Preparation method of quaternary ammonium salt fluorescent probe and application thereof in fingerprint development
Patent Information
- Application Number
- CN202610622215.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-08
- Publication Date
- 2026-09-25
AI Technical Summary
[0002]现有荧光类指纹显影材料普遍水溶性较差，通常需要借助有机溶剂、表面活性剂、盐类或聚合物助剂改善分散性，不仅操作繁琐、环境友好性低，还易造成纹线扩散、背景干扰增强等问题
[0062]本发明提供的一种具有聚集诱导发光（AIE）效应的季铵盐类荧光探针可在纯水中均匀分散，无需有机溶剂、表面活性剂、无机盐或高分子助剂，绿色环保，采用喷雾法即可对新鲜指纹、不同时长老化指纹、梯度高温胁迫指纹、梯度水泡胁迫指纹及重复按压指纹实现高对比度显现，水溶液稳定性优异，适用于多种非渗透性客体现场快速检测。
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Figure CN122810064A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of fluorescent probe technology, and in particular to a method for preparing a quaternary ammonium salt fluorescent probe and its application in fingerprint imaging. Background Technology
[0002] Existing fluorescent fingerprint developing materials generally have poor water solubility, typically requiring the use of organic solvents, surfactants, salts, or polymer additives to improve dispersibility. This not only makes the process cumbersome and environmentally unfriendly but also easily leads to problems such as ridge diffusion and enhanced background interference. Furthermore, most materials only show good results for fresh fingerprints, lacking applicability to fingerprints subjected to natural aging, high-temperature baking, water immersion, and repeated pressing. The developed solutions also exhibit poor stability, easily agglomerating and quenching fluorescence over long periods, failing to meet the needs of practical field investigations. In addition, traditional fluorescent probes often suffer from aggregation-quenching (ACQ) effects, where fluorescence weakens or even quenches in an aggregated state, further limiting their sensitivity and contrast in fingerprint development. Summary of the Invention
[0003] Based on the technical problems existing in the background art, the present invention proposes a method for preparing quaternary ammonium salt fluorescent probes and their application in fingerprint development.
[0004] The present invention proposes a method for preparing a quaternary ammonium salt fluorescent probe, comprising the following steps:
[0005] S1. Under a protective atmosphere, 4-bromotriphenylamine, 4'-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-[1,1'-biphenyl]-4-carboxaldehyde, K2CO3, Pd(PPh3)4, and solvent A are mixed and heated to react. After post-treatment, intermediate 1 is obtained.
[0006] S2. Under a protective atmosphere, intermediate 1, acetylglycine, sodium acetate and solvent B are mixed and heated to react. After post-treatment, intermediate 2 is obtained.
[0007] S3. Under a protective atmosphere, intermediate 2 is mixed with solvent C to obtain intermediate 2 solution. N,N-dimethylethylenediamine and K2CO3 are added to intermediate 2 solution, and the mixture is heated to react. After post-treatment, intermediate 3 is obtained.
[0008] S4. Mix intermediate 3 and solvent D under heating, add iodomethane, react, and after post-processing, obtain quaternary ammonium salt fluorescent probe.
[0009] Preferably, in S1, the molar ratio of 4-bromotriphenylamine, 4'-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-[1,1'-biphenyl]-4-carboxaldehyde, K2CO3, and Pd(PPh3)4 is (10-15):10:(20-30):0.5.
[0010] Preferably, in S1, solvent A is selected from one or more of tetrahydrofuran and water.
[0011] More preferably, the volume ratio of the tetrahydrofuran to water is (5-6):1.
[0012] Preferably, in S1, the heating reaction temperature is 50-70°C, and the heating reaction time is 6-8 hours.
[0013] Preferably, in S1, the heating reaction is monitored by thin-layer chromatography, and the eluent for thin-layer chromatography monitoring includes petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of (20-30):1.
[0014] Preferably, in S1, the post-processing includes cooling, pH adjustment, filtration, extraction, drying, concentration, and purification.
[0015] More preferably, the cooling temperature is 20-35°C.
[0016] More preferably, the pH adjustment is to adjust the pH to 6-7.
[0017] The purpose of adjusting the pH in this invention is to enable longer storage time at room temperature, and it can also improve the yield of column chromatography.
[0018] More preferably, the extractant used in the extraction is ethyl acetate.
[0019] More preferably, the purification is performed by silica gel column chromatography, using a flash column chromatography system manufactured by Jiangsu Ronglipu Scientific Instruments Co., Ltd., with silica gel powder of 200-300 mesh. The eluent for silica gel column chromatography purification includes petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of (20-30):1.
