immunosensing interface based on diazonium cations ...gold nanoparticles (gnp). the reductive...

Immunosensing Interface Based on Diazonium

Cations Modified Indium Tin Oxide

Nadiya T. Darwish, Yatimah B. Alias, and Khor S. Mei Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

Email: [email protected]; {yatimah70, naomikhor}@um.edu.my

Abstract—An electrochemical biosensing interface –was

fabricated for immunological based detection. Biosensing

interface contains linker molecule 1,4-Phenyl diamine (PPD)

for immunosensing application was grafted via

electrochemical reduction/in situ deposition of aryl

diazonium salts on indium tin oxide electrode (ITO) . The

Reductive adsorption behaviour through electrochemical

measurement for (PPD) were studied .The deposition of

PPD was also confirmed by using Fourier transform

infrared spectroscopy (FTIR) and Field emission scanning

electron microscopy (FE-SEM) to characterize the

morphology of the grafted films and the distribution of the

linker molecules 1,4-Phenylenediamine which bound to the

gold nanoparticles (GNP). The reductive adsorbtion peak of

PPD was observed at –0.36 V and the electrode surface

modified with diazonium salts shows suppression of the

peak current when tested in both the Fe(CN)63− and

Ru(NH3)63+solution which attributed to the formed layer

on the electrode surface repell/resist those redox probes to

be diffused to the electrode surface. FTIR spectra of

modified electrode shows a broad peak at 3505 cm__1

which clearly indicates that the NH2 group of diazonium

salt were presence on the surface of the modified surface.

FE_SEM images of modified ITO shows granular -like

structures indicate the presence of a diazonium salt with

excellent distribution of GNP.

Index Terms—biosensor, indium tin oxide, diazonium

cations

I. INTRODUCTION

Diagnostic tests are necessary to detect different

bacterial and viral diseases consistently and rapidly, and

consequently to treat these infections in an early phase.

The methods at present used to cultivate the

microorganism or to identify the specific antibody are

both unwieldy and time consuming [1], [2]. The Reverse

transcription polymerase chain reaction (RT-PCR)

method although fast for diagnosis but RT-PCR protocols

suffer from two limitations: a false negative result due to

the variation of serotypes and the absent of standard

protocol [3], [4]. The electrochemical reductive

adsorption of aryl diazonium salts has been receiving

rising consideration due to the easiness and strong

bonding between the substrate and the aryl functional

group. Moreover, the grafted robust thin films have

thermal stabilityand electrochemical stability over a wide

Manuscript received July 23, 2014; revised December 8, 2014.

potential range. Large varieties of substituent aryl groups

can be grafted to the electrode surface and impart useful

properties to the modified electrodes [5], [6].

Biosensors and more specifically electrochemical

immuno-biosensors have presented as potential

alternatives to overcome the limitations of conventional

diagnosis methods, because it is cost effective, robust,

able to detect very low concentration of analytes from

different specimen mixtures and due to specific

interaction to the analyte [7]-[9]. The objectives of this

study are: to investigate the electrochemical deposition

behavior of 1,4-Phenylenediamine (PPD) on indium tin

oxide (ITO) electrodes and to examine its

electrodeposition by electrochemical

measurement ,Fourier transform infrared spectroscopy

(FTIR) and Field emission scanning electron microscopy

(FE-SEM).

II. MATERIALS AND METHODS

In this study a sensing interface for immunological

application has been fabricated by the modification of

indium tin oxide coated glass electrode (ITO)(as a

working electrode) via using 5mM of 1,4-

Phenylenediamine (PPD) first ITO substrates were

soaked and cleaned in an ultrasonicator using

dichloromethane and then 99% methanol for 10 min each,

followed by treatment with 0.5 M K2CO3 in a 3:1

methanol:Milli-Q water mixture for 30 min under

sonication to remove any residual organic contaminants.

The ITO substrates were then rinsed with copious

amounts of Milli-Q water, dried and stored in an

nitrogen-filled container. Secondly, 10mM of NaNO2 and

0.5M of HCl. The electrochemical deposition was done

by using cyclic voltammetry poteintiostatic for two

cycles. The electrochemical measurements were

conducted by testing the modified surfaces in two

different type of redox active species Ru(NH3)6Cl3

(1mM) and K3Fe(CN)6 (1mM), respectively. Platinum

and a Ag/AgCl electrode were used as the counter and

reference electrodes, respectively. Indium tin oxide / 1,4

Phenyl diamine / GNP were prepared by immersing ITO-

phenylNH2 surfaces in GNP solution for 3 h at room

temperature [10]. Morphological characterizations of

bare ITO/ gold nanoparticles (GNP) , ITO/ PPD / GNP

were carried out by using Field emission scanning

electron microscopy (FE-SEM) (Tokyo, Japan) model

Hitachi- SU8000. The slides were tested at slow scan

652014 Engineering and Technology Publishing

Journal of Life Sciences and Technologies Vol. 2, No. 2, December 2014

doi: 10.12720/jolst.2.2.65-68

speed under 2kV vaccum, Magnification 50.00k and the

work destination was 2.1mm at an accelerating voltage of

2kV.Gold nanoparticles were used to indicate the

presence of the terminal amine groups in PPD due to

covalent bond forms between GNP-NH .GNP were

immobilezed by immersing ITO / PPD and bare ITO in

GNP solution for 3 h at room temperature [10].

