Accepted Manuscript

Electrochemical deposition of gold nanoparticles on reduced graphene oxidemodified glassy carbon electrode for simultaneous determination of levodopa,uric acid and folic acid

Ali Benvidi, Afsaneh Dehghani-Firouzabadi, Mohammad Mazloum-Ardakani,Bi-Bi Fatemeh Mirjalili, Reza zare

PII: S1572-6657(14)00466-4DOI: http://dx.doi.org/10.1016/j.jelechem.2014.10.020Reference: JEAC 1863

To appear in: Journal of Electroanalytical Chemistry

Received Date: 2 June 2014Revised Date: 14 September 2014Accepted Date: 19 October 2014

Please cite this article as: A. Benvidi, A. Dehghani-Firouzabadi, M. Mazloum-Ardakani, B.F. Mirjalili, R. zare,Electrochemical deposition of gold nanoparticles on reduced graphene oxide modified glassy carbon electrode forsimultaneous determination of levodopa, uric acid and folic acid, Journal of Electroanalytical Chemistry (2014),doi: http://dx.doi.org/10.1016/j.jelechem.2014.10.020

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Electrochemical deposition of gold nanoparticles on reduced graphene oxide modified

glassy carbon electrode for simultaneous determination of levodopa, uric acid and folic

acid

Ali Benvidi1*

, Afsaneh Dehghani-Firouzabadi, Mohammad Mazloum-Ardakani, Bi-Bi Fatemeh

Mirjalili, Reza zare

Department of Chemistry, Faculty of Science, Yazd University, Yazd 89195-741, Iran

Abstract:

A novel assembly of graphene oxide (RGO), gold nanoparticles and 2-(3, 4-dihydroxy phenyl)

benzothiazole (DHB) modified glassy carbon electrode (DHB/AuNPs/RGO/GCE) was

successfully fabricated by chemical and electrochemical deposition of gold nanoparticles at

RGO/GCE and characterized by scanning electron microscopy (SEM), electrochemical

impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques. This sensor was applied

to simultaneous determine levodopa (LD), uric acid (UA) and folic acid (FA). It was observed

that an electrochemical deposition of gold nanoparticle has higher electrocatalytic activity for the

oxidation of LD, UA and FA compared with chemical deposition. Square wave voltammograms

of these compounds showed three well defined and fully resolved anodic oxidation peaks with

large current at DHB/AuNPs/RGO/GCE. The proposed electrode was successfully applied for

determination of LD, UA and FA in some real samples (such as tablet of Madopar, urine and

human blood serum) by the standard addition method.

*1Corresponding author: Tel.: +98 351 8122645; Fax: 98 351 8210644.

e-mail address: abenvidi@yazduni.ac.ir, benvidi89@gmail.com

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Keyword: Reduced graphene oxide, Self assembled, Gold nanoparticles, Levodopa, Uric Acid,

Folic acid.

1. Introduction

Some biological compounds have an overlapping oxidation potential on the bare

electrodes. Modification of electrodes using nanomaterial and redox modifiers due to reduction

of the overpotential essential for electrochemical reaction is an interesting field in

electroanalytical chemistry. A precise modification of the surface structure on a nanometer-scale,

namely self-assembly of organo-sulfur compounds on gold surface, has been employed in the

fabrication of sensors and biosensors.

It was known that patients with Parkinson's disorder have low concentration of dopamine

(DA) in their brains [1-2]. Levodopa (LD) is a vital catecholamine in a biological system which

is used to reduce the symptoms of Parkinson's disease by increasing of DA in the brain. But if

LD is taken at high dosages, some effects of systemic DA can appear. Several methods for

determination of LD have been described such as HPLC [3], capillary electrophoresis [4-5] and

spectrophotometry [6-7]. Some electrochemical methods for determination of LD have also been

reported due to LD is an electroactive component with two OH groups (scheme 1) which are the

active sites for oxidation of levodopa [8-14].

Monitoring of uric acid (UA), a product of the metabolic breakdown of purine

nucleotides, is essential because high blood concentrations of uric acid can lead to several

diseases such as gout and hyperuricaemia [15]. Several technologies have been employed for UA

analysis such as chemilumineces [16], enzymatic–spectrophotometric [17] and electrochemical

methods [18-21].

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Folic acid (FA) or vitamin B9 can help to produce healthy cells and is an agent for cancer

prevention by antioxidant activity [22]. Its biological importance comes from the conversion of

tetrahydrofolate into dihydrofolic acid in the liver. Because of being electro activity of FA, some

electrochemical methods have been used for its determination [23, 24].