[0020] Step S1 of this invention involves screening reactant raw materials, proportions, and reaction conditions, resulting in low cost and low energy consumption. The intermediate 1 is an aldehyde derivative that can be used in organic synthesis and scientific research experiments and exhibits strong fluorescence.
[0021] Preferably, in S2, the molar ratio of intermediate 1, acetylglycine, and sodium acetate is 1:1:(1.2-1.3).
[0022] Controlling the molar ratio of intermediate 1, acetylglycine, and sodium acetate within a certain range helps to improve yield, reduce cost, and increase product purity.
[0023] Preferably, in S2, solvent B is acetic anhydride.
[0024] Preferably, in step S2, the heating reaction temperature is 100-120°C, and the heating reaction time is 3-4 hours.
[0025] Preferably, in S2, the post-processing includes pH adjustment, extraction, drying, concentration, and purification.
[0026] More preferably, the pH adjustment is to adjust the pH to 7-8.
[0027] More preferably, the extractant used in the extraction is ethyl acetate.
[0028] More preferably, the purification is carried out by silica gel column chromatography, the silica gel column is a flash column chromatography of Jiangsu Ronglipu Scientific Instruments Co., Ltd., the silica gel powder is 200-300 mesh, and the eluent for silica gel column chromatography purification includes petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of (20-30):1.
[0029] In this invention, intermediate 1 is prepared to obtain intermediate 2, which exhibits a red shift in fluorescence.
[0030] Preferably, in S3, solvent C is selected from one or more of ethanol and methanol.
[0031] Preferably, in S3, the molar ratio of intermediate 2, N,N-dimethylethylenediamine, and K2CO3 is 1:2:(2-3).
[0032] Controlling the molar ratio of intermediate 2, N,N-dimethylethylenediamine, and K2CO3 within a certain range helps to improve the reaction rate and product purity.
[0033] Preferably, in step S3, the heating reaction temperature is 70-80°C, and the heating reaction time is 8-12 hours.
[0034] Preferably, in step S3, the post-processing includes filtration and purification.
[0035] More preferably, the purification is carried out by silica gel column chromatography, the silica gel column is a flash column chromatography of Jiangsu Ronglipu Scientific Instruments Co., Ltd., the silica gel powder is 200-300 mesh, and the eluent for silica gel column chromatography purification includes dichloromethane and methanol, with a volume ratio of dichloromethane to methanol of (20-30):1.
[0036] Preferably, in S4, solvent D is selected from one or more of dichloromethane and diethyl ether.
[0037] Preferably, in S4, the molar ratio of intermediate 3 to iodomethane is 1:(26-27).
[0038] Preferably, in step S4, the heating and mixing temperature is 20-35°C, and the heating and mixing time is 24-48 hours.
[0039] Preferably, in step S4, the reaction temperature is 20-35°C and the reaction time is 24-48 hours.
[0040] Preferably, in step S4, the post-processing includes centrifugation, washing, and drying.
[0041] More preferably, the detergent used for washing is ether.
[0042] More preferably, the drying is vacuum drying, the drying temperature is 60-80℃, and the drying time is 6-8 hours.
[0043] Preferably, in S1, S2, and S3, the protective gas is selected from one or more of ammonia and argon.
[0044] A method for preparing a quaternary ammonium salt fluorescent probe, the synthetic route is as follows:
[0045]
[0046] A quaternary ammonium salt fluorescent probe, prepared by the above method, has the chemical structural formula shown in Formula 1:
[0047]
[0048] Formula 1.
[0049] Application of the above-mentioned quaternary ammonium salt fluorescent probe or the quaternary ammonium salt fluorescent probe prepared by the above-mentioned preparation method in fingerprint development.
[0050] Preferably, the application method includes: mixing the quaternary ammonium salt fluorescent probe and the solvent evenly to obtain a quaternary ammonium salt fluorescent probe solution; spraying the quaternary ammonium salt fluorescent probe solution onto a carrier containing latent fingerprints; observing the fluorescence and acquiring images under excitation light.
[0051] More preferably, the excitation light is 450 nm.
[0052] More preferably, the solvent is selected from one or more of water, 1,4-dioxane, sodium dodecyl sulfate solution, and sodium chloride solution.
[0053] More preferably, the solvent is water.
[0054] More preferably, the concentration of the quaternary ammonium salt fluorescent probe solution is 10-100 μM.
[0055] More preferably, the carrier is selected from one or more of the following: tin foil, stainless steel, tape, plastic, coin, mortar and pestle, and ceramic tile.
