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Immunochromatographic quantitative detection reagent based on near-infrared fluorescent nanosphere markers

An immunochromatographic test strip and fluorescent nanotechnology, applied in measurement devices, analytical materials, instruments, etc., can solve the problems of reducing detection sensitivity, interfere with detection signals, etc., and achieve the effect of shortening the completion time of chromatography

Active Publication Date: 2015-11-25
BEIJING RUNBO FUDE BIOLOGICAL TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because biological proteins, nucleic acids, etc. will produce fluorescence under the excitation of ultraviolet light, the use of fluorescent markers in the visible light region will produce strong background fluorescence, which will interfere with the detection signal and reduce the detection sensitivity.

Method used

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  • Immunochromatographic quantitative detection reagent based on near-infrared fluorescent nanosphere markers
  • Immunochromatographic quantitative detection reagent based on near-infrared fluorescent nanosphere markers
  • Immunochromatographic quantitative detection reagent based on near-infrared fluorescent nanosphere markers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Preparation method of near-infrared fluorescent nanospheres

[0048] In this application, the following two methods are mainly used to prepare near-infrared fluorescent nanospheres:

[0049] Method 1: The fluorescent dye is connected to the nanosphere through the intermediate molecule lysine (Lys) or BSA through the amino-carboxyl condensation reaction, and then the detection molecule is connected to the nanosphere through the condensation reaction.

[0050] Follow the steps below to label nanospheres with near-infrared fluorescent dyes and connect antibodies:

[0051] Draw 1ml0.01mMNaACpH5.0 into the glass test tube;

[0052] Add 25 μl of 10% carboxyl nanospheres (ACME microspheres, USA) and mix well;

[0053] Add 10mg carbodiimide (EDAC) and mix well;

[0054] Add 100μl 100mg / mlLys (or 10mg / mlBSA) and mix well;

[0055] Add 3.5 μl Dylight800 near-infrared fluorescent dye (ThermoScientific, USA), mix well, and shake at room temperature for 1 hour;

[00...

Embodiment 2

[0068] Embodiment 2: make near-infrared fluorescent nanometer microsphere hemoglobin antigen immunochromatography detection reagent

[0069] 1) Label the antibody.

[0070] After hemoglobin monoclonal antibody (Zhuhai Bomei Biotechnology Co., Ltd.) and chicken IgY antibody (Abcam, UK) were dialyzed with PBS, the antibody was labeled according to method 1 or 2 in Example 1. Store at 4°C.

[0071] 2) Make bonding pads

[0072] Select glass cellulose tape as the solid-phase material of the binding pad, spray hemoglobin monoclonal antibody and chicken IgY antibody labeled with near-infrared fluorescent nanospheres on the tape, and air-dry the tape at room temperature.

[0073] 3) Make the sample pad

[0074] Select the cellulose membrane strip, spray the blocking solution (5% BSA, 0.1% Tween20, PBS) on the membrane strip, and let it dry at room temperature before use.

[0075] 4) Making immunochromatographic test strips

[0076] Select nitrocellulose membrane, use hemoglobin ...

Embodiment 3

[0079] Embodiment 3 detects hemoglobin sample

[0080] Serially dilute the hemoglobin antigen standard (Zhuhai Bomei Biotechnology Co., Ltd.), use the diluent (5%BSA, 0.1%Tween20, PBS) to make 2-fold serial dilutions of the standard to make 2μg / ml, 1μg / ml, 500ng / ml, 250ng / ml, 125ng / ml, 62.5ng / ml, 31.3 / ml, 15.6ng / ml, 7.8ng / ml, 3.9ng / ml, 1.9ng / ml samples. Use a micro-sampler to draw more than 50 μl of the sample and drop it on the sample pad. After the sample is absorbed, add 50 μl of the sample diluent dropwise with a dropper. After standing at room temperature for 10 minutes, put the sample card into a portable high-sensitivity near-infrared spot fluorescence scanner (Beijing Runboford Technology Development Co., Ltd.) for reading. Divide the fluorescence value of the detection line by the fluorescence value of the quality control line to measure the value. Each sample concentration was measured twice, and after taking the average value, the measured value was plotted again...

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Abstract

The invention relates to a method based on near infrared fluorescence molecules and nano particle marks as well as an immunochromatography quantitative detection reagent based on near infrared fluorescence nanoparticles. According to the invention, infrared fluorescence is connected with nanoparticles to prepare an immunochromatography test strip based on the near infrared fluorescence nanoparticles. During the detection, an infrared light scanner is utilized, and a quality control line and a sample line are respectively scanned by utilizing near infrared light; and after the fluorescence intensity of a detection line is rectified by utilizing the fluorescence intensity of a quality control line, and a standard curve in a fluorescence analyzer is taken in, the concentration of a to-be-detected matter in a sample can be analyzed and detected. Compared with the direct connection of the near infrared fluorescence molecules and detecting molecules, the near infrared fluorescence nanoparticle immunochromatography improves the detection flexibility obviously, and the lowers the background fluorescence intensity. The marking method and the reagent can be applied to microorganism detection, food safety detection, poison detection as well as rapid dangerous chemical product detection.

Description

technical field [0001] The invention establishes an immunochromatographic quantitative detection reagent based on near-infrared fluorescent nanometer microsphere markers, including a preparation method of near-infrared fluorescent nanometer microsphere test strips and corresponding reagents. The present invention uses near-infrared fluorescent nanometer microspheres as markers, adopts immunochromatography technology to prepare near-infrared fluorescent immunochromatography test strips, and then forms a detection card including sample pad / glass fiber membrane / nitrocellulose membrane and absorbent paper, Wherein, a test line and a quality control line are fixed on the nitrocellulose membrane. In the detection process, the near-infrared light is used to scan the quality control line and the sample line respectively, and the fluorescent molecules are excited to emit light. The emitted fluorescence is converted into a digital signal by a filter and a photomultiplier tube, and the d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/558
Inventor 李志刚陈唯军
Owner BEIJING RUNBO FUDE BIOLOGICAL TECH DEV
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