Jul 2026· Talanta: The International Journal of Pure and Applied Analytical Chemistry· Vol 312 Pt A, pp.
130369
· 0 citations· 52 references
Medicine
TL;DR
A homogeneous fluorescence polarisation immunoassay for the quantification of EVs from whole blood using an antibody tracer using a portable fluorescence polarisation analyser with microfluidic devices to simplify EV quantification after pretreatment.
Abstract
Extracellular vesicles (EVs) are important biomarkers in disease diagnosis and monitoring. However, their quantification typically relies on laboratory instruments, limiting their application in point-of-care testing. In this study, we report a homogeneous fluorescence polarisation immunoassay for the quantification of EVs from whole blood using an antibody tracer. The assay uses a portable fluorescence polarisation analyser with microfluidic devices to simplify EV quantification after pretreatment. To reduce nonspecific binding of the tracer to the microfluidic channel walls, we employed polyethylene glycol-modified PDMS, which eliminates the need for blocking agents. Consequently, this study enabled homogeneous quantification using a portable fluorescence polarisation analyser, achieving a limit of detection of 5.0 × 107 particles/mL and a limit of quantification of 6.6 × 107 particles/mL. Overall, this approach provides a simple, and reagent-efficient method for EV quantification from complex biological matrices, highlighting the potential of fluorescence polarisation immunoassay as a practical tool for future EV-based clinical studies.
This proof-of-concept-study presents a novel microsensor based on specifically grafted functionalized glass for detection of low concentrated biomarkers in human fluids, gels, and for liquid biopsy. The device integrates functionalized nanofibers with grafted glass, creating an easy-to-use, rapid, and cost-effective biosensor. The approach combines fractionated nanofiber membranes with functionalized surfaces and light-scattering analysis on grafted glass which serves as a signal enhancer for data evaluation by light scattering. This method offers a novel tool for a quick evaluation of low concentrated protein samples. Experimental validation in this study confirmed its ability to identify low-abundance markers, supporting applications in precision medicine, tissue functional age assessment, and bacterial monitoring. Compared with conventional laboratory assays, optical sensors built on functionalized glass enable rapid, sensitive, and multiplexed biomarker detection directly at the point of care. We observed distinct changes in light transmittance caused by specific interactions between microsized particles and antibodies immobilized on the glass surface. This principle can also be applied to the direct detection of micrometer-scale systems such as bacteria, enabling fast urine analysis. Key words Biosensors " Nanofibers " Biomarkers " Precision medicine " Tissue aging.
Rossella Sperlongano, M. Vjaclovsky, Si Chen et al.· Physiological Research· 0 citations
The LIGMA assay is established as a viable electrokinetic strategy for improving paper-based biosensors, providing a foundation for expanded applications in clinical diagnostics, multiplexed detection, and portable field-deployable biosensing platforms.
D. McCornack, C. Ivory, Zhihong Zhang et al.· Analytical Chemistry· 0 citations
Electrochemical Lateral Flow Immunoassays (eLFIA) are emerging as powerful possibility for in vitro diagnostic tools due to their sensitivity, small size and affordable price. However, the development of non-toxic and biodegradable nanolabels that enable sensitive quantification remains a challenge. In this study, three nanocarriers (liposomes, niosomes, and lipid-polymer micelles) were synthesized and characterized, encapsulating the starch-polyiodide complex as electrochemical probe for eLFIA. After evaluating their size, morphology, molecular structure, encapsulation efficiency, and colloidal stability, micelles were selected as the ideal nanolabel for this eLFIA. A proof of concept was performed using the biotin-neutravidin model system, thus evaluating its sensitivity and detection limit, reaching a detectable concentration of 17 nM by performing a fixed-potential coulometry for three minutes, hence, employing just a little over 20 min for the total assay. This system constitutes, to the authors' knowledge, the first in vitro diagnostic device based on sustainable bio-derived nanoparticles, paving the way for future environmentally friendly tests.
