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Simulation and assay of protein biotinylation with electrochemical technique
Biosensors and Bioelectronics 26 (2011) 4610–4613
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Simulation and assay of protein biotinylation with electrochemical technique Zhaoyin Wang a , Lei Liu a,b , Yuanyuan Xu a , Lizhou Sun c , Genxi Li a,b,∗ a
Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 210093 Nanjing, China Laboratory of Biosensing Technology, School of Life Science, Shanghai University, 200444 Shanghai, China c Department of Obstetrics, The First Affiliated Hospital with Nanjing Medical University, Jiangsu Province Hospital, 210036 Nanjing, China b
a r t i c l e
i n f o
Article history: Received 26 January 2011 Received in revised form 25 April 2011 Accepted 28 April 2011 Available online 6 May 2011 Keywords: Protein biotinylation BirA Electrochemical technique Assay method
a b s t r a c t Protein biotinylation plays an important role in metabolism and transcription regulation, so study of protein biotinylation has received more and more interests. In this work, the bifunctional Escherichia coli biotin-inducible repressor protein A (BirA) and its substrate for protein biotinylation, a unique peptide with a specific sequence, are introduced as a model to electrochemically simulate the committed step in fatty acid biosynthesis. With the help of gold nanoparticles and peroxidase-labeled streptavidin involved in the electrochemical system, protein biotinylation is achieved on the surface of the working electrode, and the process of protein biotinylation can be electrochemically assayed by the obtained electrochemical response. Therefore, a new method to assay protein biotinylation is proposed and this work may provide a new perspective for understanding protein biotinylation in vitro. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Protein biotinylation is an important biological process both in transcription regulation and metabolism (Pacheco-Alvarez et al., 2002; Gravel and Narang, 2005; Lee et al., 2008). Usually, biotin protein ligase (BPL) fulfills the demand for protein biotinylation by binding biotin to biotin-dependent carboxylases (Ng et al., 2008; Pendini et al., 2008). Among all BPLs, the bifunctional Escherichia coli biotin-inducible repressor protein A (BirA) has been well studied. It may attach biotin to a biotin carboxyl carrier protein (BCCP) subunit to catalyze the first committed step in fatty acid biosynthesis. Previous studies have revealed that the sequence of the biotinylated substrate for BirA is conserved and the specific sequence of BCCP subunit can be minimized to a 14 residuepeptide named acceptor peptide (AP peptide). This 14-mer peptide can efficiently mimic the biotin acceptor function of the much larger protein domain normally recognized by BirA (Beckett et al., 1999). Therefore, a new methodology for protein–protein interaction detection and a general method to site-specifically label enveloped viruses have been reported by utilizing BirA/AP peptide as an enzyme/substrate pair (Fernandez-Suarez et al., 2008; Joo et al., 2008). Meanwhile, since the methods to assay protein biotinylation based on fluorescent, electrophoresis and mass spectrometric techniques are expensive, complex and time-consuming,
∗ Corresponding author at: Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 210093 Nanjing, China. Fax: +86 25 83592510. E-mail address: [email protected] (G. Li). 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.04.052
it has received more and more research interests to develop new approaches (Athavankar and Peterson, 2003; Brown et al., 2004; Howarth et al., 2005; Beckett, 2009). In this work, by using BirA/AP peptide pair, we have simulated protein biotinylation with electrochemical technique. Meanwhile, by making use of the excellent characters of gold nanoparticles (AuNPs) such as large surface area, good biocompatibility, high conductivity, etc. (Mani et al., 2009; Ahirwal and Mitra, 2009), and the extraordinary affinity between streptavidin and biotin (Choi-Rhee et al., 2004; Liu et al., 2005; Djellouli et al., 2007; Reichel et al., 2007; Fang et al., 2008; Haddad et al., 2009; Sheng et al., 2010), we have also proposed a new method for the assay of protein biotinylation.
