Person: Benito Peña, María Elena
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First Name
María Elena
Last Name
Benito Peña
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Químicas
Department
Química Analítica
Area
Química Analítica
Identifiers
11 results
Search Results
Now showing 1 - 10 of 11
Item Molecular super-gluing: a straightforward tool for antibody labelling and its application to mycotoxin biosensing(Analytical and Bioanalytical Chemistry, 2022) Prádanas González, Fernando; Glahn Martínez, Ana Bettina; Benito Peña, María Elena; Arola, Henri O.; Nevanen, Tarja K.; Moreno Bondi, María CruzMycotoxins are low molecular weight toxic compounds, which can cause severe health problems in animals and humans. Immunoassays allow rapid, simple and cost-efective screening of mycotoxins. Sandwich assays with a direct readout provide great improvement in terms of selectivity and sensitivity, compared to the widely used competitive assay formats, for the analysis of low molecular weight molecules. In this work, we report a non-competitive fuorescence anti-immune complex (IC) immunoassay, based on the specifc recognition of HT-2 toxin with a pair of recombinant antibody fragments, namely antigen-binding fragment (Fab) (anti-HT-2 (10) Fab) and single-chain variable fragment (scFv) (anti-IC HT-2 (10) scFv). The SpyTag and SpyCatcher glue proteins were applied for the frst time as a bioconjugation tool for the analysis of mycotoxins. To this aim, a SpyTag-mScarlet-I (fuorescent protein) and scFv-SpyCatcher fusion proteins were constructed, produced and fused in situ during the assay by spontaneous Tag-Catcher binding. The assay showed an excellent sensitivity with an EC50 of 4.8±0.4 ng mL−1 and a dynamic range from 1.7±0.3 to 13±2 ng mL−1, an inter-day reproducibility of 8.5% and a high selectivity towards HT-2 toxin without cross-reactivity with other Fusarium toxins. The bioassay was applied to the analysis of the toxin in an oat reference material and in oat samples, with a LOD of 0.6 µg kg−1, and the results were validated by analysing a certifcate reference material and by HPLC–MS/MS.Item Comparative Study of the Performance of Two Different Luciferases for the Analysis of Fumonisin B1 in Wheat Samples(Analysis & Sensing, 2022) Luque Uría, Álvaro; Peltomaa, Riikka; Navarro Duro, Marina; Fikacek, Sabrina; Head, Trajen; Deo, Sapna K.; Daunert, Sylvia; Benito Peña, María Elena; Moreno Bondi, María CruzThe development of two different immunoassays for the determination of fumonisin B1 in wheat samples is reported. A previously described mimopeptide for fumonisin B1 (FB1) was used to produce fusion proteins in combination with two different luciferases: Gaussia luciferase (GLuc) and NanoLuc luciferase (NLuc). The production, expression and the development of two immunoassays based on these fusion proteins (A2- GLuc and A2-NLuc) is detailed. The assay showing the best performance, A2-NLuc, with a limit of detection of 0.61 ngmL 1 and a dynamic range from 1.9 to 95 ngmL 1 , was employed for the analysis of spiked wheat samples, a reference matrix material, as well as naturally contaminated wheat samples. The recoveries obtained in the spiked samples were acceptable, between 81.5 and 109%, with relative standard deviations lower than 14%. The analysis of naturally contaminated wheat was validated by a liquid chromatography coupled to tandem mass detection method.- Project number: 179
Item "Chem-game", el juego como estrategia para la dinamización del aprendizaje y la evaluación de conocimientos en Química General(2020) Moreno Bondi, María Cruz; Gutiérrez Alonso, Angel; Lainez Ferrando, Alfredo; Benito Peña, María Elena; Glahn Martínez, Ana Bettina; García Pinar, Gioele; Castellano Perdomo, Raúl; Navarro Duro, Marina; Peng, Wenrong; Morales Benítez, Noelia; Rubio Ramón, Pablo Ignacio; Olmos Alonso, InésEste proyecto pretende aplicar la "gamificación" en la enseñanza de la asignatura de Química General de primer curso del grado en Química para fomentar la formación, creatividad, compromiso y la capacidad de trabajo en equipo de los estudiantes. Item Extracting mycotoxins from edible vegetable oils by using green, ecofriendly deep eutectic solvents(Food Chemistry, 2023) Pradanas-González, Fernando; Aragoneses-Cazorla, Rubén; Ángel Merino-Sierra, Miguel; Andrade-Bartolomé, Elena; Navarro-Villoslada, Fernando; Benito-Peña, Elena; Moreno-Bondi, María Cruz; Moreno Bondi, María Cruz; Benito Peña, María Elena; Navarro Villoslada, Fernando; Prádanas González, FernandoIn this work, we developed an environmentally friendly liquid–liquid microextraction method using a natural deep eutectic solvent in combination with liquid chromatography for the simultaneous determination of four mycotoxins (deoxynivalenol, alternariol, ochratoxin A and zearalenone) in edible vegetable oils. A chemometric approach assessed the effect of the operational parameters on the mycotoxin extraction efficiency. The extracts were analyzed by HPLC coupled with a diode array and fluorescence detector. The optimum NADES composition resulted in the highest extraction recoveries, and it was