posted on 2017-08-21, 00:00authored byPhilippe Abdel-Sayed, Kevin A. Yamauchi, Rachel E. Gerver, Amy E. Herr
Given the wide adoption
of polydimethylsiloxane (PDMS) for the
rapid fabrication of microfluidic networks and the utility of polyacrylamide
gel electrophoresis (PAGE), we develop a technique for fabrication
of PAGE molecular sieving gels in PDMS microchannel networks. In developing
the fabrication protocol, we trade-off constraints on materials properties
of these two polymer materials: PDMS is permeable to O2 and the presence of O2 inhibits the polymerization of
polyacrylamide. We present a fabrication method compatible with performing
PAGE protein separations in a composite PDMS-glass microdevice, that
toggles from an “enclosed” microchannel for PAGE and
blotting to an “open” PA gel lane for immunoprobing
and readout. To overcome the inhibitory effects of O2,
we coat the PDMS channel with a 10% benzophenone solution, which quenches
the inhibiting effect of O2 when exposed to UV, resulting
in a PAGE-in-PDMS device. We then characterize the PAGE separation
performance. Using a ladder of small-to-mid mass proteins (Trypsin
Inhibitor (TI); Ovalbumin (OVA); Bovine Serum Albumin (BSA)), we observe
resolution of the markers in <60 s, with separation resolution
exceeding 1.0 and CVs of 8.4% for BSA-OVA and 2.4% for OVA-TI, with
comparable reproducibility to glass microdevice PAGE. We show that
benzophenone groups incorporated into the gel through methacrylamide
can be UV-activated multiple times to photocapture protein. PDMS microchannel
network is reversibly bonded to a glass slide allowing direct access
to separated proteins and subsequent in situ diffusion-driven immunoprobing
and total protein Sypro red staining. We see this PAGE-in-PDMS fabrication
technique as expanding the application and use of microfluidic PAGE
without the need for a glass microfabrication infrastructure.