NEW YORK (GenomeWeb) – Researchers at the University of Cambridge have developed a diffusion-based technique that combines microfluidics and fluorescent detection to determine the size of native, unmodified proteins and their complexes under physiological conditions.
The approach, published online in Nature Chemistry last month, represents an alternative to conventional methods for sizing native protein complexes that is fast, requires no surfaces or gels that might alter the protein behavior, and works at low concentrations.
Fluidic Analytics, a Cambridge University spinout, has licensed the technology and plans to commercialize an instrument for the research market in mid-2016.
According to Tuomas Knowles, a professor in the Department of Chemistry at Cambridge and the senior author of the study, current methods to physically characterize proteins, such as gel electrophoresis, often require the use of matrices that can affect the behavior of proteins and make the analysis quite slow.
Instead, he and his colleagues determine the size and concentration of native proteins by measuring their diffusion inside a microfluidic device. The reason this works, he explained, is that there is no convective mixing but only diffusion in small volumes like the ones used in microfluidic channels.
"This takes seconds or even fractions of a second, so it's very fast," he said. "It's also under completely native conditions, so you don't denature the proteins. You can use essentially use any buffer you want."
In conducting the method, researchers stream a solution containing the protein of interest and a buffer solution into a microfluidic channel. When they meet, there is initially a sharp interface between them. But over time, as the solutions pass through the channel, the proteins will diffuse into the buffer, with small proteins diffusing more quickly than larger ones. After the solutions have passed through the channel for a while, the channel splits into two, and proteins in the upper part and the lower part are collected separately for analysis.
The scientists then label the proteins with a dye, using a chemistry that targets primary amines, and measure them by fluorescence microscopy. From the ratio of the fluorescence intensity in the two samples, which indicates how much of the protein has diffused across the channel, they can work out the size of the native protein or protein complex. And if they know the amino acid sequence of the protein, they can also determine its concentration from the total fluorescent signal.
The fact that the proteins are only labeled at the very end of the process means that the scientists measure their diffusion behavior in their native, unlabeled state. "We can even use disruptive labeling chemistries because at that stage, it's only for detection — we have already sorted them according to their sizes," Knowles explained.
The entire workflow takes only seconds and requires low protein concentrations, in the nanomolar range, which sets it apart from centrifugation-based methods that can also be used to study native protein complexes.
Also, by multiplexing several microfluidic chips, it might be possible to study several hundred proteins in parallel, "but that's not something we have demonstrated yet in the lab," Knowles said.
In theory, fluorescent detection could be replaced by mass spectrometry-based detection, which would make the method even more sensitive but would also require more costly equipment.
For now, the technique only works with purified proteins, and it cannot currently distinguish between different proteins but will provide an average size across all molecules present. "We're working on extending the chemistry to be protein-specific, to pick out specific proteins, but that's still under development," Knowles said. In addition, he and his colleagues are developing a new chip architecture that would allow sub-populations in a mixture to be analyzed.
In their paper, the researchers showed that they can accurately size molecules ranging from single amino acids, with a molecular weight of 146 daltons, to protein complexes up to 464 kilodaltons in size. There does not seem to be an upper limit, though. "We have sized structures from 1 nanometer to 100 nanometers, covering the known range for protein and protein complexes," Knowles said.
His team is currently using the method to study proteins that form misassembled aggregates and to look at interactions between two proteins that form functional complexes. "These are all events that are characterized by a change in their effective molecular weight, and therefore the size," he said. "This is something we can probe now very rapidly under completely native conditions on our chip."
They are also working on increasing the resolution of the method in order to deal with heterogeneous samples, and on further improving sensitivity.
Replicating the workflow described in their paper would be quite difficult for other researchers because the flows need to be controlled precisely, the labeling chemistry is tricky, and buffers need to be prepared freshly. Also, setup costs for manufacturing the microfluidic devices are quite high. "We really do want these methods to be widely used by the protein science community," Knowles said. "But because of the fact that it brings together so many types of technologies, we do realize that there is a barrier to entry."
To ensure that other researchers have access to the technology, in 2013, the researchers founded a company called Fluidic Analytics.
The spinout, which is based in Cambridge and currently has six employees, holds exclusive and worldwide licenses to two patent applications related to the technology — one covering the diffusive separation, the other the post-separation labeling.
According to CEO Andrew Lynn, the company is working towards launching its first product, called Flow Mk-1, in mid-2016. This instrument will enable researchers to measure the size and concentration of proteins under physiological conditions, similar to the experiments described in the paper.
While pricing information is not yet available, academic users will be able to purchase the instrument "without paying anywhere near the tens of thousands of dollars required to buy some lab instruments," Lynn said, and the cost of the disposable chips will be "well under" $10. Lease options for the instrument will also be available.
Late last year, Fluidic Analytics closed a £1.5 million Series A financing round. In the near future, the company plans to launch a Series B round, in which it will seek to raise between £5 million and £7 million to support the development of other products that "offer enhanced capabilities akin to those currently addressed by immunoassays and surface plasmon resonance," Lynn said. Those products are currently scheduled for launch in 2017 and 2018.