CN111690005A - Sensing array with mitochondrial targeting and aggregation-induced emission effects and application of sensing array in cell identification - Google Patents
Sensing array with mitochondrial targeting and aggregation-induced emission effects and application of sensing array in cell identification Download PDFInfo
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Abstract
The present invention relates to a sensor array capable of cell discrimination. The sensing array comprises five tetraphenyl ethylene derivatives with mitochondrion targeting and aggregation-induced emission effects. The tetraphenylethylene derivative provided by the invention adjusts a donor group and different cationic groups to further adjust a molecular probe D-A structure, so that different fluorescence spectrum properties and different electrostatic and hydrophobic interactions with cells are realized; the synthesis is easy; has good biocompatibility and light stability; the cell imaging can be effectively carried out by targeting cell mitochondria; different probes produce differential fluorescent responses with different cells; by analyzing the different fluorescent responses, different cancer cells can be distinguished and cell types can be predicted. A new method for detecting early cancer is designed, and the method has potential application in the aspect of clinical detection of cancer cells.
Description
Technical Field
The invention relates to a structural design and a preparation method of a sensing array with mitochondrial targeting and aggregation-induced emission effects and application of the sensing array in cell identification. The compound is characterized by comprising a series of tetraphenylethylene derivatives which can target mitochondria and have aggregation-induced emission properties, and forming a sensing array by the tetraphenylethylene derivatives, and the application of distinguishing different cells is realized through the charge property difference, the fluorescence signal difference and the spectral property difference of the compounds.
Background
In recent years, aggregation-induced emission fluorescent probes (AIEgens) have been developed vigorously in the fields of chemistry, physics, and biology, and have shown unique advantages in living cells. Intracellular environments affect the aggregation state of AIEgens, such as the microenvironment, proteins and plasmids. The AIEgens using tetraphenylethylene as a main body are easy to adjust molecular structures, have good biocompatibility, excellent light stability and larger Stokes shift, and the positively charged AIEgens have good mitochondrion targeting characteristics. Has wide application in tracking mitochondria, monitoring the membrane potential of mitochondria and regulating the functions of mitochondria. (adv.Mater.2018,30,1802105; ChemPhotoChem 2019,3, 129-2000; chem.Sci.2019,10,1994-2000)
Cancer cells are normal cells that reproduce maliciously due to certain mutations, the distinction between normal cells and cancer cells is often subtle, and the identification of cellular features is a major obstacle to early diagnosis of cancer. (nat. rev. cancer 2008,8,329-340) the recently emerging methods for identifying cancer cells (e.g. image diagnostics, biochemical analysis, pathology and molecular biology methods) are based on biomarkers, mainly proteins and plasmids, which require knowledge of DNA/RNA mutations or protein changes in cancer cells. (acad. sci.2000,906,8-12) however, cells do not always express unique biomarkers and no single marker or combination of multiple biomarkers has sufficient sensitivity and specificity to distinguish cells. (Clin. chem.2006,52,1651-1659) therefore, it is necessary to develop a new detection system for detecting different cancer cells and normal cells.
The unique selectivity of chemical probes is a challenge in identifying complex analytes. Sensor arrays are useful for composites due to their multi-dimensional analysis mechanism. The sensor array generates a response signal for each analyte through different receptor-analyte binding interactions. At present, no sensor array based on molecular probes for discriminating cells has been reported. The molecular probe has better repeatable output signals and can be used for constructing a database. Mitochondria play a crucial role in living cells. Mitochondrial dysfunction is associated with many diseases, such as cancer, muscle disease, cardiovascular disease and diabetes. Because mitochondria vary in number and morphology from normal cells to cancer cells, probes will exhibit different fluorescent responses after entering mitochondria. Doubly charged molecules are able to enter mitochondria faster and with better photostability than singly charged molecules, and therefore, different doubly charged groups are chosen to modulate the luminescent properties of AIEgens, discriminating cancer cells through mitochondrial targeting.
Disclosure of Invention
The invention aims to provide a sensing array structure design with mitochondrial targeting and aggregation-induced emission effects, a preparation method and application thereof in cancer cell discrimination. The method solves the problems of low sensitivity, low accuracy and complex operation steps of early cancer monitoring in the prior art, and provides a novel method for distinguishing cancer cells.
The technical scheme adopted by the invention for solving the technical problem is as follows: the Tetraphenylethylene (TPE) molecules are modified by double positive charges to regulate and control the fluorescence spectrum property of the material and realize multicolor fluorescence. Electrostatic and hydrophobic interactions between aiegens and cells are modulated by the selection of different hydrophobic groups to achieve cellular mitochondrial targeting and multi-channel imaging. Meanwhile, because the shapes and the quantity of mitochondria in different normal cells and different cancer cells are different, different probes interact with different cells to generate different fluorescence responses. By analyzing different fluorescence responses, different cancer cells are distinguished.
The structure of a series of tetraphenylethylene derivatives with mitochondrion targeting and aggregation-induced emission effects provided by the invention comprises a group selected from any one of the following structural formulas:
wherein R is1、R2Are respectively selected from: -H, alkoxy, alkyl, carboxyl, hydroxyl, azido, alkylamino, haloalkyl, ester group,
R3 is selected from:
wherein R ═ PF6 -、CF3 -、BF4 -、ClO3 -，Cl-，I-Or Br-Etc. are any of the negatively charged groups.
