A single-component organic afterglow compound and its use for biological detection

Abstract

This invention discloses a single-component organic afterglow compound and its application in biodetection. It is a wavelength-tunable biomimetic organic afterglow compound and its application in aqueous biodetection, belonging to the field of luminescence imaging and diagnostic detection. The single-component organic afterglow compound provided by this invention has the advantage of high luminescence efficiency in physiological environments, with an emission peak between 580-720 nm and strong tissue penetration. This afterglow compound has the general molecular structure shown in Formula I, and the presence of electron-rich substituted double bonds is an essential molecular structural feature. This single-component afterglow compound can be observed to emit afterglow at different wavelengths in aqueous phase and can be used for luminescence imaging or luminescence diagnostic markers.

Description

Technical Field

[0001] This invention belongs to the field of luminescence imaging and diagnostic detection, specifically relating to a small-molecule organic afterglow compound with different emission wavelengths and its application in afterglow imaging. Background Technology

[0002] Afterglow luminescence refers to the phenomenon where luminescent materials continue to emit light after the excitation light is removed. Because it avoids the interference of light scattering and background fluorescence caused by real-time light excitation, it has gained popularity among researchers. Currently, inorganic afterglow luminescent quantum dots are widely used in anti-counterfeiting, inks, security indicators, and decoration. However, due to the limitations of inorganic quantum dots, especially the potential biosafety risks posed by heavy metal ions, highly biocompatible organic afterglow materials are preferred for bioimaging and luminescence detection.

[0003] Current reports on organic afterglow materials mainly fall into two categories: The first is oxygen-quenched afterglow luminescent materials, primarily formed by physical encapsulation of phosphorescent or thermally induced delayed fluorescence materials, such as host-guest coordination or microcrystalline encapsulation, resulting in afterglow luminescent nanoparticles. These materials lose their afterglow luminescence properties upon contact with dissolved oxygen in a fully dissolved state (Chem. Eur. J., 2022, 28, e2022008). The second is oxygen-consuming afterglow luminescent materials, mainly formed by multi-component doping of photosensitizers, energy storage units, and luminescent units into afterglow luminescent nanoparticles or microspheres. The generation of afterglow luminescence depends on the singlet oxygen produced by the photosensitizer under illumination, which is a characteristic of these materials (J. Am. Chem. Soc., 2022, 144, 3429). However, both of these traditional afterglow luminescent materials require the preparation of nanoparticles and do not exhibit homogeneous luminescence in aqueous solutions, making it difficult to achieve consistent luminescence effects between batches.

[0004] To address the aforementioned problems of traditional organic afterglow luminescent materials, a single small-molecule afterglow luminescent compound in aqueous phase was reported in 2020 (Angew. Chem. Int. Ed. 2020, 59, 9059), achieving afterglow luminescence of a single-component small-molecule compound in a dispersed state independent of singlet oxygen, as per Chinese invention patent (CN 110804009 A). This invention proposes a new generation of small-molecule organic afterglow compounds, employing light irradiation to simulate luciferase and Mg... 2+ ATP catalyzes the efficient reaction of small molecule compounds with oxygen to generate a 1,2-dioxane structure, which then rapidly decomposes, releasing a high-intensity afterglow luminescence in aqueous solution.

[0005] Compared to multi-component organic afterglow nanoparticles, the afterglow luminescent compounds provided by this invention can avoid the nanoparticle preparation process and be used directly in solution. Furthermore, the afterglow luminescence process of the compounds provided by this invention does not rely on singlet oxygen oxidation to generate afterglow luminescence; they can react with oxygen under light in an environment with a pH greater than 5.0, thus eliminating the need for photosensitizers and achieving single-component dispersed afterglow luminescence. In addition, the afterglow luminescent compounds provided by this invention have excellent scalability; by replacing the electron-withdrawing group A, various small-molecule organic afterglow luminescent compounds with different emission wavelengths and lifetimes can be constructed, and they have been successfully used in aqueous solutions, cells, and in vivo afterglow luminescence imaging. Summary of the Invention

[0006] The purpose of this invention is to provide a small-molecule organic afterglow compound whose molecular structure includes an electron-pull system and a double bond group substituted with an electron-rich group. In a microenvironment with a pH above 5.0, the afterglow compound rapidly undergoes electron transfer under light irradiation, forming a diradical, which further adds oxygen to form a high-energy unstable 1,2-dioxane structure. This structure then rapidly decomposes to produce afterglow luminescence. By controlling the electron-withdrawing group A, organic afterglow compounds with different emission wavelengths can be obtained.

