Fabrication of an Open Microfluidic Device for Immunoblotting

Abstract

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.

Paper‐based open microfluidic platform for protein electrophoresis and immunoprobing

“…Different stacking efficiencies have been reported in previous microchip-based protein electrophoresis systems, depending on microchip design and geometry, gel matrices, buffers, and the electrophoretic conditions employed. Typically, sample injection dispersion was reduced by 50% to more than 90% in these systems [13,14,16,28,33,49,50], essentially similar or slightly better than our open microfluidic approach. Taken together, the implementation of a stacking boundary both increased the local protein concentration and reduced the sample injection dispersion, which may be favorable for the sensitivity of the immunoassay and the electrophoretic resolution, respectively.…”

“…We speculate that this was caused by the detrimental effect of oxygen on the polymerization process and the effective compartmentalization of the precursor solution within the pores of the CA membrane. Including benzophenone or glycerol into the precursor solution, which were reported to act as oxygen scavengers during acrylamide polymerization [16,40], did not improve the reproducibility of the polymerization process (data not shown). Linear polyacrylamide derivatives were also tested, but protein migration was rather poor in these matrices, owing to severe evaporation effects during electrophoresis (including premature current breakdown) as a consequence of using linear polymers that do not form hydrogels.…”

“…In CE and microfluidic chips, proteins are electrophoretically separated in fully enclosed microchannels, severely limiting protein accessibility for consecutive analyses. Several strategies have been proposed to allow for immunoprobing upon separation in enclosed microchannels, including protein immobilization to the capillary wall (and flushing the capillaries with antibody solutions [9]), bead-based immunoassays in CE [10], electrophoretic antibody loading [11][12][13][14][15], and reversible microchip bonding [16]. Alternatively, the capillary/microchip outlet can be interfaced with a conventional Western Blot membrane (laterally moving at a constant speed) to deposit the separated proteins onto the membrane for immunoprobing [17][18][19][20].…”

A Photoclick Hydrogel for Enhanced Single‐Cell Immunoblotting

“…After 30 s of SDS‐PAGE, the FITC‐BSA was quickly immobilized onto the photoclick gel under various ultraviolet light conditions (ranging from 20 to 100 s). The protein immobilization efficiency, [ 13 ] η, was defined as the ratio of FITC's fluorescence intensity before and after photoactive gel rinsing. Imaging data of the negative control results from UV irradiation time of 0 s in Figure 2c showed that the protein photoimmobilization under no UV condition is negligible, which indicated that the MMP gel remained inert without UV exposure under normal electrophoretic conditions.…”

Single cell quantification of ribosome occupancy in early mouse development

“…We implemented numerous innovations that enable a chemistry required to achieve single-cell ribosome profiling by coupling ITP with an optimized on-chip size selection. Specifically, we leveraged pretreatment of the channel with benzophenone to enable light-induced polymerization of polyacrylamide inside PDMS chips 14 . To aid visualization, we included DNA oligonucleotide markers containing a 5' fluorophore and 3' dideoxycytosine modification to prevent marker amplification in downstream library preparation (Extended Data Fig.…”