Nitric oxide (NO) is a fundamental signaling molecule that regulates virtually every critical cellular function, and it is also a potent mediator of cellular damage in a wide range of conditions mainly via its secondary metabolite peroxynitrite (ONOO−). In this work, we present an o-phenylenediamine (OPD)-locked Si-rhodamine deoxylactam, i.e.deOxy-DALSiR, as a near-infrared fluorescent probe for the selective and sensitive detection of NO in living cells and bodies. Not only could the probe overcome the limitations suffered by widely used and commercialized OPD-type fluorescent NO probes, such as the possible interferences by dehydroascorbic acid/ascorbic acid/methylglyoxal (DHA/AA/MGO), pH-sensitive fluorescence output, and short excitation and emission wavelengths, but it can also avoid serious interference from cysteine (Cys) found in the rhodamine lactam-based fluorescent NO probes developed later. What’s more, the probe is fairly sensitive for NO, as evidenced by its rapid fluorescence response rate (within seconds), huge fluorescence off–on ratio (6300-fold), and ultra-low detection limit (0.12 nM). Its effectiveness and practicability have been demonstrated by the successful imaging of endogenous NO in RAW 264.7 macrophages, pancreatic β-cells, and endothelial EA.hy926 cells, as well as in inflamed and diabetic mouse models.

•A new fluorescent probe 1 based on a 2-(benzothiazol-2-yl) phenol (HBT) derivative was synthesized and characterized.•The limit of detection of probe 1 was as low as 3.78 × 10− 8 M for Cys.•The probe 1 exhibited a large Stock's shifts in aqueous solution.•The probe 1 was used as practical probe for imaging of Cys in living cell.In this study, a new fluorescent probe 2-(2′-hydroxy-5′-N-maleimide phenyl)-benzothiazole (probe 1), was designed and synthesized by linking the excited state intramolecular proton transfer (ESIPT) fluorophore to the maleimide group for selective detection of thiols in aqueous solution. The fluorescence of probe 1 is strongly quenched by maleimide group through the photo-induced electron transfer (PET) mechanism, but after reaction with thiol, the fluorescence of ESIPT fluorophore is restored, affording a large Stokes shifts. Upon addition of cysteine (Cys), probe 1 exhibited a fast response time (complete within 30 s) and a high signal-to-noise ratio (up to 23-fold). It showed a high selectivity and excellent sensitivity to thiols over other relevant biological species, with a detection limit of 3.78 × 10− 8 M (S/N = 3). Moreover, the probe was successfully applied to the imaging of thiols in living cells.

•A new reaction-type fluorescent probe 1 for detection of H2S was exploited.•The probe displayed excellent sensing performances for H2S.•The successful imaging application of the probe for H2S in living cell level indicates its potential for probing H2S biology in biological systems.Based on the thiolysis of 7-nitro-1,2,3-benzoxadiazole (NBD) amine, a novel fluorescent probe for hydrogen sulfide was designed and synthesized, which contains 5 (6)-carboxy-rhodamine as fluorophore and NBD as hydrogen sulfide reaction group. The probe is non-fluorescent, and could selectively detect hydrogen sulfide with a big fluorescence enhancement of 160-fold and a low detection limit of 4.80 × 10−8 mol/L. Furthermore, coupled with its good biocompatibility, the probe has been used for fluorescent bioimaging of H2S in living cells.

Nitric oxide (NO) is an important cellular signaling molecule involved in many physiological and pathological processes. To probe its spatiotemporal information in biosystems, a large number of NO fluorescent probes have been exploited in the past ten years. Among them, the o-phenylenediamine-based probes are the earliest developed and most versatile NO fluorescent probes to date. However, there are still limitations such as relatively long response time, possible interference by dehydroascorbic acid (DHA)/methylglyoxal (MGO), and pH-sensitivity of their fluorescence signals. In this work, we present two aromatic primary monoamine-based NO fluorescent probes, MA and NIR-MA, and explore the reductive deamination reaction of the electron-rich p-methoxyaniline group with NO under aerobic conditions. The superiority of both probes is illustrated by their quick, stable, sensitive, and specific fluorescence off–on responses for NO over a series of biologically relevant interfering species, including reactive oxygen species, DHA/MGO, biothiols, and metal ions. Coupled with good cell permeability and low cytotoxicity, the two probes have successfully been applied to imaging the endogenous NO in RAW 264.7 macrophages stimulated by LPS/IFN-γ. Moreover, the fluorescence response of NIR-MA for NO occurs in the physiologically favorable NIR region, enabling its further use to image endogenous NO in an inflamed mouse model.

