Visible-Light-Stimulated Enzymelike Activity of Graphene Oxide and Its

Article pubs.acs.org/JPCC

Visible-Light-Stimulated Enzymelike Activity of Graphene Oxide and Its Application for Facile Glucose Sensing Guang-Li Wang,*,†,‡ Xiufang Xu,† Xiuming Wu,† Genxia Cao,† Yuming Dong,† and Zaijun Li† †

The Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, People’s Republic of China ‡ State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing 210093, People’s Republic of China S Supporting Information *

ABSTRACT: A novel and interesting enzyme-mimicking activity of chitosanfunctionalized graphene oxide (CS-GO) upon phototriggering was demonstrated. CS-GO could catalyze oxidation of a typical chromogenic substrate (3,3′,5,5′-tetramethylbenzidine, TMB) under visible light (λ ≥ 400 nm) stimulation, which was independent of destructive H2O2. Visible light triggering was a rapid, clean, and versatile means for inducing the enzymelike activity of CS-GO, which was superior to the current peroxidase or peroxidase mimetics that use H2O2 as an oxidant. Compared to the natural enzyme horseradish peroxidase (HRP), CS-GO had a higher affinity for TMB. The mechanism of the phototriggered enzyme-mimicking activity of CS-GO was investigated in detail. In addition, a novel and facile colorimetric method was realized for detection of glucose based on the photoactivated CS-GO enzyme-mimicking system and the competitive interaction of concanavalin A with chitosan or glucose. The methodology showed a linear response for glucose in the range 2.5−5.0 mmol/L with a detection limit as low as 0.5 μmol/L. The sensing system was applied for detection of glucose in human serum with satisfactory results.

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INTRODUCTION Because of their high catalytic efficiency and high specificity for substrates, natural enzymes have found extensive applications in various domains including biosensing, agrochemical production, pharmaceutical processes, and the food industry.1 Unfortunately, natural enzymes have many intrinsic drawbacks. For example, they are easily denatured by environmental change and can be digested by protease. Their catalytic activities are sensitive to environmental conditions, and their preparation and purification processes are rigorous and expensive. These drawbacks limit the applications of natural enzymes seriously. For example, horseradish peroxidase (HRP), as a labeling biocatalyst, is widely used in traditional enzymelinked immunosorbent assays (ELISA) to produce amplified signal. The immunoreaction is evaluated by the catalytic ability of HRP to oxidize a typical chromogenic substrate such as 3,3′,5,5′-tetramethylbenzydine (TMB) by use of hydrogen peroxide as an oxidant. However, this method often suffers from inaccuracy due to the instability of HRP and the H2O2induced inactivation of bimolecules.2 Thus, the use of enzyme mimetics is highly desirable. Especially, nanotechnology opens up new possibilities for exploration of enzyme mimetics. Different materials including metal oxide,3,4 carbon,5−7 and noble metal8,9 nanostructures were demonstrated to possess peroxidase-like activity. Enzyme mimetics based on nanomaterials, as promising candidates to mimic natural enzymes, exhibit the merits of low cost in preparation, high stability, ease of storage and treating, and tunability in catalytic activities. © 2014 American Chemical Society

However, problems in applications of the natural peroxidases or peroxidase mimetics based on nanomaterials arise from harsh reaction conditions due to the presence of a large amount of H2O2 (as an oxidant), which makes in vivo applications of natural peroxidases/peroxidase mimetics difficult.10−12 To develop enzyme mimetics that have good catalytic activity under mild reaction conditions is appealing.13 Recently, we found that light-stimulated nanomaterials were more biocompatible enzyme mimetics than natural HRP or peroxidase mimetics, which could work well without the use of destructive H2O2.14 In recent years, graphene and its derivatives has become a hot spot in the field of material science. Graphene oxide (GO) contains a series of oxygen functional groups on the nanosheet surface and still retains some good characteristics of graphene, including high charge carrier mobility, high transparency, large specific surface area, and excellent mechanical strength.15−17 Compared to graphene, GO is readily available in bulk quantities, easily functionalized, dispersible in water, and biocompatible.18,19 With the development of research, more and more intriguing properties of GO, such as the variable fluorescence dependence on functionality, solvent, and excitation wavelength, have been uncovered.20−22 In this work, interestingly, a novel phototriggered enzymelike activity Received: September 2, 2014 Revised: November 8, 2014 Published: November 11, 2014 28109

