A ratiometric fluorescent smart label for visually detecting meat freshness, and its preparation method and application

Abstract

The present invention discloses a ratiometric fluorescent smart label for visually detecting the freshness of meat, and its preparation method and application. The smart label preparation method is specifically as follows: first, a ratiometric fluorescent indicator of gold-copper bimetallic nanoclusters (indicator) with red fluorescence and dual-ligand gold nanoclusters (internal standard) with green fluorescence is prepared; then, by a solution doping method, the ratiometric fluorescent indicator is added to a polyvinyl alcohol / polyethylene glycol solution to prepare a ratiometric fluorescent smart label; finally, a fluorescent colorimetric card is made based on the ratiometric fluorescent smart label, and the smart label is applied to the prediction of meat freshness grade and total volatile basic nitrogen content in combination with a smart phone. The ratiometric fluorescent smart label prepared by the present invention has higher accuracy and sensitivity than a colorimetric sensing indicator film; and the smart label is safe, biocompatible, biodegradable, and has strong mechanical properties, and has the function of real-time monitoring of the freshness of fresh meat and aquatic products.

Description

Technical Field

[0001] The present invention belongs to the field of non-destructive testing of food, and in particular relates to a ratiometric fluorescent smart label for visually detecting the freshness of meat, and a preparation method and application thereof. Background Art

[0002] During storage, the proteins in meat and aquatic products undergo biochemical changes due to the breakdown of proteins by proteolytic enzymes, forming small peptides or amino acids. These compounds further decompose to form volatile nitrogen compounds. These compounds often produce odors and are a major cause of spoilage and odor in fresh meat and seafood. Therefore, the content of volatile nitrogen compounds is an important indicator for evaluating the freshness of meat and aquatic products.

[0003] Currently, there are many methods for detecting total volatile basic nitrogen (TVB-N). Among them, optical sensors have attracted increasing attention due to their high sensitivity and good selectivity.

[0004] Chinese patent CN202010746884.0 discloses "A meat freshness indicator array, its preparation method and application". This indicator array uses 9 chemical synthetic indicators such as methylation, phenol red, and bromocresol green to prepare a colorimetric array for meat freshness detection. Although it can obtain relatively comprehensive color information, the chemical synthetic indicators used are all toxic to a certain extent, and there are safety risks in practical applications. Chinese patent CN202111172154.5 discloses "A colorimetric food freshness intelligent indicator label and its preparation method". Although this indicator label is made of non-toxic anthocyanin as an indicator, natural pigments are light-instable. Long-term sunlight exposure or long-term storage will affect the display effect of the indicator label. In addition, the indicator used in the above-mentioned research changes color based on changes in the acidity and alkalinity of the packaging environment. This is a non-specific recognition process and is therefore affected by gaseous substances other than biogenic amines. Therefore, when using colorimetric sensor array tags to detect the freshness of meat under visible light conditions, problems such as insufficient color change, long response time, and easy identification errors may occur.

[0005] Compared with traditional colorimetric sensors, fluorescence sensors have been widely used in the detection of meat freshness due to their advantages such as good sensitivity, short response time, abundant and easy modification of fluorescent materials, and rich signals.

[0006] Chinese patent CN202110725956.8 discloses a "thin-film fluorescence sensor, its preparation method, and application." This sensor uses nitrogen and sulfur co-doped carbon quantum dots as response molecules for determining the freshness of fish. Chinese patent CN201911396936.X discloses a "dual-signal indicator card for determining the freshness of red snapper." This card, made from a polyaniline / tetraphenylethylene composite, can monitor the freshness of red snapper in real time. Furthermore, Tang Benzhong et al. used molecules that can be converted to aggregation-induced emission (AIE) under the action of cadaverine to detect seafood freshness (ACS Sens, 2016, 1, 179-184). However, these methods use only a single luminophore for visual detection, making detection accuracy susceptible to factors such as photobleaching, ambient microenvironmental stability, and stability under illumination. Furthermore, changes in fluorescence intensity in most tests are difficult to detect with the naked eye, which can increase errors.

[0007] Compared with single fluorescence signal sensing, the new ratio fluorescence sensor with ratio fluorescence characteristics can weaken the interference of many difficult-to-control factors such as detection probe concentration, temperature, solvent polarity, excitation intensity, and environmental pH value, making the results more accurate and the response range wider. It is also easier to visualize with the naked eye.

[0008] Chinese patent CN202210227350.6 discloses "a method for monitoring the freshness of shrimp meat and a colorimetric card used therefor", and uses rhodamine B and fluorescein to prepare a ratiometric fluorescent colorimetric card for real-time in situ monitoring of shrimp meat freshness. Chinese patent 202110346921.3 discloses "a preparation method and application of a ratiometric fluorescent response sensor label for salmon freshness", and uses rhodamine B and tetraphenylethylene with AIE phenomenon to prepare a ratiometric fluorescent response label for salmon freshness monitoring. Chinese patent CN202010321751.9 discloses "an application of a ratiometric fluorescent polymer hydrogel in seafood freshness detection", and this method synthesizes 1,8-naphthalimide fluorescent compounds as internal standard molecules and pyridine carboxylic acid compounds and Eu 3+ The ratiometric fluorescent polymer hydrogel used as an indicator. Although the ratiometric fluorescent freshness indicator labels prepared above all have certain sensitivity and accuracy, the fluorescent indicators used are toxic or carcinogenic, posing safety risks, and the synthesis method is complex and difficult to operate.

[0009] At present, fluorescent sensors prepared based on metal nanoclusters (NCs) have broad application prospects in the field of food safety due to their advantages such as good fluorescence, catalysis, electrochemiluminescence, aggregation-induced fluorescence enhancement, low cost and good biocompatibility. In addition, some metal clusters have aggregation-induced emission effect. Unlike traditional organic light-emitting materials facing the "aggregation-induced emission quenching" effect, AIE-type metal clusters do not emit fluorescence in dilute solutions, but are enhanced in aggregation or solid state, showing great potential in the field of food detection. Zhao Xianen et al. designed a pH-driven aggregation-induced luminescence gold-copper bimetallic nanocluster for the determination of dissolved ammonia in water samples. From the experimental results, it can be seen that the metal cluster is feasible for detecting the freshness of meat (Microchimica Acta, 2021, 188, 113). However, the detection system of this method is still limited to the solution environment and is based on single-signal sensing, which is not conducive to practical application and is easily affected by the environment. Summary of the Invention

[0010] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a method for preparing a ratiometric fluorescent smart label for visually detecting the freshness of meat. The preparation method prepares a ratiometric fluorescent indicator by using gold-copper bimetallic nanoclusters as indicators and simultaneously preparing dual-ligand gold nanoclusters as internal standard molecules. The ratiometric fluorescent indicator is further loaded onto a polyvinyl alcohol / polyethylene glycol (PVA / PEG) film to prepare a ratiometric fluorescent smart indicator label.

[0011] The second purpose of the present invention is to provide a ratiometric fluorescent smart label for visually detecting the freshness of meat. The ratiometric fluorescent smart label is simple to use and has high detection accuracy. The material prepared by the present invention has higher accuracy and high sensitivity compared to colorimetric sensing indicator films. In addition, the indicator of the present invention is safe, biocompatible, biodegradable, and has strong mechanical properties. It has the function of real-time monitoring of the freshness of fresh meat and aquatic products, thereby realizing non-destructive, rapid, and real-time detection of the freshness of fresh meat and aquatic products.

[0012] The third purpose of the present invention is to provide an application of a ratiometric fluorescent smart label for visually detecting the freshness of meat. The specific application method is to combine the ratiometric fluorescent label with a smartphone APP to realize quantitative and qualitative detection of the freshness of meat. This method is accurate and reliable and does not require expensive detection equipment.

[0013] The primary purpose of the present invention can be achieved through the following technical solutions:

[0014] A method for preparing a ratiometric fluorescent smart label for visually detecting meat freshness includes the following steps:

[0015] (1) The preparation steps of gold-copper bimetallic nanoclusters (DPA-AuCuNCs) are as follows:

[0016] The prepared copper nitrate solution is slowly added dropwise to a D-penicillamine (DPA) solution at room temperature, and the reaction is carried out under magnetic stirring to obtain a first precursor solution. A tetrachloroauric acid solution is slowly added dropwise to the first precursor solution, and then hydrochloric acid is added dropwise to adjust the pH to 4.0-5.0 to form a mixed solution. The mixed solution is then heated in a water bath to obtain a reaction solution of gold-copper bimetallic nanoclusters (DPA-AuCuNCs). The reaction solution is centrifuged, filtered, washed, and freeze-dried to obtain gold-copper bimetallic nanoclusters (DPA-AuCuNCs).

[0017] (2) The preparation steps of dual-ligand gold nanoclusters (GSH-AuNCs@His) are as follows:

[0018] At room temperature, the prepared tetrachloroauric acid solution is slowly added dropwise to the histidine (His) solution. Under magnetic stirring, the color of the solution changes from colorless and transparent to light yellow, thereby obtaining a second precursor solution. Glutathione solution is then added dropwise to the second precursor solution. After the reaction is complete, the resulting reaction solution is dialyzed and freeze-dried to obtain dual-ligand gold nanoclusters (GSH-AuNCs@His).

[0019] (3) The steps for preparing the ratiometric fluorescent indicator solution are as follows:

[0020] The DPA-AuCuNCs prepared in step (1) are prepared into a DPA-AuCuNCs solution, and the GSH-AuNCs@His prepared in step (2) are prepared into a GSH-AuNCs@His solution, and the two solutions are mixed to prepare a ratiometric fluorescent indicator solution;

[0021] (4) The steps for preparing a ratiometric fluorescent smart label for visually detecting meat freshness are as follows:

[0022] The ratiometric fluorescent indicator solution prepared in step (3) is mixed with a polyvinyl alcohol / polyethylene glycol (PVA / PEG) solution by a solution doping method under magnetic stirring; after being fully mixed, the mixed solution is slowly poured into a polytetrafluoroethylene plate mold until a thin film is formed, and then the film is gently peeled off to obtain a ratiometric fluorescent smart label (ratiometric metal nanocluster-doped polyvinyl alcohol / polyethylene glycol fluorescent composite film) for visually detecting the freshness of meat.

[0023] Preferably, the molar concentration of the copper nitrate solution in step (1) is 0-0.2 mol / L; the molar concentration of the DPA solution is 0-0.4 mol / L; the molar concentration of the tetrachloroauric acid solution is 0-0.30 mol / L; and the volume ratio of the copper nitrate, tetrachloroauric acid, and DPA solution is 5:4:1000-2000.

[0024] Preferably, in step (1), the reaction time of the copper nitrate solution and DPA is 0 to 10 min; the heating temperature of the water bath is 30 to 40° C.; the heating time is 30 to 60 min; the centrifugal speed is 8000 to 10000 rpm; and the centrifugal time is 0 to 10 min.

[0025] Preferably, the molar concentration of the tetrachloroauric acid solution in step (2) is 0-0.30 mol / L; the molar concentration of the histidine (His) solution is 0-0.05 mol / L; the molar concentration of the glutathione aqueous solution is 0-0.015 mol / L; and the volume ratio of the tetrachloroauric acid, His, and glutathione solutions is 1:50-100:50-100.

[0026] Preferably, in step (2), the reaction time of the tetrachloroauric acid solution and the His solution is 0 to 4 hours; the reaction time of the second precursor solution and the glutathione solution is 0 to 1 hour; and the dialysis uses a dialysis bag with a molecular weight cutoff of 3500 and deionized water dialysis for 24 to 48 hours.

[0027] Preferably, the mass concentration of the DPA-AuCuNCs solution in step (3) is 0-0.01 g / mL; the mass concentration of the GSH-AuNCs@His solution is 0-0.01 g / mL; and the volume ratio of the DPA-AuCuNCs solution to the GSH-AuNCs@His solution is 3:2.

[0028] Preferably, the polyvinyl alcohol / polyethylene glycol (PVA / PEG) solution in step (4) is prepared as follows:

[0029] Weigh polyvinyl alcohol (PVA), add 0-10% PVA, phosphate-buffered saline, glycerol, and distilled water to it, connect the condensed water, and continue heating under mechanical stirring until the PVA powder is completely dissolved. Finally, heat under reflux and cool to room temperature to form a polyvinyl alcohol / polyethylene glycol (PVA / PEG) solution.

[0030] Preferably, the PVA: phosphate buffered saline: glycerol: distilled water is 0-2 g: 0-2 mL: 0-1 mL: 0-2 mL: 0-20 mL; the heating temperature is 60-80° C., and the reflux time is 30-60 min.

[0031] The second object of the present invention can be achieved through the following technical solutions:

[0032] A ratiometric fluorescent smart label for visually detecting the freshness of meat is prepared by the above preparation method.

[0033] The third object of the present invention can be achieved through the following technical solutions:

[0034] Application of a ratiometric fluorescent smart label for visually detecting meat freshness in visual monitoring of meat freshness.

[0035] Preferably, the application includes the production of a meat freshness intelligent indicator fluorescent colorimetric card and visual monitoring of meat freshness based on a ratiometric fluorescent intelligent label, and the specific steps are as follows:

[0036] (1) The prepared ratiometric fluorescent smart label for visually detecting the freshness of meat is pasted on the top of the inner side of a transparent fresh-keeping box and stored in the same space with fresh meat for m days, where m is a positive integer; and the TVB-N standard value of the meat is measured according to the physical and chemical indicators specified in the national standard GB5009.228-2016 to judge the freshness of the meat, observe the fluorescent color change of the indicator label during the storage process, and use a smart phone to regularly record the fluorescent photos of the freshness fluorescent smart indicator label under ultraviolet light at different storage times, and arrange the fluorescent photos from small to large according to the number of storage days to form an intelligent indicator of meat freshness fluorescent colorimetric card; or, the fluorescent photos of different colors are converted into RGB values through a mobile phone color picker APP, and the linearity of the ratio (R / G) of the red (R) to green (G) channels depends on the TVB-N content, ranging from the initial meat TVB-N value to the TVB-N value of the meat at the end of the storage period (the mth day);

[0037] (2) The prepared ratiometric fluorescent smart label for visually detecting the freshness of meat is pasted on the inner top of the fresh-keeping box, and stored in the same space as the meat sample. Then, a fluorescent color image of the smart indicator label in the packaging bag is obtained, and the observed fluorescent color image is compared with the obtained meat freshness smart indicator fluorescent colorimetric card. The visual detection of the freshness of the meat can be achieved by color comparison; or, the fluorescent color image is converted into RGB value through the color picker APP of a smartphone, and the ratio of the red to green channels (R / G) is substituted into the linear equation established in step (1) to obtain the TVB-N content value of the meat at that time point.

