Andrew W. Lantz

The use of cationic surfactants and ionic liquids in the detection of microbial contamination by capillary electrophoresis

A rapid test of whether a laboratory sample contains any microorganisms is important and necessary for many areas of science and technology. Currently, most of the standard procedures for the detection of aerobic bacteria, anaerobic bacteria and fungi, require the preparation of microbial cultures in respective growth media, which are dramatically slow. Different approaches providing fast analysis such as CE are becoming more desired. To compensate for the natural electrophoretic heterogeneity of microbes, various buffer additives were examined to stack all bacteria and fungi in a sample plug into a single peak. This peak was removed from the molecular contaminants in the sample to prevent false positives. Both cationic surfactants and ionic liquids (IL) were investigated as run buffer additives and they are both widely applicable to different species of bacteria and fungi. Given that high concentrations of surfactants can potentially lyse cells, dicationic IL offer attractive auxiliary buffer additives for use in CE‐based sterility tests. The analysis can be completed in 10 min, thus providing a great advantage over traditional direct inoculation methods that require several weeks to complete.

Combined capillary electrophoresis and DNA‐fluorescence in situ hybridization for rapid molecular identification of Salmonella Typhimurium in mixed culture

CE, long a staple in analytical chemistry for molecular separations, has recently been adapted for separating heterogeneous mixtures of microbial cells based on intrinsic differences in cell morphology and surface charge. In this application, CE enables effective separations of both relatively broad categories of cells, as well as of more similar cell types. As a phenotypic approach, CE may be less applicable to certain populations, including those comprised of pleiomorphic cells or chain‐forming cells, where differences in cell size, shape, or chain length may lead to broad, “unfocusable” distributions in cell surface charge. At the other end of the spectrum, closely related species having similar surface charge profiles may not be separable via CE alone. Successful combination of microbial CE with a compatible method for generating cell‐specific signals could address these limitations, increasing the diagnostic power of this approach. Fluorescence in situ hybridization (FISH) is a rapid molecular technique for fluorescence‐based labeling of whole target cells. In this work, we combined a simple CE‐based presence/absence test with FISH to develop a bacterial detection assay having an additional “layer” of molecular specificity. Using this approach, we were able to differentiate Salmonella Typhimurium from Escherichia coli in mixed populations via CE. Both hybridizations and CE run times were short (10–15 min), bacterial populations were highly focused (∼2–3 s peak width) and there was no need for a posthybridization wash step. As few as three injected cells of S. Typhimurium were detected against a background of ∼300 injected E. coli cells, suggesting the possibility for single‐cell detection of pathogens using this technique. This proof of concept study highlights the potential of CE‐FISH as a promising new tool for molecular detection of specific bacterial cells within mixtures of closely related, physiologically inseparable populations.

Cyclodextrins as complexation and extraction agents for pesticides from contaminated soil.

The binding constants of seven commonly used pesticides (2,4-D, acetochlor, alachlor, dicamba, dimethenamid, metolachlor, and propanil) with native and derivatized cyclodextrins (α-CD, β-CD, γ-CD, hydroxypropyl-β-CD, methyl-β-CD, sulfated-β-CD, and carboxymethyl-β-CD) were measured using affinity capillary electrophoresis. All cyclodextrins showed significant binding interactions with each of the seven pesticides investigated, with the exception of sulfated-β-CD which exhibited negligible binding to acetochlor, alachlor, and metolachlor. Propanil was found to bind most strongly to the cyclodextrins in this study. The ability of cyclodextrins to extract these pesticides from contaminated soil was also assessed. A general correlation between the pesticide-cyclodextrin binding constants and the percent extraction enhancements was found. In most cases, aqueous cyclodextrin extraction of pesticides from soil produced soluble pesticide-cyclodextrin complexes with a Type AL solubility diagram. Hydroxypropyl-β-CD and methyl-β-CD generally displayed the greatest levels of extraction enhancement. However, most pesticides with γ-CD (and a few cases with α-CD and β-CD) produced relatively insoluble pesticide-cyclodextrin complexes in these soil extraction studies, resulting in Type BS solubility diagrams. Therefore, the measured aqueous extraction level for these pesticide-cyclodextrin combinations was lower relative to the control (1.0mM phosphate at pH=7.0). The results of this study may be used for future novel methods of contaminated soil remediation, which overcome the disadvantages of organic solvent and surfactant use. In addition, such binding studies may be applicable toward the development of pesticide-cyclodextrin formulations.

High Efficiency Liquid and Super‐/Subcritical Fluid‐Based Enantiomeric Separations: An Overview

Abstract This overview covers chiral separations using high‐performance liquid chromatography, supercritical fluid chromatography, and capillary electrophoresis. This is not intended to be a comprehensive review of all papers published in these areas. Chiral selectors can be categorized into a few classes: i.e., macrocyclic chiral selectors, polymeric chiral selectors, π–π interaction chiral selectors, ligand exchange chiral selectors, and miscellaneous/hybrid chiral selectors. The focus of this work is to outline the basic principles, structural features, and usefulness of each class of molecules. The evolution and major developments within each class of chiral selectors will be examined as well.

Rapid identification of Candida albicans in blood by combined capillary electrophoresis and fluorescence in situ hybridization.

A CE method based on whole‐cell molecular labeling via fluorescence in situ hybridization was developed for the detection of Candida albicans in whole blood. Removal of potentially interfering red blood cells (RBC) with a simple hypotonic/detergent lysis step enabled us to detect and quantitate contaminating C. albicans cells at concentrations that were orders of magnitude lower than background RBC counts (∼7.0×109 RBC/mL). In the presence of the lysed blood matrix, yeast cells aggregated without the use of a blocking plug to stack the cells. Short (15 min) hybridizations yielded bright Candida‐specific fluorescence in situ hybridization signals, enabling us to detect as few as a single injected cell. The peak area response of the stacked Candida cells showed a strong linear correlation with cell concentrations determined by plate counts, up to ∼107 CFU/mL (or ∼1×104 injected cells). This rapid and quantitative method for detecting Candida in blood may have advantageous applications in both human and veterinary diagnostics.

Enantiomeric Separation of Synthetic Amino Acids Using Capillary Zone Electrophoresis

Abstract Three chiral selectors, sulfated α‐cyclodextrin (SAC), sulfated β‐cyclodextrin (SBC), and carboxymethyl β‐cyclodextrin (CMBC) were examined as run buffer additives for the separation of sixteen racemic synthetic amino acids and three prepared mixtures of chiral synthetic amino acids, using capillary zone electrophoresis. Seventeen of the nineteen synthetic amino acids were enantiomerically separated and fourteen of them were optimized to baseline using one or more chiral running buffer additives. SAC, with eleven baseline and three partial separations, and SBC, with ten baseline and four partial separations, were found to be more broadly useful than CMBC. Increasing the chiral selector concentration improved the enantioresolution, but also produced longer analyses times. Addition of organic modifier (ethanol) increased migration times and decreased enantiomeric resolution. Increasing the pH of the run buffer decreased analyses time as well as resolution. Decreasing the applied voltage generally improved resolution.