Fluorescent probe for rapid non-destructive detection of food freshness and preparation method thereof

Abstract

The application discloses a compound Cl-BODIPY used as a fluorescent probe for rapid nondestructive detection of food freshness, and also establishes a corresponding preparation method and detection method. The probe produces a color and fluorescent dual optical signal change through specific interaction of a recognition group with volatile biogenic amines, and provides a simple and convenient detection method for rapid detection of volatile biogenic amines. The fluorescent probe is simple to prepare, low in cost, and has the characteristics of high sensitivity, high selectivity and rapid response, can in-situ rapidly detect volatile biogenic amines produced by fish and meat food spoilage, and judges the freshness (biogenic amine content) of fish and meat through colorimetric and fluorescent dual visual signal changes, thereby providing an effective means for rapid nondestructive detection of food freshness, having great application prospect and great popularization significance.

Description

Technical Field

[0001] This invention belongs to the field of food testing technology, and in particular relates to a fluorescent probe for rapid and non-destructive testing of food freshness and its preparation method. Background Technology

[0002] Food quality and safety are closely related to public health. According to the World Health Organization (WHO), consuming spoiled food can lead to more than 200 diseases, including diarrhea and cancer. Meat is an important source of nutrition, rich in various proteins and fats needed by the human body; however, it is also prone to spoilage during storage and transportation due to enzymatic reactions and microbial contamination. During the spoilage of protein-rich foods (meat, fish, and seafood), various biogenic amines are typically produced, such as cadaverine, putrescine, histamine, tyramine, tryptamine, spermine, and spermidine. Cadaverine and putrescine are highly volatile and can be inhaled, ingested, or absorbed through the skin, causing strong irritation to the eyes, mucous membranes, skin, and respiratory tract. Inhalation of volatile biogenic amines can cause inflammation of the throat and bronchi, spasms, edema, pulmonary edema, or chemical pneumonia, and even death. Therefore, the content of volatile cadaverine and putrescine is the most significant indicator of food freshness, and some countries have begun to establish limits based on the characteristics of different foods. Therefore, in order to ensure the quality and safety of meat products, it is necessary to develop simple and effective methods to test the freshness of fish and meat products.

[0003] Currently, food safety issues are receiving increasing attention from all sectors of society. Ensuring food safety relies on reliable technical detection methods. To date, methods for detecting volatile biogenic amines include gas chromatography-mass spectrometry, electrochemical methods, spectrophotometry, and liquid chromatography. However, these methods still have some limitations. For example, sample preparation is complex, requiring expensive instruments, sensitivity is low, and on-site non-destructive testing is not possible. In recent years, fluorescent probe technology has been evaluated as a powerful tool for detecting biogenic amines due to its advantages such as high sensitivity, high selectivity, real-time detection, and ease of operation. Since biogenic amines themselves do not emit fluorescence, the determination of biogenic amines using fluorescence spectrophotometry mainly relies on the reaction between the reaction site of the fluorescent probe and the amino group of the biogenic amine under certain conditions to generate a product with different fluorescence properties than the fluorescent probe itself. The content of biogenic amines can be detected based on changes in both fluorescence and color signals. Considering the importance of food safety, developing novel fluorescent probes for rapid, highly sensitive, non-destructive, and visual determination of volatile biogenic amine content in fish and meat products is both crucial and extremely challenging.

[0004] Therefore, there is an urgent need to develop a simple and low-cost technical detection method for detecting volatile biogenic amines in fish and meat. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a fluorescent probe with stable structure, excellent performance, and high sensitivity for rapid and non-destructive detection of food freshness and its preparation method.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] The compound Cl-BODIPY is used in rapid, non-destructive testing of food freshness. The structural formula of the compound Cl-BODIPY is as follows:

[0008]

[0009] The food consists of fish and meat.

[0010] The compound Cl-BODIPY is used as a fluorescent probe for rapid and non-destructive testing of food freshness.

