Dendritic chemiluminescent substrates

• US 7,883,903 B2
• Filed: 04/03/2007
• Issued: 02/08/2011
• Est. Priority Date: 01/08/2001
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Sparks, Alison L.
• Primary Examiner(s): Haq; Shafiqul
• Application Number:
  US11/730,715
• Publication Number:
  US 20070184491A1
• Time in Patent Office: 1,407 Days
• Field of Search: None
• US Class Current: 436/546
• 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 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, wherein the substrate moiety comprises a dioxetane having an enzyme labile group; and

an enzyme capable of cleaving the enzyme labile group of the dioxetane to produce a peroxygenated intermediate that decomposes to produce light;

wherein the dendrimer comprising at least one enzymatically active chemiluminescent substrate moiety is represented by the formula selected from the group consisting of

2. The kit of claim 1, wherein the assay is an immunoassay and the enzyme is complexed with an agent capable of binding to the analyte.

3. The kit of claim 1, wherein the assay is a DNA probe assay, the kit further comprising a membrane on which the assay can be conducted.

4. The kit of claim 3, wherein the enzyme is complexed with an agent and wherein the agent can form a complex with the analyte.

5. The kit of claim 1, 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.

6. The kit of claim 5, wherein the enzyme is complexed with an agent permitting attachment of the enzyme to the DNA to be sequenced in the assay.

7. The kit of claim 1, wherein the enzyme is capable of oxidizing the chemiluminescent substrate to produce the peroxygenated intermediate.

14. The kit of claim 1, wherein, the linker moiety is represented by the formula

8. 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;

removing the enzyme complex which did not bind with the analyte prior to the measuring step;

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 which is conjugated to the dendrimer, wherein the substrate comprises a dioxetane having an enzyme labile group which is capable of being cleaved by the enzyme to produce a peroxygenated intermediate,

wherein the amount of chemiluminescent emissions measured indicates the presence and/or the concentration of the analyte in the sample, and

wherein the chemical delivery system is represented by the formula selected from the group consisting of

9. The method of claim 8, wherein the substance capable of binding to the analyte is an antigen, an antibody or a nucleic acid probe.

10. The method of claim 8, wherein the assay is conducted on a solid support.

11. The method of claim 10, wherein the presence of an analyte is detected in a plurality of samples arranged in an array on the solid support.

12. The method of claim 10, further comprising binding the analyte to the solid support.

13. The method of claim 8, wherein the enzyme is selected from the group consisting of glycosidases, esterases, proteases, oxidases, peptidases and phosphatases.

15. The method of claim 8, wherein, the linker moiety is represented by the formula