Development Of Novel DNA Electrochemical Biosensors Based On Metal Complex

DNA biosensors are rapidly developed and have received considerable attentions, which have taken a more and more important role in the areas of clinical diagnosis, medical research, epidemic prevention, environmental protection and bioengineering. Such devices rely on the conversion of the DNA base-pair recognition event into a useful signal and offer a promising alternative for fast, lower cost and simpler nucleic acid assays. Electrochemical biosensor based on DNA hybridization is a novel and developing technique that combining biochemical, electrochemical, medical and electronic techniques with the advantages of being simple, reliable, cheap, sensitive and selective for genetic detection, and more important it can be compatible with DNA biochip. It is not only qualified for meeting the size, cost and power requirements of decentralized genetic testing but offers an elegant route for interfacing at the molecular-level the DNA recognition and signal transduction element. Therefore, they are expected to have a broad prospect of application in clinic examination of inherited diseases and drug screening.The DNA electrochemical biosensor was prepared by self-assembling the thiol group modified GAPDH specific probe on the surface of Au electrode, metal complex as electro-active indicator. The condition for self-assemble single-molecular film was chosen during experiments as the following: the probe concentration was 100 ug/ml, self-assemble at 4°C for 12 hours. The voltammetry characteristic of single-strand DNA modified electrode was observed in 0.5 mol/L KOH solutions, and tested the electron-transfer capability of modified electrode in 5mmol/L Fe(CN)₆^3-/Fe(CN)₆^4-.The prepared DNA electrochemical biosensor was used to determine the nature and quality of complementary DNA of standard concentration. Then the DNA sensor was used to diagnose the PCR sample containing GAPDH segment (226bp). While detecting the hybridization of DNA sensor with complementary DNA of standard concentration, the effect of hybridization time, hybridization temperature, selection of indicator, adsorption condition, regeneration time and electrochemical scan condition to the responding signal was discussed. The result shows that cobalt complex is a kind of indicator with high specificity. Electro-adsorption: at -0.3V, metal complex was adsorbed to the modified electrode surface for 30s in 5mM indicator buffer solution. Hybridization: hybridization time is 60min, the optimized response of modified electrode can be got while hybridization temperature is 40℃and Na⁺ in solution is 0.3mol/L. While detecting the complementary DNA of standard concentration, the lower limit is 7.5×10^-8mol/L. In the range of 5-15 (×10^-8mol.L^-1), there was a simple equation between the respond signal of the modified electrode and the concentration of complementary DNA. The fundamental characteristic such as stability, specificity, life span was also tested. The detection for PCR segments shows that the DNA electrochemical biosensor can find out the segments existed in the solution.