Dual-emission fluorescent nano-coassemblies, preparation method thereof and application thereof in ATP detection

Abstract

This invention discloses a dual-emission fluorescent nano-assembly, its preparation method, and its application in ATP detection. The co-assembly achieves the detection of adenosine triphosphate (ATP). This invention uses an amphiphilic TPE derivative as the energy donor and an amphiphilic imidazole compound as the anion acceptor to form a dual-emission fluorescent nano-assembly with sodium fluorescein. Significant energy transfer occurs, resulting in strong fluorescence from the sodium fluorescein. After sodium fluorescein is replaced by ATP, the donor regains strong fluorescence emission while the light from the sodium fluorescein is quenched, allowing for the fluorescence detection of ATP with a detection limit as low as 0.735 nM. This method is simple, sensitive, and enables rapid and accurate specific quantitative detection of ATP, and can be widely used in the biomedical field.

Description

Technical Field

[0001] This invention relates to the technical field of fluorescent probe preparation, and in particular to a dual-emission fluorescent nanocomposite, its preparation method, and its application in ATP detection. Background Technology

[0002] Adenosine triphosphate (ATP) is an essential energy storage currency within cells, participating in the regulation of cellular metabolic activities and biochemical pathways, including energy transfer, enzyme catalysis, and biosynthesis. ATP molecules also play a signal-mediating role in regulating neurotransmission, ion channels, and cell movement. Therefore, the synthesis and consumption of ATP occur continuously in organisms to coordinate signal transduction and maintain energy homeostasis. Blood ATP concentration levels serve as an important indicator of human health; some diseases, such as Parkinson's disease, Alzheimer's disease, cardiovascular disease, colitis, and malignant tumors, are closely related to abnormal fluctuations in ATP concentration. Considering the widespread impact of ATP on human health, efficient ATP detection is of great significance in health screening and disease diagnosis.

[0003] Therefore, rapid, sensitive, and specific detection of ATP is crucial. Various methods exist for determining ATP concentration, such as electrochemical methods, high-performance liquid chromatography (HPLC), and colorimetric methods; however, these methods suffer from low sensitivity, significant interference, and complex procedures. Therefore, a solution is needed to address these issues. Summary of the Invention

[0004] The purpose of this invention is to provide a dual-emission fluorescent nano-assembly, its preparation method, and its application in ATP detection. Using an amphiphilic TPE derivative as an energy donor and an amphiphilic imidazole compound as an anion acceptor, a dual-emission fluorescent nano-assembly is formed with sodium fluorescein. Significant energy transfer occurs, resulting in strong fluorescence from the sodium fluorescein. When the sodium fluorescein is replaced by the analyte, the energy donor resumes strong fluorescence emission while the light from the sodium fluorescein is quenched, thus enabling rapid and accurate qualitative and quantitative detection.

[0005] In a first aspect, the present invention provides a dual-emission fluorescent nanocomposite comprising an amphiphilic imidazole compound, sodium fluorescein, and an amphiphilic TPE derivative, wherein the amphiphilic imidazole compound is complexed with the sodium fluorescein, and the amphiphilic imidazole compound is assembled with the amphiphilic TPE derivative.

[0006] The structural formula of the amphiphilic imidazole compound is shown in Formula I:

[0007]

[0008] The structural formula of the amphiphilic TPE derivative is shown in Formula II:

[0009]

[0010] The present invention provides a dual-emission fluorescent nanocomposite by introducing polyethylene glycol chains and alkyl chains into an amphiphilic TPE derivative, thereby giving the amphiphilic TPE derivative good water solubility. At the same time, it introduces a dicharged imidazole salt and alkyl chains into an amphiphilic imidazole compound, thereby giving the amphiphilic imidazole compound good recognition ability for anions.

[0011] The detection principle of dual-emission fluorescent nano-assemblies is as follows: After an amphiphilic imidazole compound forms an assembly with sodium fluorescein, which has strong fluorescence emission, it is then co-assembled with an amphiphilic TPE derivative. Under the excitation light of the energy donor, the amphiphilic TPE derivative, energy transfer occurs, thereby quenching the fluorescence of the amphiphilic TPE derivative and enhancing the fluorescence of the energy acceptor, sodium fluorescein. When the analyte is added, it can replace the sodium fluorescein, thus interrupting the energy transfer phenomenon under the excitation light of the amphiphilic TPE derivative. As a result, the fluorescence of the amphiphilic TPE derivative is restored, and the fluorescence of the sodium fluorescein is quenched, thereby completing the qualitative detection of the analyte.

[0012] Optionally, the molar ratio of the amphiphilic imidazole compound, the sodium fluorescein, and the amphiphilic TPE derivative is 1:1:10.

[0013] Optionally, the preparation method of the amphiphilic imidazole compound includes the following steps:

[0014] In a solvent environment, isophthalic acid was reacted with 1-(3-aminopropyl)imidazolium under the first catalyst and dehydrating agent, and then the mixture was separated and purified to obtain compound one.

[0015] Compound II was obtained by reacting tetradecylamine with bromoacetyl bromide in a solvent and alkaline environment and then separating and purifying the mixture.

[0016] In a solvent environment, compound 1 and compound 2 were reacted under a second catalyst and then separated and purified to obtain an amphiphilic imidazole compound.

[0017] Optionally, the process of separating and purifying compound one after reacting isophthalic acid with 1-(3-aminopropyl)imidazole under the first catalyst and dehydrating agent includes the following steps:

[0018] The first mixture is prepared by dissolving isophthalic acid, 1-(3-aminopropyl)imidazolium, the first catalyst, and the dehydrating agent in the first solvent.

