Preparation method of intelligent label for rapidly detecting freshness of prawns

Abstract

The invention discloses a preparation method of an intelligent label for rapidly detecting freshness of prawns, and belongs to the field of synthetic materials and the technical field of rapid food detection. According to the preparation method of the intelligent label for rapidly detecting the freshness of the prawns, fluorescein isothiocyanate and BTB-BSA-AuNCs modified gold nanoclusters are mixed, the mixture is settled and attached to a filter membrane, the ratiometric fluorescence and colorimetric intelligent label is manufactured, a ratiometric fluorescence and colorimetric double-developing signal can be formed through mutual verification of two kinds of signals, and the sensitivity and the sensitivity of the intelligent label for rapidly detecting the freshness of the prawns are improved. Visual qualitative detection of the freshness of the prawns is carried out, and the method is more convenient.

Description

Technical Field

[0001] The invention relates to a method for preparing an intelligent label for quickly detecting the freshness of prawns, and belongs to the field of synthetic materials and the technical field of rapid food detection. Background Art

[0002] Shrimp is a kind of shrimp belonging to the family Penaeidae, order Decapoda, which is widely distributed in tropical and subtropical areas around the world. It is deeply loved by people because of its delicious taste and high nutritional value. However, shrimp is easily contaminated by microorganisms during storage and transportation. These microorganisms will metabolize the nutrients in the shrimp, produce bad odor and taste, and cause the shrimp to spoil. At the same time, the endogenous enzyme activity of the shrimp still exists after the death of the shrimp, which will further accelerate the spoilage of the shrimp, causing it to volatilize nitrogen-containing compounds (volatile basic nitrogen, TVB-N), and at the same time increase the pH in the surrounding environment to a certain extent. Therefore, when the shrimp is sealed, the change of pH value in the container can be used to evaluate the freshness of the shrimp.

[0003] When shrimps spoil during storage and transportation but are not detected in time, the spoiled shrimps may be accidentally eaten by people and cause discomfort. Therefore, it is of great significance to develop an efficient, fast and intuitive method, especially one that can be used for detection under sunlight, for detecting the freshness of shrimps.

[0004] Nowadays, fluorescent materials (such as nanoclusters, carbon dots, metal organic frameworks, dyes, etc.) are a kind of emerging materials, which are widely used in the detection of freshness of aquatic products due to their advantages of high sensitivity, diverse colors, simple operation, and diverse material construction methods. For example, patent CN114624229A discloses a method for monitoring the freshness of shrimp meat and a colorimetric card used therein, which uses rhodamine B and fluorescein to prepare a ratiometric fluorescent colorimetric card for real-time in-situ monitoring of the content of biogenic amines in shrimp meat, thereby determining the freshness of shrimp meat; although this method can detect the freshness of shrimp to a certain extent, the fluorescent indicator used therein has certain toxicity and poses a safety risk. Patent CN116046732A discloses a ratiometric fluorescent smart label for visually detecting the freshness of meat, and its preparation method and application. A gold-copper bimetallic nanocluster with red fluorescence and a dual-ligand gold nanocluster with green fluorescence are compounded to obtain a ratiometric fluorescent smart label. This label can be combined with a smartphone to detect the freshness of meat and aquatic products, but the detection operation combined with a smartphone is relatively cumbersome. This smart label only uses a ratiometric fluorescent smart label and does not perform specific and targeted detection on shrimp. At the same time, the above two methods must be equipped with ultraviolet light for observation and confirmation, and the freshness of shrimp meat cannot be intuitively judged. Therefore, there is an urgent need for a method that can intuitively detect the freshness of shrimp under both sunlight and ultraviolet light, especially under sunlight. Summary of the invention

[0005] At present, most of the research and application of fluorescent materials for detecting freshness are in the form of single emission fluorescence or ratio fluorescence smart labels, which have a single color change and are easily affected by subjectivity. The present invention synthesizes a fluorescent substance that responds to changes in pH under sunlight and ultraviolet light, mixes it with fluorescein isothiocyanate, and loads the mixture onto a filter membrane to produce a ratio fluorescence + colorimetric smart label. The ratio fluorescence and colorimetric signals are deposited in the same filter membrane in parallel, and the two signals can be mutually verified to form a ratio fluorescence + colorimetric dual color development signal, so as to perform visual qualitative detection of the freshness of shrimp, which is more convenient. At the same time, quantitative measurement of the volatile basic nitrogen content (TVB-N) in shrimp can be achieved within a certain range, making the visualization result more reliable.

[0006] The specific technical solutions are as follows:

[0007] The first object of the present invention is to provide a method for preparing a smart label for quickly detecting the freshness of shrimp, the specific steps comprising:

[0008] (1) taking a bovine serum albumin solution, stirring it, adding a tetrachloroauric acid tetrahydrate (HAuCl4·4H2O) aqueous solution, and then adding a sodium hydroxide solution, reacting to obtain a first-stage solution;

[0009] (2) dialyzing the first-stage solution obtained in step (1), adding an acid-base indicator, bromothymol blue sodium (C 27 H 27 Br2NaO5S, English abbreviation BTB) salt solution, let it stand to obtain the second stage solution;

[0010] (3) dialyzing the second-stage solution obtained in step (2) to obtain a BTB-modified gold nanocluster (BTB-BSA@Au NCs) solution; mixing the solution with fluorescein isothiocyanate (C 21 H 11 The NO5S, English abbreviation FITC) solution is stirred and mixed, the microporous filter membrane is immersed in it, and then the microporous filter membrane is dried to obtain a smart label.

[0011] In one embodiment, the concentration of the bovine serum albumin solution in step (1) is 50 to 80 mg / mL; preferably, the concentration of the bovine serum albumin solution is 50 mg / mL;

[0012] In one embodiment, the stirring condition in step (1) is 35-38° C. and 500-1000 r / min for 5-10 min;

[0013] In one embodiment, the concentration of the tetrachloroauric acid tetrahydrate aqueous solution in step (1) is 10-15 mM; preferably, the concentration of the tetrachloroauric acid tetrahydrate aqueous solution is 10 mM;

[0014] In one embodiment, the tetrachloroauric acid tetrahydrate aqueous solution is added dropwise in step (1).

[0015] In one embodiment, the concentration of the sodium hydroxide solution (NaOH) in step (1) is 1.0 to 1.2 M; preferably, the concentration of the sodium hydroxide solution (NaOH) is 1.0 M;

[0016] In one embodiment, the reaction time in step (1) is 12 to 14 hours; preferably, the reaction time is 12 hours;

[0017] In one embodiment, in step (1), the volume ratio of bovine serum albumin solution to tetrachloroauric acid tetrahydrate aqueous solution to sodium hydroxide solution is (10-12):(9-11):(0.8-1.2); preferably, the volume ratio is 10:10:1.

[0018] In one embodiment, the acid-base indicator is a bromothymol blue sodium salt solution; the concentration of the bromothymol blue sodium salt solution is 5 to 10 mg / mL; preferably, the concentration of the bromothymol blue sodium salt solution is 5 mg / mL;

[0019] In one embodiment, the molecular weight cut-off of the dialysis in step (2) is above 14 kDa, the dialysis time is 24 to 30 hours, and the water is changed every 8 to 10 hours;

[0020] In one embodiment, the standing condition in step (2) is 15-45° C. for 1-2 hours;

[0021] In one embodiment, the volume ratio of the first-stage solution to the acid-base indicator in step (2) is (6-8):(1-1.2); preferably, the volume ratio of the first-stage solution to the acid-base indicator is 6:1.

[0022] In one embodiment, the molecular weight cutoff for dialysis in step (3) is 14 kDa, and the dialysis time is 20 to 24 h;

[0023] In one embodiment, the concentration of the fluorescein isothiocyanate solution is 4-5 mg / L; preferably, the concentration of the fluorescein isothiocyanate solution is 5.0 mg / L;

[0024] In one embodiment, the stirring condition in step (3) is 500 to 800 r / min for 1 to 10 min;

[0025] In one embodiment, the volume ratio of the BTB-modified gold nanocluster solution to the fluorescein isothiocyanate solution in step (3) is (1-4): (1-4); preferably, the volume ratio of the BTB-modified gold nanocluster solution to the fluorescein isothiocyanate solution is 3:1;

[0026] In one embodiment, the diameter of the microporous filter membrane in step (3) is 13 to 15 mm, and the pore size is 0.22 to 0.45 μm; the immersion time is 1 to 1.5 h;

[0027] In one embodiment, the drying condition in step (3) is drying at 30-50° C. for 3-5 minutes.

[0028] The second object of the present invention is to provide a smart label for quickly detecting the freshness of shrimps, wherein the smart label is prepared by the above preparation method.

[0029] The third object of the present invention is to provide a packaging material or a degradable material, wherein the packaging material or the degradable material comprises the smart label or the smart label prepared by the above-mentioned preparation method.

[0030] The fourth object of the present invention is to provide the application of the above-mentioned smart tag in the rapid detection of aquatic fish and shrimp.

[0031] In one embodiment, the application is to use the above-mentioned smart label or the smart label prepared by the above-mentioned preparation method as a detection matrix, and determine the freshness of aquatic fish and shrimp according to the color change of the label.

[0032] In one embodiment, the aquatic fish and shrimp include but are not limited to freshwater shrimp.

[0033] In one embodiment, the application is to place the smart label or the smart label prepared by the preparation method together with meat or seafood for 12 to 96 hours, and then determine the freshness of the meat or seafood based on the color change under sunlight or fluorescence;

[0034] When qualitatively analyzing the color changes under sunlight or fluorescence, the color blocks are divided into three groups:

[0035] Under normal daylight, yellow to light yellow means that the fruit is fresh and can be eaten; light green means that it is no longer fresh and can be eaten, but it is not recommended to be eaten; light blue means that the fruit is not fresh and cannot be eaten.

[0036] Under fluorescent light, orange-yellow means the fruit is fresh and edible; light yellow-light green means it is no longer fresh and can be eaten, but it is not recommended; green means the fruit is not fresh and cannot be eaten.

[0037] When quantifying the color change under sunlight, a total of 5 color blocks were selected and divided into 3 groups:

[0038] Under normal daylight, a color difference of ≤11.3 or a TVB-N (mg / 100g) value of ≤15.0 indicates good freshness and can be eaten; 11.3 < color difference ≤19.7 or 15.0 < TVB-N (mg / 100g) value ≤18.8 indicates that it is no longer fresh and can only be eaten, but it is not recommended to eat it; color difference >19.7 (19.7-34.5) or TVB-N (mg / 100g) value >18.8 indicates that the freshness is poor and cannot be eaten;

[0039] When quantifying the color change under fluorescence, a total of 6 color blocks were selected and divided into 3 groups:

[0040] Under fluorescence, a color difference of ≤37.7 or a TVB-N (mg / 100g) value of ≤15.0 indicates that the fruit is fresh and edible; a color difference of 37.7 < 46.8 or a TVB-N (mg / 100g) value of 15.0 < 18.8 indicates that the fruit is no longer fresh and can be eaten, but is not recommended; a color difference of >46.8 (46.8-67.2) or a TVB-N (mg / 100g) value of >18.8 indicates that the fruit is poorly fresh and cannot be eaten.

[0041] A fifth object of the present invention is to provide application of the above-mentioned smart label in the field of food or agricultural products or in the preparation of smart label products.

[0042] Beneficial Effects

[0043] (1) The present invention provides a method for preparing a smart label for quickly detecting the freshness of shrimp. BTB-modified gold nanoclusters (BTB-BSA@AuNCs) are mixed with fluorescein isothiocyanate and precipitated on a filter membrane to prepare a ratiometric fluorescence + colorimetric smart label. The two signals can be mutually verified to form a ratiometric fluorescence + colorimetric dual colorimetric signal, which can be used for visual qualitative detection of the freshness of shrimp, which is more convenient.

[0044] (2) The smart label prepared by the present invention can perform quantitative analysis in a partial range while visualizing qualitative analysis, and has a high linear correlation.

[0045] (3) The smart label prepared by the present invention reduces some negative influences that would reduce the accuracy of the two output signals at the same time. The smart label has higher stability and can be refrigerated for more than 3 months to ensure that the indication result is not affected. At the same time, it has good specificity and has no obvious response to other non-nitrogen volatile substances.

[0046] (4) Compared with the national standard method GB 5009.228-2016 "National Food Safety Standard Determination of Volatile Basic Nitrogen in Food", the smart label prepared by the present invention has a fast detection speed when detecting the freshness of shrimp, and the results can be observed immediately. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 :The color development results of fluorescein isothiocyanate smart label under sunlight.

[0048] Figure 2 :The color development results of fluorescein isothiocyanate smart label under fluorescence.

[0049] Figure 3 :Color development results of gold nanocluster (BSA@AuNCs) smart label under sunlight.

[0050] Figure 4 : Color development results of gold nanocluster (BSA@AuNCs) smart label under fluorescence.

[0051] Figure 5 :Color development results of gold nanocluster (BTB-BSA@AuNCs) smart label under sunlight.

[0052] Figure 6 : Color development results of gold nanocluster (BTB-BSA@AuNCs) smart label under fluorescence.

[0053] Figure 7 :Colorimetric results of fluorescein isothiocyanate and gold nanoclusters (BSA@AuNCs) smart labels under sunlight.

[0054] Figure 8 : Colorimetric results of fluorescein isothiocyanate and gold nanoclusters (BSA@AuNCs) smart label under fluorescence.

[0055] Fig. 9 : Color development results of methyl red smart label under sunlight.

[0056] Fig.10 : Color development results of methyl red smart label under fluorescence.

[0057] Fig.11 : The color development results of bromocresol green smart label under sunlight.

[0058] Fig.12 : Color development results under bromocresol green smart label fluorescence.

[0059] Fig.13 : The color development results of bromocresol purple smart label under sunlight.

[0060] Fig.14 : The color development results of bromocresol purple smart label under fluorescence.

[0061] Fig.15 : UV-visible spectra and fluorescence spectra of different indicator solutions.

[0062] Fig.16 : Color development results of smart label under sunlight after standing for 10 minutes.

[0063] Fig.17 : The color development results of the smart label were obtained after standing for 10 minutes under fluorescence.

[0064] Fig.18 : Color development results of smart label under sunlight after standing for 50 minutes.

[0065] Fig.19 : The color development results of the smart label under fluorescence were obtained after standing for 50 minutes.

[0066] Fig. 20 : Color development results of smart label under sunlight after standing for 2.5 hours.

[0067] Fig.21 : The color development results of the smart label under fluorescence were obtained after standing for 2.5 hours.

[0068] Fig. 22 :Color development results of smart label with addition amount of 0.1mL under sunlight.

[0069] Fig.23 :Color development results of smart label under fluorescence with addition amount of 0.1mL.

[0070] Fig.24 :Color development results of smart label with addition amount of 0.5mL under sunlight.

[0071] Fig.25 :Color development results of smart label under fluorescence with addition amount of 0.5mL.

[0072] Fig.26 :Color development results of smart label with 2mL added under sunlight.

[0073] Fig. 27 :Color development results of smart label under fluorescence with addition amount of 2mL.

[0074] Fig.28 :Color development results of smart label under sunlight at static temperature of 15℃.

[0075] Fig.29 :Color development results of smart label under fluorescence at static temperature of 15℃.

[0076] Fig.30 : Color development results of smart label under sunlight at static temperature of 35℃.

[0077] Fig.31 : Color development results of smart label under fluorescence at static temperature of 35℃.

