Integración de técnicas basadas en ADN para el desarrollo de biosensores aplicados en seguridad alimentaria

Tesis por compendio[EN] Food security is guaranteed when there is sufficient, safe and nutritious food. This assurance must be satisfied throughout the entire production process, which is known as "safety from farm to fork". This results in a new way of addressing the problem with a global and comprehensive approach. To address this challenge, molecular techniques based on the use of nucleic acids are used in the analysis of certain food threatens, such as allergens, microorganisms, genetically modified organisms (GMOs), or food authentication. However, some of the described methods still have limitations, since they are expensive, complicated, and require specialized staff and equipment. Alternatively, biosensor technology provides reliable results in a simpler and faster way and with and added capabilities such as portability and automation, allowing to perform the analysis directly at points-of-control (POC). This thesis has focused on developing a biosensor system, based on compact disc technology, for the detection of nucleic acids in food safety applications and adaptable to POC needs. The carried out investigations have yielded new insights into gene technology, making interesting methodological contributions characterized by miniaturization, integration and automation. The first part of the research deals with the simplification of the amplification step, eluding the thermocycling by using alternatives to the polymerase chain reaction (PCR). To this end, two isothermal amplification techniques have been studied: the recombinase polymerase amplification (RPA) and the multiple displacement amplification (MDA). The detection was performed by hybridization assays with DNA probes immobilized in microarray format on the polycarbonate surface of a DVD. Furthermore, RPA amplification has been combined with detection by an immunoenzymatic assay (ELISA) for the simultaneous detection of multiple analytes. In another approach, amplification and hybridization have been integrated in a single step, further simplifying the analytical process. For that, the isothermal RPA amplification is performed in solid phase on the surface of the disc in different formats: drop, microfluidic chambers or micro-reactors. In the first two formats, the reactions take place at the surface of the DVD and the measurement is performed by recording the intensity of the reflected laser. In the third case the reaction is carried out in micro-wells embedded in the DVD substrate and the measurement is performed by measuring the intensity of the transmitted signal. Finally, a method to real time monitoring DNA synthesis has been developed, integrating the amplification and quantification steps. To this end, the isothermal loop mediated isothermal amplification reaction (LAMP) has been used. The monitoring of the reaction progress is performed by sequentially measuring colorimetric or turbidimetric changes in the reaction mixture. Thus each profile is related to the number of copies of each target gene, allowing their quantitation. The analytical properties (have been established for each methodology and the obtained results have been validated by comparison with reference techniques and by using certified samples. As proof of concept, the different developments have been applied to the detection and determination of the presence of allergens (hazelnut, peanut, soybean, tomato and maize), genetically modified organisms (p35S, tNOS and Bt-11), pathogenic bacteria (Salmonella spp., Cronobacter spp. and Campylobacter spp.), fungi (Fusarium spp.), as well as meat authentication.[ES] La seguridad alimentaria está garantizada cuando se dispone de alimentos suficientes, nutritivos e inocuos. Esta garantía, además, ha de cumplirse a lo largo de todo el proceso productivo, lo que se conoce como "seguridad de la granja a la mesa". Nace así una nueva forma de abordar el problema, con un enfoque global y un tratamiento integral. Para afrontar este reto, las técnicas moleculares basadas en el empleo de ácidos nucleicos son, actualmente, utilizadas en la detección de amenazas alimentarias, como por ejemplo, alérgenos, microorganismos, organismos genéticamente modificados (OGM), o la autentificación de especies. Sin embargo, muchos de los métodos en uso presentan limitaciones, ya que son costosos, complicados, y requieren personal y equipamiento especializado. La tecnología de biosensores es una aproximación adecuada, ya que proporciona resultados fiables de manera sencilla y rápida, y con capacidades añadidas como portabilidad y automatización para llevar a cabo los ensayos directamente en puntos de control (POC). Esta tesis se ha centrado en el desarrollo de un sistema biosensor, basado en la tecnología de disco compacto, para la detección de ácidos nucleicos en aplicaciones de seguridad alimentaria adaptable a POC. Las investigaciones llevadas a cabo han permitido obtener nuevos conocimientos en tecnologías génicas, pudiendo efectuar aportaciones metodológicas de interés caracterizadas por su miniaturización, integración y automatización. En este sentido, una parte de la investigación aborda la simplificación de la etapa de amplificación del ADN diana, eludiendo el termociclado mediante el empleo de técnicas alternativas a la reacción en cadena de la polimerasa (PCR). Para ello, se han estudiado dos técnicas de amplificación isoterma, la amplificación por recombinasa polimerasa (RPA) y la amplificación por desplazamiento múltiple (MDA). La detección se lleva a cabo mediante ensayos de hibridación con sondas de ADN inmovilizadas en formato micromatriz sobre la superficie de policarbonato de un DVD. Además, la amplificación por RPA se ha combinado con la detección mediante inmunoensayo ezimático (ELISA) para la