An electrochemical sensor for homocysteine detection using gold nanoparticle incorporated reduced graphene oxide

An electrochemical sensor for homocysteine detection using gold nanoparticle incorporated reduced graphene oxide

Accepted Manuscript Title: An electrochemical sensor for homocysteine detection using gold nanoparticle incorporated reduced graphene oxide Authors: R...

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Accepted Manuscript Title: An electrochemical sensor for homocysteine detection using gold nanoparticle incorporated reduced graphene oxide Authors: Rajendran Rajaram, Jayaraman Mathiyarasu PII: DOI: Reference:

S0927-7765(18)30366-7 https://doi.org/10.1016/j.colsurfb.2018.05.066 COLSUB 9385

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

6-3-2018 28-5-2018 29-5-2018

Please cite this article as: Rajendran Rajaram, Jayaraman Mathiyarasu, An electrochemical sensor for homocysteine detection using gold nanoparticle incorporated reduced graphene oxide, Colloids and Surfaces B: Biointerfaces https://doi.org/10.1016/j.colsurfb.2018.05.066 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

An electrochemical sensor for homocysteine detection using gold nanoparticle incorporated reduced graphene oxide

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Rajendran Rajarama,b* , Jayaraman Mathiyarasua,b

Academy of Scientific and Innovative Research (AcSIR), CSIR - Central Electrochemical

Research Institute (CECRI) campus, Chennai - 600113, India. b

Electrodics and Electrocatalysis Division, CSIR-CECRI, Karaikudi - 630 003, Tamilnadu,

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India.

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*Corresponding author: [email protected]

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Tel: + 91-4565-241340; Fax: + 91-4565-227779

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Graphical Abstract:

Abstract

In this work, we report a methodology for the quantification of Homocysteine (HcySH) at neutral pH (pH-7.0) using Au nanoparticles incorporated reduced graphene oxide (AuNP / rGO / GCE) modified glassy carbon electrode. Glassy carbon electrode (GCE) is modified with

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graphene oxide - Au nanoparticles and characterized using SEM and XRD techniques. The electrode exhibited a typical behavior against the standard redox probe [Fe(CN)6]3-/4- and resulted in 0.06 V

peak to peak potential value. The modified electrode exhibited

electrocatalytic activity towards electrochemical biosensing of HcySH, which is established using voltammetric studies. HcySH oxidation peak potential is observed at 0.12 V on AuNP / rGO / GCE, which is 0.7 V cathodic than bare glassy carbon electrode (0.82 V). The large peak

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potential shift observed is reasoned as the interaction of –SH group of HcySH with the gold

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nanoparticles and the electrocatalytic property of graphene oxide that enhances the electrochemical detection at reduced overpotential. Further, successive addition of HcySH

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showed a linear increment in the sensitivity within the concentration range of 2-14 mM. From an

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amperometric protocol, the limit of detection is found to 6.9 µM with a sensitivity of 14.8 nA / µM. From a set of cyclic voltammetric measurements, it is observed that the electrode produces a

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linear signal on the concentration of HcySH in the presence of hydrogen peroxide. Thus it can be

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concluded that the matrix can detect HcySH even in the presence of hydrogen peroxide.

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Keywords: Homocysteine, AuNP / rGO / GCE, electrocatalytic detection

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1. Introduction

Homocysteine (2-Amino-4-sulfanylbutanoic acid) is an important essential amino acid

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present in human blood which cannot acquire through food intake. It can be obtained from methionine, where food material containing methionine is converted into cysteine via Homocysteine [1]. The reaction can revert to methionine from HcySH. If the reaction stops at HcySH, then the reaction cannot proceed neither towards cysteine nor methionine, due to which the level of HcySH in biological systems may exceed the normal level, 5-16 µM. This phenomenon is known as hyperhomocysteinemia [1,2]. As a

consequence, people are forced to face a range of health risks such as heart attack, pregnancy complications, osteoporosis, etc. [1-7]. Hence, development of biosensors for the quantitative estimation of HcySH will be a crucial one to save an individual from such health issues. Several efforts are in vogue for the successful quantification of HcySH such as HPLC, capillary electrophoresis and gas chromatography [3], fluorescence [3,6] and

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phosphorescent [7], luminescent [8], capillary electrophoresis with laser-induced fluorescence [9,10], immunoassay [11] and fluorescent sensing by molecular imprinting [12]. In this way, electrochemical measurements are also found an essential role in estimating the biomolecules. Since, the electrochemical protocol provides excellent sensitivity, selectivity, continuous reliability, portability and therefore electrochemical

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measurements have essential avenues in the biosensor research.

