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 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.     

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.     

‘Turn‐on’ fluorescence sensing of hydrogen peroxide in marine food samples using a carbon dots–MnO2 probe

A ‘turn‐on’ fluorescence method for detection of hydrogen peroxide (H₂O₂) in marine food samples is presented in this article. Using this method, a carbon dots (CDs)–MnO₂ probe was formed in which fluorescence intensity (FI) of CDs was quenched through fluorescence resonance energy transfer by addition of MnO₂ nanosheets. When H₂O₂ was added into the CDs–MnO₂ solution, the MnO₂ nanosheets formed Mn²⁺ ions due to a redox reaction between H₂O₂ and MnO₂ nanosheets, and CD FI was recovered. Under optimized conditions, the detection limit for H₂O₂ was 0.87 μM, and analytical linear range was 4–100 μM. Furthermore, this developed fluorescence sensing system was successfully used with satisfactory results to determine trace H₂O₂ content in marine food samples.     

Determination of 5-Fluorouracil in Plasma with HPLC-Tandem Mass Spectrometry

In this article, we describe a fast and specific method to measure 5FU with HPLC tandem-mass spectrometry. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled 5FU (1,3–¹⁵N₂–5FU) was used as an internal standard. 5FU was measured within a single analytical run of 16 min with a lower limit of detection of 0.05 μM. The intra-assay variation and inter-assay variation of plasma with added 5FU (1 μM, 10 μM, 100 μM) was less then 6%. Recoveries of the added 5FU in plasma were > 97%. Analysis of the 5FU levels in plasma samples from patients with the HPLC tandem mass spectrometry method and a HPLC-UV method yielded comparable results (r² = 0.98). Thus, HPLC with electrospray ionization tandem mass spectrometry allows the rapid analysis of 5FU levels in plasma and could, therefore, be used for therapeutic drug monitoring.     

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.     

Novel diaroylhydrazine ligands as iron chelators: coordination chemistry and biological activity

The search for orally effective drugs for the treatment of iron overload disorders is an important goal in improving the health of patients suffering diseases such as β-thalassemia major. Herein, we report the syntheses and characterization of some new members of a series of N-aroyl-N′-picolinoyl hydrazine chelators (the H₂IPH analogs). Both 1:1 and 1:2 Fe^III:L complexes were isolated and the crystal structures of Fe(HPPH)Cl₂, Fe(4BBPH)Cl₂, Fe(HAPH)(APH) and Fe(H3BBPH)(3BBPH) were determined (H₂PPH=N,N′-bis-picolinoyl hydrazine; H₂APH=N-4-aminobenzoyl-N′-picolinoyl hydrazine, H₂3BBPH=N-3-bromobenzoyl-N′-picolinoylhydrazine and H₂4BBPH=N-(4-bromobenzoyl)-N′-(picolinoyl)hydrazine). In each case, a tridentate N,N,O coordination mode of each chelator with Fe was observed. The Fe^III complexes of these ligands have been synthesized and their structural, spectroscopic and electrochemical characterization are reported. Five of these new chelators, namely H₂BPH (N-(benzoyl)-N′-(picolinoyl)hydrazine), H₂TPH (N-(2-thienyl)-N′-(picolinoyl)-hydrazine), H₂PPH, H₂3BBPH and H₂4BBPH, showed high efficacy at mobilizing ⁵⁹Fe from cells and inhibiting ⁵⁹Fe uptake from the serum Fe transport protein, transferrin (Tf). Indeed, their activity was much greater than that found for the chelator in current clinical use, desferrioxamine (DFO), and similar to that observed for the orally active chelator, pyridoxal isonicotinoyl hydrazone (H₂PIH). The ability of the chelators to inhibit ⁵⁹Fe uptake could not be accounted for by direct chelation of ⁵⁹Fe from ⁵⁹Fe–Tf. The most effective chelators also showed low antiproliferative activity which was similar to or less than that observed with DFO, which is important in terms of their potential use as agents to treat Fe-overload disease.     