Lysosomes have recently been regarded as the attractive pharmacological targets for selectively killing of cancer cells via lysosomal cell death (LCD) pathway that is closely associated with reactive oxygen species (ROS). However, the details on the ROS-induced LCD of cancer cells are still poorly understood, partially due to the absence of a lysosome-targetable, robust, and biocompatible imaging tool for ROS. In this work, we brought forward a Si-rhodamine-based fluorescent probe, named PSiR, which could selectively and sensitively image the pathologically more relavent highly reactive oxygen species (hROS: HClO, HO, and ONOO−) in lysosomes of cancer cells. Compared with many of the existing hROS fluorescent probes, its superiorities are mainly embodied in the high stability against autoxidation and photoxidation, near-infrared exitation and emission, fast fluorescence off−on response, and specific lysosomal localization. Its practicality has been demonstrated by the real-time imaging of hROS generation in lysosomes of human non-small-cell lung cancer cells stimulated by anticancer drug β-lapachone. Moreover, the probe was sensitive enough for basal hROS in cancer cells, allowing its further imaging applications to discriminate not only cancer cells from normal cells, but also tumors from healthy tissues. Overall, our results strongly indicated that PSiR is a very promising imaging tool for the studies of ROS-related LCD of cancer cells, screening of new anticancer drugs, and early diagnosis of cancers.Download high-res image (328KB)Download full-size image

Given the wavelength dependence of tissue transparency and the requirement for sufficiently low background autofluorescence, the development of fluorescent dyes with excitation and emission maxima beyond 700 nm is highly desired, but it is a challenging task. Herein, a new class of fluorescent dyes, named sulfone-rhodamines (SO2Rs), was developed on the basis of the one-atom replacement of the rhodamine 10-position O atom by a sulfone group. Such a modification makes their absorption and emission maxima surprisingly reach up to 700–710 and 728–752 nm, respectively, much longer than their O-, C-, and Si-rhodamine analogs, due to the unusual d*−π* conjugation. Among these dyes, SO2R4 and SO2R5, bearing disubstituted meso-phenyl groups, show the greatest potentials for bioimaging applications in view of their wide pH range of application, high photostability, and big extinction coefficients and fluorescence quantum yields. They could quickly penetrate cells to give stable NIR fluorescence, even after continuous irradiation by a semiconductor laser, making them suitable candidates for time-lapse and long-term bioimaging applications. Moreover, they could specifically localize in lysosomes independent of alkylmorpholine targeted group, thus avoiding the problematic alkalization effect suffered by most LysoTrackers. Further imaging assays of frozen slices of rat kidney reveal that their tissue imaging depth is suprior to the widely used NIR labeling agent Cy5.5.Keywords: fluorescent dyes; imaging agents; near-infrared; rhodamines; sulfones

•A new π-conjugation extended naphthorhodamine dye 1 with a carboxylic acid-functional group was designed and synthesized.•The single crystal of 1 was obtained and characterized.•The photochemical properties of the dye were investigated in different solvent polarity.•The dye was successfully used to construct a new fluorescent probe and a biomarker.A new extended-π-conjugated naphthorhodamine dye, 9′-diethylamino-2′-hydroxyl-benzo[a]fluoran (1) with a carboxylic acid-functional group was designed and synthesized. Compared to Rhodamine B, the emission wavelength of 1 was extended to ca. 600 nm, and its fluorescence properties could be controlled by the key carboxylic acid-functional group. By taking advantage of 1, we synthesized a new far-red bioimaging dye 2-ethyl-9′-diethylimino-2′-hydroxyl- benzo[a]fluoran (2) by esterification of 1, which could selectively stain mitochondria. Moreover, encouraged by the spirocyclization switching mechanism platform, we successfully constructed a new fluorescent probe 9′-diethylamino-2′-hydroxyl-benzo[a]fluoran hydrazine (3), the hydrazide derivative of 1, for Hg2+ with good selectivity. These results suggest that the new naphthorhodamine dye could be used as a platform to construct fluorescent probes and bioimaging reagents.

