Dark-state rhodamine probe, preparation method thereof and application of dark-state rhodamine probe in cell imaging

Abstract

The invention relates to the field of molecular fluorescent probes, in particular to a dark-state rhodamine probe, a preparation method thereof and application of the dark-state rhodamine probe in cell imaging, and the dark-state rhodamine probe has the following molecular structure expression (I): # imgabs0 #; the activation process of the dark-state rhodamine probe is an oxidation process, the dark-state rhodamine probe reacts with singlet oxygen and then is activated to become a bright-state rhodamine probe, and the wavelength of the optimal exciting light is 560 nm. Compared with a conventional rhodamine probe, the rhodamine probe has longer light excited state duration time, is an organic dye molecule with higher light stability, and solves the problem that the stability of the existing organic dye molecule is lower.

Description

Technical Field

[0001] The present application relates to the field of molecular fluorescent probes, and in particular to a dark-state rhodamine probe and a preparation method thereof and application in cell imaging. Background Art

[0002] Photostability is one of the important parameters that characterize organic dye molecules. It refers to the ability of organic dye molecules to withstand multiple excitations and return to the ground state without irreversible damage. Under strong light irradiation, most organic dyes are susceptible to photobleaching. This photobleaching is the result of the reaction between the dye's long-lived photoexcited state (such as the triplet state) and molecules in the environment (such as molecular oxygen). Photobleaching is an irreversible photochemical process that renders the fluorophore permanently non-luminescent, limiting the observation time of the molecule. In general, the fluorescence loss that accompanies photobleaching has an adverse effect by reducing the signal-to-noise ratio. Low photostability is a common weakness of organic fluorescent dyes, fluorescent cytochromes, and fluorescent proteins, which causes great problems. These problems can be seen in many fluorescence techniques, especially those that require long-time scale observations, such as fluorescence correlation spectroscopy or two-photon microscopy and super-resolution imaging. Therefore, photobleaching interferes with most fluorescence spectroscopy and microscopy. Simply put, organic dye molecules with low stability have shorter photoexcited states.

[0003] There is currently no effective solution to the problem of low stability of organic dye molecules. Summary of the invention

[0004] The present invention provides a dark-state rhodamine probe and a preparation method thereof and application in cell imaging to solve the problem of low stability of current organic dye molecules.

[0005] In a first aspect, the present invention provides a dark state rhodamine probe having the following molecular structure expression (I): .

[0006] In a second aspect, the present invention provides a method for preparing a dark-state rhodamine probe, which is used to prepare the dark-state rhodamine probe described in the first aspect, and the preparation method comprises: A bright-state rhodamine probe is prepared. The bright-state rhodamine probe has the following molecular structure expression (II): ; Dissolving the bright rhodamine probe in dichloromethane and water, and adding an excess of solid sodium borohydride and mixing well to obtain a third solution; After washing the third solution, anhydrous sodium sulfate was added to dry the solution and then evaporated under reduced pressure to obtain a fourth solution. The fourth solution was subjected to sample column chromatography to obtain a dark rhodamine probe.

[0007] In a third aspect, the present invention provides a dark state rhodamine probe bound to a Halo ligand, which is obtained by binding the dark state rhodamine probe described in the first aspect to a Halo ligand and has the following molecular structure expression (III): .

[0008] In a fourth aspect, the present invention provides a method for preparing a dark-state rhodamine probe, which is used to prepare the dark-state rhodamine probe bound to the Halo ligand described in the third aspect, and the preparation method comprises: A bright-state rhodamine probe bound to a Halo ligand is prepared. The bright-state rhodamine probe bound to a Halo ligand has the following molecular structure expression (IV): ; Dissolving the bright rhodamine probe bound to the Halo ligand in dichloromethane and water, and adding an excess of solid sodium borohydride and mixing well to obtain a seventh solution; The seventh solution was washed, added with anhydrous sodium sulfate, dried, and then evaporated under reduced pressure to obtain an eighth solution. The eighth solution was subjected to sample column chromatography to obtain a dark-state rhodamine probe bound to the Halo ligand.

