Electrochemical Methods For The Detection Of Nucleic Acids Based On DNA Walkers

DNA walker is a kind of DNA nanomachine,in which DNA walking strand moves along a specific track driven by strand displacement,enzyme catalyzed reaction or other reactions.Generally,it is composed of track,walking and driving components.The continuous movement of walking strand is realized by designing a biased system state.The automatic transition from high-energy state to low-energy state drives the operation of nanomachines.The realization of this process is accompanied by energy consumption.DNA walker has broad application prospects in the fields of biosensor,logic operation,diagnosis and therapy of diseases.Herein,an electrochemical biosensor is firstly developed based on nicking endonuclease catalysis.By modifying DNA track and walking strands with a certain ratio at the electrode,a sensing interface is established.DNA three-way junction structure is then assembled in the presence of target DNA.Since the duplex of track/target contains the cleavage site of nicking endonuclease,cleavage reaction is proceeded and target strand is recycled for additional walking reaction.The detection of target DNA is realized by the measurements of the labeled electrochemical speices.Next,a label-free self-propelled impedance sensor is designed.After target DNA initiated conformation change,autonomous walking is achieved around the DNA tetrahedron,which is also powered by nicking endonuclease.Further hybridization with thiolated auxiliary probe facilitates the immobilization of the DNA nanostructure on the gold electrode interface.After a simple electrochemical impedance spectroscopic analysis,the calculated interfacial resistance is found to be linearly related to logarithmic concentration of target nucleic acids.Subsequently,a number of tetrahedral DNA-supported tracks and walkers are comodified on the electrode surface.After the interaction between a DNA walker strand and target DNA,a single-stranded primer sequence could be released,which initiates subsequent rolling circle amplification.The generated long single-stranded product contains multiple DNAzyme cores,which facilitate highly efficient cleavage of track strands and subsequent DNA walking,which improves the sensitivity significantly.In order to improve the walking efficiency,a bipedal DNA walker is further designed.Magnetic nanomaterials are employed to enrich target DNA.Strand displacement amplification initiated by target DNA is then designed on the surface of the nanomaterials,the products of which can be used to trigger bipedal DNA walking on the interface of an electrode.By detecting the stripping voltammetric response of the adsorbed silver nanoparticles,high-sensitivity nucleic acid detection was realized.We next put forward the concept of circular bipedal walker.Primer and template for strand replacement polymerization are firstly integrated into one DNA strand.A large number of single-strand products produced upon target recongtion and enzymes catalysis.Next,the DNA interacts with another dumbbell DNA probe to form a circular DNA.Two single-stranded regions can be used as a bipedal walker to react at the electrode interface realizing ultrasensitive detection of target nucleic acids.On the other hand,the pH regulated DNA triple strand structure is used for electrode interface regeneration,which greatly improves the practicability of biosensor.In order to achieve multiple analysis,a novel logic circuit system is developed based on DNA bipedal walker.Target triggered strand displacement polymerization is able to produce a large number of strands for the fabrication of three-way junction-structured bipedal walkers.The following catalytic hairpin assembly ensures the walking event and the immobilization of signal reporters.In addition,under logic function controls by input triggered cascade strand displacement reactions,A series of logic gates are successfully established.The as-developed DNA logic system can also be extended to multi-input modes,which holds great promise in the fields of DNA computing,multiplex analysis,and clinical diagnosis.These electrochemical methods based on DNA walkers can not only realize the specific detection of low abundant nucleic acids,but also have the advantages of mild reaction conditions,low cost and strong scalability.They not only show great application potential in biological analysis and disease diagnosis,but also provide new ideas for the research work in the fields of biomolecular information control,communication,biological computer and so on.