[0056] The application of the above-mentioned quaternary ammonium salt fluorescent probe or the quaternary ammonium salt fluorescent probe prepared by the above-mentioned method in inhibiting human liver cancer cells.
[0057] Preferably, the human liver cancer cells are selected from one or more of HEPG2 cells, Huh7 cells, and MHCC97 cells.
[0058] Preferably, the application method includes adding a solution containing a quaternary ammonium salt fluorescent probe to a culture medium containing human liver cancer cells to obtain a mixed solution, culturing it, and testing its cancer inhibition rate.
[0059] More preferably, the concentration of the quaternary ammonium salt fluorescent probe in the mixed solution is 6.25-200 µg / mL.
[0060] More preferably, the tumor inhibition rate is tested according to the MTT assay.
[0061] The beneficial effects of this invention are as follows:
[0062] The present invention provides a quaternary ammonium salt fluorescent probe with aggregation-induced emission (AIE) effect that can be uniformly dispersed in pure water without the need for organic solvents, surfactants, inorganic salts or polymeric additives. It is green and environmentally friendly. Using a spray method, it can achieve high-contrast display of fresh fingerprints, fingerprints aged for different durations, fingerprints under gradient high temperature stress, fingerprints under gradient water blister stress, and fingerprints under repeated pressure. It has excellent stability in aqueous solution and is suitable for rapid on-site detection of various non-permeable objects.
[0063] 1. A four-step synthesis method is adopted, which is mild and highly reproducible, and can stably prepare quaternary ammonium salt fluorescent probes with aggregation-induced emission (AIE) effect.
[0064] 2. It can be used with pure water system, without the need for organic solvents, surfactants and other additives, making it green, environmentally friendly and easy to operate; pure water is a good solvent for probes, which can ensure that the probes are evenly dispersed and avoid premature aggregation that affects the development effect.
[0065] 3. Relying on aggregation-induced emission properties, the fluorescence of the probe is significantly enhanced after aggregation in fingerprint grease. It has high development sensitivity and good contrast, and can achieve fingerprint development under multiple conditions such as gradient aging of 0-30 days, high temperature of 0-8h at 60℃, water immersion of 0-24h, and continuous repeated pressing of 5 times, making it suitable for a wide range of scenarios.
[0066] 4. 450 nm blue light excitation is safe and gentle, suitable for rapid on-site investigation.
[0067] 5. The pure water developer exhibits excellent stability after being stored at room temperature for 2 months, demonstrating strong practicality and potential for real-world applications in public security. Attached Figure Description
[0068] Figure 1 The image shows the ¹H NMR spectrum of intermediate 1 in Example 1 of this invention.
[0069] Figure 2 The image shows the ¹H NMR spectrum of intermediate 2 in Example 1 of this invention.
[0070] Figure 3 The image shows the ¹H NMR spectrum of intermediate 3 in Example 1 of this invention.
[0071] Figure 4 The image shows the ¹H NMR spectrum of the quaternary ammonium salt fluorescent probe of Example 1 of this invention.
[0072] Figure 5 The image shows the FT-IR spectrum of the quaternary ammonium salt fluorescent probe of Example 1 of this invention.
[0073] Figure 6 The fluorescence emission spectrum of the quaternary ammonium salt fluorescent probe of Example 1 of the present invention in a pure water system (excitation light 450 nm, voltage 750 V).
[0074] Figure 7 The fluorescence emission spectrum of the solid form of the quaternary ammonium salt fluorescent probe of Example 1 of the present invention (excitation light 450nm, 500V).
[0075] Figure 8 The fluorescence emission spectrum of the quaternary ammonium salt fluorescent probe of Example 1 of the present invention in the water-1,4-dioxane system (excitation light 450 nm, voltage 750 V).
[0076] Figure 9 The images show (A) of the quaternary ammonium salt fluorescent probe of Example 1 of the present invention under sunlight and (B) under 450 nm excitation.
[0077] Figure 10 The images show sunlight (A) and fluorescence (B) of the freshly prepared aqueous solution (1) and the solution (2) stored for 2 months of the quaternary ammonium salt fluorescent probe of Example 1 of the present invention.
[0078] Figure 11 This diagram illustrates the application of different concentrations of aqueous solutions of the quaternary ammonium salt fluorescent probe from Example 1 of this invention in fingerprint development.
[0079] Figure 12 The application of the aqueous solution of the quaternary ammonium salt fluorescent probe of Example 1 of the present invention in fingerprint development: fingerprint development effect diagram of gradient aging from 0 to 30 days.
[0080] Figure 13 The application of the aqueous solution of the quaternary ammonium salt fluorescent probe of Example 1 of the present invention in fingerprint development: fingerprint development effect under high temperature stress at 60℃ for 0-8h.
