Cell tracking probe as well as preparation method and application thereof

Abstract

The invention discloses a cell tracking probe as well as a preparation method and application thereof. The retention time of the existing dye is relatively short (generally only several hours); the dye is not tolerant to fixation treatment, so that the dye leaks from cells, and the problem of efflux also exists; more seriously, the novel cell tracking and cell viability fluorescent dye has fluorescence, and aims to provide the novel cell tracking and cell viability fluorescent dye which is longer in retention time, does not need an additional cleaning step and is smaller in toxicity aiming at the technical problems existing in the existing main calcein orange red dye.

Description

Technical Field

[0001] This invention belongs to the fields of chemical synthesis and biotechnology, specifically relating to a fluorescent dye that can be used for cell tracking and cell viability detection, and more specifically to the synthesis and application of a series of cell tracking probes based on the AM ester system principle. Background Technology

[0002] Cell tracking technology is an important tool in modern life science research, playing a crucial role in areas such as immune cell function research, stem cell differentiation monitoring, and exploration of tumor metastasis mechanisms. Traditional cell tracking dyes are created by adding an acetylmethoxymethyl ester (AM) group to a fluorescent dye, transforming it from a polar, non-membrane-permeable compound into a hydrophobic, membrane-permeable probe precursor. This design allows the dye to passively diffuse into living cells, where it is hydrolyzed by non-specific esterases (including carboxylesterases and lipases) to form a fluorescent dye. The hydrolysis products exhibit strong fluorescence, and the fluorescence intensity is directly proportional to the intracellular esterase activity and cell number, thus achieving the purpose of cell tracking and testing cell viability. A representative dye of this type is Calcein AM.

[0003] Traditional cell-tracking dyes, such as Calcein AM, while performing excellently in cell viability detection, limit their application in multicolor imaging due to their single green fluorescence characteristic. In particular, the spectral overlap with GFP-transfected cells severely restricts the flexibility of experimental design, as this complete overlap makes the dye unusable in multicolor analysis of GFP-labeled cells. CellTrace Calcein Red-Orange AM, as a new generation of cell-tracking dyes, represents a significant breakthrough compared to traditional Calcein AM technology. Most importantly, this dye possesses a unique spectral characteristic of 577 / 590 nm, exhibiting excellent spectral separation from green fluorescent proteins such as GFP. This spectral characteristic allows it to be compatible with standard 561 nm lasers and PE channels, resulting in broader instrument applicability and making it considered the best-performing product currently available. However, this product also has some serious drawbacks, mainly including: 1) a relatively short dye retention time (typically only a few hours); and 2) intolerance to fixation treatment, leading to dye leakage from cells. This deficiency severely limits its application in immunofluorescence staining or other experiments requiring fixation. For example, researchers cannot use this dye to label cells for antibody staining, which limits its application in cell phenotype analysis; 3) the dye has an efflux problem; 4) more seriously, the dye itself is fluorescent, a characteristic that requires an additional washing step during use to reduce background fluorescence caused by dye not taken up by cells. This not only complicates the operation, but also often greatly reduces the experimental results. If washing is insufficient, background fluorescence will affect the sensitivity and specificity of detection. Summary of the Invention

[0004] In view of the technical problems existing in the main CellTrace Calcein Red-Orange AM dyes, the purpose of this invention is to provide a novel cell tracking and cell viability fluorescent dye with longer retention time (more than 48 hours, further more than 72 hours, and even more than 96 hours), no additional washing steps required, and less toxicity.

[0005] The present invention adopts the following technical solution.

[0006] A cell tracking probe has the following chemical structural formula:

[0007] Where n is 1 to 4, for example, n is 1, 2, 3 or 4.

[0008] This invention discloses a method for preparing the above-mentioned cell tracking probe, comprising the following steps: reacting compound J with O-(N-succinimide)-N,N,N',N'-tetramethylurea tetrafluoroborate to prepare the cell tracking probe; compound J has the following chemical structural formula: .

[0009] This invention discloses a cell tracking and / or cell viability assay kit, comprising the aforementioned cell tracking probe. Furthermore, the kit also includes conventional reagents that meet the requirements for conventional cell tracking and / or cell viability assays.

[0010] This invention discloses the application of the above-mentioned cell tracking probe in cell tracking and / or cell viability detection.

[0011] This invention discloses the application of the above-mentioned cell tracking probe in the preparation of cell tracking and / or cell viability detection reagents.

