Fluorescent probe for detecting biogenic amine and visual detection of food spoilage thereof

Abstract

The invention belongs to the technical field of synthetic chemistry and food detection, and provides a fluorescent probe for detecting biogenic amine and visual detection of food spoilage of the fluorescent probe. The fluorescent probe has the following structure. According to the fluorescent probe provided by the invention, fluorescence is obviously enhanced after BAs is added into the fluorescent probe, and the fluorescent probe can be applied to detection of biogenic amines in vegetables, dairy products and meat and has potential application value in the field of actual food sample decay detection.

Description

Technical Field

[0001] The invention belongs to the technical field of synthetic chemistry and food detection, and particularly relates to a fluorescent probe for detecting biogenic amines and application thereof. Background Art

[0002] Microbial activity in protein-rich foods like meat, fish, and dairy products can also lead to the production of biogenic amines (BAs). When food is contaminated by bacteria or mold, these microorganisms metabolize the amino acids in the food, producing biogenic amines. Microbial growth and reproduction in protein-rich foods like meat, fish, and dairy products break down proteins into biogenic amines. Excessive biogenic amines can be harmful to the human body, causing symptoms of poisoning such as headaches, nausea, vomiting, and diarrhea. They may also be associated with the development of some chronic diseases. Their metabolites can interact with biomacromolecules such as intracellular DNA, causing gene mutations and chromosomal damage.

[0003] Several conventional detection techniques are currently used to measure BAs, such as gas chromatography-mass spectrometry (GC-MS), enzyme assays, electrochemical analysis, and chromatography. Although these methods can determine BA levels, they require complex and time-consuming sample preparation procedures, hindering rapid and convenient on-site detection. In contrast, fluorescent probes are highly attractive due to their small molecular weight, high sensitivity, and non-destructive testing capabilities. Currently, a variety of fluorescent materials / probes have been developed for rapid detection of bioassays, including MOFs, fluorescein dyes, and nanoparticles. However, these probes / materials exhibit background fluorescence, making even faint fluorescence changes difficult for untrained users to discern. Therefore, there is an urgent need to design a fluorescent probe that can be used for non-destructive detection, is simple and portable, requires convenient testing conditions, offers low testing difficulty, and allows for visual monitoring. Summary of the Invention

[0004] In view of the problems in the prior art, the present invention provides a fluorescent probe for detecting biogenic amines, which can be used for the detection of actual samples.

[0005] Another object of the present invention is to provide an application of the fluorescent probe mentioned above in detecting biogenic amines in food.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions.

[0007] A fluorescent probe for detecting biogenic amines, 3-oxo-3H-phenoxazin-7-yl-4-methylbenzoate, referred to as DBP-X, has a chemical structure as shown in Formula (I): Formula (I).

[0008] A method for preparing the above fluorescent probe comprises the following steps: In a protective atmosphere, methyl benzoyl chloride and resorufin react in the presence of an acid binding agent, and the reaction liquid is separated and purified to obtain the product.

[0009] The protective atmosphere is provided by nitrogen or argon.

[0010] The acid binding agent is preferably triethylamine.

[0011] The reaction temperature is room temperature.

[0012] The steps of separation and purification are as follows: adding saturated brine and ethyl acetate to the reaction solution for extraction; subjecting the ethyl acetate extract to silica gel column chromatography using petroleum ether and ethyl acetate in a volume ratio of 5:1 as eluent, and the obtained orange solid is the fluorescent probe.

[0013] An application of the fluorescent probe in detecting biogenic amines in solutions and foods.

[0014] The biogenic amine is selected from cadaverine or putrescine.

[0015] A method for detecting biogenic amines in food comprises the following steps: The food or the food extract is reacted with the fluorescent probe, and then the fluorescence intensity of the fluorescent probe or the R / (B+G) value of the analytical system is measured.

[0016] Preferably, the extracting solution uses dimethyl sulfoxide as the extraction solvent.

[0017] The R / (B+G) value is obtained by analyzing the RGB values ​​of a picture obtained under sunlight or ultraviolet light.

[0018] The mechanism of the present invention is as follows: The fluorescent probe DBP-X for detecting BAs described in the present invention itself is ICT-forbidden due to the weak electron-donating ability of the methyl group, resulting in no fluorescence. When the probe interacts with BAs molecules, the acetyl ester group on the probe DBP-X is converted to a hydroxyl group by BAs. Since the hydroxyl group is a strong electron-donating group, the ICT-forbidden effect disappears, and the fluorescence intensity is greatly improved.

[0019] The present invention has the following advantages: The DBP-X fluorescent probe provided by the present invention belongs to the small molecule class of fluorescent probes. Its fluorescence is significantly enhanced upon addition of BAs. This result and phenomenon establish a theoretical foundation for the detection of biogenic amines in liquid samples and suggests potential application in the field of food sample spoilage detection. The probe of the present invention can not only detect the spoilage process of vegetables using fluorescence imaging technology, but also assess the degree of food spoilage in meat and milk samples under sunlight and natural light using RGB analysis on a smartphone. This allows for rapid and portable detection and has potential application in the research of non-destructive food spoilage detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the hydrogen spectrum of probe DBP-X; Figure 2 is the carbon spectrum of probe DBP-X; Figure 3 This is a titration experiment of the interaction between the probe DBP-X and cadaverine; the excitation wavelength is 540 nm; the probe concentration is 10µM; Figure 4 This is a titration experiment of the interaction between the probe DBP-X and putrescine; the excitation wavelength is 540 nm; the probe concentration is 10µM; Figure 5 This is a kinetic experiment of the interaction of probe DBP-X with cadaverine and putrescine. The excitation wavelength is 540 nm, the probe concentration is 10 µM, the concentration of cadaverine and putrescine is 100 µM, and the test time is 30 min. Figure 6 The selectivity of the probe DBP-X in the organic phase is shown in Figure 5. The excitation wavelength is 540 nm. The probe concentration is 10 µM, the concentration of the selective ion (or amino acid) is 200 µM, the concentration of the reactive oxygen (or reactive nitrogen) is 200 μM, the concentration of the aldehyde and ketone is 200 μM, and the emission wavelength is 598 nm. Figure 7 This is an imaging application of the probe DBP-X and onion cells stored at different temperatures, with an excitation wavelength of 540 nm. Figure 8 The DBP-X probe is used to detect fresh meat products; RGB color is used to draw a difference analysis diagram; Figure 9 This is the food detection of fresh milk using the probe DBP-X; and the RGB color is used to draw a difference analysis diagram. DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to the embodiments and drawings, but the present invention is not limited to the following embodiments.

[0022] Example 1 Synthesis of fluorescent probe p-Toluyl chloride (0.914 mL, 6.049 mmol) was added to 11 mL of anhydrous DMF and added dropwise to resorufin (0.644 mg, 3.025 mmol) and triethylamine (1.35 mL). The reaction was allowed to react at room temperature under nitrogen protection and traced by TLC. After the reaction was completed (about 30 h), 20 mL of saturated brine and 100 mL of ethyl acetate were added for extraction. The ethyl acetate extract was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1 v / v). The solvent was removed from the eluent to obtain an orange powdery substance, which was the probe DBP-X (345 mg, 34.5%). 1 H NMR (400 MHz, CDCl3) δ 8.13 (d, J = 8.2 Hz, 2H), 7.89 (d, J = 8.5 Hz, 1H), 7.49 (d, J = 9.8 Hz, 1H), 7.38 (d, J = 8.0 Hz, 2H), 7.32 (s, 2H), 6.91 (dd, J = 9.8, 2.0 Hz, 1H), 6.38 (d, J = 2.0 Hz, 1H), 2.51 (s, 3H); 13 C NMR(101 MHz, CDCl3) δ 160.04, 155.20, 150.56, 149.35, 132.81, 128.24, 122.40,118.99, 116.85, 110.83, 109.48, 108.98, 96.68, 65.89, 44.91, 24.73, 20.77,12.66; 1 H NMR spectrum Figure 1 ; 13 C NMR spectrum Figure 2 .

