Person: Campuzano Ruiz, Susana
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First Name
Susana
Last Name
Campuzano Ruiz
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Químicas
Department
Química Analítica
Area
Química Analítica
Identifiers
17 results
Search Results
Now showing 1 - 10 of 17
Item p53 and p63 Proteoforms Derived from Alternative Splicing Possess Differential Seroreactivity in Colorectal Cancer with Distinct Diagnostic Ability from the Canonical Proteins(Cancers, 2023) Montero Calle, Ana; Garranzo Asensio, María; Torrente Rodríguez, Rebeca Magnolia; Ruiz Valdepeñas Montiel, Víctor; Poves, Carmen; Dziakova, Jana; Sanz, Rodrigo; Díaz Del Arco, Cristina; Pingarrón Carrazón, José Manuel; Fernández Aceñero, María Jesús; Campuzano Ruiz, Susana; Barderas Manchado, RodrigoColorectal cancer (CRC) is the third most common cancer and the second most frequent cause of cancer-related death worldwide. The detection in plasma samples of autoantibodies against specific tumor-associated antigens has been demonstrated to be useful for the early diagnosis of CRC by liquid biopsy. However, new studies related to the humoral immune response in cancer are needed to enable blood-based diagnosis of the disease. Here, our aim was to characterize the humoral immune response associated with the different p53 and p63 proteoforms derived from alternative splicing and previously described as aberrantly expressed in CRC. Thus, here we investigated the diagnostic ability of the twelve p53 proteoforms and the eight p63 proteoforms described to date, and their specific N-terminal and C-terminal end peptides, by means of luminescence HaloTag beads immunoassays. Full-length proteoforms or specific peptides were cloned as HaloTag fusion proteins and their seroreactivity analyzed using plasma from CRC patients at stages I-IV (n = 31), individuals with premalignant lesions (n = 31), and healthy individuals (n = 48). p53γ, Δ40p53β, Δ40p53γ, Δ133p53γ, Δ160p53γ, TAp63α, TAp63δ, ΔNp63α, and ΔNp63δ, together with the specific C-terminal end α and δ p63 peptides, were found to be more seroreactive against plasma from CRC patients and/or individuals with premalignant lesions than from healthy individuals. In addition, ROC (receiver operating characteristic) curves revealed a high diagnostic ability of those p53 and p63 proteoforms to detect CRC and premalignant individuals (AUC higher than 85%). Finally, electrochemical biosensing platforms were employed in POC-like devices to investigate their usefulness for CRC detection using selected p53 and p63 proteoforms. Our results demonstrate not only the potential of these biosensors for the simultaneous analysis of proteoforms’ seroreactivity, but also their convenience and versatility for the clinical detection of CRC by liquid biopsy. In conclusion, we here show that p53 and p63 proteoforms possess differential seroreactivity in CRC patients in comparison to controls, distinctive from canonical proteins, which should improve the diagnostic panels for obtaining a blood-based biomarker signature for CRC detection.Item Disposable electrochemical immunoplatform to shed light on the role of the multifunctional glycoprotein TIM-1 in cancer cells invasion(Talanta, 2024) Quinchia, Jennifer; Blázquez-García, Marina; Torrente Rodríguez, Rebeca Magnolia; Ruiz Valdepeñas Montiel, Víctor; Serafín González-Carrato, Verónica; Rejas-González, Raquel; Montero-Calle, Ana; Orozco, Jahir; Pingarrón Carrazón, José Manuel; Barderas Manchado, Rodrigo; Campuzano Ruiz, SusanaDetecting overexpression of cancer biomarkers is an excellent tool for diagnostic/prognostic and follow-up of patients with cancer or their response to treatment. This work illustrates the relevance of interrogating the levels of T-cell immunoglobulin and mucin domain 1 (TIM-1) protein as a diagnostic/prognostic biomarker of high-prevalence breast and lung cancers by using an amperometric disposable magnetic microparticles-assisted immunoplatform. The developed method integrates the inherent advantages of carboxylic acid-functionalized magnetic beads (HOOC-MBs) as pre-concentrator support and the amperometric transduction at screen-printed carbon electrodes (SPCEs). The immunoplatform involves a sandwich-type immunoassay assembled on HOOC-MBs through the specific capture/labeling of TIM-1 using capture antibodies and horseradish peroxidase (HRP)-conjugated biotinylated detection antibodies as biorecognition elements. The magnetic immunoconjugates were confined onto the working electrode (WE) surface of the SPCEs for amperometric detection using the hydroquinone/hydrogen peroxide/HRP (HQ/H2O2/HRP) redox system. The method allows the selective detection of TIM-1 protein over the 87–7500 pg mL−1 concentration range in only 45 min, with a limit of detection of 26 pg mL−1. The developed bioplatform was successfully applied to the analysis of breast and lung cancer cell extracts, providing the first quantitative results of the target glycoprotein in these types of samples.Item Electrochemical immunoplatform to help managing