Automated Digital Microfluidic Platform for Magnetic-Particle-Based Immunoassays with Optimization by Design of Experiments

Abstract

We introduce an automated digital microfluidic (DMF) platform capable of performing immunoassays from sample to analysis with minimal manual intervention. This platform features (a) a 90 Pogo pin interface for digital microfluidic control, (b) an integrated (and motorized) photomultiplier tube for chemiluminescent detection, and (c) a magnetic lens assembly which focuses magnetic fields into a narrow region on the surface of the DMF device, facilitating up to eight simultaneous digital microfluidic magnetic separations. The new platform was used to implement a three-level full factorial design of experiments (DOE) optimization for thyroid-stimulating hormone immunoassays, varying (1) the analyte concentration, (2) the sample incubation time, and (3) the sample volume, resulting in an optimized protocol that reduced the detection limit and sample incubation time by up to 5-fold and 2-fold, respectively, relative to those from previous work. To our knowledge, this is the first report of a DOE optimization for immunoassays in a microfluidic system of any format. We propose that this new platform paves the way for a benchtop tool that is useful for implementing immunoassays in near-patient settings, including community hospitals, physicians' offices, and small clinical laboratories.

Digital Microfluidic Platform for the Detection of Rubella Infection and Immunity: A Proof of Concept

“…We prepared custom digital microfluidic compatible wash buffer and conjugate diluent as described previously (32,33 ). Conjugate working solutions for RV IgG or RV IgM assays were formed by diluting horseradish peroxidase (HRP)-conjugated goat polyclonal antihuman IgG (21 ng/mL) or HRP-conjugated goat antihuman IgM (23 ng/mL), respectively, in conjugate diluent.…”

“…1A) (30,32 ). Digital microfluidic devices, comprising a top plate and bottom plate, were fabricated in the University of Toronto Nanofabrication Centre cleanroom facility and assembled as described previously (32 ) (Fig.…”

“…For on-chip particle separation, a motorized magnet system called a magnetic lens (32 ) is positioned approximately 150 m underneath the device. In this activated state, Ͼ600 N of magnetic force is sufficient to focus particles into a pellet, immobilizing them on the surface ( Fig.…”

“…1C; also see Supplemental Video 1, which accompanies the online version of this article at http:// www.clinchem.org/content/vol61/issue2). With digital microfluidic actuation, the supernatant can be separated from the particles, provided that the immobilization force exceeds the minimal threshold (approximately 500 N) required to overcome the surface tension of the droplet (32,33 ). The particle pellet is resuspended by deactivating the magnetic lens (i.e., magnet is positioned 3.8 cm below the device) and moving and mixing a droplet over the particles (Fig.…”

“…DMF devices can be cost-effectively fabricated on paper by inkjet printing (29 ) and can be operated with simple and compact instrumentation (30,31 ) with no need for pumps, interconnects, valves, or fittings. Although these advantages have motivated the development of DMF-powered immunoassays for smallmolecule and protein biomarkers (32)(33)(34)(35), we are not aware of DMF immunoassays being used for diagnosis of infectious diseases.…”