[0078] Fig.32 : Color development results of smart label under sunlight at static temperature of 45℃.

[0079] Fig.33 : Color development results of smart label under fluorescence at static temperature of 45℃.

[0080] Fig.34 Figure 1 shows the spectra and performance diagrams of the BTB-modified gold nanoclusters (BTB-BSA@AuNCs) prepared in Example 1 of the present invention, wherein (a) is a fluorescence excitation spectrum, (b) is a fluorescence emission spectrum, (c) is a powder state diagram under sunlight and fluorescence, (d) is a solution state diagram (pH=7) under sunlight and fluorescence, (e) on the left is the color of the BTB-modified gold nanoclusters (BTB-BSA@AuNCs) solution under fluorescence at pH=4, 6, and 8, and (e) on the right is the color of the BTB-modified gold nanoclusters (BTB-BSA@AuNCs) solution under fluorescence at pH=4, 6, and 8. , (f) is the UV-visible absorption spectrum of bromothymol blue sodium salt (BTB), gold nanoclusters (BSA@AuNCs) without BTB modification, and gold nanoclusters (BTB-BSA@AuNCs) modified with BTB, and (g) is the fluorescence emission spectrum of gold nanoclusters (BSA@AuNCs) without BTB modification and gold nanoclusters (BTB-BSA@AuNCs) modified with BTB.

[0081] Fig.35 Spectra of fluorescein isothiocyanate used in Example 1 of the present invention, wherein (a) is the fluorescence excitation spectrum and (b) is the fluorescence emission spectrum.

[0082] Fig.36 1 is a graph showing the response of the mixed solution obtained in Example 1 of the present invention to pH, wherein (a) is the fluorescence excitation spectrum of the mixed solution, (b) is the fluorescence emission spectrum of the mixed solution, (c) is the color development under sunlight, (d) is the color development under fluorescence, (e) is the solution color space (CIELab) (hereinafter referred to as color difference) change curve under sunlight (ΔE), and (f) is the color difference change curve (ΔE) of the solution under fluorescence.

[0083] Fig.37 The figure is a color change diagram of the smart label of the present invention as the freshness of the shrimp decreases, wherein (a) is the color development under sunlight, (b) is the color development under fluorescence, (c) is the color difference change curve (ΔE) of the smart label under sunlight, and (d) is the color difference change curve (ΔE) of the smart label under fluorescence.

[0084] Fig.38The figures are related to the quantitative detection of the present invention, (a) is the curve of the change of TVB-N of fresh shrimp with the number of days, (b) is the relationship curve between TVB-N and ΔE under sunlight, (c) is the relationship curve between TVB-N and ΔE under fluorescence, (d) is the linear relationship fitting curve between TVB-N and ΔE under sunlight, and (e) is the linear relationship fitting curve between TVB-N and ΔE under fluorescence.

[0085] Fig.39 A sample of a smart label card designed for practical application.

[0086] Fig.40 The freshness comparison diagram of the intelligent label for rapid detection of freshness of shrimps of the present invention, (a) to (b) are qualitative comparison tables, wherein (a) is a qualitative comparison table under sunlight, and (b) is a qualitative comparison table under fluorescence; (c) to (d) are quantitative comparison tables, and the linear range of the quantitative comparison is under sunlight (TVB-N concentration is 14.2-20.7 mg / 100 g, and the color difference ΔE is 10.6-26.0), under fluorescence (TVB-N concentration is 9.9-20.7 mg / 100 g, and the color difference ΔE is 21.4-54.9), (c) is a quantitative comparison table under sunlight, and (d) is a quantitative comparison table under fluorescence.

[0087] Fig.41 This is the color development result of anthocyanin + gold nanoclusters (BSA@AuNCs) + fluorescein isothiocyanate smart label under sunlight.

[0088] Fig.42 This is the color development result of anthocyanin + gold nanoclusters (BSA@AuNCs) + fluorescein isothiocyanate smart label under fluorescence.

[0089] Fig.43 This is the UV-visible absorption spectrum of anthocyanins.

[0090] Fig.44 Quantitative test results analysis under sunlight.

[0091] Fig.45 For quantitative test result analysis under fluorescence. DETAILED DESCRIPTION

[0092] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention. In the following embodiments, unless otherwise specified, the solutions mentioned are all water-based as solvent.

[0093] The color change mechanism of the smart label for rapid detection of shrimp freshness prepared in the embodiment of the present invention:

[0094] (1) Color change mechanism under sunlight: Since bromothymol blue sodium salt is a pH indicator, the bromothymol blue sodium salt molecule modified on BSA still has the function of a pH indicator. When encountering an acidic medium, the phenolphthalein ring in the molecule combines with H + Protonation appears yellow, and when encountering alkaline medium, it loses H + Deprotonated, it appears blue, but the fluorescent pigment in the smart tag does not show color under sunlight. Therefore, during the shrimp spoilage process, the environmental pH gradually rises, and the smart tag changes color due to the influence of the environmental pH.

[0095] (2) Color change mechanism under fluorescence: FITC exhibits green fluorescence, and the fluorescence intensity at 521 nm increases with increasing pH. The ultraviolet absorption spectrum of sodium bromothymol blue overlaps with the fluorescence emission spectrum of BSA@AuNCs at 647 nm, and the absorption peak of sodium bromothymol blue at 647 nm gradually increases with increasing pH, resulting in a decrease in the fluorescence intensity of BSA@AuNCs modified with BTB. Therefore, the fluorescence intensity of the mixed solution of BTB-BSA@AuNCs and FITC shows a phenomenon of decreased red fluorescence and increased green fluorescence with increasing environmental pH, resulting in a change from red to green.

[0096] The test methods involved in the following embodiments are as follows:

[0097] 1. Fluorescence excitation and emission spectrum detection method

[0098] 1) Add the prepared solution to a cuvette that is translucent on all four sides, and add 2 / 3 of the volume of the cuvette; 2) Place the cuvette in a fluorescence spectrophotometer, open the fluorescence spectrophotometer setting page, select the excitation spectrum, set the appropriate excitation and emission wavelengths, adjust to zero, and select test to obtain the fluorescence excitation spectrum of the liquid being tested; 3) Open the fluorescence spectrophotometer setting page again, select the excitation spectrum, set the appropriate excitation and emission wavelengths, adjust to zero, and select test to obtain the fluorescence emission spectrum of the liquid being tested; 4) Use origin software to import data, use light wavelength as the horizontal axis and fluorescence intensity as the vertical axis, and draw the fluorescence excitation and emission spectra.

[0099] 2. UV-visible spectroscopic detection method

[0100] 1) Add the prepared solution into the quartz cuvette, filling 2 / 3 of the cuvette volume; 2) Place the cuvette into the UV-visible spectrophotometer, open the UV-visible spectrophotometer setting page, select spectrum scan, set the spectrum scan range, spectrum scan type, adjust the baseline, select test, and you can get the UV-visible spectrum of the liquid being tested; 3) Use origin software to import data, use the light wavelength as the horizontal axis and the absorbance as the vertical axis, and draw the UV-visible spectrum.

[0101] 3. Color rendering pictures under sunlight and fluorescence

[0102] 1) Prepare different pH 4-8 solutions, with a value of 0.5 and a total of 9 pH solutions. Take 3 mL of each pH solution in a test tube, add 50 μL of the mixed solution or gold nanocluster (BTB-BSA@AuNCs) solution or gold nanocluster (BSA@AuNCs) solution obtained in step (5) of Example 1, and shake evenly; 2) Pour the solutions in the test tubes into 9 glass cuvettes, and take color pictures with a mobile phone under sunlight and 365 nm ultraviolet light.

