Amperometric Immunosensors Based on Rigid Conducting

Anal. Chem. 1997, 69, 2080-2085

Amperometric Immunosensors Based on Rigid Conducting Immunocomposites Marta Santandreu, Francisco Ce´spedes, Salvador Alegret, and Esteve Martı´nez-Fa`bregas*

Grup de Sensors & Biosensors, Departament de Quı´mica, Universitat Auto` noma de Barcelona, 08193 Bellaterra, Catalonia, Spain

Novel polishable immunosensors based on rigid biocomposite materials have been constructed. These biocomposites contain graphite powder, rabbit IgG, and methacrylate or epoxy resins. This material acts as a reservoir for the biological molecules and as a transducer at the same time. In order to study the potential analytical properties of this new type of material, a competitive binding assay was developed to determine the RIgG present in a sample with the aid of goat anti-rabbit IgG labeled with alkaline phosphatase. Using phenyl phosphate as a substrate, the phenol produced by the enzymatic reaction was amperometrically detected at 800 mV (vs Ag/AgCl). The surface of the immunosensor can be regenerated by simply polishing, obtaining fresh immunocomposite ready to be used in a new competitive assay. The need to generate fast, reliable, and precise analytical information on biomedical, industrial, or environmental processes has resulted in an intensive search for more selective systems of molecular recognition. This selectivity can be provided by biological systems such as antigen-antibody pairs. Immunochemical methods1-4 couple high selectivity with highly sensitive measurement systems, such as voltammetric techniques.5,6 These methods involve the use of antibodies labeled with enzymes that are capable of generating electroactive species,7,8 providing more robust, sensitive, and selective analytical methodologies. The immunochemical materials can be integrated within the electrochemical system to yield robust and compact immunosensors.9,10 The biological material is immobilized on the transducer surface, by means of a support (membrane), a physical interaction, or a direct chemical bond. The stability of this immobilization will determine the sensitivity and reproducibility of the resulting device. The use of immunosensors require washing steps, so immunological material must be perfectly immobilized in order to avoid losses. Adsorption has been the widely used technique

because of the simplicity of the procedure involved. However, the adsorbed biomaterial could leach off from the surface and limit the sensitivity and reproducibility of the assay.7 The use of adsorbed immunoreagents have therefore been confined to singleuse and disposable immunosensors. Wang et al.11,12 proposed a new immobilization technique based on the incorporation of enzymes in the bulk of a conducting composite material. At the same time, our working group developed a great deal of conducting biocomposites based on different polymeric rigid matrices13-17 and enzymes.18-22 These materials, which have important electrochemical features, are adequate for the construction and development of enzymatic amperometric biosensors. This work presents a novel technique for the construction of immunosensors. One component of the immunological pair is easily incorporated in the bulk of rigid graphite-polymer composites which act as reservoirs of this immunological material. Such immobilization in the matrix of the composite transducer has interesting advantages, especially in reference to carbon paste immunosensors,2 which are characterized by limited physical and chemical stability. The analytical potential of the immunocomposite electrode is illustrated through a competitive binding assay for rabbit IgG, using alkaline phosphatase-labeled antibody. EXPERIMENTAL SECTION Apparatus. Amperometric measurements were performed with a 641 VA-Detector amperometric unit (Metrohm) connected

(1) Holme, D. Analytical Biochemistry; Longman: New York, 1993; Chapter 7. (2) Van Emon, J. M.; Lopez-Avila, V. Anal.Chem. 1992, 64, 79A-88A. (3) Open Universiteit (The Netherlands) and University of Greenwich (Thames Polytechnic, U.K.). Technological Applications of Immunochemicals; Biotechnology by Open Learning, Betterworth-Heinemann, Ltd: Oxford, U.K., 1994. (4) Cooper, T. G. Instrumentos y te´ cnicas de bioquı´mica; Reverte´: Barcelona, 1984; Chapter 8. (5) Heineman, W.; Halsall, H. B. Anal. Chem. 1985, 57, 1321A-1331A. (6) Wang, J. Electroanalytical technology in clinical chemistry and laboratory medicine; VCH Publishers: New York, 1988; Chapter 1. (7) Blake, C.; Gould, B. J. Analyst 1984, 109, 533-547. (8) Monroe, D. Anal. Chem. 1984, 56, 8, 920A-931A. (9) North, J. R. Trends Biotechnol. 1985, 3, 180-186. (10) Marco, M. P.; Gee, S.; Hammock, B. D. Trends Anal. Chem. 1995, 14, 341350.