[0019] After refluxing the first mixture at 75-80℃ for 12-15 hours, compound one was obtained by separation and purification.

[0020] Optionally, during the process of dissolving isophthalic acid, 1-(3-aminopropyl)imidazole, the first catalyst, and the dehydrating agent in the first solvent, the molar ratio of isophthalic acid, 1-(3-aminopropyl)imidazole, the first catalyst, and the dehydrating agent is 1∶2∶(1.5-2)∶(1.5-2).

[0021] Optionally, the process of reacting tetradecylamine with bromoacetyl bromide and then separating and purifying the resulting compound II in a solvent or alkaline environment includes the following steps:

[0022] The first alkaline accelerator, tetradecylamine, and bromoacetyl bromide are dissolved in the second solvent to prepare the second mixture;

[0023] The second mixture was stirred at 20-30℃ for 4-5 hours, and then separated and purified to obtain compound two.

[0024] Optionally, during the process of dissolving the first alkaline promoter, tetradecylamine, and bromoacetyl bromide in the second solvent, the molar ratio of the first alkaline promoter, the tetradecylamine, and the bromoacetyl bromide is (1-2):1:(1.2-1.5).

[0025] Optionally, the process of separating and purifying the amphiphilic imidazole compound after reacting compound one and compound two under a second catalyst includes the following steps:

[0026] Compound 1, Compound 2, and the second catalyst were dissolved in a third solvent to prepare a third mixture;

[0027] After refluxing the third mixture at 75-85℃ for 8-10 hours, the amphiphilic imidazole compound was obtained by separation and purification.

[0028] Optionally, during the process of dissolving compound one, compound two, and the second catalyst in the third solvent, the molar ratio of compound one, compound two, and the second catalyst is 1:(2.2-2.6):(0.8-1).

[0029] Optionally, the preparation reaction formula for the amphiphilic imidazole compound is shown below:

[0030]

[0031] Optionally, the preparation method of the amphiphilic TPE derivative includes the following steps:

[0032] Compound 3 was obtained by reacting raw material 1 with hexadecyl bromide in a solvent and alkaline environment and then separating and purifying the reaction. The raw material 1 was [1-(4-bromophenyl)-2,2-di(4-hydroxyphenyl)]ethylene.

[0033] Compound 3 was reacted with p-hydroxyphenylboronic acid in a solvent and alkaline environment under a third catalyst, and then purified to obtain compound 4.

[0034] In a solvent and alkaline environment, compound four was reacted with raw material two and then separated and purified to obtain an amphiphilic TPE derivative; the raw material two was 2-methoxy polyethyl p-toluenesulfonic acid.

[0035] Optionally, in the process of reacting raw material one with hexadecyl bromide and then separating and purifying it to obtain compound three in a solvent or alkaline environment, the following steps are included:

[0036] The second alkaline accelerator, raw material one, and hexadecyl bromide are dissolved in the fourth solvent to prepare the fourth mixture;

[0037] After refluxing the fourth mixture at 85-90℃ for 16-20 hours, compound three was obtained by separation and purification.

[0038] Optionally, during the process of dissolving the second alkaline accelerator, the first raw material, and the hexadecyl bromide in the fourth solvent, the molar ratio of the second alkaline accelerator, the first raw material, and the hexadecyl bromide is (1-2):1:(2.2-3.0).

[0039] Optionally, in the process of reacting compound three with p-hydroxyphenylboronic acid under a third catalyst in a solvent and alkaline environment to separate and purify compound four, the following steps are included:

[0040] The fifth mixture was prepared by dissolving the third catalyst, compound three, p-hydroxyphenylboronic acid, and the third basic promoter in the fifth solvent;

[0041] After reacting the fifth mixture at 85-90℃ for 23-25 ​​hours, compound four was obtained by separation and purification.

[0042] Optionally, during the process of dissolving the third catalyst, compound three, p-hydroxyphenylboronic acid, and the third basic promoter in the fifth solvent, the molar ratio of the third catalyst, compound three, p-hydroxyphenylboronic acid, and the third basic promoter is (0.1-0.5):1:(1.5-2):(1-2).

[0043] Optionally, the process of reacting compound four with starting material two and then separating and purifying the amphiphilic TPE derivative in a solvent or alkaline environment includes the following steps:

[0044] The sixth mixture is prepared by dissolving the fourth alkaline accelerator, compound four, and raw material two in the sixth solvent;

[0045] After refluxing the sixth mixture at 75-80℃ for 23-25 ​​hours, the amphiphilic TPE derivative was obtained by separation and purification.

[0046] Optionally, during the process of dissolving the fourth alkaline accelerator, compound four, and raw material two in the sixth solvent, the molar ratio of the fourth alkaline accelerator, compound four, and raw material two is (1-2):1:(1.2-1.5).

[0047] Optionally, the preparation reaction formula for the amphiphilic TPE derivative is as follows:

[0048]

[0049] Secondly, the present invention also provides a method for preparing any of the above-mentioned optional dual-emission fluorescent nanocomposites, comprising the following steps:

[0050] The assembly mother liquor was prepared by dissolving sodium fluorescein and an amphiphilic imidazole compound in a first polar solvent;

[0051] After dissolving the amphiphilic TPE derivative in a second polar solvent, it is blended with the assembly mother liquor to obtain an assembly solution containing the dual-emission fluorescent nano-assemblies.

[0052] Optionally, the assembly solution includes 4 μmol / L sodium fluorescein, 4 μmol / L amphiphilic imidazole compound, and 40 μmol / L amphiphilic TPE derivative.

