Development Of Multipedal DNA Walker-based Electrochemical Biosensor For Disease Diagnosis Application

As an emerging dynamic DNA nanotechnology,DNA walker has developed from monopedal and bipedal to multipedal.Multipedal DNA walker has been widely used in the field of biosensing due to their high reaction efficiency.In recent years,a variety of analysis methods based on multipedal DNA walker have been proposed.Among them,electrochemical biosensor has the advantages of high sensitivity,fast response,simplicity and portability.More importantly,electrochemical biosensor can be well combined with multipedal DNA walker because its electrode interface can be used as an ideal walking track.However,in the actual analysis process,the composition of the analyte is complex,which poses a huge challenge to the anti-interference ability of the sensor.Compared with traditional electrochemical biosensors in single-response signal output mode,ratiometric electrochemical biosensor shows strong anti-interference ability and reliability due to its inherent self-calibration capabilities.To further improve its response speed and sensitivity,efficient signal amplification strategy is worth exploring.Based on this,we designed a ratiometric electrochemical biosensor based on multipedal DNA walker to achieve ultrasensitive ratiometric detection of nucleic acids through a cascaded signal amplification strategy.The main research contents are as follows:(1)Strand displacement reaction-driven dynamic DNA nanosystem for ultrasensitive ratiometric detection of nucleic acid.We proposed a ratiometric electrochemical biosensor based on dynamic DNA nanosystem for the detection of human immunodeficiency virus-associated DNA fragment(HIV-DNA).The dynamic DNA nanosystem included target recycling unit and multipedal DNA walker unit.In addition to identifying the target,the former unit also undertook the task of dynamically activating the latter unit to realize cascade signal amplification.Both of the two units were driven by toehold-mediated strand displacement reaction,enabling an enzyme-free and isothermal amplification strategy.With the synergistic effect of the dynamic DNA nanosystem and the ratiometric output mode,the ultrasensitive detection of HIV-DNA was realized with an ultralow detection limit down to 36.71 aM in a wide linear range of five orders of magnitude.Finally,we verified the analytical performance of the sensor in complex biological samples.The research results showed that the sensor had a great practical application prospect in the development of reliable point-of-care diagnosis device.(2)Dual-DNA walker-based efficient dynamic DNA nanosystem for nucleic acid detection.We prepared a multipedal DNA walker by using magnetic nanoparticle as a carrier,where the walking strands were designed with a hairpin structure to keep a blocked state.To improve the activation degree and efficiency of the multipedal DNA walker in the presence of the target,blocked tethered walking strands were simultaneously modified on the surface of the same particle.As a proof-of-concept,kirsten rat sarcoma viral oncogene(KRAS)was chosen as the target.In the presence of KRAS,the cyclic target recognition and the 3D walking of the tethered walking strand were realized with the assistance of exonuclease Ⅲ,which led to the full activation of the multipedal DNA walker.Further,the ultrasensitive electrochemical ratiometric detection of KRAS was realized by the bidirectional signal conversion caused by the rolling of the activated multipedal DNA walker on the electrode surface.The linear range of the sensor was 10 aM to 1 pM with a detection limit as low as 3.880 aM.The analysis of complex biological samples demonstrated that the proposed sensor had a great potential for practical applications.