Analytical Sciences

Direct Determination of Horseradish Peroxidase Encapsulated in Liposomes by Using Luminol Chemiluminescence

Tamio KAMIDATE, Kanako KOMATSU, Hirofumi TANI, and Akihiko ISHIDA

Division of Biotechnology and Molecular Chemistry, Graduate School of Engineering, Hokkaido University, Sapporo 060-8628, Japan

Horseradish peroxidase (HRP) encapsulated in liposomes was directly detected by using luminol chemiluminescence (CL) with H₂O₂ without lysis of liposomes. At a low concentration of H₂O₂, the initial rate of HRP-catalyzed luminol CL in liposomes was slower than that of HRP-catalyzed luminol CL in a lipid-free bulk solution. The decrease in the initial rate of the CL reaction in liposomes was due to the membrane permeation of luminol and H₂O₂. At a high concentration of H₂O₂, the initial rate of the CL reaction in liposomes was the same as that in a lipid-free bulk solution. The CL measurement conditions in both a lipid-free bulk solution and in liposomes were optimized in the concentrations of luminol and H₂O₂ by measuring the CL response curves, in which only one peak appeared and the CL intensity was maximal. The CL intensity observed in HRP-catalyzed luminol CL in liposomes was a factor of seven greater than that observed in a lipid-free bulk solution. The CL intensity was dependent on the amount of HRP-encapsulated liposomes used. The detection limit in the direct detection of HRP encapsulated in liposomes was sensitive by a factor of 3 compared with that in HRP-catalyzed luminol CL in a lipid-free bulk solution.

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