sample throughput and low consumption of reagents. In such combination microfluidic technology can simplify or speed up antigen-antibody or enzyme-substrate interactions in bioanalytical systems. Decisive advantage of microfluidic systems lies in the fact that small dimensions of such systems, composed of capillaries and micro-reactors with dimensions from about 10 to 100 µm, significantly reduce diffusion time, which is inversely proportional to second power of distance. However, highly sensitive detection techniques are needed in microfludic systems, because the amounts of analytes in detection volumes are generally small and optical interaction lengths are two to three orders of magnitude shorter than in conventional spectrometric techniques. By combining microscopic TLS (TLM) with microfluidic technique it is possible to reach very low limits of detection and at the same time shorten ELISA analysis time from 20 h to 20 minutes as was described before in the literature for detection of BNP (brain natriuretic peptide). TLM furthermore allows measurements of extremely small volumes (sub-microliter) as well as fast signal response (milliseconds). In this Dissertation specific goals were the development of new methods for detection of selected bioactive substances and infectious agents: -iodinated contrast agents -NGAL (neutrophil gelatinase associated lipocalin) as a new biomarker of contrast induced nephropathy (CIN) -antibodies against human papilloma viruses (HPV) viruses and HPV-16 pseudovirions. For the development of new method for detection of iodinated contrast agents chemical degradation of iodinated contrast agents was investigated as well, as a potential method for their removal from waste water. I

5 For the determination of NGAL, a commercially available ELISA kit was used as the basis for method development. In the initial experiments the final product of the reaction of substrate with enzyme HRP (horse radish peroxidase) was transferred from microtiter plate into a microfluidic system, which served just for the sample transport to TLM detector on microchip. With comparable speed analysis we achieved LOD of 1.4 pg/ml which is 7 times lower in comparison to commercial ELISA test (LOD=10 pg/ml). For further development of the method for detection of NGAL with µfia-tlm magnetic nanobeads were used as a solid support for primary antibodies of ELISA assay. By applying appropriate magnetic field the antibodies were kept in microfluidic system, which also enabled binding of NGAL, secondary antibodies and reaction of substrate with HRP. Developed method for NGAL detection with LOD of 2.3 pg/ml compares favorably with LOD for commercial ELISA tests (10 pg/ml) in standard microtiter plates and significantly reduces the analysis time. TLM in combination with microchip for NGAL detection reduces the duration of individual incubation steps (from one hour to 5 minutes) and at the same time shortens total analysis time from four hours for commercial ELISA test to 35 minutes allowing higher sample throughput. Analysis of real blood samples was also performed and it has shown good agreement between NGAL concentrations measured by magnetic nanobeads based µfia- TLM with the concentrations measured by a commercial ELISA test. Such short analysis time of analysis and possible further optimizations are opening new possibilities for application of µfia-tlm in medical diagnostics and clinical research. By using appropriate antibodies the method for developed NGAL detection could be easily adopted for detection of different pharmaceuticals or pollutants in environmental samples. We have also developed a magnetic nanobeads based ELISA assay for detection of anti-hpv-16 L1 antibodies in the sera of HPV-16 infected women. To ensure the selectivity, HPV-16 pseudovirions were used as an antigen for anti-hpv-16 L1 antibodies, which were detected with secondary HRP labeled antibodies. Initially the ELISA assay for antibodies against HPV pseudovirions was performed on a microtiter plate and an LOD of 3.8 ng/ml was achieved by measurement on a microtiter plate reader. When performing a µfia-tlm measurement of the final ELISA solution the LOD was reduced to 0.9 ng/ml. Similar to the method for NGAL detection based on magnetic nanobeads, these were used as solid support for HPV pseudovirions and after carrying out all the incubation steps of the ELISA test in microfluidic chip the final product of the reaction of substrate with HRP was detected on TLM. With magnetic nanobeads based ELISA assay with µfia-tlm for measurement of antibodies against PsVs of HPV-16 virus an LOD of 0.6 ng/ml was achieved, which is six times lower in comparison to classic ELISA on microtiter plate. Furthermore, the analysis time was reduced from ten hours to 30 minutes. The novel method was successfully validated by analysis of real sera samples from women who were previously diagnosed for infection with HPV-16 virus. For determination of iodinated MRI contrast agents we developed a new method based on the measurement of concentration of released iodide which allows indirectly semiquantitative detection of concentration of iodinated contrast agents. For iodide release from parent molecule of contrast agent we applied a chemical reaction with Cu 2+ ions in the presence of H 2 O 2. Released iodide was first oxidized into iodine and then extracted into chloroform. Contrast agents degradation reaction showed 70 % of efficiency for removal of iomeprol, taking into account the 60 % overall efficiency of iodide oxidation and extraction. II

6 The extract was injected into microfluidic chip and iodine concentration was determined with TLM. Chloroform as organic solvent with low thermal conductivity and high temperature coefficient of refractive index is a good choice for TLM measurement due to high TLS enhancement factor, which theoretically provides 40 times higher sensitivity of TLM measurements as compared to water and a four time improvement in sensitivity for each milliwatts of excitation power, when compared to spectrophotometry. The developed µfia- TLM method for indirect determination of contrast agents based on detection of iodine provides around 60 times lower LOD, with low reagent and sample consumption in comparison to spectrophotometry. The LOD of 18 ng/ml for iomeprol achieved with TLM is 16 times lower in comparison to LOD of 294 ng/ml for iomeprol determination with HPLC. In comparison to LOD of 133 ng/ml for iomeprol achieved with detection of released iodide by ion chromatography, µfia-tlm enables around 7 times lower LOD. HPLC and HPLC/MS analysis showed that the parent compounds is completely removed after 120 min. of chemical degradation and that different degradation products are formed by cleavage of one or two iodine atoms. By this we have shown that the applied chemical degradation is efficient for removal of iomeprol and could be applied for treatment of waste waters after further optimization and reduction of reaction time. New analytical methods developed within this work provide limits of detection for the selected compounds which are significantly lower (up to 60 times) in comparison to conventional analytical techniques based on transmission mode measurements. At the same time the new methods allows shorter time of analysis and higher sample throughput for the purpose of fast screening methods. Magnetic nanobeads based µfia-tlm ELISA assays developed within this work offer several advantages in comparison to commercial ELISA tests on microtiter plates such as: higher surface for antibody binding, lower reagent consumption, and shorter analysis time. Although the TLS technique didn t reach appropriate stage of development and applicability for routine chemical analysis, improved methods for detection of NGAL and antibodies against HPV viruses could be applied for clinical studies and development of commercial tests for detection of viruses or other bioactive substances, which are needed for diagnostic purposes in hospitals. Keywords: ELISA, NGAL, PsVs, contrast agent, TLM. III

7 POVZETEK Bioaktivne spojine so esencialne in neesencialne spojine, ki so del prehranske verige in vplivajo na delovanje organizmov, ali pa v njih povzročijo določene reakcije, vplivajo pa tudi na zdravje ljudi. Med različnimi bioaktivnimi spojinami, kot so tudi encimi in vitamini, so farmacevtske učinkovine ene najbolj poznanih. Najbolj široko uporabljena so različna protivnetna zdravila, antibiotiki, β-blokatori proti povišanemu krvnemu pritisku in statini proti holesterolu, od ostalih bioaktivnih snovi, povezanih z medicino, pa tudi kontrastna sredstva, ki jih uporabljamo za slikanje organov ter krvnih žil in pogosto povzročajo številne neželene učinke, kot je na primer pokontrastna nefropatija (angl. contrast induced nephropathy). Samo porabo antibiotikov ocenjujejo v svetovnem merilu na ton letno. V preteklosti in tudi danes v postopku proizvodnje, ali še pogosteje po uporabi, farmacevtske učinkovine spuščamo v vodno okolje preko industrijskih izpustov, komunalnih odplak in bolnišničnih odpadnih vod. Nekatere farmacevtske učinkovine so zelo obstojne in jih zato v bioloških čistilnih napravah lahko samo delno odstranimo iz odpadne vode. Zato jih najdemo tudi v površinskih vodah in podtalnici ter posledično v pitni vodi. V okolju so prisotne v majhnih koncentracijah, vplivi dolgotrajne izpostavljenosti organizmov različnim farmacevtskim izdelkom ali njihovim mešanicam pa še niso dovolj raziskani. Komunalne in bolnišnične odpadne vode so tudi vir infektivnih organizmov, kot so npr. virusi, ki lahko prehajajo v vodno okolje tudi zaradi neprimernega ravnanja z infektivnimi odpadki. Biološke čistilne naprave iz vode odstranijo v povprečju le 50 % virusov, zato je voda kot»vir življenja«, lahko ključna za razširjanje nekaterih infektivnih organizmov, in z njimi povezanih bolezni, to pa zahteva stalen nadzor mikrobiološke kakovosti vode. Ker so koncentracije bioaktivnih snovi in infektivnih organizmov v telesu in okolju običajno zelo nizke, potrebujemo za njihovo določevanje visoko občutljive metode, predvsem kromatografske analizne tehnike sklopljene z masno spektrometrijo (GC/MS, LC/MS in LC MS/MS) in transmisijsko elektronsko mikroskopijo (TEM). Te metode so visoko občutljive, zahtevajo pa veliko časa in uporabo dragih aparatur in velikih količin reagentov in organskih topil, ki so škodljivi za okolje. Zaradi vse večje potrebe po analizah velikega števila okoljskih vzorcev, je nujen razvoj novih, tako imenovanih naprednih metod, ki omogočajo hitro in zanesljivo pregledovanje velikega števila vzorcev v čimkrajšem času. Analize s takimi "presejalnimi" metodami so praviloma manj natančne ali celo semikvantitativne, a kljub temu omogočajo zanesljivo identifikacijo neproblematičnih vzorcev in v praksi omejijo potrebo po uporabi zahtevnih klasičnih analiznih metod le na nekaj odstotkov vseh vzorcev. Zato je bil splošen cilj disertacije razvoj novih metod za občutljivo, hitro in cenovno učinkovito detekcijo farmacevtskih učinkovin, virusov in virusnih delcev v vodah in bioloških tekočinah in za ugotavljanje njihovih učinkov na organizme. Nove metode smo zasnovali na kombinaciji spektrometrije TLS (spektrometrija s toplotnimi lečami), mikrofluidike in imunoloških metod kot je ELISA. TLS kot visoko občutljiva tehnika (omogoča meritev absorbanc pod 10-6 ) lahko v kombinaciji z mikrofluidnimi tehnologijami zagotovi detekcijo zelo nizkih koncentracij analitov, krajši čas analize in manjšo porabo reagentov. V takih kombinacijah mikrofluidna tehnologija olajša in pospeši interakcije antigen-protitelesa ali encim-substrat v bioanaliznih sistemih, kar izhaja iz majhnih dimenzij kapilar in mikroreaktorjev (premer 10 do 100 µm), ki znatno skrajšajo IV

8 difuzijski čas velikih biomolekul. Obenem pa mikrofluidni sistemi zaradi 100 do 1000-krat krajših optičnih poti v primerjavi s konvencionalnimi spektrometrijskimi tehnikami, zahtevajo zelo občutljivo detekcijo, ki jo v primeru nefluorescenčnih analitov lahko zagotavlja le TLS. Tako je bil npr. s kombinacijo mikrofluidne ELISA tehnike in mikroskopske TLS detekcije (TLM) za določevanje B-natriuretičnega peptida (ang. brain natriuretic peptide) skrajšan čas analize z 20 ur na 20 minut, kot je opisano v literaturi. Pomembno je tudi, da je odziv TLS signala hiter (milisekunde), kar je zaradi relativno visokih linearnih hitrosti pretokov v mikrofluidnih sistemih nujen pogoj za učinkovito detekcijo v submikroliterskih vzorcih s koncentracijami v območju pg/ml. Za dosego konkretnih ciljev doktorske disertacije smo razvili nove analizne metode za naslednje izbrane analite: - jodirana kontrastna sredstva, - NGAL-na nevtrofilno gelatinazo vezani lipokalin, kot novi biomarker pokontrastne nefropatije, oblike akutne poškodbe ledvic, - protitelesa proti humanim papiloma virusom (HPV) in HPV-16 pseudovirionom. Pri razvoju metod za detekcijo jodiranih kontrastnih sredstev smo raziskali tudi kemijsko razgradnjo jodiranih kontrastnih sredstev ter tako istočasno razvili metodo za njihovo morebitno odstranjevanje iz odpadnih vod. Pri uvodnih raziskavah za razvoj metode za določevanje NGAL kot osnovo uporabili komercialno dostopne ELISA teste. Končni produkt reakcije substrata s hrenovo peroksidazo (HRP) v ELISA testu smo injicirali v mikrofluidni sistem, ki je služil le za transport vzorca do TLM detektorja na mikročipu. Tako smo ob primerljivi hitrosti analize dosegli mejo detekcije 1.4 pg/ml kar je 7 krat nižje v primerjavi s komercialnim ELISA testom (10 pg/ml). V nadaljevanju smo za razvoj metode za določevanje NGAL z µfia- TLM namesto komercialno dostopnih mikrotitrskih ploščic uporabili magnetne nanodelce kot trdne nosilce za primarna protitelesa ELISA testa. Protitelesa smo tako lahko z magnetnim poljem zadrževali v mikrofluidnem sistemu in v njem izvedli vezavo NGAL in sekundarnih protiteles, kot tudi reakcijo substrata s HRP. Metoda za določevanje NGAL je glede na meje detekcije (LOD je 2.3 pg/ml) občutljivejša v primerjavi s komercialnimi ELISA testi v standardnih mikrotitrskih ploščicah (LOD je 10 pg/ml) in bistveno skrajša čas analize. TLM v kombinaciji z mikročipom namreč omogoča krajši čas posameznega koraka inkubacije (z ene ure na le 5 min) v postopku pred samo detekcijo NGAL in s tem skrajša čas trajanja analize s štirih ur v primeru komercialnega ELISA testa na 35 minut ter tako omogoča analizo večjega števila vzorcev. Opravili smo tudi analizo realnih vzorcev krvi, ki je pokazala, da so vrednosti NGAL izmerjene z µfia-tlm ob uporabi magnetnih nanodelcev primerljive z vrednostmi izmerjenim s komercialnim ELISA testom. Kratek čas analize ob nadaljnji optimizaciji odpirajo nove možnosti za uporabo testa v medicinski diagnostiki in kliničnih raziskavah. Razvita metoda za določevanje NGAL je ob uporabi ustreznih protiteles lahko primerna tudi za detekcijo drugih farmacevtskih učinkovin ali različnih onesnaževal v okolju. V okviru disertacije smo razvili tudi metodo za določevanje anti-hpv-16 L1 protiteles v serumu žensk, okuženih s HPV-16 virusom. Pri tem smo za zagotavljanje selektivnosti uporabili HPV/16 pseudovirione kot modelni sistem virusov za razvoj ELISA testa na mikrofluidnem čipu. Imobilizirani HPV-16 pseudovirioni služijo kot antigen na V

9 katerega se vežejo anti-hpv-16 L1 protitelesa, ki smo jih zaznavali s sekundarnimi, s HRP označenimi protitelesi in TLM detekcijo produkta encimske reakcije substrata in HRP. Ob izvedbi klasičnega ELISA testa na mikrotitrski plošči in merjenju na čitalcu mikrotitrskih plošč smo dosegli spodnjo mejo detekcije 3.8 ng/ml. Ob izvedbi meritve končne raztopine v mikrofluidnem čipu s TLM smo spodnjo mejo detekcije znižali na 0.9 ng/ml. Podobno kot pri metodi za določevanje NGAL smo v nadaljevanju raziskav uporabljali magnetne nanodelce kot trdne nosilce za pseudovirione in vse korake ELISA postopka izvedli v mikrofluidnem čipu kjer smo končni produkt reakcije substrata s HRP tudi detektirali s TLM. Tako smo za določevanje protiteles proti pseudovirionom HPV-16 virusom dosegli spodnjo mejo detekcije 0.6 ng/ml, kar je šestkrat nižje v primerjavi s klasičnim ELISA testom. Obenem smo trajanje analize skrajšali z 10 ur na 30 minut. Metodo smo uspešno validirali tudi z analizami realnih vzorcev serumov žensk pri katerih je bila predhodno potrjena okužba s HPV-16 virusom. Za detekcijo kontrastnih sredstev smo razvili metodo, ki preko meritve koncentracije sproščenega jodida omogoča posredno semikvantitativno določitev koncentracije jodiranih kontrastnih sredstev. Za sproščanje jodida iz matične molekule kontrastnega sredstva iomeprola smo uporabili kemično reakcijo z bakrovimi ioni Cu2+ v prisotnosti H2O2. Po predhodni oksidaciji sproščenega jodida smo jod ekstrahirali v kloroform. Reakcija razgradnje kontrastnih sredstev je pokazala približno 70 % učinkovitost za odstranjevanje joda z izhodne spojine, če upoštevamo 60 % skupni izmerjeni izkoristek oksidacije jodida in ekstrakcije joda s kloroformom. Kloroform kot organsko topilo je zelo primeren za TLM meritve zaradi visokega ojačitvenega faktorja, ki je povezan z nizko toplotno prevodnostjo in visokim temperaturnim koeficientom lomnega količnika kloroforma, in teoretično zagotavlja približno 40-krat višjo občutljivost kot pri TLM meritvah v vodi, ter za vsak milivat vzbujevalne moči štirikrat višjo občutljivost kot spektrofotometrija. Ekstrakt joda smo injicirali v mikrofluidni čip in določili koncentracijo joda z optotermično mikroskopsko tehniko TLM. Razvita metoda μfia-tlm za posredno določevanje kontrastnih sredstev, ki temelji na detekciji joda, zagotavlja okrog 60-krat nižjo mejo detekcije v primerjavi s spektrofotometrijo. S kombiniranjem mikrofluidnih tehnologij in TLM smo uspeli izboljšati mejo detekcije ob manjši porabi reagenta in vzorca v primerjavi s spektrofotometrijo. Spodnja meja detekcije 18 ng/ml za iomeprol, ki smo jo dosegli z μfia-tlm, je 16-krat nižja v primerjavi z LOD za detekcijo iomeprola s tehniko HPLC (294 ng/ml). V primerjavi z LOD za določevanje iomeprola preko detekcije jodida z ionsko kromatografijo (133 ng/ml), omogoča µfia-tlm detekcijo okrog 7-krat nižjih koncentracij. S HPLC in HPLC/MS meritvami vzorcev raztopine po končanem postopku razgradnje kontrastnih sredstev pa smo pokazali, da izhodna spojina ni več prisotna in da nastajajo produkti, z različnim številom odcepljenih jodovih atomov iz izhodne molekule. S tem smo pokazali, da je uporabljena kemična reakcija razgradnje kontrastnih sredstev, kot je iomeprol, učinkovita in da bi jo lahko uporabili za odstranjevanje kontrastnih sredstev iz odpadnih vod. Reakcijo razgradnje kontrastnih sredstev, ki traja okrog 120 min lahko še skrajšamo z nadaljnjo optimizacijo reakcijskih pogojev. Nove analizne metode, razvite v tej disertaciji, zagotavljalo spodnje meje detekcije za izbrane spojine, ki so bistveno (tudi do 60-krat) nižje v primerjavi s klasičnimi analiznimi metodami s transmisijskim načinom merjenja. Istočasno nove metode omogočajo krajši čas analize in analizo večjega števila vzorcev za hitrejše izvajanje (fast screening) presejalnih VI

10 testov. µfia-tlm z uporabo magnetnih nanodelcev za določevanje NGAL in protiteles proti HPV virusom imajo v primerjavi s komercialnim ELISA testom na mikrotitrski ploščici številne prednosti, med katerimi so večja površina za vezavo protiteles, manjša poraba reagentov in tudi krajši čas analize. ELISA testi v mikročipih z uporabo mikrofluidnih črpalk omogočajo tudi lažje izvajanje korakov izpiranja in nanosa reagentov, ki so časovno zahtevni in pogosto prinašajo velike napake, če jih izvajamo z ročnim pipetiranjem. Čeprav tehnika TLS še ni dosegla primerne stopnje razvoja in uporabnosti za rutinske kemijske analize, lahko izboljšano metodo za detekcijo NGAL ali virusnih protiteles, ki smo jo razvili v okviru te disertacije, uporabimo za klinične študije in razvoj komercialnih testov za detekcijo virusov ali drugih bioaktivnih spojin za uporabo v bolnišnični diagnostiki. Ključne besede: ELISA, NGAL, PsVs, kontrastna sredstva, TLM VII

11 TABLE OF CONTENTS Page Abstract I Povzetek IV Table of Contents VIII List of Tables XIII List of Figures XIV Abbreviations XVIII Symbols XX 1. INTRODUCTION Environmental pollution with bioactive substances Fate in the environment Occurrence in water resources Human biomonitoring and biological effects of pharmaceuticals Removal of pharmaceuticals from waste water Detection of pharmaceuticals in the environment 5 2. THEORETICAL BACKGROUND Contrast agents Contrast agents structure and properties Contrast agents in the environment Detection of iodine contrast media in environmental samples Contrast agents and their effects on human health Contrast agents clearence Contrast agents detection in biological samples Contrast induced nephropathy Biomarkers of acute kidney injury Cystatin C NGAL NGAL level in blood and urine NGAL detection techniques Infective agents: virus like particles HPV viruses Virus like particles (VLPs) and pseudovirions (PsVs) Methods for detection of HPV viruses and HPV VLPs ELISA Microfluidics Thermal Lens Spectrometry Thermal Lens Microscope Nanobeads application Research objectives EXPERIMENTAL AND INSTRUMENTATION Chemicals and Reagents NGAL Procedure for measurement of NGAL with commercial ELISA kit Calibration curve Procedure for real sample analysis with commercial ELISA kit Sample collection Spiking experiment Measurement of final ELISA product on spectrophotometer Measurement of final ELISA product on TLS and FIA-TLS 41 VIII

12 3.2.5 Thermal Lens Microscope Clinical study-measurement of final product after ELISA test on µfia-tlm system Chessboard titration Incubation time, buffers, temperature, reagent volume SDS-PAGE and Western blot SDS-PAGE electrophoresis Western blot Nanobeads based ELISA assay Measurement of nanobeads ζ-potential Nanobeads number Determination of antibody concentration bound to nanobeads Number of bound antibody per nanobeads Nanobeads based ELISA test performed in plastic eppendorf tube Nanobeads based ELISA test in microfluidic chip Testing the flow rates, magnetic field strength Nanobeads based ELISA test with TLM detection Calibration curve Real sample analysis CONTRAST AGENTS Contrast agent detection Contrast agent degradation conditions Optimization of ph, buffer concentration Optimization of incubation time in the oven Calibration curve made on spectrophotometer Extraction efficiency Stability of chloroform iodine solution released by contrast agent 57 degradation Contrast agents measurement on µfia-tlm Calibration curve made on µfia-tlm Oxidation efficiency HPLC analysis of contrast agents and their degradation product Iomeprol calibration curve prepared on HPLC Contrast agent detection with ion chromatography HPLC-MS analysis of contrast agents degradation products HPV pseudovirions PsVs production SDS-PAGE of PsVs samples Silver staining procedure Western blot of PsVs samples L1 and L2 concentration determination by densitometry Optimization of conditions for PsVs based ELISA assay on microtiter plate 62 IX

13 Choice of buffers, temperature, incubation time Determination of PsVs and primary antibody 63 concentration Sample collection Real sample analysis with PsVs based ELISA assay on microtiter 64 plate µfia-tlm measurement of final ELISA product Western blot analysis of serum samples Dot blot analysis of serum samples PsVs nanobeads based ELISA assay in eppendorf tube Measurement of PsVs binding to nanobeads surface by Western 66 blot Measurement of PsVs binding to nanobeads surface with 67 commercial kit Nanobeads based ELISA assay in microfluidic chip with µfia- TLM detection Calibration curve of PsVs nanobeads based ELISA 68 assay Real sample analysis RESULTS AND DISCUSSION Development of TLS/TLM methods for determination of NGAL Determination of NGAL with commercial ELISA kit Real sample analysis with commercial ELISA kit Spiking experiment Measurement of final ELISA product on TLS Choosing appropriate wavelength for TLS 73 measurement FIA-TLS Impact of different solvents on TLS signal FIA-TLS calibration curve Measurement of final ELISA product on µfia-tlm Determination of optimal injection volume and flow 76 rate Calibration curve Reproducibility Application of developed µfia-tlm method in clinical study Clinical study measurement of final ELISA product on µfia-tlm Development of µfia-tlm ELISA for NGAL detection Optimization of primary Ab concentration Optimization of secondary Ab concentration Optimization of HRP-conjugate concentration Optimization of NGAL antigen concentration range 85 for calibration Optimization of buffers, blocking step, incubation time Nonspecific binding Sandwich ELISA protocol ELISA calibration curve ELISA assay of real samples SDS-PAGE and Western blot SDS-PAGE electrophoresis of plasma samples 89 X

14 Western blot of plasma samples ζ-potential of nanobeads Measurement of nanobeads number Determination of antibody concentration on nanobeads Number of immobilized antibody molecule per nanobeads Nanobeads based ELISA test in eppendorf tube Nanobeads based ELISA test in microfluidic chip Testing magnetic field, the flow rates, reagents volume, incubation time Nanobeads based ELISA test with TLM detection Calibration curve Real sample analysis Development of TLM method for contrast agent detection Calibration curve made on spectrophotometer Contrast agent degradation Choice of buffer, optimization of buffer ph, buffer 103 concentration Optimization of incubation time in the oven Extraction efficiency Stability of chloroform iodine solution released by contrast agent degradation Contrast agents measurement on µfia-tlm Calibration curve obtained on µfia-tlm Comparison of measurement of contrast agents degradation 109 product on spectrophotometer and on µfia-tlm Measurement of released iodine on µfia-tlm HPLC analysis of contrast agents and their degradation products HPLC analysis of contrast agents iomeprol Calibration curve Contrast agent detection with ion chromatography HPLC-MS/MS analysis of contrast agents degradation products Development of TLM method for detection of anti HPV-16 antibodies SDS-PAGE analysis of PsVs samples Silver staining of the gel after SDS-PAGE 126 electrophoresis Western blot of PsVs samples L1 and L2 protein concentration determination PsVs based ELISA assay on microtiter plate Choice of buffer, temperature, incubation time Optimization of the reagents concentration ELISA calibration curve Measurement of final ELISA product on µfia-tlm Comparison of the results obtained with ELISA and µfia-tlm Western blot analysis of serum samples 132 XI

15 4.3.8 Dot-blot analysis of serum samples PsVs nanobeads based ELISA in eppendorf tube Measurement of PsVs binding to nanobeads surface by Western 134 blot Measurement of PsVs binding to nanobeads surface with 135 commercial kit Nanobeads based ELISA assay with µfia-tlm detection Calibration curve Real sample analysis CONCLUSIONS AND FUTURE OUTLOOK REFERENCES 141 XII

16 LIST OF TABLES Table 1 Contrast media classification according to osmolality Table 2 Contrast agents selected for present study Table 3 Methods for analysis of iodinated contrast media in water and solid matrices Table 4 Methods for contrast media detection in biological samples Table 5 Comparison of different sandwich NGAL ELISA kits and assay NGAL detection techniques (Clerico et al., 2013) Table 6 Methods for detection of VLPs and antibodies against VLPs published in the literature Table 7 Molecular methods for HPV detection Table 8 Thermooptical properties of some organic solvents (Bialkowski, 1996) Table 9 Chip characteristics of Y-joint microfluidic chip (The Dolomite Centre Ltd., UK) Table 10 Chip characteristics of four channel spotting chip PMMA Table 11 HPLC conditions for different contrast agent detection Table 12 Silver staining procedure Table 13 NGAL concentration in serum and plasma samples expressed as mean value and corresponding standard deviation measured with commercial ELISA kit, N is number of samples. (number of replicate for each sample n=3). Table 14 Analyte concentration in serum, spiked serum and corresponding recoveries (number of replicate for each sample n=3). Table 15 NGAL concentrations at different times after application of contrast agent (for 30 patients enrolled in clinical study) determined by ELISA with microtiter plate reader and by µfia-tlm expressed as mean value with corresponding standard deviation. Sample from each patient was measured in triplicate (n=3) Table 16 Summary of optimized conditions for sandwich ELISA assay Table 17 NGAL concentrations in different plasma and urine samples measured with optimized ELISA assay and commercial ELISA kit. (number of replicate for each sample n=3). Table 18 Calculated nanobeads volumes Table 19 Optimized conditions for nanobeads based ELISA assay Table 20 NGAL concentrations in five plasma samples measured by nanobeads ELISA with µfia-tlm detection and by a commercial ELISA kit. (n=3) Table 21 Extraction+oxidation efficiency calculated with M KI Table 22 Calculated iodine concentrations released from contrast agents after different incubation times measured on spectrophotometer Table 23 Calculated iodine concentrations released from contrast agents after different incubation times measured on µfia-tlm Table 24 Fragmentation ions obtained after MS/MS analysis of precursor ions Table 25 Optimized conditions for PsVs based ELISA assay on microtiter plate Table 26 Concentration of anti L1 HPV 16 antibody measured by ELISA, µfia-tlm and nanobeads PsVs ELISA with TLM detection. (n=3) Table 27 Optimized conditions for nanobeads based PsVs ELISA assay XIII

17 LIST OF FIGURES Figure 1-Routes of pharmaceuticals entering the environment (Boxall, 2004) Figure 2-Diagrammatic representation of the relationship between environmental distress signal detectability and ecological relevance (Moore et al., 2004) Figure 3-Evolution of acute kidney injury (modified from Bellomo et al., 2012) Figure 4-Creation of Human Papilloma Virus VLP (Berzofsky et al., 2004) Figure 5-Schematic presentation of sandwich ELISA test on microtiter plate Figure 6-TLM schematic presentation of interaction between pump and probe beam and sample Figure 7-Schematic illustration of the immunoassay in micro-titer plate (left) and immunoassay chip (right) Figure 8-FIA-TLS experimental setup consisting of one a HPLC pump, an injection valve (200 µl injection loop), flow through cell, pump/probe unit, TLS detection unit Figure 9-Photography of a TLM system used for measurement of final product after ELISA test and for nanobeads based ELISA assay on a microchip. The photodiode detector is not seen behind the microfluidic chip. Figure 10-a) Schematic drawing of a microfluidic chip used in this work for NGAL detection. To schematically show the μfia-tlm detection in the microchip, the microchannel is zoomed in. b) Y-joint microfluidic chip (The Dolomite Centre Ltd., UK) Figure 11-Schematic presentation of chessboard titration on microtiter plate (a-first titration, b-second titration) Figure 12-Schematic presentation of magnetic nanobeads Figure 13-a) nanobeads solution in eppendorf tube attached by magnets b) magnetic rack applied in this work Figure 14-a) four channel spotting chip made from PMMA (ChipShop, Jena, Germany) b) foil, c) cover lid, d) fluid connectors, e) silicone tubings Figure 15-a) Permanent magnets applied in this work b) schematic presentation how magnets stick together Figure 16-Assembling microfluidic chip a) injection of nanobeads solution with connector and syringes, b) testing of different magnets combination, c) combination of two magnets positioned above and below microfluidic chip keeping nanobeads solution inside the channel Figure 17-Steps in nanobeads based ELISA assay Figure 18-Schematic presentation of nanobeads based ELISA for NGAL detection. Microchips were connected with silicone tubings. To schematically show part of the fourchannel chip with nanobeads trapped between magnets, the microchannel is zoomed. Figure 19-Schematic presentation of microfluidic chip applied in this work for contrast agents detection on µfia-tlm Figure 20-Calibration curve for NGAL obtained with four-parameter logistic curve fitting Figure 21-NGAL concentrations measured with commercial ELISA kit in serum and plasma samples of 7 healthy individuals (control) and 11 patients undergoing coronarography Figure 22-Absorbance spectra of final ELISA product (50 pg/ml of NGAL) measured on spectrophotometer XIV

18 Figure 23-TLS signals for two replicates of final ELISA product undiluted (NGAL 1000 pg/ml) Figure 24-TLS signals of a) two replicates of final ELISA product (100 pg/ml of NGAL antigen) diluted two times in 60 % ethanol with water as mobile phase, b) final ELISA product (10 pg/ml of NGAL antigen) diluted two times in 60 % ethanol with ethanol as mobile phase c) final product (100 pg/ml of NGAL antigen) diluted two times in water with water as mobile phase Figure 25-Linear calibration curve obtained on FIA-TLS (R 2 = ) Figure 26-TLM signals for different injection volumes of final ELISA product from NGAL standard (100 pg/ml) (water was used as a mobile phase with flow rate of 10 µl/min, sample injection flow rate was 200 µl/min) Figure 27-ELISA calibration curve for NGAL obtained with linear fitting (R 2 = ) Figure 28-Calibration curve for µfia-tlm system obtained with NGAL standards of known concentration (R 2 =0.9948, for pg/ml range) Figure 29-μFIA-TLM signals for four replicate injections of final ELISA product of three replicates of 100 pg/ml standard of NGAL Figure 30-NGAL dynamics measured with Tecan and µfia-tlm in plasma samples collected from four patients before the coronary angiography and up to 12 hours after injection of the contrast medium Figure 31-NGAL concentrations measured with commercial ELISA kit in 30 patients before the coronary angiography and 1, 2, 4, 6, 12 h after injection of the contrast medium. Figure 32-NGAL concentrations measured with µfia-tlm in 30 patients before the coronary angiography and 1, 2, 4, 6, 12 h after injection of the contrast medium. Figure 33-Comparison of NGAL level measured with ELISA and TLM Figure 34-Calibration curves for optimization of ELISA assay obtained with different primary Ab concentrations and different antigen (NGAL) concentration Figure 35-Calibration curves for optimized ELISA assay obtained with different secondary Ab and antigen (NGAL) concentration Figure 36-Calibration curves for optimized ELISA assay obtained with different enzymeconjugate and antigen (NGAL) concentration Figure 37-NGAL ELISA calibration curve obtained with four-parameter logistic curve fitting using PBS and carbonate buffer for immobilization of primary Ab. Each data point represents mean of three replicates with corresponding standard deviation. Figure 38-NGAL concentrations in 16 plasma samples measured with ELISA kit and homemade ELISA Figure 39-SDS-PAGE electrophoresis of a) Line 1-NGAL standard (5 µg/ml), Line 2-NGAL (2.5 µg/ml), Line 3-NGAL (1 µg/ml), Line 4- protein marker; b) plasma samples collected from six healthy individuals Line 1-NGAL standard (5 µg/ml), Line 2-7 plasma sample of six healthy persons, Line 8-protein marker Figure 40-Western blot of a) plasma sample from patient undergoing coronary angiography Lines 1-6 NGAL level in plasma samples before and 1, 2, 4, 6, 12 hours after injection of contrast agents, Line 7-NGAL standard, Line 8-protein marker b) Lines 1-6 NGAL forms in urine samples from six healthy individuals respectively, Line 7-NGAL standard; Line 8- protein marker Figure 41-ζ potential of nanobeads suspension applied in this work, as a function of ph Figure 42-Calibration curve of Easy Titer Mouse IgG Assay kit XV

