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Schistosoma japonicum antibody assay by immunosensing with fluorescence detection using 3,3′,5,5′-tetramethylbenzidine as substrate
Talanta 58 (2002) 611 /618 www.elsevier.com/locate/talanta
Schistosoma japonicum antibody assay by immunosensing with fluorescence detection using 3,3?,5,5?-tetramethylbenzidine as substrate Fu-Chun Gong a, Zhi-Jun Zhou b, Guo-Li Shen a, Ru-Qin Yu a,* a
State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China b Xiangya Medical College, Central South University, Changsha 410008, PR China Received 10 October 2001; received in revised form 15 April 2002; accepted 25 April 2002
Abstract An immunosensing system for Schistosoma Japonicum antibody (SjAb) assay has been developed which is useful for the diagnosis of schistosomaisis. To circumvent the difficulty of regeneration of the immunosensing device, the sol-gel technique is used which results in a considerable retention of the activity of the encapsulated antigen (SjAb) and easily diffusing into the pores of the polymeric silica matrix. The surface of the immunosensing device prepared can easily be renewed by simply polishing. The regenerated surface serves as a platform for the competitive immuno-reaction of HRP-SjAb and SjAb with SjAg bound at the surface. By using 3, 3?, 5, 5?-tetramethylbenzidine (TMB) as the substrate, the amount of HRP-SjAb bound is quantitated fluorimetrically, which is in turn related with the SjAb content. An amplification effect is obtained by using the enzymatic reaction, and an improved detection limit of 4.5 ng ml 1 is thus realized. The optimum analytical conditions such as pH, amount of the labeled antibody and flow rates of substrate carrier solution were established. The immunosensing procedure shows a pseudo linear response range from 4.5 to 55 ng ml 1. The proposed procedure has been employed to determine SjAb in serum samples. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Schistosoma japonicum ; fluorescence immunosensing; 3,3?,5,5?-tetramethylbenzidine
1. Introduction Among the known analytical techniques, only few of them can cope with the challenges of bioanalytical application. Schistosoma japonium antibody (SjAb) assay, for example, is a routine
* Corresponding author. Tel./fax: /86-731-8822-782 E-mail address: [email protected] (Y. Ru-Qin).
method for diagnosis of schistosomaisis, which threatens the health of millions of people in developing countries. The procedures used include indirect hemagglutination (IH) [1], immunoelectrophoretic assay (IEA) [2], enzyme-linked immunosorbent assay (ELISA) [3], radioimmunoassay (RIA) [4] and indirect fluorescent antibody test (IFAT) [5]. Unfortunately, these methods are mainly employed only for qualitative and semi-
0039-9140/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 2 ) 0 0 3 1 3 - 2
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F-C. Gong et al. / Talanta 58 (2002) 611 /618
quantitative analysis for SiAb. Moreover, in order to obtain reliable results, most of them are usually coupled with other independent tests, otherwise there are about 10% of infected samples escaping the screening test [6,7]. Searching new simple and sensitive quantitative methods for SjAb assay is of considerable interest. Among the aforementioned analytical procedures, RIA possesses the lowest detection limit, though the use of radioactive isotopes is a disgusting procedure difficult to popularize in field clinics. Various direct assay procedures suffer from the common problem of insufficient sensitivity. The enzyme-linked immunoassay (ELISA) introduced in early seventies using the amplification effect of enzymatic reaction can provide a sensitivity comparable with that of RIA. Inspired by the success of the ELISA method, the present authors tried to develop a renewable immunosensing device for SjAb assay with a fluorimetric finish. The sensing device provides a renewable surface layer containing SjAg encapsulated by solgel matrix. This layer serves as a platform for competitive immuno-reaction of HRP-SjAb and the analyte SjAb in the incubation solution with SjAg at the surface layer. The amount of HRPSjAb bound to this surface layer is related to the content of SjAb in the samples assayed, and it is quantitated by measuring the decrease of fluorescence of a substrate (TMB) caused by the HRPcatalyzed reaction. An improved detection limit for SjAb assay comparing to electrochemical methods could be obtained [8]. The proposed procedure has been applied to the determination of SjAb in serum samples with results in good agreement with those obtained by another methods
2. Experimental 2.1. Reagents Horseradish peroxidase (HRP, EC 1. 11. 1. 7) and 3, 3?, 5, 5?- tetramethylbenzidine (TMB) were purchased from Sigma, and methyltrimethoxysilane (MTEOS) from Wuda Chemicals (Wuhan, China). A 32 kDa molecular antigen of schisto-
