Construction Of Electrochemiluminescence Biosensor Based On DNA Walking Machine

Cancer has caused great trouble to patients,and even directly threatened human health and life.Early detection and timely treatment are the most effective means to prevent cancer.In recent years,with the rapid development and in-depth study of clinical laboratory medicine,some cancer markers have come out one after another.The length of microRNA（miRNA）is between 18 and 24nucleotides.MiRNA has attracted the attention of scientists because of its excellent regulation ability in gene expression,and has become a focus of cancer markers.Conventional methods such as quantitative reverse transcription polymerase chain reaction,fluorescence and Northern blotting have been used for the detection of miRNA,however the sensitivity and specificity of these methods cannotmeettheincreasinglystringentdetectionrequirements.Atpresent,electrochemiluminescence（ECL）is widely used in various fields of bioassay due to its high stability,low background and wide detection range.It is an effective way for biomarker analysis and early cancer detection.In order to improve the sensitivity of ECL detection,some nucleic acid amplification methods,such as nucleic acid amplification and polymerase chain reaction,are widely used in sensor construction.However,these methods not only need strict reaction conditions,but also have very high requirements for reaction instruments and operation.At the same time,these methods may also increase false positive or negative signals of biosensors.DNA walking machine is an artificial molecular machine synthesized from intelligently editable DNA.Because of its highly efficient self-assembly nanostructures,excellent cargo transport capability and good biomimetic phenomena,it has attracted a lot attentions.In order to improve the sensitivity of ECL biosensors,we designed several DNA nanomachines,combined with DNA amplification strategy and a variety of nanocomplexes to construct ECL biosensors to achieve ultra-sensitive detection of biomarkers.The studies of this thesis are mainly divided into the following sections:1.An“on-off-super on”ECL regenerated biosensor based on 3D DNA walking machine for ultrasensitive detection of microRNA from cancer cellsDNA walking machine has broad prospects in biological applications due to its high efficiency of self-assembly nanostructures,cargo handling capacity and biological simulation phenomena.However,the effect of cargoes transit from 1D or 2D DNA walking machines are unsatisfactory due to the inadequate ability of 1D or 2D walking space and low fluxes of the substrate DNA in nanostructures.Herein,3D DNA walking machine with high walking efficiency and strong signal amplification ability was introduced in this system to transform a small number of targets miRNA into a large number of intermediate DNA for signal amplification,which lay a foundation for the application of 3D DNA walking machine in ECL.Secondly,the unique distance-controlled energy transfer phenomenon of gold nanoparticles（AuNPs）and CdS:Mn quantum dots is employed to propose an“on-off-super on”strategy,which overcomes the shortcoming of high background signal in conventional direct detection,and also solves the problem of low second“on”state signal in the conventional“on-off-on”mode,thus improving the detection sensitivity.The biosensor based on 3D DNA walking machine proposes a new signal amplification method in ECL field and broadens its application.2.A biosensor based on 3D DNA walking machine network and distance-controlled ECL energy transfer for ultrasensitive detection of tenascin C and lead ionThe 3D DNA walking machine has high walking efficiency,strong signal amplification ability,which significantly improves the sensitivity of the biosensor.However,the limited interface of the biosensor leads to the problem of insufficient loading of the3D DNA walking machine.Based on this,a 3D-DNA walking machine network was proposed through simple base pairing between DNA linkers（DNA H1,H2）,which significantly improved the loading efficiency of nanomachines and the sensitivity of the biosensor.Secondly,the biosensor achieved different types of targets detection in account of distance-controlled ECL energy transfer between AuNPs and CdS:Mn QDs,which was of great significance in clinical diagnosis and paved a promising avenue of the DNA nanodevices in the wide application of clinical diagnosis.3.An ultrasensitive electrochemiluminescence biosensor for speedy detection of microRNA based on DNA rolling machine and target recyclingIntelligent DNA walking machine have become a hotspot in biosensing.However,due to the low concentration of DNA components in DNA walking machines and the trend of derailment induced by Brownian motion,the walking efficiency is still limited.Herein,a Zn²⁺-driven DNA rolling machine was proposed to overcome these shortages.The original-designed DNA rolling machine simply assembled by large number of leg DNA attracted on the AuNP provided a high local concentration of leg DNA matched with track DNA,which not only overcame the derail of leg DNA,but also changed the motion mode from step-by-step walking to high-speed rolling to overcome the shortages of traditional DNA walking machines.Such an excellent DNA molecular machine is used to construct biosensor in the system,which provides a super sensitive and controllable strategy for rapid detection of cancer markers.