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An electroosmotic flow (EOF)-based pump, integrated with a sol--gel stationary phase located in the electric field-free region of a microchip, enabled the separation of six nitroaromatic and nitramine explosives and their degradation products via liquid chromatography (LC). The integrated pump and LC system were fabricated within a single quartz substrate. The pump region consisted of a straight channel (3.0 cm x 230 [micro]m x 100 [micro]m) packed with 5-[micro]m porous silica beads. The sol--gel stationary phase was derived from a precursor mixture of methyltrimethoxy-and phenethyltrimethoxysilanes and was synthesized in the downstream, field-free region of the microchip, resulting in a stationary-phase monolith with dimensions of 2.6 cm x 230 [micro]m x 100 [micro]m. Fluid dynamic design considerations are discussed, especially as they relate to integrating the EOF pump with the LC system. Pump and separation performance, as characterized by flow rate measurements, injection, elution, separation, and detection, point to a viable analytical chemistry platform that encompasses all of the benefits expected of portable, laboratory-on-chip systems, including reduced sample requirements and small packaging.

In order to understand the role of stacked micelles in sample preconcentration, it is necessary to understand the factors that contribute to the micelle stacking phenomenon. Various MEKC background electrolyte (BGE) solutions were prepared in the presence of Sudan III in order to monitor the micelle stacking phenomenon in the anionic sodium dodecyl sulfate and sodium cholate micelle systems. The data show that micelle stacking is a dynamic process that is strongly dependent upon the relative conductivities of the sample matrix and BGE, the relative column length of the sample plug, and the mobilities of the ions involved in the stacking process regardless of electric field conditions (i.e., field-amplified stacking, sweeping, or high-salt stacking). Conditions under which micelle stacking can be expected to occur are presented, and the extent of micelle stacking is quantified. The micelle stacking phenomenon is correlated to the separation performance of a series of neutral alkaloids. It is shown that neutral analytes migrate rapidly through the evolving stacked micelle region in the initial moments of the separation. As a consequence of this transient interaction, analytes with small retention factors spend less time in the stacked micelle region and experience lower stacked micelle concentrations than analytes with large retention factors that spend more time in the growing stacked micelle region. It is also demonstrated that the extent of analyte enrichment generally increases with injection length, by facilitating greater interaction time with stacked micelles; however, enrichment will eventually plateau with increasing injection length as a function of an analyte's affinity for the micelle. Finally, it is shown that, in contrast to conventional wisdom, a range of long injection plugs exist where separation efficiency can be dramatically improved due to analyte interaction with an actively growing stacked micelle region.

Effective microchip extraction of deoxyribonucleic acid (DNA) from crude biological matrixes has been demonstrated using silica beads or hybrid phases composed of beads and sol--gel. However, the use of monolithic sol--gels alone for extraction of human genomic DNA has been more difficult to define. Here we describe, for the first time, the successful use of monolithic tetramethyl ortho-silicate-based sol--gels for effective micro-solid-phase extraction ([micro]SPE) of DNA in a glass microchip format. A functional monolithic silica phase with micrometer-scale pores in the silica matrix resulted from addition of poly-(ethylene glycol), a poragen, to the precursor mixture. This allowed a monolithic sol--gel bed to be established in a microchip channel that provided large surface area for DNA extraction with little flow-induced back pressure. DNA extraction efficiencies for simple systems ([lambda]-phage DNA) were ~85%, while efficiencies for the reproducible extraction of human genomic DNA from complex biological matrixes (human blood) were ~70%. Blockage of the sol--gel pores by components in the lysed blood was observed in repeat extraction on a single device as a decrease in the extraction efficiency. The developed [micro]SPE protocol was further evaluated to show applicability to clinical samples and bacterial cultures, through extraction of PCR-amplifiable DNA.

The ability to detect sub-nanomolar concentrations of ricin using fluorescently tagged RNA aptamers is demonstrated. Aptamers rival the specificity of antibodies and have the power to simplify immunoassays using capillary electrophoresis. Under nonequilibrium conditions, a dissociation constant, [K.sub.d], of 134 nM has been monitored between the RNA aptamer and ricin A-chain. With use of this free-solution assay, the detection of 500 pM (~14 ng/mL) or 7.1 amol of ricin is demonstrated. The presence of interfering proteins such as bovine serum albumin and casein do not inhibit this interaction at sub-nanomolar concentrations. When spiked with RNAse A, ricin can still be detected down to 1 nM concentrations despite severe aptamer degradation. This approach offers a promising method for the rapid, selective, and sensitive detection of biowarfare agents.