The tetraphenylethylene derivative with mitochondrion targeting and aggregation-induced emission effects in the invention has a molecular structure comprising a main structure of tetraphenylethylene and an R1Structure R2is-H, R3Is one of the above groups.
The tetraphenylethylene derivative with mitochondrion targeting and aggregation-induced emission effects in the invention has a tetraphenylethylene main structure and contains the same R1And R2Group, R3Is one of the above groups.
The invention relates to a method for applying a sensing array with mitochondrial targeting and aggregation-induced emission effects in cancer cell identification, which mainly adjusts the structural difference of a molecular probe D-A, realizes different fluorescence spectrum properties, and has different electrostatic and hydrophobic interactions with cells. Because of differences in the morphology and number of mitochondria between different normal cells and different cancer cells, different probes produce different fluorescent responses from different cells. By analyzing different fluorescence responses, different cancer cells are distinguished. The fluorescent molecular structure involved in the method is not limited to the designed double-charge aggregation-induced emission molecule, and comprises a series of tetraphenylethylene derivatives with two positive charges and aggregation-induced emission properties, and the tetraphenylethylene derivatives can target cell mitochondria.
The cells involved in the present invention comprise the following: normal cells include HUVEC, 293T, HFF, HK-2, HKC, etc., and cancer cells include human-derived cancer cells such as Colo205, A375, A549, BT474, HCT116, HeLa, HepG2, Hep-2, MCF-7, MDA-MB-157, SH-SY5Y, SK-OV-3, SW480, SW1990, GBC-SD, HCCC9810, MGC-803, SNU-5, AGS, MKN45, etc.
The invention also provides a preparation method and characterization of the tetraphenylethylene derivative with the mitochondria targeting and aggregation-induced emission properties.
The invention also provides application of the tetraphenylethylene derivative with the mitochondria targeting and aggregation-induced emission properties in intracellular imaging and organelle imaging.
The invention also provides fluorescence result statistics of the interaction of the tetraphenylethylene derivative with the mitochondria targeting and aggregation-induced emission properties and different cells.
The invention also provides a sensing array consisting of the tetraphenylethylene derivatives with the mitochondrial targeting and aggregation-induced emission properties, and a fluorescence result of the interaction between the probe and different cells is analyzed by a mathematical analysis method.
The invention also provides a sensing array consisting of the tetraphenyl ethylene derivatives with the mitochondrial targeting and aggregation-induced emission properties, and different cell discrimination and blind sample prediction are carried out.
The method for applying a series of sensing arrays with the mitochondrial targeting and aggregation-induced emission effects in cancer cell identification has the following beneficial effects: the tetraphenylethylene derivative capable of carrying out mitochondrion targeting is easy to synthesize; has good biocompatibility and light stability; the cell imaging can be effectively carried out by targeting cell mitochondria; different fluorescence spectrum properties and different electrostatic and hydrophobic interactions with cells are realized through the structural difference of the molecular probe D-A, and meanwhile, different probes and different cells generate different fluorescence responses due to the difference of the shapes and the number of mitochondria in different normal cells and different cancer cells; by analyzing different fluorescence responses, different cancer cells can be distinguished and cell types can be predicted, and a new method for early cancer detection is designed.
Drawings
FIG. 1 shows the synthesis route of tetraphenylethylene derivative PT1 with mitochondrial targeting and aggregation-induced emission effects in the examples of the present invention.
FIG. 2 shows the synthesis route of tetraphenylethylene derivative PT2 with mitochondrial targeting and aggregation-induced emission effects in the examples of the present invention.
FIG. 3 shows the synthesis route of tetraphenylethylene derivative PT3 with mitochondrial targeting and aggregation-induced emission effects in the examples of the present invention.
FIG. 4 shows the synthesis route of tetraphenylethylene derivative PT4 with mitochondrial targeting and aggregation-induced emission effects in the examples of the present invention.
FIG. 5 shows the synthesis route of tetraphenylethylene derivative PT5 with mitochondrial targeting and aggregation-induced emission effects in the examples of the present invention.
FIG. 6 is a graph showing the ultraviolet absorption intensity curve and fluorescence intensity curve of tetraphenylethylene derivatives having mitochondrial targeting and aggregation-induced emission effects in examples of the present invention.
FIG. 7 is a graph showing fluorescence intensity curves of tetraphenylethylene derivatives having mitochondrial targeting and aggregation-induced emission effects in different glycerol ratios according to examples of the present invention.
FIG. 8 is a graph showing fluorescence intensity curves of tetraphenylethylene derivatives having mitochondrial targeting and aggregation-induced emission effects in different bovine serum albumin ratios according to the examples of the present invention.
FIG. 9 is a graph showing the comparison of fluorescence intensities of tetraphenylethylene derivatives having mitochondrial targeting and aggregation-induced emission effects before and after interaction with different cells, respectively, in the examples of the present invention.
FIG. 10 is a graph showing the cytotoxicity of tetraphenylethylene derivatives having mitochondrial targeting and aggregation-induced emission effects in examples of the present invention.
FIG. 11 is a graph of photostability curve of tetraphenyl ethylene derivatives with mitochondrial targeting and aggregation-induced emission effects and the corresponding cellular images in accordance with the examples of the present invention.
