title: A highly selective biosensor with nanomolar sensitivity based on cytokinin dehydrogenase.
abstract: We have developed a N6-dimethylallyladenine (cytokinin) dehydrogenase-based microbiosensor for real-time determination of the family of hormones known as cytokinins. Cytokinin dehydrogenase from Zea mays (ZmCKX1) 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 N6- side chain of cytokinins. The rapid electron shuffling between freely diffusible DCPIP and the FAD redox group in ZmCKX1 endowed the microbiosensor with a fast response time of less than 10 s. The immobilised ZmCKX1 retained a high affinity for its preferred substrate N6-(Δ2-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-1cm-2 (n = 4, R2 = 0.9999). Excellent selectivity was displayed for several other aliphatic cytokinins and their ribosides, including N6-(Δ2-isopentenyl) adenine, N6-(Δ2-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.
authors.affiliation: Warwick Biosensor Group, School of Life Sciences, University of Warwick, Coventry, United Kingdom.
authors.forename: Faming
authors.initials: F
authors.lastname: Tian
authors.affiliation: Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University, Olomouc, Czech Republic.
authors.forename: Marta
authors.initials: M
authors.lastname: Greplová
authors.affiliation: Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University, Olomouc, Czech Republic.
authors.forename: Ivo
authors.initials: I
authors.lastname: Frébort
authors.affiliation: Warwick Biosensor Group, School of Life Sciences, University of Warwick, Coventry, United Kingdom.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Dale
authors.affiliation: Warwick Biosensor Group, School of Life Sciences, University of Warwick, Coventry, United Kingdom.
authors.forename: Richard
authors.initials: R
authors.lastname: Napier
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D003583:Cytokinins / Q000032:analysis* / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D007541:Isopentenyladenosine / Q000031:analogs & derivatives / Q000032:analysis* / Q000378:metabolism; D057230:Limit of Detection; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D003313:Zea mays / Q000201:enzymology*
chemical_list: D003583:Cytokinins; D004800:Enzymes, Immobilized; C001478:N(6)-(delta(2)-isopentenyl)adenine; D007541:Isopentenyladenosine; C009699:zeatin riboside; D010088:Oxidoreductases; C019984:cytokinin oxidase