GLUCOSE-6-PHOSPHATE DEHYDROGENASE ASSAYS

• US 20140335069A1
• Filed: 11/20/2012
• Published: 11/13/2014
• 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: Eckhardt, Allen E., Graham, Carrie A.
• Granted Patent: US 10,731,199 B2
• Time in Patent Office: Days
• US Class Current: 424/94.4
• 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;

(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.

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 reading NADPH fluorescence.

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

8. The method of claim 1 further comprising wherein the substrate formulation has a pH of about 7.8, and 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.

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

10. The method of claim 1 further comprising wherein the time interval is from about 0 seconds to about 300 seconds.

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

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

13. 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.

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

15. The method of claim 1 further comprising wherein the signal measured corresponds to glucose-6-phosphate dehydrogenase (G6PD) activity.

16. A computer readable medium programmed to cause a droplet actuator to perform any of the method steps of the methods of claim 1.

17. A system comprising a droplet actuator coupled to and controlled by a computer programmed to cause the droplet actuator to perform any of the method steps of the methods of claim 1.

18. A treatment method comprising:

(a) providing a droplet actuator, sample, and glucose-6-phosphate dehydrogenase (G6PD) substrate formulation;

(b) executing electrowetting-mediated droplet operations to detect glucose-6-phosphate dehydrogenase (G6PD) activity in a sample;

(c) detecting glucose-6-phosphate dehydrogenase (G6PD) activity; and

(d) discontinuing the administration of one or more of Dapsone, Flutamide (Eulexin), Mafenide cream (Sulfamylon), Methylene blue (Urolene Blue), Nalidixic acid (NegGram), Nitrofurantoin (Macrodantin), Phenazopyridine (Pyridium), Primaquine, Rasburicase (Elitek), Sulfacetamide (Klaron), Sulfamethoxazole (Gantanol), and Sulfanilamide (AVC).

19. A treatment method comprising providing a droplet actuator, sample, and glucose-6-phosphate dehydrogenase substrate formulation, executing electrowetting-mediated droplet operations to detect glucose-6-phosphate dehydrogenase (G6PD) activity in a sample;

detecting glucose-6-phosphate dehydrogenase (G6PD) activity; and

administration of one or more medicaments.