| Fluorescent biosensor technology has many advantages,such as high sensitivity,easy operation and fast response.With the continuous advancement of generations of scientific research workers,fluorescent biosensor technology has been rapidly developed,and it has become one of the methods widely used and studied in the field of bioanalytical method research.It has been widely used in disease diagnosis,food safety,and environmental protection.aspect.Oligonucleotide fragments are widely used by analytical chemists for constructing nucleic acid-mediated fluorescence biosensor analysis methods due to their advantages of being easily synthesized in vitro,chemically stable,and capable of performing various chemical modifications.This paper makes full use of the many advantages of nucleic acid-mediated fluorescent biosensors and develops three nucleic acid-based fluorescence biosensor strategies for depurination/apyrimidinic endonuclease I（APE1）and polynucleoside kinases（PNK）and biotin molecule detection.The specific research content is as follows:1.In this paper,the fluorescent dye 2-amino-5,6,7-trimethyl-1,8-naphthyridine（ATMND）was used as a signal molecule,and the dye molecule could be inserted into the abasic site（AP site）in double-stranded DNA.The nature of the point,using a double-stranded DNA containing AP sites as a substrate,a non-labeled fluorescence assay with high sensitivity,good selectivity,and one step reaction to detect APE1 activity was designed.The method has a good linearity between0 and 0.8 U/m L enzyme concentrations with a detection limit of 0.04 U/m L.In addition,the effect of golden tricarboxylic acid on the activity of APE1 was also studied using the analytical method proposed in this paper.Therefore,the methods established in this chapter have a good application prospect in disease diagnosis,drug screening and other APE1-related biological studies.2.In this paper,a specific cleavage of 5′-phosphorylated ds DNA based on λ exonuclease was established,and the use of ATMND to intercalate C-C mismatch sites in double-stranded DNA and result in its fluorescence quenching property,with one containing CC mismatches Site-based double-stranded DNA is a substrate,and a novel fluorescence-free molecular marker homogeneous fluorescence detection method has been developed for rapid and sensitive determination of PNK activity.In the study,T4 PNK was used as the test object.The sensitivity of the established method to T4 PNK assay was 0.012 U/m L,and the linear range was 0.05 U/m L-0.8 U/m L.It also showed good selectivity.Moreover,the research methods proposed in this paper are equally applicable to the screening of PNK inhibitors.Therefore,the research in this chapter will provide important technical references and support for PNK-related biomedical research.3.In this paper,E6 DNAzyme,which is an intermediate small molecule modified biotin,is used as a signal transduction and amplifying catalytic element.Based on the specific binding between biotin and streptavidin,the strategy of inhibiting the catalytic activity of DNAzyme is combined with competitive reaction.A simple,homogeneous fluorescence method was designed to detect biotin.The detection limit of the method is as low as 7.73×10-9 mol/L,and the linear range is 0 to 1.8×10-7 mol/L,and it has better selectivity.The establishment of the amplification in this study has certain versatility,and it has potential application value in the analysis and detection of other biological small molecules. |