FIG. 12 is an image of the mitochondrial co-localization of tetraphenylethylene derivatives with mitochondrial targeting and aggregation-induced emission effects in an example of the invention.
FIG. 13 is a fluorescent image of interaction of tetraphenylethylene derivatives with different cells having mitochondrial targeting and aggregation-induced emission effects in the examples of the present invention.
FIG. 14 is a fluorescent image of interaction of tetraphenylethylene derivatives with mitochondrial targeting and aggregation-induced emission effects with different digestive tract cancer cells in an example of the present invention.
FIG. 15 is a fluorescent image of interaction of tetraphenylethylene derivatives with mitochondrial targeting and aggregation-induced emission effects with different gastric cells in an example of the present invention.
FIG. 16 is a fluorescence statistical result diagram, a mathematical analysis result diagram and an unknown sample prediction diagram of the tetraphenylethylene derivative with mitochondrion targeting and aggregation-induced emission effects and various cells in the embodiment of the invention.
Detailed Description
The structure design, preparation method and application in cell discrimination of the sensing array with mitochondrial targeting and aggregation-induced emission effects of the present invention are further illustrated below with reference to the accompanying drawings and examples: the structure design and preparation process of the sensing array with mitochondrial targeting and aggregation-induced emission effects of the present invention are illustrated by the following examples. It should be noted that the tetraphenylethylene derivatives prepared below are only one or more of those represented by each of the structural formulas as claimed in the claims, but the aggregation-induced emission of the tetraphenylethylene derivatives as claimed in the present invention is not limited thereto;
example 1: synthesis of PT1
Structural formula (xvi):
the synthetic process is shown in the synthetic route shown in figure 1.
(1) Synthesizing TPE-2 OH: 4,4 '-dihydroxybenzophenone (3.00g, 14mmol), 4,4' -dimethoxybenzophenone (3.39g, 14mmol) and zinc powder (7.28g, 112 mmol)) Adding into a 250mL two-neck flask, adding magneton, adding anhydrous THF 150mL, removing oxygen and keeping N2And (4) environment. Slowly under acetone bath to N2Titanium tetrachloride (6.15mL, 56mmol) was added dropwise to the protected two-necked flask and stirred in an acetone bath for 0.5h, allowed to return to room temperature naturally, and then refluxed for 9 h. After the reaction was completed, the reaction was returned to room temperature, and a saturated potassium carbonate solution was added to the mixture to quench the reaction. The zinc dust was removed by suction filtration and the filtrate was extracted with water and dichloromethane (100mL x 3). The organic phase was collected, washed with water, and then dried over anhydrous sodium sulfate. The product was purified by column chromatography using an eluent of petroleum ether/ethyl acetate (4: 1, v/v) to give a white solid (3.38g, yield: 57%).1H NMR(400MHz,DMSO-d6)9.27(s,2H),6.84(d,J＝8.5Hz,4H),6.74(d,J＝8.3Hz,4H),6.68(d,J＝8.5Hz,4H),6.50(d,J＝8.5Hz,4H),3.68(s,6H).13C NMR(100MHz,DMSO-d6)157.23,155.51,138.74,136.88,136.56,134.69,131.93,114.54,113.06,54.83.HR-MS(MALDI-TOF)calcd for C28H24O4[M+]424.17,found 424.1672。
(2) Synthetic TPE-2OC3Br: taking TPE-2OH (1.00g, 2.36mmol), 1, 3-dibromopropane (1.65mL, 11.8mmol) and K2CO3(0.98g, 7.07mmol) was charged into a 50mL single-neck flask, 30mL of anhydrous acetone was added, oxygen was removed, and the mixture was heated under N2Reflux for 48h at ambient. After the reaction was completed, it was cooled to room temperature and extracted with water and dichloromethane (20mL × 3). The organic layer was collected, dried over anhydrous sodium sulfate, and purified by column chromatography using an eluent of petroleum ether/ethyl acetate (10: 1, v/v) to give a white solid (1.13g, yield: 72%).1H NMR(400MHz,Chloroform-d)7.03-6.87(m,8H),6.67(dd,J＝8.8,3.0Hz,8H),4.06(t,J＝5.8Hz,4H),3.78(s,6H),3.61(t,J＝6.4Hz,4H),2.31(p,J＝6.1Hz,4H).13C NMR(100MHz,Chloroform-d)157.83,156.92,137.15,136.84,132.55,113.64,113.05,65.14,55.11,32.51,30.11.HR-MS(MALDI-TOF)calcd for C34H34Br2O4[M+2H]+666.08,found 666.079
(3) Synthesis of PT 1: taking TPE-2OC3Br (100.00mg, 0.15mmol) and Triphenylphosphine (TPP) (393.13mg, 1.5mmol) were added to 50mL of monoIn a neck flask, 30mL of acetonitrile was added, deoxygenated, and N was added2Reflux for 48h at ambient. After the reaction, the mixture was cooled to room temperature and distilled under reduced pressure to obtain a solid. Recrystallization from methanol and hexane gave a pale green solid (76.64mg, yield: 43%).1HNMR(400MHz Chloroform-d)7.79-7.74(m,8H),7.69(d,J＝7.3Hz,6H),7.60(t,J＝2.6Hz,8H),7.48-7.36(m,8H),6.86-6.74(m,8H),6.60-6.43(m,8H),4.14(s,4H),3.64(q,J＝2.4Hz,6H),2.10(d,J＝13.9Hz,4H),1.19(d,J＝10.7Hz,4H).13C NMR(100MHz Chloroform-d)157.82,156.38,138.51,138.15,137.23,137.12,136.73,135.13,133.84,133.65,132.55,132.16,132.06,132.01,131.98,130.63,130.50,128.74,128.55,128.48,118.68,117.82,113.71,113.10,77.46,77.15,76.83,66.45,66.28,55.16,23.06,20.05,19.53.HR-MS(ESI,positive)calcd for C70H64Br2O4P2(M-2Br)/2 515.21,found515.21355。
Example 2: synthesis of PT2
Structural formula (xvi):
the synthetic process is shown in the synthetic route shown in figure 2.
