Fluorescent probe capable of rapidly responding to amine and application of sensing label of fluorescent probe and gel dual-channel indication of freshness of fish meat

Abstract

The invention discloses a fluorescent probe capable of rapidly responding to amine and application of a sensing label and gel double-channel indication of freshness of fish meat. The structural formula of the fluorescent probe is as follows: # imgabs0 #. The fluorescent probe has the advantages of simple synthesis route, high response speed, colorimetric and fluorescent double-channel response and the like. A fish freshness standard colorimetric card prepared by using the probe is divided into a sunlight and ultraviolet light fresh colorimetric zone, a sunlight and ultraviolet light qualified colorimetric zone and a sunlight and ultraviolet light decay colorimetric zone, and the sensing label and the sensing gel are combined with the standard colorimetric card to realize real-time monitoring of the fish freshness without destroying a sample and complex pretreatment. The result is accurate and reliable.

Description

Technical Field

[0001] The present invention relates to an amine-rapid-response fluorescent probe, its sensing label, and the application of a gel dual-channel for indicating the freshness of fish. Background Art

[0002] China is rich in seafood resources. In particular, seawater fish are delicious and highly nutritious, being an indispensable delicacy on people's dinner tables. However, seawater fish are extremely prone to spoilage and deterioration during long-distance transportation, processing, and storage, and consumption by consumers can cause harm to the body. Currently, most deep-sea fish are sold after being sliced or cut into sections. To extend the shelf life of the fish, they are generally sealed in plastic bags or vacuum-packed. To determine the freshness of the fish, the packaging often needs to be opened, and electronic noses, electronic tongues, or other instruments are used to test the freshness indicators for a comprehensive determination of spoilage. However, these operations must damage the product packaging, require complex instruments, and take a long time, and cannot meet on-site detection requirements, etc. Since fish spoilage produces a large amount of volatile amines, a simple and convenient method can be developed to detect volatile amines, thereby realizing the monitoring of the freshness of fish.

[0003] In recent years, fluorescence analysis has attracted wide attention due to its high sensitivity, rapid response, good selectivity, the need for no complex instruments, and real-time detection capabilities. In the past thirty years, a large number of fluorescent probes for the sole detection of volatile amines have been developed, and these probes also exhibit good application performance. However, compared with dual-channel probes, colorimetric probes often require higher R & D costs and lower utilization rates. Therefore, dual-channel probes have more application prospects. In view of the need for food safety detection, there is an urgent need to develop a dual-channel fluorescent probe. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an amine-rapid-response fluorescent probe, its sensing label, and the application of a gel dual-channel for indicating the freshness of fish. This fluorescent probe has the advantages of a simple synthesis route, fast recognition speed, excellent selectivity, and colorimetric and fluorescence dual-channel responses. At the same time, by preparing the probe into a sensing label and a sensing gel, qualitative analysis of the freshness of fish can be achieved, without damaging the sample or requiring complex pretreatment, and the results are accurate and reliable, and can be used for non-destructive, rapid, and real-time detection of the freshness of salmon fish.

[0005] The technical solution of the present invention is as follows:

[0006] An amine-rapid-response fluorescent probe, the structural formula of this fluorescent probe is as follows:

[0007]

[0008] Furthermore, the specific synthesis steps of this fluorescent probe are as follows:

[0009] Using 10 - 100 mL of ethanol as a solvent, charge the julolidine derivative and 2 - thiobarbituric acid according to a molar ratio of 1:(2 - 3.5), and the julolidine derivative is 8 - hydroxyjulolidine - 9 - formaldehyde Then, add acetic acid according to a mass ratio of 25:1 of the julolidine derivative to acetic acid; heat under reflux and stir in an oil bath for 1 hour to 8 hours, cool to room temperature, filter by suction, collect the solid and wash it with ethanol to obtain a fluorescence probe with rapid response to amines

[0010] An application of a fluorescence probe with rapid response to amines, characterized in that: detecting biogenic amines in an EtOH:H 2 O (v / v, 5 / 5) system with pH = 7.4, and the application is for non - disease diagnosis and non - disease treatment purposes

[0011] An application of a fluorescence probe with rapid response to amines, characterized in that: it can recognize diethylamine within 6 s in an EtOH:H 2 O (v / v, 5 / 5) system with pH = 7.4, and the application is for non - disease diagnosis and non - disease treatment purposes

[0012] A freshness sensing label for fish meat prepared from a fluorescence probe with rapid response to amines, characterized in that: the filter paper loaded with the fluorescence probe is used as the sensing label, which can monitor the freshness of salmon fish meat, and the application is for non - disease diagnosis and non - disease treatment purposes

[0013] A freshness sensing gel for fish meat prepared from a fluorescence probe with rapid response to amines, characterized in that: the agarose loaded with the fluorescence probe is used as the sensing gel, which can monitor the freshness of salmon fish meat, and the application is for non - disease diagnosis and non - disease treatment purposes

