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Enhanced direct electron transfer of glucose oxidase based on gold nanoprism and its application in biosensing
Colloids and Surfaces A 529 (2017) 113–118
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Research Paper
Enhanced direct electron transfer of glucose oxidase based on gold nanoprism and its application in biosensing ⁎
Jinqiong Xu, Qinglin Sheng , Yu Shen, Jianbin Zheng
MARK
⁎
Institute of Analytical Science, Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Northwest University, Xi’an, Shaanxi 710069, China
G RA P H I C A L AB S T R A C T A pair of distinct and well-defined redox peaks is observed at the GOD–AuNP–chitosan/GCE (curve c) with the formal potential of −0.460 V (vs. SCE) and the peak to peak separation was 52 mV.
A R T I C L E I N F O
A B S T R A C T
Keywords: Enhanced electron transfer Gold nanoprism Glucose oxidase Biosensing Glucose
Gold nanoprism (AuNP) was used for immobilization of glucose oxidase (GOD), and the direct electrochemistry of GOD–AuNP–chitosan modified GCE and glucose biosensing were studied. Transmission electron microscopy, UV–vis spectroscopy and electrochemical impendence spectroscopy were employed to confirm the morphology and film modification changes of the prepared biosensor. Results showed that the AuNP can provide a favorable and biocompatible microenvironment for facilitating the direct electron transfer between proteins and electrode surface. It was found that the special structure of gold nanoprism exhibited enhanced performances in direct electron transfer of GOD and glucose sensing. The adsorbed GOD displayed an apparent electron transfer rate constant (ks) of 24.92 s−1. The constructed biosensor exhibited a good response to glucose with linear range from 0.05 to 1.2 mM (R2 = 0.9975), low detection limit of 0.01 mM and high sensitivity of 11.83 μA mM−1 cm−2. The proposed biosensor offers an alternative method for the determination of glucose in real samples and has potential applications in the fabrication of other biosensors with redox proteins.
⁎
Corresponding authors. E-mail addresses: [email protected] (Q. Sheng), [email protected] (J. Zheng).
http://dx.doi.org/10.1016/j.colsurfa.2017.05.049 Received 2 March 2017; Received in revised form 5 May 2017; Accepted 20 May 2017 Available online 25 May 2017 0927-7757/ © 2017 Elsevier B.V. All rights reserved.
Colloids and Surfaces A 529 (2017) 113–118
J. Xu et al.
1. Introduction
20.0 kV. Transmission electron micrographs (TEM) were carried out by E.M. 912 Ω energy filtering TEM (120 kV). UV–vis adsorption spectra were investigated by Cary 50 Scan UV–vis spectrophotometer (Varian, Australia). The dynamic light scattering (DLS) experiments were performed on a particle size analyzer, model Zetasizer 1000HS (Malvern instruments, UK). Fourier transform infrared spectroscopy (FTIR) was recorded with TENSIR 27 (Bruker, German).
Studies on direct electrochemistry of redox protein/enzyme have attracted increasing interest, which provide a desirable model for designing electrochemical biosensors [1,2], biofuel cell [3] and bioreactor devices [4] and studying mechanisms of proteins and enzymes in biological systems [5]. However, it is difficult for redox protein to exchange electrons directly with bare solid electrodes, because its redox center is deeply immersed in the insulated protein shells, which makes it hard to realize direct electron transfer (DET) of between GOD and the bare electrode [6,7]. Thus, some efforts have been devoted to retain the biological activity and promote DET behaviors of GOD via selected matrix [8,9]. Many types of nanomaterials have been applied in bioelectrochemical analysis due to their large specific surface area and excellent biocompatibility [10], such as gold nanoparticles [11], graphene [12], carbon nanotube (CNT) [13] and so on. Among them, gold nanomaterials has been extensively exploited as biosensors due to it can provide a suitable microenvironment to immobilize enzyme and facilitate the electron transfer between the immobilized enzyme and electrode surface [14]. Compared to other biocompatible materials, chitosan has good biocompatible, biodegradable, non-toxic and excellent film forming ability [15]. So it was commonly used to disperse nanostructured materials and immobilize proteins for constructing biosensor [16]. Glucose oxidase (GOD) is an ideal mode enzyme for studying the electron transfer properties and has been widely applied to monitor glucose owing to its high specificity toward glucose [17–21]. Nanomaterial could facilitate the electron transfer between the immobilized enzyme and electrode surface. The electron transfer rate is related to structure of nanomaterial [22]. In this work, gold nanoprisms (AuNP) was synthesized successfully, and dispersed in chitosan solution and then mixed with GOD. The as-prepared GOD–AuNP–chitosan composite was modified on the surface of glassy carbon electrode (GCE) to construct a novel electrochemical biosensor. UV–vis displayed that the AuNP–chitosan provided a favorable microenvironment for GOD to retain its original structural confirmation and bioactivity. The constructed electrode showed excellent direct electrochemical behavior and was successfully applied in glucose detection, indicating that gold nanoprisms can provide a promising alternative material for immobilizing biomolecules.
