A formaldehyde fluorescent probe based on bicine isophorone and a synthesis method and application thereof

Abstract

The application discloses a formaldehyde fluorescent probe based on biscalicyl isophorone and a synthesis method and application thereof, and belongs to the field of organic small-molecule fluorescent probes. The probe is based on a 2-aza-Cope rearrangement reaction mechanism, takes biscalicyl isophorone as a fluorophore, introduces a high allylamine as a specific recognition unit through chemical modification, and can realize high selectivity and rapid response of a ratio type fluorescent-colorimetric dual-mode detection on formaldehyde. The probe FL-FP-4 can realize rapid and high-selectivity detection on formaldehyde, and the detection limit is as low as 0.84 µM. Experiments show that the probe can be successfully applied to quantitative analysis of formaldehyde in various food samples, has good recovery rate, can realize real-time fluorescent imaging of exogenous formaldehyde in living cells, and provides an efficient and reliable detection tool for food safety monitoring and life science research.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule fluorescent probes, specifically relating to a formaldehyde fluorescent probe based on dicyanoisophorone, its synthesis method, and its application. Background Technology

[0002] Formaldehyde (FA), as a widely distributed endogenous reactive carbonyl compound and exogenous environmental pollutant, is of vital importance in biomedical research and food safety monitoring due to its rapid, sensitive, and highly selective detection. Traditional formaldehyde detection methods, such as chromatography and spectroscopy, while highly accurate, typically rely on large instruments and suffer from inherent limitations such as complex sample pretreatment, long analysis cycles, and difficulty in achieving real-time in-situ detection. Furthermore, they cannot be applied to in vivo biological imaging. Currently, fluorescence probe-based detection technologies have attracted significant attention due to their high sensitivity, ease of operation, and real-time imaging potential; however, further development in this field still faces a series of serious challenges.

[0003] The response speed of a probe is fundamentally controlled by the kinetics of its group's reaction with formaldehyde. Many probes have weak nucleophilicity in their recognition groups, or steric hindrance from nearby substituents, resulting in a high initial nucleophilic condensation barrier and a slow reaction rate. This becomes a limiting factor for the entire response process, leading to response times of tens of minutes or even hours, failing to meet the needs of real-time monitoring. Regarding selectivity, many fluorescent probes lack precise molecular recognition capabilities for formaldehyde in their recognition groups. Their reactivity is not specifically designed for the unique structure of formaldehyde (the smallest aldehyde molecule), thus leading to side reactions similar to those with other aldehydes or highly reactive α-dicarbonyl compounds (such as glyoxal and methylglyoxal) commonly found in the system, producing false positive signals. This lack of selectivity, caused by the inherent broadness of the recognition group's chemical reaction mechanism, is the primary reason for the reduced reliability of probes in complex biological or food matrices. Furthermore, the overall performance of the probe is also limited by the electronic coupling and energy matching relationship between the fluorophore and the recognition group. If the connection between the two is inappropriate, or if the electronic rearrangement process triggered by the recognition reaction is insufficient to effectively modulate the photophysical properties of the fluorophore, the probe will exhibit weak signal changes and small Stokes shifts before and after the response, or it will be difficult to generate ratiometric fluorescence signals that can be used for precise quantification. Simultaneously, an imbalance in the overall hydrophilicity / hydrophobicity of some probe molecules, or the presence of easily oxidized functional groups, can lead to their easy aggregation and quenching in aqueous environments or poor chemical stability in complex samples, resulting in performance in practical applications that is lower than in ideal environments.

[0004] The patent CN115784910B, entitled "A Ratio-Modulating Fluorescent Probe for Formaldehyde Detection, Its Preparation Method and Application," specifically discloses a method for ratio-modulating fluorescence detection of formaldehyde using 1-hydroxy-2-pyrenecarboxaldehyde as the fluorophore. This method involves a one-step reaction to connect an allyl amino group as the recognition unit, based on aza-Cope rearrangement. The probe's response time in acetonitrile / HEPES buffer is in the hundreds of seconds, and it has been successfully used for qualitative and quantitative analysis of formaldehyde in food samples. However, the pyrene fluorophore used in this probe emits light in the blue-violet region, resulting in weak tissue penetration and strong background autofluorescence interference in bioimaging, limiting its application in in vivo and deep tissue detection. Summary of the Invention

[0005] To address the prominent problem that existing formaldehyde fluorescent probes struggle to balance response speed, selectivity, and applicability to practical samples, this invention provides a novel fluorescent probe, FL-FP-4, based on dicyanoisophorone and a highly specific recognition group. The aim is to obtain a reliable analytical tool that can achieve a rapid response within seconds, possesses extremely high anti-interference capabilities, and can be directly used for accurate quantification of formaldehyde in complex food samples and living cells.

