Gold nanoparticles based sandwich electrochemical immunosensor

Biosensors and Bioelectronics 25 (2010) 2016–2020

Contents lists available at ScienceDirect

Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

Gold nanoparticles based sandwich electrochemical immunosensor Gautham Kumar Ahirwal, Chanchal K. Mitra ∗ Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, Andhra Pradesh, 500046, India

a r t i c l e

i n f o

Article history: Received 27 November 2009 Received in revised form 13 January 2010 Accepted 22 January 2010 Available online 2 February 2010 Keywords: Biosensor Immunosensor Electrochemical immunoassay Gold nanoparticles Tetramethylbenzidine (TMB)

a b s t r a c t In this report we have used gold nanoparticles (AuNPs) to covalently attach an antibody (Ab1 ) using a spacer arm. The AuNPs/Ab1 modified gold electrode was used for a sandwich electrochemical immunoassay. The detection was done using cyclic voltammetry and impedance measurements using Horse Radish Peroxidase (HRP) as enzyme label on secondary antibody (Ab2 ) and 3,3 , 5,5 -tertramethyl benzidine (TMB) as an electroactive dye. The cyclic voltammetric experiments showed three clear peaks at potentials 154 mV, −33 mV and −156 mV. There was an increase in the both anodic and cathodic current values for the peak at potential −33 mV, when H2 O2 was added and the other peaks observed at potential 154 mV and −156 mV resulted due to the oxidation and reduction of TMB. The detection limit of this electrode was 2 ng/mL or 10 pg/5 ␮L of the analyte. The electrochemical impedance spectroscopy studies demonstrate that the formation of antigen–antibody complexes increases the series resistance and thus confirms the assembly on the electrode. This study showed that AuNPs was efficient in preserving the activity and orientation of the antibody and it can form a major platform in many clinical immunoassays. © 2010 Elsevier B.V. All rights reserved.

1. Introduction In recent years, the field of electrochemical biosensors design has been capable of providing better analytical characteristics in terms of sensitivity, selectivity reliability, ease of use and low cost. Among the various types of electrochemical sensors, immunosensors are attractive tools for the detection of analytes based on the binding of antibodies with antigens. Unlike other techniques, electrochemical methods are not usually affected by sample impurities and the equipment required is relatively simple and are of low cost. Most of the studies in immunosensors concentrate on the electrochemical detection using labeled immunoagent (enzymes are most commonly been used as the labels). The achievement of high sensitivity requires different amplification platforms and processes. It has been shown that, we can enhance the sensitivity of the signal and the detection ability (Storhoff et al., 1998; Hu et al., 2003; Kumar et al., 2008; Parak et al., 2003; Tkachenko et al., 2003) of the biosensing devices by using nanoparticles in their construction. Although different types of nanoparticles have been used earlier for the construction of electrochemical sensors, but the use of gold nanoparticles (AuNPs) allows enhanced analytical detection with respect to others. Further flow through system can be used to enhance the binding of the antigen to the given antibody.

∗ Corresponding author. Tel.: +91 40 23134668; fax: +91 40 23010120/145. E-mail address: c [email protected] (C.K. Mitra). 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.01.029

The ability of AuNPs to provide a stable immobilization of biomolecules, in order to retain their bioactivity is a major advantage for the preparation of sensors (it is also an highly conducting material with a high surface to volume ratio). Various approaches like, layer by layer self assembly using nafion (Tang et al., 2004a), dithiothreitol (Chatrathi et al., 2007), cysteamine (Wang et al., 2004a), 4-aminothiophenol (Wang et al., 2004b), polyvinyl butyral (Tang et al., 2004b) have been used earlier for the construction of immunosensors. In the recent years construction of immunosensors using AuNPs have received significant attention. Antibodies were also used in sol–gel matrix (Wu et al., 2005) and carbon paste electrode (Dana et al., 2007) immunosensors. Some of the earlier works haves demonstrated the direct assembly of antibody (Chen et al., 2007), antigen (Huang et al., 2007) onto the electrode. Taking an account of the advantages and methods used for gold nanoparticles modified electrode preparation, we have prepared modified gold nanoparticles surface by the direct covalent linking of the antibody to the nanoparticles and assembling them onto the electrode surface. In the current study, we have shown that direct linking of antibody to the AuNPs using covalent methods can be used for the development of immunosensors. This kind of approach provides a right orientation of the antibody with the free antigen binding sites for further immunoreaction without affecting the structure and function of the antibody. The AuNPs also helps in better immobilization of the molecules onto the electrode preventing them from dissolving back into the bulk solution. Horse Radish Peroxidase (HRP) has been used for the detection purpose in this study because of its small size and high stability to

