A phenothiazine derivative, its synthesis method and its application in food freshness detection

Abstract

The present invention provides a phenothiazine derivative, a synthesis method thereof, and its application in food freshness detection. The present invention designs and synthesizes a fluorescent molecule based on a phenothiazine derivative with protonation / deprotonation behavior. Using the protonated phenothiazine derivative as the detection target, and 1,4-butanediamine and 1,5-pentanediamine produced during food spoilage as the target, the fluorescent on-state behavior enables accurate and rapid detection of these targets. The target is then loaded onto a test strip to prepare a fluorescent label, enabling freshness detection of actual food samples at room temperature using the solution / test strip. Furthermore, for further convenience and practicality, an intelligent detection method has been constructed.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic fluorescent material detection, and in particular relates to a phenothiazine derivative and a synthesis method thereof, and application thereof 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 produce sensitive responses to specific substance molecules, which are reflected by significant changes in fluorescence color or intensity, thereby achieving rapid detection and accurate identification of target molecules. This characteristic gives them huge 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 achieve a wider range of application scenarios, thereby enhancing the economic value of organic fluorescent materials.

[0004] In daily life, the freshness of food is crucial to our health, making it a crucial consideration. Different types of food have different criteria for measuring freshness. For example, the freshness of fruits and vegetables can be assessed by observing the apparent loss of moisture and signs of spoilage; meat products require observation for odor and color. Food freshness is crucial to its quality and safety. Fresh food contains more nutrients, while stale food is more likely to harbor harmful microorganisms, posing a threat to human health. Consequently, numerous food freshness assessment methods have been developed, including sensory, microbiological, and physical testing. While these methods can assess food freshness, they still present several challenges. Sensory testing is subject to significant subjective influence from the tester, resulting in poor accuracy and reproducibility. Microbiological testing requires 24–48 hours to cultivate microorganisms to a sufficient number, resulting in a long testing cycle. Physical testing methods are expensive and require extensive data collection to build reliable models, making the entire process cumbersome. Therefore, developing a testing method and approach that balances accuracy and timeliness is crucial. Summary of the Invention

[0005] To overcome the shortcomings of the aforementioned prior art, the present invention designed and synthesized a fluorescent molecule based on a phenothiazine derivative with protonation / deprotonation behavior. Using the protonated phenothiazine derivative as the detection target, the 1,4-butanediamine and 1,5-pentanediamine produced during food spoilage were detected accurately and rapidly through fluorescence activation. The fluorescent label was then loaded onto a test strip to prepare a fluorescent tag, enabling freshness testing of actual samples at room temperature using the solution / test strip. Furthermore, for added convenience and practicality, an intelligent detection method was constructed.

[0006] 1. Phenothiazine derivatives and their synthesis

[0007] The present invention provides a phenothiazine derivative, the structural formula of which is as follows:

[0008] .

[0009] The synthesis method of the phenothiazine derivative (PyPTZ) of the present invention comprises the following steps:

[0010] (1) Phenothiazine was added to anhydrous dichloromethane to obtain a phenothiazine solution. N-bromosuccinimide was dissolved in dichloromethane and mixed with the phenothiazine solution. The reaction system was reacted in an ice bath at 0°C for 2-4 hours. After the reaction was complete, the reaction solution was extracted with water and dichloromethane, the liquids were separated, the organic phases were combined, dried over anhydrous sodium sulfate, and separated by column chromatography to obtain the compound diBr-PTZ.

[0011] The structural formula of the compound diBr-PTZ is ; The molar ratio of phenothiazine to N-bromosuccinimide is 1:2~1:3.

[0012] (2) Compound diBr-PTZ, potassium carbonate and 4-pyridineboronic acid were dissolved in a mixed solvent of 1,4-dioxane / water to obtain a mixed solution. Tetrakis(triphenylphosphine)palladium was dissolved in 1,4-dioxane and quickly added to the mixed solution. The mixture was reacted at 60-80 °C under nitrogen protection for 10-15 h. After the reaction was completed, the mixture was cooled to room temperature and extracted with dichloromethane and water. The organic phase was taken, dried over anhydrous sodium sulfate and the solvent was removed to obtain a crude product, which was then recrystallized from ethanol / water to obtain the target compound PyPTZ.

