Renewable Amperometric Immunosensor for Schistosoma japonium

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Anal. Chem. 2001, 73, 3219-3226

Renewable Amperometric Immunosensor for Schistosoma japonium Antibody Assay Guo-Dong Liu,† Zhao-Yang Wu,† Shi-Ping Wang,‡ Guo-Li Shen,† and Ru-Qin Yu*,†

College of Chemistry and Chemical Engineering, Institute for Chemometrics and Sensing Technology, Hunan University, Changsha 410082, People’s Republic of China, Department of Parasitology, Hunan Medical University, Changsha 410078, People’s Republic of China.

A renewable amperometric immunosensor has been proposed for the determination of Schistosoma japonium antibody (SjAb) in rabbit serum. A paraffingraphite-Schistosoma japonium antigen (SjAg) biocomposite, which needs no additional curing, was directly used to construct the immunosensors. The analytical sample containing the desired SjAb was mixed with SjAb labeled with horseradish peroxidase (HRP) to form the incubation solution for the competitive binding assay. Amperometry was used to determine the amount of HRP fixed on the sensor surface, which was related to the content of desired SjAb. Assay conditions were optimized, including the selection of substrate, the loading of SjAg in the biocomposite, the amount of labeled SjAb in the incubation solution, the incubation time, and the temperature. Using o-aminophenol (o-AP) as a substrate, amperometric detection at -200 mV (vs SCE) resulted in a pseudolinear detection range of about 0.36 to 14 µg/mL, with a detection limit of 0.36 µg/mL. Rabbit serum samples with varying infection degrees were analyzed, and the results demonstrated that the concentration that is detectable in this system meets the demands of clinical analyses. A new surface on the immunosensor for use in another competitive assay can be obtained by removing the original one and polishing the surface. Schistosoma japonium (Sj) is one of the widespreading parasitic diseases, mainly found in some parts of Africa, Asia, and Latin America,1 that are threatening human health. According to the statistics of World Health Organization (WHO), the disease threatened about 5.6 billion people in 76 countries; among them, about 1.6 billion persons were infected. Some diagnostic procedures, such as enzyme-linked immunosorbent assay (ELISA), electrophoretic immunoassays (EI), radio immunoassays (RIA), dot-immunobinding assay (DIBA), etc., have been adapted to clinical analyses.2,3 Most methods, unfortunately, require highly qualified personnel, tedious assay time, or sophisticated instru* Corresponding author. Fax: +86-731-8824525. E-mail: [email protected]. † Hunan University. ‡ Hunan Medical University. (1) Liu, Y. H.; Liu, X. X.; Song, C. C.; Yu, X. H. Immunology and Immunodiagnosis Parasitic Diseases (in Chinese), Jiangsu Science-Technology Press, Nanjing, China, 1991. (2) Maeda, M.; Tsuji, A. Anal. Chim. Acta 1985, 167, 241-248. (3) Bossuyt, X.; Bogaerts, A.; Schiettekatte, G.; Blankaert, N. Clin. Chem. 1998, 44, 760-766. 10.1021/ac0101048 CCC: $20.00 Published on Web 06/13/2001

