Design and Optimization Methods for Pin-Limited and Cyberphysical Digital Microfluidic Biochips

<p>Microfluidic biochips have now come of age, with applications to biomolecular recognition for high-throughput DNA sequencing, immunoassays, and point-of-care clinical diagnostics. In particular, digital microfluidic biochips, which use electrowetting-on-dielectric to manipulate discrete droplets (or "packets of biochemical payload") of picoliter volumes under clock control, are especially promising. The potential applications of biochips include real-time analysis for biochemical reagents, clinical diagnostics, flash chemistry, and on-chip DNA sequencing. The ease of reconfigurability and software-based control in digital microfluidics has motivated research on various aspects of automated chip design and optimization.</p><p>This thesis research is focused on facilitating advances in on-chip bioassays, enhancing the automated use of digital microfluidic biochips, and developing an "intelligent" microfluidic system that has the capability of making on-line re-synthesis while a bioassay is being executed. This thesis includes the concept of a "cyberphysical microfluidic biochip" based on the digital microfluidics hardware platform and on-chip sensing technique. In such a biochip, the control software, on-chip sensing, and the microfluidic operations are tightly coupled. The status of the droplets is dynamically monitored by on-chip sensors. If an error is detected, the control software performs dynamic re-synthesis procedure and error recovery.</p><p>In order to minimize the size and cost of the system, a hardware-assisted error-recovery method, which relies on an error dictionary for rapid error recovery, is also presented. The error-recovery procedure is controlled by a finite-state-machine implemented on a field-programmable gate array (FPGA) instead of a software running on a separate computer. Each state of the FSM represents a possible error that may occur on the biochip; for each of these errors, the corresponding sequence of error-recovery signals is stored inside the memory of the FPGA before the bioassay is conducted. When an error occurs, the FSM transitions from one state to another, and the corresponding control signals are updated. Therefore, by using inexpensive FPGA, a portable cyberphysical system can be implemented.</p><p>In addition to errors in fluid-handling operations, bioassay outcomes can also be erroneous due the uncertainty in the completion time for fluidic operations. Due to the inherent randomness of biochemical reactions, the time required to complete each step of the bioassay is a random variable. To address this issue, a new "operation-interdependence-aware" synthesis algorithm is proposed in this thesis. The start and stop time of each operation are dynamically determined based on feedback from the on-chip sensors. Unlike previous synthesis algorithms that execute bioassays based on pre-determined start and end times of each operation, the proposed method facilitates "self-adaptive" bioassays on cyberphysical microfluidic biochips.</p><p>Another design problem addressed in this thesis is the development of a layout-design algorithm that can minimize the interference between devices on a biochip. A probabilistic model for the polymerase chain reaction (PCR) has been developed; based on the model, the control software can make on-line decisions regarding the number of thermal cycles that must be performed during PCR. Therefore, PCR can be controlled more precisely using cyberphysical integration.</p><p>To reduce the fabrication cost of biochips, yet maintain application flexibility, the concept of a "general-purpose pin-limited biochip" is proposed. Using a graph model for pin-assignment, we develop the theoretical basis and a heuristic algorithm to generate optimized pin-assignment configurations. The associated scheduling algorithm for on-chip biochemistry synthesis has also been developed. Based on the theoretical framework, a complete design flow for pin-limited cyberphysical microfluidic biochips is presented.</p><p>In summary, this thesis research has led to an algorithmic infrastructure and optimization tools for cyberphysical system design and technology demonstrations. The results of this thesis research are expected to enable the hardware/software co-design of a new class of digital microfluidic biochips with tight coupling between microfluidics, sensors, and control software.</p>Dissertatio

