A DIGITAL MICROFLUIDIC PLATFORM FOR AUTOMATED IMMUNOASSAYS OPTIMIZED USING “DESIGN OF EXPERIMENTS” (DOE) METHODS Kihwan Choi 1 *, Alphonsus H.C. Ng 2 , Ryan Fobel 2 , David A. Chang-Yen 3 , Lyle E. Yarnell 3 , Elroy L. Pearson 3 , Carl M. Oleksak 3 , Andrew T. Fischer 3 , Robert P. Luoma 3 , John M. Robinson 3 , and Aaron R. Wheeler 1,2 1 Department of Chemistry, University of Toronto, Canada 2 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Canada 3 Abbott Diagnostics, USA ABSTRACT We introduce an automated digital microfluidic platform capable of performing immunoassays from sample-to- analysis with minimal manual intervention. This platform features a 90 Pogo pin interface, an integrated photomultiplier tube, and an adjustable magnet. The new platform was used to implement a design of experiments optimization for immunoassays, resulting in an optimized protocol that reduced detection limit and sample incubation time by up to 5-fold and 2-fold, respectively, relative to previous work. We propose that this new platform paves the way for a benchtop tool that is useful for implementing immunoassays in near-patient settings around the world. KEYWORDS: Digital Microfluidics, Immunoassay, Magnetic Separation, Design of Experiments INTRODUCTION The in-vitro diagnostic industry is dominated by robotic immunoanalyzers—the gold standard for high-throughput protein and small molecule quantitation [1]. These instruments are capable of quantifying disease biomarkers from patient samples at clinically relevant concentrations at a rate of hundreds of tests per hour. Importantly, the throughput of these instruments allows developers to rapidly determine optimal assay parameters via a Design of Experiments (DOE) approach, leading to reduced assay development and optimization timelines. Unfortunately, robotic immunoanalyzers are large, complex instruments found only in well-funded centralized facilities such as hospital reference laboratories, to which patient samples are transported after collection. As health care costs continue to rise, the global in-vitro diagnostic market is gradually shifting from centralized facilities to point of care testing. This market trend is facilitated by technological advances in nanomaterials, integrated sensors, and microfluidics. In particular, microfluidics is proving useful for the miniaturization of liquid handling, leading to the development of various microfluidic immunoassay systems and the commercialization of these platforms. Digital microfluidics (DMF), a technique in which fluids are manipulated as discrete droplets on devices bearing an array of electrodes buried under an insulating dielectric [2], is a promising format for immunoassays. Recent work has seen DMF applied to implementing immunoassays in a format using an oil carrier fluid [3] and in an oil-free format [4]. Here, we report a significant advance over the state-of-the-art for DMF immunoassays, featuring three new characteristics: complete sample-to-analysis automation, parallel sample processing, and full factorial DOE optimization. The latter is a particularly significant advance; DOE is becoming increasingly important for maximizing information output from minimum experimental effort [5]. To date, however, there have been no reports of DOE optimization of microfluidic immunoassays (of any format), likely because of a lack of automation, parallelization, and control. We propose that this report will be a useful touchstone for future work applying microfluidic DOE to a wide range of applications. EXPERIMENTAL Device fabrication and operation Digital microfluidic devices were formed by standard photolithography and wet etching as described previously [4]. The device design features an array of 80 actuation electrodes (2.2 × 2.2 mm ea.) connected to 8 reservoir electrodes (16.4 × 6.7 mm ea.) with inter-electrode gaps of 30-80 µm. Devices were assembled with an unpatterned ITO–glass top- plate and a patterned bottom-plate separated by a spacer formed from two pieces of double-sided tape (total spacer thickness 180 µm). Unit droplet and reservoir droplet volumes on these devices were ~800 nL and ~3.5 µL, respectively. An automated platform was designed and built to manage droplet operation, magnet and photomultiplier tube (PMT) position, and data collection. Droplet movement was controlled via the open-source Microdrop software and an Arduino- based (Smart Projects, Italy) high-voltage switching instrument described in detail elsewhere [6]. A custom plugin for the Microdrop software was used to control the motors in the platform, read signals from their respective optical limit switches, and trigger PMT reading. To drive droplet movement, an AC sine wave (100-120 V RMS , 10 kHz) was applied between the top-plate (ground) and sequential electrodes on the bottom-plate. 978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 491 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences 27-31 October 2013, Freiburg, Germany