[0011] The above-mentioned fluorescent probes are prepared according to the following synthetic route:

[0012]

[0013] The preparation method of the above-mentioned fluorescent probe includes the following steps:

[0014] (1) Preparation of compound 1: Pyrrole, N,N-diisopropylethylamine and organic solvent 1,2-dichloroethane were added sequentially to a three-necked round-bottom flask, and the reaction mixture was stirred in a nitrogen atmosphere for 20 min; phosgene was dissolved in 1,2-dichloroethane and added dropwise to the above mixture at 0 °C; after reacting for 2 h, pyrrole was added, and the mixture was heated under reflux at 70 °C for 1 h; excess solvent was removed under reduced pressure, and the crude product was finally purified by silica gel chromatography to obtain a white solid as target compound 1;

[0015] (2) Preparation of fluorescent probe: Compound 1, 1,2-dichloroethane and phosphorus oxychloride were added sequentially to a three-necked round-bottom flask. The reaction mixture was then heated and stirred under nitrogen atmosphere at 80°C for 3-5 h under reflux. After the reaction was complete as detected by TCI, the reaction mixture was cooled in an ice bath and triethylamine was slowly added. After stirring for 5 min, boron trifluoride diethyl ether was slowly added. The reaction mixture was then heated to room temperature and the reaction was continued for 2-5 h. Excess solvent was removed under reduced pressure. The crude product was finally purified by silica gel column chromatography to obtain a red solid as the target fluorescent probe.

[0016] In step (1), the molar ratio of triphosgene to pyrrole and N,N-diisopropylethylamine is 1-5.

[0017] In step (2), the molar ratio of compound 1 to phosphorus oxychloride, triethylamine and boron trifluoride diethyl ether is 0.5-15.

[0018] A rapid, non-destructive test strip for detecting food freshness loaded with the aforementioned fluorescent probes.

[0019] A rapid and non-destructive method for testing the freshness of food involves placing the aforementioned test paper into a sealed dish containing a fish or meat sample.

[0020] The temperatures in the sealed dish were -16℃, 0℃, and 25℃.

[0021] To address the current challenge of rapid, non-destructive, and visually detectable freshness of fish, meat, and other foods, the inventors have developed a fluorescent probe using the compound Cl-BODIPY for rapid, non-destructive testing of food freshness. Based on this, corresponding preparation and detection methods have been established. This probe interacts specifically with volatile biogenic amines through a recognition group, generating changes in both color and fluorescence optical signals, providing a simple and convenient method for the rapid detection of volatile biogenic amines. The fluorescent probe of this invention is simple to prepare, inexpensive, and features high sensitivity, high selectivity, and rapid response. It can rapidly detect volatile biogenic amines produced by spoilage in fish and meat in situ, and determine the freshness (biogenic amine content) of fish and meat through changes in both colorimetric and fluorescence visual signals. This provides an effective means for rapid, non-destructive testing of food freshness, with significant application prospects and widespread applicability. It is the preferred method for rapid detection of volatile biogenic amine content in fish and meat products now and for some time to come, and provides strong technical support for food manufacturers' self-monitoring and market supervision. The test strips prepared accordingly can achieve highly sensitive, non-destructive, and visual monitoring of the freshness of fish and meat, and can become the mainstream technology for rapid on-site detection of volatile biogenic amines in food.

