3D-DNA Walker Signal Amplification Strategy For Electrochemical Aptasensor Construction

Research background and purpose:DNA walker is a self-propelled DNA molecule composed of driving force,walking chain,and walking track.It can move along specific tracks(1D,2D,3D)with high controllability and precision.As an efficient signal amplifier in electrochemical sensors,DNA walkers significantly improve the detection sensitivity and selectivity of sensors.Electrochemical aptasensor constructed by them have been widely used in biological analysis and medical diagnosis.In this study,two electrochemical aptasensors were constructed using nanomaterials and DNA walkers to detect biomarkers c Tn I and Sars-Cov-2 RBD.The aim was to explore the application of the 3D-DNA walker signal amplification strategy in electrochemical aptasensor as a reference for developing new biosensors and biological analysis technologies.Materials and Methods:(1)Construction of c Tn I electrochemical aptasensor based on walker-walkers serial amplification strategyAu/Ni Co2S4 composite was prepared by hydrothermal and electrochemical reduction methods.Two thiolated hairpin probes,HP I(containing c Tn I aptamer and DNA walker)and HP II(containing substrate chain and trigger DNA),were designed and immobilized on the electrode surface based on Au-S bond using Au/Ni Co2S4 composite as the substrate.When c Tn I is present,HP I is specifically opened to activate DNA walker.The DNA walker recognizes and cleaves the substrate chain of HP II,releasing trigger DNA(complementary sequence of c Tn I inducer),leaving a segment of substrate chain on the electrode surface.Trigger DNA hybridizes with another HP I,activating the DNA walker again to form a new cycle,generating a large amount of substrate chains.By capturing probe(complementary sequence of substrate chain)hybridization with the substrate chain on the electrode surface to form double-stranded hybrids,a large amount of hybrid indicator methylene blue(MB)can be captured,resulting in electrical signal changes for highly sensitive and specific detection of c Tn I.Electrochemical impedance spectroscopy(EIS),cyclic voltammetry(CV),differential pulse voltammetry(DPV)were used to study and analyze the successful preparation and detection conditions of aptasensor.SEM,TEM,EDS,XRD were used to characterize morphologyand element distributionof Ni Co2S4 and Au/Ni Co2S4 composite to verify that Au/Ni Co2S4 was successfully prepared.(2)Construction of SARS-Co V-2 RBD ultra-sensitive electrochemical aptamer sensor based on 3D framework nucleic acid molecular machineA gold electrode was prepared using electrochemical reduction.D1,D2,D3,D4,aptamers(APT),and hairpin probes(HP-MB)were designed and synthesized for nucleic acid structure self-assembly through annealing.When detecting SARS-Co V-2RBD,the SARS-Co V-2 Spike RBD competitively binds with the aptamer to activate four-legged DNA walkers.Driven by DNAzyme,the four-legged DNA walkers move on the surface of the gold electrode and hybridize with MB-labeled hairpin probes(MB-HP),which cuts and releases MB to rapidly decrease electrical signals.In this study,impedance spectroscopy(EIS),cyclic voltammetry(CV),and differential pulse voltammetry(DPV)were used to analyze and evaluate the preparation process and detection performance of the aptamer sensor.Atomic force microscopy(AFM)and agarose gel electrophoresis(AGE)were used to characterize and evaluate successful nucleic acid structure self-assembly.Result:1.DNAzyme-driven walker-walkers serial amplification based c Tn I electrochemical aptasensor.The limit of detection(LOD)was 0.26 pg/m L(S/N=3)with a linear range of 0.01 ng/m L to 60 ng/m L.The c Tn I electrochemical aptasensor exhibited excellent stability(RSD=4.40 %),reproducibility(RSD=3.80 %)and selectivity.The c Tn I was analysed in normal human serum using a spiked recovery method with recoveries ranging from 97.0 % to 106.8 %,and the relative errors and RSD values were within acceptable limits,indicating the ability of the sensor to detect c Tn I in human serum.2.An electrochemical aptasensor for SARS-Co V-2 RBD based on tetrapod DNA walkers@ DTNs.The limit of detection(LOD)was 0.21 pg/m L(S/N=3)and the linear range was 0.01 ng/m L to 80 ng/m L.The c Tn I electrochemical aptasensor showed excellent stability(RSD=3.45 %),reproducibility(RSD=1.95 %)and selectivity.The recovery of the aptasensor was 98.63 % ~ 104.76 % with a relative standard error of3.11 % ~ 7.08 % for the detection of Sars-Cov-2 RBD in human saliva samples,demonstrating the potential of the tetrapod DNA walkers@ DTNs aptasensor for the diagnosis of COVID-19.Conclusion:1.A novel electrochemical aptasensor was designed for ultra-sensitive detection of c Tn I using the walker-walkers serial amplification strategy.Compared with traditional single-foot DNA walker methods,the biosensor using the walker-walkers cascade amplification strategy can effectively avoid short moving distances,long reaction times and complex modification steps,and can amplify signals by nearly 20 times,greatly enhancing detection sensitivity.In addition,Au/Ni Co2S4 nanocomposites have excellent electronic conductivity,environmental stability and a larger specific surface area,making them an efficient electrode support material.Compared with other c Tn I sensors(aptamer sensors,immune sensors,fluorescence sensors),this sensor has lower detection limits and wider detection ranges.2.Construction of SARS-Co V-2 RBD electrochemical aptamer sensor based on 3D framework nucleic acid molecular machine.Unlike other DNA walker sensors,this sensor can effectively increase the running steps of DNA walker movement and improve the detection efficiency by controlling the stereospecificity of DNA walker movement through tetrahedral control.Furthermore,compared with other multi-step DNA walker sensors,it can accumulate more signal changes under the same conditions,improving the overall sensitivity of the sensor and having a lower detection limit.3.DNA walkers can be used as signal amplifiers,which can significantly improve the sensitivity and specificity of the sensor,as well as further enhance the detection performance of the sensor by forming high affinity pairings between the aptamer and the target molecule.In addition,DNA walkers are simple and inexpensive to prepare,and their motility behaviour and selectivity can be modulated by designing sequences,making the use of DNA walkers in electrochemical aptasensor promising for a wide range of applications.