A fast-responsive fluorescent turn-on probe for nitroreductase imaging in living cells

Nitroreductase (NTR) may be more active under the environment of hypoxic conditions, which are the distinctive features of the multiphase solid tumor. It is of great significance to effectively detect and monitor NTR in the living cells for the diagnosis of hypoxia in a tumor. Here, we synthesized a novel turn-on fluorescent probe NTR-NO₂ based on a fused four-ring quinoxaline skeleton for NTR detection. The highly efficient probe can be easily synthesized. The probe NTR-NO₂ showed satisfactory sensitivity and selectivity to NTR. Upon incubation with NTR, NTR-NO₂ could successively undergo a nitro reduction reaction and then generate NTR-NH₂ along with significant fluorescence enhancement (30 folds). Moreover, the fluorescent dye NTR-NH₂ exhibits a large Stokes shift (Δλ = 111 nm) due to the intramolecular charge transfer (ICT) process. As a result, NTR-NO₂ displayed a wide linear range (0–4.5 μg mL⁻¹) and low detection limit (LOD = 58 ng mL⁻¹) after responding to NTR. In addition, this probe was adopted for the detection of endogenous NTR within hypoxic HeLa cells.

Probe NTR-NO₂ was effectively reduced in the presence of NTR generating a highly fluorescent product.     

A fast-responsive fluorescent probe based on a styrylcoumarin dye for visualizing hydrogen sulfide in living MCF-7 cells and zebrafish

As a vital antioxidant molecule, H₂S can make an important contribution to regulating blood vessels and inhibiting apoptosis when present at an appropriate concentration. Higher levels of H₂S can interfere with the physiological responses of the respiratory system and central nervous system carried out by mammalian cells. This is associated with many illnesses, such as diabetes, mental decline, cardiovascular diseases, and cancer. Therefore, the accurate measurement of H₂S in organisms and the environment is of great significance for in-depth studies of the pathogenesis of related diseases. In this contribution, a new coumarin-carbazole-based fluorescent probe, COZ-DNBS, showing a rapid response and large Stokes shift was rationally devised and applied to effectively sense H₂S in vivo and in vitro. Upon using the probe COZ-DNBS, the established fluorescent platform could detect H₂S with excellent selectivity, showing 62-fold fluorescence enhancement, a fast-response time (<1 min), high sensitivity (38.6 nM), a large Stokes shift (173 nm), and bright-yellow emission. Importantly, the probe COZ-DNBS works well for monitoring levels of H₂S in realistic samples, living MCF-7 cells, and zebrafish, showing that COZ-DNBS is a promising signaling tool for H₂S detection in biosystems.

The probe COZ-DNBS displayed excellent selectivity, a fast response, high sensitivity, a large Stokes shift, and bright-yellow emission in response to H₂S.     

Near-infrared turn-on fluorescent probe for discriminative detection of Cys and application in in vivo imaging

Near-infrared (NIR) fluorescent probes are widely employed in biological detection because of their lower damage to biological samples, low background interference, and high signal-to-noise ratio. Herein, a highly water-soluble NIR probe (NIRHA) based on a hemicyanine skeleton and bearing an acrylate moiety was synthesized. The probe showed high selectivity toward cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH). The probe also had low cytotoxicity and was successfully applied in HeLa cells and mouse experiments. Results of bioimaging experiments indicated that the probe was effective for visualizing endogenous Cys in vitro and in vivo.

Near-infrared (NIR) fluorescent probes are widely employed in biological detection because of their lower damage to biological samples, low background interference, and high signal-to-noise ratio.     

A coumarin Schiff's base two-photon fluorescent probe for hypochlorite in living cells and zebrafish

Selective and sensitive fluorescent probes for ClO⁻ are desirable due to the importance of ClO⁻ in biological processes. Here, a coumarin Schiff''s base, compound 1, has been developed and successfully used as a one- and two-photon fluorescent probe for ClO⁻ with high selectivity. This probe can recognize ClO⁻ with obvious color change from yellow-green to colorless and green to blue fluorescence emission, which can be observed by the naked eye. The properties of low cytotoxicity and good cell permeability allow it to be used for ClO⁻ detection in living cells and zebrafish by both one- and two-photon microscopy imaging. All these results indicate that the compound is a sensitive probe with potential for analysis of ClO⁻ in biological samples. The mechanism by which probe 1 recognizes ClO⁻ is possibly nucleophilic addition followed by hydrolysis.