2. Materials and methods 2.1. Materials and chemicals Biotin Ligase was obtained from GeneCopoeia Inc. (USA), which contains Biotin Ligase store buffer, 10× Biotin Ligase buffer A and 10× Biotin Ligase buffer B. The sequence of the substrate for biotinylation is N-CGLNDIFEAQKIEWR-C (target lysine for biotinylation is in boldface). The peptide was synthesized and purified (HPLC Analysis = 95.62%) by Shanghai Science Peptide Biological Technology Co., Ltd. Peroxidase-labeled streptavidin (SAv-HRP), 3 ,3 ,5 ,5 tetramethylbenzidine, cysteamine, 1-mercaptohexanol (MCH) were purchased from Sigma-Aldrich Inc. Chloroauric acid (HAuCl4 ) was purchased from Shanghai Jiushan Chemicals Co., Ltd. The buffer solutions used in this work were as follows. Peptide buffer: 10 mM Tris, 200 mM KCl, 2.5 mM MgCl2 , pH 7.5.
Z. Wang et al. / Biosensors and Bioelectronics 26 (2011) 4610–4613
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Scheme 1. Illustration of electrochemical simulation and detection of protein biotinylation. In order to exhibit the process of protein biotinylation clearly, elements in the scheme are not drawn in proportion.
Phosphate buffer solution: 10 mM Na2 HPO4 , 10 mM NaH2 PO4 , pH 7.4. The chemicals were all analytical reagents and used without further purification. All buffers were prepared with water purified with a Milli-Q purification system (Branstead, USA).
2.4. Electrochemical measurements
The AuNPs were prepared by citrate reduction of HAuCl4 according to the literatures (Storhoff et al., 1998; Liu and Lu, 2006). Briefly, a 100 mL aqueous solution of 0.01% (w/v) HAuCl4 was added into a round-bottom flask and stirred to boil. Then 3.5 mL 1% trisodium citrate was added rapidly into the boiling solution, the color of which became wine red from colorless after boiling for another 15 min with vigorous stirring. The size of the nanoparticles was 12.5 ± 2.3 nm determined by transmission electron microscopy (TEM) with the concentration of 2.3 nM, which was calculated from the quantity of starting material (HAuCl4 ) and the size of AuNPs at the wavelength of 519 nm.
Electrochemical measurements were carried out with a threeelectrode configuration which consisted of the modified gold electrode as the working electrode, a saturated calomel reference electrode (SCE), and a platinum auxiliary electrode. Electrochemical impedance spectrum (EIS) was recorded on a Potentiostat/Galvanostat Model 283-Amplifier Model 5210 (PG&G, PARC, USA), with the frequency range of 0.01 Hz–100 kHz, by using 10 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] couple as the electrochemical probe. Amperometric measurement was performed with a CHI660 C Potentiostat (CH Instruments) at −0.3 V for 60 s. A series of H2 O2 solutions with the concentration gradient from 6 × 10−5 M to 5 × 10−3 M were employed as the test solution, while 3 ,3 ,5 ,5 tetramethylbenzidine (TMB) was added into the solution with the final concentration of 100 M. Before the measurements, the solution was firstly bubbled thoroughly with high purity nitrogen for 30 min. Then a stream of nitrogen was blown gently across the surface of the solution in order to maintain the solution anaerobic throughout the experiment.
2.3. Protein biotinylation on the electrode surface
3. Results and discussion
The working electrode, a disk gold electrode, was firstly cleaned with freshly prepared piranha solution (1:3 mixture of 30% H2 O2 and concentrated H2 SO4 ) for 5 min and rinsed thoroughly with double-distilled water. Then it was polished carefully with rough and fine sand papers and alumina slurries (1.0, 0.3, and 0.05 mm), followed by sonication in absolute alcohol and double-distilled water for 5 min, respectively. Finally, the electrode was electrochemically cleaned in 0.5 M H2 SO4 until a stable cyclic voltammogram was obtained, and dried with purified nitrogen. After the pretreatment, the working electrode was immersed in 50 mM cysteamine solution for 1 h in darkness (Xiao et al., 2008). Then, the cysteamine-modified electrode was incubated in AuNPs solution at 4 ◦ C for 1 h. After that, the electrode was further incubated in 100 M peptide solution overnight. After having been backfilled with 1 mM MCH to obtain a well aligned peptide monolayer, the electrode was then treated with the mixed solution (enzyme system) of 3 L Biotin Ligase store buffer, 5 L 10× Biotin Ligase buffer A, 5 L 10× Biotin Ligase buffer B and 37 L double-distilled water at 37 ◦ C for 45 min for biotinylation. Finally, the electrode was immersed in 50 L peroxidase-labeled streptavidin (excessive to maximize the effect of biotinylation) at 37 ◦ C for 1 h so as to immobilize the compound onto the electrode surface.
Scheme 1 may illustrate the mechanism of the electrochemical simulation of protein biotinylation and its detection with electrochemical technique. Firstly, cysteamine with a thiol group in one end is immobilized onto the electrode surface by forming Au–S bond. Then, negatively charged AuNPs are further immobilized onto the electrode surface, since the other end of cysteamine is positively charged. Furthermore, since the peptide has been synthesized with a cysteine in N-terminal which may provide a free thiol group, a monolayer of peptide is thus immobilized onto the surface of nanoparticles. After that, the electrode surface is treated by MCH, a smaller molecule than peptide, which will replace some uncovalent peptides and make the monolayer uniformed. Finally, the peptides covalently immobilized on the electrode surface are biotinylated by the enzyme system, making biotin combined to the lysine residue, followed by the interaction between the biotin and peroxidase-labeled streptavidin. Consequently, the peroxidase-labeled streptavidin is immobilized onto the electrode surface, and the catalytic reaction towards H2 O2 can be achieved. The process of the modification of the electrode surface has been characterized by electrochemical impedance spectroscopy, which is a useful tool for studying the interface properties of surface-modified electrodes. The Nyquist plot of impedance spectra includes a semicircle portion and a linear portion, in which
2.2. Preparation of gold nanoparticles
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Z. Wang et al. / Biosensors and Bioelectronics 26 (2011) 4610–4613
Fig. 1. Amperometric curves for a range of H2 O2 . From bottom to top, concentration of H2 O2 varies from (a) 6 × 10−5 M, (b) 7 × 10−5 M, (c) 8 × 10−5 M, (d) 9 × 10−5 M, (e) 1 × 10−4 M, (f) 62 × 10−4 M, (g) 3 × 10−4 M, (h) 4 × 10−4 M, (i) 5 × 10−4 M, (j) 6 × 10−4 M, (k) 7 × 10−4 M, (l) 8 × 10−4 M, (m) 9 × 10−4 M, (n) 1 × 10−3 M, (o) 2 × 10−3 M, (p) 3 × 10−3 M, (q) 4 × 10−3 M.
the electron-transfer limited process can be reflected by the semicircle portion at higher frequencies, and the diffusion process can be reflected by the linear portion at lower frequencies. The electron-transfer resistance (RCT ) is equal to the semicircle diameter, which can be used to describe the interface properties of the electrode (Cao et al., 2010). Experimental results reveal that the Nyquist diameter does increase after the modification of the bare gold electrode with peptide/AuNPs/cysteamine, compared with the data of the bare electrode (Fig. S.1, Supplementary Information). Also, as is expected, the electron-transfer resistance (RCT ) is markedly decreased after the peptide/AuNPs/cysteamine modified electrode has been treated by MCH, due to the removal of the uncovalent-bound peptides. Moreover, the further loading of biotin and peroxidase-labeled streptavidin will again result in the increase of RCT due to the fact that the redox probe of Fe(CN)6 3−/4− will be further blocked to get access to the electrode surface after the immobilization of these species onto the electrode surface. On the other hand, in order to demonstrate the function of BirA in the process of biotinylation, we have conducted the control experiments by not using the enzyme, and different EIS will be obtained if the peptide/AuNPs/cysteamine modified electrode is treated by BirA or by water (Fig. S.2, Supplementary Information). Without the catalyzation by BirA, biotin cannot be linked to peptide, thus the peroxidase-labeled streptavidin cannot be further immobilized onto the electrode surface based on the interaction between the biotin and the streptavidin. Consequently, the curve will not be changed after the peptide/AuNPs/cysteamine modified electrode is further incubated with the solution containing peroxidase-labeled streptavidin (Fig. S.2B), while a significant increase of RCT can be observed if the enzyme is involved in the process of biotinylation (Fig. S.2A). Protein biotinylation can be not only simulated but also revealed by electrochemical technique, thus we have proposed an electrochemical method as well in this paper to assay protein biotinylation. As is illustrated in Scheme 1, horseradish peroxidase (HRP) has been labeled onto streptavidin, thus the enzyme may exhibit electrocatalytic response to the reduction of H2 O2 with good sensitivity. With the help of TMB, the catalytic reaction may provide a rapid and direct measurement of the current, reflecting the HRP-catalyzed electrochemical process (Ahirwal and Mitra, 2009; Liu et al., 2008). In fact, as is shown in Fig. 1, at different concentration of H2 O2 , decay curves for current vs time can show
Fig. 2. Plot for amperometric current vs concentration of H2 O2 . Inset shows a linear range from 6 × 10−5 M to 1 × 10−3 M (n = 14, R = 0.9992).
similar trends, which are a rapid decrease from the top 10 s followed by a plateau (steady-state current) in about 60 s. We then chart Fig. 2 by setting H2 O2 concentration as x-axis and current in 60 s as y-axis. As is expected, there is an increase in the current corresponding to H2 O2 concentration and a plateau in the range of higher concentration. The linear relationship can be obtained in the range from 60 M to 1 mM (n = 14, R = 0.9992). H2 O2 can be self-decomposed in some degree, so we have conducted control experiments by not using BirA in the process of biotinylation (Fig. S.3, Supplementary Information). Experimental results reveal that the current also increases with the addition of the substrate; however it is not a hyperbolic plot, which is not coincident with Michaelis–Menten (MM) kinetics. Moreover, if BirA has been used in the process of biotinylation, the current will be much higher at each concentration, which may display the efficiency of the biotinylation catalyzed by BirA. Therefore, a new method to assay protein biotinylation is proposed in this work firstly based on electrochemical technique.
4. Conclusions In summary, BirA/AP peptide pair is firstly employed for electrochemical study of protein biotinylation. Not only the process of protein biotinylation has been simulated with electrochemical technique, but a new method to assay protein biotinylation is also proposed. The electrochemical system should be also useful to the study of other proteins that can be biotinylated and the study of protein interaction which takes biotin as intermediate. This work may provide a new prospective to understand protein biotinylation in the future.
Acknowledgements This work is supported by the National Science Fund for Distinguished Young Scholars (Grant No. 20925520) and Shanghai Science and Technology Committee (Grant No. 09DZ2271800).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2011.04.052.
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Simulation and assay of protein biotinylation with electrochemical technique Zhaoyin Wang a , Lei Liu a,b , Yuanyuan Xu a , Lizhou Sun c , Genxi Li a,b,∗ a
Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 210093 Nanjing, China Laboratory of Biosensing Technology, School of Life Science, Shanghai University, 200444 Shanghai, China c Department of Obstetrics, The First Affiliated Hospital with Nanjing Medical University, Jiangsu Province Hospital, 210036 Nanjing, China b
a r t i c l e
i n f o
Article history: Received 26 January 2011 Received in revised form 25 April 2011 Accepted 28 April 2011 Available online 6 May 2011 Keywords: Protein biotinylation BirA Electrochemical technique Assay method
a b s t r a c t Protein biotinylation plays an important role in metabolism and transcription regulation, so study of protein biotinylation has received more and more interests. In this work, the bifunctional Escherichia coli biotin-inducible repressor protein A (BirA) and its substrate for protein biotinylation, a unique peptide with a specific sequence, are introduced as a model to electrochemically simulate the committed step in fatty acid biosynthesis. With the help of gold nanoparticles and peroxidase-labeled streptavidin involved in the electrochemical system, protein biotinylation is achieved on the surface of the working electrode, and the process of protein biotinylation can be electrochemically assayed by the obtained electrochemical response. Therefore, a new method to assay protein biotinylation is proposed and this work may provide a new perspective for understanding protein biotinylation in vitro. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Protein biotinylation is an important biological process both in transcription regulation and metabolism (Pacheco-Alvarez et al., 2002; Gravel and Narang, 2005; Lee et al., 2008). Usually, biotin protein ligase (BPL) fulfills the demand for protein biotinylation by binding biotin to biotin-dependent carboxylases (Ng et al., 2008; Pendini et al., 2008). Among all BPLs, the bifunctional Escherichia coli biotin-inducible repressor protein A (BirA) has been well studied. It may attach biotin to a biotin carboxyl carrier protein (BCCP) subunit to catalyze the first committed step in fatty acid biosynthesis. Previous studies have revealed that the sequence of the biotinylated substrate for BirA is conserved and the specific sequence of BCCP subunit can be minimized to a 14 residuepeptide named acceptor peptide (AP peptide). This 14-mer peptide can efficiently mimic the biotin acceptor function of the much larger protein domain normally recognized by BirA (Beckett et al., 1999). Therefore, a new methodology for protein–protein interaction detection and a general method to site-specifically label enveloped viruses have been reported by utilizing BirA/AP peptide as an enzyme/substrate pair (Fernandez-Suarez et al., 2008; Joo et al., 2008). Meanwhile, since the methods to assay protein biotinylation based on fluorescent, electrophoresis and mass spectrometric techniques are expensive, complex and time-consuming,
∗ Corresponding author at: Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 210093 Nanjing, China. Fax: +86 25 83592510. E-mail address: [email protected] (G. Li). 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.04.052
it has received more and more research interests to develop new approaches (Athavankar and Peterson, 2003; Brown et al., 2004; Howarth et al., 2005; Beckett, 2009). In this work, by using BirA/AP peptide pair, we have simulated protein biotinylation with electrochemical technique. Meanwhile, by making use of the excellent characters of gold nanoparticles (AuNPs) such as large surface area, good biocompatibility, high conductivity, etc. (Mani et al., 2009; Ahirwal and Mitra, 2009), and the extraordinary affinity between streptavidin and biotin (Choi-Rhee et al., 2004; Liu et al., 2005; Djellouli et al., 2007; Reichel et al., 2007; Fang et al., 2008; Haddad et al., 2009; Sheng et al., 2010), we have also proposed a new method for the assay of protein biotinylation.
2. Materials and methods 2.1. Materials and chemicals Biotin Ligase was obtained from GeneCopoeia Inc. (USA), which contains Biotin Ligase store buffer, 10× Biotin Ligase buffer A and 10× Biotin Ligase buffer B. The sequence of the substrate for biotinylation is N-CGLNDIFEAQKIEWR-C (target lysine for biotinylation is in boldface). The peptide was synthesized and purified (HPLC Analysis = 95.62%) by Shanghai Science Peptide Biological Technology Co., Ltd. Peroxidase-labeled streptavidin (SAv-HRP), 3 ,3 ,5 ,5 tetramethylbenzidine, cysteamine, 1-mercaptohexanol (MCH) were purchased from Sigma-Aldrich Inc. Chloroauric acid (HAuCl4 ) was purchased from Shanghai Jiushan Chemicals Co., Ltd. The buffer solutions used in this work were as follows. Peptide buffer: 10 mM Tris, 200 mM KCl, 2.5 mM MgCl2 , pH 7.5.
Z. Wang et al. / Biosensors and Bioelectronics 26 (2011) 4610–4613
4611
Scheme 1. Illustration of electrochemical simulation and detection of protein biotinylation. In order to exhibit the process of protein biotinylation clearly, elements in the scheme are not drawn in proportion.
Phosphate buffer solution: 10 mM Na2 HPO4 , 10 mM NaH2 PO4 , pH 7.4. The chemicals were all analytical reagents and used without further purification. All buffers were prepared with water purified with a Milli-Q purification system (Branstead, USA).
2.4. Electrochemical measurements
The AuNPs were prepared by citrate reduction of HAuCl4 according to the literatures (Storhoff et al., 1998; Liu and Lu, 2006). Briefly, a 100 mL aqueous solution of 0.01% (w/v) HAuCl4 was added into a round-bottom flask and stirred to boil. Then 3.5 mL 1% trisodium citrate was added rapidly into the boiling solution, the color of which became wine red from colorless after boiling for another 15 min with vigorous stirring. The size of the nanoparticles was 12.5 ± 2.3 nm determined by transmission electron microscopy (TEM) with the concentration of 2.3 nM, which was calculated from the quantity of starting material (HAuCl4 ) and the size of AuNPs at the wavelength of 519 nm.
