日本血吸虫病免疫传感技术检测方法的研究
摘要
研究背景:
日本血吸虫病是一种危害严重的人兽共患寄生虫病,主要流行在发展中国家,目前,在我国湖区5省和山区2省仍在流行。血吸虫病的快速诊断的研究对于传染源的监测,疫情的控制将起到积极作用。一般认为,血吸虫病现场应用的理想诊断、筛查方法应具备以下特点:特异、敏感、价廉、简便、稳定和快速。但是,我国血吸虫病的现场查病方法仍然以粪检(kato-katz法)和IHA、ELISA等方法为主。由于粪检费时费力,阳性检出率较低,尤其在轻度流行区漏检率高,已不适宜于大面积疫区人群的筛查。常规ELISA方法操作复杂、现场难以推广应用。因此,研究快速、灵敏、准确、成本较低、简便易行的新型诊断技术和方法,已成为当前血吸虫病诊断迫切需要解决的问题。
研究目的:
将免疫方法和传感技术相结合,研究免疫传感技术检测日本血吸虫病的不同测试体系,为研制灵敏、简便、自动、定量分析的免疫传感检测装置奠定基础。
研究方法:
(1)利用巯基混合自组装单层膜(mixed SAMs)技术,以金硫键将巯基丙酸(MPA)和巯基乙醇(ME)混合物自组装在石英晶振表面,通过偶联试剂将日本血吸虫成熟虫卵可溶性抗原(SEA)交联到晶振上,从而开发了一种新的压电免疫传感器,用于血清中日本血吸虫抗体(SjAb)的直接检测。
(2)采用自组装膜技术在石英晶振表面制备巯基丙酸(MPA)和巯基乙醇(ME)混合基底膜,通过偶联剂活化膜上羧基用于日本血吸虫抗原(SjAg)的高效共价固定。通过夹心式免疫反应引入辣根过氧化酶(HRP)标记二抗,利用HRP催化H_2O_2氧化底物4-氯-1-萘酚,在晶振表面形成不溶性沉积物,使免疫检测信号得以显著放大,提高检测的灵敏度。
(3)采用印刷电极开发便携式、一次性的电化学生物传感器。将碳浆和银浆印刷在PET板,做成二电极的试条,工作电极为碳电极,在工作电极上以不同的方法固定SEA,参比电极为银和氯化银电极,通过循环伏安法(CV法)和示差脉冲伏安法(DPV法)来检测抗体的效价,比较几种不同固定方法的检测效果。
(4)采用磁性颗粒为载体固定日本血吸虫抗原的方法,将日本血吸虫抗原通过共价吸附到核壳结构的磁性颗粒表面,与待测抗体反应后,再与酶标二抗夹心反应,最后以3,3',5,5'-四甲基联苯胺(TMB)为底物,通过测定酶催化下荧光强度的变化来检测抗体的浓度。
(5)利用双抗原夹心法,采用日本血吸虫可溶性虫卵抗原(SEA)包被硝酸纤维素膜(NC膜)作为捕获剂,日本血吸虫抗体(SjAb)包被NC膜作对照线,以胶体碳标记SEA作为显色剂,建立快速检测血清中血吸虫抗体的胶体碳试条法,并用灰度传感技术检测测试带灰度来完成半定量分析。
研究结果:
(1)与传统采用单一自组装单层膜固定化方法相比,发现基于混合自组装单层膜固化SjAg具有更高的免疫反应活性。在优化的实验条件下,该传感器检测血清稀释范围为1:1 500-1:60,比较了不同SiAb浓度的IRS实际样本,与经典ELISA法相比,相关系数达0.960。
(2)开发了一种基于酶生物催化沉积信号放大的日本血吸虫压电免疫传感器。酶催化底物反应生成不溶物沉积显著放大免疫检测信号,提高了方法的检测灵敏度,传感器检测血清稀释范围为1:10000-1:200。