[0023] Example 2 Response of fluorescent probe to different concentrations of BAs Prepare 4 mL of 10 mM aqueous solution of cadaverine and putrescine and 1 mM fluorescent probe stock solution prepared in Example 1. Prepare the probe at a concentration of 10 μM and interact with different concentrations of cadaverine and putrescine (0-100 μM) for fluorescence detection (λ ex = 540 nm, λ em= 603 nm), calculated the fluorescence intensity in each system, and established a standard curve between fluorescence intensity and BAs concentration.

[0024] The results are as follows Figure 3 , as shown in Figure 4: Within the test concentration range, as the concentration of BAs increases, the fluorescence intensity of the reaction system gradually increases linearly. Under the above conditions, the detection limits (LODs) of the fluorescent probe for cadaverine and putrescine are 9.08×10 -7 M and 1.52×10 -7 M.

[0025] Example 3 Response kinetics of fluorescent probe to different concentrations of BAs Prepare 4 mL of a 10 mM aqueous solution of BAs (cadaverine and putrescine) and a 1 mM fluorescent probe stock solution prepared in Example 1. Prepare solutions of probes and BAs at concentrations of: 10 µM probe; 100 µM cadaverine and 100 µM putrescine. Perform fluorescence detection (λ ex = 540 nm, λ em = 603 nm), test once every 1 min for 30 min, calculate the fluorescence intensity of each system over time, and establish a standard curve of fluorescence intensity and action time. Figure 5 As shown, after about 10 min of reaction, the fluorescence intensity of the reaction system reached saturation.

[0026] Example 4 Selectivity of fluorescent probes for different ions Prepare 4 mL of 10 mM aqueous solutions of various common ions and amino acids and 1 mM fluorescent probe stock solution prepared in Example 1. Add 30 μL of probe stock solution, 2910 μL of DMSO, and 20 equivalents of each ion (or amino acid) solution, shake well, and perform fluorescence detection (λ ex = 540 nm, λ em = 598nm), and establish a curve diagram of fluorescence intensity and each ion (or amino acid, active oxygen, active nitrogen). The results are shown in Figure 6 , where ions added in 1-31 are: probe, formaldehyde, chloral hydrate, acetone, hydrogen peroxide, hypochlorous acid, ferric sulfate, zinc nitrate, sodium bisulfite, sodium chloride, magnesium sulfate, potassium chloride, copper sulfate, L-leucine, L-arginine, glutathione, glycine, L-cysteine, L-histidine, L-tryptophan, L-glutamic acid, isoleucine, L-valine, L-aspartic acid, L-threonine, serine, L-alanine, L-methionine, L-proline, putrescine, cadaverine. Figure 6It can be found that other ions (or amino acids) have almost no effect on the fluorescence of compound DBP-X, while the addition of BAs significantly enhances the fluorescence of compound DBP-X.

[0027] Application Example 1: Detection of vegetables in different storage states The epidermal cells of the same location of the purchased fresh onion cells were divided into three equal parts and stored at 4°C, ambient temperature, and 32°C for 3 days. On the day of the test, the epidermal cells of the same location of the purchased new onion were tested. The four groups of onion cells were immersed in a test system of DBP-X 10 µM added to 1 mL of DMSO:PBS = 1:9, incubated for 20 minutes, then washed 3 times with PBS, placed on a slide, and covered with a cover glass to remove bubbles. The cells were then imaged on a confocal microscope. Figure 7 It can be seen that no matter at which temperature the onion cells are stored, they all produce red fluorescence compared with fresh onion cells, that is, they all produce biogenic amines. Among them, the fluorescence intensity at 4°C is the weakest, indicating that low temperature can delay the decay process; the fluorescence intensity at 32°C is the strongest, which means the degree of decay is the greatest.

[0028] Application Example 2: Detection of meat in different storage states Fresh pork (alternating fat and lean) was purchased, cut into pieces (1 cm × 1 cm), and divided into three equal parts. The pork was stored at 4°C, ambient temperature, and 32°C respectively. Filter paper of the same size (1 cm × 3 cm) soaked in the probe solution (1 mM) was placed next to the pork. The pork and filter paper were sealed in a sealed box. The pork was photographed at 365 nm at 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 60 h, and the RGB values ​​were obtained by combining the color capture with a smartphone.