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of GO was demonstrated for the first time. GO alone could not be used as an enzyme mimetic because it easily aggregated in the reaction process. Chitosan (CS) modification was demonstrated to ensure good enzymelike activity of GO under visible light (λ ≥ 400 nm) triggering. Upon visible light (λ ≥ 400 nm) stimulation, CS-GO showed catalytic activity for oxidation of a typical substrate (such as TMB), and its function was identical to that of the natural peroxidase. In comparison with the natural peroxidase, the photostimulated CS-GO demonstrated enzymelike activity without the need for damaging hydrogen peroxide, demonstrating that it is more biocompatible for promising applications in biosensing systems and biotechnology. Higher affinity for the substrate (TMB) was observed for the phototriggered CS-GO in comparison with that of the natural HRP or some peroxidase mimetics based on nanomatrials. Visible light triggering was a rapid, clean, and versatile means for inducing the enzymelike activity of CS-GO, which was superior to the currently peroxidase or peroxidase mimetics using H2O2 as an oxidant. To validate application of the as-obtained enzyme mimetics in biosensing, the photoactivated CS-GO with enzyme-mimicking activity was employed as a robust nanoprobe for colorimetric detection of glucose with good sensitivity, selectivity, and rapidity. This study provides new insight on the enzyme-mimicking properties of GO, which may find wide applications in biosensing, biotechnology, and clinical diagnosis.

constant potential of 0 V (vs saturated Ag/AgCl) in 0.1 mol/L Na2SO4 solution as the supporting electrolyte. A linear potential scan was conducted in the same three-electrode system with potential scanning from −0.8 to 0.4 V and 0.4 to 2.2 V at 5 mV/s to determine the conduction/valence band edge of the CS-GO. Fourier transform infrared (FTIR) spectra of the KBr pellets of the samples were recorded on a FTLA 2000−104 spectrometer (ABB Bomem, Canada). Synthesis of GO and CS-GO Composite. GO was prepared from natural graphite powder according to the method of Hummers and Offeman23 with some modifications. In detail, 10 g of graphite was added into 230 mL of 98% H2SO4, followed by stirring for about 24 h at room temperature. Subsequently, the mixture was kept at 0 °C in an ice bath, and then 5 g of NaNO3 and 30 g of KMnO4 were added into the mixture slowly and it was stirred for 2 h. The mixture was heated to 35−40 °C and stirred for another 30 min. Then the temperature was kept at 98 °C, and 460 mL of water was added into the mixture over 15 min. Finally, 1400 mL of water and 100 mL of 30% H2O2 were added into the mixture to stop the reaction. Subsequently, the product was collected by filter flask and washed repeatedly with 5% HCl aqueous solution. The product was dried at 50 °C in a vacuum drying apparatus. GO synthesized by the above method dispersed in water easily. On the basis ofthis phenomenon, 0.01 g of GO powder was dispersed in 20 mL of ultrapure water with the aid of ultrasonication to form a homogeneous solution (0.5 mg/mL). The solution was then mixed thoroughly with CS solution (2.0 mg/mL in acetate buffer, pH = 4.0) with a volume ratio of 1:1 under stirring for 6 h at room temperature. Finally, the solution was treated by ultrasound for 30 min, resulting in the CS-GO composite. Process for Oxidation of Substrate with Visible Light as Irradiation Source (λ ≥ 400 nm). The enzymelike activity of CS-GO was examined under visible light irradiation. That is, 5 mL of acetate buffer solution (pH = 4.0) containing 15 μg/ mL CS-GO and 250 μmol/L TMB was irradiated under visible light provided by a 300 W Xe lamp equipped with an ultraviolet cutoff filter (λ ≥ 400 nm). Glucose Detection by Use of CS-GO as Mimicking Enzyme. Detection of glucose was carried out as follows: 10 mg of concanavalin A (Con A) was dissolved in 10 mL of phosphate-buffered saline (PBS; pH = 7.0, containing 0.1 mmol/L NaCl, 0.5 mmol/L CaCl2, and 0.1 mol/L MnCl2) to form a 1.0 mg/mL Con A solution. To activate the conformation of Con A, CaCl2 and MnCl2 were added, which allowed the effective binding of ConA to carbohydrates.24−26 Con A (100 μL of 1.0 mg/mL solution) was added to 500 μL of Tris-HCl buffer (pH = 7.4), and then 100 μL aliquots of different concentration of glucose and 200 μL of 0.25 mg/mL CS-GO were added, and mixtures were allowed to react for 10 min. Subsequently, 1 mL of 0.2 mol/L acetate buffer (pH = 4.0) and 100 μL of 5 mmol/L TMB were added into the above solutions, which were irradiated under visible light (λ ≥ 400 nm) for 3 min. The method was also used to detect glucose in human serum samples. The human serum samples were provided by the Hospital of Jiangnan University and their glucose concentrations were examined by an Automatic Biochemical Instrument (FH-400). With the same experimental procedure described above for glucose detection, the reaction solution consisted of 100 μL of human serum, 100 μL of 1.0 g/L Con A,

