基于适配体构象变化及核酸酶信号放大的抗生素电化学生物传感器研究
发布时间：2018-10-23 07:23
【摘要】：抗生素在蛋、乳、肉等动物源性食品中的残留主要来源于其在畜牧业和农业的滥用,当人体长期摄入含有抗生素残留的食物,在体内累积一定量的抗生素残留时,会对人体产生药毒性,同时会导致抗药性的产生,对人体健康构成极大威胁。抗生素残留一直是动物源性食品中最重要的污染源之一。因此,当前亟需一个高效、快速的检测抗生素残留的方法。由于核酸适配体对目标靶分子的特异性和亲和性,成为检测方法中常用的工具。核酸酶具有稳定、高效的优点,被广泛地应用于各种分析方法中。本文选择卡那霉素作为目标物模板,基于核酸适配体的构象变化结合了核酸酶信号放大技术,我们构建两个检测抗生素的电化学生物传感器,均表现出很宽的线性范围和较低的检出限,并在实际样品检测中表现了很好的准确性。1、我们设计了基于核酸适配体构象变化及λ外切酶和限制性核酸内切酶辅助循环信号放大策略的电化学传感器实现了对卡那霉素超灵敏、特异性地检测。本实验均相中,巧妙设计的由目标物的核酸适配体和引物链通过杂交形成的拱形探针,当目标物和适配体进行特异性结合,释放的引物链打开5’端修饰磷酸基团的发夹探针1(HAP1),形成稳定的双链结构,在λ外切酶作用下,引物链能循环利用,继续打开下一个发夹探针1,产生大量的trigger链,能和修饰在电极表面上的发夹探针2(HAP2)的环状部分进行结合,在内切酶的剪切作用下,trigger得到释放和循环利用,继续结合下一个发夹探针2,暴露出富含鸟嘌呤的序列,在血红素和钾离子的存在下,形成具有过氧化物酶作用的G四联体/血红素复合物,通过测量G四联体/血红素复合物催化双氧水产生的电流信号达到定量检测卡那霉素的目的。通过两次酶辅助循环,显著地提高了反应效率和传感器的灵敏性,这种方法可以从1 pM到10 nM的动态范围定量检测卡那霉素,最低检出限是0.5 pM。2、第二个体系中,我们设计了基于核酸适配体构象变化和外切酶Ⅲ辅助双重循环信号放大策略的电化学生物传感器来检测卡那霉素。实验中,电极表面上Helper能打开修饰了在5’端标记了亚甲基蓝的发夹探针2(HAP2)形成双链结构,使亚甲基蓝远离电极表面。均相中,在拱形探针遇到目标物时,目标物和适配体特异性结合,引起自身构象变化和引物链的释放,引物链能和发夹探针1(HAP1)结合,在外切酶Ⅲ作用下,引物链能循环利用,继续打开下一个发夹探针1,产生的trigger能置换下Helper,发夹探针2自身折叠呈“关闭”状态,使得亚甲基蓝靠近电极表面,通过测量增强的电流强度来定量的检测卡那霉素。这种方法可以从1 pM到10 nM的动态范围定量检测卡那霉素,最低检出限是0.76 pM。
[Abstract]:The residues of antibiotics in animal-derived foods such as eggs, milk and meat are mainly due to their abuse in animal husbandry and agriculture. When the human body takes in food containing antibiotic residues for a long time, it accumulates a certain amount of antibiotic residues in the body. It can cause drug toxicity and drug resistance, which is a great threat to human health. Antibiotic residues have been one of the most important sources of pollution in animal-derived foods. Therefore, there is an urgent need for an efficient and rapid method for the detection of antibiotic residues. Because of the specificity and affinity of aptamers to target molecules, aptamers are commonly used in detection methods. Nuclease has been widely used in various analytical methods for its advantages of stability and high efficiency. In this paper, kanamycin was selected as the target template. Based on the conformation change of nucleic acid aptamer and nuclease signal amplification technique, we constructed two electrochemical biosensors to detect antibiotics. Both showed wide linear range and low detection limit. We have designed the electrochemical sensor based on the conformation change of nucleic acid aptamer and 位 exonuclease and restriction endonuclease assisted cyclic signal amplification strategy. Kanamycin is hypersensitive, To detect specifically. In this experiment, arched probes formed by hybridization of the aptamers and primer chains of the target were designed, when the target and the aptamer were specifically bound. The released primer chain opened the hairpin probe 1 (HAP1) with a 5'end modified phosphate group to form a stable double-stranded structure. Under the action of 位 exonuclease, the primer chain could be reused and continued to open the next hairpin probe 1 to produce a large number of trigger strands. It can bind to the circular part of the hairpin probe 2 (HAP2) modified on the surface of the electrode. The trigger is released and recycled under the shear action of the endonuclease, and continues to bind to the next hairpin probe 2, exposing the sequence rich in guanine. In the presence of heme and potassium ions, a G tetraad / heme complex with peroxidase action was formed. The quantitative detection of kanamycin was achieved by measuring the current