Scanning Probe Lithography (SPL) is a versatile nanofabrication platform that leverages microfluidic “ink” delivery systems with Scanning Probe Microscopy (SPM) for generating surface-patterned chemical functionality on the sub-100 nm length scale. One of the prolific SPL techniques is Dip Pen Nanolithography™ (DPN™). High resolution, multiplexed registration and parallel direct-write capabilities make DPN (and other SPL techniques) a power tool for applications that are envisioned in micro/nano-electronics, molecular electronics, catalysis, cryptography (brand protection), combinatorial synthesis (nano-materials discovery and characterization), biological recognition, genomics, and proteomics. One of the greatest challenges for the successful performance of the DPN process is the delivery of multiple inks to the scanning probe tips for nano-patterning. The purpose of the present work is to fabricate a microfluidic ink delivery device (called “Centiwell”) for DPN (and other SPL) applications. The device described in this study maximizes the number of chemical species (inks) for nanofabrication that can be patterned simultaneously by DPN to conform the industrial standards for fluid handling for biochemical assays (e.g., genomic and proteomic). Alternate applications of Centiwell are also feasible for the various envisioned applications of DPN (and other SPL techniques) that were listed above. The Centiwell consists of a two-dimensional array of 96 microwells that are bulk micromachined on a silicon substrate. A thermoelectric module is attached to the back side of the silicon substrate and is used to cool the silicon substrate to temperatures below the dew point. By reducing the temperature of the substrate to below the dew point, water droplets are condensed in the microwell array. Microbeads of a hygroscopic material (e.g., poly-ethylene glycol) are dispensed into the microwells to prevent evaporation of the condensed water. Furthermore, since poly-ethylene glycol (PEG) is water soluble, it forms a solution inside the microwells which is subsequently used as the ink for the DPN process. The delivery of the ink to the scanning probe tip is performed by dipping the tip (or multiple tips in an array) into the microwells containing the PEG solution. This thesis describes the various development steps for the Centiwell. These steps include the mask design, the bulk micromachining processes explored for the micro-fabrication of the microwell array, the thermal design calculations performed for the selection of the commercially available thermoelectric coolers, the techniques explored for the synthesis of the PEG microbeads, and the assembly of all the components for integration into a functional Centiwell. Finally, the successful implementation of the Centiwell for nanolithography of PEG solutions is also demonstrated.

Performance and manufacturability are two important issues that must be taken into account during MEMS design. Existing MEMS design models or systems follow a process-driven design paradigm, that is, design starts from the specification of process sequence or the customization of foundry-ready process template. There has been essentially no methodology or model that supports generic, high-level design synthesis for MEMS conceptual design. As a result, there lacks a basis for specifying the initial process sequences. To address this problem, this paper proposes a performance-driven, microfabrication-oriented methodology for MEMS conceptual design. A unified behaviour representation method is proposed which incorporates information of both physical interactions and chemical/biological/other reactions. Based on this method, a behavioural process based design synthesis model is proposed, which exploits multidisciplinary phenomena for design solutions, including both the structural components and their configuration for the MEMS device, as well as the necessary substances for the chemical/biological/other reactions. The model supports both forward and backward synthetic search for suitable phenomena. To ensure manufacturability, a strategy of using microfabrication-oriented phenomena as design knowledge is proposed, where the phenomena are developed from existing MEMS devices that have associated MEMS-specific microfabrication processes or foundry-ready process templates. To test the applicability of the proposed methodology, the paper also studies microfluidic device design and uses a micro-pump design for the case study. / Singapore-MIT Alliance (SMA)

