title: Single-Step Imprinting of Femtoliter Microwell Arrays Allows Digital Bioassays with Attomolar Limit of Detection.
abstract: 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.
authors.affiliation: Department of Biosystems, KU Leuven-University of Leuven , Willem de Croylaan 42, 3001 Leuven, Belgium.
authors.forename: Deborah
authors.identifier: http://orcid.org/0000-0002-4948-9981
authors.initials: D
authors.lastname: Decrop
authors.affiliation: Department of Micro- and Nanosystems, KTH Royal Institute of Technology , Stockholm, Sweden.
authors.forename: Gaspard
authors.initials: G
authors.lastname: Pardon
authors.affiliation: Department of Electrotechnical Engineering (ESAT-MICAS), KU Leuven-University of Leuven , Kasteelpark Arenberg 10, 3001 Leuven, Belgium.
authors.forename: Luigi
authors.initials: L
authors.lastname: Brancato
authors.affiliation: Department of Electrotechnical Engineering (ESAT-MICAS), KU Leuven-University of Leuven , Kasteelpark Arenberg 10, 3001 Leuven, Belgium.
authors.forename: Dries
authors.initials: D
authors.lastname: Kil
authors.affiliation: Department of Micro- and Nanosystems, KTH Royal Institute of Technology , Stockholm, Sweden.
authors.forename: Reza
authors.initials: R
authors.lastname: Zandi Shafagh
authors.affiliation: Department of Biosystems, KU Leuven-University of Leuven , Willem de Croylaan 42, 3001 Leuven, Belgium.
authors.forename: Tadej
authors.initials: T
authors.lastname: Kokalj
authors.affiliation: Department of Micro- and Nanosystems, KTH Royal Institute of Technology , Stockholm, Sweden.
authors.forename: Tommy
authors.initials: T
authors.lastname: Haraldsson
authors.affiliation: Department of Electrotechnical Engineering (ESAT-MICAS), KU Leuven-University of Leuven , Kasteelpark Arenberg 10, 3001 Leuven, Belgium.
authors.forename: Robert
authors.initials: R
authors.lastname: Puers
authors.affiliation: Department of Micro- and Nanosystems, KTH Royal Institute of Technology , Stockholm, Sweden.
authors.forename: Wouter
authors.identifier: http://orcid.org/0000-0001-8248-6670
authors.initials: W
authors.lastname: van der Wijngaart
authors.affiliation: Department of Biosystems, KU Leuven-University of Leuven , Willem de Croylaan 42, 3001 Leuven, Belgium.
authors.forename: Jeroen
authors.identifier: http://orcid.org/0000-0001-8143-6794
authors.initials: J
authors.lastname: Lammertyn
mesh_terms: D001681:Biological Assay; D057927:Hydrophobic and Hydrophilic Interactions; D057230:Limit of Detection; D036103:Nanotechnology; D019809:Streptavidin
chemical_list: D019809:Streptavidin
keywords: OSTE+; digital bioassay; femtoliter droplets; lab-on-chip; microwell array; polymer microfluidics; surface energy replication; thiol-ene-epoxy polymer