[0053] Thirdly, the present invention also provides the application of any of the above-mentioned optional dual-emission fluorescent nanocomposites in ATP detection, which can be used for qualitative and quantitative detection of adenosine triphosphate. Attached Figure Description

[0054] Figure 1 A flowchart illustrating a method for preparing a dual-emission fluorescent nanocomposite according to an embodiment of the present invention;

[0055] Figure 2 The hydrogen spectrum of the amphiphilic imidazole compound prepared in Example 1 of this invention;

[0056] Figure 3 The carbon spectrum of the amphiphilic imidazole compound prepared in Example 1 of this invention;

[0057] Figure 4 The 1H NMR spectrum of the amphiphilic TPE derivative prepared in Example 2 of this invention;

[0058] Figure 5 The carbon spectrum of the amphiphilic TPE derivative prepared in Example 2 of this invention;

[0059] Figure 6 The UV fluorescence normalization plots of the amphiphilic TPE derivative prepared in Example 2 of this invention as an energy donor and sodium fluorescein as an energy acceptor are shown.

[0060] Figure 7 This is a particle size distribution diagram of the assembly formed by the amphiphilic imidazole compound and sodium fluorescein prepared in Example 1 of this invention.

[0061] Figure 8 The selective fluorescence spectrum of the assembly of the amphiphilic imidazole compound prepared in Example 1 of this invention and sodium fluorescein for adenosine triphosphate (ATP) and other ions.

[0062] Figure 9 The diagram shows the energy transfer when sodium fluorescein acts as an energy acceptor in the amphiphilic TPE derivative prepared in Example 2 of this invention as an energy donor assembly.

[0063] Figure 10 This is a particle size distribution diagram of the dual-emission fluorescent nanocomposites prepared in Example 1 of this invention.

[0064] Figure 11 The graph shows the fluorescence response of the dual-emission fluorescent nanocomposite prepared in Example 1 of this invention to different concentrations of adenosine triphosphate.

[0065] Figure 12 The image shows the fluorescence intensity curve of the dual-emission fluorescent nanocomposite prepared in Example 1 of this invention for adenosine triphosphate in the range of 4-16 μmol / L. Detailed Implementation

[0066] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art. The terms "comprising" and similar expressions used herein mean that the element or object preceding the word covers the element or object listed after the word and its equivalents, but does not exclude other elements or objects.

[0067] This invention provides a dual-emission fluorescent nanocomposite comprising an amphiphilic imidazole compound, sodium fluorescein (UD), and an amphiphilic TPE derivative, wherein the amphiphilic imidazole compound is complexed with sodium fluorescein (UD), and the amphiphilic imidazole compound is assembled with the amphiphilic TPE derivative, thereby forming a single dual-emission fluorescent nanocomposite.

[0068] Specifically, the structural formula of the amphiphilic imidazole compound is shown in Formula I:

[0069]

[0070] Specifically, the structural formula of the amphiphilic TPE derivative is shown in Formula II:

[0071]

[0072] In some embodiments, the molar ratio of the amphiphilic imidazole compound, sodium fluorescein (UD), and the amphiphilic TPE derivative in the dual-emission fluorescent nanocomposite is 1:1:10.

[0073] Specifically, the absorption peak of sodium fluorescein overlaps well with the emission peak of amphiphilic TPE derivatives, thus sodium fluorescein can serve as an energy donor for amphiphilic TPE derivatives.

[0074] In fact, see Figure 1 This invention also provides a method for preparing an amphiphilic imidazole compound, comprising the following steps:

[0075] S1. Preparation of compound one: In a solvent environment, isophthalic acid and 1-(3-aminopropyl)imidazolium were reacted and then purified to obtain compound one.

[0076] S2. Preparation of compound two: Compound two was obtained by reacting tetradecylamine with bromoacetyl bromide in a solvent and alkaline environment, followed by separation and purification.

[0077] S3. Preparation of amphiphilic imidazole compounds: Compound 1 and Compound 2 were reacted in a solvent environment under a second catalyst and then separated and purified to obtain amphiphilic imidazole compounds.

[0078] Specifically, in the preparation of amphiphilic imidazole compounds, the execution order of steps S1 and S2 can be interchanged in any way. For example, steps S1 and S2 can be executed simultaneously, or step S2 can be executed first and then step S1 can be executed.

[0079] In some embodiments, step S1 includes the following sub-steps:

[0080] S11. Dissolve isophthalic acid, 1-(3-aminopropyl)imidazolium, the first catalyst, and the dehydrating agent in the first solvent to prepare the first mixture;

[0081] S12. After refluxing the first mixture at 75-80℃ for 12-15 hours, the mixture is separated and purified to obtain compound one.

[0082] Specifically, in step S11, the molar ratio of isophthalic acid, 1-(3-aminopropyl)imidazolium, the first catalyst, and the dehydrating agent is 1:2:(1.5-2):(1.5-2).

[0083] Specifically, in step S11, the dehydrating agent may be at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dicyclohexylcarbodiimide, and diisopropylcarbodiimide.

[0084] Specifically, in step S11, the first catalyst may be 4-dimethylaminopyridine.

[0085] In fact, the reaction that occurs during step S1 is as follows:

[0086]

[0087] In some embodiments, step S2 includes the following sub-steps:

[0088] S21. Dissolve the first alkaline accelerator, tetradecylamine, and bromoacetyl bromide in the second solvent to prepare the second mixture;

[0089] S22. After stirring the second mixture at 20-30℃ for 4-5 hours, the mixture is separated and purified to obtain compound two.

