Near-infrared Fluorescence Based On Detection Of Biologically Active Molecules Probe Design And Bioimaging Research

Fluorescence imaging technology is widely used in biomedical imaging research because of its advantages such as visualization,high sensitivity and real-time signal feedback.Near-infrared fluorescent dyes have long emission wavelength(650-900 nm),lower background interference,better tissue penetration and less light damage,which promote the application of near-infrared fluorescent probes in biomedicine,but the reported near-infrared fluorescent probes have some problems,such as poor light stability,small Stokes shift and so on.Based on this,a series of NIR fluorescence probes with excellent performance were designed and developed around the detection of bioactive molecules.Three kinds of NIR fluorescence probes with excellent performance were obtained through the study of their spectral characteristics,which were applied to the specific detection and biological imaging of peroxynitrite(ONOO~-),cysteine(Cys)and pH,respectively.The specific contents are as follows:(1)Based on the detection of ONOO~-,two kinds of near-infrared ratio fluorescent probes were developed.The hemicyanine structure was selected as the main structural skeleton of the dye,and the dicyanophorone derivative structure was introduced.Two kinds of fluorescent probes DCN-Cy-OH and DCN-Cy-OMe,with different substituents were obtained,and the probe DCN-Cy-OMe showed better specific recognition ability to ONOO~-.The oxidative cleavage of the probe by ONOO~-leads to the breakage of the?-conjugated system,accompanied by the blue shift of the emission peak from 715 nm to565 nm and 670 nm.The probe has a large Stokes shift(175 nm),and its fluorescence intensity ratio has a good linear relationship with ONOO~-in the range of 1 to 20?M.the detection limit is 0.71?M.the probe has been successfully used for ratio fluorescence imaging of ONOO~-in cells,and the probe can evaluate the ONOO~-produced by rifampicin enhanced liver injury induced by isoniazid.(2)A new type of near-infrared ratio fluorescent probe was designed and synthesized based on the selective detection of Cys.The near-infrared fluorescent dye DCN-Cy-OH was selected as the fluorophore and acryloyl chloride was used as the specific recognition site of cysteine.The probe has a large emission wavelength shift(147 nm)and a short response time.The fluorescence intensity ratio has a good linear relationship with Cys in the range of 1 to 30?M,and the detection limit is 0.41?M.The probe was successfully applied to the imaging analysis of Cys in cells,and further detected the changes of cysteine levels in normal tissues and cancerous tissues by photoacoustic imaging.(3)A"self-calibration"ratio fluorescence probe Cy-Cl-OH is synthesized based on pH detection design,which is a fluorine structure expressed as a probe signal,triggers the opening of the indole ring by protonation,resulting in the expansion of?conjugation to promote the intramolecular charge transfer(ICT)process in the fluorophore,resulting in the weakening of the 642 nm emission peak signal,while the emission peak signal at818nm does not change,which can be used as an internal reference peak.Thus the"self-tuning"response to pH is realized.Within the range of pH=4.5?7.5,the fluorescence intensity ratio of the probe has a good linear relationship with pH.The calculated p Ka value of the probe is 6.2,and it has good sensitivity and photostability,it has been successfully used for fluorescence imaging of different pH in the cell.