(11) Wang, J.; Varughese, K. Anal. Chem. 1990, 62, 318-320. (12) Wang, J.; Gonza´lez-Romero, E.; Ozsoz, M. Electroanalysis 1992, 4, 539544. (13) Alegret, S.; Alonso, J.; Bartrolı´, J.; Martı´nez-Fa`bregas, E.; Valde´s-Perezgasga, F. In Uses of immobilized biological compounds; Guilbault, G. G., Mascini, M., Eds.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1993; pp 67-79. (14) Alegret, S.; Alonso, J.; Bartrolı´, J.; Ce´spedes, F.; Martı´nez-Fa`bregas, E.; Del Valle, M. Sens. Mater. 1996, 8, 147-153. (15) Alegret, S.; Ce´spedes, F.; Martı´nez-Fa`bregas, E.; Martorell, D.; Morales A.; Centelles, E.; Mun ˜oz, J. Biosens. Bioelectron. 1996, 11, 35-44. (16) Gala´n-Vidal, C. A.; Mun ˜oz, J.; Domı´nguez, C.; Alegret, S. Trends Anal. Chem. 1995, 14, 225-231. (17) Ce´spedes, F.; Martı´nez-Fa`bregas, E.; Alegret, S. Trends Anal. Chem. 1996, 15, 296-304. (18) Martorell, D.; Ce´spedes, F.; Martı´nez-Fa`bregas, E.; Alegret, S. Anal. Chim. Acta 1994, 290, 343-348. (19) Ce´spedes, F.; Martı´nez-Fa`bregas, E.; Alegret, S. Anal. Chim. Acta 1993, 284, 21-26. (20) Ce´spedes, F.; Martı´nez-Fa`bregas, E.; Alegret, S. Electroanalysis 1994, 6, 759-763. (21) Morales, A.; Ce´spedes, F.; Mun ˜oz, J.; Martı´nez-Fa`bregas, E.; Alegret, S. Anal. Chim. Acta 1996, 332, 131-138. (22) Ce´spedes, F.; Valero, F.; Martı´nez-Fa`bregas, E.; Bartrolı´, J.; Alegret, S. Analyst 1995, 120, 2255-2258.

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Figure 1. Construction of the amperometric immunosensor: (1) female connector formed by a 2 mm gold-plated beryllium-copper contact and a molding in polyimide, (2) Bakelite washer, (3) metallic nut, (4) circular piece cut from a conducting solid piece of copper (copper sheet) soldered to the connector end, (5) PVC tube (6 mm i.d., 18 mm length), and (6) final aspect of the supporting assembly with a cup of 3 mm thickness which seats the immunocomposite.

to a Digilab 517 potentiometer (Crison). Response curves were recorded with a Labograph E586 recorder (Metrohm). A platinum auxiliary electrode and a double-junction Ag/AgCl reference electrode (Orion 900200), with 0.1 M KCl as external reference solution, were used to characterize and evaluate the working electrodes. Cyclic voltammetry scans were performed in a PGSTAT20 Autolab (Eco Chemie), using the prepared composite materials as working electrodes, jointly with a reference electrode (saturated calomel electrode) and an auxiliary platinum electrode. Reagents. The immunological system formed by goat antirabbit IgG (whole molecule) alkaline phosphatase conjugate (GaRIgG) (A-7778) and rabbit IgG (RIgG) (I-5006) was obtained from Sigma (St.Louis, MO). Immunocomposites were prepared using graphite powder with a particle size of 50 µm (Merck), Epotek H77 epoxy resin and hardener from Epoxy Technology (Billerica, MA) or Sealer-Healer 1540 commercial methacrylate (monomer QM-57T-D) from Rohm and Haas (Croydon, England), and benzoyl peroxide (Fluka). Bovine serum albumin (BSA) was purchased from BDH, Tris(hydroxymethyl)aminomethane and EDTA from Merck, and potassium chloride from Fluka Chemie. Phenyl phosphate disodium salt was obtained from Sigma, and fresh solutions of this compound were prepared daily. Dialysis membranes used were obtained from a dialysis tube (Medicell International, London, U.K.) made of regenerated cellulose which contains glycerine, water, and ∼0.1% sulfur. All other reagents were of the highest grade available. Aqueous solutions were prepared using doubly distilled water. Construction of the Amperometric Immunosensors. The construction of the immunosensor is illustrated in Figure 1. Two different immunocomposites were prepared. An RIgG-methacrylate-graphite composite was prepared by mixing graphite and deaerated methacrylate monomer in a ratio of 1:1 (w/w) and then adding RIgG to obtain a final composition of 0.9% (w/w). The biocomposite obtained was cured at room temperature in a nitrogen atmosphere for three days. An RIgG-epoxy-graphite composite was also prepared. Graphite powder and epoxy resin were mixed in a ratio of 1:4

