Pancreatic Ductal Adenocarcinoma (PDAC) continues to have a dismally low survival rate due to late diagnosis and poor treatment options. Therefore, there is a need to understand the early stages and progression of the disease. PDAC is known to have two types of cells of origin: ductal cells or acinar cells. Since acinar-derived PDAC is thought to be the more malignant of the two, it was chosen as the focus of this work. Most studies of acinar cells as they relate to PDAC are accomplished by using animal models such as genetically engineered mouse models. While this method yields a large amount of insight into the progression of the disease and the role of specific genes, it has the drawbacks of being very time and resource intensive. The quicker and less costly alternative is <i>in vitro </i>culture. Specifically, here we have developed a microfluidic model which can incorporate a key aspect of the extracellular matrix (ECM), type I collagen, and mimics the 3D geometry of an <i>in vivo </i>acinus. Most attempts at <i>in vitro </i>culture have been limited by the fact that isolated acinar cells show a decrease in the amount of digestive enzymes they secrete as culture continues. For this reason, we are using a reprogrammed cancer cell line. These cells can be induced with doxycycline to express PTF1a, which allows the cells to adapt acinar characteristics, such as the production of digestive enzymes. We were able to successfully culture and induce PTF1a in these cells within our chip. We showed that the cells exhibit no invasion into the collagen matrix once PTF1a is expressed, thus eliminating a key aspect of cancer cell culture. The cells grown in the chip are confirmed to be producing PRSS2, the digestive enzyme trypsinogen. Collectively, this suggests that we have produced healthy acinar cells growing in the same configuration that they would <i>in vivo. </i>This has many applications in the study of pancreatic ductal adenocarcinoma, as we have developed way to culture reprogramed cancer cells as their benign precursors and maintain acinar characteristics <i>in vitro.</i> It will also have applications in the study of many other pancreatic diseases by providing an <i>in vitro</i> model of a healthy, functional acinus.

Thesis advisor: Michelle Meyer / The primary bacterial targets for most antibiotics are well known. To survive the stress of an antibiotic a bacterium must decrease the antibiotic to target binding ratio to escape from harmful effects. This can occur through a number of different functions including down-regulation of the target, mutation of the binding site on the target, and decreasing the intake or increasing the efflux of the antibiotic. However, it is becoming more evident that an antibiotic stress response influences more than just the primary target, and that a wave of secondary responses can be triggered throughout the bacterium. As a result resistance mutations may arise in genes that are indirectly affected by the initial interaction between the antibiotic and target. These indirect responses have been found to be associated with metabolism, regulation, cell division, oxidative stress, and other critical pathways. One technique recently developed in our lab, called transposon insertion sequencing (Tn-seq), can be used to further understand the complexity of these indirect responses by profiling growth rates (fitness) of mutants at a genome-wide level. However, Tn-seq is normally performed with large libraries of pooled mutants and thus it remains unclear how this may influence fitness of some independent mutants that may be compensated by others in the population. Additionally, since the original method has only utilized planktonic culture, it is also not clear how higher order bacterial structures, such as biofilms or microcolonies, influence bacterial fitness. To better understand the dynamics of pooled versus individual mutant culture, as well as the effect of community structure in microcolony development on the influence of fitness, we adapted a droplet microfluidics-based technique to encapsulate and culture single mutants. We were able to successfully encapsulate at least 7 different species of bacterial pathogens, including Streptococcus pneumoniae, and culture them planktonically, or as microcolonies, in either monodisperse liquid or agarose droplets. These experiments, however, raised an important challenge: the DNA yield from one encapsulation experiment is insufficient to generate samples for sequencing by means of the traditional Tn-seq method. This led us to develop a novel Tn-seq DNA library preparation method, which is able to generate functional Tn-seq library molecules from picogram amounts of DNA. This method is not ideal yet because fitness data generated through the new method currently does not correlate well with data from traditional Tn-seq library preparation. However, we have identified one major culprit that should be easily solvable. We expect by modifying the binding site of the primer used for linear amplification of transposon ends that the new preparation method will be able recapitulate results from the traditional Illumina preparation method for Tn-seq. This will enable us to prepare robust Tn-seq samples from very small amounts of DNA in order to probe stress responses in single mutants as well as in microcolonies in a high-throughput manner. / Thesis (MS) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Micromixer is one of the most significant components of microfluidic systems, which manifest essential applications in the field of chemistry