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Rolling circle amplification-gold tetrahedron colorimetric detection method and kit for detecting creatine kinase isoenzyme

A technology of creatine kinase and detection method, applied in the field of protein detection, can solve the problems of high price, strict storage conditions, inability to detect creatine kinase isoenzyme, etc., and achieve the effects of low cost, high sensitivity and good stability

Active Publication Date: 2021-08-17
INST OF ENVIRONMENTAL MEDICINE & OCCUPATIONAL MEDICINE ACAD OF MILITARY MEDICINE ACAD OF MILITARY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional detection methods, such as enzyme-linked immunoassay (ELISA), radioimmunoassay, and enzyme activity method, are often used to detect the level of CK-MB in clinical diagnosis and treatment, but often require complex sample pretreatment processes , professional technical operators and long detection time (5-6 hours), and often limited by the laboratory environment, it is impossible to realize the timely and rapid detection of creatine kinase isozyme
In addition, although antibodies can specifically recognize creatine kinase isozymes, they are more expensive and require stricter storage conditions.

Method used

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  • Rolling circle amplification-gold tetrahedron colorimetric detection method and kit for detecting creatine kinase isoenzyme
  • Rolling circle amplification-gold tetrahedron colorimetric detection method and kit for detecting creatine kinase isoenzyme
  • Rolling circle amplification-gold tetrahedron colorimetric detection method and kit for detecting creatine kinase isoenzyme

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] This embodiment is used to illustrate the rolling circle amplification-gold tetrahedron colorimetric detection method for the detection of creatine kinase isozyme in human body, which specifically includes the following steps:

[0052] 1) Human serum sample pretreatment: take a human serum sample, dilute it 30 times with TM buffer, and use it as a sample detection matrix. Add creatine kinase isoenzyme to the diluted serum sample to make the final concentration 10nM, as the target solution to be detected.

[0053] 2) Preparation of magnetic beads-aptamer-complementary chain: Take 100 μL of MNP-SA in a centrifuge tube, separate with a magnetic stand for 10 min, and remove the supernatant. Add 100 μL PBS buffer and 100 μL biotinylated aptamer, shake and mix for 3 min, incubate at 30°C for 1 h, then magnetically separate the supernatant, wash three times with PBS buffer, and finally resuspend in PBS buffer.

[0054] 3) Preparation of DNA tetrahedral solution: prepare DNA-A...

Embodiment 2

[0062] The actual sample was measured according to steps 1)-8) of Example 1, and the value obtained in step 8) was substituted into the measured standard curve to calculate the content of the biomarker creatine kinase isoenzyme. The results are shown in Table 1.

[0063] Table 1

[0064]

Embodiment 3

[0066] 1) Human serum sample pretreatment: take a human serum sample, dilute it 30 times with TM buffer, and use it as a sample detection matrix. Cardiac troponin, c-reactive protein, heart-type fatty acid binding protein, and calcitonin were added to the diluted serum samples to make the final concentration 500nM. The above-mentioned interfering substances were added to the diluted serum samples, and CK-MB was added to make the final concentration 50nM to obtain a mixed sample Mixture. The above 5 groups of test samples were prepared to test the specificity of the designed method.

[0067] 2) According to steps 2)-7) of Example 1, the color reaction of rolling circle amplification was completed.

[0068] 3) Measure 2) the ultraviolet absorption spectrum of the reaction solution respectively, and take A650 / A520 as an index for testing the specificity of the reaction. The result is as image 3 shown.

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Abstract

The invention belongs to the field of protein detection, and relates to a rolling circle amplification-gold tetrahedron colorimetric detection method and kit for detecting creatine kinase isoenzyme. The detection method comprises the following steps: S1, preparing a magnetic bead-aptamer-complementary chain compound; S2, preparing a DNA-AuNPs compound; S3, preparing an AuNPs tetrahedron; S4, preparing a rolling circle amplification template; S5, carrying out competitive reaction on creatine kinase isoenzyme and a complementary chain; S6, performing rolling circle amplification; S7, carrying out aggregation chromogenic reaction on the gold tetrahedron; and S8, conducting reading. According to the invention, an isothermal amplification technology is utilized, a complicated temperature change process is not needed, stability is relatively good, and sensitivity is relatively high. Compared with other detection methods, the colorimetric method disclosed by the invention does not need a large instrument for detection, can obtain a detection result through imaginJ software, is relatively low in cost, has a detection limit of 0.05 pM, and is suitable for on-site screening and rapid detection processes.

Description

technical field [0001] The invention belongs to the field of protein detection, and in particular relates to a rolling circle amplification-gold tetrahedron colorimetric detection method and a kit for detecting creatine kinase isoenzymes. Background technique [0002] Excessive physical labor often induces various diseases. Myocardial infarction has the characteristics of high morbidity and mortality, and has been widely valued by people. Under normal conditions, the content of creatine kinase isoenzyme (CK-MB) in human blood is 1-20ng / ml, but it will increase immediately after myocardial infarction, and abnormalities will appear within 4-6 hours, and the content will increase to Ten times the normal condition, it can be detected in the early stage of disease, which makes creatine kinase isoenzyme a reliable and effective biomarker for detecting abnormalities in the body. Therefore, timely, rapid and sensitive detection of CK-MB is of great significance for the prevention ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844C12Q1/50G01N21/31
CPCC12Q1/6844C12Q1/485G01N21/31G01N2333/9123C12Q2525/205C12Q2531/125C12Q2563/137C12Q2565/519C12Q2563/143C12Q2563/149C12Q2521/501C12Q2521/319
Inventor 高志贤王瑜彭媛李双韩殿鹏任舒悦秦康韩铁张靖扬
Owner INST OF ENVIRONMENTAL MEDICINE & OCCUPATIONAL MEDICINE ACAD OF MILITARY MEDICINE ACAD OF MILITARY SCI
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