A fluorescent probe for identifying biogenic amines, a preparation method thereof, and applications thereof

Abstract

The present invention discloses a fluorescent probe for identifying biogenic amines, its preparation method and application, which relates to the technical field of biogenic amine detection. In the present invention, phenothiazine with a high fluorescence quantum yield is used as the fluorophore to ensure that the probe has obvious fluorescence signals for visual detection. An amine substance recognition group 2,4-thiazolidinedione is introduced to synthesize a novel "OFF-ON" type fluorescent probe. This fluorescent probe has a rapid response to biogenic amines and a low detection limit. The prepared sensing label loaded with the fluorescent probe realizes non-destructive visual monitoring of the freshness of fish meat, with obvious changes in sunlight and fluorescence color signals.

Description

Technical Field

[0001] The present invention relates to the technical field of biogenic amine detection, and particularly relates to a fluorescent probe for identifying biogenic amines, a preparation method thereof, and an application thereof. Background Art

[0002] Food safety issues have become important factors affecting human health and have attracted worldwide attention. Incorrect handling methods of food during production, transportation, storage, and sales can cause a decrease in food quality and ultimately lead to food spoilage. Especially for protein-rich foods, the action of endogenous enzymes and microorganisms in the food can cause amino acids to degrade, forming toxic biogenic amines. Biogenic amines not only affect the quality of food but also endanger human health, causing various diseases, such as headaches, diarrhea, etc., and even leading to shock or death in severe cases.

[0003] Salmon is deeply loved by people because of its plump meat, rich in high-quality polyunsaturated fatty acids such as DHA and EPA, trace elements, and a large amount of high-quality protein. However, due to its soft tissue, high water and protein content and other internal factors, fish foods such as salmon have become one of the foods that are extremely prone to spoilage. Therefore, in order to ensure human health, it is crucial to accurately evaluate the freshness of fish.

[0004] At present, traditional methods for evaluating the freshness of fish include total volatile basic nitrogen (TVB-N), total viable count (TVC), pH value, thiobarbituric acid value (TBA), etc. These methods all require contact with and destruction of samples, and at the same time require complex pretreatment steps. Although new detection methods such as electrochemistry, chromatography, and biosensor arrays can judge the freshness of fish by directly detecting the biogenic amine content, they usually require expensive and complex equipment and cumbersome operations, which severely limit their applicability in real-time monitoring of fish freshness. Therefore, developing a simple, non-contact, fast, and accurate method for detecting fish freshness is crucial for food safety supervision and human health protection.

[0005] In recent years, small molecule fluorescent probes have been widely used in fields such as environmental monitoring and food safety analysis due to their advantages of non-destructive detection, simplicity, high efficiency, and sensitivity. In this regard, the inventors believe that how to design a fluorescent probe with a fast response to biogenic amines, a low detection limit, and capable of realizing the visual detection of biogenic amines is a technical problem that needs to be urgently solved by those skilled in the art.

[0006] The information disclosed in this background art section is only intended to increase the understanding of the overall background of the present invention and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art already known to those of ordinary skill in the art. Summary of the Invention

[0007] In view of the above technical problems, an embodiment of the present invention provides a fluorescent probe for identifying biogenic amines, a preparation method thereof, and an application thereof, so as to solve the problems raised in the above background art.

[0008] A fluorescent probe, characterized in that the structural formula is as follows:

[0009] ; The above fluorescent probe is named MPZ.

[0010] A preparation method of a fluorescent probe as described above, comprising the following steps:

[0011] S21: Add compound a, 2,4-thiazolidinedione, and piperidine to dry ethanol, and heat under reflux for 4 h for reaction;

[0012] S22: After the reflux reaction is completed, filter the precipitate and wash it with ethanol to prepare the fluorescent probe;

[0013] Among them, the structural formula of compound a is as follows:

[0014] .

[0015] Preferably, the synthetic route of the fluorescent probe is as follows:

[0016] .

[0017] An application of a fluorescent probe as described above in the recognition of biogenic amines.

[0018] Specifically, the biogenic amines include any one of cyclohexanediamine, diethylamine, n-propylamine, triethylamine, ethylamine, spermine, cadaverine, putrescine, 2-phenylethylamine, tyramine, trimethylamine, dimethylamine, ammonia water, and hydrazine.

[0019] Preferably, the fluorescent probe is used to detect biogenic amines in fish to evaluate the freshness of fish.

[0020] A sensing label, comprising a filter paper soaked with a fluorescent probe as described above.

