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An electrochemical amplification immunoassay using bi-electrode signal transduction system
Talanta 71 (2007) 2029–2033
An electrochemical amplification immunoassay using bi-electrode signal transduction system Zhao-Peng Chen, Jian-Hui Jiang, Xiao-Bing Zhang, Guo-Li Shen, Ru-Qin Yu ∗ State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China Received 10 May 2006; received in revised form 9 September 2006; accepted 19 September 2006 Available online 17 October 2006
Abstract An electrochemical immunoassay technique has been developed based on the sensitive detection of the enzyme-generated product with a bielectrode signal transduction system. The system uses two separate electrodes, an immunoelectrode and a detection electrode to form a galvanic cell to implement the redox reactions on two different electrodes, that is the enzyme-generated reductant in the anode region is electrochemically oxidized by an oxidant (silver ions) in the cathode apartment. Based on a sandwich procedure, after immunoelectrode with antibody immobilized on its surface bound with the corresponding antigen and alkaline phosphatase conjugated antibody successively, the immunoelectrode was placed in enzyme reaction solution and wired to the detection electrode which was immerged into a silver deposition solution. These two solutions are connected with a salt bridge. Thus a bi-electrode signal transduction system device is constructed in which the immunoelectrode acts as anode and the detection electrode serves as cathode. The enzyme bound on the anode surface initiates the hydrolysis of ascorbic acid 2-phosphate to produce ascorbic acid in the anode region. The ascorbic acid produced in the anodic apartment is electrochemically oxidized by silver ions coupled with the deposition of silver metal on the cathode. Via a period of 30 min deposition, silver will deposited on the detection electrode in an amount corresponding to the quantity of ascorbic acid produced, leading to a great enhancement in the electrochemical stripping signal due to the accumulation of metallic silver by enzyme-generated product. Compared with the method using chemical deposition of silver, the electrochemical deposition of silver on a separate detection electrode apartment avoids the possible influence of silver deposition on the enzyme activity. © 2006 Elsevier B.V. All rights reserved. Keywords: Immunoassay; Bi-electrode signal transduction system; Immunoelectrode
1. Introduction The development of sensitive immunoassay and DNA assay techniques has been a long-sought goal in biomedical studies. Many strategies have been proposed to enhance the sensitivity of immuno or DNA sensing. Typical examples include the use of gold nanoparticles as signal amplifier [1–7], the incorporation of silver staining into gold nanoparticle labeling [8,9] the utilization of ferrocene capped gold nanoparticle tags [10], the application of carbon nanotubes to load more enzyme labels and to accumulate enzyme-generated product [11] as well as the implementation of enzyme conjugate-catalyzed precipitation of insoluble products [12–15]. Enzyme conjugated biochemistry is routinely used as the amplifier in electrochemical immunoassay. A common method
∗
Corresponding author. Fax: +86 731 8822577. E-mail address: [email protected] (R.-Q. Yu).
0039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2006.09.011
for electrochemical detection of enzymes is based on the voltammetric behavior of the enzyme-generated product. This method, however, is not sensitive enough for clinical diagnosis applications in some cases. Recently a novel method for electrochemical detection of DNA hybridization was proposed based on chemical accumulation of silver metal by an enzyme-catalyzed product of p-aminophenol. The subsequent anodic stripping voltammetry provided a DNA detection method with high sensitivity [16]. The coupling of enzyme catalysis and metal deposition seems to be a promising strategy for sensitive immunoassay. However, the chemical deposition of metal on the transducer on which the enzyme is present would block the catalytic centers of enzyme molecules, hindering a continual accumulation of the metal. The heavy metal ions coexisting in the substrate solution might also inhibit the activity of the enzyme [17–19]. These factors may all lead to a loss of detection sensitivity. On the other hand, the deposition of metal on the transducer with immobilized biomolecules makes it impossible to construct a renewable biosensor in which the biointerface should be regenerated without loss of activity.
