Near-infrared ratiometric fluorescence and colorimetric dual-channel smart label and its preparation method and application

Abstract

The present invention discloses a method for preparing a near-infrared ratiometric fluorescence and colorimetric dual-channel smart label, which is prepared by mixing NIR820 with near-infrared fluorescence and ND550 with yellow fluorescence and then adding the mixture to a Whatman No. 1 test paper. NIR820 is prepared by introducing 2-chloro-1-formyl-3-hydroxymethylcyclohexene with chlorine atoms into a C=N + ND550 is prepared from indole iodide, and ND550 is prepared by introducing 2,4-dinitrobromobenzene into naphthalic anhydride to quench its fluorescence. The present invention also discloses a near-infrared ratiometric fluorescence and colorimetric dual-channel smart label and its application. The near-infrared ratiometric fluorescence and colorimetric dual-channel smart label of the present invention can achieve accurate, real-time visual monitoring of the freshness of agricultural and livestock products such as fresh meat during storage by quantitatively detecting H2S gas, avoiding complex and tedious operating steps, long detection time, and expensive equipment and experimental consumables.

Description

Technical Field

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

[0002] Fresh meat has become the most important category in my country's meat consumption market. However, high rates of deterioration and a lack of real-time monitoring technology during logistics not only result in hundreds of billions of yuan in storage and transportation losses but also pose potential risks to human health. As a crucial line of defense for ensuring safety, real-time freshness monitoring can provide early warnings and mitigate food safety issues.

[0003] Smart tags are currently the most commonly used intelligent freshness monitoring technology for fresh meat due to their low cost, miniaturization, fast response, non-destructive nature, and real-time performance. Their principle is that gaseous markers generated during the freshness process enter the smart tag, causing chemical changes in the indicator, resulting in color or fluorescence changes. The indicator is the core of smart tags. Existing technologies mostly use pH-sensing and indicator films that detect volatile biogenic amines in meat. For example, patent application number 202211424199.1 discloses a method for preparing and applying a fluorescence ratiometric meat freshness indicator film. Dual-emission carbon quantum dots are synthesized via a one-step hydrothermal method and then electrospinned into a hydrophobic polymer film to create a highly stable portable indicator tag. This tag responds to volatile biogenic amines, enabling monitoring of the freshness of chilled meat. However, there are few reports on smart tags that respond to H2S in meat. Hydrogen sulfide (H2S) is a colorless, toxic gas with the odor of rotten eggs. At the same time, H2S is also considered a key gaseous compound to characterize 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. The ratiometric fluorescence indicator label constructed in the near-infrared and ultraviolet light regions can realize the changes of multiple fluorescence signals or colorimetric signals, effectively avoiding interference from the excitation light source, detector or test conditions, and thus 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:

[0007] 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 of formula (I) NIR820;

[0008] Step 2: 4-bromo-1,8-naphthalene anhydride and -alanine as a raw material to prepare the compound of formula (II) ND550 through multi-step reaction;

[0009] 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;

[0010]

[0011] (I),

[0012]

[0013] (II).

[0014] Preferably, step 1 specifically includes:

[0015] 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);

[0016] 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);

[0017] 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;

[0018]

[0019] (III) (IV).

[0020] Preferably, step 2 specifically includes:

[0021] 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 the intermediate product compound of formula (V);

[0022] S22, refluxing the intermediate product compound of formula (V) with potassium carbonate in ethanol to obtain the intermediate product compound of formula (VI);

[0023] 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, reacting under reflux under nitrogen environment, and purifying to obtain the compound of formula (II) ND550;

[0024]

[0025] (V) (VI) (VII).

[0026] 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) and methyl iodide are stirred and refluxed in a mixture of toluene and acetonitrile for 18 to 36 hours, and then cooled and washed with a mixture 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.

[0027] Preferably, in step S21, 4-bromo-1,8-naphthalene anhydride and -alanine is heated under reflux in dioxane and water for 10 to 16 hours; in step S22, the intermediate product compound of formula (V) is deprotonated by potassium carbonate, and the reflux reaction time is 20 to 36 hours; in step S23, the intermediate product compound of formula (VII) is refluxed with 2,4-dinitrobromobenzene under nitrogen for 5 to 10 hours, and purification is performed by column chromatography using petroleum ether and ethyl acetate.

[0028] 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.

[0029] The present invention further realizes this through near-infrared ratio fluorescence and colorimetric dual-channel smart labels.

