CHAPTER 6. SPECIFIC DETECTION OF Mycobacterium tuberculosis sp. GENOMIC DNA USING DUAL LABELED GOLD NANOPARTICLE BASED ELECTROCHEMICAL DNA BIOSENSOR

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1 76 CHAPTER 6 SPECIFIC DETECTION OF Mycobacterium tuberculosis sp. GENOMIC DNA USING DUAL LABELED GOLD NANOPARTICLE BASED ELECTROCHEMICAL DNA BIOSENSOR 6.1 INTRODUCTION The global impact of the converging dual epidemics of tuberculosis (TB) is one of the major public health challenges in recent years. Currently, about 54 million peoples around the worldwide are infected with the Mycobacterium tuberculosis (MTB). Each year approximately 8 million new infections are occurred and nearly about 2.4 million people are died. The occurrences of TB are mainly found in the developing countries, particularly in Africa, South East Asia and the countries of the former Soviet Union. According to the World Health Organization (WHO) the TB infection may escalate in coming decades, nearly about 1 billion people would become newly infected, over 150 million would become sick and 36 million would die worldwide from 2011 to 2020 (World Health Organization. Global TB Control Report, 2003). Due to its vulnerability and rapid spreading of MTB, highly sensitive detection methods are required in clinical diagnostics. At present, acid fast staining and culture of bacilli are used in diagnosis of MTB. Few nucleic acid based assays are also employed for the diagnosis of MTB like nucleic acid amplification test (NAAT) (Yun et al 2005 and Restrepo et

2 77 al 2006) and DNA probes (Park et al 2005). In addition, immunological based methods such as enzyme linked immuno sorbent assay (ELISA) (Bouda et al 2003, Mustafa et al 2005), immuno chromatographic assay (Abe et al 1999) and latex agglutination assay (Bhaskar et al 2002) have also been used to diagnose MTB. Gold nanoparticle based biosensors also performed for Mycobacterium tuberculosis detection for simple and rapid diagnosis in the real time samples (Baptista et al 2006, Chen et al 2008, Kaittanis et al 2007 and Upadhyay et al 2006). Recently, AuNPs probe based detection procedures have been reported for more sensitive and accurate detection of Mycobacterium tuberculosis (Soo et al 2009 and Liandris et al 2009). Though all the analytical methods can detect nano molar level, it entails various disadvantages including high cost, long time of assay and using highly toxic substances. However, sandwich type enzyme linked DNA based electrochemical biosensors are being considered as an effective and sensitive analytical tool to detect the biomolecules (Knopp et al 2006). Li et al 2010 have reported that the DNA probe, enzyme alkaline phosphatase (ALP) and horseradish peroxidase (HRP) labeled gold nanoparticle based biosensors show high specificity and sensitivity. Similarly, dual labeled gold nanoparticle (ALP and DNA probe) based lateral flow strip biosensors also reported by He et al (2010). However, these methods have the advantages of being simple, time saving, easily automated, and also can avoid a strict stripping procedure. The sensitivity and feasibility of this protocol needs to be further improved. In the present study an attempt has been made to develop a simple DNA probe and alkaline phosphatase (ALP) labeled gold nanoparticle based electrochemical DNA sensor for Mycobacterium sp. genomic DNA detection. The proposed electrochemical DNA biosensor was fabricated using

3 78 a sandwich detection strategy involving two types of specific DNA probe to mycobacterium sp. genomic DNA. The dual labeled gold nanoparticle probe (DNA probe and ALP) was introduced through sandwich DNA hybridization. The detection sensitivity was enhanced by gold nanoparticle, where it can carries the more number of ALP molecules per hybridization reaction. The electrochemical signal was generated by electroactive molecules produced through enzymatic catalytic reactions in the electrolytic solution. 6.2 PRINCIPLE OF DUAL LABELED GOLD NANOPARTICLE PROBE (PROBE DNA AND ALP) BASED ELECTROCHEMICAL DNA BIOSENSOR TO DETECT Mycobacterium. sp. GENOMIC DNA The dual labeled gold nanoparticle (Probe DNA AuNP ALP) facilitated electrochemical DNA biosensor was devoleped based on the sandwich DNA hybridization and an enzymatic catalytic reaction. Both the enzyme alkaline phosphatase and detector probe DNA were conjugated on the gold nanoparticle and subsequently hybridized with target genomic DNA immobilized on capture probe functionalized SAM/ITO electrode. The electrochemical signal of the electro active p NP on hydrolysis of p-npp was produced by ALP, which was measured by differential pulse voltammetry (DPV). Consequently, enhanced sensitivity was obtained due to the large number of ALP molecules bounded with gold nanoparticle present in the hybridization reaction. The gold nanoparticle acted as a platform for the anchorage of both the DNA probe as well as enzyme ALP and is clearly shown in the Schematic diagram 6.1.

