基于PAMAM/聚2,6-吡啶二甲酸膜DNA电化学生物传感器的制备及对禽病毒基因检测的研究

Preparation of DNA Electrochemical Biosensor Based on PAMAM/Poly(2,6-pyridinedicarboxylic acid) Composite Film and Its Application to the Detection to Avian Virus Gene

以铂电极上聚合的2,6-吡啶二甲酸(PDC)膜组装G5.0树状高分子(PAMAM)固定ssDNA探针,制备了一种新型的DNA电化学生物传感器.用[Fe(CN)6]3-/4-作氧化还原指示剂,以电化学交流阻抗和循环伏安技术对探针ssDNA的固定和杂交进行了表征.实验表明,当ssDNA在复合膜上固定及与其互补序列杂交后,电极表面的传递电阻(Ret)依次增大.因此,可以利用Ret的明显差异,以此固定探针的修饰电极,对互补序列DNA进行无标记交流阻抗检测.基于该生物传感器结合交流阻抗技术对禽病毒基因进行检测,在优化实验条件下,靶基因ssDNA-2在2.0×10-11～1.0×10-8mol·L-1线性范围内,其浓度与电极表面的电子传递电阻(Ret)之间呈良好的线性关系,检测限为3.6×10-12mol·L-1.表明该方法为病毒灵敏地检测提供了一个有益的传感平台.

A composite film has been prepared by electropolymerization of 2,6-pyridinedicarboxylic acid （PDC） on the platinum electrode and combined with G5.0 poly（amidoamine） dendrimer （PAMAM）. Then, a new DNA electrochemical biosensor was developed by immobilizing probe ssDNA on the composite film electrode. The immobilization and hybridization of probe ssDNA were studied by electrochemical impedance spectroscopy （EIS） and cyclic voltammetry using [Fe（CN）6]3-/4-as the redox indicator. The experimental results showed that the electron transfer resistance （Ret） of the electrode surface increased after the immobilization of probe ssDNA on the composite film and rose further after the hybridization of the probe ssDNA with its complementary sequence. The remarkable difference between the Ret value at the probe DNA-immobilized electrode and that at the hybridized electrode could be used for label-free EIS detection of the target DNA. The avian virus gene sequences were detected by this DNA electrochemical biosensor. Under optimal conditions, the detection range of the biosensor to avian virus gene target sequences was from 2.0×10-11 to 1.0×10-8 mol·L-1 with the detection limit of 3.6×10·12 mol·L-1, suggesting that the biosensor provides a perfect platform for the sensitive detection of virus gene sequences.