[0103] 4. Rapid detection of shrimp freshness Smart label is used to test the freshness of shrimp

[0104] The smart label prepared in the embodiment is used to detect the color of shrimps at different freshness levels. The specific steps are as follows: 1) Buy a certain number of shrimps in the market, take a fresh shrimp, wipe off the water, and place it in a Φ90mm culture dish; 2) Place the smart label in the embodiment in a blank space of the culture dish, seal the culture dish with a sealing film, and place it in a 4°C refrigerator for refrigeration; 3) Use a mobile phone to take pictures of the color of the smart label under sunlight and ultraviolet light every 12 hours (respectively on the 0th, 0.5th, 1st, 1.5th, 2nd, 2.5th, 3rd, 3.5th, and 4th day, the same below), for a total of 9 photos (including the initial). 4) The shooting conditions are: Under sunlight: Just shoot under ordinary sunlight, no special requirements; Under fluorescence: Use a UV365nm purple light lamp, and take pictures at a distance of about 20cm from the label.

[0105] 5. Color difference value curve (ΔE) drawing method

[0106] 1) Import the pictures taken by mobile phone in the above method "Color development pictures under sunlight and fluorescence" or "Rapid detection of shrimp freshness smart label for shrimp freshness test" into ColorMeter software, and record the Lab (L x 、a x 、b x ) value; 2) based on the color difference value of the liquid in the glass dish with pH = 4 solution or the smart label just made, recorded as L0, a0, b0, use the formula in turn:

[0107] Calculate the color difference change (ΔE) of the solution at pH 4.5-8 or the smart label at 0-4 days; 3) Use origin software to import the calculation results, use pH as the horizontal axis and ΔE as the vertical axis, and draw the change curve under sunlight and fluorescence. Among them, the Lab value of color is the color measurement established by the International Commission on Illumination (CIE) based on international standards. It is named CIELab. The L component in the Lab color measurement is used to indicate the brightness of the color, with a value range of [0,100], representing from pure black to pure white; a represents the range from red to green, with a value range of [127,-128]; b represents the range from yellow to blue, with a value range of [127,-128].

[0108] 6. Volatile nitrogen detection method

[0109] 1) The shrimps purchased in the same batch as the test method "Rapid detection of shrimp freshness smart label for testing shrimp freshness" were wiped dry and placed in a 4°C refrigerator for 4 days. During this period, the volatile basic nitrogen content was tested every 12 hours, for a total of 9 tests (including the initial test). The test method refers to the first method semi-micro nitrogen determination method test of GB 5009.228-2016 "National Food Safety Standard Determination of Volatile Basic Nitrogen in Food". 2) According to the requirements of GB 2733-2015 "National Food Safety Standard Fresh and Frozen Animal Aquatic Products", the volatile basic nitrogen content of edible freshwater fish and shrimp is ≤20mg / 100g. 3) The test results are listed in a table as shown in Table 1. At the same time, the test results are imported using the origin software, with the number of refrigeration days as the horizontal axis and the volatile basic nitrogen content in the shrimp as the vertical axis, and the change curve is drawn, as shown in Table 1. Fig.38 (a).

[0110] 7. TVB-N and ΔE relationship curve

[0111] 1) The color difference values ​​ΔE (9 groups in total) of the smart label under sunlight and fluorescence obtained in the above method "color difference change curve (ΔE) drawing method" and the TVB-N (9 groups in total) values ​​obtained in the method "volatile nitrogen detection method" are imported into the origin software, and the change curves under sunlight and fluorescence are drawn with TVB-N as the horizontal axis and ΔE as the vertical axis respectively; 2) From the data in 1), it can be seen that the color difference values ​​of the smart label on the 1st to 3rd day under sunlight and the TVB-N value (5 groups of data in total) are basically proportional, so the 5 groups of data are imported into the origin software, and a scatter plot is drawn with TVB-N as the horizontal axis and ΔE as the vertical axis, and a linear fitting curve is obtained; 3) From the data in 1), it can be seen that the color difference values ​​of the smart label on the 0.5-3rd day under fluorescence and the TVB-N value (6 groups of data in total) are basically proportional, so the 5 groups of data are imported into the origin software, and a scatter plot is drawn with TVB-N as the horizontal axis and ΔE as the vertical axis, and a linear fitting curve is obtained.

[0112] 8. Making color blocks

[0113] Import the photos taken by mobile phone in the above method "Pictures of color development under sunlight and fluorescence" or "Rapid detection of shrimp freshness smart label for testing shrimp freshness" into ColorMeter software to obtain the standard color of the corresponding liquid or smart label, and crop these color screenshots to generate color blocks of the same size.

[0114] 9. Method for making color chart for intelligent label for rapid detection of shrimp freshness

[0115] 1) For Example 1, the color blocks of the smart labels of the shrimps in each stage (0-4 days, one photo taken every 12 hours, a total of 9 photos) were obtained according to the method "production of color blocks"; the obtained color blocks and color correspondences are as follows Fig.40 As shown, according to the corresponding relationship, a color chart is made. There are 4 types, divided into qualitative color charts under daylight and fluorescence and quantitative color charts under daylight and fluorescence:

[0116] Qualitative analysis: refer to the results of volatile basic nitrogen determination and the number of days of storage, and divide the color blocks into 3 groups on average. Under normal sunlight, yellow-light yellow means good freshness and edible; light green means no longer fresh and can only be eaten, but not recommended; light blue means poor freshness and inedible; under fluorescent light, orange-light yellow means good freshness and edible; light yellow-light green means no longer fresh and can only be eaten, but not recommended; green means poor freshness and inedible;

[0117] 2) For other implementation examples, refer to the method for preparing the qualitative color chart in Implementation Example 1, and divide the color blocks under sunlight and fluorescence into three groups, namely, good freshness and edible; no longer fresh and only edible, but not recommended for consumption; poor freshness and inedible; and mark the ΔE value of each color block below.

[0118] 10. Production of smart label cards for rapid detection of shrimp freshness

[0119] 1) According to the qualitative color chart under daylight and fluorescence in method 9, the color cards in the middle of each group are taken as the control colors of the smart label product cards according to the groups, and edible, not recommended for consumption, and inedible are marked under the corresponding colors. 2) The smart label prepared in Example 1 is fixed to the "indicator card" position of the product card, which can be used to detect the freshness of shrimp.

[0120] The raw materials involved in the following examples are as follows: bovine serum albumin was purchased from Shanghai Myrrel Biochemical Technology Co., Ltd. with the item number A850221-10g; tetrachloroauric acid tetrahydrate (HAuCl4·4H2O) was purchased from Suzhou Great Pharmaceutical Technology Co., Ltd. with the item number HWG00249-5g; bromothymol blue sodium salt was purchased from Shanghai Titan Technology Co., Ltd. with the item number 50330A; fluorescein isothiocyanate was purchased from Shanghai Myrrel Biochemical Technology Co., Ltd. with the item number F6120-100mg.

[0121] The solution preparation method involved in the following embodiments is as follows:

[0122] Bovine serum albumin solution (50 mg / mL): Take 1 g of solid bovine serum albumin and place it in a 50 mL test tube. Add 20 mL of distilled water and place it at 38°C. Stir at 500 r / min for 10 min until the bovine serum albumin is completely dissolved to obtain a 50 mg / mL bovine serum albumin solution.

[0123] Tetrachloroauric acid tetrahydrate aqueous solution (10 mM): Take 10.295 g of tetrachloroauric acid tetrahydrate solid and place it in a 50 mL test tube, add 25 mL of distilled water, and stir until the solid is completely dissolved to obtain a solution with a concentration of 10 mM.

[0124] Sodium hydroxide solution (1.0 M): Place 0.4 g of solid sodium hydroxide in a 20 mL test tube, add 10 mL of distilled water, and stir until the solid is completely dissolved to obtain a 1.0 M sodium hydroxide solution.

[0125] Bromothymol blue sodium salt solution (5.0 mg / mL): Take 10 mg of bromothymol blue sodium salt solid and place it in a 10 mL test tube, add 2 mL of distilled water, and stir until the solid is completely dissolved to obtain a 5.0 mg / mL concentration of bromothymol blue sodium salt solution.