Fluigi: an end-to-end software workflow for microfluidic design

One goal of synthetic biology is to design and build genetic circuits in living cells for a range of applications with implications in health, materials, and sensing. Computational design methodologies allow for increased performance and reliability of these circuits. Major challenges that remain include increasing the scalability and robustness of engineered biological systems and streamlining and automating the synthetic biology workflow of “specify-design-build-test.” I summarize the advances in microfluidic technology, particularly microfluidic large scale integration, that can be used to address the challenges facing each step of the synthetic biology workflow for genetic circuits. Microfluidic technologies allow precise control over the flow of biological content within microscale devices, and thus may provide more reliable and scalable construction of synthetic biological systems. However, adoption of microfluidics for synthetic biology has been slow due to the expert knowledge and equipment needed to fabricate and control devices. I present an end-to-end workflow for a computer-aided-design (CAD) tool, Fluigi, for designing microfluidic devices and for integrating biological Boolean genetic circuits with microfluidics. The workflow starts with a ``netlist" input describing the connectivity of microfluidic device to be designed, and proceeds through placement, routing, and design rule checking in a process analogous to electronic computer aided design (CAD). The output is an image of the device for printing as a mask for photolithography or for computer numerical control (CNC) machining. I also introduced a second workflow to allocate biological circuits to microfluidic devices and to generate the valve control scheme to enable biological computation on the device. I used the CAD workflow to generate 15 designs including gradient generators, rotary pumps, and devices for housing biological circuits. I fabricated two designs, a gradient generator with CNC machining and a device for computing a biological XOR function with multilayer soft lithography, and verified their functions with dye. My efforts here show a first end-to-end demonstration of an extensible and foundational microfluidic CAD tool from design concept to fabricated device. This work provides a platform that when completed will automatically synthesize high level functional and performance specifications into fully realized microfluidic hardware, control software, and synthetic biological wetware

Developing microelectrode and microfluidic devices for studying neuronal development

In this thesis technologies are developed to make integrated devices to study neural activity in vitro: microelectrode arrays (MEAs), microfluidic modules and human neural stem cells (hNSCs). My aim was to develop novel in vitro models for neurodegenerative diseases such as multiple sclerosis (MS). As MS is a chronic neuroinflammatory disease that results in loss of myelin it evokes changes in neuronal conduction velocity in the central nervous system (CNS). Therefore, developing a system that could electrically follow the progress of myelination through immunocytochemistry and via conduction velocity would be of great value in MS research. As a step towards this goal, I developed and fabricated functional custom MEAs on which the electrical activity of human dopaminergic neurons (differentiated from hNSCs) and mouse spinal cord cells was recorded. Microfluidic microchannels measuring 5 μm wide were successful in separating the cell bodies of human cerebral cortical neurons (hCCNs) from the axons in two different compartments. Mouse spinal cord cultures were electrically active from 2 days in vitro (DIV) and remained active up until 52 DIV. Nominal conduction velocity (NCV) measurements were recorded from these cultures on commercial MEAs from 6 to 24 DIV. NCV increased from 0.03 m/s at 2 DIV to 15.00 m/s at 24 DIV indicating increasing myelination. Combining this data with molecular methodologies promises new approaches in developing more human-relevant models for MS research and will provide a deeper understanding of the process of myelination and possible new treatments that may one day cure MS

A modular multi electrode array system for electrogenic cell characterisation and cardiotoxicity applications

Multi electrode array (MEA) systems have evolved from custom-made experimental tools, exploited for neural research, into commercially available systems that are used throughout non-invasive electrophysiological study. MEA systems are used in conjunction with cells and tissues from a number of differing organisms (e.g. mice, monkeys, chickens, plants). The development of MEA systems has been incremental over the past 30 years due to constantly changing specific bioscientific requirements in research. As the application of MEA systems continues to diversify contemporary commercial systems are requiring increased levels of sophistication and greater throughput capabilities. [Continues.

