Programmable High-speed And Hyper-efficiency Nucleic Acid Signal Amplification For Construction Of Ultrasensitive Biosensing Platform

In general,the biosensor is a platform that can convert the invisible properties of a special target into a readable digital signal.In the amounts of biosensors,the electrochemical biosensors combining the high specificity of biorecognition system and high sensitivity of electrochemical conversion device possessed the disadvantages of simple construction,low cost,rapid response,high stability and so on,which have been widely introduced in the fields of analytical chemistry,bioanalysis,food chemistry,and clinical diagnosis.To further improve the analytical performance of the electrochemical biosensing platform for biomarker detection,lots of signal amplification methods have been developed to enhance the corresponding sensitivity.Among these methods,because of the outstanding specificity and sensitivity,programmability and self-reactivity,the nucleic acid signal amplifications have been widly applied in the fied of biosensing.Neverthless,these strategies also suffered from the low efficiency and time-consuming problems due to the limitation of inherent properties.To address these obstacles above,we aimed at developing the nucleic acid signal amplification with much faster reaction rate and quite higher efficiency by different approaches.And we systematically explored its’reaction rate and conversion efficiency through conctructing special DNA nanostructure,introducing mismatches to decrease the reaction free energy（ΔG）,adding dual catalysts and so on,which could enhance the corresponding reactivity and reaction threshold to dramtically improve the kinetic and thermaldynamic performances.This way,the study can not only solve the problems of nucleic acid signal amplification:long reaction time and low conversion efficiency,but also provide new insight and sufficient theory support for the detection of other proteins,biomarkers,heavy matal ions in the fields of food safety testing,early disease screening and therapeutic observation.The specific research work is divided into the following parts:1.Engineering A Rolling-Circle Strand Displacement Amplification Mediated Label-Free Ultrasensitive Electrochemical Biosensing PlatformWe firstly focused on the typical strand displacenment amplification（SDA）that was driven by enzyme.In the reaction of traditional SDA,the phi29 polymerase would link with the liner DNA template and then fall off from it over and over,limiting the improvement of the reaction efficiency of SDA.To solve this problem,in this study,a novel rolling-circle strand displacement amplification（RC-SDA）was carried out by introducing a circle DNA with two recognition domains as a template instead of the limited liner DNA template in traditional strand displacement amplification（SDA）,which showed quite shorter reaction time down to 30 min and quite higher conversion efficiency compared with those of traditional SDA（reaction time:120 min）,achiving the continous reaction of enzyme.Moreover,the RC-SDA could also be used to construct a label-free biosensor for ultrasensitive detection of an HIV DNA fragment.In the presence of the target HIV DNA fragment that could be recognited by the template circle DNA,the RC-SDA could be activated to dramatically output amounts of mimic target DNA based on he Phi29 DNA polymerase and Nb.Bbv CI enzyme.In application,once the output products were captured by the DNA tetrahedral nanoprobe（DTNP）modified on the electrode surface,the electrochemical tag silver nanoclusters（Ag NCs）on DTNP would be released easily with a dramatically decreased electrochemical signal.As a result,the proposed signal-off biosensing platform was successfully applied to achieve the rapid and ultrasensitive detection of HIV DNA fragment with a detection limit down to 0.21 f M,which exploits the new generation of a universal strategy beyond the traditional ones for applications in biosensing assay,clinic diagnosis,and DNA nanobiotechnology.2.Novel 2D-DNA-Nanoprobe-Mediated Enzyme-Free-Target-Recycling Amplification for the Construction of Ultrasensitive Electrochemical Biosensing PlatformAlthough the previous work achieved some improvement of reaction rate and conversion efficiency,the weak stability and high cost of protein enzyme hinder it to be more universal.Alternatively,enzyme-free target-recycling amplification based on target-catalyzed hairpin assembly and toehold-mediated strand-displacement reactions（TSDRs）was developed in recently reported studiesthe.However,some inevitable problems like low sensitivity and weak driving force still existed.In order to overcome these obstacles,herein,on the basis of a new 2D DNA nanoprobe（DNP）,an enzyme-free-target-recycling amplification was developed to construct an electrochemical biosensor for the ultrasensitive detection of mi RNA-21（mi RNA-21）.On the one hand,in the electrochemical signal amplification,enzyme-free target recycling amplification can simply and efficiently realize the 1:N ratio pattern of target-probe molecular recognition,while avoiding the poor stability and the high cost of nuclease.On the other hand,in DNA nanostructure,2D DNA nanostructures with dual-thiol labeling not only possess lower steric effects compared with 3D DNA nanostructures but also overcome the shortcomings of the nonspecific interactions and lower stabilities of 1D DNA nanostructures,resulting the improved immobilization efficiency of it on the electrode surface.Thus,the DNP possesses better flexibility,higher stability and enhanced buildability,which