[0022] The basic principle of this invention is as follows: the chlorine atom in the compound Cl-BODIPY acts as a recognition group, capable of undergoing a substitution reaction with the amino group of volatile biogenic amines. This change in molecular structure transmits the signal to the BODIPY fluorophore, altering its fluorescence emission wavelength and solution color. This allows for both colorimetric and fluorescence-based dual-signal response for volatile biogenic amines. Specifically, the fluorescent probe exhibits a distinct absorption peak at 499 nm. Upon addition of cadaverine, a new absorption peak appears at 395 nm. With increasing cadaverine concentration, the probe's original absorption peak gradually weakens while the absorption peak at 395 nm gradually strengthens, and a noticeable color change from green to pale yellow is observed. Furthermore, under excitation light, the probe exhibits significant green fluorescence at approximately 512 nm. The addition of cadaverine causes the recognition group to bind to the amino group, disrupting the original electron transfer process of the probe molecule. This weakens the green fluorescence at approximately 512 nm and strengthens the blue fluorescence at approximately 452 nm, thus achieving ratiometric fluorescence detection of cadaverine. In ratio-based detection and recognition events, external factors have the same effect on the fluorescence signal intensity of both, so external factors are unlikely to affect the ratio of the two fluorescence intensities. This mode can greatly reduce the influence of external factors and improve detection precision and accuracy. Attached Figure Description

[0023] Figure 1 This is a one-dimensional proton NMR spectrum of the fluorescent probe of this invention. In the figure, the horizontal axis represents the chemical shift and the vertical axis represents the signal intensity.

[0024] Figure 2 This is the ultraviolet titration spectrum of the fluorescent probe of the present invention on cadaverine. In the figure, the horizontal axis is wavelength and the vertical axis is absorption intensity.

[0025] Figure 3 This is the fluorescence titration spectrum of cadaverine by the fluorescent probe of this invention. In the figure, the horizontal axis represents wavelength and the vertical axis represents fluorescence intensity.

[0026] Figure 4 This is a graph showing the response time of the fluorescent probe of this invention to cadaverine. In the graph, the horizontal axis represents different times, and the vertical axis represents fluorescence intensity.

[0027] Figure 5 This is a graph showing the selectivity of the test paper loaded with fluorescent probes of the present invention for volatile biogenic amines.

[0028] Figure 6 This is a graph showing the response of the test paper loaded with fluorescent probes of the present invention to cadaverine vapor at different concentrations (0, 3, 5, 7, 11, 14, 22, 27, 68, 137, 274, 685 ppm).

[0029] Figure 7This is a graph showing the results of measuring the freshness of beef samples at different times under different temperatures using the test paper loaded with fluorescent probes of this invention. In the graph: a is an image taken under a fluorescent lamp at 365nm, b is a bar graph of volatile biogenic amine content obtained at 25℃ using the test paper in (a) and the TVBN method, c is a bar graph of volatile biogenic amine content obtained at 0℃ using the test paper in (a) and the TVBN method, and d is a bar graph of volatile biogenic amine content obtained at -16℃ using the test paper in (a) and the TVBN method.

[0030] Figure 8 This is a graph showing the results of measuring the freshness of shrimp at different times under different temperatures using the test paper loaded with fluorescent probes according to the present invention.

[0031] Figure 9 This is a bar chart showing the fluorescence response of the fluorescent probe of this invention and other BODIPY derivatives to cadaverine. In the figure, Cl-BODIPY is the fluorescent probe of this invention.

[0032] Figure 10 This is a flowchart illustrating the preparation process of the fluorescent probe Cl-BODIPY of this invention. Detailed Implementation

[0033] Example 1: Preparation of the fluorescent probe Cl-BODIPY

[0034] The fluorescent probe Cl-BODIPY used in this invention for rapid and non-destructive detection of food freshness has the chemical formula C9H6BClF2N2 and its structural formula is as follows:

[0035]

[0036] like Figure 10 As shown, the preparation steps of Cl-BODIPY are as follows:

[0037] Compound 1 (1 mmol) and phosphorus oxychloride (8 mmol) were dissolved in 1,2-dichloroethane (16 mL). The reaction mixture was then heated under nitrogen atmosphere at 80 °C with stirring for 4 h. After the reaction was complete as detected by TCI, the reaction mixture was cooled in an ice bath and triethylamine (10 mmol) was slowly added. After stirring for 5 min, boron trifluoride diethyl ether (10 mmol) was slowly added. The reaction mixture was then heated to room temperature and the reaction was continued for 4 h. Excess solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a red solid as the target fluorescent probe. Its one-dimensional proton NMR spectrum is shown below. Figure 1 .

