A novel sensor based on electrodeposited Au–Pt bimetallic nano-clusters decorated on graphene oxide (GO)–electrochemically reduced GO for sensitive detection of dopamine and uric acid

Accepted Manuscript Title: A novel sensor based on electrodeposited Au-Pt bimetallic nano-clusters decorated on graphene oxide (GO)-electrochemically reduced GO for sensitive detection of dopamine and uric acid Author: Yang Liu Pei She Jin Gong Weiping Wu Shouming Xu Jianguo Li Kang Zhao Anping Deng PII: DOI: Reference:

S0925-4005(15)30143-X http://dx.doi.org/doi:10.1016/j.snb.2015.07.086 SNB 18817

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

7-4-2015 13-7-2015 20-7-2015

Please cite this article as: Y. Liu, P. She, J. Gong, W. Wu, S. Xu, J. Li, K. Zhao, A. Deng, A novel sensor based on electrodeposited Au-Pt bimetallic nanoclusters decorated on graphene oxide (GO)-electrochemically reduced GO for sensitive detection of dopamine and uric acid, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.07.086 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.

DA A

UA A

Ac c

ep te

d

M

an

us

cr

ip t

Graphical Abstract (for review)

A novel sensor based on electrodeposited Au-Pt bimetallic nano-clusters decorated graphene oxide (GO)/electrochemically reduced GO for ultrasensitive detection of dopamine and uric acid

Page 1 of 72

Research Highlights

Highlights Au-Pt

nano-clusters

anchored

at

GO/

ERGO

were

achieved

through

electrochemicalreduction process. Synergistic electrocatalytic effect of Au-Pt nano-clusters modified graphene oxide

ip t

was investigated and discussed. An excellent dopamine and uric acid biosensor based on Au-Pt/ GO- ERGO

cr

modified glassy carbon electrode was developed.

us

Electro-analytical parameters of the sensor based on Au-Pt/ GO- ERGO modified glassy carbon electrode for detection of dopamine and uric acid were evaluated in

Ac

ce pt

ed

M

an

detail.

Page 2 of 72

*Manuscript Click here to view linked References

The highlighted version-0711

A novel sensor based on electrodeposited Au-Pt bimetallic

ip t

nano-clusters decorated on graphene oxide (GO)-electrochemically

us

cr

reduced GO for sensitive detection of dopamine and uric acid

Yang Liua,b, Pei Shea, Jin Gongb, Weiping Wub, Shouming Xua, Jianguo Lia,*, Kang

a

ed

M

an

Zhaoa, AnpingDeng a

The Key Lab of Health Chemistry & Molecular Diagnosis of Suzhou, College of

ce pt

Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China

School of Public Health, Nantong University, Nantong 226019, PR China

Ac

b



Corresponding authors at: The Key Lab of Health Chemistry & Molecular Diagnosis of Suzhou, College of

Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China. Tel.: +86 512 65882362; fax: +86 512 65882362.E-mail addresses: [email protected], [email protected]

1

Page 3 of 72

ABSTRACT Sensitive and simultaneous detection of dopamine (DA) and uric acid (UA) by a sensor based on in-situ electrodeposited Au-Pt bimetallic nano-clusters decorated on graphene oxide (GO)-electrochemically reduced GO (ERGO) modified glassy carbon

ip t

electrode (GCE) was presented. The synergistic electrocatalytic effect of Au-Pt bimetallic nano-clusters and GO-REGO was investigated. Firstly, the comparisons of

cr

electrochemical properties for GO, GO-REGO, Au (or Pt) nanoparticles, Au-Pt

us

bimetallic nano-clusters decorated on GO-REGO were studied in detail. Secondly, electrochemical parameters of DA and UA were evaluated. It was observed that for

an

the novel Au-Pt/GO-ERGO nanocomposites, GO-ERGO could provide much wider separation of the oxidation peak potentials of DA and UA, while Au-Pt bimetallic

M

nano-clusters could speed up the electron transfer and enhance the electro-active areas. The linear range of detecting DA was from 6.82×10-8 to 4.98×10-2 M and limit of

ed

detection (LOD) was 2.07×10-8 M (S/N=3). The linear range of detecting UA was from 1.25×10-7 to 8.28×10-2 M and LOD was 4.07×10-8 M (S/N=3). The sensor was

ce pt

applied for the detection of DA and UA in human serum with good results. The sensor suggested that 3D metal-GO nanocomposites were superior materials for the

Ac

fabrication of novel electrochemical sensors.

Key words: Graphene oxide, Au-Pt nano-clusters, Dopamine, Uric acid, Electrochemical sensor

2

Page 4 of 72

1. Introduction Graphene, showing excellent physical, chemical and electronic properties as well as unusual charge-carrier mobility [1-6], quickly raised the attention of physicists, chemists and engineers over the world, eventually resulted in the award of the Nobel

ip t

Prize in Physics endowed to professor Geim and Novoselov in 2010 [7]. GO, oxygenated derivative of graphene, is a very vital intermediate and precursor

cr

compound in the process for chemically preparing graphene and graphene-based

us

composite materials, respectively [8-9]. The key towards the successful application of graphene materials lies in its modification and/or integration into high quality

an

composite nanomaterials [10-13]. This is firstly because graphene has a zero band gap which can be used to exploit the non-zero band gaps in different inorganic graphene

M

analogues to influent the state-of-the-art composites for electronic applications [14-15]. Secondly, it is possible to categorize GN-based nanomaterials in terms of

ed

their dimensions in the x-y-z axes because such limitations in the different axes

ce pt

significantly influence the electronic properties of GN [16]. The third reason is the possible environmental impacts [17]. To overcome those obstacles, the incorporation of polymer and/or nanostructured metal catalysts continues to be to be a significant research focus [19-20].

Ac

It is known that common Pt catalysts usually suffer from several disadvantages, such as high cost, easily being poisoned by intermediate species and kinetic limitation of the oxygen reduction [21-24]. It was reported that such Pt-based nano-catalysts modified GO not only maximize the availability of nano-sized electro-active surface area for electron transfer but also provide better mass transport of reactants to the electro-catalyst [25-27]. It was used in fuel cells and sensors for DA, ascorbic acid (AA), UA and acetaminophen (AP) [28-29]. As the nanotechnology developed, 3

Page 5 of 72

compared with homologous monometallic counterparts, the bimetallic-NP-based catalysts such as novel Pt-Pd [30], Pt-Ag [31], Pt-Fe [32], Pt-Fe3O4 [33], Pt-Sn [34], Pt-Au [35] and Pt-Cu [36] modified GO often show superior catalytic activity from the synergistic effects of both moieties.

ip t

Pt and Au, which were used to fabricate bimetallic nano-clusters GO are based on the following two major factors. Firstly, pure metals (including Pt or Au) show

cr

unsatisfactory sensitivity, poor selectivity and easy poisoning by adsorbing

us

intermediates, which are critical issues for practical applications [37-38]. Secondly, the literatures [39-40] indicate that excellent elctrocatalytic activity and high

an

sensitivity of Pt can be enhanced by alloying with Au. The Pt-Au nanocomposites modified GO were reported in some literatures, which was synthesized by chemical

M

reactions and then anchored onto GO by physical attentions [41-42]. Controlling the folding, crumpling and bending were the main problems in chemical functionalization

ed

of GO-based materials [43-44]. But the electrochemical synthesis of catalysts is very attractive because such NPs nucleate at the electro-active sites [45-46] and a

ce pt

promising approach to reversibly tune 2D GO electronic properties. However, one-step electrodeposition of Pt-Au bimetallic nano-clusters loaded onto graphene or GO as electrocatalyst have not been reported in details.

Ac

The electrochemical detection of DA and UA has been investigated on graphene

or GO-based in some papers [47-53], but the necessity of continuing further studies with an intention to attain lower detection limits exists now [54-60]. And one-step electrodeposition of Pt-Au bimetallic nano-clusters loaded onto GO for simultaneous DA and UA has not been reported. In this research, the nanocomposites composed by Au-Pt hybrid bimetallic nanoclusters anchored at GO-ERGO was achieved by a simple electrochemical 4

Page 6 of 72

reduction process, which was simply as “Au-Pt/GO-ERGO”. The main aim of recent work, therefore, is to enhance the sensitivity/selectivity of the sensor by investigating more active and lower cost replacements for pure Pt and graphene, hence the interest in bimetallic systems, which bring interesting physical and chemical properties into

ip t

effect from the inter metallic combinations of different metals.

cr

2. Experimental

us

2.1. Materials

All reagents and materials were of analytical grade. All the solutions were prepared

an

using double-distilled water. GO was purchased from Nanjing Xian Feng Nano Technology Co., Ltd. (Nanjing, China). Chloroplatinic acid (H2PtCl6∙6H2O) and

M

chloroauric acid (HAuCl4∙6H2O) was from Sigma Chemical Co. China. DA and UA

ed

were obtained from Fluka Co. Phosphate buffer solutions (PBS, 0.2 M) with different pH values were prepared by mixing the standard stock solutions of Na2HPO4 and

ce pt

NaH2PO4 by adjusting pH with 1.0 M H3PO4 or NaOH. A recovery study was conducted utilizing healthy human serum without any pretreatments and dilution. 2.2. Instruments

Ac

All electrochemical measurements were performed with a CHI 660 electrochemical workstation (CH Instruments Co., USA). The electrochemical cell consisted of a three electrode system with a modified GCE (3 mm in diameter) as a working electrode, a platinum wire as a counter electrode and a saturated calomel electrode (SCE) as a reference electrode. The operation parameter of cyclic voltammetry (CV) was from -0.1 to +0.6V at the scan rate of 50 mV s-1 with the quiet time of 120 s. The operation parameter of differential pulse voltammetry (DPV) was from -0.1 to 0.6 V at the 5

Page 7 of 72

amplitude of 50 mV with the quiet time of 120 s. The operation parameter of chronoamperometry was from 0.0 to 0.3V for DA and from 0.1 to 0.4V for UA. All solutions were deoxygenated by bubbling highly pure nitrogen for at least 15 min and a nitrogen atmosphere was maintained during the measurements. Scanning electron

ip t

microscope (SEM) was performed in the secondary electron imaging model with the 20 KeV working voltage, 3mm laser spot diameter and different magnification times

cr

by Quanta 250FEG-Field emission environmental scanning electron microscope (FEI

us

Ltd., USA). Energy spectrum analysis (EDS) was performed in the secondary electron imaging model with the 20 KeV working voltage, 3mm laser spot and 1250

an

magnification times by Apollo-40 EDS (Edax, Ltd., USA). Electrochemical impedance spectroscopy (EIS) measurements were carried out at open circuit

M

potentials and were performed in 5.0 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) mixture containing 0.10 M KCl with the Autolab 302N electrochemical working station

ed

(Metrohm China Co. Ltd., Switzerland), and a sinusoidal potential modulation with an amplitude of ±5 mV and a frequency from 105 to 1.0 Hz was superimposed on the

ce pt

formal potential of the Fe(CN)63-/4-redox couple at 0.17 V vs SCE. 2.3 Preparation of modified electrode

Ac

The bare GCE with 3 mm diameter was polished to a mirror using 1.0, 0.3, and 0.05 μm alumina slurry and sonication in 1:1 nitric acid, acetone, and deionized water As shown in Scheme 1, 5μL of 0.5 mg/mL-1 GO solution was dropped on the GCE and dried in air to form “GO/GCE”. After electrochemical reduction of GO at -1.0 V in pH 8.0 PBS, the modified electrode (“GO-ERGO/GCE”) was washed with deionized water. To electrochemically deposit Au-Pt nano-clusters, the modified GCE was immersed into an electrolyte consisting of 1.0 mM HAuCl4 and 1.0 mM H2PtCl6 with minor modifications [61]. Au-Pt nano-clusters electrodeposition was performed 6

Page 8 of 72

by CV with a potential range of -0.1 to -0.9 V for 20 circles at scan rate of 50 mV s-1 to form “Au-Pt/GO-ERGO” modified GCE. For comparison, Au nanoparticles (NPs) and Pt NPs were synthesized under similar conditions in 1.0 mM HAuCl4 or 1.0 mM H2PtCl6.

cr

ip t

(Scheme1 should be placed here)

us

3. Results and discussion 3.1 Characterizations of Au-Pt/GO-ERGO

an

3.1.1 UV-Vis characterizations

M

Fig.S1. shows the UV-vis spectrum of the Au-Pt nano-clusters (curve a), Au NPs (curve b) and Pt NPs (curve c) modified ITO. The spectrum of Au NPs presents a

ed

strong peak with maximum absorption wavelength of 526nm, while the absorption peak for Pt NPs was not discovered from 300 to 800 nm. The broad absorption peak

ce pt

appeared at the maximum wavelength of 430 nm (curve a) for Au-Pt nano-clusters modified ITO. The blue shift was found when Au-Pt nano-clusters were formed, because the sizes and elements of Au-Pt nano-clusters were different from Au NPs

Ac

[60, 62 and 63].

3.1.2 Surface topography characterizations Fig. 1A showed the SEM images of GO-ERGO modified ITO and a typical 2D network just like silk was observed in the images. Fig.1B and Fig.1C showed the Au and Pt NPs. Fig.1D and Fig.1E showed that Au-Pt NPs assembled together and formed the cluster. Au-Pt nano-clusters on GO-EGRO modified GCE appeared to be more well-dispersed and dense. With the participation of GO, the deposited metal NPs 7

Page 9 of 72

show better dispersion, in good agreement with the literature [51-53]. It has been confirmed that GO or ERGO, which could be physico-chemically and structurally regarded as unconventional polymeric surfactant structures, may act as capping agent or stabilizer to disperse and hamper the growth of the NPs [21]. Also, the remaining

ip t

oxygen-containing groups of GO could provide binding sites for anchoring precursor metal ions or metal NPs [22]. The energy spectrum analysis (EDS) in Fig S2 results

cr

the existence of Au element and Pt element in Au-Pt/GO-ERGO.

an

3.1.3. Charge transfer resistivity characterizations

us

(Figure 1 should be placed here)

EIS is a powerful and facile electrochemical technique to monitor the changes of

M

interfacial properties in the assembly processes of the modified electrodes step by step [28, 29]. EIS of the bare GCE was shown as “curve f” in Fig.4. When GO-ERGO

ed

(curve d) was formed, the diameter of semicircles was lower than that of GO modified GCE (curve e). When Au-Pt, Pt and Au (curve a, b and c) nanomaterials were

ce pt

immobilized on GO-ERGO, the diameters of semicircles were obviously lower than that of GO-ERGO (curve d). The electron transfer resistance values for Au/GO-ERGO (curve c), Pt/GO-ERGO (curve b), and Au-Pt/GO-ERGO (curve a)

Ac

decorated on the GCEs were 308, 247, and 198Ω, respectively. These data indicated that nanomaterails play an important role similarly to a conducting wire or electron-conducting tunnel [29]. (Figure 2 should be placed here)

3.1.4. Redox probe characterizations Fig. 3A displays cyclic voltammograms of the GO, GO-ERGO, Pt/GO-ERGO, 8

Page 10 of 72

Au/GO-ERGO and Au-Pt/GO-ERGO modified GCE in 0.1 mM ferricyanide at the scan rate of 50 mV/s. The values of peak separation (ΔEp) for GO-GCE (curve a), GO-ERGO/GCE (curve b), Pt/GO-ERGO/GCE (curve c), Au/GO-ERGO/GCE (curve d) and Au-Pt/GO-ERGO (curve b) for exhibiting CV response for [Fe(CN)6]3-/4-were

ip t

204, 150, 100, 90 and 67 mV. This indicated that a good electronic communication was achieved between Au-Pt/GO-ERGO and the underlying GCE and the studied

cr

process was approximately reversible, which was similar as the properties of some

us

other excellent NPs [64-65].

Fig. 3B showed a series of CVs for Au-Pt/GO-ERGO modified GCE in 0.1 mM

an

redox probe with different scan rates. The peak current (Ip) increases linearly with the increasing of the square root of the potential scan rate (v1/2), which suggests that the

M

mass transfer phenomenon in the double layer region of the electrodes is mainly diffusion controlled [66]. Under semi-infinite linear diffusion conditions and a

ce pt

i p  kAn3/ 2 AD1/ 2C 1/ 2

ed

reversible reaction, the Randles-Sevcik equation [67] was described by:

ip refers to the peak current in ampere, A is the electrode surface area in cm2; n is the number of electrons; D is diffusion coefficient of electro-active materials; Co is bulk concentration electro-active materials in cm2 s-1, and v is the scan rate in V s-1. For

Ac

potassium ferricyanide, n is 1; D is 7.6×10-6cm2 s-1; Co is the concentration. The slope of ip versus v1/2 plot was 2.78×10-6 (R2= 0.9997), and then used to calculate the effective working surface area. The value of A calculated for Au-Pt/GO-ERGO, Pt/GO-ERGO and Au/GO-ERGO modified GCE is 10.01, 5.55 and 4.00 cm2, respectively.

