Localized Chemical Detection with Maintained Single Protein Channel Recordings on Nanoneedle Probes

在化学学部化学测量和成像（CMI）项目的支持下，以及分子和细胞生物科学部细胞动力学和功能集群的部分资助下，辛辛那提大学主校区的Ryan White教授正在研究在单个生物细胞和亚细胞尺寸尺度上测量空间分辨率界面上小分子释放的新方法。这种测量方法在测量细胞信号方面特别重要，其中小分子信使在细胞之间传播，决定了它们如何与邻近细胞和环境相互作用。由于传感方法的通用性，该方法可以应用于广泛的细胞类型，包括大脑中的非神经元细胞。怀特教授和他的团队提出的方法包括在纳米针电极的末端嵌入细胞膜蛋白质受体或蛋白质通道，纳米针电极可以放置在各种界面附近进行局部检测。为了实现这种检测，该项目将克服用于分析目的的蛋白质通道测量的主要挑战-在实验期间控制和维持单个通道的能力。该研究项目的长期目标是开发能够产生“人工突触”的探针，以监测细胞膜和细胞微环境的动态和异质性。这项工作将对测量科学和测量大脑细胞通讯的新知识产生广泛的影响。通过建立一个强大的社区和文化，通过开发一个旨在增加和持续参与本科研究的第一代大学生的项目，这种影响将影响到不同的学生和科学家群体。本提案的总体目标是开发用于高空间分辨率（纳米-微米）界面成像和局部分子和离子传感的纳米级离子通道探针。这项有影响力的研究将扩大局部检测能力，以分析物无法用当前的方法获得，因为分析物既不具有光学活性也不具有电化学活性。这种需求在测量细胞信号方面尤为重要，因为信使在细胞之间传递，决定了它们如何与环境相互作用。新方法的测量将有助于发现星形胶质细胞是如何与神经血管系统和神经元接触的，从而在两者之间发出不同的信号，这对大脑中的网络通信至关重要。然而，目前还缺乏可用于测量细胞表面分子的通用工具。将离子通道探针用于扫描离子电导显微镜（SICM），其中蛋白质通道嵌入探针末端的脂质双分子层中，是利用离子通道活性进行离子和分子检测的一种很有前途的方法，具有SICM的成像能力。然而，阻碍它们使用的主要障碍仍然存在。为了克服这些挑战，White教授和他的团队将开发一种新的SICM成像模式，该模式采用金属纳米针探针，支持离子通道测量，与现有的界面局部检测方法相比，探针尺寸大大减小。他们将展示在试验台无机基质和星形胶质细胞表面进行局部检测的能力。这些测量结果有望实现对星形胶质细胞中三磷酸腺苷（ATP）释放的首次直接、局部检测。这项工作更广泛的影响在于，所提出的测量方法能够提供关于生物系统（单细胞、组织切片）如何通过释放小分子进行交流的新知识。通过“本科生研究之路”项目的发展，这种影响将影响到不同的学生和科学家群体，该项目旨在通过创建一个强大的社区和文化，增加和持续地参与第一代大学生的本科生研究。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemical Measurement and Imaging (CMI) Program in the Division of Chemistry, and partial funding from the Cellular Dynamics and Function Cluster in the Division of Molecular and Cellular Biosciences, Professor Ryan White of the University of Cincinnati Main Campus is studying new ways for measuring the release of small molecules at an interface with spatial resolution on the single biological cell and sub-cellular size scales. This measurement method is particularly significant in measuring cell signaling where small molecule messengers travel between cells dictating how they interact with their neighboring cells and environment. Because of the generality of the sensing method, the approach can be applied to a wide range of cell types, including non-neuronal cells in the brain. The proposed approach by Professor White and his team involves embedding a cellular membrane protein receptor, or protein channel, at the end of a nanoneedle electrode that can be placed in proximity to various interfaces for localized detection. In order the achieve this detection, the project will overcome the major challenge in protein channel measurements for analytical purposes – the ability to control and maintain a single channel through the duration of an experiment. The long-term goal of the research program is to develop probes that create “artificial synapses” to monitor the dynamics and heterogeneity of the cell membrane and cellular microenvironment. The work will provide broad impact in measurement science and new knowledge in the measure of cellular communication in the brain. This impact will reach a diverse group of students and scientists through the development of a program targeting the increased and sustained participation of first-generation college students in undergraduate research, through the creation of a strong community and culture.The overarching goal of this proposal is to develop nanoscale ion channel probes for imaging and localized molecular and ion sensing at interfaces with high spatial resolution (nanometer-micrometer). The impactful research will expand localized detection abilities to analytes that are not accessible with current methodologies, because the analytes are neither optically nor electrochemically active. This need is particularly significant in measuring cell signaling where messengers travel between cells, dictating how they interact with their environment. The measurements enabled by the new method will facilitate discoveries of how astrocytes, with contacts to the neurovascular system and neurons, differentially signal between the two which is critical for networked communication in the brain. Currently, however, there is a lack of generalizable tools that can measure molecules localized at a cell surface. The use of ion channel probes for scanning ion conductance microscopy (SICM), where protein channels are embedded in a lipid bilayer at the end of a probe, is a promising method to leverage ion channel activity for ion and molecular detection with the imaging capabilities of SICM. However, major hurdles exist that hinder their use. To overcome these challenges, Professor White and his team will develop a new SICM imaging mode that employs metal nanoneedle probes that support ion channel measurements with dramatically reduced probe size over existing approaches for localized detection at interfaces. They will demonstrate the ability to perform localized detection on test-bed inorganic substrates and the surface of astrocyte cells. These measurements are anticipated to enable the first direct, localized detection of adenosine triphosphate (ATP) release from astrocytes. The broader impacts of the work lie in the ability of the proposed measurement method to provide new knowledge about how biological systems (single cells, tissue slices) communicate via the release of small molecules. This impact will reach a diverse group of students and scientists through the development of a "Pathway to Undergraduate Research" program that targets increased and sustained participation of first-generation college students in undergraduate research through the creation of a strong community and culture.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.