Fluorescent probe for detecting cadaverine as well as preparation method and application of fluorescent probe

Abstract

In meat freshness evaluation, biogenic amines, especially cadaverine, are key indexes. The novel fluorescent probe ZY1 provided by the invention reacts with cadaverine to realize efficient and rapid detection of meat freshness. The probe has excellent selectivity and ultralow detection limit (LDD = 13.8 nM), and is accompanied by remarkable fluorescence color conversion (light red to bright green). Based on the characteristic, the ZY1 is innovatively integrated with RGB analysis software of a smart phone, and a reliable linear relation between a G / B value and food preservation time is established by shooting a meat extracting solution loaded with the ZY1, so that real-time and portable detection of the food freshness is realized. In view of specific up-regulation of cadaverine biosynthesis in ulcerative colitis (UC), the research further proves the application potential of ZY1 in UC noninvasive auxiliary diagnosis. In addition, ZY1 also shows expanded application in the technical fields of preparation of novel fluorescent materials and information transmission.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food freshness detection, and relates to a fluorescent probe for detecting cadaverine as well as a preparation method and application. BACKGROUND

[0002] In recent years, food safety has become an important factor threatening human health and has attracted more and more attention. A large number of studies have shown that the intake of non-fresh food is the cause of many diseases. Meat, as the main source of protein intake for humans, is prone to spoilage during storage, transportation and other processes, producing a series of substances harmful to the human body. For example, after the spoilage of shrimp and meat rich in protein, microorganisms produce biological amines and other substances through decarboxylation, and after human consumption, health problems such as headache and diarrhea occur. Therefore, there is an urgent need to develop a technology that can quickly and simply detect biological amines to ensure the freshness of meat food and help maintain human health.

[0003] Among the numerous biological amines, cadaverine is a key indicator of meat spoilage. Therefore, by detecting the content of cadaverine in meat, the freshness of meat has become a new direction for scientists to develop. In recent years, the technology for detecting biological amines has developed rapidly, including chromatography, electrochemical method, hyperspectral imaging method, etc. However, these methods rely on expensive equipment and complex pretreatment processes, limiting their use in real-time detection of meat freshness. Among the numerous detection methods, fluorescent sensor technology has gradually attracted more and more attention due to its high sensitivity, fast response speed, strong selectivity and strong economy. A fluorescent probe that can specifically detect cadaverine has become a new solution for real-time monitoring of meat freshness.

[0004] In addition, a fluorescent probe that can detect cadaverine has application value not only limited to real-time monitoring of meat freshness. Studies have shown that the content of cadaverine in the feces of patients with ulcerative colitis is much higher than the normal value. Therefore, the application of the cadaverine probe to detect the content of cadaverine in feces is expected to assist in the diagnosis of ulcerative colitis. This method has a fast response speed, low price and strong selectivity, and has great potential for popularization and application. SUMMARY

[0005] Therefore, one of the purposes of the present application is to provide a fluorescent probe for detecting cadaverine; the second purpose of the present application is to provide a preparation method of the fluorescent probe for detecting cadaverine; and the third purpose of the present application is to provide applications of the fluorescent probe for detecting cadaverine in detecting meat freshness, diagnosing ulcerative colitis and preparing fluorescent materials.

[0006] To achieve the above-mentioned purposes, the present application provides the following technical solutions.

[0007] Further, the structural formula of the fluorescent probe XY1 is as follows:

[0008]

[0009] Further, the preparation method should include the following steps

[0010] Step (1): 5-bromothiophene-2-carboxaldehyde and 4-boronic acid triphenylamine are dissolved in tetrahydrofuran / water (9 / 1), cesium carbonate, tetrakis(triphenylphosphine)palladium are added as catalyst, stirred under nitrogen atmosphere, refluxed for 18h, remove tetrahydrofuran by rotary evaporation, then add water and dilute hydrochloric acid to adjust the pH to weakly acidic, filter, and separate and purify the filter cake to obtain compound 1.

[0011] The structural formula of the compound 1 is as follows:

[0012]

[0013] Step (2): Compound 1 obtained in step (1) is dissolved in anhydrous ethanol, then ethyl cyanoacetate and triethylamine are added, and stirred and refluxed for 12 hours. After the reaction is completed, ethanol is removed by rotary evaporation, and separation and purification are performed to obtain the fluorescent probe (ZY1).

[0014] Further, the 5-bromo-2-thiophene carboxaldehyde and [4-(diphenylamino) phenyl] boronic acid in step (1) are characterized in that the molar ratio is 1:1.1.

[0015] Further, the separation and purification in step (1) is characterized in that silica gel column chromatography is used, and the mobile phase is ethyl acetate / petroleum ether (1 / 8-20), preferably 1 / 10.

[0016] Further, the compound 1 and ethyl cyanoacetate in step (2) are characterized in that the molar ratio is 1:1-1.5.

[0017] Further, the separation and purification in step (2) is characterized in that silica gel column chromatography is used, and the mobile phase is ethyl acetate / petroleum ether (1 / 6-20), preferably 1 / 8.

[0018] The above-mentioned fluorescent probe ZY1 has applications in food freshness detection, ulcerative colitis diagnosis, and preparation of fluorescent materials.

[0019] The present application has the beneficial effect that the present application provides a fluorescent probe for detecting cadaverine. The probe takes triphenylamine thiophene as an electron donor (Donor, D), ethyl cyanoacetate as an electron acceptor (Acceptor, A), and a carbon-carbon double bond (olefin bond) as a pi bridge, thereby obtaining the above-mentioned fluorescent probe. The probe can specifically detect cadaverine / putrescine, and has high sensitivity, significant fluorescence color change, and strong anti-interference to other biological amines. Based on the above excellent properties of the fluorescent probe, we associate the fluorescence color change of the probe with the freshness of meat by using the RGB analysis software of a smart phone, and realize real-time monitoring of the freshness of meat. In addition, we also apply the fluorescent probe to the detection of cadaverine in the feces of mice with ulcerative colitis, which is expected to realize the auxiliary diagnosis of ulcerative colitis; based on the difference in fluorescence color of the fluorescent probe under ultraviolet light and visible light and the change in fluorescence color after reaction with cadaverine, we also successfully use it to prepare a series of fluorescent materials.

[0020] Other advantages, objects, and features of the present application will be in part apparent and in part pointed out hereinafter in the specification, and will be learned from a reading of the following specification and by practicing the present application. The objects and other advantages of the present application will be realized and attained by the structure particularly pointed out in the written description and claims hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to make the purposes, technical solutions and advantages of the present application clearer, the preferred detailed description of the present application will be combined with the drawings, in which:

[0022] Figure 1 The H NMR chart of compound 1 prepared in step (1) of Example 1 1 H NMR chart;

[0023] Figure 2 The C NMR chart of compound 1 prepared in step (1) of Example 1 13 C NMR chart;

[0024] Figure 3 The H NMR chart of fluorescent probe (ZY1) prepared in step (2) of Example 1 1 H NMR chart;

[0025] Figure 4 The C NMR chart of fluorescent probe (ZY1) prepared in step (2) of Example 1 13 C NMR chart;

[0026] Figure 5UV absorption spectrum, fluorescence performance test chart, CIE 1931 fluorescence spectrum chromaticity chart of the fluorescent probe (ZY1) prepared in step (2) of Example 1 after reacting with different concentrations of cadaverine, and fluorescence performance test chart of the fluorescent probe (ZY1) after reacting with different kinds of biological amines, ions, etc. Among them, (a) is the fluorescence spectrum chart of the fluorescent probe (ZY1) with a concentration of 10 μM after reacting with cadaverine with a concentration ranging from 0 to 100 μM; (b) is the linear relationship chart of the fluorescence intensity-cadaverine concentration in (a); (c) is the UV absorption spectrum chart of the fluorescent probe (ZY1) with a concentration of 10 μM after reacting with cadaverine with a concentration ranging from 0 to 100 μM; (d) is the CIE 1931 fluorescence spectrum chromaticity chart of the fluorescent probe (ZY1) with a concentration of 10 μM after reacting with cadaverine with a concentration ranging from 0 to 100 μM; (e) is the fluorescence performance test chart of the fluorescent probe (ZY1) with a concentration of 10 μM after reacting with different kinds of biological amines and ions with a concentration of 100 μM; (f) is the fluorescence intensity of the reaction solution in (e);