[0014] The specific preparation process of the sensing label is as follows:

[0015] Weigh 3.43 mg of the fluorescence probe and dissolve it in 10 mL of DMSO. Place the cut round filter paper with a diameter of 2 cm in the above solution and soak it overnight, then take it out and dry it in an oven to obtain the sensing label

[0016] An application of a freshness sensing label for salmon fish meat in determining the freshness of salmon fish meat

[0017] An application of a freshness sensing label for salmon fish meat in determining the freshness of salmon. Place the sensing label and the fish meat sample in a packaging box, and the sensing label does not directly contact the sample. Compare the color change of the sensing label with a standard colorimetric card to monitor the freshness of salmon fish meat in real - time and identify the freshness of salmon fish meat

[0018] Under visible light, a pinkish-purple color of the sensing label indicates freshness, a gray color indicates qualification, and a wheat color indicates unqualified.

[0019] Under ultraviolet light at 365 nm, when the color of the sensing label is bluish-purple without fluorescence, it indicates freshness; when the color is weak blue fluorescence, it indicates qualification; when the color is strong green fluorescence, it indicates unqualified.

[0020] The specific preparation process of the sensing gel is as follows:

[0021] Dissolve 0.3 g of agarose in 15 mL of distilled water, add 0.2 mL of glycerol, heat the mixed solution with stirring until it becomes clear and transparent. When cooled to 60 °C, dissolve 5 mg of the fluorescent probe in 1 mL of DMSO, quickly add it to the agarose solution, continue stirring until evenly mixed, then quickly pour the mixture into a petri dish with a diameter of 9 cm. Use a glass rod to drive the bubbles to the edge of the petri dish. After the agarose solution cools to room temperature, it solidifies into a block. Divide it into agarose with a diameter of 1 cm to obtain the sensing gel.

[0022] Application of a freshness sensing gel for salmon fish in determining the freshness of salmon fish.

[0023] Application of a freshness sensing gel for salmon fish in determining the freshness of salmon. Place the sensing gel and the fish sample in the packaging box, and ensure that the sensing gel does not directly contact the sample. Compare the color change of the sensing gel with the standard colorimetric card to monitor the freshness of the salmon fish in real time and identify the freshness of the salmon fish.

[0024] Under visible light, a wine-red color of the sensing gel indicates freshness, an orange-red color indicates qualification, and a dark khaki color indicates unqualified.

[0025] Under ultraviolet light at 365 nm, when the color of the sensing gel is purple without fluorescence, it indicates freshness; when the color is weak blue fluorescence, it indicates qualification; when the color is strong green fluorescence, it indicates unqualified.

[0026] Advantages of the present invention:

[0027] (1) The fluorescent probe can rapidly respond to various volatile amines in the EtOH:H 2 O (v / v, 5 / 5) system at pH = 7.4, with a low detection limit and obvious color change.

[0028] (2) The sensing tag loaded with the fluorescent probe and the sensing gel, in combination with a standard colorimetric card, can indicate the freshness of fish through dual-channel colorimetric and fluorescence methods. A single probe enables dual-channel indication of fish freshness, enhancing the reliability of the results. Dual-channel detection of fish freshness can be achieved in various media, indicating that the probe is not interfered by the media, saving R & D costs and improving the utilization rate of the probe.

[0029] In summary, the fluorescent probe designed and synthesized in the present invention can identify various amine solutions in the EtOH:H 2 O (v / v, 5 / 5) system with pH = 7.4. Meanwhile, it can also be prepared into a sensing tag and a sensing gel to identify various volatile amines. The designed sensing tag and sensing gel are simple to prepare, and can achieve dual-channel colorimetric and fluorescence detection of fish freshness, with more accurate and reliable qualitative analysis. The sensing tag and the sensing gel can non-contact and non-destructively monitor the freshness of seawater fish in real time, providing effective freshness information for producers, retailers and consumers in a timely manner, and having good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the 1 H NMR spectrum of the fluorescent probe HT of the present invention;

[0031] Figure 2 is the 1 H NMR spectrum of the fluorescent probe HTB of the present invention;

[0032] Figure 3 is the 1 H NMR spectrum of the fluorescent probe HTBR of the present invention;

[0033] Figure 4 is the 13 C NMR spectrum of the fluorescent probe HTBR of the present invention;

[0034] Figure 5 is the mass spectrum of the fluorescent probe HTBR of the present invention;

[0035] Figure 6 is the ultraviolet absorption spectrum and daylight color change diagram of the fluorescent probe HTBR of the present invention before and after adding various amine (1. cyclohexanediamine, 2. diethylamine, 3. n-propylamine, 5. triethylamine, 6. ethylamine, 7. spermine, 8. cadaverine, 9. putrescine, 10. 2-phenylethylamine, 11. tyramine, 12. tryptamine, 13. aniline, 14. trimethylamine, 15. dimethylamine, 16. ammonia water, 17. hydrazine, 18. histamine) compounds;