[0006] To achieve this objective, the following solution is provided: This invention provides a formaldehyde fluorescent probe based on dicyanoisophorone, wherein the formaldehyde fluorescent probe based on dicyanoisophorone is FL-FP-4, with the molecular formula C 23 H 25 N3O, chemical structural formula as shown in (Ⅰ): (I).

[0007] This invention provides a method for synthesizing a formaldehyde fluorescent probe based on dicyanoisophorone, comprising the following steps: (1) Dissolve 3,5,5-trimethyl-2-cyclohexen-1-one and malononitrile in anhydrous ethanol, add piperidine to react, and separate and purify to obtain intermediate compound A; (2) Intermediate compound A and p-hydroxybenzaldehyde were dissolved in anhydrous ethanol, and piperidine was added to react. The intermediate compound B was obtained by separation and purification. (3) The intermediate compound B was reacted with hexamethylenetetramine in trifluoroacetic acid. After the reaction was completed, deionized water was added to precipitate the mixture, and the intermediate compound C was obtained by separation and purification. (4) Dissolve potassium allyl trifluoroborate in ammonia methanol solution, then add intermediate compound C to react, and separate and purify to obtain formaldehyde fluorescent probe FL-FP-4 based on dicyanoisophorone.

[0008] Further, in step (1), the molar ratio of malononitrile to 3,5,5-trimethyl-2-cyclohexen-1-one is (1.0~1.5):1; the amount of piperidine used is 5%~10% of the molar amount of 3,5,5-trimethyl-2-cyclohexen-1-one; the reaction temperature is 50~60℃, and the reaction time is 10~14 h.

[0009] Further, in step (2), the molar ratio of p-hydroxybenzaldehyde to intermediate compound A is (1.2~1.8):1; the amount of piperidine used is 10%~15% of the molar number of intermediate compound A; the reaction temperature is 80~85℃, and the reaction time is 3~5 h.

[0010] Furthermore, the separation and purification in steps (1) and (2) is carried out by column chromatography purification, and the eluent used is a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of (10:1) to (15:1).

[0011] Further, in step (3), the molar ratio of hexamethylenetetramine to intermediate compound B is (2.0~4.0):1; the reaction temperature is 85~95℃, and the reaction time is 10~14 h.

[0012] Furthermore, in step (3), the separation and purification is carried out by column chromatography, and the eluent used is a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of (7:1) ~ (9:1).

[0013] Further, in step (4), the molar ratio of potassium allyl trifluoroborate to intermediate compound C is (2.0~3.0):1; the reaction starts at 0°C, and after adding intermediate compound C, the temperature is raised to 25~30°C for 8~12 h.

[0014] Further, in step (4), the separation and purification is carried out by column chromatography, and the eluent used is a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of (12:1) ~ (18:1).

[0015] This invention also provides an application of a formaldehyde fluorescent probe based on dicyanoisophorone in cell imaging and food sample detection.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The formaldehyde fluorescent probe FL-FP-4 of this invention employs a specific high-allylamine structure. Its primary amine group exhibits extremely high nucleophilicity, significantly reducing the energy barrier for the initial condensation reaction with formaldehyde, thus ensuring an ultrafast response from the molecular structure perspective. Simultaneously, this recognition group demonstrates a precise recognition preference for the smallest formaldehyde molecule in terms of spatial structure and electronic effects, fundamentally eliminating side reactions with larger aldehydes such as acetaldehyde and propionaldehyde, as well as common reactive carbonyl compounds, thereby endowing the probe with higher selectivity than existing technologies.

[0017] 2. The fluorescent probe provided by this invention has a large conjugated system and a significant intramolecular charge transfer (ICT) effect. When it is linked with a recognition group that acts as an electron acceptor and subsequent reaction products, it can produce a sufficient spectral shift. This characteristic ensures that the fluorescent probe can produce a visible color change (from brownish-red to yellow) and a significant change in fluorescence intensity ratio after the reaction, realizing sensitive colorimetric-ratio fluorescence dual-mode detection, effectively avoiding environmental interference and improving quantitative accuracy.