It

was

investigated that

the electro-active surface area

of

Au-Pt/GO-ERGO modified GCE was 2 times larger than that only Pt or Au modified GCE. 9

Page 11 of 72

Nicholson has reported [41] that CV has been extended to include electron transfer reactions described by the electrochemical rate equation. Results of theoretical calculations made it possible to use CV to measure standard rate constants for electron transfer-ks. It means a system, which appears reversible at one frequency,

ip t

may be made to exhibit kinetic behaviour at higher frequencies, as indicated by increased separation of cathodic and anodic peak potentials. ks can be understood very

cr

simply as a difficulty level to dynamics of redox probe. The system with higher ks

us

means it can be balanced by shorter time. Oppositely, it can be used for a longer time to balance when the electrochemical system hold a lower ks. The ks is determined from

an

this peak potential separation and frequency. The ks provides an extremely rapid and simple way to evaluate electrode kinetics as Nicholson theory:

M

( D / DR ) a / 2 ks  [ D (nF / RT )]1/ 2

ed

The value of ψ for [Fe(CN)6]3-/4- redox couple from the Ref [67] was 1. n is the number of electrons; α is transfer coefficient; D is diffusion coefficient of

ce pt

electro-active materials; v is the scan rate in V s-1. Under these conditions of quasi-reversibility, it may be possible to study the kinetics of the electrode reaction,

Ac

and the separation of the peak potentials (△Ep), should be a measure of the standard rate constant for electron transfer. For 1 mM K3Fe(CN)6 at 298 K, n = 1, D= 7.6 × 10-6 cm2 s-1, ψ= 0.5, v is scan rate of 0.05 V s-1. When △Ep = 69 mV, ψ= 3; when △Ep = 90 mV, ψ= 0.75; when △Ep = 100 mV, ψ= 0.5; The ks value for Au-Pt/GO-ERGO, Pt/GO-ERGO, Au/GO-ERGO, GO-ERGO and GO immobilized on GCE is calculated 4.3, 2.1, 0.99, 0.76 and 0.55 cm s-1. It indicated that a good electronic communication was achieved between Au-Pt/GO-ERGO and the underlying GCE and the studied 10

Page 12 of 72

process was approximately reversible. (Figure 3 should be placed here) 3.2 Electrochemical parameters of DA and UA at Au-Pt/GO-ERGO modified GCE

ip t

3.2.1. The number of electrons Fig.4. presents the cyclic voltammograms of DA and UA at the Au-Pt/GO-ERGO in

cr

pH 7.0 PBS at different scan rates in presence of 32.3 μM DA (shown in Fig.4A ) and

nFQv 4 RT

an

ip 

us

96.9μ M UA (shown in Fig.4C). According to the equation [67]:

ipa is on behalf of anodic peak current and ipc is on behalf of cathode peak current; n is

M

the number of electrons; Q is electric quantity; F is faraday constant; T is the thermodynamic temperature. At the condition of 297K, from Fig.4, ip increased with

ed

the increasing of scan rates either. It was observed that value of E0’ in the scan range

ce pt

were almost independent on the scan rates, because the values of Epa and Epc remained almost unchangeable with scan rates. As shown in Fig. 4B, ipa (DA, A) = -6.1638×10-6+6.45905×10-7v (mV s-1) with R2 = 0.999, and ipc (DA, A) = 5.52148×10-6-4.25688×10-7v (mV s-1) with R2=0.999. As shown in Fig. 4D, ipc (UA, A)

Ac

= 4.96652×10-6+2.62438×10-7 v (mV s-1) with R2= 0.998. The anodic and cathodic peak currents were linearly proportional to scan rates, which indicate the electro-catalytic behaviors of DA and UA are surface electron transfer processes [67]. From the slope of linear regression equation, n is calculated to be 2.02 for DA and 2.13 for UA. Therefore, the electro-catalytic behaviors of DA and UA on the modified electrode are considered to be two electrons involved. (Figure 4 should be placed here) 11

Page 13 of 72

3.2.2. Electro diffusion coefficient (D) Chronoamperometry could be used for investigation of electrode processes at chemically modified electrodes. Fig. 5 shows the well defined chronoamperograms

ip t

obtained from Au-Pt/GO-ERGO immobilized on GCE in 0.2 M pH 7.0 PBS with

cr

various concentrations of DA (Fig.5A) and UA (Fig.5C). The Cottrell equation [67-68] is aimed to calculate a diffusion coefficient (D) by investigating the relation between

us

the currents for the electrochemical reaction and the concentrations, which is

an

described as:

M

nFAD1/ 2 c I  1/ 2 1/ 2  t

C is the bulk concentration (mol cm-3) (the electro-active material as DA and UA in

ed

the research). In Fig. 5 of chronoamperometric studies, the slopes of the resulting straight line were then plotted versus the DA (Fig.5B) or UA concentration (Fig.5D).

ce pt

From the slope ofFig.5B and Fig.5D, we calculated D of 8.2×10-5 cm2 s-1 for DA and 4.2×10-6 cm2 s-1 for UA, which was a little different from the references [68-69]. Diffusion coefficient (D) is the physical properties of the material, which respected its

Ac

diffusion capacity. According to Fick’s law [69], D is the amount or the mole number, which the materials vertically diffuse in the condition of unit concentration gradient per the unit area and unit time along the direction of diffusion. The value of D mainly depends on the types of materials, diffusion medium, temperature and pressure. Generally speaking, the value of D is detected by experimental measurement. According the above discussion, the D value is a little different from the reference [68]. The reason may be that diffusion medium in our research is different from the

12

Page 14 of 72

reports. The diffusion medium in this research is pH 7.0 PBS, and the other supporting electrolytes were acetate buffer solutions [68-69]. (Figure 5 should be placed here) 3.2.3. Effect of the supporting electrolyte and pH values

ip t

The PBS was found to induce the best response after comparing the

cr

electrochemical behaviors in four different buffer solutions (B-R buffer solution, acetate buffer solution, Tris and PBS). As an important factor, pH values influenced

us

determination of DA and UA directly. Thus, the effect of pH on the voltammetric

an

response of DA and UA were studied over the pH range from 5.0 to 8.0.An increase of pH in solution led to a negative shift in the reduction potential peaks. A shift of

M

“-60.9 mV/pH” of DA was observed in the pH from 5.0 to 8.0. Since the number of electrons was 2 as determined in Section 3.2.1, the number of hydrogen ions involved

ed

in the electrode reaction is 2. A possible scheme for electrochemical oxidation of DA

ce pt

was thus proposed as below

A shift of “-68.7mV/pH” of UA was observed in the pH from 5.0 to 8.0. Since the

Ac

number of electrons was 2 as determined in Section 3.2.1, the number of hydrogen ions involved in the electrode reaction is 2 for UA. A possible scheme for electrochemical oxidation of UA was thus proposed as below

The possible scheme for electrochemical oxidation of DA and UA were similar as the refs [70-73]. The PBS 7.0 was chosen as supporting electrolyte because it was the 13

Page 15 of 72

same as human fluid condition. 3.3 DA and UA detection The excellent quiet time of detecting DA and UA, the experiment was carried at the technology of DPV as Fig.S3 when after injection of highest concentration of analyte.

ip t

From Fig.S3, we found with the increasing of quite time, the current was increasing

us

choose 120s as the excellent quiet time of this research.

cr

either. At the quiet time of 120 s, the current was nearly at the stable condition. So we

Fig. 6A was shown to illustrate that the anodic peak currents of DA are obviously

an

increased with the increase of the DA concentrations. From Fig.6B, the linear regression equation of DA detection was Ipa (μA) = -23.94-1.238CDopamine (μM) with a

M

correlation of 0.997 (n=8) and the range of linearity was from 6.82×10-2 μM to 4.98×10-2 M. Limit of detection (LOD) refers the corresponding amount at the three

ed

times of sensitivity (S) in the background signals (N) produced by the instruments at the matrix blank (S/N=3), which is one of the important indicators which reflected the

ce pt

sensitivity of instruments and methods. S was calculated from the slope of linear regression equations. N was the instrumental signal of matrix blank, which was calculated as the standard deviation obtained by detecting the matrix blank for 10

Ac

times. The LOD of DA at the Au-Pt/GO-ERGO modified GCE in 0.2 M pH 7.0 PBS was 2.07×10-2 μM at 3 folds of the signal-to-noise ratio (S/N=3) from the above discussion.

As similar trends shown in Fig. 6C and Fig. 6D for UA detection, the linear regression equation is Ipa (μA) = -2.820-0.6913Curic acid (μM) with a correlation of 0.998 (n=8). The range of linearity for UA was from 0.125 μΜ to 8.28×10-2 M and LOD was 4.07×10-2 μM. 14

Page 16 of 72

(Figure 6 should be placed here) 3.4 Discussion of Au-Pt/GO-ERGO as a novel nanomaterial for constructing sensors Fig.7 was aimed at illustrating electrochemical behaviors of the mixture of DA and UA on the Au-Pt/GO-ERGO modified GCE. At the bare GCE (curve f), only one

ip t

oxidation peak was observed at near 325 mV, and the peak potentials of DA and UA

cr

were indistinguishable, indicating poor selectivity and sensitivity. In contrast, two

anodic peaks related with oxidation of DA and UA were observed on five different

us

nanomaterials modified GCEs (other curves). The oxidation currents were weaker and

an

the peak was wider on GO/GCE (curve e). The peak current at the GO-ERGO (curve d) modified GCE was slightly increased. When Au NPs (curve c), Pt NPs (curve b),

M

and Au-Pt nano-clusters (curve a) anchored onto GO-ERGO, the peak currents were increased to different extends. Among those, Au-Pt/GO-ERGO modified GCE were

ed

remarkably enhanced. The oxidation peak potentials of DA and UA located at near 230mV and 355 mV, and the peak separations for DA and UA were 182 mV,

ce pt

respectively, which indicated that the Au-Pt/GO-ERGO showed a good catalytic activity for the oxidation of DA and UA. The superior electrochemical performance of the modified electrode could be attributed to the fact that there were significant

Ac

edge-plane-like defective sites existing on the surface of Au-Pt/GO-ERGO.

GO,

graphene

(Figure 7 should be placed here) decorated

with

randomly

distributed

oxygen-containing

functionalities on both sides of the plane, has also been widely reported as a biosensor material due to its high chemical and electrochemical activity. These oxidized areas on the GO plane break the long-range conjugated network and π-electron cloud, leading to a deg radation of carrier mobility and conductivity [73-74]. The reduced

15

Page 17 of 72

GO (rGO) [75] is believed to be one of candidates due to its reasonably reduced number of functionalities, a large number of remaining electro-active sites and the structural similarity with natural graphene. There have been reports of chemically reduced GO by ultra-high vacuum heat [76], chemical treatments (with

ip t

hydrazine/hydrazine derivatives [77] or high-concentration alkaline solution [78], which may lead some disadvantages such as considered to be dangerous substances

cr

labeled and required special equipments. A promising, green, efficient, inexpensive

us

and rapid method based on electrochemistry to produce electrochemically reduced graphene oxide (ERGO) has been introduced [79-80]. Moreover, due to the stability

an

of film on the electrode surface, time saving of fabrication and more excellent physic-chemical properties than pure GO and pure ERGO, the GO-ERGO

M

nanocomposites was reported in recent time [81-82]. Herein, we report a simple, efficient, low-cost and environmentally friendly electrochemical method to

ed

GO-ERGO nanocomposites with mini modification according the references [81-83]. In this research, ERGO was obtained by electrochemical reduction of partly GO

ce pt

immobilized on GCE surface operated in electrolytic cell. In other words, the part of GO was reduced into ERGO and formed to be the novel nanomaterials: GO-ERGO nanocomposites. The electrochemical property of GO-ERGO compared to pure GO

Ac

was explored at the aspects of EIS (Fig.2, curve d), redox probe (Fig.3A, curve b), ks (Section 3.1.4) and the electrochemical response of DA and AA (Fig.7, curve d). From

the

above

discussion,

we

found

the

GO-ERGO

has

higher

chemical/electrochemical activity and bigger surface area of electro-active sites, which was more superior performance than pure GO. GO, not only provides a substrate

to

anchor

Au-Pt

bimetallic

nano-clusters,

but

also

provides

oxygen-containing functional groups of DA and UA electrochemical-oxidation. 16

Page 18 of 72

From Fig. 7 and Table 1, the linear range of DA and UA was wider and the LOD was lower on Au-Pt/GO-ERGO than on Au, Pt, graphene and other similar nanomaterials. The reason may be that the chemical reaction rate depends on the rate which the reactants adhere to the catalyst surface. When the 3D or porous catalyst

ip t

could increase the specific surface area, the reaction was more easily occurred on the catalyst surface adhesion to accelerate the chemical reaction speed. As illustrated in

cr

3.1.4., Au-Pt nano-clusters could provide larger electrochemically active surface area

us

than that of Au and Pt NPs. From Section 3.1.2 (SEM), Au-Pt nano-clusters could make the film more porous for facilitating electron transfer, which facilitates the

an

adsorption of detection molecules. Pt, as excellent catalyst, was higher prices and the global reserves are relatively small. Recently, a number of studies have shown that the

M

catalytic properties of noble metal catalyst can be added to the second nanoporous metal materials [89-91]. They could provide larger electrochemically active surface

ed

area, which would be benefit of improving electron transfer rates for detection

ce pt

molecules.

(Table 1 should be placed here)

From the above discussion, we found this modified GCE has the wider line range and lower LOD of detecting DA and UA compared with that of other sensors reported

Ac

recently. And as a hot-topic of carbon material, Au-Pt/GO-ERGO showed excellent physical, chemical and electronic properties as well as unusual charge-carrier mobility as the above investigation. Compared with pure graphene or GO modified GCE, Au-Pt/GO-ERGO in terms of their dimensions in the x-y-z axes significantly enhances the electronic properties [14, 15, and 17]. Compared with other metal anchored GO, Au-Pt/GO-ERGO not only maximize the availability of nano-sized electrocatalyst surface area for electron transfer but also provide better mass transport 17

Page 19 of 72

of reactants to the electrocatalyst [31-34]. Compared with other chemical methods to synthesize Pt/Au grappled on the GO [41-42], one-step electrodeposition of Pt-Au bimetallic nano-clusters loaded onto GO has not been reported, and electrochemical method owned the sufficient advantages, such as low cost, green and environmental

ip t

protection, saving time and ease to control the experiment conditions.

cr

3.5. The analyzing of real samples, interference test, stability and reproducibility

practicability of Au-Pt/GO-ERGO modified GCE.

us

The tests were carried out in real samples such as human serum to illustrate the

an

Human serum was obtained from human whole blood by precipitation and centrifugation at 3000 rotations per minute for 10 minutes at 4 °C to remove red cells

M

and other interfering proteins. The concentration of DA in serum is not stable in the high nano-molar to low micro-molar range in healthy individuals, which was the same

ed

in their urine and saliva [92]. The concentration of DA has been reported as 21.8 ± 9.5 ng/Lin healthy serum [92-94]. It means the normal level of DA in healthy serum is

ce pt

about 1.15±0.05×10-10 M, which was lower than the limit of detection (LOD) of this research for DA. Just as some literatures was reported, DA was no found in the serum and the real sample detecting was based on the spiked sample analysis [95-96]. The

Ac

normal lever of UA in serum was from 149 to 445 mM [95-98], and UA can be detected in this proposed method according the LOD. The dilution folds of detecting real samples were an important factor. The diluted folds of serum were investigated by recovery experiments just as show in Table S1. From the Table S1, we found the recovery of detecting DA and UA was better than that of undiluted samples. The reason was probably the interference of other complicated

components

of

the

blood

serum,

e.g.

ascorbic

acid

and

18

Page 20 of 72

5-hydroxytryptamine (5-HT) [92]. The main reason was properly when the serum was diluted by buffered solution, the concentration of interfering substance decreased gradually by the dilution folds, which reduced the interfering of recovery experiments [99]. In contrast to the sensor previously reported [100], the optimized assay was able

ip t

to detect endogenous DA or UA in un-diluted serum samples. But some literature has suggested that larger peaks which displayed higher DA or UA concentrations were not

cr

linear in the whole sample without or less dilution [99, 101-102]. These reported

us

suggested that interference due to serum complicated composition (especially ascorbic acid) was proportionally higher for less diluted samples, which was in

an

accordance with the results described by other authors. But in the current electrochemical assay, serum samples were processed and analyzed straightforward,

M

without any additional pretreatment. The high concentration of UA in serum, detected by uricase method which was authorized in clinical analysis standardization, was

ed

determined by adding ascorbic acid oxidase to eliminate AA interfering [103]. The detection by established methodologies, such as chromatography and capillary

ce pt

electrophoresis, is usually preceded by sample pretreatment (e.g. microdialysis, acidification, cation exchange purification, chemical derivatization, etc.)[104-105]. In this experiment, the best and acceptable results were obtained for serum diluted

Ac

1:10 (PBS 7.0) with the averaged DA recoveries (%) of 99.6 ±0.84 and the averaged DA recoveries (%) of 102.6 ±0.71. So, the supernatant from whole blood was diluted by 1:10 with pH 7.0 PBS as serum samples and then were detected in the electrochemical cell. The DPV responses of human serum were shown in Fig.S5 at 1:10 dilution and the experiments were repeated for three times (n=3) and the recovery experiments were analyzed as Table 2. The recoveries in the range from 98.4% to 103.9% and 101.1% to 104.2% were attained for DA and UA, respectively. These 19

Page 21 of 72

results indicate the prospective application of the developed sensor for simultaneously detecting DA and UA in real samples.

(Table 2 should be placed here)

ip t

In addition, there is a systematic error in the recovery experiment (more than 100 recovery of UA) from Table S1 because of the interference of other complicated

cr

components of the blood serum (e.g. ascorbic acid or 5-hydroxytryptamine (5-HT)

us

[98, 92 and 101]). Human serum is a very complex mixture by removal of fibrinogen from plasma. The large part of compositions is known by science studies, but there

an

are part of them is not clear. The composition and contents in human serum were affected by sex, age, nutrient condition, drugs intake and other physical conditions

substance to be examined [98].

M

obtained from different people. Some interfering components may influence the

ed

Several coexisting compounds were selected to evaluate the ability of anti-interference. In Fig.S4, we investigated the CV (Fig.S4A) and DPV (Fig.S4A) of

ce pt

200 μM ascorbic acid (AA), 80.00 μM DA, 80.00 μM UA, 200 μM acetaminophen (AP), 200 μM epinephrine, 200 μM norepinephrine, 200 mM nitrite, 200 mM H2O2, 200 mM NaCl, 200 mM KCl, 200 mM KNO3, 200 mM Na2SO4, 200 mM ZnCl2, 200

Ac

mM CaCl2, 200 mM citric acid, 200 mM glucose, 200 mM bovine serum albumin, 100 mM immunoglobulin, and 200 mM hemoglobin. From the In Fig.S4, the peak potentials of AA and AP was very different from that of DA and UA, and other significant interference had no responses at the Au-Pt/GO-ERGO modified GCE. The stability of the Au-Pt/GO-ERGO modified GCE was also investigated. The modified GCE was stored at 4 °C in a refrigerator for 7 days and then carried in the recovery experiment. The recovery experiment was carried out at the technology of 20

Page 22 of 72

CV in 7.0 PBS, which parameter was from -0.1 to +0.6V at the scan rate of 50 mV s-1 for 20 sweep segments. It would take up for 280 s. The response of Au-Pt/GO-ERGO modified GCE to DA and UA lost 5.3% and 4.8% of its original response after storage for 7 days, respectively. The reproducibility of the proposed sensor was tested using

ip t

eight different electrodes. The relative standard deviations (RSD) of the DPV response currents for these species were less than 5.8%. Thus, the modified electrode

us

cr

showed a high stability and good reproducibility and anti-interference ability.