(w/w) and then enough RIgG was added to get a final immunocomposite containing 0.9% (w/w) RIgG. The resulting paste was placed in a PVC tube (6 mm i.d.) to a depth of 3 mm. The biocomposite material was cured at 40 °C during a week. When not in use, the immunosensors were stored at 5 °C. Regeneration of the Immunosensor Surface. Before each use, the surface of the electrode was wet with doubly distilled water and then thoroughly polished for a few seconds, first with abrasive paper and then with alumina paper (polishing strips 30144-001, Orion). The immunosensor surface was finally cleaned by sonication in doubly distilled water for 2 min. Preparation of Incubating Solutions. The methodology used in the incubating procedure was similar to the one described by Glazier and Rechnitz.23 A blocking buffer containing BSA 0.1% (w/v) in 0.1 M Tris-HCl and 0.001 M EDTA at pH 7.5 was used. RIgG standard solution was prepared by dissolving 7.48 mg of RIgG in 1 mL of blocking buffer. The commercial solution of GaRIgG-alkaline phosphatase conjugate was diluted using BSA 1% (w/v) prepared in the same Tris-EDTA buffer, obtaining a final concentration of 0.99% (v/v). A 0.1 M Tris-HCl, 0.1 M KCl pH 7.5 buffer was used as a washing solution. Competitive Binding Assay of Rabbit IgG. Figure 2 shows a schematic diagram of the procedure followed. Definite volumes of RIgG solution (0.83 mg/mL) were mixed with 300 µL of the solution of GaRIgG-alkaline phosphatase conjugate and enough 0.1% (w/v) BSA solution to give a final volume of 1200 µL. The mixture was incubated at room temperature for 30 min with continuous stirring. The immunosensor was immersed in the solution, and the system was allowed to stand for 2 h at room temperature. At the end of the period, the immunosensor was rinsed with the washing buffer. At this point, the biosensor should have had some conjugate bound to its surface. A piece of dialysis membrane was placed to cover the surface of the immunosensor in order to provide a diffusion barrier in the microenvironment of the electrode surface and contribute to an enhanced signal-tonoise ratio. This happens because the membrane stabilizes the signal and reduces the noise. The membrane is added after the incubation and before the calibration in phenyl phosphate. Amperometric Measurements of Phosphatase Activity. In order to quantify the amount of the antigen-antibody complex on the electrode surface, the activity of the alkaline phosphatase label in the antibody was measured through an amperometric technique. A three-electrode system and an applied potential of 0.8 V was used. Several increments of a stock solution of phenyl phosphate were added to 20 mL of a 0.1 M Tris-HCl, 0.1 M KCl buffer mixture (pH 7.5) to give solutions of increasing concentration. The steady-state response of the amperometric system was noted for each increment. RESULTS AND DISCUSSION Choice of Biocomposite. The incorporation of the biological material in a conducting composite enhances the performance characteristic of electrochemical biosensors.24 The biocomposite acts not only as a support for the biomaterial but also as a transducer. The product of the enzyme reaction is formed on the surface of the conducting composite and can therefore be detected immediately with great sensitivity. In our study, the RIgG immunosensors based on an epoxy and on a methacrylate composite, and incubated in a GaRIgG-alkaline (23) Glazier S. A.; Rechnitz, G. A. Anal. Lett. 1991, 24, 1347-1362. (24) Alegret, S. Analyst 1996, 121, 1751-1758.

