In Situ Growth of Surfactant-Free Gold Nanoparticles on Nitrogen

Dec 23, 2014 - metal ions to metal nanoparticles,34,39 and the N atoms can increase the number of ... Aladin Ltd. (Shanghai, China). Ultrahigh-purity ...
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In Situ Growth of Surfactant-Free Gold Nanoparticles on NitrogenDoped Graphene Quantum Dots for Electrochemical Detection of Hydrogen Peroxide in Biological Environments Jian Ju and Wei Chen* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, Jilin, China S Supporting Information *

ABSTRACT: In this work, we report a green and simple strategy for the in situ growth of surfactant-free Au nanoparticles (Au NPs) on nitrogen-doped graphene quantum dots (Au NPs−N-GQDs). The formation of hybrid was achieved by just mixing the N-GQDs and HAuCl4·4H2O without addition of any other reductant and surfactant. Highresolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) characterizations clearly showed the formation of Au nanoparticles with predominantly exposed (111) facets which can provide more adsorption sites. Such nonsurfactant-capped Au NPs can provide naked catalytic surface with highly electrocatalytic activity. The Au NPs−N-GQDs exhibit high sensitivity and selectivity for electrochemical detection of hydrogen peroxide (H2O2) with a low detection limit of 0.12 μM and sensitivity of 186.22 μA/mM cm2. Importantly, the Au NPs−NGQDs-based electrochemical biosensor has shown great potential applications for detection of H2O2 levels in human serum samples and that released from human cervical cancer cells with satisfactory results. The present study demonstrates that such novel Au NPs−N-GQDs nanocomposite is promising for fabrication of nonenzymatic H2O2 biosensors. coexisting in biological fluids can also be oxidized or reduced simultaneously at the high overpotential. Therefore, to improve the catalytic activity of electrode for H2O2 oxidation/reduction, various nanomaterials-modified electrodes such as noble metals, alloys, and metal oxides have been developed for sensitive and selective detection of H2O2.10,17−20 Among the metal nanocatalysts, gold-based electrocatalytic materials have been widely used to modify electrode due to their high stability, high electrocatalytic activity, and the ability to enhance the electrode conductivity and facilitate the electron transfer.21,22 It is well-known that the electrocatalytic activity and stability of metal nanoparticles are strongly dependent on the exposed facets.23,24 Up to present, some Au(111)-like gold electrodes have been successfully prepared for the construction of electrochemical sensors.25,26 Therefore, Au NPs with exposed Au(111) planes can be applied to the design of novel H2O2 electrochemical sensors. On the other hand, in electrocatalysis and electrochemical sensors applications, to improve the electrocatalytically active surface area, catalytic activity, and stability, metal nanoparticles are usually dispersed on supporting materials with high electrical conductivity, large surface area, and high stability.27,28 Among the supporting

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ydrogen peroxide (H2O2) plays critical roles in industrial, biological systems, pharmaceutical, and many other fields.1−3 Specially, the concentration level of H2O2 is a significant biological parameter in studying Alzheimer’s disease, myocardial infarction, Parkinson’s disease, cancer, etc.4−6 Therefore, it is of great importance to develop an efficient method for sensitive and selective detection of H2O2 level under physiological conditions. Up to now, many techniques have been developed for determining H2O2 concentration, such as spectrophotometry,7 fluorescence,4,8 chemiluminescence,9 electrochemical method,10 etc. Among these developed detection methods, electrochemical sensors can offer simple, rapid, sensitive, and cost-efficient quantification of H2O2.11,12 In the past decades, many studies have focused on enzyme-based sensors. However, the enzyme-based biosensors are limited by some serious disadvantages, such as environmental instability, tedious immobilization procedures, and high cost.13,14 In addition, the denaturation of enzyme under certain temperature, pH, and electrochemical detection conditions inevitably affects the stability and reproducibility of the sensors.15 To overcome these obstacles, increasing attention has been focused on the nonenzymatic biosensors.16 In a nonenzymatic biosensor system, H2O2 can be directly oxidized or reduced at solid electrodes. However, electrochemically direct oxidation or reduction of H2O2 usually requires a relatively high overpotential, and meanwhile some electroactive species © XXXX American Chemical Society

