Naphthalimide formaldehyde fluorescent probe and preparation method and application thereof

Abstract

This invention discloses a naphthalimide-based formaldehyde fluorescent probe, its preparation method, and its applications. The synthesis method includes the following steps: mixing 4-(4-ethoxycarbonylbenzene)-N-(2-aminoethoxy)ethanolnaphthalimide, an aqueous solution of hydrazine hydrate, and methanol; reacting at 60-120°C for 12-24 hours under stirring; cooling to room temperature; filtering under reduced pressure; washing the filter cake 2-3 times with methanol; and vacuum drying to obtain a white substance, which is the naphthalimide-based formaldehyde fluorescent probe. The naphthalimide-based formaldehyde fluorescent probe of this invention, when applied to the detection of formaldehyde content in water, exhibits advantages such as rapid detection, small error, and more accurate results. It can also be used for fluorescence imaging of formaldehyde in live cells.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule fluorescent probe technology, specifically relating to a naphthalimide formaldehyde fluorescent probe, its preparation method, and its application. Background Technology

[0002] Formaldehyde is an important industrial raw material with wide applications in chemical engineering, wood processing, textiles, and preservation. It has a strong binding capacity to proteins and DNA and is widely considered a potent carcinogen. Excessive formaldehyde levels in the human body can lead to heart disease, Alzheimer's disease, cancer, diabetes, and various neurological disorders. Therefore, developing simple, low-cost, highly selective, and highly sensitive formaldehyde detection methods is essential.

[0003] Currently, common methods for detecting formaldehyde include radiometric assays, high-performance liquid chromatography (HPLC), mass spectrometry (MS), and colorimetry. However, these traditional methods all have some drawbacks, such as expensive equipment, cumbersome and time-consuming detection processes, potential irreparable damage to the sample, and inability to perform in-situ detection and imaging of intracellular formaldehyde levels. In contrast, the use of fluorescent probes for formaldehyde detection offers advantages such as low cost, high sensitivity and selectivity, and high spatiotemporal resolution, making it a hot research topic.

[0004] 1,8-Naphthalimide is a classic fluorescent dye with good photostability, a large Stokes shift, tunable photophysical properties, and ease of functionalization. Based on this fluorophore, a series of formaldehyde fluorescent probes have been developed. For example:

[0005] CN106008342A discloses a 1,8-naphthylimide formaldehyde probe (Na-Lyso) with lysosomal targeting capability. This probe uses 1,8-naphthylimide as the fluorophore, morpholine as the lysosomal targeting group, and hydrazine as the formaldehyde recognition site. Through a PET mechanism, formaldehyde is detected in PBS (10 mM, containing 5% DMSO). The detection system contains 5% DMSO as a co-solvent, and the response time is approximately 30 min. The probe fluorescence intensity shows a good linear relationship with formaldehyde concentration in the range of 0-100 μM, and the detection limit is approximately 5.02 μM. This probe can be used for formaldehyde detection in cellular lysosomes.

[0006] CN111793029A discloses a naphthimide-based fluorescent probe (Na) for formaldehyde, composed of 1,8-naphthimide modified with 2-ethoxyethanol and hydrazine. Using 1,8-naphthimide as the fluorophore and hydrazine as the formaldehyde recognition site, the probe achieves formaldehyde detection in PBS (10 mM, pH 7.4) via a PET mechanism, with a response time of approximately 32 min. The probe fluorescence intensity shows a good linear relationship with formaldehyde concentration in the range of 0-400 μM, with a detection limit of approximately 0.76 μM. This probe can be used for trace detection of free formaldehyde in fur products.

[0007] CN109134371A discloses a naphthalimide-based formaldehyde ratio probe (CDs-NA). This probe consists of a 6-aminohexanoic acid-modified naphthalimide structure linked to a carbon dot surface. Through a PET mechanism, it achieves ratio detection of formaldehyde in PBS (10 mM, pH 7.4), with a response time of approximately 30 min, a linear detection range of 0-160 μM, and a detection limit of approximately 0.338 μM. This probe can be used for formaldehyde detection in aquatic environments and within cells.

[0008] In summary, the reported naphthalimide-based fluorescent probes for formaldehyde still suffer from problems such as poor water solubility and long response time, making it difficult to achieve rapid detection of formaldehyde in pure aqueous solutions. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a naphthalimide-based formaldehyde fluorescent probe, which features fast response, high selectivity, and high sensitivity, and can be used for rapid detection of formaldehyde content in different water qualities and foods, as well as formaldehyde fluorescence imaging in live cells using fluorescence methods.

[0010] Another object of the present invention is to provide a method for synthesizing the above-mentioned naphthalimide formaldehyde fluorescent probe.

[0011] The objective of this invention is achieved through the following technical solution.

[0012] A naphthalimide-based formaldehyde fluorescent probe (NIPh) has the following structural formula:

[0013]

[0014] The synthesis method of the above-mentioned naphthalimide formaldehyde fluorescent probe includes the following steps:

[0015] Step 1: 4-bromo-1,8-naphthalenedicarboxylic anhydride, 2-(2-aminoethoxy)ethanol, and ethanol are mixed and reacted at 80-100℃ for 2-10 h under stirring. After cooling to room temperature, the ethanol is removed by rotary evaporation under reduced pressure. Dichloromethane is added, and the mixture is washed with water to remove inorganic salts dissolved in dichloromethane. The organic phase obtained after washing with water is dried to remove water, and then evaporated to dryness. The crude product is purified by column chromatography and then dried under vacuum to obtain a yellow substance, which is 4-bromo-N-(2-aminoethoxy)ethanol-naphthalimide. The ratio of 4-bromo-1,8-naphthalenedicarboxylic anhydride to 2-(2-aminoethoxy)ethanol is (1.0-1.5):1 by weight.