[0018] 3. The catalytic synthesis system and the substituents introduced into the molecule used in this invention not only ensure the efficient synthesis of the key intermediate and the final product FL-FP-4, but also, through their electronic and steric effects, these groups further stabilize the transition state of the 2-aza-Cope rearrangement, synergistically promoting the overall reaction rate.

[0019] 4. This invention integrates a high-allylamine recognition group with a high-performance fluorophore through an optimized connection method, resulting in a significant synergistic effect. Simultaneously, at the preparation process level, the fluorophore, linker arm, and recognition group are synthesized and integrated, and the synergistic optimization of reaction conditions at each step provides a fundamental guarantee for obtaining a high-performance probe. Ultimately, a comprehensive solution achieving an optimal balance in response speed, selectivity, sensitivity, and reliability in practical applications is obtained. This probe can be directly applied to the quantitative detection of formaldehyde in complex real food samples, exhibiting high recovery rates and low relative standard deviations. Furthermore, it successfully achieves rapid, ratiometric fluorescence imaging of formaldehyde in various live cells. Attached Figure Description

[0020] Figure 1 The 1H NMR spectrum of the formaldehyde fluorescent probe FL-FP-4 based on dicyanoisophorone in Example 1 ( 1 H NMR spectrum; Figure 2 The carbon NMR spectrum of the formaldehyde fluorescent probe FL-FP-4 based on dicyanoisophorone in Example 1 ( 13 C NMR spectrum; Figure 3This is a UV absorption spectrum of the formaldehyde fluorescent probe FL-FP-4 based on dicyanoisophorone reacting with different concentrations of formaldehyde in Example 4. Figure 4 This is a fluorescence spectrum diagram of the interaction between the formaldehyde fluorescent probe FL-FP-4 based on dicyanoisophorone and different concentrations of formaldehyde in Example 4. Figure 5 This is a schematic diagram of the UV absorption response of the formaldehyde fluorescent probe FL-FP-4 based on dicyanoisophorone to different analytes in Example 4; Figure 6 This is a schematic diagram of the fluorescence spectrum response of the formaldehyde fluorescent probe FL-FP-4 based on dicyanoisophorone to different analytes in Example 4; Figure 7 This is a schematic diagram of the time response of the formaldehyde fluorescent probe FL-FP-4 based on dicyanoisophorone in the ultraviolet absorption and fluorescence spectra in Example 4. Figure 8 This is a schematic diagram illustrating the cytotoxicity analysis of the formaldehyde fluorescent probe FL-FP-4 based on dicyandioxone in Example 5. Figure 9 The image shows an image of exogenous formaldehyde and a bar chart of average fluorescence intensity for the dicyanoisophorone-based formaldehyde fluorescent probe FL-FP-4 in HeLa cells, as described in Example 5. Detailed Implementation

[0021] The present invention will be further described below with reference to preferred embodiments. The endpoints and any values ​​of the ranges disclosed in the present invention are not limited to the precise ranges or values. These ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed herein.

[0022] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0023] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0024] Argon was chosen as the protective gas for all inert atmospheres used in the reaction. 1 The H-s spectra were recorded on a JEOL ECZ600S (600 MHz) spectrometer using DMSO-d6 as the solvent. 13The C-spectrum was recorded on a JEOL ECZ600S (150 MHz) spectrometer using DMSO-d6 as the solvent; The column chromatography used in the reaction employed a thick-walled glass column and silica gel (300-400 mesh); thin-layer chromatography (TLC) was performed using commercially available 0.25 mm silica gel plates. The ultraviolet absorption spectrum of the solution was obtained using a Shimadzu UV-1900 UV-Vis-NIR spectrophotometer. Fluorescence spectra were measured using a Spectrofluorometer FS5 fluorescence spectrometer under the following conditions: λex = 420 nm, Step: 1.00 nm, Dwell: 0.1 s, Repeats: 2, and slit width: 4.