Biofilms are highly organized bacterial structures that are attached to a surface. They are ubiquitous in nature and may be detrimental, causing numerous types of illnesses in living organisms. Biofilms in the human oral cavity are the main cause of dental caries and periodontal diseases and can act as a source for pathogenic organisms to spread within the body and cause various types of systemic diseases. Streptococcus mutans is the primary etiological agent of dental caries, the single most chronic childhood disease. In many cases, quorum sensing (QS) is required for initial formation and subsequent development of biofilms and the signaling molecule autoinducer 2 (AI- 2) has been well studied as an inter-species QS signaling molecule. However, recent reports also suggest that AI-2-mediated signaling is important for intra-species biofilm formation in both Gram-negative and positive bacteria. Therefore, there is significant interest in understanding the role of different QS signals such as AI-2 in oral biofilm formation. Microfluidic devices provide biomimetic environments and offer a simple method for executing multiple stimuli experiments simultaneously, thus, can be an extremely powerful tool in the study of QS in biofilms. In this study, we report conditions that support the development of S. mutans biofilms in microchannel microfluidic devices, and the effects of extracellular addition of chemically synthesized (S)-4,5-dihydroxy-2,3-pentanedione (DPD; precursor of AI-2) on mono-species S. mutans luxS (AI-2 deficient strain) biofilm formation using a gradient generating microfluidic device. S. mutans wild type (WT) and luxS biofilms were developed in nutrient rich medium (25% brain heart infusion medium, BHI + 1% sucrose) for up to 48 h. Maximum biofilm formation with both strains was observed after 24 h, with distinct structure and organization. No changes in S. mutans luxS biofilm growth or structure were observed upon exposure to different concentrations of AI-2 in a gradient generating device (0 to 5 M). These results were also validated by using a standard 96-well plate assay and by verifying the uptake of AI-2 by S. mutans luxS. Our data suggest that extracellular addition of AI-2 does not complement the luxS deletion in S. mutans with respect to biofilm formation.

Sepsis is an uncontrolled response to infection. Severe sepsis is associated with organ dysfunction, and has mortality rate of 30-50%. Identification of severity of sepsis and prediction on mortality is crucial in making clinical decisions. Recently, cell-free DNA (cfDNA) in blood was found to have high discriminative power in predicting ICU mortality in patients with severe sepsis. In an analysis of 80 severely septic patients, the mean cfDNA level in survivors (1.16±0.13μg/ml) was similar to that of healthy volunteers (0.93±0.76μg/ml), while that of non-survivors (4.65±0.48μg/ml) was notably higher. Therefore, rapid quantification of cfDNA concentration in blood will enable physicians to quickly predict mortality of sepsis and decide on treatment. Current methods for quantification of cfDNA involve multiple steps including centrifugation, DNA-extraction from plasma, and its quantification either through spectroscopic methods or quantitative PCR. The whole process is time consuming, thus is not suitable for immediate bedside assessment. To solve the problems, a microfluidic device is designed and fabricated in this thesis, which is potential for cfDNA quantification directly using blood in 5 minutes. The goal is to use this device for distinguishing survivors or healthy donors from non-survivors in patients with severe sepsis. The two-layer device consists of a sample channel (top) and an accumulation channel (bottom) that intersect each other. The accumulation channel is preloaded with 1% agarose gel, and the blood containing cfDNA and intercalating fluorescent dye is loaded in the sample channel. Fluorescently labeled DNA is able to be trapped and concentrated at the intersection using a DC electric field, and fluorescent intensity of the accumulated DNA is representative of its concentration in the blood. The simulated electric field in the sample channel reveals that both the magnitude and the gradient of electric field reach their maximum values at the intersection. Force analysis shows that DNA was driven into the gel by the dominate electrophoretic force, while red blood cells moved away from the gel due to a strong dielectrophoretic force. In this thesis, 4 types of samples have been used to characterize the performance of the device. It showed that DNA was efficiently accumulated at the intersection, and the fluorescent intensity could be measured using a fluorescent microscope. Samples from healthy donors were able to be distinguished from that of severely septic patients in 5 minutes. However, better resolution was needed for differentiating various cfDNA concentrations in patient samples. The discussion on the effect of applied voltage showed that 9V is an optimized setting compared with 3V and 15V. In addition, it has been proved that the fluorescent reagent could be immobilized in the device and the sample preparation could be absolutely eliminated. In summary, the device proposed in this thesis is capable of distinguishing severely septic patients from healthy donors using clinical plasma in 5 minutes, and is potential to be applied in clinical blood samples. It has low cost, and is ready to be developed into a fully functioned system. This tool can be a valuable addition to the ICU to rapidly assess the severity of sepsis for informed decision making. / Thesis / Master of Applied Science (MASc)