[0090] Specifically, in step S21, the molar ratio of the first alkaline promoter, tetradecylamine, and bromoacetyl bromide is (1-2):1:(1.2-1.5).

[0091] Specifically, in step S21, the first alkaline promoter can be an inorganic base, specifically potassium carbonate.

[0092] Specifically, the reaction that occurs during the execution of S2 is shown below:

[0093]

[0094] In some embodiments, step S3 includes the following sub-steps:

[0095] S31. Compound 1, Compound 2, and the second catalyst are dissolved in a third solvent to prepare a third mixture;

[0096] S32. After refluxing the third mixture at 75-85℃ for 8-10 hours, the amphiphilic imidazole compound is obtained by separation and purification.

[0097] Specifically, in step S31, the molar ratio of compound one, compound two, and the second catalyst is 1:(2.2-2.6):(0.8-1).

[0098] Specifically, in step S31, the second catalyst can be potassium iodide.

[0099] Specifically, the reaction that occurs during the execution of S3 is shown below:

[0100]

[0101] In fact, see Figure 1 This invention also provides a method for preparing an amphiphilic TPE derivative, comprising the following steps:

[0102] S4. Preparation of Compound 3: Compound 3 is obtained by reacting raw material 1 with hexadecyl bromide in a solvent and alkaline environment, followed by separation and purification; the raw material 1 is [1-(4-bromophenyl)-2,2-di(4-hydroxyphenyl)]ethylene;

[0103] S5. Preparation of compound four: Compound three was reacted with p-hydroxyphenylboronic acid in a solvent and alkaline environment under a third catalyst, and then the mixture was separated and purified to obtain compound four.

[0104] S6. Preparation of amphiphilic TPE derivative: Compound 4 is reacted with raw material 2 in a solvent and alkaline environment, and then separated and purified to obtain amphiphilic TPE derivative; the raw material 2 is 2-methoxy polyethylene p-toluenesulfonic acid.

[0105] Specifically, when performing the separation and purification processes in steps S1, S2, S3, S4, S5, and S5, the selected separation and purification processes include common chemical separation and purification techniques, including but not limited to one or more of vacuum filtration, washing, drying, extraction, filtration, drying, and column chromatography, which can be used in an adaptive combination depending on the scenario.

[0106] Specifically, during the separation and purification process in steps S1, S2, S3, S4, S5, and S5, if an extraction operation is performed, the reagent used for extraction can be at least one of ethyl acetate, dichloromethane, petroleum ether, and methanol.

[0107] Specifically, the solvent environment selected in steps S1, S2, S3, S4, S5, and S5 can be a solvent commonly used in organic chemical reactions, including but not limited to one of dichloromethane, tetrahydrofuran, water, and acetonitrile. In practice, the solvent environment should be capable of dissolving the raw materials or products used in the reaction process, and the solvent itself should not undergo a chemical reaction.

[0108] Specifically, step S4 includes the following sub-steps:

[0109] S41. The second alkaline accelerator, raw material one and hexadecyl bromide are dissolved in the fourth solvent to prepare the fourth mixture;

[0110] S44. After refluxing the fourth mixture at 85-90℃ for 16-20h, the mixture is separated and purified to obtain compound three.

[0111] Specifically, in step S41, the molar ratio of the second alkaline accelerator, raw material one, and hexadecyl bromide is (1-2):1:(2.2-3.0).

[0112] Specifically, in step S41, the second alkaline promoter can be an inorganic base, specifically potassium carbonate.

[0113] Specifically, in step S41, the selected raw material can be synthesized using the following reaction formula:

[0114]

[0115] Specifically, the reaction that occurs during step S4 is shown below:

[0116]

[0117] Specifically, step S5 includes the following sub-steps:

[0118] S51. The third catalyst, compound three, p-hydroxyphenylboronic acid, and the third basic promoter are dissolved in the fifth solvent to prepare the fifth mixture;

[0119] S52. After reacting the fifth mixture at 85-90℃ for 23-25h, the mixture is separated and purified to obtain compound four.

[0120] Specifically, in step S51, the third alkaline promoter can be an inorganic base, specifically potassium carbonate.

[0121] Specifically, in step S51, the third catalyst can be tetra(triphenylphosphine)palladium.

[0122] Specifically, in step S51, the molar ratio of the third catalyst, compound III, p-hydroxyphenylboronic acid, and the third basic promoter is (0.1-0.5):1:(1.5-2):(1-2).

[0123] Specifically, the reaction that occurs during step S5 is shown below:

[0124]

[0125] In some embodiments, step S6 includes the following sub-steps:

[0126] S61. Dissolve the fourth alkaline accelerator, compound four, and raw material two in the sixth solvent to prepare the sixth mixture;

[0127] S62. After refluxing the sixth mixture at 75-80℃ for 23-25 ​​hours, the amphiphilic TPE derivative is obtained by separation and purification.

[0128] Specifically, in step S61, the fourth alkaline promoter can be an inorganic base, specifically potassium carbonate.

[0129] Specifically, in step S61, the molar ratio of the fourth alkaline promoter, compound four, and raw material two is (1-2):1:(1.2-1.5).

[0130] Specifically, the reaction that occurs during step S6 is shown below:

[0131]

[0132] In fact, see Figure 1 This invention also provides a method for preparing a dual-emission fluorescent nanocomposite, comprising the following steps:

[0133] An assembly solution was prepared by dissolving sodium fluorescein and an amphiphilic imidazole compound in a first polar solvent.

