Ultrasensitive Potentiometric Immunosensor Based on SAand 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 ChemicalEngineering, 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 beendeveloped by means of self-assembly (SA) and opposite-charged adsorption (OCA) techniques to immobilizehepatitis B surface antibody (HBsAb) on a platinum electrode. A cleaned platinum electrode was firstpretreated in the presence of 10% HNO3 and 2.5% K2CrO4 solution and held at -1.5 V (vs SCE) for 1 minto make it negatively charged and then immersed in a mixing solution containing hepatitis B surfaceantibody, colloidal gold (Au), and polyvinyl butyral (PVB). Finally, HBsAb was successfully immobilizedonto the surface of the negatively charged platinum electrode modified nanosized gold and PVB sol-gelmatrixes. The modified procedure was characterized by electrochemical impedance spectroscopy (EIS) andcyclic voltammetry (CV). The immobilized hepatitis B surface antibody exhibited direct electrochemicalbehavior toward hepatitis B surface antigen (HBsAg). The performance and factors influencing theperformance of the resulting immunosensor were studied in detail. More than 95.7% of the results of thehuman serum samples obtained by this method were in agreement with those obtained by enzyme-linkedimmunosorbent assays (ELISAs). The resulting immunosensor exhibited fast potentiometric response (<3min) to HBsAg. The detection limit of the immunosensor was 2.3 ng‚mL-1, and the linear range was from8 to 1280 ng‚mL-1. Moreover, the studied immunosensor exhibited high sensitivity, good reproducibility,and long-term stability (>6 months).

Introduction

There has been great interest in the development ofnew, simple, sensitive, and specific immunoassays for thequantitative determination of analytes of clinical orbiological importance recently. Besides the use of radioac-tive labels,1 nonradioactive labels, such as enzymes,2-5

fluorescent molecules6 bio- and chemluminogenic re-agents,7 amperometric, potentiometric, and photometric,for immunoassay have been developed. Of these, opticaldetection methods are most developed in terms of com-mercial applications,8 but the electrochemical detectionmethod using immunoreactions has not been applied muchto date. Although optical detection methods are widelyused for the detection of enzymatic products resulting fromthe antigen-antibody reaction in ELISA, electrochemicalmethods can provide capabilities of in vivo monitoring,free from color and turbid interferences, that opticaldetection methods cannot compete with.9,10 Thus, elec-trochemical detection methods appear to be very promising

due to the relatively simple and inexpensive equipmentrequired.9,11

Amperometric immunosensors were initially based onELISAs, and measurement of the electrochemically activeproduct was carried out using redox enzymes.12 Therefore,amperometric immunosensing requires labeling of eitherantigen or antibody. This requires highly qualified per-sonnel, tedious assay time, or sophisticated instrumenta-tion.13 In addition, immunoreagents and enzymes arecustomarily expensive in ELISAs. In contrast, because ofits good sensitivity and selectivity, low cost, small size,and ease in use,14 the potentiometric immunoassays playan ever-increasing role in immunosensors.15,16 Thus,searching for a new immobilization method for potentio-metric immunosensor with substantial improvement insensitivity, selectivity, and response time is of considerableinterest.

In the present paper we describe a new method to usecolloidal gold and polyvinyl butyral to immobilize hepatitisB surface antibody upon platinum electrode by the self-assembly (SA) and the opposite-charged adsorption (OCA)techniques. The immunosensors studied exhibit excellentsensitivity, rapid response, good reproducibility, and long-

* Corresponding author. Phone: +86-23-68252277. Fax: +86-23-68254000. E-mail: yuanruo@swnu.edu.cn.

(1) Edwards, R. In Principles and Practice of Immunoassay, 2nd ed.;Price, C. P., Newman, D. J., Eds.; Stockton: New York, 1997; pp 325-348.

(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.

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(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.

(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 inBiosensors. 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.

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

term stability toward hepatitis B surface antigen. Hepa-titis B surface antibody was selected as a model since itis well studied and is commercially available in a highlypurified form. The immunosensor fabrication procedurewas optimized with respect to the size of the goldnanoparticles and the assembling time. In addition, theperformance and factors influencing the performance ofthe resulting immunosensor have been studied in detail.

