Cytoplasmic Protein Enhanced Fluorescence Molecule Probe Based On Anthocyanin Analogue

The aim of chemical biosensing is the effective acquisition of important chemical and biological information in biological systems,which could be used to further explore the nature of various chemical reaction processes and life activities.However,Fluorescent probes,as the cornerstone of chemical biosensing technology,due to their simple operation,high sensitivity,real-time in-situ detection and non-destructive analysis has been widely used in the fields of chemistry,biochemistry,modern life sciences and clinical medicine.Unfortunately,most of the existing fluorescent probes are based on a "turn-on" or "ratio-type" one-step signal output mode,which is limited when facing with low-abundance substances in complex biological systems,and the low-abundance substances are undetected because of the "one-step" signal output has a limited magnification.So,developing a high-sensitivity probe capable of efficiently detecting low-abundance substances in complex environments is significant both in chemical biosensing and clinical medicine.The works of this thesis are aimed at the bottleneck problem of the low sensitivity of small-molecule fluorescent probes in the detection of low-abundance substances in complex environments,and the probes by means of intracellular inherently high-abundance substances,cytoplasmic proteins.This scheme can perform another signal amplification on the existing"one-step" signal output mode,effectively improving the sensitivity of the probe,solving the problem that the current probe is usefulness for low-abundance species detection.At the same time,the general strategy for efficient amplification of intracellular inherently high abundance substances proposed in this paper can also as new methods and ideas be applied to other small molecule fluorescent probes,providing further development and application of small molecule fluorescent probes in the field of biosensing.Based on this strategy,this paper has carried out the following works:(1)Inspired by natural anthocyanin dyes in life,20 kinds of anthocyanin analogue fluorescent dyes with stable,near-infrared fluorescence emission and easy to functionalize were be synthesized by one-step synthesis.It was found that the fluorescence intensity of some of the synthesized anthocyanin analogue fluorescent dyes in the cell lysate system was several times higher than that in the PBS system under the same conditions.Just like PEF-16,which has 6 fold increase in cell lysate system than in PBS.At the same time,there is no such phenomenon in commercialization of fluorescent dyes with similar structures or obvious structural differences.The fluorescence enhancement effect is also substantially the same by using cell lysates of different cells.It was finally determined that the cytoplasmic protein in the cell lysate significantly enhanced the fluorescence intensity of the dye when analyzing the components of the common components of the cell lysate.Further investigated the fluorescence properties of the dye under different pH conditions,which found the dye has good fluorescence stability,and its fluorescence increase effect is basically not affected by the change of pH under physiological conditions.The "optimized" cytoplasmic protein enhanced dye of PEF-16 for bioimaging was finally determined.(2)With the "optimized" cytoplasmic protein enhanced fluorescent dye of PEF-16 in hand,which using it as the fluorescent reporter unit of the probe.Through the related literature investigation,the H2S probe with the dye as the fluorescence reporting unit was designed and synthesized,and the control probe with the same reaction site was designed with the commercial coumarin dye as the fluorescence reporting unit.After the probe reacts with the target H2S,its azide group were reduced to an amino group,it means that reduced from the azidation probe PEF-16-N3 to a PEF-16 fluorescent dye,and the fluorescence is turned on.Furthermore,the fluorescent signal is further "amplified"(4.12 fold)in the presence of cytoplasmic proteins,to achieve efficient detection of low concentrations of H2S.Whereas the control probe reacts with the target even in the presence of cytoplasmic proteins,it is also substantially consistent with the PBS solution.Which indicating that the probes of this paper are significantly different from other H2S probes.Furthermore,the probe selectivity,pH performance and cytotoxicity were investigated,and satisfactory results were obtained.Subsequently,the probe were used for cell and tissue imaging,and the results fully demonstrated that the probe can further amplify the output signal by utilizing the inherent high-abundance substance cytoplasmic protein in the cell,breaking through the "one-step"signal output of the existing probe.The mode can effectively improve the sensitivity of the probe and achieve effective detection of low abundance H2S in cells and living tissues.