2.3. Preparation of gold nanoprism and modified electrodes Gold nanoprism was simply synthesized according to previous method [23]. Briefly, 1 mL of 0.1 M NaBH4 was added to the mixture of 1 mL of 0.01 M HAuCl4, 1 mL of 0.01 M sodium citrate and 36 mL of water while stirring vigorously. The resulting mixture was aged for 4 h in order to allow the hydrolysis of unreacted NaBH4. Then, the gold nanoparticle seeds were prepared. The gold nanoprism was obtained by three-step growth of seeds. 1 mL of seeds solution was added to solution containing 0.25 mL of 10 mM HAuCl4, 0.05 mL of 100 mM NaOH, 0.05 mL of 100 mM ascorbic acid, and the mixed solution was gently shaken. Then, 1 mL of mixed solution was added to 9 mL of CTAB prepared solution containing 50 μM NaI under shaking. The resulting solution was added to solution including 2.5 mL of 10 mM HAuCl4, 0.50 mL of 100 mM NaOH, 0.50 mL of 100 mM ascorbic acid, and 90 mL of 0.05 M CTAB prepared solution. Finally, the gold nanoprism was obtained when the color of solution changed from clear to deep magenta-purple. Prior to the modification, the GCE was polished successively with 0.3 and 0.05 mm alumina slurry to obtained mirror like surface, and rinsed with doubly distilled water, followed by sonication in 1:1 ethanol and deionized distilled water. Then, the GCE was allowed to dry in a stream of nitrogen. 200 μL gold nanoprism solution and 1.0 mL 0.5 wt% chitosan solution (containing 2.0 mg mL−1 GOD) were mixed to form GOD–AuNP–chitosan mixed solution. Subsequently, 6 μL of the mixture was dropped on the pretreated GCE surface. Then, the modified electrodes were dried at 4 °C in a refrigerator. 3. Results and discussion 3.1. Characterization of the AuNP and GOD–AuNP–chitosan The morphology and microstructure of the as-synthesized AuNP were first investigated by SEM in Fig. 1A. Meanwhile, these nanoparticles were further characterized by transmission electron microscope (TEM), are shown in Fig. 1B and C, which exhibits an uniform prism morphology with about 150 nm side length. The size distribution can be estimated by dynamic light scattering (DLS) studies. The spectrum showes the presence of two distinct peaks (Fig. 1D). The peaks at 13 nm and 155 nm are attributed to thickness and side length of the AuNP structure, respectively [24]. These measurements agreed with TEM results. The UV–vis absorption spectra of the AuNP–chitosan, native GOD and GOD–AuNP–chitosan are shown in Fig. 2. From curve a, the surface plasmon resonance due to gold could be seen at about 526 nm [23]. The UV–visible spectrum of GOD (curve b) exhibited two well-defined absorption peaks at 380 and 455 nm which are attributed to the oxidized form of the flavin groups present in GOD [25]. The two peaks for GOD in GOD–AuNP–chitosan composite (curve c) are almost the same as those for native GOD, indicating that the original structural confirmation and native structure of GOD has not been altered during the immobilization process. Fig. 2B shows FTIR spectra of the AuNP–chitosan, native GOD and GOD–AuNP–chitosan. The adsorption peak of chitosan at 1058 cm−1 (curve a) is attributed to frame symmetric and asymmetric flexible vibrations, and peak at 1411 cm−1 is assigned to CH2 bending vibration [26]. The two characteristic peaks of native GOD are displayed at 1640 and 1544 cm−1, which are ascribed to amide I and II bands of GOD
2. Experimental 2.1. Materials Glucose oxidase (E.C. 1.1.3.4, 182 U/mg, Type X-S from Aspergillus niger), Chitosan (> 90% deacetylation) was got from Shanghai Yuanju Biotechnology Co., Ltd. Cetyltrimethlyammonium bromide, hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O, 99.9%), sodium borohydride (NaBH4, 99.995%), sodium hydroxide (NaOH, 99.998%), and sodium iodide (99%) were obtained from Aldrich. All other chemicals were of analytical grade and used without further purification. All aqueous solutions were prepared with ultrapure water from a Milli-Q Plus system (Millipore). 2.2. Apparatus All of the electrochemical experiments were performed with a CHI660D electrochemical workstation (Shanghai Chenhua Instrument Co. Ltd., China) using a three-electrode system, where a standard saturated calomel electrode (SCE) served as the reference electrode, a platinum wire electrode as the auxiliary electrode, and the modified electrode GCE (3 mm in diameter) as the working electrode. Scanning electron micrographs (SEM) were carried out on a JSM–6390A (JEOL, Japan) scanning electron microscope using an accelerating voltage of 114
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Fig. 1. SEM images of (A) AuNP, TEM images of (B, C) AuNP, and DLS size distribution of (D) AuNP.
Fig. 2. UV–vis absorption spectra (A) and FTIR spectra (B) of AuNP–chitosan (a), GOD (b) and GOD–AuNP–chitosan (c).
[27]. The FTIR spectrum of GOD–AuNP–chitosan also shows two adsorption peaks at 1642 and 1546 cm−1, suggesting that GOD has been successfully immobilized on the AuNP–chitosan film. Fig. 3. Nyquist plots of EIS for bare (a), AuNP–chitosan (b) and GOD–AuNP–chitosan (c) modified GCEs.
3.2. Electrochemical impedance spectroscopy study Electrochemical impedance spectroscopy (EIS) is a powerful tool for studying properties of surface-modified electrodes. The semicircles obtained at a lower frequency correspond to a diffusion limited electron-transfer process and those obtained at a higher frequency represent a charge-transfer limited process. Generally, the semicircle diameter equaled to the electron transfer resistance (Rct). Fig. 3 shows the Nyquist plots of the different electrodes in 5 mM Fe(CN)63−/4− containing 0.1 M KCl as the supporting electrolyte. The low Rct on the bare GCE (curve a) indicated the fast electron transfer rate between GCE and Fe (CN)63−/4−. The diameter of the semicircle for GOD–chitosan/GCE (curve b) was larger than that of bare GCE, indicating that the GOD–chitosan on the surface of GCE decreased the electron transfer rate between the redox probe of Fe(CN)63−/4− and the electrode surface, which can be attribute to the insulating bulky protein structure of the GOD. Decreased electron transfer resistance was observed when AuNP was modified onto the GOD–chitosan/GCE surface (curve c), which indicates that the conductivity of the GOD–AuNP–chitosan modified electrode is improved compared with that of chitosan modified GCE
[28]. 3.3. Direct electrochemistry of GOD–AuNP–chitosan on GCE As a part of the GOD molecule, flavin adenine dinucleotide (FAD) undergoes the redox reaction [29,30]. The direct electrochemical behavior of GOD adsorbed on the substrate can be observed from the electrochemical response under optimal conditions. Fig. 4 showed the cyclic voltammograms of different modified electrodes in nitrogen-saturated PBS (pH 7.2) at scan rate of 100 mV s−1. No redox peak was observed at bare GCE (curve a), indicating that electrodes were electroinactive in the investigated potential range. Compared to bare GCE, the GOD–chitosan/GCE (curve b) showed a pair of distinct and well-defined redox peaks. However, the GOD–AuNP–chitosan modified electrode (curve c) offered a pair of more distinct and better-defined redox peaks. These suggest that GOD retained bioactivity and the prepared GOD–AuNP–chitosan electrode had fast DET during the conversion of GOD 115
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Laviron's formula (nΔEp > 200 mV), ks = αnFv/RT, where α is the charge transfer coefficient; n is the number of electron transfer, R, T and F symbols have their conventional meanings [33]. The values of ks is higher than those of 2.83 s−1 at graphene [34], 1.3 s−1 at gold nanoparticles [35] and 2.76 s−1 at carbon nanotubes modified GCE [36]. The result implies that gold nanoprism facilitates the fast electron transfer between the redox center of enzyme and the surface of electrode. According to the equation Ip = n2F2vAГ/4RT, the surface coverage (Г) of GOD on the electrode surface was estimated to be 3.55 × 10−9 mol cm−2 from the slope of the Ip ∼ v curve [37]. This value is much larger than the theoretical value (2.86 × 10−12 mol cm−2) for the monolayer of GOD on the bare electrode surface, suggesting that a multilayer and three-dimensional coverage of GOD has been formed on AuNP [38]. Good biocompatibility of AuNP has increased the absorption of GOD. 3.4. Performance of the GOD–AuNP–chitosan glucose biosensor Fig. 4. CVs of the bare GCE (a), GOD–chitosan/GCE (b), GOD–AuNP–chitosan/GCE (c) in 0.1 M pH 7.2 N2-saturated PBS at a scan rate of 100 mV s−1.
To investigate the electrocatalytic activity of the GOD–AuNP–chitosan/GCE, cyclic voltammetry experiments were carried out under the condition of oxygen and glucose. Fig. 6A shows the CVs of GOD–AuNP–chitosan/GCE in N2 saturated (curve a), O2 saturated (curve b) and O2-saturated PBS containing 0.2 mM glucose (curve c). As shown in Fig. 6A, a pair of symmetrical redox peaks appeared. When PBS was saturated with air, a great increase in reduction peak current and a simultaneous decrease in oxidation peak current can be observed, indicating that FADH2 reacted with O2 to form H2O2 and FAD (Eq. (1)) [39]. In the presence of O2, regenerated FAD resulted in the increase of the reduction peak current of FAD (Eq. (2)). GOD(FADH2) + O2 → GOD(FAD) + H2O2
(1)
GOD(FAD) + 2e− + 2H+ → GOD(FADH2)
(2)
when 0.2 mM glucose was added into this system, the reduction peak current decreased due to glucose triggered the decrease of GOD(FAD) form (curve c) [40]. The electrocatalytic reaction is expressed as follow: GOD(FAD) + Glucose → GOD(FADH2) + gluconolactone Fig. 5. CVs of GOD–AuNP–chitosan modified GCE in 0.1 M pH 7.2 N2-saturated PBS at different scan rates. Scan rates (a–h): 100, 200, 300, 400, 500, 600, 700, and 800 mV s−1. Inset: the plots of anodic and cathodic peak currents vs. scan rates, respectively.
(3)
Fig. 6B shows the CVs of GOD–AuNP–chitosan/GCE in a solution containing different concentrations of glucose under the condition of air saturation at a scan rate of 100 mV s−1. It was found that the logarithmic reduction currents were linearly decreased with the increased glucose concentrations. Linear range spaned the concentration of glucose from 0.05 to 1.2 mM with a correlation coefficient (R2) of 0.9975 and a high sensitivity of about 11.83 μA mM−1 cm−2 (inset of Fig. 6). Moreover, the detection limit could reach 0.01 mM (S/N = 3). Therefore, this GOD–AuNP–chitosan/GCE can serve as a glucose sensor. The comparison of the analytical performance of the GOD–AuNP–chitosan/GCE with some other materials modified electrode is summarized in Table 1, which indicates that the proposed biosensor have advantages over previously reported sensors regarding
(FAD) to GOD(FADH2) [31]. Accordingly, AuNP could act as matrix for stable loading of protein molecules. The effect of the scan rate on the cyclic voltammetric performance at GOD-AuNP-chitosan/GCE was shown in Fig. 5. The anodic peak currents and cathodic peak currents linearly increased with the increasing scan rate in the range from 100 to 800 mV s−1 (Fig. 5, inset), indicating that the electrochemical behavior of GOD has the typical property of a surface electrochemical process [32]. The electron transfer rate constant ks was estimated to be 24.92 s−1, according to the
Fig. 6. (A) CVs of GOD–AuNP–chitosan modified GCE in 0.1 M, pH 7.2 PBS N2-saturated (a), air-saturated (b) and air-saturated PBS including 0.2 mM glucose (c) at a scan rate of 100 mV s−1. (B) CVs of GOD–AuNP–chitosan modified GCE to glucose with various concentrations of 0.05 (a), 0.2 (b), 0.4 (c), 0.6 (d), 0.8 (e), 1.0 (f) and 1.2 (g) mM in 0.1 M, pH 7.2 air-saturated PBS.
