Application Of Microfluidic Paper Chip In Typical Biomarker Analysis Based On Tetrahedral DNA Nanostructure

Health of life is a prerequisite for a happy life of all mankind.The early detection and treatment of various diseases is currently a major problem.Disease diagnosis mainly relies on biomarkers in the body.Therefore,developing a biomarker detection platform with high sensitivity,good selectivity and accurate results is currently a hot research topic for scholars.The optical biosensor has become a powerful tool for the real-time analysis of biomarkers due to its advantages of fast detection speed,short response time and high sensitivity.In this paper,a highly sensitive optical sensing platform based on microfluidic paper chip was constructed using aptamer,tetrahedral DNA nanostructure（TDN）and combined with signal amplification technique.The research contents are summarized as following:1.TDN was combined with a rotational microfluidic paper chip,and the color change generated by the reaction between streptavitin coupled horseradish peroxidase（s-HRP）and3,3’,5,5’-tetramethylbenzidine（TMB）-H₂O₂ was used for grayscale signal analysis by Image J software.The colorimetric analysis platform realized the quantitative detection of SARS-Co V-2virus S protein and influenza A H1N1 antigen were realized simultaneously.Under the optimal conditions,the paper-based analytical device showed a good linear relationship between the two viruses in the range of 10^-14～10^-8 g/m L,and the two viruses were not affected by cross reaction.It has been successfully applied to the detection of actual sample swab virus preservation solutions.This sensor provides a convenient and reliable method for clinical rapid differentiation and diagnosis of COVID-19 and influenza A.2.An origami microfluidic paper chip was developed for highly sensitive detection of adenosine triphosphate（ATP）using TDN.In this work,one kind of TDN（B-TDN）was used as scaffold to controllably fixation probes in well-defined orientation and spacing;the other TDN（D-TDN）was employed as cargo for carrying SYBR Green I（SG I）.Here,SG I was introduced to bind with double-stranded DNA in D-TDN to produce enhanced fluorescence signal.To construct the sensor,D-TDN that associated with SG I and consisted of ATP aptamer was acted as detection probe,and the B-TDN contained sequences that are partially complementary to ATP aptamer was used as capture probe.In the absence of ATP,detection probe would hybridize with capture probe,and thus strong fluorescence signal could be observed.However,in the presence of ATP,detection probe would preferentially bind to ATP which disturbing the binding between detection and capture probes,thus resulting in decreased signal.The detection limit of this method is 0.43 p M and the detection range is 10^-12～10^-7 M.Our strategy proved an attractive paper-based fluorescence platform for monitoring of ATP and clinical diagnosis applications.3.A double channel origami microfluidic paper chip modified with G-quadruplexe TDN（G4-TDN）was developed for the analysis of DNA methyltransferase（DNMT）.The G-quadruplexe structure of detection probe G4-TDN can specifically bind to the fluorescent dye Thioflavin T（Th T）to produce significant fluorescence signal.When DNMT is present,G4-TDN can be methylated,and restriction endonuclease（Dpn I）can recognize and cut the methylation site,which changes the rigid structure of G4-TDN.At this time,the G-quadruplexe breaks away from the paper base,resulting in a decrease in fluorescence signal.Under optimal conditions,the platform shows a good linear relationship in the range of 1～90 U.This DNMT paper-based detection device has the advantages of fast,good reproducibility and accuracy,which provides a new idea for its application in the field of molecular medicine.