Materials Science and Engineering C 38 (2014) 292–298
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Electrochemical sensing platform for L-CySH based on nearly uniform Au nanoparticles decorated graphene nanosheets Fugui Xu a, Fan Wang a, Duanguang Yang a, Yong Gao a,b,c,⁎, Huaming Li a,b,c,d a
College of Chemistry, Xiangtan University, Xiangtan 411105, Hunan Province, PR China Key Laboratory of Polymeric Materials & Application Technology of Hunan Province, Xiangtan University, Xiangtan 411105, Hunan Province, PR China Key Laboratory of Advanced Functional Polymeric Materials of College of Hunan Province, Xiangtan University, Xiangtan 411105, Hunan Province, PR China d Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, Xiangtan University, Xiangtan 411105, Hunan Province, PR China b c
a r t i c l e
i n f o
Article history: Received 23 October 2013 Received in revised form 23 December 2013 Accepted 13 February 2014 Available online 20 February 2014 Keywords: Au nanoparticles Graphene Modified electrode L-Cysteine Grafting
a b s t r a c t In this study, Au nanoparticles decorated graphene nanosheets were prepared using poly(vinylpyrrolidone) (PVP) covalently functionalized graphene oxide and chloroauric acid as template and Au precursor, respectively. Both the density and the size of Au nanoparticles deposited on the surface of graphene could be adjusted by the PVP grafting density. The graphene–Au hybrid nanosheets were then applied to fabricate a highly sensitive L-cysteine (L-CySH) electrochemical sensing platform. The cyclic voltammetry results showed that the modified glassy carbon electrode with graphene–Au hybrid nanosheets exhibited strong catalytic activity toward the electrooxidation of L-CySH. The current exhibited a widely linear response ranging from 0.1 to 24 μM with a low detection limit under the optimized conditions. The detection limit of L-CySH could reach as low as 20.5 nM (S/N = 3). The enhanced electrochemical performance of the fabricated sensor was attributed to the combination of the excellent conductivity of graphene and strong catalytic property of uniform Au nanoparticles. © 2014 Elsevier B.V. All rights reserved.
1. Introduction L -Cysteine, (R)-2-amino-3-mercaptopropanoic acid, a sulfurcontaining R-amino acid, is one of about 20 amino acids commonly found in natural proteins. L-Cysteine (L-CySH) plays an important role in biological and medical systems as well as in food industries. For example, some clinical situations, such as slowed growth, hair depigmentation, edema, lethargy, and liver damage, are associated with the deficiency in L-CySH [1–4]. Therefore, developing a rapid and selective detection of L-CySH in biological samples is very significant work in both clinical and industrial applications. So far, many methods such as chemiluminescence [5], high performance liquid chromatography (HPLC) [6], fluorimetry [7,8], and electrochemical method [9–11] have been developed for the measurement of L-CySH. By comparison with other technologies, electrochemical method has received considerable attention in recent years due to its inherent virtues, such as simple operation, high sensitivity, excellent selectivity and especially in vivo real-time determination. However, early studies indicated that bare carbon surfaces show very poor activity, even no response for L-CySH determination [11]. Bulk Au and Pt electrodes usually exhibited high overpotential due to surface oxide formation, resulting in a narrow linear range and a low selectivity [12]. An effective way to improve the electrochemical performance is modifying the electrode surface by
⁎ Corresponding author: Tel.: +86 731 58298572; fax: +86 731 58293264. E-mail addresses: [email protected] (Y. Gao), [email protected] (H. Li).
http://dx.doi.org/10.1016/j.msec.2014.02.017 0928-4931/© 2014 Elsevier B.V. All rights reserved.
new functional materials with high effective surface area and excellent catalytic activity. Recently, various chemical modified electrodes such as nanoporous gold [13], boron-doped carbon nanotube [14], polymer film [15] and copper oxide nanoparticles [16] were exploited and successfully applied to detect L-CySH with satisfactory results. Graphene, a single layer of carbon atoms in a closely packed honeycomb two-dimensional lattice, has attracted considerable attention since its discovery in 2004 [17–20]. Owing to remarkable electronic properties and excellent chemical and mechanical stability, graphene was widely used in biosensors, supercapacitors, transistors, flexible electrodes and polymer nanocomposites [21]. Among these, fabrication of sensors with graphene-based hybrid nanostructures, especially graphene–noble metal hybrid nanostructures, is one of the most promising applications, combining the excellent properties of graphene, such as the large surface area and good conductivity, together with the extraordinary catalytic activity of metal nanoparticles. Different noble metals supported on graphene, including Pt, Au, Ag, Pd and Ru, demonstrated enhanced catalytic activities when applied in electroanalysis as catalysts, and these have been summarized in recent reviews [22]. Of all the noble metals, Au nanoparticles are particularly attractive due to their unexpected electronic and catalytic properties as well as biocompatible nature. Graphene–Au hybrid nanomaterials prepared through various strategies were thus widely applied to fabricate electrochemical sensor for many substrates [23–28]. It is well known that the electrochemical performance of a modified electrode is strongly relative with the morphology of a hybrid nanostructure. Therefore,
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fine control over the uniformity and density of Au nanoparticles on the graphene plane as well as the dimension of graphene was crucial for the electrochemical performance of the modified electrode. Herein, we reported a robust method for the synthesis of Au nanoparticles decorated graphene nanosheets using chloroauric acid (HAuCl4) and poly(vinylpyrrolidone) (PVP) covalently functionalized graphene oxide (PGO) as precursor and template, respectively. The AuCl− 4 anions were absorbed first on the PVP chains through coordination effect between PVP and AuCl− 4 [29] and then reduced in situ, generating graphene–Au hybrid nanosheets (Scheme 1). Therefore, the density and the size of Au NPs could be expected to be adjusted by the available PVP polymer chains. The aqueous dispersion of graphene–Au hybrid nanosheets was then directly coated onto the surface of the glassy carbon electrode (GCE) to prepare chemical modified electrode, which was then utilized to detect L-CySH. The electrochemical behaviors of L-CySH on the modified electrode were investigated. 2. Experimental 2.1. Materials PVP with average molecular weight (Mn = 5700 g/mol, PDI = 1.67) and PGO were synthesized according to our report earlier [30]. Sodium borohydride (NaBH4) and L-CySH were purchased from Aldrich. HAuCl4 (47%, Au basis) was purchased from Aladdin (China), which was diluted into 0.01% aqueous solution prior to use. All the solutions were prepared with doubly distilled water.
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NaBH4 . The final concentration of the dispersion was fixed to be 0.2 mg/mL. 2.3. Preparation of graphene–Au/GCE The GCE was polished sequentially with sandpaper and 0.05 mm alumina slurry on polishing cloth to produce a mirror-like surface. It was then sonicated sequently in HNO3/H2O (1:1, v/v), EtOH/H2O (1:1, v/v) and doubly distilled water, respectively. For the preparation of the graphene–Au modified GCE (graphene–Au/GCE), 10 μL of the graphene–Au hybrid nanosheet aqueous suspension (0.2 mg/mL) was deposited onto the freshly prepared GCE surface with a pipette. The modified electrode was dried under ambient conditions. 2.4. Characterization and test Thermogravimetric analysis (TGA) was carried out on a STA 449C instrument under a flowing nitrogen atmosphere. Transmission electron microscopy (TEM) analysis was performed on a JEOL JEM-2010 electron microscope. All electrochemical experiments were performed with CHI760D electrochemical workstation (Shanghai Chenhua Co., China) with a conventional three-electrode cell. A bare GCE or modified GCE was used as the working electrode. A saturated calomel electrode and a platinum wire electrode were used as the reference electrode and auxiliary electrode, respectively. 3. Results and discussion
2.2. Synthesis of graphene–Au hybrid nanosheets
3.1. Synthesis of graphene–Au hybrid nanosheets
In a typical experiment, 10 mg of PGO was dispersed first in 20 mL of water, affording a dark and stabilized dispersion. And then, 0.2 mL of HAuCl4 aqueous solution (0.01%) was slowly added into the dispersion, followed by continuous stirring for 2 h at room temperature. Subsequently, 5 mL of fresh NaBH4 aqueous solution (200 mM) was added with assistance of ultrasound. After 1 h of treating, the obtained black dispersion was further dialyzed against water to remove the remaining
At present, graphene can be obtained in bulk quantity by chemical reduction of GO in a solution. However, the as-synthesized graphene sheets tend to form irreversible agglomerates or even restack to form graphite through π–π stacking and van der Waals interactions [31]. Grafting is an efficient approach to obtain single graphene sheet, and the grafted functional small molecules or polymer chain could act as “spacer” to prevent the restack of produced graphene during chemical
Scheme 1. Synthesis of graphene–Au hybrid nanosheets.
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reduction process. In our previous study [30], we reported a facile method for the preparation of PGO, which was then used as template to prepare graphene/Ni(OH)2 (PG/Ni(OH)2) hybrid nanomaterials. Here, we followed that procedure to prepare graphene–Au hybrid nanosheets using PGO and HAuCl4 as template and precursor, respectively. The detailed operation was described in the Experimental section. The AuCl− 4 anions were coordinated first with PVP [29] and then in situ transferred into Au nanoparticles (NPs), which were stabilized by grafted PVP polymer chains. Therefore, the further aggregation of the formed Au NPs was prohibited due to the steric hindrance, leading to a controllable particle size. On the other hand, the density of Au nanoparticles on the surface of graphene was also determined by the available PVP polymer chains. Accompanying the reduction of AuCl− 4 , GO was also reduced to graphene simultaneously, and the overall procedure was described in Scheme 1. For simplicity, the other oxygencontaining groups such as hydroxyl group, carboxyl group as well as carbonyl group were omitted. The obtained graphene–Au hybrid nanosheets could be readily dispersed in water. Fig. 1 shows the TGA curves of GO, PGO and PVP homopolymer. As shown in Fig. 1A, GO is thermally unstable and exhibits more than 30% of mass loss below 200 °C, which is attributed to the pyrolysis of labile oxygen-containing groups [32]. As for PGO (Fig. 1B and C), TGA analysis all showed a two-step thermal decomposition. A continuous mass loss from 200 to 450 °C was caused by the pyrolysis of the physically bonded water molecules and remaining oxygen-containing groups. The second mass loss, from 480 to 550 °C for curve B and from 505 to 560 °C for curve C, was attributed to the degradation of the grafted PVP polymer. Clearly, the mass loss of the two PGO samples was different. According to TGA analysis, the content of grafted PVP was about 8.0% and 16.0%, respectively. The two PGO samples were thus designated as PGO5700/8 and PGO5700/16, respectively. The subscript number denoted the average molecular weight of grafted PVP polymer and the mass percent of PVP in PGO sample. The morphologies of pristine GO and graphene–Au hybrid nanosheets were characterized by TEM, which were shown in Fig. 2. As shown in Fig. 2A, GO sheets are very thin and have wrinkles and folded regions, indicating the random overlay of the individual sheets. As for graphene–Au hybrid nanosheets, Au NPs deposited on the surface of graphene nanosheets (Fig. 2B and C) were clearly observed. However, the size of Au nanoparticles synthesized with different PGO templates exhibited distinctive difference. Almost uniform Au NPs whose mean size was estimated to be about 9 nm according to TEM based on the sizes of more than 100 NPs (inset in Fig. 2B) were obtained using PGO5700/16 as template. In contrast, the size of Au NPs was almost 30 nm when PGO5700/8 was used as template. At the same time, an
obvious reduction in the density of Au NPs on the surface of graphene nanosheets was observed (Fig. 2C). These suggested that both the density and the size of Au NPs could be adjusted by the PVP grafted on the surface of graphene. For convenience, the obtained two kinds of graphene–Au nanosheets obtained were designated as graphene–Au9 and graphene–Au30, respectively. The subscript number denoted the average size of Au nanoparticles. 