A method for detecting freshness of meat based on a dual-signal zwitterionic hydrogel colorimetric-fluorescent smart label

Abstract

The application provides a method for detecting meat freshness based on a double-signal zwitterionic hydrogel colorimetric-fluorescent intelligent label, and belongs to the technical field of food safety detection. The intelligent label takes SBMA hydrogel as a substrate, covalently bonds fluorescent monomer AOMC, and physically embeds colorimetric indicator alizarin. When meat is decomposed to produce TVB-N, the label presents a colorimetric response of alizarin from yellow to purple red and a double signal of the change of the fluorescence intensity of AOMC. In combination with the image acquisition of a smart phone and the RGB / fluorescent analysis, a quantitative model of the double signal and TVB-N is established, so that the grading detection of freshness (≤15 mg / 100g), sub-freshness (15-25 mg / 100g) and corruption (>25 mg / 100g) is realized. The double-signal output mode effectively improves the reliability and anti-interference ability of detection, is simple and convenient to operate, and is low in cost, and the detection can be completed within 10 minutes, so that an effective platform is provided for the on-site rapid detection of meat freshness.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically involving a method for detecting meat freshness based on a dual-signal zwitterionic hydrogel colorimetric-fluorescent smart tag. It uses total volatile basic nitrogen (TVB-N) as a spoilage marker and achieves rapid detection and freshness grading through the dual response of the fluorescence signal of AOMC and the colorimetric signal of alizarin in the hydrogel. Background Technology

[0002] Meat is rich in protein, fat, and water, making it highly susceptible to spoilage due to microbial activity during storage and transportation. Microorganisms decompose proteins, producing alkaline nitrogenous volatile substances such as ammonia, trimethylamine, dimethylamine, and histamine, collectively known as total volatile basic nitrogen (TVB-N). TVB-N content is an internationally recognized core indicator for evaluating meat freshness. my country's national food safety standard GB 2707-2016 stipulates that the TVB-N limit for fresh (frozen) meat is 15 mg / 100g; exceeding this value indicates staleness.

[0003] The traditional method for TVB-N detection is the semi-micro Kjeldahl method (GB 5009.228-2016). This method requires cumbersome steps such as distillation and titration, takes 2-3 hours, and requires specialized glassware and chemical reagents, making it unsuitable for rapid on-site screening. While recent developments in detection methods such as pH indicator strips and gas sensors offer greater ease of operation, they generally suffer from low sensitivity, susceptibility to environmental humidity and interfering gases, and difficulty in quantification.

[0004] Hydrogel smart tags have become a research hotspot for food freshness monitoring due to their advantages such as visualization, quantification, and ease of integration. Zwitterionic hydrogels (such as polysulfobetaine SBMA) possess excellent anti-fouling capabilities and hydrophilicity. Their molecular chains simultaneously contain positively charged quaternary ammonium groups and negatively charged sulfonic acid groups, which can form a robust hydration layer on the material surface through electrostatic interactions, effectively resisting the non-specific adsorption of biomolecules such as proteins and fats. This characteristic enables them to maintain stable sensing performance in complex food systems, avoiding signal misinterpretation caused by surface contamination.

[0005] Coumarin-based fluorescent compounds are an important class of organic fluorescent materials, possessing advantages such as high fluorescence quantum yield, good photostability, and ease of chemical modification. 7-Acryloyloxy-4-methylcoumarin (AOMC), as a fluorescent functional monomer containing terminal double bonds, can be covalently bonded to a hydrogel network via free radical copolymerization, achieving stable fixation of fluorescent signal units and avoiding leakage problems of fluorescent indicators. Furthermore, the fluorescence intensity of AOMC changes with environmental pH, making it suitable as a pH-responsive fluorescent signal output unit.

[0006] Alizarin (1,2-dihydroxyanthraquinone) is a natural anthraquinone pH indicator. Its phenolic hydroxyl group undergoes deprotonation under alkaline conditions, altering its conjugated molecular structure and causing a red shift in its absorption spectrum. This results in a distinct color change from yellow to purplish-red within a pH range of 6.0-11.0. This pH range precisely covers the pH rise caused by TVB-N accumulation during meat spoilage.

