Search Results (214)

Direct antibody labeling is essential for immunoassays, multiplexed imaging, and biosensing; however, current methods are often time-consuming, restricted by antibody source, risk compromising protein performance, or vary with multiple steps. We introduce multiplex in situ tagging (MIST) Linker, a rapid and Fc-site-specific labeling tool that conjugates fluorophores or DNA oligonucleotides to antibodies from diverse commercial sources in as fast as 10 min using minimal starting material. MIST Linker achieves >90% cleavage upon UV exposure, facilitating rapid cyclic imaging on a single specimen. Validated across multiple species and sources, the platform outperforms conventional two-step immunofluorescence and immunohistochemistry in various tissues and cell lines. By enabling the rapid, cost-effective customization of antibody panels, MIST Linker significantly lowers the barrier to accessing antibody–DNA conjugates for spatial biology. When integrated with the spatial MIST platform and MIST-Explorer, it enables high-plex, single-cell spatial proteomics at high signal-to-noise ratios in human clinical biopsies, mouse specimens and cell lines. This toolkit provides an efficient, accessible solution for high-resolution spatial mapping, allowing for the in-depth analysis of cell subpopulations, biomarker distributions, and signaling events in complex biological specimens. Thus, MIST Linker offers a versatile, accessible, and scalable solution for antibody-labeling-based research and clinical diagnosis. Full article

In this study, a novel and sensitive fluorescent sensing platform was rationally developed for the rapid and specific detection of dopamine. The proposed strategy ingeniously integrates the superior adsorption behavior and extraordinary fluorescence quenching effect of oxidized single-walled carbon nanohorns toward FAM-modified aptamers, as well as the highly efficient cleavage property of the cryonase enzyme. Specifically, the fluorescently labeled aptamer is efficiently adsorbed and shielded by oxidized single-walled carbon nanohorns through strong supramolecular interactions, thereby triggering an evident fluorescence quenching response. Upon the introduction of target dopamine, the specific recognition event between dopamine and its corresponding aptamer takes place, accompanied by the formation of stable aptamer–dopamine complexes. Subsequently, these complexes are selectively hydrolyzed under the catalytic action of the cryonase enzyme, which contributes to the distinct release of fluorophores and the obvious recovery of fluorescence signals. Under the optimized experimental conditions, the aptamer exhibits good linearity toward dopamine in the concentration range of 50–400 ng/mL, with a correlation coefficient of 0.9957 and a low detection limit of 26.12 ng/mL. Therefore, the established analytical method offers a rapid, convenient, and reliable tool for the accurate determination of dopamine in complex human serum samples. Full article

Background/Objectives: The blood–brain barrier (BBB) presents a major challenge for delivering therapeutics to the central nervous system (CNS) due to its highly selective permeability. Human brain microvascular endothelial cells (hBMECs), the principal cellular component of the BBB, tightly regulate molecular transport and restrict the entry of many CNS-targeted therapies. Lipid-based nanoparticles have emerged as promising carriers for BBB transport because of their biocompatibility, tunable surface properties, and cargo encapsulation capabilities. One strategy to enhance nanoparticle transport involves surface functionalization with ligands that exploit endogenous transcytosis pathways. Mannose-6-phosphate (M6P), a glycan implicated in the brain entry of certain proteins and viruses, represents a potential targeting ligand for this purpose. Methods: In this study, we established a physiologically relevant in vitro BBB model using human-induced pluripotent stem cell-derived brain microvascular endothelial cells (hiPSC-BMECs) to evaluate M6P-functionalized liposomes for BBB transport. Fluorophore-labeled liposomes were used to monitor nanoparticle uptake and transcytosis. Results: M6P-functionalized liposomes exhibited significantly enhanced uptake in hiPSC-BMECs compared with non-functionalized control liposomes. Pharmacological inhibition studies supported the involvement of a clathrin-sensitive endocytic pathway. Transcytosis assays demonstrated enhanced BBB crossing of M6P-functionalized liposomes, with transport increasing according to ligand density and reaching approximately 55% of the transport observed for transferrin under the same experimental conditions. Following transcytosis, intact M6P-functionalized liposomes showed significantly higher uptake by downstream hiPSC-derived neurons and astrocytoma cells compared with control formulations. Conclusions: Together, these findings support M6P-functionalization as a promising strategy to enhance liposome uptake and transcytosis across a human-relevant in vitro BBB model. This work provides a proof-of-concept framework for the development and optimization of glycan-functionalized nanocarriers for CNS-directed delivery. Full article