[0126] Other indicators, anthocyanin solution (5.0 mg / mL): Take 10 mg of solid and place it in a 10 mL test tube, add 2 mL of distilled water, and stir until the solid is completely dissolved to obtain a 5.0 mg / mL concentration indicator and anthocyanin solution.

[0127] Fluorescein isothiocyanate solution (5.0 mg / L): Take 0.2 mg of fluorescein isothiocyanate solid and place it in a 100 mL test tube. Add 40 mL of distilled water and stir until the solid is completely dissolved to obtain a 5.0 mg / L concentration of fluorescein isothiocyanate solution.

[0128] Example 1: Preparation method of intelligent label for rapid detection of shrimp freshness

[0129] The experimental steps are as follows:

[0130] (1) Preparation of the first stage solution:

[0131] Prepare 5 mL of bovine serum albumin solution (BSA) (50 mg / mL), heat to 38°C, add 5 mL of tetrachloroauric acid tetrahydrate aqueous solution (10 mM) dropwise under stirring at 800 r / min, add 0.5 mL of sodium hydroxide solution (1.0 M) after 2 minutes, and continue the reaction for 12 h to obtain the first stage solution;

[0132] (2) One-time dialysis purification:

[0133] The first-stage solution obtained in step (1) was placed in a dialysis bag with a molecular weight cutoff of 14 kDa, and the dialysis bag was placed in ultrapure water for 24 hours, with the water being changed every 8 hours;

[0134] (3) Preparation of the second stage solution:

[0135] 6 mL of the dialyzed solution obtained in step (2) was taken, 1 mL of bromothymol blue sodium salt solution (BTB) (5.0 mg / mL) was added, and the mixture was allowed to stand at room temperature of 25° C. for 2 hours to obtain a second-stage solution;

[0136] (4) Secondary dialysis purification:

[0137] The second-stage solution obtained in step (3) was placed in a dialysis bag with a molecular weight cutoff of 14 kDa, and the dialysis bag was placed in ultrapure water for 24 hours to obtain a BTB-modified gold nanocluster (BTB-BSA@AuNCs) solution;

[0138] (5) Preparation of smart labels:

[0139] Take 2 mL of the BTB-modified gold nanocluster solution obtained in step (4) and 1 mL of fluorescein isothiocyanate solution (5.0 mg / L), stir at 500 r / min for 1 min, mix evenly to obtain a mixed solution, use hydrochloric acid (0.1 mol / L) to adjust the pH of the mixed solution to 4, soak a microporous filter membrane with a diameter of 13 mm and a pore size of 0.22 μm in it for 1.5 h, dry the soaked microporous filter membrane at 50°C for 5 minutes, and finally prepare a smart label for rapid detection of shrimp freshness.

[0140] Example 2: Characterization of different gold nanocluster solutions and smart tags

[0141] 1. The fluorescence spectra and optical properties of the BTB-modified gold nanocluster (BTB-BSA@AuNCs) solution obtained in step (4) of Example 1 and the BTB-modified gold nanocluster (BSA@AuNCs) solution obtained in step (2) were characterized. The results are as follows: Fig.34 As shown, the results show:

[0142] (1) By Fig.34 (a) The fluorescence excitation spectrum shows that the excitation curve of the BTB-modified gold nanoclusters (BTB-BSA@AuNCs) has a strong excitation peak at 491 nm. Fig.34 From the fluorescence emission spectrum of (b), it can be seen that the BTB-modified gold nanoclusters (BSA@AuNCs) have a strong fluorescence emission peak at 658 nm;

[0143] (2) Fig.34 As shown in (e), compared with BSA@AuNCs (right), the fluorescence intensity of BTB-BSA@AuNCs modified with BTB (left) was significantly reduced at pH = 4, 6, and 8; Fig.34 As shown in (f), the UV-visible absorption spectrum of bromothymol blue sodium salt (BTB) has 6 absorption peaks (194nm, 200nm, 283nm, 310nm, 416nm, 616nm), the UV-visible absorption spectrum of gold nanoclusters (BSA@AuNCs) has 2 absorption peaks (213nm, 276nm), and the UV-visible absorption spectrum of BTB-modified gold nanoclusters (BTB-BSA@AuNCs) has 4 absorption peaks (209nm, 270nm, 438nm, 624nm). There are no overlapping peaks in the UV-visible absorption spectra of the three. Fig.34 As shown in (g), the fluorescence emission spectrum of gold nanoclusters (BSA@AuNCs) has an emission peak at 647nm, and the fluorescence emission spectrum of BTB-modified gold nanoclusters (BTB-BSA@AuNCs) has an emission peak at 658nm. There is an 11nm shift in the fluorescence emission spectrum of gold nanoclusters (BSA@AuNCs). Fig.34 (e), (f), and (g) all indicate that the BTB-modified gold nanoclusters (BTB-BSA@AuNCs) have been successfully modified by bromothymol blue sodium salt.

[0144] (3) Take 30 mL of the BTB-modified gold nanocluster (BTB-BSA@AuNCs) solution obtained in step (4) or the unmodified gold nanocluster (BSA@AuNCs) solution obtained in step (2), place it in a weighing dish, and dry it in a freeze drying oven for 24 h. Take out the dry solid and grind it into powder in a mortar to obtain BTB-modified gold nanocluster (BTB-BSA@AuNCs) powder or unmodified gold nanocluster (BSA@AuNCs) powder. Take color pictures with a mobile phone under sunlight and 365 nm ultraviolet light, respectively.

[0145] The results show that: Fig.34 As shown in (c), the BTB-modified gold nanocluster (BTB-BSA@AuNCs) powder appears black under sunlight (left) and brown-red under fluorescence (right); Fig.34 As shown in (d), the gold nanocluster (BSA@AuNCs) powder without BTB modification appears green under sunlight and red under fluorescence.

[0146] 2. Fluorescence spectroscopy characterization of fluorescein isothiocyanate solution

[0147] The fluorescence spectrum of the fluorescein isothiocyanate solution used in step (5) is characterized as follows:

[0148] The results are as follows Fig.35 As shown by Fig.35 (a) The fluorescence excitation spectrum shows that the excitation curve of FITC has a strong excitation peak at 494 nm. Fig.35 From the fluorescence emission spectrum of (b), it can be seen that fluorescein isothiocyanate has a strong fluorescence emission peak at 521 nm.

[0149] 3. Characterization of the fluorescence spectrum of the mixed solution after the reaction of BTB-modified gold nanocluster solution and fluorescein isothiocyanate solution

[0150] Testing the fluorescence excitation spectrum and fluorescence emission spectrum of the mixed solution obtained in step (5) of Example 1;

[0151] The results are as follows Fig.36 As shown by Fig.36 The fluorescence excitation spectrum of (a) shows that the mixed solution has only one excitation peak at 492 nm. The fluorescence excitation peaks of BTB-modified gold nanoclusters (BTB-BSA@AuNCs) and FITC are basically consistent. Fig.36 From the fluorescence emission spectrum of (b), it can be seen that the mixed solution has strong fluorescence emission peaks at 519nm and 671nm. Comparing the fluorescence emission spectra of BTB-modified gold nanoclusters (BTB-BSA@AuNCs) and FITC, the emission peak at 519nm belongs to FITC, and the emission peak at 671nm belongs to BTB-modified gold nanoclusters. Compared with BTB-modified gold nanoclusters (BTB-BSA@AuNCs), there is a 13nm red shift phenomenon. It is speculated that fluorescence resonance energy transfer may have occurred between FITC and BTB-modified gold nanoclusters (BTB-BSA@AuNCs).

