CN113466015A - Staining reagent for distinguishing normal cells from cancer cells and preparation method and application thereof - Google Patents
Staining reagent for distinguishing normal cells from cancer cells and preparation method and application thereof Download PDFInfo
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- CN113466015A CN113466015A CN202110815980.0A CN202110815980A CN113466015A CN 113466015 A CN113466015 A CN 113466015A CN 202110815980 A CN202110815980 A CN 202110815980A CN 113466015 A CN113466015 A CN 113466015A
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- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 33
- 239000012128 staining reagent Substances 0.000 title claims abstract description 16
- 238000002360 preparation method Methods 0.000 title abstract description 6
- MPVDXIMFBOLMNW-ISLYRVAYSA-N 7-hydroxy-8-[(E)-phenyldiazenyl]naphthalene-1,3-disulfonic acid Chemical compound OC1=CC=C2C=C(S(O)(=O)=O)C=C(S(O)(=O)=O)C2=C1\N=N\C1=CC=CC=C1 MPVDXIMFBOLMNW-ISLYRVAYSA-N 0.000 claims abstract description 35
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 33
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
Abstract
The invention belongs to the technical field of biomedical detection, and particularly relates to a staining reagent for distinguishing normal cells from cancer cells, and a preparation method and application thereof. The invention takes an orange G solution as a carbon source, and prepares the orange G carbon dot solution by an electrolytic method, wherein the orange G carbon dot solution is a staining reagent for distinguishing normal cells from cancer cells. The detected cell sap treated by the staining reagent and the normal cell sap are placed under a fluorescence differential interference microscope, and the cell morphology is compared, so that whether the detected cell is cancerated or not can be judged. The method has the advantages of simplicity and quickness.
Description
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to a staining reagent for distinguishing normal cells from cancer cells, and a preparation method and application thereof.
Background
Medically, cancer refers to a malignant tumor that originates in epithelial tissue, and is the most common type of malignant tumor. Correspondingly, malignant tumors originating in mesenchymal tissue are collectively referred to as sarcomas. There are a few malignant tumors that are not named according to the above principles, such as wilms' tumor, malignant teratoma, etc. The general term "cancer" is used to generally refer to all malignant tumors. Tumor is a new organism formed by abnormal proliferation and differentiation caused by that cells of local tissues lose normal regulation and control on the growth of the cells at the gene level under the action of various tumorigenic factors of an organism. Once formed, the new organism does not stop growing because of elimination of the disease, and the growth of the new organism is not regulated by the physiology of a normal organism but destroys normal tissues and organs, which is particularly obvious in malignant tumors.
Normally, the size and shape of cells from the same tissue are substantially the same, and cancer cells are generally larger than corresponding normal cells, and the size and shape of cancer cells are also very different from each other, and "giant cells" of tumor having a large volume may appear. The cells contain cell nucleuses and cytoplasm, and under the normal condition, most cells only have one cell nucleus, and the cell nucleus and the cytoplasm have a certain proportion; the volume of the cell nucleus of the cancer cell is increased, the shape is inconsistent, and a megakaryocyte, a binuclear nucleus, a multikaryocyte or a heteromorphic nucleus can appear; cancer cells grow rapidly with obvious nucleoli, while normal cells have no apparent nucleoli.
There are many methods for detecting cancer cells, such as B-ultrasonic examination, CT examination, nuclear magnetic resonance examination, blood drawing examination of tumor markers, needle biopsy, endoscopic examination, etc. However, there are few reports in the prior art on methods for distinguishing normal cells and cells to be detected of the same tissue by using a staining agent and judging whether a canceration phenomenon occurs or not according to the morphology of the cells to be detected.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a staining reagent for distinguishing normal cells from cancer cells and a preparation method and application thereof, aiming at judging whether the detected cells are cancerated or not by placing detected cell sap treated by the staining reagent and normal cell sap under a fluorescence differential interference microscope and comparing cell forms, and the staining reagent has the advantages of simplicity and quickness.
A staining reagent for distinguishing normal cells from cancer cells is a carbon dot solution of exocarpium Citri rubrum G, wherein the mass concentration of exocarpium Citri rubrum G is 0.125%, the fluorescence excitation wavelength is 517nm, and the emission wavelength is 304 nm.
