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Preparation method of fluorescent/ultraviolet molecular probe for alpha-ketoglutarate and its application in biological samples

A technology of ketoglutaric acid and molecular probes, applied in the field of analytical chemistry, to achieve high accuracy, avoid addition, and reduce background interference

Inactive Publication Date: 2018-01-30
QUFU NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this technology and application are not involved in the methods reported by the predecessors.

Method used

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  • Preparation method of fluorescent/ultraviolet molecular probe for alpha-ketoglutarate and its application in biological samples
  • Preparation method of fluorescent/ultraviolet molecular probe for alpha-ketoglutarate and its application in biological samples
  • Preparation method of fluorescent/ultraviolet molecular probe for alpha-ketoglutarate and its application in biological samples

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1: Preparation of fluorescent / ultraviolet molecular probes for alpha-ketoglutarate

[0078] Dissolve 0.3g of NBD-Cl into 50mL of chloroform, then add 50mL of hydrazine hydrate-methanol solution with a volume concentration of 0.6%, mix well, and stir at room temperature for 30min to obtain a brown precipitate, filter, wash the filter cake with ethyl acetate, and dry , to obtain 0.29g of a brown product, which is a fluorescent / ultraviolet molecular probe for alpha-ketoglutarate (probe HNBD), with a yield of 97%.

[0079] The fluorescent / ultraviolet molecular probe effect judgment index of the prepared alpha-ketoglutarate is as follows:

[0080] Detection sensitivity: The detection limit is 0.9µmol / L; the fluorescence enhancement factor is 20-60 times;

[0081] Absorption wavelength changes: Absorption shifts 100 nm toward red light during detection;

[0082] Dual-channel color change: the color changes from light green to red under fluorescent light; the color c...

Embodiment 2

[0087] Example 2: Detection of alpha-ketoglutarate content in serum (using the fluorescent / ultraviolet molecular probe for alpha-ketoglutarate prepared in Example 1)

[0088] Serum samples from cancer patients (esophageal cancer, right breast cancer, right colon cancer, and rectal cancer) were added to alpha-ketoglutarate standards to make alpha-ketoglutarate serum models (100 µM) as test objects:

[0089] Prepare the solution

[0090] Preparation of probe HNBD stock solution: Accurately weigh the fluorescent / ultraviolet molecular probe of alpha-ketoglutaric acid and dissolve it in anhydrous acetonitrile to prepare a concentration of 2×10 -4 Probe HNBD stock solution of M;

[0091] Preparation of alpha-ketoglutaric acid (α-KA) stock solution of the target to be tested: Accurately weigh alpha-ketoglutarate, the target to be tested, and dissolve it in anhydrous acetonitrile to prepare a concentration of 1×10 -3 alpha-ketoglutarate stock solution of M;

[0092] Preparation o...

Embodiment 3

[0102] Example 3: Visual monitoring of alpha-ketoglutarate in serum samples (samples are the same as in Example 2)

[0103] The method is: mix the serum sample to be tested with anhydrous acetonitrile at a volume ratio of 5:1, centrifuge at 5000 rpm for 20 min, take the supernatant and pass it through a 0.2 µm membrane, take 200 µL of the obtained supernatant and add 100 µL of the probe HNBD reserve solution and 200 µL of alpha-ketoglutarate stock solution, then dilute to 1000 µL with PBS buffer solution of pH 7.4, store the resulting solution at 37°C for 60 min, and observe and image under a fluorescent microscope. The colors are: green under ultraviolet light, It is light red under the fluorescent lamp, which proves that it contains alpha-ketoglutarate.

[0104]The preparation method of the probe HNBD stock solution and the alpha-ketoglutarate stock solution is the same as in Example 2.

[0105] Depend on Figure 7 For blank + HNBD samples, it can be seen that there is no ...

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Abstract

The invention relates to a preparation method of an alpha-oxoglutarate fluorescent / ultraviolet molecular probe and application of the alpha-oxoglutarate fluorescent / ultraviolet molecular probe to biological samples. The preparation method includes the steps that NBD-Cl is dissolved in chloroform, the dissolution concentration is 0002-0.012 g / ml, then a hydrazine hydrate-methanol solution with the volume concentration being 0.2-1.2% is added in and uniformly mixed, brown precipitate is obtained through stirring at room temperature and filtered, a filter cake is washed through ethyl acetate and dried, and the brown product alpha-oxoglutarate fluorescent / ultraviolet molecular probe is obtained. The alpha-oxoglutarate fluorescent / ultraviolet molecular probe is applicable to qualitatively and quantitatively analyzing alpha-oxoglutarate in the biological samples, and detection is sensitive, accurate and fast. The biological samples mainly comprise serum, living cells, muscle tissue and the like. The alpha-oxoglutarate fluorescent / ultraviolet molecular probe can be applied to analytical chemistry, life organic analysis, disease pre-diagnosis, medical clinical inspection and other related fields. The alpha-oxoglutarate fluorescent / ultraviolet molecular probe has the advantages of being good in response performance and high in data accuracy, repeatability and precision, equipment is convenient, fast and easy to operate, operability is high, and the alpha-oxoglutarate fluorescent / ultraviolet molecular probe is particularly suitable for large-batch sample combined screening and other large-data research.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, and relates to a method for preparing a fluorescent / ultraviolet molecular probe of alpha-ketoglutarate and its application in biological samples. Background technique [0002] At present, the analytical chemistry methods for alpha-ketoglutarate mainly include gas phase (GC), high performance liquid chromatography (HPLC) and mass spectrometry (MS), ultraviolet detector (UV-detector), etc. However, the number of fluorescent molecular probe detection methods is very small, including the use of nitrobenzothiadiazole hydrazine DT and hydrazino-substituted naphthalimide fluorescent molecular probes for the detection of alpha-ketoglutadiene acid monitoring. [0003] Chromatographic detection methods have been applied in many fields, but they are not the best choice for rapid batch detection of clinical medical biological samples. The reasons are as follows: firstly, the technical steps are complex,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D271/12G01N21/33G01N21/64
CPCC07D271/12G01N21/33G01N21/6428
Inventor 陈光周伟赵晨阳付强王亦琳李秀刘玉霞王桦张书圣尤进茂
Owner QUFU NORMAL UNIV
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