Theses and Dissertations at Montana State University (MSU)
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Item Development of drop-based microfluidic methods for high-throughput biological assays(Montana State University - Bozeman, College of Engineering, 2021) Zath, Geoffrey Kane; Chairperson, Graduate Committee: Connie Chang; This is a manuscript style paper that includes co-authored chapters.Drop-based microfluidics allows single-cell biological assays to be performed by encapsulating samples in picoliter scale drops. Adapting biological assays to drop-based microfluidics requires novel approaches to meet the method requirements of each assay. For example, microtiter plates are a common tool for storing many unique samples in some assays. An equivalent strategy for drops involves labeling samples with a barcode prior to drop encapsulation and storing the barcoded drops in a single mixture, thereby creating a drop library. Other assay adaptions, such as drop-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) require that drops be stabilized during the high temperatures used for thermal cycling. Drop-based RT-qPCR is useful for studying single-cell dynamics in drops, such as influenza A virus (IAV) infection. Conventional methods for measuring IAV output from individual cells are labor intensive and low-throughput. Thus, there is a need to adapt RT-qPCR to drop-based microfluidics for the purpose of high-throughput single cell analysis of infected cells. The research presented here focuses on the characterization of the Pressure Cooker Chip (PCC) to rapidly encapsulate drop libraries and the development of a drop-based RT-qPCR method to measure IAV output from infected cells. The PCC was used to make drop libraries by rapidly generating drops of up to 96 different conditions in parallel by interfacing individual drop makers with a standard microtiter well plate. The drop library was optically barcoded using a two-color combination of fluorescent microbeads or quantum dots with 24 or 192 unique combinations, respectively. To adapt RT-qPCR in drops, known PCR additives were systematically tested to optimize drop stability and limit dye diffusion during thermocycling. A novel qPCR data analysis method was developed to convert drop fluorescence data collected at a single thermocycle to an initial RNA template concentration. Together, the additive screening and novel qPCR data analsyis method enabled the use of drop-based RT-qPCR to quantify the highly heterogeneous IAV burst size from single cells in thousands of drops. Our method is the first to measure single cell IAV burst size using a high-throughput, drop-based RT-qPCR assay.Item A high-throughput, multiplexed microfluidic method utilizing an optically barcoded drop library(Montana State University - Bozeman, College of Engineering, 2016) Zath, Geoffrey Kane; Chairperson, Graduate Committee: Connie ChangThe power of drop-based microfluidics promises reduced biological assaying times and greater sample throughput; however, current drop-based microfluidic methods focus on single-input single-output techniques to provide these benefits. In order to achieve truly high-throughput analysis of biological assays, a multiple-input approach must be taken. This thesis is focused on developing and validating a drop-based microfluidic method that is capable of encapsulating, in parallel, 96 assay samples in drops and optically tracking them in a barcoded drop library. The advantage of the method presented here is its ability to be integrated with current biological assays performed on a 384-well plate. The first step was to fabricate a three-dimensional microfluidic device capable of accepting 96 sample inputs. Second, formation of drops within the device was characterized by creating a state diagram using Capillary and Weber numbers of the two phase flow. Finally, the use of fluorescent microbeads was investigated for the purpose of optically barcoding drops. A barcoding scheme was developed to allow for fluorescent and spatial labeling of 96 wells of a 384-well plate. The three-dimensional microfluidic device was successfully used to encapsulate 50 microns diameter drops from 24 wells barcoded with fluorescent microbeads at a drop formation rate of 3 kHz per well. Fluorescent detection of the barcoded drop mixture was performed at a rate of 200 Hz and density-based clustering algorithm DBSCAN was used to identify barcoded drop clusters from the fluorescent signal data. Validation of this method was achieved by adding known concentrations of fluorescent blue microbeads to barcoded wells and detecting for their presence in barcoded drop clusters. The barcoding method can be expanded to fully incorporate the 96 inputs of the microfluidic device by adding a spatial barcoding component to each quadrant of 24 optically barcoded wells. The results presented here show the microfluidic platform has the potential to be a useful tool in biological assays involved with tracking a large number of samples in a well plate format.Item A study of the East Gallatin River, Montana, using an algal bioassay (batch method) and some problems encountered(Montana State University - Bozeman, College of Agriculture, 1971) Griffin, Daniel PatrickItem Estimating toxicity curves by fitting a compartment-based model to median survival times(Montana State University - Bozeman, College of Letters & Science, 1982) Chew, Robert D.