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EXPERIMENTAL SECTION Chemicals and Materials. Chitosan (CS), 3,3′,5,5′tetramethylbenzidine (TMB), tert-butanol (TBA), 2-propanol (IPA), glucose, sucrose, D-fuctose, α-lactose, tryptophan, graphite, NaNO3, KMnO4, 30% H2O2, H2SO4, NaCl, KCl, CaCl2, and MnCl2 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Horseradish peroxidase (HRP) was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Superoxide dismutase (SOD), catalase, and concanavalin A (type VI) were purchased from Sigma. Human serum samples were provided by the Hospital of Jiangnan University. Instrumentation. A JEOL JEM-2100 transmission electron microscope (TEM, Hitachi, Japan) was utilized to characterize the morphology of GO. X-ray diffraction (XRD) measurement was carried out at room temperature on an X-ray powder diffractometer with Cu Kα radiation (λ = 0.154 178 nm) and a scanning speed of 4°/min (Brooke AXS, Germany). Scanning electron microscopy (SEM) images were acquired on a Hitachi S-4800 high-resolution scanning electron microscope (Hitachi, Japan). Absorption spectra were carried out on a TU-1901 spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., China). The visible light irradiation source was a 300 W Xe lamp (NBeT, China) equipped with an ultraviolet cutoff filter (λ ≥ 400 nm). The X-ray photoelectron spectra (XPS) of GO were attained by a Phi 5000 VersaProbe X-ray photoelectron spectroscopy (Ulvac-Phi, Japan). Raman spectra were recorded on a confocal micro-Raman spectrometer (Renishaw, Britain) excitated by a 532 nm laser. Fluorescence spectra were detected on a Cary Eclipse fluorescence spectrophotometer (Varian Co., Ltd.). A Chi 800C electrochemical workstation was used to record the photocurrent of the CS-GO-modified indium tin oxide (ITO) electrode (with an area of 0.25 cm2). In the photocurrent detection system, a Pt wire and saturated Ag/ AgCl was used as the counter and reference electrode, respectively. Photocurrent measurements were performed at a 28110

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500 mL of 0.1 mol/L Tris-HCl buffer (pH = 7.4), 200 μL of 0.25 mg/mL CS-GO, 100 μL of 5.0 mmol/L TMB, and 1 mL of acetate buffer (pH = 4.0). The above mixed solution was irradiated by visible light (λ ≥ 400 nm) for 3 min.

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RESULTS AND DISCUSSION Characterization of GO and CS-GO Composites and Their Enzyme-Mimetic Activities. A modified method of

Figure 4. Steady-state kinetic assay of CS-GO as a catalyst illuminated under visible light with TMB as substrate. (Inset) Lineweaver−Burk double-reciprocal plot of the Michaelis−Menten equation.

Figure 1. XRD patterns and (inset) TEM image of GO.

Figure 5. Effect of different scavengers on catalytic activity of visiblelight-illuminated CS-GO for oxidation of TMB. Reaction conditions: [TMB] = 250 μmol/L, [GO] = 15 μg/mL, [TBA] = 0.01 mol/L, [IPA] = 0.01 mol/L, [KI] = 0.01 mol/L, [EDTA] = 1 mmol/L, [SOD] = 50 units/mL, [catalase] = 50 units/mL, reaction time 10 min.

