was added into each tube and transferred
into a 96-well plate. The absorbance was
read at 340 nm, 450 nm, or 414 nm for
the nickel sulfate, copper sulfate and
cobalt chloride hexahydrate assay wells,
respectively, and metal concentration determined using a standard curve.
Fig 6. Trace elemental concentration in seawater broken into parts per billion (ppb).
used. His6 is a peptide composed of six
histidine amino acids and was produced
chemically by 21st Century Biochemicals.
Glidden Vinyl Latex (1424) was used to
create the rechargeable coating. Nickel
sulfate (Sigma 656895), copper sulfate
(Sigma 451657) and cobalt chloride
hexahydrate (Sigma 255599) were used
for metal assay solutions. The
organophosphorus compound paraoxon
(Toronto Research Chemicals) was the reactant (substrate) used for OPDtox TM
(Reactive Surfaces Ltd., Texas).
Preparation of Peptide/Enzyme Blended
Films and Coatings. To create a coating
with 3% dry weight, 93 mg/ml peptide or
enzyme was added into water and mixed
into Glidden vinyl latex coating at a ratio
of 1 part water to 7 parts coating. The
coating was mixed until it was evenly dispersed. A draw down bar was used to
make films on a polypropylene block at 8
mils thickness (wet). Films were allowed to
cure for 48 hrs before use. Control films
were made in the same manner without the
added peptide or enzyme.
Functional Activity Assessment for
Metal Chelating Proteins. Activity was de-
termined using 1-3 cm² free films cut from
OPDtox blended coatings that had been
applied and dried on a polypropylene
sheet. The free films were incubated in the
appropriate reaction buffer under agitation
and analyzed. The reactions were per-
formed as described for solution phase.
Control samples, which included both re-
actions without films and films without en-
zyme, were treated identically. To measure
the efficiency of the enzymes in solid phase
chemistry, the reactive coatings were chal-
lenged with substrate under dry conditions
using coated aluminum panels. All assays
were performed in triplicate.
References
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