Prussian Blue acts as a mediator in a reagentless cytokinin biosensor

An electrochemical biosensor for detection of the plant hormone cytokinin is introduced. Cytokinin homeostasis in tissues of many lower and higher plants is controlled largely by the activity of cytokinin dehydrogenase (CKX, EC 1.5.99.12) that catalyzes an irreversible cleavage of N6-side chain of cytokinins. Expression of Arabidopsis thaliana CKX2 from Pichia pastoris was used to prepare purified AtCKX2 as the basis of the cytokinin biosensor. Prussian Blue (PrB) was electrodeposited on Pt microelectrodes prior to deposition of the enzyme in a sol–gel matrix. The biosensor gave amperometric responses to several cytokinins. These responses depended on the presence of both the enzyme and the Prussian Blue. Thus Prussian Blue must act as an electron mediator between the FAD centre in CKX2 and the Pt surface.

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• An electrochemical biosensor for detection of the plant hormone cytokinin.
• Constitutive expression system for large-scale protein production.
• CKX enzyme entrapment in sol–gel film on the surface of a PrB-modified electrode.
• Prussian Blue as an electron mediator between the enzyme and the electrode.
• The biosensor was sensitive to micromolar concentrations of several cytokinins.