[0012] According to the above applications, the cell tracking probe disclosed in this invention has a longer retention time when tracking cells and / or detecting cell viability, which is more than 48 hours, preferably more than 60 hours, and more preferably more than 72 hours.

[0013] According to the above applications, the cell tracking probe disclosed in this invention has low cytotoxicity when used for cell tracking and / or cell viability detection, and has no significant effect on cell viability within the working concentration range, i.e., cell survival rate exceeds 95%.

[0014] According to the above applications, the cell tracking probe disclosed in this invention has a working concentration range of 0.1-20.0 μM when used for cell tracking and / or cell viability detection.

[0015] According to the above applications, the cell tracking probe disclosed in this invention does not require washing when tracking cells and / or detecting cell viability.

[0016] This invention discloses the application of the above-mentioned cell tracking probe in the preparation of diagnostic reagents.

[0017] This invention discloses a method for cell tracking and / or cell viability detection, comprising the following steps: using the aforementioned cell tracking probe to perform cell tracking and / or cell viability detection.

[0018] To address the shortcomings of existing mainstream calcein red-orange AM dyes (CellTrace Calcein Red-Orange AM), this invention discloses a novel dye with a longer retention time (over 72 hours) while maintaining the same spectral characteristics. In particular, it eliminates the problem of dye leakage from cells due to any membrane disruption caused by fixation and permeabilization, thus allowing it to withstand fixation and permeabilization treatments. Furthermore, this invention avoids the possibility of cells actively pumping the dye out of the cells, greatly improving its applicability in long-term cell tracking and preventing errors caused by rapid dye leakage. The novel dye itself is non-fluorescent, eliminating the need for additional washing steps and greatly simplifying the process. In addition, regarding cytotoxicity, a core indicator for evaluating the biosafety of live cell dyes, the new dye of this invention is stronger than existing calcein orange-red dyes. According to official technical data of existing dyes, it can be determined that at the recommended working concentration (usually 0.5-5.0 μM), the dye has no significant effect on cell viability and the cell survival rate is over 95%, meaning that the toxicity to cells is negligible. The working concentration range of the new dye of this invention is 0.1-20.0 μM, and within this range, it has no significant effect on cell viability (cell survival rate over 95%). Attached Figure Description

[0019] Figure 1 This is the mass spectrum of compound K.

[0020] Figure 2 Mass spectrometry of another cell-tracking probe compound.

[0021] Figure 3 This is a histogram of flow cytometry data. Detailed Implementation

[0022] Existing mainstream dyes have several serious shortcomings, including: 1) relatively short dye retention time (usually only a few hours); 2) intolerance to fixation treatment, leading to dye leakage from cells, which severely limits their application in immunofluorescence staining or other experiments requiring fixation; 3) efflux problems; and 4) more seriously, the dye itself is fluorescent, requiring additional washing steps to reduce background fluorescence from unabsorbed dye, complicating the process and often significantly reducing experimental results. Insufficient washing can also affect the sensitivity and specificity of detection. To address the technical problems of existing main CellTrace Calcein Red-Orange AM dyes, this invention aims to provide a novel cell tracking and cell viability fluorescent dye with longer retention time (over 72 hours), no need for additional washing steps, and lower toxicity.

[0023] The following specific experiments illustrate the technological advancements of this invention. The raw materials used are conventional products, which can be commercially available or prepared using conventional methods. The specific preparation operations and performance testing follow conventional techniques. DMF: N,N-dimethylformamide; EA: ethyl acetate; hexane: n-hexane; DIPEA: N,N-diisopropylethylamine; TSTU: O-(N-succinimide)-N,N,N',N'-tetramethylurea tetrafluoroborate; ACN: acetonitrile; TEA: triethylamine.

[0024] Synthesis example Compound 2 can be purchased or prepared using the following method.

[0025]

[0026] 306 g of 4-bromo-1-butanol, 2.5 L of DMF, 53 g of NaH, and 359 g of benzyl bromide were added to a 3 L three-necked flask. The mixture was stirred and cooled to -70 °C under nitrogen protection. After reacting for 1 h, TLC (Hex / EA = 5 / 1) showed that the reaction was complete. The entire reaction solution was brought to room temperature, the solvent was removed by vacuum, 500 g of ice was added first, and after the ice melted, 5 L of EA was added. The mixture was stirred overnight. The organic phase was collected, washed once with 1 L of saturated sodium bicarbonate, twice with water, once with saturated brine, dried over anhydrous sodium sulfate, and filtered dry. The sample was mixed with silica gel and passed through a column at Hex / EA = 50 / 1-20 / 1. The product spot was collected, and the product was vacuum dried overnight at room temperature to constant weight, yielding 430.5 g of compound (a).