© 2001 American Chemical Society

mentation. Until now, these methods were mainly used for qualitative and semiquantitative analysis for SjAb. According to clinical experience, only .∼90% of infected samples tested show positive results; a considerable percentage escaped the screening test. Searching for new, simple, and sensitive diagnostic methods with real-time output and a low cost is of considerable interest. In recent years, with the extension of the application field of electrochemical analysis, there has been growing interest in the development of electrochemical immunosensors for diagnostic assay of biological analytes.4-6 On the basis of a specific reaction of the antibody and antigen, immunosensors provide a sensitive and selective tool for the determination of immunoreagents. Here, the immunologic material is immobilized on a transducer; the analyte is measured through a label species conjugated with one of the immunoreagents. Enzymes such as cholinesterase,7 alkaline phosphatase,8-10 and horseradish peroxidase (HRP)11,12 are used extensively as labels, and the sensitivity is increased by chemical amplification of signals. One of the obstacles retarding the practical application of immunosensors is their reusability. The difficulty of repeated use of the sensing surface arises from the high affinity constants derived from the strong antigen-antibody reactions. The treatment of antigen-antibody interfaces at high ionic strength or acidic pH provides a means to dissociate the antigen-antibody complexes and eventually allows the regeneration of the sensing surface. The use of these methods is rather limited, however, because the extreme conditions often degrade or chemically deactivate the antigen surface.13 Furthermore, such a regeneration scheme is useful only for few measurement cycles. The development of disposable immunosensors is a possible way to circumvent (4) Heineman, W. R.; Halsall, H. B. Anal. Chem. 1985, 57, 1321A-1331A. (5) Hage, D. S. Anal. Chem. 1999, 71, 294R-304R. (6) Wang, J. Anal. Chem. 1999, 71, 328R-332R. (7) Babkina, S. S.; Medyantseva, E. P.; Budnikov, H. C.; Tyshlek, M. P. Anal. Chem. 1996, 68, 3827-3823. (8) Wehmeyer, K. R.; Halsall, H. B.; Heineman, W. R.; Volle, C. P.; Chen, I. W. Anal. Chem. 1986, 58, 135-139. (9) Kreuzer, M. P.; Sullivan, C. K.; Guilbault, G. G. Anal. Chim. Acta 1999, 393, 95-102. (10) Lu, B.; Iwuoha, E. I.; Smyth, M. R.; Kennedy, R. Anal. Chim. Acta 1997, 345, 59-66. (11) He, Y. N.; Chen, H. Y.; Zheng, J. J.; Zhang, G. Y. Talanta 1997, 44, 823830. (12) Niwa, O.; Xu, Y.; Halsall, H. B.; Heineman, W. R. Anal. Chem. 1993, 65, 1559-1563. (13) Blonder, R.; Levi, S.; Tao, G. L.; Ben-Dov, I.; Willner, I. J. Am. Chem. Soc. 1997, 119, 10467-10478.

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Figure 1. Configuration of amperometric immunosensor: (1) PVC nut, (2) PVC tube (6-mm i.d., 50-mm length), (3) conducting graphite rod, (4) biocomposite, (5) electric wire connected with graphite rod.

the problem, although it is undesirable that the prepared immunosensors are used for only one assay.14-17 Fa`bregas et al.18 first reported the design of renewable immunosensors using magnetic immunoparticles. This technique seems very attractive, although the construction of magneto-immunosensors requires sufficient experimental skills, and the immobilization of magnetic particles with immunologic material requires a relatively long period of incubation. Fa`bregas19 also reported renewable amperometric immunosensors based on rigid conducting immunocomposites consisting of graphite powder, rabbit IgG, and methacrylate or epoxy resins. The surface of immunosensors can be renewed by simply polishing to obtain a fresh layer of immunocomposite that is ready to be used in a new competitive assay. In this paper, we tried to use Fa`bregas’ approach with some modification of the binding material to construct immunosensors for Schistosoma japonium antibody (SjAb) assay. Instead of methacrylate or an epoxy resin, paraffin is used as the binding material to simplify the immunosensor preparation procedure, reduce the background current, and improve the sensitivity and reproducibility. The prepared immunosensors exhibit good physical and chemical stability, low background current, and a wide working potential range. The proposed paraffin-graphite-Schistosoma japonium antigen immunosensor was applied to the determination of SjAb using a competitive binding assay with HRPlabeled SjAb. EXPERIMENTAL SECTION Apparatus. Amperometric measurements and cyclic voltammetric experiments were performed using a model 273 electrochemistry system (EG & PARC). A three-electrode system consisted of an immunosensor, a saturated calomel reference electrode (SCE), and a platinum wire auxiliary electrode. All potentials are referenced to SCE. The cell with a stirrer bar was placed on a magnetic stirrer. A model CSS501 thermostat (Chongqing) was used to control the incubating temperature. Materials. A 32 kDa molecular antigen of Schistosoma japonium from adult worm antigen (AWA) was isolated and purified to homogeneity according to the reported method.20 The concentration of SjAg was 2.8 mg/mL. The aqueous solution was (14) Wang, J.; Tian, B. M.; Rogers, K. R. Anal. Chem. 1998, 70, 1682-1685. (15) Wang, J.; Pamidi, P. V. A.; Rogers, K. R. Anal. Chem. 1998, 70, 11711175. (16) Lee, K. S.; Kim, T. H.; Shin, M. C.; Lee, W. Y.; Park, J. K. Anal. Chim. Acta 1999, 380, 17-26. (17) Gonza´lez-Martı´nez, M. A.; Puchades, R.; Maquieira, A.; Ferrer, I.; Marco, M. P.; Barcelo´, D. Anal. Chim. Acta 1999, 386, 201-210. (18) Santandreu, M.; Ce´spedes, F.; Alegret, S.; Martı´nez-Fa`bregas, E. Anal. Chem. 1997, 69, 2080-2085. (19) Sole´, S.; Alegret, S.; Ce´spedes, F.; Fa`bregas, E.; Dı´ez-Caballero, T. Anal. Chem. 1998, 70, 1462-1467.

