Fluorescence ratio type method and visual detection method for detecting histamine
Through the reaction system of copper nanoclusters and silicon quantum dots and histamine, the fluorescence ratio method is used to detect histamine, which solves the complexity and insensitiveness of detecting histamine in the prior art, and achieves rapid and accurate histamine detection, which is suitable for food freshness evaluation.
Patent Information
- Application Number
- CN202510004014.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-02
- Publication Date
- 2025-04-04
AI Technical Summary
In the detection of histamine, the prior art has problems such as long sample preparation time, cumbersome operation, expensive instruments, complex synthesis of fluorescent probes, and susceptible to environmental factors. It is difficult to achieve simple and efficient on-site detection.
A reaction system was formed by copper nanoclusters and silicon quantum dots and histamine, and histamine was detected by fluorescence ratio method, and a standard curve was made using the fluorescence intensity ratio F615/F437 at wavelengths of 615nm and 437nm to achieve rapid detection of histamine concentration.
This method is simple to operate, has high sensitivity, good specificity, has low detection limit, has good repeatability, can quickly and accurately detect histamine, and is suitable for the evaluation of food freshness.
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Figure CN119757305A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biogenic amine detection, and in particular relates to a fluorescence ratio method and a visual detection method for detecting histamine. Background Art
[0002] Food safety is closely related to human health. The content of biogenic amines increases as food spoils, and is therefore considered one of the indicators of food freshness and quality. Histamine is a biogenic amine that is associated with allergic inflammatory reactions and foodborne diseases caused by eating spoiled food, and is widely present in food. Improper fermentation or preservation of food may produce high levels of histamine. Ingestion of foods containing large amounts of histamine can lead to histamine poisoning, causing symptoms such as nausea and vomiting. Therefore, it is crucial to explore reliable and efficient methods for detecting biogenic amines, especially histamine analysis methods.
[0003] To date, a variety of analytical methods for detecting histamine have been established, including fluorescence, colorimetry, chromatography, electrochemistry, capillary electrophoresis, and enzyme-linked methods. Although some of these methods have high sensitivity, they often have some disadvantages, such as long sample preparation time, cumbersome operation, expensive instruments, etc., and they are not suitable for on-site detection. In contrast, fluorescent sensors have unique attractions such as high sensitivity, simple operation, and low cost. However, most of them have disadvantages such as complex synthesis process of organic small molecule fluorescent probes, and single-wavelength fluorescence changes are easily affected by environmental factors and produce false positive signals. Therefore, it is very necessary to provide a simple, ratiometric fluorescence and visualization detection method for histamine detection. Summary of the invention
[0004] In view of the shortcomings of the prior art, the present invention provides a fluorescence ratio method and a visual detection method for detecting histamine.
[0005] The technical solution of the present invention is as follows:
[0006] A fluorescence ratio method for detecting histamine comprises the following steps:
[0007] The copper nanocluster solution, silicon quantum dot solution and histamine solution of different concentrations were mixed and fixed to the same volume. After the reaction was allowed to stand, the mixed solutions of each system were obtained. The fluorescence emission spectra of each system under an excitation wavelength of 380 nm were detected. The ratio of the fluorescence intensity at wavelengths of 615 nm and 437 nm was used as F. 615 / F 437 A standard curve is prepared with the concentration of histamine solution as the vertical axis and the concentration of histamine solution as the horizontal axis, which is used to detect the concentration of histamine in the sample to be tested.
[0008] Preferably, according to the present invention, in the fluorescence ratio method, the volume is fixed to the same volume with deionized water, and the reaction is allowed to stand at room temperature for more than 60 minutes to obtain a mixed solution of each system.
[0009] Further preferably, in the fluorescence ratio method, the volume is fixed to the same volume with deionized water, and the reaction is allowed to stand at room temperature for 60 minutes to obtain a mixed solution of each system.