[0030] The present invention is further realized through the application of near-infrared ratiometric fluorescence and colorimetric dual-channel smart labels in monitoring the freshness of fresh agricultural and livestock products, wherein fresh agricultural and livestock products include but are not limited to pork, beef, mutton, chicken, goose, duck, shrimp, and crabs.

[0031] 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.

[0032] The present invention has at least the following beneficial effects:

[0033] 1. The preparation method of the near-infrared ratio fluorescence and colorimetric dual-channel smart label of the present invention is to first introduce 2-chloro-1-formyl-3-hydroxymethylcyclohexene with chlorine atoms into a C=N + The researchers successfully prepared NIR820 with near-infrared fluorescence by adding indole iodide. Secondly, 2,4-dinitrobromobenzene was introduced into naphthalene dicarboxylic anhydride to quench its fluorescence, thereby preparing 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.

[0034] 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, real-time visual monitoring of the freshness of fresh meat during storage, and has potential application prospects in the fields of meat freshness indication.

[0035] 3. The near-infrared ratiometric fluorescence and colorimetric dual-channel smart label of the present invention realizes quantitative detection of H2S gas through changes in ratiometric fluorescence and color difference values, and a simple and fast fluorescence response, avoiding complex and tedious operating steps, long detection time, and expensive equipment and experimental consumables.

[0036] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe NIR820 in Example 1 of the present invention;

[0038] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe ND550 in Example 1 of the present invention;

[0039] Figure 3 This is the time stability test result of the near-infrared ratiometric fluorescence and colorimetric dual-channel smart label prepared in Example 1 in Example 2 of the present invention;

[0040] Figure 4 This is a graph showing the near-infrared ratiometric fluorescence and colorimetric linear regularity of H2S gas actually detected by the near-infrared ratiometric fluorescence and colorimetric dual-channel smart tag prepared in Example 1 in Example 3 of the present invention;

[0041] Figure 5This is a diagram showing the near-infrared ratiometric fluorescence and colorimetric regularity of meat freshness actually detected using the near-infrared ratiometric fluorescence and colorimetric dual-channel smart label prepared in Example 1 in Example 4 of the present invention;

[0042] Figure 6 In Example 4 of the present invention, the near-infrared ratiometric fluorescence and colorimetric dual-channel smart label prepared in Example 1 was used to evaluate the monitoring effect of meat freshness;

[0043] Figure 7 Graph showing the cytotoxicity test results of two fluorescent probes, NIR820 and ND550, tested in Example 5 of the present invention;

[0044] Figure 8 Graphs comparing the fluorescence intensity changes at emission wavelengths of 550 nm (a) and 820 nm (b) for a single fluorescent probe and a mixed ratiometric fluorescent probe in Example 6 of the present invention before and after reaction with H2S;

[0045] Figure 9 The label color signal is analyzed 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

[0046] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0047] It should be understood that terms such as “having”, “including” and “comprising” used herein do not preclude the existence or addition of one or more other elements or combinations thereof.

[0048] 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.

[0049] In recent years, fluorescent probes have attracted widespread attention due to their high sensitivity and rapid response. However, existing H2S-responsive fluorescent probes mostly emit in the visible-ultraviolet region, which is close to the emission wavelengths of fluorescent substances such as porphyrin and tyrosine in meat. This makes them susceptible to interference and hinders accurate freshness detection. While near-infrared fluorescent probes have longer emission wavelengths, they are difficult to visualize. Therefore, the construction of ratiometric fluorescent indicator tags in the near-infrared and ultraviolet regions can detect changes in multiple fluorescent or colorimetric signals, effectively avoiding interference from the excitation light source, detector, or test conditions, thereby enabling real-time visual monitoring of the freshness of fresh meat.

[0050] Example 1

[0051] A method for preparing a near-infrared ratiometric fluorescence and colorimetric dual-channel smart label, the specific synthesis route is as follows:

[0052]

[0053]

[0054] The specific synthesis steps are as follows:

[0055] Step 1: 3-Methyl-2-butanone (36 mmol) and 3-hydrazinobenzoic acid (1a, 33.0 mmol) were mixed and heated under 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);

[0056] Step 2: Compound 1b (20.0 mmol) and iodomethane (21.0 mmol) were dissolved in 100 mL of toluene-acetonitrile (v / v, 2:1), stirred and refluxed for 24 h, and cooled and washed 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);

[0057] 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 a solution of triethylamine, n-butanol, and toluene (v / v, 1:20:20). The mixture was refluxed under nitrogen for 5 h. The near-infrared fluorescent probe NIR820 was prepared by column chromatography using methanol and dichloromethane in a volume ratio of 1:5 with a yield of 36.56%. 1H 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;

[0058] Step 4: 4-bromo-1, 8-naphthalene anhydride (2a, 18.0 mmol) and 2-Alanine (18.0 mmol) was dissolved in 150.0 mL of a mixture 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);

[0059] 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);

[0060] Step 5: Compound 2d was obtained by heating 6.7 mmol of 2c in 50 mL of hydroiodic acid (57 wt%) under reflux for 12 h with a yield of 78.67%. 1H 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);

[0061] 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).