G.K. Ahirwal, C.K. Mitra / Biosensors and Bioelectronics 25 (2010) 2016–2020

the chemical modifications. Peroxidases are enzymes of the class EC 1.11, which are defined as oxidoreductases that use hydrogen peroxides as electron acceptor. Shown below is the reaction mechanism for the enzymatic catalytic cycle of HRP (Ruzgas et al., 1996). Native peroxidase (Fe3+ ) + H2 O2 → Compound-I + H2 O

(1a)

Compound-I + AH2 → Compound-II + AH∗

(1b)

2017

2.3. Preparation of glutathione capped AuNPs To the gold colloid solution (25 mL; 9 ␮mol) glutathione (20 mg; 65 ␮mol) was added and was stirred for 10–15 min. After the stirring was complete the mixture was centrifuged at 4500 rpm to separate the capped AuNPs. The pellet obtained was resuspended in 1 mL of phosphate buffer (pH 7). 2.4. Preparation of AuNP–antibody conjugate

Compound-II + AH2 → Native peroxidase (Fe

3+

) + AH ∗ + H2 O

(1c)

The first reaction (1a) involves the two-electron oxidation of the ferriheme prosthetic group of the native peroxidase by H2 O2 (or organic hydroperoxides). This reaction results in the formation of an intermediate, compound-I (oxidation state +5), consisting of oxyferryl iron (Fe(IV) 0 = O) and a porphyrin ␲ cation radical. In the next reaction (1b), compound-I loses one oxidizing equivalent upon one-electron reduction by the first electron donor AH2 and forms the compound-II (oxidation state +4). The later in turn accepts an additional electron from the second donor molecule AH2 in the third step (1c), whereby the enzyme is returned to its native resting state, ferriperoxidase. 3,3 , 5,5 -Tertramethyl benzidine or TMB is a chromogenic substrate used in the staining procedure of immunohistochemistry and also as a visualizing agent in ELISA. In solution, TMB forms a blue product when allowed to react with peroxidase enzyme. The resulting color change can be read at 370 nm or 655 nm. The reaction is stopped using acid and the color changes to yellow and can be read at 450 nm.

2. Materials and methods 2.1. Materials and instrumentation We have used anti-human serum albumin (HSA) (Ab1 ), HSA (Ag) and HRP labeled antibody (Ab2 ) from Bangalore Genei (India) and N-Ethyl-N -(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was purchased from Sigma. Gold chloride [AuCl4 , 4H2 O, Au% > 49%] and all other chemicals were of the analytical grade and were used without further purification. All the solutions were prepared using double distilled water. The absorption spectrum of the samples was recorded in UV-1601 PC (Shimadzu). Electrochemical measurements were performed using CH Instruments 660A with three electrode system, comprising of platinum auxiliary electrode, an Ag/AgCl reference electrode and a gold electrode (as the working electrode). The 1:1 ratio of 0.1 M phosphate buffer (PB) pH 7.0 and 0.1 M KCl was used as electrolyte for all the measurements. TEM pictures of gold nanoparticles were taken at IICT (Hyderabad).

2.2. Preparation of glutathione coated AuNPs All the glassware used for the preparation were soaked in freshly prepared HNO3 /HCl mixture, rinsed thoroughly with distilled water and dried. In a typical preparation of gold colloid, glutathione was used for the capping of gold nanoparticles. The gold colloid was prepared by dissolving aurate salt (400 ␮mol) in double distilled water (100 mL) and then freshly prepared NaBH4 solution (66 ␮mol, 400 ␮L) was added dropwise with stirring. The solution was stirred for around 5 min. The prepared AuNPs were stored at 4 ◦ C in refrigerator for further use.