[0038] Among them, when preparing the meat freshness intelligent indicator fluorescent colorimetric card, when TVB-N <15 mg / 100 g is considered a fresh sample; when 15 mg / 100 g ≤ TVB-N <20 mg / 100 g is considered a sub-fresh sample, and the storage days at this time are recorded as j days, where j is a positive integer ≤ m; when TVB-N ≥20 mg / 100 g is considered a corrupted sample, and the storage days at this time are recorded as n days, where j ≤ n ≤ m and are positive integers; at this time, meat stored for 1 to j-1 days is a fresh product, stored for j to n-1 days is a sub-fresh product, and stored for n to m days is a stale product;

[0039] Observe the fluorescent color changes of the indicator label during storage, use a smartphone to regularly record the fluorescent photos of the freshness fluorescent smart indicator label under ultraviolet light at different storage times, and arrange the fluorescent photos in ascending order according to the number of storage days, and record them as F1, F2, F3, ..., F j 、F j+1 ,……,F n 、F n+1 ,……,F m , forming a fluorescent color comparison card for freshness indication.

[0040] The present invention first prepares a ratiometric fluorescent indicator composed of a composite of red-fluorescent gold-copper bimetallic nanoclusters (indicator) and green-fluorescent dual-ligand gold nanoclusters (internal standard); then, using a solution doping method, the composite ratiometric fluorescent indicator is added to a polyvinyl alcohol / polyethylene glycol (PVA / PEG) solution via a pipette to prepare a ratiometric fluorescent smart label (a ratiometric metal nanocluster-doped polyvinyl alcohol / polyethylene glycol fluorescent composite film) for visually detecting meat freshness; finally, a fluorescent colorimetric card is manufactured based on the ratiometric fluorescent smart label, and combined with a smartphone, the ratiometric fluorescent smart label is applied to the prediction of meat freshness grade and total volatile basic nitrogen content.

[0041] Compared with the prior art, the present invention is beneficial in that:

[0042] (1) The gold-copper bimetallic nanoclusters (DPA-AuCuNCs) and dual-ligand gold nanoclusters (GSH-AuNCs@His) prepared in the present invention are AIE-type metal nanocluster molecules. The synthesis method is simple, non-toxic, and they have good fluorescence properties under excitation. The ratiometric fluorescent indicator formed by the composite can effectively avoid the influence of the detection environment on the detection of amine substances. The indicator can be used to detect dissolved ammonia substances in water samples with accurate and sensitive results. The indicator also has a certain self-correction ability and has the advantages of high sensitivity, high signal-to-noise ratio, fast response rate, and good detection effect.

[0043] (2) Based on the AIE properties of the prepared metal nanoclusters, the present invention further loads the ratiometric fluorescent indicator made of the two metal clusters onto a polyvinyl alcohol / polyethylene glycol (PVA / PEG) film to prepare a ratiometric fluorescent smart label. The label can be placed in food packaging to realize the intelligent indication of meat freshness. Compared with other smart indicator labels, the indicator label is non-toxic, low-cost, highly stable, has good film-forming properties, antibacterial properties and biocompatibility, and is an excellent degradable green material. In combination with the prepared fluorescent colorimetric card and smartphone APP, it can realize the qualitative and quantitative detection of the freshness grade of meat products and the TVB-N content contained. The detection method is accurate and reliable and does not require expensive detection equipment. The prepared ratiometric fluorescent smart label has obvious changes in fluorescence color during the monitoring process, which is visible to the naked eye. It has the characteristics of high sensitivity, high accuracy, good film-forming properties, high antibacterial properties, low cost, degradability and good biocompatibility. It can be widely used for non-destructive, rapid and real-time detection of the freshness of fresh meat and aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Flow chart of the preparation and application of ratiometric fluorescent smart labels for visual detection of meat freshness;

[0045] Figure 2 (a) is the image of the DPA-AuCuNCs solution in Example 1 under visible light and ultraviolet light conditions; Figure 2 (b) is the UV absorption spectrum of DPA-AuCuNCs; Figure 2 (c) is the fluorescence excitation and emission spectra of DPA-AuCuNCs;

