Micro/nano devices for blood analysis

[Excerpt] The development of microdevices for blood analysis is an interdisciplinary subject that demandsan integration of several research fields such as biotechnology, medicine, chemistry, informatics, optics,electronics, mechanics, and micro/nanotechnologies.Over the last few decades, there has been a notably fast development in the miniaturization ofmechanical microdevices, later known as microelectromechanical systems (MEMS), which combineelectrical and mechanical components at a microscale level. The integration of microflow and opticalcomponents in MEMS microdevices, as well as the development of micropumps and microvalves,have promoted the interest of several research fields dealing with fluid flow and transport phenomenahappening at microscale devices. [...

Microswimmers and Microfluidics: Understanding and Manipulating the Locomotion of Undulatory Microswimmers

Undulatory microswimmers, such as nematodes, are of great importance to agriculture, animal and human health, and fundamental biological research. The nematode Caenorhabditis (C.) elegans is widely used as a model organism for medical studies. My work focuses on studying the locomotion of nematodes; their interactions with surfaces, fluid flow, and each other; and developing new tools to manipulate their motion for diverse applications. In the first half (chapters 2-4) of this dissertation, I investigate experimentally and theoretically the effects of flat solid surfaces, external channel flow, and other swimmers on the swimming dynamics of undulatory microswimmers. I discovered that 1) when swimming in close proximity, undulatory microswimmers synchronize their swimming gait. This synchronization is facilitated by direct collisions among the swimmers, rather than by long-range hydrodynamic interactions or deliberate actions of the swimmers; 2) undulatory micro-swimmers have a tendency to accumulate near and swim along surfaces. This behavior does not require touch sensation ability of the swimmers, and can be explained by a short-range hydrodynamic interaction between the swimmers and adjacent surface; 3) undulatory microswimmers exhibit positive rheotaxis (upstream swimming behavior) near solid surfaces. This behavior is induced by the combination of a hydrodynamic surface attraction effect and the velocity gradient of external flow near solid surfaces. These findings help explain certain intriguing behaviors of undulatory microswimmers, highlight the diverse roles of hydrodynamic forces in microswimmers\u27 life cycles, and lay the foundations for novel microswimmer manipulation methods for fundamental biological research and clinical applications. In the second half (chapters 5-7) of this dissertation, I present the design, fabrication, characterization, and applications of a few engineering devices/methods for dynamic trapping, motility measurement, motility-based sorting, and directing the motion of microswimmers. These new devices/methods enabled many studies that would be impossible or impractical with conventional methods

Unconventional elastomeric microsystems: fabrication and application

Elastomer-based microsystems hold great promise for a diverse range of applications such as rapid prototyping of lab-on-a-chip device, soft-MEMS, and soft-robotics. For better performance in such applications, unconventional elastomeric structures in terms of size, shape, and patterning trajectory, have been intensely sought after in microtechnology but their realization has been a continuous challenge. Here, as my dissertation work, I present new microfabrication schemes which enable the realizations of unconventional PDMS structures, and their applications, which will enrich the field of soft-microsystems. First, I present a new fabrication scheme for the realization of cylindrical microfluidic (MF) channels with 3D trajectories based on shaping, bonding, and assembly of sucrose fibers. Due to the high water-solubility of the sucrose templates, the scheme is a simple and environment-friendly. Also, it is cleanroom-free and cost-effective. Despite its simplicity, it enables the realization of essential 3D MF channel architectures such as highly curved MF channels, internal loops, and proper end-to-side junctions. It can, also, realize tapered junctions and stenosis which can benefit vaso-mimetic lab-on-a-chip applications. Secondly, as a practical application of the sucrose-based MF channel, I report the implementation of the bokeh-effect-based microfluidic microscopy scheme for point-of care health monitoring in highly resource-limited environment. For this work, I integrated a single polymer microlens over the sucrose-templated MF channel and retrieved magnified intra-channel images with a commercial, off-the-shelf camera. The bokeh microscope exhibited 10∼40 in magnification and 67∼252 μm of field-of-view extent, confirming their utility for point-of-care monitoring of micro-scale objects in MF channels Third, I present a new technique that enables facile fabrications of high aspect-ratio PDMS micropillars exceeding 2400 m in height and 100 in aspect-ratio. The key enabling factor is the adoption of the direct drawing technique incorporated with the in situ heating for simultaneous hardening and solidification of PDMS. The technique also allows self-aligned installation of highly reflective microspheres at the tips of the micropillars. Using the transparent PDMS micropillar as a flexible waveguide and the microsphere as a self-aligned reflector, I transformed the microsphere-tipped PDMS micropillars into all optically interrogated air-flow sensors and successfully demonstrated its air-flow sensing capability. Lastly, I present a microscale soft-robotic tentacle with spiral bending capability based on pneumatically driven bending motion of a hollow PDMS microtube. For this work, I establish a new, direct peeling-based technique for building long and thin, highly deformable microtubes and a semi-analytical model for their shape-engineering. Based on them, the artificial microrobotic tentacle exhibits the multi-turn spiraling motion with the final radius of 185 μm and squeezing force of ~ 0.78 mN. Thanks to the softness of PDMS and the spiraling motion, the micro-tentacle can function as a soft-robotic grabber of fragile micro-objects. The spiraling tentacle-based grabbing modality, the elastomeric microtube fabrication technique, and the concept of microtube shape-engineering will constitute very valuable additions to future microscale soft-robotics. Here, I organized my dissertation based on four published journal papers of which I am the first/primary author