[0016] In step 1, the molar ratio of the 4-bromo-1,8-naphthalenedicarboxylic anhydride to the volume ratio of the ethanol is (0.1 to 1.0):1, where the molar ratio is in mmol and the volume ratio is in mL.

[0017] In step 1, the ratio of the volume fraction of dichloromethane to the molar fraction of 4-bromo-1,8-naphthalenedicarboxylic anhydride is (1.0 to 10.0):1, where the molar fraction is in mmol and the volume fraction is in mL.

[0018] Step 2: Mix the 4-bromo-N-(2-aminoethoxy)ethanol-naphthalimide, 4-ethoxycarbonylphenylboronic acid, water, and 1,4-dioxane, add an alkali and a palladium catalyst, and react at 80-120°C for 3-18 hours under nitrogen or inert gas protection and stirring. Cool to room temperature, remove water and 1,4-dioxane by rotary evaporation under reduced pressure, add dichloromethane, wash with water to remove inorganic salts soluble in dichloromethane, dry the organic phase obtained by washing with water to remove water, and rotary dry. The crude product is purified by column chromatography and then vacuum dried to obtain a pale yellow substance, which is 4-(4-ethoxycarbonylphenyl)-N-(2-aminoethoxy)ethanol-naphthalimide. The ratio of 4-bromo-N-(2-aminoethoxy)ethanol-naphthalimide to 4-ethoxycarbonylphenylboronic acid is (1.0-1.5):1 by molar amount.

[0019] In step 2, the base is K2CO3, Na2CO3, NaHCO3, Cs2CO3 or Li2CO3, and the palladium catalyst is one of Pd(PPh3)4, Pd(OAc)2, PdCl2, Hermann's catalyst, PdCl2(PPh3)2, Pd(dppf)Cl2, Pd2(dba)3 and Pd(TFA)2. The ratio of 4-bromo-N-(2-aminoethoxy)ethanol-naphthalimide, base and palladium catalyst is 1:(1.0-10.0):(0.01-0.10) by molar amount.

[0020] In step 2, the ratio of the molar amount of 4-bromo-N-(2-aminoethoxy)ethanol-naphthalimide, the volume amount of water, and the volume amount of 1,4-dioxane is (0.1-1.0):(0.1-1.0):1, where the molar amount is in mmol and the volume amount is in mL.

[0021] In step 2, the ratio of the volume fraction of dichloromethane to the molar fraction of 4-ethoxycarbonylphenylboronic acid is (10.0–20.0):1, where the molar fraction is in mmol and the volume fraction is in mL.

[0022] In the above technical solution, the method for drying the organic phase obtained by water washing is: adding anhydrous magnesium sulfate and then filtering.

[0023] In the above technical solution, the eluent used in column chromatography purification is a mixture of dichloromethane and methanol, and the ratio of dichloromethane to methanol is 40:1 by volume.

[0024] Step 3: Mix the 4-(4-ethoxycarbonylbenzene)-N-(2-aminoethoxy)ethanolnaphthalimide obtained in Step 2, an aqueous solution of hydrazine hydrate, and methanol. Under stirring conditions, react at 60-120℃ for 12-24 hours. Cool to room temperature, filter under reduced pressure, wash the filter cake 2-3 times with methanol, and then dry under vacuum to obtain a white substance, which is the naphthalimide-based formaldehyde fluorescent probe. The ratio of the 4-(4-ethoxycarbonylbenzene)-N-(2-aminoethoxy)ethanolnaphthalimide to the aqueous solution of hydrazine hydrate is (1.0-10.0):1.

[0025] In step 3, the ratio of the molar amount of hydrazine hydrate to the volume fraction of methanol used to mix with the aqueous solution of hydrazine hydrate is (0.01 to 0.10):1, where the molar amount is in mmol and the volume fraction is in mL.

[0026] In step 3, the concentration of hydrazine hydrate in the aqueous hydrazine hydrate solution is 80 wt%.

[0027] In the above technical solution, the temperature of vacuum drying is 30-50℃, and the time of vacuum drying is 12-24h.

[0028] The application of the above-mentioned naphthalimide formaldehyde fluorescent probes in formaldehyde detection.

[0029] In the above technical solution, the method for detecting formaldehyde includes: adjusting the pH of the test solution to p, where p = 4-8; adding a naphthalimide-based formaldehyde fluorescent probe or an aqueous solution of the naphthalimide-based formaldehyde fluorescent probe to make the concentration of the naphthalimide-based formaldehyde fluorescent probe in the test solution X mol / L; obtaining the fluorescence spectrum of the test solution by photoexcitation; and substituting the fluorescence intensity at wavelength λ in the fluorescence spectrum into the standard curve equation to obtain the formaldehyde concentration of the test solution. The test solution is a mixture of liquid A and formaldehyde. The method for obtaining the standard curve equation is as follows: preparing a mixture of liquid A and different masses of formaldehyde as a standard solution; adjusting the pH of the standard solution to p; adding a naphthalimide-based formaldehyde fluorescent probe or an aqueous solution of the naphthalimide-based formaldehyde fluorescent probe to make the concentration of the naphthalimide-based formaldehyde fluorescent probe in the standard solution X mol / L; obtaining the fluorescence spectrum of the standard solution by photoexcitation; establishing a standard curve by combining the formaldehyde concentration of the standard solution and the fluorescence intensity at wavelength λ in the fluorescence spectrum; and obtaining the standard curve equation from the standard curve.

[0030] In the above technical solution, light with a wavelength of 330-350nm is used for excitation.

[0031] In the above technical solution, λ = 440~470nm.

