Search Results (335)

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

Achieving long-lived room-temperature phosphorescence (RTP) with high quantum efficiency is of significant interest for applications in anti-counterfeiting, flexible optoelectronic displays, and multi-level information encryption. Here, we presented a hydrogen-bond engineering strategy to enhance RTP performance by progressively increasing the number of hydrogen-bonding sites within a polyvinyl alcohol (PVA) matrix. A series of carbazole-based chromophores (Cz, ICz and 2ICz) were embedded into the PVA network, and their photophysical properties were systematically characterized using steady-state photoluminescence spectra, time-decay spectra, Fourier-transform infrared (FTIR), and Raman and X-ray photoelectron spectroscopy (XPS). Spectroscopic analysis revealed that the increased number of N-H groups significantly strengthened hydrogen-bonding interactions, effectively suppressing non-radiative decay pathways and stabilizing triplet excitons. As a result, the phosphorescence lifetime was prolonged up to 1.68 s with a quantum yield of 38.63%. Furthermore, leveraging the spectral overlap integral between the phosphorescent emission and dye absorption, efficient Förster resonance energy transfer (FRET) was realized, enabling tunable multi-color afterglow emissions. This study establishes a design strategy validated by spectroscopy for high-performance RTP materials and highlights their promising potential in advanced optical encryption and flexible photonic applications. Full article

The efficacy of X-ray-induced photodynamic therapy (X-PDT) for deep tumors is often hindered by conventional scintillators, typically rare-earth nanoparticles plagued by long-term toxicity and suboptimal scintillation yields. Here, we introduce a copper iodide (Cu-I) cluster, Cu₂I₂(PPh₃)₂(pz), composed of earth-abundant elements, as an efficient and biocompatible energy transducer for X-PDT. A theranostic nanoplatform, CuI@PcNP, was engineered by co-encapsulating the Cu-I cluster and a phthalocyanine photosensitizer (Pc4OH) within a 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-2000 (DSPE-PEG2K) matrix, which confers excellent physiological stability. This nano-architecture ensures nanoscale proximity between the cluster (donor) and photosensitizer (acceptor), facilitating efficient (58%) Förster resonance energy transfer (FRET) while overcoming aggregation-induced quenching. Upon X-ray irradiation, the platform effectively converted X-rays to visible light, activating Pc4OH to generate potent reactive oxygen species (ROS) and inducing significant dose-dependent cytotoxicity in human hepatocellular carcinoma (HepG2) cells. In a murine hepatoma model, enabling image-guided X-PDT that resulted in a 77.4% tumor inhibition rate with negligible systemic toxicity. Collectively, this work pioneers the integration of phthalocyanine with Cu-I clusters, providing a stable and versatile nanoplatform for image-guided X-PDT. Full article

This study presents a novel ratiometric fluorescent sensor based on Förster resonance energy transfer (FRET) between glutathione (GSH)-coated CdTe quantum dots (CdTe/GSH QDs) and bovine serum albumin (BSA)-coated Au nanoclusters (AuNCs/BSA) for dopamine (DA) detection. The nanoparticles were characterized using transmission electron microscopy (TEM), zeta potential measurements, Fourier transform infrared (FTIR) spectroscopy, UV-Vis absorption and fluorescence spectroscopy. Key FRET parameters, including energy transfer efficiency (E), donor–acceptor distance (r), Förster distance (R₀), and the overlap integral (J), were determined. The interactions between the CdTe/GSH-AuNCs/BSA conjugate and DA were investigated, revealing a dual mechanism of QDs fluorescence quenching that involves both energy and electron transfer. The average lifetime values and spectral profiles of CdTe/GSH QDs, both in the absence and presence of DA, suggest a dynamic fluorescence quenching process. The variation in the ratiometric signal with increasing DA concentration demonstrated a linear response within the range of 0–250 µM, with a correlation coefficient of 0.9963 and a detection limit of 6.9 nM. This proposed nanosensor exhibited selectivity against potential interfering substances, including urea, glucose, BSA, GSH, citric acid, and metal ions such as Na⁺ and Ca²⁺. The conjugate also demonstrates excellent cytocompatibility and enhances cell proliferation in HeLa epithelial cells, making it suitable for biological applications. It was successfully employed for DA detection in urine samples, achieving recoveries ranging from 99.1% to 104.2%. The sensor is highly sensitive, selective, rapid, and cost-effective, representing a promising alternative for DA detection across various sample types. Full article

Most biomarkers exhibit abnormal expression in more than one disease, making conventional single-biomarker detection strategies prone to false-negative results. Detecting multiple biomarkers associated with a single disease can therefore substantially improve diagnostic accuracy. Accordingly, recent research has focused on precise multiplex detection, leading to the development of sensors employing various readout methods, including electrochemical, fluorescence, Raman, and colorimetric approaches. This review focuses on optical sensing applications, such as fluorescence, Raman spectroscopy, and colorimetry, which offer rapid and straightforward detection and are well suited for point-of-care testing (POCT). These optical sensors exploit nanoscale phenomena derived from the intrinsic properties of nanomaterials, including metal-enhanced fluorescence (MEF), Förster resonance energy transfer (FRET), and surface-enhanced Raman scattering (SERS), which can be tailored through modifications in material type and structure. We summarize the types and properties of commonly used nanomaterials, including plasmonic and carbon-based nanoparticles, and provide a comprehensive overview of recent advances in multiplex biomarker detection. Furthermore, we address the potential of these nanosensors for clinical translation and POCT applications, highlighting their relevance for next-generation disease diagnostic platforms. Full article