Near-infrared ratio fluorescence and colorimetric dual-channel intelligent label and preparation method and application thereof

Abstract

The invention discloses a preparation method of a near-infrared ratiometric fluorescence and colorimetric dual-channel intelligent label. The near-infrared ratiometric fluorescence and colorimetric dual-channel intelligent label is prepared by mixing NIR820 with near-infrared fluorescence and ND550 with yellow fluorescence and then dropwise adding the mixture to whatmanNo.1 test paper. Wherein the NIR820 is prepared by introducing 2-chloro-1-formyl-3-hydroxymethyl cyclohexene with chlorine atoms into indole iodized salt containing C = N +, and the ND550 is prepared by introducing 2, 4-dinitrobromobenzene into naphthalic anhydride to quench fluorescence of the naphthalic anhydride. The invention further discloses a near-infrared ratio fluorescence and colorimetric two-channel intelligent label and application thereof. The near-infrared ratio fluorescence and colorimetric dual-channel intelligent label can realize accurate and real-time visual monitoring of the freshness of agricultural and animal products such as fresh meat in the storage process through quantitative detection of H2S gas, and avoids complex and tedious operation steps, longer detection time and loss of expensive equipment and experimental consumables.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety, and more specifically, to a near-infrared ratiometric fluorescence and colorimetric dual-channel intelligent label and a preparation method and application thereof. Background Art

[0002] At present, fresh meat has become the most important category in my country's meat consumption market. However, in the logistics process, the high deterioration rate and the lack of real-time monitoring technology have not only caused hundreds of billions of yuan in storage and transportation losses, but also caused potential harm to human health. As an important line of defense to ensure safety, real-time monitoring of freshness can achieve early warning to reduce food safety issues.

[0003] Smart labels have become the most commonly used intelligent monitoring technology for the freshness of fresh meat due to their advantages of being cheap, miniaturized, fast-response, non-destructive, and real-time. The principle is that the gaseous markers produced during the change of freshness of fresh meat enter the smart label, causing the chemical structure of the indicator to change, thereby producing color or fluorescence changes. The indicator is the core of the smart label. In the prior art, most of them are indicator films based on pH and the detection of volatile biogenic amines in meat. For example, the patent with application number 202211424199.1 discloses a preparation method and application of a fluorescence ratio-type meat freshness indicator film, which synthesizes dual-emission carbon quantum dots by a one-step hydrothermal method, and then fixes the carbon quantum dots in a hydrophobic polymer film by an electrospinning method to prepare a portable indicator label with high stability; the indicator label responds to volatile biogenic amines, and can monitor the freshness of chilled meat. However, there are few literature reports on smart labels that respond to H2S in meat. Hydrogen sulfide (H2S) is a colorless, toxic gas with the smell of rotten eggs. At the same time, H2S is also considered to be a key gaseous compound that characterizes the freshness of meat. Therefore, finding an effective and responsive indicator tag to monitor H2S concentration is a feasible method to achieve real-time monitoring of meat freshness. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide a method for preparing a near-infrared ratiometric fluorescence and colorimetric dual-channel smart label, which constructs a ratiometric fluorescence indicator label in the near-infrared and ultraviolet light regions to realize changes in multiple fluorescence signals or colorimetric signals, effectively avoiding interference from excitation light sources, detectors or test conditions, and thereby realizing real-time visual monitoring of the freshness of fresh meat.

[0006] In order to achieve these purposes and other advantages according to the present invention, a method for preparing a near-infrared ratiometric fluorescence and colorimetric dual-channel smart label is provided, comprising the following steps: Step 1: 3-methyl-2-butanone and 3-hydrazinobenzoic acid are used as raw materials and a multi-step reaction is performed to prepare the compound NIR820 of formula (I); Step 2: 4-bromo-1,8-naphthalene anhydride and -alanine as a raw material to prepare the compound of formula (II) ND550 through multiple steps of reaction; Step 3: Mix NIR820 and ND550 to prepare a fluorescent probe mixture, and drop the fluorescent probe mixture onto a test paper to prepare a near-infrared ratiometric fluorescence and colorimetric dual-channel smart label; (I), (II).

