A long-lasting molecule, its preparation method and application, and detection reagent

Abstract

The present invention discloses a long afterglow molecule, comprising a structure as shown in formula (I): #imgabs0# wherein R1 is a saturated hydrocarbon group, including one of a straight-chain saturated hydrocarbon group, a branched saturated hydrocarbon group and a cyclic saturated hydrocarbon group; R2 is one of hydrogen, halogen, -R, -OH, -CHO, -C=O, -COOH, -NO2, -NH2 and -SH; R3 comprises a substituted or unsubstituted aromatic heterocyclic group, wherein the aromatic heterocyclic group is one of a quinolyl group, an isoquinolyl group and a benzothiazolyl group; R4 is hydrogen, or R4 is a group or probe that can specifically identify a specific substance or can produce a specific response to a specific substance. The fluorescence quantum efficiency of the long afterglow molecule is greatly improved, especially in the long wavelength range. In addition, the lifetime of the long afterglow molecule can be controlled over a wider range or within the same wavelength range. The present invention also discloses its preparation method and application, as well as a detection reagent.

Description

Technical Field

[0001] The present invention mainly relates to the field of photochemistry, and in particular to a long afterglow molecule, a preparation method and application thereof, and a detection reagent. Background Art

[0002] Long-afterglow molecules are a special class of organic compounds that can form unstable 1,2-oxetane structures when excited by external light. After de-excitation, these structures slowly decompose, releasing energy and producing sustained luminescence, a phenomenon known as the long-afterglow effect. These molecules are characterized by their ability to maintain luminescence for a period of time after external excitation ceases, thus offering potential applications in a variety of fields, such as bioimaging, information storage, anti-counterfeiting marking, and optoelectronic displays.

[0003] Existing long-lasting luminescence molecules primarily include conjugated polymers based on poly(p-phenylene vinylene) (PPV), dimethylaniline molecules and their derivatives with oxygen-sulfur heterocycles or oxygen-oxadiene structures, and Schaap's adamantane-oxetane precursor molecules. These molecules achieve long-lasting luminescence by forming unstable 1,2-oxetane and then decomposing it. While PPV-based semiconducting polymer nanoparticles exhibit some afterglow, the lifetime of the afterglow is difficult to control, with reported half-lives as low as 6.6 minutes. On the other hand, while oxygen-sulfur heterocycles or oxygen-oxygen heterocycles containing hexadiene-dimethylaniline can achieve afterglow emission through energy transfer, their inherent luminescence efficiency is relatively weak and their afterglow lifetime is short, typically only 1.08 seconds. Compared to the aforementioned two types of molecules, Schaap's adamantane-oxetane molecules are favored due to their ease of synthesis and modification, easily tunable emission wavelength, and relatively long afterglow lifetime. The transformation and modification of the protecting group (PG) of this type of molecule achieves long-lived luminescence in response to molecules, ions or enzymes, providing new solutions for the detection and quantification of analytes, in vitro and in vivo monitoring, and clinical sample testing.

[0004] However, the emission wavelength of this type of molecule is mostly blue or green, with poor penetration, which limits its practical application. Currently, there are studies on the red shift of the wavelength of Schaap's adamantane-oxetane molecules, but the luminescence efficiency is very low, which also causes inconvenience for practical applications. There are also studies on the red shift of the emission wavelength by co-doping Schaap's adamantane-oxetane molecules with red-emitting dye molecules in nanoparticles, while utilizing the energy transfer between molecules ( Resonance energy transfer (FRET) is used to improve the efficiency of afterglow light. However, during the energy transfer process, some energy may be lost in non-radiative forms (such as heat energy) rather than converted into light energy. This energy loss will reduce the luminous efficiency, and the luminous efficiency is still low. The luminous efficiency of the existing Schaap's adamantane-oxetane molecule that emits red light is less than 5%.

[0005] At the same time, while there are currently methods for regulating the afterglow lifetime of long-lasting materials, this also causes changes in wavelength. This is particularly cumbersome when using afterglow lifetime for material detection across multiple substances. If the wavelength changes, the instrument must switch to a different wavelength receiving channel when collecting the luminescence signal, a process that is quite complex. Furthermore, the narrow range of afterglow lifetime regulation also hinders the practical application of long-lasting materials. Summary of the Invention

[0006] In order to solve the above technical problems, an embodiment of the present invention discloses a long-lasting molecule, which includes a structure as shown in formula (I):

[0007]

[0008] Wherein, R1 is a saturated hydrocarbon group, and the saturated hydrocarbon group includes one of a straight-chain saturated hydrocarbon group, a branched-chain saturated hydrocarbon group and a cyclic saturated hydrocarbon group;

[0009] R2 is one of hydrogen, halogen, -R, -OH, -CHO, -C=O, -COOH, -NO2, -NH2 and -SH;

[0010] R3 comprises a substituted or unsubstituted aromatic heterocyclic group, wherein the aromatic heterocyclic group is one of quinolyl, isoquinolyl and benzothiazolyl;

[0011] R3 comprises a substituted or unsubstituted aromatic heterocyclic group, wherein the aromatic heterocyclic group is one of quinolyl, isoquinolyl and benzothiazolyl;

[0012] R4 is hydrogen, or R4 is a group or probe that can specifically recognize a specific substance or produce a specific response to a specific substance.

[0013] By adopting the above technical solution, long-afterglow molecules with greatly improved fluorescence quantum efficiency can be obtained, especially in the long-wavelength region, where the fluorescence quantum efficiency is significantly improved. High fluorescence quantum efficiency can reduce energy consumption and lower application costs. Improving fluorescence efficiency in the long-wavelength range can also enhance signal intensity and signal-to-noise ratio, which helps to improve the sensitivity and reliability of imaging and detection. In addition, it is possible to achieve lifetime regulation of long-afterglow molecules over a wide range, and lifetime regulation within the same wavelength range at the same time, which is suitable for different application scenarios, greatly simplifies the process of collecting afterglow lifetime signals, and expands the application range of long-afterglow materials.

[0014] Optionally, the long afterglow molecule has a structure shown in formula (III) or formula (III):

[0015]

[0016] Wherein, R1 is a saturated hydrocarbon group, and the saturated hydrocarbon group includes one of a straight-chain saturated hydrocarbon group, a branched-chain saturated hydrocarbon group and a cyclic saturated hydrocarbon group;

[0017] R2 is one of hydrogen, halogen, -R, -OH, -CHO, -C=O, -COOH, -NO2, -NH2 and -SH;

[0018] R5 is a substituted or unsubstituted aromatic heterocyclic group, wherein the aromatic heterocyclic group is one of quinolyl, isoquinolyl and benzothiazolyl;

[0019] R6 is a substituted or unsubstituted aromatic heterocyclic group, wherein the aromatic heterocyclic group is one of quinolyl, isoquinolyl and benzothiazolyl;

[0020] R4 is hydrogen, or R4 is a group or probe that can specifically recognize a specific substance or produce a specific response to a specific substance.

[0021] Optionally, R4 is one of formula (I-4-1) to formula (I-4-11):

[0022]

[0023]

[0024] Among them, in formula (I-4-1) to formula (I-4-11) It represents the linking site of formula (I-4-1) to formula (I-4-11) in formula (I).

[0025] Optionally, the saturated hydrocarbon group is one of methyl, ethyl, propyl, n-butyl, n-pentyl, isopropyl, isobutyl, neopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0026] Optionally, R3 is one of formula (I-3-1) to formula (I-3-4):

[0027]

[0028] Among them, in formula (I-3-1) to formula (I-3-4) It represents the linking site of formula (I-3-1) to formula (I-3-4) in formula (I).

[0029] Optionally, the long afterglow molecule includes one of the structures shown in formula (I-a) to formula (I-d):

[0030]

[0031]

[0032] Optionally, the emission peak wavelength of the long afterglow molecule is in the range of 472-620 nm, and the fluorescence quantum efficiency is in the range of 30%-50%.

[0033] Optionally, the afterglow lifetime of the long afterglow molecule is in the range of 2.83 min-2.5 h.

[0034] According to another specific embodiment of the present invention, an embodiment of the present invention discloses a method for preparing a long-lasting molecule, comprising the following steps:

[0035] Preparation of mixed solution: raw material A and raw material B are dissolved in an organic solvent, wherein raw material A includes a molecule having a structure as shown in formula (0), and raw material B can undergo a nucleophilic substitution reaction with raw material A to obtain a molecule having a structure as shown in formula (1).

[0036]

[0037] Adding a catalyst and stirring: stirring under heating and catalyst catalysis conditions;

[0038] Post-treatment: cooling, diluting, washing, drying, or cooling, allowing the product to precipitate, filtering and washing.

[0039] By adopting the above technical solution, long-afterglow molecules with high quantum luminescence efficiency and controllable luminescence lifetime can be easily prepared.

