Tesis Doctorals - Departament - Enginyeria Electrònica i Biomèdica

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Advanced Machine Learning for EELS Spectroscopy: Magnetic Characterization, Classification and Data Generation
(Universitat de Barcelona, 2024-12-19) Pozo Bueno, Daniel del; Peiró Martínez, Francisca; Estradé Albiol, Sònia; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] This PhD thesis explores the use of Machine Learning (ML) techniques to develop advanced methods for the analysis of Electron Energy Loss Spectroscopy (EELS) spectra. EELS is mainly used in Scanning Transmission Electron Microscopy (STEM) to measure the energy loss by electrons as they pass through a thin sample (typically a few tens of nanometers thick). This energy loss provides valuable information about the composition, chemical bonding, and electronic structure of the sample being studied. However, the complexity and large volume of data generated by EELS make manual analysis time-consuming and inaccurate. To address these challenges, this thesis proposes the integration of ML techniques with EELS data to automate and improve the analysis. First, the thesis provides a comprehensive review of ML applications in EELS, analyzing supervised and unsupervised methods. Following this, the study employs Electron Magnetic Circular Dichroism (EMCD) to characterize bi-magnetic nanocubes with a core/shell (FeO/Fe3O4) structure. By combining EMCD with unsupervised ML, particularly K-means clustering, we identified magnetic regions and found a correlation between the structural and magnetic properties of the material. This analysis revealed an onion-like concentric structure in the nanocubes, with a decreasing magnetic moment from the surface to the core, linked to oxidation gradients and composition changes. These results demonstrate that combining the compositional mapping of EELS with EMCD gives valuable understanding of the magnetic interfaces in nanomaterials. Next, ML is applied, specifically, the soft-margin Support Vector Machine (SVM), to classify the oxidation states of transition metals such as iron and manganese. These metals have characteristic EELS features known as white lines, which are sensitive to the oxidation state. By training SVM models on these spectral features, an accurate classification of the oxidation states was achieved. This analysis also highlights the challenges posed by noise and energy shifts in the data, which can complicate classification. To address these issues, this thesis proposes techniques to reduce their impact, improving the robustness of the classification models. In addition to SVMs, this thesis also investigates the application of Artificial Neural Networks (ANNs) to classify oxidation states in EELS spectra. By directly comparing the performance of SVMs and ANNs, the study emphasizes the strengths and weaknesses of each approach. ANN models are refined through an extensive search for optimal hyperparameters and network architectures, focusing on both dense and convolutional structures. These models demonstrate a strong accuracy for classifying EELS spectra. Moreover, convolutional ANNs emerge as the most effective architecture, achieving precision similar to SVMs but requiring significantly more training data and computational power. CNNs have been found to need a large amount of training data to obtain robust and generalizable models, which implies a higher computational effort and longer training times. In contrast, SVMs, with fewer parameters to optimize, are more suitable for situations with limited data volumes, maintaining efficient and precise performance. To further enhance data diversity, this thesis proposes a data augmentation strategy using Generative Adversarial Networks (GANs). Given that EELS can sometimes provide a limited amount of experimental data (beam-sensitive samples) for training supervised ML models, GANs are employed to generate synthetic EELS spectra. These synthetic datasets help expand the training dataset, improving the performance and precision of the classification models. The thesis presents a functional architecture for both the GAN generator and the discriminator, as well as the process of generating and validating synthetic data. By augmenting the dataset in this way, the research demonstrates how GANs can be a powerful tool to overcome data scarcity, leading to a more robust training of supervised ML models for EELS spectra. In conclusion, this thesis presents a significant advancement in the integration of ML with EELS for material characterization. Through the use of SVMs, ANNs, and GANs, this research improves the classification and analysis of complex EELS data and opens new possibilities for generating synthetic data to support supervised ML models.
CMOS/GaN integration in micro/nanoLED arrays for biomedical applications
(Universitat de Barcelona, 2024-12-13) Moro Moreno, Víctor; Diéguez Barrientos, Àngel; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] Since their inception, LEDs have replaced traditional incandescent and fluorescent lighting in numerous sectors, ranging from general illumination and display technologies to specialized applications, such as in the biomedical field. LEDs provide significant energy savings, reduced environmental impact, and greater design flexibility, marking a significant shift in how light is utilized across different domains, even being able to substitute lasers in a wide range of applications. Building on the success of conventional LEDs, microLED technology has emerged as a groundbreaking advancement. MicroLEDs are significantly smaller than traditional LEDs, typically ranging from a few micrometers to a few hundred micrometers in size. This miniaturization opens new possibilities for high-resolution displays, where microLEDs offer superior brightness, contrast, and energy efficiency compared to OLED and LCD technologies. Additionally, the fast response times and robustness of microLEDs make them suitable for dynamic and demanding applications, such as augmented reality and virtual reality displays, as well as advanced biomedical devices. The integration of CMOS technology with GaN microLEDs in the development of products based on micro or nanoLED arrays is a significant step forward for all the applications where microLEDs can be used. In this work, we will focus on their potential usage in the biomedical field. CMOS technology is known for its scalability, low power consumption and high integration density, which are essential characteristics for creating compact and efficient electronic systems. On the other hand, GaN is valued for its high electron mobility, thermal stability and direct wide bandgap properties, enabling efficient light emission and operation under high power conditions. Combining these technologies, it is possible to create lighting devices with micro or nanoLEDs that leverage the best attributes of both materials, resulting in a compact device with enhanced performance and functionality. This PhD thesis explores the development of CMOS drivers to be integrated with GaN microLED arrays, addressing the inherent challenges posed by the material and processing incompatibilities between silicon-based CMOS and GaN. This research provides a detailed analysis of the optical, electrical, and thermal performance of these devices, demonstrating their superior characteristics compared to conventional LED arrays and other light sources. In the context of biomedical applications, the thesis focuses on microscopy and PoC diagnostics. In advanced microscopy, integrated micro/nano LED arrays can provide high-intensity, uniform illumination with precise control over wavelength and intensity. This capability enhances imaging resolution and contrast, allowing for more detailed and accurate observations at the cellular and molecular levels. The miniaturized form factor of these LEDs also facilitates the development of compact and portable microscopy devices, broadening their accessibility and utility in various medical and research settings. Furthermore, in this thesis we explore the use of micro and nanoLED devices combined with CMOS electronics to create a new type of microscopy technique, Nano-Illumination Microscopy (NIM). For PoC diagnostics, the high speed and high optical power that microLEDs can deliver when driven by CMOS IC is crucial to develop fluorescence based PoC. These arrays can be used to develop portable diagnostic devices that offer real-time monitoring and rapid analysis of biological markers. This is crucial for early disease detection and personalized medicine, where timely and accurate diagnostics can significantly improve patient outcomes. The integration of these LEDs into PoC devices ensures that they are not only effective but also energy-efficient and cost-effective, making them suitable for widespread use, including in resource-limited settings. Furthermore, microLED arrays are a perfect fit to accomplish the ASSURED criteria provided by WHO for PoC devices. In conclusion, this PhD thesis shows that the integration of CMOS and GaN technologies in micro/nano LED arrays offers a transformative approach for advancing biomedical applications, specifically in microscopy and PoC diagnostics. The enhanced performance, combined with the reliability and scalability of these integrated systems, holds significant promise for future innovations in medical diagnostics, treatment, and research. This work lays the groundwork for further exploration and development in the field, potentially leading to new breakthroughs in biomedical technology.