4. Conclusion

an

Significantly enhanced catalyticactivity of nanocomposites composed by Au-Pt hybrid bimetallic nano-clusters was achieved through a simple electrochemical

M

reduction process anchored at GO-ERGO modified GCE. Synergistic electrocatalytic effect of in-situ electrodeposited Au-Pt bimetallic nano-clusters and GO for detection

ed

of dopamine and uric acid was investigated. Based on those researches, it is believed

ce pt

that for the Au-Pt/GO-ERGO nanocomposites, GO-ERGO could provide much wider separation of the oxidation peak potentials for simultaneously detecting DA and UA while Au-Pt hybrid bimetallic nanocrystals could speed up the electron transfer for increase the sensitivity. This investigation suggested that 3D metal-graphene oxide

Ac

nanocomposites were superior for the fabrication of novel electrochemical sensors. Acknowledgments

The authors thank the National Natural Science Foundation of China (No. 21075087, 21175097 and 81202249) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions for financial support of this study.

21

Page 23 of 72

References

Ac

ce pt

ed

M

an

us

cr

ip t

[1]K.S. Novoselov, A.K. Geim, S.V.Morozov, D. Jiang, Y. Zhang, S.V. Dubonos, I.V. Grigorieva, A.A.Firsov, Electric field effect in atomically thin carbon films, Science 306 (2004) 666-669. [2]A.K.Geim, K.S.Novoselov, The rise of graphene, Nature Mater. 6 (2007) 183-191. [3]K.S. Novoselov, A.K. Geim, S.V. Morozov, D. Jiang, M.I. Katsnelson, I.V.Grigorieva, S.V. Dubonos, A.A. Firsov, Two-dimensional gas of massless Dirac fermions in graphene, Nature438 (2005) 197-200. [4]A.K.Geim, Graphene: Status and Prospects, Science324 (2009) 1530-1534. [5]X.Li, X. Wang, L. Zhang, S. Lee, H. Dai, Chemically derived, ultrasmooth graphene nano ribbon semiconductors, Science 319 (2008) 1229-1232. [6]R.R.Nair, P. Blake, A.N. Grigorenko, KS. Novoselov, T.J. Booth, T. Stauber, N.M. Peres, A.K. Geim, Fine structure constant defines visual transparency of graphene, Science 320 (2008) 1308-1308. [7]The Nobel Prize in Physics 2010. Nobelprize.org, The Nobel Prize in Physics 2010. Andre Geim, KonstantinNovoselov.http://www.nobelprize.org/nobelprizes/physics/laureates/2010/(accessedSeptember 2012). [8]S. Stankovich, D.A.Dikin, R.D.Piner, K.A. Kohlhaas, A.Kleinhammes, Y. Jia, Y. Wu, S.T.Nguyen, R.S.Ruoff, Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide, Carbon 45 (2007) 1558-1565. [9]S.Stankovich, D.A. Dikin, G.B. Dommett, K.M. Kohlhaas, E.J. Zimney, E.A. Stach, R.D. Piner, S.T. Nguyen, R.S. Ruoff, Graphene-based composite materials, Nature442(2006) 282-286. [10]K.S. Novoselov1, V.I. Falko, L. Colombo, P.-R. Gellert, M.G. Schwab, K. Kim, A roadmap for graphene, Nature 490 (2012) 192-200. [11]M.-H. Chakrabarti, C.T.J. Low, N.P. Brandon, V. Yufit, M.A. Hashim, M.F. Irfan,J. Akhtar, E. Ruiz-Trejo, M.-A.Hussain,Progress in the electrochemical modification of graphene-based materials and their applications, Electrochim. Acta 107 (2013) 425-440. [12]V.V. Singh, G. Gupta, A. Batra, A.K. Nigam, M. Boopathi, P.K. Gutch, B.K. Tripathi,A. Srivastava, M. Samuel, G.-S. Agarwal, B. Singh, R. Vijayaraghavan, Green erelectrochemical synthesis of high quality graphene nanosheets directly frompencil and its SPR sensing application, Adv. Funct. Mater. 22 (2012) 2352-2362. [13]C.T.J. Low, F.C. Walsh, M.H. Chakrabarti, M.A. Hashim, M.A. Hussain, Electrochemical approaches to the production of graphene flakes and their potential applications, Carbon 54 (2013) 1-21. [14]C.J. Shih, Q.H. Wang, Y. Son, Z. Jin, D. Blankschtein, M.S. Strano, Tuning on-off current ratio and field-effect mobility in a MoS2-Graphene heterostructure via schottky barrier modulation, ACS Nano 8 (2014) 5790-5798. [15]H. Bu, M. Zhao, A. Wang, X. Wang, First-principles prediction of the transition from graphdiyne to a superlattice of carbon nanotubes and graphene nanoribbons, Carbon65 (2013)341-348. [16]S.A.Jensen, R. Ulbricht, A.Narita, X. Feng, K. Müllen, T.Hertel, Ultra fast photoconductivity of graphene nanoribbons andcarbon Nanotubes, Nano Lett. 13 (2013) 5925-5930. [17]K.S. Kim, Y. Zhao, H. Jang, S.Y. Lee, J. M. Kim, K.S. Kim, J.H. Ahn, P.Kim, J.Y. Choi, B.H. Hong, Large-scale pattern growth of graphene films for stretchable transparent electrodes, Nature 457 (2009) 706-710. [18]D.R. Dreyer, S.Park, C.W. Bielawski, R.S. Ruoff, The chemistry of graphene oxide, Chem. Soc. Rev. 39 (2010) 228-240. [19]H. S.Han, H. Seol, D. H, Kang, M. S. Ahmed,J.M. You, S. Jeon, Electrochemical oxidation and determination of dopamine in thepresence of AA using ferulic acid functionalized electrochemicallyreduced graphene, Sens. Actuators, B 204 (2014) 289-296. [20]C.Wu, H. Zhang, Y.X. Wu, Q.C. Zhuang, L.L. Tian, X.X. Zhang, Synthesis and characterization of Fe@Fe 2O3 core-shell nanoparticles/graphene anode material for lithium-ion batteries, Electrochim. Acta 134(2014) 18-27. [21]C. Xu, X. Wang, J. Zhu, Graphene-metal particle nanocomposites, J. Phys. Chem. C 112 (2008) 19841-19845. [22]Y. Li, W. Gao, L. Ci, C. Wang, P.M. Ajayan,Catalytic performance of Pt nanoparticles on reduced graphene oxide for methanol electro-oxidation, Carbon48 (2010)1124-1130. [23]J. Zhang , J. Yu, M. Jaroniec , J.R. Gong, Noble metal-free reduced graphene oxide-ZnxCd1-xS nanocomposite with enhanced solar photocatalytic H-2-production performance, Nano let. 12(2012) 4584-4589. [24]H.Wang, S. Li, Y. Si,N. Zhang,Z. Sun,H. Wu,Y. Lin, Platinum nanocatalysts loaded on graphene oxide-dispersed carbon nanotubes with greatly enhanced peroxidase-like catalysis and electrocatalysis activities, Nanoscale 6 (2014) 8107-8116. [25]E.J. Yoo, T. Okata, T. Akita,M. Kohyama, J. Nakamura,I. Honma, Enhanced electrocatalytic activity of Pt subnanoclusters on graphene nanosheet surface, Nano Lett. 9 (2009) 2255-2259. [26]R.Kou, Y. Shao, D.Wang, M.H. Engelhard, J.H. Kwak, J. Wang,V.V. Viswanathan , C. Wang, Y. Lin, Y. Wang, I.A. Aksay, J. Liu, enhanced activity and stability of Pt catalysts on functionalized graphene sheets for electrocatalytic oxygen reduction, Electrochem. Commun. 11 (2009) 954-957. [27]R. Kou, Y.Shao, D. Mei, Z. Nie, D. Wang, C. Wang,V.V Viswanathan, S. Park,I.A. Aksay,Y. Lin,Y. Wang, J. Liu, Stabilization of electrocatalytic metal nanoparticles at metal-metal oxide-graphene triple junction points, J. Am. Chem. Soc. 133 (2011) 2541-2547 . [28]Y. Li, L.Tang, J.Li, Preparation and electrochemical performance for methanol oxidation of Pt/graphene 22

Page 24 of 72

Ac

ce pt

ed

M

an

us

cr

ip t

nanocomposites, Electrochem. Commun. 11(2009) 846-849. [29]C.L. Sun,H.H. Lee, J.M. Yang, C.C. Wu, The simultaneous electrochemical detection of ascorbic acid, dopamine, and uric acid using graphene/size-selected Pt nanocomposites, Biosens. Bioelectron. 26 (2011) 3450-2455. [30] W.Y.Liu, H.M. Yang, S.G. Ge,L.Shen,J.H. Yu, M. Yan, J.D. Huang, Application of bimetallic PtPd alloy decorated graphene in peroxydisulfate electrochemiluminescence aptasensor based on Ag dendrites decorated indium tin oxide device, Sens. Actuators, B 209 (2015) 32-39. [31]J.J. Lv, J.X. Feng, S.S. Li, Y.Y. Wang, A.J. Wang,Q.L. Zhang, J.R. Chen, J.J. Feng, Ionic liquid crystal-assisted synthesis of PtAg nanoflowers on reduced graphene oxide and their enhanced electrocatalytic activity toward oxygen reduction reaction, Electrochim. Acta, 133 (2014) 407-413. [32]Y.G. Wang, Q. Wei, Y. Zhang, D. Wu, H.M. Ma,A.P. Guo , B. Du, A sandwichtype immunosensor using Pd-Pt nanocrystals as labels for sensitive detection of human tissue polypeptide antigen, Nanotechnol. 25 (2014) 055102 (7pp). [33]Y.Q. Huang, Y.J. Liu, Z.H. Yang, J.L. Jia, X. Li, Y. Luo, Y.P. Fang, Synthesis of yolk/shell Fe3O4-polydopamine-graphene-Pt nanocomposite with high electrocatalytic activity for fuel cells, J. Power Sources, 246 (2014) 868-875. [34]Y. Wang, G.C. Wu, Y.Z. Wang, X.M. Wang,Effect of water content on the ethanol electro-oxidation activity of Pt-Sn/graphene catalysts prepared by the polyalcohol method, Electrochim. Acta 130 (2014) 135-140. [35]W.B. Lu, J. Ge, T. Lin, X.W. Cao, J. Dong, W.P. Qian, Large-scale synthesis of ultrathin Au-Pt nanowires assembled on thionine/graphene with high conductivity and sensitivity for electrochemical immunosensor, Electrochim. Acta 130 (2014) 335-242. [36]H. Mao, R.S. Li, K. Jiang, T. Huang, A.S. Yu, Facile preparation of Cu@Pt/rGO hybrids and their electrocatalytic activities for methanol oxidation, Electrochim. Acta 107 (2013) 419-424. [37]M. Giovanni, H.L. Poh, A. Ambrosi, G.J. Zhao, Z. Sofer, F. Sanek, B. Khezri, R.D. Webster, M. Pumera, Noble metal (Pd, Ru, Rh, Pt, Au, Ag) doped graphene hybrids for electrocatalysis, Nanoscale 4 (2012) 5002-5008. [38]H.J. Yin, H.J. Tang, D. Wang, Y. Gao, Z.Y. Tang, Facile Synthesis of surfactant-free Au cluster/graphene hybrids for high-performance oxygen reduction reaction, ACS Nano 6 (2012) 8288-8297. [39]V.D. Dao, Y. Choi, K. Yong, L.L. Larina, H.S. Choi, Graphene-based nanohybrid materials as the counter electrode for highly efficient quantum-dot-sensitized solar cells, Carbon, 84 (2015) 383-389. [40]L.L Feng, G. Gao, P. Huang, X.S. Wang, C.L, Zhang, J.L. Zhang, S.W. Guo, D.X, Cui, Preparation of Pt Ag alloy nanoisland/graphene hybrid composites and its high stability and catalytic activity in methanol electro-oxidation, Nanoscale 6 (2011) 551-561. [41]W. Lu, J.Ge, L.Tao, X.Cao, J.ong, W.Qian, Large-scale synthesis of ultrathin Au-Pt nanowires assembled on thionine/graphene with high conductivity and sensitivity for electrochemical immunosenso, Electrochim. Acta 130 (2014) 335-343. [42]X. Cui, S.-N. Wu, S. Jungwirth, Z.B. Chen, Z.H. Wang, L. Wang, Y.X. Li, The deposition of Au-Pt core-shell nanoparticles on reduced graphene oxide and their catalytic activity, Nanotechnol. 24 (2013) 295402. [43]W.-B. Lu, J. Ge, T. Lin, X.W. Cao, J. Dong, W.P. Qian, Large-scale synthesis of ultrathin Au-Pt nanowires assembled on thionine/graphene with high conductivity and sensitivity for electrochemical immunosensor, Electrochim. Acta 130 (2014) 335-343. [44]H.H. Yin, S.L. Liu, C.L. Zhang, J.C. Bao, Y.L. Zheng, M. Han, Z.H. Dai, Well-coupled graphene and Pd-based bimetallic nanocrystals nanocomposites for electrocatalytic oxygen reduction reaction, ACS Appl. Mater. Inter.6 (2014) 2086-2094. [45]Z.Q. Yao, R.R. Yue, F.X. Jiang, C.Y. Zhai, F.F. Ren Y.K Du, Electrochemical-reduced graphene oxide-modified carbon fiber as Pt-Au nanoparticle support and its high efficient electrocatalytic activity for formic acid oxidation, J. Solid State Electrochem.17 (2013)2511-2519. [46]C. Du, Z.Y. Yao, Y.Q. Chen, H. Bai, L. Li, Synthesis of metal nanoparticle@graphene hydrogel composites by substrate-enhanced electroless deposition and their application in, Electrochem. sens. 4 (2014) 9133-9138. [47]X.-F. Wang, Z. You, H.L. Sha, Y. Cheng, H.H. Zhu, W. Sun, Sensitive electrochemical detection of dopamine with a DNA/graphemebi-layer modified carbon ionic liquid electrode, Talanta 128 (2014) 373-378. [48]Y. Ma, M.G. Zhao, B. Cai, W. Wang, Z.Z. Ye, J.Y. Huang, 3D graphene foams decorated by CuO nanoflowers for ultrasensitive ascorbic acid detection, Biosens. Bioelectron.59 (2014) 384-388. [49]H.X. Li, Y. Wang, D.X. Ye, J. Luo, B.Q. Su, S. Zhang, J.L Kong, An electrochemical sensor for simultaneous determination of ascorbic acid, dopamine, uric acid and tryptophan based on MWNTs bridged mesocellular graphene foam nanocomposite, Talanta 127 (2014) 255-261. [50]S.H. Hwang, D.W. Kang, R.S. Ruoff, H.S. Shin, Y.B. Park, Poly(vinyl alcohol) reinforced and toughened with poly(dopamine)-treated graphene oxide, and its use for humidity sensing, ACS Nano 8 (2014) 6739-6747. [51] X. Liu, R.Yan, J. Zhua, J. Zhang, X. Liu, Growing TiO2 nanotubes on graphene nanoplatelets and applying the nanonanocomposite as scaffold of electrochemical tyrosinase biosensor, Sens. Actuators, B 209 (2015) 328-335 . [52] H. L. Zou, B. L. Li, H. Q. Luo, N. B. Li, A novel electrochemical biosensor based on hemin functionalized graphene oxide sheets for simultaneous determination of ascorbic acid, dopamine and uric acid, Sens. Actuators, B 207 (2015) 535-541. [53]B. Yu, D. Kuang, S. Liu,C. Liu, T. Zhang,Template-assisted self-assembly method to prepare three-dimensional reduced graphene oxide for dopamine sensing. Sens. Actuators, B 205 (2014) 120-126. 23