determinación simultánea de múltiples analitos. En otra aproximación, se han integrado las fases de amplificación e hibridación en una única etapa, simplificando aún más el proceso analítico. Para ello, la amplificación isoterma por RPA se realiza en fase sólida sobre la superficie del disco en diferentes formatos: gota, cámaras microfluídicas o microreactores. En los dos primeros formatos, las reacciones tienen lugar en la superficie del DVD y la medición se realiza registrando la intensidad del haz láser del lector reflejado, mientras que en el tercer caso la reacción se lleva a cabo en micropocillos integrados en la estructura del DVD y la lectura se realiza midiendo la intensidad de la señal transmitida. Finalmente, se ha desarrollado una metodología para monitorizar la síntesis de ADN en tiempo real, integrando así las etapas de amplificación y cuantificación. Para ello, se emplea la reacción de amplificación isoterma mediada por bucle (LAMP), registrando secuencialmente el avance de la reacción mediante cambios turbidimétricos o colorimétricos en el medio de reacción. De este modo, se relaciona el perfil de cada ensayo con el número de copias de cada gen diana, permitiendo así su cuantificación. Para cada metodología se han establecido las propiedades analíticas, y los resultados obtenidos han sido validados por comparación con técnicas de referencia y mediante el empleo de muestras certificadas. Como prueba de concepto, los diferentes desarrollos se han aplicado a la detección y determinación de la presencia de alérgenos (avellana, cacahuete, soja, tomate y maíz), organismos modificados genéticamente (p35S, tNOS y Bt-11), bacterias patógenas (Salmonella spp., Cronobacter spp. y Campylobacter spp.) y hongos (Fusariu[CA] La seguretat alimentària està garantida quan es disposa d'aliments suficients, nutritius i innocus. Esta garantia, a més, ha de complir-se al llarg de tot el procés productiu, el que es coneix com a "seguretat de la granja a la taula". Naix així una nova forma d'abordar el problema, amb un enfocament global i un tractament integral. Per a afrontar este repte, les tècniques moleculars basades en la utilització d'àcids nucleics són, actualment, usades en l'anàlisi de certes amenaces alimentàries, com per exemple, la detecció d'al·lèrgens, microorganismes, organismes genèticament modificats (OGM), o l'autentificació de determinades espècies. No obstant això, molts dels mètodes en ús presenten limitacions, ja que són costosos, complicats, i requerixen personal i equipament especialitzat. La tecnologia de biosensors és una aproximació adequada, ja que proporciona resultats fiables de manera senzilla i ràpida, i amb capacitats afegides com portabilitat i automatització per a dur a terme els assajos directament en punts de control (POC). Esta tesi s'ha centrat en el desenvolupament d'un sistema biosensor, basat en la tecnologia de disc compacte, per a la detecció d'àcids nucleics en aplicacions de seguretat alimentària adaptable a POC. Les investigacions dutes a terme han permés obtindre nous coneixements en tecnologies gèniques, podent efectuar aportacions metodològiques d'interés caracteritzades per la seua miniaturització, integració i automatització. En este sentit, una part de la investigació aborda la simplificació de l'etapa d'amplificació de l'ADN diana, eludint el termociclat per mitjà de l'utilització de tècniques alternatives a la reacció en cadena de la polimerasa (PCR). Per a això, s'han estudiat dos tècniques d'amplificació isoterma, l'amplificació per recombinasa polimerasa (RPA) i l'amplificació per desplaçament múltiple (MDA). La detecció es du a terme per mitjà d'assajos d'hibridació amb sondes d'ADN immobilitzades en format micromatriu sobre la superfície de policarbonat d'un DVD. A més, l'amplificació per RPA s'ha combinat amb la detecció per mitjà d'inmunoassaig ezimátic (ELISA) per a la determinació simultània de múltiples anàlits. En una altra aproximació, s'han integrat les fases d'amplificació i hibridació en una única etapa, simplificant encara més el procés analític. Per a això, l'amplificació isoterma per RPA es realitza en fase sòlida sobre la superfície del disc en diferents formats: gota, cambres microfluídicas o microreactors. En els dos primers formats, les reaccions tenen lloc en la superfície del DVD i el mesurament es realitza registrant la intensitat del feix làser del lector reflectit, mentres que en el tercer cas la reacció es du a terme en micropouets integrats en l'estructura del DVD i la lectura es realitza mesurant la intensitat del senyal transmesa. Finalment, s'ha desenvolupat una metodologia per a monitoritzar la síntesi d'ADN en temps real, integrant així les etapes d'amplificació i quantificació. Per a això, s'utilitza la reacció d'amplificació isoterma mitjançada per bucle (LAMP), registrant sequencialment l'avanç de la reacció per mitjà de canvis turbidimétrics o colorimétrics en la reacció. D'esta manera, es relaciona el perfil de cada assaig amb el nombre de còpies de cada gen diana, permetent així la seua quantificació. Per a cada metodologia s'han establit les propietats analítiques i els resultats obtinguts han sigut validats per comparació amb tècniques de referència i per mitjà de l'utilització de mostres certificades. Com a prova de concepte, els diferents desenvolupaments s'han aplicat a la detección i determinació de la presència de al¿lèrgens (avellana, cacauet, soja, tomata i dacsa), organismes modificats genèticament (p35S, tNOS i Bt-11), bacteris patògens (Salmonella spp., Cronobacter spp. i Campylobacter spp.), fongs (Fusarium spp.), així com en la identificSantiago Felipe, S. (2015). Integración de técnicas basadas en ADN para el desarrollo de biosensores aplicados en seguridad alimentaria [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/56464TESISPremios Extraordinarios de tesis doctoralesCompendi

Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc

An integrated method for the parallelized detection of multiple DNA target sequences is presented by using microstructures in a digital versatile disc (DVD). Samples and reagents were managed by using both the capillary and centrifugal forces induced by disc rotation. Recombinase polymerase amplification (RPA), in a bridge solid phase format, took place in separate wells, which thereby modified their optical properties. Then the DVD drive reader recorded the modifications of the transmitted laser beam. The strategy allowed tens of genetic determinations to be made simultaneously within < 2 h, with small sample volumes (3 mu L), low manipulation and at low cost. The method was applied to high-throughput screening of relevant safety threats (allergens, GMOs and pathogenic bacteria) in food samples. Satisfactory results were obtained in terms of sensitivity (48.7 fg of DNA) and reproducibility (below 18 %). This scheme warrants cost-effective multiplex amplification and detection and is perceived to represent a viable tool for screening of nucleic acid targets.This research has been funded through projects FEDER PrometeoII/2014/040 (GVA), and CTQ/2013/45875-R (MINECO). The Spanish Ministry of Education and Science provided S.S.F. with a grant for PhD studies.Santiago Felipe, S.; Tortajada-Genaro, LA.; Puchades, R.; Maquieira Catala, Á. (2016). Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc. Microchimica Acta. 183(3):1195-1202. https://doi.org/10.1007/s00604-016-1745-3S119512021833Li Y, Guo SJ, Shao N, Tu S, Xu M, Ren ZR, Ling X, Wang GQ, Lin ZX, Tao SC (2011) A universal multiplex PCR strategy for 100-plex amplification using a hydrophobically patterned microarray. Lab Chip 11:3609–3618Ng JK, Chong S (2011) Multiplexing Capabilities of Biosensors for Clinical Diagnostics. 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Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis

[EN] Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR-ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA-ELISA combination is proposed for amplification at a low, constant temperature (40 degrees C) in a short time (40 min), for the hybridisation of labelled products to specific 5'-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA-ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA-ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings. (C) 2013 Elsevier B.V. All rights reserved.This research has been funded through Projects GV/2009/028 (Generalitat Valenciana) and CTQ/2010/15943 (MICINN). The Spanish Ministry of Education and Science provided S.S.F. with a grant for her PhD studies. The fungal cultures were supplied by B. Mora-Sala and J. Garcia-Jimenez from the Instituto Agroforestal Mediterraneo, UPV.Santiago Felipe, S.; Tortajada-Genaro, LA.; Puchades, R.; Maquieira Catala, Á. (2014). Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis. Analytica Chimica Acta. 811:81-87. https://doi.org/10.1016/j.aca.2013.12.017818781

Design of oligonucleotide-capped mesoporous silica nanoparticles for the detection of miRNA-145 by duplex and triplex formation

[EN] The development of new strategies to detect microRNAs (miRNAS) has become an important challenge in thebiomedical ¿eld. We report herein the use of oligonucleotide-gated silica nanoparticles for the detection ofmiRNA-145. In the proposed design, mesoporous silica nanoparticles (MSNs) are loaded with a ¿uorescentreporter (rhodamine B) and pores are blocked by speci¿c DNA oligonucleotides. The opening of the gated systemand dye delivery is selectively controlled by DNA-miRNA recognition. Moreover, the use of DNA capture probesable to form duplex or triplex structures between target miRNA and the complementary oligonucleotides hasbeen studied. By this simple procedure a limit of detection as low as 0.25 pM was found for miRNA. The methodwas successfully applied to detect miRNA-145 in serum samples, which demonstrates the high potential of thesecapped materials to detect miRNA for diagnostic purposesWe thank the Spanish Government (projects MAT2015-64139-C4-1-R and CTQ2014-52588-R (MINECO/FEDER)), the Generalitat Valenciana (project PROMETEOII/2014/047) and the Generalitat de Catalunya (2014/SGR/624) for support. A.R. thanks UPV for her predoctoral fellowship. S.S. thanks the Instituto de Salud Carlos III and the European Social Fund for the financial support "Sara Borrell" (CD16/000237). V.C.-N. thanks Ministerio de Educacion, Cultura y Deporte for his predoctoral grant. The authors also thank the Electron Microscopy Service at the UPV for support.Ribes, À.; Santiago Felipe, S.; Aviñó, A.; Candela-Noguera, V.; Eritja, R.; Sancenón Galarza, F.; Martínez-Máñez, R.... (2018). Design of oligonucleotide-capped mesoporous silica nanoparticles for the detection of miRNA-145 by duplex and triplex formation. Sensors and Actuators B Chemical. 277:598-603. https://doi.org/10.1016/j.snb.2018.09.026S59860327

Oligonucleotide-capped nanoporous anodic alumina biosensor as diagnostic tool for rapid and accurate detection of Candida auris in clinical samples

