Fluorescence based metabisulfite sensing: New aspects of ion sensing by a styryl benzothiazolium dye and understanding nitrite interference.

Detection of specific ions using fluorescent probes has relevance in several areas of therapeutics development and environmental science. Here, we provide new perspectives to the sensing of a styryl benzothiazolium-based fluorescent compound 1 and report that sensing properties are for sulfite ions in general with highest preference for metabisulfite ions (S2O52-) adding to its previously determined role as a bisulfite ion sensor. This probe exhibits its sensing action via an addition reaction in which the styryl double bond gets reduced. The interference studies highlighted that the sequence of addition of nitrite and metabisulfite has a bearing on the overall interference outcome. Spectroscopic studies revealed that the order of preferential sensing of sulfites and sulfide ion is S2O52- > HSO3- > SO32- > S2-. Although this probe displays robust sensing on its own through fluorescence quenching, its fluorescence emission can be enhanced at much lower concentrations in the presence of a G-quadruplex DNA without compromising the outcome of the sensing.

A multifunctional near-infrared fluorescent probe based on benzothiazole structure for fluoride-ion detection.

Fluoride ions (F-) are one of the essential trace elements for the human body and are widely existed in nature. In this study, we present a novel fluorescent probe (YF-SZ-F) designed and synthesized for the specific detection of F-. The probe exhibits high sensitivity, excellent selectivity, and low cytotoxicity, making it a promising tool for biomedical applications. Imaging experiments conducted on zebrafish and Arabidopsis roots demonstrate the probe's remarkable cellular permeability and biocompatibility, laying a solid foundation for its potential biomedical utility. Furthermore, the probe holds potential for practical applications in environmental monitoring and public health through its capability to detect fluoride ions in water samples and via mobile phone software. This multifaceted functionality underscores the broad applicability and significance of the fluorescent probe, not only in scientific research but also in real-world scenarios, contributing to the development of more convenient and precise methods for fluoride detection.

A near-infrared ratiometric fluorescent probe for the sensing and imaging of sulfur dioxide derivatives in living systems.

As a reactive sulfur species, sulfur dioxide (SO2) and its derivatives play crucial role in various physiological processes, which can maintain redox homeostasis at normal levels and lead to the occurrence of many diseases at abnormal levels. So, the development of a suitable fluorescent probe is a crucial step in advancing our understanding of the role of SO2 derivatives in living organisms. Herein, we developed a near-infrared fluorescent probe (SP) based on the ICT mechanism to monitor SO2 derivatives in living organisms in a ratiometric manner. The probe SP exhibited excellent selectivity, good sensitivity, fast response rate (within 50 s), and low detection limit (1.79 µM). In addition, the cell experiment results suggested that the SP has been successfully employed for the real-time monitoring of endogenous and exogenous SO2 derivatives with negligible cytotoxicity. Moreover, SP was effective in detecting SO2 derivatives in mice.

Fluorescent probes based on N-CQDs: For direct detection of food additives STPP and Al3.

Sodium tripolyphosphate (STPP), as one of the many food additives, can cause gastrointestinal discomfort and a variety of adverse reactions when ingested by the human body, which is a great potential threat to human health. Therefore, it is necessary to develop a fast, sensitive and simple method to detect STPP in food. In this study, we synthesized a kind of nitrogen-doped carbon quantum dots (N-CQDs), and were surprised to find that the addition of STPP led to the gradual enhancement of the emission peaks of the N-CQDs, with a good linearity in the range of 0.067-1.96 µM and a low detection limit as low as 0.024 µM. Up to now, there is no report on the use of carbon quantum dots for the direct detection of STPP. Meanwhile, we found that the addition of Al3+ effectively bursts the fluorescence intensity of N-CQDs@STPP solution and has a good linear relationship in the range of 0.33-6.25 µM with a lower detection limit of 0.24 µM. To this end, we developed a fluorescent probe to detect STPP and Al3+. In addition, the probe was successfully applied to the detection of bread samples, which has great potential for practical application.