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Table 1 Comparison of the analytical performances of the proposed glucose biosensor with other biosensors. electrode
Linear range (mM)
Detection limit (mM)
Sensitivity (μA mM−1 cm−2)
Reference
Graphene–CdS–GOD/GCE AuNPs/GOD–MWCNTs–PVA/GCE GOD/colloidal Au/CPE Nafion/GOD/Ag-Pdop@CNT/GCE GOD/Chitosan/GCE GOD-AuNP-chitosan/GCE
2.0–16.0 0.5–8.0 0.04–0.28 0.05–1.0 0.6–2.8 0.05–1.2
0.07 0.02 0.03 0.02 0.1 0.01
1.76 16.6 8.4 3.1 0.23 11.83
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to the linear range, the detection limit or the sensitivity. The reproducibility of the proposed sensor was measured by detecting 0.6 mM glucose 5 times using the same electrode with the relative standard deviation (RSD) of 1.7%, indicating a good reproducibility. The selectivity of the sensor was evaluated in the presence of 0.4 mM uric acid (UA) and 0.4 mM ascorbic acid (AA) in oxygenated PBS. The cyclic voltammetry response of the proposed biosensor towards 0.4 mM glucose could be obtained. The current variations were less than 10%, indicating that the selectivity of the proposed sensor was not interfered by cooxidizable substances such as AA and UA. 4. Conclusions Direct GOD electrochemistry and sensitive glucose sensor were simply achieved by immobilizing GOD on gold nanoprism. Gold nanoprism can provide a unique microenvironment for the direct electrochemistry of GOD immobilized on the surface of modified electrode, which can keep its high electrocatalytic activities. A glucose biosensor based on AuNP shows satisfactory analytical performance with high sensitivity and the large apparent electron transfer rate constant. Therefore, the proposed biosensor offers an alternative method for the determination of glucose in real samples and has potential applications in the fabrication of other biosensors with redox proteins/enzymes. Acknowledgements The authors gratefully acknowledge the financial support of this project by the National Science Foundation of China (No. 21575113), the Specialized Research Foundation for the Doctoral Program of Higher Education (No. 20126101110013), the Natural Science Foundation of Shaanxi Province in China (No. 2013KJXX-25), and the Scientific Research Foundation of Shaanxi Provincial Key Laboratory (Nos. 15JS100, 16JS099). References [1] S.Y. Dong, N. Li, G.C. Suo, T.L. Huang, Inorganic/organic doped carbon aerogels as new biosensing materials for the detection of hydrogen peroxide, Anal. Chem. 85 (2013) 11739–11746, http://dx.doi.org/10.1021/ac4015098. [2] A. Brajter-Toth, J.Q. Chambers, Electroanalytical Methods for Biological Materials, Marcel Dekker, New York, 2002. [3] H.U. Lee, Y. Yoo, T.L. Khnagvasuren, Y.S. Song, C. Park, J. Kim, S.W. Kim, Enzymatic fuel cells based on electrodeposited graphite oxide/cobalt hydroxide/ chitosan composite–enzyme electrode, Biosens. Bioelectron. (2013), http://dx.doi. org/10.1016/j. [4] S.L. Zhao, X.W. Ji, P.T. Lin, Y.M. Liu, A gold nanoparticle-mediated enzyme bioreactor for inhibitor screening by capillary electrophoresis, Anal. Biochem. 411 (2011) 88–93, http://dx.doi.org/10.1016/j.ab.2010.12.025. [5] C. Leger, P. Bertrand, Direct electrochemistry of redox enzymes as a tool for mechanistic studies, Chem. Rev. 108 (2008) 2379–2438. [6] S. Shleev, J. Tkac, A. Christenson, T. Ruzgas, A.I. Yaropolov, J.W. Whittakerd, L. Gorton, Direct electron transfer between copper-containing proteins and electrodes, Biosens. Bioelectron. 20 (2005) 2517–2554, http://dx.doi.org/10.1016/j. bios.2004.10.003. [7] L. Meng, J. Jin, G.X. Yang, T.H. Lu, H. Zhang, C.X. Cai, Nonenzymatic electrochemical detection of glucose based on palladium-single-walled carbon nanotube hybrid nanostructures, Anal. Chem. 81 (17) (2009) 7271–7280, http://dx.doi.org/ 10.1021/ac901005p. [8] C. Shan, H. Yang, D. Han, Q. Zhang, A. Ivaska, L. Niu, Graphene/AuNPs/chitosan nanocomposites film for glucose biosensing, Biosens. Bioelectron. 25 (2010)
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Contents lists available at ScienceDirect
Colloids and Surfaces A journal homepage: www.elsevier.com/locate/colsurfa
Research Paper
Enhanced direct electron transfer of glucose oxidase based on gold nanoprism and its application in biosensing ⁎
Jinqiong Xu, Qinglin Sheng , Yu Shen, Jianbin Zheng
MARK
⁎
Institute of Analytical Science, Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Northwest University, Xi’an, Shaanxi 710069, China
G RA P H I C A L AB S T R A C T A pair of distinct and well-defined redox peaks is observed at the GOD–AuNP–chitosan/GCE (curve c) with the formal potential of −0.460 V (vs. SCE) and the peak to peak separation was 52 mV.
A R T I C L E I N F O
A B S T R A C T
Keywords: Enhanced electron transfer Gold nanoprism Glucose oxidase Biosensing Glucose
Gold nanoprism (AuNP) was used for immobilization of glucose oxidase (GOD), and the direct electrochemistry of GOD–AuNP–chitosan modified GCE and glucose biosensing were studied. Transmission electron microscopy, UV–vis spectroscopy and electrochemical impendence spectroscopy were employed to confirm the morphology and film modification changes of the prepared biosensor. Results showed that the AuNP can provide a favorable and biocompatible microenvironment for facilitating the direct electron transfer between proteins and electrode surface. It was found that the special structure of gold nanoprism exhibited enhanced performances in direct electron transfer of GOD and glucose sensing. The adsorbed GOD displayed an apparent electron transfer rate constant (ks) of 24.92 s−1. The constructed biosensor exhibited a good response to glucose with linear range from 0.05 to 1.2 mM (R2 = 0.9975), low detection limit of 0.01 mM and high sensitivity of 11.83 μA mM−1 cm−2. The proposed biosensor offers an alternative method for the determination of glucose in real samples and has potential applications in the fabrication of other biosensors with redox proteins.
⁎
Corresponding authors. E-mail addresses: [email protected] (Q. Sheng), [email protected] (J. Zheng).
http://dx.doi.org/10.1016/j.colsurfa.2017.05.049 Received 2 March 2017; Received in revised form 5 May 2017; Accepted 20 May 2017 Available online 25 May 2017 0927-7757/ © 2017 Elsevier B.V. All rights reserved.
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1. Introduction
20.0 kV. Transmission electron micrographs (TEM) were carried out by E.M. 912 Ω energy filtering TEM (120 kV). UV–vis adsorption spectra were investigated by Cary 50 Scan UV–vis spectrophotometer (Varian, Australia). The dynamic light scattering (DLS) experiments were performed on a particle size analyzer, model Zetasizer 1000HS (Malvern instruments, UK). Fourier transform infrared spectroscopy (FTIR) was recorded with TENSIR 27 (Bruker, German).
Studies on direct electrochemistry of redox protein/enzyme have attracted increasing interest, which provide a desirable model for designing electrochemical biosensors [1,2], biofuel cell [3] and bioreactor devices [4] and studying mechanisms of proteins and enzymes in biological systems [5]. However, it is difficult for redox protein to exchange electrons directly with bare solid electrodes, because its redox center is deeply immersed in the insulated protein shells, which makes it hard to realize direct electron transfer (DET) of between GOD and the bare electrode [6,7]. Thus, some efforts have been devoted to retain the biological activity and promote DET behaviors of GOD via selected matrix [8,9]. Many types of nanomaterials have been applied in bioelectrochemical analysis due to their large specific surface area and excellent biocompatibility [10], such as gold nanoparticles [11], graphene [12], carbon nanotube (CNT) [13] and so on. Among them, gold nanomaterials has been extensively exploited as biosensors due to it can provide a suitable microenvironment to immobilize enzyme and facilitate the electron transfer between the immobilized enzyme and electrode surface [14]. Compared to other biocompatible materials, chitosan has good biocompatible, biodegradable, non-toxic and excellent film forming ability [15]. So it was commonly used to disperse nanostructured materials and immobilize proteins for constructing biosensor [16]. Glucose oxidase (GOD) is an ideal mode enzyme for studying the electron transfer properties and has been widely applied to monitor glucose owing to its high specificity toward glucose [17–21]. Nanomaterial could facilitate the electron transfer between the immobilized enzyme and electrode surface. The electron transfer rate is related to structure of nanomaterial [22]. In this work, gold nanoprisms (AuNP) was synthesized successfully, and dispersed in chitosan solution and then mixed with GOD. The as-prepared GOD–AuNP–chitosan composite was modified on the surface of glassy carbon electrode (GCE) to construct a novel electrochemical biosensor. UV–vis displayed that the AuNP–chitosan provided a favorable microenvironment for GOD to retain its original structural confirmation and bioactivity. The constructed electrode showed excellent direct electrochemical behavior and was successfully applied in glucose detection, indicating that gold nanoprisms can provide a promising alternative material for immobilizing biomolecules.
