检测腹泻性贝类毒素大田软海绵酸的免疫传感器的研究

发布时间：2018-01-01 14:30

本文关键词：检测腹泻性贝类毒素大田软海绵酸的免疫传感器的研究　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：随着世界范围内环境恶化,海洋赤潮频发,赤潮藻类会产生多种海洋生物毒素,其中主要包括腹泻性、麻痹性和神经性毒素。其中,腹泻性毒素经过动物实验证明具有潜在的致癌性、致畸性和致突变性等,而短期内会出现腹泻、呕吐、晕眩等症状,对人体健康具有极大得威胁。因此急需快速、准确、灵敏的方法来检测腹泻性毒素中的主要种类-大田软海绵酸。本文首先简单介绍了腹泻性贝类毒素以及针对大田如海绵酸的检测方法,并简单阐述生物素-亲和素系统及纳米金在免疫传感器中的应用。然后设计研发了三种免疫传感器,包括将简单的免疫原理应用到研发试剂盒,将生物素-亲和素系统引入传统的免疫传感器实现信号放大,以及将纳米金和基于生物素-亲和素系统以及辣根辣根过氧化物酶抗原抗体的循环放大系统应用于构建更为灵敏的生物免疫传感器。三种传感器都能有效的应用于检测大田软海绵酸。本论文完成的主要工作和创新点如下:1.研制了新型的快速检测大田软海绵酸贝类毒素的试剂盒,设计了基于试剂盒的现场检测仪器。利用竞争性免疫反应,设计了一种用于检测大田软海绵酸(OA)的试剂盒。通过优化实验,该试剂盒的检测限为0.19 μg/L、线性范围0.2-10 μg/L、与鳍藻毒素(DTX)的交叉反应率为23.6%,对其他毒素的交叉反应率几乎为0,加标回收实验说明该试剂盒具有较好的回收率。该试剂盒对重金属离子具有较好的抗干扰性,且目前该试剂盒能保存到6个月。该试剂盒的检测时间在90min内,且成本是Beacon试剂盒的1/30。基于开发的试剂盒,完成了现场光学检测仪器Bionic e-Eye的设计,该仪器操作简单,直接显示结果,结合试剂盒非常适用于现场快速检测和筛查,实际样品的检测结果与高效液相-质谱联用((HPLC-MS)和酶标仪都有很好的一致性。2.提出了结合生物素-亲和素的免疫传感器设计方法,提高检测OA灵敏度的设计方法在试剂盒的基础上,引入生物素-亲和素系统,设计了信号放大的生物传感器。该传感器的检测限为0.04 μg/L,线性范围为0.01-10 μg/L,相比于试剂盒,该传感器的检测限降低了 5倍,线性范围的下限降低了 20倍;另外,该传感器对DTX1的交叉反应率为44.14%,对其他毒素的交叉反应率几乎为0,加标回收实验证明该传感器用于检测OA的回收率范围在91.09%-111.86%之间,变异系数CV低于15%。检测OA与DTX1混合加标的回收率范围在81.98-132.61%,说明该传感器对于检测OA与DTX1都具有较好的准确性。3、提出了结合纳米金和生物素-亲和素的级联放大的免疫传感器,实现了对OA超低浓度的检测。纳米金具有很大的比表面积且与生物分子具有较高的亲和力,因此,使其表面结合二抗-辣根过氧化物酶(SAb-HRP)和HRP,有了很大程度的信号放大。另外,通过引入生物素-亲和素系统以及辣根过氧化物酶及其抗体形成的免疫复合物构成三级级联放大,形成具有更强放大作用的生物传感器,该级联放大免疫传感器的检测限为0.0025 μg/L,线性范围为0.002-5 μg/L,线性区间的下限0.002μg/L比试剂盒0.2μg/L低了 100倍,并且,该传感器对DTX1的交叉反应率为19.79%,对其他毒素的交叉反应率几乎为0;该传感器的回收率的范围约在76.07%-94.63%之间,变异系数CV也低于15%,实现了对大田软海绵酸的高灵敏检测。
[Abstract]:With the worldwide environmental deterioration, frequent red tide, red tide algae will produce a variety of marine biological toxins, including diarrhea, paralysis and neurogenic toxin. The diarrhea of toxins through animal experiments that are potentially carcinogenic, teratogenic and mutagenic, but in the short term there will be diarrhea, vomiting. Dizziness and other symptoms, has a great threat to human health. So it is urgent to fast, accurate and sensitive method to detect the main field types diarrhetic toxin in okadaic acid. This paper introduces the diarrhetic shellfish toxoid and on the field detection method such as sponge acid, and briefly expounds the application of biotin avidin gold nanoparticles in the immune system and sensor. Then the design and development of three kinds of immune sensor, including the simple immune principle is applied to the development kit, the biotin avidin system The introduction of the traditional immune sensor signal amplification, and the gold nanoparticles and based on biotin avidin system and horseradish HRP antibody cycle amplification system was used to build more sensitive immunobiosensor. Three kinds of sensors can effectively be used for detecting okadaic acid. This paper completed the main work and innovation are as follows: 1. the development kit for rapid detection of okadaic acid shellfish toxin model, designed the instrument kit based on using competitive immune response, a