Microfluidic Immunoblotting using Multi-Purposed Soft Materials

The miniaturization of biological assays for basic science and clinical diagnostics is a strong focus of the microfluidics field. Substantial impacts on assay time, throughput, and sample requirement, for example, can be achieved through integrating process workflows for the detection of protein analytes. Yet to permeate the broader scientific community, a strong focus on simplified device architectures, modularity, and adaptability should be at the forefront of endeavors in the microfluidics field./nHere, I describe thesis contributions covering a number of embodiments of a microfluidic immunoblotting toolbox. Central to the themes of each facet of the toolbox are the integration of assay stages from separations to quantitative protein analyte detection using microfabricated polymer structures. These structures immobilize proteins after weight or charge-based separations from complex clinical samples or cell lysates, preserving separation resolution for often protracted immunoprobing or activity-based detection stages./nAfter describing methods to separate, immobilize, and assay for enzyme activity within polyacrylamide gradient gels, I describe the development of photoactive polyacrylamide matrices that yield rapid, highly efficient capture of separated protein bands upon the application of UV light with performance that is robust to a wide range of assay conditions./nThese materials form the basis of microfluidic immunoblotting methods based on charge and size separations, culminating in microfluidic western blotting architectures that support analysis of low-zeptomole quantities of target proteins from cell lysates, blood sera, and single cells in integrated, automated assay workflows complete in tens of minutes./nAmong specific contributions of this thesis are, firstly, the discovery and characterization of an isoelectric point photoswitching phenomenon in green fluorescent protein variants from the jellyfish Aequorea victoria in response to different wavelengths of light that may have applications in the engineering of biomimetic smart materials with light-actuated transitions in zeta potential, hydrophilicity / wetting behavior, and adhesion properties./nSecondly, I describe an immunoprobed isoelectric focusing technology that quantifies the isoforms of the prostate cancer biomarker prostate specific antigen in less than 120 min with detection limits in the low pg./nFinally, I describe two variants of an integrated microfluidic western blotting assay that reduce 6-12 hr analysis times of traditional techniques to as little as 10 min, with detection sensitivities as low as 104-105 molecules. The first is applied to the confirmatory testing of HIV+ status in clinical patient serum, addressing the traditional western blotting bottleneck in infectious disease diagnostics. The second is a single-cell western blotting technique in a standard microscope slide format that has the hallmarks of design for application and uptake in a range of life science fields. This microarray-like tool leverages timescale separation at the microscale to link lysis, separation, capture, and immunoblotting of the protein contents of more than 103 single cells per assay. I detail the nascent application of this exciting addition to the rather limited single-cell proteomics pipeline to neural stem cells in a case study of cell-to-cell heterogeneity over signaling and differentiation timescales.

Advisor: Herr, Amy E