Experimental SectionReagent 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 boughtfrom Shanghai Chemical Reagent Co. (China). Bovine serumalbumin (BSA, 96-99%), gold chloride, and tannic acid wereobtained from Sigma Chemical Co. (St. Louis, MO). All otherchemicals and solvents used were of analytical grade and usedas received. Double-distilled water was used throughout thisstudy. The standard HBsAg stock solutions were prepared withphosphate buffer solution (PBS, pH 7.4) and stored at 4 °C. TheHBsAb was stored in the frozen state, and standard solutions ofit 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) werecarried out on a CHI 660A electrochemical analyzer (ShanghaiCH Instruments Co., China) using a conventional three-electrodeelectrochemical cell. The electrodes were a platinum workingelectrode modified hepatitis B surface antibody (Φ ) 1 mm), asaturated calomel reference electrode (SCE), and a Pt coil counterelectrode. AC impedance measurement was performed with amodel IM6e (ZAHNER Elektrick Co., Germany). The size of theAu colloids was estimated by transmission electron microscopy(TEM) (H600, Hitachi Instrument Co., Japan). All potentiometricand pH measurements were made with a pH meter (MP 230,Mettler-Toledo Co, Switzerland) and a digital ion analyzer (modelPHS-3C, Dazhong Instruments, Shanghai, China).

Preparation of Au Colloids. All glassware used in thefollowing procedures was cleaned in a bath of freshly preparedsolution (3:1 K2Cr2O7-H2SO4), thoroughly rinsed with double-distilled water, and dried prior to use. The 16-nm-diameter Aucolloid was prepared according to the literature17 by adding 2mL of 1% (w/w) sodium citrate solution into 50 mL of 0.01%(w/w) HAuCl4 boiling solution. The maximum adsorption of thesynthesized colloidal Au in the UV-vis spectra was at 520 nm,and the solution was stored in a refrigerator in a dark-coloredglass bottle before use. The particle sizes were confirmed bytransmission electron microscopy (TEM).

Preparation of the Immunosensor. The platinum electrode(1-mm diameter) was first polished carefully with abrasive paperand then rinsed thoroughly twice with water, boiled in nitricacid (1:1) for 10 min, and ultrasonicated in acetone and absoluteethanol. The cleaned platinum electrode was pretreated byimmersing in a solution containing 10% HNO3 and 2.5% K2CrO4and held at -1.5 V (vs SCE) for 1 min in order to make it negativelycharged. The platinum electrode charged positively was obtainedwhen the platinum electrode was held at +1.5 V (vs SCE) for 1min. Then a sol-gel method was adopted to prepare the electrode.An appropriate amount (unless otherwise specified, 60 µL wasused) of the standard hepatitis B surface antibody solution wasmixed 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 pretreatedplatinum electrodes were dipped into the homogeneous mixingsolution containing HBsAb, PVB, and gold nanoparticles. After10 min, the electrodes were removed and stored for about 24 hat 4 °C. In the last step, the modified electrodes were treatedwith a solution of 0.25 wt % BSA for 60 min at 37 °C to eliminatenonspecific effect, followed by washing carefully 3 times withPBS. The finished HBsAb-Au-PVB-modified platinum elec-trodes were stored at 4 °C when not in use. The schematic diagram

of the immunosensor and the structure of the modified electrodecoating are shown in Figure 1.

Measurement of HBsAb Activity on the Immunosensor.The apparent activity of hepatitis B surface antibody immobilizedon the immunosensor was relative to the potentiometric responsetoward hepatitis B surface antigen. The potentiometric responseof immunosensor toward hepatitis B surface antigen is evaluatedas following the equation

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 potentiometricresponse (vs SCE) after an appropriate amount of the standardor serum sample solution is added into the same stirred phosphatebuffer solution. It is clear that the shift of the potentiometricresponse (∆E) of the immunosensor-immobilized hepatitis Bsurface antibody depends on the concentration of hepatitis Bsurface antigen in the standard or sample solutions.

Results and DiscussionElectrochemical Characteristics on the Electrode

Surface. The cyclic voltammogram (CV) of ferricyanideis a valuable and convenient tool to monitor the barrierof the modified electrode because electron transfer betweenthe solution species and the electrode must occur bytunneling either through the barrier or through the defectsin the barrier. Therefore, it was chosen as a marker toinvestigate the changes of the electrode behavior aftereach assembly step. When the electrode surface has beenmodified by some materials, the electron-transfer kineticsof Fe(CN)6

4-/3- is perturbed. Figure 2 shows cyclic vol-tammograms of Fe(CN)6

4-/3- at a bare platinum electrode(curve a), HBsAb-Au-PVB-modified electrode (curve b),HBsAb-Au-PVB-modified platinum electrode obturated(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 potentiometricimmunosensor and (b) the configuration of the potentiometricimmunosensor.

