Assays utilizing sensitizer-induced production of detectable signals

• US 5,516,636 A
• Filed: 12/01/1992
• Issued: 05/14/1996
• Est. Priority Date: 06/08/1988
• Status: Expired due to Term

• Current Assignee: Nichols Institute Diagnostics (Quest Diagnostics Incorporated)
• Original Assignee: Diagnostic Group LLC (Finn Reinholdts ApS)
• Inventors: McCapra, Frank
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Sisson, Bradley L.
• Application Number:
  US07/984,296
• Time in Patent Office: 1,260 Days
• Field of Search: 435/6, 435/164, 435/171, 435/805, 435/904, 435/91.1, 935/76-78, 436/94, 436/537, 436/546, 436/905, 536/23.1, 536/24.3, 536/25.32, 536/26.6
• US Class Current: 435/6.12
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/583 with non-fluorescent dye label
• CPC Class Codes: Y10S 436/905 Photochemical activation of reactions
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Specific binding assays are disclosed which utilize a sensitizer as a label. Such sensitizers include any moiety which, when stimulated by "excitation" with radiation of one or more wavelengths or other chemical or physical stimulus (e.g., electron transfer, electrolysis, electroluminescence or energy transfer), will achieve an excited state which (a) upon interaction with molecular oxygen will produce singlet molecular oxygen, or (b) upon interaction with a leucodye will assume a reduced form which can then be returned to its original unexcited state by interaction with molecular oxygen resulting in the production of hydrogen peroxide. Either interaction with the excited sensitizer will, with the addition of other reagents, produce a detectable signal.

Claims

1. A specific binding assay comprising a photosensitizer as a label to a specific binding material that is employed in a specific binding reaction for the presence of an analyte in a sample undergoing the assay, binding the labeled specific binding material and the analyte in the sample, exposing the sample to an energy source to bring the photosensitizer to an excited donor state where it will donate its excess energy, provide another molecule in the assay as an acceptor for the energy transmitted by the donor, transmitting the energy to the other molecule to effect a reaction therewith that results in the formation of a detectable product, the signal from which is correlated to the presence and/or amount of analyte in the sample.

2. The specific binding assay of claim 1 wherein the energy source is light of a wave-length sufficient to bring the photosensitizer to the excited triplet donor state.

3. The specific binding assay of claim 2 wherein the light is filtered light.

4. The specific binding assay of claim 1 wherein the acceptor is molecular oxygen in the ground state.

5. The specific binding assay of claim 4 wherein the energy transmitted converts the molecular oxygen in the ground state to singlet oxygen.

6. The specific binding assay of claim 5 wherein the singlet oxygen creates the signal.

7. The specific binding assay of claim 6 wherein the signal created is the oxidation of a dye.

8. The specific binding assay of claim 6 wherein there is an olefin present and singlet oxygen converts the olefin to a dioxetan.

9. The specific binding assay of claim 8 wherein the dioxetan is reacted to effect a chemiluminescent reaction.

10. The specific binding assay of claim 9 wherein the olefin is a substituted acridan.

11. The specific binding assay of claim 1 wherein the specific binding material is capable of specifically binding with the analyte and the specific binding reaction product is an analyte-photosensitizer conjugate complex.

12. The specific binding assay of claim 1 wherein the assay further utilizes a reactant that is capable of specifically binding (i) with the analyte to form an analyte-reactant complex and (ii) with the specific binding material to form a photosensitizer conjugate-reactant complex, and wherein the one or more specific binding reaction products is the photosensitizer conjugate-reactant complex.

13. The specific binding assay of claim 1 wherein the specific binding material is capable of specifically binding with the analyte and the assay further uses a reactant capable of specifically binding with the analyte to form a reactant-analyte-photosensitizer conjugate complex, and wherein a specific binding reaction product is the reactant-analyte-photosensitizer conjugate complex.

14. The specific binding assay of claim 1 wherein the photosensitizer is a dye.

15. The specific binding assay of claim 14 wherein the dye is a porphyrin, a metalloporphyrin, an aromatic hydrocarbon, a heterocyclic compound or a flavin derivative.

16. The specific binding assay of claim 15 wherein the porphyrin is protoporphyrin dimethyl ester, protoporphyrin disodium salt, methyl pyrroporphine, ethyl ester, methyl pyrroporphine, tetraphenylporphine, coporphyrin, hematoporphyrin, N-hydroxysuccinimide substituted porphyrin or sulfonyl chloride substituted porphyrin.

17. The specific binding assay of claim 14 wherein the dye is a pyrene or methylene blue.

18. The specific binding assay of claim 14 wherein the dye is phthalocyanine, hemin or rhodamine.

19. The specific binding assay of claim 1 wherein the photosensitizer conjugate comprises a photosensitizer attached to a first single-stranded polynucleotide segment.

