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Hydrogen peroxide biosensor based on direct electron transfer of horseradish peroxidase with vapor deposited quantum dots
Sensors and Actuators B 138 (2009) 278–282
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Hydrogen peroxide biosensor based on direct electron transfer of horseradish peroxidase with vapor deposited quantum dots Zhan Wang, Qiao Xu, Hai-Qiao Wang, Qin Yang, Jiu-Hong Yu, Yuan-Di Zhao ∗ Key Laboratory of Biomedical Photonics of Ministry of Education - Wuhan National Laboratory for Optoelectronics - Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, Hubei, PR China
a r t i c l e
i n f o
Article history: Received 14 November 2008 Received in revised form 15 December 2008 Accepted 18 December 2008 Available online 31 December 2008 Keywords: Horseradish peroxidase Quantum dots Vapor deposition Direct electron transfer
a b s t r a c t Horseradish peroxidase (HRP) and lipophilic quantum dots (QDs) were incorporated onto the surface of glassy carbon (GC) electrodes in various ways. It was found that HRP transfers electron directly onto the GC electrode only when the electrode was modified with QDs through vapor deposition. The response currents were linearly correlated with scan rate, indicating that the reaction is a surface controlled process. The average coverage of HRP on the electrode surface can be calculated as 2.29 × 10−11 mol cm−2 , and the heterogeneous electron transfer rate constant k was 5.80 ± 0.70 s−1 . Absorption spectra and Fouriertransform infrared spectra showed that the conformation of HRP immobilized on the electrode has no obvious change. Further studies indicated that immobilized HRP retains excellent catalytic activity to H2 O2 . The apparent Michaelis–Menten constant was calculated as 0.152 mM. It was also found that the modified electrode could be used as a sensor for H2 O2 , and the linear range of detection was 5.0 × 10−6 to 1.0 × 10−4 M, with a detection limit of 2.84 × 10−7 M. The sensor exhibited reproducibility, stability. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The electron transfer of redox enzyme onto the surface of an electrode is an important topic in the field of bioelectrochemistry [1]. It can be used to investigate the mechanism of biological electron transfer [2,3], and also provide an important platform for the development of biosensors and catalytic bioreactors [4,5]. Horseradish peroxidase (HRP) is one of the heme-containing redox enzymes with molecular weight of approximately 42,000 Da. The electron transfer converts Fe(III) in the heme of HRP to Fe(II). This process can catalyze some chemical reaction such as the reduction of H2 O2 [6]. Unfortunately, strong adsorption of HRP on the electrode surface could cause denaturation. On the other hand, the electrochemically active centers in HRP are always buried deeply in its extended three-dimensional structure, which makes direct electron transfer between HRP and the electrode surface very difficult. Some materials, such as self-assembled monolayers [7] and surfactants [8] were introduced to realize direct electron transfer between HRP and electrode. In recent years, some nanomaterials have been used in electrochemical sensors based on enzymes. For example, colloidal gold nanoparticles were used to modify a carbon paste electrode to explore the direct electrochemistry of HRP and the determination of H2 O2 in the absence of mediator [9]; TiO2
∗ Corresponding author. Tel.: +86 27 87792235; fax: +86 27 87792202. E-mail address: [email protected] (Y.-D. Zhao). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.12.040
nanoparticles were also used to modify a pyrolytic graphite electrode to realize the direct electrochemistry and electrocatalysis of HRP [10]. In our previous work, carbon nanotube material was chosen to facilitate direct electron transfer between HRP and electrode surface [11]. Quantum dots (QDs), also called semiconductor nanocrystals, are a class of nanoparticles containing group II–VI elements or group III–V elements, with diameter between 1 and 100 nm. Due to their size dependent properties and dimensional similarities with biological macromolecules, QDs show the potential application for the investigation of the bioelectrochemistry. Recently, QDs have been widely used for the study of the protein electrochemistry and used in the field of biosensor [12–14]. In this work, a novel method, vapor deposition, was used to immobilize HRP with lipophilic CdSe/ZnS QDs on a GC electrode surface. The results showed that lipophilic CdSe/ZnS QDs can be successfully used to immobilize HRP, and facilitate direct electron transfer onto the GC electrode surface. Base on the direct electrochemistry of HRP, a H2 O2 biosensor has been fabricated. 2. Experimental 2.1. Reagents HRP was purchased from Roche Corporation, and CdSe/ZnS QDs were synthesized in our laboratory. Phosphate buffer solutions (0.1 M) were prepared by mixing stock standard solutions of
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Na2 HPO4 and NaH2 PO4 , and adjusted with HCl or NaOH to various pH values. The stock solution of H2 O2 (0.1 M) was calibrated with permanganate and diluted freshly before use. All chemicals were of analytical grade and triple-distilled water was used for preparing all of the solutions. 2.2. Preparation of CdSe/ZnS QDs CdSe/ZnS QDs were prepared according to reference [15]. In brief, a mixture of tri-n-octylphosphine oxide (TOPO) and hexadecyl amine (HDA) was heated, then Cd(Ac)2 was added into the solution. The stock solution of TOP/Se prepared by dissolving 0.2 g selenium in 5 g of tri-n-octylphosphine (TOP) was injected quickly into the reaction solution under vigorous stirring, resulting in nucleation of CdSe nanocrystals. Then, Zn(Ac)2 and bis(trimethylsilyl) sulfide ((TMS)2 S) were added, to permit the inorganic epitaxial growth of the shell on the surface of the core for about 2 h, then CdSe/ZnS QDs with diameter of about 3 nm were synthesized. QDs were dissolved in chloroform and preserved in sealed condition. 2.3. Preparation of modified electrodes Glassy carbon electrodes (3 mm diameter) were polished first with 0.3–0.05 m alumina slurry. After rinsing thoroughly with triple-distilled water, the electrodes were successively sonicated in absolute ethanol and triple-distilled water for about 1 min and dried at room temperature. HRP solution was obtained by dissolving 5.0 mg of HRP in 1 ml PBS (pH 7.0), then 10 l solution was dropped onto the surface of a cleaned GC electrode with a microsyringe, the electrode was hung above the QD solution in a sealed container overnight, and the distance between the electrode and the solution was about 2–5 mm, then the electrode modified with HRP and QDs by vapor deposition was obtained as a QD + HRP/GC electrode. As a comparison, the HRP–GC electrode was fabricated with chloroform instead of QD solution and the QD–GC electrode was fabricated with PBS instead of HRP solution by the same vapor deposition method. Another method employed to modify the electrode was direct dropwise. A QD–HRP–GC electrode was obtained by adding 10 l HRP solution onto the surface of a cleaned GC electrode, then dropping 10 l QD solution on it after air-drying directly, and an HRP–QD–GC electrode was obtained by an inverse dropping sequence. Both of these electrodes were used in comparison experiments. All of the electrodes were stored at 4 ◦ C when not in use.