2.3. Preparation of gold nanoprism and modified electrodes Gold nanoprism was simply synthesized according to previous method [23]. Briefly, 1 mL of 0.1 M NaBH4 was added to the mixture of 1 mL of 0.01 M HAuCl4, 1 mL of 0.01 M sodium citrate and 36 mL of water while stirring vigorously. The resulting mixture was aged for 4 h in order to allow the hydrolysis of unreacted NaBH4. Then, the gold nanoparticle seeds were prepared. The gold nanoprism was obtained by three-step growth of seeds. 1 mL of seeds solution was added to solution containing 0.25 mL of 10 mM HAuCl4, 0.05 mL of 100 mM NaOH, 0.05 mL of 100 mM ascorbic acid, and the mixed solution was gently shaken. Then, 1 mL of mixed solution was added to 9 mL of CTAB prepared solution containing 50 μM NaI under shaking. The resulting solution was added to solution including 2.5 mL of 10 mM HAuCl4, 0.50 mL of 100 mM NaOH, 0.50 mL of 100 mM ascorbic acid, and 90 mL of 0.05 M CTAB prepared solution. Finally, the gold nanoprism was obtained when the color of solution changed from clear to deep magenta-purple. Prior to the modification, the GCE was polished successively with 0.3 and 0.05 mm alumina slurry to obtained mirror like surface, and rinsed with doubly distilled water, followed by sonication in 1:1 ethanol and deionized distilled water. Then, the GCE was allowed to dry in a stream of nitrogen. 200 μL gold nanoprism solution and 1.0 mL 0.5 wt% chitosan solution (containing 2.0 mg mL−1 GOD) were mixed to form GOD–AuNP–chitosan mixed solution. Subsequently, 6 μL of the mixture was dropped on the pretreated GCE surface. Then, the modified electrodes were dried at 4 °C in a refrigerator. 3. Results and discussion 3.1. Characterization of the AuNP and GOD–AuNP–chitosan The morphology and microstructure of the as-synthesized AuNP were first investigated by SEM in Fig. 1A. Meanwhile, these nanoparticles were further characterized by transmission electron microscope (TEM), are shown in Fig. 1B and C, which exhibits an uniform prism morphology with about 150 nm side length. The size distribution can be estimated by dynamic light scattering (DLS) studies. The spectrum showes the presence of two distinct peaks (Fig. 1D). The peaks at 13 nm and 155 nm are attributed to thickness and side length of the AuNP structure, respectively [24]. These measurements agreed with TEM results. The UV–vis absorption spectra of the AuNP–chitosan, native GOD and GOD–AuNP–chitosan are shown in Fig. 2. From curve a, the surface plasmon resonance due to gold could be seen at about 526 nm [23]. The UV–visible spectrum of GOD (curve b) exhibited two well-defined absorption peaks at 380 and 455 nm which are attributed to the oxidized form of the flavin groups present in GOD [25]. The two peaks for GOD in GOD–AuNP–chitosan composite (curve c) are almost the same as those for native GOD, indicating that the original structural confirmation and native structure of GOD has not been altered during the immobilization process. Fig. 2B shows FTIR spectra of the AuNP–chitosan, native GOD and GOD–AuNP–chitosan. The adsorption peak of chitosan at 1058 cm−1 (curve a) is attributed to frame symmetric and asymmetric flexible vibrations, and peak at 1411 cm−1 is assigned to CH2 bending vibration [26]. The two characteristic peaks of native GOD are displayed at 1640 and 1544 cm−1, which are ascribed to amide I and II bands of GOD
2. Experimental 2.1. Materials Glucose oxidase (E.C. 1.1.3.4, 182 U/mg, Type X-S from Aspergillus niger), Chitosan (> 90% deacetylation) was got from Shanghai Yuanju Biotechnology Co., Ltd. Cetyltrimethlyammonium bromide, hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O, 99.9%), sodium borohydride (NaBH4, 99.995%), sodium hydroxide (NaOH, 99.998%), and sodium iodide (99%) were obtained from Aldrich. All other chemicals were of analytical grade and used without further purification. All aqueous solutions were prepared with ultrapure water from a Milli-Q Plus system (Millipore). 2.2. Apparatus All of the electrochemical experiments were performed with a CHI660D electrochemical workstation (Shanghai Chenhua Instrument Co. Ltd., China) using a three-electrode system, where a standard saturated calomel electrode (SCE) served as the reference electrode, a platinum wire electrode as the auxiliary electrode, and the modified electrode GCE (3 mm in diameter) as the working electrode. Scanning electron micrographs (SEM) were carried out on a JSM–6390A (JEOL, Japan) scanning electron microscope using an accelerating voltage of 114
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Fig. 1. SEM images of (A) AuNP, TEM images of (B, C) AuNP, and DLS size distribution of (D) AuNP.
Fig. 2. UV–vis absorption spectra (A) and FTIR spectra (B) of AuNP–chitosan (a), GOD (b) and GOD–AuNP–chitosan (c).
[27]. The FTIR spectrum of GOD–AuNP–chitosan also shows two adsorption peaks at 1642 and 1546 cm−1, suggesting that GOD has been successfully immobilized on the AuNP–chitosan film. Fig. 3. Nyquist plots of EIS for bare (a), AuNP–chitosan (b) and GOD–AuNP–chitosan (c) modified GCEs.
