A multi-organellar targeted aie fluorescent sensing array and its application in cancer cell metastasis detection

Abstract

The application discloses a multi-organ targeting AIE fluorescent sensing array and application thereof in cancer cell metastasis detection. The application designs and synthesizes a multi-color fluorescent sensing array based on tetraphenyl ethylene, which has multi-color fluorescent emission and AIE characteristics, can give unique fluorescent fingerprint images of different kinds of cancer cells and cell balls, and overcomes the spectral crosstalk defect of single-color fluorescence. By combining convolutional neural network to learn image features and training the network, image features are completely extracted without manual intervention, and errors caused by objective factors in traditional machine learning methods are overcome. The method is convenient to operate, low in cost, non-invasive, fast and accurate.

Description

Technical Field

[0001] This invention belongs to the field of cancer cell detection technology, and particularly relates to a multi-organelle targeted AIE fluorescence sensing array and its application in cancer cell metastasis detection. Background Technology

[0002] Accurate cell type identification is crucial for cancer diagnosis and treatment. Fluorescence imaging, as a strategy for cell identification and analysis, is characterized by high specificity and sensitivity. Organelles are essential for maintaining normal cellular function and are important targets for cell imaging. Some studies have shown significant differences between cancer cells and normal cells in organelle morphology, quantity, and microenvironment. Organelle-targeted imaging can reflect these differences between normal and cancer cells. When normal and cancer cells are stained with the same probe, their fluorescence differs. Aggregation-induced emission (AIE) probes are widely used in organelle imaging due to their excellent photostability and high signal-to-noise ratio. Differences in organelles lead to varying degrees of AIE probe aggregation, resulting in a unique fluorescence fingerprint for each cell type. Cell spheroids are self-assembled aggregates of cells arranged in spherical clusters, typically composed of tens of thousands of cells. Cell spheroid models can replicate some tumor tissue characteristics, making the identification and analysis of cell spheroids particularly meaningful for clinical diagnosis.

[0003] Traditional fluorescent probe analysis often targets a single analyte, making it unsuitable for analyzing complex substances. Fluorescent sensor arrays, combining the high selectivity and sensitivity of fluorescent probes, are more suitable for multi-target imaging and analysis, including cells, microorganisms, and biomolecules. The combination of fluorescent sensor arrays and machine learning methods has been applied in the biological and medical fields, offering advantages in identifying complex samples and diseases. Traditional machine learning methods include the k-nearest neighbor algorithm (KNN), support vector machines (SVM), and logistic regression. While these methods have been used for cell identification, they rely on subjective factors to define and select color, texture, and shape features of lesion images, making the analytical results dependent on the quality of the selected features. Summary of the Invention

[0004] To address the shortcomings and deficiencies of existing technologies, this invention provides a method combining a multi-organelle-targeted AIE fluorescence sensing array with a convolutional neural network (CNN) – Residual Network (ResNet), achieving rapid and accurate detection of cells and cell spheroids of different metastatic types. The sensing array designed and synthesized in this invention possesses multi-color fluorescence emission and AIE characteristics, capable of providing unique fluorescence fingerprint images for different types of cancer cells. Different organelles emit fingerprint fluorescence of different colors, overcoming the spectral crosstalk defects of monochromatic fluorescence. By combining convolutional neural networks to learn image features and train the network, no manual image feature extraction is required, overcoming errors caused by objective factors in traditional machine learning methods. This method is convenient, low-cost, non-invasive, fast, and accurate.

[0005] The multi-organelle-targeted AIE fluorescence sensing array consists of at least three of the fluorescent probes TPE-X, TPE-G, TPE-Z, TPE-N, TPE-R, and TPE-M. The structural formulas of the six fluorescent probes are as follows:

[0006]

[0007] A kit comprising the aforementioned multi-organelle-targeting AIE fluorescence sensing array.

[0008] One method for detecting cancer cell metastasis is as follows:

[0009] (1) Cancer cells or cancer cell spheres from different locations were added to the solutions of various fluorescent probes of the multi-organ organelle-targeting AIE fluorescence sensing array and incubated. Then, fingerprint fluorescence images were acquired by confocal imaging using a laser confocal fluorescence microscope.

[0010] (2) The collected fingerprint fluorescence images are enhanced and then input into the ResNet convolutional neural network for network training. The trained neural network has the ability to identify the corresponding cancer cells or cancer cell spheres.

[0011] (3) Under the same conditions as in step (1), the cells to be tested are incubated with each fluorescent probe solution, and confocal imaging is performed by laser confocal fluorescence microscopy. The collected fingerprint fluorescence image is input into the neural network trained in step (2), and the network outputs the recognition result within one second.

[0012] The concentration of the fluorescent probe solution is 5-20 μM, and the incubation time is 5-20 minutes.

