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Magnetic particle chemiluminescent microfluidic chip for quantitatively detecting troponin I in whole blood

A microfluidic chip, quantitative detection technology, applied in chemical instruments and methods, laboratory containers, laboratory utensils, etc., can solve the problems of interference, expensive instruments, low sensitivity, etc.

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

[0008] The technical problem to be solved by the present invention is to provide a method for the quantitative detection of troponin in whole blood for the problems of low sensitivity, poor repeatability, obvious interference, and the existing chemiluminescence supporting equipment and long detection time of the existing rapid diagnostic method. I's magnetic particle chemiluminescence 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 fast, accurate and high-speed detection of cTnI in the whole blood sample on site. Sensitive Quantitative Detection

Method used

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  • Magnetic particle chemiluminescent microfluidic chip for quantitatively detecting troponin I in whole blood
  • Magnetic particle chemiluminescent microfluidic chip for quantitatively detecting troponin I in whole blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Enzymatic chemiluminescent assay of cTnI

[0062] (1) Antibody labeling

[0063] Dissolve 50 μg of HRP in 1 mL of distilled water, then add 10 μmol of fresh 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 pH 9.5 carbonate buffer, add 100 μg of anti-cTnI monoclonal antibody, and react at room temperature for 2 h in the dark. Add 0.1mL4mg / mL new NaBH 4 , Mixed and reacted at 4°C for 2h. Put the above solution into a dialysis bag, dialyze against 0.15MpH7.4PBS, overnight at 4°C, and obtain HRP-labeled cTnI antibody.

[0064]Add 1 mg magnetic particles (diameter 2 μm), 10 μg EDC and 15 μg NHS solution and 10-30 μg anti-cTnI monoclonal antibody (different from HRP-labeled antibody) solution to pH 7.4 phosphate buffer, mix well and react at room temperature for 4 h, add 1 mg glycine blocked. Enrichment and purificati...

Embodiment 2

[0075] Example 2: Direct chemiluminescent assay of cTnI

[0076] (1) Antibody labeling

[0077] Add an appropriate amount of activated acridinium ester and 100 μg anti-cTnI monoclonal antibody solution to the phosphate buffer, mix well, react at room temperature for 3 hours, and add 1 mg glycine to block. Separation and purification by dialysis to obtain the acridinium ester-labeled cTnI antibody.

[0078] Add 1 mg of magnetic particles (1 μm in diameter), 20 μg of EDC, 20 μg of NHS solution and 30 μg of streptavidin to 1 ml of 10 mM pH7.4 phosphate buffer, mix well and react at room temperature for 4 h, add 1 mg of glycine to block. Enrichment by magnet adsorption to remove unreacted streptavidin to obtain magnetic particle-labeled streptavidin.

[0079] Add 20 μg of anti-cTnI monoclonal antibody to 10 μL of 0.25 mg / mL Sulfo-NHS-LC-biotin solution, and react for 1 h. Purify by ultrafiltration and centrifugation to remove unreacted biotin to obtain biotin-cTnI antibody.

...

Embodiment 3

[0090] Example 3: Magnetic Particle Size Screening

[0091] 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.

[0092] The particle size is 0.1 μm, 0.5 μm, 1.0 μm, 2.0 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.

[0093] The experimental results show that when the 0.1μm magnetic particles are combined with the 500 Gauss magnet, the minimum detection limit is 500pg / ml, the quantitative detection range is 0.5~50ng / ml, and the linear correlation coefficient R 2 >0.95; 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.

[0094] When 10μm magnetic particles and 30,0...

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Abstract

The present invention discloses a magnetic particle chemiluminescent microfluidic chip for quantitatively detecting troponin I 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 cTnI in whole blood samples by using magnetic particle chemiluminescence technology and microfluidic chip technology, and particularly discloses a magnetic particle chemiluminescence microparticle quantitative detection method for troponin I in whole blood. The fluid control chip can realize accurate and highly sensitive quantitative detection of cTnI 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 l...

Claims

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Application Information

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