Biomedical subjects

N J Munro

Publications and source records attributed to N J Munro.

5 recordsLinked to original sources

PubMed2001

Robust polymeric microchannel coatings for microchip-based analysis of neat PCR products.

Several silica coatings have been evaluated for replicate PCR product analysis in capillaries and electrophoretic microchips. Silica coatings are an essential component to many electrophoretic separations, and this importance is magnified in microchips, where separation distances are minimized. Increasing the resistance of coatings to separation conditions improves the reproducibility and longevity of the coated microchip, which allows for the full potential of these devices (rapid separations, high through-put, and longevity) to be realized. In this study, several coating parameters have been evaluated experimentally and through the literature to produce a coating with high resistance to the separation conditions of interest, neat PCR product separations. Coating degradation induced under these conditions was tested for several coatings, and the influence of surface hydroxylation, surface hydration, silanization solvent, silanizing reagent, catalysis, endcapping, and polymerization procedure are discussed. Under the testing conditions, a coating (coating E) prepared by silanization with chlorodimethyloctylsilane in toluene with a polymer layer of poly(vinylpyrrolidone) attached by a hydrogen abstraction method [Srinivasan, K.; Pohl, C.; Avdalovic, N. Anal. Chem. 1997, 69, 2798-2805] was most resistant. This coating was tested for longevity on electrophoretic microchips and was compared to the traditional coating of polyacrylamide. The coatings produced similar resolution and efficiencies; however, coating E provided more reproducible migration times and had performed for 635 analyses when testing was terminated. This procedure provides a reproducible, resistant surface coating, thus allowing for replicate analysis of neat PCR product on microchips.

Electrophoresis, Polyacrylamide Gel↗

PubMed2001

Electrokinetic injection for stacking neutral analytes in capillary and microchip electrophoresis.

An on-column mechanism for electrokinetically injecting long sample plugs with simultaneous stacking of neutral analytes in capillary electrokinetic chromatography is presented. On-column stacking methods allow for the direct injection of long sample plugs into the capillary, with narrowing of the analyte peak width to allow for an increase in the detected signal. Low-pressure injections (approximately 50 mbar) are commonly used to introduce sample plugs containing neutral analytes. We demonstrate that injection can be accomplished by applying an electric field from the sample vial directly into the capillary, with neutral analytes injected by electroosmotic flow at up to 1 order of magnitude faster than the corresponding pressure injections. Since stacking occurs simultaneously with electrokinetic injection, stacking is initiated at the capillary inlet, resulting in an increased length of capillary remaining for separation. Reproducibility obtained for peak height and peak area with electroosmotic flow injection is comparable to that obtained with the pressure injection mode, while reproducibility of analysis time is markedly improved. Electrokinetic stacking of neutral analytes utilizing electroosmotic flow is demonstrated with discontinuous (high conductivity, high mobility) as well as continuous (equal conductivity, equal mobility) sample electrolytes. Injecting neutral analytes by electroosmotic flow affords a 10-fold or greater decrease in analysis times when capillaries of 50-microm i.d. or smaller are used. This stacking method should be exportable to dynamic pH junction stacking and electrokinetic chromatography with capillary arrays. Equations describing this electrokinetic injection mode are introduced and stacking of a neutral analyte on a microchip by electrokinetic injection using a simple cross-T channel configuration is demonstrated.

Journal Article↗

PubMed2000

Indirect fluorescence detection of amino acids on electrophoretic microchips.

