Fluorescence probe for detecting beta-galactosidase as well as preparation method and application of fluorescence probe

A technology of galactosidase and fluorescent probe, which is applied in the field of analysis and detection, can solve problems such as difficulty in ensuring detection accuracy, and achieve the effects of eliminating background interference, good detection effect, and good detection accuracy

Inactive Publication Date: 2015-09-30
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current conventional fluorescence detection methods for detecting β-galactosidase are mainly fluorescence-enhanced probes. These methods are susceptible to interference from excitation light intensity, sample concentration, background fluorescence in the sample, etc., so it is difficult to guarantee the accuracy of detection. sex

Method used

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  • Fluorescence probe for detecting beta-galactosidase as well as preparation method and application of fluorescence probe
  • Fluorescence probe for detecting beta-galactosidase as well as preparation method and application of fluorescence probe
  • Fluorescence probe for detecting beta-galactosidase as well as preparation method and application of fluorescence probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: the preparation of probe compound NG-GAL

[0064] Synthetic route such as figure 1 As shown, 1035 mg of 4-bromo-1,8-naphthalene dicarboxylic anhydride (3.75 mmol) was dissolved in 56 mL of ethanol, and under stirring conditions, 396 mg of N,N-dimethylethylenediamine was added dropwise under nitrogen protection (4.50mmol), the mixed solution was heated to reflux at 80°C, and after cooling to room temperature, the precipitate was filtered and collected, and the precipitate was recrystallized with ethanol to obtain solid 6-bromo-2-(2-(dimethylamino)ethyl Base)-benzisoquinoline-diketone 1066mg (yield rate is 82.2%); By proton nuclear magnetic resonance spectrum ( figure 2 ) to characterize the product, 1 H NMR (CDCl 3 ,400MHz,ppm):2.36(s,6H),2.65(t,J=6.4Hz,2H),4.34(t,J=6.8Hz,2H),7.86(t,J=6.8Hz,1H),8.03 (d,1H),8.42(d,1H),8.56(d,1H),8.67(d,1H). Among them, 8.67ppm, 8.56ppm, 8.42ppm, 8.03ppm and 7.86ppm correspond to the protons on the naphthalene ring 4.34...

Embodiment 2

[0069] Embodiment 2: the preparation of probe compound NG-GAL

[0070] Dissolve 2070mg of 4-bromo-1,8-naphthalene dicarboxylic anhydride (7.50mmol) in 90mL of ethanol, and add 760mg of N,N-dimethylethylenediamine (8.62mmol ), the mixed solution was heated to reflux at 82°C, and after cooling to room temperature, the precipitate was filtered and collected, and the precipitate was recrystallized with ethanol to obtain solid 6-bromo-2-(2-(dimethylamino)ethyl)- Benzisoquinoline-dione 1988 mg (76.6% yield).

[0071] Dissolve 1038mg of the above white solid (3.0mmol) and 1620mg of sodium methoxide (30mmol) in 21mL of methanol, add 102mg of CuSO 4 ·5H 2 O, under stirring conditions, the solution was heated to reflux at 72°C and maintained for 11 hours; cooled to room temperature, added deionized water, and then extracted with ethyl acetate; the organic phase was collected, and the organic phase was dried with 1800 mg of anhydrous magnesium sulfate, Filtration, rotary evaporation t...

Embodiment 3

[0076] Embodiment 3: the preparation of probe compound NG-GAL

[0077] Dissolve 552mg of 4-bromo-1,8-naphthalene dicarboxylic anhydride (2.0mmol) in 30mL of ethanol, and add 194mg of N,N-dimethylethylenediamine (2.2mmol ), the mixed solution was heated to reflux at 85°C, and after cooling to room temperature, the precipitate was filtered and collected, and the precipitate was recrystallized with ethanol to obtain solid 6-bromo-2-(2-(dimethylamino)ethyl)- Benzisoquinoline-dione 535 mg (76.7% yield).

[0078] Dissolve 346mg of the above white solid (1.0mmol) and 486mg of sodium methoxide (9mmol) in 9mL of methanol, add 34mg of CuSO 4 ·5H 2 O, under stirring conditions, the solution was heated to reflux at 68°C and kept for 11 hours; cooled to room temperature, added deionized water, and then extracted with ethyl acetate; collected the organic phase, and dried the organic phase with 1000 mg of anhydrous magnesium sulfate, Filtration, rotary evaporation to remove the organic so...

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Abstract

The invention belongs to the technical field of analysis and detection, and discloses a fluorescence probe for detecting beta-galactosidase as well as a preparation method and application of the fluorescence probe. The substrate of the fluorescence probe is 2-(2-(dimethylamino)ethyl-6-(((3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxidation)-1H-benzisoquinoline diketone, and has a structural formula shown in the description. The fluorescence probe has the advantages that naphthalic anhydride is taken as a fluorophore of the substrate of the fluorescence probe, and lined with a galactose group which can respond to the specificity of beta-galactosidase; the ratio of double fluorescence signals being I554/I445 is taken as a detection signal, and thus the detection accuracy is higher; under the condition of 1U/L enzyme concentration, obvious fluorescence alteration can be detected, and the lower detection limit is 0.35 U/L; the fluorescence probe is excited (418 nm) in a visible light region, so that the background interference can be eliminated to achieve a better detection effect.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a fluorescent probe for detecting β-galactosidase and its preparation method and application. Background technique [0002] β-galactosidase (β-gal) is an enzyme capable of hydrolyzing β-galactoside into galactose and glycosides. With the rapid development of science and technology, β-galactosidase has more and more applications in the fields of environment, biology, medicine, chemistry, etc. In the field of food industry, β-galactosidase can hydrolyze lactose to reduce the content of lactose in dairy products. In the biological field, β-galactosidase is a widely used gene marker enzyme, which can be used to study gene transcription regulation and gene expression. The β-galactosidase gene is the most commonly used reporter gene in genetic engineering, and its expression product β-galactosidase is used to study the expression regulation of the target ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C07H17/02G01N21/64
Inventor 吴水珠胡清华曾鈁
Owner SOUTH CHINA UNIV OF TECH
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