The Application Of Self-doped Polyaniline In Indicator-free And Label Free DNA Electrochemical Biosensing

Self-doped polyaniline (SPAN) has been widely used in DNA electrochemical biosensing based on the unique molecular structure containing conjugatedπbond, the particular"doped"mechanism, and the self-redox reaction. This dissertation mainly focuses on (1) fabricating the self-doped polyaniline as a highly sensitive platform for DNA hybridization detection, (2) major interfacial and electrochemical property changes between SPAN bound to dsDNA and ssDNA serving as the electrochemical signals to monitor DNA hybridization event. The paper can be summarized as follows:(1) Conductive poly(m-aminobenzenesulfonic acid)(PABSA)/TiO2 nanosheet membranes were successfully electropolymerized with pulse potentiostatic method under optimized conditions. Due to the unique properties of TiO2 nanoparticles, m-aminobenzenesulfonic acid monomers tended to be adsorbed around the particles and the electropolymerization efficiency was greatly improved. The combination of TiO2 nanoparticles and PABSA resulted in a nanocomposite membrane with unique and novel nanosheet morphology, providing more activation sites and enhancing surface electron transfer rate. DNA probes could be covalently attached to the sulfonic groups through the amines of DNA sequences based on the acyl chloride cross-linking reaction. After immobilization of probe DNA, the electrochemical impedance value increased significantly compared to that of PABSA/TiO2 nanosheet membranes, and then decreased dramatically after hybridization reaction of probe DNA with the complementary DNA sequence compared to that of the probe-immobilized electrode. Electrochemical impedance spectroscopy was adopted for indicator-free DNA biosensing, which had an eminent ability for the recognition between double-base mismatched sequence or non-complementary DNA sequence and complementary DNA sequence. The gene fragment related to one of the screening genes for the transgenically modified plants, cauliflower mosaic virus 35 S gene, was satisfactorily detected.(2) The highly sensitive and label-free electrochemical DNA hybridization assay with a self-signal amplifying procedure based on the sensing platform of conjugated SPAN-Al(Ш)-DNA hybrid was described. Due to the essential property differences between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), such as rigidity,π-stacked bases, charge distribution and long-range electron transfer, SPAN bound to dsDNA underwent major interfacial and electrochemical property changes in comparison to when it bound to ssDNA. These changes served as the electrochemical signals to monitor hybridization event. After hybridization between the surface-immobilized probe DNA and the complementary DNA sequence, an amplified redox signal of SPAN was observed. Unlike most signal amplifying processes using labeled targets, this procedure needed no fussy labeling steps. The gene fragment related to one of the screening genes for the genetically modified plants, cauliflower mosaic virus 35 S gene, was satisfactorily detected with this strategy. Under optimal conditions, the dynamic range for the DNA assay was from 1.0×10-14 mol/L to 1.0×10-8 mol/L with the detection limit of 2.3×10-15 mol/L.