Microfabricated devices enable rapid separations of a variety of clinically significant analytes, including DNA, proteins, and amino acids. However, absorbance detection has been difficult to achieve on these devices, prohibiting analysis of nonfluorophore-bearing or nonfluorescently tagged analytes. An alternative detection technique exploiting indirect fluorescence has been adapted to the electrophoretic microchip to provide fast analysis of amino acids, bypassing the need for absorbance detection or fluorescence derivitization procedures. Nineteen of the standard amino acids could be detected with an average detection limit of 32.9 microM (approximately 1.6 amol). Despite the fact that the detection sensitivity was lower than that achievable by labeling the amino acids with fluorescein isothiocyanate (approximately 1 nM), circumventing sample preparation and the difficulties inherent with tagging complex samples make this technique attractive for a variety of assays where sensitivity is not critical. To demonstrate the applicability to real sample matrixes, the analysis of urine with elevated amino acid levels is used as a model system where the elevated levels are indicative of a variety of pathologies including amino acid metabolism disorders and kidney malfunction. The minimal sample handling and rapid separations achievable by employing indirect detection on microchips provides the potential for high-throughput applications for certain amino acid analyses.

Amino Acids↗

PubMed1999

Charged cyclodextrin-mediated sample stacking in micellar capillary electrophoresis. A simple method for enhancing the detection sensitivity of hydrophobic compounds.

The development of on-line sample stacking techniques for enhancing limits of detection of neutral analytes in micellar capillary electrophoresis (MCE) has recently gained much attention. Utilizing high-conductivity sample matrices to invoke sample stacking is promising, but requires the limited use of sample solubilizing agents such as alcohols in the sample matrix. In this study, we show how simple replacement of the sample solvent (methanol) with a solution of sulfated beta-cyclodextrin (s beta-CD) allows a significant increase in the sensitivity of detection of model hydrophobic analytes. This increase in sensitivity is accompanied by significant peak sharpening. Sulfated CDs in the sample matrix allow for effective solubilization of hydrophobic analytes without the use of organic solvents such as methanol. The testing of various sample matrix s beta-CD concentrations for their effect on peak sharpening identified 3 to 5% as optimal for the estrogen standards. The use of a s beta-CD sample matrix allowed for hydrostatic injections (3.5 kPa) of 297 s, compared with 4 s when the analytes were dissolved in methanol. A mechanism explaining the s beta-CD-induced effect involves an analyte transfer mechanism where the s beta-CDs, despite providing anodic mobility to analytes in the sample zone, are able to transfer analytes to trailing separation buffer micelles for "recycling" back into the sample zone without compromising the stacking process. The overall improvement in sensitivity allows detection of estrogens in the parts-per-billion range and stands to improve the utility of MCE as a bioanalytical technique.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed1999

Molecular diagnostics on microfabricated electrophoretic devices: from slab gel- to capillary- to microchip-based assays for T- and B-cell lymphoproliferative disorders.

BACKGROUND: Current methods for molecular-based diagnosis of disease rely heavily on modern molecular biology techniques for interrogating the genome for aberrant DNA sequences. These techniques typically include amplification of the target DNA sequences followed by separation of the amplified fragments by slab gel electrophoresis. As a result of the labor-intensive, time-consuming nature of slab gel electrophoresis, alternative electrophoretic formats have been developed in the form of capillary electrophoresis and, more recently, multichannel microchip electrophoresis. METHODS: Capillary electrophoresis was explored as an alternative to slab gel electrophoresis for the analysis of PCR-amplified products indicative of T- and B-cell malignancies as a means of defining the elements for silica microchip-based diagnosis. Capillary-based separations were replicated on electrophoretic microchips. RESULTS: The microchip-based electrophoretic separation effectively resolved PCR-amplified fragments from the variable region of the T-cell receptor-gamma gene (150-250 bp range) and the immunoglobulin heavy chain gene (80-140 bp range), yielding diagnostically relevant information regarding the presence of clonal DNA populations. Although hydroxyethylcellulose provided adequate separation power, the need for a coated microchannel for effective resolution necessitated additional preparative steps. In addition, preliminary data are shown indicating that polyvinylpyrrolidone may provide an adequate matrix without the need for microchannel coating. CONCLUSIONS: Separation of B- and T-cell gene rearrangement PCR products on microchips provides diagnostic information in dramatically reduced time (160 s vs 2.5 h) with no loss of diagnostic capacity when compared with current methodologies. As illustrated, this technology and methodology holds great potential for extrapolation to the abundance of similar molecular biology-based techniques.

DNA, Neoplasm↗