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Analytical scientists are just about getting the hang of separating analytes at the tiny scales of capillary electrophoresis (CE) microchips. But detecting them is something else entirely. Conventional detection techniques, such as UV detection and laser-induced fluorescence detection, require bulky equipment that can be difficult to shrink down to small scales. Electrochemical detection techniques like conductivity detection and amperometric detection are more naturally suited to small scales but can be fiddly to set up and tend to suffer from interference from the separation current. But now David Ross and Jason Kralj at the US National Institute of Standards & Technology in Gaithersburg have turned this small scale to their advantage. They have shown that if the separation channel is short enough the separation current itself can be used to detect analytes, thereby completely doing away with the need for a separate detector. Ross and Kralj are able to conduct separations in such short channels by utilising a novel CE technique recently developed by Ross termed gradient elution moving boundary electrophoresis (GEMBE; see Paddle your own canoe). This involves applying a flow in the opposite direction to an analyte's natural direction of travel during electrophoresis. The flow is generated as a result of both normal electroosmosis and by applying air pressure to one end of the channel, giving the scientists fine control over the strength of the flow. The idea is gradually to reduce this flow during the course of the separation, so that at any point in time only those analytes with electrophoretic mobilities higher than the flow rate can migrate down the channel. It's analogous to canoeists trying to paddle up-river, only those strong enough to overcome the current are able to move. Indeed, Ross has such fine control over the flow that he can get analytes to enter the separation channel one at a time. This means that no actual separation takes place in the separation channel, and so it can be very short. Ross quickly realised that this set-up allowed him to do away with the need for a separate detector. 'Because the channel is so short and because of the separation method that we use (GEMBE), the separation current can be used in place of a detector signal,' explains Ross. All he needs to do is monitor the characteristic changes to the separation current as each analyte migrates in turn along the channel. To test whether this technique would work in practice, Ross and Kralj constructed a device consisting of 16 separation channels, each just 3mm long. They then used it to study enzyme activity: specifically the phospohorylation of the protein kemptide by the enzyme protein kinase A (PKA), which is commonly utilised in drug discovery research. This process is powered by the conversion of adenosine triphosphate (ATP) to adensosine diphosphate (ADP). So Ross and Kralj decided to use their device to determine the relative concentrations of each of these molecules: the greater the ADP concentration relative to ATP, the greater the enzyme activity. First off, the researchers monitored this process in just one channel, mixing together kemptide, PKA and ATP at one end of the channel and then gradually reducing the flow. ATP and ADP both migrate fairly quickly and, aside from two impurities, were the first molecules to make their way through the channel. As they were the only molecules of interest, the whole process was over in a few minutes. Ross and Kralj found that the two molecules migrated through the channel separately, generating distinct changes in the separation current that could be displayed as chromatographic peaks. Following this success, the researchers conducted the reaction in all 16 channels with different concentrations of PKA. As expected, low PKA concentrations produced chromatograms with high ATP peaks and low ADP peaks, while high concentrations produced low ATP peaks and high ADP peaks. The reverse happened when they used the same enzyme concentration in each channel but 15 different concentrations of an enzyme inhibitor. Ross is now building on these results. 'Currently we're working on the basic theory for the technique to better understand how different device geometries affect the performance and on developing better ways to fabricate devices,' he told separationsNOW. Related links:
The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd. |
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