soma-japonicum from adult worm antigen (AWA) was isolated and purified to homogeneity according to the reported method [9]. The concentration of SjAg is 4.5 mg ml1. The SjAb used in calibration procedure was prepared by immunizing rabbits for 45 days with schistosoma-japonicum 2500 [6]. The antibody in the infected rabbit serum was isolated by precipitation from saturated ammonium sulfate solution and purified as described in literature [10]. The actual concentration of stock SjAb solution (0.45mg ml 1) was determined by using ELISA method [11]. A blocking buffer for incubation containing 0.1% of BSA (w/ w) in 0.1M Tris /HCl and 103 M EDTA at pH 7.5 was used. A 0.1 M Tris /HCl-0.1 M KCl buffer of pH 7.5 was employed as a washing solution. The substrate solution was TMB-H2O2 in pH 4.2 phosphate /citric acid-DMSO buffer solution. The ratio of the DMSO to the phosphate-citric acid buffer was 1%, and it was constant in all the experiments. All solutions were prepared in doubly distilled deionized water, most chemicals used were of reagent grade. 2.2. Apparatus Fluorescence measurements were made on a HITACHI M850 fluorescence spectrophotometer (Japan). A peristatic pump was used to generate flowing stream (Jiangsu, China). A model CSS501 thermostat (Chongqing, China) was employed to control the incubating temperature. 2.3. Preparation of HRP-SjAb An appropriate amount of HRP (10 mg) was dissolved in 0.2 ml of 1% glutaraldehyde in pH 6.8 PBS and mixed thoroughly, then left to incubate 12 h at room temperature. The resulting solution was dialyzed against 0.01 M PBS-0.15 M NaCl solution of pH 7.2 overnight at 4 8C. SjAb (5 mg) dissolved in a mixture of 1 ml of 0.15 M NaCl solution and 0.1 ml of 1 M carbonate buffer at pH 9.6 were combined with a dialyzed HRP-glutaradehyde solution, and then incubated for 24 h at 4 8C. The resulting solution was dialyzed against 0.01 M phosphate buffer solution at pH 7.2. Further purification was conducted by gel filtra-
F-C. Gong et al. / Talanta 58 (2002) 611 /618
tion on Sephadex G-200 column to give the HRPSjAb conjugate (0.48 mg ml 1) which was used as stock working solution. 2.4. Immobilization of SjAg The mixture solution containing 0.8 ml of methyltrimethoxysilane (MTEOS), 0.5 ml of ethanol, 0.5 ml of water and 0.2 ml of 0.05 M HCl was sonicated for about 20 min and stored for 2 days at room temperature, and a silicate polymer gel paste (0.6 g) was obtained. An amount of 5 mg of SjAg and 13 mg of bovine serum albumin (BSA) were dissolved in 0.5 ml of cold (4 8C) water and mixed with 0.9 g of graphite powder. The resulting mixture was left to dry in a desiccator at 4 8C. The sol-gel and the graphite powder with SjAg-BSA adsorbed were thoroughly mixed with a weight ratio of 2:3. The paste was then squeezed into the PVC tube of 6 mm i.d to a depth of 1 cm with screw of thread at one end. An immunosensing device with a supported biocomposite was formed after gelatination and drying for 3 days at 4 8C. The configuration of the immunosensing device is illustrated in Fig. 1A. 2.5. Renewal of supported biocomposite surface The surface of supported biocomposite can be renewed by turning the nut to extrude 0.1 mmthick outer silicate glass-graphite matrix layer and polishing with an alumina paper (0.05 mm) wetted with water to obtain a smooth, shiny surface. The new surface was finally cleaned with doubly distilled water. 2.6. Measurement system and procedure The immunofluorescence assay protocol is schematically illustrated in Fig. 1. The first step involved a 30-min-incubation in the buffer solution (0.1 M Tris-HCl-0.1% BSA, pH 7.5) which contains 100 ml of HRP-SjAb tracer (48 mg ml1) and different volumes of SjAb (analyte) to give a final volume of 1 ml, (not shown in Fig. 1). The incubated surface of biocomposite was then rinsed thoroughly in a washing buffer solution (0.1 M Tris-HCl-KCl, pH 7.5) and stored in a same
613
solution prior to the fluorescence measurement. Next, the supported biocomposite was fixed onto the flow-through reaction cell in the path of flowing stream (Fig. 1B). A substrate solution containing 2.5 /107 M TMB and 5 /107 M H2O2 was pumped through the flow-through path and detector cell. The fluorescence of TMB is recorded at excitation and emission wavelengths of 305 nm and 415 nm, respectively. The biocomposite incubated in sample solution is inserted into the flow-through reaction cell in the flowing stream path, where the HRP of the HRP-Ab-Ag complexes bound on the biocomposite surface catalyzes the reaction turning part of TMB into nonfluorescence form. The fluorescence signal was recorded again. The decease of fluorescence intensity is calculated.