Synthesis of PT 2: taking TPE-CN-2OC3Br (100.00mg,0.13 mmol) and Me3N (76.83mg, 1.3mmol) was charged to a 50mL single neck flask, 30mL anhydrous THF was added, oxygen removed, and stirred at 25 ℃ for 2 days. At the end of the reaction, the mixture was filtered and the residue was washed with excess THF to give a red solid (52.61mg, 0.06mmol, yield: 45%).1H NMR(400MHz,CD3OD,):7.71(d,J＝7.2Hz,1H),7.64–7.55(m,1H),7.54–7.49(m,2H),7.48–7.41(m,1H),7.40–7.28(m,1H),7.26–7.09(m,4H),7.08–6.98(m,3H),6.98–6.82(m,5H),6.79–6.65(m,4H),4.04(t,J＝4.8Hz,4H),3.56(t,J＝8.0Hz,4H),3.17(s,18H),2.31–2.17(m,4H)；HR-MS(ESI,positive)m/z:[M-2Br]2+/2calcd.for[C48H52N4O2]2+/2,358.20396；found,358.20413。
Example 3: synthesis of PT3
Structural formula (xvi):
the synthetic process is shown in the synthetic route shown in figure 3.
(1) Synthesizing TPE-2 Br: 4,4 '-dibromobenzophenone (3.00g, 8.82mmol), 4,4' -dimethoxybenzophenone (2.13g, 8.82mmol) and zinc powder (7.28g, 70.56mmol) were charged into a 250mL two-necked flask, magnetons were added, 150mL of anhydrous THF was added, and N was maintained by removing oxygen2And (4) environment. Slowly under acetone bath to N2Titanium tetrachloride (6.15mL, 35.82mmol) was added dropwise to the protected two-necked flask and stirred in an acetone bath for 0.5h, allowed to return to room temperature naturally, and then refluxed for 9 h. After the reaction was completed, the reaction was returned to room temperature, and a saturated potassium carbonate solution was added to the mixture to quench the reaction. The zinc dust was removed by suction filtration and the filtrate was extracted with water and dichloromethane (100mL x 3). The organic phase was collected, washed with water, and then dried over anhydrous sodium sulfate. The product was purified by column chromatography using an eluent of petroleum ether/dichloromethane (1: 5, v/v) to give a white solid (4.47g, yield: 92%).1H NMR(400MHzCDCl3,):7.80-7.70(4H,d,J＝8.4Hz),7.68-7.53(4H,m),7.53-7.44(4H,m),6.97-6.95(4H,d,J＝8.7Hz),3.88(6H,s).MALDI-TOF calcd for C28H22O2Br2(M+2H+)550.29,found550.3
(2) Synthesis of TPE-2 CHO: TPE-2Br (2.19g, 4mmol) was charged into a 50mL two-neck flask, anhydrous THF 30mL was added, and N was maintained by deoxygenation2And (4) environment. Slowly under acetone bath to N2A protected two-necked flask was charged dropwise with N-butyllithium solution (9.00mL, 14.4mmol) in an acetone bath and N2Stirring for 1 h. N, N-dimethylformamide (1.86mL, 24mmol) was then added and the mixture was stirred at room temperature overnight. At the end of the reaction, the mixture was extracted with water and dichloromethane (20mL x 3). The organic phase was collected, then dried over anhydrous sodium sulfate and concentrated in vacuo. The product was purified by column chromatography using petroleum ether/dichloromethane (1: 1, v/v) as eluent to give a yellow solid (0.73g, yield: 41%).1H NMR(400MHz CDCl3,):9.90(2H,s),7.65-7.62(4H,m),7.17-7.15(4H,m),6.94-6.91(4H,m),6.70-6.68(2H,d,J＝2.2Hz),6.68-6.65(2H,d,J＝2.6Hz),3.74(6H,s).13C NMR(400MHz CDCl3,):191.76,158.96,150.33,148.52,134.92,134.30,132.70,132.65,132.00,129.41,129.34,113.35,113.32,113.30,55.08.HRMS calcd for C30H24O4(M)448.17,found 448.1669。
(3) Synthesis of PT 3: TPE-2CHO (50.00mg, 0.11mmol) and 1, 4-dimethyl iodide (103.40mg, 0.44mmol) were added to a 50mL single-neck flask, 20mL of absolute ethanol was added, and several drops of triethylamine were added dropwise. Removing oxygen in N2Reflux for 48h at ambient temperature, then cool to room temperature and extract with water and dichloromethane (20mL × 3). Dried over anhydrous sodium sulfate and concentrated in vacuo. Column chromatography was performed using dichloromethane/methanol (15: 1, v/v) as eluent to give an orange solid (20.38mg, yield: 21%).1H NMR(400MHz,DMSO-d6)8.86(d,J＝6.3Hz,4H),8.21(d,J＝6.7Hz,2H),8.02-7.29(m,8H),7.10-6.79(m,10H),6.74(d,J＝8.8Hz,4H),4.29(d,J＝19.1Hz,6H),3.75-3.67(m,6H).13C NMR(100MHz,DMSO-d6)158.14,152.40,145.80,145.02,144.44,140.11,135.21,133.20,132.18,132.08,131.59,127.90,127.84,127.78,123.39,113.36,55.19,55.02,55.01,47.27,46.93.HR-MS(ESI,positive)calcd for C44H40I2N2O2(M-2I)/2314.15,found 314.15408
Example 4: synthesis of PT4
Structural formula (xvi):
the synthetic process is shown in the synthetic route shown in figure 4.
