Preparation method of metal ion mediated copper nano aggregate and application of metal ion mediated copper nano aggregate to food monitoring and intelligent preservation

Abstract

The invention discloses a preparation method of a metal ion mediated copper nano aggregate and application of food monitoring and intelligent preservation. According to the method, amino acid or oligopeptide is used as a ligand, Cu < 2 + > is reduced and subjected to end sealing, essential oil is added, then metal ions (La < 3 + >, Ce < 3 + > and Al < 3 + >) are introduced into a mixed solution of the essential oil and CuNCs, the metal ions serve as bridges for connection between the CuNCs, the fluorescence intensity of the solution is remarkably enhanced, and rapid embedding of the essential oil is synchronously achieved. And dropwise adding the solution into a substrate with a fiber or porous structure to prepare the bifunctional sensor. The sensor triggers aggregate structure collapse by responding to total volatile basic nitrogen generated by food deterioration, synchronously realizes fluorescence signal conversion and essential oil controlled release, and prolongs the shelf life of food. The system integrates the functions of fluorescence monitoring and intelligent fresh-keeping, overcomes the technical bottlenecks of fresh-keeping lag, excessive corrosion prevention and the like, has the advantages of greenness, low cost and high efficiency, and has a wide market application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of non-destructive testing of food and the field of intelligent preservation of food, and in particular to a method for preparing copper nano-aggregates mediated by metal ions and applications of food monitoring and intelligent preservation. BACKGROUND

[0002] Aquatic products are rich in high-quality protein, containing 9 essential amino acids required by the human body; trace elements (vitamin D, iodine, selenium) and omega-3 unsaturated fatty acids, which can reduce cardiovascular disease. In addition, aquatic products are favored by consumers because of their delicate meat texture, short muscle fibers, and rich in glutamate, inosinic acid and other umami substances. Meat such as beef and pork is an important source of protein intake for people, which can achieve muscle growth and tissue repair in the human body. Meat is rich in B vitamins, which is beneficial to maintaining the health of the nervous system. However, aquatic products, meat and other foods are prone to spoilage during production, transportation, processing, storage and sales due to endogenous enzyme system autolysis, microbial contamination, and fat oxidation. Consumers who mistakenly eat spoiled food can cause adverse physiological reactions such as headache, low blood pressure, arrhythmia, and digestive disorders, and even respiratory, circulatory, and nervous system symptoms, which can be life-threatening. In addition, low-level amines produced during food spoilage can combine with nitrite to form carcinogenic substances (such as nitrosamines and nitrosamides), increasing the risk of carcinogenesis. Therefore, rapid monitoring of food freshness is an important link to protect the health of consumers.

[0003] Traditional methods for detecting food freshness include microbiological methods, physicochemical detection methods, and sensory evaluation methods. However, the above conventional methods have the disadvantages of complicated operation, long detection period, and high cost. Therefore, it is necessary to develop a rapid, non-destructive, and convenient detection technology for food freshness to achieve real-time monitoring of food freshness. Gas sensing tag technology can quickly sense changes in physicochemical indicators inside the package, has high sensitivity, fast response speed, low cost, and does not damage the sample, and has a broad application prospect. During food spoilage, proteins are gradually broken down into peptides and amino acids under the action of microorganisms and enzymes, and further degraded into methyl indole, ammonia, amine, and other total volatile basic nitrogen (TVB-N). Therefore, the development of a gas sensor for rapid detection of TVB-N can achieve visual monitoring of food freshness.

[0004] Compared with colorimetric sensors, fluorescent sensors have the advantages of high sensitivity, fast response speed, good selectivity and strong anti-interference ability. Traditional fluorescent dye molecules (rhodamine, fluorescein, etc.) generally have rigid skeletons. Due to the intermolecular Π-Π stacking effect, under the condition of aggregation or high concentration, the aggregation-induced fluorescence quenching (ACQ) phenomenon is easy to occur, which limits its application in the construction of solid-state fluorescent tags. In addition, traditional fluorescent dyes rely on organic solvents for dispersion, which has a great safety hazard. A class of organic molecules with twisted structure (tetraphenylethylene, hexaphenylthiazole, etc.) in the aggregated state, due to the restriction of intramolecular motion (RIM), the energy dissipation channel of non-radiative relaxation is inhibited, showing an aggregation-induced emission (AIE) phenomenon, which can be used for the construction of solid-state sensors. However, AIE organic small molecules face the challenges of difficult synthesis, short luminescence lifetime, and the need for short-wavelength excitation. In recent years, metal nanoclusters (MNCs) with AIE properties have attracted widespread attention due to their excellent optical properties, such as large stoke shift, good photo-stability, strong anti-photobleaching, good biocompatibility and low toxicity. Among them, copper nanoclusters (CuNCs) have the advantages of abundant raw material reserves, low cost and low toxicity, and are widely used in detection and sensing fields. For example, patent CN202210797399.5 uses glutathione (GSH) as a ligand to prepare GSH-CuNCs with AIE properties. The GSH-CuNCs solution is added to the paper label to prepare a fluorescent tag with intelligent response to TVB-N, realizing the visualization of food freshness. However, the sensing tag in the above patent application is only limited to the single function of freshness monitoring, and no preservation measures are taken according to the monitored quality information, which cannot effectively reduce the economic loss caused by food spoilage.

