Construction Of Novel DNA Electrochemical Biosensor Based On Nano Metal Oxide And Its Composite Nanomaterial

DNA is the basic genetic material of life. With the development of the research of gene structure and function, the analysis of specific DNA sequences in tissue, blood, microorganism and virus of the human body and the detection of base mutation and damage of DNA are of great significance in the development of life science. As compared with other detection techniques, the main advantages of electrochemical techniques are their low cost, small size, and convenience for integration and microminiaturization. Hence, DNA electrochemical biosensors have been rapidly developed recently.The advent of nanotechnology and construction of nanointerface have opened a new scope and stage of DNA biosensing. The excellent chemical and physical features of nanointerface will have extensive applications in biosensing field. The explore of the construction methods of different nanointerfaces, the achievement of the ultrasensitive determination of DNA sequences and damage and the study of novel biosensors have vital theoretic and instructive meaning in the understanding of energy transfer and substance metabolism of life system, the acquisition of the relations between the structure of biomacromolecules and their chemical and physical properties, and the research of physiological function and mechanism of biomacromolecules such as DNA in life.In this paper, nano metal oxide and its composite nanomaterial were used to immobilize probe DNA for the electrochemical detection of DNA specific sequences with a high sensitivity. In addition, the genotoxicity of nano metal oxide was also researched by electrochemistry. The paper can be summarized as follows:(1) A novel nanocomposite membrane, comprising of nanosized shuttle-shaped CeO₂, single-walled carbon nanotubes (SWNTs) and hydrophobic room temperature ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF₆), was developed on the glassy carbon electrode (GCE) for electrochemical sensing of the immobilization and hybridization of DNA. The properties of the CeO₂-SWNTs-BMIMPF₆/GCE, the characteristics of the immobilization and hybridization of DNA were studied by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). 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 sequence-specific DNA of phosphoenolpyruvate carboxylase (PEPCase) gene from transgenically modified rape and nopaline synthase (NOS) gene from the real sample of one kind of transgenic soybeans were detected by this DNA electrochemical biosensor. Under optimal conditions, the dynamic range for detecting the sequence-specific DNA of the PEPCase gene was from 1.0×10^-12 mol/L to 1.0×10^-7 mol/L, and the detection limit was 2.3×10^-13 mol/L.(2) A novel architecture was designed by combining the strong adsorption ability of Fe₂O₃ microspheres to the DNA probes and excellent conductivity of self-doped polyaniline (SPAN) nanofibers (copolymer of aniline and m-aminobenzenesulfonic acid) on carbon ionic liquid electrode (CILE) for electrochemical impedance sensing of the immobilization and hybridization of DNA. The immobilization of the probe DNA on the surface of electrode and the sensitivity of DNA hybridization recognition were dramatically enhanced due to the unique synergistic effect of Fe₂O₃ microspheres, SPAN nanofibers and ionic liquid. The DNA hybridization events were monitored with a label-free EIS strategy. Under optimal conditions, the dynamic range of this DNA biosensor for detecting the sequence-specific DNA of PEPCase gene from transgenically modified rape was from 1.0×10^-13 mol/L to 1.0×10^-7 mol/L, and the detection limit was 2.1×10^-14 mol/L.(3) A novel electrochemical DNA biosensor for simple, rapid, and specific detection of PML/RARA fusion gene in acute promyelocytic leukemia by using single-stranded deoxyribonucleic acid as the capture probe was constructed. Nanosized Fe₂O₃ was first immobilized on the surface of a carbon paste electrode. Then poly-xanthurenic acid (PXa), a new electroactive material, was electrogenerated by using the pulse potentiostatic method on the Fe₂O₃ substrate to form a unique and uniform nanorhombus structure. Due to the unique binding ability of xanthurenic acid (Xa) with Fe₂O₃, Xa monomers tended to be adsorbed around nanosized Fe₂O₃, and the electropolymerization efficiency was greatly improved. Owing to the presence of abundant carboxyl groups, the capture probe was covalently attached on the carboxyl-terminated PXa/Fe₂O₃ nanorhombus membranes through the free amines of DNA sequences based on the EDC/NHS cross-linking reaction. EIS was adopted for indicator-free monitoring of the hybridization reaction on the probe-captured electrode. The decrease of the impedance value was observed upon hybridization of the probe with the target DNA. As a result, the efficient probe immobilization platform, coupled with the ultrasensitive indicator-free impedance measurement, gave rise to a detection limit of 2.8 fmol/L and a dynamic range from 1.0×10^-14 mol/L to 1.0×10^-7 mol/L.(4) A novel membrane comprising of hollow TiO₂ nanocubes and poly(m-aminobenzene sulfonic acid) (PABSA) nanofibres was constructed on the GCE for detecting the oxidative damage of natural dsDNA induced by TiO₂ under UV radiation. The TiO₂/PABSA membrane was used for efficient dsDNA immobilization, dsDNA oxidation through photogenerated hydroxyl radicals, and as the platform for electrochemical sensing. The properties of the TiO₂/PABSA membrane and the immobilization of dsDNA were characterized by CV and EIS using [Fe(CN)₆]^3-/4- as the indicator. The resulting oxidative damage of dsDNA was detected by monitoring the CV response of an intercalated electroactive probe, namely, Co(phen)₃³⁺. PABSA has a profound synergistic effect with the hollow TiO₂ on the DNA damage detection.(5) Oppositely charged natural dsDNA and poly(diallyldimethyl ammonium chloride) (PDDA) were assembled into (PDDA/dsDNA)3 layer-by-layer films on electrode surface, and Ru(bpy)₃²⁺ and Co(phen)₃³⁺ in solution were used as electroactive probes to detect oxidative damage of natural dsDNA in the films after incubation of the films in V₂O₅ nanobelts/HCl/H₂O₂ solution. The mechanism of DNA oxidative damage caused by the V₂O₅ nanobelts/HCl/H₂O₂ system was similar to that of Fenton-type reaction. The reaction of V₂O₅ nanobelts with HCl would produce V(â…£), and the produced V(â…£) would further react with H₂O₂, generating hydroxyl radicals (OH·) as in the Fenton-type reaction, which could severely damage DNA in the films. The present work provided an in vitro model system to mimic the pathway of DNA damage in real bioprocess through a simple electrochemical approach combined with layer-by-layer assembly. This approach also showed promising applications in rapid and sensitive screening of new nanomaterials and chemicals in vitro for their potential genotoxicity.