The preparation method of the staining reagent for distinguishing the normal cells from the cancer cells comprises the following steps:
(1) adopting PBS buffer solution as electrolyte to prepare 0.002mol/L orange G solution;
(2) and (3) electrolyzing by taking the orange G solution as a carbon source, and preparing the orange G carbon dot solution when the solution is gradually changed from a non-fluorescent solution to a yellow-green fluorescent solution.
Wherein the pH value of the PBS buffer solution is 7.
The voltage of the electrolysis process is 10V, and the electrolysis time is 8 min.
The invention also provides application of the staining reagent for distinguishing normal cells from cancer cells in cancer cell detection.
A method for detecting cancer cells, comprising the steps of:
(1) and (3) staining the detected cells: taking 100 mu L of detected cells, adding 1mL of PBS solution for rinsing, centrifuging at 3000rpm for 5min, discarding supernatant, replacing new PBS solution, fixing the volume to 1mL, sucking 20-50 mu L of orange G carbon dots with the mass concentration of 0.125%, adding the orange G carbon dots into the PBS solution, incubating for 10min, then sucking, rinsing with the PBS solution, and sucking to obtain detected cell sap;
(2) normal cell staining: adding normal cells of the same tissue into a centrifuge tube filled with PBS buffer solution, centrifuging, removing supernatant, sucking 20-50 μ L of the exocarpium citri grandis G carbon dot solution, placing in a centrifuged cell tube, incubating for 10min, completely sucking, rinsing with PBS solution, removing supernatant, and leaving the bottom suspended normal cell solution for later use;
(3) dripping 100 mu L of detected cell sap on the glass slide, then reversely buckling the stained detected cell slide on the glass slide, and placing the glass slide under a fluorescence microscope to observe cell imaging; meanwhile, 100 mu L of the normal cell solution after the dyeing treatment is absorbed and dropped on a glass slide, and the cell imaging is observed under a fluorescence microscope; and (4) judging whether the detected cells are cancerated or not by comparing the two groups of cell images.
Wherein the pH value of the PBS solution is 7.2-7.4.
Compared with the prior art, the invention has the characteristics and beneficial effects that:
the staining reagent for distinguishing normal cells from cancer cells, provided by the invention, can judge whether the detected cells are cancerated or not by placing the detected cell sap and the normal cell sap which are stained under a fluorescence differential interference microscope and comparing the cell morphology showing fluorescence.
The cancer cell detection method is simple and can detect under a common optical microscope.
Drawings
FIG. 1 is a graph of the UV and fluorescence spectra of a orange G carbon dot solution of the present invention;
(a) ultraviolet-visible spectrograms; (b) is a fluorescence spectrum;
wavelet: a wavelength; absorbance: absorption rate; PL intensity: the intensity of fluorescence; EX: a fluorescence emission wavelength; EM: a fluorescence excitation wavelength; chrysoidine G CDs: orange G carbon dots;
FIG. 2 is a TEM image of a chrysoidine G carbon dot solution of the present invention;
FIG. 3 is a comparison of cell images of the examined cells and normal cells after staining treatment;
(a1) a cell morphology diagram of the detected cell under a bright field; (a2) a cell morphology chart of the detected cells under a fluorescent condition; (b1) a cell morphology map showing normal cells in a bright field; (b2) shows the cell morphology of normal cells under fluorescent conditions.
Detailed Description
In order to make the technical solutions in the embodiments of the present invention better understood and make the above objects, features, and advantages of the present invention more comprehensible, specific embodiments of the present invention are described below with reference to the accompanying drawings.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual values, and between the individual values may be combined with each other to yield one or more new ranges of values, which ranges of values should be considered as specifically disclosed herein.
Example 1
Preparing orange G carbon dots:
in this example, exocarpium G (C) is used12H12N4HCl) as a carbon source, and preparing a red tangerine G carbon dot solution by an electrolysis method, wherein the electrolysis device consists of a 25mL glass bottle container and two platinum electrodes which are fixed on a bottle cover and have the length of 4cm, and the voltage is (220V/10V).
Preparing a 0.002mol/L orange G solution by using PBS buffer solution (pH 7) as an electrolyte, adding 16ml of the orange G solution into an electrolytic glass bottle, inserting an electrode, electrifying for 8min, and gradually changing the solution from the initial non-fluorescence to a yellow-green fluorescence solution, which indicates that carbon dots are formed, namely preparing the orange G carbon dot solution.
