We have synthesized and studied t w o 1.2-dioxetane-based chemiluminescent enzyme substrates:
3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD), and, 3-(2'-spiroadamantane)-4-methoxy-4-(3-~-~'-galactopyrano-yloxy)phenyl-1,2-dioxetane (AMPGD), which can be activated to chemiluminescence at 470 nm by alkaline phosphatase and B-D-galactosidase, respectively. In addition, we observed that certain macromolecules enhance the luminescence of AMPPD. For example, the addition of 0.1% bovine serum albumin amplifies the luminescent signal and improves the detection limit for alkaline phosphatase by approximately one order of magnitude under certain conditions. This effect is due t o the presence of a hydrophobic microenvironment provided by the enhancer which 'stabilizes' the dephosphorylated AMPPD emitter.
Alkaline phosphatase catalysed chemiluminescence from AMPPD is constant for a prolonged period of time. Using AMPPD w e were able t o detect 0.01 attomole quantities of alkaline phosphatase immobilized on membrane supports and imaged on photographic film and, in solution, measured in a luminometer.
AMPPD and AMPGD offer alternatives t o colorimetric and fluorescent subsrates for alkaline phosphatase and 6-D-galactosidase labels used in enzyme immunoassays. The simplicity and sensitivity of this chemiluminescent readout allowed the development of rapid clinical assays (e.g. 6-hCG, LH, TSH and others).