Driving CE round the bend: Exploring biomolecule interactions with bent capillaries

Close proximity

A team of Chinese and US scientists has found a bendy new way to make capillary electrophoresis (CE) more effective at analyzing the interaction between biomolecules.

Although many research groups have tried using CE to investigate the interaction between biomolecules such as proteins and their respective antibodies, it can prove difficult to bring the biomolecules in close enough proximity to interact as they travel through the capillary. Because of this limitation, scientists are often forced to turn to other, more complex and time-consuming methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance.

Recently, scientists have also started using CE microchips to study biomolecule interactions. Such microchips tend to employ separation channels with bends in them, so they can fit onto the chip, and this seems to have the added advantage of enhancing the mixing and thus the interaction between the biomolecules. Because CE microchips can still be fairly expensive and complicated, a team led by Pengju Jiang at Changzhou University in China decided to see whether the same approach of adding bends could work with conventional CE.

Semicircular bends

They took normal fused-silica capillaries and bent them to form semicircular-shaped turns, either one, two or four. Next, they introduced a fluorescently-labeled peptide known as DYKD, followed by its antibody, into these capillaries and allowed them to migrate to a fluorescence detector at the far end. This produced three peaks: the peptide and antibody on their own, and the peptide-antibody complex formed by their interaction.

When using a capillary without any bends, Jiang and his colleagues found that the peak produced by the peptide-antibody complex was fairly small, showing that the peptide and antibody weren’t coming into contact much as they travelled through the capillary. Adding a single semicircular bend, however, increased the size of the peak produced by the complex, and this peak grew larger with more bends.This suggests that the turbulence generated by the passage through the bends brings the peptide and antibody into close proximity, enhancing their interaction. Jiang and his colleagues calculated that the proportion of the introduced peptide binding to the antibody increased from 15% in the straight capillary to 18% for one bend, 24% for two bends and 35% for four bends.

But it wasn’t just the number of bends that dictated the level of interaction between the peptide and antibody, because Jiang and his colleagues found that the distance of the bend from the start of the capillary also played a part. They introduced a single bend at various distances from the start of a capillary and found that the peak produced by the peptide-antibody complex grew, and the peaks for the peptide and antibody on their own shrank, the further the bend was from the start of the capillary. At a distance of 45cm, the peaks for the peptide and antibody disappeared entirely, leaving just the peak for the peptide-antibody complex.

Complex investigations

Because the peptide and antibody were injected at slightly different times, with the antibody injected around 20 seconds after the peptide, this suggests that the antibody needs a bit of time to catch up with the peptide. If it encounters the bend before this time, then only some of the antibody will have managed to catch up with the peptide. But if it encounters the bend after this time, then almost all the antibody will have caught up with the peptide and can mix with it as they pass through.

Finally, Jiang and his colleagues showed that conducting CE with a bent capillary can be used to assess the relative strength of the interaction between multiple biomolecules and the same target biomolecule, which is a common topic of study. They allowed the peptide and antibody to bind with each other and then injected the peptide-antibody complex into a capillary with four-bends, followed by another related peptide that can bind with the antibody to form a new complex. By monitoring the peaks produced by the original peptide-antibody complex and the new complex, they were able to determine the rate at which the new peptide displaced the original one.

Jiang and his colleagues think their novel approach of using capillaries with semicircular bends could prove particularly useful for investigating the role of biomolecule interactions in disease.