[0009] Compared with the related art, the present invention provides a new dark-state rhodamine probe 5-RB, which has a longer photoexcited state duration than the conventional rhodamine probe RB and is an organic dye molecule with stronger photostability, thus solving the problem of low stability of current organic dye molecules.

[0010] Details of one or more embodiments of the present application are set forth in the following drawings and description to make other features, objects, and advantages of the present application more readily apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Schematic diagram of the preparation of the dark state rhodamine probe in this embodiment; Figure 2 is the H NMR spectrum of the dark state rhodamine probe in this embodiment; Figure 3 is a normalized graph of the UV-visible absorption spectrum and fluorescence spectrum of the dark-state rhodamine probe in this embodiment; Figure 4 is the fluorescence spectrum of the dark state rhodamine probe in this embodiment in response to singlet oxygen; Figure 5 is a fluorescence intensity scatter plot of the dark state rhodamine probe 5-RB and the conventional rhodamine probe RB irradiated for 0-100 min in this embodiment; Figure 6 It is the spectrum that sodium ascorbate hinders the opening of the dark state rhodamine probe in this example; Figure 7 Schematic diagram of the preparation of the dark state rhodamine probe combined with the Halo ligand in this embodiment; Figure 8 is the H NMR spectrum of the dark state rhodamine probe bound to the Halo ligand in this embodiment; Fig. 9 is the cell survival rate at different concentrations of 5-RB and 5-RB-L provided in this example; Fig.10 It is the cell imaging picture of 5-RB provided in this embodiment and RB in the prior art. DETAILED DESCRIPTION

[0012] In order to more clearly understand the purpose, technical solutions and advantages of the present application, the present application is described and illustrated below in conjunction with the accompanying drawings and embodiments.

[0013] Unless otherwise defined, the technical terms or scientific terms involved in this application shall have the general meaning understood by people with general skills in the technical field to which this application belongs. The words "one", "a", "the", "these" and the like in this application do not indicate a quantitative limitation, and they may be singular or plural. The terms "include", "comprise", "have" and any variants thereof involved in this application are intended to cover non-exclusive inclusions; for example, a process, method and system, product or device comprising a series of steps or modules (units) is not limited to the listed steps or modules (units), but may include unlisted steps or modules (units), or may include other steps or modules (units) inherent to these processes, methods, products or devices. The words "connect", "connected", "coupled" and the like involved in this application are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. The "multiple" involved in this application refers to two or more. "And / or" describes the association relationship of associated objects, indicating that there may be three relationships, for example, "A and / or B" may mean: A exists alone, A and B exist at the same time, and B exists alone. Generally, the character " / " indicates that the objects associated with each other are in an "or" relationship. The terms "first", "second", "third", etc. in this application are only used to distinguish similar objects and do not represent a specific ordering of the objects.

[0014] In this embodiment, a dark state rhodamine probe 5-RB is provided, and its preparation process is as follows Figure 1 , which has the following molecular structure expression (I): .

[0015] The high-resolution mass spectrum of the dark-state rhodamine probe 5-RB is: HR-MS(m / z,ESI):Cal.for[C 28 H 32 N2O3]m / z=445.2486[M+H].Foundm / z=445.2467.

[0016] Reference Figure 2 , the H NMR spectrum of the dark state rhodamine probe 5-RB is: 1HNMR(400MHz,Chloroform-d)δ8.02–7.96(m,2H),7.33–7.26(m,2H),6.77(d,J=8.5Hz,2H),6.40(t, J=1.9Hz,2H),6.32(dt,J=8.5,1.8Hz,2H),5.12(s,1H),3.32(q,J=7.0Hz,8H),1.15(t,J=7.0Hz,12H).