Received: November 7, 2014 Accepted: December 23, 2014

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electrolyte in electrochemical experiments. The 0.02 M PBS buffer used in cell culture was purchased from Invitrogen (10010). A fresh solution of H2O2 was prepared daily. Synthesis of N-GQDs. On the basis of our previous work,41 N-GQDs were prepared by a hydrothermal treatment of the mixture of CA and DCD. In a typical synthesis, 2.0 g of CA and 1.0 g of DCD were mixed with nanopure water (5 mL). The mixture was then transferred into a 25 mL Teflon-lined autoclave and heated at 180 °C for 12 h. The product was dispersed in 100 mL of nanopure water, and the N-GQDs were collected by removing the large dots through centrifugation at 10 000 rpm for 10 min. The concentration of the obtained NGQDs aqueous solution is about 20 mg/mL. Preparation of Surfactant-Free Au Nanoparticles Supported on N-GQDs (Au NPs−N-GQDs). Au NPs−NGQDs were synthesized through a simple refluxing process. Typically, 2 mL of the as-synthesized N-GQDs (20 mg/mL) and 2 mL of nanopure water were added to 25 mL dual-port flask, and the solution was heated at 100 °C under stirring for 10 min. Then, 0.5 mL of HAuCl4·4H2O (0.1 M) was added into the above solution and stirred for 20 min. The color of the solution changed from yellow to black, indicating the formation of Au NPs. Solid precipitate was then separated by centrifuging the aqueous solution at 10 000 rpm for 10 min. The product was dried at 60 °C for 12 h. Characterizations. High-resolution transmission electron microscopy (HRTEM) measurements were carried out by using a FEI TECNAI F20 EM with an accelerating voltage of 200 kV equipped with an energy-dispersive spectrometer. X-ray photoelectron spectroscopy (XPS) measurements were performed on a Thermo ESCALAB VG Scientific 250 spectrometer equipped with monochromatized Al Kα excitation. The crystal phases of the samples were identified by powder X-ray diffraction (XRD) analysis using an X-ray D/ max-2200vpc (Rigaku Corporation, Japan) instrument operated at 40 kV and 20 mA and using Cu Kα radiation (λ = 0.15406 nm). UV−vis absorption spectra were obtained on a UV-1700 (MAPADA, China) spectrometer with a 1 cm quartz cuvette. Electrochemical experiments were performed with a CHI750D electrochemical workstation (CH Instruments, Shanghai Chenhua Instrument Corporation, China) in a conventional three-electrode cell. A glassy carbon electrode (GC, 3 mm in diameter) modified by the prepared samples was used as working electrode. A platinum coil and a Ag/AgCl (in saturated KCl solution) electrode were used as counter and reference electrode, respectively. Preparation of Au NPs/N-GQDs-Modified Glassy Carbon Electrode and the Electrochemical Sensing of Hydrogen Peroxide. Prior to the modification, a glassy carbon (GC) electrode (model CHI104, 3 mm) was polished with 1, 0.3, and 0.05 μm alumina powder, then washed successively in 0.1 M nitric acid, absolute alcohol, and doubledistilled water, respectively. The cleaned electrode was dried with high-purity nitrogen steam. An amount of 2 mg of the sample was dispersed in 1 mL of aqueous ethanol solution (1:1). The Au NPs−N-GQDs-modified GC electrode was prepared by casting 5 μL of Au NPs−N-GQDs suspension (2 mg/mL) on a GC electrode surface and drying in air. The prepared electrode was denoted as Au NPs−N-GQDs/GC. A N2-saturated 0.1 M PBS solution was used as the base electrolyte. Cyclic voltammograms (CVs) were recorded from 0.2 to −0.8 V at a scan rate of 50 mV/s. For the electrochemical detection of H2O2, different concentrations of H2O2 were

