Antonio Abad-Fuentes

Sensitive enough detection of small-molecule toxins and many other target analytes remains challenging for immunoassays that rely on stoichiometric reporters or enzymatic cascades. Here we introduce an enzyme-free, light-gated amplification strategy that teams up stabilized leucomethylene blue (LMB) derivatives with antibody recognition and photoredox cycling. As a proof-of-concept, we designed a stable LMB precursor (δ-BLMB) and a BLMB–zearalenone (ZON) conjugate that undergo rapid, selective photo-oxidation to methylene blue (MB) only under the assay illumination. Electrochemical analysis has established δ-BLMB as a redox precursor to MB, while time-resolved luminescence of [Ru­(phen)₃]²⁺ (RP3) photocatalyst revealed diffusion-limited quenching (k _q = 2.68 × 10⁹ M^–1 s^–1) consistent with dynamic electron transfer to the precursor. Mechanistic studies show that blue light activates RP3 to generate acyl-radical and MB^– intermediates via O₂-independent pathways; red light is then used to excite nascent MB to drive a secondary photocatalytic reductive cycle that regenerates MB through radical/anionic intermediates, yielding exponential signal growth. We have validated immunoassay integration using ZON-specific monoclonal antibodies in bead-based and plate formats, demonstrating specific, light-triggered fluorescence that reports competitive binding. Its implementation with sequential two-color illumination demonstrates selective system activation and precise control of the signal amplification (300-fold). This photochemical approach provides stable, long-term reagents that can be tailored to the target species and offers a potentially general strategy for developing enzyme-free, high sensitivity immunoassays for small analytes.

Immunoassays have been developed for all regulated mycotoxins, including citrinin, whose presence in food supplements has recently undergone additional restrictions in Europe. Immunoassays for mycotoxins most commonly use monoclonal antibodies alongside an enzyme tracer, resulting in the so-called direct antibody-coated competitive ELISA format. Notably, no immunoassay of this kind has been published for citrinin to date. To address this, a novel hapten featuring an azide-functionalised linker for click chemistry conjugation has been designed. Following confirmation by molecular modelling that this derivative is an excellent structural mimic of the mycotoxin, a functional enzyme tracer and an immunising conjugate were prepared and characterised by MALDI-TOF. Subsequently, high-affinity monoclonal antibodies were produced through a double screening process for hybridoma selection. In combination with the click chemistry tracer, these novel antibodies show promise as immunoreagents for setting up innovative direct immunoassays for the sensitive determination of citrinin in food supplements containing fermented red rice

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.

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.

Mycotoxins are globally pervasive contaminants that threaten food safety worldwide. Regulatory authorities have established maximum permissible levels for certain mycotoxins, and their presence is routinely monitored throughout the food chain to ensure the provision of healthy food and safe feed for humans and animals. While immunoanalytical methods are essential for mycotoxin screening, monoclonal antibodies for the detection of patulin are notably absent. Moreover, leading immunodiagnostic companies currently do not offer rapid tests for patulin in their product portfolios. This deficiency in mycotoxin testing is primarily due to the electrophilic reactivity of patulin. In this study, we exploit this reactivity to develop an innovative strategy that targets the stable adduct formed by the reaction of patulin with aryl-1,2-dithiolates, rather than analyzing the mycotoxin itself. Based on this previously unknown reaction, we present the first collection of monoclonal antibodies, enabling the long-sought goal of sensitive, simple, and user-friendly immunosensing of patulin.

Fungal plant pathogens have posed a significant threat to crop production. However, the large-scale application of pesticides is associated with possible risks for human health and the environment. Boscalid is a widely used fungicide, consistently implemented for the management of significant plant pathogens. Conventionally, the detection and determination of boscalid residues is based on chromatographic separations. In the present study, a Bioelectric Recognition Assay (BERA)-based experimental approach combined with MIME technology was used, where changes in the electric properties of the membrane-engineering cells with anti-boscalid antibodies were recorded in response to the presence of boscalid at different concentrations based on the maximum residue level (MRL) for lettuce. The membrane-engineering Vero cells with 0.5 μg/mL of antibody in their surface were selected as the best cell line in combination with the lowest antibody concentration. Furthermore, the biosensor was tested against another fungicide in order to prove its selectivity. Finally, the BERA cell-based biosensor was able to detect the boscalid residue, below and above the MRL, in spiked lettuce leaf extracts in an entirely distinct and reproducible manner. This study indicates that the BERA-based biosensor, after further development and optimization, could be used for the routine, high-throughput detection of boscalid residue in lettuce, and not only that.

Highly active antiretroviral therapy (HAART) includes very potent drugs that are often characterized by high toxicity. Tenofovir (TFV) is a widely used drug prescribed mainly for pre-exposure prophylaxis (PreP) and the treatment of human immunodeficiency virus (HIV). The therapeutic range of TFV is narrow, and adverse effects occur with both underdose and overdose. The main factor contributing to therapeutic failure is the improper management of TFV, which may be caused by low compliance or patient variability. An important tool to prevent inappropriate administration is therapeutic drug monitoring (TDM) of compliance-relevant concentrations (ARCs) of TFV. TDM is performed routinely using time-consuming and expensive chromatographic methods coupled with mass spectrometry. Immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFIAs), are based on antibody–antigen specific recognition and represent key tools for real-time quantitative and qualitative screening for point-of-care testing (POCT). Since saliva is a non-invasive and non-infectious biological sample, it is well-suited for TDM. However, saliva is expected to have a very low ARC for TFV, so tests with high sensitivity are required. Here, we have developed and validated a highly sensitive ELISA (IC50 1.2 ng/mL, dynamic range 0.4–10 ng/mL) that allows the quantification of TFV in saliva at ARCs and an extremely sensitive LFIA (visual LOD 0.5 ng/mL) that is able to distinguish between optimal and suboptimal ARCs of TFV in untreated saliva.