Ultrasensitive Potentiometric Immunosensor Based on SA and OCA

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Ultrasensitive Potentiometric Immunosensor Based on SA and OCA Techniques for Immobilization of HBsAb with Colloidal Au and Polyvinyl Butyral as Matrixes Ruo Yuan,* Dianping Tang, Yaqin Chai, Xia Zhong, Yan Liu, and Jianyuan Dai Chong Qing Key Laboratory of Analytical Chemistry, College of Chemistry and Chemical Engineering, Southwest China Normal University, Chongqing 400715, China Received December 1, 2003. In Final Form: April 1, 2004 A novel potentiometric immunosensor for detection of hepatitis B surface antigen (HBsAg) has been developed by means of self-assembly (SA) and opposite-charged adsorption (OCA) techniques to immobilize hepatitis B surface antibody (HBsAb) on a platinum electrode. A cleaned platinum electrode was first pretreated in the presence of 10% HNO3 and 2.5% K2CrO4 solution and held at -1.5 V (vs SCE) for 1 min to make it negatively charged and then immersed in a mixing solution containing hepatitis B surface antibody, colloidal gold (Au), and polyvinyl butyral (PVB). Finally, HBsAb was successfully immobilized onto the surface of the negatively charged platinum electrode modified nanosized gold and PVB sol-gel matrixes. The modified procedure was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The immobilized hepatitis B surface antibody exhibited direct electrochemical behavior toward hepatitis B surface antigen (HBsAg). The performance and factors influencing the performance of the resulting immunosensor were studied in detail. More than 95.7% of the results of the human serum samples obtained by this method were in agreement with those obtained by enzyme-linked immunosorbent assays (ELISAs). The resulting immunosensor exhibited fast potentiometric response (6 months).

Introduction There has been great interest in the development of new, simple, sensitive, and specific immunoassays for the quantitative determination of analytes of clinical or biological importance recently. Besides the use of radioactive labels,1 nonradioactive labels, such as enzymes,2-5 fluorescent molecules6 bio- and chemluminogenic reagents,7 amperometric, potentiometric, and photometric, for immunoassay have been developed. Of these, optical detection methods are most developed in terms of commercial applications,8 but the electrochemical detection method using immunoreactions has not been applied much to date. Although optical detection methods are widely used for the detection of enzymatic products resulting from the antigen-antibody reaction in ELISA, electrochemical methods can provide capabilities of in vivo monitoring, free from color and turbid interferences, that optical detection methods cannot compete with.9,10 Thus, electrochemical detection methods appear to be very promising * Corresponding author. Phone: +86-23-68252277. Fax: +8623-68254000. E-mail: [email protected]. (1) Edwards, R. In Principles and Practice of Immunoassay, 2nd ed.; Price, C. P., Newman, D. J., Eds.; Stockton: New York, 1997; pp 325348. (2) Santandreu, M.; Cespedes, F.; Alegret, S.; Martinez-Fabregas, E. Anal. Chem. 1997, 69, 1245. (3) Wang, J.; Pamidi, P. V. A.; Rogers, K. R. Anal. Chem. 1998, 70, 1171. (4) Sole, S.; Alegret, S.; Cespedes, F.; Martinez-Fabregas, E.; DiezCaballero, T. Anal. Chem. 1998, 70, 1462. (5) Kokado, A.; Tsuji, A.; Maeda, M. Anal. Chim. Acta 1997, 337, 335. (6) Matveeva, E. G.; Savitski, A. P.; Gomez-Hens, A. Anal. Chim. Acta 1998, 361, 27 (7) Brown, R. C.; Weeks, I.; Fisher, M.; Harbron, S.; Taylorson, C. J.; Woodhead, J. S. Anal. Biochem. 1998, 259, 142. (8) Byfield, M. P.; Abuknesha, R. A. Biosens. Bioelectron. 1994, 9, 373-399. (9) Skladal, P. Electroanalysis 1997, 9, 737-745.

