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Fluorescent probe for selectively identifying ATP based on aggregation-induced fluorescence enhancement characteristic, synthetic method and application thereof

A technology of fluorescent probe and synthesis method, which is applied in fluorescence/phosphorescence, material analysis by optical means, measurement device, etc., can solve the problems of low detection signal-to-noise ratio, fluorescence quenching, and limiting the practical application of ATP fluorescent probe. , to achieve the effect of simple synthesis steps, overcoming easy bleaching, and easier large-scale production and application

Active Publication Date: 2017-06-09
GANNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the fluorescent molecules used in traditional ATP fluorescent probes 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 in the detection process, resulting in a low detection signal-to-noise ratio, which limits the practical application of traditional ATP fluorescent probes.

Method used

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  • Fluorescent probe for selectively identifying ATP based on aggregation-induced fluorescence enhancement characteristic, synthetic method and application thereof
  • Fluorescent probe for selectively identifying ATP based on aggregation-induced fluorescence enhancement characteristic, synthetic method and application thereof
  • Fluorescent probe for selectively identifying ATP based on aggregation-induced fluorescence enhancement characteristic, synthetic method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Synthesis of fluorescent probe 1

[0026]

[0027] (1) Synthesis of Compound 2: Add zinc powder (1.56g, 24mmol) and 4,4'-dibromobenzophenone (408mg, 1.2mmol) into anhydrous tetrahydrofuran, and cool the suspension to 0 ℃. 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 to separate liquid and extracted, 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 86%.

[0028] (2) Synthesis of Compound 3: Compound 2 (200mg, 0.31mmol), 4-pyridineboronic acid (305mg, 2.48mmol), Pd(dppf)Cl 2 (200mg, 0.245mmol), CH 2 Cl 2 (1mL), Bu 4 NI (25mg, 0.068mmol) and potass...

Embodiment 2

[0031] ATP probe performance test

[0032] (1) Fluorescent titration test of fluorescent probe 1 to ATP: Take 5 μL of fluorescent probe 1 in DMSO solution (1 mM) in 2 mL of HEPES buffer solution, and then add different amounts of ATP aqueous solution (1 mM). Fluorescence emission spectra (E x =344nm), the fluorescence intensity of the mixed solution in the concentration range of 1.0 μM to 4.0 μM ATP presents a good linear relationship (such as figure 1 shown in b). When changing the concentration of fluorescent probe 1 to 20.0 μM, the system can show a good linear relationship with the fluorescence intensity of fluorescent probe 1 in the range of 2.0 μM to 20.0 μM ATP concentration (such as figure 1 shown in c). Similarly, when the concentration of fluorescent probe 1 continues to increase to 40.0 μM, the system can present a good linear relationship with the fluorescence intensity of fluorescent probe 1 within the concentration range of 20.0 μM to 40.0 μM ATP (such as fig...

Embodiment 3

[0035] Monitoring of an ATP hydrolase Apyrase reaction process in the solution: Add different concentrations of ATP hydrolase Apyrase (0,5,10,15,25,50mU) to the HEPES buffer solution containing 20 μM fluorescent probe 1 and 20 μM ATP, respectively, The monitoring system changes the fluorescence intensity with the reaction time at 500nm, and the results show that when there is no Apyrase, the fluorescence intensity of fluorescent probe 1 changes less (3 VO 4 After repeating the above experiments, it was found that due to the activity of the enzyme being controlled by Na 3 VO 4 Inhibition, the decrease rate of the fluorescence intensity of the system with the reaction time is obviously slower (such as image 3 shown in b). It shows that fluorescent probe 1 can be used to monitor the reaction process of ATP hydrolase Apyrase in solution.

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Abstract

The invention discloses a fluorescent probe for selectively identifying ATP (adenosine triphosphate) based on an aggregation-induced fluorescence enhancement characteristic, a synthetic method and an application thereof. The fluorescent probe 1 is synthesized by taking a benzophenone derivative as a starting material. The invention carries out a detection research on ATP, ADP (adenosine diphosphate), AMP (adenosine monophosphate) and pyrophosphoric acid (PPi) with the fluorescent probe 1, and discoveries that the fluorescent probe has very good sensitivity and selectivity to ATP; compared with the prior art, the invention has the advantages that the synthetic raw material is easy to obtain, the fluorescent probe has high fluorescence quantum yield and strong photobleaching resistance, and the like, and avoids the defect that a conventional fluorescent dye is not suitable for detection under a high concentration; in addition, the fluorescent probe 1 is successfully used for monitoring a reaction process of ATP hydrolase Apyrase in a solution, and is also used for imaging research of ATP in cells. Therefore, the fluorescent probe 1 has a great application prospect in the aspect of detecting the content of in vivo ATP.

Description

technical field [0001] The invention belongs to the field of biochemical materials, and relates to a fluorescent probe for selectively recognizing ATP based on aggregation-induced fluorescence enhancement characteristics, a synthesis method and an application. Background technique [0002] ATP is a kind of adenosine triphosphate, which exists in the form of coenzyme in the cell and is the smallest molecular unit of the energy transfer process in the cell. ATP has important physiological functions in cells, such as cell signal transduction, maintenance of cell structure, and biosynthesis of DNA or RNA. Usually, healthy adults contain an average of 250g of ATP in their bodies. Abnormal ATP content is usually closely related to cardiovascular disease, local ischemia, tissue hypoxia, hypoglycemia, Parkinson's disease, Alzheimer's disease and other diseases. The important physiological role of ATP has inspired scientists to study its detection methods. For example, the commonly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 王建国姜国玉吴勇权范小林李勋
Owner GANNAN NORMAL UNIV
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