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Active cell imaging method by using fluorescence probe under extreme pH value

A fluorescent imaging and fluorescent probe technology, applied in the field of analytical chemistry, can solve the problems of fluorescence intensity interference, easy quenching stability, and short fluorescence lifetime, and achieve good stability, good membrane penetration, and strong visibility Effect

Inactive Publication Date: 2016-05-04
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In practical applications, these molecules have the disadvantages of short fluorescence lifetime, easy quenching and poor stability.
In particular, the fluorescence of most protonated fluorescent probes shows a single weakening change with the increase of pH value. It is shown as acidic "Off" and alkaline "On", and it is rare to realize cell imaging under the extreme medium conditions of strong acid and strong alkali
It has been reported that the fluorescence intensity of some types of fluorescent probes will be interfered to varying degrees by the biologically relevant metal ions, anions, amino acids, sugars and other biological small molecules that coexist in the system.

Method used

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  • Active cell imaging method by using fluorescence probe under extreme pH value
  • Active cell imaging method by using fluorescence probe under extreme pH value
  • Active cell imaging method by using fluorescence probe under extreme pH value

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment one: the preparation method of each solution and reagent in the present invention.

[0032] (1) Preparation method of probe solution: weigh 2.8 mg of probe (molecular formula: C 17 h 13 NO 3 Molecular weight: 279.09), dissolved in acetonitrile / water (v / v, 1 / 20), and prepared into a 100mL solution with a concentration of 100mM.

[0033] (2) Preparation method of probe solutions with different pH values: Weigh 2.8 mg of probe (molecular formula: C 17 h 13 NO 3 Molecular weight: 279.09), dissolved in acetonitrile / water (v / v, 1 / 20), adjusted to pH 2.0, 4.0 and 10.0, 11.4 with Tris-HCl and HEPES-NaOH (50mM) respectively, and prepared into a 100mL solution with a concentration of 100mM.

[0034] (3) Tris-HCl buffer solution: Prepare with a concentration of 50mM Tris (Tris) and 50mM HCl, and adjust the pH to the required value;

[0035] (4) HEPES buffer solution: Prepare with 50mM 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) and 5mM NaOH, adjust the pH...

Embodiment 2

[0043] Example 2: Preparation of probe compounds.

[0044] Using 8-hydroxyquinaldine and 2,4-dihydroxybenzaldehyde as raw materials, using acetic anhydride and pyridine / water as solvents, firstly synthesize the intermediate, and then hydrolyze the intermediate in a mixed solvent of pyridine / water. The synthetic route is as follows:

[0045]

[0046] In the there-necked flask, in the acetic anhydride solution that is dissolved with 8-hydroxyquinaldine, add 2,4-dihydroxybenzaldehyde, by molar ratio 8-hydroxyquinaldine:2,4-dihydroxybenzaldehyde equals 1: 2. Under the protection of nitrogen, reflux, the reaction is completed, concentrated to remove the solvent acetic anhydride, and eluted by silica gel column chromatography to obtain an intermediate. Reaction temperature: 139°C (reflux), reaction time: 5h, reaction solvent: acetic anhydride, eluent: volume ratio chloroform: ethyl acetate (3:1).

[0047] N 2 Under protection, add the intermediate in the three-necked flask, py...

Embodiment 3

[0048] Example 3: Fluorescence microscopy imaging of living PC3 cells.

[0049] (1) Recovery cells

[0050] Take out the PC3 cells from the -80°C refrigerator, place them in 37°C water and shake the cell cryopreservation tube quickly, and thaw them completely within 1-2 minutes. In the aseptic operating table, suck them into the centrifuge tube, and add about 11ml of culture medium (Containing 10% fetal bovine serum, 1% double antibody solution) mix well, centrifuge the cell suspension at 1000r / min centrifuge for 5min, remove the supernatant, blow and mix the precipitated cells at the bottom with culture medium Transfer to a culture bottle so that the volume of the culture solution in the culture bottle is within 5-7mL, and place it at 37°C, containing 5% CO 2 cultured in an incubator.

[0051] (2) Observation, subculture, plate transfer

[0052] Change the culture medium once a day, and observe the cell growth under a microscope until the PC3 cells adhere to the wall and c...

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Abstract

The invention discloses an active cell imaging method by using a fluorescence probe under an extreme pH value, and belongs to the field of analytical chemistry. A quinaldine derivative namely (E)-2-(2,4-dihydroxylphenyl)vinyl-8-hydroxyl quinoline is taken as the fluorescence imaging probe (probe for short) for detecting pH of active cells. The method comprises the following steps: preparing a probe solution with a pH of 2-4 and a probe solution with a pH of 10-12 from an acetonitrile buffer solution; incubating the probe with active cells, and detecting the cells by a fluorescence inverted microscope to obtain a cell image giving off green fluorescence and a cell image giving off red fluorescence, wherein the color and strength of the fluorescence of cells change along with the change of pH value; and the fluorescent image of cells cannot be observed when the pH is in a range of 5 to 9. The chemical structure of the probe is represented in the description.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, in particular to a method for detecting pH in active cells by using a fluorescent probe. Background technique [0002] The combination of fluorescent probes and fluorescence microscopy has made fluorescent probes widely used in the detection of biologically active substances and cell imaging. For biologically active substances, the detection sensitivity limit is a single molecule, which requires higher sensitivity of the testing technology and more stringent testing conditions. Fluorescence imaging analysis is a highly sensitive visualization analysis technique widely used in live cell analysis. Live cell imaging technology is an important research method in the field of life science and technology. Combined with fixed cell research, it can explain a variety of life phenomena in living cells. In cell biology, the measurement of intracellular pH, cell proliferation and apoptosis, endocytosis,...

Claims

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Application Information

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IPC IPC(8): G01N21/64C07D215/26C09K11/06
CPCC07D215/26C09K11/06C09K2211/1029G01N21/6428G01N21/6458
Inventor 曾晞朱勤牟兰吴玉田曾莉张红
Owner GUIZHOU UNIV
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