Figure 2. Color evolution of TMB by visible-light-irradiated (λ ≥ 400 nm) GO or chitosan-modified GO (CS-GO) in a pH = 4.0 acetate buffer at room temperature.

Figure 6. (A) Fluorescence emission profile of CS-GO. (B) Photocurrent−time performance of CS-GO-modified electrode in 0.1 mol/L Na2SO4 aqueous solutions under visible light irradiation (λ ≥ 400 nm).

Hummers and Offeman23 was used to prepare GO. Chemical oxidation of graphite by this method has been proven to be a relatively mild, convenient, and effective means to produce GO with good dispersion in aqueous solutions.23,27 The polar and hydrophilic characteristics, resulting from a number of reactive oxygen functional groups including epoxide, hydroxyl, and carboxylic acid present on the basal planes and edges of GO, are responsible for its good dispersibility. Morphology and composition of the as-synthesized GO were initially characterized by transmission electron microscopy (TEM) and X-ray diffraction (XRD) measurements. The TEM image of GO demonstrated its sheetlike morphology with some corrugations

Figure 3. UV−vis spectra of (a) TMB, (b) mixture of CS-GO and TMB without visible light irradiation, and (c) mixture of CS-GO and TMB under visible light irradiation. Reaction condition: [TMB] = 250 μmol/L and irradiation time = 10 min. (Inset) Corresponding color of the solutions.

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Figure 7. Cartoon illustration of the oxidation mechanism of TMB catalyzed by CS-GO under visible light irradiation.

Figure 10. Effect of different substances on the detection of glucose, all at a concentration of 1.0 mmol/L except ascorbic acid (AA) at 10 μmol/L. The absorbance of oxTMB catalyzed by Con A/CS-GO under visible light irradiation is defined as A0, and A is the absorbance of oxTMB at 652 nm in the presence of different substances. [Con A] = 50 μg/mL, [TMB] = 250 μmol/L, irradiation time = 3 min, and the pH of the colorimetric reaction is 4.0.

Table 1. Results for Detection of Glucose in Human Serum Samples content determined (mmol·L−1)

a

Figure 8. Fabrication process and detection principle of the system to detect glucose.

sample

by local hospital

by this method

RSDa (%)

1 2 3

4.87 5.43 5.10

4.97 5.39 5.24

+2.09 −0.74 +2.75

RSD = relative standard deviation.

binding energies for the C 1s peak in the range of 280−290 eV (Figure S1B, Supporting Information), which were contributed by several oxygen functionalities including C−C (284.6 eV), C−O (286.3 eV), and CO (288.1 eV). The atomic concentrations of C 1s and O 1s are 70.39% and 29.61%, respectively, and the O 1s/C 1s ratio is 0.42 for the GO specimen. To investigate the enzymelike activity of GO under visible light illumination, we chose TMB as a typical chromogenic substrate,29 because it is a benign and noncarcinogenic color reagent. As shown in Figure 2, under visible light triggering (λ ≥ 400 nm), GO catalyzed oxidation of TMB to produce the

on the edge (Figure 1). XRD traces of GO revealed a typical diffraction peak at about 2θ = 10.25°, corresponding to an interlayer spacing of 0.88 nm formed by the insertion of hydroxyl and epoxy groups between the graphite sheets.28 This study also used X-ray photoelectron spectra (XPS) to analyze the composition of GO. Spectra shown in Figure S1A (Supporting Information) exhibit peaks with binding energies of approximately 284.6 (C 1s) and 531.4 eV (O 1s). The oxygen content in GO resulted from oxygenation of the graphene basal plane. There were three peaks with different

Figure 9. (A) UV−vis spectra of oxTMB catalyzed by Con A/CS-GO complex in the presence of different concentrations of glucose. (B) Linear relationship between (A − A0) of oxTMB at 652 nm and the concentration of glucose. The absorbance of oxTMB catalyzed by Con A/CS-GO under visible light irradiation is defined as A0, and A is the absorbance of oxTMB at 652 nm catalyzed by Con A/CS-GO under visible light irradiation in the presence of different amounts of glucose. [Con A] = 50 μg/mL, [TMB] = 250 μmol/L, irradiation time = 3 min, and the pH of colorimetric reaction is 4.0. 28112

