Browsing by Author "Fair, Richard B"
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Item Open Access Accelerated Sepsis Diagnosis by Seamless Integration of Nucleic Acid Purification and Detection(2014) Hsu, BangNingBackground 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.
Item Open Access Digital Microfluidics for the Detection of Inorganic Ions in Aerosols(2018) Huang, ShuquanThe quantitative measurement of inorganic ions in the atmosphere is an important aspect in environmental science. The three most important inorganic ions are sulfate, nitrate and ammonium, which are the most abundant components of atmospheric pollutants and have a significant impact on rainfall, atmospheric visibility and human health. To accurately and quickly measure the distribution of inorganic ions in the vertical and horizontal directions of the atmosphere, a compact and automatic real-time detection system is in need.
The research performed in this study is aimed at developing the science and technology for an aerosol detection system that combines digital microfluidics technology, aerosol impaction and chemical detection on the same chip. The system will be smaller and faster with respect to current aerosol analyzing instruments. The chip in this study performs the integrated functions of aerosol collection, extraction, and quantitative detection in real-time, unlike current benchtop methods that require operator handling and laboratory equipment. All functions are realized in dedicated sections on a digital microfluidic platform.
This thesis will present the design and test of individual components of the aforementioned functions. The digital microfluidics chip design includes transparent top and bottom plates for light absorbance measurement. The droplets are dispensed, transported and mixed on chip with other droplets by activating electrodes individually with a 50V AC sine voltage.
In Chapters 3 and 4, the issues involving droplet transportation are addressed, including droplet movement between the air and silicone oil media and droplet transport across the aerosol impaction area. Next, an aerosol impactor and a chip-to-world chamber are demonstrated and tested with lab generated sulfate aerosol. The collected aerosol showed a clear pattern on the impaction plate, and the collection efficiency inside the chip was 96%.
In Chapter 5, the development of colorimetric methods are described as well as experimental testing for inorganic ion detection. Three well-known tests for detecting sulfate, nitrate and ammonium were first adjusted to adapt to on-chip measurement conditions, the adjustments including the choices of solvent, concentration ranges and mixing ratios. The particle measurement results using a conventional spectrometer were compared with on-chip measurements in terms of absorbance range, limit of detection, sensitivity (based on the coefficient of determination and the slope of the linear regression) and signal-to- noise ratio (presented with standard deviation/average of absorbance measurements).
The thin oil film between the droplet and the top/bottom plate, which is naturally formed, plays an important role in lubrication and reduces contact angle hysteresis. However, these oil films are not always uniform in thickness. During the absorbance measurement tests, varied sizes of oil lenses were observed at the oil/top plate interface, and the size and position of the oil lenses randomly changed when a droplet moved between electrodes. The absorbance measured in the normal direction to the chip’s surface was affected by these oil lenses and, thus, not stable for multiple measurements of the same droplet or for different droplets. To solve this problem, optical fibers were introduced horizontally inside the chip, and measurements taken in this direction proved to produce stable results.
Prototypes of the chip have been fabricated, and the impaction and on-chip colorimetric tests for sulfate and ammonium were successful. Although this study was designed to build the fundamentals of a novel detection system of inorganic ions in aerosol, the potential use of the designed system is not limited to atmospheric studies. Applications can extend to testing the quality of drinking water, detection of nitroaromatic explosives or other experiments based on colorimetry.
Item Open Access Enhanced Biomolecular Binding to Beads on a Digital Microfluidic Device(2022) Preetam, ShrutiDigital microfluidic (DMF) technology is being utilized for commercial applications such as point-of-care diagnostics, sample processing and genomic library preparation. Advances in this field offer exciting possibilities into immunoassays, enzymatic analysis, and next-generation sequencing. However, typical biomolecular protocols performed in laboratories are far more complex, requiring large reagent volumes, long processing times and provide a low throughput analysis. Using a DMF platform enables overcoming experimental barriers of manual laboratory protocol execution, allowing for scaling the platform geometry, assay times, volumes of reagents used, and minimizing the use of external mechanical equipment. The DMF platform also allows for easy integration with detection systems enabling real-time data analysis and efficient resource allocation. This thesis explores theoretical and experimental approaches for carrying out enhanced biomolecular binding to magnetic beads on a DMF platform as part of a sample preparation protocol. Different DMF prototypes were designed using standard microfabrication procedures to compare passive mixing, active mixing on electrode arrays and local bead actuation on an integrated current-wire electrode, whereby current is sequenced through copper wires fabricated on the electrode to generate magnetic-fields on-chip that cause the magnetic beads in the assay to move relative to the antibody. By making use of an integrated fluorescence detection system, the binding efficiency for each of these approaches is determined. The current-wire device design proves to be a valuable tool in creating an integrated DMF system to carry out intensive bead binding in an assay allowing for lower reagent volumes, shorter assay times and reduced surface area, thus impacting device yield.
