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

• US 5,004,565 A
• Filed: 07/27/1988
• Issued: 04/02/1991
• Est. Priority Date: 07/17/1986
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Wayne State University
• Inventors: Schaap, Arthur P.
• Primary Examiner(s): Lee, Mary C.
• Assistant Examiner(s): SCALZO, CATHERINE KILBY
• Application Number:
  US07/224,681
• Time in Patent Office: 979 Days
• Field of Search: 549/510, 549/511, 549/332, 252/700, 252/301.16, 252/301.34, 252/301.35, 252/311, 435/147, 435/148, 436/829, 436/905, 424/450, 428/402
• US Class Current: 252/7
• CPC Class Codes: C07C 2603/18 Fluorenes; Hydrogenated fluorenes
• CPC Class Codes: C07C 2603/74 Adamantanes
• CPC Class Codes: C07C 51/15 by reaction of organic compounds with carbon dioxide, e.g. Kolbe-Schmitt synthesis
• CPC Class Codes: C07C 65/11 with carboxyl groups on a condensed ring system containing two rings
• CPC Class Codes: C07D 207/46 with hetero atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07D 321/00 Heterocyclic compounds containing rings having two oxygen atoms as the only ring hetero atoms, not provided for by groups C07D317/00 - C07D319/00
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 407/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 417/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 493/10 Spiro-condensed systems
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C07F 7/1804 Compounds having Si-O-C linkages Si-O-acyl linkages C07F7/1896
• CPC Class Codes: C07F 9/12 with hydroxyaryl compounds
• CPC Class Codes: C07F 9/65512 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/65522 condensed with carbocyclic rings or carbocyclic ring systems
• CPC Class Codes: C07F 9/6561 containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
• CPC Class Codes: C07H 15/203 Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 2334/00 O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
• CPC Class Codes: G01N 33/533 : with fluorescent label
• CPC Class Codes: G01N 33/582 : with fluorescent label
• CPC Class Codes: Y10S 436/829 : Liposomes, e.g. encapsulation
• CPC Class Codes: Y10S 436/905 : Photochemical activation of reactions
• CPC Class Codes: Y10T 428/2982 : Particulate matter [e.g., sphere, flake, etc.]

Abstract

A method and compositions including a 1,2-dioxetane and a fluorescent compound is described. In particular, enzymatic triggering of a triggerable 1,2-dioxetane admixed with a surfactant and the fluorescent compound attached to a hydrocarbon to provide a co-surfactant in a micelle or other structure providing close association of these molecules is described. The method and compositions are useful in immunoassays and in DNA probes used for various purposes.

Claims

1. A composition which generates light upon triggering which comprises in admixture:

(a) a fluorescent compound; and

(b) a stable 1,2-dioxetane of the formula;

##STR32## wherein ArOX represents an aryl group substituted with an X-oxy group which forms an unstable oxide intermediate 1,2-dioxetane compound when triggered to remove X by an activating agent so that the unstable 1,2-dioxetane compound decomposes and releases electronic energy to form light and two carbonyl containing compounds of the formula;

##STR33## wherein X is a chemically labile group which is removed by the activating agent to form the unstable oxide intermediate 1,2-dioxetane and wherein A are passive organic groups which allow the light to be produced wherein the stable 1,2-dioxetane is decomposed with the activating agent wherein the dioxetane and the fluorescent compound are provided in a closely spaced relationship in the presence of a surfactant, micelle, liposome, reversed micelle, microemulsion, film, including a monolayer, or polymer and wherein the fluorescent compound accepts the electronic energy generated upon decomposition of the unstable oxide intermediate and produces a more intense light than is produced by the triggering of the dioxetane alone.

4. The composition of claim 1 wherein the mole ratio of dioxetane to fluorescent compound is between about 1,000 to 1 and 1 to 1.

5. The composition of claim 1 wherein the fluorescent compound is a fluorescein compound.

6. The composition of claim 1 wherein in addition a surfactant is provided in the composition.

10. The composition of claim 1 wherein a cationic surfactant is provided in the composition, wherein the fluorescent compound is chemically linked with a hydrocarbon chain to provide a fluorescent hydrocarbon which acts as a co-surfactant with a cationic surfactant to increase the light from the dioxetane and fluorescent compound and wherein the dioxetane is present in an amount between about 10^-2 to 10^-6 M, the surfactant is present in an amount greater than 10^-4 M and the co-surfactant is present in an amount between 10^-3 to 10^-6 M.

11. The composition of claim 10 wherein the cationic surfactant is a cetyltrimethylammonium salt.

12. The composition of claim 1 wherein the dioxetane and fluorescent compound are in a closely spaced relationship in a micelle, liposome, reversed micelle, microemulsion, film, monolayer or polymer.

13. The composition of claim 1 wherein the 1,2-dioxetane has the formula:

##STR38## ps wherein X is a group reactive with the activating agent to produce the two carbonyl containing compounds ##STR39## and light.

14. The composition of claim 13 wherein OX is a phosphate group and the activating agent is alkaline phosphatase.

15. The composition of claim 13 wherein in addition a surfactant is admixed in the composition.

16. The composition of claim 15 wherein the surfactant is a cetyltrimethylammonium salt.

17. The composition of claim 13 wherein the fluorescent compound is a fluorescein compound.

18. The composition of claim 17 wherein in addition a surfactant is admixed in the composition.

19. The composition of claim 18 wherein the surfactant is a cetyltrimethylammonium salt.

20. The composition of claim 17 wherein the fluorescein compound is fluorescein chemically linked to a hydrocarbon to provide a fluorescein surfactant.

