CN108484621B - Dye probe for tracking and detecting morphological change of single cell and preparation method and application thereof - Google Patents
Dye probe for tracking and detecting morphological change of single cell and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/10—Spiro-condensed systems
- C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B57/00—Other synthetic dyes of known constitution
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
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- C09K2211/1014—Carbocyclic compounds bridged by heteroatoms, e.g. N, P, Si or B
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
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- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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- C09K2211/1059—Heterocyclic compounds characterised by ligands containing three nitrogen atoms as heteroatoms
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
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Abstract
The invention relates to a dye probe for tracking and detecting the morphological change of a single cell, a preparation method and application thereof, wherein the structural formula of the dye probe is shown in the specification
The method is applied to tracking and detecting the morphological change of the single cell polluted by the mercury ions. The invention has good tracking effect on the morphological change of the single cell caused by the concentration of the mercury ions; the product is in the form of solid powder, is convenient to use and store, and has the advantages of simple synthesis method, high yield, low cost and good application prospect.
Description
Technical Field
The invention belongs to the field of tracking and detecting single cells, and particularly relates to a dye probe for tracking and detecting morphological changes of single cells, and a preparation method and application thereof.
Background
The heavy metal mercury is a substance forming the earth crust and is very widely distributed in nature. The natural environment has the lowest content in each part of the natural environment, and if the content exceeds the standard, immeasurable pollution and harm can be caused to water, soil and human bodies. The normal mercury content in human body is lower than 0.01mg/L, the mercury is chronic poisoning in the period of 0.01mg/L-0.5mg/L, the incidence rate is accelerated along with the increase of the mercury content accumulated in the human body, and when the mercury content reaches 0.5mg/L, the concentration is acute poisoning, and the human body can lose life even in a few hours. Chronic poisoning can cause corresponding diseases such as canceration and the like along with the accumulation of mercury ions in the body.
The mercury element in the environment enters the human body through the action of a food chain, the content of the mercury element taken by the human body through food is directly related to the content of the mercury element in soil at the site of the human body in blood, and the increase of the content of the mercury element can cause a series of canceration of cells, particularly the mortality rate of liver cancer, nasopharyngeal cancer, breast cancer and leukemia and the content of the mercury element are directly related, and the higher the content is, the faster the morbidity is. Cancer begins with the change of cells in the human body, and the canceration of the cells is caused by the possibility that normal cells are converted into cancer cells when the cells are exposed to radioactive substances for a long time, or chemical substances or certain viruses. The most prominent feature of normal cell canceration is that the morphology and structure of the normal cell changes, for example, normal fibroblasts are in the shape of a flat spindle and become spherical when the cell becomes cancerous. This change is gradual, with the morphology of normal cells slowly changing into cancer cells.
Chinese patent '201110211911.5' reports in the multifunctional probe for single cell detection: temperature measurement, membrane potential measurement, ion channel detection, pH detection and ion concentration detection are utilized. The probe comprises an intracellular temperature measurement thin film thermocouple and three microelectrodes, wherein the three microelectrodes are as follows: a membrane potential measuring and ion channel detecting microelectrode, a pH value measuring microelectrode and an ion concentration detecting microelectrode; the three microelectrodes are all conical, so that the common measurement of parameters can be realized, and the structure and the property of a cell layer can be better researched.
Chinese patent '201310052402.1' reports a unicellular detector based on nano-fiber probe and its probe manufacturing method, including nano-probe, light source unit, micro-manipulation system, point location detection unit, cell positioning system and photon detection unit, the innermost layer of nano-probe is fiber layer, the outer wall of fiber layer is wrapped with nano-ring electrode layer, and the outer wall is wrapped with insulating layer. The invention has high sensitivity, can realize single cell level detection, greatly reduces the required cell number and improves the success rate of early disease detection compared with the traditional detection means which needs to crush tens of thousands of cells.
The invention relates to an electrode sensor, in particular to a potential micro-electrode sensor for single cell detection and application thereof, belonging to the Chinese patent '201710700839. X'. The potential microelectrode sensor is a polymer film ion selective microelectrode and consists of a carbon fiber microelectrode, a PEDOT transfer layer and a polymer ion selective film. The invention has the advantages of high sensitivity, simple and convenient manufacture, low cost, easy miniaturization and the like, simultaneously has the advantages of high mass transfer rate of the microelectrode, high current density, high response speed and the like, and can realize the detection of various ion concentration changes on the surfaces of various single cells under various environmental stimuli.
