Research Progress of Fluorescent Probes for Detection of Glutathione (GSH): Fluorophore, Photophysical Properties, Biological Applications.

Intracellular biothiols, including cysteine (Cys), glutathione (GSH), and homocysteine (Hcy), play a critical role in many physiological and pathological processes. Among them, GSH is the most abundant non-protein mercaptan (1-10 mM) in cells, and the change in GSH concentration level is closely related to the occurrence of many diseases, such as Parkinson's disease, Alzheimer's disease, and neurological diseases. Fluorescent probes have attracted much attention due to their advantages of high specificity, high sensitivity, high selectivity, low cost, and high quantum yield. Methods that use optical probes for selective detection of GSH in vitro and in vivo are in high demand. In this paper, we reviewed the most recent five years of research on fluorescence probes for the detection of GSH, including the specific detection of GSH, dual-channel identification of GSH and other substances, and the detection of GSH and other biothiols. According to the type of fluorophore, we classified GSH fluorescent probes into eight classes, including BODIPY, 1,8-Naphthalimide, coumarin, xanthene, rhodamine, cyanine, benzothiazoles, and others. In addition, we roundly discuss the synthesis, detection mechanism, photophysical properties, and biological applications of fluorescent probes. We hope that this review will inspire the exploration of new fluorescent probes for GSH and other related analyses.

Towards portable kits for on-site colorimetric detection of aqueous hydrazine using piperidine-thiophene-barbituric acid push-pull probe.

Owing to the carcinogenicity and environmental risks as well as the wide industrial use of hydrazine, we report herein a colorimetric probe for its ratiometric detection in pure water. The developed probe possesses push-pull architecture with 2-(piperidyn-1-yl)thiophene as the donor, N,N'-dibutylbarbituric as the acceptor, and butadiene as the spacer. In contrast to weak solvatochromic behavior in organic solvents, the probe showed distinct optical photophysical properties in water resulting from the formation of nanoscopic aggregates. The probe underwent pronounced spectral changes upon the addition of hydrazine including an 11.5-fold decrease in absorbance and ~2.4-fold fluorescence quenching. The mechanistic investigation revealed the rapid formation of hydrazone upon the interaction of the probe with hydrazine via retro-Knoevenagel reaction as confirmed experimentally and corroborated with DFT calculations. The induced colorimetric and fluorometric changes were utilized in hydrazine sensing with excellent selectivity over other biologically relevant analytes with a detection limit of 0.76 µM in aqueous media. The practical utility of the probe was assessed in real-life natural water samples, while we have also developed a cost-effective portable kit for the on-site hydrazine detection both in the solution and vapor phases.

Determination of ligand selectivity to G-tetrad through an AMCA fluorescence quenching approach.

The selective binding of ligand molecules towards the 5' and 3' ends of G-quadruplex (G4) may differentially affect the physiological function of G4s. However, there is still a lack of sensitive and low-cost approaches to accurately measure the binding preference of ligands on G4s, although multiple ways have been developed to evaluate the interaction between ligands and G4s. Here, we propose a new protocol named G4-AFQ to test the selectivity of ligands towards the two terminal G-tetrads of G4s. In this protocol, the fluorophore AMCA is respectively modified at the 5' or 3' end of G4, and which end of AMCA fluorescence is quenched means that the ligand binds to the G-tetrad at that end. Through G4-AFQ, the affinity constant of ligands towards the binding site can also be obtained. Compared with the commonly used nuclear magnetic resonance (NMR) method, G4-AFQ is more convenient, sensitive, cost-effective, and suitable for the measurement of the vast majority of G4 ligands, with a great potential for widespread application.

Generic Reporter Sets for Colorimetric Multiplex dPCR Demonstrated with 6-Plex SNP Quantification Panels.

Digital PCR (dPCR) is a powerful method for highly sensitive and precise quantification of nucleic acids. However, designing and optimizing new multiplex dPCR assays using target sequence specific probes remains cumbersome, since fluorescent signals must be optimized for every new target panel. As a solution, we established a generic fluorogenic 6-plex reporter set, based on mediator probe technology, that decouples target detection from signal generation. This generic reporter set is compatible with different target panels and thus provides already optimized fluorescence signals from the start of new assay development. Generic reporters showed high population separability in a colorimetric 6-plex mediator probe dPCR, due to their tailored fluorophore and quencher selection. These reporters were further tested using different KRAS, NRAS and BRAF single-nucleotide polymorphisms (SNP), which are frequent point mutation targets in liquid biopsy. We specifically quantified SNP targets in our multiplex approach down to 0.4 copies per microliter (cp/µL) reaction mix, equaling 10 copies per reaction, on a wild-type background of 400 cp/µL for each, equaling 0.1% variant allele frequencies. We also demonstrated the design of an alternative generic reporter set from scratch in order to give detailed step-by-step guidance on how to systematically establish and optimize novel generic reporter sets. Those generic reporter sets can be customized for various digital PCR platforms or target panels with different degrees of multiplexing.