A modular approach to high throughput microsystems

A modular high throughput microsystem was developed using microfabrication technology and nucleic acid analysis. The purpose of developing this microsystem is to identify acute infectious disease and prevent contagious outbreak in a matter of time. This microsystem included three major components, a reagent distribution device, a 96 CFPCR array, and a multi-zone thermal system, to efficiently amplify specific DNA fragments to determine the disease status or precisely pinpoint a disease from multiple patients. Polymerase chain reaction (PCR) is the key component in the nucleic acid analysis for disease because it can be used to amplify interested DNA fragments by repeatedly thermal cycles. Temperature distribution is critical to the PCR reaction, and thermal management was studied; the approaches which reached good biochemical results were applied to the design of a 96 CFPCR array and a multi-zone thermal system. Protein adsorption is another issue when PCR is realized in a micro device because of the increased surface-to-volume ratio, which might terminate the biochemical reaction. A series of experiments were carried out to understand the protein adsorption in the microchannels with different geometries. A disposable 96 CFPCR was designed and fabricated on a polycarbonate substrate by double-sided hot embossing and its requirement of multiple temperature zones was fulfilled by building a multi-zone thermal system. Their thermal performance was characterized by numerical simulations and validated by infrared camera experiments. To evenly distribute the analyte with reducing pipetting steps, a reagent distribution device was designed to assemble with the 96 CFPCR array by using passive alignment structures to perform a systematic performance. The passive alignment structures including three pairs of v-groove and hemisphere-tipped post was studied and modified to ensure a smooth passage for mass, momentum, and energy of chemical analyte. Biochemical experiments demonstrated parallel amplifications of both identical and different DNA fragments from the multiple CFPCRs on the multi-zone thermal system, which implied the potential to detect acute infectious disease with acceleration, accuracy, specificity, and high throughput

Development of an acousto-electric biochemical sensor (AEBS) for monitoring biological and chemical processes

Ph.D., Electrical Engineering -- Drexel University, 200

Optimization of Continuous Flow Polymerase Chain Reaction with Microfluidic Reactors

The polymerase chain reaction (PCR) is an enzyme catalyzed technique, used to amplify the number of copies of a specific ~gion ofDNA. This technique can be used to identify, with high-probability, disease-causing viruses and/or bacteria, the identity of a deceased person, or a criminal suspect. Even though PCR has had a tremendous impact in clinical diagnostics, medical sciences and forensics, the technique presents several drawbacks. For example, the costs associated with each reaction are high and the reaction is prone to cont,amination due its inherent efficiency and high sensitivity. By employing microfluidic' systems to perform PCR these advantages can be circumvented. This thesis addresses implementation issues that adversely affect PCR . in microdevices and aims to improve the efficiency of the reaction by introducing novel materials and methods to existing protocols. Molecule-surface-interactions and ,' temperature control/determination are the main focus within this work. Microchannels and microreactors are char:acterized by extremely high surface-tovolume ratios. This dictates that surfaces play a dominant role in defining the efficiency ofPCR (and other synthetic processes) through increased molecule-surface interactions. In a multicomponent reaction system where the concentration of several components needs to be maintained the situation is particularly complicated. For example, inhibition of PCR is commonly observed due to polymerase adsorption on channel walls. Within??????? this work a number of different surface treatments have been investigated with a view to minimizing adsorption effects on microfluidic channels. In addition, novel studies introducing the use of superhydrophobic coatings on microfluidic channels are presented. Specifically superhydrophobic surfaces exhibiting contact angles in excess of 1500 have been created by growing Copper oxide and Zinc oxide' nanoneedles and silica-sol gel micropores on microfluidic channels. Such surfaces utilize additional surface roughness to promote hydrophobicity. Aqueous solutions in contact with superhydrophobic surfaces are suspended by bridging-type wetting, and therefore the fraction of the surface in contact with the aqueous layer is significantly lower than for a flat surface. An additional difficulty associated with PCR on microscale is the detennination and control of temperature. When perfonning PCR, the ability to accurately control system temperatures is especially important since both primer annealing to singlestranded DNA and the catalytic extension of this primer to fonn the complementary strand will only proceed in an efficient manner within relatively narrow temperature ranges. It is therefore imperative to be able to accurately monitor the temperature distributions in such microfluidic channels. In this thesis, fluorescence lifetime imaging (FLIM) is used as a novel method to directly quantify temperature within microchannel environments. The approach, which includes the use of multiphoton e'xcitation to achieve optical sectioning, allows for high spatial and temporal resolution, operates over a wide temperature range and can be used to rapidly quantify local temperatures with high precision.Imperial Users onl