19 Figure 43-Bound primary Ab concentration at different incubation times Figure 44-a) Supernatant with unbounded enzyme collected after every washing step. First tube is the enzyme solution that was added in the tubes with nanobeads for incubation and removed before first washing step. Tubes 2-6 represents supernatant after 1 st -5 th washing step respectively b) final blue colored product (NGAL concentration 2 ng/ml) in eppendorf tube after addition of TMB substrate Figure 45-Nanobeads in the microchannel retained with permanent magnet (picture made with optical microscope with 80x magnification) Figure 46-Combination of six magnets positioned at three channels of microfluidic chip Figure 47-Nanobeads based ELISA test performed in two channels with a) buffer as negative control (upper channel) and NGAL standard (2 ng/ml) and b) plasma sample in upper channel and NGAL standard (2 ng/ml) in the lower channel Figure 48-Calibration curve for nanobeads based ELISA assay obtained with four-parameter logistic curve fitting Figure 49-µFIA-TLM signals for 8 replicate pulses of final ELISA product (from NGAL standard 500 pg/ml) obtained on nanobeads based ELISA assay Figure 50-Spectra recorded with different iodine concentration in chloroform Figure 51-Calibration curve of different iodine concentration in chloroform (R 2 = ) measured by spectrophotometer Figure 52-Iodine extraction with chloroform after contrast agent degradation Figure 53-Spectra of iodine released from iohexol (initial concentration of 7.55 mg/ml iohexol which corresponds to 3.50 mg/ml of iodine) in reaction with buffers of different ph (in chloroform) Figure 54-Iodine concentration as function of incubation time in the oven Figure 55-Spectrophotometric calibration curves for iodine (standard solutions in chloroform) and for KI (KI standard solutions were first oxidized and then iodine was extracted with chloroform) (R 2 = , R 2 = ) Figure 56-a) Stability of iodine in chloroform released by degradation of iohexol measured with spectrophotometer ( M I 2 initial iodine concentration) b) Stability of iodine in chloroform released by degradation of diatrizoate measured with spectrophotometer ( M I 2 initial iodine concentration) Figure 57-Influence of different injection volume of the sample on µfia-tlm signal for the iodine solution ( M) in chloroform (sample flow rate 10 µl/min, chloroform flow rate 10 µl/min) Figure 58-Linear calibration curve obtained on µfia-tlm with iodine solutions in chloroform Figure 59-µFIA-TLM signals in chloroform for iodine released from iohexol after 60, 90, 120 min incubation in the oven Figure 60-HPLC analysis of a) diatrizoate solution (0.705 mg/ml) and b) diatrizoate solution after 180 minutes of degradation (C 18 Purospher, 250 x 4.6 mm, 5 µm column; AcN : H 2 O, (5:95 %) ph= 3; injection volume 20 µl, flow rate 1 ml/min) Figure 61-HPLC analysis of a) iohexol contrast agent solution (1.82 mg/ml) and b) iohexol solution after degradation (C 18 Purospher, 250 x 4.6 mm, 5 µm column; AcN : H 2 O, (5:95 %) ph= 3; injection volume 20 µl, flow rate 1 ml/min) Figure 62-a) HPLC chromatogram of iomeprol mg/ml b) HPLC chromatogram of iomeprol solution after degradation (C 18 Purospher, 250 x 4.6 mm, 5 µm column; AcN : H 2 O, (5:95 %) ph= 3; injection volume 20 µl, flow rate 1 ml/min) Figure 63-Calibration curve made on HPLC with iomeprol standards XVI

20 Figure 64-Calibration curve made on ion chromatograph with iodide standards Figure 65-MS spectrum of iohexol standard solution ( M) Figure 66-HPLC/MS chromatogram of analyzed iohexol standard solution ( M) Figure 67-MS spectra of pseudomolecular ions of iohexol Figure 68-HPLC/MS chromatogram of iohexol after degradation with marked four fractions Figure 69-MS spectrum of fraction IV collected during HPLC analysis Figure 70-Proposed degradation pathway of iohexol with calculated m/z values Figure 71-Proposed fragmentation pathway for pseudomolecular ion with m/z value of Figure 72-Proposed fragmentation pathway for pseudomolecular ion with m/z value of Figure 73-Proposed fragmentation pathway for pseudomolecular ion with m/z value of Figure 74-Proposed fragmentation pathway for precursor ion with m/z value of Figure 75-SDS-PAGE analysis; Line 1-10 mg/ml BSA, Line 2-4 mg/ml BSA, Line 3-2 mg/ml BSA, Line 4-1 mg/ml BSA, Line mg/ml BSA, Line 6-PsVs sample, Line 7- protein marker Figure 76-Silver staining of SDS-PAGE gel, Line 1-BSA 8 mg/ml, Line 2-BSA 4 mg/ml, Line 3-BSA 2 mg/ml, Line 4-BSA 1 mg/ml, Line 5-PsVs containing sample, Line 6-protein markers Figure 77-a) Western blot results of PsVs samples incubated with anti L1 HPV 16 antibody, Line 1-PsVs 100 times diluted, Line 2-PsVs 150 times diluted, Line times diluted, Line times diluted, Line 5-protein marker b) Western blot of PsVs sample incubated with HPV 16 L2 mouse monoclonal antibody Line 1-PsVs 100 times diluted, Line 2-PsVs 150 times diluted, Line times diluted, Line 4-PsVs 1000 times diluted, Line 5-protein marker Figure 78-ELISA calibration curve made with two different PsVs concentrations constructed with four-parameter logistic curve fitting Figure 79-PsVs ELISA calibration curve Figure 80-Calibration curve obtained on µfia-tlm with four-parameter logistic curve fitting Figure 81-Line 1-11 serum sample numbers respectively from 1 to 11, Line c-is the positive control with anti L1 HPV 16 antibody, Line m-protein markers Figure 82-Dot blot analysis of 11 serum samples respectively. Numbers represents serum numbers while c is positive control Figure 83-Nanobeads based ELISA assay in eppendorf tube. Tube 1, 2, 3 represents aliquots of supernatant after 1 st, 2 nd, 3 rd washing step where substrate solution was added, Tube 4- nanobeads after 3 rd washing step after addition of substrate Figure 84-Western blot of PsVs samples before and after incubation with nanobeads Line 1- PsVs sample 60 times diluted in PBS, Line 2-aliquot of PsVs samples immediately after mixing with nanobeads, Line 3-aliquot containing free PsVs after 1st washing step, Line 4-7 aliquot containing free PsVs after 2 nd, 3 rd, 4 th and 5 th washing step respectively, Line 8- nanobeads with PsVs proteins on the surface, Line 9-protein marker Figure 85-Nanobeads based PsVs ELISA test performed in one channel with anti L1 HPV 16 antibody as positive control (150 ng/ml) Figure 86-Calibration curve for nanobeads based PsVs ELISA assay XVII

21 ABBREVIATIONS 1 Ab Primary antibody 2 Ab Secondary antibody AA Acrylamide AKI Acute kidney injury APS Ammonium persulfate AOX Adsorbable organic halogen AUC Analytical ultracentrifugation BAA Bis-Acrylamide BSA Bovine serum albumin CBB-R-250 Coomassie Briliant Blue-R-250 CIN Contrast induced nephropathy CMIA Chemiluminescent Microparticle Immuno Assay Cys C Cystatin C DLS Dynamic light scattering EDTA Ethylenediaminetetraacetic acid ELISA Enzyme linked immunosorbent assay ESI Electrospray ionization FIA Flow injection analysis GFR Glomerular filtration rate Hsp-72 Heat shock protein-72 HRP Horseradish peroxidase HPV Human Papilloma Virus HPLC-DAD High Performance Liquid Chromatography-Diode Array detector HPLC-UV High Performance Liquid Chromatography- ultraviolet detection ICP/MS Inductively coupled plasma-mass spectrometry IC ICP-MS Ion chromatography-inductively coupled plasma-mass spectrometry ICM Iodinated Contrast Media IEC Ion-exchange chromatography IgG Immunoglobulin G IL-6 Interleukin-6 IL-8 Interleukin-8 IL-18 Interleukin-18 KIM-1 Kidney injury molecule-1 L-FABP Liver fatty acid binding protein LOD Limit of detection LOQ Limit of quantification LC/MS Liquid chromatography-mass spectrometry LC-ESI-MS Liquid chromatography-electrospray ionization-mass spectrometry LC-UV Liquid chromatography ultraviolet detection kda kilo Dalton khz kilo Hertz NAG N-acetyl-β-D-glucosaminidase NdFeB Neodymium-Iron-Boron NGAL Neutrophil gelatinase-associated lipocalin Md Median XVIII

22 MECC mv PBS π-gst PMMA POPLC PsVs RSD RP-HPLC SEC SCr SDS-PAGE SDS SEM SPE STPs TAOI TEM TEMED TFA TMB TNF-α TLM TLS UPLC USE WHO WWTP VLP Micellar electrokinetic capillary chromatography mili Volt Phosphate buffer saline π-glutathione s-transferase Polymethylmetacrylate Phase optimized liquid chromatography Pseudovirions Relative standard deviation Reversed-phase high performance liquid chromatography Size exclusion chromatography Serum creatinine Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sodium Dodecyl Sulfate Scanning Electron Microscopy Solid Phase Extraction Sewage treatment plants Total adsorbable organic iodine Transmission Electron Microscopy Tetramethylethylendiamin Trifluoroacetic acid 3,3',5,5'-Tetramethylbenzidine Tumor necrosis factor-α Thermal lens microscopy Thermal lens spectrometry Ultra Performance Liquid Chromatography Ultrasonic Solvent Extraction World Health Organization Wastewater treatment plants Virus like particles XIX

23 SYMBOLS A A 1 A 2 D - dn dt E Ɛ f F I I bc I bc k l λ m M m 1, m 2, m 3 n N Na ρ σ P R r sa SA T V x x 0 y Absorbance, concentration of the added analyte in the spiked portion minimum asymptote maximum asymptote Thermal diffusivity Temperature coefficient of the sample Enhancement factor Molar absorption (Extinction) coefficient Modulation frequency, or focal length of lens Analyte concentration in spiked sample Analyte concentration in serum sample Relative change in the beam center intensity Thermal conductivity of the sample Sample length, length of fluid Wavelength of the probe beam, wavelength of the excitation beam, thermal wavelength Mass Molecular weight Mass of nanobeads core, mass of silicone coat with amino groups, mass of one nanobead n-number of moles, mole quantity, number of measurements Number of nanobeads present in 1 g, number of IgG 1 molecule per ml of nanobeads, number of samples Avogadro s number Density Standard deviation Excitation laser power, Hill slope Recovery in percent Radius Surface area of one nanobead Total surface of all nanobeads SA present in 1g transmittance through a sample Volume Concentration in pg/ml Inflection point Response value expressed in absorbance, lock-in signal XX

24 1. INTRODUCTION 1.1 Environmental pollution with bioactive substances A bioactive substance is any substance that has a biological activity and the ability to interact with a living tissue or system. It is also defined as a substance that has an effect on a living organism, tissue or cell including interactions at molecular level. Bioactive compounds are essential and nonessential compounds that occur in nature, are part of the food chain, and can be shown to have an effect on human health (Biesalski et al., 2009) such as for example antibiotics, enzymes, vitamins etc... Among them pharmaceuticals are one of the most known group of substances having an effect on, or causing a reaction in living organisms. In the past decades various pharmaceuticals and personal care products (PPCPs) were produced and released into the aqueous environment extensively. Among them are antibiotics, steroids, antidepressants, antimicrobials, disinfectants, stimulants and many other chemicals which are used on a daily basis for various purposes. Concerns about the presence and possible harmful effects of these compounds are constantly increasing (García et al., 2013). Natural and anthropogenic impacts on ecosystem and human health are both an urgent and international problem (Moore et al., 2004). Pharmaceuticals have been identified as emerging contaminants of concern already fifteen years ago (Daughton and Ternes, 1999). They are traditionally applied in human and veterinary medicine, but their usage and release into the environment is still not strictly controlled. In countries with growing economy such as China, pharmaceuticals were considered as rising micropollutants as they may have significant adverse environmental and human health effects (Jiang et al., 2013) Fate in the environment Pharmaceuticals are designed to have a specific mode of action and once they end up in the environment they can cause unwanted effects in non-target organisms. They are potentially persistent, hazardous and ubiquitous pollutants (Mendoza et al., 2015). Since they are biologically active compounds that are made to be hardly biodegradable and are often water soluble, they can be found in waste waters and can easily end up in natural waters. The behavior and fate of pharmaceuticals and their metabolites in the aquatic environment is still not well understood. The low volatility of pharmaceuticals indicates that they enter the environment primarily through aqueous transport (Fent et al., 2006). Due to their large consumption, pharmaceuticals may enter the aqueous environment (see Fig 1.) directly or indirectly through anthropogenic activities such as industries, sewage discharge, livestock breeding, fertilizing with manure and landfill leachate, resulting in their presence in surface water and groundwater (Sui et al., 2015; Matamoros et al., 2012). 1

25 Figure 1-Routes of pharmaceuticals entering the environment (Boxall, 2004) Occurrence in water resources Beside households and industries, hospitals are particularly important and interesting among various sources discharging pharmaceuticals into the environment. In a hospital numerous procedures (anaesthesia, anticancer treatment, diagnosis, etc.) are performed which lead to the consumption of large quantities of pharmaceuticals. Among them antibiotics, antiinflammatory drugs, β-blockers and X-ray contrast media have been used extensively (Jiang et al., 2013), and are consumed in the ton-range per year (Pérez and Barceló, 2007). Since they are designed to have a physiological effect on humans and animals already in trace concentrations, amounts released by excretion via the toilette seems to be at first instance of minor importance. But, because of frequent use of pharmaceuticals in large quantities they became one of the main pollutants in the environment. Medical substances are excreted through urine or faeces as a mixture of metabolites, as unchanged substances or conjugated with an inactivating substituent attached to the molecule, depending on the pharmacology of the substance of interest (Rang et al., 2015). Usually they are excreted only slightly transformed or even unchanged mainly conjugated to polar molecules (e.g. as glucuronides). These conjugates can simply be split during sewage treatment and the original pharmaceuticals will then be released into the aquatic environment mostly by effluents from municipal sewage treatment plants (STPs) (Al Aukidy et al., 2014). Since they are persistent to biological degradation because of continuous input they could remain in the environment for a long time and their presence there is considered to be harmful in both low and high concentration (Klavarioti et al., 2009). Physicochemical analyses have confirmed the presence of drug residues and their metabolites in all different compartments of the aquatic 2

26 environment: surface water, wastewater, groundwater, and drinking water (Houeto et al. 2012; Mompelat et al., 2009; Cunningham et al., 2009). Pharmaceuticals end up in soil, surface waters and eventually in ground and drinking water after their excretion through the sewage system and into the influent of wastewater treatment plants (Darlymple et al., 2007). Verlicchi et al., (2010) showed that high concentrations of pharmaceuticals have been found in hospital wastewater in amounts greater than in urban wastewater Human biomonitoring and biological effects of pharmaceuticals Since humans are daily exposed to a complex mixture of chemicals in their lives through the environment, products, food and drinking water, the impact of pollutants on ecosystems and human health became an urgent and international issue (Moore, 2000). Human biomonitoring allows us to measure our exposure to chemicals by measuring either the substances themselves, their metabolites or markers of subsequent health effects in body fluids or tissues (Angerer et al., 2011). Crucial issue with pharmaceuticals as pollutants is not their acute toxic effects but their chronic toxicity. These compounds are commonly present at low levels in the lifecycle of many aquatic organisms and are especially substantial for those living in waters receiving sewage effluent (rivers) (Jiang et al., 2013). The need to detect and evaluate the impact of pollution on the quality of environment, particularly in case of low concentrations of increasingly complex mixtures of contaminants, has led to studies and development of the so-called biomarkers, which are molecular markers of the biological effects of contaminants on organisms. Biomarkers include a variety of specific molecular, cellular and physiological responses of key species to contaminant exposure. A response is generally indicative of either contaminant exposure or poor health. The challenge is to integrate individual biomarker responses into a set of tools and indices capable of detecting and monitoring the deterioration in health of a particular organism (Allen and Moore, 2004). In environmental terms a biomarker was originally defined as a change in a biological response (ranging from molecular through cellular and physiological responses to behavioural changes) which can be related to exposure to/or toxic effects of environmental chemicals (Peakall, 1994). Environmental contamination problem could be partly resolved in the use of diagnostic clinical-type laboratory-based ecotoxicological tests or biomarkers combined with direct immunochemical tests for contaminants. These rapid ecotoxicological tests give information about health status of individuals based on relatively small samples. For the purpose of environmental monitoring contaminant's concentrations can be measured in air, soil, water, plants, organisms and food. For human biomonitoring the analysis biomarker can be performed in different matrixes such as: blood, urine, breast milk, nails, saliva, meconium, adipose tissue, teeth, hair etc Biomarkers were also applied to connect effects from molecular and cellular level up to higher levels of biological organization. The sequential order of responses to pollutant stress within a biological system is presented on Fig. 2. To connect adequately impact of pollutants to ecosystem and human health through various hierarchical levels requires an integrated approach based on available information about pollutant uptake, detoxication and pathology with each other and with other complex effects (Moore et al., 2004). 3

27 Figure 2-Diagrammatic representation of the relationship between environmental distress signal detectability and ecological relevance (Moore et al., 2004) Pharmaceuticals released in the environment may have toxic impact on any level of the biological hierarchy, i.e. cells, organs, organisms, population, ecosystems, or the ecosphere (Klavarioti et al., 2009). Beside toxic effects, certain classes of pharmaceuticals like antibiotics may cause long-term and irreversible change to the micro-organisms genome, making them resistant in their presence, even at low concentrations (Guardabassi et al., 1998). Pal et al., (2010) found that pharmaceuticals have negative influence on freshwater fish and invertebrates. Thus the medical substances have many of the required properties to bioaccumulate and provoke effects in the aquatic or terrestrial ecosystems Removal of pharmaceuticals from waste water When pharmaceuticals or any other xenobiotics enter aquatic environment there are mainly three possible fates: (a) mineralization to carbon dioxide and water (b) if the compound entering the plant is lipophilic it can t be degraded easily and partly retain in the sediment sludge (c) metabolize to a more hydrophilic molecule, passes through the wastewater treatment plant and ends up in the receiving waters (which are surface waters, mainly rivers) (Jiang et al., 2013). Since surface water is broadly used as a water source for drinking water, the widespread distribution of pharmaceuticals in surface waters may cause a problem to water utilities. Several studies (Sui et al., 2015; Matamoros et al., 2012; Putschew et al., 2000) reported about the presence of a considerable range of pharmaceuticals and contrast media in surface waters and groundwater at concentration levels of ng/l to μg/l. Several investigations (Ternes et al., 2004; Joss et al., 2006; Verliefde et al., 2007; Vieno et al., 2006) showed that substances of pharmaceuticals origin are often not removed quantitatively by current techniques during waste water treatment and also not biodegraded in environment. Therefore, it is important to evaluate water treatment processes considering their potential for 4

28 eliminating pharmaceuticals (Doll and Frimmel, 2004). In waste water treatment, two removing processes are generally important: adsorption to suspended solids (sewage sludge) and biodegradation (Fent et al., 2006). According to Larsen et al., (2004) there are four approaches to remove micropollutants: optimization of existing technology at wastewater treatment plants (WWTP), upgrade WWTP with new technology, source control, and source separation. The main focus usually lies on end-of-pipe measures, and ozonation of the WWTP effluent or addition of powdered activated carbon were evaluated as promising tertiary treatment step. Currently for removing of pharmaceuticals from waste water are used advance oxidation processes including: heterogeneous photo-catalysis, ozonation (Dantas et al., 2008; Lei and Snyder et al., 2007), Fenton and photo-fenton reaction (Kulik et al., 2008; Gonzalez et al., 2007), sonolysis (Hartmann et al., 2008), treatment with UV/H 2 O 2 (Pereira et al., 2007), wet air oxidation, electrolysis (Sirés et al., 2007) Detection of pharmaceuticals in the environment Currently, most common applied analytical techniques for monitoring of micropollutants such as pharmaceuticals are solid phase extraction (SPE) and chromatography technology coupled with mass spectrometry, GC/MS, LC/MS and LC MS/MS (Wille et al., 2012; Cimetiere et al., 2013; Hu et al., 2011). These techniques require greatly specialized equipment, long time and relatively high cost of analysis. Sample preparation prior to injection on the analytical instrument is time- and labor-intensive step. Matrix effect is one of the main disadvantages associated with LC MS/MS, especially when working in the electrospray ionization (ESI) mode (Jiang et al., 2013). Since the matrix is such a complex mixture containing several pollutants with similar or different properties there is a clear need for new rapid cost-effective analytical methods for environmental application, with an emphasis on reduced cost in comparison to existing techniques. Clinical chemists have applied immunoassay techniques to detect and quantify various compounds of physiological relevance. As the most sensitive immunoassay and at the same time a relatively rapid screening tools for determination of trace organic pollutants are considered enzyme-linked immunosorbent assays (ELISA) (Levine and Asano, 2004). In environmental monitoring and research ELISA method based on antigen-antibody interaction is used to measure and quantify target compounds in field samples. Therefore, with appropriate calibration and careful comparison with the results obtained by conventional analytical methods immunoassays were used for rapid low cost pollutant determination in soil, sediment, water and body fluids (Deng et al., 2003; Calisto et al., 2011; Shelver et al., 2008). Nowadays immunoassay-based sensors, usually called immunosensors became an interesting alternative for chromatographic techniques. Immunoassays offer several advantages such as: simplicity, inherent sensitivity, specificity against particular compound of interest. Beside this they are rapid, with low cost of assay, little sample pre-treatment, possibility of simultaneous detection of large number of samples making them as a potential analytical tools for screening purposes. Their application for environmental monitoring is thus justified and has been reported in several studies (Salvador et al., 2007; Valera et al., 2013; Sanvicens et al., 2011). 5

29 Chemical analysis field is still not adequately investigated for applying of the flowinjection analysis (FIA) and its diverse variants for the determination of trace organic pollutants in environment. Numerous such applications have been already reported, e.g. for determination of pesticide residues with biosensors used as detectors in flow systems (Prieto- Simon et al., 2006; Wong and Sotomayor, 2014). Flow-injection analysis methods require suitable method for on-line sample processing and the use of highly sensitive detection method. Therefore FIA coupled with several analytical techniques such as ELISA and Thermal Lens Spectroscopy (TLM) could be highly promising in the future for monitoring and determination of low concentration of environmental contaminants. In this dissertation environmental pollution with pharmaceuticals as one of the main pollutants was first described and then research goals and methods applied in this study were described. In the experimental part experimental design of the new methods for detection of biologically active compounds was introduced. In the next chapter of results and discussion obtained results of optimized methods were presented and compared with standard analytical methods. At the end conclusions and future perspectives are presented. 6

30 2. THEORETICAL BACKGROUND In the introduction chapter environmental pollution with pharmaceuticals was described and some analytical methods for their detection were mentioned. In this chapter biologically active compounds of interest and most commonly used methods for their detection in human and in the environment are described. As representative of pharmaceuticals contrast agents were investigated, their impact in humans, environmental pollution and analytical methods for their detection were reviewed. Among different analytical methods that could be applied for this purpose special interest is put on one of the photothermal techniques called thermal lens spectrometry (TLS) and its miniaturized instrumentation - thermal lens microscope (TLM). This method was combined with microfluidic devices for development of methods for detection of biologically active compounds. At the end of this chapter research goals were presented. 2.1 Contrast agents Contrast agents structure and properties Contrast agents are one of the most widely applied pharmaceutical compounds in medicine which are frequently used for intravascular administration in radiographic procedures, since they create distinction between the organs, blood vessels and the surrounding tissue (Christiansen, 2005). Most of these highly hydrophilic compounds are derivatives of 2, 4, 6-triiodobenzoic acid containing on benzene ring three iodine atoms which magnify X-ray absorption and on the other side they have polar carboxyl and hydroxyl moieties to allow high water solubility. Contrast agents are chemically inert drugs which are administrated intravenous, designed to be very stable in human metabolism and excreted mainly through urine within one day (Seitz et al., 2006). Different types of contrast agents exist. Some contrast media are ionic having one or more carboxyl groups, others are amide derivatives and, as such are neutral compounds. All types of compounds are of low molecular weight, are highly water-soluble, have low protein binding capacity and are non-reactive (Christiansen, 2005). Contrast media is classified according to osmolality, which represents the total particle concentration of the solution (molecule number dissolved in a specific volume). Contrast media classification described by Barrett and Parfrey, (2006) is presented in Table 1. Table 1-Contrast media classification according to osmolality Contrast medium class Osmolality (mosm/kg) Example high-osmolar Diatrizoate low-osmolar iohexol iso-osmolar 290 iodixanol Iodinated contrast media is divided according to molecular structure into ionic (1 st generation of contrast media) and non-ionic forms (2 nd generation of contrast media) which could be monomeric and dimeric (Sacher et al., 2005; Jost et al. 2009). 7

31 The ionic monomers (three iodine atoms: two particles) dissociate into anion and cation, causing an increase of the osmolality value that becomes double. Therefore if ionic monomer is used there is a problem for patient safety. The non-ionic monomers (three iodine atoms: one particle) consist of benzene ring substituted in position 1, 3, 5 to allow adequate solubility. The ionic dimers (six iodine atoms: two particles) consists of two monomers one ionic and one non-ionic bounded through covalent bond. Third generation of contrast agents such as iodixanol are non-ionic dimeric (six iodine atoms: one particle) contrast media (Morcos and Thomsen, 2001) with osmolality equal to the blood. Nowadays more than 80 % of medical examinations for diagnosis of soft tissues are performed with non-ionic contrast media, since they showed considerably better tolerability as compared to the older class of ionic contrast media (Kim et al., 2015). In Table 2 contrast media selected for present study are presented. 8

32 Table 2-Contrast agents selected for present study Contrast agent Iohexol Diatrizoate Chemical structure IUPAC name 5-(N-2,3)- dihydroxypropylacetami do-2,4,6-triiodo-n,n - bis(2,3- dihydroxypropyl) isophthalamide 3,5-diacetamido-2,4,6- triiodobenzoic acid (sodium salt) Trade name, class Omnipaque Low osmolar, Nonionic (monomer) Hypaque, Gastrografin Urografin Ionic (monomer) Molecular weight (g/mol) Iodine concentration (mg/ml) CAS number Iomeprol N,N -bis (2,3- dihydroxypropyl)-5- [(hydroxyacetyl) (methyl)amino]-2,4,6- triiodobenzene-1,3- dicarboxamide Iomeron Low osmolar, Nonionic (monomer) Iodixanol 5-[acetyl-[3-[N-acetyl- 3,5-bis(2,3- dihydroxypropylcarbam oyl)-2,4,6- triiodoanilino]-2- hydroxypropyl]amino]- 1-N,3-N-bis(2,3- dihydroxypropyl)-2,4,6- triiodobenzene-1,3- dicarboxamide Visipaque Iso-osmolar Nonionic (dimer)

33 2.1.2 Contrast agents in the environment Iodinated contrast media are the most commonly applied substances among all pharmaceuticals used in hospitals. Worldwide consumption of iodinated contrast media is approximately 3500 t per year (Pérez and Barceló, 2007). After intake, contrast media are applied in high dosages (up to 200 g of contrast media, corresponding to approximately 100 g iodine), excreted non-metabolized and released in large quantities into the aquatic environment (Steger-Hartmann et al., 2002). Contrast media exhibit high polarity and they are very persistent against metabolism by the organism and environmental degradation (Hirsch et al., 2000). Municipal treatment plants are not able to remove the contrast agents and they accumulate in drinking water (Ternes and Hirsch, 2000). Since iodinated contrast media are halogenated compounds, they are main contributors to the load of total adsorbable organic halogen (AOX) in clinical wastewater (Weissbrodt et al., 2009). Several contrast agents have been found at relatively high concentrations (> 1 µg/ml) in different aqueous environment, including groundwaters, water treatment plant effluent, rivers, creeks, and drinking water (Ternes and Hirsch, 2000). Several studies have shown that iodinated contrast media are released almost quantitatively in unmetabolized form by a municipal sewage treatment plant (Steger-Hartmann et al., 1998; Kalsch, 1999; Ternes and Hirsch, 2000). Although they are considered as harmless to the human body, their long-term effects on the environment are still unknown, for which reason their removal is mandatory. The behaviour and even the occurrence of iodinated X-ray contrast media in rivers, groundwater, and drinking water are mostly unknown. Based on detection of total adsorbable organic iodine (TAOI) Fono and Sedlak, (2007) showed that organic compounds containing iodine are present in the aquatic environment and in drinking water. In the last years different analytical techniques have improved markedly allowing the detection of these compounds at ng and sub-ng/l levels in environmental waters. Scientific efforts dedicated to investigate their occurrence and potential impact in the aquatic environment is constantly increasing (Fatta-Kassinos et al., 2011; Verlicchi et al., 2012) Detection of iodine contrast media in environmental samples In the study of Klavarioti et al., (2009) it was confirmed that presence of pharmaceuticals in the environment and in aquatic systems present serious environmental problem due to several reasons such as: a) they are greatly resistant to degradation processes and they are lost from treatment plants b) can cause different toxic and other effects to human and other living organisms c) their presence in low concentrations, therefore require highly sensitive and laborious analytical methods for accurate determination. Most of the methods for the environmental analysis of contrast media previously described in the literature are based on the use of liquid chromatography and tandem mass spectrometry with previous enrichment of the samples by solid-phase extraction procedure. In the Table 3 are presented different methods for analysis of iodinated contrast media in water and solid matrices published in the literature. With respect to 10

34 aqueous matrices, the analytical method usually includes solid-phase extraction (SPE), in order to enrich the analytes and clean them up from the matrix sample, followed by liquid chromatography (LC) with mass spectrometry in tandem (MS/MS) using electro spray ionization (ESI) as the detection technique (LC (ESI) MS/MS), in order to quantify the analytes at low levels. Although all these methods achieve the determination of ICM (iodinated contrast media) at ng/l levels, the polarity of these compounds make them difficult to extract using SPE (Echeverría et al., 2013). ICM have been detected in environmental waters at concentrations between several hundred ng/l (Seitz et al., 2006; Boleda et al., 2011) and several mg/l (Ternes and Hirsch, 2000; Duirk et al., 2011), which are concentrations a couple of orders of magnitude higher than those of other pharmaceuticals. These high concentrations are attributable to the high dosages of ICM used in medical interventions. Most of the methods for determination of contrast agents described in the literature are based on direct measurement by HPLC and LC/MS (Table 3). Just few methods based on iodine measurement were described in the literature including colorimetric measurement involving deiodination by alkaline hydrolysis and measurement of released iodine by Sandell Kolthoff reaction with cerium and arsenite (Bäck et al., 1988; Dasgupta et al., 2008) by ion chromatography (Fono and Sedlak, 2007), ICP/MS (Braselton et al., 1997), by FIA (Ratanawimarnwong et al., 2005; Waseem et al., 2008), X-ray fluorescence spectrometry (Iyengar, 1989). All of these analytical methods require complex procedures and timeconsuming sample pre-treatment. Some of the above mentioned techniques such as colorimetric measurement is not sensitive enough for iodine determination at low levels (<0.1 μg/l) and most of them are not appropriately selective and suffer from various interferences (Andrási et al., 2007). In the study of Fono and Sedlak, (2007) the content of contrast agents was measured in different water samples indirectly by measurement of iodide released in degradation reaction. Contrast media were extracted with solid phase extraction, dry extract containing contrast agents was treated in chemical reaction with Cu 2+ /H 2 O 2 and released iodide was measured by ion chromatography. Degradation reaction applied in their work could be potentially used for removal of contrast agents from aquatic environment. However, iodide released in this reaction could also be oxidized to iodine with H 2 SO 4 and NaNO 2 (Adotey et al., 2011, Andrási et al., 2007) and exploited for indirect determination of iodinated contrast agents. Extraction with organic solvents is the most reliable method to extract traces of elemental iodine from aqueous solutions. Chloroform, carbon tetrachloride, diethyl ether, benzene, toluene, carbon disulfide, and other solvents could be used for this purpose (Tunali et al., 1979) since iodine forms a pink coloured charge transfer complex with them in these solvents. 11

35 12 Table 3-Methods for analysis of iodinated contrast media in water and solid matrices Compound Matrix LOD (ng/l) LOQ Extraction procedure Separation and detection method Reference SPE: Elution with Mendoza et al., Iomeprol HPLC-MS/MS methanol (2015) Wastewater effluent, Comparison of SPE Tap water: 10 surface water, cartridges. ENV+, Diatrizoate, iomeprol / WWTP LC (+)-ESI-MS/MS Hirsch et al., (2000) groundwater, ph 2.8. Eluting solvent: effluents 50 drinking water methanol Carballa et al., Iopromide Wastewater 6.7 / SPE: ENV+, ph 2.8. LC (+)-ESI-MS/MS (2004) Wastewater, SPE: ENV+ and Envi- Putschew et al., Diatrizoate, iohexol / 50 LC (+)-ESI-MS/MS surface water Carb ph 3.5 (2001) Diatrizoate Wastewater / 27 SPE: ENV+, ph 2 LC (+)-ESI-MS/MS Kanda et al., (2003) Diatrizoate, Iotrolane, Surface water, SPE: ENV+ and Envi- Putschew et al., Iopromide, / LC (+)-ESI-MS drinking water Carb, ph 3.5 (2000) Iotroxin acid Diatrizoate, Iopamidol, Iomeprol, Iopromide Daitrizoate, Iothalamic acid, Ioxithalamic acid, Iopamidol, Iomeprol, Iopromide, Iohexol Diatrizoate, Iothalamic acid, Ioxithalamic acid, Iopamidol, Iomeprol, Iopromide, Iohexol Iomeprol, Iohexol, Iopamidol, Ioxitalamic acid, Iopromide, Diatrizoate Diatrizoate, Iomeprol Iopromide Iopamidol Groundwater / SPE: ENV, ph 3 LC (+)-ESI-MS/MS Sacher et al., (2001) Surface and drinking water Sludge 50 / Hospital wastewater, WWTP wastewater German Municipal STP Effluents German Rivers and Creeks / / / Direct injection µg/l µg/l USE (ultrasonic solvent extraction), SPE: ENV+ and RP-C18ec, ph 2.8 USE 0.05 µg/l 0.05 µg/l SPE: Isolute ENV+/ 0.01 µg/l ph µg/l LC (+)-ESI-MS/MS IC ICP-MS Sacher et al., (2005) LC (+)-ESI-MS/MS Ternes et al., (2005) Time resolved mass spectrometry LC-electrospray tandem MS detection Weissbrodt et al., (2009) Ternes and Hirsch, (2000) 12

36 2.1.4 Contrast agents and their effects on human health Among different pharmaceuticals used for intravascular administration contrast media are applied in much higher concentrations and doses in comparison to other compounds. According to this it is not surprising that these products cause various adverse reactions. These reactions are either immediate occurring within the first hours after injection or they are late which become apparent from several hours up to 7 days after contrast media exposure. The most frequent immediate adverse reactions are urticaria, nausea, itching, vomiting, and sneezing while nausea and different types of skin reactions often combined with fever constitute the most frequent late reactions (Singh and Daftari, 2008). Beside these adverse effects there are evidences that iodinated contrast media also have toxic effects on renal cells (Heinrich et al., 2005; Romano et al., 2008) but the exact mechanism of contrast media cytotoxicity remains unclear. Acute exposure to contrast media cause more serious effects such as: contrast induced nephropathy (CIN), effects on thyroid gland and neurotoxicity. Iodinated contrast media exposure could be connected with development of either hyperthyroidism or hypothyroidism, presumably due to the effect of free, biologically active iodide ions present in the contrast media preparation (Rhee et al., 2012). In the study of Leung and Braverman, (2012) it was found that contrast media influence secretion of thyroxine by thyroid gland and on peripheral thyroidal deiodination of thyroxine to tri-iodothyronine. Many studies have reported that nonionic contrast media is less nephrotoxic than ionic one and that dimeric contrast media has more attractive properties than monomeric with regard to cytotoxicity (Heinrich et al., 2005; Aspelin et al., 2003). Ion charges formed by dissociation of ionic contrast agents can disrupt electrical charges within the brain and heart, which is known as neurotoxicity. Ionic contrast agents also could cross the blood-brain barrier, causing patient dizziness and confusion (Law et al., 2012; Chisci et al., 2011) Contrast agents clearence During percutaneous coronary interventions contrast media are injected intravenously in amount depends on procedure. In some cases contrast media injection can cause CIN and renal function is ruined as was described above. GFR (glomerular filtration rate) is the most important parameter for estimation of renal function. The GFR is determined by measuring plasma clearance of a GFR marker. Serum creatinine as endogenous metabolic product or exogenously administered substance could be used as a GFR marker. Since contrast media has toxic effects such as CIN it is important to measure how fast they are eliminated from human body after application. Plasma clearance is the ability of the living organism to eliminate a drug. The clearance of substance from a compartment is the ratio between the elimination rate of the substance (mg/min) from that compartment and the concentration of the substance (mg/ml) in the same compartment. Ideal filtration marker is the one that is not bounded to plasma proteins, secreted or metabolized by the renal tubules, and that is not affecting GFR. Therefore contrast media such as diatrizoate, iohexol, iopromide, iopamidol are used as exogenous GFR markers as was described before by Thomsen et al., (1991), 13

37 Gaspari et al., (1995), Frennby, (1997). Iomeprol contrast agent has a similar chemical structure and properties as iohexol so it may have potential to become another applicable marker to evaluate GFR. Currently several HPLC and LC MS/MS methods have been introduced to determine iomeprol (Casase and Bester, 2015; Echeverría et al., 2013). With contrast agents clearence methods it is possible to estimate the amount of contrast agents released from human body to the environment that is causing environmental pollution Contrast agents detection in biological samples Due to the side effects that could be caused by retention of contrast media in the human body determination of their concentration has a crucial meaning. Most publications concerning the analysis of contrast media are designed for biological fluids such as blood or urine. Liquid/liquid extraction was the predominant enrichment procedure described in the literature. Most published methods for contrast media detection in biological samples have used liquid chromatography ultraviolet detection (LC-UV), LC/MS, or LC tandem mass spectrometry (LC-MS/MS) with gradient elution profiles. Because of the high selectivity of mass spectrometry compared to ultraviolet detection, specimen cleanup is often simplified and shorter chromatographic times can be achieved. The limits of detection for these methods are in the range of about 0.2 µg/ml for the non-enriched serum or urine samples. In Table 4 methods for contrast media detection in biological fluids are presented. Disadvantages of these methods are time consuming sample preparation and they are using expensive and laborious apparatus. 14

38 15 Table 4-Methods for contrast media detection in biological samples Compound Matrix LOD (ng/l) LOQ (ng/l) Iohexol Plasma 6 µg/ml 10 µg/ml Iohexol Serum / 2.5 µg/ml Sample preparation Protein removing with 5 % of perchloric acid Protein precipitation with zinc sulfate Iohexol Serum 0.5 mg/l / / Iohexol Serum, urine 12 µg/ml / Iodixanol Iohexol, Iothalamate Plasma 2 µg/ml 10 µg/ml Plasma, urine 1 µg/ml / Protein removing with 5 % of perchloric acid Protein precipitation with 20 % perchloric acid Protein precipitation with 0.1 % TFA Iomeprol Plasma 0.68 µg/ml 2.26 µg/ml SPE: LiChlorut Iohexol Serum 0.5 µg/ml Protein precipitation with zinc sulfate Separation and detection method HPLC-UV (UPLC) triple quadrupole MS/MS micellar electrokinetic capillary chromatography (MECC) HPLC HPLC-UV HPLC-UV POPLC (Phase optimized liquid chromatography) Reference Soman et al., (2005) Annesley and Clayton, (2009) Kitahashi and Furuta, (2004) Cavalier et al., (2008) Chitnis and Akhlaghi, (2008) Farthing et al., (2005) Zhou et al., (2015) HPLC-MS/MS Lee et al., (2006) 15

39 2.2 Contrast induced nephropathy Contrast-induced nephropathy (CIN) is also known under the name contrast induced acute kidney injury (AKI) is the third leading cause of acute kidney injury in hospitalized patients (accounting for around 11 % of cases) (McCullough et al., 2005). CIN can occur as a result of intravenous or intra-arterial application of iodine contrast media during medical procedures which include injection of nephrotoxic contrast media (Chang and Lin, 2013). Among all procedures utilizing contrast media for diagnostic or therapeutic purposes, coronary angiography and percutaneous coronary intervention (PCI) are associated with the highest rates of CIN (Mehran and Nikolsky, 2006). The incidence of acute kidney injury (AKI) is increasing worldwide and it is associated with increased morbidity and mortality. Identifying patients early is of paramount importance in order to offer a prompt intervention and to improve the prognosis in both settings. CIN is defined as a rise in serum creatinine level of more than 25 %. The current practice to diagnose AKI involves measuring the surrogate markers of glomerular filtration rate (GFR) such as an increase in SCr and/or a decrease in urine output with the RIFLE and/or AKIN criteria (Wasung et al., 2015). Despite several known limitations in current clinical practice, serum creatinine level and urine output are the most frequently used indicators of renal impairment. They have limited sensitivity and specificity and creatinine level has a slow rate of change, thus limiting their usefulness in the early detection of AKI (Haase et al., 2009). First of all serum creatinine concentration might not change until about 50 % of kidney function has already been lost and creatinine does not accurately depict kidney function until a steady state has been reached, which could take several days (Mishra et al., 2005). Therefore there is an urgent need for a biomarker of AKI that could accurately detect AKI much before a rise in serum creatinine. 2.3 Biomarkers of acute kidney injury A biomarker is a characteristic that is objectively measured and defined as a parameter of structural, biochemical, physiologic or genetic change that indicates the presence, severity or progress of a disease (Schiffl and Lang, 2012). In AKI, cells produce biomarkers up to 3 days before the clinical syndrome develops (Soni et al., 2009) and therefore a biomarker should be measured in this period. According to Annigeri et al., (2013) the biomarkers of AKI can be classified as: a) functional markers: SCr, serum Cys C b) upregulated proteins: neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecute-1 (KIM-1), interleukin-18 (IL-18), Liver fatty acid binding protein (L- FABP), heat shock protein-72 (Hsp-72), c) low molecular weight proteins: urine Cys C and d) enzymes: N-acetyl-β-D-glucosaminidase (NAG), α-glutathione transferase (α-gst) and π-glutathione s-transferase (π-gst). NGAL and Cys C are already being applied in clinical studies and have shown positive results. Biomarkers are studied in the serum and urine of patients (Ghatanatti et al., 2014). Among these, urine NGAL, plasma NGAL and cystatin C concentrations are by far 16

40 the most widely studied in clinical setting. On Fig. 3 it is shown evolution of acute kidney injury and biomarkers for different level of kidney injury. Figure 3-Evolution of acute kidney injury (modified from Bellomo et al., 2012) 2.4 Cystatin C Cystatin C is a 13 kda serum protein which is originated in all types of nucleated cells. It is freely filtered out of the blood by the glomerular membrane in the kidney (Taglieri et al., 2009). Its concentration in the blood associate with the glomerular filtration rate and since it serves as a measure of kidney function, it is considered to be a marker of early kidney dysfunction. The levels of cystatin C are independent of weight and height, muscle mass, age, and gender. In comparison to creatinine cystatin C is a better marker of the glomerular filtration rate and kidney function and it is expected that will replace SCr in the future as the blood marker of renal filtration function (Shlipak et al., 2013). 2.5 NGAL NGAL (neutrophil gelatinase associated lipocalin) which belongs to a lipocalin family of proteins (also known as human neutrophil lipocalin (HNL), lipocalin-2, siderocalin, uterocalin, 24p3 or LCN2) is a 25 kda glycoprotein originally isolated from human neutrophils and localized in neutrophil granules (Cai et al., 2010). Lipocalin share the same three-dimensional structure in a single eight stranded anti-parallel β-barrel surrounding a central pocket. This central calix determines the main function of lipocalins: to act as 17

41 transporters of small hydrophobic substances such as prostaglandins, retinoids, arachidonic acid, hormones and fatty acids (Bolignano et al., 2010). NGAL is present in urine and blood. NGAL exists in three different forms as a 25 kda monomer, 45 kda disulfide-linked homodimer, and covalently conjugated with gelatinase MMP9 (matrix metalloproteinase 9) through intermolecular disulfide bridge as a 135 kda heterodimer (Kjeldsen et al., 1993). Beside neutrophils NGAL is also expressed in kidney, liver and epithelial cells and his level is rising in some pathologic and stressful conditions such as infection, inflammation, cancer, intoxication, ischemia, kidney injury (Bauer et al., 2008; Roudkenar et al., 2007). Kidney tubular epithelial cells produce mainly the monomeric form while neutrophils secrete the dimeric form (Cai et al., 2010). Dominant characteristic of NGAL, on the other hand, is to capture the iron containing particles, the siderophores, and transport them to the inner cell after interacting with specific membrane receptors (24p3R, megalin), thus causing an increase in the cytoplasmic levels of iron (Devireddy et al., 2005; Hvidberg et al., 2005). This is believed to be the mechanism that underlies the main, multiple effects attributed to NGAL. In the study of Flo et al., (2004) it was shown that the main biological function of NGAL is inhibition of bacterial growth. This protein was initially known only as an antibacterial factor of natural immunity (released by activated neutrophils, this protein can block bacterial cells growth by causing iron depletion). On the other hand NGAL is a true and proper stress protein induced and hyperproduced by different types of cells after exposure to various stress conditions, probably in order to trigger iron-dependent enzymatic defence systems (Yang et al., 2002; Nairz et al., 2009). Diverse inflammatory conditions influencing human tissues, such as those in the respiratory, gastro-enteric and urinary tracts, are associated with a substantial increase in the local and systemic expression of NGAL and, in some cases (e.g. during the course of some renal diseases), the evaluation of serum and urinary levels of this protein has been found to be particularly useful as an early and specific biomarker of organ damage and clinical diagnostics (Bolignano et al., 2008). In the study of Mishra et al., (2005) it was found that plasma and urinary NGAL level increase 2 h after kidney injury and several hours before plasma creatinine increase. In experimental and clinical studies, NGAL appears as an excellent biomarker, the most frequently investigated and most promising for the early diagnosis of contrast-induced AKI (Nguyen and Devarajan, 2008). Its crucial advantage is that it responds earlier than other renal status markers like serum creatinine and shows a comparable response to injury. NGAL determination thus permits the early diagnosis and prognostic stratification of patients with AKI. NGAL is released from the specific granules of activated neutrophils and plasma levels grow in inflammatory or infective conditions. Monitoring NGAL levels provides crucial information on the possible kidney injury that may outcome from the use of contrast agents in diagnostic imaging (Devarajan, 2010). In several prospective studies (Bachorzewska- Gajewska et al., 2007; Ling et al., 2008; Hirsch et al., 2007) on adults or children who received contrast media, urine and plasma NGAL level rise within 2 to 4 hours after contrast media administration. 18

42 2.5.1 NGAL level in blood and urine The NGAL concentrations in urine are in the ranges of 1-20 ng/ml, in plasma of healthy adults ng/ml, or ng/ml in children. It rises immediately after renal injury in urine and plasma, where its concentrations are in the range of ng/ml and ng/ml, respectively. However, in case of acute renal failure, the NGAL concentrations in urine and plasma are over 350 ng/ml and above 400 ng/ml, respectively (Moore et al., 2010; Vashist et al., 2012) NGAL detection techniques The majority of NGAL results described in the literature have been obtained using research-based ELISA assays that are currently available on the market used for NGAL measurement in urine, plasma and serum samples. These assays are accurate, but are not practical for clinical usage and most of the assays cannot distinguish between the different forms of NGAL (monomeric, homodimeric, heterodimeric). Besides hemolysis affecting plasma NGAL measurement, other factors that can affect serum and urine NGAL measurement include production from neutrophils (Clerico et al, 2012). Beside ELISA there are also various other immunoassays developed for the measurement of NGAL in various body fluids (Table 5). The assays are based on different formats and include radioimmunoassay (RIA), Western-blotting, Triage device and the Architect platform. The problem with different assay formats involve differences in antibodies that recognize different forms of NGAL in urine and plasma and whether differences had any impact on the clinical performance of the assay (Cai et al., 2009). 19

43 20 Table 5-Comparison of different sandwich NGAL ELISA kits and assay NGAL detection techniques (modified from Clerico et al., 2012) Method, manufacturer Antibody immobilization technique Sample LOD (pg/ml) Time for test Reference GNP based assay Chemical crosslinking Blood, serum, plasma h Vashist, (2014) ELIA covalent assay Chemical crosslinking 2.5 ~6 h Vashist et al., (2012) Passive assay Passive adsorption 40.0 ~20 h Vashist et al., (2012) ELISA kit, R&D systems Passive adsorption Serum, plasma, urine, saliva 40.0 ~4.5 h ELISA kit, Boster biological Serum, plasma, urine, Passive adsorption technology saliva 10.0 ~4 h ELISA kit, Antibody and immunoassay services Passive adsorption Serum, plasma 0.4 ~3h Meso scale diagnostics Passive adsorption 2.9 / Elisa kit, BioPorto diagnostics Passive adsorption Serum, plasma, urine 0.2 <1 h ELISA kit, CycLex Passive adsorption Serum, plasma, urine 26.7 / ELISA kit, BioVendor Passive adsorption Serum, plasma 20.0 / Argutus medical Passive adsorption 0.4 / Manual technique Passive adsorption Urine 1 24 h Mishra et al., (2005) Manual technique Passive adsorption Plasma, urine 1 <3h Bachorzewska-Gajewska et al., (2006) POCT Triage Passive adsorption Blood, plasma 60 <30 min Dent et al., (2007) Manual technique Passive adsorption Urine 0.1 ~24 h Cai et al., (2009) Manual technique Passive adsorption Serum, plasma, urine 0.15 <4 h Stejskal et al., (2008) ELISA kit, manual Passive adsorption Lithium-heparinized plasma and urine ng/ml <3 h Pedersen et al., (2010) CMIA (Chemiluminescent Microparticle Immuno Assay) Architect i1000, Abott Diagnostics / Urine ng/ml <30 min Grenier et al., (2010) Covalent attaching Urine 0.95 ng/ml / Cangemi et al., (2013) 20

44 2.6 Infective agents: virus like particles As a crucial element for human life water could be under certain circumstances a transmission vehicle for microorganisms. It has been proposed that more than 140 different types of viruses, obviously not eliminated by massive purification treatments, can be found in drinking water (Gutiérrez et al., 2007). Wastewater treatment plants, remove only around % of enteric viruses (Ottoson et al., 2006; La Rosa et al., 2012; Okoh et al., 2010) allowing releasing of a significant viral load in effluent discharge and spread in the environment, transported through groundwater, seawater and rivers. Human excreta are the main sources for water contamination with viruses. Actually, viruses infiltrate the ground, penetrating to depths larger than 60 m where they can remain hidden for several months, until the temperature remains low and the environment humid. Under these conditions they can easily reach aquifers. Viruses as microscopic infectious agents ( nm) are exclusively intracellular organisms that depend on animal, plant, or bacterial cells to multiply. All viruses contain one nucleic acid, RNA or DNA, enclosed within a protein coat made from many homogeneous subunits spontaneously aggregated to protect the viral genes. They also possess a viral envelope covering the nucleocapsids, consisting of host cell membrane lipids and proteins, and viral glycoproteins that protects viruses from degradation outside the cell and helps their attachment to host cell membranes. Viruses can be inactivated in the environment by the capsid damage that causes the liberation of the contained nucleic acid (required for productive infection), which can be easily degraded when capsid protection is not present (Gutiérrez et al., 2007). Although virus concentration in the water is relatively low, these microorganisms possess health risks, since they have very low infectious doses ( virions) so that even a few viral particles in water can pose health risks. Among different virus groups enteric viruses that belong to the families Caliciviridae (norovirus), Picornaviridae (enterovirus and hepatitis A virus) and Adenoviridae (adenovirus) have been detected in sewage, surface water, groundwater and drinking water sources around the world. Other virus groups are considered to be potentially emerging waterborne pathogens and include hepatitis E virus, the viral agent of avian influenza, coronavirus, polyomavirus, picobirnavirus, and papillomavirus (La Rossa et al., 2012; Rzeżutka and Cook, 2004; Dann et al., 2015). In this study HPV (human papilloma viruses) that belongs to group of papillomavirus were investigated and used as a virus model system. There are evidences (Strauss et al., 2002; Cantalupo et al., 2011; La Rosa et al., 2013) that these viruses are not just present in human body. They are also found on environmental surfaces in the hospitals (treatment rooms, toilets) so therefore they could also enter environment via the toilets and occur in water sources as potential infectious agents. Genital human papillomaviruses (HPV) infections are spread predominantly through sexual intercourse, although other routes of transmission have been postulated, and papillomaviruses may stay infectious within cells for up to 7 days, even after desiccation (Roden et al., 1997). 21

45 2.6.1 HPV viruses Papillomaviruses are the members of the Papovaviridae family of nonenveloped, doublestranded DNA viruses, which are both species and tissue type specific. They are a large group of viruses, and over 170 different genotypes have been identified and collectively categorized into high-risk or low-risk genotypes depending on their oncogenic capacity (Clifford et al., 2006; Schiffman et al., 2009). HPV infection is the most common sexually transmitted infection worldwide and causes approximately new cases of cervical cancer per year, of which more than 80 % occur in developing countries (Zhao et al., 2014). HPV is responsible for 5 % of all cancers worldwide, including cervical cancer and the majority of vaginal, vulval, penile, anal and a subset of certain head and neck cancers (Parkin and Bray, 2006). Approximately one-third of the human papillomaviruses (HPVs) are targeted to genital mucosal epithelium. Low-risk HPVs (HPV-6 and HPV-11) preferentially infect cutaneous skin and usually cause only benign warts. Other HPV types, most significantly HPV-16 and -18, have been implicated as etiologic agents of cervical cancer in 70 % of cases and they belong to the group of high-risk papillomaviruses (Mc Carthy et al., 1998). HPV infection is usually identified by detection of viral nucleic acids in clinical specimens and these methods are called molecular methods (see Table 7). In clinical practice HPV infection is detected by PAP test (Papanicolau test) during colposcopy procedure which detects abnormal cervix cells formed from normal one due to HPV infection (Akhter et al., 2015). Human papillomavirions are nonenveloped, icosahedral, and smaller in size about 55 nm in diameter. The viral capsid is composed of 72 pentamers (capsomeres) of the major L1 capsid protein (53 to 57 kda) which is associated with 12 or more copies of a minor L2 capsid protein (76 to 86 kda). It has been proved that neutralizing epitopes are presented on the surface of L1 major capsid protein. Both conformational and linear epitopes have been identified on the surface of HPV 16 virus-like particles (VLPs). The L1 pentamers are being capable of forming virus capsid in vitro by self-assembly into empty capsids referred to as (VLPs) (see Fig. 4) (Vidyasagar et al., 2014). These VLPs are immunogenic in humans and are capable of inducing neutralizing antibodies (Kirnbauer et al., 1992; Christensen et al., 1996a). Presently available prophylactic vaccines for HPV are composed of VLPs formed by self-assembly of the recombinant L1 capsid proteins of HPV-6, HPV-11, HPV-16, and HPV-18 (Vidyasagar et al., 2014). 22

46 Figure 4-Creation of Human Papilloma Virus VLP (Berzofsky et al., 2004) Virus-like particles (VLPs) and pseudovirions (PsVs) Virus like particles (VLPs) and pseudovirions (PsVs) are multiprotein structures composed of viral structural proteins that, when expressed in recombinant systems, form multimeric protein complexes mimicking the organization and conformation of authentic native viruses but lacking the viral genome and they have similar characteristics to the virions. The only difference between VLPs and pseudovirions (PsVs) is that the latter contain also a reporter DNA. VLPs and PsVs are, like true virions, made from L1 and L2 proteins. Several applications have been proposed for viruses, VLPs and PsVs: vaccination, gene therapy, drug delivery, nanotechnology, and bioweapons, among others. VLPs have been widely used in vaccine development in the last decades as they can trigger a protective immune response with lower doses and are obtained by harmless processes (Roldäo et al., 2010; Braun et al., 2011). If a person is vaccinated with VLPs then an immune response is generated as if the immune system has been presented with a real virus. To date, different types of viruses have been mimicked by VLPs and PsVs: viruses with single or multiple capsid proteins and with or without lipid envelopes (Roldäo et al., 2011) Methods for detection of HPV viruses and HPV VLPs Assays for HPV antibody detection are still limited for clinical applications due to the lack of highly sensitive and reproducible assay systems. Most of the serum specimens have low titers of antibodies to HPV, probably because of the immune isolation of the cervix and/or due to the fact that infection with HPV has no viremic phase. In addition, low titers of antibodies to conformational epitopes of HPV make background reactivity a major barrier to the development 23

47 of a clinically useful assay for HPV antibody screening (Studentsov et al., 2002). However, one problem with VLP-based ELISAs is that while the immunodominant neutralization epitopes of HPVs have been shown to be genotype-specific, ELISAs may also detect non-neutralizing and cross-genotype reactive antibodies, thus complicating the interpretation of some results (Pastrana et al., 2004). After natural HPV infection, women with HPV-16 infection may not necessarily have detectable antibodies against HPV-16 VLPs (Ho et al., 2004). Measurement of HPV capsid antibodies for epidemiologic and clinical investigations requires assays that are capable of measuring antibodies to multiple HPV types simultaneously with high sensitivity and type specificity in a high-throughput format. In the absence of efficient methods of harvesting native antigen from culture, serologic detection of HPV has largely relied on the use of HPV VLPs (Hernandez et al., 2012). In the Table 6 are presented most common used methods for HPV detection based on VLP measurement or antibodies against VLPs. VLP-based enzyme-linked immunosorbent assays (ELISAs) are among the most commonly used assays for HPV research. In productive infections, such as warts, virus particles about 50 nm in diameter can be detected by electron microscopy and by immune detection of the virus capsid proteins (L1, L2). Immunological detection of HPV in human cells or tissues has been hindered by three main reasons: (1) the late, capsid proteins are only expressed in productive infections; (2) the early proteins are often expressed in low amounts in infected tissues; and (3) there is a lack of sensitive and specific antibodies of high quality against the viral proteins. Beside above mentioned methods molecular methods presented in Table 7 are also used for HPV detection. 24

48 25 Table 6-Methods for detection of VLPs and antibodies against VLPs published in the literature Immobilization technique Time for analysis Technique Disadvantage Reference Direct coating with VLPs ~16 h ELISA / Christensen et al., (1996b) / / SEC (Size exclusion chromatography) / Park et al., (2008) / / DLS (dynamic light scattering) Low resolution Mach et al., (2006) / / TEM Not quantitative sample heterogeneity could give incorrect results Pease et al., (2009) / / AUC (Analytical ultracentrifugation) / Mukherjee et al., (2008) L1 from VLPs of HPV-18 injected on C 4 or C 8 column Agarose media 35 min RP-HPLC / Yuan et al., (1998) / IEC (ion-exchange chromatography) Relies on the viral particle charge as well as that of the contaminants Yu et al., (2014) Direct coating with VLPs ~16 h VLP-based ELISA for detection of HPV-16 antibodies / Karem et al., (2002) Direct coating with VLPs ~20 h HPV 16 L1 ELISA / Touze et al., (1998) L1-L2 VLPs coupled with carboxyl groups to the fluorescence polystyrene beads Direct coating with pseudovirions / L1-L2 serologic assay / Hernandez et al., (2012) / Pseudovirus-based papillomavirus neutralization assay for HPV-16 and HPV-18 / Pastrana et al., (2004) Capture assay ~20 h HPV 16 L1 ELISA / Le Cann et al., (1995) Direct coating with pseudovirions ~72 h L2-based neutralization assay / Day et al., (2012) 25

49 26 Table 7 Molecular methods for HPV detection Assay Methods Benefits Disadvantages Reference Nucleic acids hybridization assay Signal amplification assay Nucleic acids amplification assays Southern blot In situ hybridization Southern blot is gold standard for HPV genomic analysis Presence of HPV in association with morphology Low sensitivity, time consuming, relatively large amounts of purified DNA Southern blot and hybridization cannot use Schiffman et al., (1991) Kelesidis et al., (2011) Dot blot hybridization degraded DNA Teng et al., (2007) Licensed and patented Cervista HPV Quantitative technologies Einstein et al., (2010) Wasn t designed to genotyping Bartholomew et al., (2011) individual FDA-approved test (hc2) Hwang and Shroyer, Hybrid capture 2 High sensitivity to genotyping Lower false-positive rate (2011) Microarray Hoheisel, (2006) Papillo Check Papillo Check allow detection Bryant et al., (2011) PCR of multiple Romero-Pastrana, (2011) PCR-RFLP infections, and may be Flexible technology (viral Lugo-Trampe et al., (2013) Real-time PCR considered a reliable screening load and genotype) Yadav et al., (2015) test Abbott Real-Time Very high sensitivity Chung et al., (2014) Lower amplification signals of COBAS 4800 HPV Multiplex analysis some HPV genotypes Bernal et al., (2014) Genome sequencing Contamination with previously Kocjan et al., (2011) CLART HPV 2 amplified material can lead to Pista et al., (2011) INNO-LiPa false positive Alberizzi et al., (2014) The Linear Array Steinau et al., (2008) 26

50 Titer of antibodies against HPV-16 is quite small even at the beginning of infection and with time and progress of virus infection antibody concentration is even decreasing. Therefore highly sensitive method for measurement of antibodies against PsVs of HPV virus is needed. ELISA method based on the use of VLPs could be potentially applied for detection of different virus types in environmental samples since their concentrations are extremely low but are enough to cause harmful effects on humans. PsVs of HPV viruses applied in this study were used as a model system to confirm possible detection of different other viruses present in the environment. 2.7 ELISA Nowadays among various immunoassays ELISA (enzyme-linked immunosorbent assay) is one of the most predominant analytical techniques for the quantitative determination of a wide variety of analytes in clinical, medical, biotechnological, and environmental significance (Ohkuma et al., 2002; Du et al., 2015; Pribowo et al., 2013). ELISA is highly specific and sensitive technique, based on the usage of antibody molecule, which recognize the corresponding antigen (analyte). The antigen is allowed to bind to a secondary, enzymecoupled antibody. The enzyme converts a colourless substrate (chromogen) to a coloured product, indicating the presence of Ag-Ab binding (Gan and Patel, 2013). Routinely ELISA is performed in a microtiter plates with 48 or 96 sample wells. ELISA has been widely used in the life science research since it allows detection of concentrations of ng/ml to pg/ml in different biological samples such as: serum, urine, sperm and culture supernatant. The most important advantages of immunoassays are their speed, sensitivity, selectivity, and cost effectiveness. These advantages appear from the selectivity of antibody-antigen reactions, the use of excess capture antibody and enzyme-antibody conjugate, and the chemicalamplification with enzyme-conjugates that allows the detection of very low concentrations of analytes. An ELISA can be used to detect the presence of antigens or antibodies in a sample, depends on experimental needs (Ma et al., 2006). Antibody-sandwich ELISA may be the most useful of the immunosorbent assays for detecting an antigen because they are frequently between two and five times more sensitive than those in which antigen is directly bound to the solid phase. 27

51 Figure 5-Schematic presentation of sandwich ELISA test on microtiter plate Although ELISA gives precise and accurate results, it has several disadvantages such as: high consumption of expensive reagents, requirement of long incubation periods, timeconsuming procedure which usually takes several hours with multiple incubation and washing steps. Such a long incubation time is mostly because of the ineffective mass transport of reagents from a solution to the surface where binding between antibody and antigen occurs while the immunoreaction itself is relatively fast. A typical heterogeneous immunoassay, however, has a relatively long assay time, and involves inconvenient liquidhandling procedures and large amount of expensive antibody reagents. Moreover, automated assay systems used for clinical diagnoses require rather large apparatuses (Lin et al., 2010). Current technological approaches have allowed the miniaturization of conventional microtiter plate ELISA to a microfluidic ELISA platform to overcome these drawbacks. The advantages of microfluidic devices are portability, small volume of samples and reagents, low chances of contamination, low cost, low power consumption, enhanced sensitivity, and reliability (Ahmed and Azzazy, 2013). There is a need to improve the sensitivity of the current ELISA method for highly sensitive detection of protein biomarker, which is important for early diagnosis of cancer, neurodegenerative and other diseases (Jia et al., 2009). Performing an ELISA assay in microfluidic devices is good choice for future work and for development lab-on-a-chip platforms that could be used in clinical practise. 2.8 Microfluidics Microfluidics refers to the science and technology of systems that operate or manipulate with small amounts (10 9 to litres) of fluids. This new and promising technology in the field of chemistry, chemical engineering and biotechnology is attracting growing attention in the past years. Microfluidic technologies involve implementation of microdevices with channel dimensions of tens to hundreds of micrometre. Due to their small scale, microdevices have many practical advantages such as large surface to volume ratio, 28

52 very low reagent consumption, short diffusion length (Whitesides, 2006; Hou et al., 2012). Microchannel reaction systems (also called lab-on-chip, micro-total-analysis-systems) offer several advantages over traditional technologies in performing chemical reactions such as rapid heat exchange and rapid mass transfer that cannot be achieved by the conventional batch system. Microfluidic systems operate mainly under laminar flow conditions, which allow strict control of reaction conditions and time (Asanomi et al., 2011). Microdevices allow improvement of analytic efficiencies by reducing the sample volume and the incubation time while increasing sensitivity and enabling processing of multiple samples via automation of multichannel microchips (Herr et al., 2007). Microfluidic technologies offer numerous useful competences such as: the ability to use very small amounts of samples and reagents, to perform separations and detections with high resolution and sensitivity, low cost, short time for analysis, and small footprints for the analytical devices. Main purpose of microfluidics research is the implementation and integration of several fluid manipulation components (e.g., pumps, valves, filters, and mixers) and analytical separation and detection techniques (e.g., electrophoresis, chromatography, fluorescence, and electrochemical detection) on individual microfabricated devices for complete on-chip analysis. To date, microfluidics has been successfully implemented in a variety of biological applications, including DNA detection allowing manipulation with nl sample volume (Mayr et al., 2016; Tian et al., 2015), protein analysis with LOD of 4.7 pm for anti-mouse IgG (Sang et al., 2016) and 20 pm of thrombin (Zhao et al., 2016), immunoassays for detection of Hepatitis B virus antigen with LOD of 0.01 IU/mL (Kamińska et al., 2015), detection of interleukin-6 and 8 (IL-6 and IL-8) with LODs of 5 fg/ml and 7 fg/ml respectively (Otieno et al., 2014), detection of TNF-α (tumor necrosis factor-α) with LOD of pg/ml (Giri and Dutta, 2014) and several others. Several ELISA microchip assays were developed based on an immunoreaction on the surface of a single microchannel (Yakovleva et al., 2002) or on nanoparticles entrapped inside the microchannels that increase the surface-to-volume ratio (Huang et al., 2012; Lin et al., 2010; Du et al., 2015) resulting in a decrease in assay time from 20 h to only 20 minutes (Sato et al., 2003). Optical detection methods based on absorption of light are frequently used in microfluidic devices because they are easy to perform and reliable. Spectrophotometric detection has broad applicability, but its sensitivity is poor due to the short optical interaction length in the microfluidic chips. Therefore, thermal lens methods showed to be a sensitive promising method for detection in microfluidics (Yamauchi et al., 2006). Thermal lens spectrometry (TLS) is a promising method to overcome the low sensitivity of the transmission mode spectrometric methods (Bialkowski, 1996). Kitamori et al., (2004) confirmed that thermal lens microscopy (TLM), is extremely sensitive method for trace analysis in microchips. As it was previously described in the literature by Sato et al., (2001) sandwich immunoassay with TLM detection in a microfluidic system was successfully implemented for detection of carcinoembryonic antigen. This resulted in reducing the reaction time for antigen-antibody reaction 90 times because of the size effect of the microspace. In the work of Liu and Franko, (2014) a combined microfluidic flow injection analysis (μfia) TLM device was developed for rapid determination of hexavalent chromium, where they achieved up to 20 sample injections in 1 minute for injection of sub- µl samples. 29

53 2.9 Thermal Lens Spectrometry Thermal lens spectrometry (TLS) belongs to a family of photothermal techniques of high sensitivity applied to measure spectroscopic and thermo-optical properties of materials (Proskurnin et al., 2010; Franko, 2001). TLS is known as one of the most sensitive spectroscopic techniques for measurements in liquids, applied for determination of weak absorbances as low as 10-7 (Bialkowski, 1996) and low concentrations of different compounds. Among different analytical techniques TLS has various advantages such as: high sensitivity analysis of biological molecules, remote analysis, on-line determination in the flow and can be presented as an analytical method with unique properties (Navas and Jiménez, 2003). Beside this TLS gives possibility of analysing very small (sub pl) sample volumes with relatively fast signal response on the millisecond scale and allows detection in flowing samples. TLS is based on an indirect measurement of absorbance by a photothermal effect, which emerged from a nonradiative relaxation of excited molecules in the sample causing defocusing of the laser beam. Irradiated sample lose the absorbed energy through deexcitation, causing heating of the sample along the beam path. The greater heat produced at the beam center cause the raises of the sample refractive index. If sample is non-fluorescent it is possible to assume that the amount of released heat is equal to the energy absorbed by the sample, where a lens-like element is created through the temperature dependence of the sample s refractive index. Formed thermo-optical element usually has a negative focal length since most materials enlarge upon heating and the refractive index is proportional to the density, which results in a negative temperature coefficient of refractive index. This negative lens causes beam divergence and the signal is detected as a time dependent decrease in intensity on the axis of the beam (Franko and Tran, 2010). Light intensity changes on the axis of the beam are directly related to the absorbance of the sample and could be described by Eq. 1 if simplified model is used dn I bc P (- = dt ) A I bc λ k (1) I bc I bc - is the relative change in the beam center intensity P- is the excitation laser power - dn - temperature coefficient of the sample dt A- is absorbance of the sample λ -is the wavelength k- thermal conductivity of the sample According to Beer-Lambert's law absorbance of an analyte is defined as: where T-transmittance through a sample A=- log T=- log I bc I bc (2) 30

54 For low absorbance values (<0.05 AU) Beer-Lambert's law is approximated and ratio I bc I bc between light intensity before and after the sample becomes: I bc I bc = A In TLS detection technique sample is irradiated periodically with a modulated pump beam (running at emission line required for the desired absorption of sample) with Gaussian radial intensity distribution. Pump beam laser (operating at higher laser power up to several hundred milliwatts) serve as the excitation beam source supplying power at the location of the detection cell. Created lens like optical element is detected with lower-power laser beam (called probe beam). The generated thermal lens causes variations in the probe beam intensity that can be sensitively monitored by signal-averaging devices, such as lock-in amplifiers, which provide that pump beam is filtered out before the detector. Convenient TL signal is detected by a photodetector through a pinhole, which is usually proportional to the concentration of analytes. Photodiode monitors the change of the probe beam intensity (Franko and Tran, 2010). TLS is more sensitive than conventional transmission spectrometry because the photothermal effect enhances the measured optical signal (Bialkowski, 1996). This amplification is known as the enhancement factor, representing the ratio of the signal achieved with photothermal spectrometry and with conventional transmission spectrometry. Because of the enhancement factor magnitude of the TLS effect depends also on the thermooptical properties of the sample which can be seen from Eq. 4: E= (- dn dt ) P λ k (4) Solvents with relatively low thermal conductivity and high temperature coefficient are good choice for TLS measurement. In the Table 8 are presented thermooptical properties of different organic solvents. Table 8-Thermooptical properties of some organic solvents (Bialkowski, 1996) Solvent k Cp -dn/dt W/m K J/g K x 10-4 K -1 water methanol ethanol acetone acetonitrile diethyl ether ethyl acetate dichloromethane thichloromethane tetrachloremethane n-hexane n-heptane n-octane benzene toluene (3)

55 Water is not appropriate solvent for TLS measurement due to the high thermal conductivity and low temperature coefficient in comparison to other organic solvents Thermal Lens Microscope TLM (thermal lens microscope) is a kind of photothermal microscope, which was developed by applying TLS detection in FIA (flow injection analysis) (Harada et al., 1993) which allows a various new applications of TLS in microchemical analysis. TLM was obtained by miniaturization of a conventional TLS spectrometer and it is a powerful detection technique for highly-sensitive determination of non-fluorescent molecules in a microchannel. TLM is one of the most adjustable detection methods for microsystems and integrated chemistry (Kitamori et al., 2004). TLM consisted of pump and probe beam with singular geometry that are aligned coaxially under the microscope and focused with a single chromatic lens (see Fig. 6). Figure 6-TLM schematic presentation of interaction between pump and probe beam and sample As a result chromatic aberration of a few micrometers was obtained allowing detection of analytes in microwells and microchannels, which cannot be carried out on analytical microchips by transverse mode TLS (Kitamori et al., 2004). FIA-TLM analysis on microchip is specifically adapted for small volume samples and can be carried out in a relatively short time. Microchip-based TLM detection proposes numerous other interesting configurations for assays that will for sure find application in the future. TLM system could be applied to a broad variety of biochemical studies and cellular analyses by coupling it with several analysis systems such as flow injection analysis, immunoassay (Sato et al., 2003), chromatography (Shimizu et al., 2011), and electrophoresis (Nedosekin et al., 2007). Among those that rely on TLM detection in flow, a microchip-based ELISA for determination of interferon-γ was developed (Sato et al., 2004). The sample volume in conventional microchannel devices is quite low (from several hundred µl to less than 1 µl). If we combine such devices with TLM with sensitivity 100 to 1000 times higher in comparison to conventional spectrophotometry (Kamaki et al., 2003) we could reduce reaction time for ELISA. As it is shown on Fig. 7 depth of solution in microtiter 32

56 plate well is around 1.5 mm which is a big difference in comparison to depth of a microfluidic chip channel of 100 µm. Figure 7-Schematic illustration of the immunoassay in micro-titer plate (left) and immunoassay chip (right) Microfluidic devices therefore allow reduction of separate incubation steps because diffusion time decreases with the square of the distance and in ELISA assays reaction between antibodies and antigen is controlled by diffusion Nanobeads application In the last years nanoscience become one of the most important research field in modern medicine. Nanotechnology found wide applications in various field such as pharmaceutical industry, medicine, cosmetics, electronics, robotics, tissue engineering etc. (Kango et al., 2013). Among different nanoparticles and nanocomposites because of their unique physical, chemical, thermal, and mechanical properties magnetic nanoparticles that can be manipulated easy by an external magnetic field offer a high potential for several biomedical applications (Umut, 2013; Mahdavi et al., 2013; Roca et al., 2009; Akbarzadeh et al., 2012). Furthermore, magnetic nanoparticles have advantages including reduced analysis time and selectivity control. With development of microfluidic systems, magnetic nanoparticles have been used for immunoassay applications that involved a simple microfluidic chip. Magnetic nanobeads have many great properties, including small size, high surface to volume ratio, surface rich with functional groups, good biocompatibility, and vigorous controllability (Zhao et al., 2012), which make them applicable for biochemical analysis and have attracted enormous concern in recent years. Nanoparticles usage as an immunological platform integrated into a microfluidic device results in effective area growth and decrease of incubation times by lowering the diffusional distances (Choi et al., 2002). Microfluidic device detection systems must be adequate to ensure sensitive measurements in low volumes (Seia et al., 2014) 33

57 2.12 Research objectives The aim of presented research is development of novel methods for detection of biologically active substances such as contrast agents, biomarkers (e.g. NGAL-neutrophil gelatinase-associated lipocalin), and virus particles and to improve: sensitivity, selectivity, and sample throughput. There is also a clear need for new rapid cost-effective analytical methods for environmental application, with an emphasis on reduced cost when compared to existing techniques. To contribute to the progress in this field we were combining methods such as: Flow injection analysis (FIA), Immunological methods (e.g. ELISA), and Thermal lens spectrometry (TLS) The primary objective of this study was to design ELISA assay on microfluidic chip for measurement of NGAL and antibodies against HPV (Human Papilloma Viruses) viral particles and to produce a rapid ELISA platform. Idea is to design an optimal and cost effective assay which allow short reaction (processing) times, small reagent consumption and portability, for further possible application in medical diagnostics. For this purpose a microfluidic device with four straight channels was used for performing ELISA assay. Instead of commercial microtiter plate magnetic nanobeads were used as a solid support for antibody/antigen binding. Reason for investigating and designing novel method for detection of NGAL biomarker of contrast induced acute kidney injury is to develop method that is better than existing ones in terms of earlier and more specific diagnosis and/or mechanism of injury. For the development of proposed novel methods for NGAL detection commercially available ELISA kit was used as the basis for method development and transferred into a FIA system on a macro scale. After optimization of basic experimental parameters (sample injection volumes, carrier solution, reagent flow rates, addition of organic solvents, etc...) the system was downscaled into a microfluidic system on microchip for TLM detection which is particularly suited for small sample volume and can be performed in a relatively short time. Application of TLM in combination with microchip for NGAL detection gives possibilities of further reduction of the total analysis time of the ELISA test by shortening individual incubation steps down to few minutes. Results obtained by a commercial ELISA kit on a microtiter plate reader were compared with results obtained by FIA-TLM. FIA gives all the necessary characteristics to gain adequate repeatability of measurements, faster analysis, and small consumption of reagents and samples. Before starting with ELISA assay on magnetic nanobeads in microchip optimization of reagent (antibodies and antigen, enzyme, substrate) concentration, reaction time, ph, buffer, etc was carried out on microtiter plate. Then the system was transferred on microfluidic chip and final optimization on magnetic nanobeads was performed with measurement of final product on µfia-tlm system. Since virus presence in the environment is still not investigated enough in this study HPV (human papilloma viruses) were used as a virus model. In the present study, we are developing an nanobead based ELISA assay using HPV-16 PsVs, as an antigen for detection 34

58 of anti-hpv-16 L1 antibodies in the sera of HPV-16 infected women. For development of nanobeads based ELISA platform for antibodies against HPV viral particles optimal concentration of reagents was determined by performing ELISA assay on microtiter plate. Final solution was measured on a microtiter plate reader and then transfer to microfluidic chip for FIA-TLM measurement. Results obtained on microtiter plate reader and µfia-tlm were compared. After optimization of basic experimental parameters nanobeads based ELISA assay was performed on microfluidic chip and final product was measured on µfia- TLM system. ELISA-TLM technique for detection of NGAL as well as PsVs of HPV viruses were used as a model system in the design and proof-of-concept for detection of different other viruses, virus-like proteins, PsVs or other analytes. By choosing different antibodies against various analytes the ELISA-TLM technique could be designed also for their detection as well as for any other analyte with adequate ELISA assay already available or expected to be developed. For detection of contrast media the development of a new method was based on the release of iodine from parent molecules by chemical reaction, and its extraction into an organic solvent such as chloroform which could be performed on-line in a microfluidic system, with a final measurement of the iodine charge transfer complex in microfluidic chip with TLM detection. We expect that the new analytical methods developed within this work will provide limits of detection for the selected compounds up to times lower than compared to analytical techniques reported in the literature. At the same time the new methods will enable shorter time of analysis and higher sample throughput for the purpose of fast screening methods. 35

59 36

60 3. EXPERIMENTAL AND INSTRUMENTATION 3.1 Chemicals and reagents Chemicals for contrast agents detection: copper (II) sulfate (1 g L -1 ) (assay 99.0 %) (Fluka, Germany), copper (II) chloride dihydrate (1 g L -1 ), (assay 99.0 %) (AppliChem, Germany), NaNO 2 (0.2 % w/v) (The British Drug House, England) prepared by dissolving 0.2 g NaNO 2 in 100 ml double deionized water, H 2 SO 4 (0.01M) prepared from % H 2 SO 4 (Fluka, Switzerland), HCl (1M) prepared from 32 % HCl (J.T. Baker, USA), iodine (assay 99.8 %) (Riedel-de Haën, Germany), chloroform (assay 99.8 %) (Merck, Germany), NaI (assay 99.5 %) (Fluka, Switzerland), KJ (assay 99.5%) (Fluka, Switzerland), NaHCO 3 (assay 99 %) (Fluka, Germany), Na 2 CO 3 (assay 99 %), diatrizoate (assay 99 %) (Sigma Aldrich, USA), Histodenz (Sigma Aldrich, USA), hydrogen peroxide (30 %) (Belinka Perkemija, Slovenija), Iomeron (Iomeron-350, iomeprol; Bracco Immaging SpA, Italy), Iodixanol (Visipaque-200, GE Healthcare, England). All solutions were prepared in double deionized water (MiliQ water) (18 MΩ cm) prepared through the NANOpure water system (Barnstead, USA). Chemicals for Ion chromatography, HPLC and LC/MS analyses: NaHCO 3 (assay 99 %) (Fluka, Germany), Na 2 CO 3 (assay 99 %), iodide standard for ion chromatography (Sigma Aldrich, USA), acetonitrile (HPLC grade, Sigma Aldrich, USA), orthophosphoric acid 85 % (Sigma Aldrich, USA), double deionized water, acetic acid glacial (Sigma Aldrich, USA). Chemicals for NGAL detection: anti-ngal mouse monoclonal antibody (BioPorto, Gentofte), anti-ngal mouse monoclonal antibody biotinylated (BioPorto, Gentofte), NGAL antigen (Cusabio Biotechnology, China), Tween-20 (Sigma Aldrich, USA), HRP-streptavidin (Invitrogen USA), TMB (Invitrogen, USA), goat anti-mouse Ig-G (Fab specific) peroxidase antibody produced in goat (Sigma Aldrich, USA), mouse IgG 1 antibody (GE Healthcare, UK) BSA (Santa Cruz, USA), skimmed milk (Pomurske mlekarne, Slovenia), Na 2 HPO 4 (assay 99 %) (Acros Chemicals, USA), KH 2 PO 4 (assay 98 %) (Sigma Aldrich, USA), NaCl (assay 99.8 %) (Sigma Aldrich, USA), KCl (assay 99.5 %) (Riedel-de Haën, Germany), 2- mercaptoethanol (Merck, Germany), TEMED (Applichem, Germany), Tris base (Chem Cruz, USA), glycine (Applichem, Germany), glycerol (100 %) (Sigma Aldrich, USA), acrylamide N,N-methylenbisacrylamide (Fluka, Germany), APS (Applichem, Germany), Coomassie Brilliant blue R-250 (Applichem, Germany), methanol (HPLC grade, J. T. Baker, USA), bromphenol blue sodium salt (Applichem, Germany), acetic acid (Sigma Aldrich, USA), SDS (Applichem, Germany), Blue Star Protein Marker (BioRad, Hungary). Chemicals for detection of antibody against PsVs: Na 2 HPO 4 (assay 99 %) (Acros Chemicals, USA) KH 2 PO 4 (assay 98 %) (Sigma Aldrich, USA), NaCl (assay 99.8 %) (Sigma Aldrich, USA), KCl (assay 99.5 %) (Riedel-de Haën, Germany), Anti-HPV 16 L1 (CamVir 1) mouse monoclonal antibody (Abcam, UK), HPV 16 L2 mouse monoclonal antibody (Santa Cruz Biotechnology, USA), goat anti-mouse Ig-G (Fab specific) peroxidase antibody produced in goat (Sigma Aldrich, USA). 37

61 Solutions and buffers for ELISA test PBS Washing buffer 10 mm Na 2 HPO 4 (1.44 g) PBS with 0.05 % Tween mm NaCl (8 g) 2 mm KH 2 PO 4 (0.278 g) Blocking buffers 2.7 mm KCl (0.2 g) 2-5 % BSA in PBS buffer dh 2 O to 1 L 5 % skimmed milk in PBS buffer ph=7.4 (1 M HCl) Carbonate buffer Sample diluent buffer NaHCO 3 (6.06 g) 3 % BSA % Tween 20 in PBS Na 2 CO 3 (3.03 g) dh 2 O to 1 L ph=9.6 (1 M HCl) SDS-PAGE buffers Stacking gel Running gel d H 2 O ml d H 2 O ml ml 40 % AA/BAA ml 40 % AA/BAA- 1 ml 1.5 M TrisHCl ph=6.8 5 ml 1.5 M TrisHCl ph= ml 10 % SDS 0.2 ml 10 % SDS 0.08 ml 10 % APS 0.2 ml 10 % APS ml TEMED TEMED ml Loading sample buffer (2x) Running buffer 1 ml 1 M TrisHCl ph= g Tris 2 ml 20 % SDS 14.4 g glycine 2 ml 100 % glycerol 1 g SDS ml 2-mercaptoethanol d H 2 O to 1 L 4 ml 0.5% bromophenol blue dh 2 O to 10 ml CBB staining solution CBB destaining solution 0.12 g CBB (0.1 %) 30 ml dh 2 O 45 ml dh 2 O 15 ml methanol (10 %) 45 ml methanol (40 %) 5 ml acetic acid (7 %) 10 ml acetic acid (10 %) CBB fix solution CBB storage solution 200 ml d H 2 O 5 % acetic acid 250 ml methanol (50 %) 50 ml acetic acid (10 %) 38

62 Silver stain solutions Fixing solution Developing solution 75 ml ethanol 6.25 g sodium carbonate 25 ml glacial acetic acid 0.2 ml formaldehyde (37 % w/v) dh 2 O to final volume of 250 ml dh 2 O to final volume of 250 ml Silver solution Stop solution 25 ml silver nitrate solution (2.5 % w/v) 3.65 g Na EDTAx2H 2 O dh 2 O to final volume of 250 ml dh 2 O to final volume of 250 ml Sensitizing solution Preserving solution 75 ml ethanol 75 ml ethanol 10 ml sodium thiosulphate (5 % w/v) 11.5 ml glycerol (87 %) 17 g sodium acetate dh 2 O to final volume of 250 ml 1.25 ml glutaraldehid (25 % w/v) Washing solution dh 2 O to final volume of 250 ml dh 2 O Western blot buffers Transfer buffer TBS-T buffer 3.03 g Tris 1 L TBS buffer 14.4 g Glycin 500 µl Tween ml methanol Washing buffer dh 2 O to 1 L TBS buffer with 0.05% Tween 20 TBS buffer Ponceau-S solution 2.8 g NaCl 1 g Ponceau S 2.4 g Trisma base 50 ml 5% acetic acid d H 2 O to final volume of 1 L VLP ELISA buffers Coating buffer Blocking buffer PBS PBS + 4 % skimmed milk % Tween Washing buffer Sample diluent buffer 0.05 % Tween 20 in PBS PBS + 2 % skimmed milk+ 0.1 % Tween 39

63 3.2 NGAL Procedure for measurement of NGAL with commercial ELISA kit NGAL levels in blood samples (plasma, serum) were determined by commercially available ELISA kit 036 (BioPorto Diagnostics, Denmark) as one of the most sensitive assays for NGAL detection. The assay is a four-step procedure: Step µl of NGAL standards or diluted samples were added to the wells on a microtiter plate which were precoated with monoclonal capture antibody against NGAL. Microtiter plate was then incubated for 1 h at room temperature on a shaking platform at 200 rpm. NGAL present in the solutions will bind to the coat, while unbound material was removed by washing with washing buffer. Step µl of biotinylated monoclonal detection antibody is added to each test well and incubated for 1 h. The detection antibodies attaches to bound NGAL antigen. Unbound detection antibodies were removed by washing with washing buffer. Step µl of HRP (horse radish peroxidase)-conjugated streptavidin was added to each test well and allowed to form a complex with the bound biotinylated antibody. Unbound conjugate was removed by washing with washing buffer. Step µl of a color-forming peroxidase substrate containing tetramethylbenzidine (TMB) was added to each test well in the dark. The bound HRP-streptavidin reacted with the substrate to generate a blue colored product. The enzymatic reaction was stopped chemically after 10 min by adding 100 µl of stop solution and the color intensity was read at 450 nm and at reference wavelength of 650 nm in a microtiter plate reader (TECAN Infinite F200, Austria). Absorbance value at 650 nm was subtracted from absorbance at 450 nm. The color intensity (absorbance) is proportional to the concentration of NGAL in each well Calibration curve NGAL concentrations in the test samples were obtained on the basis of a calibration curve prepared with NGAL standards (from 0 to 1000 pg/ml). Absorbance of NGAL standards were plotted against concentrations and according to supplier s indications the calibration curve was obtained by four-parameter logistic curve fitting. Serum and plasma samples were diluted 500 times with sample diluent buffer before measurement. All measurements were done in duplicate or triplicates and samples with absorbance readings out of the calibration range were diluted appropriately by sample diluent buffer and the assay was repeated Procedure for real sample analysis with commercial ELISA kit Sample collection Blood samples were collected from patients admitted to General Hospital Dr. Franca Derganca (Šempeter pri Novi Gorici, Slovenia) for percutaneous coronary intervention (PCI). 40

64 As control group to determine normal NGAL level in population, serum and plasma samples were collected from individuals (11 patients) with normal serum creatinine before percutaneous coronary angiography and from healthy individuals (7 healthy persons). NGAL level was measured in plasma ad serum and results were compared. Plasma samples were collected into EDTA anti-coagulant tubes and then left for spontaneous clotting, aliquoted to avoid repeated freeze-thaw cycles and stored at -20º C until measurement, or at -80º C for longer storage. Serum samples were collected in vacuum tubes with clot activator at same time intervals as plasma samples and were centrifuged immediately after collection. This study was approved by the National Ethics Committee (approval number 47/03/15) of the Republic of Slovenia. All patients were informed about the aim of the study and gave their written consent. All research activities were done with adheres to fundamental ethics principles, as laid out in the Declaration of Helsinki, the Charter of Fundamental Rights of the European Union and the European Code of Conduct for Research Integrity. This work was performed in accordance with all regulations of the Directive 2004/23/EC of the European Parliament and of the Council of 31 March 2004 on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells. Because of the complex and time-consuming procedure of approval of the National Ethics Committee, the number of plasma samples collected in this study was small Spiking experiment To determine NGAL recovery serum sample was spiked with known amount of NGAL standard provided with the kit. 10 µl of NGAL standards of 10 pg/ml and 50 pg/ml were added into serum and incubated for 1 h. 100 µl of serum and spiked serum was then analyzed in triplicate on commercial ELISA kit Measurement of final ELISA product on spectrophotometer For commercial ELISA test HRP was used as enzyme and TMB as enzyme substrate. Substrate is transformed by enzyme into blue colored compound having absorbance maximum at 650 nm as was already described by Josephy et al., (1982). By adding stop solution blue colored compound is transformed into final yellow product with absorbance maximum at 450 nm. After ELISA was finished final yellow product was measured on spectrophotometer (Beckman) to confirm if absorbance maximum is at 450 nm. According to absorbance maximum we will choose which wavelength on the laser is suitable for measurement of final ELISA product on TLS Measurement of final ELISA product on TLS and FIA-TLS Final yellow product after ELISA assay was then measured on TLS. First measurements were done in batch mode in 1 cm optical path length cell. Then we tried to improve the LOD with FIA-TLS. NGAL detection was performed on a dual beam, mode 41

65 mismatched TLS spectrometer consisting of an argon ion laser (Innova 90, Coherent Inc., Santa Clara, CA, USA) as a pump beam laser (130 mw, nm) and a He-Ne laser (Uniphase, Carlsbad, CA, USA) as a probe beam laser operating at nm (4 mw). The pump beam laser was modulated at a frequency of 25 Hz by a mechanical chopper (Scitec Instruments 310CD, Redruth, Cornwall, UK). The changes in probe-beam intensity after passing a 1 cm optical path flow cell (8 µl volume) were monitored behind a pinhole by a photodiode equipped with an interference filter (MellesGriot) and connected to a lock-in amplifier (Model SR830 DSP, Stanford Research Instruments). The lock-in amplifier was further connected to a computer, where the data were collected using a Matlab program. For NGAL detection FIA-TLS system (Fig. 8) was built in-house and consists of a HPLC pump (Shimatzu LC-10Ai) pumping the deionized water as a mobile phase (flow rate 1 ml/min) and one injection valve (Knauer, Berlin, Germany) with 200 µl injection loop. For the first measurement in FIA-TLS samples were diluted in deionized water 10 times and more. Figure 8-FIA-TLS experimental setup consisting of one a HPLC pump, an injection valve (200 µl injection loop), flow through cell, pump/probe unit, TLS detection unit Thermal Lens Microscope After completing the reading on the microtiter plate reader products after ELISA (NGAL standards and samples with unknown concentration of NGAL) were injected into a microfluidic system connected to a microchip for TLM detection. For this purpose we used a TLM built in our laboratory (see Fig. 9), as was previously described in the literature (Franko and Tran, 2010). In brief, two pump beam lasers: argon ion laser (100 mw power, nm, Innova 90, Coherent Inc.) or a solid-state laser (Coherent Inc., USA) operating at 447 nm (power of 50 mw) modulated by a mechanical chopper (Thorlabs Inc., USA) at 1 khz were used for excitation and generation of thermal lens, depending on the required excitation wavelength. As a probe beam source two lasers were used: He-Ne laser (4 mw, MellesGriot) operating at nm or diode laser (Coherent Inc., USA) operating at 532 nm (with power of 2 mw). The thermal lens-induced changes of probe-beam intensity were monitored behind a pinhole by a photodiode (PDA36A, Thorlabs Inc.) equipped with an interference filter 42

66 (MellesGriot). The thermal lens signal was retrieved by a lock-in amplifier (SR830, Stanford Research Instruments) and then treated on a PC using a Matlab programme. Figure 9-Photography of a TLM system used for measurement of final product after ELISA test and for nanobeads based ELISA assay on a microchip. The photodiode detector is not seen behind the microfluidic chip. For NGAL detection on TLM we used a microfluidic chip (Fig. 10) (Y-junction, with one Y-junction channel and two straight channels, The Dolomite Centre Ltd., UK). Chip characteristics are presented in the Table 9. Two microsyringe pumps (NE-1000, New Era Pump Systems Inc., USA) (see Fig. 9b) were used for pumping the carrier liquid and samples through the microchannel. Figure 10-a) Schematic drawing of a microfluidic chip used in this work for NGAL detection. To schematically show the μfia-tlm detection in the microchip, the microchannel is zoomed in. b) Y-joint microfluidic chip (The Dolomite Centre Ltd., UK) 43

67 Table 9: Chip characteristics of Y-joint microfluidic chip (The Dolomite Centre Ltd., UK) Chip specification Value Number of inputs 4 Number of outputs 4 Internal channel cross-section 100 µm x 205 µm (depth x width) Channel length between Y junction 12.5 mm Volume between Y junction 0.2 µl Channel length of straight channels 22.5 mm Volume of each straight channel 0.36 µl Back pressure with 100 µl/min flow (water) through one of the straight channels 0.05 bar Surface roughness of channels (R a ) 5 nm Chip size (length x width x height) 22.5 mm x 15.0 mm x 4 mm Chip top layer thickness 2.0 mm Chip base layer thickness 2.0 mm Surface roughness of channels 5 nm Material Glass Fabrication process HF etching and thermal bonding system Clinical study-measurement of final product after ELISA test on µfia-tlm To check the applicability of TLM method for monitoring of NGAL dynamics, blood samples were collected from four patients who received low-osmolar contrast medium (Iomeron-350, iomeprol; Bracco Immaging SpA, Italy) in doses adjusted to the requirements of each intervention. Plasma samples from these patients were collected before and 1, 2, 3, 4, 5, 6, 12 h after contrast media administration. For the clinical study of NGAL dynamics in patients undergoing PCI blood samples were collected from 30 patients admitted to general hospital Dr. Franca Derganca. Patients underwent percutaneous coronary angiography during May 2014 to February 2015 and they receive different doses of contrast agents (iomeprol or iodixanol). Plasma samples were collected before and 1, 2, 4, 6, 12 hours after coronarography. Samples were diluted 500 times and NGAL level was measured with commercial kit. Every sample was analyzed in triplicate and results were expressed as mean value ± standard deviation. Final yellow product after ELISA test was analyzed on TLM and NGAL levels obtained by these two methods were compared. Measurements were done on TLM system (Fig. 9) as was already described in section A solid state laser operating at 447 nm and a diode laser as probe beam source operating at 532 nm was used. For NGAL detection it was used a microfluidic chip presented on Fig. 10. Two microsyringe pumps NE-1000, New Era Pump Systems Inc., USA) were used for pumping the carrier liquid and to introduce the analyzed solutions into the microchannel. To obtain optimal TLM signal with low background different injection volumes were tested. Samples for µfia-tlm measurement were not diluted. Water was used as a carrier liquid with a flow rate of 10 µl/min. The injection volume was 0.5 µl and injection flow rate was 200 µl/min. 44

68 Statistical analysis was performed to see if there is significant difference between the NGAL values measured by ELISA and TLM method. A two way repeated measures ANOVA test (with SPSS Statistics program) was used to check if there is a statistically significant difference between NGAL values measured by commercial ELISA and TLM. NGAL values at different time intervals measured by ELISA and TLM were compared to validate TLM for determination of NGAL against commercial ELISA on a larger set of samples. In all applied statistical analyses the results are given for 95 % confidence level, i.e. if p value is lower than 0.05 there is a statistically significant difference between the compared results, and when p is higher than 0.05 there is no statistically significant difference between the compared results. Optimization of the reagents concentration for ELISA test with nanobeads Aim of the study described in this chapter is development of a fast ELISA test for measurement of NGAL using nanobeads as solid support. Principle of the assay is sandwich ELISA as previously described by Kjeldsen et al., (1996), Cai et al., (2009), Mishra et al., (2005), with some modifications. Since the pair of antibodies that is used in the commercial ELISA kit is not available on the market, we choose other capture and detection antibodies which might bind to different epitopes on NGAL molecule than antibodies from the commercial ELISA kit. Since this combination of antibodies and antigen was never tested before ELISA optimization need to be performed. Optimization procedure includes determination of concentration of different reagents and other parameters such as: a) Capture antibody concentration (primary Ab) b) Antigen concentration c) Detection antibody concentration (secondary Ab) d) Enzyme conjugate concentration e) Substrate concentration f) Incubation time g) Temperature h) Blocking buffer i) Reagent volume Optimal concentration of reagents (capture and detection antibodies, antigen, enzyme conjugate and substrate) was determined by chessboard titration Chessboard titration Chessboard titration involves dilution of two reagents against each other to determine concentrations giving optimal reaction. This procedure is performed on microtiter plate and principle is the same as sandwich ELISA. For chessboard titration Greiner Microlon 600 (high binding) microtiter plates with binding capacity of about 600 ng/cm 2 of protein were used. To obtain the most controllable results in ELISA, we should choose reagents dilutions which give absorbance signal between AU, since values above this are inaccurate. As was already described by Crowther, (2001) optimal primary antibody concentration between 1-10 µg/ml and secondary antibody between µg/ml showed to be optimal for ELISA. Chessboard titration includes two titration experiments. In first titration experiment capture antibody and antigen were varied with constant amount of detection antibody. In 45