20. A specific binding assay for an analyte comprising a sample containing the analyte, a specific binding material conjugated with a label containing a photosensitizer that, when excited to the triplet state, reacts the photosensitizer with one or more of

(a) molecular oxygen to produce singlet molecular oxygen,

(b) a leucodye to evoke a color change, or

(c) a leucodye followed by reaction of the reduced photosensitizer of the reaction with the leucodye with molecular oxygen to return the photosensitizer to its original state and to produce hydrogen peroxide, and invoking a signal that measures the presence of the analyte.

21. The specific binding assay of claim 20 wherein the singlet molecular oxygen reacts

(i) with olefin to form a dioxetan which decays upon heating to emit a detectable photon, or

(ii) with an olefin to form a peroxide which can either

(1) decay upon heating to emit a detectable photon or

(2) oxidize a chromogen to produce a detectable color change or fluorescence or

(iii) with a leucodye to induce a color change.

22. The specific binding assay of claim 20 wherein the leucodye is oxidized and produces a detectable color change or fluoresces.

23. The specific binding assay of claim 21 wherein the olefin is a substituted olefin.

24. The specific binding assay of claim 23 wherein the substituted olefin has at least one substitution which is an electron donating group.

25. The specific binding assay of claim 23 wherein at least two of the substitutions of the substituted olefin are joined to form a ringed moiety which is fluorescent.

26. The specific binding assay of claim 20 wherein hydrogen peroxide produced by recycling the reduced photosensitizer to the presence of molecular oxygen, provides the signal by its oxidation of a chromogen resulting in a detectable color change or fluorescence or the oxidation of a chemiluminescent compound producing a detectable photon.

27. The specific binding assay of claim 20 wherein the singlet molecular oxygen reacts with an olefin to produce a dioxetan or a peroxide.

28. The specific binding assay of claim 27 wherein the dioxetan decays upon heating to produce the detectable signal as a photon.

29. The specific binding assay of claim 20 wherein the peroxide oxidizes a chromogen to produce a detectable signal as a color change or fluorescence by the chromogen.

30. A specific binding assay for the presence of an analyte in a sample which comprises providing with the sample a photosensitizer conjugated with a specific binding material which photosensitizer contains a moiety that is induced to the triplet excited state by exposure to light such that it is reactable with molecular oxygen to produce singlet molecular oxygen, the presence of analyte in the sample is proportional to the formation of one or more specific binding reaction products containing the photosensitizer conjugate, exciting the photosensitizer in the presence of oxygen in the triplet state to form oxygen in the singlet, causing the singlet oxygen to react with a leucodye to produce color or fluorescence, and measuring to determine the presence of the analyte in the sample based on the reaction of the singlet oxygen and the leucodye.

31. The specific binding assay of claim 30 wherein the specific binding material is capable of specifically binding with the analyte and the specific binding reaction product is an analyte-photosensitizer conjugate complex.

32. The specific binding assay of claim 30 wherein the assay further utilizes a reactant which is capable of specifically binding (i) with the analyte to form an analyte-reactant complex and (ii) with the specific binding material to form a photosensitizer conjugate-reactant complex, and wherein the specific binding reaction product is the photosensitizer conjugate-reactant complex.

33. The specific binding assay of claim 30 wherein the specific binding material is capable of specifically binding with the analyte and the assay further utilizes a reactant capable of specifically binding with the analyte to form a reactant-analyte-photosensitizer conjugate complex, and wherein the specific binding reaction producte is the reactant-analyte-photosensitizer conjugate complex.

34. The specific binding assay of claim 30 wherein the photosensitizer is a dye.

35. The specific binding assay of claim 34 wherein the dye is a porphyrin, a metalloporphyrin, an aromatic hydrocarbon, a pyrene, phthalocyanine, hemin, rhodamine heterocyclic compound, methylene blue or a flavin derivative.

36. The specific binding assay of claim 35 wherein the porphyrin is protoporphyrin dimethyl ester, protoporphyrin disodium salt, methyl pyrroporphine ethyl ester, methyl pyrroporphine, tetraphenylporphine, coporphyrin, hematoporphyrin, NHS substituted porphyrin or sulfonyl chloride substituted porphyrin.

37. The specific binding assay of claim 30 wherein a leucodye is oxidized to produce the detectable signal as a color change or fluorescence by the leucodye.

38. The specific binding assay of claim 30 wherein the excited photosensitizer is reduced by the leucodye and is sub-sequently oxidized by reaction with molecular oxygen to produce hydrogen peroxide.

39. The specific binding assay of claim 38 wherein the hydrogen peroxide oxidizes a chromogen to produce the detectable signal as a color change or fluorescence by the chromogen.

40. The specific binding assay of claims 30 wherein hydrogen peroxide is formed and oxidizes a chemiluminescent moiety to produce the detectable signal as a photon.