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To circumvent the aforementioned drawbacks associated with chemical deposition of silver metal, we have developed herein a sensitive electrochemical immunoassay technique that utilizes two separate electrodes, an immunoelectrode with immobilized antibody for antigen and antibody-enzyme conjugate recognition at the anode and a detection electrode for electrochemical deposition of silver as the cathode to form a galvanic cell. The immunochemical recognition event between human IgG and goat anti-human IgG antibody is chosen as the model system to illustrate the proposed immunoassay approach. After the human IgG was sandwiched between the immobilized antibodies and the ALP conjugated antibodies, the ALP conjugated antibodies bound to the immunoelectrode surface catalyzed the generation of ascorbic acid, which in turn was oxidized at the anode to dehydroascorbic acid while the silver ions was reduced at the cathode. This resulted in silver deposition on the detection electrode, enabling a sensitive detection of the immunorecognition event by stripping analysis. 2. Experimental 2.1. Reagents Goat-anti-human IgG antibody (affinity purification), human IgG and anti-human IgG-alkaline phosphatase conjugate were obtained from Sino-Americal Biotechnology Company (Shanghai). Ascorbic acid 2-phosphate AA-p was purchased from Express Technology Co., Ltd. (Japan). Other reagents were of analytical purity, and doubly distilled water was used throughout all experiments. 2.2. Apparatus Electrochemical measurements were performed with a threeelectrode system comprising a platinum foil as auxiliary electrode, a saturated calomel electrode as reference electrode, and the glass carbon electrode or the carbon paste electrode as working electrode. All electrochemical experiments were performed on CH660 electrochemical workstation (Shanghai Chenhua Instruments, Shanghai) connecting with a personal computer. 2.3. Immunoassay procedure Carbon paste electrode was fabricated by pressing a mixture of graphite powder and melting paraffin into a Teflon tube (6 mm inner diameter). Before antibody adsorption, the carbon paste electrode was pretreated in 0.1 mol/l sodium hydroxide solution with a potential of 2.0 V versus SCE reference electrode for 10 min [20]. Then the electrochemically pretreated carbon paste electrode was exposed to 0.2 mg ml−1 anti-human IgG in tris buffer (pH 7.4). Through 1 h incubation at room temperature, the anti-human IgG were immobilized through passive adsorption, and an immunoelectrode was fabricated. After the immunoelectrode was immersed in 1% BSA for 30 min to block the nonoccupied adsorption sites, the anti-human IgG modified carbon paste electrode was exposed to human IgG solutions of different
Scheme 1. The bi-electrode signal transduction system.
concentrations for 40 min at 37 ◦ C, and subsequently contacted with 0.02 mg ml−1 anti-human IgG-alkaline phosphatase (ALP) conjugate for 40 min at 37 ◦ C. The human IgG was sandwiched between the immobilized antibodies and the ALP conjugated antibodies. To detect ALP bound on the immunoelectrode with high sensitivity, a novel bi-electrode signal transduction device was designed. As shown in Scheme 1, after the immunoreactions were completed, the immunoelectrode was immersed into 1 ml enzyme reaction solution (pH 9.0) composed of 80 mM glycin and 1 mM ascorbic acid 2-phosphate. The immunoelectrode was wired to the detection electrode, which was a clean glassy carbon electrode (4 mm in diameter) immerged in 1 ml silver deposition solution (pH 9.0) composed of 80 mM glycin and 1 mM AgNO3 . These two solutions were connected with a salt bridge. Thus a bi-electrode signal transduction device was constructed with the immunoelectrode as the anode and the detection electrode as the cathode. The signal transduction device was maintained to work at 37 ◦ C for a fixed time. Then, the detection electrode was taken out of the cell and rinsed with water. Anodic stripping voltammetry (ASV) measurement was applied to the electrode in a solution of 0.6 M KNO3 and 0.1 M HNO3 with a scan rate of 100 mV/s to detect silver deposited on cathode. 3. Results and discussion It is well known, if a shiny piece of copper is placed into a solution of silver nitrate, a spontaneous reaction occurs. Grayish white silver will deposit on the copper and the solution will turn blue because of the oxidation of copper to ions(II). The redox reaction can be described as follow: 2Ag+ + Cu ⇒ Cu2+ + 2Ag The same chemical reaction can occur and produce electricity in a galvanic cell. A galvanic cell consists of two containers with a salt bridge between them. The two containers each encase the half-reactions of the equation above. Ag+ + e− → Ag Cu → Cu2+ + 2e−
Z.-P. Chen et al. / Talanta 71 (2007) 2029–2033
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The electron flow from the anode to the cathode is what creates electricity, resulting in the deposition of Ag on anode. In a galvanic cell, the cathode is potential positive with respect to that of the anode. In the present study, the interaction between antibody and antigen was measured indirectly via detection of enzyme labeled antibody after sandwich immunoreactions using a bi-electrode transduction system. In the system, the ALP bound on the immunoelectrode catalyzed the generation of ascorbic acid which was oxidized at the anode to dehydroascorbic acid while the silver ions was reduced at the cathode, resulting in silver deposition on the detection electrode. The ALP catalysis [21] and the electrochemical reactions can be expressed as follows: ALP
Ascorbic acid 2-phosphate −→ Ascorbic acid +phosphoric acid
Fig. 2. Effect of AA-p concentration on ip . The deposition time is 15 min and the concentration of Ag+ is 1 mM.
