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RESEARCH PROFILES Integrating micromixer and microcoils for time-resolved NMR Pairing diminutive microfluidic devices and enormous NMR magnets makes an odd couple. However, if you need structural data on mass-limited samples, it makes sense to “marry” microfluidics to NMR, says researcher Jonathan Sweedler. In the February 15 issue of Analytical Chemistry (pp 956–960), Masaya Kakuta, Dimuthu Jayawickrama, Andrew Wolters, Andreas Manz, and Sweedler report that they have successfully inter-
is if you don’t have enough material. The way that you improve the S/N is signalaverage multiple scans.” Taking a lot of scans takes time, and the first scan won’t see the same mixture as the last scan. To address these issues, the researchers converted the reaction’s time coordinate, which is the time after mixing, to a distance coordinate. “Normally, [reaction starting time and acquisition time] are related when you mix things together.
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Time-resolved NMR spectrometry system showing (a) syringe pumping system, (b) mixer, (c) microcoil NMR cell, and (d) outlet reservoir. The capillary bubble cell is inside the microcoil in the magnet. L is the length of the reaction coordinate. Arrows indicate the flow of the ubiquitin and methanol solutions into the mixer.
faced a low-pressure microfluidic mixer to an NMR instrument with a microcoil probe and acquired time-resolved spectra of protein folding. This marriage united Manz’s microfluidic group at the Imperial College of Science (United Kingdom) with Sweedler’s group at the Beckman Institute at the University of Illinois, which, over the past 6 years, has shown that shrinking the size of the NMR receiver coil improves the mass sensitivity (Anal. Chem. 1998, 70, 257 A–264 A). “The normal way you do time resolution, if you want to see what happens when two things mix, [is that] you take [an NMR] spectrum at every time point,” explains Sweedler. “The problem there 94 A
In this case, we have completely separated those two parameters,” says Sweedler. To do this, the researchers kept the syringe pumps and mixer outside the magnet but ran a 72-cm capillary containing the blended reaction solutions from the mixer past the microcoil inside the magnet. Because the fluid flows until the syringes are empty, “at every point along the capillary, you have a different reaction time,” explains Sweedler. So, depending on the flow rate, the time point at 2 cm from the mixing point is different from the time point at 5 cm. He adds, “If 2 cm corresponds to a 20-s time point, you can look at 20 s after mixing for an hour,” provided an infinite source of starting materials.
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Controlling the flow rate or moving the millimeter-sized microcoil to a specific location along the capillary determines the acquisition point, he says. The authors decided to manipulate the pumping speeds so they didn’t have to take the probe out every time they wanted to look at a different reaction time. In addition, they created a bubble cell—a section of the capillary etched out with hydrofluoric acid to create an 800-nL cell—to give a little more residence time and data. The researchers demonstrated their new interface by observing folding changes in the well-characterized protein ubiquitin. Under acidic conditions, proton NMR spectral data of two amino acids in ubiquitin, His-68 and Tyr-59, show that methanol induces a transition from the protein’s native state to a partially unfolded state. The results suggest that conformational changes in ubiquitin follow second-order kinetics. Mixing is also an issue in these systems. The researchers experimented with a now commercially available microfabricated mixer and a simple Y-type capillary connector, which both performed equally well with reaction times as short as ~3.8 s. To observe subsecond kinetics, they believe that the micromixer would be the better choice because it mixes faster than the Y-connector. More microcoils in a single NMR probe may also be useful in studying the fast kinetics. According to Sweedler, microcoils could conceivably pave the way for smaller NMR magnets, but he says there is no commercial product yet. For now, research groups can take advantage of the “extra space” inside the standard magnet. “You could put 4 or even 8 or 16 coils in a [standard] NMR probe so you can actually measure what was happening at 4 different [time] points simultaneously, so that you can have a probe at 20, 40, 60, and 80 cm after the mixer.” Other research groups are working on combining the microfluidic mixer and microcoil into one device. a —Rachel Petkewich
