A ratiometric fluorescent probe for rapid and specific detection of hypochlorite

Hypochlorite (ClO⁻), as a kind of essential reactive oxygen species, plays a crucial role in vitro and in vivo. Here, a ratiometric fluorescent probe ( TPAM ) was designed and constructed for sensing ClO⁻ based on substituted triphenylamine and malononitrile, which exhibited obvious colour transfer from orange to colourless under daylight accompanied by noticeable fluorescence change from red to green in response to ClO⁻. TPAM could effectively monitor ClO⁻ with the merits of fast response, excellent selectivity, high sensitivity and a low detection limit of 0.1014 μM. ¹H NMR, mass spectra and theoretical calculations proved that ClO⁻ caused the oxidation of the carbon–carbon double bond in TPAM , resulting in compound 1 and marked changes in colour and fluorescence. In addition, TPAM was utilized for imaging ClO⁻ in living cells successfully with good photostability and biocompatibility.     

A ratiometric fluorescent probe for sensing hydrogen peroxide based on a hemicyanine–naphthol fluorophore

The synthesis, properties and applications of a water‐soluble boronate‐functioned hemicyanine–naphthol hybrid as a novel ratiometric fluorescent sensor for hydrogen peroxide are presented. The dye displayed remarkable a colour change from pale orange (λ_em = 590 nm) to pink (λ_em = 690 nm) in the presence of H₂O₂, which could be rationalized by the chemoselective H₂O₂‐mediated transformation of arylboronate to phenolate with high selectivity and a fast response (within 2 min). A good linear relationship (R² = 0.9951) was obtained with the H₂O₂ concentration ranging from 0 to 25 μM, with a limit of detection of 0.09 μM according to the signal‐to‐noise ratio (S/N = 3). The advantages of this fluorophore include easy modification, excellent aqueous solubility and superior photostability, and it has been applied to the detection of trace amounts of hydrogen peroxide in water samples. Copyright © 2015 John Wiley & Sons, Ltd.     

A highly sensitive ratiometric fluorescent probe based on fluorescein coumarin for detecting hydrazine in actual water and biological samples

Hydrazine (N₂H₄) is a highly toxic and harmful chemical reagent. Fluorescent probes are simple and efficient tools for sensitive monitoring of N₂H₄ enrichment in the environment, humans, animals, and plants. In this work, a ratiometric fluorescent probe (FP-1) containing coumarin was used for hydrazine detection. The proposed FP-1 probe had a linear detection range of 0–250 μM and a limit of detection (LOD) of 0.059 μM (1.89 ppb). A large red Stokes shift was observed in fluorescence and UV–vis absorption spectra due to the hydrolysis of ester bonds between FP-1 and hydrazine. The hydrazine detection mechanism of FP-1 was also investigated using density functional theory (DFT) calculations. Finally, FP-1 could sensitively and selectively monitor hydrazine in actual water samples and BEAS-2B cells. Therefore, it has great application potential in environmental monitoring and disease diagnosis.     

A ratiometric fluorescent probe for bisulphite anion,employing intramolecular charge transfer

4‐(1H‐benzimidazol‐2‐yl)benzaldehyde (1) has been developed as a new ratiometric fluorescent probe for bisulphite, based on the modulation of intramolecular charge transfer (ICT). Upon mixing with bisulphite in aqueous ethanol, an aldehyde–bisulphite adduct was formed and the ICT of the probe was switched off, which resulted in a ratiometric fluorescence response with an enhancement of the ratios of emission intensities at 368 and 498 nm. The detection range of the probe for bisulphite is in the 2.0–200 µmol/L concentration range and the detection limit is 0.4 µmol/L. Probe 1 produces a ratiometric fluorescent response to bisulphite with a marked emission wavelength shift (130 nm) and displays high selectivity for bisulphite over other anions. Copyright © 2012 John Wiley & Sons, Ltd.     

