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Novel method for detecting activity of hydrogen sulfide synthetase by using hydrogen sulfide fluorescence probe and application of method

A technology for fluorescent probes and detection methods, applied in fluorescence/phosphorescence, material excitation analysis, etc., can solve problems such as cumbersome steps, and achieve the effects of good repeatability, good stability, and high quantum yield

Inactive Publication Date: 2014-03-12
蔡典其 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The traditional method for detecting the activity of hydrogen sulfide synthase is the reaction bottle-methylene blue method, but the step of lysing tissue cells by repeated freezing and thawing in this method is particularly cumbersome, and the detection of H in the reaction bottle absorption system 2 S content is also not as intuitive, simple and sensitive as the fluorescence imaging method of the present invention

Method used

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  • Novel method for detecting activity of hydrogen sulfide synthetase by using hydrogen sulfide fluorescence probe and application of method
  • Novel method for detecting activity of hydrogen sulfide synthetase by using hydrogen sulfide fluorescence probe and application of method
  • Novel method for detecting activity of hydrogen sulfide synthetase by using hydrogen sulfide fluorescence probe and application of method

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Embodiment 1

[0059] Embodiment 1: Novel absorption system detects the H released by the NaHS donor 2 S content

[0060] It has been shown in the literature that HS in NaHS solution - / H2S ratio is 3:1, so most of the NaHS solution is HS - . The control group added a certain volume of PBS to the reaction pool, and the experimental group added the same volume of different concentrations of NaHS donors to the reaction pool. 2 S fluorescent probe working solution), shake lightly for 1 hour at 37°C, use the reaction bottle-fluorescent probe method to detect the fluorescence intensity, the fluorescence intensity can reflect the H in the absorption solution 2 S content. The result is as figure 2 It was shown that the fluorescence intensity of the high concentration group (600 μmol / L) was greater than that of the low concentration group (200 μmol / L), and the fluorescence intensity of the experimental group was significantly greater than that of the control group.

Embodiment 2

[0061] Example 2: Different absorption times, H 2 The S fluorescent probe detects the H released from the NaHS donor 2 S content

[0062] The control group added a certain volume of PBS to the reaction pool, and the experimental group added the same volume of NaHS donors with different concentrations to the reaction pool. 2 S fluorescent probe working solution), at 37°C, shake gently for different times, and use the reaction bottle-fluorescent probe method to detect the fluorescence intensity, which can reflect the H in the absorption solution 2 S content. The result is as image 3 It shows that the fluorescence intensity of the 600μmol / L NaHS group is greater than that of the 200μmol / L NaHS group, and the fluorescence intensity of the experimental group is significantly greater than that of the control group. In the experimental group, with the same NaHS concentration, the fluorescence intensity of the 1h group is slightly greater than that of the 1 / 2h group. ** P## P<0.0...

Embodiment 3

[0063] Example 3: H 2 The S fluorescent probe directly detects the H released from the NaHS donor 2 S content

[0064] Prepare different concentrations of NaHS donors, add the same H 2 S fluorescent probe, using a full-wavelength scanning multifunctional reader (Varioskan Flash3001) (maximum excitation wavelength λex(max)=476nm, maximum emission wavelength λem(max)=513nm). The result is as Figure 4 display, with the H 2 As the concentration of S increased, the fluorescence intensity increased correspondingly in a dose-dependent manner. ** P##P<0.01 compared with 0 μmol / L NaHS group.

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Abstract

The invention belongs to the technical field of biological detection and relates to a novel method for detecting the activity of hydrogen sulfide synthetase by using a hydrogen sulfide fluorescence probe as well as an application approach of the method. The method is suitable for detection of the activity of hydrogen sulfide synthetase such as CBS, CSE and CL, and comprises the following steps: shearing a tissue sample into small blocks, grinding, performing cell lysis in a tissue cell lysis solution, performing protein quantification on a lysis product, as the drawings, adding a reaction system (comprising the tissue cell lysis product, L-cysteine and 5-pyridoxal phosphate) into a reaction pool on an outer ring of a reaction bottle, and adding an absorption system (comprising operating fluid of the H2S fluorescence probe containing a reaction adjuvant cetrimonium bromide) into an absorption pool on an inner ring of the reaction bottle, wherein H2S gas molecules generated in the reaction system are combined with the fluorescence probe in the absorption system, the system can be used for fluorescence assay and fluorescence imaging, the H2S content is quantitatively detected, and the activity of the H2S synthetase is intuitively reflected. The invention can provide a good method for research of H2S-related signal paths and significance and diagnosis of H2S in disease processes.

Description

1. Technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a new method for detecting the activity of hydrogen sulfide synthase with a hydrogen sulfide fluorescent probe and an application thereof. This method of detecting enzyme activity with fluorescent probes can be used to study the mechanism of some important enzymatic processes in tissue cells, clarify some important signaling pathways in biological systems, and provide good assistance for the early diagnosis and prevention of some major diseases means. 2. Background technology [0002] For a long time, hydrogen sulfide (H 2 S) is considered a toxic gas, however, with the 2 With the in-depth study of S, it is found that some tissue cells in the body can produce H 2 S, it is the third endogenous gas signal molecule discovered after nitric oxide (NO) and carbon monoxide (CO). More and more experiments show that H 2 S is not only an endogenous gas sign...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 蔡典其杨春涛俞晓立江伟炽郑洁蓉
Owner 蔡典其
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