Browsing by Subject "Digital microfluidics"

Background The diagnosis of sepsis is challenging because the infection can be caused by more than 50 species of pathogens that might exist in the bloodstream in very low concentrations, e.g., less than 1 colony-forming unit/ml. As a result, among the current sepsis diagnostic methods there is an unsatisfactory trade-off between the assay time and the specificity of the derived diagnostic information. Although the present qPCR-based test is more specific than biomarker detection and faster than culturing, its 6 ~ 10 hr turnaround remains suboptimal relative to the 7.6%/hr rapid deterioration of the survival rate, and the 3 hr hands-on time is labor-intensive. To address these issues, this work aims to utilize the advances in microfluidic technologies to expedite and automate the ``nucleic acid purification - qPCR sequence detection'' workflow.

Methods and Results This task is evaluated to be best approached by combining immiscible phase filtration (IPF) and digital microfluidic droplet actuation (DM) on a fluidic device. In IPF, as nucleic acid-bound magnetic beads are transported from an aqueous phase to an immiscible phase, the carryover of aqueous contaminants is minimized by the high interfacial tension. Thus, unlike a conventional bead-based assay, the necessary degree of purification can be attained in a few wash steps. After IPF reduces the sample volume from a milliliter-sized lysate to a microliter-sized eluent, DM can be used to automatically prepare the PCR mixture. This begins with compartmenting the eluent in accordance with the desired number of multiplex qPCR reactions, and then transporting droplets of the PCR reagents to mix with the eluent droplets. Under the outlined approach, the IPF - DM integration should lead to a notably reduced turnaround and a hands-free ``lysate-to-answer'' operation.

As the first step towards such a diagnostic device, the primary objective of this thesis is to verify the feasibility of the IPF - DM integration. This is achieved in four phases. First, the suitable assays, fluidic device, and auxiliary systems are developed. Second, the extent of purification obtained per IPF wash, and hence the number of washes needed for uninhibited qPCR, are estimated via off-chip UV absorbance measurement and on-chip qPCR. Third, the performance of on-chip qPCR, particularly the copy number - threshold cycle correlation, is characterized. Lastly, the above developments accumulate to an experiment that includes the following on-chip steps: DNA purification by IPF, PCR mixture preparation via DM, and target quantification using qPCR - thereby demonstrating the core procedures in the proposed approach.

Conclusions It is proposed to expedite and automate qPCR-based multiplex sparse pathogen detection by combining IPF and DM on a fluidic device. As a start, this work demonstrated the feasibility of the IPF - DM integration. However, a more thermally robust device structure will be needed for later quantitative investigations, e.g., improving the bead - buffer mixing. Importantly, evidences indicate that future iterations of the IPF - DM fluidic device could reduce the sample-to-answer time by 75% to 1.5 hr and decrease the hands-on time by 90% to approximately 20 min.

Data acquisition and diagnostics for chemical and biological analytes are critical to medicine, security, and the environment. Miniaturized and portable sensing systems are especially important for medical and environmental diagnostics and monitoring applications. Chip scale integrated planar photonic sensing systems that can combine optical, electrical and fluidic functions are especially attractive to address sensing applications, because of their high sensitivity, compactness, high surface specificity after surface customization, and easy patterning for reagents. The purpose of this dissertation research is to make progress toward a chip scale integrated sensing system that realizes a high functionality optical system integration with a digital microfluidics platform for medical diagnostics and environmental monitoring.

This thesis describes the details of the design, fabrication, experimental measurement, and theoretical modeling of chip scale optical sensing systems integrated with electrowetting-on-dielectric digital microfluidic systems. Heterogeneous integration, a technology that integrates multiple optical thin film semiconductor devices onto arbitrary host substrates, has been utilized for this thesis. Three different integrated sensing systems were explored and realized. First, an integrated optical sensor based upon the heterogeneous integration of an InGaAs thin film photodetector with a digital microfluidic system was demonstrated. This integrated sensing system detected the chemiluminescent signals generated by a pyrogallol droplet solution mixed with H2O2 delivered by the digital microfluidic system.

Second, polymer microresonator sensors were explored. Polymer microresonators are useful components for chip scale integrated sensing because they can be integrated in a planar format using standard semiconductor manufacturing technologies. Therefore, as a second step, chip scale optical microdisk/ring sensors integrated with digital microfluidic systems were fabricated and measured. . The response of the microdisk and microring sensing systems to the change index of refraction, due to the glucose solutions in different concentrations presented by the digital microfluidic to the resonator surface, were measured to be 95 nm/RIU and 87nm/RIU, respectively. This is a first step toward chip-scale, low power, fully portable integrated sensing systems.

