Physical and Biophysical Chemistry, Department of Chemistry , Bielefeld University , Universitätsstr. 25 , 33615 Bielefeld , Germany.
IRsweep AG , Laubisruetistrasse 44 , 8712 Staefa , Switzerland.
Anal Chem. 2018 Sep 4;90(17):10494-10500. doi: 10.1021/acs.analchem.8b02531. Epub 2018 Aug 15.
The kinetic analysis of irreversible protein reactions requires an analytical technique that provides access to time-dependent infrared spectra in a single shot. Here, we present a spectrometer based on dual-frequency-comb spectroscopy using mid-infrared frequency combs generated by quantum cascade lasers. Attenuation of the intensity of the combs by molecular vibrational resonances results in absorption spectra covering 55 cm in the fingerprint region. The setup has a native resolution of 0.3 cm, noise levels in the μOD range, and achieves sub-microsecond time resolution. We demonstrate the simultaneous recording of both spectra and transients of the photoactivated proton pump bacteriorhodopsin. More importantly, a single shot, i.e., a single visible light excitation, is sufficient to extract spectral and kinetic characteristics of several intermediates in the bacteriorhodopsin photocycle. This development paves the way for the noninvasive analysis of enzymatic conversions with high time resolution, broad spectral coverage, and minimal sample consumption.
不可逆蛋白质反应的动力学分析需要一种分析技术，能够在单次拍摄中提供时间相关的红外光谱。在这里，我们提出了一种基于双频梳光谱学的光谱仪，使用由量子级联激光器产生的中红外频率梳。梳状光的强度由于分子振动共振而衰减，导致在指纹区域覆盖 55cm 的吸收光谱。该装置具有 0.3cm 的固有分辨率、μOD 范围内的噪声水平和亚微秒的时间分辨率。我们演示了光激活质子泵菌紫质的两种光谱和瞬态的同时记录。更重要的是，单次拍摄，即单次可见光激发，足以提取菌紫质光循环中几个中间产物的光谱和动力学特征。这一发展为具有高时间分辨率、宽光谱覆盖范围和最小样品消耗的酶转化的非侵入性分析铺平了道路。