could further improved the efficiency of the enzyme-free-target-recycling amplification.Based on this,the developed electrochemical biosensing platform could achieve the ultrasensitive detection of mi RNA-21.3.Programmable mismatch-fueled high-efficiency DNA signal converter for the Construction of Ultrasensitive Electrochemical Biosensing PlatformAlthough the last work improved the sensitivity of biosensing platform by combining the DNA nanostructure and the enzyme-free target recycling amplification,the accurate efficiency of the target recycling was hard to obtain and limited by the basis of reversible toehold-mediated strand-displacement reactions（TSDRs）with weak driving force.To address these challenges,an enzyme-free target recycling amplification（EFTRA）with high-reactivity and high-threshold is explored by directly introducing mismatched reactant DNA.And the developed high-efficiency EFTRA（HEEFTRA）was harnessed as a programmable DNA signal converter,possessing higher conversion efficiency than the traditional one with perfect complement owing to the more negative reaction standard free energy（ΔG）.Once traces of input target mi RNA hybridize with the mismatched reactant DNA,amounts of ferrocene（Fc）-labeled DNA could be outputted via the EFTRA.Impressively,the Fc-labeled output DNA could be easily captured by the DNA tetrahedron nanoprobes（DTNPs）on the electrode to form triplex-forming oligonucleotide（TFO）at p H=7.0 for sensitive electrochemical signal generation,additionally,the DTNPs could also be regenerated at p H=10.0,from which the conversion efficiency（N）will be accurately monitored,benefiting the selection of suitable mismatches to obtain the HEEFTRA.As a application,the HEEFTRA was successfully used as an evolved DNA signal converter for the ultrasensitive detection of mi RNA-21,which gives impetus to the design of other signal converters with excellent efficiency for ultimate applications in sensing analysis,clinical diagnosis,and other areas.4.Dual 3D DNA Nanomachine-Mediated Catalytic Hairpin Assembly for the Construction of Ultrasensitive Immobilization-free Electrochemical Biosensing PlatformThe last work obtained satisfactory results in the improved performance of nucleic acid signal amplification by enhancing the driving force,however,the less effective reaction between various of reactants and resultants still cause more side reactions and limited conversion efficiency.To solve these problems while inheriting the advantages of enzyme-free nucleic acid signal amplification,in this work,we proposed a dual 3D DNA nanomachine（DDNM）mediated catalytic hairpin assembly（DDNM-CHA）to construct the ultrasensitive electrochemical biosensor for mi RNA detection,which obtaines quite faster reaction speed and much higher conversion efficiency than those of traditional catalytic hairpin assembly（CHA）.Impressively,because the DDNM decreases the steric hindrance of substrates and increases the local concentration of reactants simultaneously,the DDNM-CHA is endowed with higher collision efficiency and more effective reaction compared with traditional CHA（reaction time:70 min,conversion efficiency:2.57×10⁵）,resulting in a hyper conversion efficiency up to2.78×10⁷ only in 25 min.Therefore,the designed DDNM-CHA could easily conquer the main predicaments:long reaction time and low efficiency.As a proof of the concept,we employed the gold nanoparticles（Au NPs）and the magnetic nanoparticle（Fe₃O₄）as the kernel of DNM-A and DNM-B,separately,and used the magnetic electrode to directly adsorb the products H1-H2/Fe₃O₄ for developing an immobilization-free biosensor for high-speed and ultrasensitive detection of mi RNA-21 with a detection limit down to0.14 f M.As a result,the DDNM-CHA we developed carves out a new insight to design the functional DNA nanomachine and evolve the analysis method for practical amplification in the biosensing area,promoting the deeper exploration of the nucleic acid signal amplification strategy and DNA nanobiotechnology.5.Programmable High-Speed and Hyper-Efficiency Dual Catalyst Hairpin Assembly for the Construction of Ultrasensitive Electrochemical Biosensing PlatformThe last study was focused on the improved reaction rate and conversion efficiency of traditional CHA with simple component,nevertheless,the single catalyst and the quite low content of it in practical application obviously limited the corresponding performance improvement of the nucleic acid aignal amplification.In terms of these obstacles,in this study,a programmable dual-catalyst hairpin assembly（DCHA）for realizing the synchronous recycle of two catalysts is designed,showing high reaction rate and outstanding conversion efficiency beyond traditional nucleic acid signal amplifications（NASA）.Once catalyst I interacts with the catalyst II,the DCHA can be activated to realize the simultaneous recycle of catalysts I and II to maintain the highly concentrated intermediate product duplex I-II instead of the steadily decreased one in typical NASA,which can be completed in about only 16 min and realizes the outstanding conversion efficiency up to 4.54×10⁸（CHA here,reaction time:78 min,conversion efficiency:2.91×10⁵）,easily overcoming the main predicaments of NASA:time-consuming and low-efficiency.As a proof of the concept,the proposed DCHA successfully applied as a high-speed and hyper-efficiency DNA signal magnifier to achieve the rapid and ultrasensitive detection of mi RNA-21 in cancer cell lysates,which exploits the new generation of universal strategy for the applications in biosensing assay,clinic diagnose,and DNA nanobiotechnology.