[0040] Optionally, the raw material B includes one or more of 2-methyl-6-quinolinecarboxylic acid, 4-methylquinoline, 1-methylisoquinoline, p-toluenesulfonic acid and 2-aminobenzenethiol, the catalyst includes one of indium trichloride and p-toluenesulfonic acid, and the organic solvent includes one of anhydrous ethanol and tetrahydrofuran.

[0041] According to another specific embodiment of the present invention, an embodiment of the present invention discloses an application of a long-afterglow molecule, which is used for biological imaging, in vitro diagnosis, sensing detection, quality control, anti-counterfeiting encryption and homogeneous detection, has a wide range of application fields and good application prospects, and has good reliability.

[0042] Optionally, long-lasting glow molecules with different structures are used for bioimaging, in vitro diagnosis, sensor detection, quality control, anti-counterfeiting encryption and homogeneous detection to simultaneously collect light signals of different long-lasting glow molecules, wherein the long-lasting glow molecules with different structures include two or more long-lasting glow molecules with the structure represented by the following formula (I):

[0043]

[0044] Wherein, R1 is a saturated hydrocarbon group, and the saturated hydrocarbon group includes one of a straight-chain saturated hydrocarbon group, a branched-chain saturated hydrocarbon group and a cyclic saturated hydrocarbon group;

[0045] R2 is one of hydrogen, halogen, -R, -OH, -CHO, -C=O, -COOH, -NO2, -NH2 and -SH;

[0046] R3 comprises a substituted or unsubstituted aromatic heterocyclic group, wherein the aromatic heterocyclic group is one of quinolyl, isoquinolyl and benzothiazolyl;

[0047] R4 is hydrogen, or R4 is a group or probe that can specifically recognize a specific substance or produce a specific response to a specific substance.

[0048] Optionally, the long afterglow molecules of different structures include two or more long afterglow molecules of the structures shown in the following formulas (I-a) to (I-c):

[0049]

[0050] According to another specific embodiment of the present invention, an embodiment of the present invention discloses a detection reagent for use in homogeneous detection, wherein the detection reagent comprises two or more long-lasting glow molecules of different structures, wherein the long-lasting glow molecules are selected from the structures shown in Formula (I-a) to Formula (I-c):

[0051]

[0052]

[0053] This detection reagent can perform homogeneous detection within the same wavelength range. When using the instrument to collect signals, the signals can be collected under the same wavelength channel. There is no need to replace the relevant parts of the instrument, which is easy to operate and can improve the detection efficiency.

[0054] Optionally, the detection reagent further comprises a long afterglow molecule having a structure as shown in formula (I-d):

[0055] BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 A schematic diagram showing the luminescence principle of a specific embodiment of the long afterglow molecule of the present invention;

[0057] FIG2( a ) shows the absorption and fluorescence spectra of the long-lasting molecule in Example 1 of the present invention;

[0058] FIG2( b ) shows the absorption spectrum and fluorescence spectrum of the long afterglow molecule in Example 2 of the present invention;

[0059] FIG2( c ) shows the absorption and fluorescence spectra of the long afterglow molecule in Example 3 of the present invention;

[0060] FIG2( d ) shows the absorption and fluorescence spectra of the long-lasting molecule in Example 4 of the present invention;

[0061] FIG3( a ) shows the afterglow spectrum of the long afterglow molecule in Example 1 of the present invention;

[0062] FIG3( b ) shows the afterglow spectrum of the long afterglow molecule in Example 1 of the present invention;

[0063] FIG3( c ) shows the afterglow spectrum of the long afterglow molecule in Example 1 of the present invention;

[0064] FIG3( d ) shows the afterglow spectrum of the long afterglow molecule in Example 1 of the present invention;

[0065] FIG4( a ) shows the afterglow decay curve and afterglow image of the long afterglow molecule in Example 1 of the present invention;

[0066] FIG4( b ) shows the afterglow decay curve and afterglow image of the long afterglow molecule in Example 1 of the present invention;

[0067] FIG4( c ) shows the afterglow decay curve and afterglow image of the long afterglow molecule in Example 1 of the present invention;

[0068] FIG4( d ) shows the afterglow decay curve and afterglow image of the long afterglow molecule in Example 1 of the present invention;

[0069] Figure 5 A schematic diagram showing the application of anti-counterfeiting encryption of information of long-lasting molecules with different lifespans in Example 5 of the present invention;

[0070] Figure 6 The afterglow photos at different time points of the long afterglow molecule applied to digital anti-counterfeiting encryption in Example 5 of the present invention are shown. DETAILED DESCRIPTION

[0071] The following is an explanation of the embodiments of the present invention by specific specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Although the description of the present invention will be introduced in conjunction with the preferred embodiment, this does not mean that the features of this invention are limited to this embodiment. On the contrary, the purpose of introducing the invention in conjunction with the embodiment is to cover other options or modifications that may be extended based on the claims of the present invention. In order to provide a deep understanding of the present invention, the following description will contain many specific details. The present invention can also be implemented without using these details. In addition, in order to avoid confusion or blurring the focus of the present invention, some specific details will be omitted in the description. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0072] It should be noted that in this specification, similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined or explained in subsequent drawings.

[0073] To make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0074] The first aspect of the present invention discloses a long afterglow molecule, the long afterglow molecule and 1 O2 reacts to form an intermediate 1,2-oxetane structure, which decomposes to remove adamantane ketone, generating a benzoate structure of a long afterglow molecule and then emitting light. Specifically, the long afterglow molecule is first excited to a higher energy state by light energy. The excited long afterglow molecule reacts with the surrounding singlet oxygen to form a 1,2-oxetane intermediate. The 1,2-oxetane intermediate is unstable and can be further decomposed, while releasing a small molecule (such as adamantane ketone). As the small molecule detaches, a benzoate structure is formed, which usually leads to a rearrangement of energy states. The newly formed benzoate structure is in an excited state and then returns to the ground state in the form of light emission. This process is slow, resulting in the long afterglow phenomenon. The specific process is as follows: Figure 1 shown.

[0075] The long afterglow molecule includes the structure shown in formula (I):

[0076]

[0077] Since the long afterglow molecule described in the structure of formula (I) generates an intermediate structure of benzoate at the part connected to R1 during the luminescence process, the group selected for the R1 part is usually selected from some stable groups that do not react with carboxylic acids. In some specific embodiments, R1 is a saturated hydrocarbon group. The saturated hydrocarbon group lacks active functional groups and will not react with carboxyl groups, which can ensure that the long afterglow molecule can function stably. More specifically, the saturated hydrocarbon group includes one of a straight-chain saturated hydrocarbon group, a branched saturated hydrocarbon group and a cyclic saturated hydrocarbon group. Preferably, the saturated hydrocarbon group is one of methyl, ethyl, propyl, n-butyl, n-pentyl, isopropyl, isobutyl, neopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. They all have stable structures and properties, can ensure the stable skeleton of the long afterglow molecule, and ensure that the long afterglow molecule can perform its function.

[0078] In certain embodiments, R1 is an aromatic group, such as a phenyl group, which is generally stable and will not react with the carboxyl group unless under specific catalytic conditions. In certain embodiments, R1 can also be a silane group, such as a trimethylsilyl group (TMS) or a triethoxysilyl group (TEOS), which can be attached to the hydroxyl group next to the carboxyl group, but do not react with the carboxylic acid itself.

[0079] R2 is one of hydrogen, halogen, -R (alkyl), -OH (hydroxy), -CHO (aldehyde), -C=O (keto), -COOH (carboxyl), -NO2 (nitro), -NH2 (amino) and -SH (thiol).

[0080] In a specific embodiment, R3 is a substituted or unsubstituted aromatic heterocyclic group, wherein the aromatic heterocyclic group is one of quinolyl, isoquinolyl, and benzothiazolyl. The inventors have discovered that when R3 has the aforementioned structure, the afterglow emission wavelength can be adjusted to the red and green regions, and the fluorescence quantum efficiency can be improved, particularly in the red region, achieving high-efficiency afterglow luminescence. Manipulating the quinoline, isoquinoline, and benzothiazole structures can also simply achieve control over a wide lifetime range, further expanding the application range and fields of long-afterglow materials based on the varying lifetimes.

[0081] In a specific embodiment, R4 is hydrogen. By introducing quinoline, isoquinoline, benzothiazole, or their substituents at the ortho position of the phenolic hydroxyl group in the Schaap's adamantane-oxetane precursor structure, a donor-acceptor electron pair is formed between the ionized phenolic hydroxyl group and the quinoline, isoquinoline, benzothiazole, or their substituents, thereby improving the fluorescence quantum efficiency. R4 can also be a group or probe that can specifically recognize or respond to a specific substance. Based on the above, the long-lasting glow molecule can be further modified according to actual application requirements.