materials, carbon materials, such as carbon nanotubes, carbon fibers, graphene etc., exhibit excellent properties and have been widely used as catalyst supports.28−30 Meanwhile, in most strategies for fabricating carbon material-supported metal nanoparticles, various templates and surfactants are generally required to load the nanoparticles and prevent them from aggregation.31 However, these methods exhibited some disadvantages, such as high cost and time-consuming processes.32,33 More importantly, such surfactant-capped metal NPs always exhibit lowered electrocatalytic performance due to the blocked surface active sites and impeded electron transfer by the surfactants covered on particle surface.34 Therefore, it is highly desirable to develop a rapid and green method to prepare surfactant-free metal NPs supported on carbon materials for electroanalysis. Recently, Chen’s group prepared GQDs-supported platinum nanoparticles by a simple hydrothermal method, and their studies showed that GQDs play a significant role in determining the electrocatalytic activity of the hybrid due to the structural defects.35 At the same time, according to the previous studies, the structure defects of GQDs may be manipulated through doping heteroatoms into the πconjugated system.36,37 For nitrogen-doped GQDs (NGQDs), the chemically bonded N atoms could drastically alter the electronic characteristics and offer more active sites, thus producing new phenomena and unexpected properties.36,38 For example, Li et al. found that the N-GQDs exhibited enhanced electrocatalytic activity for the oxygen reduction reaction (ORR).36 On the other hand, carbon materials can also serve as reducing agents for the reduction of metal ions to metal nanoparticles,34,39 and the N atoms can increase the number of anchoring sites for the adsorption of metal ions.40 Therefore, N-GQDs might also function as both reducing agents and supports for the nucleation and growth of metal NPs due to the abundant anchoring sites from the doped N atoms and various oxygen-containing functional groups on the surface. In this work, N-GQDs were first prepared and the surfactantfree Au NPs can be automatically produced on the N-GQDs (Au NPs−N-GQDs) by mixing the prepared N-GQDs and AuCl4− without the addition of any reducing agent and surfactant. Such nonsurfactant-capped Au NPs provide naked catalytic surfaces, which renders them highly active electrocatalysts. The present study demonstrates that the Au NPs−NGQDs can be used as an electrochemical probe to sensitively and selectively detect H2O2 in human serum and live cells. Such electrochemical sensor has also promising application in physiological and pathological studies.

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EXPERIMENTAL SECTION Materials. Citric acid (CA), dicyandiamide (DCD), NaH2PO4, Na2HPO4·7H2O, and H2O2 (30 wt %) were purchased from Beijing Chemical Reagent Co., China. Chloroauric acid (HAuCl4·4H2O, 99.9%) was purchased from Aladin Ltd. (Shanghai, China). Ultrahigh-purity N2 (99.999%) was used for deaeration. Dulbecco’s modified Eagle’s medium (DMEM), penicillin, fetal bovine serum (FBS), and streptomycin were purchased from Beijing Dingguo Biotechnology Co. All reagents are of analytical grade and used as received. Nanopure water (18.3 MΩ cm) was used throughout the experiments. The 0.1 M phosphate buffer solution (PBS) was prepared by mixing 19 mL of 0.1 M NaH2PO4 and 81 mL of 0.1 M Na2HPO4·7H2O and was employed as a supporting B

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Analytical Chemistry added to the PBS solution and the corresponding current was recorded. Current−time curve was conducted at −0.3 V in 10 mL of stirred solution. The modified electrode was washed with pure water and then dried in nitrogen between each measurement. Detection of H2O2 in Living Cells. HeLa cells were seeded in a 12-well plate for 24 h, and they were cultured in a DMEM medium containing 10% fetal bovine serum (FBS) supplemented with 100 units/mL penicillin and 100 mg/mL streptomycin in an incubator at 37 °C with 5% CO2.41 The incubation solution was removed and washed with PBS (0.02 M, pH = 7.4) for three times. An amount of 3 mL of PBS (0.02 M, pH = 7.4) was added to the plates for real sample measurements. The prepared Au NPs−N-GQDs/GC electrode, counter, and reference electrodes were placed inside the above plates. Upon addition of CdTe QDs into the system, the amperometric (current−time) responses to H2O2 produced from the HeLa cells were recorded.