A coumarin Schiff''s base compound can selectively recognize ClO⁻ and can be successfully applied to the detection of ClO⁻ in living cells and zebrafish by one- and two-photon fluorescence modes.     

A turn-on fluorescent probe with a dansyl fluorophore for hydrogen sulfide sensing

Hydrogen sulfide (H₂S) is a biologically relevant molecule that has been newly identified as a gasotransmitter and is also a toxic gaseous pollutant. In this study, we report on a metal complex fluorescent probe to achieve the sensitive detection of H₂S in a fluorescent “turn-on” mode. The probe bears a dansyl fluorophore with multidentate ligands for coordination with copper ions. The fluorescent “turn-on” mode is facilitated by the strong bonding between H₂S and the Cu(ii) ions to form insoluble copper sulfide, which leads to the release of a strongly fluorescent product. The H₂S limit of detection (LOD) for the proposed probe is estimated to be 11 nM in the aqueous solution, and the utilization of the probe is demonstrated for detecting H₂S in actual lake and mineral water samples with good reproducibility. Furthermore, we designed detector vials and presented their successful application for the visual detection of gaseous H₂S.

H₂S turn on the fluorescence of DNS–Cu complex probe.     

A simple two-photon turn-on fluorescent probe for the selective detection of cysteine based on a dual PeT/ICT mechanism

Herein, a simple two-photon turn-on fluorescent probe, N-(6-acyl-2-naphthayl)-maleimide (1), based on a dual PeT/ICT quenching mechanism is reported for the highly sensitive and selective detection of cysteine (Cys) over other biothiols. The probe was applied in the two-photon imaging of Cys in cultured HeLa cells, excited by a near-infrared laser at 690 nm.

N-(6-acyl-2-naphthayl)-maleimide (1) is a simple two-photon fluorescent probe with selectivity for cysteine, based on a thiol-Michael-addition-transcyclization cascade and dual PeT/ICT quenching mechanism.

Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are structurally similar biothiols, but their biological functions are quite different from one another.^1–6 Among these biothiols, Cys functions as one of the twenty-one amino acids for peptide and protein synthesis, and Cys deficiency is also associated with certain disease symptoms.^7–10 Methods for the selective detection and differentiation of Cys among different biothiols, including high performance liquid chromatography (HPLC),¹¹ capillary electrophoresis,¹² electrochemical assay,¹³ UV-vis spectroscopy,¹⁴ and fluorescence-based methods,^15–17 are important for its biological studies. Recently, fluorescent probes have attracted much attention as vital chemical biology tools due to their high sensitivity, convenient operation, and real-time imaging capabilities.^18–20 A number of Cys-selective fluorescent probes have been reported,²¹ which utilize Cys-selective recognition groups such as aldehydes,^11,22 acrylates,²³ thioesters,²⁴ and electron-deficient aromatic halides^25–27 in their structures. However, many of them have relatively long response times and low sensitivity due to a slow cyclization process. In addition, fluorescent probes with high selectivity for Cys over Hcy are difficult to achieve because they differ by only one methylene group.²⁸ Recently, we reported that N-(N′-butyl-1,8-naphthalimide-4-yl)-maleimide, containing a single maleimide group as the recognition group, is a fast, sensitive, and selective fluorescent probe for Cys based on a dual photo-induced electron transfer (PeT) and photo-induced intramolecular charge transfer (ICT) quenching mechanism.²⁸ Different from many other maleimide-based fluorescent probes that only undergo a PeT mechanism,¹⁵ the additional ICT quenching mechanism keeps the 1,8-naphthalimide (NAP) fluorophore in the thiol-Michael adduct in a low fluorescence emission state due to the strong electron-withdrawing effects of the succinimide group at its 4-position. Then, a subsequent transcyclization step, involving the formation of a six-membered thiomorpholinone ring and cleavage of a five-membered succinimide ring, converts the non-fluorescent thiol-Michael adduct into the major fluorescent product, in which the ICT quenching is removed, resulting in a drastic fluorescence turn-on response.²⁸ A similar transcyclization process and the simultaneous removal of ICT quenching allowed us to design a NAP-based turn-on fluorescent probe for γ-glutamyltranspeptidase²⁹ and a coumarin-based turn-on fluorescent probe with dual recognition groups and dual cyclization for the selective detection of Cys.³⁰ In addition, another NAP-based dual PeT/ICT probe was recently reported by Meka and Heagy for the detection of hydrogen sulfide, although two recognition groups instead of one were adopted in their probe to achieve the dual quenching mechanism.³¹Our previous work and that of other groups has demonstrated that the combination of PeT and ICT mechanisms is particularly suitable for the design of fluorescent probes with a significant fluorescence turn-on response.^30–33 However, many of these probes have a short excitation wavelength in the UV or visible range, which is not optimal for biological applications due to enhanced phototoxicity and/or autofluorescence.^34,35 Considering that two-photon fluorescence imaging has advantages such as the excitation process being carried out by a near-infrared (NIR) laser that has a reduced cell toxicity and low fluorescence background,³⁶ in this work, we aimed to introduce a similar dual PeT/ICT quenching mechanism to the known two-photon fluorophore 6-acyl-2-naphthylamine^37–39 in order to design a simple maleimide-based two-photon fluorescent probe, 1, for the selective detection of Cys over Hcy and GSH. It was also tested to determine whether it is a turn-on fluorescent probe with high sensitivity and selectivity, which reacts with Cys via a fast two-step thiol-Michael addition and transcyclization cascade reaction.²⁸ The structure of probe 1 is shown in Fig. 1. It has a maleimide group at its 2-position, which promotes the PeT quenching effect. It also has an additional electron-withdrawing methylcarbonyl group at its 6-position to ensure a pull–pull ICT quenching effect.Open in a separate windowFig. 1Design rationale of the fluorescent probe 1 for the selective turn-on detection of Cys over Hcy and GSH.Probe 1 was conveniently synthesized from 6-acyl-2-naphthylamine (3)³⁹ in a two-step process with a total yield of 38% (see Scheme S1 in the ESI^†). First, the amine 3 was reacted with maleic anhydride to form the maleic amide acid 4. Then, the amide acid 4 was cyclized to afford the maleimide 1 in the presence of acetic anhydride (see the ESI^† for more details).We then investigated the absorption and fluorescence emission response of the probe towards just 1 equiv. of Cys. The time-dependent absorption spectra upon the addition of 1 equiv. of Cys are shown in Fig. 2a. Probe 1 has a maximum absorption peak at 292 nm. Upon addition of Cys, the maximum absorption peak shifts to 314 nm, a red-shift of 22 nm. Notably, an isosbestic point can be seen at 295 nm after 2 min, indicating the formation of an intermediate within 2 min, which is then converted into the final product. The UV spectral changes supported the presence of a proposed cascade reaction sequence for the fast formation of a thiol-Michael adduct intermediate, which then underwent a relatively slow intramolecular transcyclization process to give the final product. From time-dependent fluorescence emission studies (Fig. 2b), probe 1 was found to have almost no fluorescence emission due to dual PeT and ICT quenching effects. Upon the addition of 1 equiv. of Cys, a drastic turn-on fluorescence response (a >3000 fold increase) was observed at 446 nm (see