[0007] The present invention designs an organic afterglow compound whose molecular structure features include a push-pull electron system and a double bond group substituted with an electron-rich group. A single-component organic afterglow compound has the following general structural formula:

[0008]

[0009] In Formula I, electron-withdrawing group A is a chromophore containing at least one cyano group, and X is a halogen atom; wherein, methoxy (electron-rich group) substitution of double bond group is a necessary structure, and this structure is located in the ortho or para position of electron-withdrawing group A, together forming a push-pull electron system.

[0010] Furthermore, the electron-withdrawing group A is any one of the following structures containing a cyano group:

[0011]

[0012] R1 is any one of cyano, acetyl, carboxymethyl ester, carboxyethyl ester, and carboxyl; R2 is any one of methyl, isopropyl, tert-butyl, and cyclopropane.

[0013] X is one of the halogen atoms F, Cl, or Br.

[0014] In the optimized selection, the electron-withdrawing group A is selected from any of the following structures:

[0015]

[0016] R1 can be any one of cyano, acetyl, carboxymethyl ester, carboxyethyl ester, or carboxyl.

[0017] This invention also proposes the biodetection applications of the above-mentioned single-component organic afterglow compounds, including their application in combination with various commercial luminescence enhancers, surfactants, cyclodextrins, skim milk powder, serum proteins, etc., which can significantly improve the luminescence intensity of the compounds.

[0018] The afterglow luminescent compounds do not require the addition of photosensitizers, nor do they need to be mixed with photosensitizers or block polymers to form nanoparticles; the compounds can be used alone or in combination with the aforementioned reinforcing agents.

[0019] These afterglow compounds can be used in aqueous solutions, living cells, tissues, and living animals, and all exhibit significant afterglow luminescence characteristics.

[0020] This invention also proposes a method for using single-component organic afterglow compounds, the method of use of which is as follows:

[0021]

[0022] Operating conditions: (1) Place the afterglow substrate in a solution or gel with a pH greater than 5.0; (2) Illumination time of the light source is 1-10 seconds, and the light source is a commercially available LED light source (100mW·cm). -2 In use, afterglow luminescence is only effectively generated when the light exposure is performed in an environment where the compound is at a pH greater than 5.0. The specific luminescence process is described as follows: in a microenvironment with a pH above 5.0, the afterglow compound generates a high-energy unstable 1,2-dioxane structure upon light exposure, which then rapidly decomposes to produce afterglow luminescence. Attached Figure Description

[0023] Figure 1 UV absorption of Rubine-1 (see Example 2) in Tris-HCl solution (containing 10% HSA) (1×10⁻⁶) -5 mol·L -1 ), fluorescence (1×10 -5 mol·L -1 ) and afterglow luminescence spectrum (1×10 -4 mol·L -1 );

[0024] Figure 2 UV absorption of Rubine-2 (see Example 2) in Tris-HCl solution (containing 10% HSA) (1×10⁻⁶) -5 mol·L -1 ), fluorescence (1×10 -5 mol·L -1) and afterglow luminescence spectrum (1×10 -4 mol·L -1 );

[0025] Figure 3 UV absorption of Rubine-3 (see Example 2) in Tris-HCl solution (containing 10% HSA) (1×10⁻⁶) -5 mol·L -1 ), fluorescence (1×10 -5 mol·L -1 ) and afterglow luminescence spectrum (1×10 -4 mol·L -1 );

[0026] Figure 4 UV absorption of Rubine-4 (see Example 2) in Tris-HCl solution (containing 10% HSA) (1×10⁻⁶) -5 mol·L -1 ), fluorescence (1×10 -5 mol·L -1 ) and afterglow luminescence spectrum (1×10 -4 mol·L -1 );

[0027] Figure 5 UV absorption of Rubine-5 (see Example 2) in Tris-HCl solution (containing 10% HSA) (1×10⁻⁶) -5 mol·L -1 ) and fluorescence spectrum (1×10 -5 mol·L -1 );

[0028] Figure 6 Summary of photophysical parameters for Rubine-1, Rubine-2, Rubine-3, Rubine-4, and Rubine-5; among which, λ abs The maximum absorption peak of the compound, ε abs The molar extinction coefficient at the maximum absorption peak of the compound, λ em The maximum emission peak of the compound, Φ FL The fluorescence quantum efficiency of the compound, λ CL The wavelength of the afterglow emission of the compound was determined by the test system, which consisted of Tris-HCl solution (containing 10% HSA) at a temperature of 25°C.