[0046] Figure 3 (a) is the image of the GSH-AuNCs@His solution under visible light and ultraviolet light in Example 2; Figure 3 (b) is the UV absorption spectrum of GSH-AuNCs@His; Figure 3 (c) is the fluorescence excitation and emission spectra of GSH-AuNCs@His;

[0047] Figure 4 (a) is the fluorescence emission spectra of the ratiometric fluorescence indicators DPA-AuCuNCs and GSH-AuNCs@His in Example 3 after responding to ammonia water at different concentrations; Figure 4 (b) is the fitting curve established by the ratio of the fluorescence intensity of the ratiometric fluorescent indicator at 622 nm and 504 nm and different concentrations of ammonia water;

[0048] Figure 5(a) is a graph showing the G / R value of the ratiometric fluorescent smart label for visually detecting meat freshness in Example 4 and the changing trend of TVB-N content in beef during storage; Figure 5 (b) is a fitting curve established between the G / R value of the ratiometric fluorescent smart label for visually detecting meat freshness and the TVB-N content in beef during storage;

[0049] Figure 6 Fluorescent photos of the ratiometric fluorescent smart label for visually detecting meat freshness prepared in Example 5 in beef of different freshness grades;

[0050] Figure 7 Fluorescence emission spectra of two DPA-AuCuNCs (A) and DPA-AuCuNCs (B) with the same concentrations prepared by reference to others' methods and Example 1, transmission electron microscopy images of DPA-AuCuNCs (B), and transmission electron microscopy images of DPA-AuCuNCs (A);

[0051] Figure 8 (a) is the fluorescence emission curve of the single-signal fluorescent sensing freshness indicator prepared in Comparative Example 1 after reacting with ammonia water of different concentrations; Figure 8 (b) is the fitting curve of the fluorescence intensity ratio of the single-signal fluorescence sensing freshness indicator before and after the reaction and different ammonia concentrations. DETAILED DESCRIPTION

[0052] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto.

[0053] according to Figure 1 The preparation and application flow chart of the ratiometric fluorescent sensing freshness indicator label, and the examples of the preparation, detection and application of the ratiometric fluorescent sensing freshness indicator label are as follows:

[0054] Example 1: Preparation of gold-copper bimetallic nanoclusters (DPA-AuCuNCs)

[0055] At room temperature and with magnetic stirring, Cu(NO₃)₂ (0.2 mol / L, 100 μL) was added dropwise to a solution of penicillamine (0.4 mol / L, 40 mL) for 10 minutes to obtain a first precursor solution. After the dropwise addition of tetrachloroauric acid (0.30 mol / L, 80 μL), 1 mol / L hydrochloric acid solution was added dropwise until the pH reached 4.5. The solution was then heated in a 35°C water bath for 30 minutes to obtain a DPA-AuCuNCs reaction solution. The resulting reaction solution was centrifuged at 8000 rpm, filtered, washed two to three times, and freeze-dried to obtain gold-copper bimetallic nanoclusters.

[0056] Characterization was carried out by UV-visible spectroscopy, fluorescence spectroscopy and optical properties, such as Figure 2 As shown in (a), the DPA-AuCuCNs aqueous solution appears milky white under visible light and emits strong orange-red light under ultraviolet light. It can be seen from the UV-visible spectrum ( Figure 2 (b)), the UV absorption curve of DPA-AuCuNCs does not have any surface plasmon resonance absorption peak of metal particles, indicating that the synthesized copper nanoclusters do not generate by-products such as metal nanoparticles with larger particle sizes; from the fluorescence spectrum ( Figure 2 As can be seen from (c), DPA-AuCuNCs has a strong fluorescence emission peak at 622nm.

[0057] In summary, the prepared DPA-AuCuNCs have good fluorescence properties.

[0058] Example 2: Preparation of dual-ligand gold nanoclusters (GSH-AuNCs@His)

[0059] At room temperature, the prepared tetrachloroauric acid solution (0.30 mol / L, 80 μL) was slowly added dropwise to a histidine solution (0.04 mol / L, 8 mL). The reaction was allowed to proceed for 3 hours under magnetic stirring, until the color of the solution changed from colorless and transparent to light yellow, thereby obtaining a second precursor solution. At this point, a glutathione solution (0.010 mol / L, 8 mL) was added dropwise to the second precursor solution under magnetic stirring. After 1 hour of reaction, the resulting reaction solution was dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 3500, and then freeze-dried to obtain dual-ligand gold nanoclusters.