Formalin-fixed paraffin-embedded (FFPE) tissues represent an invaluable resource for both basic and clinical research due to their stable preservation of tissue architecture and molecular integrity. Multiplex immunofluorescence (mIF) using tyramide signal amplification (TSA) enables the simultaneous detection of multiple antigens within a single FFPE section. Here, we describe a kit-independent and customizable TSA-based mIF protocol that utilizes commercially available horseradish peroxidase (HRP)-conjugated secondary antibodies and tyramide–fluorophore reagents. The method was applied using FFPE endometriosis tissue, targeting estrogen receptor alpha (ERα), progesterone receptor (PR), α-smooth muscle actin (αSMA), CD20 and CD31. Each staining round was followed by heat-induced epitope removal (HIER) of the bound antibodies while preserving covalently deposited signals. Fluorescence imaging was performed using a multi-channel slide scanner with carefully selected fluorophores to enable optical separation between detection channels. Under the conditions described, the protocol enabled clear visualization of maker-specific staining patterns with preserved tissue morphology. This study provides a practical and flexible TSA-based mIF protocol as a qualitative proof of concept, offering an accessible alternative to commercial kit-based approaches. Further studies will be required to establish quantitative performance and a broader applicability across tissue types. Full article

Background/Objectives: The F3 peptide, a tumor-homing peptide known to bind cell-surface nucleolin, is frequently employed as a targeting vector in cancer research. However, the impact of the modification site on its cellular binding properties has not been investigated yet. In this work, we aimed to design an improved F3-based radioconjugate by identifying the optimal conjugation site and establishing a protocol for its biological evaluation in vitro. To achieve this, we compared F3 peptide derivatives modified at their N- or C-termini with DOTA for complexation of indium-111 (¹¹¹In) for SPECT or Auger electron therapy or a fluorophore (FITC) for optical imaging. Methods: N-and C-terminal DOTA-modified F3 peptides were radiolabeled with indium-111 and compared for their in vitro stability in different physiologically relevant media. Suitable nucleolin-positive cell lines for further in vitro studies were identified by confocal microscopy of a FITC-labeled F3 peptide derivative. The radioconjugates were then investigated on MDA-MB-231 (breast cancer) and PC-3 (prostate cancer) cells for nucleolin-specific cell binding and uptake, and several parameters of the in vitro assays were varied to establish a suitable protocol. Results: In general, in vitro assays with F3 peptide conjugates are challenging, as the outcome depends on a number of experimental parameters, leading, in some cases, to varying results. In particular, the presence of Ca²⁺ and Mg²⁺ had a decisive impact on the results, likely because the metal ions compete with the binding of F3 conjugates to nucleolin. The C-terminal modified, ¹¹¹In-labeled F3 radioconjugate performed better than the N-terminal modified analog. While several parameters of the in vitro experiments were optimized, the overall cell uptake in vitro of radioactivity was still low (<2% of applied radioactivity). Conclusions: A standardized in vitro protocol for evaluating F3 peptide conjugates on cancer cells was established, revealing that the C-terminus is the preferred site for modification. Because the cellular uptake of the radiotracer was shown to likely not be sufficient for radiotracer development, further studies on the optimization of the F3 peptide conjugates, including structural modifications, are required. Full article

