| As an important biocatalyst in living organisms,enzymes play an important role in maintaining life activities,and different types of enzymes exercise different functions.For example:uracil DNA glycosidase（UDG）recognizes and excises uracil produced by damaged DNA and forms a depurine/pyrimidine site（AP site）that works with other repair enzymes to complete the entire base excision repair（BER）process;human alkyladenine DNA glycosidase（h AAG）is an important protease that specifically recognizes and initiates the repair of a variety of alkylated purines and hypoxanthine repair;human flap nucleic acid endonuclease 1（FEN1）is a structure-specific nuclease capable of cleaving single strands protruding from the 5’end in Okazaki fragments.Studies have shown that when the above enzyme activity is abnormal,it is usually closely associated with a number of human diseases,such as human immunodeficiency,neurodegeneration,lymphoma,and cancer.Therefore,it is important to establish a sensitive method to detect enzyme activity for disease diagnosis.At present,the following key problems exist in enzyme detection:1.the complexity of living organisms,large interference,and extremely low levels of target enzymes in the early stage of diseases;2.due to the overlapping of fluorescence spectra with each other,fluorescence-based detection methods can generally detect only two target enzymes at the same time,and at most three,making it difficult to achieve simultaneous detection of multiple target enzymes.To address the above problems,this thesis is based on isothermal nucleic acid amplification technology,which amplifies low-abundance target enzymes into a large amount of DNA under constant temperature conditions to achieve signal amplification,and combines different analytical tools to achieve the detection of single or multiple different enzymes.The specific studies are as follows:1.a nucleic acid probe without modification of burst groups was designed for sensitive detection of UDG.the method reduces experimental cost by introducing 2-aminopurine into the nucleic acid chain,which makes its own fluorescence burst due to the base stacking effect,and the fluorescence is restored when the nucleic acid chain is ablated into individual nucleotides,thus avoiding the modification of burst groups.In addition,the design of the cascade amplification reaction can effectively improve the amplification efficiency,which makes the method highly sensitive with the detection limit of 4.57×10-5 U/m L,and has good selectivity and practical application ability to complete the quantitative detection of low concentration of UDG in cell extracts.2.A six-fold cyclic cascade signal amplification method based on double exponential amplification of enzyme repair combined with multi-terminal signal output was constructed to achieve rapid and sensitive detection of FEN1.In the presence of FEN1,the double-branched substrate is sheared to produce 5’single-stranded DNA（ss DNA）,and ss DNA is used as a primer to induce a double-exponential amplification reaction to generate a large amount of ss DNA（X’and Y’）,which is then X’and Y’were hybridized to the 3’and 5’ends of the signal probes,respectively,to form partially complementary double strands.Subsequently,under the action of Bst.polymerase and T7 nucleic acid exonuclease,the signal probe on the double-stranded DNA is ablated and the fluorescent signal is released,and the whole process is repeatedly cycled,making the signal amplified.The method is highly sensitive,with a detection limit of9.7×10-3 U/m L,and can be used to distinguish normal cells from cancer cell extracts with good selectivity.In addition,the method can be successfully applied to the screening of FEN1 inhibitors,which has great application prospects in the screening of FEN1-related drugs.The method is sensitive,selective,easy to operate and does not require complex synthesis and modification of nanomaterials for the detection of FEN1,which has great potential for application in the diagnosis of FEN1-related diseases.3.A method for the simultaneous detection of three enzymes,FEN1,h AAG and UDG,was developed by taking advantage of the strong separation ability of capillary electrophoresis and low sample usage in combination with isothermal nucleic acid amplification technology.A double-branched substrate containing the recognition sites of the three target enzymes was designed,which was composed of three single strands,and the amino groups were modified to their 3’ends to prevent non-specific extension.The three enzymes recognize and shear the corresponding enzymatic sites on the double-branched substrate,and under the action of terminal transferase（Td T）,the sheared products undergo polymeric extension reaction to produce three different extension products,which are hybridized to their corresponding signal probes to form double-stranded,and the signal probes on the double-stranded are recognized and sheared by the enzymes to produce three single-stranded nucleic acids of different lengths and labeled with FAM fluorophores,which are then The three enzymes are separated and detected by capillary electrophoresis to achieve the simultaneous detection of the three enzymes in the same system.Based on this,a technique based on isothermal nucleic acid amplification-capillary electrophoresis was developed for the simultaneous detection of the three enzymes,which enabled the simultaneous analysis of multiple enzymes in cell extracts and overcame the decrease in enzyme activity at different times.The method is highly sensitive and has high throughput. |
