Last Month's Most Accessed Feature: A gap opens up: Analyzing protein binding with CE and surface plasmon resonance

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  • Published: Dec 1, 2017
  • Categories: Detectors
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A gap opens up: Analyzing protein binding with CE and surface plasmon resonanceBinding and scattering

For studying protein binding in complex biological samples, a combination of capillary electrophoresis (CE) and surface plasmon resonance (SPR) detection would seem to be a fairly good option. CE can separate target proteins from all the other proteins and biomolecules in a sample under gentle conditions that won’t harm them, while SPR can sensitively detect when these target proteins subsequently bind to corresponding proteins attached to a gold surface.

This, at least, is what a team of Dutch scientists led by Elena Domínguez-Vega from Vrije Universiteit Amsterdam thought, which is why they developed a way to couple CE with SPR. Unfortunately, they quickly discovered a serious problem with their approach. Rather more fortunately, they then quickly came up with a solution.

SPR detection takes advantage of surface plasmons, which are are virtual particles produced by the collective oscillation of electrons at the surface of metals. These oscillations interact with incoming light of matching frequencies, scattering it. For most metals, this happens with light at infrared frequencies, but for gold it happens at visible frequencies, which is why gold glitters so attractively. This scattering is also hampered if anything interacts with the surface of the metal, such as a binding protein, offering a sensitive means of detection.

Non-specific interactions

To couple SPR with CE, Domínguez-Vega and her colleagues utilized a commercially available SPR detector, comprising a flat gold sensor, a prism, a laser and a photodetector, produced by a Finnish company called Bionavis. They then attached the gold sensor to a specially-made polymer flow cell with two open parallel channels connected by a capillary, such that the gold surface formed the floor of the channels. Finally, they inserted the far end of a separation capillary into the inlet of one of the channels and another capillary into the outlet of the other channel.

The idea is to attach a protein such as an antibody for a specific protein to the gold floor of the channel receiving the separation capillary, by immobilizing them on a hydrogel coating. A protein-containing sample is then separated by CE before flowing into the channel with the gold floor, where any binding between the target protein in the sample and the attached antibody is detected by a reduction in the scattered light.

The separated sample then flows into the second channel, which has a bare gold floor, before flowing out the second capillary to a waste vial. The advantage of having a second channel is that it can act as a reference. Any non-specific interactions between biomolecules in the sample and the gold surface, which will also affect the scattering, will occur in both channels, whereas the protein binding with only occur in the first channel. This allows the signal from the protein binding to be distinguished from any background noise caused by the non-specific interactions.

Interfering voltage

When Domínguez-Vega and her colleagues tried this with dye molecules and bare gold surfaces, it worked fine, with the dye molecules producing clear signals when they interacted with the gold surfaces. When they tried it with the protein human serum albumin (HSA) and its antibody, however, it proved far less successful, with the binding failing to produce a clear signal.

The reason for this was that Domínguez-Vega and her colleagues were using the gold sensor both to detect the proteins and as an electrode for CE, and the CE voltage was interfering with the detection signal. They tried several other arrangements, but all of them experienced interference from the CE voltage. That was until they scraped off a thin section of the gold sensor to produce a gap between two sections of the sensor. They then used the smaller section as the CE electrode and the larger section as the SPR detector.

With a physical gap between the electrode and the detector, the CE voltage no longer caused any interference. As a result, they were able to monitor the binding between HSA and its antibody, in a sample containing several other proteins, at concentrations as low as 100ng/μL. They also demonstrated the ability to distinguish signals from two different proteins interacting with the same attached enzyme.

Sensors and Actuators B, 2018, 254, 1040–1047: "Development of a surface plasmon resonance sensor for coupling to capillary electrophoresis allowing affinity assessment of protein mixture components"

Article by Jon Evans

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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