Studies On The Detection Methods Of Schistosomiasis Japonicum By Immunosensing Techniques

BackgroundSchistosomiasis japonicum remains to be a serious zoonosis and mostly prevalent in developing country. In our country it mainly locates in 5 provinces around lake and 2 provinces at mountain area. Researches of rapid diagnosis on schistosomiasis are significant for monitoring the source of infection and epidemic control. It is generally believed that the ideal diagnostic methods on spot should be specific, sensitive, cheap, convenient, stable and rapid. Nowadays the diagnostic methods are Kato-Katz, IHA and ELISA yet. Kato-Kotz is time costing and labor consuming with a poor positive recall rate which is easy to show false-negative in lightly epidemic areas, so it is not suitable for screening in a large area. Normal ELISA is hard to generalize for its relatively complicate manipulation of the equipment. Therefore, establishing new, fast, accurate, low-costing and convenient techniques or methods comes to be the key concerns in schistosomiasis diagnosis.ObjectiveIn this research, we aimed to combine electrochemical sensor with immune method to explore some different testing systems of schistosomiasis japonicum diagnosis, which would lay the foundation for the development of a sensitive, convenient, automatic and quantitative immunosense equipment.Methods(1) To develope a new piezoelectric immunosensor that detects the Schistosoma japonicum antibodies (SjAb) of serum directly. According to the technology of mixed self-assembled monolayer membrane (mixed SAMs), the mix of mercaptopropionic acid (MPA) and mercaptoethanol (ME) was self assembled on the surface of quartz crystal by gold- sulphur-bond, and soluble egg antigen(SEA) of Schistosoma japonicum (Sj) was cross link to the quartz crystal through special coupling agent.(2) Prepare composite basal membrane of mercaptopropionic acid (MPA) and mercaptoethanol (ME) on the surface of quartz crystal according to self-assembled monolayer membrane technique, then activate the carboxy group on the membrane through coupling agent to covalently immobilize target Schistosoma japonicum antigens (SjAg) with high efficiency. After the presentation of horseradish peroxidase (HRP)-labeled goat-anti-rabbit secondary antibodies in immune reaction, which HRP catalyze the reaction that H₂O₂ oxidize its substrate 4-chloro-1-naphthol into insoluble deposit on the quartz crystal surface, the immune detection signal of the SjAg-SjAb reaction was significantly amplified, which the detection sensitivity was also improved.(3) Using printed electrode to developed a portable and disposable electrochemical biosensor. The carbon inks and silver/silver chloride inks were printed on the PET board to make a two electrode's test strip, carbon was working electrode and SEA was fixed on working electrode by different methods, silver/ silver chloride electrode was used as control. We tested the valency of the antibody by cyclic voltammetry and differential pulse voltammetry in the electrochemistry workstation, and conducted comparison of the effects of several different methods.(4) The SjAg was bound covalently on the surface of magnetic particles of core-shell structure reacted with the SjAb, and then the SjAg-SjAb complex was bound with the HRP-Ab by a sandwich reaction. Using 3,3',5,5'-tetramethyl benzidine(TMB) as a fluorescent substrate, the concentration of antibodies was detected indirectly by determining the variance in fluorescent intensity resulting from the catalytic oxidation of TMB.(5) Developed a rapid colloid carbon dipstick method to detect SjAb based on double antigen sandwich method. SEA was conjugated with colloidal carbon, and the conjugated antigen could react with the antibodies in the patients' sera of schistosomiasis, then the carbon-antigen-antibodies complex would be captured by SEA which had been absorbed on nitrocellulose membrane and show a gray band as a result. And we realized a semi-quantitative analysis by detecting the gray of the test band with gray sensing technology.Results(1) Compared with the traditional immobilization method by single self-assembled monolayers, the SjAg immobilization based on mixed self-assembled monolayers has a much better immunoreactivity. Under the optimal experimental conditions, its detection range is 1:1 500 to 1:60 (infected rabbit serum dilution ratios), we compared several practical samples of infected rabbit serum with varying SjAb concentration, and then made a correlation analysis that compared with classic ELISA, the correlation coefficients reached to 0.960.