3.2. Electrooxidation of L-CySH on graphene–Au/GCE The electronic conductivities of the solid electrodes including bare GCE, graphene modified GCE (G/GCE; note: graphene means the reduction product of PGO5700/16) and graphene–Au/GCE were evaluated first by electrochemical impedance spectroscopy (EIS). Generally, the linear part in the EIS represents the diffusion-limited process, while the semicircle portion corresponds to the electron transfer-limited process. The electron transfer resistance (Ret) at electrode surface is equal to the semicircle diameter [33]. The Nyquist diagrams of different electrodes 4− solution containing 0.1 M KCl were shown in in 5.0 mM Fe(CN)3− 6 / Fig. 3. As can be seen, the Ret of the bare GCE was about 300 Ω, while the Ret values of G/GCE, graphene–Au9/GCE and graphene–Au30/GCE were 1340, 880 and 580 Ω after the bare electrode was coated with graphene and graphene–Au hybrid nanosheets, respectively. The remarkable increase in Ret value suggested that the bare GCE has been successfully modified. The electrooxidation behavior of L-CySH on the solid electrodes was investigated with cyclic voltammograms (CVs). Fig. 4 showed the CV responses of the bare GCE, G/GCE, graphene–Au9/GCE, and graphene– Au30/GCE in the presence of 2.0 mM L-CySH in phosphate buffered saline (PBS) (pH 7.5, 0.10 M) at a scan rate of 50 mV/s. As shown in Fig. 4, the electrooxidation of L-CySH took place at a relatively high potential (N0.6 V) with a small current response on bare GCE and G/GCE (curves a and d), while a well defined oxidation peak with an increased current response for graphene–Au/GCE was observed (curves c and e), and the enhanced response was ascribed to the improved electronic transferring rate on electrode surface and an enlarged surface area caused by the three-dimensional graphene–Au hybrid nanosheets. However, the electrochemical behavior of L-CySH on different graphene–Au/GCE was distinct. With the decrease in size and the increase in density of Au NPs on graphene, the oxidation peak potential (Ep) of L-CySH on electrodes shifted toward negative potential accompanied with an enhanced current response. For example, by comparison with the curves c and e, the differences of Ep and peak current (Ip) were about 60 mV and 5.1 μA, respectively. This might be owing to the size-dependent behavior of Au NPs [34]. Therefore, the following electrochemical measurements of L-CySH were carried out using graphene–Au9/GCE. On the other hand, no signal was observed on the graphene–Au9/GCE in the absence of L-CySH under identical conditions (curve b), indicating a strong catalytic effect of graphene–Au hybrid nanosheets toward the electrooxidation of L-CySH. No reduction peak of L-CySH was observed in all CV curves, suggesting a completely irreversible oxidation process of L-CySH on the solid electrodes. These results are consistent with some previous reports [14,16,28]. The effective surface area of the GCE and graphene–Au9/GCE was also estimated by CV based on Randles–Sevcik equation [35]. The experiments were carried out in 0.1 mM K3[Fe(CN)6] solution containing 1.0 mM KCl. According to Randles–Sevcik: 5
Ip ¼ 2:69 10 n
Fig. 1. TGA curves of GO (A), PGO (B and C) with different contents of PVP and PVP homopolymer (D).
3=2
AD0
1=2
Cυ
1=2
where the standard diffusion coefficient (D0) of K3[Fe(CN)6] at 25 °C is 7.6 × 10−6 cm2/s, and the number of transferred electrons (n) is 1. The effective surface areas of the bare GCE and graphene–Au/GCE were estimated to be 0.143 cm2 and 0.563 cm2, respectively, a 293% increase in surface area after coating by graphene–Au9 hybrid nanosheets.
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Fig. 2. TEM images of GO (A) and graphene–Au hybrid nanosheets (B, C) synthesized with different PGO templates. Inset: The size distribution of Au nanoparticles deposited on the surface of graphene.
3.3. Effect of pH The electrochemical oxidation mechanism of L-CySH on a solid electrode was proposed by Ralph et al. [36] as the following process: CySH ↔ CyS− + H+
CyS− ↔ CyS• + e
decrease of Ip with the further increase of pH from 6.5 to 8 might be ascribed to the electrostatic repelling effect between the negative graphene and CyS−. At the same time, Ep shifted constantly toward negative potential with the progressive increase of pH value ranging from 6.0 to 8.0. This phenomenon agreed with the previous reports [38,39]. The reason was that the oxidation process involved the deprotonation process, which is facilitated at higher pH values [38]. Considering the high current response and low potential conditions as well as physiologic condition, pH 7.5 was chosen for detecting the L-CySH. 3.4. Effect of scanning rate
•
2CyS ↔ CySSCy. According to the mechanism, the proton takes part in the electrooxidation process of L-CySH. Therefore, the pH of solution should have a profound effect on the electrochemical behavior of L-CySH. The followed CV measurements also suggested that L-CySH oxidation on an electrode exhibited a pH dependence, and the results were shown in Fig. 5. As shown in Fig. 5, Ip increased first with the increase of pH from 6 to 6.5, and then decreased with the further increase of pH from 6.5 to 8. The maximum peak current was observed at pH 7.0. Moreover, the voltammetric response tends toward a plateau like curve at low pH and well-shaped curve at high pH (Fig. 5A), and the reason was that the chemical reaction on electrode surface was dominated by the supply of CyS− [37]. Since the ionization of L-CySH in aqueous solution largely depends on pH, the main electroactive species is in the CyS− form when the pH value is higher than the isoelectric point of L-CySH of 5.02. Therefore, Ip increased with the increase of pH from 6 to 6.5. The
Fig. 3. The Nyquist plots for bare GCE, graphene/GCE, graphene–Au9/GCE, and graphene– Au30/GCE.
Cyclic voltammogram behaviors of L-CySH on graphene–Au9/GCE at different scan rates (υ) have been investigated, and the results were shown in Fig. 6A. It is found that Ep shifted positively accompanied with the increase of peak current upon the increase of υ. The deviation of Ep was a result of incomplete reaction under higher υ. A good linear relationship between the peak current (Ip ) and υ1/2 was obtained within the range of 40–300 mV/s, as indicated in Fig. 6B. The result indicated that the L-CySH electrooxidation was controlled by a diffusion process. The linear regression equation was I (μA) = 0.1341υ1/2 (mV/s) 1/2 − 3.0136 (R = 0.997). As a totally irreversible oxidation process, the Ep could be represented by the equation [40]:
Ep ¼ A þ
2:3RT lnυ ð1−αÞnF
Fig. 4. Cyclic voltammograms of GCE (a), graphene–Au9/GCE (c), graphene–Au30/GCE (e) and graphene/GCE (d) in the presence of 2 mmol/L-CySH and cyclic voltammogram of graphene–Au9/GCE (b) in the absence of L-CySH at a scan rate of 50 mV/s.
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Fig. 5. (A) Cyclic voltammogram response of graphene–Au9/GCE on 2.0 mmol/L L-CySH in 0.1 M PBS of different pH: 6.0, 6.5, 7.0, 7.5, and 8.0; (B) effects of pH on the peak current and potential. Scan rate: 50 mV/s.
where A is a constant, which is related with the formal electrode potential (E0) and standard rate constant at E0. The transfer coefficient (α), which characterizes the effect of electrochemical potential on activation energy of an electrochemical reaction, can be calculated according to the slope of Ep-ln υ line. The peak potential for L-CySH oxidation at the graphene–Au9/GCE versus ln υ was plotted, as indicated in Fig. 6C. A linear equation of E (V) = 0.2518 + 0.0611 ln υ (R = 0.999) was obtained. The transfer coefficient (α) is thus calculated to be 0.52 (n = 2), which was smaller than those reported earlier [38,39,41].