Electrochemical measurements were carried out with a threeelectrode configuration which consisted of the modified gold electrode as the working electrode, a saturated calomel reference electrode (SCE), and a platinum auxiliary electrode. Electrochemical impedance spectrum (EIS) was recorded on a Potentiostat/Galvanostat Model 283-Amplifier Model 5210 (PG&G, PARC, USA), with the frequency range of 0.01 Hz–100 kHz, by using 10 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] couple as the electrochemical probe. Amperometric measurement was performed with a CHI660 C Potentiostat (CH Instruments) at −0.3 V for 60 s. A series of H2 O2 solutions with the concentration gradient from 6 × 10−5 M to 5 × 10−3 M were employed as the test solution, while 3 ,3 ,5 ,5 tetramethylbenzidine (TMB) was added into the solution with the final concentration of 100 M. Before the measurements, the solution was firstly bubbled thoroughly with high purity nitrogen for 30 min. Then a stream of nitrogen was blown gently across the surface of the solution in order to maintain the solution anaerobic throughout the experiment.
2.3. Protein biotinylation on the electrode surface
3. Results and discussion
The working electrode, a disk gold electrode, was firstly cleaned with freshly prepared piranha solution (1:3 mixture of 30% H2 O2 and concentrated H2 SO4 ) for 5 min and rinsed thoroughly with double-distilled water. Then it was polished carefully with rough and fine sand papers and alumina slurries (1.0, 0.3, and 0.05 mm), followed by sonication in absolute alcohol and double-distilled water for 5 min, respectively. Finally, the electrode was electrochemically cleaned in 0.5 M H2 SO4 until a stable cyclic voltammogram was obtained, and dried with purified nitrogen. After the pretreatment, the working electrode was immersed in 50 mM cysteamine solution for 1 h in darkness (Xiao et al., 2008). Then, the cysteamine-modified electrode was incubated in AuNPs solution at 4 ◦ C for 1 h. After that, the electrode was further incubated in 100 M peptide solution overnight. After having been backfilled with 1 mM MCH to obtain a well aligned peptide monolayer, the electrode was then treated with the mixed solution (enzyme system) of 3 L Biotin Ligase store buffer, 5 L 10× Biotin Ligase buffer A, 5 L 10× Biotin Ligase buffer B and 37 L double-distilled water at 37 ◦ C for 45 min for biotinylation. Finally, the electrode was immersed in 50 L peroxidase-labeled streptavidin (excessive to maximize the effect of biotinylation) at 37 ◦ C for 1 h so as to immobilize the compound onto the electrode surface.
Scheme 1 may illustrate the mechanism of the electrochemical simulation of protein biotinylation and its detection with electrochemical technique. Firstly, cysteamine with a thiol group in one end is immobilized onto the electrode surface by forming Au–S bond. Then, negatively charged AuNPs are further immobilized onto the electrode surface, since the other end of cysteamine is positively charged. Furthermore, since the peptide has been synthesized with a cysteine in N-terminal which may provide a free thiol group, a monolayer of peptide is thus immobilized onto the surface of nanoparticles. After that, the electrode surface is treated by MCH, a smaller molecule than peptide, which will replace some uncovalent peptides and make the monolayer uniformed. Finally, the peptides covalently immobilized on the electrode surface are biotinylated by the enzyme system, making biotin combined to the lysine residue, followed by the interaction between the biotin and peroxidase-labeled streptavidin. Consequently, the peroxidase-labeled streptavidin is immobilized onto the electrode surface, and the catalytic reaction towards H2 O2 can be achieved. The process of the modification of the electrode surface has been characterized by electrochemical impedance spectroscopy, which is a useful tool for studying the interface properties of surface-modified electrodes. The Nyquist plot of impedance spectra includes a semicircle portion and a linear portion, in which
2.2. Preparation of gold nanoparticles
4612
Z. Wang et al. / Biosensors and Bioelectronics 26 (2011) 4610–4613
Fig. 1. Amperometric curves for a range of H2 O2 . From bottom to top, concentration of H2 O2 varies from (a) 6 × 10−5 M, (b) 7 × 10−5 M, (c) 8 × 10−5 M, (d) 9 × 10−5 M, (e) 1 × 10−4 M, (f) 62 × 10−4 M, (g) 3 × 10−4 M, (h) 4 × 10−4 M, (i) 5 × 10−4 M, (j) 6 × 10−4 M, (k) 7 × 10−4 M, (l) 8 × 10−4 M, (m) 9 × 10−4 M, (n) 1 × 10−3 M, (o) 2 × 10−3 M, (p) 3 × 10−3 M, (q) 4 × 10−3 M.