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evaporated under vacuum at a temperature near 0 °C to obtain a pale yellow dried powder. Schistosoma japonium antibody used as the calibration standard was prepared by immunizing rabbits for 45 days with Schistosoma japonium 2500. The antibody in the infected rabbit serum was then purified by precipitation from saturated ammonium sulfate solution, as described in the literature.21 The actual concentration of SjAb was determined by using the ELISA method. HRP (1000 U/mg Rz ) 3.0), HRP-SjAb (1200 U/mg Rz ) 3.0), and all other reagents and solvents were of analytical reagent grade. Double-distilled water was used throughout. Preparation of HRP-SjAb Conjugate. The conjugation was performed according to a modification of a reported method.1 The HRP (5 mg dissolved in 0.5 mL of 0.2 M acetate buffer at pH 5.6) was combined with 0.25 mL of 0.1 M NaIO4 and incubated for 30 min at 4 °C. Glycol (0.5 mL, 2.5% v/v) was added, and the solution incubated for an additional 30 min at room temperature. SjAb (5 mg) was added, and the pH of the solution was adjusted to 9.0 using sodium carbonate buffer. The resulting solution was allowed to stand overnight at 4 °C. NaBH4 (0.1 mL of 5 mg/mL solution) was slowly added, and the solution was allowed to incubate 2 h at 4 °C. The resulting solution was dialyzed against 0.1 M phosphate buffer (pH 7.4) overnight at 4 °C. Further purification was conducted by gel filtration on a Sepharose G-25 column to give the HRP-SjAb conjugate. Fabrication of Amperometric Immunosensors. The configuration of the immunosensor is illustrated in Figure 1. Three different immunocomposites were prepared. A paraffin-graphiteSjAg composite was prepared from graphite powder dispersed in paraffin in a weight ratio of 1:1, which was dissolved in a small amount of ether, with SjAg and bovine serum albumin (BSA) added at 1% (w/w) each to obtain a homogeneous paste. The latter was kept at room temperature for 10 min until the most of the ether had evaporated, and then it was squeezed into the PVC tube (Figure 1) to a depth of 1 cm. The preparation of methacrylategraphite-SjAg and epoxy-graphite-SjAg composites was similar to the one described by Fa`bregas et al.19 The biocomposites obtained were placed in PVC tubes (6 mm i.d.) to a depth of 1 cm. When not in use, the immunosensors were stored in a dry state at 4 °C. Renewal of the Immunosensors Surface. The immunosensor surface was renewed by turning the nut to extrude a 0.1-mmthick outer paste layer and by polishing with an alumina paper (0.05 µm) wetted with water to produce a smooth shiny surface. (20) Wang, S. P.; Zeng, X. F.; Yi, X. Y. Chinese J. Parasitol. Parasitic Dis. 1995, 13, 25-31. (21) Wang, S. P.; Zeng, X. F.; Yi, X. Y. Chinese J. Zoonoses 1992, 4, 14-16.

Figure 2. Procedure for the competitive binding immunoassay. Paraffin-graphite-SjAg immunosensor is incubated 50 min at 27 °C in the incubation solution containing SjAb and HRP-SjAb conjugate, then the immunosensor is rinsed thoroughly with a washing solution. Amperometric measurement is performed using a three-electrode system with an applied potential of -200 mV versus SCE. After the background current is stabilized, the response is subsequently recorded after the addition of o-AP-H2O2 solution.