Conceptually innovative fluorophores for functional bioimaging.

Fluorophore chemistry is at the forefront of bioimaging, revolutionizing the visualization of biological processes with unparalleled precision. From the serendipitous discovery of mauveine in 1856 to cutting-edge fluorophore engineering, this field has undergone transformative evolution. Today, the synergy of chemistry, biology, and imaging technologies has produced diverse, specialized fluorophores that enhance brightness, photostability, and targeting capabilities. This review delves into the history and innovation of fluorescent probes, showcasing their pivotal role in advancing our understanding of cellular dynamics and disease mechanisms. We highlight groundbreaking molecules and their applications, envisioning future breakthroughs that promise to redefine biomedical research and diagnostics.

Comparative analysis of new mScarlet-based red fluorescent tags in Caenorhabditis elegans.

One problem that has hampered the use of red fluorescent proteins in the fast-developing nematode Caenorhabditis elegans has been the substantial time delay in maturation of several generations of red fluorophores. The recently described mScarlet-I3 protein has properties that may overcome this limitation. We compare here the brightness and onset of expression of CRISPR/Cas9 genome-engineered mScarlet, mScarlet3, mScarlet-I3, and GFP reporter knock-ins. Comparing the onset and brightness of expression of reporter alleles of C. elegans golg-4, encoding a broadly expressed Golgi resident protein, we found that the onset of detection of mScarlet-I3 in the embryo is several hours earlier than older versions of mScarlet and comparable to GFP. These findings were further supported by comparing mScarlet-I3 and GFP reporter alleles for pks-1, a gene expressed in the CAN neuron and cells of the alimentary system, as well as reporter alleles for the pan-neuronal, nuclear marker unc-75. Hence, the relative properties of mScarlet-I3 and GFP do not depend on cellular or subcellular context. In all cases, mScarlet-I3 reporters also show improved signal-to-noise ratio compared to GFP.

Comparison of EV characterization by commercial high-sensitivity flow cytometers and a custom single-molecule flow cytometer.

High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross-calibrated, hard-dyed beads were ∼10 PE/∼80 nm EV diameter for CytoFLEX and ∼10 PEs/∼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di-8-ANEPPS+/PE+ particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin-labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super-resolution/single-molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter-platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter-instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi-quantitative LOD values for other flow cytometers.

Benzo-fused BOPAM Fluorophores: Synthesis, Post-functionalization, Photophysical Properties, and Acid Sensing Applications.

A novel category of asymmetric boron chromophores with the attachment of two BF2 moieties denoted as BOPAM, has been successfully synthesized via a one-pot three-step reaction starting from N-phenylbenzothioamide. This synthetic route results in the production of [a] and [b]benzo-fused BOPAMs along with post-functionalization of the [a]benzo-fused BOPAMs. The photophysical properties of these compounds have been systematically investigated through steady-state absorption and fluorescence emission measurements in solvents at both ambient and cryogenic temperatures, as well as in the solid state. Computational methods have been employed to elucidate the emissive characteristics of the benzo-fused BOPAMs, revealing distinctive photophysical attributes, including solvent-dependent fluorescence intensity. Remarkably, certain BOPAM derivatives exhibit noteworthy photophysical phenomena, such as the induction of off-on fluorescence emission under specific solvent conditions and the manifestation of intermolecular charge transfer states in solid-state matrices. Through post-functionalization strategies involving the introduction of electron-donating groups onto the [a]benzo-fused BOPAM scaffold, an intramolecular charge transfer (ICT) pathway is activated, leading to substantial fluorescence quenching via non-radiative decay processes. Notably, one [a]benzo-fused BOPAM variant exhibits a pronounced fluorescence enhancement upon exposure to acidic conditions, thereby underscoring its potential utility in pH-sensing applications.

Ligand-selective turn-off sensing, harvesting and post-adsorptive use of Dy(III) and Yb(III) by intrinsically fluorescent flower-shaped Gum Acacia-grafted hydrogels.