Inkjet printing next-generation flexible devices: memristors, photodetectors and perovskite LEDs
(Universitat de Barcelona, 2024-05-15) González Torres, Sergio; Garrido Fernández, Blas; Vescio, Giovanni; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] New pressures due to emerging trends in device manufacturing are driving research efforts in new materials and manufacturing processes to achieve new combinations of properties, including capabilities such as physical and chemical sensing, conductivity, flexibility, transparency and those related to the internet of things. To face these challenges, printed electronics allows the deposition of next generation materials in ink form, at low cost, with the potential for scalable manufacturing. Inkjet printing is a solution-based deposition technology that allows the deposition of a multitude of functional materials. Thanks to its ability to form patterns digitally, device geometry can be defined without the need for a mask or photolithographic processes, reducing manufacturing costs and enabling rapid prototyping of device architectures. Inkjet printing can be used to manufacture complete device structures, or to deposit innovative materials as a complement to other more established deposition technologies. This thesis tries to show the versatility of inkjet printing as a device manufacturing technology to address future challenges. After the study and validation of various families of nanostructured materials printed by inkjet, three different types of devices are manufactured and characterized. The devices cover several application fields-, highlighting the adaptability of inkjet printing: h-BN 2D memristors, metal oxide nanoparticle photodetectors, and light emitting diodes (LEDs). The first experiments in this thesis deal with 2D inkjet printed h-BN nanoflake memristors for hardware security applications. Memristors are a family of devices whose electrical resistance can be adjusted by electrical manipulation. Although they have shown promising results as information storage units, the applicability of memristors is still subject to their limited device-to-device and cycle-to-cycle repeatability. In this thesis, the inherent stochasticity of memristors is exploited for use as true random number generators (TRNGs) and their application as physical non-clonable functions (PUFs). Next, inkjet printed metal oxide nanoparticle photodetectors are demonstrated. As wide-bandgap materials, metal oxides can play a promising role in selective, transparent, mechanically flexible, and low-cost UV photodetectors. In the final sections, the rapidly developing field of perovskite LEDs (PeLEDs) is surveyed, focusing on inkjet printing of CsPbBr3 inorganic perovskite quantum dot LEDs. Although efforts in the PeLEDs literature have focused on achieving record efficiencies with lab-scale techniques such as spin coating, few researchers have demonstrated scalable fabrication technologies for perovskite LEDs through solution processing such as inkjet printing. Here, the feasibility of inkjet printing is validated by showing fully printed PeLEDs (except the contacts) on flexible and rigid substrates, achieving pure green emission centered at 517 nm with a narrow half maximum down to 22 nm, consistent with the literature results of perovskite layers obtained by more established techniques, demonstrating that the properties of the perovskite layers are maintained in the inkjet deposition process. In this thesis, luminances of up to 17920 cd/m2 have been achieved for devices manufactured using a combination of thermal evaporation and inkjet, and of up to 324 cd/m2 for fully inkjet printed structures. In addition, low-temperature post-processing inorganic metal oxide transport layers are demonstrated, which replace widely used organic materials as a validation step towards more stable fully inorganic device structures.
Microphysiological Systems for the Evaluation of Biomaterials in Regenerative Therapies
(Universitat de Barcelona, 2022-12-12) López Canosa, Adrián; Engel, Elisabeth; Castaño Linares, Óscar; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] The design of bioresponsive materials capable of stimulating the body’s innate regenerative potential is opening unprecedented possibilities to treat tissue and organ failure, which is one of the most important burdens of healthcare systems worldwide. Unfortunately, their development is hampered by the lack of adequate preclinical models, which are essential in the successful transition of a biomaterial to the clinical trials phase. Most of the experiments rely on animal models, which usually fail to predict the material interactions with the human body, as they are unable to recapitulate the complexities of our physiology. During the last decades, the advancements in the field of microtechnology have allowed to create advanced cell culture systems capable of replicating tissue and organ-level physiology by mimicking relevant conditions such as cell organization or microenvironmental cues. These platforms, known as microphysiological systems (MPS), have shown in different studies their great potential in predicting mechanisms of action, safety, and efficacy of different drugs, attracting a lot of attention from the pharmaceutical industry and regulatory agencies. However, few studies have explored the possibility of using microphysiological systems for the preclinical testing of biomaterials. The goal of this thesis is to fill this knowledge gap by developing microfluidic cell culture systems that allow to reliably predict the actual in vivo response of different materials. One of the proposed platforms is aimed at assessing the potential of a biomaterial to stimulate endothelial progenitor cell recruitment in a bone tissue microenvironment. This is a critical step in the neovascularization and bone regeneration process that has not been properly studied due to the lack of adequate models. The proposed device allowed to identify the role of calcium ions in stimulating the recruitment of rat endothelial progenitor cells (rEPC) to the site of injury, which is mediated by an increase in the release of osteopontin, a chemotactic and mitogenic protein produced by rat bone-marrow mesenchymal stromal cells (BM-rMSC). The platform was also used to evaluate a calcium-releasing biomaterial based on electrospun polylactic acid (PLA) fibers with calcium-phosphate (CaP) nanoparticles. The results show a significant increase in terms of rEPC recruitment and the release of osteopontin and other pro-angiogenic and inflammatory proteins by BM-rMSC with respect to a regular PLA control, which is in close agreement with previous experiments performed in a murine in vivo model. The other platform proposed in this thesis is aimed at providing a physiologically relevant model of cardiac tissue to study a myocardial ischemia-reperfusion injury. There are currently no reliable in vitro models to mimic this disease, making these contributions extremely relevant for cardiac regeneration studies. A first prototype of the platform based on the combination of aligned electrospun PLA fibers with a user-friendly electrical stimulation setup in a microfluidic cell culture platform produced a biomimetic cardiac tissue in 2D. This was confirmed by the high anisotropy of the tissue constructs, based on the co- culture of neonatal mouse cardiomyocytes with cardiac fibroblasts, as well as the upregulation of several key cardiac markers such as contractile and structural proteins. In order to make the model more physiologically relevant, a second device was developed to obtain human-derived 3D tissues. This platform is based on the self-assembling of primary cardiac fibroblasts (hCF) co-cultured with human pluripotent stem cell-derived cardiomyocytes (hPSC-CM) in a fibrin-based hydrogel around two microposts structures, which exert a passive mechanical tension that stimulates tissue maturation and cell alignment. We first performed a screening using 2D assays based on hPSC-CM monolayers to select the best environmental conditions to mimic an ischemia-reperfusion injury. We then characterized the response of the human- derived cardiac organoids to an ischemia-reperfusion injury, consisting of an 8 h culture period at 0 % oxygen in an ischemic solution that replicates the acidic and hyperkalemic conditions observed in vivo, followed by a refreshment with fully supplemented cell media and recovery of 21 % environmental oxygen concentrations. We observed a drastic increase in cell death by necrosis and apoptosis as well as a strong fibrotic response, characterized by an increase in hCF proliferation, differentiation towards myofibroblasts and collagen I deposition. Taken together, we believe that the platforms developed in this thesis constitute an extremely valuable and versatile tool to perform preclinical studies, offering a promising alternative to animal studies for the development of new biomaterials and drug discovery.
Development of a Nano-Illumination Microscope
(Universitat de Barcelona, 2022-07-21) Franch Masdeu, Nil; Diéguez Barrientos, Àngel; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] This doctoral thesis proposes and explores a new approach to lensless microscopy, focusing on making high resolution imaging ubiquitous and low cost. A short introduction to microscopy frames the state of current techniques: Abbe’s law limits the resolving power for visible light microscopes with lenses, techniques using UV, X-rays, or electrons are incompatible with live samples and all of them, including super-resolution microscopy methods, are complex devices not suitable for being used in the field as mobile devices. Some lensless microscopy methods try to solve these issues. The microscopy method is named Nano Illumination Microscopy (NIM) because it relies on using nanometric light sources in an ordered array to illuminate a sample placed in close proximity to them, and a photodetector at the other side to measure the amount of light arriving from each LED. In a setup like this, the resolving power is provided by the nano-LEDs and their distribution instead of the sensing devices, as is the case in the other methods. Since the resolving power depends on the pitch of the LED array, this method also opens a path to obtain super-resolution images, depending only on obtaining LED arrays with pitches smaller than Abbe’s limit for the wavelength. After the introduction to microscopy setting the context of the thesis, the thesis continues explaining the main components used to build the microscope: a SPAD camera, designed within the context of this work, and the electronics to control the nano-LED array. The third chapter of this thesis provides an overview of the microscopy method and its fundaments, exploring the requirements and capabilities. Image formation is first introduced with simulations, and this information is then used to build the very first prototype, a microscope capable of forming 8x8 pixel images -since that is the form factor of the LED array used, with LEDs of 5 μm in size (and 10 μm in pitch). The first results from this technique are presented and compared with the simulations, showing the agreement between both, validating the method, and offering insight on building the next prototypes, which will use smaller LEDs in an attempt to study the technological limits. The thesis continues with the work done in search of the limits of the technique, building and testing new improved versions of the microscope and confronting the limitations which arise. Some of those came from the structure of the LED arrays themselves: while nano-LEDs well below the sizes used have been reported, those have been isolated structures or non individually addressable. Selecting exactly which LED will emit is one of the main problems to solve since with increasingly large arrays, the connections required increase exponentially until routing is impossible. The thesis also studies this problem, as the LED arrays were changed in search of the proper solution. This implied moving from a direct addressing strategy, in which each LED was selected individually, towards a matrix-addressing format, in which the LEDs are selected by polarising the appropriate row and columns. The microscopy technique is validated and the more advanced prototypes presented. Images with a maximum resolving power of 800 nm are shown, and the results discussed, since the physical limitations on fabricating the chips limit the maximum resolving power below what was theoretically expected. The thesis also offers a short overview into the future of the Nano Illumination Microscopy technique.
