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Synthesis and applications of beta-galactosidase sensor with aggregation induced emission enhancement performance

A galactosidase and fluorescence sensor technology, which is applied in the field of biochemical materials, can solve the problems of fluorescence quenching, less fluorescence sensors, and limit the practical application of β-galactosidase fluorescence sensors, and achieves easy mass production and Application, simple synthesis steps, overcoming the easy bleaching effect of dye molecules

Active Publication Date: 2016-12-21
GANNAN NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few fluorescent sensors for detecting β-galactosidase, and the fluorescent molecules used in most β-galactosidase fluorescent sensors are prone to fluorescence quenching at high concentrations, that is, aggregation-induced fluorescence quenching. Aggregation caused quenching (ACQ)
This phenomenon forces researchers to use only dilute solutions during the detection process, resulting in a low detection signal-to-noise ratio, which limits the practical application of these β-galactosidase fluorescent sensors.

Method used

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  • Synthesis and applications of beta-galactosidase sensor with aggregation induced emission enhancement performance
  • Synthesis and applications of beta-galactosidase sensor with aggregation induced emission enhancement performance
  • Synthesis and applications of beta-galactosidase sensor with aggregation induced emission enhancement performance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Synthesis of target compound 1

[0028]

[0029] (1) Synthesis of compound 2: Zinc powder (1.56g, 24mmol), 4-bromobenzophenone (313mg, 1.2mmol) 4,4'-dimethoxybenzophenone (242mg, 1.0mmol) It was added to anhydrous tetrahydrofuran, and the suspension was cooled to 0°C with stirring. Then titanium tetrachloride (2.276g, 12mmol) was slowly dropped into the mixture with a syringe, and the mixture was refluxed for 4h after stirring at 0°C for 30min. Cool and add sodium carbonate solution dropwise with stirring until no bubbles are produced. Dichloromethane and saturated brine were added for separation and extraction, the organic phase was washed three times with saturated brine, the organic phase was dried over anhydrous sodium sulfate, concentrated, and separated by column chromatography to obtain compound 2 with a yield of 29%. The NMR characterization data are: 1 H NMR (400MHz, CDCl 3 )δ7.21(d,J=4.6Hz,2H),7.20(m,3H),7.00(m,2H),6.95–6.87(m,6H),6.67–6.61(m,4H),3.76(s...

Embodiment 2

[0035] β-galactosidase sensor performance test

[0036] (1) Determination of the response time of the β-galactosidase sensor: Take 20 μL of DMSO solution (1 mM) of compound 1 in 2 mL of PBS buffer solution, add 8.0 U / mL β-galactosidase, and incubate the mixture at 37 °C At different times, the fluorescence intensity of the mixed solution was measured as a function of the incubation time. The results showed that the fluorescence intensity was basically saturated after 10 minutes.

[0037] (2) Fluorescent titration test of β-galactosidase sensor: Take 20 μL of compound 1 in DMSO solution (1 mM) in 2 mL of PBS buffer solution, then add different amounts of β-galactosidase in PBS solution, and mix the solution at 37 ° C After incubation for 10 min, measure the fluorescence emission spectrum (E x =344nm), the fluorescence intensity of the mixed solution presents a good linear relationship in the concentration range of 0.8U / mL to 4.8U / mL β-galactosidase (such as figure 1 shown). ...

Embodiment 3

[0040] Human ovarian cancer cell (OVCAR3) β-galactosidase fluorescence imaging: OVCAR3 cells were revived and inoculated in RPMI 1640 medium containing 10% fetal bovine serum. HeLa cells were revived and inoculated in DEME medium containing 10% fetal bovine serum. Both cells were incubated at 37°C, 5% CO 2 , 100% saturated humidity incubator. Then cultured on 18mm coverslip for 24h, ready to use.

[0041] The two kinds of cells were respectively immersed in the medium containing 5 μM compound 1, at 37 °C, 5% CO 2 After culturing for 40 minutes in an incubator with 100% saturated humidity, pour out the medium, and wash the cells 3 times with fresh medium. Observe under laser confocal fluorescence microscope respectively, and take pictures under bright field and dark field (such as image 3 shown). The results showed that the target compound 1 only showed a strong fluorescent signal in OVCAR3 cells overexpressing β-galactosidase, while under the same conditions, the fluores...

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Abstract

The invention relates to a beta-galactosidase sensor with aggregation induced emission enhancement performance, and a synthesis method and applications of the beta-galactosidase sensor. According to the synthesis method, a diphenyl ketone derivative is taken as a starting material to synthesize a target compound 1. Detection study of the target compound 1 on beta-galactosidase is carried out, and it is found that the target compound 1 possesses high sensitivity and selectivity in beta-galactosidase detection. Compared with the prior art, the synthesis raw materials are easily available, target compound fluorescence quantum yield is high, and photobleaching resistance is excellent, a defect of conventional fluorescent dyes that detection at a high concentration is difficult to realize is avoided, the target compound 1 is successively applied to fluorescence imaging of beta-galactosidase in OVCAR3, and application prospect of the target compound 1 in detection of content of beta-galactosidase in cells is promising.

Description

technical field [0001] The invention belongs to the field of biochemical materials, and relates to a β-galactosidase fluorescent sensor, a synthesis method and an application. Background technique [0002] β-galactosidase is a commonly used enzyme to detect transcription efficiency and gene transfection efficiency, and it is also an important biomarker of cellular senescence and primary ovarian cancer. β-galactosidase deficiency results in β-galactosialidosis and Morquio B syndrome. Moreover, in recent years, β-galactosidase has been widely used in the research of alternative therapy for lactose intolerance. The important physiological role of β-galactosidase has inspired scientists to study its detection methods. For example, the commonly used methods for detecting β-galactosidase are: biochemiluminescence, magnetic resonance, single photon emission computed tomography Scanning method (SPECT), positron emission tomography (PET), colorimetry and fluorescence method, etc. ...

Claims

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Application Information

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IPC IPC(8): C07H15/26C07H1/00C09K11/06G01N21/64
CPCC07H1/00C07H15/26C09K11/06C09K2211/1029G01N21/6428G01N2021/6432
Inventor 王建国姜国玉吴勇权范小林
Owner GANNAN NORMAL UNIV
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