4 79 APTMS/AuNP Probe 1 Genomic.DNA ITO electrode p-np Dual labeled AuNP p-npp i/µa P/V Scheme 6.1 Schematic diagram represents the newly developed dual labeled gold nanoparticle based electrochemical DNA biosensor for the detection of mycobacterium sp. genomic DNA 6.3 CHARACTERIZATION OF DUAL LABELED GOLD NANOPARTICLE PROBE UV-vis Spectrophotometric Analysis of Gold nanoparticle and dual labeled Gold nanoparticle UV-vis spectrophotometric analysis of the prepared gold nanoparticle and dual labeled (DNA probe and ALP) gold nanoparticle is shown in Figure 6.1. It was ascertained that the absorption maximum of gold nanoparticle is 518 nm and the surface plasmon was red shifted to 526 nm

5 80 upon conjugation of both DNA probe and ALP on gold nanoparticle surfaces. It was confirmed the formation of gold nanoparticle/dna probe/ ALP conjugate. Absorbance (a.u) 0.5 AuNP ALP/AuNP/P Wavelength (nm) Figure 6.1 UV-Vis Spectral analysis of gold nanoparticle and dual labeled gold nanoparticle probe High Resolution-Transmission Electron Microscopy (HR-TEM) Analysis of Gold nanoparticle and dual labeld Gold nanoparticle Conjugate HR-TEM (Figure 6.2) images display the gold nanoparticle and gold nanoparticle conjugate. It was observed that the colloidal gold nanoparticle has an average diameter of 16 ±0.2 nm, after the DNA probe 2 and ALP coupled on nanoparticles surface have an average diameter was increased to 18 ±0.2nm. Under higher magnification grayish halo around the modified nanoparticles surface was observed, which indicates the coupling of biomolecules on the nanoparticles surface (Li et al 2010).

6 81 (A) (B) 50 nm 50 nm Figure 6.2 HR-TEM images of (A) gold nanoparticle and (B) dual labeled (Probe 2 and ALP) gold nanoparticle conjugate 6.4 CHARACTERIZATION OF MODIFIED ITO ELECTRODE Cyclic Voltammetry (CV) Analysis The sequential modification of ITO electrodes such APTMS, AuNP, Probe 1, genomic DNA and dual labeled gold nanoparticle were characterized by cyclic voltammogram using PBS containing 2 mm K 3 [Fe(CN) 6 ]. The CV responses of the modified electrodes are shown in Figure 6.3. The Fe(CN) /4 6 shows a reversible one electron redox peak in bare ITO electrode with a peak to peak separation ( E p = E PA (anodic peak potential) E PC (cathodic peak potential) ) of 82 mv at a scan rate of (v) 50mVs 1. After the self assembly of APTMS on the electrode surface shows E P of 107 mv. This may due to the presence of APTMS on ITO electrode surface, which reduces the electron transfer rate of redox couple in the PBS solution. Susequent immobilization of gold nanoparticle on APTMS/ ITO electrode surface, the electron transfer rate of Fe(CN) /3 6 was increased. This indicated that AuNP was successfully immobilized and facilitates the required conduction on the electrode surface. Furthermore upon immobilization of probe 1 the current response of the electrode was decreased. The shift in peak potentials of the

