Amplite® Fluorimetric Glucose Oxidase Assay Kit Red Fluorescence

The glucose oxidase is a dimeric protein that catalyzes the oxidation of beta-D-glucose into hydrogen peroxide and D-glucono-1,5-lactone, which is hydrolyzed to gluconic acid. It is widely used for the determination of glucose in body fluids and in removing residual glucose and oxygen from beverages, food and other agricultural products. Furthermore, glucose oxidase is commonly used in biosensors to detect glucose. The Amplite® Glucose Oxidase Assay Kit provides a quick and sensitive method for the measurement of glucose oxidase in solution. It can be performed in a convenient 96-well or 384-well microtiter-plate format and is easily adapted to automation without a separation step. The kit uses our Amplite® Red substrate which enables a dual recordable mode. The fluorescent signal can be easily read by either a fluorescence microplate reader or an absorbance microplate reader. With the Amplite® Fluorimetric Glucose Oxidase Assay Kit, we have detected as little as 0.05 mU/mL glucose oxidase in a 100 µL reaction volume.

Example protocol

AT A GLANCE

Protocol Summary

Prepare glucose oxidase standards or test samples (50 µL)
Add GO working solution (50 µL)
Incubate at 37 °C for 10 - 30 minutes
Monitor fluorescence intensity at Ex/Em = 540/590 nm

Important Thaw all the kit components to room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Amplite™ Red stock solution (250X)

Add 100 µL of DMSO (Component E) into the vial of Amplite™ Red (Component A). The stock solution should be used promptly.
Note The Amplite™ Red 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 Amplite™ Red is also unstable at high pH (>8.5). Therefore, the reaction should be performed at pH 7 – 8. The provided assay buffer (pH 7.4) is recommended.

2. HRP stock solution (50X)

Add 1 mL of Assay Buffer (Component B) into the vial of Horseradish Peroxidase (Component C).

3. Glucose oxidase standard solution (100 U/mL)

Add 1 mL of Assay Buffer into the vial of Glucose Oxidase (Component D).

4. Glucose stock solution (10X)

Add 5 mL of Assay Buffer into the vial of Glucose (Component F).

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/11300

Glucose Oxidase standard

Prepare a glucose oxidase standard by diluting 2 µL of the 100 U/mL glucose oxidase standard solution into 200 µL of Assay Buffer (Component B) to have 1000 mU/mL glucose oxidase standard solution. And then take 10 µL of 1000 mU/mL glucose oxidase standard solution and perform 1:100 dilution to obtain 10 mU/mL glucose oxidase standard solution (GOS7). Then perform 1:3 serial dilutions to get remaining serially diluted glucose oxidase standards (GOS6-GOS1). A non-glucose oxidase buffer is included as blank control. The final glucose oxidase concentrations should be twofold lower (i.e., 0 to 5 mU/mL). Note: High concentrations of glucose oxidase may cause reduced fluorescence signal due to the overoxidation of Amplite™ Red (to a non-fluorescent product).

PREPARATION OF WORKING SOLUTION

Add 20 μL of Amplite™ Red stock solution (250X), 100 μL of HRP stock solution (50X), and 500 μL of Glucose stock solution (10X) into 4.4 mL of Assay Buffer (Component B) to make a total volume of 5 mL Glucose Oxidase (GO) working solution. Protect from light.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of glucose oxidase standards and test samples in a solid black 96-well microplate. GOS = Glucose Oxidase Standard (GOS1-GOS7, 0.01 to 10 mU/mL), BL = Blank Control, TS = Test Samples.

BL	BL	TS	TS
GOS1	GOS1	...	...
GOS2	GOS2	...	...
GOS3	GOS3
GOS4	GOS4
GOS5	GOS5
GOS6	GOS6
GOS7	GOS7

Table 2. Reagent composition for each well.

Well	Volume	Reagent
GOS1-GOS7	50 µL	serial diltuion (0.01 to 10 mU/mL)
BL	50 µL	Assay Buffer (Component B)
TS	50 µL	Sample

Prepare glucose oxidase standards (GOS), blank control (BL), and test samples (TS) according ot the layout proided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
Add 50 µL of GO working solution into each well of glucose oxidase standards, blank control, and test samples to make the total glucose oxidase assay volume of 100 µL/well. For a 384-well plate, add 25 µL of GO working solution into each well instead, for a total volume of 50 µL/well.
Incubate the reaction for 10 to 30 minutes at 37°C, protected from light.
Monitor the fluorescence intensity with a fluorescence plate reader at Excitation = 530 - 570 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. However, the absorption detection will have a lower sensitivity compared to the fluorescence reading.

Spectrum

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Product family

Name	Excitation (nm)	Emission (nm)
Amplite® Colorimetric Glucose Oxidase Assay Kit	571	584
Amplite® Fluorimetric Glutamate Oxidase Assay Kit Red Fluorescence	571	584
Amplite® Fluorimetric Monoamine Oxidase Assay Kit Red Fluorescence	571	584
Amplite® Fluorimetric Xanthine Oxidase Assay Kit Red Fluorescence	571	584
Amplite® Fluorimetric Lysyl Oxidase Assay Kit Red Fluorescence	571	584

Citations

View all 3 citations: Citation Explorer

A reassessment of the carnivorous status of salmonids: hepatic glucokinase is expressed in wild fish in Kerguelen Islands
Authors: Marandel, Lucie and Gaudin, Philippe and Gu{\'e}raud, Fran{\c{c}}ois and Glise, St{\'e}phane and Herman, Alexandre and Plagnes-Juan, Elisabeth and V{\'e}ron, Vincent and Panserat, St{\'e}phane and Labonne, Jacques
Journal: Science of the Total Environment (2018): 276--285

A reassessment of the carnivorous status of salmonids: Hepatic glucokinase is expressed in wild fish in Kerguelen Islands
Authors: Mar, undefined and el, Lucie and Gaudin, Philippe and Guéraud, Fran&ccaron;ois and Glise, Stéphane and Herman, Alex and re , undefined and Plagnes-Juan, Elisabeth and Véron, Vincent and Panserat, Stéphane and Labonne, Jacques
Journal: Science of The Total Environment (2018): 276--285

Overcoming 5-Fu resistance in human non-small cell lung cancer cells by the combination of 5-Fu and cisplatin through the inhibition of glucose metabolism
Authors: Zhao, Jun-gang and Ren, Kai-ming and Tang, Jun
Journal: Tumor Biology (2014): 12305--12315

References

View all 29 references: Citation Explorer

Factors affecting accuracy and time requirements of a glucose oxidase-peroxidase assay for determination of glucose
Authors: Hall MB, Keuler NS.
Journal: J AOAC Int (2009): 50

Conformation and activity dependent interaction of glucose oxidase with CdTe quantum dots: towards developing a nanoparticle based enzymatic assay
Authors: Priyam A, Chatterjee A, Bhattacharya SC, Saha A.
Journal: Photochem Photobiol Sci (2009): 362

Using an indicator displacement assay to monitor glucose oxidase activity in blood serum
Authors: Zhang T, Anslyn EV.
Journal: Org Lett (2007): 1627

Glucose oxidase assisted homogeneous electrochemical receptor binding assay for drug screening
Authors: Funabashi H, Tanaka Y, Imamura Y, Mie M, Manabe T, Tanaka H, Takahashi T, H and a H, Aizawa M, Kobatake E.
Journal: Biosens Bioelectron (2006): 1675

1,4-Benzoquinone-based electrophoretic assay for glucose oxidase
Authors: Urban PL, Goodall DM, Bergstrom ET, Bruce NC.
Journal: Anal Biochem (2006): 35

Page updated on November 20, 2024

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