pancreatic cancer(Journal of Electroanalytical Chemistry, 2023) Pérez Ginés, Víctor; Torrente Rodríguez, Rebeca Magnolia; Pedrero Muñoz, María; Martínez-Bosch, Neus; García de Frutos, Pablo; Navarro, Pilar; Pingarrón Carrazón, José Manuel; Campuzano Ruiz, SusanaPancreatic ductal adenocarcinoma (PDAC) is the solid tumor with the worst prognosis, representing today the third cause of cancer-related deaths in developed countries and expected to be the second in 2030. Today, CA19-9 remains the only clinically used marker for management of PDAC (FDA-approved as a disease moni- toring marker). This work reports a disposable amperometric immunoplatform for the determination of CA19-9. The immunoplatform skilfully combines the advantages of magnetic microsupports (MBs) for implementation of the immunoassay and amperometric transduction on screen-printed carbon electrodes (SPCEs). The method involves the preparation of sándwich immunocomplexes enzymatically labeled with the enzyme horseradish peroxidase (HRP) on the MBs and uses a detection antibody conjugated to HRP. Once the HRP- labeled sandwich immunocomplexes-bearing MBs were trapped on the SPCE surface, the variation of the catho- dic current was measured in the presence of H2O2 and hydroquinone (HQ), which was directly proportional to the concentration of CA19-9. Under the optimized experimental conditions, the immunoplatform allowed the amperometric determination of CA19-9 standards over the 5.0 to 500 U mL−1 concentration range, with a limit of detection (LOD) value of 1.5 U mL−1 in 1 h. The method exhibits good reproducibility and selectivity and the magnetic immunoconjugates shows a good storage stability. The immunoplatform was applied to the deter- mination of CA19-9 in serum samples of a medium-sized cohort (22 individuals) of healthy subjects and patients diagnosed with PDAC. The obtained results demonstrated the immunoplatform ability to discriminateboth types of individuals within 1 h after sample dilution. The developed immunoplatform represents an improvement in terms of cost, applicability and accessibility compared to the ELISA-based techniques currently used in the clinic.Item Towards Control and Oversight of SARS-CoV-2 Diagnosis and Monitoring through Multiplexed Quantitative Electroanalytical Immune Response Biosensors(Angewandte Chemie International Edition, 2022) Torrente Rodríguez, Rebeca Magnolia; Montero Calle, Ana; San Bartolomé, Clara; Cano, Olga; Vazquez, Monica; Iglesias Caballero, María; Corral Lugo, Andrés; McConnell, Michael J.; Pascal, Mariona; Mas, Vicente; Pingarrón Carrazón, José Manuel; Barderas, Rodrigo; Campuzano Ruiz, SusanaThe development of versatile and sensitive biotools to quantify specific SARS-CoV-2 immunoglobulins in SARS-CoV-2 infected and non-infected individuals, built-on the surface of magnetic microbeads functionalized with nucleocapsid (N) and inhouse expressed recombinant spike (S) proteins is reported. Amperometric interrogation of captured N- and S-specific circulating total or individual immunoglobulin (Ig) isotypes (IgG, IgM, and IgA), subsequently labelled with HRP-conjugated secondary antibodies, was performed at disposable single or multiplexed (8) screen-printed electrodes using the HQ/HRP/H2O2 system. The obtained results using N and in-house expressed S ectodomains of five SARS-CoV-2 variants of concern (including the latest Delta and Omicron) allow identification of vulnerable populations from those with natural or acquired immunity, monitoring of infection, evaluation of vaccine efficiency and even identification of the variant responsible for the infection.Item Amperometric magnetoimmunoassay for the determination of lipoprotein(a)(Microchimica Acta, 2015) Kaçar, Ceren; Torrente Rodríguez, Rebeca Magnolia; Pedrero Muñoz, María; Campuzano Ruiz, Susana; Kilic, Esma; Pingarrón Carrazón, José ManuelA highly sensitive amperometric magnetoimmunoassay for rapid determination of lipoprotein(a) (Lp(a)), an important predictor of cardiovascular disease risk, in human serum, is described. It uses a sandwich configuration involving selective capture antibody [antiLp(a)] and biotinylated detector antibody [biotinantiLp(a)], and a streptavidin-HRP conjugate on carboxymodified magnetic beads (HOOC-MBs). The resulting MBs bearing the sandwiched immunoconjugates were captured by a magnet placed under the working electrode surface of a disposable screen-printed carbon electrode and the extent of the affinity reaction was monitored amperometrically at −0.20 V (vs a silver pseudo-reference electrode) in the presence of hydroquinone as an electron transfer mediator and upon addition of H2O2 as the enzyme substrate. The method exhibited a wide linear response range (from 0.01 to 0.5 μgmL−1), a detection limit of 4 ng mL−1, and an excellent selectivity over other serum components. The utility of the immunoassay was demonstrated by analyzing a reference serum containing a certified quantity of Lp(a). The performance of this magnetoimmunoassay compares favorably to that of an integrated amperometric immunoassay described earlier.