Enzymatic Assays Using Umbelliferone Substrates with Cyclodextrins in Droplets of Oil

• US 20110118132A1
• Filed: 01/25/2011
• Published: 05/19/2011
• Est. Priority Date: 03/22/2007
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Wang, Tong, Xiong, Yalin, Wu, Ning, Raolji, Gajendrasinh Balvantsinh, Winger, Theodore, Saha, Sanjay, Dayam, Raveendra, Sista, Ramakrishna, Pamula, Vamsee K., Graham, Carrie, Eckhardt, Allen E.
• Granted Patent: US 8,093,062 B2
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions

Abstract

The invention is directed to droplet actuator devices and assay methods. The method may include, among other things, a method of conducting enzymatic assays using umbelliferone substrates with cyclodextrins in droplets in oil, the method including incubating a droplet in oil, the droplet preferably comprising an umbelliferone substrate, a sample, and a cyclodextrin compound. The methods may further include a method of substantially eliminating cross-contamination between droplets in enzymatic containing substrate-based bioassays. The method may include immobilization of the enzymatic substrate including forming an inclusion complex with the substrate for stabilizing the substrate within an aqueous environment, wherein the inclusion complex may be formed using cyclodextrins. In yet another embodiment, the invention may provide a method of enhancing hydrolysis of enzymatic substrates. The methods may further include a method of forming an inclusion complex with the substrate for stabilizing the substrate within an aqueous environment, wherein the substrate may be a 4-MU- or HMU containing substrate. The inclusion complex may be formed using cyclodextrin.

Claims

1. A method of conducting an assay, the method comprising incubating a droplet in oil, the droplet comprising an umbelliferone substrate, a sample, and a cyclodextrin compound.

2. The method of claim 1 wherein the cyclodextrin compound is selected from the group consisting of α

-cyclodextrins, β

-cyclodextrins, and γ

-cyclodextrins, and analogs and derivatives of the foregoing.

3. The method of claim 1 wherein the umbilliferone substrate is selected from the group consisting of alkylumbelliferyl-α

-L-iduronides, 4-methylumbelliferyl-α

-L-iduronide, 4-methylumbelliferyl-α

-L-iduronide-2-sulfate, 4-methylumbelliferyl-α

-L-idopyranosiduronic acid, 4-methylumbelliferyl-α

-L-fucoside, 4-methylumbelliferyl-α

-L-mannoside, 4-methylumbelliferyl-β

-D-mannoside, 4-methylumbelliferyl β

-D-N-acetylglucosaminide, 4-methylumbelliferyl β

-D-N-acetylglucosaminide sulfate, alkylumbelliferyl-β

-D-glycosides, methylumbelliferyl-β

-D-glycosides, 4-methylumbelliferyl-α

-D-galactoside, 4-methylumbelliferyl-β

-D-galactoside, 4-methylumbelliferyl-β

-D-glucuronic acid, phenolphthalein-β

-D-glucuronic acid, ethylumbelliferyl-β

-D-glycosides, multrifluoroethylumbelliferyl-β

-D-glycosides, pentafluoroethylumbelliferyl-β

-D-glycosides, pentafluoroethylumbelliferyl-β

-D-glucoside, umbelliferylchiotriosides, 4-alkyumbelliferylchiotrioside, 4-methylumbelliferylchiotrioside, 4-methylumbelliferyl-β

-galactose, 4-alkyumbeliferrone phosphates, 4-methylumbeliferrone phosphate, 6-alkanoylamido-4-methylumbelliferones, substrates comprising a 4-methyllumbelliferyl group, 6-hexadecanoylamido-4-methylumbelliferone, 4-methyllumbelliferyl-β

-D-glucosaminide, 4-methylumbelliferyl-α

-neuraminic acid, 4-methylumbelliferyl-α

-D-N-acetylgalactosaminide, and their functional analogs and derivatives.

4. The method of claim 1 wherein the steps are performed within droplets controlled by a droplet actuator.

5. The method of claim 4 wherein the droplet actuator controls the steps using electrode mediated droplet operations.

6. The method of claim 4 wherein the droplet actuator controls the steps using electrowetting mediated droplet operations.

7. The method of claim 4 wherein the droplet actuator controls the steps using dielectrophoresis mediated droplet operations.

8. A method of substantially eliminating cross-contamination between droplets in enzymatic containing substrate-based bioassays, comprising immobilizing the enzymatic containing substrate onto a solid support.

9. The method of claim 8 wherein the enzymatic containing substrate comprises a 4-MU-containing substrate.

10. The method of claim 9 wherein the 4-MU-containing substrate is immobilized onto a solid support via the 4-MU component of the substrate molecule.

11. The method of claim 8 wherein the solid support comprises magnetically responsive beads.

12. The method of claim 8 wherein the solid support comprises hydrogel beads.

13. The method of claim 8 wherein immobilization of the enzymatic substrate comprises forming an inclusion complex with the substrate for stabilizing the substrate within an aqueous environment.

14. The method of claim 13 wherein the inclusion complex is formed using cyclodextrins.

15. The method claim 13 wherein forming the inclusion complex comprises combing and mixing a sample droplet with a reagent droplet using droplet operations, wherein the reagent droplet comprises an additive to facilitate the inclusion complex.

16. The method claim 15 wherein the additive comprises cyclodextrin.

17. The method of claim 8 wherein immobilization of the enzymatic substrate comprises using surfactants for stabilizing the substrate within an aqueous environment.

18. A method of enhancing hydrolysis of enzymatic substrates, the method comprising:

forming an inclusion complex with the substrate for stabilizing the substrate within an aqueous environment.

19. The method of claim 18 wherein the substrate is a 4-MU- or HMU containing substrate.

20. The method of claim 18 wherein the inclusion complex is formed using cyclodextrin.

21. The method of claim 20 wherein the cyclodextrin comprises hydroxypropyl-β

-cyclodextrin.

22. The method of claim 21 wherein the concentration of hydroxypropyl-β

-cyclodextrin comprises from about 5 mM to about 15 mM.

23. The method of claim 21 wherein the concentration of hydroxypropyl-β

-cyclodextrin comprises from about 7 mM to about 13 mM.

24. The method of claim 21 wherein the concentration of hydroxypropyl-β

-cyclodextrin comprises from about 9 mM to about 11 mM.

25. The method of claim 21 wherein the concentration of hydroxypropyl-β

-cyclodextrin comprises from about 9.5 mM to about 11.5 mM.

26. The method of claim 20 wherein the cyclodextrin comprises methyl-β

-cyclodextrin.

27. The method of claim 26 wherein the concentration of methyl-β

-cyclodextrin comprises from about 1 mM to about 20 mM.

28. The method of claim 26 wherein the concentration of methyl-β

-cyclodextrin comprises from about 5 mM to about 15 mM.

29. The method of claim 26 wherein the concentration of methyl-β

-cyclodextrin comprises from about 7 mM to about 13 mM.

30. The method of claim 26 wherein the concentration of methyl-β

-cyclodextrin comprises from about 9 mM to about 11 mM.

31. The method of claim 26 wherein the concentration of methyl-β

-cyclodextrin comprises from about 9.5 mM to about 11.5 mM.