Peroxynitrite (ONOO−) is an extremely reactive oxygen species in biological systems, and can react with a wide variety of molecular targets including proteins, lipids, and nucleic acids, eventually resulting in mitochondria dysfunction and cell death. In this work, we designed and synthesized a new arylboronate-based fluorescent probe, which could detect ONOO− with a big fluorescence off-on ratio (155-fold), fast response rate (50 s), high sensitivity (detection limit:0.83 nM), and excellent selectivity over a series of biologically relevant reactive oxygen species as well as anions, cations, and biothiols. The sensing mechanism has proved to be the ONOO−-triggered covalent assembly of a bright iminocourmarin fluorophore. With the probe, the stimulation-induced ONOO− in RAW264.7 murine macrophages as well as the endogenous ONOO− in endothelial cells after oxygen-glucose deprivation (OGD) has been successfully visualized.An arylboronate-based fluorescent probe for sensitive and selective detection of peroxynitrite has been developed and applied to image the stimulation-induced peroxynitrite in RAW264.7 murine macrophages as well as the endogenous peroxynitrite in endothelial cells after oxygen-glucose deprivation.

A mitochondria-targetable fluorescence probe, methyl(4-hydroxyphenyl)amino-substituted pyronin (1), was exploited, which could highly selectively sense peroxynitrite (ONOO−) within seconds.

In this work, a cysteine (Cys)-triggered dual Michael addition/retro-aza-aldol cascade reaction has been exploited and utilized to construct a fluorescent probe for Cys for the first time. The resulting fluorescent probe 8-alkynylBodipy 1 contains an activated alkynyl unit as Michael receptor and a Bodipy dye as fluorescence reporter and can highly selectively detect Cys over homocysteine (Hcy)/glutathione (GSH) as well as other amino acids with a significant fluorescence off–on response (∼4500-fold) and an ultralow detection limit (0.38 nM). The high selectivity of 1 for Cys could be attributed to a kinetically favored five-membered cyclic intermediate produced by the dual Michael addition of Cys with the activated alkynyl unit of 1. The big fluorescence off–on response is due to the subsequent retro-aza-aldol reaction of the five-membered cyclic intermediate that results in the release of a highly fluorescent 8-methylBodipy dye 2. The probe has been successfully used to detect and image Cys in serum and cells, respectively.

An arylboronate-based fluorescent probe for detection of peroxynitrite with fast fluorescence response and high selectivity was developed based on the peroxynitrite-triggered assembly of aminocoumarin fluorescent dye.

A mitochondria-specific fluorescent probe for NO (1) was synthesized by the direct conjugation of a pyronin dye with one of the amino groups of o-phenylenediamino (OPD). The probe could selectively detect NO over dehydroascorbic acid (DHA), ascorbic acid (AA), and methylglyoxal (MGO) as well as the reactive oxygen/nitrogen species (ROS/RNS) with the significant off–on response due to the production of a red-emission triazole 2. In the presence of cysteine/glutathione (Cys/GSH), 2 could be further transformed into a green-emission aminopyronin 4 and a red-emission thiopyronin 5, respectively. Assisted by intracellular Cys and GSH, the probe demonstrated its potential to monitor mitochondrial NO in a dual-channel mode.

A 4-methoxythiophenol-substituted pyronin dye 1 was exploited as reaction-type fluorescent probe for biothiols Cys/Hcy and GSH. The probe itself is nonfluorescent due to the photoinduced electron transfer (PET) process. The Cys (or Hcy)-induced substitution–rearrangement cascade reaction and GSH-induced substitution reaction with the probe lead to the corresponding aminopyronin and thiopyronin dyes with distinct photophysical properties, enabling Cys/Hcy and GSH to be detected from visible and near-infrared (NIR) emission channels, respectively, in pure PB buffer with relatively fast kinetics and obvious fluorescence turn-on response. Assisted by laser scanning confocal microscope, we also demonstrated that probe 1 could simultaneously sense Cys/Hcy and GSH in B16 cells in multicolor imaging.

