Luis González Valle

Colon cancer is one of the leading causes of death, requiring advanced therapies that need models for developing new drugs. Conventional cell culture models do not accurately and precisely reproduce the complexity of the tumor microenvironment, limiting their usefulness in research and therapy development. To address this weakness, patient-derived organoids have emerged as promising in vitro models. The implementation of these organoid-based models into more physiologically relevant systems is expected to improve their clinical relevance. Thus, integrating these organoids into microfluidic chips acting as bioreactors will likely improve the predictive response of therapies in personalized medicine. In this article, we report the development of a new colon cancer organoid-on-a-chip model that enables the in vitro culture of patient-derived colon cancer organoids under continuous culture media flow. We demonstrate how the developed organoids were derived from tumor biopsies of patients with colorectal cancer, expanded in standard three-dimensional (3D) culture, and cultured inside the microfluidic chips. The microfluidic chip chambers are designed to house organoids in a controlled environment, allowing the injection of therapies and monitoring by optical microscopy in real time. The in vitro therapies tested were a combination of drugs based on 5-fluorouracil and oxaliplatin at different concentrations. As a result, we demonstrate for the first time that this model proves the capability of this technology for in vitro testing colon cancer therapies.

Biomarker detection in clinical diagnostics has been a breakthrough in modern medicine. Thanks to these biomarkers, many diseases can be better understood, detect them earlier and study patients evolution, allowing a more personalized and effective treatment. These markers are very heterogeneous, with many types, ranging from macromolecules such as proteins or oligonucleotides, to simple molecules. Due to this wide variety, it is possible to obtain information on multiple biological processes, pathological mechanisms or the response to a treatment. This thesis has two main objectives, the first one is the development of in-vitro detection system technologies by improving diagnostic kits in order to detect biomarkers to study different diseases. These kits are composed by interferometric biosensors by means of which COVID-19 (Coronavirus disease 2019), Alzheimer's disease and colorrectal cancer will be studied. In the colorrectal cancer study, there is also a second objective, which is to carry out a continuous culture model using Organ-on-a-chip technology by manufacturing microfluidic chips. For the first objective, the diagnostic kits used during the thesis evolution are based on cells that act as optical biosensors, these cells consist of Fabry-Pérot interferometers. Information about the binding between antibody and antigen is obtained through a process of vertical optical interrogation of the biosensor, called Interferometric Optical Detection Methodology (IODM), thus detecting the desired analyte. This interferometric signal can be optimized by using two types of nanoparticles in order to increase the biosensor sensitivity, specifically gold nanoparticles and magnetic iron oxide nanoparticles, which will be used in the immunoassays. For the second objective, biopsies culture are performed, obtained from patients suffering from colon cancer, both from affected and unaffected tissue. Organoids derived from these biopsies are then developed and cultured on both plate and microfluidic chip. Once the organoids are generated, a way to characterize them is proposed; in addition, a competitive assay is proposed to detect the tumor biomarker presence in the culture medium. In short, in this thesis, three different diseases studies are carried out, in which several biomarkers characteristic of these pathologies are analyzed. The diseases studied are COVID-19, detecting specific antibodies against SARS-CoV-2; Alzheimer's disease, detecting Tau and Lactoferrin proteins; and colon cancer, detecting a possible biomarker, the protein IGFBP2 (Insulin-like growth factor-binding protein 2). RESUMEN La detección de marcadores biológicos en el campo de la clínica ha significado un gran avance en la medicina moderna. Gracias a estos biomarcadores, se pueden comprender mejor numerosas enfermedades, detectarlas de manera más temprana y estudiar la evolución de la enfermedad, permitiendo una medicina más personalizada y un tratamiento más efectivo. Estos marcadores son muy heterogéneos, existiendo multitud de tipos, desde macromoléculas como proteínas u oligonucleótidos, hasta moléculas simples. Gracias a esta gran variedad se puede obtener información de múltiples procesos biológicos, de mecanismos patológicos o de la respuesta a un tratamiento. Esta tesis tiene dos objetivos principales, el primero es el desarrollo de tecnologías de sistemas de detección in vitro mediante la mejora de kits de diagnóstico con el fin de detectar biomarcadores que permitan estudiar diferentes enfermedades. Estos kits están formados por biosensores interferométricos mediante los cuales se estudiarán la COVID-19, la enfermedad de Alzheimer, así como el cáncer de colon. En esta última enfermedad, además, existe un segundo objetivo, que consiste en realizar un modelo de cultivo en continuo empleando la tecnología Organ-on-a-chip mediante la fabricación de chips microfluídicos. Para el primer objetivo, los kits de diagnóstico utilizados durante el desarrollo de la tesis están basados en celdas que actúan como biosensores ópticos, estas celdas consisten en interferómetros Fabry-Pérot. Mediante un proceso de interrogación óptica vertical del biosensor, denominado Metodología de Detección Óptica Interferométrica (IODM por sus siglas en inglés), se obtiene información de la unión entre anticuerpo y antígeno, detectando así el analito deseado. Esta señal interferométrica puede ser optimizada mediante el empleo de dos tipos de nanopartículas con el fin de aumentar la sensibilidad del biosensor, en concreto, nanopartículas de oro y nanopartículas magnéticas de óxido de hierro, que serán usadas en los inmunoensayos. Para el segundo objetivo, se realizan cultivos de biopsias obtenidas de pacientes que padecen cáncer de colon, tanto de tejido afectado como no afectado. Seguidamente, se desarrollan organoides derivados de estas biopsias, los cuales se cultivan tanto en placa como en chip microfluídico. Una vez generados los organoides, se propone una forma de caracterizarlos; además se plantea un ensayo competitivo con el cual detectar la presencia de un biomarcador tumoral en el medio de cultivo. En definitiva, en esta tesis se llevan a cabo estudios de tres enfermedades diferentes, en ellos se analizan diversos biomarcadores característicos de estas patologías. Las enfermedades estudiadas son la COVID-19, detectando anticuerpos específicos frente a SARS-CoV-2; la enfermedad de Alzheimer, detectando las proteínas Tau y Lactoferrina; y el cáncer de colon, detectando un posible marcador, la proteína IGFBP2 (siglas del nombre en inglés Insulin-like growth factor-binding protein 2).

