RT Journal Article
T1 CdS quantum dots generated in-situ for fluorometric determination of thrombin activity
A1 Saa, Laura
A1 Díez-Buitrago, Beatriz
A1 Briz, Nerea
A1 Pavlov, Valeri
AB A method is presented for sensitive determination of thrombin activity. It is based on (a) the interaction between fibrinogen after activation with thrombin, and (b) an enzymatic amplification step consisting of in-situ growth of CdS quantum dots (QDs). Fibrinogen is immobilized on the surface of the wells of a microplate and then incubated with a mixture of biotinylated fibrinogen and thrombin. Thrombin activates immobilized fibrinogen and free biotinylated fibrinogen. This leads to the formation of insoluble biotinylated fibrin that remains bound on the surface of the wells. Afterwards, the samples are incubated with avidin-labeled alkaline phosphatase (ALP) which binds to biotinylated fibrin. ALP hydrolyzes the substrate p-nitrophenyl phosphate (pNPP) under formation of phosphate ions which stabilize CdS QDs that are grown in-situ from cadmium(II) and sulfide. The generation of fibrin is correlated with the activity of thrombin. Increased thrombin concentration results in increased fluorescence that can be measured at excitation/emission wavelengths of 300/510 nm. The introduction of such an amplification step (the enzyme-triggered growth of QDs) allows for the quantification of thrombin in the picomolar concentration range, with a linear response up to 2.5 pM and a detection limit of 0.05 pM. The method was applied to the determination of thrombin activity in human plasma and of the thrombin inhibitor argatroban. [Figure not available: see fulltext.]
SN 0026-3672
YR 2019
FD 2019-09-01
LA eng
NO Saa , L , Díez-Buitrago , B , Briz , N & Pavlov , V 2019 , ' CdS quantum dots generated in-situ for fluorometric determination of thrombin activity ' , Mikrochimica Acta , vol. 186 , no. 9 , 657 . https://doi.org/10.1007/s00604-019-3765-2
NO Publisher Copyright: © 2019, Springer-Verlag GmbH Austria, part of Springer Nature.
NO This work was supported by the Ministry of Science, Innovation and Universities/AEI/FEDER, UE (RETOS I + D – Grant No.BIO2017-88030-R and the Maria de Maeztu Units of Excellence Programme – Grant No. MDM-2017-0720). This work was supported by the Ministry of Science, Innovation and Universities/AEI/FEDER, UE (RETOS I + D ? Grant No.BIO2017-88030-R and the Maria de Maeztu Units of Excellence Programme ? Grant No. MDM-2017-0720).
DS TECNALIA Publications
RD 29 sept 2024