[0081] Figure 14 The application of the aqueous solution of the quaternary ammonium salt fluorescent probe of Example 1 of the present invention in fingerprint development: 0-24h blister stress fingerprint development effect diagram.
[0082] Figure 15 The application of the aqueous solution of the quaternary ammonium salt fluorescent probe of Example 1 of the present invention in fingerprint development: the fingerprint development effect after five consecutive repeated pressing.
[0083] Figure 16 The application of the aqueous solution of the quaternary ammonium salt fluorescent probe of Example 1 of the present invention in fingerprint development: fingerprint development effect diagrams of different matrices.
[0084] Figure 17 The application of the aqueous solution of the quaternary ammonium salt fluorescent probe of Example 1 of the present invention in fingerprint development: a comparison of fingerprint development of freshly prepared solution (A) and solution (B) stored for 2 months.
[0085] Figure 18 This is a diagram illustrating the effect of a high-concentration quaternary ammonium salt fluorescent probe solution in inhibiting human liver cancer cells, as shown in Example 1 of this invention. Detailed Implementation
[0086] The technical solution of the present invention will be described in detail through specific embodiments.
[0087] In the following examples and comparative examples, the specific information regarding the raw materials used is as follows:
[0088] HEPG2 cells: Shangen Bio (Catalog No.: SNL-083).
[0089] The silica gel column used for purification by silica gel column chromatography was a flash column chromatography column from Jiangsu Ronglipu Scientific Instruments Co., Ltd., and the silica gel powder was 200 mesh.
[0090] Unless otherwise specified, all materials and reagents used in the following examples and comparative examples are commercially available.
[0091] Example 1
[0092] A method for preparing a quaternary ammonium salt fluorescent probe includes the following steps:
[0093] Preparation of S1 and Intermediate 1
[0094] Under nitrogen protection, 4-bromotriphenylamine (3.24 g, 10.0 mmol), 4'-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-[1,1'-biphenyl]-4-carboxaldehyde (3.08 g, 10.0 mmol), K2CO3 (4.14 g, 30.0 mmol), and Pd(PPh3)4 (0.58 g, 0.5 mmol) were added sequentially to a 100 mL dry round-bottom flask. Then, 50 mL of tetrahydrofuran and 10 mL of ultrapure water (V(THF):V(H2O) = 5:1) were added. The mixture was heated to 70 °C with magnetic stirring and reacted for 8 h. The reaction progress was monitored by thin-layer chromatography (TLC) (eluent: petroleum ether / ethyl acetate = 30:1). After the reaction was completed and cooled to room temperature, the solution was adjusted to pH 6 with 1M HCl. The insoluble solids were removed by filtration, and the filtrate was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a pale yellow crude product. The crude product was purified by silica gel column chromatography (eluent: V(petroleum ether):V(ethyl acetate) = 30:1) to give 1, 2.55 g of a pale yellow-green solid intermediate, with a yield of 72%.
[0095] Preparation of S2 and intermediate 2
[0096] Acetylglycine (500 mg, 4.27 mmol), intermediate 1 (1.49 g, 4.27 mmol), sodium acetate (437 mg, 5.33 mmol), and acetic anhydride (6 mL) were added to a 100 mL round-bottom flask. The mixture was heated to 110 °C under nitrogen protection and stirred for 3.5 h. The pH was adjusted to 7. 20 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain an orange-yellow crude product. The crude product was purified by silica gel column chromatography (eluent: V(petroleum ether):V(ethyl acetate) = 20:1). After cooling to room temperature, a dark red precipitate formed. This precipitate was dried under vacuum at 60 °C overnight to obtain an orange-yellow solid intermediate 2, 1.11 g, in 50% yield.
[0097] Preparation of S3 and intermediate 3
[0098] To 15 mL of anhydrous ethanol solution of intermediate 2 (500 mg, 1.0 mmol), N,N-dimethylethylenediamine (217 μL, 2.0 mmol) and K₂CO₃ (276 mg, 2.0 mmol) were added, and the mixture was stirred at 80 °C for 12 h under nitrogen protection. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: V(CH₂Cl₂):V(CH₃OH) = 30:1) to give intermediate 3, a pale yellow solid, 250 mg, in 43% yield.