Based on the published patent, the current single cell detection method is realized based on electrochemical probes, and the method generally has the defects of simple operation and complexity. The change of ion concentration on the surface of a single cell is detected, but the change of cell morphology is not detected, but the most basic characteristic of diseases such as cancer is that the change is very large with the morphology of normal cells, and the morphology of the normal cells is very weak at the beginning of the attack. Therefore, the detection of the morphological change of the single cell is very important, and the occurrence and the development of the treatment diseases can be predicted.
Disclosure of Invention
The invention aims to solve the technical problem of providing a dye probe for tracking and detecting the morphological change of single cells, a preparation method and application thereof, wherein the dye probe has a good tracking effect on the morphological change of the single cells caused by the concentration of mercury ions; the product is in the form of solid powder, is convenient to use and store, and has the advantages of simple synthesis method, high yield, low cost and good application prospect.
The invention provides a dye probe for tracking and detecting the morphological change of single cells, wherein the structural formula of the dye probe is shown in the specification
The invention also provides a preparation method of the dye probe for tracking and detecting the morphological change of the single cell, which comprises the following steps:
(1) dissolving p-aminobenzoyl hydrazine in a solvent, heating to 40-50 ℃ under the protection of inert gas, stirring for dissolving, dropwise adding phenyl isothiocyanate, refluxing for 1-2h, cooling, and recrystallizing to obtain an intermediate; wherein the molar ratio of the p-aminobenzoyl hydrazine to the phenyl isothiocyanate is 1.0: 1.0-1.1;
(2) dissolving the intermediate obtained in the step (1) in a solvent to obtain a solution, and controlling the temperature in an ice bath within 0-5 ℃; dissolving cyanuric chloride, controlling the pH value of a reaction system to be 6.5-7.5, and stirring and reacting at 0-5 ℃ for 5-7h to obtain a mixed solution; dissolving fluorescein hydrazide, heating to 60-70 ℃, stirring for 1-1.5h, continuously controlling the pH value of the reaction system, reacting for 2-4h, performing suction filtration, rotary evaporation, column chromatography separation, and recrystallizing to obtain the required product; wherein the molar ratio of the intermediate, cyanuric chloride to fluorescein hydrazide is 1.0: 1.0-1.1.
The solvent of the p-aminobenzoyl hydrazine in the step (1) is ethanol; the inert gas is nitrogen.
The solvent of the intermediate in the step (2) is anhydrous tetrahydrofuran; the solvent of the cyanuric chloride is acetone; the solvent of the fluorescein hydrazide is anhydrous tetrahydrofuran.
NaHCO is adopted in the step (2)3The solution controls the pH value of the reaction system.
And (3) recrystallizing in the steps (1) and (2) by adopting absolute ethyl alcohol.
The solvents adopted for column chromatography separation in the step (2) are chloroform and ethanol with the volume ratio of 28:1-32: 1.
The invention also provides an application of the dye probe for tracking and detecting the morphological change of the single cell, and the application method of the dye probe comprises the following steps:
(1) the dye probe was dissolved in a solvent to make 0.9 × 10-2M-1.1×10-2A dye probe stock solution of M; weighing mercury ion salts, dissolving the mercury ion salts in a solvent, and preparing a mercury ion solution;
(2) diluting the dye probe stock solution in the step (1), adding a BSA solution, and fixing the volume to obtain a dye probe solution; after the cells are cultured, mercury ion solutions with different concentrations are added into different pore plates, after the cells are cultured for 4-6h, a dye probe solution is added for culturing for 20-30min, and the cells are imaged by an inverted fluorescence microscope, so that tracking observation can be carried out.
The solvent in the step (1) is absolute ethyl alcohol; the mercury ion salt is mercury perchlorate.
The concentration of the BSA solution in the step (2) is 0.9-1.1 g/L; the adding amount is 1.6-1.8% of the volume of the probe liquid with the concentration to be used.
The volumes of the mercury ion solutions with different concentrations added in the step (2) are all 100 uL.
And (3) adding a dye probe solution into the step (2), culturing for 20-30min, and washing for 3-5 times by using a PBS (phosphate buffer solution).
The principle of the invention is as follows:
cyanuric chloride is used as a bridging group to link thiourea and fluorescein hydrazide, and mercury ions are complexed with N-O bonds of fluorescein lactam and S-O bonds in thiosemicarbazide, so that the ring opening of the lactam is caused, the fluorescence is enhanced, and the 'on-off' type dye probe is formed. The dye probe is dissolved in ethanol to prepare a probe solution, a mercury ion solution is prepared in the same way, BSA is used for dropwise adding to adjust the fluorescence intensity of a complexing system after complexing (when bovine serum albumin is added into probe molecules, the probe molecules can be regularly arranged in a protein double-helix structure due to the hydrogen bond induction effect in the protein double-helix structure, and the aggregation-induced luminescence effect is achieved. The invention can apply the amount of mercury ions with different concentrations and the same volume when culturing cells to realize the tracking imaging of the morphological change of single cells.
