Ratio-type pH fluorescence probe for water-soluble locating lysosome as well as preparation method, application and test method of ratio-type pH fluorescence probe

A technology of fluorescent probes and lysosomes, which is applied in the field of ratiometric pH fluorescent probes, can solve problems affecting the accuracy of detection results, etc., and achieve the effects of significant changes in fluorescence intensity, good photostability, and strong anti-interference ability

Active Publication Date: 2016-04-27
TAISHAN MEDICAL UNIV
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  • Description
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  • Application Information

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Problems solved by technology

Therefore, the ratiometric pH fluorescent probe for water-soluble positioning lysosomes, its preparation method, application and testing method are methods for quantitative detection of the pH value of lysosomes for cell analysis or diagnosis. In the pH fluorescent probe and its preparation method and application, the methods for detecting intracellular pH mainly include nuclear magnetic resonance, absorption spectroscopy and fluorescence spectroscopy, etc. Fluorescence detection technology has been widely used due to its high sensitivity, easy operation, and real-time monitoring. application, but most fluorescent probes detect the change of fluorescence intensity at a single wavelength, and the output signal of such probes will be affected by temperature, solvent polarity, and probe concentration, which will affect the accuracy of the detection results

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  • Ratio-type pH fluorescence probe for water-soluble locating lysosome as well as preparation method, application and test method of ratio-type pH fluorescence probe
  • Ratio-type pH fluorescence probe for water-soluble locating lysosome as well as preparation method, application and test method of ratio-type pH fluorescence probe
  • Ratio-type pH fluorescence probe for water-soluble locating lysosome as well as preparation method, application and test method of ratio-type pH fluorescence probe

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Experimental program
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Embodiment 1

[0106] Prepare 20 mL of the above-mentioned fluorescent probe RMPM (10 μM) at different pH values ​​(ethanol / Britton--Robinson buffer solution, 40 mM acetic acid, phosphoric acid, boric acid, 1:4, volume ratio), and measure the fluorescence spectrum by fluorescence spectrophotometry . figure 2 (a) and the fluorescent intensity ratio (I) of the probe in the pH value (5.6-7.2) region 580 / I 426 ) figure 2 (b).

[0107] The results show that: the ratiometric pH fluorescent probe used for water-soluble localization of lysosomes, that is, the terminal compound RMPM, has a significant change in fluorescence intensity in solutions with a pH of 5.8 and 6.8, and the fluorescence emission ratio I 580 / I 426 It reached the peak at 4 min and remained stable at room temperature, which indicated that RMPM can respond quickly to pH changes with high detection accuracy. ( figure 2 , 4 )

Embodiment 2

[0109] Add AgNO respectively in the solution (ethanol / Britton-Robinson buffer solution, 40mM acetic acid, phosphoric acid, boric acid, 1:4, volume ratio) containing pH fluorescent probe RMPM (10 μ M) under the condition of pH value 5.6 and 7.2, Al(NO3)3, Ba(NO3)2, Cd(NO3)2, Co(NO3)2, Cu(NO3)2, Fe(NO3)3, HgCl2, Ni(NO3)2, Pb(NO3)2, The aqueous solution of Zn(NO3)2, the concentration of all metal cations is 100 μ M, test the fluorescence intensity ratio I in the solution that contains different metal ions 580 / I 426 (λ ex =340nm, image 3 ).

[0110] The results show that the ratiometric pH fluorescent probe used for water-soluble localization of lysosomes, namely the terminal compound RMPM, has little response to the above metal ions under the conditions of pH=5.6 and pH=7.2, which proves that the probe is sensitive to H + Has high selectivity. The probe RMPM has strong anti-interference ability to common metal ions.

Embodiment 4

[0112] Hela cells were incubated with probe RMPM (1, 2 or 5 μM) at 37°C for 2h. Observed under a confocal laser microscope. The excitation wavelength is fixed at 405nm, and the emission bands are respectively the fluorescence images of the blue channel (400-555nm) and the fluorescence images of the red channel (560-700nm). Both the blue and red fluorescence intensities in Hela cells increased with the increase of the probe concentration, which indicated that the ratiometric pH fluorescent probe for water-soluble localization of lysosomes, the terminal compound RMPM, had the ability to stain living cells ( Figure 5 ).

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Abstract

The invention provides a ratio-type pH fluorescence probe for water-soluble locating lysosome as well as a preparation method, an application and a test method of the ratio-type pH fluorescence probe. The chemical structural formula of the ratio-type pH fluorescence probe is shown in the specification. The probe can detect changes of strength of fluorescence with two emission wavelengths simultaneously, external influences are eliminated through self-calibration, the detection accuracy is high, the speed of response to pH changes is high, and the probe has excellent water solubility and has very good anti-interference capacity in the existence of various metal ions. The fluorescence strength changes remarkably in the pH range from 5.8 to 6.8, and the probe is suitable for pH detection under a weak acidic condition. Experiments prove that the probe can selectively dye lysosome in cells, the light stability in the cells is good, and the probe is non-toxic to the cells and can be used for pH detection of live cells. The probe has very important application value in cell imaging and detection of changes of the pH value in lysosome.

Description

1. Technical field [0001] The invention relates to a ratio-type pH fluorescent probe, its preparation method, application and testing method, especially a ratio-type pH fluorescent probe for water-soluble localization of lysosomes, its preparation method, application and testing method. 2. Background technology [0002] Intracellular pH plays an important role in many cellular behaviors, such as cell growth, apoptosis, signal transduction and cell proliferation, etc. As an important indicator of cellular health, small changes in intracellular pH can lead to cellular abnormalities and even cellular dysfunction and serious diseases such as cancer and Alzheimer's disease. The pH inside the cells is not the same, and the cytoplasm is slightly alkaline. The internal pH of lysosomes is about 4.0-6.0, which is weakly acidic. The mildly acidic environment inside the lysosome activates the function of internal hydrolases to break down proteins in cellular metabolism. Therefore, th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D519/00C09K11/06G01N21/64G01N21/80
CPCC07D519/00C09K11/06C09K2211/1088G01N21/6428G01N21/80
Inventor 曹晓群宋光杰罗京赵宝祥
Owner TAISHAN MEDICAL UNIV
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