A simple boronic acid-based fluorescent probe for selective detection of hydrogen peroxide in solutions and living cells

An approach of high sensitivity and selectivity for hydrogen peroxide (H₂O₂) detection is highly demanded due to its important roles in regulating diverse biological process. In this work, we introduced an easily synthesized fluorescent “turn off” probe, BNBD. It is designed based on the core structure of 4-chloro-7-nitrobenzofurazan as a fluorophore and incorporated with a specific H₂O₂-reactive group, aryl boronate, for sensitive and selective detection of H₂O₂. We demonstrated its selectivity by incubating the probe with other types of ROS, and measured the limit of detection of BNBD as 1.8 nM. BNBD is also conducive to H₂O₂ detection at physiological conditions. We thus applied it to detect both exogenous and endogenous changes of H₂O₂ in living cells by confocal microscopy, supporting its future applications to selectively monitor H₂O₂ levels and identify H₂O₂-related physiological or pathological responses from live cells or tissues in the near future.     

Zinc(II)‐selective ratiometric fluorescent probe based on perylene bisimide derivative

A fluorescent probe of N,N′‐biscyclohexyl‐1,7‐di(3‐pyridoxy)‐perylene‐3,4:9,10‐tetracarboxylic acid diimide has been synthesized, and exhibited excellent selectivity and sensitivity for Zn²⁺ over other competing biological cations. The Zn²⁺‐selective fluorescence blue‐shift and enhancing property in conjunction with a visible colorimetric change from orange to green could be observed. With favorable photophysical properties in the visible region, the perylene bisimide derivatives remarkably improved the performance of the probe. Copyright © 2010 John Wiley & Sons, Ltd.     

A new fluorescent pH probe for imaging lysosomes in living cells

A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5–4.1 with the pK_a value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no ‘alkalizing effect’ on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells.     

A ratiometric fluorescent probe for alkaline phosphatase via regulation of excited‐state intramolecular proton transfer

A ratiometric fluorescent probe 2‐(benzimidazol‐2‐yl)phenyl phosphoric acid (1) for alkaline phosphatase (ALP) is designed and synthesized. The method employs the modulation of the excited‐state intramolecular proton transfer (ESIPT) process of 2‐(2'‐hydroxyphenyl)benzimidazole (HPBI) through the hydroxyl group protection/deprotection reaction. Upon phosphorylated with POCl₃, HPBI shows only an emission peak at 363 nm due to the blockage of ESIPT. However, once selective enzymatic hydrolysis with alkaline phosphatase (ALP) in Tris–HCl buffer occurs, the probe 1 is returned to HPBI and the ESIPT process is switched on, which results in a decrease in the emission band at 363 nm and an increase in a new fluorescence peak around 430 nm. The fluorescence intensity ratio at 430 and 360 nm (I₄₃₀/I₃₆₀) increases linearly with the activity of ALP up to 0.050 U/mL and the detection limit is 0.0013 U/mL. The proposed probe shows excellent specificity toward ALP. Copyright © 2015 John Wiley & Sons, Ltd.     

Recognition of thymine by triazine fluorescent probe through intermolecular multiple hydrogen bonding

In order to design a fluorescent probe with a long wavelength and multiple hydrogen bond sites for thymine, 3-(4-chloro-6-methylamino-1,3,5-triazinylamino)-7-dimethylamino-2-methylphenazine is synthesized by subsequently reacting neutral red and methylamine with cyanuric chloride. Its recognition behavior for thymine and its spectroscopic properties in different solvents are studied. The probe's fluorescence can be selectively quenched by thymine instead of guanine, indicating that fully complementary hydrogen bonding plays a key role in such recognition processes. In addition, the recognition mechanism of the probe for thymine and the bathochromic shift of its fluorescence spectra with the solvent polarity are also discussed.     

