Yield Enhancement of Digital Microfluidics-Based Biochips Using Space Redundancy and Local Reconfiguration

As microfluidics-based biochips become more complex, manufacturing yield will have significant influence on production volume and product cost. We propose an interstitial redundancy approach to enhance the yield of biochips that are based on droplet-based microfluidics. In this design method, spare cells are placed in the interstitial sites within the microfluidic array, and they replace neighboring faulty cells via local reconfiguration. The proposed design method is evaluated using a set of concurrent real-life bioassays.Comment: Submitted on behalf of EDAA (http://www.edaa.com/

CONTINUOUS FLOW, HIGH-THROUGHPUT MICROFLUIDIC PLATFORMS FOR GENOMIC DISCRIMINATION ASSAYS

DNA technologies from PCR to allele discrimination are now common and indispensable techniques utilized in a myriad of fields such as healthcare and agriculture. While traditional bench top methodologies are well refined, the standard techniques often require sample volumes and costs that are prohibitive for high throughput applications. Thus genetic screening at rapid rates and low costs is a requirement for further propagation of DNA technologies in large scale operations. Microfluidic platforms are particular well-suited to meet the challenge of creating technologies capable of high throughput, continuous flow, multiplexed allelic discrimination. Specifically, by reducing a typical reaction system from a milliliter scale Eppendorf tube down to nanoliter sized droplets, genetic screening may be performed at a fraction the cost. Here, we present two novel poly(dimethylsiloxane) (PDMS) microfluidic platforms capable of multiplexing single nucleotide polymorphism (SNP) detection. Utilizing unique SNP assays (Invader and Taqman PCR) well suited for microfluidics applications, both platforms include onchip optical detection of fluorophores that allow for direct allelic read out. Utilizing benchtop amplified target DNA, successful SNP detection on-chip was achieved in the first device with unambiguous signal readout spanning nearly 80 target DNA/probe combinations. In the second device, both target DNA amplification and allele detection were performed on-chip. Taken together, our novel PDMS microfluidic platforms provide a key advance in microfluidic devices for allele discrimination. Device capable of high throughput and affordable genomic screening now looms

A centrifugal microfluidic platform for capturing, assaying and manipulation of beads and biological cells

Microfluidics is deemed a field with great opportunities, especially for applications in medical diagnostics. The vision is to miniaturize processes typically performed in a central clinical lab into small, simple to use devices - so called lab-on-a-chip (LOC) systems. A wide variety of concepts for liquid actuation have been developed, including pressure driven flow, electro-osmotic actuation or capillary driven methods. This work is based on the centrifugal platform (lab-on-a-disc). Fluid actuation is performed by the forces induced due to the rotation of the disc, thus eliminating the need for external pumps since only a spindle motor is necessary to rotate the disc and propel the liquids inside of the micro structures. Lab-on-a-disc systems are especially promising for point-of-care applications involving particles or cells due to the centrifugal force present in a rotating system. Capturing, assaying and identification of biological cells and microparticles are important operations for lab-on-a-disc platforms, and the focus of this work is to provide novel building blocks towards an integrated system for cell and particle based assays. As a main outcome of my work, a novel particle capturing and manipulation scheme on a centrifugal microfluidic platform has been developed. To capture particles (biological cells or micro-beads) I designed an array of V-shaped micro cups and characterized it. Particles sediment under stagnant flow conditions into the array where they are then mechanically trapped in spatially well-defined locations. Due to the absence of flow during the capturing process, i.e. particle sedimentation is driven by the artificial gravity field on the centrifugal platform, the capture efficiency of this approach is close to 100% which is notably higher than values reported for typical pressure driven systems. After capturing the particles, the surrounding medium can easily be exchanged to expose them to various conditions such as staining solutions or washing buffers, and thus perform assays on the captured particles. By scale matching the size of the capturing elements to the size of the particles, sharply peaked single occupancy can be achieved. Since all particles are arrayed in the same focal plane in spatially well defined locations, operations such as counting or fluorescent detection can be performed easily. The application of this platform to perform multiplexed bead-based immunoassays as well as the discrimination of various cell types based on intra cellular and membrane based markers using fluorescently tagged antibodies is demonstrated. Additionally, methods to manipulate captured particles either in batch mode or on an individual particle level have been developed and characterized. Batch release of captured particles is performed by a novel magnetic actuator which is solely controlled by the rotation frequency of the disc. Furthermore, the application of this actuator to rapidly mix liquids is shown. Manipulation of individual particles is performed using an optical tweezers setup which has been developed as part of this work. Additionally, this optical module also provides fluorescence detection capabilities. This is the first time that optical tweezers have been combined with a centrifugal microfluidic system. This work presents the core technology for an integrated centrifugal platform to perform cell and particle based assays for fundamental research as well as for point-of- care applications. The key outputs of my specific work are: 1. Design, fabrication and characterization of a novel particle capturing scheme on a centrifugal microfluidic platform (V-cups) with very high capture efficiency (close to 100%) and sharply peaked single occupancy (up to 99.7% single occupancy). 2. A novel rotation frequency controlled magnetic actuator for releasing captured particles as well as for rapidly mixing liquids has been developed, manufactured and characterized. 3. The V-cup platform has successfully been employed to capture cells and perform multi-step antibody staining assays for cell discrimination. 4. An optical tweezers setup has been built and integrated into a centrifugal teststand, and successful manipulation of individual particles trapped in the V-cup array is demonstrated

Advanced Platforms for Multiplexed Nucleic Acid Research

Nucleic acid is the basic genetic material of all life forms on earth. In recent years, nucleic acid technology has assumed an essential role in widespread fields ranging from medicine to agriculture. In the field of molecular diagnostics, nucleic acid analysis has become widely adopted due to their high specificity, sensitivity and their capability for multiplexing. The critical first step in nucleic acid analysis is sample preparation, which involves isolation and purification of DNA/RNA from diverse samples, such as blood and serum. Compared to rapid advances in genomic analysis methods, DNA extraction techniques have remained nearly unchanged over the past 20 years. While spin columns and magnetic microparticles dominate due to their speed and ease-of-use, these methods fragment DNA and are incapable of sufficient DNA quality for the newest long-read sequencing and genome mapping technologies. Moreover, despite all the technological advancements, nucleic acid techniques that allow large-scale multiplexed analysis of polymorphic genetic loci remain needed for theoretical and practical nucleic acid research, such as the study of oncogenic mutations and genetic disease diagnosis. In addition, the cost-effectiveness of infectious disease diagnosis by nucleic acid analysis counts on the multiplex scale in panel testing. So far, solid-phase probe hybridization, PCR and LCR are the three domain assays for locus-specific analysis but none of them can be adapted to the practical needs. Therefore, developing novel and effective strategies for large-scale multiplexed analysis is still a compelling need. In this thesis, we present advanced platforms for multiplexed analysis of nucleic acids including 1) a simple silica nanomembrane-based method to extract high molecular weight, high purity DNA, 2) a Ratiometric Fluorescence Coding strategy for multiplexed detection of nucleic acids and 3) an easy single-molecule, fluorescence spectroscopic method to measure absolute telomere lengths in a variety of DNA samples. We start by discussing the existing DNA extraction methods and current strategies for expanding the multiplexed detection capacity of nucleic acid amplification testing. We also introduce the confocal single-molecule spectroscopy and its capability for screening analytes that are not amendable to amplification-based analysis (Chapter 1). Then, we introduce the thermoplastic silica nanomaterial named Nanobind and demonstrate its capability of extracting DNA > 5.7 Mb within 45 mins surpassing any existing methods (Chapter 2). Next, we present and demonstrate our Ratiometric Fluorescence Coding strategy for multiplexed detection of DNA targets from six infectious diseases and the potential for further expanding its multiplexing capability (Chapter 3). Finally, we implement the confocal single-molecule fluorescence spectroscopy to measure the absolute telomere lengths and demonstrate the accurate detection of telomeres as short as 100 bp with high reproducibility and profiling of telomere lengths in human cancer cell lines and primary cells (Chapter 4)

Microfabricated Sampling Probes for Monitoring Brain Chemistry at High Spatial and Temporal Resolution

Monitoring neurochemical dynamics has played a crucial role in elucidating brain function and related disorders. An essential approach for monitoring neurochemicals is to couple sampling probes to analytical measurements; however, this approach is inherently limited by poor spatial and temporal resolution. In this work, we have developed miniaturized sampling probes and analytical technology to overcome these limitations. Conventional sampling probes were handmade and have several disadvantages, including large sizes (over 220 µm in diameter) and limited design flexibility. To address these disadvantages, we have used microfabrication to manufacture sampling probes. By bulk micromachining of Si, microchannels and small sampling regions can be fabricated within a probe, with an overall dimension of ~100 µm. For development of a dialysis probe, nanoporous anodic aluminum oxide was adapted for monolithically embedding a membrane. Coupling the probe to liquid chromatography-mass spectrometry, multiple neurochemicals were measured at basal conditions, including dopamine and acetylcholine. Comparing to conventional dialysis probes, the microfabricated dialysis probe provided at least 6-fold improvement in spatial resolution and potentially had lower tissue disruption. Furthermore, we have continued the development of a microfabricated push-pull probe. We enhanced functionality of the probe by integrating an additional channel into the probe for chemical delivery. Further, we demonstrated that the probe can feasibly be coupled to droplet microfluidic devices for improved temporal resolution. Nanospray ionization mass spectrometry was used for multiplexed measurements of neurochemicals in nanoliter droplet samples. Utility of the integrated system was demonstrated by monitoring in vivo dynamics during potassium stimulation of 4 neurochemicals, including glutamate and GABA. The probe provided unprecedented spatial resolution and temporal resolution as high as ~5 s. Additionally, we highlighted versatility of the method by coupling the probe to another high-throughput assay, i.e., droplet-based microchip capillary electrophoresis for rapid separation (less than 3 s) and measurement of multiple amino acid neurochemicals. This collection of work illustrates that development of the microfabricated sampling probes and their compatible microfluidic systems are highly beneficial for studying brain chemistry. The integrated miniaturized analytical technology can potentially be useful for solving other problems of biological significance.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144094/1/nonngern_1.pd

Microfluidics: reframing biological enquiry

The underlying physical properties of microfluidic tools have led to new biological insights through the development of microsystems that can manipulate, mimic and measure biology at a resolution that has not been possible with macroscale tools. Microsystems readily handle sub-microlitre volumes, precisely route predictable laminar fluid flows and match both perturbations and measurements to the length scales and timescales of biological systems. The advent of fabrication techniques that do not require highly specialized engineering facilities is fuelling the broad dissemination of microfluidic systems and their adaptation to specific biological questions. We describe how our understanding of molecular and cell biology is being and will continue to be advanced by precision microfluidic approaches and posit that microfluidic tools - in conjunction with advanced imaging, bioinformatics and molecular biology approaches - will transform biology into a precision science