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.     

Fluorescence detection of hydrazine in an aqueous environment by a corrole derivative

Broadly, the industrial applications of hydrazine cause environmental pollution and damage to living organisms because of the high toxicity of hydrazine. Therefore, monitoring hydrazine in the environmental system is of great significance to human health. Here, a new fluorescent probe PC-N ₂ H ₄ based on corrole dye was developed for the detection of hydrazine that had excellent specificity, low limit of detection (LOD: 88 nM), and a wide pH range (6–12). Upon addition of hydrazine into the probe solution, the strong red fluorescence was ‘turned on’ centred at 653 nm with a 127-fold fluorescence intensity enhancement. The detection mechanism was proved using ESI-MS, ¹H NMR, and density functional theoretical calculations. Importantly, the probe was utilized to fabricate a ready-to-use test strip to realize the visual inspection of hydrazine. Furthermore, PC-N ₂ H ₄ was successfully applied for practical detection of hydrazine in water samples with satisfactory recoveries ranging from 96.2% to 105.0%, and indicating that the designed PC-N ₂ H ₄ is highly promising for hydrazine detection in an aqueous environment. Considering the diverse toxicological functions of hydrazine, PC-N ₂ H ₄ was also successfully used to image exogenous hydrazine in HeLa cells and zebrafish.     

A new colorimetric and far‐red fluorescent probe for hydrazine with a large red‐shifted absorption spectrum

Recently, growing attention has been paid to the detection of hydrazine (NH₂NH₂) because of its important roles in industrial chemical and high toxicity to human beings. Herein, we have constructed a new colorimetric and far‐red fluorescent probe containing a receptor of 4‐bromobutanoate to selectively detect hydrazine. The probe could detect hydrazine quantitatively in the range of 40–500 μM with the detection limit of 2.9 μM. In addition, the probe could monitor hydrazine by the ratiometric method with a large (185 nm) red‐shifted absorption spectrum, and the color changes from yellow to blue make it as a ‘naked‐eye’ indicator for hydrazine. Consequently, our proposed probe would be of great benefit for monitoring hydrazine in aqueous solution.     

β-Carboline-Based pH Fluorescent Probe and Its Application for Monitoring Enzymatic Ester Hydrolysis

A novel pH-activatable fluorescent probe, 1-(propan-2-yl)-9H-pyrido[3,4-b]indole-3-carboxylic acid ( L-1 ), based on β-carboline derivatives, has been developed, which displays significant fluorescent response toward pH variation with high selectivity, good photo-stability and favorable pKa value. Moreover, L-1 can dynamically monitor the release of protons during ester hydrolysis reaction in consistent with enzymatic kinetics manner.     

Sensitive detection of acetylcholine based on a novel boronate intramolecular charge transfer fluorescence probe

A highly sensitive and selective fluorescence method for the detection of acetylcholine (ACh) based on enzyme-generated hydrogen peroxide (H₂O₂) and a new boronate intramolecular charge transfer (ICT) fluorescence probe, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-N-butyl-1,8-naphthalimide (BN), was developed. This strategy involves the reaction of ACh with acetylcholinesterase (AChE) to produce choline, which is further oxidized by choline oxidase (ChOx) to obtain betaine and H₂O₂. The enzyme-generated H₂O₂ reacts with BN and results in hydrolytic deprotection of BN to generate fluorescent product (4-hydroxyl-N-butyl-1,8-naphthalimide, ON). Two consecutive linear response ranges allow determining ACh in a wide concentration range with a low detection limit of 2.7 nM (signal/noise = 3). Compared with other fluorescent probes based on the mechanism of nonspecific oxidation, this reported boronate probe has the advantage of no interference from other biologically relevant reactive oxygen species (ROS) on the detection of ACh. This study provides a new method for the detection of ACh with high selectivity and sensitivity.     