[0099] S4. Preparation of the target quaternary ammonium salt fluorescent probe
[0100] Intermediate 3 (300 mg, 0.52 mmol) was dissolved in 150 mL of dichloromethane and heated until completely dissolved. Iodomethane (0.87 mL, 13.9 mmol) was then added, and the mixture was stirred at room temperature for 48 h. After the reaction was complete, the crude product was collected by centrifugation, resuspended in diethyl ether, and washed twice by centrifugation (equivalent to one wash with dichloromethane and two washes with diethyl ether). The product was then dried under vacuum at 60 °C to obtain 300 mg of orange-red solid powder LFP-TPA, with a yield of 71%. The final product, a quaternary ammonium salt fluorescent probe exhibiting aggregation-induced emission (AIE) was obtained.
[0101] Comparative Example 1
[0102] The only difference between Comparative Example 1 and Example 1 is that the content of 4-bromotriphenylamine in step S1 is adjusted to 2.70 g and 8.33 mmol, while the rest is the same as in Example 1.
[0103] Comparative Example 2
[0104] The only difference between Comparative Example 2 and Example 1 is that the content of acetylglycine in step S2 is adjusted to 1.0g and 8.54mmol, while the rest is the same as in Example 1.
[0105] Comparative Example 3
[0106] The only difference between Comparative Example 3 and Example 1 is that N,N-dimethylethylenediamine (130 μL, 1.2 mmol) was used in step S3, while the rest was the same as in Example 1.
[0107] Comparative Example 4
[0108] The only difference between Comparative Example 4 and Example 1 is that in step S4, the resuspension and centrifugation washing twice with dichloromethane was changed to centrifugation washing twice with dichloromethane. The rest is the same as Example 1.
[0109] The yield and purity results of intermediates 1, 2, 3 and quaternary ammonium salt fluorescent probe products in the above examples and comparative examples are shown in Table 1.
[0110] Table 1
[0111]
[0112] As can be seen from the yield of the fluorescent probes prepared above, the fluorescent probes prepared by this invention through screening raw materials, controlling the ratio, and adjusting the reaction conditions have a higher yield. Dichloromethane can dissolve byproducts and unreacted substances, but it can also dissolve the target product, the fluorescent probe. The more times it is washed with dichloromethane, the greater the loss, but the purer it becomes, reaching over 99%. Ether is almost insoluble in the target product; washing with ether can reduce losses, but the purity can only reach 95%. The fluorescent probe is slightly soluble in dichloromethane and almost insoluble in ether. This invention, by washing with dichloromethane first and then with ether, ensures both high purity and high yield.
[0113] Intermediate 1, Intermediate 2, and Intermediate 3 prepared in Example 1 were all characterized by 1H NMR spectroscopy, and the test results are as follows: Figure 1-3 As can be seen, the spectrum is consistent with the corresponding structure, proving that the intermediates were successfully synthesized in each step. The quaternary ammonium salt fluorescent probe prepared in Example 1 was characterized by 1H NMR and FT-IR spectroscopy, and the test results are as follows. Figure 4-5 As shown, the characteristic proton signal and functional group absorption peaks are consistent with the molecular structure of the target quaternary ammonium salt; at the same time, combined with fluorescence spectroscopy, its aggregation-induced emission (AIE) characteristics are verified.
[0114] Application examples
[0115] The quaternary ammonium salt fluorescent probe prepared in Example 1 was added to a solvent and ultrasonically dispersed to obtain a quaternary ammonium salt fluorescent probe solution. The quaternary ammonium salt fluorescent probe solution was evenly and gently sprayed onto a carrier containing latent fingerprints, and the fluorescence was observed and images were acquired under 450 nm blue light excitation.
[0116] (1) The spectral properties of the quaternary ammonium salt fluorescent probe were tested:
[0117] Solvent system and fluorescence intensity changes:
[0118] ① When the solvent is pure water, pure water is a good solvent for quaternary ammonium salt fluorescent probes. In the quaternary ammonium salt fluorescent probe solution, the quaternary ammonium salt fluorescent probe is in a dispersed state, and the fluorescence intensity is relatively weak. Figure 6 As shown.
[0119] ② When the solvent is a mixture of water and 1,4-dioxane, 1,4-dioxane is a poor solvent for quaternary ammonium fluorescent probes. As the volume fraction of 1,4-dioxane increases in the quaternary ammonium fluorescent probe solution, the probes gradually aggregate, and the fluorescence intensity shows a regular change—when the volume fraction is 0-40%, the quaternary ammonium fluorescent probes are still mainly dispersed, and the fluorescence intensity gradually weakens; when the volume fraction is 40%-95%, the probes aggregate, and the fluorescence intensity increases. Above 85%, the aggregation-induced emission characteristics are fully demonstrated, indicating that the probes already have loose aggregates in pure water. Adding a small amount of poor solvent causes the aggregates to dissociate and recombine. Further increasing the proportion of poor solvent induces the formation of larger aggregates, such as... Figure 8 As shown.
