Phenothiazine derivative as well as synthesis method and application thereof in food freshness detection

Abstract

The invention provides a phenothiazine derivative as well as a synthesis method and application thereof in food freshness detection. The invention designs and synthesizes a fluorescent molecule based on a phenothiazine derivative with proton / deprotonation behavior, the protonated phenothiazine derivative is taken as a detection object, 1, 4-butanediamine and 1, 5-pentamethylene diamine generated in a food spoilage process are taken as detected objects, accurate and rapid detection is realized through a fluorescence opening behavior, and the detection sensitivity is high. And then the fluorescent label is prepared by loading the fluorescent label on a test strip, so that the freshness of an actual food sample is detected by the solution / test paper at normal temperature. In addition, in order to be convenient and practical, an intelligent detection method is constructed.

Description

Technical Field

[0001] The invention belongs to the technical field of organic fluorescent material detection, and specifically relates to a phenothiazine derivative and a synthesis method thereof and application in food freshness detection. Background Art

[0002] Fluorescence response has broad application prospects in optoelectronic devices, food safety, environmental testing and biomedicine due to its high sensitivity, stability and specificity. However, traditional fluorescent materials are often doped with transition metals or rare earth metals, and there is a problem of heavy metal toxicity. The production cost of inorganic phosphors is relatively high and the processability of inorganic compounds is not as good as that of organic molecules, which limits their use in certain application fields. Moreover, most fluorescent materials have low quantum yields and long fluorescence lifetimes, which limits their efficiency in detection. In view of this, the development of pure organic fluorescent materials is a very effective idea and method. Organic fluorescent materials have the advantages of good biocompatibility, low cost, and good solution processability. In the past few decades, scientists have done a lot of work in synthesizing fluorescent molecules to achieve specific detection and identification of certain substances, and various responsive organic fluorescent materials have been greatly expanded and developed.

[0003] Organic fluorescent materials have become an ideal material in the field of detection technology due to their high sensitivity and easy processing. Specific fluorescent molecular structures will respond sensitively to specific material molecules, which is reflected by significant changes in fluorescence color or intensity, thereby achieving rapid detection and accurate identification of target molecules. This characteristic makes them have great application potential in detection and identification. However, most fluorescent response behaviors only occur in the liquid phase, which greatly limits their application scenarios. Therefore, it is particularly important to develop fluorescent sensor devices with high applicability. Based on the easy processing characteristics of organic molecules, it is very meaningful to try to load them onto carriers such as test strips and silica gel plates to prepare detection devices, broaden their practical applications in life, and enhance their practicality. Designing and synthesizing new fluorescent molecules and combining liquid phase detection and device detection can obtain a wider range of application scenarios, thereby enhancing the economic value of organic fluorescent materials.

[0004] In daily life, the freshness of food is related to physical health, so people pay special attention to it. Different types of food have different standards for measuring freshness. For example, the freshness of fruit and vegetable foods can be grasped by observing the apparent water loss and corruption of the food; meat products need to observe whether there is any peculiar smell and whether the color is normal. Food freshness is crucial to food quality and safety. Fresh food contains more nutrients, and unfresh food is more likely to breed harmful microorganisms and endanger human health. Therefore, people have developed many methods for food freshness detection, such as sensory detection, microbial detection, and physical detection. These methods can evaluate the freshness of food, but there are still some problems. The sensory detection method is greatly affected by the subjective factors of the inspector, and the accuracy and repeatability of the results are poor; the microbial detection method requires 24 to 48 hours to cultivate microorganisms to reach a certain number before successful detection, and has a long detection cycle; the equipment cost of the physical detection method is high, and a large amount of data needs to be collected to establish a reliable data model, and the whole process is cumbersome. Therefore, it is of great significance to develop a detection method and method that takes into account both accuracy and timeliness. Summary of the invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the present invention designs and synthesizes a fluorescent molecule based on a phenothiazine derivative with protonation / deprotonation behavior, takes the protonated phenothiazine derivative as the detection object, and the 1,4-butanediamine and 1,5-pentanediamine produced during food spoilage as the detected object, and realizes accurate and rapid detection of them through fluorescence opening behavior, and then loads them on the test strip to prepare a fluorescent label, realizing the freshness detection of the actual sample at room temperature of the solution / test strip. Moreover, for further convenience and practicality, an intelligent detection method is constructed.

