| The immobilization of the DNA probe on the solid electrode was investigated mainly using electrochemical methods. With methylene bule (MB) as hybridization indicator, the electrochemical DNA biosensors were prepared and used to detect the products of the transgenic plant. The interaction between DNA and two dinuclear copper (II) complexes [Cu2(hdmg)4 and Cu2(bpy)2(HAO)22+] were studied to implore the possibilities of them to be used as electrochemical indicators for electrochemical DNA sensors.(1)The carbon paste electrode was fabricated with appropriate comparison of graphite powder, solid paraffin and stearic acid. An aluminum ion thin film was assembled on the surface of the electrode for the immobilization of DNA. Cyclic voltammetry was used to characterize the immobilization and hybridization of ssDNA by using MB as indicator. The conditions for preparing the basal carbon paste electrode, and the conditions for the immobilization and hybridization of the DNA probe were optimized with cyclic voltammetric method. This kind of DNA biosensor was successfully used to detect the PCR amplification of NOS gene from the sample of transgenic bean. The gene sequence related to the transgenic BAR gene in the transgenic corn was quantitatively detected by differential pulse voltammetry with the dynamic range comprised between 1.0×10-7 mol/L to 1.0×10-4 mol/L. A detection limit of 2.25×10-8 mol/L of oligonucleotides can be estimated.(2)Carbon paste electrode (CPE) with satisfactory mechanical stability and reproducibility was fabricated using solid paraffin as binder. Cetyltrimethyl ammonium bromide (CTAB) was self-assembled on the surface of the CPE to form compact CTAB mono-layer with high density of positive charges oriented outside for the immobilization of DNA through electrostatic affinity. The optimization of the factors affecting the property of CPE, stability of self-assembled CTAB, immobilization of ssDNA and electrochemical detection of the hybridization were monitored by cyclic voltammetry and differential pulse voltammetry using MB or K3Fe(CN)6 as indicator. In weak alkaline medium, both ssDNA and dsDNA could be immobilized on the CTAB/CPE. The immobilized ssDNA could selectively hybridize with its complementary DNA sequence. This kind of DNA biosensor was successfully used to detect the gene sequence related to the exogenous NPT II gene from the transgenic cole with a detection limit of 1.0×10-8 mol/L. Two-base-mismatch gene sequence also could be recognized by this method.(3)Carboxyl was formed on the surface of glassy carbon electrode (GCE) by electrochemical oxidation. Ethylenediamine (En) or ethylene glycol(Eg)as an connector was introduced onto the oxidized GCE using N-hydroxysuccinimide (NHS) and water-soluble 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride (EDC). DNA was then covalently immobilized onto the En or Eg modified GCE with the prolonged active center (surface-bound primary amino or hydroxyl group) in the presence of EDC. Cyclic voltammetry was used to characterize the denaturalization and hybridization capability of the immobilized DNA using MB as electro-active indicator. The results showed that the efficiency of ssDNA immobilization and hybridization both can be improved using En or Eg as connector. ssDNA/Eg/GCE was successfully applied to the recognition of NOS gene fragment of 20-base oligodeoxynucleotides sample. The ssDNA/En/GCE can be regenerated repeatedly to give repeatable results. ssDNA/En/GCE was successfully employed for the selective detection of CaMV35S gene (presented in almost all the genetically modified plants) in a fragment of 20-base oligodeoxynucleotides sample. The electro-reduction signal of MB was related to the CaMV35S gene concentration over the range of 5.0×10-9～1.2×10-7 mol/L. The PCR amplification of NOS gene from the sample of transgenic bean also can be detected by this method.(4)Cyclic voltammetry, absorption spectra and viscosity measurements were used to study the electrochemical properties of dinuclear copper (II) complexes: Cu2(hdmg)4 and Cu2(bpy)2(HAO)22+. Influences of pH, temperature, concentration of the complex, and ionic strength on the interaction between the complexes and DNA were described, and the mechanism of the interaction was also discussed. The results showed that Cu2(Hdmg)4 could interact with the bases of DNA to form non-electroactive complex, which could decrease the anodic peak current of Cu2(Hdmg)4. The interaction between Cu2(Hdmg)4 and ssDNA was stronger than that between Cu2(Hdmg)4 and dsDNA. The combining ratios between Cu2(Hdmg)4 and ssDNA, dsDNA were 2:1, 1:1, and the combining constants were 3.56×109, 2.75×105, respectively. The gene sequence related to the BAR gene in the transgenic corn was detected using Cu2(Hdmg)4 as electro-active indicator. Cu2(bpy)2(HAO)22+ interacts with DNA mainly through electrostatic affinity to make tiny difference between Cu2(bpy)2(HAO)22+-ssDNA and Cu2(bpy)2(HAO)22+-dsDNA. The redox peak current of Cu2(bpy)2(HAO)22+ decreased markedly after its interaction with DNA. This can be used to detect the concentration of DNA quantitatively.
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