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.Item Electrochemical detection of peanuts at trace levels in foods using a magnetoimmunosensor for the allergenic protein Ara h 2(Sensors and Actuators B: Chemical, 2016) Ruiz Valdepeñas Montiel, Víctor; Pellicanò, Alessandro; Campuzano Ruiz, Susana; Torrente Rodríguez, Rebeca Magnolia; Reviejo García, Ángel Julio; Cosio, Maria Stella; Pingarrón Carrazón, José ManuelA highly sensitive disposable amperometric magnetoimmunosensor for the rapid determination of Ara h 2 protein, one of the major peanut allergens, is reported. The approach uses a sandwich configuration involving selective capture and detector antibodies and carboxylic acid-modified magnetic beads (HOOC-MBs). Detector antibodies are labeled with HRP-conjugated secondary antibodies and the MBs bearing the immunoconjugates are magnetically captured on surface of a disposable screen-printed carbon electrode (SPCE). The affinity reactions are monitored amperometrically at −0.20 V (vs a Ag pseudo-reference electrode) in the presence of hydroquinone (HQ) as electron transfer mediator and upon addition of hydrogen peroxide as the enzyme substrate. The developed magnetoimmunosensor exhibits a wide range of linearity between 87 and 10,000 pg/mL Ara h 2 with a detection limit of 26 pg/mL as well as a great selectivity against other non-target proteins. The magnetoimmunosensing platform was successfully applied for the detection of Ara h 2 in different food extracts. After an appropriate sample dilution no matrix effects were observable. The developed methodology was able to detect trace amounts of the peanut allergen (0.0005% or 5.0 mg/kg) in wheat flour spiked samples. The results correlated properly with those provided by a commercial ELISA kit.Item 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 ManuelA 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.Item Electrochemical bioplatforms for the simultaneous determination of interleukin (IL)-8 mRNA and IL-8 protein oral cancer biomarkers in raw saliva(Biosensors & Bioelectronics, 2016) Torrente Rodríguez, Rebeca Magnolia; Campuzano Ruiz, Susana; Ruiz Valdepeñas Montiel, Víctor; Gamella Carballo, María; Pingarrón Carrazón, José ManuelThe development of electrochemical magnetobiosensors for the simultaneous determination of two biomarkers associated with salivary oral cancer, protein IL-8 and its messenger RNA (IL-8 mRNA) associated, in undiluted human saliva samples is reported in this work. The implemented methodology involves the use of functionalized magnetic beads, specific antibodies against IL-8 protein, a specific hairpin DNA sequence for IL-8 mRNA and amperometric detection at disposable dual screen printed carbon electrodes. This methodology exhibits high sensitivity and selectivity for the target analytes providing detection limits of 0.21 nM for IL-8 mRNA and 72.4 pgmL(-1) (far below the clinical established cut-off of 600 pgmL(-1)) for IL-8 protein in undiluted saliva samples. The dual amperometric magnetobiosensor was applied to the direct determination of both biomarkers in spiked raw saliva samples and to determine the endogenous content of IL-8 protein in saliva samples from 7 healthy individuals. The obtained results were statistically in agreement with those provided by a commercial ELISA kit.Item Fast Electrochemical miRNAs Determination in Cancer Cells and Tumor Tissues with Antibody-Functionalized Magnetic Microcarriers(ACS Sensors, 2016) Torrente Rodríguez, Rebeca Magnolia; Ruiz Valdepeñas Montiel, Víctor; Campuzano Ruiz, Susana; Farchado-Dinia, Meryem; Barderas Manchado, Rodrigo; San Segundo Acosta, Pablo; Montoya, Juan ; Pingarrón Carrazón, José ManuelMicroribonucleic acids (miRNAs) have been linked with various regulatory functions and diseases and constitute important targets in future medical diagnostics and prognostics. We report here a novel sensitive and rapid bioelectrochemical strategy for miRNA determination. This strategy involves the development of a sensing approach making use of magnetic beads (MBs) modified with a specific DNA-RNA antibody as capture bioreceptor and amperometric detection implying the H2O2/hydroquinone (HQ) system at disposable screen-printed carbon electrodes (SPCEs). The developed biosensor exhibits a dynamic range from 8.2 to 250 pM and a detection limit of 2.4 pM (60 amol) of a synthetic target without any amplification step in 2 h. The usefulness of the approach was evaluated by analyzing total RNA (RNAt) extracted from metastatic cancer cell lines and human tumor tissues, which demonstrated its potential to perform determination of mature miRNAs in these complex samples. Moreover, the feasibility of the developed methodology to detect simultaneously the expression of two different miRNAs at dual SPCEs (SPdCEs) in one single experiment was also explored. The feasibility to capture and release target miRNAs make the developed methodology also an attractive tool to isolate, purify, and determine target miRNAs with great applicability in the clinical field.