基于核酸适体传感器的赭曲霉毒素A检测方法研究
发布时间：2018-11-03 06:58
【摘要】：在农作物的生长、收获、储存和运输过程中,很容易受到真菌毒素的污染。赭曲霉毒素A(Ochratoxin A,OTA)是毒性强的真菌毒素之一,由于其对人体有致畸、致癌作用而受到特别的关注。常规的仪器分析法如高效液相色谱法因灵敏度高、结果准确可靠而受到检测机构的青睐,但样品前处理过程复杂,仪器昂贵,无法满足批量样品的现场快速检测,难以在基层实验室推广使用;基于抗原-抗体结合的酶联免疫分析方法具有简单、快速、易于推广等优点,然而,抗体制备过程复杂耗时、成本高,且抗体本身具有不稳定性,受免疫源性和假阳性等限制,不能作为最终的确证方法,妨碍了其更广泛的应用。本文利用核酸适体亲和力强、稳定性高、可进行官能团修饰等优势,结合电化学方法具有响应快、操作简单、仪器易于微型化等优点,利用碳基材料比表面积大、导电性好等特性,构建了基于炭气凝胶和羧基化多孔碳-纳米金的核酸适体电化学传感器用于快速检测粮食作物中OTA。具体的研究内容如下:1、将核酸适体作为识别元件固定在金电极(AuE)表面,利用炭气凝胶生物相容性好、比表面积大、导电性强等特性固载互补DNA(cDNA),并通过与核酸适体杂交连接到电极上,制备炭气凝胶-cDNA/适体/AuE电流型传感器,以亚甲基蓝(MB)为电化学信号探针,进行OTA的检测研究。研究发现,MB在炭气凝胶-cDNA/适体/AuE传感器上的电流值比在cDNA/适体/AuE传感器上增大100.4%,说明炭气凝胶具有良好的信号放大作用。对实验参数进行优化,得到最佳实验条件:核酸适体最佳浓度为4μmol/L,核酸适体和cDNA杂交时间为2h,MB的最佳固定方式为浸泡15min,OTA的最佳孵育时间为18min。在最优条件下,利用构建的传感器对OTA进行检测,当OTA浓度在0.01-20ng/mL时具有良好的线性关系,检出限是1.0×10-4ng/mL,相关系数是0.996。利用加标回收法对玉米样品进行检测,测得平均回收率为89.3%。2、前期实验发现,在AuE上固定核酸适体可能会影响其与OTA结合时的构象变化,因此本实验改变方案,采用在AuE上固定cDNA,同时利用多孔碳具有开放的孔隙结构、比表面积大、导电性强等优点,结合纳米导电性好及生物亲和能力强等特性,制备羧基化多孔碳-纳米金复合材料修饰AuE,用于提高cDNA的固载量并实现信号放大,通过杂交连接核酸适体,构建适体/cDNA/羧基化多孔碳-纳米金/AuE电流型传感器。MB在适体/cDNA/羧基化多孔碳-纳米金/AuE上的电流值比在适体/cDNA/AuE电流值增大了175.6%,说明羧基化多孔碳-纳米金具有良好的信号放大作用。利用该传感器对OTA进行检测,当OTA浓度为5.0×10-6-5.0×10-4ng/mL时具有良好的线性关系,相关系数是0.997,检出限是5.0×10-6ng/mL。对玉米样品进行检测,平均回收率为103%。对传感器进行性能研究,实验结果表明该传感器具有良好的重复性、稳定性和特异性。
[Abstract]:During the growth, harvest, storage and transportation of crops, they are easily contaminated by mycotoxins. Ochratoxin (A (Ochratoxin) is one of the most toxic mycotoxins, and has attracted special attention because of its teratogenic and carcinogenic effects. Conventional instrument analysis methods such as high performance liquid chromatography (HPLC) are favored by the detection organizations because of their high sensitivity and accuracy and reliability. However, the process of sample pretreatment is complicated and the instrument is expensive, so it can not meet the needs of rapid field detection of batch samples. It is difficult to popularize it in the basic laboratory; The enzyme-linked immunosorbent assay (Elisa) based on antigen-antibody binding has the advantages of simple, rapid and easy to be popularized. However, the preparation process of antibody is complicated, time-consuming and costly, and the antibody itself is unstable and restricted by immunogenic and false positive. It cannot be used as a final confirmation method, which hinders its wider application. In this paper, the advantages of strong aptamer affinity, high stability, functional group modification and electrochemical method are used, such as fast response, simple operation, easy miniaturization of instruments, and large specific surface area of carbon based materials. The aptamer electrochemical