[0006] 1. Phenothiazine derivatives and their synthesis The present invention provides a phenothiazine derivative, the structural formula of which is as follows: .

[0007] The synthesis method of the phenothiazine derivative (PyPTZ) of the present invention comprises the following steps: (1) Phenothiazine is added to anhydrous dichloromethane to obtain a phenothiazine solution, N-bromosuccinimide is dissolved in dichloromethane and then mixed with the phenothiazine solution, and the reaction system is reacted in an ice bath at 0°C for 2 to 4 hours. After the reaction is complete, the reaction solution is extracted with water and dichloromethane, the liquids are separated, the organic phases are combined, dried over anhydrous sodium sulfate, and separated by column chromatography to obtain a compound diBr-PTZ; The structural formula of the compound diBr-PTZ is ; The molar ratio of phenothiazine to N-bromosuccinimide is 1:2~1:3.

[0008] (2) The compound diBr-PTZ, potassium carbonate and 4-pyridineboronic acid are dissolved in a mixed solvent of 1,4-dioxane / water to obtain a mixed solution, tetrakis(triphenylphosphine)palladium is dissolved in 1,4-dioxane and quickly added to the mixed solution, and the mixture is reacted at 60-80°C for 10-15 h under nitrogen protection. After the reaction is completed, the mixture is cooled to room temperature and extracted with dichloromethane and water. The organic phase is taken, dried over anhydrous sodium sulfate and the solvent is removed to obtain a crude product, which is then recrystallized from ethanol / water to obtain the target compound PyPTZ.

[0009] The molar ratio of the compound diBr-PTZ to 4-pyridineboronic acid is 1:2~1:3; the molar ratio of the compound diBr-PTZ to potassium carbonate is 1:5~1:8; the molar ratio of the compound diBr-PTZ to tetrakis(triphenylphosphine)palladium is 15:1~25:1. The volume ratio of 1,4-dioxane / water mixed solvent is 4:1.

[0010] The synthetic route of PyPTZ is as follows: 2. Detection of food freshness by phenothiazine derivatives 1. Fluorescence responsiveness of protonated phenothiazine derivatives to biogenic amines PyPTZ was dissolved in acetonitrile to prepare 1mM and 10 μM PyPTZ solutions for later use, and 0.01 M hydrochloric acid solution, 1,4-butanediamine solution (BDA) and 1,5-pentanediamine (PDA) solution were prepared. The effects of acid and base on the fluorescence of PyPTZ were then studied. It was found that after the hydrochloric acid solution was dripped into the PyPTZ solution, as the amount of dripping increased, PyPTZ was gradually protonated, and the fluorescence of the PyPTZ solution, which originally had cyan fluorescence, gradually weakened to completely quenched, as shown in Figure 2. Figure 2 , calculated by fluorescence titration, H + The binding constant with PyPTZ is large, 1.6*10 6 M -1 The non-fluorescent H-PyPTZ system was used as the detection object, and 0.01 M 1,4-butanediamine solution and 1,5-pentanediamine solution were used as the detected objects for the next step of detection. 1,4-Butanediamine and 1,5-pentanediamine were added to the H-PyPTZ system respectively, and it was found that the fluorescence could be successfully turned on again, indicating that H-PyPTZ was deprotonated. The binding constants and minimum detection limits of the two with H-PyPTZ were 2.8*10 5 M -1 、6.96*10 -6 M and 7.0*10 6 M -1 ,9.01*10 -6 M Figure 3 , Figure 4 The above experimental results show that the H-PyPTZ system has good fluorescence response performance to biogenic amines.

