Preparation method of alpha-oxoglutarate fluorescent/ultraviolet molecular probe and application of alpha-oxoglutarate fluorescent/ultraviolet molecular probe to biological samples

A ketoglutaric acid and molecular probe technology, applied in the field of analytical chemistry, to achieve the effect of reducing error sources, sensitive detection, and reducing background interference

Inactive Publication Date: 2016-04-20
QUFU NORMAL UNIV
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  • 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.

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  • Preparation method of alpha-oxoglutarate fluorescent/ultraviolet molecular probe and application of alpha-oxoglutarate fluorescent/ultraviolet molecular probe to biological samples
  • Preparation method of alpha-oxoglutarate fluorescent/ultraviolet molecular probe and application of alpha-oxoglutarate fluorescent/ultraviolet molecular probe to biological samples
  • Preparation method of alpha-oxoglutarate fluorescent/ultraviolet molecular probe and application of alpha-oxoglutarate fluorescent/ultraviolet molecular probe to 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, which is filtered, and the filter cake is washed with ethyl acetate and dried. 0.29 g of a brown product was obtained, which was a fluorescent / ultraviolet molecular probe for alpha-ketoglutarate (probe HNBD), and the yield was 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 0.9 μmol / L; the fluorescence enhancement factor is 20-60 times;

[0081] Absorption wavelength changes: Absorption moves 100nm to red light during detection;

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

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 standard products 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: accurately weigh alpha-ketoglutaric acid and dissolve it in anhydrous acetonitrile to prepare a concentration of 1×10 -3 alpha-ketoglutarate stock solution of M;

[0092] Preparation of serum stock solution: mix serum and anhyd...

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 5000rpm for 20min, take the supernatant and pass it through a 0.2μm membrane, take 200μL of the obtained supernatant and add 100μL of the probe HNBD stock solution in turn and 200 μLalpha-ketoglutaric acid 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, fluorescent light The bottom is light red, 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 nitrobenzothiadiazolehydrazineDT and hydrazino-substitutednaphthalimide fluorescent molecular probes to monitor alpha-ketoglutarate . [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, the sample processing steps ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D271/12G01N21/33G01N21/64
CPCC07D271/12G01N21/33G01N21/6428
Inventor 陈光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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