[0032] In the above technical solution, p = 4 to 6.

[0033] In the above technical solution, X = 5 × 10 -6 ~10×10 -6 .

[0034] The above-mentioned naphthalimide formaldehyde fluorescent probes were applied to formaldehyde fluorescence imaging in live cells.

[0035] Compared with the prior art, the advantages of the present invention are:

[0036] (1) Due to the photoinduced electron transfer (PET) process from amino to naphthalimide, the aqueous solution of NIPh emits only weak fluorescence; when the amino group in NIPh reacts with formaldehyde to generate imide, the PET process is blocked and the fluorescence is restored.

[0037]

[0038] (2) Naphthalimide formaldehyde fluorescent probes use benzoyl hydrazine as the formaldehyde recognition group and have a fast response to formaldehyde, about 3 minutes.

[0039] (3) Naphthalimide formaldehyde fluorescent probes use 1,8-naphthalimide modified with 2-ethoxyethanol as the fluorophore. Naphthalimide formaldehyde fluorescent probes have good water solubility and can be used to detect formaldehyde in 100% water systems, which is environmentally friendly.

[0040] (4) Naphthalimide formaldehyde fluorescent probes have a wide range of applications. They can be used to detect formaldehyde content in different water qualities and foods, and can also be used for fluorescent imaging of formaldehyde in live cells.

[0041] (5) Compared with the probes disclosed in CN104792759A and CN114149369A, the naphthalimide formaldehyde fluorescent probe provided by the present invention has the advantages of rapid detection, small error and more accurate detection results in the application of detecting formaldehyde content in water. Attached Figure Description

[0042] Figure 1 Normalized curves of excitation and emission wavelengths of the aqueous solution of the naphthalimide formaldehyde fluorescent probe in Example 1;

[0043] Figure 2 The λ of naphthalimide-based formaldehyde fluorescent probes in aqueous solution (pH=5.0) at different formaldehyde concentrations was determined. em Reaction kinetics curve of fluorescence intensity at 450 nm;

[0044] Figure 3 a represents the fluorescence intensity of NIPh as a function of formaldehyde concentration, b represents the relationship between fluorescence intensity at 450 nm and formaldehyde concentration (formaldehyde concentration in the range of 0–1600 μM), and c represents the relationship between fluorescence intensity at 450 nm and formaldehyde concentration (formaldehyde concentration in the range of 0–100 μM).

[0045] Figure 4 The fluorescence intensity of NIPh under the influence of different analytes;

[0046] Figure 5 a is the fluorescence spectrum of NIPh for different concentrations of formaldehyde in pond water, b is the spiked and recovered values ​​in Example 5, and c is the standard curve of fluorescence intensity at 450 nm and formaldehyde concentration in Example 5.

[0047] Figure 6 a is the fluorescence spectrum of NIPh in Chinese cabbage extract for different concentrations of formaldehyde, b is the spiked and recovered values ​​in Example 6, and c is the standard curve of fluorescence intensity at 450 nm and formaldehyde concentration in Example 6.

[0048] Figure 7 Fluorescence imaging of exogenous formaldehyde in NIPh live cells;

[0049] Figure 8 'a' represents the λ of the aqueous solution of naphthalimide-based formaldehyde fluorescent probe (deionized water with pH = 6.8, without additional pH adjustment) at different formaldehyde concentrations. emThe reaction kinetics curve of fluorescence intensity at 450 nm is shown in Figure b. The fluorescence changes of NIPh in aqueous solutions at different pH values ​​with excitation wavelengths of 350 nm and emission wavelengths of 450 nm after the addition of 0 mM and 0.4 mM formaldehyde concentrations. Detailed Implementation

[0050] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0051] The drug purchase sources in the following examples are as follows:

[0052] 4-Bromo-1,8-naphthylimide, 2-(2-aminoethoxy)ethanol, and 4-ethoxycarbonylphenylboronic acid were purchased from Tianjin Xiens Biochemical Technology Co., Ltd.

[0053] Anhydrous methanol, anhydrous ethanol, dimethyl sulfoxide, 1,4-dioxane, and acetic acid were purchased from Tianjin Fuchen Chemical Reagent Co., Ltd.

[0054] Dichloromethane and trichloromethane were purchased from Tianjin Bohua Chemical Reagent Co., Ltd.

[0055] Anhydrous magnesium sulfate and potassium carbonate were purchased from Tianjin Hengshan Chemical Technology Co., Ltd.

[0056] Tetraphenylphosphine palladium was purchased from Bailingwei Technology Co., Ltd.

[0057] The hydrazine hydrate aqueous solution (mass fraction 80%) was purchased from Chengdu Huaxia Chemical Reagent Co., Ltd.

[0058] DMEM culture medium and PBS buffer solution (pH=7.4) were purchased from Tianjin Dingguo Biotechnology Co., Ltd.

[0059] The models of the instruments involved in the following embodiments are as follows:

[0060] UV-Vis spectrometer UV 2550;

[0061] Fluorescence spectrometer F4600 FL;

[0062] Varian Unity plus 400MHz NMR spectrometer;

[0063] Electronic balance ML204 / 02;

[0064] FV-1000 laser confocal microscope.

[0065] In the following examples, the units of molar amounts are mmol and the units of volume parts are mL.

[0066] Example 1

[0067] Naphthalimide formaldehyde fluorescent probe (NIPh), its structural formula is as follows:

[0068]

[0069] 1,8-Naphthylimide is the fluorophore; 2-ethoxyethanol is the hydrophilic group, which improves the water solubility of the probe; benzoyl hydrazine is the formaldehyde recognition group.