By incorporation of a specific NO-binding group, 2-amino-3′-dimethylaminobiphenyl, into a Bodipy dye, fluorescent probe 1 was constructed, which exhibited high selectivity for NO over other ROS/RNS as well as DHA, AA and MGO.

A fluorescent off–on probe for H2S was exploited by coupling the azide-based strategy with the excited-state intramolecular proton transfer (ESIPT) sensing mechanism, which exhibits a considerably high fluorescence enhancement (1150-fold), an extremely low detection limit (0.78 nM), and a relatively fast response time (3–10 min) as well as excellent selectivity.

A but-3-yn-2-one-based 7-diethylaminocoumarin dye was exploited as a fluorescent probe to specifically detect Cys over Hcy/GSH in pure PBS buffer. The probe itself is nonfluorescent due to the donor-excited photoinduced electron transfer (d-PET) process. The Cys-induced Michael addition–rearrangement cascade reaction leads to an amino-substituted product 3a with strong fluorescence due to inhibiting CC isomerization induced fluorescence quenching by a produced intramolecular N–H⋯O hydrogen bond. The Hcy (or GSH)-induced Michael addition reaction leads to a thiol-substituted product 2b (or 4), which lacks any intramolecular hydrogen-bonding interaction, and thus displays very poor fluorescence due to the efficient CC isomerization induced fluorescence quenching. Even in the presence of Hcy (or GSH), the probe could also detect Cys with the obvious fluorescence enhancement. Assisted by using a laser scanning confocal microscope, we demonstrated that the probe could selectively image Cys in the human renal cell carcinoma 786-0 cells.

A carboxylic acid-functionalized coumarin-hemicyanine near-infrared (NIR) dye 1 was exploited, which possesses good water solubility (more than 50 μM) and favorable photophysical properties, especially a large Stokes shift (around 90 nm), and has been proved to be a suitable imaging agent for targeting mitochondria. With the dye platform, fluorescent probe 2, a thioester derivative of 1, was constructed for biothiols. Probe 2 can react with cysteine (Cys) via the native-chemical-ligation (NCL) and cyclization cascade reactions to lead to coumarin 2-Cys. However, the reaction of 2 with homocysteine (Hcy) or glutathione (GSH) only stays at the stage of the initial transthioesterification reaction, producing coumarin-hemicyanines 2-Hcy or 2-GSH, due to an electrostatic interaction in 2-Hcy and an unstable macrocyclic transition state in 2-GSH, both inhibiting their subsequent S,N-acyl shift. Given the distinct photophysical properties between 2-Cys and 2-Hcy (or 2-GSH), probe 2 could highly selectively discriminate Cys from Hcy/GSH. Even in the presence of Hcy or GSH, probe 2 still works well for Cys due to the reversible transthioesterification and the irreversible S,N-acyl shift in the NCL reaction. The cell imaging assays revealed that probe 2 is cell permeable and could selectively image Cys in living cells.

A chlorinated coumarin-hemicyanine dye with three potential reaction sites was exploited as fluorescent probe for biothiols. The Cys-induced substitution–rearrangement–cyclization, Hcy-induced substitution–rearrangement, and GSH-induced substitution–cyclizatioin cascades lead to the corresponding amino-coumarin, amino-coumarin-hemicyanine, thiol-coumarin with distinct photophysical properties, enabling Cys and GSH to be selectively detected from different emission channels at two different excitation wavelengths.

A fluorescent turn-on probe for H2S was exploited based on a H2S-induced substitution–cyclization cascade reaction towards the bis-electrophilic centers of a new H2S trap group 2-(iodomethyl)benzoate.

A coumarin−hemicyanine dye 1 was reported for ratiometric fluorescent detection of SO2 derivatives HSO3− and SO32− based on a novel addition-rearrangement cascade reaction.

