| Small-molecule fluorescent probes are powerful tools for studying biological systems.They possess a great number of advantages such as high sensitivity,good selectivity,low-cost,fast response and high spatial resolution.Moreover,the binding affinity,kinetics,excitation/emission wavelength,and localization to a specific organelle can easily be optimized by using well-established design strategies.Thus,a diverse array of probes can be designed to target various biomolecules and organelles.Compared with traditional one-photon fluorescent probes,two-photon fluorescent probes allow deeper penetration,higher spatial resolution,decreased photobleaching and photodamage of biological samples,which exhibit a wide range for practical applications in complex biosystems.Therefore,it is of great significance in clinical diagnosis and biomedical research to develop various two-photon fluorescent probes for different targets and implementation of biosensing and bioimaging of targets with high sensitivity and specificity.In light of the considerations above and many relevant documents,we have designed and synthesized a series of two-photon fluorescent probes for analysis of endogenous substances and detection in living systems.The details are as follows:(1)In the second chapter,we have designed and synthesized the two-photon probe named L1 for specific detection of superoxide anion radical(O2·-).D-?-A-type naphthalene derivative is an efficient two-photon platform by virtue of its high fluorescence quanta yield and considerable two-photon properties,such as an effective two-photon active absorption cross-section and good biostability.With the phenol OH group of naphthalene derivative protected as a trifluoromethanesulfonate group via a sulfonate ester bond,L1 exhibited weak fluorescence.The triflate group activated the sulfonate ester toward nucleophilic attack by O2·-,to yield the free fluorophore,which resulted in greatly enhanced fluorescent emission.Probe L1 displayed excellent in vitro analytical performance,such as a detection limit as low as 1 nM,good linearity over a wide range of O2·-concentrations.At the same time,Probe L1 exhibited excellent specificity for O2·-over other ROS,RNS,and common cellular reductants.Furthermore,probe 1 was successfully applied for in vivo two-photon bioimaging of endogenous O2·-in live cells and tissue slices.(2)In the third chapter,we have further developed the two-photon fluorescent dye TP-NIR-OH and two-photon fluorescent probe TP-NIR-HS which possess fluorescent emission in the near-infrared(NIR)region.Inspired by A-?-D-?-D-structured oganic molecule which exhibits the absorption at long wavelength,we have designed and synthesized a novel two-photon fluorescent dye TP-NIR-OH.This dye TP-NIR-OH shows two-photon in and near-infrared out,large two-photon acton absorption across-section(>180 GM),far stokes shift(>70 nm)as well as deeper penetration,which is capable for high spatial resolution in vivo bioimaging and provide a favorable platform for building NIR emission of two-photon fluorescent probes.Based on the two-photon fluorescent dye TP-NIR-OH,we modify TP-NIR-OH with 4-dinitrobenzene-ether moiety which can respond to hydrogen sulfide(H2S)specifically.As a result,a new two-photon near-infrared fluorescent probe,TP-NIR-HS,has been developed.Probe TP-NIR-HS displays high sensitivity and selectivity.Subsequently,the probe was successfully applied to the detection of gaseous signal molecules H2S for bioimaging in both living cells and living nude mice.(3)In the fourth chapter,considering the unique physical properties of cobalt oxyhydroxide(CoOOH)nanoflakes and unparalleled advantages of two-photo dye(TPdye)in bioimaging,we herein developed an efficient CoOOH/TPdye nanoprobe for ascorbic acid(AA)imaging in living cells.Benifiting from the unique physical properties and optical properties of CoOOH nanoflakes,the TPdye is firstly absorbed onto the CoOOH nanoflakes.Due to the great overlapping of the absorption spectrum of CoOOH nanoflakes and the fluorescent emission spectrum of TPdye,the efficient energy transfer occured between CoOOH nanoflakes and TPdye.Thus,the TPdye could be greatly quenched by CoOOH nanoflakes for TPdye adjacent to its surface.After the CoOOH/TPdye nanoprobe was incubated with analyte or transported into cells,AA could specifically react with CoOOH nanoflakes,and the nanoflakes were damaged in an inreversible way followed by the release of TPdye from nanoflakes surface.Thus,the energy transfer between CoOOH nanoflakes and TPdye was prohibited and the fluorescence of TPdye was greatly restored.The in vitro experimental data have demonstrated the high sensitivity and selectivity of CoOOH/TPdye nanoprobe.Furthermore,the CoOOH/TPdye nanoprobe is biocompatible and membrane-penetratable,and can be applied for AA bioimaging in living cells.(4)In many conventional systems,luminophores experience some effects of emission quenching,partially or completely.This phenomenon is called aggregation-caused quenching(ACQ).The existence of ACQ phenomenon has seriously restricted the applications of fluorescent chromophores.Different from ACQ phenomenon,aggregation-induced emission(AIE)is another photophysical phenomenon associated with chromophore aggregation.In the AIE process,AIE fluorogens exhibit non-emissive in solution but become strongly luminescent as the aggregate happened.Traditional AIE fluorogens include tetraphenylethylene(TPE),hexaphenylsilole(HPS),and their derivatives.In the fifth chapter,we for the first time built a novel AIE-based "turn-on" fluorescent probe forinsitu K+ sensing and imaging in living cells by conjugating the TPE derivative to a G-rich oligonucleotide via the amide bond to form a TPE-oligonucleotide probe.The TPE-oligonucleotide probe displayed significant sensitivity over other G-quadruplex probes(the limited detection is 5 ?M).Furthermore,the TPE-oligonucleotide probe was successfully applied to the in situ K+ sensing and imaging in living cells.On the basis of these advantages,the TPE-oligonucleotide probe was a promising candidate for the functional study and analysis of K+. |
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