Masuo Aizawa

Electrochemical luminescence-based homogeneous immunoassay

Aromatic hydrocarbon such as pyrene capable of generating electrochemical luminescence was employed as a label of immunoassay. Pyrene labeled antigen generated luminescence upon electrolytic reduction, while the luminescence decreased remarkably in the presence of antibody. The labeled antigen (constant) and free antigen were competitively reacted to the constant amount of antibody. The luminescence was correlated to the antigen concentration as little as 10(-6)M antigen. The proposed method is a very unique immunochemical technique which requires no BF separation.

Solid-phase luminescent catalyst immunoassay for human serum albumin with hemin as labeling catalyst

Abstract A solid-phase luminescent catalyst immunoassay is described for the determination of human serum albumin (HSA) in solution; hemin is used as a label which catalytically amplifies the sensitivity. The method is essentially a non-radioactive and non-enzymatic sandwich immunoassay. Anti-HSA antibody is covalently bound to a transparent plate, which then undergoes the immunochemical reaction with HSA in the test solution, and with the fixed amount of hemin-labeled anti-HSA antibody. After the two-step immunoreaction, the immunochemically-adsorbed hemin-antibody conjugate is quantified by means of the luminescence produced in a solution containing luminol and hydrogen peroxide. The luminescence intensity is correlated with the amount of HSA. The limit of detection for HSA is 1 ng ml -1 .

Luminescence catalyst immunoassay of β2-microglobulin with haemin as a chemically amplifiable label

Luminescence catalyst immunoassay, a nonradioactive and nonenzymatic immunoassay, has been applied to the determination of β2-microglobulin (β2-MG). The method is characterized by high sensitivity and simple handling, since the analytical method employs a catalytically amplifiable label and solid-phase sandwich technique, respectively. In the first stage, an antibody-immobilized plate is reacted with the analyte (β2-MG). In the second stage, the bound β2-MG undergoes successive binding with haemin-labelled antibody. In the last stage, the plate is placed in the luminol-H2O2 system to generate luminescence. A calibration curve with a β2-MG concentration at the midpoint of 100 ng ml−1 was obtained. The 10 and 90% response levels in the curve are 10 and 1000 ng ml−1.

Delayed luminescence of luminol initiated by a membrane-bound peroxidase.

The luminescense of the luminol-H2O2 system was initiated by either free or membrane-bound horseradish peroxideae (HRP). The instantaneous luminescene decayed rapidly and was followed by the delayed luminescence in the presence of excess luminol. The delayed luminescence was characterized by a chain reaction, in which luminescence intensity increased exponentially. Membrane-bound HRP demonstrated that the delayed luminescence took place even in the absence of HRP if the instantaneous luminescence was initiated by HRP. A mechanism for the nonenzymatic luminescence is proposed and discussed.

Electrochemiluminescent Sensing for the Investigation of the Binding Modes of Anticancer, Antiviral Agents

This chapter describes electrochemiluminescent sensing for the investigation of the binding modes of anti-cancer antiviral agents. A series of DNA-binding antiviral or antitumor pharmaceuticals have been currently developed, however, the detailed binding modes of these drugs are not necessarily well known. It is strongly required to clarify the binding mechanism of these agents, as the binding mechanism is very important for the drug design of more effective pharmaceuticals. The luminescence is remarkably enhanced in the presence of oxalate. It has recently been shown that one of the phenanthroline ligands intercalates between the base pair of double helical DNA. The bound Ru-complex suffers from steric hindrance of the DNA molecule, when the DNA–Ru complex is in contact with electrode. If no considerable change occurs, the drug may bind to DNA through minor groove. The details of sensing mechanism are discussed in the chapter.

Solvent effects of dimethyl sulfoxide on the chemiluminescent reaction of luminol−H2O2 system catalyzed by horseradish peroxidase

Abstract Dimethyl sulfoxide (DMSO) shows a remarkable promoting effect on the luminescent reaction of the luminol-H 2 O 2 system catalyzed by horseradish peroxidase. Two luminescent processes occur when a mixed solvent of water and DMSO is used. The total luminescence increases exponentially with increasing DMSO concentration. The initial-phase luminescence decreases when the DMSO concentration is increased, while the delayed luminescence increases. Participation of molecular oxygen and its reactive species (O 2 − , O 2 2− ) dissolved in the reaction medium was studied enzymatically. In the medium where oxygen had been removed, the rate of luminescent reaction decreased markedly. In the presence of superoxide dismutase the luminescence decreased by 15%. Addition of catalase to the medium during the course of the reaction resulted in complete disappearance of the luminescence. These facts suggest a new reaction scheme for the enzyme-induced luminescent reaction involving oxygen as well as its reactive species.