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Protein chip, preparation method and usage

A protein chip and small-molecule protein technology, applied in the field of protein chips, can solve the problem that there is no protein chip for detecting papaverine residues, etc., and achieve the effects of improving the inability to perform high-throughput detection, and improving the cumbersome and rapid detection of pretreatment processes.

Inactive Publication Date: 2005-06-15
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] So far, there is no report on the use of protein chips for the detection of small molecule pesticides such as atrazine, parathion, and papaverine residues

Method used

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  • Protein chip, preparation method and usage
  • Protein chip, preparation method and usage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Preparation of small molecule protein conjugates

[0055] Material:

[0056] Atrazine (purity ≥ 99.7%), produced by Shandong Pesticide Factory; rabbit anti-atrazine: Biodesign; N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS), Aldrich Chemical, Co.; dicyclohexyl carbon Dicyclohexylcarbodiimide (DCC): Aldrich Chemical, Co.; 3-Mercaptopropionic acid: Fluka Chemie; OVA, Sephadex G-25: Huamei Bioengineering Company; Thion crude oil (80%): Tianjin Pesticide Co., Ltd.; rabbit anti-parathion: Biodesign; papaverine amino derivatives, rabbit anti-papaverine: Jiangsu Institute of Microbiology; EDC (1-ethyl-3-(3 -dimethylaminopropyl)carbodiimidehydrochloride): Aldrich Chemical, Co.; Sephadex G-50: Sigma; Cy3 monofunctional dye: Amersham Pharmacia Biotech; Cy5 bisfunctional dye:: Amersham Pharmacia Biotech;

[0057] 1. Preparation of atrazine-ovalbumin (OVA) conjugate

[0058] (1) Dissolve 1.01g of atrazine solid powder with a purity of 99.7% in 100mL o...

Embodiment 2

[0088] Example 2. Labeling and Identification of Atrazine, Parathion Antigen and Papaverine Antigen

[0089] 1. Configuration of some solutions

[0090]Phosphate buffer solution: Solution A (0.2mol / L NaH2PO4): Weigh 31.2g of NaH2PO4 2H2O, add double distilled water to fully dissolve and set the volume to 1000mL; solution B (0.2mol / L Na2HPO4): weigh Na2HPO4 12H2O71. Add 632g of double-distilled water to fully dissolve and set the volume to 1000mL; the phosphate buffer solution of each molar concentration and pH value required in the experiment is prepared by mixing the above-mentioned A and B solutions in different proportions.

[0091] Carbonate buffer solution: Accurately weigh 21.2g of anhydrous sodium carbonate (Na2CO3) and 16.8g of sodium bicarbonate (NaHCO3), add 1000mL of pure water to make a 0.2mol / L solution, then add 0.2mol / L Na2CO3 and 0.2mol / L NaHCO3 is mixed in different proportions as the buffer solution with the required pH value.

[0092] Preparation of 2...

Embodiment 3

[0125] Example 3 Detection of Small Molecule Pesticides and Papaverine Residues by Protein Chip

[0126] 1. Take the protein chip prepared in the above example, mix the analyte and a certain amount of Cy3-labeled competitive reporter molecule in equal amounts, take 20 μl of the mixed solution and add it dropwise to the reaction grid, and place it at 37°C under saturated humidity After 2 hours, rinse with lotion for 5 times, each time for 10s, and rinse with distilled water for 3 times. After drying, use a scanner for fluorescence measurement.

[0127] 2. Signal fluorescence detection and data processing

[0128] After the antigen-antibody reaction or antigen competition reaction is completed, it is detected with a laser confocal scanner GenePixTM4000B with a scanning resolution of 10 μm. The excitation wavelength is 530nm, and the detection wavelength is 585nm. Fluorescent signal quantification was analyzed using GenePix Pro3.0 software.

[0129] 3. Results

[0130]...

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Abstract

This invention discloses a protein chip, its preparation method and its usage. The chip is composed of processed carriers and small molecule protein conjugates, among which, the carrier surface is processed by aldehyde group reagent, pesticide and papaverine anti-body molecular are fixed on the carrier with a covalent bond by double-function crosslinker. This invented protein chip is prepared by preparing small molecule protein conjugates, aminating the carrier glass carrying chip, crosslinking double function reagenrt, fixing and packaging anti-body, useing in testing residuals of pesticide and papaverine.

Description

technical field [0001] The invention relates to a protein chip, in particular to a protein chip capable of detecting pesticide and papaverine residues, and also relates to its preparation method and application. Background technique [0002] Pesticides are widely used because of their important role in resisting pests and diseases and increasing crop yields. With the extensive use of pesticides, their residues and degradation products have polluted water sources and soil, and the resulting environmental problems have also aroused people's attention. In addition, through bioaccumulation, they can also remain in agricultural products and pose a threat to human health. [0003] The detection of pesticide residues in water, soil and other environments and food, as well as other toxic and harmful substances, plays an important role in our better assessment and control of pollution, and the maintenance of human health and ecological balance. With the rapid development of modern s...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N33/533G01N33/552
Inventor 高志贤王艳王升启戴树桂
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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