Resistive Switching in Nano-Optoelectronic Devices: Towards an Optical Memristor
(Universitat de Barcelona, 2022-07-12) Frieiro Castro, Juan Luis; Garrido Fernández, Blas; Hernández Márquez, Sergi; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] Resistive switching devices have been a topic of great interest in the last two decades, as they could lead the next generation of memories and processors. These devices present a behavior that allows them to modify their electrical resistance between two or more states and retain them without the need for external energy. The possibility of having two resistance states (high resistance interpreted as 0 and low resistance as 1) already serves as a digital memory, with the advantage of faster switching speeds, lower dimensions for single devices and lower power consumption, when compared to current memories. Since their first physical realization in 2008, great advances have been made in terms of materials employed, device structure, modelling and integration and scaling into arrays and chips. In addition, the properties of resistive switching devices have opened the door for other applications beyond pure memory and the conventional von Neumann architecture. Within the context of resistive switching research, this Doctoral Thesis proposes one new field that can be benefited in the future by the inclusion of such devices: Optoelectronics. The main objective of this Doctoral Thesis is the development of a new concept of devices, which we have called optical memristors. Two types of devices have been attempted and realized: memristors with light emission or absorption. Notwithstanding, both had a particular requirement: transparent materials where necessary for light to be transmitted not only through the electrodes but also through the active layers of the devices. The first approach to light emitting memristors presented explores the possibilities of light emitting devices based on rare earth ions. These elements are commonly employed in displays for the fabrication of phosphorus layers that are excited by a blue emitting device. When properly used as dopants, these elements are optically active and can be electrically excited within a matrix of an oxide material. Thus, the emission of such devices based on Al/Tb/Al/SiO2 layers is studied. A reduction of emission efficiency is also identified with resistive switching capabilities of these devices, though a low number of cycles is possible. A second approach starts from an already transparent conductive oxide (TCO) that has shown resistive switching properties in the literature: ZnO. This material presents advantages when compared to the most employed TCO, ITO, in the form of a non-toxic and abundant compound. In addition, it can be doped with rare earth ions that are optically active. In the same way as the previous approach, resistive switching of these devices is possible, but the inclusion of rare earth ions highly diminishes their endurance. Finally, a different strategy allows for the objective results to be achieved. Silicon oxide is employed as an already reported material with resistive switching properties, where Si nanocrystals (NCs) are embedded as luminescent centers. Their combination results optimal for the target application, yielding durable devices with differentiated emissions dependent on the resistance state and that avoid its overwriting when read. Furthermore, the range of optical properties that become available to these devices through the presence of Si NCs is extended to that of light absorption. The devices become optically-readable taking profit of the photovoltaic effect of their tandem solar cell structure, distinguishing high and low current extractions dependent on the resistance state. Last, the effect of resistive switching and the presence of conductive filaments in these solar cells is explored, achieving increased efficiencies when compared to pristine devices.
Full-3D Printed Electronics Fabrication of Radiofrequency Circuits and Passive Components
(Universitat de Barcelona, 2021-12-15) Salas Barenys, Arnau; López Villegas, José María; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] This doctoral thesis raises the idea that 3D printing can change the paradigm of radio- frequency electronics, which has been traditionally developed mainly conceiving planar topologies. A review on additive manufacturing and the different existing technologies is reported. To focus on the concerning topic, several applications of 3D-printed electronics in the RF field are collected to elaborate the State-of-the-Art. The main objectives of this project is to develop a 3D manufacturing technology for RF electronics passive components and circuits and to generate innovative research about the possibilities of AM in this area. Once the context is exposed, the manufacturing process for 3D-printed electronics developed within the frame of this project is described and characterized. This technology consists of three different steps. First of all, the 3D model of the prototype is designed using a CAD environment with electromagnetic simulation features, hence size parameters are adjusted to fit the specifications. Hereon, the 3D polymer substrate is printed by using either stereolithography or material jetting techniques. Stereolithography is found to be a cheaper AM technology while material jetting offers a better printing resolution and softer surface endings. Finally the object is partially metallized to obtain the conductive layer of the component or circuit using an electrolytic process, such as electroless plating or electroplating. The characterization includes the electromagnetic specifications of the dielectric substrates (i.e. the dielectric constant and the loss tangent) and the quality of the metallization (i.e. the resistivity and the layer thickness). The results of the plating resitivity are found to be competitive compared to the SoA. In order to demonstrate the possibilities of the developed technology, several devices are designed and tested. The key factor of these prototypes is that they would be very difficult, costly or impossible to manufacture using conventional technologies. As a preliminary demonstration, a hello-world circuit to turn on a LED proves that almost any kind of shape can be plated, including vias; both through hole and SMD components can be soldered and that mechanical stress such as USB plugging is resisted by the metal layer. In addition, a study on conical inductors is carried out showing the advantages of these components for broadband applications with compact devices. They offer a larger bandwidth cylindrical solenoids and are more compact than planar coils. As an application example, they are used in the manufacturing of 3D passive filters. The prototypes present agreement with simulations and the ideal response. Slight discrepancies are caused by the manufacturing tolerances. Moreover, 3D filters are also designed as one single-printed part, a new technique for 3D discrete component integration. That permits to reduce the number of components to assembly so that it does not increase with the order of the filter. These single 3D-printed prototypes present improvement in performance and compactness as well. In addition to the lumped circuits, a whole chapter is dedicated to distributed-element devices. A study on helical-microstrip transmission lines is carried out showing an important enhancement for line segment miniaturization. Hereon, they are implemented on the design of impedance transformers, which also benefit from bandwidth broadening. Another proposed device is the hybrid branch-line coupler, which, besides the implementation of helical lines, it has been designed conceiving a capacitively loaded folded structure. This coupler gives very interesting results in compactness improvement, without significant reduction of the bandwidth. The prototypes have been compared to the conventional topology as well as to other designs found within the SoA. Finally, helical-microstrip coupled-line couplers have also been designed, fabricated and studied. They offer a good enhancement in terms of compactness though it goes in slight detriment of the coupling factor due to the manufacturing tolerances.
Nanoscale dielectric mapping of biomembranes with in-liquid Scanning Dielectric Microscopy
(Universitat de Barcelona, 2021-05-11) Muzio, Martina Di; Gomila Lluch, Gabriel; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] The structure and physicochemical properties of biomembranes are fundamental for the functioning of cells, and many pathologies have been associated with their alteration (cancer, neurodegenerations, obesity, etc.) 1, 2. For this reason, biomembranes have been the subject of intensive research. Yet, there is still limited knowledge of biomembranes, which show a heterogeneous structure at the nanoscale that is naturally present in cells, and determines many of the phenomena occurring through them at the molecular level 3, 4. Due to their prominent role in Electrophysiology, electrical properties are among the more relevant physical properties of biomembranes. Most often, attention is paid to biomembranes' conduction properties, and the role played in them by ionic channels. However, biomembranes' dielectric properties are also of central interest in bioelectric phenomena, and a powerful reporter of membranes' composition, which can be exploited to develop label-free mapping methods. Yet, most of the available techniques have addressed the dielectric membrane properties in bulk solutions and at the level of single cells (micrometers), thus lacking spatial resolution. In other cases, they make use of exogenous labels, as in the case of spin paramagnetic resonance 5, 6 and fluorescence microscopy 7, 8, 9, 10, 11. In recent years, the Nanoscale Bioelectric Characterization group at IBEC, as well as other groups, have developed some Scanning Probe Microscopies (SPMs) based techniques to attempt the dielectrical characterization at the nanoscale 12, 13, 14, 15, 16 and applied them to biomembranes 17, 18, 19, 20, 21 and other biosystems 22, 12, 23, 24, 25, 26, 27. Initially, these techniques were implemented to be operated in air environment, but lately they were also extended to liquid environment 28, 29. The implementation of in-liquid Scanning Dielectric Microscopy (SDM) paved the way to the accurate dielectric characterization of biomembranes at the nanoscale, in their physiological environment and in a label-free way 28, 29. In-liquid SDM is based on measuring the electrostatic force acting on a nanometric probe under application of a modulated voltage between the tip and a conductive substrate, on top of which the sample is sitting. As compared to the standard SDM in air, operation in liquid environment requires several modifications. In terms of set-up, one needs to apply frequencies above the dielectric relaxation frequency of the electrolyte. Significant changes are also necessary for the modelling part 30. This work of thesis takes advantage of the latest developments of in-liquid SDM to characterize the dielectric properties of heterogeneous model and natural purified membranes systems in liquid. In this framework, new knowledge has been gained about imaging in liquid conditions with SDM, e.g. about the prominent electrostatic finite size effect and different models have been tested and optimized for the analysis of the measurements. First, I