As mentioned above, LD, UA and FA are compounds of great biomedical interest. LD

and UA can cause hyperuricaemia and gout diseases. When production of UA increases because

of some disease like kidneys deficiency, levels of UA build up in the blood. These conditions

lead to hyperuricemia. Then gout can happen as a result of hyperuricemia [25]. LD, UA and FA

have similar oxidation potential and coexistence in pharmaceutical preparations, Then UA and

FA show serious interference for the determination of LD [26]. These are the reasons that the

development of a new method for the simultaneous determination of LD, UA and FA has

appeared to be of great importance. It is very necessary to control the content of LD, UA and FA

in real samples to achieve better medicinal effect and lower toxicity. Therefore, it is important to

introduce a simple, low cost, fast, sensitive and selective detection method for the simultaneous

determination of these substances.

Recently, modified electrodes with graphene–metal nanoparticles composite have

attracted extensive attention in many fields because of their high electron conductivity and good

biocompatibility [27-28]. Graphene, a one-atom-thick sp2 bonded carbon sheet, has high surface

area, excellent electrical conductivity and electron mobility at room temperature, which can be

used to fabricate novel sensors for virtual applications.

Gold nanoparticles (AuNPs) have found many applications in biosensors due to their

excellent characteristics such as high catalytic activity, huge surface area, small dimensional

size, effective mass transport, and having compatible environment [29-30]. Modification of

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electrode with reduced graphene oxide (RGO) and AuNPs could increase the surface area of

electrode; enhance optical, electronic and catalytic properties. These modified electrodes can

react with organo-sulfur compounds to form S-Au covalent bonds resulting in monolayer

molecular assemblies. These types of modifications can greatly increase the immobilized amount

of S-functionalized compounds and enhance the stability of S-Au bond and self-assembled

monolayer (SAMs). DHB has s-functional group that can easily adhere to AuNPs and produce S-

Au bond for the formation of self-assembling and modification of the electrode surface. Also,

DHB is a catechol with two OH groups that are the active sites for oxidation. Therefore, we used

the combination of AuNPs and RGO to increase the surface area of electrode and immobilize

more DHB on the electrode surface.

Several works have been reported for electroanalytical determination of LD [8,14, 31-

34]. Babaei et al. fabricated a carbon paste electrode modified with ionic liquid, multi-walled

carbon nanotubes and cobalt hydroxide nanoparticles for simultaneous determination of

levodopa and serotonin [8]. Wang et al. used reduced graphene oxide modified glassy carbon

electrode (RGO/GCE) for determination of LD in the presence of carbidopa with a detection

limit of 0.8 µM [31]. Yi et al [32] used RGO for determination of LD in the presence of ascorbic

acid and uric acid. Weichao et al. used gold nanoparticle /MWCNT/L-cysteine in modification of

glassy carbon electrode for determination of LD [33]. Hu et al. used gold nanoparticle and

carbon nanotube for modification of pyrolytic graphite electrode for determination of LD in the

presence of uric acid and ascorbic acid [34]. They obtained a detection limit of 0.05 nM for LD.

Some of these methods have short linear range [31-34] and some of them have high detection

limit [8, 31, 32]. In the present work we used combination of RGO, AuNPs and DHB for

detection of LD. Due to unique properties of RGO and AuNPs, lower detection limit and wider

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linear range were obtained compared with other similar sensors. Also to the best of our

knowledge, there have been no report on the fabrication of SAMs by synthesized RGO, gold

nanoparticles and 2-(3, 4-dihydroxy phenyl) benzothiazole (DHB) for simultaneous detection of

LD, UA and FA. In this work, AuNPs were prepared by using two methods: chemical and

electrochemical methods. The modified electrode was characterized by cyclic voltammetry (CV),

scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS)

techniques. Modification of electrode by electrochemical deposition of AuNPs shows more

advantages in comparison with modification by chemical method. We also report the application

of DHB/AuNPs/RGO/GCE as a new electrode for the electro catalytic determination of LD in

phosphate buffer solution. The analytical performance of the modified electrode has been also

evaluated during LD quantitation in the presence of UA and FA. The applicability of the

modified electrode has successfully demonstrated by voltammetric determination of LD in real

samples.

2. Experimental

2.1. Apparatus and chemicals

Electrochemical measurements were performed with a potentiostat/galvanostat Autolab

model 302N (Eco Chemic, Utrecht, Netherlands) and a NOVA 1.7 software at laboratory

temperature (25±1°C). An Ag/AgCl (KCl, sat) electrode, a platinum wire and Glassy carbon

electrode (Azar electrode Co., Urmia, I.R. Iran, 2 mm diameter) modified with RGO, Au

nanoparticle and DHB self-assembled monolayer (DHB/AuNPs/RGO/GCE) were used as

reference, auxiliary and working electrodes, respectively. pH measurements were carried out

with a Metrohm model 691 pH/mV meter. All solutions were prepared with doubly distilled

water. LD, UA, FA and other reagents were analytical grade (Merck). The buffer solutions (0.1

6

M) were prepared from phosphoric acid and sodium hydroxide solutions in the pH range of 4.0–

10.0.

