Chemiluminescence flow biosensor for determination of total d-amino acid in serum with immobilized reagents

A flow-injection chemiluminescence (CL) biosensor for the determination of total d-amino acid is described in this paper. d-amino acid oxidase was immobilized onto amine-modified silica gel via glutaraldehyde activation and packed in a column. The other analytical reagents, including luminol and ferricyanide, were electrostatically co-immobilized on an anion-exchange column. d-amino acid was sensed by the CL reaction between hydrogen peroxide produced from the enzyme column and luminol and ferricyanide, which were eluted from the ion-exchange column through sodium phosphate injection. The linear range of d-alanine concentration is 1.1×10−6 to 1.1×10−4 mol/l and the detection limit was 4.5×10−7 mol/l (3σ). The proposed method has been successfully applied to the determination of d-amino acid in human serum.