After the formation of Au nanoparticles on the N-GQDs, the average size of the Au NPs−N-GQDs increased to 5.2 nm. Moreover, from the HRTEM image of Au NPs−N-GQDs shown in Figure 1E, each particle is composed of five singlecrystalline domains. The fivefold pentagonal symmetry can also be seen from the corresponding FFT pattern (Figure 1F). Moreover, as shown in Figure 1E, except for the lattice fringes from GQDs, the surface of the Au NPs−N-GQDs is predominated by exposed Au(111) facets.43 The size and morphology changes clearly indicate the successfully formation of Au nanoparticles on the surface of the presynthesized NGQDs without the aid of any reducing agent and surfactant. Such surface-clean Au NPs supported on N-GQDs are advantageous for their application in electrochemical sensing and electrocatalysis. The compositions of the produced N-GQDs and Au NPs− N-GQDs were then characterized by XPS. Figure 2A shows the survey spectra of N-GQDs and Au NPs−N-GQDs. In both spectra, there are three predominant peaks at 284.0, 400.0, and 530.6 eV, corresponding to the C1s, N1s, and O1s, respectively.44 The concentrations of nitrogen and oxygen were calculated to be 18.13% and 23.02%, respectively. However, compared to the spectrum of N-GQDs, additional three peaks at around 85.0, 334.0, and 353.0 eV can be observed in the spectrum of Au NPs−N-GQDs, corresponding to the binding energies of Au 4f, Au 4d5, and Au 4d3. The high-resolution XPS of Au 4f shown in Figure 2B indicates the metallic state of the formed gold nanoparticles attached on the N-GQDs.45 In the highresolution C1s spectra of N-GQDs (top) and Au NPs−NGQDs (bottom) shown in Figure 2C, three components corresponding to carbon atoms in different functional group can be fitted. The peaks at 284.4, 285.6, and 288.4 eV are assigned to C−C, C−OH, and carbonyl C or C−N, respectively. The N1s spectra of N-GQDs (top) and Au NPs−N-GQDs (bottom) are shown in Figure 2D. The two fitted peaks at 399.2 and 400.5 eV are assigned to C−N−C and N−(C)3 bonds, respectively. The high-resolution O1s spectra of N-GQDs (top) and Au NPs−N-GQDs (down) are shown in Figure 2E, in which the deconvoluted two peaks around 531.0 and 533.0 eV can be assigned to CO and C−OH/C−O−C groups, respectively. It should be noted that, compared to the value of 84.0 eV from bulk metallic gold, the binding energy of Au 4f7/2 shown in Figure 2B displays a down-shift to 83.25 eV, suggesting the strong interaction between Au nanoparticles and N-GQDs. The XPS analyses clearly demonstrate that nitrogen atoms have been successfully doped into the framework of GQDs with rich N and O elements, and surfactant-free Au nanoparticles have been formed on the surface of N-GQDs. Figure 3A shows the UV−vis absorption change of the NGQDs before and after addition of AuCl4−. The absorption spectrum of N-GQDs shows two narrow peaks at 230 and 330 nm, which could be assigned to the π―π* transition of aromatic CC sp2 domains and the trapping of excited state energy of the surface states, respectively.42,46 After the addition of AuCl4− and the following reduction reaction, the absorption centered at 230 nm disappeared, and the absorption at 330 nm became weaker. Furthermore, the UV−vis spectrum shows an absorption peak at about 450 nm, which is assigned to the surface plasmon absorption of small Au NPs.47,48 As shown in Figure 3B, the XRD pattern of the Au−N-GQDs exhibits several diffraction peaks which can be indexed to diffractions from the (111), (200), (220), and (311) facets of face-centered cubic (fcc) Au. It should be noted that the broad diffraction

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RESULTS AND DISCUSSION Characterization of the Au NPs−N-GQDs. Figure 1, parts A and B, and Figure S1, parts A and B (Supporting

Figure 1. TEM images of the as-synthesized N-GQDs (A) and Au NPs−N-GQDs (B). HRTEM images and FFT patterns of the NGQDs(C and D) and the Au NPs−N-GQDs (E and F).

Information), display the TEM and HRTEM images of the NGQDs and the Au NPs−N-GQDs, respectively. From the TEM characterizations, the prepared N-GQDs and Au NPs−NGQDs show good dispersion without obvious aggregation. Figure 1, parts C and D, shows the HRTEM image and fast Fourier transform (FFT) pattern of the N-GQDs. From the TEM characterizations, the N-GQDs have an average diameter of 3.8 nm. It can be seen from the HRTEM image that each NGQD is a single crystal with a lattice spacing of 0.22 nm, corresponding to the (1 1 2̅ 0) lattice fringes of graphene.42 C

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Figure 2. (A) XPS survey spectra of N-GQDs (line a) and Au NPs−N-GQDs (line b). (B) High-resolution XPS spectrum of Au 4f. (C−E) Highresolution XPS spectra of C1s (C), N1s (D), and O1s (E) of the N-GQDs (top) and Au NPs−N-GQDs (bottom).