7 82 Fe(CN) 6 /4 redox reaction at the probe-1 immobilized electrode was observed. Since, its insulating behavior on the electrode surface and the repulsive electrostatic interactions between negatively charged DNA and ferricyanide ions. Subsequently, the current response was decreased significantly upon immobilization of genomic DNA and dual labeled gold nanoparticle probe on the electrode surface (Cho et al 2006). These noteworthy changes in the CV response of AuNP/probe 1/genomic DNA/Dual AuNP ITO electrodes indicates the occurance of an efficient electrostatic and DNA hybridization on the SAM modified ITO electrode surface. Current/µA Bare ITO ITO/AuNP ITO/APTMS AuNP/P1 AuNP/P1/DNA AuNP/P1/DNA/Dual AuNP Potential/V Figure 6.3 Cyclic voltammetry analysis of bare ITO electrode, AuNP immobilized ITO, ssdna Probe 1, genomic DNA (10 ng /ml) and dual labeled AuNP modified ITO electrode in the presence of 2 mm K 3 [Fe(CN) 6 ] in 0.1 M KCl

8 Electrochemical Impedance Spectroscopic (EIS) Analysis The surface modified ITO electrode upon immobilization of AuNP, APTMS, capture probe 1, genomic DNA and dual labeled AuNP were also characterized by electrochemical impedance spectroscopy (EIS). The Nyquist plots for various modified electrodes response are shown in Figure 6.4. Significant differences in the impedance spectra were observed during various modification of the electrode. The bare ITO electrode shows the low interfacial charge-transfer resistances (R ct ). Upon immobilization of the APTMS on bare ITO electrode, the charge transfer resistance (R ct ) was increased significantly. This was attributed to the self assembled APTMS layer formed on the electrode surface, which reduced the interfacial electron transfer rate. The immobilization of AuNP on APTMS/ITO electrode the value of R ct was decreased significantly, which indicates the formation of conducting layer on the electrode surface. Further immobilization of probe 1 on electrode surface, the value of R ct was increased significantly. This may correspond to the immobilization of negatively charged oligo nucleotide probes on the electrode surface resulting in a negatively charged interface which electrostatically repels the negatively charged redox probe [Fe(CN) 6 ] /4 and blocks interfacial charged transfer. Thus, the diameter of the Nyquist plot semicircle was increased (Cho et al 2006). Similar trend was also appeared that the value of R ct was increased upon immobilization of genomic DNA and dual labeled AuNP probe on the electrode surface. The prevailed outcome of EIS measurements are in good agreement with that of CV measurement (Figure 6.3). Data obtained from the above studies that the surface of gold nanoparticle modified ITO electrodes were immobilized with different species.

9 84 Z"/ohm Z'/ohm AuNP/P1/DNA/Dual AuNP AuNP/P1/DNA AuNP/P1 ITO/APTMS ITO/AuNP Bare ITO Figure 6.4 Electro chemical Impedance spectroscopic (EIS) analysis of modified ITO electrodes 6.5 OPTIMIZATION OF ASSAY CONDITION FOR THE DETECTION OF Mycobacterium. sp The sensitivity of the electrochemical DNA sensor was based on the concentration of the detector probe 2 used in the assay. Various concentration of probe 2 containing dual labeled gold conjugate (10 to 100 ng/ml) was used to find out the optimum concentration needed to perform the assay. The differential pulse voltammetry (DPV) analysis was performed by using the various concentration of genomic DNA (1 to 50 ng/ml) assayed with each concentration of the probe 2 nano conjugate and probe 1 was kept at fixed concentration (50 ng/ml). Figure 6.5 explians the correlation of DPV signal with the concentration of the probe 2 coupled nano conjugate. The higher concentration of 100 ng/ml and 50 ng/ml of probe 2 nano conjugates could detect the least concentration of 1.25 ng/ml genomic DNA. Whereas, 2.5 ng/ml and 10 ng/ml of genomic DNA were detected using very low concentration of 25 ng/ml and 10 ng/ml probe 2 conjugates respectively. It

10 85 was confirmed that 50 ng/ml of probe 2 containing dual labeled nano conjugates was able to detect the lowest concentration of 1.25 ng/ml genomic DNA. Current/µA ng/ml 50 ng/ml 25 ng/ml 10 ng/ml [Genomic DNA] (ng/ml) Figure 6.5 Optimization of assay condition for genomic DNA detection using various concentration of probe 2 coupled dual labeled gold nanoparticle conjugates (n=3) 6.6 SPECIFICITY AND SENSITIVITY OF THE DNA BIOSENSOR FOR THE DETECTION OF Mycobacterium. sp Differential Pulse Voltammeter (DPV) is an efficient electrochemical technique used to detect the biomolecules. Various concentrations of genomic DNA were used from top to bottom 0.5 to 50 ng/ml (electrolyte: 0.1M Tris HCl ph 9.4 solution containing 3mM p-npp and incubated for 10 min). DPV signals supported the electro active species produced in the electrolytic solutions during the enzymatic catalytic reaction introduced in the electrode surface. ALP can catalyze the hydrolysis reaction of p NPP to produce the electroactive species of p NP and DPV responses for the detection of various concentrations of genomic DNA using dual