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Until now, electrochemical methods for the estimation of HcySH/Cysteine are reported using carbon nanotube modified electrodes [1], carbon nanotube paste electrodes [13],

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gold deposited CNT modified carbon paste electrodes [14], colloidal gold - cysteamine -

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carbon paste electrode [15], conducting polymer/gold nanoparticle hybrid nanocomposite modified screen-printed carbon electrodes [16] and poly(3,4-ethylene dioxythiophene)

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modified electrodes [17]. The carbon paste electrodes are prepared manually, and the

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response of the electrodes depends on the individual skill and the electrodes require pretreatment also. Fabrication of uniform surface electrodes is also a challenging one. Apart from carbon paste electrodes, conducting polymer modified electrodes are also reported

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for electrochemical sensing of HcySH/Cysteine. However, the deposition of conducting polymers of multiple cycles increases the film thickness, which may enhance the

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resistivity of the electrodes. Despite the availability of various modified electrodes, there is still a need for smart and novel materials for HcySH estimation. These materials can

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easily be synthesized and utilized to overcome the difficulties arise during the electrochemical sensing of HcySH such as large overpotential, attaining low limit of detection, interference from other possible biomolecules. To achieve these tasks, the present study proposes the simple preparation of AuNP / rGO / GCE for the successful electrochemical detection of HcySH. Metal nanoparticles incorporated solid substrates have been identified as an effective tool for electrocatalysis and biosensor applications

[18,19]. Such kind of materials have been utilised for the sensing of Hg2+ ions [20] and Indomethacin [21]. In the present work, it has been demonstrated that simple preparation of AuNP / rGO / GCE for the successful electrochemical biosensing of HcySH. The proposed platform is more desirable, reliable for electrochemical biosensing of HcySH.

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2 Experimental

2.1 Reagents

DL - Homocysteine, HAuCl4 used in this work was procured from Sigma-Aldrich and used as received. 0.1 M phosphate buffer (pH = 7.0) was prepared from sodium dihydrogen phosphate and disodium hydrogen phosphate received from Merck. All the

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aqueous solutions were prepared in milli-Q water having the resistivity of 18.2 MΩ and

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were prepared immediately before to use.

2.2 Apparatus

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All the electrochemical measurements were performed in Palmsens BV, Netherlands,

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portable electrochemical analyzer. A traditional three electrode system consisting of AuNP / rGO / GCE, platinum wire, saturated Ag/AgCl electrodes were used as working, counter and reference electrodes respectively. Elico Li 120 meter is used to maintain the

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pH of the solution, and SEM instrument from TESCAN is used to characterize the surface morphology of the modified electrodes. XRD measurements were carried out using X-

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Ray Diffractometer from Bruker.

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2.3 Preparation of AuNP / rGO / GCE A glassy carbon electrode of 3 mm diameter is polished with emery paper and polishing clothes containing aqueous alumina slurries of 0.3 and 0.05 µm. Then, the electrode is rinsed with ethanol and Milli-Q water and sonicated for 5 minutes. The thoroughly cleaned glassy carbon electrode is modified with GO which is prepared by modified hummer’s method. One milligram of GO is dispersed in 0.5 ml of deionized water and

subjected to ultrasonication for one hour. 4 µl of the GO is taken with the help of a micropipette and drop casted over the surface of the polished glassy carbon electrode. The GO dropcasted glassy carbon electrode is left at room temperature for drying. Thus, GO modified glassy carbon electrode (GO/GCE) was subjected to electrochemical cycling in a PBS (pH = 7.0) solution containing 6.5 mM HAuCl4 for about 40 cycles for the

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successful electrochemical incorporation of Au nanoparticles into GO [22]. During the deposition, GO was reduced to rGO, and Au nanoparticles were deposited on the rGO / GCE. Thus, the obtained Au nanoparticles incorporated rGO modified glassy carbon electrode (AuNP / rGO / GCE) was utilized for the estimation of HcySH.