sensor based on carbon aerogel and carboxylated porous carbon-nano gold was constructed for rapid detection of OTA. in grain crops. The specific research contents are as follows: 1. Nucleic acid aptamer is immobilized on the surface of gold electrode (AuE) by using carbon aerogel as a recognition element. The carbon aerogels have good biocompatibility, large specific surface area and strong conductivity to immobilize complementary DNA (cDNA),. Carbon aerogel-cDNA/ aptamer / AuE current mode sensor was prepared by hybridization with aptamer of nucleic acid. The detection of OTA was carried out using methylene blue (MB) as electrochemical signal probe. It is found that the current value of MB on the carbon aerogel-cDNA/ aptamer / AuE sensor is 100.4 higher than that on the cDNA/ aptamer / AuE sensor, which indicates that the carbon aerogel has a good signal amplification effect. The optimum conditions were obtained as follows: the optimal concentration of aptamer was 4 渭 mol/L, aptamer and cDNA hybridization time was 2hmb. The best way of immobilization was that the optimal incubation time was 18 mins after soaking for 15 min. Under the optimal conditions, the OTA was detected by using the constructed sensor. When the concentration of OTA was in 0.01-20ng/mL, there was a good linear relationship. The detection limit was 1.0 脳 10 ~ (-4) ng / mL, and the correlation coefficient was 0.996. The average recovery rate of maize samples was 89.30.2 by using the method of adding standard recovery. It was found in previous experiments that fixing aptamers on AuE might affect the conformation changes of AuE when it was combined with OTA, so the scheme was changed in this experiment. Using cDNA, fixed on AuE while using porous carbon has the advantages of open pore structure, large specific surface area, strong conductivity and so on. Carboxylated porous carbon-nano-gold composite modified AuE, was prepared to increase the amount of cDNA and to amplify the signal to connect the aptamer of nucleic acid by hybridization. An aptamer / cDNA/ carboxylation porous carbon-nanocrystalline gold / AuE current-mode sensor was constructed. The current value of MB on aptamer / cDNA/ carboxylated porous carbon-nanocrystalline gold / AuE was 175.6% higher than that on aptamer / cDNA/AuE. The results show that carboxylated porous carbon-nanocrystalline gold has a good signal amplification effect. When the concentration of OTA is 5.0 脳 10 ~ (-6) ~ 5.0 脳 10-4ng/mL, the correlation coefficient is 0.997 and the detection limit is 5.0 脳 10 ~ (-6) ng / mL. The average recovery of corn sample was 103. The experimental results show that the sensor has good repeatability, stability and specificity.