[0013] When acquiring fingerprint fluorescence images using confocal imaging with laser confocal fluorescence microscopy, the excitation band is 405-543nm, and the corresponding fluorescence receiving band covers the maximum emission wavelength of the fluorescent probe.

[0014] The beneficial effects of this invention are as follows:

[0015] (1) The multicolor fluorescent sensing array based on tetraphenylethylene provided by the present invention modifies the benzene ring of tetraphenylethylene with different functional substituents to obtain a series of organelle imaging probes with AIE characteristics. The fluorophore can be functionalized to target multiple organelles, thereby obtaining a series of organelle-targeting probes. Three or more probes can be combined to form the required sensing array for recognizing different types of cells (including normal cells and various cancer cells).

[0016] (2) This invention achieves multicolor fluorescence emission of the sensing array by introducing groups with different electron-donating and electron-withdrawing capabilities onto the AIE fluorophore tetraphenylethylene, thereby regulating the intramolecular electron transfer effect. This strategy allows the emission spectrum of the AIE probe to cover the entire spectrum from the ultraviolet region to the near-infrared II region. The maximum absorption wavelengths of TPE-X, TPE-G, TPE-Z, TPE-N, TPE-R, and TPE-M are 355, 377, 368, 425, 459, and 463 nm, respectively. The maximum emission wavelengths of TPE-X, TPE-G, TPE-Z, TPE-N, TPE-R, and TPE-M are 419, 495, 505, 578, 629, and 592 nm, respectively. The maximum emission wavelength of the sensing array is in the range of 400-600 nm, ensuring its use for multicolor imaging of cells. Multicolor imaging avoids the influence of fluorescence signals between sensing elements. The Stokes shifts are 64, 118, 137, 153, 170, and 66 nm. A large Stokes shift ensures the accuracy of cell imaging.

[0017] (3) The sensor array designed in this invention achieves multi-organ targeting by introducing different targeting groups, thereby achieving the purpose of exploring the structure-activity relationship between molecular structure and targeting function. TPE-X, TPE-G, TPE-Z, TPE-N, TPE-R and TPE-M are used for targeted imaging of mitochondria, Golgi apparatus, lipid droplets, endoplasmic reticulum, lysosomes and cell membranes, respectively.

[0018] (4) The sensor array designed in this study exhibits low cytotoxicity and high resistance to photobleaching. Within the concentration range used, the cell viability was consistently above 80%, indicating that the sensor array has good biocompatibility and is suitable for cell imaging. After 300 seconds of laser irradiation, the fluorescence intensity remained above 70%, indicating that the sensor array has good photostability. Good photostability ensures stable cell imaging analysis.

[0019] (5) The sensor array designed in this study can exhibit differential fluorescence responses to different types of cancer cells and cell spheres. After staining and confocal imaging of the cells by the sensor array, a series of fluorescence fingerprint images were obtained for each type of cell or cell sphere. The obtained images were used for training a ResNet convolutional neural network. After successful training, the ResNet convolutional neural network showed high recognition accuracy for blind-sample cancer cells and cell spheres. Attached Figure Description

[0020] Figure 1 Co-localization images of a fluorescence sensing array and a commercial organelle-targeting probe;

[0021] Figure 2 (a) The process of identifying blind cell spheres, (b) The result of identifying blind cell spheres. Detailed Implementation

[0022] Example 1:

[0023] 1. The synthesis route and characterization results of the six fluorescent probes for the multi-organelle-targeting AIE sensing array are as follows:

[0024] (1) Synthesis of TPE-X:

[0025] Synthesis of TPE-OH: Dry THF (100 mL) was added to a mixture of 4-hydroxybenzophenone (2.77 g, 14 mmol), benzophenone (2.55 g, 14 mol), and zinc powder (7.28 g, 112 mmol), and the resulting mixture was purged with N2 at 0 °C. Titanium tetrachloride (6.15 mL, 56 mmol) was added to the cold mixture, and the mixture was stirred at 0 °C for 0.5 h, then refluxed for 9 h. Saturated potassium carbonate solution was added to the mixture to quench the titanium tetrachloride. The zinc powder was filtered, and the extract was obtained by water and dichloromethane (DCM) (3 × 100 mL). The organic phase was collected, washed with water, and dried over anhydrous sodium sulfate. The crude product was purified by chromatography with petroleum ether / dichloromethane (4:1, v / v) eluent to give a white solid (1.64 g, yield: 31%). 1 H NMR (400MHz, CDCl3) δ7.16-7.09 (m, 9H), 7.06 (dd, J=8.8, 3.7Hz, 6H), 6.92 (d, J=8.7Hz, 2H), 6.59 (d, J=8.6Hz, 2H).