A rhodamine-inspired fluorescence dye (2) bearing 7-diethylaminocoumarin fluorophore was designed as a platform for the construction of an NIR and ratiometric fluorescent probe. The ring-open form of 2 shows NIR absorption and emission; however, its ring-closed form displays the visible absorption and emission because an intact 7-diethylaminocoumarin fluorophore was involved in the structure, providing the basis for an NIR and ratiometric fluorescent platform based on the spirocyclization-induced fluorescence switching mechanism. With the platform, we developed a novel NIR and ratiometric fluorescent probe R1, a thiolactone of 2, for sensing Hg2+, an environmentally and biologically concerned species. R1 displays an emission peak with the maximum at 480 nm, which is the typical emission of 7-diethylaminocoumarin moiety; however, upon addition of Hg2+ ions, the emission intensity at 480 nm gradually decreased with the simultaneous appearance of a new NIR emission band centred at 695 nm. Thus, the Hg2+-promoted ratiometric fluorescence response can be realized. The high selectivity towards Hg2+ over various cations and the high stability in a wide pH range of 1–12 indicate its potential for applications in biological systems. The subsequent cell imaging experiment revealed that R1 is cell permeable, and could be employed for ratiometric fluorescence imaging of Hg2+ in living cells.

A fluorescein-based biothiol probe 1 was exploited based on a novel thiol-specific cascade reaction, which could detect and image biothiols in both aqueous solution and cells with high selectivity, sensitivity and signal-to-noise ratio.

A Cu(II)-based 2:2 chemosensing ensemble bearing rhodamine B fluorophore was developed for fluorescence turn-on detection of CN− with high selectivity and sensitivity based on the CN− complexation to the Cu2+ centre in the nearly planar central binding moiety.

A new strategy for enhancing the brightness of the NIR distyryl Bodipy derivatives was proposed based on the indirect S0 → S2 excitation, and applied to fluorescence turn-on detection of Hg2+, by which the marked fluorescence enhancement (2.5-fold) and the high sensitivity for Hg2+ (up to 3 ppb) could be realized.

A fluorescent turn-on probe for Cys/Hcy based on inhibiting the C═N isomerization quenching process by an intramolecular hydrogen bond was reported. The probe exhibited higher selectivity toward Cys/Hcy over other amino acids as well as thiol-containing compounds.

A coumarin–indanedione conjugate as an activated Michael addition type probe was prepared for cyanide ion detection. The colorimetric and ratiometric fluorescent response of the probe to cyanide ion is due to the Michael addition of cyanide to the doubly activated Michael receptor of the probe which blocks an intramolecular charge transfer process. The probe displays a fast response to cyanide ion at room temperature, and a maximal ratiometric fluorescent signal is achieved in the presence of only 2 equivalents of cyanide ion. Moreover, competitive species did not induce any significant changes both in color and emission intensity ratio (I485/I609), demonstrating the high selectivity of the probe to cyanide.Highlights► A coumarin–indanedione conjugate was prepared for CN− detection. ► It exhibits ratiometric fluorescent response to CN− due to the Michael addition. ► High selectivity and sensitivity for detecting CN− are noted for this system.

A fluorescence turn-on probe for bisulfite has been developed by taking advantage of the specific reaction of bisulfite and aldehyde in combination with the hydrogen bond inhibited CN isomerization mechanism. The practical value of this selective and sensitive fluorescent probe was confirmed by its application to detection of bisulfite in granulated sugar.

On the basis of fluorescent resonance energy transfer from 1,8-naphthalimide to rhodamine B, a new fluorophore dyads (4) containing rhodamine B and a naphthalimide moiety was synthesized as a ratiometric fluorescent probe for detecting Hg2+ with a broad pH range 5.7–11.0. The selective fluorescence response of 4 to Hg2+ is due to the Hg2+-promoted desulfurization of the thiocarbonyl moiety, leading to the ring-opening of rhodamine B moiety of 4. When 4 was employed at 0.1 μM with the slit size being 20 nm/20 nm, a low level of Hg2+ (up to 3 × 10−8 M) can be detected using the system.Highlights► A fluorescent probe for Hg2+ based on Hg2+-promoted desulfurization reaction. ► Ratiometric fluorescent sensing for Hg2+ based on FRET. ► Excellent selectivity and high sensitivity toward detecting Hg2+. ► The large Stokes shift (185 nm) can be realized in this system.

The energy transfer cassettes composed of coumarin and Bodipy as well as NIR distyryl Bodipys were designed to capture photonic energy and convert it to longer wavelength fluorescence emission; large pseudo-Stokes' shifts (up to 410 nm) and efficient energy transfer efficiencies (up to 98.6%) as well as a marked antenna effect were demonstrated.

