Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 109, Issue 52, Pages 21450-21455Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1207754110
Keywords
immunoblotting; medical diagnostics; protein microarrays; systems biology; electrophoresis
Categories
Funding
- National Institutes of Health [1DP2OD007294]
Ask authors/readers for more resources
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 mu Western 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 mu Western blot to analyses of human sera (HIV immunoreactivity) and cell lysate (NF kappa B). 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 separationmatrix 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 mu Western blot in a standard microscope slide form factor. Taken together, the mu Western 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
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available