Dual excitation channel ratiometric fluorescent probes for visual and fluorescent detection of anthrax spore biomarker and tetracycline hydrochloride.

Long-term and excessive use of tetracycline hydrochloride (TC) can lead to its accumulation in the environment, which can cause water contamination, bacterial resistance, and food safety problems. 2,6-Pyridine dicarboxylic acid (DPA) is a major biomarker of Bacillus anthracis spores, and its rapid and sensitive detection is of great significance for disease prevention and counter-terrorism. A bifunctional ratiometric fluorescent nanoprobe has been fabricated to detect DPA and TC. 3,5-dicarboxyphenylboronic acid (BOP) was intercalated into layered europium hydroxide (LEuH) by the ion-exchange method and exfoliated into nanosheets as a fluorescent nanoprobe (PNP). DPA and TC could significantly enhance the red fluorescence of Eu3+ through the antenna effect under different excitation wavelengths, while the fluorescence of BOP can be used as a reference based on the constant emission intensity, realizing ratiometric detection. A low limit of detection (LOD) for the target (DPA: 9.7 nM, TC: 21.9 nM) can be achieved. In addition, visual detection of DPA and TC was realized using color recognition software based on the obvious color changes. This is the first ratiometric fluorescent nanoprobe based on layered rare-earth hydroxide (LRH) for the detection of DPA and TC simultaneously, which opens new ideas in the design of multifunctional probes.

Highly selective and visual detection of vanillin based on fluorescence Cd-MOF sensor in milk powders.

Vanillin is a commonly used synthetic flavoring agent in daily life. However, excessive intake of vanillin may pose risks to human health. Therefore, there is an urgent need for rapid and sensitive detection methods for vanillin. In this study, we developed a fluorescent sensor based on Cd-MOF for the sensitive and selective recognition of vanillin. The presence of vanillin leads to significant fluorescence quenching of Cd-MOF due to competitive absorption and photoinduced electron transfer (PET). The limit of detection was determined to be 39.6 nM, which is the lowest-among the reported fluorescent probes. The sensor was successfully applied for the detection of vanillin in real samples such as powdered milk and milk, with a recovery rate ranging from 96.88 % to 104.83 %. Furthermore, by immobilizing the Cd-MOF probe into a polyvinyl alcohol (PVA) film, we achieved a portable and visual detection composite materials for vanillin.

Cysteine triggered cascade reaction forming coumarin: Visualization of cysteine fluctuation in alcoholic liver disease by a NIR fluorescent probe.

Alcoholic liver disease (ALD) is a chronic toxic liver injury caused by long-term heavy drinking. Due to the increasing incidence, ALD is becoming one of important medical tasks. Many studies have shown that the main mechanism of liver damage caused by large amounts of alcohol may be related to antioxidant stress. As an important antioxidant, cysteine (Cys) is involved in maintaining the normal redox balance and detoxifying metabolic function of the liver, which may be closely related to the pathogenesis of ALD. Therefore, it is necessary to develop a simple non-invasive method for rapid monitoring of Cys in liver. Thus, a near-infrared (NIR) fluorescent probe DCI-Ac-Cys which undergoes Cys triggered cascade reaction to form coumarin fluorophore is developed. Using the DCI-Ac-Cys, decreased Cys was observed in the liver of ALD mice. Importantly, different levels of Cys were monitored in the livers of ALD mice taking silybin and curcumin with the antioxidant effects, indicating the excellent therapeutic effect on ALD. This study provides the important references for the accurate diagnosis of ALD and the pharmacodynamic evaluation of silybin and curcumin in the treatment of ALD, and support new ideas for the pathogenesis of ALD.

Automated In Vivo Phenotypic Screening Platform for Identifying Factors that Affect Cell Regeneration Kinetics.

