Department of Physics, Cavendish Laboratory, University of Cambridge, United Kingdom.
Yusuf Hamied Department of Chemistry, University of Cambridge, United Kingdom.
Methods Appl Fluoresc. 2022 Sep 20;10(4). doi: 10.1088/2050-6120/ac9113.
The fluorescent detection of proteins without labels or stains, which affect their behaviour and require additional genetic or chemical preparation, has broad applications to biological research. However, standard approaches require large sample volumes or analyse only a small fraction of the sample. Here we use optofluidic hollow-core photonic crystal fibres to detect and quantify sub-microlitre volumes of unmodified bovine serum albumin (BSA) protein down to 100 nM concentrations. The optofluidic fibre's waveguiding properties are optimised for guidance at the (auto)fluorescence emission wavelength, enabling fluorescence collection from a 10 cm long excitation region, increasing sensitivity. The observed spectra agree with spectra taken from a conventional cuvette-based fluorimeter, corrected for the guidance properties of the fibre. The BSA fluorescence depended linearly on BSA concentration, while only a small hysteresis effect was observed, suggesting limited biofouling of the fibre sensor. Finally, we briefly discuss how this method could be used to study aggregation kinetics. With small sample volumes, the ability to use unlabelled proteins, and continuous flow, the method will be of interest to a broad range of protein-related research.
无需标记或染色即可检测蛋白质的荧光，这不会影响其行为，也无需额外的遗传或化学准备，在生物研究中有广泛的应用。然而，标准方法需要大量的样品体积，或者只能分析样品的一小部分。在这里，我们使用基于空芯光子晶体光纤的光流体技术来检测和定量分析亚微升体积的未经修饰的牛血清白蛋白（BSA）蛋白，其浓度低至 100 nM。该光流体光纤的波导特性经过优化，可在（自）荧光发射波长处进行引导，从而能够从 10 cm 长的激发区域收集荧光，提高了灵敏度。观察到的光谱与从传统基于比色皿的荧光计获得的光谱一致，并对光纤的引导特性进行了校正。BSA 的荧光强度与 BSA 浓度呈线性关系，而观察到的滞后效应很小，这表明纤维传感器的生物污垢有限。最后，我们简要讨论了这种方法如何用于研究聚集动力学。该方法需要的样品体积小，能够使用未标记的蛋白质，并实现连续流动，因此将引起广泛的蛋白质相关研究的兴趣。