Ascorbic acid → Dehydroascorbic acid + 2H+ + 2e− ,
3.1. Effect of the AA-p concentration
pH 9.0
E0 = 0.390 versus NHE Ag+ + e− → Ag,
E0 = 0.7995 versus NHE
The deposited silver metal was measured by anodic stripping voltammetry. Curves a–d in Fig. 1 depict the anodic stripping voltammograms for determination of 0, 10, 100 and 1000 ng/ml human IgG obtained using the developed immunoassay technique with a deposition time of 30 min. The concentration of AA-p in enzyme reaction solution and the silver ions in silver deposition solution were both 1 mM. Obviously, the presence of human IgG results in the binding of ALP conjugated antihuman IgG on the immunoelectrode, producing relatively large stripping currents (curves b–d). In contrast, with the immunoelectrode exposed to ALP conjugated anti-human IgG in the absence of analyte human IgG, only a small stripping current is obtained due to non-specific adsorption of ALP conjugate (curve a).
Since the amount of deposited silver metal on the detection electrode is depended on the amount of enzyme-generated ascorbic acid which is controlled by the concentration of AA-p, the dependence of the amount of deposited silver metal on the concentration of AA-p should be investigated. As shown in Fig. 2, with a fixed deposition time of 15 min, the silver metal stripping current for detection of 100 ng/ml human IgG increased with the increase of the concentration of AA-p in enzyme reaction solution up to 0.8 mM, and became stable for AA-p concentration over 0.8 mM (Fig. 2). As a result, an AA-p concentration of 1.0 mM was selected in the subsequent work. No appreciable stripping current was obtained in the absence of AA-p. This confirmed that sliver deposition is attributed to the enzyme-mediated generation of ascorbic acid. 3.2. Effect of the deposition time Obviously, the amount of silver metal deposited on the detection electrode is related to the deposition time. One can predict that more deposition time should result in more silver deposition. This would affect the sensitivity of the immunoassay and should be optimized. As shown in Fig. 3, the stripping current for determination of 100 ng/ml human IgG increases as the deposition time increases up to 20 min. With a deposition time more than 30 min, the stripping current tends to level off, indicating that the enzyme activity becomes low and enzyme catalytic reaction becomes very slow over 20 min. A deposition period of 30 min was adopted in this work to obtain a better reproducibility because the effect of time under this condition is insignificant. 3.3. Effect of the Ag+ concentration
Fig. 1. The anodic stripping voltammograms obtained using the developed immunoassay technique with a deposition time for determination of different concentration of human IgG. (a) 0 ng/ml; (b) 10 ng/ml; (c) 100 ng/ml and (d) 1000 ng/ml. The anode solution contained 1 mM of AA-p and the cathode solution had 1 mM AgNO3 .
To determination quantitatively the amount of the enzyme bound on the immunoelectrode, the enzyme-generated ascorbic acid was measured by the amount of the deposited silver on the detection electrode. Sufficient Ag+ should be added in the cathodic solution to ensure ascorbic acid generated at the
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Fig. 3. Effect of the deposition time on ip . The concentration of AA-p is 1 mM and the concentration of Ag+ is 1 mM.
immunoelectrode to be electrochemically oxidized as quickly as possible. Two different concentrations of Ag+ , 1 mM and 10 mM, were investigated. The results showed that the stripping currents had no obvious difference in these two conditions. Then, Ag+ concentration of 1 mM was used for complete oxidation of ascorbic acid. No ascorbic acid remained in the enzyme reaction solution was detectable with differential pulse voltammetry or the bi-electrodes system in which the immunoelectrode was replaced by a glassy carbon electrode, evidencing that ascorbic acid generated at the immunoelectrode was practically completely oxidized by silver ions. This also indicates 1 mM silver ions is sufficient to oxidize enzyme-generated product. 