Method for quantifying transferrin in human serum by nanogold labeling and liquid chromatography mass spectrometry
A technology of transferrin and liquid mass spectrometry, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problem of lack of transferrin, etc.
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Embodiment 1
[0050] Preparation of gold nanoparticles:
[0051] The three-necked flask used for synthesizing gold nanoparticles was thoroughly washed with freshly prepared aqua regia and soaked overnight, then thoroughly rinsed with ultrapure water and dried in an oven. On a heated magnetic stirrer, 50ml of 0.01% chloroauric acid solution was heated to boiling, then 1, 1.5, 1.75, 2.5, 3.75ml of different volumes of 1% sodium citrate solution were quickly added to the flask, and the stirring was continued for 30 minutes. The color of the solution changed from pale yellow to bright red, after which stirring was continued at room temperature for 20 minutes to cool. The prepared gold nanoparticles were filtered through a 0.45um membrane and stored at 4°C in the dark.
[0052] The particle size of AuNPs was observed by high-resolution transmission electron microscopy (TEM). The concentration of AuNPs was determined by ICP-MS in 1% HNO 3 Prepare an Au single-element gradient standard solution...
Embodiment 2
[0056] In the preparation of nano-gold-transferrin conjugates, the choice of buffer for labeling transferrin:
[0057] Gold nanoparticles with a particle size of 15 nm were centrifuged at 9000 rpm for 20 min, the supernatant was carefully discarded, and resuspended in buffers A, B and C. Then, the gold nanoparticles in different buffers were aliquoted (5×200ul) into a 96-well plate, and 1ul, 2ul, 4ul, 6ul, and 8ul of 0.1 mg / ml standard transferrin were added respectively. The 96-well plate was slightly shaken at room temperature for 30 minutes, and after incubation, 10ul of 12% sodium chloride solution was added to each well, and the stability of gold nanoparticles in different buffers was tested by a salt spray test. The gold nanoparticles purified by centrifugation were resuspended in different kinds of buffers. If the color of the gold nanoparticles solution changed from the initial red to blue, it indicated that the stability of the gold nanoparticles was destroyed and cau...
Embodiment 3
[0064] Preparation of nano-gold-transferrin conjugates
[0065] The gold nanoparticles with a particle size of 15 nm were centrifuged at 9000 rpm for 20 min, and the supernatant was carefully discarded and resuspended in phosphate buffer at pH 7.8. Then, the gold nanoparticles in the buffer were aliquoted (5×200ul) into a 96-well plate, and 1ul, 2ul, 4ul, 6ul, and 8ul of 0.1 mg / ml standard transferrin were added respectively. The 96-well plate was shaken gently for 30 minutes at room temperature.
[0066] Electron microscopy images of gold nanoparticles and gold nanoparticles-transferrin conjugates ( Figure 5 ), UV spectra of gold nanoparticles and gold nanoparticles-transferrin conjugates at 400-700 nm ( Figure 4 ) to determine whether the nano-gold-transferrin conjugate was labeled.
[0067] like Figure 5 As shown in (a), we selected gold nanoparticles with an average particle size of 15 nm, which is relatively stable when the particle size is small. Due to the face-...
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