[0025] Example 1 This embodiment provides a method for synthesizing a formaldehyde fluorescent probe based on dicyanoisophorone, including the following steps: (1) 3,5,5-trimethyl-2-cyclohexen-1-one (1.56 mL, 10 mmol) and malononitrile (0.66 g, 10 mmol) were added to a two-necked flask and dissolved together in 10 mL of anhydrous ethanol. 50 μL of piperidine was added dropwise. The mixture was stirred at 50 °C for 12 h under an argon atmosphere, and the reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, and the solvent was evaporated under reduced pressure using a rotary evaporator. The mixture was purified by column chromatography using PE / EA = 10:1 as the eluent to obtain a white solid, namely intermediate compound A (1.43 g, 76.8%). The chemical structure of intermediate compound A is shown in (II). The preparation route is as follows: (II); (2) Weigh intermediate compound A (0.74 g, 4 mmol) and p-hydroxybenzaldehyde (0.73 g, 6 mmol) and dissolve them in 10 mL of anhydrous ethanol. Add 40 μL of piperidine to the mixture and reflux at 80 °C for 4 h under an argon atmosphere. Monitor the reaction progress using TLC. After the reaction is complete, cool to room temperature, evaporate the solvent under reduced pressure, and purify by column chromatography using PE / EA = 10:1 as the eluent to obtain an orange solid, namely intermediate compound B (0.89 g, 76.7%). The chemical structure of intermediate compound B is shown in (III), and the preparation route is as follows: (III); (3) Weigh intermediate compound B (0.43 g, 1.5 mmol) and hexamethylenetetramine (0.63 g, 4.5 mmol) into a flask, seal the flask, introduce argon gas, and slowly add 10 mL of trifluoroacetic acid while stirring. Heat to 90 °C and reflux for 14 h. Monitor the reaction progress using TLC. After the reaction is complete, adding ice water will precipitate a yellow precipitate. Filter, wash with water, and dry. Dissolve the product in dichloromethane, and evaporate the solvent under reduced pressure using a rotary evaporator. Purify by column chromatography using PE / EA = 8:1 as the eluent to obtain a yellow solid, namely intermediate compound C (0.21 g, 44.0%). The chemical structure of intermediate compound C is shown in (Ⅳ). The preparation route is as follows: (Ⅳ); (4) Potassium allyl trifluoroborate (0.14 g, 1 mmol) was dissolved in 4 mL of ammonia-methanol solution. Under an argon atmosphere, the mixture was stirred at 0 °C for 30 min. Then, intermediate compound C (0.15 g, 0.5 mmol) was added to the mixture, and the temperature was raised to 25 °C for 10 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was evaporated under reduced pressure using a rotary evaporator. The mixture was purified by column chromatography with PE / EA = 15:1 as the eluent to obtain a yellow solid FL-FP-4 (81.31 mg, 45.3%). The chemical structure of the formaldehyde fluorescent probe FL-FP-4 is shown in (I). The preparation route is as follows: (I).

[0026] The final formaldehyde fluorescent probe FL-FP-4 based on dicyanoisophorone was subjected to 1H NMR spectroscopy. 1 H NMR and carbon spectroscopy 13 The results were obtained by C NMR (cell NMR) detection. Figure 1 and Figure 2 As shown, 1 The proton signals in the H NMR spectrum all match well. 13 The CNMR spectrum showed 21 clear carbon signals, consistent with the target molecule's C14. 23 H 25 The structure of N3O is consistent, and the difference in the number of signals is due to the overlap of signals from carbon atoms with similar chemical environments in some molecules.

