鼠精细胞味觉传感器及其在苦味检测中的应用研究

发布时间：2018-03-02 01:26

本文关键词： 苦味检测细胞阻抗传感器精细胞钙离子检测　出处：《浙江大学》2017年硕士论文　论文类型：学位论文

【摘要】：苦味感受作为人们的一种防御机制,在人的生活中起到很重要的作用,如今苦味评价广泛应用于食品药品中,用于检测毒性,研究苦味掩味剂等方面。由于传统的人尝味的方法具有很大的不确定性,近年来基于化学和生物的电子舌得到广泛的发展,然而存在用于检测的敏感性膜检测的单一性和细胞敏感元件较难获取等缺陷。味觉细胞是通过G蛋白偶联受体(GPCR)来检测苦味的,研究发现小鼠精细胞中含有大量的苦味基因的表达,可以用来检测苦味化合物,当苦味化合物激活GPCR后,一系列的级联反应会引起细胞骨架的变化以及胞内钙离子浓度的变化,细胞阻抗传感器可以检测出GPCR引起的细胞形态的变化,利用钙离子荧光探针可以检测胞内的钙离子的浓度变化。基于以上,本文详细研究了检测苦味化合物浓度的方法。本论文的主要工作和创新点如下:1.深入研究了细胞阻抗传感器及其检测系统的设计和应用本文研究了细胞阻抗传感器的细胞阻抗模型,阻抗传感器检测细胞的状态的原理,阻抗传感器在GPCR检测中的应用。研究了细胞阻抗传感器芯片的设计和加工流程,以及叉指电极的尺寸选取,接着介绍了整个阻抗传感器系统的构成。为后续实验提供检测系统。2.设计了基于鼠精细胞的阻抗传感器,提出了检测苦味化合物的新方法。本文构建了一种基于鼠精细胞的阻抗传感器系统用于检测苦味化合物。利用鼠精细胞作为初级敏感元件,阻抗传感器作为换能元件来检测苦味物质。利用该传感器检测了三种常见的化合物,该方法对苯硫脲线性检测浓度范围为10-200μmol/L,检测限为4μmol/L;奎宁检测线性范围为62.5-1000μmol/L,检测限为40μmol/L。苯甲地那铵的线性范围为50-1000μmol/L,检测限为13.5μmol/L,并且对苦味检测有良好的特异性。相对于传统的利用重组细胞来检测苦味物质来说,原代的精细胞具有获取简单,数量大的优势,并且阻抗传感器可以对细胞的变化进行实时无损的监测,操作简单,在苦味评价方面具有很大的应用潜力。3.提出结合鼠精细胞的流式细胞仪和钙离子荧光探针检测苦味物质的方法本文提出一种用鼠精细胞作为初级敏感元件,利用Fluo4-AM和流式细胞仪来检测胞内钙离子浓度从而检测苦味化合物浓度的方法。该方法对苯硫脲的线性检测浓度范围为25μmol/L-1000μmol/L,检测限为25.1μmol/L;奎宁的检测线性范围为50μmol/L-20000μmol/L,检测限为5.7μmol/L。苯甲地那铵的线性范围为50μmol/L-4000μmol/L,检测限为15.6μmol/L。并且对苦味检测有良好的特异性。该方法与传统的钙离子荧光成像方法相比,具有操作简单,容易定量计算的优点,具有一定的应用前景。
[Abstract]:Bitter taste, as a defense mechanism, plays a very important role in people's life. Nowadays, bitter taste evaluation is widely used in food and medicine to detect toxicity. In recent years, electronic tongue based on chemistry and biology has been widely developed because of the great uncertainty of traditional human taste methods. However, there are defects such as the singularity of sensitive membrane detection for detection and the difficulty in obtaining cell sensitive elements. Taste cells are used to detect bitter taste through G-protein-coupled receptor (GPCR). It was found that mouse spermatocytes contain a large number of bitter taste genes, which can be used to detect bitter compounds. When the bitter compounds activate GPCR, a series of cascade reactions will cause changes in cytoskeleton and intracellular calcium concentration. Cell impedance sensors can detect changes in cell morphology caused by GPCR, and calcium fluorescence probes can be used to detect changes in intracellular calcium concentrations. The main work and innovation of this thesis are as follows: 1. The design and application of cell impedance sensor and its detection system are studied. Cell impedance model, The principle of impedance sensor for cell state detection, the application of impedance sensor in GPCR detection, the design and fabrication process of cell impedance sensor chip, and the size selection of cross finger electrode are studied. Secondly, the constitution of the whole impedance sensor system is introduced. The impedance sensor based on mouse spermatocyte is designed. A new method for the detection of bitter compounds was proposed. An impedance sensor system based on mouse spermatocytes was constructed for the detection of bitter compounds. The impedance sensor is used as an energy transfer element to detect bitter substances. The sensor is used to detect three common compounds. The linear detection range for phenylthiourea is 10-200 渭 mol / L, the detection limit is 4 渭 mol / L, the linear range of quinine is 62.5-1000 渭 mol / L, the detection limit is 40 渭 mol / L, the linear range of bendenaammonium is 50-1000 渭 mol / L, the detection limit is 13.5 渭 mol / L, and has good specificity for bitter taste detection. For the traditional use of recombinant cells to detect bitter substances, The primary spermatocytes have the advantages of simple acquisition and large quantity, and the impedance sensor can monitor the changes of the cells in real time and lossless, and the operation is simple. There is great application potential in bitter taste evaluation. 3. A method for detecting bitter substances by flow cytometry combined with mouse spermatogonia and calcium fluorescence probe is proposed in this paper, which uses mouse spermatogonia as a primary sensitive element. Fluo4-AM and flow cytometry were used to detect intracellular calcium concentration and the concentration of bitter compounds. The linear detection range for phenylthiourea was 25 渭 mol/L-1000 渭 mol / L, the detection limit was 25.1 渭 mol / L, and the linear range for quinine was 50 渭 mol/L-20000 渭 mol / L, respectively. The linear range of benzodimethylammonium is 50 渭 mol/L-4000 渭 mol / L, and the detection limit is 15.6 渭 mol / L. this method has good specificity for bitter taste detection. It has the advantages of simple operation, easy quantitative calculation, and has a certain application prospect.
【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】：R318;TP212

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