[0026] Synthesis of TPE-OBr: Dry acetone was added to a mixture of TPE-OH (0.88 g, 2.36 mmol), 1,3-dibromopropane (0.82 mL, 5.9 mmol), and K₂CO₃ (0.98 g, 7.07 mmol), and the resulting mixture was refluxed for 48 hours. The reaction mixture was cooled to room temperature and extracted with water and DCM (3 × 20 mL). The solvent was evaporated under reduced pressure. The crude product was used directly for the next reaction without further purification.

[0027] Synthesis of TPE-ON: TPE-OBr (0.27 g, dissolved in 5 mL CH2Cl2) was added dropwise to a mixture of glacial acetic acid (HAc, 5 mL) and concentrated nitric acid (HNO3, 8 mL) in a two-necked flask. The reaction mixture was then stirred at room temperature for 3 h. The reaction mixture was extracted with water and dichloromethane (3 × 30 mL). The combined organic phases were dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography using petroleum ether:ethyl acetate = 10:1 (v / v) as eluent to give a pale yellow solid (0.23 g, yield: 60%). 1 H NMR (400MHz, CDCl3) δ 8.16-8.04 (m, 6H), 7.50 (d, J=2.3Hz, 1H=5.8Hz, 2H).

[0028] Synthesis of TPE-X: THF (20 mL) was added to a mixture of TPE-ON (0.23 g, 0.35 mmol) and trimethylamine (0.06 g, 1.05 mmol) under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 24 hours. The solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography with DCM / methanol = 20:1 (v / v) as the eluent to give a pale yellow solid (0.15 g, yield: 62%). 1H NMR (400MHz, DMSO-d6) δ8.19-8.04 (m, 6H), 7.59 (d, J=2.3Hz, 1H), 7.44-7.31 (m, 7H), 7.27 (d, J=8.9Hz, 1H), 4.20 (t, J=5.9Hz, 2H), 3.47-3.41(m), 3.10(s, 9H), 2.25-2.12(m, 2H. 13C NMR (101MHz, DMSO-d6) δ151.08, 148.66, 148.34, 140.87, 140.41, 139.03, 13 7.33, 133.69, 132.54, 127.85, 124.03, 115.68, 67.11, 63.16, 52.78, 22.81.

[0029]

[0030] (2) Synthesis of TPE-R

[0031] Synthesis of Br-TPE-OH: Dry THF (100 mL) was added to a mixture of 4-hydroxybenzophenone (2.77 g, 14 mmol), 4-bromobenzophenone (3.66 g, 14 mmol), and zinc powder (7.28 g, 112 mmol). The reaction mixture was stored at 0 °C under a N2 atmosphere. Titanium tetrachloride (6.15 mL, 56 mmol) was added to the cold mixture, and the mixture was stirred at 0 °C for 0.5 h, then refluxed for 9 h. A saturated potassium carbonate solution was added to the mixture to quench the titanium tetrachloride. The zinc powder was filtered, and the extract was obtained with water and dichloromethane (DCM) (3 × 100 mL). The organic phase was collected, washed with water, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography using petroleum ether / DCM (1:3, v / v) eluent to give a white solid (2.52 g, yield: 29%). 1 H NMR (400MHz, CDCl3) δ7.24 (dd, J=13.9, 8.5Hz, 2H), 7.17-7.09 (m, 6H), 7.09-6.99 (m, 4H), 6.96-6.86 (m, 4H), 6.60 (dd, J=14.9, 8.6Hz, 2H).

[0032] Br-TPE-R: A mixture of Br-TPE-OH (0.21 g, 0.5 mmol), 2-(4-morpholinoethyl bromide) (0.29 g, 1.5 mmol), and K₂CO₃ (0.69 g, 5.0 mmol) was dissolved in acetone. The reaction mixture was heated to 80 °C and reacted under N₂ for 4 hours. The mixture was cooled to room temperature, and the solvent was removed by vacuum. The resulting solid mixture was dissolved in DCM (50 mL) and washed with water (3 × 25 mL). The combined organic phases were dried over anhydrous Na₂SO₄. The solvent was removed under vacuum to obtain a crude product, which was further purified by silica gel column chromatography with DCM / methanol eluent of 20:1 (v / v) to give a white oily liquid (0.24 g, yield: 90%). 1 H NMR (400MHz, CDCl3) δ7.24 (dd, J=13.7, 8.5Hz, 2H), 7.18-7.09 (m, 7H), 7.03 (ddd, J=9.0, 6.0, 2.9Hz, 4H), 6.95-6.87 (m,3H),6.67(dd,J=14.7,8.7Hz,2H),4.11(t,J=5.9Hz,2H),2.99-2.71(m,4H),2.63(s,4H),2.54(d,J=4.8Hz,2H).