[0017] For the above-mentioned dark-state rhodamine probe 5-RB, its oxidation properties can be used to change the probe structure, thereby causing corresponding changes in its optical properties. The probe changes from a dark state to a bright state, and the probe is activated. Correspondingly, in the present embodiment, the activation process of the dark-state rhodamine probe is an oxidation process. Specifically, the dark-state rhodamine probe can be activated and transformed into a bright-state rhodamine probe after reacting with singlet oxygen. The fluorescence intensity of the activated rhodamine probe can be enhanced by 7.13 times. The activation process is: the dark-state rhodamine probe solution is fused with the configured singlet oxygen solution, and then the solution is irradiated with an excitation light source, and singlet oxygen is generated in the solution through excitation, thereby activating the dark-state rhodamine probe. Among them, the optimal excitation light wavelength of the dark-state rhodamine probe is 560nm. The dark-state rhodamine probe is completely transformed from a dark state to a bright state when irradiated with 560nm light for 100min. This feature can be seen by Figure 3 It is concluded that Figure 3 : is a normalized graph of the UV-visible absorption spectrum and fluorescence spectrum of the dark state rhodamine probe in this embodiment, and the ordinate represents the normalized values ​​of the absorption intensity and the fluorescence intensity, corresponding to excitation and emission, respectively. Figure 3 , the UV-visible spectrometer measured that the absorbance of 5-RB-L for 560nm excitation light is the largest, and the emission wavelength with the maximum fluorescence intensity obtained by exciting the probe with 560nm excitation light is 586nm. Since 5-RB has not been activated, the absorbance and fluorescence intensity are almost undetectable. Therefore, the optimal excitation wavelength of the dark state rhodamine probe is 560 nm and the optimal emission wavelength is 586 nm. Also refer to Figure 4 It can be seen that when singlet oxygen is added to the probe solution, the fluorescence intensity increases significantly compared to when no singlet oxygen is added, proving that the probe can be activated by singlet oxygen.

[0018] After the dark state rhodamine probe is activated, the fluorescence quantum yield changes from 0.5% to 65%. It should be noted that the activation process of the dark state rhodamine probe can be affected by the antioxidant substance sodium ascorbate. The sodium ascorbate in the probe solution makes the conversion rate of the rhodamine probe from the dark state to the bright state change to 32.7%, making it unable to be completely converted, indicating that the activation process of the dark state rhodamine probe is an oxidation process.

[0019] As described above, the properties of the dark-state rhodamine probe 5-RB provided in this embodiment are described, and its properties will be specifically described below through some specific experimental data.

[0020] In this embodiment, a method for preparing a dark-state rhodamine probe is also provided, which is used to prepare the dark-state rhodamine probe in this embodiment. The preparation method includes steps S110, S120 and S130.

[0021] Step S110, preparing a bright state rhodamine probe, the bright state rhodamine probe has the following molecular structure expression (II): .

[0022] Specifically, step S110 includes step S111, step S112, step S113 and step S114.

[0023] Step S111, grinding 4-carboxybenzaldehyde and 3-diethylaminophenol to obtain powder.

[0024] Step S112, heating the powder and the sulfuric acid aqueous solution under nitrogen flow to obtain a first solution, wherein the heating temperature is 150-180° C. and the heating time is 8-12 hours.

[0025] Step S113, neutralizing the first solution to obtain a solid precipitate.

[0026] Step S114, adding anhydrous ethanol to the solid precipitate for ultrasonic dissolution to obtain a second solution, and performing column chromatography on the second solution to obtain a bright rhodamine probe.

[0027] Step S120, dissolving the bright rhodamine probe in dichloromethane and water, and adding an excess of sodium borohydride solid and mixing well to obtain a third solution; Step S130, washing the third solution, adding anhydrous sodium sulfate, drying, and evaporating under reduced pressure to obtain a fourth solution, and performing sample column chromatography on the fourth solution to obtain a dark-state rhodamine probe.