Fig. S1b in the ESI^†). The fluorescence intensity at 446 nm reached its maximum value after around 30 min indicating that the cascade reaction finished in about 30 min (Fig. 2b, and S2a in the ESI^†). The pseudo-first-order reaction kinetic constant based on the fluorescence enhancement was calculated as 0.123 min⁻¹ (half-time = 5.64 min, Fig. S2b in the ESI^†), indicating an overall fast cascade reaction. Fluorescence titration experiments using an increasing amount of Cys from 0 to 4.0 equiv. over 30 min showed a steady increase in the fluorescence intensity and the maximum intensity was reached at exactly 1.0 equiv. of Cys. Further Cys addition did not increase the fluorescence intensity, indicating that probe 1 reacts with Cys in a 1 : 1 molar ratio (Fig. 2c and S3 in the ESI^†), which was also supported by the Job plot (see Fig. S4 in the ESI^†). From the linear relationship of the fluorescence intensity at 446 nm versus the Cys concentrations, the detection limit of probe 1 (2 μM) for Cys was calculated as 1.4 nM (S/N = 3, Fig. 3d), indicating that 1 is a highly sensitive probe for Cys. Moreover, the probe showed excellent selectivity for the detection of Cys over many other species (Fig. 2e, and S5 in the ESI^†), including the structurally similar thiols Hcy, GSH, and N-acetylcysteine (NAC). The fluorescence intensity at 446 nm for 1 equiv. of Cys was significantly higher (12.2-fold, 9.1-fold, and 17.7-fold, respectively) than that of 10 equiv. of Hcy, GSH, or NAC. To further confirm the reaction mechanism, the reaction product, 2, from the reaction of probe 1 with Cys, was isolated and its structure was confirmed using ¹H nuclear magnetic resonance (NMR) spectroscopy, ¹³C NMR spectroscopy, 2D-rotating-frame nuclear Overhauser effect spectroscopy (ROESY), and high-resolution mass spectrometry (HRMS) (see the ESI for more details^†). The fluorescence quantum yields of probe 1 and product 2 were measured as 0.002 and 0.782, respectively (see the ESI for more details^†). Therefore, the formation of the transcyclization product 2 was determined to be responsible for the observed fluorescence turn-on response. For the other thiols, the transcyclization steps of the thiol-Michael adducts were much slower, resulting in the observed high selectivity. Overall, we have shown here that probe 1 is a highly sensitive and selective turn-on fluorescent probe for Cys.Open in a separate windowFig. 2(a) Time-dependent UV-vis spectra of probe 1 (10 μM) upon the addition of 1 equiv. of Cys (a spectrum was recorded every 2 minutes); (b) time-dependent fluorescence emission spectra of probe 1 (2 μM) upon the addition of 1 equiv. of Cys (a spectrum was recorded every 3 minutes); (c) time-dependent fluorescence emission intensity at 446 nm of probe 1 (2 μM) upon addition of Cys (0 to 4 equiv.); (d) a linear relationship of the fluorescence intensity at 446 nm versus the Cys concentration (0.2–2.0 μM); (e) fluorescence response of probe 1 (2 μM) at 446 nm toward various species in PBS buffer (10 mM, pH 7.4): (1) blank; (2) Cys; (3) Hcy; (4) GSH; (5) NAC; (6) valine; (7) glycine; (8) isoleucine; (9) lysine; (10) leucine; (11) histidine; (12) asparagine; (13) methionine; (14) proline; (15) serine; (16) alanine; (17) threonine; (18) arginine; (19) glutamine; (20) aspartic acid; (21) glutamic acid; (22) tyrosine; (23) tryptophan; (24) phenylalanine; (25) glucose; (26) H₂O₂; (27) Na⁺; (28) K⁺; (29) Ca²⁺; (30) Mg²⁺; (31) Fe³⁺; (32) Fe²⁺; (33); Cu²⁺; (34) Zn²⁺ (All measurements were made in 10 mM PBS buffer, pH 7.4, 25 °C, and λ_ex = 314 nm).Open in a separate windowFig. 3Two-photon fluorescence images (b, e, h, k) of HeLa cells collected at 410–510 nm (blue to cyan-blue, λ_ex = 690 nm), the corresponding bright field view (a, d, g, j), and overlap of the fluorescence channel and the bright field view (c, f, i, l) after different treatments: (a–c) the cells were pretreated with 0.5 mM of N-ethylmaleimide (NEM) for 30 min and then incubated with 10 μM of probe 1 for 30 min; (d–f) cells were first pretreated with 0.5 mM of NEM for 30 min, then after addition of 1 mM of Cys were incubated for 30 min, and finally, incubated with 10 μM of probe 1 for 30 min (scale bar = 10 μm); the conditions for (g–i) and (j–l) were similar to those of (d–f), except that 10 μM of Hcy and 10 μM of GSH were used instead of 10 μM of Cys.Encouraged by the fast, selective, and sensitive in vitro fluorescence response of probe 1 for the detection of Cys, we further evaluated its potential use as a two-photon imaging agent for Cys in biological systems, such as in living cells. The fluorescence response of probe 1 towards Cys at different pH values was evaluated and a suitable pH range for Cys detection was determined to be 7.0 to 10.0, which is a good range for cell imaging applications because physiological conditions have a pH of around 7.4 (see Fig. S7 in the ESI^†). HeLa cells were then pretreated with N-ethylmaleimide (NEM, 0.5 mM) for 30 min to remove the endogenous cellular thiols, and incubated with Cys (1 mM), Hcy (1 mM), or GSH (1 mM), respectively for 30 min to increase the specific thiol levels. The samples were then further incubated with probe 1 (10 μM) for 30 min and were then washed with PBS buffer before two-photon fluorescence cell images and the corresponding bright-field view images were taken (Fig. 3(d–l)). Control images were also taken for samples pretreated with NEM (0.5 mM) and then incubated with probe 1 (10 μM) (Fig. 3a–c). Only cells pretreated with NEM and then Cys showed a distinctive blue fluorescence (Fig. 3e). The above cell imaging studies clearly demonstrated that probe 1 is capable of the selective detection and imaging of intracellular Cys over Hcy and GSH in living cells by two-photon fluorescence imaging with low background fluorescence interference.     