3. Results and discussions 3.1. Characteristic performance of the immunosensing system A major obstacle of the practical use of immunosensing devices is the difficulty associated with the regeneration of the sensing surface because the binding force between antigen and antibody is relatively strong. The use of sol-gel technique results in a considerable retention of the activity of the encapsulated SjAg and the surface of the biocomposite can easily be renewed by simply polishing. The BSA is added to reduce the nonspecific absorption of HRP-SjAb onto the surface sites without SjAg. The porous structure of polymeric silica matrix is favorable for the substrate molecules smoothly reaching the sites of HRP-SjAb-SjAg complexes. The supported biocomposite can be used as a disposable device without the need for regeneration. The comparative experiments for SjAb determinations using disposable biocomposite and renewed ones were carried out with the results shown in Table 1. One notices that a better reproducibility could be obtained with the same supported biocomposite with renewed surface comparing to disposable biocomposites even from the same batch of preparation. The reprodu-
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Fig. 1. A: Configuration of immunosensing device. (1) PVC nut, (2) PVC tube, (3) PVC screw, (4) sol-gel-graphite matrix, (5) SjAg. B: Schematic diagram of flow injection system coupling with fluometry.
cibility of regenerated biocomposite after twomonth usage is still comparable with newly prepared disposable biocomposites. An additional advantage of the surface regeneration procedure is the lower cost of assay due to repeated use of a biocomposite. 3.2. Quantitative basis TMB is a methylated derivative of benzidine. The extended unsaturated ring system of TMB can emit strong fluorescence in the ultraviolet region, which may be related to resonance interaction at different positions of the molecule and the formation of a quinoid structure with a planar configuration in the excited stated [12] (Fig. 2). As a substrate of HRP, TMB has been used for the
helicobacter pylori specific immunoglobulir G (IgG) antibody assay with electrochemical detection [13,14]. In the incubation procedure of competitive binding immunoassay, the SjAb from the analytical sample and HRP-SjAb added would compete to bind on the supported biocomposite surface. The amount of HRP-SjAb bound on the biocomposite surface after incubation would be reversely proportional to the amount of SjAb in the analytical samples. The concentration of SjAb in samples (analyte) is consequently reversely proportional to the fluorescence decrease of TMB solution. The enzymatic coupling reaction scheme and fluorescent emission scheme are shown in Fig. 2. Fig. 3 shows the change of the fluorescence of 2.5 /10 7 M TMB-5/107 M H2O2 solution
F-C. Gong et al. / Talanta 58 (2002) 611 /618
615
Fig. 2. Scheme of enzymatic reaction and fluorescent emission.
Table 1 Comparison of reproducibility and repeatability for SjAb determined with disposable and renewed biocomposites Experiments Fluorescence decreasea b
I Ic IId
172, 169, 170, 173, 167, 175 128, 136, 130, 144, 140, 142 184, 166, 178, 173, 162, 169
Mean RSD (%) 171 135 172
2.1 4.6 4.7
a Each fluorescence reading corresponds to a measurement cycle when the newly prepared or regenerated biocomposite is incubated followed by fluorimetric measurement. Concentration of SjAb in sample is 0.062 mg ml 1. b Measurements with the same newly prepared biocomposite after repeated regeneration by polishing. c Measurements with a biocomposite after 89 h discrete usage (78 measurements) during a two month period. Each measurement involves one polishing. d Measurements with biocomposites newly prepared by the same procedure and in the same batch, each biocomposite used as a disposable one without regeneration.
after addition of different amount of HRP-SjAb. It was observed that HRP-SjAb caused a decrease
Fig. 3. Fluorescence decrease of TMB obtained with the addition of different HRP-SjAb concentration in phosphatecitric acid buffer solution containing 2.5/10 7 mol ml 1 TMB and 5 /10 7 mol ml 1 H2O2 at pH 4.2. Concentration of HRP-SjAb was (mg ml 1): (a) 0; (b) 1.2/10 2; (c) 2.4 / 10 2; (d) 4.8/10 2.
of fluorescence intensity in dependence of its concentration. One would expect a similar phenomenon by sweeping a TMB-H2O2 solution over
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the supported biocomposite surface with bound HRP-SjAb-SjAg. This serves as the quantitative basis of the proposed sensing system for SjAb assay. 3.3. Effect of reaction pH The fluorescence intensity of TMB solution is pH dependent, moreover, the activity of HRPSjAb and the binding stability of HRP-SjAb-SjAg complexes are also affected by pH. The influence of pH on the fluorescence signal of TMB was investigated. Fig. 4 shows the dependence of the fluorescence signal of 2.5 /107 M TMB solution on pH. Fluorescence intensity increases with pH up to pH 3.6, then tend to decrease. It seems that the pH affects the equilibrium between ground and excited states. The effect of pH on the catalysis of HRP-SjAb and HRP itself was also examined. As TMB has maximum absorption only in ultraviolet region, and the product of enzymatic reaction shows an absorption peak at 450 nm [15]. The absorption at 450 nm is taken to monitor the enzyme-catalyzed reaction of TMB as shown in Fig. 5. For HRPSjAb, the absorption increases with pH up to 3.6, then tends to decrease at higher pH values. It
Fig. 5. Effect of pH on the activity of HRP (m) and SjAb-HRP (^). Absorbance measured at 450 nm.