Rare earth metals (REMs), such as Dysprosium (Dy) and Ytterbium (Yb), have experienced unprecedented demand in recent times due to their applications in high-end technologies. REMs are found only in select geographic locations placing tremendous economic constraints on their use. In this work, we have developed Gum Acacia-grafted hydrogels (GmAc-FluoroTerPs) that are capable of selective detection and capture of Dy and Yb. The intrinsically blue fluorescent polymer hydrogel GmAc-FluoroTerP has been optimized for Dy(III) and Yb(III) specific quenching, enabling limit of detection of the REMs at 0.13 nM and 60.8 pM, respectively. A comprehensive structural characterization of the fluorescent hydrogel has been performed via NMR, FTIR, XPS, EPR, TGA, XRD, TEM, SEM, EDX, TCSPC, and DLS. In addition to an in situ generated fluorophore, GmAc-FluoroTerP displays a distinctive aggregation induced emission enhancement in mixed solvents. The complexation of Dy(III)/Yb(III) with GmAc-FluoroTerP hydrogel has been characterized by XPS, TCSPC, and logic gate analyses, and the adsorptive capacity for Dy(III) and Yb(III) are found to be best reported till date as 125.57 mg g-1 and 102.27 mg g-1, respectively. Desorption at acidic pH allows recovery of the REMs. We also report semiconducting behaviour of the native fluorescent hydrogel, that is enhanced upon adsorptive capture of Dy(III) and Yb(III), with calculated band gaps at 1.37, 0.77, and 0.49 eV, respectively. The convergent sensing, capture, and reuse of Dy(III) and Yb(III) presented in this work promises a hitherto unreported template for application on other REMs.

Substituted Maleimides: Self-Reportable Linkers and Tags in Bioconjugation, Materials Science, and Nanotechnology.

Substituted maleimides are customisable fluorescent linkers and probes with adaptable reactivity and optical properties. Their compact nature and tunability has led to their use in various applications. For example, their solvatochromic properties offer real-time feedback on linking chemistry and environmental changes, essential for applications in material labelling, drug delivery, and nanoparticle functionalisation. This review focusses on developing, synthesising, and modifying substituted maleimides as environment-sensitive fluorescent linkers and probes. It delves into their photophysical dynamics and strategic applications, highlighting their significant contributions to macromolecule conjugation and the development of self-reporting materials.

Melanocortin-4 Receptor PLC Activation Is Modulated by an Interaction with the Monocarboxylate Transporter 8.

The melanocortin-4 receptor (MC4R) is a key player in the hypothalamic leptin-melanocortin pathway that regulates satiety and hunger. MC4R belongs to the G protein-coupled receptors (GPCRs), which are known to form heterodimers with other membrane proteins, potentially modulating receptor function or characteristics. Like MC4R, thyroid hormones (TH) are also essential for energy homeostasis control. TH transport across membranes is facilitated by the monocarboxylate transporter 8 (MCT8), which is also known to form heterodimers with GPCRs. Based on the finding in single-cell RNA-sequencing data that both proteins are simultaneously expressed in hypothalamic neurons, we investigated a putative interplay between MC4R and MCT8. We developed a novel staining protocol utilizing a fluorophore-labeled MC4R ligand and demonstrated a co-localization of MC4R and MCT8 in human brain tissue. Using in vitro assays such as BRET, IP1, and cAMP determination, we found that MCT8 modulates MC4R-mediated phospholipase C activation but not cAMP formation via a direct interaction, an effect that does not require a functional MCT8 as it was not altered by a specific MCT8 inhibitor. This suggests an extended functional spectrum of MCT8 as a GPCR signaling modulator and argues for the investigation of further GPCR-protein interactions with hitherto underrepresented physiological functions.

Fluorescence-Based Multimodal DNA Logic Gates.

The use of DNA structures in creating multimodal logic gates bears high potential for building molecular devices and computation systems. However, due to the complex designs or complicated working principles, the implementation of DNA logic gates within molecular devices and circuits is still quite limited. Here, we designed simple four-way DNA logic gates that can serve as multimodal platforms for simple to complex operations. Using the proximity quenching of the fluorophore-quencher pair in combination with the toehold-mediated strand displacement (TMSD) strategy, we have successfully demonstrated that the fluorescence output, which is a result of gate opening, solely relies on the oligonucleotide(s) input. We further demonstrated that this strategy can be used to create multimodal (tunable displacement initiation sites on the four-way platform) logic gates including YES, AND, OR, and the combinations thereof. The four-way DNA logic gates developed here bear high promise for building biological computers and next-generation smart molecular circuits with biosensing capabilities.

Shining a Light on Venom-Peptide Receptors: Venom Peptides as Targeted Agents for In Vivo Molecular Imaging.