[0120] ③ When the solvent is 1,4-dioxane, the quaternary ammonium salt fluorescent probe does not dissolve in 1,4-dioxane. Therefore, the solid form of the quaternary ammonium salt fluorescent probe is tested. Figure 7 As shown, due to the strong fluorescence of the solid, the excitation light was 450nm and 500V for testing.
[0121] In pure aqueous solutions, quaternary ammonium fluorescent probes are in a dispersed state with weak fluorescence intensity, which corresponds to the dispersed state characteristics of the AIE effect.
[0122] (2) The optical properties of the quaternary ammonium salt fluorescent probe were tested:
[0123] ①For example Figure 9 As shown, the solid morphology of the quaternary ammonium salt fluorescent probe is orange-red under sunlight and exhibits orange-yellow fluorescence under 450 nm blue light excitation; the fluorescence intensity is the strongest (reflecting the aggregation state characteristics of the AIE effect, the solid is in a fully aggregated state).
[0124] ②For example Figure 10 As shown, the quaternary ammonium salt fluorescent probe exhibits yellow-green fluorescence after dispersion in pure water, with relatively weak fluorescence intensity (dispersed state, corresponding to the AIE effect); it also shows good fluorescence stability.
[0125] A 60 μM quaternary ammonium salt fluorescent probe aqueous solution was stored at room temperature in a sealed environment for 2 months. Compared with the freshly prepared solution, under 450 nm blue light excitation, both the freshly prepared solution and the solution stored at room temperature in a sealed environment for 2 months showed a small amount of aggregation, and the amount of aggregation did not increase after 2 months of storage. This small amount of aggregation could not be removed by filtration through a 0.44 μm filter and did not affect the normal use of the solution. The fluorescence color and intensity of the two solutions did not show significant attenuation, and there was no obvious aggregation, precipitation or quenching phenomenon. The fingerprint development effect was basically the same. Figure 17 The results indicate that its aqueous solution has excellent stability and can be prepared in advance and stored for long-term use; at the same time, the amount of probe aggregation did not increase during storage, further verifying its dispersion stability in a pure water system (good solvent).
[0126] ③ For example Figure 11 As shown, the fingerprint papillary ridges exhibit bright yellow-green fluorescence with a clean background and clear ridges; the enhanced fluorescence originates from the accumulation of the probe in the fingerprint grease, fully utilizing the aggregation-induced emission effect.
[0127] (3) Testing the potential fingerprint development performance of the quaternary ammonium salt fluorescent probe:
[0128] The performance of a quaternary ammonium fluorescent probe solution with a concentration of 60 μM was prepared by reacting the quaternary ammonium fluorescent probe with pure water.
[0129] ① Gradient aging fingerprint (0-30 days)
[0130] After imprinting fingerprints on the surface of tin foil, the foil was left at room temperature in the dark for 0, 1, 3, 9, 15, and 30 days, respectively, before being sprayed with a quaternary ammonium salt fluorescent probe solution. Figure 12 The results show that fingerprints can be effectively developed within the 0-30 day aging range. As the aging time increases, the integrity of the ridges decreases slightly, but the overall outline remains clear and the main ridges are continuous. Fingerprints aged for 30 days still have good recognizability, indicating that the probe has excellent development ability for old fingerprints. At the same time, thanks to its aggregation-induced emission properties, even if the fingerprint residue decreases, the probe can still achieve fluorescence enhancement by aggregating in the residue, ensuring development sensitivity.
[0131] ② Gradient high temperature stress fingerprints (60℃, 0-8 h)
[0132] After fingerprinting, the fingerprint samples were heated at 60℃ for 0 h, 2 h, 4 h, 6 h, and 8 h, respectively, and then sprayed with quaternary ammonium salt fluorescent probe solution. For example... Figure 13 The results show that the probe can effectively develop fingerprints within the high-temperature treatment range of 0-8 hours. As the heating time increases, some fingerprint residue is lost, but the continuity of the ridges remains good. The overall ridges can still be clearly identified after 8 hours of high-temperature stress, demonstrating excellent resistance to high-temperature interference.
[0133] ③ Gradient bubbling stress fingerprints (0-24 h)
[0134] The objects from which fingerprints were taken were immersed in water for 0 h, 2 h, 4 h, 6 h, 8 h, and 24 h, respectively, and then sprayed with a quaternary ammonium salt fluorescent probe solution. For example... Figure 14 The results show that fingerprints can be displayed with high contrast under 0-24 h immersion conditions, with no diffusion or blurring of ridges and stable fluorescence signals. This indicates that the probe has good dispersibility in aqueous environment and strong resistance to moisture interference, making it suitable for water stains and humid environments.
