| DNA electrochemical biosensor,designed by basic principle of interaction with complementary DNA,is a novel genetic detection technology.For the advantages of rapid,simple,low cost and sensitive,it shows the significant value in the analysis of nucleic acid and other applications.In recent years,due to the rapid development of electrochemical technology,many excellent hybrid indicators are widely used in DNA biosensors.In order to improve the detection capability of biosensor,how to screen high sensitivity electrochemical hybridization indicators is becoming an important task in the research of DNA biosensors.Therefore,different charge properties’ hybridization indicators are studied,and the high sensitivity hybridization indicators are selected.Meanwhile,the hybridization indicators are used in the detection of major disease,and constructed a series of high sensitivity and specific DNA electrochemical biosensors.This thesis uses electrochemistry and ultraviolet-visible spectra method to study the interaction of different charge properties and DNA,such as cationic celestine bule,neutral kaempferol and anionic nuclear fast red.The results of the electrochemical study indicate,all of the electroactive materials can combine with DNA to form non-electroactive supermolecule material leading to decreasing of the peak current.The uv-vis spectroscopy shows that the absorption peak decrease and accompanied with red shift after the accession of salmon sperm DNA.The experimental results show that different charge properties’ materials are able to implant the construction of DNA double helix in the optimized condition.The the binding constants of celestine blue,kaempferol and nuclear fast red are estimated to be 1012.87,109.68 and 1011.71 respectively.The DNA electrochemical biosensors are constructed by the three different charge properties’ hybridization indicators and use in the detection of osteosarcoma related gene,hand-foot and mouth disease and west nile virus.The details are given as follows:1 、 Using cationic celestine blue as hybridization indicator,we construct a DNA biosensor to detect the osteosarcoma gene.On the optimized experimental conditions,the sensors can distinguish complementary sequence,single-base mismatch and completely mismatch sequence well.In artificial simulated samples,the peak current shows a excellent linear relation with the amount of the complementary oligonucleotides in the range from1.0×10-9 5×10-7mol/L.The regression equation was ΔIμA=0.62038 +0.36576 lg CDNA（nM）,and the regression coefficient of the linear curve was 0.9905.A detection limit of0.046 nmol/L of the target DNA can be estimated using 3σ.The biosensor can detect osteosarcoma gene with sensitive,specific and quantitative.In simulating samples,the complementary sequences can be detected accurately,and will not affected by interference sequences.2 、 Eelectrochemical DNA biosensor,which prepared with neutral kaempferol as hybidization indicator can rapidly detect hand-foot and mouth disease.The results show that the biosensor used kaempferol as hybridization indicator is highly sensitivity and specificity.In the serum simulated samples,the peak current showed an excellent linear relation with the amount of the complementary oligonucleotides in the range from 5.0×10-9 5×10-7mol/L.The regression equation was ΔIμA=0.7192+0.5012lgCDNA（nM）,and the regression coefficient of the linear curve was 0.9908.A detection limit of 0.167 nmol/L of the target DNA can be estimated using 3σ.The proposed biosensor realize the rapid detection of hand-foot and mouth disease in the simulated samples.3 、 The electrochemical DNA biosensor prepared by anionic nuclear fast red as electroactive indicator,can be used to detect WNV in the simulated samples.The peak current showed an excellent linear relation with the concentration of the complementary oligonucleotides in the range from 5.0×10-8 5×10-7mol/L.The regression equation wasΔIμA=-0.823+ 0.7676 lg CDNA（nM）,and the regression coefficient of the linear curve was0.9910.A detection limit of 3.513 nmol/L of the target DNA can be estimated using 3σ. |
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