Glucose-6-phosphate dehydrogenase assays

• US 10,731,199 B2
• Filed: 11/20/2012
• Issued: 08/04/2020
• Est. Priority Date: 11/21/2011
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Graham, Carrie A., Eckhardt, Allen E.
• Primary Examiner(s): Visone, Thomas J.
• Application Number:
  US14/359,177
• Publication Number:
  US 20140335069A1
• Time in Patent Office: 2,814 Days
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: G01N 2333/904 acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)

Abstract

Aspects of embodiments may include methods for automated enzymatic detection of glucose-6-phosphate dehydrogenase (G6PD) activity. Aspects of embodiments may include methods for enzymatic detection of G6PD activity in droplets in oil. Aspects of embodiments may include a system including a droplet actuator. Aspects of embodiments may include a treatment method.

Claims

1. A method of detecting glucose-6-phosphate dehydrogenase (G6PD) activity, the method comprising:

(a) mixing an aliquot of prepared sample with an aliquot of substrate formulation, wherein the substrate formulation includes about 100 mM Tris HCL, about 26 mM maleimide, about 2.6 mM NADP+, about 2.4 mM magnesium chloride, and about 2.0 mM glucose-6-phosphate, said substrate formulation having a pH of 7.8;

(b) incubating the sample at about 37°

C. for a time interval; and

(c) detecting glucose-6-phosphate dehydrogenase (G6PD) activity present in the sample, wherein the detecting step comprises reading NADPH fluorescence at different times within the time interval wherein the time interval is about 0 seconds to about 300 seconds.

2. The method of claim 1 further comprising:

(a) executing electrowetting-mediated droplet operations using droplets on a droplet microactuator to effect an assay;

(b) combining one or more substrate formulation droplets with one or more prepared sample droplets; and

(c) generating and detecting a signal which corresponds to the conversion of NADP+ to NADPH in the sample.

3. The method of claim 1 further comprising conducting the method on a droplet that is partially or completely surrounded by a filler fluid on a droplet actuator.

4. The method of claim 1 further comprising providing a microfluidic actuator.

5. The method of claim 1 further comprising detecting the enzymatic conversion of NADP+ to NADPH.

6. The method of claim 1 further comprising wherein reading NADPH fluorescence is done at 340 nm excitation/460 nm emission.

7. The method of claim 1 further comprising wherein the time interval is from about 0 seconds to about 10 minutes.

8. The method of claim 1 further comprising wherein the sample comprises a biological sample.

9. The method of claim 1 further comprising wherein the sample comprises an aliquot of one or more dried blood spots.

10. The method of claim 1 further comprising wherein the sample has been isolated from a patient less than about 30 days old at the time of sample collection.

11. The method of claim 1 further comprising wherein the detecting step comprises detecting a signal from a droplet on a droplet microactuator.

12. The method of claim 11 further comprising wherein the signal detected corresponds to glucose-6-phosphate dehydrogenase (G6PD) activity.

13. A method of detecting glucose-6-phosphate dehydrogenase (G6PD) activity, the method comprising:

(a) mixing sample with reactant, wherein said reactant includes about 100 mM Tris HCL, about 26 mM maleimide, about 2.6 mM NADP+, about 2.4 mM magnesium chloride, and about 2.0 mM glucose-6-phosphate, said reactant having a pH of 7.8;

(b) incubating the sample for a time interval;

(c) detecting glucose-6-phosphate dehydrogenase (G6PD) activity present in the sample over time t=0 seconds to time t=300 seconds; and

(d) conducting the method on a droplet that is partially or completely surrounded by a filler fluid on a droplet actuator.

14. A method to assay a reaction, the method comprising:

(a) executing droplet operations using droplets on a droplet microactuator to effect an assay;

(b) combining one or more reactant droplets with one or more sample droplets, wherein the reactants are about 100 mM Tris HCL, about 26 mM maleimide, about 2.6 mM NADP+, about 2.4 mM magnesium chloride, and about 2.0 mM glucose-6-phosphate, said reactants having a pH of 7.8; and

(c) generating and eventually detecting a signal which corresponds to the conversion of NAD(P)+ to NAD(P)H in the sample, wherein the detecting step comprises reading NADPH fluorescence at different times starting from time t=0 seconds to t=300 seconds.

15. A method of performing a redox reaction, the method comprising:

(a) providing reactants in an aqueous droplet;

(b) oxidizing or reducing the reactants, wherein the reactants are about 100 mM Tris HCL, about 26 mM maleimide, about 2.6 mM NADP+, about 2.4 mM magnesium chloride, and about 2.0 mM glucose-6-phosphate, said reactants having a pH of 7.8; and

(c) generating and eventually detecting a signal which corresponds to the oxidizing or reducing step, wherein the detecting step comprises reading NADPH fluorescence at intervals of 50 seconds over time t=0 seconds to time t=300 seconds.

16. A method of detecting glucose-6-phosphate dehydrogenase (G6PD) activity, the method comprising:

(a) mixing an aliquot of prepared sample with an aliquot of substrate formulation, wherein the substrate formulation includes about 100 mM Tris HCL, about 26 mM maleimide, about 2.6 mM NADP+, about 2.4 mM magnesium chloride, and about 2.0 mM glucose-6-phosphate;

(b) incubating the sample at about 37°

C. for a time interval; and

(c) detecting glucose-6-phosphate dehydrogenase (G6PD) activity present in the sample, wherein the detecting step comprises reading NADPH fluorescence at different times within the time interval, said time interval starting at about t=0 seconds and ending at about t=300 seconds.