[0028] In this example, the bright rhodamine probe 5-RB-L was first prepared: 4-carboxybenzaldehyde (5.5 g, 36.4 mmol) and 3-diethylaminophenol (12.12 g, 71.7 mmol) were added to a mortar and ground for 20 minutes to make it a brown powder. The powder was then added to a 250 ml three-necked flask, followed by an aqueous sulfuric acid solution (55 ml of concentrated sulfuric acid and 40 ml of water). The flask was filled with nitrogen, the reaction temperature was slowly raised to 180°C, and heated to reflux for 10 hours to stop the reaction. The reaction solution (first solution) was cooled to room temperature, poured into 200 ml of ice water, and the mixed solution was neutralized with 60% potassium hydroxide solution. The mixture was stirred continuously with a glass rod to dissipate heat, and a purple precipitate was generated. The solution was filtered with a 250 ml Buchner funnel, and the obtained solid was washed with deionized water 3 times. After vacuum drying, it was taken out and anhydrous ethanol (300 ml) was added and ultrasonically dissolved (the second solution was obtained). The mixture was filtered again, and the filtrate was collected. The anhydrous ethanol was removed by evaporation under reduced pressure, and column chromatography (dichloromethane: methanol = 100: 7) was performed to obtain 10.48 g of a dark purple solid with a yield of 65%.

[0029] The H NMR spectrum of the bright state rhodamine probe 5-RB-L is: 1HNMR(400MHz,DMSO-d6)δ8.17(d,J=7.8Hz,2H), 7.61(d,J=7.8Hz,2H), 7.17(d,J=9.5Hz,2 H)7.13–7.06(m,2H), 6.95(d,J=2.7Hz,2H), 3.62(q,J=7.2Hz,8H), 1.17(t,J=7.1Hz,12H).

[0030] The high-resolution mass spectrum of the bright-state rhodamine probe 5-RB-L is: HR-MS(m / z,ESI):Cal.for[C 28 H 31 N2O3]+m / z=443.2329[M]+.Foundm / z=443.2314.

[0031] Next, prepare the dark state rhodamine probe 5-RB: take 5-RB-L (0.5g, 1.13mmol) and dissolve it in 20ml of dichloromethane and 20ml of deionized water, add excess sodium borohydride solid, stir for 30min, the solution color changes from rose red to light pink (the third solution is obtained), separate dichloromethane with a separatory funnel, wash dichloromethane twice with deionized water, add anhydrous sodium sulfate to dry the dichloromethane solution, evaporate the dichloromethane solution under reduced pressure, transfer it to a brown 50ml sample vial, evacuate it with nitrogen, and store it at -20℃. 5-RB (0.45g) is obtained with a yield of 90%.

[0032] With respect to the dark-state rhodamine probe 5-RB prepared in this example, this example continues to provide some experiments to characterize and analyze its properties.

[0033] First, prepare the singlet oxygen solution: prepare the mother solution to 10 -2 M sodium hypochlorite solution and 10 -2 The mother solution was diluted to 100µM hydrogen peroxide solution, mixed together, added to a 10µM 5-RB probe solution, and the fluorescence spectrum of the probe 5-RB in response to singlet oxygen was measured using a fluorescence spectrometer at 560nm excitation.

[0034] The operation of measuring the fluorescence spectrum of the probe 5-RB in response to singlet oxygen is as follows: irradiate the probe solution of 5-RB with a concentration of 10µM in a fluorescent dish with a light source of the best excitation light of 560nm, detect it every 5 minutes under the light source of 560nm, and continue irradiation for 100 minutes. 5-RB uses rhodamine B (RB, an existing rhodamine probe) as a standard reference, and irradiates it with the best excitation light of rhodamine B for 100 minutes, and detects it every 5 minutes to obtain the fluorescence change comparison of 5-RB and RB. Figure 5 5-RB and the conventional rhodamine probe RB in the dark state of this embodiment are fluorescence intensity scatter plots when irradiated for 0-100 min. Figure 5 , the conventional rhodamine probe RB has a certain brightness that can be observed in the 0-40min interval, and the luminescence time lasts for about 40min. The dark state rhodamine probe 5-RB in this embodiment has a certain brightness that can be observed in the 20-100min interval, and the luminescence time can last at least 80min, because it can be reasonably inferred that after 100min, 5-RB can continue to be observed for a certain time. It can be seen that the dark state rhodamine probe 5-RB in this embodiment has a longer duration of light excitation state and is an organic dye molecule with strong light stability.

