Structure And Performance Regulation Of Oxygenated And Nitrogen Heterocyclic Fluorescent Dyes And Their Applications In Sensing And Imaging

Optical probe-based imaging techniques have been widely used in life science research due to the advantages of high sensitivity,simple operation,and minor damage to organisms.Organic small-molecule fluorescent probes have the advantages of strong structural design,rapid response,easy metabolism,and slight damage to samples,and are powerful tools for obtaining biochemical information at the cellular and living levels.With the gradual deepening of research,it is extremely urgent to acquire biochemical information efficiently in the deep tissues of living organisms.However,most organic fluorescent dyes suffer from short excitation and emission wavelengths,environmental sensitivity,and small Stokes shift,which limit the application of traditional organic small molecule fluorescent probes in in vivo sensing and imaging.In recent years,with the development of large-scale instruments such as two-photon,near-infrared I,and near-infrared II small animal imagers,the development of high-performance long-wavelength excitation and emission dyes are crucial for in vivo imaging research.In response to the above problems,a series of long-wavelength excitation and emission fluorescent dyes containing oxygen and nitrogen heterocycles were constructed,and their fluorescence regulation mechanism was revealed.On this basis,a series of high-performance fluorescent probes were constructed to realize high-fidelity imaging detection of some important biomolecules during zebrafish liver development and rapid assessment of the cholestatic liver injury.The specific works are as follows:（1）Electron donor-acceptor（D-A）type molecules are the most widely employed two-photon scaffolds.Unfortunately,current D-A type fluorophores suffer from serious solvent-dependent emission wavelength and brightness,thwarting their use for high-fidelity imaging in complicated biological systems.In chapter two,with single-atom replacement and acceptor fusing in Acedan,we devise a new class of D-A type fluorophore（TPQL dyes）.Our design results in TPQL fluorophores with a slight emission redshift(Δλ_TPQL1<32 nm VsΔλ_acedan-NH2=91 nm)and high brightness(εΦ_TPQL1=7600 to 6923 VsεΦ_acedan-NH2=7372 to 1314)in different solvent systems.Such features provide bright and unbiased fluorescent images of TPQL fluorophores in the cell and bring deeper tissue penetration and a higher signal-to-background ratio in tissue.As a proof-of-concept demonstration,using probe TPQL-N₃ and TPQL-APN,we monitor the dynamic changes of H₂S and APN during zebrafish liver development with high fidelity for the first time.（2）Although the excitation wavelength of the two-photon in the previous chapter was relatively long,the emission wavelengths of the TPQLs were still relatively short.Rhodamine dyes have been widely employed in biological imaging and sensing.However,it is always challenging to design rhodamine derivatives with a massive Stokes shift to address the draconian requirements of single-excitation multicolor imaging.This work described a general strategy to enhance the Stokes shift of rhodamine dyes by completely breaking their electronic symmetry.As a result,the Stokes shift of novel rhodamine dye DQF-RB-Cl is up to 205 nm in PBS,which is the largest in all the reported rhodamine derivatives.In addition,we successfully realized the single excitation trichromatic imaging of mitochondria,lysosomes,and cell membranes by combining DQF-RB-Cl with commercial lysosomal targeting probe Lyso-Tracker Green and membrane targeting dye Dil.This is the organic synthetic dyes for SLE-trichromatic imaging in cells for the first time.These results demonstrate the potential of our design as a useful strategy to develop a huge Stokes shift fluorophore for bioimaging.（3）In the third chapter,although we significantly improved the Stokes shift of rhodamine-based dyes and extended their emission wavelengths,the introduction of electron-deficient groups instead led to a blue-shift of their absorption wavelengths.To solve this problem,we first fused rhodamine dyes and phenazine derivatives to obtain a series of phenazine-rhodamine hybrid dyes.Spectral performance studies showed that the maxima absorption and fluorescence of DPN-RB(λ_ab/λ_em=674/766 nm)were red-shifted by 122 nm and 184 nm compared to that of rhodamine B.We further extended this strategy to dyes such as fluorescein,coumarin,and fluoroborane,and developed a series of phenazine hybrid dyes with significantly red-shifted wavelengths.Finally,we explore the potential of DPN-RB for in vivo imaging.The results show that the long-wavelength absorbing and emitting dyes have good imaging signal-to-noise ratios in both in vivo NIR fluorescence and photoacoustic imaging.（4）Based on the method developed in Chapter 4,we further developed a near-infrared II dye（DPN-OX）with the smallest molecular weight to date.The experimental results show that DPN-OX has good chemical stability,photostability and photothermal conversion efficiency,and has good application potential in in vivo near-infrared II fluorescence,photoacoustic dual-mode imaging and photothermal therapy.The results of the pharmacokinetic study showed that:after intravenous injection,DPN-OX was first taken up by the liver,rapidly secreted into the intestine through the gallbladder,and finally excreted with the feces.In addition,we can also track the progression of cholestatic liver injury in vitro by NIR II fluorescence imaging of stool.Due to the superior photophysical properties of DPN-OX,we designed it as a lung-specific targeting agent using polymer encapsulation technology and realized real-time monitoring of heart rate in normal mice and hypoxic states.