[0033] Synthesis of O-TPE-R: A mixture of Br-TPE-R (0.27 g, 0.5 mmol), 5-aldehyde-2-thiopheneboronic acid (0.20 g, 1.25 mmol), K₂CO₃ (0.69 g, 5 mmol), and Pd(PPh₃)₄ (0.03 g, 0.03 mmol) was dissolved in a mixed solvent (THF:H₂O = 10:1 mL). The reaction mixture was heated to 80°C and reacted for 16 hours. The mixture was cooled to room temperature and filtered upon completion of the reaction. The solvent was removed under reduced pressure, and the residue was dissolved in DCM (50 mL) and washed with water (3 × 25 mL). The combined organic phases were dried over anhydrous Na₂SO₄. The solvent was removed under reduced pressure to obtain a crude product, which was further purified by silica gel column chromatography with eluent DCM / methanol = 15:1 (v / v) to obtain a yellow oily liquid (0.20 g, yield: 69%). 1 HNMR (400MHz, CDCl3) δ9.88(d,J=4.5Hz,1H),7.44(dd,J=14.5,8.3Hz,1H),7.36(dd,J=10.3,4.0Hz,1H),7.27-7.1 6(m,2H),7.15-7.09(m,7H),7.07-7.01(m,4H),7.00-6.87(m,3H),6.72-6.62(m,2H),3.80-3.67(m,2H),2.84(d,J=

[0034] 16.6Hz, 4H), 2.65-2.60 (m, 4H), 2.54 (t, J = 4.7Hz, 2H).

[0035] Synthesis of TPE-R: DCM (10 mL) was added to a mixture of O-TPE-R (0.20 g, 0.35 mmol) and malononitrile (0.07 g, 1.04 mmol). The reaction mixture was stirred at room temperature for 2 hours. When the reaction was complete, the reactants were extracted with water and DCM (3 × 50 mL). The combined organic phases were dried over anhydrous Na₂SO₄. The solvent was removed under reduced pressure to obtain the crude product, which was further purified by silica gel column chromatography with DCM / methanol eluent = 15:1 (v / v) to obtain a red solid (0.18 g, yield: 86%). 1H NMR (400MHz, DMSO-d6) δ7.93 (t, J=4.2Hz, 1H), 7.76 (dd, J=8.0, 4.1Hz, 1H), 7.63-7.60 (m, 4H), 7.56 (ddd, J=7.1, 3.2, 1.1Hz, 4H 2H), 6.79-6.63 (m, 2H), 4.03-3.86 (m, 2H), 3.55 (dd, J=9.4, 6.4Hz, 4H), 2.63 (d, J=4.8Hz, 2H), 2.48-2.36 (m, 4H). 13 C NMR (101MHz, DMSO-d6) δ157.76, 155.01, 153.12, 146.17, 143.67, 143.52 ,142.77, 141.83, 139.18, 135.55, 134.48, 132.44, 132.32, 131.25, 130. 18, 128.52, 128.41, 128.24, 127.37, 127.16, 126.43, 126.31, 126.03, 11 5.07, 114.45, 114.32, 114.24, 75.18, 66.58, 59.99, 57.47, 54.06, 53.49.

[0036]

[0037] (3) Synthesis of TPE-M

[0038] Synthesis of TPE-Br: Dry THF (100 mL) was added to a mixture of 4,4'-dimethoxybenzophenone (3.40 g, 14 mmol), 4-bromobenzophenone (3.66 g, 14 mol), and zinc powder (7.28 g, 112 mmol). The reaction mixture was stirred at 0 °C under N2. Titanium tetrachloride (6.15 mL, 56 mmol) was added to the cold mixture, and the mixture was stirred at 0 °C for 0.5 h, then refluxed for 9 h. Saturated potassium carbonate solution was added to the mixture to quench the titanium tetrachloride. The zinc powder was filtered off, and the solution was extracted with water and DCM (3 × 100 mL). The organic phase was collected, washed with water, and dried over anhydrous sodium sulfate. The product was purified by silica gel column chromatography with petroleum ether / DCM (3:1, v / v) eluent to give a white solid (1.98 g, yield: 29%). 1 H NMR (400MHz, CDCl3) δ7.24(d,J=8.5Hz,2H),7.13(d,J=7.0Hz,3H),7.03(d,J=7.6 Hz,2H),7.00-6.88(m,6H),6.67(dd,J=14.4,8.8Hz,4H),3.78(d,J=14.0Hz,6H).