Page 25 of 72

Ac

ce pt

ed

M

an

us

cr

ip t

[54]D. Keerthy, S. John , T. Ramachandran, G.N. Bipin, B.T.G. Satheesh, Pt-CuO nanoparticles decorated reduced graphene oxide for the fabrication of highly sensitive non-enzymatic disposable glucose sensor, Sens. Actuators B 195 (2014) 197-205. [55]L.J. Yan, X.J. Bo, D.X. Zhu, L.P. Guo, Well-dispersed Pt nanoparticles on polydopamine-coated ordered mesoporous carbons and their electrocatalytic application, Talanta 120 (2014) 304-311. [56]T.Q. Xu, Q.L. Zhang, J.N. Zheng, Z.Y. Lv, J. Wei, A.J. Wang, J.J. Feng, Simultaneous determination of dopamine and uric acid in the presence of ascorbic acid using Pt nanoparticles supported on reduced graphene oxide, Electrochim. Acta 115 (2014) 109-115. [57]J. Yan, S. Liu, Z.Q Zhang, G.W. He, P. Zhou, H.Y. Liang, L.L. Tian, X.M. Zou, H.J. Jiang, Simultaneous electrochemical detection of ascorbic acid, dopamine and uric acid based on graphene anchored with Pd-Pt nanoparticles, Colloids and Surf. B 111 (2013) 392-397. [58]X.J. Bo, L.P. Guo, Simple synthesis of macroporous carbon-graphene composites and their use as a support for Pt electrocatalysts, Electrochim. Acta 90 (2013) 283-290. [59]M.H. Lin, H.L. Huang, Y.J. Liu, C.J. Liang, S.D. Fei, S.F. Chen, C.L. Ni, High loading of uniformly dispersed Pt nanoparticles on polydopamine coated carbon nanotubes and its application in simultaneous determination of dopamine and uric acid, Nanotechnol. 24 (2013) pp065501. [60]C.L. Sun, H.H. Lee, J.M. Yang, J.M. Yang, C.C. Wu, The simultaneous electrochemical detection of ascorbic acid, dopamine, and uric acid using graphene/size-selected Pt nanocomposites, Biosens. Bioelectron. 26 (2011) 3450-3455. [61]Y. Song, Y. Ma, Y. Wang, J. Di, Y. Tu, Electrochemical deposition of gold-platinum alloy nanoparticles on an indiumtin oxide electrode and their electrocatalytic applications, Electrochim. Acta 55 (2010) 4909-4914. [62]S. Basu, S. Pande, S. Jana, S. Bolisetty, T. Pal, Controlled Interparticle Spacing for Surface-Modified Gold Nanoparticle Aggregates, Langmuir 24 (2008) 5562-5568. [63]T.J. Norman Jr., C.D. Grant, A.M. Schwartzberg, J.Z. Zhang, Structural correlations with shift in the extended plasma resonance of gold nanoparticle aggregates. Opt. Mater. 27 (2005) 1197-1203. [64]Y.Q. Lin, L.B. Li, L.L. Hu, K.Y. Liu, Y.N. Xu, Multifunctional poly(dopamine)-assisted synthesis of silver nano particles/carbon nanotubes nanocomposite: toward electrochemical sensing of hydrogen peroxide with enhanced sensitivity, Sens. Actuators B 202 (2014) 527-535. [65]C.L. Zhou, S. Li, W. Zhu, H.-J. Pang, H.Y. Ma, A sensor of a polyoxometalate and Au-Pd alloy for simultaneously detection of dopamine and ascorbic acid, Electrochim.Acta 113 (2013) 454-463. [66]L. Yu, G. Zhang, Y. Wu, X. Bai, D. Guo, Cupric oxide nanoflowers synthesized with a simple solution route and their field emission, J. Cryst. Growth. 310 (2008) 3125-3130. [67]A.J. Bard, Elecreochemical methods, New York, Chichester Bribane Toronto (1980) Ch. 9. [68]S.M .Ghoreishi, M. Behpour, M.Motaghedi, Electrochemical methods for simultaneous determination of trace amounts of dopamine and uric acid using a carbon paste electrode incorporated with multiwall carbon nanotubes and modified with α-cyclodextrine, J.Solid State Electrochem.16 (2012) 16:179-189. [69]F. Gao, X. Cai, X. Wang, C. Gao, S. Liu, F.Gao, Q. Wang, Highly sensitive and selective detection of dopamine in the presence of ascorbic acid at graphene oxide modified electrode, Sens. Actuators, B 186 (2013) 380-387. [70] L. Huang, Shoufeng Jiao, Maoguo Li, Determination of uric acid in human urine by eliminating ascorbic acid interference on copper(II)-polydopamine immobilized electrode surface, Electrochim. Acta 121 (2014) 233-239. [71] M. Roushani, M.Shamsipur, H. R. Rajabi, Highly selective detection of dopamine in the presence of ascorbic acid and uric acid using thioglycolic acid capped CdTe quantum dots modified electrode, J. Electroanal.Chem. 712 (2014) 19-24 [72] J.S. N. Dutt, M.F. Cardosi, J. Davis, Electrochemical tagging of urate: developing new redox probes, Analyst, 128 (2003) 811-813. [73] Y. Bo, W.Wang, J. Qi, S. Huang, A DNA biosensor based on graphene paste electrode modified with prussian blue and chitosan, Analyst, 136 (2011 )1946-1951 [74]B. Li, G.Pan, N. Avent, R.B.Lowry, T.E.Madgett, P.L.Waines, Graphene electrode modified with electrochemically reduced graphene oxide for label-free DNA detection, Biosens. Bioelectron. 72 (2015) 313-319. [74]W.Gao, L.B.Alemany, L. Ci, P.M. Ajayan, New insights into the structure and reduction of graphite oxide, Nat.Chem. 1 (2009) 403-408. [75] Y. Zhang, L. Stan, P. Xu, H.L. Wang, S. K. Doorn, H. Htoon, Y. Zhu, Q. Jia. A double-layered carbon nanotube array with super-hydrophobicity, Carbon 47 (2009) 68-72. [76] V. C. Tung, M. J. Allen, Y. Yang, R. B. Kaner, High-throughput solution processing of large-scale grapheme, Nat. Nanotechnol. 4 (2009) 25-29. [77] X. Fan, W. Peng, Y. Li, X. Li, S. Wang, G. Zhang, F. Zhang, A green route to graphene preparation, Adv.Mater. 20 (2008) 4490-4493. [78] J. F. Ping, Y. X. Wang, Y. B.Ying, J. Wu, Application of electrochemically reduced graphene oxide on screen-printed ion-selective electrode. Anal. Chim., 84 (2012) 3473-3479. [79] J. Ping, Y. Wang, J. Wu, Y. Ying, Development of an electrochemically reduced graphene oxide modified disposable bismuth film electrode and its application for stripping analysis of heavy metals in milk, Food Chem. 151 (2014) 65-71.

24

Page 26 of 72

Ac

ce pt

ed

M

an

us

cr

ip t

[80] Y.Wang, Y.Zhao, W. He, J. Yin, Y. Su, Palladium nanoparticles supported on reduced graphene oxide: Facile synthesis and highly efficient electrocatalytic performance for methanol oxidation, Thin Solid Films 544 (2013) 88-92. [81] S.S.Tuteja, V.Bhalla, G.Shekhawat,V. Dravid, C.R. Suri, Bio-functionalized graphene-graphene oxide nanocomposite based electrochemical immunosensing Priyanka, Biosens. Bioelectron. 39 (2013) 99-105. [82] D. Lin, J. Wu, M. Wang, F. Yan, H.X. Ju, Triple Signal amplification of graphene film, polybead carried Gold nanoparticles as tracing tag and silver deposition for ultrasensitive electrochemical immunosensing, Anal. Chim. 84 (2012) 3662-3668. [83]X. Zhu, Y. Liang, X. Zuo, R. Hu, X. Xiao, J. Nan, Novel water-soluble multi-nanopore graphene modified glassy carbon electrode for simultaneous determination of dopamine and uric acid in the presence of ascorbic acid, Electrochim. Acta 143 (2014) 366-373. [84]C.L. Sun, H.H. Lee, J.M. Yang, C.C. Wu, The simultaneous electrochemical detection of ascorbic acid, dopamine, and uric acid using graphene/size-selected Pt nanocomposites, Biosens. Bioelectron. 26 (2011) 3450-3455. [85]C. Wang, J. Du, H. Wang, C. Zou, F. Jiang, P. Yang, Y. Du, A facile electrochemical sensor based on reduced graphene oxide and Au nanoplates modified glassy carbon electrode for simultaneous detection of ascorbic acid, dopamine and uric acid, Sens. Actuators B. 204 (2014) 302-309. [86]S. Pruneanu , A.R. Biris, F. Pogacean, C. Socaci, M. Coros, M.C. Rosu, F. Watanabe, A.S. Biris,The influence of uric and ascorbic acid on the electrochemical detection of dopamine using graphene-modified electrodes, Electrochim. Acta 154 (2015) 197-204. [87]M. Liu,Q. Chen,C. Lai,Y. Zhang, J. Deng, H. Li, S.Yao, A double signal amplification platform for ultra sensitive and simultaneous detection of ascorbic acid, dopamine, uric acid and acetaminophen based on a nanocomposite of ferrocene thiolate stabilized Fe3O4@Au nanoparticles with graphene sheet, Biosens. Bioelectron. 48 (2013) 75-81. [88]Y.C. Bai, W.D. Zhang, Highly Sensitive and Selective Determination of dopamine in the presence of ascorbic acid using Pt@Au/MWNTs modified electrode, Electroanalysis 22 (2010) 237-243 [89]Y.H. Zhu, L.P. Stubbs, F. Ho, R.Z. Liu, C.P. Ship, J.A. Maguire, N.S. Hosmane,Magnetic nanocomposites: a n ew perspective in catalysis, Chem. Cat. Chem. 2(2010) 365-374. [90]M.Y. Duan, R. Liang, N. Tian, Y.J. Li, E.S. Yeung, Self-assembly of Au-Pt core-shell nanoparticles for effective enhancement of methanol electrooxida-tion, Electrochim. Acta 87 (2013) 432-437. [91]A.K. Shukla, R.K. Raman, N.A. Choudhury, K.R. Priolkar, P.R. Sarode, S.Emura, R. Kumashiro, Carbon-supported Pt-Fe alloy as a methanol-resistantoxygen-reduction catalyst for direct methanol fuel cells, J. Electroanal. Chem. 563 (2000) 181-190. [92] M. Lee, F. Cheng, H. Yeh, T. Liou, J. Liu, Determination of plasma serotonin and 5-hydroxyindoleacetic acid in healthy subjects and cancer patients, Clin. Chem.46 (2003) 422-423. [93] I. Eichler, H.G. Eichler, M. Rotter, P.A. Kyrle, S. Gasic, A. Korn, Plasma concentrations of free and sulfoconjugated dopamine, epinephrine and norepinephrinein healthy infants and children, Kiln. Wochenschr. 67 (1989) 672-675. [94] V. Ambade, B.M. Arora, P. Singh, B.L. Somani, D. Basannar, Adrenaline, noradren-aline and dopamine level estimation in depression: does it help? Med. J. Armed Forces India 65 (2009) 216-220. [95] X. Ouyang, L. Luo, Y. Ding, B. Liu, D. Xu, A. Huang, Simultaneous determination of uric acid, dopamine and ascorbic acid based on poly(bromocresol green) modified glassy carbon electrode, J. Electroanal. Chem. 748 (2015) 1-7 [96] M.Wang, Y. Gao, J. Zhang, J. Zhao, Highly dispersed carbon nanotube in new ionic liquid-graphene oxides aqueous dispersions for ultrasensitive dopamine detection, Electrochim. Acta 155 (2015) 236-243. [97] B.K. Kim, J. Y. Lee, J.-H. Park, J. Kwak, Electrochemical detection of dopamine using a bare indiumtin oxide electrode and scan rate control, J.Electroanal.Chem. 708 (2013) 7-12. [98] G. Desideri, G. Castaldo, A. Lombardi, M. Mussap, A. Testa, R. Pontremoli, L. Punzi, C. Borghi, Is it time to revise the normal range of serum uric acid levels?, Eur. Rev. Med. Pharmacol.Sci.18 (2014) 1295-1306. [99] P. Gupta, R. N. Goyal, Y.B. Shim, Simultaneous analysis of dopamine and 5-hydroxyindoleacetic acid at nanogold modified screen printed carbon electrodes, Sens. Actuators B 213 (2015) 72-81 [100]C.Xue, Q. Han,Y. Wang, J.Wu, T. Wen, R. Wang, J. Hong, X. Zhou, H. Jiang, Amperometric detection of dopamine in human serum by electrochemical sensor based on gold nanoparticles doped molecularly imprinted polymers, Biosens. Bioelectron. 49 (2013) 199-203 [101] Z. Herrasti, F. Martínez, Eva Baldrich, Electrochemical detection of dopamine using streptavidin-coated magnetic particles and carbon nanotube wiring, Sens. Actuators B 203 (2014) 891-898 [102]T.P. Taylor, M.G. Janech, E.H. Slate, E.C. Lewis, J.M. Arthur, J.C. Oates, Overcoming the effects of matrix interference in the measurement of urine protein analytes, Biomark Insights 7 (2012) 1-8. [103] G. Lippi, M. Montagnana, M. Franchini, E. J. Favaloro, G.Targher, The paradoxical relationship between serum uric acid and cardiovascular disease, Clin. Chim. Acta, 392 (2008) 1-7. [104] K. Vuorensola, H. Siren, U. Karjalainen, Determination of dopamine and methoxycatecholamines in patient urine by liquid chromatography with electrochemical detection and by capillary electrophoresis coupled with spectrophotometry and mass spectrometry, J. Chromatogr. B 788 (2003) 277-289. [105] J. Bicker, A. Fortuna, G. Alves, A. Falcao, Liquid chromatographic methods forthe quantification of catecholamines and their metabolites in several biological samples -a review, Anal. Chim. Acta 768 (2013) 12-34. 25

Page 27 of 72

Figures Captions: Scheme 1 The schematic illustration of Au-Pt/GO-ERGO modified GCE

Fig.1. SEM images of the GO-ERGO (a), Au/GO-ERGO (b), Pt/GO-ERGO(c) and Au-Pt/GO-ERGO (d) modified ITO at 10,000× magnification; Au-Pt/GO-ERGO (e)

ip t

modified ITO at 50,000× magnification

Fig.2. EIS of different materials modified GCE (a-e) and bare GCE (f): Au-Pt/GO-ERGO (a),

cr

Pt/GO-ERGO (b), Au/GO-ERGO(c), GO-ERGO (d) and GO (e) modified GCE

us

Fig.3. (A)Cyclic voltammograms obtained for GO(a), GO-ERGO(b), Pt/GO-ERGO(c), Au/GO-ERGO(d) and Au-Pt/GO-ERGO modified GCE(e)1 mM K3Fe(CN)6

an

containing 0.1 M KCl at the scan rate of 50 mV/s

(B)Cyclic voltammograms of Au-Pt/GO-ERGO modified in 1 mM K3Fe(CN)6 containing 0.1 M KCl at the different scan rates, from a to h: 10, 30, 50, 80, 100, 120 and 140 mV

M

s-1; Inset: Peak currents as a function of scan rate for the determination of the effective working surface area.

ed

Fig.4. (A)Effect of the scan rates on cyclic voltammograms for the Au-Pt/GO-ERGO immobilized on GCE in 0.2 M pH 7.0 PBS in presence of 5.25 μM DA; the scan rates from a (inner) to j (outer) were 30, 40, 50, 60,70, 80, 90 and 100 mV/s.

ce pt

(B) Plots of the peak currents for vs. the scan rates in Fig.4A (C) Effect of the scan rates on cyclic voltammograms for the Au-Pt/GO-ERGO immobilized on GCE in 0.2 M pH 7.0 PBS in presence of 96.9 μM UA; the scan rates from a (inner) to j (outer) were 30, 40, 50, 60,70, 80, 90 and 100 mV/s.

Ac

(D) The plots of the peak currents for vs. the scan rates in Fig.4C

Fig.5. (A) Chronoamperograms obtained from Au-Pt/GO-ERGO immobilized on GCE in 0.2 M pH 7.0 PBS with various concentration of DA: 3.25 (a), 6.50 (b), 9.75 (c), 13.0 (d), 16.2 (e), 19.5 (f), 22.7 (g) and 26.0 (h) μM; (B) The plot of the slope from Fig.5A versus the concentrations of DA; (C) Chronoamperograms obtained from Au-Pt/GO-ERGO immobilized on GCE in 0.2 M pH 7.0 PBS with various concentration of UA: 96.9 (a), 129.0 (b), 151.0 (c), 194.0 (d), 261.0 (e), 258.0 (f) , 292.0 (g) and 322.0 (h) μM; (D) The plot of the slope from Fig.5C versus the concentration of UA

26

Page 28 of 72

Fig.6. (A) Differential pulse voltammograms of Au-Pt/GO-ERGO immobilized on GCE in 0.2 M pH 7.0 PBS with various concentration of DA: 9.75 (a), 19.75 (b), 29.75 (c), 39.75 (d), 49.75 (e) , 59.75(f) , 69.75 (g) and 79.75 (h) μM; (B) The plot of current vs. different concentrations of DA. (C) Differential pulse voltammograms of Au-Pt/GO-ERGO immobilized on GCE in 0.2 M PBS 7.0 PBS with various concentration of UA: 27.15 (a), 50.15 (b), 67.15 (c), 77.15

(D) The plot of current vs. different concentrations of UA.

ip t

(d), 100.15 (e) ,130.15 (f) ,140.15 (g) and 160.15 (h) μM;

cr

Fig.7. Cyclic voltammograms of bare GCE(a) and different materials modified GCE (b-f): GO (b)

GO-ERGO (c), Au/GO-ERGO (d), Pt/GO-ERGO (e) and Pt-Au/GO-ERGO (f) modified

us

GCEs in 0.2 M pH 7.0 PBS in the presence of 5.00 μM DA and 10.00 μM UA ; scan rate:

Ac

ce pt

ed

M

an

50 mV/s

27

Page 29 of 72

*Manuscript Click here to view linked References

The corrected version-0711

A novel sensor based on electrodeposited Au-Pt bimetallic

ip t

nano-clusters decorated on graphene oxide (GO)-electrochemically

us

cr

reduced GO for sensitive detection of dopamine and uric acid

Yang Liua,b, Pei Shea, Jin Gongb, Weiping Wub, Shouming Xua, Jianguo Lia,*, Kang

a

ed

M

an

Zhaoa, AnpingDeng a

The Key Lab of Health Chemistry & Molecular Diagnosis of Suzhou, College of

ce pt

Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China

School of Public Health, Nantong University, Nantong 226019, PR China

Ac

b



Corresponding authors: The Key Lab of Health Chemistry & Molecular Diagnosis of Suzhou, College of

Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China. Tel.: +86 512 65882362; fax: +86 512 65882362.E-mail addresses: [email protected], [email protected]

1

Page 30 of 72

ABSTRACT Sensitive and simultaneous detection of dopamine (DA) and uric acid (UA) by a sensor based on in-situ electrodeposited Au-Pt bimetallic nano-clusters decorated on graphene oxide (GO)-electrochemically reduced GO (ERGO) modified glassy carbon

ip t

electrode (GCE) was presented. The synergistic electrocatalytic effect of Au-Pt bimetallic nano-clusters and GO-REGO was investigated. Firstly, the comparisons of

cr

electrochemical properties for GO, GO-REGO, Au (or Pt) nanoparticles, Au-Pt

us

bimetallic nano-clusters decorated on GO-REGO were studied in detail. Secondly, electrochemical parameters of DA and UA were evaluated. It was observed that for

an

the novel Au-Pt/GO-ERGO nanocomposites, GO-ERGO could provide much wider separation of the oxidation peak potentials of DA and UA, while Au-Pt bimetallic

M

nano-clusters could speed up the electron transfer and enhance the electro-active areas. The linear range of detecting DA was from 6.82×10-8 to 4.98×10-2 M and limit of

ed

detection (LOD) was 2.07×10-8 M (S/N=3). The linear range of detecting UA was from 1.25×10-7 to 8.28×10-2 M and LOD was 4.07×10-8 M (S/N=3). The sensor was

ce pt

applied for the detection of DA and UA in human serum with good results. The sensor suggested that 3D metal-GO nanocomposites were superior materials for the

Ac

fabrication of novel electrochemical sensors.

