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A method for detecting lipase activity based on fluorescent probe

A fluorescent probe and detection method technology, applied in the field of lipase activity detection, can solve the problems of high detection limit, limited effect, low sensitivity and the like

Active Publication Date: 2021-05-04
湖南格美特生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the curcumin used in this patent is a hydrophobic substance with poor water solubility. Although its compound use with Tween can improve its hydrophilicity to a certain extent, the effect is limited, and the coating of Tween has no effect on curcuma longa. The release of the fluorescence effect of curcumin has a certain influence, which leads to inaccurate test results; in addition, because the fluorescence efficiency of curcumin itself is not high enough, the sensitivity is relatively low when detecting lipase activity, resulting in a high detection limit, which is difficult to detect. Accurate detection of less active lipases

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  • A method for detecting lipase activity based on fluorescent probe
  • A method for detecting lipase activity based on fluorescent probe
  • A method for detecting lipase activity based on fluorescent probe

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Experimental program
Comparison scheme
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Embodiment 1

[0056] The present embodiment provides a method for testing lipase activity based on a fluorescent probe, comprising the following steps:

[0057] S1, after 1.92g citric acid, 0.72g ethylenediamine and 0.61g chloroauric acid are mixed, be dissolved in 30mL deionized water, obtain mixed solution, now the mol ratio of citric acid, ethylenediamine and chloroauric acid is 100: 12:1.5; the mixed solution was heated and stirred at 80°C for 10min, then moved into an autoclave lined with polytetrafluoroethylene, heated at 160°C for 12h, and then naturally cooled to room temperature to obtain nitrogen gold Co-doped carbon nano-dot crude product; centrifuge the nitrogen-gold co-doped carbon nano-dot crude product at a speed of 10000r / min for 10min to remove larger particles, and then use a 0.22μm microporous membrane to centrifuge The supernatant was filtered, and the obtained filtrate was purified nitrogen-gold co-doped carbon nanodots.

[0058] S2. The 2mL activity was respectively 0...

Embodiment 2~3 and comparative example 1~2

[0068] Embodiment 2 ~ 3 and comparative example 1 ~ 2 provide a kind of preparation method of carbon nano-dot respectively, compare with the step S1 of embodiment 1, difference is to change nitrogen source ethylenediamine and gold source chloroauric acid The dosage. The molar ratios of citric acid, ethylenediamine and chloroauric acid corresponding to Examples 2 to 3 and Comparative Examples 1 to 2 are shown in Table 1.

[0069] Table 1 The mol ratio of citric acid, ethylenediamine and chloroauric acid corresponding to Examples 2~3 and Comparative Examples 1~2

[0070] Example / Comparative example Molar ratio of citric acid, ethylenediamine and chloroauric acid Example 2 100:10:1 Example 3 100:15:2 Comparative example 1 100:12:0 Comparative example 2 100:0:0

[0071] The carbon nano-dots prepared in Examples 2-3 and Comparative Examples 1-2 were detected by fluorescence. When the excitation wavelength was 360nm, the fluorescence spectra ...

Embodiment 4~6

[0074] Embodiments 4 to 6 respectively provide a method for testing lipase activity based on fluorescent probes. Compared with Example 1, the difference is that the excitation wavelength of the fluorescence detection process in step S3 is changed. Embodiments 4 to 6 correspond to The excitation wavelengths are 340nm, 350nm and 370nm, respectively.

[0075]The fluorescence spectra of the nitrogen-gold co-doped carbon nano-dots prepared in Examples 4-6 and Example 1 under different excitation wavelengths are as follows: Figure 5 shown. Figure 5 Among them, the excitation wavelengths corresponding to a, b, c, and d are 340nm (Example 4), 350nm (Example 5), 360nm (Example 1) and 370nm (Example 6), respectively. Depend on Figure 5 It can be seen that with the increase of the excitation wavelength, the maximum fluorescence intensity value of the obtained fluorescence spectrogram first increases and then decreases. Therefore, in order to make nitrogen-gold co-doped carbon nano-...

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Abstract

The invention discloses a method for testing lipase activity based on a fluorescent probe. In the method, citric acid is used as a carbon source, ethylenediamine is used as a carbon and nitrogen source, and chloroauric acid is used as a gold source. Doping carbon nanodots as fluorescent probes, and mixing the fluorescent probes with copper ions and lipase hydrolyzate, using the difference in strength of the fluorescent probe and lipase hydrolyzate on copper ions, the lipase activity was established The standard curve between the fluorescence intensity of the probe and the fluorescence intensity of the probe realizes the efficient, sensitive and accurate detection of lipase activity. Through the above method, the present invention can effectively improve the optical performance and fluorescence quantum yield of carbon nano-dots, so that the test has higher sensitivity; and the test method is simple and easy to operate, and has the advantages of fast detection speed, wide detection range and low detection limit. It can also accurately detect low-activity lipase, has a wide range of applications, and has a good application prospect.

Description

technical field [0001] The invention relates to the technical field of lipase activity testing, in particular to a method for testing lipase activity based on a fluorescent probe. Background technique [0002] Lipase is an enzyme with a variety of catalytic capabilities that is ubiquitous in living organisms. It can catalyze the hydrolysis, alcoholysis, esterification, transesterification and reverse synthesis of triacylglycerides and other water-insoluble esters. , not only has important physiological significance, but also has potential value for industrial application, and has been widely used in food, medicine, leather, daily chemical and other fields. Since the catalytic ability of lipase is closely related to its activity, it is very important to accurately test the activity of lipase to study the catalytic performance and application of lipase. [0003] At present, lipase activity detection methods mainly include plate method, turbidity analysis, titration, colorimet...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6428
Inventor 孙旭东
Owner 湖南格美特生物科技有限公司
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