Search Results (339)

Hydrogen sulfide (H₂S) has emerged as an important gaseous signaling molecule involved in numerous physiological and pathological processes, including redox regulation, inflammation, and cellular signaling. Accurate detection of H₂S in biological systems is therefore essential for understanding its roles in health and disease. Among various fluorescent platforms, the 1,8-naphthalimide scaffold has attracted considerable attention due to its unique photophysical properties, structural tunability, and high sensitivity to substituent modification. This review summarizes recent advances in the development of 1,8-naphthalimide-based fluorescent probes for H₂S detection. The fundamental photophysical mechanisms associated with the 1,8-naphthalimide fluorophore, including intramolecular charge transfer (ICT), photoinduced electron transfer (PET), and Förster resonance energy transfer (FRET), are discussed in relation to probe design. Emphasis is placed on reaction-based sensing strategies, highlighting organic reactions that enable selective recognition of H₂S, such as reduction, nucleophilic addition, nucleophilic substitution, and intramolecular cyclization. Representative probes are summarized and compared with respect to sensing mechanisms and photophysical behavior. In addition, the current challenges of 1,8-naphthalimide-based H₂S probes are discussed. We hope this review will provide useful perspectives for the development of efficient 1,8-naphthalimide-based fluorescent probes for H₂S detection. Full article

The extensive use of pesticides in modern agriculture has resulted in their persistent accumulation in environmental systems, posing significant risks to ecosystems and human health. Consequently, the development of integrated strategies for the sensitive detection and efficient removal of pesticide residues has become critically important. In this context, fluorescent inorganic–organic hybrid nanoparticles have emerged as versatile platforms owing to their tunable physicochemical properties and distinctive optical behavior. This review provides a comprehensive overview of recent advances in these hybrid nanomaterials for pesticide sensing and remediation. Particular emphasis is placed on the underlying photophysical mechanisms governing detection, including fluorescence quenching, Förster resonance energy transfer (FRET), inner filter effect (IFE), and photoinduced electron transfer (PET). In parallel, the role of interfacial interactions such as hydrogen bonding, electrostatic attraction, and π–π stacking in adsorption processes is critically discussed. Furthermore, these hybrid systems exhibit high adsorption capacities and rapid removal kinetics, enabling efficient pesticide elimination using both adsorption and catalytic degradation pathways. Overall, this review underscores the potential of fluorescent inorganic–organic hybrid nanoparticles as next-generation materials for sustainable environmental monitoring and remediation of pesticide contaminants. Full article

The timing of HIV-1 protease (PR) activation and the determinants of orderly Gag and Gag-Pol polyprotein cleavage, leading to structural maturation of virions, are not well-understood. Here, we employed total internal reflection microscopy to visualize PR activation and cleavage of a FRET-based fluorescent substrate in single HIV-1 particles assembled on the cell’s ventral membrane. PR activity manifested as a reduction in FRET signal in a relatively small fraction of nascent viral particles. By contrast, most virions released from cells contained a processed FRET substrate, suggesting that PR activation may be delayed or suppressed in most virions assembled at the ventral membrane. A combination of single-particle tracking and FRET measurements detected PR activity, on average, within ~20 min after the onset of particle assembly, while cleavage of a FRET substrate in single virions was completed within a few minutes. Importantly, substrates containing distinct PR cleavage sequences derived from Gag and Gag-Pol polyproteins were processed with different rates and efficiency. Our findings validate the use of surrogate FRET substrates to assess the timing of PR activation in single virions and elucidate the determinants of the tightly orchestrated order of Gag and Gag-Pol cleavage required for the formation of infectious virions. Full article

Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger downstream of G protein-coupled receptors (GPCRs) and a central regulator of cellular signaling. Genetically encoded exchange proteins directly activated by cAMP (Epac)-based Förster resonance energy transfer (FRET) biosensors enable real-time monitoring of cAMP dynamics in living cells, but commonly used cyan/yellow FRET pairs require short-wavelength excitation, limiting compatibility with multiplex imaging and blue-light optogenetic tools such as bacterial photoactivated adenylyl cyclases (bPACs). Here, we engineered and systematically characterized four red-shifted Epac-based single-chain FRET cAMP sensors combining yellow or orange FRET donors with red fluorescent FRET acceptors. Using ratiometric live-cell imaging, we quantified stimulus-evoked FRET responses and identified Epac_red4 as the best-performing variant, showing an approximately 55% decrease in normalized FRET after forskolin stimulation. Epac_red4 also reliably detected G_i/o-mediated decreases in cAMP following μ-opioid receptor activation. Brief 405 nm light pulses induced graded and reversible cAMP elevations using the low dark-activity variant bPAC-F198Y. Furthermore, Epac_red4 enabled analysis of cAMP recovery kinetics during phosphodiesterase inhibition and multiplex imaging of cAMP and intracellular Ca²⁺ using Fura-2 with minimal spectral and pH-related interference under physiological imaging conditions. Together, Epac_red4 represents a robust red-shifted cAMP sensor for optogenetic and multiplex signaling studies. Full article