[0035] Reference Figure 6 , 10 times the amount of sodium ascorbate was added to the 10 µM probe solution, and the final concentration of 5-RB was 10 as measured by HPLC. -4M, detector 260nm, mobile phase: acetonitrile: water = 9:1. As the irradiation time of 365 nm ultraviolet light increases, 5-RB is gradually converted into 5-RB-L. When sodium ascorbate is added to the solution and compared with that without addition, 5-RB without sodium ascorbate is completely converted into 5-RB-L after irradiation, while only a small amount of conversion is added after sodium ascorbate. The conversion rate of 5-RB-L in the probe solution with sodium ascorbate added is 32.7%, indicating that sodium ascorbate can remove singlet oxygen generated by light irradiation, thereby inhibiting the activation of the probe, further illustrating that the activation process of the dark state rhodamine probe 5-RB in this embodiment is an oxidation process.

[0036] As described above, the dark state rhodamine probe and its preparation method provided in this embodiment are specifically described. It can be seen from the above description that the dark state rhodamine probe provided in this embodiment has strong light stability, which solves the problem of low stability of current organic dye molecules.

[0037] The dark-state rhodamine probe provided in this embodiment can be used in cell imaging. In this case, in order for the dark-state rhodamine probe to effectively track proteins in cells, it needs to be combined with a Halo ligand.

[0038] Therefore, this embodiment also provides a dark state rhodamine probe combined with Halo ligand, which is obtained by combining the dark state rhodamine probe in this embodiment with Halo ligand. The preparation process thereof is as follows Figure 7 , has the following molecular structure expression (III): .

[0039] When the dark-state rhodamine probe combined with the Halo ligand is used in cell imaging, it can effectively track proteins in cells, thereby making it easier for experimenters to observe cells.

[0040] This embodiment also provides a method for preparing the above-mentioned dark-state rhodamine probe bound to the Halo ligand, which includes step S210, step S220 and step S230.

[0041] Step S210, preparing a bright-state rhodamine probe bound to the Halo ligand, wherein the bright-state rhodamine probe bound to the Halo ligand has the following molecular structure expression (IV): .

[0042] Among them, step S210 specifically includes step S211, step S212, step S213 and step S214.

[0043] Step S211, 60% NaH is passed through N2 under ice bath conditions, and DMF and diglycolamine are added and mixed thoroughly, and then 1-chloro-6-iodohexane and DMF (dimethylformamide) are added to react and solid-liquid separation is performed to obtain a fifth solution; Step S212, the fifth solution is quenched with water and then extracted with dichloromethane, and then washed and dried with anhydrous sodium sulfate to obtain 2-(2-((6-chlorohexyl)oxy)ethoxy)ethylamine; Step S213, using dichloromethane to fully dissolve the bright rhodamine probe, N,N,N',N'-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate and N,N-diisopropylethylamine to obtain a sixth solution; Step S214, adding 2-(2-((6-chlorohexyl)oxy)ethoxy)ethylamine to the sixth solution, evaporating dichloromethane under reduced pressure, and performing column chromatography to obtain a bright rhodamine probe bound to the Halo ligand.

[0044] Step S220, dissolving the bright-state rhodamine probe bound to the Halo ligand in dichloromethane and water, and adding an excess of solid sodium borohydride and mixing thoroughly to obtain a seventh solution.

[0045] Step S230, washing the seventh solution, adding anhydrous sodium sulfate, drying, and evaporating under reduced pressure to obtain an eighth solution, and subjecting the eighth solution to sample column chromatography to obtain a dark-state rhodamine probe bound to the Halo ligand.

