Terminal Deoxynucleotide Transferase And Alkaline Phosphatase Were Detected By Fluorescence Enhanced Assay

Terminal deoxynucleotidyl transferase?TdT?is a unique template-free DNA polymerase that allows random addition of deoxynucleoside triphosphates?dNTPs?to the 3'-OH end of single-stranded DNA?ssDNA?to extend the ssDNA strand.A large number of clinical studies have shown that abnormal expression of TdT plays an important role in the occurrence and development of tumors,which may reduce the response of tumors to cancer chemotherapy.In addition,TdT is also an important biomarker in early bone marrow cells.Therefore,the simple,rapid and label-free method for measuring TdT activity is of great significance for the rapid diagnosis of tumors and the development of related drugs.Alkaline phosphatase?ALP?is the most important hydrolase in the phosphatase family and is widely distributed in bacteria and eukaryotes.ALP can hydrolyze phosphate groups in proteins and phosphate groups in non-protein compounds.It plays an important regulatory role in the biogeochemical cycle of phosphorus due to its function as a compound of the decomposition machine phosphoric acid.In addition,ALP is also an external enzyme of osteoblasts.Its main physiological function is to provide phosphoric acid and hydrolyze pyrophosphate to inhibit the formation of bone salt by hydrolysis of phosphate esters into hydroxyapatite during osteogenesis.effect.At the same time,it has been found clinically that the occurrence and development of many diseases are often accompanied by abnormal ALP expression,such as certain hepatobiliary or bone diseases can cause changes in serum ALP levels.Therefore,the versatile method for detecting ALP is of great significance for basic research and clinical testing of liver and gallbladder,bones,and related diseases.In this paper,we developed a novel fluorescence-enhanced sensing method for TdT activity assay and ALP activity assay based on TdT-mediated nucleic acid amplification technology combined with the properties of coralyne and T-Hg²⁺-T structures.The main contents are as follows:1.In the second chapter of the thesis,we established a novel fluorescence enhancement sensing method for the determination of TdT activity based on TdT-mediated nucleic acid amplification technology and the properties of coralyne.The summary is as follows:1)Design a short-chain ssDNA probe containing 3'-OH end as a TdT reaction substrate;2)Add TdT and dATP,and use TDT as raw material to extend the ssDNA 3'end with thousands of bases.a poly-A tail;3)a dye-containing calalyne,a ssDNA with a poly-A tail interacting with coralyne to form a polyA-coralyne fluorescence enhancing complex;4)adding potassium thiocyanate?KSCN?,KSCN can quench the fluorescence produced by free coralyne and reduce the background signal.Under the optimal experimental conditions,the system can detect TdT activity within 55 minutes with a detection limit of 0.025 mU mL^-1,which is consistent with previous detection methods.Comparing this method is simpler,faster,and requires no marking.Moreover,this system also has good sensing performance for the detection of endogenous TdT activity in RBL-2H3 and Reh cell lysates,indicating its potential application in biochemical research and clinical diagnosis.2.In the third chapter of the paper,we established a new fluorescence enhancement sensing method based on the characteristics of ALP,TdT and T-Hg²⁺-T.The summary is as follows:1)Design a single-stranded DNA?ssDNA?probe that does not contain thymidine deoxyribonucleotides?dTTP?.The 3'end of the probe is phosphorylated and cannot be extended by TdT.In the presence of ALP,the3'-phosphate end of ssDNA-p is hydrolyzed to form the 3'-OH end,which produces a TdT reaction substrate;2)TdT and dTTP are added,and TdT extends the ssDNA to form a polymer-free template-T?poly-T?tail;3)Hg²⁺is added,ssDNA with poly-T tail interacts with Hg²⁺to form T-Hg²⁺-T mediated metal DNA duplex;4)Fluorescent dye SYBR green 1 is added for detection A sensitive measurement of alkaline phosphatase can be achieved by a change in fluorescence intensity.Under the optimal experimental conditions,the linear range of ALP activity detection was 0-2500 mU mL^-1,and the detection limit was 0.025 mU mL^-1,which was lower than the previous detection methods.Moreover,this system showed good sensory performance in detecting ALP activity in human serum samples and MCF-7 cell lysates,indicating its potential application value in biochemical research and clinical diagnosis.