[0021] A preparation method of a sensing label as described above, comprising the following steps:

[0022] S61: Cut the filter paper into circular thin slices with a diameter of 1 cm;

[0023] S61: Soak the circular thin slice of filter paper in a 1 mM stock solution of the fluorescent probe for 10 minutes, and dry it at 40 °C for 3 h to prepare the sensing label.

[0024] An application of a sensing label as described above in the evaluation of fish freshness.

[0025] Preferably, after the sensing label detects the fish meat, there are color changes under both sunlight and fluorescence, and the above color changes can be used as detection indicators for evaluating the freshness of fish meat.

[0026] A freshness level indicating card is provided with a first color comparison area and a second color comparison area; the first color comparison area includes various colors displayed under sunlight after the fish meat is detected by the sensing label described above, and the second color comparison area includes various colors displayed under fluorescence after the fish meat is detected by the sensing label described above; the color changes on the first color comparison area and the second color comparison area are used to indicate different levels of freshness of the fish meat.

[0027] Preferably, a yellow color comparison area, a light pink color comparison area, and a pink color comparison area are provided on the first color comparison area; the yellow color comparison area indicates that the fish meat is at the first-level freshness and belongs to premium products, the light pink color comparison area indicates that the fish meat is at the second-level freshness and belongs to qualified products, and the pink color comparison area indicates that the fish meat has deteriorated and is inedible;

[0028] An orange-red color comparison area, a purple color comparison area, and a gray-blue color comparison area are provided on the second color comparison area; the orange-red color comparison area indicates that the fish meat is at the first-level freshness and belongs to premium products, the purple color comparison area indicates that the fish meat is at the second-level freshness and belongs to qualified products, and the gray-blue color comparison area indicates that the fish meat has deteriorated and is inedible.

[0029] A fluorescence probe for identifying biogenic amines, its preparation method and application provided by an embodiment of the present invention have the following beneficial effects: The present invention uses phenothiazine with a high fluorescence quantum yield as a fluorophore to ensure that the probe has obvious fluorescence signals for visual detection. By introducing an amine substance recognition group 2,4-thiazolidinedione, a novel "OFF-ON" type fluorescence probe MPZ is synthesized. The fluorescence probe MPZ responds rapidly (7 s) to biogenic amines in an EtOH / H2O (4 / 6, v / v) solution, has a low detection limit (0.72 μM), and emits red fluorescence (620 nm); the prepared sensing label loaded with MPZ (MPZ / FPS) realizes non-destructive visual monitoring of the freshness of fish meat, with obvious color signal changes under sunlight and fluorescence. Description of the Drawings

[0030] Figure 1 It is a UV-visible absorption and fluorescence spectrum test chart of probe MPZ (10 μM) in an EtOH / H2O (4 / 6, v / v) solution after adding various biogenic amines (50 μM);

[0031] Among them, biogenic amines include 1: cyclohexanediamine, 2: diethylamine, 3: n-propylamine, 4: triethylamine, 5: ethylamine, 6: spermine, 7: cadaverine, 8: putrescine, 9: 2-phenylethylamine, 10: tyramine, 11: trimethylamine, 12: dimethylamine, 13: ammonia water, 14: hydrazine;

[0032] Figure 1 a is the ultraviolet-visible absorption spectrum, inset: daylight color change;

[0033] Figure 1 b is the fluorescence spectrum, inset: fluorescence color change;

[0034] Figure 1 c is the fluorescence response of MPZ to different concentrations of Cad (0 - 50 μM), inset: linear relationship between Cad concentration (0 - 50 μM) and fluorescence intensity of MPZ at 620 nm;

[0035] Figure 1 d is the change in fluorescence intensity of the probe MPZ and MPZ + Cad at different pH values;

[0036] Figure 1 e is the relationship between fluorescence color and time after adding Cad (50 μM) to the probe MPZ (10 μM);

[0037] Figure 2 It is the result diagram of the reaction mechanism study of MPZ responding to Cad;

[0038] Among them, Figure 2 a is the speculated reaction mechanism of MPZ and Cad;

[0039] Figure 2 b is the ultraviolet spectra of MPZ + Cad, MPZ + NaOH, and MPZ;

[0040] Figure 2 c is the fluorescence spectra of MPZ + Cad, MPZ + NaOH, and MPZ;

[0041] Figure 2 d is the frontier molecular orbital energy diagram of MPZ and MPi calculated based on B3LYP / 6-31G;