[0062] 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 dropwise to a 1.5 cm × 1.5 cm Whatman No.1 test paper.

[0063] Example 2

[0064] Temporal stability of the near-infrared ratiometric fluorescence and colorimetric dual-channel smart label prepared in Example 1.

[0065] The temporal stability of the material was determined by recording the temporal variation of the fluorescence signal of the smart tag using a fluorescence spectrophotometer (excitation wavelength: 450 nm, slit width: 5 nm). Figure 3 shown.

[0066] from Figure 3 It can be seen that the near-infrared ratio fluorescence and colorimetric dual-channel smart label can be stored stably. After storage for 7 days, the fluorescence ratio still does not change significantly. Therefore, it can be inferred that the near-infrared ratio fluorescence and colorimetric dual-channel smart label has high stability.

[0067] Example 3

[0068] The near-infrared ratiometric fluorescence and colorimetric dual-channel smart label prepared in Example 1 is applied to the quantitative detection of H2S, specifically comprising the following steps:

[0069] Step 1: Add 10 mM NaHS solution to the sample cell and calculate the H2S gas concentration according to the formula;

[0070] Step 2: The smart label prepared in Example 1 was attached to the headspace of the sample cell, and different volumes of the above-mentioned H2S solution were added. Subsequently, distilled water was added to ensure that the solution volume was 2 mL, and an aqueous solution with a gradient of H2S concentration was obtained. The fluorescence and color of the smart label were then detected using a fluorescence spectrophotometer (F380, Tianjin Gangdong Technology), a colorimeter (CM-600d, Konica Minolta, Japan), and a UV analyzer (WFH-203C, Shanghai Chitang Industrial). Finally, a linear law of the change in H2S concentration, fluorescence ratio, and color difference of the fluorescent label was obtained. Figure 4 shown.

[0071] Figure 4 The horizontal axis in a 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 Figure a, as the H2S gas concentration increases, the label fluorescence ratio increases, and the two show a good linear law.

[0072] Figure 4 The horizontal axis in b represents the H2S gas concentration, and the vertical axis represents the color difference value of the smart label. Figure 4 As can be seen in Figure b, as the H2S gas concentration increases, the label color difference value increases, and the two show a good linear law.

[0073] 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 the -OH group, activating the ICT process and enhancing fluorescence.

[0074] Example 4

[0075] The near-infrared ratiometric fluorescence and colorimetric dual-channel smart label prepared in Example 1 is applied to the real-time monitoring of the freshness of fresh meat, specifically comprising the following steps:

[0076] Fresh meat was placed in a packaging box, and the smart label prepared in Example 1 was attached to the inner side of the packaging cover. The freshness of the fresh meat during storage was monitored based on the fluorescence and color changes of the indicator label. The monitoring results were as follows: Figure 5 shown.

[0077] 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 tag. Figure 5 As can be seen in Figure a, as the freshness of the fresh meat decreases, the label fluorescence ratio gradually increases.

[0078] Figure 5 The horizontal axis in b 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 fresh meat decreases, the color difference value of the label increases.

[0079] Actual fresh meat monitoring sample photos Figure 6 As shown, from Figure 6 As can be seen in the figure, the fluorescence and color of the smart label change significantly as freshness decreases. Within 0-16 hours of storage, the label changes from bluish-green to gray-green and exhibits yellow fluorescence. The H2S concentration is <120 ppb, indicating that the meat has not yet spoiled. After 16-32 hours of storage, the label changes from gray-green to off-white, and the yellow fluorescence increases. The H2S concentration is between 120 ppb and 600 ppb, indicating that the meat has reached a state of spoilage. Therefore, the fluorescence and color changes of the smart label of this invention can be used to determine the freshness of fresh meat. Whether it can be seen with the naked eye indicates its applicability.

