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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

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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

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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.

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Dual labeled

AuNP

APTMS/AuNP Probe 1 Genomic.DNA

ITO electrode

p-NP

p-NPP

P/V

i/µ

A

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

6.3.1 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

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.

450 500 550 600 650 7000.0

0.1

0.2

0.3

0.4

0.5 AuNP

ALP/AuNP/P2

Wavelength (nm)

Ab

sorb

an

ce (

a.u

)

Figure 6.1 UV-Vis Spectral analysis of gold nanoparticle and dual

labeled gold nanoparticle probe

6.3.2 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).

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50 nm50 nm

(B)(A)

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

6.4.1 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

K3[Fe(CN)6]. The CV responses of the modified electrodes are shown in

Figure 6.3. The Fe(CN)6/4

shows a reversible one–electron redox peak in

bare ITO electrode with a peak to peak separation ( Ep= EPA (anodic peak potential)–

EPC (cathodic peak potential)) of 82 mV at a scan rate of (v) 50mVs1. After the self

assembly of APTMS on the electrode surface shows EP 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)6/3

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

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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.

Potential/V

Cu

rren

t/µ

A

-30

-20

-10

0

20

10

30

0.40.30.20.10

Bare ITO

ITO/APTMS

ITO/AuNP

AuNP/P1AuNP/P1/DNA

AuNP/P1/DNA/Dual AuNP

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 K3[Fe(CN)6] in 0.1 M KCl

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6.4.2 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 (Rct). Upon immobilization of the

APTMS on bare ITO electrode, the charge transfer resistance (Rct) 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 Rct 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 Rct 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 Rct 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.

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100 200 300 400 500 600 700 8000

-50

-100

-150

-200

-250

-300

-350

-400

Bare ITO

ITO/APTMS

ITO/AuNP

AuNP/P1

AuNP/P1/DNA

AuNP/P1/DNA/Dual AuNP

Z'/ohm

Z"

/oh

m

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

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.

[Genomic DNA] (ng/mL)

Cu

rren

t/µ

A

0 20 40 600.0

0.4

0.8

1.2

1.6100 ng/mL 50 ng/mL

25 ng/mL 10 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

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.

0.0 0.1 0.2 0.3 0.40.0

-0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

-1.6

Potential/V

50 ng/mL

40

05

10

20

2.51.25

E.coli

0.5

Cu

rren

t/µ

A

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

87

Cu

rren

t/µ

A

[Genomic DNA ](ng/mL)

0 20 40 600.0

-0.4

-0.8

-1.2

-1.6

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.

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M +Ve-Ve 1 2 3 4 5 6 7 8 9 10

Sputum Samples

317 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

0.0 0.1 0.2 0.3 0.40.0

-0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4

SS 2

SS 1

SS 6

SS 3

SS 10

SS 8

SS 7

SS 9

SS 5

SS 4

+Ve

Potential/V

Cu

rren

t/µ

A

Figure 6.9 Dual labeled AuNP based electrochemical DNA biosensor

for the detection of Mycobacterium genomic DNA detection

in sputum samples

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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 PCRAuNP-DNA

sensor

+Ve -Ve +Ve -Ve +Ve -Ve +Ve -Ve

41 2 18 25 39 4 39 4