Naturally occurring protein switches have been repurposed for the development of biosensors and reporters for cellular and clinical applications¹. However, the number of such switches is limited, and reengineering them is challenging. Here we show that a general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which the binding of a peptide key triggers biological outputs of interest². The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; analyte binding drives the switch from the closed to the open state. Because the sensor is based on the thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We create biosensors that can sensitively detect the anti-apoptosis protein BCL-2, the IgG1 Fc domain, the HER2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac troponin I and an anti-hepatitis B virus antibody with the high sensitivity required to detect these molecules clinically. Given the need for diagnostic tools to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)³, we used the approach to design sensors for the SARS-CoV-2 spike protein and antibodies against the membrane and nucleocapsid proteins. The former, which incorporates a de novo designed spike receptor binding domain (RBD) binder⁴, has a limit of detection of 15 pM and a luminescence signal 50-fold higher than the background level. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes, and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.

天然存在的蛋白质开关已被重新用于开发用于细胞和临床应用的生物传感器和报告基因¹。然而，此类开关的数量有限，重新设计它们具有挑战性。在这里，我们表明可以通过从头设计的蛋白质开关反转信息流来创建一类基于蛋白质的生物传感器，其中肽键的结合会触发感兴趣的生物输出². 所设计的传感器是具有封闭暗态和开放发光态的模块化分子器件；分析物结合驱动开关从关闭状态到打开状态。由于传感器基于分析物结合与传感器激活的热力学耦合，因此只需要一个目标结合域，这简化了传感器设计并允许在溶液中直接读出。我们创造了可以灵敏检测抗凋亡蛋白 BCL-2、IgG1 Fc 域、HER2 受体和肉毒杆菌神经毒素 B 的生物传感器，以及高灵敏度的心肌肌钙蛋白 I 和抗乙型肝炎病毒抗体的生物传感器临床上需要检测这些分子。鉴于需要诊断工具来追踪严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) ³，我们使用该方法为 SARS-CoV-2 刺突蛋白和针对膜和核衣壳蛋白的抗体设计传感器。前者结合了从头设计的刺突受体结合域 (RBD) 粘合剂⁴，具有 15 pM 的检测限和比背景水平高 50 倍的发光信号。该平台的模块化和灵敏度应该能够为广泛的分析物快速构建传感器，并突出从头蛋白质设计的力量，以创建具有新的和有用的功能的多状态蛋白质系统。