基于复合纳米材料和酶切信号放大电化学适体传感器检测沙门氏菌

An Electrochemical Aptasensor for Detection of Salmonella Based on Composite Nanomaterial and Enzymatic Recycling for Amplification

【目的】沙门氏菌是食品中致病菌检测的一项重要指标。本研究拟构建一种实用性更强的用于沙门氏菌检测的新型电化学适配体传感器,以克服各种沙门氏菌传统检测方法的缺陷。【方法】通过混合还原氧化石墨烯(rGO)溶液与甲苯胺蓝(Tb)溶液制得Tb-rGO复合物,再将此复合物分散于纳米金(Au NPs)溶胶中得到Au NPs-Tb-rGO复合物。最后将Au NPs-Tb-rGO复合物与带有氨基的DNA链(S1)孵育得DNA-复合纳米材料(S1-Au NPs-Tb-rGO)。通过金硫键将沙门氏菌适配体互补链(S2)修饰在金电极表面,以己硫醇为封闭剂消除非特异性吸附后,滴涂沙门氏菌适配体(Apt)于电极表面,使Apt与S2杂交结合。将修饰好的电极浸入含有沙门氏菌与核酸外切酶I(Exo I)的混合液中,基于Exo I信号放大效应,利用适配体对沙门氏菌的特异性结合作用,循环带离适配体,再通过S1-Au NPs-Tb-rGO中的S1与S2杂交将S1-Au NPs-Tb-rGO负载到电极表面,监测电极表面的电化学信号,并对在菌液中的孵育时间、Exo I浓度和S1-Au NPs-Tb-rGO浓度进行优化,构建沙门氏菌电化学适配体传感器。使用该传感器,对大肠杆菌、金黄色葡萄球菌、志贺氏菌、单增李斯特菌和阪崎肠杆菌进行检测,以确定沙门氏菌电化学适配体的特异性;对6×10~2—6×10~6 cfu/mL的沙门氏菌进行检测,以确定沙门氏菌电化学适配体传感器的敏感性;对羊奶样品进行检测,以确定沙门氏菌电化学适配体传感器的实用性。【结果】所建立的沙门氏菌电化学适配体传感器在菌液中的最佳孵育时间为1 h,Exo I的最适浓度为0.6 U·μL^(-1),S1-Au NPs-Tb-rGO的最适浓度为200 nmol·L^(-1)。在进行沙门氏菌的检测时,沙门氏菌与Apt特异结合,S1-Au NPs-Tb-rGO复合纳米材料被结合到电极表面使其线性伏安曲线氧化峰升高。特异性试验结果表明,所建立的方法仅对沙门氏菌的检测有电信号响应,而对非目标菌无响应。敏感性试验结果表明,所构建的沙门氏菌电化学适配体传感器,具有很高的敏感性,对沙门氏菌检测的敏感性达200 cfu/mL。使用建立的沙门氏菌电化学适配体传感器对羊奶中的沙门氏菌含量进行测定,加标回收率在91.6%—106.3%,结果令人满意。【结论】所建立的沙门氏菌电化学适配体传感器具有操作简便、检测范围宽、检出限低和成本低廉等优点,有望应用于食品工业中沙门氏菌的现场快速定量检测。

【Objective】Salmonella is an important detection target of pathogeny bacteria in food.In order to overcome the shortcomings of traditional Salmonella detection methods,a novel assay of electrochemical aptasensor for quantitative detection of Salmonella with better practicability was established.【Method】Reduced Graphene Oxide（rGO） solution and toluidine blue（Tb） solution were mixed together to obtain the Tb-rGO nanocomposite,and then the Tb-rGO nanocomposite was dispersed in gold nanoparticles（Au NPs） colloidal solution to obtain the Au NPs-Tb-rGO nanocomposite.Then,the as-prepared Au NPs-Tb-rGO nanocomposite was incubated with amino-DNA to obtain the DNA-nanocomposite（S1-Au NPs-Tb-rGO）.The complementary strands of the aptamers of Salmonella（S2） were attached to the surface of gold electrode by Au-S-bond,and then the electrode surface was blocked with HT.Subsequently,the aptamers of Salmonella（Apt） were dripped onto the modified electrode to make Apt bind with S2.The modified electrode was immersed into the mixture containing Salmonella and exonuclease I（Exo I）.In terms of the characteristics of Exo I that could amplify electrical signals and the aptamers that could exclusively bind with Salmonella,the aptamers were taken away from S2 circularly.Then,the S1-Au NPs-Tb-rGO composite was attached to the surface of electrode by the hybridization of S1 and S2.Finally,the conditions of the incubation time in bacteria liquid,the Exo I concentration and the S1-Au NPs-Tb-r Go composite concentration were optimized and the electrical signals of the electrode surface was monitored to construct the aptasensor.This electrochemical aptasensor was used to test Escherichia coli,Staphylococcus aureus,Shigella,Listeria monocytogenes and Enterobacter sakazakii to ensure the electrochemical aptasensor＇s specificity.The electrochemical aptasensor was used to detect 6×102-6×106 cfu/mL Salmonella to ensure the electrochemical aptasensor＇s sensitivity.Then this electrochemical aptasensor was used to detect the goat milk to evaluate the practical use of electrochemical aptasensor.【Result】The optimization of the electrochemical aptasensor incubation time in bacterial liquid,the Exo I concentration and the S1-Au NPs-Tb-r Go composite concentration were studied in detail,and the optimal conditions were 1 h,0.6 U·μL^（-1） and 200 nmol·L^（-1）.When Salmonellas were tested existent,they had specific binding with Apt and the S1-Au NPs-Tb-r Go composite was attached to the electrode surface.So the linear sweep voltammetry curve of the electrochemical aptasensor showed a rise of oxidation peak.The developed aptasensor was specific to Salmonella and did not react with non-target bacteria.The electrochemical aptasensor detected the Salmonella target at a titer higher than 200 cfu/mL.A good recovery of Salmonella in the range of 91.6%-106.3% was obtained in goat milk by electrochemical aptasensor assays developed.【Conclusion】This electrochemical aptasensor can detect Salmonella with a easy operation,a wide linear range,a high sensitivity and a low cost,which provide good application prospects in the field of rapid quantitative detection of salmonella.