电化学免疫传感器检测TET1蛋白  

An Electrochemical Immunosensor for Detection of Ten-eleven Translocation 1 Protein

作　　者：丁佳 梁文煦 周云雷[1] 殷焕顺[1] 李怡靖 艾仕云[1] DING Jia;LIANG Wen-Xu;ZHOU Yun-Lei;YIN Huan-Shun;LI Yi-Jing;AI Shi-Yun(College of Chemistry and Material Science,Food Safety Analysis and Test Engineering Technology Research Center of Shandong Province,Shandong Agricultural University,Taian 271018,China)

机构地区：[1]山东农业大学化学与材料科学学院,山东省食品安全分析与测试工程技术研究中心,泰安271018

出　　处：《分析化学》2022年第2期217-224,共8页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(Nos.21775090,41807484,41977345,42077363)资助。

摘　　要：基于Ten-eleven translocation 1(TET1)蛋白催化5-甲基胞嘧啶(5-Methylcytosine,5mC)生成5-羟甲基胞嘧啶(5-Hydroxymethylcytosine,5hmC),结合5hmC抗体的免疫识别反应,建立了一种电化学分析方法用于TET1蛋白的特异性检测。以金纳米颗粒(AuNPs)修饰的金电极为基体电极,通过AuNPs与探针DNA 3′端巯基之间形成的Au—S键,将探针DNA与含有5mC的互补DNA杂交形成的双链DNA自组装到电极表面。在α-酮戊二酸和Fe^(2+)存在下,TET1催化氧化5mC生成5hmC。然后,5hmC抗体特异识别5hmC,5hmC抗体被修饰于电极表面。由于抗体的修饰,电极表面的电子传输速率明显下降,阻抗增大,导致此修饰电极在含有氧化还原探针的检测液里面的电化学信号明显降低。在0.5~10μg/mL浓度范围内,传感器的电化学信号降低值与TET1蛋白浓度的对数值呈良好的线性关系,检出限为0.17μg/mL(3σ)。采用本方法考察了环境污染物Pb^(2+)对TET1蛋白活性的影响,结果表明,Pb^(2+)对TET1蛋白的活性具有抑制作用,IC 50=41.72 nmol/L。本方法操作简单、仪器成本低、灵敏度高、特异性好,为TET1蛋白的检测提供了新方法,也为研究环境污染物的生态毒理效应提供了可供选择的生物标志物和评价方法。An electrochemical method for sensitive and specific detection of ten-eleven translocation 1(TET1)protein was developed based on TET1 protein catalyzing the oxidation of 5-methylcytosine(5mC)to generate 5-hydroxymethylcytosine(5hmC)and the immunological recognition of 5hmC by anti-5hmC antibody(Ab).The bare gold electrode modified with gold nanoparticles(AuNPs/Au)was employed as the substrate electrode,while the double-stranded DNA(dsDNA,formed from the hybridization reaction between probe DNA and its complementary DNA that contains 5mC base)was self-assembled onto the AuNPs/Au electrode through the formation of Au—S bond between AuNPs and—SH at 3′-end of probe DNA.In the presence ofα-ketoglutaric acid and Fe^(2+),TET1 protein catalyzed the oxidation of 5mC to produce 5hmC,which triggered the immune-recognition reaction,leading to the immobilization of anti-5hmC antibody.Based on the increasing steric hindrance effect,the electron transmission rate on the electrode surface decreased,and the electrochemical resistance increased,resulting in a decreased electrochemical reduction signal in detection buffer solution.On the basis of the relationship between the decreased electrochemical signal and the concentration of TET1 protein,the detection of TET1 protein was realized.Under the optimal conditions,the developed method presented a wide linear range from 0.5 to 10μg/mL,with a low detection limit of 0.17μg/mL(3σ).Moreover,the effect of Pb^(2+)on the activity of TET1 protein was investigated.The results showed that Pb^(2+)could inhibit the activity of TET1 protein with IC 50 value of 41.72 nmol/L.In addition,this method showed many advantages such as simple operation,inexpensive instrument,high detection sensitivity and selectivity.This work not only provided a new method for detection of TET1 protein,but also presented alternative biomarker and evaluation method for the investigation of ecotoxicological effects of environmental pollutants.

关 键 词：DNA羟甲基化 TET1蛋白 电化学免疫传感器 5-羟甲基胞嘧啶抗体 

分 类 号：O657.1[理学—分析化学] X830[理学—化学]