peak at 2θ ≈ 25.80° could be assigned to the graphite(002) planes.49 The size of the Au NPs was also evaluated based on the XRD data and the Scherrer’s equation. The average size of the Au NPs was calculated to be about 5.1 nm, which agrees well with the result from TEM measurements. Above X-ray characterizations further demonstrate that the AuCl4− reduction can be achieved by N-GQDs without the addition of any additional reducing agent. The driving force for the in situ reduction of AuCl4− to Au NPs is attributed to the inherent reducing property of carbon materials, which has also been reported previously.34,39 Electrochemical Detection of Hydrogen Peroxide (H2O2) by the Au NPs−N-GQDs. Figure 4A shows typical cyclic voltammetry (CV) of the Au NPs−N-GQDs/GC in N2saturated 0.5 M H2SO4 solution within the potential range of −0.2 to 1.6 V. The CV features of the Au electrode can be observed clearly, that is the oxidation peaks in the range of 1.2 to 1.5 V in the anodic direction and a sharp reduction peak around 0.9 V in the reversed scan.50 The electrochemical behavior of the Au NPs−N-GQDs/GC electrode in 0.1 M PBS solution was also investigated by CV. Figure 4B shows the CVs of Au NPs−N-GQDs/GC electrode at various scan rates. It can be seen that both anodic peak potential (Epa) and cathodic peak potential (Epc) remained almost unchanged with the increase of potential scan rate, indicating the good electrochemical reaction ability and fast electron transfer kinetics of the Au NPs−NGQDs/GC electrode. Moreover, Figure 4C shows that with the

increase of scan rate (ν), the anodic and cathodic peak current increase linearly with scan rate in the range from 0.01 to 0.2 V/ s, implying the dominance of a surface-confined redox process. The electrocatalytic activity of the Au NPs−N-GQDs NPs toward H2O2 reduction was then investigated. Figure 4D−F shows the CVs of blank GC electrode (Figure 4D), N-GQDs/ GC (Figure 4E), and Au NPs−N-GQDs NPs/GC (Figure 4F) electrodes in the absence (black curves) and presence (red curves) of 4 mM H2O2 in N2-saturated 0.1 M PBS solution. As can be seen from Figure 4, parts D and E, the blank GC electrode and the N-GQDs show low electrocatalytic activities for H2O2 reduction with weak reduction current and onset potentials negative than −0.1 V. However, significantly improved reduction current (at least 5 times larger than those obtained from GC and N-GQDs/GC electrodes) and more positive onset potential (∼0.2 V) were obtained for H2O2 reduction at the Au NPs−N-GQDs/GC electrode. The effect of scan rate on the cathodic current from H2O2 reduction on the Au NPs−N-GQDs/GC electrode was also investigated. As shown in Figure 4, parts G and H, the reduction current increases with increasing scan rate and a liner relationship can be obtained between cathodic peak current (ipc) versus square root of scan rate (v1/2) in the range from 0.02 to 1 V/s, demonstrating the diffusion-controlled irreversible electrochemical process of H2O2 reduction on the Au NPs−NGQDs/GC electrode. D

