CdS quantum dots generated in-situ for fluorometric determination of thrombin activity
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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.
KeywordsFibrinogen Biosensing Nanoparticles Human plasma Coagulation Ecarin Bioassay Emission Fibrin Alkaline phosphatase
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).
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Compliance with ethical standards
All procedures performed in studies did not involve neither human participants nor animals. Lyophilized human plasma and other blood derived materials were obtained from Sigma Inc.
- 6.Chang J-Y (1985) Thrombin specificity. Eur J Biochem 151:217–224. https://doi.org/10.1111/j.1432-1033.1985.tb09091.x CrossRefPubMedGoogle Scholar
- 13.Oberfrank S, Drechsel H, Sinn S, Northoff H, Gehring FK (2016) Utilisation of quartz crystal microbalance sensors with dissipation (QCM-D) for a clauss fibrinogen assay in comparison with common coagulation reference methods. Sensors 16:282. https://doi.org/10.3390/s16030282 CrossRefPubMedGoogle Scholar
- 15.Li YJ, Chiu WJ, Unnikrishnan B, Huang CC (2014) Monitoring thrombin generation and screening anticoagulants through pulse laser-induced fragmentation of biofunctional nanogold on cellulose membranes. ACS Appl Mater Interfaces 6:15253–15261. https://doi.org/10.1021/am503615c CrossRefPubMedGoogle Scholar
- 18.Lin J-H, Huang K-H, Zhan S-W, Yu C-J, Tseng W-L, Hsieh M-M (2019) Inhibition of catalytic activity of fibrinogen-stabilized gold nanoparticles via thrombin-induced inclusion of nanoparticle into fibrin: application for thrombin sensing with more than 104-fold selectivity. Spectrochim Acta Part A Mol Biomol Spectrosc 210:59–65. https://doi.org/10.1016/j.saa.2018.11.013 CrossRefGoogle Scholar
- 27.Cheng T, Li X, Huang P, Wang H, Wang M, Yang W (2019) Colorimetric and electrochemical (dual) thrombin assay based on the use of a platinum nanoparticle modified metal-organic framework (type Fe-MIL-88) acting as a peroxidase mimic. Microchim Acta 186:94. https://doi.org/10.1007/s00604-018-3209-4 CrossRefGoogle Scholar
- 32.Hursting MJ, Alford KL, Becker JP, Brooks RL, Joffrion JL, Knappenberger GD, Kogan PW, Kogan TP, Mckinney AA, Schwarz RP (1997) Novastan® (brand of Argatroban): a small-molecule, direct thrombin inhibitor. Semin Thromb Hemost 23:503–516. https://doi.org/10.1055/s-2007-996128 CrossRefPubMedGoogle Scholar
- 33.Morita T, Iwanaga S. (1981) Prothrombin activator from Echis carinatus venom. Methods Enzymol 80:303–311. https://doi.org/10.1016/S0076-6879(81)80026-2