Analysis Methods On The Fluorescence Lifetime Of Novel Biosensors

Compared with small molecule-based probes,quantum dots and nanomaterial-based probes,genetically encoded fluorescent protein probes are widely used to study the function and dynamics of biomolecules in living cells due to the advantages of endogenous non-toxicity,stable fluorescence properties and good compatibility with biomolecules.The researchers have analyzed the three-dimensional structure of the fluorescent protein,and can specifically develop probes with stable performance and outstanding spectral properties by directly or randomly mutated the amino acid residues near the chromophore according to the characteristics of the analyte.In this dissertation,we studied the fluorescence properties of the probes by steady-state fluorescence spectroscopy and time-resolved fluorescence spectroscopy,and studied a fluorescence lifetime analysis method combining genetic algorithm.We inserted the aminostyrene fluorescent molecule p-DASPMI into BSA to form the p-DASPMI-BSA chimeric probe which can sense and reflect the redox state of the environment through the changes of the fluorescence.Under different redox states,the reducing agent can increase the fluorescence intensity by about 3 times and the average lifetime increases from 1.47 ns to 2.12 ns.The oxidant quenches the fluorescence of the whole sample,and the average lifetime decreases from 2.15 ns to 1.47 ns.Then we studied the fluorescence properties of the iNap1,a fluorescent protein-based probe.The response of the probe to NADPH showed the characteristics of the intensity ratiometric probe.And the data of the time-resolved fluorescence spectrum indicated that the fluorescence lifetime also increased with the increase of NADPH concentration.We also expanded the dynamic detection range of the probe by the lifetime ratiometric method.Finally,based on the iNap1 and Peredox fluorescent protein-based probes,we fitted the data by the life distribution analysis method combining with the genetic algorithm.The data shows that the method uses fewer parameters for the fluorescence lifetime decay curves than the classical e-exponential decay model,and has nice fitting results;when analyzing the mixed samples,the type and the concentration of the analytes can be identified according to the characteristic parameters of the analytes.Several groups of experiments have proved that the analysis method has good fitting results and has good application prospects in distinguishing different analytes.