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Fluorescence sensing method and kit for simultaneously detecting cortisol, serum testosterone and creatine kinase isoenzyme

A serum testosterone and creatine kinase technology, applied in biological testing, biochemical equipment and methods, measuring devices, etc., can solve the problems of inability to fully reflect the state of the human body, detection of a single target, long detection time in the sample pretreatment process, etc. problems, achieve the effect of low cost, short response time and high sensitivity

Active Publication Date: 2021-12-17
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 widely used in clinical diagnosis and treatment, but they often require complex sample pretreatment processes and professional technical operations. Personnel and longer testing time (5-6 hours)
In addition, these detection methods can only be used to detect a single target, but cannot fully reflect the physical state of the human body

Method used

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  • Fluorescence sensing method and kit for simultaneously detecting cortisol, serum testosterone and creatine kinase isoenzyme
  • Fluorescence sensing method and kit for simultaneously detecting cortisol, serum testosterone and creatine kinase isoenzyme
  • Fluorescence sensing method and kit for simultaneously detecting cortisol, serum testosterone and creatine kinase isoenzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Exploration of the optimal experimental conditions for the complementary strand-responsive DNA tetrahedral tweezers fluorescent sensing method, including the following steps:

[0059] 1) Optimization of DNA tetrahedral tweezers incubation time: Mix the four DNA tetrahedral tweezers chains with each other, shake and mix, incubate at 95°C for 5 minutes, and then plot the fluorescence intensity changes of DNA tetrahedral tweezers under different time conditions, and plot the time-fluorescence intensity Curve.

[0060] 2) Complementary strand-DNA tetrahedral tweezer response time optimization: Mix the four DNA tetrahedral tweezer strands with each other and oscillate to mix. Prepare DNA tetrahedral tweezers under the optimized DNA tetrahedral tweezer incubation time. The complementary strand was then added at a concentration of 1 μM. Detect the change of its fluorescence value at different concentrations, and draw the time-fluorescence value change curve.

[0061] 3) Dos...

Embodiment 2

[0065] The complementary strand-responsive DNA tetrahedral tweezers fluorescent sensing detection method for detecting cortisol, serum testosterone, and creatine kinase isoenzyme detection specifically includes the following steps:

[0066] 1) Pretreatment of human serum samples: take the inactivated human serum samples and dilute them 30 times with TM buffer as the sample detection matrix. Add cortisol, serum testosterone, and creatine kinase isoenzyme to the diluted serum sample to make the final concentration 10nM, 100nM, 500nM, as the sample solution to be detected.

[0067] 2) Preparation of magnetic beads-aptamer-complementary chain: put streptavidinized magnetic beads (50 μL, 10 mg / μL) on a shaker, shake and mix for 20 seconds, and resuspend the magnetic beads. Take 100 μL streptavidin-coated magnetic beads in a 1.5ml centrifuge tube, let stand on the magnetic stand for 2min, and remove the supernatant. Add 1ml of PBS reagent to the centrifuge tube, shake to resuspend ...

Embodiment 3

[0079] 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. Serum testosterone, cortisol, and creatine kinase isoenzymes of different concentrations were added to the diluted serum samples to make the final concentrations 10nM, 100nM, and 500nM to obtain actual samples. To test the feasibility of the designed method in the actual blood sample detection.

[0080] 2) Measure the actual sample according to steps 1)-7) of Example 2, and substitute the value obtained in step 7) into the measured standard curve to complete the DNA tetrahedral tweezers fluorescence recovery reaction.

[0081] 3) Measure the fluorescence spectrum of the reaction solution in 2) respectively, taking the fluorescence values ​​at 522, 602, and 667 nm as detection standards. The content of biomarkers cortisol, serum testosterone, creatine kinase isoenzyme was calculated. The results are shown in Table 1.

[0082] The r...

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Abstract

The invention discloses a fluorescence sensing method and kit for simultaneously detecting cortisol, serum testosterone and creatine kinase isoenzyme, and belongs to the field of biomarker detection. The fluorescence sensing method comprises the following steps: (1), preparing a magnetic bead-aptamer-complementary chain compound; (2), preparing DNA tetrahedral tweezers; (3), performing competition between the target object and the complementary chain; (4), carrying out EXPAR amplification reaction; (5), carrying out complementary chain-DNA tweezers reaction; and (6), performing reading: measuring the fluorescence value of the reaction system. The EXPAR isothermal amplification technology is utilized, a complex temperature change process is not needed, stability is good, sensitivity is high, compared with other detection methods, the content of three target substances including testosterone, cortisol and creatine kinase isoenzyme in serum can be detected at the same time, the content of the target substances is detected according to the fluorescence recovery degree, cost is low. And the detection limits can respectively reach 41pM, 68pM and 8pM.

Description

technical field [0001] The invention relates to the field of biomarker detection, in particular to a fluorescent sensing method and kit for simultaneously detecting cortisol, serum testosterone and creatine kinase isoenzymes. Background technique [0002] Excessive physical labor and mental stress often induce a variety of diseases, and even endanger human health in severe cases, and are widely valued by people. Under normal circumstances, the ratio of cortisol / serum testosterone in the human body is about 3, and when physiological changes such as fatigue occur in the human body, the level of cortisol increases and the level of serum testosterone decreases, and the corresponding ratio increases accordingly; in addition, normal 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 normal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/74G01N33/573C12Q1/6844
CPCG01N33/743G01N33/573C12Q1/6844G01N2333/9123C12Q2521/307C12Q2563/107
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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