[0060] Characterization was carried out by UV-visible spectroscopy, fluorescence spectroscopy and optical properties, such as Figure 3 As shown in (a), the GSH-AuNCs@His aqueous solution shows light yellow under visible light and emits strong green light under ultraviolet light. It can be seen from the UV-visible spectrum ( Figure 3 (b)), the UV absorption curve of GSH-AuNCs@His does not have any surface plasmon resonance absorption peak of metal particles, indicating that the synthesized gold nanoclusters do not generate by-products such as metal nanoparticles with larger particle sizes; from the fluorescence spectrum ( Figure 3 As can be seen from (c), GSH-AuNCs@His has a strong fluorescence emission peak at 504 nm.

[0061] In summary, the prepared GSH-AuNCs@His has good fluorescence properties.

[0062] Example 3: Detection of Amines of Different Concentrations and Types by Ratio-Type Fluorescent Indicators

[0063] 0.2g of DPA-AuCuNCs and GSH-AuNCs@His were weighed and dissolved in 20mL of deionized water. Then, 3mL of DPA-AuCuNCs and 2mL of GSH-AuNCs@His were pipetted and mixed. The mixture was stirred under magnetic stirring for 20 minutes to obtain a uniformly mixed ratiometric fluorescent indicator. 3mL of the mixture was pipetted into a cuvette. 2mL of a 20-400ppm ammonia solution was then added to the cuvette and reacted for 3 minutes. The excitation and emission slit widths of the fluorescence spectrophotometer were set to 5nm, the excitation wavelength was 360nm, and the scanning range was 450nm-750nm. The fluorescence intensity ratio (F) of the fluorescence emission peak at 622nm and 504nm was recorded. 622nm / F 504nm ) to explore whether the ratiometric fluorescent indicator has a good response to ammonia. Figure 4 As shown in the figure, the ratiometric fluorescent indicator has good response characteristics to biogenic amines. The ratio of the fluorescence intensity at 622 nm to 504 nm is linearly related to the ammonia concentration, and the determination coefficient can reach R 2 =0.9775.

[0064] In this example, when exploring whether the ratiometric fluorescent indicator has a good response to N, the ammonia water can also be replaced by trimethylamine, dimethylamine, putrescine, histamine, cadaverine, etc., and the concentration is 20 to 400 ppm; the fluorescence emission intensity is measured using a fluorescence spectrophotometer with an excitation slit width of 5 to 10 nm, an emission slit width of 5 to 10 nm, and an excitation wavelength set to 340 to 360 nm.

[0065] Example 4: Ratio-based fluorescent smart label for visual detection of meat freshness

[0066] First, weigh 1.5g of PVA powder and pour it into a three-necked flask. Then, add 2mL of 5% polyvinyl alcohol, 1mL of phosphate-buffered saline, 2mL of glycerol, and 20mL of distilled water. After connecting the condenser, adjust the speed to 350rpm and continue heating to 75°C with mechanical stirring until the PVA powder is completely dissolved. Finally, heat under reflux for 45 minutes and cool to room temperature to form a uniform, transparent PVA / PEG mixture.

[0067] Using a pipette, 3 mL of the PVA / PEG mixed solution and 2 mL of the ratiometric fluorescent composite indicator prepared in Example 3 were transferred to a conical flask. The PVA / PEG and indicator were mixed under magnetic stirring for 30 minutes. The mixture was sonicated at room temperature for 20 minutes, allowed to stand at room temperature for 2 hours, and then slowly and evenly applied to a polytetrafluoroethylene mold using a pipette. The mold was allowed to stand at room temperature for 24 hours to produce a ratiometric fluorescent smart label (ratiometric metal nanocluster-doped polyvinyl alcohol / polyethylene glycol fluorescent composite film) for visually detecting meat freshness.

[0068] Example 5: Visual detection of meat freshness using a ratiometric fluorescent smart tag for visual monitoring of beef freshness

[0069] (1) Preparation of intelligent fluorescent colorimetric card for beef freshness: The ratiometric fluorescent intelligent label for visually detecting meat freshness prepared in Example 4 was cut into a 2 cm × 2 cm square and pasted on the top of the inner side of a transparent 500 mL fresh-keeping box. The card was stored in a constant temperature and humidity chamber at 4°C together with 35 g of beef sample. The TVB-N standard value of beef stored for different days was measured according to the physical and chemical indicators specified in the national standard GB5009.228-2016. Figure 5 As shown in (a), it can be found that the TVB-N value of beef with a storage period of 1 to 12 days is less than 15 mg / 100 g, and the beef at this time is fresh beef; when the storage period is greater than or equal to 12 days and less than 16 days, the TVB-N value is greater than or equal to 15 mg / 100 g and less than 20 mg / 100 g, and the beef is sub-fresh beef; when the storage period is greater than or equal to 16 days, the TVB-N value is greater than or equal to 20 mg / 100 g, and the beef is not fresh beef.

