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A rolling circle amplification-gold tetrahedron colorimetric detection method and kit for detecting creatine kinase isoenzymes

A detection kit and rolling circle amplification technology, 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 low cost, high sensitivity and good stability. Effect

Active Publication Date: 2022-06-21
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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  • A rolling circle amplification-gold tetrahedron colorimetric detection method and kit for detecting creatine kinase isoenzymes
  • A rolling circle amplification-gold tetrahedron colorimetric detection method and kit for detecting creatine kinase isoenzymes
  • A rolling circle amplification-gold tetrahedron colorimetric detection method and kit for detecting creatine kinase isoenzymes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0052] 1) Pretreatment of human serum samples: Take human serum samples and dilute them by 30 times with TM buffer as a sample detection matrix. The creatine kinase isoenzyme was added to the diluted serum sample to make the final concentration of 10nM as the target solution to be detected.

[0053] 2) Preparation of magnetic bead-aptamer-complementary strand: take 100 μL of MNP-SA into 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 soluti...

Embodiment 2

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

[0063] Table 1

[0064]

Embodiment 3

[0066] 1) Pretreatment of human serum samples: Take human serum samples and dilute them by 30 times with TM buffer 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 a final concentration of 500 nM. The above-mentioned interfering substances were added to the diluted serum samples, and CK-MB was added to make the final concentration of 50 nM 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 is completed.

[0068] 3) Measure the ultraviolet absorption spectrum of the reaction solution in 2) 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 a kit for detecting creatine kinase isoenzymes. The method comprises the following steps: S1. preparing magnetic bead-adapter-complementary chain complex; S2. preparing DNA-AuNPs complex; S3. preparing AuNPs tetrahedron; S4. preparing rolling circle amplification template; S5. creatine kinase Competitive reaction between enzyme and complementary strand; S6. Rolling circle amplification; S7. Gold tetrahedron aggregation color reaction; S8. Readout. The invention utilizes the isothermal amplification technology, does not need complicated temperature changing process, has good stability and high sensitivity. Compared with other detection methods, the colorimetric method of the present invention does not require large-scale instrument detection, and the detection results can be obtained by imaginJ software, the cost is low, and the detection limit can reach 0.05pM, which is suitable for on-site screening and rapid detection process.

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 kit for detecting creatine kinase isoenzymes. Background technique [0002] Excessive physical work often induces a variety of diseases. Myocardial infarction has the characteristics of high morbidity and mortality, and has received extensive attention. 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 abnormality occurs within 4-6 hours, and the content increases to It is ten times higher than normal and can be detected at an early stage, making creatine kinase isoenzyme a reliable and effective biomarker for detecting abnormality in the body. Therefore, the timely, rapid and sensitive detection of CK-MB is of great significance for preventing myocardial infarction an...

Claims

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Application Information

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Patent Type & Authority Patents(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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