[0029] Figure 7 Photoactivated afterglow luminescence imaging of Rubine-1, Rubine-2, Rubine-3, and Rubine-4 in Tris-HCl solution (containing 10% HSA), with an afterglow compound concentration of 1 × 10⁻⁶.-4 mol·L -1 Shot with Honor 30 Pro;

[0030] Figure 8 Afterglow luminescence kinetics of Rubine-2 were measured using a Tris-HCl solution (containing 10% HSA) at a concentration of 1×10⁻⁶. -5 mol·L -1 Excitation light intensity 100mW·cm -2 (5s); where the upper part is the image acquired by the Imaging Quant4000 system, and the lower part is the quantitative representation of the light intensity in the image;

[0031] Figure 9 Confocal fluorescence imaging of Rubine-2 in A549 cells;

[0032] Figure 10 Application of Rubine-2 in in vivo afterglow imaging in A549 subcutaneous tumor model mice, with a tested concentration of 1×10⁻⁶. - 5 mol·L -1 Excitation light intensity 400mW·cm -2 (10s) Detailed Implementation

[0033] Definition of relevant descriptive phrases:

[0034] Small molecules: In this invention patent, small molecules refer to a class of organic compounds with a molecular weight of less than 700.

[0035] Dispersed state: Under the action of additives such as surfactants, cyclodextrins or proteins, the afterglow compound is uniformly dispersed in the aqueous solution in the form of single molecules, without the obvious phenomenon of fluorescence redshift or quenching caused by the π-π stacking of two or more afterglow luminescent molecules.

[0036] Diradical: In this invention patent, it refers to a luminescent molecule in an excited state under light illumination, where one electron from its electron-donating portion transfers to its electron-withdrawing portion, forming a charge-separated state within a single molecule. Electron-withdrawing group: In this invention patent, it refers to a group with significant electron-withdrawing ability that is linked to a phenolic hydroxyl group via a vinyl group. The presence of a cyano group is a common characteristic of this type of group.

[0037] Enhancer: In this invention patent, it refers to the addition of surfactants, cyclodextrins, proteins, and other substances to the afterglow luminescence system, which bind with the afterglow luminescence substrate through hydrophobic interactions to enhance the luminescence intensity.

[0038] This invention aims to provide a small-molecule organic afterglow compound and its method of use. It can be used for antigen or antibody labeling in immunoassays, and also as an afterglow contrast agent for in vivo luminescent imaging in mice and other organisms. In potential applications, the hydroxyl groups of the compound can be modified to prepare various activated afterglow luminescent probes, such as those containing metal ions, thiols, reactive oxygen species, and enzymes. Furthermore, the generation of a charge-separated state under illumination is crucial for initiating the reaction; therefore, such compounds need to possess a push-pull electron structure.

[0039] It is important to emphasize in the usage instructions that the phenolic hydroxyl groups of the compound need to be deprotonated to maintain its photoactivation performance. Ensuring that the pH of the operating environment allows for partial or complete deprotonation of the phenolic hydroxyl groups is crucial. Furthermore, these compounds are highly hydrophobic, and the π-π stacking between molecules is detrimental to subsequent photochemical reactions. Using them in conjunction with additives can effectively resolve molecular aggregation and enhance luminescence intensity.

[0040] As an example of compounds that emit afterglow light at various emission wavelengths, the compound structures are as follows:

[0041]

[0042] Taking Rubine-1 as an example, with human serum albumin as an additive, the activation process of afterglow luminescence is as follows: Take 10 μL of 1×10 -3 mol·L -1 Rubine-1 dimethyl sulfoxide solution was mixed with 890 μL Tris buffer and 100 μL human serum albumin (40 mg / mL), and the solution was thoroughly mixed. Then, 200 μL of the mixture (1 × 10⁻⁶) was taken. -5 mol·L -1 Placed in a black 96-well plate, using 100mW·cm -2 Irradiate the solution with white light for 5 seconds, and immediately after illumination, record the luminescence intensity or images at different times using an ImageQuant LAS 4000 system. It is important to note that to avoid decomposition of the afterglow-emitting substrate and to reduce background noise in the luminescent solution, the solution should be strictly protected from light before photoactivation. Alternatively, a final concentration of 1×10⁻⁶ can be used. -5 mol·L - 1 Rubine-1 solution is added to the surface of hydrogels or coated onto agarose gels, and then used at 100 mW·cm⁻¹. -2 A clear luminescence signal can also be observed when exposed to white light for 5 seconds.

[0043] The following are specific embodiments, which are intended to illustrate rather than limit the invention.

