适配体识别—分子马达传感器的构建及在食源性致病菌检测中的应用研究

发布时间：2018-07-22 16:04

【摘要】：食源性疾病已成为全球性的公共卫生问题,其中由食源性致病菌引起的食源性疾病发病率最高,对人类健康造成了巨大威胁。传统的食源性致病菌检测方法在检测时间、灵敏度和特异性上存在一定的缺陷,不能满足日益严格的检测需要。因此,发展更为快捷,方便、灵敏度高和特异性好的检测方法对保障食品安全具有重要意义。分子马达是一种生物大分子,广泛存在于细胞内,近年来得到国内外的普遍关注,其中旋转分子马达F_0F_1-ATPase作为生物传感器被多次应用于食源性致病菌的检测。该马达具有灵敏度高、特异性强等特点。因此本研究以分子马达F_0F_1-ATPase作为信号分子,以适配体作为识别分子,构建基于适配体识别的分子马达生物传感系统,并应用于食源性致病菌的检测,主要工作内容包括:首先,构建了一种基于适配体识别-F_0F_1-ATPase生物传感检测鼠伤寒沙门氏菌的方法。通过F_0F_1-ATPase表面ε亚基-ε亚基抗体-生物素-亲和素-生物素化的鼠伤寒沙门氏菌适配体,将适配体固定于载色体表面;同时利用p H敏感型荧光染料F-DHPE监测载色体膜外荧光强度的变化。当体系中存在沙门氏菌时,适配体特异性与沙门氏菌结合,使得F_0F_1-ATPase酶的负荷增加,引起旋转速度下降,从而导致泵出载色体外的质子流速率下降,继而p H值升高,最终导致荧光强度增强,通过监测荧光强度的变化实现对沙门氏菌的检测。结果显示,沙门氏菌在10~1-10~4 cfu/m L范围内与相对荧光强度具有良好的线性关系(y=1085.13x+363.98,R~2=0.9944),最低检测限为10 cfu/m L。该方法已成功应用于牛奶实际样品的检测。其次,构建了以量子点为荧光指示物的F_0F_1-ATPase-适配体生物传感检测副溶血性弧菌的方法。通过F_0F_1-ATPase表面ε亚基-ε亚基抗体-生物素-亲和素-生物素化的副溶血性弧菌适配体,将适配体固定于载色体表面;同时将p H敏感型量子点标记到载色体膜外,形成基于适配体识别的分子马达生物传感系统。当体系中存在副溶血性弧菌时,适配体特异性与副溶血性弧菌结合,使得F_0F_1-ATPase酶的负荷增加,引起旋转速度下降,继而引起H~+由载色体膜内泵出膜外的速率变慢,膜外的H~+浓度变小,p H值升高,量子点荧光强度增强,通过监测荧光强度的变化实现对副溶血性弧菌的检测。结果显示,副溶血性弧菌在50-10~(6 )cfu/m L范围内与体系相对荧光强度具有良好的线性关系(y=138.3x+13.05,R~2=0.9962),最低检测限为5 cfu/m L。所建立方法已经成功应用于市售虾的检测。
[Abstract]:Foodborne diseases have become a global public health problem, in which foodborne pathogens cause the highest incidence of foodborne diseases, which pose a great threat to human health. There are some defects in the detection time, sensitivity and specificity of the traditional methods for the detection of foodborne pathogenic bacteria, which can not meet the need of increasingly stringent detection. Therefore, it is important to develop a more rapid, convenient, sensitive and specific detection method to ensure food safety. Molecular motor is a kind of biological macromolecule, which widely exists in cells. In recent years, the molecular motor F0F1-ATPase has been widely used as a biosensor for the detection of foodborne pathogenic bacteria, among which the rotating molecular motor F0F1-ATPase has been widely concerned at home and abroad. The motor has the characteristics of high sensitivity and strong specificity. Therefore, in this study, the molecular motor FSP / F1-ATPase is used as signal molecule and the aptamer as recognition molecule to construct a molecular motor biosensor system based on aptamer recognition, and it is applied to the detection of foodborne pathogens. The main work includes: first of all, A biosensor method for detection of Salmonella typhimurium based on aptamer recognition-F0 F1-ATPase was developed. The aptamer of Salmonella typhimurium was immobilized on the surface of Color-carrying Salmonella typhimurium by using the antibody of 蔚 subunit 蔚 subunit on the surface of F0F1-ATPase-biotin-avidin-biotin-biotin aptamer of Salmonella typhimurium. At the same time, F-DHPE, a p-H sensitive fluorescent dye, was used to monitor the outside fluorescence intensity of the carrier. When salmonella was present in the system, the specific binding of aptamer to Salmonella caused the increase of the enzyme load of FSP _ 0F1-ATPase, which resulted in the decrease of rotation speed, which resulted in the decrease of proton velocity and the increase of pH value. Finally, the fluorescence intensity was enhanced, and the detection of Salmonella was realized by monitoring the change of fluorescence intensity. The results showed that there was a good linear relationship between the relative fluorescence intensity and salmonella in the range of 10 ~ 1010 cfu/m / L (yttrium 1085.13x 363.98 ~ 0.9944), and the detection limit was 10 cfu/m / L. The method has been successfully applied to the detection of milk samples. Secondly, a method for detecting Vibrio parahaemolyticus by using quantum dots as fluorescent indicator F0FSTs 1-ATPase- aptamer biosensor was constructed. The aptamer of Vibrio parahaemolyticus was immobilized on the surface of Vibrio parahaemolyticus by using the antibody of 蔚 subunit 蔚 subunit on the surface of F0F1-ATPase, and the pH-sensitive quantum dot was labeled outside the carrier membrane. A molecular motor biosensor system based on aptamer recognition is formed. When Vibrio parahaemolyticus exists in the system, the aptamer specificity binds to Vibrio parahaemolyticus, which increases the load of FSP _ 0F1-ATPase enzyme, decreases the speed of rotation, and then causes the rate of H ~ to pump out of the membrane of the chromogenic body to become slower. The fluorescence intensity of quantum dots increased with the increase of H concentration outside the film. The detection of Vibrio parahaemolyticus was realized by monitoring the change of fluorescence intensity. The results showed that the relative fluorescence intensity of Vibrio parahaemolyticus was linear in the range of 50-10 ~ (6) cfu/m L (yttrium 138.3x 13.05 cfu/m / L, 0.9962), and the lowest detection limit was 5 cfu/m / L. The established method has been successfully applied to the detection of commercial shrimp.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R155.3

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