Multi-functional quick fluorescence immunoassay test method using functional substrate as media and using single color and multi-color quantum dots as mark

A fluorescent immune and functionalized technology, applied in fluorescence/phosphorescence, measurement devices, instruments, etc., can solve the problems of cumbersome operation, time-consuming, harmful to operators, etc., and achieve the effect of intuitive visual effect, rich fluorescence characteristics, and improved sensitivity.

Inactive Publication Date: 2013-06-12
HENAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These traditional methods have their own shortcomings: radioimmunoassay, enzyme-linked immunoassay, time-resolved fluorescence immunoassay and chemiluminescence immunoassay all have the problems of cumbersome operation, long time-consuming and expensive detection equipment, which are harmful to the operators and the working environment. Certain requirements; the luminescence time of the chemiluminescence method is short, and repeated measurements of the same sample cannot be realized; the markers of the radioimmunoassay method are harmful to the operator and have poor environmental friendliness, which limits its general promotion and grass-roots use; although colloidal gold technology is simple Fast but can only achieve semi-quantitative, can not be used for compound detection with higher sensitivity and multiple antigens

Method used

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  • Multi-functional quick fluorescence immunoassay test method using functional substrate as media and using single color and multi-color quantum dots as mark
  • Multi-functional quick fluorescence immunoassay test method using functional substrate as media and using single color and multi-color quantum dots as mark
  • Multi-functional quick fluorescence immunoassay test method using functional substrate as media and using single color and multi-color quantum dots as mark

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Taking HCG monoclonal antibody as an example, prepare red fluorescent quantum dot-labeled antibody: take 100 μl of red water-soluble fluorescent quantum dots (emission wavelength is 620nm) modified with carboxyl groups in 900 μl MES buffer solution (0.1mol / L, pH= 4.7) Dissolve evenly in the medium, add EDC 2mmol and Sulfo-NHS 5mmol respectively, react at 20°C for 40min, then centrifuge at 10,000rpm for 90min to remove the supernatant, and dissolve the red fluorescent quantum dot solution in the lower layer in 1ml of boric acid buffer solution (0.1 mol / L, pH=8.5), and centrifuged again at 15000rpm for 30min, then discarded the supernatant, dispersed the red quantum dot solution in the lower layer into 1ml PBS buffer solution (0.01mol / L, pH=7.4) and added 0.1 mg of HCG monoclonal antibody for labeling, and react at 20°C to 25°C for 3 hours. After the reaction, add 1ml of 1% BSA solution and incubate at 37°C for 30min, centrifuge at 15,000rpm for 60min to disperse the red ...

Embodiment 2

[0029] Taking the AFP monoclonal antibody as an example, prepare the antibody labeled with green fluorescent quantum dots: take 100 μl of green water-soluble fluorescent quantum dots (emission wavelength is 540nm) modified with carboxyl groups in 900 μl MES buffer solution (0.1mol / L, pH= 4.7) Dissolve evenly in the medium, add EDC 2mmol and Sulfo-NHS 5mmol respectively, react at 20°C for 40min, then centrifuge at 40,000rpm for 40min to remove the supernatant, and dissolve the green fluorescent quantum dot solution in the lower layer in 1ml of boric acid buffer solution (0.1 mol / L, pH=8.5), and centrifuge again at 50000rpm for 30min, then discard the supernatant, disperse the green quantum dot solution in the lower layer into 1ml PBS buffer solution (0.01mol / L, pH=7.4) and add 0.1 mg AFP monoclonal antibody for labeling, react at 20°C to 25°C for 3 hours. After the reaction, add 1ml of 1% BSA solution and incubate at 37°C for 30min, centrifuge at 50,000rpm for 30min to disperse...

Embodiment 3

[0031] Taking HCG monoclonal antibody as an example, prepare HCG-coated antibody-immobilized microporous membrane: place the surface carboxylated filter membrane in a clean glass bottle, add 1ml MES buffer solution (0.1mol / L, pH= 4.7), add 2mmol EDC and 5mmol Sulfo-NHS respectively and react at 20°C for 40min, then take out the membrane, rinse the residual reaction solution on the surface of the membrane with PBS buffer solution (0.01mol / L, pH=7.4), and put Place it in 1ml of PBS buffer solution (0.01mol / L, pH=7.4), add HCG-coated antibody for fixation at the same time, so that the final concentration of antibody in the solution is 10μg / ml, and react at 20℃~25℃ for 3 hours After the reaction, take out the filter membrane, rinse the remaining reaction solution on the surface of the membrane with PBS buffer solution (0.01mol / L, pH=7.4) again, add 1ml of 1% BSA solution and incubate at 37°C for 30 minutes, then remove the membrane The slices were washed three times with PBS buffe...

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Abstract

The invention relates to a novel, quick, high-sensitivity, qualitative and quantitative clinical test method which uses a chemically modified functional polymorphic substrate as media and combines inorganic fluorescent semiconductor nano materials (quantum dots) and immunology means. The multi-functional quick fluorescence immunoassay test method using a functional substrate as media and using single color and multi-color quantum dots as a mark is suitable for a single or compound qualitative and quantitative test of various hormone, protein, nucleic acid and pathogens. Compared with traditional enzyme-linked immunoassay and colloidal gold test paper, the multi-functional quick fluorescence immunoassay test method has the advantages of being simple, quick, high in sensitivity, long in storage time and the like. The multi-functional quick fluorescence immunoassay test method can meet requirements of different testing conditions and samples, can conduct the single or compound qualitative and quantitative test on different antigens on the same substrate media at the same time, and has great potential on aspects of clinical applications.

Description

technical field [0001] The present invention relates to an in vitro multifunctional compound immune system for various hormones, proteins, nucleic acids and pathogens, which uses a new functional substrate as a detection platform, uses multi-monochrome and color fluorescent quantum dots as labels, and combines immunological means. diagnosis method. Background technique [0002] Fluorescent quantum dots (Quantum Dots, hereinafter referred to as QDs) are II-VI and III-V semiconductor nanocrystal particles with a particle size smaller than or close to the exciton Bohr radius. Its excitation spectrum is wide and continuously distributed, its emission spectrum is narrow and symmetrical, its emission light stability is strong, and it is not easy to be photobleached. By changing the size and composition of the particles, the spectrum at any point from ultraviolet to near infrared can be obtained, so it is relatively traditional organic Fluorescent reagents have incomparable advant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N21/64
Inventor 李林松刘丹
Owner HENAN UNIVERSITY
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