(2) Explored a high sensitive piezoelectric immunosensor based on enzyme-biocatalyzed precipitation amplification for the determination of SjAb. Insoluble products precipitate generated by enzyme catalysis substrate can significantly magnify the signal of immunity detection, and improve the detection sensitivity. Its detection range is 1:10 000 to 1:200.(3) Developed four immobilization print electrode immunosensors based on different methods to fix SjAg: glutaraldehyde cross-linking transducer, chitosan-glutaraldehyde cross-linking transducer, Sol-gel method transducer, and carbon nano-tube modified Sol-gel method transducer. Detection ranges of glutaraldehyde cross-linking immunosensors is 1:1 000 to 1:400; chitosan-glutaraldehyde cross-linking immunosensors is 1:1 000 to 1:500; Sol-gel immunosensors is 1:1 000 to 1:500; carbon nano-tube modified Sol-gel immunosensors is 1:2.000 to 1:100.(4) Developed a fluoroimmunosensing assay to detected SjAb concentration for the first time. The method was applied to the determination of SjAb in rabbit serum. This transducer exhibits a good linear relationship to the dilution 1: 2×10⁵ to 1:10⁴ of SjAb in infected rabbit serum.(5) Established a rapid colloidal-carbon dipstick immunoassay for detecting antibodies of Sj in the serum. Gray sensing of the testing band (T band), which has a good curvilinear relationship with the sera valence was applied to make a semi-quantitative analysis. 137 sera were tested and the consistency of the results to IHA is 98.54%. Taking detection results of the sera by IHA as a reference standard, the sensitivity of colloidal carbon dipstick assay was 98.99% while the specificity was 97.37%.Conclusion(1) Piezoelectricity immunosensor based on hydrosulfide group mixed self-assembly monolayer membrane is sensitive and convenient. The technique has satisfaction results in practical detection. And it is capable of the ELISA. It would be used in the diagnosis of schistosomiasis after grading-up.(2) The SjAg can be efficiently immobilized by immobilization procedure of immune material based on mixed self-assembled layers of mercapto, and the insoluble products precipitate generated by enzyme catalysis substrate can significantly magnify the signal of immunity detection (even at the SjAb dilution ratio of 1:10 000). Compared with the conventional immunoassays, this method has many advantages, just as higher sensitivity, wider linear range and lower cost. It is hoped to be used in the diagnosis of the Schistosomiasis japonicum.(3) Based on different methods to fix SjAg, we have developed four immobilization print electrode immunosensors. Comparing four methods, carbon nano-tube modified sol-gel method transducer is more sensitive, high detection range, less biomaterials using, and lay a foundation for developing a rapid electrode test trip.(4) A fluoroimmunosensing assay of SjAb detection based on magnetic nanoparticle immobilization was developed. This assay is sensitive, simple and with high selectivity and reproducibility, and explored a new way of the study on optical sensing detection.(5) Connected gray sensor technology with colloidal carbon dipstick for the first time, a feasible semi-quantitative detection method of schistosomiasis japonicum has been established. This method is simple, rapid, and high selectivity and specificity, it is suitable for schisto-somiasis field diagnosis and open up the direction for the immunosensing technology.(6) We compared the different immunosensors in chart 1. Magnetic nanoparticle immobilization fluoroimmunosense and enzyme-catalyzed depositing enlargement piezoelectric immunosensor have high sensitivity, but both of them need long assay time and complex to operation; Self-assembled monolayers piezoelectric immunosensor is easy to operate and the sensitivity meets the need of diagnostic of schistosomiasis japonicum, but the cost of immunosensors and equipment is high and the equipment is easy to interference; Carbon nano-tube modified Sol-gel immunosensor is convenient to operate and the sensitivity meets the need of diagnostic of schistosomiasis japonicum, the cost of immunosensors and equipment is low, but the repeatability doesn't satisfy the application of clinical. Colloidal-carbon dipstick is convenient and rapid to operate, and the sensitivity meets the need of diagnostic of schistosomiasis japonicum, but it is confined to semi-quantivity detection. chart 1: Comparison of different immunosensors...