Ecole Polytechnique Fédérale de Lausanne (EPFL), Institute of Chemical Sciences and Engineering (ISIC), NCCR in Chemical Biology, 1015, Lausanne, Switzerland.
Lucentix SA, EPFL Innovation Park, Bâtiment C, 1015, Lausanne, Switzerland.
Angew Chem Int Ed Engl. 2017 Jun 12;56(25):7112-7116. doi: 10.1002/anie.201702403. Epub 2017 May 16.
We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no-wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP-tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔR >500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera.
我们介绍了一种将抗体转化为比率型生物发光传感器蛋白的通用方法，可用于无洗涤分析物定量。我们的方法基于抗体片段与 NanoLuc 荧光素酶和 SNAP 标签的基因融合，后者用抗原的合成荧光竞争物标记。传感器与抗原（这里是合成药物）结合，将连接的荧光竞争物从抗体上置换下来，并破坏荧光素酶和荧光团之间的生物发光共振能量转移（BRET）。半合成传感器具有可调节的响应范围（亚毫摩尔至亚毫摩尔）和大动态范围（ΔR>500%），它们可以通过将样品点在纸上，然后用数码相机进行分析，来定量分析物。
