Glucose-6-phosphate dehydrogenase assays
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.
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.