Biochemical

Communication

Biosci. Biotech. Res. Comm. 10(1): 83-90 (2017)

Highly sensitive gold nanoparticle based

electrochemical biosensor for detection of Antigen

antibody interactions

Anita Rawat

, K. P. Singh

, Pashupat Vasmatkar*

and Pratibha Baral

Nanobiosensor Research Laboratory, Biophysics Unit,

Department of Biochemistry, College of Basic Sciences and Humanities, G. B Pant University of Agriculture

and Technology, Uttarakhand-263145, India

ABSTRACT

The electrochemical biosensor was designed for label-free detection of bovine serum albumin (BSA). In the developed

electrochemical sensor gold coated polycarbonate membrane of different 30, 50 and 100 nm pore sizes. were used for

detection. The gold coated polycarbonate membranes were thiolated by 16-Mercaptohexadecanoic acid (MHDA) and

then activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) for the

binding of the antibody (BSA). After activation of the membranes, the BSA antibody was immobilized and the mem-

branes were subjected to the detection of BSA antigen with the help of a homemade electrochemical setup. The speci-

 city of the antibody was cross-checked with a non-corresponding Prostate Speci c Antigen (PSA). The speci city

and sensitivity of the designed biosensor along with the signal ampli cation due to binding of antigen-gold nano-

particle conjugate was also determined. The impedance corresponding to each step of membrane modi cation was

observed and it was found that there was a sharp increase in the measured impedance from modi cation to detection.

Also, there was multiple fold increase in signal due to tagging of GNP. The impedance corresponding to different pore

sized membranes was used to  nd a suitable pored membrane for the sensitive detection and it was observed that 30

nm pore size membrane showed comparatively better result than 50 and 100 nm pore size membrane.

KEY WORDS: NANOPARTICLE, IMMUNOASSAY, ELECTROCHEMICAL BIOSENSOR, ANTIGEN-ANTIBODY

ARTICLE INFORMATION:

*Corresponding Author: vasmatkar-bcm@pau.edu

Received 21

Feb, 2017

Accepted after revision 27

March, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

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Online Contents Available at: http//www.bbrc.in/

84 HIGHLY SENSITIVE GOLD NANOPARTICLE BASED ELECTROCHEMICAL BIOSENSOR BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Anita Rawat et al.

INTRODUCTION

Since the development of the  rst glucose biosensor by

Clark in 1962. Electrochemical biosensors have been

of great interest because of the properties like simple

instrumentation, low cost, rapid and sensitive response

(Lin et al., 2005, Grieshaber et al., 2008). Recently, the

emergence of nanoparticles helped in improving the

performance of electrochemical sensors. Some particular

nanomaterials, such as gold and semiconductor quan-

tum-dot nanoparticles, have already been widely used.

Biosensors are being developed by integrating func-

tional biomolecules with different types of nanomate-

rials, including metallic nanoparticles, semiconduc-

tor nanoparticles, magnetic nanoparticles, inorganic/

organic hybrid, dendrimers, and carbon nanotubes/

grapheme (Singh, 2011). Many biomolecules includ-

ing proteins/enzymes/oligo-peptides (Shen et al., 2003,

Crespilho et al., 2009), antibody/antigens (Haes et al.,

2004, Pengo et al., 2007), biotin/streptavidin (Zhu et al.,

2008) and DNA/oligonucleotides/aptamers (Nykypan-

chuk et al., 2008) have been immobilized on the surface

of nanoparticles to form noble metal nanoparticle–bio-

molecule conjugates which are then used for biosensing

(Willner et al., 2007, Guo and Dong, 2009, Li et al., 2010,

Song et al., 2010) . Gold nanoparticles (AuNP) are widely

used due to their biocompatibility, high surface to vol-

ume ratio, conductivity and catalytic properties (Wang

et al., 2002, Azzazy et al., 2009). AuNPs are employed

for signal ampli cation (Cao et al., 2011), as a label in

bioanalytical devices (Omidfar et al., 2013) especially

in the case of optical and electrochemical detection

method.

Biosensors based on the selective and speci c binding

properties of antigen and antibody are known as immu-

nosensors. The basic principle of immunosensors is the

speci city of the molecular recognition of antigens by

antibodies to form a stable complex. The sensitive and

selective binding nature of antigen and antibody is very

useful in various applications such as medical detection,

processing quality control, and environmental monitor-

ing (Tang et al., 2002).

Recently different electrochemical immunosensors

have been developed for selective detection of proteins,

pathogens or toxins such as a atoxin M1 (Vig et al.,

2009), enro oxacin (Wu et al., 2009), prostate speci c

antigen (Wang et al., 2012), pathogenic Staphylococcus

aureus ATCC25923 (Braiek et al., 2012).

This study of gold nanoparticle-based electrochemi-

cal biosensors for antibody-antibody interaction aims

to compare the detection performances of different pore

sized gold coated polycarbonate membrane. Here a sim-

ple low-cost electrochemical set-up was prepared to

measure the change in impedance on antigen-antibody

interactions and also to measure the effect of gold nano-

particle and antigen conjugate in signal ampli cation.

