Development of A Hydrophobicity Controlled Microfluidic Dispenser

Ph.DDOCTOR OF PHILOSOPH

Factories of the Future

Engineering; Industrial engineering; Production engineerin

Reactive inkjet printing and functional inks : a versatile route to new programmed materials

Starting as an ink dispensing tool for documents and images, inkjet printing has emerged as an important instrument for delivering reactive fluids, into a means for creating new, programmed materials. Inkjet is a processing technology with some very unique capabilities, which allows the handling of materials in the picoliter range, and the creation of functionality in new, previously unexplored ways. In particular, drop-on-demand technology provides the chance to dispense liquids in picoliter/nanoliter quantities to very specific locations, with minimal material loss, and in a contact-free manner. This dramatic scale-down of production, not just miniaturization but "nanonization", affords materials that would be either too costly or otherwise inaccessible by other manners. As this is still an emerging technology, there remain a lot of opportunities to pioneer new applications. The underlying, unifying concept behind the chapters of this thesis has been an interest in investigating how inkjet printing, combined with reactive inks, can lead to new applications, new devices, and new materials, wherein unique functionality is imparted as a direct result of the confluence between microfluidic processing, chemistry, and life science. The ability to deliver uniform, sub-nanoliter droplets to specific locations opens up new possibilities that did not exist before. Inherent in the geometry of such droplets, the volume of liquid dispensed also offers some special utility. Based on the aforementioned diameters, drop-on-demand inkjet printing can deliver volumes in the range of approximately 0.5 to 1,000 pL; the direct writing attributes of inkjet ensure that these droplets are not only precise, but can be delivered to a specific location, giving them a "home address". This combination of precise, reproducible, small aliquots and precision deposition is especially important for preparing high-density analytical arrays, as discussed in Chapter 1 on inkjet printing of proteins. In the case of highly specialized proteins such as reactive enzymes or antibodies, where available materials are often limited, the ability to dispense precise quantities in a reproducible fashion means that small amounts of precious material can be used parsimoniously to perform thousands of experiments without compromising the quality of the data. For drop-on-demand printing, droplets normally produced by inkjet printing are commonly in the range of 10 to 125 µm in diameter, depending on the physical characteristics of the fluid, the nozzle used, and the printing conditions; taking advantage of the this aliquot size has some unique attributes that make dispensing highly suitable to materials science challenges that have gone unmet. In the second chapter of this thesis, this size domain is taken advantage of for use in tissue engineering, where it is used to create soft, cell-scale porogenic structures by the use of a reversible, rapid alginate gelation reaction to freeze droplet structures in place. By switching to a continuous inkjet device, larger volumes of beads in the size domain of 100 to 500 µm can be achieved, opening up prospects for pore sizes matching those needed for hosting capillaries. By incorporating reversible hydrogels as a motif in these applications, these controlled cell-scale dimensions can be retained during key processing steps, and then removed (or eroded) later after they have served their function. Extending the concept to the task of dispensing living cells, in Chapter 3, printed alginate structures are used for cell encapsulation. By adjusting the printing conditions to prevent jet break-up before alginate hardening, continuous, one-dimensional "living threads" can be created, which allow for cell cultures to be handled and woven into desired complex patterns. In addition to their role as basic building materials for tissue engineering scaffolds, the alginate threads provide a stable, bio-friendly environment for culturing different cell types, with cells exhibiting a high post-processing viability rate. In Chapter 4, the lower limits of single cell printing are explored, in the concept of "one cell-one well", where the attributes of inkjet printing are used to dispense individual cells. By careful selection of droplet size and accounting of cell concentrations, the statistical probability of single cell printing can be optimized, yielding spatially addressable arrays of isolated living cell cultures on a surface. Additional steps necessary to prevent cells from dehydration are also outlined, offering access to high density arrays of isolated living single cells on glass slides, where each individual droplet acts as independent nanoincubator, hosting intrinsically monoseptic cell cultures in parallel. In addition to describing the theoretical limits of single-pass cell printing experiment designs, an outline is given for experimental designs for tuning single particulate dispensing probability to any value desired between 0 and 1. The focus of Chapter 5 relates to reactive inkjet printing of ultrathin films on surfaces. For systems with moderately good surface wetting, such as polar solvents on glass or metal oxides, inkjet printed droplets result in features ranging approximately from 20 to 300 µm in diameter per droplet. By first printing a thiol-functionalized heterochelic linker and covalently bonding it to the print surface, the surface will accommodate subsequent thiol-ene click reactions only with original monolayer, and only where the first and second deposition features overlap. This combination of spatial selectivity as well as chemoselectivity allows for the preparation of a wide range of monolayers on a printed surface, in a format well-suited to automated surface characterization techniques, as was illustrated using XPS. In Chapters 6, two different categories of irreversible polymerization reactions are described, where print features are reacted in a specific pattern that is process unique. Printable ionogels are developed, which impart conductivity to printed patterns, and consequently, functionality to only those locations where the material has been deposited. Also in Chapter 6, the first example of a moisture-sensitive reactive printing is outlined, where a diisocyanate is combined with different polyols within seconds to create highly crosslinked, ultra-stiff surfaces, which can be built up into three dimensions by successive layering. The topics outlined in this thesis are intended to illustrate the breadth of how inkjet technology can be utilized to support a diverse field of materials science applications — particularly when coupled with modular, off-the-shelf synthetic transformations. The incorporation of synthetic chemistry into inkjet extends the application of inkjet from dispensing static materials merely from a cartridge onto a target, into a dynamic tool for transforming these materials into something new. At the same time, inkjet printing and other allied microfluidics tools enable chemistry experiments (and by extension, life science experiments) on a scale that would otherwise be challenging to realize by other means. The two driving forces of high-throughput experiment design, miniaturization and automation, are both embodied in this dispensing technique, and consequently inkjet printing is a rapidly evolving discipline; it is the intent of this work and the examples given to underscore the diversity offered by this technology