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GQDs electrode has linear chronoamperometric response to H2O2 concentration in the range of 0.25−13327 μM, as shown in Figure 5 inset. The sensitivity was calculated to be 186.22 μA/mM cm2 (the slope of the calibration plot divided by geometric surface area of GC electrode). The linear regression equation is as follows: I (μA) = −0.01311C (μM) − 0.5322 (R2 = 0.998). The limit of detection (LOD) of the H2O2 was calculated as three times the standard deviation for the average measurement of blank sample [LOD = 3(RSD/slope)]. The LOD was estimated to be 0.12 μM, which is lower than some Au nanomaterials-based biosensors, as shown in Table S1 (Supporting Information). The reproducibility of the sensor was also evaluated. Five Au NPs−N-GQDs modified electrodes were fabricated and their current responses at −0.3 V to 0.2 mM H2O2 were measured, resulting a relative standard deviation of 5.6%. The stability measurements showed that the Au NPs−N-GQDs electrode can retain 89% of its initial response after 3 weeks of storage at 4 °C. Therefore, the Au NPs−N-GQDs nanocomposite is an ideal electrochemical sensing material for H2O2 detection with low detection potential, high sensitivity, and wide linear range. The influence of some other physiological-level electroactive species of 0.3 mM ascorbic acid (AA), uric acid (UA), dopamine (DA), glucose, and 0.2 mM dissolved oxygen on the detection of H2O2 was evaluated at the potential of −0.3 V. As shown in Figure 6A, the relevant physiological level of AA, UA, DA, and glucose have no interference for H2O2 detection, while the addition of 0.2 mM O2 exhibits a little influence. Therefore, N 2 -saturated PBS solution was used to avoid the O 2 interference and maintain the stability of this sensor system. To explore the application of the present Au NPs−N-GQDs hybrid nanomaterials in the physiological environment, the standard titration method was also carried out to detect the H2O2 in human serum samples, as shown in Figure 6B. Here, 10 mL of PBS solution was first added in a small electrochemical cell and the solution was saturated with N2. Certain concentrations of N2-saturated serum samples were then added, and standard concentration of H2O2 was spiked into the solution, as shown in Table 1. The concentration of H2O2 in serum samples was finally calculated using the linear relationship as shown in Figure 5 inset. The results are satisfactory and agree closely with those reported previously.52 These results demonstrate that the fabricated sensor based on the Au NPs−N-GQDs hybrid has a potential application in nonenzymatic detection of H2O2 in biological samples. With the high sensitivity achieved, the Au NPs−N-GQDs/ GC electrode was used to detect the H2O2 produced from living cells. The cells were first washed by PBS solution (0.02 M, pH = 7.4) for three times to remove DMEM. As shown in Figure 7A, for the Au NPs−N-GQDs/GC electrode in PBS (0.02 M, pH = 7.4) without HeLa cells, no current response was recorded after addition of CdTe QDs. For comparison, Figure 7B depicts the amperometric responses obtained from the Au NPs−N-GQDs/GC electrode located near the living cells in PBS (0.02 M, pH = 7.4) containing 50 mM glucose after the injection of 25 μL of CdTe QDs (0.5 mg/mL). An increased cathodic current can be observed upon the addition of CdTe QDs in PBS solution. According to the previous reports, due to the high cytotoxicity, CdTe QDs can induce cells death by generating hydrogen peroxide.52,53 In order to verify whether the current response was due to the H2O2 produced from the living cells or not, 100 μL of catalase (800 U/mL) was also added into the plate; the reduction current

Figure 3. (A) UV−vis absorption spectra of N-GQDs (black line) and the as-synthesized Au NPs−N-GQDs in PBS solution. (B) XRD pattern of the Au NPs−N-GQDs.

On the other hand, from Figure 4I, one can see that the cathodic peak current of H2O2 reduction increases with the concentration of H2O2 increasing from 1 to 10 mM at the Au NPs−N-GQDs electrode. Figure 4I inset indicates that there is a good linear relationship between peak current and concentration of H2O2. These results strongly indicate that the Au NPs−N-GQDs show a good electrocatalytic activity toward H2O2 reduction. The enhanced electrocatalytic property of Au NPs−N-GQDs/GC electrode could be ascribed to the following three possible aspects. First, the formed Au NPs with Au(111) domains can provide plenty of adsorption sites for −OH species of H2O2.24 Second, the surfactant-free Au NPs provide a naked catalytic surface, which renders them the highly active electrocatalysts.34,39 Third, similar to graphene nanosheet, the N-GQDs support can improve the electron transfer from N-GQDs-Au surface to GC electrode.51 These three structural features together contribute to the enhanced electrochemical response of the Au NPs−N-GQDs/GC electrode for the reduction of hydrogen peroxide. The high electrocatalytic performance of the Au NPs−N-GQDs makes them promising electrochemical sensing materials for H2O2 detection. In order to ensure large current density and avoid the interference from other biologically active molecules, −0.3 V was chosen in the following study of H2O2 detection. Figure 5 shows the amperometric current−time curve of the Au NPs− N-GQDs/GC electrode with successive addition of varying concentrations of H2O2 in 0.1 M PBS solution. It can be observed that the Au NPs−N-GQDs sensor exhibits quick response to the change of H2O2 concentration and can reach the steady-state current within 5 s. Moreover, the Au NPs−NE