[0070] The reaction process of the above synthesis method is as follows:

[0071]

[0072] The synthesis method of the above-mentioned naphthalimide formaldehyde fluorescent probe includes the following steps:

[0073] Step 1: In a 250 mL round-bottom flask, 4-bromo-1,8-naphthalenedicarboxylic anhydride (2.7 g, 10 mmol), 2-(2-aminoethoxy)ethanol (1 g, 10 mmol), and ethanol (60 mL) were mixed and reacted at 80 °C for 2 h with magnetic stirring at 300 rpm. After cooling to room temperature (25 °C), the ethanol was removed by rotary evaporation under reduced pressure. Dichloromethane was added, and the mixture was washed three times with water to remove inorganic salts dissolved in dichloromethane. Anhydrous magnesium sulfate was added, and the mixture was filtered to dry the organic phase obtained from the water washing to remove water. The mixture was then evaporated to dryness, and the crude product was purified by column chromatography (the eluent used for column chromatography was a mixture of dichloromethane and methanol, with a volume ratio of 40:1). The product was then dried under vacuum at 37 °C for 12 h to obtain a yellow substance, 4-bromo-N-(2-aminoethoxy)ethanol-naphthalimide (2.76 g, 76%). 1 H NMR (400MHz, CDCl3) δ8.59(d,J=4.0Hz,1H),8.48(d,J=8.0Hz,1H),8.33(d,J=8.0Hz,1H),7.98(d,J=8.0Hz,1H),7.7 7(t, J1=4.0Hz, J2=8.0Hz, 1H), 4.37 (t, J1=4.0Hz, J2=8.0Hz, 2H), 3.79 (t, J1=J2=4.0Hz, 2H), 3.61 (d, J=8.0Hz, 4H). Of which, by molar fraction, the ratio of 4-bromo-1,8-naphthalenedicarboxylic anhydride to 2-(2-aminoethoxy)ethanol is 1.0:1, the ratio of the molar fraction of 4-bromo-1,8-naphthalenedicarboxylic anhydride to the volume fraction of ethanol is 0.16:1, and the ratio of the volume fraction of dichloromethane to the molar fraction of 4-bromo-1,8-naphthalenedicarboxylic anhydride is 6.0:1;

[0074] Step 2: In a 25 mL three-necked flask, 4-bromo-N-(2-aminoethoxy)ethanol-naphthalimide (117 mg, 0.6 mmol), 4-ethoxycarbonylphenylboronic acid (200 mg, 0.5 mmol), 2 mL of deionized water, and 6 mL of 1,4-dioxane were mixed. A base (152 mg, 1.1 mmol) and a palladium catalyst (39.1 mg, 0.034 mmol) were added. Under nitrogen protection and with stirring in the dark, the mixture was reacted at 100 °C for 3.5 h. After cooling to room temperature, the mixture was rotary evaporated under reduced pressure. After removing water and 1,4-dioxane, dichloromethane was added, and the mixture was washed three times with water to remove inorganic salts dissolved in dichloromethane. Anhydrous magnesium sulfate was added, and the mixture was filtered. The organic phase obtained after washing with water was dried to remove water, and then evaporated to dryness. The crude product was purified by column chromatography (the eluent used for column chromatography was a mixture of dichloromethane and methanol, with a volume ratio of 40:1). The product was then dried under vacuum at 37°C for 12 hours to obtain a pale yellow substance, 4-(4-ethoxycarbonylbenzene)-N-(2-aminoethoxy)ethanolnaphthalimide (104 mg, 44%). 1 H NMR(400MHz, CDCl3)δ8.59(t,J1=J2=8.0Hz,2H),8.15(dd,J=12.0,8.0Hz,3H),7.65(m,2H),7.52(d,J=8.0 Hz, 2H), 4.41 (m, 4H), 3.82 (t, J1 = 4.0Hz, J2 = 8.0Hz, 2H), 3.63 (d, J = 4.0Hz, 4H), 1.38 (t, J1 = J2 = 8.0Hz, 3H). In this process, the ratio of 4-bromo-N-(2-aminoethoxy)ethanol-naphthalimide to 4-ethoxycarbonylphenylboronic acid is 1.2:1 by molar amount; the base is K2CO3; the palladium catalyst is Pd(PPh3)4 (tetraphenylphosphine palladium); the ratio of the molar amount of 4-bromo-N-(2-aminoethoxy)ethanol-naphthalimide, the volume amount of water, and the volume amount of 1,4-dioxane is 0.1:0.3:1; the ratio of the volume amount of dichloromethane to the molar amount of 4-ethoxycarbonylphenylboronic acid in step 2 is 16.0:1; and the ratio of 4-bromo-N-(2-aminoethoxy)ethanol-naphthalimide, the base, and the palladium catalyst is 1:1.83:0.056 by molar amount.

[0075] Step 3: In a 25 mL three-necked flask, 97 mg (0.2 mmol) of 4-(4-ethoxycarbonylbenzene)-N-(2-aminoethoxy)ethanolnaphthalimide obtained in Step 2, an aqueous solution of hydrazine hydrate, and 8 mL of methanol were mixed. The mixture was reacted at 80 °C for 12 h under stirring. After cooling to room temperature, the mixture was filtered under reduced pressure. The filter cake was washed three times with methanol and then dried under vacuum at 37 °C for 12 h to obtain a white substance, which was a naphthalimide-based formaldehyde fluorescent probe (71 mg, 73%). 1 H NMR(400MHz, DMSO-d6)δ9.98(s,1H),8.58(d,J=4.0Hz,2H),8.23(d,J=8.0Hz,1H),8.04(d,J=8.0Hz,2H),7.86(m,2H),7.68(d,J=8.0Hz,2H), 5.87(s,1.5H),4.6(s,2H),4.28(t,J1=J2=8.0Hz,1H),3.68(t,J1=8.0Hz,J2=4.0Hz,0.5H),3.47(s,1H),3.17(t,1H),3.31(t,J=4.0Hz,1H). The ratio of hydrazine hydrate in the aqueous solution of 4-(4-ethoxycarbonylbenzene)-N-(2-aminoethoxy)ethanolnaphthalimide to hydrazine hydrate is 1.0:1, the ratio of the molar amount of hydrazine hydrate to the volume fraction of methanol used to mix with the aqueous solution of hydrazine hydrate is 0.025:1, and the concentration of hydrazine hydrate in the aqueous solution of hydrazine hydrate is 80 wt%.

