Gold nanocluster-encapsulated glucoamylase as a biolabel for sensitive detection of thrombin with glucometer readout

This article reports on a sensitive aptamer-based assay for thrombin. The assay includes the following steps: (a) a first thrombin-specific aptamer (P-1) was immobilized on the surface of the wells of a microtiter plate via biotin-streptavidin interaction; (b) gold nanoclusters (GNCs) were used to cover the enzyme glucoamylase via a reverse micelle method; (c) the GNCs were then coated with the second aptamer (P-2) via thiol chemistry; (d) addition of a solution containing thrombin to the well, and (e) subsequent addition of amylopectin. The glucoamylase in the GNC label catalytically hydrolyzes the amylopectin to form glucose which then is quantified with a glucometer. Under optimal conditions, the signal for glucose increases with the concentration of thrombin in range from 0.05 to 100 nM, and the detection limit is as low as 10 pM. The assay has a good repeatability and displays an intermediate precision of down to 11 %. Nonspecific adsorption was not observed in a series of analyses. The method was applied to the determination of thrombin in spiked serum samples, and the recoveries ranged from 94 to 110 %. The method is assumed to have a wide scope in that it may be extended to numerous other analytes for which appropriate aptamers are available.