Vascular pro-oxidant effects secondary to the autoxidation of gallic acid in rat aorta

Gallic acid autoxidation was monitored by absorption spectroscopy and H₂O₂ production; vascular effects related to the autoxidation process were studied on intact and rubbed aortic rings from WKY rats. Gallic acid autoxidation in an oxygenated physiological salt solution (37°C, pH=7.4) mostly occurred in a 2-h time period. Superoxide anions, H₂O₂ and gallic acid quinones were produced during gallic acid autoxidation. In rings partially precontracted with phenylephrine, 0.1–3 μM gallic acid induced marked and largely endothelium-dependent contractions, 10–30 μM gallic acid induced endothelium-independent contractions and 0.1–0.3 mM gallic acid induced complete, fast-developing, endothelium-independent relaxations. Superoxide dismutase (SOD) shifted the endothelium-dependent gallic acid contractions to the right, and N^G-nitro-l-arginine abolished them. Indomethacin suppressed the endothelium-independent gallic acid contractions, and catalase abolished the endothelium-independent contractions and relaxations. Gallic acid (30 μM) inhibited the relaxant effects of acetylcholine and sodium nitroprusside. In rings maximally precontracted with KCl, 0.1–100 μM gallic acid did not modify the tone, whereas 0.3 mM induced complete, slow-developing, endothelium-independent relaxations. Moreover, 0.3 mM gallic acid induced an irreversible impairment of ring reactivity and the release of lactate dehydrogenase. Catalase and N-acetyl cysteine suppressed the deleterious effects induced by gallic acid in the rings. In conclusion: (a) gallic acid is rapidly and nonenzymatically oxidized in physiological solutions, generating superoxide anions, H₂O₂ and quinones; (b) superoxide anions (by destroying NO) and low H₂O₂ levels (by activating cyclooxygenase) both increase vascular tone; (c) moderate H₂O₂ levels decrease vascular tone; (d) high H₂O₂ and quinone levels cause irreversible relaxations due to cellular damage.     

Discriminative detection of mercury (II) and hydrazine using a dual‐function fluorescent probe

A dual‐function fluorescent probe (Probe 1 ) was developed for discriminative detection of Hg²⁺ and N₂H₄. Probe 1 could discriminatively detect Hg²⁺ and N₂H₄ through two different reaction sites, with the mechanism for Probe 1 for Hg²⁺ depending on a desulfurization reaction and for N₂H₄ depending on the Schiff‐base reaction. N₂H₄ had minimal effect on Hg²⁺ detection in dimethyl sulfoxide (DMSO)/H₂O solution, but Hg²⁺ could interfere with N₂H₄ detection in DMSO/buffer solution. Different concentrations of Hg²⁺ and N₂H₄ resulted in different blue shades of Probe 1 test strips, and the shade of blue was different with the same concentration of Hg²⁺ or N₂H₄, as observed under ultraviolet light at 365 nm wavelength.     

Oxidative stress induces alkaloid production in Uncaria tomentosa root and cell cultures in bioreactors

The effect of oxidative stress on indole alkaloids accumulation by cell suspensions and root cultures of Uncaria tomentosa in bioreactors was investigated. Hydrogen peroxide (H₂O₂, 200 μM) added to U. tomentosa cell suspension cultures in shaken flasks induced the production of monoterpenoid oxindole alkaloids (MOA) up to 40.0 μg/L. In a stirred tank bioreactor, MOA were enhanced by exogenous H₂O₂ (200 μM) from no detection up to 59.3 μg/L. Root cultures grew linearly in shaken flasks with a μ=0.045 days^?1 and maximum biomass of 12.08±1.24 g DW/L (at day 30). Roots accumulated 3α‐dihydrocadambine (DHC) 2354.3±244.8 μg/g DW (at day 40) and MOA 348.2±32.1 μg/g DW (at day 18). Exogenous addition of H₂O₂ had a differential effect on DHC and MOA production in shaken flasks. At 200 μM H₂O₂, MOA were enhanced by 56% and DHC by 30%; while addition of 800 and 1000 μM H₂O₂, reduced by 30–40% DHC accumulation without change in MOA. Root cultures in the airlift reactor produced extracellular H₂O₂ with a characteristic biphasic profile after changing aeration. Maximum MOA was 9.06 mg/L at day 60 while at this time roots reached ca. 1 mg/L of DHC. Intracellular H₂O₂ in root cultures growing in the bioreactor was 0.87 μmol/g DW compared to 0.26 μmol/g DW of shaken flasks cultures. These results were in agreement with a higher activity of the antioxidant enzymes superoxide dismutase and peroxidase by 6‐ and 2‐times, respectively. U. tomentosa roots growing in the airlift bioreactor were exposed to an oxidative stress and their antioxidant system was active allowing them to produce oxindole alkaloids.     

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.     

Hydrogen peroxide fluorescent probe–monitored butyric acid inhibition of the ferroptosis process

Short-chain fatty acids, such as butyrate, play pivotal roles in various physiological processes within the human body. Recent advances in understanding cell death pathways, specifically ferroptosis, have unveiled unique opportunities for therapeutic development. Ferroptosis is linked to iron accumulation and oxidative stress, whereas butyrate has emerged as a cellular protector against oxidative stress, potentially inhibiting ferroptosis. Hydrogen peroxide (H₂O₂) is a key player in oxidative stress, and its monitoring has gained significance in disease mechanisms. We present an innovative fluorescent probe, HOP , capable of dynamically tracking intracellular H₂O₂ levels, enabling spatial and temporal visualization. The probe exhibits high accuracy (limit of detection = 0.14 μM) and sensitivity, paving the way for disease diagnosis and treatment innovations. Importantly, HOP displayed minimal toxicity, making it suitable for cellular applications. Cellular imaging experiments demonstrated its ability to penetrate cells and monitor intracellular H₂O₂ levels accurately. The HOP probe confirmed H₂O₂ as a critical marker in ferroptosis. Our innovative HOP provides a powerful tool for tracking intracellular H₂O₂ levels and offers insights into the modulation of ferroptosis, potentially opening new avenues for disease research and therapeutic interventions.     

Sensitive determination of bromhexine hydrochloride based on its quenching effect on luminol/H2O2 electrochemiluminescence system

In this paper, the electrochemiluminescence (ECL) behavior of luminol/H₂O₂ system in the presence of bromhexine hydrochloride (BrH) was investigated. It was found that the ECL intensity of luminol/H₂O₂ system on a platinum electrode could be intensely quenched by BrH owing to the scavenging superoxide radical ability of BrH, and therefore the sensitive determination of BrH was possible. Under optimal conditions, the quenched ECL intensity was linear to the concentration of BrH in a wide range of 0.08 to 500 μM, with a detection limit of 0.02 μM (signal‐to‐noise ratio (S/N) = 3). This ECL method possessed the merits of rapid, simple and sensitive, and was successfully applied to the BrH quantification in pharmaceutical preparations with satisfactory recoveries of 91.0 ± 4.0 to 106.5 ± 3.4%. The possible route of the quenched ECL of luminol/H₂O₂ in the presence of BrH was also discussed.     

Gas chromatographic–mass spectrometric determination of α-ketoisocaproic acid and [2H7]α-ketoisocaproic acid in plasma after derivatization with N-phenyl-1,2-phenylenediamine

A method for determination of α-ketoisocaproic acid (KIC) and [4,5,5,5,6,6,6-²H₇]α-ketoisocaproic acid ([²H₇]KIC) in rat plasma was developed using gas chromatography–mass spectrometry-selected ion monitoring (GC–MS-SIM). [5,5,5-²H₃]α-Ketoisocaproic acid ([²H₃]KIC) was used as an analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. The keto acids were extracted by cation-exchange chromatography using BondElut SCX cartridge and derivatized with N-phenyl-1,2-phenylenediamine to form N-phenylquinoxalinone derivatives. Quantitation was performed by SIM of the respective molecular ions at m/z 278, 281 and 285 for the derivatives of KIC, [²H₃]KIC and [²H₇]KIC on the electron impact method. The limit of detection was found to be 70 fmol per injection (S/N=3) and the limit of quantitation for [²H₇]KIC was around 50 nM in rat plasma. Endogenous KIC concentrations in 50 μl of rat plasma were measured with relative intra- and inter-day precision of 4.0% and 3.3%, respectively. The intra- and inter-day precision for [²H₇]KIC spiked to rat plasma in the range of 0.1 to 10 μM gave good reproducibility with relative standard deviation (RSD) of 6.5% and 5.4%, respectively. The intra- and inter-day relative errors (RE) for [²H₇]KIC were less than 6.4% and 3.8%, respectively. The method was applied to determine the plasma concentration of [²H₇]KIC after an intravenous administration of [²H₇]KIC in rat.     

A dual-mode optical assay for iron(II) and gallic acid based on Fenton reaction

The hydroxyl radicals ( · OH) produced by the Fenton reaction of iron(II) and hydrogen peroxide (H₂O₂) can oxidize the colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxidized TMB (Ox-TMB), resulting in a decrease in the fluorescence intensity of the reaction system and an increase in ultraviolet absorption. Ox-TMB had a visible absorption peak at 625 nm and a fluorescence peak around 420 nm. When gallic acid (GA) was added to the system, Ox-TMB was reduced to TMB, which made the color of the system disappear and the fluorescence recover. The linear ranges for determination of iron(II) were 0.5–10 μM (fluorometric) and 0.5–20 μM (colorimetric), and the detection limits were 0.25 μM (fluorometric) and 0.28 μM (colorimetric). The linear ranges for determination of GA were 0–80 μM (fluorometric) and 0–60 μM (colorimetric), and the detection limits were 0.31 μM (fluorometric) and 0.8 μM (colorimetric). The results of anti-interference experiments shew that this dual-mode assay had very good selectivity for the determination of iron(II) and GA.     