[0152] 4. Test of pH response of the mixed solution after the reaction between the BTB-modified gold nanocluster solution and the fluorescein isothiocyanate solution

[0153] 1) Prepare different pH 4-8 solutions, with a value of 0.5 and a total of 9 pH solutions. Take 3 mL of each pH solution in a test tube, and add 50 μL of the mixed solution obtained in step (5) of Example 1 to each test tube; 2) Pour the solutions in the test tubes into 9 glass cuvettes, and take color pictures with a mobile phone under sunlight and 365 nm ultraviolet light.

[0154] The pH response results are as follows Fig.36 (c) Fig.36 As shown in (d), the mixed solution changes from orange to green under fluorescence and from yellow to blue under daylight as the pH changes.

[0155] It can be seen that after the two substances are mixed, there is an obvious response (color change) as the pH changes in the solution, which can be used as the theoretical basis for the smart label in Example 1 to detect the freshness of shrimps, and it is expected that it will also have a good performance in the detection process.

[0156] 5. Using the above-mentioned “color difference change curve (ΔE) drawing method”, the color difference value of the mixed solution obtained in step (5) of this embodiment is tested;

[0157] The results are as follows Fig.36 (e) Fig.36 As shown in (f), with the change of pH value, the color difference value (ΔE) of the mixed solution under sunlight and fluorescence both shows an upward trend, indicating that the color of the solution has obvious changes. The color difference value under sunlight is in the range of 0-52.0; the color difference value under fluorescence is in the range of 0-82.3, indicating that the degree of color change under fluorescence is greater than that under sunlight.

[0158] 6. Using the smart label prepared in step (5) of this embodiment, color blocks are produced according to the above methods "Smart label for rapid detection of shrimp freshness for testing shrimp freshness" and "Production of color blocks":

[0159] like Fig.37 As shown in (a) and (b), after 0-4 days, the shrimp gradually deteriorated, and the smart tag changed from yellow to light blue under sunlight and from orange to green under fluorescence.

[0160] 7. Use the above-mentioned “color difference change curve (ΔE) drawing method” to test the color difference of the smart label obtained in step (5) of this embodiment:

[0161] The results are as follows Fig.37 (c) Fig.37As shown in (d), the color difference values ​​of the smart tag under daylight and fluorescence show an upward trend as the number of days changes, indicating that the color of the smart tag changes significantly with the change of shrimp freshness. The color difference value under daylight is in the range of 0-34.5; the color difference value under fluorescence is in the range of 0-67.2.

[0162] 8. Use the above-mentioned "Volatile Basic Nitrogen Detection Method" to test the volatile basic nitrogen (TVB-N) of shrimp:

[0163] The results are as follows Fig.38 (a) As shown in Table 1, the volatile basic nitrogen content in shrimps showed an upward trend with the change of days, ranging from 7.5-29.7 mg / 100g. According to the requirements of GB 2733-2015 "National Food Safety Standard Fresh and Frozen Animal Aquatic Products", the volatile basic nitrogen content of edible freshwater fish and shrimps is ≤20 mg / 100g. The shrimps used in the example can be stored at -4°C for about 3 days.

[0164] Table 1

[0165] Number of days TVB-N(mg / 100g) 0 7.5 0.5 9.9 1 14.2 1.5 15.0 2 15.7 2.5 18.8 3 20.7 3.5 24.5 4 29.7

[0166] 9. Use the above “TVB-N and ΔE relationship curve preparation method” to integrate the above results:

[0167] The results are as follows Fig.38 As shown in (b) and (d), under sunlight, as the number of days increases, TVB-N and the color difference ΔE of the smart label show a positive correlation. On the 1st to 3rd day, the positive correlation between the two sets of data is high, and they are basically linearly correlated. The fitting equation is y = -23.0125 + 2.3352x, with a high correlation coefficient (R 2 =0.998); Fig.38 As shown in (c) and (e), under fluorescence, as the number of days increases, TVB-N and the color difference ΔE of the smart label show a positive correlation. On the 0.5-3 day, the positive correlation between the two sets of data is high, and they are basically linearly correlated. The fitting equation is y = -10.5548 + 3.1517x, with a high correlation coefficient (R 2 =0.997).

[0168] 10. Make a color card table using the above-mentioned “making of color blocks” and “making method of color card table for quick detection of shrimp freshness smart label”:

[0169] Qualitative results such as Fig.40As shown in (a) and (b), for qualitative analysis: under normal daylight, yellow to light yellow means good freshness and can be eaten; light green means no longer fresh and can only be eaten, but not recommended; light blue means poor freshness and cannot be eaten; under fluorescent light, orange to yellow means good freshness and can be eaten; light yellow to light green means no longer fresh and can only be eaten, but not recommended; green means poor freshness and cannot be eaten;

[0170] 11. Verification method for quantitative detection

[0171] 1) Buy some shrimps from the market, take 6 fresh shrimps (1 group), wipe them dry, and place them in 6 Φ90mm culture dishes respectively;

[0172] 2) Place the smart label in Example 1 in a blank space of a culture dish, seal the culture dish with a sealing film, and store it in a refrigerator at 4° C., and take color photos of the smart label in its initial state under sunlight and fluorescence.

[0173] 3) The above 6 shrimps were divided into 2 portions, each with 3 shrimps. One portion was photographed with a mobile phone on the first day to show the color of the smart tag under sunlight and ultraviolet light, 2 photos for each shrimp, for a total of 6 photos; the other portion was photographed in the same way on the second day, 2 photos for each shrimp, for a total of 6 photos.

[0174] 4) According to the method "TVB-N and ΔE relationship curve", the color difference values ​​under sunlight and fluorescence on the 1st and 2nd day were tested, and the average values ​​were taken respectively. The closest photo was selected according to the average value, and the color block was made according to method 8. At the same time, the average color difference value was brought into the fitting curve under sunlight and fluorescence obtained in Example 1 to obtain the TVB-N prediction value on the 1st and 2nd day; 5) After each photo was taken, the actual TVB-N value was obtained according to method 6 using the national standard method, and the average value was taken. 6) A total of 3 groups of experiments were carried out, and the significant differences were calculated. The data table records are as follows: Figures 44-45 .

[0175] The results show:

[0176] (1) In the first group of experiments, under sunlight, the measured values ​​(average) of TVB-N content were 13.62 mg / 100 g on the first day and 17.27 mg / 100 g on the second day. The predicted values ​​of TVB-N calculated according to the calibration equation were 14.74 and 16.11 mg / 100 g on the first and second days, respectively. The color of the smart label also changed from yellow to light green and then to green, with a significant change. Under fluorescence, the predicted values ​​of TVB-N calculated according to the calibration equation were 13.06 and 17.69 mg / 100 g on the first and second days, respectively. The color of the smart label also changed from red to yellow and then to green, with a significant change. At the same time, the measured values ​​of TVB-N were analyzed with the predicted values, and no significant difference was found (P < 0.05).

[0177] (2) The same analysis was performed on Group 2 and Group 3, and similar results were obtained. The overall results showed that within a certain range, the smart tag in Example 1 can be used to quantitatively detect the content of TVB-N in shrimp.

[0178] Make a quantitative color chart such as Fig.40 As shown in (c) and (d), when quantified:

[0179] Under normal daylight, color difference ≤ 11.3 and TVB-N (mg / 100g) value ≤ 15.0 means that the freshness is good and can be eaten; 11.3 < color difference ≤ 19.7 and 15.0 < TVB-N (mg / 100g) value ≤ 18.8 means that it is no longer fresh and can only be eaten, but it is not recommended to eat it; color difference > 19.7 and TVB-N (mg / 100g) value > 18.8 means that the freshness is poor and cannot be eaten;

[0180] Under fluorescence, color difference ≤37.7 and TVB-N (mg / 100g) value ≤15.0 means that the fruit is fresh and edible; 37.7<color difference ≤46.8 and 15.0<TVB-N (mg / 100g) value ≤18.8 means that the fruit is no longer fresh and can be eaten but is not recommended; color difference>46.8 and TVB-N (mg / 100g) value>18.8 means that the fruit is poorly fresh and cannot be eaten.

