TY - JOUR A1 - Tobias, Charlie A1 - López-Puertollano, Daniel A1 - Abad-Somovilla, Antonio A1 - Mercader, J. V. A1 - Abad-Fuentes, A. A1 - Rurack, Knut T1 - Development of Simple and Rapid Bead-Based Cytometric Immunoassays Using Superparamagnetic Hybrid Core−Shell Microparticles N2 - Flow cytometry-based immunoassays are valuable in biomedical research and clinical applications due to their high throughput and multianalyte capability, but their adoption in areas such as food safety and environmental monitoring is limited by long assay times and complex workflows. Rapid, simplified bead-based cytometric immunoassays are needed to make these methods viable for point-of-need applications, especially with the increasing accessibility of miniaturized cytometers. This work introduces superparamagnetic hybrid polystyrene-silica core−shell microparticles as promising alternatives to conventional polymer beads in competitive cytometric immunoassays. These beads, featuring high specificity, sensitivity, and excellent handling capabilities via magnetic separation, were evaluated with three different antibodies and binding methods, showing variations in signal intensity based on the antibody and its attachment method. The optimal performance was achieved through a secondary antibody binding approach, providing strong and consistent signals with minimal uncertainty. The optimized protocol made it possible to achieve a detection limit of 0.025 nM in a total assay time of only 15 min and was successfully used to detect ochratoxin A (OTA) in raw flour samples. This work highlights the potential of these beads as versatile tools for flow cytometry-based immunoassays, with significant implications for food safety, animal health, environmental monitoring, and clinical diagnostics. KW - Immunoassays KW - Bead-based KW - Core-shell particles KW - Cytometry KW - Mycotoxins PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-610593 DO - https://doi.org/10.1021/acsmeasuresciau.4c00038 SP - 1 EP - 11 PB - American Chemical Society CY - Washington, D.C. AN - OPUS4-61059 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - López-Puertollano, Daniel A1 - Duncan, Hadyn A1 - Abad-Somovilla, Antonio A1 - Abad-Fuentes, Antonio A1 - Rurack, Knut T1 - Competitive cytometry-based immunoassay for patulin determination in apple juice N2 - Patulin is a mycotoxin that is frequently found in apples and apple-derived products. Given the potential harm it can cause to humans, maximum levels for patulin in food have been set worldwide. Conventional methods for the detection of patulin are often time-consuming or lack sensitivity. In this study, a novel cytometry approach based on specific monoclonal antibodies is presented. These high-affinity binders do not target patulin itself, but a stable derivative (adduct) that is rapidly obtained in an aqueous medium at room temperature. To develop the assay, a specific fluorescent competitor was designed and synthesized. After optimizing the assay conditions, including the concentration of the fluorescent competitor and of the antibody bound to polystyrene-silica core–shell microparticles, a detection limit of 0.03 μg L-1 in buffer was achieved. Finally, validation according to Commission Regulation (EU) 2023/2782 demonstrated that apple juice samples spiked with patulin at 25 or 50 μg L-1 (permissible limits set by the EU) were properly scored as non-compliant without any additional treatment other than a simple dilution step in buffer. The developed assay offers several key advantages, including rapid analysis, high sensitivity and specificity, and the potential for multiplexing, making it a promising analytical tool for routine monitoring of patulin contamination in food. KW - Bead-based assay KW - Cytometry KW - Derivatization KW - Mycotoxin KW - Patulin PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-627659 DO - https://doi.org/10.1016/j.microc.2025.113287 SN - 1095-9149 VL - 212 SP - 1 EP - 8 PB - Elsevier B.V. AN - OPUS4-62765 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - López-Puertollano, Daniel A1 - Tobias, Charlie A1 - Bell, Jérémy A1 - Abad-Somovilla, A. A1 - Abad-Fuentes, A. A1 - Rurack, Knut T1 - Superparamagnetic Bead-Based Microfluidic Fluoroimmunoassay Platform for Rapid Ochratoxin A Detection in Flour N2 - Simplification and reduction of time and costs are the primary goals in the development and use of onsite methods in diagnostics and food safety. To facilitate the transition from laboratory techniques to simple, miniaturized devices, we have developed a modular microfluidic platform. This platform integrates a competitive fluorescence immunoassay on the surface of superparamagnetic beads, serving as a complementary technique to traditional cytometry assays. In the first chip module, a fast competitive reaction (5 min) occurs, after which the particles are retained in the second module. This module consists of a PDMS chip and a permanent magnet, allowing only the fluorescent competitor to reach the detection module. Ochratoxin A (OTA) was chosen as the model analyte for device development, using fluorescein-labeled OTA as a competitor. The system efficiently separates particles, with OTA concentration directly correlated to the amount of fluorescent competitor remaining in solution after the competitive reaction. This innovative setup allows to perform rapid measurements with small sample volumes in a short time (10 min), achieving a limit of detection for OTA of 1.2 μg L–1. The system was successfully applied to the accurate determination of OTA in wheat flour spiked at regulatorily relevant concentrations. Using this device, conventional cytometry immunoassays can be seamlessly transformed into user-friendly, miniaturized analytical methods at reduced cost for applications outside of a laboratory directly at the point of need. KW - Bead-based assay KW - Fluorescence KW - Immunoassay KW - Microfluidics KW - mycotoxins PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-638472 DO - https://doi.org/10.1021/acssensors.5c01119 SN - 2379-3694 SP - 1 EP - 10 PB - American Chemical Society CY - Washington, D.C. AN - OPUS4-63847 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - López-Puertollano, Daniel T1 - Superparamagnetic core-shell particles application: from cytometry assay to simplified fluidic system N2 - Superparamagnetic hybrid polystyrene-core silica-shell beads have emerged as promising alternatives to traditional in flow cytometry-based competitive antibody assays [1]. These materials consist of a polystyrene core and a silica shell, in which magnetic nanoparticles are embedded, facilitating the handling and retention in tests. The outer silica surface allows for easy modification through silane chemistry, allowing the attachment of antibodies, or other molecules of interest. Ochratoxin A (OTA), a mycotoxin that can be found in grain products, coffee, cacao, or grapes, was chosen as the main target analyte to detect [2]. In this study, previously in house produced anti-OTA antibodies [3] were attached to the surface of the particles and the whole system was used as detection entity. In a first approach, the system was used for the development of a competitive cytometry assay using an OTA-fluorescein (OTA-F) adduct as competitor and marker. In this assay the fluorescence emitted by the OTA-F competitor on the surface of the particle was detected at a wavelength of 518 nm using a 533/30.H filter and was correlated to the forward scatter (FSC) to distinguish it from the excess of competitor still in solution. Under optimised conditions, the final assay showed a limit of detection of 0.03 nM. In a second approach, a simplified ready-to-inject fluidic system was built based on a laser (488 nm) and a photomultiplier detector to measure the signal of competitor still in solution. The competition step was carried out in a vial and the whole mixture was injected into the fluidic system. To avoid signal scattering, the particles were separated in-line using a magnet and only the OTA-F competitor still in solution was detected, reaching a limit of detection of 1.2 nM. With the aim to reduce user manipulation, the final assay is still under development for in-line incubation during the competitive step. T2 - 15th Rapid Methods Europe Conference CY - Amsterdam, Netherlands DA - 06.11.2023 KW - Microfluidics KW - Flow cytometry KW - Bead-based assays KW - Magnetic beads KW - Core-shell particles KW - Immunoassays PY - 2023 AN - OPUS4-58817 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -