Uncured PDMS inhibits myosin in vitro motility in a microfluidic flow cell

• Published in: Analytical biochemistry Vol. 563; pp. 56 - 60
• Main Authors: Wang, Yihua, Burghardt, Thomas P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.12.2018
• Subjects: Actins - chemistry; Animals; Dimethylpolysiloxanes - chemistry; Extended curing recovers myosin motility; Microfluidics - methods; Muscle, Skeletal - metabolism; Myosin in vitro motility; Myosins - chemistry; Nanoliter volume motility assay; Polydimethylsiloxane/glass hybrid microfluidic; Unpolymerized PDMS inhibits myosin; Myosin in vitro motility; Unpolymerized PDMS inhibits myosin; Extended curing recovers myosin motility; Nanoliter volume motility assay; Polydimethylsiloxane/glass hybrid microfluidic
• ISSN: 0003-2697; 1096-0309; 1096-0309
• DOI: 10.1016/j.ab.2018.10.007

The myosin motor powers cardiac contraction and is frequently implicated in hereditary heart disease by its mutation. Principal motor function characteristics include myosin unitary step size, duty cycle, and force-velocity relationship for translating actin under load. These characteristics are sometimes measured in vitro with a motility assay detecting fluorescent labeled actin filament gliding velocity over a planar array of surface immobilized myosin. Assay miniaturization in a polydimethylsiloxane/glass (PDMS/glass) hybrid microfluidic flow channel is an essential component to a small sample volume assay applicable to costly protein samples however the PDMS substrate dramatically inhibits myosin motility. Myosin in vitro motility in a PDMS/glass hybrid microfluidic flow cell was tested under a variety of conditions to identify and mitigate the effect of PDMS on myosin. Substantial contamination by unpolymerized species in the PDMS flow cells is shown to be the cause of myosin motility inhibition. Normal myosin motility recovers by either extended cell aging (~20 days) to allow more complete polymerization or by direct chemical extraction of the unpolymerized species from the polymer substrate. PDMS flow cell aging is the low cost alternative compatible with the other PDMS and glass modifications needed for in vitro myosin motility assaying. •Uncured polymer in a microfluidic flow cell inhibits myosin based actin motility•Simple and low cost alternatives restore normal myosin functionality including:•Extended flow cell aging of ~20 days or more•Chemical extraction of free monomers from the polymer substrate