Green synthesis of silver nanoparticles–graphene oxide nanocomposite and its application in electrochemical sensing oftryptophan

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Green synthesis of silver nanoparticles-graphene oxide nanocomposite and its application in electrochemical sensing of tryptophan Junhua Li, Daizhi Kuang, Yonglan Feng, Fuxing Zhang, Zhifeng Xu, Mengqin Liu, Deping Wang

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28 July 2012 2 October 2012 9 October 2012

Cite this article as: Junhua Li, Daizhi Kuang, Yonglan Feng, Fuxing Zhang, Zhifeng Xu, Mengqin Liu and Deping Wang, Green synthesis of silver nanoparticles-graphene oxide nanocomposite and its application in electrochemical sensing of tryptophan, Biosensors and Bioelectronics, http://dx.doi.org/10.1016/j.bios.2012.10.029 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 galley proof before it is published in its final citable 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.

Green synthesis of silver nanoparticles-graphene oxide nanocomposite and its application in electrochemical sensing of tryptophan

Junhua Lia,b,*, Daizhi Kuanga,b, Yonglan Fenga,b, Fuxing Zhanga,b, Zhifeng Xua,b, Mengqin Liua,b, Deping Wanga,b

a

Department of Chemistry and Material Science, Hengyang Normal University,

Hengyang, 421008, Hunan, PR China b

Key Laboratory of Functional Organometallic Materials of Hunan Province College,

Hengyang Normal University, Hengyang, 421008, Hunan, PR China

*

Corresponding author. Tel.: + 86 734 8486779

E-mail address: [email protected] (J. Li).

Abstract: A new kind of nanocomposite based on silver nanoparticles (AgNPs)/graphene oxide (GO) was conveniently achieved through a green and low-cost synthesis approach using glucose as a reducing and stabilizing agent, and the synthetic procedure can be easily used for the construction of a disposable electrochemical sensor on glassy carbon electrode (GCE). The nanocomposite was detailedly characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR) and electrochemical impedance spectroscopy (EIS). The

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experimental results demonstrated that the nanocomposite possessed the specific features of both silver nanoparticles and graphene, and the intrinsic high specific area and the fast electron transfer rate ascribed to the nanohybrid structure could improve its electrocatalytic performance greatly. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were employed to evaluate the electrochemical properties of AgNPs/GO/GCE towards tryptophan, and the AgNPs/GO film exhibited a distinctly higher activity for the electro-oxidation of tryptophan than GO film with tenfold enhancement of peak current. The oxidation mechanism and the kinetic parameters were investigated, and analysis operation conditions were optimized. Under the selected experimental conditions, the oxidation peak currents were proportional to tryptophan concentrations over the range of 0.01 μM to 50.0 μM and 50.0 μM to 800.0 μM, respectively. The detection limit was 2.0 nM (S/N = 3). Moreover, the proposed method is free of interference from tyrosine and other coexisting species. The resulting sensor displays excellent repeatability and long-term stability; finally it was successfully applied to detect tryptophan in real samples with good recoveries, ranging from 99.0% to 103.0%. Keywords: Graphene oxide; Silver nanoparticles; Tryptophan sensor; Electrochemical tryptophan detection

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1. Introduction

Tryptophan is an essential amino acid with diverse physiological roles, functioning both independently or via incorporation into the structure of larger molecules or polymers (Mazloum-Ardakani et al., 2011). It is a vital constituent of proteins and indispensable in human nutrition for establishing and maintaining a positive nitrogen balance (Fang et al., 2007). According to the World Health Organization (WHO), the tryptophan requirement is set at 4 mg per kg of body per day. Unfortunately, it can not be synthesized directly in human body and therefore must be taken from dietary, food products, pharmaceutical formulas, in which tryptophan sometimes is added because the presence of tryptophan in vegetables is scarce (Li et al., 2010). Tryptophan also serves as a precursor for serotonin and melatonin which can improve the sleep, mood and mental health (Fiorucci and Cavalheiro, 2002). Abnormal levels of serotonin and melatonin have been shown to be associated with Alzheimer’s and Parkinson’s diseases. When tryptophan is improperly metabolized, it creates a waste product in the brain that is toxic, causing hallucinations and delusions (Kochen and Steinhart, 1994). In general, it has been implicated as a possible cause of schizophrenia in people who cannot metabolize it properly. Therefore, development of a simple, inexpensive, sensitive and accurate analytical method for determination of tryptophan is of great significance and urgency to people’s health. Nowadays, many methods have been developed for the determination of tryptophan, including high-performance liquid chromatography (HPLC) (Lian et al., 2012), HPLC with fluorescence detection (Zhang et al., 2012), liquid chromatography–tandem mass spectrometry (Zhu et al., 2011), spectrophotometry (Yu et al., 2004), spectrofluorometry 3