2.2. General procedure for the synthesis of 2-(3, 4-dihydroxy phenyl) benzothiazole

A mixture of 2-aminothiophenol (1.2 mmol), 3, 4-dihydroxybenzalaldehyde (1mmol) and

Al(HSO4)3 (0.02g) was heated at 80 °C for 10 minutes. The progress of the reaction was

monitored by thin layer chromatography (TLC). After completion of the reaction, the mixture

was cooled to room temperature, dissolved in acetone followed by addition of water, the solid

product was appeared. The product was re-crystallized in hot ethanol. FT-IR (ATR, neat),

ν=3478(OH), 1470 (C=N), 1264 (C-O), 1175(C-O), 1364, 1200, 1071, 1055, 765, 721 cm-1

.

1HNMR (400 MHz, Acetone-d6): δ: 6.98 (d, J=8.4 Hz, 1H), 7.38 (t, J=7.2 Hz, 1H), 7.51 (m, 2H),

7.67 (d, J=2 Hz, 1H), 7.96 (d, J=8.0 Hz, 1H), 8.02 (d, J=8.0 Hz, 1H), 8.50 (s, 1H), 8.69 (s, 1H).

2.3. Preparation of electrode

Graphene nanosheets were prepared by oxidizing graphite using a new method [35]. For

the reduction, GO suspension in purified water (150 mg/50 mL) was added to 50 µL of hydrazine

solution (98%) with 200 µL of ammonia solution (30%). After refluxing at 90°C for 12 h,

solution was cooled down. Subsequently, solution was centrifuged and precipitates were washed

with deionized water and then dried at 60°C in vacuum for 24 h [36]. SEM was applied for

characterizing of RGO (Fig. 1) which can easily adhere to a GCE surface via interaction between

graphene and glassy carbon [37]. Two following methods were used for modification of

RGO/GCE with AuNPs.

2.3.1. Chemical method

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In the chemical method, AuNPs were prepared according to Lee and Mesial’s method

with sodium citrate reduction of HauCl4 [38] and 20 µL of this suspension was casted on the

surface of RGO/GCE and dried in air to form an AuNPs film at the electrode surface.

2.3.2. Electrochemical method

In the electrochemical method, gold nanoparticles were deposited on the RGO/GCE by

reducing 0.3 mM HAuCl4 solution at a constant potential of -0.2 V. This electrode was

characterized by SEM (Fig. 1) which was used to evaluate the physical appearance and surface

characteristics of AuNPs on the electrode surface at a higher magnification.

2.4. Preparation of DHB self-assembled on AuNPs/RGO/GCE

DHB/AuNPs/RGO/GCE was prepared by immersing the GCE modified with AuNPs and

RGO in 0.1 mM ethanol solution of DHB for 24 h at room temperature. The electrode was

thoroughly rinsed with twice-distilled water to remove the physically adsorbed species of DHB.

Scheme 2 shows preparation procedure of DHB/AuNPs/RGO/GCE.

2.5. Preparation of real samples

Five tablets of LD (modopar) were weighted and grounded. Enough amount of the

obtained fine powder, equivalent to a stock solution of concentration about 0.01 M, was

dissolved in 0.1 M the phosphate buffer solution (PBS) in an ultrasonic bath. After filtering with

filter paper (Whatman, No. 1) and adjusting the pH using phosphate buffer (pH 7.0), further

dilution was performed to reach the calibration range of LD.

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Urine samples were stored in a refrigerator after collection. 10.0 mL of the sample was

centrifuged for 10 min and diluted to 50.0 mL with PBS. Ten mL of solution was transferred into

the voltammetric cell to be analyzed without any further pretreatment.

The serum sample was centrifuged for 10 min, filtered with filter paper, and then diluted

with PBS (pH 7.0). Ten mL of solution was transferred into the voltammetric cell to be analyzed

without any further pretreatment.

3. Results and discussion

3.1. Characterizing of RGO and AuNPs by SEM

Fig. 1A shows the SEM image of bare GCE. The electrode has a smooth and

homogeneous surface which can act as an ideal template. Fig. 1B shows the SEM image of

RGO/GCE. This image demonstrates a large surface area made of petal-like graphene

nanoflakes, random directions and sharp edges. Fig. 1C is the SEM image of electrochemically

deposited AuNPs on the RGO/GCE. This image shows many spherical AuNPs are

electrodeposited on the surface of RGO/GCE but SEM of AuNPs/RGO/GCE (AuNPs were

produced by chemical method) (Fig. 1E) shows the assembly of AuNPs on RGO/GCE is not

uniform. Fig. 1D shows the SEM image of DHB/AuNPs/RGO/GCE. Since the molecular size of

DHB is smaller than 10 nm, the SEM image has not enough resolution for distinguishing this

size.

