Construction And Performance Control Of Electrochemical Nucleic Acid Biosensors

In recent years,electrochemical biosensors have attracted much attention from researchers because of their unique charm of low production cost,simple operation,fast response and strong portability.Using biometrics technology to meet application requirements is the core goal of various biosensors.In this work,toe-hold-mediated chain replacement reaction(TSDRs),enzyme-assisted signal amplification and entropy-driven signal amplification strategies were used to construct two new electrochemical biosensors,and the performance of the sensors was studied to achieve a wide detection range and real-time detection.The main contents are as follows:1.We report an electrochemical DNA biosensor based on a signal amplification strategy of enzyme-assisted Toe-hod mediated chain displacement reaction.We modelled the dynamic detection range of the DNA biosensor with a single DNA capture probe(CP)at a single sensing interface.In the absence of Eco RI endonuclides,the capture probe can only be opened by fewer targets due to its thermodynamic stability,resulting in a lower electrochemical response signal.In the presence of endonucase,the capture probe is truncated,the contact binding region is released,and the binding affinity with the target is enhanced.A large number of signal probes(5’ labeled methylene blue)are bound,and the REDOX active substance is close to the electrode surface,resulting in a larger intensity of electron transfer,resulting in a higher electrochemical response signal.Therefore,the Eco RI endonuclease treated DNA biosensor platform provides a detection mode for relatively low concentration targets.Compared with other biosensors,this biosensor is realized by using only one acquisition probe,avoiding complex probe design or using multiple probes to achieve the adjustable dynamic range of target analysis,showing good selectivity and sensitivity in different buffer solutions and serums,significantly reducing the detection limit,and improving the ability to recognize the mismatch sequence.The current manufacturing of enzyme-assisted biosensors opens up a promising path for the development of biosensors with controllable detection performance and holds great potential for diagnostic applications and drug monitoring.2.We report an electrochemical DNA biosensor based on an entropy-driven,enzyme-assisted signal amplification strategy.In the entropy-driven process,enzymes are introduced to detect signal changes in a multi-step,real-time manner,and at the same time realize grid element recovery.As a DNA initiator,the target triggers the chain shift reaction and replaces S1 chain,generating a metastable structure containing an exposed 4 nt region.This metastable structure allows a full-length fuel connected to F1(5’ labeled electroactive substance methylene blue)to simultaneously replace T and S2 chains,resulting in the recircuation of the target chain and the generation of F1:H products.During this process,the electrochemical response increases from zero.After treatment with Nt.Bbv CI endonuclease,a new H:S1:S2’notch DNA complex is created to participate in the next reaction.At this time,the target continues to act as the DNA initiator chain,replacing the S1 chain of the H:S1:S2’ DNA complex to generate the metastable structure of T:S2’:H.The substitution of both T and S2’ chains with F2(which is not modified with any electrochemically active substance)results in the recircuation of the target chain and the F2:H product,which again returns to the original process at the electrode surface and continues to be recycled in the next reaction,in which the electrochemical response goes from high to low.The electrochemical signals still changed significantly during the repeated cycle,indicating that the prepared electrochemical biosensor can detect the target repeatedly and in real time in multiple steps.In the reaction process,the circuit consumes excess F fuel units,so as to complete the entropy driven DNA programming cycle circuit and realize a high cyclic reaction rate.This method provides a simple and convenient idea for DNA programming circuit.