[0007] Preferably, step one specifically includes: S11, 3-methyl-2-butanone and 3-hydrazinobenzoic acid are mixed and dissolved in a mixture of ethanol and concentrated sulfuric acid and refluxed to obtain an intermediate product of formula (III); S12, dissolving the intermediate product compound of formula (III) and methyl iodide in an organic solvent, stirring and refluxing, cooling and washing to obtain the intermediate product compound of formula (IV); S13, mixing the intermediate product compound of formula (IV) and 2-chloro-1-formyl-3-hydroxymethylcyclohexene in an organic solvent, reacting under alkali and nitrogen protection under reflux, and purifying to obtain the compound of formula (I) NIR820; (III) (IV).

[0008] Preferably, step 2 specifically includes: S21, 4-bromo-1,8-naphthalene anhydride and - Alanine is dissolved in a mixed solvent of dioxane and water, and heated under reflux to obtain an intermediate product of formula (V); S22, refluxing the intermediate product compound of formula (V) with potassium carbonate in ethanol to obtain the intermediate product compound of formula (VI); S23, heating the intermediate product compound of formula (VI) and hydroiodic acid to reflux to obtain the intermediate product compound of formula (VII), dissolving the intermediate product compound of formula (VII) in dioxane, adding 2,4-dinitrobromobenzene to reflux reaction under nitrogen environment, and purifying to obtain the compound of formula (II) ND550; (V) (VI) (VII).

[0009] Preferably, in step S11, 3-methyl-2-butanone and 3-hydrazinobenzoic acid are refluxed in ethanol for 12 to 24 hours; in step S12, the intermediate product of formula (III) compound and iodomethane are stirred and refluxed in a mixed solution of toluene and acetonitrile for 18 to 36 hours, and cooled and washed with a mixed solution of acetonitrile and n-hexane; in step S13, the intermediate product of formula (IV) compound and 2-chloro-1-formyl-3-hydroxymethylcyclohexene are refluxed in the presence of triethylamine for 3 to 8 hours, and purified by column chromatography using methanol and dichloromethane as eluents.

[0010] Preferably, in step S21, 4-bromo-1,8-naphthalene anhydride and -alanine is heated to reflux in dioxane and water for 10 to 16 h; in step S22, the intermediate product compound of formula (V) is deprotonated by potassium carbonate, and the reflux reaction time is 20 to 36 h; in step S23, the intermediate product compound of formula (VII) is refluxed with 2,4-dinitrobromobenzene under nitrogen environment for 5 to 10 h, and purification is performed by column chromatography using petroleum ether and ethyl acetate.

[0011] Preferably, in step 3, the test paper is whatman No. 1 test paper, and in the fluorescent probe mixture, the molar ratio of compound NIR820 to compound ND550 is 1 to 10:1.

[0012] The present invention further realizes this through near-infrared ratio fluorescence and colorimetric dual-channel smart labels. The present invention is further realized by the application of near-infrared ratio fluorescence and colorimetric dual-channel intelligent labels in the monitoring of the freshness of fresh agricultural and livestock products, wherein the fresh agricultural and livestock products include but are not limited to pork, beef, mutton, chicken, goose, duck, shrimps, and crabs.

[0013] Preferably, the application scenarios of the near-infrared ratio fluorescence and colorimetric dual-channel smart label include but are not limited to the following methods: affixed to the inner side of the fresh meat package without contacting the fresh meat.

[0014] The present invention has at least the following beneficial effects: 1. The preparation method of the near-infrared ratio fluorescence and colorimetric dual-channel smart label of the present invention first introduces 2-chloro-1-formyl-3-hydroxymethylcyclohexene with chlorine atoms into a C=N + Indole iodide salt was added to successfully prepare NIR820 with near-infrared fluorescence. Secondly, 2,4-dinitrobromobenzene was introduced into naphthalene dicarboxylic anhydride to quench its fluorescence to prepare ND550 with yellow fluorescence. Finally, NIR820 and ND550 were mixed and added dropwise to whatman No.1 test paper to prepare a smart label with dual fluorescence signals and colorimetric signals.

[0015] 2. The near-infrared ratiometric fluorescence and colorimetric dual-channel smart label of the present invention can realize ratiometric fluorescence and colorimetric dual-channel detection of meat freshness, and realize accurate and real-time visual monitoring of the freshness of fresh meat during storage, and has potential application prospects in the fields of meat freshness indication.