indicates that the optimal pH of catalysis is 3.6, which is slightly different from the optimal pH for the reaction catalyzed by HRP alone (4.2). It seems that HRP is cross-linked with charged SjAb making the HRP-SjAb positively charged. This makes the appropriate pH lower than the original state for enzymatic reaction. A low pH might be unfavorable for the tight binding between SjAb and SjAg which causes loss of HRP-SjAb when substrate solution sweeps over the biocomposite surface. A pH of 4.2 was employed in most experiments. 3.4. Effect of flow rate on fluorescence signal As the TMB and H2O2 are continuously delivered to the reaction cell, the fluorescence signal value should be dependent on the flow rate of the substrate. The effect of flow rate of the carrier solution containing TMB and H2O2 on the fluorescence signal was investigated as shown in Fig. 6. As expected, lower flow rate led to greater fluorescence signal decrease. A flow rate of 30 ml/h was used. 3.5. Optimization of experimental parameters
Fig. 4. Effect of pH on TMB fluorescence intensity.
Fig. 7 displays the effect of the amount of HRPSjAb by incubating the biocomposite with increasing amount of HRP-SjAb. The response increases with the increase of the amount of conjugate up to 100 ml of HRP-SjAb (48 mg ml 1) in 1 ml of
F-C. Gong et al. / Talanta 58 (2002) 611 /618
Fig. 6. Effect of the flow rate of the substrate carrier solution on fluorescence decrease.
Fig. 7. Effect of the amount of HRP-SjAb.
incubating solution and then tend to saturate. This is due to the fact that the number of the epitopes of the antigen in the biocomposite surface is limited. Consequently, 100 ml of HRP-SjAb solution added to 1 ml of final incubation solution was routinely employed for the assays. An experimental study shows that the optimum incubation temperature is 27 8C with an incubation of 30 min.
Fig. 8. Calibration curve for SjAb determination obtained by the competitive immunoassay. Biocomposite incubated in 0.1 M tris /HCl-1 mM EDTA buffer solution of pH 7.5 containing different amount of SjAb (analyte), 1.2 mg ml 1 SjAb-HRP conjugate and 0.1% (v/v) BSA. Each point represents the mean9/SD of four determinations.
detection limit of 4.5 ng ml 1 (determined as 3 times of the SD of the measurement blank). The detection limit of proposed immunosensing system is lower than that obtained by immunosensing with a piezoelectric approach (7.2 mg ml1) [16]. The SjAg immunosensing system was used to determine the infected serum samples of rabbits, which were obtained from Xiangya Medical College, Central South University, Changsha, China. The results are shown in Table 2. It indicates that the results obtained by the proposed procedure were in good agreement with those obtained by ELISA method. The proposed immunosensing procedure can be used to detect the SjAb in serum samples. Table 2 SjAb determination with the prepared sensor in rabbit serum samples Samplea
3.6. Measurement with immunsensing system The calibration curve for Sj antibody detection is shown in Fig. 8. A nearly linear dependence was observed between the fluorescence decrease and the SjAb concentration in the initial incubation. The pseudo-linear detection range for assay extended between 4.5 ng ml 1 to 50 ng ml 1, with a
617
#
1 (31 days) 2#(44 days) 3#(55 days) 4#(280 days) a
Sensor method (g l 1) b
5.319/0.27 8.859/0.36 10.879/0.51 13.379/0.57
ELISA 5.52 8.24 11.26 13.48
The infected degree of samples is expressed by days of infection by schistosoma japonicum 2000. The serum samples were diluted 100 times. b Mean9/SD of four measurements.
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4. Conclusions The proposed procedure offers several advantages over conventional immunoassys for Schistosoma japonicum antibody detection since it is based on a compact biocomposite with a renewable reaction interface. This approach implies a higher sensitivity and sufficient simplicity. The proposed methodology is useful for the immunoassay of others immunoreagents.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grants Nos. 20975006 and 29975006) and the Foundation for Scientific Committee of Hunan province.
References [1] Y.Q. Xie, Y.R. Chen, C.Y. Qing, Chinese Journal of Preventive Medicine 24 (1990) 52. [2] J.W. Hong, X.H. Lin, Shanghai Journal of Immunology 60 (1984) 92.