Molecular imaging has revolutionised the field of biomedical research by providing a non-invasive means to visualise and understand biochemical processes within living organisms. Optical fluorescent imaging in particular allows researchers to gain valuable insights into the dynamic behaviour of a target of interest in real time. Ion channels play a fundamental role in cellular signalling, and they are implicated in diverse pathological conditions, making them an attractive target in the field of molecular imaging. Many venom peptides exhibit exquisite selectivity and potency towards ion channels, rendering them ideal agents for molecular imaging applications. In this review, we illustrate the use of fluorescently-labelled venom peptides for disease diagnostics and intraoperative imaging of brain tumours and peripheral nerves. Finally, we address challenges for the development and clinical translation of venom peptides as nerve-targeted imaging agents.

A Fluorometric Method for Zinc Estimation: Applications in the Estimation of Plasma Zinc and in Assessing Zinc Bioaccessibility from Rice.

Sensitive and precise methods for the estimation of zinc (Zn) in biological fluids and foods are important tools in understanding the various aspects related to Zn nutrition. Estimation of serum/plasma Zn was suggested for assessing the population Zn status while assessing the bioaccessible Zn following simulated gastrointestinal digestion of crop varieties such as rice helps in ranking the crops. Atomic absorption spectrometry (AAS) or inductively coupled plasma-mass spectrometry (ICP-MS) are widely used for Zn estimation. Zinquin, a Zn fluorophore, has been used for the localization of cellular Zn and labile Zn pools in biological fluids with extremely high sensitivity. However, it was not tested for its use in Zn estimation in serum/plasma or in assessing the Zn bioaccessibility from foods. In the current study, we demonstrate a sensitive method for Zn estimation in human plasma and validate it against the reference method (AAS) by comparing the paired measurements on the same samples. The method-related bias between zinquin with AAS was negligible (0.48 µg/dL), and the precision (CV) of the assay was < 5% across different Zn concentrations. In addition, we also demonstrated the utility of zinquin assay in estimating the bioaccessibility of Zn from rice varieties and showed that the method is again comparable to AAS. The zinquin method is capable of discriminating the differences in zinc bioaccessibility between polished and unpolished rice varieties. In the context of required low plasma volume (100 µL Vs 400 µL), excellent comparability of the results with the reference method and analytical simplicity could be particularly useful.

Single-Molecule RNA Imaging in Live Cells with an Avidity-Based Fluorescent Light-Up Aptamer biRhoBAST.

Observing individual RNA molecules provides valuable insights into their regulation, interactions with other cellular components, organization, and functions. Although fluorescent light-up aptamers (FLAPs) have recently shown promise for RNA imaging, their wider applications have been mostly hindered by poor brightness and photostability. We recently developed an avidity-based FLAP known as biRhoBAST that allows for single-molecule RNA imaging in live or fixed cells and tracking individual mRNA molecules in living cells due to its excellent photostability and high brightness. Here, we present step-by-step detailed protocols starting from cloning biRhoBAST repeats into the target RNA sequence, to imaging dynamics of single mRNA molecules. Additionally, we address the validation of single-molecule imaging experiments through single-molecule fluorescence in situ hybridization (smFISH) and colocalization studies.

Applying Flow Virometry to Study the HIV Envelope Glycoprotein and Differences Across HIV Model Systems.

The HIV envelope glycoprotein (Env) is a trimeric protein that facilitates viral binding and fusion with target cells. As the sole viral protein on the HIV surface, Env is important both for immune responses to HIV and in vaccine designs. Targeting Env in clinical applications is challenging due to its heavy glycosylation, high genetic variability, conformational camouflage, and its low abundance on virions. Thus, there is a critical need to better understand this protein. Flow virometry (FV) is a useful methodology for phenotyping the virion surface in a high-throughput, single virion manner. To demonstrate the utility of FV to characterize Env, we stained HIV virions with a panel of 85 monoclonal antibodies targeting different regions of Env. A broad range of antibodies yielded robust staining of Env, with V3 antibodies showing the highest quantitative staining. A subset of antibodies tested in parallel on viruses produced in CD4+ T cell lines, HEK293T cells, and primary cells showed that the cellular model of virus production can impact Env detection. Finally, in addition to being able to highlight Env heterogeneity on virions, we show FV can sensitively detect differences in Env conformation when soluble CD4 is added to virions before staining.

Exploring the twin potential of nanostructured TiO2:SeO2 as a promising visible light photocatalyst and selective fluorosensing platform.