A colorimetric and ratiometric fluorescent probe for sulfite based on an intramolecular cleavage mechanism

A colorimetric and ratiometric fluorescent sulfite probe, the levulinate of 4‐hydroxynaphthalimide, was successfully synthesized from 4‐hydroxy‐naphthalimide and levulinic acid. Through sulfite‐mediated intramolecular cleavage, the probe was converted into 4‐hydroxynaphthalimide, which when excited at 450 nm, displayed a large Stokes shift due to the intramolecular charge transfer process. The probe exhibited high selectivity and sensitivity towards sulfite over other typical anionic species (F^–, Cl^–, Br^–, I^–, HPO₄^2–, SO₄^2–, NO₃^–, AcO^–, ClO₄^–, HCO₃^–) in HEPES‐buffered solution (25 mm , pH 7.4, 50% acetonitrile, v/v). Copyright © 2013 John Wiley & Sons, Ltd.     

8-Aminoquinoline-based ratiometric zinc probe: Unexpected binding mode and its application in living cells

PMQA, an 8-aminoquinoline-based ratiometric fluorescent sensor, demonstrates the Zn²⁺-induced red-shift of emission (85 nm), and was successfully applied to image zinc in living cells. Compared to 2:1 stoichiometry in PMQA–Zn²⁺, PMQA–Cu²⁺ shows 1:1 composition. Both nitrogen atoms from the aminoquinoline are missing in binding of zinc, while they are critically involved in Cu²⁺ chelation. The structure difference between PMQA–Zn²⁺ and PMQA–Cu²⁺ might shed light in designing novel zinc probes without suffering from copper interference.     

TMA-DPH A fluorescent probe of membrane dynamics in living cells

Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess a variety of particular properties in interaction with intact living cells —specific and rapid incorporation into the plasma membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here for quantitative monitoring of phago-cytosis kinetics, by means of simple fluorescence intensity measurements. This method is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process. It has been tested on mouse bone marrow macrophages.     

Ratiometric fluorimetric and colorimetric probe for sensing and imaging pH changes in living cells

Cell, enzyme, and tissue activity in living organisms are closely related to intracellular pH. Detecting the changes of intracellular pH is important to understanding the physiological and pathological changes in the process of crucial cell metabolism. A pH probe (HTBI) based on hemicyanine was synthesized. The probe solution displayed a marked colour change from yellow to amaranth with the pH increase from neutral to basic; simultaneously, the emission spectra showed a significant red shift. The probe exhibited a ratiometric fluorescence emission (F_586nm/F_542nm) characteristic of pK_a 8.82. As expected, HTBI exhibited high sensitivity and selectivity for pH, fine photostability, reversibility, and low cytotoxicity. Therefore, it would be a very useful tool for measuring the intracellular pH changes.     

A novel fluorescent probe with large Stokes shift for the detection of viscosity changes and its imaging in living cells

As an important cellular microenvironmental parameter, viscosity could reflect the status of living cells. Small molecular fluorescent probes are a vital tool to measure the change of viscosity in living cells. A novel fluorescence probe ZL-1 with a large Stokes shift (in methanol it reached to 153 nm and in glycerol it reached to 125 nm) and excellent sensitivity toward viscosity was developed. The sharp enhancement of the emission intensity for the probe ZL-1 from low viscous methanol to high viscous glycerol indicated that the probe ZL-1 could respond to the viscosity variations. Moreover, the probe ZL-1 has been successfully utilized to detect of the viscosity variations in living cells.     

A fast‐responsive fluorescent probe for sulfite and its bioimaging

A turn‐on fluorescent probe Coumarin‐SO2 based on a nucleophilic addition reaction was developed for the rapid detection of SO₃^2– in aqueous media. The probe Coumarin‐SO2 displays excellent water solubility, fast response, highly sensitivity and highly selectivity over other biological related species. More importantly, living cell imaging experiments indicate the feasibility of using the probe for the detection of SO₃^2– in biological systems. Copyright © 2015 John Wiley & Sons, Ltd.     

