论文标题
无标签蛋白上自荧光相关光谱的紫外线光稳定性提高
Ultraviolet Photostability Improvement for Autofluorescence Correlation Spectroscopy on Label-Free Proteins
论文作者
论文摘要
蛋白质自动荧光的光稳定性差和亮度低是主要的局限性，可阻止单分子水平检测无标记的蛋白质。克服这些问题，我们在这里报告了一种促进蛋白质光稳定性并在紫外线（UV）中使用其天然色氨酸自动荧光的策略，以进行荧光相关光谱（FCS）。将酶氧气清除剂与抗氧化剂和三重态淬火剂相结合，可以极大地促进蛋白质光稳定性，降低光漂白概率并提高净自荧光检测率。我们的结果表明，最初针对有机可见荧光染料得出的基本光化学概念是相当一般的。使用这种方法，我们在无标签的链霉亲和素蛋白上实现了UV荧光相关光谱，该蛋白仅包含24个色氨酸残基，比当前的最新面积少6.5倍。该策略极大地扩展了单个分子荧光的多功能性检测无标记蛋白的可能性，而无需存在潜在的干扰外部荧光标记。它还开辟了新的观点，以提高有机设备的紫外线耐用性。
The poor photostability and low brightness of protein autofluorescence have been major limitations preventing the detection of label-free proteins at the single molecule level. Overcoming these issues, we report here a strategy to promote the photostability of proteins and use their natural tryptophan autofluorescence in the ultraviolet (UV) for fluorescence correlation spectroscopy (FCS). Combining enzymatic oxygen scavengers with antioxidants and triplet state quenchers greatly promotes the protein photostability, reduces the photobleaching probability and improves the net autofluorescence detection rate. Our results show that the underlying photochemical concepts initially derived for organic visible fluorescent dyes are quite general. Using this approach, we achieved UV fluorescence correlation spectroscopy on label-free streptavidin proteins containing only 24 tryptophan residues, 6.5x less than the current state-of-the-art. This strategy greatly extends the possibility to detect single label-free proteins with the versatility of single molecule fluorescence without requiring the presence of a potentially disturbing external fluorescent marker. It also opens new perspectives to improve the UV durability of organic devices.