Finally, the electrode surface was cleaned with double distilled water. Solutions. A blocking buffer containing 0.1% (w/w) BSA in 0.1 M tris-HCl and 1 mM EDTA at pH 7.5 was used. A 0.1 M tris-HCl/0.1 M KCl buffer of pH 7.5 was used as a washing solution. A Britton-Robinson (BR) buffer with an ionic strength of 0.5 was prepared from an acidic solution that contained 0.04 M each of H3PO4, HOAc, and H3BO3 by adjusting to appropriate pH using 0.2 M NaOH.22 A pH 5.7 BR buffer was used to prepare a 5 × 10-3 M o-aminophenol (o-AP)-2 × 10-3 M H2O2 solution. Standard SjAb and HRP-SjAb solutions were prepared by diluting stock SjAb and HRP-SjAb solutions using 1% (w/v) BSA prepared in the same tris-HCl buffer. Measurement Procedure. Figure 2 shows a schematic diagram of the procedure that was followed. Different volumes of SjAb solution (1.25 mg/mL) were mixed with 100 µL of the solution of HRP-SjAb conjugate (14 µg/mL) and enough blocking buffer solution to give a final volume of 1 mL. The immunosensor was immersed in the solution and incubated 50 min at 27 °C. The immunosensor was then rinsed thoroughly with the washing buffer and stored in the blocking buffer prior to the amperometric measurement. Amperometric measurements were performed in 20 mL of BR buffer, pH 5.7. A three-electrode system was used at an applied potential of -200 mV versus SCE. After the background current was stabilized, the response was subsequently recorded after addition of 2.3 mL of o-AP-H2O2 solution. RESULTS AND DISCUSSION Comparison of Biocomposites. A biocomposite is formed by combination of two or more phases of different natures. It acts (22) Britton, H. T. S.; Robinson, R. A. J. Chem. Soc. 1931, 458, 1456-1462.

Figure 3. Background current of immunosensors at different applied potentials: a, paraffin-graphite-SjAg; b, epoxy-graphite-SjAg; c, methacrylate-graphite-SjAg; solution, pH 7.5 BR buffer.

not only as a support for the immunologic material but also as a transducer. The binding material is essential in the biocomposite formation. The use of methacrylate or epoxy for the preparation of biocomposites requires several hours or days of curing at room temperature or higher. This might deactivate the biomaterial, thus leading to a deterioration of the sensor performance. The SjAg immunosensors based on paraffin, methacrylate, and epoxy composites were prepared, and their performance characteristics were compared. Figure 3 shows the amperometric responses at different applied working potentials for immunosensors with different composites in pH 5.7 BR buffer in the absence of the substrate. A low background current and a wide working potential range obtained with a paraffin-graphite-BSA-SjAg immunosenAnalytical Chemistry, Vol. 73, No. 14, July 15, 2001

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Table 1. Effect of Incorporation of BSA in Biocomposite on the Amperometric Response Current

a

electrode composition

response current (nA)a

SD (%)

paraffin-graphite (50/50, w/w) paraffin-graphite-BSA (49.5/49.5/1, w/w) paraffin-graphite-SjAg (49.5/49.5/1, w/w) paraffin-graphite-BSA-SjAg (49/49/1/1, w/w)

150 25 910 820

5.8 5.2 6.3 4.8

Experimental conditions: electrodes were incubated 50 min at 27 °C in the incubation solution containing 1.4 µg/mL HRP-SjAb conjugate.

sor show an additional advantage of the use of paraffin to replace methacrylate or epoxy for SjAb assay. Immunosensors with three tested composites incubated in HRP-SjAb solution with zero dose of free SjAb exhibited similar amperometric responses (∼820 nA) toward the substrate solution; however, a better reproducibility was obtained with paraffin-graphite-BSA-SjAg immunosensor. Evaporation of SjAg solution under vacuum to remove water and evaporation of ether from the paraffin-graphite-SjAg composite substantially improved the response characteristics and reproducibility of the immunosensor that is prepared. Nonspecific adsorption was examined using electrodes with biocomposites containing BSA but without SjAg. Table 1 shows that the current response for a blank paraffin-graphite composite was 150 nA. This current due to nonspecific adsorption can be reduced by the addition of BSA into the blank composite (Table 1). A comparison of biocomposites with and without an addition of BSA further confirms the role BSA in reducing the nonspecific adsorption. The biocomposite paraffin-graphite-SjAg provided apparently the highest amperometric response, which contained the contribution of a nonspecific adsorption effect. Incorporation of BSA into the biocomposite seems to reduce the generally accessible surface area in addition to the available SjAg epitopes. BSA might also alter the hydrophicility characteristics of the biocomposite surface, which in turn affects the adsorption properties of the electrode. The preparation of paraffin biocomposite takes less time than that of epoxy and methacrylate biocomposites. Selection of Substrate for Enzyme Reaction. Enzyme catalyzes the redox reaction of the following type that involve hydrogen peroxide

sub(red) + H2O2 f sub(ox) + H2O

(1)