[0027] Example 2 This embodiment provides a method for synthesizing a formaldehyde fluorescent probe based on dicyanoisophorone, including the following steps: (1) 3,5,5-trimethyl-2-cyclohexen-1-one (1.56 mL, 10 mmol) and malononitrile (0.79 g, 12 mmol) were added to a two-necked flask and dissolved together in 10 mL of anhydrous ethanol. 79 μL of piperidine was added dropwise. The mixture was stirred at 55 °C for 14 h under an argon atmosphere, and the reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature and the solvent was evaporated under reduced pressure using a rotary evaporator. The mixture was purified by column chromatography with PE / EA = 15:1 as the eluent to obtain a white solid, namely intermediate compound A (1.14 g, 61.3%). (2) Weigh intermediate compound A (0.74 g, 4 mmol) and p-hydroxybenzaldehyde (0.73 g, 4.8 mmol) and dissolve them in 10 mL of anhydrous ethanol. Add 48 μL of piperidine to the mixture and reflux at 83 °C for 5 h under an argon atmosphere. Monitor the reaction progress using TLC. After the reaction is complete, cool to room temperature, evaporate the solvent under reduced pressure, and purify by column chromatography using PE / EA = 15:1 as the eluent to obtain an orange solid (0.84 g, 72.4%), which is intermediate compound B. (3) Weigh intermediate compound B (0.43 g, 1.5 mmol) and hexamethylenetetramine (0.42 g, 3.0 mmol) into a flask, seal the flask, introduce argon gas, and slowly add 10 mL of trifluoroacetic acid while stirring. Heat to 85 °C and reflux for 10 h. Monitor the reaction progress using TLC. After the reaction is complete, adding ice water will precipitate a yellow precipitate. Filter, wash with water, and dry. Dissolve the product in dichloromethane, and evaporate the solvent under reduced pressure using a rotary evaporator. Purify by column chromatography using PE / EA = 7:1 as the eluent to obtain a yellow solid, namely intermediate compound C (0.17 g, 35.8%). (4) Allyl trifluoroborate potassium (0.18 g, 1.25 mmol) was dissolved in 4 mL of ammonia methanol solution. Under an argon atmosphere, the mixture was stirred at 0 °C for 30 min. Then, intermediate compound C (0.15 g, 0.5 mmol) was added to the mixture, and the temperature was raised to 30 °C for 8 h. The reaction process was monitored by TLC. After the reaction was completed, the solvent was evaporated under reduced pressure using a rotary evaporator. The mixture was purified by column chromatography with PE / EA = 18:1 as the eluent to obtain a yellow solid FL-FP-4 (83.50 mg, 46.3%).

[0028] Example 3 This embodiment provides a method for synthesizing a formaldehyde fluorescent probe based on dicyanoisophorone, including the following steps: (1) 3,5,5-trimethyl-2-cyclohexen-1-one (1.56 mL, 10 mmol) and malononitrile (0.99 g, 15 mmol) were added to a two-necked flask and dissolved together in 10 mL of anhydrous ethanol. 99 μL of piperidine was added dropwise. The mixture was stirred at 60 °C for 10 h under an argon atmosphere, and the reaction progress was monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, and the solvent was evaporated under reduced pressure using a rotary evaporator. The mixture was purified by column chromatography with PE / EA = 13:1 as the eluent to obtain a white solid, namely intermediate compound A (1.03 g, 55.3%). (2) Weigh intermediate compound A (0.74 g, 4 mmol) and p-hydroxybenzaldehyde (0.88 g, 7.2 mmol) and dissolve them in 10 mL of anhydrous ethanol. Add 59 μL of piperidine to the mixture and reflux at 85 °C for 3 h under an argon atmosphere. Monitor the reaction progress using TLC. After the reaction is complete, cool to room temperature, evaporate the solvent under reduced pressure, and purify by column chromatography using PE / EA = 13:1 as the eluent to obtain an orange solid, namely intermediate compound B (0.93 g, 80.1%). (3) Weigh intermediate compound B (0.435 g, 1.5 mmol) and hexamethylenetetramine (0.84 g, 6.0 mmol) into a flask, seal the flask, introduce argon gas, and slowly add 10 mL of trifluoroacetic acid while stirring. Heat to 95 °C and reflux for 12 h. Monitor the reaction progress using TLC. After the reaction is complete, adding ice water will precipitate a yellow precipitate. Filter, wash with water, and dry. Dissolve the product in dichloromethane, and evaporate the solvent under reduced pressure using a rotary evaporator. Purify by column chromatography using PE / EA = 9:1 as the eluent to obtain a yellow solid, namely intermediate compound C (0.23 g, 48.5%). (4) Allyl trifluoroborate potassium (0.22 g, 1.5 mmol) was dissolved in 4 mL of ammonia methanol solution. Under an argon atmosphere, the mixture was stirred at 0 °C for 30 min. Then, intermediate compound C (0.15 g, 0.5 mmol) was added to the mixture, and the temperature was raised to 27 °C for 12 h. The reaction process was monitored by TLC. After the reaction was completed, the solvent was evaporated under reduced pressure using a rotary evaporator. The mixture was purified by column chromatography with PE / EA = 12:1 as the eluent to obtain a yellow solid FL-FP-4 (85.92 mg, 47.7%).

[0029] Example 4 In this embodiment, the formaldehyde fluorescent probe FL-FP-4 based on dicyanoisophorone prepared according to Example 1 is used to detect formaldehyde in a solvent. When formaldehyde is present, the formaldehyde fluorescent probe FL-FP-4 based on dicyanoisophorone reacts with formaldehyde, producing a significant color change, thus achieving detection.