[0011] 2. Fluorescent label preparation PyPTZ was dissolved in acetonitrile and prepared into 1mM and 10 μM solutions for later use. The filter paper was cut into appropriate sizes and soaked in DMSO for 12 h in advance, and then air-dried for use to obtain the test strips. The pre-treated test strips were then soaked in 1mM H-PyPTZ solution for 6 h and naturally air-dried as fluorescent labels. First, the effect of adding different concentrations (1×10 -6 M ~1×10 -1 M) Fluorescence changes of H-PyPTZ-based fluorescent labels after BDA and PDA. Figure 5 It can be seen that as the concentration of the detected object dropped on the fluorescent label increases, its fluorescence is significantly enhanced.

[0012] 3. Testing the freshness of actual food samples During the food spoilage process, there is a large amount of microbial metabolic activity, accompanied by decarboxylation reactions, which leads to the release of biogenic amine gases (BDA and PDA gases). As the food is placed for a longer time, the degree of food spoilage increases, and the amount of biogenic amine gas released will increase significantly. Prepare live shrimp as the actual test sample, put the live shrimp, 10 μM H-PyPTZ solution, and the prepared fluorescent label in a small box with a lid, place it at room temperature, and monitor the fluorescence changes of the solution and the fluorescent label in time. Figure 6 . It was found that after 4 hours, the fluorescence of the solution increased slightly, and after 6 hours, the fluorescent label showed obvious yellow fluorescence. After 48 hours, the fluorescence intensity no longer increased, indicating that the detection endpoint was reached and the shrimp was completely corrupted. As time goes by, the shrimp releases biogenic amine gases (BDA and PDA gases), which increase the fluorescence of the H-PyPTZ solution and the fluorescent label. The above experimental phenomena show that the process of shrimp from freshness to corruption has been successfully detected.

[0013] 4. Intelligent detection of freshness of actual food samples In order to further enhance the practicality and reliability of the detection method, we considered applying smartphones to the detection. The shrimp meat and 10 μM H-PyPTZ detection solution were placed in a small box with a lid and placed in a dark room. Every two hours, the detection solution was photographed under 365nm ultraviolet light to obtain fluorescent photos. The fluorescent photos of the detection solution at different times were analyzed through the smartphone APP color picker to analyze their RGB values, and then G / B was further obtained. Finally, a good linear relationship between the G / B of the shrimp meat sample and the detection time was established ( Figure 7 ), the linear equation of shrimp meat sample G / B and detection time is G / B=0.050t+0.41, R 2 =0.98, t-time, unit: h.

[0014] To verify the universality of the detection system, we selected fish meat as the object to be detected and conducted the detection experiment according to the live shrimp detection method. We found that there was not much difference between the phenomenon and the result, which showed the reliability of this method. Fish meat and 10 μM H-PyPTZ solution were placed in a small box with a lid and placed at room temperature. The detection results are as follows: Figure 8 As shown, the change of fish freshness over time can be successfully detected. The phenomenon is consistent with the deterioration process of shrimp. As the fish is placed for a longer time, the fluorescence turns on.

[0015] The fish meat and 10 μM H-PyPTZ detection solution were placed in a small box with a lid and placed in a dark room at room temperature. The detection solution was photographed every two hours under 365nm ultraviolet light to obtain fluorescent photos. The RGB values ​​of the fluorescent photos taken at different times were analyzed by the smartphone APP color picker, and then the G / B was further obtained. The linear relationship between the G / B of the fish meat sample and the detection time was established ( Figure 8 ), the actual sample G / B has a good linear relationship with the detection time, and the linear equation is G / B=0.01922t+0.7117, R 2 =0.99, t-time, unit: h.

[0016] Finally, we used the detection solution to test the purchased poultry meat, obtained G / B during the detection process, substituted G / B into the linear relationship, calculated the experimental measurement time, and compared it with the actual time, and found that the difference between the two was small, which further proved the universality and accuracy of this method.