Here, sub is substrate, and red and ox refer to reduced and oxidated states of the substrate, respectively. The enzymatic activity can be assayed by amperometric or voltammetric detection of the reduction current generated by sub(ox). In the presence of H2O2, HRP can catalyze oxidation of o-aminophenol (o-AP), m-aminophenol (m-AP) and p-aminophenol (p-AP) that have already been used as substrates for voltammetric ELISA.23,24 Cyclic voltammetric studies of substrates and their catalytic products were carried out at a paraffin-graphite electrode. Whether or not H2O2 is added to the solution, one irreversible oxidation peak in cyclic voltamgrams of o-AP and p-AP appears at the potential of 666 and 620 mV versus SCE, respectively. The peak current decreases slightly with the addition of H2O2 (Figure 4 a,b, shown (23) Zhang, S. S.; Jiao, K.; Chen, H. Y.; Wang, M. X. Chin. J. Anal. Chem. 1999, 9, 993-996. (24) Zhang, S. S.; Jiao, K.; Chen, H. Y. Anal. Lab (in Chinese) 1999, 18, 19-23.

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Figure 4. Cyclic voltammograms of o-AP in the absence and presence of H2O2 and with added HRP using a paraffin-graphite electrode in a pH 5.7 BR buffered solution: a, 5 × 10-4 M o-AP; b, a + 2 × 10-4 M H2O2; c, b + 1.0 × 10-10 g/mL HRP.; sweep rate, 100 mV/s.

only for o-AP). No electrochemical activity was observed for m-AP. The solution pH was adjusted to the optimum value for the corresponding aminophenol isomer as determined experimentally or taken from literature: o-AP, 5.7; p-AP, 5.0; m-AP, 7.5 (BR buffer). The addition of HRP (1.0 × 10-9 g/mL) to the above solutions results in the disappearance of the aforementioned oxidation peak and the appearance of a new redox peak for all three of the aminophenols at -200 to 0 mV versus SCE (Figure 4c, shown

Table 2. Amperometric Response of Catalytic Products of Substrates at Different Applied Potentials

applied potential mV

background current nA

-300 -200 -100 0

250 80 65 40

current response (nA) of catalytic productsa o-AP m-AP p-AP 600 820 0 0

50 20 10 0

300 210 140 20

a The pH values of BR buffer used were 5.7, 5.0, and 7.5 for ο-AP, p-AP and m-AP, respectively. The concentrations of the oxidized aminophenols were 3 × 10-4 M. The background current was substracted.

only for o-AP). The peak current of o-AP is higher than that of p-AP and m-AP. For quantitative comparison of the amperometric responses of the catalytic products of three aminophenols, the following experiments were performed. First, the aminophenols were oxidized enzymatically using an excess of H2O2 (5× the stoichiometric value). The concentrations of the oxidized aminophenol obtained were determined by spectrophotometry using the molar absorptivities of these species.25 On the basis of these experiments, solutions of three aminophenols of the same concentration (3 × 10-4 M) were prepared. The current responses using a paraffingraphite electrode were recorded by amperometric measurement at different applied potentials ranging from -300 to 0 mV versus SCE, where the background current is not too large to allow precise current recording. After the background current was stabilized, the response was obtained after the addition of the same amounts of the catalytic products of the corresponding aminophenol. The optimum working potential for o-AP, p-AP, and m-AP turned out to be -200, -300, and -300 mV versus SCE, respectively. The current responses of catalytic products of the three aminophenols are shown in Table 2. One notices that the best sensitivity of amperometric response is obtained for the catalytic product of o-AP, which was selected as the substrate in this study. For three aminophenol isomers, the hydrogen atoms located in the ortho and para positions on the benzene ring tend to show higher activity as compared to that of the meta position. Because both amino and hydroxy groups are electron-repelling ones, as a result of space considerations one would expect the electron density in the vicinity of the carbon atom attaching the hydroxy group to be higher for o-AP in comparison with p-AP. When HRP catalyzes the oxidation of aminophenol in the presence of H2O2, the aminophenol is first oxidized to quinone compounds.26 The redox reaction involves the carbon atom attaching the hydroxy group. The ability of HRP to catalyze the oxidation of aminophenols in the presence of H2O2 should show the following order: o-AP>p-AP>m-AP, which is in agreement with our experimental observations. Zhang et al.23,24 also used o-AP and m-AP as substrates in conjunction with HRP-H2O2 for ELISA of thyroxine. These authors reported that o-AP can provide a much lower detection limit for thyroxine as compared to m-AP, and the (25) Volpe, G.; Compagnone, D.; Draisci, R.; Palleschi, G. Analyst 1998, 123, 1303-1307. (26) Zhang, S. S.; Chen, H. Y.; Jiao, K. Science in China (Series B) 1992, 29 (1), 83-90.