[0016] 3. The near-infrared ratio fluorescence and colorimetric dual-channel smart label of the present invention realizes quantitative detection of H2S gas through changes in ratio fluorescence and color difference values, and simple and fast fluorescence response, avoiding complicated and tedious operating steps, long detection time, and expensive equipment and experimental consumables.

[0017] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe NIR820 in Example 1 of the present invention; Figure 2 is the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe ND550 in Example 1 of the present invention; Figure 3 This is the time stability test result of the near-infrared ratio fluorescence and colorimetric dual-channel smart label prepared in Example 1 in Example 2 of the present invention; Figure 4 This is a near-infrared ratio fluorescence and colorimetric linear regularity diagram of the actual detection of H2S gas by the near-infrared ratio fluorescence and colorimetric dual-channel smart label prepared in Example 1 in Example 3 of the present invention; Figure 5 This is a near-infrared ratio fluorescence and colorimetric regularity diagram of the near-infrared ratio fluorescence and colorimetric dual-channel smart label prepared in Example 1 in Example 4 of the present invention for actually detecting the freshness of meat; Figure 6 In Example 4 of the present invention, the near-infrared ratio fluorescence and colorimetric dual-channel smart label prepared in Example 1 is used to evaluate the monitoring effect of meat freshness; Figure 7 The figure is a result diagram of the cytotoxicity experiment of two fluorescent probes NIR820 and ND550 tested in Example 5 of the present invention; Figure 8 The graphs are comparison results of the fluorescence intensity changes at emission wavelengths of 550 nm (a) and 820 nm (b) before and after the single fluorescent probe and the mixed ratio fluorescent probe react with H2S in Example 6 of the present invention; Fig. 9 The label color signal is obtained by linear regression in Example 7 of the present invention. E value, ratio fluorescence signal I 550 / I 820 The value is fitted with the freshness index TVB-N to establish a quantitative prediction model for freshness. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0020] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0021] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0022] In recent years, fluorescent probes have attracted extensive attention due to their high sensitivity and rapid response. However, the emission wavelengths of existing H2S-responsive fluorescent probes are mostly in the visible-ultraviolet region, which is similar to the emission wavelengths of fluorescent substances such as porphyrin and tyrosine in meat. They are easily interfered with and difficult to accurately detect the freshness. Although near-infrared fluorescent probes have longer emission wavelengths, they are difficult to achieve visual monitoring. Therefore, constructing a ratiometric fluorescent indicator label in the near-infrared and ultraviolet regions can realize the changes of multiple fluorescent signals or colorimetric signals, effectively avoiding interference from excitation light sources, detectors or test conditions, and thus achieving real-time visual monitoring of the freshness of fresh meat.