[0017] Beneficial effects of the present invention: In the present invention, PyPTZ is used as a basic fluorescent molecule and H +Then the original fluorescence properties were changed to quench it, and a non-fluorescent H-PyPTZ system was obtained. Subsequently, the fluorescence was restored through the deprotonation process to achieve the detection purpose. This process has excellent fluorescence response performance and short response time, and is a sensing molecule with good properties. In addition, loading it on the test paper to prepare it as a fluorescent label enhances its practicality and convenience. Finally, we successfully established a detection method based on smartphones, which further enhanced its practicality and reliability. We also expanded the detection substrate and verified its universality. This liquid phase detection, solid phase detection and intelligent detection method has a higher utilization value and provides more possibilities for the development of new fluorescent molecules, detection devices and intelligent detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the H-NMR spectrum characterization of PyPTZ.

[0019] Figure 2 Fluorescence titration curve of PyPTZ with hydrochloric acid solution (a) and detection limit calculation (b).

[0020] Figure 3 Fluorescence titration curve of BDA against H-PyPTZ (a) and detection limit calculation (b).

[0021] Figure 4 Fluorescence titration curve of PDA to H-PyPTZ (a) and detection limit calculation (b).

[0022] Figure 5 The responses of the fluorescent label to different concentrations of BDA and PDA.

[0023] Figure 6 Schematic diagram of shrimp sample detection and the final actual effect diagram; (a) the preparation method of fluorescent labels and the actual sample detection method, (b) the detection effect at room temperature and low temperature.

[0024] Figure 7 Flow chart of intelligent detection of shrimp meat samples and the established linear equation; (a) detection process, (b) (c) linear relationship between G / B and detection time.

[0025] Figure 8 To detect the freshness of fish and establish a linear relationship. DETAILED DESCRIPTION

[0026] Example 1 Synthesis of Phenothiazine Derivatives (PyPTZ) (1) Phenothiazine (1.99 g, 10 mmmol) was placed in a 250 mL two-necked flask, 30 mL of dichloromethane was added, N-bromosuccinimide (NBS) (4.27 g, 24 mmol) was dissolved in 50 mL of dichloromethane, and then the NBS solution was slowly dripped into the phenothiazine solution through a dropping funnel within 1 h. The reaction system was reacted in an ice bath at 0°C, and the reaction progress was monitored by TLC. After the phenothiazine reaction was complete, the reaction solution was extracted three times, separated, and the organic phase was combined. The organic phase was dried over anhydrous sodium sulfate and separated by column chromatography. The eluent was ethyl acetate / petroleum ether = 1 / 40. 2.51 g of light green solid diBr-PTZ was obtained with a yield of 71.4%.

[0027] (2) Take diBr-PTZ (1.00 g, 3 mmol), potassium carbonate (2.77 g, 20 mmol) and 4-pyridine boronic acid (0.89 g, 7.2 mmol) prepared in step (1) and dissolve them in 10 mL of a mixed solvent of 1,4-dioxane / water (v / v=4 / 1) to obtain a mixed solution. Take tetrakis(triphenylphosphine)palladium (0.17 g, 0.15 mmol) and dissolve it in 5 mL of 1,4-dioxane. Quickly add it to the first mixed solution, immediately evacuate, react at 70 °C under nitrogen protection for 12 h, cool to room temperature after the reaction, extract with dichloromethane and water, take the organic phase, dry the organic phase with anhydrous sodium sulfate, and remove the solvent to obtain a crude product. Then recrystallize it with ethanol / water to obtain the target compound PyPTZ as a yellow solid of 0.75 g, with a yield of 74.8%.

[0028] Example 2 Qualitative detection of food samples (1) Weigh 3.54 mg of PyPTZ and dissolve it in 10 mL of acetonitrile to obtain a 1 mM PyPTZ solution. Take 100 μL of the solution and add 9.9 mL of acetonitrile to dilute it to obtain a 10 μM PyPTZ acetonitrile solution. Prepare 0.01 M hydrochloric acid solution and standardize it with sodium hydroxide solution. Then prepare 0.01 M 1,4-butanediamine solution and 1,5-pentanediamine solution.

