A Rapid and Ultrasensitive Thrombin Biosensor Based on a Rationally Designed Trifunctional Protein

Abstract

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.

Structural and photophysical characterization of the small ultra-red fluorescent protein

“…These properties make purified smURFP useful for immediate fluorescence imaging without requiring time-consuming protein translation, folding, and chromophore incorporation. Thus far, purified smURFP sensors and probes have been used in numerous applications, for example, to sense BV (detection limit of 0.4 nanomolar) 24 , detect thrombin protease activity (detection limit 0.2 attomolar) 25 , label membrane proteins using sortase-mediated conjugation 26 , label virus-like nanoparticles for imaging animal biodistribution 27 , and for noninvasive in vivo imaging of cancer when incorporated into protein nanoparticles 21 . In addition to these applications, smURFP should be compatible with advanced imaging modalities, such as two-photon, fluorescence lifetime, and single-molecule imaging.…”

Proteolytic Biosensors with Functional Nanomaterials: Current Approaches and Future Challenges

“…In addition, c-nucleophile (C-Nu) attached to the pyronin precursor can be screened for the optimization of fluorescent signal generation for sensitive protease measurement. Zhang et al developed an ultrasensitive and rapid thrombin biosensor composed of trifunctional protein (Figure 1a) [57]. This particular trifunctional protein consists of three functional parts: a thrombin cleavage site (TCS), far-red fluorescent protein (smURFP), and hydrophobin (HGFI).…”

Innovative surface bio-functionalization by fungal hydrophobins and their engineered variants

“…Since the two proteins are linked by the specific cleavage site of thrombin, the higher the protease concentration, the lower the fluorescence intensity measured in the well, due to the release of GFP from the surface, thus developing an ultrasensitive biosensor. A very similar approach was used by Zhang and coworkers by using a HGFI-fusion protein ( Zhang et al, 2020 ). Mirzaei et al coated glassy carbon electrodes with HFBI and used it as a glue to immobilize the lactate dehydrogenase enzyme developing a biosensor for pyruvate, whose concentration is an index of circulatory disorders ( Mirzaei et al, 2019 ).…”