Proc. Natl Acad. Sci. USA 109, 21450–21455 (2012)

Credit: © 2012 NAS

The Western blot is a highly sensitive analytical technique that is used to identify specific proteins. Unlike simple chromatographic techniques such as size-exclusion chromatography, Western blots separate and identify different entities based on a combination of two properties, namely molecular size and how strongly they are bound by a particular antibody. Despite the undoubted sensitivity, Western blots suffer from several drawbacks — they are quite labour intensive, not suitable for high-throughput, and the process can be slow.

Now, Alex J. Hughes and Amy E. Herr, both at the University of California at Berkeley, have developed a microfluidic version of the Western blot technique. Their method allows for 48 simultaneous runs on a single chip and greatly reduces the volume of expensive antibody reagents used and the time required to detect a target protein. The chip is laid out with a series of individual microchannels that are filled with a polyacrylamide gel and connected to electrodes at both ends. For each micro-Western blot, the mixture of proteins is added to a channel and separated by molecular size using gel electrophoresis. Instead of blotting the proteins to a separate membrane — as is normally required in a Western blot — the gel is irradiated with UV light triggering a photo-reaction in which benzophenone groups in the gel covalently bond to the proteins and thereby immobilize them. Antibody probes are then added to the microfluidic channel and electrophoretically migrated along it until they bind to their target or pass through the gel.

The microfluidic approach works with multiple antibodies, including the normal arrangement of primary and secondary ones. The highly selective primary antibody binds solely to the target protein, and the secondary antibody — which is modified to contain a detection label — binds to the primary antibody. This combination enables selective detection of the target protein. The sample separation and probing are completed in 10 to 60 minutes. Hughes and Herr say the process offers near-complete analyte capture and is capable of detecting low picomolar concentrations.