Livermore—If a cell typically contains 10,000 to 100,000 proteins or subcomponent peptides, then determining the distinct identities of only a few can be like looking for a proverbial needle in a haystack.
The task is even harder if the starting material is hardly more than a few cells. Then, provided the target has been sorted out, how does one manipulate that small amount to analyze it further? Standard screening currently requires cumbersome and lengthy processing steps, using equipment the size of kitchen appliances.
Sandia researcher Anup Singh and colleagues in the Labs’ Biosystems Research department are developing microfabricated devices for protein and peptide analysis, dubbed a mProLab. The work is funded as part of the Molecular Integrated Microsystems (MIMS) Grand Challenge by the project.
The team has found, Singh says, that "by miniaturizing, we can actually do better." Using microchannels a few centimeters long, and in some cases just a few millimeters long, on glass chips, Sandia has demonstrated separation of multiple proteins and peptides in 30-45 seconds.
That’s one-tenth the time it would take if performed in longer columns or gels, and with only a thousandth of the starting sample amount needed for laboratory-bench-topscale separations.
Liquid chromatography uses porous matrix-filled tubes called chromatography columns to separate molecules based on their interaction as liquid passes through under applied pressure or electric field. Different types of proteins interact differently with the column materials and drip out at different times, forming isolated "peaks."
Researchers have learned that separations can be tailored to a protein’s different physical properties by selection of the material used for the spongelike matrix and the liquid used to rinse it through the column.
MIMS aims to integrate steps needed to sort and identify small amounts of proteins or peptides by "addressing" smart materials on chip assemblies to "do certain things at certain times in a certain place," Singh explains. In addition to running chromatography and other separations at the microscale, the chips will include components such as valves to control movement of fluids and concentrators. This will permit pre- and post-analysis concentration of dilute samples.
Singh hit upon his patent-applied preconcentrator invention by serendipity. He was working determinedly to get ready for a conference presentation. A minuscule, picoliter—sized protein sample he'd injected onto a microchannel that had been carefully packed with porous beads should have emerged, based on theory, after an electric field was applied. Anup suspected the initial sample injection didn’t work. He used a hand-held syringe to push the fluid out of the channel. The detector happened to still be on, and to his surprise it registered a huge peak of concentrated protein. "If not for that conference, I might not have discovered it," he says.
He and collaborators termed the technique "electrokinetic trapping." Sharp, concentrated peaks form by using an electric field to focus charged analytes into a small spot in the separation channel. The preconcentration technique is addressable and reversible. Proteins can be trapped and concentrated at specific locations by turning the voltage on and released by turning the voltage off. Investigators, including Tim Shepodd of Sandia's Materials Chemistry department, have created in-place sieving gels by using ultraviolet light. The light polymerizes a porous matrix, whose composition can be fine-tuned for various separations. Select locations can be polymerized by using a mask.
For controlling flow through a branching array of intersecting channels, Sandia Microfluidics department researcher Brian Kirby has used a moving plug of polymerized material to shuttle flow through a bypass, thus creating a sort of check valve.
The team plans to combine separation techniques to "fingerprint" proteins, such as cytokines, as is currently done in bench-top processes, separating by both charge and size dimensions. Already, Singh and Biosystems Research coworkers Jongyoon Han and Dan Throckmorton have seen separation speed and efficiencies in a single dimension 10—fold greater than the larger techniques allow.
"We hope to cut the time, overall, 10—to 100—fold," he says, "and work with a small sample—possibly a few cells." The ultimate goal is that, once sorted by charge and size, a spot of protein can be microfluidically transported to a mass spectrometer for analysis of its constituent elements. Ideally this will occur through a completely automatic transfer when the device integration is complete.