(Reynolds, 2003), capillary electrophoresis technique (Malone et al., 1995), and especially infrared optical sensor (Huang et al., 2011). Although these methods are very important for quantitative analysis, the majority of them suffer from some disadvantages such as high costs, tedious extraction process, long analysis time and requirement for complicated instruments, and in some cases low sensitivity and selectivity that makes them unsuitable for routine analysis. In order to overcome these inherent shortcomings, electrochemical method may be a good option due to its elegant and sensitive properties in analysis field. Particularly, electrochemical technique offers a wide dynamic range and requires only small sample volumes, often in the microliter range, that coupled with the low detection limits, allows analysis on subpicogram amounts of analyte. Also, the selectivity of electrochemical detection in complex samples is excellent, because relatively fewer electroactive interferents are often encountered than spectroscopic interferents (Lunte and Osbourn, 2007). However, the direct electrochemical oxidation of tryptophan is known to be kinetically sluggish, and a relatively high overpotential is required for its oxidation at bare electrodes. Therefore, the working electrodes employed in conventional electrochemical method always need surface modification, so that it can decrease the overpotential and enhance the electrochemical response of the objective molecules (Li et al., 2012). During recent years, great efforts have been devoted to the development of electrochemical sensors for tryptophan using different kinds of modified electrodes, most of which are based on multi-walled carbon nanotubes (MWCNT) (Wu et al., 2004), particularly MWCNT hybrid composite (Fang et al., 2007; Goyal et al., 2011; Güney and Yıldız, 2011; Noroozifar et al., 2011; Shahrokhian and Fotouhi, 2007) and

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metal nanoparticles including gold (Li et al., 2011), copper-cobalt hexacyanoferrate (Liu and Xu, 2007), cerium hexacyanoferrate (Fang et al., 2007), TiO2 (Raoof et al., 2009; Fan et al., 2011) and Co3O4 (Ye et al., 2012). Among these mediators, there are few graphene and graphene derivative used for electrochemical sensing of tryptophan. In addition, the oxidation peak potential for another electroactive amino acid, tyrosine, is very close to that of tryptophan at conventional electrodes. Therefore, interest continues in the development of interference-free electrochemical sensor for tryptophan. Graphene, as a new form of carbon, is a two-dimensional sheet of carbon atoms bonded through sp2 hybridization. Owing to its novel properties such as large specific surface area (2630 m2 g−1), high electrical conductivity (103~104 S m−1), high thermal and mechanical properties, graphene is an ideal two-dimensional catalyst support to anchor metal and semiconductor catalyst nanoparticles, offering versatile selective catalytic or sensing performances (Xing et al., 2012). However, graphene sheets, unless well separated from each other, tend to form irreversible agglomerates or even restack to form graphite through van der Waals interactions. Aggregation would occur with any change under the different conditions of solution, such as addition of salts, acids or organic dispersants. This restricts the synthesis of many hybrid graphene materials and application fields of graphene sheets. Graphene oxide (GO), the oxidized form of graphene, bears two-dimensional plane and a large number of oxygen-containing functional groups with disorder on the basal planes and edges (Stankovich et al., 2006). It possesses not only the similar properties with graphene, but also the excellent dispersibility and film-forming features. The covalent oxygen functional groups in GO

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give rise to remarkable mechanical strength along with molecular-level chemical sensing capability (Dikin et al., 2007; Robinson et al., 2008). Thus, GO is exceptionally suitable for use in chemical and biological sensors owing to the nature of the atoms on its surface, scalable solution-based preparation process, and controllable surface defect density that modulates the sensor sensitivity and specificity (Guo et al., 2011). Nanocomposites based on nanosized inorganic particles and clusters represent an attractive field of research activity because of the possibility to tailor and optimize the properties of the resulting materials for various applications (Li et al., 2012; Liu et al., 2011). GO nanosheets have already been employed as supports to disperse and stabilize numerous nanoparticles (Au (Liu et al., 2011), ZnO (Li et al., 2012), MnO2 (Li et al., 2010), TiO2 (Zhang and Choi, 2011), Fe3O4 (Bai et al., 2012)). This is because that the abundant surface functional groups (–OH, C–O–C, and –COOH) on GO can provide reactive sites for the nucleation and binding of metal nanoparticles (Zhang et al., 2011). Silver nanoparticles (AgNPs) possess high quantum characteristics of small granule diameter and large specific surface area as well as the ability of quick electron transfer and the strong antibacterial activity, so they have been applied in the preparation of biosensors and bactericidal agents (Bao et al., 2011). In this work, the AgNPs/GO nanocomposite was expediently synthesized by the reduction of silver ions on GO surface using glucose as reducing and stabilizing agent. The synthetic process was carried out only in aqueous solution, which is versatile and environmentally friendly. The resultants can be dispersed into water and common organic solvents to form a stable system without any additional protection by polymeric or

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surfactant stabilizers. The AgNPs/GO composite was then used to fabricate a novel electrochemical sensor for tryptophan, and it could provide larger electrochemically active surface area for the adsorption of tryptophan and effectively accelerate the electron transfer between electrode and solution, which could lead to a more rapid and sensitive current response. The physical characteristics and electrochemical performances of the prepared sensor were examined, and kinetic parameters and analysis conditions were also discussed. This developed sensor was successfully applied for the selective determination of tryptophan in the presence of a high concentration of tyrosine. A complete resolution of peak-to-peak separation between tryptophan and its coexisting substances provides a very suitable and effective method for determination of tryptophan in human fluids and pharmaceutical preparations.

