Flow-through optical biosensor based on the permanent immobilization of an enzyme and transient retention of a reaction product

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Abstract
A new sensor based on the twofold immobilization of a catalyst and a reaction product in an optical flow cell is proposed. The packing material of the flow cell consists of two physically distinct layers: an upper layer of controlled-pore glass (CPG) onto which the enzyme is immobilized, and a lower layer of suitable exchanger where the product of the enzymatic reaction is temporarily retained. The performance of the biosensor was tested by immobilizing peroxidase on CPG and retaining the product of the reaction between hydrogen peroxide, 4-aminophenazone, and an aniline derivative on a Sephadex resin. The sensor can be used to monitor hydrogen peroxide and any previous reaction which yields it (e.g., any reaction catalyzed by an oxidase). The biosensor was applied to the determination of hydrogen peroxide and glucose in serum. The features of the determinations (linear ranges between 10 and 200 ng/mL, and RSD values smaller than 2%), in addition to the great consistency of the results with those obtained in the clinical determination of glucose by an AACC method and analyte recoveries between 95 and 105% testify to the excellent performance of the sensor.
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