Batch-fabrication of novel nanoprobes for SPM

A micromachining method has been developed for fabricating 20µm tall silicon atomic force tips with flat tops less than 2µm wide suitable for defining nanosensors upon, and with low aspect ratio sides suitable for defining electrical connections to the sensor. Methods have been developed to allow flat substrate processing techniques to be applied to such non-planar micromachined substrates. This has necessitated the development of a novel resist-coating technique and the use of defocused electron-beam lithography. Methods for through-wafer alignment by electron-beam lithography and accurate alignment to the tips using micromachined alignment markers have also had to be developed. The fabrication process has been designed to enable a wide variety of sub-micron sensors to be defined on the atomic force probes, with little additional development beyond that of : sensors themselves. This flexibility has enabled very different sensors meant for very different scanning probe microscopy techniques to be designed without significant redevelopment of the underlying fabrication process. The main restrictions on the type of sensor that can be used are the physical dimensions of the sensor, the number of alignment levels necessary, the degree of alignment accuracy required and the choice of sensor materials. However, within these constraints it has been found that probes optimised for scanning near-field optical microscopy (SNOM), scanning thermal microscopy, modulation differential scanning calorimetry (MDSC) and scanning Hall-probe microscopy can be fabricated. For the SNOM probes three methods for fabricating sub-l00nm diameter apertures have been developed, analysed and compared with each other to evaluate both the process latitude. and, the size and reproducibility of apertures that can be fabricated, as a function of electron beam dose, pattern shape and size, and metallisation material and thickness. Two methods, both utilising multilayer 'resist' schemes have been found suitable for this purpose, one based on conventional electron-beam lithography with PMMA and a new dry etching process for titanium, and the other based on a novel electron-beam lithography technique utilising cross-linked PMMA for lifting off nichrome. A simple analytical model has also been developed for these probes allowing the effects of changes in the sensor design parameters on the light throughput to be compared qualitatively, if not quantitatively. For the scanning thermal probes a method for lifting-off sub-l00nm, thin-film thermocouple sensors on silicon tips without the loss of electrical continuity has been developed. For the MDSC probes, a similar method has been developed for defining thermal resistors. A method has also been presented for fabricating sensors for scanning Hall-probe microscopy based on an evaporated germanium sensing layer. This has been found to require annealing and optimisation of sensor design and geometry to reduce sensor resistance to acceptable levels

A nanostructured porous silicon based drug delivery device

Targeted and controlled delivery of therapeutic agents on demand is pivotal in realising the efficacy of many pharmaceuticals. The design and fabrication of a novel, electrically-addressable, porous structure-based drug delivery device for the controlled release of therapeutic proteins and peptides, are described in this thesis. The initial prototype microdevice design incorporates a porous polysilicon (PPSi) structure as a drug reservoir. Two alternative methods were investigated to fabricate the PPSi structure: i) the chemical stain etching method; ii) a reactive ion etching (RIE) method through a masking template. Random pores, with irregular pore shape and size in the micro- to mesoporous regime (< 50 nm), were obtained using the stain etching method but this method suffered from poor reproducibility and non-uniformity. Two novel RIE approaches were investigated to fabricate ordered PPSi structures; two different masking templates were investigated – a porous anodic alumina (PAA) and a metal mask with hexagonally arranged holes produced by a novel nanosphere lithography (NSL) technique. A quasi-ordered PAA template with pore diameters in the region of 50 nm was fabricated but was not suitable for the subsequent proposed RIE process. By using the NSL technique, quasi-ordered PPSi structures with tapered pore profiles, were obtained. This is the first demonstration of the fabrication of PPSi with ordered pores of sizes in the macropore range of ~ 370 nm.A revised silicon-based prototype microdevice was designed and fabricated. The microdevice incorporates a nanostructured, quasi-ordered porous silicon (PSi) as a drug reservoir and an integrated heater and temperature sensor as an active control mechanism. The PSi structure was fabricated using a modified NSL technique and a Bosch-based RIE process. Hexagonally arranged cylindrical pores with diameters between ~75 nm and ~120 nm, and depths in the range of ~330 nm and 500 nm, were obtained. The novel fabrication techniques investigated here are simple and versatile; both p-type and n-type PSi structures have been successfully fabricated. Proof-of-concept studies, using the revised prototype drug delivery microdevices, suggested that the nanostructured PSi would be suitable for the passive release of an intermediate-sized (~23,000 Dalton) model protein. It is envisaged that the microdevice has the potential to deliver osteoinductive growth factors, on demand, to the site of fracture, in a controlled and sustainable manner, as a first step to an intelligent therapeutic system for skeletal regeneration

