A method for ultrasensitive detection of dopamine based on nucleic acid aptamer

A nucleic acid aptamer and sensitive detection technology, applied in the field of biomedicine, can solve the problems of large trauma, poor sensitivity, and complicated operation, and achieve the effects of reducing burden, low cost and high sensitivity

Active Publication Date: 2021-11-09
HEBEI MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a method for supersensitive detection of dopamine based on nucleic acid aptamers, so as to solve the problems of poor sensitivity, complicated operation and large trauma of the existing detection methods.

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  • A method for ultrasensitive detection of dopamine based on nucleic acid aptamer
  • A method for ultrasensitive detection of dopamine based on nucleic acid aptamer
  • A method for ultrasensitive detection of dopamine based on nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1 The drawing of standard curve

[0023] (a) Prepare dopamine standard solutions with concentrations of 0nM, 0.1nM, 0.3nM, 0.6nM, 0.9nM, 3.0nM, 4.0nM, 5.0nM and 10.0nM respectively.

[0024] (b) According to the concentration gradient of the dopamine solution, the detection test was performed in groups, and the test operation in each group was the same, specifically: 6 μL ssDNA1 (5 μM), 6 μL ssDNA2 (5 μM), 6 μL dopamine aptamer (5 μM ) was added to 230 μL Tris-HCl (10 mM) and incubated at room temperature for 5 hours; 20 μL of corresponding concentration of dopamine solution was added to react at room temperature for 1 hour, and 6 μL of hairpin DNA (15 μM) and 19 μL of SYBR Green Ⅰ (purchased from Sangon Bioengineering (Shanghai) Co., Ltd., diluted 1000 times with water) reacted at room temperature for 45 minutes; then added 70U exonuclease III (Exo-Ш) and incubated at 37°C for 1 hour (this was verified by polyacrylamide gel electrophoresis) Enzyme digestion...

Embodiment 2

[0028] Example 2 Detection of Dopamine Content in Mouse Brain Tissue

[0029] The mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium, and the anesthesia dose was 0.1ml / 10g. After complete anesthesia, the brain tissue was collected under low temperature conditions. Add tissue lysate to the brain tissue according to 1g:7.5mL, homogenize for 30s, centrifuge at low temperature (14000r / min, 4°C), take the supernatant and centrifuge again under the same conditions. Fluorescence detection was performed on the supernatant according to the detection steps in Example 1, and enzyme-linked immunosorbent assay was performed according to conventional methods. The test results are shown in Table 1. After statistical analysis, P=0.235>0.1, the difference between the two methods is not statistically significant, so it is considered that the two methods have good consistency.

[0030] Table 1:

[0031]

[0032]The sensitivity of the detection method of the prese...

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Abstract

The present invention provides a method for ultrasensitive detection of dopamine based on nucleic acid aptamers. First, ssDNA1 is combined with dopamine aptamers and ssDNA2 through the principle of complementary base pairing to form double-stranded DNA1 with two 3' protruding ends, wherein , ssDNA2 has fewer bases than ssDNA1 and is complementary to ssDNA1, and the dopamine aptamer starts from the 3' end of ssDNA2 to partially complement ssDNA1; then add the sample to be tested, hairpin DNA, fluorescent dye SYBR GreenⅠ, and exonucleic acid Enzyme III, wherein, the number of bases of the hairpin DNA is less than that of ssDNA1 and can form a double-stranded DNA2 with a recess at the 3' end of the hairpin DNA chain with the free ssDNA1 through the principle of complementary base pairing. In the present invention, the dopamine aptamer recognizes dopamine, so that the aptamer is detached from the double-stranded DNA1, thereby continuously triggering the enzyme cutting reaction, reducing the fluorescence signal, and realizing the supersensitive detection of dopamine by using the cyclic amplification technology.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for ultrasensitive detection of dopamine based on nucleic acid aptamers. Background technique [0002] Parkinson's disease is a common neurological dysfunction disease that mainly affects middle-aged and elderly people. It is the "third killer" of middle-aged and elderly people after tumors and cardiovascular and cerebrovascular diseases. Surprisingly, of the approximately 4.5 million Parkinson's disease patients worldwide, nearly half are in China. The main pathological change of Parkinson's disease is the degeneration and death of dopaminergic neurons in the substantia nigra of the midbrain, which leads to a significant decrease in the content of dopamine (DA) in the striatum. Therefore, the detection of neurotransmitters such as dopamine is of great significance for the protection of national health and the treatment and prevention of diseases. [0003] However, due to th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/94G01N33/533G01N21/64
CPCG01N21/64G01N21/6428G01N33/533G01N33/9413G01N2021/6417G01N2021/6439
Inventor 牛凌梅康维钧王艳仙
Owner HEBEI MEDICAL UNIVERSITY
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