[0005] Plant essential oils contain active ingredients such as phenols and terpenes, and have antioxidant and broad-spectrum antibacterial properties, which are low-cost and safe natural biological preservatives. However, its application in the field of long-acting food preservation is limited due to its limitations such as light and heat sensitivity, easy volatility and poor water solubility. The slow-release technology reduces the release rate of essential oils through carriers and prolongs their shelf life to achieve the long-acting preservation effect of essential oils. Common carriers include microcapsules, nano-emulsion, film, hydrogel, etc. For example, patent application 202510222283.2 introduces a porous gel substrate to embed plant essential oils in the inner layer of the gel network and attach copper nano-aggregates on the surface of the gel. Based on the responsiveness of copper nano-aggregates to TVB-N, the gel sensor realizes the rapid monitoring and intelligent preservation of the freshness of black fish. However, the synthesis cycle of the gel substrate of the sensor is long, and the loading process of the preservative and indicator is complicated, making it difficult to standardize and mass-produce, limiting its market application. Therefore, it is urgent to develop an easy-to-prepare, low-cost and efficient freshness monitoring and preservation integrated sensor. SUMMARY

[0006] To solve the above problems, the primary object of the present application is to induce CuNCs aggregation mechanism based on metal ions (La 3+ , Ce 3+ , Al 3+ ) to enhance the fluorescence intensity of copper nanoclusters and achieve rapid encapsulation of plant essential oils. Secondly, a sensor based on copper nano-aggregates is constructed, which is convenient and efficient in preparation. In addition, by triggering the collapse of the copper nano-aggregate structure through TVB-N, the fluorescence signal of the sensor is changed, realizing the visual output of the freshness of food, and accelerating the release of plant essential oils embedded in the aggregate, realizing the intelligent linkage of food freshness monitoring and preservation.

[0007] The object of the present application is achieved by the following technical solutions:

[0008] A preparation method of metal ion-mediated copper nano-aggregates, comprising the following steps:

[0009] (1) The preparation steps of copper nanoclusters (CuNCs) solution are as follows:

[0010] Under room temperature conditions, disperse the amino acid or oligopeptide ligand in the NaOH solution, add the Cu 2+ solution, shake and mix to prepare the CuNCs solution.

[0011] (2) The preparation steps of copper nano-aggregate solution based on metal ion-induced aggregation are as follows:

[0012] First, a plant essential oil solution of a certain concentration is prepared, a certain amount of the essential oil solution is taken to the CuNCs solution prepared in step (1), ultrasonic dispersion is performed, a metal ion solution of a certain concentration is added to the above mixed solution, and oscillation is performed to uniformly mix the solution. The carboxyl groups on the surface ligands of the clusters are coordinated and combined with the metal ions, and the CuNCs are induced to aggregate to form copper nano-aggregates embedding the plant essential oil.

[0013] The metal ion is selected from one or more of lanthanum ion (La 3+ ), cerium ion (Ce 3+ ), and aluminum ion (Al 3+ ).

[0014] Preferably, the amino acid or oligopeptide ligand in step (1) is selected from N-acetyl-L-cysteine, L-cysteine, D-penicillamine, and glutathione, the concentration of the ligand in the NaOH solution is 0.2-1.0 mol / L; the concentration of the Cu 2+ solution is 0.02-0.1 mol / L, the volume of the NaOH solution is 5-10 mL, the concentration of the NaOH solution in step (1) is 0.1-1.0 mol / L, the oscillation time is 2-10 min, and the molar ratio of the ligand to Cu 2+ is 3-20:1.

[0015] Preferably, the plant essential oil in step (2) is selected from phenolic compounds, terpene compounds, or aldehyde compounds, including but not limited to eugenol, carvacrol, thymol, cinnamaldehyde, and citral, the essential oil dispersant is 10%-95% (w / w) ethanol, the concentration of the plant essential oil is 0.125-5 μg / mL, the concentration of the metal ion is 0.01-0.2 mol / L, the ultrasonic treatment time is 5-10 min, the ultrasonic power is 50-150 W, the ultrasonic water bath temperature is 25-37℃, the volume ratio of the CuNCs solution, the plant essential oil solution, and the metal ion solution is 2.0-8.0:0.5-1.0:0.5-1.0, and the oscillation time is 2-5 min.

[0016] A copper nano-aggregate prepared by any one of the preparation methods described above.

[0017] A sensor with freshness monitoring and preservation dual functions, comprising the following steps:

[0018] A copper nano-aggregate solution described above is added to a fiber / porous substrate, and a sensor with freshness monitoring and preservation dual functions is prepared by vacuum freeze drying.

[0019] Preferably, the fiber / porous substrate is one or more of filter paper, tissue paper, non-woven fabric, and porous gel, and preferably is tissue paper; and the loading amount of the copper nano-aggregate solution on the fiber / porous substrate is 15-100 μL / cm2 .

[0020] Preferably, the duration of the vacuum freeze-drying is 24-48h.

[0021] The sensor described above is applied in the freshness monitoring and preservation of food.

[0022] Preferably, the application method comprises the following steps: fixing the sensor in the food packaging, the total volatile basic nitrogen (TVB-N) generated by the spoilage of the food responds to the copper nano-aggregates in the sensor, destroys the nano-aggregate structure, causes the fluorescence intensity to decrease to realize the freshness visual monitoring, and triggers the controlled release of the plant essential oil in the sensor to prolong the shelf life of the food.

[0023] Preferably, the monitoring method of the fluorescence intensity is: constructing a freshness grading color card based on the fluorescence picture of the sensor to realize the rapid evaluation of the freshness; establishing a linear regression model of the color channel numerical value of the fluorescence picture and the TVB-N value of the food to more accurately determine the freshness of the food.

[0024] A freshness monitoring method based on a copper nano-aggregate sensor is constructed, comprising the following steps:

[0025] The sensor described above is fixed on the inner side of the top of a culture dish, a certain mass of commercially available South American white prawns is weighed and placed on the bottom of the culture dish, the culture dish is sealed with a sealing film, and then it is placed in a constant temperature and humidity incubator. Every other time, the culture dish is placed in a dark box, and under the irradiation of a 365nm ultraviolet lamp, the fluorescence picture of the sensor is taken by a smart phone. In addition, every other time, about 50g of prawn samples are taken out from the culture dish, and the total volatile basic nitrogen (TVB-N) value of the prawns at different storage times is determined according to GB 5009.228-2016. A freshness grading color card based on the fluorescence picture of the sensor is constructed to rapidly evaluate the freshness grade of the prawns. In addition, the average value of the color channel intensity of the region of interest (ROI) of the fluorescence picture of the sensor in the RGB space is extracted, the average intensity ratio of the red (R) and blue (B) in the ROI region is calculated respectively (R / B), which is used as the independent variable, and the TVB-N value is used as the dependent variable, and a linear regression model of the two is established.