Chemolithoautotrophy and mixotrophy in the thiophene-2-carboxylic acid-utilizing Xanthobacter tagetidis

Xanthobacter tagetidis grew as a chemolithotrophic autotroph on thiosulfate and other inorganic sulfur compounds, as a heterotroph on thiophene-2-carboxylic acid, acetic acid and α-ketoglutaric acid, and as a mixotroph on thiosulfate in combination with thiophene-2-carboxylic acid and/or acetic acid. Autotrophic growth on one-carbon organosulfur compounds, and intermediates in their oxidation are also reported. Thiosulfate enhanced the growth yields in mixotrophic cultures, presumably by acting as a supplementary energy source, since ribulose bisphosphate carboxylase was only active in thiosulfate-grown cells and was not detected in mixotrophic cultures using thiosulfate with thiophene-2-carboxylic acid. Bacteria grown on thiophene-2-carboxylic acid also oxidized sulfide, thiosulfate and tetrathionate, indicating these as possible sulfur intermediates in thiophene-2-carboxylic acid degradation. Thiosulfate and tetrathionate were oxidized completely to sulfate and, consequently, did not accumulate as products of thiophene-2-carboxylic acid oxidation in growing cultures. K _m and V _max values for the oxidation of thiosulfate, tetrathionate or sulfide were 13 μM and 83 nmol O₂ min^–1 (mg dry wt.)^–1, respectively; thiosulfate and tetrathionate became autoinhibitory at concentrations above 100 μM. The true growth yield (Y_max) on thiophene-2-carboxylic acid was estimated from chemostat cultures (at dilution rates of 0.034–0.094 h^–1) to be 112.2 g mol^–1, with a maintenance coefficient (m) of 0.3 mmol thiophene-2-carboxylic acid (g dry wt.)^–1 h^–1, and the maximum specific growth rate (μ_max) was 0.116 h^–1. Growth in chemostat culture at a dilution rate of 0.041 h^–1 indicated growth yields [g dry wt. (mol substrate)^–1] of 8.1 g (mol thiosulfate)^–1, 60.9 g (mol thiophene-2-carboxylic acid)^–1, and 17.5 g (mol acetic acid)^–1, with additive yields for growth on mixtures of these substrates. At a dilution rate of 0.034 h^–1, yields of 57.8 g (mol α-ketoglutaric acid)^–1 and 60.7 g (mol thiophene-2-carboxylic acid)^–1 indicated some additional energy conservation from oxidation of the thiophene-sulfur. SDS-PAGE of cell-free preparations indicated a polypeptide (M _r, 21.0 kDa) specific to growth on thiophene-2-carboxylic acid for which no function can yet be ascribed: no metabolism of thiophene-2-carboxylic acid by cell-free extracts was detected. It was shown that X. tagetidis exhibits a remarkable degree of metabolic versatility and is representative of facultatively methylotrophic and chemolithotrophic autotrophs that contribute significantly to the turnover of simple inorganic and organic sulfur compounds (including substituted thiophenes) in the natural environment. Received: 1 July 1997 / Accepted: 3 November 1997     

4-Azido-7-nitrobenzoxadiazole as innovative clickable fluorescence probe for trace and selective quantification of ethinylestradiol in human plasma

Quantification of ethinylestradiol (EE) in biological matrices is challenging as it is a very potent drug with a very low C_max (75 pg.ml⁻¹). Despite the high sensitivity of fluorometric methods, the detection of EE was confined because its structure exhibited very limited fluorescence. Therefore, it must be derivatized first using a fluorogenic agent to produce a more potent fluorescence derivative to achieve the desired ultrasensitive bioanalysis. Here, for the first time, we proposed a promising click fluorescent probe, 4-azido-7-nitrobenzoxadiazole (NBD-AZ) to react with the alkyne group of EE, with the help of copper sulphate and l -ascorbic acid to give a highly fluorescent and stable 1,2,3-triazole derivative. Density functional theory calculation revealed how the triazole formation affects the quantum yield and fluorescence of click reaction product when compared with NBD-AZ. The resulting triazole exhibited a strong signal at a wavelength of 540 nm after excitation at 470 nm. Reaction parameters impacting the intensity of fluorescence were cautiously studied and optimized. The suggested approach has shown outstanding performance, high linearity (25–300 pg.ml⁻¹) and a low detection limit of 7.5 pg.ml⁻¹_. The enhanced sensitivity and selectivity were exploited for analyzing EE in plasma using liquid–liquid extraction for samples cleaning up without interference from any biological components and with a mean % recovery of 100.13 ± 0.39. Accuracy, sensitivity, selectivity, simplicity, and cost–effectiveness make this approach a convincing, promising, and appealing alternative to the reported analytical methods for EE bioanalysis in different matrices.     