[EN] Candida auris has arisen as an important multidrug-resistant fungus because of several nosocomial outbreaks and elevated rates of mortality. Accurate and rapid diagnosis of C. auris is highly desired; nevertheless, current methods often present severe limitations and produce misidentification. Herein a sensitive, selective, and time-competitive biosensor based on oligonucleotide-gated nanomaterials for effective detection of C. auris is presented. In the proposed design, a nanoporous anodic alumina scaffold is filled with the fluorescent indicator rhodamine B and the pores blocked with different oligonucleotides capable of specifically recognize C. auris genomic DNA. Gate opening modulation and cargo delivery is controlled by successful DNA recognition. C. auris is detected at a concentration as low as 6 CFU/mL allowing obtaining a diagnostic result in clinical samples in one hour with no prior DNA extraction or amplification steps.The authors want to thank the Spanish Government (project RTI2018-100910-B-C41 (MCIU/AEI/FEDER, UE)), the Generalitat Valenciana (project PROMETEO/2018/024), Universitat Politecnica de Valencia-Instituto de Investigacion Sanitaria La Fe (AURISGATE project) and Centro de Investigacion Biomedica en Red en Bioingenieria, Biomateriales y Nanomedicina (NANOPATH AND CANDI-EYE projects) for support. L.P thanks MINECO for his predoctoral fellowship. S.S. thanks the Instituto de Salud Carlos III and the European Social Fund for the financial support "Sara Borrell" (CD16/000237).Pla, L.; Santiago-Felipe, S.; Tormo-Mas, MÁ.; Ruiz-Gaitán, A.; Peman, J.; Valentín, E.; Sancenón Galarza, F.... (2021). Oligonucleotide-capped nanoporous anodic alumina biosensor as diagnostic tool for rapid and accurate detection of Candida auris in clinical samples. Emerging microbes & infections. 10(1):407-415. https://doi.org/10.1080/22221751.2020.187041140741510

INSIGHTS INTO THE HABITS OF THE ELUSIVE NOCTURNAL CURASSOW (NOTHOCRAX URUMUTUM)

Nocturnal curassows (Nothocrax urumutum) are one of the most enigmatic birds from South American rainforests. Their elusive habits and their nocturnal vocal behavior have led to the generalized assumption that they differ from other curassows in their presumable nocturnal habits. Here, we compiled camera trap data from long term projects in the Amazon rainforest and the Andes Cordillera piedmont to describe the temporal activity of Nocturnal Curassows. Based on an overall sampling effort of 68838 camera nights we obtained 274 independent records of their activity. The results of this study evidence that Nothocrax has diurnal habits and resembles in activity patterns to all other cracids more than previously expected. This study highlights the use of novel technologies and collaborative research towards the understanding of the natural history, ecology and behavior of animals with cryptic behaviors such as the Nocturnal Curassows

Isothermal DNA amplification strategies for duplex microorganism detection

[EN] A valid solution for micro-analytical systems is the selection of a compatible amplification reaction with a simple, highly-integrated efficient design that allows the detection of multiple genomic targets. Two approaches under isothermal conditions are presented: recombinase polymerase amplification (RPA) and multiple displacement amplification (MDA). Both methods were applied to a duplex assay specific for Salmonella spp. and Cronobacter spp., with excellent amplification yields (0.2 8.6 108 fold). The proposed approaches were successfully compared to conventional PCR and tested for the milk sample analysis as a microarray format on a compact disc (support and driver). Satisfactory results were obtained in terms of resistance to inhibition, selectivity, sensitivity (101 102 CFU/mL) and reproducibility (below 12.5%). The methods studied are efficient and cost-effective, with a high potential to automate microorganisms detection by integrated analytical systems working at a constant low temperature.Funding projects MINECO CTQ2013-45875-R and GV PrometeoII/2014/040. MECD provided S.S.F with a PhD grant.Santiago Felipe, S.; Tortajada-Genaro, LA.; Morais, S.; Puchades, R.; Maquieira Catala, Á. (2015). Isothermal DNA amplification strategies for duplex microorganism detection. Food Chemistry. 174:509-515. https://doi.org/10.1016/j.foodchem.2014.11.080S50951517