Fig. 1. SEM views with magnification of 5000: (a) QD–HRP–GC electrode and (b) QD + HRP/GC electrode.
A QD–HRP film was made by dropping HRP solution onto a glass slide, then QDs incorporated onto it by a vapor deposition method. Fourier transform infrared (FTIR) spectra of the QD–HRP film were recorded with horizontal attenuated total reflectance (HATR) and HRP powders were recorded with a potassium bromide wafer using a Vertex 70 spectrometer (Bruker, Germany). UV–vis absorption spectra of QD–HRP film and HRP solution were obtained on a UV2500 spectrometer (Shimadzu, Japan). 3. Results and discussion
2.4. Apparatus and methods A three-electrode cell was used in the electrochemical measurement, with HRP–GC, QD–GC, QD–HRP–GC, HRP–QD–GC and QD + HRP/GC electrodes as working electrode, and a saturated calomel reference electrode (SCE) and a platinum wire counter electrode; all of the potentials were reported versus SCE. The electrochemical experiments were carried out with an IM6e electrochemical workstation (Zahner Co., Germany). Cyclic voltammogram experiments were carried out in a static electrochemical cell. Amperometric experiments were carried out in a stirred system by controlling the potential of the working electrode at −400 mV, and an aliquot of H2 O2 standard solution was added successively to the solution and the steady-state current was recorded. All experimental solutions were deoxygenated by bubbling highly pure nitrogen for 20 min and a nitrogen atmosphere maintained during measurements. The surface morphology of the modified electrodes was observed by S-3000N scanning electron microscopy (Hitachi, Japan).
The surface morphology of the modified electrodes was observed by scanning electron microscopy (SEM) after preparation. The results showed that, compared with electrode modified by dropping directly (Fig. 1a), a porous membrane structure formed on the surface of the electrode modified by vapor deposition (Fig. 1b), and this structure should favor electrochemical reactions. Fig. 2 shows the cyclic voltammograms obtained with the above modified electrodes in 0.1 M PBS (pH 7.0). It can be seen that only a weak reduction peak appeared at the HRP–GC electrode (curve a). At the electrodes modified by dropping directly, QD–HRP–GC and HRP–QD–GC, although there was HRP on the surface, only very weak redox peaks could be observed (curves c and d). However, when QDs were introduced into this system in the form of vapor deposition, a well-defined quasi-reversible peak could be observed at the QD + HRP/GC electrode (curve e). Since there were no redox peaks at the QD–GC electrode (curve b), the redox peaks in curve e were produced by HRP and caused by addition of QDs. These results showed that the direct electron transfer of HRP onto the surface of the electrode is enhanced by the introduction of QDs.
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Fig. 2. Cyclic voltammograms of: (a) HRP–GC electrode; (b) QD–GC electrode; (c) QD–HRP–GC electrode; (d) HRP–QD–GC electrode; (e) QD + HRP/GC electrode in 0.1 M PBS (pH 7.0) at a scan rate of 100 mV s−1 .
That is, HRP transfers electrons directly to the GC electrode only when QDs are modified through vapor deposition. This may be due to the slow vapor deposition process of QDs, which results in good combination of HRP and QDs, and HRP adjusts its molecule to an appropriate orientation for direct electron transfer. Thus QDs may play an important role in the electron transfer between HRP and electrode. It is also found that capacitive currents at mixed modified electrodes (curves c–e) are larger, these are due to the enhanced accessible surface area to the electrolyte. As shown in Fig. 2, curve e, the oxidation peak potential (Epa ) and reduction peak potential (Epc ) are between −177 and −242 mV, respectively, and the peak-to-peak separation (Ep ) is 65 mV. The ratio of reduction peak current (Ipc ) to oxidation peak current (Ipa ) is close to 1. All of these electrochemical parameters indicate that the reaction of HRP occurring at the electrode modified by QDs in the form of vapor deposition is a quasi-reversible redox process. The formal potential (E◦ ), which is estimated as the midpoint between Epa and Epc , is about −210 mV, and close to the −220 mV of native HRP in solution [16], suggesting that most HRP molecules preserve their native structure after being incorporated with QDs by way of vapor deposition. The effect of scan rate on the response of immobilized HRP at the QD + HRP/GC electrode is shown in Fig. 3. With the increasing of scan rate, Epa and Epc were shifted slightly toward the positive
Fig. 3. Cyclic voltammograms of QD + HRP/GC electrode at different scan rates (from a to j: 50, 100, 200, 300, 400, 500, 600, 7000, 800, and 900 mV s−1 ). Inset: the plots of (a) anodic peak current and (b) cathodic peak current versus scan rate.