A biotin-guided hydrogen sulfide fluorescent probe and its application in living cell imaging

Hydrogen sulfide (H₂S), a well-known signaling molecule, exerts significant regulatory effects on the cardiovascular and nervous systems. Therefore, monitoring the metabolism of H₂S offers a potential mechanism to detect various diseases. In addition, biotin is significantly used as a targeting group to detect cancer cells exclusively. In this work, a biotin-guided benzoxadizole-based fluorescent probe, NP-biotin, was developed for H₂S detection and evaluated in normal liver cell (LO2) and liver cancer cell (HepG2) lines. Results reveal that NP-biotin can detect cellular H₂S with high sensitivity and selectivity. Moreover, NP-biotin has been confirmed to possess the ability to target cancer cells under the guidance of the biotin group.

A biotin-guided hydrogen sulfide fluorescent probe has been shown clearly to possess the ability to target cancer cells.     

A novel ratiometric fluorescent probe for the selective determination of HClO based on the ESIPT mechanism and its application in real samples

Based on the ESIPT fluorescence mechanism, herein, a novel ratiometric fluorescent probe was designed and synthesized for the detection of HClO. The reaction site of diaminomaleonitrile at the ortho-position of the phenolic hydroxyl group made the probe exhibit a ratiometric fluorescence response towards hypochlorous acid (HClO). The specific sensing mechanism was verified via MS, HPLC and ¹H NMR spectroscopy. Moreover, the probe showed excellent performance with high sensitivity and good selectivity towards HClO in the presence of other reactive oxygen species. In addition, the probe was successfully applied to detect HClO spiked in tap water, river water and diluted human serum with good recoveries.

Based on the ESIPT fluorescence mechanism, herein, a novel ratiometric fluorescent probe was designed and synthesized for the detection of HClO.     

A two-photon lysosome-targeted probe for endogenous formaldehyde in living cells

Formaldehyde (FA) is a gaseous signaling molecule that plays a vital role in various biological processes as well as neurodegenerative diseases. Therefore, it is of great practical significance to develop effective and reliable chemical sensors for the monitoring of endogenous FA. Here, we designed and synthesized a two-photon (810 nm) turn-on chemosensor AMNT (aminomorpholine naphthalimide) that accurately localizes lysosomes in cells for imaging of cellular endogenous FA. The fluorescence emission peak of AMNT was at ∼540 nm, with a slight blue shift (∼528 nm) in response to FA, while the green fluorescence intensity increased. The probe exhibits excellent selectivity for FA among other biological interference species and a fast response time for FA. It is worth mentioning that the probe successfully imaged endogenous FA in cells in two-photon mode, making the probe an effective research tool in the biomedical field to study diseases related to abnormal FA expression.

A turn-on two-photon lysosome-targeted probe based on the ICT mechanism has been synthesized and was successfully used not only to monitor and image formaldehyde exogenously but also endogenously with excellent performance in living cells.     

