用于DNA杂交检测的丝网印刷型生物传感器的研究

Screen-Printed Electrochemical Biosensor for Detection of DNA Hybridization

将单链DNA(ssDNA)固定到丝网印刷碳电极上构成电化学DNA传感器,采用电化学指示剂,建立DNA杂交的检测方法。Co(phen)33+电化学指示剂通过钴盐与配体邻菲罗啉络合制备,采用等离子发射光谱法(ICP-AES)和核磁共振法(NMR)表征功能基团,采用循环伏安法(CV)分析指示剂的电化学特性,并以此为基础研究ssDNA在电极表面的固定及DNA杂交过程。本研究探讨了直接吸附、静电吸附与键合等3种ssDNA在电极表面的固定方法,结果表明,静电吸附法和键合法具有较高的ssDNA固定量,采用静电吸附法固定探针的电极杂交目标DNA后,Co(phen)33+易于嵌入双链DNA(dsDNA)中,CV峰电流(ip)信号随目标DNA浓度增加。本研究采用静电吸附ssDNA的电极检测DNA杂交,实验表明,当探针固定液中ssDNA浓度为5 mg/L时,目标DNA浓度在6.65×10-8～4.26×10-6mol/L范围内,Co(phen)33+在dsDNA修饰电极上ip值与DNA浓度呈良好的线性关系,R2为0.9819。本研究为建立新的微生物分子分型手段提供了初步依据。

The method for detection of DNA hybridization process on the screen-printed electrochemical biosensor was investigated.The screen-printed electrochemical DNA biosensor was immobilized with single-stranded DNA（ssDNA）,then adsorbed with electroactive indicator Co（phen）33＋.Inductively coupled plasma-Atomic emission spectrometry（ICP-AES） and 1H nuclear magnetic resonance（NMR） were employed to characterize the electrochemical indicator complex which was prepared by the method of complexation reaction.Then cyclic voltammogram（CV） was used to investigate its electrochemical characteristics on the surface of electrodes.On this basis,the process of ssDNA fixed and DNA hybridization event on the electrodes were also studied.Here three immobilization methods,direct absorption,covalent binding and electrostatic adsorption,were also discussed.The results showed that ssDNA was immobilized well by the methods of covalent binding and static adsorption.Meanwhile,The peak current of CV was rising as target DNA hybridized with the probe,which indicating a higher affinity of dsDNA to Co（phen）33＋ when the probe was immobilized with electrostatic adsorption.Besides,it displayed peak current is strongly related with target DNA concentration in range of 6.65×10-8~4.26×10-6 mol/L,R2=0.9819.Based on the above mechanism,the calibration experiment was constructed,and was expected to be used in the detection of disease and pathogenic microorganisms