[0029] (2) Take 5 mL of 10 μM PyPTZ acetonitrile solution, add 100 μL of hydrochloric acid solution according to the amount of hydrochloric acid solution required by fluorescence titration, so that the fluorescence of the PyPTZ solution is completely quenched, and a protonated 10 μM H-PyPTZ system is obtained. The obtained H-PyPTZ system is placed in a vial. The same method is used to obtain a 1 mM H-PyPTZ system.

[0030] (3) The test strips previously soaked in DMSO were immersed in a 1 mM H-PyPTZ solution for 6 h and air-dried to obtain the fluorescent label.

[0031] (4) Qualitative detection of samples: Place the food sample to be tested, H-PyPTZ solution system or fluorescent label in a small box with a lid for 4 to 6 minutes. If the fluorescence of the H-PyPTZ solution system or fluorescent label turns on, it means that the food is spoiled. The degree of food spoilage is positively correlated with the degree of fluorescence turn-on. If the fluorescence of the H-PyPTZ solution system or fluorescent label does not change, it means that the food is fresh.

[0032] Example 3 Quantitative detection of food samples Combined with the linear relationship established based on the detection of fish and shrimp freshness, we tested the freshness of poultry meat. We placed the pork, beef, mutton and poultry meat purchased from the market with 10 μM H-PyPTZ detection solution in a small box with a lid and placed it in a dark room at room temperature. After 4-6 minutes, we took a photo of the detection solution under 365nm ultraviolet light to obtain a fluorescent photo. The fluorescent photo was analyzed by the smartphone APP color picker to further obtain G / B. According to the linear relationship G / B=0.050t+0.41 (shrimp) and G / B=0.01922t+0.7117 (fish) obtained from the detection of shrimp and fish, the G / B value of the detection solution in the detection of pork, beef, mutton and poultry meat was substituted into the linear relationship, and the measurement time a and b were calculated respectively. They were compared with the actual storage time c of the sample. As shown in Table 1, it can be found that the difference between the measurement time and the real time is not large, indicating that this detection method is applicable to fish, shrimp and poultry foods.

[0033] surface Comparison table of measured time and real time for poultry meat experiment In summary, the synthesized fluorescent molecule PyPTZ can be used as a system for detecting freshness in the detection of actual samples after protonation. The present invention provides a detection technology for detecting the degree of food deterioration or freshness based on the fluorescence on / off phenomenon. The fluorescence of the phenothiazine derivative is quenched by protonation, and then the fluorescence is reopened in the environment of food corruption by relying on the biogenic amines released when the food is corrupted. In order to enhance its practicality, we load the detection object on the test strip and successfully prepare the intelligent detection label to realize the multi-channel detection of solution and test strip. The detection method shows excellent response performance in the detection of actual samples. Taking live shrimp as the detection object, the shrimp can be detected to be corrupted after being placed at room temperature for 6 hours, and the fluorescence change is more obvious. This performance is outstanding among similar materials. In addition, we also use it to detect the freshness of fish and poultry meat, and both have good responsiveness. This detection method based on the fluorescence change of phenothiazine derivatives is widely used. Due to the high fluorescence response sensitivity and stability, it has good application value and great potential for further development in the fields of detection and identification. In addition, we combined this detection method with a smartphone to construct an intelligent detection method. The RGB value of the solution was extracted by the mobile phone to obtain G / B. The corresponding freshness was obtained based on the linear relationship between G / B and storage time.

Claims

1. A phenothiazine derivative having the following structural formula: 。 2. The method for synthesizing the phenothiazine derivative according to claim 1, comprising the following steps: (1) adding phenothiazine to anhydrous dichloromethane to obtain a phenothiazine solution, dissolving N-bromosuccinimide in dichloromethane and mixing with the phenothiazine solution, reacting the reaction system in an ice bath for 2 to 4 hours, and after the reaction is complete, extracting the reaction solution with water and dichloromethane, separating the liquids, combining the organic phases, drying with anhydrous sodium sulfate, and separating by column chromatography to obtain a compound diBr-PTZ; The structural formula of the compound diBr-PTZ is ; (2) The compound diBr-PTZ, potassium carbonate and 4-pyridineboronic acid are dissolved in a mixed solvent of 1,4-dioxane / water to obtain a mixed solution, tetrakis(triphenylphosphine)palladium is dissolved in 1,4-dioxane and quickly added to the mixed solution, and the mixture is reacted at 60-80°C for 10-15 h under nitrogen protection. After the reaction is completed, the mixture is cooled to room temperature and extracted with dichloromethane and water. The organic phase is taken, dried over anhydrous sodium sulfate and the solvent is removed to obtain a crude product, which is then recrystallized from ethanol / water to obtain the target compound PyPTZ.

