Amperometric microcells for alkaline phosphatase assay

Abstract

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.

A novel system combining biocatalytic dephosphorylation of l-ascorbic acid 2-phosphate and electrochemical oxidation of resulting ascorbic acid

“…It has been reported that AA2P can be dephosphorylated to AA with alkali phosphatase (Kokado et al, 2000;Gyurcsányi et al, 2002;Kazakevičienė et al, 2008). Indeed, electrochemical measurement of its activity has been investigated in weak alkali solution on the basis of this dephosphorylation reaction.…”

Self‐Assembled Redox System for Bioelectrocatalytic Assay of L‐Ascorbylphosphate and Alkaline Phosphatase Activity

“…This result, most likely, indicates that hydrolysis of p-NPP and AAP by ALP at the molecular level occurs through the same ratelimiting step. Here, it is also worth noting that the single K M value for AAP reported in work [18] does not adequately reflect mechanism of ALP enzymatic action, which as it is seen from data presented in this work, occurs through binding of AAP to two catalytic centers.…”

Towards Protein Assays on Paper Platforms with Potentiometric Detection

“…The detection limit for solution-based calibrations was found to approach 10 À8 M while for paper-based calibration was somewhat better than 10 À5 M. The exact reason for this behavior is presently not clear and will form the subject of an independent work, but sluggish mass transport [34][35][36] and the contingent presence of Ag + complexing sites within the nitrocellulose paper are likely to affect paper-based calibrations.…”