Development of microfluidic tools for cancer single cell encapsulation and proliferation in microdroplets

The role of microfluidics in liquid biopsy as a more capable solution to address the monitoring of cancer progression in patients is gaining increasing attention. One out of the several difficulties in can-cer monitoring resides with the offset between current cell growth techniques in vitro and the influence of the cellular microenvironment in proliferation. One application of microfluidics consists in the use of microdroplets to replicate the complex dynamic microenvironment that can accurately describe factual 3D models of cancer cell growth. The goal of this thesis was to develop a set of microfluidic-based tools that would enable the encapsulation, proliferation and monitoring of single cancer cells in micro-droplets. For this, a set of microfluidic devices made of PDMS for droplet generation and containment were developed by photo- and soft-lithography techniques, being tested and optimized to ensure single cancer cell encapsulation. After the optimization of the droplet generation parameters in terms of droplet size and long-term stability on-chip, the best performance conditions were selected for cell growth ex-periments. Different densities of MDA-MB-435S cancer cells were combined with various percentages of Matrigel®, an extracellular matrix supplement, to promote cell proliferation. As a result, it was possi-ble to monitor droplets with cancer cells for a range of 1-20 days. A preliminary observation showed signs of cell aggregation, indicating that the tools developed during the thesis have the potential of developing 3D cancer spheroids from cancer single cells

Development of a microphysiological system with integrated electrodes for cardiac cell culture, stimulation and sensing

Treballs Finals de Grau d'Enginyeria Biomèdica. Facultat de Medicina i Ciències de la Salut. Universitat de Barcelona. Curs: 2022-2023. Tutor/Director: Rodríguez Trujillo, RomenThis project focuses on the development and experimentation of a microfluidic device with integrated electrodes, specifically designed to support the growth and maturation of a 3D cardiac cell matrix. The goal is to create a functional system that not only enables the cells to thrive and pump, but also facilitates the propagation of electrical stimuli, mimicking the behavior of natural cardiovascular tissue. The motivation behind this research stems from the limited regenerative capacity of adult heart tissue, particularly when it comes to cardiomyocytes. Traditional healing methods are often inadequate, necessitating heart transplants as the only definitive treatment option. To address this challenge, scientists are exploring the potential of biomaterial scaffolds to regenerate cardiovascular tissue by replacing damaged or necrotic tissue. The microfluidic device developed in this project holds great promise for researchers in the pharmaceutical field, offering a valuable tool for drug testing and disease modeling. Despite facing challenges in incorporating gold electrodes into the device, the team has successfully characterized it using an EIS machine. The design of the microelectrodes and microchannels, along with the overall functionality of the microchip, have been accomplished. While the current focus has been on a 2D layer of cells, the future objectives involve achieving a fully functional 3D matrix to fulfill the original research goals. Overall, this project aims to represent a significant step towards the advancement of regenerative medicine and the potential for innovative solutions in treating cardiovascular diseases

Nanostructuration of innovative molecular imprinted polymers for their use in protein detection