2.4.1. AuNP–antibody was prepared in two steps Glutathione capped AuNPs were first activated by adding 58 mM EDC (300 ␮L) in 0.1 M pH 5.0 MES buffer (3 mL) and the solution was kept at 4 ◦ C for 1.5 h on a rocker. The solution was next centrifuged at 15,000 rpm for 1 h to separate activated AuNPs and the pellet was resuspended in 0.1 M phosphate buffer. 50 ␮L of 500 ␮M 1,6diaminohexane (DAH) was added to the activated AuNPs and mixed for 30 min at room temperature and the reaction mixture was again centrifuged and the pellet obtained was redissolved in phosphate buffer. In the first step 500 ␮L of 10 ␮g/mL primary antibody was added to pH 5.0 MES buffer (3 mL) and 58 mM EDC (300 ␮L) was added and the reaction mixture was kept at 4 ◦ C for 1.5 h on a rocker. The activated primary antibody was added to AuNPs/DAH and the reaction mixture was kept at 4 ◦ C for 6 h. The coupled AuNP–antibody (AuNPs/Ab1 ) conjugate was separated by centrifugation (Scheme 1). Scheme 1 (upper part) shows the broad outline of the preparation of the modified gold nanoparticles. 2.5. Antibody immobilization and immunoassay procedure AuNPs/Ab1 was immobilized (by adsorption) onto a gold electrode. 5 ␮L of AuNPs/Ab1 solution was spread onto the cleaned and polished gold electrode and the electrode was incubated at 4 ◦ C. After the incubation, the electrode was rinsed with distilled water and the electrode was blocked with 1% BSA + 0.05% tween for 30 min at room temperature. The electrode was again rinsed with distilled water to remove any residuals. The AuNPs/Ab1 electrode was then incubated with a 5 ␮L of antigen for 30 min at room temperature. After the binding of antigen, the electrode was incubated in 50 ␮L of 2 mg/mL Ab2 /HRP conjugate. Finally the electrode was washed to remove unbound conjugate. Scheme 1 (lower part) shows the schematic representation of the immobilization procedure. We would like to mention here that the gold electrode is basically “painted over” with a suspension of modified gold nanoparticles. 2.6. Measurement procedure The modified electrode was then placed in electrochemical cell containing 2.0 mL of 0.1 M PB (pH 7.0) and 0.1 M KCl. TMB (15 ␮M) was used as electron mediator and H2 O2 (20 ␮M) was used as substrate. The cyclic voltammetry measurements were done at the potential range of 450 mV to −300 mV at a scan rate of 10 mV/s and Ag/AgCl as the reference electrode. The impedance measurements were done at the frequency range of 0.1 Hz to 1.0 × 10−5 Hz at room temperature (Zre vs. Zim at 160 mV vs. Ag/AgCl reference electrode). All the electrochemical measurements were performed at room temperature. 3. Results and discussions 3.1. UV–vis spectroscopy studies The gold nanoparticles synthesized by borohydride reduction of aurate salt are relatively monodisperse in colloidal solution, which is confirmed by a single peak in the absorbance spectra

2018

G.K. Ahirwal, C.K. Mitra / Biosensors and Bioelectronics 25 (2010) 2016–2020

Scheme 1. Steps involved in the preparation of antibody conjugated gold nanoparticles on a gold electrode prior to analysis. The top reactions show (i) glutathione coated gold nanoparticles activated with EDC (ii) followed by coupling with EDC activated primary antibody. Below is the representation of immunoassay steps done onto the electrode. The secondary antibody can bind only to the primary antibody–antigen binary complex. The ternary complex containing HRP is detected electrochemically using TMB and H2 O2 .

