| Adenosine triphosphate（ATP）,as a direct energy source for organisms,plays a crucial role in most physiological processes.At the same time,abnormal ATP concentration in the human body can also serve as a reference indicator for the occurrence of some diseases.Therefore,it is of great significance to construct highly sensitive ATP detection methods in fields such as life science research and medical diagnosis.Silver nanoclusters（DNA-AgNCs）synthesized using DNA as a template have advantages such as simple synthesis,low cost,high fluorescence signal and ease of further modification,making them widely used in fields such as biological detection.This thesis utilizes DNA-AgNCs as fluorescence output signals to construct two fluorescent aptasensors for detecting ATP,providing more effective methods for sensitive detection of ATP.Firstly,a fluorescent aptasensor based on DNA-AgNCs and exonuclease Ⅲ（Exo Ⅲ）assisted signal amplification was constructed.ATP can compete to open the double stranded Apt/cDNA formed by ATP aptamer（Apt）and partial complementary DNA（cDNA）.The competing cDNA binds to the cleverly designed hairpin probe（HP）,triggering Exo Ⅲ to cleave HP,forming a large number of DNAzyme sequences.DNAzyme cleaves DNA-AgNCs fluorescent probes,causing a significant decrease in their fluorescence intensity,which reflects the concentration of ATP.Under optimal conditions,the linear response range of the sensor to ATP was 25μmol·L-1 to 1000μmol·L-1,detection limit 4.46μmol·L-1.The recovery rate of ATP detection in human serum samples using the constructed sensor was90.36～105.03%,with a relative standard deviation of 6.07～10.62%.Secondly,a fluorescent aptasensor combining DNA-AgNCs and DNAzyme/walker nucleic acid molecular mechanisms was constructed.S1 sequence containing the DNAzyme substrate chain and S2 sequence comprising the ATP split aptamer Apt1 were connencted to the surface of Fe3O4@Au.The multifunctional oligonucleotide sequence S3 containing the ATP split aptamer Apt2 and DNAzyme/walker was designed.ATP can induce the formation of complete aptamers and immobilize S3 on the surface of Fe3O4@Au.DNAzyme sequence on S3 cleaves substrate chain S1,hindering the terminal deoxynucleotide transfer（TDT）utilizing deoxycytidine triphosphate（d CTP）to polymerize poly C at the 3’end of S1,resulting in the failure of synthesizing DNA-AgNCs.The fluorescence response of DNA-AgNCs reflected ATP concentration.Under optimal conditions,the aptasensor exhibited a linear response range to ATP from 5 nmol·L-1 to 10000 nmol·L-1,with a detection limit of 0.27 nmol·L-1.The constructed sensor was successfully applied to the determination of ATP levels in human serum samples,with a recovery rate of 96.10～104.56%and a relative standard deviation of 2.08～6.71%.The two sensors constructed in this article are based on different signal amplification and output techniques.One is based on signal changes caused by changes in the secondary structure of DNA-AgNCs fluorescent probes,and the other is based on fluorescence signals generated by the synthesis of DNA-AgNCs.In contrast,the aptasensor the synthesis of DNA-AgNCs have achieved higher sensitivity and lower detection limits.Both aptasensors have good detection performance and specificity and actual sample testing has verified their promising application prospects. |