A dual-emission fluorescent probe for discriminating cysteine from homocysteine and glutathione in living cells and zebrafish models

Cellular biothiols function crucially and differently in physiological and pathological processes. However, it is still challenging to detect and discriminate thiols within a single one molecule, especially for cysteine (Cys) and homocysteine (Hcy). In this study, a simple two-emission turn-on fluorescent biothiol probe (ICN-NBD) was rationally designed and synthesized through a facile ether bond linking 7-nitro-1,2,3-benzoxadiazole (NBD) and phenanthroimidazole containing a cyano tail. The probe in the presence of Cys elicited two fluorescence responses at 470 nm and 550 nm under excitation at 365 nm and 480 nm, respectively, because of the concomitant generation of both the fluorophore and NBD-N-Cys. In contrast, addition of Hcy and glutathione (GSH) could result in only a blue fluorescence enhancement at 470 nm. which was reasonably attributed to rearrangement from NBD-S-Hcy/GSH to NBD-N-Hcy/GSH as a result of geometrical constraints or solvent effects. Therefore, the fluorescent probe with the NBD scaffold could detect biothiols and simultaneously discriminate Cys from Hcy/GSH in both blue and green channels. The probe has been successfully applied for visualizing biothiols in living cells and zebrafish.     

A novel colorimetric fluorescent probe for detecting hydrazine in living cells and zebrafish

Hydrazine (NH₂NH₂) is a highly toxic organic substance that poses a threat to human health. Monitoring hydrazine with high sensitivity and selectivity is very important. Here, a simple colorimetric fluorescent probe for hydrazine detection, which is a seminaphthorhodafluor derivative containing thiophene-2-carboxylic acid ester reaction site, was rationally constructed. The probe itself exhibits weak fluorescence. The fluorescence is significantly enhanced when hydrazine is added. The probe exhibited a broad linear range (0–1 mM) with satisfactory selectivity and sensitivity (limit of detection 36.4 μM), which turned out to be an excellent fluorescent probe for monitoring hydrazine. Additionally, the probe was used to track hydrazine in living cells and zebrafish with great success, and the detection performance was satisfying. These results proved that this type of fluorescent probe with the thiophene-2-carboxylic acid ester structure can detect hydrazine with higher selectivity and sensitivity.     

A novel 1,8-naphthalimide-based fluorescent chemosensor for the detection of HSA in living cells

Human serum albumin (HSA) is an essential protein for maintaining human health. Accurate detection and quantification of HSA are of great significance for disease diagnosis and biochemical research. Here, a new HSA fluorescent probe BNPE based on the 1,8-naphthalimide fluorophore was designed and synthesized. The probe could recognize HSA through a twisted intramolecular charge transfer mechanism, effectively avoid the interference of most substances, and realize HSA fluorescence imaging in living cells.     

A red-emitting naphthofluorescein-based fluorescent probe for selective detection of hydrogen peroxide in living cells

We report the synthesis, properties, and cellular application of Naphtho-Peroxyfluor-1 (NPF1), a new fluorescent indicator for hydrogen peroxide based on a red-emitting naphthofluorescein platform. Owing to its boronate cages, NPF1 features high selectivity for hydrogen peroxide over a panel of biologically relevant reactive oxygen species (ROS), including superoxide and nitric oxide, as well as excitation and emission profiles in the far-red region of the visible spectrum (>600nm). Flow cytometry experiments in RAW264.7 macrophages establish that NPF1 can report changes in peroxide levels in living cells.     

A near‐infrared fluorescent probe based on boric acid hydrolysis for hydrogen peroxide detection and imaging in HeLa cells

Using the characteristics of hydrogen peroxide that are able to cleave phenyl‐boric acid selectively and efficiently, we here report a dicyanoisophorone‐boric acid ( DCP‐BA )‐based near‐infrared (NIR) fluorescent probe for detection of hydrogen peroxide. This probe shows a rapid, highly selective, and sensitive detection process for hydrogen peroxide with a significant NIR fluorescent turn‐on response that has been successfully applied to detect exogenous hydrogen peroxide in HeLa cells.