Fig. 4. Effect of pH on cathodic peak current of QD + HRP/GC electrode in 0.1 M PBS at 100 mV s−1 . Inset: plot of anodic peak potential (a), formal potential (b) and cathodic peak potential (c) versus pH value.
and the negative direction of potential, respectively, while Ipa and Ipc increasing linearly. The results show that the electrochemical reaction is a surface-controlled process. For one surface-controlled process, surface coverage concentration ( ) and the number of electrons transported (n) can be obtained according to Lavrion’s equation [17]. From the curves in Fig. 3, Q can be calculated as 2.21 × 10−7 C. The effective surface area A of the electrode was obtained as 0.089 cm2 by using K3 Fe(CN)6 as a probe to characterize the QD + HRP/GC electrode. So, n can be calculated as 1.12 from the slope of Fig. 3(inset), indicating that a single electron reaction occurs at the interface. Then the average surface coverage of HRP on the surface, can be obtained as 2.29 × 10−11 mol cm−2 . The electron transfer rate ks of HRP can be estimated with the formula ks = nmF v/RT when nEp < 200 mV, where m is a parameter associated with nEp [18]. Taking the charge transfer coefficient ˛ as 0.5, an average ks value of 5.80 ± 0.70 s−1 could be produced with the peak-to-peak separation at various scan rates. This result indicates that an effective promotion could be performed by QDs in the process of electron transfer between HRP and GC electrode. It is also found that this ks value is close to the 6.04 ± 0.18 s−1 of HRP immobilized on a carbon paste electrode modified by gold colloids [19], and much larger than the 1.13 s−1 of HRP immobilized on DNA films [20]. By comparison, it can be seen that ks value depends on the materials used to immobilize the proteins. For nano-structured electrodes, the results are maybe better. Following that, the effect of pH on the electrochemical process of QD + HRP/GC electrode was observed. Clearly, a maximum peak current is present at pH 7.0, which is similar with soluble HRP [21], indicating that the optimal pH value for electron transfer of immobilized HRP on QD + HRP/GC electrode does not change. With increasing pH value from 4.0 to 9.0, Epa , Epc and E◦ all shift negatively and linearly with pH value (Fig. 4); the slopes are −52.7, −42.9 and −47.8 mV pH−1 , respectively. These values are approximately close to the theoretical value of −57.6 mV pH−1 at 18 ◦ C for a reversible, one-proton-coupled single electron transfer, indicating that a proton participates in the process of redox reaction [22]. After obtaining the direct chemistry of HRP, the other question was whether the HRP structure was changed significantly and then influenced its bioactivity or not. The shape and position of the Soret absorption band of HRP can provide structural information about HRP, especially about heme groups, so the UV–vis absorption spectra of modified films were investigated. It could be seen that the Soret band of QD + HRP film at 403.4 nm (Fig. 5, curve b) is similar to that of HRP in solution (Fig. 5, curve a), indicating that HRP retained its structure in QD + HRP film.
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Fig. 5. UV–vis absorption spectra of (a) HRP solution and (b) QD + HRP films.
As is well known, amide I (1700–1600 cm−1 ) and amide II (1620–1500 cm−1 ) can provide detailed information on the secondary structure of the polypeptide chain, so the QD + HRP film was also studied by FTIR. As shown in Fig. 6, the amide I and amide II bands of HRP are located at 1657 and 1535 cm−1 in the FTIR spectrum (curve a), and the FTIR spectrum of QD + HRP film (curve b) has similar shapes to that of free HRP, with the amide I and amide II bands only shifted slightly to 1644 and 1537 cm−1 , respectively. The results further prove that HRP is not denatured in QD + HRP film. In order to inspect the catalytic activity of HRP, cyclic voltammograms of the QD + HRP/GC electrode were studied in various solutions with different concentration of H2 O2 . The results indicate that the cathodic peak currents increase along with increasing H2 O2 concentration (Fig. 7A). However, the controlled experiments show that, although the electrodes are modified with HRP, no relevant catalytic peak currents could be found by the direct dropping method (Fig. 7B). This proves that the catalytic activity of HRP to H2 O2 is attributed to the introduction route of QDs, that is to say, the immobilized HRP on the electrode still maintains its electrocatalytic activity only when QDs are immobilized by vapor deposition. The steady-state current response of H2 O2 on the QD + HRP/GC electrode was applied at the potential of −400 mV. The results showed that the catalytic currents increased with the successive addition of H2 O2 . The current is linear with H2 O2 concentration from 5.0 × 10−6 to 1.0 × 10−5 M with a correlation coefficient of 0.987, and the detection limit was 2.84 × 10−7 M.
Fig. 7. Cyclic voltammograms of QD + HRP/GC electrode (A) and HRP–QD–GC electrode (B) in different concentration of H2 O2 : (a) 0 M; (b) 25 M; (c) 75 M.
The reaction has typical kinetic characteristics of the enzymecatalyzed reaction because the current tends toward stability when the concentration is above 1.0 × 10−4 M. The reciprocals of steady-state current and H2 O2 concentration have a good linear relationship with a correlation coefficient of 0.9998. So, the app apparent Michaelis–Menten constant (KM ) for the electrocatalytic activity of the QD + HRP/GC electrode can be calculated as 0.152 mM from the Lineweaver–Burk equation [23], which implies that HRP immobilized on the QD + HRP/GC electrode exhibits a high degree of catalysis. It is also confirmed that HRP could retain its biological activity when exchanging electrons with the electrode directly. The repeatability of the QD + HRP/GC electrode was examined at 10 M H2 O2 . The electrode showed a fairly good repeatability with a relative standard deviation (R.S.D.) of 2.9% for nine successive measurements. Four electrodes made independently showed an acceptable reproducibility with a R.S.D. of 3.5% for the current determined at 10 M H2 O2 . When the modified electrode was stored at 4 ◦ C for about 1 week, it retained more than 89% of its initial sensitivity to the reduction of H2 O2 , showing that the modified electrode possesses good stability. 4. Conclusions
Fig. 6. FT-IR spectra of (a) HRP and (b) QD + HRP films.