(3)探索了四种不同的血吸虫抗原固定方法,并研制了四种血吸虫印刷电极免疫传感器:戊二醛交联(GA)传感器、壳聚糖-戊二醛(Chit-GA)交联法传感器、溶胶-凝胶法(Sol-gel)传感器、碳纳米管修饰的溶胶-凝胶法(Sol-gel)传感器。采用阳性兔血清测试戊二醛交联(GA)传感器的检测血清稀释范围为:1:1 000-1:400;壳聚糖-戊二醛(Chit-GA)交联法传感器的检测范围为:1:1 000-1:500;溶胶-凝胶法(Sol-gel)传感器的检测范围为:1:1 000-1:500;碳纳米管修饰的溶胶-凝胶法(Sol-gel)传感器检测范围为:1:2 000-1:100。
(4)首次采用荧光免疫传感分析方法,检测了兔血清中日本血吸虫抗体的浓度,抗体稀释度在1:2×10~5-1:10~4范围内荧光强度响应呈良好的线性关系。
(5)建立了一种快速检测血清中日本血吸虫抗体的胶体碳试纸条免疫诊断方法,引入灰度传感以得出检测带(T带)的灰度,而T带灰度与血清效价具有较好曲线关系,因而可以根据曲线对待测血清作出半定量分析。检测137份血清,与间接血凝法(indirecthaemagglutination assay,IHA)检测结果一致性为98.54%。以IHA试剂盒为参照,该方法的检测敏感性为98.99%,特异性为97.37%。
结论:
(1)基于巯基混合自组装单层膜的压电免疫传感器,检测方法灵敏、简便,在实际样品的检测中,取得了满意结果。且检测能力与经典ELISA法相接近,经进一步优化可望用于日本血吸虫病的诊断。
(2)基于巯基化合物混合自组装膜的免疫材料固定化程序可实现SjAg在压电晶振表面的高效固定化,引入的酶催化底物反应生成不溶物步骤可显著放大免疫检测信号,从而提高了检测灵敏度(SjAb浓度稀释比达1:10 000),与常规免疫测定方法相比,该压电免疫传感技术具有灵敏度较高、线性范围较宽、成本低廉等优点,可望用于日本血吸虫病的检测分析。
(3)基于不同的血吸虫抗原固定方法,研制了四种血吸虫印刷电极免疫传感器。四种方法比较,碳纳米管修饰的溶胶-凝胶法(Sol-gel)传感器灵敏度高,线性范围宽,生物材料抗原的使用量少,成本低,为发展成一次性电极快速测试条奠定了基础。
(4)建立了一种荧光免疫传感分析方法并用于日本血吸虫抗体的检测,方法简单实用且具有良好的选择性和重现性,为光学传感检测的研究探索了新的途径。
(5)首次将灰度传感技术应用于日本血吸虫病的诊断,结合胶体碳试纸条,建立一种可实现日本血吸虫抗体半定量检测的方法,该法操作简便,快速,具有较高的敏感性和特异性,适合作血吸虫病的现场诊断,也为免疫传感技术的应用开辟了新的方向。
(6)表1为本文中研究的几种免疫传感器性能比较,免疫荧光检测方法和酶催化沉积放大的压电免疫传感器的灵敏度高,但存在测试时间相对较长,操作复杂的弊端;混合自组装单层膜压电免疫传感器操作简单,检测的灵敏度可以满足血吸虫检测的需要,但存在传感器和测试仪器成本较高,仪器抗干扰性差的问题;基于印刷电极的碳纳米管修饰的溶胶-凝胶传感器检测时间短,仪器和传感器成本低,检测的灵敏度可以满足血吸虫病检测的需要,但目前传感器的重现性与实际应用还存在一定差距;纳米碳颗粒标记的层析免疫试条使用方便,测试时间短,检测的灵敏度可以满足血吸虫筛查的需要,具有一定的实用性,但目前的研究成果尚限于半定量的检测。
Background
Schistosomiasis 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.
