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Application of a two-photon ratiometric fluorescent probe in the detection of monoamine oxidase b

A technology of monoamine oxidase and fluorescent probe, which is applied in the fields of fluorescence/phosphorescence, organic chemistry, measuring devices, etc., can solve the problems of poor spatial resolution and failure to provide real-time information of enzymes, and achieve high sensitivity, high photostability, and good selection sexual effect

Active Publication Date: 2021-06-15
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

SPECT and PET have high sensitivity and tomographic capabilities, but poor spatial resolution (1-2 mm)
Other assays, such as the use of labeled antibodies or proteomic approaches, can be used to detect and track enzymes in fixed cells or in vitro, but fail to provide real-time information on enzymes in living cells

Method used

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  • Application of a two-photon ratiometric fluorescent probe in the detection of monoamine oxidase b
  • Application of a two-photon ratiometric fluorescent probe in the detection of monoamine oxidase b

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1) Synthesis of compound FTP-B1:

[0028] Dissolve Np-Rhod dye (1.2 mmol, 1 eq), potassium carbonate (3 mmol, 2.5 eq) in 20 mL of anhydrous N,N-dimethylformamide, and then add 1,3-bis Propane bromide (3.5 mmol, 3 eq), stirred at room temperature for 4 h, then heated to 60°C and refluxed for 12 h. Monitor the reaction with a TCL plate. After the reaction is complete, extract with dichloromethane, dry over anhydrous sodium sulfate, evaporate under reduced pressure, and separate with a silica gel column. The silica gel particle size is 200-300 mesh to obtain compound FTP-B1, which is light red Solid, the ratio of eluent is methanol:dichloromethane=1:120, and the yield is 80%.

[0029] 2) Synthesis of Compound FTP-B:

[0030] Add sodium azide (3.4 mmol, 4 eq), potassium carbonate (3.6 mmol, 4.2 eq) to 10 mL of FTP-B1 (0.85 mmol, 1 eq) in anhydrous N,N-dimethylformamide to make The reaction mixture was stirred at 70 °C for 10 h. The reaction was monitored with a TCL plat...

Embodiment 2

[0032] Compound FTP-B fluorescent probe with different concentrations of MAO-B (0-30 U) I 542 nm / I 448 nm Ratiometric Fluorescence Emission Changes

[0033] The FTP-B fluorescent probe prepared in Example 1 was dissolved in PBS (10 mM, pH 7.4) containing EtOH as a co-solvent (PBS / EtOH=10:1, v / v) to prepare a 1 mmol / L stock solution. Take 10 μL from the stock solution and add it to a 5 mL centrifuge tube, add different amounts (0-30 U) of MAO-B standard solution, and use PBS buffer solution (0.1 mol / L, pH 7.5) and EtOH at a volume ratio of The 10:1 solution was diluted to 3 mL and its fluorescence properties were measured. Using 370 nm as the excitation light, as the amount of MAO-B increased, the fluorescence intensity at 448 nm decreased sharply, and a red-shifted fluorescence peak appeared at 542 nm, providing a visualized ratiometric fluorescence signal (I 542 nm / I 448 nm ).

Embodiment 3

[0035] Measurement of Fluorescent Linear Range of Compound FTP-B Fluorescent Probe

[0036]Prepare 10 parts of 2 mL FTP-B fluorescent probe solution (10 μM), add MAO-B (0-20 U) respectively, and perform fluorescence detection (λ ex =370 nm), calculate the fluorescence intensity in each system, by analyzing the I 542 nm / I 448 nm The relationship between the fluorescence intensity and the concentration of MAO-B was used to evaluate the response performance of the probe to MAO-B. It shows that the probe has a linear relationship in the range of MAO-B concentration 0.1-1.5 U, and the linear correlation coefficient is R 2 =0.99688, and the detection limit of the probe for MAO-B was 10 ng / mL.

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Abstract

The invention discloses a preparation method and application of a two-photon ratio fluorescent probe for accurately measuring monoamine oxidase B in tumor cells. Based on the mechanism of fluorescence resonance energy transfer, the probe uses enzymatic oxidative deamination reaction to generate corresponding iminium ions, which are eliminated after hydrolysis and re-release hydroxyl groups, resulting in proportional changes in fluorescence emission signals. The advantage is that the fluorescent spectrum distance between the energy donor and the acceptor is larger, the interference between the spectra is smaller, and the sensitivity is higher, effectively avoiding the interference of various environmental conditions on the analysis. It provides a potential tool for clinical detection of important marker enzymes of Alzheimer's disease in vivo.

Description

technical field [0001] The present invention relates to small molecule fluorescent probes to identify the location and expression level of abnormal biological enzyme activity in cancer cells, more specifically, a two-photon naphthalene derivative fluorescent probe based on fluorescence resonance energy transfer for Alzheimer's disease Detection of monoamine oxidase B (MAO-B), an important marker of the disease. Background technique [0002] Alzheimer's disease (AD) is the most common dementia among older adults, currently affecting more than 35 million people worldwide. In particular, the clinical detection of related biomarkers is of great significance for the early diagnosis, treatment and management of diseases. Monoamine oxidase B (MAO-B) is a mitochondria-associated flavin enzyme highly expressed in the human brain, mainly in astrocytes, where it helps maintain the homeostasis of neurotransmitters in the brain, thereby ensuring proper neural and behavioral outcomes. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D493/10G01N21/64
CPCC07D493/10G01N21/6428G01N21/6447
Inventor 颜梅卫先哲张晶王建荣张乐淘武虹乐
Owner UNIV OF JINAN
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