[0042] Figure 3 a is the preparation process of the sensing label MPZ / FPS;

[0043] Figure 3b shows the colorimetric and fluorescence responses of MPZ / FPS to different amine vapors (1: MPZ, 2: cyclohexanediamine, 3: diethylamine, 4: n-propylamine, 5: triethylamine, 6: ethylamine, 7: spermine, 8: cadaverine, 9: putrescine, 10: 2-phenylethylamine, 11: tyramine, 12: trimethylamine, 13: dimethylamine, 14: ammonia water, 15: hydrazine, 50 mM);

[0044] Figure 4 are the fluorescence monitoring results of the stability of MPZ / FPS for 7 consecutive days;

[0045] Figure 5 are the fluorescence (R + G) / B values of MPZ / FPS for 7 consecutive days

[0046] Figure 6 are the daylight and fluorescence color changes of MPZ / FPS during the storage of fish meat at 4°C;

[0047] Figure 7 a shows the changing trends of TVB-N value and pH value with time in salmon meat stored at 4°C;

[0048] Figure 7 b shows the changing trends of TVC value and TBA value with time in salmon meat stored at 4°C;

[0049] Figure 7 c are the daylight and fluorescence color changes of MPZ / FPS within 8 days during storage at 4°C;

[0050] Figure 8 is a schematic diagram of the freshness grade indicator card. Detailed implementation manners

[0051] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the protection scope of the present invention.

[0052] In view of the above technical problems, the embodiments of the present invention provide a fluorescence probe for identifying biogenic amines, its preparation method and application to solve the problems raised in the above background technology.

[0053] 1. Experimental part

[0054] 1.1. Synthesis of probe MPZ

[0055] Compound a (3.5 mmol, 1.00 g), 2,4-thiazolidinedione (2 mmol, 0.82 g) and piperidine (0.5 mL) were added to dry ethanol (15 mL), and the mixture was heated under reflux for 4 h. After the reaction was completed, the precipitate was filtered and washed with ethanol three times to obtain a red solid (0.68 g, 50.4%). M.p. 288.4 - 289.3 °C, QY = 0.01% (in EtOH / H2O = 4 / 6). 1H NMR (400 MHz, DMSO- d 6 ) δ 12.48 (s, 1H), 7.83 (s, 1H), 7.22 (t, J = 8.4 Hz, 6.8 Hz, 1H), 7.16 (d, J = 7.7 Hz, 1H), 7.08 (d, J = 7.6 Hz, 1H), 7.03 (s, 1H), 6.99 (t, J = 7.6 Hz, 1H), 6.67 (s, 1H), 4.04 (q, J = 6.8 Hz, 2H), 3.93 (s, 3H), 1.33 (t, J = 7.2 Hz, 6.8 Hz, 3H). 13C NMR (101 MHz, DMSO- d 6 ) δ 168.04, 167.64, 158.94, 148.47, 142.76, 127.82, 127.18, 126.14, 125.46, 123.33, 122.65, 120.12, 116.20, 115.49, 113.68, 99.82, 56.17, 41.64, 12.75. HRMS (ESI - ) m / z, calculated for C 19 H 15 N2O3S2 - [M-H] - calcd: 383.0530, found: 383.0515。

[0056] 1.2. Preparation for spectroscopic tests

[0057] Weigh 3.84 mg of the probe MPZ and dissolve it in DMSO to make a stock solution of 1 mM. Then dilute it with EtOH / H2O (v / v = 4 / 6) to a final concentration of 10 μM before use. The amine solutions required for the experiment are prepared with secondary distilled water (1: cyclohexanediamine, 2: diethylamine, 3: n-propylamine, 4: triethylamine, 5: ethylamine, 6: spermine, 7: cadaverine, 8: putrescine, 9: 2-phenylethylamine, 10: tyramine, 11: trimethylamine, 12: dimethylamine, 13: ammonia water, 14: hydrazine, 50 mM). The prepared ionic solutions are stored in the refrigerator and diluted to the required concentration when used. Test solution: EtOH / H2O (v / v = 4 / 6, pH = 7.4); Test conditions: λex = 410 nm, λem = 620 nm, slit = 5 / 10.

[0058] 1.3. Preparation of filter paper (MPZ / FPS) sensing tags

[0059] Cut the filter paper (FPS) into circular slices with a diameter of 1 cm. Then, soak the FPS in the stock solution of the probe with a concentration of 1 mM for 10 minutes and dry it at 40 °C for 3 h to obtain the MPZ / FPS sensing tag.

