Seeking to improve on the performance of conventional multi-stage proteomics separations, a University of California, Berkeley, researcher has developed a new microfluidics technology that could streamline such assays while also reducing information loss.
The technology, called iMosaic, uses standard glass chips regionally photopatterned with discrete nanostructured separation materials to integrate all the steps of a given multistage assay within a single chamber.
The researcher, Amy Herr, said she hopes that by combining all the stages in one location, researchers can avoid the separation loss associated with current microfluidic devices, which typically use networks of intersecting channels.
The loss occurs at the points of intersection where analytes separated in the first stage of the assay are channeled into the second stage of the assay. Because all analytes funneled into a given second-stage channel are starting from the same point, any separation between them achieved in the first stage is negated.
"If you have a three-dimensional plug of material and you drive it into a perpendicular array, basically everything in that [second channel] is one unit," Herr told ProteoMonitor this week. "It doesn't matter if there are five peaks in [that group of analytes] or one-third of a peak – it all becomes one piece of information."
To an extent this problem can be combated by oversampling, but "a lot of the 2D technologies that have been developed in microfluidic devices have a hard time with that," she noted. "I think that's because this sort of discretized geometry makes it inherently difficult to get a continuum of information."
To solve this problem, Herr is building devices that perform multistage separations via single chambers functionalized throughout with different separation media. This enables samples to, transfer from one medium to the other without any compression.
By doing "a continuous transfer of material from the first dimension into the second dimension" researchers can get "a continuum of information from that first dimension transferred in a low-dispersion way to maintain a continuum of information in the second dimension, as well," she said.
In February, Herr received a National Foundation of Science career grant for the iMosaic work. Such grants are for a maximum of five years and $400,000, and thus far her lab has received $74,450 under the award.
She's also received funding from the Rogers Family Foundation and industry players like Bio-Rad and Eli Lilly.
Much of her work under the NSF grant will involve devising interfaces between the different dimensions of the separation assays, she said, noting that this was probably the most challenging part of the project.
"You have two separation dimensions, so how do you get that material from the first dimension to the second without losing any information?" she said. "How do we use either the device geometry design, the electric fields that we apply, or the structure of the materials inside the chamber to help mitigate those losses?"
Among the first assays her team attempted to use this strategy was native Western blotting, for which they combined native polyacrylamide gel electrophoresis with an immunoassay. Results of this effort were published in the February 2010 edition of the Journal of the American Chemical Society.
Since then they've explored applying the technology to true Western blotting, Eastern blotting, 2D electrophoresis, and a multi-analyte blotting approach. According to Herr, the latter approach combines "a Western blot with a protein microarray to allow people to probe one sample separation against multiple antibody targets in a quantitative manner." A paper on that technique has been accepted and is forthcoming in Analytic Chemistry.
In addition to reducing information loss, the integration of the separation steps combined with the microfluidic format for faster assay runtimes than do traditional platforms. Between doing the separation and the immunoassay, a conventional Western blot can take from hours to a day. According to Herr, a Western blot can take about one minute on the iMosiac platform.
Recognizing the need for higher-throughput assays was what initially spurred the research, said Herr. She said inspiration for the device came from an oncologist at Stanford University Medical Center who had a repository of roughly 50,000 prostate cancer samples she wanted to run in order to quantify their levels of the PSA protein biomarker.
The oncologist "has this longitudinal information archived in serum samples from the patients, and she said, 'I just wish I could do Western blotting or some sort of protein separation on all of those samples,'" Herr said. "And I realized that there was just no way from the standpoint of her resources – be it people, equipment, money, or time – that she could do that sort of large-scale measurement.
"So I started thinking about how we could help her make that measurement, and I thought if we could do this in a way that doesn't lose information but allows her to walk away from the instrument, that could be a really big contribution," she said.
Herr's lab is currently working with the Stanford oncologist on using the iMosaic system to analyze prostate cancer samples. The researchers are also collaborating with another UC-Berkeley team to measure protein acetylation in stem-cell populations with the aim of identifying pathways involved in aging.
"I definitely have an interest in translational research, and that means getting these approaches out of our lab and into biology and clinical research labs," said Herr.
In particular, the multi-analyte blotting technique could be useful as a platform for studying protein signaling pathways, allowing researchers to quantify the levels of various protein isoforms in a sample, she noted.
In the future, the researchers plan to look at ways to pair the system with a mass spectrometer, and might also look into mating it with a pressure-driven, as opposed to an electrophoretically driven.flow to enable liquid chromatography assays, Herr said.
She added that she's currently in licensing talks over the technology, though she declined to elaborate.
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