[0044] Example 1:

[0045] Synthesis of norborneol aldehyde intermediate

[0046] 1) Synthesis of norborneol aldehyde precursor

[0047]

[0048] In a 100 mL dry three-necked flask, a phospholipid intermediate (10.0 g, 25.3 mmol) and 35 mL of ultra-dry N,N-dimethylformamide were added. Sodium hydride (0.67 g, 27.9 mmol) was added under ice bath conditions and stirred until homogeneous. Then, a THF solution of 2-norborneol (3.1 g, 27.9 mmol) was added dropwise, and the reaction was allowed to proceed for 12 h at room temperature. The reaction was monitored using a TCL. After the reaction was complete, 100 mL of saturated sodium bicarbonate solution was added to the reaction mixture. The mixture was extracted with n-hexane (150 mL × 3). The organic phase was dried over anhydrous Na₂SO₄ and separated by column chromatography to give a white solid (4.9 g, yield: 51%). 1 H NMR (400MHz, CDCl3, ppm): δ7.13-7.08(m,1H,-Ph-H),6.91-6.84(m,2H,-Ph-H),3.30-3.34(d,3H,-O-CH3),2.41-2.28(s,2H,-Norcamphor -H),2.07-1.90(s,1H,-Norcamphor-H),1.52-1.19(m,7H,-Norcamphor-H),1.04(d,9H,-Si-C-(CH3)3),0.24-0.22(d,6H,-Si-CH3).Mass spectrometry(ESI-MS,m / z):[M+H] + calcd for C 21 H 32 ClO2Si,349.1860; found,349.1868.

[0049] 2) Deprotection of norborneol aldehyde precursor from TBS

[0050]

[0051] The above product (3.0 g, 7.9 mmol) was added to a 100 mL dry single-necked flask and dissolved in 30 mL THF. Tetrabutylammonium fluoride (1.0 M in THF, 8.7 mL, 8.7 mmol) was then added, and the mixture was stirred in an ice bath for 2 h. The reaction was monitored using a TCL. After the reaction was complete, the reaction solution was diluted with ethyl acetate (150 mL) and washed with 1 M HCl (100 mL). The organic phase was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to remove the organic solvent. The solution was purified by column chromatography (PE:EtOAc = 85:15) to give a milky white solid (2.0 g, yield: 95%). 1 H NMR (400MHz, CDCl3, ppm): δ7.19-7.14(m,1H,-Ph-H),7.02-6.97(m,1H,-Ph-H),6.88-6.84(m,1H,-Ph-H),3.35-3.31(d,3H ,-O-CH3),2.42-2.30(m,2H,-Norcamphor-H),2.08-1.93(m,1H,-Norcamphor-H),1.59-1.22(m,7H,-Norcamphor-H).Mass spectrometry(ESI-MS,m / z):[M–H]-calcd for C 16 H 20 ClO2,263.0839; found,263.0842.

[0052] 3) Synthesis of norborneol aldehyde intermediate

[0053]

[0054] In a 250 mL dry double-necked flask, MgCl2 (1.75 g, 18.4 mmol), paraformaldehyde (2.47 g, 82.3 mmol), Et3N (2.73 g, 27.0 mmol), a deprotected intermediate (3.25 g, 12.3 mmol), and 150 mL of acetonitrile were added sequentially and stirred until homogeneous. The reaction mixture was then heated overnight at 80 °C under nitrogen protection. The reaction progress was monitored by TLC (PE:EtOAc = 95:10). After the reaction was complete, the reaction solution was cooled to room temperature and then diluted with 100 mL of DCM. The mixture was filtered, and the filtrate was washed with brine, dried over Na2SO4, and concentrated under reduced pressure to remove the organic solvent. The crude product was purified by silica gel column chromatography (PE:EtOAc = 95:5) to give a pale yellow solid (1.80 g, yield: 60%). 1H NMR (400MHz, CDCl3, ppm): δ11.65(s,1H,-Ar-OH),9.91-9.90(d,J=5.6Hz,1H,-CHO),7.51-7.48(m,1H,-Ph-H),7.03-6.99(m,1H,-Ph-H),3 .38-3.34(d,3H,-O-CH3),2.44-2.34(m,2H,-Norcamphor-H),2.10-1.96(m,1H,-Norcamphor-H),1.56-1.27(m,7H,-Norcamphor-H).Mass spectrometry(ESI-MS,m / z):[MH] - calcd for C 16 H 16 ClO3,291.0788; found,291.0794.