Figure 2. Cyclic voltammograms (CVs) of the electrode atdifferent stages: (a) bare platinum electrode, (b) HBsAb-Au-PVB-modified platinum electrode, (c) HBsAb-Au-PVB-modi-fied platinum electrode incubated with BSA, and (d) HBsAb-Au-PVB-modified platinum electrode combined with HBsAg.Supporting electrolyte, 10 mM PBS (pH 7.4) + 0.1 M KCl + 2.5mM Fe(CN)6

4-/3- solution; scan rate, 100 mV‚s-1.

∆E ) E2 - E1

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with BSA (curve c), and HBsAb-Au-PVB-modifiedelectrode combined with HBsAg (curve d). As shown inFigure 2, stepwise modification on the platinum electrodeis accompanied by a decrease in the amperometricresponse of the electrode and an increase in the peak-to-peak separation between the cathodic and anodic wavesof the redox probe. This is consistent with the enhancedelectron-transfer barriers introduced upon assembly ofthese layers. In particular, after hepatitis B surfaceantigen molecules were combined with hepatitis B surfaceantibody molecules, an obvious disappearance of theanodic peak and cathodic peak was obtained (Figure 2,curve d). The reason is that the antigen-antibody complexacts as the inert electron and mass-transfer blocking layer,and it hinders the diffusion of ferricyanide toward theelectrode surface.

Electrochemical impedance spectroscopy (EIS) can givefurther information on the impedance changes of theimmunosensor surface in the modification process. In EIS,the semicircle diameter of EIS equals the electron-transferresistance, Ret. This resistance controls the electron-transfer kinetics of the redox probe at the electrodeinterface. Curve a in Figure 3 shows EIS of the bareplatinum electrode. There is a very small semicircledomain, implying very low eT resistance (Ret ) 121 Ω) tothe redox probe dissolved in the electrolyte solution. Whenthe bare platinum electrode was dipped into the colloidalgold, we were surprised to find that the EIS of the goldcolloid-modified electrode is similar to that of the bareplatinum electrode (Ret )147 Ω, Figure 3, curve b). Thisimplied that the conductivity of the gold colloid-modifiedplatinum electrode was essentially equivalent to a bulkPt electrode. The reason may be that the nanometer-sizedgold colloids immobilized on the platinum electrode playan important role similar to a conducting wire or electron-conduction tunnel, which makes it easier for the electrontransfer to take place. After the platinum electrode wasimmersed in gold colloid and PVB ethanol solution, theEIS of the resulting assembled layer shows a highinterfacial eT resistance (Ret ) 1197 Ω, Figure 3, curvec), indicating that the PVB obstructed eT of the electro-chemical probe. The HBsAb-Au-PVB-modified platinumelectrode was finally obtained by dipping the platinumelectrode into the homogeneous mixing solution containinghepatitis B surface antibody, gold colloids, and polyvinylbutyral. After immobilization of hepatitis B surfaceantibody, the interfacial resistance of the electrode

increased again (Ret ) 1986 Ω, Figure 3, curve d), whichwas approximately twice that of the Au-PVB-modifiedplatinum electrode. On the basis of the CVs and EISresults, we conclude that hepatitis B surface antibody issuccessfully immobilized on the surface of the platinumelectrode via colloidal gold and polyvinyl butyral.

Effect of Platinum Electrodes with DifferentCharges on Response Characteristics of the Immu-nosensors. Figure 4 shows the potentiometric responseof the differently charged platinum electrodes modifiedwith HBsAb-Au-PVB toward hepatitis B surface anti-gen. As shown in Figure 4, the platinum electrodes chargednegatively could enhance the sensitivity of the immun-osensors. The reason for this could be from the fact thatthe isoelectric point of hepatitis B surface antibody is morethan 7.4, which is positively charged in PBS solution, pH7.4. In addition, the gold nanoparticles are negativelycharged species as a result of the adsorption of citrate inthe fabrication process,21 which can connect with -NH2between the hepatitis B surface antibody molecules.

