Recombinant fructosyl peptide oxidase preparation and its immobilization on polydopamine coating for colorimetric determination of HbA1c.

Recombinant fructosyl peptide oxidase (FPO) from Phaeosphaeria nodorum SN15 was functionally expressed by Escherichia coli cells and one-step purified from crude cells extract using immobilized metal affinity chromatography (IMAC) to achieve a specific activity of 26 U/mg. A ready-use colorimetric detection of HbA1c level in blood sample was developed based on FPO immobilized membrane. Facile bio-inspired polydopamine coating on the surface of a microporous membrane was employed for effective FPO immobilization. Glutaraldehyde activation of the polydopamine coating significantly enhanced FPO immobilization yield that at least 5-fold higher activity could be achieved. The stability of FPO membrane was also enhanced by glutaraldehyde activation that 85% activity could be maintained after 7 repeated uses. Highly correlated optical densities at 727 nm (OD727) against fructosylvaline (FV) in the range of 0.02 mM to 0.7 mM (R2 = 0.988) could be achieved using FPO membrane. At least 80% of the initial activity was maintained for FPO membrane stored at 4 °C for 7 days. Rather low OD727 but good correlation still could be obtained by using FPO membrane for the detection HbA1c levels (6-14%) in blood samples.