Single-molecule Förster resonance energy transfer (smFRET) enables direct measurement of nanoscale conformational dynamics and heterogeneity in biomolecules, but quantitative interpretation of smFRET data critically depends on well-controlled excitation geometry, low background fluorescence, robust calibration, and reproducible data-analysis workflows. Prism-based total internal reflection fluorescence (pTIRF) microscopy provides important advantages for such measurements by physically separating excitation and emission paths and generating a highly confined evanescent field, yet practical guidance for implementing reproducible, quantitative pTIRF systems remains fragmented. Here we present a comprehensive, standardized framework for the design, alignment, calibration, validation, and operation of a prism-based TIRF microscope optimized for single-molecule fluorescence measurements. We describe the complete optical architecture for dual-color excitation and detection, establish alignment invariants that ensure reproducible evanescent excitation and stable donor–acceptor channel registration, and detail surface preparation, flow control, and photostabilization strategies required for reliable long-term imaging. Quantitative benchmarking protocols are introduced to evaluate signal-to-noise ratio, photobleaching kinetics, and spectral crosstalk, providing objective criteria for defining optimal operating conditions and instrument performance limits. Finally, we integrate these experimental procedures with an end-to-end single-molecule data-analysis workflow encompassing channel registration, automated and manual trajectory selection, FRET calculation, and kinetic analysis using hidden Markov modeling. The utility of the platform is demonstrated through smFRET measurements of conformational dynamics in a model nucleic acid system. Together, this work provides a reproducible and accessible methodology for implementing prism-based TIRF microscopy as a robust quantitative platform for single-molecule fluorescence studies across a wide range of biomolecular systems. Full article

Carbon dots (CDs) are zero-dimensional carbon nanomaterials with sizes below 10 nm, with high fluorescence quantum yields, variable emission colours, and excellent photostability. Due to their different structural origins and complex surface chemicals, CDs display complex photoluminescence behaviors (PL) and different fluorescence suppression responses. This review systematically summarizes recent advances in understanding the PL mechanisms of CDs, including carbon-core emission, surface emission, molecular emission and crosslink emission. In addition, fluorescence quenching processes triggered by various analytical techniques are discussed, including dynamic quenching, static quenching, Förster resonance energy transfer (FRET), photoinduced electron transfer (PET), and the inner filter effect (IFE). Emphasis is placed on mechanistic understanding and experimental differentiation strategies. A clear understanding of these fundamental mechanisms is essential for optimizing the fluorescence properties of CDs and the design of highly sensitive and selective fluorescence sensors. Finally, potential research directions and applications of CDs based on these mechanical insights are also highlighted. Full article

We report the synthesis and characterization of the photophysical characterization of a series of rhodamine (Rho)–perylenebisimide (PBI) electron donor–acceptor dyad/triads containing flexible alkyl spacers (ethylene or hexylene chains). Steady-state absorption and emission, femtosecond and nanosecond transient absorption (fs-TA and ns-TA), cyclic voltammetry, triplet–triplet energy transfer (TTET) experiments and DFT/TD-DFT calculations were combined to elucidate the excited-state dynamics. fs-TA spectral study indicates fast decay of the S₁ state and formation of the ³PBI state (0.32–663 ps), which is supported by the ns-TA spectra. The localized PBI triplet (³PBI*) exhibits unusually long lifetimes (up to 272 μs) as determined by the TTET experiment. No long-lived charge-separated (CS) state was observed. While a Förster resonance energy transfer (FRET) probably occurs between PBI and the open-ring rhodamine, a photo-induced electron transfer is proposed to be responsible for the quenching of the fluorescence of the PBI moiety. Full article

All forms of neural communications, from cognition to emotion, are regulated by neurotransmitters, which are otherwise the chemical language of the brain. Precise detection of these neurotransmitters is essential for the perception of neurophysiology and diagnosis of neurodegenerative diseases as well. Among the existing techniques for the detection of these molecules, fluorescence sensing is evolving as a powerful approach in terms of high sensitivity, rapid response, and real-time visualization of the chemical events occurring in the neural system. In recent years, nanomaterials have transformed this field by integrating tunable optical properties, excellent photostability, and modifiable surface chemistry into biocompatible nanostructures. We summarize the recent advances of these architectures to show how the material type and dimensionality, as well as the surface functionality, play roles in sensing through the mechanisms of Förster resonance energy transfer (FRET), photoinduced electron transfer (PET), inner filter effect (IFE), and aggregation-induced emission (AIE). The discussion has also been extended to the correlation of fluorescence modulation with the selectivity and sensitivity in the mechanism-to-function relationship. The potential utility of such innovative technologies, including artificial intelligence, spectral deconvolution analysis via big data algorithms, and chip-integrated sensing, was explored as a means to enable real-time neurochemical detection. This converging area of nanotechnology and neuroscience leaves a mark not just in analytical accuracy, but also parallels human brain rhythms. Full article