A coumarin-based thiol probe featuring the 1,4-addition reaction of thiols to nitroolefin was reported. The molecular probe exhibited higher selectivity toward biothiols (Cys, Hcy and GSH) than other amino acids.

A new ratiometric fluorescent cyanide probe was developed based on the nucleophilic attack of CN− toward the indolium group of a hybrid coumarin–hemicyanine dye, by which high selectivity as well as large emission shift could be achieved.

A water-soluble fluorescent sensor, 1, based on the “receptor-spacer-fluorophore” [2-(2′-aminophenyl)benzoxazole-amide-2-picolylamine] sensor platform, demonstrates the high sensitivity for Zn2+ with a 25-fold fluorescence enhancement upon chelation to Zn2+ and also exhibits high selectivity to Zn2+ over other metal ions. X-ray crystal structure of Zn2+ complex reveals that the amide oxygen (O2) cooperates with 2-picolylamine unit (N3, N4) as a receptor bind Zn2+.

A new rhodamine B-based dual-function chromo- and fluorogenic probe for Cu2+ and ClO− has been designed, synthesized, and characterized. The probe comprises a spectroscopic unit of rhodamine B and a Cu2+-specific chelating unit of pyridinecarboxamide as well as a ClO−-specific reactive moiety of diacylhydrazine, and is a highly selective and extremely sensitive fluorescent and colorimetric sensor for Cu2+ and ClO− in different pH conditions. Compared with the reported probes for Cu2+ or ClO−, this is the first chemosensor based on a small molecule that can detect both Cu2+ and ClO−, respectively, at 1 nM level.

On the basis of FRET from 4-(N,N-dimethylamino)benzamide to fluorescein, a new ratiometric fluorescence probe bearing a hydrazone binding unit was developed for highly selective and sensitive detection of CN− in aqueous solution.

A new fluorescent probe, rhodamine B derivative (1) bearing an 8-hydroxyquinoline group, was synthesized and displayed highly selective and sensitive Hg2+-amplified absorbance and fluorescence emission above 500 nm in aqueous solution with a broad pH range 4–9. It was found that mercury ions coordinate reversibly to 1 and the spirolactam ring of 1 was opened, forming a 1:1 metal–ligand complex. Furthermore, this sensor was applied for in vivo imaging in HeLa cells to confirm that 1 can be used as a fluorescent probe for monitoring Hg2+ in living cells.

Two highly selective cyanide probes with a salicylaldehyde hydrazone functionality as binding site were reported with fast response. The detection of cyanide was performed via the nucleophilic attack of cyanide anion on the imine group of the probes with a 1:1 binding stoichiometry, which could be confirmed by 1H NMR and MS studies. The specific reaction results in a prominent fluorescence enhancement for one of the probes and a color change from pale yellow to dark red for the other. The cyanide detection method described here should have potential application as a new family of probes for detecting cyanide in aqueous solution.

Peroxynitrite (ONOO−) is an extremely powerful oxidant in biological systems, and can react with a wide variety of molecular targets including proteins, lipids, and nucleic acids, eventually resulting in a series of disease states such as diabetes, Alzheimer's disease, cancer, arthritis, autoimmune, and other disorders. In this work, we present a new class of ONOO− fluorescent probes by exploiting the ONOO−-triggered N-oxidation and N-nitrosation reactions of aromatic tertiary amine for the first time. The as-obtained fluorescent probe A2 could detect ONOO− with quite fast fluorescence off-on response (within seconds), ultrasensitivity (detection limit: <2 nM), and excellent selectivity over a series of biologically relevant reactive oxygen species as well as metal cations. With the probe, the endogenous ONOO− in activated RAW264.7 murine macrophage, EA.hy926 endothelial cells after oxygen glucose deprivation and reoxygenation (OGD/RO), and kidney tissue of diabetic rats has been successfully visualized. Based on the molecular platform of A2, we further develop its mitochondria- and lysosome-targetable fluorescent probes Mito-A2 and Lyso-A2 by installing the corresponding targeting groups to alkoxy unit of A2, and confirm their abilities to image ONOO− in mitochondria and lysosomes, respectively, by co-localization assays. It is greatly expected that these probes can serve as useful imaging tools for clarifying the distribution and pathophysiological functions of ONOO− in cells, subcellular organelles, and animal tissues.

