Novel microfluidic tools & theoretical fundamentals: a synergistic approach for untangling proteomics

With the ever accumulating understanding of the complexity of biological systems, the design of novel research tools is becoming increasingly important for critical advances in biological and medical technology. Innovation is required not only in the development of better alternatives to existing assays, which are plagued by issues such as low throughput and low reproducibility, but also for the creating of completely new tools that can lead to biological inquiry not previously accessible. Theoretical understanding of mass transport and analytical chemistry not only guides the optimal design of highly efficient devices, but also enables insight of the fundamental processes underlying the system being studied. Accordingly, we have paired a strong understanding of theory with the precise material transport afforded by microfluidics to address technological gaps in a wide range of topics relevant to biological research./nUtilizing rational device design and simulations for precise electric field control and polyacrylamide photopatterning to control local chemical conditions, we developed a microfluidic version of the traditional two-dimensional electrophoresis (2DE) assay. 2DE yields the isoelectric point and molecular weight of the components in a sample and is therefore an extremely powerful analytical tool for the study of proteins, yet it is underutilized due to its lack of reproducibility, long run times (up to a day), and the high expertise required perform the assay. Prior attempts to develop microfluidic alternatives have had difficulties integrating the two distinct separations that constitute the assay: isoelectric focusing (IEF) for charge-based separations and molecular sizing. By employing a microchamber geometry and using polyacrylamide gels to localize the chemical conditions required for IEF and molecular sizing, we were able to perform 2DE separations under one hour./nOne of the challenges about IEF separations is that once proteins reach their isoelectric point, they have no electrophoretic mobility and therefore these focused proteins are difficult to transfer for subsequent separations, analysis, and detection. One approach to mobilize focused proteins is chemical mobilization, in which ions are introduced into the separation column in order to disrupt the local pH gradient required for IEF. While functional, chemical mobilization introduces distortions and information losses to the separation. The degree and the mechanisms of these losses as well as which conditions would minimize the losses, were not well understood. Given this knowledge gap, we performed a study leveraging the rapid and reproducible separations possible in microfluidics in conjunction with a theoretical model to further understand this chemical mobilization process./nThis synergistic approach also led to insight into the biophysics of green fluorescent protein photoswitching. Green fluorescent protein (GFP) is utilized ubiquitously in biological research, and yet its ability to switch isolectric points when stimulated with different wavelengths of light had not been described. We performed real-time imaging of GFP during microfluidic IEF with benzophenone-modified polyacrylamide gels to enable covalent protein capture and immunoprobing. By interpreting the results within the framework of a mass transport and reaction theoretical model we were able to describe the photoswitching process./nSimulations and experiments were used to quantify the performance of degas-driven flow in polydimethylsiloxane (PDMS) microchannels. Future device design can be guided by the theoretical model developed. Electrophoresis simulations informed assay design for microfluidic separations of functional riboswitches and non-equilibrium electrophoresis and IEF of proteins denatured with urea./nThe described examples showcase how theory in conjunction with novel tools can lead to insight in a wide variety of topics. In more recent research, we have worked on developing a platform for performing proteomic analysis from the contents of individual mitochondria. Mitochondrial heterogeneity and dysfunction are implicated in aging and various neurodegenerative diseases, and thus we hope our work will address a critical technological gap that could lead to breakthroughs in our understanding of these diseases and the development of new therapies. Finally, theoretical understanding of IEF was utilized for designing assays for IEF separations from single cells. We see these advances as forming a new design paradigm for automated multi-stage assays with relevance spanning from fundamental biological inquiry to medical research.

Advisor: Herr, Amy E