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Abstract
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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
electronmicroscopy 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.
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