Third, a chip scale sensing system, which is composed of a planar integrated optical microdisk resonator and a thin film InGaAs photodetector, integrated with a digital microfluidic system, was fabricated and experimentally characterized. The measured sensitivity of this sensing system was 69 nm/RIU. Estimates of the resonant spectrum for the fabricated systems show good agreement with the theoretical calculations. These three systems yielded results that have led to a better understanding of the design and operation of chip scale optical sensing systems integrated with microfluidics.

Microfluidic biochips are now being used for biochemical applications such as high-throughput DNA sequencing, point-of-care clinical diagnostics, and immunoassays. In particular, digital microfluidic biochips (DMFBs) are especially promising. They manipulate liquid as discrete droplets of nanoliter or picoliter volumes based on the principle of electrowetting-on-dielectric under voltage-based electrode actuation. DMFBs have been commercially adopted for sample preparation and clinical diagnostics. Techniques have also been developed for high-level synthesis, module placement, and droplet routing.

However, reliability is a major concern in the use of DMFBs for laboratory protocols. In addition to manufacturing defects and imperfections, faults can also arise during a bioassay. For example, excessive or prolonged actuation voltage may lead to electrode breakdown and charge trapping, and DNA fouling may lead to the malfunction of electrodes. Faults may eventually result in errors in droplet operations. If an unexpected error appears during an experiment, the outcome of the experiment will be incorrect. The repetition of an experiment leads to wastage of valuable reagents and time.

Therefore, it is necessary to ensure the correctness of the hardware and bioassay execution on the biochip. In this thesis, we focus on three types of reliability: biochip testing, error/fault recovery, and fault-tolerant synthesis. First, when a biochip is fabricated, defects might occur in parts of the biochip. Therefore, our objective is to develop biochip testing methods to detect and locate faults. Second, to faults that appear during droplet operation or in the hardware, we develop error-recovery procedures and redundancy solutions. Finally, we develop fault-tolerant synthesis techniques so that even if faults occur during droplet operations (e.g., unbalance splitting), the bioassay can proceed unimpeded. The proposed solutions are applied to two new types of biochip platforms, namely micro-electrode-dot-array (MEDA) and digital acoustofluidics.

Digital microfluidic biochips (DMFBs) are revolutionizing many biochemical analysis procedures, e.g., high-throughput DNA sequencing and point-of-care clinical diagnosis. However, today's DMFBs suffer from several limitations: (1) constraints on droplet size and the inability to vary droplet volume in a fine-grained manner; (2) the lack of integrated sensors for real-time detection; (3) the need for special fabrication processes and the associated reliability/yield concerns.

To overcome the above limitations, DMFBs based on a micro-electrode-dot-array (MEDA) architecture have recently been proposed. Unlike conventional digital microfluidics, where electrodes of equal size are arranged in a regular pattern, the MEDA architecture is based on the concept of a sea-of-micro-electrodes. The MEDA architecture allows microelectrodes to be dynamically grouped to form a micro-component that can perform different microfluidic operations on the chip.

Design-automation tools can reduce the difficulty of MEDA biochip design and help to ensure that the manufactured biochips are versatile and reliable. In order to fully exploit MEDA-specific advantages (e.g., real-time droplet sensing), new design, optimization, and test problems are tackled in this dissertation.

The dissertation first presents a droplet-size aware synthesis approach that can configure the target bioassay on a MEDA biochip. The proposed synthesis method targets reservoir placement, operation scheduling, module placement, and routing of droplets of various sizes. An analytical model for droplet velocity is proposed and experimentally validated using a fabricated MEDA chip.

Next, this dissertation presents an efficient error-recovery strategy to ensure the correctness of assays executed on MEDA biochips. By exploiting MEDA-specific advances in droplet sensing, the dissertation presents a novel probabilistic timed automata (PTA)-based error-recovery technique to dynamically reconfigure the biochip using real-time data provided by on-chip sensors. An on-line synthesis technique and a control flow are also proposed to connect local-recovery procedures with global error recovery for the complete bioassay.

A potentially important application of MEDA biochips lies in sample preparation via a series of dilution steps. Sample preparation in digital microfluidic biochips refers to the generation of droplets with target concentrations for on-chip biochemical applications. The dissertation presents the first droplet size-aware and error-correcting sample-preparation method for MEDA biochips. In contrast to previous methods, the proposed approach considers droplet sizes and incorporates various mixing models in sample preparation.

In order to ensure high confidence in the outcome of biochemical experiments, MEDA biochips must be adequately tested before they can be used for bioassay execution. The dissertation presents efficient structural and functional test techniques for MEDA biochips. The proposed structural test techniques can effectively detect defects and identify faulty microcells, and the proposed functional test techniques address fundamental fluidic operations on MEDA biochips.

In summary, the dissertation tackles important problems related to key stages of MEDA chip design and usage. The results emerging from this dissertation provide the first set of comprehensive design-automation solutions for MEDA biochips. It is anticipated that MEDA chip users will also benefit from these optimization methods.