The lower α value suggested that the oxidation of L-CySH proceeded more easily on graphene–Au 9 /GCE, and this could be explained that conductive graphene facilitated the electrons to transfer into the electrode. 3.5. Amperometric response of graphene–Au9/GCE to L-CySH The amperometric response of the modified electrode to L-CySH was investigated by successive additions of L-CySH solution to a continuous stirring electrolyte solution in an electrochemical cell under the optimized conditions. The applied constant potential for amperometry at the working electrode was chosen first on the basis of the electrochemical behavior of the graphene–Au9/GCE. Fig. 7A showed the amperometric response of 10 μM L-CySH over a prolonged duration (600 s) at four different applied potentials of 0.2, 0.35, 0.45 and 0.6 V. Rapid and well-defined current responses could all be observed from the amperometric curves at different applied potentials. The stable current response with low background noise could be obtained at 0.45 V. Therefore, the subsequent amperometric measurement was performed at applied constant potential of 0.45 V. Fig. 7B showed the typical current–time responses of the modified electrodes upon successive addition of L-CySH at low concentrations to PBS solution. An obvious steady-state current response was observed when L-CySH concentration reached 0.1 μM, as shown in Fig. 7B (inset). The asprepared sensors showed a very rapid response to the change of L-CySH concentrations, and a steady-state current can be achieved within 5 s. This is attributed to the rapid diffusion of L-CySH molecules from the solution to the modified electrode. The calibration curve for the graphene–Au9/GCE sensor was shown in Fig. 7C. The calibration curve between the peak current (Ip) and the L-CySH concentration (C) can be described by the equation I (μA) = 0.0138 + 0.0250C (μM) (R2 = 0.999) for the 0.1–25 μM. The detection limit could reach as low as 20.5 nM (S/N = 3) using the calibration equation obtained for low concentration range, which was lower than or close to those reported previously [15,16,39,41]. Such a low detection limit was further verified by chronoamperometric response measurements. As shown in Fig. 7D, an obvious current response was found by successively adding 20.5 nM of L-CySH in the 0.1 M PBS of pH 7.5 solution. 3.6. Reproducibility, stability and anti-interference property of the graphene–Au9/GCE
Fig. 6. (A) Cyclic voltammograms of graphene–Au9/GCE in 0.1 M PBS containing 2.0 mmol/L L-CySH at various scan rates: 40, 50, 60, 80, 100, 120, 140, 160, 180, 200, 250 and 300 mV/s. (B) The plot of peak current vs. υ1/2. (C) The relationship between the peak potential (Ep) and the ln υ.
The reproducibility and stability of the sensor were evaluated by cyclic voltammetry. The relative standard deviation of the modified electrode response to 2 mM L-CySH is 3.3% after 10 successive measurements with the same modified electrode, showing high reproducibility. When the fabricated sensor was stored at 4 °C in a refrigerator for
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Fig. 7. (A) Current–time curve at different applied potentials: 0.2, 0.35, 0.45 and 0.6 V; (B) current–time response obtained at graphene–Au9/GCE for the addition of 0.1–39 μM L-CySH into acutely stirred 0.1 M pH 7.5 PBS. Applied potential: 0.45 V; (C) linear calibration curve in the range of 0.1–39 μM; (D) current–time response obtained at graphene–Au9/GCE for the addition of 20.5 nM of L-CySH into acutely stirred 0.1 M pH 7.5 PBS. Applied potential: 0.45 V. Inset: the magnification of 1560–1820 s in (B).
10 days, the current response was approximately 93% of its original response, which could be mainly attributed to the chemical stability of Au NPs and graphene. To demonstrate the selectivity of our fabricated sensor, some interference found in biological fluids was investigated. The interference tests were carried out by amperometric method at a constant applied potential of 0.45 V. Typical organic compounds such as arginine, tyrosine, glutamic acid, glucose, urea, and K+, Na+, and Ca2 + were examined during amperometric response for L-CySH. As shown in Fig. 8, the influences of these existing species on current responses of L-CySH were negligible.
3.7. Determination of L-CySH in real samples The graphene–Au9/GCE was applied to determine L-CySH in the presence of human urine. The recoveries of L-CySH are determined by standard addition, and the corresponding results are given in Table 1. The recovery of four samples is found to be in the range of 96.2%– 103.6%, revealing that the graphene–Au9/GCE is very reliable and sensitive in the determination of L-CySH.
4. Conclusions In this study, Au nanoparticles were successfully deposited on the surface of graphene using PGO as template. The density and size of Au nanoparticles on graphene nanosheets could be tuned by the content of grafted PVP polymer. Nearly uniform Au NPs with high density could be obtained using PGO5700/16 as template. The graphene–Au hybrid nanosheets showed excellent catalytic capability toward the electrooxidation of L-CySH. The fabricated L-CySH electrochemical sensor, graphene–Au/GCE, exhibited enhanced electrochemical properties, such as low detection limit, good stability, resistance to interference and satisfactory recovery, and these were attributed to the synergistic effects of the conductive graphene and the uniform Au nanoparticles.
Table 1 Determination of L-CySH in urine samples using the graphene–Au9/GCE.
Fig. 8. Current responses obtained at the graphene–Au9/GCE in 0.1 M pH 7.5 PBS for the ordinal additions (indicated by arrows) of 5 μM of L-CySH, 5 μM of arginine, 5 μM of glutamic acid, 5 μM of tyrosine, 5 mM of urea, 5 mM of glucose, and 5 mM of Ca2+, K+, and Na+, 5 μM of L-CySH. Applied potential: 0.45 V.