Elemental mapping and EDX Energy Dispersive X-Ray

and FTIR spectrometer (model: Bruker IFS 66/S,

Germany) were also used to characterize the grafted 1,4-

Phenylenediamine and gold nanoparticles.

III. RESULTS AND DISCUSSION

A. Electrochemical Measurement

Electrodeposition of PPD (5mM) caused a completed

electode coverage even after two cycles and the reductive

adsorbtion peak was obtained at –0.36 [11] and this

might be due to PPD has low molecular weight and it has

faster diffusion rate on the ITO surface [12]. The

accumulation and the deposition of PPD was also

confirmed by finding the kinetic parameters via using the

cyclic voltammograms of PPD in 1mM Fe(CN)63− at

different scan rates (10-2000) mv/sec. the result showed

the oxidation peak was directly proportional to the scan

rate as the peak current increased linearly with the root of

scan rate between 10 and 2000 mV s_1 and this indicates

a diffusion controlled process on working electrode

surface with R2 value 0.98 [5], [13] (Fig. 1 A-D).

Figure 1. Cyclic voltammograms of 1,4-Phenylenediamine (PPD) on ITO. Reductive adsorption (A ) comparison between before (Blue) and after (Red) modification in 1 mM Fe(CN)6 (B) comparison between before (Blue) and after (Red) modification in 1 mM Ru(NH3)6 3+ (C) in 0.05 M

phosphate buffer, pH 7.4, at 100 mV/s. Electrodes were modified in (5 mM 1,4-Phenylenediamine (PPD) , 10mM NaNo2 and 0.5M HCl ) by 2 scans

of cyclic voltammetry on ITO electrode. Effect of scan rate on the peak current responses (D).

Figure 2. Infrared Spectra for bare ITO (Blue) and ITO/PPD (Red).

Figure 3. FESEM image of bare ITO electrode (A) ITO-PPD (20nm) (B). EDX spectra ITO-PPD-GNP (c)



B. FTIR Analysis

FTIR spectra of modified electrode shows broad peak

at 3505 cm_1 which may be attributed to N-H stretching

vibrations of PPD in the composite [14], [15] while no

such peak is observed on bare indium tin oxide electrode

(Fig. 2).

C. FESEM Study

The FESEM image of the bare ITO electrode was in

grainy nature [16] and showed a absence of GNP while

the ITO modified PPD/GNP image showed uniform and

homogenous distribution of gold nanoparticles (Fig. 3).

And this successful attachment confirms the grafting

of linker molecules PPD. The linkage of amines to GNP

can be by the easy molecular cluster (NH2-GNP) or the

dehydrogenated form (NH-GNP) The linkage of amines

to GNPs was more likely to occur by dehydrogenation

from NH-GNP because of its stronger covalent bond [10],

[17].[18]. EDX results for the ITO 1,4

Phenylenediamine/ AuNP nanocomposites exhibited

atomic composition ratio of 7.36 C, 20.24 O, 3.33 N,

0.12 S and 2.11 Au (Fig. 3.C).and this result correlates

the FTIR findings that the nanocomposites were

successfully deposited on ITO electrode.

IV. CONCLUSION

A biosensing interface contains linker molecules is

described based on in situ generation diazonium salt.

Indium tin oxide surface was successfully coated with 1,4

Phenyl diamine then it further coated with GNP via

hydrogen bond between PPD and GNP. The electrostatic

behavior of the grafted surfaces and the FE_SEM and

EDX and FTIR results suggests the deposition of this

molecules. The dense and homogenous distribution of

GNP, indicating the interface was sensitive and useful for

analyte detection and biosensing applications. The output

of this study highlights the prospective for using aryl

diazonium salt as potential and easy to prepare for

biosensing interface in detecting analyte in different

samples.

AKNOWLEDGMENT

This research is supported by High Impact Research

Grant UM-MOE UM.C/625/1/HIR/MOE/F00004-21001

from the Ministry of Education Malaysia, RG159-12SUS,

FP014-2013A, PG120-2012B.

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Prof Yatimah Alias received the BSC and M.Sc. degrees in chemistry from university of

Malaya and the Ph.D. degree from East

Anglia university in UK. Areas of expertise are Electrochemistry (Electrosynthesis,

Electrodeposition) and coordination

chemistry (Supramolecular).



Nadiay Taha Darwish received the BSC degree in Microbiology from university of Al

Anbar in Iraq and M.Sc. degrees in

biomedical Science from university of Malaya and currently she is PhD student in

university of Malaya. Areas of expertise are

Molecular bacteriology, electrochemical Biosensors, Electroanalytical Chemistry).



immunosensing interface based on diazonium cations ...gold nanoparticles (gnp). the reductive...

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