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Method for detecting pyruvic acid and concentration thereof by fluorescent gold nanocluster probe, method for detecting pyruvate oxidase and concentration thereof

A technology of pyruvate oxidase and fluorescent gold nanometers, which is applied in the direction of material analysis, fluorescence/phosphorescence, and measurement devices by optical means, can solve the problems of expensive equipment, increase the cost of users, and complicated operations, and achieve excellent fluorescence properties. , good selectivity, wide linear range effect

Inactive Publication Date: 2019-04-02
CHANGCHUN UNIV OF CHINESE MEDICINE
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  • Abstract
  • Description
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Problems solved by technology

[0005] At present, the methods for detecting pyruvate recorded in the existing literature include dinitrophenylhydrazone colorimetry, ultraviolet enzyme coupling spectrophotometry, gas chromatography, etc., but there are disadvantages such as poor specificity and complicated operation, especially sometimes requiring large-scale However, there are disadvantages such as complex operation, expensive equipment, long time consumption, and low sensitivity, which make it difficult to meet the daily detection requirements, which greatly limits the promotion and application of these detection methods, and also increases the cost of the user.

Method used

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  • Method for detecting pyruvic acid and concentration thereof by fluorescent gold nanocluster probe, method for detecting pyruvate oxidase and concentration thereof
  • Method for detecting pyruvic acid and concentration thereof by fluorescent gold nanocluster probe, method for detecting pyruvate oxidase and concentration thereof
  • Method for detecting pyruvic acid and concentration thereof by fluorescent gold nanocluster probe, method for detecting pyruvate oxidase and concentration thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0161] (1) Take 3700 μL of pyruvate solution (600 μM) and blend it with 250 μL of pyruvate oxidase at a concentration of 1 mg / mL. Then place it in a 43°C water bath and incubate for 2.5 hours;

[0162] (2) Add 350 μL of disodium hydrogen phosphate-citric acid buffer solution (concentration: 1 mM, pH=4.3) and 250 μL of N-2-pyrrolidone (concentration: 40 mM) to the solutions of various concentrations in the above step (1). , Cu 2 SO 4 60μL (concentration is 50mM). Shake well and react at room temperature for 65 minutes. Then dialyze with a dialysis bag with a molecular weight of 1000Da for 18 hours;

[0163] (3) Take 600 μL of the solution prepared in step (2), and add HAuCl in sequence 4 250 μL (100 mM), 75 μL of acetic acid-sodium acetate buffer solution (pH=4.5), and shake to mix evenly. The sample was placed in a microwave oven, the power was set to 600w, and the time was 9 minutes to obtain gold nanoclusters with strong fluorescence;

[0164] (4) Steps (1)-(3) were...

Embodiment 2

[0172] (1) Take 3500 μL of pyruvate solution (100 μM), and blend it with 100 μL of pyruvate oxidase at a concentration of 0.5 mg / mL. Then place it in a 40°C water bath and incubate for 2 hours;

[0173] (2) Add 100 μL of disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (concentration: 0.5 mM, pH=5.5) and 100 μL of N-2-pyrrolidone (concentration: 20mM), oxalic acid 10μL (30mM concentration). Shake well and react at room temperature for 10 minutes. Then dialyze for 12 hours with a dialysis bag with a molecular weight of 500 Da;

[0174] (3) Take 200 μL of the solution prepared in step (2), and add HAuCl in sequence 4 100 μL (50 mM), 50 μL of disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH=5.2), and shake to mix evenly. Put the sample in a microwave oven with the power set to 400w for 15 minutes to obtain gold nanoclusters with strong fluorescence;

[0175] (4) Steps (1)-(3) were repeated to measure the fluorescence e...

Embodiment 3

[0177] (1) Take 4000 μL of pyruvate solution (1600 μM) and blend it with 500 μL of pyruvate oxidase at a concentration of 2 mg / mL. Then place it in a 45°C water bath and incubate for 3 hours;

[0178] (2) Add 500 μL of glycine-hydrochloric acid buffer solution (concentration: 2 mM, pH=3.2), 400 μL of N-2-pyrrolidone (concentration: 60 mM), NaHSO 3 100μL (concentration is 70mM). Shake well and react at room temperature for 120min. Then dialyze with a dialysis bag with a molecular weight of 1500Da for 24 hours;

[0179] (3) Take 1000 μL of the solution prepared in step (2), and add HAuCl in sequence 4 500 μL (200 mM), 100 μL of citric acid-sodium hydroxide-hydrochloric acid buffer solution (pH=4.2), and shake to mix evenly. Put the sample in a microwave oven with the power set to 800w for 3 minutes to obtain gold nanoclusters with strong fluorescence;

[0180] (4) Steps (1)-(3) were repeated to measure the fluorescence emission spectra of other pyruvate solutions (1800 μM...

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Abstract

The invention provides an application of a fluorescent gold nanocluster in fluorescent detection of pyruvic acid and / or pyruvate oxidase. The fluorescent gold nanocluster probe is used for detecting the pyruvate and / or the pyruvate oxidase, has the characteristics of high sensitivity, good selectivity, wide linear range and the like. According to the application of the fluorescent gold nanoclusterin the fluorescent detection of the pyruvic acid and / or the pyruvate oxidase, the detection of the pyruvic acid by a method for emitting fluorescence by the fluorescent nanocluster not only avoids interference of other factors of biology, but also selectively detects the concentration of the pyruvic acid in blood and urine, and can detect the concentration of the pyruvic acid non-invasively. A fluorescence emission method is used to detect the concentration of the pyruvic acid instead of using pre-prepared fluorescent metal nanocluster to minimize the interference that may be caused by falsesignals in the experiment. The fluorescent gold nanocluster generated simultaneously during the detection process has excellent fluorescence properties and great significance in applications in the fields of optical materials, biomedicine and the like.

Description

technical field [0001] The invention relates to the technical field of pyruvate detection, in particular to an application of fluorescent gold nanoclusters in the detection of pyruvate and / or pyruvate oxidase by fluorescence method, a method for detecting pyruvate concentration by fluorescence method of fluorescent gold nanoclusters, and a method for detecting pyruvate concentration by fluorescence method. A method for detecting the concentration of pyruvate oxidase by a gold nanocluster fluorescence method and a method for detecting whether pyruvate or pyruvate oxidase is contained in a sample by a fluorescent gold nanocluster fluorescence method, especially involving a fluorescent gold nanocluster in the detection of pyruvate by a fluorescence method And / or application in pyruvate oxidase, method for detecting pyruvate and its concentration by fluorescent gold nanocluster probe, method for detecting pyruvate oxidase and its concentration by fluorescent gold nanocluster probe,...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6428
Inventor 姜英男孙天霞辛精卫刘美欣刘宇昕王新宇吉世禹张阳
Owner CHANGCHUN UNIV OF CHINESE MEDICINE
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