Fluorescent probe method for monitoring Zn<2+> in cancer cells

A fluorescent probe, cancer cell technology, applied in the field of analytical chemistry, can solve many problems and achieve the effects of high luminescence yield, good membrane penetration, and good water solubility

Active Publication Date: 2015-04-08
广西平果润民脱贫发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been many reports on the use of probes to detect zinc ions, but there are not many that can be applied to imaging in living cells

Method used

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  • Fluorescent probe method for monitoring Zn&lt;2+&gt; in cancer cells
  • Fluorescent probe method for monitoring Zn&lt;2+&gt; in cancer cells
  • Fluorescent probe method for monitoring Zn&lt;2+&gt; in cancer cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment one: the preparation method of each solution, reagent among the present invention

[0038] (1) Preparation of probe solution: Weigh 40.4 mg of probe, dissolve it in DMF, and prepare a 100 mL solution with a concentration of 1.00×10 -3 mol L -1 ; According to the need, gradually dilute with saline to the appropriate concentration.

[0039] (2) Zn 2+ Standard solution: Weigh 59.5mg of analytically pure Zn(NO 3 ) 2 , dissolved in normal saline, and prepared into 100mL solution with a concentration of 2.00×10 -3 mol L -1 ; According to the need, gradually dilute with saline to the appropriate concentration.

[0040] (3) Preparation of other coexisting ionic solutions: Take analytically pure nitrates or hydrochlorides of various metals, dissolve them in double distilled water, and prepare them at a concentration of 2.00×10 -3 mol L -1 double distilled aqueous solution.

[0041] (4) 75% ethanol solution: Add 75mL of absolute ethanol to 100mL with distill...

Embodiment 2

[0048] Embodiment 2: Probe pair Zn 2+Selective fluorescence detection of ions

[0049] Add the DMF stock solution of the probe (1.00×10 -4 mol L -1 , 1mL), metal ion Zn 2+ (2.00×10 -3 mol L -1 , 1 mL), with DMF / H 2 Dilute the O (v / v, 1 / 4) solution to the mark, shake well, and transfer it into a 1cm quartz cuvette for fluorescence spectrum measurement.

[0050] Set the fluorescence excitation wavelength to 360nm, add about 3ml probe (1.00×10 -5 mol L -1 ) DMF / H 2 O (v / v, 1 / 4) solution was tested for fluorescence spectrum, and the probe had no fluorescence emission at a wavelength of 457nm. Join Zn 2+ (2.00×10 -4 mol L -1 ), the probe solution emits strong blue fluorescence. Under the same conditions, Li was added to the probe solution respectively + , Na + , K + , Mg 2+ , Ca 2+ , Ba 2+ ,Sr 2+ , Hg 2+ ,Co 2+ , Ni 2+ , Cu 2+ , Ag + , Pb 2+ , Cd 2+ , Al 3+ , Cr 3+ , Fe 3+ After adding metal ions, the fluorescence spectrum and intensity of the pro...

Embodiment 3

[0052] Example 3: Fluorescence microscopy imaging of living PC3 cells

[0053] (1) Recovery cells

[0054] 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 evenly, centrifuge the cell suspension at 1000r / min for 5min, remove the supernatant ， Gently blow and break up the cells and culture medium precipitated at the bottom and transfer them to the culture bottle so that the volume of the culture medium in the culture bottle is within 5-7mL, and place it at 37 ℃, containing 5% CO 2 cultured in an incubator.

[0055] (2) Observation → passage → transfer to plate

[0056] Change the culture medium once a day, and observe the cell growth under a microscope until the PC3 cells adhere to t...

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Abstract

The invention discloses a fluorescent probe method for monitoring Zn<2+> in cancer cells. The fluorescent probe method utilizes bis-(7-hydroxy-coumarin-8-aldehyde)ethylenediamine as a fluorescent probe of a trace amount of Zn<2+>. Through the selectively-formed probe-Zn<2+> complex characteristic of emitting 457nm wavelength strong fluorescence, the probe and Zn<2+> are respectively permeated into active cancer cells so that fusion with the cells is realized. After Zn<2+> in cells is dyed by the probe, a distinct blue fluorescent cell image is detected and showed by an inverted fluorescence microscope. The probe is used as a fluorescent imaging reagent of Zn<2+> in active cancer cells. The probe has a chemical structural formula shown in the following description.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, in particular to a method for Zn in living cancer cells 2+ Fluorescent probe method of detection. Background technique [0002] Cell is the basic unit of life activities and the basic unit of all organic life forms. Cell research is the starting point of all life science research, and its growth, aging and other periodic phenomena will directly affect the life activities of organisms. Some heavy metals and transition metal elements have very important physiological roles and functions because of their participation in cell growth and development, gene transcription, neurotransmission, etc. It will cause obvious damage to the shape and structure. Therefore, to study the effects of various ions on organisms, a very necessary step is the online visualization and real-time monitoring of ions in living cells. Live cell imaging technology is an important research method in the field of life scien...

Claims

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Application Information

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IPC IPC(8): C12Q1/02G01N21/64C07D311/16
Inventor 袁剑英李丽沈韵曾晞牟兰
Owner 广西平果润民脱贫发展有限公司
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