[3] G.V. Hillyer, F.J. Caresd, H.S. Crinston, Amer. J. Trop. Med. Hyg. 28 (1979) 237. [4] Y.M. Jiang, Z.P. Wang, J.A. Zhang, Chinese Journal of Radiologic Medicine 1 (1981) 22. [5] W.S. Aronstein, R.D. Lowe, Amer. J. Trop. Med. Hyg. 34 (1985) 879. [6] Y.H. Liu, X.X. Liu, C.C. Song, X.H. Yu, Immunology and Immunodiagnosis of Parasitic Diseases, Jiangsu Science and Technology Press, 1991, pp. 128 /157. [7] S.P. Wang, X.F. Zeng, X.Y. Yi, Chinese, J. Zoonoses 4 (1992) 14. [8] G.D. Liu, Z.Y. Wu, S.P. Wang, G.L. Shen, R.Q. Yu, Anal. Chem. 73 (2001) 3219. [9] S.P. Wang, X.F. Zeng, X.Y. Yi, Chinese, J. Parasitiol. Parasitic Dis. 13 (1995) 25. [10] S.S. Zhang, H.Y. Chen, K. Jiao, Science in China (Series B) 123 (1992) 1303. [11] S.S. Zhang, K. Jiao, H.Y. Chen, M.X. Wang, Chinese, J. Anal. Chem. 9 (1999) 993. [12] R.H. Yang, K.M. Wang, D. Xiao, K. Luo, X.H. Yang, Analyst 125 (2000) 877. [13] Y.N. He, H.Y. Chen, J.J. Zhong, G.Y. Zhang, Z.L. Chen, Talanta 44 (1997) 823. [14] G. Volpe, D. Compangnone, R. Draisci, G. Palleschi, Analyst 123 (1998) 1303. [15] K. Morimune, Y. Yamaguchi, Y. Beppu, S. Miyake, Anal. Chim. Acta 376 (1998) 37. [16] Z.Y. Wu, G.L. Shen, Z.Q. Li, S.P. Wang, R.Q. Yu, Anal. Chim. Acta 398 (1999) 57.
Schistosoma japonicum antibody assay by immunosensing with fluorescence detection using 3,3?,5,5?-tetramethylbenzidine as substrate Fu-Chun Gong a, Zhi-Jun Zhou b, Guo-Li Shen a, Ru-Qin Yu a,* a
State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China b Xiangya Medical College, Central South University, Changsha 410008, PR China Received 10 October 2001; received in revised form 15 April 2002; accepted 25 April 2002
Abstract An immunosensing system for Schistosoma Japonicum antibody (SjAb) assay has been developed which is useful for the diagnosis of schistosomaisis. To circumvent the difficulty of regeneration of the immunosensing device, the sol-gel technique is used which results in a considerable retention of the activity of the encapsulated antigen (SjAb) and easily diffusing into the pores of the polymeric silica matrix. The surface of the immunosensing device prepared can easily be renewed by simply polishing. The regenerated surface serves as a platform for the competitive immuno-reaction of HRP-SjAb and SjAb with SjAg bound at the surface. By using 3, 3?, 5, 5?-tetramethylbenzidine (TMB) as the substrate, the amount of HRP-SjAb bound is quantitated fluorimetrically, which is in turn related with the SjAb content. An amplification effect is obtained by using the enzymatic reaction, and an improved detection limit of 4.5 ng ml 1 is thus realized. The optimum analytical conditions such as pH, amount of the labeled antibody and flow rates of substrate carrier solution were established. The immunosensing procedure shows a pseudo linear response range from 4.5 to 55 ng ml 1. The proposed procedure has been employed to determine SjAb in serum samples. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Schistosoma japonicum ; fluorescence immunosensing; 3,3?,5,5?-tetramethylbenzidine
1. Introduction Among the known analytical techniques, only few of them can cope with the challenges of bioanalytical application. Schistosoma japonium antibody (SjAb) assay, for example, is a routine
* Corresponding author. Tel./fax: /86-731-8822-782 E-mail address: [email protected] (Y. Ru-Qin).
method for diagnosis of schistosomaisis, which threatens the health of millions of people in developing countries. The procedures used include indirect hemagglutination (IH) [1], immunoelectrophoretic assay (IEA) [2], enzyme-linked immunosorbent assay (ELISA) [3], radioimmunoassay (RIA) [4] and indirect fluorescent antibody test (IFAT) [5]. Unfortunately, these methods are mainly employed only for qualitative and semi-
0039-9140/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 9 - 9 1 4 0 ( 0 2 ) 0 0 3 1 3 - 2
612
F-C. Gong et al. / Talanta 58 (2002) 611 /618
quantitative analysis for SiAb. Moreover, in order to obtain reliable results, most of them are usually coupled with other independent tests, otherwise there are about 10% of infected samples escaping the screening test [6,7]. Searching new simple and sensitive quantitative methods for SjAb assay is of considerable interest. Among the aforementioned analytical procedures, RIA possesses the lowest detection limit, though the use of radioactive isotopes is a disgusting procedure difficult to popularize in field clinics. Various direct assay procedures suffer from the common problem of insufficient sensitivity. The enzyme-linked immunoassay (ELISA) introduced in early seventies using the amplification effect of enzymatic reaction can provide a sensitivity comparable with that of RIA. Inspired by the success of the ELISA method, the present authors tried to develop a renewable immunosensing device for SjAb assay with a fluorimetric finish. The sensing device provides a renewable surface layer containing SjAg encapsulated by solgel matrix. This layer serves as a platform for competitive immuno-reaction of HRP-SjAb and the analyte SjAb in the incubation solution with SjAg at the surface layer. The amount of HRPSjAb bound to this surface layer is related to the content of SjAb in the samples assayed, and it is quantitated by measuring the decrease of fluorescence of a substrate (TMB) caused by the HRPcatalyzed reaction. An improved detection limit for SjAb assay comparing to electrochemical methods could be obtained [8]. The proposed procedure has been applied to the determination of SjAb in serum samples with results in good agreement with those obtained by another methods