[0134] An amphiphilic TPE derivative was dissolved in a second polar solvent and then blended with the assembly solution to obtain an assembly solution containing a dual-emission fluorescent nano-assembly.

[0135] Preparation Example 1

[0136] Example 1 of this preparation provides a method for preparing an amphiphilic imidazole compound, comprising the following steps:

[0137] Y1. Preparation of Compound 1:

[0138] Y11. The first precursor solution was prepared by dissolving 1 g (6 mmol) of isophthalic acid and 1.5 mL (12 mmol) of 1-(3-aminopropyl)imidazol in 35 mL of acetonitrile.

[0139] Y12. Under a nitrogen atmosphere, 1.86 g (9 mmol) of dicyclohexylcarbodiimide and 1.10 g (9 mmol) of 4-dimethylaminopyridine were dissolved in the first precursor solution to prepare the first mixture.

[0140] Y13. The first reflux reaction solution is obtained by refluxing the first mixture at 80°C for 15 hours.

[0141] Y14. Add 30 mL of distilled water and 60 mL of chloroform to the first reflux reaction solution for extraction. After extraction, evaporate the solvent to dryness and collect the first crude residue. Purify the first crude residue by silica gel column chromatography (dichloromethane / methanol = 50:1, V / V) to obtain 2.0 g of compound one, with a calculated yield of 87.7%.

[0142] Y2, Preparation of Compound Two:

[0143] Y21. Dissolve 1 g (4.70 mmol) of tetradecylamine, 0.60 mL (7.00 mmol) of bromoacetyl bromide and 0.65 g (4.70 mmol) of K2CO3 in 20 mL of dichloromethane to prepare a second mixture.

[0144] Y22. Under a nitrogen atmosphere, the second mixture was stirred at room temperature (25°C) for 5 hours to obtain the first reaction solution. The solvent was evaporated and the second crude residue was collected. The second crude residue was purified by silica gel column chromatography (dichloromethane / petroleum ether = 1:1, V / V) to obtain 1.22 g of compound II, with a calculated yield of 78.2%.

[0145] Y3. Preparation of amphiphilic imidazole compounds:

[0146] Y31. Dissolve 1 g (3 mmol) of compound I, 0.156 g (1.36 mmol) of compound II and 0.225 g (1.36 mmol) of potassium iodide in 35 mL of acetonitrile to prepare a third mixture;

[0147] Y32. The third mixture was refluxed at 85°C for 10 hours to obtain the second reflux reaction solution.

[0148] Y33. After adding 30 mL of distilled water to the second reflux reaction solution, 80 mL of dichloromethane was added for extraction. The organic solvent was evaporated under vacuum and the third crude residue was collected. The third crude residue was purified by silica gel column chromatography (dichloromethane / methanol = 50:2, V / V) to obtain 1.05 g of amphiphilic imidazole compound, with a calculated yield of 74.1%.

[0149] See Figure 2 and Figure 3 The amphiphilic imidazole compound prepared in Preparation Example 1 was analyzed using a nuclear magnetic resonance instrument (Varian instrument 400MHz). The results are as follows:

[0150] 1 H NMR (400MHz, Chloroform-d) δ9.88 (s, 2H), 8.86 (s, 1H), 8.56 (q, J=4.0, 2.8Hz, 4H), 8.14 (d, J=7.7Hz, 2H), 7.53 (d, J=7.5Hz, 3H), 7.31 (s, 2H), 5.27 (d, J =14.4Hz, 4H), 4.44 (t, J = 6.4Hz, 4H), 3.48 (q, J = 5.5Hz, 4H), 3.22 (q, J = 7.2H z, 4H), 2.35 (q, J=5.9Hz, 4H), 1.23 (d, J=4.5Hz, 48H), 0.86 (t, J=6.7Hz, 6H).

[0151] 13 C NMR (101MHz, Chloroform-d) δ167.23, 163.94, 137.77, 133.31, 131.83, 129.09, 124.77, 123.98, 120.88, 51.8 6, 47.54, 40.16, 35.84, 31.89, 29.68, 29.66, 29.63, 29.58, 29.33, 29.27, 29.17, 29.04, 27.07, 22.66, 14.09.

[0152] Preparation Example 2

[0153] Example 2 of this preparation provides a method for preparing an amphiphilic TPE derivative, comprising the following steps:

[0154] D1. Preparation of Compound Three:

[0155] D11. Dissolve 1.0 g (2.26 mmol) of [1-(4-bromophenyl)-2,2-di(4-hydroxyphenyl)]ethylene, 1.5 mL (4.972 mmol) of hexadecyl bromide and 0.31 g (2.26 mmol) of K2CO3 in 20 mL of acetonitrile to prepare the fourth mixture.

[0156] D12. The third reflux reaction solution was prepared by refluxing the fourth mixture at 80°C for 24 hours.

[0157] D13. The solvent of the third reflux reaction solution was evaporated to collect the fourth crude residue. The fourth crude residue was purified by silica gel column chromatography (dichloromethane / petroleum ether = 1:25, V / V) to obtain 1.50 g of compound III, and the yield was calculated to be 75%.

[0158] D2. Preparation of Compound Four:

[0159] D21. Dissolve 1.0 g (1.12 mmol) of compound III, 0.31 g (2.24 mmol) of p-hydroxyphenylboronic acid, 0.31 g (2.24 mmol) of K2CO3, and 0.13 g (0.112 mmol) of tetra(triphenylphosphine)palladium in a mixed solution of 20 mL of dichloromethane and 20 mL of water to prepare the fifth mixture.