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vibrations, respectively.34 The spectra of CS showed a broad absorption band at 3400 cm−1, attributed to -NH2 stretching vibration, and a peak at 1640 cm−1, corresponding to CO stretching vibration of the amide groups. After the immobilization of CS on GO, the peak of GO at 1725 cm−1 almost disappeared and a new band emerged at 1680 cm−1, corresponding to COOH groups from GO, and was downshifted because of the formation of hydrogen bonds between GO and CS.35 The FTIR spectra proved that CS was immobilized on GO through the carboxyl groups of GO. Raman spectroscopy (Figure S2B, Supporting Information) further confirmed the successful modification of CS on GO. In the GO spectrum, two peaks were observed: at 1349 cm−1, assigned to D band (the symmetry A1g mode), and at 1591 cm−1, assigned to G band (the E2g mode of sp2 carbon atoms).36 The CS-GO composites exhibited a slight shift of D and G bands to 1340 and 1602 cm−1, respectively. The blue shift in the G band could be attributed to strong interaction between the oxygenated functional groups of GO and the amino groups of CS.37 It was also found that the D/G intensity ratio of CS-GO (0.97) was higher than that of GO (0.84). The Raman D/G intensity ratio was inversely proportional to the average size of the sp2 domains; the increase of the D/G intensity ratio actually resulted from the decrease in the number of in-plane sp2 domains of CS-GO.38 The Raman spectra also demonstrated the effective modification of CS on GO. SEM measurements provided direct information about the interfacial interactions between CS and GO. As shown in Figure S3 (Supporting Information), unlike GO, with a rough structure exhibiting many wrinkles, the morphology of CS-GO became much smoother, also indicating that CS was modified on the surface of CS-GO. As shown in Figure S4 (Supporting Information), under visible light (λ ≥ 400 nm) stimulation, CS-GO catalyzed the oxidation of TMB effectively and fast. The absorbance at 652 nm of oxTMB increased with time and then reached a plateau at 10 min. Similar to natural enzymes, the catalytic activity of the CS-GO composite was dependent on pH and temperature. As shown in Figure S5 (Supporting Information), the optimal pH was 4.0 and the optimal temperature was 30 °C. The optimal temperature and pH were similar to those observed with other nanomaterial-based enzyme mimetics and natural HRP.8,39 The absorbance of oxTMB (652 nm) increased gradually with TMB concentration (from 10 to 250 μmol/L) until it reached a maximum at high concentrations of TMB (about 250 μmol/L) (Figure S6, Supporting Information), indicating that the catalytic reaction was substrate-concentration-dependent. The catalytically kinetic data of CS-GO were further investigated by varying the concentration of the substrate. As shown in Figure 4, typical Michaelis−Menten curves could be obtained in a certain range of TMB concentration. The Michaelis−Menten constant (Km), indicating affinity of an enzyme for its substrate, was obtained by use of a Lineweaver−Burk plot:

oxidized product of TMB (oxTMB) with typical blue color. However, the above reaction solution formed flocculent precipitate immediately. The simple mixture of TMB and GO did not produce precipitates. In order to further explore the reason for the aggregation, oxTMB produced by horseradish peroxidase (HRP) with H2O2 as an oxidant was added into the GO solution and it was observed that the solution formed flocculent precipitate. So, it may be concluded that oxTMB led to the precipitation of GO. We introduced different surface modifiers such as chitosan (CS), poly(ethylene glycol) (PEG), and dextran on the surface of GO to improve the stability of GO and avoid the formation of flocculent precipitates of the solution. It was found that GO modified by CS showed efficient catalytic activity for TMB oxidation and the reaction solution was homogeneous and clear without the formation of flocculent precipitates (Figure 2). However, GO modified by PEG or dextran still precipitated after TMB oxidation. These results indicated that CS was an effective modifier for GO to make GO demonstrate good catalytic activity. By monitoring the absorption spectra, it was found that the oxTMB had characteristic absorbance peaks at 370 and 652 nm originating from its distinctive blue charge-transfer complex of diamine and diimine.30 The phototriggered oxidation reaction for TMB by CS-GO was similar to that of natural peroxidase with H2O2 as an oxidant,31 indicating the enzyme-mimicking activity of CSGO. In contrast, TMB alone illuminated by visible light (λ ≥ 400 nm) did not produce color change, and the mixture of TMB and CS-GO solution without visible light irradiation also showed no oxidative reaction (Figure 3), indicating that CSGO demonstrated enzymelike activity for TMB oxidation when triggered by visible light. Different from the feature that natural peroxidase or peroxidase mimetics based on nanomaterials usually need a high concentration of H2O2 to achieve high catalytic activity,2−9 the visible-light-triggered enzyme-mimicking activity of CS-GO was independent of destructive H2O2, which indicated that CS-GO is a more green and biocompatible enzyme mimetic. The oxTMB is a charge-transfer (CT) complex of TMB and TMB2+.32 More importantly, the CT complex is prone to assemble into aggregates in solution via stacking of its aromatic molecular structure.32 The positively charged CT complex of oxTMB may easily conjugate with negatively charged GO through electrostatic interactions. In addition, the assembling property of oxTMB on the surface of GO was prone to make GO aggregate. As a result, the formation of oxTMB resulted in the flocculent precipitates of GO. It was noteworthy that the resulting dispersion of CS-GO was stable as a catalyst for oxidation of TMB under visible light irradiation, indicating that the CS stabilized GO. The -OH, -NH2, and OC−NH2 groups on the macromolecular chains of CS could interact with the residual oxygen-containing groups of GO through electrostatic interactions and formation of hydrogen bonds,33 which led to widespread adsorption of CS macromolecules onto GO nanosheets. So the GO nanosheets were stably dispersed in water. On the other hand, modification by CS (pKa ∼6.5) with positive charges prevented direct interaction between GO and the positively charged oxTMB, avoiding the formation of flocculent precipitates. Figure S2A (Supporting Information) shows FTIR spectra of CS, GO, and the asprepared CS-GO composite. A typical strong band at 1725 cm−1, assigned to stretching vibrations from carbonyl and carboxyl groups, was observed for GO. The peaks at 1622 and 1398 cm−1 were attributed to CC and C−OH stretching

K 1 1 1 = m + v vmax [S] vmax

where v, vmax, [S] and Km are initial velocity, maximum reaction velocity, concentration of substrate, and Michaelis constant, respectively. Km and vmax for photoactivated CS-GO composite with TMB as the substrate are 32.6 μmol/L and 1.0 × 10−7 mol·L−1·s−1. The Km value of photoactivated CS-GO with TMB 28113

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observed for the as-synthesized CS-GO (Figure 6A), which provided further evidence for the generation of photogenerated holes (h+) for CS-GO under visible light irradiation. We also used photoelectrochemistry to investigate photoinduced charge generation and transfer characteristics of semiconductors.51,52 The CS-GO promptly generated switchable and stable photocurrent in response to on/off cycles of visible light irradiation (λ ≥ 400 nm) in 0.1 mol/L Na2SO4 solution, proving effective charge generation and transfer of CS-GO (Figure 6B). We used electrochemical methods to study the conduction and valence band edge of GO.53 Figure S8 (Supporting Information) shows that CS-GO had a conduction band edge at −0.68 V and a valence band edge at 1.67 V (vs Ag/AgCl). The oxidation potential of h+ in the CS-GO valence band was higher than the redox potential of TMB in the range 0.22−0.7 V (vs standard hydrogen electrode, SHE).30 So, the h+ in the CS-GO valence band could directly oxidize TMB. GO is naturally a 2D network, which is highly anisotropic with extremely high surface area exposed after being dispersed in water to molecular scale readily accessible to substrates.54 Furthermore, it has high mobility of charge carriers,55 which are sufficiently large to migrate the holes quickly toward the surface for efficient photocatalytic application. As a result, CS-GO demonstrated high enzymelike catalytic activity for TMB oxidation. A schematic illustration for oxidation of TMB catalyzed by photoactivated CS-GO is shown in Figure 7. Glucose Detection by Use of Photoactivated CS-GO Composite. Glucose, the major energy source in cellular metabolism, has an important effect on cells’ natural growth. It has been proven that there is a correlation between the breakdown of glucose transport in the human body and the occurrence of certain diseases, such as diabetes and some cancers. So, the detection of glucose has been an active area of research.56−59 Based on the enzymelike catalytic activity of CSGO, a novel biosensing platform for glucose was developed. As shown in Figure S9 (Supporting Information), the absorbance of oxTMB at 652 nm was reduced from 0.68 to 0.23 when Con A was added in CS-GO solution, indicating that the visiblelight-induced catalytic activity of Con A/CS-GO was reduced greatly in the presence of Con A. This phenomenon mainly was attributed to agglutination of GO caused by the high-affinity and multivalent interaction of Con A with CS on the surface of GO. The enhanced resonance light scattering intensity of CSGO after addition of Con A (Figure S10, Supporting Information) confirmed the aggregation of GO due to the interaction of Con A with CS on the surface of GO. As is wellknown, concanavalin A is a metalloprotein that is capable of noncovalently and reversibly binding to specific carbohydrates including α-D-mannose and α-D-glucose as well as N-acetyl-Dglucosamine.60,61 CS is a polysaccharide made up of β(1 → 4)linked 2-amino-2-deoxy-β-D-glucopyranose (N-acetylglucosamine).62 That is, CS is a kind of cationic polysaccharide with a large amount of N-acetyl-D-glucosamine groups in its main chain, enabling it to specifically recognize Con A.63−65 If Con A and glucose were mixed first, the catalytic activity of glucose/Con A/CS-GO system was recovered compared to that in the presence of Con A without glucose. This may be due to the stronger affinity between Con A and glucose than between Con A and CS,65 leading to less Con A interaction with CS on the surface of GO. This off/on mode resulting from the competition interaction between glucose and CS for Con A was the main foundation of glucose sensing (as shown in Figure

