基于核酸適體傳感器的赭曲霉毒素A檢測方法研究
發(fā)布時(shí)間：2018-11-03 06:58
【摘要】：在農(nóng)作物的生長、收獲、儲(chǔ)存和運(yùn)輸過程中,很容易受到真菌毒素的污染。赭曲霉毒素A(Ochratoxin A,OTA)是毒性強(qiáng)的真菌毒素之一,由于其對人體有致畸、致癌作用而受到特別的關(guān)注。常規(guī)的儀器分析法如高效液相色譜法因靈敏度高、結(jié)果準(zhǔn)確可靠而受到檢測機(jī)構(gòu)的青睞,但樣品前處理過程復(fù)雜,儀器昂貴,無法滿足批量樣品的現(xiàn)場快速檢測,難以在基層實(shí)驗(yàn)室推廣使用;基于抗原-抗體結(jié)合的酶聯(lián)免疫分析方法具有簡單、快速、易于推廣等優(yōu)點(diǎn),然而,抗體制備過程復(fù)雜耗時(shí)、成本高,且抗體本身具有不穩(wěn)定性,受免疫源性和假陽性等限制,不能作為最終的確證方法,妨礙了其更廣泛的應(yīng)用。本文利用核酸適體親和力強(qiáng)、穩(wěn)定性高、可進(jìn)行官能團(tuán)修飾等優(yōu)勢,結(jié)合電化學(xué)方法具有響應(yīng)快、操作簡單、儀器易于微型化等優(yōu)點(diǎn),利用碳基材料比表面積大、導(dǎo)電性好等特性,構(gòu)建了基于炭氣凝膠和羧基化多孔碳-納米金的核酸適體電化學(xué)傳感器用于快速檢測糧食作物中OTA。具體的研究內(nèi)容如下:1、將核酸適體作為識(shí)別元件固定在金電極(AuE)表面,利用炭氣凝膠生物相容性好、比表面積大、導(dǎo)電性強(qiáng)等特性固載互補(bǔ)DNA(cDNA),并通過與核酸適體雜交連接到電極上,制備炭氣凝膠-cDNA/適體/AuE電流型傳感器,以亞甲基藍(lán)(MB)為電化學(xué)信號(hào)探針,進(jìn)行OTA的檢測研究。研究發(fā)現(xiàn),MB在炭氣凝膠-cDNA/適體/AuE傳感器上的電流值比在cDNA/適體/AuE傳感器上增大100.4%,說明炭氣凝膠具有良好的信號(hào)放大作用。對實(shí)驗(yàn)參數(shù)進(jìn)行優(yōu)化,得到最佳實(shí)驗(yàn)條件:核酸適體最佳濃度為4μmol/L,核酸適體和cDNA雜交時(shí)間為2h,MB的最佳固定方式為浸泡15min,OTA的最佳孵育時(shí)間為18min。在最優(yōu)條件下,利用構(gòu)建的傳感器對OTA進(jìn)行檢測,當(dāng)OTA濃度在0.01-20ng/mL時(shí)具有良好的線性關(guān)系,檢出限是1.0×10-4ng/mL,相關(guān)系數(shù)是0.996。利用加標(biāo)回收法對玉米樣品進(jìn)行檢測,測得平均回收率為89.3%。2、前期實(shí)驗(yàn)發(fā)現(xiàn),在AuE上固定核酸適體可能會(huì)影響其與OTA結(jié)合時(shí)的構(gòu)象變化,因此本實(shí)驗(yàn)改變方案,采用在AuE上固定cDNA,同時(shí)利用多孔碳具有開放的孔隙結(jié)構(gòu)、比表面積大、導(dǎo)電性強(qiáng)等優(yōu)點(diǎn),結(jié)合納米導(dǎo)電性好及生物親和能力強(qiáng)等特性,制備羧基化多孔碳-納米金復(fù)合材料修飾AuE,用于提高cDNA的固載量并實(shí)現(xiàn)信號(hào)放大,通過雜交連接核酸適體,構(gòu)建適體/cDNA/羧基化多孔碳-納米金/AuE電流型傳感器。MB在適體/cDNA/羧基化多孔碳-納米金/AuE上的電流值比在適體/cDNA/AuE電流值增大了175.6%,說明羧基化多孔碳-納米金具有良好的信號(hào)放大作用。利用該傳感器對OTA進(jìn)行檢測,當(dāng)OTA濃度為5.0×10-6-5.0×10-4ng/mL時(shí)具有良好的線性關(guān)系,相關(guān)系數(shù)是0.997,檢出限是5.0×10-6ng/mL。對玉米樣品進(jìn)行檢測,平均回收率為103%。對傳感器進(jìn)行性能研究,實(shí)驗(yàn)結(jié)果表明該傳感器具有良好的重復(fù)性、穩(wěn)定性和特異性。
[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.
【學(xué)位授予單位】：河南工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS210.7;TP212.9
本文編號(hào)：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.
【學(xué)位授予單位】：河南工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TS210.7;TP212.9
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