2. Experimental

2.1. Reagents

Graphite powder (320 mesh, spectrum pure), H2SO4, KMnO4, and H2O2 (30 wt%) were purchased from Shanghai Chemical Reagent Co. and used for synthesizing GO. AgNO3, glucose and NH3·H2O were obtained from Alfa Aesar. Tryptophan was purchased from Sigma–Aldrich, and its standard stock solutions of 1.0 mM were prepared daily by dissolving the right amount of tryptophan in dilute HCl. An acetate buffer (pH 4.5) of 0.1 M was employed as a supporting electrolyte. All other reagents were of analytical grade and used without further purification. Doubly distilled water was used throughout. 7

2.2. Apparatus

Scanning electron microscopy (SEM) images were obtained from S-4800 field emission SEM system (Hitachi, Japan) operating at 5.0 kV. Transmission electron microscopy (TEM) micrographs were recorded on a JEM 2100 transmission electron microscopy (JEOL, Japan) operating at 200 kV, equipped to perform elemental chemical analysis by energy dispersive X-ray spectroscopy (EDX). For SEM, TEM, and EDX measurements, the samples were coated onto the surfaces of small copper plates and measured directly. Atomic force microscopy (AFM) study was carried out using a SPI3800N microscope (Seiko, Japan). Fourier transform infrared (FTIR) spectra were recorded on a FTIR-8700 Spectrometer (Shimadzu, Japan) with KBr pressing plate method, and the Raman spectra were recorded on a Renishaw inVia plus Raman microscope using a 514.5 nm argon ion laser. All electrochemical experiments were performed with a CHI 660D electrochemical workstation (CH Instruments, Shanghai, China). The conventional three-electrode geometry was adopted. The working electrode was a bare or modified glassy carbon electrode (GCE, 3 mm in diameter), and the auxiliary and reference electrodes were platinum wire and saturated calomel electrode (SCE), respectively. Electrochemical impedance measurements were performed in 10.0 mL of 0.1 M KCl containing 1.0 mM Fe(CN)63− and Fe(CN)64− (1:1 mixture). The impedance spectra were measured in the frequency range from 105 Hz to 0.1 Hz at open circuit potential, with voltage amplitude of 0.005 V. All electrochemical experiments were carried out under high purity nitrogen, at room temperature.

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2.3. Synthesis of AgNPs/GO nanocomposite

In this work, GO was synthesized from natural graphite powder by a modified Hummers’ method as originally presented by Hirata et al (Hirata et al., 2004). In general, noble metal nanoparticles were easily obtained using the strong reducing agents such as hydrazine and sodium borohydride, but these reducing agents were poisonous and not friendly to environment. Besides, they can also reduce the oxygen-containing functional groups of GO, resulting in the decrease of sensing performances of the prepared sensor. For green and inexpensive chemical synthesis, the AgNPs/GO nanocomposite was prepared by reducing silver ions directly on GO with glucose as reducing and stabilizing agent. The typical procedure for nanocomposite synthesis was described as follows (Xu and Wang, 2009). Firstly, GO powder (15.0 mg) was dispersed in water (15.0 mL) by sonication for 1 h to form a stable GO colloid and then glucose (0.75 g) was dissolved in this solution under stirring. Second, ammonia (0.55 mol L−1) was added slowly to a silver nitrate aqueous solution (10.0 mL, 0.06 mol L−1) until the AgOH/Ag2O precipitate dissolved. Subsequently, the Ag(NH3)2OH solution was mixed with the GO and glucose-containing solution. After being stirred for 0.5 h, the mixture was allowed to sit undisturbed at room temperature for 2 h. The slurry-like product was centrifugated and washed with water repeatedly to remove any impurities. Finally, the obtained product was dried overnight in an oven at 60 0C, and then AgNPs/GO nanocomposite was obtained.

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2.4. Preparation of the AgNPs/GO/GCE

Firstly, GCEs were mechanically polished with 0.3 and 0.05 mm alumina slurry and then sequentially sonicated in dilute nitric acid, anhydrous ethanol and redistilled water for 15 min. Finally, the cleaned GCEs were dried under nitrogen stream. 5.0 mg of AgNPs/GO nanohybrids were added to 2.5 mL DMF solution, which was sonicated in ultrasound bath for 30 min to form a stable suspension. The mixed suspension of 5.0 µL was cast onto GCE surface by a micropipette, and then the suspension was thoroughly dried out under an infrared lamp. After that, the electrode was rinsed by distilled water for several times and further dried in air before use, and the final obtained electrode was denoted as AgNPs/GO/GCE. For comparison, the GO/GCE was prepared using GO dispersion only by the same procedure as described previously.

