TITLE: Measuring urea concentrations in water samples
AUTHORS: Jacob   Waldbauer, Amy Amy Zimmerman

[DESCRIPTION]
Colorimetric assay for direct (vs. enzymatic) measurement of urea to a detection limit of 0.4 µM concentration. The reaction of urea with diacetylmonoxime (DAM) to form a colored product is enhanced by addition of thiosemicarbazide (TSC). The original direct method was adapted for use with a single mixed reagent and incubation at room temperature (vs. the “high temperature direct method” that incubates at 85ºC for 30 minutes). NOTE: This protocol is written for measurement in 24-well plates.

[STEPS]
2. Making Reagents and Solutions
2.1. Diacetylmonoxime (DAM) solution: Dissolve 3.4 g in 100 mL nanopure or LC-MS water (34 g L-1 or 0.3363 M stock). Store solution at 4°C in dark. Stable for at least 1 month. 
a. Also known as 2,3-butanedione monoxime.
b. Dissolve using rotisserie (hybridization oven) set to room temp (prop open door).
2.2. Thiosemicarbazide (TSC) solution: Dissolve 0.19 g in 20 mL nanopure water (9.5 g L-1 or 0.104235 M stock). Store solution at 4°C in dark. Stable for at least 1 month. a.Dissolve using rotisserie (hybridization oven) set to room temp (prop open door).
2.4. Reagent A solution: Mix 25 parts DAM with 1 part TSC. Make fresh prior to each analysis. (32.7 g L-1 or 0.3234 M DAM and 0.365 g L-1or 0.004 M TSC) 
2.3. Ferric chloride (hexahydrate) solution: Dissolve 0.15 g ferric chloride (FeCl3, 6H2O) in 10 mL nanopure water (15 g L-1 or 0.0554877 M stock). Store solution at 4°C in dark.
2.5. Reagent B solution: Add 300 mL concentrated sulfuric acid (~98% or 18.4 M) to 535 mL nanopure water (final concentration H2SO4 = 35.2% or 6.6 M). Add 0.5 mL ferric chloride solution to diluted acid (8.977 mg L-1 or 0.03321 mM stock). Store solution at 4°C in dark. Stable for at least 1 month.
2.6. Color developing reagent (COLDER): Mix 1 part of Reagent A with 3.2 parts of Reagent B. Use within 15 minutes. a.Turbidity blank solution: Substitute nanopure water for Reagent A above for determination of optical turbidity blank.
3. Assay set-up.
3.1. Label four polypropylene tubes for each sample and all standards (7) (includes triplicate reactions and single turbidity blanks).NOTE: Assay requires >8 mL of each sample and standard (includes replication).
3.2. Calculate total volume of COLDER reagent needed to run triplicate reactions for each standard and sample: (7 standards + # samples + 1 extra) x 3 x 0.6 mL = total vol (mL).a.If quantifying urea from 2 samples: 7+2+1 = 10 x3x0.6 = 18 mL reagent.
3.3. Calculate total volume of turbidity blank solution needed to run duplicates for each standard and sample: (7 standards + # samples + 1 extra) x 2 x 0.6 mL = total vol (mL). a.If quantifying urea from 2 samples: 7+2+1 = 10 x2x0.6 = 12 mL blank solution.
3.4. Aliquot 2 mL of each sample and all standards into corresponding reaction tubes. 
3.5. Prepare the volumes of COLDER reagent and turbidity blank solution needed by mixing 1 part of Reagent A (or nanopure water) with 3.2 parts of Reagent B as described below. Use reagent within 15 minutes. 
a.If need 18 mL, mix 4.5 mL of Reagent A with 14.4 mL of Reagent B (18.9 mL). 
b.If need 12 mL, mix 3 mL of nanopure water with 6 mL of Reagent B (12.6 mL).
3.6. Add 0.6 mL of COLDER reagent (or turbidity blank) to each reaction tube. 
a. DAM: 1.7963 g L-1 or 0.01777 M final concentration
b.TSC: 0.02008 g L-1 or 0.0002203 M final concentration
c.FeCl3: 0.001578 g L-1 or 0.00000584 M (5.84 µM)
d.H2SO4: 6.2% or 1.16 M final concentration

3.7. Mix (vortex) and incubate in the dark at room temperature (~22ºC) for 3 days.
3.8. After 72 hours, transfer/pour entire 2.6 mL volume of each reaction into corresponding wells of 24-well plates and measure absorbance on plate reader.

4. Reading plates.
4.1. Turn on Tecan Infinite 200 PRO plate reader 20-30 minutes prior to use.
4.2. Once warmed up, open the iControl software on MLCLab-PC.
4.3. Open file “Revilla_urea_24well”.
4.4. Load the plate—check whether the “plate with cover” box is checked (since using clear plates for this, can be read with lid on).
4.5. Read absorbance at 520±9 nm (25 flashes). Program automatically opens an Excel file that documents read parameters and data. ‘Save as’ before measuring 2nd plate.
5. Analyzing data.
5.1. Subtract the average absorbance of sample turbidity blanks from the absorbance of the samples treated with COLDER reagent (= corrected sample absorbance).
5.2. Subtract the average absorbance of the nanopure water tubes (i.e., 0 µM urea) treated with turbidity blank solution from the absorbances of all the standards. 
5.3. Plot corrected absorbance (y) vs. concentration (x) for all standards to establish a standard curve with linear regression. 
5.4. Use the equation of the standard curve to calculate sample concentration from absorbance.
1. Making Standards.
1.1. Prepare 200 µM stock solution. Dilute 1:500 from 0.1 M solution => 20 µL + 9.980 mL nanopure water.

1.2. Dilute the stock solution to the following concentrations in nanopure water: 0, 0.5, 1.25, 2.5, 5, 7.5, 10  µM.