细胞和分子传感器及其在海洋生物毒素检测中的应用研究

发布时间：2018-04-18 18:53

本文选题：细胞阻抗传感器 + 声表面波传感器　；参考：《浙江大学》2015年博士论文

【摘要】：海洋生物毒素是一类由赤潮藻产生的小分子化合物,通过贝类滤食后进入贝肉体内并且完成生物富集。一旦人们误食了被毒素污染的贝肉,就会引起中毒,严重时甚至会导致死亡。近年来,随着环境污染的加剧,由于误食被污染的贝类而引起的人类死亡事件也常有报道。目前用来检测海洋毒素的方法主要有小鼠生物法(MBA)、酶联免疫吸附法(ELISA)、高效液相色谱法(HPLC)和液相色谱质谱联用法(LC-MS/MS)等。然而,这些方法在实际应用中都有一定的局限性,例如重复性差、设备昂贵以及涉及动物伦理等问题。作为一门新兴的检测技术,生物传感器是一种以细胞分子等生物材料为敏感元件,结合了物理化学等二级传感器来实现对于各种物质进行检测的分析系统。由于其具有操作简单、分析速度快、灵敏度高、小型化和容易实现自动化等优点,生物传感器在细胞生理、药物筛选以及食物检测等领域都有着广泛的应用。本论文基于细胞分子水平上检测海洋毒素的机理不同,结合了特定类型的二级传感器与检测系统,分别从细胞、抗体和适配体三个方面分别研究了生物传感器在海洋毒素检测中的应用潜力。本论文的主要的创新点工作包括：1.提出了一种基于细胞阻抗传感器的用于检测贝类腹泻性毒素的新方法构建了一种基于人类细胞株的细胞阻抗传感器,并将其应用到腹泻性毒素的检测中。当毒素浓度在10-100μg/L的范围内,该传感器检测到的细胞阻抗值与毒素的浓度呈良好的线性关系。并且通过检测其他毒素与贝肉基质的干扰实验,可以证明该方法具有很好的特异性与重复性,且与小鼠生物法有着很好的相关性。说明该方法对于贝肉中腹泻性毒素的检测方面具有很好的应用潜力。2.提出了一种基于神经瘤细胞的阻抗传感器的麻痹性毒素检测的改进方法基于麻痹性毒素能够特异性结合细胞膜上钠离子通道的特性,建立了一种新的基于小鼠神经瘤细胞的阻抗传感器检测麻痹性毒素的方法。采用了细胞钠离子通道激活剂的藜芦定和钠钾泵抑制剂的乌本苷共同作用小鼠神经瘤细胞,细胞会因为钠离子持续性内流而引起死亡,但是麻痹性毒素可以通过阻碍钠离子内流来延缓藜芦定和乌本苷引起的细胞死亡。当麻痹性毒素的代表性毒素石房蛤毒素(Saxitoxin, STX)浓度在0.1-1000nM的范围内,细胞的相对存活率与STX浓度的对数呈良好的线性关系。且该方法具有很好的特异性与灵敏度,可以用于麻痹性毒素的实际样品检测。3.提出了一种基于纳米金修饰的声表面波免疫传感器,实现了大田软海绵酸(OA)的特异性检测由于OA的分子量很小,因此本章采用免疫竞争分析法来检测贝肉中OA的含量。纳米金标记的二抗能够放大传感器的检测信号,且传感器检测信号与OA浓度在10-150ng/mL之间有着很好的线性关系,检测限为5.45ng/mL。并且通过与ELISA方法的对比实验,说明该方法具有很好的特异性和准确性,且能够用于贝肉实际样品中OA的特异性检测。4.设计了一种基于核酸适配体的声表面波传感器用于OA的高灵敏检测基于核酸适配体能够特异性结合OA分子以及DNA双链互补杂交的特性,设计一种基于核酸适配体的三明治结构用来检测样品中的OA分子。当存在OA分子时,适配体链便会与OA形成特异的结构,从而从传感器表面脱落下来,引起传感器的相位幅值增大。当OA浓度在1-100ng/mL范围内,传感器相位幅值的增大与OA浓度之间呈一定的线性关系,且检测限为0.3ng/mL。该方法为生物毒素的检测提供了一个新的思路,且三明治结构的设计也具有一定的通用性。此外,适配体作为敏感元件不仅降低了检测成本也缩短了检测时间。
[Abstract]:Marine biological toxins are small molecular compounds produced by algae and shellfish by Filter Feeding Shellfish after entering the body and complete biological enrichment. Once people are eating the contaminated shellfish toxins, can cause poisoning, even death. In recent years, with the aggravation of environmental pollution, due to the ingestion of the event the contaminated shellfish caused human death are often reported. Current methods for detecting marine toxins include mouse bioassay (MBA), enzyme linked immunosorbent assay (ELISA), high performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS/MS). However, these methods are there are some limitations in the practical application, such as poor repeatability, expensive equipment and relates to animal ethics problems. As a new detection technology, biosensor is a kind of cell molecular biological materials as a sensitive element, combined with The physical and chemical sensors to achieve level two for all kinds of material for detection and analysis system. Because of its simple operation, fast analysis, high sensitivity, miniaturization and easy to realize the advantages of automated biosensors in the field of cell physiology, drug screening and detection of food have extensive application. The mechanism of cell molecular detection of marine toxin based on the different combination of two level sensor and detection of specific types of systems, respectively, from the three aspects of cells, antibodies and aptamers were studied using potential biosensor in marine toxin detection. The main innovation work of this dissertation includes: 1. propose a new the method of cell impedance sensor for