the electron-transfer limited process can be reflected by the semicircle portion at higher frequencies, and the diffusion process can be reflected by the linear portion at lower frequencies. The electron-transfer resistance (RCT ) is equal to the semicircle diameter, which can be used to describe the interface properties of the electrode (Cao et al., 2010). Experimental results reveal that the Nyquist diameter does increase after the modification of the bare gold electrode with peptide/AuNPs/cysteamine, compared with the data of the bare electrode (Fig. S.1, Supplementary Information). Also, as is expected, the electron-transfer resistance (RCT ) is markedly decreased after the peptide/AuNPs/cysteamine modified electrode has been treated by MCH, due to the removal of the uncovalent-bound peptides. Moreover, the further loading of biotin and peroxidase-labeled streptavidin will again result in the increase of RCT due to the fact that the redox probe of Fe(CN)6 3−/4− will be further blocked to get access to the electrode surface after the immobilization of these species onto the electrode surface. On the other hand, in order to demonstrate the function of BirA in the process of biotinylation, we have conducted the control experiments by not using the enzyme, and different EIS will be obtained if the peptide/AuNPs/cysteamine modified electrode is treated by BirA or by water (Fig. S.2, Supplementary Information). Without the catalyzation by BirA, biotin cannot be linked to peptide, thus the peroxidase-labeled streptavidin cannot be further immobilized onto the electrode surface based on the interaction between the biotin and the streptavidin. Consequently, the curve will not be changed after the peptide/AuNPs/cysteamine modified electrode is further incubated with the solution containing peroxidase-labeled streptavidin (Fig. S.2B), while a significant increase of RCT can be observed if the enzyme is involved in the process of biotinylation (Fig. S.2A). Protein biotinylation can be not only simulated but also revealed by electrochemical technique, thus we have proposed an electrochemical method as well in this paper to assay protein biotinylation. As is illustrated in Scheme 1, horseradish peroxidase (HRP) has been labeled onto streptavidin, thus the enzyme may exhibit electrocatalytic response to the reduction of H2 O2 with good sensitivity. With the help of TMB, the catalytic reaction may provide a rapid and direct measurement of the current, reflecting the HRP-catalyzed electrochemical process (Ahirwal and Mitra, 2009; Liu et al., 2008). In fact, as is shown in Fig. 1, at different concentration of H2 O2 , decay curves for current vs time can show
Fig. 2. Plot for amperometric current vs concentration of H2 O2 . Inset shows a linear range from 6 × 10−5 M to 1 × 10−3 M (n = 14, R = 0.9992).
similar trends, which are a rapid decrease from the top 10 s followed by a plateau (steady-state current) in about 60 s. We then chart Fig. 2 by setting H2 O2 concentration as x-axis and current in 60 s as y-axis. As is expected, there is an increase in the current corresponding to H2 O2 concentration and a plateau in the range of higher concentration. The linear relationship can be obtained in the range from 60 M to 1 mM (n = 14, R = 0.9992). H2 O2 can be self-decomposed in some degree, so we have conducted control experiments by not using BirA in the process of biotinylation (Fig. S.3, Supplementary Information). Experimental results reveal that the current also increases with the addition of the substrate; however it is not a hyperbolic plot, which is not coincident with Michaelis–Menten (MM) kinetics. Moreover, if BirA has been used in the process of biotinylation, the current will be much higher at each concentration, which may display the efficiency of the biotinylation catalyzed by BirA. Therefore, a new method to assay protein biotinylation is proposed in this work firstly based on electrochemical technique.
4. Conclusions In summary, BirA/AP peptide pair is firstly employed for electrochemical study of protein biotinylation. Not only the process of protein biotinylation has been simulated with electrochemical technique, but a new method to assay protein biotinylation is also proposed. The electrochemical system should be also useful to the study of other proteins that can be biotinylated and the study of protein interaction which takes biotin as intermediate. This work may provide a new prospective to understand protein biotinylation in the future.
Acknowledgements This work is supported by the National Science Fund for Distinguished Young Scholars (Grant No. 20925520) and Shanghai Science and Technology Committee (Grant No. 09DZ2271800).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2011.04.052.
Z. Wang et al. / Biosensors and Bioelectronics 26 (2011) 4610–4613
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