Integrated Lithographic Molding for Microneedle-Based Devices

This paper presents a new fabrication method consisting of lithographically defining multiple layers of high aspect-ratio photoresist onto preprocessed silicon substrates and release of the polymer by the lost mold or sacrificial layer technique, coined by us as lithographic molding. The process methodology was demonstrated fabricating out-of-plane polymeric hollow microneedles. First, the fabrication of needle tips was demonstrated for polymeric microneedles with an outer diameter of 250 mum, through-hole capillaries of 75-mum diameter and a needle shaft length of 430 mum by lithographic processing of SU-8 onto simple v-grooves. Second, the technique was extended to gain more freedom in tip shape design, needle shaft length and use of filling materials. A novel combination of silicon dry and wet etching is introduced that allows highly accurate and repetitive lithographic molding of a complex shape. Both techniques consent to the lithographic integration of microfluidic back plates forming a patch-type device. These microneedle-integrated patches offer a feasible solution for medical applications that demand an easy to use point-of-care sample collector, for example, in blood diagnostics for lithium therapy. Although microchip capillary electrophoresis glass devices were addressed earlier, here, we show for the first time the complete diagnostic method based on microneedles made from SU-8

Streamline-Based Microfluidic Devices for Erythrocytes and Leukocytes Separation

In this paper, we report two devices for the continuous size-based separation of particles, such as blood cells, which is an important step for on-chip blood preparation. Unlike previously demonstrated passive fluidic devices for particle separation, the local geometry of the bifurcated side channels was used as a design parameter. The design of the devices was based on 2-D fluidic simulation of a T-shaped model. This novel approach was proved to be effective in predicting device performance. The critical particle size for separation was clearly defined in the bifurcated region by simulation under the established theoretical framework. We validated the operation principle of the devices by separating 5- and 10-μm polystyrene beads. Human leukocytes were also successfully separated from erythrocytes with 97% efficiency. The separation region of the device had a small footprint for the separation of particles in micrometer range, which makes this device a good candidate to be integrated into a lab-on-a-chip system. The particles were collected in different exit channels after they were separated, which facilitated further sensing and processing. Similar to cross-flow filters, particles were separated perpendicular to the flow direction. The filtering effect was achieved with the collection zones established by the fluidic field. Clogging was minimized by designing the minimal channel width of the devices larger than the largest particle diameter. Solvent exchange could be accomplished for particles

Spatiotemporal Dynamics of Dilute Red Blood Cell Suspensions in Low-Inertia Microchannel Flow

Microfluidic technologies are commonly used for the manipulation of red blood cell (RBC) suspensions and analyses of flow-mediated biomechanics. To enhance the performance of microfluidic devices, understanding the dynamics of the suspensions processed within is crucial. We report novel, to our knowledge, aspects of the spatiotemporal dynamics of RBC suspensions flowing through a typical microchannel at low Reynolds number. Through experiments with dilute RBC suspensions, we find an off-center two-peak (OCTP) profile of cells contrary to the centralized distribution commonly reported for low-inertia flows. This is reminiscent of the well-known “tubular pinch effect,” which arises from inertial effects. However, given the conditions of negligible inertia in our experiments, an alternative explanation is needed for this OCTP profile. Our massively parallel simulations of RBC flow in real-size microfluidic dimensions using the immersed-boundary-lattice-Boltzmann method confirm the experimental findings and elucidate the underlying mechanism for the counterintuitive RBC pattern. By analyzing the RBC migration and cell-free layer development within a high-aspect-ratio channel, we show that such a distribution is co-determined by the spatial decay of hydrodynamic lift and the global deficiency of cell dispersion in dilute suspensions. We find a cell-free layer development length greater than 46 and 28 hydraulic diameters in the experiment and simulation, respectively, exceeding typical lengths of microfluidic designs. Our work highlights the key role of transient cell distribution in dilute suspensions, which may negatively affect the reliability of experimental results if not taken into account

