<p>The overall objective of this applicationis to stabilize oxidase-based biosensors. The specific objectives of this proejct are: </p>
<p>Objective 1: Determine the optimal pressure for maximal stability and activity of xanthine oxidase, pyruvate oxidase, alcohol oxidase, and glucose oxidase. The working hypothesis, strongly supported by our preliminary data on glucose oxidase and other enzymes is that HHP below 400 MPa stabilizes oxidases in buffered solutions. Free oxidase activity will be monitored with spectrophotometric assays. Enzymes, in buffered solutions will be treated at selected HHP. </p>
<p>Objective 2: To identify the effect of derivatizing hydrophobic compounds at Patm or HHP. The working hypothesis is that covalently attaching hydrophobic molecules of benzene carboxylic acid or aniline to amino or carboxylate side groups respectively will stabilize the enzymes. Stability and structural changes will be determined as described in Objective 1. </p>
<p>Objective 3: To fabricate highly stable amperometric enzyme biosensors using nano-scaffolds of enzymes entrapped and crosslinked in nanofilms electrochemically generated at HHP. The working hypothesis is that under HHP, the enzyme is at its most stable conformation and polymerization under these conditions results in stability retention upon depressurization. Stability of resulting biosensors will be characterized in terms of the amperometric response under continuous operation.</p>
<p>NON-TECHNICAL SUMMARY: <br/>Rapid detection of pathogen biomarkers, pesticides, fertilizer residues, sugars, alcohol and other compounds is very important to the food and agricultural industries to ensure food safety and quality, as well as to diagnose plant diseases. Because food and agricultural samples are complex mixtures of hundreds of compounds, analyses typically require tedious sample treatment, compound separation, and identification. Enzymes that specifically interact with compounds of interest can be coupled to electrodes to make biosensors that directly and rapidly measure such compounds in complex mixtures. Enzymes are very large proteins consisting of series of amino acids and are folded in a complex manner. Arguably the most difficult challenge that prevents the development of practical, reliable enzyme biosensors is that, over time and under the effect of
temperature, enzymes unfold and lose their activity. The stability of enzymes depends in part on the balance between hydrophobic and hydrophilic groups in the protein. In addition, application of high hydrostatic pressure often stabilizes enzymes. This research focuses on the stabilization of enzyme biosensors relevant to food and agriculture using a combination of nanoscale and chemical manipulations of enzymes at high hydrostatic pressure (HHP). Chemical attachment of hydrophobic groups to hydrophilic residues on the enzyme, followed by attachment or entrapment in nanofilms as they grow under of HHP is expected to maximize the stability of the selected enzyme biosensors. The enzymes selected for this research are xanthine oxidase, pyruvate oxidase, alcohol oxidase, and glucose oxidase. These enzymes can be used, for example to measure xanthine in fish to determine freshness, phosphates
in water to determine the presence of fertilizer residues, alcohol and glucose in beverages. The specific objectives of this research are: 1) to determine the optimal pressure for maximal stability and activity for each of the selected enzymes; 2) to characterize the effect of attaching hydrophobic groups to the enzymes at atmospheric pressure or at HHP on the stability of each enzyme; and 3) to fabricate stable enzyme biosensors by entrapping modified enzymes and attaching them to nanofilms grown on the surface of platinum electrodes.
<p>APPROACH: <br/>The main milestones of this project are the accomplishement of each objective. In Objective 1 of this project, the selected enzymes will beexposed to selectedhigh pressuresand temperatures combinations. The residual activity aftertreatments for selected amounts of timewill be determined to determine the optimal presure for each enzyme.The kinetics of enzyme inactivation will be characterized. Protein unfolding willbe monitoredby in-situfluorescence determinations in ahigh pressure optical cell. Enzyme conformation after selected treaments willbe studiedbycircular dichroism and differential scanning calorimetry.Although the stability of several enzymes under high hydrostatic pressure has been studied, to the best of our knowledge there are no studies for alcohol oxidase, xanthine oxidase, pyruvate oxidase or glucoseoxidase.Objective 1 will be completed at
the end of the first year of this project. Success consists in the determination of the optimal pressure for each enzyme and the relative increase in stability with respect to treatments at atmospheric pressures. Accomplishement of Objective 1 will be documented as publications in refereed journals and in the annual report of this project. <p>In Objective 2, the enzymes will be chemically modified at the optimal presure from objective1 or atmospheric pressure,tochange their stability.Stabilization upon chemical modification will bedeterminedby treating the enzymes atmoderate temperaturesand determining the residual activity at atmospheric pressure.Changes in enzyme structure will be characterzed by circular dichroism. Protein unfolding will be monitored by fluorescence at ambient pressure.To the best of our knowledge, enzymes have not been chemically modified under pressure. Objective2 will
be completed at the end of thesecond year of this project. Success consists in enzymes with increased stability as a result of chemica modificationfor each enzymeThe relative increase in stability will be determinedwith respect to native enzymes. Accomplishement of Objective2 will be documented as publications in refereed journals and in the annual report of this project <p>In Objective 3, enzyme biosensors will be fabricated under high hydrostatic pressure by immobilizing the enzymes (native and chemically modified) in electrochemically generated polymers. The stability of the resulting biosensors will be determined by monitoring the sensor amperometric response at constant substrate concentration. Biosensors fabricated using native enzymes will be used as controls.Biosensors that display the greatest stability will be further caracterized in terms of linearity, limit of detection,
sensitivity, sensor-to-sensor reproductiblity and drift. To the best of our knowledge, there has been no research on immobilizing enzymes at hight pressure in electrochemically generated polymers. Objective3 will be completed at themiddleof the third year of this project. Success consistsin the development of highly stable enzyme biosensors.Two to ten fold more stable biosensorsare expected. Accomplishement of Objective3 will be documented as publications in refereed journals and in the final report of this project.