Person: Benito Peña, María Elena
Loading...
First Name
María Elena
Last Name
Benito Peña
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Químicas
Department
Química Analítica
Area
Química Analítica
Identifiers
3 results
Search Results
Now showing 1 - 3 of 3
Item Homogeneous quenching immunoassay for fumonisin B1 based on gold nanoparticles and an epitope-mimicking yellow fluorescent protein(ACS Nano, 2018) Peltomaa, Riikka Johanna; Amaro Torres, Francisco; Carrasco, Sergio; Orellana Moraleda, Guillermo; Benito Peña, María Elena; Moreno Bondi, María CruzHomogeneous immunoassays represent an attractive alternative to traditional heterogeneous assays due to their simplicity, sensitivity, and speed. On the basis of a previously identified epitope-mimicking peptide, or mimotope, we developed a homogeneous fluorescence quenching immunoassay based on gold nanoparticles (AuNPs) and a recombinant epitope-mimicking fusion protein for the detection of mycotoxin fumonisin B1 (FB1). The fumonisin mimotope was cloned as a fusion protein with a yellow fluorescent protein that could be used directly as the tracer for FB1 detection without the need of labeling or a secondary antibody. Furthermore, owing to the fluorescence quenching ability of AuNPs, a homogeneous immunoassay could be performed in a single step without washing steps to separate the unbound tracer. The homogeneous quenching assay showed negligible matrix effects in 5% wheat extract and high sensitivity for FB1 detection, with a dynamic range from 7.3 to 22.6 ng/mL, a detection limit of 1.1 ng/mL, and IC50 value of 12.9 ng/mL, which was significantly lower than the IC50 value of the previously reported assay using the synthetic counterpart of the same mimotope in a microarray format. The homogeneous assay was demonstrated to be specific for fumonisins B1 and B2, as no significant cross-reactivity with other mycotoxins was observed, and acceptable recoveries (86% for FB1 2000 μg/kg and 103% for FB1 4000 μg/kg), with relative standard deviation less than 6.5%, were reported from spiked wheat samples, proving that the method could provide a valuable tool for simple analysis of mycotoxin-contaminated food samples.Item Highly fluorescent magnetic nanobeads with a remarkable stokes shift as labels for enhanced detection in immunoassays(Small, 2018) Salis, Francesca; Descalzo López, Ana Belén; Benito Peña, María Elena; Moreno Bondi, María Cruz; Orellana Moraleda, GuillermoFluorescence immunoassays are popular for achieving high sensitivity, but they display limitations in biological samples due to strong absorption of light, background fluorescence from matrix components, or light scattering by the biomacromolecules. A powerful strategy to overcome these problems is introduced here by using fluorescent magnetic nanobeads doped with two boron-dipyrromethane dyes displaying intense emission in the visible and near-infrared regions, respectively. Careful matching of the emission and absorption features of the dopants leads to a virtual Stokes shift larger than 150 nm achieved by an intraparticle Förster resonance energy transfer (FRET) process between the donor and the acceptor dyes. Additionally, the magnetic properties of the fluorescent beads allow preconcentration of the sample. To illustrate the usefulness of this approach to increase the sensitivity of fluorescence immunoassays, the novel nanoparticles are employed as labels for quantification of the widely used Tacrolimus (FK506) immunosuppressive drug. The FRET-based competitive inhibition immunoassay yields a limit of detection (LOD) of 0.08 ng/mL, with a dynamic range (DR) of 0.15–2.0 ng/mL, compared to a LOD of 2.7 ng/mL and a DR between 4.1 and 130 ng/mL for the immunoassay carried out with direct excitation of the acceptor dye.Item Sensitive rapid fluorescence polarization immunoassay for free mycophenolic acid determination in human serum and plasma(Analytical Chemistry, 2018) Glahn Martínez, Ana Bettina; Benito Peña, María Elena; Salis, Francesca; Descalzo López, Ana Belén; Orellana Moraleda, Guillermo; Moreno Bondi, María CruzIn this Article, we describe a fluorescence polarization immunoassay (FPIA) using a new label-near infrared fluorescent dye. The developed FPIA method was optimized for the rapid analysis of free mycophenolic acid (MPA) in plasma of transplanted patients. The approach is based on the fluorescence competitive assay between the target immunosuppressant and a novel emissive near-infrared fluorescent dye-tagged MPA and MPA-AO for the binding sites of the anti-MPA antibody. The fluorescent analogue of MPA exhibits emission at 654 nm upon excitation at 629 nm (λexcmax) and shows a good photochemical stability and a significant emission quantum yield (0.16) in phosphate buffer media. Free mycophenolic acid was isolated from blood or plasma samples using ultrafiltration prior to analysis. The sample was incubated for 20 min with 5 μg/mL of anti-MPA antibody and 1 nM of MPA-AO before the measurements. The developed FPIA displays a limit of detection of 0.8 ng/mL (10% binding inhibition) and a dynamic range of 1.7−39 ng/mL (20%−80% binding inhibition) in a PBST buffer, fitting the therapeutic requirements. The immunoassay selectivity was evaluated by measuring the cross-reactivity to other immunosuppressive drugs administered in combination with MPA (cyclosporin A and tacrolimus), as well as for the metabolite MPA glucuronide. The assay has been successfully applied to the analysis of free MPA in the blood of a heart-transplanted patient after oral administration of both mycophenolate mofetil (MMF) and tacrolimus, and the results have been compared with those obtained by rapid-resolution liquid chromatography with diode array detection (RRLC-DAD).