3.4. Detection of human IgG Under the optimized conditions, the stripping current was dynamically correlated to the concentration of human IgG. The current showed a linear correlation to human IgG concentration in the range of 10–1000 ng/ml with a regression equation i (mA) = −0.338 + 0.66 log CIgG (ng/ml) (r = 0.9963). The detection limit was 3.6 ng/ml. The sensitivity of this immunoassay technique was comparable to those for the immunosensors based on iridium oxide matrices [22] and the immunoassay using of nanoparticles as amplifier [4]. 4. Conclusion We have developed a new immunoassay technique that using a bi-electrode signal transduction device where the reductant catalytically generated by enzyme bound on the anode electrochemically initiates the deposition of silver metal on the cathode. The developed immunoassay technique embodies several attractive features. First, the accumulation of the enzyme conjugate-catalyzed product in a relatively long period via the silver deposition enables a highly sensitive stripping analysis of the metal in a relative short time, thereby offering a substantial signal amplification of the immunorecognition events. Second,
compared with the procedure based on chemical deposition of silver, the silver deposition on a separate detection electrode instead of the immunoelectrode itself circumvents possible influence of silver deposition and heavy metal ion inhibitors on the enzyme activity. Finally, the deposition of silver on the separate detection electrode instead of the immunoelectrode makes it possible to fabricate renewable immunosensors by regenerating the immunoelectrode under mild conditions [23–25]. To our knowledge, this is the first report demonstrating the implementation of a bi-electrode signal transduction system for sensitive immunoassay. The device can also be applied for sensitive detection of ascorbic acid, hydroquinone, aminophenol or other reductants, which, in some sense, can be regarded as the stripping voltammetry for detecting organic substance. Some preliminary experiments showed that ascorbic acid as low as 10−7 M could be detected using this device, which is more sensitive than some other electrochemical methods for ascorbic acid detection [26,27]. It was expected that this device would hold great promise in detecting enzyme-catalyzed products for immunoassay. Acknowledgment The work was financially supported by the National Natural Science Foundation of China (Grant Nos. 20435010, 20375012, 20205005). References [1] L. He, M.D. Musick, S.R. Nicewarner, F.G. Salinas, S.J. Benkovic, M.J. Natan, C.D. Keating, J. Am. Chem. Soc. 122 (2000) 9071. [2] L.A. Lyon, M.D. Musick, M.J. Natan, Anal. Chem. 70 (1998) 5177. [3] X.C. Zhou, S.J. O’Shea, S.F.Y. Li, Chem. Commun. 11 (2000) 953. [4] C. Zhang, Z. Zhang, B. Yu, J. Shi, X. Zhang, Anal. Chem. 74 (2002) 96. [5] M. Dequaire, C. Degrand, B. Limoges, Anal. Chem. 72 (2000) 5521. [6] T.A. Taton, R.C. Mucic, C.A. Mirkin, R.L. letsinger, J. Am. Chem. Soc. 122 (2000) 6305. [7] F. Patolsky, K.T. Ranjit, A. Lichtentein, I. Willner, Chem. Commun. 12 (2000) 1025. [8] X.D. Su, S.F.Y. Li, S.J. O’Shea, Chem. Commun. 8 (2001) 755. [9] X. Chu, X. Fu, K. Chen, G.L. Shen, R.Q. Yu, Biosens. Bioelectron. 20 (2005) 1805. [10] J. Wang, J. Li, A.J. Baca, J. Hu, F. Zhou, W. Yan, D.W. Pang, Anal. Chem. 75 (2003) 3941. [11] J. Wang, G. Liu, M.R. Jan, J. Am. Chem. Soc. 126 (2004) 3010. [12] L. Alfonta, I. Willer, D.J. Throchmorton, A.K. Singh, Anal. Chem. 73 (2001) 5287. [13] C. Ruan, K. Zeng, O.K. Varghese, C.A. Grimes, Anal. Chem. 75 (2003) 6494. [14] E. Katz, L. Alfonta, I. Willner, Sens. Actuators B 76 (2001) 134. [15] L. Alfonta, A.K. Singh, I. Willner, Anal. Chem. 73 (2001) 91. [16] S. Hwang, E. Kim, J. Kwak, Anal. Chem. 77 (2005) 579. [17] C. Durrieu, C. Tran-Minhw, Ecotoxicol. Environ. Saf. 51 (2002) 206. [18] S.D. Kamtekar, R. Pande, M.S. Ayyagari, K.A. Marx, D.L. Kaplan, J. Kumar, S. Tripathy, Anal. Chem. 68 (1996) 216. [19] Q.X. Chen, W.Z. Zheng, J.Y. Lin, Y. Shi, W.Z. Xie, H.M. Zhou, Int. J. Biochem. Cell Biol. 32 (2000) 879. [20] Y.M. Zhou, S.Q. Hu, G.L. Shen, R.Q. Yu, Biosens. Bioelectron. 18 (2003) 473. [21] Kokado, H. Arakawa, M. Maeda, Anal. Chim. Acta 407 (2000) 119. [22] M.S. Wilson, R.D. Rauh, Biosens. Bioelectron. 19 (2004) 693.
Z.-P. Chen et al. / Talanta 71 (2007) 2029–2033 [23] V.B. Kandimalla, N.S. Neeta, N.G. Karanth, M.S. Thakur, K.R. Roshini, B.E.A. Rani, A. Pasha, N.G.K. Karanth, Biosens. Bioelectron. 20 (2004) 903. [24] Z. Gao, F. Chao, Z. Chao, G. Li, Sens. Actuators B 66 (2000) 193.