[0135] ④ Press 5 times consecutively
[0136] In different areas of the tin foil substrate, the same finger was pressed five times consecutively to simulate the real-world scenario where fingerprint sweat / oil residue gradually fades after multiple contacts. Figure 15 The results showed that after development with the quaternary ammonium salt fluorescent probe solution, all five fingerprint samples were effectively illuminated: Fingerprints from the first and second presses exhibited continuous and complete ridges with clear details, and tertiary features (sweat pores) were discernible; fingerprints from the third press showed good ridge integrity, with a slight decrease in fluorescence intensity; fingerprints from the fourth and fifth presses, although the fluorescence intensity further weakened due to a significant reduction in sweat residue, the ridge outline and main flow direction were still clearly identifiable, demonstrating recognizability. These results indicate that the probe possesses high sensitivity for recognizing low-residue, weak-signal fingerprints on non-permeable surfaces such as tin foil, making it suitable for detecting trace decay after multiple contacts in complex environments.
[0137] ⑤ Multi-matrix applicability
[0138] like Figure 16 The probe can effectively reveal potential fingerprints on various common non-porous surfaces such as tin foil, stainless steel, plastic, and ceramic tiles, making it widely applicable.
[0139] (4) Testing the inhibitory performance of the quaternary ammonium fluorescent probe on human hepatocellular carcinoma cells: A solution containing the quaternary ammonium fluorescent probe was added to a culture medium containing human hepatocellular carcinoma cells (HEPG2 cells), resulting in final concentrations of the quaternary ammonium fluorescent probe in the culture medium of 200, 100, 50, 25, 12.5, and 6.25 µg / mL, respectively. The tumor inhibition rate of the quaternary ammonium fluorescent probe provided in this invention against HepG2 cells was detected using the MTT assay.
[0140] The solution containing the quaternary ammonium fluorescent probe consists of mixing 0.02 g of the quaternary ammonium fluorescent probe with 0.5 ml of DMSO to obtain a stock solution concentration of 40 mg / mL, which is then diluted with PBS.
[0141] Cell seeding procedure: ① Remove HEPG2 cells from a 37°C 5% CO2 incubator and observe cell growth under an inverted microscope. ② Discard the culture medium and wash twice with 1ml PBS. ③ Digest HEPG2 cells with 400-500ml trypsin and incubate at a constant temperature for 4-5 minutes. ④ Remove the culture dish and observe whether the cells have been digested. ⑤ Prepare an empty sterile tube, add 2ml of 1640 ppm and gently pipette the cells, transfer to a sterile tube, and centrifuge for 5 minutes at 800 rpm. ⑥ Discard the separation solution and add 1-2ml of... Mix 1640 by pipetting, and aspirate 100 μL to seed on a Newton-Bauer counting plate. ⑦ Count the cells under an inverted microscope, and take the average of the total number of cells in the four large squares (Note: about 200 cells per large square is appropriate). ⑧ ÷ 24 × 7 ÷ the average number of cells counted in the large squares, then add 7 minus the amount of 1640 just added, mix well, ⑨ seed the plate, add 100 μL of cell suspension to each well (make sure to mix well), add 100 μL of PBS around the outside of the plate, ⑩ label the seeded plate, and incubate at 37 degrees Celsius in a 5% CO2 incubator for 4 hours.
[0142] Absorbance measurement: Weigh 0.0075 g MTT, add 1.5 mL PBS solution, mix thoroughly by pipetting, and sonicate if necessary. Filter through a 0.22 µL microporous membrane and use the filtrate for subsequent experiments. First, mix 0.5% MTT solution with 1640 complete culture medium at a ratio of 1:4 to obtain 1640 complete culture medium containing 0.5% MTT solution. After culturing cells for 24 h, aspirate the 1640 complete culture medium from the wells, then add 100 µL of the above-mentioned 1640 complete culture medium containing 0.5% MTT solution, and continue culturing in a 37℃, 5% CO2 incubator for 4 h. Then, aspirate the liquid from the wells and add 150 µL PBS to each well. Shake at 400 r / min for 2 min on a microplate shaker to dissolve. Measure the absorbance (OD) value at 490 nm using a microplate spectrophotometer. The tumor inhibition rate of different drugs on HepG2 cells was calculated by the following formula: Tumor inhibition rate (%) = (absorbance 1 - absorbance 2) / absorbance 1 × 100%, Note: absorbance 1 is the absorbance value of the blank control group, and absorbance 2 is the absorbance value of the drug treatment.