Lipophilic QDs with a core–shell structure have been extensively applied to bioanalysis. The results of our experiments indicate that lipophilic QDs could promote the electron transfer between proteins and electrode, and this electrochemical behavior relies on the way that QDs modify the surface of the electrode. Accordingly, this work has important consequences for providing a new way for the investigation of direct electron transfer between proteins and
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electrode, and it also expands the application prospect of lipophilic nanoparticles. Acknowledgements The work was supported by the National Natural Science Foundation of China (Grant No. 30670553, 90717101). We also thank Analytical and Testing Center (Huazhong University of Science & Technology) for the help in measurement. References [1] Y.H. Wu, S.S. Hu, Biosensors based on direct electron transfer in redox proteins, Microchim. Acta 159 (2007) 1–17. [2] L. Zhu, X.C. Zhou, Y.W. Zhang, W. Xing, T.H. Lu, A bifunctional structure for the direct electron transfer of cytochrome c, Electrochim. Acta 53 (2008) 7726–7729. [3] R. Andreu, E.E. Ferapontova, L. Gorton, J.J. Calvente, Direct electron transfer kinetics in horseradish peroxidase electrocatalysis, J. Phys. Chem. B 111 (2007) 469–477. [4] W.J. Zhang, G.X. Li, Third-generation biosensors based on the direct electron transfer of proteins, Anal. Sci. 20 (2004) 603–609. [5] A.K.M. Kafi, D.Y. Lee, S.H. Park, Y.S. Kwon, Development of peroxide biosensor of thiolated-viologen and hemoglobin-modified gold electrode, Microchem. J. 85 (2007) 308–313. [6] J.G. Zhao, R.W. Henkens, J. Stonehuerner, J.P.O. Daly, A.L. Crumbliss, Direct electron transfer at horseradish peroxidase–colloidal gold modified electrodes, J. Electroanal. Chem. 327 (1992) 109–119. [7] F.H. Wu, Z.H. Hua, J.J. Xua, Y. Tian, L.W. Wang, Y.Z. Xian, L.T. Jin, Immobilization of horseradish peroxidase on self-assembled (3-mercaptopropyl) trimethoxysilane film: Characterization, direct electrochemistry, redox thermodynamics and biosensing, Electrochim. Acta 53 (2008) 8238–8244. [8] L.H. Liua, F.Q. Zhao, L.Q. Liua, J. Lia, B.Z. Zeng, Improved direct electron transfer and electrocatalytic activity of horseradish peroxidase immobilized on gemini surfactant–polyvinyl alcohol composite film, Colloid Surf. B 68 (2009) 93–97. [9] Y. Liu, R. Yuan, Y.Q. Chai, D.P. Tang, J.Y. Dai, X. Zhong, Direct electrochemistry of horseradish peroxidase immobilized on gold colloid/cysteine/nafion-modified platinum disk electrode, Sens. Actuators B 115 (2006) 109–115. [10] Y. Zhang, P.L. He, N.F. Hu, Horseradish peroxidase immobilized in TiO2 nanoparticle films on pyrolytic graphite electrodes: direct electrochemistry and bioelectrocatalysis, Electrochim. Acta 49 (2004) 1981–1988. [11] Y.D. Zhao, W.D. Zhang, H. Chen, Q.M. Luo, S.F.Y. Li, Direct electrochemistry of horseradish peroxidase at carbon nanotube powder microelectrode, Sens. Actuators B: Chem. 87 (2002) 168–172. [12] Y.X. Xu, J.G. Liang, C.G. Hu, F. Wang, S.S. Hu, Z.K. He, A hydrogen peroxide biosensor based on the direct electrochemistry of hemoglobin modified with quantum dots, J. Biol. Inorg. Chem. 12 (2007) 421–427. [13] Q. Liu, X.B. Lu, J. Li, X. Yao, J.H. Li, Direct electrochemistry of glucose oxidase and electrochemical biosensing of glucose on quantum dots/carbon nanotubes electrodes, Biosens. Bioelectron. 22 (2007) 3203–3209. [14] D. Du, W.J. Chen, J. Cai, J. Zhang, F.G. Qu, H.B. Li, Development of acetylcholinesterase biosensor based on CdTe quantum dots modied cysteamine self-assembled monolayers, J. Electroanal. Chem. 623 (2008) 81–85. [15] H.Q. Wang, T.C. Liu, Y.C. Cao, Z.L. Huang, J.H. Wang, X.Q. Li, Y.D. Zhao, A flow cytometric assay technology based on quantum dots-encoded beads, Anal. Chim. Acta 580 (2006) 18–23.
[16] H.A. Harbury, Oxidation–reduction potentials of horseradish peroxidase, J. Biol. Chem. 225 (1957) 1009–1024. [17] E. Laviron, The use of linear potential sweep voltammetry and of A.C. voltammetry for the study of the surface electrochemical reaction of strongly adsorbed systems and of redox modified electrodes, J. Electroanal. Chem. 100 (1979) 263–270. [18] E. Laviron, General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems, J. Electroanal. Chem. 101 (1979) 19–28. [19] S.Q. Liu, H.X. Ju, Renewable reagentless hydrogen peroxide sensor based on direct electron transfer of horseradish peroxidase immobilized on colloidal gold-modified electrode, Anal. Biochem. 307 (2002) 110–116. [20] X. Chen, C. Ruan, J. Kong, J. Deng, Characterization of the direct electron transfer and bioelectrocatalysis of horseradish peroxidase in DNA film at pyrolyltic graphite electrode, Anal. Chim. Acta 412 (2000) 89–98. [21] A.C. Maehly, Plant peroxidase, in: S.P. Colowick, N.O. Kaplan (Eds.), Methods in Enzymology, vol. 11, Academic Press, New York, 1955, p. 807. [22] A.M. Bond, Modern polarographic methods, in: Analytical Chemistry, Marcel Dekker, New York, 1980, pp. 27–45. [23] H. Lineweaver, D.J. Burk, Determination of enzyme dissociation constants, J. Am. Chem. Soc. 56 (1934) 658–666.