[0082] In a specific embodiment, the long afterglow molecule has a structure shown in formula (III) or formula (III):

[0083]

[0084] Wherein, R1 is a saturated hydrocarbon group, and the saturated hydrocarbon group includes one of a straight-chain saturated hydrocarbon group, a branched-chain saturated hydrocarbon group and a cyclic saturated hydrocarbon group;

[0085] R2 is one of hydrogen, halogen, -R, -OH, -CHO, -C=O, -COOH, -NO2, -NH2 and -SH;

[0086] R5 is a substituted or unsubstituted aromatic heterocyclic group, wherein the aromatic heterocyclic group is one of quinolyl, isoquinolyl and benzothiazolyl;

[0087] R6 is a substituted or unsubstituted aromatic heterocyclic group, wherein the aromatic heterocyclic group is one of quinolyl, isoquinolyl and benzothiazolyl;

[0088] R4 is hydrogen, or R4 is a group or probe that can specifically recognize a specific substance or produce a specific response to a specific substance.

[0089] Specifically, R4 is one of formula (I-4-1) to formula (I-4-11):

[0090]

[0091]

[0092] Among them, in formula (I-4-1) to formula (I-4-11) It represents the linking site of formula (I-4-1) to formula (I-4-11) in formula (I).

[0093] Formula (I-4-1) responds to β-galactosidase, formula (I-4-2) responds to membrane-linked proteinase B, formula (I-4-3) responds to NAD(P)H:quinolone oxidoreductase 1, formula (I-4-4) responds to H2O2, formula (I-4-5) responds to nitroreductase, and formulas (I-4-6 to (I-4-11) respond to RSH, ONOO, etc. - , alkaline phosphatase, HNO, formaldehyde, and cysteine ​​to meet different usage needs.

[0094] The long-afterglow molecule has an emission peak wavelength range of 472-620 nm, with wavelengths tuned to the red and green regions. Its absorption peak is red-shifted by approximately 94-148 nm compared to long-afterglow molecules reported in the prior art, and its emission peak is at least 140 nm red-shifted. Its fluorescence quantum efficiency ranges from 30% to 50%, demonstrating a high fluorescence quantum efficiency even in the long-wavelength range. The long-afterglow molecule has an afterglow lifetime of 2.83 min to 2.5 h, offering a wide range of lifetime control, broadening the application possibilities for long-afterglow materials. Furthermore, by combining different R4 groups or probes, it can respond to specific structures to meet diverse application needs.

[0095] In certain specific embodiments, R3 is one of formula (I-3-1) to formula (I-3-4):

[0096]

[0097] Among them, in formula (I-3-1) to formula (I-3-4) It represents the linking site of formula (I-3-1) to formula (I-3-4) in formula (I).

[0098] In the above structures, the olefin groups in formulas (I-3-1) to (I-3-3) are conjugated with the quinoline or isoquinoline structure. That is, the carbon-carbon double bond shown in the formula can be conjugated with the quinoline or isoquinoline structure, thereby enhancing electron delocalization of the entire molecule and thus affecting the electronic properties of the entire molecule. In formula (I-3-1), the quinoline is also connected to an electron-withdrawing group (such as a nitro, cyano, or carboxyl group). This electron-withdrawing group pulls electron density away from the quinoline, increasing the electron cloud offset of the entire conjugated system. This, in turn, achieves a change in the photochemical properties of the entire long-lasting glow report.

[0099] In certain specific embodiments, the long afterglow molecule comprises one of the structures shown in formula (I-a) to formula (I-d):

[0100]

[0101] In particular, the inventors unexpectedly discovered that the fluorescence quantum efficiency of the luminescent body is greatly improved by using the long afterglow molecules of the above formula (I-a) to (I-d), especially compared with the long-wavelength long afterglow molecules reported previously, the fluorescence quantum efficiency is improved by more than one order of magnitude. Specifically, the fluorescence quantum efficiency is 30%-50%, and the emission peak wavelength range is 472-620nm. The reason is that in formula (I-a) to (I-d), the introduction of quinoline, isoquinoline and the corresponding substituent structures can form donor-acceptor electron pairs, which greatly improves the fluorescence quantum efficiency of the luminescent body. Especially for the molecules with structures shown in formula (I-a) to (I-c), they can achieve high-efficiency afterglow luminescence in the long wavelength region, which may also be due to the formation of special donor-acceptor electron pairs and conjugated structures, realizing a special distribution of electron clouds, and realizing efficiency improvement in the red light region through the mutual cooperation of various structures. Although existing studies have also produced long-lasting red-emitting molecules that form donor-acceptor electron pairs, they do not form the donor-acceptor effect and conjugation effect of formula (I-a) to formula (I-c), the electron cloud distribution is also different, the fluorescence quantum efficiency is very low, and practical applications are limited.

[0102] In addition, for formula (I-d), although it emits blue-green light, its fluorescence quantum efficiency is also improved compared with the existing blue-green afterglow molecules, which may also be caused by the formation of a special donor-acceptor structure.

[0103] In addition, in terms of controlling the afterglow lifetime, the inventors also found that the afterglow lifetime can be particularly effectively controlled by introducing quinoline, isoquinoline, benzothiazole or their substituent structures. During the research process, the inventors unexpectedly discovered that when the adamantane molecule is modified by a quinoline group and a benzothiazole group, the electron cloud density of the quinoline group changes with the change of the structure, resulting in differences in electron-withdrawing properties, which can affect the afterglow lifetime of the long afterglow molecule. The reason for this may be that in the luminescence process of the long afterglow molecule of this structure of the present invention, among the two reactions of the reaction of the long afterglow molecule with singlet oxygen (the first step) and the decomposition of 1,2-oxetane into the benzoate structure of the long afterglow molecule (the second step), the second step is the decisive step for the afterglow lifetime of the long afterglow molecule of the present invention, and changing the electron-withdrawing property of the quinoline group can change the speed of the second step, thereby controlling the afterglow lifetime. Specifically, in Formulas (I-a) through (I-c), due to the varying electron cloud densities (i.e., electron-withdrawing properties) of different quinoline groups, the stronger the electron-withdrawing quinoline group, the more favorable the decomposition reaction of 1,2-oxetane, and the shorter the afterglow lifetime of the corresponding long-lasting molecule. This can be controlled on a minute-by-minute scale. For example, in Formula (I-a), connecting the quinoline group via a carboxyl group further enhances the electron-withdrawing property of the quinoline, resulting in a relatively shorter lifetime. Furthermore, while different groups can be used to control afterglow lifetimes, this approach also alters the wavelength. For applications requiring lifetime signal collection, this wavelength change requires changing the instrument's wavelength receiving channel, such as by replacing a different filter. This is cumbersome and places high demands on the instrument, significantly increasing collection time and cost, especially with large sample volumes. Through the quinoline, isoquinoline and their substituent structures in formula (I-a) to formula (I-c), the afterglow lifetime can also be controlled within the same wavelength range, which can be beneficial to the application of long afterglow materials. For example, a time-resolved fluorescence microscope or imaging camera can be used to collect and split different lifetime signals in the same wavelength channel. By setting different delay time gates to collect different lifetime signals, there is no need to change different wavelength channels, and the requirements for test instruments are relatively low. The operation is simple, the collection efficiency is high, and the collection cost is reduced.

[0104] For formula (I-d), the inventors found that it additionally formed intramolecular hydrogen bonds, which could slow down the ionization rate of the phenolic hydroxyl group. The slower ionization rate further affected the decomposition rate of 1,2-oxetane, resulting in longer afterglow luminescence, with an afterglow lifetime of several hours, which further broadened the application range of long afterglow materials.

[0105] Through formula (I-a) to formula (I-d), the step of decomposing 1,2-oxetane into the benzoate structure of the long-lasting molecule is mainly controlled, achieving regulation from minutes to hours, and can also meet a wide range of application needs.

[0106] The second aspect of the present application discloses a method for preparing a long-lasting molecule, comprising the following steps:

[0107] Preparation of mixed solution: raw material A and raw material B are dissolved in an organic solvent, raw material A includes a molecule with a structure as shown in formula (0), and raw material B can undergo a nucleophilic substitution reaction with raw material A to obtain a molecule with a structure as shown in formula (I).

[0108]

[0109] Adding a catalyst and stirring: stirring under heating and catalyst catalysis conditions;

[0110] Post-treatment: cooling, diluting, washing, drying, or cooling, allowing the product to precipitate, filtering and washing.

[0111] In this way, the long-lasting molecule of formula (1) can be prepared relatively simply.