[0036] Figure 7It is the fluorescence emission spectrum and fluorescence color change diagram of the fluorescence probe HTBR of the present invention before and after adding various amine (cyclohexanediamine, diethylamine, n-propylamine, triethylamine, ethylamine, spermine, cadaverine, putrescine, 2-phenylethylamine, tyramine, tryptamine, aniline, trimethylamine, dimethylamine, ammonia water, hydrazine, histamine) compounds;

[0037] Figure 8 It is the ultraviolet absorption spectrum of the fluorescence probe HTBR of the present invention after adding different concentrations of diethylamine;

[0038] Figure 9 It is the fluorescence emission spectrum of the fluorescence probe HTBR of the present invention after adding different concentrations of diethylamine;

[0039] Figure 10 It is the detection limit diagram of the fluorescence probe HTBR of the present invention for recognizing diethylamine;

[0040] Figure 11 It is the fluorescence intensity change diagram of the fluorescence probe HTBR of the present invention at different pH values before and after adding diethylamine;

[0041] Figure 12 It is the change diagram of the fluorescence intensity of the fluorescence probe HTBR of the present invention over time after adding diethylamine;

[0042] Figure 13 It is the screenshot of the change of daylight color over time after adding diethylamine to the fluorescence probe HTBR of the present invention;

[0043] Figure 14 It is the photos of daylight color change (a, c) and fluorescence color (b, d) before and after the prepared sensing label HTBR-L and sensing gel HTBR-G of the present invention recognize volatile amines;

[0044] Figure 15 It is the change diagram of the TVB-N content of salmon fish meat at 4°C with the extension of storage time;

[0045] Figure 16 It is the change diagram of the number of colony species of salmon fish meat at 4°C with the extension of storage time;

[0046] Figure 17 It is the change diagram of the color difference value of the sensing label and the sensing gel of salmon fish meat at 4°C with the extension of storage time;

[0047] Figure 18 It is the color photos of the TVB-N content of salmon fish meat at 4°C with the extension of storage time and the corresponding sensing label and sensing gel under natural light and ultraviolet light irradiation;

[0048] Figure 19It is a standard color comparison card made according to the TVB-N value of fish meat and the colors of the corresponding sensing label HTBR-L and sensing gel HTBR-G under natural light and ultraviolet light irradiation; from left to right on the upper layer of the standard color comparison card are the fresh natural light color comparison area, the qualified natural light color comparison area, and the spoiled natural light color comparison area, and from left to right on the lower layer of the standard color comparison card are the fresh ultraviolet light color comparison area, the qualified ultraviolet light color comparison area, and the spoiled ultraviolet light color comparison area;

[0049] Figure 20 It is the monitoring of the freshness of salmon fish meat by combining the color changes of the sensing label HTBR-L and sensing gel HTBR-G prepared in the present invention with salmon samples stored at 4°C under sunlight and ultraviolet light and the standard color comparison card; the fish meat on the 0th day of storage at 4°C belongs to fresh products, when stored for 4 days, the fish meat belongs to qualified products at this time, and when stored for 8 days, the fish meat belongs to unqualified products;

[0050] Figure 21 It is a graph showing the change of the TVB-N content of salmon fish meat with the extension of storage time at 25°C;

[0051] Figure 22 It is the monitoring of the freshness of salmon fish meat by combining the color changes of the sensing label HTBR-L and sensing gel HTBR-G prepared in the present invention with salmon samples stored at 25°C under sunlight and ultraviolet light and the standard color comparison card; the fish meat on the 0th hour of storage at 25°C belongs to fresh products, when stored for 11 hours, the fish meat belongs to qualified products at this time, and when stored for 21 hours, the fish meat belongs to unqualified products;

[0052] Figure 23 It is the application of the fluorescent probe HTBR of the present invention as a fluorescent ink, and the English letter imprints, Chinese characters and fingerprints collected under natural light (a, c) and ultraviolet light (b, d);

[0053] Figure 24 It is a photograph of the sensing label HTBR-L (fresh, qualified, spoiled) prepared in the present invention under the magnification of a scanning electron microscope (1.00K, 10.00K, 25.00K);

[0054] Figure 25 It is the RGB value (a), the linear relationship between TVB-N and the G / B ratio (b), and the smartphone verification (c) of the standard color comparison card prepared in the present invention. Detailed implementation manners

[0055] The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments.

[0056] Example 1

[0057] The specific synthesis steps of compound HT are as follows:

[0058]

[0059] Compound 3-aminophenol (109.13 mg, 1 mmol), 1-bromo-3-chloropropane (629.76 mg, 4 mmol), and sodium bicarbonate (336.04 mg, 4 mmol) were dissolved in N,N-dimethylformamide (2 mL). The mixture was refluxed and stirred in an oil bath for 12 hours, cooled to room temperature, the reaction solution was poured into ice water, the solution was extracted with ethyl acetate, and then washed with water. The remaining solution was dried over anhydrous sodium sulfate, the organic phase was evaporated under reduced pressure, and the residue was purified by column chromatography (ethyl acetate: petroleum ether = 1:5) to obtain white compound HT with a yield of 68.3%; 1 The H NMR spectrum is as Figure 1 shown.