[0027] 283 g of diethyl malonate, 430 g of compound (a), 2.5 L of anhydrous THF, and 120 g of sodium ethoxide were added to a 5 L three-necked flask. The mixture was stirred at room temperature under nitrogen protection for 20 h, and TLC (Hex / EA = 5 / 2) showed that the reaction was complete. The entire reaction solution was concentrated. After concentration, 300 g of ice was added. After the ice melted, 200 mL of 0.1 N HCl solution and 4 L of EA were added, and the mixture was stirred overnight. The organic phase was collected, washed once with 1 L of saturated sodium bicarbonate, once with saturated brine, dried over anhydrous sodium sulfate, and filtered dry. The sample was mixed with silica gel and passed through a column (Hex / EA = 10 / 1-5 / 2). The product spot was collected and dried under vacuum at room temperature overnight to constant weight, yielding 490.8 g of compound (b).

[0028] 70.7 g of sodium borohydride (NaBH4) was added in portions to a suspension of 510 g zinc chloride in 5000 mL anhydrous tetrahydrofuran (THF) under nitrogen protection at 0–5 °C. The reaction mixture was stirred at 20–25 °C for 30 minutes, then cooled to 0–5 °C, and an anhydrous THF solution containing 483 g of compound (b) was added dropwise at this temperature. After the addition was complete, the system was heated to 45 °C and stirred for 18 hours. TLC (ACN / H2O = 10:1) showed that the reaction was complete. The reaction solution was cooled to -15 to -10 °C, and the reaction was quenched by adding 1000 mL of water dropwise at this temperature. The entire solution was then heated to room temperature, and 500 g of diatomaceous earth was added. The solid was removed by filtration. The filtrate was concentrated under reduced pressure to remove tetrahydrofuran; the filter cake was washed with chloroform (1000 mL × 3 times), and the organic phases were combined and precipitated with 1000 mL of chloroform. The mixture was washed with water and allowed to stand to separate into layers. The organic phase was dried over anhydrous sodium sulfate (Na2SO4) and concentrated under reduced pressure to give 290.9 g of compound (C).

[0029] A solution of 276 g of compound (c) and 850 g of carbon tetrabromide in dichloromethane (3000 mL) was prepared in a 5 L flask and cooled to 0 °C. 670 g of triphenylphosphine was added in portions through a powder funnel with stirring over a period of 60 minutes. After the addition of triphenylphosphine, the colorless solution turned light brown, and stirring continued for 2 hours at room temperature. TLC (ACN / H₂O = 10:1) showed that the reaction was complete. The reaction mixture was then concentrated to obtain a brown oily substance, which was filtered through a silica gel column (Hex / EA = 15 / 1–3 / 1). The product was collected and dried under vacuum overnight at room temperature to constant weight, yielding 331.6 g of compound (d).

[0030] At 25°C, 1800 g of N,N-dimethylaniline dissolved in dichloromethane was added dropwise through a dropping funnel to a solution of 270 g of compound (d) dissolved in 2000 mL of dichloromethane. After the addition was complete, 1600 g of solid aluminum trichloride was added in one go. The reaction mixture was stirred at 25°C for 1 hour, then cooled to 0°C, and 2 L of 2 mol / L sodium bisulfate aqueous solution was added. The reaction mixture was heated to 25°C and extracted separately with 2 L of chloroform and 500 mL of saturated sodium bicarbonate aqueous solution. The aqueous phase was extracted again with 2 L of chloroform. The combined organic phases were washed with 500 mL of saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The crude oil obtained was purified by silica gel rapid column chromatography (eluting with CHCl3 / EtOAc / hex = 10 / 10 / 80 gradient to CHCl3 / EtOAc / hex = 30 / 30 / 40) to give 168.7 g of colorless oily compound (e); Under an oxygen atmosphere, 25 g of ferric nitrate nonahydrate, 9.3 g of 2,2,6,6-tetramethylpiperidine oxide, 4.5 g of potassium chloride, 163 g of substrate compound (e), and 2000 mL of 1,2-dichloroethane (DCE) were added sequentially to a 5 L single-necked flask. The flask was then stirred at room temperature for 18 h. The reaction progress was monitored by thin-layer chromatography (TLC, developing solvent: CHCl3 / hex = 5 / 1), and the reaction was terminated after 12 hours. The entire reaction solution was filtered and then concentrated. The crude product was filtered through a column with silica gel (ACN / H2O = 15 / 1-5 / 1), and the product spot was collected. The product was vacuum dried overnight at room temperature to constant weight, yielding 145.3 g of compound (f).