[0181] Example 3: Preparation of different smart labels

[0182] 1. Preparation of fluorescein isothiocyanate smart label

[0183] Take 4 mL of fluorescein isothiocyanate solution (5.0 mg / L) in a test tube, soak a microporous filter membrane with a diameter of 13 mm and a pore size of 0.22 μm in it for 1.5 hours, dry the soaked microporous filter membrane at 50°C for 5 minutes, and prepare a fluorescein isothiocyanate smart label.

[0184] 2. Preparation of gold nanocluster (BSA@AuNCs) smart labels

[0185] 4 mL of the dialyzed solution obtained in step (2) of Example 1 was placed in a test tube, and a microporous filter membrane with a diameter of 13 mm and a pore size of 0.22 μm was immersed therein for 1.5 h. The immersed microporous filter membrane was dried at 50° C. for 5 min to prepare a gold nanocluster (BSA@AuNCs) smart label.

[0186] 3. Prepare BTB-modified gold nanocluster (BTB-BSA@AuNCs) smart label according to the method of Example 1

[0187] 4. Preparation of fluorescein isothiocyanate and gold nanoclusters (BSA@AuNCs) smart labels

[0188] Take 3 mL of the dialyzed solution obtained in step (2) of Example 1, i.e., the gold nanocluster (BSA@AuNCs) solution, and 1 mL of fluorescein isothiocyanate solution (5.0 mg / L), stir at 500 r / min for 1 min, and after mixing evenly, soak a microporous filter membrane with a diameter of 13 mm and a pore size of 0.22 μm in a test tube for 1.5 h. The soaked microporous filter membrane is dried at 50°C for 5 minutes to finally prepare fluorescein isothiocyanate and gold nanocluster (BSA@AuNCs) smart labels.

[0189] The four smart tags obtained above were used to prepare qualitative color charts for testing shrimp according to the above test methods 4, 8, and 9.

[0190] The results are as follows Figures 1 to 8 As shown:

[0191] The results show:

[0192] (1) Under sunlight, the color of the FITC smart label did not change significantly with the change of shrimp freshness, and it was all yellow, with a color difference value (ΔE) in the range of 0-5.3; under fluorescence, the color of the smart label changed significantly with the change of shrimp freshness, from dark green to bright green, with a color difference value in the range of 0-30.7. Under fluorescence, FITC had a certain effect of indicating freshness, but there was no obvious change in color, and the visibility was poor;

[0193] (2) Under sunlight, the gold nanocluster (BSA@AuNCs) smart tag did not show obvious color changes with the change of shrimp freshness, and was gray, with a color difference value in the range of 0-4.6; under fluorescence, the smart tag did not show obvious color changes with the change of shrimp freshness, and was red, with a color difference value in the range of 0-5.1; under sunlight and fluorescence, it had no function of indicating freshness;

[0194] (3) Under sunlight, the color of the gold nanocluster (BTB-BSA@AuNCs) smart tag changes with the freshness of the shrimp, from yellow to light green to light blue to blue, with a color difference value ranging from 0 to 28.6; under fluorescence, the color of the smart tag also changes with the freshness of the shrimp, from purple-red to blue-purple to dark blue, with a color difference value ranging from 0 to 25.8. The gold nanocluster (BTB-BSA@AuNCs) smart tag has basically no function of indicating freshness under sunlight and fluorescence, especially after the second day under sunlight and the second day under fluorescence, the value changes are small, the color is basically the same, and it is impossible to distinguish;

[0195] (4) Under sunlight, the color of the BSA@AuNCs smart tag did not change significantly with the change of shrimp freshness, and the color was all yellow, with a color difference value ranging from 0 to 5.9. Under fluorescence, the smart tag showed a more obvious color change with the change of shrimp freshness, from yellow to light yellow to dark green to green, with a color difference value ranging from 0 to 31.4. Under fluorescence, it had a certain effect of indicating freshness, but the color change was not obvious on the 2nd to 3.5th day, and it was relatively messy and fluctuating, with poor visibility.

[0196] It can be seen that only the gold nanocluster (BTB-BSA@AuNCs) + fluorescein smart label of the present invention can achieve a relatively obvious indication effect on the change of shrimp freshness under sunlight and fluorescence.

[0197] Example 4: Effect of different color developers

[0198] 1. Specific implementation method The same as Example 1, except that the bromothymol blue sodium salt solution in step (3) is adjusted to methyl red, bromocresol green and bromocresol purple, respectively, and finally three smart labels are obtained respectively; the labels are used to prepare qualitative color charts according to the above test methods 4, 8, and 9 to detect shrimps.

[0199] The results are as follows Figures 9 to 14 As shown:

[0200] The results show:

[0201] (1) Under sunlight and fluorescence, the visual range of methyl red and bromocresol green smart labels is small, and the color change is relatively single, with poor visibility;

[0202] (2) Under sunlight, the color of the bromocresol purple smart label changes with the freshness of the shrimp, from brown-green to dark blue to blue, with a color difference value ranging from 0 to 38.1; but from the second day on, the color change is not obvious and cannot indicate freshness; under fluorescent light, the color change is not obvious and visibility is poor;

[0203] At the same time, the UV spectra of the three substituted indicator solutions, gold nanocluster (BSA@AuNCs) solution, and substituted indicator modified gold nanocluster (BSA@AuNCs) solution and the fluorescence spectra of the gold nanocluster (BSA@AuNCs) solution and substituted indicator modified gold nanocluster (BSA@AuNCs) solution were obtained using the above test methods 1 and 2. The results Fig.15 shown.

[0204] The results show:

[0205] The UV-visible absorption spectrum of methyl red has two absorption peaks (194nm, 524nm), the UV-visible absorption spectrum of gold nanoclusters (BSA@AuNCs) has two absorption peaks (213nm, 276nm), and the UV-visible absorption spectrum of gold nanoclusters modified with methyl red (methyl red-BSA@AuNCs) has three absorption peaks (213nm, 278nm, 589nm), which are basically consistent with the absorption peak of gold nanoclusters (BSA@AuNCs) at ultraviolet light (≤365nm);

[0206] The UV-visible absorption spectrum of bromocresol purple has three absorption peaks (198nm, 276nm, 432nm), the UV-visible absorption spectrum of gold nanoclusters (BSA@AuNCs) has two absorption peaks (213nm, 276nm), and the UV-visible absorption spectrum of gold nanoclusters modified with bromocresol purple (bromocresol purple-BSA@AuNCs) has three absorption peaks (213nm, 277nm, 430nm), which are basically consistent with the absorption peak of gold nanoclusters (BSA@AuNCs) at ultraviolet light (≤365nm);

[0207] The UV-visible absorption spectrum of bromocresol green has two absorption peaks (221nm, 276nm), the UV-visible absorption spectrum of gold nanoclusters (BSA@AuNCs) has two absorption peaks (213nm, 276nm), and the UV-visible absorption spectrum of gold nanoclusters modified with bromocresol green (bromocresol green-BSA@AuNCs) has two absorption peaks (213nm, 274nm), which are basically consistent with the absorption peak of gold nanoclusters (BSA@AuNCs) at ultraviolet light (≤365nm);

[0208] The fluorescence emission spectra of gold nanoclusters (BSA@AuNCs) modified with three indicators showed emission peaks at 647nm, 648nm, and 648nm, respectively, which was consistent with the fluorescence emission spectrum of gold nanoclusters (BTB-BSA@AuNCs).

[0209] ad shows that methyl red, bromocresol green, bromocresol purple, etc. did not successfully modify gold nanoclusters (BSA@AuNCs).

