Volumeless reagent delivery: a liquid handling method for adding reagents to microscale droplets without increasing volume

The addition of reagents for assays in digital microfluidic (DMF) systems is traditionally done by merging of droplets containing different analytes or reagents in solution. However, this process significantly increases droplet volume after each step, resulting in dilution of the analyte and reagents. Here, we report a new technique for performing reagent additions to aqueous droplets without significantly increasing the droplet's volume: volume-less reagent delivery (VRD). VRD is enabled by a physical phenomenon we call "exclusive liquid repellency" (ELR), which describes an aqueous/oil/solid 3-phase system where the aqueous phase can be fully repelled from a solid phase (contact angle ∼180°). When performing VRD, a reagent of interest in solution is deposited onto the ELR solid surface and allowed to dry. The ELR surface containing the dried reagent is then immersed under oil, followed by introduction of an aqueous droplet. By dragging the aqueous droplet over the spot of dried reagent using paramagnetic particles or via a physical sliding wall, the droplet can then recover and reconstitute the reagent with negligible increase in its total volume, returning the ELR surface to its initial liquid repellent state in the process. We demonstrate that VRD can be performed across a wide range of reagent types including sugars, proteins (antibodies), nucleic acids (DNA), antibiotics, and even complex enzyme/substrate/buffer "kit" mixtures. We believe VRD is a flexible and powerful technique which can further the development of self-contained, multi-step assays in DMF and other microfluidic systems.