Preparation Of Tris (2,2'-bipyridyl) Ruthenium Encapsulated Liposome And Its Applications In Electrochemiluminescent Immunoassay

Recently, sensitive detection of disease-related proteins is critical to many areas such as modern biomedical research and clinical diagnosis. Immunoassay is one of the most important analytical techniques in the quantitative detection of disease markers with selectivity due to the highly specific molecular recognition between antigen and antibody. In comparison with other immunological methods such as radioimmunoassay, fluorescence immunoassay and chemiluminescence immunoassay, electrochemiluminescence immunoassay (ECLIA) possesses intrinsic advantages of low cost, high sensitivity, and good portability and has attracted considerable interest. Various ECL immunosensors have been developed for the determination of tumor markers. However, the sensitivity of the immunosensors is low, thus restricts their wide applications. At the same time, the demand for early and ultrasensitive screening of cancer biomarkers is increasing, therefore lots of signal amplification strategies arised. Among them, liposomes as the signal amplification of immunosensors aroused great interest because of its simple preparation, high specific surface area and large internal volume to contain a great number of signal molecules.In this dissertation, tris(2,2′-bipyridyl)ruthenium (Ru(bpy)32+)- encapsulated liposomes were prepared and ultrasensitive ECL immunosensors were constructed by combining the advantages of ECL technique and signal amplification function of liposomes. Four main work is involved: (1) Ru(bpy)32+-encapsulated liposomes was synthesized and the effect of ratio of ingredients, post-treatments such as ultrasound-treatment, extrusion and dialysis on the encapsulation efficiency (EE) and stability of the liposomes were investigated. Moreover, a new method based on ECL for the determination of EE of liposome was established; (2) A simple and sensitive electrochemiluminescent immunosensor was fabricated by immobilizing goat-anti-human immunoglobulin G (hIgG) antibody on a multi-walled carbon nanotubes (MWCNTs) modified glassy carbon electrode. Using antibody-tagged Ru(bpy)32+-encapsulated liposome as a second antibody, The hIgG antigen and antibody-tagged liposome can be sequentially bound on the immunosensor through antigen-antibody specific interaction, forming a sandwich-type immunocomplex. The ECL intensity of the immunosensor increased linearly with the hIgG concentration from 0.01 to 0.8 ng/mL with a detection limit of 0.004 ng/mL (S/N=3). In addition, it was successfully applied for the detection of hIgG in human serum; (3) Ru(bpy)32+-encapsulated, antibody-tagged liposomes (liposomal biolabels) was used as the label for the ECL immunosensor based on an electrodeposited gold nanoparticles (NPs) modified electrode. The use of liposomal biolabels with large internal volume to carry a great number of reporter molecules, together with the gold NPs with large surface for high antibody loading, could lead to enormous dual signal amplification, thus result in high sensitivity. With a sandwich type immunoassay format, a linear response to hIgG from 0.1 to 25 pg/mL (R=0.992) with a detection limit of 0.01 pg/mL (S/N=3) was obtained, which is five orders magnitude lower than that in the previous reports. In addition, the immunosensor showed some good performance such as high sensitivity, high selectivity, long-term stability and could be regenerated easily; (4) Preliminary studies on the the application of Ru(bpy)32+-encapsulated liposome in competitive ECL immunoassay were carried out. The assay was based on the competition between the antigen and antigen-tagged liposomes. This detection model could be used for the determination of antigen with a single antibody. The developed protocols provide new ways of designing immunosensors with high sensitivity, which may find potentially broad applications in clinical diagnosis.