the detection of diarrhetic shellfish toxin was constructed based on a cell impedance sensor based on human cell lines, and its application to the diarrhea of Toxin detection. When the toxin concentration in the range of 10-100 g/L, the cell impedance sensor detects the value of a good linear relationship between concentration and toxin. And through the experiment of other shellfish toxins and interference detection matrix, it can be proved that the method has good specificity and repeatability, and a good correlation with mouse bioassay. The method presents an improved method based on the paralytic toxin detection of the cell impedance sensor neuroma paralysis toxin binds to the cell membrane properties of sodium channels based on the detection of diarrhetic shellfish meat toxin.2. has very good application potential, established a new method for impedance sensor Neuro-2a cells based on the detection of paralytic toxin. The cell sodium channel inhibitor and veratridine activated sodium potassium pump inhibitor ouabain In the interaction of Neuro-2a cells, cells because of persistent sodium ion influx and cause death, but the PSP can prevent the deaths to delay veratridine and ouabain induced cell flow of sodium ions. Representative toxin saxitoxin as paralytic toxin (Saxitoxin, STX) concentration in 0.1-1000nM within the range of relative log survival rate and STX concentration showed a good linear relationship between the cells. And this method has good specificity and sensitivity, can be used for real sample detection of.3. paralytic toxin presents a surface acoustic wave immunosensor based on gold nanoparticles, the okadaic acid (OA) the specificity of detection due to the molecular weight of OA is very small, so this chapter uses immune competition to detect content of shellfish meat OA analysis method. Nano gold labeled anti two can amplify the detection signal and the sensing sensor. For detecting signal and the concentration of OA has a good linear relationship between 10-150ng/mL, the detection limit is 5.45ng/mL. and by comparison with the ELISA method, this method has good specificity and accuracy, and can be used for specific detection of.4. OA in actual samples of shellfish meat to design a surface acoustic wave sensor nucleic acid aptamer based on OA for highly sensitive detection of nucleic acid aptamer capable of specific binding of OA molecules and DNA double strand complementary hybridization based on the characteristics of the design of a sandwich structure of the aptamer for detection of OA molecules. Based on the presence of OA molecules, the aptamer chain will form a specific structure and OA, which dropped from the sensor surface, caused by phase amplitude of the sensor increases. When the concentration of OA in the range of 1-100ng/mL, the phase and amplitude increase between sensor and OA concentration showed a linear relationship The detection limit is 0.3ng/mL.. This method provides a new idea for the detection of biological toxins, and the design of sandwich structure is also universal. In addition, aptamers as sensitive components not only reduce the cost of detection, but also shorten the detection time.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R99;TP212.3

【参考文献】