Enzyme-mediated DNA Walker Biosensor For Cancer Cell Marker Detection

Biosensors can combine biosensing elements and chemical-physical sensors to convert the recognition events of biomolecules into output signals for the detection of some cancer cell markers.Traditional biosensors have great limitations in achieving trace and sensitive detection of cancer cell markers in vitro and in vivo without the help of signal amplification techniques,so amplification detection of cancer markers can be achieved under certain reaction conditions with the help of some non-enzymatic-mediated or enzyme-mediated signal amplification techniques.DNA Walker is a novel artificial dynamic DNA nanodevice that relies on the continuous movement of DNA walking strands along a specific walking track with various driving forces as energy supply to achieve continuous signal amplification.Based on the rich variety and diverse driving forces of DNA Walker,its application to the design of biosensors is also very promising.Therefore,this thesis is dedicated to constructing a cascade amplification signal amplification system by combining nucleic acid signal amplification technology with nuclease-driven DNA Walker nanodevices,and then constructing biosensors of different signal types with excellent specific recognition and practical sample applicability and low detection limits for highly sensitive detection and analysis of cancer cell markers.To be specific,this study included the following two sections:（1）At the first section of the work,we mainly construct an APE1 enzyme-driven tripedal DNA Walker regenerable electrochemical biosensor for ultrasensitive detection of mi RNA-155.In this work,we select the mi RNA-155overexpressed in cancer cells as the target and acted as the initiation switch of the DNA Walker.The target triggers the CHA reaction between H1,H2 and H3hairpins to form a tripedal DNA Walker,which not only realizes the cyclic amplification of the target,but also integrates the three legs of the DNA Walker into one structure,which has a larger walking rate and amplification efficiency compared to the unipedal DNA Walker.The tripedal DNA Walker recognizes the track strands on the surface of the gold electrode,and the nuclease APE1drives the continuous walking of the tripedal DNA Walker.The enzymatic cleavage and hydrolysis of the track strands results in a shift in MB signaling molecules embedded in the DNA strand on the electrode surface,thus causing an electrochemical signal alteration in correlation to the concentration of the target.The combination of CHA,APE1 enzymatic-driven DNA Walker walking in tandem to construct a cascade amplification signal amplification strategy and a fast response electrochemical detection method enables ultra-sensitive detection of mi RNA-155 with a detection limit as low as 10 p M and it shows a good linearity in a relatively wide concentration range.It also exhibited excellent differentiation and anti-cross-talk ability,and shows good analytical ability in 10%and 20%of serum samples.This work further improved the electrical signal response of the detection system by rational design of strand replacement strategy,and also achieves multiple cycles of electrode regeneration,which reduces the cost of detection.Therefore,this work presents a novel thought for the regeneration of electrodes,which has a wide application prospect in the detection of various markers related to cancer cells.（2）At the second section of the work,we construct a dual-mode biosensor constructed by a cellular endogenous dual enzyme that synergistically turns on and drives DNA walker motion.In this scheme,we construct a 3D DNA Walker nanodevice by taking advantage of the good biocompatibility,abundant loading sites and fluorescence burst properties of Au NP,combined with the amplification strategy of continuous walking motion of DNA Walker.The target APE1 is used as the initiation switch of DNA Walker to initiate the binding of DNA Walker to the fluorescent track strand,and the endogenous nuclease FEN1 is selected as the driving force to drive the continuous movement of DNA Walker on the 3D track to bring about the recovery of fluorescence signal,which realizes the sensitive detection of APE1 by the fluorescence detection mode.As the APE1 concentration rose,the fluorescence signal increases in the 0-500 U m L^-1 range,with a linearity of 0.01-10 U m L^-1and a detection limit as low as 0.01 U m L^-1,exhibits high sensitivity.The cascaded upstream DNA Walker and downstream HCR amplification strategy,combined with SWV,an electrochemical detection method,further enhanced the sensitivity of the APE1.The linear range is extended to 0.01-50 U m L^-1,and the limit of detection is down to 0.002 U m L^-1.Moreover,the dual detection modes achieves the specific detection of APE1,and can effectively discriminate the APE1 content in cancer cells and normal cells.To sum up,this dual-mode biosensor has the capacity to be sensitive,precise,and stable,and it will be widely utilizes in the realm of biological analysis and early diagnosis of illness.