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 (R(2)=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).
Chen
Chuan-Kuo
CK
Department of Chemistry, National Taiwan University, 1, Section 4, Roosevelt Road, Taipei 10617, Taiwan.
Huang
Chih-Ching
CC
Chang
Huan-Tsung
HT
D015374:Biosensing Techniques / Q000295:instrumentation*; D001774:Blood Chemical Analysis / Q000295:instrumentation*; D003124:Colorimetry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006046:Gold / Q000737:chemistry*; D006801:Humans; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D013917:Thrombin / Q000032:analysis*
D006046:Gold; D013917:Thrombin