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Figure 2. Procedure for the competitive binding immunoassay.

phosphatase solution, exhibited similar responses to phenyl phosphate. However, better reproducibility and a higher sensitivity were obtained with the methacrylate immunosensors. Deaeration of the methacrylate and curing the composite in a nitrogen atmosphere greatly improved the response characteristics of the methacrylate immunocomposite. This is because polymerizations based on acrylate groups are inhibited by oxygen, and when this procedure is followed, the quality and the reproducibility of the response is enhanced. Consequently, the methacrylate composites were used in the immunosensors developed for RIgG. Optimization of the Assay for the Enzyme-Label. Alkaline phosphatase catalyzes the hydrolysis of phenyl phosphate into phenol and phosphoric acid or phosphate ions, depending on the pH. The activity of the enzyme could be assayed through an amperometric measurement of the phenol produced by this reaction. The cyclic voltammogram obtained with phenol and phenyl phosphate, using a graphite-methacrylate electrode as the working electrode is shown in Figure 3. Phenol exhibited a welldefined oxidation plateau at ∼800 mV vs SCE, whereas phenyl phosphate showed no electrochemical activity. A potential of 800 mV was consequently used in the amperometric measurements. During successive measurements using the same surface of the immunocomposite sensor, the signal was observed to deteriorate when used continuously. This behavior could be attributed 2082

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to the fouling of the immunosensor, caused by the electropolymerization of phenolic radicals on the electrode surface.25,26 The use of an immunocomposite sensor provides a solution to this problem, since the surface of the immunosensor can be regenerated by polishing it to produce a fresh surface which exhibits a reproducible behavior. Once the immunosensor is incubated, the activity of the alkaline phosphatase enzyme label is affected by the pH of the assay medium. The attached enzyme label exhibited maximum activity at pH 9. In solution, this enzyme has the ability to hydrolyze phosphomonoesters at more basic pH.25,27,28 However, a pH of 7.5 was selected as the optimum pH, since at this pH the composite matrix is not physically altered by the working medium.18 A multiple addition of phenyl phosphate standard was done during the assay of the attached enzyme label. This method provided sufficient data for a kinetic evaluation of the enzyme activity. According to the Michaelis-Menten equation, the enzyme activity is proportional to the maximum velocity of the (25) Tang, H. T.; Lunte, C. E.; Halsall, H. B.; Heineman, W. R. Anal. Chim. Acta 1988, 214, 187-195. (26) Meusel, M.; Renneberg, R.; Spener, F.; Schmitz, G. Biosens. Bioelectron. 1995, 10, 577-586. (27) Thompson, R. Q.; Porter, M.; Stuver, C.; Halsall, H. B.; Heineman W. R.; Buckley, E.; Smyth M. R. Anal. Chim. Acta 1993, 271, 223-229. (28) Foulds, N. C.; Frew, J. E.; Green M. J. Biosensors: a practical approach; Cass, A. E. G., Ed., Oxford University Press: New York, 1990.

a

b Figure 3. Cyclic voltammograms for phenol 1.7 × 10-4 M (- - -) and phenyl phosphate 2.7 × 10-4 M (- ‚‚ -), using a graphitemethacrylate electrode in a Tris 0.1 M and KCl 0.1 M pH 7.5 buffered solution. Sweep rate is 0.05 V/s.

enzyme reaction. A typical Michaelis-Menten curve obtained during the assay of the enzyme label in an incubated immunosensor is shown in Figure 4a. The response at the plateau (Imax) corresponds to the maximum velocity of the enzyme reaction and is proportional to the activity of the enzyme and, consequently, to the amount of GaRIgG-alkaline phosphatase conjugate present in the surface of the immunosensor. A more accurate method of determining the maximum velocity (Imax) is through the Lineweaver-Burk method, which is based on the following equation: app