A colorimetric fluorescent probe for rapid and specific detection of nitrite

The method of fluorescent probes has been an important technique for detection of nitrite (NO₂^?). As an important inorganic salt, excessive nitrite would threaten humans and the environment. In this paper, a colorimetric fluorescent probe P‐N (1,2‐diaminoanthraquinone) with rapid response and high selectivity, which could detect NO₂^? by visual colour changes and fluorescence spectroscopy is presented. The probe P‐N solution (pH 1) changed from pink to colourless with the addition of NO₂^? and fluorescence intensity at 639 nm clearly decreased. Good linear exists between fluorescence intensities and NO₂^? concentrations for the range 0–16 μM, and the detection limit was 54 nM (based on a 3σ/slope). Moreover, probe P‐N could also detect NO₂^? in real water samples, and results were all satisfactory. Probe P‐N shows great practical application value for detecting NO₂^? in the environment.     

Terbium (III) coordination polymer–copper (II) compound as fluorescent probe for time‐resolved fluorescence ‘turn‐on’ detection of hydrogen sulfide

With recognition of the biological importance of hydrogen sulfide (H₂S), we present a simple and effective fluorescent probe for H₂S using a Tb³⁺ coordination polymer–Cu²⁺ compound (DPA/Tb/G–Cu²⁺). Dipicolinic acid (DPA) and guanosine (G) can coordinate with Tb³⁺ to form a macromolecular coordination polymer (DPA/Tb/G). DPA/Tb/G specifically binds to Cu²⁺ in the presence of coexisting cations, and obvious fluorescence quenching is observed. The quenched fluorescence can be exclusively recovered upon the addition of sulfide, which is measured in the mode of time‐resolved fluorescence. The fluorescence intensities of the DPA/Tb/G–Cu²⁺ compound enhance linearly with increasing sulfide concentrations from 1 to 30 μM. The detection limit for sulfide in aqueous solution is estimated to be 0.3 μM (at 3σ). The DPA/Tb/G–Cu²⁺ compound was successfully applied to sense H₂S in human serum samples and exhibited a satisfactory result. It displays some desirable properties, such as fast detection procedure, high selectivity and excellent sensitivity. This method is very promising to be utilized for practical detection of H₂S in biological and environmental samples.     

Glutathione sulfinamide serves as a selective,endogenous biomarker for nitroxyl after exposure to therapeutic levels of donors

Nitroxyl (HNO) donors exhibit promising pharmacological characteristics for treatment of cardiovascular disorders, cancer, and alcoholism. However, whether HNO also serves as an endogenous signaling agent is currently unknown, largely because of the inability to selectively and sensitively detect HNO in a cellular environment. Although a number of methods to detect HNO have been developed recently, sensitivity and selectivity against other nitrogen oxides or biological reductants remain problematic. To improve selectivity, the electrophilic nature of HNO has been harnessed to generate modifications of thiols and phosphines that are unique to HNO, especially compared to nitric oxide (NO). Given high bioavailability, glutathione (GSH) is expected to be a major target of HNO. As a result, the putative selective product glutathione sulfinamide (GS(O)NH₂) may serve as a high-yield biomarker of HNO production. In this work, the formation of GS(O)NH₂ after exposure to HNO donors was investigated. Fluorescent labeling followed by separation and detection using capillary zone electrophoresis with laser-induced fluorescence allowed quantitation of GS(O)NH₂ with nanomolar sensitivity, even in the presence of GSH and derivatives. Formation of GS(O)NH₂ was found to occur exclusively upon exposure of GSH to HNO donors, thus confirming selectivity. GS(O)NH₂ was detected in the lysate of cells treated with low-micromolar concentrations of HNO donors, verifying that this species has sufficient stability to server as a biomarker of HNO. Additionally, the concentration-dependent formation of GS(O)NH₂ in cells treated with an HNO donor suggests that the concentration of GS(O)NH₂ can be correlated to intracellular levels of HNO.     

Synthesis and application of a “turn on” fluorescent probe for glutathione based on a copper complex of coumarin hydrazide Schiff base derivative

Discrimination and quantification of intracellular biothiols, such as cysteine (Cys), homocysteine (Hcy), glutathione (GSH) under physiological conditions is significant for academic research and disease diagnosis. A new fluorescent probe (complex 1-Cu²⁺) for discriminate detection of GSH was prepared by copper ions coordinate with coumarin carbohydrazide Schiff base derivative 1. In suitable buffer solution (CH₃CN: HEPES = 3:2, v/v) and under appropriate pH condition (pH = 7.2–7.4), the UV–vis spectroscopy experiments showed that compound 1 and copper ion exhibited a 1:1 ratio binding mode and moderate binding ability. Fluorescence quenching of compound 1 was observed when it complexed with Cu²⁺ ions. An obviously fluorescence restoration appeared after addition of GSH to the solution of probe, which also exhibited a highly selectivity relative to cysteine (Cys) and homocysteine (Hcy) in the amino acid competitive experiments. The minimum detection limit was calculated to 0.12 μM by fluorescent method, which was distinctly below the physiological concentration of GSH in live cells. Its biological application to detect the endogenous GSH was further proved by the HepG2 cell fluorescence image test.     

