Research And Application Of DNA Nanostructure And Nucleic Acid Signal Amplification Strategy In Electrochemical Biosensor

As a new type of biological analysis and medical diagnosis tool,electrochemical biosensors was widely used in many fields,such as medicine,environmental monitoring,food analysis,industry and agriculture,which possessed the characteristics of simple preparation,easy miniaturization,fast detection speed,and low consumption cost.With the continuous development of nanotechnology,researchers have creatively combined the latest research results with electrochemical biosensors and try to develop a sensing platform with better perfomance.In this paper,based on nucleic acid signal amplification strategies and DNA nanostructures,several electrochemical biosensors was innovatively constructed to achieve more accurate,efficient and sensitive detection of targets,which was expected to provide new ideas for the biosensor research.The specific research content was exhibited as follows:1.One DNA Circle Capture Probe with Multiple Target Recognition Domains for Simultaneous Electrochemical Detection of miRNA-21 and miRNA-155Previous studies have found that the occurrence of a disease was often related to the simultaneous changes of multiple tumor markers.Therefore,it was necessary to develop a biosensor that can be used to detect multiple targets at the same time.In this work,a novel DNA circle capture probe with multiple target recognition domains was designed to develop an electrochemical biosensor for ultrasensitive detection of micro RNA-21(miRNA-21)and miRNA-155 simultaneously.The DNA circle capture probe was anchored at the top of the tetrahedron DNA nanostructure(TDN)to simultaneously recognize miRNA-21 and miRNA-155 through multiple target recognition domains under the assistance of Helper strands,which could trigger mimetic proximity ligation assay(m PLA)for capturing the beacons ferrocene(Fc)-A1 and methylene blue(MB)-A2 to achieve multiple miRNAs detection.In this way,the local reaction concentration could be enhanced and avoid the interference of various capture probes compared with the traditional multiplexed electrochemical biosensor with the use of different capture probes,resulting in the significantly improvement of detection sensitivity.As a result,this proposed biosensor showed wide linearity ranging from 0.1 fmol/L to 10 nmol/L with detection limits of miRNA-21 and miRNA-155 as 18.9 amol/L and 39.6 amol/L respectively.The present strategy paved a new path in the design of capture probes for achieving more efficient and sensitive multiple biomarkers detections and possessed the potential applications in clinical diagnostic of diseases.2.A DNA Three-Way Junction-Mediated DNA Walker for Electrochemical Ultrasensitive Detection of miRNA-182-5pSince the content of miRNA in real samples was very low,it was difficult to actually perform detection,and the sensitivity of the biosensor need to be relatively high.Therefore,it was necessary to develop a biosensor with high sensitivity and low detection limit in combination with signal amplification strategies.Among them,the signal amplification technology that used enzymes to achieve target recycling has attracted wide attention,which not only effectively achieved target recycling,but also increased the electrical signals and reduced the detection limit.In this work,a DNA three-way junction(TWJ)possessed multiple recognition regions was intelligently engineered,which could be applied as DNA walker with rapid walking speed assisted by endonuclease for electrochemical miRNA-182-5p detection.Once the target miRNA presented,the hairpins on TWJ could be successively opened to form an annular DNA walker,which could walk on the full filed of electrode surface without the confine of DNA walker legs compared with traditional DNA walker,further improved the walking efficiency.In addition,this DNA walker accompanied with multi-recognition segments could obviously increase the local concentration and realize the constantly cyclic utilization of it,which significantly enhanced the detection speed and magnified electrochemical signals.As a result,this proposed biosensor could dexterously address the fundamental challenges resulted from restricted walking space,single recognition site and complicated operation in traditional DNA walker strategies,which finally achieved the high-efficiency and ultrasensitive detection of miRNA-182-5p with a detection limit of 31.13 amol/L ranging from 0.1 fmol/L to 1 nmol/L.As a proof of concept,this strategy explored an innovative path in the exploit of new DNA walker nanostructure with multiple recognition functions for accomplishing more efficient and sensitive biomarkers detection.3.Strand Displacement Amplication(SDA)-Powered Free 3D DNA Walker for Electrochemical Ultrasensitive Detection of miRNA-21Due to the disadvantages of enzyme-assisted target cyclic amplification,such as: macromolecular enzymes caused steric hindrance as interference,and need to control temperature during operation.Therefore,signal amplification technology without enzyme assistance was required.In this work,we designed a newly three-dimensional(3D)free DNA walker driven by DNA strand displacement amplication(SDA)to construct an electrochemical biosensor combing with DNA functionalized gold nanoparticles(Au NPs)to achieve ultrasensitive and rapid detection of the target miRNA-21.When the target miRNA was introduced into the DNA walker system,the target could be released and walked around the entire 3D nanoparticle tracks after the DNA SDA.In the process of walking,each step of the reaction would release a doublestranded structure labeled with the electroactive substance ferrocene(Fc),which can eventually be captured on the electrode surface by hairpin DNA to generate an electrochemical signal.Compared with the traditional DNA walker,the free 3D DNA walker was more efficient because all the DNA reaction substrates were immobilized on the same Au NPs,which possessed a high local effective concentration and accelerated the reaction speed severely.Moreover,the continuous recycling of the target can be achieved through the DNA strand displacement,which was beneficial to increase the reaction signal and reduce the detection limit.As a result,through this ingenious designing,this proposed electrochemical biosensor realized the sensitive,rapid and efficient detection of the target miRNA-21 with the detection range ranging from 100 amol/L to 100 pmol/L,and the detection limit was 26.50 amol/L.More meaningfully,the enzyme-free DNA walker proposed in this project as a novel DNA machine provided a reference for the simple assembly of DNA machines with high efficiency.