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[用于活细胞成像的荧光探针的开发]

[Development of a Fluorescence Probe for Live Cell Imaging].

作者信息

Shibata Aya

机构信息

Faculty of Engineering, Gifu University.

出版信息

Yakugaku Zasshi. 2017;137(11):1323-1337. doi: 10.1248/yakushi.17-00132.

Abstract

Probes that detect specific biological materials are indispensable tools for deepening our understanding of various cellular phenomena. In live cell imaging, the probe must emit fluorescence only when a specific substance is detected. In this paper, we introduce a new probe we developed for live cell imaging. Glutathione S-transferase (GST) activity is higher in tumor cells than in normal cells and is involved in the development of resistance to various anticancer drugs. We previously reported the development of a general strategy for the synthesis of probes for detection of GST enzymes, including fluorogenic, bioluminogenic, and F-NMR probes. Arylsulfonyl groups were used as caging groups during probe design. The fluorogenic probes were successfully used to quantitate very low levels of GST activity in cell extracts and were also successfully applied to the imaging of microsomal MGST1 activity in living cells. The bioluminogenic and F-NMR probes were able to detect GST activity in Escherichia coli cells. Oligonucleotide-templated reactions are powerful tools for nucleic acid sensing. This strategy exploits the target strand as a template for two functionalized probes and provides a simple molecular mechanism for multiple turnover reactions. We developed a nucleophilic aromatic substitution reaction-triggered fluorescent probe. The probe completed its reaction within 30 s of initiation and amplified the fluorescence signal from 0.5 pM target oligonucleotide by 1500 fold under isothermal conditions. Additionally, we applied the oligonucleotide-templated reaction for molecular releasing and peptide detection.

摘要

检测特定生物材料的探针是深化我们对各种细胞现象理解的不可或缺的工具。在活细胞成像中,探针必须仅在检测到特定物质时才发出荧光。在本文中,我们介绍一种我们为活细胞成像开发的新探针。谷胱甘肽S-转移酶(GST)在肿瘤细胞中的活性高于正常细胞,并且参与对各种抗癌药物的耐药性发展。我们之前报道了一种用于合成检测GST酶的探针的通用策略,包括荧光探针、生物发光探针和F-核磁共振探针。在探针设计过程中,芳基磺酰基被用作封闭基团。荧光探针成功用于定量细胞提取物中极低水平的GST活性,并且还成功应用于活细胞中微粒体MGST1活性的成像。生物发光探针和F-核磁共振探针能够检测大肠杆菌细胞中的GST活性。寡核苷酸模板反应是核酸传感的强大工具。该策略利用靶链作为两个功能化探针的模板,并为多次周转反应提供了一种简单的分子机制。我们开发了一种亲核芳香取代反应触发的荧光探针。该探针在起始后30秒内完成反应,并在等温条件下将来自0.5 pM靶寡核苷酸的荧光信号放大了1500倍。此外,我们将寡核苷酸模板反应应用于分子释放和肽检测。

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