Key

words:

Gaphene

oxide,

Au-Pt

nano-clusters,

Dopamine,

Uric

acid,

Electrochemical sensor

2

Page 31 of 72

1. Introduction Graphene, showing excellent physical, chemical and electronic properties as well as unusual charge-carrier mobility [1-6], quickly raised the attention of physicists, chemists and engineers over the world, eventually resulted in the award of the Nobel

ip t

Prize in Physics endowed to professor Geim and Novoselov in 2010 [7]. GO, oxygenated derivative of graphene, is a very vital intermediate and precursor

cr

compound in the process for chemically preparing graphene and graphene-based

us

composite materials, respectively [8-9]. The key towards the successful application of graphene materials lies in its modification and/or integration into high quality

an

composite nanomaterials [10-13]. This is firstly because graphene has a zero band gap which can be used to exploit the non-zero band gaps in different inorganic graphene

M

analogues to influent the state-of-the-art composites for electronic applications [14-15]. Secondly, it is possible to categorize GN-based nanomaterials in terms of

ed

their dimensions in the x-y-z axes because such limitations in the different axes

ce pt

significantly influence the electronic properties of GN [16]. The third reason is the possible environmental impacts [17]. To overcome those obstacles, the incorporation of polymer and/or nanostructured metal catalysts continues to be to be a significant research focus [19-20].

Ac

It is known that common Pt catalysts usually suffer from several disadvantages, such as high cost, easily being poisoned by intermediate species and kinetic limitation of the oxygen reduction [21-24]. It was reported that such Pt-based nano-catalysts modified GO not only maximize the availability of nano-sized electro-active surface area for electron transfer but also provide better mass transport of reactants to the electro-catalyst [25-27]. It was used in fuel cells and sensors for DA, ascorbic acid (AA), UA and acetaminophen (AP) [28-29]. As the nanotechnology developed, 3

Page 32 of 72

compared with homologous monometallic counterparts, the bimetallic-NP-based catalysts such as novel Pt-Pd [30], Pt-Ag [31], Pt-Fe [32], Pt-Fe3O4 [33], Pt-Sn [34], Pt-Au [35] and Pt-Cu [36] modified GO often show superior catalytic activity from the synergistic effects of both moieties.

ip t

Pt and Au, which were used to fabricate bimetallic nano-clusters GO are based on the following two major factors. Firstly, pure metals (including Pt or Au) show

cr

unsatisfactory sensitivity, poor selectivity and easy poisoning by adsorbing

us

intermediates, which are critical issues for practical applications [37-38]. Secondly, the literatures [39-40] indicate that excellent elctrocatalytic activity and high

an

sensitivity of Pt can be enhanced by alloying with Au. The Pt-Au nanocomposites modified GO were reported in some literatures, which was synthesized by chemical

M

reactions and then anchored onto GO by physical attentions [41-42]. Controlling the folding, crumpling and bending were the main problems in chemical functionalization

ed

of GO-based materials [43-44]. But the electrochemical synthesis of catalysts is very attractive because such NPs nucleate at the electro-active sites [45-46] and a

ce pt

promising approach to reversibly tune 2D GO electronic properties. However, one-step electrodeposition of Pt-Au bimetallic nano-clusters loaded onto graphene or GO as electrocatalyst have not been reported in details.

Ac

The electrochemical detection of DA and UA has been investigated on graphene

or GO-based in some papers [47-53], but the necessity of continuing further studies with an intention to attain lower detection limits exists now [54-60]. And one-step electrodeposition of Pt-Au bimetallic nano-clusters loaded onto GO for simultaneous DA and UA has not been reported. In this research, the nanocomposites composed by Au-Pt hybrid bimetallic nanoclusters anchored at GO-ERGO was achieved by a simple electrochemical 4

Page 33 of 72

reduction process, which was simply as “Au-Pt/GO-ERGO”. The main aim of recent work, therefore, is to enhance the sensitivity/selectivity of the sensor by investigating more active and lower cost replacements for pure Pt and graphene, hence the interest in bimetallic systems, which bring interesting physical and chemical properties into

ip t

effect from the inter metallic combinations of different metals.

cr

2. Experimental

us

2.1. Materials

All reagents and materials were of analytical grade. All the solutions were prepared

an

using double-distilled water. GO was purchased from Nanjing Xian Feng Nano Technology Co., Ltd. (Nanjing, China). Chloroplatinic acid (H2PtCl6∙6H2O) and

M

chloroauric acid (HAuCl4∙6H2O) was from Sigma Chemical Co. China. DA and UA

ed

were obtained from Fluka Co. Phosphate buffer solutions (PBS, 0.2 M) with different pH values were prepared by mixing the standard stock solutions of Na2HPO4 and

ce pt

NaH2PO4 by adjusting pH with 1.0 M H3PO4 or NaOH. A recovery study was conducted utilizing healthy human serum without any pretreatments and dilution. 2.2. Instruments

Ac

All electrochemical measurements were performed with a CHI 660 electrochemical workstation (CH Instruments Co., USA). The electrochemical cell consisted of a three electrode system with a modified GCE (3 mm in diameter) as a working electrode, a platinum wire as a counter electrode and a saturated calomel electrode (SCE) as a reference electrode. The operation parameter of cyclic voltammetry (CV) was from -0.1 to +0.6V at the scan rate of 50 mV s-1 with the quiet time of 120 s. The operation parameter of differential pulse voltammetry (DPV) was from -0.1 to 0.6 V at the 5

Page 34 of 72

amplitude of 50 mV with the quiet time of 120 s. The operation parameter of chronoamperometry was from 0.0 to 0.3V for DA and from 0.1 to 0.4V for UA. All solutions were deoxygenated by bubbling highly pure nitrogen for at least 15 min and a nitrogen atmosphere was maintained during the measurements. Scanning electron

ip t

microscope (SEM) was performed in the secondary electron imaging model with the 20 KeV working voltage, 3mm laser spot diameter and different magnification times

cr

by Quanta 250FEG-Field emission environmental scanning electron microscope (FEI

us

Ltd., USA). Energy spectrum analysis (EDS) was performed in the secondary electron imaging model with the 20 KeV working voltage, 3mm laser spot and 1250

an

magnification times by Apollo-40 EDS (Edax, Ltd., USA). Electrochemical impedance spectroscopy (EIS) measurements were carried out at open circuit

M

potentials and were performed in 5.0 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) mixture containing 0.10 M KCl with the Autolab 302N electrochemical working station

ed

(Metrohm China Co. Ltd., Switzerland), and a sinusoidal potential modulation with an amplitude of ±5 mV and a frequency from 105 to 1.0 Hz was superimposed on the

ce pt

formal potential of the Fe(CN)63-/4-redox couple at 0.17 V vs SCE. 2.3 Preparation of modified electrode

Ac

The bare GCE with 3 mm diameter was polished to a mirror using 1.0, 0.3, and 0.05 μm alumina slurry and sonication in 1:1 nitric acid, acetone, and deionized water As shown in Scheme 1, 5μL of 0.5 mg/mL-1 GO solution was dropped on the GCE and dried in air to form “GO/GCE”. After electrochemical reduction of GO at -1.0 V in pH 8.0 PBS, the modified electrode (“GO-ERGO/GCE”) was washed with deionized water. To electrochemically deposit Au-Pt nano-clusters, the modified GCE was immersed into an electrolyte consisting of 1.0 mM HAuCl4 and 1.0 mM H2PtCl6 with minor modifications [61]. Au-Pt nano-clusters electrodeposition was performed 6

Page 35 of 72

by CV with a potential range of -0.1 to -0.9 V for 20 circles at scan rate of 50 mV s-1 to form “Au-Pt/GO-ERGO” modified GCE. For comparison, Au nanoparticles (NPs) and Pt NPs were synthesized under similar conditions in 1.0 mM HAuCl4 or 1.0 mM H2PtCl6.

cr

ip t

(Scheme1 should be placed here)

us

3. Results and discussion 3.1 Characterizations of Au-Pt/GO-ERGO

an

3.1.1 UV-Vis characterizations

M

Fig.S1. shows the UV-vis spectrum of the Au-Pt nano-clusters (curve a), Au NPs (curve b) and Pt NPs (curve c) modified ITO. The spectrum of Au NPs presents a

ed

strong peak with maximum absorption wavelength of 526nm, while the absorption peak for Pt NPs was not discovered from 300 to 800 nm. The broad absorption peak

ce pt

appeared at the maximum wavelength of 430 nm (curve a) for Au-Pt nano-clusters modified ITO. The blue shift was found when Au-Pt nano-clusters were formed, because the sizes and elements of Au-Pt nano-clusters were different from Au NPs

Ac

[60, 62 and 63].

3.1.2 Surface topography characterizations Fig. 1A showed the SEM images of GO-ERGO modified ITO and a typical 2D network just like silk was observed in the images. Fig.1B and Fig.1C showed the Au and Pt NPs. Fig.1D and Fig.1E showed that Au-Pt NPs assembled together and formed the cluster. Au-Pt nano-clusters on GO-EGRO modified GCE appeared to be more well-dispersed and dense. With the participation of GO, the deposited metal NPs 7

Page 36 of 72

show better dispersion, in good agreement with the literature [51-53]. It has been confirmed that GO or ERGO, which could be physico-chemically and structurally regarded as unconventional polymeric surfactant structures, may act as capping agent or stabilizer to disperse and hamper the growth of the NPs [21]. Also, the remaining

ip t

oxygen-containing groups of GO could provide binding sites for anchoring precursor metal ions or metal NPs [22]. The energy spectrum analysis (EDS) in Fig S2 results

cr

the existence of Au element and Pt element in Au-Pt/GO-ERGO.

an

3.1.3. Charge transfer resistivity characterizations

us

(Figure 1 should be placed here)

EIS is a powerful and facile electrochemical technique to monitor the changes of

M

interfacial properties in the assembly processes of the modified electrodes step by step [28, 29]. EIS of the bare GCE was shown as “curve f” in Fig.4. When GO-ERGO

ed

(curve d) was formed, the diameter of semicircles was lower than that of GO modified GCE (curve e). When Au-Pt, Pt and Au (curve a, b and c) nanomaterials were

ce pt

immobilized on GO-ERGO, the diameters of semicircles were obviously lower than that of GO-ERGO (curve d). The electron transfer resistance values for Au/GO-ERGO (curve c), Pt/GO-ERGO (curve b), and Au-Pt/GO-ERGO (curve a)

Ac

decorated on the GCEs were 308, 247, and 198Ω, respectively. These data indicated that nanomaterails play an important role similarly to a conducting wire or electron-conducting tunnel [29]. (Figure 2 should be placed here)

3.1.4. Redox probe characterizations Fig. 3A displays cyclic voltammograms of the GO, GO-ERGO, Pt/GO-ERGO, 8

Page 37 of 72

Au/GO-ERGO and Au-Pt/GO-ERGO modified GCE in 0.1 mM ferricyanide at the scan rate of 50 mV/s. The values of peak separation (ΔEp) for GO-GCE (curve a), GO-ERGO/GCE (curve b), Pt/GO-ERGO/GCE (curve c), Au/GO-ERGO/GCE (curve d) and Au-Pt/GO-ERGO (curve b) for exhibiting CV response for [Fe(CN)6]3-/4-were

ip t

204, 150, 100, 90 and 67 mV. This indicated that a good electronic communication was achieved between Au-Pt/GO-ERGO and the underlying GCE and the studied

cr

process was approximately reversible, which was similar as the properties of some

us

other excellent NPs [64-65].

Fig. 3B showed a series of CVs for Au-Pt/GO-ERGO modified GCE in 0.1 mM

an

redox probe with different scan rates. The peak current (Ip) increases linearly with the increasing of the square root of the potential scan rate (v1/2), which suggests that the

M

mass transfer phenomenon in the double layer region of the electrodes is mainly diffusion controlled [66]. Under semi-infinite linear diffusion conditions and a

ce pt

i p  kAn3/ 2 AD1/ 2C 1/ 2

ed

reversible reaction, the Randles-Sevcik equation [67] was described by:

ip refers to the peak current in ampere, A is the electrode surface area in cm2; n is the number of electrons; D is diffusion coefficient of electro-active materials; Co is bulk concentration electro-active materials in cm2 s-1, and v is the scan rate in V s-1. For

Ac

potassium ferricyanide, n is 1; D is 7.6×10-6cm2 s-1; Co is the concentration. The slope of ip versus v1/2 plot was 2.78×10-6 (R2= 0.9997), and then used to calculate the effective working surface area. The value of A calculated for Au-Pt/GO-ERGO, Pt/GO-ERGO and Au/GO-ERGO modified GCE is 10.01, 5.55 and 4.00 cm2, respectively.

It

was

investigated that

the electro-active surface area

of

Au-Pt/GO-ERGO modified GCE was 2 times larger than that only Pt or Au modified GCE. 9

Page 38 of 72

Nicholson has reported [41] that CV has been extended to include electron transfer reactions described by the electrochemical rate equation. Results of theoretical calculations made it possible to use CV to measure standard rate constants for electron transfer-ks. It means a system, which appears reversible at one frequency,

ip t

may be made to exhibit kinetic behaviour at higher frequencies, as indicated by increased separation of cathodic and anodic peak potentials. ks can be understood very

cr

simply as a difficulty level to dynamics of redox probe. The system with higher ks

us

means it can be balanced by shorter time. Oppositely, it can be used for a longer time to balance when the electrochemical system hold a lower ks. The ks is determined from

an

this peak potential separation and frequency. The ks provides an extremely rapid and simple way to evaluate electrode kinetics as Nicholson theory:

M

( D / DR ) a / 2 ks  [ D (nF / RT )]1/ 2

ed

The value of ψ for [Fe(CN)6]3-/4- redox couple from the Ref [67] was 1. n is the number of electrons; α is transfer coefficient; D is diffusion coefficient of

ce pt

electro-active materials; v is the scan rate in V s-1. Under these conditions of quasi-reversibility, it may be possible to study the kinetics of the electrode reaction,

Ac

and the separation of the peak potentials (△Ep), should be a measure of the standard rate constant for electron transfer. For 1 mM K3Fe(CN)6 at 298 K, n = 1, D= 7.6 × 10-6 cm2 s-1, ψ= 0.5, v is scan rate of 0.05 V s-1. When △Ep = 69 mV, ψ= 3; when △Ep = 90 mV, ψ= 0.75; when △Ep = 100 mV, ψ= 0.5; The ks value for Au-Pt/GO-ERGO, Pt/GO-ERGO, Au/GO-ERGO, GO-ERGO and GO immobilized on GCE is calculated 4.3, 2.1, 0.99, 0.76 and 0.55 cm s-1. It indicated that a good electronic communication was achieved between Au-Pt/GO-ERGO and the underlying GCE and the studied 10

Page 39 of 72

process was approximately reversible. (Figure 3 should be placed here) 3.2 Electrochemical parameters of DA and UA at Au-Pt/GO-ERGO modified GCE

ip t

3.2.1. The number of electrons Fig.4. presents the cyclic voltammograms of DA and UA at the Au-Pt/GO-ERGO in

cr

pH 7.0 PBS at different scan rates in presence of 32.3 μM DA (shown in Fig.4A ) and

nFQv 4 RT

an

ip 

us

96.9μ M UA (shown in Fig.4C). According to the equation [67]:

ipa is on behalf of anodic peak current and ipc is on behalf of cathode peak current; n is

M

the number of electrons; Q is electric quantity; F is faraday constant; T is the thermodynamic temperature. At the condition of 297K, from Fig.4, ip increased with

ed

the increasing of scan rates either. It was observed that value of E0’ in the scan range

ce pt

were almost independent on the scan rates, because the values of Epa and Epc remained almost unchangeable with scan rates. As shown in Fig. 4B, ipa (DA, A) = -6.1638×10-6+6.45905×10-7v (mV s-1) with R2 = 0.999, and ipc (DA, A) = 5.52148×10-6-4.25688×10-7v (mV s-1) with R2=0.999. As shown in Fig. 4D, ipc (UA, A)

Ac

= 4.96652×10-6+2.62438×10-7 v (mV s-1) with R2= 0.998. The anodic and cathodic peak currents were linearly proportional to scan rates, which indicate the electro-catalytic behaviors of DA and UA are surface electron transfer processes [67]. From the slope of linear regression equation, n is calculated to be 2.02 for DA and 2.13 for UA. Therefore, the electro-catalytic behaviors of DA and UA on the modified electrode are considered to be two electrons involved. (Figure 4 should be placed here) 11

Page 40 of 72

3.2.2. Electro diffusion coefficient (D) Chronoamperometry could be used for investigation of electrode processes at chemically modified electrodes. Fig. 5 shows the well defined chronoamperograms

ip t

obtained from Au-Pt/GO-ERGO immobilized on GCE in 0.2 M pH 7.0 PBS with

cr

various concentrations of DA (Fig.5A) and UA (Fig.5C). The Cottrell equation [67-68] is aimed to calculate a diffusion coefficient (D) by investigating the relation between

us

the currents for the electrochemical reaction and the concentrations, which is

an

described as:

M

nFAD1/ 2 c I  1/ 2 1/ 2  t

C is the bulk concentration (mol cm-3) (the electro-active material as DA and UA in

ed

the research). In Fig. 5 of chronoamperometric studies, the slopes of the resulting straight line were then plotted versus the DA (Fig.5B) or UA concentration (Fig.5D).

ce pt

From the slope ofFig.5B and Fig.5D, we calculated D of 8.2×10-5 cm2 s-1 for DA and 4.2×10-6 cm2 s-1 for UA, which was a little different from the references [68-69]. Diffusion coefficient (D) is the physical properties of the material, which respected its