applied to coextract the target mycotoxins in several types of edible vegetable oils without using hazardous solvents or requiring further clean-up. The limits of detection ranged from 0.07 to 300μg/kg, and recoveries were close to 100%, except for zearalenone(viz. 35%), with relative standard deviations below 9% in all cases. The proposed method was validated following the European Commission 2002/657/EC and 2006/401/EC.Item Magnetic Janus micromotors for fluorescence biosensing of tacrolimus in oral fluids(Biosensors and Bioelectronics, 2024) Glahn Martínez, Ana Bettina; Jurado-Sánchez, Beatriz; Benito Peña, María Elena; Escarpa, Alberto; Moreno Bondi, María CruzTacrolimus (FK506) is a macrolide lactone immunosuppressive drug that is commonly used in transplanted patients to avoid organ rejection. FK506 exhibits high inter- and intra-patient pharmacokinetic variability, making monitoring necessary for organ graft survival. This work describes the development of a novel bioassay for monitoring FK506. The bioassay is based on using polycaprolactone-based (PCL) magnetic Janus micromotors and a recombinant chimera receptor that incorporates the immunophilin tacrolimus binding protein 1A (FKBP1A) tagged with Emerald Green Fluorescent Protein (EmGFP). The approach relies on a fluorescence competitive bioassay between the drug and the micromotors decorated with a carboxylated FK506 toward the specific site of the fluorescent immunophilin. The proposed homogeneous assay could be performed in a single step without washing steps to separate the unbound receptor. The proposed approach fits the therapeutic requirements, showing a limit of detection of 0.8 ng/mL and a wide dynamic range of up to 90 ng/mL. Assay selectivity was evaluated by measuring the competitive inhibition curves with other immunosuppressive drugs usually co-administered with FK506. The magnetic propulsion mechanism allows for efficient operation in raw samples without damaging the biological binding receptor (FKBP1A-EmGFP). The enhanced target recognition and micromixing strategies hold considerable potential for FK506 monitoring in practical clinical use.Item Homogeneous immunoassay for cyclopiazonic acid based upon mimotopes and upconversion-resonance energy transfer(Biosensors and Bioelectronics, 2023) Prádanas González, Fernando; Peltomaa, Riikka Johanna; Satu Lahtinen; Luque Uria, Álvaro; Vicente Más; Rodrigo Barderas; Chris M. Maragos; Canales Mayordomo, María Ángeles; Tero Soukka; Benito Peña, María Elena; Moreno Bondi, María CruzStrains of Penicillium spp. are used for fungi-ripened cheeses and Aspergillus spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin α-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel epitope mimicking peptide, or mimotope, selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved, and the analysis of individual phage clones revealed four different mimotope peptide sequences. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, surface plasmon resonance binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion nanoparticles (UCNP, type NaYF4:Yb3+, Er3+) were used as energy donors and coated with streptavidin to anchor the synthetic biotinylated mimotope. Alexa Fluor 555, used as an energy acceptor, was conjugated to the anti-CPA antibody fragment. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg/mL (1.5 μg/kg) and an IC50 value of 0.36 ng/mL. No significant cross-reactivity was observed with other co-produced mycotoxins. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) as a reference method.Item Competitive upconversion-linked immunoassay using peptide mimetics for the detection of the mycotoxin zearalenone(Biosensors and Bioelectronics, 2020) Peltomaa, Riikka Johanna; Farka, Zdeněk; Mickert, Matthias ; Brandmeier, Julian ; Pastucha, Matěj; Hlaváček, Antonín; Martínez Orts, Mónica; Canales Mayordomo, María Ángeles; Skládal, Petr; Benito Peña, María Elena; Moreno Bondi, María Cruz; Gorris, HansDue to increasing food safety standards, the analysis of mycotoxins has become essential in the food industry. In this work, we have developed a competitive upconversion-linked immunosorbent assay (ULISA) for the analysis of zearalenone (ZEA), one of the most frequently encountered mycotoxins in food worldwide. Instead of a toxin-conjugate conventionally used in competitive immunoassays, we designed a ZEA mimicking peptide extended by a biotin-linker and confirmed its excellent suitability to mimic ZEA by nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) analysis. Upconversion nanoparticles (UCNP, type NaYF4:Yb,Tm) served as background-free optical label for the detection of the peptide mimetic in the competitive ULISA. Streptavidin-conjugated UCNPs were prepared by click reaction using an alkyne-PEG-neridronate linker. The UCNP conjugate clearly outperformed conventional labels such as enzymes or fluorescent dyes. With a limit of detection of 20 pg mL−1 (63 pM), the competitive ULISA is well applicable to the detection of ZEA at the levels set by the European legislation. Moreover, the ULISA is specific for ZEA and its metabolites (α- and