title: Microfluidic Western blotting.
abstract: Rapid, quantitative Western blotting is a long-sought bioanalytical goal in the life sciences. To this end, we describe a Western blotting assay conducted in a single glass microchannel under purely electronic control. The μWestern blot is comprised of multiple steps: sample enrichment, protein sizing, protein immobilization (blotting), and in situ antibody probing. To validate the microfluidic assay, we apply the μWestern blot to analyses of human sera (HIV immunoreactivity) and cell lysate (NFκB). Analytical performance advances are achieved, including: short durations of 10-60 min, multiplexed analyte detection, mass sensitivity at the femtogram level, high-sensitivity 50-pM detection limits, and quantitation capability over a 3.6-log dynamic range. Performance gains are attributed to favorable transport and reaction conditions on the microscale. The multistep assay design relies on a photopatternable (blue light) and photoreactive (UV light) polyacrylamide gel. This hydrophilic polymer constitutes both a separation matrix for protein sizing and, after brief UV exposure, a protein immobilization scaffold for subsequent antibody probing of immobilized protein bands. We observe protein capture efficiencies exceeding 75% under sizing conditions. This compact microfluidic design supports demonstration of a 48-plex μWestern blot in a standard microscope slide form factor. Taken together, the μWestern blot establishes a foundation for rapid, targeted proteomics by merging exceptional specificity with the throughput advantages of multiplexing, as is relevant to a broad range of biological inquiry.
authors.affiliation: Department of Bioengineering and University of California at Berkeley-University of California at San Francisco Graduate Program in Bioengineering, University of California, Berkeley, CA 94720, USA.
authors.forename: Alex J
authors.initials: AJ
authors.lastname: Hughes
authors.forename: Amy E
authors.initials: AE
authors.lastname: Herr
mesh_terms: D015153:Blotting, Western / Q000379:methods*; D002457:Cell Extracts; D004591:Electrophoresis, Polyacrylamide Gel; D015483:HIV Antibodies / Q000097:blood; D006801:Humans; D044085:Microfluidics / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D015203:Reproducibility of Results
chemical_list: D002457:Cell Extracts; D015483:HIV Antibodies; D011506:Proteins