[0060] The specific synthesis steps of compound HTB are as follows:

[0061]

[0062] Dry N,N-dimethylformamide (2 mL) was added dropwise to phosphorus oxychloride (2 mL) under nitrogen at room temperature, and the mixture was stirred for 30 minutes. Compound HT (189 mg) was dissolved in dry N,N-dimethylformamide (2 mL) and poured into this solution. After stirring at room temperature for 30 minutes, the solution was heated to 60 °C. After 30 minutes, the reactant was slowly added to ice water and aged for 2 hours. The precipitate HTB was collected by filtration with a yield of 78%; 1 The H NMR spectrum is as Figure 2 shown.

[0063] The specific synthesis steps of the fluorescent probe HTBR are as follows:

[0064]

[0065] Compound HTB (217 mg, 1 mmol), 2-thiobarbituric acid (433 mg, 3 mmol), and piperidine (0.08 mL) were dissolved in ethanol (5 mL). The mixture was stirred at room temperature for 1 hour, refluxed for 2 hours, cooled to room temperature, filtered by suction, and the crude product, a black-purple solid, was collected and washed with ice ethanol to obtain the fluorescent probe HTBR with a yield of 58%; 1 The H NMR spectrum, 13 the C NMR spectrum, and the mass spectrum are as Figures 3 - 5 shown.

[0066] 1 H NMR (400 MHz, DMSO-d 6) δ 11.86 (s, 2H), 9.94 (s, 1H), 8.59 (s, 1H), 8.48 (s, 1H), 3.41–3.36 (m, 4H), 2.60–2.56 (m, 4H), 1.85–1.82 (m, 4H).

[0067] 13 C NMR (100 MHz, DMSO-d 6 ) δ 177.12, 159.53, 152.69, 149.23, 133.99, 115.68, 113.26, 106.06, 104.58, 56.47, 50.72, 49.89, 27.32, 21.44, 21.10, 20.37, 19.01.

[0068] HRMS (ESI – ) for C 17 H 16 N 3 O 3 S – [M–H] – calcd: 342.0918, found: 342.0867.

[0069] Example 2

[0070] Compound HTB (2.17 g, 10 mmol), 2-thiobarbituric acid (4.33 g, 3 mmol), piperidine (0.16 mL) were dissolved in ethanol (30 mL). Stirred at room temperature for 1 h, refluxed for 4 h, cooled to room temperature, filtered by suction, and the crude product, a black-purple solid, was collected and washed with ice ethanol to obtain the fluorescent probe HTBR with a yield of 47.7%; the 1 1H NMR spectrum of the fluorescent probe HTBR in this example is as shown in Figure 3 , 13 the 13C NMR spectrum is as shown in Figure 4 , and the high-resolution mass spectrum is as shown in Figure 5 .

[0071] Example 3

[0072] Compound HTB (21.7 mg, 0.1 mmol), 2-thiobarbituric acid (43.3 mg, 0.3 mmol), piperidine (0.08 mL) were dissolved in ethanol (3 mL). Stirred at room temperature for 1 h, refluxed for 2 h, cooled to room temperature, filtered by suction, and the crude product, a black-purple solid, was collected and washed with ice ethanol to obtain the fluorescent probe HTBR with a yield of 66.2%; the 1 1H NMR spectrum of the fluorescent probe HTBR in this example is as shown in Figure 3 , 13 the 13C NMR spectrum is as shown inFigure 4 , high-resolution mass spectrometry is as Figure 5 shown.

[0073] I. Selective detection of diethylamine by fluorescence probe HTBR:

[0074] In a 2 mL EtOH:H 2 O (v / v, 5 / 5) solution, add a solution of fluorescence probe HTBR to prepare a 10 μmol / L solution for use. Then, add 20 μL of 50 mmol / L solutions of various amines (1. cyclohexanediamine, 2. diethylamine, 3. n-propylamine, 5. triethylamine, 6. ethylamine, 7. spermine, 8. cadaverine, 9. putrescine, 10. 2-phenylethylamine, 11. tyramine, 12. tryptamine, 13. aniline, 14. trimethylamine, 15. dimethylamine, 16. ammonia water, 17. hydrazine, 18. histamine) respectively, and observe the changes in ultraviolet and fluorescence. From Figure 6 the ultraviolet spectrum, it can be seen that except for aniline, the solutions of the other 16 amine compounds cause the maximum absorption peak of HTBR at 530 nm to blue-shift to 470 nm, and the solution color changes from rose pink to yellow. From Figure 7 the fluorescence spectrum, it can be seen that except for aniline, the solutions of the other 17 amine compounds cause a significant increase in the fluorescence emission of HTBR at 510 nm. These results indicate that HTBR can perform colorimetric and fluorescence recognition of various amine compounds in the EtOH:H 2 O (v / v, 5 / 5) solution, with a wide recognition range.

