Intracellular pH is an important parameter associated with cellular behaviors and pathological conditions. Quantitative sensing pH and monitoring its changes by near-infrared (NIR) fluorescence imaging with high resolution in living systems are essential but challenging due to the lack of effective probes. To achieve good adaptability, in this study, a class of resolution-tunable ratiometric NIR fluorescent probes, which possess a stable NIR hemicyanine skeleton bearing different substituents, are rationally designed and synthesized, enabling detection through noninvasive optical imaging of organisms. Based on the protonation/deprotonation of the hydroxy group, a marked NIR emission shift provides a ratio signal in response to pH. Meanwhile, two states exhibit good photostability, sensitivity and reversibility, conducive to longtime monitoring of persistent pH changes without disturbance of other biological active species. Among the series, NIR-Ratio-BTZ modified with an electron-withdrawing substituent of benzothiazole exhibited the largest emission shift of about 76 nm from 672 to 748 nm with the pH environment changing from acidic to basic, which could be considered as a good candidate for high resolution pH imaging in live cells, tissues and organisms. Moreover, NIR-Ratio-BTZ has an ideal pK(a) value (pK(a) ≈ 7.2) for monitoring the minor fluctuations of physiological pH near neutrality. The ratiometric fluorescence measurement is beneficial to ensure the accuracy of quantitative measuring pH changes, as well as the real-time monitoring pH-related physiological effects both in living cells and living mice. The results demonstrate that NIR-Ratio-BTZ is a highly sensitive ratiometric pH probe in vivo, giving it potential for biological applications.

Molecular tools capable of providing information on a target analyte in an organelle of interest are especially appreciated. Traditionally, organelle-targetable probes are designed by incorporating an organelle-specific guiding unit to target the probe molecules into the organelle. The imperfect targeting function of the guiding unit and nonspecific distribution of the analyte in cytosol and each organelle would lead to low spatiotemporal resolution and limited sensitivity. To solve this problem, we report herein a new approach for detection of a target analyte in a specific organelle by engineering a target and location dual-controlled molecular switch. For this proof-of-concept study, fluorescent detection of H2S in lysosomes was performed with a simultaneous H2S and proton-activatable probe based on the acidic environment of lysosomes. 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 lysosomal pH environment but is out of operation in neutral cytosol and other organelles. Fluorescence imaging shows that SulpHensor is membrane-permeable and suitable for visualization of both the exogenous and endogenous H2S in lysosomes of living cells. The good performance of our proposed approach for H2S sensing demonstrates that this strategy might open up new opportunities for the development of efficient subcellular molecular tools for bioanalytical and biomedical applications.

No abstract

Fluorescent chemodosimeters for a fluoride ion (F(-)) based on a specifically F(-)-triggered chemical reaction are characterized by high selectivity. However, they are also subjected to intrinsic limits, such as long response time, poor stability under aqueous solution, and unpredictable cell-member penetration. To address these issues, we reported here that the self-assembly of fluorescent chemodosimeter molecules on a graphene oxide (GO) surface can solve these problems by taking advantage of the excellent chemical catalysis and nanocarrier functions of GO. As a proof of concept, a new F(-)-specific fluorescent chemodosimeter molecule, FC-A, and the GO self-assembly structure of GO/FC-A were synthesized and characterized. Fluorescent sensing and imaging of F(-) with FC-A and GO/FC-A were performed. The results showed that the reaction rate constant of GO/FC-A for F(-) is about 5-fold larger than that of FC-A, so that the response time was shortened from 4 h to about 30 min, while for F(-), the response sensitivity of GO/FC-A was >2-fold higher than that of FC-A. Furthermore, GO/FC-A showed a better bioimaging performance for F(-) than FC-A because of the nanocarrier function of GO for cells. It is demonstrated that this GO-based strategy is feasible and general, which could help in the exploration of the development of more effective fluorescent nanodosimeters for other analytes of interest.

Inner filter effect (IFE), a well-known phenomenon of fluorescence quenching resulting from absorption of the excitation or emission light of luminescent species by absorbent, has been used as a smart approach to design fluorescent sensors, which are characterized by the simplicity and flexibility with high sensitivity. However, further application of IFE-based sensors in complex environment is hampered by the insufficient IFE efficiency and low sensitivity resulting from interference of the external environment. In this paper, we report that IFE occurring on a solid substrate surface would solve this problem. As a proof of concept, a fluorescent sensor for intracellular biothiols has been developed on the basis of the absorption of a newly designed thiols-specific chromogenic probe (CP) coupled with the use of a thiols-independent fluorophore, rhodamine 6G (R6G), operative on the IFE on graphene oxide (GO). To construct an efficient IFE system, R6G was covalently attached to GO, and the CP molecules were adsorbed on the surface of R6G-GO via π-π stacking interaction. The reaction of thiols with CP on R6G-GO decreases the absorption of CP, resulting in the increase of the intensity of R6G fluorescence. The results showed that the IFE efficiency, sensitivity, and dynamic response time of R6G-GO/CP for biothiols could be significantly improved compared with R6G/CP, and furthermore, R6G-GO/CP functioned under complex system and could be used for assaying biothiols in living cells and in human serum samples. This new strategy would be general to explore the development of more effective IFE-based sensors for other analytes of interest.