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We have developed a  N   6 -dimethylallyladenine (cytokinin) dehydrogenase-based microbiosensor for real-time determination of the family of hormones known as cytokinins. Cytokinin dehydrogenase from  Zea mays  ( Zm CKX1) was immobilised concurrently with electrodeposition of a silica gel film on the surface of a Pt microelectrode, which was further functionalized by free electron mediator 2,6-dichlorophenolindophenol (DCPIP) in supporting electrolyte to give a bioactive film capable of selective oxidative cleavage of the  N   6 - side chain of cytokinins. The rapid electron shuffling between freely diffusible DCPIP and the FAD redox group in  Zm CKX1 endowed the microbiosensor with a fast response time of less than 10 s. The immobilised  Zm CKX1 retained a high affinity for its preferred substrate  N   6 -(Δ 2 -isopentenyl) adenine (iP), and gave the miniaturized biosensor a large linear dynamic range from 10 nM to 10 µM, a detection limit of 3.9 nM and a high sensitivity to iP of 603.3 µAmM −1 cm −2  (n = 4, R 2  = 0.9999). Excellent selectivity was displayed for several other aliphatic cytokinins and their ribosides, including  N   6 -(Δ 2 -isopentenyl) adenine,  N   6 -(Δ 2 -isopentenyl) adenosine,  cis -zeatin,  trans -zeatin and  trans -zeatin riboside. Aromatic cytokinins and metabolites such as cytokinin glucosides were generally poor substrates. The microbiosensors exhibited excellent stability in terms of pH and long-term storage and have been used successfully to determine low nanomolar cytokinin concentrations in tomato xylem sap exudates.

A Highly Selective Biosensor with Nanomolar Sensitivity Based on Cytokinin Dehydrogenase