1.  The supernatant after centrifugation must be clarified in in pretreatment step. Otherwise take the turbid supernatant to another centrifuge tube and centrifuge again.

2.  As the concentration of Zn2+ in serum is very low, avoid zinc contamination of vessels and reagents used in the experiment.

3.  Prevent the formulation of bubbles when the supernatant is transferred into the microplate.

                                [Note]: A, blank well; B-H：standard wells; S1-S80: sample wells.

a)    The pretreatment of sample

Mix the sample and reagent 2 at a ratio of 1:1 and centrifuge at 13780 g for 10 min at 4℃, then take the supernatant for detection.

b)    Standard wells: Add 0.05 mL of Standard solution with different concentrations.

Sample wells: Add 0.05 mL of pretreated supernatant of sample in Step a.

c)    Add 0.2 mL of chromogenic agent working solution into each well of Step b.

d)    Shake with Microplate with for 30 s and stand for 5 min at room temperature.

e)    Measure the OD value at 560 nm with Microplate Reader.

Dilute 1.54 mmol/L Zinc standard solution with double distilled water to a serial concentration. The recommended dilution gradient is as follows: 46.20, 30.80, 23.10, 15.40, 11.55, 7.70, 3.85, 0 μmol/L. Then carry the assay according to the operation table.

Plot the standard curve by using OD value of standard and correspondent concentration as y-axis and x-axis respectively. Create the standard curve with graph software (or EXCEL). The concentration of the sample can be calculated according to the formula based on the OD value of sample. The standard curve is: y= ax + b.

For human serum sample, take 0.1 mL of sample and add 0.1 mL of reagent 2, centrifuge at 13780 g for 10 min at 4℃, then take the supernatant and carry the assay according to the operation table.

The results are as follows:

standard curve: y = 0.0152 x + 0.0023, the average OD value of the sample well is 0.217, the average OD value of the blank well is 0.105, and the calculation result is:

Zn content (μmol/L)=(0.217-0.105-0.0023)÷0.0152×2=14.43 μmol/L

Detect human serum, human urine, human milk and rat serum according to the protocol, the result is as follows: