Better beads Molecularly imprinted polymers are usually prepared as a monolith, which is ground and sieved to the desired size. The grinding and sieving process has several disadvantages—it is time-consuming, only moderate amounts of useful polymers are created, the particles are irregular, and some of the binding sites may be damaged. Suspension polymerization in perfluorocarbon solves some of those problems and produces good yields of monodisperse spherical particles, but the procedure is expensive and requires extensive optimization. Klaus Mosbach and colleagues at Lund University (Sweden) instead use precipitation polymerization to prepare cross-linked microspheres that incorporate molecularly imprinted binding sites. They prepared molecularly imprinted affinity beads
against theophylline and 17p-estradiol. The specific binding sites created were analyzed by radioligand-binding analysis. They found that the imprinted microspheres were easy to prepare. Imprinted microspheres bound three to four times more of the original print molecule than the corresponding nonimprinted microspheres. In addition, the microspheres had higher binding-site densities than conventional imprinted particles, and the smaller bead diameter made ligand transfer to the binding sites significantly faster. They also found that they needed less of the print molecule during the imprinting process. When suspended in an appropriate solvent, the resulting sorbent suspenresistant to precipitation and aggregation However upon centrifugation the microspheres formed a dense
pellet, which readily allows separation. (Anal. Commun. 1999, 36, 35-38)
rent cleanup buffer. 'The cleanup buffer is there to encourage diffusion of salts and low-molecular-weight substances through the lower membrane," says Smith. 'The cleanest or most highly desalted sample is exposed to the cleanest buffer, so it ends up producing a very clean sample very rapidly." The overall cleanup time per sample is on the order of 2 min. In this particular version of the device, the molecular weight cutoffs are 50,000 and 500 Da for the high and low molecu-
lar weight cutoffs, respectively. Smith says that the choice of molecular weight cutoffs is primarily a matter of experience. 'The way the device works," he says, "the effective molecular weight cutoffs are different than they would be in the conventional ultrafiltration application. They're as much as a factor of 10 lower than the nominal values quoted by the manufacturer." Smith speculates that the differences are primarily due to the limited amount of time allowed for diffusion. Added benefits of the device are that it can clean small samples and work at reduced flow rates. The sample flow rate, which is roughly equal to the outlet flow rate, is 200 nL/min. "By reducing the flow rates, we more effectively utilize the sample for mass spectrometry," says Smith. "The closest previous device required roughly an order of magnitude higher flow rate. Basically, that extra order of magnitude is all wasted sample." Experience has shown that the membranes can often be used for several days without problems. However, for more difficult appllcattons, and when the membranes do need to be changed, the process is simple. "Once [the device] has been properly built and aligned, you open it up, slap in a new membrane, and quickly reassemble. Commercial implementation would probably be even smoother, likely using a clamping ar-
Scanning electron micrographs of molecularly imprinted microspheres against 17$-estradiol. (Adapted with permission. Copyright 1999 Royal Society of Chemistry.)
SCIENCE
Shorter sample cleanup times For many analyses, the sample preparation is the bottleneck, and the entire process would be greatly improved by a fast, easy way to clean up samples. In the April 15 issue of Analytical Chemistry (p. 1485)8 Richard D. Smith and co-workers at Pacific Northwest National Laboratory describe a microfabricated device that uses dual microdialysis to do just that. In the device, which is 30 x 30 x 6 mm, two microdialysis membranes are sandwiched between three pieces of polycarbonate, which have serpentine channels etched in them. Channel 3 is connected at one end to channel 2 by a hole drilled through the middle of the polycarbonate device and at the other end to the outside edge of the device, where an emitter tip for electrospray ionization MS can be placed. Alternatively, the sample can be collected for analysis by other methods. When the sample is injected into the first channel, the membrane with a high molecular weight cutoff prevents all the species above that molecular weight from diffusing to the second channel. Sample flows from the second channel to the third channel. On the other side of the third channel is a membrane with a low molecular weight cutoff. Low molecular weight components, such as salts, diffuse across this membrane to the fourth channel and are swept away by a countercur-
An exploded view showing the construction of the device.
Analytical Chemistry News & Features, May 1, 1999 309 A
News rangement that would be more efficient and allow you to change a membrane in 30 s, perhaps." Smith is already thinking ahead to possible improvements to the device. "We've thought of stacking additional stages, doingfinerfractionation, and having multiple outlets," he says. "You can imagine a whole stack of membranes using the same sandwich concept with 5 or 10 outlets and collecting fractions." In addition, microfabrication holds the promise of even greater speed, lower flow rates, and the ability to process multiple samples simultaneously on a single device. "You can have 5,10, or 20 of these on a single device. It depends on the size of the channels, the path they follow, and the size of the overall device," says Smith. "For the mass spectrometer one channel is much all can handle at the ment but down the road there will likely be options for multiplexing " As far as commercial implementation, Bioanalytical Systems is now offering a single-stage earlier version of the device for rapid desalting. "There is also interest being expressed by others in the more complex multistage microfabricated devices," says Smith. Celia Henry
The dope on dopamine Every handyman knows the importance of having the right tool. So does every analytical chemist. For Mark Wightman, the right tool is cyclic voltammetry, especially when it comes to studying dopamine. In a recent Nature paper (1999, 398, ,7-69), he end his colleagues at the University of North Carolina-Chapel Hill and Illinois State University suggest that dopamine is not the brain's main reward—the driving force behind addiction—but probably plays a role in learning. "[Dopamine] seems to be a transient signal that shows up when something new is going on," Wightman says, "[rather than being] the kind of continuous reward you would expect in the case of addiction." Dopamine's association with addiction has been assumed for a long time, and many researchers have studied the link with a technique called intracranial selfstimulation (ICS). ICS was discovered in 1954 by James Olds and Peter Milner in studies of rats. An electrode is used to stimulate regions of the brain near dopamine 310 A
neurons, inducing a pleasurable sensation. If the electrode is rigged so that a rat can stimulate it by pressing a lever, the animal will do so over and over until it is exhausted. Because this behavior is similar to patterns seen with drug abuse, ICS is a paradigm for studying addiction. Researchers seeking evidence that ICS causes dopamine release have placed probes in the nucleus accumbens—an area rich in dopamine terminals, where the neurotransmitter is released—and have conducted microdialysis measurements. They have seen dopamine levels rise, supporting the idea that dopamine and addiction are linked. Years later, Wightman's lab developed fast-scan cyclic voltammetry, which rapidly measures (on the order of 10 ms) specific neurotransmitters in precise places. Early on, the group studied anesthetized rats, where they followed dopamine uptake and release in various regions of the brain and examined the effects of drugs of abuse and potential treatments for diseases thought to involve dopamine. Eventually, Paul Garris (now at Illinois State University), a postdoctoral fellow in Wightman's lab, found a way to monitor dopamine in freely moving animals. The researchers chose to recreate the ICS experiments, hoping to correlate neurotransmitter release with changes in the animals' behavior. Initially, Wightman's graduate students Melissa Bunin and Michaux Kil-
patrick found that stimulating the rats evoked large dopamine signals, which seemed to reinforce what had previously been hypothesized. But when the researchers trained the rats for ICS, they no longer saw a big dopamine signal. "[T]he animal would push the bar, and the signal was just feeble," says Wightman. 'We just couldn't understand [it]." Wightman's group tentatively concluded that, contrary to all other evidence, dopamine was not needed for continuous reward in ICS. But they could not explain why the dopamine signal was different during training. Then, by chance, they trained a rat on a Friday but didn't conduct the experiment until the following Tuesday. This time, the dopamine signal was huge. "The difference between this experiment and every other experiment we'd ever done was we had always trained animals the day before and conducted the experiment the next day. And we've always had much less dopamine than we would expect," Wightman explains. "This time, we let the rat rest over the weekend, and he had tons and tons of dopamine." The researchers quickly realized that this result supported their tentative conclusion. "Dopamine is only transiently released at the beginning of the experiment, and then the supply of dopamine either runs out or gets turned off," he says. "So I like to think of dopamine, not as a reward, but as signaling something novel. If you learn something one day and do it the next it's not novel. There's no dopamine. But if you have to re-learn how to do something, it's novel Then dopamine sets involved" The 2roup also found that rats that
Voltammetric changes measured (a) during ICS training and (b) 30 minutes later, when the level of dopamine released is much lower. Dopamine concentration changes are shown at the top, and topographical data is at the bottom. The vertical lines represent occasions when the animal self-stimulated by pressing the bar. (Adapted with permission. Copyright 1999 Macmillan Magazines.)
Analytical Chemistry News & Features, May 1, 1999
were prevented from releasing pamine couldn't learn the behavior for the idea that dopamine is involved in learning.