Various strategies for replacing retinal neurons lost in degenerative diseases are under investigation, including stimulating the endogenous regenerative capacity of Müller Glia (MG) as injury-inducible retinal stem cells. Inherently regenerative species, such as zebrafish, have provided key insights into mechanisms regulating MG dedifferentiation to a stem-like state and the proliferation of MG and MG-derived progenitor cells (MGPCs). Interestingly, promoting MG/MGPC proliferation is not sufficient for regeneration, yet mechanistic studies are often focused on this measure. To fully account for the regenerative process, and facilitate screens for factors regulating cell regeneration, an assay for quantifying cell replacement is required. Accordingly, we adapted an automated reporter-assisted phenotypic screening platform to quantify the pace of cellular regeneration kinetics following selective cell ablation in larval zebrafish. Here, we detail a method for using this approach to identify chemicals and genes that control the rate of retinal cell regeneration following selective retinal cell ablation.

Assessment of Viability of Listeria monocytogenes by Flow Cytometry.

In food industry, Listeria monocytogenes contamination can occur accidentally despite the quality control of raw materials and factory. Decontamination processes or inhibitory effects of ingredients/additives in food products are set up to ensure compliance with hygiene and microbiological criteria. These actions represent stresses for the pathogenic agent, causing fluctuations in its physiological states. Moreover, during these environmental stresses, Listeria monocytogenes can enter in a viable but nonculturable (VBNC) state which is not detected by plate counting but by flow cytometry. This technique coupled with cell staining by fluorescent dyes offers the possibility to assess different physiological states based on different cellular parameters: enzymatic activity, transmembrane integrity, membrane potential, and respiratory activity. In this chapter, we present a method to assess the viability of foodborne pathogens using a double-staining principle based on the assessment of membrane integrity and intracellular esterase activity.

The first ER-targeting flavone-based fluorescent probe for Cys: Applications in real-time tracking in an epilepsy model and food analysis.

Epilepsy is one of the most commonly-seen neurological disorders, and both endoplasmic reticulum stress (ERS) and oxidative stress (OS) have been demonstrated to be associated with epileptic seizures. As one of the three endogenous thiol-containing amino acids, cysteine (Cys) is recognized not only as an important biomarker of various biological processes but also widely used as a significant additive in the food industry. However, the exact role that Cys plays in ERS has not been well answered up to now. In this paper, we reported the first flavone-based fluorescent probe (namely BFC) with nice endoplasmic reticulum (ER)-targeting ability, which was capable of monitoring Cys in a fast response (3.0 min), large stokes shift (130 nm) and low detection limit (10.4 nM). The recognition mechanism of Cys could be attributed to the addition-cyclization reaction involving a Cys residue and an acrylate group, resulting in the release of the strong excited-state intramolecular proton transfer (ESIPT) emission molecule of benzoflavonol (BF). The low cytotoxicity and good biocompatibility of the probe BFC allowed for monitoring the fluctuation of endogenous Cys levels under both ERS and OS processes, as well as in zebrafish models of epilepsy. Quantitative determination of Cys with the probe BFC was also achieved in three different food samples. Additionally, a probe-immersed test strips integrated with a smartphone device was successfully constructed for on-site colorimetric detection of Cys. Undoubtedly, our work provided a valuable tool for tracking Cys levels in both an epilepsy model and real food samples.

A novel structurally modified isophorone fluorescent probe for H2S detection.

Hydrogen sulfide (H2S) has a comprehensive contribution to the normal operation and stability of organisms and is also present in environmental water samples and food deterioration. Thus, it is exceedingly promising and significant to develop a highly sensitive detection technique for tracing H2S. Inspired by this, we designed and synthesized a new fluorescent probe 2-[3-[2-[3-bromo-4-(2,4- dinitrobenzenesulfonate)] ethenyl]-5,5-dimethyl-2-cyclohexen-1-ylidene]propanedinitrile (SP-Br) for hydrosulfide ion detection by introducing bromine atom. Compared with reported H2S probes based on the same fluorescent parent, SP-Br has longer fluorescence emission (λem = 670 nm), shorter response time (3 min), lower detection limit (149 nM), and wider detection range (0-30 nM). SP-Br can emit weak yellow fluorescence, and the emission intensity at 670 nm is considerably enhanced in the presence of hydrosulfide ions. The identification mechanism of hydrosulfide ion by SP-Br was verified by high-resolution mass spectrometry, fluorescence, and UV-vis absorption spectroscopy. In addition, SP-Br has been successfully applied to the monitoring of actual water samples and beer samples and has certain development prospects and value in the fields of environmental pollution and food quality analysis.

