Rapid and sensitive detection of thrombin is imperative for the early diagnosis, prevention, and treatment of thrombin‐related diseases. Here, an ultrasensitive and rapid thrombin biosensor is developed based on rationally designed trifunctional protein HTs, comprising three functional units, including a far‐red fluorescent protein smURFP, hydrophobin HGFI, and a thrombin cleavage site (TCS). smURFP is used as a detection signal to eliminate any interference from the autofluorescence of sample matrix to increase detection sensitivity. HGFI serve as an adhesive unit to allow rapid immobilization of HTs on a multiwall plate. The TCS linking HGFI and smURFP function as a sensing element to recognize and detect thrombin. HTs immobilization is symmetrically optimized and characterized. Thrombin assay reveals the specific recognition of active thrombin in samples and the hydrolysis of the immobilized HTs, resulting in a decrease in the fluorescence intensity of the sample in a thrombin concentration‐dependent manner. The limit of detection (LOD) is as low as 0.2 am in the serum. To the authors’ knowledge, this is the lowest LOD ever reported for any thrombin biosensor. This study sheds light on the engineering of multifunctional proteins for biosensing.

中文翻译：

---

基于合理设计的三功能蛋白的快速，超灵敏的凝血酶生物传感器。

对于凝血酶相关疾病的早期诊断，预防和治疗，必须快速，灵敏地检测凝血酶。在这里，基于合理设计的三功能蛋白HTs开发了一种超灵敏，快速的凝血酶生物传感器，该传感器包含三个功能单元，包括远红色荧光蛋白smURFP，疏水蛋白HGFI和凝血酶裂解位点（TCS）。smURFP用作检测信号，可消除样品基质自发荧光的任何干扰，从而提高检测灵敏度。HGFI用作粘合单元，可将HT快速固定在多层板上。连接HGFI和smURFP的TCS充当识别和检测凝血酶的传感元件。HTs固定化得到对称优化和表征。凝血酶测定法揭示了样品中活性凝血酶的特异性识别和固定化HT的水解，从而导致凝血酶浓度依赖性地降低了样品的荧光强度。检出限（LOD）低至0.2 a血清中的m。据作者所知，这是任何凝血酶生物传感器报告的最低LOD。这项研究为多功能蛋白质的生物传感工程提供了启示。