Microfluidic Bioanalytical Device and Assay Development for High-Throughput Electrophoretic Protein Analysis
- Author(s): Duncombe, Todd Andrew
- Advisor(s): Herr, Amy E
- et al.
Seminal bioanalytical technologies for high-throughput analysis, such as flow cytometry and capillary electrophoresis, were leveraging microfluidic physical phenomena long before the advent of the term “microfluidics”. Transitioning from the initial solid-state micro-electronic fabrication approaches, microfluidic fabrication has moved towards polymer based technologies that are amenable to a rapid design, prototype, and test development cycle. In my dissertation, I took advantage of these features to create new tools for performing electrophoresis-based protein assays over a range of applications, including, rapid low-power electrophoretic immunoassays, open-microfluidic ‘soft-MEMS’ platform for high-throughput protein analysis, and spatially & temporally controlled separation media for enhanced single-cell western blotting assays.
Rapid low-power electrophoretic immunoassay: To reduce the power requirements for a portable electrophoresis platform, we developed a new assay format that minimized the separation length to 1 mm for an electrophoretic immunoassay. The polyacrylamide gel moving boundary electrophoresis (PAGMBE) assay consumed just 3 μW and was completed in less than 30 seconds using only a 9 V battery - the lowest voltage reported for an electrophoretic protein separation.
Open-microfluidic ‘soft-MEMS’ platform: We developed an open-channel hydrogel architecture for rapid protein analysis. Directly photo-patterned free-standing polyacrylamide gel (fsPAG) microstructures support electrophoretic performance rivalling that of microfluidic platforms while maintaining easy interfacing with automated robotic controllers and downstream processing (e.g., sample spotters, immunological probing). We demonstrated 96 concurrent SDS protein fsPAGE separations in under 5 minutes.
Spatially varied separation media: Grayscale mask photo-patterning of gel density realized periodic 1 mm gradient gels arrays for single cell analysis. These dense arrays with a spatially varied sieving medium enabled concurrent protein sizing for 1000s of single cells in parallel. We demonstrated the single cell gradient gel western blotting to analyze multiple targets in the Her2 signaling pathway in a primary breast cancer tissue that varied by an order of magnitude in molecular weight. This broad-range protein sizing 1 mm gradient gel condition corresponds to the molecular weight range for 78% of the human proteome.
Pore-expansion for enhanced intra-gel assays: Protein separations in dense sieving media followed by in-gel immobilization enable high performance separations and minimal protein loss. This assay integration is necessary for processing low abundance samples – for example the protein content of a single cell. Unfortunately, for dense sieving medium prevents easy access for downstream assays on the immobilized proteins. We developed an active hydrogel matrix that could maintain a dense form to achieve a high-resolution protein separation and immobilization, then after a simulation, could transition to a lower-density form that is more accessible to antibody probing and the delivery of other large-reagents.
In summary, this dissertation focused on high-throughput protein analysis platforms that are designed towards the goal of massively parallelized proteomics, a major unrealized goal from bioanalytical technology.