Integrated sensing systems are designed to address a variety of problems, including clinical diagnosis, water quality testing, and air quality testing. The growing prevalence of tropical diseases in the developing world, such as malaria, trypanosomiasis (sleeping sickness), and tuberculosis, provides a clear and present impetus for portable, low cost diagnostics both to improve treatment outcomes and to prevent the development of drug resistance in a population. The increasing scarcity of available clean, fresh water, especially noticeable in the developing world, also presents a motivation for low-cost water quality diagnostic tools to prevent exposure of people to contaminated water supplies and to monitor those water supplies to effectively mitigate their contamination. In the developed world, the impact of second-hand cigarette smoke is receiving increased attention, and measuring its effects on public health have become a priority. The `point-of-need' technologies required to address these sensing problems cannot achieve a widespread and effective level of use unless low-cost, small form-factor, portable sensing devices can be realized. Optical sensors based on low cost polymer materials have the potential to address the aforementioned `point-of-need' sensing problems by leveraging low-cost materials and fabrication processes. For portable clinical diagnostics and water quality testing in particular, on-chip sample preparation is a necessity. Electrowetting-on-dielectric (EWD) technology is an enabling technology for chip-scale sample preparation, due to its very low power consumption compared to other microfluidics technologies and the ability to move fluids without bulky external pumps. Potentially, these technologies could be combined into a cell phone sized portable sensing device.

Towards the goal of developing a portable diagnostic device using EWD microfluidics with an embedded polymer microresonator sensor, this thesis describes a viable fabrication process for the system and explores the design trade-offs of such a system. The main design challenges for this system are optimization of the sensor's limit-of-detection, minimization of the insertion loss of the optical system, and maintaining the ability to actuate droplets onto and off of the sensor embedded in the microfluidic system. The polymer microresonator sensor was designed to optimize the limit-of-detection (LOD) using SU-8 polymer as the bus waveguide and microresonator material and SiO2 as the substrate cladding material. The fabrication process and methodology were explored with test devices using a tunable laser system working around a wavelength of 1550 nm using glucose solutions as a refractive index standard. This sensor design was then utilized to embed the sensor and bus waveguides into an EWD top plate in order to minimize the impact of the sensor integration on microfluidic operations. Finally, the performance of the embedded sensor embedded was evaluated in the same manner and compared to the performance of the sensor without the microfluidic system.

The primary result of this research was the successful demonstration of a high performance polymer microresonator sensor embedded in the top plate of an electrowetting microfluidic device. The embedded sensor had the highest reported figure-of-merit for any microresonator integrated with electrowetting microfluidics. The embedded microresonator sensor was also the first fully-embedded microresonator in an EWD system. Because the sensor was embedded in the top plate, full functionality of the EWD system was maintained, including the ability to move droplets onto and off of the sensor and to address the sensor with single droplets. Furthermore, the highest figure-of-merit for an SU-8 microresonator sensor yet reported at a probe wavelength of 1550 nm was measured on a test device fabricated with the embedded sensor structure described herein. Optimization of the sensor sensitivity utilized recently developed waveguide sensor design theory, which accurately predicted the measured sensitivity of the sensors. Altogether, the results show that embedding of a microresonator sensor in an EWD microfluidics system is a viable approach to develop a portable diagnostic system with the high efficiency sample preparation capability provided by EWD microfluidics and the versatile sensing capability of the microresonator sensor.

Electrowetting-on-dielectric (EWD) digital microfluidics is a droplet-based fluid handling technology capable of radically accelerating the pace of genome engineering research. EWD-based laboratory-on-chip (LoC) platforms demonstrate excellent performance in automating labor-intensive laboratory protocols at ever smaller scales. Until now, there has not been an effective means of gene transfer demonstrated in EWD microfluidic platforms. This thesis describes the theoretical and experimental approaches developed in the demonstration of an EWD-enabled electrotransfer device. Standard microfabrication methods were employed in the integration of electroporation (EP) and EWD device architectures. These devices enabled the droplet-based bulk transformation of E. coli with plasmid and oligo DNA. Peak on-chip transformation efficiencies for the EP/EWD device rivaled that of comparable benchtop protocols. Additionally, ultrasound induced in-droplet microstreaming was developed as a means of improving on-chip electroporation. The advent of electroporation in an EWD platform offers synthetic biologists a reconfigurable, programmable, and scalable fluid handling platform capable of automating next-generation genome engineering methods. This capability will drive the discovery and production of exotic biomaterials by providing the instrumentation necessary for rapidly generating ultra-rich genomic diversity at arbitrary volumetric scales.