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[25] R.D. Vaughan, C.K. O’Sullivan, G.G. Guilbault, Enzyme Microb. Technol. 29 (2001) 635. [26] J.B. Raoof, R. Ojani, S. Rashid-Nadimi, Electrochem. Acta 49 (2004) 271. [27] S. Wang, D. Du, Sens. Actuators 97 (2004) 371.
An electrochemical amplification immunoassay using bi-electrode signal transduction system Zhao-Peng Chen, Jian-Hui Jiang, Xiao-Bing Zhang, Guo-Li Shen, Ru-Qin Yu ∗ State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China Received 10 May 2006; received in revised form 9 September 2006; accepted 19 September 2006 Available online 17 October 2006
Abstract An electrochemical immunoassay technique has been developed based on the sensitive detection of the enzyme-generated product with a bielectrode signal transduction system. The system uses two separate electrodes, an immunoelectrode and a detection electrode to form a galvanic cell to implement the redox reactions on two different electrodes, that is the enzyme-generated reductant in the anode region is electrochemically oxidized by an oxidant (silver ions) in the cathode apartment. Based on a sandwich procedure, after immunoelectrode with antibody immobilized on its surface bound with the corresponding antigen and alkaline phosphatase conjugated antibody successively, the immunoelectrode was placed in enzyme reaction solution and wired to the detection electrode which was immerged into a silver deposition solution. These two solutions are connected with a salt bridge. Thus a bi-electrode signal transduction system device is constructed in which the immunoelectrode acts as anode and the detection electrode serves as cathode. The enzyme bound on the anode surface initiates the hydrolysis of ascorbic acid 2-phosphate to produce ascorbic acid in the anode region. The ascorbic acid produced in the anodic apartment is electrochemically oxidized by silver ions coupled with the deposition of silver metal on the cathode. Via a period of 30 min deposition, silver will deposited on the detection electrode in an amount corresponding to the quantity of ascorbic acid produced, leading to a great enhancement in the electrochemical stripping signal due to the accumulation of metallic silver by enzyme-generated product. Compared with the method using chemical deposition of silver, the electrochemical deposition of silver on a separate detection electrode apartment avoids the possible influence of silver deposition on the enzyme activity. © 2006 Elsevier B.V. All rights reserved. Keywords: Immunoassay; Bi-electrode signal transduction system; Immunoelectrode
1. Introduction The development of sensitive immunoassay and DNA assay techniques has been a long-sought goal in biomedical studies. Many strategies have been proposed to enhance the sensitivity of immuno or DNA sensing. Typical examples include the use of gold nanoparticles as signal amplifier [1–7], the incorporation of silver staining into gold nanoparticle labeling [8,9] the utilization of ferrocene capped gold nanoparticle tags [10], the application of carbon nanotubes to load more enzyme labels and to accumulate enzyme-generated product [11] as well as the implementation of enzyme conjugate-catalyzed precipitation of insoluble products [12–15]. Enzyme conjugated biochemistry is routinely used as the amplifier in electrochemical immunoassay. A common method
∗
Corresponding author. Fax: +86 731 8822577. E-mail address: [email protected] (R.-Q. Yu).
0039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2006.09.011
for electrochemical detection of enzymes is based on the voltammetric behavior of the enzyme-generated product. This method, however, is not sensitive enough for clinical diagnosis applications in some cases. Recently a novel method for electrochemical detection of DNA hybridization was proposed based on chemical accumulation of silver metal by an enzyme-catalyzed product of p-aminophenol. The subsequent anodic stripping voltammetry provided a DNA detection method with high sensitivity [16]. The coupling of enzyme catalysis and metal deposition seems to be a promising strategy for sensitive immunoassay. However, the chemical deposition of metal on the transducer on which the enzyme is present would block the catalytic centers of enzyme molecules, hindering a continual accumulation of the metal. The heavy metal ions coexisting in the substrate solution might also inhibit the activity of the enzyme [17–19]. These factors may all lead to a loss of detection sensitivity. On the other hand, the deposition of metal on the transducer with immobilized biomolecules makes it impossible to construct a renewable biosensor in which the biointerface should be regenerated without loss of activity.
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To circumvent the aforementioned drawbacks associated with chemical deposition of silver metal, we have developed herein a sensitive electrochemical immunoassay technique that utilizes two separate electrodes, an immunoelectrode with immobilized antibody for antigen and antibody-enzyme conjugate recognition at the anode and a detection electrode for electrochemical deposition of silver as the cathode to form a galvanic cell. The immunochemical recognition event between human IgG and goat anti-human IgG antibody is chosen as the model system to illustrate the proposed immunoassay approach. After the human IgG was sandwiched between the immobilized antibodies and the ALP conjugated antibodies, the ALP conjugated antibodies bound to the immunoelectrode surface catalyzed the generation of ascorbic acid, which in turn was oxidized at the anode to dehydroascorbic acid while the silver ions was reduced at the cathode. This resulted in silver deposition on the detection electrode, enabling a sensitive detection of the immunorecognition event by stripping analysis. 2. Experimental 2.1. Reagents Goat-anti-human IgG antibody (affinity purification), human IgG and anti-human IgG-alkaline phosphatase conjugate were obtained from Sino-Americal Biotechnology Company (Shanghai). Ascorbic acid 2-phosphate AA-p was purchased from Express Technology Co., Ltd. (Japan). Other reagents were of analytical purity, and doubly distilled water was used throughout all experiments. 2.2. Apparatus Electrochemical measurements were performed with a threeelectrode system comprising a platinum foil as auxiliary electrode, a saturated calomel electrode as reference electrode, and the glass carbon electrode or the carbon paste electrode as working electrode. All electrochemical experiments were performed on CH660 electrochemical workstation (Shanghai Chenhua Instruments, Shanghai) connecting with a personal computer. 2.3. Immunoassay procedure Carbon paste electrode was fabricated by pressing a mixture of graphite powder and melting paraffin into a Teflon tube (6 mm inner diameter). Before antibody adsorption, the carbon paste electrode was pretreated in 0.1 mol/l sodium hydroxide solution with a potential of 2.0 V versus SCE reference electrode for 10 min [20]. Then the electrochemically pretreated carbon paste electrode was exposed to 0.2 mg ml−1 anti-human IgG in tris buffer (pH 7.4). Through 1 h incubation at room temperature, the anti-human IgG were immobilized through passive adsorption, and an immunoelectrode was fabricated. After the immunoelectrode was immersed in 1% BSA for 30 min to block the nonoccupied adsorption sites, the anti-human IgG modified carbon paste electrode was exposed to human IgG solutions of different
Scheme 1. The bi-electrode signal transduction system.
concentrations for 40 min at 37 ◦ C, and subsequently contacted with 0.02 mg ml−1 anti-human IgG-alkaline phosphatase (ALP) conjugate for 40 min at 37 ◦ C. The human IgG was sandwiched between the immobilized antibodies and the ALP conjugated antibodies. To detect ALP bound on the immunoelectrode with high sensitivity, a novel bi-electrode signal transduction device was designed. As shown in Scheme 1, after the immunoreactions were completed, the immunoelectrode was immersed into 1 ml enzyme reaction solution (pH 9.0) composed of 80 mM glycin and 1 mM ascorbic acid 2-phosphate. The immunoelectrode was wired to the detection electrode, which was a clean glassy carbon electrode (4 mm in diameter) immerged in 1 ml silver deposition solution (pH 9.0) composed of 80 mM glycin and 1 mM AgNO3 . These two solutions were connected with a salt bridge. Thus a bi-electrode signal transduction device was constructed with the immunoelectrode as the anode and the detection electrode as the cathode. The signal transduction device was maintained to work at 37 ◦ C for a fixed time. Then, the detection electrode was taken out of the cell and rinsed with water. Anodic stripping voltammetry (ASV) measurement was applied to the electrode in a solution of 0.6 M KNO3 and 0.1 M HNO3 with a scan rate of 100 mV/s to detect silver deposited on cathode. 3. Results and discussion It is well known, if a shiny piece of copper is placed into a solution of silver nitrate, a spontaneous reaction occurs. Grayish white silver will deposit on the copper and the solution will turn blue because of the oxidation of copper to ions(II). The redox reaction can be described as follow: 2Ag+ + Cu ⇒ Cu2+ + 2Ag The same chemical reaction can occur and produce electricity in a galvanic cell. A galvanic cell consists of two containers with a salt bridge between them. The two containers each encase the half-reactions of the equation above. Ag+ + e− → Ag Cu → Cu2+ + 2e−
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The electron flow from the anode to the cathode is what creates electricity, resulting in the deposition of Ag on anode. In a galvanic cell, the cathode is potential positive with respect to that of the anode. In the present study, the interaction between antibody and antigen was measured indirectly via detection of enzyme labeled antibody after sandwich immunoreactions using a bi-electrode transduction system. In the system, the ALP bound on the immunoelectrode catalyzed the generation of ascorbic acid which was oxidized at the anode to dehydroascorbic acid while the silver ions was reduced at the cathode, resulting in silver deposition on the detection electrode. The ALP catalysis [21] and the electrochemical reactions can be expressed as follows: ALP
Ascorbic acid 2-phosphate −→ Ascorbic acid +phosphoric acid
Fig. 2. Effect of AA-p concentration on ip . The deposition time is 15 min and the concentration of Ag+ is 1 mM.
Ascorbic acid → Dehydroascorbic acid + 2H+ + 2e− ,
3.1. Effect of the AA-p concentration
pH 9.0
E0 = 0.390 versus NHE Ag+ + e− → Ag,
E0 = 0.7995 versus NHE
The deposited silver metal was measured by anodic stripping voltammetry. Curves a–d in Fig. 1 depict the anodic stripping voltammograms for determination of 0, 10, 100 and 1000 ng/ml human IgG obtained using the developed immunoassay technique with a deposition time of 30 min. The concentration of AA-p in enzyme reaction solution and the silver ions in silver deposition solution were both 1 mM. Obviously, the presence of human IgG results in the binding of ALP conjugated antihuman IgG on the immunoelectrode, producing relatively large stripping currents (curves b–d). In contrast, with the immunoelectrode exposed to ALP conjugated anti-human IgG in the absence of analyte human IgG, only a small stripping current is obtained due to non-specific adsorption of ALP conjugate (curve a).
Since the amount of deposited silver metal on the detection electrode is depended on the amount of enzyme-generated ascorbic acid which is controlled by the concentration of AA-p, the dependence of the amount of deposited silver metal on the concentration of AA-p should be investigated. As shown in Fig. 2, with a fixed deposition time of 15 min, the silver metal stripping current for detection of 100 ng/ml human IgG increased with the increase of the concentration of AA-p in enzyme reaction solution up to 0.8 mM, and became stable for AA-p concentration over 0.8 mM (Fig. 2). As a result, an AA-p concentration of 1.0 mM was selected in the subsequent work. No appreciable stripping current was obtained in the absence of AA-p. This confirmed that sliver deposition is attributed to the enzyme-mediated generation of ascorbic acid. 3.2. Effect of the deposition time Obviously, the amount of silver metal deposited on the detection electrode is related to the deposition time. One can predict that more deposition time should result in more silver deposition. This would affect the sensitivity of the immunoassay and should be optimized. As shown in Fig. 3, the stripping current for determination of 100 ng/ml human IgG increases as the deposition time increases up to 20 min. With a deposition time more than 30 min, the stripping current tends to level off, indicating that the enzyme activity becomes low and enzyme catalytic reaction becomes very slow over 20 min. A deposition period of 30 min was adopted in this work to obtain a better reproducibility because the effect of time under this condition is insignificant. 3.3. Effect of the Ag+ concentration
Fig. 1. The anodic stripping voltammograms obtained using the developed immunoassay technique with a deposition time for determination of different concentration of human IgG. (a) 0 ng/ml; (b) 10 ng/ml; (c) 100 ng/ml and (d) 1000 ng/ml. The anode solution contained 1 mM of AA-p and the cathode solution had 1 mM AgNO3 .
To determination quantitatively the amount of the enzyme bound on the immunoelectrode, the enzyme-generated ascorbic acid was measured by the amount of the deposited silver on the detection electrode. Sufficient Ag+ should be added in the cathodic solution to ensure ascorbic acid generated at the
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Fig. 3. Effect of the deposition time on ip . The concentration of AA-p is 1 mM and the concentration of Ag+ is 1 mM.