focused on characterizing mono- and bicomponent planar supported bilayer lipid mixtures containing cholesterol, providing a first proof-of-concept of the label-free mapping capabilities of the technique in liquid media, extending earlier work done in air on nanoparticles 12. This study allowed gaining information on the composition of sub- micrometric membrane domains in liquid environment 31 and to provide reliable values of the intrinsic dielectric properties of DOPC and DOPC/cholesterol compositions, about which there was some debate in the literature. The low values obtained are responsible for membranes’ low permeability to ions, in agreement with previous studies on monocomponent biomembranes 29. Our results allow speculating on fundamental properties of lipid bilayers like viscosity and hydration of cholesterol-containing layers. Afterwards, we extended the methods to deal with more complex biomembrane 3D structures, such as liposomes 32. Liposomes with few hundred nanometers in height have been successfully imaged by in-liquid SDM, showing a sensitivity comparable to the one for flat biomembranes only a few nanometers thin. Once again, the dielectric properties of the liposomes’ membrane were precisely extracted, this time in a more natural configuration of the biomembrane. This study also highlighted the technique’s sub- surface capabilities in the liquid environment, demonstrated earlier only in air measurements 33, 34, 35, 36, 37, 38, 39. This capability enabled to obtain in a label-free way the lamellarity of liposomes, a crucial parameter in liposomes technology. The developed methodology has the potential to be used to screen a myriad of different compositions of liposomes (shell and core), since in-liquid SDM was shown to be sensitive to the dielectric properties of the membrane but also to the conductivity of the medium inside the liposomes. This accomplishment was essential to evaluate its future application to living cells and constitutes one of the main achievements of this work. During the thesis, I also draw my attention to the dielectric characterization of natural purified membranes in liquid environment. As a test example, we focused on the case of the purple membrane (PM), which had previously been studied in air environment 40, 20, 19. PMs are constituted by the protein bacteriorhodopsin (BR) arranged in a crystal lattice, and lipids in a ratio 10:1 lipids:proteins. However, an unsolved uncertainty in determining the real topography of supported PM patches in the liquid environment, which can also display a concave surface, made the dielectric quantification problematic, and further explorations will be necessary to provide reliable values of the permittivity of these layers in liquid media. In summary, the objective and common thread connecting all the chapters of this work has been implementing and demonstrating the capabilities of in-liquid SDM in the dielectric characterization of bio-membranes, model and natural systems, with nanoscale spatial resolution. I believe that this work laid the ground for elucidating the structure and dielectric properties of more complex membranes systems and their associated electric phenomena, e.g. conduction. Preliminary studies of the cell membrane directly on living neuroblastoma cells, in low concentration MOPS buffer, were carried out in collaboration with Maria Elena Piersimoni, PhD student at Imperial College, London. The group is now collaborating with experts in the field and trying to develop new algorithms, fundamental to extend the methods to living cells. In addition to the main objective of my thesis, I also participated in a side project concerning the in-liquid SDM characterization of an operative EGOFET transistor 41, crucial for optimizing the devices and gain a better understanding of the transduction mechanism with biological entities. One of the newest frontiers of such technology is indeed to use supported lipid bilayers for bio-sensing purposes 42. References: (1) Lauwers, E.; Goodchild, R.; Verstreken, P. Membrane Lipids in Presynaptic Function and Disease. Neuron 2016, 90 (1), 11–25. https://doi.org/10.1016/j.neuron.2016.02.033. (2) Ashrafuzzaman, M., Tuszynski, J. Membrane-Related Diseases, Springer-V.; Springer- Verlag Berlin Heidelberg 2012, 2012. (3) Mueller, P.; Rudin, D. O. Resting and Action Potentials in Experimental Bimolecular Lipid Membranes. J. Theor. Biol. 1968, 18 (2), 222–258. https://doi.org/10.1016/0022- 5193(68)90163-x. (4) Hodgkin, A. L.; Huxley, A. F. A Quantitative Description of Membrane Current and Its Application to Conduction and Excitation in Nerve. J Physiol. 1952, 117, 500–544. https://doi.org/10.1109/ICCCT2.2017.7972284. (5) Kurad, D.; Jeschke, G.; Marsh, D. Lipid Membrane Polarity Profiles by High-Field EPR. Biophys. J. 2003, 85 (2), 1025–1033. https://doi.org/10.1016/S0006-3495(03)74541-X. (6) Marsh, D. Polarity and Permeation Profiles in Lipid Membranes. Proc. Natl. Acad. Sci. U. S. A. 2001, 98 (14), 7777–7782. https://doi.org/10.1073/pnas.131023798. (7) Huang, H.; McIntosh, A. L.; Atshaves, B. P.; Ohno-Iwashita, Y.; Kier, A. B.; Schroeder, F. Use of Dansyl-Cholestanol as a Probe of Cholesterol Behavior in Membranes of Living Cells. J. Lipid Res. 2010, 51 (5), 1157–1172. https://doi.org/10.1194/jlr.M003244. (8) Parasassi, T.; De Stasio, G.; Ravagnan, G.; Rusch, R. M.; Gratton, E. Quantitation of Lipid Phases in Phospholipid Vesicles by the Generalized Polarization of Laurdan Fluorescence. Biophys. 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Nanoscale Electric Polarizability of Ultrathin Biolayers on Insulating Substrates by Electrostatic Force Microscopy. Nanoscale 2015, 7, 18327–18336. https://doi.org/10.1039/x0xx00000x. (22) Cuervo, A.; Dans, P. D.; Carrascosa, J. L.; Orozco, M.; Gomila, G.; Fumagalli, L. Direct Measurement of the Dielectric Polarization Properties of DNA. Proc. Natl. Acad. Sci. U. S. A. 2014, 111 (35). https://doi.org/10.1073/pnas.1405702111. (23) Biagi, M. C.; Fabregas, R.; Gramse, G.; Van Der Hofstadt, M.; Juárez, A.; Kienberger, F.; Fumagalli, L.; Gomila, G. Nanoscale Electric Permittivity of Single Bacterial Cells at Gigahertz Frequencies by Scanning Microwave Microscopy. ACS Nano 2016, 10 (1), 280–288. https://doi.org/10.1021/acsnano.5b04279. (24) Esteban-Ferrer, D.; Edwards, M. A.; Fumagalli, L.; Juárez, A.; Gomila, G. Electric Polarization Properties of Single Bacteria Measured with Electrostatic Force Microscopy. ACS Nano 2014, 8 (10), 9843–9849. https://doi.org/10.1021/nn5041476. (25) Van Der Hofstadt, M.; Fabregas, R.; Millan-Solsona, R.; Juarez, A.; Fumagalli, L.; Gomila, G. Internal Hydration Properties of Single Bacterial Endospores Probed by Electrostatic Force Microscopy. ACS Nano 2016, 10 (12), 11327–11336. https://doi.org/10.1021/acsnano.6b06578. (26) Checa, M.; Millan-Solsona, R.; Blanco, N.; Torrents, E.; Fabregas, R.; Gomila, G. Mapping the Dielectric Constant of a Single Bacterial Cell at the Nanoscale with Scanning Dielectric Force Volume Microscopy. Nanoscale 2019, 11 (43), 20809–20819. https://doi.org/10.1039/c9nr07659j. (27) Lozano, H.; Fabregas, R.; Blanco-Cabra, N.; Millán-Solsona, R.; Torrents, E.; Fumagalli, L.; Gomila, G. Dielectric Constant of Flagellin Proteins Measured by Scanning Dielectric Microscopy. Nanoscale 2018, 10 (40), 19188–19194. https://doi.org/10.1039/c8nr06190d. (28) Gramse, G.; Edwards, M. A.; Fumagalli, L.; Gomila, G. Dynamic Electrostatic Force Microscopy in Liquid Media. Appl. Phys. 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Electrical Properties and Lamellarity of Single Liposomes Measured by In-Liquid SDM. [in Prep. (33) Fumagalli, L.; Esfandiar, A.; Fabregas, R.; Hu, S.; Ares, P.; Janardanan, A.; Yang, Q.; Radha, B.; Taniguchi, T.; Watanabe, K.; et al. Anomalously Low Dielectric Constant of Confined Water. Science (80-. ). 2018, 360 (6395), 1339–1342. https://doi.org/10.1126/science.aat4191. (34) Fabregas, R.; Gomila, G. Dielectric Nanotomography Based on Electrostatic Force Microscopy: A Numerical Analysis. J. Appl. Phys. 2020, 127 (2). https://doi.org/10.1063/1.5122984. (35) Castañeda-Uribe, O. A.; Reifenberger, R.; Raman, A.; Avila, A. Depth-Sensitive Subsurface Imaging of Polymer Nanocomposites Using Second Harmonic Kelvin Probe Force Microscopy. ACS Nano 2015, 9 (3), 2938–2947. https://doi.org/10.1021/nn507019c. (36) Riedel, C.; Alegra, A.; Schwartz, G. A.; Arinero, R.; Colmenero, J.; Senz, J. J. 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Nanoscale Tomography Based in Electrostatic Force Microscopy
(Universitat de Barcelona, 2021-05-12) Balakishan, Harishankar; Gomila Lluch, Gabriel; Izquierdo Fábregas, Lazaro René; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] The ability to characterize the elements beneath the surface has been a dire necessity in the fields of materials science, polymer technology, biology, and medical sciences. Scanning Probe Microscopies are the family of microscopies that scans the surface using a nanometric probe and the acquired data is used to reconstruct the physical properties of the samples in nanometric resolution (e.g., topography). Since the measurements could be carried out in non-contact mode, the ability to study tomography have made them a better contender. SPM also possess the relative advantage of being non-invasive, non-destructive, requires relatively minimal sample preparation, can be extended into any environment (inert, ambient vacuum), and also be measured in air, water, or any biological medium. Among them, Electrostatic Force Microscopy, has been successfully used in subsurface investigations to study the compositional modifications below the organic layers, imaging below the organic layers, imaging water molecules in confined nanometric channels, imaging of carbon nanotubes, graphene networks and nanoparticles inside the polymeric nanocomposites. Nanocomposites, which consist of nanostructures in their bulk matrix to improve the matrix efficiency, have been one of the successfully incorporated material science application of the last two decades. Silver nanoparticle especially have a barrage of applications to its credit ranging from solar cell applications, touch screens, LEDs to flexible wearable devices. Understanding the subsurface features or tomography of these nanocomposites could help us in understanding their properties, interpreting them based on their parametric dependence which would later aid us in tuning them for our desired applications. In this thesis. Individual computational studies have been carried out of nanowires buried in a dielectric matrix to observe the effects of various parameters influencing the subsurface imaging. Spatial resolution is given prime importance as its behavior of two parallel nanowires is studied along with two nanowires overlapped one on top of each other. Also, the analysis of silver nanowire nanocomposites has been investigated with the help of Scanning Dielectric Force Volume Microscopy, a technique proposed recently with EFM. The bulk matrix is composed of gelatin which can offer a range of permittivities depending on the degree of hydration, for e.g., here εr ~ 5 to εr ~ 14 . This sample is experimentally analyzed, imaged and the depth of nanowires in the matrix inside the bulk matrix is mapped with the theoretical analysis. This thesis research provides us with subsurface information that would help us in understanding and tuning the parameters to achieve desired applications.