Yes / We report the first application of a microfluidic device to observe chemotactic migration in multicellular spheroids. A microfluidic device was designed comprising a central microchamber and two lateral channels through which reagents can be introduced. Multicellular spheroids were embedded in collagen and introduced to the microchamber. A gradient of fetal bovine serum (FBS) was established across the central chamber by addition of growth media containing serum into one of the lateral channels. We observe that spheroids of oral squamous carcinoma cells OSC–19 invade collectively in the direction of the gradient of FBS. This invasion is more directional and aggressive than that observed for individual cells in the same experimental setup. In contrast to spheroids of OSC–19, U87-MG multicellular spheroids migrate as individual cells. A study of the exposure of spheroids to the chemoattractant shows that the rate of diffusion into the spheroid is slow and thus, the chemoattractant wave engulfs the spheroid before diffusing through it. / This work has been supported by National Research Program of Spain (DPI2011-28262-c04-01) and by the project "MICROANGIOTHECAN" (CIBERBBN, IMIBIC and SEOM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

There is a clear need for testing targeted drug carrier systems in a more realistic microenvironment where both biochemical interactions and shear forces are present. This is critical both for understanding of the molecular mechanisms involved in this process and during the drug discovery process. Current in vitro models of the leukocyte adhesion cascade cannot be used for real-time studies of the entire leukocyte adhesion cascade including rolling, adhesion and migration in a single assay. In this study, we have developed and validated a novel bioinspired microfluidic device (bMFD) and used it to test the hypothesis that blocking of specific steps in the adhesion/migration cascade significantly affects other steps of the cascade. The bMFD consists of an endothelialized microvascular network in communication with a tissue compartment via a 3 µm porous barrier. Human neutrophils in bMFD preferentially adhered to activated human endothelial cells near bifurcations with rolling and adhesion patterns in close agreement with in vivo observations. Treating endothelial cells with monoclonal antibodies to E-selectin or ICAM-1 or treating neutrophils with wortmannin reduced rolling, adhesion, and migration of neutrophils to 60%, 20% and 18% of their respective control values. Antibody blocking of specific steps in the adhesion/migration cascade (e.g. mAb to E-selectin) significantly downregulated other steps of the cascade (e.g. migration). This novel in vitro assay provides a realistic human cell based model for basic science studies, identification of new treatment targets, selection of pathways to target validation, and rapid screening of candidate agents. / Mechanical Engineering

Master of Science / Department of Chemistry / Christopher T. Culbertson / A microfluidic device integrated with valves and a peristaltic pump was fabricated using multilayer soft lithography to analyze single cells. Fluid flow was generated and mammalian cells were transported through the channel manifold using the peristaltic pump. A laser beam was focused at the cross-section of the channels so fluorescence of individual labeled intact cells could be detected. Triggered by the fluorescence signals of intact cells, valves could be actuated so fluid flow was stopped and a single cell was trapped at the intersection. The cell was then rapidly lysed through the application of large electric fields and injected into a separation channel. Various conditions such as channel geometry, pumping frequency, control channel size, and pump location were optimized for cell transport. A Labview program was developed to control the actuation of the trapping valves and a control device was fabricated for operation of the peristaltic pump. Cells were labeled with a cytosolic dye, Calcein AM or Oregon Green, and cell transport and lysis were visualized using epi-fluorescent microscope. The cells were transported at rates of [simular to] 1mm/s. This rate was optimized to obtain both high throughput and single cell trapping. An electric field of 850-900 V/cm was applied so cells could be efficiently lysed and cell lysate could be electrophoretically separated. Calcein AM and Oregon Green released from single cells were separated and detected by laser-induced fluorescence. The fluorescence signals were collected by PMT and sampled with a multi-function I/O card. This analyzing method using microchip may be applied to explore other cellular contents from single cells in the future.

