Application of Electrochemical Biosensor Based on DNA Structure and Nucleic Acid Amplification Technique in Detection of Tumor Markers

Author:Chang Yuan Yuan

Supervisor:yuan ruo xiao zuo


Degree Year:2019





The emergence of biosensor has triggered a revolution in many fields.It is an analytical device that uses biological or biochemical reactions to detect analytes and mainly consists of biological component and sensor.The electrochemical biosensor developed by combining with electrochemistry has become research hotspot in the fields of clinical diagnosis,environmental monitoring,food analysis and pathogenic microorganisms exploration in recent years due to its advantages of simplicity,low cost and high sensitivity.In the construction of electrochemical biosensor,it is of great scientific significance and practical value to improve the performance of electrochemical biosensor with some new methods and technologies for the specific and sensitive detection of various biological molecules.In this study,multiple DNA structures were designed by using DNA nano-assembly technology,and applied in several electrochemical biosensors with high throughput,simple operation and good biocompatibility by combining with a variety of high-efficiency signal amplification strategies.The specific works are as follows:1.Homogeneous entropy catalytic-driven DNA hydrogel as strong signal blocker for highly sensitive electrochemical detection of platelet-derived growth factorIn the construction of biosensor,the flux of signal input or output will affect the sensitive detection of the target.Therefore,in order to improve the flux of signal input and output simultaneously for achieving sensitive detection of the target,we first synthesized g-C3N4@Au Fc-NH2 nanomaterials as the signal substance,which have the properties of low impedance,high conductivity and good film-forming,and can be directly attached to the electrode surface to generate strong initial electrochemical signal.When the DNA hydrogel generated by target-induced homogeneous entropy catalytic amplification was captured to the electrode surface as a strong signal blocker,the electrochemical signal was significantly reduced.Thereout,a novel electrochemical biosensor was developed to detect platelet growth factor(PDGF).As a result,he detection limit could reach 3.5 fmol/L at the range of 0.01 pmol/L to 10 nmol/L.This strategy simultaneously improves the signal input and output flux by means of nanomaterial probe and DNA macromolecular structure,providing a new thought for the highly sensitive detection of biomarkers and possessing potential application in clinical diagnosis,sensing and other related subjects.2.Construction of renewable biosensor based on host-guest recognition-assisted electrochemical release DNA cross configurationAt present,the reusable of biosensor is generally with low efficiency,and usually requires the addition of external substances,which wastes resources and increases target detection cost to some extent.In this work,an elegantly designed host-guest recognition-assisted electrochemical release was established and applied in a reusable electrochemical biosensor for the detection of microRNA-182-5p(miRNA-182-5p),a prostate cancer biomarker in prostate cancer,based on the DNA cross configuration-fueled target cycling amplification and strand displacement reaction(SDR).With such design,the single target miRNA input could be converted to large numbers of single-stranded DNA(S1-Trp and S2-Trp)output,which could be trapped by cucurbit[8]uril methyl viologen(CB-8-MV2+)based on the host-guest recognition,significantly enhancing the sensitivity for miRNA detection.Moreover,the nucleic acids products obtained from the process of cycling amplification could be utilized sufficiently,avoiding the waste and saving the experiment cost.Impressively,by resetting a settled voltage,the proposed biosensor could release S1-Trp and S2-Trp from the electrode surface,attributing that the guest ion methyl viologen(MV2+)was reduced to MV+·under this settled voltage and formed a more-stable CB-8-MV+·-MV+·complex.Once O2 was introduced in this system,MV+·could be oxidized to MV2+,generating the complex of CB-8-MV2+for capturing S1-Trp and S2-Trp again in only 5 min.As a result,the simple and fast regeneration of biosensor for target detection was realized on the base of electrochemical redox-driven assembly and release,overcoming the challenges of time-consuming,burdensome operations and expensive experimental cost in traditional reusable