ESTABLISHMENT OF THE OPTICALLY PUMPED CHEMILUMINESCENCE TECHNIQUE FOR DIAGNOSTICS

A new chemiluminescence procedure was established that eliminates the need to use a separate oxidant such as hydrogen peroxide for chemiluminescence detection reactions. In the procedure called optically pumped chemiluminescence (OPC), red light energy is used by a photosensitive dye to oxidize luminol and generate blue light. By pulsing the excitation light and measuring chemiluminescence during the off time, light scattering background was eliminated and sensitivity enhanced 10-fold. The OPC reaction mechanism was determined to be type I (direct reaction of excited dye with substrate), and the dye catalyst is recycled in the reaction. Of the popular photosensitive dyes tested, methylene blue showed the best detection limit in the OPC system. A methylene blue dye derivative was synthesized and covalently attached to protein. Antibody proteins were labeled with up to 16 dyes per antibody. An alpha-fetoprotein immunoassay was developed having a detection limit of 17 pg. This assay had sensitivity of 1.5 ng of alphafetoprotein/mL of plasma and showed good correlation (R = 0.98). The OPC catalysts are more stable and less temperature sensitive than enzyme catalyst, and the substrate solution needed is very stable. Thus, OPC may be valuable in applications where convenience and reagent stability are important.