69 second titration detection antibody and antigen were varied with constant amount of capture antibody and HRP conjugate. In first titration two-fold serial dilution of capture antibody (starting from 10 µg/ml) was prepared in coating buffer across the 12 columns of an ELISA plate (Fig.11a). Microtiter plate was coated with capture antibody (starting from 10 µg/ml) and the plates were incubated at 4 C overnight. The next day capture antibody solution was discarded and the wells were blocked with 300 µl blocking buffer. To test optimal concentration of antigen (NGAL molecule) two fold serial dilution of antigen (starting from 2 µg/ml) was performed down to 8 rows. Then fixed concentration of secondary antibody, enzyme conjugate and substrate were added respectively. Between all incubation steps wells were washed manually six times with 300 µl of washing buffer. Finally TMB substrate was then added into the wells. Color was developed within 10 min and reaction was stopped with 0.5 M H 2 SO 4. For the negative control solution without antigen (PBS buffer) was used. Figure 11-Schematic presentation of chessboard titration on microtiter plate (a-first titration, b-second titration) In second titration (Fig. 11b) microtiter plate was coated with capture antibody concentration determined in first titration. Further blocking step was performed and NGAL antigen was serially diluted down the 8 rows as was done in first titration. Two-fold serial dilution of detection antibody (starting from 2 µg/ml) was prepared across the 12 columns of an ELISA plate. Then enzyme conjugate and substrate were added respectively. Final yellow product was then measured on microtiter plate reader at 450 nm and at 650 nm as reference wavelength Incubation time, buffers, temperature, reagent volume To determine optimal conditions for ELISA assay different blocking solutions were tested to see which of them will gives lower background by preventing nonspecific binding. PBS buffer with different content of BSA from 1-5 % and PBS with skimmed milk were tested. For immobilization with primary antibody carbonate buffer ph=9.6 and PBS buffer ph=7.4 were tested. 46

70 Different incubation time and temperature were checked in order to allow optimal conditions for ELISA assay. For all incubation steps adequate time is required to allow binding of capture antibody to the plate as well as antigen to primary and secondary antibody, and enzymatic reaction for color development. Incubation with primary and secondary antibody, antigen, and HRP-conjugate were tested at room temperature and 37 C for 1 h and 2 h and at 4 C overnight. Blocking step was performed at 37 C and at room temperature SDS-PAGE and Western blot Since antibodies that are used in commercial ELISA kit are not commercially available, NGAL antibodies from the producer of commercial kit were used. There are no evidences that these two antibodies in pair were tested before and also it was not confirmed that they are recognizing applied NGAL standard or NGAL antigen present in serum, plasma or urine samples. SDS-PAGE with immunoblotting was applied to determine immunoreaction between antibody and antigen. These techniques are immensely useful for screening for the presence of antigen in a sample and may be used to check if the right antibody was chosen for the use in used immunological method such as ELISA SDS-PAGE electrophoresis SDS-PAGE was performed according to protocol described by Laemmli, (1970) on vertical electrophoresis system from Cleaver (Cleaver Scientific Ltd., UK). SDS-PAGE was performed on 10 % running gel. Thickness of the gel was 1 mm. NGAL standards and plasma samples were diluted with loading buffer and heated for five minutes at 95 C. Samples and protein markers were loaded into the wells. Samples were run at 75 V until color reach stacking gel, then gel was run at 130 V for 1 h. To visualize protein bands after SDS-PAGE gel was stained with CBB-R-250 (Coomassie Briliant Blue-R-250) according to procedure described by Bradford, (1976) with some modifications. In brief gel after SDS-PAGE was first washed with deionized water for 1 minute. Subsequently gel was treated in gel fix solution for 20 minutes; CBB R-250 solution for 20 min and in destains solution for 20 minutes. Gel was resolved until bands appear and background is clear, and then it was kept in gel storage solution overnight at 4 C. Gels that were further used for Western blot transfer were not stained Western blot Gels after SDS-PAGE were transferred to nitrocellulose membrane. Wet transfer was done according to protocol described by Mahmood and Yang, (2012) on Western blotting system from BioRad (BioRad, USA). Gel after SDS-PAGE was washed in deionized H 2 O and soaked in the blotting buffer. The nitrocellulose membrane and filter papers were cut to the size of the gel and soaked in blotting buffer. Sandwich containing membrane and the gel between filter papers were put in blotting unit. Western blot transfer was done at 100 V, 1 h at 4 C. Membrane was colored with Ponceau-S solution for quick and reversible staining. To remove the excess of Ponceau-S solution membrane was washed away with washing 47

71 solution. Each washing step last five minutes. Then blocking solution (5 % BSA in TBS-T) was added for 1 h at room temperature. Membrane was then washed with washing buffer and then incubated in primary Ab solution (anti-ngal monoclonal antibody) 1:2000 times diluted in TBS-T buffer at 4 C overnight. After washing step membrane was incubated with secondary Ab solution (anti mouse IgG Fab specific peroxidase antibody produced in goat) diluted 1:2000 times. Membrane was then washed and substrate (Immobilon Western Chemiluminescent HRP Substrate, Merck Millipore) solution was added. Membrane was visualized with imager (UVITEC Cambridge Imager, UK) Nanobeads based ELISA assay After optimization of basic experimental parameters (sample and reagents volumes, reagents concentration, reaction times, ph, and buffer composition) the system was downscaled from ELISA on microtiter plate into a microfluidic system for nanobeads based ELISA assay with TLM detection. Instead of performing assay in the well of microtiter plate nanobeads were used as a solid support. Magnetic nanobeads that were used in this study were provided by Nanos SCI (inanovative BIO amine, Ljubljana), according to procedure described by Tadic et al., (2014). Magnetic nanobeads were previously characterized by producer with SEM and TEM analysis and results revealed a core-shell structure. TEM measurement showed that core is composed of ~ 80 superparamagnetic maghemite nanobeads cluster (~100 nm in diameter) which is covered by a silica shell, ~ 20 nm thick and it is further functionalized with covalently bonded terminal amine groups Measurement of ζ-potential of nanobeads Nanobeads applied in this work have silica coat with amino groups. Amino groups on nanobeads surface have different charge in dependence of ph as well as protein. Binding of primary Ab to nanobeads is done by electrostatic interaction between carboxyl groups on protein surface and amino groups on nanobeads surface. Therefore ζ-potential was measure on Brookhaven Instruments Corporation ZetaPALS (New York, USA) to get information about charge on nanobeads surface and choice of buffers and experimental conditions. Diluted nanobeads suspension was prepared in KCl (10 mm) solution. Concentration of the beads was around 50 µg/ml. ph of nanobeads solution was then adjusted from ph 3 to ph 10 with 0.1 M NaOH and 0.1 M HCl. Six ph values in this range were chosen for the measurement of zeta potential. For every ph value potential was measured 10 times and results were present as mean value ± standard deviation Nanobeads number The first step in nanobeads based ELISA is binding of primary Ab to nanobeads surface. This step is the most critical one and it is necessary to determine binding of antibodies to nanobeads surface. To test binding efficiency nanobeads number in presented 48

72 volume was calculated. Nanobeads suspension in water with concentration of 6 mg/ml was used. On Fig. 12 magnetic nanobeads applied in this work were schematically presented. Figure 12-Schematic presentation of magnetic nanobeads Applied nanobeads consist of core from iron oxide and silicone surface with amino groups. Mass of one particle m 3 is equal to: m 3 =m 1 + m 2 (5) where m 1 is mass of core with radius r 1 of 50 nm and m 2 mass of silicone coat with amino groups with radius r 2 of 20 nm. Radius of one nanobead is equal to: r 3 =r 1 + r 2 (6) Since mass is equal to m=ρ V mass of one nanobead could be expressed as: m 3 =ρ 1 V 1 +ρ 2 V 2 (7) where ρ 1 is the density of iron core and ρ 2 the density of silicone coat with the values 5.2 g/cm 3 and 2.2 g/cm 3 respectively and V 1 and V 2 volume of iron core and silicone coat V 2 =V 3 -V 1 (8) V 1 = 4 3 r 1 3 π (9) V 3 = 4 3 r 3 3 π density of one nanobead ρ 3 was calculated by formula: ρ 3 = m 3 V 3 (11) Total volume of nanobeads present in 1 g is equal to: V tot =N V (12) where N is the number of nanobeads and V is the volume of one nanobeads which we labelled with V 3 (V=V 3 ). V tot could be also expressed as: where m is equal to 1 g and ρ 3 density of one nanobead. Number of nanobeads present in 1 g is: 49 (10) V tot = m ρ 3 (13) N= V tot V 3 (14)

73 Surface area of one bead sa is equal to sa=4r 2 3 π (15) Total surface of all nanobeads SA present in 1 g is equal to: SA=N sa (16) Determination of antibody concentration bound to nanobeads The first step in nanobeads based ELISA assay is binding of anti-ngal mouse monoclonal antibody to nanobeads surface. It is necessary to determine efficiency of antibody binding and optimal time for binding. The amount of bound antibody was quantified by a supernatant assay using a commercial Easy Titer Mouse IgG Assay kit (Thermo Scientific, USA). This assay allows quick and accurate determination of IgG concentration in the range from ng/ml. In this assay monodispersed polystyrene beads coated with anti IgG are used. These beads absorb light at 340 and 405 nm wavelengths. When they are mixed with a sample containing IgG, they aggregate, causing decreased absorption of light and, therefore, low IgG concentrations yield high absorbance values and high IgG concentrations yield low absorbance values. The decrease in absorption is proportional to IgG concentration and a standard curve can be generated to accurately quantify levels of IgG in different samples according to supplier suggestion. Nanobeads were mixed with primary Ab as it follows: 1 µl primary Ab (1 mg/ml) 49 µl PBS buffer 50 µl nanobeads (6 mg/ml) primary Ab in this mixture were diluted 100 times (10 µg/ml primary Ab concentration in the mixture) and nanobeads were diluted twice (3 mg/ml concentration in the mixture). Primary Ab were incubated for 5, 10, 15, 20, 30 and 60 minutes respectively on shaking platform. After incubation nanobeads were removed with magnetic rack and supernatant with unbounded primary Ab was measured with commercial Easy Titer Mouse IgG Assay according to procedure provided by supplier. With assumption that all the amount of primary Ab was unbound, supernatant was diluted 50 times (10 µg/ml 50 times diluted) to be in the range of calibration curve. Since primary Ab (mouse anti-ngal monoclonal antibody) belongs to group of IgG 1 (immunoglobulin G 1 ) mouse IgG 1 antibody standard (GE Healthcare, UK) of known concentration in the range of ng/ml was used for construction of calibration curve. Curve was made by plotting the concentration of IgG standards (natural logarithm Ln of concentration was used) in dependence of absorbance values of the standards. Assay protocol: 1. Monodispersed polystyrene beads were shaken for 10 minutes. Before adding those into the well of microtiter plate beads were vortexed for 60 s vigorously µl of sensitized beads was added into appropriate number of wells of 96-well microtiter plate µl of sample (with unknown concentration of IgG) and IgG standards were added in appropriate number of wells containing beads. 4. Microtiter plate was mixed on a plate mixer at a 350 rpm for 5 minutes. 50

74 µl of blocking buffer was added to each well. Plate was shaken for 5 minutes at moderate speed (200 rpm). 6. Before measurement all the bubbles were removed. Absorbance was measured at 405 nm on a microtiter plate reader (Tecan, Infinite F200) Number of bound antibody per nanobeads The number of bounded primary antibodies per nanobeads was calculated by dividing the amount of bounded antibodies (as was described in section ) by the known amount of nanobeads in the solution, using the antibody molecular weight and nanobeads concentration Nanobeads based ELISA test performed in plastic eppendorf tube For nanobeads based ELISA assay concentration of antibodies, antigen, enzyme conjugate and substrate were determined previously by chessboard titration on microtiter plate. Nanobeads based ELISA assay was first performed in a plastic eppendorf tube to check if primary antibodies are active after immobilization and then assay was performed in microfluidic chip. Primary Ab were added in a final antibody to mass particle concentration of 10 µg/ml per 0.3 mg of nanobeads. These concentrations were chosen randomly since with this experiment we were just testing the activity of immobilized antibodies. Nanobeads were diluted in PBS buffer and mixed with antibodies as it follows: 3 μl of primary Ab (10 µg/ml of primary Ab in nanobeads solution) 297 μl of nanobeads solution (0.3 mg/ml concentration) in PBS ELISA assay in eppendorf tube was performed as it follows: 1. In the first step negatively charged antibodies were electrostatically associated with the positively charged amine-functionalized magnetic nanobeads during incubation time with constant shaking on vortex. Nanobeads solution was incubated (10 minutes) with primary Ab in eppendorf tube (Fig. 13a). For washing and removing of unbound primary antibody a magnetic rack was used (Fig. 13b) which keeps magnetic nanobeads inside the tube. Eppendorf tube was placed into magnetic rack and nanobeads were washed three times with 1 ml of washing buffer to remove unbound primary Ab ml of blocking buffer was added into eppendorf tube. Blocking buffer was removed by washing ml of NGAL antigen (2 ng/ml) solution was added into eppendorf tube. After incubation nanobeads were washed three times with 1 ml of washing buffer to remove unbound antigen. 4. Then 1 ml of secondary Ab (0.25 μg/ml) was added into plastic tube. After incubation nanobeads were washed three times with 1 ml of washing buffer to remove unbound secondary Ab ml of HRP-streptavidin (0.5 μg/ml) was added into plastic tube. After incubation nanobeads were washed three times with 1 ml of washing buffer to remove unbound enzyme μl of TMB substrate was added into eppendorf tube. 51

75 Incubations with blocking buffer, NGAL antigen, secondary Ab, and HRP-conjugate were first performed for 30 minutes and then the same experiment was repeated by lowering all incubation times to 15 and further to 5 minutes. Figure 13-a) nanobeads solution in eppendorf tube attached by magnets b) magnetic rack applied in this work Nanobeads based ELISA test in microfluidic chip After plastic eppendorf tube nanobeads based ELISA was performed in microfluidic chip (four channel spotting chip PMMA (polymethylmetacrylate), Chip Shop, Jena, Germany) (Fig. 14) with chip characteristics presented in Table 10. Chip with such a simple design was chosen since it allows to perform four separate assays at the same time. Figure 14-a) four channel spotting chip made from PMMA (ChipShop, Jena, Germany) b) foil, c) cover lid, d) fluid connectors, e) silicone tubings Table 10-Chip characteristics of four channel spotting chip PMMA Chip specification Value Number of inputs 4 Number of outputs 4 Width 1000 µm Channel length 58.5 mm Volume of each channel 20 µl Chip size (Length x width x height) 58.5 mm x 1000 µm x 340 µm Chip lid thickness 250 µm Chip base layer thickness 2.0 mm Material PMMA Fabrication process HF etching and thermal bonding 52

76 Nanobeads were kept in the channel of microfluidic chip with magnetic field produced by sintered NdFeB (Neodymium-Iron-Boron) block magnets 2 mm (ChenYang Technologies GmbH & Co. KG, Finsing, Germany) (Fig. 15). Magnets were placed on the outer side of microfluidic chip like it is shown on the Fig. 16. Figure 15-a) Permanent magnets applied in this work b) schematic presentation how magnets stick together Testing the flow rates, magnetic field strength Nanobeads were introduced into the channel of microfluidic chip with syringe pump. Applied magnets provides very strong magnetic field, which retains magnetic nanobeads inside the channel. Different combinations of magnets (Fig. 16) were tested from five and more (Fig. 16b) in one line and even more to just one pair of magnets (Fig. 16c) positioned below and above microfluidic chip. Figure 16-Assembling microfluidic chip a) injection of nanobeads solution with connector and syringes, b) testing of different magnets combination, c) combination of two magnets positioned above and below microfluidic chip keeping nanobeads solution inside the channel Different flow rates were tested to find optimal one for keeping nanobeads inside the channel. Initial flow rate was 100 µl/min and we tried with higher until we notice that nanobeads were washed away and pressure cause pulling of silicone tubes. After optimization of basic experimental parameters (flow rates, magnetic field strength, incubation time) nanobeads based ELISA was transferred to microfluidic chip. In first step it was mixed: 1 µl primary Ab (1 mg/ml) 99 µl PBS buffer 50 µl nanobeads (6 mg/ml) 53

77 Steps in nanobeads based ELISA assay are shown on Fig. 17. Figure 17-Steps in nanobeads based ELISA assay Nanobeads based ELISA includes the following steps as was presented on Fig. 17: 1. Nanobeads were introduced from one inlet hole into the channel of microfluidic chip with a syringe pump (flow rate 150 µl/min) and kept inside the channel with magnetic field produced by magnets positioned above and below microfluidic chip. Unbound primary antibodies were removed by washing buffer from a syringe pump (flow rate 200 µl/min) 2. To prevent unspecific binding the blocking buffer was injected into the channel with syringe pump (flow rate 150 µl/min) and kept in a channel to allow binding of BSA to nanobeads surface. During the incubation time magnets were released to allow higher surface area coming in contact with reagent. Blocking solution was removed by washing buffer (flow rate 200 µl/min) 3. Then NGAL-antigen solution was injected with a syringe pump (flow rate 150 µl/min) into the microchip. Unbound antigen was removed by washing buffer from a syringe pump (flow rate 200 µl/min). 4. Secondary antibody was then introduced into the channel of microchip with syringe pump (flow rate 150 µl/min) and unbound antibodies were removed by washing buffer (flow rate 200 µl/min). 5. HRP-conjugate was injected into the channel of microchip with a syringe pump (flow rate 150 µl/min) and unbound species were washed away by washing buffer (flow rate 200 µl/min). 6. Substrate solution was then injected into the channel of microchip with a syringe pump (flow rate 150 µl/min) and after incubation final colored product was sent with a syringe pump to Y-joint chip for TLM detection. Different incubation times were tested as was previously described in section Nanobeads based ELISA test with TLM detection Nanobeads based ELISA assay was performed on TLM system (see Fig. 9) previously described in section As pump and probe beam sources a solid-state laser (operating at 54

78 660 nm) and a diode laser (operating at 532 nm) were used. Nanobeads based ELISA assay was performed on four-channel chip (as was described in section ) and after enzymatic reaction was completed the reaction product was washed into a Y-joint microchip for TLM detection (Fig. 18). The two microfluidic chips were connected with silicone tubings. Figure 18-Schematic presentation of nanobeads based ELISA for NGAL detection. Microchips were connected with silicone tubings. To schematically show part of the fourchannel chip with nanobeads trapped between magnets, the microchannel is zoomed. To schematically show the μfia-tlm detection in the microchip, the microchannel is zoomed in. Syringe pump was used to send a final colored product to Y-joint chip. Water was used as a mobile phase with flow rate of 50 µl/min and sample flow rate was 150 µl/min Calibration curve For construction of calibration curve ELISA assay was performed with the same NGAL standard (same concentration) in three replicates in three channels of microfluidic with one minute time delay between incubation in separate channels. Every standard replicate was injected six times with syringe pump by shifting the pump from one channel to another with one minute delay. Obtained signal was expressed as mean value ± standard deviation Calibration curve was made with four-parameter logistic curve fitting. For calculation of LOD just the linear part of calibration curve was used Real sample analysis To improve test sensitivity and selectivity we tested five real plasma samples from the patients undergoing PCI. Plasma samples were diluted 100 times in sample diluent buffer. Same sample was injected with syringe pump into three and analyzed the same way as the NGAL standard (see section ). Enzymatic reaction product from each sample replicate was injected six times into the Y-joint microchip by the syringe pump after changing the flow rate during the last incubation stage. NGAL concentration in plasma sample was determined from calibration curve prepared with NGAL standards (see section ). 55

79 3.3 CONTRAST AGENTS Contrast agents detection Four contrast agents (Table 2) of different type were used as model compounds. Iohexol and diatrizoate were bought as analytical standards. Iomeprol and iodixanol were gift from General Hospital Dr. Franc Derganc and they were applied in hospital during the percutaneous coronary intervention Contrast agents degradation conditions Contrast agents degradation was done according to procedure described by Fono and Sedlak, (2007) with some modifications. In this study contrast media solution prepared from standards or solutions applied in hospital for PCI intervention were used for experiments. Since we didn t work with environmental samples containing contrast media and other compounds sample pretreatment step is not acquired. Reaction mixture was prepared as follows: 100 µl contrast agent iohexol, diatrizoate or NaI of appropriate dilution in water 500 µl NaHCO 3 /Na 2 CO 3 buffer 200 µl CuCl 2 (1 g/l) 200 µl H 2 O 2 (30 %) Different factors have influence on degradation efficiency such as: buffer ph, reaction time, reagent concentration, temperature, choice of buffer and reagents volume and all these factors were optimized. After addition of reagents, reaction mixture was further heated in the oven at 80 C in HPLC vials for different incubation times (15, 30, 45, 60, 90, 120, 180 min). After cooling to room temperature iodide containing samples were oxidized as it follows: 1 ml reaction mixture 1 ml H 2 SO 4 (0.01 M) 100 µl NaNO 2 (0.2 %) Different concentrations of H 2 SO 4 (0.01 M, 2.5 M, 3 M) and NaNO 2 (0.2 %, 10 %) were tested. In the first reactions low concentrations were used and then we tried also with higher concentrations to see if we could obtain higher oxidation efficiency. After reduction with hydrogen peroxide and oxidation with sulfuric acid and sodium nitrate, mixture containing iodine was extracted with chloroform Optimization of ph, buffer concentration To find optimal ph for degradation of contrast agents buffer ph was adjusted to different values between 4-9 with 1 M HCl. For this experiment solution of contrast agent iohexol used in hospital for PCI intervention was used and diluted 100 times (initial iohexol concentration was 7.55 mg/ml which correspond to iodine concentration of 3.50 mg/ml). According to Fono and Sedlak, (2007) for contrast agent degradation carbonate buffer with 56

80 concentration of at least M should be used. In this study we tested M and 0.1 M carbonate buffer Optimization of incubation time in the oven To determine what is the optimal incubation time in the oven contrast agent iohexol (7.55 mg/ml iohexol, 3.5 mg/ml of iodine) was treated in chemical reaction with Cu 2+ /H 2 O 2. Reaction mixture was prepared as was described above and then incubated for 15, 30, 45, 60, 75, 90, 120 and 180 minutes respectively. Released iodine was then oxidized and extracted with chloroform and final pink complex was measured on spectrophotometer Calibration curve made on spectrophotometer Concentration of iodine released from contrast agent was obtained from calibration curve. Iodine solutions in the concentration range M were prepared in chloroform and measured on spectrophotometer (Perkin Elmer) in 1 cm optical path length cell. Iodine concentration was plotted against absorbance of pink colored complexes. To determine absorbance maximum of pink colored complex, spectra of iodine solutions in chloroform were recorded Extraction efficiency To determine extraction efficiency with chloroform KI solutions in the concentration range M (with twice higher concentration than iodine standards used for construction of calibration curve) were prepared. Reaction mixture was prepared as follows: 1 ml H 2 SO 4 (0.01 M) 1 ml KI solution ( M) 100 µl NaNO 2 (0.2 %) After oxidation, KI reaction mixture was further extracted with 1 ml of chloroform and reextracted with 1 ml of chloroform. Water phase was discarded and pink chloroform phase was measured on spectrophotometer in 1 cm optical path-length cell. If oxidation efficiency and extraction efficiency are around 100 %, curve obtained with KI in chloroform should be similar to the calibration curve made with iodine standards in chloroform Stability of chloroform iodine solution released by contrast agents degradation Since iodine solutions are light sensitive, pink coloured iodine solutions in chloroform were measured on spectrophotometer for four hours to check their stability. Final solution containing iodine that was released from contrast agents iohexol and diatrizoate ( M I 2 ) after degradation reaction was extracted with chloroform for this purpose. To check stability of iodine in chloroform M I 2 solution was used. 57

81 3.3.6 Contrast agents measurement on µfia-tlm After measurement on spectrophotometer pink coloured iodine solution in chloroform was measured on µfia-tlm system which was also used for NGAL detection (see section 3.2.5). System consisted of argon laser (operating at 514 nm, 50 mw power) and pink coloured iodine charge transfer complex was measured on custom made microfluidic chip (Micronit, Netherland) presented on Fig. 19. Chloroform with flow rate of 10 µl/min was first used as a carrier. Sample flow rate was 10 µl/min and different injection volumes were tested (0.2, 0.4, 0.8 µl). Figure 19-Schematic presentation of microfluidic chip applied in this work for contrast agents detection on µfia-tlm Calibration curve made on µfia-tlm Iodine solutions in chloroform (range M) that were used for construction of calibration curve on spectrophotometer were also measured on µfia-tlm. Calibration curve was prepared by plotting iodine concentration against TLM signal Oxidation efficiency Oxidation efficiency was determined by oxidizing NaI solution ( M) as follows: 1 ml NaI solution ( M) 1 ml 3 M H 2 SO µl NaNO 2 (10 %) After oxidation iodine was extracted with 1 ml of chloroform and concentration of extracted iodine was determined spectrophotometrically. Oxidation efficiency in % was calculated by dividing the measured iodine concentration (formed from iodide by oxidation) with calculated iodine concentration ( M). Reaction efficiency refers to cumulative efficiency of oxidation and extraction. measured iodine concentration Reaction eff. %= calculated iodine concentration 100 (17) 58

82 3.3.7 HPLC analysis of contrast agents and their degradation products To check degradation efficiency of cooper/peroxide chemical reaction contrast agent solutions were analyzed on HPLC before and after degradation reaction. Chromatograms obtained in both cases were compared to verify if contrast agent is completely degraded. The HPLC system consisted of a HP 1100 Series chromatograph, coupled with a DAD detector. The analytical column was thermostated at 35 C. Detection and quantification was performed spectrophotometrically at 254 nm. Contrast agents solutions were filtered through 0.45 µm teflon filters before analysis on HPLC. Reaction conditions for HPLC analysis of different contrast agents are presented on Table 11. Table 11-HPLC conditions for different contrast agent detection Contrast agent Column C 18 Purospher STAR RP-18 end Iohexol caped C 18 (250x4.6 mm, 5 µm) C Diatrizoate 18 Purospher STAR RP-18 (250x4.6 mm, 5 µm) Supelco Discovery C 18 (250x4.6 mm, 5 µm), Iomeprol precolumn:opti-guard, 1mm Guard Column C 18 Supelco Discovery C18 (250x4.6 mm, 5 µm), Iodixanol precolumn:opti-guard, 1mm Guard Column C 18 *ph was adjusted with orto-phosphoric acid Mobile phase AcN: H 2 O (5:95 %), ph= 3* AcN: H 2 O (5:95 %), ph= 3* AcN: H 2 O (5:95 %), ph= 3* 5 min AcN:H 2 O (8:92 %) 5 min AcN:H 2 O (16:84 %) 5 min AcN:H 2 O(16:84 %) Injection volume (µl) Flow rate (ml/min) Iomeprol calibration curve prepared on HPLC Calibration curve was prepared from iomeprol solution in the range µg/ml and analyzed on HPLC (as was described in section 3.3.7) Contrast agent detection with ion chromatography Ion chromatographic system consisted of Autosampler (SHIMADZU, SIL 10 Ai) with a CDD-6A conductivity detector. Iodide concentration was determined on a SHODEX IC SI-90 4E (250 mm x 4 mm) column. A mixture of sodium carbonate and sodium bicarbonate (1.8:1.7 mole ratio) at a flow rate of 1.5 ml/min was used as mobile phase. The injection volume was 20 µl. Calibration curve was constructed with iodide solutions in the range of µg/ml. 59

83 HPLC-MS analysis of contrast agent degradation products Contrast agents are degraded by a reaction which releases iodide atoms from the benzene ring (Steger-Hartmann et al., 2002; Jeong et al., 2010). Each molecule of the selected contrast agents has three iodide atoms and based only on the type of the reaction it is difficult to confirm if all iodide atoms were released during degradation process. Additional structural elucidation of formed degradation products was obtained by HPLC-MS/MS. Four solutions of contrast agents (1 mm) were prepared for HPLC-MS/MS analysis. Contrast media were degraded as was described above (see section 3.3.2). HPLC fractions were collected and analyzed on HPLC-MS/MS to see which degradation products were formed and to determine the number of iodine atoms released from the benzene ring. HPLC-MS/MS analysis was performed on Applied Biosystems 4000 hybrid linear ion trap-triple-quadrupole mass spectrometer (QTrap; AB SCIEX, Concord, ON, Canada). HPLC column Machery Nagel Nucleodur (150 mm 4.6 mm with 3 µm particles) was thermostated at 30 C. Injection volume was 20 µl, and mobile phase consisting of acetonitrile and water (5:95 % v/v) (details were described in section 3.3.7). Flow rate was 0.7 ml/min. Run time was 15 min. The analysis was performed in positive electrospray ionization mode. Temperature source was set at 500 C. De-clustering potential of 100 V, entrance potential of 10 V and electron spray voltage of 5000 V were used. 60

84 3.4 HPV pseudovirions PsVs (pseudovirions) HPV-16 virus type were used as a model system of HPV in our study PsVs production PsVs (pseudovirions) were obtained from the Center for biomedical research and engineering, University of Nova Gorica, Slovenia) as luciferase reporter transducing HPV-16 pseudovirions (PsVs) and were generated in 293 TT cells as previously described (Smith et al., 2007; Buck et al., 2005). Different sets of PsVs with different concentrations of PsVs proteins were applied in this study. The main components of PsVs samples that we were using were L1 and L2 capsid proteins, with some impurities SDS-PAGE of PsVs samples SDS-PAGE was used to test the content and purity of applied PsVs samples and to verify the presence of capsid proteins L1 and L2 in samples. SDS-PAGE was performed on 10 % running gel. Thickness of the gel was 1 mm. PsVs samples were mixed with loading buffer and heated for five minutes at 95 C. Samples and protein markers were loaded into the wells, run at 75 V until color reach stacking gel, then gel was run at 130 V for 1 h. To visualize protein bands after SDS-PAGE, gel was stained with CBB-R-250. Gels that were further used for Western blot transfer were not stained. More sensitive silver staining as was also applied to test the protein content of the samples Silver staining procedure After SDS-PAGE gel with different PsVs was stained with silver using commercial silver staining kit (Silver staining kit, Protein, GE Healthcare). Staining procedure includes several steps is presented in Table 12. Table 12-Silver staining procedure Reaction step fixation sensitizing washing Silver reaction washing developing stopping preserving Procedure Gel was soaked in fixing solution for 60 minutes Fixation solution was first removed and gel was left for 60 minutes in sensitizing solution Sensitizing solution was removed and gel was washed 4 times with distilled water for 15 minutes each time. Silver solution was added and left for 60 minutes on shaking platform. Silver solution was removed and washed twice in distilled water for 1 minute each time. Developing solution was added and left for shaking for 4 to 6 minutes. Gel was then transferred to stopping solution until the bands reached desired intensity. Gel was put in stopping solution and left for shaking for 60 minutes. Preserving solution was added and left for shaking for 60 minutes 61

85 3.4.3 Western blot of PsVs samples Western blot analysis was applied to test the presence of L1 and L2 proteins and their reaction with antibodies used for ELISA assay. PsVs samples were diluted 100, 150, 500, 1000 times respectively, mixed with loading buffer (2x) and 20 µl was loaded on two SDS- PAGE gels and then transferred to nitrocellulose membrane. Western blot was performed according to procedure described by Mahmood and Yang, (2012). Transfer was done at 100 V, 1 h at 4 C. Membranes were colored with Ponceau-S solution for quick and reversible staining. Excess of Ponceau-S solution from the membranes was washed away three times with washing solution. Each washing step last five minutes. Then blocking solution (5 % BSA in TBS-T) was added for 1 h at room temperature. One membrane was then incubated in primary Ab solution (anti-l1 HPV 16 (CamVir1) mouse monoclonal antibody) diluted 2000 times in TBS-T buffer (with 5 % BSA) at 4º C overnight. To confirm the presence of L2 protein second membrane was incubated with HPV 16 L2 mouse monoclonal antibody diluted 400 times in TBS-T buffer (with 5 % BSA). After washing step both membranes were incubated with 2 Ab solution (goat anti mouse IgG Fab specific peroxidase antibody) 2000 times diluted in TBS-T buffer (with 5 % BSA). Membrane was then washed three times with washing solution and chemiluminescence substrate solution was added for blot development. Membrane was visualized with UVITEC CAMBRIDGE Imager (Chemil Imager CB 164) L1 and L2 concentration determination by densitometry PsVs samples applied in this study contained also other production cell-related proteins. Therefore it is complicated to determine concentration of L1 and L2 proteins alone. Bradford test for determination of protein concentration measure total protein content and in our case is not useful. L1 and L2 protein concentration in the sample was determined by densitometry which is a semi-quantitative technique based on measurement of band intensity and comparing with protein standards of known concentration. For determination of L1 and L2 protein content in PsVs samples, BSA solutions with known protein concentration were used as standards. 5 µl of BSA standards and PsVs samples were mixed with 5 µl of loading buffer (2x) and loaded on SDS-PAGE gel. Gel after SDS-PAGE was colored with CBB R-250 (as was described in section ) and imaged with UVITEC CAMBRIDGE Imager (Chemil Imager CB 164). Intensity of the bands was measured with UVISPOT program which allows measurement of density by measuring the surface or height of the protein band and making comparison with protein band of BSA protein of known concentration Optimization of conditions for PsVs based ELISA assay on microtiter plate Optimal concentrations of the PsVs, primary antibody, secondary antibody which will give optimal reaction for nanobeads based ELISA were determined with ELISA test on microtiter plate. 62