[0013] 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 the mixed solvent of 1,4-dioxane / water is 4:1.

[0014] The synthetic route of PyPTZ is as follows:

[0015]

[0016] 2. Detection of food freshness using phenothiazine derivatives

[0017] 1. Fluorescence responsiveness of protonated phenothiazine derivatives to biogenic amines

[0018] PyPTZ was dissolved in acetonitrile to prepare 1 mM and 10 μM PyPTZ solutions for later use. 0.01 M hydrochloric acid solution, 1,4-butanediamine solution (BDA), and 1,5-pentanediamine (PDA) solutions were also prepared. The effects of acid and base on the fluorescence of PyPTZ were then investigated. It was found that as the amount of hydrochloric acid solution added to the PyPTZ solution increased, PyPTZ was gradually protonated. The fluorescence of the originally cyan fluorescent PyPTZ solution gradually weakened until it was completely quenched, as shown in Figure 3. Figure 2 , calculated by fluorescence titration to obtain H + It has a large binding constant of 1.6*10 with PyPTZ. 6 M -1 The non-fluorescent H-PyPTZ system was used as the detector, and 0.01 M 1,4-butanediamine solution and 1,5-pentanediamine solution were used as the analytes for the next step of detection. 1,4-Butanediamine and 1,5-pentanediamine were added to the H-PyPTZ system, 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*105 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.

[0019] 2. Fluorescent Label Preparation

[0020] PyPTZ was dissolved in acetonitrile to prepare a solution with a concentration of 1mM and 10 μM. Filter paper was cut into appropriate sizes and soaked in DMSO for 12 hours in advance, and then air-dried to obtain a test strip. The pre-treated test strip was then soaked in 1mM H-PyPTZ solution for 6 hours and naturally air-dried as a fluorescent label. 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, the effect is as follows Figure 5 , it can be seen that as the concentration of the detected substance dropped on the fluorescent label increases, its fluorescence is significantly enhanced.

[0021] 3. Testing the freshness of actual food samples

[0022] During food spoilage, a large amount of microbial metabolic activity occurs, accompanied by decarboxylation reactions, leading to the release of biogenic amine gases (BDA and PDA gases). As food is stored for longer, the degree of food spoilage increases, and the amount of biogenic amine gas released increases significantly. Live shrimp were prepared as the actual test sample. Live shrimp, 10 μM H-PyPTZ solution, and the prepared fluorescent label were placed in a small box with a lid and left at room temperature. Fluorescence changes in the solution and the fluorescent label were monitored in real time. Figure 6 After 4 hours, the solution's fluorescence increased slightly. After 6 hours, the fluorescent tag exhibited a distinct yellow fluorescence. After 48 hours, the fluorescence intensity ceased to increase, indicating the detection endpoint and the shrimp were completely spoiled. As the shrimp decayed from freshness to decay, the biogenic amine gases (BDA and PDA) released over time caused the fluorescence of the H-PyPTZ solution and the fluorescent tag to increase over time. These experimental observations demonstrate the successful detection of the shrimp's decay process.

[0023] 4. Intelligent detection of the freshness of actual food samples

[0024] To further enhance the practicality and reliability of this detection method, we considered using smartphones in the test. Shrimp meat and a 10 μM H-PyPTZ test solution were placed in a small, covered box in a darkroom. Every two hours, the test solution was photographed under 365nm UV light to obtain fluorescence images. The fluorescence images of the test solution at different times were analyzed using a smartphone app color picker to analyze their RGB values. The G / B ratio was then further calculated. Finally, a good linear relationship between the G / B ratio of the shrimp meat sample and the test 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.

[0025] To verify the universality of the detection system, we selected fish as the test object and conducted the detection experiment according to the live shrimp detection method. We found that the phenomenon and results were not much different, which shows the reliability of the method. Fish meat and 10 μM H-PyPTZ solution were placed in a small box with a lid and left at room temperature. The test 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.

