TY - JOUR A1 - Gregório, Bruno J.R. A1 - Ramos, Inês I. A1 - Marques, Sara S. A1 - Barreiros, Luísa A1 - Magalhães, Luís M. A1 - Schneider, Rudolf A1 - Segundo, Marcela A. T1 - Microcarrier-based fluorescent yeast estrogen screen assay for fast determination of endocrine disrupting compounds N2 - The presence of endocrine-disrupting compounds (EDCs) in water poses a significant threat to human and animal health, as recognized by regulatory agencies throughout the world. The Yeast Estrogen Screen (YES) assay is an excellent method to evaluate the presence of these compounds in water due to its simplicity and capacity to assess the bioaccessible forms/fractions of these compounds. In the presence of a compound with estrogenic activity, Saccharomyces cerevisiae cells, containing a lacZ reporter gene encoding the enzyme β-galactosidase, are induced, the enzyme is synthesised, and released to the extracellular medium. In this work, a YES-based approach encompassing the use of a lacZ reporter gene modified strain of S. cerevisiae, microcarriers as solid support, and a fluorescent substrate, fluorescein di-β-D-galactopyranoside, is proposed, allowing for the assessment of EDCs’ presence after only 2 h of incubation. The proposed method provided an EC50 of 0.17 ± 0.03 nM and an LLOQ of 0.03 nM, expressed as 17β-estradiol. The assessment of different EDCs provided EC50 values between 0.16 and 1.2 × 103 nM. After application to wastewaters, similar results were obtained for EDCs screening, much faster, compared to the conventional 45 h spectrophotometric procedure using a commercial kit, showing potential for onsite high-throughput screening of environmental contamination. KW - Biosensoren KW - YES assay KW - Endokrine Disruptoren PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-602315 DO - https://doi.org/10.1016/j.talanta.2024.125665 VL - 271 SP - 1 EP - 7 PB - Elsevier B.V. CY - Amsterdam AN - OPUS4-60231 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Ramos, Inês I. A1 - Carl, Peter A1 - Schneider, Rudolf A1 - Segundo, Marcela A. T1 - Development of automated immunosensing methods for clinical and environmental Analysis using lab-on-valve platforms N2 - The lab-on-valve (LOV) is a mesofluidic platform that has been recently exploited for the automation and miniaturization of bioanalytical assays, resorting namely to molecular recognition schemes based on immunosensing. Due to its high versatility for reagent accommodation, it is possible to establish immunoassays under several formats (eg. direct competitive ELISA, sandwich ELISA or even label-free immunoaffinity chromatography). For instance, the LOV has been used as a manifold for UV-vis micro-Bead Injection Spectroscopy (μ-BIS), a technique that involves the quantification of the target analyte by direct measurement on the surface of a solid phase capable of retaining the target analyte by molecular recognition. The μ-BIS-LOV strategy affords several analytical advantages, namely short time-toresult intervals (3 to 15 min), low sample volume (1-20 μL), automated solution handling and washing steps, downscaling of reagents’ consumption, low-cost analysis and little generation of waste. Additionally, the solid support is renewed before each determination, minimizing surface fouling, cross-contamination issues and functional group deactivation. No sample clean-up steps are required because interferences are separated from the target analyte upon quantification mediated by a molecular recognition element attached to the micro-bead column. The portability of the LOV device makes it compatible with point-of-care testing. To our knowledge, this technique has been mainly employed for the evaluation and optimization of bioaffinity processes, but its potential for clinical and environmental analysis remains underexploited. Hence, in this communication, different immunosensing strategies using the LOV platform will be addressed, namely the determination of autoimmune IgG in human serum, and the assessment of drug (carbamazepine) levels in wastewater samples. T2 - EBS 2017 CY - Potsdam, Germany DA - 20.03.2017 KW - Lab-on-valve KW - Automation KW - Immunoassay PY - 2017 AN - OPUS4-39521 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Carl, Peter A1 - Ramos, Inês I. A1 - Segundo, Marcela A. A1 - Schneider, Rudolf T1 - Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure N2 - Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagentsmare expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via Nhydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficient coupling. It is demonstrated, that the hydrolysis of N-hydroxysuccinimide esters occurs at similar rates as in solution, limiting the utilizable time for coupling. Finally, applicability of the generated material for automated affinity extraction is demonstrated on the mesofluidic platform lab-on-valve. KW - Mesofluidics KW - Bead injections KW - ELISA KW - Carbamazepine KW - Biomarkers PY - 2019 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-483919 DO - https://doi.org/10.1371/journal.pone.0218686 SN - 1932-6203 VL - 14 IS - 6 SP - e0218686, 1 EP - 18 PB - Public Library of Science CY - San Francisco, California, USA AN - OPUS4-48391 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -