Illustration of the assembly, distribution, and point-of-care use of a rapidly-deployable, cell-free COVID-19 biosensor: 1) Assemble: Assembling CFPS reagents by mixing E. coli lysate, murine RNase Inhibitor (mRI), energy sources, cofactors, and toehold switch riboregulator plasmid. 2) Print: aliquoting CFPS reagents onto paper substrates housed in a plastic test cassette. 3) Dehydrate: lyophilizing CFPS reagents on paper substrates. 4) Distribute. 5) Saliva sample: applying saliva samples onto cassette without pretreatments. 6) Reaction: bioluminescent protein expression in presence of target RNA (+), or ribosome detachment in absence of target RNA (-). 7) Visual result: bioluminescent output in the presence of target RNA and NanoLuc luciferase expression.

Droplet generation has been widely used in conventional two-dimensional (2D) microfluidic devices, and has recently begun to be explored for 3D-printed droplet generators. A major challenge for 3D-printed devices is preventing water-in-oil droplets from sticking to the interior surfaces of the droplet generator when the device is not made from hydrophobic materials. In this study, two approaches were investigated and shown to successfully form droplets in 3D-printed microfluidic devices. First, several printing resin candidates were tested to evaluate their suitability for droplet formation and material properties. We determined that a hexanediol diacrylate/lauryl acrylate (HDDA/LA) resin forms a solid polymer that is sufficiently hydrophobic to prevent aqueous droplets (in a continuous oil flow) from attaching to the device walls. The second approach uses a fully 3D annular channel-in-channel geometry to form microfluidic droplets that do not contact channel walls, and thus, this geometry can be used with hydrophilic resins. Stable droplets were shown to form using the channel-in-channel geometry, and the droplet size and generation frequency for this geometry were explored for various flow rates for the continuous and dispersed phases.

Microfluidic devices (MFDs) printed in 3-D geometry using digital light projection to polymerize monomers often have surfaces that are not as hydrophobic as MFDs made from polydimethylsiloxane. Droplet microfluidics in these types of devices are subject to droplet adhesion and aqueous spreading on less hydrophobic MFD surfaces. We have developed a post-processing technique using hydrophobic monomers that renders the surfaces of these devices much more hydrophobic. The technique is fast and easy, and involves flowing monomer without initiator into the channels and then exposing the entire device to UV light that generates radicals from the initiator molecules remaining in the original 3-D polymerization. After treatment the channels can be cleared and the surface is more hydrophobic, as evidenced by higher contact angles with aqueous droplets. We hypothesize that radicals generated near the previously printed surfaces initiate polymerization of the hydrophobic monomers on the surfaces without bulk polymerization extending into the channels. The most hydrophobic surfaces were produced by treatment with an alkyl acrylate and a fluorinated acrylate. This technique could be used for surface treatment with other types of monomers to impart unique characteristics to channels in MFDs.