How to compare and contrast biosensor performance
Stephanie Sutton |
Researchers working on a European Metrology Research Programme project called BioSurf have developed a reference biosensor surface that they believe will help to benchmark different biosensors by assessing accuracy.
“Diagnostic tests, as implemented in a point-of-care setting, have the advantage of providing rapid disease identification,” says Alex Shard, a researcher at the National Physical Laboratory (UK) and one of the authors of the work (1). “The current issues revolve around sensitivity, reliability and quality control. Since many of the tests rely in some way upon having active probes attached to a surface it is important to be able to assess and verify how many probes are attached, how many are active and whether other species that interfere with the test have become attached to the surface.”
Detection of biomolecules relies on flat surfaces or nanoparticles that are designed to specifically capture one type of molecule from a huge diversity of others. In many instances, researchers want to know if a biomolecule is present above a critical concentration; therefore, the sensitivity of the method used to detect it must also be known. The aim of the reference biosensor is to provide a surface that could be reproduced as part of a wide range of detection strategies to provide a benchmark for sensitivity.
The reference biosensor works by attaching two types of molecule to a gold surface: one is based on polyethylene glycol and resists non-specific attachment of biomolecules; the other has a biotin group at the end, to which avidin specifically binds. So far, the reference biosensor surface has been tested with serum proteins to confirm that there is little or no ‘non-specific’ binding.
“The key property of the surface we developed is its excellent repeatability in binding – and it appears to be robust to minor changes in surface composition. The amount of protein attached is now well understood and it can be used to compare the sensitivity of different methods,” says Shard. “For example, we showed that quartz crystal oscillators are very sensitive to low amounts of protein attachment, but rapidly lose sensitivity as more protein binds. As the protein layer approaches full coverage, the sensitivity is reduced tenfold.”
Development wasn’t completely straightforward. Shard says that one of the key problems was measuring the concentration of the probe at the surface. The group developed a novel form of mass spectrometry to address this issue. “We were able to measure the concentration of probes with a detection limit ten times better than traditional methods. This was a major advance, since we were able to demonstrate that the biotin molecules were randomly spaced on the surface and that the manner in which avidin bound to the surface changed when the spacing between the biotin groups was about 5 nm, which is similar to the size of the avidin molecule,” he says.
Now, Shard and the rest of the group are looking to investigate more forms of avidin and streptavidin to assess some of the more “intriguing” details of the response of the surface to different modes of binding. They are also adapting the surface to nanoparticle-based assays to investigate their sensitivity in colorimetric detection.
- S.Ray et al., “Neutralized Chimeric Avidin Binding at a Reference Biosensor Surface,” Langmuir, 31(6), 1921-1930 (2015).