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Photocatalytic visual immunoassay method for on-line labeling of photosensitizer

An immune analysis and photosensitizer technology, applied in the field of biological analysis, can solve the problems of expensive reagents, low sensitivity, and unstable biological enzymes, and achieve the effects of good stability, high sensitivity, and cost reduction

Inactive Publication Date: 2018-02-16
CHENGDU UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Radioimmunoassay has high sensitivity, strong specificity, and mature technology, but it has radioactive pollution and expensive reagents; fluorescent immunoassay uses fluorescein instead of radioactive elements for labeling, which has low sensitivity and is difficult to use for the determination of trace proteins; The resolution fluorescent immunoassay method has high sensitivity, but the instrument is expensive; enzyme-linked immunoassay uses biological enzymes instead of radioactive elements, and utilizes the biomagnification of enzyme-catalyzed substrates to achieve high-sensitivity analysis. It is currently the most commonly used protein quantitative analysis in clinical practice. method, but the biological enzyme is unstable and the enzyme-labeled antibody or antigen is expensive, and the test cost is high
In order to avoid the use of unstable biological enzymes (such as horseradish peroxidase HRP), many scientists have used nanoparticles to mimic enzymes instead of biological enzymes, but they also face problems such as labeling and uneven particle sizes.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Add 100 µL of CEA antibody at a concentration of 0.5mg / L to a 300 µL detachable microtiter plate and incubate overnight for about 12 hours to absorb the antibody on the microtiter plate; wash the microtiter plate lightly with ultrapure water 4 times, 20 Wash twice with mM pH 7.2 sodium dihydrogen phosphate buffer; add 20% BSA to block the remaining sites, pour out the liquid in the well after incubation for 2 h, wash the microplate with ultrapure water 4 times, and wash with 20 mM pH 7.2 sodium dihydrogen phosphate Wash with buffer twice; add carcinoembryonic antigen (CEA) standard solution or sample solution, pour out the liquid in the well after incubation for 2 h, wash lightly with ultrapure water four times, wash twice with 100mM pH 7.2 sodium dihydrogen phosphate buffer; add 500 nM extended CEA aptamer chain (final concentration), incubated for 2 h, then poured out the liquid in the well, gently washed 4 times with ultrapure water, washed 2 times with 20 mM pH 7.2 s...

Embodiment 2

[0018] Add 100 µL of vascular endothelial growth factor (VEGF) antibody at a concentration of 0.5 mg / L to a 300 µL detachable microtiter plate and incubate overnight for about 12 hours to absorb the antibody on the microtiter plate; lightly wash the enzyme with ultrapure water Mark the plate 4 times, wash 2 times with 20 mM pH 7.2 sodium dihydrogen phosphate buffer; add 20% BSA to block the remaining sites, incubate for 2 h, pour out the liquid in the well, and wash the microtiter plate 4 times with ultrapure water, 20 Wash twice with mM pH 7.2 sodium dihydrogen phosphate buffer; add vascular endothelial growth factor (VEGF) standard solution or sample solution, incubate for 2 h, pour out the liquid in the well, wash lightly with ultrapure water 4 times, 100mM pH 7.2 diphosphate Wash 2 times with sodium hydrogen buffer; add 500 nM extended VEGF aptamer chain (final concentration), incubate for 2 h, then pour out the liquid in the well, gently wash 4 times with ultrapure water, ...

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PUM

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Abstract

The invention relates to a photocatalytic visual immunoassay method for on-line labeling of a photosensitizer. The method utilizes the special performance that graphene can adsorb nucleic acid and photosensitizers simultaneously to achieve on-line labeling of a photosensitizer on a nucleic acid aptamer in an immunoassay process, under the irradiation of an LED lamp, the photosensitizer generates singlet oxygen to oxidize colorless TMB into blue, thus realizing visual semiquantitative analysis, and at the same time realizing quantitative analysis through spectrophotometry. The method avoids useof bio-enzyme and has good stability, has no need for labelling the used antibody or aptamer, and has the low cost advantage, and can detect carcino-embryonic antigens low to 0.1 ng mL<-1>, vascularendothelial growth factors and other protein. The method can greatly reduce the immunoassay cost of hospitals, and is also suitable for on-site diagnosis of diseases in remote areas short of equipment.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, in particular to a method for quantitative analysis of proteins in biological samples. Background technique [0002] Immunoassay is an analytical method that uses antigen-antibody specific binding reactions to detect various analytes, and is widely used in the quantitative analysis of various proteins, especially disease marker proteins. Simple and highly sensitive immunoassay methods are of great significance for clinical diagnosis. According to the classification of detection methods, immunoassay methods mainly include radioimmunoassay, fluorescence immunoassay, and enzyme-linked immunoassay. Radioimmunoassay has high sensitivity, strong specificity, and mature technology, but it has radioactive pollution and expensive reagents; fluorescent immunoassay uses fluorescein instead of radioactive elements for labeling, which has low sensitivity and is difficult to use for the determinat...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/533
Inventor 张信凤吴鹏侯贤灯邓莉黄承鹏
Owner CHENGDU UNIVERSITY OF TECHNOLOGY