The preparation route is as follows:
advantageous effects
The invention has good tracking effect on the morphological change of the single cell caused by the concentration of the mercury ions; the product is in the form of solid powder, is convenient to use and store, and has the advantages of simple synthesis method, high yield, low cost and good application prospect.
Drawings
FIG. 1 is a graph showing the results of the toxicity test of the dye probe to the cells in example 3, wherein the abscissa represents the concentration of the added dye probe and the ordinate represents the survival rate of the cells.
FIG. 2 shows the results of the selectivity test of the dye probe for metal ions in example 4, with the abscissa representing the wavelength of the fluorescence spectrum and the ordinate representing the fluorescence intensity.
FIG. 3 is a graph of the effect of coexisting ions on the fluorescence intensity of a mercury ion dye probe system in example 5; the abscissa is the metal ion species and the ordinate is the fluorescence intensity; the black column is the fluorescence intensity of the probe complexed with the metal ion, and the gray column is the dye probe/Hg2+The fluorescence intensity of the system after different metal ions are added.
FIGS. 4a-d are fluorescence images of the cells of example 6 with different concentrations of mercury ions resulting in morphological changes.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
Synthesis of 2-benzyl-N- (phenyl) thiosemicarbazide
1.6g (10.6mmol) of p-aminobenzoyl hydrazine is dissolved in 30mL of ethanol, heated and stirred to be dissolved under the protection of nitrogen, and 0.47mL (11.0mmol) of phenyl isothiocyanate is slowly added dropwise. Reflux for 1.5h until the solution was a pale yellow solution and the mixture cooled to room temperature to precipitate a large amount of white solid. Recrystallizing the absolute ethyl alcohol for many times to obtain a pure productAnd carrying out suction filtration to obtain a white powdery solid, thus obtaining an intermediate probe with the yield of 53%. mp:136-137 ℃. FTIR (KBr): v-3440 cm-1,3296cm-1(-NH2),2069cm-1(C＝S),1541cm-1(-NH),1609cm(C＝O).1H NMR(600MHz,DMSO,298K,/ppm):3.84(s,-NH2,2H),7.92-7.71(m,ArH,9H),10.38-10.35(d,-NH,3H).13C NMR (600MHz, DMSO,298K,/ppm) 179.7,166.2,144.4,131.6,130.2,125.1,130.2,125.1,122.9,113.1,52.3, 51.6. Elemental analysis (C)14H14N4SO): theoretical value: c, 58.72%; h, 4.93%; n, 19.57%; experimental values: c, 57.44%; h, 4.33%; n,18.93 percent.
Example 2
Synthesis of dye Probe
1.144g (4.0mmol) of the intermediate probe are dissolved in 20mL of tetrahydrofuran, the temperature is brought to 0-5 ℃ in an ice bath, then 0.730g (4.0mmol) of 99% cyanuric chloride is dissolved in 15mL of acetone, transferred to a constant pressure dropping funnel and slowly added dropwise to a three-necked flask, after stirring for 1h, 0.330g (4.0mmol) of NaHCO are added by means of the dropping funnel3The solution was added dropwise to prevent the solution from becoming acidic (the pH of the reaction system was controlled to 6.5 to 7.5). The mixture is stirred at 0-5 ℃ and reacts for 6 h. Dissolving 1.396g (4.0mmol) of fluorescein hydrazide with 10ml of THF, pouring into a constant pressure dropping funnel, slowly dropping into a three-neck flask, heating to 65 ℃, stirring for 1h, and adding 0.34g (4mmol) of NaHCO with the constant pressure dropping funnel3The solution was added dropwise. After 3h of reaction, the resulting solid-liquid mixture was filtered with suction and the collected filtrate was rotary evaporated, the target product was isolated by silica gel column chromatography (chloroform/ethanol ═ 30:1) and recrystallized from ethanol to give a pale yellow powder solid with a yield of 62%. mp:149-150 ℃. FTIR (KBr): v 3441cm-1(OH),3123cm-1(-NH-),2085cm-1(C＝S),1708cm-1(C＝O),1607cm-1,1543cm-1(Ar-H),1499cm-1,1435cm-1,1283cm-1(triazine).1H NMR(600MHz,DMSO,298K,/ppm):＝3.82(s,-NH-,1H),6.44(m,ArH,2H),6.55(m,ArH,2H),6.61(m,ArH,2H),7.22(m,ArH,4H),7.51-7.94(m,ArH,10H),9.99(s,-OH,2H),10.45(s,-NH-,1H),10.91(s,-NH-,1H),11.11(s,-NH-,1H).13C NMR (600MHz, DMSO,298K,/ppm): 102.55,123.66,124.75,129.14,129.85,132.02,134.57,150.21,153.14,159.32,163.67,168.06,169.52,170.23(C ═ S),66.89 (quaternary C); elemental analysis (C)37H26ClN9O5S) theoretical value C, 59.72%; h, 3.52%; n, 16.94%, experimental value C, 60.90%; h, 3.62%; n,16.25 percent.
