Construction And Application Of Electrochemiluminescence Biosensors Based On Enzyme-free DNA Walker

DNA walker,a type of special dynamic DNA nanomachine which can move along pre-designed tracks with the assistance of driving forces,has been utilized in various fields including the assembly and synthesis of nanomaterials,drug delivery,biocomputing and biosensing.Especially in the field of biosensing,DNA walkers exhibit excellent signal transduction and stable signal amplification capability,and thus have been widely applied in the analysis and detection of biomarkers.However,current DNA walker-based biosensors mainly face with the following problems.Firstly,for the biological enzymes-driven DNA walkers,the biological enzymes are impressionable to reaction environment such as temperature,humidity,p H and so forth,which may affect the normal operation of DNA walkers and further limit the performance of biosensors.Besides,DNA walkers still remain in relatively low running rate.Moreover,the DNA walker-based biosensors are incapable to accurately analyze trace amount biomarker on account of their low sensitivity.In response to the problems above mentioned,in this paper,we integrated enzyme-free DNA walkers into electrochemiluminescence(ECL)technology and utilized Au@CDs,Au@luminol and Au NPs@CDs@PEDOT:PSS@ZIF-8 nanomaterials as ECL signal output.Then,we recruited biomarkers p53 DNA and miRNA-21 as target model to assess the performance of constructed ECL biosensors.These enzyme-free DNA walkers-based ECL biosensor provide a new tool and thought for the clinical diagnosis of early diseases and the assessment of the prognostic effects and provide some valuable reference for the design and development of drugs.The detailed contents are as follows:Part 1.Catalytic hairpin assembly-driven DNA walker-based ratiometric ECL biosensor for p53 DNA detection.We designed catalytic hairpin assembly(CHA)-driven DNA walker,coupling Au@CDs and Au@luminol,and constructed a novel ratiometric ECL biosensor for highly sensitive detection of p53 DNA.In this work,CHA-driven DNA walker exhibited excellent signal amplification ability and relatively rapid running rate,improving the sensitivity of the ECL biosensor.Meanwhile,the ratio strategy improved the accuracy of the biosensor,achieving the detection of p53 DNA ranging from 1 f M to 10 p M with the detection limit of 0.34 f M.Part 2."One-to-more"opening mode of DNAzyme walker-based ECL biosensor for miRNA-21 detectionIn this section,a novel DNAzyme walker with"one-to-more"opening mode was proposed and the DNAzyme walker-based ECL biosensor was assessed toward miRNA detection.Entropy toehold-mediated strand displacement amplification(E-TMSDA)was first employed to amplify target miRNA-21,and the generated output DNA opened DNAzyme strand.Subsequently,the DNAzyme could walk along the track with the assistance of Mn²⁺.Based on the excellent ECL capability of Au@CDs@PEDOT:PSS@ZIF-8,a rapid and ultrasensitive detection of miRNA-21target was achieved within 100 min with a detection limit of 50 a M.Part 3.Tripedal DNA walker prepared by target-triggered catalytic hairpin assembly for ECL detection of miRNA-21In this section,we adopted a simple target-triggered CHA circuit to synthesize a tripedal DNA walker.Then,the tripedal DNA walker could rapidly move along the track-functionalized electrode under the driving force of CHA.the tripedal DNA walker-based ECL biosensor could achieve the ultrasensitive detection of miRNA-21 in the broad linear range of 10 aM to 1 pM with a detection limit of 4 aM.