Tian Faming, Greplová Marta, Frébort Ivo, Dale Nicholas, Napier Richard
Warwick Biosensor Group, School of Life Sciences, University of Warwick, Coventry, United Kingdom.
Department of Molecular Biology, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University, Olomouc, Czech Republic.
PLoS One. 2014 Mar 4;9(3):e90877. doi: 10.1371/journal.pone.0090877. eCollection 2014.
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.
我们开发了一种基于N6-二甲基烯丙基腺嘌呤（细胞分裂素）脱氢酶的微生物传感器，用于实时测定被称为细胞分裂素的激素家族。来自玉米的细胞分裂素脱氢酶（ZmCKX1）与硅胶膜在铂微电极表面的电沉积同时固定，该铂微电极在支持电解质中通过游离电子介质2,6-二氯酚靛酚（DCPIP）进一步功能化，以形成能够选择性氧化裂解细胞分裂素N6-侧链的生物活性膜。自由扩散的DCPIP与ZmCKX1中的FAD氧化还原基团之间的快速电子转移赋予了微生物传感器小于10秒的快速响应时间。固定化的ZmCKX1对其优选底物N6-（Δ2-异戊烯基）腺嘌呤（iP）保持高亲和力，并赋予微型化生物传感器10 nM至10 μM的大线性动态范围、3.9 nM的检测限以及对iP的603.3 μAmM-1cm-2的高灵敏度（n = 4，R2 = 0.9999）。对其他几种脂肪族细胞分裂素及其核糖苷，包括N6-（Δ2-异戊烯基）腺嘌呤、N6-（Δ2-异戊烯基）腺苷、顺式玉米素、反式玉米素和反式玉米素核糖苷，表现出优异的选择性。芳香族细胞分裂素及其代谢物如细胞分裂素葡萄糖苷通常是较差的底物。这些微生物传感器在pH值和长期储存方面表现出优异的稳定性，并已成功用于测定番茄木质部汁液渗出物中低纳摩尔浓度的细胞分裂素。