Brucellosis remains a major global public health concern, with B. melitensis and B. abortus being the primary causative agents in China. This study describes the development of a multiplex droplet digital PCR (ddPCR) assay for the simultaneous detection and differentiation of B. melitensis and B. abortus. The assay employs TaqMan probes targeting the bcsp31 gene (genus-specific), a transposase gene (B. melitensis-specific) and an autotransporter-associated beta strand repeat-containing protein gene (B. abortus-specific), each labeled with distinct fluorophores (FAM, HEX, ROX). The optimized assay exhibited no cross-reactivity with other pathogens and exhibited significantly higher sensitivity than both qPCR and conventional PCR, with detection limits as low as 2.54–3.11 copies/reaction. Repeatability was excellent, with intra- and inter-assay coefficients of variation below 9%. When validated on a panel of clinical nucleic acid samples, the ddPCR assay showed strong agreement with qPCR (kappa = 0.85), with a sensitivity of 100% (79.42%~100%, 95%CI) and specificity of 95.96% (92.08%~99.84%, 95%CI). These findings establish the multiplex ddPCR as a rapid, sensitive, and highly specific diagnostic platform that improves brucellosis detection accuracy and supports targeted control strategies. Full article

Paralytic shellfish toxins (PSTs) are produced by several toxic species of the dinoflagellate genera Alexandrium and Gymnodinium, and they pose significant threats to marine ecosystems and public health. Rapid and accurate detection of harmful algal blooms (HABs) is essential for effective management. In this study, we developed a multiplex quantitative real-time PCR (qPCR) assay targeting the 28S ribosomal DNA region to simultaneously detect three PST-producing dinoflagellates, Alexandrium catenella, A. pacificum, and Gymnodinium catenatum, in the East China Sea off southern Korea. Species-specific primers and hydrolysis probes labeled with distinct fluorophores were validated for simultaneous detection. The standard curves showed strong linearity (R² > 0.99) and high amplification efficiencies (95.268–99.325%). No cross-reactivity was observed among the 20 non-target microalgal species. Field application of the assay using environmental DNA (eDNA) samples collected during spring successfully detected A. catenella and A. pacificum, whereas G. catenatum was not detected during the survey period. This multiplex qPCR assay provides a rapid and reliable molecular tool for early detection and spatial monitoring of potentially PST-producing dinoflagellates, supporting sustainable HAB management in East Asian coastal ecosystems. Full article

Oral squamous cell carcinoma (OSCC) is an aggressive disease with a glycoproteomically unmapped progression and a low five-year survival rate. Thus, the aim of this pilot study was to explore the N-glycosylation pattern differences in malignant, adjacent mucosal and healthy tissues in the context of OSCC. Oral mucosal soft tissue samples was obtained by incisional biopsy from five patients with OSCC, both from the malignant and the opposite healthy gingival sides, and from seven age-sex-matched healthy controls. The collected tissues were homogenized, followed by N-glycan profiling of the endoglycosidase-released and fluorophore-labeled carbohydrates using capillary electrophoresis with ultra-sensitive laser-induced fluorescent detection (CE-LIF). Six out of the twenty-two identified N-glycan structures, including glycogens, showed significant (p < 0.05) differences between the malignant tissue samples of the OSCC patients and the healthy controls. Comparing the healthy and the positive control oral mucosal samples, differences in four N-glycan structures were revealed, while only one alteration was observed between the N-glycan profiles of the malignant tumor and positive control samples. However, the results are presented descriptively, reflecting the limited sample size of the pilot study, it shows the potential of high-resolution CE-LIF-based glyocoanalytical protocol to be highly efficient and sensitive for glycobiomarker-based molecular diagnostics of oral malignant lesions. Full article

Homogeneous fluorescence immunoassays are in high demand due to their simplicity, rapidity, sensitivity, and specificity. These methods typically utilize immune-induced changes in the rotational mobility of the fluorophore with depolarization of plane-polarized excitation light (FPIA, etc.) or fluorescence quenching based on intramolecular energy transfer (FRET, etc.). This article presents an immunoassay based on enhanced emission of the fluorescein label in immune complexes. Over the entire history of fluorescence immunoassay research, this effect has been described in a few papers, while it allows overcoming the limitations of prevailing approaches. We discovered the assay for detecting aflatoxin B1 (AFB1), a widespread toxic contaminant of agricultural products. The one-step assay procedure consists of mixing the sample with antibodies and fluorescently labeled AFB1, accompanied by fluorescence measurement. This method enables the detection of AFB1 at concentrations up to 200 pg/mL in 10 min, including measurements in complex samples (corn extracts). Minimal manipulations in the course of the testing also provide high accuracy. The AFB1 revealed in contaminated corn samples was in the range of 76–136%. The influence of immune complex formation on the fluorescent label’s emission can be easily tested and serve as a basis for applying this principle to other diverse analytes and various kinds of samples. Full article