11 86 labeled gold nanoparticle probe (Figure 6.6). The DPV signals evidently enhanced the presence of target DNA and a linear relationship observed between the background subtracted peaks current versus the concentration of target DNA shown in Figure 6.7. The linear response over the range from 1.25 to 50 ng/ml and the detection limit about 1.25 ng/ml genomic DNA was noted. Besides, negative control of non specific E. coli genomic DNA employed and observed no DPV signal. It evidently shows that the DPV signal was not suitable to non specific adherence of the gold nanoparticle to the electrode surface and suggested that the dual labeled gold nanoparticle probe based DNA sensor assay ensures highly sensitive and specific to mycobacterium sp. Current/µA Potential/V 50 ng/ml E.coli Figure 6.6 Differential Pulse Voltametric (DPV) analysis of electrochemical DNA biosensor using dual labeled gold nanoparticle for the detection of Mycobacterium sp. genomic DNA

12 Current/µA [Genomic DNA ](ng/ml) Figure 6.7 Calibration plots of peak current versus the concentration of genomic DNA of the electrochemical DNA biosensor (n=3) 6.7 APPLICATION OF THE AuNP BASED ELECTROCHEMICAL DNA BIOSENSOR IN ANALYSIS OF CLINICAL SAMPLES The practicability of applying AuNP based electrochemical DNA biosensor in clinical samples investigated by analyzing sputum samples of suspected TB patients. Initially, ten sputum samples were assayed with both PCR and AuNPs based electrochemical sensor methods to demonstrate the efficiency of the enhanced sensor. PCR analysis of sputum sample of 1, 2, 3, 5, 6, 7, 9 and 10 infers the presence of Mycobacterium. Whereas, sample 4 and 8 were devoid to Mycobacterium presence (Figure 6.8). All the PCR positive samples were also detected with AuNPs based electrochemical DNA biosensor and peak area of DPV detectably correlated with the intensity of PCR bands obtained (Figure 6.9). It was clearly suggested that the proposed nanoparticles based electrochemical sensor was successfully detected Mycobacterium sp. in clinical samples which was comparable to that of PCR detected level.

13 88 Sputum Samples M -Ve +Ve bp Lane 1 Marker, lane 2 -ve control E.coli genomic DNA, lane 3 +ve control and lane 4-13 sputum samples No.1 to 10 Figure 6.8 PCR analysis for the detection of Mycobacterium. sp genomic DNA in sputum samples Current/µA Potential/V +Ve SS 2 SS 7 SS 6 SS 9 SS 3 SS 5 SS 1 SS 10 SS 4 SS 8 Figure 6.9 Dual labeled AuNP based electrochemical DNA biosensor for the detection of Mycobacterium genomic DNA detection in sputum samples

14 89 From the observation from experiments, it is inferred that the newly developed sensor was comparable with microscopic, bacterial culture and PCR analysis and can be used as effective and efficient diagnostic tool. Total of 43 suspected TB patients sputum samples were analyzed using all the methods reffrred above and the result are presented in Table 6.1. Over all of 41 sputum samples were culture positive and among this 90% sputum samples was detected by PCR and AuNP based electrochemical DNA biosensors, whereas only 43% of samples was detected using microscopic analysis and none of the culture negative samples convinced by other methods. It was also confirmed and demonstrated that the proposed AuNPs based electrochemical DNA biosensors can be practically applied in monitoring and detecting the Mycobacterium sp. for clinical diagnostics. Table 6.1 Comparison of efficiency of gold nanoparticle based electrochemical DNA biosensor with different analytical method for the detection of Mycobacterium sp. genomic DNA Total sputum samples = 43 AFB culture Microscopy PCR AuNP-DNA sensor +Ve -Ve +Ve -Ve +Ve -Ve +Ve -Ve