[0046] In this example, a bright rhodamine probe bound to a Halo ligand was first prepared: 60% NaH (3.54 g, 88.4 mmol) was added to a 250 ml three-necked round-bottom flask, N2 was introduced into the flask under ice bath conditions, 30 ml of ultra-dry DMF was added, diglycolamine (7.4 ml, 70,4 mmol) was slowly added with a syringe, and after slow stirring for 50 min, 1-chloro-6-iodohexane (12.5 ml, 81.1 mmol) was added, and 30 ml of ultra-dry DMF was added. The reaction was allowed to stand for solid-liquid separation, and the reaction solution (the fifth solution) was quenched with water. The reaction solution was extracted with dichloromethane, and the dichloromethane phase was retained. The dichloromethane phase was washed once with saturated sodium bicarbonate (100 ml) and twice with water, and then the washed dichloromethane was dried with anhydrous sodium sulfate. The dichloromethane was evaporated under reduced pressure (the temperature did not exceed 30° C.) to obtain a light yellow oily substance 2-(2-((6-chlorohexyl)oxy)ethoxy)ethylamine.

[0047] 5-RB-L (0.5 g, 1.13 mmol, 1 eq), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)urea hexafluorophosphate (0.64 g, 1.69 mmol, 1.5 eq) and N,N-diisopropylethylamine (240 µL, 1.36 mmol, 1.2 eq) were added to a 50 ml round-bottom flask and dissolved in 20 ml of dichloromethane. After stirring at room temperature for 1 h (to obtain the sixth solution), 2-(2-((6-chlorohexyl)oxy)ethoxy)ethylamine (2.5 g, 10 eq) was added. The reaction was allowed to proceed overnight, and dichloromethane was evaporated under reduced pressure. Column chromatography (dichloromethane:methanol=100:1) was performed to obtain the bright rhodamine probe 5-RB-HT (0.32 g, yield 43.8%) bound to the Halo ligand.

[0048] The high-resolution mass spectrum of the bright-state rhodamine probe bound to the Halo ligand is: HR-MS (m / z, APCl): Cal.for[C 38 H 52 C l N3O4]m / z=650.3719[M+H].Foundm / z=650.3684.

[0049] H NMR spectrum of bright rhodamine probe bound to Halo ligand: 1H NMR (400 MHz, Chloroform-d) δ7.97 (s, 2H), 7.65 (d, J = 8.2 Hz, 1H), 7.23–7.17 (m, 2H), 6.81 (d, J = 8.6 Hz, 2H), 6.59–6.37 (m, 4H), 5.25 (s, 1H), 3.58 ( d,J=4.8Hz,2H), 3.53–3.48(m,2H), 3.35–3.32(m,4H), 2.87(d,J=33.9Hz,12H), 1.99(s, 2H), 1.73–1.62(m,2H), 1.50(p,J=6.9Hz,2H), 1.38–1.31(m,2H), 1.09(t,J=7.1Hz,12H).

[0050] Next, prepare the dark state rhodamine probe 5-RB: take 5-RB-L (0.5g, 0.77mmol) and dissolve it in 20ml of dichloromethane and 20ml of deionized water, add excess sodium borohydride solid, stir for 30min, the solution color changes from rose red to light pink (the seventh solution is obtained), separate dichloromethane with a separatory funnel, wash dichloromethane twice with deionized water, add anhydrous sodium sulfate to dry the dichloromethane solution, evaporate the dichloromethane solution under reduced pressure, transfer it to a brown 50ml sample vial, evacuate it with nitrogen, and store it at -20℃. The dark state rhodamine probe 5-RB-HT (208mg) bound to the Halo ligand is obtained with a yield of 42%.

[0051] High-resolution mass spectrometry of the dark state rhodamine probe 5-RB-HT bound to the Halo ligand: HR-MS (m / z, APCl): Cal.for[C 38 H 52 C l N3O4]m / z=650.3719[M+H].Foundm / z=650.3684.

[0052] Reference Figure 8 , H NMR spectrum of the dark state rhodamine probe 5-RB-HT bound to Halo ligand: 1H NMR (400 MHz, Chloroform-d) δ7.97 (s, 2H), 7.65 (d, J = 8.2 Hz, 1H), 7.23–7.17 (m, 2H), 6.81 (d, J = 8.6 Hz, 2H), 6.59–6.37 (m, 4H), 5.25 (s, 1H), 3 .58(d,J=4.8Hz,2H),3.53–3.48(m,2H),3.35–3.32(m,4H),2.87(d,J=33.9Hz,12H),1.99( s,2H),1.73–1.62(m,2H),1.50(p,J=6.9Hz,2H),1.38–1.31(m,2H),1.09(t,J=7.1Hz,12H).

