Isolation And Detection Of Single Molecules On Paramagnetic Beads Using Sequential Fluid Flows In Microfabricated Polymer Array Assemblies
Lab On A Chip | December 11, 2011
Cheuk W. Kan, Andrew J. Rivnak, Todd G. Campbell, Tomasz Piech, David M. Rissin, Matthias Mösl, Andrej Peterça, Hans-Peter N iederberger, Kaitlin A. Minnehan, Purvish P. Patel, Evan P. Ferrell, Raymond E. Meyer, Lei Chang, David H. Wilson, David R. Fournier, David C. Duffy.
Lab on a Chip
We report a method for isolating individual paramagnetic beads in arrays of femtoliter-sized wells and detecting single enzyme-labeled proteins on these beads using sequential fluid flows in microfabricated polymer array assemblies. Arrays of femtoliter-sized wells were fabricated in cyclic olefin polymer (COP) using injection moudling 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 femtoliter-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 approach compared favourably to the standard method, i.e., glass arrays mechanically sealed against a silicone gasket, such that prostate specific antigen (PSA) could be detected from 0.02 pg/mL up to 100 pg/mL. 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.