[0023] Example 1 A method for preparing a near-infrared ratiometric fluorescence and colorimetric dual-channel smart label, the specific synthesis route is as follows: The specific synthesis steps are as follows: Step 1: Mix 3-methyl-2-butanone (36 mmol) and 3-hydrazinobenzoic acid (1a, 33.0 mmol), heat and reflux in 120 mL of ethanol and 1 mL of concentrated sulfuric acid (98%) for 18 h to prepare compound 1b with a yield of 80.25%. 1 H NMR (400MHz, DMSO-d6) δ 8.38 (s, 1H), 8.19 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 3.99 (s, 3H), 2.80 (s, 3H), 1.56 (s, 6H); Step 2: Dissolve compound 1b (20.0 mmol) and iodomethane (21.0 mmol) in 100 mL of toluene-acetonitrile (v / v, 2:1), stir and reflux for 24 h, and cool and wash three times with 30 mL of a mixed solvent of acetonitrile and n-hexane (v / v, 1:1) to obtain compound 1c with a yield of 64.33%. 1 H NMR (400 MHz, DMSO-d6) δ 8.37 (s, 1H), 8.19 (d, J=8.4 Hz, 1H), 8.01 (d, J=8.4 Hz, 1H), 3.99 (s, 3H), 2.80 (s, 3H), 1.56 (s, 6H); Step 3: Compound 1c (8.0 mmol) and 2-chloro-1-formyl-3-hydroxymethylcyclohexene (1d, 4.0 mmol) were dissolved and mixed in 20.0 mL of triethylamine, n-butanol and toluene (v / v, 1:20:20) solution, and refluxed under nitrogen protection for 5 h. Column chromatography was performed using methanol and dichloromethane in a volume ratio of 1:5 to prepare the near-infrared fluorescent probe NIR820 with a yield of 36.56%. 1 H NMR (400 MHz, ) δ 12.43 (s, 2H), 8.29 (d, J = 14.2 Hz,2H), 8.15(s, 2H), 8.03 (d, J = 8.3 Hz, 2H), 7.52 (s, 2H), 6.39 (d, J = 14.2 Hz, 2H),2.73 (t, J = 7.0 Hz, 6H), 1.89 (d, J = 16.6 Hz, 3H), 1.71 (s, 12H); Compound 1d is a commercial product and was purchased directly for use; Step 4: 4-bromo-1, 8-naphthoic anhydride (2a, 18.0 mmol) and -Alanine (18.0 mmol) was dissolved in 150.0 mL of a mixed solution of dioxane and water (v / v, 8:2) and heated under reflux for 14 h to obtain compound 2b with a yield of 82.34%. 1 HNMR (400 MHz, DMSO-d6) δ 8.60 – 8.52 (m, 2H), 8.33 (d, J = 7.9 Hz, 1H), 8.22(d, J = 7.9Hz, 1H), 8.00 (dd, J = 8.5, 7.3 Hz, 1H), 4.29 – 4.20 (m, 2H), 2.63– 2.54 (m, 2H); Subsequently, compound 2b (14.0 mmol) and K2CO3 (114.0 mmol) were refluxed in 200.0 mL of ethanol for 24 h to prepare compound 2c with a yield of 85.57%. 1 H NMR (400 MHz, DMSO-d6) δ 12.35 (s, 1H), 8.57 – 8.43 (m, 3H), 7.82 (dd, J = 8.4, 7.4 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 4.29 –4.20 (m, 2H), 4.13 (s, 3H), 2.62 – 2.54 (m, 2H); Step 5: 6.7 mmol 2c was heated to reflux in 50 mL hydroiodic acid (57 wt%) for 12 h to obtain compound 2d with a yield of 78.67%. 1 H NMR (400 MHz, DMSO-d6) δ 8.52 – 8.45 (m, 1H), 8.37 (d, J= 7.3 Hz,1H), 8.18 (d, J = 8.6 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 6.75 (d, J = 8.6 Hz,1H), 4.27 – 4.19 (m, 2H), 2.55 (d, J = 7.9 Hz, 2H); Subsequently, compound 2d (3.5 mmol) and 2,4-dinitrobromobenzene (7.0 mmol) were dissolved in 50.0 mL of dioxane, and KHCO3 (70.0 mmol) was dissolved in 50.0 mL of water. The two were mixed and refluxed under nitrogen for 8 h. The fluorescent probe ND550 was prepared by column chromatography using petroleum ether and ethyl acetate in a volume ratio of 5:1 with a yield of 36.66%. 1 H NMR (400 MHz, DMSO-d6) δ 8.59 (d, J = 3.2 Hz, 1H), 8.34 (d, J = 7.8 Hz, 1H), 8.18 (d, J = 7.5 Hz, 1H), 7.92 (d, J = 8.9 Hz, 1H), 7.81 – 7.65 (m, 3H), 7.24 (d,J = 7.5 Hz, 1H), 4.22 (s, 2H), 2.52 (s, 2H).

[0024] Step 6: Mix the two fluorescent probes NIR820 and ND550 in a molar ratio of 5:1 to obtain a mixed ratio fluorescent probe, so that the final concentration of ND550 is 5 M, NIR820 is 25 M, as the working solution, take 10 The near-infrared ratiometric fluorescent smart label was prepared by adding L working solution to a 1.5 cm × 1.5 cm whatman No.1 test paper.

[0025] Example 2 The temporal stability of the near-infrared ratiometric fluorescence and colorimetric dual-channel smart label prepared in Example 1.

[0026] The temporal changes of the fluorescence signal of the smart tag were recorded by a fluorescence spectrophotometer (excitation wavelength: 450 nm, slit width: 5 nm) to determine the temporal stability of the material, such as Figure 3 shown.

[0027] from Figure 3 It can be seen that the near-infrared ratio fluorescence and colorimetric dual-channel smart label can be stored stably. When stored for 7 days, the fluorescence ratio still has no obvious change. Therefore, it can be inferred that the near-infrared ratio fluorescence and colorimetric dual-channel smart label has high stability.

