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Hydrogen peroxide (H 2 O 2) plays a significant role in regulating a variety of biological processes. Dysregulation of H 2 O 2 can lead to various diseases. Although numerous fluorescent imaging probes for H 2 O 2 have been reported, the development of H 2 O 2 ratiometric fluorescent probe with large Stokes shift remains rather limited. Such probes have shown distinct advantages, such as minimized interference from environment and improved signal-to noise ratio. In this work, we reported a new pyrene-based compound Py-VPB as H 2 O 2 fluorescent probe in vitro. The probe demonstrated ratiometric detection behavior, large Stokes shift and large emission shift. In addition, the probe showed high sensitivity and selectivity towards H 2 O 2 in vitro. Based on these excellent properties, we successfully applied Py-VPB to the visualization of exogenous and endogenous H 2 O 2 in living cells. Cell imaging study also showed that our probe was localized in the mitochondria. We envision that the probe can provide a useful tool for unmasking the biological roles of mitochondrial H 2 O 2 in living systems. Image 1 • The first pyrene-based fluorescent probe for H 2 O 2 detection with ratiometric readout was presented. • The probe has shown prominent properties in detecting H 2 O 2 , such as high sensitivity & selectivity and large Stokes shift. • The probe was successfully applied to visualizing exogenous and endogenous H 2 O 2 in living cells. [ABSTRACT FROM AUTHOR]

• Introduction of a novel ratiometric fluorescent probe for the differential detection of Cys-and GSH. • Detection limits of the probe PYR-CG were determined to be 88 nM for Cys-and 164 nM for GSH. • Rapid response time of less than 3 min for both Cys-and GSH. • PYR-CG selectively recognizes C-terminal Cys-in peptides. Cysteine (Cys) and glutathione (GSH) are crucial biothiols implicated in cellular redox balance, necessitating accurate detection methods. Herein, we developed a novel fluorescent probe based on the fluorescent dye pyronin (PYR), capable of monitoring both Cys-and GSH using ratiometric fluorescence signals. The probe, named PYR-CG , where "C" stands for Cys-and "G" stands for GSH, demonstrates selective detection capabilities for Cys-and GSH. UV absorption and fluorescence emission spectra changes revealed distinct shifts upon interaction with Cys-and GSH. Theoretical and experimental studies validated PYR-CG 's responsiveness to Cys-and GSH, elucidating its mechanism as a ratiometric probe. Notably, PYR-CG exhibited rapid response kinetics for Cys-and GSH, with low detection limits of 88 nM and 164 nM, respectively. Importantly, PYR-CG demonstrated exceptional selectivity for Cys-and GSH over interfering analytes. Screening of molecules with similar structures to Cys-further affirmed PYR-CG 's specificity, particularly towards N-terminal Cys-in peptides. Furthermore, recovery experiments underscored PYR-CG 's reliability for quantitative Cys-detection in complex food samples. Our findings highlight the innovative design and practical applicability of PYR-CG , offering a valuable tool for sensitive and selective detection of Cys-in diverse biological and food samples. [Display omitted] [ABSTRACT FROM AUTHOR]

• A novel mitochondrion-targeting turn-on fluorescent probe RThy for detecting •OH. • Its turn on mechanism is based on the cleavage of azo-bond by •OH. • The azo group acts not only as a quenching-group but also as recognizing-group of •OH. • It successfully tracked mitochondrion •OH in live cells and zebrafish. Detection and imaging of hydroxyl radical (•OH) with high sensitivity and selectivity in biological systems is of great importance in evaluating the role of •OH plays in the physiological and pathological processes. Herein, a novel mitochondrion-targeting turn-on fluorescent probe RThy , has been developed to detect •OH in living cells and zebrafish. Probe's turn on mechanism is based on the cleavage of azo-bond by •OH. In RThy sensing system, possessing two groups: a lipophilic cation as a targeting-group, and an azo group acts not only as a quenching-group for tunable light absorption but also as recognizing-group of •OH. In the absence of •OH, almost non-fluorescence emission at 550 nm was observed. However, after reaction with •OH, bright fluorescence emission at 550 nm gradually increased with the continuous addition of •OH, displaying ˜50 folds fluorescence intensity enhancements and a quite low detection limit of 8 nM. The selectivity test showed RThy exhibits excellent selectivity toward •OH among all the ROS/RNS species tested. Furthermore, the probe has been successfully employed for imaging •OH in living cells and zebrafish, thus demonstrating RThy as a useful tool for studying •OH in practical sample application, and providing powerful theoretical basis and technical support for investigation of the interaction •OH with physiological and pathology. [ABSTRACT FROM AUTHOR]