[0070] In addition, during the storage period, a smartphone was used to obtain fluorescent color images of the smart indicator label in the packaging bag under ultraviolet light every day. When the beef was stored for 0, 2, 4, 6, 8, 10, 12, 14, and 16 days, the collected fluorescent color images were arranged from small to large according to the storage days, and recorded as F0, F2, F4, F6, F8, F9, F10, F11, F12, F13, F14, F15, F16, F17, F18, F19, F20, F21, F22, F30, F31, F41, F50, F61, F70, F80, F90, F100, F110, F120, F130, F140, F150, F160 10 、F 12 、F 14 and F 16 , forming a meat freshness intelligent indicator fluorescent colorimetric card that intelligently indicates the freshness of beef.

[0071] In addition, the RGB values of the fluorescent color pictures arranged in ascending order of storage days were extracted through the smartphone color picker APP, and the ratio of the green to red channels (G / R) (y) was fitted with the TVB-N value (C) during storage, as shown in the following figure: Figure 5As shown in (b), the fitting equation is y = 0.0538C-0.2131, and the determination coefficient R 2 =0.9653. Using this equation, consumers can not only determine the freshness of beef using the meat freshness intelligent indicator fluorescent colorimetric card, but also calculate the TVB-N content of beef by extracting RGB information from the image, thereby accurately determining the freshness level of the beef.

[0072] Example 6: Visual detection of meat freshness using a ratiometric fluorescent smart label for visual and intelligent detection of beef freshness

[0073] Three beef samples of different freshness were selected, and the ratio-type fluorescent smart labels for visual detection of meat freshness were attached to the top of the inner side of the packaging boxes of the three beef samples of different freshness. Under ultraviolet light, the fluorescent color changes of the indicator labels can be clearly observed from the outside of the packaging boxes. Fluorescent color images of the three types of beef were obtained and further tested in combination with the fluorescent images. The colors of the fluorescent images were compared with the fluorescent colorimetric card. The results are as follows: Figure 6 As shown in the figure, it was found that the three types of beef with different freshness levels could be distinguished by freshness category using the fluorescent colorimetric card.

[0074] To further verify the accuracy of the indication results, the actual volatile basic nitrogen (TVB-N) content of three types of beef of varying freshness was measured according to the national standard method. The measured values were 9.72±0.67, 16.23±0.76, and 24.26±1.44 mg / 100g, respectively. These results demonstrate that the ratiometric fluorescent smart label can indicate beef freshness. Furthermore, the TVB-N content was quantitatively calculated by extracting the RGB values of the ratiometric fluorescent smart label for the three types of beef freshness and applying a linear equation. The calculated TVB-N values for the three types of beef were 9.37, 16.81, and 23.47 mg / 100g, respectively. A significant difference analysis between the calculated TVB-N values and the actual values measured according to the national standard method revealed no significant differences, confirming the effectiveness of the ratiometric fluorescent smart label for quantitative TVB-N analysis.

[0075] Comparative Example 1: Two gold-copper bimetallic nanoclusters (DPA-AuCuNCs) with different synthesis conditions were prepared as single-signal fluorescence sensing freshness indicators and used to detect ammonia solutions of different concentrations.

[0076] Two gold-copper bimetallic nanoclusters were prepared according to the methods described in Zhao Xianen et al. (Microchimica Acta, 2021, 188, 113) and Example 1, respectively, and were labeled as DPA-AuCuNCs (A) and DPA-AuCuNCs (B). Their morphological characteristics and fluorescence properties were analyzed by transmission electron microscopy scanning and fluorescence spectrophotometry.

[0077] like Figure 7 As shown, the fluorescence emission peak of DPA-AuCuNCs (B) prepared in Example 1 has a fluorescence intensity of 622 nm higher than that of DPA-AuCuNCs (A) prepared in the study of Zhao Xianen et al. (Microchimica Acta, 2021, 188, 113), and as Figure 7 As shown in the transmission electron micrographs of DPA-AuCuNCs (A) and DPA-AuCuNCs (B), at the same concentration, the clusters prepared by the method of the present invention exhibited superior aggregation. Therefore, the DPA-AuCuNCs prepared by the method of the present invention exhibited improved fluorescence properties and stability.

[0078] Based on this, a 10 g / L DPA-AuCuNCs (B) solution was prepared, and the two indicators were reacted with ammonia water of different concentrations (0-400 ppm) according to the method in Example 3. The results are as follows Figure 8 (a) and Figure 8 As shown in (b), the single-signal fluorescence sensing freshness indicator has good response characteristics with different concentrations of ammonia as a reaction substance, and the fluorescence intensity at 622nm also shows a linear relationship with the ammonia concentration, but the determination coefficient R 2 =0.9245, which is lower than the ratiometric fluorescent indicator prepared in Example 3.