[0031]

[0032] In a 5L single-necked flask, 145 g of compound (f), 68 g of allyl bromide, and 500 g of cesium carbonate were added, followed by 2000 mL of anhydrous dimethylformamide (DMF). The reaction mixture was stirred at room temperature for 16 hours. After the reaction was complete, the mixture was concentrated under reduced pressure to a gel state, 1000 mL of water was added, and the mixture was extracted three times with 500 mL of dichloromethane each time. The organic phases were combined, concentrated under reduced pressure, and the crude product was purified by rapid column chromatography (EtOAc / CHCl3 / hex = 3 / 1 / 6). The product spot was collected and dried under vacuum overnight at room temperature to constant weight, yielding compound 2, 135.6 g.

[0033] By replacing the 4-bromo-1-butanol with bromools of different numbers of methylene groups, products with different numbers of methylene groups were obtained.

[0034] Example 1: Synthesis of Fluorescent Probes

[0035] 40.6 g of compound (1), 1.5 L of acetonitrile, 30 g of sodium iodide, 131.2 g of compound (2), and 84.8 g of sodium carbonate were added to a 3 L reaction flask. The mixture was heated to reflux at 85 °C and mechanically stirred overnight. The mixture was then filtered at room temperature, evaporated to dryness, and a large amount of solid was precipitated. Ethyl acetate was added and stirred for 30 min, then filtered. The filtrate was evaporated to dryness, mixed with silica gel, and purified by column chromatography with a PE / DCM ratio of 20 / 1-5 / 1. The filtrate was collected and evaporated to dryness to obtain 42.7 g of compound A.

[0036] 32.7 g of compound A, 27.2 g of sodium ethanethiol, 600 mL of DMF, and 3 g of NaH 60% dispersion in mineral oil were added to a 2 L reaction flask. The mixture was heated to 55 °C and reacted for 48 h. TLC (DCM:MeOH = 10:1) showed that the reaction was complete. After most of the DMF was evaporated at room temperature using a rotary evaporator, a yellowish-brown viscous liquid remained. 1 L of EA:hexane (1:1) was added, and the mixture was refluxed at 50 °C for 1 h. The mixture was then filtered at room temperature. The filter cake was acidified with 300 mL of 5% hydrochloric acid solution and then extracted with chloroform. The chloroform layer was first washed with water, then with 1% sodium hydroxide solution, washed again with water, dried, and then evaporated and concentrated to obtain a light brown oily solid. After drying, 22.3 g of brown gel-like compound B was obtained.

[0037]

[0038] 16 g of compound B and 200 mL of water were added to a 2 L three-necked flask. With stirring, 10 mL of 98% concentrated sulfuric acid was added at -20 °C, followed by 9.8 g of 1,2,4-benzoic anhydride at -5 °C. The reaction was carried out at room temperature for 3 days, then at 32 °C for 24 h. TLC (DCM:MeOH=9:1) showed complete reaction of the starting material. 4.4 g of 3-aminophenol was added at room temperature and the reaction was carried out at room temperature for 2 days. TLC (DCM:MeOH=9:1) showed complete reaction of the 3-aminophenol. Then, 100 mL of 98% concentrated sulfuric acid was added at -20 °C, air was introduced, and the reaction was carried out at -20 °C for 0.5 h, then at room temperature for 0.5 h, then at 55 °C for 0.5 h, and finally at 125 °C for 8 days. TLC (ACN:H2O=3:1) showed complete reaction of the starting material. (Detection) The reaction of the raw materials was completed; then ammonia water was added at -10℃ for neutralization. After neutralization, the reaction solution was evaporated and a large amount of salt was precipitated. The solid was dissolved in methanol and filtered. The filtrate was collected, mixed with alumina, and column-fed with alumina. The eluent was (ACN:H2O=10:1-5:1). The product band was collected and freeze-dried to obtain 18.6 g of orange-red solid, which was compound C.