due to the relatively simple and inexpensive equipment required.9,11 Amperometric immunosensors were initially based on ELISAs, and measurement of the electrochemically active product was carried out using redox enzymes.12 Therefore, amperometric immunosensing requires labeling of either antigen or antibody. This requires highly qualified personnel, tedious assay time, or sophisticated instrumentation.13 In addition, immunoreagents and enzymes are customarily expensive in ELISAs. In contrast, because of its good sensitivity and selectivity, low cost, small size, and ease in use,14 the potentiometric immunoassays play an ever-increasing role in immunosensors.15,16 Thus, searching for a new immobilization method for potentiometric immunosensor with substantial improvement in sensitivity, selectivity, and response time is of considerable interest. In the present paper we describe a new method to use colloidal gold and polyvinyl butyral to immobilize hepatitis B surface antibody upon platinum electrode by the selfassembly (SA) and the opposite-charged adsorption (OCA) techniques. The immunosensors studied exhibit excellent sensitivity, rapid response, good reproducibility, and long(10) Electrochemical sensors in Immunological Analysis; Ngo, T. T., Ed.; Plenum Press: New York, 1987. (11) McNeil, C. J.; Athey, D.; Rennerberg, R. In Frontiers in Biosensors. 2. Practical Applications; Scheller, F. W., Scheubert, F., Skladal, P., Fedrowitz, J., Eds.; Birkhauser: Basel, 1997; p 17. (12) Tiefenauer, L. X.; Kossek, S.; Padese, C.; Thiebaud, P. Biosens. Bioelectron. 1997, 12, 213-223. (13) Maunaert, E.; Daenens, P. Analysis 1994, 119, 2221-2226. (14) Biosensor: Fundamentals and Applications; Turner, A. P. F., Karube, I., Wilson, G. S., Eds.; Oxford University Press: Oxford, U.K., 1987. Applied Biosensors; Wise, D. L., Ed.; Butterworth: Boston, 1989. (15) Blackburn, G. F.; Talley, D. B.; Booth, P. M.; Durfor, C. N.; Martin, M. T. Anal. Chem. 1990, 62, 2211-2216. (16) Purvis, D.; Leonardovab, A. O.; Farmakovskyb, D.; Cherkasovb, V. Biosens. Bioelectron. 2003, 18, 1385-1390.

10.1021/la030428m CCC: $27.50 © 2004 American Chemical Society Published on Web 07/24/2004

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term stability toward hepatitis B surface antigen. Hepatitis B surface antibody was selected as a model since it is well studied and is commercially available in a highly purified form. The immunosensor fabrication procedure was optimized with respect to the size of the gold nanoparticles and the assembling time. In addition, the performance and factors influencing the performance of the resulting immunosensor have been studied in detail. Experimental Section Reagent and Materials. Hepatitis B surface antibody (HBsAb) and hepatitis B surface antigen (HBsAg) (E.C 1.1.3.4, 1.28 µg‚mL-1) were purchased from Kehua Bioengineering Co. (Shanghai China). Polyvinyl butyral (PVB, 99.8%) was bought from Shanghai Chemical Reagent Co. (China). Bovine serum albumin (BSA, 96-99%), gold chloride, and tannic acid were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals and solvents used were of analytical grade and used as received. Double-distilled water was used throughout this study. The standard HBsAg stock solutions were prepared with phosphate buffer solution (PBS, pH 7.4) and stored at 4 °C. The HBsAb was stored in the frozen state, and standard solutions of it were prepared daily in PBS solution. In the preparation of a phosphate buffer solution of pH 7.4, NaCl (8.0 g), Na2HPO4 (1.15 g), KH2PO4 (0.2 g), and KCl (0.2 g) were dissolved in 1000 mL of double-distilled water. Apparatus. Cyclic voltammetric measurements (CVs) were carried out on a CHI 660A electrochemical analyzer (Shanghai CH Instruments Co., China) using a conventional three-electrode electrochemical cell. The electrodes were a platinum working electrode modified hepatitis B surface antibody (Φ ) 1 mm), a saturated calomel reference electrode (SCE), and a Pt coil counter electrode. AC impedance measurement was performed with a model IM6e (ZAHNER Elektrick Co., Germany). The size of the Au colloids was estimated by transmission electron microscopy (TEM) (H600, Hitachi Instrument Co., Japan). All potentiometric and pH measurements were made with a pH meter (MP 230, Mettler-Toledo Co, Switzerland) and a digital ion analyzer (model PHS-3C, Dazhong Instruments, Shanghai, China). Preparation of Au Colloids. All glassware used in the following procedures was cleaned in a bath of freshly prepared solution (3:1 K2Cr2O7-H2SO4), thoroughly rinsed with doubledistilled water, and dried prior to use. The 16-nm-diameter Au colloid was prepared according to the literature17 by adding 2 mL of 1% (w/w) sodium citrate solution into 50 mL of 0.01% (w/w) HAuCl4 boiling solution. The maximum adsorption of the synthesized colloidal Au in the UV-vis spectra was at 520 nm, and the solution was stored in a refrigerator in a dark-colored glass bottle before use. The particle sizes were confirmed by transmission electron microscopy (TEM). Preparation of the Immunosensor. The platinum electrode (1-mm diameter) was first polished carefully with abrasive paper and then rinsed thoroughly twice with water, boiled in nitric acid (1:1) for 10 min, and ultrasonicated in acetone and absolute ethanol. The cleaned platinum electrode was pretreated by immersing in a solution containing 10% HNO3 and 2.5% K2CrO4 and held at -1.5 V (vs SCE) for 1 min in order to make it negatively charged. The platinum electrode charged positively was obtained when the platinum electrode was held at +1.5 V (vs SCE) for 1 min. Then a sol-gel method was adopted to prepare the electrode. An appropriate amount (unless otherwise specified, 60 µL was used) of the standard hepatitis B surface antibody solution was mixed with 0.3 mL of gold nanoparticles in a beaker in ice water. Ten minutes later, 3 mL of polyvinyl butyral ethanol solution (2%, v/v) was added to the beaker quickly. The pretreated platinum electrodes were dipped into the homogeneous mixing solution containing HBsAb, PVB, and gold nanoparticles. After 10 min, the electrodes were removed and stored for about 24 h at 4 °C. In the last step, the modified electrodes were treated with a solution of 0.25 wt % BSA for 60 min at 37 °C to eliminate nonspecific effect, followed by washing carefully 3 times with PBS. The finished HBsAb-Au-PVB-modified platinum electrodes were stored at 4 °C when not in use. The schematic diagram (17) Frens, G. Nat. Phys. Sci. 1973, 241, 20-22.