Example 3
Toxicity testing of dye probes
Cytotoxicity was tested by MTT method (fig. 1). The specific operation is as follows:
1. adding appropriate culture medium to the digested cells to obtain cell suspension, and planting in cell well plate to maintain the cell density in each well substantially consistent 5 × 105The wells were then encoded according to the established protocol and the marginal wells were filled with sterile PBS.
2. Standing at 37 deg.C with 5% CO2Pre-culturing for more than 12h in an incubator, and taking out after the cells are completely attached to the wall. Completely sucking the culture medium in the well plate with disposable pipette, repeatedly washing the well plate with PBS, and adding probes with concentration gradient (concentration is 0M, 10M in sequence) under the condition of keeping out of the sun-8M、10-7M、10-6M、10-5M and 10-4M), 5 wells per gradient. Adding 40 μ LMTT and culture medium with the same concentration into each well, and standing at 37 deg.C with 5% CO2Culturing in an incubator for 4 h.
3. Taking out the cell well plate, completely sucking the solution in the well plate under the condition of keeping out of the sun, adding 400 mu L of DMSO into each air, and shaking in a constant-temperature shaking table at 37 ℃ for 30 min;
4. the solution in each well was sequentially removed and placed in a 96-well plate, and the OD at 490nm of each well was measured using a microplate reader. The measurement result is processed and calculated to obtain that the toxicity of the dye probe to the cells completely meets the first-level standard.
Example 4
Selectivity of dye probes for ions in ethanol/BSA
In a system of ethanol and BSA solution with a volume ratio of 85:15, the dye probe is measured in the presence of added metalIon Zn2+,Ca2+,Cd2+,Sn2+,Mg2+,Fe3+,Co2+,Ni2+,Ba2+,Pb2+,Al3+,Cr3+,Mn2+,Cu2+,Hg2+Fluorescence spectra before and after.
The dye probe is found to have good response to lead ions in the fluorescence spectrum through the experimental result (figure 2).
Example 5
Ionic interference test in ethanol/BSA
Exploring biologically relevant coexisting ion-pair dye probes/Hg2+Influence of the fluorescence intensity at 530nm of the ethanol/BSA (85: 15) solution. Wherein, the concentration: 10 μ M (dye probe), 100 μ M (Hg)2+) 100 μ M (other ions), black column for adding different metal ions in the solvent system, gray column for dye probe-Hg2+Different metal ions are added into the system.
step 2: dissolving lead salt, ferric salt, cadmium salt, zinc salt, magnesium salt, chromium salt, calcium salt, barium salt, sodium salt, manganese salt and mercury salt in solvent ethanol, and metering volume in a 100ml volumetric flask by using the solvent ethanol to obtain concentrated solutionDegree of 1.0 × 10-2Each metal ion stock solution of M;
step 4, 0.1ml of the solution with the concentration of 1.0 × 10 is removed-2Adding 1ml of the probe stock solution obtained in the step 1 into the mercury ion stock solution of M, and respectively adding 0.1ml of 1.0 × 10-2Fixing the volume of each metal ion stock solution of M in a 10ml volumetric flask by using a solvent ethanol, standing for 2min, and detecting the fluorescence spectrum intensity of the metal ion stock solution;
as can be seen from FIG. 3, other ions have little influence on the dye probe, and therefore, the dye probe has good anti-interference performance.
Example 6
Dye probe tracking detection of cell morphological change
Adding appropriate culture medium into the digested cells to obtain 5 × 104Cell suspension/ml, 400. mu.L of suspension extracted, plated in cell well plates, and placed in 5% CO2The temperature of the incubator is 37 ℃ for 24h under saturated humidity. The experiment was performed until the cells were fully adherent.