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 triphenylamine-based carbohydrazide hydrazone fluorescent probe for selective detection of hypochlorite and sensing acidic gases

Hypochlorous acid and its hypochlorite are important reactive oxygen species in the body, and are involved in various physiological processes related to immunity; their rapid detection is of great significance. Here, we synthesized a fluorescent probe (TPAS) by condensation of 4-(diphenylamino)benzaldehyde, carbohydrazide, and salicylaldehyde, which can be used for the detection of ClO⁻ in water and sensing of acidic gas in its solid state. The probe showed strong selective recognition of ClO⁻ in acetonitrile and good tolerance to interference ions. There were good linear responses between the intensity of absorbance and fluorescence and the amount of ClO⁻. The TPAS solid and its paper strips can emit red fluorescence when exposed to volatile acidic vapours. After being treated with NH₃, the red fluorescence can be restored to yellow. The response process of TPAS to ClO⁻ and acid gases was characterized using nuclear magnetic resonance, electrospray ionisation mass spectrometry, transmission electron microscopy, and density functional theory calculations. Furthermore, it can be utilized in analyzing ClO⁻ in commercially available bleaching products; the detection results were basically compatible with the labelled values. In addition, the probe is biocompatible and can be applied for imaging ClO⁻ in zebrafish.     

A deep-red xanthene-based highly sensitive fluorescent probe for detection of hypochlorite

As an oxidant, deodorant and bleaching agent, the hypochlorous acid (HClO) and hypochlorite (ClO⁻) are widely used in corrosion inhibitors, textile dyes, pharmaceutical intermediates and in our daily lives. However, excess usage or aberrant accumulation of ClO⁻ leads to tissue damage or some diseases and even cancer. Therefore, it is necessary to develop a fluorescent probe that specifically identifies ClO⁻. In this article, we synthesized a deep-red xanthene-based fluorescent probe (XA-CN). The strong electron deficient group dicyano endows the probe XA-CN deep-red fluorescent emission with high solubility, selectivity and sensitivity for ClO⁻ detection. Studies showed that the probe demonstrated turn-off fluorescence (643 nm) at the presence of ClO⁻ in dimethylsulfoxide/phosphate-buffered saline 1:1 (pH 9) solution with a limit of detection of 1.64 μM. Detection mechanism investigation revealed that the electron deficient group -CN and the hydroxyl group was oxidized into aldehyde or carbonyl groups at the presence of ClO⁻, resulting ultraviolet-visible absorption of the probe blue shifted and turned-off fluorescence. Furthermore, XA-CN was successfully used for the detection of ClO⁻ in tap water samples.     

A novel sialidase-activatable fluorescence probe with improved stability for the sensitive detection of sialidase

Sialidases catalyse the hydrolysis of terminal sialic acid residues of various glycoconjugates and visualising sialidase activity is important for understanding its function in the biological and pathological context. Upon developing a novel fluorescence probe for sialidase with improved fluorescence characteristics based on our previously reported fluorophore, HMRef, an inherent instability of sialic acid conjugates was found to both reduce selectivity and sensitivity. We aimed at increasing the stability of the probes by incorporating a self-immolative spacer with a higher pK_a between the sialic acid residue and HMRef to develop HMRef-S-Neu5Ac, which shows superior stability allowing for the specific detection of sialidase.     