Biographies Zhan Wang received her MS from Wuhan Polytechnic Uniersity in 2002. Presently, she is a PhD candidate in Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science & Technology, PR China. Her current fields of interest are bioelectrochemistry and biosensor. Qiao Xu received her MS from HuBei Uniersity in 2006. Presently, she is a PhD candidate in Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science & Technology, PR China. Her current fields of interest are bioelectrochemistry and biosensor. Hai-Qiao Wang received his Bachelor from Huazhong Uniersity of Science & Technology in 2003. Presently, he is a PhD candidate in Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science & Technology, PR China. His current fields of interest are bionanomaterials and biosensor. Qin Yang received her BA from Huazhong Uniersity of Science & Technology in 2008. Presently, she is a graduate student in Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science & Technology, PR China. Her current fields of interest are bioelectrochemistry and biosensor. Jiu-Hong Yu received his BSc from Hubei University, PR China in 2000 and PhD from Nanjing University, PR China in 2003. Presently, he is a postdoctoral candidate in Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science & Technology, PR China. Her current fields of interest are bioelectrochemistry and biosensor. Yuan-Di Zhao received his BSc in chemistry from Wuhan University, PR China in 1994 and PhD of science from the same University in 1999. Presently, he is a professor in the Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science & Technology, PR China. His current fields of interest are bioelectrochemistry, nanobiotechnology, and biosensor.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Hydrogen peroxide biosensor based on direct electron transfer of horseradish peroxidase with vapor deposited quantum dots Zhan Wang, Qiao Xu, Hai-Qiao Wang, Qin Yang, Jiu-Hong Yu, Yuan-Di Zhao ∗ Key Laboratory of Biomedical Photonics of Ministry of Education - Wuhan National Laboratory for Optoelectronics - Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, Hubei, PR China
a r t i c l e
i n f o
Article history: Received 14 November 2008 Received in revised form 15 December 2008 Accepted 18 December 2008 Available online 31 December 2008 Keywords: Horseradish peroxidase Quantum dots Vapor deposition Direct electron transfer
a b s t r a c t Horseradish peroxidase (HRP) and lipophilic quantum dots (QDs) were incorporated onto the surface of glassy carbon (GC) electrodes in various ways. It was found that HRP transfers electron directly onto the GC electrode only when the electrode was modified with QDs through vapor deposition. The response currents were linearly correlated with scan rate, indicating that the reaction is a surface controlled process. The average coverage of HRP on the electrode surface can be calculated as 2.29 × 10−11 mol cm−2 , and the heterogeneous electron transfer rate constant k was 5.80 ± 0.70 s−1 . Absorption spectra and Fouriertransform infrared spectra showed that the conformation of HRP immobilized on the electrode has no obvious change. Further studies indicated that immobilized HRP retains excellent catalytic activity to H2 O2 . The apparent Michaelis–Menten constant was calculated as 0.152 mM. It was also found that the modified electrode could be used as a sensor for H2 O2 , and the linear range of detection was 5.0 × 10−6 to 1.0 × 10−4 M, with a detection limit of 2.84 × 10−7 M. The sensor exhibited reproducibility, stability. © 2008 Elsevier B.V. All rights reserved.
1. Introduction The electron transfer of redox enzyme onto the surface of an electrode is an important topic in the field of bioelectrochemistry [1]. It can be used to investigate the mechanism of biological electron transfer [2,3], and also provide an important platform for the development of biosensors and catalytic bioreactors [4,5]. Horseradish peroxidase (HRP) is one of the heme-containing redox enzymes with molecular weight of approximately 42,000 Da. The electron transfer converts Fe(III) in the heme of HRP to Fe(II). This process can catalyze some chemical reaction such as the reduction of H2 O2 [6]. Unfortunately, strong adsorption of HRP on the electrode surface could cause denaturation. On the other hand, the electrochemically active centers in HRP are always buried deeply in its extended three-dimensional structure, which makes direct electron transfer between HRP and the electrode surface very difficult. Some materials, such as self-assembled monolayers [7] and surfactants [8] were introduced to realize direct electron transfer between HRP and electrode. In recent years, some nanomaterials have been used in electrochemical sensors based on enzymes. For example, colloidal gold nanoparticles were used to modify a carbon paste electrode to explore the direct electrochemistry of HRP and the determination of H2 O2 in the absence of mediator [9]; TiO2
∗ Corresponding author. Tel.: +86 27 87792235; fax: +86 27 87792202. E-mail address: [email protected] (Y.-D. Zhao). 0925-4005/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2008.12.040
nanoparticles were also used to modify a pyrolytic graphite electrode to realize the direct electrochemistry and electrocatalysis of HRP [10]. In our previous work, carbon nanotube material was chosen to facilitate direct electron transfer between HRP and electrode surface [11]. Quantum dots (QDs), also called semiconductor nanocrystals, are a class of nanoparticles containing group II–VI elements or group III–V elements, with diameter between 1 and 100 nm. Due to their size dependent properties and dimensional similarities with biological macromolecules, QDs show the potential application for the investigation of the bioelectrochemistry. Recently, QDs have been widely used for the study of the protein electrochemistry and used in the field of biosensor [12–14]. In this work, a novel method, vapor deposition, was used to immobilize HRP with lipophilic CdSe/ZnS QDs on a GC electrode surface. The results showed that lipophilic CdSe/ZnS QDs can be successfully used to immobilize HRP, and facilitate direct electron transfer onto the GC electrode surface. Base on the direct electrochemistry of HRP, a H2 O2 biosensor has been fabricated. 2. Experimental 2.1. Reagents HRP was purchased from Roche Corporation, and CdSe/ZnS QDs were synthesized in our laboratory. Phosphate buffer solutions (0.1 M) were prepared by mixing stock standard solutions of
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Na2 HPO4 and NaH2 PO4 , and adjusted with HCl or NaOH to various pH values. The stock solution of H2 O2 (0.1 M) was calibrated with permanganate and diluted freshly before use. All chemicals were of analytical grade and triple-distilled water was used for preparing all of the solutions. 2.2. Preparation of CdSe/ZnS QDs CdSe/ZnS QDs were prepared according to reference [15]. In brief, a mixture of tri-n-octylphosphine oxide (TOPO) and hexadecyl amine (HDA) was heated, then Cd(Ac)2 was added into the solution. The stock solution of TOP/Se prepared by dissolving 0.2 g selenium in 5 g of tri-n-octylphosphine (TOP) was injected quickly into the reaction solution under vigorous stirring, resulting in nucleation of CdSe nanocrystals. Then, Zn(Ac)2 and bis(trimethylsilyl) sulfide ((TMS)2 S) were added, to permit the inorganic epitaxial growth of the shell on the surface of the core for about 2 h, then CdSe/ZnS QDs with diameter of about 3 nm were synthesized. QDs were dissolved in chloroform and preserved in sealed condition. 2.3. Preparation of modified electrodes Glassy carbon electrodes (3 mm diameter) were polished first with 0.3–0.05 m alumina slurry. After rinsing thoroughly with triple-distilled water, the electrodes were successively sonicated in absolute ethanol and triple-distilled water for about 1 min and dried at room temperature. HRP solution was obtained by dissolving 5.0 mg of HRP in 1 ml PBS (pH 7.0), then 10 l solution was dropped onto the surface of a cleaned GC electrode with a microsyringe, the electrode was hung above the QD solution in a sealed container overnight, and the distance between the electrode and the solution was about 2–5 mm, then the electrode modified with HRP and QDs by vapor deposition was obtained as a QD + HRP/GC electrode. As a comparison, the HRP–GC electrode was fabricated with chloroform instead of QD solution and the QD–GC electrode was fabricated with PBS instead of HRP solution by the same vapor deposition method. Another method employed to modify the electrode was direct dropwise. A QD–HRP–GC electrode was obtained by adding 10 l HRP solution onto the surface of a cleaned GC electrode, then dropping 10 l QD solution on it after air-drying directly, and an HRP–QD–GC electrode was obtained by an inverse dropping sequence. Both of these electrodes were used in comparison experiments. All of the electrodes were stored at 4 ◦ C when not in use.
