Construction Of Electrochemical Biosensors Based On Efficient Enzyme-Free Target Cyclic Amplification Technology For The Detection Of MiRNA-21

Electrochemical biosensors are considered one of the most promising devices for detecting biomarkers due to their advantages of fast detection,simple operation,high sensitivity,and intelligence.They have been widely used in fields such as food detection,environmental monitoring,and drug analysis.Numerous studies have confirmed that the abnormal regulation of miRNAs is usually associated with the occurrence of cancer.Based on their strong correlation with cancer,miRNAs could serve as promising biomarkers for cancer diagnosis and treatment.Therefore,accurate,rapid and highly sensitive detection of intracellular miRNA remains a huge challenge.In order to solve these problems and further improve the analytical performance of biosensors,in recent years,with the development of DNA nanotechnology,various nucleic acid signal amplification methods have been introduced by researchers to improve the sensitivity of biosensors ascribe to their strong programmability and high amplification efficiency.In comparison,the enzyme-free target recycling amplification（EFTRA）method has been more widely used ascribe to its simple operation.However,because of its weak driving force and the easy degradation of target RNA during the cycling process,there are still problems with low efficiency and slow reaction rate.In order to solve the above problems,this article starts from the perspective of improving the speed and efficiency of EFTRA to research.By introducing appropriate mismatched bases and increasing the number of simulated targets in EFTRA,the efficiency and rate of EFTRA reaction are improved to construct an electrochemical biosensor for rapid,efficient,and ultra sensitive detection of miRNA.The research content of this article will provide new ideas for the subsequent research of highly sensitive biosensors.The specific research content is as follows:1.Mismatch-fueled catalytic hairpin assembly mediated biosensor for ultrasensitive detection of miRNA.Because of the favourable amplification rate,stability,and applicability of traditional catalytic hairpin assembly（CHA）reactions,it is a highly promising enzyme free signal amplification strategy in enzyme free target cycle amplification methods.However,the CHA reaction is still limited by weak driving forces which hindering the improvement of amplification efficiency.To solve this problem,we introduced suitable mismatched bases in the neck region of H2,which increased the free energy difference before and after the CHA reaction,thereby improving the reaction driving force,increasing the reaction limit,and further improving the overall reaction amplification efficiency.Therefore,we designed a mismatch driven catalytic hairpin assembly strategy（MCHA）with higher amplification efficiency for constructing an electrochemical biosensing platform for ultra-sensitive detection of miRNA-21.According to experimental verification,compared to traditional CHA（50 minutes,conversion efficiency:2.42×10⁵）,MCHA could generate up to 9.56×10⁶ in just 30 minutes amplification efficiency.The electrochemical biosensor developed based on MCHA has achieved ultra-sensitive detection of miRNA-21 with a detection limit of 0.17 fmol·L^-1,and could further achieve the detection of miRNA-21 in tumor cell lysate.Therefore,this method opens up a new strategy for sensitive detection of biomarkers in sensing analysis.2.Self-accelerated DNA walker mediated electrochemical biosensor for rapid and ultrasensitive detection of miRNA.DNA walkers are a new type of man-made nanomachine that has attracted considerable attention in the analytical community for the detection of nucleic acids,proteins and cells.However,the efficiency of signal amplification is limited by the low walking efficiency and few walking arms of traditional DNA walkers.On the other hand,although the introduction of appropriate mismatched bases into the CHA in the previous system could increase the driving force before and after the reaction,and thus improve the amplication efficiency of the reaction,the degradation of target miRNA still exists in the system of target-recycled.To comprehensively solve the above problems,we developed a novel three-dimensional（3D）self-accelerated DNA walker（SADW）which progressively expedite walking rate by unlocking the more walking arm continuously in walker process to construct electrochemical biosensor for ultra-sensitive detection of miRNA-21.Particularly,we skillfully introduced a target analogue sequence in the double-loop hairpin,which could be released in the walking process of SADW,then rapidly activating more silenced walking strands to achieve the continuous self-acceleration,resulting in the expedited reaction rate.Surprisingly,the experimental results indicate,compared to DW,SADW significantly reduced its reaction time and improved walking rate,which was ascribed to the outstanding acceleration process of the SADW.This way,the elaborated SADW was favorably applied in the ultrasensitive and rapid detection of miRNA-21 in tumor cancer cell lysates with a detection limit down to5.81 amol·L^-1 which was far from lower than the detection limit of biosensor based on DW.This method has opened up broad prospects for clinical diagnosis,biosensing detection,and the application of DNA nanobiotechnology.