| There are three parts in this paper, an eletroactive complex was synthesized in the first part and this complex was used as indicator to develop a novel electrochemical DNA biosensor; we also developd two label-free DNA biosensors in the second part and third part.1. The complex diaquabis[N-(2-pyridinylmethyl) benzamide-κ2 N,O]-cadmium(II) dinitrate {[CdL2(H2O)2](NO3)2, where L = N-(2-pyridinylmethyl) benzamide} was synthesized and characterized by x-ray diffraction analysis. Fluorescence spectroscopy and voltammetry were used to probe the interaction between [CdL2]2+ and salmon sperm DNA. Results showed that [CdL2]2+ had excellent electrochemical activity on glassy carbon electrode (GCD) and could intercalate into the double helix of double-stranded DNA (dsDNA). In 0.2 mol·L-1 NaAc-HAc media (pH 7.02), the binding ratio between [CdL2]2+ and salmon sperm DNA was calculated to be 2:1 and the binding constant was 25.56 L1/2 mol-1/2. An electrochemical DNA biosensor for the detection of human hepatitis B virus (HBV) DNA fragment was developed. The biosensor relied on the covalent immobilization of the 21-mer single-stranded DNA (ssDNA) related to HBV gene on the modified glassy carbon electrode (GCE). Using [CdL2]2+ as novel electroactive indicator, the hybridization between the probe and its complementary ssDNA, as the target, was investigated by differential pulse voltammetry (DPV). Experiment with non-complementary oligonucleotides was carried out to assess the selectivity of the developed electrochemical DNA biosensor. The target HBV DNA could be quantified ranged from 1.01×10-8 to 1.62×10-6 mol·L-1with good linearity (γ= 0.9962). The detection limit was 7.19×10-9 mol·L-1 (3σ, n = 11).2. A novel method for sensitive detection of single stranded DNA (ssDNA) using 2-mercaptobenzothiazole (MBT) self-assembled monolayer modified gold electrode has been developed. Based on the intercalating effect of MBT into double stranded DNA (dsDNA), Au-MBT electrode possessed the ability to distinguish ssDNA and dsDNA. Cyclic voltammetry (CV) and scan electron microscope (SEM) were used to preliminarily characterize the MBT self-assembled monolayer on gold electrode. In the mixture of excessive probe ssDNA and target ssDNA, current signal recorded by differential pulse voltammetry (DPV) after DNA hybridization reflected the extent of the hybridization. Definite relations were observed between the reductive peak currents and the concentration of target ssDNA. The quantitative detection of target ssDNA was achieved with a linear range from 1.59×10-8 to 2.4×10-6 mol·L-1. The detection limit was 2.38×10-9 mol·L-1 (3σ, n = 11). The Au-MBT electrode possessed high reusability and reproducibility, no significant deterioration of peak currents occurring over 10 detection/regeneration cycles. Combined with the advantages of simplicity and low-cost, the developed electrochemical biosensors show potential in continuous and quantitative detection of ssDNA in biological samples.3. A method for sensitive detection of ssDNA using 2-mercaptobenzimidazole (MBI) self-assembled monolayer modified gold electrode has been developed. Firstly, a glassy carbon electrod which had been modified with probe ssDNA was used to separate the target ssDNA from sample and then target ssDNA was transfer to a new solution, Au-MBI electrod detect target ssDNA in the solution. The quantitative detection of target ssDNA was achieved with a linear range from 1.32×10-8 1.59×10-6 mol·L-1,the detection limit was 4.58×10-9 mol·L-1. (3σ, n = 11). |
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