Application of a Liposomal Bioluminescent Label in the Development of a Flow Injection Immunoanalytical System

Abstract

A flow injection liposome immunoanalytical system was developed using biotin as the model analyte and liposomal aequorin as the label. Aequorin is a photoprotein isolated from luminescent jellyfish (notably Aequorea victoria) and other marine organisms that emits visible light in the presence of a trace of Ca2+. Because of this characteristic, the aequorin complex has been used as an intracellular Ca2+ indicator. In this study, a bioluminescent label was designed by encapsulating aequorin inside the cavity of the liposome, whose outer surface was sensitized with the analyte of interest. The analyte-tagged liposomal aequorin was employed in the development of a heterogeneous bioluminescence immunoassay for the model analyte biotin. The proposed immunoassay was based on the competition between the model biotin and aequorin-encapsulating, biotin-tagged liposomes for a limited number of anti-biotin antibody-binding sites. The anti-biotin antibodies were immobilized via protein A in a capillary immunoreactor column, and 30% MeOH was used for the regeneration of antibody-binding sites after each measurement, which allowed the immunoreactor to be used for up to 50 sequential sample injections without any loss of reactivity. The calibration curve for biotin in Tris-buffered saline solution had a linear range of 1 x 10(-11)-1 x 10(-3) M. The detection limit of the assay was 50 pg (equivalent to 200-microL injection of 1 x 10(-9) M). This study demonstrates the procedures for the encapsulation of the photoprotein aequorin into the liposome, which can be used as a sensitive label in bioluminescence immunoassays for biotin or in other applications.

Electrochemiluminescence immunosensor for α-fetoprotein using Ru(bpy)32+-encapsulated liposome as labels

“…Molecules or nanoparticles could be entrapped in the cavity or immobilized onto the surface of liposomes via covalent and non-covalent interactions, which leads to a variety of applications such as gene delivery, drug delivery, and immunoassay [1][2][3]. Since a great number of reporter molecules could be contained in the cavity of the liposomes, great signal amplification could be obtained [2,3].…”

Gold-Nanostructured Immunosensor for the Electrochemical Sensing of Biotin Based on Liposomal Competitive Assay

“…The sensitivity of the nano-Au structured SPE-type immunosensor for model biotin is more than five times that of the liposome-based FILIA-BL (bioluminescence detection) system (LOD: 50 pg), previously reported by our group. 33 This significant improvement in the LOD is assumed to be caused by the nano-structure pattern modified on the screen-printed electrode, which increases the efficiency of electron transfer. The proposed electrochemical immunosensor can detect biotin at room temperature and a single assay can be conducted in 10 min.…”

Silica-coated liposomes loaded with quantum dots as labels for multiplex fluorescent immunoassay

“…Loaded liposomes were successfully applied in different immunassays, such as the microtiter-plate sorbent assay [8][9][10][11], flow-injection analysis [12][13][14], lateral flow on-site tests [15], chemiluminescent [16] and electrochemical [17] biosensors and also microarray [18].…”