[0039]

[0040] 11.6 g of compound C, 200 mL of pyridine, and 400 mL of acetic anhydride were added to a 1 L single-necked flask equipped with a condenser. Under nitrogen protection, the mixture was reacted in an oil bath at 90 °C for 3 h. TLC (acetonitrile / water = 5 / 1) showed that the reaction was complete. At room temperature, the mixture was poured into 4 L of ice-cold 1 N hydrochloric acid and stirred for 0.5 h. A large amount of solid precipitated out. The mixture was filtered, washed with water, and the wet filter cake was directly added to a 3 L reaction flask. 1.6 L of acetonitrile and 320 mL of water were added and stirred at room temperature until dissolved. The mixture was left to stand overnight. The next day, a large amount of solid precipitated out. The mixture was filtered, the filter cake was washed with a small amount of water, and lyophilized to obtain 6.8 g of grayish-white solid compound D.

[0041]

[0042] At room temperature, 13 g of N-BOC-L-lysine and 200 mL of acetonitrile were added to a 1 L three-necked flask. The mixture was magnetically stirred, and 40 mL of DIPEA was added dropwise, followed by 10 g of NaI and 50 g of methyl bromoacetate. The mixture was then reacted at 75 °C for 24 h. TLC analysis (PE:EA = 1:1) showed complete reaction and formation of a disubstituted product. The reaction was stopped, and 420 mL of ethyl acetate and 350 mL of water were added at room temperature. The mixture was separated. 200 mL of EA was added to the aqueous phase, and the mixture was extracted again. The organic phases were then combined. The organic phase was washed twice with water (2 x 210 mL), once with 200 mL of saturated brine, and then dried over anhydrous sodium sulfate. The filtrate was collected after filtration, evaporated to dryness, and then 10 mL of ethyl acetate and 100 mL of petroleum ether were added. The mixture was then stirred and dried under vacuum to obtain 11.2 g of a grayish-white gel solid product, which was compound E.

[0043]

[0044] In a 500 mL three-necked flask, 8.08 g of compound E, 200 mL of dichloromethane, and 10 mL of trifluoroacetic acid were added and stirred at room temperature for 2 h. TLC analysis (CH2Cl2:MeOH = 1:1) indicated that the reactants had basically reacted completely. The entire reaction solution was concentrated, and the trifluoroacetic acid was dried under vacuum to obtain a viscous gel-like compound F. This compound was directly dissolved in 200 mL of methanol and 50 mL of 6 M sodium hydroxide aqueous solution and stirred at room temperature for 2 h. TLC analysis (CH2Cl2:MeOH:CH3COOH = 1:1:0.2) indicated that the reaction was complete. After evaporating the solvent, the compound was dissolved in 200 mL of distilled water, and the pH was adjusted to 6.8 with 3 M hydrochloric acid. The solution was then concentrated to a volume of 20 mL and placed in a refrigerator at 4°C overnight to crystallize. The solid was collected after filtration and freeze-dried to obtain 2.92 g of a grayish-white solid compound G.

[0045]

[0046] To a 500 mL single-necked flask, add 6.21 g of intermediate compound D, 100 mL of DMF, and 20 mL of DIPEA. After 30 min in an ice-water bath, add 6.62 g of TSTU and let it sit overnight at room temperature. TLC (ACN / H2O = 7 / 1) showed the reaction was complete. After removing the solvent, add 50 mL of a solution with an EA / ACN ratio of 5 / 1 and stir for 3 h. Stop stirring, decant the supernatant, and dry the precipitate under vacuum at room temperature overnight. Add the obtained solid and 200 mL of DMF to a 1000 mL single-necked flask, stir magnetically, and let it sit in an ice-water bath for 10 min. Add 20 mL of TEA and stir for 0.5 h. Then, continue to add 2.62 g of compound G while maintaining the temperature. After the addition is complete, remove the ice bath and allow it to cool to room temperature naturally. Let it react overnight. Take a sample and detect the reaction by TLC (acetonitrile / water = 4 / 1), which showed the reaction was complete. The reaction solution was evaporated, then slurried twice with 200 mL of acetonitrile, and the solid was dried under vacuum to obtain 5.89 g of grayish-white crude product H.

