Method and compositions providing enhanced chemiluminescence from 1,2-dioxetanes

A method for generating light which comprises:

• (a) providing in a setting where the light is to be produced a fluorescent compound dispersed in or on one of a surfactant, a micelle, liposome, reversed micelle, microemulsion, film, including a monolayer or a polymer which brings the fluorescent compound into a closely spaced relationship with and in admixture with a 1,2-dioxetane compound which is stable in the setting where the light is to be produced of the formula ##STR34## wherein R.sub.1 and R.sub.2 together and R.sub.3 and R.sub.4 together can be joined as spirofused cycloalkylene which can obtain hereto atoms selected from the group consisting of N, S, O and P in addition to carbon and aryl rings, wherein at least one of R.sub.1 and R.sub.2 or R.sub.3 and R.sub.4 is an aryl group substituted with an X-oxy group which forms an oxide intermediate 1,2dioxetane compound when triggered by removing X with an activating agent selected from acids, bases, salts, enzymes, inorganic and organic catalysts and other electron donors so that the oxide intermediate 1,2-dioxetane compound decomposes and releases electronic energy to form light and two carbonyl containing compounds of the formula:
• ##STR35## wherein those of R.sub.1, R.sub.2, R.sub.3 or R.sub.4 which are unsubstituted by an X-oxy group are carbon and hereto atom containing organic groups which provide stability for the stable 1,2-dioxetane compound and wherein X is a chemically labile group which is removed by the activating agent to form the oxide intermediate 1,2-dioxetane;
• and
• (b) triggering the stable 1,2-dioxetane with the activating agent to remove X wherein the fluorescent compound accepts the electronic energy generated upon decomposition of the oxide intermediate 1,2-dioxetane compound and produces a more intense light than is produced by the triggering of the dioxetane alone.

A method for generating light which comprises:

• (a) providing in a setting where the light is to be produced a fluorescent compound dispersed in or on one of a surfactant, a micelle, liposome, reversed micelle, microemulsion, film, including a monolayer or a polymer which brings the fluorescent compound and the dioxetane into a closely spaced relationship with and in admixture with a dioxetane compound which is stable in the setting where the light is to be produced of the formula:
• ##STR36## wherein R.sub.1 is selected from alkyl, alkoxy, aryloxy dialkylamino, trialkyl or aryl silyloxy and aryl groups including spirofused aryl groups joined with R.sub.2, wherein R.sub.2 is an aryl group which can include R.sub.1 and is substituted with an X-oxy group which forms an oxide intermediate 1,2-dioxetane compound when triggered by removing X with an activating agent selected from acids, bases, salts, enzymes, inorganic and organic catalysts and other electron donors so that the oxide intermediate 1,2-dioxetane compound decomposes and releases electronic energy to form light and two carbonyl containing compounds of the formula:
• ##STR37## wherein X is a chemically labile group which is removed by the activating agent to form the unstable oxide intermediate 1,2-dioxetane compound and wherein R.sub.3 and R.sub.4 are selected from aryl, heteroalkyl and alkyl groups which can be joined together as spirofused polycyclic alkyl and polycyclic aryl groups;
• and
• (b) triggering the stable 1,2-dioxetane with the activating agent to remove X wherein the fluorescent compound accepts the electronic energy generated upon decomposition of the oxide intermediate 1,2-dioxetane compound and produces a more intense light than is produced by the triggering of the dioxetane alone.

In a method for detecting DNA by hybridization of the DNA with a DNA probe having an enzyme label and then generating a detectable signal by means of the enzyme label, the improvement which comprises:

• (a) providing a 1,2-dioxetane of the formula:
• ##STR38## wherein X is a group reactive with the enzyme label to produce light;
• (b) providing a fluorescent compound which enhances the light from the 1,2-dioxetane in admixture with the hybrid of the probe and the DNA;
• and
• (c) detecting the light from the hybrid produced by the reaction of the enzyme label with the dioxetane.

In a method for generating light by reacting a 1,2-dioxetane with an activating agent, the improvement which comprises:

• (a) providing in admixture in a setting where light is to be produced a 1,2-dioxetane of the formula:
• ##STR39## wherein OX is a phosphate group reactive with alkaline phosphatase to produce light and a fluorescent compound which enhances the light from the 1,2-dioxetane in admixture with the dioxetane;
• and
• (b) triggering the 1,2-dioxetane with alkaline phosphatase to generate the light.