Synthesis and evaluation of a novel ‘off-on’ chemical sensor based on rhodamine B and the 2,5-pyrrolidinedione moiety for selective discrimination of glutathione and its bioimaging in living cells

A new “turn-on” fluorescent probe, RDMBM, based on the rhodamine B dye and the 2,5-pyrrolidinedione moiety was synthesized and characterized. Its sensing behavior toward various amino acids was evaluated via UV–vis and fluorescence spectroscopic techniques. The observed spectral changes showed that RDMBM displays high selectivity and sensitivity toward GSH in MeOH/H₂O (1:2, v/v, pH 7.40, Tris-HCl buffer, 1?mM) solution and that it undergoes 1:1 covalent binding with GSH. More importantly, the hydrogenation and ring-opening of the nitrogen atom in the spirane structure of rhodamine B derivatives were tightly bound to the induction effects of different groups. Furthermore, fluorescence imaging applications demonstrated that RDMBM can be successfully used for the detection of GSH in human breast cancer cells MCF-7.     

A Novel Fluorescent GFP Chromophore Analog-Based Dye for Quantitative PCR

This is the first report describing the possibility of using a green fluorescent protein chromophore synthetic analog, P-HOBDI-BF₂, as a fluorescent dye for a linear hydrolysis probe used in qPCR. The study was carried out on a system for detection of the plant pathogenic fungus Fusarium avenaceum using a plasmid containing translation elongation factor 1α fragment as a template. To estimate fluorogenic properties of P-HOBDI-BF₂, 6-FAM-and BDP-FL-labeled probes were used. It was demonstrated that a synthetic dye based on the P-HOBDI-BF₂ chromophore can be used for labeling hydrolysis probes for qPCR, but fluorescence increase levels for P-HOBDI-BF₂-labeled probes were slightly lower than those for 6-FAM-labeled ones. At the same time, the sensitivity of P-HOBDI-BF₂-based assays remained high, and this fact together with acceptable fluorescence levels suggests that this dye can be considered as an efficient alternative for reporters traditionally used for fluorescence detection in the FAM channel.     

Development of europium(III) complex fluorescent probe for hydrogen sulfide detection and its application in water samples

Hydrogen sulfide (H₂S) is a crucial endogenous signaling component in organisms that is involved in redox homeostasis and numerous biological processes. Modern medical research has confirmed that hydrogen sulfide plays an important role in the pathogenesis of many diseases. Herein, a fluorescent probe Eu(ttbd)₃abt based on europium(III) complex was designed and synthesized for the detection of H₂S. Eu(ttbd)₃abt exhibited significant quenching for H₂S at long emission wavelength (625 nm), with rapid detection ability (less than 2 min), high sensitivity [limit of detection (LOD) = 0.41 μM], and massive Stokes shift (300 nm). Additionally, this probe showed superior selectivity for H₂S despite the presence of other possible interference species such as biothiols. Furthermore, the probe Eu(ttbd)₃abt was successfully applied to detect H₂S in water samples.     

A new Rhodamine B-based ‘on–off’ chemical sensor with high selectivity and sensitivity toward Fe3+ and its imaging in living cells

A new fluorescent chemosensor based on a Rhodamine B and pyrrole conjugate (RBPY) has been designed and synthesized. UV–vis absorption and fluorescence spectroscopic studies show that RBPY exhibits a high selectivity and sensitivity toward Fe³⁺ among many other metal cations in a MeOH/H₂O solution (3:2, v/v, pH 7.10, HEPES buffer, 0.1 mM) by forming a 1:1 complex with Fe³⁺. Furthermore, results reveal that the formation of the RBPY–Fe³⁺ complex is fully reversible in the presence of sulfide anions and could also be used as an efficient sensor for S²⁻. Importantly, fluorescence microscopy experiments further demonstrated that RBPY can be utilized as a fluorescent probe for the detection of Fe³⁺ in human liver (L-02) cells.     

Methylene blue-based 7-nitro-1,2,3-benzoxadiazole NIR fluorescent probe triggered by H2S

A new Methylene blue–based 7-nitro-1,2,3-benzoxadiazole NIR fluorescent probe 3, 7-bis-dimethylamino-10-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)-10H-phenothiazine (leuco-MB-NBD) was designed and synthesized. Leuco-MB-NBD showed high sensitivity and selectivity for H₂S as a fluorescent probe in C₂H₅OH-PBS (9:1, v/v, pH = 7.4) solution, this fluorescent assay showed a linear range of 0–50.0 μM and a LOD (limit of detection) of 0.43 μM. Moreover, the probe leuco-MB-NBD has lower toxicity at low concentrations to HCT-116 cells and can be used for cell imaging. Additionally, Leuco-MB-NBD is triggered by hydrogen sulfide to generate methylene blue, methylene blue which has potential rescuing effects on the mitochondrial activity can act as an antidote against sulfide intoxication.