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ANALYTICAL CURRENTS
Natural channel meets cyclodextrin In a variation on a natural theme, Orit Braha, Hagan Bayley, and co-workers at Texas A&M University created a unique sensor by placing a cyclodextrin inside a cellular transmembrane channel. The result is a stochastic sensor, which detects and quantifies molecules by observing individual binding events between analyte molecules and a single receptor. The sensor in this study was built with the polypeptide a-hemolysin, an exotoxin secreted by the bacterium Staphylococcus aureus. a-Hemolysin self-assembles in bii layers to form a 100-A-long channel—in essence, a tunnel through a cell wall. At micromolar concentrations, cyclodextrins enter this channel and form a reversible partial block, which affects the ionic current. In the case of fS-cyclodextrin, kinetics indicate that there is a single binding site for the molecule inside the channel. The addition of 2-adamantamine or 1-adamantanecarboxylic acid to the cyclodextrin channel further reduced the current, with the two analytes producing different responses. Residence times inside the partially blocked channel for the two analytes also differed, providing another means for identifying the compounds. However, the residence time was independent of analyte concentration; instead, the frequency of association was linearly dependent on concentration. Moreover, in a mixture, individual binding events could be distinguished, indicating that the analytes compete for a single binding site in the oc-hemolysin-p-cyclodextrin complex The concept of stochastic sensing with this approach was demonstrated by using fcyclodextrin to identify and quantify the tricyclic antidepressant imipramine from the antihistaminic promethazine, which have similar structures. This novel sensor approach can obviously be extended to other systems, and the authors draw an analogy to the process of olfaction, in which carrier molecules deliver odorants to membrane-bound receptors. (Nature 1999, 398, 686-90)
natorial library samples, which is designed to overcome all three problems. At the heart of the system is a capilCombinatorial chemistry has presented lary precolumn coupled directly to a the pharmaceutical industry with a whole valve-switching device. Analytes are new set of analytical challenges. Large trapped in the precolumn, and an aquenumbers of samples must be analyzed ous mobile phase is passed through to rapidly, and in many cases, single beads remove ion-suppressing contaminants. must be analyzed, where the amount of With the flick of a switch, the trapped material is very small (pico-/subnanoanalytes are back-flushed into the mole level). In addition, samples often source of the mass spectrometer with contain cleaving material from the beads an organic mobile phase, and the aqueand solvents (e.g., dimethyl sulfoxide), ous phase goes out to waste. After the which interfere with MS detection. Peter back-flush, the valve is switched back to S. Marshall of Glaxo Wellcome (U.K.) re-equilibrate the precolumn with aquereports on a rapid back-flush microsepaous mobile phase. In addition to providration system for the analysis of combiing rapid, in-line removal of interfering components, the back-flush system can be used to concentrate samples. Multiple injections can be made before switching' the backflush valve When switching back and forth from aqueous to organic phases, there is the danger of the sample precipitating out of solution; however, the authors saw no indication of it. The system remained unclogged after analyzing 1056 samples (11 microwell plates x 96 Schematic of a rapid back-flush microseparation wells each). In addition, masystem. On the left, the precolumn is back-flushed terial from 23 single beads with an organic phase (solvent B), and the aqueous from a core library phase (solvent A) is directed to waste. On the right, cessfully identified. (Rapid valve 2 has been switched to re-equilibrate the Commun. Mass Spectrom. precolumn with the aqueous phase. (Adapted with 1999 13 778-81) permission. Copyright 1999 John Wiley & Sons, Ltd.)
Back-flush micro-LC/MS
Investigating on-column fluorescence lifetimes Fluorescent dyes used in DNA sequencing are usually discriminated by their wavelengths, but they can also be discriminated by their fluorescence lifetimes. Few commercial dyes are optimized for the latter approach, however, so Lijuan Li and Linda B.
McGown at Duke University investigated the effects of gel matrixes and experimental conditions on fluorescence lifetimes. Using DNA primers labeled with various fluorescent dyes, the researchers evaluated the sieving buffer hydroxyethylenecellulose with several organic modifiers. Better S/B ratios (the signal compared to the background level) were obtained in the presence of water, methanol, formamide, or glycerol
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