Development of Digital Mocrofludic Heterogeneous Immunossays

Digital microfluidics (DMF) is an emerging liquid-handling technique in which picoliter to microliter-sized droplets are manipulated on a hydrophobic surface. The most common DMF systems actuate droplets through the application of electrical potentials on a generic (m × n) array of insulated electrodes. The generic geometry and simple actuation scheme allows users to adopt a "programming" approach to microfluidics, calling a series of "functions" comprising various combinations of droplet operations (e.g. metering from reservoirs, splitting, merging, and mixing). While this configuration is perfect for implementing homogeneous chemical and enzymatic reactions, this format is not an obvious match to applications that require functionalized surfaces. In particular, heterogeneous immunoassays, which require a solid support to immobilize antibodies or antigens, represent an important class of tests that can benefit from miniaturization and automation. Thus, this thesis describes the development, optimization, and validation two new digital microfluidic methods for performing heterogeneous immunoassays. The first method enables efficient on-chip separation and resuspension of magnetically activated particles. While previous embodiments of DMF/magnetic separations have been limited by the particular format used (including suspension in immiscible oils), the method developed here was much more flexible, and was found to be very efficient at removing unbound reagents in the supernatant. The method was validated by application to quantifying a wide range of different analytes, including thyroid-stimulating hormone, 17 beta-estradiol, and rubella virus immunoglobulins. Based on this technique, we designed and built a shoebox-sized integrated instrument capable of performing immunoassays from sample-to-analysis with minimal manual intervention. The diagnostic performance of this system was evaluated using serum and plasma samples from a commercial anti-rubella mixed titer performance panel, yielding 100% sensitivity and specificity. The second method was given the moniker, "digital microfluidic immunocytochemistry in single-cells" (DISC), which can stimulate and fix adherent cells for single-cell immunoassays. The use of DMF facilitated rapid stimulation and fixation of cells, enabling the interrogation protein phosphorylation in the seconds to minutes timescale. Fixed and stained cells on the device were scanned using a microarray reader, which has the ability to resolve signaling networks in heterogeneous cell populations. Using this technique, we interrogated the phosphorylation state of PDGFR and Akt in NIH 3T3, MCF-7, and MDA-MB-231 cell lines resulting from various time, frequency, and concentration dependent PDGF-BB stimulation. These studies revealed detailed kinetic behaviour and population heterogeneity of PDGFR stimulation, and uncovered a possible Akt-dependent relationship to breast cancer cell aggressiveness. Unlike flow cytometry, this technique evaluates and visualizes adherent cells in situ, precluding the need for disruptive disassociation protocols. We propose that the new methods described in this thesis may eventually become useful for disease surveillance and diagnostics, and life science research.

Keywords: Cell-based assay
digital microfluidics
electrowetting-on-dielectric
Immunoassay
Immunocytochemistry
Lab-on-a-chip
Advisor: Wheeler, Aaron R