Compartmentalised microfluidic culture systems for in vitro modelling of neurological and neuromuscular microenvironments
(Universitat de Barcelona, 2021-03-10) Badiola Mateos, Maider; Samitier i Martí, Josep; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] Movement of skeletal-muscle fibres is generated by the locomotion circuit, in which many cells play a different role. Failures in any part of the circuit can cause or define the severity of neuromuscular diseases (NMD), such as amyotrophic lateral sclerosis (ALS). Conventional in vitro study models are based on cocultures of motoneurons and skeletal muscle cells from animal origin in 2D. These models have proved to be quite limited for the understanding of neuromuscular connection and NMD. They do not consider that: i) neural somas and muscles or peripheral glia are physically separated in vivo and have different microenvironment requirements; ii) both sensory and motor neurons can be altered in particular NMD; iii) glial cells are also affected and involved in several neuromuscular pathologies; iv) 2D cultures do not mimic physiological conditions; v) rodent models offer limited benefit translated into clinic research, as they do not carry human genetic background. Later progresses in neuromuscular-mimicking in vitro systems, have been achieved incorporating increasingly evolving technologies, such as 3D cell-culture techniques, human induced pluripotent stem cells (hiPSC) and compartmentalised microfluidic culture systems (cµFCS). The later ones are microfluidic devices for 2D or 3D cell- cultures, with several interconnected compartments, each mimicking different microenvironments or functional units in organ or tissue level. 3D cell culture techniques make cells acquire more in vivo like phenotype and genotype patterns. And finally, hiPSC serve to create study models that mimic the human physiology in both healthy and pathological conditions. This thesis, entitled “Compartmentalised microfluidic culture systems for in vitro modelling of neurological and neuromuscular microenvironments”, aims to study the neuromuscular context in vitro through cµFCS and to create physiologically relevant models. It offers an evolving prospective of in vitro models, moving from mice to human cells, from 2D to 3D cell cultures, from primary cells to hiPSC, and analysing both healthy and diseased cells. Chapter 1 reviews the state of the art in the neuromuscular circuit, amyotrophic lateral sclerosis, and the evolution of in vitro techniques available for their study. Chapter 2 presents the first approach of the neuromuscular in vitro connection model on a chip, showing the relevance of myelin in the peripheral nervous system and in the neuromuscular circuit. Chapter 3 moves to study the proprioception, the differentiation of human neural stem cells to proprioceptive sensory neurons, and their role in ALS. These concepts, together with the ones introduced in Chapter 2, are integrated in Chapter 4, presenting the development of a physiological human neuromuscular circuit on a microfluidic device, that integrates neuromuscular motor and sensory pathways in a 3D cell culture system. Lastly, in a context of neuromuscular vascularisation, Chapter 5 studies the blood-brain barrier and the techniques to monitor its permeability, known to be affected in some NMD such as ALS. This thesis presents the use of several cµFCS for different purposes, incorporating the study of several neuromuscular key role players and obtaining the following results. Myelination induction was successfully incorporated in a designed and fabricated compartmentalised microfluidic culture system (a PDMS device with two compartments connected through microchannels). This system was capable of a simplified mimicking of both peripheral nervous system and neuromuscular afferent or efferent pathways. To move onto human models, first proprioceptive sensory neuron (pSN) differentiation protocol was established. Genetic comparative analysis between healthy and ALS diseased samples revealed differences among pSN related genetic patterns and those involved in the communication between pSN and motoneurons (MN). Human pSN differentiation was combined with skeletal muscle cells to create sensorimotor units in a two-compartment commercial microfluidic device, showing for the first time the formation of synaptic bouton like structures in the contact points of an annulospiral wrapping. Then, a human neuromuscular circuit model was created, integrating for the first time human motor and sensory pathways in 3D cultures in tailored microfluidic devices. Finally, the blood-brain barrier was studied as an example of neural vascularisation, within the framework of potentially affected components in NMD. To that end, a new technology for the in vitro monitorisation of blood-brain barrier permeability was created and implemented in a device previously developed in the lab. This system could easily be translated for blood-spinal cord barrier studies. This thesis gathers many technological innovations from a Bioengineering point of view, paving the way for future studies in the neuromuscular field. It shows that the integration of the entire neuromuscular circuit components in the developed in vitro systems provides a wider view of the neuromuscular physiology and the pathological processes. These results show first steps towards future 3D physiological neuromuscular circuit models on a chip for NMD studies.
Electrochemical Plug-and-Power e-readers for Point-of-Care Applications
(Universitat de Barcelona, 2020-12-15) Montes Cebrián, Yaiza; Miribel-Català, Pere Ll. (Pere Lluís); Colomer i Farrarons, Jordi; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] Point-of-Care diagnostic tests enable monitor health conditions and obtain fast results close to the patient, reducing medical costs, and allowing the control of infectious outbreaks. The interest in developing Point-of-Care devices is increasing due to they are suitable for a wide variety of applications. This doctoral thesis focuses on the development of Plug-and-Power electronic readers (e- readers) for electrochemical detections and the demonstration of their possibilities as Point-of-Care diagnostic testing. The solutions proposed in this study make it possible to improve Point-of-Care tests whose premises are laboratory decentralization, personalized medicine, rapid diagnosis, and improvement of patient care. Developed electronic readers can be powered from a conventional system, such as a USB port or a lithium battery, or can be defined as self-powered systems, capable of extracting energy from alternative energy sources, such as fuel cells, defining Plug-and-Power systems. The designed electrochemical detection devices in this thesis are based on low-power consumption electronic instrumentation circuits. These circuits are capable of controlling the sensing element, measuring its response, and representing the result quantitatively. The implemented devices can work with both electrochemical sensors and fuel cells. Furthermore, it is possible to adapt its measurement range, enabling its use in a wide variety of applications. Thanks to their reduced energy consumption, some of these developments can be defined as self-powered platforms able to operate only with the energy extracted from the biological sample, which in turn is monitored. These devices are easy-to-use and plug-and-play, enabling those unskilled individuals to carry out tests after prior training. Moreover, thanks to their user-friendly interface, results are clear and easy to understand. This doctoral dissertation is presented as an article compendium and composed of three publications detailed in chronological order of publication. The first contribution describes an innovative portable Point-of-Care device able to provide a quantitative result of the glucose concentration of a sample. The proposed system combines an e-reader and a disposable device based on two elements: a glucose paper-based power source, and a glucose fuel cell-based sensor. The battery-less e-reader extracts the energy from the disposable unit, acquires the signal, processes it, and shows the glucose concentration on a numerical display. Due to low-power consumption of the e-reader, the whole electronic system can operate only with the energy extracted from the disposable element. Furthermore, the proposed system minimizes the user interaction, which only must deposit the sample on the strip and wait a few seconds to see the test result. The second publication validates the e-reader in other scenarios following two approaches: using fuel cells as a power element, and as a dual powering and sensing element. The device was tested with glucose, urine, methanol, and ethanol fuel cells and electrochemical sensors in order to show the adaptability of this versatile concept to a wide variety of fields beyond clinical diagnostics, such as veterinary or environmental fields. The third study presents a low-cost, miniaturized, and customizable electronic reader for amperometric detections. The USB-powered portable device is composed of a full- custom electronic board for signal acquisition, and software, which controls the systems, represents and saves the results. In this study, the performance of the device was compared against three commercial potentiostats, showing comparable results to those obtained using three commercial systems, which were significantly more expensive. As proof of concept, the system was validated by detecting horseradish peroxidase samples. However, it could be easily extended its scope and measure other types of analytes or biological matrices since it can be easily adapted to detect currents a wide range of currents.