and biochemistry. Achieving complete mixing performance at the shortest micro channel length is essential for a successful micromixer design. We have developed five novel micromixers which have advantages of high efficiency, simple fabrication, easy integration and ease for mass production. The design principle is based on the concept of splitting-recombination and chaotic advection. Numerical models of these micromixers are developed to characterize the mixing performance. Experiments are also carried out to fabricate the micromixers and evaluate the mixing performance. Numerical simulation for different parameters such as fluids properties, inlet velocities and microchannel cross sectional sizes are also conducted to investigate their effects on the mixing performance. The results show that critical inlet velocities can be predicted for normal fluid flow in the micromixers. When the inlet velocity is smaller than the critical value, the fluids mixing is dominated by mechanism of splitting-recombination, otherwise, it is dominated by chaotic advection. If the micromixer can tolerate higher inlet velocity, the complete mixing length can be further reduced. Our simulation results will provide valuable information for engineers to design a micromixer by choosing appropriate geometry to boost mixing performance and broaden implicational range to fit their specific needs. Accurate and complicated fluidic control, such as flow mixing or reaction, solution preparation, large scale combination of different reagents is also important for bio-application of microfluidics. A proposal microfluidic system is capable of creating 1024 kinds of combination mixtures. The system is composed of a high density integrated microfluidic chip and control system. The high density microfluidic chip, which is simply fabricated through soft lithography technique, contains a pair of 32 flow channels that can be specifically addressed by each 10 actuation channels based on principle of multiplexor in electronic circuits. The corresponding hardware and software compose the control system, which can be easy fabricated and modified, especially for prototype machine developing. Moreover, the control system has general application. Experiments are conducted to verify the feasibility of this microfluidic system for multi-optional solution combination. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2013.

In order for microfluidics devices to be marketable, they must be inexpensive and easy to use. Two projects were pursued in this study for this purpose. The first was the design of a chip alignment system for visual feedback, in which a two-layer microfluidic chip was placed under a camera and an image processing and linear algebra program aligned a computer model to it. The system then translated the new locations of air valves and could detect valve activation in a chip filled with food coloring. The second was the design of a cheap, portable system to detect phosphorus in water. This system could not be completed due to time constraints, but the methods were detailed, and design ideas were laid out for future work. / by Ian Gerstel. / Thesis (M.S.C.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.

DNA arrays, capable of detecting specific DNA sequences from a sample have become widely used. They rely on DNA heterogeneous hybridization, which is the binding between a single strand of DNA immobilized on a surface (probe) and its complementary strand present in the bulk (target). In order to improve the hybridization time in DNA arrays, it is crucial to understand the kinetics of DNA hybridization. The study of the Damkohler number that compares the DNA supply by diffusion to the DNA consumption by reaction (hybridization) shows that in many cases we can expect DNA hybridization to be a diffusion limited process. This is verified by a finite element study, where a whole microfluidic chamber (bulk and reacting surface) is simulated. In these cases, the formation of a depletion zone above the sensing zone is observed. The reaction rate is much lower than in the ideal case where the reaction would be reaction rate limited. A better DNA transport could be a solution to overcome the diffusion barrier. Therefore, the influence of convection on DNA hybridization was studied. Finite element simulation shows that even a small DNA velocity (10 ƒ�m/s) can greatly enhance the overall reaction rate and help preventing the formation of a depletion zone. These observations are valid when one kind of probe reacts with one kind of target. In reality, non specific hybridization can happen between a probe and a non complementary target. We show that in some cases, non specific hybridization can slow down the kinetics and reduce the fraction of specifically hybridized probes at equilibrium. The fraction of non specific hybrids can reach a maximum before decreasing and reaching equilibrium, suggesting that a longer hybridization time would lead to a better specificity. The addition of convective transport does not affect the equilibrium, but allows to reach it faster and with a better ratio between specific and non specific hybrids during the process. Therefore, convective transport of DNA appears to be beneficial. Another possibility is to act on the DNA itself to focus it near the sensing zone. Our study of the different electrokinetic forces leads us to derive the expression of the dielectrophoretic force in a field resulting from the combination of a DC field and an AC field. This could be a novel way to act on polarizable particles like DNA.