Figure 5. Current-versus-o-AP concentration plot. Immunosensor incubated in 0.1 M tris-HCl/1 mM EDAT buffered solution of pH 7.5 containing 1 µg/mL SjAb (analyte), 1.4 µg/mL SjAb-HRP conjugate, and 0.1% (v/v) BSA. Other conditions are the same as for Figure 2. Inset: a, Linewaver-Burk plot; b, the effect of pH of substrate solution on response current.

reaction product of HRP-catalyzed oxidation of o-AP by H2O2 is 3-aminophenophenoxazine.26 The experimental results obtained with aminophenol isomers in the present study seem to be in fair agreement with the aforementioned reports. Assay of the Enzyme Label Activity Attached to the Immunosensor Surface. A current-versus-o-AP concentration plot is useful for kinetics studies of enzymatic reactions. The response current generated by the product of enzymatic catalytic reaction corresponds to the velocity of the enzymatic reaction and, according to the Michaelis-Menten equation, is proportional to the activity of the enzyme and consequently, to the amount of SjAb-HRP conjugate bound to the surface of the immunosensor of interest. Figure 5 presents a current-versus-o-AP concentration plot obtained during the assay of the enzyme label in an incubated immunosensor. One notices that the current increases rapidly with the increase of the concentration of substrate. When the o-AP concentration reaches 0.5 mM or higher, the current response value tends to increase more slowly. The apparent MichaelisMenten constant (Kappm) was estimated by using the LineweaverBurk plot of 1/I vs 1/[o-AP] (Inset a of Figure 5, employing experimental values used in Figure 5). A value of 0.275 (mM) for Kappm was obtained. The activity of the enzyme label attached to the immunosensor surface is influenced by the pH of reaction medium. The response currents of o-AP obtained at pH 4.0 to 9.0 are shown in inset b of Figure 5. Although the current response obtained at pH 4.0 is the highest, a pH of 5.7 was selected as the optimum one, because at pH 4.0 or lower, the antigen-antibody complex may tend to decompose. Optimization of the Assay Conditions. Parameters of the assay procedure would affect the response of the immunosensor. The amount of SjAg incorporated into the biocomposite influences the amount of HRP-SjAb bound to the surface of the immunoAnalytical Chemistry, Vol. 73, No. 14, July 15, 2001

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Figure 6. Effect of experimental parameters on sensor response: a, SjAg loading in the biocomposite, immunosensors with different amounts of SjAg loading in the biocomposite incubated 50 min in a solution containing 1.4 µg/mL SjAb-HRP conjugate at 25 °C; b, amount of labeled SjAb, an immunosensor with 5 mg SjAg in the biocomposite incubated in incubation solutions containing different amounts of SjAb-HRP conjugate at 25 °C; c, incubation time, an immunosensor with 5 mg SjAg in the biocomposite incubated different times in the incubation solution containing 1.4 µg/mL HRP-SjAb conjugate at 25 °C; d, incubation temperature, an immunosensor with 5 mg SjAg in the biocomposite incubated 50 min in the incubation solution containing 1.4 µg/mL HRP-SjAb conjugate at different temperatures. Inset Figure 6d, the effect of temperature on pH of the incubation solution. Error bars represent SD, n ) 4.

