基于適配體構象變化及核酸酶信號放大的抗生素電化學生物傳感器研究
發(fā)布時間：2018-10-23 07:23
【摘要】：抗生素在蛋、乳、肉等動物源性食品中的殘留主要來源于其在畜牧業(yè)和農業(yè)的濫用,當人體長期攝入含有抗生素殘留的食物,在體內累積一定量的抗生素殘留時,會對人體產生藥毒性,同時會導致抗藥性的產生,對人體健康構成極大威脅�？股貧埩粢恢笔莿游镌葱允称分凶钪匾奈廴驹粗�。因此,當前亟需一個高效、快速的檢測抗生素殘留的方法。由于核酸適配體對目標靶分子的特異性和親和性,成為檢測方法中常用的工具。核酸酶具有穩(wěn)定、高效的優(yōu)點,被廣泛地應用于各種分析方法中。本文選擇卡那霉素作為目標物模板,基于核酸適配體的構象變化結合了核酸酶信號放大技術,我們構建兩個檢測抗生素的電化學生物傳感器,均表現(xiàn)出很寬的線性范圍和較低的檢出限,并在實際樣品檢測中表現(xiàn)了很好的準確性。1、我們設計了基于核酸適配體構象變化及λ外切酶和限制性核酸內切酶輔助循環(huán)信號放大策略的電化學傳感器實現(xiàn)了對卡那霉素超靈敏、特異性地檢測。本實驗均相中,巧妙設計的由目標物的核酸適配體和引物鏈通過雜交形成的拱形探針,當目標物和適配體進行特異性結合,釋放的引物鏈打開5’端修飾磷酸基團的發(fā)夾探針1(HAP1),形成穩(wěn)定的雙鏈結構,在λ外切酶作用下,引物鏈能循環(huán)利用,繼續(xù)打開下一個發(fā)夾探針1,產生大量的trigger鏈,能和修飾在電極表面上的發(fā)夾探針2(HAP2)的環(huán)狀部分進行結合,在內切酶的剪切作用下,trigger得到釋放和循環(huán)利用,繼續(xù)結合下一個發(fā)夾探針2,暴露出富含鳥嘌呤的序列,在血紅素和鉀離子的存在下,形成具有過氧化物酶作用的G四聯(lián)體/血紅素復合物,通過測量G四聯(lián)體/血紅素復合物催化雙氧水產生的電流信號達到定量檢測卡那霉素的目的。通過兩次酶輔助循環(huán),顯著地提高了反應效率和傳感器的靈敏性,這種方法可以從1 pM到10 nM的動態(tài)范圍定量檢測卡那霉素,最低檢出限是0.5 pM。2、第二個體系中,我們設計了基于核酸適配體構象變化和外切酶Ⅲ輔助雙重循環(huán)信號放大策略的電化學生物傳感器來檢測卡那霉素。實驗中,電極表面上Helper能打開修飾了在5’端標記了亞甲基藍的發(fā)夾探針2(HAP2)形成雙鏈結構,使亞甲基藍遠離電極表面。均相中,在拱形探針遇到目標物時,目標物和適配體特異性結合,引起自身構象變化和引物鏈的釋放,引物鏈能和發(fā)夾探針1(HAP1)結合,在外切酶Ⅲ作用下,引物鏈能循環(huán)利用,繼續(xù)打開下一個發(fā)夾探針1,產生的trigger能置換下Helper,發(fā)夾探針2自身折疊呈“關閉”狀態(tài),使得亞甲基藍靠近電極表面,通過測量增強的電流強度來定量的檢測卡那霉素。這種方法可以從1 pM到10 nM的動態(tài)范圍定量檢測卡那霉素,最低檢出限是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，
本文編號：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，
本文編號：2288487
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