New Strategies For Improving Performances Of Fluorescent Probes And Their Applications

Chemo/biosensing is a critical technical means for acquiring important chemicaland biological information to explore the essence in a variety of chemical reactionprocesses and life phenomena. As the cornerstone of chemo/biosensing, fluorescentprobe combining with the flurescence imaging technology is increasingly becomingan indispensable tool in modern life science and disease diagnosis fields, which canbe readily used for detecting and monitoring target molecules and biologicalprocesses in real-time, non-destructive and in situ. However, with the progress anddevelopment of mordern science and technology, sensing techniques are faced withnew challenges and more requriments. Although fluorescent probes embraced rapiddevelopment and widely application in recent years, their performances were gettingless satisfactory in dealing with some intractable problems. Design and application ofnovel fluorescent probes are still on the way, the key challenge is how to improvetheir propeties by keeping pace with the times so as to meet the demands of new era.Therefore, in this thesis, we are focusing on the improvement of the sensing andimaging performances of fluorescent probes by the integration of multi-disciplinesincluding analytical chemistry, organic chemistry, biology and nanotechnique, toeffectively solve the bottleneck problems. For this purpose, we had proposed somenovel improved strategies on the design of several typical fluorescent probes forsensing and bioimaging analysis, which provide novel approaches and new methodsfor the development and further application of fluorescent probes. The fiveidiographic works involved three aspects are as follows:(I) Investigations on graphene oxide assisted fluorescent probes based on innerfilter effect and chemical reaction and their applications.(1) Inner filter effect (IFE) has been used as a smart approach to designfluorescent sensors, which are characterized by the simplicity and flexibility with highsensitivity. However, further application of IFE-based sensors in complexenvironment is hampered by the insufficient IFE efficiency and low sensitivityresulting from interference of the external environment. In this section, we report thatIFE occurring on a solid substrate surface would solve this problem. As a proof ofconcept, a fluorescent probe for intracellular biothiols has been developed based onthe absorption of a new designed thiols-specific chromogenic probe (CP) coupledwith the use of a thiols-independent fluorophore, rhodamine6G (R6G), operative on the IFE on graphene oxide (GO). To construct an efficient IFE system, R6G wascovalently attached to GO, and the CP molecules were adsorbed on the surface ofR6G-GO via π-π stacking interaction. The results showed that not only the IFEefficiency, sensitivity, and dynamic response time of R6G-GO/CP for biothiols couldbe significantly improved compared with R6G/CP, but also R6G-GO/CP functionedunder complex system and could be used for assaying biothiols in living cells and inhuman serum samples (Chapter2).(2) To address some defects of chemical reaction based fluorescent probes forfluoride ion (F-) such as long response time and poor bioimaging performance, wereported herein that self-assembly of the fluorescent probe molecules on the surfaceof GO can solve these problems by taking advantage of the excellent chemicalcatalysis and nanocarrier functions of GO. Therefore, a new F--specific fluorescentchemodosimeter molecule, FC-A, and the GO self-assembly structure of GO/FC-Awere synthesized and characterized. With the help of chemical catalysis function ofGO, the transformation efficiency of reaction from FC-A to IC-B was increased,more F-were participated in this reaction, and the reaction rate was accelerated. Sothat the response sensitivity of GO/FC-A was>2-fold higher than that of FC-A, thereaction rate constant of GO/FC-A for F-was about5-fold larger than that of FC-A,the response time was shortened from4h to about30min, and the dynamic responserange of GO/FC-A to F-was enlarged from0-900μM to0-5mM. Furthermore,GO/FC-A showed a better intracellular imaging performance for F-than FC-A due tothe nanocarrier function of GO (Chapter3).(II) Investigation on simultaneous organelle and analyte-activatable fluorescentprobe and its application.Molecular tools capable of providing information on a target analyte in anorganelle of interest are especially appreciated. Currently, organelle-targetable probesare designed by incorporating an organelle-specific guiding unit to target the probemolecules into the organelle. The imperfect targeting function of the guiding unit andnon-specific distribution of the analyte within the cells would lead to lowspatiotemporal resolution and false signal. To solve this problem, we report herein anew approach for detection of a target analyte in a specific organelle by engineering atarget and location dual-controlled molecular switch. For this proof-of-concept study,fluorescent detection of hydrogen sulfide (H2S) in lysosomes was performed with asimultaneous H2S and proton-activatable probe based on the acidic environment oflysosomes. The new synthesized fluorescent sensor, SulpHensor, which contains a spirolactam moiety to bind hydrogen protons and an azide group to react with H2S,displays highly sensitive and selective fluorescence response to H2S under lysosomalpH environment, but is out of operation in neutral cytosol and other organelles.Fluorescence imaging showed that SulpHensor was membrane permeable andsuitable for visualization of both the exogenous and endogenous H2S in lysosomes ofliving cells (Chapter4).(II) Investigation on fluorescence resonance energy transfer based fluorescentprobe and its application.(1) To address some limits of two-photon fluorescent probes for H2S at presentincluding liable photolysis, long response time and interference by environment, wedesign a new intramolecular resonance energy transfer based two-photon fluorescentprobe for H2S using a hybrid of benzo[h]chromene and nitrobenzofurazan through thepiperazine link. In comparison with these reported ones based on PET and ICTmechanisms, the proposed FRET based one showed improved stability, sensitivity andresponse rate for H2S and was applied for H2S detection in serum samples and in vivoimaging of H2S in living cells and zebrafish, all with satisfactory results (Chapter5).(2) Although recent fluorescence nanosensing systems based on GO using FRETmechanism were developed quickly, most of the fluorescent dyes labeled in thesenanosensing systems are traditional one-photon excited fluorophores, which’sabsorption and fluorescence emission located in the ultraviolet-visible spectral regionwould render them difficult to be employed in complicated biological samples andliving tissues. To address this issue, we proposed the improved strategy thatconstruction of two-photon aptamer/GO nanosensing platform by substitutingone-photon fluorescent dyes with two-photon ones. As a proof of concept, wesynthesized a two-photon dye benzo[h]chromene labeled aptamer of ATP, which wasself-assembled with GO to prepare a new two-photon fluorescent nanoprobeGO/Aptamer-TPdye. Because two-photon dyes with near infrared photons as theexcitation source have the unique properties of lower auto-fluorescence andself-absorption, reduced photodamage, higher spatial resolution and deeperpenetration depth, GO/Aptamer-TPdye had been successfully used for sensitive andselective detection or imaging of ATP in the complex biological environment, livingcells and zebrafish (Chapter6).