Sipior J, Bambot S, Romauld M, Carter G M, Lakowicz J R, Rao G
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201-1503, USA.
Anal Biochem. 1995 May 20;227(2):309-18. doi: 10.1006/abio.1995.1286.
We describe the fabrication and characterization of an optical CO2 sensor based on the change in fluorescence lifetimes due to fluorescence resonance energy transfer from a pH-insensitive donor, sulforhodamine 101, to a pH-sensitive acceptor, either m-cresol purple or thymol blue, entrapped in an ethyl cellulose film. A phase transfer agent allows incorporation of the dyes and water into the film, while providing an initially basic environment for the acceptor. Diffusion of CO2 into the water entrapped in the film produced carbonic acid, causing a pH-dependent decrease in the spectral overlap of the acceptor absorbance with the donor emission, and decreased energy transfer, resulting in increased SR101 donor lifetimes. The lifetime changes were detected as a change in the phase of the emission, relative to the modulated excitation, and were insensitive to excitation intensities and emission signal levels. In addition to an externally modulated 442-nm light source, we excited the sensor with a directly modulated 635-nm laser diode and detected the anti-Stokes emission. The CO2 sensor is not fragile and can provide stable readings for weeks. The use of fluorescence resonance energy transfer, along with the simple entrainment procedure, allows facile change of the CO2 response range through change of the acceptor dye and the use of laser diode excitation sources.
我们描述了一种基于荧光寿命变化的光学二氧化碳传感器的制备与表征。该变化源于荧光共振能量转移,即从对pH不敏感的供体磺基罗丹明101转移到包裹在乙基纤维素膜中的对pH敏感的受体间甲酚紫或百里酚蓝。一种相转移剂可使染料和水掺入膜中,同时为受体提供初始碱性环境。二氧化碳扩散到膜中截留的水中会生成碳酸,导致受体吸光度与供体发射光谱的重叠随pH降低,能量转移减少,从而使磺基罗丹明101供体的寿命增加。寿命变化通过发射相位相对于调制激发的变化来检测,且对激发强度和发射信号水平不敏感。除了外部调制的442 nm光源外,我们还用直接调制的635 nm激光二极管激发传感器并检测反斯托克斯发射。该二氧化碳传感器不易损坏,可在数周内提供稳定读数。利用荧光共振能量转移以及简单的包埋程序,通过改变受体染料和使用激光二极管激发源,可轻松改变二氧化碳响应范围。