As shown in fig. 1 and fig. 2, wherein fig. 1 is the ultraviolet and fluorescence spectra of the orange G carbon dot solution of the invention, and fig. 2 is the TEM electron micrograph of the orange G carbon dot solution of the invention. Wherein FIG. 1a shows a UV-visible spectrum; FIG. 1b shows the fluorescence spectrum, and it can be seen from FIG. 1b that the fluorescence excitation wavelength of the orange G carbon dot solution is 517nm, and the emission wavelength is 304 nm.
Example 2
Cell staining:
and (3) cancer cell staining: and (3) rinsing the cultured liver cancer cell HepG2 reptile with a PBS (pH 7.2-7.4) solution for 2 times, sucking 1.5ml of prepared exocarpium citri rubrum G carbon dot solution into the cell, incubating for 10min, rinsing twice with the PBS solution, and sucking off. And dripping 10 mu L of cell culture solution on the glass slide, then reversely buckling the processed cell slide on the glass slide, and observing cell imaging under a fluorescence microscope.
Staining oral epithelial cells: taking buccal mucosa cells twice by using a clean cotton swab, immersing the buccal mucosa cells in a centrifuge tube filled with 1.5ml of PBS (pH 7.2-7.4), centrifuging the cells for 6min at 500rpm, discarding supernatant, sucking 1.5ml of prepared orange G carbon dot solution, placing the orange G carbon dot solution in a centrifuged cell test tube, incubating the orange G carbon dot solution for 10min, completely sucking the orange G carbon dot solution, rinsing the orange G carbon dot solution twice by using PBS solution, discarding supernatant liquid, and leaving cell solution of a bottom suspension part. Then, 30. mu.L of the treated cell solution was pipetted onto a glass slide and placed under a fluorescent microscope to observe cell imaging.
Example 3
And (3) microscopic observation test:
placing cancer cells and oral epithelial cell slides treated by the orange G carbon dot solution under a fluorescence microscope, finding clear cell forms in a bright field by adopting a 40X objective lens, taking a picture, then switching on a mercury lamp to an optical filter corresponding to the excitation wavelength and the emission wavelength of the orange G carbon dot material, turning off an external light source, observing cells showing fluorescence after the orange G carbon dot solution is dyed in a dark environment, and comparing after taking the picture.
FIG. 3 shows a contrast image of the cell images of the examined cells and normal cells after staining treatment. Wherein FIG. 3a1 shows the cell morphology of the tested cell under bright field, and FIG. 3a2 shows the cell morphology of the tested cell under fluorescent condition; FIG. 3b1 shows the cell morphology of normal cells under bright field, and FIG. 3b2 shows the cell morphology of normal cells under fluorescent condition.
As can be seen from FIG. 3, after the cancer cells and the normal cells are stained by the orange G carbon dot solution, the cell nucleus volume of the cancer cells can be seen to be increased through microscope observation, 2-4 cell nuclei are shown, the cell morphology is inconsistent, the cell nuclei of the normal cells are not obvious, and the size and the shape are consistent; moreover, the cancer cells are propagated quickly, the nucleoli of the cells are obvious, and the nucleoli of the cells of the normal cells are not obvious.
Therefore, after the detected cells and normal cells are dyed by the orange G carbon dot solution, the cell morphology is observed and compared by a fluorescence microscope, whether the detected cells are cancerated or not can be judged, and the detection method is simple to operate.
The staining reagent for distinguishing normal cells from cancer cells provided by the present invention can be applied to the detection of liver cancer cells in example 2, and is also suitable for the detection of other cancer cells.
The embodiments of the present invention are described in detail above with reference to the drawings, but the present invention is not limited to the described embodiments. Various changes, modifications, substitutions and alterations to these embodiments will occur to those skilled in the art without departing from the spirit and scope of the present invention.
Claims (6)
1. A staining reagent for distinguishing normal cells from cancer cells is characterized in that a carbon dot solution is prepared by taking orange G as a carbon source, the mass concentration of the orange G is 0.125%, the fluorescence excitation wavelength is 517nm, and the emission wavelength is 304 nm.
2. A method for preparing a staining reagent for distinguishing between normal cells and cancer cells according to claim 1, comprising the steps of:
(1) adopting PBS buffer solution as electrolyte to prepare 0.002mol/L orange G solution;
(2) and (3) electrolyzing by taking the orange G solution as a carbon source, and preparing the orange G carbon dot solution when the solution is gradually changed from a non-fluorescent solution to a yellow-green fluorescent solution.