[0038] 1 H NMR (500MHz, CDCl3): d=7.89(brs,2H),7.41(d, 3 J = 4.3 Hz, 2H), 6.58 ppm (d,3 J = 4.3 Hz, 2H).

[0039] Preparation of test reagents:

[0040] (1) Preparation of probe stock solution: Weigh 5.0 mg of probe, dissolve it in dry dichloromethane, and prepare a probe solution with a probe concentration of 5 mM.

[0041] (2) Preparation of cadaverine stock solution: Take 5.0 mL of 1,5-diaminopentane, dissolve it in HPLC-grade acetonitrile, and prepare a solution with a concentration of 1 mM.

[0042] (3) Preparation of test strips loaded with probes: Soak filter paper strips (3×3cm) in 1mM probe solution for 30min, then remove them with tweezers and place them in a cool, dark place to air dry naturally to prepare test strips loaded with probes.

[0043] Example 2: Ultraviolet titration of cadaverine with a fluorescent probe

[0044] The UV spectral response of the fluorescent probe to cadaverine was verified using the fluorescent probe from Example 1. Figure 2 The fluorescent probe (concentration of 0.5 × 10⁻⁶) was shown. -5 The change in UV wavelength of an acetonitrile solution (mol / L) when cadaverine (0.78 molar amounts of the fluorescent probe) is added is shown. The probe initially exhibits a distinct absorption peak at 499 nm; upon addition of cadaverine, a new absorption peak appears at 395 nm. With increasing cadaverine concentration, the original absorption peak gradually weakens while the absorption peak at 395 nm gradually strengthens, and a noticeable color change from green to pale yellow is observed. This indicates that the fluorescent probe can achieve colorimetric detection of cadaverine.

[0045] Example 3: Fluorescent titration of cadaverine with a fluorescent probe

[0046] The fluorescence spectral response of the fluorescent probe to cadaverine was verified using the fluorescent probe from Example 1. Figure 3 The fluorescent probe (concentration of 0.5 × 10⁻⁶) was shown. -5 The fluorescence wavelength of the fluorescent probe was measured in acetonitrile solution (mol / L) when 0.78 molar amounts of cadaverine were added. The emission wavelength of the fluorescent probe changed from 512 nm to 452 nm, and a significant change in fluorescence from green to blue was observed under UV light. This indicates that the fluorescent probe can achieve ratiometric fluorescence detection of cadaverine.

[0047] Example 4: Response time of the fluorescent probe to cadaverine

[0048] The response time of the fluorescent probe to cadaverine was evaluated using the fluorescent probe from Example 1. Figure 4The fluorescent probe (concentration of 0.5 × 10⁻⁶) was shown. -5 The fluorescence intensity of an acetonitrile solution (mol / L) was measured when 0.78 molar amounts of cadaverine (the fluorescent probe) were added over time. The excitation wavelength of the fluorescent probe was 392 nm, and the emission wavelength was 452 nm. The fluorescence intensity stabilized within 120 s after the addition of 0.78 molar amounts of cadaverine to the fluorescent probe solution, indicating that the fluorescent probe has a good response speed to cadaverine and good practical applicability.

[0049] Example 5: Selectivity of fluorescent probes for volatile biogenic amines

[0050] The selectivity of the fluorescent probe for volatile biogenic amines was evaluated using the fluorescent probe from Example 1. The fluorescent probe (concentration 1.0 × 10⁻⁶) was used. -3 Test paper loaded with probes was prepared in a dichloromethane solution (mol / L). Figure 5 The results show changes in fluorescence and colorimetry of the test strips when various common biogenic amine-related interfering substances (such as EA, DEA, TEA, NH3, N2H4, H2O2, HCOOH, SO2, H2S, HCHO, TMP, Tyr, His, Put, and Cad) vapors are added. When the probe-loaded test strips respond to cadaverine and putrescine, a significant fluorescence change from green to blue and a colorimetric change from dark brown to pale yellow are observed. However, no significant fluorescence or colorimetric changes are observed when various common biogenic amine interfering species are added, indicating that the fluorescent probe has good selectivity for cadaverine and putrescine and has good practical applicability.

