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    Single-Step Imprinting of Femtoliter Microwell Arrays Allows Digital Bioassays with Attomolar Limit of Detection

    Year: 2017

    Journal: ACS Appl. Mater. Interfaces, Volume 9, nov-29, page 10418–10426

    Authors: Decrop, Deborah; Pardon, Gaspard; Brancato, Luigi; Kil, Dries; Shafagh, Reza Zandi; Kokalj, Tadej; Haraldsson, Tommy; Puers, Robert; van der Wijngaart, Wouter; Lammertyn, Jeroen

    Organizations: Research Foundation Flanders [FWO G086114N, G080016N]; De Vlaamse Liga tegen Kanker [EXM C8744-A]; KU Leuven [OT 13/058, C32/15/005, IOF KP/12/009]; EU project Norosensor [FP7-NMP-2013-SMALL7604244]; Agency for Innovation by Science and Technology in Flanders [IWT 121615]

    Keywords: microwell array; femtoliter droplets; digital bioassay; lab-on-chip; polymer microfluidics; surface energy replication; thiol-ene-epoxy polymer; OSTE

    Bead-based microwell array technology is growing as an ultrasensitive analysis tool as exemplified by the successful commercial applications from Illumina and Quanterix for nucleic acid analysis and ultrasensitive protein measurements, respectively. High-efficiency seeding of magnetic beads is key for these applications and is enhanced by hydrophilic-in-hydrophobic microwell arrays, which are unfortunately often expensive or labor-intensive to manufacture. Here, we demonstrate a new single-step manufacturing approach for imprinting cheap and disposable hydrophilic-in- hydrophobic microwell arrays suitable for digital bioassays. Imprinting of arrays with hydrophilic-in-hydrophobic microwells is made possible using an innovative surface energy replication approach by means of a hydrophobic thiol-ene polymer formulation. In this polymer, hydrophobic-moiety-containing monomers self-assemble at the hydrophobic surface of the imprinting stamp, which results in a hydrophobic replica surface after polymerization. After removing, the stamp, microwells with hydrophobic walls and a hydrophilic bottom are obtained. We demonstrate that the hydrophilic-in-hydrophobic imprinted microwell arrays enable successful and efficient self-assembly of individual water droplets and seeding of magnetic beads with loading efficiencies up to 96%. We also demonstrate the suitability of the microwell arrays for the isolation and digital counting of single molecules achieving a limit of detection of 17.4 aM when performing a streptavidin biotin binding assay as model system. Since this approach is up-scalable through reaction injection molding, we expect it will contribute substantially to the translation of ultrasensitive digital microwell array technology toward diagnostic applications.
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