2. Experimental 2.1. Reagents Horseradish peroxidase (HRP, EC 1. 11. 1. 7) and 3, 3?, 5, 5?- tetramethylbenzidine (TMB) were purchased from Sigma, and methyltrimethoxysilane (MTEOS) from Wuda Chemicals (Wuhan, China). A 32 kDa molecular antigen of schisto-
soma-japonicum from adult worm antigen (AWA) was isolated and purified to homogeneity according to the reported method [9]. The concentration of SjAg is 4.5 mg ml1. The SjAb used in calibration procedure was prepared by immunizing rabbits for 45 days with schistosoma-japonicum 2500 [6]. The antibody in the infected rabbit serum was isolated by precipitation from saturated ammonium sulfate solution and purified as described in literature [10]. The actual concentration of stock SjAb solution (0.45mg ml 1) was determined by using ELISA method [11]. A blocking buffer for incubation containing 0.1% of BSA (w/ w) in 0.1M Tris /HCl and 103 M EDTA at pH 7.5 was used. A 0.1 M Tris /HCl-0.1 M KCl buffer of pH 7.5 was employed as a washing solution. The substrate solution was TMB-H2O2 in pH 4.2 phosphate /citric acid-DMSO buffer solution. The ratio of the DMSO to the phosphate-citric acid buffer was 1%, and it was constant in all the experiments. All solutions were prepared in doubly distilled deionized water, most chemicals used were of reagent grade. 2.2. Apparatus Fluorescence measurements were made on a HITACHI M850 fluorescence spectrophotometer (Japan). A peristatic pump was used to generate flowing stream (Jiangsu, China). A model CSS501 thermostat (Chongqing, China) was employed to control the incubating temperature. 2.3. Preparation of HRP-SjAb An appropriate amount of HRP (10 mg) was dissolved in 0.2 ml of 1% glutaraldehyde in pH 6.8 PBS and mixed thoroughly, then left to incubate 12 h at room temperature. The resulting solution was dialyzed against 0.01 M PBS-0.15 M NaCl solution of pH 7.2 overnight at 4 8C. SjAb (5 mg) dissolved in a mixture of 1 ml of 0.15 M NaCl solution and 0.1 ml of 1 M carbonate buffer at pH 9.6 were combined with a dialyzed HRP-glutaradehyde solution, and then incubated for 24 h at 4 8C. The resulting solution was dialyzed against 0.01 M phosphate buffer solution at pH 7.2. Further purification was conducted by gel filtra-
F-C. Gong et al. / Talanta 58 (2002) 611 /618
tion on Sephadex G-200 column to give the HRPSjAb conjugate (0.48 mg ml 1) which was used as stock working solution. 2.4. Immobilization of SjAg The mixture solution containing 0.8 ml of methyltrimethoxysilane (MTEOS), 0.5 ml of ethanol, 0.5 ml of water and 0.2 ml of 0.05 M HCl was sonicated for about 20 min and stored for 2 days at room temperature, and a silicate polymer gel paste (0.6 g) was obtained. An amount of 5 mg of SjAg and 13 mg of bovine serum albumin (BSA) were dissolved in 0.5 ml of cold (4 8C) water and mixed with 0.9 g of graphite powder. The resulting mixture was left to dry in a desiccator at 4 8C. The sol-gel and the graphite powder with SjAg-BSA adsorbed were thoroughly mixed with a weight ratio of 2:3. The paste was then squeezed into the PVC tube of 6 mm i.d to a depth of 1 cm with screw of thread at one end. An immunosensing device with a supported biocomposite was formed after gelatination and drying for 3 days at 4 8C. The configuration of the immunosensing device is illustrated in Fig. 1A. 2.5. Renewal of supported biocomposite surface The surface of supported biocomposite can be renewed by turning the nut to extrude 0.1 mmthick outer silicate glass-graphite matrix layer and polishing with an alumina paper (0.05 mm) wetted with water to obtain a smooth, shiny surface. The new surface was finally cleaned with doubly distilled water. 2.6. Measurement system and procedure The immunofluorescence assay protocol is schematically illustrated in Fig. 1. The first step involved a 30-min-incubation in the buffer solution (0.1 M Tris-HCl-0.1% BSA, pH 7.5) which contains 100 ml of HRP-SjAb tracer (48 mg ml1) and different volumes of SjAb (analyte) to give a final volume of 1 ml, (not shown in Fig. 1). The incubated surface of biocomposite was then rinsed thoroughly in a washing buffer solution (0.1 M Tris-HCl-KCl, pH 7.5) and stored in a same
613
solution prior to the fluorescence measurement. Next, the supported biocomposite was fixed onto the flow-through reaction cell in the path of flowing stream (Fig. 1B). A substrate solution containing 2.5 /107 M TMB and 5 /107 M H2O2 was pumped through the flow-through path and detector cell. The fluorescence of TMB is recorded at excitation and emission wavelengths of 305 nm and 415 nm, respectively. The biocomposite incubated in sample solution is inserted into the flow-through reaction cell in the flowing stream path, where the HRP of the HRP-Ab-Ag complexes bound on the biocomposite surface catalyzes the reaction turning part of TMB into nonfluorescence form. The fluorescence signal was recorded again. The decease of fluorescence intensity is calculated.
