Studies On Electrochemiluminescence Biosensors Based On DNA Nanomachine For The Detection Of MicroRNA

DNA acts as an exciting functional biomaterial,which has various advantages such as specific base-pairing,structural flexibility,and programmed assembly.Inspired by these advantages,scientists had successfully fabricated several nanomachines including“tweezers”,“walkers”and“motors”with the involvement of DNA as the fundamental engineering materials in recent dacades.In response to external stimuli,synthetic DNA nanomachines could accomplish mechanical movement,suggesting the huge furture of constructing a DNA nanomachine that is being used in the field of biosensors.Cancer has became one of common clinical diseases as the leading cause of mortality,which is often no early symptoms and difficult to diagnose.In addition,realizing early diagnosis of cancer correctly has a positive vuale to prevent and reduce the mortality.Amazingly,a lot of reports have demonstated that certain miRNAs have been recognized as the key biomarkers for early diagnosis of various cancers due to the close association with cancer occurrence.Thus,building reliable and efficient analytical methods for sensitive miRNA biomarkers detection is critical to the development of clinical diagnosis in different cancers.Hererin,based on the advantage of DNA nanomchines,the preparation of electrochemiluminescence?ECL?nanomaterials and the application of the strand displacement reaction,we constructed the novel biosensor for sensitive detection of miRNA.In our paper,the research contents are carried out as follows:Part 1 Bi-directional DNA walking machine and its application in an enzyme-free electrochemiluminescence biosensor for sensitive detection of microRNAsDNA walking machine which consists of a DNA walker and a DNA track is attracting substantial attention in recent years and the development of artificial DNA walking machine for performing biological function is still in urgent need.In this work,a novel DNA walking machine with bi-directional movement which was powered by microRNAs?miRNAs?was designed for the first time and applied in an enzyme-free biosensor for electrochemiluminescence?ECL?determination of two miRNAs.Firstly,the DNA track was assembled on the electrode which was modified with Mn²⁺doped CdS nanocrystals?CdS:Mn NCs?as the ECL emitters.Subsequently,the Au nanoparticles?AuNPs?-labeled walkers were introduced into the sensor with the help of miRNA-21 which led to the long-distance between AuNPs and CdS:Mn NCs.Interestingly,the appropriate large separation of AuNPs and CdS:Mn NCs,which triggered surface plasmon resonance?SPR?,achieved an ECL enhancement.Next,the strand displacement strategy prompted stepwise movements of the DNA walker on the DNA track,resulting in the proximity of AuNPs and CdS:Mn NCs.Simultaneously,the proximity realized ECL quenching due to energy transfer between AuNPs and CdS:Mn NCs,thereby achieving the sensitive detection of miRNA-21.Impressively,with the introduction of miRNA-155,the walker could move back along the track automatically,and SPR occurred between distant AuNPs and CdS:Mn NCs,achieving an ECL enhancement and realizing the sensitive detection of miRNA-155.With the DNA walker bi-directionally moving along the DNA track,the proposed biosensor for miRNA-21 and miRNA-155 detection achieved the low detection limits of 1.51 fmol·L^-1 and 1.67 fmol·L^-1 respectively.Part 2 A switching reversible and distance-controllable DNA Scissor:A regenerated electrochemiluminescence biosensor for ultrasensitive detection of microRNAArtificial DNA machine representing a new and exciting platform of nanotechnology has been extensively explored.While DNA scissor which has rotational free sticky ends has been developed,reversible switching and precise control still remain a great challenge.Herein,a novel DNA scissor-based regenerated biosensor has been constructed upon the PTCA-PEI-Ru?II?complex as electrochemiluminescence?ECL?signal for sensitive detection of microRNA?miRNA?.With rational design,miRNA-21 could transform the configuration of DNA scissor labeled with the coreactant diethylenetriamine?DETA?based on toehold-mediated strand displacement reaction?TSDR?,realizing ultra-high sensitive analysis of miRNA-21.Simultaneously,the successive switching of DNA scissor could influence luminous efficiency due to the distance change between DETA and PTCA-PEI-Ru?II?film,significantly increasing the sensitivity for miRNA determination.Impressively,the elaborated biosensor was able to be regenerated by adding Waste DNA?W?based on TSDR.The superior sensitive DNA scissor biosensor was constructed successfully with a detection limit of 0.17 fmol·L^-1.Amazingly,the ECL assay could be used for miRNA detection from different cancer cells,which hold a great future for applications of dynamic machines in clinical analysis and early disease diagnosis.Part 3 DNA transporter-based a novel electrochemiluminescence resonance energy transfer system and its application for ultrasensitive detection of microRNA in cancer cellsIn this work,a DNA transporter which was composed of a track containing central axis and three surrounding branchs and a walker containing two legs,was construced to realize programmed circular by using lambda exonuclease.Herein,a novel electrochemiluminescence resonance energy transfer?ERET?strategy between the acceptor of CdS:Mn NCs for acting as the ECL emitter and the donor of tetracycline?TET?,which is a type of small molecule dye,was applied for the construction of a DNA transporter-based biosensor for the ultra-high sensitive determination of cancer cells.First,a programmable DNA cyclic amplification was employed to achieve the conversion of a small number of miRNA-21 into a large amount of universal DNA reporters?TET-S?.Second,TET-S hybridized with the track and the walker to form dsDNA?TET-S:W_a:SA?.Simultaneously,the ECL intensity of the QDs was decreased remarkably because ERET occured between QDs and TET.Impressively,once lambda exonuclease was introduced,the walker was enlabled to programmed circulate along the track because the dsDNA?TET-S:W_a:SA?is selectively hydrolyzed by lambda exonuclease from 5'phosphate in the 5'-to-3'direction.The successive circular of the walker on the track led to continuous removal of massive TET from the sensing electrode,which resulted in ECL enhancement and realized the highly sensitive detection of miRNA-21 down to 5.3 amol·L^-1.Impressively,this method could sensitively measure the miRNA-21 of different cancer cells and provide a promising platform for the monitor of other biomarkers in early cancer diagnosis.