Search Results (568)

Background: Lysosomal-associated membrane protein 1 (LAMP1), typically localized to the lysosomal membrane, is increasingly implicated as a marker of cancer aggressiveness and metastasis when expressed on the cell surface. This study aimed to develop a LAMP1-targeted antibody-based PET tracer and assess its efficacy in mouse models of human breast and colon adenocarcinoma. Methods: To determine the source of LAMP1 expression, we utilized human single-cell RNA sequencing and spatial transcriptomics, complemented by in-house flow cytometry on xenografted mouse models. Tissue microarrays of multiple epithelial cancers and normal tissue were stained for LAMP-1, and staining was quantified. An anti-LAMP1 monoclonal antibody was conjugated with desferrioxamine (DFO) and labeled with zirconium-89 (⁸⁹Zr). Human triple-negative breast cancer (MDA-MB-231) and colon cancer (Caco-2) cell lines were implanted in nude mice. PET/CT imaging was conducted at 24, 72, and 168 h post-intravenous injection of ⁸⁹Zr-DFO-anti-LAMP1 and ⁸⁹Zr-DFO-IgG (negative control), followed by organ-specific biodistribution analyses at the final imaging time point. Results: Integrated single-cell and spatial RNA sequencing demonstrated that LAMP1 expression was localized to myeloid-derived suppressor cells (MDSCs) and cancer-associated fibroblasts (CAFs) in addition to the cancer cells. Tissue microarray showed significantly higher staining for LAMP-1 in tumor tissue compared to normal tissue (3986 ± 2635 vs. 1299 ± 1291, p < 0.001). Additionally, xenograft models showed a significantly higher contribution of cancer cells than the immune cells to cell surface LAMP1 expression. In vivo, PET imaging with ⁸⁹Zr-DFO-anti-LAMP1 PET/CT revealed detectable tumor uptake as early as 24 h post-injection. The ⁸⁹Zr-DFO-anti-LAMP1 tracer demonstrated significantly higher uptake than the control ⁸⁹Zr-DFO-IgG in both models across all time points (MDA-MB-231 SUV_max at 168 h: 12.9 ± 5.7 vs. 4.4 ± 2.4, p = 0.003; Caco-2 SUV_max at 168 h: 8.53 ± 3.03 vs. 3.38 ± 1.25, p < 0.01). Conclusions: Imaging of cell surface LAMP-1 in breast and colon adenocarcinoma is feasible by immuno-PET. LAMP-1 imaging can be expanded to adenocarcinomas of other origins, such as prostate and pancreas. Full article

Exosomes are nanoscale extracellular vesicles (EVs) that carry biomolecular signatures reflective of their parent cells, making them powerful tools for non-invasive diagnostics and therapeutic monitoring. Despite their potential, clinical application is hindered by challenges such as low abundance, heterogeneity, and the complexity of biological samples. To address these limitations, plasmonic biosensing technologies—particularly propagating surface plasmon resonance (PSPR), localized surface plasmon resonance (LSPR), and surface-enhanced Raman scattering (SERS)—have been developed to enable label-free, highly sensitive, and multiplexed detection at the single-vesicle level. This review outlines recent advancements in nanoplasmonic platforms for exosome detection and profiling, emphasizing innovations in nanostructure engineering, microfluidic integration, and signal enhancement. Representative applications in oncology, neurology, and immunology are discussed, along with the increasingly critical role of artificial intelligence (AI) in spectral interpretation and diagnostic classification. Key technical and translational challenges—such as assay standardization, substrate reproducibility, and clinical validation—are also addressed. Overall, this review highlights the synergy between exosome biology and plasmonic nanotechnology, offering a path toward real-time, precision diagnostics via sub-femtomolar detection of exosomal miRNAs through next-generation biosensing strategies. Full article

Extracellular vesicles (EVs), including small EVs (sEVs) such as exosomes, play crucial roles in intercellular communication and disease pathology. Their heterogeneous nature, shaped by cellular origin and activation state, requires precise and multiplexed profiling of surface markers for effective characterization. Despite recent advances, current analytical methods remain complex, costly, or inaccessible for routine laboratory use. Here, we present a simple and cost-effective flow cytometry-based assay for the multiplexed analysis of tetraspanin markers (CD63, CD81, CD9) on fluorescently labeled sEVs. Our method combines metabolic labeling with paraformaldehyde fixation and low-speed centrifugation using a benchtop centrifuge, enabling efficient removal of unbound antibodies and minimizing nonspecific signals while preserving vesicle integrity. Using either metabolically labeled exosomes or bulk sEVs stained with carboxyfluorescein succinimidyl ester (CFSE), we demonstrate robust recovery and accurate, semi-quantitative profiling of tetraspanin expression. The assay reveals substantial variability in tetraspanin distribution across different cell lines and does not require ultracentrifugation or immunocapture. Notably, this versatile and reproducible method supports high sEV recovery and is adaptable to additional protein markers. Its compatibility with standard laboratory equipment makes it a practical and scalable alternative to more complex techniques, expanding access to multiplex sEV analysis for both research and clinical applications. Full article