[0060] 1.4. Application of the sensing tag (MPZ / FPS) in monitoring the freshness of salmon

[0061] Purchase fresh salmon from Jinzhou Aquatic Products Market, quickly transport it to the laboratory for cutting and packaging it into transparent petri dishes or lunch boxes. Place the sensing tag (MPZ / FPS) in the petri dish or lunch box containing fresh fish. The experiment is carried out under the condition of 4 °C (refrigeration). Under the refrigeration condition, the TVB-N, pH, TBA, and TAC values of the salmon meat are measured every 24 h by the national standard method, and the daylight and fluorescence changes of MPZ / FPS are photographed and recorded.

[0062] The determination of the TVB-N value refers to GB / T 18108-2019, the determination of the TVC value refers to GB 4789.2-2022, the determination of the TBA value refers to GB 5009.181-2016, and the pH value is measured using a pH meter.

[0063] 2. Results and discussion

[0064] 2.1. Spectral characteristics of MPZ for detecting biogenic amines

[0065] To study the sensing characteristics of the probe MPZ for biogenic amines, ultraviolet-visible absorption and fluorescence spectroscopy tests were carried out in an EtOH / H2O (v / v = 4 / 6) system solution. As Figure 1As shown in Fig. a, MPZ has a weak absorption peak at 437 nm. When different amines are added to the MPZ solution, the absorption peak at 437 nm blue-shifts to 416 nm and is significantly enhanced. At the same time, the color of the solution changes from colorless to yellow ( Figure 1 inset in Fig. a).

[0066] The addition of the amine solution also causes obvious changes in the fluorescence spectrum of MPZ. As Figure 1 shown in Fig. b, MPZ has a very weak emission peak at 640 nm. When different amines are added, the emission peak at 640 nm blue-shifts to 620 nm, and at the same time, the fluorescence intensity is greatly enhanced. The fluorescence color of the solution changes from weak dark red to bright orange-red ( Figure 1 inset in Fig. b). The results show that the probe MPZ has a good response ability to biogenic amines, and at the same time has obvious colorimetric and fluorescence changes, enabling the naked-eye detection of biogenic amines.

[0067] A large amount of biogenic amines are produced during the spoilage of aquatic products. During the late storage of salmon, the contents of cadaverine (Cad) and putrescine increase by 32.65 and 15.81 times respectively, which are much higher than the changes of other biogenic amines. Therefore, the content changes of cadaverine and putrescine, as representative biogenic amines, can be used to evaluate the freshness of salmon. According to the spectral test results, cadaverine was finally selected for the titration experiment to study the response sensitivity of MPZ to Cad. As Figure 1 shown in Fig. c, as the concentration of cadaverine increases, the fluorescence intensity of the probe MPZ at 620 nm gradually increases. When the Cad concentration is 50 μM, the fluorescence intensity reaches a steady state, and the fluorescence quantum yield changes from 0.01% to 5.49%. From the fluorescence titration data, it can be seen that there is a good linear relationship between the change in the fluorescence intensity of MPZ and the concentration of Cad (0 - 50 μM) (Y = 238.72 + 102.03X, R 2 = 0.999). According to the equation LOD = 3S / K, the detection limit of the probe MPZ for Cad is calculated to be 0.72 mM( Figure 1 inset in Fig. c). This indicates that MPZ has a high response sensitivity to Cad.

[0068] In addition, different pH environments may affect the detection of Cad by MPZ. To explore the pH applicable range of MPZ, the fluorescence changes of MPZ and MPZ + Cad in solutions with different pH systems were tested. As Figure 1 shown in Fig. d, MPZ can produce good recognition effects on Cad in the pH range of 4 - 7. Subsequently, we explored the response time of MPZ to Cad. As Figure 1As shown in e, when Cad (50 M) was added, the fluorescence color of MPZ changed from dark red to orange - red completely within 7 s. The results indicate that MPZ can rapidly detect Cad.