[0055] Example 2:

[0056] Synthesis of afterglow luminescent compounds

[0057] 1) Synthesis of Rubine-1

[0058]

[0059] Add 2-(2,3-dihydro-1H-inden-1-yl)malononitrile (246 mg, 1.37 mmol), norborneol intermediate (200 mg, 0.68 mmol), acetonitrile (30 mL), and piperidine (0.5 mL) to a 100 mL dry double-necked flask. Reflux the mixture for 11 h, cool, and concentrate under reduced pressure to remove the organic solvent. Purify the crude solid by silica gel column chromatography (PE:EtOAc = 65:35) to give a yellowish-brown solid (79 mg, yield: 25%). Rubine-1: 1 H NMR (400MHz, CDCl3, ppm): δ8.42-8.40(d,J=8.0Hz,1H),7.85(s,1H),7.68-7.66(d,J=7.6Hz,1H),7.50-7.39(m,3H),6. 97-6.95(d,J=7.6Hz,1H),3.76(s,2H),3.38-3.34(d,3H),2.47-2.34(m,2H),2.11-1.99(m,1H),1.55-1.25(m,7H).Mass spectrometry(ESI-MS,m / z):[MH] - calcd for C 28 H 22ClN2O2,453.1370; found,453.1372.

[0060] 2) Synthesis of Rubine-2

[0061]

[0062] Add 2-(2,3-dihydro-3-oxo-1H-inden-1-yl)malononitrile (266 mg, 1.37 mmol), norborneol intermediate (200 mg, 0.68 mmol), acetonitrile (25 mL), and piperidine (0.5 mL) to a 100 mL dry double-necked flask. Reflux the mixture for 6 h, cool, and concentrate under reduced pressure to remove the organic solvent. Purify the crude solid by silica gel column chromatography (PE:EtOAc = 65:35) to give a yellowish-brown solid (61 mg, yield: 19%). Rubine-2: 1 H NMR (400MHz, CDCl3, ppm): δ8.69-8.67(d,J=7.6Hz,1H),8.32(s,1H),8.01-7.99(d,J=7.6Hz,1H),7.92-7.83(m,2H),7.71-7.69 (d,J=8.0Hz,1H),7.01-6.99(d,J=8.0Hz,1H),3.39-3.35(d,3H),2.46-2.34(m,2H),2.11-1.99(m,1H),1.57-1.28(m,7H).Mass spectrometry(ESI-MS,m / z):[MH] - calcd forC 28 H 20 ClN2O3,467.1162; found,467.119968.

[0063] 3) Synthesis of Rubine-3

[0064]

[0065] Add 4-(dicyanomethylene)-2,6-dimethyl-4H-pyran (235 mg, 1.37 mmol), norborneol intermediate (200 mg, 0.68 mmol), acetonitrile (30 mL), and piperidine (0.5 mL) to a 100 mL dry double-necked flask. Reflux the mixture for 6 h, cool, and concentrate under reduced pressure to remove the organic solvent. Purify the crude solid by silica gel column chromatography (PE:EtOAc = 65:35) to give a yellowish-brown solid (100 mg, yield: 33%). Rubine-3: 1H NMR (400MHz, CDCl3, ppm): δ7.69-7.63(dd, J1=16.0Hz, J2=5.8Hz, 1H), 7.41-7.37(t, J=7.2Hz, 2H), 6.96-6.91(m ,2H),6.71(s,1H),6.72(s,1H),3.37-3.33(d,2H),2.46-2.34(m,5H),2.10-1.98(m,1H),1.43-1.25(m,7H).Mass spectrometry(ESI-MS,m / z):[MH] - calcd for C 26 H 22 ClN2O3,445.1319; found,445.1318.

[0066] 4) Synthesis of Rubine-4

[0067]

[0068] In a 100 mL dry single-necked flask, norborneol intermediate (200 mg, 0.68 mmol), isoflurane (255 mg, 1.37 mmol), piperidine (0.5 mL), and an appropriate amount of acetonitrile were added sequentially. The mixture was heated under argon protection at reflux for 6 h at a reaction temperature of 90 °C. After the reaction was complete, a red liquid with orange-red fluorescence was obtained. The solvent was removed by concentration under reduced pressure, and the product was purified by column chromatography (DCM:MeOH = 98:2) to obtain an orange solid (210 mg, yield: 67%). 1 H NMR (400MHz, CDCl3, ppm): δ7.45-7.43 (d, J=8.0Hz, 1H), 7.39-7.35 (d, J=16.4 Hz,1H),7.12-7.08(d,J=16.4Hz,1H),6.90-6.88(d,J=8.0Hz,1H),6.85(s,1H) ,3.37-3.33(d,2H),2.61(s,2H),2.50(s,2H),2.46-2.34(m,2H),2.10-1.98( m,1H),1.57-1.28(m,7H),1.09(s,6H).Massspectrometry(ESI-MS,m / z):[MH] - calcd for C 26 H 22 ClN2O3,445.1319; found,445.1318.