[0027] Figure 6 Reaction mechanism chart of the fluorescent probe (ZY1) prepared in step (2) of Example 1 after reacting with cadaverine, electrostatic potential surface chart of the fluorescent probe (ZY1), and HOMO-LUMO energy level calculation energy chart and corresponding electron density distribution. Among them, (a) is the reaction mechanism chart of the fluorescent probe (ZY1) after reacting with cadaverine; (b) is the electrostatic potential surface chart of the fluorescent probe (ZY1); (c) is the HOMO-LUMO energy level calculation energy chart and corresponding electron density distribution of the fluorescent probe (ZY1);

[0028] Figure 7 Chart of the fluorescent probe (ZY1) prepared in step (2) of Example 1 for detecting the freshness of shrimp meat. Among them, (a) is the chart of the fluorescent probe (ZY1) prepared in step (2) of Example 1 for detecting the shrimp meat over time; (b) is the chart of the solution after reacting with the fluorescent probe (ZY1) under the ultraviolet lamp; (c) is the chart of the G / R value-time relationship of the color of the solution after reacting in (b) analyzed by the RGB analysis software of the smart phone;

[0029] Figure 8Figure for the fluorescent probe (ZY1) prepared in step (2) of Example 1 for the auxiliary diagnosis of ulcerative colitis. Among them, (a) is a schematic diagram of the fluorescent probe (ZY1) for the auxiliary diagnosis of ulcerative colitis; (b) is a display diagram under ultraviolet light after the fecal extract of mice in the 3% DSS modeling group reacts with the fluorescent probe (ZY1). The left figure is the diagram after the fecal extract of mice on the first day reacts with the probe, and the right figure is the diagram after the fecal extract of mice on the fourth day reacts with the probe; (c) is a diagram of the G value after the color of the solution obtained in (b) is analyzed by the RGB analysis software of a smartphone; (d) is a diagram of the body weight change of mice in the 3% DSS modeling group and the control group from 0 to 7 days; (e) is a diagram of the colon of mice in the 3% DSS modeling group and the control group; (f) is a schematic diagram of the DAI index of mice in the 3% DSS modeling group and the control group; (g) is a diagram of the colon length of mice in the 3% DSS modeling group and the control group;

[0030] Figure 9 Figure for making a fluorescent material by using the fluorescent probe (ZY1) prepared in step (2) of Example 1 and its properties. Among them, (a) is a schematic diagram of using the ZY1-DMSO solution to stain filter paper and then cut it into a certain shape; (b) is a schematic diagram of using the DMSO solution of cadaverine as ink to write the character "ding" on a silica gel plate paved with ZY1-EtOH; (c) is a schematic diagram of using the DMSO solution of cadaverine as ink to write on a silica gel plate paved with ZY1-DMSO; Detailed implementation manners

[0031] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the diagrams provided in the following examples only illustrate the basic concept of the present invention in a schematic manner. Without conflict, the following examples and the features in the examples can be combined with each other.

[0032] Example 1

[0033] (1) Preparation of Compound 1

[0034] A mixture of 4-boronic acid triphenylamine (0.64 g, 2.20 mmol) and 5-bromothiophene-2-carboxaldehyde (0.38 g, 2.00 mmol), cesium carbonate (0.24 g, 4.00 mmol), tetrakis(triphenylphosphine)palladium (0.04 g, 0.2 mmol), tetrahydrofuran (18 mL) and water (2 mL) was degassed for 2 minutes, and the reaction was heated to reflux under a nitrogen atmosphere. The reaction progress was monitored by TLC (ethyl acetate / petroleum ether = 1 / 7), and the reaction was complete after about 18 hours. The solution was cooled to room temperature, and the THF was evaporated. The residue was diluted with 10 mL of water, and the pH was adjusted to weakly acidic with dilute hydrochloric acid. The crude product was obtained by suction filtration. The crude product was separated by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1 / 10), and the yield was 55%. 1 HNMR (600 MHz, DMSO-d6) δ 9.86 (s, 1H), 7.99 (d, J = 4.0 Hz, 1H), 7.69-7.65 (m, 2H), 7.59 (d, J = 4.0 Hz, 1H), 7.36-7.32 (m, 4H), 7.12 (t, J = 7.4 Hz, 2H), 7.08 (dd, J = 8.5, 1.1 Hz, 4H), 6.96-6.93 (m, 2H) (see Figure 1 ). 13 C NMR (151 MHz, DMSO-d6) δ 183.66, 152.90, 148.53, 146.40, 140.86, 139.46, 129.75, 127.39, 125.52, 124.96, 124.08, 123.95, 121.69 (see Figure 2 ).

[0035] (2) Preparation of fluorescent probe (ZY1): The compound 1 obtained in step (1) (0.355 g, 1.00 mmol) and ethyl cyanoacetate (0.113 g, 1.00 mmol) were dissolved in anhydrous ethanol (5 mL), and triethylamine (0.01 g, 0.1 mmol) was added. After mixing, the reaction was carried out at 78°C under reflux, and the reaction progress was monitored by TLC (ethyl acetate / petroleum ether = 1 / 3). The reaction was complete after about 12 hours. After the reaction was complete, the ethanol was removed by a rotary evaporator, and the crude product was separated by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1 / 8). The yield was 75%. 1HNMR (600MHz, DMSO-d6) δ8.51(s,1H),8.03(d,J=4.1Hz,1H),7.68(d,J=8.7Hz,2H),7.64(d,J=4.1Hz,1H),7.36(t,J=7.9H z, 4H), 7.14 (t, J = 7.4Hz, 2H), 7.10 (d, J = 7.6Hz, 4H), 6.96 (d, J = 8.7Hz, 2H), 4.28 (q, J = 7.1Hz, 2H), 1.29 (t, J = 7.1Hz, 3H) (see Figure 3 ); 13 C NMR (151MHz, DMSO) δ 162.40, 153.96, 148.79, 147.10, 146.29, 142.50, 133.35, 129.78, 127.53, 125.10, 124.25, 124.05, 121.50, 116.19, 95.77, 61.96, 14.07 (see...) Figure 4 ).

[0036] Example 2

[0037] 1. Study on the responsiveness of probe (ZY1) to cadaverine

[0038] ZY1 and cadaverine were dissolved and diluted in DMSO to prepare solutions with ZY1 concentration of 10 μM and cadaverine concentrations of 0, 1, 3, 5, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μM. These solutions were heated at 40 °C for 3 hours, and UV-Vis absorption spectroscopy (UV-Vis) and fluorescence spectroscopy (PL) were performed. UV-Vis spectroscopy results (5c) showed that, at a fixed ZY1 concentration, the maximum absorption peak intensity of ZY1 at 475 nm gradually decreased with increasing cadaverine concentration, while the absorption at 350 nm gradually increased. This phenomenon indicates that probe ZY1 specifically reacted with different concentrations of cadaverine, proving its feasibility as a cadaverine detection probe. Fluorescence spectroscopy (PL) showed ( Figure 5 a) Under a fixed excitation wavelength, after the probe ZY1 reacts with cadaverine, a maximum fluorescence emission peak is generated at 505 nm (λex = 390 nm). The fluorescence intensity of this emission peak increases significantly with increasing cadaverine concentration. Within the cadaverine concentration range of 1-100 μM, the fluorescence intensity shows a good linear relationship with the cadaverine concentration, and the linear regression equation is y = 250.5961x - 347.7344 (R0). 2 =0.99234), the limit of detection (LOD) is 13.8 nM ( ). Figure 5 b). Simultaneously, the CIE 1931 XYZ chromaticity coordinate diagram ( Figure 5d) Further confirmed that the luminescence color of the solution after reaction changed obviously under the irradiation of ultraviolet light or visible light.

[0039] 2. Interference study of cadaverine by probe (ZY1)

[0040] To systematically evaluate the specific recognition ability of probe ZY1 to cadaverine, mixed solutions of ZY1 concentration of 10 μM and various interference substances of 100 μM were prepared, and the solution of cadaverine with the same concentration was used as a positive control. After constant temperature reaction at 40 °C for 30 minutes, fluorescence spectrum scanning was carried out under excitation at λex=390 nm. The results showed that Figure 5 e) Only cadaverine and its structural analog putrescine triggered significant enhancement of fluorescence intensity at 505 nm (cadaverine > 170 times, putrescine > 180 times), while the rest of the interference substances (including biological amines: histamine, ethylamine, spermine, tyramine, cyclohexylamine, aniline; amino acids: methionine, phenylalanine, lysine; inorganic substances: HCO3 - , Mg 2+ , Zn 2+ , Cl - , I - , Ca 2+ ; small molecules: ammonia, hydrazine hydrate, glucose) maintained low fluorescence emission level at 505 nm Figure 5 f), indicating that ZY1 has high selectivity to cadaverine, and its cross-reactivity to putrescine is due to the similar structure of fatty diamines, but the probe can effectively exclude the influence of 18 common interference substances in complex biological systems.

[0041] 3. Sensing mechanism of probe (ZY1) for detecting cadaverine

[0042] To clarify the sensing mechanism of probe ZY1, Gaussian 09W and GaussView 5.0 software were used to optimize the molecular configuration of ZY1 and its reaction product with cadaverine (Cad) Figure 6 b), indicating that the ethylene double bond of ZY1 structure has the highest electrostatic potential, which makes it easy to react with cadaverine by nucleophilic addition. Based on density functional theory (DFT), the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energy levels of the two were calculated. The results showed that the HOMO-LUMO energy gap (ΔE) of ZY1-Cad reactant Figure 6 a) is significantly larger than that of free ZY1 Figure 6c) This trend is consistent with the fluorescence attenuation phenomenon. Orbital distribution analysis further revealed that the HOMO / LUMO electron clouds of ZY1 are widely distributed in the benzothiophene (D) and ethyl cyanoacetate (A) units, confirming that its D-π-A structure has a strong delocalized conjugated system; while the frontier orbital electron clouds of the ZY1-Cad reactant are concentrated in the benzothiophene fragment, and the destruction of the conjugated system leads to a blue shift in the emission wavelength.

[0043] Example 3

[0044] To investigate the application potential of this product in food freshness testing, commercially available fresh shrimp were used as samples and allowed to naturally decompose at room temperature (25℃). Samples were taken at 0h, 8h, 20h, 30h, and 46h. Figure 7 a) Take 10g of shrimp meat from each group, add 10mL of DMSO to wash away the cadaverine on the surface of the shrimp meat, then dissolve and dilute ZY1 in the washing solution to a concentration of 10μM. Incubate this solution at 25℃ in the dark for 15min, then capture fluorescence images under a 365nm UV lamp. Quantify the intensity ratio (G / R) of the green and red channels using RGB analysis software on a smartphone. Figure 7 b). The results showed that as the putrefaction time increased, the solution color gradually changed from light blue to bright green, and the G / R value was significantly positively linearly correlated with the putrefaction time (y = 0.0045x + 0.75339, R). 2 =0.96, P<0.001)( Figure 7 c). This phenomenon stems from the continuous accumulation of biogenic amines such as cadaverine during the spoilage of shrimp meat, confirming that ZY1 can achieve rapid and convenient detection of food freshness through a visual semi-quantitative strategy.

[0045] Example 4

[0046] To explore the potential application of probe ZY1 in the auxiliary diagnosis of ulcerative colitis (UC), a disease model was established using C57BL / 6 mice: the experimental group (n=5) was given drinking water containing 3% dextran sulfate sodium (DSS) to induce UC, while the control group (n=5) received normal drinking water; both groups had free access to food. Fecal samples were collected daily in the morning, homogenized in DMSO, centrifuged (12000 rpm, 10 min), filtered, and the supernatant was added to adjust the ZY1 concentration to 10 μM. Figure 8 a) After incubation at 25°C in the dark for 15 min, fluorescence images were captured under a 365nm UV lamp, and the green channel intensity (G value) was quantified using RGB software on a smartphone. Results showed that the experimental group mice exhibited a typical UC phenotype on day 4, with significant weight loss. Figure 8 d) Significantly shortened colon length ( Figure 8 e, 8g), and the Disease Activity Index (DAI) score was significantly increased ( Figure 8f), which confirmed the success of modeling. The synchronous fluorescence analysis showed that the G value of the fecal extract of the experimental group on the 4th day was significantly higher than that on the 1st day, and the color of the solution changed from light blue to bright green Figure 8 b, Figure 8 c), while there was no significant change in the control group Figure 8 b). This phenomenon is directly related to the up-regulation of cadaverine biosynthesis caused by intestinal flora imbalance in the development of UC, which confirms that ZY1 can dynamically monitor the severity of UC by detecting the level of cadaverine in feces, providing a new strategy for non-invasive diagnosis.