Electron paramagnetic resonance (EPR) spectroscopy is gaining increasing recognition in research on various nanostructures. In the case of iron oxide nanoparticles, EPR measurements offer the possibility of determining the magnetic phase and the exact type (Fe₃O₄, γ-Fe₂O₃, α-Fe₂O₃, or a combination) of the core material. Furthermore, the EPR technique enables the study of relaxation processes, estimation of the effective and surface anisotropy constants, and assessment of the influence of sample aging on the magnetic properties of nanoparticles. The scope of the information obtained can be further expanded by utilizing spin labeling of polymer-coated nanoparticles. By analyzing the signals from the attached nitroxide, one can determine certain properties of the coating and its interactions with the environment (e.g., body fluids, cells, tissues) and also perform imaging of nanoparticles in various media. In some cases, EPR can help monitor the encapsulation of active substances and their release processes. Unfortunately, despite the enormous potential, not all of the possibilities offered by EPR are routinely used in nanoscience. Therefore, the present article aims not only to present the current applications and existing trends but also to indicate directions for future EPR research, constituting a road map. Full article

Nanoquenchers with a single quenching cofactor exhibit limited fluorescence quenching efficiency. In this work, a metal–organic hybrid with dual quenching cofactors (Cu²⁺ and pyrroloquinoline quinone or PQQ) was prepared by metal-coordinated assembly and used as a nanoquencher for a protease assay with enhanced quenching efficiency. The peptide substrate with an oligohistidine (His₆) tag was labeled with a fluorophore. Caspase-3 was determined as a protease example. The substrate was attached onto the surface of the Cu-PQQ nanoquencher by a metal coordination interaction between the unsaturated Cu²⁺ on the nanoparticle surface and the His₆ tag in the peptide. The cleavage of the peptide substrate by enzymatic hydrolysis led to the release of a fluorophore-conjugated segment from the nanoquencher surface, thus turning on the fluorescence. The nanoprobe was used to determine caspase-3 with a linear range of 0.01–5 ng/mL and a detection limit of 7 pg/mL. Furthermore, the method was used to evaluate inhibition efficiency and monitor drug-induced cell apoptosis. In contrast to other means of peptide immobilization, such as physical adsorption and covalent coupling, the strategy based on the metal coordination interaction is simple and powerful, thereby achieving assays of caspase-3 activity in lysates with a satisfactory result. The work should be valuable for the design of nanoquenchers with multiple quenching cofactors and the development of novel biosensors. Full article

Microfluidics-based mixing methods have attracted increasing attention due to their great potential in bio-related and material science fields. The combination of acoustics and microfluidics, called acoustofluidics, has been shown to be a promising tool for precise manipulation of microfluids and micro-objects. In general, achieving robust mixing performance in an efficient and simple manner is crucial for microfluidics-based on-chip devices. When surface acoustic waves (SAWs) are introduced into microfluidic devices, the acoustic field can drive highly controllable acoustic streaming flows through acoustofluidic interactions with micro-solid structures, which have the advantages of label-free operation, flexible control, contactless force, fast-response kinetics, and good biocompatibility. Therefore, the design and application of various SAW micromixers have been demonstrated. Herein, we present a comprehensive overview of the latest research and development of SAW-based micromixers. Specifically, we discuss the design principles and underlying physics of SAW-based acoustic micromixing, summarize the distinct types of existing SAW micromixers, and highlight established applications of SAW micromixing technology in chemical synthesis, nanoparticle fabrication, cell culture, biochemical analysis, and cell lysis. Finally, we present current challenges and some perspectives to motivate further research in this area. The purpose of this work is to provide an in-depth understanding of SAW micromixers and inspire readers who are interested in making some innovations in this research field. Full article

