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.
Keywords: Colorimetric assay; FPO (fructosyl peptide oxidase); Fructosyl valine (FV); HbA1c; Polydopamine.
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