[0075] II. Titration test of fluorescence probe HTBR for diethylamine:

[0076] In a 10 μmol / L solution of fluorescence probe HTBR in EtOH:H 2 O (v / v, 5 / 5), add 0 - 300 μmol / L of diethylamine respectively, and test the changes in the ultraviolet spectrum and fluorescence emission spectrum of the solution, as Figure 8 , 9 shown. When the concentration of the diethylamine solution gradually increases, the absorbance of the HTBR solution gradually decreases and the fluorescence emission intensity gradually decreases and then increases. When the concentration of diethylamine is 300 μmol / L, the absorbance and fluorescence emission intensity no longer change, indicating that the saturation state has been reached at this time.

[0077] III. Detection limit test of fluorescence probe HTBR for diethylamine:

[0078] In the EtOH:H 2 O (v / v, 5 / 5) solution of probe HTBR, test the fluorescence intensity of no less than 11 parallel samples. According to the formula ∑(X i -X) 2 =(X 1 -X)2 +(X 2 -X) 2 +……+(X n -X) 2 Find the sum of the squared differences (X i is the fluorescence intensity value of the receptor itself for each measurement, X is the average fluorescence intensity, n is the number of tests, n ≥ 11), and then according to the formula: S = [∑(X i -X) 2 / (n - 1)] 0.5 calculate the sensitivity S, and then according to the detection limit formula: Detection limit = 3S / K, where K is the slope of the selected linear part (Note: The straight line is plotted based on titration, with the ion concentration on the abscissa and the fluorescence intensity on the ordinate), and the detection limit is found to be 6.22 μmol / L (see Figure 10 ), indicating that the probe has a low detection limit and certain practical application value.

[0079] IV. pH response test of the fluorescent probe HTBR to diethylamine:

[0080] To confirm the practicality of HTBR, we investigated the influence of different pH values. As Figure 11 shown, HTBR is insensitive in the pH range of 2 - 8. After adding a saturated multiple of diethylamine, the fluorescence intensity increases significantly in the pH range of 5 - 10. HTBR can recognize diethylamine in a relatively wide pH range, indicating that the probe HTBR can detect diethylamine in biological systems and has potential application value.

[0081] V. Response time test of the fluorescent probe HTBR to diethylamine:

[0082] To explore the photostability of the fluorescent probe ( Figure 12 ) and the response time for recognizing amine compounds, in an EtOH:H 2 O (v / v, 5 / 5) solution, after adding 300 μmol / L of diethylamine solution to the probe HTBR (10 μmol / L), the color of the solution changes rapidly within 6 s under sunlight ( Figure 13 ), and the fluorescence signal of the HTBR solution increases significantly within 2 s until it levels off ( Figure 12 ). Such an ultrafast response speed lays the foundation for the subsequent preparation of sensing tags for real-time monitoring of the freshness of fish.

[0083] VI. Recognition effect of the sensing tag HTBR-L and the sensing gel HTBR-G on volatile amines in a simulated environment (a cylindrical glass bottle with a height of 65 mm and a diameter of 18 mm)

[0084] The preparation process of the sensing label HTBR-L is as follows: Weigh 3.43 mg of the fluorescent probe and dissolve it in 10 mL of DMSO. Place the cut circular filter paper with a diameter of 2 cm in the above solution and soak it overnight. Then take it out and dry it in an oven to obtain the sensing label HTBR-L.

[0085] The specific preparation process of the sensing gel HTBR-G is as follows: Dissolve 0.3 g of agarose in 15 mL of distilled water, add 0.2 mL of glycerol, heat the mixed solution with stirring until it is clear and transparent. When it cools to 60 °C, dissolve 5 mg of the fluorescent probe in 1 mL of DMSO, and quickly add it to the agarose solution. Continue to stir until it is evenly mixed. Then quickly pour the mixed solution into a petri dish with a diameter of 9 cm, and use a glass rod to drive the bubbles to the edge of the petri dish. After the agarose solution cools to room temperature, it solidifies into a block. Divide it into agarose with a diameter of 1 cm to obtain the sensing gel HTBR-G.

[0086] During the spoilage process of fish, some metabolic volatiles will be produced, such as triethylamine, n-propylamine, diethylamine, etc. Detecting these metabolic volatiles can monitor the freshness of fish. In order to verify whether the fabricated sensing label HTBR-L and sensing gel HTBR-G are selective to volatile amines, we placed the sensing label HTBR-L and sensing gel HTBR-G in the headspace of a 0.5% aqueous solution of volatile amines to simulate whether they respond to volatile amines in the fish spoilage environment.

