A method for enriching prealbumin in human plasma with solid-phase extractant
A technology of solid-phase extraction agent and prealbumin, which is applied in the field of solid-phase extraction agent enrichment of prealbumin in human plasma, can solve the problems of difficult to achieve satisfactory results, low sensitivity, complicated process, etc., and achieve low cost, large ratio Surface area, simple operation effect
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Embodiment 1
[0028] Preparation of graphene microsphere solid phase extractant.
[0029] Step 1: Add ultrapure water to the graphene oxide to prepare a graphene oxide solution with a mass concentration of 6.0 wt%, stir for 15 hours under a magnetic stirrer at a speed of 1000r / min, and put the resulting solution into an ultrasonic machine Shake for 10 hours to obtain a dispersed graphene oxide solution;
[0030] Step 2: Put the mixed solution in an ultrasonic nebulizer connected with a quartz tube, and the ultrasonic frequency is 1.5MHz. Make it atomized into aerosol droplets, in N 2 Driven by a vacuum pump and a suction filter, it slowly passes through a quartz tube heated to 450°C, and the end is collected with a PTFE filter membrane. Dry at room temperature for 4 hours, then put it in a vacuum desiccator and dry at 70°C for 24 hours to obtain a graphene microsphere solid-phase extraction agent with a particle size of 1 μm to 3 μm, such as figure 1 shown.
Embodiment 2
[0032] Pretreatment of blood plasma.
[0033] Step 1: Take the plasma out of the refrigerator to thaw, add the preservative sodium azide according to the standard of 0.5g / L, centrifuge at room temperature, set the speed at 8000rpm, and set the time for 10min, take the supernatant, and store it at room temperature Filter under suction, collect the filtrate and store it in a refrigerator at 4°C for later use;
[0034] Step 2: phenol is placed in 60 ℃ of water baths and melted 2h in advance, and preparation volume ratio is the phenol solution of 5.5% in fume hood; Under magnetic stirring, add sodium chloride (mass fraction of sodium chloride in blood plasma) in filtrate 20%), after being completely dissolved, the phenol solution was added dropwise to the above solution with a peristaltic pump at a volume ratio of 1:1, and after stirring for 2 hours, the solution was left to stand at 4°C for 20 hours; the solution was taken out and centrifuged , the rotational speed is set to 800...
Embodiment 3
[0036] Using the graphene microspheres obtained in Example 1 as a solid-phase extractant to adsorb prealbumin in plasma, the specific steps are:
[0037] Step 1: Weigh 30 mg of graphene microspheres and add them to a solid-phase extraction column with a volume of 3 ml, seal the upper and lower sieve plates, and compress them with a sieve plate insertion tool; activate the column with 8 ml of 50% methanol solution by volume Treatment, control the flow rate to 0.8ml / min;
[0038]Step 2: Dilute the supernatant in Example 2 by 40 times, use a constant flow pump to pass the diluted 40ml plasma solution through the solid phase extraction column, control the flow rate to 1.0ml / min, add 2ml after the sample is loaded, and the volume percentage is 10 % methanol solution, wash to remove unadsorbed protein, and vacuum pump until dry. Use 3ml of acetonitrile as the protein eluent, control the flow rate to 0.1ml / min, collect the eluate and blow it to near dryness with a nitrogen blowing i...
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