[0069] 5) Synthesis of Rubine-5

[0070]

[0071] Add 1,3-bis(diacyanomethylene)indane (331 mg, 1.37 mmol), norborneol intermediate (200 mg, 0.67 mmol), acetonitrile (25 mL), and piperidine (0.5 mL) to a 100 mL dry double-necked flask. Reflux the mixture for 1 h, cool, and concentrate under reduced pressure to remove the organic solvent. Purify the crude solid by silica gel column chromatography (PE:EtOAc = 65:35) to give a blue solid (37 mg, yield: 10%). Rubine-1: 1 H NMR (400MHz, CDCl3, ppm): δ8.72-8.70(m,2H),8.40(s,1H),8.11-8.08(m,2H),7.71-7.69(d,J=8.0Hz,1H),7 .12-7.10(d,J=8.0Hz,1H),3.38-3.34(d,3H),2.44-2.32(m,2H),2.09-1.79(m,1H),1.55-1.28(m,7H).Mass spectrometry(ESI-MS,m / z):[MH] - calcd for C 31 H 20 ClN4O2,515.1275; found,515.1279.

[0072] Example 3:

[0073] The UV absorption, fluorescence, and afterglow emission spectra of the afterglow compound Rubine-1. Rubine-1 prepared in Example 2 was dissolved in analytical grade dimethyl sulfoxide to prepare a 1.0 × 10⁻⁶ solution. -2 The stock solution of M was prepared. Then, 2 mL of a mixed solvent was prepared by taking 1.8 mL of Tris buffer solution and 0.2 mL of human serum albumin solution. Subsequently, 2 μL of Rubine-1 stock solution was added to the prepared mixed solvent, mixed thoroughly, and then transferred to an optical quartz cuvette (10 × 10 mm) to test its absorption and fluorescence spectra. Figure 1 As shown, its maximum absorption wavelength is located at 463 nm, and a fluorescence spectrum with a maximum emission wavelength of 582 nm was obtained by excitation at this wavelength. Similarly, 20 μL of Rubine-1 stock solution was added to the prepared mixed solvent, and after thorough mixing, the mixture was transferred to an optical quartz cuvette (10 × 10 mm) to test the afterglow emission spectrum at 100 mW / cm². -2 The afterglow emission spectrum of white light irradiated for 5 seconds showed an emission peak of 589 nm.

[0074] Example 4:

[0075] The UV absorption, fluorescence, and afterglow emission spectra of the afterglow compound Rubine-2. Rubine-2 prepared in Example 2 was dissolved in analytical grade dimethyl sulfoxide to prepare a 1.0 × 10⁻⁶ solution. -2 The stock solution of M was prepared. Then, 2 mL of a mixed solvent was prepared by taking 1.8 mL of Tris buffer solution and 0.2 mL of human serum albumin solution. Subsequently, 2 μL of Rubine-2 stock solution was added to the prepared mixed solvent, mixed thoroughly, and then transferred to an optical quartz cuvette (10 × 10 mm) to test its absorption and fluorescence spectra. Figure 2 As shown, its maximum absorption wavelength is at 475 nm, and a fluorescence spectrum with a maximum emission wavelength of 618 nm was obtained by excitation at this wavelength. Similarly, 20 μL of Rubine-2 stock solution was added to the prepared mixed solvent, and after thorough mixing, the mixture was transferred to an optical quartz cuvette (10 × 10 mm) to test the afterglow emission spectrum at 100 mW / cm². -2 The afterglow emission spectrum with an emission peak of 635 nm was obtained after 5 seconds of white light illumination.

[0076] Example 5:

[0077] The UV absorption, fluorescence, and afterglow emission spectra of the afterglow compound Rubine-3. Rubine-3 prepared in Example 2 was dissolved in analytical grade dimethyl sulfoxide to prepare a 1.0 × 10⁻⁶ solution. -2 The stock solution of M was prepared. Then, 2 mL of a mixed solvent was prepared by taking Tris buffer solution (1.8 mL) and human serum albumin solution (0.2 mL). Subsequently, 2 μL of Rubine-3 stock solution was added to the prepared mixed solvent, mixed thoroughly, and then transferred to an optical quartz cuvette (10 × 10 mm) to test its absorption and fluorescence spectra. Figure 3 As shown, its maximum absorption wavelength is at 494 nm, and a fluorescence spectrum with a maximum emission wavelength of 621 nm was obtained by excitation at this wavelength. Similarly, 20 μL of Rubine-3 stock solution was added to the prepared mixed solvent, and after thorough mixing, the mixture was transferred to an optical quartz cuvette (10 × 10 mm) to test the afterglow emission spectrum at 100 mW / cm². -2 The afterglow emission spectrum with an emission peak of 638 nm was obtained after 5 seconds of white light illumination.

