A highly selective biosensor with nanomolar sensitivity based on cytokinin dehydrogenase.
A highly selective biosensor with nanomolar sensitivity based on cytokinin dehydrogenase.
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DOI：
10.1371/journal.pone.0090877
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Napier R
中科院分区：
文献类型：
--
作者：
Tian F;Greplová M;Frébort I;Dale N;Napier R
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，用于实时检测称为细胞分裂素的激素家族。将玉米细胞分裂素脱氢酶(ZmCKX1)固定化的同时，在铂微电极表面沉积了一层硅胶膜，再用支持电解液中的自由电子介体2，6-二氯苯基吲哚(DCPIP)对其进行功能化，得到了一种能够选择性氧化裂解细胞分裂素N-6侧链的生物活性薄膜。ZmCKX1上可自由扩散的DCPIP和FAD氧化还原基团之间的快速电子洗牌使其具有小于10 S的快速响应时间。固定化ZmCKX1与其首选底物N6-(Δ2-异戊烯基)腺嘌呤(IP)保持了高亲和力，并使小型生物传感器在10~10µM范围内具有很大的线性动态范围，检测下限为3.9nM，对IP的高灵敏度为603.3µamM−1 cm−2(n = 4，R2 = 0.9999)。对其他几种脂肪族细胞分裂素及其核苷，包括N-6-(Δ-2-异戊烯基)腺嘌呤、N-6-(Δ-2-异戊烯基)腺苷、顺式-玉米素、反式-玉米素和反式-玉米素核苷都表现出很好的选择性。芳香的细胞分裂素和代谢物，如细胞分裂素和葡萄糖苷，通常是较差的底物。该微生物传感器具有良好的pH稳定性和长期保存性，已成功用于测定番茄木质部汁液中低浓度的纳米分子细胞分裂素。
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.
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