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

发布时间：2018-01-16 16:15

本文关键词：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　出处：《中国农业科学》2017年21期 　论文类型：期刊论文

【摘要】：【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。
[Abstract]:[objective] Salmonella is an important indicator for the detection of pathogenic bacteria in food. This study aims to construct a new electrochemical aptamer sensor for the detection of salmonella. In order to overcome the defects of traditional methods for detection of salmonella, Tb-rGO complexes were prepared by mixed reduction of graphene oxide and toluidine blue (TB) solution. The composite was then dispersed in nano-gold au NPs. Au NPs-Tb-rGO complexes were obtained from sols. Finally, DNA-composite nanomaterials were prepared by incubating au NPs-Tb-rGO complexes with amino DNA chain S1. S1-Au NPs-Tb-rGO.Salmonella aptamer complementary chain S2) was modified on the surface of gold electrode by gold-sulfur bond. After the nonspecific adsorption was eliminated with hexanethiol as the sealant, salmonella aptamer (Apt) was dripped on the electrode surface. The modified electrode was immersed in the mixed solution containing Salmonella and nucleic acid exonuclease I, based on the amplification effect of Exo I signal. Using the specific binding effect of aptamer to Salmonella, the aptamer was separated from the aptamer. Then S1-Au NPs-Tb-rGO was loaded on the electrode surface by S1 and S2 hybridization in S1-Au NPs-Tb-rGO to monitor the electrochemical signal on the electrode surface. The incubation time of Exo I and the concentration of S1-au NPs-Tb-rGO were optimized to construct the salmonella electrochemical aptamer sensor. E. coli, Staphylococcus aureus, Shigella, Listeria monocytogenes and Enterobacter sakazakii were detected to determine the specificity of the electrochemical adaptor of Salmonella. In order to determine the sensitivity of salmonella electrochemical aptamer biosensor, 6 脳 10 ~ (2) ~ 6 脳 10 ~ (6) cfu/mL salmonella was detected. In order to determine the practicability of salmonella electrochemical aptamer biosensor, the optimal incubation time of salmonella electrochemical aptamer sensor was 1 h. The optimum concentration of Exo I was 0.6 U 路渭 L ~ (-1). The optimum concentration of S1-Au NPs-Tb-rGO was 200 nmol 路L ~ (-1) 路L ~ (-1). In the detection of Salmonella, Salmonella specifically binds to Apt. The S1-Au NPs-Tb-rGO composite nanomaterials were bound to the electrode surface to increase the oxidation peak of the linear voltammetric curve. The sensitivity test results showed that the electrochemical adaptor biosensor of Salmonella was highly sensitive to the detection of salmonella, but not to non-target bacteria. The sensitivity of salmonella detection was up to 200 cfur / mL. The salmonella content in goat milk was determined using the established electrochemical aptamer sensor. The recovery rate of salmonella was 91.6-106.3, and the results were satisfactory. [conclusion] the electrochemical aptamer biosensor of Salmonella has a simple operation and a wide detection range. The advantages of low detection limit and low cost are expected to be applied to rapid quantitative detection of salmonella in food industry.
【作者单位】：陕西师范大学食品工程与营养科学学院;
【基金】：陕西省科技统筹创新工程计划重大成果转化引导专项(2016KTCG01-12) 中央高校基本科研业务费专项资金(GK201703063)
【分类号】：O657.1;TP212;TS207.4
【正文快照】： 0引言【研究意义】沙门氏菌是一种常见的食源性致病菌,其主要传播方式为人摄入被污染的牛奶、肉类和鸡蛋等动物性食品[1]。大量对沙门氏菌疫情的调查和对志愿者的研究显示出一些特定的沙门氏菌菌株在极低的剂量下即可引发疾病[2],因此,开展沙门氏菌相关检测技术研究,对食源

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