An Indirect Perfluorosulfonated Ionomer-Coated Electrochemical

An Indirect Perfluorosulfonated Ionomer-Coated Electrochemical Immunosensor for the Detection of the Protein Human Chorionic Gonadotrophin. Albert F. ...
0 downloads 6 Views 116KB Size
Download PDF
Anal. Chem. 1999, 71, 4088-4094

An Indirect Perfluorosulfonated Ionomer-Coated Electrochemical Immunosensor for the Detection of the Protein Human Chorionic Gonadotrophin Albert F. Chetcuti and Danny K. Y. Wong*

Department of Chemistry, Macquarie University, Sydney, New South Wales 2109, Australia Margaret C. Stuart

Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia

The development of an amperometric immunosensor for the detection of human chorionic gonadotrophin (hCG) is described. In this immunosensor, Nafion was used to immobilize an anti-hCG monoclonal antibody onto a glassy carbon electrode. A systematic study on the effects of experimental parameters such as the quantity of ethanol present in the Nafion solution, the percentage composition of Nafion, the pH of the immobilization buffer, and the concentration of antibody used for entrapment experiments on the binding between the immobilized antibody and 125I-labeled hCG has been carried out. Two immobilization methods, coimmobilization and adsorption immobilization, have then been attempted. A binding of approximately 3% was obtained in the former method, while 5.5% binding was achieved in the latter. On the basis of these results, adsorption immobilization was employed to entrap antibody on the electrode surface. A sandwich assay was then developed for hCG in which the enzyme horseradish peroxidase was conjugated to a second anti-hCG monoclonal antibody. The activity of the enzyme was determined electrochemically by the reduction of benzoquinone to hydroquinone. Binding of hCG to immobilized antibody determines the quantity of enzymeconjugated antibody at the electrode surface, permitting the quantification of hCG. By a standard additions calibration method of hCG performed in blank human serum samples, the immunosensor exhibits a limit of linearity at 200 mIU mL-1 and a detection limit of 11.2 mIU mL-1 (based on twice the standard deviation of the blank solution). The interactions between an antibody and an antigen are known to be very specific chemical reactions. Such a specific molecular recognition of antigens by antibodies has been exploited in immunoassays to develop highly selective detection methods in many clinical analyses and medical diagnostics as well as for environmental monitoring. In these immunoassays, enzyme* To whom correspondence should be addressed. Fax: +61 2 98508313; E-mail: [email protected].

4088 Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

catalyzed reactions are often used to provide the chemical amplification necessary for the detection of extremely low concentrations of analyte. Electrochemical detection is particularly well suited for such assays owing to its ease and sensitivity in detecting minute quantities of enzyme-generated product. Hence, in conjunction with the specificity of antibody reactions and the amplification feature of an enzyme label, electrochemical immunoassays1,2 are being developed to provide a highly selective, sensitive, and low-cost detection system. Currently, a lot of work3-7 is being focused on the development of rapid, simple, sensitive, automated, and on-site electrochemical immunoassays. Here, we are interested in developing an electrochemical immunosensor for the detection of human chorionic gonadotrophin (hCG), which is a 37 kDa glycoprotein hormone. Briefly, hCG consists of two subunits, designated R and β, which are noncovalently bonded and are synthesized separately. The R-subunit of hCG is identical to the R-subunit of other pituitary glycoprotein hormones, but the biological activity of hCG is conferred by the β-subunit. Both R- and β-subunits are species specific. This is the first glycoprotein produced by trophoblasts of the placenta during pregnancy8,9 and is secreted by trophoblastic neoplasms10 and a variety of nontrophoblastic tumors.11 Detection of hCG in urine or serum is thus employed in all modern immunological pregnancy tests and for monitoring of trophoblastic diseases. (1) Ghindilis, A. L.; Atanasov, P.; Wilkins, M.; Wilkins, E. Biosens. Bioelectron. 1998, 13, 113. (2) Skla´dal, P. Electroanalysis 1997, 9, 737. (3) Tiefenauer, L. X.; Kossek, S.; Padeste, C.; Thiebaud, P. Biosens. Bioelectron. 1997, 12, 213. (4) Ghindilis, A. L.; Krishnan, R.; Atanasov, P.; Wilkins, E. Biosens. Bioelectron. 1997, 12, 415. (5) Santandreu, M.; Ce´spedies, F.; Alegret, S.; Martı´nez-Fa`bregas, E. Anal. Chem. 1997, 69, 2080. (6) Rishpon, J.; Ivnitski, D. Bionsens. Bioelectron. 1997, 12, 195. (7) Ba¨umner, A. J.; Schmid, R. D. Bionsens. Bioelectron. 1998, 13, 519. (8) Braunstein, G. D.; Rasor, J.; Alder, D.; Danzer, H.; Wade, M. Am. J. Obstet. Gynecol. 1976, 126, 678. (9) Vaitukaitis, J. L.; Ross, G. T.; Braunstein, G. D.; Rayford, P. L. Recent Prog. Hormone Res. 1976, 32, 280. (10) Pastofide, G. B.; Goldstein, D. P.; Kosasa, T. S. Am. J. Obstet. Gynecol 1974, 120, 1025. (11) Braunstein, G. D.; Vaitukaitis, J. L.; Carbone, P. P.; Ross, G. T. Ann. Intern. Med. 1973, 78, 39. 10.1021/ac981216a CCC: $18.00

