Dendritic chemiluminescent substrates

• US 20020155523A1
• Filed: 01/08/2002
• Published: 10/24/2002
• Est. Priority Date: 01/08/2001
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Sparks, Alison L.
• Granted Patent: US 7,214,546 B2
• Time in Patent Office: Days
• US Class Current: 435/68.1
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 436/805 Optical property

Abstract

Chemiluminescent substrate delivery systems comprising a conjugate a dendrimer and at least one chemiluminescent substrate are provided. The substrate delivery systems can also include a chemiluminescence enhancer. The dendrimer/chemiluminescent substrate conjugates can be used in kits including an enzyme capable of activating the chemiluminescent substrate to produce a peroxygenated intermediate that decomposes to produce light. The dendrimer/chemiluminescent substrate conjugates can be used in assays to detect the presence of an analyte (e.g., an enzyme, an antibody, an antigen or a nucleic acid) in a sample.

Claims

1. A chemiluminescent substrate delivery system comprising:

a dendrimer; and

at least one enzymatically activated chemiluminescent substrate conjugated to the dendrimer.

2. The chemiluminescent substrate delivery system of claim 1, wherein the chemiluminescent substrate comprises a moiety selected from the group consisting of a dioxetane moiety, a luminol moiety, an isoluminol moiety, an acridinium ester moiety, an acridinium sulfonylamide moiety, a luciferin moiety and combinations thereof.

3. The chemiluminescent substrate delivery system of claim 1, wherein a plurality of enzymatically activated chemiluminescent substrates are conjugated to the dendrimer.

4. The chemiluminescent substrate delivery system of claim 3, wherein at least two different enzymatically activated chemiluminescent substrates are conjugated to the dendrimer.

5. The chemiluminescent substrate delivery system of claim 1, wherein the substrate delivery system is made by a method comprising:

covalently bonding one or more molecules comprising an enzymatically activated chemiluminescent moiety to the dendrimer;

or

covalently bonding one or more molecules comprising an enzymatically activated chemiluminescent moiety precursor to the dendrimer and thereafter converting the chemiluminescent moiety precursor to a chemiluminescent moiety.

6. The chemiluminescent substrate delivery system of claim 5, wherein the dendrimer is selected from the group consisting of polyamidoamine dendrimers with amino surface groups, polyamidoamine dendrimers with carboxylic acid surface groups, polyamidoamine dendrimers with hydroxyl surface. groups, and polypropyleneimine dendrimers with amino surface groups.

7. The chemiluminescent substrate delivery system of claim 1, wherein the chemiluminescent substrate comprises a dioxetane moiety selected from the group consisting of:

3-(2′

-spiroadamantane) 4-methoxy-4-(3″

-phosphoryloxy)phenyl-1,2-dioxetane;

disodium 3(4-methoxyspiro[1,2-dioxetane-3,2′

-(5′

-chloro) tricyclo[3.3.1.1³⁷]decan]-4-yl)phenyl phosphate dioxetane; and

disodium-2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2′

(5′

-chloro-)tricyclo-{3.3.1.1^3,7]-decan]-4yl)-phenyl phosphate.

8. The chemiluminescent substrate delivery system of claim 1, wherein the chemiluminescent substrate comprises a dioxetane moiety and wherein the substrate delivery system is represented by the formula:

9. The chemiluminescent substrate delivery system of claim 8, wherein the linker moiety is represented by the formula:

10. The chemiluminescent substrate delivery system of claim 1, wherein the chemiluminescent substrate comprises a dioxetane moiety and the delivery system is represented by the formula:

11. The chemiluminescent substrate delivery system of claim 10, wherein the linker moiety is represented by the formula:

12. The chemiluminescent substrate delivery system of claim 1, wherein the chemiluminescent substrate comprises a dioxetane moiety and the delivery system is represented by the formula:

13. The chemiluminescent substrate delivery system of claim 12, wherein the linker moiety is represented by the formula:

14. The chemiluminescent substrate delivery system of claim 1, wherein the chemiluminescent substrate comprises a dioxetane moiety and the delivery system is represented by the formula:

