| DNA electrochemical biosensor is noted widely in recent years for rapid development. It has been used in many fields such as food industry, disease diagnosis, pharmaceutical analysis and environmental monitoring due to the high sensitivity, better specificity, rapid response, easy handling, no pollution and low cost. However, the method and material used to immobilize biomolecules is one of the crucial factors for improving the stability, selectivity and sensitivity of biosensor in the preparation. Three New-typed DNA electrochemical biosensors were prepared, and the detection of Avian Influenza Virus genotype based on the guanine oxidation signal was studied in this paper. The dissertation is divided into three main parts:(1) A novel electrochemical hybridization biosensor for label-free determination of sequence-specific DNA was described in this paper. First, cobalt hexacyanoferrate films and poly (amidoamine) (PAMAM) dendrimer (generation 4.0) were modified on the glassy carbon electrode (GCE) sequentially. Then, DNA probes were successfully immobilized on the modified electrode with G4 PAMAM dendrimer acting as the coupling agent. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement based on the oxidation signals of guanine without any external labels. This biosensor exhibited a superior electrochemical stability, selectivity and sensitivity in detection of the oligonucleotide sequence. Under the optimal conditions, the guanine oxidation signal was directly proportional to the mutant gene sequence concentration from 7.6×10-11 to 3.05×10-8 mol/L with a correlation coefficient of 0.9975 and a detection limit of 1.0×10-11mol/L (6.6×10-2 ng/mL)(S/N=3).(2) In this paper, a novel and reliable electrochemical sensor based on PbO2-carbon nanotubes-room temperature ionic liquid (i.e., 1-butyl-3-methylimidazolium hexafluorophosphate, BMIMPF6) composites film modified glassy carbon electrode (GCE) (PbO2-MWNT-RTIL/GCE) was proposed for simultaneous and individual determination of guanine and adenine. The guanine and adenine oxidation responses were monitored by differential pulse voltammetry (DPV) measurement. Compared with the bare electrode, the PbO2-MWNT-RTIL/GCE not only significantly enhanced the oxidation peak currents of guanine and adenine, but also lowered their oxidation over potentials, suggesting that the synergistic effect of PbO2, MWNT and RTIL could dramatically improve the determining sensitivity of guanine and adenine. The results demonstrated that the PbO2-MWNT- RTIL/GCE showed good stability, high accumulation efficiency and enhanced electrocatalytic ability for the detection of guanine and adenine. Besides, the modified electrode also exhibited good behaviors in the simultaneous detection of adenine and guanine with the peak separation as 0.29 V in 0.1 M pH 7.0 phos/phate buffer solutions (PBS). Under the optimal conditions, the detection limit for individual determination of guanine and adenine was 1.0 ng/mL and 4.0 ng/mL (S/N=3), respectively. The proposed method for the measurements of guanine and adenine in herring sperm DNA were successfully applied with satisfactory results.(3) A novel DNA electrochemical biosensor for label-free determination of DNA sequence related to the Avian Influenza Virus (AIV) genotype was demonstrated in this paper. First, the multi-walled carbon nanotubes-cobalt phthalocyanine (MWNTs-CoPc) nanocomposite and poly (amidoamine) (PAMAM) dendrimer (generation 4.0) were modified on the glassy carbon electrode (GCE) sequentially. Then, DNA probes were successfully immobilized on the modified electrode with G4 PAMAM dendrimer acting as the coupling agent. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement based on the oxidation signals of guanine without any external labels. Under the optimal conditions, the difference of guanine oxidation signal of the probe modified GCE in the absence and presence of complementary target (ΔIp) was linear with the logarithmic value of the complementary target concentration from 0.01 to 500 ng/mL with a correlation coefficient of 0.998 and a detection limit of 1.0 pg/mL.
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