TY - JOUR
T1 - Uncured PDMS inhibits myosin in vitro motility in a microfluidic flow cell
AU - Wang, Yihua
AU - Burghardt, Thomas P.
N1 - Funding Information:
This work was supported by NIH grants R01AR049277 and R01HL095572 and by the Mayo Foundation .
Publisher Copyright:
© 2018 Elsevier Inc.
PY - 2018/12/15
Y1 - 2018/12/15
N2 - 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.
AB - 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.
KW - Extended curing recovers myosin motility
KW - Myosin in vitro motility
KW - Nanoliter volume motility assay
KW - Polydimethylsiloxane/glass hybrid microfluidic
KW - Unpolymerized PDMS inhibits myosin
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U2 - 10.1016/j.ab.2018.10.007
DO - 10.1016/j.ab.2018.10.007
M3 - Article
C2 - 30300623
AN - SCOPUS:85054839413
SN - 0003-2697
VL - 563
SP - 56
EP - 60
JO - Analytical Biochemistry
JF - Analytical Biochemistry
ER -