Single cell heterogeneity plays an important role in the onset and progression of a variety of disease pathologies. One of the most notable examples of the impact of heterogeneity in the complexity of a disease is cancer. Traditionally, molecular analyses on cancer-related samples have been performed on bulk populations of cells, with the resultant data only representative of an average of the population, thereby concealing potentially relevant information about individual cells. Performing these studies at the single cell level is proposed to address this issue. However, current methods for the isolation and analysis of single cells often require specialized and expensive equipment that may be prohibitive to labs wishing to perform such analyses. Herein, a method for the isolation and gene expression analysis of single cells is described that (1) relies only on readily available, inexpensive materials, (2) is compatible with phase and fluorescent microscopy, and (3) allows for the ability to track specific cells throughout all measurements. This method utilizes random seeding of single cells on 72-well Terasaki plates (also called microtest plates) that have 20 µl, optically clear flat-bottomed wells in order to circumvent the need for specific hardware for cell isolation. Suspensions of the Barrett’s esophagus epithelial cell line CP-D stably expressing turboGFP and a related, GFP-negative BE cell line, CP-A, were prepared, seeded at a concentration of approximately 1-2 cells/well and incubated overnight. Wells containing single cells were visually identified using phase-contrast and fluorescent microscopy. Single cells were then lysed directly in the well, total RNA was isolated, and RT-qPCR was performed. RT-qPCR results reflected the ability to distinguish between turboGFP-expressing and non-expressing cells that matched previous identification by microscopy. These results indicate that this is a convenient and cost-effective method for studying gene expression in single cells.

A major goal of the Center for Biosignatures Discovery Automation (CBDA) is to design a diagnostic tool that detects novel cancer biosignatures at the single-cell level. We designed the Single-cell QUantitative In situ Reverse Transcription-Polymerase Chain Reaction (SQUIRT-PCR) system by combining a two-photon laser lysis (2PLL) system with a microfluidic reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) platform. It is important to identify early molecular changes from intact tissues as prognosis for premalignant conditions and develop new molecular targets for prevention of cancer progression and improved therapies. This project analyzes RNA expression at the single-cell level and presents itself with two major challenges: (1) detecting low levels of RNA and (2) minimizing RNA absorption in the polydimethylsiloxane (PDMS) microfluidic channel. The first challenge was overcome by successfully detecting picogram (pg) levels of RNA using the Fluidigm (FD) BioMark™ HD System (Fluidigm Corporation, South San Francisco, CA) for RT-qPCR analysis. This technology incorporates a highly precise integrated fluidic circuit (IFC) that allows for high-throughput genetic screening using microarrays. The second challenge entailed collecting data from RNA flow-through samples that were passed through microfluidic channels. One channel was treated with a coating of polyethylene glycol (PEG) and the other remained untreated. Various flow-through samples were subjected to RT-qPCR and analyzed using the FD FLEXsix™ Gene Expression IFC. As predicted, the results showed that the treated PDMS channel absorbed less RNA than the untreated PDMS channel. Once the optimization of the PDMS microfluidic platform is complete, it will be implemented into the 2PLL system. This novel technology will be able to identify cell populations in situ and could have a large impact on cancer diagnostics.

Filtration for microfluidic sample-collection devices is desirable for sample selection, concentration, preprocessing, and downstream manipulation, but microfabricating the required sub-micrometer filtration structure is an elaborate process. This thesis presents a simple method to fabricate polydimethylsiloxane (PDMS) devices with an integrated membrane filter that will sample, lyse, and extract the DNA from microorganisms in aqueous environments. An off-the-shelf membrane filter disc was embedded in a PDMS layer and sequentially bound with other PDMS channel layers. No leakage was observed during filtration. This device was validated by concentrating a large amount of cyanobacterium Synechocystis in simulated sample water with consistent performance across devices. After accumulating sufficient biomass on the filter, a sequential electrochemical lysing process was performed by applying 5VDC across the filter. This device was further evaluated by delivering several samples of differing concentrations of cyanobacterium Synechocystis then quantifying the DNA using real-time PCR. Lastly, an environmental sample was run through the device and the amount of photosynthetic microorganisms present in the water was determined. The major breakthroughs in this design are low energy demand, cheap materials, simple design, straightforward fabrication, and robust performance, together enabling wide-utility of similar chip-based devices for field-deployable operations in environmental micro-biotechnology.