The active surface area of the modified electrode was obtained using cyclic

voltammograms of K4Fe(CN)6 solution at different scan rates. For a reversible process, the peak

current is obtained by Randles–Sevcik equation:

Ipeak= 2.69 × 105 n

2/3AD

1/2Cν

1/2 Eq(1)

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where Ip refers to the anodic peak current, n is the total number of transferring electron, A is the

microscopic surface area of the electrode, D is the diffusion coefficient of Ferrocyanate and C is

the concentration of K4Fe(CN)6. Using the slope of Ipeak versus root of scan rate (ν1/2

), assuming

n = 1 and D= 6.20×10−6

cm s−1

[39], the microscopic surface areas for GCE and

AuNPs/RGO/GCE were calculated to be 0.0579 and 0.1194 cm2, respectively (the geometric

area of the GCE was 0.0314 cm2). The results indicate that the presence of RGO and AuNPs

increased the active surface of the electrode.

3.2. Electrochemical characterizing of modified electrode

In this work, EIS and CV were used to monitor the fabricating of the biosensor processes

and the results are presented in Fig. 2. The Cyclic voltammograms of bare and modified GCEs

were plotted in solution of 0.5 mM [Fe(CN)6]3−/4

and 0.1 M phosphate buffer solution (PBS).

The CV obtained at GCE shows a peak separation of ∆E≈75 mV in the presence of a reversible

marker [Fe(CN)6]3−/4−

, (Fig. 2, curve a). By coating GCE with RGO (RGO/GCE), peak current

increases in the presence of a reversible marker [Fe(CN)6]3−/4

(Fig. 2, curve b). This exhibits the

graphene nanosheet modification enables to increase the effective electrode surface area and the

rate of electron transfer. After the modification of RGO/GCE with AuNPs by electrochemical

method, an obvious increase in the redox peak current and a decrease in the peak potential

difference are observed in the CV response of AuNPs/RGO/GCE, suggesting that the

introduction of gold nanoparticles can enhance the electrochemical activity of obtained electrode

(Fig. 2, curve c). Formation of thiol SAMs on AuNPs/RGO/GCE electrodes generally leads to a

decrease in the current and increase peak-to-peak potential separation, indicating the DHB has

been successfully immobilized on the surface of the modified electrode (Fig. 2, curve d).

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The results of impedance experiments for different modification steps were in good

agreement with the results obtained by CV experiments. According to curve b in inset of Fig. 2,

by modification of the bare electrode with RGO, Rct decreases compared with bare GCE

indicating an easy electronic transfer at the electrode surface upon modification with RGO. Rct of

electrode is decreased with gold nanoparticle electrodeposition at RGO/GCE (curve c in inset of

Fig. 2). The respective semicircle diameter, corresponding to the Rct of the electrode surface, is

increased upon DHB-SAM formation on the AuNPs/RGO/GCE surface (curve d in inset of Fig.

2). This process has introduced a larger barrier to the interfacial charge transfer, which has been

revealed by increasing diameter of the semicircle in the spectrum.

3.3. Electrochemical behavior of the DHB/AuNPs/RGO/GCE

We prepared modified electrodes based on DHB/AuNPs/RGO/GCE and studied their

electrochemical properties in a buffered aqueous solution (pH = 7.0) using cyclic voltammetry.

CV experiments showed anodic and cathodic peaks with Ea and Ec of 0.285 and 0.169 V vs.

Ag/AgCl, respectively whit ∆E=0.116 V. These observations demonstrate that the electrode

behavior is quasi reversible [40]. The effect of sweep rate on electrochemical properties of the

DHB was investigated by CV (Fig. 3). The result showed that plots of both anodic and cathodic

peak currents were linearly dependent on scan rate (ν) in the range of 10 to 600 mV s-1

(Fig. 3,

inset B) indicating that the nature of redox process is controlled in a diffusion-independent

manner for a surface-confined redox process [41]. According to the procedure demonstrated by

Laviron [42], the apparent heterogeneous charge transfer rate constant (ks) and the transfer

coefficient (α) of a surface-confined redox couple can be estimated from CV experiments. At

high scan rates (above of 100 mV s-1

) peak potentials vs logarithm of scan rate are linear (Fig. 3,

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inset C). We can extract kinetic parameters αc and αa from the slopes of these linear segments.

According to the slopes of the linear segments equal to -2.303RT/αnF and 2.303RT/(1 – α)nF for

the cathodic and anodic peaks respectively, a value of 0.41 was obtained for αa. Also, the

electron transfer rate constant can be extracted from Eq. (2)

logks = α log (1 – α) + (1 – α) log α – log (RT/ Fnν) – αnαF∆Ep (1 – α)/2.3RT (2)

Where ν is the sweep rate, n is the number of electrons involved in the redox reaction of the

modifier (DBH) and all the other symbols have their conventional meanings. The value of ks was

evaluated to be 0.9 ± 0.1 s-1

using Eq. (2).