Samples
Amount added (μM)
Amount found (μM)
Recovery (%)
Human urine
5 10 15 20
5.18 10.27 14.76 19.24
103.6% 102.7% 98.4% 96.2%
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Acknowledgments The authors thank the financial supports from the National Natural Science Foundation of China (21174118), the Key Project of Education Department of Hunan Province (12A134), the Department of Science and Technology of Hunan Province Project (2013FJ3035), the Open Project Program of Key Laboratory of Advanced Functional Polymeric Materials of Hunan Province (12K050) and the High-tech Project of Development and Reform Commission of Hunan Province. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Electrochemical sensing platform for L-CySH based on nearly uniform Au nanoparticles decorated graphene nanosheets Fugui Xu a, Fan Wang a, Duanguang Yang a, Yong Gao a,b,c,⁎, Huaming Li a,b,c,d a
College of Chemistry, Xiangtan University, Xiangtan 411105, Hunan Province, PR China Key Laboratory of Polymeric Materials & Application Technology of Hunan Province, Xiangtan University, Xiangtan 411105, Hunan Province, PR China Key Laboratory of Advanced Functional Polymeric Materials of College of Hunan Province, Xiangtan University, Xiangtan 411105, Hunan Province, PR China d Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, Xiangtan University, Xiangtan 411105, Hunan Province, PR China b c
a r t i c l e
i n f o
Article history: Received 23 October 2013 Received in revised form 23 December 2013 Accepted 13 February 2014 Available online 20 February 2014 Keywords: Au nanoparticles Graphene Modified electrode L-Cysteine Grafting
a b s t r a c t In this study, Au nanoparticles decorated graphene nanosheets were prepared using poly(vinylpyrrolidone) (PVP) covalently functionalized graphene oxide and chloroauric acid as template and Au precursor, respectively. Both the density and the size of Au nanoparticles deposited on the surface of graphene could be adjusted by the PVP grafting density. The graphene–Au hybrid nanosheets were then applied to fabricate a highly sensitive L-cysteine (L-CySH) electrochemical sensing platform. The cyclic voltammetry results showed that the modified glassy carbon electrode with graphene–Au hybrid nanosheets exhibited strong catalytic activity toward the electrooxidation of L-CySH. The current exhibited a widely linear response ranging from 0.1 to 24 μM with a low detection limit under the optimized conditions. The detection limit of L-CySH could reach as low as 20.5 nM (S/N = 3). The enhanced electrochemical performance of the fabricated sensor was attributed to the combination of the excellent conductivity of graphene and strong catalytic property of uniform Au nanoparticles. © 2014 Elsevier B.V. All rights reserved.
1. Introduction L -Cysteine, (R)-2-amino-3-mercaptopropanoic acid, a sulfurcontaining R-amino acid, is one of about 20 amino acids commonly found in natural proteins. L-Cysteine (L-CySH) plays an important role in biological and medical systems as well as in food industries. For example, some clinical situations, such as slowed growth, hair depigmentation, edema, lethargy, and liver damage, are associated with the deficiency in L-CySH [1–4]. Therefore, developing a rapid and selective detection of L-CySH in biological samples is very significant work in both clinical and industrial applications. So far, many methods such as chemiluminescence [5], high performance liquid chromatography (HPLC) [6], fluorimetry [7,8], and electrochemical method [9–11] have been developed for the measurement of L-CySH. By comparison with other technologies, electrochemical method has received considerable attention in recent years due to its inherent virtues, such as simple operation, high sensitivity, excellent selectivity and especially in vivo real-time determination. However, early studies indicated that bare carbon surfaces show very poor activity, even no response for L-CySH determination [11]. Bulk Au and Pt electrodes usually exhibited high overpotential due to surface oxide formation, resulting in a narrow linear range and a low selectivity [12]. An effective way to improve the electrochemical performance is modifying the electrode surface by
⁎ Corresponding author: Tel.: +86 731 58298572; fax: +86 731 58293264. E-mail addresses: [email protected] (Y. Gao), [email protected] (H. Li).
http://dx.doi.org/10.1016/j.msec.2014.02.017 0928-4931/© 2014 Elsevier B.V. All rights reserved.
new functional materials with high effective surface area and excellent catalytic activity. Recently, various chemical modified electrodes such as nanoporous gold [13], boron-doped carbon nanotube [14], polymer film [15] and copper oxide nanoparticles [16] were exploited and successfully applied to detect L-CySH with satisfactory results. Graphene, a single layer of carbon atoms in a closely packed honeycomb two-dimensional lattice, has attracted considerable attention since its discovery in 2004 [17–20]. Owing to remarkable electronic properties and excellent chemical and mechanical stability, graphene was widely used in biosensors, supercapacitors, transistors, flexible electrodes and polymer nanocomposites [21]. Among these, fabrication of sensors with graphene-based hybrid nanostructures, especially graphene–noble metal hybrid nanostructures, is one of the most promising applications, combining the excellent properties of graphene, such as the large surface area and good conductivity, together with the extraordinary catalytic activity of metal nanoparticles. Different noble metals supported on graphene, including Pt, Au, Ag, Pd and Ru, demonstrated enhanced catalytic activities when applied in electroanalysis as catalysts, and these have been summarized in recent reviews [22]. Of all the noble metals, Au nanoparticles are particularly attractive due to their unexpected electronic and catalytic properties as well as biocompatible nature. Graphene–Au hybrid nanomaterials prepared through various strategies were thus widely applied to fabricate electrochemical sensor for many substrates [23–28]. It is well known that the electrochemical performance of a modified electrode is strongly relative with the morphology of a hybrid nanostructure. Therefore,
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fine control over the uniformity and density of Au nanoparticles on the graphene plane as well as the dimension of graphene was crucial for the electrochemical performance of the modified electrode. Herein, we reported a robust method for the synthesis of Au nanoparticles decorated graphene nanosheets using chloroauric acid (HAuCl4) and poly(vinylpyrrolidone) (PVP) covalently functionalized graphene oxide (PGO) as precursor and template, respectively. The AuCl− 4 anions were absorbed first on the PVP chains through coordination effect between PVP and AuCl− 4 [29] and then reduced in situ, generating graphene–Au hybrid nanosheets (Scheme 1). Therefore, the density and the size of Au NPs could be expected to be adjusted by the available PVP polymer chains. The aqueous dispersion of graphene–Au hybrid nanosheets was then directly coated onto the surface of the glassy carbon electrode (GCE) to prepare chemical modified electrode, which was then utilized to detect L-CySH. The electrochemical behaviors of L-CySH on the modified electrode were investigated. 2. Experimental 2.1. Materials PVP with average molecular weight (Mn = 5700 g/mol, PDI = 1.67) and PGO were synthesized according to our report earlier [30]. Sodium borohydride (NaBH4) and L-CySH were purchased from Aldrich. HAuCl4 (47%, Au basis) was purchased from Aladdin (China), which was diluted into 0.01% aqueous solution prior to use. All the solutions were prepared with doubly distilled water.