[0087] First, take 1 mL of volatile amine with a concentration of 50 mmol / L and add it to 1 mL of distilled water to prepare a 25 mmol / L volatile amine solution; Select seventeen amines including cyclohexanediamine, diethylamine, n-propylamine, triethylamine, ethylamine, spermine, cadaverine, putrescine, 2-phenylethylamine, tyramine, tryptamine, aniline, trimethylamine, dimethylamine, ammonia water, hydrazine, and histamine as the experimental group, and the empty bottle of the sensing label as the blank control group. As Figure 14 shown, the daylight color of the sensing label HTBR-L is rose red and there is no fluorescence. When it comes into contact with different amines, except that the daylight colors of aniline and histamine do not change significantly, the other 15 amines turn into pink, orange, brown, etc. under daylight, and the fluorescence colors turn into pink, green, blue, yellowish brown, etc.; The daylight color of the sensing gel HTBR-G is dark red and there is no fluorescence. When it comes into contact with different amines, except that the daylight colors of aniline and histamine do not change significantly, the other 15 amines turn into red, yellow, orange, brown, etc. under daylight, and the fluorescence colors turn into pink, green, blue, yellow, etc. Both the daylight and fluorescence colors change significantly, indicating that the sensing label HTBR-L and sensing gel HTBR-G are sensitive to volatile amines and have good colorimetric and fluorescence dual responses, and also indicating that the sensing label HTBR-L and sensing gel HTBR-G have the potential to indicate the freshness of fish.

[0088] VII. Preparation of the Label Standard Color Card

[0089] To prepare the standard color card, we need to monitor the changes in the natural light color and the color under fluorescent lamp irradiation of the sensing label HTBR-L and the sensing gel HTBR-G during the process of fish meat spoilage from fresh to spoiled. We selected salmon fish meat as the experimental object, removed the skin from the back meat, divided the fish meat into small pieces of 5-6 g each, placed them in disposable round transparent plastic petri dishes respectively, put the sensing label in the headspace of the petri dish lid, tightened the lid, and stored them in a 4°C refrigerator. The TVB-N content, the number of colony species, and the color difference value of the salmon fish meat were measured every 24 hours (see Figures 15 - 17 ), and natural light photos and fluorescence photos of the sensing label HTBR-L and the sensing gel HTBR-G were collected. The obtained data are as shown in Figure 18 . The TVB-N content of fresh salmon fish meat showed an overall upward trend starting from the initial 6.25±0.06 mg / 100 g. The TVB-N content reached 15.12±0.06 mg / 100 g on the 4th day and 31.73±0.06 mg / 100 g on the 8th day, while the acceptable limit value of TVB-N is 30 mg / 100 g. Therefore, the 8th day of storage of salmon fish meat at 4°C can be regarded as the critical value of storage spoilage. The sensing label HTBR-L changed from the initial rose red to blue and finally to yellow under natural light; under 365 nm ultraviolet light, the sensing label HTBR-L changed from initially non-fluorescent to weak blue fluorescence and finally to green fluorescence; the sensing gel HTBR-G changed from the initial dark red to orange red and finally to yellow under natural light; under 365 nm ultraviolet light, the sensing gel HTBR-G changed from initially non-fluorescent to weak blue fluorescence and finally to green fluorescence.

[0090] According to the regulations in the General Rules for Fresh Seawater Fish (GB / T 18108-2019) of the People's Republic of China National Standard, the maximum limit of TVB-N content in seawater fish is 30 mg / 100 g. When the TVB-N value ≤ 15 mg / 100 g, the sample is of excellent grade; when 15 mg / 100 g < TVB-N value ≤ 30 mg / 100 g, the sample is qualified; when the TVB-N value > 30 mg / 100 g, the sample is unqualified.

[0091] Based on the measured TVB-N values and the corresponding photos of the sensing label HTBR-L's natural light color and ultraviolet light color, we selected the sensing labels corresponding to TVB-N values of 6.25 mg / 100 g and 7.93 mg / 100 g, which presented as purple and magenta respectively under natural light, and showed blue-violet without fluorescence and blue without fluorescence under ultraviolet light irradiation. The sensing gel HTBR-G presented as dark red under natural light and purple without fluorescence under ultraviolet light irradiation. As the reference standard for the fresh part in the color card, when the sensing label or gel shows one of the above color states, it can be judged that this sample is a fresh product (see Figure 19 the fresh area in Figure 19 ). We selected the sensing labels HTBR-L corresponding to TVB-N values of 17.45 mg / 100 g and 20.44 mg / 100 g, which presented as blue and light blue respectively under natural light, and showed weak blue fluorescence under ultraviolet light irradiation. The sensing gel HTBR-G presented as orange-red under natural light and showed weak fluorescence of blue and blue-green under ultraviolet light irradiation. As the reference standard for the qualified part in the standard color card, when the sensing label or gel shows one of the above color states, it can be judged that this sample is a qualified product (see Figure 18 the qualified area in

[0092] VIII. Practical Application of the Sensing Label

[0093] The sensing label HTBR-L and the sensing gel HTBR-G were used for the actual monitoring application of the freshness of salmon. The salmon samples were processed in the same way as described above. Figure 20The color changes of the sensing label HTBR-L and the sensing gel HTBR-G of salmon with increasing storage time during storage in a 4 °C refrigerator are shown. In newly purchased salmon, the sensing label HTBR-L appears pink-purple under natural light and emits strong blue-violet non-fluorescence under ultraviolet light. The sensing gel HTBR-G appears wine-red under natural light and emits strong purple non-fluorescence under ultraviolet light, corresponding to the fresh part in the standard color comparison card. When the storage time is 4 days, the natural light color of the sensing label HTBR-L is gray and it is blue non-fluorescent under ultraviolet light. The sensing gel HTBR-G appears orange-red under natural light and emits strong blue weak fluorescence under ultraviolet light, indicating that it is still a qualified product at this time. When the storage time is further increased to 8 days, the natural light color of the sensing label HTBR-L is wheat-colored and it emits strong green fluorescence under ultraviolet light. The sensing gel HTBR-G appears dark khaki under natural light and emits strong green fluorescence under ultraviolet light, indicating that the salmon has spoiled at this time and the unqualified product is inedible. This sensing label can accurately judge the actual freshness of salmon fish through the change of dual-mode signals and by comparing with the standard color comparison card, which can help consumers purchase qualified fish products and is of great significance for the safety detection of seafood.

[0094] To explore the corresponding relationship between the color of the sensing label HTBR-L and the freshness of salmon, the sensing label and salmon fish were stored at 25 °C, and the TVB-N value was measured. The results are as Figure 21 shown. The TVB-N value showed an overall upward trend starting from (9.2 ± 0.15) mg / 100 g and reached (14.6 ± 0.42) mg / 100 g at the 11th h, indicating that the fish sample was qualified at this time. It reached (30.52 ± 0.16) mg / 100 g at the 21st h, indicating that the fish sample had spoiled at this time. Then, we stored the sensing label HTBR-L and the fish together at 25 °C and monitored the color change of the sensing label. The results are as Figure 22 shown. When the daylight color of the sensing label HTBR-L is pink-purple and the fluorescent color is blue-violet non-fluorescent, it indicates that the salmon fish is in a fresh state. When the daylight color of the sensing label HTBR-L is gray and the fluorescent color is blue weak fluorescence, it indicates that the salmon fish is in a qualified state. When the daylight color of the sensing label HTBR-L is wheat-colored and the fluorescent color is strong green fluorescence, it means that the salmon fish has spoiled. These results show that the sensing label HTBR-L can accurately judge the freshness level of salmon fish through the color change of the dual channels of daylight and fluorescence.

[0095] IX. Application of the Probe HTBR as a Fluorescent Ink

[0096] Dissolve HTBR in ethanol and a small amount of glycerol. After stirring for 5 minutes, HTBR ink is obtained. Dip your fingertip and a stamp into the HTBR ink and print it on a silica gel plate. Fingerprints, English letters, and Chinese characters are printed as magenta imprints observed under sunlight and orange weak fluorescence imprints observed under ultraviolet light ( Figure 23 a,b). Add 20 μL of 50 mmol / L diethylamine to the HTBR ink. After stirring evenly, HTBR fluorescent ink is obtained. Then dip your fingertip and a stamp into the HTBR fluorescent ink and print it on a silica gel plate. Fingerprints, English letters, and Chinese characters are printed as yellow imprints observed under sunlight and strong green fluorescence imprints presented under 365 nm ultraviolet light ( Figure 23 c,d). In summary, it shows that HTBR can be used as a fluorescent ink.

[0097] X. Scanning Electron Microscope (SEM) Test of the Sensing Label

[0098] Collect the fresh, qualified, and spoiled sensing label HTBR-L after the salmon freshness experiment for SEM test. The images are as Figure 24 shown. It can be clearly seen at magnifications of 1.00K, 10.00K, and 25.00K that when fresh, the surface of the sensing label HTBR-L is relatively tight and smooth; when qualified, the surface of the sensing label HTBR-L starts to become loose and holes appear; when spoiled, the number of holes on the surface of the sensing label HTBR-L increases sharply. The test results show that the sensing label can indicate the freshness grade of salmon.

[0099] XI. Application of Smartphones

[0100] To increase the convenience, reliability, and practicality of sensing label detection, we use the color recognition software of a smartphone to extract the RGB values of the standard colorimetric card of the sensing label HTBR-L ( Figure 25 a). According to the curve fitting software, there is a good linear relationship between the G / B ratio and the TVB-N value (R 2 = 0.991, Figure 25 b). Then, we carried out verification ( Figure 25 c). Extract the G / B ratio of the sensing label for verification at 4 °C. Substitute the ratio into the linear equation Y = 0.018 + 0.748X to obtain the TVB-N value. The freshness judged by the color of the sensing label at 4 °C is consistent with the freshness of the TVB-N value. The results show that the sensing label combined with a smartphone can conveniently and reliably evaluate the freshness of fish.

