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limiting the correct use of these drugs. As Rosamund Williams, head of WHO’s team working on drug resistance, says, “If we fail to make full and proper use of medicines discovered in our lifetime, many of these drugs will slip through our grasp.” “Proper use” involves choosing an agent that will wipe out the infection completely rather than only thin out the more susceptible cells, which promotes antibacterial resistance.
Because antibiotics are often specific to either Gram-negative or Gram-positive bacteria, Mikkelsen and her group chose Escherichia coli (Gram-negative) and Clostridium sporogenes (Gram-positive) as their subjects. E. coli was cultured in the presence of air, and C. sporogenes, an anaerobe, was grown in its absence. Thirteen antibiotics were evaluated with the electrochemical method, comparing results against the
conventional disk diffusion method. Currently, this approach is being extended to provide quantitative results, and it is applied to other microorganisms. A patent is pending, and more than one company is interested in commercial sales. And for those without electrochemistry skills, “we’re working on automation with electrochemical arrays at the moment,” says Mikkelsen. Gerald Keller
MEETING NEWS ACS National Meeting—Elizabeth Zubritsky reports from Washington, DC Monitoring gene expression with SPR The researchers report a detection limit of 10 nM for the 18-nt-long synthetic target and 2 nM for the 1500-ntlong natural sequence. Although the researchers acknowledge that fluorescence detection is more sensitive for small numbers of molecules, they note that SPR imaging can detect the adsorption of biological molecules at nanomolar concentrations and has the advantage of being a label-free method.
IR SPR imaging. In this approach, both the wavelength and incident angle of the light are fixed, and the reflectivity at various points on the surface is measured, allowing the researchers to look at the entire microarray at once. Two types of targets, which were derived from the 16S fraction of the ribosomal RNA (rRNA) in bacteria—a sequence that can be used to distinguish among bacterial species—were used. The first target was a synthetic, 18-ntlong rRNA oligo. The second target was a natural, full-length 16S rRNA (~1500-nt long) sequence, which was denatured to eliminate the troublesome secondary structure that often occurs in single-stranded RNA.
CE for single cell analysis
ROBERT CORN
Surface plasmon resonance (SPR) seems intent on being a player in the genomics arena. It has already been used for DNA hybridization studies and, more recently, for single nucleotide polymorphism detection. Now Robert Corn and colleagues at the University of Wisconsin– Madison are using it for microarraybased measurements of RNA–DNA hybridization, the most popular application of which is gene expression monitoring. Although some early work describing the use of SPR to detect RNA hybridization has been published, Corn and colleagues note that these studies did not use an array format. In contrast, the new work was carried out using microarrays that were fabricated on gold-coated glass microscope slides. Thiol-modified DNA probes—each 18 nucleotides (nt) long with a 15-T spacer—were arranged in a grid of 500 µm 3 500 µm squares, with a surface coverage of 1 3 1012 molecules/cm2. The area surrounding the probe spots was coated with polyethylene glycol to prevent the nonspecific binding of target oligonucleotides (oligos). Rather than use traditional SPR—in which the wavelength of the light is fixed and the angle of incidence is varied, or the angle is fixed and the wavelength varied—the researchers use near-
Microarray with an RNA target binding onto two of four DNA probes.
Eager to move beyond “bulk” studies of cell populations, analytical chemists are hard at work on single cell analysis. And as one might expect, the technique of choice is often CE. Although the foundation for these studies was laid several years ago, single cell analyses are still no easy task. For example, if a cell contains ~5 fmol of total protein and expresses ~10,000 different proteins, then the average expression level is in the zmol range. One approach to this detection problem is “chemical cytometry”, developed by Norm Dovichi at the University of Alberta (Canada) and colleagues. Unlike its namesake, flow cytometry, chemical cytometry emphasizes the comprehensive characterization
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MEETING NEWS of a cell’s contents rather than a few components. A cell is drawn into a capillary and lysed, and the liberated proteins are fluorescently labeled, separated using CE, and detected with laser-induced fluorescence. Researchers using this method have already reported a resolution of ~30 components from individual cultured human cancer cells (Anal. Chem. 2000, 72, 872–877). They also have noted significant variations in the protein levels from cell to cell—even for proteins that are expressed at high levels—which they attribute, in part, to cells being in different stages of the cell cycle at the time of analysis. In more recent work on the embryogenesis of Caenorhabditis elegans, the researchers have detected differential
protein expression in the AB and P1 cells, which are the products of the very first division of the fertilized P0 cell. CE-based single cell studies using laser-induced native fluorescence detection are being performed by Sheri J. Lillard at the University of California– Riverside and colleagues. Using a system that Lillard worked with while in Edward Yeung’s lab at Ames Laboratory–USDOE and Iowa State University, her group is now pushing for higher throughput. For the analysis of hemoglobin and carbonic anhydrase, they have achieved an average run time of