Salivary hydrogen peroxide produced by holding or chewing green tea in the oral cavity

Tea (Camellia sinensis) catechins have been studied for disease prevention. These compounds undergo oxidation and produce H₂O₂. We have previously shown that holding tea solution or chewing tea leaves generates high salivary catechin levels. Herein, we examined the generation of H₂O₂ in the oral cavity by green tea solution or leaves. Human volunteers holding green tea solution (0.1–0.6%) developed salivary H₂O₂ with C_max = 2.9–9.6 μM and AUC_{0 → ∞} = 8.5–285.3 μM min. Chewing 2 g green tea leaves produced higher levels of H₂O₂ (C_max = 31.2 μM, AUC_{0 → ∞} = 1290.9 μM min). Salivary H₂O₂ correlated with catechin levels and with predicted levels of H₂O₂ (C_{max(expected)} = 36 μM vs C_{max(determined)} = 31.2 μM). Salivary H₂O₂ and catechin concentrations were similar to those that are biologically active in vitro. Catechin-generated H₂O₂ may, therefore, have a role in disease prevention by green tea.     

Relationship between changes in ploidy and stable cellular resistance to hydrogen peroxide

Stable hydrogen peroxide (H₂O₂)-resistant variants of the Chinese hamster ovary HA-1 line have been isolated by culturing cells in progressively increasing concentrations of H₂O₂ (>200 days, in 50–800 μM H₂O₂). Increases in catalase activity in these variant cell lines were shown to correlate with increased H₂O₂ resistance. Stable (>240 days) H₂O₂-resistant cell lines, seven quasidiploid (21–22 chromosomes/cell) and six quasitetraploid (40–44 chromosomes/cell) were clonally isolated from the 800 μM adapted H₂O₂-resistant variants which were heterogeneous with respect to ploidy. The H₂O₂ dose-modifying factors (DMFs) were 3, 5, 8, 13, 15, 26, and 27 for the seven quasidiploid cell lines, and 21, 32, 38, 40, 42, and 49 for the six quasitetraploid cell lines. The mean DMF was 14±10 for the former and 37±10 for the latter. Our data show that on the average the quasitetraploid cell lines were significantly more resistant to H₂O₂-mediated cell killing than the quasidiploid cell lines derived from the same mixed population of 800 μM H₂O₂-adapted cells. When catalase activities (k units/cell) of the HA-1 cells and three of the clonally derived cell lines (two quasidiploid and one quasitetraploid) were determined and plotted vs. H₂O₂–DMF, a positive linear correlation was obtained (correlation coefficient = 0.99). This result was further confirmed when immunoreactive catalase protein/cell was detected by Western blots. Our data show that chronic exposure of cells to H₂O₂ stress (800 μM) was accompanied by increases in quasitetraploid cells within the population. Quasitetraploid cell lines derived from this population demonstrated increased stable H₂O₂-resistance which may be related to stable increases in the expression of catalase.     

Cytochrome P450 oxidase activity and its role in NADPH dependent lipid peroxidation

A comparison is made between microsomal NADPH-dependent H₂O₂ production and malondialdehyde (MDA) formation in rat liver microsomes, obtained from phenobarbital pretreated rats. An increase in H₂O₂ formation was observed during NADPH-dependent disposition (10 min) of 100 μM diazepam (33%) and 2 mM hexobarbital (69%). In contrast orphenadrine (100 μM) and its mono-N-demethylated metabolite tofenacine (100 μM) decreased the H₂O₂ formation (35% and 55%, respectively). However, all these substrates were found to inhibit NADPH-dependent lipid peroxidation (60 min), estimated by measuring MDA formation, to various extents. These data strongly suggest that the oxidase activity of cytochrome P450 (H₂O₂ production) is not involved in a rate-limiting step in NADPH-dependent lipid peroxidation.     

A colorimetric probe for detection of Cu2+ by the naked eye and application in test paper

A novel colorimetric probe RP1 was synthesized using rhodamine derivatives and heterocyclic compounds for the purpose of detecting Cu²⁺. RP1 showed good selectivity, high sensitivity and affinity toward Cu²⁺ over other competing ions in CH₃OH–H₂O (1/1, v/v) solution. Absorbance intensity showed a good linear fit between probe R1 and Cu²⁺ over the concentration range 1–8 μM and the association constant was also calculated to be 1.145 × 10⁵ M^?1. The sensing mechanism was deduced using Job's plot, Fourier transform infrared spectroscopy, and density functional theory studies. In addition, the colorimetric experiment indicated that probe RP1 could be made into test paper to detect Cu²⁺ with a colour change from colourless to pink.     

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.