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- Title
Fluorometric determination of paraoxon in human serum using a gold nanoparticle-immobilized organophosphorus hydrolase and coumarin 1 as a competitive inhibitor.
- Authors
Kamelipour, Nahid; Mohsenifar, Afshin; Tabatabaei, Meisam; Rahmani-Cherati, Tavoos; Khoshnevisan, Kamyar; Allameh, Abdolamir; Milani, Majid; Najavand, Saeid; Etemadikia, Batool
- 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 M. [Figure not available: see fulltext.]
- Subjects
FLUORIMETRY; PARAOXON; SERUM; GOLD nanoparticles; ORGANOPHOSPHORUS compounds; HYDROLASES; COUMARINS; ENZYME inhibitors; BIOSENSORS
- Publication
Microchimica Acta, 2014, Vol 181, Issue 1/2, p239
- ISSN
0026-3672
- Publication type
Article
- DOI
10.1007/s00604-013-1103-7