C. Saweres-Argüelles, A. Sánchez-Calvo, Gemma Gutiérrez et al.· Colloids and Surfaces B: Bio...· 0 citations
Fluorescence-based nanoparticle tracking analysis (Fl.NTA) enables selective detection and quantification of labelled nanoparticles. However, the lack of a standardized workflow has hindered the reproducibility and comparability of concentration measurements across different studies. Here, we established a comprehensive Fl.NTA protocol using fluorescent polystyrene bead standards (Fluoresbrite 50/100 nm; FluoSpheres 100/200 nm). We systematically optimized the standardized workflow by aligning excitation modules and long-pass emission filters with fluorophore spectra, and by rigorously defining key acquisition and processing parameters, such as camera level, camera shutter, and detection threshold. To ensure reliable quantification, the proposed protocol was validated through day-to-day repeatability tests and an intra-laboratory comparison involving five independent analysts. Our results demonstrate that while particle sizing remains robust (inter-analyst CVs 0.0-8.0%), concentration recovery is highly sensitive to optical configurations and signal detectability, particularly for sub-100 nm particles. The workflow was further applied to biological extracellular vesicles (EVs), including CD63-EGFP and CD9-QD625 labelled EVs. The significant variance in Fl:Sc concentration ratios (13.8% for EGFP vs. 124.5% for QD625) underscores the necessity of considering fluorophore photostability and labelling efficiency for reliable quantification. Overall, this study provides a practical, performance-driven standardized workflow for Fl.NTA that addresses existing operational gaps in ISO 19430:2024 and provides a practical foundation for the consistent and reproducible characterization of heterogeneous bio-nanoparticles in EV research and quality control.
Yewon Han, Minseop Kim, Zayakhuu Gerelkhuu et al.· Methods· 0 citations
Aim: High-sensitivity flow cytometry (FC) allows multiparametric analysis of nanoparticles (NPs) and extracellular vesicles (EVs). With new instruments available, studies that evaluate their performance using the same materials in a controlled environment are required. Here, we performed a comparative study to investigate the capabilities of three flow cytometers, the NanoFCM (NF), BD Influx (IF) and CytoFLEX LX (CF).
Methods: Firstly, we analyzed a mixed population of silica NPs (SiNPs, 68, 91, 113 and 155 nm) by using light-scatter-based detection thresholds [side scatter (SSC), forward scatter (FSC), violet side scatter (VSSC)] across a concentration range from 106 to 109 particles/mL. Next, we analyzed fluorescent recombinant EVs (rEVs) by comparing light-scatter-based thresholding (488 nm SSC available for all platforms), the combination of SSC thresholding with a fluorescent gate, and fluorescent thresholding.
Results: Upon qualitative and quantitative analysis, we observed that instruments differed in sensitivity, the NF could detect 68 nm SiNPs, while both IF and CF were able to detect down to 91 nm SiNPs when using a scatter-based threshold, which was improved by using FSC and VSSC compared to SSC, respectively. We show that the NF required a higher sample concentration to ensure optimal detection, while IF and CF benefited from more diluted samples. Next, we defined a single particle detection range measuring fluorescent rEV and demonstrated that fluorescence-based detection improved the detection of particles of interest due to lower background interference.
Conclusion: We here provide the strengths and limitations for each platform regarding the analysis of differently sized NPs at different sample concentrations.
E. Lozano-Andrés, Ye Tian, S. Libregts et al.· Extracellular Vesicles and C...· 0 citations
Extracellular vesicles (EVs) are recognized as important mediators of intercellular communication and potential diagnostic biomarkers. However, their precise characterization, particularly from complex biological samples like platelet-poor plasma (PPP), demands highly standardized methods. Our work establishes a complete process for the enrichment, fluorescent staining, and multicolor flow cytometric analysis of EVs from PPP using the CytoFLEX nano Flow Cytometer, leveraging its improved violet side scatter (VSSC-1) for sensitive detection.
Izon qEV SEC fractionation was used to enrich EVs from PPP, followed by serial dilutions of EV-enriched fractions to determine the optimal dilution at which the swarming effect is avoided. Thorough antibody titration was performed to minimize antibody aggregation and maintain background noise at the level set at the beginning of acquisition.
During our investigation, we determined three crucial factors that could affect EV staining and thus their characterization:
1. Variation between different donors within the same fraction.
2. Differences among CD9 antibody clones in both the detected amount of PPP EVs and their Median Fluorescence Intensity (MFI), highlighting the importance of selecting the optimal clone for staining.
3. Confirmation of the importance of proper clone selection in multicolor analysis, along with demonstration of fluorochrome combinations using different fluorochromes conjugated to the same CD9 clone. In our multicolor flow cytometry analysis, we further showed that besides CD9, PPP EVs also express CD61, whereas CD63 and CD81 exhibited very low–if any–expression levels.
In conclusion, our standardized process demonstrates the importance of optimizing EV and sample preparation, along with careful attention to the EV fraction, donor variability, and antibody choices (clone, fluorochrome, and dilutions). These factors influence the accuracy and consistency of EV staining and characterization.
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Technological Innovations in Immunology (TECH)
A. Jose, Rita K. Bowers, Evgenia Kim et al.· Journal of Immunology· 0 citations