3. Results and Discussion

3.1. Characterizations of AgNPs/GO nanocomposite

In order to verify the successful synthesis of the AgNPs/GO nanocomposite, various characterization methods were employed, including SEM, TEM, EDX and FTIR. Fig. 1 displays the SEM images of GO film (A, B) and AgNPs/GO composite film (C, D) at low and high magnifications. It is clearly seen in Fig. 1A and B that the GO sheets displayed a typical crumpled and wrinkled surface of grapheme, and the sheets stacked together to form a typical multi-layer structure. The geometric wrinkling arising from π–π interaction within sheets of GO not only minimizes the surface energy but also induces mechanical

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integrity with tensile strength, so the film-forming ability could improve greatly. In addition, the wrinkled structure of GO sheets provides a large rough surface as scaffold for further modification. After the reduction of silver ions, AgNPs were completely distributed on GO sheets (Fig.1C and D) and no particles scattered out of the supports, indicating a strong interaction between GO support and AgNPs. The surface of nanocomposite is much rougher than that of GO nanosheets, which might be attributed to the growth of nanoparticles on GO sheets. Furthermore, due to the crumpled surface and large surface areas of GO nanosheets, AgNPs can be deposited on both sides of these sheets. Highly dispersed AgNPs on supports with larger surface area would be beneficial to improve the catalytic activity and sensor sensitivity.

Fig. 1. The SEM images of GO film (A, B) and AgNPs/GO composite film (C, D) at different magnifications.

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Fig. 2 A and B show the typical TEM images of the synthesized GO and AgNPs/GO nanocomposite. It can be seen that a large number of AgNPs are attached onto the surface of GO nanosheets. However, the size of these nanoparticles is between 30–80 nm, and the dispersion is heterogeneous. The reason might be attributed to the stirring inhomogeneity while the formation of AgNPs on GO nanosheets. Some AgNPs were slightly aggregated due to the loading degree close to saturation. Because of GO and silver ions with opposite charges, AgNPs can interact with the GO sheets through physisorption, electrostatic binding or through charge transfer interactions. In addition, the AFM image (inset of Fig. 2B) displays that the surface of nanocomposite is full of bumps and hollows, and several large parts maybe form from the saturated deposition of AgNPs on GO. This can increase the extent of surface roughness greatly, and then result in the increase of surface active site. The formation of AgNPs/GO nanocomposite was further characterized by EDX. The corresponding EDX spectra of GO (Fig. 2C) and GO/AgNPs (Fig. 2D) show the peaks corresponding to C, O, S, Cu and Ag elements, confirming the existence of metallic AgNPs onto the surface of GO nanosheets. The existing O element demonstrated oxygen-containing groups were generated in GO preparation, while most of them weren’t reduced during AgNPs/GO preparation process. The Cu element was from the basement membrane and trace amounts of S may be from the reactant. The atomic and weight ratio of C/Ag in nanocomposite is 1.5/1 and 16/100 respectively, demonstrating a higher loading of AgNPs on GO sheet surface, which is consistent with the SEM and TEM observation. The results of FT-IR spectra (See Fig. S1 in the Supporting information) also

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confirmed that oxygen-containing groups have been generated on GO during its preparation process, and most of them existed after AgNPs were reduced onto the surface of GO, indicating glucose is a mild reductant. Typical features for the GO in Raman spectra (See Fig. S2 in the Supporting information) are the G line around 1600 cm−1 and the D line around 1350 cm−1. The G line is usually assigned to the first-order scattering of the E2g phonons of sp2 C atoms; the D line is the breathing mode of the k-point phonons of A1g symmetry (Kudin et al., 2008). Due to the surface-enhanced Raman scattering (SERS) activity of AgNPs, both the Raman intensities of the D and G bands obviously increase for AgNPs/GO, so it indicates AgNPs have been successfully deposited on GO. The result of Raman spectra is in agreement with the previous conclusion from EDX.

Fig. 2. The TEM images and EDX spectra of GO film (A, C) and AgNPs/GO composite film (B, D), and the insert of B is AFM image of AgNPs/GO composite.

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3.2. Electrochemical characteristics of AgNPs/GO/GCE

Electrochemical characterization of AgNPs/GO/GCE was carried out by electrochemical impedance spectroscopy (EIS) and chronoamperometry (CA). The EIS results (See Fig. S3 in the Supporting information) show that the charge transfer resistance (Rct = 420 Ω) of AgNPs/GO/GCE is lower that of GCE (Rct = 600 Ω) and GO/GCE (Rct = 1000 Ω). Electrochemically active surface areas obtained by CA are 0.1803 cm2 and 0.2315 cm2 for GO/GCE and AgNPs/GO/GCE, which are 2.5 times and 3.3 times of that of GCE (0.0711 cm2), respectively.