as the substrate was lower than that of HRP (Km = 434 μmmol/L),2 demonstrating that CS-GO had a higher affinity for TMB. In comparison with other nanoparticles exhibiting peroxidase-like activity (with H2O2 as an oxidant) in previously published reports, CS-GO possessed remarkable advantages as indicated by Km (Table S1, Supporting Information). Catalytic Mechanism of CS-GO under Visible Light Irradiation. In order to investigate the reactive species generated during the oxidation process of TMB catalyzed by CS-GO, different quenchers that can scavenge the relevant reactive species including hydroxyl radicals (•OH), superoxide anions (O2• −), and photogenerated holes (h+) were added to the catalytic reaction system. Here, 2-propanol (IPA) or tertbutanol (TBA) was utilized to scavenge •OH in solution40,41 while superoxide dismutase (SOD) was used for O2• −42 and ethylenediaminetetraacetic acid (EDTA) or KI was used for the photogenerated holes (h+).41,43 As shown in Figure 5, the catalytic activity of CS-GO illuminated by visible light (λ ≥ 400 nm) for TMB oxidation showed no obvious decline in the presence of IPA or TBA, indicating that no •OH existed in the process. The presence of SOD also did not show any obvious inhibition for TMB oxidation, demonstrating that superoxide anion (O2• −) was not the main active species. In addition, the catalytic activity of photoactivated CS-GO for oxidation of TMB was not influenced by anaerobic condition with nitrogen. This implied that oxygen was not involved in the catalytic oxidation process. However, the visible-light-stimulated catalytic activity of CS-GO decreased obviously in the presence of EDTA or KI, indicating that photogenerated holes (h+) were the main reactive species responsible for TMB oxidation. In order to verify whether H2O2 was produced by CS-GO for TMB oxidation under irradiation, catalase was added in the catalytic system. As is known, one catalase molecule can convert millions of molecules of H2O2 to water and oxygen per second.44 In our experiment, oxygen hardly affected the catalytic activity of CS-GO, while the presence of H2O2 could lead to oxidation of TMB by CS-GO even without light (data not shown) due to the peroxidase-like activity of GO.31 If H2O2 were produced in the catalytic process, a decrease in the catalytic activity would be expected after the addition of catalase. It was found that the photostimulated catalytic activity was unaffected by catalase (Figure 5), indicating that H2O2 was not produced by CS-GO under irradiation. From the absorption spectrum of CS-GO (Figure S7, Supporting Information), we can see that a characteristic shoulder at 305 nm resulted from n → π* transitions of CO bonds45 and the absorption peak at 230 nm due to the π → π* transitions was observed. GO consists of two main regions: hydrophobic π-conjugated sp2 domains and hydrophilic sp3 domains with oxygen-containing functional groups.46,47 In GO nanosheets, the islands of sp2 domains with high conductivity are surrounded by an insulating matrix of the sp3 domains. When excitated by light with energy exceeding the band gap of GO, its sp2 domains acted as semiconductors, exhibiting photoreactivity. The smaller O/C ratio (0.42) of GO confirmed by XPS (Figure S1, Supporting Information) also indicated that the as-synthesized GO by our method was partially oxidized and had semiconductor properties.48 An electron is excited into the π* conduction band and a hole is generated in the π valence band in CS-GO when excitated by light. The formation of localized electron−hole (e−−h+) pairs could be confirmed by fluorescence properties.47,49,50 A weak fluorescence emission peak centered at 576 nm under excitation at 420 nm was 28114