A novel highly specific and ultrasensitive fluorescent probe for monitoring hypochlorous acid and its application in live cells

Based on the important role of hypochlorous acid (HOCl) in the immune system and numerous physiological processes, the detection of intracellular basal HOCl is of significant interest. In this work, we present a simple thiocarbamate-protected fluorescein fluorescent probe, TCFL, for imaging basal HOCl in live cells. Surprisingly, probe TCFL could determine HOCl quantitatively in a large concentration range with a detection limit of 0.65 nM. In addition, probe TCFL showed excellent specificity for HOCl in the presence of other higher concentration analytes (1 mM). Moreover, probe TCFL exhibited a rapid response (within 3 s) to HOCl and thus could provide a tool for real-time monitoring of HOCl. Importantly, probe TCFL with outstanding response features could be applied for monitoring basal HOCl in live cells.

A highly specific, ultrasensitive, thiocarbamate-caged fluorescein probe was developed for real-time detection of HOCl in live cells.     

A naphthalimide-based turn-on fluorescence probe for peroxynitrite detection and imaging in living cells

Peroxynitrite (ONOO⁻) is a potent biological oxidant that plays a significant role in diverse physiological and pathological processes. A novel fluorescent probe HCA-OH was developed for specific and sensitive detection of peroxynitrite, and displayed a significant fluorescence turn-on signal. With low cytotoxicity and good photostability, the probe HCA-OH could be applied in imaging ONOO⁻ distribution in HepG2 cells and live C. elegans in real time. Therefore, the probe can be a promising tool for imaging in vivo.

A novel fluorescent probe HCA-OH was designed for selective detection of peroxynitrite and imaging in HepG2 cells and C. elegans.     

Development of a turn-on graphene quantum dot-based fluorescent probe for sensing of pyrene in water

Polycyclic aromatic hydrocarbons (PAHs) are potentially harmful pollutants that are emitted into the environment from a range of sources largely due to incomplete combustion. The potential toxicity and carcinogenic effects of these compounds warrants the development of rapid and cost-effective methods for their detection. This work reports on the synthesis and use of graphene quantum dots (GQDs) as rapid fluorescence sensors for detecting PAHs in water. The GQDs were prepared from two sources, i.e. graphene oxide (GO) and citric acid (CA) – denoted GO-GQDs and CA-GQDs, respectively. Structural and optical properties of the GQDs were studied using TEM, Raman, and fluorescence and UV-vis spectroscopy. The GQDs were then applied for detection of pyrene in environmental water samples based on a “turn-off-on” mechanism where ferric ions were used for turn-off and pyrene for turn-on of fluorescence emission. The fluorescence intensity of both GQDs was switched on linearly within the 2–10 × 10⁻⁶ mol L⁻¹ range and the limits of detection were found to be 0.325 × 10⁻⁶ mol L⁻¹ and 0.242 × 10⁻⁶ mol L⁻¹ for GO-GQDs and CA-GQDs, respectively. Finally, the potential application of the sensor for environmental water samples was investigated using lake water and satisfactory recoveries (97–107%) were obtained. The promising results from this work demonstrate the feasibility of pursuing cheaper and greener environmental monitoring techniques.

Graphene quantum dots provide a more environmentally friendly fluorescence sensor for pyrene.     

In vivo micro-vascular imaging and flow cytometry in zebrafish using two-photon excited endogenous fluorescence

Zebrafish has rapidly evolved as a powerful vertebrate model organism for studying human diseases. Here we first demonstrate a new label-free approach for in vivo imaging of microvasculature, based on the recent discovery and detailed characterization of the two-photon excited endogenous fluorescence in the blood plasma of zebrafish. In particular, three-dimensional reconstruction of the microvascular networks was achieved with the depth-resolved two-photon excitation fluorescence (TPEF) imaging. Secondly, the blood flow images, obtained by perpendicularly scanning the focal point across the blood vessel, provided accurate information for characterizing the hemodynamics of the circulatory system. The endogenous fluorescent signals of reduced nicotinamide adenine dinucleotide (NADH) enabled visualization of the circulating granulocytes (neutrophils) in the blood vessel. The development of acute sterile inflammation could be detected by the quantitative counting of circulating neutrophils. Finally, we found that by utilizing a short wavelength excitation at 650 nm, the commonly used fluorescent proteins, such as GFP and DsRed, could be efficiently excited together with the endogenous fluorophores to achieve four-color TPEF imaging of the vascular structures and blood cells. The results demonstrated that the multi-color imaging could potentially yield multiple view angles of important processes in living biological systems.OCIS codes: (170.1470) Blood or tissue constituent monitoring, (180.4315) Nonlinear microscopy, (180.5810) Scanning microscopy     