(Figure 1Sa). The max was observed at around 530 nm. In next step, which involved the protection of nanoparticles for stability, we have used glutathione as the capping or protecting agent. As shown in Figure 1Sb, the peak is shifted towards the higher wavelength after capping with glutathione. The max was observed at around 540–580 nm and the change of color was also seen from wine red to blue color after capping. 3.2. TEM measurements Figure 2S (supplementary information) shows the image of the neat gold nanoparticles. The results show the uniform and average synthesis of gold nanoparticles with an average size of ∼25 nm. 3.3. Electrochemical studies HRP catalyzes the reaction, in which reduced TMB is oxidized in the presence of H2 O2 . TMB (red) + H2 O2 → TMB (ox) + 2H2 O In absence of the catalyst (HRP), the reaction takes place exceedingly slowly. The mechanism of HRP catalysis is well known and not reproduced here (Chance, 1951a,b). The primary step is the oxidation of the iron atom and the porphyrin moiety by hydrogen peroxide in a 2e transfer reaction. The oxidized heme then can oxidize a suitable acceptor in either one two-electron transfer

Fig. 1. (A) Voltammogram of modified electrode (containing primary antibody–antigen–secondary antibody attached to the gold nanoparticles) (1) in presence of only 20 ␮M H2 O2 , (2) only 15 ␮M TMB, and (3) 15 ␮M TMB + 20 ␮M H2 O2 . (B) Voltammogram of (1) bare gold electrode, after stepwise immobilization of (2) primary antibody, (3) antigen and (4) HRP coupled secondary antibody, in 0.1 M PB/0.1 M KCl (pH 7.0) at a potential range of 450 mV to −300 mV. Scan rate 10 mV/s vs. Ag/AgCl reference electrode. All the solutions had same TMB and H2 O2 concentration.

process or in two one-electron transfer process. For TMB, the overall reaction is shown in Figure 3S (Joesphy et al., 1982, 1983). The enzyme activity can be measured by the voltammetric detection of the reduction current generated by TMB at the gold electrode. Electrochemical investigation of the TMB and the modified electrode was carried out using gold electrode. The most interesting results in terms of the generated electrocatalytic current were observed with TMB in the presence of the enzyme. 3.3.1. Cyclic voltammetry of the immunosensors The cyclic voltammetry of the (AuNP–Ab1 )(Ag)(Ab2 –HRP) modified gold electrode (MGE) was done in PB/KCl at a scan rate of 10 mV/s and sweeping range of 450 mV to −300 mV (Fig. 1A). The results in Fig. 1A(1) show no change in response with the presence of only H2 O2 in the solution. The CV of TMB alone in the solution using the modified electrode, showed two peaks at the potential 240 mV and −35 mV [Fig. 1A(2)]. The voltammogram in the presence of both H2 O2 and TMB [Fig. 1A(3)] showed two well-defined new peaks at the potential 154 and −156 mV. The current value of the peak at −33 mV was increased. The new peaks developed were due to the further electrochemical oxidation of oxidized TMB (Figure 3S in supplementary information) When compared to CV without adding H2 O2 , the CV of TMB showed two peaks at 240 mV and 35 mV, which was due to the electrochemical oxidation of TMB. Fig. 1B shows the voltammogram of various blanks at various stages of modification of the gold electrode using 15 ␮M TMB/20 ␮M H2 O2 in 0.1 M PB/KCl at potential range of 450 mV to −300 mV and 10 mV/s scan rate. There were no peaks for the blank gold electrode, AuNP–Ab1 , and AuNP–Ab1 /Ag. But when secondary antibody HRP conjugate was added to the AuNP–Ab1 /Ag electrode, the voltammogram showed two well-defined peaks. In voltammo-

G.K. Ahirwal, C.K. Mitra / Biosensors and Bioelectronics 25 (2010) 2016–2020

2019

Fig. 2. Voltammogram of modified electrode using different concentrations of antigen (2 ng/mL, 4 ng/mL, 8 ng/mL, 16 ng/mL and 32 ng/mL, respectively) in 0.1 M PB/0.1 M KCl (pH 7.0) at a potential range of 450 mV to −300 mV, vs. Ag/AgCl reference electrode. Scan rate 10 mV/s. A plot of log[antigen] vs. peak current (second anodic) shows a linear graph (inset).

