Preparation And Application Of An ATP Fluorescent Aptamer Sensor Based On An Enzymatic Amplification Strategy

The sensing of small molecules involed in life processes has become a hot spot in the fields of cell imaging,disease diagnosis,and research on life peocesses.Establish a fluorescent aptamer sensor that can detect low concentrations of small molecules,which can prevent or diagnose diseases such as tumors and cancers.There are very complex communication mechanisms within and between cells,which trigger corresponding life processes and phenomena.Therefore,the development of a fluorescent biosensor with high sensitivity and high selectivity is of great significance in clinical diagnosis and treatment.This paper mainly carried out the following three aspects of work:1.The choice of energy receptors.Fluorescence resonance energy transfer(FRET)is a common detection principle for fluorescent DNA/aptamer sensing(probe).Generally,the hybridization of DNA is used to narrow the distance between the energy donor(fluorophore)and the energy acceptor(quencher)to force the fluorescence to be quenched,and to detect the change in fluorescence.In this paper,gold nanoparticles are selected as the energy acceptor,four Au NPs with different particle sizes are prepared by two methods,and 16 nm diameter Au nanoparticles with the best diameter are selected as the energy acceptor in this experiment.2.Construct a fluorescent ATP aptamer sensor.Fluorescence signal is used as a transmission method to detect ATP with high sensitivity and selectivity.ATP aptamer molecular beacons modified with gold nanoparticles(Au NPs)are hybridized with its complementary single-stranded 3?-FAM-DNA₂ to prepare fluorescent aptamer probes.The detection limit of ATP was 14.6 nmol/L.3.Construct a fluorescent aptamer sensing system based on enzyme amplification strategy.The Exo III enzyme can hydrolyze the blunt or embedded3'end sequence in double-stranded DNA base by base,The fluorescent ATP aptamer probe hybridizes with FAM-DNA₂ to form a fluorescence resonance energy transfer system,After adding the target ATP,the fluorescent ATP aptamer automatically forms a molecular beacon to release FAM-DNA₂,After adding Exo III enzyme,the molecular beacon embedded in the 3'end is hydrolyzed by the enzyme to release ATP,which enters the Exo enzyme amplification cycle and releases more fluorescent signals.The detection limit of ATP was 3.35 nmol/L.Therefore,the sensitivity is greatly improved,and the sensory detection of ATP can be realized at a very low concentration.