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Detection reagent and quantitative detection method of human serum albumin

A technology for human serum albumin and detection reagents, which is applied in measurement devices, instruments, fluorescence/phosphorescence, etc., can solve problems such as inability to determine binding sites, low content of S-nitroso albumin, etc., and achieves strong anti-interference ability. , high selectivity, good specificity

Active Publication Date: 2016-05-11
王铮
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nitric oxide (NO) can also form S-nitrosoalbumin through electrophilic combination with cysteine-34, resulting in vasodilator effect, but under physiological conditions, the content of S-nitrosoalbumin in plasma is extremely low (<10nmol / L), it is unclear to what extent HSA can bind NO and its derivatives under pathological conditions
In addition, there are some drugs and compounds for which the specific binding site cannot be determined

Method used

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  • Detection reagent and quantitative detection method of human serum albumin
  • Detection reagent and quantitative detection method of human serum albumin
  • Detection reagent and quantitative detection method of human serum albumin

Examples

Experimental program
Comparison scheme
Effect test

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 hours, 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, and the yield is 50% . 1H NMR of probe 1 (400MHz, CDCl3): δ13.19(s, 1H), 7.91(d, J=7.0Hz, 2H), 7.57(d, J=8.9Hz, 2H), 7.37-7.49(m, 2H), 7.00(d, J=8.4Hz, 1H), 6.85-6.96(m, 1H), 6.69(d, J=8.9Hz, 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, then warmed to room ...

Embodiment 2

[0044] Example 2. Fluorescence changes before and after probe 1 binds to HSA

[0045] (1) Use a standard solution of 1mg / mL human serum albumin (HSA), dilute it to 300mg / L with phosphate buffer), shake and mix at 37°C;

[0046] (2) Add 2 μL of fluorescent substrate molecule 1 with a concentration of 0.2 mM to each solution sample (198 μL) to make the final concentration 2 μM; shake and mix at 37°C for 5 seconds;

[0047] (3) Detect the fluorescence intensity (λ ex =424nm, λ 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 1mg / mL human serum albumin (HSA), dilute it to 300mg / L with phosphate buffer), shake and mix at 37°C;

[0050] (2) Add 2 μL of fluorescent substrate molecule 2 with a concentration of 0.2 mM 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 (λ ex =424nm, λ em =560nm) the fluorescence intensity value at the collection wavelength (see Figure 5 ).

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Abstract

The invention provides a detection reagent and a quantitative detection method of human serum albumin and belongs to the technical field of fine chemical engineering. Based on a combining attribute of albumin and small molecules, the specific principle of the method is that HCAB can be specifically combined with the albumin, a charge transferring phenomenon in HCAB twisting molecules is inhibited and non-radiation transition is reduced, so that fluorescent light is recovered and quantitative detection of the albumin is realized. After the human serum albumin is excited under a physiological pH value condition (an excitation wavelength lambdaex is equal to 424nm), a stable fluorescent signal (an emission wavelength lambdaem is equal to 526nm) is generated. The fluorescent value intensity is in direct proportion to the concentration of the albumin; and according to a detected fluorescent value, the content of the albumin in a biological sample can be calculated. The method can be used for determining absolute content of the albumin in a plurality of types of biological samples including blood serum, urine and the like; and meanwhile, the method also has the advantages of high sensitivity, high accuracy, high environment interference resisting capability, easiness of carrying out high-flux detection and the like.

Description

technical field [0001] The invention belongs to the field of fine chemical industry, and in particular relates to a method and application for quantitatively measuring albumin content in biological samples. Background technique [0002] Human serum albumin (humanserumalbumin, HSA) is a small molecular weight, non-glycosylated, negatively charged serum protein with multiple functions such as substance binding, transport, antioxidant and enzyme activity [1]. Under physiological conditions, albumin is the most abundant protein in extracellular fluid, among which the plasma albumin content is 35-50 g / L, accounting for about 60% of the total plasma protein, much higher than the protein content in interstitial fluid. Albumin is mainly synthesized in the liver, but not stored in the liver. It is directly secreted by the liver cells into the sinusoidal space and enters the blood circulation with a half-life of 17-19 days. [0003] HSA is a small molecule globular protein containing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/64G01N21/6486
Inventor 崔京南李鹏波冯磊
Owner 王铮
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