gram, the redox peaks at potential 154 mV, −33 mV and −156 mV corresponds to the oxidized TMB. The electrochemical study was done using different concentrations of antigen to check the sensitivity of the modified electrode. The antigen concentration used was 2 ng/mL, 4 ng/mL, 8 ng/mL, 16 ng/mL and 32 ng/mL. 5 ␮L each of the analyte was immobilized onto the electrode and the electrochemical measurement was done. Fig. 2 shows that, up to 2 ng/mL sample, which equals to 10 pg/5 ␮L of the antigen was easily detected and showed a defined CV. Peak currents (for all the three peaks) show a linear dependence on log[antigen] but the second peak is most sensitive and we have shown this as an inset in Fig. 2. 3.3.2. Identification of the peaks As shown in Fig. 1, the cyclic voltammogram of the TMB in the presence of H2 O2 showed three well-defined peaks. Two sets of experiments were performed to confirm that the peaks observed are due to the oxidation of TMB. First set: Using low dye concentration, the cyclic voltammetry was done using two different concentrations of H2 O2 and different scan rates. One experiment was performed using 1 ␮M of TMB concentration in the solution and the H2 O2 concentration was 10 ␮M and 100 ␮M using the modified gold electrode at the potential range of 450 mV to −300 mV and 10 mV/s scan rate (Fig. 3B). The other experiment was done at different scan rates of 10 mV/s, 30 mV/s, 50 mV/s and 100 mV/s (Figure 4S(a) supplementary information). Second set: In this set of experiments, we have used lower concentration of H2 O2 . The cyclic voltammetry was done at two different concentrations of the dye and two different scan rates. The experiment was performed using 5 ␮M of H2 O2 in the solution and 2.5 ␮M and 25 ␮M of TMB using the modified gold electrode at the potential range of 450 mV to −300 mV and 10 mV/s scan rate (Fig. 3A). The other experiment was done at different scan rates of 10 mV/s, 30 mV/s, 50 mV/s and 100 mV/s (Figure 4S(b) supplementary information). If the potential difference (Epc − Epa ) is ∼57 mV, we can say that it is a single electron transfer with the diffusion of the redox species, but if it is ∼28 mV than the process is two-electron transfer and the redox species is in the solution. To check this we have calculated the potential differences from the CV (Fig. 3A and B) and represented it in the table below. The potential difference of both the voltammogram, using different concentrations of the dye and H2 O2

Fig. 3. (A) Voltammogram of (1) 2.5 ␮M and (2) 25 ␮M TMB, (B) voltammogram of (1) 10 ␮M and (2) 100 ␮M H2 O2 using modified electrode (containing primary antibody–antigen–secondary antibody attached to the gold nanoparticles). All voltammograms are recorded in 0.1 M PB (pH 7.0) at a potential range of 450 mV to −300 mV, scan rate 10 mV/s vs. Ag/AgCl reference electrode.

shows that the peak at 154 mV (Fig. 3A and B) is a two-electron process (which represents the reaction (1a)), and the peak at −33 mV (Fig. 3A and B) and peak at −156 mV (Fig. 3A and B) (which represents the reactions (1b) and (1c)), are one-electron process. It is clear (from the peak values) from Table 1 that one of the peaks (first from the left: around 150 mV) corresponds to a two-electron transfer process while the remaining two peaks correspond to oneelectron transfer processes. The CV at different scan rates as shown in Figure 4S (supplementary information) for both 1 ␮M of TMB and 5 ␮M of H2 O2 showed a linear increase in the peak current values with increasing scan rates. To show that the dye and the H2 O2 are in the solution and the rate is diffusion controlled, we have calculated the ratio of TMB and H2 O2 peak cathodic current values for the 100 mV/s and 10 mV/s scan rates as shown in Table 1 above. The value should be approximately 3.162 (i.e., square root of 10). The above values in Table 1 are nearly equal to 3.16, which suggest that the substrates are in the solution and the electron transfer is diffusion controlled. 3.4. Electrochemical impedance spectroscopy (EIS) studies EIS is an effective method to monitor the changes of the surface modified electrodes allowing the understanding of the chemical transformation and processes associated with the conductive electrode (Bard and Faulkner, 2001). The impedance spectra include a semicircle portion and a linear portion. The semicircle portion at higher frequencies corresponds to the electron transfer limited process and the linear part at lower frequencies corresponds to the diffusion process. The semicircle also corresponds to the electron transfer resistance. Fig. 4 exhibits the Nyquist plot of the impedance spectroscopy of the bare and modified electrodes. In EIS, the bare electrode exhibited an almost straight line (Fig. 4a),