(1) Synthesis of PT 4: taking TPE-CN-2OC3Br (100.00mg,0.132mmol) and Triphenylphosphine (TPP) (345.96mg,1.32mmol) were charged to a 50mL single-neck flask, 30mL acetonitrile was added, oxygen was removed, and the mixture was stirred under N2Reflux for 48h at ambient. After the reaction, the mixture was cooled to room temperature and distilled under reduced pressure to obtain a solid. Recrystallization from methanol and hexane gave an orange solid (76.64mg, yield: 36%).1HNMR(400MHz Chloroform-d,):7.76(d,J＝18.9Hz,10H),7.61(d,J＝7.6Hz,10H),7.48(t,J＝7.5Hz,4H),7.43-7.29(m,10H),7.13-6.93(m,10H),6.80(d,J＝8.5Hz,4H),6.54(dd,J＝21.6,7.6Hz,4H),4.19(s,4H),2.11(d,J＝7.5Hz,4H),1.18(s,4H).13C NMR(100MHzChloroform-d,):174.72,157.46,149.75,143.10,142.72,137.73,136.02,135.88,135.16,133.92,133.82,133.35,133.01,132.74,132.66,132.16,132.06,131.97,131.66,131.37,130.67,130.55,130.12,128.86,128.59,128.47,128.10,126.72,118.68,117.82,114.34,114.09,113.82,77.38,77.06,76.74,66.70,23.16,20.35,19.83.HR-MS(ESI,positive)calcd for C78H64Br2N2O2P2(M-2Br)/2 561.22,found 561.22179
Example 5: synthesis of PT5
Structural formula (xvi):
the synthetic process is shown in the synthetic route shown in FIG. 5.
(1) Synthesis of PT 5: TPE-2CHO (44.82mg, 0.1mmol) and 1,2,3, 3-tetramethyl-3H-indole-1-iodoiodide (120.41mg, 0.4mmol) were charged into a 50mL single-neck flask, 20mL of anhydrous toluene was added, oxygen was removed, and the mixture was charged in N2Reflux for 48h at ambient temperature, then cool to room temperature and extract with water and dichloromethane (20mL × 3). Dried over anhydrous sodium sulfate and concentrated in vacuo. Column chromatography was performed using dichloromethane/methanol (30: 1, v/v) as an eluent to give a red solid (30.42mg, yield: 28%).1H NMR(400MHz CDCl3,):8.18(d,J＝16.1Hz,2H),7.95(d,J＝8.3Hz,4H),7.72(d,J＝16.1Hz,2H),7.63-7.56(m,8H),7.16(d,J＝8.2Hz,4H),6.99(d,J＝8.7Hz,4H),6.71(d,J＝8.8Hz,4H),4.43(s,6H),3.79(s,6H),1.88(s,12H).13C NMR(400MHz CDCl3,):182.22,159.21,154.10,150.53,142.99,141.50,134.95,132.97,132.65,131.98,131.16,129.92,129.67,122.66,114.81,113.52,112.60,77.37,77.05,76.73,55.29,52.56,37.20,26.96.HR-MS(ESI,positive)calcd for C54H52N2O2(M-2I)/2 380.2,found 380.2010.
Example 6: the molecular probe is characterized by ultraviolet spectral property, fluorescence spectral property and AIE property
(1) Ultraviolet spectral properties of the molecular probe: as shown in FIG. 6A, the maximum absorption peaks corresponding to 10. mu.M molecular probe in 65% glycerol solution were 310nm, 415nm, 417nm, 420nm and 488nm, respectively. And connecting groups with different conjugated structures on a tetraphenyl ethylene benzene ring to obtain molecules with different absorption peaks. The absorption peak shifted from 310nm for the probe consisting of methoxy and bistriphenylphosphine groups to 488nm for the probe consisting of methoxy and bistriphenylphosphine groups, the absorption peak changed mainly because of the difference in donor-acceptor structure, and the D- π -A structure successfully shifted the absorption of the doubly charged probe to more red wavelengths.
(2) Fluorescence spectrum property of molecular probe: as shown in FIG. 6B, the fluorescence spectra of the five molecules also showed significant differences, with maximum emission peaks corresponding to 10. mu.M molecular probes in 65% glycerol solution at 475nm, 625nm, 625nm, 640nm and 690nm, respectively. By adjusting the substituents on the TPE backbone, a difference in fluorescence signal from blue to red can be achieved.