[0026] Preferably, the mass of the shrimp in the culture dish is 150-250 g, and the diameter of the culture dish bottom is 150 mm; the mass of the shrimp in the sealed box is 200-500 g, the volume of the sealed box is 500-750 mL, the temperature of the constant-temperature and constant-humidity incubator is 4℃ and 25℃, and the interval time is 1-2 days (4℃) and 2-3 h (25℃); the mobile phone shooting parameter settings are: WB is 6400K, ISO is 3200, and 1 / S is 1 / 50.

[0027] Verification of TVB-N triggering the release effect of plant essential oil in copper nano-aggregates

[0028] (1) A certain amount of copper nano-aggregate solution was taken in a centrifuge tube, vacuum freeze-dried to prepare multiple copper nano-aggregate solid samples containing eugenol.

[0029] (2) Ammonia was gradiently diluted with distilled water to prepare ammonia solutions with different concentrations, a certain volume of ammonia solution was taken in a culture dish, and the culture dish was then placed at the bottom of a sealed barrel. Multiple centrifuge tubes containing copper nano-aggregates were fixed in each barrel, and the centrifuge tubes were placed open. The sealed barrel was placed in a constant-temperature and constant-humidity incubator, and after a certain interval, a sample was taken out, dissolved in a large volume of ethanol-water solution, and treated with ultrasonic treatment and water bath oscillation for a certain time to destroy the spherical structure of the copper nano-aggregates, extract the residual plant essential oil from the sample, and then a certain volume of solution was taken into a cuvette, and the optical density (OD) value of the solution at the characteristic peak of the plant essential oil was measured by ultraviolet spectrophotometry. Referring to the standard curve of plant essential oil concentration and OD value at the characteristic peak prepared in advance, the concentration of plant essential oil in the solution was calculated. The residual rate (R%) of the plant essential oil was calculated according to formula (1), and the plant essential oil residual rate-time curve of the copper nano-aggregates exposed to different ammonia concentrations was drawn.

[0030] Preferably, the volume of the copper nano-aggregate solution in step (1) is 0.5-2 mL, and the volume of the centrifuge tube is 0.8-2.5 mL.

[0031] Preferably, in step (2), the diameter of the culture dish is 30-90 mm, the concentration of ammonia is 5-5000 ppm, the volume of ammonia is 0.5-2 mL, the volume of the sealed barrel is 1-3 L, the temperature of the constant-temperature and constant-humidity incubator is 25-37℃, the interval time is 2-8 h, the mass concentration of the ethanol solution is 20%-50%, the volume of the ethanol solution is 10-30 mL, the ultrasonic treatment time is 5-10 min, the ultrasonic power is 100-300 W, the water bath temperature during ultrasonic treatment is 25-30℃, the water bath oscillation time is 1-3 h, and the temperature is 25-30℃.

[0032] Preferably, in step (2), formula (1) is as follows:

[0033] The calculation formula of the residual rate (R%) of the plant essential oil is:

[0034]

[0035] Wherein, R%: the residual rate of the plant essential oil; C t : the concentration of the plant essential oil in the sample at time t; C0: the concentration of the plant essential oil in the sample at the initial time.

[0036] Step four: preservation application test based on the copper nanocluster sensor.

[0037] The copper nanocluster sensor containing the plant essential oil is prepared according to step (3), serving as an experimental group A; and the sensor without the plant essential oil is prepared without adding the plant essential oil solution during the preparation, serving as a control group B. Each group contains multiple sealed boxes, and the same mass of the Penaeus vannamei samples are placed at the bottom of the boxes. In addition, the sensor containing the plant essential oil is pasted on the top of each sealed box in the group A, and the sensor without the plant essential oil is pasted on the top of each sealed box in the group B. The two groups of sealed boxes are placed in a constant temperature and humidity box, and at intervals, a certain mass of the shrimp is taken out from the sealed boxes, and the TVB-N values of the shrimp at different storage times are determined according to GB 5009.228-2016. The difference between the TVB-N values of the samples in the groups A and B is compared, so as to evaluate the actual preservation effect of the sensor loaded with the plant essential oil.

[0038] Preferably, the mass of the Penaeus vannamei is 50-200 g, the temperature of the constant temperature and humidity box is 4℃ and 25℃, and the interval length is 1-2 days (4℃) and 1-3 hours (25℃).

[0039] The present application synthesizes the copper nanocluster (CuNCs) solution responsive to the spoilage gas by using the amino acid or oligopeptide as the ligand through the "one-pot method", reducing and end-capping. 2+ The plant essential oil is dispersed into the CuNCs solution, which is combined with the CuNCs through the hydrophobic interaction. Then, the metal ions (La 3+ , Ce 3+ , Al 3+)The mixed solution of plant essential oil and CuNCs is introduced, metal ions act as a bridge connecting CuNCs, and are coordinated with the carboxyl group of the surface ligand of CuNCs, so as to induce the rapid aggregation and rearrangement of CuNCs, and the copper nano aggregate solution is prepared, the fluorescence intensity of the solution is significantly enhanced, and the rapid embedding of the plant essential oil is realized at the same time. The solution is added dropwise into a fiber or a porous structure substrate, and vacuum freeze drying is carried out, so that the dual-functional sensor is prepared. The sensor triggers the collapse of the aggregate structure by responding to the total volatile nitrogen generated by food spoilage, and realizes the fluorescence signal conversion and essential oil controlled release at the same time, thereby prolonging the shelf life of food.

[0040] Compared with the prior art, the present application has the advantages that:

[0041] (1) The copper nano aggregate embedded with plant essential oil prepared by the present application fully excavates the metal ion induced CuNCs aggregation mechanism, and the fluorescence signal and stability of CuNCs are significantly improved due to the improvement of the aggregation degree; the rapid embedding of plant essential oil is realized synchronously in the aggregation process, and no chemical reagents such as surfactants and emulsifiers need to be introduced, so that the preparation process is green and energy-saving.