[0076] A 1 mM aqueous solution of a naphthalimide-based formaldehyde fluorescent probe was dissolved in deionized water. This solution was then diluted with deionized water to obtain a 10 μM aqueous solution, which was used as the test solution. The absorption and emission spectra were measured using a UV spectrophotometer and a fluorescence spectrometer, respectively. Figure 1 As shown, where, Figure 1 The vertical axis (Normalized intensity) represents the normalized intensity.

[0077] Example 2

[0078] NIPh Reaction Kinetics Test

[0079] Prepare 10 mL of a 250 mM formaldehyde aqueous solution and a 2.5 mM naphthalimide-based formaldehyde fluorescent probe aqueous solution. Take several portions of the 2.5 mM naphthalimide-based formaldehyde fluorescent probe aqueous solution and dilute with deionized water to a concentration of 10 μM. Adjust the pH to 5.0 with 5 mM acetic acid solution. Then add formaldehyde aqueous solution to achieve formaldehyde concentrations of 0, 0.4, 1.0, and 2.0 mM in the naphthalimide-based formaldehyde fluorescent probe aqueous solution, respectively. Measure the fluorescence intensity every 1 min using an excitation wavelength of 355 nm for 15 min. Record the position λ of the maximum emission peak. em The fluorescence intensity at 450 nm was plotted as a kinetic curve over time, as shown below. Figure 2 As shown. By Figure 2 It can be seen that the fluorescence intensity did not change significantly within 15 minutes, while the fluorescence intensity of the naphthalimide formaldehyde fluorescent probe tended to saturate at about 3 minutes in the presence of different concentrations of formaldehyde. This indicates that the naphthalimide formaldehyde fluorescent probe responds quickly to formaldehyde and the reaction with formaldehyde is basically complete within 3 minutes.

[0080] Example 3

[0081] Study on the variation of NIPh fluorescence intensity with formaldehyde concentration

[0082] Prepare 2.5 mM formaldehyde aqueous solution and 2.5 mM naphthalimide formaldehyde fluorescent probe aqueous solution for later use.

[0083] A 2.5 mM aqueous solution of naphthalimide-based formaldehyde fluorescent probe was diluted to 10 μM with deionized water, and the pH was adjusted to 5.0 with 5 mM acetic acid solution. Then, 2.5 mM formaldehyde solution was gradually added to this solution to achieve formaldehyde concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 170, 190, 210, 230, 250, 270, 300, 350, 380, 450, 530, 600, 700, 800, 1000, 1200, 1400, and 1600 μM. After gentle shaking and standing for 5 min, the fluorescence spectrum (λ) of the system was measured. ex =355nm, λ em =380-680nm) and record, such as Figure 3 As shown in Figure a. A coordinate system is set up with 355 nm as the excitation wavelength, fluorescence intensity at 450 nm as the ordinate, and formaldehyde concentration in the system after adding formaldehyde aqueous solution as the abscissa, as shown in Figure a. Figure 3 As shown in b, the fluorescence intensity of the system gradually increases with increasing formaldehyde concentration, reaching saturation when the formaldehyde concentration reaches 1000 μM. Figure 3As shown in Figure c, the fluorescence intensity of NIPh exhibits a good linear relationship with formaldehyde concentration in the range of 0-100 μM. The equation of the standard curve is: F = 239.73 + 16.22 [HCHO], R 2 =0.9978, detection range: [HCHO] =0~100μM ([HCHO] represents formaldehyde concentration, F is fluorescence intensity).

[0084] Example 4

[0085] NIPh Selectivity Study

[0086] Prepare an aqueous solution of the analyte with a concentration of 250 mM. The aqueous solution of the analyte is a mixture of the analyte and water. The analyte is one of the following: a naphthalimide formaldehyde fluorescent probe, 4-methylbenzaldehyde, p-hydroxybenzaldehyde, 4-methoxybenzaldehyde, 4-nitrobenzaldehyde, propionaldehyde, acetone, methylglyoxal, benzoic acid, acetic acid, glucose, acetaldehyde, and formaldehyde.

[0087] Prepare an aqueous solution of naphthalimide-based formaldehyde fluorescent probe with a concentration of 2.5 mM.

[0088] A 2.5 mM aqueous solution of naphthalimide-based formaldehyde fluorescent probe was diluted with deionized water to a concentration of 10 μM, and the pH was adjusted to 5.0 with 5 mM acetic acid solution. Then, 250 mM of the analyte aqueous solution was added to this solution to bring the analyte concentration to 1 mM. After gentle mixing, the solution was allowed to stand for 5 min before fluorescence spectroscopy analysis. ex =355nm, λ em =380-680nm, record the fluorescence spectrum, such as Figure 4 As shown. By Figure 4 It is evident that only when the analyte is formaldehyde ( Figure 4 The "probe + formaldehyde" curve shows that the naphthalimide-based formaldehyde fluorescent probe significantly enhances the fluorescence of the aqueous solution, while other analytes have little effect on the fluorescence intensity of the aqueous solution, indicating that the naphthalimide-based formaldehyde fluorescent probe has a high selective recognition ability for formaldehyde.