(3) AIE properties of molecular probes: in order to investigate the AIE properties of the molecular probes, the ratio of glycerol was adjusted in an aqueous solution of glycerol, and as shown in FIG. 7, the fluorescence intensities of five molecular probes gradually increased with the increase in the ratio of glycerol, demonstrating that all five molecules had significant AIE properties. Proteins and other biomolecules also caused an increase in the fluorescence of AIE compounds, and to mimic the environment in living cells, the addition of varying amounts of Bovine Serum Albumin (BSA) (from 0 to 1mg/mL) showed a concentration-dependent increase in the fluorescence of five molecules (fig. 8), indicating that five molecular probes all had significant AIE properties.
Example 7: spectral characterization of molecular probes and five representative cells
The probes were dissolved in a small amount of DMSO to obtain a stock solution, and then an amount of PTx was added to PBS to obtain the corresponding concentration, in the case of HeLa, the cells were cultured, after the cells were confluent, the cells were collected and 3 × 105Was inoculated in 6-well plates and 1mL of complete medium was added. After 24h of incubation, the medium was removed and the cells were incubated with 2mL of the prepared probe solution for 5 minutes. The solution was transferred from the 6-well plate to a centrifuge tube (5 mL). The cells were digested with 0.5mL of trypsin,digestion was stopped with 1mL of cell culture medium, the medium was transferred to a centrifuge tube (15mL), the well plate was rinsed with 1mL PBS to ensure that all cells were transferred into the centrifuge tube, and the cells were harvested by centrifugation (1000rpm for 2 minutes). The previously removed solution from the 5mL centrifuge tube was added to the 15mL centrifuge tube and the cells were resuspended. Finally, fluorescence intensity was measured using a fluorescence spectrometer. Each set was used in triplicate and the different probe-cell interaction procedures were identical. Incubation with cells using PBS served as a control experiment.
The invention will be used for the discrimination of different cells. To determine that different cells have different interactions with the probe, the fluorescence spectra of the different probes after their interaction with the cell are determined. Five cells, including normal cells (293T), cervical cancer cells (HeLa), lung cancer cells (A549), pancreatic cancer cells (SW1990) and gastric cancer cells (SNU-5) were selected for fluorescence intensity comparison. As shown in fig. 9, the same PTx interacts with different cells and the fluorescence intensity differs; different PTx interact with the same cell and the fluorescence intensity also differs. Taking the interaction results of different cells with PT1 and PT2 as examples, after incubation with 10. mu.M PT1, the fluorescence intensity of different cells is only 1.2-1.6 times higher than that of PBS, and different fluorescence responses are shown from 293T (1.6 times) to SNU-5(1.2 times). For PT2, the fluorescence intensities of the different cells were 16.9(HeLa), 10.3(293T), 7.4(SW1990), 4.6(SNU-5) and 1.7(A549), respectively, times that of PBS. Fluorescence occurred in HeLa (16.9 fold) and the lowest fluorescence in a549 (1.7 fold). The fluorescence intensity of other probes after interaction with the cell also varied and the maximum emission wavelength of PTx did not change much. The different fluorescence intensities of PTx upon cell interaction provide a database for cell discrimination.
Example 8: cytotoxicity experiment, photostability experiment and mitochondria targeting co-location experiment of molecular probe.
(1) Characterization of the cytotoxicity of the molecular probes: the present invention will be applied to cellular imaging, first to assess the cytotoxicity of PTx using the conventional MTT method. HeLa was incubated with fresh medium at different concentrations (0, 5, 8, 10, 15 and 20. mu.M) of PTx, after 24h, the medium containing MTT reagent was replaced and cultured for another 4h, after which the supernatant was aspirated off and 150. mu.L of DMSO was added, and the optical density at 490nm was read with a microplate reader to obtain the cell viability. As shown in fig. 10, the cytotoxicity was low despite the higher concentration, indicating that the molecular probe is promising as a candidate molecule for cell identification.
(2) Characterization of the photostability of the molecular probes to further characterize the advantages of the molecular probes, a photostability test under continuous light irradiation was performed, the cells were collected and tested at 3 × 105Is seeded in a confocal dish. After 24h, 10 μ M1 mL PTx in DMEM was added to the culture dish and incubated for 5 minutes, and cell images at various bleaching times were taken. The excitation wavelengths used for PTx were 405nm, 405nm, 405nm, 405nm, 488nm, respectively. As in fig. 11, PTx maintained good fluorescence intensity after 300 exposures and the maximum signal loss of PTx was less than 20%. Fluorescence images of cells stained with PTx from 0, 100 and 300 bleaching times showed no significant change in fluorescence intensity. Thus, PTx has excellent photostability, and can provide long-term mitochondrial imaging and morphological analysis capabilities for various cells.
(3) Mitochondrial targeting Co-localization experiments with molecular probes the present invention performs cell identification based on mitochondrial targeting fluorescent probes, thus determining mitochondrial targeting Co-localization experiments with molecular probes, cells were collected and tested as 3 × 105Is seeded in a confocal dish. After 24h, HeLa cells were first incubated with 10 μ M PTx for 5 min and then with Rhod 123 for 20 min. Cells were washed with PBS and then imaged. The excitation wavelengths for PTx and Rhod 123 were 405nm, 405nm, 405nm, 405nm, 552nm and 488nm, respectively. HeLa cells were stained with PTx and rhodamine 123(Rhod 123) for confocal imaging. Rhod 123 is a commercial fluorescent dye that targets mitochondria. As shown in fig. 12, PTx staining was consistent with Rhod 123 in cells with pearson correlation coefficients of 0.8796, 0.8940, 0.8842, 0.8786, 0.9070, respectively. Confocal fluorescence images indicate that PTx can selectively stain mitochondria in living cells.