Near-infrared fluorescent probe visual detection of Hg2+ and its application in biological system and ecological system.

Mercury ion (Hg2+), a heavy metal cation with greater toxicity, is widely present in the ecological environment and has become a serious threat to human health and environmental safety. Currently, developing a solution to simultaneously visualize and monitor Hg2+ in environmental samples, including water, soil, and plants, remains a great challenge. In this work, we created and synthesized a near-infrared fluorescent probe, BBN-Hg, and utilized Hg2+ to trigger the partial cleavage of the carbon sulfate ester in BBN-Hg as a sensing mechanism, and the fluorescence intensity of BBN-Hg was significantly enhanced at 650 nm, thus realizing the visualization of Hg2+ with good selectivity (detection limit, 53 nM). In live cells and zebrafish, the probe BBN-Hg enhances the red fluorescence signal in the presence of Hg2+, and successfully performs 3D imaging on zebrafish, making it a powerful tool for detecting Hg2+ in living systems. More importantly, with BBN-Hg, we are able to detect Hg2+ in actual water samples, soil and plant seedling roots. Furthermore, the probe was prepared as a test strip for on-site determination of Hg2+ with the assistance of a smartphone. Therefore, this study offers an easy-to-use and useful method for tracking Hg2+ levels in living organisms and their surroundings.

Fluorescent/colorimetric probe for the detection of Cr(â ¥) based on MIL-101(Fe)-NH2 with peroxidase-like activity.

In the present research, Fe-based metal-organic frameworks (MIL-101(Fe)-NH2) nanoparticles were synthesized by simple solvothermal methods and used to assay Cr(Ⅵ). The MIL-101(Fe)-NH2 performs dual functions: the 2-aminoterephthalic acid (NH2-BDC) ligand endows a strong fluorescence emission, and the Fe metal nodes are able to facilitate the oxidation of 3,3',5,5'- tetramethylbenzidine (TMB) directly, resulting in the generation of oxidized-TMB (ox-TMB). Our research results showed that reducing agents such as ascorbic acid (AA) can collapse the structures of MIL-101(Fe)-NH2 because of the reduction of Fe3+ by AA, resulting in release of NH2-BDC. In the presence of Cr(Ⅵ), the fluorescence intensity of the MIL-101(Fe)-NH2 + AA system will be decreased due to the competitive reduction of Fe3+ and Cr(Ⅵ). Nevertheless, Cr(Ⅵ) can significantly accelerate the oxidation of TMB by MIL-101(Fe)-NH2 as it boosts the electron transfer rate between Fe3+ and Fe2+. Therefore, a fluorescent/colorimetric dual-mode platform was developed for the detection of Cr(Ⅵ) with an extensive linear range (7.5-750 µg/L and 13.3-1000 µg/L) as well as a remarkably low detection limit (0.99 µg/L and 1.98 µg/L). This MOF with the ability to release ligands not only provides inspiration for the design of new luminescent materials, but also offers a novel and reliable solution for the detection of Cr(Ⅵ).

Rational design of a high-affinity fluorescent probe for visualizing monitoring the amyloid ß clearance effect of anti-Alzheimer's disease drug candidates.