21. The composition of claim 13 wherein the fluorescent compound is selected from the group consisting of 5-N-tetradecanoylaminofluorescein and 1-hexadecyl-6-hydroxy-2-benzothiazamide.

22. The composition of claim 21 wherein in addition a non-fluorescent surfactant is admixed in the composition.

23. The composition of claim 22 wherein the surfactant is a cetyltrimethylammonium salt.

24. The composition of claim 1 wherein the fluorescent compound is fluorescein chemically linked to a hydrocarbon to provide a fluorescein surfactant which increases the light from the fluorescein compound and the dioxetane.

25. The composition of claim 1 wherein the fluorescent compound is selected from the group consisting of 5-N-tetradecanoylaminofluorescein and 1-hexadecyl-6-hydroxy-2-benzothiazamide.

26. The composition of claim 25 wherein in addition a surfactant is admixed in the composition.

27. The composition of claim 26 wherein the surfactant is a cetyltrimethylammonium salt.

32. The composition of claim 1 wherein the dioxetane and the fluorescent compound are provided in a closely spaced relationship in a solid or liquid medium.

2. A composition which generates light upon triggering which comprises in admixture:

(a) a fluorescent compound; and

(b) a stable 1,2-dioxetane compound of the formula;

##STR34## wherein one of R₁ and R₂ together and R₃ and R₄ together can be joined as spirofused alkylene which can contain hetero atoms selected from the group consisting of N, S, O and P in addition to carbon and aryl rings, wherein at least one of R₁ and R₂ or R₃ and R₄ is an aryl group substituted with an X-oxy group which forms an unstable oxide intermediate 1,2 dioxetane compound when triggered to remove X by an activating agent selected from acids, bases, salts, enzymes, inorganic and organic catalysts and electron donors so that the unstable 1,2-dioxetane compound decomposes and releases electronic energy to form light and two carbonyl containing compounds of the formula;

##STR35## wherein those of R₁, R₂, R₃ or R₄ which are unsubstituted by an X-oxy group are 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 unstable oxide intermediate wherein the stable 1,2-dioxetane is decomposed with the activating agent, wherein the dioxetane and the fluorescent compound are provided n a closely spaced relationship in the presence of a surfactant, micelle, liposome, reversed micelle, microemulsion, film, including a monolayer, or polymer and wherein the fluorescent compound accepts the electronic energy generated upon decomposition of the unstable oxide intermediate and produces a more intense light than is produced by the triggering of the dioxetane alone.

7. The composition of claim 2 wherein R₁ is a methoxy group and R₂ is a phenyl group substituted with an X-oxy group and wherein R₃ and R₄ are joined together as an adamantyl group.

8. The composition of claim 7 wherein the X-oxy group is selected from hydroxyl, trialkyl or aryl silyloxy, inorganic oxyacid salt, oxygen-pyranoside, arylcarboxyl esters and alkylcarboxyl esters and wherein X is removed by the activating agent.

9. The composition of claim 8 wherein the fluorescent compound is a fluorescein compound.

33. The composition of claim 2 wherein the dioxetane and the fluorescent compound are provided in a closely spaced relationship in a solid or liquid medium.

3. A composition which generates light upon triggering which comprises in admixture:

(a) a fluorescent compound; and

(b) a stable dioxetane compound of the formula;

##STR36## wherein R₁ is selected from alkyl, alkoxy, aryloxy dialkylamino, trialkyl or aryl silyloxy and aryl groups including spirofused aryl groups with R₂, wherein R₂ si an aryl group which can include R₁ and is substituted with an X-oxy group which forms an unstable oxide intermediate 1,2-dioxetane compound when triggered to remove X by an activating agent selected from acids, bases, salts, enzymes, inorganic and organic catalysts and electron donors so that the unstable 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 and wherein R₃ and R₄ are selected from aryl, heteroalkyl and alkyl groups which can be joined together as spirofused polycyclic alkyl and polycyclic aryl groups wherein the stable 1,2-dioxetane is decomposed with the activating agent, wherein the dioxetane and the fluorescent compound are provided in a closely spaced relationship in the presence of a surfactant, micelle, liposome, reversed micelle, microemulsion, film, including a monolayer, or polymer and wherein the fluorescent compound accepts the electronic energy generated upon decomposition of the unstable oxide intermediate and produces a more intense light than is produced by the triggering of the dioxetane alone.

28. The composition of claim 3 wherein the fluorescent compound is fluorescein chemically linked to a hydrocarbon to provide a fluorescein surfactant.

29. The composition of claim 3 wherein the fluorescent compound is selected from the group consisting of 5-N-tetradecanoylaminofluorescein and 1-hexadecyl-6-hydroxy-2-benzothiazamide.

30. The composition of claim 29 wherein in addition a surfactant is admixed in the composition.

31. The composition of claim 30 wherein the surfactant is a cetyltrimethylammonium salt.

34. The composition of claim 3 wherein the dioxetane and the fluorescent compound are provided in a closely spaced relationship in a solid or liquid medium.

35. The composition of claim 3 wherein in addition a surfactant is provided in the composition.