Results and discussion

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3.1 Microscopic analysis of the modified electrodes

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Surface morphologies of the GO, and AuNP / rGO modified GCE were analyzed with the

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help of scanning electron microscope. The images obtained were shown in the figure 1A

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& B. Graphene oxide modified glassy carbon electrode (GO / GCE) shows a shape containing folded, wrinkled, disordered and flexible solid lines due to which it can be

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confirmed that few layers of graphene have occupied the surface of the electrode. When

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the electrode is treated in a solution containing 6.5 mM of HAuCl4 in 0.1 M PBS, a significant number of Au nanoparticles were incorporated into the system which is shown in figure 1B. The average Au nanoparticles size is found less than 100 nm, and it is spread

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over the GO lattice. The EDAX analysis further confirms the presence of significant amount of Au nanoparticles. From the SEM images and EDAX spectrum (1C), it is

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confirmed that Au nanoparticles were successfully incorporated into the GO matrix and

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the size of Au nanoparticles is in nano dimensions [22].

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2 µm

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2 µm

Figure – 1: SEM images of GO / GCE (A) and AuNP / rGO / GCE. EDAX spectrum of

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AuNP / rGO / GCE (C).

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3.2 XRD analysis

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Figure – 2: XRD patterns of GO (A) rGO/GCE (B), AuNP/GCE (C), AuNP/ rGO/GCE (D).

GO, rGO, AuNP and AuNP/rGO modified GCE were characterized by XRD analysis, and the spectrum is shown in Figure 2. The figure compares the XRD pattern of

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AuNP/rGO/GCE with GO/GCE, rGO/GCE and AuNP/GCE. In Figure 2A, a sharp peak observed at a 2θ value of ~11º is the characteristic peak of GO, and the peak at ~42º

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confirms the presence of GO at the surface glassy carbon electrode. These two peaks are corresponding to the diffraction planes, (001) and (111) respectively and these values

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resemble previous report [23]. The figure 2B is corresponding to the XRD pattern of rGO. Two prominent peaks can be observed at the 2θ value of ~25º and ~43º and the peak due to graphene oxide at ~11º disappears. As a consequence, it can be concluded that all the functional groups present in the graphene oxide matrix are reduced and converted into reduced graphene oxide. Figure 2C shows the XRD pattern of AuNP/GCE. In the pattern, five discrete peaks can clearly be observed at various 2θ values such as ~38º, ~44º,

~64.5º, ~77.5º and ~81.5º. They were assigned to the various planes of Au nanoparticles including (111), (002), (022), (113), (222). The XRD pattern of AuNP/rGO/GCE (Figure 2D) exhibited peaks at different 2θ involve values such as ~39º, ~44.7º, ~65.9º, ~78.8º, ~82.7º, which are corresponding to different planes (111), (002), (022), (113), (222) [(JCPDS-98-006-1206)] of Au nanoparticles [24] with a broad peak at ~25º. From the

are successfully incorporated into the matrix.

Cyclic voltammetric response of AuNP / rGO / GCE

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analysis, one can clearly point out that GO is converted into rGO and Au nanoparticles

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Figure – 3: Cyclic voltammetric response rGO/GCE (a), AuNP/GCE (b), AuNP/ rGO/GCE (c) in the solution containing 0.5M H2SO4.

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To confirm the presence of Au incorporation in the modified electrode, a traditional experiment, (i.e.) electrochemical cycling of the modified electrode in an electrolyte, ca.

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0.5 M H2SO4 was performed (Figure. 3). The figure compares the cyclic voltammograms of AuNP/rGO/GCE with rGO/GCE and AuNP/GCE in the electrolyte of 0.5 M H2SO4. In case of rGO/GCE, there is a strong oxidative wave is observed at around 1.6 V vs NHE. This may be due to the oxidation of functional groups present in rGO. At the meantime, AuNP/rGO/GCE and AuNP/GCE produce peaks at ~1.37 V and ~1.59 V which are due to the formation of gold oxides. In the reverse scan, the formed gold oxides are reduced at

~0.11 V vs. NHE. The increase in current at AuNP/rGO/GCE is due to the formation of gold oxides as well as the oxidation of reduced functional groups present in the matrix. From the electrochemical confirmed that the spectrum, it is electrode is modified by the incorporation of gold nanoparticles. Thus, it can be proved that Au is successfully

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incorporated into the GO matrix.

