A detection reagent and quantitative detection method for human serum albumin
A technology of human serum albumin and detection reagents, applied in the field of fine chemicals, can solve the problems of uncertain binding sites, low content of S-nitrosoalbumin, etc., and achieve strong anti-interference ability, high selectivity, and low cost Effect
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Embodiment 1
[0036] Example 1. Synthesis of probes 1, 2 or HCAB
[0037]
[0038] Synthesis of Probe 1:
[0039] 1.3 mmol of compound 3 (2'-hydroxyacetophenone) and 1.2 mmol of compound 4 (4-dimethylaminobenzaldehyde) were dissolved in 30 mL of ethanol, and 4.9 mmol of potassium hydroxide solution was added dropwise thereto. Stir for 8 h, remove the solvent by rotary evaporation, and purify the remaining solid (silica gel, ethyl acetate-n-hexane as eluent, 1:5, v / v) to obtain 160 mg of red solid, which is probe 1, with a yield of 50 %. 1H NMR of probe 1 (400 MHz, CDCl3): δ 13.19 (s, 1H), 7.91 (d, J = 7.0 Hz,2H), 7.57 (d, J = 8.9 Hz, 2H), 7.37-7.49 (m , 2H), 7.00 (d, J = 8.4 Hz, 1H), 6.85-6.96 (m, 1H), 6.69 (d, J = 8.9 Hz, 2H), 3.05 (s, 6H).
[0040] Synthesis of probe HCAB:
[0041] Add 0.5 mmol of Probe 1 and 0.5 mmol of triethylamine to 10 mL of THF, then add benzoyl chloride (0.6 mmol, mixed with 5 mL of THF) dropwise to the reaction mixture over 30 min and stir at 0 °C 1 h, the...
Embodiment 2
[0044] Example 2. Fluorescence changes before and after probe 1 binds to HSA
[0045] (1) Use a standard solution of 1 mg / mL human serum albumin (HSA), dilute it to 300 mg / L with phosphate buffer), shake and mix at 37 °C;
[0046] (2) Add 2 µL of 0.2 mM fluorescent substrate molecule 1 to each solution sample (198 µL) to make the final concentration 2 µM; shake and mix at 37°C for 5 s;
[0047] (3) Detect the fluorescence intensity generated by the combination of substrate molecules and albumin ( lambda ex = 424nm, lambda em = 560nm) the fluorescence intensity value at the collection wavelength (see Figure 4 ).
Embodiment 3
[0048] Example 3. Fluorescence changes before and after probe 2 binds to HSA
[0049] (1) Use a standard solution of 1 mg / mL human serum albumin (HSA), dilute it to 300 mg / L with phosphate buffer), shake and mix at 37 °C;
[0050] (2) Add 2 µL of 0.2 mM fluorescent substrate molecule 2 to each solution sample (198 µL) to make the final concentration 2 µM; shake and mix at 37°C for 5 s;
[0051] (3) Detect the fluorescence intensity generated by the combination of substrate molecules and albumin ( lambda ex = 424nm, lambda em = 560nm) the fluorescence intensity value at the collection wavelength (see Figure 5 ).
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