Magnetic particle chemiluminescence microfluidic chip for quantitative detection of D-dimer

A microfluidic chip, quantitative detection technology, applied in chemical instruments and methods, measuring devices, scientific instruments, etc., can solve the problems of high non-specific background, low degree of integration, low sensitivity, etc., and reduce non-specific interference. , Improve the efficiency of immune response, the results are accurate and reliable

Active Publication Date: 2016-05-25
SHENZHEN HUAMAIXINGWEI MEDICAL TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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
[0007] 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 particle chemiluminescence microfluidic chip for quantitative detection of D-dimer
  • Magnetic particle chemiluminescence microfluidic chip for quantitative detection of D-dimer
  • Magnetic particle chemiluminescence microfluidic chip for quantitative detection of D-dimer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1: horseradish peroxidase-luminol (HRP-luminol) system is used for the detection of D-dimer

[0062] 1. Fabrication of microfluidic chip

[0063] 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 D-dimer antibody to be labeled to 5ml with normal saline, add dropwise to the HRP solution while stirring; use 0.25ml of 1M pH9.5 carbonate buffer, continue Stir for 3 hours; add 0.25ml of 0.2M lysine, mix well, and place 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 0.15MpH7.4; Use Naphtha...

Embodiment 2

[0071] Embodiment 2: alkaline phosphatase-adamantane (ALP-AMPPD) system is used for the detection of D-dimer

[0072] 1. Fabrication of microfluidic chip

[0073] 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; dissolve 1.5mgD-dimer antibody in 100uL 1M carbonate solution (pH9.0); add the activated AP to the prepared protein liquid In medium, mix well, react at 4°C for 24 hours, add 10 μL of 200 mM lysine solution, mix well, react at 22°C for 2 hours; dialyze to 50 mMPBS (pH7.2) at 4°C for 12 hours, change the medium 4 times; centrifuge, take the supernatant, wash with 50mMTB7.4+0.6%BSA+0.05%NaN 3 Dilute 10 times and store at -20°C. ii) Magnetically labeled antibody: accurately pipette 30 μl of streptavidin-labeled magnetic particles with a concentration of 2 mg / ml, wher...

Embodiment 3

[0081] Example 3: Screening of Magnetic Particle Size

[0082] 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.

[0083] Refer to Example 2 for other experimental conditions, and the particle size of the magnetic particles is determined according to the following scheme.

[0084] 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.

[0085] The experimental results are as follows:

[0086] The particle size of magnetic particles increases sequentially from 0.1μm, 0.5μm, 1.6μm, 2μm, and 3μm. The interference increases at 3μm, an...

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Abstract

The invention discloses a magnetic particle chemiluminescence microfluidic chip for quantitative detection of a D-dimer. The structure of the chip mainly comprises a cover plate (1) and a base plate (11), wherein a sample introduction opening (2), a sample liquid flow channel (6), a first biomarker storage pool (4), a micro-mixer (7) and a transition zone (10) which are arranged on the cover plate (1) are sequentially connected; and a filter (12), a reaction pool (13), a cleaning pool (14), a detection pool (15) and a solution release channel (18) which are arranged on the base plate (11) are sequentially connected, and the detection pool (15) on the base plate (11) is connected with a cleaning liquid storage pool (16) and a luminescence 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 immunity, in particular to a magnetic particle chemiluminescent microfluidic chip for quantitative detection of D-dimer, which can realize quantitative detection of D-dimer in biological samples in a short time, and has the advantages of operation Simple, high sensitivity, low cost and so on. technical background [0002] The fibrinolysis system (fibrinolysis system) is the most important anticoagulant system of the human body, which consists of four main parts: plasminogen (plasminogen), plasminogen activator (plasminogenactivator, such as t-PA, u-PA), Plasmin (plasmin), plasmin inhibitor (plasminactivator inhibitor, PAI-1, antiplasmin). When a fibrin clot (fibrinclot) is formed, in the presence of tPA, plasminogen is activated and converted into plasmin, the process of fibrinolysis begins, and plasmin degrades the fibrin clot to form various soluble fragments, forming fibers Protein produc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01L3/00G01N33/543
Inventor 范玉霞李泉
Owner SHENZHEN HUAMAIXINGWEI MEDICAL TECH CO LTD
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