© 1999 American Chemical Society Published on Web 08/12/1999

There have hitherto been several workers reporting on electrochemical immunoassays for hCG. More than a decade ago, Robinson et al.12 covalently immobilized a hCG antibody on the surface of glassy carbon electrodes to establish a sandwich assay with a second antibody conjugated to the enzyme glucose oxidase. In their work, (dimethylaminomethyl)ferrocene was used to mediate the electron-transfer process in the oxidation of glucose oxidase. The sensitivity of such a biosensor was estimated to be 9 mIU mL-1 (Note that 1 mg ≡ 9825 international units (IU).). However, the response curve of the biosensor showed a saturation point at a relatively low hCG concentration of 75 mIU mL-1. In an alternative design, Robinson et al.13 attempted to coimmobilize both glucose oxidase and the hCG antibody onto a glassy carbon electrode. The binding of hCG to the antibody was used to modulate the electrochemical current arising from the catalytic activity of the enzyme, in the presence of (dimethylaminomethyl)ferrocene and glucose. The sensitivity was then enhanced to 7 mIU mL-1. Similarly, Thompson et al.14 described an immunoassay in which hCG is sandwiched between a capture antibody and a second urease-labeled antibody. Following an urease-catalyzed reaction of urea, conductance of hCG in serum solutions was measured. Concentrations as low as 11 mIU mL-1 hCG with a 30-s rate measurement were detected. Duan and Meyerhoff15 designed an electrochemical detection system for hCG in which the glycoprotein was sandwiched between a capture antibody immobilized on a microporous membrane gold electrode and an alkaline phosphatase-labeled anti-hCG. The enzyme substrate, 4-aminophenyl phosphate, was introduced through the backside of the porous membrane to facilitate immediate oxidation of the enzymatically generated product, 4-aminophenol, at the gold electrode. Such a separation-free electrochemical immunoassay exhibited a detection limit of 2.5 mIU mL-1. Unfortunately, the limit of linearity in the calibration plot was reached at a relatively low hCG concentration of approximately 40 mIU mL-1. More recently, Shiku et al.16 carried out a sandwich assay for hCG using scanning electrochemical microscopy. In this work, the tunneling current was measured as a result of the oxidation of ferrocenylmethanol ion generated by the horseradish peroxidase-catalyzed reaction between ferrocenylmethanol and hydrogen peroxide. A detection limit of 100 mIU mL-1 and a limit of linearity of 300 mIU mL-1 were reported. In the manufacture of biosensors, conducting polymers17-19 (e.g., polypyrrole, polythiophene) are often used to act as a transduction matrix support enabling the desired chemical reactions to take place on the electrode surface prior to electrode reactions. Similarly, entrapment of enzymes on a biosensor surface is often carried out using ionomers such as Nafion.20-22 Notably, (12) Robinson, G. A.; Cole, V. M.; Rattle, S. J.; Forrest, G. C. Biosensors 1986, 2, 45. (13) Robinson, G. A.; Cole, V. M.; Forrest, G. C. Biosensors 1987/88, 3, 147. (14) Thompson, J. C.; Mazoh, J. A.; Hochberg, A.; Tseng, S. Y., Seago, J. L. Anal. Biochem. 1991, 194, 295. (15) Duan, C.; Meyerhoff, M. E. Anal. Chem. 1994, 66, 1369. (16) Shiku, H.; Hara, Y.; Matsue, T.; Uchida, I.; Yamauchi, T. J. Electroanal. Chem. 1997, 438, 187. (17) Vidal, J.-C.; Garcia, E.; Castillo, J.-R. Bionsens. Bioelectron. 1998, 13, 371. (18) Fare, T. L.; Cabelli, M. D.; Dallas, S. M.; Herzog, D. P. Bionsens. Bioelectron. 1998, 13, 459. (19) Rohde, E.; Dempsey, E.; Smyth, M. R.; Vos, J. G.; Emons, H. Anal. Chim. Acta 1993, 278, 5. (20) Liu, H.; Yang, T.; Sun, K.; Li, H.; Qi, D. Anal. Chim. Acta 1997, 344, 187.