15. The chemiluminescent substrate delivery system of claim 14, wherein the linker moiety is represented by the formula:

16. The chemiluminescent substrate delivery system of claim 1, wherein the chemiluminescent substrate comprises an isoluminol moiety and the delivery system is represented by the formula:

17. The chemiluminescent substrate delivery system of claim 16, wherein the linker moiety is represented by the formula:

18. The chemiluminescent substrate delivery system of claim 1, wherein the chemiluminescent substrate comprises luciferin moiety and the delivery system is represented by the formula:

19. The chemiluminescent substrate delivery system of claim 18, wherein the linker moiety is represented by:

—

—

COO(CH₂)_n—

NA—

C(O)—

—

;

—

—

OO(CH₂)_n—

C(O)—

NH—

—

;

or

—

—

COO(CH₂)_n—

B—

—

;

wherein;

n is a positive integer;

A is hydrogen or an alkyl group; and

B is NA, O, S or CH₂.

20. The chemiluminescent substrate delivery system of claim 1, wherein the chemiluminescent substrate comprises an acridinium ester moiety and the delivery system is represented by the formula:

21. The chemiluminescent substrate delivery system of claim 20, wherein the linker moiety is represented by:

—

—

C(O)—

NA—

(CH₂)_n—

—

;

—

—

NH—

C(O)—

(CH₂)_n—

—

;

or

—

—

B—

(CH₂)_n—

—

;

wherein;

n is a positive integer;

A is hydrogen, an alkyl group or an aryl group; and

B is NA, O, S or CH₂.

22. The chemiluminescent substrate delivery system of claim 1, wherein the chemiluminescent substrate comprises an acridinium sulfonylamide moiety and the delivery system is represented by the formula:

23. The chemiluminescent substrate delivery system of claim 22, wherein the linker moiety is represented by:

—

—

C(O)—

NA—

(CH₂)_n—

—

;

—

—

NH—

C(O)—

(CH₂)_n—

—

;

—

—

B—

(CH₂)_n—

—

;

—

—

C(O)—

NA—

(CH₂)_n—

C(O)NH—

—

;

—

—

NH—

C(O)—

(CH₂)_n—

C(O)NH—

;

or

—

—

B—

(CH₂)_n—

C(O)NH—

;

wherein;

n is a positive integer;

A is hydrogen, an alkyl group or an aryl group; and

B is NA, O, S or CH₂.

24. The chemiluminescent substrate delivery system of claim 1, wherein the chemiluminescent substrate comprises an acridan moiety and the delivery system is represented by the formula:

25. The chemiluminescent substrate delivery system of claim 24, wherein the linker moiety is represented by:

—

—

(CH₂)_n—

NA—

C(O)—

—

;

—

—

(CH₂)_n—

C(O)—

NA—

—

;

or

—

—

(CH₂)_n—

B—

—

;

wherein;

n is a positive integer;

A is hydrogen or an alkyl group; and

B is NA, O, S or CH₂.

26. The chemiluminescent substrate delivery system of claim 1, wherein the dendrimer is covalently or ionically associated with one or more additional dendrimers.

27. The chemiluminescent substrate delivery system of claim 1, wherein the substrate delivery system further comprises a chemiluminescence enhancer moiety.

28. The chemiluminescent substrate delivery system of claim 27, wherein the enhancer moiety is formed by coupling a chemiluminescent enhancing molecule to a reactive site on the dendrimer or by chemically modifying a reactive site on the dendrimer to an enhancing moiety.

29. The chemiluminescent substrate delivery system of claim 28, wherein the enhancer moiety is formed by peralkylation of amino groups on the dendrimer or by peralkylcarbonylation of amino groupds on the dendrimer by alkylation of amide groups on the dendrimer.

30. The chemiluminescent substrate delivery system of claim 28, wherein the enhancer moiety is formed by reaction of carboxylate groups on the dendrimer with an amino linked ammonium, phosphonium or sulfonium salt.

