A Highly Selective Biosensor with Nanomolar Sensitivity Based on Cytokinin Dehydrogenase

Abstract

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 (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 N 6- 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 N 6-(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 N 6-(Δ2-isopentenyl) adenine, N 6-(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.

Zooming In on Plant Hormone Analysis: Tissue- and Cell-Specific Approaches

“…Because biosensors are calibrated, their output value is concentration, but this value may not represent the concentration of a single molecular species. For example, Tian et al (124) showed that the use of CKX confers high selectivity for CKs, excluding other prevalent purines; however, there are many CKs, and a biosensor gives a value that is the integral of all substrates present. The CKX biosensor was calibrated using isopentenyladenosine (iP), and a unit of iP equivalents was then defined to account for the CKX signal gained from CK ribosides as well as free bases (124).…”

“…For example, Tian et al (124) showed that the use of CKX confers high selectivity for CKs, excluding other prevalent purines; however, there are many CKs, and a biosensor gives a value that is the integral of all substrates present. The CKX biosensor was calibrated using isopentenyladenosine (iP), and a unit of iP equivalents was then defined to account for the CKX signal gained from CK ribosides as well as free bases (124). Validation by MS showed very good agreement between the biosensor iP equivalent value and the MS data once the latter had been converted to iP equivalents by summing the concentrations of each component compound.…”

Perspectives of Nano-Materials and Nanobiosensors in Food Safety and Agriculture

“…Simple amperometric biosensor developed by graphite coated with polypyrrole (PPy) for the determination of salicylic acid in samples of plasma and milk [100]. A real time highly selective nanobiosensor developed for determination of cytokinins and auxion concentrations in tomato xylem sap exudates [101,102]. It is recommended that precautions are better than cure or remedies in case of plant stresses otherwise at the calamity stage it will cause huge crop loss and poor quality of the produce.…”