The regulatory mechanisms of collagen formation and cell adhesion pathways during African Swine Fever Virus (ASFV) infection remain poorly understood. This study aims to investigate whether ASFV manipulates these pathways by targeting host transcriptional regulators. Through weighted Kendall correlation analysis of transcription factor binding sites (TFBSs) in differentially expressed genes (DEGs) from the lung tissue of ASFV-recovered pigs, we identified SP2 and KLF6 as key transcription factors (TFs) associated with collagen synthesis and cell adhesion, respectively. Domain–domain interaction prediction, followed by Förster resonance energy transfer (FRET) assays, confirmed that the ASFV proteins R238L and R298L directly bind to the zf-C2H2 domains of SP2 and KLF6. Furthermore, overexpression of R238L and R298L in HeLa and 3D4/21 cells significantly downregulated SP2 and KLF6 target genes involved in these pathways. Our findings reveal a novel mechanism by which ASFV proteins R238L and R298L interfere with host transcription factors SP2 and KLF6, potentially disrupting collagen matrix integrity and cell adhesion to facilitate viral pathogenesis. Full article

RhoB is an atypical Rho GTPase whose function is tightly linked to its subcellular localization and membrane trafficking, reflecting its unique post-translational modifications and association with endosomal membranes in addition to the plasma membrane. Despite its implication in membrane trafficking and cytoskeletal regulation, tools to directly monitor RhoB activity in space and time have been lacking. Here, we describe the development and validation of a single-chain, genetically encoded Förster resonance energy transfer (FRET) biosensor that enables direct visualization of RhoB activity in living cells while preserving its native membrane-targeting determinants. The biosensor exhibits a large dynamic range and resolves spatially heterogeneous RhoB activity during leading-edge protrusion–retraction cycles in migrating mouse embryonic fibroblasts. To demonstrate the utility of this tool, we performed multiplex live-cell imaging with a previously developed near-infrared FRET biosensor for the exocytic Rho GTPase TC10. Quantitative morphodynamic and cross-correlation analyses reveal coordinated yet antagonistic spatiotemporal patterns of RhoB and TC10 activities at the leading edge and show that perturbation of TC10 regulation reorganizes their spatial coupling. Together, this work introduces a robust biosensor for RhoB and establishes a multiplex imaging framework to study the coordination of trafficking and signaling during cell migration. Full article

The quality of cow’s milk is critical for human nutrition; thus, it is important to develop rapid, sensitive, and cost-effective methods to monitor milk quality. Volatile Organic Compounds (VOCs) from milk are odorant molecules that can be used as key indicators of milk quality, since their presence is influenced by important factors such as animal metabolism, animal diet, and farming practices. In this work, we used the porcine odorant-binding protein (pOBP) and the bovine odorant-binding protein (bOBP) as molecular recognition elements (MREs) of an innovative fluorescence biosensor to detect the presence of odorant molecules in (a) milk produced by intensive livestock farming and (b) milk produced by extensive livestock farming. For biosensors, it is important to use proteins that are stable under operative conditions; therefore, we used fluorescence spectroscopy for a biophysical characterization of the pOBP and of the bOBP at different temperatures. The proposed biosensor employs a system to capture the odorant molecules from milk, which are then transferred to a liquid phase for quantitative and qualitative analyses. The binding of the odorant molecules to the OBPs triggers a Förster Resonance Energy Transfer (FRET) signal, allowing for real-time VOC quantification. The performance of the assays was evaluated by Headspace Solid-Phase Microextraction coupled with Gas Chromatography–Mass Spectrometry (HS-SPME/GC-MS) experiments. The experimental approach used for the development of the biosensor demonstrated high sensitivity and specificity, enabling the differentiation of milk from intensive and extensive farming systems. The results indicate the potential of this method for the real-time monitoring of VOCs in milk samples for food traceability and quality control. Full article