[0047]

[0048] 4.32 g of compound H and 100 mL of DMF were added to a 250 mL single-necked flask. Under nitrogen protection, the mixture was cooled in an ice bath. 10 mL of DIEA was added, and the mixture was stirred for 15 min. 4.58 g of bromomethyl acetate was added, and the mixture was allowed to warm naturally overnight. The reaction was confirmed to be complete by HPLC. The reaction solution was poured into 1 L of 0.1 N hydrochloric acid at ice temperature, stirred for 10 min, filtered, and the filter cake was dissolved with EA. The mixture was washed once with saturated brine, dried over anhydrous sodium sulfate, and then passed through a silica gel column with eluent PE / EA = 3 / 1 to 1 / 1. The product was collected and dried to obtain 2.97 g of a grayish-white solid, compound I.

[0049] Add 2.16 g of intermediate compound I, 50 mL of THF, and 50 mL of methanol to a 250 mL single-necked flask. Incubate in an ice-water bath for 10 min. Then add 200 mg of Pd(PPh3)4 and 1.5 g of p-toluenesulfinate sodium (sodium p-toluenesulfinate). Continue to react in an ice-water bath for 2 h. After removing the ice bath, allow the entire reaction solution to return to room temperature overnight. TLC (ACN / H2O = 20 / 1) showed that the reaction was complete. After drying the solvent, add 100 mL of a solution with a ratio of EA / ACN / H2O = 20 / 1 / 1. Stir for 3 h. Stop stirring, separate the organic phase and the aqueous phase, concentrate the organic phase, and then dry to obtain crude product J.

[0050] The crude product J obtained above was dissolved in 50 mL of DMF and then placed in a 250 mL single-necked flask. The entire solution was then in an ice-water bath for 30 min, followed by the addition of 5 mL of DIPEA. The mixture was then kept in an ice-water bath for another 30 min, after which 1.6 g of TSTU was added. The entire reaction solution was allowed to return to room temperature overnight. TLC analysis (PE:EA = 1:1) showed complete conversion of the starting material to the product. After the solvent was removed, the mixture was slurried once with 200 mL of diethyl ether, and then slurried again with 100 mL of a mixture of diethyl ether and acetonitrile in a 10:0.5 ratio. Finally, 920 mg of a grayish-white gelatinous solid compound K was obtained.

[0051] Figure 1 For compound K and its mass spectrum, Chemical Formula: C 56 H 58 N4O 22 Exact Mass: 1138.35.

[0052] Example 2 Referring to Example 1, by changing the number of methylene groups in compound 2, another cell-tracking probe was obtained. Figure 2 Here is the molecular structural formula and mass spectrum of this compound. Chemical Formula: C 58 H 62 N4O 22 Exact Mass: 1166.39.

[0053] Example 3 (1) The cytotoxicity test experiment is as follows: CCK-8 assay for compound K toxicity test Test principle: Dehydrogenases in living cells can reduce WST-8 in CCK-8 reagent to orange formazan. The formazan concentration is positively correlated with the number of living cells. Cell viability is reflected by absorbance (OD value), which indirectly determines toxicity. (2) Experimental materials: adherent cells HeLa, CCK 8 (CK04), culture medium (D6429) and compound K; (3) Experimental steps: (1) Blank control group: only complete culture medium is added, no cells are inoculated, and no compound K is added, which is used to subtract the fluorescence background of the culture medium itself; (2) Negative control group: cells are inoculated, complete culture medium is added, and no compound K is added, which serves as a control for normal cell growth; (3) Compound K treatment group: cells are inoculated and complete culture medium containing different concentrations of compound K is added. The concentration gradient is set as 0.1μM, 1μM, 5μM, 10μM, 20μM, and the solvent is culture medium. Three replicates are set for each concentration to ensure the reproducibility of the experiment; (4) Experimental time settings: 1h, 4h, 24h, 48h groups; (5) 1. Cell plating: Add 100 μL of cell suspension (2 × 10⁻⁶) to each well of a 96-well plate according to the grouping. 4 1. Add 100 μL of culture medium to the blank control group and place the 96-well plate in the incubator to allow the cells to adhere; 2. Prepare compound K solutions of various concentrations using culture medium; 3. Remove the culture plate, aspirate the old culture medium from the wells, and add culture medium containing the corresponding concentration of compound K to each well according to the grouping, 100 μL per well of the 96-well plate, and add the same amount of culture medium to the negative control group and blank control group; 4. Place the culture plate back in the incubator and perform detection at 1h, 4h, 24h, and 48h time points; at the set time points, add 10 μL of CCK-8 reagent to each well, gently shake, and then return to the incubator for 1-2h, and detect the absorbance at 450nm wavelength using a microplate reader; 5. Toxicity assessment: relative cell viability = (OD value of compound K treatment group - OD value of blank group) / (OD value of negative group - OD value of blank group) × 100%; if the relative viability is < 80%, it can be considered that there is significant toxicity at this concentration.

