Person:
Torrente Rodríguez, Rebeca Magnolia

First Name

Rebeca Magnolia

Last Name

Torrente Rodríguez

URI

https://hdl.handle.net/20.500.14352/85645

Affiliation

Universidad Complutense de Madrid

Faculty / Institute

Ciencias Químicas

Department

Química Analítica

Identifiers

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Now showing 1 - 10 of 23

Sensitive electrochemical determination of miRNAs based on a sandwich assay onto magnetic microcarriers and hybridization chain reaction amplification
(Biosensors & Bioelectronics, 2016) Torrente Rodríguez, Rebeca Magnolia; Campuzano Ruiz, Susana; Ruiz Valdepeñas Montiel, Víctor; Montoya, Juan José; Pingarrón Carrazón, José Manuel
A novel electrochemical approach for determination of miRNAs involving a sandwich hybridization assay onto streptavidin-magnetic beads (Strep-MBs), hybridization chain reaction (HCR) amplification and amperometric detection at disposable screen-printed carbon electrodes is reported. Using miRNA-21 as the target analyte, a dynamic linear range from 0.2 to 5.0 nM with a 60 pM (1.5 fmol in 25 μL) detection limit was obtained. The achieved sensitivity is 24-fold higher than a non-HCR amplification approach involving conventional sandwich type assay onto MBs. Moreover, the whole assay time lasted 1 h 45 min which is remarkably shorter than other reported methodologies. The methodology exhibited full selectivity against other non-complementary miRNAs as well as an acceptable discrimination between homologous miRNA family members. The applicability of this novel approach was demonstrated by determining mature miRNA-21 in total RNA (RNAt) extracted from tumor cells and human tissues.
Electrochemical magnetoimmunosensing platform for determination of the milk allergen β-lactoglobulin
(Talanta, 2015) Ruiz Valdepeñas Montiel, Víctor; Campuzano Ruiz, Susana; Conzuelo, Felipe; Torrente Rodríguez, Rebeca Magnolia; Gamella Carballo, María; Reviejo García, Ángel Julio; Pingarrón Carrazón, José Manuel
A very sensitive magnetoimmunosensor for the determination of β-lactoglobulin (β-LG) is reported in this work. A sandwich configuration involving covalent immobilization of the capture antibody (antiβ-LG) onto activated carboxylic-modified magnetic beads (HOOC-MBs) and incubation of the modified MBs with a horseradish peroxidase labeled antibody (HRP-antiβ-LG), is used. The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE) and the amperometric responses are measured at −0.20 V (vs. Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as electron transfer mediator and hydrogen peroxide as the enzyme substrate. The β-LG magnetoimmunosensor exhibited a wide range of linearity (2.8–100 ng/mL) and a low detection limit of 0.8 ng/mL (20 pg in 25 μL sample). The magnetoimmunosensing platform was successfully applied for the detection of β-LG in different types of milk without any matrix effect after just a sample dilution. The results correlated properly with those provided by a commercial ELISA method offering a truthful analytical screening tool. These features make the developed methodology a promising alternative in the development of user-friendly devices for on-site determination of β-LG in dairy products.
Comparative evaluation of the performance of electrochemical immunosensors using magnetic microparticles and nanoparticles. Application to the determination of tyrosine kinase receptor AXL
(Microchimica Acta, 2017) Serafín González-Carrato, Verónica; Torrente Rodríguez, Rebeca Magnolia; Batlle, Montserrat; García de Frutos, Pablo; Campuzano Ruiz, Susana; Yáñez-Sedeño Orive, Paloma; Pingarrón Carrazón, José Manuel