[0112] In a specific embodiment, raw material B includes one or more of 2-methyl-6-quinolinecarboxylic acid, 4-methylquinoline, 1-methylisoquinoline, p-toluenesulfonic acid and 2-aminobenzenethiol. For example, raw material B is a 2-methyl-6-quinolinecarboxylic acid catalyst, or raw material B is 4-methylquinoline, or raw material B is 1-methylisoquinoline, or raw material B is p-toluenesulfonic acid and 2-aminobenzenethiol. In a specific embodiment, the catalyst is indium trichloride (InCl3) or p-toluenesulfonic acid (p-TsOH). Wherein, InCl3 is a strong Lewis acid that can effectively catalyze many types of chemical reactions, including Friedel-Crafts alkylation and acylation reactions, cycloaddition reactions, etc. Reactions using InCl3 as a catalyst can often be carried out under mild conditions, which helps to protect sensitive functional groups. In some cases, InCl3 can be recovered from the reaction mixture and can be reused in some cases. P-toluenesulfonic acid is a strong acid that very effectively catalyzes reactions that require acidic conditions, such as esterification, hydrolysis, and cyclization reactions. Unlike some inorganic acids, p-TsOH generally does not induce oxidative effects, making it relatively mild towards sensitive groups. p-TsOH has good solubility in many organic solvents, allowing it to be used in a variety of solvent systems. After the reaction, p-TsOH can usually be removed from the product through simple neutralization or extraction steps.

[0113] In a specific embodiment, the organic solvent is anhydrous ethanol or tetrahydrofuran, which can effectively dissolve raw material A and raw material B.

[0114] Preferably, the synthesis route of the compound having the structural molecule of formula (0) is as follows:

[0115]

[0116] The third aspect of the present invention discloses an application of a long-lasting glow molecule having a structure shown in formula (I) and can be used for biological imaging, in vitro diagnosis, sensor detection, quality control, anti-counterfeiting encryption and homogeneous detection.

[0117] Preferably, long-lasting glow molecules with different structures are used for bioimaging, in vitro diagnosis, sensor detection, quality control, anti-counterfeiting encryption and homogeneous detection to simultaneously collect light signals of different long-lasting glow molecules, wherein the long-lasting glow molecules with different structures include two or more long-lasting glow molecules with the structure represented by the following formula (I):

[0118]

[0119] Wherein, R1 is a saturated hydrocarbon group, and the saturated hydrocarbon group includes one of a straight-chain saturated hydrocarbon group, a branched-chain saturated hydrocarbon group and a cyclic saturated hydrocarbon group;

[0120] R2 is one of hydrogen, halogen, -R, -OH, -CHO, -C=O, -COOH, -NO2, -NH2 and -SH;

[0121] R3 contains a substituted or unsubstituted aromatic heterocyclic group, wherein the aromatic heterocyclic group is one of quinolyl, isoquinolyl and benzothiazolyl;

[0122] R4 is hydrogen, or R4 is a group or probe that can specifically recognize a specific substance or produce a specific response to a specific substance.

[0123] Optionally, the long afterglow molecules of different structures include two or more long afterglow molecules of the structures shown in the following formulas (I-a) to (I-c):

[0124]

[0125] A fourth aspect of the present invention discloses a detection reagent for simultaneously detecting the contents of different components in a homogeneous detection, for example, a detection kit, wherein the detection reagent comprises two or more long-lasting molecules of different structures, wherein the long-lasting molecules are selected from the structures shown in Formula (I-a) to Formula (I-c):

[0126]

[0127]

[0128] This detection reagent can be used for homogeneous detection in the red light region. When a photon detector is used to collect signals, the signals can be collected in the same wavelength channel, such as the red light channel. There is no need to improve the components of the detector, which is easy to operate and can improve the detection efficiency. For example, when it is necessary to detect the concentration of a marker in a sample to be tested (such as blood or urine, etc.), the above-mentioned long afterglow molecule can be coated in a carrier microsphere (such as polystyrene) and coupled to an antibody to prepare a part of the detection reagent (another part requires a sensitizer to be coated in the carrier microsphere to form a donor microsphere). In the detection reagent, any one of the long afterglow molecules (Ia) to (Ic) can be selected for use, or these three molecules can be used at the same time. When a single molecule is used, if there is only one sensitizer to be selected, one marker is generally detected. When molecules of multiple structures are used at the same time, they can be coded and combined to achieve multi-color coding. In this way, the microspheres of different long afterglow molecules are connected to antibody pairs with different markers. When the sample to be tested, for example, through a blood test, the user's inflammatory markers and kidney disease markers are both high (or not within the normal range), it may mean that the user's body may have problems with inflammation and kidney disease, or in some scenarios, when a doctor needs to combine multiple markers for analysis to make an accurate diagnosis, there is no need to send samples for each marker multiple times for testing, but it is possible to get the results of multiple markers with one sample. In particular, when collecting light signals, there is no need to replace the filter, because the afterglow emitted by the molecules of the above three structures belongs to the same wavelength range and can be collected together, which greatly reduces the workload and facilitates rapid detection.

[0129] Furthermore, the detection reagent may also contain a long-lasting molecule having a structure represented by formula (I-d):

[0130]

[0131] The following will be described in conjunction with more specific embodiments 1-4:

[0132] Examples 1-4 are compounds of long-lasting molecules having the structure shown in formula (I). FDAG mentioned below refers to a molecule having the structure of formula (0). First, FDAG compounds were prepared, and then Examples 1-4 were prepared respectively:

[0133] (1) Preparation of FDAG compounds

[0134] The overall synthetic route of FDAG compounds is as follows:

[0135]

[0136] (1) Synthesis of compound 1b

[0137] Compound 1a (2-chloro-3-hydroxybenzaldehyde, 1 g, 6.38 mmol) was weighed and dissolved in 10 mL of methanol. Once dissolved, trimethyl orthoformate (1.12 mL, 10.22 mmol) and tetrabutylammonium tribromide (154 mg, 0.32 mmol) were added. The mixture was stirred at room temperature for 4 h and monitored by thin-layer chromatography (TLC). The reaction was terminated after the starting material spot disappeared. The mixture was diluted with ethyl acetate (100 mL), and impurities were washed away by adding 0.01 M sodium bicarbonate solution (100 mL). The resulting organic phase was dried over anhydrous sodium sulfate and then purified by column chromatography (petroleum ether:ethyl acetate = 80:20). The product was collected to obtain 1b (1.28 g, 99% yield) as a colorless oil.

[0138] 1H NMR (400MHz, DMSO-d6) δ7.21 (d, J = 1.5Hz, 2H), 7.05-7.02 (m, 1H), 5.59 (s, 1H), 3.39 (s, 6H).

[0139] (2) Synthesis of compound 1c

[0140] Compound 1b (1.08 g, 5.32 mmol) and imidazole (0.79 g, 11.56 mmol) were dissolved in 7.5 mL of dichloromethane. Once fully dissolved, tert-butyldimethylsilyl chloride (TBSCl, 1.04 g, 6.89 mmol) was added, resulting in the formation of a white precipitate. Stirring was continued at room temperature for 3 h, monitored by TLC. The reaction was terminated upon disappearance of the starting material spot. The white precipitate was filtered, and the filtrate was rotary evaporated and subjected to column chromatography (petroleum ether:ethyl acetate = 95:5) to collect the product, yielding 1c (1.56 g, 93% yield) as a colorless oil.

[0141] 1H NMR (400MHz, DMSO-d6) δ7.26 (t, J=7.9Hz, 1H), 7.16 (dd, J=7.9, 1.5Hz, 1H),

[0142] 7.02(dd,J=8.0,1.6Hz,1H),5.54(s,1H),3.28(s,6H),1.01(s,9H),0.23(s,6H).

[0143] (3) Synthesis of compound 1d

[0144] Compound 1c (1.50 g, 4.74 mmol) and trimethoxyphosphine (0.75 mL, 6.18 mmol) were weighed and dissolved in 15 mL of anhydrous dichloromethane. The reaction flask was placed in an ice bath to dissolve and cooled to 0°C. Titanium tetrachloride (5.70 mL, 5.70 mmol) was added via syringe and stirred at 0°C for 3 h. The reaction was monitored by TLC. The reaction was terminated after the starting material spot disappeared. Saturated sodium bicarbonate solution (60 mL) was added to the system, which produced abundant bubbles and turned white. After stirring for 10 min, dichloromethane (60 mL) was added for extraction. The organic phase was then dried over anhydrous sodium sulfate and purified by column chromatography (petroleum ether:ethyl acetate = 40:60) to obtain 1d (1.80 g, 96% yield) as a colorless oil.

[0145] 1H NMR (400MHz, DMSO-d6) δ7.31(t,J=7.5Hz,1H),7.17(d,J=7.5Hz,1H),7.03(d,J=7.9Hz,1H),5. 09(d,J=15.1Hz,1H),3.68(d,J=10.5Hz,3H),3.55(d,J=10.3Hz,3H),1.01(s,9H),0.24(s,6H).

