Development of enzyme-based sensor arrays

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Curey, Theodore Edward

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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.


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