Objective
In 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 H_2O_2 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: 2×10~5 to 1:10~4 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
日本血吸虫病是一种危害严重的人兽共患寄生虫病,主要流行在发展中国家,目前,在我国湖区5省和山区2省仍在流行。血吸虫病的快速诊断的研究对于传染源的监测,疫情的控制将起到积极作用。一般认为,血吸虫病现场应用的理想诊断、筛查方法应具备以下特点:特异、敏感、价廉、简便、稳定和快速。但是,我国血吸虫病的现场查病方法仍然以粪检(kato-katz法)和IHA、ELISA等方法为主。由于粪检费时费力,阳性检出率较低,尤其在轻度流行区漏检率高,已不适宜于大面积疫区人群的筛查。常规ELISA方法操作复杂、现场难以推广应用。因此,研究快速、灵敏、准确、成本较低、简便易行的新型诊断技术和方法,已成为当前血吸虫病诊断迫切需要解决的问题。
研究目的:
将免疫方法和传感技术相结合,研究免疫传感技术检测日本血吸虫病的不同测试体系,为研制灵敏、简便、自动、定量分析的免疫传感检测装置奠定基础。
研究方法:
(1)利用巯基混合自组装单层膜(mixed SAMs)技术,以金硫键将巯基丙酸(MPA)和巯基乙醇(ME)混合物自组装在石英晶振表面,通过偶联试剂将日本血吸虫成熟虫卵可溶性抗原(SEA)交联到晶振上,从而开发了一种新的压电免疫传感器,用于血清中日本血吸虫抗体(SjAb)的直接检测。
(2)采用自组装膜技术在石英晶振表面制备巯基丙酸(MPA)和巯基乙醇(ME)混合基底膜,通过偶联剂活化膜上羧基用于日本血吸虫抗原(SjAg)的高效共价固定。通过夹心式免疫反应引入辣根过氧化酶(HRP)标记二抗,利用HRP催化H_2O_2氧化底物4-氯-1-萘酚,在晶振表面形成不溶性沉积物,使免疫检测信号得以显著放大,提高检测的灵敏度。
(3)采用印刷电极开发便携式、一次性的电化学生物传感器。将碳浆和银浆印刷在PET板,做成二电极的试条,工作电极为碳电极,在工作电极上以不同的方法固定SEA,参比电极为银和氯化银电极,通过循环伏安法(CV法)和示差脉冲伏安法(DPV法)来检测抗体的效价,比较几种不同固定方法的检测效果。
(4)采用磁性颗粒为载体固定日本血吸虫抗原的方法,将日本血吸虫抗原通过共价吸附到核壳结构的磁性颗粒表面,与待测抗体反应后,再与酶标二抗夹心反应,最后以3,3',5,5'-四甲基联苯胺(TMB)为底物,通过测定酶催化下荧光强度的变化来检测抗体的浓度。
(5)利用双抗原夹心法,采用日本血吸虫可溶性虫卵抗原(SEA)包被硝酸纤维素膜(NC膜)作为捕获剂,日本血吸虫抗体(SjAb)包被NC膜作对照线,以胶体碳标记SEA作为显色剂,建立快速检测血清中血吸虫抗体的胶体碳试条法,并用灰度传感技术检测测试带灰度来完成半定量分析。
研究结果:
(1)与传统采用单一自组装单层膜固定化方法相比,发现基于混合自组装单层膜固化SjAg具有更高的免疫反应活性。在优化的实验条件下,该传感器检测血清稀释范围为1:1 500-1:60,比较了不同SiAb浓度的IRS实际样本,与经典ELISA法相比,相关系数达0.960。
(2)开发了一种基于酶生物催化沉积信号放大的日本血吸虫压电免疫传感器。酶催化底物反应生成不溶物沉积显著放大免疫检测信号,提高了方法的检测灵敏度,传感器检测血清稀释范围为1:10000-1:200。