Le but de ce travail de thèse était de concevoir et de développer un nouveau type de matériau nanostructuré qui pourrait être utilisé dans une biopuce capable de détecter sélectivement des protéines telles que des biomarqueurs cancéreux. La méthode choisie pour atteindre cet objectif a été la technique des polymères à empreinte moléculaire (MIP). Le MIP a dû être structuré en lignes nanométriques pour être couplé par la suite avec un système de détection sans marquage utilisant la diffraction de la lumière. L'ensemble pourrait être ensuite mis en forme sur des biopuces. Pendant la première partie de ce projet, les différentes formulations d'hydrogel ont été évaluées, ce qui nécessite de répondre à plusieurs spécifications: processus de polymérisation à 25-37 °C, dans une solution tampon phosphate et temps de polymérisation de moins de 15 minutes. En outre, l'hydrogel devait porter des groupes fonctionnels pouvant interagir avec une protéine, être transparent et biocompatible. Enfin, ces matériaux devaient présenter d'une part des tailles de pores compatibles avec celle de la protéine afin d'assurer une reconnaissance en surface et d'autre part des propriétés mécaniques qui soient compatibles avec les procédés technologiques usuels. Trois formulations ont été sélectionnées pour la synthèse d'hydrogel, ayant des groupes fonctionnels présentant soit une charge positive ou négative, ou sans charge du tout. Ces matériaux ont été caractérisés par des techniques telles que piézorhéométrie, calorimétrie différentielle à balayage (DSC), microscopie électronique à balayage (MEB, MET et cryoSEM), microscopie à force atomique (AFM) et profilométrie. En suivant la formation de l'hydrogel sous irradiation UV par piézorhéométrie, nous avons montré que la réticulation maximale était atteinte en moins de 5 minutes en utilisant une lampe avec une puissance de 150 mW/cm2. En outre, nous avons également confirmé que ces formulations sont compatibles avec la lithographie par nanoimpression UV et que des réseaux périodiques de taille sub-micrométriques pouvaient être obtenus. Les MIP à protéine réalisés à partir des conditions optimisées ont été évalués par fluorescence après les expériences de ré-incubation, indiquant une reconnaissance de la streptavidine avec un facteur d'impression de F.I = 1,7.The aim of this PhD work was to design and develop a new type of nanostructured material that could be further used in a biochip capable of selectively detecting proteins such cancer biomarkers. The chosen method to achieve this goal was the molecularly imprinted polymer (MIP) technique. The MIP had to be structured in nanometric lines to be coupled subsequently with the diffracting label-free detection. During the first part of this project, different hydrogel formulations were assessed, which needed to respond to several specifications: polymerization process at 25-37°C in phosphate buffer solution and a polymerization time of less than 15 minutes. In addition, the hydrogel required functional groups that can interact with the protein, it needed to be transparent and biocompatible. Finally, these materials had to have pore sizes compatible with that of the protein for successful surface recognition and exhibit mechanical properties which are compatible with routine technological processes. Three formulations for hydrogel synthesis were selected, including functional groups presenting either a positive or negative charge, or no charge at all. These materials were characterized by techniques such as piezorheometry, differential scanning calorimetry (DSC), electron microscopy (SEM, TEM and cryoSEM), atomic force microscopy (AFM) and profilometry. By following the formation of the hydrogel under UV irradiation by piezorheometry, we showed that maximal crosslinking was achieved in less than 5 minutes when using a lamp with a power of 150 mW/cm2. In addition we also confirmed that these formulations were compatible with UV-nanoimprint lithography and that sub-micron periodic gratings could be obtained. The protein MIPs after batch rebinding experiments were evaluated by fluorescence, showing recognition for streptavidin with an imprinting factor of I.F= 1.7

Development of a generic multi-analyte optical sensor platform for fluorescence-based sensing

This work describes the development of two advanced sensor platforms based on different spectroscopic techniques. The first, and the primary focus of this work, is an enhanced generic multi-analyte sensor platform for fluorescence-based sensors and the second is an absorbance-based portable sensor for the detection of nitrates in groundwater. A generic multi-analyte sensor platform can be applied to a broad range of areas such as food packaging and blood gas analysis. A multi-analyte optical sensor platform for enhanced capture of fluorescence was modelled, designed and fabricated. The sensor platform was developed using a range of microfabrication techniques. Films sensitive to oxygen, relative humidity and carbon dioxide respectively were developed for the context of indoor air-quality monitoring. Deposition methods for printing the sensor solutions onto the sensor platforms were also investigated. The sensor films and platforms were integrated into a working sensor chip with both a fluorescence intensity and phase fluorometric detection system. An absorbance-based portable sensor for the detection of nitrates in groundwater was also developed. This was based on the direct absorbance of UV-light by the nitrate ion. Other contaminants, which could be found in groundwater and interfere with the nitrate detection, such as humic acid and chlorides, were investigated and compensated for

Clay minerals in Enhanced Oil Recovery; Implications for fines migration as a redox controlled process during Low Salinity Water Flooding