Microfabrication has given rise to numerous technologies and has resulted in new paradigms for how science and technology has advanced in recent years. Having originated from the microelectronics industry, microfabrication techniques have increasingly been leveraged in the development of various other fields. Such techniques have an increasing presence in the field of medical devices, especially with the advent of microfluidics. The capability that microfluidics lends to miniaturizing and making portable analytical tools was, and still is, extremely useful in the advancement of medical technologies. In this dissertation, we explore novel microfabrication techniques towards the development of next-generation medical devices. We can broadly classify these devices as devices that function in in vitro and in vivo settings. In vitro devices typically function in a non-invasive manner such as when patient samples are processed externally for diagnostic purposes. In vivo medical devices, on the other hand, normally play a role in disease treatment upon implantation into a patient, such as with stents, pacemakers and drug delivery devices. Here we demonstrate how microfabrication techniques can be implemented in the improvement of devices involved in diagnosis and treatment; two important branches of medical sciences that go hand in hand. Firstly, microfabrication and microfluidic techniques were implemented in developing a CD4+ T helper cell counter. This integrated device, where capture and analysis are performed on the same platform, also employs a chemiluminescence-based method of detection. This a rather simple and elegant technique that is amenable for miniaturization in future as it does not require the use of external complex light source (such as for fluorescence imaging) nor the use of image/data analysis methods. The second part of this dissertation describes novel microfabrication techniques for the development of a new class of implantable devices- hydrogel MEMS devices. This technique is comparable to additive manufacturing techniques such as 3D printing. Current 3D printing or fabrication techniques for biocompatible materials normally result in standalone structures. Using our technique, we are not only able to construct microcomponents entirely out of hydrogels but also have the capability to assemble and align various moving components to form a robust MEMS-like device. As these MEMS devices are constructed entirely out of biocompatible PEG-based hydrogels, they are ideal candidates for implantable devices. Once implanted, they can be wirelessly actuated using simple permanent magnets and the operation of the devices do not require onboard power-sources or electronics, which is common for current MEMS-based implantable devices. These devices can also be designed to deliver payloads and this delivery can be actively controlled. We also explore the use of hydrogel MEMS in the in vivo delivery of therapeutics, and assess its efficacy in delivering local, low-doses of a chemotherapeutic drug in a disease model. We envision that these devices, and the technology from which they are borne, will open up a new paradigm in the way implantable devices are developed.

Many droplet microfluidic assays have been described in the literature over the last decade of research, however, there has been little reported industrial use of droplet microfluidics in drug discovery compound screening, and in particular that of P450 enzyme inhibition assays for profiling drug-drug interactions. This is partly for Intellectual Property reasons, since Pharmaceutical companies do not wish to give away trade secrets in a competitive market, but also because the technology is not yet 'proven' and remains in the proof-of-concept stage. In droplet microfluidics, where at least two liquid phases are encountered, it is important that leakage of material between phases is addressed. This effect has been extensively reported in the literature using fluorescent dyes, however there is very little evidence of research using large compound sets of diverse chemistry. This is probably because few researchers have access to the large pharmaceutical libraries necessary for this work. This project assessed the feasibility of translating a widely used microtitre plate-based P450 enzyme inhibition assay to droplet format; determined the extent of partitioning from droplets using a large pharmaceutical library set and attempted to model this behaviour, and thirdly, considered the pharmacological impact the droplet format may have on the assay. The P450 cytochrome 1A2 enzyme type (isoform) was chosen for translation to the micro-droplet format. Assays of this type are often conducted using fluorogenic substrates, making them favourable for relatively easy fluorescent detection in droplet format using simple optical detection assemblies. Oil selection was investigated to determine which oil systems would be better suited in respect of droplet formation. The use of surfactants in the oil phase and its impact on droplet formation was studied and the synthesis, preparation and characterisation of a custom perfluoropolyether (PFPE) surfactant ('AZF') conducted. Droplet chips were designed and fabricated to produce droplets of 200-300 µm diameter using novel channel designs and sealing techniques. The droplets were analysed by fluorescence spectroscopy using bespoke detector apparatus. Partitioning from aqueous to oil phase was studied for a small range of compounds and oils (with and without surfactant for fluorous oils). Partitioning was lowest using fluorous oils alone, and increased substantially when surfactant was included. Results from the large pharmaceutical test set suggested the percentage of compounds that may partition readily to the oil phase is low even when using surfactant. However, attempts to correlate this to known physicochemical properties and to develop a predictive model for fluorous solubility proved largely unsuccessful. Partitioning in the droplet chip using a droplet collection pooling method was difficult to quantify as a consequence of the profound impact turbulence had on partitioning. Miniaturisation of the P450 cytochrome inhibition assay to the droplet format initially gave poorly reproducible low signals. Possible causes included detector insensitivity, partitioning of reagent and/or fluorescent metabolite over longer incubation times, and binding of the 1A2 P450 cytochrome enzyme-protein at the droplet interface. Protein interaction at the droplet-oil boundary was studied by fluorescence labelling a protein contained in 200µm droplets and observing the extent of fluorescence localisation at the interface by epifluorescent and confocal fluorescence microscopy. The data from this work indicates a pronounced localisation of protein at the droplet interface, possibly leading to enzyme deactivation and the loss of signal seen for the assay in the droplet chip. A number of protein titrations were co-added to the droplets as 'blocking proteins' which were found to improve the reaction output, however were also noted to affect the pharmacology of the assay, noted by an order of magnitude shift in the reported IC50 for the test inhibitor used (fluvoxamine). The effects of compound leakage from droplets, and the possible detrimental impact on biological reagents by interaction at the droplet-oil interface, is a challenge that may limit widespread adoption of droplet MF systems in drug screening operations. Appropriate control measures and/or a means to reduce these effects are essential to enable accurate quantification with industrial drug discovery environments. The findings in this work highlight the challenges that have to be addressed for droplet microfluidic technology to be successfully incorporated into key areas of assay screening within drug discovery. In terms of further research, there is a significant requirement for the research community to delve further into these challenges and work closely with the industry sector to understand the beneficial role microfluidics can have and how to develop effective robust strategies the industry can easily adopt to progress this area of science.