Nafion is a polyanionic perfluorosulfonated ionomer with permselectivity due to accumulation of large hydrophobic cations rather than small hydrophilic ones. In general, acting as a matrix support, Nafion offers the advantage of speed and ease in the immobilization procedure. Nonetheless, Nafion has only recently been employed to immobilize antibody on the surface of electrochemical biosensors. For example, competitive immunoassays at Nafioncoated electrodes23 were developed for the detection of the antiepileptic drug, phenytoin. In this paper, we describe a fabrication methodology of an amperometric immunosensor for the detection of hCG involving the use of Nafion as an entrapment medium for antibody on an electrode surface. Initially, we investigated the effects of several experimental parameters on the binding between hCG and its antibody in solution and at the electrochemical immunosensor surface. The performance of this Nafion-coated electrochemical immunosensor in a sandwich assay of hCG analyte solutions was then demonstrated. EXPERIMENTAL SECTION Reagents. Purified murine monoclonal anti-hCG immunoglobulin 1 (IgG1) antibody 1 (Ab1) and anti-hCG IgG1 antibody 2 (Ab2) were prepared as described previously.24 Human chorionic gonadotrophin (hCG) was purchased from UCB Bioproducts (Brussels, Belgium), prepared in deionized water (purified by a Milli-Q water system), and stored at -20 °C. Horseradish peroxidase (EC 1.111.7, activity approximately 335 units/mg) was purchased from Sigma. Nafion perfluorinated ion-exchange powder supplied as a 5 wt % mixture in lower aliphatic alcohols and 10% water was purchased from Aldrich. Absolute ethanol was used throughout unless specified otherwise. A pH 7.6 phosphatebuffered saline (PBS), consisting of 10 mM phosphate (KH2PO4/ Na2HPO4), 150 mM NaCl, 10 mM EDTA (disodium salt), and 0.1% sodium azide, was used throughout. However, azide-free PBS was used in the presence of antibody conjugate because azide can deactivate the enzyme horseradish peroxidase. All bovine serum albumin (BSA)/PBS solutions used were prepared by addition of 0.5% (w/v) BSA in PBS unless otherwise stated. Ab2-horseradish peroxidase conjugate was prepared as described previously.25,26 Purification of the conjugates was achieved in a Sephacryl S-200 column (Pharmacia) using azide-free PBS as the elution buffer. Fractions containing suitable conjugated molecules were diluted in 1% BSA/PBS (azide free), aliquoted, and stored at 4 °C. All chemicals used were of analytical grade unless otherwise stated, and all aqueous solutions were prepared with Milli-Q deionized water. Human blood sera were obtained from St. Vincent’s Hospital (Sydney, Australia). Serum samples containing no hCG were pooled, aliquoted, and stored at -20 °C. (21) Smyth, M. R.; Manning, F.; O’Fagain, C.; O’Kennedy, R.; Deasy, B. Anal. Proc. 1994, 31, 13. (22) Fortier, G.; Vaillancourt, M.; Belanger, D. Electroanalysis 1992, 4, 275. (23) Le Gal La Salle, A.; Limoges, B.; Rapicault, S.; Degrand, C.; Brossier, P. Anal. Chim. Acta 1995, 311, 301. (24) Stuart, M. C.; Underwood, P. A.; Harman, D. F.; Payne, K. L.; Rathjen, D. A.; Razziudin, S.; Von Sturmer, S. R.; Vines, K. J. Endocrinol. 1983, 98, 323. (25) Tijssen, P.; Kurstak, E. Anal. Biochem. 1984, 136, 451. (26) Wilson, M. B.; Nakane, P. K. Recent developments in the periodate method of conjugating horeradish peroxidase to antibodies. In Immunofluorescence and related staining techniques; Knapp, W., Holubar, K., Wick, G., Eds.; Elsevier: New York, 1978; pp 215-224.

Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

4089

Preparation of 125I-Labeled hCG. Five micrograms of hCG was labeled with sodium [125I] iodine (ANSTO, Sydney, Australia) using the Chloramine T technique.27 The reaction was quenched by addition of sodium metabisulfite. The reaction mixture was transferred to a G-25 Sephadex size exclusion column (Pharmacia) and fractionated. Fractions containing 125I-labeled hCG were aliquoted and stored at -20 °C until required. Before use, [125I]hCG was purified in an Ab-1 immunoaffinity chromatography column.28 Electrode Preparation. Glassy carbon electrodes were initially polished smooth with a P-1200 emery paper. After being rinsed with water, the electrode was polished with 1.0-, 0.3-, and 0.05-µm alumina powder to a mirror finish, sonicated in water for 1 min, rinsed, and allowed to dry in air. Two methods of immobilization of antibody onto glassy carbon electrodes were investigated. In coimmobilization, the casting solution was prepared by diluting the Nafion stock solution with PBS. A 10-µL aliquot of Nafion/Ab1 solution was then spread onto the electrode surface and allowed to evaporate in air for 35 min. Dry electrodes were rinsed with water before use. In the second methodology, an adsorption immobilization technique was investigated. In this technique, 5 µL of 1% Nafion (in PBS) was cast onto a clean electrode and was allowed to dry. An equivalent volume of antibody was spread over the dry Nafion film and allowed to evaporate in air until dry. An additional coat of Nafion was placed on top of the dried antibody to assist in maintaining the antibody on the electrode surface. Immunological Activity of Immobilized Antibody. The availability of antibody binding sites on an electrode immobilized with Ab1 was determined by incubation of the prepared electrode in 250 µL of 125I-labeled hCG (224 000 counts min-1 (cpm)/300 mIU mL-1). After a 1-h incubation, the electrode was washed with deionized water for 1 min. Antibody-bound hCG was quantified by counting on the electrode surface in a LKB-Wallac 1260 Multigamma II gamma counter. Instrumentation. A 10-mL electrochemical cell accommodated with a three-electrode system was used throughout. In this system, the working electrode is a 3-mm-diameter glassy carbon electrode, an Ag/AgCl reference electrode (both purchased from BioAnalytical Systems, West Lafayette, IN), and a platinum wire auxiliary electrode. All electrochemical experiments were performed with a MacLab/4e Potentiostat (ADInstruments Pty Ltd, Castle Hill, Australia). Electrochemical conditions were controlled using a Macintosh computer using EChem v1.3.1 and Chart v3.4.3 software. Immunosensor Assay. Antibody immobilized onto glassy carbon electrodes was washed in water before incubation with 250 µL of hCG (in 0.2% BSA/PBS) for 1 h at room temperature. Electrodes were then rinsed in water to remove unbound hCG and incubated in 250 µL of conjugate (1 µg mL-1 in 0.2% BSA/ azide-free PBS) for 1 h. This corresponds to a sandwich immunoassay configuration on the immunosensor. Following the removal of unbound conjugate, the amount of enzyme-labeled antibody bound to the electrode surface was determined using the electrochemical detection procedure described below. (27) Greenwood, F. C.; Hunter, W. H. Biochem. J. 1963, 89, 114. (28) Stuart, M. C.; Boscato, L. M.; Underwood, P. A. Clin. Chem. 1983, 29, 241.