[0054] (4) Specific experimental results

[0055] Conclusion: The toxicity of the newly invented dye compound K to cells in the range of 0.1-20.0 μM is negligible (cell survival rate exceeds 95%).

[0056] Example 4 Experimental methods: (1) Coat the flat-bottomed 96-well plates with anti-CD3 antibody (anti-CD3) and anti-CD28 antibody (anti-CD28), with both antibodies at a concentration of 2 μg / mL in 4 mL of phosphate-buffered saline (PBS) containing bovine serum albumin (BSA). Incubate the plates overnight at 4°C before use; (2) Peripheral blood mononuclear cells (PBMNCs) were thawed at 37°C and resuspended in phosphate-buffered saline (PBS) containing 20% ​​fetal bovine serum (FCS). The cells were then centrifuged at 1200 rpm for 10 minutes at 20°C. Staining was then performed using compound K. The final concentration of compound K was adjusted to 2.5 μM with PBS, and the cells were immediately incubated for 10 minutes. Cells were washed with pre-chilled RPMI-1640 medium (Sigma, catalog number R8758) containing 0.1% FCS to terminate the staining reaction, and then incubated on ice for 5 minutes. The cells were then washed twice with RPMI-1640 medium containing 10% FCS, and the cell concentration was adjusted to 2 × 10⁶ cells / mL. 6Cells / mL. Finally, the cells were seeded into pre-coated 96-well plates, with 2.5 × 10⁶ cells per well. 5 Cells were incubated at 37°C and 5% CO2 for 96 hours, and analyzed by flow cytometry daily during this period. A 561 nm (yellow-orange laser) was used to excite the cells, and a 585 / 15 nm bandpass filter was used to collect the signal. This was repeated five times in total. No washing was required before flow cytometry analysis. Figure 3 The results showed that compound K remained an excellent cell tracking agent even after 96 hours of continuous incubation.

[0057] Existing dyes possess intrinsic fluorescence; therefore, an additional washing step is required to minimize background fluorescence emitted by dyes not absorbed by cells. Furthermore, cross-linking with aldehyde fixatives is not possible, as the dyes are lost during fixation. Importantly, they can be pumped out by normal cellular efflux mechanisms in a short period. This invention effectively solves these problems. After incubation with cells, the dye requires no washing and can be directly used for testing without background interference, and offers an exceptionally long retention time (over 96 hours), exceeding previous expectations.

Claims

1. A cell tracking probe having the following chemical structural formula: ； in, n can be 1 to 4, for example, n can be 1, 2, 3 or 4.

2. A method for preparing the cell tracking probe according to claim 1, comprising the following steps: reacting compound J with O-(N-succinimide)-N,N,N',N'-tetramethylurea tetrafluoroborate to prepare the cell tracking probe; compound J has the following chemical structural formula: 。 3. A cell tracking and / or cell viability assay kit, comprising the cell tracking probe of claim 1.

4. The use of the cell tracking probe of claim 1 or the kit of claim 3 in cell tracking and / or cell viability detection.

5. The use of the cell tracking probe of claim 1 or the kit of claim 3 in the preparation of cell tracking and / or cell viability assay reagents.

6. The application according to claim 4 or 5, characterized in that, The cell tracking probe has a longer retention time when used for cell tracking and / or cell viability detection.

7. The application according to claim 4 or 5, characterized in that, The cell tracking probe exhibits low cytotoxicity during cell tracking and / or cell viability detection, and has no significant impact on cell viability within the working concentration range, i.e., cell survival rate exceeds 95%.

8. The application according to claim 4 or 5, characterized in that, The cell tracking probe operates at a concentration range of 0.1–20.0 μM for cell tracking and / or cell viability detection.

9. The use of the cell tracking probe of claim 1 or the kit of claim 3 in the preparation of diagnostic reagents.

10. A method for cell tracking and / or cell viability detection, comprising the steps of using the cell tracking probe of claim 1 or the kit of claim 3 to perform cell tracking and / or cell viability detection.

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