CdS quantum dots generated in-situ for fluorometric determination of thrombin activity

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ISSN: 0026-3672
DOI: 10.1007/s00604-019-3765-2
Publication date
2019-09-01
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Abstract
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.]
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Publisher Copyright: © 2019, Springer-Verlag GmbH Austria, part of Springer Nature.
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Alkaline phosphatase , Bioassay , Biosensing , Coagulation , Ecarin , Emission , Fibrin , Fibrinogen , Human plasma , Nanoparticles , Analytical Chemistry
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journal article
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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