Method of hydrolyzing an enzymatic substrate

• US 8,394,641 B2
• Filed: 11/29/2011
• Issued: 03/12/2013
• Est. Priority Date: 12/21/2009
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Winger, Theodore
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): COLE, MONIQUE T
• Application Number:
  US13/305,820
• Publication Number:
  US 20120136147A1
• Time in Patent Office: 469 Days
• Field of Search: 436/86, 435/7.72, 435/183
• US Class Current: 436/86
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/18 Means for temperature control
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: C07D 311/16 substituted in position 7
• CPC Class Codes: C07D 311/18 substituted otherwise than in position 3 or 7
• CPC Class Codes: C07D 311/20 hydrogenated in the hetero ring
• CPC Class Codes: C07D 405/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 413/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07H 15/203 Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
• CPC Class Codes: C07H 17/075 Benzo[b]pyran-2-ones
• CPC Class Codes: C12Q 1/25 involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/40 involving amylase
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 2333/916 : acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
• CPC Class Codes: G01N 2333/926 : acting on alpha -1, 4-glucosidic bonds, e.g. hyaluronidase, invertase, amylase
• CPC Class Codes: G01N 2333/938 : acting on beta-galactose-glycoside bonds, e.g. beta-galactosidase
• CPC Class Codes: G01N 2800/04 : Endocrine or metabolic disorders
• CPC Class Codes: G01N 33/5438 : Electrodes

Abstract

The invention is directed to droplet actuator devices and assay methods. The method may include immobilization of the enzymatic substrate including forming an inclusion complex with the substrate within an aqueous environment in contact with an oil.

Claims

1. A method of hydrolyzing an enzymatic substrate, the method comprising:

(a) forming an inclusion complex with the substrate in an aqueous droplet in contact with oil;

(b) hydrolyzing the substrate; and

wherein the substrate is a 4-MU- or HMU containing substrate.

2. The method of claim 1 wherein the inclusion complex is formed using cyclodextrin.

3. The method of claim 1 wherein the cyclodextrin comprises hydroxypropyl-β

-cyclodextrin.

4. The method of claim 3 wherein the concentration of hydroxypropyl-β

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

5. The method of claim 3 wherein the concentration of hydroxypropyl-β

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

6. The method of claim 3 wherein the concentration of hydroxypropyl-β

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

7. The method of claim 3 wherein the concentration of hydroxypropyl-β

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

8. The method of claim 3 wherein the cyclodextrin comprises methyl-β

-cyclodextrin.

9. The method of claim 8 wherein the concentration of methyl-β

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

10. The method of claim 8 wherein the concentration of methyl-β

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

11. The method of claim 8 wherein the concentration of methyl-β

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

12. The method of claim 8 wherein the concentration of methyl-β

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

13. The method of claim 8 wherein the concentration of methyl-β

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

14. The method of claim 1, wherein the aqueous droplet is surrounded by oil.