Fig. 1. SEM views with magnification of 5000: (a) QD–HRP–GC electrode and (b) QD + HRP/GC electrode.
A QD–HRP film was made by dropping HRP solution onto a glass slide, then QDs incorporated onto it by a vapor deposition method. Fourier transform infrared (FTIR) spectra of the QD–HRP film were recorded with horizontal attenuated total reflectance (HATR) and HRP powders were recorded with a potassium bromide wafer using a Vertex 70 spectrometer (Bruker, Germany). UV–vis absorption spectra of QD–HRP film and HRP solution were obtained on a UV2500 spectrometer (Shimadzu, Japan). 3. Results and discussion
2.4. Apparatus and methods A three-electrode cell was used in the electrochemical measurement, with HRP–GC, QD–GC, QD–HRP–GC, HRP–QD–GC and QD + HRP/GC electrodes as working electrode, and a saturated calomel reference electrode (SCE) and a platinum wire counter electrode; all of the potentials were reported versus SCE. The electrochemical experiments were carried out with an IM6e electrochemical workstation (Zahner Co., Germany). Cyclic voltammogram experiments were carried out in a static electrochemical cell. Amperometric experiments were carried out in a stirred system by controlling the potential of the working electrode at −400 mV, and an aliquot of H2 O2 standard solution was added successively to the solution and the steady-state current was recorded. All experimental solutions were deoxygenated by bubbling highly pure nitrogen for 20 min and a nitrogen atmosphere maintained during measurements. The surface morphology of the modified electrodes was observed by S-3000N scanning electron microscopy (Hitachi, Japan).
The surface morphology of the modified electrodes was observed by scanning electron microscopy (SEM) after preparation. The results showed that, compared with electrode modified by dropping directly (Fig. 1a), a porous membrane structure formed on the surface of the electrode modified by vapor deposition (Fig. 1b), and this structure should favor electrochemical reactions. Fig. 2 shows the cyclic voltammograms obtained with the above modified electrodes in 0.1 M PBS (pH 7.0). It can be seen that only a weak reduction peak appeared at the HRP–GC electrode (curve a). At the electrodes modified by dropping directly, QD–HRP–GC and HRP–QD–GC, although there was HRP on the surface, only very weak redox peaks could be observed (curves c and d). However, when QDs were introduced into this system in the form of vapor deposition, a well-defined quasi-reversible peak could be observed at the QD + HRP/GC electrode (curve e). Since there were no redox peaks at the QD–GC electrode (curve b), the redox peaks in curve e were produced by HRP and caused by addition of QDs. These results showed that the direct electron transfer of HRP onto the surface of the electrode is enhanced by the introduction of QDs.
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Fig. 2. Cyclic voltammograms of: (a) HRP–GC electrode; (b) QD–GC electrode; (c) QD–HRP–GC electrode; (d) HRP–QD–GC electrode; (e) QD + HRP/GC electrode in 0.1 M PBS (pH 7.0) at a scan rate of 100 mV s−1 .
That is, HRP transfers electrons directly to the GC electrode only when QDs are modified through vapor deposition. This may be due to the slow vapor deposition process of QDs, which results in good combination of HRP and QDs, and HRP adjusts its molecule to an appropriate orientation for direct electron transfer. Thus QDs may play an important role in the electron transfer between HRP and electrode. It is also found that capacitive currents at mixed modified electrodes (curves c–e) are larger, these are due to the enhanced accessible surface area to the electrolyte. As shown in Fig. 2, curve e, the oxidation peak potential (Epa ) and reduction peak potential (Epc ) are between −177 and −242 mV, respectively, and the peak-to-peak separation (Ep ) is 65 mV. The ratio of reduction peak current (Ipc ) to oxidation peak current (Ipa ) is close to 1. All of these electrochemical parameters indicate that the reaction of HRP occurring at the electrode modified by QDs in the form of vapor deposition is a quasi-reversible redox process. The formal potential (E◦ ), which is estimated as the midpoint between Epa and Epc , is about −210 mV, and close to the −220 mV of native HRP in solution [16], suggesting that most HRP molecules preserve their native structure after being incorporated with QDs by way of vapor deposition. The effect of scan rate on the response of immobilized HRP at the QD + HRP/GC electrode is shown in Fig. 3. With the increasing of scan rate, Epa and Epc were shifted slightly toward the positive
Fig. 3. Cyclic voltammograms of QD + HRP/GC electrode at different scan rates (from a to j: 50, 100, 200, 300, 400, 500, 600, 7000, 800, and 900 mV s−1 ). Inset: the plots of (a) anodic peak current and (b) cathodic peak current versus scan rate.