The effect of pH on the electrochemical behavior of DHB/AuNPs/RGO/GCE in 0.1 M

PBS was studied by cyclic voltammetry. It was observed that the anodic and cathodic peak

potentials of the DHB/AuNPs/RGO/GCE shift to negative values with increasing pH. The

potential-pH diagram was constructed by plotting the calculated E1/2 values as a function of pH.

This diagram is composed of a straight line with a slope of 62.0 mV pH-1

. The Nernstian slope

indicates that the oxidation of DHB is an equal electron-proton reaction in the pH range of 4.0–

10.0 [40].

3.4. Electrocatalytic properties of the DHB/AuNPs/RGO/GCE for levodopa

The cyclic voltammograms of electrocatalytic oxidation of 100 µM levodopa in the PBS

(pH = 7.0) at different electrodes are displayed in Fig. 4. Anodic peak potential for levodopa at

GCE is about 430 mV (Fig. 4 curve a), while corresponding potential at RGO/GCE (Fig. 4 curve

b) is 320 mV. Modification of this electrode by AuNPs and DHB shows an enhancement in the

anodic peak current (curves c and d). Curves e and f in Fig. 4 introduces the cyclic

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voltammograms obtained at the DHB/AuNPs/RGO/GCE and GCE in the phosphate buffer at a

scan rate of 30 mV s-1

. Based on these results, we propose an EC' mechanism to describe the

electrochemical oxidation of LD at DHB/AuNPs/RGO/GCE. The LD is oxidized at the potential

of 280 mV at the DHB/AuNPs/RGO/GCE while it is oxidized at 430 mV at the bare electrode.

The effect of scan rate on the electrocatalytic oxidation of LD at the DHB/AuNPs/RGO/GCE

was investigated by cyclic voltammetry (Fig. 5). The oxidation peak potential shifts towards

more positive with increasing scan rates, confirming the kinetic limitation of the electrochemical

reaction [40]. Also a plot of peak height (Ip) against square root of scan rate (ν1/2

) (Fig. 5A) was

found to be linear, suggesting at a convenient potential the oxidation of LD is mass transport

limited. The inset B of Fig. 5 illustrates the Tafel plot for the sharp rising part of the current–

voltage curve. Using the Tafel slope of 0.1261V decade-1

and assuming nα = 1, the electron

transfer coefficient (α) was obtained to be 0.46 which is in agreement with values reported by

other works such as: 0.48 [43, 9] and 0.49 [44].

3.5. Chronoamperometric measurements

Chronoamperometric measurements of LD at DHB/AuNPs/RGO/GCE were carried out

at the working electrode potential of 280 mV for various concentrations of LD (Fig. 6).

Experimental plots of I vs. t-1/2

were constructed at different concentrations of LD (inset A of

Fig. 6) in phosphate buffer media. The slopes of the resultant straight lines were then plotted vs.

LD concentration (inset B of Fig. 6). The mean value of the diffusion coefficient for LD was

found to be (7.4±0.3) ×10-6

cm2 s

-1 using Cottrell equation [40]. From the reduced form of the

Galus equation (Eq. 3) [45], we can evaluate the catalytic rate constant, k, for the reaction

between LD and DHB/AuNPs/RGO/GCE.

ICat/IL = π1/2γ

1/2 = π

1/2(KCbt)

1/2 (3)

13

where t is elapsed time, IL and IC are the limited current and catalytic current of the modified

electrode in the absence and presence of LD, respectively. The value of k can be obtained for a

given LD concentration based on the slope of the IC/IL vs. t1/2

plot that shown in inset C of Fig. 6.

The average value of k was found to be k =3.2 × 104 M

-1s

-1.

3.6. Selectivity

Under optimum experimental condition, the influence of various foreign species on the

determination of LD was investigated. The tolerance limit was taken as the maximum

concentration of the foreign agents, which caused an approximately ±5% relative error in the

determination of analyte. The influence of various foreign species on the determination of 1.0 ×

10-4

M LD was investigated. Table 1 shows the obtained results of possible interferences.

3.7. Calibration plot and detection limit

Calibration graph and detection limit for LD can be evaluated by DPV and SWV methods

using the proposed sensor. Similar to DPV, SWV is a sensitive technique that permits

determining analytes in a short time and sometimes with better results. So, we compared the

electrochemical response for the detection of LD using DPV and SWV techniques. SWV in

optimized condition (square-wave frequency of 25 s-1

, pulse amplitude of 30 mV and voltage

step of 25 mV), showed a linear range of 0.05-1200.0 µM with a detection limit (based on 3s/m)

of 18 nM for LD. The plot of peak current versus LD concentration is constituted of two linear

segments with different slopes (slope: 0.0631 µA µM-1

for the first linear segment and 0.0112 µA

µM-1

for the second linear segment), corresponding to two different ranges of LD concentration.