2020

G.K. Ahirwal, C.K. Mitra / Biosensors and Bioelectronics 25 (2010) 2016–2020

Table 1 (a) The cathodic and anodic potentials and the potential differences for the three peaks (left to right) in Fig. 3. (b) Peak current ratios calculated from the CVs at different scan rate of dye and hydrogen peroxide (CVs are given in supplementary information 4S). (a) [TMB] (␮M)

Peak 1 (mV)*

Peak 2 (mV)*

Peak 3 (mV)*

[H2 O2 ] (␮M)

Peak 1 (mV)*

Peak 2 (mV)*

Peak 3 (mV)*

2.5 25

152 − 165 = −13 145 − 160 = −15

(−38.47) − (−13.17) = −51 51 − 0.816 = 50

(−163) − (−132) = −31 (−180) − (−115) = 65

10 100

146 − 166 = −20 151 − 164 = −13

(−49.5) − (0.094) = −49 (−51) − (−0.906) = −50

(−196) − (−120) = −76 (−193) − (−121) = −72

(b) (Ip @ 100 mV/s)/(Ip @ 10 mV/s)

(Ip @ 100 mV/s)/(Ip @ 10 mV/s)

TMB

Peak 1

Peak 2

Peak 3

H2 O2

Peak 1

Peak 2

Peak 3

1 ␮M Cathodic 1 ␮M Anodic

2.8 2.8

2.7 2.2

3.1 2.8

5 ␮M Cathodic 5 ␮M Anodic

2.5 2.7

2.2 2.1

2.4 2.8

*

Peak = Epc − Epa ; peaks 1. . .3 are from left to right as seen in the plots in Fig. 3.

ments showed the formation of the antibody–antigen interaction. We suggest that the sensitivity of the sensors is good but can be increased using flow injection system. We propose that this kind of approach is simple, faster and cost effective and can be used for developing an immunosensor. Acknowledgements One of the authors (GKA) thanks CSIR (Council of Scientific and Industrial Research, Govt. of India) for a fellowship (Senior Research Fellowship). We thank Lo Gorton (University of Lund) for various suggestions and discussions. The work was supported by a grant from the Department of Biotechnology (Govt of India). Appendix A. Supplementary data Fig. 4. EIS of (a) bare gold electrode, (b) glutathione coated gold nanoparticles, (c) AuNP–Ab conjugate, (d) AuNP–Ab/antigen, (e) AuNPs–Ab/antigen/antibody–HRP modified gold electrode in the background solution of TMB and PB/KCl (0.1 M, pH 7.0). The frequency range used was 0.1 Hz to 1.0 × 10−5 Hz at room temperature (Zre vs. Zim at 160 mV vs. Ag/AgCl). The series resistance is seen to increase from 100k to about 350k as the electrode is successively modified.