[0089] Example 5

[0090] NIPh detection of formaldehyde in pond water.

[0091] The pond water was taken from a pond near Xiuchuan Road in Tianjin.

[0092] Pond water sample preparation: Take 50 mL of pond water, add 2 mg of activated carbon, stir well, centrifuge, filter through a 0.2 μM filter membrane, and store in a 4℃ refrigerator for later use.

[0093] Prepare an aqueous solution of naphthalimide-based formaldehyde fluorescent probe with a concentration of 2.5 mM for later use.

[0094] The pH of the pond water was adjusted to 5.0 using a 5mM acetic acid solution. Then, an appropriate amount of naphthalimide-based formaldehyde fluorescent probe aqueous solution was added to achieve a concentration of 10 μM for the probe. Different concentrations of formaldehyde aqueous solution were added to this solution, resulting in formaldehyde concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μM. After standing for 5 minutes, the fluorescence spectra at an excitation wavelength of 355 nm were recorded. Figure 5 As shown in Figure a, a standard curve with a linear relationship was established by plotting the fluorescence intensity at 450 nm as the ordinate and the formaldehyde concentration in the system as the abscissa. Figure 5 As shown in c. The standard curve equation is: F = 237.68 + 15.56 [HCHO], R 2 =0.9974, detection range: [HCHO] =0~100μM ([HCHO] represents formaldehyde concentration, F is fluorescence intensity).

[0095] Prepare pond water, then add an appropriate amount of naphthalimide-based formaldehyde fluorescent probe aqueous solution to make the concentration of naphthalimide-based formaldehyde fluorescent probe in the system 10 μM. Add different concentrations of formaldehyde to the pond water to make the formaldehyde concentrations in the pond water 32, 46, and 74 μM (defined as spiked), which are used as the test pond water. Record the fluorescence intensity of the test pond water at 450 nm under 355 nm excitation using a fluorescence spectrometer. Substitute the fluorescence intensity into F in the above standard curve equation to obtain the formaldehyde concentration in the test pond water. The test shows that the fluorescence intensity of the test pond water at 450 nm under 355 nm excitation is 744, 961, and 1410, respectively. Substituting these values ​​into F in the standard curve equation F = 237.68 + 15.56 [HCHO], the formaldehyde concentrations in the test pond water are 32.5, 46.5, and 75.3 μM (defined as recovery values). The spiked and recovery values ​​are as follows: Figure 5 As shown in b, the error between the recovery value calculated by the standard curve equation and the actual formaldehyde concentration in the pond water being tested is within 1.8%.

[0096] Example 6

[0097] NIPh is used to detect formaldehyde content in food.

[0098] The cabbage was purchased from a local supermarket in Tianjin.

[0099] Preparation of Chinese cabbage extract: Accurately weigh 5.0 g of Chinese cabbage, place it in a mortar, add 50 mL of deionized water, crush the Chinese cabbage, then sonicate and centrifuge, and collect the supernatant. Filter the supernatant through a 0.2 μM filter membrane, and then dilute to 50 mL with deionized water to obtain the Chinese cabbage extract, which is stored at 4℃ for later use.

[0100] Prepare an aqueous solution of naphthalimide-based formaldehyde fluorescent probe with a concentration of 2.5 mM for later use.

[0101] Adjust the pH of a suitable amount of cabbage extract to 5.0 with acetic acid aqueous solution (5 mM). Then, add a suitable amount of naphthalimide-based formaldehyde fluorescent probe aqueous solution to the cabbage extract to make the concentration of naphthalimide-based formaldehyde fluorescent probe in the system 10 μM. Add formaldehyde aqueous solution of different concentrations to the system to make the formaldehyde concentration in the system 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μM, respectively. Let it stand for 5 min, and test the fluorescence spectrum with 355 nm as the excitation wavelength. Figure 6 As shown in Figure a, a standard curve with a linear relationship was established by plotting the fluorescence intensity at 450 nm as the ordinate and the formaldehyde concentration in the system as the abscissa. Figure 6 As shown in c. The standard curve equation is: F = 234.95 + 16.51 [HCHO], R 2 =0.9978, detection range is [HCHO]=0~100μM ([FA] represents formaldehyde concentration, F is fluorescence intensity).

[0102] Prepare a cabbage extract, then add an appropriate amount of naphthalimide-based formaldehyde fluorescent probe aqueous solution to the cabbage extract to make the concentration of the naphthalimide-based formaldehyde fluorescent probe in the system 10 μM. Add formaldehyde aqueous solutions of different concentrations to the cabbage extract to make the formaldehyde concentration in the cabbage extract 32, 46, and 74 μM (as spiked). Record the fluorescence intensity at 450 nm under 355 nm excitation using a fluorescence spectrometer. Substitute F into the standard curve equation: F = 234.95 + 16.51[HCHO], and the formaldehyde concentration in the cabbage extract can be calculated as the recovery value. Figure 6 As shown in b, the fluorescence intensity at 450 nm was 759, 1011 and 1461 when the formaldehyde concentration in the cabbage extract was 32, 46 and 74 μM, respectively. After substituting into the standard curve equation, the formaldehyde concentrations in the cabbage extract were found to be 31.7, 47.0 and 74.2 μM, respectively, with an error within 2.1%.

[0103] Example 7

[0104] Fluorescent imaging of formaldehyde in living cells using NIPh.

[0105] HeLa cells (purchased from Wuhan Pronosai Life Science Technology Co., Ltd.) were mixed with DMEM medium to achieve a HeLa cell density of 3 × 10⁶ cells / mL in the DMEM medium. 5 The cells were cultured at a density of 1 / mL in DMEM medium containing HeLa cells into sterile 35mm culture dishes and incubated in a CO2 incubator (37℃, 5% CO2). After cell attachment, the cells were divided into two experimental groups for further culture.

