铂电极表面生物素-亲和素固载单链脱氧核糖核酸的电化学传感器  

Electrochemical Sensor Based on Immobilization of Single Stranded Deoxyribonucleic Acid on Pt Electrode Surface by Avidin-biotin System

作　　者：王保珍[1] 杜晓燕[1] 郑晶[1] 金滨锋[1] 

机构地区：[1]哈尔滨医科大学公共卫生学院,哈尔滨150001

出　　处：《分析化学》2005年第6期789-792,共4页Chinese Journal of Analytical Chemistry

基　　金：黑龙江省教育厅科技项目(No.10541107);黑龙江省自然科学基金(No.D9705)资助课题

摘　　要：通过直接吸附将亲和素固定在Pt电极表面,联于生物素标记的脱氧核糖核酸(DNA)探针,制备了电化学基因传感器,建立了Pt电极表面修饰单链脱氧核糖核酸(ssDNA)的方法。修饰电极与待测溶液中人工合成的转基因食品中常有的花椰菜花叶病毒35S启动子(CaMV35S)或根癌农杆菌终止子(NOS)DNA片段进行杂交,以邻菲罗林钴络合物[Co(phen)3+3]为杂交指示剂,循环伏安法测量,通过杂交前后指示剂峰电流的变化检测DNA杂交的量。研究了电极修饰、杂交反应及测量的适宜条件,在优化实验条件下,峰电流的差值与DNA杂交量之间有良好的线性关系,相关系数r=0.9996。杂交后的电极经热变性再生,可重复使用多次。An electrochemical sensor for detection of specific deoxyribonucleic acid (DNA) sequences based on ssDNA-modified Pt electrode by avidin-biotin has been developed. The method is simple, convenient, reagent-saving and practicale for DNA studies. The experimental steps are as follows: ( i) avidin was immobilized on the Pt electrode surface via direct adsorption, ( ii) biotinylated ssDNA was modified onto the electrode surface by avidin-biotin system, ( iii) the ssDNA-electrode was immersed into 2 mL of the 2 x saline-sodium citrate (SSC) buffer containing complementary DNA, and the hybridization reaction was carried out at certain temperature, (iv) the electrode was immersed into a Co (phen)(3)(3+) solution (5mmol/L Tris-HCI + 5 mmol/L NaCl pH7. 05 and detected voltammetrically. The immobilized probes were specific for most inserted sequences in genetically modified food: the promoter 35 S or the terminator NOS. The sensor signal dependent on external conditions (pH, temperature, the time of hybridization and immobilization) were studied for clarification of their effect on the assay and to find the optimum condition for measurements. The results showed that it was better for avidin to be adsorbed onto the electrode surface twice, 50 minutes every time and the optimum conditions of hybridization reaction were pH 7.0, 50 degrees C, 60 min. Under optimum conditions, the sensor's output signals present primely a relation of linearity with the concentration of complementary DNA sequences between 7.0 x 10(-8) mol/L and 2.3 x 10(-7) mol/L. The linear equation of probe 35 S was y = 1.16x + 0.137, and the correlation coefficient was 0.9996. The sensor can be reused over 6 times by electrode regeneration.

关 键 词：脱氧核糖核酸 电极表面 电化学传感器 亲和素 单链 花椰菜花叶病毒 DNA杂交 固载 35S启动子 生物素标记 基因传感器 DNA片段 根癌农杆菌 转基因食品 循环伏安法 可重复使用 表面修饰 PT电极 人工合成 修饰电极 钴络合物 

分 类 号：TP212.2[自动化与计算机技术—检测技术与自动化装置]