[0160] D22. After refluxing the fifth mixture at 90°C for 24 hours, the fourth reflux reaction solution is obtained.

[0161] D23. After adding 30 mL of distilled water to the fourth reflux reaction solution, 80 mL of dichloromethane was added for extraction. The organic solvent was evaporated under vacuum and the fifth crude residue was collected. The fifth crude residue was purified by silica gel column chromatography (petroleum ether / dichloromethane = 1:1, V / V) to obtain 0.81 g of compound four, and the yield was calculated to be 79%.

[0162] D3. Preparation of amphiphilic TPE derivatives:

[0163] D31. Dissolve 0.35 g (0.387 mmol) of compound IV, 0.53 g (0.58 mmol) of 2-methoxy polyethylene p-toluenesulfonic acid, and 0.053 g (0.387 mmol) of K2CO3 in 25 mL of acetonitrile to prepare the sixth mixture.

[0164] D32. After refluxing the sixth mixture at 90°C for 24 hours, the fifth reflux reaction solution is obtained.

[0165] D33. After adding 40 mL of distilled water to the fifth reflux reaction solution, 120 mL of dichloromethane was added for extraction. The organic solvent was evaporated under vacuum and the sixth crude residue was collected. The sixth crude residue was purified by silica gel column chromatography (dichloromethane / methanol = 40:1, V / V) to obtain 0.47 g of amphiphilic TPE derivative, and the yield was calculated to be 71%.

[0166] See Figure 4 and Figure 5 The amphiphilic TPE derivative prepared in Preparation Example 2 was characterized using a nuclear magnetic resonance instrument (Varian instrument 400MHz), and the characterization data are as follows:

[0167] 1H NMR (400MHz, Chloroform-d) δ7.56-7.38 (m, 2H), 7.28 (d, J=8.3Hz, 2H), 7.15-6.98 (m, 7H), 6.98-6.87 (m, 7H), 6.61 (dd, J=8.8, 7.3Hz, 3H), 4.13 (dt, J=9. 4, 4.9Hz, 3H), 3.85 (q, J=4.1, 2.7Hz, 6H), 3.65-3.62 (m, 64H), 3.54 (dd, J=5. 7, 3.6Hz, 3H), 1.84-1.64 (m, 4H), 1.52-1.13 (m, 56H), 0.87 (t, J=6.7Hz, 6H).

[0168] 13C NMR (101MHz, Chloroform-d) δ158.13, 157.63, 142.91, 140.01, 132.57, 131.76, 131.45, 127.73, 127.64, 125.65, 114.75, 113.51, 113 .42, 71.88, 70.78, 70.57, 70.51, 69.72, 67.75, 67.40, 59.02, 31.90, 29.67, 29.64, 29.58, 29.55, 29.41, 29.35, 29.29, 22.68, 14.13.

[0169] Example 1

[0170] This embodiment 1 provides a method for preparing a dual-emission fluorescent nanocomposite, including the following steps:

[0171] F0. Preparation of stock solution: Dissolve sodium fluorescein (UD) in methanol to prepare a solution with a concentration of 2×10⁻⁶. -3 UD stock solution of mol / L; dissolve the amphiphilic imidazole compound prepared in Example 1 in dimethyl sulfoxide to prepare a concentration of 2×10 mol / L. -3 A stock solution of an amphiphilic imidazole compound at a concentration of mol / L; the amphiphilic TPE derivative was dissolved in dimethyl sulfoxide to prepare a concentration of 2 × 10⁻⁶ mol / L. - 3 mol / L amphiphilic TPE derivative stock solution;

[0172] F1. Preparation of assembly solution: The assembly mother solution is prepared by mixing the UD mother solution and the amphiphilic imidazole compound mother solution in equal volumes. The assembly mother solution is diluted with HEPES buffer at pH 7.4 to an assembly solution with UD concentration of 4 μmol / L and amphiphilic imidazole compound concentration of 4 μmol / L.

[0173] F2. Preparation of dual-emission fluorescent nanocomposites: The amphiphilic TPE derivative stock solution was diluted to a concentration of 40 μmol / L using HEPES buffer solution at pH 7.4, and then mixed with an equal volume of the assembly solution to obtain an assembly solution containing the dual-emission fluorescent nanocomposites; wherein the molar ratio of the amphiphilic TPE derivative, the amphiphilic imidazole compound and UD in the assembly solution was 10:1:1.

[0174] Performance Characterization

[0175] 1. Fluorescence UV analysis of amphiphilic TPE derivatives and sodium fluorescein (UD):

[0176] The amphiphilic TPE derivative prepared in Preparation Example 2 was dissolved in dimethyl sulfoxide to prepare a concentration of 4 × 10⁻⁶. -3 The stock solution was prepared by diluting the stock solution with HEPES buffer at pH 7.4 to prepare a 20 μmol / L amphiphilic TPE derivative test solution 1.

[0177] Sodium fluorescein (UD) was dissolved in methanol to prepare a concentration of 4 × 10⁻⁶. -3 The stock solution was prepared by diluting the stock solution with HEPES buffer at pH 7.4 to prepare UD test solution with a concentration of 20 μmol / L.

[0178] The fluorescence intensity and UV absorbance of the amphiphilic TPE derivative test solution 1 and UD test solution 1 were measured, and the results are as follows: Figure 6 As shown.