3. Results and discussions 3.1. Characteristic performance of the immunosensing system A major obstacle of the practical use of immunosensing devices is the difficulty associated with the regeneration of the sensing surface because the binding force between antigen and antibody is relatively strong. The use of sol-gel technique results in a considerable retention of the activity of the encapsulated SjAg and the surface of the biocomposite can easily be renewed by simply polishing. The BSA is added to reduce the nonspecific absorption of HRP-SjAb onto the surface sites without SjAg. The porous structure of polymeric silica matrix is favorable for the substrate molecules smoothly reaching the sites of HRP-SjAb-SjAg complexes. The supported biocomposite can be used as a disposable device without the need for regeneration. The comparative experiments for SjAb determinations using disposable biocomposite and renewed ones were carried out with the results shown in Table 1. One notices that a better reproducibility could be obtained with the same supported biocomposite with renewed surface comparing to disposable biocomposites even from the same batch of preparation. The reprodu-
614
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Fig. 1. A: Configuration of immunosensing device. (1) PVC nut, (2) PVC tube, (3) PVC screw, (4) sol-gel-graphite matrix, (5) SjAg. B: Schematic diagram of flow injection system coupling with fluometry.
cibility of regenerated biocomposite after twomonth usage is still comparable with newly prepared disposable biocomposites. An additional advantage of the surface regeneration procedure is the lower cost of assay due to repeated use of a biocomposite. 3.2. Quantitative basis TMB is a methylated derivative of benzidine. The extended unsaturated ring system of TMB can emit strong fluorescence in the ultraviolet region, which may be related to resonance interaction at different positions of the molecule and the formation of a quinoid structure with a planar configuration in the excited stated [12] (Fig. 2). As a substrate of HRP, TMB has been used for the
helicobacter pylori specific immunoglobulir G (IgG) antibody assay with electrochemical detection [13,14]. In the incubation procedure of competitive binding immunoassay, the SjAb from the analytical sample and HRP-SjAb added would compete to bind on the supported biocomposite surface. The amount of HRP-SjAb bound on the biocomposite surface after incubation would be reversely proportional to the amount of SjAb in the analytical samples. The concentration of SjAb in samples (analyte) is consequently reversely proportional to the fluorescence decrease of TMB solution. The enzymatic coupling reaction scheme and fluorescent emission scheme are shown in Fig. 2. Fig. 3 shows the change of the fluorescence of 2.5 /10 7 M TMB-5/107 M H2O2 solution
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615
Fig. 2. Scheme of enzymatic reaction and fluorescent emission.
Table 1 Comparison of reproducibility and repeatability for SjAb determined with disposable and renewed biocomposites Experiments Fluorescence decreasea b
I Ic IId
172, 169, 170, 173, 167, 175 128, 136, 130, 144, 140, 142 184, 166, 178, 173, 162, 169
Mean RSD (%) 171 135 172
2.1 4.6 4.7
a Each fluorescence reading corresponds to a measurement cycle when the newly prepared or regenerated biocomposite is incubated followed by fluorimetric measurement. Concentration of SjAb in sample is 0.062 mg ml 1. b Measurements with the same newly prepared biocomposite after repeated regeneration by polishing. c Measurements with a biocomposite after 89 h discrete usage (78 measurements) during a two month period. Each measurement involves one polishing. d Measurements with biocomposites newly prepared by the same procedure and in the same batch, each biocomposite used as a disposable one without regeneration.
after addition of different amount of HRP-SjAb. It was observed that HRP-SjAb caused a decrease
Fig. 3. Fluorescence decrease of TMB obtained with the addition of different HRP-SjAb concentration in phosphatecitric acid buffer solution containing 2.5/10 7 mol ml 1 TMB and 5 /10 7 mol ml 1 H2O2 at pH 4.2. Concentration of HRP-SjAb was (mg ml 1): (a) 0; (b) 1.2/10 2; (c) 2.4 / 10 2; (d) 4.8/10 2.
