Department of Molecular Genetics , University of Toronto, Charles Best Institute , 112 College Street , Toronto , Ontario M5G 1L6 , Canada.
Bioconjug Chem. 2020 Jan 15;31(1):16-27. doi: 10.1021/acs.bioconjchem.9b00710. Epub 2019 Dec 13.
Since their discovery, fluorescent probes have found widespread use in biological research. Over time, multiple next-generation probes increased the fluorescence catalog by offering novel capabilities of detection that have been previously difficult or lacking with conventional probes. One of such probes is called a fluorogen-activating protein (FAP). These are bimodular sensors, composed of a single-chain antibody that exhibits high-affinity and selectivity for small-molecule fluorogens. Because fluorogens are inherently nonfluorescent unless sterically restricted, upon the formation of the noncovalent FAP-fluorogen complex the fluorogen module emits fluorescence when excited by light. More interestingly, these bimodular sensors permit improvement of their biophysical properties. For instance, the fluorescence spectra and environmental sensing capabilities of fluorogens may be altered by the method of chemical modification at the fluorogen structural level. Also, optimizations of the single-chain antibody scaffold, via amino acid substitutions at the selectivity regions, may improve the detection brightness and affinities of fluorogens; this may also improve the biophysical stability of FAPs in different cellular environments. Additionally, when utilized as biological discovery probes, FAP biosensors exhibit functional activity as genetic fusion tags with cellular proteins; this results in high fluorescent sensitivities of cell surface and intracellular targets. Also, FAPs allow the monitoring of cellular traffic of surface receptors by fluorescence methods of real-time color switching, or signal onset and offset. They find application as biological probes integrated into biomaterials, or as soluble affinity reagents for whole live animal studies. Overall, this noncovalent activation of fluorogen particles results in advanced strategies of fluorescence detection.
自发现以来，荧光探针在生物研究中得到了广泛应用。随着时间的推移，多种下一代探针通过提供以前用传统探针难以实现或缺乏的新型检测能力，增加了荧光目录。其中一种探针称为荧光基团激活蛋白 (FAP)。这些是双模块传感器，由单链抗体组成，对小分子荧光基团表现出高亲和力和选择性。由于荧光基团在没有空间限制的情况下本质上是非荧光的，因此在形成非共价 FAP-荧光基团复合物后，当用光激发时，荧光基团模块会发出荧光。更有趣的是，这些双模块传感器允许改善它们的物理特性。例如，通过在荧光基团结构水平上进行化学修饰，可以改变荧光基团的荧光光谱和环境感应能力。此外，通过在选择性区域进行氨基酸取代来优化单链抗体支架，可能会提高荧光基团的检测亮度和亲和力；这也可能提高 FAP 在不同细胞环境中的物理稳定性。此外，当用作生物发现探针时，FAP 生物传感器作为细胞蛋白的遗传融合标签表现出功能活性；这导致细胞表面和细胞内靶标的荧光灵敏度很高。此外，FAP 允许通过实时颜色切换、信号起始和结束的荧光方法监测表面受体的细胞内运输。它们可作为整合到生物材料中的生物探针或作为用于整个活体动物研究的可溶性亲和试剂应用。总的来说，这种对荧光基团颗粒的非共价激活导致了荧光检测的先进策略。