Ac

diffusion capacity. According to Fick’s law [69], D is the amount or the mole number, which the materials vertically diffuse in the condition of unit concentration gradient per the unit area and unit time along the direction of diffusion. The value of D mainly depends on the types of materials, diffusion medium, temperature and pressure. Generally speaking, the value of D is detected by experimental measurement. According the above discussion, the D value is a little different from the reference [68]. The reason may be that diffusion medium in our research is different from the

12

Page 41 of 72

reports. The diffusion medium in this research is pH 7.0 PBS, and the other supporting electrolytes were acetate buffer solutions [68-69]. (Figure 5 should be placed here) 3.2.3. Effect of the supporting electrolyte and pH values

ip t

The PBS was found to induce the best response after comparing the

cr

electrochemical behaviors in four different buffer solutions (B-R buffer solution, acetate buffer solution, Tris and PBS). As an important factor, pH values influenced

us

determination of DA and UA directly. Thus, the effect of pH on the voltammetric

an

response of DA and UA were studied over the pH range from 5.0 to 8.0.An increase of pH in solution led to a negative shift in the reduction potential peaks. A shift of

M

“-60.9 mV/pH” of DA was observed in the pH from 5.0 to 8.0. Since the number of electrons was 2 as determined in Section 3.2.1, the number of hydrogen ions involved

ed

in the electrode reaction is 2. A possible scheme for electrochemical oxidation of DA

ce pt

was thus proposed as below

A shift of “-68.7mV/pH” of UA was observed in the pH from 5.0 to 8.0. Since the

Ac

number of electrons was 2 as determined in Section 3.2.1, the number of hydrogen ions involved in the electrode reaction is 2 for UA. A possible scheme for electrochemical oxidation of UA was thus proposed as below

The possible scheme for electrochemical oxidation of DA and UA were similar as the refs [70-73]. The PBS 7.0 was chosen as supporting electrolyte because it was the 13

Page 42 of 72

same as human fluid condition. 3.3 DA and UA detection The excellent quiet time of detecting DA and UA, the experiment was carried at the technology of DPV as Fig.S3 when after injection of highest concentration of analyte.

ip t

From Fig.S3, we found with the increasing of quite time, the current was increasing

us

choose 120s as the excellent quiet time of this research.

cr

either. At the quiet time of 120 s, the current was nearly at the stable condition. So we

Fig. 6A was shown to illustrate that the anodic peak currents of DA are obviously

an

increased with the increase of the DA concentrations. From Fig.6B, the linear regression equation of DA detection was Ipa (μA) = -23.94-1.238CDopamine (μM) with a

M

correlation of 0.997 (n=8) and the range of linearity was from 6.82×10-2 μM to 4.98×10-2 M. Limit of detection (LOD) refers the corresponding amount at the three

ed

times of sensitivity (S) in the background signals (N) produced by the instruments at the matrix blank (S/N=3), which is one of the important indicators which reflected the

ce pt

sensitivity of instruments and methods. S was calculated from the slope of linear regression equations. N was the instrumental signal of matrix blank, which was calculated as the standard deviation obtained by detecting the matrix blank for 10

Ac

times. The LOD of DA at the Au-Pt/GO-ERGO modified GCE in 0.2 M pH 7.0 PBS was 2.07×10-2 μM at 3 folds of the signal-to-noise ratio (S/N=3) from the above discussion.

As similar trends shown in Fig. 6C and Fig. 6D for UA detection, the linear regression equation is Ipa (μA) = -2.820-0.6913Curic acid (μM) with a correlation of 0.998 (n=8). The range of linearity for UA was from 0.125 μΜ to 8.28×10-2 M and LOD was 4.07×10-2 μM. 14

Page 43 of 72

(Figure 6 should be placed here) 3.4 Discussion of Au-Pt/GO-ERGO as a novel nanomaterial for constructing sensors Fig.7 was aimed at illustrating electrochemical behaviors of the mixture of DA and UA on the Au-Pt/GO-ERGO modified GCE. At the bare GCE (curve f), only one

ip t

oxidation peak was observed at near 325 mV, and the peak potentials of DA and UA

cr

were indistinguishable, indicating poor selectivity and sensitivity. In contrast, two

anodic peaks related with oxidation of DA and UA were observed on five different

us

nanomaterials modified GCEs (other curves). The oxidation currents were weaker and

an

the peak was wider on GO/GCE (curve e). The peak current at the GO-ERGO (curve d) modified GCE was slightly increased. When Au NPs (curve c), Pt NPs (curve b),

M

and Au-Pt nano-clusters (curve a) anchored onto GO-ERGO, the peak currents were increased to different extends. Among those, Au-Pt/GO-ERGO modified GCE were

ed

remarkably enhanced. The oxidation peak potentials of DA and UA located at near 230mV and 355 mV, and the peak separations for DA and UA were 182 mV,

ce pt

respectively, which indicated that the Au-Pt/GO-ERGO showed a good catalytic activity for the oxidation of DA and UA. The superior electrochemical performance of the modified electrode could be attributed to the fact that there were significant

Ac

edge-plane-like defective sites existing on the surface of Au-Pt/GO-ERGO.

GO,

graphene

(Figure 7 should be placed here) decorated

with

randomly

distributed

oxygen-containing

functionalities on both sides of the plane, has also been widely reported as a biosensor material due to its high chemical and electrochemical activity. These oxidized areas on the GO plane break the long-range conjugated network and π-electron cloud, leading to a deg radation of carrier mobility and conductivity [73-74]. The reduced

15

Page 44 of 72

GO (rGO) [75] is believed to be one of candidates due to its reasonably reduced number of functionalities, a large number of remaining electro-active sites and the structural similarity with natural graphene. There have been reports of chemically reduced GO by ultra-high vacuum heat [76], chemical treatments (with

ip t

hydrazine/hydrazine derivatives [77] or high-concentration alkaline solution [78], which may lead some disadvantages such as considered to be dangerous substances

cr

labeled and required special equipments. A promising, green, efficient, inexpensive

us

and rapid method based on electrochemistry to produce electrochemically reduced graphene oxide (ERGO) has been introduced [79-80]. Moreover, due to the stability

an

of film on the electrode surface, time saving of fabrication and more excellent physic-chemical properties than pure GO and pure ERGO, the GO-ERGO

M

nanocomposites was reported in recent time [81-82]. Herein, we report a simple, efficient, low-cost and environmentally friendly electrochemical method to

ed

GO-ERGO nanocomposites with mini modification according the references [81-83]. In this research, ERGO was obtained by electrochemical reduction of partly GO

ce pt

immobilized on GCE surface operated in electrolytic cell. In other words, the part of GO was reduced into ERGO and formed to be the novel nanomaterials: GO-ERGO nanocomposites. The electrochemical property of GO-ERGO compared to pure GO

Ac

was explored at the aspects of EIS (Fig.2, curve d), redox probe (Fig.3A, curve b), ks (Section 3.1.4) and the electrochemical response of DA and AA (Fig.7, curve d). From

the

above

discussion,

we

found

the

GO-ERGO

has

higher

chemical/electrochemical activity and bigger surface area of electro-active sites, which was more superior performance than pure GO. GO, not only provides a substrate

to

anchor

Au-Pt

bimetallic

nano-clusters,

but

also

provides

oxygen-containing functional groups of DA and UA electrochemical-oxidation. 16

Page 45 of 72

From Fig. 7 and Table 1, the linear range of DA and UA was wider and the LOD was lower on Au-Pt/GO-ERGO than on Au, Pt, graphene and other similar nanomaterials. The reason may be that the chemical reaction rate depends on the rate which the reactants adhere to the catalyst surface. When the 3D or porous catalyst

ip t

could increase the specific surface area, the reaction was more easily occurred on the catalyst surface adhesion to accelerate the chemical reaction speed. As illustrated in

cr

3.1.4., Au-Pt nano-clusters could provide larger electrochemically active surface area

us

than that of Au and Pt NPs. From Section 3.1.2 (SEM), Au-Pt nano-clusters could make the film more porous for facilitating electron transfer, which facilitates the

an

adsorption of detection molecules. Pt, as excellent catalyst, was higher prices and the global reserves are relatively small. Recently, a number of studies have shown that the

M

catalytic properties of noble metal catalyst can be added to the second nanoporous metal materials [89-91]. They could provide larger electrochemically active surface

ed

area, which would be benefit of improving electron transfer rates for detection

ce pt

molecules.

(Table 1 should be placed here)

From the above discussion, we found this modified GCE has the wider line range and lower LOD of detecting DA and UA compared with that of other sensors reported

Ac

recently. And as a hot-topic of carbon material, Au-Pt/GO-ERGO showed excellent physical, chemical and electronic properties as well as unusual charge-carrier mobility as the above investigation. Compared with pure graphene or GO modified GCE, Au-Pt/GO-ERGO in terms of their dimensions in the x-y-z axes significantly enhances the electronic properties [14, 15, and 17]. Compared with other metal anchored GO, Au-Pt/GO-ERGO not only maximize the availability of nano-sized electrocatalyst surface area for electron transfer but also provide better mass transport 17

Page 46 of 72

of reactants to the electrocatalyst [31-34]. Compared with other chemical methods to synthesize Pt/Au grappled on the GO [41-42], one-step electrodeposition of Pt-Au bimetallic nano-clusters loaded onto GO has not been reported, and electrochemical method owned the sufficient advantages, such as low cost, green and environmental

ip t

protection, saving time and ease to control the experiment conditions.

cr

3.5. The analyzing of real samples, interference test, stability and reproducibility

practicability of Au-Pt/GO-ERGO modified GCE.

us

The tests were carried out in real samples such as human serum to illustrate the

an

Human serum was obtained from human whole blood by precipitation and centrifugation at 3000 rotations per minute for 10 minutes at 4 °C to remove red cells

M

and other interfering proteins. The concentration of DA in serum is not stable in the high nano-molar to low micro-molar range in healthy individuals, which was the same

ed

in their urine and saliva [92]. The concentration of DA has been reported as 21.8 ± 9.5 ng/Lin healthy serum [92-94]. It means the normal level of DA in healthy serum is

ce pt

about 1.15±0.05×10-10 M, which was lower than the limit of detection (LOD) of this research for DA. Just as some literatures was reported, DA was no found in the serum and the real sample detecting was based on the spiked sample analysis [95-96]. The

Ac

normal lever of UA in serum was from 149 to 445 mM [95-98], and UA can be detected in this proposed method according the LOD. The dilution folds of detecting real samples were an important factor. The diluted folds of serum were investigated by recovery experiments just as show in Table S1. From the Table S1, we found the recovery of detecting DA and UA was better than that of undiluted samples. The reason was probably the interference of other complicated

components

of

the

blood

serum,

e.g.

ascorbic

acid

and

18

Page 47 of 72

5-hydroxytryptamine (5-HT) [92]. The main reason was properly when the serum was diluted by buffered solution, the concentration of interfering substance decreased gradually by the dilution folds, which reduced the interfering of recovery experiments [99]. In contrast to the sensor previously reported [100], the optimized assay was able

ip t

to detect endogenous DA or UA in un-diluted serum samples. But some literature has suggested that larger peaks which displayed higher DA or UA concentrations were not

cr

linear in the whole sample without or less dilution [99, 101-102]. These reported

us

suggested that interference due to serum complicated composition (especially ascorbic acid) was proportionally higher for less diluted samples, which was in

an

accordance with the results described by other authors. But in the current electrochemical assay, serum samples were processed and analyzed straightforward,

M

without any additional pretreatment. The high concentration of UA in serum, detected by uricase method which was authorized in clinical analysis standardization, was

ed

determined by adding ascorbic acid oxidase to eliminate AA interfering [103]. The detection by established methodologies, such as chromatography and capillary

ce pt

electrophoresis, is usually preceded by sample pretreatment (e.g. microdialysis, acidification, cation exchange purification, chemical derivatization, etc.)[104-105]. In this experiment, the best and acceptable results were obtained for serum diluted

Ac

1:10 (PBS 7.0) with the averaged DA recoveries (%) of 99.6 ±0.84 and the averaged DA recoveries (%) of 102.6 ±0.71. So, the supernatant from whole blood was diluted by 1:10 with pH 7.0 PBS as serum samples and then were detected in the electrochemical cell. The DPV responses of human serum were shown in Fig.S5 at 1:10 dilution and the experiments were repeated for three times (n=3) and the recovery experiments were analyzed as Table 2. The recoveries in the range from 98.4% to 103.9% and 101.1% to 104.2% were attained for DA and UA, respectively. These 19

Page 48 of 72

results indicate the prospective application of the developed sensor for simultaneously detecting DA and UA in real samples.

(Table 2 should be placed here)

ip t

In addition, there is a systematic error in the recovery experiment (more than 100 recovery of UA) from Table S1 because of the interference of other complicated

cr

components of the blood serum (e.g. ascorbic acid or 5-hydroxytryptamine (5-HT)

us

[98, 92 and 101]). Human serum is a very complex mixture by removal of fibrinogen from plasma. The large part of compositions is known by science studies, but there

an

are part of them is not clear. The composition and contents in human serum were affected by sex, age, nutrient condition, drugs intake and other physical conditions

substance to be examined [98].

M

obtained from different people. Some interfering components may influence the

ed

Several coexisting compounds were selected to evaluate the ability of anti-interference. In Fig.S4, we investigated the CV (Fig.S4A) and DPV (Fig.S4A) of

ce pt

200 μM ascorbic acid (AA), 80.00 μM DA, 80.00 μM UA, 200 μM acetaminophen (AP), 200 μM epinephrine, 200 μM norepinephrine, 200 mM nitrite, 200 mM H2O2, 200 mM NaCl, 200 mM KCl, 200 mM KNO3, 200 mM Na2SO4, 200 mM ZnCl2, 200

Ac

mM CaCl2, 200 mM citric acid, 200 mM glucose, 200 mM bovine serum albumin, 100 mM immunoglobulin, and 200 mM hemoglobin. From the In Fig.S4, the peak potentials of AA and AP was very different from that of DA and UA, and other significant interference had no responses at the Au-Pt/GO-ERGO modified GCE. The stability of the Au-Pt/GO-ERGO modified GCE was also investigated. The modified GCE was stored at 4 °C in a refrigerator for 7 days and then carried in the recovery experiment. The recovery experiment was carried out at the technology of 20

Page 49 of 72

CV in 7.0 PBS, which parameter was from -0.1 to +0.6V at the scan rate of 50 mV s-1 for 20 sweep segments. It would take up for 280 s. The response of Au-Pt/GO-ERGO modified GCE to DA and UA lost 5.3% and 4.8% of its original response after storage for 7 days, respectively. The reproducibility of the proposed sensor was tested using

ip t

eight different electrodes. The relative standard deviations (RSD) of the DPV response currents for these species were less than 5.8%. Thus, the modified electrode

us

cr

showed a high stability and good reproducibility and anti-interference ability.

4. Conclusion

an

Significantly enhanced catalyticactivity of nanocomposites composed by Au-Pt hybrid bimetallic nano-clusters was achieved through a simple electrochemical

M

reduction process anchored at GO-ERGO modified GCE. Synergistic electrocatalytic effect of in-situ electrodeposited Au-Pt bimetallic nano-clusters and GO for detection

ed

of dopamine and uric acid was investigated. Based on those researches, it is believed

ce pt

that for the Au-Pt/GO-ERGO nanocomposites, GO-ERGO could provide much wider separation of the oxidation peak potentials for simultaneously detecting DA and UA while Au-Pt hybrid bimetallic nanocrystals could speed up the electron transfer for increase the sensitivity. This investigation suggested that 3D metal-graphene oxide

Ac

nanocomposites were superior for the fabrication of novel electrochemical sensors. Acknowledgments

The authors thank the National Natural Science Foundation of China (No. 21075087, 21175097 and 81202249) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions for financial support of this study.