[0051] Example 6: Colorimetric and fluorescence responses of test paper loaded with fluorescent probes to different concentrations of cadaverine vapor.

[0052] The fluorescent probes from Example 1 were used to evaluate the colorimetric and fluorescence responses of the test paper loaded with the fluorescent probes to different concentrations of cadaverine vapor. For example... Figure 6 As shown, with the increase of cadaverine vapor concentration, a significant fluorescence change of the test paper gradually from green to blue and a colorimetric change gradually from dark brown to pale yellow can be observed. The results indicate that the probe-loaded test paper can achieve rapid and visual detection of cadaverine vapor, demonstrating good practical analytical applicability.

[0053] Example 7: Determination of the freshness of beef samples stored for different times at different temperatures using probe-loaded test paper.

[0054] The fluorescent probes from Example 1 were used to evaluate the response of probe-loaded test paper to beef samples placed for different durations at different temperatures. Figure 7The fluorescence response of probe-loaded test paper to beef samples placed in sealed dishes for 0, 1, 3, 5, 7, 9, 12, and 24 hours at three temperatures: -16℃, 0℃, and 25℃ is shown. The results indicate that the probe-loaded test paper demonstrates good practical applicability and can achieve non-destructive, visual detection of beef sample freshness.

[0055] Example 8: Determination of shrimp freshness at different temperatures and storage times using probe-loaded test paper.

[0056] The fluorescent probes from Example 1 were used to evaluate the response of probe-loaded test paper to shrimp samples placed for different durations at different temperatures. Figure 8 The fluorescence response of probe-loaded test paper to shrimp placed in sealed dishes for 0, 1, 3, 5, 7, 9, 12, and 24 hours at three temperatures: -16℃, 0℃, and 25℃. The results indicate that the probe-loaded test paper demonstrates good practical applicability and can achieve non-destructive, visual detection of shrimp sample freshness.

[0057] Example 9: Fluorescence response of various BODIPY derivatives to cadaverine

[0058] To verify the specificity of the fluorescent probe Cl-BODIPY of the present invention, the inventors also conducted fluorescence spectral response tests on the following other BODIPY derivatives in response to cadaverine.

[0059]

[0060] Figure 9 Other BODIPY derivatives (concentration 0.5 × 10⁻⁶) were shown. -5 The change in fluorescence intensity of an acetonitrile solution (mol / L) when cadaverine (0.78 molar amounts of the fluorescent probe) is added is measured. The emission wavelength of the fluorescent probe Cl-BODIPY of this invention changes from 512 nm to 452 nm, and a significant change in fluorescence from green to blue is observed under UV light, while other BODIPY derivatives show no significant wavelength change. This indicates that cadaverine only responds to the fluorescent probe Cl-BODIPY of this invention; other BODIPY derivatives show neither colorimetric nor fluorescent responses to volatile biogenic amines.

Claims

1. The application of compound Cl-BODIPY in rapid, non-destructive testing of food freshness, characterized by: The structural formula of the compound Cl-BODIPY is as follows: ； The food items mentioned are fish and meat.

2. A rapid and non-destructive method for detecting the freshness of food, characterized in that... Test paper loaded with compound Cl-BODIPY as a fluorescent probe was placed in a sealed dish containing a fish or meat sample; the structural formula of the compound Cl-BODIPY is as follows. 。 3. The detection method according to claim 2, characterized in that: The temperature in the sealed dish is -16℃, 0℃, or 25℃.

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