[0007] Fluorescent hydrogels are a novel type of polymeric composite material with a three-dimensional cross-linked network and fluorescence emission properties. They can change their fluorescence intensity or fluorescence emission color in response to external analyte stimuli, making them an important tool for the quantitative detection of analyte concentrations. Integrating the fluorescence signal of AOMC and the colorimetric signal of alizarin into the same hydrogel system can construct a smart sensor tag with dual signal output. The mutual verification of the two signals can effectively improve the reliability and anti-interference ability of the detection.

[0008] However, the release of TVB-N in actual meat samples is significantly affected by sample inhomogeneity and matrix interference, and direct headspace or surface contact detection using smart tags often results in poor reproducibility. Therefore, developing a simple and standardized sample pretreatment method, combined with highly sensitive smart tag detection, is of great significance for achieving accurate on-site evaluation of meat freshness.

[0009] Based on the above background, this invention combines the antifouling properties of SBMA zwitterionic hydrogel, the pH-responsive fluorescence characteristics of AOMC, and the colorimetric response characteristics of alizarin to develop a dual-signal output colorimetric-fluorescence smart tag, enabling rapid, accurate, and visual detection of TVB-N in meat. Summary of the Invention

[0010] The purpose of this invention is to provide a method for detecting meat freshness based on a dual-signal zwitterionic hydrogel colorimetric-fluorescent smart tag. Using TVB-N as the detection target, the method utilizes the dual response of the fluorescence signal of AOMC and the colorimetric signal of alizarin in the hydrogel, combined with smartphone image acquisition and RGB / fluorescence analysis, to achieve rapid quantitative detection and grading of meat freshness.

[0011] On one hand, this invention provides a method for detecting meat freshness using a dual-signal zwitterionic hydrogel colorimetric-fluorescent smart tag. The smart tag comprises a zwitterionic SBMA hydrogel, a fluorescent monomer AOMC covalently bonded to the gel network, and a physically embedded colorimetric indicator, alizarin. AOMC undergoes a free radical copolymerization reaction with the SBMA monomer under UV light initiation via its terminal double bonds, becoming covalently fixed in the three-dimensional hydrogel network; alizarin is physically embedded in the gel network through hydrogen bonding between its phenolic hydroxyl groups and the sulfonic acid groups of the hydrogel. When the tag comes into contact with a meat extract containing TVB-N, the alkaline amines cause the solution pH to rise, alizarin undergoes deprotonation, and its color gradually changes from yellow to purplish-red (colorimetric signal); simultaneously, the fluorescence intensity of AOMC changes regularly with increasing pH (fluorescence signal), and both signals are positively correlated with the TVB-N concentration.

[0012] The detection method also includes using a smartphone to collect colorimetric images (under visible light) and fluorescence images (under 365 nm ultraviolet light excitation) of the label, extracting the colorimetric RGB values ​​and fluorescence intensity values ​​using RGB analysis software, calculating the color difference ΔE (ΔE = √[(ΔL)²+(ΔA)²+(ΔB)²]) and fluorescence intensity change ΔF respectively, substituting them into a pre-established dual-signal-TVB-N standard curve, and outputting the TVB-N content and freshness grade.

[0013] On the other hand, this invention provides a method for synthesizing AOMC fluorescent monomers. 7-hydroxy-4-methylcoumarin is reacted with acryloyl chloride in the presence of a potassium hydroxide / polyethylene glycol composite catalyst to obtain 7-acryloyloxy-4-methylcoumarin (AOMC). Specifically, 7-hydroxy-4-methylcoumarin (1.0 mmol) and sodium hydroxide (1.0 mmol) are mixed in 50 mL of ethanol and stirred at 60°C until completely dissolved. After cooling to 0-5°C, acryloyl chloride (1.1 mmol) is slowly added, and stirring continues for 90 minutes. The resulting white solid is filtered, dried, and purified by recrystallization from methanol to obtain AOMC.

[0014] On the other hand, the present invention provides a method for preparing a dual-signal smart tag, comprising the following steps: (1) Preparation of precursor solution: Dissolve SBMA monomer and AOMC fluorescent monomer in a solution containing crosslinking agent N,N'-methylenebisacrylamide (MBAA) and photoinitiator Irgacure 2959, add alizarin stock solution (solvent is 30% glycerol aqueous solution), and vortex mix. The mass-volume ratio of SBMA in the precursor solution is 25% (w / v), the concentration of AOMC is 10 mg / mL, the volume fraction of MBAA is 1%, the volume fraction of Irgacure 2959 is 1%, and the final concentration of alizarin is 0.5 mg / mL; (2) Pour the above mixed solution into a mold (20 mm in diameter and 2 mm in thickness); (3) Irradiate under a 365 nm UV lamp for 10-15 minutes to initiate the free radical copolymerization reaction of SBMA and AOMC, forming a three-dimensional cross-linked network structure hydrogel tag in which AOMC is covalently bonded. (4) Wash the label thoroughly with deionized water three times to remove unreacted monomers and free alizarin, and store it in a sealed container at 4°C to obtain a dual-signal smart label.

