title: Fluorometric immunoassay based on pH-sensitive dye-encapsulating liposomes and gramicidin channels.
abstract: 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 (1.0mM) 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 x 10(-8) to 1.2 x 10(-6)g/ml, avidin ranging from 1.0 x 10(-8) to 1.0 x 10(-6)g/ml, substance P ranging from 1.0 x 10(-8) to 1.0 x 10(-6)g/ml, and neurokinin A ranging from 1.0 x 10(-8) to 1.0 x 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.
authors: Department of Chemistry, College of Humanities and Sciences, Nihon University, Sakurajosui, Setagaya-ku, Tokyo 156-8550, Japan.
authors: Mieko
authors: M
authors: Horie
authors: Hiroyuki
authors: H
authors: Yanagisawa
authors: Masao
authors: M
authors: Sugawara
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D001360:Avidin / Q000378:metabolism; D019921:Biotinylation; D002784:Cholesterol / Q000737:chemistry; D004140:Dinitrophenols / Q000378:metabolism; D005452:Fluoresceins; D005470:Fluorometry / Q000379:methods*; D006096:Gramicidin / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007118:Immunoassay / Q000379:methods; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000276:immunology; D015640:Ion Channel Gating / Q000502:physiology*; D008081:Liposomes / Q000737:chemistry*; D015288:Neurokinin A / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013373:Substance P / Q000378:metabolism; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D004140:Dinitrophenols; D005452:Fluoresceins; D007140:Immunoglobulin Fab Fragments; D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D001360:Avidin; D006096:Gramicidin; D013373:Substance P; C483858:phosphatidylethanolamine; D015288:Neurokinin A; D002784:Cholesterol