[0146] (4) Synthesis of compound 1e

[0147] Compound 1d (1.50 g, 3.80 mmol) was dissolved in 2 mL of anhydrous tetrahydrofuran and cooled to -78°C in an ethanol bath. A solution of lithium diisopropylamide (LDA) (2.0 M in THF, 2.30 mL, 4.60 mmol) was added to the reaction flask via syringe and stirred for 20 min. 2-Adamantane ketone was dissolved in 7.5 mL of anhydrous tetrahydrofuran and added to the reaction system. The mixture was stirred in an ethanol bath at -78°C for 15 min and then allowed to cool to room temperature. The reaction was monitored by TLC spot plate. The reaction was terminated after the starting material spot disappeared. The mixture was diluted with 60 mL of ethyl acetate, followed by the addition of 60 mL of saturated sodium chloride solution to remove impurities from the organic phase. The resulting organic phase was dried over anhydrous sodium sulfate and then purified by column chromatography (petroleum ether:ethyl acetate = 95:5) to yield 1e (826 mg, 52% yield) as a white solid.

[0148] 1H NMR (400MHz, CDCl3) δ7.16-7.08 (m, 1H), 6.89 (dd, J = 7.8, 1.7Hz, 2H), 3.33 (s, 3H),3.29(s,1H),2.08(s,1H),2.02-1.62(m,12H),1.06(s,8H),0.25(s,5H).

[0149] (5) Synthesis of Compound 1f

[0150] Compound 1e (800 mg, 1.91 mmol) was weighed and dissolved in 10 mL of anhydrous tetrahydrofuran. Stir at room temperature until completely dissolved. Tetrabutylammonium fluoride (TBAF, 1.0 M in THF, 2.50 mL, 2.50 mmol) was pipetted into the reaction flask using a syringe and stirred at room temperature for 3 h. Monitor the reaction using a TLC plate and stop the reaction once the starting material spot disappears. After the solvent in the system was completely evaporated, 100 mL of ethyl acetate was added to dissolve the mixture, followed by extraction with 1 M hydrochloric acid (100 mL). The resulting organic phase was dried over anhydrous sodium sulfate and then purified by column chromatography (petroleum ether:ethyl acetate = 85:15) to obtain compound 1f (574 mg, 99% yield).

[0151] 1H NMR (400MHz, DMSO-d6) δ10.20 (s, 1H), 7.17-7.10 (m, 1H), 6.95 (dd, J = 8.1, 1.5Hz, 1H), 6 .70(dd,J=7.5,1.5Hz,1H),3.18(s,3H),3.17(s,1H),1.97(s,1H),1.94-1.59(m,14H).

[0152] (6) Synthesis of FDAG compounds

[0153] Compound 1f (500 mg, 1.64 mmol) was weighed and dissolved in ultra-dry tetrahydrofuran (THF, 4 mL). Magnesium chloride (344 mg, 3.61 mmol), triethylamine (500 μL, 3.61 mmol), and paraformaldehyde (395 mg, 13.10 mmol) were added to the solution in a reaction flask. The mixture was heated to 80°C and stirred under nitrogen for 2 hours. Monitored by TLC (petroleum ether:ethyl acetate = 90:10). After the starting material spot disappeared, the reaction was terminated. The mixture was diluted with ethyl acetate (100 mL) and extracted with 0.1 M hydrochloric acid (100 mL). The resulting organic phase was dried over anhydrous sodium sulfate and then purified by column chromatography (petroleum ether:ethyl acetate = 95:5) to obtain Compound FDAG (475 mg, 95% yield) as a pale yellow solid.

[0154] 1H NMR (400MHz, CDCl3) δ9.90 (s, 1H), 7.48 (d, J = 7.9Hz, 1H), 6.98 (d, J = 7.9Hz, 1H), 3.34 (s, 3H), 3.28 (s, 1H), 2.08 (s, 1H), 2.00-1.67 (m, 12H).

[0155] 13C NMR (101MHz, CDCl3) δ195.84,157.70,143.66,139.03,133.54,131.05,123.03,120.53,57.68,39.37,39.18,38.71,37.13,33.11,29.89,28.45.

[0156] MS(ES-):m / z calc.for C19H21ClO3:332.1; found:331.2[MH]-.

[0157] (II) Preparation Examples 1-4. FDAG-1 to FDAG-4 mentioned below refer to the molecules having the structure of formula (I) prepared by using the FDAG compound in Examples 1-4, respectively.

[0158] (1) Synthesis of Example 1.

[0159]

[0160] FDAG compound (200 mg, 0.60 mmol) and 2-methyl-6-quinolinecarboxylic acid (112 mg, 0.60 mmol) were dissolved in ultra-dry tetrahydrofuran (THF, 0.3 mL). Catalyst indium trichloride (13 mg, 0.06 mmol) was added to the mixed solution and stirred. The reaction tube was sealed, and the mixture was heated at 120°C and stirred for 24 hours. After 24 hours of reaction, the reaction was monitored by TLC (petroleum ether: ethyl acetate = 50:50), and the reactants were almost gone. The mixture was cooled to room temperature and diluted with ethyl acetate (10 mL). Then, it was washed with saturated brine and dried over anhydrous sodium sulfate. Finally, it was purified by column chromatography (petroleum ether: ethyl acetate = 50:50) to obtain FDAG-1 compound (240 mg, 80% yield) as a dark yellow solid.

[0161] 1H NMR (400MHz, DMSO-d6) δ8.62(s,1H),8.54(d,J=8.5Hz,1H),8.22-8.13(m,2H),8.07(d,J=8.8Hz,1H),7.90(d,J=8.7Hz,1H),7.75(d,J=8. 0Hz,1H),7.55(d,J=16.3Hz,1H),6.86(d,J=7.9Hz,1H),3.24(s,3H),3.18(s,1H),2.05(s,1H),1.91(d,J=23.1Hz,4H),1.85-1.59(m,8H).

[0162] 13C NMR(101MHz,DMSO-d6)δ167.47,158.18,151.66,149.94,140.53,138.49,135.56,131.06,130.18,129.86,129.65,129.45,12 8.47,126.79,125.93,125.42,123.43,122.93,121.55,56.80,38.99,38.92,38.67,38.57,36.99,32.89,29.47,28.22,28.08.

[0163] HRMS(ES-):m / z calc.for C30H28ClNO4:501.2,found 500.2[MH]-.

[0164] (2) Synthesis of Example 2

[0165]

[0166] The FDAG compound (200 mg, 0.60 mmol) and 4-methylquinoline (80 μL, 0.60 mmol) were dissolved in ultra-dry tetrahydrofuran (THF, 0.3 mL). Catalyst indium trichloride (13 mg, 0.06 mmol) was added to the mixed solution and stirred. The reaction tube was sealed, and the mixture was heated at 120°C and stirred for 24 hours. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 80:20). After the reaction was completed, the mixture was cooled to room temperature and diluted with ethyl acetate (10 mL). Then, it was washed with saturated brine and dried over anhydrous sodium sulfate. Finally, it was purified by column chromatography (petroleum ether: ethyl acetate = 80:20) to obtain the light yellow solid FDAG-2 compound (233 mg, 85% yield).

[0167] 1H NMR(400MHz, DMSO-d6)δ9.83(s,1H),8.91(d,J=4.6Hz,1H),8.49(d,J=7.9Hz,1H),8.14-8.03(m,2H),7.91(d,J=8.0Hz,1H), 7.81(dd,J=14.9,8.8Hz,3H),7.67(s,1H),6.88(d,J=8.0Hz,1H),3.25(s,3H),3.20(s,1H),2.06(s,1H),1.98-1.65(m,13H).

[0168] 13C NMR(101MHz,DMSO-d6)δ28.09,28.23,29.45,32.91,37.01,38.58,38.69,38.90,38.97,56.72,117.28,122.87,123.33,12 4.16,124.54,125.65,126.24,127.07,129.59,129.73,129.91,130.02,135.36,140.57,142.87,148.77,150.79,151.38.

[0169] MS(Maldi-TOF):m / z calc.for C29H28ClNO2:457.2; found:458.2[M+H]+.

[0170] (3) Synthesis of Example 3

[0171]

[0172] The FDAG compound (200 mg, 0.60 mmol) and 1-methylisoquinoline (80 μL, 0.60 mmol) were dissolved in ultra-dry tetrahydrofuran (THF, 0.3 mL). Catalyst indium trichloride (13 mg, 0.06 mmol) was added to the mixed solution and stirred. The reaction tube was sealed, and the mixture was heated at 120°C and stirred for 24 hours. The reaction was monitored by TLC (petroleum ether: ethyl acetate = 80:20). After the reaction was completed, the mixture was cooled to room temperature and diluted with ethyl acetate (10 mL). Then, it was washed with saturated brine and dried over anhydrous sodium sulfate. Finally, it was purified by column chromatography (petroleum ether: ethyl acetate = 80:20) to obtain the FDAG-3 compound as a light yellow solid (197 mg, yield 72%).

[0173] 1H NMR (400MHz, CDCl3) δ8.58(d,J=5.6Hz,1H),8.37(d,J=9.1Hz,1H),8.19(s,2H),7.83(d,J=8.1Hz,1H),7.69(m,J=8.2,6.8,1.2Hz,1H),7.62(m,J=8 .3,6.9,1.4Hz,1H),7.58(d,J=5.1Hz,1H),7.54(d,J=8.0Hz,1H),6.88(d, J=7.9Hz,1H),3.34(s,3H),3.29(s,1H),2.18(s,1H),2.01-1.70(m,12H).

[0174] 13C NMR (101MHz, CDCl3) δ28.26,28.39,29.69,32.89,37.12,38.76,39.10,57.16,120.06,121.87,123.42,124.72,124.7 3,124.88,126.12,126.87,127.29,127.30,130.09,131.29,131.99,134.72,136.74,139.82,142.15,150.47,155.02.

[0175] MS(Maldi-TOF):m / z calc.for C29H28ClNO2:457.2; found:458.2[M+H]+.

[0176] (4) Synthesis of Example 4

[0177]

[0178] Dissolve the FDAG compound (200 mg, 0.60 mmol) and p-toluenesulfonic acid (11.4 mg, 0.06 mmol) in 5 mL of anhydrous ethanol. Add 2-aminobenzenethiol (75 mg, 0.60 mmol) to the mixture for reaction. The reaction was carried out at 80°C. Monitor the reaction using a spot plate. Once the starting material spot disappears, turn off the heat. After the reaction mixture cools to room temperature, add 5 mL of H2O to precipitate a solid. Filter and wash the mixture to obtain the white solid product, FDAG-4 (185 mg, 70% yield).

[0179] 1H NMR (400MHz, DMSO-d6) δ12.73(s,1H),8.23(d,J=8.3Hz,1H),8.14(d,J=8.1Hz,1H),7.98(d,J=8.1Hz,1H),7.61(t,J= 7.7Hz,1H),7.53(t,J=7.6Hz,1H),6.99(d,J=8.0Hz,1H),3.27(s,3H),3.21(s,1H),2.06(s,1H),1.99-1.63(m,12H).

[0180] 13C NMR(101MHz,DMSO-d6)δ28.24,28.41,29.72,32.97,37.10,38.59,38.63,39.07,39.26,57.30,116.89,121.6 3,122.35,122.38,123.37,125.74,125.98,127.02,132.25,132.64,139.06,139.45,151.35,154.10,168.45.

[0181] MS(Maldi-TOF):m / z calc.for C25H24ClNO2S:437.1; found:438.2[M+H]+.

[0182] Comparative Examples 1-3 are compounds having the following molecular structures:

[0183] Comparative Example 1:

[0184]

[0185] Comparative Example 2:

[0186]

[0187] Comparative Example 3:

[0188]

[0189] Among them, comparative example 1 is a compound without introducing an R3 group molecule, and the above compound 1f is taken as comparative example 1.

[0190] The preparation methods of Comparative Example 2 and Comparative Example 3 are as follows:

[0191] Preparation method of comparative example 2:

[0192]

[0193] FDAG compound (200mg, 0.60mmol), piperidine (88μL, 0.72mmol) and dicyanomethyl chromium (DCMC) (152mg, 0.72mmol) are dissolved in 12m1 ACN. The reaction mixture is stirred at 80°C for 1 hour and monitored by thin layer chromatography (Hex: EtOAc 70:30). After completion, the reaction mixture is diluted with ethyl acetate (100mL) and washed with a saturated solution of 0.5M HCl (500ml). The organic layer is separated, washed with brine, dried over Na2SO4, and evaporated under reduced pressure. After completion, the solvent is concentrated under reduced pressure and the product is purified by reversed-phase high performance liquid chromatography (ACN gradient in water). The product comparison 2 compound is an orange solid (260mg, yield 79%).

[0194] 1 H-NMR (400MHz, CDCl3) δ8.93(dd,J=8.4,1.2Hz,1H),7.85(d,J=16.1Hz,1H),7.76(m,2H),7.61(m,2H),7.51–7.41(m ,1H),7.12(d,J=16.1Hz,1H),6.92(s,1H),6.60(s,1H),3.25(s,3H),3.03(s,1H),2.07(s,1H),1.94–1.34(m,12H).

[0195] 13 C-NMR (400MHz, CDCl3) δ157.30,152.90,152.46,150.87,134.87,134.07,132.78,126.96,126.19,125.97,125.27,124.35,122.1 9,118.85,117.96,116.71,115.65,111.66,107.79,96.45,49.82,36.63,34.20,33.61,32.99,32.28,31.69,29.80,26.24,25.92.

[0196] MS(Maldi-TOF):m / z calc.for C32H27ClN2O3:523.03; found:522.17[MH]-.

[0197] Preparation method of comparative example 3:

[0198]

[0199] Take compound 1g (441mg, 0.14mmol) with the above structure, piperidine (28μL, 0.28mmol) and DCMC (29mg, 0.17mmol) and dissolve in 3mL ACN. The reaction mixture is refluxed and stirred for 1h and monitored by RP-HPLC. After completion, the reaction mixture is diluted with ethyl acetate (100ml) and washed with a saturated solution of 0.5M HCl (100mL). The organic layer is separated, washed with brine, dried over Na2SO4, and evaporated under reduced pressure. After completion, the solvent is concentrated under reduced pressure and the product is purified by preparative reverse phase high performance liquid chromatography (ACN gradient in water). The product comparison 3 compound is an orange solid (60mg, yield 76%).

[0200] 1 H-NMR (400MHz, DMSO-d6) δ8.72(d,J=8.3Hz,1H),8.30(t,J=7.8Hz,2H),7.92(t,J=7.8Hz,1H),7.81(d,J=16.2Hz,1H),7.64–7.48(m ,4H),6.95(s,1H),6.81(d,J=16.2Hz,1H),3.09(s,3H),3.05(s,1H),2.27–2.16(m,1H),1.95(d,J=15.4Hz,3H),1.80–1.45(m,9H).

[0201] 13 C-NMR(400MHz,DMSO-d6)δ168.37,158.91,153.38,152.48,138.29,136.29,136.13,128.80,126.84,125.41,124.91,123.43,121.55,1 20.01,119.51,119.10,117.72,116.42,112.30,107.09,95.70,55.48,50.24,36.39,34.48,33.64,32.11,31.00,29.56,26.07,25.79.

[0202] MS(Maldi-TOF):m / z calc.for C35H29ClN2O5:593.08; found:592.18[MH]-.

[0203] Optical performance tests were performed on Examples 1-4 and Comparative Examples 1-3:

[0204] The compounds of Examples 1-4 (FDAG-1-FDAG-4) were dissolved in dimethyl sulfoxide (DMSO) to prepare 1 mM solutions as stock solutions. 5% tetrabutylammonium fluoride (TBAF, 1 mM) was added to adjust the solution to alkalinity. A photosensitizer (SiPc, 3 μM) was then added to the solution (500 μM, 200 μL). The solution was excited with a 680 nm laser for 20 seconds, then the laser was turned off. Afterglow spectra and kinetic decay curves corresponding to different afterglow emission peaks for different molecules were collected. The afterglow decay curves for the long-afterglow molecules of Examples 1-4 were collected for 1 hour, 3 hours, 1.3 hours, and 2.5 hours, respectively, with an integration time of 0.5 seconds.

[0205] The compounds of Comparative Examples 1-3 were respectively dissolved in dimethyl sulfoxide (DMSO) to prepare 1 mM solutions as stock solutions. 5% tetrabutylammonium fluoride (TBAF 1 mM) was added to adjust the solution to alkalinity. Subsequently, a photosensitizer (SiPc, 3 μM) was added to the solution (500 μM, 200 μL). After excitation with a 680 nm laser for 20 s, the laser light source was turned off and the afterglow spectrum was collected. The absorption spectrum, fluorescence spectrum, and fluorescence quantum efficiency were also collected.

[0206] The specific test results are shown in Table 1, and combined with Figure 2(a)-Figure 2(d) as well as Figure 3(a)-Figure 3(d) The results show that the maximum absorption peak and maximum emission peak of Comparative Example 2 are 520nm and 690nm respectively, and the fluorescence quantum efficiency is almost zero; the maximum absorption peak and maximum emission peak of Comparative Example 3 are 550nm and 700nm respectively, and the fluorescence quantum efficiency is only 1.12%.

[0207] The absorption and fluorescence spectra of the long afterglow molecule of Example 1 (FDAG-1 compound) show that the maximum absorption peak and maximum emission peak of the molecule are 475 nm and 600 nm, respectively, and the corresponding molar extinction coefficient ε is 1.57×10 4 The maximum absorption peak and maximum emission peak of the long afterglow molecule of Example 2 (FDAG-2 compound) are 478 nm and 620 nm respectively, and the molar extinction coefficient ε is 2.32×10 4 The maximum absorption peak and maximum emission peak of the long afterglow molecule of Example 3 (FDAG-3 compound) are 477nm and 600nm, and the corresponding molar extinction coefficient ε is 2.31×10 4 The maximum absorption peak and maximum emission peak of the long afterglow molecule of Example 4 (FDAG-4 compound) are 424 nm and 472 nm, and the molar extinction coefficient ε is 1.38×10 4L / (mol cm). The maximum absorption peaks of the molecules in Examples 1-3 (FDAG-1-FDAG-3 compounds) are respectively at 475nm, 478nm, and 477nm, which is due to the twisted intramolecular charge-transfer (TICT) caused by the molecules modified with the EWG group. The maximum emission peaks of the molecules in Examples 1-3 (FDAG-1-FDAG-3 compounds) are respectively at 600nm, 620nm, and 600nm, with a large Stokes shift (125-142nm). At the same time, compared with the molecules to which the R3 group has not been introduced (the molecular structure possessed by the compound of Comparative Example 1), due to the increase in the degree of conjugation of the molecules in Examples 1-3 (FDAG-1-FDAG-3 compounds), the emission peak position moves significantly, red-shifting 140-160nm to the red light region. Furthermore, the formation of donor-acceptor electron pairs, electron cloud distribution, and conjugation effects also enhance the fluorescence quantum efficiency of the molecules in Examples 1-3 (FDAG-1-FDAG-3 compounds), reaching 35.21%, 30.24%, and 36.06%, respectively. Compared to the comparative example, the molecules in Examples 1-3 (FDAG-1-FDAG-3 compounds) achieve significantly improved fluorescence quantum efficiency within the same long-wavelength range.

[0208] In the long-lasting molecule of Example 4 (FDAG-4 compound), the benzothiazole group is modified as an electron acceptor at the ortho position of the phenolic hydroxyl group. The degree of conjugation is relatively small, so the emission wavelength does not change much. However, due to the formation of donor-acceptor electron pairs, the fluorescence quantum efficiency is further improved to 49.97%.

[0209] Although comparative examples 2 and 3 form donor-acceptor electron pairs and are fluorescent molecules of the donor-acceptor structure type, their luminous efficiency is still low.

[0210] In addition, the afterglow emission of the molecules in each example was evaluated after adding the photosensitizer SiPc. Figure 4(a)-Figure 4(d)The afterglow emission peak positions of the long afterglow molecules of Examples 1-4 (FDAG-1-FDAG-4 compounds) are at 600nm, 605nm, 590nm and 490nm, respectively. It can be observed that the long afterglow molecules of Examples 1-3 emit orange-red afterglow light; the long afterglow molecule of Example 4 (FDAG-4 compound) emits green afterglow light. By fitting the decay curve lifetime, the lifetime of the long afterglow molecule of Example 1 (FDAG-1 compound) is 2.83min, and the afterglow light decay image within 30min is collected. The afterglow lifetime of the long afterglow molecule of Example 2 (FDAG-2 compound) obtained by fitting is 12.6min, which is longer than that of the molecule of Example 1 (FDAG-1 compound), and the afterglow light decay image of the molecule within 2h is observed. The afterglow decay lifetime of the long afterglow molecule of Example 3 (FDAG-3 compound) is 4.75 min, which is longer than that of Example 1 (FDAG-1 compound) but slightly shorter than that of Example 2 (FDAG-2 compound). At the same time, an afterglow decay image of the long afterglow molecule of Example 3 (FDAG-3 compound) within 1 hour was captured. In comparison, the afterglow decay of the long afterglow molecule of Example 4 (FDAG-4 compound) is relatively slow, and the afterglow intensity does not undergo significant change within the 2.5 hours of testing. Examples 1-3 (FDAG-1-FDAG-3 compounds) of the present invention can achieve regulation of the afterglow lifetime within the same wavelength range, and the afterglow lifetime of Example 4 (FDAG-4 compound) reaches 2.5 hours, achieving regulation of the afterglow lifetime from minutes to hours.

[0211] Table 1: Performance test table

[0212]

[0213] Examples 5-10 are specific applications of Examples 1-4 (FDAG-1-FDAG-4 compounds).

[0214] Example 5: Application of long afterglow molecules in anti-counterfeiting encryption.

[0215] like Figure 5 As shown, a mixed solution of long-lasting molecules of Examples 1-3 (FDAG-1-FDAG-3 compounds) (3 μM SiPc, 1 mM long-lasting molecules of Examples 1-3) was added to different wells of the porous steel plate in a set pattern. After irradiation with a 680 nm LED light, afterglow photos were collected at different time points. Figure 6As shown, when the LED light is turned off, the number "8" can be read. After half an hour, the afterglow of the long-afterglow molecule of Example 1 essentially disappears, and only the number "7" can be read. After an hour, only the number "1" can be read, resulting in the true information number "1". This method is simple, easy to use, fast, and intuitive for digital anti-counterfeiting encryption, and has great application potential.

[0216] Example 6: Application of long-lasting glow molecules in biological imaging.

[0217] The long afterglow molecules of Example 1-4 (FDAG-1-FDAG-4 compounds) were dissolved in tetrahydrofuran to form an organic phase, and 1 g of polystyrene microspheres was redissolved in 50 mL of deionized water and fully sonicated to form a polystyrene sphere aqueous solution; 1 mL each of 20% sodium dodecylbenzenesulfonate and 10% ethylenediamine polyoxyethylene polyoxypropylene block polyether was added to the polystyrene sphere aqueous solution, and a 2 mL magnetic stirrer was placed and stirred to obtain solution A; the organic phase was quickly added to solution A, and then the temperature was gradually increased to 50° C. and stirred for 10 h; excess fluorescent dye was removed by centrifugation, and the mixture was washed twice with deionized water and ethanol, respectively, and stored in deionized water, protected from light at room temperature for later use. The fluorescent microspheres prepared by the above method were centrifuged, redissolved in 18 mL of BBS buffer (pH 7.4), and thoroughly sonicated to obtain a dispersion. 10 mg of EDC and 5 mg of NHSS were added to the dispersion, and the mixture was allowed to react at room temperature for 2 hours. After the reaction, the microspheres were washed by centrifugation, redissolved in 10 mL of BBS buffer (pH 7.4), and stored at 4°C until further use. The imaging reagents prepared above were used to label cancer cells, enabling long-term imaging without the need for continuous illumination. These molecules were modified by targeting specific receptors or proteins on the surface of cancer cells, enabling specific labeling of cancer cells. Results showed that these nanoparticles, with their long-lasting luminescence properties, could emit light for extended periods in vivo.

[0218] Example 7: Application of long afterglow molecules in quality control.

[0219] The long-lasting molecules of Examples 1-4 (FDAG-1-FDAG-4 compounds) can be used in quality control (QC) to detect and quantify trace amounts of biomarkers or chemicals. The following is a specific example of using long-lasting molecules in quality control to detect the presence and concentration of a specific protein.

[0220] Prepare a series of dilutions of the standard protein solution for constructing a calibration curve, and prepare protein samples of unknown concentration. Add the long-lasting molecule of Example 1 (FDAG-1 compound) to all standard protein solutions and test samples, ensuring that the amount of reporter molecule in each sample is the same. Use a UV light source to irradiate the solution containing PUC nanoparticles for a certain period of time to achieve an excited state. Turn off the light source and immediately use a spectrometer to detect the long-lasting luminescence, recording the luminescence intensity and time.

[0221] All standard protein solutions undergo the same excitation and detection process, and the data is recorded. A calibration curve is created based on the luminescence intensity of the standard solutions. The long-lasting luminescence intensity of the unknown sample is compared with the calibration curve to determine the protein concentration in the sample. Repeat this process for multiple batches of samples to assess the consistency and stability of protein quality. The results show that the measurement results of the standard protein solution display a curve in which the luminescence intensity increases with increasing protein concentration, and this curve should show a good linear relationship. Multiple measurements of the same sample show a high degree of reproducibility, with a standard deviation of 0.5%.

[0222] Example 8: Application of long afterglow molecules in in vitro diagnosis.

[0223] In this example, the long-lasting molecule of Example 2 (FDAG-2 compound) is used to detect biomarkers such as tumor markers.

[0224] The long afterglow molecule of Example 2 (FDAG-2 compound) is coated in polystyrene nanospheres by a swelling method, 10% BSA solution is added thereto, and an antibody is added, shaken and allowed to stand to prepare an antibody-coated nanosphere marker with the molecule of Example 2 (FDAG-2 compound). The prepared antibody-biomarker solution is centrifuged at low temperature, the supernatant is removed, and the solution is diluted for later use. An appropriate light source is used to excite the nanoparticles to a luminescent state. Within a certain period of time after stopping the excitation, the luminescence intensity is measured using a fluorescence spectrometer or similar instrument. The results show that the luminescence intensity of the luminescent microspheres should be proportional to the concentration of the biomarker in the sample.

[0225] Example 9: Application of long afterglow molecules in homogeneous detection.