86 Choice of buffers, temperature, incubation time PBS and carbonate buffer were tested for coating with PsVs. As a blocking buffer we used PBS buffer with 4 % skimmed milk and 0.2 % Tween 20. For PsVs based ELISA assay different incubation time and conditions were tested, starting at room temperature for 1 h and 2 h, at 4 C overnight, 1 h at 37 C. First experiments were performed at 37 C and then incubation was performed at room temperature. Blocking step was performed at 37 C, and then incubation at room temperature was also tested Determination of PsVs and primary antibody concentration In the previous studies described in the literature (Rajendar et al., (2013); Dessy et al. (2008); Karem et al., (2002); Vidyasagar et al., (2014); Jagu et al., (2010)) PsVs concentration used for coating of microtiter plate was in the range of ng/well. According to this literature data PsVs concentration in the range ng/well were tested (400 ng/well, 267 ng/well, 200 ng/well, 100 ng/well). Principle of the applied PsVs based ELISA assay is direct. Test includes several steps as described below: 1. Coating with PsVs PsVs were diluted (in the range of ng/well) with PBS coating buffer (in fume hood) and transferred to each well of a high affinity binding ELISA microtiter plate (Greiner Microlon 600). Plate was covered with parafilm to prevent evaporation and incubation at different temperatures was tested as was described above in section Blocking step Microtiter plate was brought to room temperature, PsVs solution was discarded, and plate was washed with washing buffer. Plate was blotted against clean paper towel. Non-specific binding sites were blocked by adding blocking buffer (PBS + 4 % skimmed milk % Tween-20). Plate was covered with parafilm and incubated at different temperatures for the purpose of testing. Plate was washed with washing buffer and blotted against clean paper towel. 3. Addition of primary Ab Anti-HPV 16 L1 (CamVir1) mouse monoclonal antibody was used as a standard for detection of titer of anti-hpv 16 antibodies in the patients serum. Anti-HPV 16 L1 antibody was diluted serially (1:2) starting from 1000 times (corresponds to 1200 ng/ml) in sample diluent buffer (PBS + 2 % skimmed milk % Tween-20) in polypropylene tubes or plate and added to the wells of microtiter plate. Plate was covered with parafilm and incubated at different temperatures (room temperature, at 4 C overnight, 1 h at 37 C) for the purpose of testing. Plate was washed with washing buffer and blotted against clean paper towel. 4. Addition of detection antibody and serum samples: HRP-labeled detection antibody (goat anti-mouse IgG (Fab specific) peroxidase Ab produced in goat-sigma Aldrich) was used as secondary antibody. Antibodies or patient serum samples were diluted in sample diluent buffer (PBS + 2 % skimmed milk % Tween-20) and added to each well of microtiter plate. Plate was covered with parafilm and incubated at 63

87 different temperatures (room temperature, at 4 C overnight, 1 h at 37 C) for the purpose of testing. Plate was washed with washing buffer and blotted against clean paper towel. 5. Addition of TMB substrate TMB substrate was added per well and incubated at room temperature for color development. Reaction was stopped with stop solution. Absorbance of the wells was measured with a microtiter plate reader (Tecan Infinite F200, Austria) at 450 nm and 650 nm as a reference wavelength Sample collection To test ELISA assay for sensitivity serum samples were collected from women undergoing colposcopy and the PAP test showed the presence of abnormal cells. These patients were also tested with Hybrid Capture II (HCII) test (Digene Corporation, Gaithersburg, USA) and just patients with positive test were enrolled in this study. HCII test confirm the presence of infection with high risk HPV viruses such as type 16, 18, 31, 33 but it cannot distinguish and determine virus type. For this purpose serum samples were collected from 11 women in vacuum tubes with clot activator, centrifugated immediately after collection and stored at -20º C until measurement. Since HPV-16 belongs to the group of high-risk HPV virus with high malignancy potential with this ELISA test we could determine if HPV infection is related to HPV-16 infection. This study was approved by the National Ethics Committee of the Republic of Slovenia (approval number 47/03/15). All patients were informed about the aim of the study and gave their written consent. All research activities carried out within the program with adhere to fundamental ethics principles, as laid out in the Declaration of Helsinki, the Charter of Fundamental Rights of the European Union and the European Code of Conduct for Research Integrity. This work was performed in accordance with all regulations of the Directive 2004/23/EC of the European Parliament and of the Council of 31 March 2004 on setting standards of quality and safety for the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells Real sample analysis with PsVs based ELISA assay on microtiter plate Eleven serum samples that were collected from patients undergoing colposcopy were analyzed with ELISA to determine presence and concentration of antibodies against HPV-16. After ELISA is finished final solution was measured on microtiter plate reader (Tecan Infinite F200, Austria) on 450 nm wavelength and 650 nm reference wavelength. Patients serum samples were diluted in sample diluent buffer (PBS buffer with 2 % skimmed milk and 0.1 % Tween 20). Sample diluent buffer without PsVs was used as negative control. Concentration of antibodies against HPV-16 was determined from calibration curve constructed by using anti HPV-16 CamVir antibody as a standard ( ng/ml). Antibody concentration was plotted against absorbance value. 64

88 3.4.7 µfia-tlm measurement of final ELISA product After completing the reading on the microtiter plate reader final products after ELISA were injected into a microfluidic chip (see Fig. 10) and measured on µfia-tlm system described in section presented on Fig. 9. Water was used as a carrier liquid with a flow rate of 10 µl/min. The injection volume was 0.5 µl and injection flow rate was 200 µl/min. Calibration curve was constructed with final solution of antibody standards obtained in ELISA assay by plotting TLM signal against antibodies concentration Western blot analysis of serum sample To test the presence of anti L1 HPV 16 antibody in the patients serum samples Western blot analysis was performed as was already described in section In brief same PsVs samples were diluted 100 times in PBS buffer and 12 µl was loaded and separated on SDS-PAGE and then proteins from the gel were transferred to nitrocellulose membrane. Membrane was cut into lines where one line contains one separated PsVs sample. Every line was then blocked with blocking buffer (TBS-T buffer with 5 % BSA) for 1 h on room temperature and incubated with different serum sample (five times diluted in blocking buffer) overnight at 4º C. As a positive control one piece of membrane was incubated with anti L1 HPV-16 antibody 2000 times diluted in blocking buffer. Next day membrane parts were incubated with goat anti-mouse IgG HRP labeled antibody (2000 times diluted in blocking buffer) for 1 h at room temperature. Between incubation steps membrane lines were washed three times with washing buffer. Blots were developed by adding chemiluminescence substrate and pictures were made with UVITEC Cambridge Imager Dot blot analysis of serum samples Collected serum samples were also analyzed with dot blot technique according to procedure described by Nadala and Loh, (2000) to evaluate the presence of anti L1 HPV 16 antibody. PsVs were diluted 100 times in PBS buffer and 1 µl was spotted several times on nitrocellulose membrane and dried on room temperature. Excess of VLPs was removed with washing buffer. Membrane was blocked with blocking buffer for 1 h at room temperature. Membrane was cut into pieces in such a way that one piece contains one PsVs sample. Then membrane pieces were incubated with different serum samples five times diluted in blocking buffer (TBS-T with 5 % BSA) and incubated overnight at 4º C. As a positive control one membrane piece was incubated with anti L1 HPV 16 antibody 2000 times diluted in blocking buffer and incubated overnight at 4º C. Next day membrane was washed three times with washing buffer to remove unbound serum antibodies or antibody standard. Membrane pieces were then incubated with goat anti-mouse IgG HRP labeled antibody 2000 times diluted in blocking buffer for 1 h at room temperature. Blot was developed with chemiluminescence substrate and visualized with UVITEC Cambridge Imager. 65

89 PsVs nanobeads based ELISA in eppendorf tube The same nanobeads used for ELISA assay for NGAL were used also for PsVs based ELISA assay for detection of antibodies against HPV-16. Nanobeads based ELISA assay was first performed in eppendorf tube to check if PsVs are active after immobilization and then assay was transferred to microfluidic chip. Concentrations used for this experiment were chosen randomly since we were just testing the activity of immobilized PsVs. For this purpose PsVs samples were diluted 200 times in PBS buffer. For ELISA in eppendorf tube it was mixed: 10 µl of PsVs (200 times diluted to 50 µg/ml) 50 µl of nanobeads (6 mg/ml) and incubated 15 min on shaking platform at 3000 rpm. After incubation time eppendorf tube was placed in magnetic rack which attracts magnetic nanobeads and supernatant was discarded. Nanobeads were washed away three times (3 x 200 µl) with washing buffer to remove unbounded virus like proteins. Blocking solution (200 µl) was added to eppendorf tube and incubated for 15 min on shaking platform at 3000 rpm to block all free places on nanobeads surface. Tube was placed in magnetic rack and supernatant was discarded. 200 µl of anti L1 HPV 16 antibody (2000 times diluted in blocking buffer) was added to nanobeads and incubated for 15 minutes on shaking platform at 3000 rpm. Unbound anti L1 HPV 16 antibody was washed away with washing buffer (3 x 200 µl). In final step 200 µl of goat anti mouse IgG HRP labeled antibody 2000 times diluted in blocking buffer was added. Unbound HRP labeled antibody was removed with washing buffer (3 x 200 µl). To be sure that all unbound free secondary antibodies were removed by washing, substrate was added to supernatant after every washing step. After the last washing step substrate was added to eppendorf tube and nanobeads were removed from solution with a magnetic rack. The solution was analyzed on spectrophotometer at 650 nm Measurement of PsVs binding to nanobeads surface by Western blot To confirm binding of PsVs to nanobeads surface VLPs were mixed with nanobeads as it follows: 10 µl of VLP 100 times diluted in PBS 10 µl PBS 20 µl nanobeads and incubated for five minutes on shaking platform at 3000 rpm. Then eppendorf tube was placed in magnetic rack and aliquot was taken for Western blot analysis. Also 5 µl of starting VLP solution 100 times diluted in PBS was mixed with 5 µl of loading buffer (2x) and loaded on SDS-PAGE for further Western blot analyses. Supernatant was removed and nanobeads were washed with washing buffer to remove unbound VLPs. Supernatants aliquots of 5 µl after every washing step were mixed with 5 µl of loading buffer (2x) and analyzed by Western blot. After five washing steps supernatant was completely removed and nanobeads were mixed with 5 µl of PBS and same amount of loading buffer (2x) and analyzed with Western blot. Transfer was done according to procedure described in section

90 Measurement of PsVs binding to nanobeads surface with commercial kit Quant-iTTM commercial Protein Assay Kit (Q3320) (ThermoFischer Scientific) was used to determine concentration of PsVs bounded to nanobeads by using Qubit fluorimeter provided by supplier. PsVs samples were mixed with nanobeads as it follows: 2 µl PsVs (50 times diluted in PBS) 48 µl PBS 50 µl nanobeads (6 mg/ml) Nanobeads were attracted by external magnetic field in magnetic rack and solution above the nanobeads which contain free PsVs proteins was measure immediately after mixing on Qubit. This concentration is initial PsVs concentration before binding. After five minutes of incubation on shaking platform at 3000 rpm nanobeads were removed with magnetic rack and supernatant solution with free PsVs was measured on Qubit. Difference between initial concentrations measured immediately and after five minutes gives mass of PsVs bound to nanobeads Nanobeads based ELISA assay in microfluidic chip with µfia-tlm detection Optimal concentration of reagents for ELISA assay in microfluidic chip was determined in microtiter plate. Reaction mixture was prepared with: 2 µl PsVs (50 times diluted in PBS) 48 µl PBS 50 µl nanobeads (6 mg/ml) After 5 minutes of incubation nanobeads were injected in microfluidic chip used for nanobeads based ELISA assay for NGAL detection (see Fig. 14). The same combination of microfluidic chips applied for design of NGAL assay was also used for PsVs (see Fig. 18). Since the same nanobeads were used for NGAL and PsVs based ELISA basic experimental parameters (flow rates, injection volumes, reagents volume, magnet positions) were tested before for NGAL assay. Nanobeads based PsVs ELISA assay was performed according to procedure previously described in section : 1. Incubation with PsVs PsVs diluted in PBS were mixed with nanobeads and incubated in eppendorf tube for 5 minutes. Mixture was injected from an inlet hole into microfluidic channel with a syringe pump (flow rate 150 µl/min). Beads were attracted in the channel with two external magnets positioned on the outer side of the chip, above and below the channel. Beads were washed with 200 µl washing buffer from syringe pump (flow rate 200 µl/min) to remove unbound PsVs. 2. Blocking step Blocking solution (flow rate 150 µl/min) was injected into microfluidic channel and incubated for five minutes after stopping the flow. During incubation magnets were released 67

91 to allow higher contact surface between nanobeads and reagent. Blocking solution was removed with 200 µl of washing buffer (flow rate 200 µl/min). 3. Incubation with anti L1 HPV 16 antibody Anti L1 HPV 16 antibody ( ng/ml) (flow rate 150 µl/min) was injected into microfluidic channel and incubated for five minutes after stopping the flow. Excess of antibody was washed away with 200 µl of washing buffer (flow rate 200 µl/min). 4. Incubation with secondary Ab HRP labeled 2 Ab HRP labeled (0.5 µg/ml) (flow rate 150 µl/min) was injected into microfluidic channel and incubated for five minutes after stopping the flow. Unbound antibody was washed away with 300 µl of washing buffer (flow rate 200 µl/min). 5. Incubation with TMB substrate TMB substrate was injected into microfluidic channel with syringe pump (flow rate 150 µl/min) and incubated for two minutes after stopping the flow. The final blue enzyme reaction product was sent to Y-joint chip for TLM detection Calibration curve of PsVs nanobeads based ELISA assay Calibration curve was constructed with the anti L1 HPV 16 antibodies (in the range ng/ml) standard solution. Every standard was injected with syringe pump in three replicates in three channels of microfluidic chip (see Fig. 14) with one minute time delay by changing the syringe pump from one channel to another. After enzymatic reaction was completed the reaction product of each replicate was washed into a Y-joint microchip for TLM detection and injected six times with syringe pump by changing the pump from one channel to another with one minute delay. Obtained signal was expressed as mean value ± standard deviation. Calibration curve was made with four-parameter logistic curve fitting. For calculation of LOD just linear part of calibration curve was used Real sample analysis Serum samples were diluted two times in sample diluent buffer and every sample was injected with syringe pump in three replicates in three separate channels of microfluidic chip used for reaction (see Fig.18). Syringe pump was shifted from one channel to another with one minute delay between the separate channels. Final blue product was injected six times to Y-joint chip for TLM detection. 68

92 4. RESULTS AND DISCUSSION 4.1 Development of TLS/TLM methods for determination of NGAL Determination of NGAL with commercial ELISA kit Calibration curve (Fig. 20) for determination of NGAL by a commercial ELISA kit and the TEKAN microtiter plate reader was prepared with NGAL standards of known concentration and four-parameter logistic curve fitting was performed Absorbance (AU) NGAL (pg/ml) Figure 20-Calibration curve for NGAL obtained with four-parameter logistic curve fitting For ELISA tests LOD expressed in terms of absorbance was calculated by the formula (Dixit et al., 2010): LOD=average(blank)+3 σ(blank) (18) where average(blank)- is average absorbance of the blank from all replicates σ (blank)-standard deviation of the blank We achieved LOD of: LOD= LOD= AU Corresponding LOD expressed in pg/ml was calculated by the formula: where: x-is concentration in pg/ml x 0 -is inflection point x = x 0 ( y-a 1 A 2 -y ) ( 1 P ) (19) 69

93 y-is response value expressed in absorbance A 1 -is minimum asymptote A 2 -is maximum asymptote P-is Hill slope And we obtain: LOD=4.4 pg/ml Usually in chemical analysis the LOD for linear calibration curves is calculated by formula: LOD= 3 σ(blank) b (20) where equation parameters represent: σ(blank)-standard deviation of the blank b- slope of the regression line Since with a highly sensitive technique such as TLS, we are interested primarily in low concentration ranges, we took just the linear part (0-250 pg/ml) of calibration curve (y= x, R 2 = ) for calculation and comparison of LODs. With σ= AU and b= AU ml/pg LOD was calculated to be: LOD=3.4 pg/ml Real sample analysis with commercial ELISA kit Results for NGAL in different serum and plasma samples expressed as mean value in ng/ml with corresponding standard deviations are presented in Table 13 and on Fig. 21. Table 13-NGAL concentration in serum and plasma samples expressed as mean value and corresponding standard deviation measured with commercial ELISA kit, N is number of samples. (number of replicate for each sample n=3) Group N Mean NGAL SD (ng/ml) (ng/ml) serum control patients plasma control patients

94 250 Serum control Serum patients Plasma control Plasma patients NGAL concentration (ng/ml) Figure 21-NGAL concentrations measured with commercial ELISA kit in serum and plasma samples of 7 healthy individuals (control) and 11 patients undergoing coronarography Results of NGAL measurements with commercial ELISA kit showed that concentrations of NGAL in the analyzed samples (control group and patients group) do not follow the normal distribution (neither in serum (Z=0.207, p=0.04), or in plasma (Z=0.265, p=0.002)) according to Kolmogorov Smirnov test. Mann Whitney test showed that there is a statistically significant difference (Z=-2.581, p=0.01) in serum NGAL levels between control (Md=89) and patients group (Md=156) and between NGAL concentrations in plasma of control (Md=7) and patient (Md=93) group (Z=-2.765, p=0.006). Wilcoxon test showed that there is a statistically significant difference in NGAL values between serum and plasma samples within control group (Z=-2.371, p=0.018) as well as within patients group (Z= , p=0.003). Wilcoxon test furthermore, showed that there is a statistically significant difference in NGAL values between serum (Md=115.5) and plasma (Md=86) samples (Z=- 3,726, p=0.000) in all samples. Wilcoxon test furthermore, showed that there is a statistically significant difference in NGAL values between serum (Md=115.5) and plasma (Md=86) samples (Z=-3.726, p=0.000) in all samples. NGAL concentrations determined in serum were higher than in plasma in both groups and in agreement with previous reports in the literature (Clerico et al. 2012; Bachorzewska et al. 2006; Malyszko et al., 2009) where NGAL concentrations between pg/ml were stated as normal level. Results showed that in group of patients undergoing coronarography NGAL concentration in serum and plasma was significantly higher in comparison to NGAL level in healthy individuals. NGAL concentration in these patients was measured before coronarography procedure with contrast agents injection. Such high level could be due to different reasons such as infection or inflammation as was already described in the literature since NGAL exists in three different forms already described in section. Different forms are released from different sources. Neutrophils synthesize the monomer and the homodimer forms while renal tubular epithelial cells synthesize monomer and in small 71

95 amount heterodimer form (Hatipoglu et al., 2011; Nickolas et al., 2012). Ideal immunoassay capable to distinguish different molecular forms of NGAL still doesn t exist. Antibodies applied in different ELISA assays does not distinguish between different forms of NGAL protein so therefore there is a need to develop immunoassay which will combine polyclonal and monoclonal antibodies that primarily recognize monomer NGAL released from kidney tubules which could, hence, be distinguished from the homodimer and monomer synthesized by neutrophils. NGAL kit 036 applied in this study is measuring total NGAL level which include monomer, dimer, trimer form. For our study monomer form is the relevant one which level could increase if acute kidney injury occurs. Commercial kit applied in this study is not specific just for monomer NGAL form and applied antibodies recognize also other NGAL forms synthesized due to several reasons. Ideal immunoassay for NGAL detection does not exist. According to Noto et al., (2013) antibodies combination used in the current NGAL immunoassays does not distinguish between different isoforms of the protein. Due to this reason there is a need to develop immunoassays that will combine monoclonal and polyclonal antibodies that favorably recognize monomeric NGAL originating from kidney tubules which could, hence, be distinguished from the homodimer and particularly from the monomeric form synthesized by neutrophils due to the different molecular structure and epitope exposure Spiking experiment Recovery was calculated from the difference between spiked and unspiked serum samples applying formula: %R= F-I A 100 (21) where symbols represents: R-is recovery in percent F-analyte concentration in spiked sample I-analyte concentration in serum sample A-concentration of the added analyte in the spiked portion Calculated NGAL recoveries are presented in Table 14. Table 14-Analyte concentration in serum, spiked serum and corresponding recoveries. (number of replicate for each sample n=3) I F A Recovery Sample (pg/ml) (pg/ml) (pg/ml) (%) Serum 88±7 / / / Serum spiked with 10 pg/ml NGAL Serum spiked with 50 pg/ml NGAL / 89±9 0.9± / 92±9 4.5± Recovery of spiked samples should be in the range of %. Obtained results for both spiked samples are in this range. According to obtained recovery values we can conclude that beside NGAL other constituents of the sample didn t influence on the results. The spike and 72

96 recovery experiment demonstrated the efficiency and specificity of commercial ELISA kit. Measurement of final ELISA product on TLS Measurement of final ELISA product on TLS Choosing appropriate wavelength for TLS measurement Since the final ELISA product exhibits an absorbance maximum at 450 nm as known from literature and also confirmed by spectrophotometric measurements (Figure 22), there are two available excitation lines from the argon laser at 458 nm and 476 nm for measurements on TLS. 0.6 Absorbance (A.U.) Wavelength (nm) Figure 22-Absorbance spectra of final ELISA product (50 pg/ml of NGAL) measured on spectrophotometer According to the spectrum (Fig. 22) absorbance at 458 nm is A= (which is 92 % of absorbance value at 450 nm). At this wavelength laser power is around 50 mw providing low sensitivity. At the wavelength of nm laser power is 150 mw and more. The corresponding absorbance at this wavelength was A= (which corresponds to 58 % of absorbance value at 450 nm). Comparison of the signal was made at these two wavelengths by Eq. 1 to choose appropriate one for NGAL measurement. At wavelength of 458 nm I 458 At wavelength of 476 nm I 458 = W K m W m K =

97 I 476 = W K =19.23 I W m m K Obtained results showed that working at 476 nm wavelength will improve TLS signal since absorbed power is 1.9 higher in comparison to the absorbed power at 458 nm wavelength. Therefore we chose 476 nm wavelength on argon laser for measurement of final NGAL product FIA-TLS Considering the fact that the final ELISA product is sensitive to the light and photo labile, measurements were done in FIA where the contact between analyte and laser light is minimal. For the flowing mode measurement repeated sample injections (at least 2 times) with a 200 μl injection loop were performed to verify the reproducibility and minimize measurement errors. Because of high absorbance values final products after ELISA were diluted in water which was used also as a mobile phase. Different flow rates were tested (0.5, 1, 2, 3 ml/min) and flow rate of 1 ml/min was most appropriate because of sample throughput and signal stability. With higher concentration (1000 pg/ml) of final product the thermal lens saturation occurs and a black spot appears on the pinhole. Consequently the corresponding TLS signal from the lock-in amplifier (Fig. 23) is lower. Figure 23-TLS signals for two replicates of final ELISA product undiluted (NGAL 1000 pg/ml) The same effect was obtained with NGAL standard of 250 and 500 pg/ml. With lower concentrations we didn t notice such effect. This effect is the result of defocusing of the probe beam causing the drop of the light intensity on the probe beam axis. For such high absorbance values the probe beam is completely defocused and the probe beam intensity drops to zero, which can be observed by a naked eye as a black spot in the center of beam image on the detector. TLS is too sensitive in comparison to Tecan reader of microtiter plate. Because of this final product with higher NGAL concentrations after ELISA test were diluted 74

98 in deionized water 10 times and more for measurement. With dilution such effect didn t occur but by diluting samples we are losing sensitivity. In our case we are interested primary in low concentration ranges which doesn t need dilution and for this purpose highly sensitive technique with low sample and reagent consumption is needed Impact of different solvents on TLS signal As it was already described in section 2.9, TLS is highly affected by the solvent used for measurements. TLS signal is proportional to (dn/dt)/k ratio. Therefore, choosing an appropriate solvent for the analyte with larger dn/dt and smaller k will increase sensitivity (see Table 8). The lowest TLS signal is obtained with water opposite to organic solvents which gives higher signals due to thermo-optical properties. Measurement of two replicates of 100 pg/ml of NGAL antigen (Fig. 24a) diluted in 60 % ethanol showed that some kind of tail structure appears in the FIA-TLS signal. Water as mobile phase gives high noise signal lowering the LOD of measurement. This measurement showed that there was a problem due to non-homogeneity between the sample and mobile phase as was already described in the literature (Kožar Logar et al., 2002). Figure 24-TLS signals of a) two replicates of final ELISA product (100 pg/ml of NGAL antigen) diluted two times in 60 % ethanol with water as mobile phase, b) final ELISA product (10 pg/ml of NGAL antigen) diluted two times in 60 % ethanol with ethanol as mobile phase c) final product (100 pg/ml of NGAL antigen) diluted two times in water with water as mobile phase Although TLS signals in samples with added ethanol are in principle higher there is a problem with high background and instability of baseline. Furthermore, precipitates can be formed after mixing of final product solution and ethanol. According to presented results (Fig. 24c) we decided to use water as a solvent for sample dilution and as a mobile phase. 75

99 FIA-TLS calibration curve After optimization of basic experimental parameters (solvent, injection volume, flow rate) calibration curve (Fig. 25) was constructed and for calculation of LOD just linear part (0-100 pg/ml) of calibration curve was used. LOD was calculated by applying Eq TLS signal (V) NGAL concentration (pg/ml) Figure 25-Linear calibration curve obtained on FIA-TLS (R 2 = ) For FIA-TLS measurement LOD expressed in pg/ml (σ= V and b= V ml/pg) was calculated to be: LOD=0.5 pg/ml If we compare FIA-TLS LOD of 0.5 pg/ml with 3.4 pg/ml obtained with ELISA there is around 7 times lower improvement with TLS. Beside such low LOD there is still need for technique which will reduce reaction time, reagent consumption Measurement of final ELISA product on µfia TLM In comparison to TLS, TLM is not sensitive to solvent composition allowing analysis of samples in microfluidic flows containing different organic solvents. Samples for TLM analysis were not diluted as was the case with FIA-TLS. Optical path length in TLM is 100 times shorter, therefore inhomogeneity has lower impact on probe beam propagation. Furthermore, as we know from own previous experience (Liu and Franko, 2014) sample throughput in µfia-tlm is affected by the flow rate and the injection volume, which at the same time governs the sensitivity of the method Determination of optimal injection volume and flow rate Different injection volumes were tested to obtain the highest possible TLM signal. As it is presented on Fig. 26 we tested three different injection volumes 0.25, 0.5 and 1 µl. 76

100 1.2x L 1.0x L Lock-in signal (V) 8.0x x x L 2.0x Time (s) Figure 26-TLM signals for different injection volumes of final ELISA product from NGAL standard (100 pg/ml) (water was used as a mobile phase with flow rate of 10 µl/min, sample injection flow rate was 200 µl/min) Time intervals between injections of samples within a series of injections for a given injection volume were not the same. As expected, there is no observable effect on the peak shape due to differences in composition of carrier flow and the injected sample. The peak height increases with injection volume. If we compare the signals for 0.25, 0.5 and 1 µl, samples, intensity of the signal for 1 µl is 20 % higher compared to signals from 0.5 µl samples, while the signal for 0.5 µl is around 40 % higher than the signal for 0.25 µl sample. Elution time for larger volumes is however longer and as a consequence the sample throughput for 0.5 µl injection volume is about two times higher in comparison to sample throughput for injection of 1 µl samples. To reduce analysis time we choose 0.5 µl as optimal injection volume for our later measurement of the final ELISA product on TLM. This injection volume allows analysis of higher number of samples (9 samples per minute) in comparison to 1 µl volume Calibration curve NGAL concentration in the test samples were obtained on the basis of the calibration curve prepared with NGAL standards (from 0 to 1000 pg/ml). Absorbance of NGAL standards was measured on microtiter plate reader and plotted against NGAL concentration (Fig. 27). Same NGAL standards after ELISA were analyzed on µfia-tlm and the obtained signals (peak heights) were plotted against NGAL concentration. For calculation of LOD and comparison of ELISA and µfia-tlm only the linear part of the calibration curve was used. 77

101 Absorbance (AU) NGAL concentration (pg/ml) Figure 27-ELISA calibration curve for NGAL obtained with linear fitting (R 2 = ) LOD for NGAL obtained on Tecan microtiter plate reader was calculated by applying Eq. 20 (σ= AU and b= AU ml/pg): LOD=10 pg/ml Fig. 28 represents calibration curve obtained by μfia-tlm detection of final ELISA product from NGAL standards of known concentration in the range (0-500 pg/ml). Regression line was calculated for the linear range pg/ml Lock-in signal (mv) NGAL concentration (pg/ml) Figure 28-Calibration curve for µfia-tlm system obtained with NGAL standards of known concentration (R 2 =0.9948, for pg/ml range) 78

102 Limit of detection calculated by applying Eq. 20 (σ= mv and b= mv ml/pg) was: LOD=1.4 pg/ml The results showed that despite about 70 times shorter optical path length µfia-tlm provides about seven times lower LODs for detection of NGAL in comparison to transmission mode UV-Vis measurements on the Tecan instrument. For illustration, the LOD on Tecan reader (~7 mm sample thickness) was 10 pg/ml, while for TLM detection on a microfluidic chip (100 µm deep microchannel) the LOD was 1.4 pg/ml Reproducibility Final products of real plasma samples were also analyzed on µfia-tlm after the ELISA assay. As demonstrated by TLM signals on Fig. 29, we obtained a sample throughput of about seven samples per minute with the μfia-tlm system with good repeatability. 1.4x x x10-3 Lock-in signal (mv) 8.0x x x x Time (s) Figure 29-μFIA-TLM signals for four replicate injections of final ELISA product of three replicates of 100 pg/ml standard of NGAL Obtained results indicate good reproducibility and repeatability of this assay platform. Relative standard deviation of three replicates of plasma sample was calculated according to Everitt, (2002). A relative standard deviation of around 2 % confirmed good repeatability of measurement. With the possible seven injections per minute and assumed triplicate injection for one sample replicate the sample throughput is comparable with measurement on Tecan which can analyze 40 samples in duplicate in around 20 minutes. 79

103 4.1.5 Application of developed µfia-tlm method in clinical study In pilot study of NGAL dynamics (Fig. 30) plasma samples from four patients undergoing coronary angiography were measured. Results showed that patients respond very differently to contrast media. NGAL concentration (ng/ml) Time (h) ELISA TLM Patient D Patient C Patient B Patient A Figure 30-NGAL dynamics measured with Tecan and µfia-tlm in plasma samples collected from four patients before the coronary angiography and up to 12 hours after injection of the contrast medium. Where observed (Patients C and D), the NGAL levels showed increase at 2 hours after injection of contrast media and returned to the initial value after 12 hours. Comparison of NGAL dynamics followed by Tecan reader and μfia-tlm in plasma samples additionally confirmed that NGAL concentrations determined by these two techniques are in good agreement Clinical study-measurement of final ELISA product on µfia-tlm In Table 15 are presented mean NGAL values for 30 patients enrolled and corresponding standard deviations measured with ELISA kit on microtiter plate reader and TLM method. 80

104 Table 15-NGAL concentrations at different times after application of contrast agent (for 30 patients enrolled in clinical study) determined by ELISA with microtiter plate reader and by µfia-tlm expressed as mean value with corresponding standard deviation. Sample from each patient was measured in triplicate (n=3) Time (h) Method Mean(ng/mL)±SD(ng/mL) ELISA 120 ± 70 µfia-tlm 130 ± 90 ELISA 110 ± 60 µfia-tlm 110 ± 60 ELISA 120 ± 70 µfia-tlm 120 ± 60 ELISA 100 ± 40 µfia-tlm 110± 40 ELISA 120 ± 60 µfia-tlm 120 ± 50 ELISA 130 ± 110 µfia-tlm 130 ± 90 NGAL values from 30 patients measured with commercial ELISA kit before and up to 12 h after injection of contrast agents are presented on Fig NGAL (ng/ml) Time (h) Figure 31-NGAL concentrations measured with commercial ELISA kit in 30 patients before the coronary angiography and 1, 2, 4, 6, 12 h after injection of the contrast medium. Fig. 32 presents NGAL values from 30 patients measured with µfia-tlm method at different time interval. 81

105 NGAL (ng/ml) Time (h) Figure 32-NGAL concentrations measured with µfia-tlm in 30 patients before the coronary angiography and 1, 2, 4, 6, 12 h after injection of the contrast medium. Comparison of NGAL concentrations obtained with ELISA and µfia-tlm by the two way repeated measures ANOVA test showed that there is no statistically significant difference (Wilks Lambda=0.994, F(1, 29)=0.173, p=0.68, Partial Eta Squared=0.006) in NGAL values, which confirms good agreement of the two methods. With the two way repeated measures ANOVA test we also found that there is statistically significant change in NGAL concentration at time intervals: 0-1 h (p=0.025), 0-4 h (p=0.48), 4-6 h (p=0.003). Results showed that after 1 h there is a drop in NGAL level and then NGAL level is slightly increasing 2 h (but not statistically significant) after injection of contrast agents at was previously described in the literature (Cai et al., 2009; Mishra et al., 2005). 12 h after administration of contrast media NGAL level is coming to the value close to the one at the beginning. Fig. 33 presents a comparison of NGAL level in 30 patients (expressed as mean value) measured with commercial ELISA kit and µfia-tlm. Figure 33-Comparison of NGAL level measured with ELISA and TLM 82

106 Patients enrolled in this study received different doses of contrast agent (according to age, height, and glomerular filtration rate) which may be a reason for different NGAL concentration measured in their blood. Since the NGAL concentrations measured by the two methods differ from patient to patient we tested correlation between measured concentration of NGAL and applied volume of contrast agent. Pearson-s correlation test showed that there is no a statistically significant correlation between applied volume of contrast agent and measured NGAL concentration Development of µfia-tlm ELISA for NGAL detection In order to develop µfia-tlm method for detection of NGAL, optimization of reagents concentrations for ELISA assay in microfluidic chip by chessboard titration on microtiter plate was performed initially to optimize primary and secondary antibody concentration, concentration of enzyme-conjugate and substrate as well as the NGAL concentration range. The summary of optimized conditions is given in Table Optimization of primary Ab concentration According to the obtained results presented on Fig. 34 capture antibody (primary Ab) concentration between 0.63 and 1.25 µg/ml is the optimal giving absorbance values between 1.5 and 1.8 AU, since the values above this range are inaccurate as was already described by Crowther, (2001). Therefore primary Ab concentration of 1 µg/ml was chosen as most appropriate for our later experiments. 2.5 A (AU) Ab 5 μg/ml 1 Ab 2.5 μg/ml 1 Ab 1.25 μg/ml 1 Ab 0.63 μg/ml 1 Ab 0.31 μg/ml NGAL concentration (pg/ml) Figure 34-Calibration curves for optimization of ELISA assay obtained with different primary Ab concentrations and different antigen (NGAL) concentration Optimization of secondary antibody concentration Chessboard titration results (Fig. 35) showed that the optimal secondary antibody concentration is between and 0.25 µg/ml. Secondary antibody concentration of

