Label-free colorimetric detection of picomolar thrombin in blood plasma using a gold nanoparticle-based assay

We unveil a novel, label-free, colorimetric assay – using fibrinogen (Fib) and gold nanoparticles (Au NPs) – for the highly selective and sensitive detection of thrombin. Addition of fibrinogen to a solution of Au NPs (average diameter: 56 nm) led to ready conjugation, forming Fib–Au NPs through electrostatic and hydrophobic interactions. Introduction of thrombin (a serine protease) into the Fib–Au NPs solutions in the presence of excess fibrinogen induced the formation of insoluble fibrillar fibrin–Au NPs agglutinates through the polymerization of the unconjugated and conjugated fibrinogen. After centrifugation, the absorbance at 532 nm of the supernatants decreased upon increasing the concentration of thrombin. This Fib–Au NP probe provides high sensitivity [limit of detection (LOD): 0.04 pM] for thrombin, with remarkable selectivity over other proteins and proteases. The range of linearity for the absorbance against the thrombin concentration was 0.1–10 pM (R2 = 0.96). This approach provides an LOD for thrombin that is lower than those obtainable using other nanomaterial- and aptamer-based detection methods. We validated the utility of this Fib–Au NP probe through separate analyses of thrombin and Factor Xa at picomolar levels in plasma samples—without the need for sample pretreatment. This technique appears to have practical potential in the diagnosis of diseases associated with coagulation abnormalities and cancers (e.g., pulmonary metastasis).