Research And Application Of Functional Nucleic Acid Nanomachines And Novel Rubrene Electrochemiluminescent Materials In Biosensors

Electrochemiluminescence（ECL）technology has been widely used to construct biosensors,due to its low background signal,high sensitivity and good selectivity.However,in clinical diagnosis,the analysis requirements for the low-abundance biomarkers,especially for low-abundance biomarkers in complex samples,are getting higher and higher.At present,the following two methods were mainly proposed to improve the accuracy and sensitivity of low-abundance target analysis:（1）increasing the target concentration,（2）enhancing ECL signal.Therefore,in this study,the novel nucleic acid nanomachines were designed and applied reasonably for the construction of ECL biosensors,achieving target amplification and improving the accuracy and sensitivity of target analysis.Addtionally,the preparation of the novel polycyclic aromatic hydrocarbons ECL materials（rubrene）could be used to increase their ECL intensity in aqueous media,thereby improving the sensitivity of target detection.The following are the main contents of this work:1.Electrochemiluminescence technology,the mechanism of electrochemiluminescence and their application in biosensors were summarized in this section.In addition,the development of functional nucleic acid nanomachines and their application in electrochemiluminescence biosensor are reviewed.2.The accurate and rapid quantitative detection of antibodies had a significant influence in controlling and preventing disease or toxin outbreaks.In general,the detection of antibodies mainly depends on the specific immune response.However,due to its limited specificity and cross-reactivity（nonspecific binding to sample matrix components）,the accuracy and sensitivity of antibody detection decreases.In addition,aptamers are often used for the detection of antibodies and other proteins due to their high binding affinity and selectivity for specific targets.However,although the use of aptamers overcomes the shortcomings of the immune response,the complicated screening process and expensive screening costs also limit the application of aptamers.Therefore,this work first utilizes the antibody-powered triplex-DNA nanomachine to convert non-nucleic acid targets（such as digoxin antibodies）into ideal amplifiable“substitute target”—nucleic acids,which can further participate in the enzyme-assisted cycling strand displacement reaction to achieve antibody conversion and the amplification of the substitute target,improving the sensitivity of antibody detection.In addition,rubrene microblocks（RubMBs）with uniform size and stable ECL signal in aqueous medium were prepared by reprecipitation.Then,porous palladium nanospheres with excellent catalytic activity used as a coreaction accelerator were introduced into the ECL system using the rubrene microblocks（RubMBs）as the emitters and dissolved oxygen as the endogenous coreactant.Finally,a novel ECL ternary system（RubMBs/dissolved O₂/pPdNSs）was successfully constructed.Therefore,based on the above ECL ternary system and signal amplification method,the sensitive detection of the target antibody was achieved via the"on-off-on"signal output mode.A wider linear range(1.0×10^-11 mol/L-2.0×10^-7 mol/L)and low limit of detection（6.7pmol/L）were obtained.This method provides a novel ECL analysis platform for trace detection of other antibodies.3.In recent years,most functional DNA nanomachines have been widely applied in the field of ECL biosensing,due to their unique signal amplification function and kinetic advantages.However,the molecular outputs generated by the operation of these functional DNA nanomachines are almost all freely dispersed in the reaction solution.Influenced by Brownian motion,the reaction of these molecular outputs on the surface of the sensing platform reflected the problems of low movement efficiency and poor sustainability.In order to solve the above problems,a novel multisite-anchored DNA nanomachine was designed and assembled for the sensitive and rapid detection of target.First,the high-density hairpin DNAs were immobilized on the surface of gold nanoparticle through Au-S covalent bonds to form a locked DNA nanomachine.Next,in the presence of the target（ochratoxin A,OTA）and its corresponding aptamer,the locked DNA nanomachine can be triggered to undergo an enzyme-assisted hairpin self-assembly cycle amplification reaction,which gradually activated the multisite-anchored DNA nanomachine.Notably,the activated DNA nanomachine processed a high local concentration of signal probe-recognizing sequence（leg DNA）on its surface with high-order,compact and stability,which could provide multiple sites to operate simultaneously on the sensing platform.This greatly promoted the movement efficiency and velocity,thereby improving the efficiency and sensitivity of the ECL biosensor for target detection.In the previous work,bulk rubrene materials were synthesized by the reprecipitation method.Because its dense molecular stacking caused the increase of inner filter effect and hindered the internal ECL luminophores from being electrochemically activated.Thus,the novel hollow rubrene-metal nanomaterials were synthesized by in-situ redox method in this work.Compared with the bulk rubrene materials,the hollow nanomaterials had a higher ECL intensity and efficiency in the aqueous medium.Therefore,based on the hollow rubrene-metal nanomaterial/dissolved oxygen/palladium nanoparticle ternary ECL system,we utilized the dynamic advantages and signal amplification functions of the multisite-anchored DNA nanomachine to construct an"on-off-supper on"-type ECL biosensor,achieving the sensitive detection of ochratoxin A（OTA）with the wide liner range from 1.0×10^-14 g/mL to 1.0×10^-10 g/mL and the detection limit of 4.7 fg/mL.