Scheme 1. All-in-one immunoassay with chemiluminescence detection operated based on the reaction of 3-(2′-spiroadamantyl)-4-methoxy-4-(3″-phosphoryloxy)-phenyl-1,2-dioxetane (Adamantyl 1,2-dioxetane aryl phosphate), which is one of adamantyl 1,2-dioxetane phosphate derivatives, and ALP.
An adamantly 1,2-dioxetane phosphate derivative (AMPPD), such as adamantyl 1,2-dioxetane aryl phosphate and adamantyl 1,2-dioxetane chlorophenyl phosphate (CSPD), reacts with alkaline phosphatase (ALP) under basic condition (pH 9 – 11) to emit glow chemiluminescence. As shown in Scheme 1, the intermediate, 2, is formed when the AMPPD derivative, 1, reacts with an ALP-conjugated antigen-binding antibody formed in an all-in-one immunoassay. Then, the intermediate, 2, is rapidly separated into a high-energy intermediate, 3, in the excited state and a stable product, 4. Finally, the high-energy intermediate, 3, emits blue chemiluminescence at 477 nm. For example, trace levels of D-dimer in human serum were quantified with the all-in-one immunoassay with chemiluminescence detection operated based on the reaction mechanism shown in Scheme 1. The brightness of chemiluminescence emitted through the all-in-one immunoassay is enhanced with the increase of D-dimer in human serum.