Intracellular delivery of macromolecules is crucial for the success of many research and clinical applications. Several conventional intracellular delivery methods have been used for many years but are still inadequate for several applications because of the issues associated with toxicity, low-throughput, and/or difficulty to target certain cell types. In this study, we developed and evaluated new high-throughput intracellular delivery methods for the efficient delivery of macromolecules while maintaining high cell viability. First, we studied the feasibility of using an array of nanoneedles, with sharp tip diameters in the range of tens of nanometers, to physically make transient holes in cell membranes for intracellular delivery. Puncture loading and centrifuge loading methods were developed and assessed for the effect of various experimental parameters on cell viability and delivery efficiency of fluorescent molecules. In both methods, high-throughput intracellular delivery was feasible by creating transient holes in cell membranes with the sharp tips of the nanoneedles. The second physical intracellular delivery method we studied was a novel microfluidic device that created transient holes in the cell membrane by mechanical deformation and shear stress to the cell. We observed efficient delivery of fluorescent molecules and studied the effect of device design and flow pressure on the delivery efficiency compared to data in the literature. We accounted for cell loss and clogging in the microfluidic devices and determined the true loss of cell viability associated with this method. Lastly, we investigated the possibility of intracellular delivery using nanoparticles on a leukemia cell line. Among number of materials for nanoparticles tested, mesoporous silica/poly-L-lysine nanoparticles were selected for further intracellular delivery study based on cell viability and intracellular delivery capability. We demonstrated the co-delivery of protein and plasmid by encapsulating into and coating onto the surface of the nanoparticles, respectively, which would be advantageous for certain therapeutic strategies. In summary, this work introduced two new intracellular delivery methods involving nanoneedles and novel nanoparticles, and provided an early, independent assessment of microfluidic delivery, showing the strengths and weaknesses of each method. These methods can be further optimized for a number of laboratory and clinical applications with continued research.

Metamaterials are artificially designed composite materials which can exhibit unique and unusual properties such as the negative refractive index, negative phase velocity, etc. The concept of metamaterials becomes prevalent in the electromagnetic society since the first experimental implementation in the early 2000s. Many fascinated potential applications, e.g. super lens, invisibility cloaking, and novel antennas that are electrically small, have been proposed based on metamaterials. However, most of the applications still remain in theory and are not suitable for practical applications mainly due to the intrinsic loss and narrow bandwidth (large dispersion) determined by the fundamental physics of metamaterials .In this dissertation, we incorporate active gain devices into conventional passive metamaterials to overcome loss and even provide gain. Two types of active gain negative refractive index metamaterials are proposed, designed and experimentally demonstrated, including an active composite left-/right-handed transmission line and an active volumetric metamaterial. In addition, we investigate the non-Foster circuits for broadband matching of electrically small antennas. A rigorous way of analyzing the stability of non-Foster circuits by normalized determinant function is proposed. We study the practical factors that may affect the stability of non-Foster circuits, including the device parasitics, DC biasing, layouts and load impedance. A stable floating negative capacitor is designed, fabricated and tested. Moreover, it is important to resolve the sign of refractive index for active gain media which can be quite challenging. We investigate the analytical solution of a gain slab system, and apply the Nyquist criterion to analyze the stability of a causal gain medium. We then emphasize that the result of frequency domain simulation has to be treated with care. Lastly, this dissertation discusses another interesting topic about THz spectroscopy of live cells. THz spectroscopy becomes an emerging technique for studying the dynamics and interactions of cells and biomolecules, but many practical challenges still remain in experimental studies. We present a prototype of simple and inexpensive cell-trapping microfluidic chip for THz spectroscopic study of live cells. Cells are transported, trapped and concentrated into the THz exposure region by applying an AC bias signal while the chip maintains a steady temperature at 37°C by resistive heating. We conduct some preliminary experiments on E. coli and T cell solution and compare the transmission spectra of empty channels, channels filled with aqueous media only, and channels filled with aqueous medium with un-concentrated and concentrated cells.