Nitric oxide (NO) is a ubiquitous biological messenger molecule, and plays the active roles in the regulation of various physiological processes. Although numerous NO fluorescent probes have also been successfully developed in the past ten years, it still remains challenging to increase the response rate for NO while having the high selectivity and sensitivity. In this work, a simple N-nitrosation reaction of the electron-rich aromatic secondary amine with NO under aerobic condition has been utilized for the first time to construct fluorescent probe for NO. The resulting probe 1, containing a N-benzyl-4-hydroxyaniline moiety as reaction group and a BODIPY dye as fluorescence reporter, could detect NO with the fast fluorescence off-on response (within seconds), high sensitivity (nM level), and excellent selectivity over various reactive oxygen species (ROS) as well as dehydroascorbic acid (DHA), ascorbic acid (AA), and methylglyoxal (MGO). Even in the presence of glutathione (GSH, a high reactive biothiol for NO), the probe still works well for NO. Further, a mitochondria-targetable probe 2 was exploited by introducing a targeted triphenylphosphonium cation into probe 1 scaffold. It's excellent NO sensing performance as well as its ability to specifically target mitochondria and image NO there have been nicely demonstrated. With the two probes, the basal and stimulation-induced NO in RAW264.7 murine macrophages as well as the endogenous NO in endothelial cells after oxygen–glucose deprivation (OGD) have been successfully visualized.

A new ratiometric fluorescent cyanide probe was developed based on the nucleophilic attack of CN− toward the indolium group of a hybrid coumarin–hemicyanine dye, by which high selectivity as well as large emission shift could be achieved.

A Cu(II)-based 2:2 chemosensing ensemble bearing rhodamine B fluorophore was developed for fluorescence turn-on detection of CN− with high selectivity and sensitivity based on the CN− complexation to the Cu2+ centre in the nearly planar central binding moiety.

A 4-methoxythiophenol-substituted pyronin dye 1 was exploited as reaction-type fluorescent probe for biothiols Cys/Hcy and GSH. The probe itself is nonfluorescent due to the photoinduced electron transfer (PET) process. The Cys (or Hcy)-induced substitution–rearrangement cascade reaction and GSH-induced substitution reaction with the probe lead to the corresponding aminopyronin and thiopyronin dyes with distinct photophysical properties, enabling Cys/Hcy and GSH to be detected from visible and near-infrared (NIR) emission channels, respectively, in pure PB buffer with relatively fast kinetics and obvious fluorescence turn-on response. Assisted by laser scanning confocal microscope, we also demonstrated that probe 1 could simultaneously sense Cys/Hcy and GSH in B16 cells in multicolor imaging.

A coumarin-based thiol probe featuring the 1,4-addition reaction of thiols to nitroolefin was reported. The molecular probe exhibited higher selectivity toward biothiols (Cys, Hcy and GSH) than other amino acids.

Nitric oxide (NO) is an important cellular signaling molecule involved in many physiological and pathological processes. To probe its spatiotemporal information in biosystems, a large number of NO fluorescent probes have been exploited in the past ten years. Among them, the o-phenylenediamine-based probes are the earliest developed and most versatile NO fluorescent probes to date. However, there are still limitations such as relatively long response time, possible interference by dehydroascorbic acid (DHA)/methylglyoxal (MGO), and pH-sensitivity of their fluorescence signals. In this work, we present two aromatic primary monoamine-based NO fluorescent probes, MA and NIR-MA, and explore the reductive deamination reaction of the electron-rich p-methoxyaniline group with NO under aerobic conditions. The superiority of both probes is illustrated by their quick, stable, sensitive, and specific fluorescence off–on responses for NO over a series of biologically relevant interfering species, including reactive oxygen species, DHA/MGO, biothiols, and metal ions. Coupled with good cell permeability and low cytotoxicity, the two probes have successfully been applied to imaging the endogenous NO in RAW 264.7 macrophages stimulated by LPS/IFN-γ. Moreover, the fluorescence response of NIR-MA for NO occurs in the physiologically favorable NIR region, enabling its further use to image endogenous NO in an inflamed mouse model.

