Amperometric microcells for alkaline phosphatase assay.
To develop simple electrochemical immunoassays, a screen printed amperometric microcell with graphite working and Ag/AgCl reference electrodes was tested for the determination of alkaline phosphatase enzyme (ALP) and anti-humanIgG conjugated ALP (alpha-hIgG-ALP) activity in 5-10 microl samples. To ensure reproducible, steady state conditions, the working electrode surface was coated with mass-transport controlling hydrogel layer. The kinetic response curves of the hydrogel coated electrodes were linear. In addition, the hydrogel layer reduced the nonspecific adsorption of the alpha-hIgG-ALP conjugate on the working electrode surface. The measurements were made in the range of 2 divided by 4000 mU ml(-1) enzyme activities using ascorbic acid 2-phosphate (AAP) as the enzyme substrate. AAP is commercially available, non-toxic and has excellent stability. The sensitivity of the determinations was about 71% of the sensitivity which could be achieved using p-aminophenylphosphate (PAPP), a not easily accessible and unstable enzyme substrate. The experimentally determined kinetic parameters of the ALP enzyme catalyzed reactions were the same with the bare and hydrogel layer coated electrodes.
Gyurcsányi
Róbert E
RE
Joint Graduate Program in Biomedical Engineering, The University of Memphis, University of Tennessee Health Science Center, 38152, USA.
Bereczki
Andrea
A
Nagy
Géza
G
Neuman
Michael R
MR
Lindner
Ernö
E
D000469:Alkaline Phosphatase / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D015374:Biosensing Techniques / Q000379:methods*; D004796:Clinical Enzyme Tests / Q000379:methods*; D004563:Electrochemistry; D006801:Humans
D015415:Biomarkers; D000469:Alkaline Phosphatase