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detection of glucose. This material is available free of charge via the Internet at http://pubs.acs.org

8). On the basis of this off/on mechanism, we realized the detection of glucose. If the concentration of glucose increased, the absorbance of oxTMB at 652 nm (Figure 9A) increased progressively and the absorbance increased linearly with the logarithmic concentrations of glucose over the range 2.5−5.0 mmol/L (Figure 9B). A detection limit of 0.5 μmol/L was obtained. The sensitivity of the method was higher than some of the previously reported peroxidase mimetics based on nanomaterials with TMB as a substrate and H2O2 (produced by glucose oxidase) as an oxidant. A detailed comparison is listed in Table S2 (Supporting Information). To explore the selectivity of the method for detection of glucose, responses of the sensor to other saccharides and potential interferents such as D-fructose, sucrose, α-lactose, ascorbic acid, tryptophan, K+, and Na+ were also investigated under the same detection conditions. As shown in Figure 10, the fabricated colorimetric sensor demonstrated high affinity and selectivity to glucose. The good selectivity was ascribed to specific recognition of glucose by ConA. The serum glucose concentration for healthy people is generally in the range of about 3−8 mmol/L, while it can reach 9−40 mmol/L for diabetic people.66,67 Thus, our method could be suitable for monitoring the concentration of glucose in human serum. Serum samples were diluted 20-fold to ensure the concentration of glucose was within the linear range. As shown in Table 1, the glucose concentrations determined by our method were close to the values provided by the Hospital of Jiangnan University, for which the glucose concentration was examined on an Automatic Biochemical Instrument (FH-400). The relative standard deviation (RSD) varied within the range −0.74% to +2.75%, which indicated that the proposed method was reliable.

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CONCLUSIONS

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; tel +86-510-85917090; fax +86-510-85917763. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Fundamental Research Funds for the Central Universities (JUSRP51314B), National Natural Science Foundation of China (21275065, 21005031), and MOE & SAFEA for the 111 Project (B13025).

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REFERENCES

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A novel enzymelike activity of graphene oxide integrated with chitosan was demonstrated and used as an efficient biosensing system for glucose detection. The CS-GO hybrid was demonstrated to be a good enzyme mimetic for oxidation of a typical substrate (TMB) under visible light (λ ≥ 400 nm) stimulation and was independent of destructive hydrogen peroxide. Catalytic activity of the photostimulated CS-GO system followed typical Michaelis−Menten kinetics, and a higher affinity of CS-GO for TMB was observed than that of HRP. The catalytic mechanism investigations indicated that photogenerated holes (h+) were the reactive species responsible for TMB oxidation. By taking advantage of the enzymemimicking activity of CS-GO and the competition interaction between glucose and CS for Con A, a facile, rapid, sensitive, and selective colorimetric method was developed to determine glucose. It can be expected that the novel enzymelike activity of CS-GO upon visible light triggering may broaden the scope of its applications in the field of catalysis, biochemistry, and biotechnology.

S Supporting Information *

Ten figures showing XPS, FT-IR, Raman, and absorbance spectra, SEM images, catalytic characteristics, cathodic and anodic linear potential scans, and resonance light scattering spectra; two tables comparing kinetic parameters of lighttriggered CS-GO with other different nanomaterials and comparing various peroxidase-mimicking nanomaterials for 28115

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