β-zearalenol) without significant cross-reactivity with other related mycotoxins. We detected ZEA in spiked and naturally contaminated maize samples using liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) as a reference method to demonstrate food analysis in real samples.Item Recombinant antibodies and their use for food immunoanalysis(Analytical & Bioanalytical Chemistry, 2021) Peltomaa, Riikka Johanna; Barderas, Rodrigo; Benito Peña, María Elena; Moreno Bondi, María CruzAntibodies are widely employed as biorecognition elements for the detection of a plethora of compounds including food and environmental contaminants, biomarkers, or illicit drugs. They are also applied in therapeutics for the treatment of several disorders. Recent recommendations from the EU on animal protection and the replacement of animal-derived antibodies by non-animal-derived ones have raised a great controversy in the scientific community. The application of recombinant antibodies is expected to achieve a high growth rate in the years to come thanks to their versatility and beneficial characteristics in comparison to monoclonal and polyclonal antibodies, such as stability in harsh conditions, small size, relatively low production costs, and batch-to-batch reproducibility. This review describes the characteristics, advantages, and disadvantages of recombinant antibodies including antigen-binding fragments (Fab), single-chain fragment variable (scFv), and single-domain antibodies (VHH) and their application in food analysis with especial emphasis on the analysis of biotoxins, antibiotics, pesticides, and foodborne pathogens. Although the wide application of recombinant antibodies has been hampered by a number of challenges, this review demonstrates their potential for the sensitive, selective, and rapid detection of food contaminants.Item Bioluminescent detection of zearalenone using recombinant peptidomimetic Gaussia luciferase fusion protein(2020) Peltomaa, Riikka Johanna; Fikacek, Sabrina; Benito Peña, María Elena; Barderas, Rodrigo; Head, Trajen; Deo, Sapna; Daunert, Sylvia; Moreno Bondi, María CruzThe development of a bioluminescent immunosensor is reported for the determination of zearalenone (ZEA) based on a peptide mimetic identified by phage display. The peptide mimetic GW, with a peptide sequence GWWGPYGEIELL, was used to create recombinant fusion proteins with the bioluminescent Gaussia luciferase (GLuc) that were directly used as tracers for toxin detection in a competitive immunoassay without the need for secondary antibodies or further labeling. The bioluminescent sensor, based on protein G–coupled magnetic beads for antibody immobilization, enabled determination of ZEA with a detection limit of 4.2 ng/mL (corresponding to 420 μg/kg in food samples) and an IC50 value of 11.0 ng/mL. The sensor performance was evaluated in spiked maize and wheat samples, with recoveries ranging from 87 to 106% (RSD < 20%, n = 3). Finally, the developed method was applied to the analysis of a naturally contaminated reference matrix material and good agreement with the reported concentrations was obtained.Item Identification of high-affinity phage-displayed VH fragments by use of a quartz crystal microbalance with dissipation monitoring(Sensors and Actuators: B. Chemical, 2021) Gómez-Arribas, Lidia ; Juste-Dolz, Augusto; Peltomaa, Riikka Johanna; Giménez-Romero, David; Morais, Sergi; Barderas, Rodrigo; Cuadrado, Carmen; Maquieira, Ángel; Benito Peña, María Elena; Moreno Bondi, María CruzPhage display has become a powerful tool for antibody discovery in a wide variety of fields. This technology allows specific binders for a given antigen to be selected from combinatorial libraries. A key step in the process is characterizing and evaluating antibody clones thus selected to reliably identify the best antigen binders. Novel characterization methods can provide essential insight into the binding mechanism and supplement the information obtained with conventional techniques. In this work, we used a quartz crystal microbalance with dissipation monitoring (QCM-D) to determine the kinetic and thermodynamic binding parameters for phage-displayed VH antibody fragments. Phytohemagglutinin (PHA), a legume lectin of analytical interest, was used as a complex model antigen to select specific VH fragments from a phage-displayed library. Eight VH fragments with a unique amino acid sequence were identified as PHA binders by using the well-established enzyme-linked immunosorbent assay (ELISA). QCM-D measurements, structural analysis and principal component analysis (PCA) were used to evaluate the antibody fragments and identify clone clusters with similar binding characteristics and molecular interaction mechanisms. This unprecedented study has enabled the identification of high-affinity phage-displayed VH antibody fragments for PHA, which could be useful for PHA analysis (apparent association constant ranged from 10e8 to 10e10 1/M). In fact, the proposed methodology provides a useful tool for evaluating and characterizing antibody fragments with capabilities beyond those of conventional techniques.