Beta-amyloid (Aß), the most pivotal pathological hallmark for Alzheimer's disease (AD) diagnosis and drug evaluation, was recognized by TZ095, a high-affinity fluorescent probe developed by rational molecular design. With a TICT mechanism, TZ095 exhibited remarkable affinity with Aß aggregates (Kd = 81.54 nM for oligomers; Kd = 66.70 nM for fibril) and substantial fluorescence enhancement (F/F0 = 44), enabling real-time monitoring of Aß in live cells and nematodes. Significantly, this work used TZ095 to construct a new protocol that can quickly and conveniently monitor Aß changes at the cellular and nematode levels to evaluate the anti-AD efficacy of candidate compounds, and four reported Aß-lowering drug candidates were administrated for validation. Imaging data demonstrated that TZ095 can visually and quantitatively track the effect of Aß elimination after drug treatment. Furthermore, TZ095 excelled in ex vivo histological staining of 12-month-old APP/PS1 mouse brains, accurately visualizing Aß plaques. Integrating CUBIC technology, TZ095 facilitated whole-brain, 3D imaging of Aß distribution in APP/PS1 mice, enabling high-resolution in situ analysis of Aß plaques. Collectively, these innovative applications of TZ095 offer a promising strategy for rapid, convenient, and real-time monitoring of Aß levels in preclinical therapeutic assessments.

Fluorescent probe for imaging N2H4 in plants, food, and living cells and for quantitative detection of N2H4 in soil and water using a smartphone.

Hydrazine is volatile and highly toxic, causing severe harm to water, soil, air, and organisms. Therefore, real-time detection and long-term monitoring of hydrazine are crucial for environmental protection and human health. Herein, an "OFF-ON" fluorescent probe 5-((10-ethyl-2-methoxy-10 H-phenothiazin-3-yl)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (MPD) for hydrazine detection through a nucleophilic addition reaction was developed. MPD could exclusively identify hydrazine through colorimetric and fluorescent dual-channel responses within 30 s, which also demonstrated high sensitivity (detection limit, 12 nM) and a wide pH range (6 -12). The sensing mechanism of MPD was confirmed using theoretical calculations, where fluorescence was emitted following the recognition of hydrazine because of the disappearance of the photoinduced electron transfer (PET) process. Using a smartphone, MPD enabled the quantitative detection of hydrazine in real water samples and sandy soil. Notably, in the process of detecting hydrazine in actual water samples, the establishment of analytical methods and the completion of rapid quantitative detection only required a smartphone and built-in apps. Additionally, we showed that MPD could recognize hydrazine in various environmental samples, including plants, food, hydrazine vapors, and cells. We believe that the fluorescent probe MPD developed in this study and the established smartphone visualization platform will provide a convenient and effective tool for detecting hydrazine in environmental monitoring, food safety assessment, biological system safety, and other fields.

Mechanism study of Dual-Emission ratiometric fluorescent pH-Sensitive carbon quantum dots and its application on mornitoring enzymatic catalysis.

Carbon dots (CQD) have received significant attention as a novel ratiometric fluorescent pH nanoprobe, owing to their favorable optical properties and excellent biocompatibility. Despite their appealing features, the precise mechanism behind the pH-sensitive photoluminescence of CQDs remains to be fully understood. This study endeavors to unravel the mechanism underlying the pH-responsive ratiometric fluorescence in dual-emission CQDs, synthesized through a one-step hydrothermal method using o-phenylenediamine and oxalic acid as precursors. The resultant CQDs exhibit inherent dual-emission at wavelengths of 383 nm and 566 nm, with the ratiometric fluorescence response tailored by the ratio of precursors, providing a robust tool for pH sensing across a range of 2 to 6. Detailed characterizations, including chemical, morphological, and optical analyses, alongside theoretical insights from time-dependent density functional theory (TD-DFT), elucidate the mechanism underlying the pH-dependent luminescence, attributed to the electron cloud transmission between amide and adjacent carboxyl groups on the CQD surface. The superior performance of these CQDs in real-time pH monitoring is demonstrated through their application in glucose oxidase-catalyzed reactions, showcasing their potential as efficient, reliable nanoprobes for biomedical research and diagnostic applications.

ExoJ: an ImageJ2/Fiji plugin for automated spatiotemporal detection and analysis of exocytosis.

