胶体金免疫层析技术快速检测水中微囊藻毒素-LR的研究
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摘要
湖泊河流富营养化导致的蓝藻水华已成为国内外普遍关注的环境问题,它所带来的主要危害之一是产生的藻毒素对环境生态系统的影响。在已发现的藻毒素中,微囊藻毒素(microcystins, MCs)因其分布广、毒性大、危害严重,而备受关注。目前尚缺乏防止蓝藻水华发生的有效措施,因此要预防和消除MCs对人畜的危害,检测和控制MCs在各种水体和藻类食品中的限量是行之有效的方法。
目前国内外检测微囊藻毒素的方法有：生物测试法,化学分析法,免疫法等。总体上看这些微囊藻毒素的检测技术都还不能适应越来越迫切的现场检测的需求。大多数藻毒素标准品价格昂贵,购买困难,而相关的检测方法需消耗的标准品多,检测成本较高,限制了许多监测机构开展该项目的检测；仪器法需配置价格昂贵,体积庞大的HPLC-MS或GC-MS等,样品制备过程复杂且耗时长,且需要专业技术人员操作,检测费用很高,限制了常规监测次数。因此迫切需要建立一种工作原理简单易行、分析速度快、灵敏度较高的统一检测方法,对水体特别是饮用水源中的微囊藻毒素进行检测。
本研究在免疫层析快速检测技术的基础上,采用竞争法建立了检测水体中微囊藻毒素-LR的检测系统,此系统由玻璃纤维膜为样品垫,玻璃纤维膜为结合垫,NC膜为层析膜,吸水纸为吸收垫,单面胶PVC板为底板组成。本文通过优化金标抗体制备条件,改变测试线、质控线位置以及其包被抗体浓度等参数,观察这些参数对检测系统的影响,以达到优化检测系统的目的。本文研制的检测系统采用试纸条的形式,能够在10min内完成对水体中的微囊藻毒素-LR的检测,最低检测限为8ng/mL。该法操作简单,无需样品前处理,无需大型仪器或专业技术人员,特别适合现场实时在线监测,快速筛选。本研究也可为其他水污染监测试纸条的研制提供借鉴。
With the deterioration of eutrophication, the problem of microcystins produced by some algae has become a highlight research topic all over the word. But routine monitoring of microcystins in natural waters is very difficult because the concentration of the toxin is low and the detection method is usually complicated. It is urgent to develop a rapid, sensitive and efficient microcystins detection technology.
At present, detecting methods for microcystins are biological testing, chemical analysis, immunization testing and so on. Overall, all these current detecting methods for microcystins can not meet the practice demands. the high cost of detection, the complex and time-consuming sample preparation, and the bulky equipments. all these limited the number of routine monitorings and its widely use.
In this study, A gold immuno-chromatographic assay(GICA)for detection of microcystin-LR in waters was developed with the sample pad, the conjugate pad, NC membrane for purified Goat-anti-mouse IgG and McAb to MC-LR. This paper aims to optimize test system by changing the antibody-colloidal gold conjugates, the test line's position, antibody's concentration on the test line and the marker material and observing the influences of these parameters on the test system.
The new dipstick sensor, in form of strips, is capable of detecting limit down to 8 ng/mL for microcystin-LR, and the samples can be detected at the test site within 10 minutes, without having to bring samples back to the laboratory or need any equipments, trained technicians. The method producing GICA for microcystin-LR could be used for reference in the development of the detetion of other environmental toxins.
目前国内外检测微囊藻毒素的方法有：生物测试法,化学分析法,免疫法等。总体上看这些微囊藻毒素的检测技术都还不能适应越来越迫切的现场检测的需求。大多数藻毒素标准品价格昂贵,购买困难,而相关的检测方法需消耗的标准品多,检测成本较高,限制了许多监测机构开展该项目的检测；仪器法需配置价格昂贵,体积庞大的HPLC-MS或GC-MS等,样品制备过程复杂且耗时长,且需要专业技术人员操作,检测费用很高,限制了常规监测次数。因此迫切需要建立一种工作原理简单易行、分析速度快、灵敏度较高的统一检测方法,对水体特别是饮用水源中的微囊藻毒素进行检测。
本研究在免疫层析快速检测技术的基础上,采用竞争法建立了检测水体中微囊藻毒素-LR的检测系统,此系统由玻璃纤维膜为样品垫,玻璃纤维膜为结合垫,NC膜为层析膜,吸水纸为吸收垫,单面胶PVC板为底板组成。本文通过优化金标抗体制备条件,改变测试线、质控线位置以及其包被抗体浓度等参数,观察这些参数对检测系统的影响,以达到优化检测系统的目的。本文研制的检测系统采用试纸条的形式,能够在10min内完成对水体中的微囊藻毒素-LR的检测,最低检测限为8ng/mL。该法操作简单,无需样品前处理,无需大型仪器或专业技术人员,特别适合现场实时在线监测,快速筛选。本研究也可为其他水污染监测试纸条的研制提供借鉴。
With the deterioration of eutrophication, the problem of microcystins produced by some algae has become a highlight research topic all over the word. But routine monitoring of microcystins in natural waters is very difficult because the concentration of the toxin is low and the detection method is usually complicated. It is urgent to develop a rapid, sensitive and efficient microcystins detection technology.
At present, detecting methods for microcystins are biological testing, chemical analysis, immunization testing and so on. Overall, all these current detecting methods for microcystins can not meet the practice demands. the high cost of detection, the complex and time-consuming sample preparation, and the bulky equipments. all these limited the number of routine monitorings and its widely use.
In this study, A gold immuno-chromatographic assay(GICA)for detection of microcystin-LR in waters was developed with the sample pad, the conjugate pad, NC membrane for purified Goat-anti-mouse IgG and McAb to MC-LR. This paper aims to optimize test system by changing the antibody-colloidal gold conjugates, the test line's position, antibody's concentration on the test line and the marker material and observing the influences of these parameters on the test system.
The new dipstick sensor, in form of strips, is capable of detecting limit down to 8 ng/mL for microcystin-LR, and the samples can be detected at the test site within 10 minutes, without having to bring samples back to the laboratory or need any equipments, trained technicians. The method producing GICA for microcystin-LR could be used for reference in the development of the detetion of other environmental toxins.
引文
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[4]张琳.饮用水源地微囊藻毒素及其高级氧化技术去除研究.贵州师范大学硕士学位论文2.
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