K M1 1 1 ) + I Imax Imax S Imax and KappM can be determined from the experimental data by plotting 1/I vs 1/S and determining the values for slope (Kapp/ Imax) and the y-intercept (1/Imax). Figure 4b shows the Lineweaver-Burk plot for the data used in Figure 4a. These values were used to start an iteration procedure for fitting the experimental data to the Michaelis-Menten equation. The kinetic evaluation of enzyme activity was employed because raw experimental measurements are subject to random experimental errors, and data obtained by adjustment to theoretical models are more significant since they include information of several measurements. Immunosensor Measurements. Figure 5 shows the calibration curve obtained for the biocomposite immunosensor. A linear dependence was observed between the response (Imax) and the concentration of RIgG in the initial incubating solution. The linear range includes RIgG concentrations from 0 to 0.014 mg/mL, which correspond to 0-87.5 nM RIgG in the incubating media. At higher RIgG concentrations, the amount of the bound antibody on the surface of the immunosensor is less, and therefore, the activity of the immobilized enzyme label will also be lower. As a result of low enzyme activity, saturation occurs at a low concentra-

Figure 4. Michaelis-Menten (a) and Lineweaver-Burk (b) adjustments for experimental data from the phenyl phosphate calibration curve using an incubated RIgG-graphite-methacrylate immunosensor, in a Tris 0.1 M, KCl 0.1 M pH 7.5 buffered solution. Applied potential 0.8 V vs SCE. Immunosensor incubated in a Tris-HCl 0.1 M, EDTA 0.001 M pH 7.5 buffered solution, containing 0.05 mg/ml RIgG (analyte), 0.33% (v/v) GaRIgG alkaline phosphatase conjugate and 0.1% (v/v) BSA.

tion of phenyl phosphate. Regarding precision, it can be observed that the dispersion of the results at high concentrations is high. This may be so because the quantity of marked antibodies (GaRIgG) capable of binding to the surface of the immunosensor is small. No loss of the immunological material to the solution was found to take place, since no change was observed in the activity of the immobilized enzyme label after immersing the immunosensor in a buffer solution of pH 7.5 using 0.1 M Tris, 0.1 M KCl for several hours. No adsorption of antibodies could occur on the surface of the immunosensor, since the antibody was found not to be adsorbed on a graphite-polymer composite. The RSD is 7% for the Imax values obtained with four different and newly polished surfaces of the same immunosensor after the corresponding incubation. Good reproducibility was obtained with two different immunosensors, as can be seen in Figure 6. In general, polishing of the immunosensor surface exposes a fresh reagent layer which exhibits responses of acceptable Analytical Chemistry, Vol. 69, No. 11, June 1, 1997

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Figure 5. RIgG calibration curve using Michaelis-Menten intensity values for a RIgG-graphite-methacrylate immunosensor after the competitive immunoassay. Error bars indicate standard deviation for each average. Regression: slope, -4.8 × 104; y-intercept, 847; r ) 0.98.

Figure 6. Standard RIgG calibration curve using corrected normalized Michaelis-Menten intensity values for two different RIgGgraphite-methacrylate immunosensors after the competitive immunoassay. (- - -) Regression for immunosensor 1: slope, -5.6 × 103; y-intercept, 105; r ) 0.95. (‚‚‚) Regression for immunosensor 2: slope, -4.6 × 103; y-intercept, 100.6; r ) 0.998.

reproducibility. This indicates that the distribution of the antigen in the bulk is uniform (also observed in enzyme biosensors18-22). This means that reproducible results can be obtained not only by using different surfaces of one single immunosensor but also by using different immunosensors. Similar values of KappM have been obtained for both polymers: 0.25 mM (RSD 8%) with epoxy and 0.10 mM (RSD 14%) with methacrylate immunosensors. This means that the interaction between enzyme and substrate is similar, regardless of the 2084 Analytical Chemistry, Vol. 69, No. 11, June 1, 1997

