IGEM:IMPERIAL/2009/M2/Assays/glu3

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Amplex® Red Glucose/Glucose Oxidase Assay Kit

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INTRODUCTION

The Amplex® Red Glucose/Glucose Oxidase Assay Kit A22189) provides a sensitive one-step method for detecting glucose or glucose oxidase. The Amplex® Red reagent (10-acetyl-3,7- dihydroxyphenoxazine) is a colorless, stable, and extremely versatile peroxidase substrate. Because peroxidase- and glucose oxidase–mediated reactions can be coupled,1,2 it is possible to measure glucose oxidase activity or the release of glucose by any glucosidase enzyme—for instance, β-glucosidase and glucocerebrosidase— in either a continuous or discontinuous assay. This assay should also be very useful for quantitation of glucose levels in foods, fermentation media, and bodily fluids.
In the assay, glucose oxidase reacts with d-glucose to form d-gluconolactone and H2O2. In the presence of horseradish peroxidase (HRP), the H2O2 then reacts with the Amplex® Red reagent in a 1:1 stoichiometry to generate the red-fluorescent oxidation product, resorufin.3 Resorufin has fluorescence excitation and emission maxima of approximately 571 nm and 585 nm, respectively (Figure 1), and because the extinction coefficient is high (54,000 cm-1M-1), the assay can be performed either fluorometrically or spectrophotometrically. Furthermore, at these long wavelengths, there is little interference from autofluorescence found in most biological samples. With the Amplex® Red Glucose/Glucose Oxidase Assay Kit, we have detected as little as 3 μM d-glucose (Figure 2) and 0.05 mU/mL glucose oxidase (Figure 3).

NOTES

Allow reagents to warm to room temperature before opening vials. The Amplex® Red reagent is somewhat air sensitive. Once a vial of Amplex® Red reagent is opened, the reagent should be used promptly. PROTECT THE AMPLEX® RED REAGENT FROM LIGHT

Please note the following restrictions on the use of the Amplex® Red reagent. The Amplex® Red

reagent is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 μM. The Amplex® Red reagent is also unstable at high pH (>8.5). Furthermore, the absorption and fluorescence of the reaction product, resorufin, are pH-dependent. Below the pKa (~6.0), the absorption maximum shifts to ~480 nm and the fluorescence quantum yield is markedly lower. For these reasons, the reactions should be performed at pH 7–8. The provided reaction buffer, pH 7.4, is recommended.

PREPARING THE STOCK SOLUTION

Preparing the Stock Solutions 1.1 Prepare a 10 mM stock solution of Amplex® Red reagent. Allow one vial of Amplex® Red reagent (Component A, blue cap) and DMSO (Component B, green cap) to warm to room temperature. Just prior to use, dissolve the contents of the vial of Amplex® Red reagent in 60 μL of DMSO. Each vial of Amplex® Red reagent is sufficient for approximately 100 assays, with a final reaction volume of 100 μL per assay.
1.2 Prepare 1X Reaction Buffer. Add 4 mL of 5X Reaction Buffer (Component C, white cap) to 16 mL of deionized water (dH2O). This 20 mL volume of 1X Reaction Buffer is sufficient for approximately 100 assays of 100 μL each with a 10 mL excess for making stock solutions.
1.3 Prepare a 10 U/mL stock solution of horseradish peroxidase (HRP). Dissolve the contents of the vial of HRP (Component D, yellow cap) in 1 mL of 1X Reaction Buffer. After the assay, any remaining unused solution should be divided into single-use aliquots and stored frozen at ≤–20°C.
1.4 Prepare a 100 U/mL glucose oxidase stock solution. Dissolve the contents of the vial of glucose oxidase (Component E, orange cap) in 1.0 mL of 1X Reaction Buffer. This stock solution should be stored frozen at ≤–20°C.
1.5 Prepare a 400 mM (72 mg/mL) glucose stock solution. Weigh out a portion of glucose (Component F, black cap), and dissolve it in the appropriate amount of 1X Reaction Buffer
1.6 Prepare a 20 mM H2O2 working solution. Dilute the ~3% H2O2 stock solution (Component G, red cap) into the appropriate volume of 1X Reaction Buffer. The actual concentration of H2O2 is indicated on the label. For instance, a 20 mM H2O2 working solution can be prepared from a 3.0% (0.88 M) H2O2 stock solution by diluting 22.7 μL of 3.0% H2O2 into 977 μL of 1X Reaction Buffer.
Note: Although the ~3% H2O2 stock solution has been stabilized to slow degradation, the 20 mM H2O2 working

Experimental protocol

glucose assay

The following procedure is designed for use with a fluorescence or absorbance microplate reader. For use with a standard fluorometer, volumes must be increased accordingly.
The following protocol describes the assay of glucose in a total volume of 100 μL per microplate well. The volumes recommended here are sufficient for ~100 assays. The kit provides sufficient material for ~500 assays.
2.1 Prepare a glucose standard curve. Dilute the appropriate amount of the 400 mM glucose stock solution (prepared in step 1.5) into 1X Reaction Buffer to produce glucose concentrations of 0 to 200 μM, each in a volume of 50 μL. Be sure to include a no-glucose control. Final glucose concentrations will be twofold lower (e.g., 0 to 100 μM).
2.2 If no standard curve is to be used, prepare positive and negative controls. For a glucosepositive control, dilute the 400 mM glucose stock solution (prepared in step 1.5) to 200 μM in 1X Reaction Buffer. For an H2O2-positive control, dilute the 20 mM H2O2 working solution (prepared in step 1.6) to 10 μM in 1X Reaction Buffer. For a negative control, use 1X Reaction Buffer without H2O2.
2.3 Dilute the glucose-containing samples in 1X Reaction Buffer. A volume of 50 μL will be used for each reaction. A variable dilution will be required depending on the total glucose present in the sample. In the first trial the samples should be serially diluted to determine the optimal amount of sample for the assay.
Note: Extremely high levels of glucose (e.g., 500 μM, final concentration) can produce lower fluorescence than moderately high levels (e.g., 100 μM), because excess H2O2 resulting from the reaction of glucose with glucose oxidase can oxidize the reaction product, resorufin, to nonfluorescent resazurin.
2.4 Load the samples. Pipet 50 μL of the standard curve samples, controls, and experimental samples into individual wells of a microplate. Amplex® Red Glucose/Glucose Oxidase Assay Kit |

2.5 Prepare a working solution of 100 μM Amplex® Red reagent, 0.2 U/mL HRP and 2 U/mL glucose oxidase. Mix the following: 50 μL of 10 mM Amplex® Red reagent stock solution (prepared in step 1.1) 100 μL of 10 U/mL HRP stock solution (prepared in step 1.3) 100 μL of 100 U/mL glucose oxidase stock solution (prepared in step 1.4) 4.75 mL of 1X Reaction Buffer This 5 mL volume is sufficient for ~100 assays. Note that the final concentration of each component will be twofold lower in the final reaction volume.
2.6 Begin the reactions. Add 50 μL of the Amplex® Red reagent/HRP/glucose oxidase working solution to each microplate well containing the standards, controls, and samples.
2.7 Incubate the reactions. Incubate at room temperature for 30 minutes, protected from light. Because the assay is continuous (not terminated), fluorescence or absorbance may be measured at multiple time points to follow the kinetics of the reactions.
2.8 Measure the fluorescence or absorbance. Use a microplate reader equipped for excitation in the range of 530–560 nm and fluorescence emission detection at ~590 nm, or for absorbance at ~560 nm (see Figure 1).
2.9 Correct for background fluorescence or absorbance. For each point, subtract the value derived from the no-glucose control.