3.2. Electrochemical impedance spectroscopy study Electrochemical impedance spectroscopy (EIS) is a powerful tool for studying properties of surface-modified electrodes. The semicircles obtained at a lower frequency correspond to a diffusion limited electron-transfer process and those obtained at a higher frequency represent a charge-transfer limited process. Generally, the semicircle diameter equaled to the electron transfer resistance (Rct). Fig. 3 shows the Nyquist plots of the different electrodes in 5 mM Fe(CN)63−/4− containing 0.1 M KCl as the supporting electrolyte. The low Rct on the bare GCE (curve a) indicated the fast electron transfer rate between GCE and Fe (CN)63−/4−. The diameter of the semicircle for GOD–chitosan/GCE (curve b) was larger than that of bare GCE, indicating that the GOD–chitosan on the surface of GCE decreased the electron transfer rate between the redox probe of Fe(CN)63−/4− and the electrode surface, which can be attribute to the insulating bulky protein structure of the GOD. Decreased electron transfer resistance was observed when AuNP was modified onto the GOD–chitosan/GCE surface (curve c), which indicates that the conductivity of the GOD–AuNP–chitosan modified electrode is improved compared with that of chitosan modified GCE
[28]. 3.3. Direct electrochemistry of GOD–AuNP–chitosan on GCE As a part of the GOD molecule, flavin adenine dinucleotide (FAD) undergoes the redox reaction [29,30]. The direct electrochemical behavior of GOD adsorbed on the substrate can be observed from the electrochemical response under optimal conditions. Fig. 4 showed the cyclic voltammograms of different modified electrodes in nitrogen-saturated PBS (pH 7.2) at scan rate of 100 mV s−1. No redox peak was observed at bare GCE (curve a), indicating that electrodes were electroinactive in the investigated potential range. Compared to bare GCE, the GOD–chitosan/GCE (curve b) showed a pair of distinct and well-defined redox peaks. However, the GOD–AuNP–chitosan modified electrode (curve c) offered a pair of more distinct and better-defined redox peaks. These suggest that GOD retained bioactivity and the prepared GOD–AuNP–chitosan electrode had fast DET during the conversion of GOD 115
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Laviron's formula (nΔEp > 200 mV), ks = αnFv/RT, where α is the charge transfer coefficient; n is the number of electron transfer, R, T and F symbols have their conventional meanings [33]. The values of ks is higher than those of 2.83 s−1 at graphene [34], 1.3 s−1 at gold nanoparticles [35] and 2.76 s−1 at carbon nanotubes modified GCE [36]. The result implies that gold nanoprism facilitates the fast electron transfer between the redox center of enzyme and the surface of electrode. According to the equation Ip = n2F2vAГ/4RT, the surface coverage (Г) of GOD on the electrode surface was estimated to be 3.55 × 10−9 mol cm−2 from the slope of the Ip ∼ v curve [37]. This value is much larger than the theoretical value (2.86 × 10−12 mol cm−2) for the monolayer of GOD on the bare electrode surface, suggesting that a multilayer and three-dimensional coverage of GOD has been formed on AuNP [38]. Good biocompatibility of AuNP has increased the absorption of GOD. 3.4. Performance of the GOD–AuNP–chitosan glucose biosensor Fig. 4. CVs of the bare GCE (a), GOD–chitosan/GCE (b), GOD–AuNP–chitosan/GCE (c) in 0.1 M pH 7.2 N2-saturated PBS at a scan rate of 100 mV s−1.
To investigate the electrocatalytic activity of the GOD–AuNP–chitosan/GCE, cyclic voltammetry experiments were carried out under the condition of oxygen and glucose. Fig. 6A shows the CVs of GOD–AuNP–chitosan/GCE in N2 saturated (curve a), O2 saturated (curve b) and O2-saturated PBS containing 0.2 mM glucose (curve c). As shown in Fig. 6A, a pair of symmetrical redox peaks appeared. When PBS was saturated with air, a great increase in reduction peak current and a simultaneous decrease in oxidation peak current can be observed, indicating that FADH2 reacted with O2 to form H2O2 and FAD (Eq. (1)) [39]. In the presence of O2, regenerated FAD resulted in the increase of the reduction peak current of FAD (Eq. (2)). GOD(FADH2) + O2 → GOD(FAD) + H2O2
(1)
GOD(FAD) + 2e− + 2H+ → GOD(FADH2)
(2)
when 0.2 mM glucose was added into this system, the reduction peak current decreased due to glucose triggered the decrease of GOD(FAD) form (curve c) [40]. The electrocatalytic reaction is expressed as follow: GOD(FAD) + Glucose → GOD(FADH2) + gluconolactone Fig. 5. CVs of GOD–AuNP–chitosan modified GCE in 0.1 M pH 7.2 N2-saturated PBS at different scan rates. Scan rates (a–h): 100, 200, 300, 400, 500, 600, 700, and 800 mV s−1. Inset: the plots of anodic and cathodic peak currents vs. scan rates, respectively.
(3)
Fig. 6B shows the CVs of GOD–AuNP–chitosan/GCE in a solution containing different concentrations of glucose under the condition of air saturation at a scan rate of 100 mV s−1. It was found that the logarithmic reduction currents were linearly decreased with the increased glucose concentrations. Linear range spaned the concentration of glucose from 0.05 to 1.2 mM with a correlation coefficient (R2) of 0.9975 and a high sensitivity of about 11.83 μA mM−1 cm−2 (inset of Fig. 6). Moreover, the detection limit could reach 0.01 mM (S/N = 3). Therefore, this GOD–AuNP–chitosan/GCE can serve as a glucose sensor. The comparison of the analytical performance of the GOD–AuNP–chitosan/GCE with some other materials modified electrode is summarized in Table 1, which indicates that the proposed biosensor have advantages over previously reported sensors regarding
(FAD) to GOD(FADH2) [31]. Accordingly, AuNP could act as matrix for stable loading of protein molecules. The effect of the scan rate on the cyclic voltammetric performance at GOD-AuNP-chitosan/GCE was shown in Fig. 5. The anodic peak currents and cathodic peak currents linearly increased with the increasing scan rate in the range from 100 to 800 mV s−1 (Fig. 5, inset), indicating that the electrochemical behavior of GOD has the typical property of a surface electrochemical process [32]. The electron transfer rate constant ks was estimated to be 24.92 s−1, according to the
Fig. 6. (A) CVs of GOD–AuNP–chitosan modified GCE in 0.1 M, pH 7.2 PBS N2-saturated (a), air-saturated (b) and air-saturated PBS including 0.2 mM glucose (c) at a scan rate of 100 mV s−1. (B) CVs of GOD–AuNP–chitosan modified GCE to glucose with various concentrations of 0.05 (a), 0.2 (b), 0.4 (c), 0.6 (d), 0.8 (e), 1.0 (f) and 1.2 (g) mM in 0.1 M, pH 7.2 air-saturated PBS.