[0015] On the other hand, the present invention provides a method for preparing and detecting sample extracts, comprising the following steps: (1) Cut the meat sample to be tested into small pieces of about 2 mm × 2 mm, accurately weigh 2.0 g and place it in a centrifuge tube, add 10 mL of deionized water or PBS buffer (pH 5.8), shake to extract for 5-10 minutes, let stand and take the supernatant or filter to obtain meat juice extract; (2) Immerse the dual-signal smart tag in the meat juice extract and let it stand at room temperature for 5-10 minutes until the colorimetric and fluorescence signals stabilize. (3) Remove the label, wipe the surface liquid with filter paper, take a colorimetric photo with a smartphone under visible light, and then take a fluorescence photo under 365 nm ultraviolet light. (4) Use RGB analysis apps such as Color Picker to extract the RGB values ​​of the visible light image of the label and calculate the color difference ΔE; use image analysis software such as ImageJ to extract the gray value or fluorescence intensity of the fluorescence image and calculate the fluorescence intensity change ΔF; (5) Substitute ΔE and ΔF into the pre-established dual-signal-TVB-N standard curve to calculate the TVB-N content of the sample; (6) Freshness is determined according to GB 2707-2016: TVB-N ≤ 15 mg / 100g is fresh; 15-25 mg / 100g is not fresh; >25 mg / 100g is spoiled.

[0016] On the other hand, the present invention performs a systematic performance characterization of dual-signal smart tags, including the following aspects: (1) Fluorescence spectral characterization: The fluorescence emission spectra of AOMC under different pH conditions were measured using a fluorescence spectrophotometer to determine its pH response characteristics and the optimal excitation / emission wavelength.

[0017] (2) Colorimetric response characterization: The labels were immersed in different pH buffer solutions (pH 2.0-12.0), and the color change photos and RGB values ​​were recorded to establish a ΔE-pH standard curve.

[0018] (3) Rheological performance characterization: The viscoelasticity of the smart tag was characterized by a rotational rheometer, including frequency scanning (measuring the changes of storage modulus G' and loss modulus G'' with angular frequency), strain scanning (determining the linear viscoelastic region) and temperature scanning (evaluating the mechanical stability at different temperatures).

[0019] (4) Characterization of swelling and water retention performance: The equilibrium swelling rate and water content were determined by weighing method. The water transport mechanism was analyzed by Fick diffusion model through swelling kinetic test.

[0020] (5) Anti-protein contamination performance test: FITC-labeled bovine serum albumin (FITC-BSA) was used to conduct protein adsorption test to evaluate the anti-contamination ability of SBMA hydrogel.

[0021] (6) TVB-N response and dual-signal correction curve establishment: The label was reacted with ammonia water of different concentrations or meat juice extracts of different spoilage degrees, and the colorimetric signal ΔE and fluorescence signal ΔF were recorded at the same time to establish a dual-signal-TVBN quantitative correction model.

[0022] (7) Long-term storage stability test: Store the label at 4°C in a dry, light-protected place for 30 days, and periodically test the pH response (colorimetric and fluorescence) and mechanical properties to assess the shelf life.

[0023] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (i) This invention is the first to covalently bond AOMC fluorescent monomers to an SBMA zwitterionic hydrogel network, and together with alizarin colorimetric indicator, construct a smart tag with dual signal output. AOMC is fixed to the gel network by covalent bonds, avoiding the leakage problem of traditional physically doped fluorescent indicators, and improving the long-term stability of the tag.

[0024] (ii) The dual-signal output mode is an important innovation of this invention: the colorimetric signal (alizarin) provides visually visible color changes, which is suitable for rapid qualitative screening; the fluorescence signal (AOMC) provides highly sensitive quantitative information. The two verify each other and complement each other, effectively improving the reliability and anti-interference ability of the detection, and overcoming the defects of the single signal mode being susceptible to environmental interference.

