Person: Povedano Muñumel, Eloy
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First Name
Eloy
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Povedano Muñumel
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Universidad Complutense de Madrid
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Item Magnetic microbeads-based amperometric immunoplatform for the rapid and sensitive detection of N6-methyladenosine to assist in metastatic cancer cells discrimination(Biosensors and Bioelectronics, 2021) Povedano Muñumel, Eloy; Gamella Carballo, María; Torrente Rodríguez, Rebeca Magnolia; Montero-Calle, Ana; Pedrero Muñoz, María; Solís-Fernández, Guillermo; Navarro Villoslada, Fernando; Barderas, Rodrigo; Campuzano Ruiz, Susana; Pingarrón, José; Pingarrón Carrazón, José ManuelThis work describes the preparation of an immunoplatform for the sensitive and selective determination of N6-methyladenosine (m6A). The simple and fast protocol involves for the first time the use of micromagnetic immunoconjugates to establish a direct competitive assay between the m6A target and a biotinylated RNA oligomer bearing a single m6A enzymatically labelled with a commercial conjugate of streptavidin-peroxidase (Strep-HRP) as tracer. The cathodic current change measured in the presence of H2O2/hydroquinone (HQ) at screen-printed carbon electrodes (SPCEs) upon surface capturing the magnetic bioconjugates is inversely proportional to the m6A target concentration. After evaluating the effect of key variables, the analytical characteristics were established for the determination of three different targets: the N6-methyladenosine-5′ -triphosphate (m6ATP) ribonucleotide, a short synthetic RNA oligomer bearing a single m6A and the positive control provided in a commercial colorimetric kit for m6A-RNA quantification. The obtained results show that this immunoplatform is competitive with other methods reported to date, achieving an improved sensitivity (limit of detection of 0.9 pM for the short synthetic oligomer) using a much simpler and faster protocol (~1 h) and disposable electrodes for the transduction. Furthermore, the applicability for discriminating the metastatic potential of cancer cells by directly analyzing a small amount of raw total RNA without enriching or fragmenting was also preliminary assessed.Item Magnetic Beads-Based Sensor with Tailored Sensitivity for Rapid and Single-Step Amperometric Determination of miRNAs(International Journal of Molecular Sciences, 2017) Vargas Orgaz, Eva; Torrente Rodríguez, Rebeca Magnolia; Ruiz Valdepeñas Montiel, Víctor; Povedano Muñumel, Eloy; Pedrero Muñoz, María; Montoya, Juan; Campuzano Ruiz, Susana; Pingarrón Carrazón, José ManuelThis work describes a sensitive amperometric magneto-biosensor for single-step and rapid determination of microRNAs (miRNAs). The developed strategy involves the use of direct hybridization of the target miRNA (miRNA-21) with a specific biotinylated DNA probe immobilized on streptavidin-modified magnetic beads (MBs), and labeling of the resulting heteroduplexes with a specific DNA–RNA antibody and the bacterial protein A (ProtA) conjugated with an horseradish peroxidase (HRP) homopolymer (Poly-HRP40) as an enzymatic label for signal amplification. Amperometric detection is performed upon magnetic capture of the modified MBs onto the working electrode surface of disposable screen-printed carbon electrodes (SPCEs) using the H2O2/hydroquinone (HQ) system. The magnitude of the cathodic signal obtained at −0.20 V (vs. the Ag pseudo-reference electrode) demonstrated linear dependence with the concentration of the synthetic target miRNA over the 1.0 to 100 pM range. The method provided a detection limit (LOD) of 10 attomoles (in a 25 μL sample) without any target miRNA amplification in just 30 min (once the DNA capture probe-MBs were prepared). This approach shows improved sensitivity compared with that of biosensors constructed with the same anti-DNA–RNA Ab as capture instead of a detector antibody and further labeling with a Strep-HRP conjugate instead of the Poly-HRP40 homopolymer. The developed strategy involves a single step working protocol, as well as the possibility to tailor the sensitivity by enlarging the length of the DNA/miRNA heteroduplexes using additional probes and/or performing the labelling with ProtA conjugated with homopolymers prepared with different numbers of HRP molecules. The practical usefulness was demonstrated by determination of the endogenous levels of the mature target miRNA in 250 ng raw total RNA (RNAt) extracted from human mammary epithelial normal (MCF-10A) and cancer (MCF-7) cells and tumor tissues.- Project number: PIMCD320/23-24