the Nyquist plot (Fig. 4b) of glutathione coated AuNPs showed a much lower resistance, implying that the AuNPs are acting as good electron conducting materials and there was also electron transfer between the redox probe and the electrode. When compared to the other electrodes the semicircle diameter was increasing with the addition of the AuNP–Ab1 (Fig. 4c) AuNP–Ab1 /Ag (Fig. 4d) and AuNP–Ab1 /Ag/Ab2 (Fig. 4e), suggesting that the series of additions blocked the electron transfer between the redox probe and the electrode. The above results clearly confirm the success of the assembly on the electrode. 4. Conclusion Many different methods have been reported widely for the preparation of electrochemical immunosensors. This study shows the direct linking of antibody to AuNPs using a spacer arm. It not only provides the right orientation of the antibody to bind with the antigen but also the AuNPs used has good conducting capabilities to pass the electron to the electrode for the detection. This electrode provides a novel means for the antibody immobilization and antibody–antigen interaction studies. The cyclic voltammetry measurements were done using TMB that had shown a couple of clear and well-defined peaks and the detection limit was found to be 2 ng/mL or 10 pg/5 ␮L of the sample. The impedance measure-

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2010.01.029. References Bard, A.J., Faulkner, L.R., 2001. Electrochemical Methods: Fundamentals and Applications, third ed. Wiley, New York. Chatrathi, M.P., Wang, J., Collins, G.E., 2007. Biosens. Bioelectron. 22, 2932–2938. Chance, B., 1951a. In: Sumner, J.B., Myrback, K. (Eds.), The Enzymes, vol. 2, Part 1. New York, Academic Press. Chance, B., 1951b. In: Nord, F.F. (Ed.), Advances in Enzymology, vol. 12. Interscience Publishers, pp. 153–190. Chen, H., Jiang, J.H., Huang, Y., Deng, T., Li, J.S., Shen, G.L., Yu, R.Q., 2007. Sens. Actuators, B 117, 211–218. Dana, D., Xiaoxing, X., Wang, S., Aidong, Z., 2007. Talanta 71, 1257–1267. Huang, H., Ran, P., Liu, Z., 2007. Bioelectrochemistry 70, 257–562. Hu, S.Q., Xie, J.W., Xu, Q.H., Rong, K.T., Shen, G.L., Yu, R.Q., 2003. Talanta 61, 769–777. Joesphy, P.D., Eling, T., Mason, R.P., 1982. J. Biol. Chem. 257 (7), 3669–3675. Joesphy, P.D., Eling, T., Mason, R.P., 1983. J. Biol. Chem. 258 (9), 5561–5569. Kumar, S., Aaron, J., Sokolov, K., 2008. Nat. Protoc. 3, 314–320. Parak, W.J., Gerion, D., Pellegrino, T., Zanchet, D., Micheel, C., Williams, S.C., Boudreau, R., Gros, M.A.L., Larabell, C.A., Alivisatos, A.P., 2003. Nanotechnology 14, R15–R27. Ruzgas, T., Csoregi, E., Emnéus, J., Gorton, L., Varga, G.M., 1996. Anal. Chim. Acta 330, 123–138. Storhoff, J.J., Elghanian, R., Mucic, R.C., Mirkin, C.A., Letsinger, R.L., 1998. J. Am. Chem. Soc. 120, 1959–1964. Tang, D.P., Yuan, R., Chai, Y.Q., Zhong, X., Liu, Y., Dai, J.Y., Zhang, L.Y., 2004a. Anal. Biochem. 333, 345–350. Tang, D., Yuan, D., Chai, Y., Dai, J., Zhong, X., Liu, Y., 2004b. Bioelectrochemistry 65, 15–22. Tkachenko, A.G., Xie, H., Coleman, D., Glomm, W., Ryan, J., Anderson, M.F., Franzen, S., Feldheim, D.L., 2003. J. Am. Chem. Soc. 125, 4700–4701. Wang, M., Wang, L., Yuan, H., Ji, H., Sun, C., Ma, L., Bai, Y., Li, T., Li, J., 2004a. Electroanalysis 16, 757–764. Wang, M., Wang, L., Yuan, H., Ji, H., Sun, C., Ma, L., Bai, Y., Li, T., Li, J., 2004b. Biosens. Bioelectron. 19, 575–582. Wu, Z.S., Li, J.S., Luo, M.H., Shen, G.L., Yu, R.Q., 2005. Anal. Chim. Acta 528, 235–242.