(3)探索了四种不同的血吸虫抗原固定方法,并研制了四种血吸虫印刷电极免疫传感器:戊二醛交联(GA)传感器、壳聚糖-戊二醛(Chit-GA)交联法传感器、溶胶-凝胶法(Sol-gel)传感器、碳纳米管修饰的溶胶-凝胶法(Sol-gel)传感器。采用阳性兔血清测试戊二醛交联(GA)传感器的检测血清稀释范围为:1:1 000-1:400;壳聚糖-戊二醛(Chit-GA)交联法传感器的检测范围为:1:1 000-1:500;溶胶-凝胶法(Sol-gel)传感器的检测范围为:1:1 000-1:500;碳纳米管修饰的溶胶-凝胶法(Sol-gel)传感器检测范围为:1:2 000-1:100。
(4)首次采用荧光免疫传感分析方法,检测了兔血清中日本血吸虫抗体的浓度,抗体稀释度在1:2×10~5-1:10~4范围内荧光强度响应呈良好的线性关系。
(5)建立了一种快速检测血清中日本血吸虫抗体的胶体碳试纸条免疫诊断方法,引入灰度传感以得出检测带(T带)的灰度,而T带灰度与血清效价具有较好曲线关系,因而可以根据曲线对待测血清作出半定量分析。检测137份血清,与间接血凝法(indirecthaemagglutination assay,IHA)检测结果一致性为98.54%。以IHA试剂盒为参照,该方法的检测敏感性为98.99%,特异性为97.37%。
结论:
(1)基于巯基混合自组装单层膜的压电免疫传感器,检测方法灵敏、简便,在实际样品的检测中,取得了满意结果。且检测能力与经典ELISA法相接近,经进一步优化可望用于日本血吸虫病的诊断。
(2)基于巯基化合物混合自组装膜的免疫材料固定化程序可实现SjAg在压电晶振表面的高效固定化,引入的酶催化底物反应生成不溶物步骤可显著放大免疫检测信号,从而提高了检测灵敏度(SjAb浓度稀释比达1:10 000),与常规免疫测定方法相比,该压电免疫传感技术具有灵敏度较高、线性范围较宽、成本低廉等优点,可望用于日本血吸虫病的检测分析。
(3)基于不同的血吸虫抗原固定方法,研制了四种血吸虫印刷电极免疫传感器。四种方法比较,碳纳米管修饰的溶胶-凝胶法(Sol-gel)传感器灵敏度高,线性范围宽,生物材料抗原的使用量少,成本低,为发展成一次性电极快速测试条奠定了基础。
(4)建立了一种荧光免疫传感分析方法并用于日本血吸虫抗体的检测,方法简单实用且具有良好的选择性和重现性,为光学传感检测的研究探索了新的途径。
(5)首次将灰度传感技术应用于日本血吸虫病的诊断,结合胶体碳试纸条,建立一种可实现日本血吸虫抗体半定量检测的方法,该法操作简便,快速,具有较高的敏感性和特异性,适合作血吸虫病的现场诊断,也为免疫传感技术的应用开辟了新的方向。
(6)表1为本文中研究的几种免疫传感器性能比较,免疫荧光检测方法和酶催化沉积放大的压电免疫传感器的灵敏度高,但存在测试时间相对较长,操作复杂的弊端;混合自组装单层膜压电免疫传感器操作简单,检测的灵敏度可以满足血吸虫检测的需要,但存在传感器和测试仪器成本较高,仪器抗干扰性差的问题;基于印刷电极的碳纳米管修饰的溶胶-凝胶传感器检测时间短,仪器和传感器成本低,检测的灵敏度可以满足血吸虫病检测的需要,但目前传感器的重现性与实际应用还存在一定差距;纳米碳颗粒标记的层析免疫试条使用方便,测试时间短,检测的灵敏度可以满足血吸虫筛查的需要,具有一定的实用性,但目前的研究成果尚限于半定量的检测。
Background
Schistosomiasis 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.
Objective
In 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 H_2O_2 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: 2×10~5 to 1:10~4 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
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