4090 Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

Electrochemical Measurements for the Immunosensor. All electrodes were stored in supporting electrolyte prior to use. Batch experiments were conducted at room temperature using 0.1 M phosphate buffer as the supporting electrolyte. The electrolyte solution was purged with nitrogen gas for 10 min before use. The following reactions are involved in determining the quantity of horseradish peroxidase at the electrode surface.29 horseradish peroxidase

hydroquinone + hydrogen peroxide 98 benzoquinone + H2O -250 mV vs Ag/AgCl

benzoquinone + 2H+ + 2e- 98 hydroquinone Cyclic voltammograms (not shown) for the reduction of benzoquinone at a glassy carbon electrode in the phosphate buffer depicted a peak potential at approximately -130 mV against Ag/ AgCl. Chronoamperometric experiments were then performed at an applied potential of -250 mV against Ag/AgCl electrode. In our experiments, freshly prepared hydroquinone in 0.1 M phosphate buffer was injected into the electrochemical cell to reach a final concentration of 1 mM. Similarly, H2O2 substrate was added to yield a final concentration of 176 µM. This concentration of H2O2 was determined by an experiment in which the largest current signal was obtained at a glassy carbon electrode in the presence of 0.1 M hydroquinone in phosphate buffer. The benzoquinone produced was reduced at -250 mV vs the Ag/AgCl electrode. The resultant current was initially measured for 10 min in the absence of H2O2 substrate. After the addition of substrate, the current was again measured for a further 10 min. The electrode response is defined as the difference in current before and after the addition of H2O2. Note that this time duration is intentionally employed to ensure maximum enzyme catalytic effects on the reactions. Analysis involving human blood serum was carried out by spiking the serum with a known quantity of hCG, followed by measurement of the cathodic current response as described above. RESULTS AND DISCUSSION Binding of hCG and Antibody. Two different antibodies, Ab1 and Ab2, were used in the present study. Stuart et al.24 previously characterized these antibodies and determined that Ab1 exhibits specific binding to free β-subunit and intact hCG dimers, while Ab2 recognizes the intact hCG molecule only. In addition, high binding affinities24 of 1.3 × 1010 and 0.91 × 1010 L mol-1 were estimated for Ab1 and Ab2, respectively, yet both showed low to insignificant cross reaction (1.9 and 4.2% with human luteinizing hormone, respectively) with other pituitary glycoprotein hormones. These are important criteria for consideration in the selection of antibodies employed in immunoassays in order to achieve maximum specificity. In this work, the binding between hCG and antibodies Ab1 and Ab2, respectively, in solution was initially investigated by radioimmunoassay. The results obtained after an incubation period of 1 and 24 h are depicted in Figure 1. In general, hCG binding increases as the concentration of Ab1 and Ab2 is increased. The monotonic relation between hCG (29) Dominguez Sanchez, P.; Tunon Blanco, P.; Fernandez Alvarez, J. M.; Smyth, M. R.; O’Kennedy, R. Electroanalysis 1990, 2, 303.

Table 1. Effect of the Presence of Ethanol on Ab1-[125I]hCG Binding Determined by Radioimmunoassay of a System Consisting of 25% (v/v) 125I-labeled hCG and 25% (v/v) Ab1 (Prepared in BSA/PBS) % (v/v) ethanol

% [125I]hCG binding

0 25 50 75 100

41.2 46.1 38.8 21.7 4.4

Table 2. Effect of Nafion on Binding of 125I-Labeled hCG to Ab1 in the Presence of 88 µg mL-1 Ab1 % (v/v) Nafion solution

% [125I]hCG binding

1 2 3 4 5

30.4 29.3 3.1 0.8 0

binding and antibody concentration in the plots is also indicative that both Ab1 and Ab2 exhibit a monoclonal binding characteristic toward hCG, as expected. Note that binding was still achieved at low antibody concentrations (e.g., 500 pg mL-1), indicating a high specificity of Ab1 and Ab2 toward hCG. In this study, very similar binding percentage is obtained after a 1- and a 24-h incubation period. For example, at an antibody concentration of 1.4 µg mL-1, a binding of 48.6 and 46.3% between hCG and Ab1 and Ab2, respectively, was obtained over a 1-h incubation period, compared to 54% for Ab1 and 44% for Ab2 after a 24-h incubation period. Although a 1-h incubation period appears to be adequate to obtain near-maximum binding for hCG with both Ab1 and Ab2, a 24-h incubation period has been used in the following experiments, unless specified otherwise, to ensure sufficient antibody-hCG equilibrium in the immunosystem. In the present work, antibody Ab1 was chosen to be immobilized on the surface of the immunosensor owing to its ability to bind to both free β-subunit and intact hCG dimers. This characteristic feature of Ab1 will ensure the capture of both forms of hCG, which are the major components in serum obtained during pregnancy and in trophoblastic disease.30 This enhanced binding with hCG in an analyte solution will then aid in increasing detection capability of the immunosensor. Here, Nafion was being employed as an entrapment medium. Nafion is supplied in lower aliphatic alcohols with ethanol being commonly used as a solvent in immobilization experiments.22 It is thus necessary to examine