Optimization of Experimental Conditions. Theeffect of the size of the Au nanoparticles on the poten-tiometric response was studied. Figure 5a shows thepotentiometric responses of the immunosensors withdifferent sizes of gold nanoparticles toward the sameconcentration of HBsAg under steady-state conditions ina phosphate buffer solution, pH 7.4. As is well known, theinteraction between protein molecules and Au colloidparticles is very strong due to the very high surface-to-volume ratio of Au colloid particles and their high surfaceenergy.18 Doron et al.19 confirmed that the larger-sizedAu colloids can discontinuously assemble and the smallersized Au colloids may generate continuous arrays ofparticles on the base monolayer. The packing of smaller-sized Au nanoparticle-bound HBsAb is denser than thatof the larger-sized Au colloids due to the very high surface-to-volume ratio of Au colloid particles.18 However, thecoulomb repulsion becomes stronger as the size of goldnanoparticles is much smaller.20 The immunosensorfabricated with 16-nm gold nanoparticles exhibits a largerresponse than that of the other sizes; therefore, a 16-nmgold nanoparticle was chosen as the immobilized matrix.

The self-assembly time between HBsAb and colloidalAu is a vital step for the fabrication of the immunosensor.

(18) Shipway, A. N.; Willner, I. Chem. Commun. 2001, 20, 2035-2045.

(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.

Figure 3. Electrochemical impedance spectroscopy (EIS) ofthe different electrodes: (a) a bare platinum electrode, (b) Au-modified platinum electrode, (c) Au-PVB-modified platinumelectrode, and (d) HBsAb-Au-PVB-modified platinum elec-trode. Supporting electrolyte, 10 mM PBS (pH 7.4) + 0.1 M KCl+ 2.5 mM Fe(CN)6

4-/3- solution. Z vs Z′ at 220 mV vs SCE.

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

7242 Langmuir, Vol. 20, No. 17, 2004 Yuan et al.

When the time is shorter, the amount of self-assembledHBsAb is smaller. It is not convenient to fabricate theimmunosensor at the following steps. The output voltageincreases rapidly with the elapse of time and convergesafter 10 min. Therefore, 10 min was chosen as the properself-assembled time (Figure 5b).

By varying the volume of HBsAb (from 20 to 100 µL)in the immobilization solution, the change in sensitivityof the HBsAb-Au-PVB-modified immunosensor wasstudied. The prepared immunoelectrodes were allowed tocombine with hepatitis B surface antigen, and the po-tentiometric responses to hepatitis B surface antigen weremeasured. Figure 5c shows the results. The potentiometricresponses of the electrode increased with the incrementof HBsAb amount on the electrode and started to level offwhen the volume of HBsAb became larger than 60 µL. Toensure enough hepatitis B surface antibody for itsimmunoreaction with hepatitis B surface antigen, 60 µLof hepatitis B surface antibody solutions were adopted forelectrode immobilization.

The effect of pH on the immunosensor behavior wasstudied between 5.5 and 8.5 in PBS. As shown in Figure5d, the potentiometric response increases from 5.5 to 7.4and decreases from pH 7.4 to 8.5. It is well known thatthe activity of the antibody or antigen is inhibited atrelatively high pH. Therefore, PBS of pH 7.4 is used asthe medium for the immunoreaction.

Dynamics Curve of the Potentiometric Response.Dynamics curves of the potentiometric response of theimmunosensor in the presence of 20 ng‚mL-1 HBsAgpositive serum and in the absence of HBsAg negativeserum in phosphate buffer solution (pH 7.4) at roomtemperature are illustrated in Figure 6. The potentio-metric responses increased with the increment of reactiontime and started to level off after 3 min. The resultindicates that reaction between immobilized antibody andfree antigen is an equilibrium process. In addition, a shiftof the potentiometric response to the negative serum isnegative (i.e., ∆E < 0) while a shift to the positive serumis positive (i.e., ∆E > 0). The reason is that as hepatitisB surface antibody complex combined with hepatitis B

surface antigen, the electrical charge of the resultingcomplex will be different from that of HBsAb or HBsAgalone. If HBsAb is immobilized on the platinum electrode,the surface charge of the immunosensor will depend onthe net charge of the immobilized HBsAb. When HBsAgis present in the solution, the immunochemical reactionwill take place at the interface with a resulting change ofthe surface charge. According to the ∆E value (∆E > 0 or∆E <0), we could qualitatively identify a positive ornegative serum specimen in the clinic.

