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Ratio-type pH fluorescent probe for water-soluble localization of lysosomes, its preparation method, application and test method

A technology of fluorescent probes and lysosomes, applied in the field of ratiometric pH fluorescent probes, can solve problems affecting the accuracy of detection results, achieve significant changes in fluorescence intensity, strong anti-interference ability, and improve detection accuracy

Active Publication Date: 2018-08-17
TAISHAN MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • 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

Method used

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  • Ratio-type pH fluorescent probe for water-soluble localization of lysosomes, its preparation method, application and test method
  • Ratio-type pH fluorescent probe for water-soluble localization of lysosomes, its preparation method, application and test method
  • Ratio-type pH fluorescent probe for water-soluble localization of lysosomes, its preparation method, application and test method

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Experimental program
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Effect test

Embodiment 1

[0106] Prepare 20 mL of the above 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 test 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 almost no 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 2 h. Observed under a confocal laser microscope. The excitation wavelength is fixed at 405nm, and the emission bands are the fluorescence images of the blue channel (400-555 nm) and the fluorescence images of the red channel (560-700 nm). 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

A ratiometric pH fluorescent probe for water-soluble localization of lysosomes, its preparation method, application and testing method, its chemical structural formula is shown in the following formula: the probe of the present invention can simultaneously detect the fluorescence intensity of two emission wavelengths Changes, eliminate external influences through self-calibration, high detection accuracy, rapid response to pH changes, excellent water solubility, and good anti-interference ability in the presence of various metal ions. Fluorescence intensity changes significantly in the pH range from 5.8 to 6.8, which is suitable for pH detection under weak acidic conditions. Experiments have proved that the probe can selectively stain lysosomes in cells, has good photostability in cells, is nontoxic to cells, and can be used for pH detection of living cells. The probe of the invention has important application value in cell imaging and detection of pH value changes in lysosomes.

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 Patents(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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