3.4 Electrochemical behavior of AuNP / rGO / GCE against the standard Redox couple Fe(CN)6]3- / 4-.

Figure 4 shows the cyclic voltammetric response of bare and modified glassy carbon electrodes for a standard redox probe [Fe(CN)6]3-/4- measured in 0.1M KNO3 supporting

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electrolyte. The peak to peak potential difference obtained for bare glassy carbon

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electrode is 85 mV with the j value of 0.081 mA cm-2. Upon modification with GO, a

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poor redox response is observed with a negligible peak current. This can be reasoned as, GO layers are functionalized with negatively charged groups have a repulsive interaction

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with the negatively charged redox couple [Fe(CN)6]3-/4-. Due to the strong charge repulsions, the peak potential difference is shifted from 0.085 to 0.3 V and hence the

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modified electrode produces sluggish electrode kinetics against the redox probe. This is

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visualized with the cyclic voltammetric response of GO modified glassy carbon electrode with the positively charged standard redox couple [Ru(NH3)6]3+. The sensitivity of

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GO/GCE is enhanced rather than obtained with [Fe(CN)6]3-/4- with a peak to peak potential difference of 0.050 V (Figure. 4 Inset). Au nanoparticles were incorporated into the GO modified glassy carbon electrode by potential cycling. The obtained Au

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nanoparticles incorporated rGO/GCE was subjected to potential cycling with [Fe(CN)6]3/4-

redox probe and the electrode shows ideal behavior with a peak current density value

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of 0.155 mA cm-2. The peak to peak potential difference obtained was 0.06 V with a ip,a/ip,c ratio of 1, which indicates the electrode behaves ideally and electrocalytic one. .At the same moment, the adsorption of Prussian blue formed during the electrochemical cycling of Au in [Fe(CN)6]3-/4- solution leads to increase in the capacitance of the system, evidenced from the voltammetric loop.

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Figure – 4: Cyclic

voltammetric responses

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of bare glassy carbon electrode (a), GO modified glassy carbon electrode (b) and Au

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nanoparticles incorporated reduced graphene oxide (c) with the standard redox couple at the scan

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rate of 20 mV s-1. Cyclic voltammetric response of GO/GCE with [Ru(NH3)6]3+ (inset).

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3.5 Electrochemical Impedance Analysis of the modified electrodes

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The electro-catalytic characteristics of the modified and unmodified glassy carbon

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electrodes were further investigated by monitoring the charge transfer resistance at the interface of the electrode while interacting with the redox couple, [Fe(CN)6]3-/4-. The

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charge transfer resistance (Rct) of the modified and unmodified GC electrodes were obtained through circuit fitting with Randles equivalent circuits, R(Q(RW)) and R(Q(R(QR))). Where RS is the solution resistance, Rct is the charge transfer resistance, W

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is the Warburg impedance of Nyquist plot, and QCPE is the constant phase element. Figure 5 shows the electrochemical impedance responses of the bare GCE, GO/GCE and

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AuNP/rGO/GCE. From the response, it can be assumed that the resistance of AuNP/rGO/GCE is much lower than that of unmodified GCE and GO modified electrodes. Rs values are found to be 39 Ω, 117 Ω and 92.99 Ω for bare GCE, GO/GCE and AuNP/rGO/GCE respectively. And the Rct values of the respective electrodes are 168 Ω, 1492 Ω, 69.81 Ω. These results revealed that AuNP/rGO/GCE could form better

electron pathway between the electrode and redox couple [Fe(CN)6]3-/4- at the interface

of bare glassy carbon

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Figure – 5: EIS response and the fitted data

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[25].

electrode (a & a’), GO modified glassy carbon electrode (b & b’) and AuNP / rGO / GCE (c &

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c’) and their equivalent circuit fits (inset)

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3.6 Electrochemical determination of HcySH at AuNP / rGO / GCE