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Figure 4. (A) CV of the Au−N-GQDs/GC electrode in N2-saturated 0.5 M H2SO4 at a scan rate of 50 mV/s. (B) CVs of the Au NPs−N-GQDs/ GC electrode in 0.1 M PBS (pH = 7.4) at different scan rates from 0.01 to 0.2 V/s. (C) The linear relationship between the anodic and cathodic peak currents and the scan rates. CVs of blank GC (D), N-GQDs/GC (E), and Au NPs−N-GQDs/GC (F) electrodes in 0.1 M PBS (pH = 7.4) with the absence (black curves) and presence (red curves) of 4 mM H2O2 at a scan rate of 50 mV/s. (G) CV curves of the Au NPs−N-GQDs/GC electrode in 0.1 M PBS (pH = 7.4) containing 1 mM H2O2 at different potential scan rates. (H) Dependence of cathodic peak current (ipc) vs square root of scan rate (v1/2). (I) CVs of the Au NPs−N-GQDs/GC electrodes in N2-saturated 0.1 M PBS (pH = 7.4) in the absence and presence of H2O2 with different concentrations from 1 to 10 mM at a scan rate of 50 mV/s. Inset shows the dependence of reduction peak current on the H2O2 concentration.

became small due to the metabolism of H2O2 by catalase to water and oxygen. Thus, above results suggest that the current change only depends on H2O2 released from the living cells. The current change is around 0.012 μA, corresponding to 0.56 μM H2O2, as calculated based on the regression equation in Figure 5. Such results show that Au NPs−N-GQDs could be used for the sensitive detection of H2O2 in biological environments.

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CONCLUSION In summary, N-GQDs were prepared by a facile one-step hydrothermal treatment, and the as-prepared N-GQDs can be used as reducing agent and support for the formation of gold nanoparticles by the reduction of HAuCl4 without additional reductant. TEM, HRTEM, XRD, and XPS characterizations clearly demonstrated the production of N-GQDs-supported Au nanoparticles with exposed (111) planes. Such surfactant-free Au NPs supported on N-GQDs provide naked catalytic surfaces, which renders them highly active electrocatalysts. The electrochemical measurements showed that the obtained Au NPs−N-GQDs have high electrocatalytic activity for H2O2 reduction and they can be used as electrochemical sensing

Figure 5. Amperometric responses of the Au-NPs/N-GQDs/GC electrode to the successive addition of H2O2 (applied potential: −0.3 V). Insets show the magnified amperometric response to the low H2O2 concentration (top) and the corresponding calibration plot for the Au NPs−N-GQDs/GC electrode (RSD = 5.6%, n = 5) (bottom).

F

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shown great potential application for H2O2 detection in biological environments.

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ASSOCIATED CONTENT

S Supporting Information *

HRTEM images of the prepared materials and Table S1. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (no. 21275136) and the Natural Science Foundation of Jilin Province, China (no.201215090).

Figure 6. (A) Effects of 0.3 mM AA, 0.3 mM UA, 0.3 mM DA, 0.3 mM glucose, and 0.2 mM O2 on the amperometric responses of Au NPs−N-GQDs/GC electrode with 0.005 mM H2O2. (B) Amperomtric responses recorded on the addition of some concentration of serum followed by successive additions of 10 μM H2O2 (three times) at the Au NPs−N-GQDs/GC electrode in N2-saturated 0.1 M PBS (pH =7.4) solution.

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Table 1. Detection of H2O2 in Human Serum Sample sample

added (μM)

found (μM)

RSD (%)

recovery (%)

serum

0 10 20 30

12.32 21.34 30.59 40.98

1.8 1.5 1.6 1.7

95.6 94.6 96.8

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Figure 7. (A) Amperometric responses of the Au NPs−N-GQDs/GC electrode in the absence (A) and presence (B) of HeLa cells in N2saturated PBS (pH = 7.4) containing 50 mM glucose at the potential of −0.3 V vs Ag/AgCl, with the addition of 0.5 mg/mL CdTe QDs and 800 U/mL catalase.

materials for sensitive and selective detection of H2O2 with a low detection limit of 0.12 μM and a wide detection linear range of 0.25−13327 μM. More importantly, the electrochemical sensor fabricated from the Au NPs−N-GQDs has G

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