In oil and gas exploration and production, the chemical and physical properties of reservoir clay minerals can have an effect on drilling operations, reservoir quality and oil recovery rates. Various methods have been used to optimize the recovery of oil from reservoirs, through technologies that are more economical, easier to apply and environmentally friendly, culminating in the development of low salinity water flooding (LSWF). LSWF is a chemical method whereby the concentration of cations, especially multivalent cations, in the injected water is carefully reduced and controlled. LSWF is used in secondary and tertiary enhanced oil recovery (EOR) operations. In this present study, we explore a new frontier in EOR research by examining the wettability and swelling capacity of reservoir clay minerals as a function of reduction extent. We investigate how changes in the redox state during a LSWF impact on the wettability of iron rich clay minerals. We make an attempt to map the roughness profile of the clay minerals, to be used as baseline for wettability and contact angle studies. For that, we introduce a novel approach to measuring the roughness with the use of Confocal Microscopy in combination with Atomic Force Microscopy (AFM) and White Light Interferometry (CCI). To further elucidate the behavior of clay minerals, we test the hydration of model clays, including swelling and non – swelling types, using infrared spectroscopy. Additionally we investigate how reduction extent impacts the surface hydration and water sorption by nitrogen BET and water vapor volumetry methods. We couple these studies with controlled humidity XRD scans of the clay minerals and measuring of the interlayer cation budget by means of the ICP-OES method. Building on the basic understanding of redox active clay mineral at the mineral level derived from the above studies, a polymer – coated silicon wafer is used as proxy to a sandstone, and anoxic – reduced conditions simulated by means of an experimental apparatus. This setup will allow direct visualization and modelling of the effects of reduction and re – oxidation within the context of low salinity water flooding, creating a better tool for understanding fines migration in EOR applications that could lead to optimizing the operations

Microfluidic tools for metabolomics

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2008.Includes bibliographical references (p. 153-160).A primary challenge in embryology is to understand the factors that govern the development of preimplantation (PI) embryos and how these factors relate to embryo viability in the field of in vitro fertilization (IVF). This is particularly important as clinical policy moving towards single embryo transfer (SET) has gained awareness to manage unprecedented numbers of multiple births, such as twins and triplets, resulting from artificial reproductive techniques. Conditions that correlate with developmental potential of candidate embryos are disputed in the field, however, as the requisite data is difficult to obtain.The metabolic profiles of embryos during in vitro culture have been suggested as a key indicator of developmental potential, and approaches have been clinically implemented to select transfer candidates which make the most efficient use of nutrients. Existing microdroplet analysis techniques are accurate and suitable for non-invasive assessment of single embryos. Unfortunately, the process of determining metabolite levels in nanoliters of culture media through fluorometric assays is low-throughput and requires specialized expertise, hindering widespread clinical use of these methods. The goal of this thesis is to develop microfluidics-based approaches for improving metabolic analysis of PI embryos and mammalian cells. This challenge necessitates two competencies: methods for automating chemical assays and methods for supporting cell cultures, which can be integrated with analysis. Contributions include a standalone platform for determining the metabolite use of single embryos. Profiles may be acquired automatically, which reduces significant technician hours and improves repeatability. Techniques are developed for performing embryo culture in the smallest culture volumes to date in microfabricated environments. Microfluidic approaches have enabled culture that outperforms the current state of art approach based on cell count measurements.(cont.) An integrated system is introduced, merging analysis and culture competencies to perform metabolic profiling of separate cultures of mammalian cells in parallel. Finally, new paradigms in microfluidic design are presented based on the concept of vertically integrated architectures, suitable for overcoming density limitations of microfluidic assays. A scalable analysis platform for refining embryo selection has been long warranted and would enable pursuit of the difficult questions relating metabolism and embryo viability as the clinical movement towards SET continues.by John Paul Urbanski.Ph.D

Design and fabrication of a next generation regenerative neural interface

A Spiral Peripheral Neural Interface (SPNI) is an electrode array that has been previously presented as a regenerative neural interface capable of receiving information from, and transmitting information to nerves. The SPNI has previously been proven in concept, however, when stimulating nerves in the device, the electrodes areinsufficiently isolated from each other and stimulations can trigger unwanted neural activity in neighbouring channels of the SPNI. Along with this, neural interfaces generally, suffer from chronic viability problems, due to biological rejection. These issues were addressed in this thesis, by the addition of a PDMS silicone membrane, into the structure of the SPNI. Improvements to the understanding and performance of structural, electrical and biocompatibility aspects of the SPNI are addressed, with the addition of the PDMS film, which is used to electrically seal SPNI channels whilst not hindering conductor integrity. The inclusion ofPDMS also provides a platform which may enable drug delivery. This work dramatically improves SPNI performance whilst providing routes to improved biocompatibility. This thesis addresses the main issues previously presented in the SPNI and brings the device up to a new standard which can once again be tested for its viability in vivo