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Multi-marker liquid chip and preparation method thereof

A solution and compound technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of reducing the content of target substances, unstable uniformity of microsphere functional groups, reducing the reactivity of activating groups, etc., and achieves easy operation , Prevent aggregation and adhesion, and the effect of small sample volume

Inactive Publication Date: 2021-04-16
UB BIOTECHNOLOGY ZHEJIANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the prior art, it is very easy to bond between microspheres, such as commonly used polystyrene microspheres, because of their small particle size, large specific surface area, strong adsorption, easy modification and modification, etc., they can be used in flow fluorescence However, because of the strong adsorption of polystyrene microspheres, it is easy to form large particle clusters, and it will also reduce the content of target substances marked on the microspheres.
[0005] The activation of microsphere functional groups usually requires the use of ester condensation reaction reagents such as NHS and EDC. Since group activation itself is an unstable process, various chemical potential energies increase, and the pH value and salt concentration of the environment will reduce the activation of the active group. The subsequent reactivity of the group, at this time, the uniformity of the functional groups of the microspheres will be unstable

Method used

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  • Multi-marker liquid chip and preparation method thereof
  • Multi-marker liquid chip and preparation method thereof
  • Multi-marker liquid chip and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] This example provides a method for preparing a primary antibody-microsphere complex:

[0047] 1. Primary antibody desalting treatment

[0048] S11: Use PBS buffer to wash the desalting column, centrifuge at 1500g for 1 min, remove the buffer, and repeat the washing operation twice;

[0049] S12: Add the primary antibody and centrifuge at 1500g for 2 minutes to obtain the desalted primary antibody.

[0050] 2. Microsphere activation treatment

[0051] S21: Take 2 mg of microspheres, use MES buffer for gradient washing, fully mix with a vortex mixer, separate and remove the supernatant, and repeat the washing step twice to obtain microspheres after washing;

[0052] S22: Mix 38 μL NHS and 19 μL EDC with the washed microspheres, and rotate them in a vortex mixer for 1 hour to obtain activated microspheres to be cleaned; the concentration of NHS and EDC is 50 mg / ml;

[0053] S23: Using the ligation buffer to wash the activated microspheres to be washed three times repeat...

Embodiment 2

[0061] This embodiment provides a method for preparing a multiple marker liquid chip:

[0062] 1. The primary antibody-microsphere complex was prepared by the preparation method provided in Example 1.

[0063] 2. Secondary Antibody Desalting Treatment

[0064] S41: wash the desalting column with PBS buffer, centrifuge at 1500g for 1 min, remove the buffer, and repeat the washing operation twice;

[0065] S42: Add secondary antibody, centrifuge at 1500 g for 2 min to obtain desalted secondary antibody.

[0066] 3. Cross-linking of Desalted Secondary Antibody with Biotin

[0067] S51: dissolving 5 mg of NHS-biotin in 100 μL of DMSO to obtain an NHS-biotin solution;

[0068] S52: Take 15 μL of NHS-biotin solution and 780 ug of desalted secondary antibody for 5 hours at room temperature and shake in the dark to obtain a secondary antibody-biotin complex.

[0069] In the preparation method provided by Example 1 and Example 2, MES buffer solution and ligation buffer were used fo...

Embodiment 3

[0071] This embodiment provides a multiple marker liquid chip prepared by the method provided in Example 2.

[0072] Using the liquid chip provided in this example to carry out intra-batch and inter-batch experiments, the steps are as follows:

[0073] 1. Take the primary antibody-microsphere complex out of the refrigerator, return to room temperature, vortex to mix, and pipette 20 μL into 96-well plate.

[0074] 2. Add 10 μL of clinical serum sample and 50 μL of diluent to each well. Shake and react at room temperature for 2 hours in the dark.

[0075] 3. Add 200 μL of washing solution to each well, shake for 30 seconds, magnetically adsorb for 1 minute, and absorb the supernatant.

[0076] 4. Add 50 μL of secondary antibody-biotin complex to each well. Shake and react at room temperature for 2 hours in the dark.

[0077] 5. Add 50 μL of PE-SA, shake and react at room temperature for 30 minutes in the dark.

[0078] 6. Add 200 μL of washing solution to each well, shake f...

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Abstract

The invention discloses a multi-marker liquid chip and a preparation method thereof. The liquid chip comprises a primary antibody microsphere compound and a secondary antibody biotin compound. The preparation method of the liquid chip comprises the following four parts: desalting treatment of the primary antibody and the secondary antibody, activation of the microspheres, crosslinking of the activated microspheres and the desalted primary antibody, and crosslinking of biotin and the desalted secondary antibody, wherein a surfactant treatment is added to the operating steps involving the microspheres to obtain uniformly dispersed microspheres. The liquid chip microsphere provided by the invention has high dispersity, is not easy to aggregate and bond, and improves the detection accuracy of the liquid core.

Description

technical field [0001] The invention relates to the technical field of biology and new medicine, in particular to a multi-marker liquid chip and a method for preparing the multi-marker liquid chip. Background technique [0002] Liquid chip technology, also known as suspended lattice technology, is mainly led by the liquid chip developed by Luminex Corporation of the United States. It combines coded microspheres, laser technology, applied fluidics, the latest high-speed digital signal processors and computer algorithms. With the advantages of high throughput, high speed, low cost, high sensitivity, high precision, wide linear range, and easy operation, it is widely used in immunoassay, nucleic acid analysis, enzymatic analysis, receptor and ligand recognition, etc. . [0003] The realization principle of the liquid chip (Luminex xMap) technology is to encode polystyrene microspheres with fluorescent dyes and adjust the ratio of two different fluorescent dyes to obtain up to ...

Claims

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Application Information

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IPC IPC(8): G01N33/543G01N33/532
Inventor 李国平严崇虎刘正权
Owner UB BIOTECHNOLOGY ZHEJIANG CO LTD
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