[0028] Example 3 The near-infrared ratiometric fluorescence and colorimetric dual-channel smart label prepared in Example 1 is applied to quantitative detection of H2S, specifically comprising the following steps: Step 1: Add 10 mM NaHS solution into the sample cell and calculate the H2S gas concentration according to the formula; Step 2: Attach the smart label prepared in Example 1 to the top space of the sample pool, add different volumes of the above H2S solution, and then use distilled water to ensure that the solution volume is 2 mL, and an aqueous solution with a gradient change in H2S concentration can be obtained. Then use a fluorescence spectrophotometer (F380, Tianjin Gangdong Technology), a colorimeter (CM-600d, Konica Minolta, Japan) and an ultraviolet analyzer (WFH-203C, Shanghai Chitang Industry) to perform fluorescence and color detection on the smart label, and finally obtain the linear law of H2S concentration and fluorescence label fluorescence ratio and color difference change. Figure 4 shown.

[0029] Figure 4 In a, the horizontal axis represents the H2S gas concentration, and the vertical axis represents the dual fluorescence ratio of the smart tag. Figure 4 As can be seen in a, as the H2S gas concentration increases, the label fluorescence ratio increases, and the two show a good linear relationship.

[0030] Figure 4 In b, the horizontal axis represents the H2S gas concentration, and the vertical axis represents the color difference value of the smart label. Figure 4As can be seen in b, as the H2S gas concentration increases, the label color difference value increases, and the two show a good linear law.

[0031] The detection principle can be explained as follows. On the one hand, H2S nucleophilically attacks the C=N + Forming -SH, breaking the NIR820 molecular structure The conjugated system activates the PET process, causing its fluorescence to be quenched. On the other hand, H2S nucleophilically attacks the COC bond of ND550, causing 2,4-dinitrophenyl ether to leave the molecular structure, exposing -OH, activating the ICT process and enhancing fluorescence.

[0032] Example 4 The near-infrared ratiometric fluorescence and colorimetric dual-channel smart label prepared in Example 1 is applied to real-time monitoring of the freshness of fresh meat, specifically comprising the following steps: The fresh meat was placed in a packaging box, and the smart label prepared in Example 1 was attached to the inner side of the upper cover of the packaging. The freshness change of the fresh meat during storage was monitored according to the fluorescence and color change of the indicator label. The monitoring results were as follows: Figure 5 shown.

[0033] Figure 5 The horizontal axis in a represents the storage time of fresh meat, and the vertical axis represents the dual fluorescence ratio of the smart label. Figure 5 As can be seen in figure a, as the freshness of the fresh meat decreases, the label fluorescence ratio gradually increases.

[0034] Figure 5 In b, the horizontal axis represents the storage time of fresh meat, and the vertical axis represents the color difference value of the smart label. Figure 5 As can be seen in figure b, as the freshness of the fresh meat decreases, the color difference value of the label increases.

[0035] Actual fresh meat monitoring sample photos are as follows Figure 6 As shown, from Figure 6 It can be seen that as the freshness decreases, the fluorescence and color of the smart label change significantly. Within 0-16 hours of storage, the label changes from blue-green to gray-green, and yellow fluorescence appears. The H2S concentration is <120 ppb, and the fresh meat has not yet been corrupted; after 16-32 hours of storage, the label changes from gray-green to gray-white, the yellow fluorescence increases, and the H2S concentration is 120 ppb ~ 600 ppb, and the fresh meat has reached a corrupt state. Therefore, the freshness of fresh meat can be judged by the fluorescence and color changes of the smart label of the present invention. Whether it can be seen by the naked eye indicates that there is a basis for application.