This investigation presents an overview of various optical biosensors utilized for the detection of cancer cells. It covers a comprehensive range of technologies, including surface plasmon resonance $\left(SPR\right)$ sensors, which exploit changes in refractive index $\left(RI\right)$ at the sensor surface to detect biomolecular interactions. Localized surface plasmon resonance $\left(LSPR\right)$ sensors offer high sensitivity and versatility in detecting cancer biomarkers. Colorimetric sensors, based on color changes induced via specific biochemical reactions, provide a cost-effective and simple approach to cancer detection. Sensors based on fluorescence work using the light emitted from fluorescent molecules detect cancer-specific targets with specificity and high sensitivity. Photonics and waveguide sensors utilize optical waveguides to detect changes in light propagation, offering real-time and label-free detection of cancer biomarkers. Raman spectroscopy-based sensors utilize surface-enhanced Raman scattering $\left(SERS\right)$to provide molecular fingerprint information for cancer diagnosis. Lastly, fiber optic sensors offer flexibility and miniaturization, making them suitable for in vivo and point-of-care applications in cancer detection. This study provides insights into the principles, applications, and advancements of these optical biosensors in cancer diagnostics, highlighting their potential in improving early detection and patient outcomes. Full article

It is known that the ciliate Paramecium cell surface including cilia is completely covered by high-molecular-mass GPI-anchored proteins named surface antigens (SAgs). However, their functions are not well understood. It was found that ciliate Paramecium caudatum reversibly changes its SAgs depending on the absence or presence of the endonuclear symbiotic bacterium Holospora obtusa in the macronucleus. Immunofluorescence microscopy with a monoclonal antibody produced SAg of the H. obtusa-free P. caudatum strain RB-1-labeled cell surface of the H. obtusa-free P. caudatum RB-1 cell but not the H. obtusa-bearing RB-1 cell. When this antibody was added to the living P. caudatum RB-1 cells, only H. obtusa-free cells were immobilized. An immunoblot with SAgs extracted from Paramecium via cold salt/ethanol treatment showed approximately 266-kDa SAgs in the extract from H. obtusa-free cells and 188 and 149-kDa SAgs in the extract from H. obtusa-bearing cells. H. obtusa-free RB-1 cells produced from H. obtusa-bearing cells via treatment with penicillin-G-potassium re-expressed 266-kDa SAg, while the 188 and 149-kDa SAgs disappeared. This phenotypic change in the SAgs was not induced by degrees of starvation or temperature shifts. These results definitively show that Paramecium SAgs have functions related to bacterial infection. Full article

Sorting particles or cells of specific sizes in complex systems has long been a focus of many researchers. Acoustic surface waves, which generate acoustic radiation forces on particles or cells and, thus, influence their motion, are commonly used for the non-destructive separation of particles or cells of specific sizes. In previous studies, the frequency of acoustic surface wave generation has been limited by the interdigitated transducer (IDT). To extend the effective operating frequency range of the IDT, a slanted-finger interdigitated transducer (SFIT) with a wide acoustic path and multiple operating frequencies was designed. Compared with traditional acoustic sorting devices, which suffer from a limited frequency range and narrow acoustic paths, this new design greatly expands both the operating frequency range and acoustic path width, and enables adjustable operating frequencies, providing a solution for sorting particles or cells with uneven sizes in complex environments. The optimal resonance frequency is distributed within the 32–42 MHz range, and the operating frequencies within this range can generate a standing wave acoustic path of approximately 200 $\mathsf{\mu }$m, thus enhancing the effectiveness of the operating frequencies. The microfluidic sorting device based on SFIT can efficiently and accurately sort polystyrene (PS) with particle sizes of 20 $\mathsf{\mu }$m, 30 $\mathsf{\mu }$m, and 50 $\mathsf{\mu }$m from mixed PS microspheres (5, 10, 20 $\mathsf{\mu }$m), (5, 10, 30 $\mathsf{\mu }$m), and (5, 10, 50 $\mathsf{\mu }$m), with a sorting efficiency and purity exceeding 96%. Additionally, the device is capable of sorting other types of mixed microspheres (5, 10, 20, 30, 50 $\mathsf{\mu }$m). This new wide-acoustic-path, multi-frequency sorting device demonstrates the ability to sort particlesin a high-purity, label-free manner, offering a more alternative to traditional sorting methods. Full article

