| DNA biosensor technology is a novel technology integrated with many disciplines includingchemistry,biology,physics,and so on.Specially,DNA biosensors are generally applied to various fields(such as medical science,food and environment)due to its short analysis time,good selectivity,high sensitivity,and so on.In this paper,DNA biosensors of fluorescence and electrochemistrywereestablished for the detection of guanine and its derivatives or miR-21 based on guanine-lost G-quadruplex(GLGq)and double signals ratio,showing promising approaches for the diagnosis,treatment and evaluation of diseases.This thesis includes three chapters.The first and second chapters introduce two fluorescent or electrochemical DNA biosensorsfor the detection of guanine and its derivatives.The third chapter is about a ratio-electrochemical DNA biosensor for the detection of the miR-21 derived from the contents of exosomes.Chapter one: In this chapter,a label-free fluorescent DNA biosensor was proposed based on guanine-lost G-quadruplex(GLGq)structure and Thioflavine T(ThT)for the detection of guanine.In the presence of guanine,guanine filled the guanine-lost in GLGq via Hoogsteen hydrogen bonds,forming stabilized G-quadruplex structure with ThT facilitated.Meanwhile,ThT specifically bound to G-quadruplex,exhibiting a prominentfluorescencesignal.Under the optimized conditions,the linear response range of biosensor is 0.05~50 μM and the detection limit is 0.018 μM.Thus,the biosensor is worth being further applied in practice owing to its excellent properties including label-free detection,low background signal,stablefluorescencesignal,good selectivity,high sensitivity and so on.Chapter two: On the basis of GLGq structure and Hemin,a label-free electrochemical DNA biosensor was developed for the detection of guanine and its derivatives.In the presence of guanine,the guanine-lost of GLGq was filled,formingstabilized G-quadruplex with Hemin promoted.Simultaneously,a G-quadruplex-Hemin DNAzyme formed by Hemin and G-quadruplex,which has horseradish peroxidase-like activity,being capable of effectively catalyzing the H2O2-mediated oxidation of TMB accompanied by an enhanced electrochemical current signal and a obvious change in color(colorless solution transformed toblue solution)of the reaction solution.The biosensor possesses a good linearity range of5~100 nM with a lower detection limit of 0.361 nM,being potential to realizing visible and portable detection because of lower background signal,higher selectivity and sensitivity.Chapter three: An electrochemical DNA biosensor was established for the detection of miR-21 derived from the contents of exosomes based on double signals and ratio method.In the presence of miR-21,a chain substitution reaction occurred in “Y” DNA structure which was assembled on the electrode,resulting the release of Fc-labeled DNA(Fc neared to electrode surface)from “Y” structure and the departure of Fc from electrode surface.Thus,the current of Fc(IFc)decreased.Meanwhile,MB-labeled DNA(MB is away from the electrode surface)which existed as the single-stranded DNA folded into hairpin structure by self-hybridization,leading MB to approach the electrode surface and increasing the current of MB(IMB).Generally,the sensor made the miR-21 detectable via the change of IMB/IFc with the linearity range of 10~70 fM and the detection limit of 2.3fM.Compared to single signal,the electrochemical biosensor employed dual signals and ratio method is more stable and reliable because it is not susceptible to the statuson the electrode surface and so on,significantly being of potentially clinicalapplication value. |