[0026] Fish meat and a 10 μM H-PyPTZ detection solution were placed in a small box with a lid and placed in a darkroom at room temperature. Every two hours, the detection solution was photographed under 365 nm ultraviolet light to obtain fluorescent photos. The RGB values ​​of the fluorescent photos taken at different times were analyzed using a smartphone APP color picker, and the G / B ratio was further calculated. A linear relationship between the G / B ratio 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.

[0027] Finally, we used the detection solution to test the purchased poultry meat. During the detection process, we obtained G / B, substituted G / B into the linear relationship, calculated the experimental measurement time, and compared it with the actual time. We found that the difference between the two was small, further proving the universality and accuracy of this method.

[0028] Beneficial effects of the present invention: In the present invention, PyPTZ is used as a basic fluorescent molecule and H +The original fluorescence properties were then changed to quench it, resulting in a non-fluorescent HPYPTZ system. Subsequently, the fluorescence was restored through a deprotonation process, achieving the detection purpose. This process has excellent fluorescence response performance and a short response time, making it a sensing molecule with good properties. In addition, loading it onto a test paper to prepare a fluorescent label enhances its practicality and convenience. Finally, we successfully established a smartphone-based detection method, further enhancing 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 higher utilization value and provides more possibilities for the development of new fluorescent molecules, detection devices, and intelligent detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the H NMR spectrum characterization of PyPTZ.

[0030] Figure 2 Fluorescence titration curve of PyPTZ with hydrochloric acid solution (a) and detection limit calculation (b).

[0031] Figure 3 Fluorescence titration curve of BDA against H-PyPTZ (a) and detection limit calculation (b).

[0032] Figure 4 Fluorescence titration curve of PDA to H-PyPTZ (a) and detection limit calculation (b).

[0033] Figure 5 The responses of the fluorescent label to different concentrations of BDA and PDA.

[0034] Figure 6 Schematic diagram of shrimp sample detection and the final actual effect diagram; (a) Preparation method of fluorescent label and actual sample detection method, (b) Detection effect at room temperature and low temperature.

[0035] Figure 7 Flowchart 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.

[0036] Figure 8 For fish freshness detection and linear relationship establishment. DETAILED DESCRIPTION

[0037] Example 1 Synthesis of Phenothiazine Derivatives (PyPTZ)

[0038] (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 the NBS solution was slowly dripped into the phenothiazine solution through a dropping funnel over 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 phases were combined and dried over anhydrous sodium sulfate. The organic phases were separated by column chromatography with an eluent of ethyl acetate / petroleum ether = 1 / 40. 2.51 g of light green solid diBr-PTZ was obtained with a yield of 71.4%.

[0039] (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 the mixture, and react at 70 °C under nitrogen protection for 12 h. After the reaction is completed, cool it to room temperature, extract it with dichloromethane and water, take the organic phase, dry it with anhydrous sodium sulfate, and remove the solvent to obtain a crude product. Then recrystallize it from ethanol / water to obtain the target compound PyPTZ as a yellow solid 0.75 g with a yield of 74.8%.

[0040] Example 2 Qualitative detection of food samples

[0041] (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 this solution and dilute it with 9.9 mL of acetonitrile to obtain a 10 μM PyPTZ solution in acetonitrile. Prepare a 0.01 M hydrochloric acid solution and standardize it with sodium hydroxide solution. Then prepare a 0.01 M solution of 1,4-butanediamine and a 1,5-pentanediamine solution.

[0042] (2) Take 5 mL of 10 μM PyPTZ in acetonitrile solution and add 100 μL of hydrochloric acid solution according to the amount of hydrochloric acid solution required by fluorescence titration to completely quench the fluorescence of the PyPTZ solution, thereby obtaining a protonated 10 μM H-PyPTZ system. The obtained H-PyPTZ system is then placed in a vial. The same method is used to obtain a 1 mM H-PyPTZ system.

[0043] (3) The test strips that had been soaked in DMSO were immersed in a 1 mM H-PyPTZ solution for 6 h and air-dried to obtain the fluorescent label.