Potentiometric Response Characteristics of Im-munosensors. Figure 7 shows the standard calibrationgraph for hepatitis B surface antigen analysis. A sigmoidalrelationship between the potentiometric responses andthe logarithm of the hepatitis B surface antigen concen-trations was obtained with a dynamic range of 3.6-1960ng‚mL-1 (Figure 7a). As shown in Figure 7b, the immu-noelectrode exhibits a linear dependence on the logarithmof HBsAg concentrations from 8 to 1280 ng‚mL-1 with adetection limit of 2.3 ng‚mL-1. The linear regressionequation was ∆E ) -51.9 + 56.0 log[HBsAg] with acorrelation coefficient of 0.9993. According to the linear

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

Figure 6. Potentiometric responses of the immunosensor vsreaction time in the presence of 20 ng‚mL-1 HBsAg positiveserum and in the absence of HBsAg negative serum in phosphatebuffer solution of pH 7.4 at room temperature.

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regression equation, we could detect HBsAg concentrationquantitatively in the serum sample.

Regeneration and Reproducibility of the Immu-nosensor. Regeneration of immunosensors is of interestto immunoanalysts. Although the antibody-antigen link-age can be broken under drastic conditions (e.g., inalkalinic or acidic solutions or with chaotropic agents),the immobilized immunoreagents could also suffer fromfunctional damage or even be released from the immu-nosorbents.22 In this experiment, 0.2 M glycine-hydro-chloric acid (Gly-HCl) buffer solution (pH 2.8) was chosento break the antibody-antigen linkage. After detecting40 ng‚mL-1 HBsAg, the immunosensor was dipped intoglycine-hydrochloric acid buffer solution for 5 min andremoved to detect 40 ng‚mL-1 HBsAg, and this wasrepeated 40 times continuously. The immunosensorretained 94.6% of the original potentiometric responsefor the initial 21 times; the relative standard deviation(RSD) was 2.8% for 21 successive assays (Figure 8a). Thereason is that the extreme conditions degraded or chemi-cally deactivated the antibody surface.23 The electrode-to-electrode reproducibility was estimated from the re-sponse to 40 ng‚mL-1 HBsAg with eight differentimmunosensors. This series yielded a mean potentiometricresponse of 35.9 mV and a RSD of 3.21%. Good reproduc-ibility may be explained by the fact gold nanoparticlesand PVB have little effect on immunoprotein activity andHBsAb molecules are firmly attached on the surface ofthe platinum electrode modified with gold nanoparticlesand PVB.

Selectivity and Lifetime of Immunosensor. Theeffect of substances that might interfere with the responseof the immunosensor was studied. The inhibition poten-tiometric response obtained for each interfering substance

presented at a concentration of 0.5 mmol‚L-1 (unlessotherwise stated) was compared to that of 0.5 mmol‚L-1

hepatitis B surface antigen, and this ratio was used as acriterion for the selectivity of the sensor. Similar to otherstudies in the literature, diphtheria antigen, glucose, aceticacid, and bovine serum albumin did not cause anyobservable interference and hepatitis B score antigeninterfered slightly.

The long-term stability of the immunosensor wasinvestigated for a 240-day period. When the immunosensorwas stored dry at 4 °C and measured intermittently (every3-5 days), no apparent change in the same HBsAgconcentration was found over 180 days (see Figure 8b).However, when the Au nanoparticles or PVB was absent,the HBsAb electrode retained ∼23.6% of its initialsensitivity to 40 ng‚mL-1 HBsAg after 3 weeks. Good long-term stability can be attributed to the strong interactionsbetween the Au nanoparticles and HBsAb.

Application of Immunosensor in Human Serum.Eighty samples of serum from college students from ouruniversity hospital were determined using the immun-osensor. These serum samples were diluted to differentconcentrations with phosphate buffer solution (pH 7.4).The potentiometric responses were determined using apotentiometer. According to the shift of the ∆E value (∆E> 0 or ∆E <0), we could qualitatively identify a positiveor negative serum specimen. The partial results obtainedare shown in Table 1. The results with human serumsamples obtained from the immunosensor were comparedwith those from an established ELISA technique. A goodcorrelation is foundbetweentheresultsof the twomethods,and there is close agreement between results at allconcentrations. More than 95.7% of the results obtainedby this method were in agreement with those obtained byenzyme-linked immunosorbent assays (ELISAs).

Potentiometric Response Mechanism of Immu-nosensor. Either antibodies or antigens in aqueoussolution have a net electrical charge polarity, which is

(22) Bright, F. B.; Betts, T. A.; Litwiler. K. S. Anal. Chem. 1990, 62,1065-1072.

(23) Blinde, R.; Levi, S.; Tao, G. L.; Ben-Dov, L.; Willner, I. L. J. Am.Chem. Soc. 1997, 119, 10467-10478.