Single-molecule detection of molecular beacons generated from LDR on thermoplastic microfluidic device for bioanalysis

Current clinical techniques for nucleic acid detection and analysis often involve PCR, lacking adequate specificity or sensitivity to meet the stringent requirements in certain applications. This research aims to develop an innovative molecular assay and the associated hardware to rapidly signal the presence of certain targets using reporter sequence found in their genome without requiring PCR. This assay coupled the sensitivity of single-pair fluorescence resonance energy transfer (spFRET) with the specificity of ligase detection reaction (LDR) to provide near real-time readout of target biomarkers. Heightened concerns on potential bioterrorism threats, such as rapid dissemination of pathogenic bacteria or viruses into water and/or food supplies, demand fast detection strategies. In this work, a pair of strain-specific primers was designed based on the 16S rRNA gene and were end-labeled with a donor (Cy5) and acceptor (Cy5.5) dyes. In the presence of the target bacterium, the primers were joined using LDR to form a reverse molecular beacon (rMB), thus bringing Cy5 and Cy5.5 into close proximity to allow FRET to occur. These rMBs were analyzed using single-molecule detection of the FRET pairs (spFRET). The LDR was performed in a Cyclic Olefin Copolymer (COC) microfluidic device equipped with 2 or 20 thermal cycles in a continuous flow format. Single-molecule photon bursts from the resulting rMBs were detected on-chip and registered using a laser-induced fluorescence (LIF) instrument. The presence of target pathogens could be reported in as little as 2.6 min using spFRET. In another development, a similar assay format was utilized to quantify mRNA expression levels of MMP-7 gene, which is highly relevant to invasion, metastasis and progression of a variety of tumors. HT-29 cells were found to express the highest levels of MMP-7 transcripts among the studied cell lines using LDR primers specific to MMP-7 gene. This observation is consistent with the results obtained with RT-qPCR. The LDR-spFRET assay was also used for stroke subtyping by designing primers specific to AMPH gene and using a microfluidic chip with tapered detection window to improve sampling efficiency. The detection could be completed in 15 min with extended readout time to glean low copy number transcripts

Three dimensional optofluidic devices for manipulation of particles and cells

Optical forces offer a powerful tool for manipulating single cells noninvasively. Integration of optical functions within microfluidic devices provides a new freedom for manipulating and studying biological samples at the micro scale. In the pursuit to realise such microfluidic devices with integrated optical components, Ultrafast Laser Inscription (ULI) fabrication technology shows great potential. The uniqueness and versatility of the technique in rapid prototyping of 3D complex microfluidic and optical elements as well as the ability to perform one step integration of these elements provides exciting opportunities in fabricating novel devices for biophotonics applications. The work described in this thesis details the development of three dimensional optofluidic devices that can be used for biophotonics applications, in particular for performing cell manipulation and particle separation. Firstly, the potential of optical forces to manipulate cells and particles in ULI microfluidic channels is investigated. The ability to controllably displace particles within a ULI microchannel using a waveguide positioned orthogonal to it is explored in detail. We then prototype a more complex 3D device with multiple functionalities in which a 3D optofluidic device containing a complex microchannel network and waveguides was used for further investigations into optical manipulation and particle separation. The microfluidic channel network and the waveguides within the device possess the capability to manipulate the injected sample fluid through hydrodynamic focusing and optically manipulate the individual particles, respectively. This geometry provided a more efficient way of investigating optical manipulation within the device. Finally, work towards developing a fully optimised 3D cell separator device is presented. Initial functional validation was performed by investigating the capability of the device to route particles through different outlet channels using optical forces. A proof of concept study demonstrates the potential of the device to use for cell separation based on the size of the cells. It was shown that both passive and active cell separation is possible using this device