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Further, voltammetric protocols are resorted for the electrochemical sensing of HcySH at neutral pH. HcySH sensing was carried out in a solution containing 5 mM of HcySH in 0.1M PBS (pH =

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7.0) with bare glassy carbon electrode, GO/GCE and AuNP/rGO/GCE. The corresponding cyclic voltammograms are shown in Figure 6A and it is understood that bare glassy carbon and GO modified electrodes exhibited the oxidative response of HcySH at 0.824 V and 0.704 V

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respectively at a scan rate of 50 mV s-1. From these observations, it is concluded that bare as well as GO modified glassy carbon electrode produces a poor oxidative signal with the HcySH molecule. However, upon modification with AuNP/rGO the GCE exhibited responses at two different peak potential viz. at 0.119 V and 0.35 V. The first peak at 0.119 V is due to the

formation of HcyS. radical and the second one at 0.35 V may be due to the adsorption of the radical at the electrode surface [1, 15]. the mechanism is well explained as follows: HcySH  HcyS•ads + H+ + e-

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The oxidative overpotential of HcySH at AuNP/rGO/GCE is shifted 0.7 V towards cathodic direction compared to the bare and GO modified glassy carbon electrodes. The drastic shift in the overpotential in the case of HcySH at the Au modified rGO electrode could be attributed to the strong interaction of Au surface with the –SH group and catalytic nature of the reduced graphene oxide. The Au nanoparticles and reduced graphene oxide hybrid brings attractive catalytic behavior together and promotes the

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shuttling of faster electron transfer between the electrode and electrolyte that pave ways

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for the better biosensing of HcySH at very low overpotential. This electrocatalytic behaviour is verified with ten identical electrodes and the reproducibility is evaluated.

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They produce signal at 0.12 V without any significant deviation in the sensitivity. Thus it

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is believed that the electrode is reproducible with HcySH. The additional peak observed at around 0.7 V during the electrochemical oxidation of

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HcySH is due to the formation of gold oxide at the electrode surface simultaneously. In

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this reaction, oxygen is transferred to the unreacted radical that is enhanced by the labile sulfur oxide. The reaction is catalyzed by oxides that produce sulfonic acid. The reaction is explained according to the following mechanism and is well supported by L. Agui et

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al., in 2007 [15]. The peak observed in the reverse scan at ~ 0.4 V is evidently due to the reduction of gold oxide.

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2HcyS•ads + 3H2O  HcySO3- + 6H+ + 5e-

From the above observation, it can be concluded that AuNP/rGO/GCE exhibited better Figure 6B

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electrocatalytic performance compared to bare and GO modified GCE.

compares the cyclic voltammetric response of AuNP/rGO/GCE in the absence and presence of HcySH under a restricted potential window, from which one can clearly understand the appearance of the peak for the oxidative electrochemical biosensing of HcySH. The sensitivity of the modified electrode varies linearly (2-14 mM) with the concentration of the analyte molecule, which is confirmed by further addition of HcySH

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Figure – 6: A: Comparison of cyclic voltammetric response of 5mM HcySH in 0.1M PBS at

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AuNP / rGO / GCE.(a) Background (b) Bare GCE (c) GO / GCE (d) AuNP / rGO / GCE. B: Comparison of cyclic voltammetric responses of AuNP / rGO / GCE in 0.1M PBS in the absence (a) as well as the presence (b) of 5mM HcySH. C: Cyclic voltammetric responses of

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Homocysteine at AuNP/rGO/GCE with different concentrations ranging from 2 – 14mM. D: Cyclic voltammetric responses of Homocysteine at AuNP / rGO / GCE with various in scan

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rates between 20 and 80mV s-1

into the electrolyte (Figure 6C). The regression coefficient value obtained with the

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increase in the concentration of HcySH is found to 0.9957. Thus, it is very clear that the electrochemical sensing of HcySH at Au nanoparticles modified reduced graphene oxide lead to a reduction in overpotential up to 0.7 V. And increase in sensitivity with different concentrations of HcySH. This confirms that the proposed platform is highly stable towards the electrochemical biosensing of HcySH.