[0025] (III) The present invention uses SBMA zwitterionic hydrogel as the label substrate. Its excellent anti-protein adsorption ability can effectively resist the non-specific adhesion of interfering substances such as proteins and fats in meat juice, keep the label surface clean, and ensure the accuracy of colorimetric and fluorescence signal reading.

[0026] (iv) The three-dimensional network structure of the hydrogel has a high water content (82%±2%), which provides a homogeneous microenvironment similar to a solution for the colorimetric reaction of alizarin and the fluorescence response of AOMC. The reaction kinetics are fast, and the signal can be stabilized in 5-10 minutes.

[0027] (v) Alizarin exhibits a continuous color gradient from yellow to purplish-red in the pH range of 6.0-11.0, which is visible to the naked eye; AOMC shows a regular change in fluorescence intensity in this pH range, and both signals have a good quantitative relationship with TVB-N content (R²≥0.95). The detection results are consistent with the national standard method and can accurately distinguish between fresh, semi-fresh and spoiled grades.

[0028] (vi) The label preparation process is simple, and the raw materials SBMA, AOMC, and alizarin can all be obtained through simple synthesis or commercially available, making it suitable for large-scale production. The label can be stored for more than 30 days under dry and light-protected conditions at 4°C.

[0029] (vii) The testing process does not require any professional equipment. Only a smartphone (with a simple ultraviolet light source) is needed to complete image acquisition and result determination. It is suitable for rapid screening of meat freshness in non-professional environments such as homes, supermarkets, farmers' markets, and catering enterprises. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the preparation process and detection principle of the dual-signal smart tag of the present invention.

[0031] Figure 2 A shows the UV-Vis absorption spectrum and color change of alizarin indicator in different pH buffers (pH 2.0-12.0); B shows the color change of AOMC fluorescent indicator in different pH buffers.

[0032] Figure 3 Scanning electron microscopy (SEM) images and Fourier transform infrared (FTIR) spectra of hydrogels with different compositions are shown. A represents the SEM cross-sectional morphology of the SM group (pure SBMA hydrogel), B represents the SEM cross-sectional morphology of the SMA group (SBMA+AOMC), C represents the SEM cross-sectional morphology of the SMQ group (SBMA+alizarin), D represents the SEM cross-sectional morphology of the SMAQ group (SBMA+AOMC+alizarin), E represents the SEM cross-sectional morphology of the SMAQG group (SBMA+AOMC+alizarin+glycerol), and F is a superimposed comparison of the FTIR spectra of the five hydrogel groups.

[0033] Figure 4 The rheological test results for the SBMA hydrogel smart tag are shown; where ABC represents strain scanning test and DEF represents frequency scanning test. Figure 5The graphs show the swelling performance of SBMA hydrogel smart tags; where A is the drying kinetics curve; B is the drying Fick diffusion model fitting curve; C is the swelling kinetics curve; and D is the swelling Fick diffusion model fitting curve. Figure 6 A comparison chart of the anti-pollution performance of smart tags (FITC-BSA adsorption fluorescence intensity, SBMA hydrogel and ordinary agarose gel as controls).

[0034] Figure 7 The two-signal pH response curves of the smart tag are shown; where A is the colorimetric signal ΔE-pH standard curve (pH 6.0-11.0) and B is the fluorescence signal ΔF-pH standard curve. Figure 8 The two-signal calibration curves for TVB-N are shown for the smart tag; where A is the colorimetric signal ΔE-TVBN curve and B is the fluorescence signal ΔF-TVBN curve. Figure 9 The chart shows the long-term storage stability test results of smart tags. In the chart, A represents the stability of the colorimetric signal ΔE response over 30 days, and B represents the stability of the fluorescence signal ΔF response over 30 days. Detailed Implementation

[0035] The following examples use pork and fish as examples for methodological verification, but those skilled in the art should understand that the present invention is also applicable to the detection of other meats such as beef, mutton, and poultry. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available products.

[0036] The preparation process and detection principle of the dual-signal smart tag of this invention are as follows: Figure 1 As shown, the present invention is designed and implemented based on this principle. Example 1 Synthesis of AOMC fluorescent monomer 7-Hydroxy-4-methylcoumarin (1.0 mmol) and sodium hydroxide (1.0 mmol) were mixed in 50 mL of ethanol and stirred continuously at 60 °C until completely dissolved. After stirring for 30 minutes, the mixture was cooled in an ice bath at 0–5 °C. Subsequently, acryloyl chloride (1.1 mmol) was slowly added, and stirring was continued for 90 minutes. The resulting white solid (7-acryloyloxy-4-methylcoumarin, AOMC) was filtered, dried, and purified by recrystallization from methanol to obtain the AOMC fluorescent monomer. The structure was confirmed by 1H NMR and 13C NMR, and the product purity was ≥98%.

