1,2‐Dioxetanes: Novel chemiluminescent enzyme substrates. Applications to immunoassays

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Abstract
We have synthesized and studied two 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″‐β‐D′‐galactopyrano‐yloxy)phenyl‐1,2‐dioxetane (AMPGD), which can be activated to chemiluminescence at 470 nm by alkaline phosphatase and βD‐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 to 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 we were able to 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 to colorimetric and fluorescent subsrates for alkaline phosphatase and β‐D‐galactosidase labels used in enzyme immunoassays. The simplicity and sensitivity of this chemiluminescent readout allowed the development of rapid clinical assays (e.g. β‐hCG, LH, TSH and others).