[0210] 2. The influence of anthocyanins

[0211] The specific implementation method is the same as Example 1, except that the bromothymol blue sodium salt solution in step (3) is adjusted to anthocyanin, and finally anthocyanin + gold nanoclusters (BSA@AuNCs) + fluorescein isothiocyanate smart labels are obtained;

[0212] The results are as follows Figures 41-43 shown.

[0213] The anthocyanin smart label has a very small visible range under sunlight, with basically no change, and fails to indicate the freshness of the shrimp well. The visibility is poor. It is speculated that anthocyanin is not suitable for this type of preparation method and cannot be well adsorbed on the filter membrane. Under fluorescence, the visible range is 0-21.9, and the color changes, but it is basically light-colored. Combined with Example 2, it may be the result of the dominant role of gold nanoclusters (BSA@AuNCs) + isothiocyanate.

[0214] At the same time, the mixed solution was characterized using methods 1 and 2. The UV-visible absorption spectrum of anthocyanin had two absorption peaks (204nm and 280nm), the UV-visible absorption spectrum of gold nanoclusters (BSA@AuNCs) had two absorption peaks (213nm and 276nm), and the UV-visible absorption spectrum of anthocyanin-modified gold nanoclusters (anthocyanin+BSA@AuNCs) had three absorption peaks (213nm and 277nm), which were basically consistent with the absorption peak of gold nanoclusters (BSA@AuNCs) under ultraviolet light (≤365nm); the anthocyanin-modified gold nanoclusters had an emission peak at 649nm, which was consistent with the fluorescence emission spectrum of gold nanoclusters (BSA@AuNCs). The two figures showed that anthocyanin had no obvious modification effect.

[0215] Example 5: Optimization of reaction conditions

[0216] 1. Optimization of reaction time (standing time) of bromothymol blue sodium salt

[0217] The specific implementation method is the same as Example 1, except that the standing time (2h) of the bromothymol blue sodium salt solution in step (3) is adjusted to 10min, 50min, and 2.5h, respectively, to obtain three types of smart labels.

[0218] The results are as follows Figures 16 to 21 shown.

[0219] The results show:

[0220] (1) The smart tag left for 10 minutes has a small visible range under sunlight, especially after 0.5 days, the color is basically the same, the visibility is poor, and the change range is small under fluorescence, which cannot be accurately judged;

[0221] (2) The smart tag left for 50 minutes has a slightly smaller visible range under sunlight, and a slight color change from yellow to light yellow to light green to green, but it is not obvious enough; under fluorescent light, the visible range is smaller, and the color change is more chaotic, with no trend. In actual application, the resolution is poor and the visibility is not good;

[0222] (3) The visible range of the smart tag after standing for 2.5 hours under sunlight and fluorescence is not much different from that of the smart tag after standing for 1.5 hours in Example 1. It is speculated that bromothymol blue sodium salt has completely combined with gold nanoclusters (BSA@AuNCs), and the visibility is no longer improved, indicating good visibility.

[0223] 2. Optimization of the amount of bromothymol blue sodium salt added

[0224] The specific implementation method is the same as Example 1, except that the amount of bromothymol blue sodium salt solution added in step (3) is adjusted to 0.1 mL, 0.5 mL, and 2.0 mL, respectively, to obtain three types of smart labels.

[0225] The results are as follows Figures 22 to 27 shown.

[0226] The results showed that: (1) the smart label with an addition amount of 0.1 ml had a small visible range under sunlight and fluorescence, and the color change was not obvious from 0 to 2 days. The color change under fluorescence was more chaotic and repeated, and the visibility was poor;

[0227] (2) The visible range of the smart label with an added amount of 0.5 ml is small under sunlight, only 0-9.5, with a slight color change from light yellow to light green. After one day, the color change is not obvious. Under fluorescence, the visible range is small, only 0-25.6. After one day, it basically turns yellow, and the color change is more chaotic, with poor visibility.

[0228] (3) Under sunlight, the visible range of the smart label with an addition amount of 2 ml is 0-32.2, which is a large visible range. The color changes from yellow-dark green-green-dark green, and the visibility is acceptable. Under fluorescence, the visible range is 0-65.4, which is a large visible range. The color changes are obvious. It is speculated that bromothymol blue sodium salt has been completely combined with gold nanoclusters (BSA@AuNCs), and its color development characteristics will no longer show a large difference with the change of addition amount; the visibility is good.

[0229] 3. Optimization of reaction temperature (standing temperature) of bromothymol blue sodium salt

[0230] The specific implementation method is the same as Example 1, except that the standing temperature of the bromothymol blue sodium salt solution in step (3) is adjusted to 15° C., 35° C., and 45° C., respectively, to obtain three types of smart labels.

[0231] The results are as follows Figures 28 to 33 shown.

[0232] The results show:

[0233] (1) The smart tag at a static temperature of 15°C has a small visible range in sunlight, especially in 0-1 day, the color change range is very small, the color is messy, and the visibility is poor. Although there is a color change under fluorescence, the color change is more messy and the visibility is poor in actual application;

[0234] (2) The smart tag at a static temperature of 35°C has a wide visible range of 0-32.3 under sunlight, and the colors range from yellow-brown-yellow-brown-green-green; under fluorescence, the visible range is 0-54.8, and the colors range from red-yellow-dark green-green, and the visibility is good. This example is not much different from Example 1. It is speculated that this may be because the temperature is not high and is still more suitable for reaction and binding;

[0235] (3) The visibility range of the smart tag at a static temperature of 45°C under sunlight and fluorescence is 0-21.1 and 0-48.6, respectively. The color transition is relatively simple, especially in the early stage under sunlight and the late stage under fluorescence. The color transition is relatively simple and the visibility is poor. It is speculated that the BSA protein in the gold nanoclusters (BSA@AuNCs) is affected by the high temperature.

[0236] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for preparing a smart label, characterized in that: The preparation method comprises: (1) taking a bovine serum albumin solution, stirring it, adding a tetrachloroauric acid tetrahydrate aqueous solution, and then adding a sodium hydroxide solution, reacting to obtain a first-stage solution; (2) dialyzing the first-stage solution obtained in step (1), adding bromothymol blue sodium salt solution, and standing to obtain a second-stage solution; (3) dialyzing the second-stage solution obtained in step (2) to obtain a gold nanocluster solution modified with bromothymol blue sodium salt; stirring and mixing the gold nanocluster solution with a fluorescein isothiocyanate solution, immersing a microporous filter membrane therein, and then drying the microporous filter membrane to obtain a smart label.

2. The method according to claim 1, characterized in that The concentration of the bromothymol blue sodium salt solution is 5-10 mg / mL; the volume ratio of the first-stage solution to the bromothymol blue sodium salt solution in step (2) is (6-8):(1-1.2).

3. The method according to claim 1, characterized in that The standing condition in step (2) is 25-30° C. for 1-2 hours.

4. The method according to claim 1, characterized in that The molecular weight cutoff of the dialysis in step (2) is above 66 kDa, the dialysis time is 24 to 30 hours, and the water is changed every 8 to 10 hours; the molecular weight cutoff of the dialysis in step (3) is above 14 kDa, and the dialysis time is 20 to 24 hours.

5. The method according to claim 1, characterized in that: In step (3), the diameter of the microporous filter membrane is 13 to 15 mm and the pore size is 0.22 to 0.45 μm; preferably, the immersion time is 1 to 1.5 hours.

6. A smart label prepared by the method according to any one of claims 1 to 5.

7. A packaging material or a degradable material comprising the smart label according to claim 6.

8. Application of the smart label according to claim 6 in rapid detection of aquatic fish and shrimp; Preferably, the aquatic fish and shrimp include but are not limited to freshwater prawns.

9. The use according to claim 8, characterized in that: The application is to use the smart label of claim 6 as a detection matrix to determine the freshness of shrimps according to the color change of the label.

10. Use of the smart label according to claim 6 in the field of food or agricultural products or in the preparation of smart label products.

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