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Researchers use TRAP to fabricate reversible protein array For Ashutosh Chilkoti, thinking outside the box has become helpful in speeding up the proteomic process. The associate professor at Duke University is a chemical engineer trained in surface chemistry and molecular biology. “It’s kind of a bizarre background, … partially by design because I wanted to learn biology,” he says. But Chilkoti has taken advantage of his “bizarre” background to design a simple way for making protein arrays using some unique polypeptides. He calls the method the “thermodynamically reversible addressing of proteins”, or “TRAP”. In the February 15 issue of Analytical Chemistry (pp 709–715), Chilkoti and assistant research professor Nidhi Nath show that it’s possible to fabricate functionally active protein arrays directly from crude cell lysate without intermediate purification steps. They do this by exploiting the unique properties of a recombinant, stimuli-responsive elastinlike polypeptide (ELP), which reversibly binds proteins to a surface. Fabricating functional protein arrays presents challenges because of the physicochemical, structural, and functional properties of proteins. The rate-limiting step, say Chilkoti and Nath, is purifying each protein from its native organism or expression system before it’s arrayed on a surface. “Typically, most proteins take at least one step to purify, if not more,” says Chilkoti. “It’s not unrealistic to say it can take three to four purification steps using chromatography.” In a big proteomics lab, this could take up to a day, he adds. To get around this problem, the researchers use ELPs, which they have been working with for about four years. The ELPs belong to a class of stimuliresponsive polymers (SRPs) that undergo a conformational phase transition in aqueous solutions, switching from a disordered, random hydrophilic polymer coil to a more ordered, collapsed hydrophobic globule. Below a lower critical solution temperature (LCST), SRPs are soluble in aqueous solution. But
when their temperature is raised above the LCST, the SRPs become hydrophobic and aggregate in solution. The Inert protein Immobilized ELP External stimuli process is reversible so that when the temRecombinant protein/ligand Protein/ligand perature falls with ELP tag below the LCST, the polymers reSchematic of protein patterning by TRAP. A covalently patterned ELP dissolve in against an inert BSA background is incubated with an ELP fusion protein. solution. The LCST phase transition is triggered by an environmental stimulus, which The recauses the ELP fusion protein to specifically bind to the ELP pattern. searchers engioff to some other instrument.” The LCST neered Escherichia coli to synthesize an transition of SRPs can also be isotherELP; another E. coli expressed thioremally triggered by other external stimdoxin-ELP (Trx-ELP), a fusion protein uli, such as changes in ionic strength, in which the same ELP is connected pH, electric field, light, and chemical or to the C-terminus of the protein Trx. biological analytes, say the researchers. Chilkoti and Nath then micropatterned “I wanted to create surfaces [that] a 10 10 array of ELP alone onto the could change, because dynamic surfaces surfaces of glass cover slips with a spot are all over biology,” says Chilkoti. “I size of ~140 µm and a center-to-center thought if one could do it artificially, distance of 250 µm. To prevent any that there’s some very interesting applicross-contamination, they filled in the cations—in vitro and perhaps in vivo in rest of the surface with an inert bovine ... materials.” serum albumin background. In another experiment, the array capThe ELP-patterned surface was incutured Trx from a cell lysate of E. coli bated for 10 min with a 1.6 µM Trxexpressing Trx-ELP, demonstrating the ELP, which also was linked to the dye selectivity of the technique. Antibodies fluorescein, in a phosphate-buffered against Trx also clamped onto the arsaline (PBS) solution at room temperarayed proteins, showing that the proture. As seen by confocal microscopy, teins remained functionally active. the Trx-ELP was captured from soluChilkoti says there’s still more to be tion onto the array. Hydrophobic interdone. “We need to do [the experiment] actions between the fusion protein and the ELP on the surface were responsible with many more proteins to see how well this works on the surface,” he says. for the binding. When the surface was “[Trx] is a pretty well-behaved proagain rinsed with PBS, this time at 4 °C tein.” The challenge will be using profor 10 min, the fusion protein desorbed teins that are structurally unstable and from the surface. tend to denature on the surface, he The reversibility is important, says Chilkoti. “Proteomics isn’t just a matter says. “Reversibility is sort of the key of something binding to something,” he here, not just the elimination or the says. “You want to then study it in great reduction of protein purification prior to arraying.” a detail. Once you have a hit, it’d be nice —Cheryl M. Harris to send it off to a mass spec, or send it M A R C H 1 , 2 0 0 3 / A N A LY T I C A L C H E M I S T R Y
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