Development And Application Of Novel Optical Fiber Biosensor Based On Nucleic Acid Specific Cutting

Rapid detection and accurate analysis of biomarkers has become a hot topic in scientific research and clinical application.Biosensors have developed rapidly in recent decades due to their good selectivity,high sensitivity,fast analysis speed and low cost.Chemiluminescence optical fiber biosensor(COFS)is a new type of sensing system that has developed rapidly in recent years,showing unique advantages in the accurate detection of disease biomarkers.COFS uses micro-sized optical fiber with excellent light guiding performance as the carrier of biological recognition molecules,as the conductor of chemiluminescence(CL)signal,and does not require additional light source system.These characteristics make COFS system have low signal-to-noise ratio,small size,easy automation,miniaturization and integration.In the construction of COFS system,the specific molecular recognition mechanism and sensitivity enhancement strategy are crucial,which determines the specificity and sensitivity of the sensor response to the target analyte.Especially for a wide variety of biomarkers,some markers have not developed specific recognition antibodies or aptamers,etc.The development of COFS with a new recognition mechanism is of great significance for expanding its application in the field of disease marker detection.The purpose of this paper is to construct a highly sensitive and highly specific chemiluminescence optical fiber biosensor with excellent analytical performance.By introducing a new specific recognition mechanism and a non-enzymatic constant temperature nucleic acid amplification strategy,a new chemiluminescence optical fiber sensor for low abundance biomarkers in biological samples is developed.The main research work is as follows:1.Site-specific cleavage-based chemiluminescent optical fiber biosensor for highly sensitive assay of myeloperoxidase activity and inhibitor screeningWe first introduced site-specific cleavage as the recognition mechanism of chemiluminescence fiber optic biosensors,and established a site-specific cleavage sensing strategy induced by myeloperoxidase(MPO).The developed sensor can be used for ultra-sensitive detection of MPO activity in blood circulation and inhibitor screening.The thiophosphate-containing hairpin probe modified on the surface of the optical fiber sensor is used as a molecular recognition element,and its thiophosphate group is a specific cutting site for recognizing HOCl catalyzed by MPO.In order to further improve the detection sensitivity,we introduced the hybridization chain reaction(HCR)amplification method into the chemiluminescence optical fiber biosensor system.This method can avoid the interference of other DNA sequences and make the HCR isothermal amplification proceed in an orderly manner,thus greatly improving the detection sensitivity.The amplified DNA sequence on the fiber surface provides a large number of biotin active sites for binding to streptavidin-modified horseradish peroxidase(SA-HRP).In the presence of luminol-hydrogen peroxide,horseradish peroxidase on the fiber surface catalyzes the generation of enhanced chemiluminescence signals to achieve the target MPO signal output.Under the optimal conditions,the detection range of MPO obtained by NSC-COFS was 5-1000 μU/m L,and the detection limit was as low as 1.7 μU/m L.The proposed NSC-COFS has ultrahigh sensitivity,high specificity and high signal-to-noise ratio,which broadens the application range of optical fiber sensors in the field of biosensors and has potential application value in the field of POCT diagnosis of disease markers in biological samples.2.Ultrasensitive detection of flap endonuclease 1 by hybrid chain reaction-based chemiluminescence optical fiber biosensorFlap endonuclease 1(FEN1)is an endogenous nuclease that can cleave the 5’-end of branched ds DNA and is of great significance in DNA replication and repair.It has been recognized as a potential biomarker in oncology research.However,there are few reports on the analysis of FEN1 with high sensitivity and specificity.In this work,we developed a novel fiber-optic biosensor for highly sensitive and specific detection of tumor marker FEN1.FEN1 specifically recognizes and cleaves the 5’-end branch structure of the DNA substrate modified on the surface of the magnetic beads.After magnetic separation,based on the principle of complementary base pairing,the free 5’flap was captured by a hairpin probe modified on the surface of the fiber,triggering HCR amplification.Based on HCR amplification,the DNA sequence on the surface of the fiber can produce a large number of biotin active sites for specific binding to streptavidin-modified horseradish peroxidase(SA-HRP),thereby catalyzing the CL substrate to produce enhanced chemiluminescence.Using this sensor,we achieved high sensitivity detection of FEN1 with a detection limit of 3.4 f M.This method shows high specificity,which makes it possible to quantify FEN1 in real samples.The fiber-optic biosensor based on hybridization chain reaction proposed by us is expected to be a diagnostic tool based on FEN1 and applied to the clinical and drug research fields related to FEN1.