[0078] Example 6:

[0079] The UV absorption, fluorescence, and afterglow emission spectra of the afterglow compound Rubine-4. Rubine-4 prepared in Example 2 was dissolved in analytical grade dimethyl sulfoxide to prepare a 1.0 × 10⁻⁶ solution. -2The stock solution of M was prepared. Then, 2 mL of a mixed solvent was prepared by taking Tris buffer solution (1.8 mL) and human serum albumin solution (0.2 mL). Subsequently, 2 μL of Rubine-4 stock solution was added to the prepared mixed solvent, mixed thoroughly, and then transferred to an optical quartz cuvette (10 × 10 mm) to test its absorption and fluorescence spectra. Figure 4 As shown, its maximum absorption wavelength is 430 / 543 nm, and a fluorescence spectrum with a maximum emission wavelength of 653 nm was obtained by excitation at 540 nm. Similarly, 20 μL of Rubine-4 stock solution was added to the prepared mixed solvent, and after thorough mixing, the mixture was transferred to an optical quartz cuvette (10 × 10 mm) to test the afterglow emission spectrum at 100 mW / cm². -2 The afterglow emission spectrum with an emission peak of 693 nm was obtained after 5 seconds of white light illumination.

[0080] Example 7:

[0081] The UV absorption, fluorescence, and afterglow emission spectra of the afterglow compound Rubine-5. Rubine-5 prepared in Example 2 was dissolved in analytical grade dimethyl sulfoxide to prepare a 1.0 × 10⁻⁶ solution. -2 The stock solution of M was prepared. Then, 2 mL of a mixed solvent was prepared by taking Tris buffer solution (1.8 mL) and human serum albumin solution (0.2 mL). Subsequently, 2 μL of Rubine-5 stock solution was added to the prepared mixed solvent, mixed thoroughly, and then transferred to an optical quartz cuvette (10 × 10 mm) to test its absorption and fluorescence spectra. Figure 5 As shown, its maximum absorption wavelength is 610 nm, and a fluorescence spectrum with a maximum emission wavelength of 670 nm is obtained by excitation with 518.

[0082] Example 8:

[0083] A summary of the photophysical parameters of afterglow compounds Rubine-1, Rubine-2, Rubine-3, Rubine-4, and Rubine-5. (For example...) Figure 6 As shown, λ abs Indicates the maximum absorption peak of the compound, ε abs The molar extinction coefficient at the maximum absorption peak of the compound, λ em Indicates the maximum emission peak of the compound, Φ FL The fluorescence quantum efficiency of a compound, λ CLThe wavelength of the afterglow emission of the compound is indicated. The test system is a Tris-HCl solution (containing 10% HSA) at a temperature of 25°C. The test instruments used in Examples 3-8 are an Agilent Cary 500 UV-Vis absorption spectrophotometer and an Agilent Cary Eclipse fluorescence spectrophotometer.

[0084] Example 9:

[0085] Afterglow luminescence photographs of the compounds Rubine-1, Rubine-2, Rubine-3, and Rubine-4. Figure 7 As shown, the Rubine series of afterglow luminescent compounds with different emission wavelengths; the test system was Tris buffer (containing 10% HSA), and the compound concentration was 1.0 × 10⁻⁶. -4 M, at 500mWcm -2 The photo was taken with a mobile phone after being exposed to white light for 5 seconds.

[0086] Example 10:

[0087] Afterglow luminescence kinetics of the afterglow compound Rubine-2. Rubine-2 prepared in Example 2 was dissolved in analytical grade dimethyl sulfoxide to prepare 1.0 × 10⁻⁶ ppm solution. -2 M's stock solution. Then, 1 mL of mixed solvent was prepared by taking Tris buffer (0.9 mL) and human serum albumin solution (0.1 mL). Subsequently, 1 μL of Rubine-2 stock solution was added to the prepared mixed solvent, mixed thoroughly, and transferred to a black 96-well plate (200 μL / well) to test the afterglow luminescence kinetics. The afterglow luminescence signal was acquired using an Imaging Quant 4000 system, with an exposure time of 1 min. Figure 8 As shown, the afterglow luminescent compound showed almost no luminescence signal before illumination; at 100 mW / cm², -2 After 5 seconds of exposure to white light, the emission signal immediately reaches its maximum (1.43*10). 8 The light emission (RLU) gradually decays over time, lasting for about 1 hour.

