Hydrogen Peroxide Droplet-Based Assays

• US 20100028920A1
• Filed: 03/05/2008
• Published: 02/04/2010
• Est. Priority Date: 03/05/2007
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Eckhardt, Allen E.
• Granted Patent: US 8,426,213 B2
• Time in Patent Office: Days
• US Class Current: 435/14
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: C12Q 1/006 for glucose
• CPC Class Codes: C12Q 1/28 involving peroxidase
• CPC Class Codes: G01N 2333/904 acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
• CPC Class Codes: Y10T 436/118339 with formation of a segmented stream
• CPC Class Codes: Y10T 436/14 Heterocyclic carbon compound [i.e. , O, S, N, Se, Te, as only ring hetero atom]
• CPC Class Codes: Y10T 436/141111 Diverse hetero atoms in same or different rings [e.g., alkaloids, opiates, etc.]
• CPC Class Codes: Y10T 436/142222 Hetero-O [e.g., ascorbic acid, etc.]
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/144444 Glucose
• CPC Class Codes: Y10T 436/145555 Hetero-N
• CPC Class Codes: Y10T 436/146666 Bile pigment
• CPC Class Codes: Y10T 436/147777 Plural nitrogen in the same ring [e.g., barbituates, creatinine, etc.]
• CPC Class Codes: Y10T 436/148888 Uric acid
• CPC Class Codes: Y10T 436/206664 Ozone or peroxide

Abstract

The present invention relates to providing improved hydrogen peroxide assays, as well as droplet actuators for conducting such assays. The droplet actuators of the invention may be used to conduct droplet-based hydrogen peroxide assays. They may also be associated with detectors for analyzing the results of the hydrogen peroxide assays of the invention. They may be provided as components of systems which control droplet operations and/or detection for conducting the hydrogen peroxide assays. Measurement by the detector may be used to quantify the presence of an analyte in a sample.

Claims

1. A method of conducting a hydrogen peroxide assay for detecting an analyte, the method comprising combining hydrogen peroxide assay reagents and sample into a droplet having a volume which ranges from about 1 nL to about 10 μ

L and conducting the assay in the droplet to yield a detectable signal.

2. The method of claim 1 wherein the hydrogen peroxide assay reagents comprise an oxidase, a peroxidase, and a substrate which produces a signal molecule in the presence of a peroxidase and hydrogen peroxide.

3. The method of claim 2 wherein the peroxidase comprises horseradish peroxidase.

4. The method of claim 2 wherein the analyte comprises glucose and the oxidase comprises glucose oxidase.

5. The method of claim 2 wherein the sample comprises a biological fluid and the peroxidase is provided in an amount which produces a substantially linear result.

6. The method of claim 5 wherein the result has a lag time which is less than about five seconds.

7. The method of claim 5 wherein the result has substantially no lag time.

8. The method of claim 1 wherein the droplet has a size which ranges from about 10 nL to about 1 μ

L.

9. The method of claim 1 wherein the droplet has a size which ranges from about 1 nL to about 500 nL.

10. The method of claim 1 wherein the combining of hydrogen peroxide assay reagents and sample comprises combining one or more sample droplets with one or more hydrogen peroxide assay reagents using one or more droplet operations.

11. The method of claim 10 wherein one or more of the droplet operations comprises an electrode mediated droplet operation.

12. The method of claim 10 wherein one or more of the droplet operations comprises an electric field mediated droplet operation.

13. The method of claim 10 wherein one or more of the droplet operations comprises an electrowetting mediated droplet operation.

14. The method of claim 10 wherein one or more of the droplet operations comprises an electrophoresis mediated droplet operation.

15. The method of claim 1 wherein the analyte is selected from a group consisting of metabolites present in the blood susceptible to production of hydrogen peroxide in the presence of an oxidase.

16. The method of claim 1 wherein the analyte is selected from a group consisting of creatinin, lactate, pyruvate, bilirubin, glutamate.

17. The method of claim 1 wherein the droplet is partially or completely surrounded by a filler fluid which is immiscible with the droplet.

18. The method of claim 17 wherein the filler fluid comprises an oil.

19. The method of claim 17 wherein the filler fluid comprises a silicon oil.

20. The method of claim 1 wherein the assay comprises a flash assay in which substantially all of the analyte is consumed.

21. The method of claim 20 wherein the sample and reagents for conducting the flash assay are combined in the presence of sensor on the droplet actuator using one or more droplet operations.

22. A method of conducting a hydrogen peroxide assay, the method comprising combining hydrogen peroxide assay reagents and sample into a droplet and conducting the assay in the droplet to yield a detectable signal, wherein the assay produces a substantially linear result.

23. A method of conducting a hydrogen peroxide assay for detecting an analyte, the method comprising:

(a) combining hydrogen peroxide assay reagents and sample into a droplet and conducting the assay in the droplet to yield a detectable signal, wherein substantially all of the analyte is consumed;

(b) detecting the signal; and

(c) analyzing the signal to determine the quantity of analyte in the sample.

24. The method of claim 23 wherein the sample and reagents for conducting the assay are combined in the presence of a sensor on the droplet actuator using one or more droplet operations.

25. The method of claim 23 wherein substantially all of the analyte is consumed in less than about 5 minutes.

26. The method of claim 23 wherein substantially all of the analyte is consumed in less than about 2 minutes.

27. The method of claim 23 wherein the sample comprises a biological sample.

28. The method of claim 23 wherein the sample is selected from the group consisting of blood, serum, and plasma.

29. A method of detecting multiple analytes on a single droplet actuator using a single detector, the method comprising:

(a) providing a droplet actuator device comprising;

(i) a substrate comprising electrodes arranged for conducting droplet operations on a droplet operations surface of the substrate; and

(ii) one or more reservoirs in fluid communication with the droplet operations surface comprising one or more reagents for conducting an assay in which hydrogen peroxide is an intermediate; and

(b) using droplet operations to combine multiple sample droplets with multiple reagents to conduct multiple hydrogen peroxide assays on a single droplet actuator.

30. The method of claim 29 wherein assay reactions are started sequentially and presentation to a detector is timed so that droplets are presented during a phase in the assay reaction having substantially linear kinetics.