Ratiometric fluorescent probe for detecting avidin, and synthesis method and application thereof

A technology of avidin and fluorescent probes, which is applied in the field of synthesis of fluorescent probes, can solve the problems of single fluorescence enhancement and rare ratiometric fluorescent probes

Active Publication Date: 2017-06-20
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, most fluorescent probes for detecting and labeling proteins are limited to a single fluorescence enhancement accompanied by blue shift, which is greatly ...

Method used

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  • Ratiometric fluorescent probe for detecting avidin, and synthesis method and application thereof
  • Ratiometric fluorescent probe for detecting avidin, and synthesis method and application thereof
  • Ratiometric fluorescent probe for detecting avidin, and synthesis method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: A method for synthesizing a ratiometric fluorescent probe for detecting avidin.

[0031] (1) Synthesis of intermediate N-Boc-ethylenediamine:

[0032] Di-tert-butyl dicarbonate (1.2 g, 5.5 mmol) was dissolved in 40 mL of dichloromethane, and 2.2 mL (33 mmol) of ethylenediamine was added dropwise under ice-cooling. The reaction solution was stirred at room temperature overnight (10-16 h), washed three times with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, filtered to obtain a clear solution, and spin-dried to obtain 845 mg of a colorless oil, with a yield of 96%. 1 HNMR (400MHz, CDCl 3 ) δ 4.91 (s, 1H), 3.20–3.14 (m, 2H), 2.80 (t, J=5.9Hz, 2H), 1.45 (s, 9H), 1.41 (s, 2H).

[0033]

[0034] (2) Synthesis of intermediate N-Boc-N-biotin ethylenediamine:

[0035] EDC.HCl (144mg, 1.0mmol), HOBt.H2O (182mg, 1.0mmol), triethylamine (350μL, 2.1mmol), biotin (110mg, 0.2mmol) were dissolved in 10mL DMF, and N -Boc-Ethylenediami...

Embodiment 2

[0041] Example 2: Fluorescent response of the fluorescent probe prepared in Example 1 under different ratios of water and methanol.

[0042] image 3 The concentration of the fluorescent probe prepared in Example 1 is 10 μM, and the excitation light is 405 nm. The probe exhibits fluorescence with a wavelength of ~470 nm in aqueous solution, and fluorescence with a wavelength of ~550 nm in methanol. When the proportion of methanol gradually increases, the wavelength The fluorescence intensity at ~550nm increases gradually, while the fluorescence intensity at ~470nm decreases gradually.

Embodiment 3

[0043] Example 3: The response of the fluorescent probe prepared in Example 1 to 0-2.0 equivalents of avidin.

[0044] Figure 4 The fluorescent probe concentration in the medium is 5 μM, the excitation light is 365 nm, 10.0 μL of the probe mother solution is dissolved in 4 mL of PBS buffer (pH=7.4, 20 mM), and a test solution with a fluorescent probe concentration of 5 μM is prepared. When the test solution is gradually added relative to the probe 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0 equivalents of avidin (0, 1.0, 2.0, 3.0, 4.0 , 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 15.0, 20.0μL), the fluorescence intensity at ~470nm gradually decreased, while the fluorescence at ~550nm gradually increased.

[0045] Figure 5 The fluorescent probe concentration in the medium is 5 μM, the excitation light is 405 nm, 10.0 μL of the probe mother solution is dissolved in 4 mL of PBS buffer (pH=7.4, 20 mM), and a test solution with a fluorescent probe concentration of 5 μM is ...

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Abstract

The invention relates to a ratiometric fluorescent probe for detecting avidin, and a synthesis method and an application thereof. The probe is obtained by carrying out a reaction of an intermediate 4-(2-N,N-diethylamino)acetyl)amino-1,8-naphthalic anhydride with biotin and the like, and the synthesis method has the advantages of simple operation and cheap raw material. The probe has two fluorescence peaks at 470 nm and 550 nm appearing at different ratios of water and methanol. A 20 mM phosphoric acid buffer solution has a fluorescence main peak at 470 nm; when 0-2.0 equivalent of avidin is added to the buffer solution, the fluorescence peak at 470 nm is weakened gradually while the fluorescence peak at 550 nm is gradually enhanced, so as to achieve the ratio detection of avidin.

Description

technical field [0001] The invention relates to the synthesis and application of a fluorescent probe for ratiometric detection of avidin. Background technique [0002] Protein is one of the main contents of life science research, and it is an important embodiment of cell and biological functions. The expression of various proteins is closely related to the normal physiological and life activities of human beings. Therefore, it is extremely important for protein detection, functional research, and real-time tracking. At present, there are mainly Kjeldahl method, biuret method, ultraviolet absorption method, Coomassie brilliant blue method, phenol reagent method, etc. for the detection of protein. These methods are cumbersome, time-consuming, and require a lot of preliminary preparation. Therefore, fluorescence detection technology has gradually been successfully applied to protein detection and functional research due to its advantages of high sensitivity, easy operation, a...

Claims

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Application Information

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IPC IPC(8): C09K11/06C07D495/04G01N21/64
Inventor 徐兆超乔庆龙
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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