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NaBH4 . The final concentration of the dispersion was fixed to be 0.2 mg/mL. 2.3. Preparation of graphene–Au/GCE The GCE was polished sequentially with sandpaper and 0.05 mm alumina slurry on polishing cloth to produce a mirror-like surface. It was then sonicated sequently in HNO3/H2O (1:1, v/v), EtOH/H2O (1:1, v/v) and doubly distilled water, respectively. For the preparation of the graphene–Au modified GCE (graphene–Au/GCE), 10 μL of the graphene–Au hybrid nanosheet aqueous suspension (0.2 mg/mL) was deposited onto the freshly prepared GCE surface with a pipette. The modified electrode was dried under ambient conditions. 2.4. Characterization and test Thermogravimetric analysis (TGA) was carried out on a STA 449C instrument under a flowing nitrogen atmosphere. Transmission electron microscopy (TEM) analysis was performed on a JEOL JEM-2010 electron microscope. All electrochemical experiments were performed with CHI760D electrochemical workstation (Shanghai Chenhua Co., China) with a conventional three-electrode cell. A bare GCE or modified GCE was used as the working electrode. A saturated calomel electrode and a platinum wire electrode were used as the reference electrode and auxiliary electrode, respectively. 3. Results and discussion
2.2. Synthesis of graphene–Au hybrid nanosheets
3.1. Synthesis of graphene–Au hybrid nanosheets
In a typical experiment, 10 mg of PGO was dispersed first in 20 mL of water, affording a dark and stabilized dispersion. And then, 0.2 mL of HAuCl4 aqueous solution (0.01%) was slowly added into the dispersion, followed by continuous stirring for 2 h at room temperature. Subsequently, 5 mL of fresh NaBH4 aqueous solution (200 mM) was added with assistance of ultrasound. After 1 h of treating, the obtained black dispersion was further dialyzed against water to remove the remaining
At present, graphene can be obtained in bulk quantity by chemical reduction of GO in a solution. However, the as-synthesized graphene sheets tend to form irreversible agglomerates or even restack to form graphite through π–π stacking and van der Waals interactions [31]. Grafting is an efficient approach to obtain single graphene sheet, and the grafted functional small molecules or polymer chain could act as “spacer” to prevent the restack of produced graphene during chemical
Scheme 1. Synthesis of graphene–Au hybrid nanosheets.
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reduction process. In our previous study [30], we reported a facile method for the preparation of PGO, which was then used as template to prepare graphene/Ni(OH)2 (PG/Ni(OH)2) hybrid nanomaterials. Here, we followed that procedure to prepare graphene–Au hybrid nanosheets using PGO and HAuCl4 as template and precursor, respectively. The detailed operation was described in the Experimental section. The AuCl− 4 anions were coordinated first with PVP [29] and then in situ transferred into Au nanoparticles (NPs), which were stabilized by grafted PVP polymer chains. Therefore, the further aggregation of the formed Au NPs was prohibited due to the steric hindrance, leading to a controllable particle size. On the other hand, the density of Au nanoparticles on the surface of graphene was also determined by the available PVP polymer chains. Accompanying the reduction of AuCl− 4 , GO was also reduced to graphene simultaneously, and the overall procedure was described in Scheme 1. For simplicity, the other oxygencontaining groups such as hydroxyl group, carboxyl group as well as carbonyl group were omitted. The obtained graphene–Au hybrid nanosheets could be readily dispersed in water. Fig. 1 shows the TGA curves of GO, PGO and PVP homopolymer. As shown in Fig. 1A, GO is thermally unstable and exhibits more than 30% of mass loss below 200 °C, which is attributed to the pyrolysis of labile oxygen-containing groups [32]. As for PGO (Fig. 1B and C), TGA analysis all showed a two-step thermal decomposition. A continuous mass loss from 200 to 450 °C was caused by the pyrolysis of the physically bonded water molecules and remaining oxygen-containing groups. The second mass loss, from 480 to 550 °C for curve B and from 505 to 560 °C for curve C, was attributed to the degradation of the grafted PVP polymer. Clearly, the mass loss of the two PGO samples was different. According to TGA analysis, the content of grafted PVP was about 8.0% and 16.0%, respectively. The two PGO samples were thus designated as PGO5700/8 and PGO5700/16, respectively. The subscript number denoted the average molecular weight of grafted PVP polymer and the mass percent of PVP in PGO sample. The morphologies of pristine GO and graphene–Au hybrid nanosheets were characterized by TEM, which were shown in Fig. 2. As shown in Fig. 2A, GO sheets are very thin and have wrinkles and folded regions, indicating the random overlay of the individual sheets. As for graphene–Au hybrid nanosheets, Au NPs deposited on the surface of graphene nanosheets (Fig. 2B and C) were clearly observed. However, the size of Au nanoparticles synthesized with different PGO templates exhibited distinctive difference. Almost uniform Au NPs whose mean size was estimated to be about 9 nm according to TEM based on the sizes of more than 100 NPs (inset in Fig. 2B) were obtained using PGO5700/16 as template. In contrast, the size of Au NPs was almost 30 nm when PGO5700/8 was used as template. At the same time, an
obvious reduction in the density of Au NPs on the surface of graphene nanosheets was observed (Fig. 2C). These suggested that both the density and the size of Au NPs could be adjusted by the PVP grafted on the surface of graphene. For convenience, the obtained two kinds of graphene–Au nanosheets obtained were designated as graphene–Au9 and graphene–Au30, respectively. The subscript number denoted the average size of Au nanoparticles. 