[0030] (1) Spectroscopic detection of formaldehyde based on dicyanoisophorone fluorescent probe FL-FP-4 reacting with different concentrations of formaldehyde in the same solvent: The formaldehyde fluorescent probe FL-FP-4 was prepared as a stock solution using dimethyl sulfoxide as solvent and contained in a 3 mL (PBS / DMSO = 1:1, v / v) system, with a final concentration of FL-FP-4 of 20 μM. Formaldehyde solutions of different concentrations were added, and their UV spectra were measured. The fluorescence spectra of FL-FP-4 reacting with different concentrations of formaldehyde were also measured under an excitation light of 420 nm. The experimental results are as follows: Figure 3 As shown in Figure a, with the increase of FA concentration from 0 to 800 μM, the intensity of the characteristic absorption peak of FL-FP-4 at 520 nm wavelength shows a significant decreasing trend, while the new absorption peak at 430 nm shows an increasing trend. When the FA concentration reaches approximately 200 μM, the absorption intensity no longer changes, indicating that the system has reached saturation. Figure 3 b. Standard curves constructed based on the quantitative relationship between different concentrations of FA and their corresponding absorbance show that FL-FP-4 and FA concentration exhibit a good linear correlation (Rb) within a specific range. 2 =0.9912), and its linear regression equation is y = 0.03318x + 0.87681. For example... Figure 4 As shown in Figure a, fluorescence spectroscopy tests revealed that with increasing FA concentration, the fluorescence signal at the emission peak of 672 nm of the FL-FP-4 probe under 420 nm excitation light gradually decreased, while the fluorescence intensity of the new emission peak at 591 nm gradually increased. When the FA concentration increased to 200 μM, the fluorescence intensity tended to stabilize at its lowest value, and the new emission peak at 591 nm tended to stabilize at its highest value. This finding was achieved by establishing the relationship between FA concentration (0-40 μM) and I... 591 nm / I 672 nm The correlation between fluorescence intensity ratios was investigated, and a concentration titration standard curve was plotted, such as... Figure 4 As shown in b, the FA concentration and the fluorescence response of FL-FP-4 have a significant linear correlation within a specific range (R0). 2 =0.9997), and its linear regression equation is y = 0.0221x + 0.28324. Based on the LOD calculation formula, the lowest detection limit of the FL-FP-4 probe for FA was finally determined to be 0.84 μM.

[0031] (2) Selective detection of formaldehyde based on dicyanoisophorone fluorescent probe FL-FP-4 Formaldehyde, acetaldehyde, glyoxal, methylglyoxal, propionaldehyde, acetylacetone, hydrogen peroxide, homocysteine, glutamic acid, sodium chloride, sodium bromide, sodium iodide, sodium fluoride, potassium chloride, calcium chloride, zinc chloride, copper sulfate pentahydrate, manganese dichloride, ferric chloride, sodium carbonate, sodium sulfate, sodium sulfite, sodium bisulfite, disodium hydrogen phosphate, and potassium dihydrogen phosphate were dissolved in deionized water to prepare a stock solution, which was controlled in a 3 mL (PBS / DMSO=1:1, v / v) system. The final concentration of FL-FP-4 was 20 μM. The above interfering substances were added to make the final concentration of all of them 200 μM. The results are as follows. Figure 5 As shown, the absorption peaks of the probe in the UV absorption spectrum did not change significantly until FA was added, at which point a significant change occurred. To investigate the specificity of the probe for FA detection in the fluorescence spectrum, interfering substances were added to the same system. Figure 6 When these analytes were added, no significant fluorescence changes were detected in the probes. However, after adding the FA stock solution, both emission peaks at 591 nm and 672 nm under 420 nm excitation showed dramatic changes accompanied by obvious colorimetric reactions. For example, Figure 6 As shown in b, 1-26 represent Br - Cl - CO3 2- F - I - SO3 2- SO4 2- HSO3 - HPO4 2- H2PO4 - Ca 2+ Fe 3+ K + Mn 2+ Cu 2+ Na + Zn 2+ The addition of FA (acetylacetone, methylglyoxal, acetaldehyde, methylglyoxal, acetylacetone, and formaldehyde) increased the ratio of the highest fluorescence intensity at 591 nm to 672 nm by approximately 8 times, demonstrating the specificity of FL-FP-4.