[0042] (2) The indicator is added dropwise on the fiber / porous substrate, such substrate is low in cost and easy to obtain, has excellent three-dimensional space structure, and the preparation process of the sensor is simple and short in cycle, so that efficient loading of the copper nano aggregate is realized.

[0043] (3) The sensor based on the copper nano aggregate prepared by the present application can realize fluorescence monitoring of freshness, and realizes intelligent controlled release of plant essential oil according to the monitored food freshness quality information, so that intelligent linkage of freshness monitoring and preservation of food is realized. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The fluorescence spectrum (A) and the columnar graph (B) of the CuNCs solution prepared in Example 2 after the introduction of metal ions;

[0045] Figure 2 The UV-vis absorption spectrum of CuNCs and CuNCs-La prepared in Examples 1 and 3; 3+ In the fluorescence picture and the fluorescence spectrum;

[0046] Figure 3 The UV-vis absorption spectrum of CuNCs-La prepared in Example 3; 3+ In the fluorescence picture and the fluorescence spectrum;

[0047] Figure 4 The XPS analysis graph of CuNCs and CuNCs-La prepared in Examples 1 and 3; 3+ In the fluorescence picture and the fluorescence spectrum;

[0048] Figure 5Eug@CuNCs-La prepared in Example 4 3+ FT-IR plot;

[0049] Figure 6 In Example 5, CuNCs and CuNCs-La 3+ Fluorescence spectra of the solution before and after the introduction of eugenol;

[0050] Figure 7 In Example 6, CuNCs and La were introduced into the eugenol solution. 3+ UV-vis absorption spectra before and after;

[0051] Figure 8 For CuNCs and Eug@CuNCs-La in Example 7 3+ Fluorescence images of the solution used in stability testing;

[0052] Figure 9 Eug@CuNCs-La in Example 8 3+ TEM images of the solution before (A) and after (B) the reaction with ammonia;

[0053] Figure 10 Eug@CuNCs-La in Example 9 3+ SEM images of the sensor before (A) and after (B) the response to ammonia gas;

[0054] Figure 11 Eug@CuNCs-La in Example 10 3+ Graph showing the test results of the sensor's sensitivity and selectivity to ammonia;

[0055] Figure 12 The graphs show the eugenol residue rate-time curves of the samples in Example 11, Comparative Example 2 and Comparative Example 3 under different concentrations of ammonia.

[0056] Figure 13 The images shown are: fluorescence photographs of the samples in Example 12 (A), line graphs showing the changes in TVB-N content in each group of shrimp (B), color charts showing the freshness grading of the sensor (C), and linear curves showing the TVB-N value of shrimp in Example 13 and the R / B value in the sensor images (D).

[0057] Figure 14 This is a schematic diagram illustrating a method for preparing metal ion-mediated copper nanoaggregates according to the present invention and their application in food monitoring and intelligent preservation. Detailed Implementation

[0058] The present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0059] Example 1: Preparation of copper nanoclusters (CuNCs) solution

[0060] 0.2613 g of N-acetyl-L-cysteine (NAC) was added to a 10 mL centrifuge tube, and a NaOH solution (0.25 mol / L, 4 mL) and a copper nitrate solution (0.1 mol / L, 1 mL) were added, and the CuNCs solution was synthesized by oscillation at room temperature for 30 s.

[0061] Example 2: Test of the effect of different metal ions on the fluorescence of CuNCs

[0062] Different kinds of metal ion solutions (La 3+ , Al 3+ and Ce 3+ ) were prepared, and the CuNCs solution prepared in Example 1 (900 μL) and the above metal ion solution (100 μL, 0.1 mol / L) were mixed, and the mixture was oscillated at room temperature for 60 s and incubated for 10 min. Distilled water (100 μL) was used instead of the metal ion solution as a blank control group. The fluorescence emission spectrum of each group of solutions was determined, and the fluorescence intensity change ratio (F / F0) of the solution was calculated, where F is the fluorescence intensity of the solution after adding the metal ion or distilled water, and F0 is the initial fluorescence intensity of the solution. As shown in (A) of Figure 1 , La 3+ , Ce 3+ and Al 3+ can significantly enhance the fluorescence intensity of the CuNCs solution. As shown in (B) of Figure 1 , the F / F0 of the CuNCs-La 3+ and CuNCs-Ce 3+ solutions were 7.47 and 6.73, respectively, and the fluorescence enhancement effect was significant. Among them, the CuNCs-Al 3+ solution had the largest fluorescence intensity increase, with a F / F0 of 15.17.

[0063] Example 3: Preparation of copper nanocluster aggregates (CuNCs-La 3+ ) solution based on La 3+ induced aggregation

[0064] The CuNCs solution (900 μL) prepared in Example 1 and a lanthanum nitrate solution (100 μL, 0.1 mol / L) were added to a centrifuge tube, and the CuNCs-La 3+ solution was synthesized by oscillation at room temperature for 2 min, and the CuNCs-La 3+Characterization was performed using fluorescence spectroscopy, ultraviolet-visible (UV-vis) absorption spectroscopy, and X-ray photoelectron spectroscopy (XPS).

[0065] CuNCs and CuNCs-La 3+ Fluorescence spectrum ( Figure 2 This indicates that the optimal excitation wavelength for CuNCs is 380 nm, and the optimal emission wavelength is 645 nm; CuNCs-La 3+ The optimal excitation wavelength is 390 nm, and the optimal emission wavelength is 620 nm. Among them, CuNCs-La... 3+ The fluorescence intensity was 7.3 times that of CuNCs, indicating a significant increase in fluorescence intensity after CuNCs aggregation. Figure 3 As shown, the UV-vis absorption spectrum of CuNCs does not show Cu. 2+ The characteristic peak at 301 nm indicates that CuNCs were successfully prepared. CuNCs-La 3+ The UV-vis absorption spectrum did not show La 3+ The characteristic peak at 297 nm indicates that La 3+ It was successfully combined with CuNCs.