the effects of ethanol on the binding between hCG and Ab1. In such a study, 25% (v/v) 125I-labeled hCG and 25% (v/v) Ab1 (prepared in BSA/PBS) were incubated for 24 h in ethanol-BSA/ PBS mixture solutions, with the ethanol composition varying between 25 and 100% (v/v). The binding between hCG and Ab1 was then determined by radioimmunoassays. The results are as shown in Table 1. Here, the blank solution contained only BSA/ PBS. A similar binding percentage (approximately 40-46%) between 125I-labeled hCG and Ab1 was observed in the presence of e50% ethanol. However, the percentage binding is diminished as the composition of ethanol exceeds 50%. It is speculated that the presence of ethanol in the solutions has reduced the mobility of both antibody and antigen, thus inhibiting the interaction between hCG and Ab1. Further, in these experiments, the intermolecular attractive forces (e.g., electrostatic interactions, van de Waals bonds, hydrophobic interactions) between hCG and Ab1 could have become weaker, giving rise to a reduction in the affinity of Ab1 for hCG. Experimentally, immediate precipitation of proteins was observed at >50% ethanol, possibly caused by deactivation of the proteins at such a level of ethanol in the system. This would clearly discourage a close contact between hCG and Ab1, leading to a gradual decrease in binding, so much so that only a low value of 4% was obtained in pure ethanol. Such results are in agreement with a study reported by Weetall31 in which the time for antibody-antigen interaction to reach equilibrium was found to increase from 6 h in an aqueous medium to 19 h in hexane. In another experiment, 88 µg mL-1 Ab1 was added to five separate Nafion solutions (in ethanol) with compositions ranging from 1 to 5%, followed by incubation of the mixtures with 100 µL of labeled hCG. Immunoassays of these solutions were carried out to investigate the effects of Nafion on the binding between hCG and Ab1. The results are tabulated in Table 2. As expected, an increase in the amount of Nafion was found to reduce binding because the ionomer would have prohibited a close interaction

(30) Cole, C. A.; Kardana, A. Clin. Chem. 1992, 38, 263.

(31) Weetall, H. H. J. Immunol. Methods 1991, 136, 139.

Figure 1. Radioimmunoassay of antibody (a) Ab1 and (b) Ab2 binding to 125I-labeled hCG over a 1- and 24-h incubation period.

Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

4091

Table 3. Effect of pH of the Buffer Solution Used in Coimmobilization of Ab1 and Nafion

a

pH of immobilization buffer

% [125I]hCG binding

4 6 7 7.6 9 10

0.86 ( 0.14a 0.60 ( 0.14a 2.20 ( 0.14a 2.25 ( 0.14a 0.48 ( 0.14a 0.65 ( 0.14a

Represents a 95% confidence intervals.