Example 9: confocal imaging of molecular probes with different cells
(1) Confocal imaging of different cells with molecular probes: to demonstrate the versatility of molecular probe arrays for staining cellsScanning confocal laser scanning imaging of the probe and cells, cells were collected and examined at 3 × 105Is seeded in a confocal dish. After 24h, 1mL of 10. mu.M PTx medium was added to the confocal dish and incubated for 5 minutes. And selecting corresponding excitation and emission according to the excitation wavelength of the molecular probe by a laser scanning confocal microscope to perform fluorescence imaging. First, different cells were confocal imaged with molecular probes, and normal cells (HUVEC, 293T), cervical cancer cells (HeLa), breast cancer cells (MCF-7), neuroblastoma cells (SH-SY5Y), lung cancer cells (A549), and cholangiocarcinoma cells (HCCC9810) were selected, as shown in FIG. 13, where the same probe had fluorescence of different intensities in different cells and the same cell in different probes.
(2) Confocal imaging of different digestive tract cancer cells with molecular probes: to further verify the utility of the probe, a fluorescence confocal experiment was performed using four digestive tract cancer cells. Bile duct cancer cells (HCCC9810), gallbladder cancer cells (GBC-SD), pancreatic cancer cells (SW1990) and gastric cancer cells (MGC-803) were selected. As shown in fig. 14, the same probe showed fluorescence with different intensities in different digestive tract cancer cells, and the different probes showed fluorescence with different intensities in the same digestive tract cancer cells.
(3) Confocal imaging of gastric cancer cells with molecular probes of different similar histologies: to further verify the versatility of the probe, fluorescence confocal experiments were performed using four similarly histologic gastric cancer cells. MGC-803, SNU-5, MKN45 and AGS are selected. As shown in fig. 15, the same probe showed different fluorescence intensities in different gastric cancer cells of similar histology, and different probes showed different fluorescence intensities in the same gastric cancer cells.
(3) Confocal imaging of mixed gastric cancer cells with molecular probes: early clinical diagnosis of cancer usually has a mixed gastric cancer cell sample. Therefore, in the fourth group of cells, ten mixed gastric cancer cell samples were identified using the molecular probe array, including six mixtures of two gastric cancer cells, three mixtures of three gastric cancer cells, and four gastric cancer cells.
Example 10: cell discrimination, including fluorescence statistics, cell discrimination and blind sample analysis, was performed using a tetrastyrene derivative with a mitochondrion-targeted aggregation-induced emission effect.
(1) Fluorescence statistics, in the data analysis and statistics process, a lot of data need to be recorded, the microplate reader is a high-throughput and easy-to-operate instrument, and can be used for recording fluorescence intensity statistics of molecular probes incubated with different cells, collecting cells and performing 1 × 104Seeded in 96-well plates. After 24h, 100. mu.L of 10. mu.M PTx dissolved in PBS was added to each well and the probes were incubated with the cells for 5 minutes. The fluorescence intensity was measured in a microplate reader with different excitation and emission wavelengths. Fluorescence intensity was recorded at the optimal excitation/emission (Ex/Em) wavelength, for PTx: Ex/Em 310/475nm (PT1), Ex/Em 415/625nm (PT2), Ex/Em 417/625nm (PT3), Ex/Em 420/640nm (PT4), Ex/Em488/690nm (PT 5). Control experiments were performed with 100. mu.L of 10. mu.M PTx in PBS without cells, and the experiments were repeated eight times per group. For all experiments with various cell types under the same conditions, we used the same volume of PBS. Thus, the changes caused by PBS alone will be the same for all experiments. For data analysis, I/I0 was calculated using Excel, where I is the fluorescence intensity of PTx with cells and I0 is the fluorescence intensity of PTx without cells.
(2) Fluorescence statistics: as shown in FIGS. 16A, D, G represent the fluorescence analysis results of five molecular probes with different cells, different digestive tract cancer cells and different gastric cancer cells of similar histology. The relative fluorescence intensity of the molecular probes (I/I0) before and after incubation with cells was shown to be used to characterize the fluorescent response of each probe to different cells. For example, the fluorescence intensity of interaction with 293T using PT5 was 17 times higher than that in PBS only, and the fluorescence intensity of cells using PT1 and SH-SY5Y was 1.2 times higher than that of PBS only. Different molecular probes produce differential fluorescent responses with different cells. The five molecular probes form a sensing array, and the sensing array generates different fluorescent fingerprints according to different response results, so that different cells are distinguished.
(3) Fluorescence statistics: early clinical diagnosis of cancer usually has a mixed gastric cancer cell sample. Thus, ten mixed gastric cancer cell samples were identified using a molecular probe sensing array, including six mixtures of two gastric cancer cells, three mixtures of three gastric cancer cells, and four mixtures of gastric cancer cells. As in fig. 16J, the fluorescence fingerprint was different for each mixed cell sample.
