Homogeneous Electrogenerated Chemiluminescence Immunoassay

This thesis includes a review and a research section. The review includes the history, principle and characteristics of electrogenerated chemiluminescence immunoassay (ECLIA), the immunoassay labeled technologies, and applications and developments of ECLIA. The research section contains four subunits. In the research section, we have investigated Electrogenerated chemiluminescence (ECL) system of luminol and lucigenin in neutral solution, and developed ECL determination method of procaine hydrochloride, isatin and riboflavin; we have proposed a novel multilabeled homogeneous ECLIA method. Digoxin, luminol and BSA are used as a model for this ECLIA system and digoxin and anti-digoxin antibody are determined in sub-trace level based on the change of diffusion coefficient.ECL involves the production of light at or near an electrode surface by electrochemically generating species capable of forming exited states. ECL analysis is becoming increasingly promising method for biomedical, environmental analysis owing to its simple instrumentation, high sensitivity, wide dynamic range, reproducibility, simpl city and rapidity analysis, its automation from electrochemical control and highlights in homogeneous immunoassay. The review describes the history, principle, characteristics, kinds of ECLIA and the application of ECLIA in analytical science. The focus of the review is the application of ECLIA in analytical science. The technologies of ECLIA with another analytical method such as flow injection, HPLC, capillary electrophoresis and magnetic particle is described.The research section includes four sections, the former three sections of which are based on the electron-transfer ECL between the luminescence and the analyzed medicines. That is to say, the principle of these methods is based on the repression or enhancement of luminescence of ECL caused by the reagents electrochemically generated at electrode to determine the medicines.Based on the enhancement effect on the ECL of luminol in neutral aqueous solution at a platinum electrode, a novel ECL method for the determination of procaine hydrochloride is developed. The linear range of procaine hydrochloride is from 4.0xlO~7g/mL to 6.0xlO~6g/mL with the detection limit of 2.0x10"7g/mL. The relativestandard derivation is 4.4%. This method is rapid and simple, and has been applied to the determination of procaine hydrochloride in injection solution.Three ECL peaks of lucigenin are observed at a platinum electrode in a neutral aqueous solution. The ECL peak of lucigenin at -0.65V(vs. Ag/AgCl) is greatly enhanced by isatin and riboflavin, which is believed produced from the chemiluminescence reaction between lucigenin and superoxide anion that is generated by the catalytic reaction of electrochemically reduced isatin or riboflavin with dissolved oxygen. The potential application to determine isatin and riboflavin is proposed. Under chosen condition, the linear range of isatin is 4.8><10"7g/mL ~1.9xlO~5g/rriL with the detection limit of isatin of 3.3xlO"8 g/mL, and the relative standard derivation for 1.0 x lO^g/mL isatin is 3.8%. The calibration graph of ECL intensity vs. riboflavin concentration is linear in the range from 2.1 x 10"8 g/mL to 3.0 x 10'7g/mL, and the regression equation is ^7=187+112C (10'8g/ml, r = 0.9968) with a detection limit of 8.2 x 10"9 g/mL (SW=3). Relative standard derivation is 4.0%This method has been applied to the determination of riboflavin in the pharmaceutical preparations.A novel homogeneous ECLIA is developed for the determination of small hapten. Digoxin (Dx), luminol and BSA are used as a model system for this immunoassay. Based on the change of diffusion coefficient, anti-digoxin antibody and digoxin are determined. The multilabeled conjugation of luminol to BSA-digoxin (Dx-BSA-Lu) is developed as a homogeneous ECLIA analyzer. After immunoreaction between digoxin antibody and Dx-BSA-Lu, the complex of Ab-Dx-BSA-Lu undergoes a less ECL quantity due to the change of diffusion coefficient. The linear range of antibody is...