3.2. Electrooxidation of L-CySH on graphene–Au/GCE The electronic conductivities of the solid electrodes including bare GCE, graphene modified GCE (G/GCE; note: graphene means the reduction product of PGO5700/16) and graphene–Au/GCE were evaluated first by electrochemical impedance spectroscopy (EIS). Generally, the linear part in the EIS represents the diffusion-limited process, while the semicircle portion corresponds to the electron transfer-limited process. The electron transfer resistance (Ret) at electrode surface is equal to the semicircle diameter [33]. The Nyquist diagrams of different electrodes 4− solution containing 0.1 M KCl were shown in in 5.0 mM Fe(CN)3− 6 / Fig. 3. As can be seen, the Ret of the bare GCE was about 300 Ω, while the Ret values of G/GCE, graphene–Au9/GCE and graphene–Au30/GCE were 1340, 880 and 580 Ω after the bare electrode was coated with graphene and graphene–Au hybrid nanosheets, respectively. The remarkable increase in Ret value suggested that the bare GCE has been successfully modified. The electrooxidation behavior of L-CySH on the solid electrodes was investigated with cyclic voltammograms (CVs). Fig. 4 showed the CV responses of the bare GCE, G/GCE, graphene–Au9/GCE, and graphene– Au30/GCE in the presence of 2.0 mM L-CySH in phosphate buffered saline (PBS) (pH 7.5, 0.10 M) at a scan rate of 50 mV/s. As shown in Fig. 4, the electrooxidation of L-CySH took place at a relatively high potential (N0.6 V) with a small current response on bare GCE and G/GCE (curves a and d), while a well defined oxidation peak with an increased current response for graphene–Au/GCE was observed (curves c and e), and the enhanced response was ascribed to the improved electronic transferring rate on electrode surface and an enlarged surface area caused by the three-dimensional graphene–Au hybrid nanosheets. However, the electrochemical behavior of L-CySH on different graphene–Au/GCE was distinct. With the decrease in size and the increase in density of Au NPs on graphene, the oxidation peak potential (Ep) of L-CySH on electrodes shifted toward negative potential accompanied with an enhanced current response. For example, by comparison with the curves c and e, the differences of Ep and peak current (Ip) were about 60 mV and 5.1 μA, respectively. This might be owing to the size-dependent behavior of Au NPs [34]. Therefore, the following electrochemical measurements of L-CySH were carried out using graphene–Au9/GCE. On the other hand, no signal was observed on the graphene–Au9/GCE in the absence of L-CySH under identical conditions (curve b), indicating a strong catalytic effect of graphene–Au hybrid nanosheets toward the electrooxidation of L-CySH. No reduction peak of L-CySH was observed in all CV curves, suggesting a completely irreversible oxidation process of L-CySH on the solid electrodes. These results are consistent with some previous reports [14,16,28]. The effective surface area of the GCE and graphene–Au9/GCE was also estimated by CV based on Randles–Sevcik equation [35]. The experiments were carried out in 0.1 mM K3[Fe(CN)6] solution containing 1.0 mM KCl. According to Randles–Sevcik: 5
Ip ¼ 2:69 10 n
Fig. 1. TGA curves of GO (A), PGO (B and C) with different contents of PVP and PVP homopolymer (D).
3=2
AD0
1=2
Cυ
1=2
where the standard diffusion coefficient (D0) of K3[Fe(CN)6] at 25 °C is 7.6 × 10−6 cm2/s, and the number of transferred electrons (n) is 1. The effective surface areas of the bare GCE and graphene–Au/GCE were estimated to be 0.143 cm2 and 0.563 cm2, respectively, a 293% increase in surface area after coating by graphene–Au9 hybrid nanosheets.
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Fig. 2. TEM images of GO (A) and graphene–Au hybrid nanosheets (B, C) synthesized with different PGO templates. Inset: The size distribution of Au nanoparticles deposited on the surface of graphene.
3.3. Effect of pH The electrochemical oxidation mechanism of L-CySH on a solid electrode was proposed by Ralph et al. [36] as the following process: CySH ↔ CyS− + H+
CyS− ↔ CyS• + e
decrease of Ip with the further increase of pH from 6.5 to 8 might be ascribed to the electrostatic repelling effect between the negative graphene and CyS−. At the same time, Ep shifted constantly toward negative potential with the progressive increase of pH value ranging from 6.0 to 8.0. This phenomenon agreed with the previous reports [38,39]. The reason was that the oxidation process involved the deprotonation process, which is facilitated at higher pH values [38]. Considering the high current response and low potential conditions as well as physiologic condition, pH 7.5 was chosen for detecting the L-CySH. 3.4. Effect of scanning rate
•
2CyS ↔ CySSCy. According to the mechanism, the proton takes part in the electrooxidation process of L-CySH. Therefore, the pH of solution should have a profound effect on the electrochemical behavior of L-CySH. The followed CV measurements also suggested that L-CySH oxidation on an electrode exhibited a pH dependence, and the results were shown in Fig. 5. As shown in Fig. 5, Ip increased first with the increase of pH from 6 to 6.5, and then decreased with the further increase of pH from 6.5 to 8. The maximum peak current was observed at pH 7.0. Moreover, the voltammetric response tends toward a plateau like curve at low pH and well-shaped curve at high pH (Fig. 5A), and the reason was that the chemical reaction on electrode surface was dominated by the supply of CyS− [37]. Since the ionization of L-CySH in aqueous solution largely depends on pH, the main electroactive species is in the CyS− form when the pH value is higher than the isoelectric point of L-CySH of 5.02. Therefore, Ip increased with the increase of pH from 6 to 6.5. The
Fig. 3. The Nyquist plots for bare GCE, graphene/GCE, graphene–Au9/GCE, and graphene– Au30/GCE.
Cyclic voltammogram behaviors of L-CySH on graphene–Au9/GCE at different scan rates (υ) have been investigated, and the results were shown in Fig. 6A. It is found that Ep shifted positively accompanied with the increase of peak current upon the increase of υ. The deviation of Ep was a result of incomplete reaction under higher υ. A good linear relationship between the peak current (Ip ) and υ1/2 was obtained within the range of 40–300 mV/s, as indicated in Fig. 6B. The result indicated that the L-CySH electrooxidation was controlled by a diffusion process. The linear regression equation was I (μA) = 0.1341υ1/2 (mV/s) 1/2 − 3.0136 (R = 0.997). As a totally irreversible oxidation process, the Ep could be represented by the equation [40]:
Ep ¼ A þ
2:3RT lnυ ð1−αÞnF
Fig. 4. Cyclic voltammograms of GCE (a), graphene–Au9/GCE (c), graphene–Au30/GCE (e) and graphene/GCE (d) in the presence of 2 mmol/L-CySH and cyclic voltammogram of graphene–Au9/GCE (b) in the absence of L-CySH at a scan rate of 50 mV/s.
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Fig. 5. (A) Cyclic voltammogram response of graphene–Au9/GCE on 2.0 mmol/L L-CySH in 0.1 M PBS of different pH: 6.0, 6.5, 7.0, 7.5, and 8.0; (B) effects of pH on the peak current and potential. Scan rate: 50 mV/s.
where A is a constant, which is related with the formal electrode potential (E0) and standard rate constant at E0. The transfer coefficient (α), which characterizes the effect of electrochemical potential on activation energy of an electrochemical reaction, can be calculated according to the slope of Ep-ln υ line. The peak potential for L-CySH oxidation at the graphene–Au9/GCE versus ln υ was plotted, as indicated in Fig. 6C. A linear equation of E (V) = 0.2518 + 0.0611 ln υ (R = 0.999) was obtained. The transfer coefficient (α) is thus calculated to be 0.52 (n = 2), which was smaller than those reported earlier [38,39,41].
