Glucose-6-phosphate dehydrogenase assays
First Claim
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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.
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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.
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Citations
16 Claims
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1. A method of detecting glucose-6-phosphate dehydrogenase (G6PD) activity, the method comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method of detecting glucose-6-phosphate dehydrogenase (G6PD) activity, the method comprising:
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(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.
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14. A method to assay a reaction, the method comprising:
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(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.
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15. A method of performing a redox reaction, the method comprising:
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(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.
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16. A method of detecting glucose-6-phosphate dehydrogenase (G6PD) activity, the method comprising:
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(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.
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Specification