Increased abundance of Segatella copri (S. copri) within the gut microbiota is associated with systemic inflammatory diseases, including rheumatoid arthritis. Although outer-membrane vesicles (OMVs) of Gram-negative bacteria are important players in microbiota–host communication, the effect of S. copri-derived OMVs on immune cells is unknown. Macrophages engulf and eliminate foreign material and are conditioned by environmental signals to promote either homeostasis or inflammation. Thus, we aimed to explore the impact of S. copri-OMVs on human macrophages in vitro, employing THP-1 and monocyte-derived macrophage models. The uptake of DiO-labeled S. copri-OMVs into macrophages was monitored by confocal microscopy and flow cytometry. Furthermore, the effect of S. copri and S. copri-OMVs on the phenotype and cytokine secretion of naïve (M0), pro-inflammatory (M1), and anti-inflammatory (M2) macrophages was analyzed by flow cytometry and ELISA, respectively. We show that S. copri-OMVs enter human macrophages through macropinocytosis and clathrin-dependent mechanisms. S. copri-OMVs, but not the parental bacterium, induced a dose-dependent increase in the expression of M1-related surface markers in M0 and M2 macrophages and activated the secretion of large amounts of pro-inflammatory cytokines in M1 macrophages. These results highlight an important role of S. copri-OMVs in promoting pro-inflammatory macrophage responses, which might contribute to systemic inflammatory diseases. Full article

Human papillomavirus type 16 (HPV16) is an etiological agent of human cancers that requires endocytosis to initiate infection. HPV16 entry into epithelial cells occurs through a non-canonical endocytic pathway that is actin-driven, but it is not well understood how HPV16–cell surface interactions trigger actin reorganization in a way that facilitates entry. This study provides evidence that Wiskott–Aldrich syndrome protein family verprolin-homologous proteins 1 and 2 (WAVE1 and WAVE2) are molecular mediators of actin protrusions that occur at the cellular surface upon HPV addition to cells, and that this stimulation is a key step prior to endocytosis and intracellular trafficking. We demonstrate through post-transcriptional gene silencing and genome editing that WAVE1 and WAVE2 are critical for efficient HPV16 infection, and that restoration of each in knockout cells rescues HPV16 infection. Cells lacking WAVE1, WAVE2, or both internalize HPV16 at a significantly reduced rate. Microscopic analysis of fluorescently labeled cells revealed that HPV16, WAVE1, WAVE2, and actin are all colocalized at the cellular dorsal surface within a timeframe that precedes endocytosis. Within that same timeframe, we also found that HPV16-treated cells express cellular dorsal surface filopodia, which does not occur in cells lacking WAVE1 and WAVE2. Taken together, this study provides evidence that WAVE1 and WAVE2 mediate a key step prior to HPV entry into cells that involves actin reorganization in the form of cellular dorsal surface protrusions. Full article

The purpose of this study was to examine whether supplementation of ultra- and nanofiltered colostrum-based products, combined with egg yolk extract, nicotinamide mononucleotide (NMN), quercetin, alpha-ketoglutarate, white button mushroom, and celery seed extracts (the formula was patented by 4Life Research Company, USA and named as AgePro), modulate the functional activity of natural killer (NK) cells in vivo. We found that this supplement, taken orally in two capsules twice a day for 30 days, significantly enhanced the cytotoxic activity of NK cells. This was evidenced by the increased NK cell-mediated killing of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled K562 human myeloid leukemia cells. As expected, this effect was dependent on the ratio between the effector (E) (e.g., peripheral blood mononuclear cells (PBMCs)) and target (T) (e.g., K562) cells, illustrating maximal killing of K562 cells at a 50:1 E/T ratio. Of note, increased NK-mediated killing of K562 cells after taking AgePro correlated with increased perforin release, evidenced by the CD107a degranulation assay. In concordance with these findings, taking of AgePro for 1 month increased production of several cytokines and chemokines, including IL-1β, IL-1Rα, IL-6, IL-8, IL-10, IFN-γ, TNF-α, G-CSF, PDGF-AA, PDGF-AB/BB, GRO, MCP-1, MCP-3, and MIP-1α, in PBMCs co-cultured with K562 cells. Of note, increased production of the cytokines correlated with the activation state of PBMCs, as evidenced by increased expression of the surface activation markers (e.g., the interleukin-2 receptor alpha chain—CD25). A strong correlation was found between NK-based cytotoxic activity and the production of IL-1β, IL-6, TNF-α, and MIP-1α. Importantly, no increase in the aforementioned soluble factors and activation markers was detected in PBMCs cultured alone, thereby illustrating the potent immunoregulatory activity of AgePro only in the presence of the harmful target cells. Hematological parameters also remained unchanged over the entire study period. Collectively, we show herein the significant enhancement of the cytotoxic activity of NK cells against target tumor cells after taking AgePro for 1 month. Notably, this effect was observed for all age groups, including young, adult, and elderly participants. Moreover, a significant improvement in NK cytotoxic activity was also detected for participants with low basal (e.g., before taking AgePro) numbers of NK-mediated killing. The enhancement of NK-based cytotoxicity was associated with an increased release of several cytokines and chemokines involved in regulating a broad spectrum of mechanisms outside the cell-mediated cytotoxicity and killing of target cells. Of note, spontaneous activation of PBMCs, particularly NK cells, was not detected after taking AgePro. Given that spontaneous activation of autoreactive lymphocytes is a feature associated with autoimmunity and taking into account our data illustrating the AgePro-induced activation of NK cells detected only in the presence of the potentially harmful cells, we conclude that our innovative product exhibits potent immunoregulatory activity and high safety profile. Full article