[0039] Synthesis of TPE-SO: A mixture of TPE-Br (0.47 g, 1 mmol), 5-aldehyde-2-thiopheneboronic acid (0.39 g, 2.5 mmol), K₂CO₃ (1.38 g, 10.0 mmol), and Pd(PPh₃)₄ (0.12 g, 0.1 mmol) was dissolved in a mixed solvent (THF:H₂O = 10:1 mL). The reaction was heated to 80 °C and reacted under N₂ for 16 h. The mixture was cooled to room temperature and filtered when the reaction was complete. The solvent was removed by vacuum. The residue was dissolved in DCM (50 mL) and washed with water (3 × 25 mL). The combined organic phases were dried over anhydrous Na₂SO₄. The filtrate was removed under vacuum to obtain the crude product, which was further purified by silica gel column chromatography with petroleum ether / ethyl acetate = 5:1 (v / v) to give a yellow solid (0.14 g, yield: 27%). 1 H NMR (400MHz, CDCl3) δ9.89 (s, 1H), 7.73 (d, J = 4.0Hz, 1H), 7.45 (d, J = 8.4Hz, 2H), 7.36 (d, J = 4.0Hz, 1H), 7.1 8-7.12(m,3H),7.11-7.04(m,4H),6.98(dd,J=15.5,8.8Hz,4H),6.68(dd,J=12.5,8.8Hz,4H),3.78(s,6H).

[0040] Synthesis of TPE-M: Piperidine (2 drops) was added to a mixture of TPE-SO (0.07 g, 0.13 mmol), pyridine (10 μL), 4-methyl-1-[3-(trimethylamino)propyl]-dibromide (0.05 g, 0.2 mmol) in ethanol (5 mL). The reaction mixture was refluxed for 24 hours and cooled to room temperature. The solvent was removed under reduced pressure to obtain a solid. The solid was further recrystallized from methanol and hexane to give a dark red solid (0.04 g, yield: 39%). 1 H NMR (400MHz, DMSO-d6) δ9.05 (d, J=6.5Hz, 1H), 8.39-8.20 (m, 2H), 7.61 (d, J= 3.9Hz, 1H), 7.57-7.45 (m, 3H), 7.25-7.08 (m, 4H), 7.01 (dd, J=11.7, 9.0Hz, 4H Hz, 6H), 3.11 (s, 9H), 2.47 (d, J=8.1Hz, 2H), 1.10 (d, J=7.0Hz, 2H). 13C NMR (101MHz, DMSO-d6) δ158.28, 153.36, 144.75, 139.96, 138.43, 135.96, 134.66, 132.57 , 132.51, 132.15, 131.29, 128.45, 126.90, 123.88, 113.85, 113.65, 55.41, 52.92, 24.63.

[0041]

[0042] (4) Synthesis of TPE-Z

[0043] Synthesis of NN-TPE-Br: Dry THF (100 mL) was added to a mixture of 4,4'-dimethylaminobenzophenone (5.34 g, 20 mmol), 4,4'-dibromobenzophenone (6.80 g, 20 mmol), and zinc powder (8.46 g, 160 mmol), and the reaction mixture was stirred in N2 at 0 °C. Titanium tetrachloride (8.78 mL, 80 mmol) was added to the cold mixture, and the mixture was stirred at 0 °C for 0.5 h, then refluxed for 9 h. A saturated potassium carbonate solution was added to the mixture to quench the titanium tetrachloride. The zinc powder was filtered off, and the extract was obtained with water and DCM (3 × 100 mL). The organic phase was collected, washed with water, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography with petroleum ether / ethyl acetate eluent of 10:1 (v / v) to give a yellow solid (3.57 g, yield: 31%). 1 H NMR (400MHz, CDCl3) δ7.24 (d, J=8.5Hz, 4H), 6.90 (t, J=8.7Hz, 8H), 6.50 (s, 4H), 2.94 (s, 12H).

[0044] Synthesis of TPE-Z: A mixture of NN-TPE-Br (0.57 g, 1 mmol), 4-methoxycarbonylphenylboronic acid (0.90 g, 5 mmol), K₂CO₃ (1.38 g, 10.0 mmol), and Pd(PPh₃)₄ (0.12 g, 0.1 mmol) was dissolved in a mixed solvent (THF:H₂O = 10:1 mL). The reaction mixture was heated to 80°C and reacted under N₂ for 16 hours. The mixture was cooled to room temperature and filtered upon completion of the reaction. The solvent was removed under reduced pressure. The residue was dissolved in DCM (50 mL) and washed with water (3 × 25 mL). The combined organic phases were dried over anhydrous Na₂SO₄ and filtered. The filtrate was removed under reduced pressure to give the crude product, which was further purified by silica gel column chromatography with petroleum ether / ethyl acetate eluent of 5:1 (v / v) to give a yellow solid (0.29 g, yield: 42%). 1HNMR (400MHz, DMSO-d6) δ 8.00 (d, J=6.7Hz, 4H), 7.82 (d, J=7.0Hz, 4H), 7.60 (s, 4H, 6.49 (d, J=6.9Hz, 4H), 3.87 (s, 6H), 2.84 (s, 12H). 13 C NMR (101MHz, CDCl3) δ167.56, 160.56, 131.96, 131.13, 128.90, 122.08, 115.33, 52.09, 29.71.

