Desarrollo de plataformas electroquímicas para biosensado directo de analitos de relevancia alimentaria y clínica en muestras de elevada complejidad

La evolución de la sociedad está generando nuevas inquietudes, como la garantía de la seguridad alimentaria y el acceso físico y económico a alimentos en cantidad suficiente, seguros y nutritivos. En este sentido, las enfermedades o trastornos asociados a la alimentación cobran cada día mayor importancia, como por ejemplo las alergias alimentarias, reacciones adversas a los alimentos producidas por un mecanismo inmunológico como consecuencia de la ingesta, contacto o inhalación de ciertas proteínas presentes en los alimentos, que pueden generar, incluso a niveles de traza, respuestas exacerbadas del sistema inmunológico. La alergia alimentaria afecta a alrededor del 5 % de la población adulta y al 80 % de la población infantil, presentando comúnmente sintomatologías locales (problemas digestivos, vómitos o dolor abdominal), sistemáticas (problemas cutáneos, respiratorios o cardiovasculares), e incluso severas, como el shock anafiláctico..

Dual detection system for cancer-associated point mutations assisted by a multiplexed LNA-based amperometric bioplatform coupled with rolling circle amplification

DNA point mutation in a BRAF proto-oncogene, V600E, is considered an important prognostic and predictive biomarker in various types of cancer, such as melanoma or colorectal cancer. We report here a novel electrochemical (EC) bioplatform for the analysis of BRAF V600E mutation coupled with rolling circle amplification (RCA) and locked nucleic acid (LNA) capture probes. A dual detection system was implemented, whereby two padlock probes complementary to either wild-type (wt) BRAF gene or DNA with V600E mutation (mut) led to amplification of wt or mut variant, respectively. Hybridization with specific LNA capture probes then increased the assay specificity, while EC detection provided rapid measurement times. The bioplatform was applied to analyze BRAF V600E mutation of cancer cells and tumor tissues from patients with melanoma or colorectal cancer. This is the first RCA-based EC bioplatform for BRAF analysis in a dual format without using PCR or sophisticated instrumentation.The financial support of the Czech Health Research Council (No. NU21-08-00078), National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU, BBMRI.cz (No. LM2023033), MH CZ - DRO (MMCI, 00209805), PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Innovacion), ´ TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (S2018/NMT-4349) and PI20CIII/00019 Grants from the AES-ISCIII Program co-founded by FEDER funds, are gratefully acknowledged. A. Valverde acknowledges a predoctoral contract from Complutense University of Madrid. Authors would like to thank Nina Libova for her technical support.S

Disposable electrochemical immunoplatform to shed light on the role of the multifunctional glycoprotein TIM-1 in cancer cells invasion

Detecting 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.The financial support of PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Innovación) Research Projects and PI20CIII/00019 Grant from the AES-ISCIII Program co-founded by FEDER funds and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged. A.M-C. was supported by a FPU predoctoral contract supported by the Spanish Ministerio de Educación, Cultura y Deporte. J.Q. was founded by Minciencias, Mineducacion, MINCIT, and ICETEX through the Program Ecosistema Cientifico Cod. FP44842-211–2018, project number 58536. J.O. thanks support from the University of Antioquia and the Max Planck Society through the cooperation agreement 566–1, 2014.S

Electrochemical affinity biosensors for fast detection of gene-specific methylations with no need for bisulfite and amplification treatments

This paper describes two different electrochemical affinity biosensing approaches for the simple, fast and bisulfite and PCR-free quantification of 5-methylated cytosines (5-mC) in DNA using the anti-5-mC antibody as biorecognition element. One of the biosensing approaches used the anti-5-mC as capture bioreceptor and a sandwich type immunoassay, while the other one involved the use of a specific DNA probe and the anti-5-mC as a detector bioreceptor of the captured methylated DNA. Both strategies, named for simplicity in the text as immunosensor and DNA sensor, respectively, were implemented on the surface of magnetic microparticles and the transduction was accomplished by amperometry at screen-printed carbon electrodes by means of the hydrogen peroxide/hydroquinone system. The resulting amperometric biosensors demonstrated reproducibility throughout the entire protocol, sensitive determination with no need for using amplification strategies, and competitiveness with the conventional enzyme-linked immunosorbent assay methodology and the few electrochemical biosensors reported so far in terms of simplicity, sensitivity and assay time. The DNA sensor exhibited higher sensitivity and allowed the detection of the gene-specific methylations conversely to the immunosensor, which detected global DNA methylation. In addition, the DNA sensor demonstrated successful applicability for 1 h-analysis of specific methylation in two relevant tumor suppressor genes in spiked biological fluids and in genomic DNA extracted from human glioblastoma cells.The financial support of the Spanish Ministerio de Economía y Competitividad CTQ2015-64402-C2-1-R and SAF2014-53209-R Research Projects, the PI17CIII/00045 research project from AESI and the NANOAVANSENS Program from the Comunidad de Madrid (S2013/MT-3029) and predoctoral contracts from the Spanish Ministerio de Economía y Competitividad (R.M. Torrente-Rodríguez and E. Povedano) and Universidad Complutense de Madrid (V. Ruiz-Valdepeñas Montiel) are also gratefully acknowledged.S

