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

总结 单细胞分析正在成为一个重要的研究领域，因为技术在灵敏度和 通过增加的通量，允许测量和理解复杂生物学中的细胞异质性， 系统.该项目的目标是创造识别和量化单个细胞内生物分子的能力。 使用电化学技术以最小的扰动和长时间的活细胞。 细胞内电化学可以使用一种新的多功能碳纳米管为基础的 一种传感器，由位于单个细胞穿透尖端的工作电极和参比电极组成，流体可通过该传感器 可以同时注射。在本项目中，构建了多功能纳米探针，能够 注入流体以便在封闭的水溶液内进行独立的电化学测量 微环境然后利用纳米探针将反应使能基质注入细胞中， 选择性定量衰老相关的β-半乳糖苷酶，一种在 许多慢性疾病。我们假设基于CNT的纳米探针将提供选择性地 实时量化活细胞异质群体中的细胞衰老;目前还没有能力 可以用最先进的技术。我们计划追求以下两个具体目标：（1）建立一个 能够注入流体以进行自给式电化学的多功能纳米探针 在封闭的含水微环境中进行测量。在这里，我们将制造基于CNT的探针 并开发利用探针进行细胞内电化学所需的技术;（2）利用 纳米探针注射对氨基苯基β-D-吡喃半乳糖苷，以选择性地量化衰老- 相关β-半乳糖苷酶。在这里，我们将使用CNT探针来电化学测量衰老- 体外单个活细胞内的相关分子，并评估探针的测量能力 用标准的检测方法该提案的结果将提供一流的基于CNT的工具， (a)建立了一种新的单细胞分析技术（细胞内电化学），并且（B）将其与 传统的技术（细胞内注射和荧光显微镜），以形成一个全新的最低限度 从单个活细胞收集定量数据的侵入性分析技术 解决方案，并在很长一段时间。此外，新的纳米生物传感工具和技术可以 有效地传播到更大的科学界，以帮助研究人员研究和阐明 细胞生物学基础实时细胞内电化学测量将具有很大的 在开发几种疾病的诊断和治疗方法方面的转化潜力， 包括慢性阻塞性肺疾病（COPD）、肺癌和心血管疾病，所有这些疾病 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.