[0179] 2. Selectivity testing and particle size determination of assemblies formed by amphiphilic imidazole compounds and sodium fluorescein (UD):

[0180] Y0. The amphiphilic imidazole compound prepared in Preparation Example 1 was dissolved in dimethyl sulfoxide to prepare a concentration of 2 × 10⁻⁶. -3 A stock solution of an amphiphilic imidazole compound at a concentration of mol / L; sodium fluorescein (UD) was dissolved in methanol to prepare a solution with a concentration of 2 × 10⁻⁶ mol / L. - 3 UD stock solution of mol / L;

[0181] Y1. Equal volumes of the amphiphilic imidazole compound stock solution from Y0 and the UD stock solution from Y0 were mixed and diluted with HEPES buffer at pH 7.4 to an assembly solution where both the amphiphilic imidazole compound and UD had a concentration of 2 μmol / L. The particle size range of the assemblies in the assembly solution was then determined as follows: Figure 7 As shown;

[0182] Y2. Adenosine triphosphate and other ions were added to the assembly solution in Y1, and the changes in fluorescence intensity were measured. The results are as follows: Figure 8 As shown; specifically, the other ions are 10 eq of ADP, AMP, GTP, UTP, TTP, PPi, Pi, and Cl. - ,Br - ACO - CO3 2- SO4 2- .

[0183] 3. Energy transfer test between amphiphilic TPE derivatives and sodium fluorescein (UD):

[0184] U1. Mix equal volumes of the amphiphilic imidazole compound stock solution in Y0 and the UD stock solution in Y0, and then dilute with HEPES buffer at H=7.4 to an assembly titration solution with a concentration of 4 μmol / L for both the amphiphilic imidazole compound and UD.

[0185] U2. The amphiphilic TPE derivative prepared in Preparation Example 2 was dissolved in dimethyl sulfoxide to prepare a concentration of 2×10⁻⁶. -3 The amphiphilic TPE derivative stock solution was prepared and diluted with HEPES buffer at pH 7.4 to a test solution with an amphiphilic TPE derivative concentration of 40 μmol / L.

[0186] U3. The assembly titrant from U1 is added dropwise to the test solution in U2, and the changes in fluorescence intensity of the test solution at 490 nm and 520 nm are measured during the addition process. The results are as follows: Figure 9 As shown.

[0187] 4. Particle size determination of the dual-emission fluorescent nanoassemblies prepared in Example 1 and their response to adenosine triphosphate:

[0188] The particle size of the dual-emission fluorescent nano-assemblies in the assembly solution prepared in Example 1 was measured, and the results are as follows: Figure 10 As shown;

[0189] ATP solution was added dropwise to the assembly solution prepared in Example 1, and the change in fluorescence intensity of the test solution during the addition process was measured, as well as the fluorescence droplets at 520 nm. The results are as follows: Figure 11 As shown, the relationship between ATP concentration and fluorescence intensity during titration is plotted, as follows. Figure 12 As shown.

[0190] Results Analysis

[0191] See Figure 6 It can be seen that the emission peak of the amphiphilic TPE derivative overlaps well with the absorption peak of sodium fluorescein (UD), satisfying the energy transfer conditions. Therefore, UD can be used as an energy acceptor for the amphiphilic TPE derivative.

[0192] See Figure 7 It can be seen that the particle size of the assembly formed by the amphiphilic imidazole compound and UD is 320 nm.

[0193] See Figure 8It was observed that after the ATP solution reacted with the assembly solution, the corresponding fluorescence intensity recovered to 5416 a.u.; however, when other ions were mixed with the assembly solution, the fluorescence intensity did not change significantly. This indicates that the assembly formed by the amphiphilic imidazole compound and UD can specifically recognize ATP. Therefore, the co-assembly formed by the assembly and the amphiphilic TPE derivative can specifically recognize ATP and can be used to detect ATP concentration.

[0194] See Figure 9 It was observed that as the amphiphilic TPE derivative solution was gradually added to the assembly titrant to form a dual-emission fluorescent nano-co-assembly, the fluorescence intensity of the amphiphilic TPE derivative at 490 nm gradually decreased, while the fluorescence intensity of UD gradually increased at 520 nm. When the molar ratio of the amphiphilic TPE derivative to the assembly reached 10:1, the fluorescence at 490 nm quenched from 2459 a.u. to 1173 a.u., and the fluorescence at 520 nm increased from 0 a.u. to 6530 a.u. Therefore, this indicates that energy transfer can occur between the amphiphilic TPE derivative and UD, which can be used for amplification and detection of ATP signals.

[0195] See Figure 10 It can be seen that the particle size of the dual-emission fluorescent co-assembly formed by the assembly of the assembly and the amphiphilic TPE derivative is 884 nm.

[0196] See Figure 11 It can be seen that as ATP is gradually added to the assembly solution, the fluorescence intensity of the amphiphilic TPE derivative at 490 nm gradually recovers, while the fluorescence intensity of UD at 520 nm gradually weakens. When the amount of ATP added reaches 8 eq, the fluorescence of the amphiphilic TPE derivative recovers from 881 a.u. to 1241 a.u., while the fluorescence of UD is quenched from 6480 a.u. to 2336 a.u.

[0197] See Figure 12 It can be seen that when the concentration of ATP is in the range of 4-16 μmol / L, the fluorescence intensity has a good linear relationship with the concentration of ATP, and thus it can be used for quantitative detection of ATP.

