New smaller, faster model: Microfluidic Western blotting

Moving from left to right

Last year, a team led by Amy Herr at the University of California, Berkeley, in the US developed an automated, microfluidic version of Western blotting that reduced the time taken to conduct this analytical technique from 12 hours to under two hours (see Microfluidics go way out west). By improving the design, they have now managed to reduce this time down to just 10 minutes.

Western blotting is a sensitive electrophoresis-based technique for detecting specific biomolecules, especially proteins. The standard version involves separating a protein-containing sample by gel electrophoresis and then manually blotting the separated proteins onto a polymer membrane. Finally, individual proteins are detected by exposing the membrane to labelled antibodies that bind with the protein of interest. But this standard version suffers from having lots of separate steps, making it time-consuming and labor-intensive, and also requires fairly large samples.

So Herr and her team set about developing a microfluidic version that would be quicker, automated and able to utilize much smaller samples. This version consisted of a central microchamber, around 1mm wide and 1.5mm long, filled with polyacrylamide (PA) gel.

A protein-containing sample is introduced into the left half of the chamber, where it is separated by electrophoresis. Next, an electric field is applied at right angles to the separation direction, pulling the proteins into the right-hand side of the chamber, where they are bound and immobilized by beta-galactosidase molecules incorporated in the gel on this side. Crucially, because the proteins are pulled at right angles, they retain their positions relative to each other when pulled across to the right-hand side. Finally, the immobilized proteins are exposed to one or more fluorescently-labelled antibodies.

 

Stay where you are

Now, Herr has brought out a new version of microfluidic Western blotting, which does away with the need to move the proteins after they’ve been separated. The whole process takes place in a channel just 70μm wide and 10μm deep, which is again filled with a PA gel. In this case, however, a light-responsive molecule known as benzophenone is incorporated into the gel.

The idea is to separate a protein-containing sample in the channel via electrophoresis and then expose the gel to UV light, which causes the benzophenone molecules to bind with the proteins, immobilizing them. Finally, fluorescently-labelled antibodies are sent down the channel via electrophoresis, binding with their target proteins.

Testing this new microfluidic version on a mixture of the proteins β-galactosidase, ovalbumin (OVA) and trypsin inhibitor, Herr and her team found it could perform all the steps in Western blotting – separating the three proteins, immobilising them and then exposing them to a fluorescently-labelled antibody targeting OVA – in 10 minutes. It could also separate and detect these proteins at concentrations as low as 50pM. To show that this new version also works with more complex samples, they used it to detect a specific transcription factor in cell lysate and to detect HIV antibodies in human blood serum.

 

Multi-tasking abilities

As well as being fast, automated and able to work with small samples, the other major advantage of this new version is that it can detect a large number of proteins at the same time. For a start, Herr showed that at least three different antibodies can be sent down the same channel simultaneously, allowing three different proteins to be detected.

On top of this, Herr came up with a design in which three of these channels stretch between a single pair of inlet and outlet ports, with 48 pairs of these ports able to fit onto a single microscope slide-sized chip. This means that each chip is able to house 144 separate channels; if three antibodies are sent down each channel, then 432 proteins can be tested for in a sample at the same time.

Herr is now exploring the multi-tasking abilities of her new multifluidic version of Western blotting in more detail and is also looking to develop it as a practical technique for diagnosing HIV infections.