Fig. 4. Effect of pH on cathodic peak current of QD + HRP/GC electrode in 0.1 M PBS at 100 mV s−1 . Inset: plot of anodic peak potential (a), formal potential (b) and cathodic peak potential (c) versus pH value.
and the negative direction of potential, respectively, while Ipa and Ipc increasing linearly. The results show that the electrochemical reaction is a surface-controlled process. For one surface-controlled process, surface coverage concentration ( ) and the number of electrons transported (n) can be obtained according to Lavrion’s equation [17]. From the curves in Fig. 3, Q can be calculated as 2.21 × 10−7 C. The effective surface area A of the electrode was obtained as 0.089 cm2 by using K3 Fe(CN)6 as a probe to characterize the QD + HRP/GC electrode. So, n can be calculated as 1.12 from the slope of Fig. 3(inset), indicating that a single electron reaction occurs at the interface. Then the average surface coverage of HRP on the surface, can be obtained as 2.29 × 10−11 mol cm−2 . The electron transfer rate ks of HRP can be estimated with the formula ks = nmF v/RT when nEp < 200 mV, where m is a parameter associated with nEp [18]. Taking the charge transfer coefficient ˛ as 0.5, an average ks value of 5.80 ± 0.70 s−1 could be produced with the peak-to-peak separation at various scan rates. This result indicates that an effective promotion could be performed by QDs in the process of electron transfer between HRP and GC electrode. It is also found that this ks value is close to the 6.04 ± 0.18 s−1 of HRP immobilized on a carbon paste electrode modified by gold colloids [19], and much larger than the 1.13 s−1 of HRP immobilized on DNA films [20]. By comparison, it can be seen that ks value depends on the materials used to immobilize the proteins. For nano-structured electrodes, the results are maybe better. Following that, the effect of pH on the electrochemical process of QD + HRP/GC electrode was observed. Clearly, a maximum peak current is present at pH 7.0, which is similar with soluble HRP [21], indicating that the optimal pH value for electron transfer of immobilized HRP on QD + HRP/GC electrode does not change. With increasing pH value from 4.0 to 9.0, Epa , Epc and E◦ all shift negatively and linearly with pH value (Fig. 4); the slopes are −52.7, −42.9 and −47.8 mV pH−1 , respectively. These values are approximately close to the theoretical value of −57.6 mV pH−1 at 18 ◦ C for a reversible, one-proton-coupled single electron transfer, indicating that a proton participates in the process of redox reaction [22]. After obtaining the direct chemistry of HRP, the other question was whether the HRP structure was changed significantly and then influenced its bioactivity or not. The shape and position of the Soret absorption band of HRP can provide structural information about HRP, especially about heme groups, so the UV–vis absorption spectra of modified films were investigated. It could be seen that the Soret band of QD + HRP film at 403.4 nm (Fig. 5, curve b) is similar to that of HRP in solution (Fig. 5, curve a), indicating that HRP retained its structure in QD + HRP film.
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Fig. 5. UV–vis absorption spectra of (a) HRP solution and (b) QD + HRP films.
As is well known, amide I (1700–1600 cm−1 ) and amide II (1620–1500 cm−1 ) can provide detailed information on the secondary structure of the polypeptide chain, so the QD + HRP film was also studied by FTIR. As shown in Fig. 6, the amide I and amide II bands of HRP are located at 1657 and 1535 cm−1 in the FTIR spectrum (curve a), and the FTIR spectrum of QD + HRP film (curve b) has similar shapes to that of free HRP, with the amide I and amide II bands only shifted slightly to 1644 and 1537 cm−1 , respectively. The results further prove that HRP is not denatured in QD + HRP film. In order to inspect the catalytic activity of HRP, cyclic voltammograms of the QD + HRP/GC electrode were studied in various solutions with different concentration of H2 O2 . The results indicate that the cathodic peak currents increase along with increasing H2 O2 concentration (Fig. 7A). However, the controlled experiments show that, although the electrodes are modified with HRP, no relevant catalytic peak currents could be found by the direct dropping method (Fig. 7B). This proves that the catalytic activity of HRP to H2 O2 is attributed to the introduction route of QDs, that is to say, the immobilized HRP on the electrode still maintains its electrocatalytic activity only when QDs are immobilized by vapor deposition. The steady-state current response of H2 O2 on the QD + HRP/GC electrode was applied at the potential of −400 mV. The results showed that the catalytic currents increased with the successive addition of H2 O2 . The current is linear with H2 O2 concentration from 5.0 × 10−6 to 1.0 × 10−5 M with a correlation coefficient of 0.987, and the detection limit was 2.84 × 10−7 M.
Fig. 7. Cyclic voltammograms of QD + HRP/GC electrode (A) and HRP–QD–GC electrode (B) in different concentration of H2 O2 : (a) 0 M; (b) 25 M; (c) 75 M.
The reaction has typical kinetic characteristics of the enzymecatalyzed reaction because the current tends toward stability when the concentration is above 1.0 × 10−4 M. The reciprocals of steady-state current and H2 O2 concentration have a good linear relationship with a correlation coefficient of 0.9998. So, the app apparent Michaelis–Menten constant (KM ) for the electrocatalytic activity of the QD + HRP/GC electrode can be calculated as 0.152 mM from the Lineweaver–Burk equation [23], which implies that HRP immobilized on the QD + HRP/GC electrode exhibits a high degree of catalysis. It is also confirmed that HRP could retain its biological activity when exchanging electrons with the electrode directly. The repeatability of the QD + HRP/GC electrode was examined at 10 M H2 O2 . The electrode showed a fairly good repeatability with a relative standard deviation (R.S.D.) of 2.9% for nine successive measurements. Four electrodes made independently showed an acceptable reproducibility with a R.S.D. of 3.5% for the current determined at 10 M H2 O2 . When the modified electrode was stored at 4 ◦ C for about 1 week, it retained more than 89% of its initial sensitivity to the reduction of H2 O2 , showing that the modified electrode possesses good stability. 4. Conclusions
Fig. 6. FT-IR spectra of (a) HRP and (b) QD + HRP films.