design for detection of okadaic acid (OA) kit. Through the optimization experiments, the detection kit the limit is 0.19 g/L, the linear range of 0.2-10 g/L, and dinophysistoxin (DTX) of the cross reaction rate was 23.6% and the cross reaction with other toxins rate is almost 0, recovery experiments that the reagent The box has a better recovery. The kit has better anti-interference of heavy metal ions, and the kit can save up to 6 months. The detection time of the kit in the 90min, and the cost is 1/30. Beacon kit kit based on the development of the designed field optical detector Bionic e-Eye, the instrument has the advantages of simple operation, direct display results, combined with the kit is very suitable for on-site rapid detection and screening, detection results of actual samples with high performance liquid chromatography - mass spectrometry ((HPLC-MS) and eliasa have good consistency with.2. made with the avidin immune sensor design in the method, improve the design method of OA based on sensitivity detection kit, the introduction of a biotin avidin system designed biosensor signal amplification. The detection limit of the sensor is 0.04 g/L, the linear range of 0.01-10 g/ Compared to the L kit, the detection limit of the sensor is reduced by 5 times, the lower limit of the linear range is reduced by 20 times; in addition, the sensor cross reaction rate of DTX1 was 44.14%. The cross reaction with other toxins rate is almost 0, recovery experiments show that the detection rate of OA sensor for the recovery in the range of 91.09%-111.86% standard addition, variation coefficient of CV is lower than that of the 15%. detection of OA and DTX1 mixed with standard recovery rate in the range of 81.98-132.61%, indicating that the sensor has good accuracy for the detection of.3 OA and DTX1, put forward a combination of gold nanoparticles and avidin immune sensor in cascade, achieves the detection of low concentration OA the nano fittings with great specific surface area and biological molecules have high affinity, so that its surface with two anti horseradish peroxidase (SAb-HRP) and HRP, a large degree of signal amplification. In addition, Through the introduction of biotin avidin system and horseradish peroxidase antibody and the formation of immune complexes composed of three cascaded amplification, the formation of biosensor has stronger amplification and detection of the immune cascade limit of the sensor is 0.0025 g/L, the linear range of 0.002-5 g/L, the lower limit of linear range of 0.002 g/L than kit 0.2 g/L 100 times lower, and the sensor cross reaction rate of DTX1 was 19.79%. The cross reaction with other toxins rate of almost 0; the recovery rate of the sensor range is about 76.07%-94.63%, the variation coefficient of CV is less than 15%, to achieve a high sensitive detection of okadaic acid on the field.

【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R155.3;TP212.3

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