[0106] Experimental group 1 involved adding a naphthalimide-based formaldehyde fluorescent probe to achieve a final concentration of 5 μM, followed by incubation for 30 min. Figure 7 The first line in the middle is "probe NIPh"

[0107] Experimental group 2 involved adding a naphthalimide-based formaldehyde fluorescent probe to achieve a final concentration of 5 μM. After incubation for 30 min, formaldehyde was added to achieve a final concentration of 200 μM, and incubation was continued for another 30 min. Figure 7 The second line in the middle reads "Probe NIPh + Formaldehyde".

[0108] After culturing, HeLa cells were washed three times with PBS buffer (pH=7.4), and then fluorescence images of HeLa cells cultured under these two conditions were taken using a fluorescence microscope, as shown below. Figure 7 As shown. By Figure 7 It is evident that HeLa cells co-cultured with naphthalimide formaldehyde fluorescent probes emit almost no fluorescence, while cells co-cultured with both naphthalimide formaldehyde fluorescent probes and formaldehyde emit blue fluorescence, indicating that naphthalimide formaldehyde fluorescent probes can be used for fluorescence imaging of formaldehyde in live cells.

[0109] Example 8

[0110] NIPh Reaction Kinetics Test

[0111] Prepare 10 mL of a 250 mM formaldehyde aqueous solution and a 2.5 mM naphthalimide-based formaldehyde fluorescent probe aqueous solution. Take several portions of the 2.5 mM naphthalimide-based formaldehyde fluorescent probe aqueous solution and dilute them with deionized water (the deionized water itself has a pH of 6.8, so no additional pH adjustment is needed) to a concentration of 10 μM for the naphthalimide-based formaldehyde fluorescent probe. Then add 250 mM formaldehyde aqueous solution to make the formaldehyde concentration in the naphthalimide-based formaldehyde fluorescent probe aqueous solution 0, 0.4, 1.0, and 2.0 mM, respectively. Measure the fluorescence intensity every 5 minutes using an excitation wavelength of 355 nm for 90 minutes. Record the position λ of the maximum emission peak. em The fluorescence intensity at 450 nm was plotted as a kinetic curve over time, as shown below. Figure 8 As shown in a. From Figure 8As can be seen from a, the fluorescence intensity did not change significantly within 90 minutes, while the fluorescence intensity of the naphthalimide formaldehyde fluorescent probe tended to saturate at about 30 minutes in the presence of different concentrations of formaldehyde, indicating that the reaction between the naphthalimide formaldehyde fluorescent probe and formaldehyde was basically complete within 30 minutes.

[0112] according to Figure 8 a and Figure 2 The comparison shows that pH has a significant impact on the response performance of NIPh, with a better response at pH 5.

[0113] Example 9

[0114] NIPh's ability to detect formaldehyde at different pH values.

[0115] Prepare an aqueous solution of naphthalimide-based formaldehyde fluorescent probe with a concentration of 2.5 mM.

[0116] NIPh+FA group: A 2.5 mM aqueous solution of naphthalimide-based formaldehyde fluorescent probe was diluted with deionized water to a concentration of 10 μM. The pH was adjusted to 4.0, 4.5, 5.0, 5.5, 6.0, and 6.5 with 5 mM acetic acid solution, and to 7.0, 7.5, and 8.0 with 5 mM sodium hydroxide solution. Then, 2.5 mM formaldehyde solution was added to the 10 μM aqueous solution of naphthalimide-based formaldehyde fluorescent probe at different pH values ​​to bring the final formaldehyde concentration in the system to 0.4 mM. After gentle shaking, the solution was allowed to stand for 30 min before fluorescence spectroscopy was performed. A coordinate system was set with 355 nm as the excitation wavelength, fluorescence intensity at 450 nm as the ordinate, and pH as the abscissa.

[0117] The NIPh group is basically the same as the "NIPh+FA group" mentioned above, except that the NIPh group does not include the step of "adding 2.5mM of formaldehyde aqueous solution to 10μM naphthalimide formaldehyde fluorescent probe aqueous solution at different pH values ​​to make the final formaldehyde concentration in the system 0.4mM".

[0118] The coordinate system of the NIPh+FA group is as follows Figure 8 The curve “NIPh+FA” in b, the coordinate system of the NIPh group is as follows Figure 8 The curve “NIPh” in b.

[0119] Experiments have shown that weakly acidic conditions are favorable for the recognition of formaldehyde by naphthalimide-based fluorescent probes. Considering that the organelle with the lowest pH value in cell imaging applications is the lysosome (usually around 5.0) and the response time, the pH experiment was conducted at pH=5.

