Design Of New Fluorescent Molecular Probes And Its Application In Biological Detection And Bioimaging

Fluorescent sensing method has prominent advantages in sensitivity,selectivity and real-time in situ detection.Combining with fluorescence imaging,the application of fluorescent probes becomes more and more widely in the fields of biological,environment,medical diagnosis and the other fields.Some new designed elements such as the design of probe with near-infrared structure,construct of ratiometric fluorescent probe for visual detection and sensitive color interval change have been introduced to develop new fluorescent probes and further broaden the scope of application.In this dissertation,our goal is to explore to construct optical sensors for the detection of gaseous signal molecules(carbon monoxide,sulfur dioxide),pH value and penicillamine.We want to improve the design of fluorescent probe through enhance the response rate and sensitivity,provide visualization signal output.These can be obtained by the synthesis of functional organic molecules,develop of principle for target recognition using chemical reaction and combined with fluorescent nanomaterials.The main contents of this dissertation are summarized as follows:(1)Design a fluorescence turn on detection method for gaseous and endogenous CO based on the combination of the specific carbonylation between CO and palladacycle compound,and the heavy-atom electronic effects of palladium.We designed a non-fluorescent benzimidazole functionalized palladacycle probe for CO and in order to improve the water solubility,the carboxyl groups were functionalized on the benzimidazole moiety.In the presence of CO,the carbonylation between CO and palladacycle compound occurred and induced the protonolysis of Pd complex,subsequently releasing the fluorescence compound and restore the fluorescence.The selective reaction was against interfering from other possible co-existing reactive oxygen species,and achieved a detection limit of?0.06 ?M.Furthermore,the fluorescence turn-on probe was demonstrated with high cellular uptake rate and successfully applied for cell imaging of exogenous and endogenous carbon monoxide in living cells.(2)A new type of nanocomposite probe(Hex-FGO-DPS)was designed for sulfur dioxide and its derivatives consists of hexamethylenediamine modified fluorescent graphene oxide(FGO)and naphthaline derived cyanine(DPS).Its construct principle based on the ?-? stack interaction between graphene oxide and aryl compound.The fluorescent graphene oxide is used as internal standard signal with blue fluorescence,and the naphthaline derived cyanine moiety display yellow fluorescence.In the nanocomposite,the fluorescence of FGO was quenched by the fluorescence resonance energy transfer and the nanocomposite has yellow fluorescence.In the presence of bisulfite,it reacts with DPS through a nucleophilic addition reaction and inhibit the FRET process,further restore the blue fluorescence.In the presence of bisulfite,the ratiometric fluorescence intensityof Hex-FGO-DPS changes up to 25 folds,accompanied by a distinct color change from yel-low to blue which can be visually identified.The detection limit was measured to be 0.44 ?M.The probecan be further applied for sensing intracellular bisulfite through the ratiometric fluorescence imaging analysis in living cells.In addition,the probe Hex-FGO-DPS also can be used for selective determination of gaseous sulfur dioxide in atmosphere and aqueous solution with high selectivity,can be further used to develop new fast visual recognition device for sulfur dioxide.(3)Through the reaction between aldehyde hemicyenine and hydroxylammonium chloride,a-C=N-OH substituted hemicyenine has been synthesized for colorimetric and near-infrared fluorescence detection of pH value.The probe has a good water solubility,excellent photostability,reversibility,low cytotoxicity andexhibits a good performance to detect pH fluctuation in both solution and live cells.When the pH value change from neutral to acid,the protonation of nitrogen atom in the probe skeleton induced the gradually decrease of absorbance at 460 nm,meanwhile a new absorption peak at 630 nm distinctly enhance.Under visible light,the color rendering of probe solution varied from yellow to green to blue with gradual change.The initial maximum emissionat 623 nm gradually shifted to 660 nm.Meanwhile,the fluorescence intensity at 660 nm gradually increased over 8 folds and theintegrated intensity increased nearly 2.1 folds.Moreover,the fluorescence intensity ratios of probe increased nearly 26 folds.The pKa value of the probe was estimated to be 5.46.The results demonstrated that the probe was more sensitive to acid environment(4.5-7.4),highly consistent with the pH value of intracellular physiological environment and is expected to be further used to monitor the cellular pH value variation induced physiological and pathological processes.(4)A new designed fluorescence "on-off-on" assay was explored for detection of penicillamine through the recognition principle of competitive complexation.MUA served as both a reducing reagent and a protecting ligand to get a red fluorescent AuNCs.The as-prepared fluorescent AuNCs are well monodispersed and with an average diameter around 2.6 nm.The as-prepared MUA functionalized AuNCs(MUA-AuNCs)has good water solubility,excellent photostability and strong fluorescence emission at 610 nm with a quantum yield of 7.28%in water.The fluorescence of MUA-AuNCs was first quenched by copper ions through electron transfer,subsequently caused obvious restoration by competitive effect after adding penicillamine.The intensity increases of emission bands were in a good linear relationship which can be calibrated for quantification.The limit of detection is found to be 0.08 ?M,which is much lower to those of other reported penicillamine probes.The probe has good selectivity for penicillamine and can be explored for the measurement of penicillamine in complex real water and urine samples with satisfactory results.