Estudio eléctrico y topográfico de apéndices bacterianos en la nanoescala
(Universitat de Barcelona, 2020-10-13) Lozano Caballero, Helena; Gomila Lluch, Gabriel; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] Recently the property of some bacteria to exchange electrons with non-soluble electron acceptors, such as minerals, has been discovered. In addition, some of these bacteria can use electrodes as the final electron acceptor. This phenomenon is called Extracellular Electron Transfer (EET) and it can be done through several mechanisms, especially through conductive bacterial nanowires. The main objective of this thesis is the investigation of the polarization properties of such electrochemically active bacteria and their appendages as the polarization plays a key role in EET. The curiosity also stems out from the increasing interest in using such bacteria as a biosensing platform. Specifically, I have studied two types of bacteria, Shewanella oneidensis MR-1 and cable bacteria, from the Desulfobulbaceae family. With this purpose, I have used the Electrostatic Force Microscopy (EFM) technique, which measures the electrostatic force using a nanometric probe, combined with finite element simulations to obtain the polarization properties of bacterial nanowires. The electrostatic force depends mainly on the geometry and dielectric constant of the probe-sample system. In order to do that, first, I have developed a way to obtain the dimensions of objects without damaging them, avoiding any physical contact, by measuring the electrostatic force. I have tested this technique on silver nanowires and bacterial flagella. In this way, I have been able to optimize the EFM technique to nanowire-like biological samples at the nanoscale. Secondly, I have analyzed one of the S. oneidensis appendages, the flagella, and I have compared their properties with the flagella of a non-electrochemically active bacteria, the Pseudomonas aeruginosa. I have obtained a dielectric constant of εSo = 4.3 ± 0.6 for S. oneidensis and εPa = 4.5 ± 0.7 for P. aeruginosa, similar results for both bacteria. In addition, this value corresponds to the dielectric constant of proteins (εr ~ 4) measured with the same technique, in agreement with the fact that flagella are composed of flagellin protein monomers. Later, I have studied the electrical properties of another S. oneidensis appendages, the outer membrane extensions (OMEs), responsible for the extracellular electron transfer. In this analysis, I have obtained a relatively low value of the dielectric constant (εOME = 3.7 ± 0.7) corresponding to a combination of lipids (εr ~ 2) and proteins (εr ~ 4). However, considering that the conduction mechanism of such OMEs is through electron hopping, and electrons are localized, these results do not contradict the literature. I have also studied the electrical properties of the cable bacteria, especially the fibers that are along this filamentous bacterium. The dielectric constant of the fibers was εr = 7 ± 1. However, this result is not compatible with the conductivity reported in the literature. Therefore, a core-shell model was proposed with a conductive core of h ~ 10 – 20 nm. Subsequently, I have finished the nanoscale analysis performing qualitative EFM measurements in liquid over living S. oneidensis bacteria and rehydrated bacteria. Finally, I have connected these nanoscale measurements in dry conditions with macroscale measurements in living S. oneidensis using a microfluidic device that I designed, fabricated and characterized at the Denmark Technical University (DTU) in Copenhagen. The microfluidic device was used to perform two-electrode impedance measurements. In these measurements, the impedance experiences an abrupt change for f ~ 102 – 103 Hz when bacteria were in anaerobiosis. However, further experiments are needed to explain this phenomenon.
A portable device for time-resolved fluorescence based on an array of CMOS SPADs with integrated microfluidics
(Universitat de Barcelona, 2020-07-16) Canals Gil, Joan; Diéguez Barrientos, Àngel; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] Traditionally, molecular analysis is performed in laboratories equipped with desktop instruments operated by specialized technicians. This paradigm has been changing in recent decades, as biosensor technology has become as accurate as desktop instruments, providing results in much shorter periods and miniaturizing the instrumentation, moving the diagnostic tests gradually out of the central laboratory. However, despite the inherent advantages of time-resolved fluorescence spectroscopy applied to molecular diagnosis, it is only in the last decade that POC (Point Of Care) devices have begun to be developed based on the detection of fluorescence, due to the challenge of developing high-performance, portable and low-cost spectroscopic sensors. This thesis presents the development of a compact, robust and low-cost system for molecular diagnosis based on time-resolved fluorescence spectroscopy, which serves as a general-purpose platform for the optical detection of a variety of biomarkers, bridging the gap between the laboratory and the POC of the fluorescence lifetime based bioassays. In particular, two systems with different levels of integration have been developed that combine a one-dimensional array of SPAD (Single-Photon Avalanch Diode) pixels capable of detecting a single photon, with an interchangeable microfluidic cartridge used to insert the sample and a laser diode Pulsed low-cost UV as a source of excitation. The contact-oriented design of the binomial formed by the sensor and the microfluidic, together with the timed operation of the sensors, makes it possible to dispense with the use of lenses and filters. In turn, custom packaging of the sensor chip allows the microfluidic cartridge to be positioned directly on the sensor array without any alignment procedure. Both systems have been validated, determining the decomposition time of quantum dots in 20 nl of solution for different concentrations, emulating a molecular test in a POC device.
Reaching the tumour: nanoscopy study of nanoparticles biological interactions
(Universitat de Barcelona, 2020-01-23) Feiner Gracia, Natalia; Albertazzi, Lorenzo; Samitier i Martí, Josep; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] The precise delivery of therapeutic agents to their specific site of action is a big challenge in cancer treatment, which would enhance the efficacy and reduce the side effects of drugs. In this framework, nanotechnology can greatly contribute to the development of novel drug delivery systems. Nowadays, a large number of nanoparticles, differing in chemical nature, have been synthesized and evaluated for their therapeutic performances. However, most of the newly developed delivery systems are ineffective in the clinic because they don’t reach the specific cancer cells. One of the main reasons of this failure is the lack of knowledge about the interactions between the designed nanosystems and the biological media before getting to the targeted cells, the so-called nanobiointeractions. In particular, undesired interactions with blood proteins and other molecules in vasculature are often responsible for the poor performance of nanocarriers. Further research about the biological interactions occurring in the blood vessels is needed in order to design novel and improved therapeutic nanoparticles. We believe that the understanding of these critical steps together with an in-depth study of the structural composition of nanoparticles will guide a rational design of systems, increasing their applicability and performance in the clinic. To accomplish a comprehensive study, in this thesis, we propose the use of advanced optical microscopy techniques to investigate the chemical and biological identity of nanomaterials and to understand their role with a nanometric precision. One of the first biological barriers nanoparticles encounter when introduced intravenously to the body are proteins which travel through the blood stream. These molecules form the so-called protein corona: a shell of proteins attached to the surface of the nanoparticle. One of the main drawbacks caused by protein corona formation is the hindering of the nanoparticle’s surface, reducing the specific interactions of nanosystems with the cancer cells they are targeting. Protein corona formation is mainly studied using ensemble techniques which give only an approximate idea of the molecules interacting with the surface of the nanoparticles. In chapter two, STORM imaging of corona is presented as a new methodology to obtain an in situ characterization of protein corona on individual nanoparticles. This study reveals a high interparticle heterogeneity regarding the number of proteins per nanoparticle, which may be one of the causes of their poor clinical performance. Protein interactions are not only responsible of reducing specific interactions but can dramatically affect the stability of nanosized delivery systems. Therefore, it is important to study their stability in the blood complex environment. Polyplexes are nanocarriers characterized by the electrostatic interactions between the carrier and the nucleic acid. These systems need to be fully complexed during their circulation in the blood vessels in order to protect their cargo from degradation. Up to now, the challenges in characterizing the molecular distribution of the individual components have limited the rational design of nanosystems. In the third chapter, dSTORM imaging is used to visualize the exact molecular composition of polyplexes. dSTORM imaging unveiled the differences in the stoichiometry of individual systems, revealing a heterogeneity inside the same population. Once the system is fully characterized, his complexation can be followed under different blood-like conditions thanks to the molecular resolution of the technique. This new method allows to determine the real molecular stability of the system in contact with serum proteins and provides mechanistic insights into the disassembly process. The stability in complex biological media is a determining factor of the good performance of drug delivery systems, especially in the use of supramolecular structures, due to their dynamic nature. Therefore, it is necessary to understand the behavior of self-assembled nanoparticles in conditions close to the ones they would confront in vivo. Serum proteins can prematurely disassemble the system, as seen in the previous chapter, and lead to non-selective release of the cargo in healthy tissues. Another critical issue of self-assembling systems is the strong dilution they undergo when injected in blood, which may severely affect the supramolecular stability. Hence, it is of crucial importance to investigate the effect of dilution in biologically relevant media. These issues are often overlooked in the literature, most likely due to the difficulties of studying supramolecular assemblies in complex biological media. In chapter four, micelles that change their fluorescent properties upon disassembly are characterized under different blood- like conditions using a combination of fluorescence spectroscopy and microscopy techniques, allowing to predict the system with the best properties. A last critical step nanoparticles face when injected into the blood vessels is the flow, which may also affect the stability of the system. Moreover, their efficiency is directly proportional to the ability of extravasation from the blood vessel across the tight endothelial layer before reaching the cancer cells. In each of these barriers, the stability of supramolecular systems may be compromised. In chapter five a microfluidic chip mimicking the vascular tumor microenvironment is optimized to study the ability and stability of supramolecular structures during extravasation. A monolayer of human umbilical vein endothelial cells are grown in the microfluidic device forming a blood-vessel-like channel. Moreover, the chip contains a second channel of tumorigenic cells to test the stability of the nanocarrier in the extracellular matrix close to cancer cells after extravasation. This device allows to screen the behavior of the different delivery systems and predicts the most stable and promising system thus optimizing and reducing the pre-clinical and clinical testing.