Exocytosis is a dynamic physiological process that enables the release of biomolecules to the surrounding environment via the fusion of membrane compartments to the plasma membrane. Understanding its mechanisms is crucial, as defects can compromise essential biological functions. The development of pH-sensitive optical reporters alongside fluorescence microscopy enables the assessment of individual vesicle exocytosis events at the cellular level. Manual annotation represents, however, a time-consuming task, prone to selection biases and human operational errors. Here, we introduce ExoJ, an automated plugin based on ImageJ2/Fiji. ExoJ identifies user-defined genuine populations of exocytosis events, recording quantitative features including intensity, apparent size and duration. We designed ExoJ to be fully user-configurable, making it suitable to study distinct forms of vesicle exocytosis regardless of the imaging quality. Our plugin demonstrates its capabilities by showcasing distinct exocytic dynamics among tetraspanins and vesicular SNAREs protein reporters. Assessment of performance on synthetic data showed ExoJ is a robust tool, capable to correctly identify exocytosis events independently of signal-to-noise ratio conditions. We propose ExoJ as a standard solution for future comparative and quantitative studies of exocytosis.

Fluorescence-Based HTS Assays for Ion Channel Modulation in Drug Discovery Pipelines.

Ion channels represent a druggable family of transmembrane pore-forming proteins with important (patho)physiological functions. While electrophysiological measurement (manual patch clamp) remains the only direct method for detection of ion currents, it is a labor-intensive technique. Although automated patch clamp instruments have become available to date, their high costs limit their use to large pharma companies or commercial screening facilities. Therefore, fluorescence-based assays are particularly important for initial screening of compound libraries. Despite their numerous disadvantages, they are highly amenable to high-throughput screening and in many cases, no sophisticated instrumentation or materials are required. These features predispose them for implementation in early phases of drug discovery pipelines (hit identification), even in an academic environment. This review summarizes the advantages and pitfalls of individual methodological approaches for identification of ion channel modulators employing fluorescent probes (i.e., membrane potential and ion flux assays) with emphasis on practical aspects of their adaptation to high-throughput format.

Innovative Fluorescent Polymers in Niosomal Carriers: A Novel Approach to Enhancing Cancer Therapy and Imaging.

Cancer is anticipated to become the pioneer reason of disease-related deaths worldwide in the next two decades, underscoring the urgent need for personalized and adaptive treatment strategies. These strategies are crucial due to the high variability in drug efficacy and the tendency of cancer cells to develop resistance. This study investigates the potential of theranostic nanotechnology using three innovative fluorescent polymers (FP-1, FP-2, and FP-3) encapsulated in niosomal carriers, combining therapy (chemotherapy and radiotherapy) with fluorescence imaging. These cargoes are assessed for their cytotoxic effects across three cancer cell lines (A549, MCF-7, and HOb), with further analysis to determine their capacity to augment the effects of radiotherapy using a Linear Accelerator (LINAC) at specific doses. Fluorescence microscopy is utilized to verify their uptake and localization in cancerous versus healthy cell lines. The results confirmed that these niosomal cargoes not only improved the antiproliferative effects of radiotherapy but also demonstrate the practical application of fluorescent polymers in in vitro imaging. This dual function underscores the importance of dose optimization to maximize therapeutic benefits while minimizing adverse effects, thereby enhancing the overall efficacy of cancer treatments.

A novel ultrafast and highly sensitive NIR fluorescent probe for the detection of organophosphorus pesticides in foods and biological systems.

The threat posed by organophosphorus pesticides (OPS) to food safety, human health, and the ecological environment is significant, which underscoring the need for the development of new detection tools. We designed and synthesized a NIR fluorescent probe PT-CES which targets carboxylesterase (CES), for the detection of OPS based on the principle of enzyme inhibition. The PT-CES is capable of instantaneous response to CES, exhibiting excellent stability, anti-interference capability. PT-CES realizes the quantitative detection of CES and OPS. It is noteworthy that PT-CES shows excellent stable and accurate detection ability in vegetable pesticide testing. It also enables the monitoring of CES activity in cells and liver tissue. This provides a novel tool for tracking the effect of OPS on CES activity in biological systems. Furthermore, it provides a useful method for ensuring food safety and enhancing pesticide residue analysis.