Electrochemical sensor for rapid determination of fibroblast growth factor receptor 4 in raw cancer cell lysates

The first electrochemical immunosensor for the determination of fibroblast growth factor receptor 4 (FGFR4) biomarker is reported in this work. The biosensor involves a sandwich configuration with covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic microcarriers (HOOC-MBs) and amperometric detection at disposable carbon screen-printed electrodes (SPCEs). The biosensor exhibits a great analytical performance regarding selectivity for the target protein and a low LOD of 48.2 pg mL-1. The electrochemical platform was successfully applied for the determination of FGFR4 in different cancer cell lysates without any apparent matrix effect after a simple sample dilution and using only 2.5 μg of the raw lysate. Comparison of the results with those provided by a commercial ELISA kit shows competitive advantages by using the developed immunosensor in terms of simplicity, analysis time, and portability and cost-affordability of the required instrumentation for the accurate determination of FGFR4 in cell lysates

Implementación de la metodología flipped classroom en los laboratorios de Química Analítica

Adaptar el sistema tradicional de aprendizaje a las necesidades actuales del alumnado empleando la metodología flipped classroom en el laboratorio de Química Analítica I, con el objetivo de fomentar el aprendizaje utilizando herramientas digitales.Depto. de Química AnalíticaFac. de Ciencias QuímicasFALSEsubmitte

Sensitive electrochemical determination of miRNAs based on a sandwich assay onto magnetic microcarriers and hybridization chain reaction amplification

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.Spanish Ministerio de Economía y CompetitividadNANOAVANSENS Program from the Comunidad de MadridCANAAN Research & Investment S.L.Universidad Complutense de MadridDepto. de Química AnalíticaFac. de Ciencias QuímicasTRUEpu

Cutting-Edge Advances in Electrochemical Affinity Biosensing at Different Molecular Level of Emerging Food Allergens and Adulterants

The presence of allergens and adulterants in food, which represents a real threat to sensitized people and a loss of consumer confidence, is one of the main current problems facing society. The detection of allergens and adulterants in food, mainly at the genetic level (characteristic fragments of genes that encode their expression) or at functional level (protein biomarkers) is a complex task due to the natural interference of the matrix and the low concentration at which they are present. Methods for the analysis of allergens are mainly divided into immunological and deoxyribonucleic acid (DNA)-based assays. In recent years, electrochemical affinity biosensors, including immunosensors and biosensors based on synthetic sequences of DNA or ribonucleic acid (RNA), linear, aptameric, peptide or switch-based probes, are gaining special importance in this field because they have proved to be competitive with the methods commonly used in terms of simplicity, test time and applicability in different environments. These unique features make them highly promising analytical tools for routine determination of allergens and food adulterations at the point of care. This review article discusses the most significant trends and developments in electrochemical affinity biosensing in this field over the past two years as well as the challenges and future prospects for this technology

Electrochemical bioplatforms for the simultaneous determination of interleukin (IL)-8 mRNA and IL-8 protein oral cancer biomarkers in raw saliva

