RESUMEN
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.
RESUMEN
We introduce a new format for particle-based immunoassays relying on digital microfluidics (DMF) and magnetic forces to separate and resuspend antibody-coated paramagnetic particles. In DMF, fluids are electrostatically controlled as discrete droplets (picoliters to microliters) on an array of insulated electrodes. By applying appropriate sequences of potentials to these electrodes, multiple droplets can be manipulated simultaneously and various droplet operations can be achieved using the same device design. This flexibility makes DMF well-suited for applications that require complex, multistep protocols such as immunoassays. Here, we report the first particle-based immunoassay on DMF without the aid of oil carrier fluid to enable droplet movement (i.e., droplets are surrounded by air instead of oil). This new format allowed the realization of a novel on-chip particle separation and resuspension method capable of removing greater than 90% of unbound reagents in one step. Using this technique, we developed methods for noncompetitive and competitive immunoassays, using thyroid stimulating hormone (TSH) and 17ß-estradiol (E2) as model analytes, respectively. We show that, compared to conventional methods, the new DMF approach reported here reduced reagent volumes and analysis time by 100-fold and 10-fold, respectively, while retaining a level of analytical performance required for clinical screening. Thus, we propose that the new technique has great potential for eventual use in a fast, low-waste, and inexpensive instrument for the quantitative analysis of proteins and small molecules in low sample volumes.