The development of a chip-based sensor array composed of individually addressable polystyrene−poly(ethylene glycol) and agarose microspheres has been demonstrated. The microspheres are selectively ...arranged in micromachined cavities localized on silicon wafers. These cavities are created with an anisotropic etch and serve as miniaturized reaction vessels and analysis chambers. A single drop of fluid provides sufficient analysis media to complete ∼100 assays in these microetch pits. The cavities possess pyramidal pit shapes with trans-wafer openings that allows for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microspheres. Identification and quantitation of analytes occurs via colorimetric and fluorescence changes to receptor and indicator molecules that are covalently attached to termination sites on the polymeric microspheres. Spectral data are extracted from the array efficiently using a charge-coupled device allowing for the near-real-time digital analysis of complex fluids. The power and utility of this new microbead array detection methodology is demonstrated here for the analysis of complex fluids containing a variety of important classes of analytes including acids, bases, metal cations, metabolic cofactors, and antibody reagents.
Minute quantities of native cellular fluorophores can be quantitatively assayed using ultraviolet fluorescence detection with microcolumn separations, but spectral diversity of biological ...chromophores imposes serious limitations on the use of this strategy to investigate biological components. We present an approach for rapid characterization of picoliter samples containing dissimilar cellular fluorophoresincluding amino acids, monoamine neurotransmitters, flavins, and pyridine nucleotidesusing multiphoton excited fluorescence detection coupled to capillary electrophoresis separations. In this highly versatile approach, biological fluorophores are excited through the nearly simultaneous absorption of different numbers of low-energy photons. Because spectrally distinct species all can be excited with a single, long-wavelength laser source, fluorescence throughout the ultraviolet and visible regions can be detected efficiently with extremely low background. Samples containing serotonin, melatonin, FAD, and NADH can be reproducibly analyzed in 5-μm and 2-μm i.d. channels. Detection limits in 5-μm capillaries range from 350 zmols (38 nM) for FAD to 27 amols (1.0 μM) for serotonin. Use of 2-μm channels is shown to improve the mass detection limit for serotonin approximately as the decrease in capillary cross-sectional area (LOD ≈ 4 amol), and further reductions in mass detection limits are projected for analysis with even smaller diameter channels that better match the submicron size of the diffraction-limited multiphoton focal spot.
An enzyme-based sensor array has been developed to detect multiple disaccharides in aqueous solutions. Porous agarose beads, derivatized with enzymes for assaying disaccharides, are localized within ...wells etched into a silicon chip in a regular 5 × 7 array. Each well is individually addressable and acts as a microanalysis chamber where sample solution passes through the agarose matrix and is exposed to the enzymes. Detection is achieved by observing the increase in absorbance of a quinoneimine dye produced during the reaction. This technique is used to quantify the disaccharides lactose, sucrose, and maltose and the monosaccharide glucose. Preexisting glucose in the sample complicates multicomponent sensing but can be accounted for by including a glucose sensor in the array. This detection strategy is applied to the simultaneous analysis of these sugars in several beverages.
We have examined the effects of dissolved molecular oxygen on multiphoton-excited (MPE) photochemical derivatization of serotonin (5HT) and related cellular metabolites in various buffer systems and ...find that oxygen has a profound effect on the formation efficiency of visible-emitting photoproducts. Previously, end-column MPE photoderivatization provided low mass detection limits for capillary electrophoretic analysis of hydroxyindoles, but relied on the use of Good's buffers to generate high-sensitivity visible signal. In the present studies, visible emission from 5HT photoderivatized in different buffers varied by 20-fold under ambient oxygen levels but less than 2-fold in the absence of oxygen; oxygen did not significantly alter the photoproduct excited-state lifetime (∼0.8 ns). These results support a model in which oxygen interferes with formation of visible-emitting photoproducts by quenching a reaction intermediate, an effect that can be suppressed by buffer molecules. Deoxygenation of capillary electrophoresis separation buffers improves mass detection limits for 5-hydroxyindoles fractionated in 600-nm channels by approximately 2-fold to ≤30 000 molecules and provides new flexibility in identifying separation conditions for resolving 5HT from molecules with similar electrophoretic mobilities, such as the catecholamine neurotransmitters.
We report the development of a sensor for rapidly and simultaneously measuring multiple sugars in aqueous samples. In this strategy, enzyme-based assays are localized within an array of individually ...addressable sites on a micromachined silicon chip. Microspheres derivatized with monosaccharide-specific dehydrogenases are distributed to pyramidal cavities anisotropically etched in a wafer of silicon (100) and are exposed to sample solution that is forced through the cavities by a liquid chromatography pumping system. Production of fluorescent reporter molecules is monitored under stopped-flow conditions when localized dehydrogenase enzyme systems are exposed to their target sugars. We demonstrate the capability of this analysis strategy to quantify β-d-glucose and β-d-galactose at low micromolar to millimolar levels, with no detectable cross-talk between assay sites. Analysis is achieved either through fluorescence detection of an initial dehydrogenase product (NADH, NADPH) or by production of a secondary fluorescent product created by hydride transfer from the reduced nicotinamide cofactor to a fluorogenic reagent. The array format of this sensor provides capabilities for redundant analysis of sugars and for monitoring levels of other solution components known to affect the activity of enzymes. The use of this strategy to normalize raw fluorescence signals is demonstrated by the determination of glucose and pH on a single chip. Alternatively, uncertainties in the activity of an immobilized enzyme can be accounted for using standard additions, an approach used here in the determination of serum glucose.
Thesis (Ph. D.)--University of Texas at Austin, 2001.
Vita. Includes bibliographical references. Available also from UMI/Dissertation Abstracts International.
Measuring multiple components in complex mixtures can present significant analytical challenges. Characterization of such samples typically requires sample “pre-treatment” steps. Separations, for ...example, commonly are used to isolate analytes spatially before being determined sequentially. This process, however, tends to limit the breadth of measurable analytes, and can suffer from poor selectivity and long analysis times. Detection strategies based on molecular recognition have in some cases improved specificity and shortened analysis times; however, these approaches typically have been designed for the detection and quantification of single components present in complex mixtures. Recently, progress has been made towards the development of sensor arrays, combining the speed and selectivity of multiple sensing methods into a single platform. These technologies, however, have largely focused on narrow groups of analytes such as nucleic acids of varying polymer length. The multiplicity of analytes and variations in analyte properties such as molecular mass, charge or hydrophilicity contribute to the challenges of comprehensive solution phase multi-component analysis. We report here the development of enzyme-based sensors that, in combination with other non-catalytic sensors, form the basis of a solution-phase sensor array. In this strategy, enzymes are immobilized onto porous agarose beads, localized within wells etched into a silicon chip, and coupled to fluorometric and colorimetric reporting schemes to form an optically based site-addressable sensor array. In these studies, we have explored factors that impact the immobilization of enzymes onto solid supports (e.g., pH, incubation time) and the changes to enzyme behavior upon immobilization. We demonstrated the concept of using individual sensors to correct other sensors on the same platform. The linear response of a biosensor is important to analytical measurements and we illustrated the extension of a glucose sensor's linear response. Finally, we demonstrated rapid multi-component sensing in the determination of monosaccharides, disaccharides, and essential wine components.
