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

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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.

Schematic representation of a fluorometric method for determination of thrombin activity in the picomolar concentration range based on the interaction between fibrinogen after activation with thrombin, and an enzymatic amplification step consisting of in-situ growth of CdS quantum dots (CdS QD).

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Acknowledgments

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).

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Authors and Affiliations

  1. Biosensing Laboratory, CIC biomaGUNE, Paseo Miramón 182, 20014, San Sebastián, Spain

    Laura Saa, Beatriz Díez-Buitrago & Valeri Pavlov

  2. Tecnalia, Paseo Mikeletegi, 20009, San Sebastián, Spain

    Beatriz Díez-Buitrago & Nerea Briz

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  1. Laura Saa
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  4. Valeri Pavlov
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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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Correspondence to Valeri Pavlov.

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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.

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ESM 1

Size distribution of the CdS QDs, comparison between colorimetric and fluorogenic methods and effect of argatroban on the detection system. (DOCX 465 kb)

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Saa, L., Díez-Buitrago, B., Briz, N. et al. CdS quantum dots generated in-situ for fluorometric determination of thrombin activity. Microchim Acta 186, 657 (2019). https://doi.org/10.1007/s00604-019-3765-2

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