sensor (Figure 6a) The response current increases upon raising the SjAg loading from 0.5 to 5 mg, and then it starts to level off, which corresponds to the saturation of the surface-exposed SjAg epitopes. A SjAg loading of 5 mg was used to prepare the immunosensors for the experiments. Furthermore, higher loading would affect the characteristic performance of the immunosensor, such as an increase in the time required for obtaining steadystate baseline, the enhancement of background current, etc. The response signal of the immunosensor depends on the amount of conjugate bound to the surface of the immunosensor, which in turn corresponds to the amount of conjugate in the incubation solution. To obtain a maximum response using a minimum amount of HRP-SjAb conjugate, the optimal amount of HRP-SjAb in the incubation solution was estimated by incubating the immunosensor with increasing amounts of HRPSjAb. The response increased linearly up to 1.4 µg/mL of HRPSjAb in solution and then tended to saturate (Figure 6b). Higher or lower concentration of HRP-SjAb leads to a decrease in detection concentration of free SjAb in the samples, because the SjAg epitopes of the immunosensor surface are limited. Consequently, 100 µL of 14 µg/mL of HRP-SjAb solution added to 1 mL of final incubation solution was routinely used for these assays. 3224 Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

The effect of the incubation time on response signals was also investigated. When the antibodies in the incubating solution reach the antigens at the surface of the immunosensor, it takes time for the contacting species to form compact immunocomplexes. The amperometric signal increases with the incubation time rapidly up to 30 min, and then the change slows down, which seems to be a consequence of mass action effects as the concentration of free SjAb decreases (Figure 6c). The response signal tends to be stable after incubating 50 min, which was used as the optimal incubation time for most experiments. One would expect that most of the surface-exposed antigens are binding with the antibodies in the incubation solution, forming compact complexes on the surface of the immunosensor. An additional parameter that affected the assay was the incubation temperature. Various incubation temperatures were reported in the literature, ranging from 25 to 37 °C.18,27 As is wellknown, an optimal temperature of immunoreaction would be 37 °C. Enzyme exhibits the best activity at the temperature range of 20 to 25 °C. A higher temperature would be harmful to its activity.1 The effect of incubation temperature on response current was (27) Wu, Z. Y.; Shen, G. L.; Li, Z. Q.; Wang, S. P.; Yu, R. Q. Anal. Chim. Acta 1999, 398, 57-63.

Table 3. Reusability and Reproducibility of Immunosensors experiment

current response, nAa

mean, nA

RSD, %

Ib IIc

713, 708, 710, 715, 702, 720 642, 640, 628, 634, 637, 625

711 634

6.2 6.7

a Each current reading corresponds to a measurement cycle when the tested immunosensor was newly polished followed by incubation and amperometric measurement procedures. Concentration of SjAb in sample was 0.72 mg/mL; other conditions as in Figure 5. b Measurement with a newly prepared paraffin-graphite-SjAg immunosensor. c Measurement with the same Paraffin-graphite-SjAg immunosensor after two-month usage. The number of assays is 100 and the working time at 27 °C is 84 hours.

Figure 7. Current-versus-concentration plot for a paraffin-graphiteSjAg immunosensor after the competitive immunoassay. Immunosensor incubated in 0.1 M tris-HCl/1 mM EDAT buffered solution of pH 7.5 containing different amounts of SjAb (analyte), 1.4 µg/mL SjAb-HRP conjugate, and 0.1%(v/v) BSA. Other conditions as in Figure 2. Error bars represent SD, n ) 6.

examined at 5 to 40 °C. It was found that the signal increases with an increase of temperature up to 27 °C, and then it decreases at higher temperature (Figure 6d). The loss of signal at higher temperatures may be attributed to the effect of temperature on the pH of the incubation solution, which decreases ∼0.3 pH units with an increase of incubation temperature ranging from 27 °C to 40 °C (inset of Figure 6d). Additionally, paraffin used in biocomposite is a heat-sensitive reagent. A higher temperature would affect its physical stability, thus leading to an increase in background current and the deterioration of the sensitivity of the sensor. So an incubation temperature of 27 °C was used in most experiments. Measurements with the Immunosensor. Figure 7 shows the calibration curve obtained using SjAb standards under optimal experimental conditions. The curve is not a linear one, as commonly observed for an immunoassay. A curve-fitting procedure could be used for the calibration procedure. A pseudolinear relationship between the current and the concentration of SjAb, however, can be fitted to the experimental points from 0.36 to 14 µg/mL. A detection limit of 0.36 µg/mL was estimated to be 3× the standard deviation of zero-dose response, which coincides with the lower bound of the pseudolinear part of the calibration curve. The detection limit obtained is lower than 7.2 µg/mL, the lowest value reported so far using a piezoelectric body acoustic wave sensor for the determination of SjAb.27 A relative standard deviation (RSD) of 6.4% was obtained in the pseudolinear range after the corresponding incubation with three newly polished surfaces of the same immunosensor. This indicates that the distribution of the SjAg in the composite bulk is uniform, and the paraffin-graphite-SjAg immunosensor can offer reliable results for the quantitative determination of SjAb. Reproducibility of the immunosensors was tested by measuring 0.72 µg/mL SjAb standard solution. RSD of normalized signals is shown in Table 3. The signals have good reproducibility, as can be observed from the RSD data. To investigate the selectivity of the paraffin-graphite-SjAg immunosensor, the sensor was incubated in incubation solutions containing separately T3, T4 and CRP. The results are shown in