And step 3: after incubation, 200uL of the total dye probe solution of step 1 was added to a, b, c and d in the well plate of step 2, and incubation was continued for 20 min. Cells were then imaged by washing 3 times with PBS buffer.
As can be seen from fig. 4, the cells in the a-well plate were normal fusiform, while the cells in the b-well plate were almost normal, but most of the cells in the c-well plate changed from fusiform to round, while the cells in the d-well plate were all round and had already undergone apoptosis. The dye probe of the invention can visually track and detect the morphological change of cells.
Claims (10)
1. A dye probe for tracking and detecting the morphological change of a single cell is characterized in that: the structural formula of the dye probe is
2. A preparation method of a dye probe for tracking and detecting the morphological change of a single cell comprises the following steps:
(1) dissolving p-aminobenzoyl hydrazine in a solvent, heating to 40-50 ℃ under the protection of inert gas, stirring for dissolving, dropwise adding phenyl isothiocyanate, refluxing for 1-2h, cooling, and recrystallizing to obtain an intermediate; wherein the molar ratio of the p-aminobenzoyl hydrazine to the phenyl isothiocyanate is 1:1.0 to 1.1;
(2) dissolving the intermediate obtained in the step (1) in a solvent to obtain a solution, and controlling the temperature in an ice bath within 0-5 ℃; dissolving cyanuric chloride, controlling the pH value of a reaction system to be 6.5-7.5, and stirring and reacting at 0-5 ℃ for 5-7h to obtain a mixed solution; dissolving fluorescein hydrazide, heating to 60-70 ℃, stirring for 1-1.5h, continuously controlling the pH value of the reaction system, reacting for 2-4h, performing suction filtration, rotary evaporation, column chromatography separation, and recrystallizing to obtain the required product; wherein the molar ratio of the intermediate, cyanuric chloride to fluorescein hydrazide is 1.0: 1.0-1.1: 1.0 to 1.1.
3. The method for preparing the dye probe for tracking and detecting the morphological change of the single cell as claimed in claim 2, wherein the dye probe comprises: the solvent of the p-aminobenzoyl hydrazine in the step (1) is ethanol; the inert gas is nitrogen.
4. The method for preparing the dye probe for tracking and detecting the morphological change of the single cell as claimed in claim 2, wherein the dye probe comprises: the solvent of the intermediate in the step (2) is anhydrous tetrahydrofuran; the solvent of the cyanuric chloride is acetone; the solvent of the fluorescein hydrazide is anhydrous tetrahydrofuran.
5. The method for preparing the dye probe for tracking and detecting the morphological change of the single cell as claimed in claim 2, wherein the dye probe comprises: NaHCO is adopted in the step (2)3The pH value of the reaction system is controlled to be 6.5-7.5 by the solution.
6. The method for preparing the dye probe for tracking and detecting the morphological change of the single cell as claimed in claim 2, wherein the dye probe comprises: and (3) recrystallizing in the steps (1) and (2) by adopting absolute ethyl alcohol.
7. The method for preparing the dye probe for tracking and detecting the morphological change of the single cell as claimed in claim 2, wherein the dye probe comprises: the solvents adopted for column chromatography separation in the step (2) are chloroform and ethanol with the volume ratio of 28:1-32: 1.
8. The use of the dye probe for tracking and detecting morphological changes of single cells as claimed in claim 1, wherein: the application method of the dye probe comprises the following steps:
(1) the dye probe was dissolved in a solvent to make 0.9 × 10-2M-1.1×10-2A dye probe stock solution of M; weighing mercury ion salts, dissolving the mercury ion salts in a solvent, and preparing a mercury ion solution;
(2) diluting the dye probe stock solution in the step (1), adding a BSA solution, and fixing the volume to obtain a dye probe solution; after the cells are cultured, mercury ion solutions with different concentrations are added into different pore plates, after the cells are cultured for 4-6h, a dye probe solution is added for culturing for 20-30min, and the cells are imaged by an inverted fluorescence microscope, so that tracking observation can be carried out.
9. The use of the dye probe for tracking and detecting the morphological change of the single cell as claimed in claim 8, wherein: the solvent in the step (1) is absolute ethyl alcohol; the mercury ion salt is mercury perchlorate.
10. The use of the dye probe for tracking and detecting the morphological change of the single cell as claimed in claim 8, wherein: the concentration of the BSA solution in the step (2) is 0.9-1.1 g/L; the adding amount is 1.6-1.8% of the volume of the stock solution of the concentration probe.
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