Los resultados derivados de esta investigación fueron empleados por la Oficina de Transferencia de Resultados de Investigación de la UCM para la elaboración de una noticia con título 'Desarrollan un biosensor capaz de identificar cáncer oral en muestras de saliva' . A continuación se incluyen las direcciones de diversos sitios webs que recogen la repercusión. tanto nacional como internacional, de los resultados derivados de esta investigación: http://www.antena3.com/noticias/salud/desarrollan-biosensor-capaz-diagnosticar-cancer-muestras-saliva_2016030700308.html http://www.lasexta.com/constantes-vitales/investigacion/desarrollan-biosensor-detectar-cancer-oral-traves-saliva_2016030700386.html http://www.farodevigo.es/sociedad/2016/03/08/crean-biosensor-capaz-identificar-cancer/1418665.html http://www.abc.es/salud/enfermedades/abci-investigadores-espanoles-disenan-test-detecta-cancer-oral-saliva-201603080404_noticia.html http://www.larazon.es/sociedad/nuevo-dispositivo-capaz-de-identificar-el-cancer-oral-a-traves-de-la-saliva-LB12138542#.Ttt1PgWSRXRJBno http://www.agenciasinc.es/Noticias/Desarrollan-un-biosensor-capaz-de-identificar-cancer-oral-en-muestras-de-saliva http://www.rtve.es/noticias/20160307/desarrollan-biosensor-capaz-identificar-cancer-oral-muestras-saliva/1314202.shtml http://www.telemadrid.es/noticias/sociedad/noticia/espanoles-desarrollan-un-prueba-para-identificar-el-cancer-oral-con-la-sal http://www.ibercampus.es/un-biosensor-identifica-canceres-orales-por-la-saliva-32557.htm http://cadenaser.com/ser/2016/03/07/ciencia/1457351053_140572.html http://www.noticiasdegipuzkoa.com/2016/03/07/ocio-y-cultura/internet/investigadores-disenan-un-biosensor-que-identifica-el-cancer-oral-en-saliva- http://eldia.es/agencias/8575907-Investigadores-espanoles-desarrollan-biosensor-capaz-identificar-cancer-oral-muestras-saliva http://www.navarrainformacion.es/2016/03/07/investigadores-espanoles-desarrollan-un-biosensor-capaz-de-identificar-el-cancer-oral-en-muestras-de-saliva/ http://www.noticiasdenavarra.com/2016/03/07/ocio-y-cultura/internet/investigadores-disenan-un-biosensor-que-identifica-el-cancer-oral-en-saliva- http://www.laopinioncoruna.es/sociedad/2016/03/08/creado-biosensor-detecta-tumores-orales/1048769.html http://politicalocal.es/investigadores-la-complutense-desarrollan-sensor-capaz-detectar-cancer-la-saliva/ http://biotech-spain.com/es/articles/desarrollan-un-biosensor-capaz-de-identificar-c-ncer-oral-en-muestras-de-saliva/ http://www.lagranepoca.com/ciencia-y-tecnologia/51733-desarrollan-biosensor-capaz-identificar-cancer-oral-muestras-saliva.html http://www.hispantv.com/newsdetail/salud/217830/biosensor-detectar-cancer-oral-saliva http://noticiasdelaciencia.com/not/18581/desarrollan-un-biosensor-capaz-de-identificar-cancer-oral-en-muestras-de-saliva/ http://www.diariodejerez.es/article/salud/2235541/cientificos/espanoles/desarrollan/biosensor/detecta/cancer/oral/muestras/saliva.html http://www.catalunyavanguardista.com/catvan/detectan-cancer-oral-mediante-saliva/ http://www.discapnet.es/Castellano/Actualidad/Linea_Social/cientificos-espanholes-desarrollan-un-biosensor-capaz-de-identificar-cancer-oral-en-muestras-de-saliva.aspx http://www.eldiariomontanes.es/agencias/201603/07/investigadores-disenian-biosensor-identifica-620936.html http://www.diariovasco.com/agencias/201603/07/investigadores-disenian-biosensor-identifica-620936.html http://www.elcorreo.com/agencias/201603/07/investigadores-disenian-biosensor-identifica-620936.html http://www.eldiadecordoba.es/article/salud/2235541/cientificos/espanoles/desarrollan/biosensor/detecta/cancer/oral/muestras/saliva.html http://www.europasur.es/article/salud/2235541/cientificos/espanoles/desarrollan/biosensor/detecta/cancer/oral/muestras/saliva.html http://www.lasprovincias.es/agencias/201603/07/investigadores-disenian-biosensor-identifica-620936.html http://www.diariosur.es/agencias/201603/07/investigadores-disenian-biosensor-identifica-620936.html http://www.elcorreogallego.es/tendencias/ecg/un-equipo-disena-un-biosensor-identifica-tumores-orales-saliva/idEdicion-2016-03-08/idNoticia-984438/ http://diariodigitaldeleon.com/sociedad-leon/salud-noticias-leon/desarrollan-un-biosensor-capaz-de-identificar-cancer-oral-en-la-saliva-16604 http://www.laopinioncoruna.es/sociedad/2016/03/08/creado-biosensor-detecta-tumores-orales/1048769.html