Una nova tècnica de microscòpia de forces atòmiques per a l'estudi de les propietats nanoelèctriques de les cèl·lules
(Universitat de Barcelona, 2020-02-24) Checa Nualart, Martí; Gomila Lluch, Gabriel; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] This work of thesis is organized into 8 different chapters: Chapter 1: introduces the nanoscale electrical properties of cells and their relevance in the different fields of science in order to establish a framework for the objectives of the project. Chapter 2: introduces the Scanning Probe Microscopy techniques, with a special interest in its electrical modes. It mainly focuses the interest on Electrostatic Force Microscopy as it is the technique used during the project. Motivation and objectives of the thesis: Once the introductory chapters have established a framework for the realization of the thesis, its motivation, and its main goals are exposed. Chapter 3: covers the theoretical modeling of electrostatic force measurements in electrolyte solutions for 1D geometry. Chapter 4: extends the theoretical modeling of electrostatic force measurements in electrolyte solutions to 3D realistic geometries, and their implications regarding EFM in liquid media are discussed. Chapter 5: presents and validates a new SPM technique called Scanning Dielectric Force Volume Microscopy (SDFVM) designed specially to deal with topographically complex and heterogeneous samples like cells. Chapter 6: exposes the application of SDFVM to obtain the first (to our knowledge) local dielectric constant map of a cell at the sub-cellular level. Chapter 7: uses the developed technique to obtain EFM images of different interesting systems in nanotechnology, showing the power of method by achieving nice images in a wide range of systems going from small nanowires to big eukaryotic cells. Chapter 8: presents the steps performedtowards implementing SDFVM into liquid environments, and some preliminary results for fixed and living eukaryotic cells in pyhsiological conditions. Then the conditions of the project and its futures perspectives are exposed. The references can be found at the end of the document.
Cell culture interfaces for different organ-on-chip applications: from photolithography to rapid-prototyping techniques with sensor embedding
(Universitat de Barcelona, 2019-12-09) Paoli, Roberto; Homs Corbera, Antoni; Samitier i Martí, Josep; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] Despite the last 60 years have seen major advances in many scientific and technological inputs of drug Research and Development, the number of new molecules hitting the market per billion US dollars of R&D spending has been declined steadily during the same period. The current scenario highlights the need for new research tools to enable reduce costly animal and clinical trials while providing a better prediction about drug efficacy and security in humans A recent emerging approach to improve the current models is emerging from the field of microfluidics, which studies systems that process or manipulate tiny amounts of fluids using channels with dimensions of tens to hundreds of micrometers. Combining microfluidics with cell culture, scientists gave rise to a new field named “Organ-on-chip” (OOC). Microfluidic OOCs are advanced platforms designed to mimic physiological structures and continuous flow conditions, thus allowing the culture of cells in a friendlier microenvironment. This thesis, titled “Cell culture interfaces for different organ-on-chip applications: from photolithography to rapid-prototyping techniques with sensor embedding”, aims to design, simulate and test new OOC devices to reproduce cell culture interface under flow conditions. The work has a focus on the exploration of novel fabrication techniques which enable rapid prototyping of OOC devices, reducing costs, time and human labor associated to the fabrication process. The final objective is to demonstrate the viability of the devices as research tools for biological problems, applying them to the tubular kidney and the blood brain barrier (BBB). To achieve the objective, at least three device version have been developed: 1) OOCv1, fabricated by multilayer PDMS soft lithography; 2) OOCv2, fabricated in thermoplastic by layered object manufacturing using both a vinyl cutter and a laser cutter, integrating standard fluidic connectors alone (OOCv2.1) or together with embedded electrodes (OOCv2.2); 3) OOCv3 using a mixed technique of laser cut and 3D printing by stereolithography. All devices are fabricated using biocompatible materials with high optical quality and an embedded commercial membrane. The biological experiments with renal tubular epithelial cells, realized on OOCv1 and OOCv2.1 devices, demonstrated the viability of the device for culturing cells under flow conditions. The study realized on fatty acid oxidation and accumulation in cells exposed to physiological and diabetogenic oscillating levels of glucose suggest a possible positive role of shear stress in activation of fatty acid metabolism. The studies were performed using a compact experimental unit with embedded flow control which reduce significatively the complexity and cost of the fluidic experimental setup. The biological experiments on the BBB confirmed viability of OOCv2.1 and OOCv2.2 for compartmentalized co-culturing of endothelial cells and pericytes. The formation and recovery of the barrier after disruptive treatment has been assessed using different techniques, including immunostaining, fluorescence and live phase contrast imaging, and electrical impedance spectroscopy. The repeatability of measurements using electrodes was verified. A model to classify measurements from different timepoints has been developed, resulting in accuracy of 100% in learning and 90% in testing case. Results are confirmed by imaging data, which also suggest a critical role of pericytes in the development, maintenance, and regulation of BBB, in accordance with the literature.
Advances in semiconducting nanowires for gas sensing: synthesis, device testing, integration and electronic nose fabrication
(Universitat de Barcelona, 2019-12-19) Domènech Gil, Guillem; Romano Rodríguez, Albert; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] The work presented in this dissertation is focused on the fabrication, integration and test of chemoresistive gas sensor devices and systems based on semiconducting nanowires (NWs). The first objective of this dissertation was to grow monocrystalline In2O3 and Ga2O3 NWs via the vapor-liquid-solid mechanism using a chemical vapor deposition furnace and solid precursors. Subsequently devices based on individual NWs were fabricated, contacted on top of microhotplates using Focused Electron Beam Induced Deposition (FEBID), and their gas sensing properties were characterized. The gas sensors based on individual In2O3 NWs present considerable response towards ethanol at 300 ºC, with a response time of about 4 minutes. On the other hand, the gas sensors based on individual Ga2O3 NWs showed high selectivity towards relative humidity at room temperature, with resistance variations up to 90% in times as short as 2 minutes, and with minimal response to other gases (NO2, CO, ethanol and H2). This behavior is completely different from that reported on this material and is a direct consequence of the NW growth method, which gives rise to a carbon shell around the NWs. Furthermore, the sensing behavior ressembles that of carbon-containing materials.. A second objective was to deepen into methodologies to integrate the sensing material in the substrates where the gas sensing devices are fabricated, with the aim of simplifying the integration procedures and increasing the throughput. With this in mind, dielectrophoretic alignment of NWs was the first methodology proposed to fabricate chemoresistors based on arrays of individual WO3 NWs. The maximum gas response of the fabricated arrays of individual NWs was towards 5 ppm of NO2 for the pristine and towards 100 ppm of EtOH for the Pt-functionalized WO3 NWs, respectively. This higher response of the Pt-functionalized WO3 NWs-based gas sensors is related to the surface decoration of these NWs, which increases the amount of oxygen adsorbed species at their surface, allowing EtOH molecules to be more easily adsorbed than on pristine NWs. The second approach proposed for contacting individual SnO2 NWs on top of suspended microhotplates was Electron Beam Lithography (EBL) in combination with lift-off. The method allows fabricating several devices sequentially but without breaking the vacuum of the EBL system and required optimization of the holder for spinning and of the electron dose used to modify the photoresist properties. The gas characterization of these devices showed higher resistance variations than that obtained for the reference fabrication technique, FEBID. This superior behavior can be the result of the better electrical characteristics of the Ti/Pt contacts in front of the FEBID Pt-deposition. This demonstrates the potentiality of this techniques for contacting individual NWs on top of micromembranes. The third objective was, to a certain point, a natural extension of the device integration activity when considering one of the major drawbacks of chemoresistors: their lack of selectivity. For this, SnO2, WO3 and Ge NWs, have been grown on well-defined and pre-specified regions of one single chip, allowing their simultaneous operation. Here, NO2, CO and relative humidity (RH), diluted in dry synthetic air, have been tested. The calibration of each individual sensor has been carried out exposing the whole chip to the individual gases but with only this particular sensor heated and biased, while the others were unheated and unbiased. This has allowed determining the optimal operation conditions for each sensor. Next, at these optimal temperatures, all the sensors have been tested, simultaneously, towards each gas specie alone. Finally, tests of the three sensors, operating simultaneously, towards mixtures of the three gases were performed. The data from all the mentioned measurements have been treated according to the Principal Component Analysis (PCA) methodology and the results demonstrate that the fabricated sensor system can discriminate and quantify the concentration of the three different studied analytes. The three sensors, made of three different materials and operating simultaneously, constitute an electronic nose, which we here call nano electronic nose.