Factories of the Future

Engineering; Industrial engineering; Production engineerin

Probing multivalent particle–surface interactions using a quartz crystal resonator

The rise in market-approved cellular therapies demands for advancements in process analytical technology (PAT) capable of fulfilling the requirements of this new industry. Unlike conventional biopharmaceuticals, cell-based therapies (CBT) are complex “live” products, with a high degree of inherent biological variability. This exacerbates the need for in-process monitoring and control of critical product attributes, in order to guarantee safety, efficacious and continuous supply of this CBT. There are therefore mutual industrial and regulatory motivations for high throughput, non-invasive and non-destructive sensors, amenable to integration in an enclosed automated cell culture system. While a plethora of analytical methods is available for direct characterization of cellular parameters, only a few satisfy the requirements for online quality monitoring of industrial-scale bioprocesses. [Continues.

SystemC Through the Looking Glass : Non-Intrusive Analysis of Electronic System Level Designs in SystemC

Due to the ever increasing complexity of hardware and hardware/software co-designs, developers strive for higher levels of abstractions in the early stages of the design flow. To address these demands, design at the Electronic System Level (ESL) has been introduced. SystemC currently is the de-facto standard for ESL design. The extraction of data from system designs written in SystemC is thereby crucial e.g. for the proper understanding of a given system. However, no satisfactory support of reflection/introspection of SystemC has been provided yet. Previously proposed methods for this purpose %introduced to achieve the goal nonetheless either focus on static aspects only, restrict the language means of SystemC, or rely on modifications of the compiler and/or parser. In this thesis, approaches that overcome these limitations are introduced, allowing the extraction of information from a given SystemC design without changing the SystemC library or the compiler. The proposed approaches retrieve both, static and dynamic (i.e. run-time) information

