BTeam, a Novel BRET-based Biosensor for the Accurate Quantification of ATP Concentration within Living Cells.
BTeam, a Novel BRET-based Biosensor for the Accurate Quantification of ATP Concentration within Living Cells.
复制标题
DOI：
10.1038/srep39618
复制
发表时间：
2016-12-21
影响因子：
4.6
通讯作者：
Imamura H
中科院分区：
文献类型：
--
作者：
Yoshida T;Kakizuka A;Imamura H
三磷酸腺苷水平可能代表细胞的基本健康状况。然而，由于缺乏合适的方法，精确测量活细胞内的ATP水平受到了阻碍。在这里，我们开发了一种名为BTeam的新型三磷酸腺苷生物传感器。BTeam由一个黄色荧光蛋白(YFP)、细菌三磷酸腺苷合成酶ε亚单位的三磷酸腺苷结合区和三磷酸腺苷非消耗性荧光素酶(NLuc)组成。为了实现发射，BTeam只需要NLuc衬底。BTeam显示了与ATP结合后生物发光共振能量转移效率的提高，导致发射光谱随ATP浓度的变化而变化。通过使用YFP/NLuc发射比的值来表示ATP水平，BTeam在发射强度改变的情况下也实现了稳定的信号输出。利用这种生物传感器，我们成功地对一组活细胞的细胞内三磷酸腺苷浓度进行了准确的定量，在一些培养细胞系的胞浆(3.7-4.1 mM)和线粒体基质(2.4-2.7 mM)中检测到轻微的分布。此外，BTeam允许连续跟踪相同细胞的胞浆ATP水平，以及对单个细胞内胞浆ATP动态的生物发光成像。该技术简单、准确，是定量测定细胞内三磷酸腺苷浓度的有效方法。
ATP levels may represent fundamental health conditions of cells. However, precise measurement of intracellular ATP levels in living cells is hindered by the lack of suitable methodologies. Here, we developed a novel ATP biosensor termed “BTeam”. BTeam comprises a yellow fluorescent protein (YFP), the ATP binding domain of the ε subunit of the bacterial ATP synthase, and an ATP-nonconsuming luciferase (NLuc). To attain emission, BTeam simply required NLuc substrate. BTeam showed elevated bioluminescence resonance energy transfer efficiency upon ATP binding, resulted in the emission spectra changes correlating with ATP concentrations. By using values of YFP/NLuc emission ratio to represent ATP levels, BTeam achieved steady signal outputs even though emission intensities were altered. With this biosensor, we succeeded in the accurate quantification of intracellular ATP concentrations of a population of living cells, as demonstrated by detecting the slight distribution in the cytosol (3.7–4.1 mM) and mitochondrial matrix (2.4–2.7 mM) within some cultured cell lines. Furthermore, BTeam allowed continuous tracing of cytosolic ATP levels of the same cells, as well as bioluminescent imaging of cytosolic ATP dynamics within individual cells. This simple and accurate technique should be an effective method for quantitative measurement of intracellular ATP concentrations.
登录
查看更多内容
影响因子：
4.8
作者：
Kennedy, HJ;Pouli, AE;Rutter, GA
通讯作者：
Rutter, GA
影响因子：
13.9
作者：
Fenno L;Yizhar O;Deisseroth K
通讯作者：
Deisseroth K
影响因子：
46.9
作者：
Nagai, T;Ibata, K;Miyawaki, A
通讯作者：
Miyawaki, A
影响因子：
3.5
作者：
SIESS, EA;WIELAND, OH
通讯作者：
WIELAND, OH
影响因子：
16.6
作者：
通讯作者：
--