signal generated by G quadruple / heme complex catalyzed hydrogen peroxide. The efficiency of the reaction and the sensitivity of the sensor were significantly improved by two enzyme assisted cycles. This method could be used to quantitatively detect kanamycin from 1 pM to 10 nM. The lowest detection limit was 0.5 pM.2, in the second system. We designed an electrochemical biosensor for the detection of kanamycin based on the conformation change of aptamer and the strategy of double cycle signal amplification assisted by exonuclease 鈪，
本文编号：2288487
[Abstract]:The residues of antibiotics in animal-derived foods such as eggs, milk and meat are mainly due to their abuse in animal husbandry and agriculture. When the human body takes in food containing antibiotic residues for a long time, it accumulates a certain amount of antibiotic residues in the body. It can cause drug toxicity and drug resistance, which is a great threat to human health. Antibiotic residues have been one of the most important sources of pollution in animal-derived foods. Therefore, there is an urgent need for an efficient and rapid method for the detection of antibiotic residues. Because of the specificity and affinity of aptamers to target molecules, aptamers are commonly used in detection methods. Nuclease has been widely used in various analytical methods for its advantages of stability and high efficiency. In this paper, kanamycin was selected as the target template. Based on the conformation change of nucleic acid aptamer and nuclease signal amplification technique, we constructed two electrochemical biosensors to detect antibiotics. Both showed wide linear range and low detection limit. We have designed the electrochemical sensor based on the conformation change of nucleic acid aptamer and 位 exonuclease and restriction endonuclease assisted cyclic signal amplification strategy. Kanamycin is hypersensitive, To detect specifically. In this experiment, arched probes formed by hybridization of the aptamers and primer chains of the target were designed, when the target and the aptamer were specifically bound. The released primer chain opened the hairpin probe 1 (HAP1) with a 5'end modified phosphate group to form a stable double-stranded structure. Under the action of 位 exonuclease, the primer chain could be reused and continued to open the next hairpin probe 1 to produce a large number of trigger strands. It can bind to the circular part of the hairpin probe 2 (HAP2) modified on the surface of the electrode. The trigger is released and recycled under the shear action of the endonuclease, and continues to bind to the next hairpin probe 2, exposing the sequence rich in guanine. In the presence of heme and potassium ions, a G tetraad / heme complex with peroxidase action was formed. The quantitative detection of kanamycin was achieved by measuring the current signal generated by G quadruple / heme complex catalyzed hydrogen peroxide. The efficiency of the reaction and the sensitivity of the sensor were significantly improved by two enzyme assisted cycles. This method could be used to quantitatively detect kanamycin from 1 pM to 10 nM. The lowest detection limit was 0.5 pM.2, in the second system. We designed an electrochemical biosensor for the detection of kanamycin based on the conformation change of aptamer and the strategy of double cycle signal amplification assisted by exonuclease 鈪，
本文编号：2288487
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