[0143] from Figure 18 As can be seen, the toxicity of the quaternary ammonium salt fluorescent probe increases with the increase of its concentration, and it has the ability to inhibit human liver cancer cells.
[0144] In summary, the quaternary ammonium salt fluorescent probes provided by this invention can be uniformly dispersed in pure water without the need for organic solvents, surfactants, inorganic salts, or polymeric additives. Using a spray method, they can achieve high-contrast visualization of fresh fingerprints, fingerprints aged for different durations, fingerprints under gradient high-temperature stress, fingerprints under gradient blister stress, and fingerprints subjected to repeated pressing. The aqueous solution exhibits excellent stability, making them suitable for rapid on-site detection of various non-permeable objects. Furthermore, the quaternary ammonium salt fluorescent probes provided by this invention can also be used to inhibit human liver cancer cells, demonstrating a high inhibition rate.
[0145] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A method for preparing a quaternary ammonium salt fluorescent probe, characterized in that, Includes the following steps: S1. Under a protective atmosphere, 4-bromotriphenylamine, 4'-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-[1,1'-biphenyl]-4-carboxaldehyde, K2CO3, Pd(PPh3)4, and solvent A are mixed and heated to react. After post-treatment, intermediate 1 is obtained. S2. Under a protective atmosphere, intermediate 1, acetylglycine, sodium acetate and solvent B are mixed and heated to react. After post-treatment, intermediate 2 is obtained. S3. Under a protective atmosphere, intermediate 2 is mixed with solvent C to obtain intermediate 2 solution. N,N-dimethylethylenediamine and K2CO3 are added to intermediate 2 solution, and the mixture is heated to react. After post-treatment, intermediate 3 is obtained. S4. Mix intermediate 3 and solvent D under heating, add iodomethane, react, and after post-processing, obtain quaternary ammonium salt fluorescent probe.
2. The preparation method according to claim 1, characterized in that, In S1, the molar ratio of 4-bromotriphenylamine, 4'-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)-[1,1'-biphenyl]-4-carboxaldehyde, K2CO3, and Pd(PPh3)4 is 10:10:30:0.5; solvent A is selected from one or more of tetrahydrofuran and water; the heating temperature is 50-70℃, and the heating time is 6-8h; the heating reaction is monitored by thin-layer chromatography, and the eluents for thin-layer chromatography monitoring include petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of (20-30):
1.
3. The preparation method according to claim 1, characterized in that, In S1, the post-processing includes cooling, pH adjustment, filtration, extraction, drying, concentration, and purification; the cooling temperature is 20-35℃; the extraction solvent is ethyl acetate; the purification is silica gel column chromatography, and the eluent for silica gel column chromatography includes petroleum ether and ethyl acetate, with a volume ratio of (20-30):
1.
4. The preparation method according to claim 1, characterized in that, In S2, the molar ratio of intermediate 1, acetylglycine, and sodium acetate is 1:1:(1.2-1.3); solvent B is acetic anhydride; the heating temperature is 100-120℃, and the heating time is 3-4h.
5. The preparation method according to claim 1, characterized in that, In S2, the post-processing includes pH adjustment, extraction, drying, concentration, and purification; the pH adjustment is to adjust the pH to 7-8; the extraction solvent is ethyl acetate; the purification is silica gel column chromatography, and the eluent for silica gel column chromatography includes petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of (20-30):
1.
6. The preparation method according to claim 1, characterized in that, In S3, solvent C is selected from one or more of ethanol and methanol; the molar ratio of intermediate 2, N,N-dimethylethylenediamine, and K2CO3 is 1:2:(2-3); the heating temperature is 70-80℃, and the heating time is 8-12h.
7. The preparation method according to claim 1, characterized in that, In S4, solvent D is selected from one or more of dichloromethane and diethyl ether; the molar ratio of intermediate 3 to iodomethane is 1:(26-27); the heating and mixing temperature is 20-35℃, and the heating and mixing time is 24-48h; the reaction temperature is 20-35℃, and the reaction time is 24-48h; the post-treatment includes centrifugation, washing, and drying; the washing agent is diethyl ether.
8. A quaternary ammonium salt fluorescent probe, characterized in that, Prepared by the preparation method according to any one of claims 1-7, the chemical structural formula is shown in Formula 1: Formula 1.
9. The application of a quaternary ammonium salt fluorescent probe according to claim 8 or a quaternary ammonium salt fluorescent probe prepared by any one of claims 1-7 in fingerprint development.
10. The use of a quaternary ammonium salt fluorescent probe according to claim 8 or a quaternary ammonium salt fluorescent probe prepared by any one of claims 1-7 in inhibiting human liver cancer cells.