R. Pande et al., A BIOTINYLATED UNDECYLTHIOPHENE COPOLYMER BIOCONJUGATE FOR SURFACE IMMOBILIZATION - CREATING AN ALKALINE-PHOSPHATASE CHEMILUMINESCENCE-BASED BIOSENSOR, Bioconjugate chemistry, 7(1), 1996, pp. 159-164
Methodology is described for the creation of a molecular assembly cons
isting of the enzyme alkaline phosphatase immobilized onto a glass sur
face using a biotinylated conjugated copolymer, y(3-undecylthiophene-c
o-3-thiophenecarboxaldehyde) 6-biotinamidohexanohydrazone. The biotiny
lated polymer is attached to the inside walls of a silanized glass cap
illary via hydrophobic interactions, and a streptavidin-conjugated alk
aline phosphatase is interfaced with the polymer through the classical
biotin-streptavidin interaction. Utilizing a simple optical setup, we
can detect the activity of as little as approximately 0.1 fmol of alk
aline phosphatase with this molecular assembly. The assembly is mechan
ically robust and retains the majority of bound enzyme activity for up
to 30 days. We have utilized this molecular assembly for the detectio
n of organophosphorus-based pesticides. Both paraoxon and methyl parat
hion inhibit the enzyme-mediated generation of chemiluminescence signa
l. We are able to detect paraoxon and methyl parathion concentrations
down to 500-700 ppb.