One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method

[EN] An integrated sensor comprising isothermal DNA amplification and in situ detection is presented. The method principle is based on recombinase polymerase amplification (RPA) and detection in the microarray format by compact disc technology as a high-throughput sensing platform. Primers were immobilised on the polycarbonate surface of digital versatile discs (DVD) and, after hemi-nested amplification, multiplexing identification of each tethered product was achieved by optical scanning with a 650 nm-laser of the DVD drive. The efficiency of one-pot hybridisation/elongation/detection depended strongly on probedensity and other factors such as the concentration of the unbound primers present in solution. The optimised conditions provided equivalent amplification factors (7.3 x 10(8) -8.9 x 10(8) fold) to those obtained by conventional reactions performed in vials. The proposed method was applied to Salmonella detection (generic by hns and oriC genes, and specific for subspecies I by STM4507 gene). A triplex assay was satisfactorily compared to the non-integrated protocols. Food and vaccine samples were analysed in a shorter time with less handling. The results indicate that the multiplex DVD assay is a simple, competitive, isothermal, portable system that is particularly useful for microbiological routine analysis. (C) 2014 Elsevier B.V. All rights reserved.This research has been funded through Projects GVA-PROMETEO/2010/008 (Generalitat Valenciana) and CTQ/2013/ 45875-R (MINECO). The Spanish Ministry of Education and Science provided S.S.F. with a grant for her PhD studies.Santiago Felipe, S.; Tortajada-Genaro, LA.; Morais, S.; Puchades, R.; Maquieira Catala, Á. (2014). One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method. Sensors and Actuators B: Chemical. 204:273-281. https://doi.org/10.1016/j.snb.2014.07.073S27328120

Detection of kinase domain mutations in BCR::ABL1 leukemia by ultra-deep sequencing of genomic DNA

The screening of the BCR::ABL1 kinase domain (KD) mutation has become a routine analysis in case of warning/failure for chronic myeloid leukemia (CML) and B-cell precursor acute lymphoblastic leukemia (ALL) Philadelphia (Ph)-positive patients. In this study, we present a novel DNA-based next-generation sequencing (NGS) methodology for KD ABL1 mutation detection and monitoring with a 1.0E-4 sensitivity. This approach was validated with a well-stablished RNA-based nested NGS method. The correlation of both techniques for the quantification of ABL1 mutations was high (Pearson r = 0.858, p < 0.001), offering DNA-DeepNGS a sensitivity of 92% and specificity of 82%. The clinical impact was studied in a cohort of 129 patients (n = 67 for CML and n = 62 for B-ALL patients). A total of 162 samples (n = 86 CML and n = 76 B-ALL) were studied. Of them, 27 out of 86 harbored mutations (6 in warning and 21 in failure) for CML, and 13 out of 76 (2 diagnostic and 11 relapse samples) did in B-ALL patients. In addition, in four cases were detected mutation despite BCR::ABL1 < 1%. In conclusion, we were able to detect KD ABL1 mutations with a 1.0E-4 sensitivity by NGS using DNA as starting material even in patients with low levels of disease

Real-life disease monitoring in follicular lymphoma patients using liquid biopsy ultra-deep sequencing and PET/CT

In the present study, we screened 84 Follicular Lymphoma patients for somatic mutations suitable as liquid biopsy MRD biomarkers using a targeted next-generation sequencing (NGS) panel. We found trackable mutations in 95% of the lymph node samples and 80% of the liquid biopsy baseline samples. Then, we used an ultra-deep sequencing approach with 2 · 10−4 sensitivity (LiqBio-MRD) to track those mutations on 151 follow-up liquid biopsy samples from 54 treated patients. Positive LiqBio-MRD at first-line therapy correlated with a higher risk of progression both at the interim evaluation (HRINT 11.0, 95% CI 2.10–57.7, p = 0.005) and at the end of treatment (HREOT, HR 19.1, 95% CI 4.10–89.4, p < 0.001). Similar results were observed by PET/CT Deauville score, with a median PFS of 19 months vs. NR (p < 0.001) at the interim and 13 months vs. NR (p < 0.001) at EOT. LiqBio-MRD and PET/CT combined identified the patients that progressed in less than two years with 88% sensitivity and 100% specificity. Our results demonstrate that LiqBio-MRD is a robust and non-invasive approach, complementary to metabolic imaging, for identifying FL patients at high risk of failure during the treatment and should be considered in future response-adapted clinical trials.This study has been funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union through the projects PI21/00314, PI19/01430, PI19/01518 and PI18/00295, PTQ2020-011372, CP19/00140, CP22/00082, Doctorado industrial CAM IND2020/TIC-17402 and CRIS cancer foundation