Development and optimization of inkjet printing based technologies for hybrid printed circuit boards
(Universitat de Barcelona, 2019-07-23) Arrese Carrasquer, Javier; Cirera Hernández, Albert; Xuriguera Martín, María Elena; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] The main goal of this doctoral thesis is the development and optimization of inkjet- based technologies for hybrid electronic circuits manufacturing, as well contribute on the development of the incoming low cost electronics. Regarding that, a novel solution for connecting regular SMDs and standard silicon SMD packages by inkjet printing is proposed. The novel connecting method allows the assembling at very low temperatures, and thus assures the compatibility with the incoming substrates. Electrical contact resistance and shear strength measurements performed by silver nanoparticle- based ink are comparable to benchmark connecting materials. In sum up, flexible hybrid circuit is successfully manufactured by silver nanoparticle-based ink on paper, where different SMDs size-shaped are assembled demonstrating the reliability and feasibility of the proposed method. Another objective of the work is to apply and adapt the print-on-slope technique to assemble directly the silicon dies on PCB, proposing a novel strategy to overcome the drawbacks of the wire bonding in the Conductive AFM measurements. Then, a novel setup for conductive AFM mode 2D materials characterization was manufactured. The 2D connection on ramp-shape terminations gives a better functionality than current wire bonding connections. The AFM tip moves over the silicon die without physical obstruction, giving a unique solution at this novel method to characterize the material degradation. In the field of multilayer hybrid PCB manufacturing, the goal is to prove the potentiality of different metal-insulator-metal structures inkjet-printed and evaluate their reliability and the electrical performance for low cost multilayer circuit based on paper substrate. In the light of the results, heterogeneous structures combining inorganic and organic dielectric material, where PVP fills the inorganic cracks and voids, possess a similar and outstanding feasibility in both paper and glass substrate without short-circuits. The greatest achievement of this work is the development and optimization of a novel capillarity-assisted SMD assembling method for the manufacturing of hybrid circuits inkjet-printed. In addition, taking advantage of print-on-slope technique, direct assembling of silicon die integrated circuits to PCB is successfully applied. Moreover, heterogeneous structures inkjet-printed open new solutions for multilayer hybrid circuits.
Transparent nanostructured metal oxides for chemical biosensors: towards point-of-care environments
(Universitat de Barcelona, 2019-09-18) Pruna Morales, Raquel; López de Miguel, Manuel; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] There is an increasing need for developing innovative, versatile and low-cost point-of-care (POC) systems capable of screening for at early stages of development. POC systems usually consist of a biosensor part integrated in an electronic circuit and eventually a microfluidic system to manage the body fluid samples. The aim of this doctoral thesis is to investigate several ways of improving POC technology. On the one hand, biosensors currently integrated into POC systems have limitations. A wide variety of important analytes cannot be properly detected and quantified, and methods supported by a powerful electronic systems that supply the necessary energy to trigger a measurable event that can be monitored are required. For this, adequate sensing substrates are required that allow the coupling of analytes and other biomolecules and enable the detection of chemical reactions occurring at their surfaces. Besides, the complex electronic circuitry capable of simultaneously exciting the sensor and monitoring its response must be redesigned into a low-cost and miniaturized format to be integrated into POC systems. Electrochemical and optical biosensors have become relevant in point-of-care technology due to the versatility of POC systems based on such transducing principles, which provide the sensors with high sensitivities and specificities. In particular, sensitivity may become badly affected by the miniaturization of sensors and devices. Thus, the need for reducing the surface of sensing electrodes and yet maintaining the sensitivity has boosted the research and development of nanostructured surfaces. The high surface-to- volume ratio (SVR) presented by nanostructures makes them extremely interesting for the detection of biomolecules, since an increase of surface enables the interaction with a big amount of small-sized molecules and this implies an increase of sensitivity and the possibility to reduce the sensor size. In this thesis, nanostructured indium tin oxide (ITO) is proposed as working electrode (WE) for electrochemical biosensors. The first part of this thesis consists in a study of ITO properties and its electrical, optical, electrochemical and structural characterization both as a thin film and as nanostructured electrodes prepared by electron beam evaporation onto silicon and glass substrates. Moreover, the interaction of nanostructured ITO with some molecules known as crosslinkers, which allow subsequent functionalization of surfaces with biomolecules, has also been studied in the frame of this thesis. Finally, several immunoassays were performed using nanostructured ITO as substrate, with special attention to the detection of several concentrations of tumour necrosis factor α (TNF-α). On the other hand, several electrochemical sensor mechanisms were studied. These were based upon different ways of electrically attacking the sensor and processing its response, and included potentiometry, amperometry and electrochemical impedance spectroscopy. A low-cost and miniaturized device implementing electrochemical impedance spectroscopy measurements was designed and developed for the detection of several concentrations of TNF–α biomarker with an array of eight parallel gold-based microelectrodes. Besides, we also designed the electronics for performing two-electrode amperometry and potentiometry. The latter was tested on nanostructured ITO electrodes covered with a doped conducting polymer, which was sensitive to pH changes in aqueous media. To synthesize, this thesis gathers several proposals for improving current POC systems, regarding both the biosensor and the electronic parts, employing an important biomarker in the biomedical area for the measurements and proofs of concept, and being such approaches extensible to the environmental field.
Development and optimization of a Low Temperature Co-fired Ceramic suspension for Mask-Image-Projection-based Stereolithography
(Universitat de Barcelona, 2019-06-20) Fernandes, Joana Gonçalves; Xuriguera Martín, María Elena; Universitat de Barcelona. Departament d'Enginyeria Electrònica i Biomèdica
[eng] This dissertation has the main goal of developing ceramic materials for Mask-Image- Projection-based Stereolithography (MIP-SLA) technology, focused on electronic applications. To accomplish this aim, a low temperature co-fired ceramic (LTCC) material was selected, mainly used for radio frequency applications. Moreover, the proof of concept of AM technology hybridization and multi-materials printing is also demonstrated, opening the door to new researchers in the field of 3D-2D printing electronic devices, from both the material and technology perspectives. To successfully achieve the main goal, the different steps of the whole process were successfully achieved, i.e, formulation of a LTCC photocurable suspension, its printability by MIP-SLA technology, and the post-thermal treatment of debinding and sintering. The photocurable LTCC suspension consists of ceramic particles dispersed in a suitable photocurable resin, which must polymerize in the visible light range, trapping the ceramic particles. The challenge of the development and optimization of a LTCC photocurable suspension for MIP-SLA with the appropriate rheological and photocurable behavior is accomplished in this work. The printed piece contains the polymeric part, which must be removed (debinding) and then sintered for the densification of the final ceramic piece. This is the most difficult and time- consuming step of the whole process. The so-called debinding is one of the most challenging steps of the SLA-based technology of ceramic materials. In this sense, a detailed study of the debinding process is carried out for a deeper understanding of the degradation of the resin during the thermal debinding. For this to happen, the optimization of the temperature rate and used atmosphere during the thermal treatment is also presented in this work. In fact, the analysis and understanding of thermal treatment parameters and their repercussion on the final results is the key to successfully achieving the main goal, which is to obtain final ceramic pieces without defects. The limits of the MIP-SLA printing process are presented, analyzing the resolution, fidelity of pattern transfer, and accuracy of the printing process using the optimized LTCC suspension. The involved phenomena during the photopolymerization such as light scattering, non- uniformities of the light projection along the building platform and shrinkage during the polymerization, are analyzed and optimized for a fruitful printing process. The greatest achievement of this work is the possibility of printing complex geometry with high resolution with a LTCC material, which has never been demonstrated before in the field of additive manufacturing. This is the beginning of new breakthroughs in multimaterial printing for electronic applications.

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