The decrease of sensitivity (slope) in the second linear range is probably due to kinetic

14

limitations. DPV in the optimum parameters (pulse height 50 mV, scan rate 30 mV s-1

and pulse

time 5.0 ms) exhibited a linear range of 0.08-1200.0 µM with a detection limit (based on 3s/m)

of 26 nM for LD. According to the obtained results, comparing between two methods, SWV

exhibits slightly wider linear range and lower detection limit for detection of LD. Table 2 shows

some analytical characteristics of proposed method such as detection limit and linear range for

determination of LD in comparison with some previously reported electrochemical methods. As

this table shows, detection limit of the proposed electrode is lower and its linear range is wider

than other works [31-34] due to use of graphene and AuNPs in the electrode modification.

3.8. Simultaneous determination of LD, UA and FA

The prepared electrode (DHB/AuNPs/RGO/GCE) was used for simultaneous

determination of LD, UA and FA. Fig. 7 shows SWV of different concentrations of LD, UA and

FA. The SWV results show three well distinguished anodic peaks at potentials of 195, 400 and

720 mV corresponding to the oxidation of LD, UA and FA, respectively (Fig. 7). The

sensitivities of the modified electrode towards LD in the absence and presence of UA and FA are

very close to each other, which indicates the oxidation processes of LD, UA and FA at the

DHB/AuNPs/RGO/GCE are independent and therefore, the simultaneous determination of the

three analytes is possible without any interference.

3.9. Applicability of proposed method

The proposed method was successfully applied for determination of LD in

pharmaceutical sample (Madopar). The LD contents in these samples were determined by the

standard addition method in order to prevent any matrix effect. The value of LD was found to be

195 mg (±0.2) (n=5) in Madopar drug sample. The obtained values were in a good agreement

15

with the labeled value (200 mg). Based on the t-test [46], the texp value was calculated to be 2.52.

Since texp is less than tcrit (tcrit = 2.78) therefore, there is no evidence for systematic error in the

obtained results. Thus, the proposed method can be efficiently used to determine LD in Madopar

drug sample. These results indicate that DHB/AuNPs/RGO/GCE could be applied for the routine

analytical control of pharmaceuticals.

In order to evaluate the analytical applicability of the proposed method, it was also

applied to determine LD, UA and FA in urine and human blood serum samples. Therefore

different amounts of LD, UA and FA were spiked to the samples and analyzed by the proposed

method. The results for the determination of three species in real samples are given in Table 3.

Satisfactory recovery percent revealed the capability of DHB/AuNPs/RGO/GCE for the

determination of LD, UA and FA in urine and human blood serum samples.

3.10. Reproducibility and stability of DHB/AuNPs/RGO/GCE

The reproducibility is one of the important characters of a biosensor. To investigate the

reproducibility, three independent DHB/AuNPs/RGO/GCE sensors were prepared on the same

day using three GCE and the same substance of DHB, RGO, AuNPs. Then, prepared modified

electrodes were used for determination of 90 µM LD solution. A relative standard deviation

(RSD) of 4.2 % was obtained for oxidation peak current of LD demonstrated a high

reproducibility of newly developed sensor. In addition, the stability of the

DHB/AuNPs/RGO/GCE was tested by determination of 90 µM LD solution in different days.

After measurement, the electrode was stored in buffer solution at room temperature. No apparent

change was observed in the LD oxidation peak current after three days. After 6 days peak current

16

of LD decreased 6.2% compared with the initial fabricated electrode. These results showed that

the electrode had the same behavior at least for three days.

3.11. Modification of RGO/GCE by chemically deposition of AuNPs

In chemical method, AuNPs were synthesized by sodium citrate reduction of HauCl4 and

used for modification of electrode. In this method, all steps for electrode modification were

repeated similar to electrochemical procedure, except that RGO/GCE was modified with AuNPs

produced by citrate reduction. Chemically produced AuNPs suspension was casted on the

surface of RGO/GCE and dried in air to form an AuNPs film at electrode surface

(AuNPs/RGO/GCE). The electrode was then modified with DHB to prepare

DHB/AuNPs/RGO/GCE. Comparison of cyclic voltammograms of reversible marker

[Fe(CN)6]3−/4

at RGO/GCE and AuNPs/RGO/GCE showed that the peak current at

AuNPs/RGO/GCE is less than that at RGO/GCE. This decrease shows that the assembly of

AuNPs on RGO/GCE is not uniform or demonstrates the presence of impurities at the surface of

electrode because of chemically prepared AuNPs [47]. Probably the interferential ions entrap in

the chemically produced AuNPs. After modification we investigated electrocatalytic properties

of the prepared DHB/AuNPs/RGO/GCE for the oxidation of LD. In this method a linear range of

1.0-500.0 µM with a detection limit (based on 3s/m) of 0.6 µM for LD was obtained. Comparing

the results obtained by the two methods, electrochemical deposition exhibits wider linear range,

lower detection limit and higher electrocatalytic activity for the oxidation of LD.