The modification of tantalum oxide (Ta₂O₅) nanoparticles (NPs) with biocompatible polymers is crucial for their biomedical use. Such modification can prolong NP circulation in the bloodstream by minimizing salt-induced aggregation and reducing nonspecific protein adsorption onto their surface. Understanding the features of polymer–NP interactions is a key issue in the fabrication of nanostructures with required characteristics. The present work aims to provide a comprehensive comparative study of bovine serum albumin (BSA) adsorption on bare and polydopamine (PDA)-coated Ta₂O₅ NPs. The synthesized NPs were characterized via transmission electron microscopy, Fourier transform infrared spectroscopy, dynamic light scattering, and zeta potential measurements. Fluorescence and circular dichroism spectroscopy were also employed for the first-time investigation of the interactions of Ta₂O₅ NPs and Ta₂O₅@PDA NPs with BSA. The results obtained show that PDA coating significantly enhances the protein-binding affinity. Time-resolved measurements revealed signatures of Förster resonance energy transfer, confirming complex formation between NPs and BSA. Moreover, colloidal stability tests in phosphate-buffered saline indicated that the presence of adsorbed BSA improves the dispersion stability of bare and PDA-coated Ta₂O₅ NPs. These findings advance the understanding of protein–NP interactions and highlight the potential of PDA coatings for designing stable and functional nanostructures for biomedical applications. Full article

Nanosurface energy transfer (NSET) has emerged as a pivotal mechanism in nanobiophotonics, facilitating the development of highly sensitive biosensors with extended dynamic ranges. Unlike conventional Förster resonance energy transfer, NSET exhibits an inverse fourth-power dependence on distance, enabling quantitative measurements over distances up to 40 nm. This review comprehensively explores the fundamental principles governing NSET, with particular emphasis on non-radiative coupling between fluorescent donors and metallic nanostructures such as gold nanoparticles. Additionally, the applications of these probes are surveyed across various bioanalytical domains, including nucleic acid assays, immunoassays, real-time intracellular monitoring, and various biomolecule detection. Additionally, the evolving integration of NSET, plasmonics, and nanophotonic architectures is discussed, focusing on emerging trends and the trajectory for developing next-generation, multiplexed, and point-of-care diagnostic platforms. Current challenges and prospective pathways for translating these advanced sensing systems into clinical and field-deployable solutions are also considered. Full article

Fibroblast activation protein (FAP), a transmembrane serine protease expressed primarily in pathological conditions, plays a pivotal role in tumor progression. Despite extensive studies on FAP in solid tumors, its role in hematologic cancers, particularly lymphoid malignancies, remains underexplored. This study aimed to investigate the level and activity of soluble FAP (sFAP) in pre-therapeutic serum samples from 120 lymphoma patients. We measured sFAP serum levels using time-resolved immunofluorometric assay and sFAP activity with Förster resonance energy transfer assay. In addition, immunohistochemistry was used to analyze intratumoral FAP expression in tissue biopsies from a subset of B-cell lymphoma patients (n = 34). Notably, the results revealed significantly reduced circulating sFAP levels (p = 0.002) and activity (p < 0.001) in aggressive disease subtypes compared with indolent subtypes and healthy individuals. At the time of diagnosis, low sFAP activity correlated with inferior overall survival (both p < 0.001) in patients with the aggressive entities, suggesting altered FAP functionality in these tumors. Interestingly, measuring intratumoral FAP levels revealed an inverse pattern, with diffuse large B-cell lymphoma showing higher tissue FAP localization compared with follicular lymphoma (p < 0.001). These findings provide new insights into the biological and clinical significance of FAP in lymphoid malignancies, particularly highlighting the importance of sFAP activity as a potential prognostic marker in aggressive lymphoid malignancies. Full article

Yeast +1 nucleosomes positioned at transcription start sites must be reorganized to allow transcription initiation. Nucleosome reorganization involves multiple factors including histone chaperone FACT (FAcilitates Chromatin Transcription), histone acetylation, and histone variant H2A.Z; however, the mechanism of this process is not fully understood. Here we investigated nucleosome unfolding in the presence of these factors by combining biochemical assays with single-particle Förster resonance energy transfer (spFRET) microscopy. The presence of the H3:K56Ac mimic (H3:K56Q) alone or together with H2A.Z (but not H2A.Z alone) facilitates the Nhp6-dependent unfolding of nucleosomes by FACT. In contrast to canonical nucleosomes, the unfolding of nucleosomes with the studied variant histones promotes the eviction of core histones from nucleosomal DNA. Furthermore, H2A.Z alone or in synergy with H3:K56Q facilitates transcription through a nucleosome as efficiently as FACT facilitates transcription through canonical nucleosomes. The data suggest that FACT, together with H3:K56 acetylation and H2A.Z, unfold promoter nucleosomes and participate in the eviction of histones to increase the accessibility of the transcription start site, thereby stimulating transcription initiation and possibly early elongation. Full article