of fluorescence intensity in dependence of its concentration. One would expect a similar phenomenon by sweeping a TMB-H2O2 solution over
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616
the supported biocomposite surface with bound HRP-SjAb-SjAg. This serves as the quantitative basis of the proposed sensing system for SjAb assay. 3.3. Effect of reaction pH The fluorescence intensity of TMB solution is pH dependent, moreover, the activity of HRPSjAb and the binding stability of HRP-SjAb-SjAg complexes are also affected by pH. The influence of pH on the fluorescence signal of TMB was investigated. Fig. 4 shows the dependence of the fluorescence signal of 2.5 /107 M TMB solution on pH. Fluorescence intensity increases with pH up to pH 3.6, then tend to decrease. It seems that the pH affects the equilibrium between ground and excited states. The effect of pH on the catalysis of HRP-SjAb and HRP itself was also examined. As TMB has maximum absorption only in ultraviolet region, and the product of enzymatic reaction shows an absorption peak at 450 nm [15]. The absorption at 450 nm is taken to monitor the enzyme-catalyzed reaction of TMB as shown in Fig. 5. For HRPSjAb, the absorption increases with pH up to 3.6, then tends to decrease at higher pH values. It
Fig. 5. Effect of pH on the activity of HRP (m) and SjAb-HRP (^). Absorbance measured at 450 nm.
indicates that the optimal pH of catalysis is 3.6, which is slightly different from the optimal pH for the reaction catalyzed by HRP alone (4.2). It seems that HRP is cross-linked with charged SjAb making the HRP-SjAb positively charged. This makes the appropriate pH lower than the original state for enzymatic reaction. A low pH might be unfavorable for the tight binding between SjAb and SjAg which causes loss of HRP-SjAb when substrate solution sweeps over the biocomposite surface. A pH of 4.2 was employed in most experiments. 3.4. Effect of flow rate on fluorescence signal As the TMB and H2O2 are continuously delivered to the reaction cell, the fluorescence signal value should be dependent on the flow rate of the substrate. The effect of flow rate of the carrier solution containing TMB and H2O2 on the fluorescence signal was investigated as shown in Fig. 6. As expected, lower flow rate led to greater fluorescence signal decrease. A flow rate of 30 ml/h was used. 3.5. Optimization of experimental parameters
Fig. 4. Effect of pH on TMB fluorescence intensity.
Fig. 7 displays the effect of the amount of HRPSjAb by incubating the biocomposite with increasing amount of HRP-SjAb. The response increases with the increase of the amount of conjugate up to 100 ml of HRP-SjAb (48 mg ml 1) in 1 ml of
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Fig. 6. Effect of the flow rate of the substrate carrier solution on fluorescence decrease.
Fig. 7. Effect of the amount of HRP-SjAb.
incubating solution and then tend to saturate. This is due to the fact that the number of the epitopes of the antigen in the biocomposite surface is limited. Consequently, 100 ml of HRP-SjAb solution added to 1 ml of final incubation solution was routinely employed for the assays. An experimental study shows that the optimum incubation temperature is 27 8C with an incubation of 30 min.
Fig. 8. Calibration curve for SjAb determination obtained by the competitive immunoassay. Biocomposite incubated in 0.1 M tris /HCl-1 mM EDTA buffer solution of pH 7.5 containing different amount of SjAb (analyte), 1.2 mg ml 1 SjAb-HRP conjugate and 0.1% (v/v) BSA. Each point represents the mean9/SD of four determinations.
detection limit of 4.5 ng ml 1 (determined as 3 times of the SD of the measurement blank). The detection limit of proposed immunosensing system is lower than that obtained by immunosensing with a piezoelectric approach (7.2 mg ml1) [16]. The SjAg immunosensing system was used to determine the infected serum samples of rabbits, which were obtained from Xiangya Medical College, Central South University, Changsha, China. The results are shown in Table 2. It indicates that the results obtained by the proposed procedure were in good agreement with those obtained by ELISA method. The proposed immunosensing procedure can be used to detect the SjAb in serum samples. Table 2 SjAb determination with the prepared sensor in rabbit serum samples Samplea
3.6. Measurement with immunsensing system The calibration curve for Sj antibody detection is shown in Fig. 8. A nearly linear dependence was observed between the fluorescence decrease and the SjAb concentration in the initial incubation. The pseudo-linear detection range for assay extended between 4.5 ng ml 1 to 50 ng ml 1, with a
617
#
1 (31 days) 2#(44 days) 3#(55 days) 4#(280 days) a
Sensor method (g l 1) b
5.319/0.27 8.859/0.36 10.879/0.51 13.379/0.57
ELISA 5.52 8.24 11.26 13.48
The infected degree of samples is expressed by days of infection by schistosoma japonicum 2000. The serum samples were diluted 100 times. b Mean9/SD of four measurements.
618
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4. Conclusions The proposed procedure offers several advantages over conventional immunoassys for Schistosoma japonicum antibody detection since it is based on a compact biocomposite with a renewable reaction interface. This approach implies a higher sensitivity and sufficient simplicity. The proposed methodology is useful for the immunoassay of others immunoreagents.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grants Nos. 20975006 and 29975006) and the Foundation for Scientific Committee of Hunan province.
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