An acrylate AIE-active dye with a two-photon fluorescent switch for fluorescent nanoparticles by RAFT polymerization: synthesis,molecular structure and application in cell imaging

Recently, AIE-active fluorescent materials have attracted extensive investigation due to their significant applications in the fields of memory devices, photomodulation, information displays, sensors, and biological imaging. In this contribution, a novel acrylate AIE-active dye of TPMA was successfully synthesized by Suzuki coupling and acylation reaction, and belongs to the monoclinic crystal system and P2₁/c space group from the crystal structure analysis, and its fluorescence intensity was stronger with an obvious red shift of emission wavelength as compared with the reported TPB dye. Moreover, the obtained TPMA dye exhibits multi-stimuli-responsivity and a two-photon fluorescent switch with excellent reversibility in the solid state. Subsequently, the corresponding fluorescent polymers of PEG-TM were successfully fabricated via RAFT polymerization of TPMA and PEGMA with a molecular weight of about 25 000 (M_n) and narrow polydispersity index (PDI). From ¹H NMR analysis, when the feeding ratio of TPMA increased to 32.2% from 19.2%, the molar fraction of TPMA in PEG-TM polymers accordingly increased to 32.8% from 19.5%. In water solution, the as-prepared PEG-TM1 polymers would self-assemble into fluorescent organic nanoparticles (FONs) with diameters ranging from 150 to 250 nm, and their maximum emission wavelength presented at 518 nm with obvious AIE phenomena. Moreover, the as-synthesized PEG-TM polymers have prospective application in biological imaging due to their good fluorescence, high water solubility and excellent biocompatibility.

This work reported the synthesis of a novel acrylate AIE-active dye with a reversible two-photon fluorescent switch and its amphiphilic PEG-TM polymers via RAFT polymerization, which are attractive for applications in cell imaging.     

A sensitive BODIPY-based fluorescent probe for detecting endogenous hydroxyl radicals in living cells

The hydroxyl radical (˙OH) has been suggested to play very vital roles in many physiological and pathological processes. However, selective detection of ˙OH is highly challenging owing to its extremely high reactivity and short lifetime. Herein, we designed and synthesized a sensitive “turn on” fluorescent probe for detecting endogenous ˙OH based on a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) platform. The probe had shown good properties including high sensitively, ideal selectively and low cytotoxicity, and was successfully employed to image endogenous hydroxyl radical in living cells.

A novel “turn-on” NIR fluorescent probe was designed and used for monitoring endogenous hydroxyl radical in living cells, which also showed higher selectivity toward hydroxyl radical over other reactive oxygen/nitrogen species.     

A dual responsive fluorescent probe for selective detection of cysteine and bisulfite and its application in bioimaging

A coumarin-based dual responsive fluorescent probe with a simple structure was developed for the detection of Cys and HSO₃⁻. Under simulated physiological conditions, Cou-F displayed an on–off fluorescence response to Cys at 521 nm and an off–on fluorescence response to HSO₃⁻ at 500 nm. Furthermore, Cou-F had the advantages of high sensitivity, strong specificity and rapid response. The detection limits of Cou-F toward Cys and HSO₃⁻ were 0.54 μM and 0.65 μM, respectively. Cou-F enabled high selective responses to Cys and HSO₃⁻ over other biologically related species. The response times of Cou-F toward Cys and HSO₃⁻ were 80 s and 100 s. The fluorescence imaging of Cys and HSO₃⁻ was achieved in living RAW246.7 cells.

A coumarin-based dual responsive fluorescent probe with a simple structure was developed for the detection of Cys and HSO₃⁻.     

