Determination of Hydrogen Peroxide by Fluorescein Chemiluminescence Catalyzed with Horseradish Peroxidase Encapsulated in Liposome

Abstract

Fluorescein (FL) and H2O2 rapidly permeated into the inner phase of liposome which trapped horseradish peroxidase (HRP), to initiate HRP-catalyzed FL chemiluminescence (CL) with H2O2. The CL intensity was dependent on the concentration of H2O2. The optimum conditions of charge-type, composition and diameter of liposome in the assay of H2O2 were determined by measuring the CL intensity, to be maximal under optimum conditions. Anionic liposome containing phosphatidylcholine, dimyristoyl-glycero-phosphocholine and cholesterol (Chol) was effective for enhancing the CL intensity and stability of liposome. The CL intensity decreased with an increase in the content of Chol in liposome. The optimal content of Chol was thus determined to be 10 mol%. The effect of the liposome size on the CL intensity was examined by preparing liposomes with a different diameter. The CL intensity increased with an increase in the diameter of liposome. The optimal diameter of liposome was thus determined to be 1000 nm. The logarithmic calibration curve of H2O2 was linear over the range from the detection limit of 4.0 × 10−8 M up to 1.0 × 10−5 M. When HRP trapped in liposome was used as a catalyst, the CL intensity was greater than that observed by using HRP dissolved in the bulk solution in the range of 4.0 × 10−7 M up to 1.0 × 10−5 M of H2O2.