MATERIALS AND METHODS

PREPARATION OF GOLD NANOPARTICLES

Synthesis of AuNPs of 20 nm was carried out according

to the procedures described by Turkevich et al., (1951).

In brief, 50 ml of 1.0 mM HAuCl

was boiled with vigor-

ous stirring for 15-20 minutes. On boiling, 5 ml of 1%

trisodium citrate was quickly added. Solution changes

its color from clear to dark blue and then to deep red.

The heat was turned off after 10-15 min, but stirring was

continued for 10 minutes. The prepared gold nanoparti-

cle suspension was cooled,  ltered and stored in a dark

bottle at 4°C.

BIOPHYSICAL CHARACTERIZATION OF GOLD

NANOPARTICLES

UV–visible characterization of the AuNP suspension was

performed using a UV-Visible spectrophotometer (Lin et

al., 2007). The absorbance measurements were made

over the wavelength range of 250-700 nm using 1 cm

path length quartz cuvette.

COUPLING OF ANTIGEN (BSA) WITH AUNP

For coupling of antigen with the citrate-stabilized

AuNPs,  rst 900 μl of AuNP suspension (dilution 1:10)

was mixed with 100 μl of 1mM MHDA solution and

incubated for 30 min a shaker. Afterward, the solution

was centrifuged at 10,000 rpm at 4°C for 20 minutes

and resuspended in 500 μl of sodium phosphate buffer.

Afterward, 10 μg/ml suspension of antigen (BSA) was

added, and the solution was further incubated for 30

min under rotation. Then the solution was made up to

1ml with PBS buffer and centrifuged at 12000 rpm at

4°C for 1 hour, and was  nally resuspended in sodium

phosphate buffer (Kleo et al., 2012).

MODIFICATION OF GOLD COATED

POLYCARBONATE TRACK ETCH (PCTE)

MEMBRANE

The PCTE membrane of different pore size (30, 50 and

100 nm) was coated by gold through sputter coating.

Each membrane was then subjected to surface modi ca-

tion (Chen et al., 2008). Firstly, gold coated polycarbon-

ate membrane is thiolated by immersing it in an etha-

nol solution of 1 Mm 16-mercaptohexadecanoic acid

(MHDA) for 24 hours to form self-assembled monolayer

and then activated by immersing in a 75 mM solution of

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS HIGHLY SENSITIVE GOLD NANOPARTICLE BASED ELECTROCHEMICAL BIOSENSOR 85

Anita Rawat et al.

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)

and 15 mM solution of N-hydroxysuccinimide (NHS)

for 1 hour to convert carboxylic group to an active

NHS ester. After the reaction, the membrane was rinsed

with de-ionised water. Immobilization of antibody was

done by 1mg/ml anti-BSA Antibody solution prepared

in phosphate buffer saline (PBS) solution. 100μl of this

solution was added on the thiolated membrane and

stored at room temperature for 1 hour and then rinsed

with PBS solution for removing excess antibody. 100μl

of antigen BSA is applied on antibody immobilized

gold coated membrane for 1 hour. The membrane was

mounted in the glass cell for measuring the change in

impedance after each step of surface modi cation.

CHARACTERIZATION OF MODIFIED MEMBRANE

Characterization of polycarbonate membrane was per-

formed by Fourier Transform Infrared Spectroscopy

(FTIR) after each step of membrane’s surface modi cation

by simply putting the dry membrane in a sample holder.

FABRICATION OF MEMBRANE BASED

ELECTROCHEMICAL BIOSENSOR

The electronic circuit designed in the laboratory is

shown in  gure 3.1 while the original photograph of

the system is also depicted in  gure 3.2. Electrochemical

measurements were carried out at room temperature in

a cell with two Ag/Ag-Cl electrodes (with a surface area

of 1 cm

) and a working volume of 6 ml divided in two

L-shaped glass cell by the membrane.

The change in the potential was recorded as a func-

tion of time using the digital multimeter (in volts) after

each step of membrane modi cation impedance was cal-

culated by the given formula:

RESULTS AND DISCUSSION

CHARACTERIZATION OF GOLD

NANOPARTICLES

The peak obtained at 516 nm (Fig. 2) depicts that the size

of nanoparticles ranges from 5- 15 nm..

CHARACTERIZATION OF MODIFIED GOLD

COATED MEMBRANE

The FTIR spectra of PCTE membrane recorded on the

computer using OMNIC software (Figure 3). The sam-

ple (membrane) was  tted into membrane sized hole in

the sample holder. After placing membrane between the

light  lters of the instrument, IR rays interact with the

sample and yield various types of stretching, bending

and vibrations forming a spectrum.

Various FTIR peaks of Ag-Ab immobilized membrane

observed (Figure 4); the peak at 3300 cm-1 was due to

the presence of amide linkage of peptide bonding (NH

stretching), the absorption band at 1655 is for the amide

I region which is due to C=O stretching of peptide link-

age. The peaks observed at 1494 and 1229 are due to CN

stretching and NH bending (Kong and Yu 2007).