Lipophilic QDs with a core–shell structure have been extensively applied to bioanalysis. The results of our experiments indicate that lipophilic QDs could promote the electron transfer between proteins and electrode, and this electrochemical behavior relies on the way that QDs modify the surface of the electrode. Accordingly, this work has important consequences for providing a new way for the investigation of direct electron transfer between proteins and
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electrode, and it also expands the application prospect of lipophilic nanoparticles. Acknowledgements The work was supported by the National Natural Science Foundation of China (Grant No. 30670553, 90717101). We also thank Analytical and Testing Center (Huazhong University of Science & Technology) for the help in measurement. References [1] Y.H. Wu, S.S. Hu, Biosensors based on direct electron transfer in redox proteins, Microchim. Acta 159 (2007) 1–17. [2] L. Zhu, X.C. Zhou, Y.W. Zhang, W. Xing, T.H. Lu, A bifunctional structure for the direct electron transfer of cytochrome c, Electrochim. Acta 53 (2008) 7726–7729. [3] R. Andreu, E.E. Ferapontova, L. Gorton, J.J. Calvente, Direct electron transfer kinetics in horseradish peroxidase electrocatalysis, J. Phys. Chem. B 111 (2007) 469–477. [4] W.J. Zhang, G.X. Li, Third-generation biosensors based on the direct electron transfer of proteins, Anal. Sci. 20 (2004) 603–609. [5] A.K.M. Kafi, D.Y. Lee, S.H. Park, Y.S. Kwon, Development of peroxide biosensor of thiolated-viologen and hemoglobin-modified gold electrode, Microchem. J. 85 (2007) 308–313. [6] J.G. Zhao, R.W. Henkens, J. Stonehuerner, J.P.O. Daly, A.L. Crumbliss, Direct electron transfer at horseradish peroxidase–colloidal gold modified electrodes, J. Electroanal. Chem. 327 (1992) 109–119. [7] F.H. Wu, Z.H. Hua, J.J. Xua, Y. Tian, L.W. Wang, Y.Z. Xian, L.T. Jin, Immobilization of horseradish peroxidase on self-assembled (3-mercaptopropyl) trimethoxysilane film: Characterization, direct electrochemistry, redox thermodynamics and biosensing, Electrochim. Acta 53 (2008) 8238–8244. [8] L.H. Liua, F.Q. Zhao, L.Q. Liua, J. Lia, B.Z. Zeng, Improved direct electron transfer and electrocatalytic activity of horseradish peroxidase immobilized on gemini surfactant–polyvinyl alcohol composite film, Colloid Surf. B 68 (2009) 93–97. [9] Y. Liu, R. Yuan, Y.Q. Chai, D.P. Tang, J.Y. Dai, X. Zhong, Direct electrochemistry of horseradish peroxidase immobilized on gold colloid/cysteine/nafion-modified platinum disk electrode, Sens. Actuators B 115 (2006) 109–115. [10] Y. Zhang, P.L. He, N.F. Hu, Horseradish peroxidase immobilized in TiO2 nanoparticle films on pyrolytic graphite electrodes: direct electrochemistry and bioelectrocatalysis, Electrochim. Acta 49 (2004) 1981–1988. [11] Y.D. Zhao, W.D. Zhang, H. Chen, Q.M. Luo, S.F.Y. Li, Direct electrochemistry of horseradish peroxidase at carbon nanotube powder microelectrode, Sens. Actuators B: Chem. 87 (2002) 168–172. [12] Y.X. Xu, J.G. Liang, C.G. Hu, F. Wang, S.S. Hu, Z.K. He, A hydrogen peroxide biosensor based on the direct electrochemistry of hemoglobin modified with quantum dots, J. Biol. Inorg. Chem. 12 (2007) 421–427. [13] Q. Liu, X.B. Lu, J. Li, X. Yao, J.H. Li, Direct electrochemistry of glucose oxidase and electrochemical biosensing of glucose on quantum dots/carbon nanotubes electrodes, Biosens. Bioelectron. 22 (2007) 3203–3209. [14] D. Du, W.J. Chen, J. Cai, J. Zhang, F.G. Qu, H.B. Li, Development of acetylcholinesterase biosensor based on CdTe quantum dots modied cysteamine self-assembled monolayers, J. Electroanal. Chem. 623 (2008) 81–85. [15] H.Q. Wang, T.C. Liu, Y.C. Cao, Z.L. Huang, J.H. Wang, X.Q. Li, Y.D. Zhao, A flow cytometric assay technology based on quantum dots-encoded beads, Anal. Chim. Acta 580 (2006) 18–23.
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Biographies Zhan Wang received her MS from Wuhan Polytechnic Uniersity in 2002. Presently, she is a PhD candidate in Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science & Technology, PR China. Her current fields of interest are bioelectrochemistry and biosensor. Qiao Xu received her MS from HuBei Uniersity in 2006. Presently, she is a PhD candidate in Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science & Technology, PR China. Her current fields of interest are bioelectrochemistry and biosensor. Hai-Qiao Wang received his Bachelor from Huazhong Uniersity of Science & Technology in 2003. Presently, he is a PhD candidate in Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science & Technology, PR China. His current fields of interest are bionanomaterials and biosensor. Qin Yang received her BA from Huazhong Uniersity of Science & Technology in 2008. Presently, she is a graduate student in Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science & Technology, PR China. Her current fields of interest are bioelectrochemistry and biosensor. Jiu-Hong Yu received his BSc from Hubei University, PR China in 2000 and PhD from Nanjing University, PR China in 2003. Presently, he is a postdoctoral candidate in Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science & Technology, PR China. Her current fields of interest are bioelectrochemistry and biosensor. Yuan-Di Zhao received his BSc in chemistry from Wuhan University, PR China in 1994 and PhD of science from the same University in 1999. Presently, he is a professor in the Key Laboratory of Biomedical Photonics of Ministry of Education, Huazhong University of Science & Technology, PR China. His current fields of interest are bioelectrochemistry, nanobiotechnology, and biosensor.