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Application of pyrene-1-carboxaldehyde and detection method of biological small molecules

A technology of small biological molecules and detection methods, which is applied in the detection of small biological molecules and the application field of 1-pyrene formaldehyde, can solve problems such as difficult simultaneous qualitative and quantitative analysis, detection structure influence, complex system, etc. High chemical efficiency, low purchase cost, and highly sensitive quantitative detection effect

Active Publication Date: 2021-02-05
JILIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The existing analysis methods for small biomolecules Cys and Hcy are mainly fluorescent methods, but due to their similar structures, after the combination of fluorescent groups, the fluorescence emission peaks of the two usually interfere with each other, which affects the detection structure , so it is difficult for existing fluorescence methods to simultaneously perform both qualitative and quantitative analysis; and in complex biological systems, false positive results may also appear due to the complexity of the system
In addition, the traditional MALDI detection method has limitations in the detection of small molecule compounds, so it is very important to establish a fast, high-throughput quantitative analysis method for Cys and Hcy

Method used

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  • Application of pyrene-1-carboxaldehyde and detection method of biological small molecules

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Embodiment 1

[0047] This embodiment provides 1-pyrene formaldehyde as the optimal condition test method for label derivatization of small biomolecules Cys and Hcy, which includes the following steps:

[0048] S1. Use PBS buffer solution to prepare Cys and Hcy stock solutions with a concentration of 5mM respectively, and store them in a refrigerator at 4°C for later use;

[0049] S2. Dissolving 1-pyreneformaldehyde in methanol to prepare a 1-pyreneformaldehyde solution with a concentration of 5mM.

[0050] S3. Take 500 μL of 1-pyrene formaldehyde solution in a 1.5 mL centrifuge tube, then add 250 μL of Cys and Hcy stock solutions obtained in step S1, and react to obtain a reaction solution.

[0051] S4, using an ultraviolet spectrophotometer to measure the absorbance of the reaction solution.

[0052] As a comparison: the pH of the PBS buffer solution in step S1 is 7, 7.5 and 8 respectively, and the test results are as follows Figure 1~2 As shown, it can be seen from the figure that the ...

Embodiment 2

[0056] This embodiment provides the method that 1-pyrene formaldehyde is used as label detection biological small molecule Cys and Hcy in MALDI mass spectrometry, and it comprises the following steps:

[0057] S1. Cys and Hcy stock solutions with a concentration of 5 mM were prepared respectively using PBS buffer solution with pH=7.5, and stored in a refrigerator at 4° C. for future use.

[0058] S2. Dissolving 1-pyreneformaldehyde in methanol to prepare a 1-pyreneformaldehyde solution with a concentration of 5mM.

[0059] S3. Take 500 μL of 1-pyrene formaldehyde solution in a 1.5 mL centrifuge tube, then add 250 μL of the stock solution obtained in step S1, and react at room temperature for 1 hour to obtain a reaction solution.

[0060] S4, the substrate 2,5-dihydroxybenzaldehyde (DHB) is dissolved in the mixed solution of ammonium dihydrogen phosphate (ADP) and acetonitrile (ACN) with a volume ratio of 7:3, and the substrate solution whose concentration of DHB is 10g / L is pr...

Embodiment 3

[0066] This embodiment provides a method for measuring the detection limit of Cys and Hcy when 1-pyrene formaldehyde is used as a label, which includes the following steps:

[0067] S1. Dilute the stock solutions of Cys and Hcy in Example 2 to concentrations of 1 mM, 0.5 mM, 0.05 mM, 0.005 mM and 0.000000005 mM respectively according to the concentration gradient, and set aside.

[0068] S2. Dissolving 1-pyreneformaldehyde in methanol to prepare a 1-pyreneformaldehyde solution with a concentration of 5mM.

[0069] S3. Take five 1.5mL centrifuge tubes numbered 1, 2, 3, 4, and 5, add 500 μL of 1-pyrene formaldehyde solution, and then add 500 μL of Cys and Hcy solutions of different concentrations in step S1, and react at room temperature for 1 hour , to obtain the reaction solution.

[0070] S4, the substrate 2,5-dihydroxybenzaldehyde (DHB) is dissolved in the mixed solution of ammonium dihydrogen phosphate (ADP) and acetonitrile (ACN) with a volume ratio of 7:3, and the substr...

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Abstract

The invention is applicable to the technical field of biology, and provides application of pyrene-1-carboxaldehyde and a detection method of biological small molecules. Pyrene-1-carboxaldehyde is taken as a label, and can selectively react with cysteine and homocysteine, so that the molecular weight of an analyte is enlarged, the analyte is far away from a severely interfered low-quality region, meanwhile, the ionization efficiency of a to-be-detected substance is improved, so that simultaneous, rapid and high-sensitivity quantitative detection of cysteine and homocysteine is realized, and thelimitation that when matrix-assisted laser desorption ionization time-of-flight mass spectrometry is used for detection, due to interference of a small molecular matrix, biological small molecular compounds cysteine and homocysteine cannot be detected can be solved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an application of 1-pyrene formaldehyde and a detection method of small biological molecules. Background technique [0002] Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) has been widely used in proteomics, metabolomics and microbial analysis in clinic. It is a fast, sensitive, high-throughput analytical method. Its principle is to absorb laser energy through a special organic small molecule compound as a matrix, carry out protonization transfer, and then ionize the analyte, so as to achieve the purpose of detection. This unique working principle leads to signal and strong matrix signal peaks in the low-mass region of the detection spectrum, which makes it difficult to detect small molecule compounds in traditional MALDI detection. [0003] Small biomolecules are a class of compounds that play important roles in living organisms. They play imp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/06G01N30/72C07C47/546
CPCC07C47/546G01N30/06G01N30/72
Inventor 国新华郭黎明
Owner JILIN UNIV
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