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Fluorescence analysis method for detecting lipase activity on basis of inner filter effect of fluorescence

A technique for lipase activity and fluorescence analysis, applied in the field of fluorescence analysis, can solve problems such as detection that has not yet been reported, and achieve the effects of improved detection limit, simple and fast operation, and low cost

Inactive Publication Date: 2018-11-06
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] At present, based on the fluorescence inner filter effect, it has been successfully applied to the enzyme activity detection of alkaline phosphatase, acetylcholinesterase, glucose oxidase, etc. detection has not yet been reported

Method used

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  • Fluorescence analysis method for detecting lipase activity on basis of inner filter effect of fluorescence
  • Fluorescence analysis method for detecting lipase activity on basis of inner filter effect of fluorescence
  • Fluorescence analysis method for detecting lipase activity on basis of inner filter effect of fluorescence

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Embodiment 1

[0019] Example 1 Mechanism Verification

[0020] Add 3μM cadmium sulfide quantum dots and 2mM PBS buffer solution (pH 6.0) to 2mL centrifuge tubes a, b, c, and d respectively, then add 20μg lipase to tube b, and add 1.61nM thioglycolic acid-modified gold nanoparticles and 20μg lipase, add 1.61nM thioglycolic acid-modified gold nanoparticles to tube d, and the others were adjusted to 1000μL with ultrapure water. The wavelength is 360nm, and the emission wavelength range is 400-650nm to detect its fluorescence intensity (such as image 3 shown).

[0021] image 3 It is a mechanism verification diagram. It can be seen from curve a and curve b that no matter whether there is lipase or not, the maximum fluorescence emission peak of cadmium sulfide quantum dots at 505nm does not change, indicating that there is no difference between cadmium sulfide quantum dots and lipase. Molecular interactions. When cadmium sulfide quantum dots are mixed with thioglycolic acid-modified gold na...

Embodiment 2

[0022] The drafting of the standard curve of embodiment 2 lipase activity detection

[0023] Add 3μM cadmium sulfide quantum dots, 1.61nM thioglycolic acid-modified nano-gold and 2mM PBS buffer solution (pH 6.0) to a 2mL centrifuge tube, respectively, add different concentrations of lipase, and the zinc ion concentration is as follows: 0μg / L, 0.1μg / L, 5μg / L, 10μg / L, 15μg / L, 20μg / L, 25μg / L, 30μg / L, 35μg / L, 40μg / L, 45μg / L, dilute to 1000 μL, measured its fluorescence intensity after bathing in water at 45°C for 14 minutes; taking the concentration of lipase as the abscissa and the fluorescence recovery intensity as the ordinate to draw a graph, the standard curve of lipase activity can be drawn (such as Figure 4 shown).

[0024] Figure 4 It shows that with the increase of the lipase concentration, the fluorescence recovery intensity of the solution is gradually enhanced and the lipase concentration has a good linear relationship between 0.1-45μg / L, and the detection limit i...

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Abstract

The invention discloses a fluorescence analysis method for detecting lipase activity on the basis of the inner filter effect of fluorescence. The detection principle is that cadmium sulfide quantum dots are taken as fluorophores, methyl thioglycolate modified nanogold is taken as an absorption group, after the cadmium sulfide quantum dots and methyl thioglycolate modified nanogold are mixed, fluorescence emission intensity of the cadmium sulfide quantum dots is reduced obviously due to the inner filter effect of fluorescence, the inner filter effect of fluorescence is weakened after lipase isadded, and fluorescence intensity of the cadmium sulfide quantum dots change obviously, which indicates that lipase activity and fluorescence emission intensity change have certain relationship, so that lipase activity can be detected by a fluorescence signal sensor. The method for detecting lipase activity is simple to operate and low in cost, and special instrument and equipment are not needed.

Description

technical field [0001] The invention belongs to the field of nano biosensors, in particular to a fluorescence analysis method for detecting lipase activity based on fluorescence inner filter effect. Background technique [0002] Lipase (EC 3.1.1), also known as triacylglyceride hydrolase, in addition to its natural function of hydrolyzing carboxylic ester bonds, lipase can also catalyze esterification, transesterification and interesterification reactions in non-aqueous media. Lipases come from a variety of sources, resulting in large differences in their catalytic properties. The main reaction of lipase occurs at the oil-water interface, which distinguishes lipase from other hydrolytic enzymes. In the discovery and application of lipase, lipase activity is an important parameter. The traditional detection of lipase activity Methods such as titration, turbidity and chromatography are not suitable for non-purified samples and large-scale analysis and detection, because they ...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N1/28
CPCG01N1/28G01N21/6428G01N21/6486
Inventor 田丹碧朱杰张凌华
Owner NANJING UNIV OF TECH
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