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A magnetic particle chemiluminescence microfluidic chip for quantitative detection of procalcitonin

A microfluidic chip, chemiluminescence technology, applied in chemical instruments and methods, measuring devices, scientific instruments, etc., can solve the problems of low integration, large consumption of reagents, complex detection process, etc., and improve immune response. Efficiency, accurate and reliable results, simple operation

Active Publication Date: 2017-10-17
SHENZHEN HUAMAIXINGWEI MEDICAL TECH CO LTD
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Problems solved by technology

Chinese patent (CN102359958 A) discloses a kit for detecting procalcitonin, which uses enzymatic chemiluminescence to achieve quantitative detection of procalcitonin. Compared with other traditional methods, this method improves the sensitivity and accuracy of detection , but requires complex pretreatment of the test sample, and requires the use of a large chemiluminescence detector, while requiring a large amount of reagent consumption and long detection time
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
[0008] 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 procalcitonin
  • A magnetic particle chemiluminescence microfluidic chip for quantitative detection of procalcitonin
  • A magnetic particle chemiluminescence microfluidic chip for quantitative detection of procalcitonin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: horseradish peroxidase-luminol (HRP-luminol) system is used for the detection of procalcitonin

[0063] 1. Fabrication of microfluidic chip

[0064] 1) Antibody labeling:

[0065] ii) Enzyme-labeled antibody: Weigh 25 mg of HRP, dissolve it in 1.25% glutaraldehyde solution, and let it stand overnight at room temperature; the reacted enzyme solution passes through a Sephadex G-25 chromatography column, and is eluted with normal saline, and the flow rate is controlled at 1ml / min, collect the brown effluent; dilute 12.5mg of PCT monoclonal antibody to 5ml with normal saline, add dropwise to the HRP solution under stirring; use 0.25ml of 1M pH9.5 carbonate buffer, continue to stir for 3 hours; add 0.2M lysine 0.25ml, after mixing, put it at room temperature for 2 hours; add an equal volume of saturated ammonium sulfate dropwise under stirring, and place it at 4°C for 1 hour; centrifuge at 3000rpm for half an hour, discard the supernatant; Wash twice with am...

Embodiment 2

[0073] Embodiment 2: Alkaline phosphatase-adamantane (ALP-AMPPD) system is used for the detection of procalcitonin

[0074] 1. Fabrication of microfluidic chip

[0075] 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 procalcitonin antibody was dissolved in 100uL 1M carbonate solution (pH 9.0); 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 TB7.4+0.6%BSA+0.05%NaN 3 Dilute to the concentration required for the test and store at -20°C. ii) Magnetically labeled antibody: Accurately pipette 50 μL of streptavidin-lab...

Embodiment 3

[0082] Example 3: Magnetic Particle Size Screening

[0083] 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.

[0084] Refer to Example 2 for other experimental conditions, and the particle size of the magnetic particles is determined according to the following scheme.

[0085] Magnetic particle sizes of 0.1 μm, 0.5 μm, 2 μm, 2.2 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.

[0086] The experimental results are as follows:

[0087] The particle size of magnetic particles increases sequentially from 0.1 μm, 0.5 μm, 2 μm, 2.2 μm, and 3 μm. The interference increases at 3 μm,...

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Abstract

The invention discloses a magnetic particulate chemiluminescent micro-fluidic chip for quantitatively detecting procalcitonin. The structure of the magnetic particulate chemiluminescent micro-fluidic chip mainly comprises a cover piece (1) and a bottom piece (11), wherein an air pump (3), an airflow micro-channel (5), a sample feeding hole (2), a sample liquid flowing channel (6), a first biological marker storage tank (4), a micro-mixer (7) and a transition region (10) on the cover piece (1) are connected in sequence; a filter (12), a reaction tank (13), a washing tank (14), a detection tank (15) and a solution releasing channel (18) on the bottom piece (11) are connected in sequence; the detection tank (15) is connected with a washing liquid storage tank (16) and a luminescent liquid storage tank (17) through the solution releasing channel (18).

Description

technical field [0001] The invention relates to the field of in vitro diagnosis of medical immunity, in particular to a magnetic particle chemiluminescence microfluidic chip for quantitative detection of procalcitonin, which can realize quantitative detection of procalcitonin in biological samples in a short time, and has the advantages of operation Simple, high sensitivity, low cost and so on. technical background [0002] Procalcitonin (PCT) is a small protein with 116 amino acid residues and a molecular weight of about 13 kDa. The amino acid sequence of PCT was first discovered in 1984 by Moullec et al. It belongs to a family of related proteins (CAPA peptide family) that includes calcitonin gene-related peptide I and II, amylin, adrenomedullin, and calcitonin. Like other peptides of the CAPA family, PCT is derived from a precursor molecule, the procalcitonin precursor. Procalcitonin precursor contains 141 amino acids, and its N-terminal removes 25 amino acid residues ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68B01L3/00
CPCB01L3/5027G01N33/6893
Inventor 范玉霞李泉
Owner SHENZHEN HUAMAIXINGWEI MEDICAL TECH CO LTD
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