[0069] 2.2. Sensing mechanism

[0070] Based on the above experimental results, the reaction mechanism of the probe MPZ responding to Cad was studied. After the pH reached 10, the fluorescence intensities of MPZ and MPZ + Cad were almost the same. Therefore, we speculated that the addition of alkaline Cad and inorganic base would both cause the H protons on 2,4 - thiazolidinedione to be removed ( Figure 2 a), which would lead to obvious changes in the UV and fluorescence spectra of MPZ. To verify this conjecture, high - resolution mass spectrometry was performed on MPZ after adding Cad. The mass spectrometry showed that MPZ + Cad produced a mass spectrometry peak at m / z = 383.0541, which was the peak corresponding to the target product [M - H] - (the theoretical value was 383.0530). In addition, the UV and fluorescence spectra of MPZ + Cad and MPZ + NaOH solutions at the same pH (9.94) were measured, and the daylight and fluorescence color changes were recorded. As Figure 2 shown in b, 2c, there were no obvious differences in the UV and fluorescence spectra of the two, and the daylight and fluorescence color changes were the same ( Figure 2 insets in b, 2c). The above experimental results confirmed that the addition of Cad caused the deprotonation of the H protons of MPZ, and a deprotonation reaction occurred. In addition, due to the different basicities of different amines, their deprotonation abilities were different, resulting in differences in UV and fluorescence spectra.

[0071] To further study the fluorescence change mechanism of MPZ before and after responding to Cad, we performed quantum chemical calculations using density functional theory (DFT) and time - dependent density functional theory (TD - DFT) methods. As Figure 2 shown in d, the frontier molecular orbitals (FMOs) on the highest occupied molecular orbital (HOMO) of MPZ were mainly located in the phenothiazine part, while the FMOs on the lowest unoccupied molecular orbital (LUMO) were mainly distributed on the 2,4 - thiazolidinedione group. After photoexcitation, electrons transitioned from the LUMO back to the HOMO, accompanied by a complete intramolecular charge transfer from the 2,4 - thiazolidinedione group to the phenothiazine, and a photoinduced electron transfer (PET) process occurred, resulting in fluorescence quenching through non - radiative transitions. While the FMOs on the HOMO and LUMO of MPi were distributed on the entire molecular skeleton, and the excited state S1 state was a local transition state. After photoexcitation, electrons could directly fall back from the LUMO to the HOMO through radiative transitions, causing fluorescence turn - on.

[0072] 2.3. Response of MPZ / FPS to biogenic amine vapors

[0073] To achieve non-destructive monitoring of fish freshness, MPZ / FPS was placed in vials containing different biogenic amines (50 mM) for fumigation (as shown in Figure 3 a). Through a simulated fish spoilage environment, the practical application value of the sensing label MPZ / FPS was explored. As shown in Figure 3 b, MPZ / FPS showed obvious daylight and fluorescence color changes for most biogenic amines, indicating that MPZ / FPS can detect gaseous biogenic amines. In addition, the stability of MPZ / FPS was tested through 7-day continuous monitoring, and it was found that there was no obvious change in the fluorescence color of MPZ / FPS ( Figure 4 ), and there was no obvious fluctuation in the fluorescence (R+G) / B value of MPZ / FPS ( Figure 5 ). The above results show that MPZ / FPS has good stability and has the potential to be a portable tool for monitoring fish freshness.

[0074] 2.4. Monitoring of Salmon Freshness

[0075] To further evaluate the practical application ability of the sensing label MPZ / FPS to monitor fish freshness, the daylight and fluorescence color changes of MPZ / FPS during the storage of fish at 4 °C were recorded ( Figure 6 ). At the same time, in order to accurately judge the corresponding relationship between the color change trend of the sensing label and fish freshness, four indicators for measuring fish freshness, namely TVB-N, TBA, TVC, and pH, were selected as the judgment basis. As shown in Figure 7 a, 7b, as the storage time prolonged, the values of TVB-N, TBA, and TVC all gradually increased. The increase in TVB-N indicates an increase in the content of ammonia substances produced by the decomposition of proteins in fish; the increase in TBA indicates that the unsaturated fatty acids in fish are being continuously oxidized; the increase in TVC indicates that the microorganisms in fish are multiplying continuously. The pH showed a trend of first decreasing and then increasing because lactic acid is produced during the initial storage of fish, and the accumulation of lactic acid leads to a decrease in pH. After a period of time, a large amount of protein decomposes, and the production of alkaline substances such as ammonia leads to an increase in pH. The changes in all four indicators indicate that the quality of fish is continuously declining.