[0080] Example 5

[0081] Cytotoxicity experiments of two fluorescent probes:

[0082] Take cells (HepG2) in the logarithmic growth phase and in good growth condition, and use 4×10 3 Cells were seeded into 96-well plates and cultured in a 37°C, 5% CO2 incubator. After the cells had grown for 24 h, the culture medium in each well was discarded and the cells were rinsed twice with PBS. 100 μl of the prepared 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 to incubate in the cell culture incubator for 4 hours; add 100 Continue incubating in a cell culture incubator with commercial formazan solution until the formazan is completely dissolved under a light microscope. Measure the absorbance at 570 nm using a microplate reader. Calculate cell viability (%) using the following formula: Cell viability = [(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 no fluorescent probe); Ab: absorbance of the white well (containing culture medium and MTT solution, but no cells or fluorescent probe).

[0083] The experimental results are as follows Figure 7 As shown, Figure 7 The figure a is the cytotoxicity result of probe ND550, and the figure b is the cytotoxicity result of 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.

[0084] Example 6

[0085] Test of the response ability of single fluorescent probe and mixed ratio fluorescent probe to H2S:

[0086] 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) was recorded by fluorescence spectrophotometer (excitation wavelength: 450 nm, slit width: 5 nm). The fluorescence intensity change of M before and after the H2S reaction for 20 min was analyzed to determine the response performance of the mixed ratio fluorescence probe.

[0087] The experimental results are as follows Figure 8 As shown, Figure 8 In a, the vertical axis of the bar graph represents the fluorescence intensity change comparison diagram at the emission wavelength of 550 nm, and in b, the vertical axis of the bar graph represents the fluorescence intensity change comparison diagram at the emission wavelength of 820 nm. The vertical axis of the broken 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.

[0088] Example 7

[0089] Construction of a quantitative prediction model for label dual-channel signals and fresh meat freshness:

[0090] 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.

[0091] The results are as follows Figure 9 As shown, Figure 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. Figure 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 freshness index TVB-N value (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.

[0092] The smart label changes from blue-green to gray-green and shows strong yellow fluorescence, indicating that the meat is changing 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 indicator TVB-N value all have a good linear relationship (R 2 >0.9), the models were y=2.5445x-1.1876 and y=9.2715x+3.5404, respectively. The use of a multi-channel signal output mechanism enables cross-validation of monitoring results. This strategy ensures the accuracy of detection results and effectively eliminates potential background fluorescence interference. These measures enhance the reliability of smart tags in practical applications.

[0093] This invention uses a physical mixing method to prepare a near-infrared ratiometric fluorescence and colorimetric dual-channel smart label. By adjusting the probe ratio and excitation wavelength, the dual-fluorescent material with different fluorescence ratios can be obtained. By using multi-channel signal changes, the present method overcomes the shortcomings of previous detection methods, such as susceptibility to interference and long detection time.

[0094] The number of devices and processing scales described here are used to simplify the description of the present invention. Modifications and variations of the near-infrared ratiometric fluorescence and colorimetric dual-channel smart label, preparation method, and application of the present invention will be apparent to those skilled in the art.

[0095] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. 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 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 multi-step reaction; Step 3: NIR820 and ND550 were mixed in a molar ratio of 5:1 to obtain a mixed ratiometric fluorescent probe with a final concentration of 5 μM ND550 and 25 μM NIR820. 10 μL of the working solution was added dropwise to the 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, wherein 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, wherein 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 the intermediate product compound of formula (V); S22, refluxing the intermediate product compound of formula (V) with potassium carbonate in methanol 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, reacting under reflux under nitrogen environment, and purifying to obtain the compound of formula (II) ND550; (V) (VI) (VII) 4. The preparation method according to claim 2, wherein 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 mixture of toluene and acetonitrile for 18 to 36 hours, and then cooled and washed with a mixture 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, wherein In step S21, 4-bromo-1,8-naphthalene anhydride and -alanine is heated under reflux in dioxane and water for 10 to 16 hours; in step S22, the intermediate product compound of formula (V) is deprotonated by potassium carbonate, and the reflux reaction time is 20 to 36 hours; in step S23, the intermediate product compound of formula (VII) is refluxed with 2,4-dinitrobromobenzene under nitrogen for 5 to 10 hours, and purification is performed by column chromatography using petroleum ether and ethyl acetate.

6. A near-infrared ratiometric fluorescence and colorimetric dual-channel smart label prepared by the preparation method according to any one of claims 1 to 5.

7. Application of the near-infrared ratiometric fluorescence and colorimetric dual-channel smart label in monitoring the freshness of fresh agricultural and livestock products as claimed in claim 6, wherein: Fresh agricultural and livestock products include pork, beef, mutton, chicken, goose, duck, shrimp or crab.

8. The use as claimed in claim 7, wherein the near-infrared ratiometric 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.

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