107 µg/ml was chosen as optimal for our experiments giving at the same time the lowest blank values ( ± AU) in comparison to other secondary antibody concentrations. A (AU) Ab 2 μg/ml 2 Ab 1 μg/ml 2 Ab 0.5 μg/ml 2 Ab 0.25 μg/ml 2 Ab μg/ml NGAL concentration (pg/ml) Figure 35-Calibration curves for optimized ELISA assay obtained with different secondary Ab and antigen (NGAL) concentration Optimization of HRP-conjugate concentration Based on the results presented on Fig. 36 optimal HRP-streptavidin concentration was determined to be 0.5 µg/ml. This concentration gives optimal absorbance between 1.5 and 1.8 AU and at the same time lower background ( ± AU) in comparison to other enzyme-conjugate concentrations. A (AU) HRP 5 g/ml HRP 2.5 g/ml HRP 1.25 g/ml HRP g/ml HRP g/ml NGAL concentration (pg/ml) Figure 36-Calibration curves for optimized ELISA assay obtained with different enzymeconjugate and antigen (NGAL) concentration 84

108 Optimization of NGAL antigen concentration range for calibration In commercial ELISA kit NGAL antigen concentration for calibration is in the range from 10 to 1000 pg/ml. According to results presented on Fig. 36 the NGAL concentration of 2000 pg/ml gives absorbance above 1.8 AU which is already too high for accurate absorbance measurement (Crowther, 2001). Furthermore, in wells with this concentration of NGAL in combination with optimal capture and detection antibody concentration black crystals appeared. This could be due to excess of antigen and related compounds which form aggregates with antibodies causing formation of TMB substrate crystals. As optimal NGAL concentration we used the range pg/ml. This concentration range gives optimal absorbance values between 1.5 and 1.8 AU Optimization of buffers, blocking step, incubation time Nonspecific binding During the optimization of reagents concentrations we observed a problem with high background signal in the negative control. A uniform color was present in the wells with standards and with the sample because of nonspecific binding. The reason for high background could be also insufficient washing, inappropriate concentration of the blocking agent and insufficient amount of Tween detergent in the buffers. Problem with nonspecific binding was resolved by increasing the percentage of blocking agent and by increasing the amount of added Tween detergent. Between different tested blocking buffers PBS with 2 % BSA was chosen for all our later experiments. This buffer showed to be the most efficient since it allowed the highest possible binding of capture antibody and the lowest background signal. For immobilization of capture antibody on microtiter plate with two buffers were tested: carbonate buffer (ph=9.6) and PBS (ph=7.4) and the resulting calibration curves are presented on Fig. 37. Calibration curves were compared with statistical analysis using Wilcoxon Signed Ranks test (for dependent samples with abnormal distribution). Obtained results (Z=-0.28, p=0.779) showed that no significant differences in the slopes between the two curves were detected. Because the carbonate buffer gives higher background we choose the PBS buffer for immobilization of primary antibody. Furthermore, nanobeads that are applied in this work are more stable at ph=7.4 of PBS buffer than in carbonate buffer with ph=9 85

109 2.0 PBS buffer carbonate buffer 1.5 A (AU) NGAL concentration (pg/ml) Figure 37-NGAL ELISA calibration curve obtained with four-parameter logistic curve fitting using PBS and carbonate buffer for immobilization of primary Ab. Each data point represents mean of three replicates with corresponding standard deviation. Different incubation times were also tested. For coating with primary antibody incubation overnight at 4º C showed to be the most efficient. For other incubation steps we try with 2 h and shorter incubation time. 1 h incubation time showed to be sufficient for optimal binding. The effect of temperature was also tested. Incubation at room temperature showed to be sufficient for optimal binding Sandwich ELISA protocol Optimized conditions for ELISA assay on microtiter plate are shown in Table 16. Table 16-Summary of optimized conditions for sandwich ELISA assay Applied Optimal Reagent volume concentration (µl) 86 Incubation time Washing step Primary Ab 1 µg/ml 50 Overnight at 4 C 5 x 300 µl Blocking buffer 2 % BSA h at room temperature 3 x 300 µl NGAL antigen pg/ml 50 1 h at room temperature 6 x 300 µl Secondary Ab 0.25 µg/ml 50 1 h at room temperature 6 x 300 µl HRP-enzyme conjugate 0.5 µg/ml 50 1 h at room temperature 6 x 300 µl 10 min at room / TMB substrate ready to use solution 50 temperature Stop solution 0.5 M H 2 SO 4 50 / / ELISA calibration curve Fig. 37 presents calibration curves prepared by NGAL standards solutions ( pg/ml). Calibration curves were obtained by applying four-parameter logistic curve

110 fitting with Origin program. All the measurements were done in triplicate and results were expressed as mean value ± standard deviation. Limit of detection for ELISA test (using calibration curve with PBS coating buffer) was calculated by Eq. 18 which gave: LOD= AU Corresponding LOD expressed in concentration in pg/ml was obtained by applying Eq. 19: LOD=2.5 pg/ml If just linear part of calibration curve (y= x, R 2 = ) was used limit of detection calculated by Eq. 20 (with σ= au b= AU ml/pg) was: LOD=1.2 pg/ml ELISA assay of real samples To test the accuracy and reliability of optimized ELISA assay NGAL concentrations were determined in plasma samples of the patients before coronary angiography and urine samples from healthy individuals and results were compared with the concentrations determined with commercial ELISA kit. Plasma samples were diluted 200 times in dilution buffer, while urine samples were diluted 100 times. In Table 17 NGAL concentrations in different plasma and urine samples are presented. Table 17-NGAL concentrations in different plasma and urine samples measured with optimized ELISA assay and commercial ELISA kit. (number of replicates for each sample n=3) ELISA kit home made ELISA Plasma sample NGAL* NGAL* SD (ng/ml) SD (ng/ml) (ng/ml) (ng/ml) * * * * Urine sample NGAL* NGAL* SD (ng/ml) (ng/ml) (ng/ml) SD (ng/ml)

111 On Fig. 38 NGAL concentrations measured with ELISA kit and home-made ELISA in 16 plasma samples are presented. 160 ELISA kit home made ELISA NGAL concentration (ng/ml) Figure 38-NGAL concentrations in 16 plasma samples measured with ELISA kit and homemade ELISA NGAL concentrations measured in plasma with home-made ELISA and commercial ELISA kit were tested for normal distribution with Kolmogorov Smirnov test. Test showed that data do not have normal distribution (Z=0.269, p=0.003 for ELISA kit and Z=0.226, p=0.028 for home-made ELISA). The Wilcoxon Signed Ranks test for dependent samples showed that there is a statistically significant difference (Z=-3.516, p=0.000) in NGAL concentrations determined by the two applied methods (ELISA (Md=89.2) and home-made ELISA (Md=60.1)), which could also be caused by the variability of NGAL concentrations and presence of different NGAL forms (Kjeldsen et al., 1993) among the patients. With exception of four samples where the results of the two methods were in good agreement (samples number 6, 8, 10, 13) the commercial ELISA kit has consistently given significantly higher concentrations for NGAL. This could be attributed to the fact that commercial kit is detecting total NGAL level because the used antibodies are recognizing all three forms of NGAL. ELISA assay that was optimized within this work is using pairs of antibodies which were not tested for recognition of different NGAL forms and might not be efficient for recognition of some forms of NGAL. According to producer (BioPorto, Denmark) primary as well as secondary Ab is recognizing NGAL in monomer and dimer forms. Also it is not specified by producer if these two antibodies recognize same epitopes on NGAL antigen molecule. Results for NGAL concentrations in urine samples obtained by previously mentioned methods were also compared by applying Wilcoxon Signed Rank test. It was observed that there is no statistically significant difference (Z=-1.826, p=0.068) in NGAL concentration measured by ELISA kit (Md=18.5) and home-made ELISA (Md=14.1). 88

112 SDS-PAGE and Western blot SDS-PAGE and Western blot were applied to test the compatibility between antibodies and NGAL antigen used in ELISA assay SDS-PAGE electrophoresis of plasma samples To investigate in which forms NGAL is present in plasma and urine samples SDS- PAGE technique was used. Plasma samples were diluted twice with loading buffer and 20 µl of diluted samples were loaded on the gel. NGAL protein which is released in response to acute kidney injury is a monomer of 25 kda. NGAL standard applied in this work is also in a monomer form what was confirmed with SDS-PAGE (Fig. 39a). Plasma samples were analyzed with SDS-PAGE to confirm the presence of NGAL protein. Figure 39-SDS-PAGE electrophoresis of a) Line 1-NGAL standard (5 µg/ml), Line 2-NGAL (2.5 µg/ml), Line 3-NGAL (1 µg/ml), Line 4-protein marker; b) plasma samples collected from six healthy individuals Line 1-NGAL standard (5 µg/ml), Line 2-7 plasma sample of six healthy persons, Line 8-protein marker SDS-PAGE results showed that NGAL standard applied in this work has molecular weight of 25 kda. SDS-PAGE of six plasma samples from six individuals showed same electrophoresis profile and bands at 25 kda. This band could be due to presence of NGAL or some other proteins with same molecular weight Western blot of plasma samples Western blot results of analyzed plasma and urine samples are presented on Fig. 40. In plasma and urine samples the NGAL monomer form (band at 25 kda) was presented. In urine sample monomer is the only existing NGAL form. In plasma samples several bands were observed. Band at 25 kda belongs to NGAL monomer protein from epithelial tubular cells. Bands at around 50 kda belong to NGAL dimeric form from neutrophils (46 kda). Band at around 100 kda and 180 kda are from heterodimer forms. These results are in agreement with those already reported by Mårtensson et al., (2012). For our measurement it will be better to use urine samples since this sample contains just the NGAL monomer (25 89

113 kda) from kidney. Because it is easier to collect plasma samples than urine while performing angiography we decided to focus on plasma samples. Fig. 40a presents a blot of plasma samples of a patient before and up to 12 h after coronary angiography with contrast agent injection. The concentration of NGAL dimer level is changing with time but it is hard to accurately evaluate the concentration and related changes, since immunoblotting is a semiquantitive technique. The NGAL pool present in blood samples belongs to three NGAL forms: monomeric from tubular epithelial cells and dimeric form from neutrophils (Kjeldsen et al., 1993a). NGAL that we are measuring with commercial ELISA assay is the combination of two forms monomeric and dimeric. Figure 40-Western blot of a) plasma sample from patient undergoing coronary angiography Lines 1-6 NGAL level in plasma samples before and 1, 2, 4, 6, 12 hours after injection of contrast agents, Line 7-NGAL standard, Line 8-protein marker b) Lines 1-6 NGAL forms in urine samples from six healthy individuals respectively, Line 7-NGAL standard; Line 8- protein marker ζ- potential of nanobeads In our case amino coated nanobeads have isoelectric point at ph=8 (Fig. 41). That means that sum of the charge on the beads surface at current ph is equal to 0 and at that ph or at the value near to current (ph between 6.5 and 9) beads are colloidally less stable. For nanobeads and antibodies dilution as well, we used the PBS buffer with ph = 7.4. At this ph value ζ-potential in water is around 30 mv. PBS buffer with high content of different salts is lowering ζ-potential to the value of around 20 mv (data not shown). Such a ζ-potential value of around 20 mv is indicating that nanobeads are positively charged. That means that amino groups on the beads surface are positively charged and they could electrostatically bind with negatively charged carboxyl groups on the antibody molecule surface. ζ-potential at ph = 7.4 confirmed that nanobeads are still colloidally stable. Even with possible decreasing of ζ- potential due to high concentration of salt in the PBS buffer ζ-potential value at this ph indicates that nanobeads surface is positively charged. 90

114 60 potential (mv) ph Figure 41-ζ potential of nanobeads suspension applied in this work, as a function of ph Measurement of nanobeads number After calculation we obtained for V 1, V 2, and V 3 values (as was described in section ) presented in Table 18. Table 18-Calculated nanobeads volumes Calculated volume Volume (cm 3 ) V cm 3 V cm 3 V cm 3 where symbols represents: V 1 -volume of iron core V 2 -volume of silicone coat V 3 -volume of one nanobead Mass of one nanobead is equal to m 3 = g and density of one nanobead calculated by Eq. 11 was obtained to be: ρ 3 =3.29 g/cm 3. V tot -total volume of nanobeads present in 1 g applying Eq. 12 was calculated to be: V tot =0.304 cm 3. Number of nanobeads present in 1 g by Eq. 14 was calculated to be particles per gram. Since beads concentration in suspension was 6 mg/ml number of nanobeads present in this mass was calculated to be: beads in 6 mg or in 1 ml. By applying Eq. 15 the total surface of all nanobeads SA was calculated to be: m 2. SA of all nanobeads present in 1g calculated by Eq. 16 is equal to: SA=12.98 m 2 91

115 Determination of antibody concentration on nanobeads Concentration of primary Ab bound to nanobeads was determined with commercial Easy Titer Mouse IgG Assay kit (Thermo Scientific, USA). Calibration curve (Fig. 42) was constructed by plotting natural logarithm of standard antibody (mouse IgG) concentration against measured absorbance value. Concentration of IgG in unknown sample was determined by interpolating between points on the calibration curve. Since unknown sample has concentration between two standards 250 and 125 ng/ml linear interpolation equation was used to calculate IgG concentration in unknown sample A 430 (AU) Natural logarithm of mouse IgG (ng/ml) Figure 42-Calibration curve of Easy Titer Mouse IgG Assay kit After incubation with nanobeads at different incubation times, supernatant was removed and analyzed to determine unbound antibody concentration. The amount of bound antibody was determined from the difference between initial primary Ab concentration and unbound antibody concentration. Fig. 43 represents changing of bound antibody concentration with time. 10 bound 1 Ab concentration ( g/ml) Incubation time (min) Figure 43-Bound primary Ab concentration at different incubation times 92

116 For the concentration 10 µg/ml of primary Ab added to nanobeads it was calculated that after 60 minutes of incubation with nanobeads more than 90 % of antibodies were bound. At the same time, Fig. 43 shows that after five minutes of incubation less than 1 µg of primary antibodies was bound per 1 ml of nanobeads (i.e. 0.5 µg/ml). Since 1 µg/ml was found to be the optimal concentration of primary Ab for ELISA assay on microtiter plate (see section ) it was concluded that for nanobeads ELISA assay incubation time should be longer than 5 min. Degree of primary Ab binding to nanobeads surface measured with commercial kit revealed that 70 % of applied antibodies was bound after 10 minutes of incubation. Since our research goal was development of fast ELISA assay for determination of NGAL, we choose 10 minutes as shortest incubation time which provided an acceptable loss of sensitivity (30 %) as confirmed by small difference (30 %) in bound antibody concentration after longer incubation time Number of immobilized antibody molecules per nanobeads Since nanobeads are used as a solid support for ELISA assay, it is important to determine how many antibody molecules are bound to nanobeads surface. Usually number of immobilized antibody molecules is expressed as micrograms or milligrams of protein per milliliter of beads. To determine the number of immobilized antibody molecules per nanobeads we mixed: 1 µl of primary Ab (1 mg/ml) 99 µl of PBS 50 µl of nanobeads (6 mg/ml) The number of moles of IgG 1 was calculated by the formula: n= m M (22) where the symbols represent: n-number of moles m-mass of IgG (1 µg in applied volume) M-molecular weight (IgG 1 = g/mol) We calculated n= mol. Number of IgG 1 molecules N was calculated by multiplying the calculated number of moles (n) by Avogadro s number (Na): N=Na n= (23) N= molecules In section it was calculated that in six mg or in one ml of nanobeads solution there was beads. In applied beads volume of 50 µl there was beads. Therefore the number of immobilized antibody molecules per nanobead is: N= IgG 1 molecule beads N=65.1 molecule 93

117 Nanobeads based ELISA test in eppendorf tube For ELISA assay in eppendorf tube three µl of primary Ab (1 mg/ml) was mixed with 297 µl of nanobeads (0.3 mg/ml) giving the final antibody concentration of 10 µg/ml. Different incubation times were tested and five minutes incubation was found to be sufficient (by absorbance measurement at 650 nm, data not shown) for ELISA assay in eppendorf tube. We could observe by naked eye the formation of colored complex (Fig. 44b) which confirmed the reaction between the enzyme and substrate on nanobeads surface. Because of low absorbance (0.187 AU) obtained with 0.3 mg/ml concentration of nanobeads and with 2 ng/ml of NGAL antigen, we used higher nanobeads concentration (> 0.3 mg/ml) for experiments on microfluidic chip. After incubation of immobilized antibodies with enzyme, nanobeads were washed to remove unbounded enzyme. To be sure that washing is sufficient, washing was repeated five times. Supernatant which contains unbounded enzyme was collected after every washing step. To confirm that enzyme level is decreasing by increasing the number of washing steps TMB substrate was added into supernatant after every washing. Obtained results (Fig. 44a) showed that intensity of the color is decreasing by increasing the number of washing step. In the last fifth washing step (tube number six on Fig. 44a) solution is transparent indicating that there is no unbounded enzyme present in solution. Figure 44-a) Supernatant with unbounded enzyme collected after every washing step. First tube is the enzyme solution that was added in the tubes with nanobeads for incubation and removed before first washing step. Tubes 2-6 represents supernatant after 1 st -5 th washing step respectively b) final blue colored product (NGAL concentration 2 ng/ml) in eppendorf tube after addition of TMB substrate According to the color in the last two eppendorf tubes (Fig. 44a tube number 5 and 6 absorbance at 650 nm was equal to and AU respectively), we can conclude that nanobeads were washed sufficiently enough to remove unbound enzyme. Absorbance in last eppendorf tube (tube number 6) was similar to the value of negative control (sample diluent buffer without NGAL antigen) giving absorbance value of AU. Therefore we can conclude that colored product was formed in the tube with nanobeads (Fig. 44b) due to the sandwich complex with enzyme on the surface of the nanobeads. 94

118 Nanobeads based ELISA test in microfluidic chip After determination of basic experimental parameters (incubation time, reagents concentration, volume) ELISA assay was downscaled from eppendorf tube to microfludic chip. For nanobeads based ELISA assay in microfluidic chip different parameters such as magnetic field, flow rates, incubation times etc. were optimized. time Testing magnetic field, the flow rates, reagents volumes, incubation Applied magnets provides sufficiently strong magnetic field, which retains magnetic nanobeads inside the channel (Fig. 45). Figure 45-Nanobeads in the microchannel retained with permanent magnet (picture made with optical microscope with 80x magnification) Different combinations of magnets were tested for keeping the nanobeads inside the channel. Problem with using more than one magnet in line is the inhomogeneity of the magnetic field produced by magnets. Fig. 46 presents a combination of six magnets positioned above and below the microchip. We want to use combination of several magnets at the same time in order to perform same reaction with same conditions in three or four channels. This combination of magnets showed that the strongest magnetic field was between first and second magnet and between last two, while the weakest was in the center. The amount of nanobeads attracted by magnetic field was the highest at the position of strong magnetic field. Figure 46-Combination of six magnets positioned at three channels of microfluidic chip 95

119 We also tried to use just one pair of magnets per channel but if they are positioned at the same place on microfluidic channel they attract each other and stick together as was presented on Fig. 46. For performing ELISA assay at the same time in few channels magnets needs to be moved slightly from each other down the channel. Therefore we choose to use just one pair of magnets above and below microfluidic chip. Different flow rates were tested and it was confirmed that at 500 µl/min nanobeads still remain inside the channel. Above this flow rate nanobeads are washed away. To ensure that nanobeads are not removed from microchannel by the carrier flow we used flow rates lower than 500 µl/min. 200 µl/min flow rate was used for washing. For injection of reagents into microfluidic channel in each incubation step we used lower flow rate of 150 µl/min to allow longer contact time between nanobeads and reagent. Also for TLM measurement to have a stable signal flow rates lower than 500 µl/min are more appropriate. Since the channel volume is 20 µl, 50 µl of reagent volumes were injected to ensure that entire channel is filled with reagent. Different incubation times were tested and it was demonstrated that five minutes incubation time is sufficient for ELISA assay in microfluidic chip. Optimized conditions for nanobeads based ELISA assay are presented in Table 19. Table 19-Optimized conditions for nanobeads based ELISA assay Incubation step Reagent concentration Reagent volume (µl) 96 Reagent flow rate (µl/min) Washing buffer (µl) Incubation time (min) Incubation with 6.7 µg/ml primary Ab Blocking step PBS + 2 % BSA Incubation with NGAL antigen pg/ml Incubation with 0.25 µg/ml secondary Ab Incubation with 0.5 µg/ml HRP conjugate Incubation with ready to use TMB substrate solution / 2 After optimization of basic experimental parameters the ELISA assay was tested in real working conditions on a microfluidic chip. An aliquot of nanobeads which were precoated with a capture antibody (details were described in section ) were injected from an inlet hole into microfluidic channel. Then blocking buffer, NGAL standard, sample or negative control (sample diluent buffer without NGAL antigen), secondary Ab, HRPconjugate were injected successively. Between these steps unbounded species were removed by washing with washing buffer. After final washing procedure substrate solution was injected and after two minutes the resulting enzyme reaction product was sent to Y-joint chip for TLM detection (see Fig. 18). Results are presented on Fig. 47. As was expected no color was obtained in the channel with negative control since NGAL antigen is not present and sandwich complex is not formed. This also confirms that blocking and washing processes were efficient and no secondary antibodies were bound in the microchannel. By injecting NGAL standard (2 ng/ml) a blue colored compound was formed in the channel which confirmed the binding between antigen and antibodies.

120 Figure 47-Nanobeads based ELISA test performed in two channels with a) buffer as negative control (upper channel) and NGAL standard (2 ng/ml) and b) plasma sample in upper channel and NGAL standard (2 ng/ml) in the lower channel In the channel with plasma sample (Fig. 47b) a colored product was formed as well, but was hardly observable by naked eye. It was however detected by the TLM system, confirming that the assay is working also with real samples Nanobeads based ELISA test with TLM detection Calibration curve TLM signals of NGAL standards were plotted against concentrations of NGAL ( pg/ml) and calibration curve was obtained by four-parameter logistic curve fitting (Fig. 48). For some concentrations of standards (500 and 1000 pg/ml) we obtained higher standard deviation for the three replicates (every replicate was injected six times) in comparison to others (see 250 pg/ml and lower concentrations on Fig. 48). Reason for this could the difference in replicates because it is difficult to provide same reaction conditions for each replicate. First of all, pipetting same number of nanobeads (same amount of primary Ab molecules) from nanobeads solution is hardly reproducible. Secondly, nanobeads need to be positioned precisely at the same position along the microfluidic channel after each refilling of nanobeads or reagents. Furthermore nanobeads surface in contact with the reagents should be always the same as well as the contact time. Finally, it is hard to position the magnets at the same place along the channel with sufficiently high reproducibility. 97

121 Lock-in signal (mv) NGAL concentration (pg/ml) Figure 48-Calibration curve for nanobeads based ELISA assay obtained with four-parameter logistic curve fitting LOD was calculated from linear part of calibration curve (y= x, R 2 =0.9649) and it was (with blank σ= mv and b= mv ml/pg): LOD=2.3 pg/ml At the current stage of development the described method enables LODs of 2.3 pg/ml which compares favorably with LODs for commercial ELISA tests (10 pg/ml) for NGAL in standard microtiter plates (about 7 mm optical path), but reduces the analysis time from 4 h to just 35 min for one sample, and enables detection rate of 12 measurements per minute as it is presented on the Fig x x x10-3 Lock-in signal (V) 1.0x x x x x Time (s) Figure 49-µFIA-TLM signals for 8 replicate pulses of final ELISA product (from NGAL standard 500 pg/ml) obtained on nanobeads based ELISA assay 98

122 Nanobeads based ELISA assay has several advantages compared to commercial ELISA assay such as minimized reagent consumption (50 µl or less depending on the chip channel size), short time of assay, and hence the potential for application in medical/pharmaceutical research and clinical testing Real sample analysis Five serum samples were analyzed as it was described in section Results are shown in Table 20. Table 20 NGAL concentrations in five plasma samples measured by nanobeads ELISA with µfia-tlm detection and by a commercial ELISA kit. (number of replicates for each sample n=3) Nanobeads ELISA on µfia-tlm ELISA kit NGAL (ng/ml) SD (ng/ml) NGAL (ng/ml) Plasma sample SD (ng/ml) NGAL concentrations measured by both techniques were in the range between 45 and 100 pg/ml, which is within the range of normal NGAL levels as stated in section NGAL concentrations measured by these two methods were compared and analyzed statistically. Kolmogorov Smirnov test showed that data follow normal distribution (for nanobeads ELISA Z=0.190, p=0.2 and for commercial ELISA kit Z=0.176, p=0.2). Paired samples t-test has showed that there is no statistically significant difference between the NGAL values obtained with these two methods (t(4)=-0.962, p=0.390). We can conclude that developed nanobeads ELISA with µfia-tlm detection is in a good agreement with commercial ELISA assay. Developed method had high standard deviation as was already described in section There is still need for optimization of conditions and reaction parameters to overcome problem with low reproducibility and repeatability. NGAL concentration measured with nanobeads ELISA and commercial kit. Commercial ELISA kits for NGAL detection present on the market (see Table 5) with LODs of around 1 pg/ml are more expensive when compared to microfluidic ELISA assay on nanobeads, which provides comparable LODs (LOD=2.3 pg/ml). The commercial NGAL ELISA kit used as reference in this work cost around 600 euros for a single microtiter plate, which allows analysis of just 40 samples in duplicate. In comparison to this nanobeads ELISA assay in microchip cost around 2000 euros (including the price of two different antibodies, substrate, and NGAL antigen) allowing analysis of around 1300 samples. In addition, the microfluidic nanobeads ELISA assay developed in this study is consuming lower reagent volumes of about 50 µl or less, while commercial ELISA kit requires 100 µl of the reagent. Furthermore, NGAL ELISA microfluidic assay doesn t require a custom made microfluidic chip since microchips with simple design can be used. Until now, there are no evidences in the literature that NGAL was detected by ELISA assay with magnetic 99

123 nanobeads as support for NGAL primary antibodies. In the study of Vashist, (2014), the assay based on gold nanoparticles provides LOD of 0.7 pg/ml. This assay, which lasts around 6 h gives three times lower LOD but it requires about 10 times more time for the analysis as compared to the microfluidic ELISA assay developed within this work. 100

124 4.2 Development of TLM method for contrast agents detection Calibration curve made on spectrophotometer Absorption spectra for solutions of different iodine concentration in chloroform are presented on Fig. 50. The spectra confirmed that iodine complex in chloroform gives absorbance maximum at 510 nm. 1.0 Absorbance (AU) M M M M M M Wavelength (nm) Figure 50-Spectra recorded with different iodine concentration in chloroform Calibration curve prepared with different iodine solutions ( M) in chloroform is presented on Fig Absorbance (AU) x x x x x10-3 Iodine concentration (mol/l) Figure 51-Calibration curve of different iodine concentration in chloroform (R 2 = ) measured by spectrophotometer 101

125 LOD was calculated by Eq. 20 and it was expressed in mol/l of iodine: LOD= mol/l I 2 which corresponds to: LOD=527 ng/ml I 2. Assuming that all iodine atoms are released from the contrast agent molecule, this LOD corresponds to an LOD for iohexol (containing three iodine atoms, M = g/mol): LOD=1.1 µg/ml for iomeprol (containing three iodine atoms, M = g/mol) the corresponding LOD is equal to: LOD=1.1 µg/ml and for diatrizoate (containing three iodine atoms, M = g/mol) LOD is equal to: LOD=0.8 µg/ml Contrast agent degradation Degradation of contrast agents applied in this work is based on Cu 2+ / H 2 O 2 system which was previously used in study of Mantzavinos, (2003) for degradation of p- hydroxybenzoic, gallic and vanillic acids (which are three benzoic acid derivatives as well as investigated contrast media). This degradation system was used in the work of Fono and Sedlak, (2007). Possible reactions occurring in degradation of contrast media with Cu 2+ / H 2 O 2 system are: 2Cu 2+ +4I 2CuI+I 2 (24) 2H + +H 2 O 2 +2I I 2 +H 2 O (25) In previous studies of Steger-Hartmann et al., (2002), Jeong et al., (2010), Hapeshi et al., (2013) it was shown that very similar degradation products (of contrast agents iohexol, iomeprol, iopromide and iopamidol) were formed by deiodination as most prominent route in the photodegradation and microbial degradation of contrast agents. After degradation of contrast agents, iodine can exist in form of many different iodine species in aqueous solution, since iodine can form compounds in all oxidation states from -1 to +7. Among them iodine, iodide and iodate are most stable forms (Del Moro et al., 2015). Because of sulfuric acid addition directly into the iodide solution, I 3 is formed by the oxidation with oxygen and the solution turns yellow (Bichsel and Gunten, 1999). According to Bichsel and Gunten, (1999) and Del Moro et al., (2015) elemental iodine is poorly extracted from aqueous solutions of potassium iodide, because triiodide ions form in solutions according to the equation I 2 +I I 3 (26) This reaction is important, since we are aiming to detect contrast agents indirectly by measurement of iodine, and this side reaction is decreasing iodine concentration in solution. 102

126 The product of side reaction I 3 - does not form pink charge transfer complex in chloroform. Fig. 52 shows extraction in separatory funnel with chloroform. Figure 52-Iodine extraction with chloroform after contrast agent degradation Pink colored organic phase confirms the formation of charge transfer complex of iodine in chloroform Choice of buffer, optimization of buffer ph, buffer concentration Carbonate buffer was chosen for our work since there are evidences (Medinas et al., 2007) that carbonate and hydrogen carbonate ions are helping in degradation of different organic compounds by forming radical species. Hydrogen peroxide was also used in reaction mixture and it is known that ph has a strong effect on hydrogen peroxide chemistry and effectiveness. ph influences the solubility of the catalyst and its reactivity against hydrogen peroxide, as well as the radicals formed, which is reflected in the degradation of target contaminants (Petri et al., 2011). Copper was also used to catalyze formation of free radicals from hydrogen peroxide. Fono and Sedlak, (2007) suggested that carbonate buffer concentration should be at least M so therefore we decided to use 0.1 M carbonate buffers of different ph. The impact of ph on iohexol degradation is shown on Fig. 53. Results showed that at ph=8 concentration of iodine released from contrast agents was the highest. According to this result for contrast agent degradation it was chosen buffer with ph=8. 103

127 Absorbance (AU) ph 4 ph 5 ph 6 ph 7 ph 8 ph Wavelength (nm) Figure 53-Spectra of iodine released from iohexol (initial concentration of 7.55 mg/ml iohexol which corresponds to 3.50 mg/ml of iodine) in reaction with buffers of different ph (in chloroform) Optimization of incubation time in the oven Changing of iodine concentration released from iohexol (3.5 mg/ml) is presented on Fig Iodine concentration (mg/ml) Time (min) Figure 54-Iodine concentration as function of incubation time in the oven As was expected the amount of released iodine is constantly increasing with time of exposure to reagents in the oven. Release of iodine reached the highest amount after 120 min 104

128 and 180 minutes of incubation. According to this we decided to incubate contrast agents for 90 minutes and more. By dividing iodine concentration at 180 min with initial iodine concentration it was calculated that after 180 minutes just 24 % of initial iodine concentration in contrast agent was released during degradation. This value includes yields of degradation, oxidation and extraction which simultaneously contribute to the observed degradation efficiency. Possible reason for the observed low degradation efficiency could be oxidation of iodide which was done with 0.01 M H 2 SO 4 and 0.2 % NaNO 2 of low concentration Extraction efficiency To determine the extraction efficiency calibration curves were prepared from KI solutions, following previously described reaction and extraction procedure (red curve) and by directly dissolving I 2 in chloroform (Fig. 55). Iodide concentration (mol/l) x x x x x A 510 (AU) I 2 in chloroform KI in chloroform x x x x x10-3 Iodine concentration (mol/l) Figure 55-Spectrophotometric calibration curves for iodine (standard solutions in chloroform) and for KI (KI standard solutions were first oxidized and then iodine was extracted with chloroform) (R 2 = , R 2 = ) Upper calibration curve made with I 2 in chloroform marked with black corresponds to I 2 concentrations at 100 % extraction efficiency. Calibration curve made with KI in chloroform represents the combination of oxidation efficiency and extraction efficiency. The product of oxidation and extraction efficiency is needed to correctly asses the degradation efficiency of contrast agents in terms of released iodine. 105

129 For determination of the oxidation-extraction efficiency, six samples of KI ( M) were oxidised and extracted in parallel. The oxidation-extraction efficiency was calculated by the formula: Calculated iodine concentration Extraction + oxidation efficiency%= expected iodine concentration 100 (27) and the results are shown in Table 21. Table 21-Extraction+oxidation efficiency calculated with M KI KI (mol/l) Mean A 510 (AU) Iodine conc. (mol/l) ± SD (mol/l) Extraction+oxidation efficiency (%) ± Results showed that the extraction efficiency is around 60 %. Since low concentrations of sulfuric acid 0.01 M and 0.2 % NaNO 2 were used for oxidation it is possible that oxidation reaction didn t transform all iodide into iodine. Obtained extraction and oxidation efficiency could be due to triiodide formation in solutions according to the Eq. 27. Therefore for further contrast agents oxidation higher concentration of sulfuric acid (3 M) and 10 % of NaNO 2 were used. These concentrations of sulfuric acid and NaNO 2 were chosen according to literature data previously described by Adotey et al., (2011) and Andrási et al., (2007) where they used 2.5 M H 2 SO 4 and 10 % of NaNO 2 for oxidation of iodide to iodine Stability of chloroform iodine solution released by contrast agents degradation Absorbances of iodine in chloroform released from iohexol and diatrizoate during degradation reaction were recorded on spectrophotometer and changes of absorbance with time are presented on Fig. 56. Absorbance of iodine in chloroform released from iohexol is constantly decreasing in first 2.5 h and after 4 h reaches some stable value with absorbance of around 70 % of initial concentration of iodine. This value was obtained by dividing initial iodine concentration with concentration after 4 h. 106