between hCG and Ab1 for binding. In fact, at the concentration of Ab1 used here, 5% Nafion was found to almost completely prevent binding between Ab1 and labeled hCG. Interestingly, the presence of 1 and 2% Nafion was both found to produce approximately half of the original binding percentage. Based on the results obtained in the above two experiments (see Table 1 and Tabel 2), it is clear that both ethanol and Nafion have a synergistic effect on the binding between Ab1 and 125I-labeled hCG. Such results need to be taken into consideration when Nafion is used as an immobilization medium for antibodies. Immobilization of Ab1 on the Electrode Surface. In this work, immobilization of Ab1 on the surface of a glassy carbon electrode was carried out by two methods, coimmobilization and adsorption immobilization. In the former, a mixture of Ab1 and Nafion solution was left to dry on the surface of a glassy carbon electrode. In adsorption immobilization, Ab1 was sandwiched between two thin Nafion films on the electrode surface. Coimmobilization Method. We initially investigated the optimum pH for immobilization in the coimmobilization method. In this experiment, antibody immobilization was conducted at pH values varying from 4.0 to 10.0, followed by determinations of the binding between labeled hCG and the immobilized Ab1. The results are shown in Table 3. A maximum binding of 2.2% was evident at pH 7-7.6. At pH values lower than 6.0 and higher than 7.6, the binding of 125[I]hCG was reduced by 50% compared to neutral pH. The effect of the amount of Ab1 on binding with hCG in the coimmobilization technique was investigated. As described above, a higher binding percentage was obtained in the presence of 1 and 2% Nafion solution. Hence, the amount of binding obtained in 1 and 2% Nafion solution as a function of concentration of Ab1 was determined. The results are shown in Figure 2. Clearly, enhanced binding (approximately 3%) was obtained in 20 µg mL-1 Ab1 in both 1 and 2% Nafion with no significant difference between the two Nafion solutions. At both higher (40 µg mL-1) and lower (1 µg mL-1) antibody loading, a lesser extent of [125I]hCG binding was evident. When a lower concentration of antibody was used, [125I]hCG binding was most likely to be lower due to limited availability of antibody. However, when a higher antibody concentration was used, low binding was most likely due to an overloading of Nafion matrix with protein. On the basis of these results, an optimum binding of hCG was achieved with a 1 or 2% Nafion solution containing 20 µg mL-1 antibody. We have also investigated the effect of PBS, instead of ethanol, as a diluent for Nafion from 5 to 1% on [125I]hCG binding with Ab1 immobilized on the electrode surface. When ethanol alone 4092 Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

Figure 2. Dependence of 125I-labeled hCG-Ab1 binding on the concentration of Ab1 in the presence of 1 (2) and 2% (4) Nafion, respectively, used in the coimmobilization method.

Figure 3. Effect of the presence of PBS as a diluent for Nafion on the binding between 125I-labeled hCG and Ab1.

was used as a Nafion diluent, the drying time of the antibody/ Nafion film was 15 min. The use of PBS had increased the drying time to 35 min. Figure 3 shows a plot of percentage binding of hCG against percentage of PBS used. An increase in the amount of PBS in the diluent was found to increase antibody at the surface, leading to an increase in hCG binding from 1% in 100% ethanol to 2.5% when PBS was used. Even though these results indicate that immobilization was improved in the presence of PBS, this value is not significantly different from the 3% binding obtained in Figure 2. Adsorption Immobilization Method. In the second method, adsorption immobilization (see above) was employed to entrap antibody onto the surface of a glassy carbon electrode. Note that only a 1-h incubation period was used in all adsorption immobilization experiments because there was no significant difference in the binding percentage compared to that obtained over a 24-h incubation period. Adsorption time of a 100-ng drop of Ab1 on a glassy carbon electrode surface was varied from 15 min to 1 h. During the shorter adsorption times (between 15 and 30 min), only 0.9% of labeled hCG was capable of binding to immobilized antibody. When adsorption times were increased to longer than

Figure 5. Schematic representation of the electrochemical immunosensor for hCG detection.

Figure 4. Dependence of 125I-labeled hCG-Ab1 binding on the volume of 1% Nafion used in the adsorption immobilization method.

45 min, 1.5% of [125I]hCG was found to bind to the antibody. Nonspecific binding of hCG to the electrode surface was 0.05%. These results demonstrate that it is necessary for the solution to dry on the electrode to obtain a higher antibody immobilization. In the adsorption immobilization method, 100% PBS was also used as a diluent to immobilize the Nafion film. Antibody (100 ng of Ab1) was added and allowed to dry on the Nafion film for 45 min. Binding between immobilized Ab1 and [125I]hCG was then determined. A 2.9% binding was estimated in the presence of Nafion + PBS, compared to 1.5% obtained in Nafion + ethanol above. In an attempt to increase the quantity of antibody available on the electrode, a second Nafion film was introduced on top to prevent loss of adsorbed antibodies from the electrode surface. This was carried out by varying the volume of 1% Nafion (in PBS) introduced on the electrode surface from 1 to 5 µL with a 1-µL increment. This additional film of Nafion was dried, followed by the determination of hCG binding. Figure 4 shows a plot of percentage binding of hCG against volume of 1% Nafion. In general, hCG binding increased as a function of the thickness of the dry Nafion, indicating that more immobilized antibody was available on the electrode surface. A maximum binding of 5.5% hCG was obtained when 4 µL of 1% Nafion was spread over adsorbed antibody. However, this binding was reduced when >4 µL of 1% Nafion was used in the experiment. From the above studies, it is apparent that the adsorption immobilization technique has resulted in approximately twice the quantity of hCG binding to Ab1 immobilized on the electrode compared to the coimmobilization technique. Adsorption of antibodies onto Nafion alone was insufficient to increase the quantity of binding sites, but the addition of a thin second coat of Nafion has increased the quantity of antibody available on the electrode. This is likely to enhance the detection limit and the dynamic range of the immunosensor. Hence, the adsorption immobilization technique was adopted here to construct an electrochemical immunosensor for hCG. Immunosensor Performance. Following the adsorption immobilization of antibodies on the glassy carbon electrode, the assemblage was incubated in a hCG analyte solution and then in

Figure 6. Calibration plot showing the electrochemical immunosensor response to increasing levels of hCG.

a solution containing the antibody Ab2, which has been conjugated to the enzyme horseradish peroxidase. This corresponds to the configuration of a two-site sandwich immunoassay in which Ab2 binds to both the hCG dimer and free β-subunit, which have been captured by Ab1.32 A schematic diagram of the design of the immunosensor is depicted in Figure 5. In this work, the enzyme-catalyzed electrochemical reduction of benzoquinone was used for quantitative determination of hCG in the analyte solutions. Initially, antibody Ab1 immobilized on the electrode surface was allowed to bind with hCG in an analyte solution, which was then sandwiched by a second antibody Ab2. An increase in concentration of added hCG would lead to a proportional increase of hCG bound to Ab1. In turn, there would be a proportional increase of enzyme conjugated Ab2 at the outer layer of the assemblage bound to the hCG, giving rise to an increase in the electrochemical reduction current of benzoquinone, which is quantitatively related to the amount of hCG present in an analyte solution. We examined the performance of the immunosensor in a series of hCG standard solutions. Figure 6 shows a plot of the electrochemical reduction current measured at the immunosensor as a function of increasing concentrations of hCG. The response of the sensor increased with increasing hCG concentration (cathodic current ) 3.2 ( 5.8 (nA mL mIU-1)[hCG concentration (mIU mL-1)] + 71.5 ( 343.8 (nA), where the errors here represent the 95% confidence intervals; correlation coefficient, 0.92, N ) 3), with the response only reaching a plateau when the (32) Boscato, L. M.; Egan, C. M.; Stuart, M. C. J. Immunol. Methods 1989, 117, 221.

Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

4093

Figure 7. Calibration plot showing the electrochemical immunosensor response to increasing levels of hCG present in a human serum sample.

hCG concentration approached 500 mIU mL-1. This represents a useful concentration range of hCG nearly 7 times wider than that reported by several previous workers.13-16 In addition, the detection limit of the immunosensor has been determined to be (33) hCG MAIAclone (Code 12304) product leaflet, Serono Diagnostics Ltd., 21 Working Business Park, Albert Drive, Working, Surrey, GU21 5JY, U.K.

4094 Analytical Chemistry, Vol. 71, No. 18, September 15, 1999

approximately 10 mIU mL-1 (defined as twice the standard deviation of the blank solution), which also compares favorably with previous work.13-16 Next, the performance of the immunosensor was examined in real serum sample solutions containing no hCG. In this experiment, by adopting a standard additions calibration method, pure hCG solutions were introduced into this serum solution and the corresponding cathodic current was then measured. The calibration plot obtained is shown in Figure 7. A linear relation (cathodic current ) 0.71 ( 0.29 (nA mL mIU-1)[hCG concentration (mIU mL-1)] + 5.8 ( 16.8 (nA), where the errors here represent the 95% confidence intervals; correlation coefficient, 0.98, N ) 4) is still obtained, however, the dynamic range has now been reduced to approximately 200 mIU mL-1. A detection limit of 11.2 mIU mL-1 based on twice the standard deviation of the blank solution is estimated. Note that the current response obtained in serum solutions is reduced compared to hCG standard solutions, likely to be due to electrode fouling often encountered in electrochemical detection of biological samples. Despite a narrower dynamic range in the analysis of real sample solutions, this level of detection is well below the threshold of 25 mIU mL-1 hCG in serum considered necessary for reliable pregnancy diagnosis.33

Received for review November 6, 1998. Accepted June 24, 1999. AC981216A