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A magnetic particle chemiluminescence microfluidic chip for quantitative detection of c-reactive protein

A microfluidic chip and reactive protein technology, applied in the field of clinical medicine in vitro diagnosis, can solve the problems of high non-specific background, complex detection process, low sensitivity, etc., to reduce non-specific interference, high degree of integration, accurate and reliable results Effect

Active Publication Date: 2017-10-31
SHENZHEN HUAMAIXINGWEI MEDICAL TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent (CN101769932) provides a full-range CRP detection kit by using styrene latex with different particle sizes, which can detect both low-concentration CRP and high-concentration CRP. This method will not affect the accuracy of detection due to the HOOK effect , but the operation is complex and the detection time is long
The microfluidic chip system has the characteristics of fast, efficient, and integrated. The combination of the two will become a new type of high-performance detection method to solve the problems of low sensitivity, complicated detection process, and difficulty in achieving trace amounts in current detection methods. The problem of sample detection is expected to further promote the development of clinical testing instruments towards portability and miniaturization
[0006] The biological microfluidic chip of immune magnetic beads is an analysis and detection method that integrates magnetic particle technology and immune analysis on the microfluidic chip. At present, the main difficulties of this comprehensive detection method are as follows: For the intelligent control of internal micro-flow, the method commonly used at present is to set multiple micro-pumps and micro-valves inside the chip, which makes the micro-fluidic system more complicated; 2) Insufficient mixing of the reaction system leads to insufficient reaction; 3) The degree of integration is not high, resulting in high non-specific background

Method used

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  • A magnetic particle chemiluminescence microfluidic chip for quantitative detection of c-reactive protein
  • A magnetic particle chemiluminescence microfluidic chip for quantitative detection of c-reactive protein
  • A magnetic particle chemiluminescence microfluidic chip for quantitative detection of c-reactive protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1: horseradish peroxidase-luminol (HRP-luminol) system is used for the detection of C-reactive protein

[0059] 1. Fabrication of microfluidic chip

[0060] 1) Antibody labeling: i) Enzyme-labeled antibody: Weigh 25 mg of HRP and dissolve it in 1.25% glutaraldehyde solution, and let it stand overnight at room temperature; the reacted enzyme solution is eluted with Sephadex G-25 chromatography column and normal saline , the flow rate was controlled at 1ml / min, and the brown effluent was collected; 12.5mg of the anti-C-reactive protein antibody to be diluted to 5ml with normal saline, and added dropwise to the HRP solution while stirring; 0.25ml of 1M pH9.5 carbonate buffer solution, continued Stir for 3 hours; add 0.25ml of 0.2M lysine, mix well, and let stand at room temperature for 2 hours; add an equal volume of saturated ammonium sulfate drop by drop under stirring, and place at 4°C for 1 hour; centrifuge at 3000rpm for half an hour, discard the supernatan...

Embodiment 2

[0069] Embodiment 2: Alkaline phosphatase-adamantane (ALP-AMPPD) system is used for the detection of C-reactive protein

[0070] 1. Fabrication of microfluidic chip

[0071]1) Antibody labeling: i) Enzyme-labeled antibody: 2.5mg ALP (50IU / mg), add 200uL 100mM PB (pH6.8) containing 1.25% glutaraldehyde, mix well, and react overnight at room temperature; Electromagnetic stirring, dialyzed to 50mM PBS (pH7.2), 12 hours, change medium 4 times; 1.5mg C-reactive protein antibody was dissolved in 100uL 1M carbonate solution (pH9.0); activated AP was added to the prepared In protein liquid, mix well, react at 4°C for 24 hours, add 10 μL of 200mM lysine solution, mix well, react at 22°C for 2 hours; dialyze to 50mM PBS (pH7.2) at 4°C, 12 hours, change the medium 4 times; centrifuge, take the supernatant, wash with 50mM TB pH7.4+0.6%BSA+0.05%NaN 3 Dilute 10 times and store at -20°C. ii) Magnetic-labeled antibody: accurately pipette 30 μL of streptavidin-labeled magnetic particles wit...

Embodiment 3

[0079] Example 3: Magnetic Particle Size Screening

[0080] The particle size of magnetic microspheres is small, the specific surface area is large, and the surface contains active groups, so the coupling capacity is large, but the size of magnetic particles is too small to be conducive to magnet collection, so the magnetic particle size screening is carried out.

[0081] Refer to Example 2 for other experimental conditions, and the particle size of the magnetic particles is determined according to the following scheme.

[0082] Magnetic particle sizes of 0.1 μm, 0.5 μm, 1.6 μm, 2 μm, 3 μm, and 10 μm were selected to label the anti-C-reactive protein antibody. The permanent magnet whose magnetic size has been optimized is used in the detection to fix the height of the magnet.

[0083] The experimental results are as follows:

[0084] The particle size of magnetic particles increases sequentially from 0.1μm, 0.5μm, 1.6μm, 2μm, and 3μm. The interference increases at 3μm, and d...

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Abstract

The invention discloses a magnetic particle chemiluminescence microfluidic chip for quantitative detection of C-reactive protein. , the sample liquid flow channel (6), the first biomarker storage pool (4), the micro-mixer (7), and the transition zone (10) are connected in sequence; the filter (12) on the bottom sheet (11), the reaction pool ( 13), the cleaning pool (14), the detection pool (15), and the solution release channel (18) are connected in sequence, and the detection pool (15) on the film (11) is connected to the cleaning solution storage pool (16) and the cleaning solution storage pool (16) through the solution release channel (18). The luminescent liquid storage tank (17) is connected.

Description

technical field [0001] The invention relates to the field of in vitro diagnosis of clinical medicine, in particular to a magnetic particle chemiluminescence microfluidic chip for quantitative detection of C-reactive protein, which can realize quantitative detection of C-reactive protein in biological samples in a short time, and has the advantages of simple operation and high sensitivity. High, low cost and other characteristics. technical background [0002] C-reactive protein (C-reaction protein, CRP) often appears in the serum of patients with acute inflammation, is synthesized by stem cells, and is a non-specific indicator of systemic inflammatory response. The content of CRP increases significantly during infection. During the acute inflammatory response, CRP is one of the most sensitive proteins in the body and is an accurate objective indicator of the body's inflammatory response. CRP is one of the independent risk factors of coronary heart disease and has important ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01L3/00G01N33/68
Inventor 范玉霞李泉
Owner SHENZHEN HUAMAIXINGWEI MEDICAL TECH CO LTD