The Construction Of Electrochemical Biosensor Via Advanced DNA Nanostructure And The Research Of Its Application For The Detection Of Clinical Biomarkers

Electrochemical biosensor is a new type of sensor that takes the advantage of the development in both biology and electrochemistry.By combining biomolecules with electrochemical signal conversion devices,electrochemical biosensors can achieve highly sensitive,selective and fast detection of biomolecules with high sensitivity,simple preparation,good selectivity,short response time and easy maintenance.It has been widely applied in various fields,such as environmental monitoring,clinical diagnoisis,medical drug development,food safety monitoring and industrial production.In recent years,with the continuous development of DNA nanomaterials,researchers have developed a variety of programmable DNA structures,which utilize their structural flexibility and high stability of DNA to enable electrochemical biosensors achieve significantly improved specificity and sensitivity.In this paper,several electrochemical biosensors have been constructed via DNA nanostructures to fabricate electrochemical biosensorsfor the detection of human disease related biomarkers with high efficiency,high precision,and high sensitivity,which provided new approach for disease diagnosis and further development of biosensing platforms.Details of the research are as follows:1.Tetrahedral DNA Nanostructure with Multiple Target-Recognition Domains and Its Application for Ultrasensitive Electrochemical Detection of Mucin 1In conventional electrochemical sensors based on DNA tetrahedral nanostructures,the low structural utilisation of DNA nanostructure often limits the sensitivity and detection limits of the constructed biosensors in the application of disease marker detection.Therefore,in this work,we designed a DNA nanostructure with multiple target recognition sites to improve the structural utilisation of DNA tetrahedra and investigated the application of the constructed electrochemical biosensor for the detection of the tumour marker protein Mucin1.Compared to conventional tetrahedral structures that use only the top endpoint as the capture domain,the design of this scheme using all four vertices as target capture domain to improve the utilisation of the DNA nanostructure and hence the capture efficiency.By closely combining this DNA nanomaterial with an electrochemical biosensor,the sensitivity and detection limits of the sensor are improved,enabling efficient and sensitive detection of Mucin-1.First,the four vertices in the tetrahedral DNA nanostructure(TDN)were designed with an aptamer sequence of the target protein,and the four vertices were first hybridised with the SP strand labelled with ferrocene(Fc).As the aptamer binds more strongly to the target protein than the SP strand,the Fc-labelled DNA single strand at the m-TDN vertex,which acts as an electrochemical signal probe(SP)in the presence of the target,will be released away from the electrode surface,resulting in a significant reduction in the electrochemical signal and thus enabling sensitive detection of the target.The results show that the sensor exhibits a wide linear range from 1fg/m L to 1 ng/m L for the detection of Mucin 1,with the limit of detection down to 0.31 fg/m L.This method opens a new pathway for the construction of nanostructures from DNA nanomaterials and provides a new approach to improve the sensitivity of electrochemical sensors for disease diagnosis.The efficient and sensitive detection of Mucin 1 was achieved,which has the potential to be practical in the analysis of real samples.2.A Biosensor Constructed Via Dual-Purpose Rolling Circle Amplification for the Detection of SARS-Co V-2 GeneTraditional designs of rolling loop amplification reaction(RCA),although allows for the amplification of DNA and RNA,still suffers from low efficiency when primer concentrations are low.In addition to this the template DNA for RCA is often excessive and not reusable,leaving further room for improvement in the efficiency of the biosensor it constructs.In this study,we based on a bifunctional RCA reaction,by regulating the reaction process,we not only achieved cyclic amplification of the target and outputted a large amount of Output DNA,but also further continued to trigger new amplification reactions through the reaction of Output DNA and the same RCA template in order to achieve a multiplicative increase in reaction efficiency,thus effectively achieving the electrochemical signal amplification,increasing the minimum detection limit of the target,and using this as an innovative point to construct a threedimensional DNA nanoscaffold,the application of which was investigated for the detection of gene fragments of SARS-Co V-2,achieving sensitive detection of Rdrp gene in the linear range from 1 fmol/L to 100 pmol/L,with the limit of detection down to 0.36 fmol/L.In addition to its use in the recent popularity of neo-crown assays,this strategy also provides a more efficient way to construct novel DNA nanostructures,opening the way to amplification strategies for low concentration targets for more efficient and sensitive detection of disease biomarker.