Figure 1. Schematic diagram of the immunosensor showing (a) the method of determination of HBsAg by the potentiometric immunosensor and (b) the configuration of the potentiometric immunosensor.

Figure 2. Cyclic voltammograms (CVs) of the electrode at different stages: (a) bare platinum electrode, (b) HBsAb-AuPVB-modified platinum electrode, (c) HBsAb-Au-PVB-modified platinum electrode incubated with BSA, and (d) HBsAbAu-PVB-modified platinum electrode combined with HBsAg. Supporting electrolyte, 10 mM PBS (pH 7.4) + 0.1 M KCl + 2.5 mM Fe(CN)64-/3- solution; scan rate, 100 mV‚s-1. of the immunosensor and the structure of the modified electrode coating are shown in Figure 1. Measurement of HBsAb Activity on the Immunosensor. The apparent activity of hepatitis B surface antibody immobilized on the immunosensor was relative to the potentiometric response toward hepatitis B surface antigen. The potentiometric response of immunosensor toward hepatitis B surface antigen is evaluated as following the equation

∆E ) E2 - E1 where E1 is the value of the steady-state potentiometric response (vs SCE) in a 5 mL stirred phosphate buffer solution (pH 7.4) and E2 represents the value of the steady-state potentiometric response (vs SCE) after an appropriate amount of the standard or serum sample solution is added into the same stirred phosphate buffer solution. It is clear that the shift of the potentiometric response (∆E) of the immunosensor-immobilized hepatitis B surface antibody depends on the concentration of hepatitis B surface antigen in the standard or sample solutions.

Results and Discussion Electrochemical Characteristics on the Electrode Surface. The cyclic voltammogram (CV) of ferricyanide is a valuable and convenient tool to monitor the barrier of the modified electrode because electron transfer between the solution species and the electrode must occur by tunneling either through the barrier or through the defects in the barrier. Therefore, it was chosen as a marker to investigate the changes of the electrode behavior after each assembly step. When the electrode surface has been modified by some materials, the electron-transfer kinetics of Fe(CN)64-/3- is perturbed. Figure 2 shows cyclic voltammograms of Fe(CN)64-/3- at a bare platinum electrode (curve a), HBsAb-Au-PVB-modified electrode (curve b), HBsAb-Au-PVB-modified platinum electrode obturated