[0226] A homogeneous detection method is developed using the long afterglow molecules of Examples 1-4 (FDAG-1-FDAG-4 compounds) for rapid and sensitive detection of biomarkers or other analytes. For example, the long afterglow molecules of Example 1 (FDAG-1 compound) are coated in polystyrene microspheres as acceptor microspheres in homogeneous detection. Among them, the long afterglow molecules of Example 1 (FDAG-1 compound) correspond to the detection of the inflammatory marker SAA. Take a quantitative SAA standard and dilute it to a series of different concentrations, such as 0μg / mL, 5μg / mL, 50μg / mL, 100μg / mL and 400μg / mL. Mix the prepared marker with these dilutions of different concentrations. Then, the silicon phthalocyanine (SiPc) photosensitizer is coated in polystyrene microspheres as donor microspheres for homogeneous detection. Among them, the donor microspheres are coupled with SAA-Ab1 antibodies, and the acceptor microspheres are coupled with SAA-Ab2 antibodies. They are blocked with bovine serum albumin, mixed in the dark by ultrasound, centrifuged and the supernatant is discarded, and then borate buffer (0.05M, pH 8.0) is added to the precipitate, and stored after ultrasonic dispersion. The sample to be tested and the diluent are added to the same reaction cup in sequence, stirred evenly to prepare an immune reaction system, and incubated for 5 minutes. The reaction cup is irradiated with excitation light, the excitation light is turned off, the long afterglow light signal value is collected, and the long afterglow light signal value is substituted into the above-mentioned long afterglow light signal-to-be-detected substance concentration standard curve to obtain the concentration value of the to-be-detected substance. The correlation between the measurement results and the Beckmann constant value is also analyzed. In addition, the sample containing SAA is diluted at multiple points with the calibration diluent, the concentration of the diluted sample is measured, and the recovery rate is calculated according to the dilution ratio. The results are shown in Tables 2 and 3.

[0227] Table 2: Test results of samples

[0228]

[0229] Table 3: Detection accuracy of the reagent kit prepared with the long-lasting luminescence molecules of Example 1 for samples

[0230]

[0231] Example 10: Application of long-lasting molecules in homogeneous detection

[0232] FDAG-1, FDAG-2, and FDAG-3 were dissolved in ethanol at a concentration of 1 mg / mL. Polystyrene microspheres (2 μm, 10 mg / mL) were dispersed in ethanol and treated in an ultrasonic bath for 10 minutes to ensure uniform dispersion. The solutions of long afterglow molecules were added to the polystyrene microsphere solutions, mixed thoroughly, and stirred at room temperature for 2 hours. The polystyrene microspheres coated with long afterglow molecules were collected by centrifugation (8000 rpm, 10 minutes) and washed 3 times with ethanol to remove unbound long afterglow molecules. Finally, the microspheres were resuspended in PBS for later use.

[0233] The polystyrene microspheres coated with long afterglow molecules were divided into three parts and washed with MES buffer (pH 6.0) respectively. EDC and NHS were dissolved in MES buffer to final concentrations of 0.2M and 0.05M, respectively, added to the microsphere suspension, and stirred at room temperature for 30 minutes to activate the carboxyl group. The activation solution was removed by centrifugation (8000rpm, 10 minutes) and PBS was added to wash 3 times. SAA-Ab1, CRP-Ab1 and PCT-Ab1 antibodies were dissolved in PBS (concentration of 1mg / mL) and added to three microsphere suspensions respectively, and incubated at room temperature for 2 hours. Unbound antibodies were removed by centrifugation (8000rpm, 10 minutes) and washed 3 times with PBS. Finally, the receptor microspheres were resuspended in PBS for standby use.

[0234] Silicon phthalocyanine was dissolved in ethanol at a concentration of 1 mg / mL. Polystyrene microspheres (2 μm, 10 mg / mL) were dispersed in ethanol and treated in an ultrasonic bath for 10 min to ensure uniform dispersion. The silicon phthalocyanine solution was added to the polystyrene microsphere solution, mixed thoroughly and stirred at room temperature for 2 hours. The polystyrene microspheres coated with silicon phthalocyanine were collected by centrifugation (8000 rpm, 10 minutes) and washed 3 times with ethanol to remove unbound silicon phthalocyanine. The microspheres were finally resuspended in PBS for later use. The polystyrene microspheres coated with silicon phthalocyanine were washed with MES buffer (pH 6.0) respectively, and SAA-Ab2, CRP-Ab2 and PCT-Ab2 antibodies were coupled to the donor microspheres according to the above-mentioned antibody coupling steps.

[0235] A whole blood sample to be tested was mixed with acceptor and donor microspheres, respectively. Afterglow signals from the mixture were collected using a photon detector. The collection time window was set from 1 to 20 minutes after the excitation was terminated to capture the afterglow signals of long-lasting molecules. The collected light signals were processed using a vector decomposition method, converting them into time-resolved light intensity decay curves. An exponential decay model was applied to fit the decay curves of each afterglow molecule, extracting their respective afterglow lifetime parameters. Based on the extracted lifetime parameters, the total signal was decomposed into independent signal components corresponding to FDAG-1, FDAG-2, and FDAG-3. Analysis revealed that significant afterglow signals were detected in the corresponding time dimensions after the acceptor microspheres conjugated with SAA-Ab1 and CRP-Ab1 antibodies in the whole blood sample bound to the corresponding donor microspheres (conjugated with SAA-Ab2 and CRP-Ab2 antibodies). This indicates that the sample contained high concentrations of SAA and CRP, as specific binding to the corresponding antibody-coupled microspheres occurs only in the presence of these inflammatory markers, resulting in an afterglow signal. In contrast, no significant afterglow signal was observed over the same timeframe when acceptor microspheres coupled to PCT-Ab1 antibodies bound to donor microspheres coupled to PCT-Ab2 antibodies. This suggests that PCT concentrations in the sample were low or absent, resulting in no specific binding or afterglow signal due to PCT.

[0236] This embodiment uses a time-resolved detection method based on the difference in afterglow lifetimes of different long afterglow molecules to achieve simultaneous detection of different markers in a whole blood sample. Since the wavelength ranges of the three molecules in this embodiment are the same, the same wavelength channel can be used to collect the afterglow signal.

[0237] Although the present invention has been illustrated and described with reference to certain preferred embodiments thereof, it should be understood by those skilled in the art that the above description is provided as a further detailed description of the present invention in conjunction with specific embodiments thereof, and that the specific implementation of the present invention is not limited to these descriptions. Those skilled in the art may make various changes in form and details, including simple deductions or substitutions, without departing from the spirit and scope of the present invention.

Claims

1. A long afterglow molecule, characterized in that The structure of the long afterglow molecule is shown in formula (I-d): Wherein, R4 is hydrogen, or R4 is a group or probe that can specifically recognize a specific substance or can produce a specific response to a specific substance.

2. A long afterglow molecule according to claim 1, characterized in that: The R4 is one of formula (I-4-1) to formula (I-4-11): Among them, in formula (I-4-1) to formula (I-4-11) It represents the linking site of formula (I-4-1) to formula (I-4-11) in formula (I-d).

3. The long-lasting molecule according to claim 1, wherein: The emission peak wavelength of the long afterglow molecule is 472 nm, and the fluorescence quantum efficiency is 49.97%.

4. The long-lasting molecule according to claim 1, wherein: The afterglow lifetime of the long afterglow molecule is 2.5h.

5. A method for preparing a long afterglow molecule according to any one of claims 1 to 4, characterized in that: The steps include: Preparation of mixed solution: raw material A and raw material B are dissolved in an organic solvent, wherein raw material A is a molecule having a structure as shown in formula (0), and raw material B can undergo a nucleophilic substitution reaction with raw material A to obtain a molecule having a structure as shown in formula (I-d). Adding a catalyst and stirring: stirring under heating and catalyst catalysis conditions; Post-treatment: cooling, diluting, washing, drying, or cooling, allowing the product to precipitate, filtering and washing; Wherein, R1 is methyl and R2 is chlorine.

6. The method for preparing a long afterglow molecule according to claim 5, characterized in that: The raw material B is 2-aminobenzenethiol, the catalyst is p-toluenesulfonic acid, and the organic solvent is anhydrous ethanol.

7. Use of a long afterglow molecule according to any one of claims 1 to 4, characterized in that: The long afterglow molecule is used for biological imaging, preparation of in vitro diagnostic reagents, sensor detection, quality control, anti-counterfeiting encryption and homogeneous detection.

8. A detection reagent for homogeneous detection, characterized in that The detection reagent comprises two or more long-lasting glow molecules of the structures shown in formula (I-a) to formula (I-c) and a long-lasting glow molecule of the structure shown in formula (I-d):

9. A detection kit, characterized in that The detection kit comprises two or more long-lasting glow molecules of the structures shown in formula (I-a) to formula (I-c) and a long-lasting glow molecule of the structure shown in formula (I-d):

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