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Homogeneous chemiluminiscence detection method and application thereof

A homogeneous chemiluminescence, detection method technology, applied in chemical instruments and methods, chemiluminescence/bioluminescence, analysis by chemical reaction of materials, etc. Label signal detection and other problems, to achieve the effect of eliminating matrix effects, low cost, and fast response

Pending Publication Date: 2020-09-15
上海索昕生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although homogeneous immunoassays do not require separation and washing steps, some substances in serum or plasma can interfere with label signal detection or interfere with chemiluminescent reactions
At the same time, in the field of chemiluminescence in the homogeneous labeling immunoassay technology, some involve oxidation-reduction reactions. For example, some oxidation-reduction drugs (such as vitamin C) in blood samples also interfere with the generation process of light signals, seriously affecting the detection results. the accuracy of
In addition, each reaction of the current homogeneous immunoassay can only target one molecule to be detected, which is time-consuming and laborious

Method used

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  • Homogeneous chemiluminiscence detection method and application thereof
  • Homogeneous chemiluminiscence detection method and application thereof
  • Homogeneous chemiluminiscence detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0145] Example 1: Preparation of a chemiluminescent microarray chip for simultaneous detection of cTnI, CK-MB, MYO, and NT-proBNP.

[0146] 1. Modification of the surface of the solid phase support.

[0147] On the glass chip with 4×4 layout, there are 16 reaction areas, and the surface of each reaction area is modified with groups that can be connected with biotin molecules.

[0148] (1) Prepare 20mg / mL biotin solution with DMSO,

[0149] (2) with 0.1M NaHCO 3 Solution Dilute the biotin solution to 1 mg / ml.

[0150] (3) Soak the modified glass chip in the above biotin solution, and let it stand at 2-8° C. for 12-16 hours to react.

[0151] (4) Take out the glass chip and use 0.1M NaHCO 3 The solution was washed to obtain a solid phase support (glass chip) whose surface was modified with biotin.

[0152] 2. Preparation of donor microspheres coated with avidin

[0153] 1) Preparation of donor microspheres

[0154] (1) Prepare a 25 mL round bottom flask, add 0.1 g of copp...

Embodiment 2

[0180] Example 2: Preparation of receptor microspheres coated with antibody II

[0181] 1) Preparation of acceptor microspheres

[0182] (1) Prepare a 25mL round-bottomed flask, add 0.1g of europium(III) complex, 10mL of 95% ethanol, stir magnetically, and raise the temperature of the water bath to 70°C to obtain a solution of europium(III) complex.

[0183] (2) Prepare a 100mL three-necked flask, add 10mL of 95% ethanol, 10mL of water and 10mL of polystyrene microspheres coated with carboxydextran hydrogel with a particle size of 10% and a particle size of 200nm, stir magnetically, and heat up the water bath to 70°C.

[0184] (3) Slowly add the europium(III) complex solution in step 1 into the three-necked flask in step 2, react at 70°C for 2 hours, stop stirring, and cool naturally to obtain an emulsion.

[0185] (4) Centrifuge the above emulsion for 1 hour at 30,000 g, discard the supernatant after centrifugation, and then resuspend with 50% ethanol. After repeated centr...

Embodiment 3

[0192] Example 3: Using the method of the present invention to simultaneously detect cTnI, CK-MB, MYO, and NT-proBNP in the analytes.

[0193] (1) Take 15ul of the 4 samples and spot them on columns 1, 2, 3, and 4 shown in Table 1.

[0194] (2) Dilute the receptor microspheres coated with cTnI antibody Ⅱ, CK-MB antibody Ⅱ, MYO antibody Ⅱ, and NT-proBNP antibody Ⅱ to 50ug / ml with buffer, respectively, and take 15ul samples and place them in Table 1. Incubate for 10 minutes at 37°C for rows A, B, C, and D shown.

[0195] (3) The glass chip is irradiated with excitation light with a wavelength of 680nm, and the donor microspheres are induced to activate and release active oxygen in a high-energy state. The active oxygen in the high-energy state is captured by the acceptor microspheres at a short distance, thereby transferring energy to activate the luminescent compound in the acceptor microspheres. After a few microseconds, the luminescent compound in the acceptor microsphere w...

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Abstract

The invention relates to a homogeneous chemiluminiscence detection method and application thereof in the field of chemiluminiscence analysis. The method comprises the following steps: S1, enabling a liquid-phase reactant containing a first microsphere in chemiluminescence pairing microspheres and a to-be-detected object suspected to contain a to-be-detected target molecule to be in contact reaction with the surface of a solid-phase support, wherein the surface of the solid-phase support is provided with a corresponding second microsphere in the chemiluminescence pairing microspheres; S2, exciting one microsphere in the chemiluminescence pairing microspheres to generate active oxygen by utilizing energy or an active compound, and reacting the other microsphere in the chemiluminescence pairing microspheres with the active oxygen contacted with the microsphere to generate a chemiluminescence signal; and S3, detecting the intensity of the chemiluminescence signal in the step S2, and analyzing whether a to-be-detected target molecule and / or the concentration of the to-be-detected target molecule exist / exists in the to-be-detected object. The method can simultaneously detect a pluralityof to-be-detected molecules, is high in reaction speed, and effectively eliminates the matrix effect in homogeneous immunoassay.

Description

technical field [0001] The invention belongs to the field of chemiluminescence analysis, and in particular relates to a homogeneous chemiluminescence detection method and application thereof. Background technique [0002] Chemiluminescence is a specific chemical reaction in which organic molecules undergo energy level transitions after absorbing chemical energy, producing a high-energy intermediate with an unstable electronically excited state. When it returns to the ground state and emits photons, it is chemiluminescence. The immunoassay technology formed by combining chemiluminescence with antigen and antibody is chemiluminescence immunoassay. Chemiluminescence Immunoassay (CLIA) is a non-radioactive immunoassay developed very rapidly in the world in the past ten years. It has the advantages of high sensitivity, wide detection range, simple and fast operation, good stability of markers, no pollution, simple and economical instrument, etc. It is a substitute for radioimmu...

Claims

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Application Information

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IPC IPC(8): G01N21/76G01N33/53G01N33/536B01L3/00
CPCG01N21/76G01N33/53G01N33/536B01L3/5027B01L2200/10B01L2300/0861B01L2300/12G01N2800/32
Inventor 刘宇卉杨阳李临
Owner 上海索昕生物科技有限公司
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