Enzyme assays on a droplet actuator

• US 9,574,220 B2
• Filed: 03/11/2015
• Issued: 02/21/2017
• 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: Raolji, Gajendrasinh, Eckhardt, Allen E, Graham, Carrie, Sista, Ramakrishna, Pamula, Vamsee K., Winger, Theodore, Wang, Tong, Xiong, Yalin, Wu, Ning, Saha, Sanjay, Dayam, Raveendra
• Primary Examiner(s): Lewis, Patrick
• Application Number:
  US14/644,989
• Publication Number:
  US 20150184219A1
• Time in Patent Office: 713 Days
• Field of Search: None
• US Class Current: 1/1
• 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 invention includes assay methods of conducting an assay comprising combining a sample with an umbelliferyl derivative, wherein the sample potentially comprises an enzyme capable of cleaving the umbelliferyl derivative and where the umbelliferyl derivative comprises an umbelliferyl core modified with one or more modifying moieties.

Claims

1. A compound having one of the following general formulas:

2. The compound of claim 1, wherein n is an integer of from 1 to 50.

3. The compound of claim 1, wherein n is an integer of from 1 to 25.

4. The compound of claim 1, wherein n is an integer of from 1 to 10.

5. The compound of claim 1, wherein the hydrophilic polymer or oligomer comprises a polyethylene glycol moiety.

6. The compound of claim 5, wherein the polyethylene glycol moiety of the oligomer has at least 2 polyethylene glycol subunits.

7. The compound of claim 5, wherein the polyethylene glycol moiety of the oligomer has at least 5 polyethylene glycol subunits.

8. The compound of claim 5, wherein the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.

9. The compound of claim 1, wherein the hydrophilic polymer or oligomer comprises polar or charged functional groups that render the polymer or oligomer soluble in water.

10. The compound of claim 9, wherein the hydrophilic polymer or oligomer is selected from the group consisting of polyacrylic acid, polyacrylamide, polymaleic anhydride polymers, polyallylamine, polyethyleneimine, polyoxazoline, polylysine, and co-polymers thereof.

11. The compound of claim 9, wherein the hydrophilic polymer or oligomer comprises one or more other moieties selected from the group consisting of additional hydrophilic moieties, lipophilic moieties, spacer moieties, linker moieties, and terminating moieties.

12. The compound of claim 11, wherein the one or more additional hydrophilic moieties are selected from the group consisting of sugars, polyhydric alcohols, polyalkylene oxides, and polyamine/PEG copolymers.

13. The compound of claim 11, wherein the spacer moieties are selected from the group consisting of sugar, cholesterol and glycerine moieties.

14. The compound of claim 11, wherein the terminating moieties are present at the one or more ends of the oligomer not coupled to the 4-HU backbone, and are selected from the group consisting of C1-6 alkyl and alkoxy moieties.

15. The compound of claim 14, wherein the terminating moiety is methyl or methoxy.

16. A compound of claim 1, having one of the following formulas:

17. The compound of claim 16, wherein n is an integer of from 1 to 50.

18. The compound of claim 16, wherein n is an integer of from 1 to 25.

19. The compound of claim 16, wherein n is an integer of from 1 to 10.

20. The compound of claim 12, wherein the sugar moieties are coupled to the 4-MU core structure at position R2, and are selected from the group consisting of:

21. The compound of claim 12, wherein the polyhydric alcohols are selected from the group consisting of glycol (2-carbon), glycerol (3-carbon), erythritol (4-carbon), threitol (4-carbon), arabitol (5-carbon), xylitol (5-carbon), ribitol (5-carbon), mannitol (6-carbon), sorbitol (6-carbon), dulcitol (6-carbon), iditol (6-carbon), isomalt (12-carbon), maltitol (12-carbon), lactitol (12-carbon), and polyglycitol.

22. A compound selected from the group consisting of:

23. A method of conducting an assay, the method comprising combining with a compound having one of the following general formulas, a blood sample potentially comprising an enzyme capable of cleaving said compound:

24. A process for preparing compounds of Formula 2

25. A compound having Formula II:

26. A compound of claim 25, having the following formula:

27. A method of conducting an assay, the method comprising combining with a compound having one of the following general formulas, a blood sample potentially comprising an enzyme capable of cleaving said compound: