TITLE: Enzymatic Ethanol Assay 
AUTHORS: Sarah Hammer, Dev Kapadia, Shuen Hon, Marybeth Maloney, Daniel Olson, Lee Lynd

[DESCRIPTION]
This protocol describes a 96-well-plate-based, enzymatic assay for reliably estimating ethanol concentrations in experimental samples in one hour. In the presence of excess NAD+, alcohol dehydrogenase (ADH) is employed to convert ethanol to acetaldehyde. The concomitant conversion of NAD+ to NADH is monitored via increased absorbance at 340 nm. When highly accurate analytical techniques (such as high performance liquid chromatography) are not necessary, or are too costly or low-throughput, this assay offers reliable, inexpensive, and rapid detection of ethanol concentrations. This assay is useful for applications such as determining relative ethanol production from microbial fermentations, and detecting ethanol evaporation from media.

Version notes:
10-27-2021. Add 50 ul water to the assay plate first, then add sample. This prevents ethanol evaporation and allows the assay to be started (by adding assay master mix) immediately before reading.

[STEPS]
SECTION: Preparation
1. Create a microplate spectrophotometer program to read absorbance at 340 nm of each well in a 96-well plate. 
The program should take absorbance readings at 340 nm every 20 - 30 seconds (or at minimum interval), shaking for 10 seconds immediately before each reading. 
The program should be set to run for 1 hour at 30 °C.
SECTION: Preparation
2. Make nicotinamide adenine dinucleotide (NAD+) stock solution
 
Vortex to ensure that NAD+ is completely dissolved. 
Store aliquots in 1.5-mL microcentrifuge tubes at -80 °C.

SECTION: Preparation
3. Make pH 9.0 glycine buffer

 
Store at 25 °C for up to 1 month. 

SECTION: Preparation
4. Make alcohol dehydrogenase (ADH) enzyme stock solution

 
Store aliquots in 1.5-mL microcentrifuge tubes at -20 °C for up to 1 month. 

SECTION: Experimental Steps
5. Make ethanol standards at eight concentrations encompassing the range of concentrations expected from the experimental samples, including a 0 g/L ethanol standard. Dilute ethanol in the same media present in the experimental samples to make the standards. 

 

SECTION: Experimental Steps
6. Make master mix for enzyme assay on ice, adding components in the order listed in the table below. If NAD+ and ADH stock solution aliquots are frozen, defrost the necessary volume on ice before proceeding.
 

 

Keep the master mix on ice during and after preparation. 
SECTION: Experimental Steps
7. Turn on the microplate spectrophotometer and open the program (defined in Step 1) to begin heating to 30 °C, the temperature at which the assay will be run.

SECTION: Experimental Steps
9. 
 

Designate two of the 12 columns in the 96-well plate for standards, and add 10 μL of each standard with a P10 pipette to the 190 μL of assay master mix. 

In the remaining wells, add 10 μL of each sample. It is recommended to use a multi-channel (8-channel) pipette for this step to fill the plate as rapidly as possible. 
SECTION: Experimental Steps
10. Start the assay by adding 190 μL of the assay master mix using a repeater pipette and a 5 mL repeater pipette tip.


 

SECTION: Experimental Steps
11. Seal the plate with a ThermalSeal RTS sealing film. Use a sealing film roller or a roll of tape to ensure that the film is adhered closely to the rim of each well, taking care to avoid any large wrinkles or gaps.
SECTION: Experimental Steps
12. Place the sealed 96-well plate (without a lid) in the microplate spectrophotometer (preheated to 30 °C) and start the program to read the absorbance at 340 nm.

 


SECTION: Data Analysis
13. 
Use the 340 nm absorbance data to calculate Vmax (change in absorbance per unit time) for each well in the plate, including wells containing experimental samples and standards. Use at least 30 data points in a range where absorbance is linearly increasing with time to calculate Vmax. 

SECTION: Data Analysis
14. Use the standard wells with known ethanol concentrations to generate a standard curve, as specified in the sub-steps below. Sample data is attached.  

SECTION: Data Analysis
14.1. User-defined standard concentrations will serve as x-axis data inputs. Vmax data at 340 nm will serve as y-axis data inputs. 
SECTION: Data Analysis
14.2. Use a four-parameter nonlinear regression curve fit to generate a standard curve with the formula Y = (A-D)/(1+(X/C)^B)+D.
  

SECTION: Data Analysis
15. Use the standard curve to calculate ethanol concentrations in the experimental samples.
SECTION: Experimental Steps
8. Fill all wells of a 96-well plate with 50 μL of water. The purpose of the water is to minimize ethanol evaporation during subsequent sample pipetting steps.