Thiophenols are widely utilized for producing pesticides, polymers and pharmaceuticals. Studies have shown that prolonged exposure of human to thiolphenol can result in serious health problems such as significant damage of the central nervous system. Herein, we have developed a novel turn-on two-photon fluorescent probe TP-ASH for selective and sensitive detection of thiophenols. We first constructed a new two-photon dye by connecting a coumarin fluorophore and a naphthalene fluorophore through 2,5- disubstituted-1,3,4-oxadiazole ring to afford d -π-A-π- d -structure. Fluorescent studies showed that this new dye possess large two-photon active absorption across-section (250 GM) and high fluorescence quantum yield (0.85). Encouraged by the results, we proceeded to conjugate this novel dye to a thiophenol recognition moiety, 4-dinitrobenzene-ether (DNB). The resulting probe displayed high selectivity towards thiophenols over their similar analogues, aliphatic thiols. The probe also exhibited high sensitivity, with a detection limit of 6.7 nM and a fluorescence enhancement of 163-fold. In addition, we have successfully applied the probe for two-photon imaging studies of thiphenols in living cells and mice tissues. We envision that the probe will add useful tools in detecting thiophenols in environmental samples and biological systems. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H 2 S), one of the biologically important gaseous signal molecules, plays an essential role in diverse normal biochemical functions and pathological processes. Herein, an efficient two-photon in and near-infrared out mitochondria-targeting dye has been designed, synthesized and characterized. It is easily synthesized by the condensation reaction (C˭C) of 4-hydroxybenzaldehyde and 6-(diethylamino)−1,2,3,4-tetrahydroxanthylium (mitochondria-targeting), which possesses large two-photon action absorption cross-section ~160 g and high fluorescence quantum yield ~0.15. Encouraged by the results, we proceeded to conjugate this new dye with a H 2 S recognition moiety (4-dinitrobenzene-ether, DNB), on the basis of the intramolecular charge transfer (ICT) strategy, to construct a novel H 2 S fluorescent probe (TP-NIR-HS), which shows a targeting ability with high sensitivity and selectivity, and low cytotoxicity. This new probe was then applied for two-photon imaging of living cells and tissues and showed high imaging resolution and a deep-tissue imaging depth of ~350 µm, thus demonstrating its practical application in biological systems, and providing a valuable theoretical basis and technical support for the study of physiological and pathological functions of H 2 S. [ABSTRACT FROM AUTHOR]

We report a novel turn-on two-photon fluorescent probe ( TPNF ) for detecting formaldehyde based on aza-Cope rearrangement and intramolecular charge transfer (ICT). The probe, containing a 1,8-naphthalimide dye with a homoallylamino group, has was shown with high selectivity, sensitivity and low cytotoxicity and it was capable of detecting formaldehyde in live cells. Moreover, a time-dependent density functional theory (TD-DFT) calculation was performed to demonstrate the turn on mechanism of the probe via ICT effect. Finally, a zebrafish larvae imaging experiment reveals that TPNF displayed a significant signal change when treated with FA in vivo. These results indicated that TPNF is a promising tool in more biomedical researches. [ABSTRACT FROM AUTHOR]

In contrast to one-photon microscopy, two-photon probe-based fluorescence imaging can provide improved three-dimensional spatial localization and increased imaging depth. Therefore, the development of new functional two-photon fluorescent dye has attracted great attention. Herein, we have adopted the fluorescence resonance energy transfer (FRET) strategy to design a unique type of two-photon ratiometric fluorescent probe platform. Specifically, a two-photon fluorophore (D-π-A-structured naphthalimide derivative) and a rhodamine B fluorophore are directly connected by a flexible piperidine linker. We further used this platform to develop a new type of two-photon ratiometric fluorescent probe, NRLys , for lysosomal pH detection and bioimaging. The experiments demonstrated that the NRLys is a reliable and specific probe for labeling lysosomes in living cells and tissues with two well-resolved emission peaks separated by 60 nm. The probe showed high ratiometric imaging resolution and deep-tissue imaging depth of over 180 μm. Based on the above results, we expect that the new platform may prompt the development of a wide variety of two-photon ratiometric fluorescent probes application in biological systems. [ABSTRACT FROM AUTHOR]

