A biotinylated undecylthiophene copolymer bioconjugate for surface immobilization: creating an alkaline phosphatase chemiluminescence-based biosensor.
Methodology is described for the creation of a molecular assembly consisting of the enzyme alkaline phosphatase immobilized onto a glass surface using a biotinylated conjugated copolymer, poly(3-undecylthiophene-co-3-thiophenecarboxaldehyde) 6-biotinamidohexanohydrazone. The biotinylated polymer is attached to the inside walls of a silanized glass capillary via hydrophobic interactions, and a streptavidin-conjugated alkaline 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 alkaline phosphatase with this molecular assembly. The assembly is mechanically robust and retains the majority of bound enzyme activity for up to 30 days. We have utilized this molecular assembly for the detection of organophosphorus-based pesticides. Both paraoxon and methyl parathion inhibit the enzyme-mediated generation of chemiluminescence signal. We are able to detect paraoxon and methyl parathion concentrations down to 500-700 ppb.
Pande
R
R
Department of Chemistry, University of Massachusetts, Lowell 01854, USA.
Kamtekar
S
S
Ayyagari
M S
MS
Kamath
M
M
Marx
K A
KA
Kumar
J
J
Tripathy
S K
SK
Kaplan
D L
DL
D000469:Alkaline Phosphatase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D001426:Bacterial Proteins; D015374:Biosensing Techniques; D001710:Biotin / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D004800:Enzymes, Immobilized / Q000032:analysis* / Q000378:metabolism; D007306:Insecticides / Q000032:analysis; D007700:Kinetics; D008163:Luminescent Measurements; D008743:Methyl Parathion / Q000032:analysis*; D010261:Paraoxon / Q000032:analysis*; D011485:Protein Binding; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods; D019809:Streptavidin; D013876:Thiophenes / Q000138:chemical synthesis
D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D007306:Insecticides; D013876:Thiophenes; C099579:poly(3-undecylthiophene-co-3-thiophenecarboxaldehyde) 6-biotinamidohexanohydrazone; D008743:Methyl Parathion; D001710:Biotin; D019809:Streptavidin; D000469:Alkaline Phosphatase; D010261:Paraoxon