[0037] Example 2 pH response spectrum characterization of alizarin indicator Accurately weigh 10 mg of alizarin standard and dissolve it in 10 mL of deionized water containing 0.1% HCl (pH≈3.0) to prepare a 1 mg / mL stock solution. Aliquot the stock solution and store it at -20°C protected from light. When using, take an appropriate amount of the stock solution and dilute it with different pH buffers (pH 2.0-12.0, interval 1.0) to a final concentration of 0.05 mg / mL. After equilibration at room temperature for 10 minutes, record the absorption spectrum at 400-700 nm using a UV-Vis spectrophotometer and take color photographs simultaneously.

[0038] The results are as follows Figure 2 As shown in Figure A, the color changes from yellow to purplish-red from pH 6.0 to 11.0, which are visible to the naked eye. The absorption spectrum shows that the absorption intensity at 520 nm gradually increases with increasing pH, indicating that the pH response range of alizarin (pH 6.0-11.0) is highly consistent with the pH change range caused by the accumulation of TVB-N during meat spoilage.

[0039] Example 3: pH-responsive fluorescence characterization of AOMC AOMC was dissolved in buffer solutions of different pH values ​​(pH 4.0-11.0) to a final concentration of 10 μg / mL. After equilibration at room temperature for 10 minutes, the fluorescence emission spectrum (excitation wavelength 320 nm) was measured using a fluorescence spectrophotometer. The results showed that the fluorescence intensity of AOMC changed regularly with increasing pH in the pH range of 6.0-11.0. Fluorescence was not significant before pH 7.0, and gradually increased with increasing pH. This pH response range highly coincides with the pH change range caused by TVB-N accumulation during meat spoilage and can be used as a fluorescence signal output unit for TVB-N detection.

[0040] The results are as follows Figure 2 As shown in Figure B, the fluorescence intensity of AOMC increases regularly with increasing pH in the pH range of 7.0-11.0. This pH response range is highly consistent with the pH change range caused by TVB-N accumulation during meat spoilage. Moreover, the sensitivity of the fluorescence signal to pH change is better than that of the colorimetric signal, and it can be used as a high-sensitivity fluorescence signal output unit for TVB-N detection.

[0041] Example 4: Fabrication of a Signal Smart Tag Dissolve 1.25 g of SBMA monomer in 2 mL of 1% MBAA crosslinking agent solution, add 2.5 mL of 1% Irgacure2959 photoinitiator solution, 0.5 mL of AOMC solution (10 mg / mL, dissolved in 30% glycerol aqueous solution), and 0.5 mL of 5 mg / mL alizarin stock solution, and add deionized water to a total volume of 5 mL. Vortex to mix. Pour 500 μL of the mixture into a circular mold (20 mm in diameter, 2-3 mm thick) and irradiate under a 365 nm UV lamp for 12 minutes to initiate the free radical copolymerization reaction of SBMA and AOMC, forming a three-dimensional crosslinked network hydrogel tag with AOMC covalently bonded within it. Wash the tag thoroughly three times with deionized water to remove unreacted substances and free alizarin, and store it sealed at 4℃ for later use.

[0042] Example 5: Comparison of SEM morphology, FTIR structural characterization, and embedding effect of hydrogels with different compositions To verify the encapsulation advantages of SBMA zwitterionic hydrogels for alizarin indicators, this embodiment designed and prepared five sets of hydrogel control samples with different compositions: SM (pure SBMA hydrogel), SMA (SBMA+AOMC), SMQ (SBMA+alizarin), SMAQ (SBMA+AOMC+alizarin), and SMAQG (SBMA+AOMC+alizarin+glycerol). The effects of different gel compositions on the indicator encapsulation effect were systematically compared through SEM morphology observation, FTIR structural analysis, and alizarin release behavior testing. After freeze-drying, the five sets of hydrogel samples were sputter-coated with gold, and the cross-sectional microstructure was observed using scanning electron microscopy.