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Figure 3. Electrochemical impedance spectroscopy (EIS) of the different electrodes: (a) a bare platinum electrode, (b) Aumodified platinum electrode, (c) Au-PVB-modified platinum electrode, and (d) HBsAb-Au-PVB-modified platinum electrode. Supporting electrolyte, 10 mM PBS (pH 7.4) + 0.1 M KCl + 2.5 mM Fe(CN)64-/3- solution. Z vs Z′ at 220 mV vs SCE.

with BSA (curve c), and HBsAb-Au-PVB-modified electrode combined with HBsAg (curve d). As shown in Figure 2, stepwise modification on the platinum electrode is accompanied by a decrease in the amperometric response of the electrode and an increase in the peakto-peak separation between the cathodic and anodic waves of the redox probe. This is consistent with the enhanced electron-transfer barriers introduced upon assembly of these layers. In particular, after hepatitis B surface antigen molecules were combined with hepatitis B surface antibody molecules, an obvious disappearance of the anodic peak and cathodic peak was obtained (Figure 2, curve d). The reason is that the antigen-antibody complex acts as the inert electron and mass-transfer blocking layer, and it hinders the diffusion of ferricyanide toward the electrode surface. Electrochemical impedance spectroscopy (EIS) can give further information on the impedance changes of the immunosensor surface in the modification process. In EIS, the semicircle diameter of EIS equals the electron-transfer resistance, Ret. This resistance controls the electrontransfer kinetics of the redox probe at the electrode interface. Curve a in Figure 3 shows EIS of the bare platinum electrode. There is a very small semicircle domain, implying very low eT resistance (Ret ) 121 Ω) to the redox probe dissolved in the electrolyte solution. When the bare platinum electrode was dipped into the colloidal gold, we were surprised to find that the EIS of the gold colloid-modified electrode is similar to that of the bare platinum electrode (Ret )147 Ω, Figure 3, curve b). This implied that the conductivity of the gold colloid-modified platinum electrode was essentially equivalent to a bulk Pt electrode. The reason may be that the nanometer-sized gold colloids immobilized on the platinum electrode play an important role similar to a conducting wire or electronconduction tunnel, which makes it easier for the electron transfer to take place. After the platinum electrode was immersed in gold colloid and PVB ethanol solution, the EIS of the resulting assembled layer shows a high interfacial eT resistance (Ret ) 1197 Ω, Figure 3, curve c), indicating that the PVB obstructed eT of the electrochemical probe. The HBsAb-Au-PVB-modified platinum electrode was finally obtained by dipping the platinum electrode into the homogeneous mixing solution containing hepatitis B surface antibody, gold colloids, and polyvinyl butyral. After immobilization of hepatitis B surface antibody, the interfacial resistance of the electrode

Yuan et al.

Figure 4. Potentiometric response characteristics of the modified electrodes based on platinum electrode with different charges for different HBsAg concentrations in phosphate buffer solution (pH 7.4): (a) HBsAb-Au-PVB-modified platinum electrode charged negatively, (b) HBsAb-Au-PVB-modified platinum electrode without charges, and (c) HBsAb-Au-PVBmodified platinum electrode charged positively.