[0079] Comparative Example 2: Detection of ammonia solutions with different concentrations based on dual-ligand gold nanoclusters (GSH-AuNCs@His)

[0080] A 10 g / L aqueous solution of GSH-AuNCs@His was prepared, and the indicator was reacted with various concentrations of ammonia (0 to 400 ppm) according to the method in Example 3. The results showed that the fluorescence signal did not change significantly. Therefore, GSH-AuNCs@His serves as an internal standard molecule and plays a self-calibration role in the ratiometric fluorescence indicator.

[0081] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing a ratiometric fluorescent smart label for visually detecting meat freshness, characterized in that: The method comprises the following preparation steps: (1) The preparation steps of DPA-AuCuNCs are as follows: The prepared copper nitrate solution was slowly added dropwise to the DPA solution at room temperature, and the mixture was reacted under magnetic stirring to obtain a first precursor solution. The tetrachloroauric acid solution was slowly added dropwise to the first precursor solution, and then hydrochloric acid was added dropwise to adjust the pH to 4.0-5.0 to form a mixed solution. The mixed solution was then placed in a water bath and heated to obtain a DPA-AuCuNCs reaction solution; The resulting reaction solution was centrifuged, filtered, washed, and freeze-dried to obtain DPA-AuCuNCs; (2) The preparation steps of GSH-AuNCs@His are as follows: At room temperature, the prepared tetrachloroauric acid solution was slowly added dropwise to the His solution. Under magnetic stirring, the color of the solution changed from colorless and transparent to light yellow, thereby obtaining a second precursor solution. At that time, glutathione solution is added dropwise to the second precursor solution. After the reaction is complete, the resulting reaction solution is dialyzed and freeze-dried to obtain GSH-AuNCs@His. (3) The steps for preparing the ratiometric fluorescent indicator solution are as follows: The DPA-AuCuNCs prepared in step (1) are prepared into a DPA-AuCuNCs solution, and the GSH-AuNCs@His prepared in step (2) are prepared into a GSH-AuNCs@His solution, and the two solutions are mixed to prepare a ratiometric fluorescent indicator solution; (4) The steps for preparing a ratiometric fluorescent smart label for visually detecting meat freshness are as follows: The ratiometric fluorescent indicator solution prepared in step (3) is mixed with the PVA / PEG solution by a solution doping method under magnetic stirring; After being fully mixed, the mixed solution is slowly poured into a polytetrafluoroethylene plate mold until a thin film is formed. Then, it is gently peeled off to obtain a ratiometric fluorescent smart label for visually detecting the freshness of meat. The molar concentration of the copper nitrate solution in step (1) is 0-0.2 mol / L; the molar concentration of the DPA solution is 0-0.4 mol / L; the molar concentration of the tetrachloroauric acid solution is 0-0.30 mol / L; the volume ratio of the copper nitrate, tetrachloroauric acid, and DPA solution is 5:4:1000-2000; The reaction time of the copper nitrate solution and DPA in step (1) is 0-10 min; the temperature of the water bath is 30-40 o C; the heating time is 30~60 min; the centrifugal speed is 8000~10000 rpm; the centrifugal time is 0~10 min; The molar concentration of the tetrachloroauric acid solution in step (2) is 0-0.30 mol / L; the molar concentration of the His solution is 0-0.05 mol / L; the molar concentration of the glutathione solution is 0-0.015 mol / L; the volume ratio of the tetrachloroauric acid, His, and glutathione solutions is 1:50-100:50-100; The reaction time of the tetrachloroauric acid solution and the His solution in step (2) is 0-4 h; the reaction time of the second precursor solution and the glutathione solution is 0-1 h; the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500 and deionized water for 24-48 h; The mass concentration of the DPA-AuCuNCs solution in step (3) is 0-0.01 g / mL; The mass concentration of the GSH-AuNCs@His solution is 0~0.01 g / mL; the volume ratio of the DPA-AuCuNCs solution to the GSH-AuNCs@His solution is 3:

2.

2. The method for preparing a ratiometric fluorescent smart label for visually detecting meat freshness according to claim 1, characterized in that: The preparation method of the PVA / PEG solution in step (4) is as follows: Weigh PVA, add 0-10% polyvinyl alcohol, phosphate buffered saline, glycerol, and distilled water to it, connect the condensed water, and continue heating under mechanical stirring until the PVA powder is completely dissolved, and finally heat under reflux and cool to room temperature to form a PVA / PEG solution; the PVA: phosphate buffered saline: glycerol: distilled water is 0-2 g: 0-2 mL: 0-1 mL: 0-2 mL: 0-20 mL; the continuous heating temperature is 60-80 o C, reflux time 30~60 min.

3. A ratiometric fluorescent smart label for visually detecting meat freshness, characterized in that: The product is prepared by the preparation method according to any one of claims 1 to 2.

4. An application of the ratiometric fluorescent smart label for visually detecting meat freshness according to claim 3 in visual monitoring of meat freshness.

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