[0088] Example 11:

[0089] Confocal fluorescence imaging of Rubine-2 in A549 cells. Human non-small cell lung cancer cells (human adenocarcinoma alveolar basal epithelial cells, A549 cell) were purchased from the Shanghai Institute of Cell Biology. F-12K cell culture medium contained 10% fetal bovine serum (FBS, Biological Industry, Kibbutz Beit Haemek, Israel) and 1% penicillin (10,000 U mL-1 penicillin and 10 mg / mL streptomycin, Solarbio Life Science, Beijing, China). Culture conditions were 37°C in a humidified atmosphere containing 5% CO2.

[0090] A549 cells were fed at a rate of 1×10 5 Cells were transferred at a density of 1.5 mL to glass culture dishes containing complete culture medium. After the cells had fully adhered, F-12K medium containing 1 μM Rubine-2 was added, and the dishes were incubated in the dark for 2 hours. After incubation, the cells were washed with PBS (1 mL × 3) and then imaged using a Leica TCS SP8 confocal laser microscope (63 × oil immersion). Excitation was at 488 nm, and fluorescence was observed at 610–650 nm. Figure 9 As shown, the cytoplasm exhibits obvious red fluorescence, indicating that Rubine-2 has good cell membrane permeability.

[0091] Example 12:

[0092] Application of Rubine-2 in live afterglow imaging. All live experiments in this invention adhered to regulations regarding the husbandry and use of laboratory animals and were approved by the Ethics Committee of East China University of Science and Technology. Tumor-bearing nude mice were purchased from East China Normal University in Shanghai and housed in sterile cages within a laminar flow fume hood in a sterile room, fed with food and water treated by high-pressure steam.

[0093] First, 20 μL of 10 μM Rubine-2 Tris-HCl solution was injected in situ into the tumor area. Fifteen minutes after injection, the tumor area was exposed to light for 10 seconds (light intensity: 400 mW·cm²). -2 Immediately afterwards, the Perkin Elmer In-Vivo Professional Imaging System was used to record the afterglow emission signal in the mice. Figure 10As shown, the mouse tumor area showed almost no afterglow emission signal before light exposure; however, after light activation, the mouse tumor area showed a much higher afterglow emission signal than other areas, indicating that Rubine-1 has excellent in vivo afterglow imaging capabilities.

Claims

1. A single-component organic afterglow compound, characterized by, The molecular structure of this organic afterglow compound features a push-pull electron system and double-bonded groups substituted with electron-rich groups, and its general structural formula is as follows: ； In Formula I, the electron-withdrawing group A can be any one of the following structures containing a cyano group: ； X is a halogen atom; the methoxy-substituted double bond group is a necessary structure, and this structure is located in the ortho or para position of the electron-withdrawing A, together forming a push-pull electron system; R1 is any one of cyano, acetyl, carboxymethyl ester, carboxyethyl ester, and carboxyl; R2 is any one of methyl, isopropyl, tert-butyl, and cyclopropane.

2. The single-component organic afterglow compound according to claim 1, characterized in that, X is one of the halogen atoms F, Cl, or Br.

3. The single-component organic afterglow compound according to claim 1, wherein Electron-withdrawing group A is selected from any of the following structures: ； R1 can be any one of cyano, acetyl, carboxymethyl ester, carboxyethyl ester, or carboxyl.

4. The non-diagnostic biological detection application of a single component organic afterglow compound according to claim 1, wherein, This afterglow luminescent compound is compounded with various commercial luminescence enhancers, surfactants, cyclodextrins, skim milk powder, or serum proteins to improve the luminescence intensity of the compound.

5. The non-diagnostic biological detection application of a single component organic afterglow compound according to claim 1, wherein, The afterglow luminescent compound does not require the addition of photosensitizers, nor does it need to be mixed with photosensitizers or block polymers to form nanoparticles; it can be used alone.

6. The method for applying a single-component organic afterglow compound according to claim 1, characterized in that, The following operating conditions are included in the use: (1) the afterglow substrate is placed in a solution or gel with a pH greater than 5.0; (2) the illumination time of the light source is 1-10 seconds, and the light source is a commercial LED light source; ； In a microenvironment with a pH above 5.0, the afterglow compound generates a high-energy unstable 1,2-dioxane structure upon illumination, which then rapidly decomposes to produce afterglow luminescence.

Images