[0053] With respect to the dark-state rhodamine probe 5-RB-HT bound to the Halo ligand prepared in this example, this example continues to provide some experiments to characterize and analyze its properties.

[0054] 1. Cytotoxicity assay.

[0055] Cell viability was assessed using the MTT (Methylthiazolyldiphenyl-tetrazolium bromide) method. Briefly, cells (5000 cells / well) were seeded into 96-well plates. They were cultured at 37°C in a 5% CO2 incubator for 24 h and then exposed to 100 μL of medium containing different concentrations of 5-RB and 5-RB-L (1, 2, 4, 6, 8, 10 μM). After 12 h of culture, the medium was removed and MTT solution (15 μL, 5 mg / mL) and 180 μL of fresh medium were added to each well. After 4 h, the medium was removed and 150 μL of dimethyl sulfoxide was added to dissolve (incubated in the dark for 10 min). The absorbance of each well at 492 nm was measured using a microplate reader and the cell viability was calculated. Cell viability = a / b×100%, where a is the absorbance of the treated well and b is the absorbance of the control well. Fig. 9is the cell survival rate under different concentrations of 5-RB and 5-RB-L provided in this example. Fig. 9 The probe concentration used in the cell experiment was 1µL. This experiment studied the cytotoxicity of the probe at a dose of 0-10µM. The experiment showed that even at a concentration 10 times higher than that used in the cell experiment, the cell survival rate was above 90%, indicating that the probe is of low toxicity and suitable for long-term live cell imaging.

[0056] 2. Cell transfection experiment.

[0057] In the transfection experiment, 293T cells were seeded in confocal dishes and cultured to 20%-30% cell confluence. The culture medium was replaced with fetal bovine serum containing 10% FBS. Then the cells were transfected with Lipofectamine3000 (Invitrogen, USA) and the plasmid Halo-TMGFP (from E.coil, by Plasmid Macro Extraction Kit (TIANGEN)). 1μg of plasmid and 2μLP3000 were gently added to 75μL of Opti-MEM culture medium. 3μL Lipo3000 was gently added to 75μL Opti-MEM culture medium. The two mixtures were gently mixed in a 1:1 ratio and incubated for 15min. The obtained transfection mixture was added to the cell culture dish and further incubated at 37°C for 6-8h to facilitate transfection. After replacing the culture medium, the cells were cultured for another 30h to express the Halo-Tag fusion protein.

[0058] Based on the same transfection conditions, Fig.10 This experiment compared the cell imaging effects of 5-RB and RB. Among them, the excitation wavelength of 5-RB is 560nm, and the cell membrane labeled with 5-RB can be observed in the confocal mode for at least 8min to 40min, that is, it can be imaged continuously for at least 32min. Similarly, the cell membrane labeled with 5-RB can be imaged continuously for 27min in the high-resolution mode. In contrast, when activated RB is added to the cells for the same experiment, the continuous imaging time in the confocal mode is about 20min (the fluorescence brightness at 20min is basically at the observable lower limit, and the fluorescence brightness at 30min is obviously not supported by observation), and the quenching time in the high-resolution mode is 10min (the fluorescence brightness at 10min is basically at the observable lower limit, and the fluorescence brightness at 15min is obviously not supported by observation), after which the cell morphology cannot be observed, and the fluorescence of 5-RB lasts 2-3 times longer than that of RB. These imaging results show that 5-RB has the unique ability to extend the imaging time.

[0059] It should be understood that the specific embodiments described herein are only used to explain the application, rather than to limit it. Based on the embodiments provided in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the protection scope of this application.

[0060] Obviously, the drawings are only some examples or embodiments of the present application. For ordinary technicians in the field, the present application can also be applied to other similar situations based on these drawings without creative work. In addition, it is understandable that although the work done in this development process may be complicated and lengthy, for ordinary technicians in the field, certain changes in design, manufacturing or production based on the technical content disclosed in this application are only conventional technical means and should not be regarded as insufficient content disclosed in this application.

