title: Microfluidic integration of Western blotting is enabled by electrotransfer-assisted sodium dodecyl sulfate dilution.
abstract: We integrate sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent antibody probing in a single, monolithic microdevice to realize microfluidic Western blotting. A hurdle to successful on-chip Western blotting lies in restoring antibody recognition of previously sized (denatured, reduced) proteins. To surmount this hurdle, we locally dilute free SDS from SDS-protein complexes using differential electromigration of the species during electrotransfer between SDS-PAGE and blotting regions of a microchamber. Local dilution of SDS minimizes re-association of SDS with proteins offering means to restore antibody binding affinity to proteins after SDS-PAGE. To achieve automated, programmable operation in a single instrument, we utilize a 1 × 2 mm(2) glass microchamber photopatterned with spatially distinct, contiguous polyacrylamide regions for SDS-PAGE, electrotransfer, and antibody blotting. Optimization of both the SDS-PAGE and electrotransfer conditions yields transfer distances of <1 mm (40 s). The Western blot is completed in 180 s, with fully automated assay operation using programmable voltage control. After SDS-PAGE and electrotransfer, we observe ~80% capture of protein band mass on the blotting region for a model protein, C-reactive protein. This novel microfluidic Western blot approach introduces fine transport control for in-transit protein handling to form the basis for an automated, rapid alternative to conventional slab-gel Western blotting.
authors.affiliation: Electrical Engineering & Computer Science, 308B Stanley Hall, University of California, Berkeley, CA, USA. hou.chenlu@gmail.com
authors.forename: Chenlu
authors.initials: C
authors.lastname: Hou
authors.forename: Amy E
authors.initials: AE
authors.lastname: Herr
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000295:instrumentation / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004867:Equipment Design; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D009038:Motion; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*
chemical_list: D011506:Proteins; D012967:Sodium Dodecyl Sulfate