[0036] Example 5 Cytotoxicity experiments of two fluorescent probes: Take cells (HepG2) in the logarithmic growth phase and in good growth condition, and use 4×10 3 Each well was inoculated into a 96-well cell culture plate and cultured in a 37°C, 5% CO2 incubator. After the cells adhered to the wall and grew for 24 h, the culture medium of each well was discarded and the cells were rinsed twice with PBS. Different concentrations of ND550 and NIR820 fluorescent probes were added to each well. L, three parallel wells were set for each concentration; after incubation for 24 h, 10 L MTT solution, continue incubation in the cell culture incubator for 4 h; add 100 L Commercial Formazan solution, continue to incubate in the cell culture incubator until the Formazan is completely dissolved under an optical microscope, and use a microplate reader to detect the absorbance value at 570 nm. Calculate according to the following formula: Cell survival rate (%) = Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%. As: absorbance of the experimental well (containing cells, culture medium, MTT solution and fluorescent probe solution); Ac: absorbance of the control well (containing cells, culture medium, MTT solution, but not containing fluorescent probe); Ab: absorbance of the white well (containing culture medium, MTT solution, but not containing cells or fluorescent probe).

[0037] The experimental results are as follows Figure 7 As shown, Figure 7 The figure a shows the cytotoxicity results of the probe ND550, and the figure b shows the cytotoxicity results of the probe NIR820. Figure 7 It can be concluded that the concentration conditions used in the present invention (5 M ND550, 25 M NIR820), the cell survival rate was greater than 80%, with low toxicity, and can be used for subsequent freshness monitoring of fresh meat.

[0038] Example 6 Test of the response ability of single fluorescent probe and mixed ratio fluorescent probe to H2S: The fluorescence of two single fluorescent probes (NIR820 and ND550) and the mixed ratio fluorescent probe (the mixed ratio fluorescent probe prepared in Example 1) with 10 The fluorescence intensity change of M before and after the H2S reaction for 20 min was measured to determine the response performance of the mixed ratio fluorescence probe.

[0039] The experimental results are as follows Figure 8 As shown, Figure 8In a, the ordinate of the bar graph represents the fluorescence intensity change comparison diagram at the emission wavelength of 550 nm, and in b, the ordinate of the bar graph represents the fluorescence intensity change comparison diagram at the emission wavelength of 820 nm. The ordinates of the line graphs in a and b both represent the fluorescence change ratio before and after the reaction with H2S. Figure 8 It can be concluded that the fluorescence change ratio of the mixed ratio fluorescent probe is more significant than that of the two single fluorescent probes. Therefore, the mixed ratio fluorescent probe has better H2S response ability.

[0040] Example 7 Label dual-channel signals and the construction of a quantitative prediction model for the freshness of fresh meat: The label color signal is transformed into E value, ratio fluorescence signal I 550 / I 820 The value was fitted with the freshness index TVB-N to establish a quantitative prediction model for freshness.

[0041] The results are as follows Fig. 9 As shown, Fig. 9 The horizontal axis of Figure a represents the color signal E value, the horizontal axis of Figure b represents the ratio fluorescence signal I 550 / I 820 The vertical axes in a and b represent the TVB-N value of freshness index. Fig. 9 It can be concluded that the color signal of the label E value, ratio fluorescence signal I 550 / I 820 The values ​​have a good linear relationship with the TVB-N value of the freshness index (R 2 >0.9), and when TVB-N exceeds 15 mg / 100 g, E>6, I 550 / I 820 >1.11, which shows a significant change. Therefore, the chemometric model can be used to realize quantitative monitoring of freshness using smart tags, which has great application value.

[0042] The smart label changes from blue-green to gray-green and shows strong yellow fluorescence, indicating that the meat changes from fresh to spoiled. Using chemometrics to build a quantitative prediction model for freshness, the smart label color, ratio fluorescence dual-channel signal and freshness index TVB-N value have a good linear relationship (R 2 >0.9), the models are y=2.5445x-1.1876 and y=9.2715x+3.5404 respectively. The application of multi-channel signal output mechanism realizes the cross-validation of monitoring results. This strategy ensures the accuracy of the detection results and effectively eliminates the potential background fluorescence interference. Through these measures, the high reliability of smart labels in practical applications is improved.

[0043] The present invention prepares a near-infrared ratio fluorescence and colorimetric dual-channel smart label by a physical mixing method, and obtains dual-fluorescence materials with different fluorescence ratios by adjusting the probe ratio, excitation wavelength, etc. The method of the present invention overcomes the shortcomings of the previous detection being susceptible to interference and having a long detection time by using a multi-channel signal change method.

[0044] The number of devices and processing scales described here are used to simplify the description of the present invention. Modifications and changes to the near-infrared ratio fluorescence and colorimetric dual-channel smart label and preparation method and application of the present invention are obvious to those skilled in the art.

[0045] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for preparing a near-infrared ratiometric fluorescence and colorimetric dual-channel smart label, characterized in that: The following steps are involved: Step 1: 3-methyl-2-butanone and 3-hydrazinobenzoic acid are used as raw materials and a multi-step reaction is performed to prepare the compound NIR820 of formula (I); Step 2: 4-bromo-1,8-naphthalene anhydride and -alanine as a raw material to prepare the compound of formula (II) ND550 through multiple steps of reaction; Step 3: Mix NIR820 and ND550 to prepare a fluorescent probe mixture, and drop the fluorescent probe mixture onto a test paper to prepare a near-infrared ratiometric fluorescence and colorimetric dual-channel smart label; （I）， (II).

2. The preparation method according to claim 1, characterized in that Step 1 specifically includes: S11, 3-methyl-2-butanone and 3-hydrazinobenzoic acid are mixed and dissolved in a mixture of ethanol and concentrated sulfuric acid and refluxed to obtain an intermediate product of formula (III); S12, dissolving the intermediate product compound of formula (III) and methyl iodide in an organic solvent, stirring and refluxing, cooling and washing to obtain the intermediate product compound of formula (IV); S13, mixing the intermediate product compound of formula (IV) and 2-chloro-1-formyl-3-hydroxymethylcyclohexene in an organic solvent, reacting under alkali and nitrogen protection under reflux, and purifying to obtain the compound of formula (I) NIR820; (III) (IV).

3. The preparation method according to claim 1, characterized in that: Step 2 specifically includes: S21, 4-bromo-1,8-naphthalene anhydride and - Alanine is dissolved in a mixed solvent of dioxane and water, and heated under reflux to obtain an intermediate product of formula (V); S22, refluxing the intermediate product compound of formula (V) with potassium carbonate in ethanol to obtain the intermediate product compound of formula (VI); S23, heating the intermediate product compound of formula (VI) and hydroiodic acid to reflux to obtain the intermediate product compound of formula (VII), dissolving the intermediate product compound of formula (VII) in dioxane, adding 2,4-dinitrobromobenzene to reflux reaction under nitrogen environment, and purifying to obtain the compound of formula (II) ND550; (V) (VI) (VII).

4. The preparation method according to claim 2, characterized in that: In step S11, 3-methyl-2-butanone and 3-hydrazinobenzoic acid are refluxed in ethanol for 12 to 24 hours; in step S12, the intermediate product of formula (III) and methyl iodide are stirred and refluxed in a mixed solution of toluene and acetonitrile for 18 to 36 hours, and cooled and washed with a mixed solution of acetonitrile and n-hexane; in step S13, the intermediate product of formula (IV) and 2-chloro-1-formyl-3-hydroxymethylcyclohexene are refluxed in the presence of triethylamine for 3 to 8 hours, and purified by column chromatography using methanol and dichloromethane as eluents.

5. The preparation method according to claim 3, characterized in that: In step S21, 4-bromo-1,8-naphthalene anhydride and -alanine is heated to reflux in dioxane and water for 10 to 16 h; in step S22, the intermediate product compound of formula (V) is deprotonated by potassium carbonate, and the reflux reaction time is 20 to 36 h; in step S23, the intermediate product compound of formula (VII) is refluxed with 2,4-dinitrobromobenzene under nitrogen environment for 5 to 10 h, and purification is performed by column chromatography using petroleum ether and ethyl acetate.

6. The preparation method according to claim 1, characterized in that: In step 3, the test paper is whatman No. 1 test paper, and in the fluorescent probe mixture, the molar ratio of compound NIR820 to compound ND550 is 1 to 10:

1.

7. A near-infrared ratiometric fluorescence and colorimetric dual-channel smart label prepared by the preparation method according to any one of claims 1 to 6.

8. Application of the near-infrared ratio fluorescence and colorimetric dual-channel smart label in monitoring the freshness of fresh agricultural and livestock products as claimed in claim 7, wherein: Fresh agricultural and livestock products include pork, beef, mutton, chicken, goose, duck, shrimp or crab.

9. The use as claimed in claim 8, wherein the near-infrared ratio fluorescence and colorimetric dual-channel smart label is attached to the inside of the fresh agricultural and livestock product packaging without contacting the fresh agricultural and livestock products.

Images