3. The method for synthesizing a phenothiazine derivative according to claim 2, characterized in that: In step (1), the molar ratio of phenothiazine to N-bromosuccinimide is 1:2 to 1:

3.

4. The method for synthesizing a phenothiazine derivative according to claim 2, characterized in that: In step (2), the molar ratio of the compound diBr-PTZ to 4-pyridineboronic acid is 1:2-1:3; the molar ratio of the compound diBr-PTZ to potassium carbonate is 1:5-1:8; and the molar ratio of the compound diBr-PTZ to tetrakis(triphenylphosphine)palladium is 15:1-25:

1.

5. The method for synthesizing a phenothiazine derivative according to claim 2, characterized in that: In step (2), the volume ratio of 1,4-dioxane to water in the mixed solvent of 1,4-dioxane / water is 4:

1.

6. The use of the phenothiazine derivative in the detection of biogenic amines according to claim 1, characterized in that: The biogenic amine is 1,4-butanediamine or 1,5-pentanediamine.

7. The use of the phenothiazine derivative in the detection of biogenic amines according to claim 6, characterized in that: A hydrochloric acid solution is added to an acetonitrile solution of a phenothiazine derivative to obtain a non-fluorescent H-PyPTZ solution system. When 1,4-butanediamine or 1,5-pentanediamine is added to the H-PyPTZ system, the fluorescence of the non-fluorescent H-PyPTZ solution system turns on.

8. Use of the phenothiazine derivative according to claim 1 in food freshness detection.

9. The use of the phenothiazine derivative in food freshness detection according to claim 8, characterized in that: A hydrochloric acid solution is added to an acetonitrile solution of a phenothiazine derivative to obtain an H-PyPTZ solution system; a test strip is immersed in the H-PyPTZ solution and naturally air-dried to obtain a fluorescent label; Place the food sample, H-PyPTZ solution system or fluorescent label in a small box with a lid for 4 to 6 minutes. If the fluorescence of the H-PyPTZ solution system or fluorescent label turns on, it means that the food is spoiled, and the degree of food spoilage is positively correlated with the degree of fluorescence turning on. If the fluorescence of the H-PyPTZ solution system or fluorescent label does not turn on, it means that the food is fresh.

10. The use of the phenothiazine derivative in food freshness detection according to claim 8, characterized in that: The method for intelligently detecting the freshness of food using phenothiazine derivatives comprises the following steps: (1) Preparation of detection solution: Add hydrochloric acid solution to the acetonitrile solution of phenothiazine derivative to obtain H-PyPTZ detection solution; (2) Establishment of a linear relationship: Food samples and H-PyPTZ detection solution were placed in a small box with a lid, placed in a dark room at room temperature, and irradiated with a 365nm ultraviolet lamp. Data was collected every two hours, and the detection solution was photographed at different times to obtain fluorescent photos. The RGB values ​​of the fluorescent photos at different detection times were analyzed using a smartphone APP color picker to further obtain the G / B, thus establishing a linear relationship between the G / B of the food sample and the detection time. (3) Sample testing: The food sample to be tested and the H-PyPTZ detection solution are placed in a small box with a lid. After being placed in a dark room at room temperature for 4 to 6 minutes, the detection solution is photographed under 365nm ultraviolet light to obtain a fluorescent photo. The RGB value of the fluorescent photo is analyzed by a smartphone APP color picker to further obtain the G / B. The G / B value is substituted into the established linear relationship between the G / B of the food sample and the detection time to obtain the storage time of the food sample, thereby realizing intelligent detection of food freshness.

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