Fluorometric immunoassay based on pH-sensitive dye-encapsulating liposomes and gramicidin channels

This article describes a new method for direct fluorometric immunoassay with a liposome array using pH-sensitive dye (BCECF [2′,7′-bis(carboxyethyl)-4 or 5-carboxyfluorescein])-encapsulating liposomes immobilized on an avidin slip and gramicidin channels. The liposomes were composed of phosphatidylcholine (PC), cholesterol (Chol), biotinylated phosphatidylethanolamine (B-cap-PE), and recognition sites (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(2,4-dinitrophenyl) [DNP-PE], Fab′ fragment of anti-substance P, and Fab′ of anti-neurokinin A). The addition of gramicidin induced release of H+ ions from the inner solution (pH 5.5) to the outer one (pH 7.8), enhancing fluorescence of BCECF (1.0 mM) encapsulated in liposome. The binding of an analyte (anti-dinitrophenyl [anti-DNP], avidin, substance P, or neurokinin A) to the membrane-bound recognition sites caused further enhancement of fluorescence of BCECF due to a local distortion of the bilayer structure that affects the channel kinetics of gramicidin. The intensity of fluorescence from the immobilized liposomes 60 min after the addition of gramicidin (10 ng/ml) increased with an increase in the concentration of anti-DNP ranging from 1.2 × 10−8 to 1.2 × 10−6 g/ml, avidin ranging from 1.0 × 10−8 to 1.0 × 10−6 g/ml, substance P ranging from 1.0 × 10−8 to 1.0 × 10−6 g/ml, and neurokinin A ranging from 1.0 × 10−8 to 1.0 × 10−6 g/ml. The direct fluorometric immunoassay with a liposome array is simple and easy to carry out. The intensity of fluorescence emitted from the immobilized liposomes is directly measured after incubation with a sample solution and a gramicidin solution in sequence without washing steps. The assay allows simultaneous quantification of multiple components without labeling of antibody or antigen with a fluorescent tag. The liposome-based assay is discussed in terms of principle, sensitivity, and selectivity.