Single Cell Analysis of Cellular Senescence using a Novel Carbon Nanotube-Based Probe

摘要 随着技术的提高，单细胞分析正在成为一个重要的研究领域 随着吞吐量的增加，可以测量和了解复杂生物中的细胞异质性 系统。这个项目的目标是创造一种能力来识别和量化单个 使用电化学技术，最小扰动和长时间的活细胞。 细胞内电化学可以用一种新型的多功能碳纳米管来完成 由工作电极和参比电极组成的传感器，在一个细胞穿透尖端，流体通过该电极 可以同时注射。在本项目中，构建了多功能纳米探针，能够 注入液体，以便在受限水中进行独立的电化学测量 微环境。然后利用纳米探针将能够进行反应的底物按顺序注入细胞 为了选择性地定量衰老相关的β-半乳糖苷酶，这是一种在 许多慢性病。我们假设，基于碳纳米管的纳米探针将提供选择性地 实时量化异质活细胞群体中的细胞衰老；目前尚不具备 采用最先进的技术。我们计划追求以下两个具体目标：(1)建设一个 一种可注入流体进行自含式电化学的多功能纳米探针 在受限的水微环境中进行测量。在这里，我们将制造基于碳纳米管的探测器 并开发利用该探针进行细胞内电化学所需的技术；(2)利用 注射对氨基苯基β-D-半乳糖苷的纳米探针，以选择性地定量衰老- 相关的β-半乳糖苷酶。在这里，我们将使用碳纳米管探针来电化学测量衰老- 体外单个活细胞内的相关分子，并评价该探针的测量能力 用标准检测方法检测。这项提议的结果将提供一流的基于CNT的工具， (A)建立一种新的单细胞分析技术(细胞内电化学)，(B)将其与 传统技术(细胞内注射和荧光显微镜)形成一个全新的最小 高时间性单个活细胞定量数据的侵入性分析技术 在很长的一段时间内，我们都有能力解决这些问题。此外，新的纳米生物传感工具和技术可以 有效地传播给更大的科学界，以帮助研究人员研究和阐明 细胞生物学基础。实时的细胞内电化学测量将会有很大的 在开发几种疾病的诊断和治疗方法方面的翻译潜力， 包括慢性阻塞性肺疾病(COPD)、肺癌和心血管疾病，所有这些 是主要的公共卫生问题。

SUMMARY Single cell analysis is emerging as an important field of research as technologies improve in sensitivity and with increased throughput to allow measurement and understanding of cell heterogeneity in complex biological systems. The goal of this project is to create the ability to identify and quantify biomolecules inside a single living cell with minimal perturbation and over long periods of time using electrochemical techniques. Intracellular electrochemistry can be accomplished using a novel multifunctional carbon nanotube-based sensor consisting of both working and reference electrodes at a single cell-penetrating tip, through which fluids can be simultaneously injected. In this project, the multifunctional nanoprobe is constructed, capable of injecting fluids in order to conduct self-contained electrochemical measurements inside confined aqueous microenvironments. The nanoprobe is then utilized to inject a reaction-enabling substrate into the cell in order to selectively quantify senescence-associated beta-galactosidase, a cytosolic enzyme of critical importance in many chronic diseases. We hypothesize that CNT-based nanoprobes will provide the ability to selectively quantify cell senescence in a heterogeneous population of living cells in real-time; capabilities not currently available with state-of-the-art technologies. We plan to pursue the following two Specific Aims: (1) construct a multifunctional nanoprobe capable of injecting fluids in order to conduct self-contained electrochemical measurements inside confined aqueous microenvironments. Here, we will manufacture the CNT-based probe and develop the techniques needed to utilize the probe for intracellular electrochemistry; (2) utilize the nanoprobe to inject p-aminophenyl β-D-galactopyranoside in order to selectively quantify senescence- associated β-galactosidase. Here, we will use the CNT probe to electrochemically measure senescence- relevant molecules inside individual living cells in vitro and evaluate the measurement capabilities of the probe with standard detection assays. The outcome of this proposal will provide a first-in-class CNT-based tool that (a) establishes a new single cell analytical technique (intracellular electrochemistry) and (b) combines it with traditional techniques (intracellular injection and fluorescence microscopy) to form an entirely new minimally invasive analytical technique for gathering quantitative data from single living cells with high temporal resolution and over long periods of time. Moreover, the new nano-biosensing tool and technique can be efficiently disseminated to the larger scientific community to assist researchers in studying and elucidating fundamentals in cell biology. Real-time intracellular electrochemistry measurements will have great translational potential in the development of diagnostic and therapeutic approaches for several diseases, including chronic obstructive pulmonary disease (COPD), lung cancer and cardiovascular disease, all of which are major public health concerns.