3. The method of claim 2, wherein the PBS buffer pH is 7.
4. The method of claim 2, wherein the electrolysis process is performed at a voltage of 10V for 8 min.
5. A method for detecting cancer cells, comprising the steps of:
(1) and (3) staining the detected cells: taking 100 mu L of detected cells, adding 1mL of PBS solution for rinsing, centrifuging at 3000rpm for 5min, discarding supernatant, replacing new PBS solution to fix the volume to 1mL, sucking 20-50 mu L of an orange G carbon dot solution with the mass concentration of 0.125% of orange G, adding the orange G carbon dot solution into the PBS solution, incubating for 10min, then completely sucking, rinsing with the PBS solution, and then completely sucking to obtain detected cell sap;
(2) normal cell staining: adding normal cells of the same tissue into a centrifuge tube filled with PBS buffer solution, centrifuging, removing supernatant, sucking 20-50 μ L of the exocarpium citri grandis G carbon dot solution, placing in a centrifuged cell tube, incubating for 10min, completely sucking, rinsing with PBS solution, removing supernatant, and leaving the bottom suspended normal cell solution for later use;
(3) dripping 100 mu L of detected cell sap on the glass slide, then reversely buckling the stained detected cell slide on the glass slide, and placing the glass slide under a fluorescence microscope to observe cell imaging; meanwhile, 100 mu L of the normal cell solution after the dyeing treatment is absorbed and dropped on a glass slide, and the cell imaging is observed under a fluorescence microscope; and (4) judging whether the detected cells are cancerated or not by comparing the two groups of cell images.
6. The method of claim 5, wherein the PBS solution has a pH of 7.2-7.4.
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Citations (7)
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|---|---|---|---|---|
| CN103232029A (en) * | 2012-12-25 | 2013-08-07 | 首都医科大学 | Preparation method and application of green fluorescent carbon dots |
| CN103436257A (en) * | 2013-08-27 | 2013-12-11 | 湖南师范大学 | Method for preparing fluorescent carbon dots (C-dots) through electrochemical carbonization of ketone |
| CN104787742A (en) * | 2014-01-16 | 2015-07-22 | 中国药科大学 | Method for preparing fluorescent carbon nanoparticles by spontaneous reaction |
| CN105316697A (en) * | 2015-12-09 | 2016-02-10 | 青岛大学 | Preparation method for solid-state carbon quantum dot |
| CN107254309A (en) * | 2017-07-01 | 2017-10-17 | 中国科学院兰州化学物理研究所 | A kind of preparation method of carbon quantum dot |
| CN110982513A (en) * | 2019-11-29 | 2020-04-10 | 郑州大学 | Preparation method of fluorescent carbon dots and application of fluorescent carbon dots in cell imaging |
| WO2021087646A1 (en) * | 2019-11-04 | 2021-05-14 | Beijing Normal University | Carbon quantum dots and uses thereof |
-
2021
- 2021-07-20 CN CN202110815980.0A patent/CN113466015B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103232029A (en) * | 2012-12-25 | 2013-08-07 | 首都医科大学 | Preparation method and application of green fluorescent carbon dots |
| CN103436257A (en) * | 2013-08-27 | 2013-12-11 | 湖南师范大学 | Method for preparing fluorescent carbon dots (C-dots) through electrochemical carbonization of ketone |
| CN104787742A (en) * | 2014-01-16 | 2015-07-22 | 中国药科大学 | Method for preparing fluorescent carbon nanoparticles by spontaneous reaction |
| CN105316697A (en) * | 2015-12-09 | 2016-02-10 | 青岛大学 | Preparation method for solid-state carbon quantum dot |
| CN107254309A (en) * | 2017-07-01 | 2017-10-17 | 中国科学院兰州化学物理研究所 | A kind of preparation method of carbon quantum dot |
| WO2021087646A1 (en) * | 2019-11-04 | 2021-05-14 | Beijing Normal University | Carbon quantum dots and uses thereof |
| CN110982513A (en) * | 2019-11-29 | 2020-04-10 | 郑州大学 | Preparation method of fluorescent carbon dots and application of fluorescent carbon dots in cell imaging |
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