[0044] (4) Qualitative testing of samples: Place the food sample to be tested, the H-PyPTZ solution system, or the fluorescent label in a small box with a lid for 4–6 min. If the fluorescence of the H-PyPTZ solution system or the fluorescent label turns on, it indicates that the food has deteriorated. The degree of food deterioration is positively correlated with the degree of fluorescence. If the fluorescence of the H-PyPTZ solution system or the fluorescent label does not change, it indicates that the food is fresh.

[0045] Example 3 Quantitative detection of food samples

[0046] Integrating the linear relationship established for freshness testing of fish and shrimp, we tested the freshness of poultry meat. Commercially available pork, beef, mutton, and poultry meat were placed in a small, covered box with a 10 μM H-PyPTZ test solution and stored in a darkroom at room temperature. After 4–6 minutes, the test solution was photographed under 365 nm UV light to obtain a fluorescence image. The fluorescence image was analyzed using a smartphone app color picker to determine the RGB ratio (G / B). Based on the linear relationship established for testing shrimp and fish (G / B = 0.050t + 0.41 for shrimp) and (G / B = 0.01922t + 0.7117 for fish), the G / B values ​​of the test solution used for testing pork, beef, mutton, and poultry meat were substituted into the linear relationship to calculate the measured times a and b, respectively. These values ​​were then compared with the actual storage time c of the samples. As shown in Table 1, the measured and actual times were similar, demonstrating the applicability of this testing method for both fish, shrimp, and poultry.

[0047] surface Comparison table of measured time and real time for poultry meat experiment

[0048]

[0049] In summary, the synthesized fluorescent molecule PyPTZ, after protonation, can be used as a freshness detection system for real-world sample testing. This invention provides a detection technology for food spoilage or freshness based on the fluorescence on / off phenomenon. By protonating a phenothiazine derivative, its fluorescence is quenched. Then, in a food spoilage environment, the fluorescence is reactivated by the release of biogenic amines. To enhance its practicality, the test substance is loaded onto a test strip, successfully preparing an intelligent detection tag, enabling multi-channel detection using both solution and test strips. This detection method demonstrated excellent response performance in real-world sample testing. Using live shrimp as the test subject, the onset of shrimp spoilage was detected after 6 hours at room temperature, with a significant fluorescence change, a performance that stands out among similar materials. Furthermore, the method was used to detect the freshness of fish and poultry meat, demonstrating good responsiveness. This detection method based on the fluorescence change of phenothiazine derivatives has a wide range of applications, thanks to its high fluorescence response sensitivity and stability, making it highly valuable in detection and identification fields and offering significant potential for further development. In addition, we combined this detection method with a smartphone to build an intelligent detection method. The RGB value of the solution was extracted by the mobile phone to obtain G / B, and 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: , named PyPTZ.

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

3. The method for synthesizing a phenothiazine derivative according to claim 2, wherein: 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, wherein: 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, wherein: 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 5, 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 7, 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 allowed to air-dry 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 has deteriorated, and the degree of food deterioration is positively correlated with the degree of fluorescence. 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 7, 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: The food sample and the H-PyPTZ detection solution were placed in a small box with a lid, placed in a darkroom at room temperature, and irradiated with a 365nm UV lamp. Data was collected every two hours, and the detection solution was photographed at different times to obtain fluorescence photos. The fluorescence photos at different detection times were analyzed for their RGB values ​​using a smartphone APP color picker to further obtain the G / B ratio. A linear relationship between the G / B ratio of the food sample and the detection time was established. (3) Product testing: Place the food sample to be tested and the H-PyPTZ test solution in a small box with a lid. Place it in a dark room at room temperature for 4 to 6 minutes. Then, take a photo of the test solution under 365nm ultraviolet light to obtain a fluorescent photo. The RGB value of the fluorescent photo is analyzed by the color picker of the smartphone APP to further obtain the G / B. Substitute it into the established linear relationship between the G / B of the food sample and the test time to obtain the storage time of the food sample, thereby realizing intelligent detection of food freshness.

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