• The structures of small-molecule fluorophores for plasma membrane staining were summarized. • The membrane targeting groups were categorized and summarized. • The mechanisms to achieve the membrane specificty were summarized and discussed. • The bioimaging applications of fluorescent probes were summarized. Plasma membrane (PM), a phospholipid bilayer that separates the interior of cell from the extracellular fluid, is closely involved in various cellular activities. However, monitoring dynamic membrane has always been a challenge due to its dynamic structure and complex constitution. Owing to the distinct advantages of fluorescence imaging technology, diverse small molecular fluorescent probes have been well developed for membrane staining to investigate membrane function and monitor its dynamic activity. Herein, representative small molecular fluorescent probes reported in the last ten years were summarized and categorized into four groups according to their functional membrane-anchoring moieties, namely hydrophobic alkyl chain, ammonium unit, cholesterol, and short peptide sequence. In particular, the underlying mechanisms to achieve membrane-specific fluorescence staining were fully discussed, including membrane targeting group approach, aggregation induced emission (AIE), disassembly light up fluorescence strategy and restricted intramolecular rotation (RIM). Furthermore, membrane-targeting probes for super-resolution imaging of membrane dynamics were presented as an emerging technology. In this review, we showcased recent advances in this research area, and outlined potential challenges and prospects with the goal of driving the generation of innovative fluorescent probes for PM staining. [ABSTRACT FROM AUTHOR]

Ratiometric two-photon fluorescent probes can not only eliminate interferences from environmental factors but also achieve deep-tissue imaging with improved spatial localization. To quantitatively track Pd 2 + in biosystems, herein, we reported a ratiometric two-photon fluorescent probe, termed as Np-Pd, which based on a D-π-A-structure two-photon fluorophore of the naphthalimide derivative and deprotection of aryl propargyl ethers by palladium species. The probe Np-Pd displayed a more than 25-fold enhancement towards palladium species with high sensitivity and selectivity. Additionally, the probe Np-Pd was further used for fluorescence imaging of Pd 2 + ions in living cells and tissues under two-photon excitation (820 nm), which showed large tissue-imaging depth (19.6–184.6 μm), and a high resolution for ratiometric imaging. [ABSTRACT FROM AUTHOR]

Hypochlorite anion, one of the biologically important reactive oxygen species, plays an essential role in diverse normal biochemical functions and abnormal pathological processes. Herein, an efficient high-resolution mitochondria-targeting ratiometric fluorescent probe for hypochlorous acid detection has been designed, synthesized and characterized. It is easily synthesized by the condensation reaction (C C) of a 2-(2-hydroxyphenyl) quinazolin-4(3H)-one fluorophore and a cyanine group (mitochondria-targeting), which made the whole molecular a large Stokes shift (210 nm) and the two well-resolved emission peaks separated by 140 nm. As a result, it is considered as a good candidate for high resolution hypochlorous acid imaging in live cells. The ratiometric fluorescent probe exhibited outstanding features of high sensitivity, high selectivity, rapid response time (within 50 s), and excellent mitochondria-targeting ability. Moreover, the probe can also be successfully applied to imaging endogenously hypochlorous acid in the mitochondria of living cells with low cytotoxicity, and high resolution. [ABSTRACT FROM AUTHOR]

Intracellular pH plays a pivotal role in various biological processes. Herein, we adopted the through bond energy transfer (TBET) strategy to design a unique type of ratiometric two-photon fluorescent probe for imaging of lysosomal pH in live cells and tissues, termed Np-Rh-Lys. Specifically, a two-photon fluorophore (D-π-A-structured naphthalimide derivative) and a rhodamine B fluorophore were directly connected, and dimethylamino moiety served as a lysosomal targeting-group. The experiments demonstrate new probe Np-Rh-Lys was a reliable and specific probe for labeling lysosomes in living cells with two well-resolved emission peaks separated by 80 nm, and which showed high ratiometric imaging resolution and deep-tissue imaging depth over 180 μm, we thus may expect the new platform prompt the development of a wide variety of ratiometric two-photon fluorescent probes application in biological systems. [ABSTRACT FROM AUTHOR]