[0032] (3) Time-response detection of formaldehyde based on dicyanoisophorone fluorescent probe FL-FP-4 like Figure 7 As shown in ab, when FA at a final concentration of 200 μM was added to the working solution (PBS / DMSO = 1:1, v / v) of the probe (20 μM), the absorption peak of FL-FP-4 at 520 nm immediately decreased, while a new absorption peak appeared at 430 nm; in the fluorescence spectroscopy characteristic tests, as shown... Figure 7As shown in cd, under the same system, the addition of FA causes a rapid decrease in the fluorescence intensity of the probe at 672 nm, while the fluorescence signal intensity of the new emission peak at 591 nm directly increases to its maximum value. Among these, the fluorescence intensity ratio (I...) 591 nm / I 672 nm It can maintain a stable state immediately when different concentrations of FA (5 μM, 200 μM) are added, accompanied by a color change of the working solution from brownish-red to yellow.

[0033] Example 5 This embodiment provides the application of the formaldehyde fluorescent probe based on dicyanoisophorone prepared according to the above embodiment in the detection of food samples.

[0034] Six samples—10 g of frozen shrimp, squid, jellyfish, chicken feet, kelp, and lotus root—were weighed and added to 10 mL of deionized water. The samples were then pulverized using a grinder, sonicated at 37°C for 30 min, and centrifuged at 1000 rpm for 10 min. The supernatant was collected, filtered, and stored at -20°C. A 20 μM FL-FP-4 probe and 100 μL of sample stock solution were added to a 3 mL DMSO / PBS (1:1, v / v) cuvette system. Preliminary UV absorption and fluorescence spectra were performed on the six samples. Detectable samples were then subjected to concentration titration. Simultaneously, spiked recovery experiments were conducted on the six samples by adding the probe stock solution, 100 μL of sample stock solution, and different concentrations of FA (0.5, 1, and 2 mmol / L) to the system. The UV absorption and fluorescence intensity changes of FL-FP-4 under 420 nm excitation were recorded using UV absorption and fluorescence spectrometry. The spiked recovery rate and mean relative deviation (RSD) were calculated using a linear regression equation between the fluorescence intensity ratio of FA and FP-4. The spiked recovery formula is: P (recovery rate) = (spiked sample amount - measured sample amount) / spiked amount. Experimental results demonstrate that, compared to jellyfish and kelp, other samples where FA was not detected contain extremely low levels of FA, indicating high food safety. The recovery rates of FA in these food samples ranged from 96.2% to 118.2%, showing good recovery rates, and the RSD was less than 5%. Calculations showed that the FA content in jellyfish and kelp was 4.5 mg / kg and 2.52 mg / kg, respectively, which are lower than the standard for FA addition in food. The experimental results also demonstrate that FL-FP-4 can be effectively applied to the quantitative detection of actual samples.

[0035] Example 6 This embodiment provides the application of the formaldehyde fluorescent probe based on dicyanoisophorone prepared according to the above embodiment in the detection of exogenous formaldehyde in cells.

[0036] The cytotoxicity of the probe to HeLa cells was detected using the Cell Counting Kit-8 (CCK-8) assay. Cells were seeded in 96-well plates and cultured for 24 h at 37°C in a CO2 cell culture incubator. Different concentrations of FL-FP-4 probe (0, 5, 8, 10, 15, 20, 30, 40 μM) were added to each well. A blank control group and a control group were established, with six replicates per group. After drug administration, incubation continued for 12 h. Then, 10 μL of CCK-8 reagent and 100 μL of culture medium were added to each well, and the cells were cultured for 3 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated using the cell viability formula. Results are as follows: Figure 8 As shown, when the probe concentration reaches 10 μM, the cell survival rate is still greater than 90%, and the cell survival rate remains highly active after increasing the concentration. Therefore, a probe concentration of 10 μM was chosen as one of the conditions for subsequent cell imaging experiments.