[0043] The results are as follows Figure 3As shown in Figures ABCDE: Group SM (pure SBMA hydrogel) exhibits a uniform and dense three-dimensional porous network structure with a uniform pore size distribution (approximately 50-150 μm) and smooth, continuous pore walls, indicating that SBMA polymerization alone can form a well-structured cross-linked network. Group SMA (SBMA+AOMC) has even smoother and more uniform pore walls, due to AOMC's covalent bonding into the gel network, forming a uniform copolymer structure with SBMA segments. Group SMQ (SBMA+Alizarin) shows a small amount of particulate deposits on the pore wall surface, reflecting alizarin molecules attaching to the gel backbone through hydrogen bonding; however, the pore structure remains intact, indicating that the physical embedding of alizarin does not significantly damage the gel network. Group SMAQ (SBMA+Alizarin+AOMC) exhibits the richest three-dimensional network structure, with fine particles (alizarin attachment) uniformly distributed on the pore wall surface while maintaining unobstructed pores, indicating that the coexistence of the two indicators (alizarin+AOMC) did not cause network collapse or phase separation. The pore size of the SMAQG group (SBMA+Alizarin+AOMC+Glycerin) is slightly larger than that of the other four groups (approximately 80-200 μm). This is because glycerin, as a plasticizer, increases the free volume of the network to a certain extent, but the overall three-dimensional porous structure remains intact, the pore walls are smooth and uniform, and alizarin is uniformly dispersed in the gel skeleton without obvious aggregation.

[0044] The chemical structures of the five hydrogels were characterized using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), and the results are as follows: Figure 3 As shown in F: SMA, SMAQ, and SMAQG groups at 3400 cm - ¹The broad peak near the 3440 cm⁻¹ changes significantly: the OH stretching vibration peak of pure SBMA hydrogel (SM group) is located at 3440 cm⁻¹. - ¹; After the introduction of alizarin (SMA group), the peak redshifted (shifted to a lower wavenumber) to 3415 cm⁻¹. - ¹ indicates that a hydrogen bond interaction exists between the phenolic hydroxyl group of alizarin and the sulfonic acid group of SBMA. In the SMAQ group, this peak further redshifts to 3405 cm⁻¹. - ¹ This is attributed to the formation of a multiple hydrogen bond network between the alizarin hydroxyl group, SBMA sulfonic acid group, and AOMC ester group, which further enhances the fixation effect of alizarin in the gel network.

[0045] Example 6 Rheological performance characterization of dual-signal smart tags The viscoelasticity of smart tags was systematically characterized using a rotational rheometer (equipped with a 25 mm parallel plate fixture with a 1 mm spacing).

[0046] The results are as follows Figure 4As shown in AF: (1) Frequency scanning test: Under the conditions of strain amplitude of 1% and temperature of 25℃, the storage modulus (G') and loss modulus (G'') were measured in the range of angular frequency of 0.1-100 rad / s. The results show that ( Figure 4 DF), G' is always greater than G'' throughout the entire frequency scanning range, and both show a slow upward trend with increasing frequency. G' is about 800-1200 Pa, and G” is about 80-150 Pa. The loss factor tanδ is between 0.08 and 0.15, indicating that the smart tag has formed a stable chemical cross-linked three-dimensional network structure.

[0047] (2) Strain scanning test: Under fixed frequency of 1 Hz and temperature of 25℃, strain scanning was performed within the strain amplitude range of 0.1%-1000%. The results show that ( Figure 4 (AC) G' and G'' remain constant within the strain amplitude range of 0.1%-10%; when the strain amplitude exceeds 10%, G' begins to decrease, and the critical failure strain is about 15%.

[0048] Example 7 Characterization of swelling properties of dual-signal smart tags The swelling properties of smart tags were determined using a gravimetric method. Dry tags were weighed (W_d), immersed in deionized water, and weighed at different time points (W_t) until the weight reached a constant (W_e). The equilibrium swelling ratio (ESR) was calculated as (W_e - W_d) / W_d × 100%, and the water content (WC) was calculated as (W_e - W_d) / W_e × 100%. The Fick diffusion model was used, with Mt / M∞ = k·t. n Analyze the water transport mechanism.