A 3D-printed microfluidic lab-on-a-disc (LOAD) device was designed and manufactured using a low cost ( ̃£1600) consumer grade fused deposition modelling (FDM) Ultimaker 2+ 3D printer with imbedded microfluidic channels 1 mm wide, 400 μm depth and with a volumetric capacity of approximate 23 μl. FDM printers are not typically used, or are capable, of producing the fine detailed structures required for microfluidic fabrication; in addition 3D-printed objects can suffer from poor optical transparency. However, in this work, imbedded microfluidic channels were produced and the optical transparency of the device was improved though manufacture optimisation to such a point that optical colourimetric assays can be performed in a microfluidic cuvette device with sample path length of 500 μm and volumetric capacity of 190 μl. When acetone vapour treatment was used, it was possible to improve transparency of plastic samples by up to a further 30%. The LOAD device is capable of being spun using an unmodified optical disc drive (ODD), demonstrating the centrifugation based separation of plasma from whole blood in a low-cost FDM 3D-printed microfluidic LOAD device. A cholesterol assay and glucose assay was developed and optimised using cholesterol oxidase (ChOx) or glucose oxidase (GlOx) respectively and horseradish peroxidase (HRP) for the oxidative coupling of chromotropic acid (CTA) and 4-aminoantipyrine (AAP). This produced a blue quinoneimine dye with a broad absorbance peaking at 590 nm for the quantification of cholesterol/glucose in solution. The colourimetric enzymatic cascade assays were developed for use within low-cost FDM 3D-printed microfluidic devices to demonstrate the capabilities and functionality of the devices. For comparison, the assay was run in standard 96 well plates with a commercial plate reader. The results demonstrated that the quantification of 0-10 mM glucose solution using a 3D-printed microfluidic optical device had a performance comparable to a plate reader assay; glucose assay in whole blood samples R2 = 0.96.

Les microsystèmes intégrant des techniques microfluidiques offrent la possibilité de réaliser des analyses biologiques directement sur le site de prélèvement de l’échantillon. Ils ont pour objectifs notamment d’augmenter l’efficacité, la rapidité et l’accessibilité de ces tests. Pour développer efficacement un tel dispositif, un ensemble de critères doit être fixé tels que la limitation du coût, la portabilité, la simplicité d’utilisation et la précision des résultats. Un objectif de cette thèse est également de proposer un nouveau système portable permettant de répondre à un maximum d’applications. Pour cela, il convient d’intégrer et d’automatiser des protocoles biologiques complexes c’est-à-dire nécessitant l’ajout de plusieurs réactifs et des réactions en parallèle. A titre d’exemple, les tests ELISA sont abordés.Pour répondre à cette problématique, une technique innovante utilisant un matériau hyperélastique est combinée à une architecture X-Y. Des chambres étirables, permettant de calibrer et de mélanger des volumes compris entre 1 µL et une centaine de µL, sont ainsi réalisées. Différents protocoles sont intégrés et validés par ordre de complexité croissante dans des cartes microfluidiques en commençant par une gamme de dilution qui est la première étape pour la calibration des protocoles biologiques, puis un test enzymatique et un test ELISA homogène, avant d’aborder le test ELISA hétérogène qui est le protocole visé.Un démonstrateur permettant de piloter les cartes microfluidiques est ensuite présenté. Cette plateforme est générique et compatible avec les cartes microfluidiques développées. Enfin, pour automatiser complétement la mise en œuvre des protocoles, une nouvelle technique d’embarquement de réactifs liquide est proposée. / Microsystems utilizing microfluidic techniques offer the possibility to perform point-of-need biological analysis. An objective of these systems is to increase the efficiency, speed and accessibility of these analyses. In order to effectively develop this kind of device, a set of criteria must be established and adhered to. This set should address cost limitations, portability, user-friendliness, and accuracy of the results. Another objective is to propose a new portable system that has the capability to address as many applications as possible. To this end, complex biological assays with multiple steps and multiple reagents must be integrated and automated. ELISA is one such assay being