Electrochemical sandwich immunoassay strategies involving the use of carboxyl-functionalized magnetic microbeads (cMBs) and magnetic nanoparticles (cMNPs) have been evaluated and compared. The proteolytically cleaved soluble Tyrosine kinase receptor sAXL was used as the target analyte. Antibodies against AXL were covalently immobilized on cMBs or cMNPs. Immunobinding of AXL was detected by means of a secondary biotinylated antibody and a streptavidinhorseradish peroxidase conjugate. The electrochemical transduction was accomplished by capturing the cMBs or cMNPs bearing the immunoconjugates onto screen-printed carbon electrodes (SPCEs) by using a small magnet. The amperometric response was measured at −0.20 V (vs the silver pseudoreference electrode of the SPCE) upon the addition of H2O2 in the presence of hydroquinone as the redox mediator. The calibration plots for AXL extended up to 7.5 ng mL−1 when cMBs were used for the preparation of the immunosensor and up to 40 ng mL−1 in the case of using cMNPs. The respective slope values were 158 (cMBs) and 43 nA mL ng−1 (cMNPs), while the achieved LODs were 74 (cMBs) and 75 pg mL−1 (cMNPs). Although the immunosensors prepared with cMBs provided a shorter range of linearity, they exhibited a 3.7-times larger sensitivity than those constructed with cMNPs. The successful application of the new strategies was demonstrated for the determination of the endogenous content of sAXL in real human serum samples (a cut-off value of 71 ng mL−1 have been established for patients with risk of heart failure). The immunosensors constructed using cMBs or cMNPs can be advanta geously compared, in terms of sensitivity and fabrication time, with the only immunosensor for AXL previously reported. In addition, these new immunosensors took approximately half time than ELISA to perform the assay.
Simultaneous detection of two breast cancer-related miRNAs in tumor tissues using p19-based disposable amperometric magnetobiosensing platforms
(Biosensors & Bioelectronics, 2015) Torrente Rodríguez, Rebeca Magnolia; Campuzano Ruiz, Susana; López-Hernández, Eva; Ruiz Valdepeñas Montiel, Víctor; Barderas Manchado, Rodrigo; Granados, Rosario; Sánchez-Puelles, Jose Maria; Pingarrón Carrazón, José Manuel
A novel magnetobiosensing approach for the rapid and simultaneous detection of two breast cancer-related miRs (miR-21 and miR-205) is reported. It involves the use of antimiR-21 and antimiR-205 specific probes, chitin-modified magnetic beads (Chitin-MBs), the p19 viral protein as capture bioreceptor and amperometric detection with the H2O2/hydroquinone (HQ) system at dual screen-printed carbon electrodes (SPdCEs). The use of SPdCEs allows the simultaneous independent amperometric readout for each target miR to be measured. The magnetosensor exhibited sensitive and selective detection with dynamic ranges from 2.0 to 10.0nM and detection limits of 0.6nM (6fmol) for both target miRs without any amplification step in less than 2h. The usefulness of the approach was evaluated by detecting the endogenous levels of both target miRs in total RNA (RNAt) extracted from metastatic breast cancer cell lines and human tissues.
Direct Determination of miR‐21 in Total RNA Extracted from Breast Cancer Samples Using Magnetosensing Platforms and the p19 Viral Protein as Detector Bioreceptor
(Electroanalysis, 2014) Torrente Rodríguez, Rebeca Magnolia; Campuzano Ruiz, Susana; López‐Hernández, Eva; Granados, Rosario; Sánchez‐Puelles, José Maria; Pingarrón Carrazón, José Manuel
A novel magnetobiosensing approach for the rapid, sensitive and selective miR‐21 detection is reported involving the use of a specific RNA probe (antimiR‐21), streptavidin‐magnetic beads (Strep‐MBs), the siRNA Binding Protein p19 as detector bioreceptor, and amperometric detection with the H2O2/hydroquinone (HQ) system at disposable screen‐printed carbon electrodes. The magnetosensor exhibited a dynamic range from 1.4 to 10 nM and a detection limit of 4.2 fmol of synthetic target miR‐21 without any amplification step in 75 min. The usefulness of the approach was evaluated by analyzing total RNA (RNAt) extracted from metastatic breast cancer cell lines, human tissues and breast cytologies.
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 Manuel
This 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.
Toward Liquid Biopsy: Determination of the Humoral Immune Response in Cancer Patients Using HaloTag Fusion Protein-Modified Electrochemical Bioplatforms
