title: Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated polymer array assemblies.
abstract: We report a method for isolating individual paramagnetic beads in arrays of femtolitre-sized wells and detecting single enzyme-labeled proteins on these beads using sequential fluid flows in microfabricated polymer array assemblies. Arrays of femtolitre-sized wells were fabricated in cyclic olefin polymer (COP) using injection moulding based on DVD manufacturing. These arrays were bonded to a complementary fluidic structure that was also moulded in COP to create an enclosed device to allow delivery of liquids to the arrays. Enzyme-associated, paramagnetic beads suspended in aqueous solutions of enzyme substrate were delivered fluidically to the array such that one bead per well was loaded by gravity. A fluorocarbon oil was then flowed into the device to remove excess beads from the surface of the array, and to seal and isolate the femtolitre-sized wells containing beads and enzyme substrate. The device was then imaged using standard fluorescence imaging to determine which wells contained single enzyme molecules. The analytical performance of this device as the detector for digital ELISA compared favourably to the standard method, i.e., glass arrays mechanically sealed against a silicone gasket; prostate specific antigen (PSA) could be detected from 0.011 pg mL(-1) up to 100 pg mL(-1). The use of an enclosed fluidic device to isolate beads in single-molecule arrays offers a multitude of advantages for low-cost manufacturing, ease of automation, and instrument development to enable applications in biomarker validation and medical diagnosis.
authors.affiliation: Quanterix Corporation, Cambridge, MA 02139, USA.
authors.forename: Cheuk W
authors.initials: CW
authors.lastname: Kan
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Rivnak
authors.forename: Todd G
authors.initials: TG
authors.lastname: Campbell
authors.forename: Tomasz
authors.initials: T
authors.lastname: Piech
authors.forename: David M
authors.initials: DM
authors.lastname: Rissin
authors.forename: Matthias
authors.initials: M
authors.lastname: Mösl
authors.forename: Andrej
authors.initials: A
authors.lastname: Peterça
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Niederberger
authors.forename: Kaitlin A
authors.initials: KA
authors.lastname: Minnehan
authors.forename: Purvish P
authors.initials: PP
authors.lastname: Patel
authors.forename: Evan P
authors.initials: EP
authors.lastname: Ferrell
authors.forename: Raymond E
authors.initials: RE
authors.lastname: Meyer
authors.forename: Lei
authors.initials: L
authors.lastname: Chang
authors.forename: David H
authors.initials: DH
authors.lastname: Wilson
authors.forename: David R
authors.initials: DR
authors.lastname: Fournier
authors.forename: David C
authors.initials: DC
authors.lastname: Duffy
mesh_terms: D003516:Cycloparaffins / Q000737:chemistry; D008280:Magnetics; D046228:Microarray Analysis; D053758:Nanoparticles / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry*; D017430:Prostate-Specific Antigen / Q000737:chemistry
chemical_list: D003516:Cycloparaffins; D011108:Polymers; D017430:Prostate-Specific Antigen