免标记DNA电化学传感器测定凝血酶  

A Label-free DNA Electrochemical Sensor Based on Modified Mesoprous Silica for Detection of Thrombin

作　　者：林洁华[1] 邵美佳 张慧慧[1] 刘大鹏 

机构地区：[1]青岛科技大学化学与分子工程学院,山东青岛266042 [2]青岛市团岛污水处理厂,山东青岛266002

出　　处：《青岛科技大学学报（自然科学版）》2015年第3期245-250,共6页Journal of Qingdao University of Science and Technology:Natural Science Edition

基　　金：山东省博士基金项目(BS2011SW008)

摘　　要：研制了一种基于介孔二氧化硅负载纳米金和适体DNA的免标记电化学传感器，用于检测凝血酶的含量。实验中先用三甲基氯硅烷（TMCS）封闭介孔二氧化硅（MPS）前驱体外壁硅羟基的活性，煅烧除去模板剂后，再用氨基丙基三乙氧基硅烷（APTS）与孔道内壁硅羟基反应，接枝氨基，最后得到内壁修饰氨基的介孔硅材料（APTS—TMCS—MPS）。金纳米粒子（GNPs）通过与APTS—TMCS—MPS氨基的静电作用力组装到介孔孔道内，然后和巯基修饰的适体DNA通过金硫键相结合，制得免标记探针（DNA／GNPs／APTS—TMCS—MPS）。将探针修饰在玻碳电极表面，制得免标记DNA电化学传感器。将该传感器与含有凝血酶的待测液温育反应后，凝血酶和固定在介孔内的适体DNA发生特异性反应，形成位阻较大的适体DNA和凝血酶的复合物，从而增加了介孔孔道内的空间位阻，导致电流响应信号降低。随着凝血酶浓度的增加，孔道内部的位阻也随之增加。根据形成的复合物对电子转移和响应电流的阻碍，可以实现对凝血酶的免标记测定。实验结果表明，APTS—TMCS—MPS介孔孔道内的GNPs，可以提高孔道内电子的传递效率。在优化的实验条件下，该免标记DNA电化学传感器对凝血酶的检测线性范围是1．0×10-8～1．0×10-6m01·L-1检测限是7．5×10-9mol·L-1（3d）。A label-free electrochemical aptamer sensor for the detection of thrombin is proposed based on controlled fabrication of gold nanoparticles （GNPs） and DNA inside the pores of mesoporous silica （MPS）. The MPS precursor was mixed with trimethyl- chlorosilane （TMCS） firstly to block the external surface silanol groups, afterwards the internal pore walls silanol groups were grafted by aminopropyltriethoxyl silane （APTS）. Then the obtained modified mesoporous silicon was named as APTS-TMCS- MPS for short. The GNPs were confined inside the mesopores of APTS-TMCS-MPS by the covalent linking with the amino groups, mercapto-group of DNA were confined in-side the mesopores of APTS-TMCS-MPS by the covalent linking with the Au-S key of GNPs, thus formed the label free probe （DNA/GNPs/APTS-TMCS-MPS）. The pre- pared probe were used to modify glassy carbon electrode （GCE） to construct a label-free electrochemical aptamer sensor. After incubating thrombin with the sensor, the specific reaction were formed on the surface of GCE, form the nonconductive conjugates of DNA and thrombin, and the spatial block increased. Thus, the peak current decreased with increasing concentration of thrombin. The nonconductive conjugates blocked the elec- tron transfer and the peak responses changed on the corresponding surface respectively. Then, the detection of thrombin achieved. The GNPs inside the mesopores could pro- mote the electron transportation through the pore channel. Under the optimal experi- mental conditions, the fabricated aptamer sensor could detect thrombin in a linear range from 1.0 × 10 8 to 1.0 × 10-6 mol. L-1 with a detection limit of 7.5 × 10 9mol. L 1（3a）. The fabricated aptamer sensor shows appropriate sensitivity and offers an al- ternative to the multianalyte detection of other bioactive molecules.

关 键 词：DNA电化学传感器 免标记 凝血酶 介孔二氧化硅 

分 类 号：O652[理学—分析化学]