Use of nanoparticles for enhancing IDE biosensor response

Transcription

1 HIGH SOPHISTICATED BIOSENSO SYSTEMS AND THEIR APPLICATIONS 2 nd Workshop of BVT TECHNOLOGIES, a.s. Use of nanoparticles for enhancing IDE biosensor response

2 Nanotechnology Nowadays nanotechnology is sharing knowledge, tools, techniques and information with electrochemistry, electroanalysis and electrochemical biosensors between other fields. Nanobiomaterials are one of the very important products of nanotechnology. Nanobiomaterials can be obtained, in general, by either the controlled assembly of nanoscale building blocks (a bottomup approach) or controlled elimination of starting materials and biomaterials to the nanoscale (a top-down approach).

3 Context stability Sensitivity food safety and quality control specificity improvement of the characteristics of the biosensor detection limit medical diagnostic Biosensors biological analysis Current Research environmental monitoring use of nanoparticles in a bottom-up approach as tools to optimize biosensors 3

4 Principles of Biosensors Biosensor design Molecules Biomolecules Bacteria = f(canalyte) 4

5 Outline In this presentation, some examples of nanomaterial-based electrochemical sensors will be presented, based on bottomup building technologies of nanomaterials, applied to conductometric transduction. gold nanoparticles (AuNPs) applied to enzymatic biosensors for detection of some metabolites (urea, glucose) magnetic nanoparticles (MNPs) applied to immunosensors for bacteria detection

6 Functionalized gold Nanoparticles amino group probe target NH 2 Enzyme Thiol Carboxylic Group Streptavidin protein Antibody covalent link or Affinity link probe minimize the diffusion problem maintain the stability of the biomolecular activity. Increase electron transfer rate Synthesized and functionalized by biological compounds 6

7 Biosensors based on the conductometric transduction A conductometric transducer is a miniaturized device with two electrodes The best design is an interdigitated structure (increase of the electrode length). 5,0 Electrode or (hauteur 0,2 µm) Contacts Substrat céramique 1,5 0,5 1,4 30,0 Electrodes interdigitées 20 µm 20 µm

8 Conductometric transducer Work electrode Reference electrode Conductometric transducers Pairs of gold interdigitated electrodes Thin film electrodes : suitable for miniaturization, large scale production, low cost Possibility of analysis a large spectrum of compounds, Driving voltage : sufficiently low to decrease the power consumption. Fabricated by Lashkaryov Institute of Semiconductor Physics (Kiev, Ukraine) W. Nouira, A. Maaref, F. Vocanson, M. Siadat, J. Saulnier, F. Lagarde, N. Jaffrezic-Renault, Electronanalysis, 2012, 24,

9 Results : conductimetric biosensor response Urea sensitive biosensor Urease CO(NH 2 ) 2 + H + + 2H 2 O 2NH HCO 3 - The hydrolysis of urea catalysed by urease increases local ph Significant increase of the local conductivity W. Nouira, A. Maaref, F. Vocanson, M. Siadat, J. Saulnier, F. Lagarde, N. Jaffrezic-Renault, Electronanalysis, 2012, 24,

10 Biofunctionalization of AuNPs + PAH + urease Gold NPs coated with citrate (negatively charged) Conductometric Transducer Frens method (23 nm) Enzyme immobilization by LbLmethod Characterization of NPs by DLS (size and zeta potential at 25 C) : NPs Au mv ; NPs Au+PAH 5.5 mv ; NPs Au+PAH+Enz mv W. Nouira, A. Maaref, F. Vocanson, M. Siadat, J. Saulnier, F. Lagarde, N. Jaffrezic-Renault, Electronanalysis, 2012, 24,

11 Electrical characterization of enzyme/aunp layer Admitance (ms) Urease with gold nanoparticles Urease without gold nanoparticles [Urea] (mm) Calibration curve for admittance measurement Equivalent circuit of the functionalized NPs urea biosensor Rs : solution resistance ; R1/CPE1 : enzyme/aunps film R2/CPE2: film/electrolyte interface Higher signal in presence of gold NP (x 3.5) Increase of conductivity film W. Nouira, A. Maaref, F. Vocanson, M. Siadat, J. Saulnier, F. Lagarde, N. Jaffrezic-Renault, Electronanalysis, 2012, 24,

12 Conductometric biosensor response (a) (b) Influence of the urea concentration Conductivity (µs) (c) (d) 50 0 (e) (f) (g) (h)(i) Time (mn) Evaluation of conductance response of the urea sensor (5mM phosphate buffer, ph=7.3) for 6 mm(a), 3 mm (b), 1 mm (c), 0.5 mm (d), 0.25 mm (e), 0.1 mm(f), 60 µm (g), 10 µm (h) and 5 µm (i) urea concentration. Response time : τ90 = 3 min Repeatability : standard deviation of 10 % W. Nouira, A. Maaref, F. Vocanson, M. Siadat, J. Saulnier, F. Lagarde, N. Jaffrezic-Renault, Electronanalysis, 2012, 24,

