Design,Synthesis And The Performance Studies Of FRET-Based Ratiometric Fluorescent Probes

Quantitative determination of specific analytes is essential for a variety of applications ranging in the fields of life sciences and environmental monitoring.Optical sensing allows non-invasive measurements with in biological milieus,parallel monitoring of multiple samples,and less invasive imaging.Among the optical sensing methods currently being explored,FRET-based ratiometric fluorescent probe has received particular attention as a technique with the potential to provide precise and quantitative analyses.Among its advantages are high sensitivity,excellent selectivity and self-calibration provided by monitoring two?or more?emissions,which improves the accuracy of detection.Abnormal concentration changes of active small molecule species such as sulfur dioxide?SO₂?,hydrogen sulfide?H₂S?and hydrogen peroxide?H₂O₂?in cells may produce diseases such as hypertension,cardiovascular disease,and cancer.Therefore,it is important to design and synthesize FRET-based mechanism ratiometric fluorescent probes to detect biological active species.In this paper,FRET-ICT-based ratiometric fluorescent probe for detecting H₂O₂,based on multi-signal mode and ICT-FRET mechanism ratiometric probe to detect H₂S concentration,and FRET-based fluorescent probe to detect sulfur dioxide with excellent signal-to-background ratio were elaborately designed and synthesized.In chapter 2,FRET-ICT-based ratiometric fluorescent probe?CNBE?for monitoring H₂O₂ was elaborately designed and synthesized by using coumarin-naphthalimide as fluorophore.To construct a FRET dyad,the coumarin chromophore with relative short emission and naphthalimide chromophore with relative long wavelength were chosen as the energy donor and acceptor,respectively.The ratio signal of fluorescent probe was manipulated by interplaying ICT-activated FRET mechanism.Under the excitation of 410 nm light,the probe initially emitted only the blue fluorescent of coumarin.After adding H₂O₂,the boronic acid pinacol ester group was converted into a hydroxyl group with strong electron donating ability,the absorption wavelength is red-shift due to ICT effect of the naphthalimide moiety,which would continue to activate FRET procedure and emit fluorescence of naphthalimide.The probe exhibited good selectivity and sensitivity to H₂O₂.The ratiometric fluorescent imaging was successfully applied to detect H₂O₂ using CNBE in living cells and zebrafish.In chapter 3,an interplaying ICT-FRET-based dual regulation mechanism ratiometric fluorescent chemosensor?NapN₃-PCM?was elaborately designed and synthesized.NapN₃-PCM contains two recognition sites for H₂S with different sensitivity,diazido naphthalimide derivative and phenylchromenylium chromophore,which is capable of succinctly determining H₂S concentration levels by dual fluorescence signal mode of two distinct emission bands.For sensing H₂S at low concentrations,NapN₃-PCM showed a synergistic changet at both 535 nm and 650 nm and less fluctuation of the ratio signal(I₅₃₅/I₆₅₀).For sensing H₂S at high concentrations,NapN₃-PCM produced an antagonistic changet aunder the same two emission bands and the ratio signal achieved a significant enhancement.NapN₃-PCM exhibited good biocompatibility and was successfully applied to detect H₂S levels in living cells and zebrafish using ratiometric fluorescence imaging.In chapter 4,we proposed a new strategy for designing the ratiometric fluorescent probes that consist of rigid-fluorophore-molecular rotor dyad and perform superior response signal-to-background ratio in viscous systems.A practical sulfur dioxide?SO₂?fluorescent probe?RFC-MRC?based on conmarin-cyanine dyad was prepared as a proof-of-concept of new design strategy.The probe undergoes a nucleophilic addition reaction with SO₂,which causes the emission wavelength of the probe to undergo a blue shift.Compared with the SO₂-induced ratiometric response signal in non-viscous environment?35.4-fold enhanced?,the fluorescence response signal in the viscous system will be much more remarkable?71.5-fold enhanced?.The probe can selectively in mitochondria and was successfully utilized to sense SO₂ in living HeLa cells through confocal fluorescence imaging.What's more,in the fluorescence imaging experiments of monitoring SO₂ in apoptotic cells using probe RFC-MRC,a more obviously superior of signal-to-background ratio was observed in the early apoptotic cells?51.2-fold increase?than in the lately apoptotic cells?37.1-fold increase?.