31. The chemiluminescent substrate delivery system of claim 1, further comprising a second dendrimer, wherein the second dendrimer comprises a chemiluminescence enhancer moiety and wherein the second dendrimer is covalently or ionically associated with the dendrimer conjugated to the at least one enzymatically active chemiluminescent substrate moiety.

32. The chemiluminescent substrate delivery system of claim 1, wherein the chemiluminescent substrate and/or the dendrimer comprises one or more water solubilizing groups.

33. The chemiluminescent substrate delivery system of claim 32, wherein the one or more water solubilizing groups are selected from the group consisting of carboxylic acids, esters, alkyl-oxides, aryl-oxides, alkyl-amides, aryl-amides, aralkyl-amides, alkyl-urethanes, aryl-urethanes, alkyl-sulfonamides, aryl-sulfonamides, alkyl-sulfonic acids, aryl-sulfonic acids, quaternary ammonium salts, and combinations thereof.

35. The kit of claim 34, wherein the enzymatically activated chemiluminescent substrate moiety comprises a dioxetane having an enzyme labile group and wherein the enzyme is capable of cleaving the enzyme labile group to produce the peroxygenated intermediate.

36. The kit of claim 35, further comprising a chemiluminescent enhancement substance.

37. The kit of claim 34, wherein the assay is an immunoassay and the enzyme is complexed with an agent capable of binding to the analyte.

38. The kit of claim 34, wherein the assay is a DNA probe assay, the kit further comprising a membrane on which the assay can be conducted.

39. The kit of claim 38, further comprising an enhancement substance for increasing the chemiluminescent signal obtained from the substrate.

40. The kit of claim 38, wherein the enzyme is complexed with an agent and wherein the agent can form a complex with the analyte.

41. The kit of claim 34, wherein the assay is a DNA sequence analysis assay and the kit further comprises a membrane on which said sequence analysis assay can be conducted.

42. The kit of claim 41, wherein the kit further comprises a chemiluminescent enhancement substance.

43. The kit of claim 41, wherein the enzyme is complexed with an agent permitting attachment of the enzyme to the DNA to be sequenced in the assay.

44. The kit of claim 34, wherein the enzyme is capable of oxidizing the chemiluminescent substrate to produce the peroxygenated intermediate.

45. The kit of claim 44, wherein the chemiluminescent substrate comprises a moiety selected from the group consisting of a luminol moiety, an isoluminol moiety, an acridinium ester moiety, an acridinium sulfonylamide moiety or a luciferin moiety.

47. The method of claim 46, further comprising a step of removing enzyme complex which did not bind with the analyte prior to the measuring step.

48. The method of claim 46, wherein the substance capable of binding to the analyte is an antigen, an antibody or a nucleic acid probe.

49. The method of claim 46, wherein the assay is conducted on a solid support.

50. The method of claim 49, wherein the presence of an analyte is detected in a plurality of samples arranged in an array on the solid support.

51. The method of claim 49, further comprising binding the analyte to the solid support.

52. The method of claim 46, wherein the enzyme is selected from the group consisting of glycosidases, esterases, proteases, oxidases, peptidases and phosphatases.

34. A kit for conducting an assay to determine the presence or concentration of an analyte in a sample, wherein the kit comprises:

a dendrimer comprising at least one enzymatically active chemiluminescent substrate moiety; and

an enzyme capable of activating the chemiluminescent substrate to produce a peroxygenated intermediate that decomposes to produce light.

46. A method for detecting the presence of an analyte in a sample comprising:

forming an enzyme complex between an enzyme and a substance capable of binding to the analyte;

adding the enzyme complex to the sample;

allowing the enzyme complex to bind with analyte present in the sample;

adding a chemiluminescent delivery system to the sample; and

measuring chemiluminescent emissions from the sample;

wherein the chemiluminescent delivery system comprises a dendrimer and at least one enzymatically active chemiluminescent substrate conjugated to the dendrimer and wherein the amount of chemiluminescent emissions measured indicates the presence and/or the concentration of the analyte in the sample.