[0101] In summary, the amine rapid response fluorescent probe HTBR we prepared can be used for the detection of various amine substances and has obvious color and fluorescence changes. In EtOH:H at pH = 7.4 2In the O(v / v,5 / 5) system, HTBR can recognize a variety of amine solutions, and it can quickly respond to diethylamine with good sensitivity. At the same time, it can also be prepared into a sensing label HTBR-L and a sensing gel HTBR-G to recognize a variety of volatile amines. The designed sensing label and sensing gel are simple to prepare, and can realize colorimetric and fluorescence dual-channel detection of fish freshness, and the qualitative analysis is more accurate and reliable. The sensing label and sensing gel can quickly and real-time monitor the freshness of seawater fish meat non-contact and non-destructively, and can provide effective freshness information for producers, retailers and consumers in a timely manner, with good practical application value.

Claims

1. A fluorescent probe that responds quickly to amines, characterized in that: The structural formula of the fluorescent probe is as follows:

2. The method for synthesizing a fluorescent probe that rapidly responds to amines according to claim 1, characterized in that: The specific synthesis process is as follows: Using 10 to 100 mL of ethanol as a solvent, a julolidine derivative and 2-thiobarbituric acid are added at a molar ratio of 1:(2 to 3.5), wherein the julolidine derivative is 8-hydroxyjulolidine-9-carboxaldehyde. Then, acetic acid was added according to the mass ratio of the julolidine derivative to acetic acid of 25:1; the mixture was heated under reflux and stirred in an oil bath for 1 to 8 hours, cooled to room temperature, filtered, and the solid was collected and washed with ethanol to obtain a dual-channel fluorescent probe.

3. The use of a fluorescent probe that rapidly responds to amines according to claim 1, characterized in that: Biogenic amines are detected in an EtOH:H2O (v / v, 5 / 5) system at pH=7.4, and the application is for non-disease diagnosis and non-disease treatment purposes.

4. The use of a dual-channel fluorescent probe for indicating the freshness of fish according to claim 1, characterized in that: In an EtOH:H2O (v / v, 5 / 5) system at pH=7.4, diethylamine can be recognized within 2 seconds, and the application is for non-disease diagnosis and non-disease treatment purposes.

5. A fish freshness sensor tag prepared with the amine fast-response fluorescent probe as claimed in claim 1, wherein the specific preparation process of the sensor tag is as follows: 3.43 mg of the fluorescent probe is weighed and dissolved in 10 mL of DMSO, and then a cut filter paper piece with a diameter of 1 cm is soaked in the solution. After overnight, it is taken out and placed in an oven to dry to obtain a sensor tag for monitoring the freshness of fish meat.

6. Use of the sensor tag according to claim 5 in determining the freshness of fish meat.

7. The use of the sensor tag according to claim 5 in determining the freshness of fish meat is characterized by: The sensor tag is placed in the package without direct contact with the food. The color change of the sensor tag is compared with the standard colorimetric card to monitor the freshness of the fish in real time and judge the freshness of the fish. Under visible light, a pink-purple color indicates freshness, a gray color indicates qualified, and a wheat-colored color indicates unqualified. Under 365nm ultraviolet light, a blue-purple color with no fluorescence indicates freshness, a blue weak fluorescence indicates qualified, and a green strong fluorescence indicates unqualified.

8. A fish freshness sensing gel prepared by using the amine fast-response fluorescent probe as claimed in claim 1, wherein the specific preparation process of the sensing gel is as follows: 0.3 g of agarose is dissolved in 15 mL of distilled water, 0.2 mL of glycerol is added, and the mixed solution is heated under stirring until it becomes clear and transparent. When it is cooled to 60° C., 5 mg of the fluorescent probe is dissolved in 1 mL of DMSO and then quickly added to the agarose solution. The mixture is stirred until it is uniformly mixed, and then the mixture is quickly poured into a culture dish with a diameter of 9 cm. The bubbles are driven to the edge of the culture dish with a glass rod. The agarose solution is cooled to room temperature and solidified into blocks, which are divided into agarose blocks with a diameter of 1 cm to obtain a sensing gel for monitoring the freshness of fish.

9. Use of the sensor gel according to claim 8 in determining the freshness of fish meat.

10. The use of the sensor gel according to claim 8 in determining the freshness of fish, characterized in that: The sensor gel is placed in the package without direct contact with food. The color change of the sensor gel is compared with the standard colorimetric card to monitor the freshness of the fish in real time and judge the freshness of the fish. Under visible light, a wine red color of the sensor gel indicates freshness, an orange red color indicates qualified, and a dark khaki color indicates unqualified. Under 365nm ultraviolet light, a purple color with no fluorescence indicates freshness, a blue color with weak fluorescence indicates qualified, and a green color with strong fluorescence indicates unqualified.

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