4. Conclusions

We explored a novel biosensor based on DHB, unique properties of reduced graphene oxide and

gold nanoparticles. Combination of RGO and gold nanoparticles especially electrochemical

17

deposition of Au can increase electrode surface and enhance electronic and catalytic properties.

The result showed that AuNPs immobilized at the RGO/GCE not only increased the electrode

surface area and consequently the amount of chemisorbed DHB, but also enhanced the charge

transfer between the oxidized compound and the GCE. This modified electrode was

characterized by CV, SEM and EIS techniques. The CV, SWV and DPV investigations showed

effective electrocatalytic activity of the modified electrode in lowering the anodic overpotential

for the oxidation of LD. High sensitivity, selectivity and good reproducibility of the

voltammetric responses, and very low detection limit (18 nM) are the advantages of modified

electrode. The sensor was applied for simultaneous determination of LD, UA and FA in real

samples such as modopar, urine and human blood serum, with satisfactory results.

Acknowledgments

The authors wish to thank the Yazd University Research Council for financial support of

this research. We also thank Dr. M. Namazian and Dr. Yingning Gao for editing the manuscript.

18

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24

Caption of figures:

Fig. 1. (A) SEM image of unmodified GCE, (B) SEM image of the RGO on GCE, (C) SEM

image of electrochemically deposited AuNPs on the RGO/GCE, (D) SEM image of

DHB/AuNPs/RGO/GCE, (E) SEM image of AuNPs/RGO/GCE (Gold nanoparticles were

prepared by chemical method).

Fig. 2. Cyclic voltammograms obtained in 0.1 M PBS containing 0.5 mM [Fe(CN)6]3−/4

at scan

rate 100 mVs-1

at (a) GCE, (b) RGO/ GCE, (c) AuNPs/ RGO/GCE and (d)

DHB/AuNPs/RGO/GCE. Inset: The Nyquist plots obtained in the same solution at (a) GCE, (b)

RGO/ GCE, (c) AuNPs/ RGO/GCE and (d) DHB/AuNPs/RGO/GCE. EIS conditions: ac

potential 5 mV, frequency range 10 kHz to 0.1 Hz.

Fig. 3. Cyclic voltammograms obtained at DHB/AuNPs/RGO/GCE in 0.1 M phosphate buffer

(pH 7.0) at various scan rates from 10 to 400 mV s-1

. Insets: (A) CVs in the same condition at

various scan rates from 400 to 600 mV s-1

. (B) Variations of Ipa and Ipc versus scan rate (C)

Variation of E versus the logarithm of scan rate, (D) Epa, Epc and E1/2 vs. pH.

Fig. 4. Cyclic voltammograms obtained at (a) GCE (b) RGO/GCE (c) AuNP/RGO/GCE (d)

DHB/AuNPs/RGO/GCE in 0.1 M phosphate buffer solution (pH 7.0) containing 100 µM LD at

a scan rate of 30 mVs-1

. Cyclic voltammograms obtained at DHB/AuNPs/RGO/GCE and (f)

GCE in 0.1 M phosphate buffer solution (pH 7.0) in the absence of LD.

Fig. 5. CVs obtained at DHB/AuNPs/RGO/GCE in 0.1 M phosphate buffer (pH 7.0) containing

100 µM LD at scan rates 5, 8, 10, 15, 20, 25, 30 and 40 mVs-1

, Insets: (A) Variation of the

electrocatalytic currents vs. the square root of the scan rate, (B) The Tafel plot derived from the

CV at scan rate 25 mV s-1

.

25

Fig. 6. Chronoamperograms obtained at DHB/AuNPs/RGO/GCE in 0.1 M phosphate buffer

solution (pH 7.0) for LD concentrations of 0.0, 0.3, 0.6, 0.8, 1.0 and 1.5 mM. Insets: (A) plots of

I vs. t-1/2

obtained from the chronoamperogram data, (B) plot of the slope of the straight lines

against the LD concentration and (C) dependence of IC/IL derived from the data of

chronoamperograms vs. t-1/2

.