[0045]

[0046] (5) Synthesis of TPE-G

[0047] Synthesis of N-TPE-Br: Dry THF (100 mL) was added to a mixture of 4,4'-bis(dimethylamino)benzophenone (5.37 g, 20 mmol), 4-bromobenzophenone (5.22 g, 20 mol), and zinc powder (8.46 g, 160 mmol), and the reaction mixture was stirred at 0 °C under a N2 atmosphere. Titanium tetrachloride (8.78 mL, 80 mmol) was added to the cold mixture, and the mixture was stirred at 0 °C for 0.5 h, then refluxed for 9 h. A saturated potassium carbonate solution was added to the mixture to quench the titanium tetrachloride. The zinc powder was filtered off, and the solution was extracted with water and DCM (3 × 100 mL). The organic phase was collected, washed with water, and dried over anhydrous sodium sulfate. The crude product was purified by silica gel column chromatography with petroleum ether / ethyl acetate = 10:1 (v / v) to give a solid (3.28 g, yield: 33%). 1HNMR (400MHz, CDCl3) δ7.23 (d, J=8.5Hz, 2H), 7.14-7.04 (m, 5H), 6.97-6.86 (m, 6H), 6.50 (d, J=11.1Hz, 4H), 2.93 (d, J=10.6Hz, 12H).

[0048] Synthesis of TPE-G: A mixture of N-TPE-Br (0.16 g, 0.32 mmol), 4-aminosulfonylphenylboronic acid (0.16 g, 0.81 mmol), K₂CO₃ (0.45 g, 3.2 mmol), and Pd(PPh₃)₄ (0.04 g, 0.03 mmol) was dissolved in a mixed solvent (THF:H₂O = 10:1 mL). The reaction mixture was heated to 80°C and reacted under N₂ for 16 h. The mixture was cooled to room temperature and filtered when the reaction was complete. The solvent was removed under reduced pressure. The residue was dissolved in DCM (50 mL) and washed with water (3 × 25 mL). The combined organic phases were dried over anhydrous Na₂SO₄ and filtered. The filtrate was removed under reduced pressure to give the crude product, which was further purified by silica gel column chromatography with petroleum ether / ethyl acetate eluent of 4:1 (v / v) to give a yellow solid (0.06 g, yield: 31%). 1 HNMR (400MHz, CDCl3) δ7.96 (d, J=8.4Hz, 2H), 7.71 (d, J=8.4Hz, 2H), 6.95 (dd, J=12.1, 8.8Hz, 4H), 6.49 (t, J=7.0Hz, 4H), 4.94 (s, 2H), 2.92 (s, 12H). 13 C NMR (101MHz, DMSO-d6) δ158.35, 158.23, 144.48, 144.18, 143.24, 143.03, 140.85, 138.54, 136.46, 131.97, 131.29, 128.41, 127.23, 126.82, 126.76, 126.70, 113.81, 113.64, 55.39.

[0049]

[0050] (6) Synthesis of TPE-N

[0051] Synthesis of TPE-B: 1,4-dioxane (10 mL) was added to a mixture of TPE-Br (0.16 g, 0.32 mmol), pinacol bisphenylboronic acid (0.81 g, 3.2 mmol), CH3COOK (0.63 g, 6.4 mmol), and [1,1'-bis(diphenylphosphine)ferrocene]palladium(II) dichloride (0.08 g, 0.11 mmol). The reaction was heated to 110°C and reacted for 12 hours. The mixture was cooled to room temperature and filtered upon completion of the reaction. The solvent was removed under reduced pressure. The residue was dissolved in DCM (50 mL) and washed with water (3 × 25 mL). The combined organic phases were dried over anhydrous Na2SO4 and filtered. The filtrate was removed under reduced pressure to give the crude product, which was further purified by silica gel column chromatography with eluent DCM / petroleum ether = 3:1 (v / v) to give a white solid (0.38 g, yield: 71%). 1 H NMR (400MHz, CDCl3) δ7.56 (d, J=8.0Hz, 2H), 7.16-6.92 (m, 11H), 6.65 (d, J=7.3Hz, 4H), 3.77 (s, 6H), 1.34 (s, 12H).

[0052] Synthesis of TPE-SZ: A mixture of TPE-B (0.10 g, 0.20 mmol), 4,7-dibromo-2,1,3-benzothiadiazole (0.14 g, 0.50 mmol), K₂CO₃ (0.20 g, 2.0 mmol), and Pd(PPh₃)₄ (0.02 g, 0.01 mmol) was dissolved in a mixed solvent (THF:H₂O = 10:1 mL). The reaction mixture was heated to 80°C and reacted for 16 hours. The mixture was cooled to room temperature and filtered upon completion of the reaction. The solvent was removed under reduced pressure. The residue was dissolved in DCM (50 mL) and washed with water (3 × 25 mL). The combined organic phases were dried over anhydrous Na₂SO₄ and filtered. The filtrate was removed under reduced pressure to obtain the crude product, which was further purified by silica gel column chromatography with petroleum ether / ethyl acetate as the eluent at a ratio of 10:1 (v / v) to give a yellow solid (0.09 g, yield: 73%). 1 H NMR (400MHz, CDCl3) δ7.98-7.88(m, 1H), 7.76-7.68(m, 2H), 7.58(d, J=7.7Hz, 1H), 7.04(d , J=8.8Hz, 2H), 6.98 (d, J=8.7Hz, 2H), 6.68 (dd, J=10.5, 8.7Hz, 4H), 3.77 (d, J / 2.2Hz, 6H).