[0029] The results are as follows Figure 8 As shown, Figure (a) is a photograph of storage at different temperatures under 365 nm light, Figure (b) is a differential analysis of the changes in R / (B+G) over time at 32°C, Figure (c) is a differential analysis of the changes in the R / (B+G) values ​​of the test strips at 4°C over time; Figure (d) is a differential analysis of the changes in R / (B+G) values ​​of the test strips at ambient temperature over time.

[0030] Depend on Figure 8 It can be seen that when stored at 32°C, the color change of the test strip is the most obvious, with the largest color difference. The color change of the test strip at 4°C is relatively the least obvious. Two-way ANOVA is used to analyze the differences between the R / (B+G) values ​​at 0 h and the R / (B+G) values ​​at 60 h at different temperatures. The results are as follows: Figure 8As shown, the analysis showed that at 32°C and ambient temperature, p<0.0001, and at 4°C, p<0.001, indicating that the three groups had extremely significant differences. Combined with the bar graph, the difference value at 32°C was the largest, indicating that the present invention can sensitively detect biogenic amines in solid food samples at different temperatures.

[0031] Application Example 3: Detection of dairy products in different storage states Prepare the fluorescent probe stock solution prepared in Example 1 at a concentration of 1 mM for later use. Commercially available fresh milk was divided into two portions and stored at ambient temperature and 4°C, respectively. For each test, the same amount of milk (30 µL) was placed in 3 mL of DMSO and 30 µL of the probe stock solution was added. Every other day, photographs were taken at 365 nm under daylight conditions. RGB color analysis was performed using a smartphone, and a differential analysis bar graph was plotted.

[0032] See the results Figure 9 Figure (a) shows photos taken at different temperatures under sunlight and 365 nm, Figures (b) and (c) are histograms of the difference analysis of R / (B+G) of sunlight and 365 nm at ambient temperature, and Figures (d) and (e) are histograms of the difference analysis of R / (B+G) of 365 nm and sunlight at 4°C.

[0033] Depend on Figure 9 It can be seen that under different temperature conditions, the color difference of ambient temperature is more obvious than that of 4℃. The R / (B+G) values ​​of fluorescence and daylight photos at different temperatures were analyzed using one-way ANOVA. The results are as follows: Figure 9 As shown, the analysis showed that there were significant differences among the four groups, that is, p<0.05, which shows that the present invention can sensitively detect biogenic amines in liquid food samples at different temperatures.

Claims

1. A fluorescent probe for detecting biogenic amines, the chemical structure of which is shown in formula (I): Formula (I).

2. A method for preparing a fluorescent probe according to claim 1, characterized in that: The following steps are involved: In a protective atmosphere, methyl benzoyl chloride and resorufin react in the presence of an acid binding agent, and the reaction liquid is separated and purified to obtain the product.

3. The preparation method according to claim 2, characterized in that The protective atmosphere is provided by nitrogen or argon; the acid binding agent is triethylamine; and the reaction temperature is room temperature.

4. The preparation method according to claim 2, characterized in that The steps of separation and purification are as follows: adding saturated brine and ethyl acetate to the reaction solution for extraction; subjecting the ethyl acetate extract to silica gel column chromatography using petroleum ether and ethyl acetate in a volume ratio of 5:1 as eluent, and the obtained orange solid is the fluorescent probe.

5. Use of the fluorescent probe according to claim 1 in detecting biogenic amines in solutions and foods.

6. The use according to claim 5, characterized in that The biogenic amine is selected from cadaverine or putrescine.

7. A method for detecting biogenic amines in food, characterized in that: The following steps are involved: The food or the food extract is reacted with the fluorescent probe, and then the fluorescence intensity of the fluorescent probe or the R / (B+G) value of the analytical system is measured.

8. The method according to claim 7, characterized in that The extracting solution uses dimethyl sulfoxide as the extraction solvent.

9. The method according to claim 7, characterized in that The R / (B+G) value is obtained by analyzing the RGB values ​​of a picture obtained under sunlight or ultraviolet light.

Images