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

使用新型碳纳米管探针进行细胞衰老的单细胞分析

基本信息

批准号：
9226901
负责人：
Michael George Schrlau
金额：
$ 23.1万
依托单位：
ROCHESTER INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-15 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9226901
关键词：
Biological Assay Biomedical Research Biosensing Techniques Carbon Carbon Nanotubes Cardiovascular Diseases Cell Aging Cell physiology Cells Cellular biology Chronic Disease Chronic Obstructive Airway Disease Communities Data Detection Development Diagnostic Disease Electrochemistry Electrodes Environment Enzymes Epithelial Cells Fluorescence Microscopy Galactose Goals Heterogeneity In Vitro Individual Injection of therapeutic agent Life Liquid substance Malignant neoplasm of lung Measurement Measures Methods Nanomanufacturing Noise Outcome Performance Pharmacotherapy Population Heterogeneity Public Health Reaction Reagent Research Research Personnel Role Signal Transduction System Techniques Technology Therapeutic Time Treatment Efficacy Work aqueous base beta-Galactosidase complex biological systems drug development implanted sensor improved innovation minimally invasive nano nanobiosensor nanoprobe novel point-of-care diagnostics research study senescence sensor single cell analysis temporal measurement time use tool

项目摘要

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.

概括随着技术灵敏度和性能的提高，单细胞分析正在成为一个重要的研究领域。提高通量，可以测量和理解复杂生物学中的细胞异质性该项目的目标是创造识别和量化单个生物分子的能力。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新型多功能碳纳米管来完成传感器由位于单个细胞穿透尖端的工作电极和参考电极组成，流体通过该尖端在这个项目中，构建了多功能纳米探针，能够同时注射。注入液体以便在密闭的水中进行独立的电化学测量然后利用纳米探针将促进反应的底物按顺序注入细胞中。选择性定量衰老相关的 β-半乳糖苷酶，这是一种在衰老过程中至关重要的胞质酶我们追求基于碳纳米管的纳米探针能够选择性地治疗许多慢性疾病。细胞实时量化异质活细胞群体的衰老情况；我们计划实现以下两个具体目标：（1）构建一个能够注入流体以进行独立电化学的多功能纳米探针在这里，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量化衰老- 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该提案的成果将提供一种基于 CNT 的一流工具。 (a) 建立了一种新的单细胞分析技术（细胞内电化学），并且 (b) 将其与传统技术（细胞内注射和荧光显微镜）形成全新的最小化用于从高时间单个活细胞收集定量数据的侵入性分析技术此外，新的纳米生物传感工具和技术可以在很长一段时间内实现。有效地传播到更大的科学界，以协助研究人员研究和阐明细胞生物学的基础知识将产生巨大的影响。开发多种疾病的诊断和治疗方法的转化潜力，包括慢性阻塞性肺病（COPD）、肺癌和心血管疾病，所有这些是主要的公共卫生问题。