Study On The Interaction Of Phenothiazine Drug With DNA And Its Electrochemical Biosensing

A DNA electrochemical biosensor has been one of the research hotspots in the present interdisciplinary fields of materials science, life science and information science. It has received wide investigation and applications in genetic engineering, medical diagnosis, new drug screening, mechanism of drug action, environmental monitoring and forensic identification due to the simple, rapid, sensitive and economic advantages. Therein, the immobilization of DNA probe on an electrode surface, the hybridization of probe DNA with target DNA, and selection of electroactive indicators are considered as key technologies. In this dissertation, Au nano film electrodes have been prepared; the self-assembly property of DNA on the Au electrodes and the interaction of DNA with phenothiazine drugs have been investigated systematically. The content of this dissertation is as follows.Au nano thin-film electrodes have been prepared by using magnetron sputtering method. The surface morphologies of electrodes produced under different processes are characterized with scanning electron microscope (SEM), energy-dispersive spectrometer (EDS), atom force microscope (AFM), X-ray diffraction (XRD) instruments. The electrochemical characterization of Au electrodes and the self-assembly of thiolated single-stranded DNA (HS-ssDNA) are carried out by means of cyclic voltammetry and Raman spectroscopy, with contrast to the others. The result shows that the thickness of the metal transition layer is not more than 50 nm; the metals as transition layers should be those which can not diffuse mutually with Au, otherwise, the electrochemical behavior of the electrodes can be affected.SH-ssDNA is immobilized on the Au nano thin-film electrode by self-assembling method. The self-assembling, hybridization and orientation of SH-ssDNA on the thin-film gold electrodes are characterized by means of X-ray photoelectron spectroscopy (XPS) and electrochemical impedance technique (EIS). The effect of assembling time, concentration and the length of SH-ssDNA on its self-assembling are investigated. The Ret and surface coverage reach the highest values when assembling time is 15h. The influence of concentration and length of SH-ssDNA and its hybridization method with complementary DNA on the hybridization reaction are also studied. The result shows the change of Ret decreases with the increase of the SH-ssDNA concentration. The data simulation and analysis of impedance spectroscopy indicate that a unique self-assembled monolayer of SH-ssDNA is formed by with an upright orientation on the Au electrode surface; the hybridization efficiency is closely associated with the coverage of SH-ssDNA. The interaction mechanisms of chlorpromazine hydrochloride (CPZ) or promethazine hydrochloride (PZ) with thiolated single-stranded DNA (HS-ssDNA) and double-stranded DNA (HS-dsDNA) self-assembled on gold electrodes have been studied with CV and chronocoulometry. The results show the binding of CPZ or PZ with ssDNA shows a mechanism containing an electrostatic interaction, and the mode of CPZ interaction with dsDNA is also the electrostatic interaction; while the mode of PZ interaction with dsDNA contains the electrostatic interaction and intercalative binding. The diffusion coefficients D, reaction standard rate constants ks, electron transfer coefficients a, binding constants K and the binding ratio are determined. The calculated thermodynamic and dynamics parameters indicate that the binding capabilities of CPZ with ssDNA and dsDNA have no differences, while those of PZ with ssDNA and dsDNA show great changes.The 1H and 13C NMR assignments of CPZ and PZ have been analysized by using 2D NMR technique. The modes of the molecular structures of CPZ and PZ are optimized. The influence of the structural difference of promethazine drugs on their interactions with DNA has been investigated.PZ is selected as an electrochemically electroactive indicator of DNA hybridization for developing novel DNA biosensors according to the different interaction mechanism of CPZ and PZ. The influence of scanning rate, pH and ionic strength on the electrochemical behavior of PZ on a dsDNA/Au electrode have been studied. PZ shows good electrochemical performance on a dsDNA/Au electrode. As an electrochemcal indicator, PZ can be used to distinguish ssDNA from dsDNA. The DNA sensor based on PZ shows good sensitivity to the complementary target DNA (with the detection limit of 3.8×10-1mol L-1) and good selectivity. The single-base mismatched and random complementary DNAcan be discriminated from the complementary target DNA.