immunoelectrode to be electrochemically oxidized as quickly as possible. Two different concentrations of Ag+ , 1 mM and 10 mM, were investigated. The results showed that the stripping currents had no obvious difference in these two conditions. Then, Ag+ concentration of 1 mM was used for complete oxidation of ascorbic acid. No ascorbic acid remained in the enzyme reaction solution was detectable with differential pulse voltammetry or the bi-electrodes system in which the immunoelectrode was replaced by a glassy carbon electrode, evidencing that ascorbic acid generated at the immunoelectrode was practically completely oxidized by silver ions. This also indicates 1 mM silver ions is sufficient to oxidize enzyme-generated product. 3.4. Detection of human IgG Under the optimized conditions, the stripping current was dynamically correlated to the concentration of human IgG. The current showed a linear correlation to human IgG concentration in the range of 10–1000 ng/ml with a regression equation i (mA) = −0.338 + 0.66 log CIgG (ng/ml) (r = 0.9963). The detection limit was 3.6 ng/ml. The sensitivity of this immunoassay technique was comparable to those for the immunosensors based on iridium oxide matrices [22] and the immunoassay using of nanoparticles as amplifier [4]. 4. Conclusion We have developed a new immunoassay technique that using a bi-electrode signal transduction device where the reductant catalytically generated by enzyme bound on the anode electrochemically initiates the deposition of silver metal on the cathode. The developed immunoassay technique embodies several attractive features. First, the accumulation of the enzyme conjugate-catalyzed product in a relatively long period via the silver deposition enables a highly sensitive stripping analysis of the metal in a relative short time, thereby offering a substantial signal amplification of the immunorecognition events. Second,
compared with the procedure based on chemical deposition of silver, the silver deposition on a separate detection electrode instead of the immunoelectrode itself circumvents possible influence of silver deposition and heavy metal ion inhibitors on the enzyme activity. Finally, the deposition of silver on the separate detection electrode instead of the immunoelectrode makes it possible to fabricate renewable immunosensors by regenerating the immunoelectrode under mild conditions [23–25]. To our knowledge, this is the first report demonstrating the implementation of a bi-electrode signal transduction system for sensitive immunoassay. The device can also be applied for sensitive detection of ascorbic acid, hydroquinone, aminophenol or other reductants, which, in some sense, can be regarded as the stripping voltammetry for detecting organic substance. Some preliminary experiments showed that ascorbic acid as low as 10−7 M could be detected using this device, which is more sensitive than some other electrochemical methods for ascorbic acid detection [26,27]. It was expected that this device would hold great promise in detecting enzyme-catalyzed products for immunoassay. Acknowledgment The work was financially supported by the National Natural Science Foundation of China (Grant Nos. 20435010, 20375012, 20205005). References [1] L. He, M.D. Musick, S.R. Nicewarner, F.G. Salinas, S.J. Benkovic, M.J. Natan, C.D. Keating, J. Am. Chem. Soc. 122 (2000) 9071. [2] L.A. Lyon, M.D. Musick, M.J. Natan, Anal. Chem. 70 (1998) 5177. [3] X.C. Zhou, S.J. O’Shea, S.F.Y. Li, Chem. Commun. 11 (2000) 953. [4] C. Zhang, Z. Zhang, B. Yu, J. Shi, X. Zhang, Anal. Chem. 74 (2002) 96. [5] M. Dequaire, C. Degrand, B. Limoges, Anal. Chem. 72 (2000) 5521. [6] T.A. Taton, R.C. Mucic, C.A. Mirkin, R.L. letsinger, J. Am. Chem. Soc. 122 (2000) 6305. [7] F. Patolsky, K.T. Ranjit, A. Lichtentein, I. Willner, Chem. Commun. 12 (2000) 1025. [8] X.D. Su, S.F.Y. Li, S.J. O’Shea, Chem. Commun. 8 (2001) 755. [9] X. Chu, X. Fu, K. Chen, G.L. Shen, R.Q. Yu, Biosens. Bioelectron. 20 (2005) 1805. [10] J. Wang, J. Li, A.J. Baca, J. Hu, F. Zhou, W. Yan, D.W. Pang, Anal. Chem. 75 (2003) 3941. [11] J. Wang, G. Liu, M.R. Jan, J. Am. Chem. Soc. 126 (2004) 3010. [12] L. Alfonta, I. Willer, D.J. Throchmorton, A.K. Singh, Anal. Chem. 73 (2001) 5287. [13] C. Ruan, K. Zeng, O.K. Varghese, C.A. Grimes, Anal. Chem. 75 (2003) 6494. [14] E. Katz, L. Alfonta, I. Willner, Sens. Actuators B 76 (2001) 134. [15] L. Alfonta, A.K. Singh, I. Willner, Anal. Chem. 73 (2001) 91. [16] S. Hwang, E. Kim, J. Kwak, Anal. Chem. 77 (2005) 579. [17] C. Durrieu, C. Tran-Minhw, Ecotoxicol. Environ. Saf. 51 (2002) 206. [18] S.D. Kamtekar, R. Pande, M.S. Ayyagari, K.A. Marx, D.L. Kaplan, J. Kumar, S. Tripathy, Anal. Chem. 68 (1996) 216. [19] Q.X. Chen, W.Z. Zheng, J.Y. Lin, Y. Shi, W.Z. Xie, H.M. Zhou, Int. J. Biochem. Cell Biol. 32 (2000) 879. [20] Y.M. Zhou, S.Q. Hu, G.L. Shen, R.Q. Yu, Biosens. Bioelectron. 18 (2003) 473. [21] Kokado, H. Arakawa, M. Maeda, Anal. Chim. Acta 407 (2000) 119. [22] M.S. Wilson, R.D. Rauh, Biosens. Bioelectron. 19 (2004) 693.
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