[0053] Synthesis of TPE-SZZ: A mixture of TPE-SZ (0.13 g, 0.21 mmol), 4-aminosulfonylphenylboronic acid (0.11 g, 0.63 mmol), K₂CO₃ (0.27 g, 2.1 mmol), and Pd(PPh₃)₄ (0.02 g, 0.02 mmol) was dissolved in a mixed solvent (THF:H₂O = 10:1 mL). The reaction was heated to 80°C and reacted for 16 hours. The mixture was cooled to room temperature and filtered upon completion of the reaction. The solvent was removed under reduced pressure. The residue was dissolved in DCM (50 mL) and washed with water (3 × 25 mL). The combined organic phases were dried over anhydrous Na₂SO₄ and filtered. The filtrate was removed under reduced pressure to obtain the crude product, which was further purified by silica gel column chromatography with petroleum ether / ethyl acetate = 5:1 (v / v) to obtain an orange solid (0.07 g, yield: 52%). 1H NMR (400MHz, CDCl3) 1 H NMR (400MHz, CDCl3) δ8.23(d,J=8.4Hz,2H),8.08(d,J=8.5Hz,2H),7.85-7.80(m,4H),7.23(s,2H),7.19-7.11(m,5 H), 7.06 (d, J = 8.7Hz, 2H), 6.99 (d, J = 8.7Hz, 2H), 6.69 (dd, J = 12.2, 8.8Hz, 4H), 3.99 (s, 3H), 3.77 (d, J = 2.2Hz, 6H).

[0054] Synthesis of TPE-SZS: A mixture of TPE-SZZ (0.18 g, 0.27 mmol) and NaOH (0.44 g, 11 mmol) was dissolved in a mixed solvent (methanol:THF = 10:10 mL). The reaction mixture was refluxed for 5 hours. The reaction mixture was cooled to room temperature, and HCl was slowly added to neutralize the NaOH. A yellow solid (0.15 g, yield: 85%) was obtained by centrifugation. 1 H NMR (400MHz, DMSO-d6) δ8.13 (q, J=8.5Hz, 4H), 8.02 (d, J=7.4Hz, 1H), 7.96 (d, J=7.4Hz, 1H), 7.88 (d, J=8.1Hz, 2H), 7.16 (dq, J=16.4 , 7.9, 7.5Hz, 5H), 7.04 (d, J=6.9Hz, 2H), 6.98 (d, J=8.7Hz, 2Hh), 6.90 (d, J / 8.7Hz, 2Hs), 6.74 (dd, J=20.9, 8.7Hz, 4H), 3.69 (s, 6H).

[0055] Synthesis of TPE-N: TPE-SZS (0.11 g, 0.17 mmol) was dissolved in 1,2-dichloroethane (20 mL) under a N2 atmosphere. Phosphorus oxychloride (0.16 g, 0.51 mmol) was slowly added to the mixture at 0 °C. The mixture was refluxed for 5 hours. The mixture was cooled to room temperature and filtered when the reaction was complete. The solvent was removed under reduced pressure to obtain TPE-XL. N-(2-aminoethyl)-4-methylbenzenesulfonamide (0.86 g, 0.85 mmol) was dissolved in acetonitrile (20 mL), followed by the addition of triethylamine (10 drops) under a N2 atmosphere. TPE-XL (dissolved in 5 mL of acetonitrile) was added dropwise to the above reaction mixture, and the reaction mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure. The residue was dissolved in DCM (50 mL) and washed with water (3 × 25 mL). The combined organic phases were dried over anhydrous Na2SO4 and filtered. The filtrate was removed under reduced pressure to obtain the crude product, which was further purified by silica gel column chromatography with petroleum ether / ethyl acetate = 5:1 (v / v) as the eluent to obtain an orange solid (0.07 g, yield: 49%). 1 H NMR (400MHz, CDCl3) δ8.04(d,J=8.3Hz,2H),7.96(d,J=8.3Hz,2H),7.78(q,J=10.7,9.6Hz,7H),7.31(s,1H),7.22(d,J=8.4Hz,2H),7.15(q,J=7.0,6 .1Hz,5H),7.06(d,J=8.8Hz,2H),7.00(d,J=6.7Hz,2H),6.69(dd,J=11.4, 8.8Hz,4H),3.77(d,J=4.9Hz,6H),3.65(s,2H),3.26(s,2H),2.41(s,3H). 13 C NMR (101MHz, CDCl3) δ167.90,158.25,158.14,153.86,144.70,144.17,1 43.61,140.79,140.56,138.71,136.64,136.29,134.61,133.60,133.26 ,132.65,131.70,131.54,129.85,129.30,128.50,128.46,127.81,127. 68,127.39,127.04,126.23,113.20,113.02,55.12,43.06,40.10,21.54.

