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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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</span><span style = "vertical-align:super;">+</span><span>, alcohol dehydrogenase (ADH) is employed to convert ethanol to acetaldehyde. The concomitant conversion of NAD</span><span style = "vertical-align:super;">+</span><span> 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.</span></div></div>

[STEPS]
?. [Preparation]
Make  glycine bufferSodium pyrophosphate33.3 g/L3.333 gGlycine1.67 g/L0.167 gSemicarbazide hydrochloride0.125 g/L12.5 mgHydrochloric acid (1N)---To pH 9.0Water---Up to 100 mLStore at  for up to 1 month.
Sodium pyrophosphate33.3 g/L3.333 gGlycine1.67 g/L0.167 gSemicarbazide hydrochloride0.125 g/L12.5 mgHydrochloric acid (1N)---To pH 9.0Water---Up to 100 mL
25 °C
?. [Preparation]
Make nicotinamide adenine dinucleotide (NAD+) stock solutionNAD50 mM0.4976 gWater---Up to 15 mLVortex to ensure that NAD+ is completely dissolved. Store aliquots in 1.5-mL microcentrifuge tubes at .
NAD50 mM0.4976 gWater---Up to 15 mL
-80 °C
?. [Preparation]
Make alcohol dehydrogenase (ADH) enzyme stock solutionPotassium phosphate dibasic83 g/L1.66 gPotassium phosphate monobasic17 g/L0.34 gBovine serum albumin1 g/L0.02 gAlcohol dehydrogenase enzyme (~300 U/mg)20 U/mL1.36 mgWater---Up to 20 mLStore aliquots in 1.5-mL microcentrifuge tubes at  for up to 1 month.
Potassium phosphate dibasic83 g/L1.66 gPotassium phosphate monobasic17 g/L0.34 gBovine serum albumin1 g/L0.02 gAlcohol dehydrogenase enzyme (~300 U/mg)20 U/mL1.36 mgWater---Up to 20 mL
-20 °C
?. [Experimental Steps]
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.NAD stock solution8 mM3.2 mLADH stock solution0.1 U/mL111 uLGlycine buffer---Up to 20 mLKeep the master mix on ice during and after preparation.
The master mix in the table below is sufficient for one full 96-well plate (with each well containing 190 μL of assay master mix). If running more or less than one plate, scale the recipe accordingly.
NAD stock solution8 mM3.2 mLADH stock solution0.1 U/mL111 uLGlycine buffer---Up to 20 mL
?. [Experimental Steps]
Turn on the microplate spectrophotometer and open the program (defined in Step 1) to begin heating to , the temperature at which the assay will be run.
30 °C
?. [Experimental Steps]
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. Add the standards in order of either increasing or decreasing concentration down the columns.
Work as quickly as possible through the next three steps (Steps 9 -11) in order to minimize ethanol evaporation and substantial progression of the enzymatic reaction before the microplate program has started.
?. [Experimental Steps]
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.
This protocol was optimized for detecting ethanol concentrations between 0.05 and 1.5 g/L in the assay solution, which corresponds concentrations between 1 and 30 g/L in the experimental samples. Measuring ethanol concentrations outside of this range will require preparing a different standard curve, and making different dilutions of samples (while maintaining 200 μL total volume in each well) to achieve final ethanol concentrations between 0.05 and 1.5 g/L in the assay solution.
?. [Preparation]
Create a microplate spectrophotometer program to read absorbance at 340 nm of each well in a 96-well plate. The program should be set to run for 1 hour at .
30 °C
?. [Experimental Steps]
To each well in the remaining 10 columns of the 96-well plate, add 10 μL of experimental sample to the 190 μL of assay master mix. It is recommended to use a multi-channel (8-channel) pipette for this step to fill the plate as rapidly as possible.
At this point, the assay has started and the plate should be read as quickly as possible.
?. [Experimental Steps]
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.
?. [Experimental Steps]
Place the sealed 96-well plate (without a lid) in the microplate spectrophotometer (preheated to ) and start the program to read the absorbance at 340 nm.
30 °C
As alcohol dehydrogenase converts NAD+ to NADH, absorbance at 340 nm will increase with time. If after 30 minutes, the absorbance at 340 nm plateaus (is no longer increasing) for all of the samples and standards, the program can be terminated. If not, let the program run for the full hour.
?. [Experimental Steps]
Fill all wells of a 96-well plate with 190 μL of the assay master mix using a repeater pipette and a 5 mL repeater pipette tip.
?. [Data Analysis]
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.
?. [Data Analysis]
Use the standard wells with known ethanol concentrations to generate a standard curve, as specified in the sub-steps below. Sample data is attached.
?. [Data Analysis]
User-defined standard concentrations will serve as x-axis data inputs. Vmax data at 340 nm will serve as y-axis data inputs.
?. [Data Analysis]
Use a four-parameter nonlinear regression curve fit to generate a standard curve with the formula Y = (A-D)/(1+(X/C)^B)+D.
?. [Data Analysis]
Use the standard curve to calculate ethanol concentrations in the experimental samples.