polymeric resins of the electrode surfaces used in the measurement. The enzyme is equally active and the immobilizing matrix or the sensing material being used does not have an influence. This demonstrates that RIgG’s from distinct fresh surfaces are equally active when binding to GaRIgG. These immunosensors are not characterized only by their easy regeneration attained by polishing their surfaces but also by their ability to preserve antibody species in the bulk of the composite during the lifetime of the device. Thus, the matrix behaves as a reservoir of biological material. On the other hand, KappM’s obtained are consistent with those found in the literature for the enzymatic detection of phenyl phosphate using alkaline phosphatase. There are not many bibliographical references on the kinetics of this enzyme when it is used as a label in immunoassays. Heineman et al.29 reported KappM values of 0.17 mM for an electrochemical enzyme immunoassay with alkaline phosphatase-labeled digoxin using a glassy carbon electrode. Additional references are found about its activity as a free enzyme. Walker and King30 reported a value of 0.6 mM for an electrochemical immunoassay procedure developed in solution. KappM values of 0.082 and 0.056 mM are reported31 for p-nitrophenyl phosphate and p-aminophenyl phosphate, respectively. The enzyme kinetic constants were evaluated in a pH 9 Tris-buffered solution using a flow injection amperometric system. A value of KappM of 0.027 mM is reported32 for 4-aminophenyl phosphate when it is detected in the same buffered solution also using a flow injection system. It may be concluded that the kinetics of the enzyme when it is used as a label of immunological assays is similar to the kinetics shown when it catalyzes reactions as a free enzyme in solution. CONCLUSIONS The immunocomposites described in this paper provide the immunosensors with excellent features to develop immunoassays. Their preparation is simple and carried out using dry chemistry techniques, so the resulting immunosensors are inexpensive and can be considered for single-use applications. The immunocomposites are highly moldable before curing, permitting the construction of amperometric immunosensors of different shapes and sizes. The construction procedure is compatible with thick-film technology. The immunocomposites are characterized by their mechanical stability and rigidity after curing, when their surface can be polished or mechanically altered. The components of the sensing surface can be controlled by adjusting their content in the bulk of the immunocomposite. The morphology, dimensions, and distribution of the conducting particles in the composite surface allow a microelectrode array behavior (efficient mass transport, high signal-to-noise ratio, fast response times, low detection limits).24 Furthermore, the close contact between the immunological material and the conducting sites on the immunosensor surface allows an effective detection of the product from the enzymatic reaction developed by the labeling enzyme. This considerably increases the signal and kinetically enhances the electronic transference. (29) Tang, H. T.; Lunte, C. E.; Halsall, H. B.; Heineman, W. R. Anal. Chim. Acta 1988, 214, 187-195. (30) Walker, P. G.; King, E. J. Biochem. J. 1950, 47, 93-95. (31) Thompson, R. Q.; Barone, G. C.; Halsall, H. B.; Heineman W. R. Anal. Biochem. 1991, 192, 90-95. (32) Thompson, R. Q.; Porter, M.; Stuver, C.; Halsall, H. B.; Heineman W. R.; Buckley, E.; Smyth M. R. Anal. Chim. Acta 1993, 271, 223-229

Immunoassay times are long because of the extended time required for incubation. Our present work is aimed at reducing the total time required for analysis. A higher detection range may be attained with immunocomposites of a larger retention capacity. This entails the use of materials that can be cured properly while a high density of immunological material is retained. An interesting advantage of these devices is their easy regeneration: their surface can be polished, obtaining fresh immunocomposite ready to be used in a new immunoassay. Each new surface yields reproducible results if all the individual components are homogeneously dispersed in the bulk of the immunocomposite. So, they are an alternative to the expensive and complex conventional procedures for the construction of immunosensors.

ACKNOWLEDGMENT M.S. acknowledges the support received from the Comissionat per a Universitats i Recerca of the Generalitat of Catalonia (FI/ 95-2117). This work has been funded by the European Comission, Directorate General for Science, Research and Development (XIID-1), Environmental Technologies (EV5V-CT94-0407), and the Comisio´n Interministerial de Ciencia y Tecnologı´a, Spain (BIO951196-CE and BIO96-0740). Received for review December 3, 1996. February 23, 1997.X

Accepted

AC961222B X

Abstract published in Advance ACS Abstracts, April 1, 1997.

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