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Magnetic particle chemiluminiscence micro-fluidic chip for detecting n-terminal portion of brain natriuretic peptide in whole blood

A microfluidic chip and chemiluminescence technology, applied in the field of sensitive determination, can solve the problems of interference, long detection time, low sensitivity, etc., and achieve the effect of high sensitivity detection

Active Publication Date: 2016-01-13
SHENZHEN HUAMAIXINGWEI MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to provide a method for detecting nitrogen-terminal brain sodium in whole blood for the problems of low sensitivity, poor repeatability, obvious interference, and expensive and long detection time of existing chemiluminescent matching instruments in the existing rapid diagnostic method. Peptide magnetic particle chemiluminescent microfluidic chip, through the integrated chip (all components except the test sample are integrated into the chip) and supporting small portable equipment, so as to realize the rapid, accurate and high-speed detection of NT-proBNP in the field sample. Sensitive Quantitative Detection

Method used

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  • Magnetic particle chemiluminiscence micro-fluidic chip for detecting n-terminal portion of brain natriuretic peptide in whole blood
  • Magnetic particle chemiluminiscence micro-fluidic chip for detecting n-terminal portion of brain natriuretic peptide in whole blood
  • Magnetic particle chemiluminiscence micro-fluidic chip for detecting n-terminal portion of brain natriuretic peptide in whole blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Enzymatic chemiluminescence determination of NT-proBNP

[0059] (1) Antibody labeling

[0060] Dissolve 5μg of HRP in 1mL of distilled water, then add 0.2mL of 0.1M freshly prepared NaIO 4 After the solution was reacted for 20 minutes in the dark at room temperature, the purified solution was dialyzed against 1 mM pH4.4 sodium acetate buffer. Then adjust the pH to 9.0 with 0.2MpH9.5 carbonate buffer, add 10μg of anti-NT-proBNP monoclonal antibody, and react for 2h at room temperature in the dark. Add 0.1mL freshly prepared 4mg / mLNaBH 4 Mix well and react at 4℃ for 2h. The above solution was put into a dialysis bag and dialyzed with 0.15MpH7.4PBS at 4°C overnight to obtain HRP-labeled NT-proBNP antibody.

[0061] Add 1mg magnetic particles (size 2μm), 10μg EDC and 15μg NHS solution and 10~30μg anti-NT-proBNP monoclonal antibody (different from HRP-labeled antibody) solution to the phosphate buffer, mix well and react at room temperature for 4h, then add 1mg Glycin...

Embodiment 2

[0072] Example 2: Direct Chemiluminescence Determination of NT-proBNP

[0073] (1) Antibody labeling

[0074] Add appropriate amount of activated acridinium ester and 100μg anti-NT-proBNP monoclonal antibody solution to the phosphate buffer, mix well and react at room temperature for 4h, and add 1mg glycine to block. After dialysis, an acridine ester labeled NT-proBNP antibody was obtained.

[0075] Add 1mg magnetic particles (size 1μm), 10μg EDC and 15μg NHS solution and 20μg streptavidin to 1ml10mMpH7.4 phosphate buffer, mix well and react at room temperature for 4h, add 1mg glycine to block. Magnet adsorption and enrichment are used to remove unreacted streptavidin to obtain magnetic particle labeled streptavidin.

[0076] Add 10μg of anti-NT-proBNP monoclonal antibody to 5μL of 0.25mg / mL Sulfo-NHS-LC-biotin solution, and react for 1h. Purify by ultrafiltration centrifuge tube to remove unreacted biotin. Obtain biotinylated anti-NT-proBNP antibody.

[0077] Through the interactio...

Embodiment 3

[0087] Example 3: Particle size screening of magnetic particles

[0088] See Example 2 for other experimental conditions. The size of the magnetic particles and the magnetic induction intensity of the magnet are carried out according to the following scheme.

[0089] The particle size is 0.1μm, 0.5μm, 0.7μm, 1μm, 2.4μm, 3μm, 10μm. The magnetic induction intensity of the magnet is 500 Gauss, 1000 Gauss, 4000 Gauss, 8000 Gauss, 12000 Gauss, and 30000 Gauss. The six types of magnets are used to drive seven types of magnetic particles.

[0090] The experimental results show that: when 0.1μm magnetic particles and 500 Gauss magnet are used in combination, the minimum detection limit is 40pg / ml, the quantitative detection range is 40~20000pg / ml, and the linear correlation coefficient is R 2 >0.90; The repeatability within and between batches is less than 20%. Namely: the chemiluminescence signal is weak, the sensitivity is not high, and the repeatability is poor.

[0091] When 10μm magnet...

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Abstract

The invention discloses a magnetic particle chemiluminiscence micro-fluidic chip for detecting the n-terminal portion of brain natriuretic peptide in whole blood. The micro-fluidic chip comprises a top plate (1) and a base plate (2), wherein an air pump (3), a sample adding port (4), a sample filling area (12), a labelled antibody storage pool (5) and a sample mixing area (13) on the top plate (1) are sequentially connected, a filtering area (6), a magnetic particle coating area (7), a cleaning area (14) and a detection area (8) on the base plate (2) are sequentially connected, and the detection area (8) on the base plate (2) is connected with a cleaning fluid storage pool (9) and a luminous substrate liquid storage pool (10) through a liquid releasing channel (16).

Description

Technical field [0001] The invention relates to a method for realizing NT-proBNP high-sensitivity quantitative detection by using magnetic particle chemiluminescence technology and microfluidic chip technology, and particularly discloses a magnetic particle chemiluminescence microfluidic control for detecting nitrogen-terminal brain natriuretic peptide in whole blood The chip realizes the rapid and accurate quantitative detection of NT-proBNP in cardiovascular and cerebrovascular diseases, especially heart failure, and can be widely used in primary hospitals and clinics, belonging to the field of microfluidic chip chemiluminescence immunodetection technology. Background technique [0002] There are two types of BNP currently used for clinical testing: NT-proBNP and BNP. Although the two have the same biological source, their biological effects and clinical significance are not exactly the same. After the cardiomyocytes are stimulated, the original gene product pre-BNP precursor ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01N33/68
Inventor 王东李泉
Owner SHENZHEN HUAMAIXINGWEI MEDICAL TECH CO LTD
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