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.
Thesefindingsdon't necessarily mean that other researchers' results were wrong, Wightman says. "Previous measurements used microdialysis," he explains. "Because of the nature of the measurements, you collect data over a 5-minute period, essentially averaging it." A big spike that falls off quickly might look like a sustained release. But with the rapid measurements of cyclic voltammetry, it's easy to tell the difference. Wightman says the task now is to find out what dopamine really does. His lab will study a variety of novelty behaviors—such as an animal's reaction to being startled—to see if dopamine is present. Somebody also has to pinpoint the neurotransmitter involved in addiction. Wightman says he has a few ideas. And with any luck, the culprit will be one he can detect with cyclic voltammetry. Elizabeth Zubritsky
dent on pH or electroosmotic flow, or on whether you use cationic or anionic micelles, and if all that's involved is adding salt to the sample, it seems that it should be applicable to most MCE systems." Landers describes the stacked micelles as "magnets for hydrophobic analytes". "By stacking the micelles at the front of the sample plug," he says, "you essentially create a super-concentrated plug of magnets that migrates through the sample plug and effectively collects all of the analytes into a very sharp zone." The width of the individual analyte peaks depends their affinity for the micelles. Palmer points out that the affinity of all of the analytes increases with micelle concentration "Our mechanism is efficient because stack the micelles before they interact with the analyte " In this system sodium chloride was used to adjust the conductivity of the sample matrix But Palmer says there is a caveat about the
MCE stacks up Field-amplified stacking, in which the separation buffer has a much higher ionic strength than the sample matrix, is a common method of reducing the width of the sample zone and concentrating analytes in CE. Sample stacking has also been used with micellar CE (MCE), but most of the existing methods are "finicky"—requiring a particular combination of micelle type, sample pH, electroosmotic flow rate, and separation-mode polarity. In addition, the difference in ionic strength between separation buffer and sample matrix is often achieved by diluting the sample, which compromises sensitivity. A remarkably simple method described by James P. Landers, James Palmer, and Nicole J. Munro of the University of Pittsburgh has no such problems {Anall Chem. 1999, 71,1679-87). .nstead of stacking the analytes in the sample matrix, they cause the micelles to stack by adding salt (in this case NaCl) to the sample so that the conductivity of the sample is 2-4 times that of the separation buffer. The stacking of the micelles leads, in turn, to sample stacking but without the need for sample dilution. Infield-amplifiedstacking, the species to be stacked starts out in a low-conductivity region where it has a higher velocity because it carries all the charge. The analytes are slowed upon reaching the region of higher conductivity, which stacks the analyte (or micelles in this case) into a very narrow zone. In normal field-amplified
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The proposed mechanism of high-salt stacking. (A) Conventional field-amplified stacking with a low ionic strength sample matrix. (B) High-salt stacking with a sample matrix of ionic strength greater than the separation buffer.
stacking, the stacking phenomenon occurs with the analytes in the sample matrix. In this method, the stacking occurs with the micelles in the separation buffer outside the sample matrix. "What we describe in this paper is really counterintuitive," says Landers. "We actually increase the ionic strength of the sample to get the opposite effect [of standard field-amplilied stacking]. The advantage of doing this is that it's always easy to add something to your sample. You essentially can keep the sample concentration intact and just add salt. You now get a stacking effect that will enhance detection sensitivity as opposed to diminishing it." They demonstrated this "universal concept" with normal MCE, meaning that the system includes an anionic micelle (sodium cholate), electroosmoticflow,and normal polarity (cathode on the detector side). However, they believe that their method is not constrained to these conditions. "I don't think you can say that [the method will work with every buffer system] until you try every buffer system," says Landers. "The bottom line is that if it's not depen-
are nonmicellar systems that have a lot of negative chare-ps on them like his-hly sulfated 1H t ' i K h'th
complexing agents have pretty high mobilities, he says. You need the ionic component of your sample matrix to have a higher ionic mobility than your micellar carner. We knew it intuitively, but we p,oved it with highly sulfated B-cyclodextrins, which could not be stacked with any conn i
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"ji IT' as a mucu u-lgu er anodic morality, were we nai y arae to stacK tne suuatea y-cyclodextnns. Landers predicts that this method will make some biological fluids, which tend to be high in ionic strength, easier to work with. "In the past, some biological samples have been a nightmare to work with by CE because the high ionic strength was counterproductive. Now, if a micellar system is in the separation mode, the naturally occurring high ionic strength in those fluids may aid you in stacking and thus improve the detection sensitivity." Celia Henry
Analytical Chemistry News & Features, May 1, 1999 311 A
