Synthesis And Bioimaging Of Organic Small Molecule Fluorescent Probes For The Detection Of PH And Hydrogen Peroxide

Fluorescent probes,as a kind of analytical tool,can monitor analytes in cells visually,noninvasively and in real time.Compared with other analytical detection tools,fluorescent probes have the advantages of high sensitivity,strong specificity and low detection limit,so they are widely used in biomedical detection and optical microscopy.At present,a variety of fluorescent probes have been developed for the detection of biological substances,but the probes have shortcomings such as short emission wavelength and poor biocompatibility.It was found that hemicyanine was a kind of fluorescent dye with strong biocompatibility in near infrared region.Naphthalic is a kind of fluorescent dyes with strong regulability,stable optical properties and good water solubility.According to the above research background of fluorophore,hemicocyanine and naphthalic dyes were selected as the fluorophore in this paper,and the performance of the fluorophore was regulated through heteroatomic modification.Two kinds of fluorescence probe structures were designed and synthesized,which were respectively used to detect the change of pH in biological microenvironment and detect hydrogen peroxide.Chapter 1:The mechanism of action of common fluorescent probes（PET,ICT,TICT,FRET,ESIPT,AIE）and reported fluorescent probes for the detection of pH and hydrogen peroxide are briefly reviewed based on literature reports.Chapter 2:A fluorescence probe（He-Cy）for pH detection was designed and synthesized based on tetrahydropyridine hemianocyanine unit,and its structure and fluorescence properties were studied.The results showed that due to ICT effect triggered by protonation and deprotonation of hydroxides,the UV absorption of the probe was different in alkaline and acidic environments.The absorbance increased at676 nm at pH 3.6-8.0,but decreased at pH 8.0-12.0.When the excitation wavelength was 650 nm,the fluorescence emission of He-Cy was 710 nm.With the increase of pH value,the fluorescence intensity of He-Cy was gradually enhanced,and the p Ka was 6.76.Interestingly,the color of He-Cy solution changed significantly with the change of pH.When pH was 3.60,the color of the solution was dark blue;when pH was 4.50-7.12,the color of the solution gradually changed from dark purple to blue;when pH was 8.00-12.00,the blue color of the solution gradually weakened,indicating that the probe He-Cy could detect pH with naked eyes.In addition,it also showed excellent biocompatibility and optical properties,realizing the response to different exogenous pH levels at the cellular level and in vivo level of zebrafish.When pH was 7.12,the fluorescence brightness was brighter,while when it was more acidic,the fluorescence brightness was lower,indicating that He-Cy could detect the change of pH level of biological microenvironment.Compared with other similar probes that only detect pH at the cellular level,this probe is expected to have better performance in biological applications.Chapter 3:A fluorescence probe（Nap-B）for detecting H₂O₂ was designed and synthesized based on 4-mercaptothenimide unit.By studying the structure and fluorescence properties,we found that the electron giving effect of sulfhydryl group changed significantly due to the protective effect of phenyl borate unit.When hydrogen peroxide was added to Nap-B system,the UV absorption peak was redshifted by 90 nm.With the increase of hydrogen peroxide concentration,the fluorescence intensity of Nap-B at 500 nm gradually increased,and showed a good linear relationship in the range of hydrogen peroxide concentration 0-600μM,and the detection limit was as low as 2.8μM.In addition,the probe has good photostability and excellent specificity.Biological imaging experiments showed that probe Nap-B can image H₂O₂ in cells and young zebrafish,which is expected to provide a powerful detection tool for the physiological and pathological studies of H₂O₂.