[0037] Human cervical cancer cells (HeLa) were cultured with 10 μM FL-FP-4 in a CO2 cell culture incubator at 37℃ for 30 min. After washing three times with PBS to remove excess probe residue, 1 mL of PBS (5% DMSO) was slowly added. Cell imaging was performed under a confocal microscope at 420 nm excitation. FA stock solution was then added, and the cells were cultured for another 30 min. After washing three times with PBS, 1 mL of PBS was added again for cell imaging. Results are as follows: Figure 9 As shown, HeLa cells incubated with probe FL-FP-4 exhibited red fluorescence under 420 nm excitation. After treatment with excess formaldehyde and incubation for 30 min, the fluorescence in the 625-690 nm red channel decreased, while the yellow light in the 550-600 nm yellow channel gradually brightened. By calculating the average fluorescence intensity of the probe in different channels and performing ratio calculations, the ratio of the average fluorescence intensity of the yellow channel to the red channel significantly increased to 1.7 times after the addition of FA. These results demonstrate that the FL-FP-4 sensor can monitor FA levels in HeLa cells proportionally and possesses bioimaging analysis capabilities.

[0038] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A formaldehyde fluorescent probe based on dicyanoisophorone, characterized in that, The formaldehyde fluorescent probe based on dicyanoisophorone is FL-FP-4, with the molecular formula C0. 23 H 25 N3O, chemical structural formula as shown in (Ⅰ): （Ⅰ）。 2. A method for synthesizing a formaldehyde fluorescent probe based on dicyanoisophorone as described in claim 1, characterized in that, Includes the following steps: (1) Dissolve 3,5,5-trimethyl-2-cyclohexen-1-one and malononitrile in anhydrous ethanol, add piperidine to react, and separate and purify to obtain intermediate compound A; (2) Intermediate compound A and p-hydroxybenzaldehyde were dissolved in anhydrous ethanol, and piperidine was added to react. The intermediate compound B was then separated and purified. (3) Intermediate compound B was reacted with hexamethylenetetramine in trifluoroacetic acid. After the reaction was completed, deionized water was added to precipitate the mixture, and intermediate compound C was obtained by separation and purification. (4) Dissolve potassium allyl trifluoroborate in ammonia methanol solution, then add intermediate compound C to react, and separate and purify to obtain formaldehyde fluorescent probe FL-FP-4 based on dicyandioxolone.

3. The method for synthesizing a formaldehyde fluorescent probe based on dicyanoisophorone according to claim 2, characterized in that, In step (1), the molar ratio of malononitrile to 3,5,5-trimethyl-2-cyclohexen-1-one is (1.0~1.5):1; the amount of piperidine used is 5%~10% of the molar amount of 3,5,5-trimethyl-2-cyclohexen-1-one; the reaction temperature is 50~60℃, and the reaction time is 10~14 h.

4. The method for synthesizing a formaldehyde fluorescent probe based on dicyanoisophorone according to claim 2, characterized in that, In step (2), the molar ratio of p-hydroxybenzaldehyde to intermediate compound A is (1.2~1.8):1; the amount of piperidine used is 10%~15% of the molar number of intermediate compound A; the reaction temperature is 80~85℃ and the reaction time is 3~5 h.

5. The method for synthesizing a formaldehyde fluorescent probe based on dicyanoisophorone according to claim 2, characterized in that, In steps (1) and (2), the separation and purification were carried out by column chromatography, and the eluent used was a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of (10:1) to (15:1).

6. The method for synthesizing a formaldehyde fluorescent probe based on dicyanoisophorone according to claim 2, characterized in that, In step (3), the molar ratio of hexamethylenetetramine to intermediate compound B is (2.0~4.0):1; the reaction temperature is 85~95℃ and the reaction time is 10~14 h.

7. The method for synthesizing a formaldehyde fluorescent probe based on dicyanoisophorone according to claim 2, characterized in that, In step (3), the separation and purification were carried out by column chromatography, and the eluent used was a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of (7:1) ~ (9:1).

8. The method for synthesizing a formaldehyde fluorescent probe based on dicyanoisophorone according to claim 2, characterized in that, In step (4), the molar ratio of potassium allyl trifluoroborate to intermediate compound C is (2.0 ~ 3.0):1; the reaction starts at 0°C and after adding intermediate compound C, the temperature is raised to 25~30°C and the reaction is carried out for 8~12 h.

9. The method for synthesizing a formaldehyde fluorescent probe based on dicyanoisophorone according to claim 2, characterized in that, In step (4), the separation and purification were carried out by column chromatography, and the eluent used was a mixed solvent of petroleum ether and ethyl acetate, with a volume ratio of petroleum ether to ethyl acetate of (12:1) ~ (18:1).

10. The application of the formaldehyde fluorescent probe based on dicyanoisophorone as described in claim 1 in cell imaging and food sample detection.

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