biosensors and updating the construction method for a reusable bisensor.Furthermore,the biosensor could be reused for more than 10 times with a regeneration rate of 93.20%-102.24%.After all,the conception of this work provides a novel thought for the construction of effective reusable biosensor to detect miRNA and other biomarkers and has great potential application in the area requiring the release of nucleic acids or proteins.3.Designing cascade reaction-fueled DNA walker and its application in electrochemical biosensor for ultrasensitive microRNA detectionThe motion of DNA walker usually depends on external forces,such as enzymes,light,chemical reactions,etc.,and can be slowed down or even stopped due to the presence of the original driving sites.Therefore,how to avoid continuously adding external driving substances and blocking the original driving sites for achieving the rapid walking of DNA walker is still a challenge.In this study,we designed cascade reaction-driven DNA walker to solve the above problems,and applied it to a novelbiosensor for the detect of microRNA with the help of DNA tetrahedral structure,which can efficiently capture DNA walker,improve the capture amount of the electroactive material ferrocene(Fc)labeled DNA,and thus improving the sensitivity of target detection.It is worth noting that this designed cascade reaction-fueled DNA walker not only realizes the rapid driving of DNA walker,but also covers the original driving sites to speed up the DNA walker for achieving rapid and sensitive detection of the target.4.Simply constructed and highly efficient classified cargo-discharge DNA robot:A novel DNA walking nanomachine platform for electrochemical ultrasensitive multiple detectionTraditional DNA walker usually has single function,while the assembly efficiency of complex DNA walker is limited to some extent.In this work,a classified cargo-discharge DNA robot with only two DNA strands was designed and driven by analogous proximity ligation assay(aPLA)-based enzyme cleaving for fast walk to construct a novel electrochemical biosensor for simultaneously ultrasensitive detection of microRNA-155 and microRNA-21.Compared with traditional DNA nanomachines,the multifunctional DNA robot possessed simple structure,high self-assembling efficiency and walking efficiency.Once it interacted with target miRNAs,this DNA robot could fast walk on the electrode surface and realize the classified cargoes discharging including beacons methylene blue(MB)and ferrocene(Fc)respectively labeled in the double stranded DNA(A1-A2)for ultrasensitive detection of multiple miRNAs simultaneously.As a result,the wide linearity ranging from 100 amol/L to 100 pmol/L and low detection limit of 35 amol/L and 39 amol/L were obtained for microRNA-155 and microRNA-21 detection respectively.As a proof of concept,the present strategy initiates a novel and high efficient walking platform to realize the ultrasensitive detection of biomarkers and possesses the potential applications in clinical diagnosis of disease.5.Programing highly efficient internal conformational-switching DNA device:Novel detection platform for ultrasensitive analysis of multiple microRNAsHerein,a highly efficient internal conformational-switching DNA(ICSD)device was tactfully designed with H DNA strand and tetrahedral DNA nanostructure,and applied in an electrochemical biosensor for simultaneously ultrasensitive and fast detection of microRNA-155 and microRNA-21.Significantly,compared with traditional conformation switching of DNA applied to biosensing,the prepared ICSD device could not only realize the multiplexed detection in one structure by targets-fueled the invert of H DNA strand labeled with methylene blue(MB)and ferrocene(Fc),but also achieve synergistic effect of two different targets for benefiting the internal signal-switching,enhancing the local reaction concentration and immobilization efficiency,improving the detection speed and sensitivity of multiple targets.As a result,the present strategy could obtain ultrasensitive detection of microRNA-155 and microRNA-21 in the range from 10 amol/L to 10 pmol/L with the detection limit of 6.9 amol/L and 5.3 amol/L respectively,as well as be applied in real cell detection and presented good selectivity,reproducibility.As a proof of concept,this internal ICSD device provides new thought for the construction of DNA structures to design late-model biosensor which achieves ultrasensitive detection of biomarkers and has the potential applications in clinical diagnosis of cancer.