130 Figure 56-a) Stability of iodine in chloroform released by degradation of iohexol measured with spectrophotometer ( M I 2 initial iodine concentration) b) Stability of iodine in chloroform released by degradation of diatrizoate measured with spectrophotometer ( M I 2 initial iodine concentration) Instability of iodine solutions in chloroform could be due to the presence of extractable reactive species formed during the degradation process. The effect on iodine in chloroform released by diatrizoate degradation is presented on Fig. 56b. After 1 h the absorbance of iodine in chloroform decreased around 80 % Contrast agents measurement on µfia-tlm According to the absorbance maximum at 510 nm (see Fig. 50), the 514 nm emission line from an argon laser was chosen for µfia-tlm measurements. Different injection volumes of iodine complex in chloroform were tested to determine optimal injection volume for µfia-tlm measurement. Different injection volumes of iodine solution in chloroform were tested: 0.2, 0.4, 0.8 µl. Results are presented on Fig

131 L 1.2 Lock-in signal (mv) L 0.4 L Time (s) Figure 57-Influence of different injection volume of the sample on µfia-tlm signal for the iodine solution ( M) in chloroform (sample flow rate 10 µl/min, chloroform flow rate 10 µl/min) As it was expected the peak height increases with injection volume. Comparison of the signal for 0.4 µl and 0.8 µl samples showed that signal from 0.8 µl sample is 37 % higher, while the signal for 0.4 µl sample is 31 % higher than the signal for 0.2 µl sample. Injection volume higher than 0.8 µl caused signal distortion and according to this 0.8 µl was used as sample injection volume for determination of iodine on µfia-tlm. With all tested injection volumes we obtained relatively good repeatability. A relative standard deviation of around 5 % for 0.8 µl injection volume confirmed good repeatability of measurement Calibration curve obtained on µfia-tlm Calibration curve on µfia-tlm was prepared by µfia-tlm measurements of different solutions of iodine in chloroform by applying linear fitting. One of the obtained linear calibration curves is presented on Fig

132 Lock-in signal (mv) x x x x x10-3 Iodine concentration (mol/l) Figure 58-Linear calibration curve obtained on µfia-tlm with iodine solutions in chloroform Limit of detection for iodine (with σ(blank)= mv and b=82.83 mv L/mol) calculated by applying Eq. 20 was LOD= mol/l I 2 which is equal to LOD=9.06 ng/ml I 2 By assuming release of all three atoms of iodine from contrast agent molecule, the limit of detection for iohexol was calculated to be LOD=20 ng/ml Calculated LOD for iomeprol was LOD=18 ng/ml and for diatrizoate LOD=15 ng/ml. If we compare LOD for TLM with LOD for spectrophotometry we obtained around 59 times lower LOD with TLM. By combining microfluidic technologies with TLM we achieved improvement of LOD with low reagent and sample consumption in comparison to spectrophotometry Comparison of measurement of contrast agents degradation product on spectrophotometer and on µfia-tlm Iohexol and diatrizoate were exposed for 60, 90 and 120 minutes respectively in the oven. NaI solution was used to determine the oxidation efficiency. Iodine concentration was determined from calibration curve for µfia-tlm (Fig. 58). As was expected reaction efficiency is increasing with longer incubation time, and/or more iodine atoms are released from contrast agents molecule. Reaction efficiency was calculated by dividing the measured 109

133 concentration of released iodine by the expected total iodine concentration from the amount of the contrast agent before degradation (see Eq. 17). Reaction efficiency refers to cumulative efficiency of oxidation efficiency and extraction efficiency (see section ) since both could not be determined simultaneously. Results for released iodine were also corrected for the oxidation and extraction efficiency. Oxidation of NaI solution ( M) as was described in section yielded iodine with concentration of M, which corresponds to oxidation efficiency of Table 22-Calculated iodine concentrations released from contrast agents after different incubation times measured on spectrophotometer Sample 110 Iodine conc. (mol/l) Released iodine (%) iohexol 60 min ( M I 2 ) iohexol 90 min iohexol 120 min diatrizoate 60 min ( M I 2 ) diatrizoate 90 min diatrizoate 120 min Obtained results showed that complete reaction efficiency (including degradation, oxidation and extraction efficiency) is increasing with longer incubation time. Degradation efficiency was low indicating that reaction conditions should be changed. The release of iodine which can be related to degradation efficiency was low Measurement of released iodine on µfia-tlm The same solutions that were measured on spectrophotometer were also measured on µfia-tlm. Results are presented in Table 23. Table 23-Calculated iodine concentrations released from contrast agents after different incubation times measured on µfia-tlm Iodine conc. Released Sample (mol/l) ± SD(mol/L) iodine (%) iohexol 60 min ± iohexol 90 min ± iohexol 120 min ± diatrizoate 60 min ± diatrizoate 90 min ± diatrizoate 120 min ± Reaction efficiency obtained by spectrophotometer and µfia-tlm were compared and analyzed statistically. Iodine concentrations were tested for normal distribution with Kolmogorov Smirnov test. Data show normal distribution (for spectrophotometer Z=0.236, p=0.2 and for TLM Z=0.358, p=0.116). Paired samples t-test has showed that there is no

134 statistically significant difference between the values obtained with these two methods (t(5)= , p=0.155). Fig. 59 shows µfia-tlm signals for chloroform complex of iodine released from contrast agent iohexol after 60, 90, 120 min incubation in the oven. Every sample was injected five times. Longer incubation time gives higher concentration of iodine as it was expected. Developed µfia-tlm method showed good repeatability with sample throughput of around 8 samples per minute. 2.0x x x x10-2 Lock-in signal (V) 1.2x x x x x x Time (s) Figure 59-µFIA-TLM signals in chloroform for iodine released from iohexol after 60, 90, 120 min incubation in the oven To correlate the determined iodine concentration to the concentration of contrast agents and therefore to the degradation efficiency we were interested if all iodine atoms are released from benzene ring of contrast agent. To obtain this information HPLC analysis of contrast agents before and after degradation reaction was performed. Beside HPLC degradation products were analyzed also with LC/MS HPLC analysis of contrast agents and their degradation products After addition of CuCl 2 and H 2 O 2 contrast agents solution of diatrizoate, iohexol and iomeprol were heated in the oven and analyzed on HPLC before and after degradation to check degradation efficiency. Unfortunately HPLC is not giving information about structure of degradation products. Since contrast agents are measured indirectly by measuring released iodine it is important to get information if all iodine atoms are released from benzene ring during degradation reaction. LC/MS analysis was also performed to get information about the structure of degradation products and eventual mechanism of degradation. Chromatograms obtained for diatrizoate before and after degradation are shown on Fig. 60. Diatrizoate standard solution gives a peak at the retention time of seven minutes as presented on Fig. 60a. The chromatogram of the diatrizoate solution after degradation is presented on Fig. 60b. 111

135 Figure 60-HPLC analysis of a) diatrizoate solution (0.705 mg/ml) and b) diatrizoate solution after 180 minutes of degradation (C 18 Purospher, 250x4.6 mm, 5 µm column; AcN: H 2 O, (5:95 %) ph= 3; injection volume 20 µl, flow rate 1 ml/min) Peak with retention time of 7 minute that belongs to diatrizoate doesn t appear in the chromatogram, which confirms that under given degradation conditions diatrizoate was degraded and that there is no peak that belongs to parent molecule. Same experiment was repeated with iohexol and HPLC chromatograms of iohexol standard and iohexol solution after degradation are presented on Fig. 61. If we compare chromatograms of iohexol solution before and after degradation we can see that after degradation of iohexol there is no peak that belongs to parent molecule. Analysis of iohexol solutions revealed two peaks at retention time of 7 and 8 minutes, which is in agreement with literature data (Baere et al., 2012). In fact, commercially available iohexol consists of two geometric isomers, i.e. endo and exo forms which explain the appearance of two peaks in the chromatogram of iohexol standard. 112

136 Figure 61-HPLC analysis of a) iohexol contrast agent solution (1.82 mg/ml) and b) iohexol solution after degradation (C 18 Purospher, 250x4.6 mm, 5 µm column; AcN: H 2 O, (5:95 %) ph= 3; injection volume 20 µl, flow rate 1 ml/min) HPLC chromatogram of iohexol solution after degradation showed no peaks belonging to iohexol isomers. A peak with retention time similar to diatrizoate degradation products was observed instead, thus indicating that degradation products of iohexol and diatrizoate could be similar or the same. Fig. 62a shows a HPLC chromatogram of iomeprol standard solution. Iomeprol has a retention time of around 6 minutes. On Fig. 62b a chromatogram of iomeprol degradation products is presented. If we compare these two chromatograms we can see that after degradation of contrast agent iomeprol there is no peak that belongs to parent molecule. This chromatogram is a confirmation that degradation reaction efficiently degraded the parent molecule. If we compare HLPC chromatogram of three contrast agents degradation product we could notice that all of them gives products with retention time between two and three minutes. 113

137 Figure 62-a) HPLC chromatogram of iomeprol mg/ml b) HPLC chromatogram of iomeprol solution after degradation (C 18 Purospher, 250x4.6 mm, 5 µm column; AcN: H 2 O, (5:95 %) ph= 3; injection volume 20 µl, flow rate 1 ml/min) HPLC analysis of contrast agent iomeprol Calibration curve Calibration curve was constructed by plotting peak heights for iomeprol standards against their concentration and the obtained calibration curve is presented on Fig Height Iomeron concentration ( g/ml) Figure 63-Calibration curve made on HPLC with iomeprol standards 114

138 Limit of detection for iomeprol by HPLC was calculated by applying Eq. 20. For calculation of the standard deviation of baseline signal σ= AU and b= AU ml/µg were used to give LOD=294 ng/ml If we compare the achieved LOD for HPLC with LOD for µfia-tlm for iomeprol, TLM gives 16 times lower LOD Contrast agent detection with ion chromatography Ion chromatography could be used as a method for indirect measurement of contrast agents by measurement of iodide released in degradation reaction. Therefore we used ion chromatography as a reference method for comparison with µfia-tlm. Calibration curve constructed with iodide standards in the range µg/ml is shown on Fig Area Iodide concentration (µg/ml) Figure 64-Calibration curve made on ion chromatograph with iodide standards LOD for iodide achieved with ion chromatography was calculated: LOD=71 ng/ml which corresponds to LOD for iomeprol of LOD=133 ng/ml If we compare LOD obtained with ion chromatography with LOD on µfia-tlm of 18 ng/ml, TLM enables around 7 times lower LOD HPLC-MS/MS analysis of contrast agents degradation products Iohexol was chosen as a model contrast agent for the structural elucidation of main degradation products. Direct injection of iohexol standard solution on MS gave the MS spectrum with m/z value of representing a precursor ion of iohexol (M + H + ) (Fig. 65). 115

139 2.0x x10 10 Intensity (cps) 1.0x x m/z (Da) Figure 65-MS spectrum of iohexol standard solution ( M) After MS analysis, iohexol standard solution was analyzed also with HPLC/MS, which gave the total ion chromatogram as shown on Fig. 66 and mass spectra of compounds in two chromatographic peaks as shown on Fig x10 12 Intensity (cps) 3x x x Time (min) Figure 66-HPLC/MS chromatogram of analyzed iohexol standard solution ( M) 116

140 Figure 67-MS spectra of precursor ions of iohexol HPLC/MS analysis of iohexol standard solution confirmed the presence of two iohexol isomers endo and exo (8.74 and 9.59 retention time) as was already shown before with HPLC analysis (see Fig. 61). Fig. 67 shows MS spectra of endo and exo iohexol isomers both with precursor ions at m/z value of After degradation iohexol solution was analyzed by HPLC/MS and the corresponding total ion chromatograph is shown on Fig. 68. Figure 68-HPLC/MS chromatogram of iohexol after degradation with marked four fractions. HPLC/MS analysis of iohexol showed that after degradation peaks that belong to exo and endo iohexol were not present, confirming that the degradation process was efficient in removal of parent compound. Four fractions of degradation products were collected during HPLC analysis and the fractions were further analyzed with MS to get information about the formed degradation products (Fig. 68). Obtained chromatogram differs from the one shown 117

141 on Fig. 61b because analysis was performed on a different HPLC instrument (also different HPLC column) coupled with MS. Since the MS detector has lower sensitivity at higher flow rates due to lower ionization yield in the ion source (ESI), we lowered the flow rate from 1 ml/min (previously used for HPLC as was described in section 3.3.7) to 0.7 ml/min. Therefore the obtained retention times of analyzed HPLC/MS chromatogram differ from the one presented in Fig. 61b. The marked fraction IV in Fig. 68 was chosen as representative example for iohexol degradation and structural elucidation of obtained degradation products and further analyzed by MS. The obtained MS spectrum shown in Fig. 69 gave mass spectrum of several precursor ions representing degradation products of both iohexol isomers. These precursor ions were further analyzed by MS/MS. 3.0x x10 7 Intensity (cps) 2.0x x x x m/z (Da) Figure 69-MS spectrum of fraction IV collected during HPLC analysis The fragmentation obtained with MS/MS analysis of precursor ions which are presented in Table 24 were used to confirm the structure of iohexol degradation products presented on Fig. 70. Table 24-Fragmentation ions obtained after MS/MS analysis of precursor ions Precursor ions with measured m/z value Fragmentation ions with m/z values after MS/MS analysis A (5 %), (15 %), (100 %), (5 %) B (10 %), (12 %), (100 %), (85 %), (20 %) C (7 %), (5 %), (5 %), (5 %) D (3 %), (15 %), (4 %), (100 %), (3 %) The aim of this additional MS analysis was to confirm if the degradation reaction process results in cleavage of all three iodine atoms from the parent molecules which would enable 118

142 stoichiometric calculation of contrast agent concentration after determination of iodine by TLM. By integrated analysis of LC and MS data we propose four potential degradation products of iohexol presented in Fig. 70. Proposed structures for iohexol degradation products that correspond to precursor ions in fraction IV are presented on Fig. 70. Degradation pathway shown on Fig. 70 clearly suggests that the iodine was cleaved under the used degradation conditions. Proposed iohexol degradation pathway (Fig. 70) showed that according to masses of four precursor ions two of them are formed by loss of two iodine atoms (products B and C). These precursor ions were further fragmented using MS/MS analysis to confirm their identity. Resolved fragmentation for these ions is shown on Figs. 71, 72, 73 and

143 120 Figure 70-Proposed degradation pathway of iohexol with calculated m/z values 120

144 121 Figure 71-Proposed fragmentation pathway for precursor ion with m/z value of

145 122 Figure 72-Proposed fragmentation pathway for precursor ion with m/z value of

146 123 Figure 73-Proposed fragmentation pathway for precursor ion with m/z value of

147 124 Figure 74-Proposed fragmentation pathway for precursor ion with m/z value of

148 HPLC-MS/MS analysis showed that different degradation products were formed by cleavage of one or two iodine atoms. It was confirmed that mechanism applied for the degradation of contrast agents releases iodine atoms from contrast agent molecule. Cleavage of other substituents from benzene ring is also possible, which can yield tri-iodide degradation products (product A in fraction IV). Formation of different iodine substituted degradation products makes it impossible to exactly calculate the concentration of iohexol on the basis of determined iodine concentration. For this reason a detailed and time consuming analysis of degradation products in other HPLC fractions was not performed. Reaction used for degradation of iohexol could be potentially applied for contrast media removal in a waste water treatment plant. It is however necessary to identify all potential degradation by-products and obtain data about their potential biological activity. According to Jeong et al., (2010) the loss of iodine from the parent molecule should result in more biodegradable products during degradation treatment. It is also unknown whether the contrast media are biologically degraded or degraded by induced photolysis with sunlight (direct or indirect). The research goal of this study was to investigate which compounds are formed during chemical degradation of contrast agents in order to estimate how many iodine atoms are released from the parent molecule, and not to develop technology for removal of contrast agents from water. Therefore, experiments and related discussion about stability of these compounds in environment were not performed since this is opening new frontiers which were not the topic of interest of this work. Methods for detection of contrast agents which were previously described in the literature (see Table 3), such as LC/MS can provide ultimate LODs as low as 0.11 ng/l (Sacher et al., 2005). This is more than 1000 times lower in comparison to the LOD of 18 ng/ml achieved with µfia-tlm method. However, expensive and laborious apparatus, requiring a time consuming step of sample pretreatment, with large consumption of toxic organic solvents, is needed in LC/MS. In the work of Hirsch et al., (2000) sample pre-treatment step was performed before LC/MS analysis with times pre-concentration factor. Chromatographic separation required elution time of around 30 minutes for one sample. On the other hand, chemical reaction for contrast agent degradation lasts around 120 minutes and could be potentially reduced in future by optimizing reaction conditions. The final detection by µfia-tlm method developed within this work gives sample throughput of around 8 samples per minute. Considering all the facts above, µfia-tlm competes favorably in overall duration of analysis, while providing comparable LODs (considering possible pre-concentration) with much low consumption of toxic organic solvents. 125

149 4.3 Development of TLM method for detection of anti HPV-16 antibodies SDS-PAGE analysis of PsVs samples SDS-PAGE gel of analyzed HPV-16 PsVs sample is shown on Fig. 75. The gel showed protein band of approximately 56 kda in elution fractions confirming the presence of HPV major capsid proteins L1. Another band at around 75 kda belongs to L2 proteins which are minor components of capsid proteins. As it is obvious from the gel intensity of the bands confirmed that L1 protein is present in higher concentration than L2 protein according to literature data (Millán et al., 2010) 12:1 ratio. Figure 75-SDS-PAGE analysis; Line 1-10 mg/ml BSA, Line 2-4 mg/ml BSA, Line 3-2 mg/ml BSA, Line 4-1 mg/ml BSA, Line mg/ml BSA, Line 6-PsVs sample, Line 7-protein marker CBB dying didn t show the presence of other proteins in the mixture but since this gel dying procedure is not sensitive, purity of used PsVs samples was also tested with silver staining of the gel Silver staining of the gel after SDS-PAGE electrophoresis Silver staining of the gel after SDS-PAGE is shown on Fig. 76. Beside two most intensive bands at around 56 kda and 75 kda that belong to L1 and L2 protein respectively, there are also bands from other proteins with molecular masses lower than 56 kda. The content of these proteins is much lower in comparison to L1 and L2 proteins amount and most likely represent degradational products of L1 protein. Almost no band of higher molecular mass confirms relative purity of the PsVs preparations applied. Therefore by incubation of PsVs samples with nanobeads we expect that predominantly L1 and L2 will bind to nanobeads surface. We expect that the percent of other contaminating proteins bounded to the nanobeads surface should be irrelevant. 126

150 Figure 76-Silver staining of SDS-PAGE gel, Line 1-BSA 8 mg/ml, Line 2-BSA 4 mg/ml, Line 3- BSA 2 mg/ml, Line 4-BSA 1 mg/ml, Line 5-PsVs containing sample, Line 6-protein markers Western blot of PsVs samples Western blot of analyzed PsVs samples was performed to quantify and confirm the presence of both proteins L1 and L2. The results of Western blot for different sample dilutions is presented on Fig. 77. Membrane incubated with anti L1 HPV 16 antibody is shown on Fig. 77a and on Fig. 77b is shown membrane incubated with HPV 16 L2 mouse monoclonal antibody. In all four samples on Fig. 77a the intensive band around 60 kda belongs to L1 protein with known molecular mass of 56 kda. In the samples with higher concentration of PsVs intensity was higher. Bands with masses lower than 56 kda are probably L1 protein degradation products. On Fig. 77b results showed the presence of band at 75 kda that belongs to L2 protein in PsVs. Band intensity is decreasing by lowering PsVs concentration. Figure 77-a) Western blot results of PsVs samples incubated with anti L1 HPV 16 antibody, Line 1-PsVs 100 times diluted, Line 2-PsVs 150 times diluted, Line times diluted, Line times diluted, Line 5-protein marker b) Western blot of PsVs sample incubated with HPV 16 L2 mouse monoclonal antibody Line 1-PsVs 100 times diluted, Line 2-PsVs 150 times diluted, Line times diluted, Line 4-PsVs 1000 times diluted, Line 5-protein marker 127

151 4.3.3 L1 and L2 protein concentration determination According to densitometric measurement of bands on Fig. 75 by comparison of band intensity of BSA protein of known concentration ( mg/ml) L1 and L2 protein concentration was determined. Calibration curve was constructed with area of BSA bands of known concentration was used to calculate L1 and L2 concentration. L1 and L2 protein concentration was calculated to be around 9.5 mg/ml and 0.8 mg/ml PsVs based ELISA assay on microtiter plate Choice of buffer, temperature, incubation time PBS buffer showed to be better coating buffer for PsVs coating in comparison to carbonate buffer. Carbonate buffer gives lower background but also lower signal for serum samples. Reason for this could be denaturing effect of carbonate buffer on PsVs conformational epitope as was described previously by Karem et al., (2002). As a blocking buffer we used PBS buffer with 4 % skimmed milk and 0.2 % Tween. Patient serum samples were diluted in sample diluent buffer (PBS buffer with 2 % skimmed milk and 0.1 % Tween) Optimization of the reagents concentrations For PsVs coating step incubation at 37 C for one hour was found to be the most efficient. Other incubation steps were performed at room temperature for one hour. For coating of microtiter plate with PsVs two concentrations in the range of ng/well were tested. Since the determined L1 concentration is around 9.5 mg/ml, 1000 and 1500 times (equal to 475 and 317 ng/well) dilution in PBS correspond to the range of HPV particles previously described in the literature (Zhao et al., 2014). Calibration curve obtained with these two PsVs concentrations are shown on Fig

152 x VLP dilution 1500 x VLP dilution 1.8 A (AU) anti L1 HPV-16 antibody concentration (ng/ml) Figure 78-ELISA calibration curve made with two different PsVs concentrations constructed with four-parameter logistic curve fitting Obtained calibration curve and absorbance values showed that these two concentrations are effective for coating. Since absorbance signals at these two PsVs concentrations are similar, we decided to use lower PsVs concentration which still allows optimal level of sensitivity for different anti L1 HPV-16 antibody. We also tested higher dilutions of PsVs with concentration of 200 ng and lower but they all give lower signal. Anti L1 HPV-16 antibody was diluted at least 1000 times giving optimal absorbance of around 1.8 AU or lower. Higher antibody concentration (dilution lower than 1000 times) gives absorbance signal higher than 2 AU and the values above this range are inaccurate as was already described by Crowther, (2001). Goat anti-mouse IgG (Fab specific) peroxidase antibody was diluted 2000 times as was suggested by supplier. Optimized conditions for PsVs based ELISA assay on microtiter plate are shown in Table 25. Table 25-Optimized conditions for PsVs based ELISA assay on microtiter plate Reagent Optimal concentration Applied volume (µl) Incubation time Washing step PsVs 300 ng/well 50 Overnight at 4 C 3 x 300 µl Blocking buffer PBS + 4 % skimmed 3 x 300 µl h at 37 C milk+ 0.2 % Tween-20 Primary Ab ng/ml 50 1 h at room 3 x 300 µl temperature HRP-labeled 1 h at room 3 x 300 µl 500 ng/ml 50 secondary Ab temperature TMB substrate ready to use solution min / Stop solution 0.5 M H 2 SO 4 50 / / 129

153 Microtiter plate based ELISA assay for antibodies against PsVs developed in this work requires 10 h for completion. This compares favorably with commercial kits used for same purpose available on the market (see Table 6) ELISA calibration curve Differently than in NGAL ELISA assay, where we measured the level of NGAL antigen, the level of anti L1 HPV-16 antibody is measured in PsVs based ELISA assay. Calibration curve was prepared by plotting absorbance against concentration of anti L1 HPV-16 antibody in the range from ng/ml. Every standard was prepared three times and results were expressed as mean value ± standard deviation. Concentration of anti L1 HPV-16 antibody in patient serum was determined from calibration curve. Calibration curve was achieved by applying fourparameter logistic curve fitting with Origin program (Fig. 79) A (AU) anti L1 HPV-16 antibody (ng/ml) Figure 79-PsVs ELISA calibration curve From the obtained calibration curve LOD was calculated by Eq. 18 (average(blank)= AU and σ(blank)= AU) LOD= AU By applying Eq. 19 LOD expressed in ng/ml was obtained: LOD=12.6 ng/ml Linear fitting was also performed with first four points ( ng/ml) from the linear part of the calibration curve (y= x , R 2 = ). LOD calculated from linear calibration curve (σ(blank)= AU, b= AU ml/ng) by applying Eq. 20 was: LOD=3.8 ng/ml 130

154 4.3.5 Measurement of final ELISA product on µfia-tlm After measurement on microtiter plate reader final yellow solution was measure on TLM. Calibration curve constructed by plotting antibody concentration against TLM signal is showed on Fig x x10-3 Lock-in signal (V) 1.5x x x anti L1 HPV 16 antibody (ng/ml) Figure 80-Calibration curve obtained on µfia-tlm with four-parameter logistic curve fitting LOD was calculated with Eq. 18 (average(blank)= V and σ(blank)= V): LOD= V. By applying Eq. 19 LOD expressed in ng/ml was obtained: LOD=1.3 ng/ml Linear fitting was also performed with the points in the linear part of calibration curve (y= x). LOD for µfia-tlm measurement was calculated by Eq. 20 (with σ(blank)= V and b= V ml/ng): LOD=0.9 ng/ml If we compare LOD obtained by ELISA and µfia-tlm we can conclude that LODs achieved by µfia-tlm are at least four times lower with respect to LODs for ELISA Comparison of the results obtained with ELISA and µfia TLM Anti L1 HPV 16 antibody concentrations in 11 serum samples measured with ELISA, µfia-tlm and nanobeads PsVs ELISA with TLM detection are shown in Table

155 Table 26-Concentration of anti L1 HPV 16 antibody measured by ELISA, µfia-tlm and nanobeads PsVs ELISA with TLM detection. (number of replicates for each sample n=3) ELISA µfia-tlm Nanobeads PsVs ELISA Serum Anti L1 HPV 16 SD Anti L1 HPV 16 SD Anti L1 HPV 16 SD sample (ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml) (ng/ml) below LOD / / / / / / / / / ELISA assay on microtiter plate showed the presence of anti L1 HPV 16 antibodies in all the samples except one. In serum sample number 4 anti L1 HPV 16 antibody concentration measured by ELISA was below the LOD. Antibody concentration in serum samples number 2, 6, 7, 9, 10, 11 were higher than in the other serum samples. Since the LOD for µfia-tlm is lower in comparison to ELISA, µfia-tlm showed the presence of anti L1 HPV 16 antibody in all the samples including sample number 4. Statistically different concentrations of HPV-16 antibody in examined patients could be explained by different time elapsed after infection or different stage of infection in different patients. Statistical analysis was performed to see if there is significant difference between the anti L1 HPV 16 antibody concentration measured by ELISA and µfia-tlm method. Anti L1 HPV 16 concentrations measured with home-made ELISA and µfia-tlm were tested for normal distribution with Kolmogorov Smirnov test. Test showed that data are following normal distribution (Z=0.174, p=0.2 for ELISA assay and Z=0.137, p=0.2 for µfia-tlm) and the paired samples t-test (t(10)=-0.371, p=0.718) confirmed that there is no statistically significant difference in anti L1 HPV 16 concentration measured with ELISA and µfia-tlm. We can conclude that the two applied methods for measurement of anti L1 HPV 16 antibodies are in good agreement Western blot analysis of serum samples Besides measurement of anti L1 HPV 16 antibodies concentrations in patient s serum samples additional methods were used to confirm the presence of antibodies. Serum samples collected from women with positive HCII test were loaded on SDS-PAGE gel and analyzed with Western blot to test the presence of anti HPV-16 L1 antibody. Obtained Western blot results for 11 serum samples are shown on Fig. 81. Within 11 analyzed serum samples, six (serum number 2, 6, 7, 9, 10, 11) showed the presence of anti-l1 HPV-16 antibodies. These results are in good 132

156 correlation with ELISA assay which also showed higher level of anti L1 HPV 16 antibodies in these six serum samples. Other serum samples didn t show the presence of anti HPV-16 antibodies. Figure 81-Line 1-11 serum sample numbers respectively from 1 to 11, Line c-is the positive control with anti L1 HPV 16 antibody, Line m-protein markers Since the level of these antibodies is changing with time, higher level is at the initial contact with the patient and depends on immune response of the patients so it is possible that they were infected long time ago and the serum samples were taken after certain time of infection. Since concentration of antibodies is such low, it is hard to determine and to be sure if infection is present or not. Therefore there is a need for sophisticated method that could determine antibodies in lower concentration Dot-blot analysis of serum samples Obtained dot-blot analysis results are shown on Fig. 82. Obtained results confirmed previous results obtained with ELISA and Western blot indicating that these individuals have anti L1 HPV 16 antibodies in the serum. The only difference is that serum number 1 on dot blot shows some small signal. This could be due to experimental error or some possible unspecific binding or insufficient washing. Positive control gives black point in the center of membrane confirming complex formation between L1 protein and anti L1 HPV-16 antibody. Figure 82-Dot blot analysis of 11 serum samples respectively. Numbers represents serum numbers while c is positive control 133

157 4.3.9 PsVs nanobeads based ELISA in eppendorf tube Results of nanobeads based ELISA assay in eppendorf tube are presented on Fig. 83. First three tubes represent supernatant aliquots after three washing steps respectively after substrate addition. Because of the presence of free goat anti-mouse IgG HRP labeled antibody in the supernatant, it becomes blue after addition of substrate (with absorbance values of and AU in first two tubes respectively). Washing was stopped when supernatant solution remained transparent after addition of substrate (with absorbance value of AU). Intensity of the color is decreasing by increasing the number of washing steps since amount of free HRPantibody is also decreasing. Blue color in the test tube number four with nanobeads is the improvement of binding of PsVs to nanobeads surface on one side and to the primary and secondary HRP labeled antibody to the other side. Figure 83-Nanobeads based ELISA assay in eppendorf tube. Tube 1, 2, 3 represents aliquots of supernatant after 1 st, 2 nd, 3 rd washing step where substrate solution was added, Tube 4- nanobeads after 3 rd washing step after addition of substrate After nanobeads ELISA assay in eppendorf tube reaction was transferred to microfluidic chip Measurement of PsVs binding to nanobeads surface by Western blot VLP binding to nanobeads surface was tested with Western blot and obtained results are presented on Fig. 84. As it was expected initial solution of PsVs in Line 1 has the highest content of L1 protein. Line 2 contain L1 band from PsVs sample immediately after mixing with nanobeads. Line 3-7 represents PsVs samples from aliquots after five washing steps. By increasing the number of washing steps L1 protein content is decreasing which can be seen by the lowering of bands intensity. L1 protein content is high in the Line 8 where just nanobeads after five washing steps were analyzed. 134

158 Figure 84-Western blot of PsVs samples before and after incubation with nanobeads Line 1- PsVs sample 60 times diluted in PBS, Line 2-aliquot of PsVs samples immediately after mixing with nanobeads, Line 3-aliquot containing free PsVs after 1 st washing step, Line 4-7 aliquot containing free PsVs after 2 nd, 3 rd, 4 th and 5 th washing step respectively, Line 8-nanobeads with PsVs proteins on the surface, Line 9-protein marker Therefore we can conclude that such high protein content is due to the fact that L1 protein is bounded to nanobeads surface and even after five washing steps significant amount stays bound to nanobeads surface Measurement of PsVs binding to nanobeads surface with commercial kit PsVs binding to nanobeads surface was performed according to procedure described in section Since L1 protein concentration in applied PsVs sample was determined to be around 9.5 mg/ml (see section 4.3.3) in this mixture L1 protein was diluted 2500 times and concentration of around 3.8 µg/ml. According to calibration curve made with standards provided by supplier of Qubit fluorimeter in the applied sample volume it was obtained that after 5 min of incubation around 1 µg/ml of proteins presented in PsVs sample are bound to nanobeads surface. L1 protein is the main component of the mixture and it was expected that at least 50 % of bound proteins (corresponds to 500 ng/ml) are L1 with exact orientation for antibody binding Nanobeads based ELISA assay with µfia-tlm detection For nanobeads based ELISA assay reaction mixture was prepared as it follows: 2 µl PsVs (50 times diluted in PBS) 48 µl PBS 50 µl nanobeads (6 mg/ml) and incubated in eppendorf tube for five minute. 135

159 Optimized conditions for nanobeads based ELISA assay are shown in the Table 27. Table 27-Optimized conditions for nanobeads based PsVs ELISA assay Reagent Reagent Reagent flow Incubation step volume concentration rate (µl/min) (µl) Washing buffer (µl) Incubation time (min) Incubation with PsVs 3.8 µg/ml Blocking step Incubation with anti L1 HPV 16 antibody Incubation with secondary Ab HRP labeled Incubation with TMB substrate PBS + 4 % skimmed milk+ 0.2 % Tween ng/ml µg/ml Ready to use solution / 2 Formation of final blue reaction product is presented on Fig. 85. Anti L1 HPV 16 antibody (150 ng/ml) was used as the positive control. Formed reaction product confirmed successful formation of antigen antibody complexes on the nanobeads surface. Figure 85-Nanobeads based PsVs ELISA test performed in one channel with anti L1 HPV 16 antibody as positive control (150 ng/ml) Complete assay time is around 30 minutes which is more than 10 times shorter in comparison to PsVs ELISA assay on microtiter plate which last around 10 hours (including all incubation steps). If we compare developed assay with commercial ELISA kits currently present on the market this assay is at least five times faster. Using microfluidic chip which is designed to contain more than four channels it is also possible to reduce analysis time and to analyse higher number of samples at the same time. 136