Fluorescence fluctuation spectroscopy (FFS) techniques rely on determination of monomeric molecular brightness, i.e., the fluorescence intensity of a single, non-aggregated fluorophore, as a critical reference for estimating protein oligomer size. By comparing measured molecular brightness of fluorescently labeled proteins of interest to this monomeric brightness benchmark, FFS enables inference of oligomerization states. However, widely used fluorescent proteins often exhibit self-association, compromising monomeric brightness calibration and introducing errors in brightness-derived oligomer-size estimates. This study compares two strategies for determining monomeric brightness: the conventional fluctuation-based method and a more recently proposed average-intensity-based alternative. The comparison uses two model fluorophores, a fluorescent protein (mCitrine) and the small-molecule dye Janelia Fluor 525 (JF₅₂₅) conjugated to HaloTag. Our results show strong agreement between intensity- and fluctuation-derived brightness values only in the minimally aggregating JF₅₂₅–HaloTag benchmark. In contrast, in the mCitrine samples, where aggregation is more prevalent, only the intensity-based method maintains a consistent estimate across sample preparation, while the fluctuation-based method overestimates brightness when aggregation effects become pronounced. This robustness makes the intensity-based approach a valuable cross-check for monomeric brightness calibration. Our results support a combined strategy, using both methods to improve the reliability of monomeric brightness calibration and protein oligomerization analysis in FFS. Full article

Microplastics (MPs) are synthetic environmental pollutants increasingly linked to adverse human health effects. To study their biological impact, researchers require access to environmentally relevant MPs that can be accurately tracked in biological systems. However, most ambient MPs are composed of non-conjugated polymers that lack intrinsic fluorescence, limiting their utility in live-cell or in vivo imaging. Addressing this challenge, we present two alternative labeling approaches that enable visualization, tracking, and quantification of MPs. First, we stained nylon and polypropylene MPs with Rhodamine 6G, a fluorescent dye known for its stability and compatibility with in vivo applications. These labeled MPs retained strong fluorescence in murine lung tissue for up to one week, as confirmed by fluorescent microscopy. Second, we conjugated aminated polystyrene microspheres with IRDye-800CW, a near-infrared fluorophore that enables high-resolution imaging with minimal tissue autofluorescence via an In Vivo Imaging System and confocal microscopy. In vivo experiments revealed organ-specific accumulation of IRDye-labeled MPs, with a 2.8-fold increase in the liver and a 5-fold increase in spleen compared to controls, detectable up to 72 h post-injection. These labeling strategies provide researchers with practical tools to visualize and study the biodistribution of MPs in biological systems, advancing efforts to understand their health implications. Full article

This study presents a comparative investigation of plasmonic sensing platforms based on colloidal gold nanoparticle (AuNP) suspensions and gold film over nanosphere (AuFoN) solid substrates for the detection of epidermal growth factor receptor (EGFR), an essential biomarker and therapeutic target in oncology. The strategy relies on fluorescence emission modulation of an Atto647N-labeled DNA oligomer competitively bound to an EGFR-specific aptamer. Our results demonstrate that the colloidal AuNPs can function as competitive binding sensors, leading to fluorescence quenching upon fluorophore attachment to the surface of the NPs and partial fluorescence recovery due to EGFR-induced displacement of the fluorophore–aptamer complex. This specificity was confirmed by reversed binding experiments. However, the system proved highly sensitive to the experimental design: excessive washing (centrifugation) led to unspecific aggregation and signal loss, while reduced washing steps improved signal retention and revealed EGFR-induced fluorophore displacement into the supernatant. On the contrary, film-based substrates exhibited strong initial fluorescence, but failed to retain the fluorophore–aptamer complex after washing, resulting in fluorescence decay independent of EGFR incubation. This indicates that AuFoN lacked the binding stability necessary for specific displacement-based sensing. These findings highlight that while colloidal AuNPs can support competitive binding detection, their reproducibility is limited by colloidal stability and protocol sensitivity, whereas AuFoN substrates require improved surface functionalization strategies. The study emphasizes the critical role of surface chemistry, aptamer–fluorophore affinity, and washing protocols in determining the success or failure of plasmon-enhanced aptamer-based biosensing systems and suggests opportunities for improving specificity and robustness in future designs. Full article