3.3. Electrochemical behaviors of tryptophan on AgNPs/GO/GCE

The electrocatalytic activity of AgNPs/GO/GCE for the oxidation of tryptophan was investigated by cyclic voltammetry (CV). Figure 3 shows CVs of GCE, GO/GCE and AgNPs/GO/GCE in the absence (A) and presence (B) of 0.1 mM tryptophan in 0.1 M acetate buffer (pH 4.5) at a scan rate of 100 mV s−1. There was no redox peak obtained at GCE and GO/GCE without tryptophan, indicating the GO film was non-electroactive in the selected potential region. The background current of GO/GCE was higher than that of GCE, which could be attributed to the large specific area of GO. A pair distinct redox peaks were observed at AgNPs/GO/GCE without tryptophan. Obviously, the observed redox peaks can be attributed to the redox of Ag+/0 in nanocomposite, in which the electroactive AgNPs are oxidized to Ag+ at 0.38 V vs. SCE on the forward anodic scan,

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with conversion of Ag+ back to Ag at 0.28 V vs. SCE on the reversed cathodic scan. This observation further demonstrated that AgNPs have been successfully immobilized onto the GO surface. When 0.1 mM tryptophan was added into the acetate buffer, a weak oxidation peak was observed on GCE at a relatively high potential of 1.05 V vs. SCE, and the peak current was lower than that of GO/GCE. When GO was immobilized on GCE surface, the oxidation peak potential shifted negatively to 0.90 V vs. SCE accompanying the oxidation peak current increased to 0.33 µA , indicating that GO can catalyze the oxidation of tryptophan. However, the oxidation peak potential of tryptophan shifted more negatively to 0.80 V vs. SCE at AgNPs/GO/GCE with the overpotential reduction of 0.25 V compared with bare GCE, and the oxidation peak current significantly increased to 3.15 µA which went up tenfold as compared to GO/GCE. The increasing peak current and lowering peak potential indicate that AgNPs/GO can effectively catalyze the electrochemical oxidation of tryptophan due to a synergistic effect. Moreover, the anodic peak current of AgNPs at 0.38 V vs. SCE decreased while its cathodic peak current at 0.28 V vs. SCE increased a little, which also indicated AgNPs/GO nanocomposite has an electrocatalytic activity for tryptophan (Zhang et al., 2012). Here, the enhanced electrochemical response at AgNPs/GO/GCE could be attributed to the following major factors: (1) Metallic AgNPs themselves have good catalytic ability (Bao et al., 2011). (2) Metallic AgNPs own superior electrical conductivity. (3) Noble metallic nanoparticles exhibit an optical phenomenon known as SERS which can produce electromagnetic field (Shin et al., 2008), and the electromagnetic field from AgNPs would improve diffusion rate of objective molecules and increase effective accumulation scope. (4) Tryptophan has

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an aromatic structure, so there may be π–π stacking between GO and tryptophan, resulting in an increase of the interaction. (5) The as-prepared AgNPs/GO possessed large surface area and excellent conductivity, which would facilitate the accumulation of the tryptophan molecules at the surface of electrode and accelerate the electron transfer between the electrode and species in solution. Finally, no corresponding reduction peaks for tryptophan were observed at the examined electrodes. Therefore, it is very likely that the oxidation of tryptophan is totally irreversible under our experimental conditions.

Fig. 3. CVs of different electrodes in the absence (A) and presence (B) of 0.1 mM tryptophan in 0.1 M acetate buffer (pH 4.5) with scan rate of 100 mV s−1.

3.4. Effect of the modifier Amount

The oxidation current of tryptophan at the prepared sensor can be affected by the amount of modifier on the electrode surface. This can be controlled by using the same volume (5.0 µL) of the suspension with the different concentrations of AgNPs/GO in DMF, casted onto the surface of the GCE. The experiments showed that the oxidation peak current for 0.1 mM tryptophan increased quickly by increasing the concentration of AgNPs/GO suspension deposited onto the surface of the GCE up to 2.0 mg mL–1. A

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further increase caused a gradual decrease in the anodic peak current of tryptophan but with an increase in background current. As a result, 5.0 µL of 2.0 mg mL–1 AgNPs/GO suspension was selected as the optimum amount for preparation of the modified electrode.

3.5. Effect of pH value

The influence of pH on the electrochemical behaviors of tryptophan was investigated by CV at different pH values in 0.1 M acetate buffer (pH: 3.6~5.8) and phosphate buffer (pH: 5.8~8.0). It was found that the peak currents of tryptophan increased from pH 3.6 to pH 4.5 first, and then decreased from pH 4.5 to pH 8.0. Therefore, considering the sensitivity of tryptophan determination, a pH value of 4.5 was chosen for the subsequent analytical experiments. Moreover, the anodic peak potential shifted toward negative potential with the increasing pH values. The phenomenon indicated that the electrochemical reaction of tryptophan is a proton-coupled electron transfer, which was in agreement with the literatures reported by Shahrokhian (Shahrokhian and Fotouhi, 2007), Güney (Güney and Yıldız, 2011), and Wu (Wu et al., 2004). Especially, a good linear relationship was observed between the Epa and pH values in the range of 3.6 to 8.0, and the linear regression equation was expressed as: Epa (V) = –0.0584 pH + 1.0538 (R2 = 0.9966). The slope is 0.058 V pH−1, which was very close to the Nernstian value of 0.059 V pH−1 at 25 0C. According to the equation: −0.058 x/n = −0.059, where n is the transferred electron number and x is the number of hydrogen ions participating in the reaction, the number of transferred electrons was equal with that of hydrogen ions taking part in the electrode reaction.