considered.To deal with this issue, an innovative technique employs a hyper-elastic material joined to an X-Y architecture. The resulting chambers are flexible, thus allowing for calibration and mixing on the range of 1 µL to hundreds of µL. Several protocols are integrated and validated in microfluidic chips in order of increasing complexity. To start, a range of dilutions is performed, which is then used to calibrate biological assay. Next, an enzymatic assay and a homogeneous ELISA are integrated. Finally, heterogeneous ELISA, which is the aimed assay, is achieved.We present here a prototype to demonstrate the handling of the microfluidic chip. This platform is versatile and compatible with those that have been previously developed. Additionally, the introduction and integration of liquid reagents is proposed in order to completely automate the protocol.

Materials on the nanoscale have very interesting properties. Hence, they are commonly used for a variety of applications such as drug delivery, bio-imaging and sensing devices. Moreover, coating these particles with other materials forming core@shell or Janus particles can further enhance their properties. However, for the particles to be used in medical and electronic devices, their properties such as size, shape and composition need to be precisely controlled. In this PhD., an emulsification technique was chosen to investigate the synthesis of nanoparticles; it is a simple process, does not require any harsh chemicals or temperature and is fast. Emulsification occurs when two or more immiscible liquids and surfactants are mixed. Here, emulsion droplets were produced using a microfluidic device which allowed for the creation of uniform droplets. These were employed as templates to synthesise and assemble nanomaterials. The main aim of the Ph.D. was to develop a droplet based synthesis process to generate nanoparticles and then assemble them into core@shell particles. This Ph.D., starts by synthesising Fe3O4 nanoparticles (~ 12 nm) and assembling them into microparticles (~ 1µm 2µm) using emulsion droplets as microreactors. By tuning the surfactant, droplet size and evaporation rate of the dispersed phase, microparticles of varying shapes and sizes, such as spherical or crumbled shapes, were produced. When these particles are compared with the commercially available particles, the magnetic content of the in-house particles, or sometimes referred to as Loughborough University Enterprises Ltd. (LUEL), are much higher and more uniform, hence resulting in faster separation when used for extraction of analytes. LUEL particles were supplied as part of commercial collaboration. The use of Pickering emulsions were then explored to create core@shell particles using gold nanoparticles instead of a surfactant to produce gold shells and the addition of pre-synthesised Fe3O4 nanoparticles results in Fe3O4@Au core@shell particles. This is the first time Pickering emulsions were used to produce Fe3O4@Au core@shell particles (~ 1.5 µm) within a microfluidic device. However, the shells were not uniform in thickness. In order to improve the coverage, nanoparticles were synthesised in situ at the droplet interface. By placing the gold chloride (AuCl4-) in the continuous phase and by varying the concentration of the electron donor in hexane droplet, single crystal gold nanoparticles and platelets were formed. The reaction is spontaneous at room temperature, creating gold nanoparticles at the interface of the emulsion droplet. The size and shape of the gold nanoparticles were controlled by varying the concentration of the reactants and the size of the droplets. By adding pre-synthesised particles (Fe3O4 nanoparticles) to the droplet, Au@Fe3O4 core@shell particles were formed with an approximate size of 250 nm. The same concept of forming core@shell particles using gold nanoparticles was further expanded by using other metal ions; palladium and silver. Unlike gold, palladium and silver only formed spherical nanoparticles, no platelets were observed. The addition of preformed iron oxide nanoparticles to the palladium results in core@shell particles. However, in the case of silver, no core@shell particles were formed. The study of the rate of reaction was conducted to understand the details of the mechanism. Overall, the process developed in this Ph.D. study allows for the facile synthesis of core@shell particles in a rapid, high throughput reaction. In the future, it is believed it could be scaled up for commercial purposes.