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Table 1 Comparison of the analytical performances of the proposed glucose biosensor with other biosensors. electrode
Linear range (mM)
Detection limit (mM)
Sensitivity (μA mM−1 cm−2)
Reference
Graphene–CdS–GOD/GCE AuNPs/GOD–MWCNTs–PVA/GCE GOD/colloidal Au/CPE Nafion/GOD/Ag-Pdop@CNT/GCE GOD/Chitosan/GCE GOD-AuNP-chitosan/GCE
2.0–16.0 0.5–8.0 0.04–0.28 0.05–1.0 0.6–2.8 0.05–1.2
0.07 0.02 0.03 0.02 0.1 0.01
1.76 16.6 8.4 3.1 0.23 11.83
[41] [42] [43] [44] [45] This work
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to the linear range, the detection limit or the sensitivity. The reproducibility of the proposed sensor was measured by detecting 0.6 mM glucose 5 times using the same electrode with the relative standard deviation (RSD) of 1.7%, indicating a good reproducibility. The selectivity of the sensor was evaluated in the presence of 0.4 mM uric acid (UA) and 0.4 mM ascorbic acid (AA) in oxygenated PBS. The cyclic voltammetry response of the proposed biosensor towards 0.4 mM glucose could be obtained. The current variations were less than 10%, indicating that the selectivity of the proposed sensor was not interfered by cooxidizable substances such as AA and UA. 4. Conclusions Direct GOD electrochemistry and sensitive glucose sensor were simply achieved by immobilizing GOD on gold nanoprism. Gold nanoprism can provide a unique microenvironment for the direct electrochemistry of GOD immobilized on the surface of modified electrode, which can keep its high electrocatalytic activities. A glucose biosensor based on AuNP shows satisfactory analytical performance with high sensitivity and the large apparent electron transfer rate constant. Therefore, the proposed biosensor offers an alternative method for the determination of glucose in real samples and has potential applications in the fabrication of other biosensors with redox proteins/enzymes. Acknowledgements The authors gratefully acknowledge the financial support of this project by the National Science Foundation of China (No. 21575113), the Specialized Research Foundation for the Doctoral Program of Higher Education (No. 20126101110013), the Natural Science Foundation of Shaanxi Province in China (No. 2013KJXX-25), and the Scientific Research Foundation of Shaanxi Provincial Key Laboratory (Nos. 15JS100, 16JS099). References [1] S.Y. Dong, N. Li, G.C. Suo, T.L. Huang, Inorganic/organic doped carbon aerogels as new biosensing materials for the detection of hydrogen peroxide, Anal. Chem. 85 (2013) 11739–11746, http://dx.doi.org/10.1021/ac4015098. [2] A. Brajter-Toth, J.Q. Chambers, Electroanalytical Methods for Biological Materials, Marcel Dekker, New York, 2002. [3] H.U. Lee, Y. Yoo, T.L. Khnagvasuren, Y.S. Song, C. Park, J. Kim, S.W. Kim, Enzymatic fuel cells based on electrodeposited graphite oxide/cobalt hydroxide/ chitosan composite–enzyme electrode, Biosens. Bioelectron. (2013), http://dx.doi. org/10.1016/j. [4] S.L. Zhao, X.W. Ji, P.T. Lin, Y.M. Liu, A gold nanoparticle-mediated enzyme bioreactor for inhibitor screening by capillary electrophoresis, Anal. Biochem. 411 (2011) 88–93, http://dx.doi.org/10.1016/j.ab.2010.12.025. [5] C. Leger, P. Bertrand, Direct electrochemistry of redox enzymes as a tool for mechanistic studies, Chem. Rev. 108 (2008) 2379–2438. [6] S. Shleev, J. Tkac, A. Christenson, T. Ruzgas, A.I. Yaropolov, J.W. Whittakerd, L. Gorton, Direct electron transfer between copper-containing proteins and electrodes, Biosens. Bioelectron. 20 (2005) 2517–2554, http://dx.doi.org/10.1016/j. bios.2004.10.003. [7] L. Meng, J. Jin, G.X. Yang, T.H. Lu, H. Zhang, C.X. Cai, Nonenzymatic electrochemical detection of glucose based on palladium-single-walled carbon nanotube hybrid nanostructures, Anal. Chem. 81 (17) (2009) 7271–7280, http://dx.doi.org/ 10.1021/ac901005p. [8] C. Shan, H. Yang, D. Han, Q. Zhang, A. Ivaska, L. Niu, Graphene/AuNPs/chitosan nanocomposites film for glucose biosensing, Biosens. Bioelectron. 25 (2010)
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