Figure 7. Calibration plots for the immunosensor in a phosphate buffer solution of pH 7.4 in the presence of different HBsAgconcentrations.

Figure 8. (a) Reproducibility and (b) lifetime of the immunosensor to 40 ng‚mL-1 HBsAg.

7244 Langmuir, Vol. 20, No. 17, 2004 Yuan et al.

correlated to the isoelectric points of the species and theionic composition of the solution. If antibody complexcombined with antigen, the electrical charge of theresulting complex will be different from that of antibodyalone.

If hepatitis B surface antibody is immobilized on the Ptelectrode, the surface charge of the Pt electrode will dependon the net charge of the immobilized HBsAb. Whenhepatitis B surface antigen is present in the solution, theimmunochemical reaction will take place at the interfacewith a resulting change of the surface charge. This changecan be measured potentiometrically against the referenceelectrode immersed in the same solution.

In addition, binding between immobilized antibody andbulk analyte is known to be a diffusion-controlled phe-nomenon, which depends on the ratio of the solid-phasegeometry surface area to the analyte volume.24-27 In thisstudy we researched the AC impedance of the immun-osensors conditioned in pH 7.4 phosphate buffer solution

containing 0.1 mol‚L-1 of K3[Fe(CN)6]/K4[Fe(CN)6] anddifferent HBsAg concentrations. A well-resolved bulk andsurface impedance in the high-frequency region in additionto Warburg impedance in the low-frequency region wereobserved (see Figure 9). The bulk resistance increasedwith the increment of HBsAg concentration. It wasindicated that the binding between antibody and bulkanalyte was a diffusion-controlled phenomenon.24,26

ConclusionIn the present paper we introduced a novel approach

for fabrication of immunosensor via self-assembled andopposite-charged adsorption techniques. The gold nano-particles and hepatitis B surface antibody have beensuccessfully immobilized on the platinum electrode toimprove the potentiometric response characteristics to-ward hepatitis B surface antigen. CV and EIS have beensuccessfully used as powerful tools to characterize theimmobilization of immunoproteins with ferricyanide as amarker. The immunosensors obtained exhibited highsensitivity, fast response, good reproducibility, and long-term stability.

Several advantages of the proposed method should behighlighted. First, this approach can become a universalmethod for immunosensor fabrication since the stronginteractions between gold nanoparticles and biologicalmacromolecules such as hepatitis B surface antibody canbe firmly immobilized on the electrode. Second, it canprovide a simple method to directly realize the potentio-metric response of antigen or antibody. Third, goldnanoparticles can be assembled into a multilayer, whichcan increase the immunoprotein loading. In addition, bothgold nanoparticles and PVB have little effect on antibodyactivity; therefore, the resulting immunosensor can havehigh sensitivity and good stability.

Acknowledgment. Financial support of this work wasprovided by the National Natural Science Foundation ofChina (29705001), the Chinese Education Ministry Foun-dation for excellent young teachers, and the NaturalScience Foundation of Chongqing City, China.

LA030428M

(24) Katz, E.; Willner, I. Electroanalysis 2003, 15, 913-947.(25) Dong, S.; Luo, G.; Feng, J.; Li, Q. W.; Gao, H. Electroanalysis

2001, 13, 30-33.(26) Bardea, A.; Katz, E.; Willner, I. Electroanalysis 2000, 12, 1097-

1106.(27) Gyss, C.; Bourdillon, C. Anal. Chem. 1987, 59, 2350-2355.

Table 1. Partial Results of Different Methods Obtained in Clinic Serum Testa

sample no. 1 9 15 21 30 42 53 62 71 80

∆E/mV 23.5 17.5 -2.5 16.9 8.7 -2.5 -3.8 -5.2 32.1 28.1by HBsAb-Au-PVB/Pt electrode + + - + + - - - + +by ELISA’s + + - + + - - + + +a + ) Positive serum. - ) Negative serum

Figure9. ACimpedanceplotsof immunoelectrodesconditionedin phosphate buffer solution of pH 7.4 containing 0.1 mol‚L-1

of K3[Fe(CN)6]/K4[Fe(CN)6] and different HBsAg concentra-tions: (a) 0, (b) 26.4 ng‚mL-1, (c) 40 ng‚mL-1 at frequency 1 ×10-2 to 1 × 106 Hz at 20 °C (Z′ vs Z′′ at 220 mV vs SCE).

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