[0066] XPS full spectrum of CuNCs ( Figure 4 -A) contains S (2p), C (1s), N (1s), O (1s), Cu (2p), and Na (1s) regions. The Cu 2p fine spectrum of CuNCs ( Figure 4 -B) There are two peaks, which are attributed to Cu 2p. 3 / 2 (932.0 eV) and Cu 2p 1 / 2 The absence of a satellite peak at 942.0 eV in the (951.8 eV) orbital indicates a lack of Cu(II) in CuNCs, which are primarily composed of Cu(0) and Cu(I). CuNCs-La 3+ XPS full spectrum ( Figure 4 -C) In addition to the regions mentioned above, there is also a La (3d) region. CuNCs-La 3+ Cu 2p fine spectrum ( Figure 4 The positions of the two characteristic peaks in -D) are not significantly different from the Cu 2p fine spectrum of CuNCs, and can be attributed to Cu 2p. 3 / 2 (932.04 eV) and Cu 2p 1 / 2 (951.83 eV), confirming La 3+ It did not undergo redox reactions with the CuNCs copper core. Furthermore, the La (3d) fine spectrum ( Figure 4 -F) There are two pairs of spin-orbit splitting peaks, which are assigned to La 3d.2 / 3 Orbits (854.47 and 851.24 eV) and La 3d 5 / 3 The orbits (837.81 and 834.60 eV) confirm La 3+ The valence state remained unchanged before and after combining with CuNCs.

[0067] Example 4: Eugenol (Eug)-loaded copper nanoaggregates (Eug@CuNCs-La) 3+ Solution preparation

[0068] Eugenol solution (1 μg / mL) was prepared using 20% ​​(w / w) ethanol, and lanthanum nitrate solution (0.1 mol / L) was prepared using distilled water. 800 μL of CuNCs solution and 100 μL (1 μg / mL) of eugenol solution prepared in Example 1 were added to a 2 mL centrifuge tube and ultrasonically dispersed at room temperature for 10 min. Then, 100 μL (0.1 mol / L) of lanthanum nitrate solution was added, and the mixture was shaken at room temperature for 2 min to synthesize Eug@CuNCs-La 3+ Solution. For Eug@CuNCs-La 3+ Fourier transform-infrared spectroscopy (FT-IR) was performed for characterization. Based on the FT-IR spectral results ( Figure 5 NAC is 2546.93cm -1 The stretching vibration peak attributed to the thiol group disappeared after the formation of CuNCs, confirming that NAC is bound to the metal core through a Cu-S bond. Adding eugenol to the CuNCs solution did not significantly change the FT-IR spectrum of the CuNCs, indicating that the addition of eugenol does not impair the properties of CuNCs. 3+ After introducing a mixed solution of CuNCs and eugenol, compared to the FT-IR spectrum of CuNCs-Eug, the Eug@CuNCs-La... 3+ At 1529.69cm -1 and 1593.27cm -1 At this point, the vibrational peaks attributed to the carboxyl group are significantly weakened, indicating that La 3+ Coordination occurred between Eug@CuNCs and carboxyl groups on the CuNCs surface, confirming that Eug@CuNCs-La 3+ Successfully prepared.

[0069] Example 5: Eugenol on CuNCs and CuNCs-La 3+ Investigating the influence of fluorescence properties

[0070] Take 900 μL of the CuNCs solution prepared in Example 1 and the CuNCs-La solution prepared in Example 3. 3+The solution (900 μL) was divided into three replicates in different centrifuge tubes. Eugenol solution (1 μg / mL) was prepared using 20% ​​(w / w) ethanol. Control group: in CuNCs and CuNCs-La 3+ 100 μL of 20% (w / w) ethanol solution was added to each; Experimental group: CuNCs and CuNCs-La 3+ Eugenol solution (100 μL, 1 μg / mL) was added to each solution. 500 μL of each solution was transferred to a microcuvette (700 μL) for fluorescence spectroscopy characterization. The test conditions were: slit width 3 nm, excitation wavelength of CuNCs series solutions 380 nm, and CuNCs-La 3+ The excitation wavelength for the series of solutions is 390 nm. For example... Figure 6 As shown, the introduction of eugenol did not significantly affect CuNCs and CuNCs-La 3+ The fluorescence intensity of the Eug@CuNCs-La preliminarily confirmed the construction of the Eug@CuNCs-La 3+ The feasibility of using the system as a fluorescent indicator.

[0071] Example 6: CuNCs and La 3+ Investigating the effect of eugenol on its properties

[0072] First, take 900 μL of the CuNCs solution prepared in Example 1 into a centrifuge tube, and then add 100 μL of eugenol solution (1 μg / mL) and La... 3+ A solution (100 μL, 0.1 mol / L) was prepared, and distilled water (100 μL) was added to the centrifuge tubes used in the control group. The OD values ​​of the three solutions were measured using a UV-Vis spectrophotometer. 281 nm Values ​​to explore CuNCs and La 3+ Effects on the properties of eugenol. Based on UV-vis absorption spectra ( Figure 7 CuNCs and La 3+ The addition of [unclear] did not change the peak position and intensity of eugenol in the UV-vis absorption spectrum, indicating that CuNCs and La [unclear] 3+ It does not damage the chemical properties of eugenol, further confirming that CuNCs, eugenol, and La... 3+ The three components construct Eug@CuNCs-La 3+ The system demonstrates the feasibility of encapsulating eugenol.

[0073] Example 7: Eug@CuNCs-La 3+ Stability test of solution

[0074] Take the CuNCs solution prepared in Example 1 and the Eug@CuNCs-La prepared in Example 4. 3+2 mL of each solution was placed in centrifuge tubes and stored at 4°C for 30 days. Fluorescence images of both solutions were then captured under a 365 nm UV lamp. Figure 8 As shown, after storage at 4℃ for 30 days, the fluorescence of CuNCs was essentially quenched. (Eug@CuNCs-La) 3+ The fluorescence intensity did not change significantly, indicating that CuNCs aggregated into Eug@CuNCs-La 3+ Afterward, stability significantly increases, making it suitable for freshness indication over longer periods.