Efficient bacterial lysis and RNA purification are essential for molecular diagnostics and biosensing applications. This study presents a piezoelectric platform integrated with gold-plated RNA capture pins (RCPs) functionalized with synthetic oligonucleotides to extract and enrich E. coli 16S ribosomal RNA (rRNA). The 3D-printed device enables selective bacterial capture using E. coli-specific aptamers and incorporates a piezoelectric transducer operating at 60 kHz to facilitate bacterial cell wall disruption. The platform demonstrated high specificity for E. coli over B. cereus, confirming aptamer selectivity. E. coli viability assessment demonstrated that positioning the piezoelectric plate in contact with the bacterial suspension significantly improved the bacterial lysis, reducing viability to 33.68% after 15 min. RNA quantification confirmed an increase in total RNA released by lysed E. coli, resulting in 10,913 ng after 15 min, compared to 4310 ng obtained via conventional sonication. RCP-extracted RNA has a threefold enrichment of 16S rRNA relative to 23S rRNA. RT-qPCR analysis indicated that the RCPs recovered, on average, 2.3 ng of 16S RNA per RCP from bacterial suspensions and 0.1 ng from aptamer-functionalized surfaces. This integrated system offers a rapid, selective, and label-free approach for bacterial lysis, RNA extraction, and enrichment for specific types of RNA with potential applications in clinical diagnostics and microbial biosensing. Full article

Surface acoustic wave (SAW)-based microfluidics has emerged as a promising technology for precisely manipulating particles and cells at the micro- and nanoscales. Acoustofluidic devices offer advantages such as low energy consumption, high throughput, and label-free operation, making them suitable for particle manipulation tasks including pumping, mixing, sorting, and separation. In this review, we provide an overview and discussion of recent advancements in SAW-based microfluidic devices for micro- and nanoparticle manipulation. Through a thorough investigation of the literature, we explore interdigitated transducer designs, materials, fabrication techniques, microfluidic channel properties, and SAW operational modes of acoustofluidic devices. SAW-based actuators are mainly based on lithium niobate piezoelectric transducers, with a plethora of wavelengths, microfluidic dimensions, and transducer configurations, applied for different fluid manipulation methods: mixing, sorting, and separation. We observed the accuracy of particle sorting across different size ranges and discussed different alternative device configurations to enhance sensitivity. Additionally, the collected data show the successful implementation of SAW devices in real-world applications in medical diagnostics and environmental monitoring. By critically analyzing different approaches, we identified common trends, challenges, and potential areas for improvement in SAW-based microfluidics. Furthermore, we discuss the current state-of-the-art and opportunities for further research and development in this field. Full article

The study of infected biological cells is crucial in modern biomedical research. This work presents a passive sensing approach using optical resonators, designed to detect malignant diseases within a refractive index (RI) range of 1 to 1.5. A comprehensive theoretical analysis is conducted, yielding an expected limit of detection (LoD) ranging from 0.03 nm/RIU to 0.92 nm/RIU. Furthermore, an in-depth investigation of DNA hybridization is performed, incorporating a 1.8 nm linker layer at the analyte boundary. The refractive indices of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) are 1.456 and 1.529, respectively. The novelty of this work lies in the renaturation process of ssDNA to dsDNA, demonstrated through a labeled sensing modality with a measurable shift in the resonance wavelength spectrum. The proposed surface-functionalized resonators, designed using Silicon-on-Insulator (SOI) technology, include (a) a Rectangular Waveguide-based Ring Resonator (RWRiR), (b) a Rectangular Waveguide-based Racetrack Resonator (RWRaR), (c) a Slot Waveguide-based Ring Resonator (SWRiR), and (d) a Slot Waveguide-based Racetrack Resonator (SWRaR). Among these, the SWRiR exhibits the best performance for DNA sensing, achieving a quality factor (Q-factor) of 2216.714, a sensitivity (S) of 54.282 nm/RIU, and a normalized sensitivity (S’) of 0.0349. Full article