21

Page 50 of 72

References

Ac

ce pt

ed

M

an

us

cr

ip t

[1]K.S. Novoselov, A.K. Geim, S.V.Morozov, D. Jiang, Y. Zhang, S.V. Dubonos, I.V. Grigorieva, A.A.Firsov, Electric field effect in atomically thin carbon films, Science 306 (2004) 666-669. [2]A.K.Geim, K.S.Novoselov, The rise of graphene, Nature Mater. 6 (2007) 183-191. [3]K.S. Novoselov, A.K. Geim, S.V. Morozov, D. Jiang, M.I. Katsnelson, I.V.Grigorieva, S.V. Dubonos, A.A. Firsov, Two-dimensional gas of massless Dirac fermions in graphene, Nature438 (2005) 197-200. [4]A.K.Geim, Graphene: Status and Prospects, Science324 (2009) 1530-1534. [5]X.Li, X. Wang, L. Zhang, S. Lee, H. Dai, Chemically derived, ultrasmooth graphene nano ribbon semiconductors, Science 319 (2008) 1229-1232. [6]R.R.Nair, P. Blake, A.N. Grigorenko, KS. Novoselov, T.J. Booth, T. Stauber, N.M. Peres, A.K. Geim, Fine structure constant defines visual transparency of graphene, Science 320 (2008) 1308-1308. [7]The Nobel Prize in Physics 2010. Nobelprize.org, The Nobel Prize in Physics 2010. Andre Geim, KonstantinNovoselov.http://www.nobelprize.org/nobelprizes/physics/laureates/2010/(accessedSeptember 2012). [8]S. Stankovich, D.A.Dikin, R.D.Piner, K.A. Kohlhaas, A.Kleinhammes, Y. Jia, Y. Wu, S.T.Nguyen, R.S.Ruoff, Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide, Carbon 45 (2007) 1558-1565. [9]S.Stankovich, D.A. Dikin, G.B. Dommett, K.M. Kohlhaas, E.J. Zimney, E.A. Stach, R.D. Piner, S.T. Nguyen, R.S. Ruoff, Graphene-based composite materials, Nature442(2006) 282-286. [10]K.S. Novoselov1, V.I. Falko, L. Colombo, P.-R. Gellert, M.G. Schwab, K. Kim, A roadmap for graphene, Nature 490 (2012) 192-200. [11]M.-H. Chakrabarti, C.T.J. Low, N.P. Brandon, V. Yufit, M.A. Hashim, M.F. Irfan,J. Akhtar, E. Ruiz-Trejo, M.-A.Hussain,Progress in the electrochemical modification of graphene-based materials and their applications, Electrochim. Acta 107 (2013) 425-440. [12]V.V. Singh, G. Gupta, A. Batra, A.K. Nigam, M. Boopathi, P.K. Gutch, B.K. Tripathi,A. Srivastava, M. Samuel, G.-S. Agarwal, B. Singh, R. Vijayaraghavan, Green erelectrochemical synthesis of high quality graphene nanosheets directly frompencil and its SPR sensing application, Adv. Funct. Mater. 22 (2012) 2352-2362. [13]C.T.J. Low, F.C. Walsh, M.H. Chakrabarti, M.A. Hashim, M.A. Hussain, Electrochemical approaches to the production of graphene flakes and their potential applications, Carbon 54 (2013) 1-21. [14]C.J. Shih, Q.H. Wang, Y. Son, Z. Jin, D. Blankschtein, M.S. Strano, Tuning on-off current ratio and field-effect mobility in a MoS2-Graphene heterostructure via schottky barrier modulation, ACS Nano 8 (2014) 5790-5798. [15]H. Bu, M. Zhao, A. Wang, X. Wang, First-principles prediction of the transition from graphdiyne to a superlattice of carbon nanotubes and graphene nanoribbons, Carbon65 (2013)341-348. [16]S.A.Jensen, R. Ulbricht, A.Narita, X. Feng, K. Müllen, T.Hertel, Ultra fast photoconductivity of graphene nanoribbons andcarbon Nanotubes, Nano Lett. 13 (2013) 5925-5930. [17]K.S. Kim, Y. Zhao, H. Jang, S.Y. Lee, J. M. Kim, K.S. Kim, J.H. Ahn, P.Kim, J.Y. Choi, B.H. Hong, Large-scale pattern growth of graphene films for stretchable transparent electrodes, Nature 457 (2009) 706-710. [18]D.R. Dreyer, S.Park, C.W. Bielawski, R.S. Ruoff, The chemistry of graphene oxide, Chem. Soc. Rev. 39 (2010) 228-240. [19]H. S.Han, H. Seol, D. H, Kang, M. S. Ahmed,J.M. You, S. Jeon, Electrochemical oxidation and determination of dopamine in thepresence of AA using ferulic acid functionalized electrochemicallyreduced graphene, Sens. Actuators, B 204 (2014) 289-296. [20]C.Wu, H. Zhang, Y.X. Wu, Q.C. Zhuang, L.L. Tian, X.X. Zhang, Synthesis and characterization of Fe@Fe2O3 core-shell nanoparticles/graphene anode material for lithium-ion batteries, Electrochim. Acta 134(2014) 18-27. [21]C. Xu, X. Wang, J. Zhu, Graphene-metal particle nanocomposites, J. Phys. Chem. C 112 (2008) 19841-19845. [22]Y. Li, W. Gao, L. Ci, C. Wang, P.M. Ajayan,Catalytic performance of Pt nanoparticles on reduced graphene oxide for methanol electro-oxidation, Carbon48 (2010)1124-1130. [23]J. Zhang , J. Yu, M. Jaroniec , J.R. Gong, Noble metal-free reduced graphene oxide-ZnxCd1-xS nanocomposite with enhanced solar photocatalytic H-2-production performance, Nano let. 12(2012) 4584-4589. [24]H.Wang, S. Li, Y. Si,N. Zhang,Z. Sun,H. Wu,Y. Lin, Platinum nanocatalysts loaded on graphene oxide-dispersed carbon nanotubes with greatly enhanced peroxidase-like catalysis and electrocatalysis activities, Nanoscale 6 (2014) 8107-8116. [25]E.J. Yoo, T. Okata, T. Akita,M. Kohyama, J. Nakamura,I. Honma, Enhanced electrocatalytic activity of Pt subnanoclusters on graphene nanosheet surface, Nano Lett. 9 (2009) 2255-2259. [26]R.Kou, Y. Shao, D.Wang, M.H. Engelhard, J.H. Kwak, J. Wang,V.V. Viswanathan , C. Wang, Y. Lin, Y. Wang, I.A. Aksay, J. Liu, enhanced activity and stability of Pt catalysts on functionalized graphene sheets for electrocatalytic oxygen reduction, Electrochem. Commun. 11 (2009) 954-957. [27]R. Kou, Y.Shao, D. Mei, Z. Nie, D. Wang, C. Wang,V.V Viswanathan, S. Park,I.A. Aksay,Y. Lin,Y. Wang, J. Liu, Stabilization of electrocatalytic metal nanoparticles at metal-metal oxide-graphene triple junction points, J. Am. Chem. Soc. 133 (2011) 2541-2547 . [28]Y. Li, L.Tang, J.Li, Preparation and electrochemical performance for methanol oxidation of Pt/graphene 22

Page 51 of 72

Ac

ce pt

ed

M

an

us

cr

ip t

nanocomposites, Electrochem. Commun. 11(2009) 846-849. [29]C.L. Sun,H.H. Lee, J.M. Yang, C.C. Wu, The simultaneous electrochemical detection of ascorbic acid, dopamine, and uric acid using graphene/size-selected Pt nanocomposites, Biosens. Bioelectron. 26 (2011) 3450-2455. [30] W.Y.Liu, H.M. Yang, S.G. Ge,L.Shen,J.H. Yu, M. Yan, J.D. Huang, Application of bimetallic PtPd alloy decorated graphene in peroxydisulfate electrochemiluminescence aptasensor based on Ag dendrites decorated indium tin oxide device, Sens. Actuators, B 209 (2015) 32-39. [31]J.J. Lv, J.X. Feng, S.S. Li, Y.Y. Wang, A.J. Wang,Q.L. Zhang, J.R. Chen, J.J. Feng, Ionic liquid crystal-assisted synthesis of PtAg nanoflowers on reduced graphene oxide and their enhanced electrocatalytic activity toward oxygen reduction reaction, Electrochim. Acta, 133 (2014) 407-413. [32]Y.G. Wang, Q. Wei, Y. Zhang, D. Wu, H.M. Ma,A.P. Guo , B. Du, A sandwichtype immunosensor using Pd-Pt nanocrystals as labels for sensitive detection of human tissue polypeptide antigen, Nanotechnol. 25 (2014) 055102 (7pp). [33]Y.Q. Huang, Y.J. Liu, Z.H. Yang, J.L. Jia, X. Li, Y. Luo, Y.P. Fang, Synthesis of yolk/shell Fe3O4-polydopamine-graphene-Pt nanocomposite with high electrocatalytic activity for fuel cells, J. Power Sources, 246 (2014) 868-875. [34]Y. Wang, G.C. Wu, Y.Z. Wang, X.M. Wang,Effect of water content on the ethanol electro-oxidation activity of Pt-Sn/graphene catalysts prepared by the polyalcohol method, Electrochim. Acta 130 (2014) 135-140. [35]W.B. Lu, J. Ge, T. Lin, X.W. Cao, J. Dong, W.P. Qian, Large-scale synthesis of ultrathin Au-Pt nanowires assembled on thionine/graphene with high conductivity and sensitivity for electrochemical immunosensor, Electrochim. Acta 130 (2014) 335-242. [36]H. Mao, R.S. Li, K. Jiang, T. Huang, A.S. Yu, Facile preparation of Cu@Pt/rGO hybrids and their electrocatalytic activities for methanol oxidation, Electrochim. Acta 107 (2013) 419-424. [37]M. Giovanni, H.L. Poh, A. Ambrosi, G.J. Zhao, Z. Sofer, F. Sanek, B. Khezri, R.D. Webster, M. Pumera, Noble metal (Pd, Ru, Rh, Pt, Au, Ag) doped graphene hybrids for electrocatalysis, Nanoscale 4 (2012) 5002-5008. [38]H.J. Yin, H.J. Tang, D. Wang, Y. Gao, Z.Y. Tang, Facile Synthesis of surfactant-free Au cluster/graphene hybrids for high-performance oxygen reduction reaction, ACS Nano 6 (2012) 8288-8297. [39]V.D. Dao, Y. Choi, K. Yong, L.L. Larina, H.S. Choi, Graphene-based nanohybrid materials as the counter electrode for highly efficient quantum-dot-sensitized solar cells, Carbon, 84 (2015) 383-389. [40]L.L Feng, G. Gao, P. Huang, X.S. Wang, C.L, Zhang, J.L. Zhang, S.W. Guo, D.X, Cui, Preparation of Pt Ag alloy nanoisland/graphene hybrid composites and its high stability and catalytic activity in methanol electro-oxidation, Nanoscale 6 (2011) 551-561. [41]W. Lu, J.Ge, L.Tao, X.Cao, J.ong, W.Qian, Large-scale synthesis of ultrathin Au-Pt nanowires assembled on thionine/graphene with high conductivity and sensitivity for electrochemical immunosenso, Electrochim. Acta 130 (2014) 335-343. [42]X. Cui, S.-N. Wu, S. Jungwirth, Z.B. Chen, Z.H. Wang, L. Wang, Y.X. Li, The deposition of Au-Pt core-shell nanoparticles on reduced graphene oxide and their catalytic activity, Nanotechnol. 24 (2013) 295402. [43]W.-B. Lu, J. Ge, T. Lin, X.W. Cao, J. Dong, W.P. Qian, Large-scale synthesis of ultrathin Au-Pt nanowires assembled on thionine/graphene with high conductivity and sensitivity for electrochemical immunosensor, Electrochim. Acta 130 (2014) 335-343. [44]H.H. Yin, S.L. Liu, C.L. Zhang, J.C. Bao, Y.L. Zheng, M. Han, Z.H. Dai, Well-coupled graphene and Pd-based bimetallic nanocrystals nanocomposites for electrocatalytic oxygen reduction reaction, ACS Appl. Mater. Inter.6 (2014) 2086-2094. [45]Z.Q. Yao, R.R. Yue, F.X. Jiang, C.Y. Zhai, F.F. Ren Y.K Du, Electrochemical-reduced graphene oxide-modified carbon fiber as Pt-Au nanoparticle support and its high efficient electrocatalytic activity for formic acid oxidation, J. Solid State Electrochem.17 (2013)2511-2519. [46]C. Du, Z.Y. Yao, Y.Q. Chen, H. Bai, L. Li, Synthesis of metal nanoparticle@graphene hydrogel composites by substrate-enhanced electroless deposition and their application in, Electrochem. sens. 4 (2014) 9133-9138. [47]X.-F. Wang, Z. You, H.L. Sha, Y. Cheng, H.H. Zhu, W. Sun, Sensitive electrochemical detection of dopamine with a DNA/graphemebi-layer modified carbon ionic liquid electrode, Talanta 128 (2014) 373-378. [48]Y. Ma, M.G. Zhao, B. Cai, W. Wang, Z.Z. Ye, J.Y. Huang, 3D graphene foams decorated by CuO nanoflowers for ultrasensitive ascorbic acid detection, Biosens. Bioelectron.59 (2014) 384-388. [49]H.X. Li, Y. Wang, D.X. Ye, J. Luo, B.Q. Su, S. Zhang, J.L Kong, An electrochemical sensor for simultaneous determination of ascorbic acid, dopamine, uric acid and tryptophan based on MWNTs bridged mesocellular graphene foam nanocomposite, Talanta 127 (2014) 255-261. [50]S.H. Hwang, D.W. Kang, R.S. Ruoff, H.S. Shin, Y.B. Park, Poly(vinyl alcohol) reinforced and toughened with poly(dopamine)-treated graphene oxide, and its use for humidity sensing, ACS Nano 8 (2014) 6739-6747. [51] X. Liu, R.Yan, J. Zhua, J. Zhang, X. Liu, Growing TiO2 nanotubes on graphene nanoplatelets and applying the nanonanocomposite as scaffold of electrochemical tyrosinase biosensor, Sens. Actuators, B 209 (2015) 328-335 . [52] H. L. Zou, B. L. Li, H. Q. Luo, N. B. Li, A novel electrochemical biosensor based on hemin functionalized graphene oxide sheets for simultaneous determination of ascorbic acid, dopamine and uric acid, Sens. Actuators, B 207 (2015) 535-541. [53]B. Yu, D. Kuang, S. Liu,C. Liu, T. Zhang,Template-assisted self-assembly method to prepare three-dimensional reduced graphene oxide for dopamine sensing. Sens. Actuators, B 205 (2014) 120-126. 23

Page 52 of 72

Ac

ce pt

ed

M

an

us

cr

ip t

[54]D. Keerthy, S. John , T. Ramachandran, G.N. Bipin, B.T.G. Satheesh, Pt-CuO nanoparticles decorated reduced graphene oxide for the fabrication of highly sensitive non-enzymatic disposable glucose sensor, Sens. Actuators B 195 (2014) 197-205. [55]L.J. Yan, X.J. Bo, D.X. Zhu, L.P. Guo, Well-dispersed Pt nanoparticles on polydopamine-coated ordered mesoporous carbons and their electrocatalytic application, Talanta 120 (2014) 304-311. [56]T.Q. Xu, Q.L. Zhang, J.N. Zheng, Z.Y. Lv, J. Wei, A.J. Wang, J.J. Feng, Simultaneous determination of dopamine and uric acid in the presence of ascorbic acid using Pt nanoparticles supported on reduced graphene oxide, Electrochim. Acta 115 (2014) 109-115. [57]J. Yan, S. Liu, Z.Q Zhang, G.W. He, P. Zhou, H.Y. Liang, L.L. Tian, X.M. Zou, H.J. Jiang, Simultaneous electrochemical detection of ascorbic acid, dopamine and uric acid based on graphene anchored with Pd-Pt nanoparticles, Colloids and Surf. B 111 (2013) 392-397. [58]X.J. Bo, L.P. Guo, Simple synthesis of macroporous carbon-graphene composites and their use as a support for Pt electrocatalysts, Electrochim. Acta 90 (2013) 283-290. [59]M.H. Lin, H.L. Huang, Y.J. Liu, C.J. Liang, S.D. Fei, S.F. Chen, C.L. Ni, High loading of uniformly dispersed Pt nanoparticles on polydopamine coated carbon nanotubes and its application in simultaneous determination of dopamine and uric acid, Nanotechnol. 24 (2013) pp065501. [60]C.L. Sun, H.H. Lee, J.M. Yang, J.M. Yang, C.C. Wu, The simultaneous electrochemical detection of ascorbic acid, dopamine, and uric acid using graphene/size-selected Pt nanocomposites, Biosens. Bioelectron. 26 (2011) 3450-3455. [61]Y. Song, Y. Ma, Y. Wang, J. Di, Y. Tu, Electrochemical deposition of gold-platinum alloy nanoparticles on an indiumtin oxide electrode and their electrocatalytic applications, Electrochim. Acta 55 (2010) 4909-4914. [62]S. Basu, S. Pande, S. Jana, S. Bolisetty, T. Pal, Controlled Interparticle Spacing for Surface-Modified Gold Nanoparticle Aggregates, Langmuir 24 (2008) 5562-5568. [63]T.J. Norman Jr., C.D. Grant, A.M. Schwartzberg, J.Z. Zhang, Structural correlations with shift in the extended plasma resonance of gold nanoparticle aggregates. Opt. Mater. 27 (2005) 1197-1203. [64]Y.Q. Lin, L.B. Li, L.L. Hu, K.Y. Liu, Y.N. Xu, Multifunctional poly(dopamine)-assisted synthesis of silver nano particles/carbon nanotubes nanocomposite: toward electrochemical sensing of hydrogen peroxide with enhanced sensitivity, Sens. Actuators B 202 (2014) 527-535. [65]C.L. Zhou, S. Li, W. Zhu, H.-J. Pang, H.Y. Ma, A sensor of a polyoxometalate and Au-Pd alloy for simultaneously detection of dopamine and ascorbic acid, Electrochim.Acta 113 (2013) 454-463. [66]L. Yu, G. Zhang, Y. Wu, X. Bai, D. Guo, Cupric oxide nanoflowers synthesized with a simple solution route and their field emission, J. Cryst. Growth. 310 (2008) 3125-3130. [67]A.J. Bard, Elecreochemical methods, New York, Chichester Bribane Toronto (1980) Ch. 9. [68]S.M .Ghoreishi, M. Behpour, M.Motaghedi, Electrochemical methods for simultaneous determination of trace amounts of dopamine and uric acid using a carbon paste electrode incorporated with multiwall carbon nanotubes and modified with α-cyclodextrine, J.Solid State Electrochem.16 (2012) 16:179-189. [69]F. Gao, X. Cai, X. Wang, C. Gao, S. Liu, F.Gao, Q. Wang, Highly sensitive and selective detection of dopamine in the presence of ascorbic acid at graphene oxide modified electrode, Sens. Actuators, B 186 (2013) 380-387. [70] L. Huang, Shoufeng Jiao, Maoguo Li, Determination of uric acid in human urine by eliminating ascorbic acid interference on copper(II)-polydopamine immobilized electrode surface, Electrochim. Acta 121 (2014) 233-239. [71] M. Roushani, M.Shamsipur, H. R. Rajabi, Highly selective detection of dopamine in the presence of ascorbic acid and uric acid using thioglycolic acid capped CdTe quantum dots modified electrode, J. Electroanal.Chem. 712 (2014) 19-24 [72] J.S. N. Dutt, M.F. Cardosi, J. Davis, Electrochemical tagging of urate: developing new redox probes, Analyst, 128 (2003) 811-813. [73] Y. Bo, W.Wang, J. Qi, S. Huang, A DNA biosensor based on graphene paste electrode modified with prussian blue and chitosan, Analyst, 136 (2011 )1946-1951 [74]B. Li, G.Pan, N. Avent, R.B.Lowry, T.E.Madgett, P.L.Waines, Graphene electrode modified with electrochemically reduced graphene oxide for label-free DNA detection, Biosens. Bioelectron. 72 (2015) 313-319. [74]W.Gao, L.B.Alemany, L. Ci, P.M. Ajayan, New insights into the structure and reduction of graphite oxide, Nat.Chem. 1 (2009) 403-408. [75] Y. Zhang, L. Stan, P. Xu, H.L. Wang, S. K. Doorn, H. Htoon, Y. Zhu, Q. Jia. A double-layered carbon nanotube array with super-hydrophobicity, Carbon 47 (2009) 68-72. [76] V. C. Tung, M. J. Allen, Y. Yang, R. B. Kaner, High-throughput solution processing of large-scale grapheme, Nat. Nanotechnol. 4 (2009) 25-29. [77] X. Fan, W. Peng, Y. Li, X. Li, S. Wang, G. Zhang, F. Zhang, A green route to graphene preparation, Adv.Mater. 20 (2008) 4490-4493. [78] J. F. Ping, Y. X. Wang, Y. B.Ying, J. Wu, Application of electrochemically reduced graphene oxide on screen-printed ion-selective electrode. Anal. Chim., 84 (2012) 3473-3479. [79] J. Ping, Y. Wang, J. Wu, Y. Ying, Development of an electrochemically reduced graphene oxide modified disposable bismuth film electrode and its application for stripping analysis of heavy metals in milk, Food Chem. 151 (2014) 65-71.

