Intracellular Cascade FRET for Temperature Imaging of Living Cells with Polymeric Ratiometric Fluorescent Thermometers
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- 作者：Xianglong Hu ; Yang Li ; Tao Liu ; Guoying Zhang ; Shiyong Liu
- 刊名：ACS Applied Materials & Interfaces
- 出版年：2015
- 出版时间：July 22, 2015
- 年：2015
- 卷：7
- 期：28
- 页码：15551-15560
- 全文大小：514K
- ISSN：1944-8252
文摘
Intracellular temperature plays a prominent role in cellular functions and biochemical activities inside living cells, but effective intracellular temperature sensing and imaging is still in its infancy. Herein, thermoresponsive double hydrophilic block copolymers (DHBCs)-based fluorescent thermometers were fabricated to investigate their application in intracellular temperature imaging. Blue-emitting coumarin monomer, CMA, green-emitting 7-nitro-2,1,3-benzoxadiazole (NBD) monomer, NBDAE, and red-emitting rhodamine B monomer, RhBEA, were copolymerized separately with N-isopropylacrylamide (NIPAM) to afford dye-labeled PEG-b-P(NIPAM-co-CMA), PEG-b-P(NIPAM-co-NBDAE), and PEG-b-P(NIPAM-co-RhBEA). Because of the favorable fluorescence resonance energy transfer (FRET) potentials between CMA and NBDAE, NBDAE and RhBEA, as well as the slight tendency between CMA and RhBEA fluorophore pairs, three polymeric thermometers based on traditional one-step FRET were fabricated by facile mixing two of these three fluorescent DHBCs, whereas exhibiting limited advantages. Thus, two-step cascade FRET among three polymeric fluorophores was further interrogated, in which NBD acted as a bridging dye by transferring energy from CMA to RhBEA. Through the delicate optimization of the molar contents of three polymeric components, a 鈭?.4-fold ratio change occurred in the temperature range of 20鈥?4 掳C, and the detection sensitivity improved significantly, reached as low as 鈭?.4 掳C, which definitely outperformed other one-step FRET thermometers in the intracellular temperature imaging of living cells. To our knowledge, this work represents the first example of polymeric ratiometric thermometer employing thermoresponsive polymer-based cascade FRET mechanism.