Analytical Currents: Investigating on-column fluorescence lifetimes

micro-LC/MS. Combinatorial chemistry has presented the pharmaceutical industry with a whole new set of analytical challenges. Large numbers of samples...
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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 capillary precolumn coupled directly to a Combinatorial chemistry has presented the pharmaceutical industry with a whole valve-switching device. Analytes are trapped in the precolumn, and an aquenew set of analytical challenges. Large ous mobile phase is passed through to numbers of samples must be analyzed remove ion-suppressing contaminants. rapidly, and in many cases, single beads With the flick of a switch, the trapped must be analyzed, where the amount of analytes are back-flushed into the material is very small (pico-/subnanosource of the mass spectrometer with mole level). In addition, samples often an organic mobile phase, and the aquecontain cleaving material from the beads ous phase goes out to waste. After the and solvents (e.g., dimethyl sulfoxide), which interfere with MS detection. Peter back-flush, the valve is switched back to re-equilibrate the precolumn with aqueS. Marshall of Glaxo Wellcome (U.K.) ous mobile phase. In addition to providreports on a rapid back-flush microsepaing rapid, in-line removal of interfering ration system for the analysis of combicomponents, 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 varrous 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

Analytical Chemistry News & Features, July 1, 1999 4 3 5 A


than in the presence of dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or 4% noncross-linked polyacrylamide gel. Organic additives also appeared to affect the fluorescence lifetimes in some cases. Although multiexponential decays were observed with batch solutions, they were usually eliminated in on-the-fly experiments. Polyacrylamide gels made with various cross-linkers, total acrylamide concentrations, and cross-linker concentrations were also tested. Fluorescence lifetimes determined in batch solution were significantly different from those determined on-the-fly during CE (1.0-1.5 ns compared with 4-5 ns), and mis variation was attributed to general effects of the interaction between the dye-labeled primers and the polyacrylamide matrix There were no significant differences due to gel composition which implies that fluorescence lifetimes cannot be optimized this way However Li and McGown note that ti also means that fluctuations in gel conroosition will not complicate analyses(J.Chromatogr. A 1999 841, 95-103)

Imprinting proteins Attempts to make molecularly imprinted polymers using proteins as the template have met with only limited success. That may be about to change. Buddy D. Ratner and co-workers at the University of Washington and Universita Degli Studi Di Trento (Italy) have developed a method that allows them to imprint protein recognition sites on surfaces. In their method, the template protein is adsorbed on mica. They chose mica because it is atomically flat (therefore, the surface topography will be due only to the

Nanotube electrode Small continues to be beautiful. In this case, what is small is an electrode constructed from a single carbon nanotube. Carbon nanotubes—a by-product of the "buckyball" series of carbon compounds—offer unique geometric, electronic, and mechanical properties. Richard M. Crooks and colleagues at Texas A&M University attach 80- to 100-nm-diameter nanotubes to 15- to 50-um long, sharpened platinum wires to fabricate the electrodes. As would be expected, the limiting current of these electrodes scaled linearly with the depth of immersion into the electrolyte solutions. The Texas researchers also electrically insulated the electrodes with polyphenol using a new procedure for preparing polyphenol that yielded thinner and less permeable coatings and

protein recognition sites), and because its negatively charged surface minimizes protein denaturation. The adsorbed proteins are coated with disaccharide, which forms multiple hydrogen bonds with the protein upon dehydration. This "sugar shell" protects the protein from drying-induced denaturation and plasma-induced degradation, preserving the protein's structural integrity so that the recognition information can be transferred to the imprinted cavity. A fluoropolymer is formed on top of the sugar-coated protein by radio-frequency glow-discharge plasma deposition. The dep-

Protocol for the template imprinting of proteins. The protein is adsorbed onto freshly cleaved mica. A solution of disaccharide is spin-cast to form a sugar overlayer. A layer of C3F6 is formed on the disaccharide by radio-frequency glow-discharge plasma deposition. The mica is then peeled away and the protein is removed. (Adapted with permission. Copyright 1999 Macmillan Magazines.) 436 A

Analytical Chemistry News & Features, July 1, 1999

Cyclic voltammograms with an uninsulated nanotube electrode as a function of immersion depth into a solution of 5 mM Ru(NH3)63+ and 0.1 M K2SO4..

limited electrochemical activity to the tip. These new electrodes are strong and have high length-to-diameter aspect ratios, which could be important for scanning electrochemical microscopy and bioanalytical applications. (J. Am. Chem. Soc. 1999, 121, 3779-80)

osition process covalently incorporates the disaccharide into the polymer film, fixing the location of the disaccharide hydroxyls that are involved in hydrogen bonding, but it does not disturb the protein. The mica is then peeled away, and the sample is soaked in a NaOH/NaCIO solution to dissolve and extract the protein. Caviites wiih recognition sites specific to the protein (in the form of the precisely positioned sugar hydroxyl groups) are left in the polymer film. The researchers used this method to imprint the proteins bovine serum albumin, immunoglobulin G, andfibrinogen.Atomic force microscopy revealed that the cavities resembled the shapes of the template proteins. For example, the cavities formed by fibrinogen were elongated trenches. Analysis of the surface chemistry by other techniques indicated that it is dominated by sugar species and that the extraction process does indeed remove the protein from the imprints. When die proteins were adsorbed onto various templates and control surfaces, the amount of protein adsorbed was slightly higher for the templated surfaces. In the absence of competition, proteins would adsorb onto any protein recognition site, indicating nonspecific protein adsorption. However, in assays of binary protein mixtures, the imprints preferentially adsorbed their templates under competitive binding conditions. (Nature 1999, 398, ,93-97)