[0049] The results are as follows Figure 5 As shown in Figure AD, under conditions of 25℃ and 40% relative humidity, the water loss rate of SBMA hydrogel gradually increased over time. A linear fit of ln(Mt / M∞) to ln(t) yielded a swelling index of 0.43, close to 0.5, indicating that the drying process was mainly controlled by internal water diffusion. The tag initially absorbed water rapidly within the first 10 molecules, reaching a swelling ratio of over 80% of the equilibrium value. Subsequently, the water absorption rate gradually slowed, reaching swelling equilibrium in approximately 40 minutes. These results demonstrate that SBMA zwitterionic hydrogel possesses excellent hydrophilicity and water retention capacity, which is beneficial for the rapid penetration of TVB-N and the uniformity of the colorimetric reaction.

[0050] Example 8: Anti-contamination performance test of dual-signal smart tags To verify the anti-protein adsorption capacity of SBMA hydrogel, FITC-labeled bovine serum albumin (FITC-BSA) was used for testing. SBMA dual-signal smart tags and ordinary agarose gel tags were incubated with 0.5 mg / mL FITC-BSA solution for 30 minutes, washed three times with PBS buffer, and observed using an inverted fluorescence microscope.

[0051] The results are as follows Figure 6 As shown, the fluorescence intensity on the surface of the SBMA gel is only about 5% of that of the agarose gel, indicating that the SBMA zwitterionic hydrogel can effectively resist non-specific protein adsorption, ensuring that the label surface is clean in the complex matrix of meat juice, and that colorimetric and fluorescence signal readings are not interfered with by stains.

[0052] Example 9: Response and Standard Curve of Dual-Signal Smart Tag Dual-signal smart tags were immersed in triethylamine at concentrations of 0, 5, 10, 15, 20, 25, 30, 40, and 60 mg / 100g for 10 minutes, respectively. After removal, the surface was blotted dry with filter paper, and images were immediately taken under visible light (colorimetric signal) and under 365 nm ultraviolet light (fluorescence signal). RGB values ​​and fluorescence intensity values ​​were extracted using a smartphone app, and ΔE and ΔF were calculated, respectively.

[0053] The results are as follows Figure 7 AB and Figure 8 As shown in Figure AB, within the range of 0-30 mg / 100g, ΔE exhibits a good linear relationship with pH (R²=0.99355), and the visually perceptible threshold ΔE>5 meets the sensitivity requirements for TVB-N detection. The label color gradually changes from yellow to orange-red and then to purplish-red with increasing pH, with distinct color gradations that are easy to identify. The fluorescence intensity of AOMC gradually increases with increasing concentration within the range of 0-30 mg / 100g, and ΔE shows a good linear relationship with pH (R²=0.99604). Both signals exhibit good linear responses within their respective ranges. The colorimetric signal is suitable for rapid qualitative discrimination by the naked eye (direct observation of color changes under visible light), while the fluorescence signal provides highly sensitive quantitative information (detection of fluorescence intensity changes under ultraviolet light). The two signals corroborate and complement each other, effectively improving the reliability of the detection.

[0054] Example 10 TVB-N Response and Dual-Signal Correction Curve Establishment Fresh pork and fish were collected, and meat extracts at different degrees of spoilage were prepared as follows: Meat samples were chopped and stored at 4℃. Samples were taken at 0, 12, 24, 36, 48, and 72 h. Deionized water was added at a ratio of 1:5 (w / v), and the samples were extracted by shaking for 10 minutes. The extracts were then filtered. A dual-signal smart tag was immersed in each extract for 8 minutes. After removal, images were taken under visible light and 365 nm ultraviolet light, and ΔE and ΔF were extracted. Simultaneously, the TVB-N content of the same meat sample was determined according to the national standard GB 5009.228-2016. The results of the smart tag detection were consistent with the national standard method.

[0055] Example 11: Monitoring the Freshness of Actual Samples and Verifying Dual Signals One portion each of fresh pork and fish was stored at 4℃ and 25℃, respectively. The tags were removed at different time points, and the colorimetric signal ΔE and fluorescence signal ΔF were recorded simultaneously. TVB-N (national standard method) was measured at each time point. The dual-signal smart tags were stored at 4℃ in a dry, light-protected environment. Every 5 days, their response to pH 7.0 buffer solution (colorimetric signal ΔE and fluorescence signal ΔF) and tensile strength were tested, and this monitoring continued for 30 days.