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3.6. Effect of scan rate

The useful information involving electrochemical mechanisms and kinetic characteristics can usually be obtained from the investigation of CVs at the different potential scan rates. Figure 4A shows the CVs of 0.1 mM tryptophan between 0.5~1.1 V vs. SCE at the AgNPs/GO/GCE when the scan rate varies from 10 to 300 mV s−1. From Fig. 4B of line a, it can be seen that the anodic peak currents (ipa) vary linearly in the selected scan rate range with a slope of (0.0481) and a correlation coefficient of 0.9918, suggesting that the kinetics of electrode reaction are controlled by an adsorption process. A similar behavior is observed at several carbon-based electrodes (Goyal et al., 2011; Güney and Yıldız, 2011; Jiang et al., 2010; Liu et al., 2011) and Fe3O4 nanoparticles modified electrode (Wang et al., 2012). It is interesting to note that the oxidation peak potential (Epa) for tryptophan shifts positively when increasing the scan rates. The relationship between Epa and the natural logarithm of scan rate (lnv) was shown in Fig. 4B of line b. It can be seen that Epa changed linearly vs. lnv with a linear regression equation of Epa (V) = 0.6796 + 0.0269 lnv (R2 = 0.9763) in the range from 10 to 300 mV s−1. As for an irreversible electrode process, Epa is given by the Laviron’s equation (Laviron, 1979):

Epa = E 0 + (

RT RTk 0 RT ) ln( )+( ) ln v (1 − α )nF (1 − α )nF (1 − α )nF

where α is the transfer coefficient, k0 is the standard heterogeneous rate constant of the reaction, n is the number of transferred electrons, v is the scan rate, and E0 is the formal redox potential. Thus, the value of (1-a)n can be easily calculated to be 0.954 from the

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slope of Epa vs. lnv. According to Bard and Faulkner (Bard and Faulkner, 2001), a transfer coefficient is generally between 0.3 and 0.7, and transferred electron number is adapted to positive integer, so α should be 0.523 while n being 2. According to the above results, the electrochemical reaction of tryptophan on this modified electrode was a two-electron two-proton process. The same results were obtained for the electro-oxidation of tryptophan at other different electrodes which were modified with MWCNT/cobalt salophen (Shahrokhian and Fotouhi, 2007), poly(9-aminoacridine) functionalized MWCNT (Güney and Yıldız, 2011), gold nanoparticles/MWCNT (Goyal et al., 2011) and carbon ionic liquid (Jiang et al., 2010). According to the correlative reports (Jin and Lin, 2004; Liu et al., 2011) and the obtained results in this work, two kinds of possible mechanisms for electro-oxidation of tryptophan were proposed (See Fig. S4 in the Supporting information).

Fig. 4. (A) CVs of 0.1 mM tryptophan at AgNPs/GO/GCE with different scan rates (10~300 mV s−1) in pH 4.5 acetate buffer, and the insert is the plot of Epa vs. v; (B) The plots for ipa vs. v (line a) and Epa vs. lnv (line b), respectively. 19

3.7. Effect of accumulation conditions

Accumulation for the electrochemical measurements is an important operation which could affect the amount of analytes on electrode surface, resulting in the improvement of current response. The oxidation peak current of 0.1 mM tryptophan at AgNPs/GO/GCE gradually increased with increasing accumulation time from 0 s to 120 s, and reached the maximum current response at 120 s. Further increasing the accumulation time, there was almost no change in the current response. This phenomenon may be related to the saturated adsorption of tryptophan on the AgNPs/GO film. Accordingly, 120 s was chosen in the following experiments. However, varied accumulation potential in the range of −0.5 to 0.5 V vs. SCE has no sufficient influence on the current response of tryptophan; therefore accumulation under open-circuit potential was used for the preconcentration step.

3.8. Analytical performance and interference study

Differential pulse voltammetry (DPV) is a widely used analytical technique for the enhancement of sensitivity and specificity in quantitative analysis (Shahrokhian and Fotouhi, 2007). Compared with conventional CV process, the DPV mode yields better signal-to-background characteristics. Besides, the peaks of DPV are sharper and better defined at lower concentration of analytes. Thus, determination of tryptophan was carried out by DPV obtained by scanning the potential in the range from 0.5 to 1.1 V vs. SCE at differential pulse step potential of 0.005 V and modulation amplitude of 0.05 V. After the background current declined to a steady value, tryptophan solutions were added into the 20

