The Preparation And Application Of The Cucumber DNA Voltammetric Biosensor

On the basis of consulting a great amount of articles about biosensor, the extraction, the purification of plant DNA and the enzyme operation of DNA. The preparation and application of cucumber DNA voltammetric biosensor have been studied.1. The procedure of the extraction of plant DNA. The optimum conditions of the extraction of cucumber DNA using SDS method were as following ( for 1.0 g cucumber ) : 4.0 mL of cell extraction solution, 1.0 mL of saturated KC1 solution, the rate between the phenol / chloroform / isoamyl alcohol solution, chloroform / isoamyl alcohol solution or isopropanol and the sample solution being 0.6, 0.6, and 0.58, respectively. Under such optimum conditions, the yield of the cucumber DNA is high and the cucumber DNA is very pure. Comparing with the reported methods, the amounts of the poisonous chemicals in the proposed procedure were decreased greatly.2. The electrophoresis conditions for the purification of cucumber DNA were optimized. The optimum conditions are as following: the agarose concentration is 1.0%, and the thickness of gel, the pH, the ionic intention and the potential, 4.0 mm, 8.0, 0.050, and 60 V, respectively. We used the gel embedded - little chamber electrically washed method to obtain the purest cucumber DNA.3. The restriction enzyme Hind III was used to cut the cucumber DNA, and the fragment of the obtained DNA is suitable to prepare the cucumber DNA voltammetric biosensor. Thus the sensor possessed excellent analytical characteristics.4. The cucumber DNA voltammetric biosensor was prepared using the stearic acid-modified carbon paste electrode, and the conditions for the determination of cucumber DNA using the sensor were optimized. These conditions include the components of the basic sensor, the solution for the activation of the basic sensor, the solution for the immobilization of cucumber ssDNA and the solution for DNA hybridization reaction. The basic biosensor consists of 60% carbon powder, 5.0% stearic acid and 35% paraffin liquid, the surface of the sensor was polished on a smooth mirror. After the activation of the basic sensor in 100 μ L of solution (pH6.0) containing 7.0 mmol/L EDC, 8.0 mmol/L NHS and 0.050 mol/L Na2HPO4, theimmobilization of cucumber ssDNA on the activized surface was performed in 100 u L of immobilization solution (pH6.9) containing 5.0 μ g/mL denaturalized cucumber DNA, 0.010 mol/L Na2HPO4. The DNA hybridization reaction and the determination of DNA was carried out in a pH7.0 hybridization solution containing 0.020 mol/L NaCl, 5.0 mmol/L Tris and 90 u mol/L Co(bpy)3(ClO4)3. Under the optimum conditions, the linear range is from 0.010 μ g/mL to 0.090 μ g/mL with the detection limit of 2.97 ng/mL and the relative standard deviation 1.8%. This kind of biosensor can be used to identify the class of samples, which has a broad application in forensic science. The cucumber DNA samples extracted in our laboratory was determined by the proposed prdecdure and the recovery is between 98% - 104%. The cyclic voltammetric data showed that the electrochemical reaction of the Co(bpy).3(ClO4)3 complex insetted to the double-stranded DNA on the voltammetric biosensor is reversible.