SCANNING ELECTRON MICROSCOPY (SEM)

The SEM micrographs of AuNP tagged antigen bound

with the corresponding antibody immobilized on the

membrane was depicted in  gure 6 at 2500 KX magni -

cation at a voltage of about 15 KV (Fig. 5).

MEASUREMENT OF IMPEDANCE BY

ELECTROCHEMICAL BIOSENSOR

Different sets of membranes of different pore size viz.

30, 50 and 100nm; simple membrane, gold-coated

membrane, gold-coated thiolated, antibody immobilized

FIGURE 1. Circuit diagram of the electrochemical biosensor.

Impedance, Z =

(Applied Potential – Potential

voltmeter

) × Resistance

Potential

voltmeter

Anita Rawat et al.

86 HIGHLY SENSITIVE GOLD NANOPARTICLE BASED ELECTROCHEMICAL BIOSENSOR BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 2. UV-Visible spectra of gold nanoparticles.

FIGURE 3. FTIR spectrum of thiolated and

plane PCTE membrane

FIGURE 4. FTIR spectrum of Ab and Ag-Ab immobilized PCTE membrane.

membrane, membrane immobilized with correspond-

ing Ag-Ab, membrane immobilized with corresponding

gold coated Ag-Ab and gold coated membrane immobi-

lized with non-corresponding Ag-Ab were  tted in the

experimental setup and measurements were taken one

by one in electrochemical biosensor. The setup was used

to measure the potential (in volts) across 1 KΩ resistance

with applied potential of 3 V at room temperature (Fig.

6a, b and c).

By using the formula of impedance, the values of

impedance across the different membrane was calcu-

lated using the values of potential measured after every

30 seconds of the time interval, resistance and applied

potential. It is evident from the graph that the values

of impedance (in KΩ) changes with time and with the

modi cation of membrane.

Figures 7, 8 and 9 shows the change in impedance

from surface modi cation of membrane to detection of

30, 50 and 100 nm of gold coated polycarbonate mem-

brane respectively.After the gold coated membrane was

functionalized with MHDA, the electron transfer across

the membrane surface was reduced due to negatively

charged carboxylic groups which cause repulsion while

the impedance of plane polycarbonate decreases sharply.

When anti-BSA and BSA were immobilized on the mem-

brane surface, the impedance was increased again. In the

case of 30 nm, best results were obtained in comparison

to 50 and 100 nm membrane which could be due to the

larger pore size of latter. The increase in impedance after

each modi cation increases successively indicating that

Anita Rawat et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS HIGHLY SENSITIVE GOLD NANOPARTICLE BASED ELECTROCHEMICAL BIOSENSOR 87

FIGURE 5. SEM Micrograph of gold coated antigen tagged with the

antibody on the gold coated PCTE membrane.

FIGURE 6a. Values of impedance with respect to time for 30

nm membrane.

FIGURE 6b. Values of impedance with respect to time for 50

nm membrane.

Anita Rawat et al.

88 HIGHLY SENSITIVE GOLD NANOPARTICLE BASED ELECTROCHEMICAL BIOSENSOR BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 8. Comparison of change in impedance on non-speci c

antigen-antibody interactions.

FIGURE 6c. Values of impedance with respect to time for 100

nm membrane.

FIGURE 7. Comparison of change in impedance of antigen-

antibody interactions.

Anita Rawat et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS HIGHLY SENSITIVE GOLD NANOPARTICLE BASED ELECTROCHEMICAL BIOSENSOR 89

membrane was modi ed successfully due to a reduction

in pore size resulting in increased membrane thickness.

This increase between each modi cation step was found

to be greater in the case of 30 nm membrane. BSA layer

immobilized on the membrane might have acted as inert

blocking layer hindering the diffusion of ions across the

membrane and thus resulting in increased impedance

(Huang et al., 2010).

For signal ampli cation, antigen-functionalized

AuNPs (size ~15 nm, modi ed with a carboxy-terminated

thiol and covalently coupled to the antibody through an

amide bond) were utilized. The antigen-functionalized

AuNPs bind to the corresponding antibody speci cally

which are already captured on the membrane surface.

This step decreases the pore size of the surface of the

membrane and leads to a further decrease in current

 ow. This additional decrease in pore size of the mem-

brane due to AuNP coupled antigen results in signal

ampli cation almost ten times than without AuNP.

The results obtained are summarized in the form of

following bar graphs given below showing the compari-

son of the change in impedance during antigen-anti-

body interactions of all the three membranes viz. 30 nm,

50 nm and 100 nm.

Comparison of impedance produced by antigen-anti-

body interaction on gold coated polycarbonate mem-

brane of different pore size shows that with the increase

in pore size the impedance increases as shown in Figure

7. This observation could be due to partial blocking of

larger pores of the membrane on protein immobilization

which cannot inhibit ion movement across the mem-

brane ef ciently as compared to the lower pore sized

membrane.

From the Fi gure 8, it is depicted that during non-

speci c antigen and antibody interaction, the imped-

ance decreases due to improper attachment as antibody

lacks the binding site for non-speci c antigen. On the

other hand, the higher value of impedance obtained in

the case of speci c antigen-antibody interaction.

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