Biomolecules Detection Based On The Interactions Between Nucleic Acid Probe And Novel Sensing Material

In recent years,the incidence of tumor has been on the rise.Highly sensitive detection of disease-related biomolecules is of great significance for early diagnosis and treatment of diseases.As a new interdisciplinary technology,biosensor has the advantages of quick response,good specificity and real-time continuous detection.The emergence of novel sensing materials with excellent properties provides many new ideas for improving the performance of biosensors.Conjugated polymers(CPs)are a class of polymer materials containing delocalized large ? bond structure,which has a "molecular wire" function to achieve significant amplification of fluorescence signals and improve the sensitivity of biosensors.Graphene oxide(GO),a derivative of Graphene,is characterized by large surface area,easy surface modification and good dispersion.Based on the fluorescence resonance energy transfer(FRET)between GO and Fluorescence molecules,an efficient biosensor can be designed.In this paper,nucleic acid probes are combined with new sensing materials to design fluorescence biosensors with high sensitivity,good specificity and convenient operation,so as to realize efficient detection of disease-related biomolecules.The research includes the following three parts:(1)Detetion of S1 nuclease and ATP based on nucleic acid probe and water-soluble conjugated polymersS1 nuclease is a single-strand specific endonuclease,which plays an important role in DNA replication,recombination,repair and other biological processes.The detection of its activity and inhibitor is of great significance in biological analysis,disease diagnosis and other aspects.Water-soluble conjugated polymers(PPE-PLL)can form aggregation structure in aqueous solution,and its fluorescence is weak due to fluorescence quenching caused by self-aggregation.The fluorescence of PPE-PLL is significantly enhanced after electrostatic interaction with ss DNA.In the presence of S1 nuclease,ss DNA is sheared by S1 nuclease,and the aggregation of PPE-PLL is formed again.Furthermore,if both ATP and S1 nuclease exist in the system,PPE-PLL will still emit strong fluorescence,because the activity of S1 nuclease is inhibited by ATP.S1 nuclease and ATP can be detected by observing the fluorescence changes of the system.The limit of detection of S1 nuclease and ATP is 0.15 U/m L and 0.068 m M,respectively.(2)Detection of T4 polynucleotide kinase based on nucleic acid probe and graphene oxideT4 polynucleotide kinase(T4 PNK)catalyzes the transfer of the ?-phosphate group on adenosine triphosphate(ATP)to the hydroxyl terminus of 5'-oligonucleotide or nucleic acid,thereby phosphorylate the 5'-of nucleic acid.Abnormal levels of T4 PNK can lead to abnormal DNA phosphorylation,which is closely related to the occurrence of some human diseases.In this study,FAM-ds DNA probe is combined with GO to realize sensitive detection of T4 PNK.The 5'-hydroxyl terminus of ds DNA can be phosphorylated by T4 PNK,which is further hydrolyzed by lambda exonuclease(? exo)to release FAM-ss DNA.FAM-ss DNA can adsorb on the GO with the fluorescence quenching of FAM.FAM-ds DNA can't be phosphorylated in the absence of T4 PNK,so it can't be hydrolyzed by ? exo.Because FAM-ds DNA can't adsorb on the GO,the fluorescence of the system is still strong.The limit of detection of the biosensor for T4 PNK is 0.08 U/m L.Compared with other methods,this strategy is simple with high specificity.(3)Detection of T4 polynucleotide kinase based on hairpin DNA probeThe "turn-on" detection of T4 PNK can be realized by using the flexible structure of hairpin DNA(hp DNA)probe.The quenching group is modified at the 3'-of the hp DNA,and the fluorescence group is modified at the T base near the 5'-.When T4 PNK is added,the 5'-hydroxyl terminus of the hp DNA is phosphorylated.Then ? exo is added,leading to the hydrolyzing of the 5'-stem with the fluorescent group released.The detection of T4 PNK is realized through the change of fluorescence from quenching state to “turn-on” after adding the target molecule,with the linear range of 0.5-7.5 U/m L and the limit of detection of 0.13 U/m L.This method is convenient and fast with wide linear range.