Item Contribuyendo a la internacionalización de la docencia práctica inclusiva y sostenible en Química Analítica(0024) Gamella Carballo, María; Pedrero Muñoz, María; Campuzano Ruiz, Susana; Serafín González-Carrato, Verónica; Torrente Rodríguez, Rebeca Magnolia; Ruiz Valdepeñas Montiel, Víctor; Valverde De La Fuente, Alejandro; Povedano Muñumel, Eloy; Muñoz San Martín, Cristina; Pérez Ginés, Víctor; Blázquez García, Marina; Tejerina Miranda, Sandra; de Valle Ávila, Marcos; Molla Escudero, David Item Versatile electroanalytical bioplatforms for dimultaneous determination of cancer-related DNA 5-hethyl- and 5-hydroxymethyl-cytosines at global and gene-specific levels in human serum and tissues(ACS Sensors, 2018) Povedano Muñumel, Eloy; Ruiz Valdepeñas Montiel, Víctor; Valverde De La Fuente, Alejandro; Navarro Villoslada, Fernando; Yáñez-Sedeño Orive, Paloma; Pedrero Muñoz, María; Montero-Calle, Ana; Barderas Manchado, Rodrigo; Peláez-García, Alberto; Mendiola, Marta; Hardisson, David; Feliú, Jaime; Camps, Jordi; Rodríguez-Tomàs, Elisabet; Joven, Jorge; Arenas, Meritxell; Campuzano Ruiz, Susana; Pingarrón Carrazón, José ManuelThis paper reports the preparation of versatile electrochemical biosensing platforms for the simple, rapid, and PCR-independent detection of the most frequent DNA methylation marks (5-methylcytosine, 5-mC, and/or 5-hydroxymethylcytosine, 5-hmC) both at global and gene-specific levels. The implemented strategies, relying on the smart coupling of immuno-magnetic beads (MBs), specific DNA probes and amperometric detection at screen-printed carbon electrodes (SPCEs), provided sensitive and selective determination of the target methylated DNAs in less than 90 min with a great reproducibility and demonstrated feasibility for the simultaneous detection of the same or different cytosine epimarks both at global level and in different loci of the same gene or in different genes. The bioplatforms were applied to determine global methylation events in paraffin-embedded colorectal tissues and specific methylation at promoters of tumor suppressor genes in genomic DNA extracted from cancer cells and paraffin-embedded colorectal tissues, and in serum without previous DNA extraction from cancer patients.Item Multiplexed magnetic beads-assisted amperometric bioplatforms for global detection of methylations in nucleic acids(Analytica Chimica Acta, 2021) Povedano Muñumel, Eloy; Gamella Carballo, María; Torrente Rodríguez, Rebeca Magnolia; Ruiz Valdepeñas Montiel, Víctor; Montero-Calle, Ana; Solís-Fernández, Guillermo; Navarro Villoslada, Fernando; Pedrero, María; Peláez-García, Alberto; Mendiola, Marta; Hardisson, David; Feliú, Jaime; Barderas, Rodrigo; Pedrero Muñoz, María; Pingarrón Carrazón, José ManuelThis work reports the first electrochemical bioplatform developed for the multidetection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in DNA, DNA N6-methyladenine (6mA) and RNA N6-methyladenosine (m6A) methylations at global level. Direct competitive immunoassays were implemented on the surface of magnetic beads (MBs) and optimized for the single amperometric determination of different targets varying in length, sequence and number of methylations on screenprinted carbon electrodes. After evaluating the sensitivity and selectivity of such determinations and the confirmation of no cross-reactivity, a multiplexed disposable platform allowing the simultaneous determination of the mentioned four methylation events in only 45 min has been prepared. The multiplexed bioplatform was successfully applied to the determination of m6A in cellular total RNA and of 5-mC, 5-hmC and 6mA in genomic DNA extracted from tissues. The developed bioplatform showed its usefulness to discriminate the aggressiveness of cancerous cells and between healthy and tumor tissues of colorectal cancer patients.Item Comparison of Different Strategies for the Development of Highly Sensitive Electrochemical Nucleic Acid Biosensors Using Neither Nanomaterials nor Nucleic Acid Amplification(ACS Sensors, 2017) Ruiz Valdepeñas Montiel, Víctor; Povedano Muñumel, Eloy; Vargas, Eva; Torrente Rodríguez, Rebeca Magnolia; Pedrero Muñoz, María; Reviejo García, Ángel Julio; Campuzano Ruiz, Susana; Pingarrón Carrazón, José ManuelCurrently, electrochemical nucleic acid-based biosensing methodologies involving hybridization assays, specific recognition of RNA/DNA and RNA/RNAduplexes, and amplification systems provide an attractive alternative to conventional quantification strategies for the routine determination of relevant nucleic acids at different settings. A particularly relevant objective in the development of such nucleic acid biosensors is the design of as many as possible affordable, quick, and simple methods while keeping the required sensitivity. With this aim in mind, this work reports, for the first time, a thorough comparison between 11 methodologies that involve different assay formats and labeling strategies for targeting the same DNA. The assayed approaches use conventional sandwich and competitive hybridization assays, direct hybridization coupled to bioreceptors with affinity for RNA/DNA duplexes, multienzyme labeling bioreagents, and DNA concatamers. All of them have been implemented on the surface of magnetic beads (MBs) and involve amperometrictransduction at screen-printed carbon electrodes (SPCEs). The influence of the formed duplex length and of the labeling strategy have also been evaluated. Results demonstrate that these strategies can provide very sensitive methods without the need for using nanomaterials or polymerase chain reaction (PCR). In addition, the sensitivity can be tailored within several orders of magnitude simply by varying the bioassay format, hybrid length or labeling strategy. This comparative study allowed us to conclude that the use of strategies involving longer hybrids, the use of antibodies with specificity for RNA/DNA heteroduplexes and labeling with bacterial antibody binding proteins conjugated with multiple enzyme molecules, provides the best sensitivity.