Establish Of Electrochemiluminescence Amplification System And Its Immunoassay With High Sensitivity

This paper has studied the electrochemiluminescence(ECL) of Ru(bpy)32+enhancedby cyclodextrin as well as ECL enhancement of SiO2nanoparticals absorbed withRu(bpy)32+by electrostatic interaction and microporous adsorption, the preparation ofRu(bpy)32+-doped SiO2nanoparticals, the synthesis and functionalization ofRu(bpy)32+-doped SiO2nanoparticals encapsulated by Nafion film mixed with anionicpolyelectrolyte and their applications in the detection of hepatitis B surfaceantigen(HBsAg). Meanwhile, a new detection device has been designed and madeaccording to detection demand, and its detection performance also has been studied.Firstly, new detection device I and II were designed and made according to detectiondemand of electrochemiluminescence immunoassay(ECLIA) based on magnetic separation,and they can be well used in ECL detecting instrument in laboratory. Through comparingtheir detection performance, it was found that detection device II was more suitable forECLIA based on magnetic separation in this paper. As the detection device II had goodelectrochemistry and ECL detection performance proved by the detection of Ru(bpy)32+and commercial reagents, it was chosen as detection device in subsequent experiments ofthis paper.The ECL of Ru(bpy)32+was enhanced when detecting with β-cyclodextrin(β-CD) orcarboxymethyl β-cyclodextrin(CM-β-CD) and the enhancement effect of CM-β-CD wasbetter. When β-CD or CM-β-CD was mixed with Ru(bpy)32+to formed supramolecularinclusion and then the supramolecular inclusion was absorbed on SiO2nanoparticals toformed nanocomposites, their ECL were enhanced and the ECL of nanocompositesformed by β-CD was better. The morphology and quality of SiO2nanoparticals werecharacterized by transmission electron microscopy (TEM) and Zeta potential analyzer. Thequality of nanocomposites was characterized by ECL detection instrument, electrochemicaldetector and Zeta potential analyzer.The Ru(bpy)32+-doped SiO2nanoparticals were synthesized by Stober method, whose ECL was good. As Nafion had good film-forming property, it was mixed with anionicpolyelectrolyte sodium polyacrylate(PAA-Na) to form film and this mixed film was used toencapsulate Ru(bpy)32+-doped SiO2nanoparticals to form new ECL nanocomposites.Electrochemical detection, ECL detection and Zeta potential detection were used tocharacterize the new ECL nanocomposites. It showed that the ECL of nanocompositesformed by Nafion film mixed with PAA-Na was better than those formed by pure Nafionfilm. On this basis, second antibody was connected with the new ECL nanocompositesformed by mixed film through hydrophobic structure of Nafion, and then, Ru(bpy)32+wasfixed on the surface of them through ion exchange and electrostatic adsorption to furtherenhance the ECL. Therefore, the nanocomposites with strong ECL could be used asdetection probe in immunoassay.The anti-HBsAg antibody labelled by above mentioned new ECL nanocompositesthrough the connection with second antibody was used as detection probe. Anotheranti-HBsAg antibody labelled by commercial immunomagnetic beads was used as captureprobe. Based on the dual-antibody sandwich-type immunoassay, the ECL detection methodfor HBsAg with high sensitivity was established. This method had a wide linear range ofHBsAg, which was0.005-1ng/mL (R2=0.995), and its detection limit was1.71pg/mL(S/N=3). This method was simple, fast and sensitive, which could be used as a newhighly sensitive detection method for HBsAg.