13 Conductometric biosensor response 400 Effect of the NPs on the amplification Conductance (µs) urease with gold nanoparticle free urease [urea](mm) Calibration curve of urea sensor (with and without functionalized NPs, 5 mm phosphate buffer, ph=7.3) Biosensor response is linear up to 3 mm Sensitivity with NPs Au funct. (107 µs/mm compared to 10 µs/mm) Detection limit of 2 µm of urea compared to 100 µm W. Nouira, A. Maaref, F. Vocanson, M. Siadat, J. Saulnier, F. Lagarde, N. Jaffrezic-Renault, Electronanalysis, 2012, 24,

14 Conclusions and future Role of gold nanoparticles Increase the density of immobilized enzyme, Amplification of the conductivity variation, NP Au behavior of microelectrodes, Interdigit distance in the range of 10 nm, Same range of probed distance by field lines from the transducer surface, Variation of conductivity when the distance to the transducer surface * *N.F. Sheppard, J.D. J. Mears, Biosens. Bioelectron. 1996, 11,

15 Oxidase based conductometric biosensor Detection of glucose according the enzymatic reaction GOD Glucose + O 2 + H 2 O Gluconolactone Enzyme immobilization Cross-linked enzyme Gluconic acid + H 2 O 2 Sensitivity 30 µs/mm 50 µm Enzyme/AuNPs 45 µs/mm 9 µm Enzyme/MNPs 70 µs/mm 3 µm Detection Limit W. Nouira, A. Maaref, H. Elaissari F. Vocanson, M. Siadat, N. Jaffrezic-Renault, MSEC 2012 (in press)

16 Magnetic Nanoparticles (MNPs) Enzyme Thiol amino group NH 2 protein Antibody MNPs consist of a paramagnetic or superparamagnetic core (mainly magnetite (Fe 3 O 4 )) surrounded by a polymeric outer layer suitable for the immobilization of biomolecules (Albers et al 2003). They can be gathered using a simple magnet. Carboxylic Group Streptavidin Strong interest in the last years for separation and detection applications

17 Magnetic Nanoparticles (MNPs) MNPs biofunctionalization has resulted in important practical advantages from an analytical point of view, including shorter reaction times between dissolved species and biomolecules immobilized on the surface of the nanoparticles which is also favoured by the easy dispersion of MNPs into solution with only gentle shaking, readily miniaturization of the assay system by using MNPs as a mobile solid phase, reduction of the required volumes of reagents and produced waste, obtaining lower detection limits with shorter assay times (Kuramitz 2009).

18 Detection of pathogenic bacteria Remains a crucial problem in numerous domains Biological Weapons Nosocomial Diseases Environnement (water-soil) Agrobusiness Biosensor development

19 Design of a Biosensor For Bacteria Detection Both bacterial strains, used in this study, come from Pasteur Institute collection : Escherichia coli (CIP 76.24) (negative gram) Staphylococcus epidermidis (CIP 68.21) (positive gram)

20 Characterisation of anti LPS antibody Gram-positive Cell Wall LTA Peptidoglycan E. coli S. epi 10 8 Protein Phospholipid Cytoplasmic membrane Gram-negative Cell Wall Protein Peptidoglycan Phospholipid LPS Porins Outer membrane Periplasmic space Inner membrane LPS Western profile blot

21 Immunosensors: Antibody/Magnetic Nanoparticles (MNP) on conductometric transducer Bactérie Bactérie MNP diameter: 200 nm COOH functionalized provided by Ademtech France 0 R--C NH- 0 R--C NH- 0 R--C NH- 0 R--C NH- Anticorps anti-lps Billes magnétiques fonctionnalisées (1%) Champ magnétique (0,3 T) Principe du biocapteur Transducers CC2 from BVT Frequency(KHz) Sensitivity(µS pm -1 )

22 A B C D SEM images of magnetic nanoparticles coated with anti-lps antibodies (A) and after interaction with E. coli (B, C and D) on the surface of the interdigitated electrodes (QuantaTM 250)

23 Real time response of the conductometric immunosensor Response time: 2 mins 40 σ (µs) ,50E+009 2,00E+009 2,50E+009 3,00E+009 3,50E+009 4,00E+009 Time (s)

24 Calibration curve of the conductometric immunosensor y = 11,162x + 13, σ(µs) 30 y = 5,5704x + 11,726 BVT 20 EI ,5 1 1,5 2 2,5 3 3,5 Log [E. coli] (Log (CFU ml -1 )) D.L. 1 CFU/mL Linear dynamic range: CFU/mL

25 Conclusion In this presentation, it was demonstrated clearly on conductometric transducers, the potentialities and advantageous features of nanomaterials-based biosensors exhibiting enhanced performances.

26 Thanks to people involved in this study Sarra El Ichi, Wided Nouira, Florence Lagarde, Nicole Jaffrezic- Renault Institute of Analytical Sciences, UMR-CNRS 5280 Claude Bernard University, Lyon 1 Abderrazak Maaref University of Monastir, Tunisia Chantal Fournier-Wirth, Joliette Coste EFS Montpellier Jan Krejci, Radka Kučerovà BVT Technologies

27 LYON UNESCO World Heritage Vincent Formica / Office du Tourisme de Lyon Sponsors: This work was supported by 7th FP of European Union (INFULOC project n ), RapidBACT project of EFS and PHC Utique project. LYON UNESCO World Heritage 27 Thank you for your attention