[0056] The results are as follows Figure 9 As shown in Figure AB, the ΔE fluctuation does not exceed ±5% and the ΔF fluctuation does not exceed ±8% within 30 days, and the colorimetric and fluorescence response performance remains stable. The dual-signal label has a shelf life of no less than 30 days under dry, light-protected conditions at 4℃.

Claims

1. A method for detecting meat freshness based on a dual-signal zwitterionic hydrogel colorimetric-fluorescent smart tag, characterized in that, include: The smart tag comprises zwitterionic polysulfonated betaine (SBMA) hydrogel, a fluorescent monomer 7-acryloyloxy-4-methylcoumarin (AOMC) covalently bonded to the gel network, and a colorimetric indicator alizarin physically embedded therein. The SBMA hydrogel was prepared by photoinitiated polymerization. AOMC was covalently fixed in the three-dimensional network of the hydrogel by copolymerization with SBMA monomer through its terminal double bonds, and alizarin was physically embedded in the gel network through hydrogen bonding. The smart tag exhibits a dual signal response when in contact with meat extract containing total volatile basic nitrogen (TVB-N): alizarin produces a colorimetric signal change from yellow to purple-red, and AOMC produces a fluorescence signal change that varies with pH. The detection method also includes using a smartphone to collect colorimetric and fluorescence images of the label, extracting RGB values ​​and fluorescence intensity values, and establishing a quantitative relationship between the dual signals and TVB-N content.

2. The method according to claim 1, characterized in that, The AOMC was synthesized by the following method: 7-hydroxy-4-methylcoumarin was prepared by esterification of 7-hydroxy-4-methylcoumarin with acryloyl chloride under alkaline conditions.

3. The method according to claim 1, characterized in that, The preparation method of the SBMA hydrogel includes the following steps: (1) Dissolve SBMA monomer and AOMC fluorescent monomer in a solution containing crosslinking agent N,N'-methylenebisacrylamide (MBAA) and photoinitiator Irgacure 2959; (2) Add alizarin stock solution, mix well and then pour into the mold; (3) After being irradiated with 365 nm ultraviolet light for 10-15 minutes, SBMA and AOMC are copolymerized and crosslinked to form a hydrogel; (4) Wash thoroughly with deionized water to remove unreacted substances and unencapsulated alizarin, and a dual-signal smart tag is obtained.

4. The method according to claim 3, characterized in that, The precursor solution contains 22-27% (w / v) SBMA by mass, 8-12 mg / mL AOMC, 0.5-1.5% MBAA by volume, 0.5-1.5% Irgacure 2959 by volume, and 0.2-0.8 mg / mL alizarin by volume.

5. The method according to claim 4, characterized in that, The precursor solution contained 25% (w / v) SBMA, 10 mg / mL AOMC, 1% MBAA, 1% Irgacure 2959, and 0.5 mg / mL alizarin.

6. The method according to claim 1, characterized in that, The detection method includes the following steps: (1) Cut the meat sample to be tested into small pieces and mix it with the extract. After standing or filtering, the meat juice extract is obtained. (2) Immerse the smart tag in the meat extract for 5-10 minutes until the colorimetric and fluorescence signals stabilize; (3) Take out the label and take visible light and fluorescence photos with a smartphone under ultraviolet light irradiation; (4) Use RGB analysis software to extract the RGB values ​​and fluorescence intensity values ​​of the label, and calculate the color difference ΔE and fluorescence intensity change ΔF; (5) Substitute ΔE and ΔF into the pre-established dual-signal-TVB-N standard curve to obtain the TVB-N content and freshness grade.

7. The method according to claim 1, characterized in that, The freshness grading standards are as follows: Fresh: TVB-N ≤ 15 mg / 100g; Less fresh: 15-25 mg / 100g; Spoiled: > 25 mg / 100g.

8. A dual-signal smart tag for implementing the method of any one of claims 1-7, characterized in that, The label comprises an SBMA zwitterionic hydrogel matrix, covalently bonded AOMC fluorescent monomers, and physically embedded alizarin colorimetric indicator; the label exhibits a dual response of colorimetric and fluorescence signals simultaneously within the pH range of 6.0-11.

0.

9. The dual-signal smart tag according to claim 8, characterized in that, The label has an equilibrium swelling rate of 450%±20%, an equilibrium water content of 82%±2%, and a shelf life of not less than 30 days under dry and light-protected conditions at 4℃.

10. The dual-signal smart tag according to claim 8 or 9, characterized in that, It is prepared by steps (1)-(4) as described in claim 3.

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