[0076] According to GB / T 18108-2019 "National Food Safety Standard General Rules for Fresh Seawater Fish", when the TVB-N content < 15 mg / 100 g, the fish is of first-class freshness and belongs to premium grade products; when the TVB-N content is in the range of 15-30 mg / 100 g, the fish is of second-class freshness and belongs to qualified products and can still be eaten; when the TVB-N content > 30 mg / 100 g, it indicates that the fish has spoiled and is inedible. As shown in Figure 7As shown in c, on the first day, MPZ / FPS showed a yellow color under daylight and orange-red fluorescence under 365 nm ultraviolet light irradiation. At this time, the TVB-N value was 4.2 mg / 100 g, and the fish meat was in the first-grade freshness and belonged to the superior grade; on the fifth day, the daylight color and fluorescence color of MPZ / FPS were light pink and purple respectively. At this time, TVB-N was 15.4 mg / 100 g, and the fish meat was in the second-grade freshness and belonged to the qualified product and was still edible; by the seventh day, the daylight color and fluorescence color of MPZ / FPS were pink and gray-blue respectively. At this time, TVB-N reached 30.8 mg / 100 g, and the fish meat had deteriorated and was inedible. In addition, on the seventh day, the TVC and TBA values were 7.02 lg (cfu / g) and 1.10 mg MDA / kg ( Figure 7 b), respectively, both exceeding their respective maximum safety limit values (7.0 lg (cfu / g) and 1.0 mg MDA / kg), which fully indicated that the fish meat had deteriorated and was inedible by the seventh day. Based on the corresponding relationship between the good colorimetric and fluorescence dual change trends of MPZ / FPS and the freshness of fish meat, a freshness grade indicator card ( Figure 8 ) was designed to help consumers and merchants judge the freshness grade of fish meat.

[0077] In summary, we successfully synthesized a new fluorescent probe MPZ, which can rapidly detect biogenic amines through daylight colorimetry and fluorescence dual channels in the EtOH / H2O (v / v = 4 / 6, pH = 7.4) system; the prepared sensing label MPZ / FPS can be used for real-time monitoring of fish meat freshness.

[0078] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of a fluorescent probe in the identification of biogenic amines; characterized in that: The fluorescent probe is used to detect biogenic amines in fish meat to evaluate the freshness of the fish meat; The structural formula of the fluorescent probe is as follows: 。 2. A method for preparing a fluorescent probe for use as claimed in claim 1, characterized in that: The following steps are involved: S21: Compound a, 2,4-thiazolidinedione and piperidine were added to dry ethanol and heated under reflux for 4 h for reaction; S22: After the heating reflux reaction is completed, the precipitate is filtered and washed with ethanol to prepare the fluorescent probe; Wherein, the structural formula of compound a is as follows: 。 3. A sensor tag, characterized in that: The invention comprises filter paper soaked with the fluorescent probe as claimed in claim 1.

4. A method for preparing a sensor tag as claimed in claim 3, characterized in that: The following steps are involved: S41: Cut the filter paper into circular slices with a diameter of 1 cm; S41: Soak the circular thin filter paper in a stock solution of the fluorescent probe at a concentration of 1 mM for 10 minutes, and dry it at 40° C. for 3 h to prepare the sensor label.

5. Use of the sensor tag as claimed in claim 3 in the evaluation of fish freshness.

6. The use of the sensor tag according to claim 3 in the evaluation of fish freshness, characterized in that: After the sensor tag detects fish meat, the color changes under both sunlight and fluorescence, and the color change can be used as a detection index for evaluating the freshness of fish meat.

7. A freshness level indicator card, characterized in that: The freshness level indicator card is provided with a first color colorimetric area and a second color colorimetric area; the first color colorimetric area includes a plurality of colors displayed by the sensor tag under sunlight after detecting the fish meat, and the second color colorimetric area includes a plurality of colors displayed by the sensor tag under fluorescence after detecting the fish meat; the color changes in the first color colorimetric area and the second color colorimetric area are used to indicate different levels of freshness of the fish meat; the sensor tag is the sensor tag described in claim 3.

8. The freshness level indicator card according to claim 7, characterized in that: The first color comparison area is provided with a yellow color comparison area, a light pink color comparison area and a pink color comparison area; the yellow color comparison area indicates that the fish meat is at the first level of freshness and is a superior product, the light pink color comparison area indicates that the fish meat is at the second level of freshness and is a qualified product, and the pink color comparison area indicates that the fish meat has been corrupted and deteriorated and is not edible; The second color comparison area is provided with an orange-red colorimetric area, a purple colorimetric area and a gray-blue colorimetric area; the orange-red colorimetric area indicates that the fish meat is at the first level of freshness and is a superior product, the purple colorimetric area indicates that the fish meat is at the second level of freshness and is a qualified product, and the gray-blue colorimetric area indicates that the fish meat has been rotten and deteriorated and is inedible.

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