Enzyme based electrochemical biosensors are divided into three generations according to their type of electron transfer from the cofactors of the enzymes to the electrodes. Although the 3rd generation sensors using direct electron transfer (DET) type enzymes are ideal, the number of enzyme types which possess DET ability is limited. In this study, we report of a glucose sensor using mediator-modified glucose dehydrogenase (GDH), that was fabricated by a new quick-and-easy method using the pre-functionalized amine reactive phenazine ethosulfate (arPES). Thus mediator-modified GDH obtained the ability to transfer electrons to bulky electron acceptors as well as electrodes. The concentration of glucose was successfully measured using electrodes with immobilized PES-modified GDH, without addition of external electron mediators. Therefore, continuous monitoring systems can be developed based on this "2.5th generation" electron transfer principle utilizing quasi-DET. Furthermore, we successfully modified two other diagnostically relevant enzymes, glucoside 3-dehydrogenase and lactate oxidase, with PES. Therefore, various kinds of diagnostic enzymes can achieve quasi-DET ability simply by modification with arPES, suggesting that continuous monitoring systems based on the 2.5th generation principle can be developed for various target molecules.
Keywords: 2.5th generation biosensor; Electrochemical enzyme sensor; Glucose dehydrogenase; Glucoside 3-dehydrogenase; Lactate oxidase; Mediator modification of enzymes.
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