This thesis demonstrates a new way to achieve sparse biological sample detection, which uses magnetic bead manipulation on a digital microfluidic device. Sparse sample detection was made possible through two steps: sparse sample capture and fluorescent signal detection. For the first step, the immunological reaction between antibody and antigen enables the binding between target cells and antibody-­‐‑ coated magnetic beads, hence achieving sample capture. For the second step, fluorescent detection is achieved via fluorescent signal measurement and magnetic bead manipulation. In those two steps, a total of three functions need to work together, namely magnetic beads manipulation, fluorescent signal measurement and immunological binding. The first function is magnetic bead manipulation, and it uses the structure of current-­‐‑carrying wires embedded in the actuation electrode of an electrowetting-­‐‑on-­‐‑dielectric (EWD) device. The current wire structure serves as a microelectromagnet, which is capable of segregating and separating magnetic beads. The device can achieve high segregation efficiency when the wire spacing is 50µμm, and it is also capable of separating two kinds of magnetic beads within a 65µμm distance. The device ensures that the magnetic bead manipulation and the EWD function can be operated simultaneously without introducing additional steps in the fabrication process. Half circle shaped current wires were designed in later devices to concentrate magnetic beads in order to increase the SNR of sample detection. The second function is immunological binding. Immunological reaction kits were selected in order to ensure the compatibility of target cells, magnetic bead function and EWD function. The magnetic bead choice ensures the binding efficiency and survivability of target cells. The magnetic bead selection and binding mechanism used in this work can be applied to a wide variety of samples with a simple switch of the type of antibody. The last function is fluorescent measurement. Fluorescent measurement of sparse samples is made possible of using fluorescent stains and a method to increase SNR. The improved SNR is achieved by target cell concentration and reduced sensing area. Theoretical limitations of the entire sparse sample detection system is as low as 1 Colony Forming Unit/mL (CFU/mL).

Digital microfluidics is an emerging technology that provides fluid-handling capability on a chip. Biochips based on digital microfluidics have therefore enabled the automation of laboratory procedures in biochemistry. By reducing the rate of sample and reagent consumption, digital microfluidic biochips allow continuous sampling and analysis for real-time biochemical analysis, with application to clinical diagnostics, immunoassays, and DNA sequencing. Recent advances in technology and applications serve as a powerful driver for research on computer-aided design (CAD) tools for biochips.

This thesis research is focused on a design automation framework that addresses chip synthesis, droplet routing, control-pin mapping, testing and diagnosis, and error recovery. In contrast to prior work on automated design techniques for digital microfluidics, the emphasis here is on practical CAD optimization methods that can target different design problems in a unified manner. Constraints arising from the underlying technology and the application domain are directly incorporated in the optimization framework.

The avoidance of cross-contamination during droplet routing is a key design challenge for biochips. As a first step in this thesis research, a droplet-routing method based on disjoint droplet routes has been developed to avoid cross-contamination during the design of droplet flow paths. A wash-operation synchronization method has been developed to synchronize wash-droplet routing steps with sample/reagent droplet-routing steps by controlling the order of arrival of droplets at cross-contamination sites.

In pin-constrained digital microfluidic biochips, concurrently-implemented fluidic operations may involve pin-actuation conflicts if they are not carefully synchronized. A two-phase optimization method has been proposed to identify and synchronize these fluidic operations. The goal is to implement these fluidic operations without pin-actuation conflict, and minimize the duration of implementing the outcome sequence after synchronization.

Due to the interdependence between droplet routing and pin-count reduction, this thesis presents two optimization methods to concurrently solve the droplet-routing and the pin-mapping design problems. First, an integer linear programming (ILP)-based optimization method has been developed to minimize the number of control pins. Next an efficient heuristic approach has been developed to tackle the co-optimization problem.

Dependability is an important system attribute for microfluidic biochips. Robust testing methods are therefore needed to ensure correct results. This thesis presents a built-in self-test (BIST) method for digital microfluidic biochips. This method utilizes digital microfluidic logic gates to implement the BIST architecture. A cost-effective fault diagnosis method has also been proposed to locate a single defective cell, multiple

rows/columns with defective cells, as well as an unknown number of rows/columns-under-test with defective cells. A BIST method for on-line testing of digital microfluidic biochips has been proposed. An automatic test pattern generation (ATPG) method has been proposed for non-regular digital microfluidic chips. A pin-count-aware online testing method has been developed for pin-constrained designs to support the execution of both fault testing and the target bioassay protocol.

To better monitor and manage the execution of bioassays, control flow has been incorporated in the design and optimization framework. A synthesis method has been developed to incorporate control paths and an error-recovery mechanism during chip design. This method addresses the problem of recovering from fluidic errors that occur

during on-chip bioassay execution.

In summary, this thesis research has led to a set of unified design tools for digital microfluidics. This work is expected to reduce human effort during biochip design and biochip usage, and enable low-cost manufacture and more widespread adoption for laboratory procedures.