Item Open Access Fluorescent Detection of Chromatin using Functionalized Magnetic Beads on a Digital Microfluidic Device(2022) Bigdeli, YaasEpigenetics is the study of inheritable mechanisms and factors that regulate gene expression. Although the underlying genetic sequence is the same in every cell, it is the epigenome that controls the expression of these genes and accounts for differences in phenotype. Epigenetic controls have clinical ramifications from cancer to autoimmune disorders to psychiatric pathologies. The main tool to study epigenetics is chromatin immunoprecipitation (ChIP), which probes the relationship between the underlying DNA and its structural histone proteins. Standard benchtop ChIP has five major drawbacks: (1) it requires a large input volume of cells, (2) it is very time consuming, work intensive, and low throughput, (3) it suffers from poor chromatin yield and sensitivity, (4) ChIP antibodies can be non-specific, vary by batch, and have low sensitivity, (5) and ChIP performs bulk tissue analysis which loses the granularity necessary to detect cell-to-cell variations. Digital microfluidic biochips (DMFBs) have proven successful at utilizing small volumes of reagents and samples to perform high throughput analyses using a variety of assaying techniques, making them an ideal platform for ChIP adaptation. Droplet manipulation using electrowetting-on-dielectric, in conjunction with magnetic bead control using magnetic field gradients generated by a current running through a wire on the device, provide all the necessary functionality to successfully run ChIP more efficiently on a DMFB. Translation of the benchtop ChIP protocol onto a DMFB addresses the issues facing epigenetic study workflow. The smaller volumes reduce reaction time, decrease reagent and sample use, and increase sensitivity and granularity towards single-cell resolution. Automation makes ChIP less labor consuming. DMFB platforms can be expanded for parallel operation and multiplexing thus increasing throughput. Finally, streamlining all the steps of ChIP onto one device greatly reduces sample loss, thereby expanding the type of studies possible. Herein, specifically modified nucleosomes and human chromatin were detected in a new semi-quantitative fluorescent immunoassay on a DMFB. Furthermore, chromatin was immunoprecipitated using a new targeted biotinylated technique. Successful chromatin capture and detection is a powerful tool for ChIP protocol development. This approach provides a rapid method to screen for antibody specificity and sensitivity as well as a confirmatory check point in the overall ChIP protocol to ensure that the target analyte has been isolated prior to any downstream analyses. Finally, a new modified ‘pull-through’ DMFB design was introduced to enhance the capture and detection of analyte-bound magnetic beads. The contributions from the studies described in this dissertation have provided the first steps towards ChIP implementation on a DMFB: 1) Developed new fluorescent confirmatory chromatin and nucleosome immunoprecipitation assays.
2) Demonstrated that the immunoprecipitation assays were detectible on-chip without any complex downstream analyses nor specialized fluoroscopy instrumentation.
3) Demonstrated that the immunoprecipitation assays performed at higher sensitivity than traditional benchtop ChIP.
4) Developed a single-channel pixel intensity measurement system for semi-quantitative analysis of chromatin and post-translationally modified nucleosomes directly on-chip.
5) Designed a new DMFB for improved capture of magnetic beads with twice the measured signal intensity using a new pull-through droplet scan method with on-chip embedded magnetic controls.
Item Open Access Low Voltage DNA Sequencing Platform Utilizing Picofluidic Electrowetting Devices(2011) Lin, YanYouDigital microfluidics as implemented in electrowetting-on-dielectric (EWD) technology has been widely used as a platform for miniaturizing the biomedical or biochemical laboratory on a chip in recent years. DNA pyrosequencing, one of the DNA sequencing-by-synthesis methods, has been successfully integrated on EWD devices. However, this platform requires microliters of reagents and 200~300V of applied voltages, which contributes to higher costs and limits the feasibility of a portable system. This dissertation proposes a low voltage EWD device using multi-layer insulators that can manipulate picoliter droplets on chip. A 300pl droplet was dispensed and actuated at voltages as low as 11.4Vrms and 7.2Vrms respectively on a 95um electrode a EWD device with a 20um SU8 gasket. The stacked insulators in the actuator consisted of 135nm tantalum pentoxide (Ta2O5) and 180nm parylene C films deposited and coated with 70 nm of CYTOP. The physical scaling of electrodes was further demonstrated for 33um and 21um electrode devices, resulting in droplets of 12pl and 5pl respectively in conjunction with 3um gaskets. Manipulation of magnetic beads during dispensing, droplet splitting and merging, and droplet transport were also demonstrated on the scaled EWD devices. The chemiluminescent light produced by the on-chip reaction of 100pl ATP-luciferin and luciferase could be detected with an external cooled CCD camera, but detecting this reaction with smaller-scale droplet reactions was limited by the external detector's sensitivity. Based on fundamental theories and experiments, the actuation voltage and dimensional scaling of EWD devices have been demonstrated, but the use of picoliter droplets in biochemical applications will required improved sensing methods.
Item Open Access Scalable Genome Engineering in Electrowetting on Dielectric Digital Microfluidic Systems(2015) Madison, Andrew CaldwellElectrowetting-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.
Item Open Access Sparse Sample Detection Using Magnetic Bead Manipulation on a Digital Microfluidic Device(2016) Chen, LijiThis 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).