Also, a series of voltammetric experiments containing a solution of 5 mM HcySH at a neutral buffer solution was carried out at different scan rates (Figure 6D). A linear increase in the sensitivity on the square root of scan rate with a regression coefficient of 0.9914 is observed, which concluded that the reaction is diffusion controlled rather than surface controlled.

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3.7 Amperometric Studies

Further, amperometric measurements were carried out to examine the AuNP/rGO/GCE towards the electrochemical sensing of HcySH. Figure 7 shows the amperometric profile for the electrochemical biosensing of HcySH at a fixed overpotential of 0.2 V. In this experiment, each addition carries 10 µM HcySH and the sensitivity found to vary linearly with the concentration of the analyte. The linearity ranges from 10 to 80 µM, and the

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regression coefficient is found to be 0.9912. From the amperometric profile, it is observed

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that the electrode was able to produce a signal on the concentration of HcySH within a short duration (< 3 sec). The limit of detection (LOD) and sensitivity obtained from the

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same amperometric experiment are found to 6.9 µM and 14.8 nA / µM respectively. The

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LOD value is comparable with the other reported values such as 3 µM [9], 4.6 µM [13], 5

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µM [27], 28.5 µM [31]

Figure – 7: Amperometric response of HcySH in 0.1 M PBS with the successful addition of 10µM HcySH and its corresponding linear fit (inset).

3.8 Stability and Interference studies The stability of the sensor matrix was examined using cyclic voltammetric experiments. The electrode produces stable response against the biomolecule at the applied overpotential of 0.12 V for 15 consecutive cyclic voltammograms without any significant change in the sensitivity with the RSD value of 8.57 %. Thus it is believed that

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the matrix is highly stable towards the electrochemical biosensing of HcySH.

Considering the interfering molecules while estimating HcySH, H2O2 and O2 are the products formed while oxidizing HcySH. So, they should be avoided given interference, and hence cyclic voltammetric experiments were carried out with 0.1 M phosphate buffer solution containing 50 mM H2O2 with and without HcySH. In the absence of HcySH, the electrode can produce a signal for the electrochemical reduction of H 2O2. As soon as

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HcySH is introduced into the system, the signal due to H2O2 disappears, and the signal of

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HcySH starts appearing. The response of the electrode against HcySH in the presence of H2O2 varies linearly with different concentrations ranging from 5 to 35 mM with a

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regression coefficient of 0.9945 which is shown in figure 8. Thus, the proposed platform

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can detect HcySH even in the presence of H2O2, a major interfering molecule.

Figure - 8: Interference studies of HcySH in the presence of electrolyte PBS (pH = 7.0) containing Hydrogen peroxide (50mM).

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Conclusions AuNP / rGO / GCE is successfully fabricated and characterized with several

characterization techniques such as SEM and XRD as well as electrochemical tools such as cyclic voltammetry and impedance measurements. From the cyclic voltammograms against the standard redox couple [Fe(CN)6]3-/4-, it can be observed that AuNP / rGO /

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GCE is more catalytic than bare glassy carbon electrode and GO alone modified glassy carbon electrode. From the cyclic voltammogram obtained with the help of 0.5 M H 2SO4 proves the presence of Au nanoparticles in the matrix. From those techniques, it was proved that Au nanoparticles were successfully incorporated into GO and matrix are now AuNP / rGO / GCE which provides a better platform for the electrochemical biosensing of HcySH at very low overpotential. Overpotential during electrochemical sensing of

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HcySH takes place at 0.12V which is 0.7 V cathodic than bare glassy carbon electrode.

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The response of the electrode against the different concentration of HcySH varies linearly with the regression coefficient value of 0.9957, and the linearity ranges from 2-14 mM.

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From a set of experiments with the varying scan, rates confirm that the reaction is

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diffusion controlled rather than surface controlled. Limit of detection and sensitivity obtained with the help of amperometric measurement were 6.9 µM and 14.8 nA / µM

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respectively. It is possible to detect HcySH in the presence of a significant interfering

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Acknowledgement

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biomolecule, Hydrogen peroxide.

The Council of Scientific and Industrial Research (CSIR), New Delhi is acknowledged for

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the financial support from the project, Molecule to Materials to Devices (M2D) networked project and the authors are grateful to our Director Dr. Vijayamohanan K.

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Pillai for his support.

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