【学位授予单位】：河南工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS210.7;TP212.9
本文编号：2307041
[Abstract]:During the growth, harvest, storage and transportation of crops, they are easily contaminated by mycotoxins. Ochratoxin (A (Ochratoxin) is one of the most toxic mycotoxins, and has attracted special attention because of its teratogenic and carcinogenic effects. Conventional instrument analysis methods such as high performance liquid chromatography (HPLC) are favored by the detection organizations because of their high sensitivity and accuracy and reliability. However, the process of sample pretreatment is complicated and the instrument is expensive, so it can not meet the needs of rapid field detection of batch samples. It is difficult to popularize it in the basic laboratory; The enzyme-linked immunosorbent assay (Elisa) based on antigen-antibody binding has the advantages of simple, rapid and easy to be popularized. However, the preparation process of antibody is complicated, time-consuming and costly, and the antibody itself is unstable and restricted by immunogenic and false positive. It cannot be used as a final confirmation method, which hinders its wider application. In this paper, the advantages of strong aptamer affinity, high stability, functional group modification and electrochemical method are used, such as fast response, simple operation, easy miniaturization of instruments, and large specific surface area of carbon based materials. The aptamer electrochemical sensor based on carbon aerogel and carboxylated porous carbon-nano gold was constructed for rapid detection of OTA. in grain crops. The specific research contents are as follows: 1. Nucleic acid aptamer is immobilized on the surface of gold electrode (AuE) by using carbon aerogel as a recognition element. The carbon aerogels have good biocompatibility, large specific surface area and strong conductivity to immobilize complementary DNA (cDNA),. Carbon aerogel-cDNA/ aptamer / AuE current mode sensor was prepared by hybridization with aptamer of nucleic acid. The detection of OTA was carried out using methylene blue (MB) as electrochemical signal probe. It is found that the current value of MB on the carbon aerogel-cDNA/ aptamer / AuE sensor is 100.4 higher than that on the cDNA/ aptamer / AuE sensor, which indicates that the carbon aerogel has a good signal amplification effect. The optimum conditions were obtained as follows: the optimal concentration of aptamer was 4 渭 mol/L, aptamer and cDNA hybridization time was 2hmb. The best way of immobilization was that the optimal incubation time was 18 mins after soaking for 15 min. Under the optimal conditions, the OTA was detected by using the constructed sensor. When the concentration of OTA was in 0.01-20ng/mL, there was a good linear relationship. The detection limit was 1.0 脳 10 ~ (-4) ng / mL, and the correlation coefficient was 0.996. The average recovery rate of maize samples was 89.30.2 by using the method of adding standard recovery. It was found in previous experiments that fixing aptamers on AuE might affect the conformation changes of AuE when it was combined with OTA, so the scheme was changed in this experiment. Using cDNA, fixed on AuE while using porous carbon has the advantages of open pore structure, large specific surface area, strong conductivity and so on. Carboxylated porous carbon-nano-gold composite modified AuE, was prepared to increase the amount of cDNA and to amplify the signal to connect the aptamer of nucleic acid by hybridization. An aptamer / cDNA/ carboxylation porous carbon-nanocrystalline gold / AuE current-mode sensor was constructed. The current value of MB on aptamer / cDNA/ carboxylated porous carbon-nanocrystalline gold / AuE was 175.6% higher than that on aptamer / cDNA/AuE. The results show that carboxylated porous carbon-nanocrystalline gold has a good signal amplification effect. When the concentration of OTA is 5.0 脳 10 ~ (-6) ~ 5.0 脳 10-4ng/mL, the correlation coefficient is 0.997 and the detection limit is 5.0 脳 10 ~ (-6) ng / mL. The average recovery of corn sample was 103. The experimental results show that the sensor has good repeatability, stability and specificity.
【学位授予单位】：河南工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS210.7;TP212.9
【参考文献】
相关期刊论文 前3条
1 段诺;吴世嘉;王周平;;核酸适配体识别-荧光法检测赭曲霉素A[J];分析化学;2011年03期
2 许世超;张纪梅;姚翠翠;代昭;郑帼;孙波;佟魏;齐鲁;;以亚甲基蓝为杂交指示剂的DNA电化学传感器[J];精细化工;2008年12期
3 吴海霞;康敬万;李志峰;董树清;苏碧泉;卢小泉;;亚甲基蓝与DNA相互作用的电化学研究[J];分析测试学报;2006年04期
相关硕士学位论文 前1条
1 袁蓓;基于电化学传感器的黄曲霉毒素B_1及赭曲霉毒素A检测方法研究[D];河南工业大学;2016年
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