[0075] Example 8: Eug@CuNCs-La 3+ Response to ammonia in solution system

[0076] Take the Eug@CuNCs-La prepared in Example 4 3+ The solution (900 μL) was added to a centrifuge tube, and ammonia solution (100 μL, 200 ppm) was added. The mixture was incubated at room temperature for 30 min. Eug@CuNCs-La solutions were then added to the carbon membrane before and after the ammonia response. 3+ The solution (20 μL) was then dried (50 °C, 20 min), and the morphological characteristics of the material were observed under a high-resolution transmission electron microscopy (TEM). Figure 9 As shown in (A), Eug@CuNCs-La 3+ The particle size is 142±15nm, exhibiting a spherical morphology. For example... Figure 9 As shown in (B) of the image, Eug@CuNCs-La 3+ Upon reaction with ammonia, the spherical nanoparticles disappeared, and only irregular, sheet-like organic matter could be observed.

[0077] Example 9: Eug@CuNCs-La 3+ Sensor fabrication

[0078] A 1.0 cm diameter hole was fabricated on a polytetrafluoroethylene (PTFE) film, and a 1.2 cm diameter cotton pad was adhered between two films to prepare a blank sensor. The Eug@CuNCs-La sensor prepared in Example 4 was then used. 3+ Solution (100 μL / cm) 2 The sample was dropped onto cotton paper and then freeze-dried under vacuum (-60°C, 48h). Based on Eug@CuNCs-La 3+ The sensor is stored at 4°C for later use.

[0079] Example 10: Eug@CuNCs-La 3+ Sensor responsiveness to ammonia and morphological characterization.

[0080] Ammonia solution with different concentrations (200, 400, 600, 800 and 1000 ppm) were prepared, and 1 mL of the above ammonia solution was added into a petri dish with a diameter of 90 mm, and the sensor prepared in Example 9 was pasted inside the top of the petri dish, and the petri dish was sealed. Every 10 min, the fluorescence picture of the sensor was taken under the ultraviolet lamp at 365 nm to explore the response sensitivity of Eug@CuNCs-La 3+ to ammonia. In addition, the morphology of Eug@CuNCs-La 3+ before and after the response to ammonia (1000 ppm, 30 min) was observed by scanning electron microscopy (SEM). The SEM image (A) before the response to ammonia in Figure 10 , a large number of spherical nanoparticles distributed on the fibers can be observed. The SEM image (B) after the response in Figure 10 , the spherical structure of Eug@CuNCs-La 3+ collapsed, which confirmed the feasibility of realizing the controlled release of eugenol by destroying the aggregate structure of Eug@CuNCs-La 3+ based on ammonia. The sensitivity results of the response to ammonia are shown in Figure 11 , the fluorescence intensity of Eug@CuNCs-La 3+ sensor decreased significantly after being exposed to 200 ppm ammonia for 10 min; and after being exposed to 1000 ppm ammonia for 30 min, the fluorescence of the sensor was almost quenched, indicating that Eug@CuNCs-La 3+ was sensitive to ammonia.

[0081] Example 11: Ammonia gas response to the slow-release mechanism and effect verification of eugenol embedded in Eug@CuNCs-La 3+

[0082] The Eug@CuNCs-La 3+ solution (1.5 mL) prepared in Example 4 was added into a 2 mL centrifuge tube, and a solid sample of Eug@CuNCs-La 3+ was prepared by vacuum freeze-drying treatment. Ammonia solutions with different concentrations (50, 100, 200, 400 and 800 ppm) were prepared, and 1 mL of the above ammonia solution was taken into a petri dish, and then the petri dish containing the ammonia water and the centrifuge tube containing Eug@CuNCs-La 3+ were fixed in a 0.5 L sealed barrel. They were placed in a constant temperature and humidity incubator at 25°C, and every 30 min, a sample was taken out, and the sample was dissolved with 10 mL of ethanol solution 20% (w / w). Ultrasonic treatment was performed for 10 min, and water bath shaking (30°C, 1 h) was performed to extract Eug@CuNCs-La 3+ ​eugenol. 500 μL of the above solution was taken into a 700 μL cuvette, and the optical density value (OD) of the solution at 281 nm was measured by a UV-vis spectrophotometer. 281 nm The concentration of eugenol in the solution was calculated by referring to the standard curve of eugenol concentration versus OD 281 nm at 281 nm. The residual rate (R%) of eugenol was calculated according to Formula (1), and the eugenol residual rate-time curve of Eug@CuNCs-La 3+ exposed to different ammonia concentrations was plotted. 3+

[0083] As shown in FIG. 13-A, with the increase of ammonia concentration in the exposure environment, the residual rate of eugenol in Eug@CuNCs-La 3+ gradually decreased. The residual rate of eugenol was 41.3% when exposed to 50 ppm ammonia for 300 min, and was 19.7% when exposed to 800 ppm ammonia for 300 min. With the increase of ammonia concentration in the environment, the time at which the residual rate of eugenol in Eug@CuNCs-La 3+ began to rapidly decrease gradually moved forward: the time at which the residual rate of eugenol began to rapidly decrease was 150 min when the ammonia concentration was 50 ppm, and the residual rate of eugenol had already shown a rapid decrease trend in the period of 0-60 min when the ammonia concentration was 800 ppm.

[0084] Example 12: Freshness monitoring and preservation application test of the sensor based on Eug@CuNCs-La 3+

[0085] The Eug@CuNCs-La 3+ sensor prepared in Example 9 was pasted on the inner side of the top of a petri dish, and about 50 g of South American white prawns were placed at the bottom of the petri dish. The petri dish was placed in a 4°C refrigerator for storage. Every other day, about 50 g of prawn samples were taken out from the petri dish, and the total volatile basic nitrogen (TVB-N) values of the prawns at different storage times were determined according to GB 5009.228-2016. Every other day, the fluorescence pictures of the sensor under the irradiation of a 365 nm ultraviolet lamp (13-A) of one of the samples were taken by a mobile phone, and a freshness grading color card based on the pictures of the Eug@CuNCs-La 3+ sensor was constructed. According to the TVB-N change broken line graph (FIG. 14), the freshness grading color card based on the Eug@CuNCs-La 3+ sensor was constructed, and the freshness grading color card was shown in FIG. 15. Figure 13 ​​TVB-N content of the Penaeus vannamei in Example 12 increased from 6.28 mg / 100 g at the beginning to 15.79 and 18.15 mg / 100 g at the 2nd and 3rd day of storage, and the label changed from orange yellow to orange red. At the 7th day of storage, the TVB-N content exceeded 30 mg / 100 g, indicating that the shrimp had been severely spoiled, the fluorescence of the sensor was almost quenched, and the label changed to brown black. Compared with the sensor without eugenol in Comparative Example 4, the Eug@CuNCs-La 3+ The sensor extended the shelf life of the shrimp by 2 days (4°C).