Fig. 7. SWVs obtained for DHB/AuNPs/RGO/GCE in 0.1 M phosphate buffer solution (pH 7.0)

containing different concentrations of LD, UA and FA (from inner to outer) mixed solutions of

0.0+0.0+0.0, 5.0+10.0+10.0, 10.0+20.0+20.0, 20.0+40.0+40.0, 60.0+80.0+80.0,

100.0+150.0+150.0, 200.0+300.0+300.0, 400.0+500.0+500.0, 600.0+700.0+700.0,

800.0+900.0+900.0, 1000.0+1100.0+1100.0 and 1200.0+1300.0+1300.0 µM, respectively.

Insets: A) plot of the peak current as a function of LD concentration, B) Plot of the peak current

as a function of UA concentration and C) Plot of the peak current as a function of FA

concentration. (Instrumental parameters: Square-wave frequency of ƒ=25 s-1

, pulse amplitude=30

mV and voltage step ∆Es= 25 mV).

26

Table 1 The effect of various foreign species on the determination of 1.0 × 10-4

M LD

Recovery % Tolerance limit Foreign species Recovery % Tolerance limit Foreign species

99.8±1.5 1000 Uric acid 98.2±1.2 1000 Na+

101.2±3.5 100 L-tryptophan 97.5±2.2 1000 K+

98.8±1.5 100 acetaminophen 99.1±1.5 1000 SO42-

103.2±2.2 100 NADH 102.0±3.2 1000 NO3-

97.4±1.5 50 Ascorbi Acid 99.8±1.5 1000 Ca+2

96.5±2.2 10 N-acetyl-L-cysteine 103.1±2.2 1000 Glucose

98.7±1.5 5 Dopamine 101.1±2.5 1000 Sucrose

98.2±3.5 5 Epinephrine 97.5±2.8 1000 Fructose

97.3±3.2 1 Isoprenaline 100.1±3.2 1000 Glycine

96.4±3.4 1 Captopril 99.6±1.5 1000 Folic Acid

27

Table 2 Comparison of some electrochemical procedures used to determine of LD

Electrode Modifier Linear range (µM)

Detection

limit (µM) Ref.

CPE Co(OH)2 0.25-220.0 0.12 [8]

CPE CP 0.1-100.0 0.069 [9]

GCE RGO 1.0-16.0 0.8 [31]

GCE RGO 2.0-100.0 1.13 [32]

GCE AuNPs/MWCNT/L-cys 0.6-120.0 0.052 [33]

PGa AuNPs 0.1-150.0 0.05 [34]

CPE Coumarin 0.1-900.0 0.041 [43]

GCE DHB/AuNPs/RGO 0.05-1200.0 0.018 This work

a Pyrolytic Graphite

28

Table 3 Determination of LD, UA and FA in urine and human blood serum samples using

DHB/AuNPs/RGO/GCE

Recovery (%) Found (µM)a Spiked (µM) Sample

FA UA LD FA UA LD FA UA LD

- - - ND 5.1 ND 0 0 0

Urine 98

98.0

106.0 106.0 9.8 15.2 5.3 10 10 5

98.5 102.0 14.7 19.8 10.2 15 15 10

101.0 107.9 99.0 20.2 25.3 19.8 20 20 20

- - - ND 10.0 ND 0 0 0

Human

blood serum

102.0 99.0 101.0 10.2 19.8 10.1 10 10 10

98.5 106.6 99.0 19.7 30.2 19.8 20 20 20

99.6 101.8 102.0 49.8 61.1 51.0 50 50 50

a mean value for five replicate measurements

29

Fig. 1

30

Fig. 2

31

Fig. 3.

32

Fig. 4

-0.4

0.15

0.7

1.25

1.8

0 0.17 0.34 0.51 0.68

I/ µ

A

E / V

a

b

c

d

e

f

33

Fig. 5

34

Fig. 6

35

Fig. 7

3

14

25

36

-0.05 0.95 1.95

I/

µA

E / V

y = 0.0375x + 9.283R² = 0.9902

y = 0.0114x + 13.83R² = 0.9857

5

20

35

0 750 1500

[UA] / µM

I/

µA B

y = 0.0601x + 8.6453R² = 0.9908

y = 0.0108x + 13.667R² = 0.9938

5

20

35

0 750 1500

I/

µA

[LD] / µM

A

y = 0.0601x + 7.7658R² = 0.9865

y = 0.0123x + 15.312R² = 0.9897

5

20

35

0 750 1500

I/

µA

[FA] / µM

C

36

HO

HO

NH2

O

OH

Scheme 1. Structure of levodopa

37

Scheme 2. Schematic illustration of preparation procedures of DHB/AuNPs/RGO/GCE.

Graphical Abstract

38

Highlight:

1- We have prepared a DHB self-assembled monolayer at GNPs/RGO/GCE to determine

levodopa

2- The proposed sensor showed a low detection limit of 18 nM for levodopa

3- A method for simultaneous determination of levodopa, uric acid and folic acid

4- Ease of preparation, high repeatability and stability are characteristics of the electrode