(4) Cell discrimination using Principal Component Analysis (PCA) analysis method: PCA was performed using I/I0 under different excitation conditions using Matlab R2014a software. PCA is a mathematical analysis method used for dimensionality reduction. A plurality of indexes are converted into a plurality of comprehensive indexes (namely, principal components) through linear conversion, problems are simplified, and more scientific and effective data are obtained. As shown in fig. 16B, E, H, K, the molecular probe sensing array was able to distinguish seven different cell lines, 4 different digestive tract cancer cells, 4 gastric cancer cells with similar histology and 10 different gastric cancer mixed cells with 100% efficiency.
(5) Blind sample analysis: one key step in chemical sensing is to challenge its repeatability. And (3) verifying the prediction capability of the molecular probe sensing array on unknown cells by using a Support Vector Machine (SVM) analysis method. The SVM is a supervised learning model and is mainly used for pattern recognition, prediction, classification and regression analysis. To predict the samples, a set of known examples is given, the mapping is performed by the SVM, and each training example is labeled as a different class to form the true class (red line of fig. 16C, F, I, L). The SVM then maps the blind sample data set to the same space and predicts a class according to its position in space to form a predicted class. (blue line of fig. 16C, F, I, L) the predicted class is compared to the actual class to obtain the predictive power of the sensing array. To test for unknown cell types, the samples were tested using the same procedure as the training samples, the resulting fluorescence was analyzed, and the fluorescence data was subjected to SVM analysis. SVM was performed using I/I0 under Matlab R2014a software to derive the cell species to which the blind sample belongs.
(6) Blind sample analysis: for different cells, the predictive ability of sensing on unknown cells was verified using a blind sample of 7 cell lines (7 cell lines × 3 replicates ═ 21 samples), as in fig. 16C, the sensing array was able to predict 100% of unknown cases of different cells. Using 4 cell lines (4 cell lines × 5 replicates ═ 20 samples) for different gut cancer cells, the sensor array was able to predict 100% of unknown cases of different gut cancer cells as shown in fig. 16F. For gastric cancer cells of similar histology, using (4 cell lines × 5 replicates ═ 20 samples), 100% unknown sample identification accuracy was shown. For mixed gastric cancer cell lines of similar histology, the sensor array was able to predict all unknown cell types with 100% accuracy using (10 cell lines × 3 replicates ═ 30 samples). Thus, the sensor array was predicted with 100% accuracy for different cell lines, different gut cancer cell lines, different gastric cancer cell lines of similar histology and mixed gastric cancer cell lines.
It should be understood to be apparent to one of ordinary skill in the art. Modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention, and all such modifications and variations are intended to be included within the scope of the following claims.
Claims (8)
1. The invention relates to a structure design and a preparation method of a sensing array with mitochondrial targeting and aggregation-induced emission effects and application thereof in cell identification; the compound is characterized by comprising a series of tetraphenylethylene derivatives which can target mitochondria and have aggregation-induced emission properties, and forming a sensing array by the tetraphenylethylene derivatives, and the application of distinguishing different cells is realized through the charge property difference, the fluorescence signal difference and the spectral property difference of the compounds.
2. The compound of tetraphenylethylene derivative with mitochondrial targeting and aggregation-induced emission effects as claimed in claim 1, wherein said compound uses tetraphenylethylene as the backbone to design aggregation-induced emission molecules with recognition groups of different positive charges, the molecular structure comprises a group selected from any of the following structural formulas:
wherein R is1、R2Are respectively selected from: -H, alkoxy, alkyl, carboxyl,Hydroxy, azido, alkylamino, haloalkyl, ester group,
R3 is selected from:
wherein R ═ PF6 -、CF3 -、BF4 -、ClO3 -，Cl-，I-Or Br-Etc. are any of the negatively charged groups.
3. The tetraphenylethylene derivative molecule with mitochondrion targeting and aggregation-induced emission effects as claimed in claims 1 and 2, characterized by comprising a main structure of tetraphenylethylene and comprising an R1Structure R2is-H, R3Is one of the above groups.
4. The tetraphenylethylene derivative molecule with mitochondrion targeting and aggregation-induced emission effects as claimed in claims 1 and 2, characterized by comprising a main structure of tetraphenylethylene and comprising the same R1And R2Group, R3Is one of the above groups.
5. The use of the tetraphenylethylene derivative with mitochondrial targeting and aggregation-induced emission effects according to any one of claims 1 to 4 for cellular imaging and imaging of organelles, for generating differential fluorescence responses by interacting with different cells, for cell discrimination and blind prediction.
6. The use of claim 5 in cell discrimination and blind prediction, wherein the array of sensors is composed of five tetraphenylethylene derivatives.
7. The cell of claim 6, comprising the following: normal cells include HUVEC, 293T, HFF, HK-2, HKC, etc., and cancer cells include any of human-derived cancer cells such as Colo205, A375, A549, BT474, HCT116, HeLa, HepG2, Hep-2, MCF-7, MDA-MB-157, SH-SY5Y, SK-OV-3, SW480, SW1990, GBC-SD, HCCC9810, MGC-803, SNU-5, AGS, MKN45, etc.
8. The application of the probe in cell discrimination and blind sample prediction as claimed in claim 6, wherein the probe interacts with the cell, the fluorescence result is counted, the cell discrimination is realized by a principal component analysis mathematical analysis method, and the cell blind sample prediction is realized by a support vector machine mathematical analysis method.
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