http://www.laprovincia.es/sociedad/2016/03/08/investigadores-disenan-biosensor-identifica/799275.html http://www.larioja.com/culturas/201603/08/biosensor-identifica-cancer-oral-20160308004933-v.html http://www.lavozdigital.es/salud/enfermedades/abci-investigadores-espanoles-disenan-test-detecta-cancer-oral-saliva-201603080404_noticia.html http://www.asturiasmundial.com/noticia/83094/un-biosensor-logra-identificar-cancer-oral-en-muestras-de-saliva/ http://www.noticiasdealava.com/2016/03/07/ocio-y-cultura/internet/investigadores-disenan-un-biosensor-que-identifica-el-cancer-oral-en-saliva- http://www.noticiasdegipuzkoa.com/2016/03/07/ocio-y-cultura/internet/investigadores-disenan-un-biosensor-que-identifica-el-cancer-oral-en-saliva- http://www.hoy.es/agencias/201603/07/investigadores-disenian-biosensor-identifica-620936.html http://www.eldigitaldeasturias.com/noticias/desarrollan-un-biosensor-capaz-de-identificar-el-cancer-oral-en-muestras-de-saliva/ http://www.noticiasgalicia.com/index.php/ng-salud/28335/desarrollan-un-biosensor-capaz-de-identificar-cancer-oral-en-muestras-de-saliva http://www.radiointereconomia.com/2016/03/07/investigadores-disenan-un-biosensor-que-identifica-el-cancer-oral-en-saliva/ http://www.burgosnoticias.com/ciencia/003000/encontrar-el-cancer-en-la-saliva http://www.cantabrialiberal.com/noticia.php?id=326551&noticia=Investigadores%20espa%C3%B1oles%20desarrollan%20un%20biosensor%20capaz%20de%20identificar%20el%20c%C3%A1ncer%20oral%20en%20muestras%20de%20saliva http://www.canarias7.es/articulo.cfm?id=411093 http://www.jano.es/noticia-desarrollan-un-biosensor-capaz-detectar-25708 http://saludiario.com/crean-sensor-para-detectar-cancer-oral-mediante-saliva/ http://elclarinweb.com/salud/investigadores-disenan-un-biosensor-que-identifica-el-cancer-oral-en-saliva?utm_source=dlvr.it&utm_medium=twitter http://criterio.hn/cientificos-espanoles-disenan-biosensor-capaz-diagnosticar-cancer-muestras-salivales/ http://infovzla.net/salud/una-pequena-muestra-de-saliva-es-suficiente-para-que-un-biosensor-identifique-cancer-oral/ http://revoluciontrespuntocero.com/desarrollan-un-biosensor-capaz-de-identificar-cancer-oral-en-muestras-de-saliva/ http://www.invdes.com.mx/salud-mobil/10705-disenan-un-biosensor-capaz-de-identificar-cancer-oral-en-muestras-de-saliva http://confirmado.com.ve/crean-un-test-que-detecta-el-cancer-oral-en-la-saliva/ http://www.prensalibre.com/vida/salud-y-familia/crean-test-que-detecta-cancer-oral-por-la-saliva http://globovision.com/article/crean-un-test-que-detecta-el-cancer-de-oral-en-la-saliva http://www.plenglish.com/index.php?option=com_content&task=view&id=4677261&Itemid=1 http://www.prensa-latina.cu/index.php?option=com_content&task=view&idioma=1&id=4677101&Itemid=1 http://www.primicias24.com/salud/este-biosensor-detecta-el-cancer-oral-en-la-saliva/The 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.Spanish Ministerio de Economía y CompetitividadComunidad de MadridSpanish Ministry of EducationDepto. de Química AnalíticaFac. de Ciencias QuímicasTRUEpu

Electrochemical magnetic beads-based immunosensing platform for the determination of α-lactalbumin in milk

Alpha-lactalbumin (α-LA) is one of the whey proteins in cows’ milk that has been identified as allergenic. In this work, we present, for the first time, a very sensitive magnetic beads (MBs)-based immunosensor for the determination of α-LA. A sandwich configuration involving selective capture and horseradish peroxidase-labeled detector antibodies was implemented on carboxylic acid-modified magnetic beads, captured magnetically under the surface of a disposable screen-printed carbon electrode for amperometric detection using the hydroquinone (HQ)/hydrogen peroxide system. The α-LA immunosensor exhibited a wide linear range (37.0–5000 pg/ml), a low limit of detection (LOD, 11.0 pg/ml) and noteworthy selectivity against other non-target proteins. The MBs-based immunosensing platform was applied successfully for the determination of α-LA in several varieties of milk (raw and UHT cows’ milk as well as human milk) and infant formulations. The results were corroborated with those obtained using a commercial ELISA method, thereby substantiating the analytical merits of this unique method.Spanish Ministerio de Economía y Competitividad Research ProjectsNANOAVANSENS Program from the Comunidad de MadridDepto. de Química AnalíticaFac. de Ciencias QuímicasTRUEpu