Quantitative sensors for phytohormone signalling systems

微生物传感器是一种非常小的探针，用于实时测量活体组织中重要信号的浓度。它们的小尺寸是有利的，因为收集的数据可以与更精确的组织放置相关联，并且在传感器放置期间引起的创伤更少。为了发挥作用，生物传感器需要对被测量的信号有选择性，并且高度敏感，以便记录活样本中这些信号的低浓度。沃里克大学电化学技术的进步最近使得一些神经递质（对大脑功能很重要的信号）的良好微生物传感器得以开发。一些植物激素与神经递质属于同一化学信号家族。细胞分裂素是植物激素，除其他作用外，有助于决定种子储存组织的发育和大小。我们已经用同样的沃里克制造技术制造了细胞分裂素的微生物传感器原型。植物中天然存在一种酶，叫做细胞分裂素脱氢酶（CKX），它与细胞分裂素发生反应，使它们变性。这种酶已被纯化，并包裹在涂层微电极上的可渗透玻璃层中。当浸入细胞分裂素时，随之产生的电信号取决于细胞分裂素的存在及其浓度。这些原型生物传感器工作良好，但需要改进。这个项目是关于我们将如何测试它们灵敏度的改进。我们的细胞分裂素微生物传感器可以通过使用具有更高活性的酶的替代形式来改进，所有这些都可以通过我们在捷克共和国奥洛穆茨的合作者获得。我们将测试不同的电极涂层，以改善连通性和测试方法，以赋予电极纳米结构，以增加其表面积而不增加其尺寸。优化细胞分裂素生物传感器后，我们将在植物样本上进行测试。最初，我们将把它浸入汁液中，这些汁液是茎从根系中切断后形成的。可以添加已知浓度的细胞分裂素来验证校准。一个更严格的测试是将其中一个微型传感器插入种子的储存组织中。在种皮上做一个微切口，我们将评估现成的ATP传感器在这个新环境中的性能，然后细胞分裂素传感器记录激素信号强度随时间的实时变化，并将其与种子大小联系起来。我们从其他工作中知道，高种子细胞分裂素会产生更大的种子，通过了解信号到达的方式和时间，我们可能能够为改善粮食安全的战略提供信息。并不是所有的植物组织都像种子储存组织那样柔软和多浆。我们将评估如何使这些生物传感器更加坚固耐用，以便它们可以直接部署到植物中。我们将测试钨丝（比铂更强），微导管用于从保护盖中输送传感器尖端，并评估在玻璃微血管中制造传感器的前景-这是已知的有利于植物细胞穿刺，包括单细胞测量。细胞分裂素微生物传感器的开发将是一个令人印象深刻的进步。基于经过验证的ATP框架的坚固生物传感器将说明这种生物传感器技术可以应用于解决植物生物学中许多其他悬而未决的问题。我们的实验还将说明细胞分裂素如何驱动种子填充，以及我们如何将这些信息用于粮食安全。简而言之，生物传感器将提供所需的精确数据，为当前和未来操纵植物种子大小和产量潜力的研究提供信息。其他传感器也将紧随其后。

Microbiosensors are very small probes used to measure the concentrations of important signals in living tissue and in real time. Their small size is advantageous because the data collected can be associated with more precise tissue placements and less trauma is induced during sensor placement. In order to be useful, biosensors need to be selective to the signal being measured and highly sensitive in order to record the low concentrations of these signals in living samples. Advances in electrochemistry technologies at Warwick have recently allowed the development of good microbiosensors for some neurotransmitters, signals important for brain function. Some plant hormones are part of the same family of chemical signals as the neurotransmitters. The cytokinins are phytohormones which, amongst other roles, help determine the development and size of the storage tissue in seeds. We have made a prototype microbiosensor for cytokinins using the same Warwick fabrication technologies. An enzyme that occurs naturally in plants known as cytokinin dehydrogenase (CKX) reacts with cytokinins to denature them. This enzyme has been purtified and encased in a permable glass layer on a coated microelectrode. When dipped into cytokinins the consequent electrical signal is dependent on both the presence of cytokinins and on their concentration. These prototype biosensors work well, but need improving. This project is about how we will test for improvements in their sensitivity. Our microbiosensors for cytokinin may be improved by using alternative forms of the enzyme with much higher activity, all available to us through our collaborators in Olomouc, Czech Republic. We will test different electrode coatings in order to improve connectivity and test methods to give nanostructure to the electrode to increase its surface area without increasing its size. Having optimised the cytokinin biosensor we will test it on plant samples. Initially we will dip it into sap droplets which form when the stem is cut off a root system. Known concentrations of cytokinin can be added to validate calibrations. A more exacting test will be to insert one of these microsensors into the storage tissue of a seed. A microincision into the seed coat will give access and we will evaluate the performance of both off-the-shelf ATP sensors in this new environment, and then the cytokinin sensors to record real-time changes in hormone signal strength with time and relate this to seed size. We know from other work that high seed cytokinins give bigger seeds, by understanding how and when the signal arrives we may be able to inform strategies for improving food security. Not all plant tissues are as soft and pulpy as seed storage tissue. We will evaluate how to make these biosensors more rugged so that they can be deployed directly into plants. We will test tungsten wires (stronger than platinum), microcatheters for delivering the sensor tip from a protected cover, and evaluate the prospects of making the sensors in glass micrcapillaries - which are known to be good for plant cell impalements, including single-cell measurements. Development of a microbiosensor for cytokinins will be a singularly impressive advance. Ruggedised biosensors based on the proven ATP framework will illustrate that this biosensor technology can be applied to address many other outstanding questions in plant biology. Our experiments will also illustrate how cytokinins drive seed filling and how we might use this information for food security. In short, the biosensors will provide the type of precision data needed to inform current and future studies on manipulating plant seed size and yield potential. Other sensors will follow.