[0086] In addition, based on the relationship between the freshness of the shrimp and the fluorescence color and TVB-N of the sensor, a freshness grading color card was constructed to realize rapid identification of freshness. As shown in Figure 13 -C, the freshness: TVB-N < 10 mg / 100 g, the color of the sensor was orange yellow; the sub-freshness: TVB-N was 10-20 mg / 100 g, the color was orange red; the beginning of spoilage: TVB-N was 20-25 mg / 100 g, the color was pink; the slight spoilage: TVB-N was 25-30 mg / 100 g, the color was pink brown; the severe spoilage: TVB-N > 30 mg / 100 g, the color was brown black.

[0087] Example 13: Eug@CuNCs-La 3+ The relationship between the color channel value of the sensor and the freshness of the Penaeus vannamei was established.

[0088] Sample preparation and photograph shooting were carried out as in Example 12, and the pictures were named as the TVB-N value when the shrimp was shot. The OpenCV library was called to standardize the pretreatment of the collected sensor images, including grayscale, Gaussian filter denoising and histogram equalization to enhance the contrast. Based on the Hough transform algorithm, the edge features of the label were detected, and the regular circular region was cut out as the region of interest (ROI), and the average values of the pixel points in the red (Red, R) and blue (Blue, B) channels in the ROI region were calculated. The value of the red-green channel average intensity (R / B) was taken as the independent variable, and the TVB-N was taken as the dependent variable, and a linear regression model was established. As shown in Figure 13 -D, in the interval of 8.02-32.10 mg / 100 g of TVB-N value, the linear regression equation Y = -0.0727X + 3.3786 can be constructed with TVB-N value as X and R / B value as Y, and the R 2 was 0.993, indicating that the two had a good linear relationship, and the equation could accurately evaluate the freshness of the Penaeus vannamei.

[0089] Comparative Example 1: Test Fe 3+ Effect of metal ions on the fluorescence performance of CuNCs

[0090] Using Fe 2+ Solution substitution of La 3+ Ce 3+ And Al 3+ The solution was prepared using the same steps as in Example 2 to prepare CuNCs-Fe. 2+ Solution, test with added Fe 2+ The ratio of the change in fluorescence intensity of the CuNCs solution after the change (F / F0). Figure 1 As shown in (A), Comparative Example 1 introduces Fe into CuNCs solution. 2+ Afterwards, the change in solution fluorescence intensity was relatively small, with an F / F0 value of 1.52. Figure 1 -B). The above results indicate that the present invention has a certain selectivity for metal ions, and the metal ions (La) in Example 2 3+ Ce 3+ Al 3+ CuNCs exhibit aggregation-induced fluorescence enhancement, while Fe... 2+ It does not have a similar effect on CuNCs solutions.

[0091] Comparative Example 2: Same as Example 11, using an ammonia-free distilled aqueous solution as a control group, the eugenol residue rate-time curve in the control group was tested. Figure 12 As shown, at 300 min, in Comparative Example 2, Eug@CuNCs-La 3+ The eugenol residue rate was 98.8%, significantly higher than that of the experimental groups containing different concentrations of ammonia in Example 11, indicating that ammonia is the trigger for Eug@CuNCs-La 3+ Key factors in the release of eugenol.

[0092] Comparative Example 3: An eugenol solution (1 μg / mL) was prepared using 20% ​​(w / w) ethanol. 2 mL of the eugenol solution was added to a 16 mL solution of 0.25 mol / L 2-methylimidazole, and the mixture was dispersed thoroughly. Zinc nitrate solution (1.95 mol / L, 2 mL) was then added dropwise to the mixture. The mixture was stirred at 500 rpm for 10 min at room temperature, centrifuged, washed, and the precipitate was freeze-dried to obtain the eugenol-encapsulated zeolite imidazolate framework-8 (Eug@ZIF-8). Similar to Example 11, the eugenol residue-time curve of Eug@ZIF-8 exposed to 200 ppm ammonia was tested. Figure 12 As shown, in Comparative Example 3, after exposure to 200 ppm ammonia for 300 min, the eugenol residue rate of Eug@ZIF-8 remained as high as 98.5%, showing no significant change. This indicates that ammonia cannot trigger the release of eugenol from Eug@ZIF-8, and ZIF-8 only has a slow-release effect on eugenol, failing to achieve an intelligent response to putrefactive gases.

[0093] Comparative Example 4: Based on CuNCs-La 3+ Sensor-based freshness monitoring and preservation application testing

[0094] CuNCs-La prepared in Example 3 3+ Solution replacement of Eug@CuNCs-La added in Example 9 3+ Solution preparation of CuNCs-La 3+ The sensor uses CuNCs-La. 3+ The sensor replaces the Eug@CuNCs-La sensor pasted in Example 12. 3+ Sensor. The TVB-N value of shrimp was measured, as in Example 12. The results are as follows: Figure 13 As shown in (B) of the table, the TVB-N value of the shrimp in Comparative Example 4 increased from an initial 6.31 mg / 100g to 18.20 and 24.55 mg / 100g on the 2nd and 3rd days of storage at 4°C, respectively. On the 5th day of storage, the TVB-N content exceeded 30 mg / 100g, indicating that the shrimp had completely spoiled and was inedible.

