Study On Electrochemical Biosensors Based On Short-Stranded DNA Inducing LAMP/H~+ To Regulate DNA Structure Switch

Loop-mediated isothermal amplification（LAMP）is a nucleic acid amplification technique that can complete rapidly under isothermal conditions.The primers can continuously extend,generating variable lengths of double-stranded DNA and a large amount of by-product hydrogen ions,achieving 10⁹ amplification rate within an hour.However,there are also disadvantages such as the requirement for complex primer design and the limitation of target template length.In addition,common detection methods based on LAMP（such as semi-quantitative gel electrophoresis,turbidity,and colorimetry）have drawbacks such as low sensitivity and poor stability,which greatly limit the practical application of LAMP reaction.Electrochemical biosensors are a detection technique that uses biological recognition elements（such as enzymes,antibodies,nucleic acids,etc.）to convert biological reactions into electrochemical signals,which have advantages such as high selectivity,high sensitivity,and fast response speed,and are widely used in disease diagnosis,environmental monitoring,agriculture,and food hygiene,and other fields.Based on this,this study used short-strand DNA to activate the LAMP reaction amplification process,using LAMP by-product hydrogen ions（referred to as LAMP/H⁺）as a signal transducer,combined with p H-respone DNA structures to construct different types of electrochemical sensors.The main research work includes the following three aspects:1.A highly sensitive electrochemical biosensor via short-stranded DNA activatingLAMP/H⁺to regulate i-motif folds for signal transductionHerein,a HIV-specific DNA segment（t DNA）was introduced to implement a variant LAMP reaction,where the generated by-products H⁺,v LAMP/H⁺,acted as signal transducers for developing a sensitive electrochemical biosensor.As proof-of-concept,the t DNA-recognizable complement was programmed in a template hairpin（TH）that was ended with a3’-terminus phosphate group to avoid non-specific elongation.Upon TH simultaneously hybridizing with t DNA and a precursor hairpin（PH）,the PH underwent a loop-mediated extension with Bst polymerase and d NTPs,forming a new stem-loop hairpin in another terminus.Then the elongated product containing a double stem-loop dumbbell-like DNA structure（d DDS）was displaced and released.Using d DDS as an initiator,the v LAMP reaction occurred via consecutively cycled strand displacement for self-primed DNA incorporation.The resulting t DNA-dependent v LAMP/H⁺was introduced in the modified electrode surface to guide the p H-sensitive i-motif（i M）folding.Thus,a signaling hairpin labeled with electroactive ferrocene（Fc）was dehybridized and closed,thereby bringing Fc close to the electrode interface for increased current signal readout that was highly sensitive to t DNA down to 0.034 fmol/L.2.An Ultrasensitive electrochemical assay for Ustilaginoidea virens DNA based on G-quadruplex stack from conformational switch of i-motif structure regulated byLAMP/H⁺In this paper,u DNA-induced v LAMP amplification technology was combined with highly sensitive electrochemical method,v LAMP amplification was triggered by double stem-loop dumbbell-like DNA structures intermediates formed by extension of u DNA,with by-product H⁺inducing i-motif conformational transformation to achieve significant changes to release G-rich Fc-GS forming G-quadruplex structure and stacking to enrich Fc in electrode surface to achieve increase of the electrochemical signal.The detection linear range was 10 amol/L～100nmol/L,and the detection limit was 4.8 amol/L.This method avoided the complexity and limitations of conventional LAMP technology,simplified templates and primers design.Moreover,the sensing surfaces without more kinds of DNA,effectively reduced background interference and further improved the sensitivity of the system.3.An electrochemical assay for SARS-Co V-19 DNA based on LAMP/H⁺as regulator ofp H-response DNA motifs for signal transductionHerein,based on LAMP/H⁺as signal transducer and hybridization chain reaction（HCR）,the SARS-Co V-19-related s DNA was introduced to initiate v LAMP reaction to produce abundant v LAMP/H⁺acting as signal transducer to develop a sensitive electrochemical biosensor.As proof of concept,s DNA and precursor hairpin（PH）was hybridized with template hairpin（TH）to form double dumbbell DNA structure（d DDS）as initiator of v LAMP reaction for continuous cycle of strand displacement by self-primed DNA incorporation.The resulting v LAMP/H⁺associated with s DNA protonated adenine in the p H-responsive strand PRM,forming A⁺-C complementary pairs,thereby causing intramolecular hybridization of PRM and releasing NH₂-M chains to trigger HCR reactions with Fc-labeled Fc-H1 and H2,thereby bringing the electroactive material Fc close to the electrode surface and enabling cascade amplification of the electrochemical signal,resulting in a sensor with a detection limit（LOD）as low as 35.4 fmol/L.