24

Page 53 of 72

Ac

ce pt

ed

M

an

us

cr

ip t

[80] Y.Wang, Y.Zhao, W. He, J. Yin, Y. Su, Palladium nanoparticles supported on reduced graphene oxide: Facile synthesis and highly efficient electrocatalytic performance for methanol oxidation, Thin Solid Films 544 (2013) 88-92. [81] S.S.Tuteja, V.Bhalla, G.Shekhawat,V. Dravid, C.R. Suri, Bio-functionalized graphene-graphene oxide nanocomposite based electrochemical immunosensing Priyanka, Biosens. Bioelectron. 39 (2013) 99-105. [82] D. Lin, J. Wu, M. Wang, F. Yan, H.X. Ju, Triple Signal amplification of graphene film, polybead carried Gold nanoparticles as tracing tag and silver deposition for ultrasensitive electrochemical immunosensing, Anal. Chim. 84 (2012) 3662-3668. [83]X. Zhu, Y. Liang, X. Zuo, R. Hu, X. Xiao, J. Nan, Novel water-soluble multi-nanopore graphene modified glassy carbon electrode for simultaneous determination of dopamine and uric acid in the presence of ascorbic acid, Electrochim. Acta 143 (2014) 366-373. [84]C.L. Sun, H.H. Lee, J.M. Yang, C.C. Wu, The simultaneous electrochemical detection of ascorbic acid, dopamine, and uric acid using graphene/size-selected Pt nanocomposites, Biosens. Bioelectron. 26 (2011) 3450-3455. [85]C. Wang, J. Du, H. Wang, C. Zou, F. Jiang, P. Yang, Y. Du, A facile electrochemical sensor based on reduced graphene oxide and Au nanoplates modified glassy carbon electrode for simultaneous detection of ascorbic acid, dopamine and uric acid, Sens. Actuators B. 204 (2014) 302-309. [86]S. Pruneanu , A.R. Biris, F. Pogacean, C. Socaci, M. Coros, M.C. Rosu, F. Watanabe, A.S. Biris,The influence of uric and ascorbic acid on the electrochemical detection of dopamine using graphene-modified electrodes, Electrochim. Acta 154 (2015) 197-204. [87]M. Liu,Q. Chen,C. Lai,Y. Zhang, J. Deng, H. Li, S.Yao, A double signal amplification platform for ultra sensitive and simultaneous detection of ascorbic acid, dopamine, uric acid and acetaminophen based on a nanocomposite of ferrocene thiolate stabilized Fe3O4@Au nanoparticles with graphene sheet, Biosens. Bioelectron. 48 (2013) 75-81. [88]Y.C. Bai, W.D. Zhang, Highly Sensitive and Selective Determination of dopamine in the presence of ascorbic acid using Pt@Au/MWNTs modified electrode, Electroanalysis 22 (2010) 237-243 [89]Y.H. Zhu, L.P. Stubbs, F. Ho, R.Z. Liu, C.P. Ship, J.A. Maguire, N.S. Hosmane,Magnetic nanocomposites: a n ew perspective in catalysis, Chem. Cat. Chem. 2(2010) 365-374. [90]M.Y. Duan, R. Liang, N. Tian, Y.J. Li, E.S. Yeung, Self-assembly of Au-Pt core-shell nanoparticles for effective enhancement of methanol electrooxida-tion, Electrochim. Acta 87 (2013) 432-437. [91]A.K. Shukla, R.K. Raman, N.A. Choudhury, K.R. Priolkar, P.R. Sarode, S.Emura, R. Kumashiro, Carbon-supported Pt-Fe alloy as a methanol-resistantoxygen-reduction catalyst for direct methanol fuel cells, J. Electroanal. Chem. 563 (2000) 181-190. [92] M. Lee, F. Cheng, H. Yeh, T. Liou, J. Liu, Determination of plasma serotonin and 5-hydroxyindoleacetic acid in healthy subjects and cancer patients, Clin. Chem.46 (2003) 422-423. [93] I. Eichler, H.G. Eichler, M. Rotter, P.A. Kyrle, S. Gasic, A. Korn, Plasma concentrations of free and sulfoconjugated dopamine, epinephrine and norepinephrinein healthy infants and children, Kiln. Wochenschr. 67 (1989) 672-675. [94] V. Ambade, B.M. Arora, P. Singh, B.L. Somani, D. Basannar, Adrenaline, noradren-aline and dopamine level estimation in depression: does it help? Med. J. Armed Forces India 65 (2009) 216-220. [95] X. Ouyang, L. Luo, Y. Ding, B. Liu, D. Xu, A. Huang, Simultaneous determination of uric acid, dopamine and ascorbic acid based on poly(bromocresol green) modified glassy carbon electrode, J. Electroanal. Chem. 748 (2015) 1-7 [96] M.Wang, Y. Gao, J. Zhang, J. Zhao, Highly dispersed carbon nanotube in new ionic liquid-graphene oxides aqueous dispersions for ultrasensitive dopamine detection, Electrochim. Acta 155 (2015) 236-243. [97] B.K. Kim, J. Y. Lee, J.-H. Park, J. Kwak, Electrochemical detection of dopamine using a bare indiumtin oxide electrode and scan rate control, J.Electroanal.Chem. 708 (2013) 7-12. [98] G. Desideri, G. Castaldo, A. Lombardi, M. Mussap, A. Testa, R. Pontremoli, L. Punzi, C. Borghi, Is it time to revise the normal range of serum uric acid levels?, Eur. Rev. Med. Pharmacol.Sci.18 (2014) 1295-1306. [99] P. Gupta, R. N. Goyal, Y.B. Shim, Simultaneous analysis of dopamine and 5-hydroxyindoleacetic acid at nanogold modified screen printed carbon electrodes, Sens. Actuators B 213 (2015) 72-81 [100]C.Xue, Q. Han,Y. Wang, J.Wu, T. Wen, R. Wang, J. Hong, X. Zhou, H. Jiang, Amperometric detection of dopamine in human serum by electrochemical sensor based on gold nanoparticles doped molecularly imprinted polymers, Biosens. Bioelectron. 49 (2013) 199-203 [101] Z. Herrasti, F. Martínez, Eva Baldrich, Electrochemical detection of dopamine using streptavidin-coated magnetic particles and carbon nanotube wiring, Sens. Actuators B 203 (2014) 891-898 [102]T.P. Taylor, M.G. Janech, E.H. Slate, E.C. Lewis, J.M. Arthur, J.C. Oates, Overcoming the effects of matrix interference in the measurement of urine protein analytes, Biomark Insights 7 (2012) 1-8. [103] G. Lippi, M. Montagnana, M. Franchini, E. J. Favaloro, G.Targher, The paradoxical relationship between serum uric acid and cardiovascular disease, Clin. Chim. Acta, 392 (2008) 1-7. [104] K. Vuorensola, H. Siren, U. Karjalainen, Determination of dopamine and methoxycatecholamines in patient urine by liquid chromatography with electrochemical detection and by capillary electrophoresis coupled with spectrophotometry and mass spectrometry, J. Chromatogr. B 788 (2003) 277-289. [105] J. Bicker, A. Fortuna, G. Alves, A. Falcao, Liquid chromatographic methods forthe quantification of catecholamines and their metabolites in several biological samples -a review, Anal. Chim. Acta 768 (2013) 12-34. 25

Page 54 of 72

Figures Captions: Scheme 1 The schematic illustration of Au-Pt/GO-ERGO modified GCE

Fig.1. SEM images of the GO-ERGO (a), Au/GO-ERGO (b), Pt/GO-ERGO(c) and Au-Pt/GO-ERGO (d) modified ITO at 10,000× magnification; Au-Pt/GO-ERGO (e)

ip t

modified ITO at 50,000× magnification

Fig.2. EIS of different materials modified GCE (a-e) and bare GCE (f): Au-Pt/GO-ERGO (a),

cr

Pt/GO-ERGO (b), Au/GO-ERGO(c), GO-ERGO (d) and GO (e) modified GCE

us

Fig.3. (A)Cyclic voltammograms obtained for GO(a), GO-ERGO(b), Pt/GO-ERGO(c), Au/GO-ERGO(d) and Au-Pt/GO-ERGO modified GCE(e)1 mM K3Fe(CN)6

an

containing 0.1 M KCl at the scan rate of 50 mV/s

(B)Cyclic voltammograms of Au-Pt/GO-ERGO modified in 1 mM K3Fe(CN)6 containing 0.1 M KCl at the different scan rates, from a to h: 10, 30, 50, 80, 100, 120 and 140 mV

M

s-1; Inset: Peak currents as a function of scan rate for the determination of the effective working surface area.

ed

Fig.4. (A)Effect of the scan rates on cyclic voltammograms for the Au-Pt/GO-ERGO immobilized on GCE in 0.2 M pH 7.0 PBS in presence of 5.25 μM DA; the scan rates from a (inner) to j (outer) were 30, 40, 50, 60,70, 80, 90 and 100 mV/s.

ce pt

(B) Plots of the peak currents for vs. the scan rates in Fig.4A (C) Effect of the scan rates on cyclic voltammograms for the Au-Pt/GO-ERGO immobilized on GCE in 0.2 M pH 7.0 PBS in presence of 96.9 μM UA; the scan rates from a (inner) to j (outer) were 30, 40, 50, 60,70, 80, 90 and 100 mV/s.

Ac

(D) The plots of the peak currents for vs. the scan rates in Fig.4C

Fig.5. (A) Chronoamperograms obtained from Au-Pt/GO-ERGO immobilized on GCE in 0.2 M pH 7.0 PBS with various concentration of DA: 3.25 (a), 6.50 (b), 9.75 (c), 13.0 (d), 16.2 (e), 19.5 (f), 22.7 (g) and 26.0 (h) μM; (B) The plot of the slope from Fig.5A versus the concentrations of DA; (C) Chronoamperograms obtained from Au-Pt/GO-ERGO immobilized on GCE in 0.2 M pH 7.0 PBS with various concentration of UA: 96.9 (a), 129.0 (b), 151.0 (c), 194.0 (d), 261.0 (e), 258.0 (f) , 292.0 (g) and 322.0 (h) μM; (D) The plot of the slope from Fig.5C versus the concentration of UA

26

Page 55 of 72

Fig.6. (A) Differential pulse voltammograms of Au-Pt/GO-ERGO immobilized on GCE in 0.2 M pH 7.0 PBS with various concentration of DA: 9.75 (a), 19.75 (b), 29.75 (c), 39.75 (d), 49.75 (e) , 59.75(f) , 69.75 (g) and 79.75 (h) μM; (B) The plot of current vs. different concentrations of DA. (C) Differential pulse voltammograms of Au-Pt/GO-ERGO immobilized on GCE in 0.2 M PBS 7.0 PBS with various concentration of UA: 27.15 (a), 50.15 (b), 67.15 (c), 77.15

(D) The plot of current vs. different concentrations of UA.

ip t

(d), 100.15 (e) ,130.15 (f) ,140.15 (g) and 160.15 (h) μM;

cr

Fig.7. Cyclic voltammograms of bare GCE(a) and different materials modified GCE (b-f): GO (b)

GO-ERGO (c), Au/GO-ERGO (d), Pt/GO-ERGO (e) and Pt-Au/GO-ERGO (f) modified

us

GCEs in 0.2 M pH 7.0 PBS in the presence of 5.00 μM DA and 10.00 μM UA ; scan rate:

Ac

ce pt

ed

M

an

50 mV/s

27

Page 56 of 72

cr

ip t

Table(s)

Au-Pt/GO-ERGO

-8

Pt/ graphene

-6

3×10 -8.13×10 6.85×10-6 – 4.15×10-5 4×10-6 -2×10-6 3×10-7-3×10-4 M 5×10-7-5×10-5, 8.0×10−8-120 ×10-6

Line Range of UA (M) 1.25×10-7 8.28×10-2 5.0×10-61.2 × 10-4 5×10-8-1.185×10-4 8.85×10-6 5.35×10-5 4×10-6-4×10-4 None 1×10-6-3×10-4 None

2.07×10-8 1.5 × 10-6 3×10

-8

1.4 5×10-6 -6

4×10 2.05×10-7 2×10-8 8.0×10−8

LOD of UA (M)

Reference

4.07×10-7

This work

2.0 × 10-6

[83]

5×10

-8

1.85×10-6 -7

1.0×10 None 5×10-8 None

[84] [85] [57] [86] [87] [88]

Ac c

ep te

d

Au /graphene

LOD of DA (M)

M

4.0×10-6- 4.0 ×10-5

Graphene

Pt-Pb/graphene Au-Ag/ graphene Au-Fe3O4 / graphene Au-Pt /carbon tubes

Line Range of DA (M) 6.82×10-8 4.98×10-2

an

Similar nanomaterials modified electrode

us

Table 1 Comparison of proposed sensor for determination of DA and UA with others.

Page 57 of 72

cr

ip t

Table(s)

us

Table 2 The recovery experiments of the determination of DA and UA in human serums with 1:10 dilution.

DA level (μM)

Spiked DA (μM)

Total found of DA (μM)

Recovery of DA (%)

RSD of DA (n=3)

UA level (μM)

Spiked UA (μM)

Total found of UA (μM)

Recovery of UA (%)

RSD of DA (n=3)

1 2 3 4 5 6

0

0

Null 4.98±0.13 9.84±0.27 14.87±1.8 20.11±3.3 30.06±1.9

Null 99.7 98.4 99.2 100.5 100.2

Null 2.0 2.3 2.1 2.4 2.0

26.10* 25.10 26.20 27.30 24.20 28.90

Null 10.00 15.00 20.00 25.00 30.00

26.93±1.3 35.11±1.3 41.42±2.3 48.41±4.3 49.90±3.7 59.68±2.3

103.2 101.1 101.6 104.2 102.8 102.6

2.2 1.8 2.2 2.5 2.4 2.0

M

d

5 10 15 20 30

an

Sample

Ac c

ep te

*The result was calculated from automatic biochemical analyzer (Hitachi-7600, Japan) by uricase method.

Page 58 of 72

Figure(s)

ed

M

an

us

cr

ip t

Fig.1A

Ac

ce pt

Fig.1B

Page 59 of 72

ed

M

an

us

cr

ip t

Fig.1C

Ac

ce pt

Fig.1D

Page 60 of 72

Ac

ce pt

ed

M

an

us

cr

ip t

Fig.1E

Page 61 of 72

Ac

ce pt

ed

M

an

us

cr

ip t

Fig.2

Page 62 of 72

M

an

us

cr

ip t

Fig.3A

10

a

Ac

I / A

5

h

I / A

15

ce pt

20

ed

Fig.3B

3.0

B

Inset

2.5 2.0

0

1.5

-5

1.0

-10

5

10

-15

1/2



15

/V

1/2

20

s

25

-1/2

-20 0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

E vs. SCE / V

Page 63 of 72

M

an

us

cr

ip t

Fig.4A

Ac

ce pt

ed

Fig.4B

Page 64 of 72

ed

M

an

us

cr

ip t

Fig.4C

Ac

ce pt

Fig.4D

Page 65 of 72

ed

M

an

us

cr

ip t

Fig.5A

Ac

ce pt

Fig.5B

Page 66 of 72

ed

M

an

us

cr

ip t

Fig.5C

Ac

ce pt

Fig.5D

Page 67 of 72

ed

M

an

us

cr

ip t

Fig.6A

Ac

ce pt

Fig.6B

Page 68 of 72

ed

M

an

us

cr

ip t

Fig.6C

Ac

ce pt

Fig.6D

Page 69 of 72

Ac

ce pt

ed

M

an

us

cr

ip t

Fig.7.

Page 70 of 72

Ac ce p

te

d

M

an

us

cr

ip t

Supplementary Material Click here to download Supplementary Material: revised supporting materals-0711.docx

Page 71 of 72

*Author Biographies

Biographies: Yang Liu obtained her bachelor of medicine from Nantong University, China, in 2006 and master degree of medicine from Nantong University, China, in 2009. Now she is a doctor student of analytical chemistry in Soochow University and an instructor teacher in Nantong University. Her research interests are in the areas of bio-electroanalytical chemistry and immunosensor.

ip t

She Pei is currently a MS student majoring in analytical chemistry of Soochow

University in China. Her current research interesting is bio-electroanalytical chemistry

cr

and immunosensor.

Jin Gong obtained her MBBS degree in 2012 from Nantong University, China. Now

us

she is a postgraduate student of Nantong University. Her research interests are in the areas of electroanalytical chemistry.

an

Weiping Wu is a senior student in Nantong University in China. His research interests are in the areas of electroanalytical chemistry.

Shouming Xu is a doctor student of Soochow University in China. His research

M

interests are in the areas of nanomaterials.

Jianguo Li obtained his PhD in 2006 from Nanjing University in China. Currently he

ed

is a professor of analytical chemistry in Soochow University, China. His research interests are preparation of quantum dots nanocomposites, electrochemiluminescence and biosensor.

ce pt

Anping Deng obtained his PhD degree from Masaryk University, Brno, Czech Republic in 1999. Currently he is a professor of analytical chemistry in Soochow University, China. His scientific interest is mainly on immunoassays and

Ac

bio-electroanalytical chemistry.

Page 72 of 72