Study On The Detection Of Nucleic Acid Based On Chemical Modified Electrodes

Nucleic acid is the most important part in the living cells, which containing thehereditary informationin, participating in genetic expression and monitoring the metabolicprocess of living nature. As the basic gene and the centre of life, DNA is the key for biologicalphenomena research. As one of two epigenetic systems, DNA methylation has attracted moreattention because it plays an important role in the regulation of gene expression. MicroRNAplays crucial role in cell growth, differentiation, morphologic change and cell apotosis. DNAelectrochemical biosensor is widely apply to disease diagnosis, biotechnology, food detection,enviromntal study and so on due to its advantages of simple operation, high sensitivity and nopollution, which is regarded as the bridge between electrochemistry and molecular biology. Inthis study, three electrochemical biosensors were proosed based on new nanomaterials forDNA hybridization, DNA methylation and microRNA expression determination. The maincontents could be outlined as follows:(1) In this study, a novel sensitive biosensor was fabricated for DNA detection based ongold nanoparticles (AuNPs) locked nucleic acid modified hairpin DNA probe(LNA-m-HpDNA) and enzymatic signal amplification. LNA-m-HpDNA dually labeled with3’-thiol and5’-biotin was efficiently assembled on the electrode via the interaction of thioland gold. The stem-loop structure of LNA-m-HpDNA was unfolded after hybridization withtarget DNA, which forced biotin away from the electrode surface. Streptavidin-labeledhorseradish peroxidase (Streptavidin-HRP) was subsequently immobilized on the electrodesurface via the specific conjugation of biotin and streptavidin. Thereafter, the resultingHRP/target DNA-LNA-m-HpDNA (cDNA-LNA)/AuNPs modified Au electrode wassuccessfully assembled for the construction of the DNA biosensor. Cyclic voltammetry andelectrochemical impedance spectroscopy were carried out for the characterization of modifiedelectrodes. Electrochemical reduction signal of benzoquinone was investigated in this work toindirectly analyze DNA hybridization via chronoamperometry. Under optimum conditions,the biosensor showed a good linear relationship between the current value and logarithm ofthe target DNA concentration ranging from10to1000pmol/L with a low detection limit of6.0pmol/L (S/N=3). Furthermore, the DNA biosensor exhibited excellent discriminationability to detect single-base mismatched DNA, three-base mismatched DNA andnon-complementary DNA sequence. This strategy could provide a new platform for diseasediagnose with high sensitivity.(2) In this study, a simple and sensitive method was proposed for5-methylcytosine determination based on sodium dodecyl sulfate functionalized graphene (SDS-GR) modifiedpyrolytic graphite electrode (PGE), where SDS-GR was synthesized by electrolyzing graphiterod in0.01mol/L SDS solution containing0.1mol/L KNO3. After electrochemical activationby cyclic voltammetry (CV), activated SDS-GR modified PGE (A-SDS-GR/PGE) wasfabricated for discriminating5-methylcytosine from other nucleic acid bases. The resultsindicated that SDS-GR could improve the conductivity of the electrode and promote theelectron transfer rate. And the process of electrochemical activation significantly enhanced theamount of active sites and oxygenated groups on electrode surface. As a result,5-methylcytosine, guanine, adenine and cytosine could be distinguished with differentoxidation potentials in a mixture solution except the overlap of the oxidation potential ofthymine and5-methylcytosine. However, the interference caused by thymine could beeliminated because the content of it is equal to adenine. The fabricated A-SDS-GR/PGEprovided an alternative route for5-methylcytosine determination when cytosine existed.(3) A simple and novel microRNA (miRNA) electrochemical biosensor was developedusing DNA-Au bio bar code (DNA-Au) and G-quadruplex-based DNAenzyme. DNA-Aucould increased the amount of microRNA-21(miRNA-21) participated in hybridization. AndHemin/G-quadruplex DNAenzyme significantly improved the catalysis of H2O2on theoxidation of hydroquinone, resulting in an obvious reduction current of benzoquinone formiRNA-21indirect detection. Under the optimum conditions, the linear relationship betweenmiRNA-21concentration and reduction response was obtained with the detection limit of0.006pmol/L, which showed a good sensitivity. Besides, the selectivity of biosensor wasinvestigated by detecting the base mismatched miRNAs. This proposed method was furtherapplied to detect miRNA-21extracted from human hepatocarcinoma BEL-7402cells andhuman mastocarcinoma MCF-7cells. Moreover, the influence of bisphenol A (BPA) on theexpression of miRNA-21in cells was also investigated. The results showed this biosensorcould be used for practical application with good performance, which provided a newplatform for gene diagnose.