Immobilization and hybridization of DNA based on magnesium ion modified 2,6-pyridinedicarboxylic acid polymer and its application for label-free PAT gene fragment detection by electrochemical impedance spectroscopy

摘要

A new approach for a simple electrochemical detection of PAT gene fragment is described. Poly(2,6-pyridinedicarboxylic acid) (PDC) modified glassy carbon electrode (GCE) was prepared by potential scan electropolymerization in an aqueous solution. Mg~(2+) ions were incorporated by immersion of the modified electrode in 0.5 mol/L aqueous solution of MgCl_2 to complete the preparation of a generic "activated" electrode ready for binding the probe DNA. The ssDNA was linked to the conducting polymer by forming a bidentate complex between the carboxyl groups on the polymer and the phosphate groups of DNA via Mg~(2+). DNA immobilization and hybridization were characterized with differential pulse voltammetry (DPV) by using methylene blue (MB) as indicator and electrochemical impedance spectroscopy (EIS). The EIS was of higher sensitivity for DNA detection as compared with voltammetric methods in our strategy. The electron transfer resistance (R_(et)) of the electrode surface in EIS in [Fe(CN)_6]~(3-/4-) solution increased after the immobilization of the DNA probe on the Mg/PDC/GCE electrode. The hybridization of the DNA probe with complementary DNA (cDNA) made R_(et) increase further. The difference between the R_(et) at ssDNA/Mg/PDC/GCE and that at hybridization DNA modified electrode (dsDNA/Mg/PDC/GCE) was applied to determine the specific sequence related to the target PAT gene with the dynamic range comprised between 1.0 × 10~(-9) and 1.0 × 10~(-5) mol/L. A detection limit of 3.4 × 10~(-10) mol/L of oligonucleotides can be estimated.