The present work describes the development of TiO2/SeO2 nanostructure as a potential candidate for visible light photocatalysis as well as selective fluorophore for the sensing of picric acid. The obtained nanostructure consists of uniform globular nanoparticles having approximate size of 170 nm and possess an optical band gap of 2.33 eV with absorption maxima at 473 nm. The photocatalyst was able to achieve 90.34% degradation efficiency for 2, 4-dichlorophenol (2,4-DCP) with rate constant of 0.0046 min-1 in the visible region. Further the nanostructure was able to serve as a selective fluorophore for sensing of Picric acid portraying more than 95% of fluorescence quenching when the concentration of PA is 10-4 M. Theoretical calculations indicate the interaction of organic pollutants with the nanostructure and reveal that both picric acid (- 66.21 kcal/mol) and 2,4-DCP (- 12.31 kcal/mol) possess more negative binding energy values demonstrating a strong interaction of both with the nanostructure, making it suitable for the degradation as well as sensing of organic pollutants. Thus this study explains the potential of prepared catalyst for waste water treatment.

Active Hydrogen Free, Z-Isomer Selective Isatin Derived "Turn on" Fluorescent Dual Anions Sensor.

An efficient and anions fluorescence "on-off" sensor of 1-(prop-2-yn-1-yl)-3-(quinolin-3-ylimino)indolin-2-one (PQI) has been developed for the selective sensing of dual anions of F- and NO3- ions in aqueous medium. Active hydrogen and Lewis acidic binding sites free, Z- isomer of isatin based π-conjugated quinoline exhibited excellent sensing activity against F- and NO3- ions in UV light. The fluorescence turns on the process accomplished via the PET "on-off" mechanism. The interaction between probe molecule and anions is thought to be a non-covalent interaction of the low electron density covalently bonded N-methylene moiety of propargyl isatin (-N-CH2-) of probe molecule with F- ion and the terminal acidic proton of propargyl group of isatin (-C≡C-H) with NO3- ions. The modes of anions binding with PQI and plausible mechanisms are proposed by 1H and 13C NMR titrations. The selectivity of anions sensing may be offered by the bucked structure of the Z-isomer. The calculated association constant values for PQI and F- and NO3- are ions 2.5 × 104 M-1 and 2.2 × 103 M-1, respectively, indicating strong binding interaction between the PQI and anions. The association nature of anions and probes was analyzed by a Jobs plot and the finding indicates both F- and NO3- ions are in 1:1 complexation with PQI. The limit of detection (LOD) of the probe with F- and NO3- ions is calculated and is to be 6.91 × 10-7 M and 9.93 × 10-7 M, respectively. The proposed PQI fluorophore possesses a low limit of detection (LOD) for both F- and NO3- ions which is within the WHO prescribed detection limit.

Multiplexing LAMP Assays: A Methodological Review and Diagnostic Application.

The loop-mediated isothermal amplification (LAMP) technique is a great alternative to PCR-based methods, as it is fast, easy to use and works with high sensitivity and specificity without the need for expensive instruments. However, one of the limitations of LAMP is difficulty in achieving the simultaneous detection of several targets in a single tube, as the methodologies that allow this rely on fluorogenic probes containing specific target sequences, complicating their adaptation and the optimization of assays. Here, we summarize different methods for the development of multiplex LAMP assays based on sequence-specific detection, illustrated with a schematic representation of the technique, and evaluate their practical application based on the real-time detection and quantification of results, the possibility to visualize the results at a glance, the prior stabilization of reaction components, promoting the point-of-care use, the maximum number of specific targets amplified, and the validation of the technique in clinical samples. The various LAMP multiplexing methodologies differ in their operating conditions and mechanism. Each methodology has its advantages and disadvantages, and the choice among them will depend on specific application interests.

Enhanced optical imaging and fluorescent labeling for visualizing drug molecules within living organisms.

The visualization of drugs in living systems has become key techniques in modern therapeutics. Recent advancements in optical imaging technologies and molecular design strategies have revolutionized drug visualization. At the subcellular level, super-resolution microscopy has allowed exploration of the molecular landscape within individual cells and the cellular response to drugs. Moving beyond subcellular imaging, researchers have integrated multiple modes, like optical near-infrared II imaging, to study the complex spatiotemporal interactions between drugs and their surroundings. By combining these visualization approaches, researchers gain supplementary information on physiological parameters, metabolic activity, and tissue composition, leading to a comprehensive understanding of drug behavior. This review focuses on cutting-edge technologies in drug visualization, particularly fluorescence imaging, and the main types of fluorescent molecules used. Additionally, we discuss current challenges and prospects in targeted drug research, emphasizing the importance of multidisciplinary cooperation in advancing drug visualization. With the integration of advanced imaging technology and molecular design, drug visualization has the potential to redefine our understanding of pharmacology, enabling the analysis of drug micro-dynamics in subcellular environments from new perspectives and deepening pharmacological research to the levels of the cell and organelles.