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Fluorometric determination of paraoxon in human serum using a gold nanoparticle-immobilized organophosphorus hydrolase and coumarin 1 as a competitive inhibitor

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Abstract

A dimeric organophosphorus hydrolase (OPH; EC 3.1.8.1; 72 kDa) was isolated from wild-type bacteria, analyzed for its 16s rRNA sequence, purified, and immobilized on gold nanoparticles (AuNPs) to form the transducer part of a biosensor. The isolated strain was identified as Pseudomonas aeruginosa. The AuNPs were characterized by transmission electron microscopy and localized surface plasmon resonance. Covalent binding of OPH to the AuNPs was confirmed by spectrophotometry, enzymatic activity assays, and FTIR spectroscopy. Coumarin 1, a competitive inhibitor of OPH, was used as a fluorogenic probe. The bioconjugates quench the emission of coumarin 1 upon binding, but the addition of paraoxon results in an enhancement of fluorescence that is directly proportional to the concentration of paraoxon. The gold-OPH conjugates were then used to determine paraoxon in serum samples spiked with varying levels of paraoxon. The method works in the 50 to 1,050 nM concentration range, has a low standard deviation (with a CV of 5.7–11 %), and a detection limit as low as 5 × 10−11 M.

Coumarin 1, a competitive inhibitor of organophosphorus hydrolase, was used as a fluorogenic probe in the bioconjugates. The gold nanoparticles contained in the bioconjugates quench the emission of coumarin 1 upon binding, but the addition of paraoxon results in an enhancement of fluorescence leading to its detection.

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Acknowledgments

This project was financially supported by Tarbiat Modares University. We also would like appreciate Mr. Shahram Pourbeyranvand for technical support in conducting TEM analyses.

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Authors and Affiliations

  1. Department of Toxicology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

    Nahid Kamelipour

  2. Research and Development Department, Nanozino, 16536-43181, Tehran, Iran

    Afshin Mohsenifar & Tavoos Rahmani-Cherati

  3. Nanosystems Research Team (NRTeam), Microbial Biotechnology and Biosafety Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj, Iran

    Afshin Mohsenifar, Meisam Tabatabaei & Kamyar Khoshnevisan

  4. Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran

    Abdolamir Allameh, Majid M. Milani & Batool Etemadikia

  5. Department of Biology, Faculty of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran

    Saeid Najavand

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  1. Nahid Kamelipour
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  2. Afshin Mohsenifar
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Correspondence to Afshin Mohsenifar.

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Kamelipour, N., Mohsenifar, A., Tabatabaei, M. et al. Fluorometric determination of paraoxon in human serum using a gold nanoparticle-immobilized organophosphorus hydrolase and coumarin 1 as a competitive inhibitor. Microchim Acta 181, 239–248 (2014). https://doi.org/10.1007/s00604-013-1103-7

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  • DOI: https://doi.org/10.1007/s00604-013-1103-7

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