(Analytical Chemistry, 2016) Garranzo Asensio, María; Guzmán Aránguez, Ana Isabel; Povés Francés, Carmen; Fernández Aceñero, María Jesús; Torrente Rodríguez, Rebeca Magnolia; Ruiz Valdepeñas Montiel, Víctor; Domínguez Muñóz, Gemma; San Frutos Llorente, Luis; Rodríguez Salas, Nuria; Villalba Díaz, María Teresa; Pingarrón Carrazón, José Manuel; Campuzano Ruiz, Susana; Barderas Manchado, Rodrigo
Autoantibodies raised against tumor-associated antigens have shown high promise as clinical biomarkers for reliable diagnosis, prognosis, and therapy monitoring of cancer. An electrochemical disposable biosensor for the specific and sensitive determination of p53-specific autoantibodies has been developed for the first time in this work. This biosensor involves the use of magnetic microcarriers (MBs) modified with covalently immobilized HaloTag fusion p53 protein as solid supports for the selective capture of specific autoantibodies. After magnetic capture of the modified MBs onto screen-printed carbon working electrodes, the amperometric signal using the system hydroquinone/H2O2 was related to the levels of p53-autoantibodies in the sample. The biosensor was applied for the analysis of sera from 24 patients with high-risk of developing colorectal cancer and 6 from patients already diagnosed with colorectal (4) and ovarian (2) cancer. The developed biosensor was able to determine p53 autoantibodies with a sensitivity higher than that of a commercial standard ELISA using a just-in-time produced protein in a simpler protocol with less sample volume and easily miniaturized and cost-effective instrumentation.
Bioplataformas electroanalíticas versátiles para diagnóstico temprano y fiable de cáncer a diferentes niveles moleculares
(2019) Torrente Rodríguez, Rebeca Magnolia; Pingarrón Carrazón, José Manuel; Campuzano Ruiz, Susana; Gamella Carballo, María
Desde los antiguos egipcios hasta nuestros días, el cáncer es uno de los peores enemigos de la salud en la historia de la humanidad.Como respuesta a la presencia, desarrollo, mitigación y reincidencia de una neoplasia, las células (tanto cancerosas como sanas) liberan entidades moleculares detectables en todo tipo de especímenes y fluidos biológicos, conocidas como biomarcadores, que incluyen proteínas, genes y alteraciones epigenéticas, y cuya alteración y/o desregulación permite detectar de forma temprana, identificar y clasificar distintas variantes de cáncer, evaluar el riesgo de recaída tras un tratamiento, y predecir la respuesta a ciertas terapias aplicadas...
Magnetobiosensors Based on Viral Protein p19 for MicroRNA Determination in Cancer Cells and Tissues
(Angewandte Chemie International Edition, 2014) Campuzano Ruiz, Susana; Torrente Rodríguez, Rebeca Magnolia; López‐Hernández, Eva; Conzuelo, Felipe; Granados, Rosario; Sánchez‐Puelles, José Maria; Pingarrón Carrazón, José Manuel
MicroRNAs (miRs) have emerged as important clinical biomarkers with both diagnostic and prognostic value for relevant diseases, such as cancer. MiRs pose unique challenges for detection and are currently detected by northern blotting, real‐time PCR, and microarray techniques. These expensive, complicated, and time‐consuming techniques are not feasible for on‐site miR determination. In this study, amperometric magnetobiosensors involving RNA‐binding viral protein p19 as a selective biorecognition element were developed for miR quantification. The p19‐based magnetosensors were able to detect 0.4 fmol of a synthetic target and endogenous miR‐21 (selected as a model for its role in a wide variety of cancers) in only 2 h in total RNA extracted from cancer cells and human breast‐tumor specimens without PCR amplification and sample preprocessing. These results open up formidable perspectives for the diagnosis and prognosis of human cancers and for drug‐discovery programs.
Amperometric Immunosensing Scaffolds for Rapid, Simple, Non-Invasive and Accurate Determination of Protein Biomarkers of Well-Accepted and Emerging Clinical Importance
(Proceedings, 2017) Pedrero Muñoz, María; Muñoz San Martín, Cristina; Torrente Rodríguez, Rebeca Magnolia; Ruiz Valdepeñas Montiel, Víctor; Vargas Orgaz, Eva; Manuel de Villena Rueda, Francisco Javier; Barderas Manchado, Rodrigo; Campuzano Ruiz, Susana; Pingarrón Carrazón, José Manuel