We have previously demonstrated on-column dynamic labeling of protein-SDS complexes on capillaries and microchips for laser-induced fluorescence (LIF) detection using both a commercially available fluor and a protein separation buffer. Upon binding to hydrophobic moieties (of the analyte or separation buffer), the fluor undergoes a conformational change allowing fluorescence detection at 590 nm following excitation with 488-nm light. Our original work showed on-chip limits of detection (LOD) comparable with those using UV detection (1 x [10.sup.-5] M) on capillaries-falling significantly short of the detection limits expected for LIF. This was largely a function of the physicochemical characteristics of the separation buffer components, which provided significant background fluorescence. Having defined the contributing factors involved, a new separation buffer was produced which reduced the background fluorescence and, consequently, increased the available dye for binding to protein--SDS complexes, improving the sensitivity in both capillaries and microchips by at least 2 orders of magnitude. The outcome is a rapid, sensitive method for protein sizing and quantitation applicable to both capillary and microchip separations with a LOD of 500 ng/mL for bovine serum albumin. Interestingly, sensitivity on microdevices was improved by inclusion of the dye in the sample matrix, while addition of dye to samples in conventional CE resulted in a drastic reduction in sensitivity and resolution. This can be explained by the differences in the injection schemes used in the two systems. The linear range for protein quantitation covered at least 2 orders of magnitude in microchip applications. On-chip analysis of human sera allowed abnormalities, specifically the presence of elevated levels of [gamma]-globulins, to be determined.

A microchip solid-phase extraction method for purification of DNA from biological samples, such as blood, is demonstrated. Silica beads were packed into glass microchips and the beads immobilized with sol-gel to provide a stable and reproducible solid phase onto which DNA could be adsorbed. Optimization of the DNA loading conditions established a higher DNA recovery at pH 6.1 than 7.6. This lower pH also allowed for the flow rate to be increased, resulting in a decrease in extraction time from 25 min to less than 15 min. Using this procedure, template genomic DNA from human whole blood was purified on the microchip platform with the only sample preparation being mixing of the blood with load buffer prior to loading on the microchip device. Comparison between the microchip SPE ([mu]chipSPE) procedure and a commercial microcentrifuge method showed comparable amounts of PCR-amplifiable DNA could be isolated from cultures of Salmonella typhimurium. The greatest potential of the [mu]chipSPE device was illustrated by purifying DNA from spores from the vaccine strain of Bacillus anthracis, where eventual integration of SPE, PCR, and separation on a single microdevice could potentially enable complete detection of the infectious agent in less than 30 min.

The continual or on-demand delivery of aerosol is important for sensor validation and the development of trace particle detection methodologies. The system presented here operates by introducing aerosolized analytical standard solutions in a heated air flow, generating vapor phase analytes that are subsequently transported and cooled to provide aerosolized particles. The notable design feature was a small concentric air flow device that blends chilled dry air with vapor phase analyte to induce a phase transition without the need for volatile organic solvents. While organic solvents accelerate solvent evaporation, they present potential interferences for downstream sensors. Cyclotrimethylenetrinitramine (RDX) particles were generated and optically counted from the exhaust plume aerosol. The solid RDX material was binned into 1–2.5, 2.5–5, 5–10, and 10+ µm diameter sizes and when the system was actively cooled, the generation of particles exhibited a relative increase of 4.9%, 27.6%, 95.6%, and 78.0% in production, respectively. When observed under a microscope, collected RDX particles ranged from about 1 to 7 µm in diameter. It was determined that the production of solid material was proportional to the temperature and mass loading of the vapor, as dictated by the feed solution concentration; cooler temperatures and higher concentrations led to the increased production of particles ranging from 1 to 10 μm in diameter for the low-vapor pressure analytes studied: RDX (cyclotrimethylenetrinitramine), PETN (pentaerythritol tetranitrate), TNT (2-methyl-1,3,5-trinitrobenzene), and 2,6-DNT (2,6-dinitrotoluene). [ABSTRACT FROM AUTHOR]