The development of low-background, facile, and robust fluorescent nanoprobes for imaging and monitoring of intracellular mRNA changes remains a great challenge. Taking advantage of the high fluorescence quenching efficiency of core-shell gold@polydopamine (Au@PDA) nanocomposites and Ca²⁺-promoting DNA adsorption stability, a simple and universal bioconjugate strategy was designed to a construct fluorescent nanoprobe for highly efficient tumor-related mRNA imaging. The fluorescence of Cy5-labeled DNA was quenched up to 92.38% by the AuNP and PDA via nanometal surface energy transfer (NSET) and photoinduced electron transfer (PET), respectively. TK1 mRNA, a biomarker of tumor growth, initiates hybridization and results in fluorescence recovery, which built the foundation for identifying the expression level changes in living cells. More importantly, three kinds of tumor-related mRNA (TK1 mRNA, GalNAc-T mRNA, and C-myc mRNA) can be detected simultaneously with different fluorophore-modified recognition sequences, which can avoid false positive signals and improve the reliability of cancer diagnostic, holding great promise for cancer diagnosis, prognosis, and therapy. Full article

Background/Objectives: Giardiasis is one of the oldest-reported infectious diseases worldwide. It affects individuals with weakened immune systems and progresses into chronic infection if untreated. Morphological analysis and visualization of cell shapes using unlabeled or fluorophore-labeled samples are commonly employed to identify the parasite. Methods: To distinguish molecular content variations between trophozoites and infectious type I cysts, the current research presents an alternative approach based on label-free Raman microscopy. Results: Constituents responsible for plasma membrane thickening and cyst wall formation during encystation, such as N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc) were detected. Although these two coexisting compounds have similar molecular structures, their spectroscopic distinction and visual localization through Raman microscopy are achievable. While immature and non-viable cysts contain a larger amount of GlcNAc, a potential transition of this moiety to GalNAc might occur as the cysts mature and become infectious. Other Raman results revealed changes in the oxidation states of heme-binding proteins and in lipid–protein metabolism, each serving as an additional protection mechanism that the parasite employs for survival. Complementary bright field and confocal fluorescence microscopy results corroborate the Raman outcomes. Conclusions: The molecular-level findings of this work, which presents a detailed spectroscopic analysis of Giardia’s encystation and excystation stages, substantiate the need to use complementary methods for monitoring the parasite’s dynamics and efficacy in terms of self-protection. This alternative method provides accurate insights for further understanding the multifaceted factors involved in Giardia’s encystation process and its acquired resistance to external stimuli. Full article

Phytoglobins (Pgbs) are plant hemoglobin-like proteins with key roles in nitric oxide (NO) scavenging, oxygen sensing, and hypoxic stress responses. Their typical hexacoordination results in unusually high affinities for gaseous ligands such as NO and carbon monoxide (CO), complicating measurement using conventional methods. Standard assays often require large sample volumes and lack sensitivity for high-affinity, low-abundance proteins like hexacoordinated Pgbs. Here, we present a microscale capillary-based fluorescence assay for the high-precision measurement of protein–gas binding. Fluorophore-labeled proteins are loaded into gas-saturated capillaries and analyzed via dual-wavelength fluorescence to monitor isothermal spectral shifts upon ligand binding. Phosphate-buffered saline with Tween20 (PBS-T20) ensures gas stability and minimizes nonspecific adsorption. Using this approach, we characterized CO and NO binding to the recombinant wildtype (rWT) of Beta vulgaris Pgb 1.2 (BvPgb 1.2) and its C86A mutant. CO titrations revealed biphasic binding, with EC₅₀ ~400 nM and ~700 μM (rWT) and ~500 nM and ~400 μM (C86A). NO binding showed K_D values of ~1600 nM (rWT) and ~400 nM (C86A), implicating Cys86 in ligand affinity. This assay provides a robust, low-volume method for high-affinity protein–gas studies and shows biphasic dynamics in BvPgbs. Full article