[0056]

[0057] 2. Localization effect detection of six fluorescent probes: HeLa cells were co-incubated with TPE-N and the commercial probe ER-Trackerred for 10 min, and confocal imaging was performed using laser confocal fluorescence microscopy, as shown in the attached figure. Figure 1 As shown, the Pearson correlation coefficient is 0.88 (TPE-N). Similarly, testing revealed that the commercial colocalization probes for TPE-R are Lysogreen, for TPE-M they are Dio, for TPE-Z they are BDP493 / 503, for TPE-G they are Golgi-Trackerred, and for TPE-X they are Rhod 123, as shown in the attached figure. Figure 1 As shown, the Pearson correlation coefficients were 0.89 (TPE-R), 0.91 (TPE-M), 0.92 (TPE-Z), 0.94 (TPE-G), and 0.98 (TPE-X). The results indicate that the probe designed in this invention has good co-localization effects with commercial organelle-targeting probes.

[0058] 3. Synthesized fluorescence sensing arrays were used for the detection of different types of pancreatic cancer cells:

[0059] (1) The concentration of the TPE-M probe solution was 5 μM, and the concentration of the other five probe solutions was 20 μM. Pancreatic ductal carcinoma cells PANC-1 and four types of metastatic pancreatic cancer cells were selected: ASPC-1 cells from ascites of cancer patients, SW1990 cells from spleen metastases, CFPAC-1 cells from liver metastases, and HS766T cells from lymph node metastases. Each of the five cell types was incubated with one of six probe solutions for 10 minutes. Confocal imaging was then performed using laser confocal fluorescence microscopy. During confocal imaging, the excitation wavelengths used for TPE-X, TPE-G, TPE-Z, TPE-N, TPE-R, and TPE-M were 405, 488, 458, 543, 458, and 488 nm, respectively, corresponding to fluorescence receiving bands of 425-500 nm, 508-528 nm, 478-500 nm, 563-583 nm, 478-580 nm, and 508-600 nm. Because different types of pancreatic cancer cells differ in the morphology, number, polarity, and viscosity of their organelles, the degree of aggregation of the probe after entering the cell will vary, ultimately resulting in differential fluorescence.

[0060] (2) The number of images in the fingerprint fluorescence images obtained through confocal imaging was increased by image cropping. A 1024-pixel image was cropped into 16 256×256-pixel images. Image cropping increased the number of images to 16 times that of the original data, which is beneficial for the learning and training of the convolutional neural network. All the obtained images were input into the ResNet convolutional neural network for network training. The convolutional neural network trained on the fingerprint fluorescence images of pancreatic cancer cells showed good recognition ability for pancreatic cancer cells.

[0061] (3) The cells to be tested, PANC-1, ASPC-1, SW1990, HS766T, and CFPAC-1, were incubated with the six probe solutions according to the conditions in step (1). The fingerprint fluorescence images obtained by confocal imaging were input into the trained network, and the network would output the recognition result within one second. The recognition accuracy was 100%.

[0062] 4. The synthesized fluorescence sensing array was used for the detection of different types of pancreatic cancer cell spheroids: PANC-1, ASPC-1, SW1990, and HS766T cells were cultured into cell spheroids and incubated with six probe solutions under the same conditions. The fingerprint fluorescence images obtained from confocal imaging were enhanced and then input into a ResNet convolutional neural network for training. The convolutional neural network trained on the pancreatic cancer cell spheroid fingerprint fluorescence images showed good recognition ability for pancreatic cancer cell spheroids. The test cell spheroids (PANC-1, ASPC-1, SW1990, and HS766T cells) were incubated with the six probe solutions under the same conditions. The fingerprint fluorescence images obtained from confocal imaging were input into the trained network, which output the recognition result within one second. The recognition accuracy was 100%. The recognition process and results are attached. Figure 2 As shown.

Claims

1. A multi-organelle-targeted AIE fluorescence sensing array, characterized in that, The array consists of fluorescent probes TPE-X, TPE-G, TPE-Z, TPE-N, TPE-R, and TPE-M, and the structural formulas of the six fluorescent probes are as follows: 。 2. A reagent kit, characterized in that, The kit comprises the multi-organelle-targeted AIE fluorescence sensing array as described in claim 1.

Images