increased again (Ret ) 1986 Ω, Figure 3, curve d), which was approximately twice that of the Au-PVB-modified platinum electrode. On the basis of the CVs and EIS results, we conclude that hepatitis B surface antibody is successfully immobilized on the surface of the platinum electrode via colloidal gold and polyvinyl butyral. Effect of Platinum Electrodes with Different Charges on Response Characteristics of the Immunosensors. Figure 4 shows the potentiometric response of the differently charged platinum electrodes modified with HBsAb-Au-PVB toward hepatitis B surface antigen. As shown in Figure 4, the platinum electrodes charged negatively could enhance the sensitivity of the immunosensors. The reason for this could be from the fact that the isoelectric point of hepatitis B surface antibody is more than 7.4, which is positively charged in PBS solution, pH 7.4. In addition, the gold nanoparticles are negatively charged species as a result of the adsorption of citrate in the fabrication process,21 which can connect with -NH2 between the hepatitis B surface antibody molecules. Optimization of Experimental Conditions. The effect of the size of the Au nanoparticles on the potentiometric response was studied. Figure 5a shows the potentiometric responses of the immunosensors with different sizes of gold nanoparticles toward the same concentration of HBsAg under steady-state conditions in a phosphate buffer solution, pH 7.4. As is well known, the interaction between protein molecules and Au colloid particles is very strong due to the very high surface-tovolume ratio of Au colloid particles and their high surface energy.18 Doron et al.19 confirmed that the larger-sized Au colloids can discontinuously assemble and the smaller sized Au colloids may generate continuous arrays of particles on the base monolayer. The packing of smallersized Au nanoparticle-bound HBsAb is denser than that of the larger-sized Au colloids due to the very high surfaceto-volume ratio of Au colloid particles.18 However, the coulomb repulsion becomes stronger as the size of gold nanoparticles is much smaller.20 The immunosensor fabricated with 16-nm gold nanoparticles exhibits a larger response than that of the other sizes; therefore, a 16-nm gold nanoparticle was chosen as the immobilized matrix. The self-assembly time between HBsAb and colloidal Au is a vital step for the fabrication of the immunosensor. (18) Shipway, A. N.; Willner, I. Chem. Commun. 2001, 20, 20352045. (19) Doron, A.; Katz, E.; Willner, I. Langmuir 1995, 11, 1313-1317. (20) Colvin, V. L.; Goldstein, A. N.; Alivisatos, A. P. J. Am. Chem. Soc. 1992, 114, 5221-5230. (21) Weitz, D. A.; Lin, M. Y.; Sandroff, C. J. Surf. Sci. 1985, 158, 147-164.

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Figure 5. Effect of experimental parameters such as (a) the size of gold nanoparticles, (b) self-assembly time, (c) amount of immobilized HbsAb-loaded gold nanoparticles, and (d) pH values in the presence of 40 ng‚mL-1 HBsAg PBS solution on potentiometric response of the immunosensor.

When the time is shorter, the amount of self-assembled HBsAb is smaller. It is not convenient to fabricate the immunosensor at the following steps. The output voltage increases rapidly with the elapse of time and converges after 10 min. Therefore, 10 min was chosen as the proper self-assembled time (Figure 5b). By varying the volume of HBsAb (from 20 to 100 µL) in the immobilization solution, the change in sensitivity of the HBsAb-Au-PVB-modified immunosensor was studied. The prepared immunoelectrodes were allowed to combine with hepatitis B surface antigen, and the potentiometric responses to hepatitis B surface antigen were measured. Figure 5c shows the results. The potentiometric responses of the electrode increased with the increment of HBsAb amount on the electrode and started to level off when the volume of HBsAb became larger than 60 µL. To ensure enough hepatitis B surface antibody for its immunoreaction with hepatitis B surface antigen, 60 µL of hepatitis B surface antibody solutions were adopted for electrode immobilization. The effect of pH on the immunosensor behavior was studied between 5.5 and 8.5 in PBS. As shown in Figure 5d, the potentiometric response increases from 5.5 to 7.4 and decreases from pH 7.4 to 8.5. It is well known that the activity of the antibody or antigen is inhibited at relatively high pH. Therefore, PBS of pH 7.4 is used as the medium for the immunoreaction. Dynamics Curve of the Potentiometric Response. Dynamics curves of the potentiometric response of the immunosensor in the presence of 20 ng‚mL-1 HBsAg positive serum and in the absence of HBsAg negative serum in phosphate buffer solution (pH 7.4) at room temperature are illustrated in Figure 6. The potentiometric responses increased with the increment of reaction time and started to level off after 3 min. The result indicates that reaction between immobilized antibody and free antigen is an equilibrium process. In addition, a shift of the potentiometric response to the negative serum is negative (i.e., ∆E < 0) while a shift to the positive serum is positive (i.e., ∆E > 0). The reason is that as hepatitis B surface antibody complex combined with hepatitis B

Figure 6. Potentiometric responses of the immunosensor vs reaction time in the presence of 20 ng‚mL-1 HBsAg positive serum and in the absence of HBsAg negative serum in phosphate buffer solution of pH 7.4 at room temperature.

surface antigen, the electrical charge of the resulting complex will be different from that of HBsAb or HBsAg alone. If HBsAb is immobilized on the platinum electrode, the surface charge of the immunosensor will depend on the net charge of the immobilized HBsAb. When HBsAg is present in the solution, the immunochemical reaction will take place at the interface with a resulting change of the surface charge. According to the ∆E value (∆E > 0 or ∆E 0 or ∆E