We report for the first time the whole development of a biosensing system based on the Interferometric Optical Detection Method (IODM) enriched with gold nanoparticles (AuNPs), acting as interferometric enhancers for improving the performance of immunoassays. For this purpose, the Lactoferrin sandwich immunoassay model was employed. We describe in detail the entire value chain from the AuNPs production, its functionalization, and characterization with anti-Lactoferrin (anti-LF), the biosensing response of these conjugates as well as their corresponding calculation of the kinetic constants, performance comparison of the readout interferometric signals versus Scanning Electron Microscopy (SEM) and the percentage of the sensing surface covered. Finally, a Lactoferrin sandwich immunoassay was carried out and correlated with Enzyme-Linked ImmunoSorbent Assay (ELISA), and the Limit of Detection and sensitivity figures were obtained. As a result, we demonstrate how the AuNPs act as interferometric amplifiers of the IODM for improving the biosensing response, opening the possibility of being applied in multiple biological detection applications.

In this scientific work, we demonstrate, for the first time, a new biosensing system and procedure to measure specifically the total Tau (T-Tau) protein in serum, one of the most relevant biomarkers of Alzheimer’s disease (AD). AD is a progressive brain disorder that produces neuronal and cognitive dysfunction and affects a high percentage of people worldwide. For this reason, diagnosing AD at the earliest possible stage involves improving diagnostic systems. We report on the use of interferometric bio-transducers integrated with 65 microwells forming diagnostic KITs read-out by using the Interferometric Optical Detection Method (IODM). Moreover, biofunctionalized silicon dioxide (SiO₂) nanoparticles (NPs) acting as interferometric enhancers of the bio-transducers signal allow for the improvement of both the optical read-out signal and its ability to work with less-invasive biological samples such as serum instead of cerebrospinal fluid (CSF). As a result, in this paper, we describe for the first time a relevant diagnostic alternative to detect Tau protein at demanding concentrations of 10 pg/mL or even better, opening the opportunity to be used for detecting other relevant AD-related biomarkers in serum, such as β-amyloid and phosphorylated Tau (P-Tau), neurofilaments, among others that can be considered relevant for AD.

Despite the remarkable development related to Point-of-Care devices based on optical technology, their difficulties when used outside of research laboratories are notable. In this sense, it would be interesting to ask ourselves what the degree of transferability of the research work to the market is, for example, by analysing the relation between the scientific work developed and the registered one, through patent. In this work, we provide an overview of the state-of-the-art in the sector of optical Point-of-Care devices, not only in the research area but also regarding their transfer to market. To this end, we explored a methodology for searching articles and patents to obtain an indicator that relates to both. This figure of merit to estimate this transfer is based on classifying the relevant research articles in the area and the patents that have been generated from these ones. To delimit the scope of this study, we researched the results of a large enough number of publications in the period from 2015 to 2020, by using keywords “biosensor”, “optic”, and “device” to obtain the most representative articles from Web of Science and Scopus. Then, we classified them according to a particular classification of the optical PoC devices. Once we had this sampling frame, we defined a patent search strategy to cross-link the article with a registered patent (by surfing Google Patents) and classified them accordingly to the categories described. Finally, we proposed a relative figure called Index of Technology Transference (IoTT), which estimates to what extent our findings in science materialized in published articles are protected by patent.

In the present work, highly multiplexed diagnostic KITs based on an Interferometric Optical Detection Method (IODM) were developed to evaluate six Coronavirus Disease 2019 (COVID-19)-related biomarkers. These biomarkers of COVID-19 were evaluated in 74 serum samples from severe, moderate, and mild patients with positive polymerase chain reaction (PCR), collected at the end of March 2020 in the Hospital Clínico San Carlos, in Madrid (Spain). The developed multiplexed diagnostic KITs were biofunctionalized to simultaneously measure different types of specific biomarkers involved in COVID-19. Thus, the serum samples were investigated by measuring the total specific Immunoglobulins (sIgT), specific Immunoglobulins G (sIgG), specific Immunoglobulins M (sIgM), specific Immunoglobulins A (sIgA), all of them against SARS-CoV-2, together with two biomarkers involved in inflammatory disorders, Ferritin (FER) and C Reactive Protein (CRP). To assess the results, a Multiple Linear Regression Model (MLRM) was carried out to study the influence of IgGs, IgMs, IgAs, FER, and CRP against the total sIgTs in these serum samples with a goodness of fit of 73.01% (Adjusted R-Squared).