In this study, Cu(II)-mediated differential alteration of cysteine (Cys) reactivity is reported by using a Cys-specific fluorescent probe. The probe could react with Cys to give out strong fluorescence. When Cys was preincubated with Cu(II), the fluorescence of the probe was decreased due to the inhibition of Cys's reactivity by Cu(II). Remarkably, experimental results reveal that the probe could detect Cu(II) at subnanomolar concentrations. In contrast, Cu(II) could only partially inhibit the reaction between Cys and Ellman's reagent (DTNB). Furthermore, selectivity experiments show that Cu(II) is a much more potent inhibitor for Cys compared to other metal ions. Cell imaging experiments also confirm the inhibitory effects of Cu(II) on Cys's reactivity in living cells. We envision that the probe could add a useful tool for sensitive and selective detection of Cu(II) for biomedical research. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H 2 S) has recently been identified as the third gaseous signaling molecule that is involved in regulating many important cellular processes. We report herein a novel fluorescent probe for detecting H 2 S based on iminocoumarin benzothiazole scaffold. The probe displayed high sensitivity and around 80-fold increment in fluorescence signal after reacting with H 2 S under physiological condition. The fluorescent intensity of the probe was linearly related to H 2 S concentration in the range of 0–100 μM with a detection limit of 0.15 μM (3σ/slope). The probe also showed excellent selectivity towards H 2 S over other biologically relevant species, including ROS, RSS and RNS. Its selectivity for H 2 S is 32 folds higher than other reactive sulfur species. Furthermore, the probe has been applied for imaging H 2 S in living cells. Cell imaging experiments demonstrated that the probe is cell-permeable and can be used to monitor the alteration of H 2 S concentrations in living cells. We envisage that this probe can provide useful tools to further elucidate the biological roles of H 2 S. [ABSTRACT FROM AUTHOR]

Abstract: We report herein a fluorescent probe for the detection of hydrogen sulfide (H2S) with high sensitivity and selectivity. The probe is installed with double azide groups and displays high sensitivity to H2S (around 120-fold turn-on response). It reacts with H2S with high selectivity over other reactive sulfur, nitrogen, and oxygen species. The probe was also applied to detect H2S in living cells. [Copyright &y& Elsevier]

Lysine crotonylation plays vital roles in gene transcription and cellular metabolism. Nevertheless, methods for dissecting the molecular mechanisms of decrotonyaltion remains limited. So far, there is no single-step fluorescent method developed for enzymatic decrotonylation activity detection. The major difficulty is that the aliphatic crotonylated lysine doesn't allow π-conjugation to a fluorophore and decrotonylation can not modulate the electronic state directly. Herein, we have designed and synthesized two activity-based single-step fluorogenic probes KTcr-I and KTcr-II for detecting enzymatic decrotonylation activity. These two probes can be recognized by histone deacetylases and undergo intramolecular nucleophilic exchange reaction to generate fluorescence signal. Notably, peptide sequence-dependent effect was observed. KTcr-I can be recognized by Sirt2 more effectively, while KTcr-II with LGKcr peptide sequence preferentially reacted with HDAC3. Compared to other methods of studying enzymatic decrotonylation activity, our single-step fluorescent method has a number of advantages, such as facileness, high sensitivity, cheap facility and little material consumed. We envision that the probes developed in this study will provide useful tools to screen inhibitors which suppress the decrotonylation activity of HDACs. Such probes will be useful for further delineating the roles of decrotonylation enzyme and aid in biomarker identification and drug discovery. The first single-step fluorescent probes to detect enzymatic decrotonylation activity. Image 1 • The first single-step fluorescent probes to detect decrotonylation activity of HDACs. • The probes undergo intramolecular nucleophilic exchange reaction to generate fluorescence signal. • Peptide sequence-dependent effect for enzyme recognition was observed. • KTcr-I can be used to screen inhibitors which suppress the decrotonylation activity of enzymes. [ABSTRACT FROM AUTHOR]

• A robust strategy for probe design with dual-quenching groups in one probe. • The strategy has been applied for designing HOCl fluorescent probe successfully. • The strategy can both enhance the probe's sensitivity and selectivity significantly. • The detection limit for HOCl is as low as 3.5 nM. • Two-photon probe has been used to detect HOCl in living cells and kidney tissues. We report herein fluorescent probes equipped with dual-quenching groups exhibiting superior sensitivity than probes with mono-quenching groups. Importantly, with this strategy, the probe with dual-quenching groups react with HOCl through two distinct reaction mechanisms, which reduce the plausible side reactions with other competing analytes and enhance the probe's selectivity. As a proof-of-concept study, we designed and synthesized a probe with dual-quenching groups DQ-HOCl to detect HOCl, which is one of the most important ROS and linked with a number of diseases. In addition, two control probes with mono-quenching groups, MQ-HOCl-1 and MQ-HOCl-2 , were also synthesized for comparison purpose. Fluorescent assays demonstrated that DQ-HOCl indeed shows ultra-high sensitivity and selectivity compared with probes with mono-quenching groups. Furthermore, the probe has been successfully utilized to imaging exogenous/endogenous HOCl in living cells. Moreover, DQ-HOCl was applied to visualize HOCl in kidney tissues from rat due to the increased penetration depth and lower tissue autofluorescence from the nature of two-photon probes. [ABSTRACT FROM AUTHOR]