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Magnetic micro-particle chemiluminescence micro-fluidic chip for quantitatively detecting C-reaction 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: 2015-12-30
SHENZHEN HUAMAIXINGWEI MEDICAL TECH CO LTD
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  • Abstract
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  • Application Information

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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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  • Magnetic micro-particle chemiluminescence micro-fluidic chip for quantitatively detecting C-reaction protein
  • Magnetic micro-particle chemiluminescence micro-fluidic chip for quantitatively detecting C-reaction protein
  • Magnetic micro-particle chemiluminescence micro-fluidic chip for quantitatively detecting C-reaction 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, dissolve it in 1.25% glutaraldehyde solution, and let it stand overnight at room temperature; Control at 1ml / min, collect the brown effluent; dilute 12.5mg of the anti-C-reactive protein antibody to 5ml with normal saline, add dropwise to the HRP solution under stirring; use 0.25ml of 1M pH9.5 carbonate buffer, and continue stirring 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 dropwise while stirring, and place at 4°C for 1 hour; centrifuge at 3000rpm for half an hour, and discard the supernatant. The precipitate was washed twice with half-saturated ammonium sulfate, and finally the precipitate was dissolved in a small amount of PBS of ...

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: 2.5mgALP (50IU / mg), add 200uL of 100mM PB (pH6.8) containing 1.25% glutaraldehyde, mix well, and react overnight at room temperature; Stir, dialyze to 50mMPBS (pH7.2), 12 hours, change the medium 4 times; 1.5mg C-reactive protein antibody is dissolved in 100uL 1M carbonate solution (pH9.0); add the activated AP to the prepared 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 at 4°C to 50mMPBS (pH7.2), 12 hours, change medium 4 times ; Centrifuge, take the supernatant, use 50mMTBpH7.4+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 with a concentration of 1 mg / L, where...

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 micro-particle chemiluminescence micro-fluidic chip for quantitatively detecting a C-reaction protein. The magnetic micro-particle chemiluminescence micro-fluidic chip structurally comprises a cover plate (1) and a bottom plate (11), wherein a sampling opening (2), a sample liquid flow channel (6), a first biomarker storage pool (4), a micro-mixer (7) and a transitional area (10) on the cover plate (1) are connected in sequence; a filter (12), a reaction pool (13), a cleaning pool (14), a detection pool (15) and a solution release channel (18) on the bottom plate (11) are connected in sequence; a detection pool (15) on the bottom plate (11) is connected with a cleaning liquid storage pool (16) and a luminous liquid storage pool (17) through the solution release channel (18).

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-reactionprotein, CRP) often appears in the serum of patients with acute inflammation. Due to cell synthesis, it 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 value in ...

Claims

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Application Information

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IPC IPC(8): B01L3/00G01N33/68
Inventor 范玉霞李泉
Owner SHENZHEN HUAMAIXINGWEI MEDICAL TECH CO LTD
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