Claims

1. A dark state rhodamine probe, characterized in that: It has the following molecular structure expression (I): 。 2. The dark state rhodamine probe according to claim 1, characterized in that: The activation process is an oxidation process.

3. The dark state rhodamine probe according to claim 2, characterized in that: After reacting with singlet oxygen, it is activated and becomes a bright-state rhodamine probe, and the optimal excitation light wavelength is 560nm.

4. A method for preparing a dark-state rhodamine probe, characterized in that: For preparing the dark state rhodamine probe according to any one of claims 1 to 3, the preparation method comprises: A bright-state rhodamine probe is prepared. The bright-state rhodamine probe has the following molecular structure expression (II): ； Dissolving the bright rhodamine probe in dichloromethane and water, and adding an excess of solid sodium borohydride and mixing well to obtain a third solution; After washing the third solution, anhydrous sodium sulfate was added to dry the solution and then evaporated under reduced pressure to obtain a fourth solution. The fourth solution was subjected to sample column chromatography to obtain a dark rhodamine probe.

5. The method for preparing the dark-state rhodamine probe according to claim 4, characterized in that: The preparation of bright state rhodamine probe includes: Grinding 4-carboxybenzaldehyde and 3-diethylaminophenol to obtain a powder; The powder and the aqueous sulfuric acid solution are heated under nitrogen flow to obtain a first solution; neutralizing the first solution to obtain a solid precipitate; The solid precipitate is added to anhydrous ethanol for ultrasonic dissolution to obtain a second solution, and the second solution is subjected to column chromatography to obtain a bright rhodamine probe.

6. The method for preparing the dark-state rhodamine probe according to claim 5, characterized in that: The heating temperature is 150-180°C and the heating time is 8-12h.

7. A dark state rhodamine probe binding to a Halo ligand, characterized in that: The dark state rhodamine probe according to any one of claims 1 to 3 is combined with the Halo ligand and has the following molecular structure expression (III): 。 8. Use of the dark-state rhodamine probe combined with Halo ligand according to claim 7 in cell imaging.

9. A method for preparing a dark-state rhodamine probe, characterized in that: The dark state rhodamine probe for binding to Halo ligand according to claim 7 is prepared by a preparation method comprising: A bright-state rhodamine probe bound to a Halo ligand is prepared. The bright-state rhodamine probe bound to a Halo ligand has the following molecular structure expression (IV): ； Dissolving the bright rhodamine probe bound to the Halo ligand in dichloromethane and water, and adding an excess of solid sodium borohydride and mixing well to obtain a seventh solution; The seventh solution was washed, added with anhydrous sodium sulfate, dried, and then evaporated under reduced pressure to obtain an eighth solution. The eighth solution was subjected to sample column chromatography to obtain a dark-state rhodamine probe bound to the Halo ligand.

10. The method for preparing a dark-state rhodamine probe according to claim 9, characterized in that: The preparation of a bright state rhodamine probe bound to a Halo ligand comprises: 60% NaH was passed through N2 under ice bath conditions, and DMF and diglycolamine were added and mixed thoroughly, and then 1-chloro-6-iodohexane and DMF were added to react and solid-liquid separation was performed to obtain a fifth solution; The fifth solution was quenched with water and extracted with dichloromethane, and then washed and dried over anhydrous sodium sulfate to obtain 2-(2-((6-chlorohexyl)oxy)ethoxy)ethylamine; The sixth solution is obtained by using dichloromethane to fully dissolve the bright rhodamine probe, N,N,N',N'-tetramethyl-O-(7-azabenzotriazole-1-yl) urea hexafluorophosphate and N,N-diisopropylethylamine; After adding 2-(2-((6-chlorohexyl)oxy)ethoxy)ethylamine to the sixth solution, dichloromethane was evaporated under reduced pressure and the solution was subjected to column chromatography to obtain a bright rhodamine probe bound to the Halo ligand.

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