Preparation of d-amino acid oxidase-immobilized polyion complex membranes

d-Amino acid oxidase (DAAO) catalyzes the d-amino acids which size around 100 Da. Polyion complex (PIC) membrane shows the permeability changes with the solute size of 110 Da. Combining the DAAO and PIC membrane, sensitivity of the enzyme electrode that depends on the molecular mass of substrates could be adjusted; three PIC-DAAO electrodes, a glassy carbon (GC)/PIC/DAAO electrode (E1), a GC/DAAO-entrapped PIC (E2) and a GC/DAAO-entrapped PIC/PIC (E3) were prepared on the GC base electrodes. The current responses to d-amino acids were measured by the oxidation of hydrogen peroxide, which was produced through the DAAO catalysis. Relative response on E2 to E1 to d-amino acids with its molecular weight over the 110 Da (d-phenylalanine (PHE), d-methionine (MET), d-valine (VAL)) was low compared with that to small d-amino acids (d-alanine (ALA) and d-serine (SER)), because of the suppression of penetration in PIC. The effect of penetration suppression was appeared remarkably in the E3: owing to the completely coverage of PIC on enzyme layer. On the E2, the sensitivity of the five d-amino acids, d-ALA, d-SER, d-VAL, d-MET and d-PHE, were almost same (175±25 nA at 0.5 mM). The E2 would be used as a biosensor for measuring total amount of these five d-amino acids.