Manipulation of Cell and Particle Trajectory in Microfluidic Devices

Microfluidics, the manipulation of fluid samples on the order of nanoliters and picoliters, is rapidly emerging as an important field of research. The ability to miniaturize existing scientific and medical tools, while also enabling entirely new ones, positions microfluidic technology at the forefront of a revolution in chemical and biological analysis. There remain, however, many hurdles to overcome before mainstream adoption of these devices is realized. One area of intense study is the control of cell motion within microfluidic channels. To perform sorting, purification, and analysis of single cells or rare populations, precise and consistent ways of directing cells through the microfluidic maze must be perfected. The aims of this study focused on developing novel and improved methods of controlling the motion of cells within microfluidic devices, while simultaneously probing their physical and chemical properties. To this end we developed protein-patterned smart surfaces capable of inducing changes in cell motion through interaction with membrane-bound ligands. By linking chemical properties to physical behavior, protein expression could then be visually identified without the need for traditional fluorescent staining. Tracking and understanding motion on cytotactic surfaces guided our development of new software tools for analyzing this motion. To enhance these cell-surface interactions, we then explored methods to adjust and measure the proximity of cells to the channel walls using electrokinetic forces and 3D printed microstructures. Combining our work with patterned substrates and 3-dimensional microfabrication, we created micro-robots capable of rapid and precise movements via magnetic actuation. The micro-robots were shown to be effective tools for mixing laminar flows, capturing or transporting individual cells, and selectively isolating cells on the basis of size. In the course of development of these microfluidic tools we gained valuable new insights into the differences and limitations of planar vs. 3D lithography, especially for fabrication of magnetic micro-machines. This work as a whole enables new mechanisms of control within microfluidics, improving our ability to detect, sort, and analyze cells in both a high throughput and high resolution manner

Gold nanoparticles for nanotheranostics in leukemia – Addressing Chronic Myeloid Leukemia

Leukemia is a type of cancer that initiates in the bone marrow and results in the unregulated production of immature white blood cells (leukemic cells). The most homogenous subgroup of the disease is chronic myeloid leukemia (CML) accounting for nearly 1.5 million patients worldwide. Virtually all cases harbor the genetic translocation t(9;22)(q34.1;q11.2) resulting in the BCR-ABL1 gene fusion, that encodes for BCR-ABL1 tyrosine kinase. CML treatment success relies on an early diagnosis and the intense research towards developing effective tyrosine kinase inhibitors (TKI). Nanotechnology offers unprecedent advantages to tackle the shortcomings of conventional procedures for the management of CML. Gold nanoparticles (AuNPs) have unique optical properties suitable for ex vivo biosensing applications, but can also function in vivo as nanocarriers in a theranostic approach that links treatment with diagnosis according to patient’s molecular profile. A gold nanoprobe (Au-nanoprobe) colorimetric assay was optimized for the detection of the most frequent BCR-ABL1 isoform (e14a2) and was validated on fully characterized clinical samples. This simple and cheap method enabled the direct detection of e14a2-expressing RNA samples, with accuracy and high specificity. The Au-nanoprobe assay was translated onto a microfluidics chip, resulting in a faster outcome with smaller sample volumes, due to the scale and design of the device. Additionally, a new therapeutic strategy was designed to overcome CML resistance to first line therapy, such as imatinib (IM). BCR-ABL1 gene silencing was effectively achieved in vitro, using AuNPs functionalized with polyethylene glycol and a hairpin-shaped antisense single stranded DNA (ssDNA) oligonucleotide. Furthermore, the nanoconstruct allowed to reduce the dose of IM, when tested in a combined approach, and potentiated a viability decrease of K562 cells resistant to IM. The results of this thesis strongly suggest that AuNPs are a suitable and flexible tool for CML nanotheranostics, improving detection and a personalized treatment strategy

Microfluidics and Nanofluidics Handbook

The Microfluidics and Nanofluidics Handbook: Two-Volume Set comprehensively captures the cross-disciplinary breadth of the fields of micro- and nanofluidics, which encompass the biological sciences, chemistry, physics and engineering applications. To fill the knowledge gap between engineering and the basic sciences, the editors pulled together key individuals, well known in their respective areas, to author chapters that help graduate students, scientists, and practicing engineers understand the overall area of microfluidics and nanofluidics. Topics covered include Finite Volume Method for Numerical Simulation Lattice Boltzmann Method and Its Applications in Microfluidics Microparticle and Nanoparticle Manipulation Methane Solubility Enhancement in Water Confined to Nanoscale Pores Volume Two: Fabrication, Implementation, and Applications focuses on topics related to experimental and numerical methods. It also covers fabrication and applications in a variety of areas, from aerospace to biological systems. Reflecting the inherent nature of microfluidics and nanofluidics, the book includes as much interdisciplinary knowledge as possible. It provides the fundamental science background for newcomers and advanced techniques and concepts for experienced researchers and professionals