[0095] Comparative Example 5: Preparation of CuNCs-La 3+ Sensor with separate load from eugenol

[0096] A stock solution of eugenol (1 μg / mL) was prepared using 20% ​​(w / w) ethanol as the solvent. The sensor was prepared in the same manner as in Example 9, except that the solvent loaded on the sensor was changed; CuNCs-La prepared in Example 3 was gradually added dropwise to the sensor. 3+ A solution (900 μL) and an eugenol essential oil solution (1 μg / mL, 100 μL) were prepared. The TVB-N value of shrimp was determined as in Example 12 to evaluate the preservation effect of the sensor without eugenol encapsulation. Figure 13 As shown in (B), the TVB-N value of shrimp in Comparative Example 5 increased from an initial 6.42 mg / 100g. On the second and third days of storage at 4°C, it increased to 11.95 and 17.52 mg / 100g, respectively. On the sixth day of storage, the TVB-N content of the shrimp exceeded 30 mg / 100g, indicating that the shrimp had completely spoiled and was inedible. Compared to Comparative Example 4, which did not contain eugenol, the sensor in this comparative example extended the shelf life of the shrimp by one day (4°C), but the preservation effect was not as good as the Eug@CuNCs-La with slow-release function in Example 12. 3+ sensor.

[0097] Comparative Example 6: Gel Sensor Loaded with Eugenol and Copper Nanoparticles

[0098] A 0.02 g gel substrate was prepared according to patent application 202510222283.2, and eugenol (1 μg / mL, 100 μL) was adsorbed into the gel. A CuNCs solution (800 μL) and La were then added dropwise to the gel surface. 3+ A solution (0.1 M, 100 μL) was freeze-dried to prepare a gel sensor for shrimp freshness testing. The TVB-N value of the shrimp was determined in the same manner as in Example 12 to evaluate the preservation effect of the gel sensor. Figure 13 As shown in (B), the TVB-N value of shrimp in Comparative Example 6 increased from an initial 6.35 mg / 100g to 14.50 and 18.63 mg / 100g on days 2 and 3 of storage at 4°C, respectively. On day 6 of storage, the TVB-N content of the shrimp was 31.25 mg / 100g, at which point the shrimp had completely spoiled. Compared to Comparative Example 4, which did not contain eugenol, the gel sensor in this comparative example extended the shelf life of the shrimp by 1 day (4°C), demonstrating better preservation than Comparative Example 4, which did not contain eugenol. However, the preservation effect was not as good as that of Eug@CuNCs-La in Example 12. 3+ The sensor, possibly due to the longer slow-release pathway of eugenol in the gel, results in a slower preservation response compared to Eug@CuNCs-La. 3+ sensor.

[0099] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for the preparation of metal ion mediated copper nanoaggregates, characterized in that, The method comprises the following steps: (1) Disperse the amino acid or oligopeptide ligand in a NaOH solution, then add Cu 2+ solutions, shake mix, prepare CuNCs solution; (2) mixing the plant essential oil solution with the CuNCs solution, adding a metal ion solution after ultrasonic dispersion, which coordinates with the carboxyl groups on the surface ligands of the clusters, and then induces the aggregation of CuNCs to form copper nano-aggregates embedding the plant essential oil; The metal ion is selected from one or more of lanthanum ion, cerium ion and aluminum ion.

2. The method for preparing metal ion-mediated copper nanoaggregates according to claim 1, characterized in that, The amino acid or oligopeptide ligand in step (1) is one or more of N-acetyl-L-cysteine, L-cysteine, D-penicillamine and glutathione; the concentration of the ligand in the NaOH solution is 0.2-1.0 mol / L, the concentration of Cu 2+ The concentration of the solution is 0.02-0.1 mol / L, the concentration of the NaOH solution is 0.1-1.0 mol / L, the oscillation time is 2-10 min, and the molar ratio of the ligand to Cu 2+ is 3-20:

1.

3. The method for preparing metal ion-mediated copper nanoaggregates according to claim 1, characterized in that, In step (2), the plant essential oil is selected from phenolic compounds, terpene compounds or aldehyde compounds, preferably eugenol, carvacrol, thymol, cinnamaldehyde or citral; the concentration of the plant essential oil solution is 0.125-5 μg / mL; the concentration of the metal ion solution is 0.01-0.2 mol / L; the ultrasonic dispersion time is 5-10 min, the ultrasonic power is 50-150 W, and the ultrasonic water bath temperature is 25-37℃; the volume ratio of the CuNCs solution, the plant essential oil solution and the metal ion solution is 2.0-8.0:0.5-1.0:0.5-1.

0.

4. A copper nano-aggregate prepared by the method of any one of claims 1-3.

5. A sensor having dual functions of freshness monitoring and preservation, characterized by, The method comprises the following steps: The copper nano-aggregate solution of claim 4 is added to a fiber / porous substrate to prepare a sensor with freshness monitoring and preservation dual functions by vacuum freeze drying.

6. The sensor with dual functions of freshness monitoring and preservation according to claim 5, wherein, The fiber / porous substrate is one or more of filter paper, tissue paper, non-woven fabric and porous gel; the loading amount of the copper nano-aggregate solution on the fiber / porous substrate is 15-100 μL / cm 2 .

7. The sensor with dual functions of freshness monitoring and preservation according to claim 5, wherein, The vacuum freeze drying time is 24-48 h.

8. The sensor of claim 5 in the freshness monitoring and preservation of food.

9. Use of the sensor according to claim 8 in the freshness monitoring and preservation of food products, characterized by the fact that, The application method comprises the following steps: fixing the sensor in the food packaging, and the total volatile nitrogen produced by the spoilage of the food responds to the copper nano-aggregate in the sensor, which destroys the nano-aggregate structure, resulting in a decrease in fluorescence intensity to realize visual monitoring of freshness, and triggers the controlled release of the plant essential oil in the sensor to prolong the shelf life of the food.

10. The method for preparing metal ion-mediated copper nanoaggregates according to claim 9, characterized in that, The monitoring method of the fluorescence intensity is: constructing a freshness grading color card based on the fluorescence pictures of the sensor to realize rapid evaluation of freshness; establishing a linear regression model of the color channel values of the fluorescence pictures and the TVB-N values of the food to more accurately determine the freshness of the food.

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