0.1 M acetate buffer (pH 4.5), and the currents produced as a result of electrocatalytic oxidation of tryptophan was recorded after a previous accumulation time of 120 s at open-circuit potential. From the Fig. 5, it can be seen that the oxidation peak currents increase linearly with tryptophan concentrations over the range of 0.01 μM to 50.0 μM, and 50.0 μM to 800.0 μM, respectively. The linear regression equations can be expressed as ipa (μA) = 2.4554 + 0.2837 c (μM) (R2 = 0.9980) and ipa (μA) = 10.8033 + 0.1456 c (μM) (R2 = 0.9982). A detection limit of 2.0 nM was obtained with the calculation based on signal/noise of 3. From the slopes of calibration plots and the active electrode surface area, the sensitivities of this sensor in the two linear ranges were calculated to be 1.225 A M−1 cm−2 and 0.629 A M−1 cm−2, respectively. In order to make a realistic comparison with previous procedures, the characteristics of different electrochemical sensors for tryptophan are summarized in Table 1. It can be seen that the proposed sensor offered reasonable linear range which is wider than that from most of other sensors. Especially, the detection limit is the lowest in the all. The comparison has confirmed that AgNPs/GO nanocomposite is an appropriate platform for the electrochemical sensing of tryptophan. More importantly, graphene-based materials have the advantages of low production cost and facile preparation procedure over other widely used carbon-based materials (Fan et al., 2011). Unquestionably, graphene derivative and its hybrid materials hold great promise for extensive applications in electrochemical sensors and biosensors. The results of tyrosine interference study (See Fig. S5 in the Supporting information) showed that a relatively high concentration of tyrosine couldn’t affect on the determination of tryptophan.

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Furthermore, uric acid, ascorbic acid, dopamine and glucose as important biological substances often also coexist with tryptophan in human fluids, food processing, pharmaceuticals and clinical analysis. The influence of these interfering substances and other available amino acids was examined by DPV in 0.1 M acetate buffer (pH 4.5) containing 1.0 µM tryptophan. The results suggested that 50-fold concentration of uric acid, ascorbic acid and dopamine had no influence on the signals of tryptophan with deviations of 3.2%, 3.6% and 2.1%, respectively. 100-fold concentration of glucose had no influence on the signal of tryptophan with deviation of 1.3%. 100-fold concentration of lysine, glutamine, valine, threonine, alanine, methionine, arginine, asparagines, serine and aspartic acid had no obvious oxidation peak at AuNPs/GO/GCE, and they had no influence on the signals of tryptophan with all deviations below 3.0%. Thus, the proposed sensor has good anti-interference property, and it clearly exhibited the executive efficiency for the determination of trace amounts of tryptophan in the present of potentially interfering species in complex matrix.

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Fig. 5. (A) DPVs obtained at AgNPs/GO/GCE in 0.1 M acetate buffer (pH 4.5) containing low concentrations of tryptophan (from a to l: 0.01, 0.05, 0.1, 1.0, 3.0, 6.0, 10.0, 15.0, 21.0, 28.0, 38.0 and 50.0 µM, respectively), inset is the calibration plot; (B) DPVs obtained at AgNPs/GO/GCE in 0.1 M acetate buffer (pH 4.5) containing high concentrations of tryptophan (from a to h: 50.0, 100.0, 150.0, 250.0, 350.0, 500.0, 650.0 and 800.0 µM, respectively), inset is the calibration plot.

23

Table 1 Comparison of major characteristics of electrochemical sensors used in the determination of tryptophan. Electrochemical sensors

Technique

MWCNT/cobalt salophen/CNTPE

a

DPV b

Linear

Detection

Correlation

Recovery

range

limit

coefficient

(%)

References

(µM)

(µM)

0.5–50

0.1

0.9988

–

Shahrokhian

0.2–100

0.02

0.9971

99.11–102.3

and

MWCNT/cerium

CA

hexacyanoferrate/GCE

DPV

0.4–14

0.124

0.9991

98.59–100.90

(2007)

DPV

0.09–50

0.08

0.999

92.2–95.6

Fang

acid/CPE

CA

0.085–43

0.023

0.998

98.11–101.70

(2007)

AuNPsd/GCE

DPV

0.5–50

0.05

0.996

–

Raoof

DPV

0.85–63.4

0.56

0.9927

–

(2009)

1–500

0.81

0.9988

92.32–95.30

Li et al. (2010)

SWV

0.5–90

0.025

0.998

96.60–103.28

Gholivand

DPV

0.25–100

0.027

0.9964

–

al. (2011)

CV

0.85–120

0.0185

0.9988

–

Özcan

SWV

5–900

4

0.995

95.6–100.6

Şahin (2012)

CV

1–100

0.2

0.9948

95.0–104.5

Raoof

CuHCF -cysteamine-AuNPs/GW

CA

10–900

6

0.994

–

(2008)

AuNPs/CILEl

DPV

5–140

0.7

0.9971

–

Güney

CA

0.03–2.5

0.01

0.9985

98–104

Yıldız. (2011)

DPV

5–100

3

0.9993

–

Goyal

Nafion/TiO2-graphene/GCE

DPV

2–60

0.6

–

97.2–105.5

(2011)

AuNPs/CNT/GCE

DPV

0.2–4

0.143

0.9992

–

Wu

nano-Au/MWCNT/GCE

DPV

0.8–300

0.5

0.998

–

(2004)

butyrylcholine/GCE

CA

0.05–10

0.01

0.996

–

Prabhu et al.