A new strategy for enhancing the brightness of the NIR distyryl Bodipy derivatives was proposed based on the indirect S0 → S2 excitation, and applied to fluorescence turn-on detection of Hg2+, by which the marked fluorescence enhancement (2.5-fold) and the high sensitivity for Hg2+ (up to 3 ppb) could be realized.

A fluorescent turn-on probe for H2S was exploited based on a H2S-induced substitution–cyclization cascade reaction towards the bis-electrophilic centers of a new H2S trap group 2-(iodomethyl)benzoate.

A mitochondria-targetable fluorescence probe, methyl(4-hydroxyphenyl)amino-substituted pyronin (1), was exploited, which could highly selectively sense peroxynitrite (ONOO−) within seconds.

The energy transfer cassettes composed of coumarin and Bodipy as well as NIR distyryl Bodipys were designed to capture photonic energy and convert it to longer wavelength fluorescence emission; large pseudo-Stokes' shifts (up to 410 nm) and efficient energy transfer efficiencies (up to 98.6%) as well as a marked antenna effect were demonstrated.

On the basis of FRET from 4-(N,N-dimethylamino)benzamide to fluorescein, a new ratiometric fluorescence probe bearing a hydrazone binding unit was developed for highly selective and sensitive detection of CN− in aqueous solution.

A new rhodamine B-based dual-function chromo- and fluorogenic probe for Cu2+ and ClO− has been designed, synthesized, and characterized. The probe comprises a spectroscopic unit of rhodamine B and a Cu2+-specific chelating unit of pyridinecarboxamide as well as a ClO−-specific reactive moiety of diacylhydrazine, and is a highly selective and extremely sensitive fluorescent and colorimetric sensor for Cu2+ and ClO− in different pH conditions. Compared with the reported probes for Cu2+ or ClO−, this is the first chemosensor based on a small molecule that can detect both Cu2+ and ClO−, respectively, at 1 nM level.

A fluorescein-based biothiol probe 1 was exploited based on a novel thiol-specific cascade reaction, which could detect and image biothiols in both aqueous solution and cells with high selectivity, sensitivity and signal-to-noise ratio.

A fluorescent off–on probe for H2S was exploited by coupling the azide-based strategy with the excited-state intramolecular proton transfer (ESIPT) sensing mechanism, which exhibits a considerably high fluorescence enhancement (1150-fold), an extremely low detection limit (0.78 nM), and a relatively fast response time (3–10 min) as well as excellent selectivity.

By incorporation of a specific NO-binding group, 2-amino-3′-dimethylaminobiphenyl, into a Bodipy dye, fluorescent probe 1 was constructed, which exhibited high selectivity for NO over other ROS/RNS as well as DHA, AA and MGO.

A coumarin−hemicyanine dye 1 was reported for ratiometric fluorescent detection of SO2 derivatives HSO3− and SO32− based on a novel addition-rearrangement cascade reaction.

A new fluorescent probe, rhodamine B derivative (1) bearing an 8-hydroxyquinoline group, was synthesized and displayed highly selective and sensitive Hg2+-amplified absorbance and fluorescence emission above 500 nm in aqueous solution with a broad pH range 4–9. It was found that mercury ions coordinate reversibly to 1 and the spirolactam ring of 1 was opened, forming a 1:1 metal–ligand complex. Furthermore, this sensor was applied for in vivo imaging in HeLa cells to confirm that 1 can be used as a fluorescent probe for monitoring Hg2+ in living cells.

An arylboronate-based fluorescent probe for detection of peroxynitrite with fast fluorescence response and high selectivity was developed based on the peroxynitrite-triggered assembly of aminocoumarin fluorescent dye.

A water-soluble fluorescent sensor, 1, based on the “receptor-spacer-fluorophore” [2-(2′-aminophenyl)benzoxazole-amide-2-picolylamine] sensor platform, demonstrates the high sensitivity for Zn2+ with a 25-fold fluorescence enhancement upon chelation to Zn2+ and also exhibits high selectivity to Zn2+ over other metal ions. X-ray crystal structure of Zn2+ complex reveals that the amide oxygen (O2) cooperates with 2-picolylamine unit (N3, N4) as a receptor bind Zn2+.