[0120] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. A naphthalimide-based formaldehyde fluorescent probe, characterized in that, The structural formula of the naphthalimide formaldehyde fluorescent probe is: 。 2. The method for synthesizing the naphthalimide-based formaldehyde fluorescent probe as described in claim 1, characterized in that, Includes the following steps: Step 1: 4-bromo-1,8-naphthalenedicarboxylic anhydride, 2-(2-aminoethoxy)ethanol, and ethanol are mixed and reacted at 80-100 °C for 2-10 h under stirring. After cooling to room temperature, the ethanol is removed by rotary evaporation under reduced pressure. Dichloromethane is added, and the mixture is washed with water to remove inorganic salts dissolved in dichloromethane. The organic phase obtained after washing with water is dried to remove water, and then evaporated to dryness. The crude product is purified by column chromatography and then dried under vacuum to obtain a yellow substance, which is 4-bromo-N-(2-hydroxyethoxy)ethyl-1,8-naphthalimide. The ratio of 4-bromo-1,8-naphthalenedicarboxylic anhydride to 2-(2-aminoethoxy)ethanol is (1.0-1.5):1 by weight. Step 2: Mix the 4-bromo-N-(2-hydroxyethoxy)ethyl-1,8-naphthalimide, 4-ethoxycarbonylphenylboronic acid, water, and 1,4-dioxane, add an alkali and a palladium catalyst, and react under nitrogen or inert gas protection and stirring at 80-120 °C for 3-18 h. Cool to room temperature, remove water and 1,4-dioxane by rotary evaporation under reduced pressure, add dichloromethane, wash with water to remove inorganic salts dissolved in dichloromethane, dry the resulting organic phase to remove water, and rotary dry. Purify the crude product by column chromatography and vacuum dry to obtain a pale yellow substance, 4-(4-ethoxycarbonylphenyl)-N-(2-hydroxyethoxy)ethyl-1,8-naphthalimide, wherein, by molar fraction, the 4-bromo-N-( The ratio of 2-hydroxyethoxy)ethyl-1,8-naphthalimide to 4-ethoxycarbonylphenylboronic acid is (1.0~1.5):1; Step 3: Mix the 4-(4-ethoxycarbonylphenyl)-N-(2-hydroxyethoxy)ethyl-1,8-naphthalimide obtained in Step 2, an aqueous solution of hydrazine hydrate, and methanol. Under stirring conditions, react at 60-120 °C for 12-24 h. Cool to room temperature, filter under reduced pressure, wash the filter cake 2-3 times with methanol, and then dry under vacuum to obtain a white substance, which is the naphthalimide-based formaldehyde fluorescent probe. The ratio of the 4-(4-ethoxycarbonylphenyl)-N-(2-hydroxyethoxy)ethyl-1,8-naphthalimide to the hydrazine hydrate in the aqueous solution is (1.0-10.0):

1.

3. The synthesis method according to claim 2, characterized in that, In step 1, the molar ratio of the 4-bromo-1,8-naphthalenedicarboxylic anhydride to the volume ratio of the ethanol is (0.1~1.0):1, the molar ratio is in mmol, and the volume ratio is in mL. In step 2, the base is K2CO3, Na2CO3, NaHCO3, Cs2CO3 or Li2CO3, and the palladium catalyst is one of Pd(PPh3)4, Pd(OAc)2, PdCl2, Hermann's catalyst, PdCl2(PPh3)2, Pd(dppf)Cl2, Pd2(dba)3 and Pd(TFA)2; In step 2, the ratio of the molar amount of 4-bromo-N-(2-hydroxyethoxy)ethyl-1,8-naphthalimide, the volume amount of water, and the volume amount of 1,4-dioxane is (0.1~1.0):(0.1~1.0):

1.

4. The synthesis method according to claim 3, characterized in that, In step 1, the ratio of the volume fraction of dichloromethane to the molar fraction of 4-bromo-1,8-naphthalenedicarboxylic anhydride is (1.0~10.0):1; In step 2, the ratio of the volume fraction of dichloromethane to the molar fraction of 4-ethoxycarbonylphenylboronic acid is (10.0~20.0):

1.

5. The synthesis method according to claim 2, characterized in that, The method for drying the organic phase obtained by water washing is as follows: add anhydrous magnesium sulfate and then filter. The eluent used in column chromatography purification is a mixture of dichloromethane and methanol, with a volume ratio of 40:

1. The vacuum drying temperature is 30~50℃, and the vacuum drying time is 12~24h.

6. The synthesis method according to claim 2, characterized in that, In step 3, the ratio of the molar amount of hydrazine hydrate to the volume fraction of methanol used to mix with the aqueous solution of hydrazine hydrate is (0.01~0.10):1, where the molar amount is in mmol and the volume fraction is in mL. In step 3, the concentration of hydrazine hydrate in the aqueous hydrazine hydrate solution is 80 wt%.

7. The application of the naphthalimide formaldehyde fluorescent probe as described in claim 1 in the detection of formaldehyde for purposes other than disease diagnosis and treatment.

8. The application according to claim 7, characterized in that, The method for detecting formaldehyde includes: adjusting the pH of the test solution to p, adding the naphthalimide-based formaldehyde fluorescent probe or an aqueous solution of the naphthalimide-based formaldehyde fluorescent probe to make the concentration of the naphthalimide-based formaldehyde fluorescent probe in the test solution X mol / L, obtaining the fluorescence spectrum of the test solution by photoexcitation, and substituting the fluorescence intensity at wavelength λ in the fluorescence spectrum into the standard curve equation to obtain the formaldehyde concentration of the test solution. The test solution is a mixture of liquid A and formaldehyde. The method for obtaining the standard curve equation is as follows: preparing a mixture of liquid A and different masses of formaldehyde as a standard solution, adjusting the pH of the standard solution to p, adding the naphthalimide-based formaldehyde fluorescent probe or an aqueous solution of the naphthalimide-based formaldehyde fluorescent probe to make the concentration of the naphthalimide-based formaldehyde fluorescent probe in the standard solution X mol / L, obtaining the fluorescence spectrum of the standard solution by photoexcitation, establishing a standard curve by combining the formaldehyde concentration of the standard solution and the fluorescence intensity at wavelength λ in the fluorescence spectrum, and obtaining the standard curve equation from the standard curve. Excited with light of wavelength 330~350 nm; λ = 440~470 nm; p=4~8;X=5×10 -6 ~10×10 -6 。 9. The application of the naphthalimide formaldehyde fluorescent probe as described in claim 1 in the preparation of formaldehyde fluorescent imaging reagents in live cells.

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