Magnetic particle chemiluminescent microfluidic chip for detecting creatine kinase isoenzyme in whole blood

A microfluidic chip and creatine kinase technology, applied in the field of CK-M, can solve problems such as low sensitivity, poor repeatability, and long detection time

Active Publication Date: 2016-03-30
SHENZHEN HUAMAIXINGWEI MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The technical problem to be solved by the present invention is to provide a method for detecting creatine kinase in whole blood in view of the problems of low sensitivity, poor repeatability, obvious interference, and the existing chemiluminescence supporting equipment and long detection time. The magnetic parti

Method used

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  • Magnetic particle chemiluminescent microfluidic chip for detecting creatine kinase isoenzyme in whole blood
  • Magnetic particle chemiluminescent microfluidic chip for detecting creatine kinase isoenzyme in whole blood
  • Magnetic particle chemiluminescent microfluidic chip for detecting creatine kinase isoenzyme in whole blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Enzymatic chemiluminescent assay of CK-MB

[0063] (1) Antibody labeling

[0064] Dissolve 5 μg of HRP in 1 mL of distilled water, then add 0.2 mL of 0.1 M freshly prepared NaIO 4 The solution was reacted at room temperature in the dark for 20 minutes, and the purified solution was dialyzed against 1 mM pH 4.4 sodium acetate buffer. Then adjust the pH to 9.0 with 0.2 M pH9.5 carbonate buffer, add 10 μg of anti-CK-MM monoclonal antibody, and react at room temperature for 2 h in the dark. Add 0.1mL freshly prepared 4mg / mLNaBH 4 solution, mixed well, and reacted at 4°C for 2h. The above solution was put into a dialysis bag, dialyzed against 0.15M pH7.4PBS, overnight at 4°C, and the HRP-labeled CK-MM antibody was obtained.

[0065] Add 1 mg magnetic particles (directly 2 μm), 10 μg EDC and 15 μg NHS solution and 10-30 μg anti-CK-BB monoclonal antibody (different from HRP-labeled antibody) solution to the phosphate buffer, mix well and react at room temperatur...

Embodiment 2

[0076] Example 2: Direct chemiluminescent assay of CK-MB

[0077] (1) Antibody labeling

[0078] Add an appropriate amount of activated acridinium ester and 100 μg anti-CK-BB monoclonal antibody solution to the phosphate buffer, mix well and react at room temperature for 4 hours, and add 1 mg glycine to block. After dialysis, an acridinium ester-labeled CK-BB antibody was obtained.

[0079] Add 1mg magnetic particles (diameter 1μm), 10μg EDC, 15μg NHS solution and 20μg streptavidin to 1ml 10mM pH7.4 phosphate buffer, mix well and react at room temperature for 4h, add 1mg glycine to block. Enrichment by magnet adsorption to remove unreacted streptavidin to obtain magnetic particle-labeled streptavidin.

[0080] Add 10 μg of anti-CK-MM monoclonal antibody to 5 μL of 0.25 mg / mL Sulfo-NHS-LC-biotin solution, and react for 1 h. Purify with an ultrafiltration centrifuge tube to remove unreacted biotin. A biotinylated anti-CK-MM antibody was obtained.

[0081] Through the intera...

Embodiment 3

[0091] Example 3: Magnetic Particle Size Screening

[0092] Refer to Example 2 for other experimental conditions, and the magnetic particle size and magnetic induction of the magnet are carried out according to the following scheme.

[0093] The particle size is 0.1 μm, 0.5 μm, 0.7 μm, 1 μm, 2.4 μm, 3 μm, 10 μm. The magnetic induction of the magnet is 500 Gauss, 1000 Gauss, 4000 Gauss, 8000 Gauss, 12000 Gauss, 30000 Gauss. Magnetic particles of seven sizes are respectively driven by the six kinds of magnets.

[0094]The experimental results show that when 0.1μm magnetic particles and 500 Gauss magnets are used in combination, the minimum detection limit is 1ng / ml, the quantitative detection range is 1-150ng / ml, and the linear correlation coefficient R 2 >0.90; both the intra-assay and inter-assay repeatability are less than 20%. That is: the chemiluminescent signal is weak, the sensitivity is not high, and the repeatability is poor.

[0095] When 10μm magnetic particles an...

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Abstract

The present invention discloses a magnetic particle chemiluminescent microfluidic chip for detecting creatine kinase isoenzyme in whole blood. The microfluidic chip comprises a head plate (1) structure and a bottom plate (2) structure, and a gas pump (3), a sampling port (4), a sample filling area (12), a marked antibody storage pool (5) and a sample mixing region (13) on the head plate (1) are connected successively; a filtration zone (6), a magnetic particle coating region (7), a washing zone (14), a detection zone (8) and a liquid release channel (16) on the bottom plate are connected successively; the detection area (8) of the bottom plate is connected with a washing fluid storage pool (9) and a lighting substrate fluid storage pool (10) through the fluid release channel (16), respectively.

Description

technical field [0001] The invention relates to a method for realizing highly sensitive quantitative detection of CK-MB by using magnetic particle chemiluminescence technology and microfluidic chip technology, and particularly discloses a magnetic particle chemiluminescence microfluidic method for detecting creatine kinase isoenzyme in whole blood The control chip can realize accurate and highly sensitive quantitative detection of CK-MB in whole blood samples, and belongs to the technical field of microfluidic chip chemiluminescence immunoassay. Background technique [0002] At present, the situation of prevention and control of cardiovascular diseases in my country is still severe, and the incidence of cardiovascular diseases is on the rise. According to statistics, the mortality rate of cardiovascular disease accounts for 40% of the population's death. Therefore, early detection, early prevention and early treatment are the key to improving the level of cardiovascular dise...

Claims

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Application Information

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IPC IPC(8): B01L3/00G01N33/573
Inventor 王东李泉
Owner SHENZHEN HUAMAIXINGWEI MEDICAL TECH CO LTD
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