Nafion/cucurbit[8]uril/GCE

DPV

0.01–50.0;

0.002

0.9980;

99.0–103.0

(2011)

nano-TiO2/ferrocene

carboxylic

c

Ni(II)/ACDA

e

/AuNPs/cysteine/gold

electrode

DPV i

pencil graphite electrode f

4FEPE /CPE g

poly(9-aminoacridine)/MWCNT /GCE AuNPs-MWCNT/ITO

h

MWCNT/GCE j

k

4-aminobenzoic acid/GCE copper-cobalt hexacyanoferrate/GE

β-Cyclodextrin/Fe3O4/GCE

m

50.0–800.0

0.9982

Fotouhi.

et

al.

et

al.

et

and

et

al.

and

et

et

Safavi

al.

al.

and

Co3O4/graphene/GCE

Momeni (2010)

AgNPs/GO/GCE

Huang et al. (2009) Liu

and

Xu

(2007) Fan

et

al.

et

al.

(2011) Guo (2010) Kooshki et al. (2011) Jin

and

Lin.

(2004) Pozo

et

al.

24

(2011) Wang

et

al.

(2012) Ye et al. (2011) This work a

CNTPE: carbon nanotube paste electrode;

b

CA: chronoamperometry;

c

CPE: carbon paste electrode;

d

AuNPs: gold nanoparticles;

e

ACDA:

2-amino-1-cyclopentene-1-dithiocarboxylic acid; f 4FEPE: 1-[4-ferrocenyl ethynyl) phenyl]-1-ethanone; g MWCNT: multi-walled carbon nanotubes; h ITO: indium tin oxide; i SWV: square wave voltammograms; j CuHCF: copper hexacyanoferrate; k GW: graphite-wax; l CILE: carbon ionic liquid electrode;

m

electrode;

3.9. Reproducibility and stability of the sensor The reproducibility of the proposed approach was evaluated by intra- and inter-assay coefficients of variation. The intra-assay precision of this method was evaluated by assaying tryptophan at two concentration levels for twenty replicative measurements using the same sensor, and the inter-assay precision was evaluated by assaying tryptophan at two concentration levels for successive measurements using twenty sensors made independently. The intra-assay variation coefficients of this method were 4.2% and 3.7% at tryptophan concentrations of 0.05 and 0.1 mM respectively, while the inter-assay variation coefficients at these concentrations were 3.9% and 4.5%. Therefore, the sensor showed good repeatability. The stability of the prepared sensor can be maintained by being stored under a clean and dry condition. No obvious decrease in the response of tryptophan was observed in the first 10-day storage. After a 90-day storage period, the sensor retained 92.1% of its initial current response. These results indicated the sensor held satisfactory performances in reproducibility and stability, and it would be applicable for the analysis of real samples.

25

GE: graphite

3.10. Determination tryptophan in real samples

Human serum and pharmaceutical injections were used as models of real samples to evaluate practical utility of the proposed method. The blood samples were pretreated according to the previous report (Gholivand et al., 2011). The amino acid injection samples were purchased from local hospital, and then 10.0 µL of the injections without any pretreatment were directly injected into 10.0 mL of acetate buffer solution (pH 4.5) for assay. The determination of tryptophan in real samples was carried out by the standard addition method in order to prevent any matrix effect, and the results observed are listed in Supplementary 6. The recoveries varied in the range from 99.0% to 102.7% in the case of serum and from 99.0% to 103.0% in the case of injection. The recovery data lie in the acceptable range, thus the developed sensor can be successfully utilized for the determination of tryptophan in complex samples with adequate accuracy.

4. Conclusion

The AuNPs/GO nanocomposite has been firstly synthesized through a green and low-cost approach, and then used for the fabrication of a novel electrochemical sensor. The hybrid nanocomposite film was characterized by SEM, TEM, EDX, FTIR and different electrochemical techniques. The electrochemical studies have demonstrated that the AuNPs/GO possessed synergetic catalytic effect on the oxidation of tryptophan, and the significant increase in peak current and a reduction in peak potential have greatly improved analytical performance of the prepared sensor. The kinetic parameters, such as transferred electron number, electron transfer coefficient, surface coverage and standard 26

heterogeneous rate constant, for the oxidation of tryptophan were achieved. Under optimized conditions, the wider linear ranges and a lower detection limit were obtained. To the best of our knowledge, it is the first time to construct an electrochemical sensor for tryptophan with such a low detection limit (2.0 nM), and its analytical characteristics outperformed the most of reported electrochemical sensors. Furthermore, the nanocomposite favored oxidation peak separation of tryptophan and tyrosine, and the on-site determination of tryptophan in the presence of a high concentration of tyrosine was realized by DPV. Furthermore, AuNPs/GO composite has other fascinating features, including ease of synthesis, low toxicity, surface functionalities and excellent stability, which also make it hold great promise in the electroanalysis and electrocatalysis of biological and organic molecules.

Acknowledgements

This work was kindly supported by the Scientific Research Project of Education Department of Hunan Province (nos.12C0536, 10K010) and the Youth Backbone Teacher Training Program of Hengyang Normal University (2010).

27

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Highlights

1. The silver nanoparticles-graphene oxide nanocomposite was prepared by a green

approach. 2. The nanocomposite was used for preparation of a tryptophan electrochemical

sensor. 3. The prepared sensor exhibited excellent electro-catalysis to tryptophan oxidation.

33