[0047] Example 5

[0048] Based on the excellent solubility and photophysical stability of the probe ZY1, this study expands its application scenarios as a multifunctional fluorescent material. First, 10 μM ZY1-DMSO solution was soaked in filter paper, and after dyeing and drying, it showed uniform fluorescence Figure 9 a), which confirmed that it can be used as a solid-state fluorescent dye; then, an information encryption material was constructed, and a solvent-regulated invisible ink system was designed based on the colorimetric response characteristics of ZY1 changing from light yellow to colorless under sunlight after reacting with Cad. Using 1 mM cadaverine solution as "ink", it was written on silica gel plates coated with 10 μM ZY1-ethanol (EtOH) or ZY1-DMSO solution, respectively Figure 9 b, Figure 9 c). Under sunlight, the writing was completely hidden (not visible to the naked eye), while under 365 nm ultraviolet light, the cadaverine writing area formed a clear contrast due to fluorescence change (EtOH system) or enhancement (DMSO system), and the writing was clearly readable. This dual-mode response mechanism realizes solvent-dependent information development control for the first time, which can be used for the development of high-level anti-counterfeiting materials.

[0049] In summary, the present application provides a high-performance fluorescent probe (ZY1) for detecting cadaverine. The probe has the following outstanding features: rapid response, low detection limit, obvious color change after reacting with cadaverine, and high selectivity. Within the range of 1-100 μM cadaverine concentration (probe concentration is 10 μM), the fluorescence intensity of the probe shows a good linear relationship with the concentration of cadaverine. Since cadaverine is generated in large quantities during food spoilage, and intestinal flora imbalance leads to up-regulation of cadaverine biosynthesis in the development of ulcerative colitis (UC), the probe (ZY1) has broad application prospects in detecting food freshness and assisting in the diagnosis of ulcerative colitis due to its excellent performance. In addition, it is further found that the fluorescent probe is not only suitable for the above-mentioned fields, but also can be used for the preparation of new fluorescent materials and information transmission technology, thereby expanding its potential application range.

[0050] Finally, it is to be explained that the above embodiments are only used to illustrate the technical solutions of the present application but not to limit the present application, and although the present application is described in detail with reference to the best embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions, and all should be covered in the scope of the claims of the present application.

Claims

1. A fluorescent probe ZY1 for detecting cadaverine, characterized in that, The structural formula of the fluorescent probe is shown below:

2. The method for preparing the fluorescent probe ZY1 for detecting cadaverine according to claim 1, characterized in that, Includes the following steps: Step (1): Dissolve 4-boronic acid triphenylamine and 5-bromothiophene-2-carboxaldehyde in tetrahydrofuran / water (9 / 1), add cesium carbonate and tetra(triphenylphosphine)palladium as catalysts, stir and reflux for 18 hours under nitrogen atmosphere, remove tetrahydrofuran by rotary evaporation, then add water and dilute hydrochloric acid to adjust the pH to weakly acidic, filter, take the filter cake, separate and purify to obtain compound 1; The structural formula of compound 1 is shown below: Step (2): Dissolve compound 1 in anhydrous ethanol, add ethyl cyanoacetate and triethylamine, stir and reflux for 12 hours, remove ethanol by rotary evaporation, and separate and purify to obtain ZY1.

3. The preparation method according to claim 2, characterized in that, The molar ratio of 5-bromothiophene-2-carboxaldehyde to triphenylamine 4-boronic acid in step (1) is 1:1.

1.

4. The preparation method according to claim 2, characterized in that, The separation and purification in step (1) is performed by silica gel column chromatography, with ethyl acetate / petroleum ether (1 / 8 to 20), preferably 1 / 10, as the mobile phase.

5. The preparation method according to claim 2, characterized in that, The molar ratio of compound 1 to ethyl cyanoacetate in step (2) is 1:1 to 1.

5.

6. The preparation method according to claim 2, characterized in that, The separation and purification in step (2) is performed using silica gel column chromatography with ethyl acetate / petroleum ether (1 / 6 to 20) as the mobile phase, preferably 1:

8.

7. The application of the fluorescent probe ZY1 for detecting cadaverine as described in claim 1 in food freshness detection, ulcerative colitis diagnosis, and fluorescent material preparation.

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