本文關(guān)鍵詞： 苦味檢測 細(xì)胞阻抗傳感器 精細(xì)胞 鈣離子檢測　出處：《浙江大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：苦味感受作為人們的一種防御機(jī)制,在人的生活中起到很重要的作用,如今苦味評價(jià)廣泛應(yīng)用于食品藥品中,用于檢測毒性,研究苦味掩味劑等方面。由于傳統(tǒng)的人嘗味的方法具有很大的不確定性,近年來基于化學(xué)和生物的電子舌得到廣泛的發(fā)展,然而存在用于檢測的敏感性膜檢測的單一性和細(xì)胞敏感元件較難獲取等缺陷。味覺細(xì)胞是通過G蛋白偶聯(lián)受體(GPCR)來檢測苦味的,研究發(fā)現(xiàn)小鼠精細(xì)胞中含有大量的苦味基因的表達(dá),可以用來檢測苦味化合物,當(dāng)苦味化合物激活GPCR后,一系列的級聯(lián)反應(yīng)會引起細(xì)胞骨架的變化以及胞內(nèi)鈣離子濃度的變化,細(xì)胞阻抗傳感器可以檢測出GPCR引起的細(xì)胞形態(tài)的變化,利用鈣離子熒光探針可以檢測胞內(nèi)的鈣離子的濃度變化�；谝陨�,本文詳細(xì)研究了檢測苦味化合物濃度的方法。本論文的主要工作和創(chuàng)新點(diǎn)如下:1.深入研究了細(xì)胞阻抗傳感器及其檢測系統(tǒng)的設(shè)計(jì)和應(yīng)用本文研究了細(xì)胞阻抗傳感器的細(xì)胞阻抗模型,阻抗傳感器檢測細(xì)胞的狀態(tài)的原理,阻抗傳感器在GPCR檢測中的應(yīng)用。研究了細(xì)胞阻抗傳感器芯片的設(shè)計(jì)和加工流程,以及叉指電極的尺寸選取,接著介紹了整個阻抗傳感器系統(tǒng)的構(gòu)成。為后續(xù)實(shí)驗(yàn)提供檢測系統(tǒng)。2.設(shè)計(jì)了基于鼠精細(xì)胞的阻抗傳感器,提出了檢測苦味化合物的新方法。本文構(gòu)建了一種基于鼠精細(xì)胞的阻抗傳感器系統(tǒng)用于檢測苦味化合物。利用鼠精細(xì)胞作為初級敏感元件,阻抗傳感器作為換能元件來檢測苦味物質(zhì)。利用該傳感器檢測了三種常見的化合物,該方法對苯硫脲線性檢測濃度范圍為10-200μmol/L,檢測限為4μmol/L;奎寧檢測線性范圍為62.5-1000μmol/L,檢測限為40μmol/L。苯甲地那銨的線性范圍為50-1000μmol/L,檢測限為13.5μmol/L,并且對苦味檢測有良好的特異性。相對于傳統(tǒng)的利用重組細(xì)胞來檢測苦味物質(zhì)來說,原代的精細(xì)胞具有獲取簡單,數(shù)量大的優(yōu)勢,并且阻抗傳感器可以對細(xì)胞的變化進(jìn)行實(shí)時無損的監(jiān)測,操作簡單,在苦味評價(jià)方面具有很大的應(yīng)用潛力。3.提出結(jié)合鼠精細(xì)胞的流式細(xì)胞儀和鈣離子熒光探針檢測苦味物質(zhì)的方法本文提出一種用鼠精細(xì)胞作為初級敏感元件,利用Fluo4-AM和流式細(xì)胞儀來檢測胞內(nèi)鈣離子濃度從而檢測苦味化合物濃度的方法。該方法對苯硫脲的線性檢測濃度范圍為25μmol/L-1000μmol/L,檢測限為25.1μmol/L;奎寧的檢測線性范圍為50μmol/L-20000μmol/L,檢測限為5.7μmol/L。苯甲地那銨的線性范圍為50μmol/L-4000μmol/L,檢測限為15.6μmol/L。并且對苦味檢測有良好的特異性。該方法與傳統(tǒng)的鈣離子熒光成像方法相比,具有操作簡單,容易定量計(jì)算的優(yōu)點(diǎn),具有一定的應(yīng)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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R318;TP212

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