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

在化学系化学测量和成像（CMI）项目的支持下，以及分子和细胞生物科学系细胞动力学和功能群的部分资助下，辛辛那提大学主校区的Ryan白色教授正在研究在单个生物细胞和亚细胞尺寸尺度上以空间分辨率测量界面处小分子释放的新方法。这种测量方法在测量细胞信号传导中特别重要，其中小分子信使在细胞之间传播，指示它们如何与相邻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。白色教授和他的团队提出的方法涉及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。该研究计划的长期目标是开发创建“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产生广泛的影响，并为测量大脑中的细胞通信提供新的知识。这种影响将通过制定一项旨在增加和持续参与第一代大学生本科研究的计划，该提案的总体目标是开发用于成像的纳米级离子通道探针，并在界面处以高空间分辨率进行局部分子和离子传感（纳米-微米）。这项有影响力的研究将扩大本地化检测能力，使其能够检测目前无法使用的分析物，因为这些分析物既不是光学活性也不是电化学活性的。这种需要在测量细胞信号传导时特别重要，其中信使在细胞之间旅行，决定它们如何与环境相互作用。新方法实现的测量将有助于发现星形胶质细胞如何与神经血管系统和神经元接触，在两者之间发出差异信号，这对大脑中的网络通信至关重要。然而，目前缺乏可以测量位于细胞表面的分子的通用工具。使用离子通道探针进行扫描离子电导显微镜（SICM），其中蛋白质通道嵌入在探针末端的脂质双层中，是一种利用离子通道活性进行离子和分子检测的有前途的方法，具有SICM的成像能力。然而，存在着阻碍其使用的主要障碍。为了克服这些挑战，白色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，该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。这些测量预计将使第一次直接，局部检测三磷酸腺苷（ATP）从星形胶质细胞释放。这项工作的更广泛影响在于所提出的测量方法能够提供有关生物系统（单细胞，组织切片）如何通过释放小分子进行通信的新知识。这种影响将通过发展“本科生研究之路”计划，针对增加和持续参与的第一，该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响进行评估，被认为值得支持审查标准。

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.