Table 4. Amperometric Response After the Incubation of the Paraffin-Graphite-SjAg Immunosensor in Normal Human Serum Solution Containing Interfering Agents interfering agent

tolerance limit, µg/mL amperometric response,a nA

T3

T4

CRP

5.4 810

15.6 760

65 790

a The current reading corresponds to a measurement cycle when the immunosensor was newly polished followed by incubation and amperometric measurement procedures. Concentration of HRP-SjAb in the incubation solution is 1.4 µg/mL, other conditions as in Figure 2.

Table 5. Amperometric Response of Rabbit Serum Samples rabbit serum samples

infection degreea

current nAb

concentration detected, µg/mL

ELISA µg/mL

1 2 3 4

31 44 55 280

640 560 490 430

5.15 8.74 11.89 14.0

5.31 8.61 12.03 14.56

a The infection degree is expressed in days of infection by Schistosoma japonium 2000. b The difference of current in the absence and presence of samples.

Table 4. T3, T4, and CRP below some tolerance limit in normal human serum samples do not interfere with the sensor’s response. The selectivity of the paraffin-graphite-SjAg immunosensor based on the highly specific antigen-antibody immunoreaction was satisfactory. Furthermore, to demonstrate the use of the paraffin-graphiteSjAg immunosensor for the determination of the SjAb in rabbit serum, four rabbit serum samples with different infection degrees were assayed. The results are shown in Table 5. One notices that the current response increases with an increase in the degree of infection. These results imply that the detectable concentration of SjAb in this system meets the requirement of clinical analysis, and the use for the direct determination of SjAb concentration in serum and the evaluation of infection degree is really feasible. The stability of the device using the paraffin-graphite-SjAg biocomposite was investigated using different immunosensors from the same batch under dry storage at 4 °C. Table 3 shows examples of the measurement with the same immunosensor when it was newly prepared and after a two-month usage. For the same Analytical Chemistry, Vol. 73, No. 14, July 15, 2001

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experimental cycle, the current readings obtained after each new polishing and incubation were very close to each other. A 10% decrease of amperometric response was observed after using it for two months, which indicates fair retention of the specific binding ability of antigens and minimal microstructure changes. The slow decrease of amperometric response seemed to be related to the gradual deactivation of SjAg incorporated in the paraffingraphite mass. Calibration with standard samples should be repeated from time to time to guarantee the precision of analytical results. A newly prepared paraffin-graphite-SjAg immunosensor can be used at least for 100 immunoassays. CONCLUSIONS The paraffin-graphite-SjAg immunosensor described in this paper provides a novel tool for directly monitoring the concentration of SjAb in serum samples. The immunocomposites containing paraffin need not be cured before being put into the PVC tube. Their preparation is simpler and faster than using curing binding materials. Physical and chemical characteristics of the immun-

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osensor, including short-term stability and reproducibility, are comparable with rigid carbon-polymer technology. The resulting paraffin-graphite-SjAg immunosensor offers a relatively fast and sensitive response using a HRP-SjAb conjugate after a relatively short incubation time. the proposed system provides a promising alternative to the diagnostic method for Schistosoma japonium disease in the clinical laboratory. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grants nos. 29735150, 29975006, and 20075006), Science Commission of Hunan Province, and the Foundation for Technological Development of Machinery Industry.

Received for review January 23, 2001. Accepted March 21, 2001. AC0101048