<p>The presence of <i>Escherichia coli</i> in water is an environmental indicator that the water is contaminated with faeces. Approximately, 30% of the world population drink water from sources contaminated with human faeces. Consequently, this percentage comprises of people that are highly vulnerable to <i>Escherichia coli</i> infection. While most strains of <i>Escherichia coli</i> are harmless or maintain a symbiotic relationship with humans, the pathogenic strains are responsible for injurious health effects, such as diarrhoea and kidney failure. The traditional method of detecting <i>Escherichia coli</i> takes about 24 – 48 hours, does not detect viable but non-culturable cells, and requires advanced equipment and great technical skills. Most other available detection techniques lack specificity, as observed with enzyme-based techniques, or are not very sensitive, as observed with most impedance-based techniques with clogged surfaces.</p> <p> </p> <p>As a result of the health effects due to this microorganism and the basic limitations of available detection techniques, there is need for a specific, sensitive and rapid detection technique to ensure a sustained and timely access to <i>E. coli</i>- free water. Therefore, the aim of this research work is to develop a detection technique devoid of the basic limitations of available methods. In this study, the antibody-antigen relationship was taken advantage of to ensure the specificity of the technique is guaranteed. This was achieved using <i>Escherichia coli</i> polyclonal antibodies that target the O and K antigens found in most pathogenic strains. These antibodies were functionalized on carboxyl group modified superparamagnetic fluorescent microparticles immobilized with streptavidin. The sensitivity of the technique was ensured by utilizing the low detection limit feature offered by the use of microfluidic devices. Two microfluidic devices, glass-based and PDMS-based, were fabricated with easily accessible materials. </p> <p> </p> <p>On introducing the sample reagents and test samples into the microfluidic devices, and passing an alternating current frequency through the system, the antibodies specifically isolated the target organisms from the pool of water contaminants and a drop in the device electric potential proportional to the bacteria concentration was observed. The success of this procedure depends on the identification of the alternating current frequency beyond which manipulation of the samples would not be easily carried out. As a result, the flow field analysis of the microparticles was carried out to study the particle behavior by varying the alternating current frequency from 15 kHz – 75 kHz. </p> <p> </p> <p>The optimum frequency observed was 35 kHz. Using the glass-based microfluidic device, the voltage drop observed for the serial dilutions, 10¹ to 10⁶ ranged from 200 mV to 420 mV while that for the serial dilutions, 10^-7 to 10^-1 ranged from 90 mV to 285 mV. To ascertain if a lower detection limit could be obtained, the PDMS-based microfluidic device, with a channel with of 300 µm, was used to analyze the response of the device to 10^-7 to 10^-1<b> </b>serial dilutions. The result ranged from 10 mV to 30 mV respectively. A comparative analysis with the conventional detection method showed that it was able to detect less than 300 <i>Escherichia coli</i> colony-forming units. This result indicates that an optimized PDMS-based microfluidic device with higher resolution microchannel could potential detect tens of bacteria colony-forming units. These results were obtained in about 60 secs of introducing the sample in the device.</p> <p> </p> <p>The rapidity and consistency of the results observed by the continuous increase in voltage drop with increasing concentrations of <i>Escherichia coli</i> indicate that this detection technique has great potential in addressing the time, specificity and sensitivity issues observed with most available detection methods.</p><br>