The lower α value suggested that the oxidation of L-CySH proceeded more easily on graphene–Au 9 /GCE, and this could be explained that conductive graphene facilitated the electrons to transfer into the electrode. 3.5. Amperometric response of graphene–Au9/GCE to L-CySH The amperometric response of the modified electrode to L-CySH was investigated by successive additions of L-CySH solution to a continuous stirring electrolyte solution in an electrochemical cell under the optimized conditions. The applied constant potential for amperometry at the working electrode was chosen first on the basis of the electrochemical behavior of the graphene–Au9/GCE. Fig. 7A showed the amperometric response of 10 μM L-CySH over a prolonged duration (600 s) at four different applied potentials of 0.2, 0.35, 0.45 and 0.6 V. Rapid and well-defined current responses could all be observed from the amperometric curves at different applied potentials. The stable current response with low background noise could be obtained at 0.45 V. Therefore, the subsequent amperometric measurement was performed at applied constant potential of 0.45 V. Fig. 7B showed the typical current–time responses of the modified electrodes upon successive addition of L-CySH at low concentrations to PBS solution. An obvious steady-state current response was observed when L-CySH concentration reached 0.1 μM, as shown in Fig. 7B (inset). The asprepared sensors showed a very rapid response to the change of L-CySH concentrations, and a steady-state current can be achieved within 5 s. This is attributed to the rapid diffusion of L-CySH molecules from the solution to the modified electrode. The calibration curve for the graphene–Au9/GCE sensor was shown in Fig. 7C. The calibration curve between the peak current (Ip) and the L-CySH concentration (C) can be described by the equation I (μA) = 0.0138 + 0.0250C (μM) (R2 = 0.999) for the 0.1–25 μM. The detection limit could reach as low as 20.5 nM (S/N = 3) using the calibration equation obtained for low concentration range, which was lower than or close to those reported previously [15,16,39,41]. Such a low detection limit was further verified by chronoamperometric response measurements. As shown in Fig. 7D, an obvious current response was found by successively adding 20.5 nM of L-CySH in the 0.1 M PBS of pH 7.5 solution. 3.6. Reproducibility, stability and anti-interference property of the graphene–Au9/GCE
Fig. 6. (A) Cyclic voltammograms of graphene–Au9/GCE in 0.1 M PBS containing 2.0 mmol/L L-CySH at various scan rates: 40, 50, 60, 80, 100, 120, 140, 160, 180, 200, 250 and 300 mV/s. (B) The plot of peak current vs. υ1/2. (C) The relationship between the peak potential (Ep) and the ln υ.
The reproducibility and stability of the sensor were evaluated by cyclic voltammetry. The relative standard deviation of the modified electrode response to 2 mM L-CySH is 3.3% after 10 successive measurements with the same modified electrode, showing high reproducibility. When the fabricated sensor was stored at 4 °C in a refrigerator for
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Fig. 7. (A) Current–time curve at different applied potentials: 0.2, 0.35, 0.45 and 0.6 V; (B) current–time response obtained at graphene–Au9/GCE for the addition of 0.1–39 μM L-CySH into acutely stirred 0.1 M pH 7.5 PBS. Applied potential: 0.45 V; (C) linear calibration curve in the range of 0.1–39 μM; (D) current–time response obtained at graphene–Au9/GCE for the addition of 20.5 nM of L-CySH into acutely stirred 0.1 M pH 7.5 PBS. Applied potential: 0.45 V. Inset: the magnification of 1560–1820 s in (B).
10 days, the current response was approximately 93% of its original response, which could be mainly attributed to the chemical stability of Au NPs and graphene. To demonstrate the selectivity of our fabricated sensor, some interference found in biological fluids was investigated. The interference tests were carried out by amperometric method at a constant applied potential of 0.45 V. Typical organic compounds such as arginine, tyrosine, glutamic acid, glucose, urea, and K+, Na+, and Ca2 + were examined during amperometric response for L-CySH. As shown in Fig. 8, the influences of these existing species on current responses of L-CySH were negligible.
3.7. Determination of L-CySH in real samples The graphene–Au9/GCE was applied to determine L-CySH in the presence of human urine. The recoveries of L-CySH are determined by standard addition, and the corresponding results are given in Table 1. The recovery of four samples is found to be in the range of 96.2%– 103.6%, revealing that the graphene–Au9/GCE is very reliable and sensitive in the determination of L-CySH.
4. Conclusions In this study, Au nanoparticles were successfully deposited on the surface of graphene using PGO as template. The density and size of Au nanoparticles on graphene nanosheets could be tuned by the content of grafted PVP polymer. Nearly uniform Au NPs with high density could be obtained using PGO5700/16 as template. The graphene–Au hybrid nanosheets showed excellent catalytic capability toward the electrooxidation of L-CySH. The fabricated L-CySH electrochemical sensor, graphene–Au/GCE, exhibited enhanced electrochemical properties, such as low detection limit, good stability, resistance to interference and satisfactory recovery, and these were attributed to the synergistic effects of the conductive graphene and the uniform Au nanoparticles.
Table 1 Determination of L-CySH in urine samples using the graphene–Au9/GCE.
Fig. 8. Current responses obtained at the graphene–Au9/GCE in 0.1 M pH 7.5 PBS for the ordinal additions (indicated by arrows) of 5 μM of L-CySH, 5 μM of arginine, 5 μM of glutamic acid, 5 μM of tyrosine, 5 mM of urea, 5 mM of glucose, and 5 mM of Ca2+, K+, and Na+, 5 μM of L-CySH. Applied potential: 0.45 V.
Samples
Amount added (μM)
Amount found (μM)
Recovery (%)
Human urine
5 10 15 20
5.18 10.27 14.76 19.24
103.6% 102.7% 98.4% 96.2%
298
F. Xu et al. / Materials Science and Engineering C 38 (2014) 292–298
Acknowledgments The authors thank the financial supports from the National Natural Science Foundation of China (21174118), the Key Project of Education Department of Hunan Province (12A134), the Department of Science and Technology of Hunan Province Project (2013FJ3035), the Open Project Program of Key Laboratory of Advanced Functional Polymeric Materials of Hunan Province (12K050) and the High-tech Project of Development and Reform Commission of Hunan Province. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
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