[0198] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A dual-emission fluorescent nanocomposite, characterized in that, The mixture includes an amphiphilic imidazole compound, sodium fluorescein, and an amphiphilic TPE derivative, wherein the amphiphilic imidazole compound is complexed with the sodium fluorescein, the amphiphilic imidazole compound is assembled with the amphiphilic TPE derivative, and the molar ratio of the amphiphilic imidazole compound, the sodium fluorescein, and the amphiphilic TPE derivative is 1:1:

10. The structural formula of the amphiphilic imidazole compound is shown in Formula I: ； The structural formula of the amphiphilic TPE derivative is shown in Formula II: 。 2. A method for preparing the dual-emission fluorescent nanocomposite as described in claim 1, characterized in that, Includes the following steps: An assembly solution was prepared by dissolving sodium fluorescein and an amphiphilic imidazole compound in a first polar solvent. An amphiphilic TPE derivative was dissolved in a second polar solvent and then blended with the assembly solution to obtain an assembly solution containing a dual-emission fluorescent nano-assembly.

3. The preparation method according to claim 2, characterized in that, The assembly solution includes 4 μmol / L sodium fluorescein, 4 μmol / L amphiphilic imidazole compound, and 40 μmol / L amphiphilic TPE derivative.

4. The preparation method according to claim 2, characterized in that, The preparation method of the amphiphilic imidazole compound includes the following steps: In a solvent environment, isophthalic acid was reacted with 1-(3-aminopropyl)imidazolium under the first catalyst and dehydrating agent, and then the mixture was separated and purified to obtain compound one. Compound II was obtained by reacting tetradecylamine with bromoacetyl bromide in a solvent and alkaline environment and then separating and purifying the mixture. In a solvent environment, compound 1 and compound 2 were reacted under a second catalyst and then separated and purified to obtain an amphiphilic imidazole compound; The reaction formula for the preparation of the amphiphilic imidazole compound is shown below: 。 5. The preparation method according to claim 4, characterized in that, The process of separating and purifying compound one after reacting isophthalic acid with 1-(3-aminopropyl)imidazolium under the first catalyst and dehydrating agent includes the following steps: A first mixture was prepared by dissolving isophthalic acid, 1-(3-aminopropyl)imidazolium, a first catalyst, and a dehydrating agent in a first solvent at a molar ratio of 1:2:(1.5-2):(1.5-2). After refluxing the first mixture at 75-80℃ for 12-15 hours, compound one was obtained by separation and purification.

6. The preparation method according to claim 4, characterized in that, The process of reacting tetradecylamine with bromoacetyl bromide in a solvent and alkaline environment, followed by separation and purification to obtain compound two, includes the following steps: A second mixture was prepared by dissolving a first alkaline accelerator, tetradecylamine, and bromoacetyl bromide in a second solvent at a molar ratio of (1-2):1:(1.2-1.5). The second mixture was stirred at 20-30℃ for 4-5 hours, and then separated and purified to obtain compound two.

7. The preparation method according to claim 4, characterized in that, The process of separating and purifying the amphiphilic imidazole compound after reacting compound 1 and compound 2 under a second catalyst includes the following steps: A third mixture was prepared by dissolving compound one, compound two, and the second catalyst in a molar ratio of 1:(2.2-2.6):(0.8-1) in a third solvent; After refluxing the third mixture at 75-85℃ for 8-10 hours, the amphiphilic imidazole compound was obtained by separation and purification.

8. The preparation method according to claim 2, characterized in that, The preparation method of the amphiphilic TPE derivative includes the following steps: Compound 3 was obtained by reacting raw material 1 with hexadecyl bromide in a solvent and alkaline environment and then separating and purifying the reaction. The raw material 1 was [1-(4-bromophenyl)-2,2-di(4-hydroxyphenyl)]ethylene. Compound 3 was reacted with p-hydroxyphenylboronic acid in a solvent and alkaline environment under a third catalyst, and then purified to obtain compound 4. In a solvent and alkaline environment, compound four was reacted with raw material two and then separated and purified to obtain an amphiphilic TPE derivative; the raw material two was 2-methoxy polyethyl p-toluenesulfonic acid. The preparation reaction formula of the amphiphilic TPE derivative is shown below: 。 9. The preparation method according to claim 8, characterized in that, In the process of reacting raw material one with hexadecyl bromide and then separating and purifying it to obtain compound three in a solvent and alkaline environment, the following steps are included: A fourth mixture was prepared by dissolving a second alkaline accelerator, a first raw material, and a hexadecyl bromide in a fourth solvent at a molar ratio of (1-2):1:(2.2-3.0); After refluxing the fourth mixture at 85-90℃ for 16-20 hours, compound three was obtained by separation and purification.

10. The preparation method according to claim 8, characterized in that, In the process of reacting compound three with p-hydroxyphenylboronic acid under a third catalyst in a solvent and alkaline environment to obtain compound four, the following steps are included: The fifth mixture was prepared by dissolving the third catalyst, compound III, p-hydroxyphenylboronic acid, and the third basic promoter in the fifth solvent at a molar ratio of (0.1-0.5):1:(1.5-2):(1-2). After reacting the fifth mixture at 85-90℃ for 23-25 ​​hours, compound four was obtained by separation and purification.

11. The preparation method according to claim 8, characterized in that, In the process of reacting compound four with raw material two and then separating and purifying the amphiphilic TPE derivative in a solvent and alkaline environment, the following steps are included: The sixth mixture was prepared by dissolving the fourth alkaline accelerator, compound four, and raw material two in the sixth solvent at a molar ratio of (1-2):1:(1.2-1.5). After refluxing the sixth mixture at 75-80℃ for 23-25 ​​hours, the amphiphilic TPE derivative was obtained by separation and purification.

12. An application of the dual-emission fluorescent nanoassembly of claim 1 in ATP detection, characterized in that, The dual-emission fluorescent nanoassembly can be used for qualitative and quantitative detection of adenosine triphosphate.

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