A nitroreductase responsive and photoactivated fluorescent probe for dual-controlled tumor hypoxia imaging

Tumor hypoxia has great importance in tumor progression and resistance to antitumor therapies. To precisely monitor tumor hypoxia, a controllable hypoxia imaging method is meaningful but still lacking. Herein, we develop a dual-controlled tumor hypoxia probe (TNB) by introducing a nitrophenol group and methyltetrazine group to the boron-dipyrromethene (BODIPY) dye. The fluorescence-quenching group nitrophenol is reduced to aminophenol by upregulated nitroreductase in hypoxic tumors, and the photocage methyltetrazine is cleaved by light irradiation. Hence the fluorescence of TNB is dual-controlled by hypoxia and photoactivation. We first evaluated TNB''s potential for controllable hypoxia imaging in solution and tumor cells. The fluorescence of TNB under nitroreductase incubation and photoactivation increased more than 60 fold over that which was untreated or only treated with nitroreductase. Furthermore, results validate that TNB possesses photo-controllable activation features in tumor sections. We believe that the probe design based on enzyme and photoactivation responsiveness provides potential for spatiotemporal detection of other biomarkers.

Tumor hypoxia has great importance in tumor progression and resistance to antitumor therapies.     

A highly specific and sensitive turn-on fluorescence probe for hypochlorite detection and its bioimaging applications

Development of high performance fluorescent chemosensors for the detection of ClO⁻in vitro and in vivo is very desirable, because many human diseases are caused by ClO⁻. In this paper, a highly selectivity and sensitive fluorescent probe, EDPC, based on 3-acetylcoumarin, was synthesized, which could respond to ClO⁻ and exhibit an “off–on” mode in Tris–HCl buffer (pH = 7.2, 10 mM, 50% C₂H₅OH) solutions. The detection limit of the EDPC probe for ClO⁻ was as low as 1.2 × 10⁻⁸ M. Moreover, the high selectivity and high sensitivity of EDPC towards ClO⁻ are attributed to the oxidation reaction between the C–O of the coumarin lactone and the C C formed by aldol condensation and the mechanism was further verified using ESI-MS and DFT. Additionally, the concentrations of ClO⁻ in real water were also calculated using the EDPC probe and showed good recovery. Finally, the distribution of intracellular endogenous ClO⁻ was gained by confocal fluorescence microscopy in living HEK293T cells.

A new probe, EDPC, was designed and synthesized for highly specific and sensitive detection of ClO⁻ with a fast response time in living cells.     

A pH tuning single fluorescent probe based on naphthalene for dual-analytes (Mg2+ and Al3+) and its application in cell imaging

In this study, a naphthalene Schiff-base P which serves as a dual-analyte probe for the quantitative detection of Al³⁺ and Mg²⁺ has been designed. The proposed probe showed an ‘‘off–on’’ fluorescent response toward Al³⁺ in ethanol–water solution (1 : 9, v/v, pH 6.3, 20 mM HEPES) over other metal ions and anions, while the detection by the probe could be switched to Mg²⁺ by regulating the pH from 6.3 to 9.4. The sensing mechanisms of P to Al³⁺/Mg²⁺ are attributed to inhibition of the photo-induced electron transfer (PET) process by the formation of 1 : 1 ligand–metal complexes. More importantly, the probe was applied successfully in living cells for the fluorescent cell-imaging of Al³⁺ and Mg²⁺.

In this study, a naphthalene Schiff-base P which serves as a dual-analyte probe for the quantitative detection of Al³⁺ and Mg²⁺ has been designed.     

A ratiometric fluorescent probe for the detection of β-galactosidase and its application

Herein, a coumarin fluorescent probe (Probe 1) was developed for the ratiometric detection of β-galactosidase (β-gal) activity. The detection range was 0–0.1 U mL⁻¹ and 0.2–0.8 U mL⁻¹, and the limit of detection (LOD) was 0.0054 U mL⁻¹. Moreover, the luminous intensity of Probe 1 increased gradually with increase in β-gal activity. It could be observed under 254 nm UV irradiation by the naked eye. Furthermore, this method only required a small amount of sample (20 μL) and a short analytical time (30 min) for the detection of β-gal activity with a low LOD. Probe 1 was successfully used to detect β-gal activity in real fruit samples, and can be applied to the quantitative and qualitative detection of β-gal activity.

A ratiometric fluorescent probe was successfully used as a tool to determine β-galactosidase activity in fruits.