Application

This kit can be used to measure the urea content in serum, plasma, urine and other samples.

Detection principle

In strong acidic and heating condition, urea can react with diacetyl to form red diazine compound. The depth of color is proportional to the content of urea. Because the instability of the diacetyl, the diacetyl oxime usually react with the strong acid firstly in the reaction system to generate diacetyl, then react with urea to generate the red diazine compound. The reaction equation is as follows:

Experimental instrument

Test tube, Micropipette, Vortex mixer, Centrifuge, Spectrometer (520 nm), Incubator (water-bath)

Pretreatment of sample

[Note]: It is recommended to take 2~3 samples which expected large difference to do pre-experiment before formal experiment. Bring all the reagents to room temperature before experiment.

1. For serum (plasma) sample:

Centrifuge the serum (plasma) at 12000 rpm for 5 min, take the supernatant for detection.

2. Urine sample:

Centrifuge the urine sample at 12000 rpm for 5 min, take the supernatant and dilute with normal saline at a ratio of 1:10~ 1:50 for detection. If the concentration of urine sample is out of the linear range, dilute it again.

Operation steps

1. Open the water bath in advance and set the temperature to 100℃.

2. Blank tube: add 0.02 mL of double-distilled water into a 10 mL glass tube.

Standard tube: add 0.02 mL of 10 mmol/L urea standard into a 10 mL glass tube.

Sample tube: add 0.02 mL of Sample into a 10 mL glass tube. 

3. Add 1 mL of Reagent 1 and 1 mL of Reagent 2 working solution into each tube. Tight the tubes with preservative film and mix fully with vortex mixer. Incubate the tubes in boiling water for 15 min. Cool the tubes with running water.

4. Set to zero with double-distilled water and measure the OD value of each tube with 1cm cuvette at 520 nm.

Note: It can be refer to the following operating table.

Note: Equilibrate the pipette tip in that reagent before pipetting each reagent, such as slowly fill the tip and gently expel the contents. 

Notes

1. This kit is for research use only.

2. Please progress strictly with operation procedures.

3. Do not use components from different batches of kit.