[0010] According to the preferred embodiment of the present invention, in the fluorescence ratio method, the copper nanocluster solution, the silicon quantum dot solution and the histamine solution of different concentrations are mixed and fixed to the same volume, and the final concentration of the copper nanocluster added to each system is 17-267 μg·mL -1 .
[0011] Preferably, the final concentration of copper nanoclusters added to each system is 130-140 μg·mL -1 .
[0012] More preferably, the final concentration of copper nanoclusters added to each system is 133.3 μg·mL -1 .
[0013] Preferably according to the present invention, in the fluorescence ratio method, when preparing the standard curve, the final concentration of histamine is 0.4-7 mM.
[0014] Preferably according to the present invention, in the fluorescence ratio method, the volumes of silicon quantum dot solutions added are equal.
[0015] Further preferably, in the fluorescence ratio method, the ratio of the volume of the silicon quantum dot solution added to the volume after constant volume is 1:(290-310).
[0016] More preferably, in the fluorescence ratio method, the ratio of the volume of the silicon quantum dot solution added to the volume after constant volume is 1:300.
[0017] Preferably according to the present invention, in the fluorescence ratio method, a standard curve method is used to detect the concentration of histamine in the sample to be tested.
[0018] Further preferably, in the fluorescence ratio method, the copper nanocluster solution, the silicon quantum dot solution and the sample solution to be tested are mixed by the same method as that for preparing the standard curve, and the volume is fixed to the same volume with deionized water. After the reaction is allowed to stand, a mixed solution of each system is obtained, and the fluorescence emission spectrum at an excitation wavelength of 380 nm is detected and recorded. The ratio of the fluorescence intensity at wavelengths of 615 nm and 437 nm is used as the value F 615 / F 437 , using the prepared standard curve, calculate the histamine content in the sample to be tested.
[0019] Preferably, according to the present invention, the fluorescence ratio method comprises the following steps:
[0020] 100 μL, 2 mg mL-1 The copper nanocluster solution, 50 μL silicon quantum dots and histamine solutions of different concentrations were mixed and fixed to 1.5 mL with deionized water. After the reaction was allowed to stand for 60 min, the mixed solutions of each system were obtained. The fluorescence emission spectra of each system were detected at an excitation wavelength of 380 nm. The ratio of the fluorescence intensity at wavelengths of 615 nm and 437 nm was used as the fluorescence intensity ratio F. 615 / F 437 A standard curve is prepared with the concentration of histamine solution as the vertical axis and the concentration of histamine solution as the horizontal axis, which is used to detect the concentration of histamine in the sample to be tested.
[0021] Preferably according to the present invention, the linear range of the detection method is 0.4-7 mM and the detection limit is 16.6 μM.
[0022] A hydrogel comprises the following components: copper nanoclusters, silicon quantum dot solution, sodium alginate and calcium chloride.
[0023] The method for preparing the hydrogel comprises the following steps:
[0024] The copper nanocluster solution, the silicon quantum dot solution and the sodium alginate solution were stirred and mixed, a calcium chloride solution was added and allowed to stand, and the solution was washed with deionized water to prepare a hydrogel.
[0025] Preferably, according to the present invention, the method for preparing the hydrogel comprises the following steps:
[0026] Take 2 mg mL -1 The copper nanocluster solution, the silicon quantum dot solution and the sodium alginate solution with a mass volume ratio of 1: (0.3-0.8): (10-15) were mixed and stirred for 55-65 minutes to obtain a mixture. The mixture was mixed with 75mM calcium chloride solution at a volume ratio of 1: (1.8-2.2), allowed to stand for more than 60 minutes, and washed with deionized water to prepare a hydrogel.
[0027] Further preferably, the method for preparing the hydrogel comprises the following steps:
[0028] Take 2 mg mL -1 The copper nanocluster solution, the silicon quantum dot solution and the 1% sodium alginate solution by mass volume ratio were mixed in a volume ratio of 1:0.5:13.5, and stirred for 60 minutes to obtain a mixture. The mixture was mixed with 75mM calcium chloride solution in a volume ratio of 1:2, allowed to stand for more than 60 minutes, and washed with deionized water to prepare a hydrogel.
[0029] Preferably according to the present invention, the hydrogel is prepared into strips or sheets using a mold.
[0030] Application of the hydrogel or the hydrogel prepared by the method in detecting histamine in a sample.
[0031] Preferably according to the present invention, the hydrogel or the hydrogel prepared by the above method is used to evaluate the freshness of a sample by detecting histamine in the sample.
[0032] Preferably according to the present invention, the sample is food.
[0033] Further preferably, the food includes fish, shrimp and dairy products.
[0034] A method for visual detection of histamine comprises the following steps:
[0035] The hydrogel or the hydrogel prepared by the above method is placed in contact with the sample to be tested or the hydrogel is immersed in the sample to be tested, and the hydrogel is irradiated with ultraviolet light, and the change of histamine in the sample to be tested is determined according to the color change.
[0036] Preferably according to the present invention, in the histamine visualization detection method, the wavelength of ultraviolet light irradiation is 365 nm.
[0037] Preferably, according to the present invention, in the histamine visualization detection method, as the histamine content in the sample to be tested increases, the color of the hydrogel gradually changes from red to blue.
[0038] In the fluorescence ratio method for detecting histamine, the hydrogel or the method for preparing the hydrogel, the solvent of the copper nanocluster solution is ethanol.
[0039] In the above-mentioned fluorescence ratio method for detecting histamine, hydrogel or method for preparing hydrogel, the pH value of the silicon quantum dot solution is adjusted to 7.0-7.4.
[0040] In the fluorescence ratio method for detecting histamine, the hydrogel or the method for preparing the hydrogel, the method for preparing the copper nanoclusters comprises the following steps:
[0041] Copper sulfate, 2,3,5,6-tetrafluorobenzenethiol and ethanol are stirred and mixed, and the precipitate is collected after centrifugation, washed with ethanol, and dried to obtain copper nanoclusters.
[0042] Preferably, according to the present invention, the method for preparing the copper nanoclusters comprises the following steps:
[0043] Copper sulfate and 2,3,5,6-tetrafluorothiophenol were mixed in ethanol at a mass volume ratio of 1:(1-3) g / mL, stirred at room temperature for more than 30 minutes, and the precipitate was collected by centrifugation, washed with ethanol for more than 3 times, and dried to obtain copper nanoclusters.
[0044] Further preferably, the method for preparing the copper nanoclusters comprises the following steps:
[0045] Copper sulfate and 2,3,5,6-tetrafluorothiophenol were mixed in ethanol at a mass volume ratio of 1:2.25 g / mL, stirred at room temperature for more than 30 minutes, and the precipitate was collected by centrifugation, washed with ethanol for more than 3 times, and dried to obtain copper nanoclusters.
[0046] In the fluorescence ratio method for detecting histamine, the hydrogel or the method for preparing the hydrogel, the method for preparing the silicon quantum dot solution comprises the following steps:
[0047] 3-Aminopropyltrimethoxysilane was mixed evenly with deionized water and transferred into an autoclave for reaction. After the reaction was completed, the mixture was cooled to room temperature, the product was collected, ethyl acetate was added, the mixture was allowed to stand until the phases were separated, and the aqueous phase at the bottom was the silicon quantum dot solution.
[0048] Further preferably, the method for preparing the silicon quantum dot solution comprises the following steps:
[0049] Mix 3-aminopropyltrimethoxysilane and deionized water evenly, with a volume ratio of 3-aminopropyltrimethoxysilane to deionized water of 1:(5-6); transfer to an autoclave for reaction at 180-200°C for more than 8 hours, cool to room temperature, and collect the product; add ethyl acetate with a volume 5-10 times that of the product, mix and let stand until the phases are separated, and the water phase at the bottom is the silicon quantum dot solution.
[0050] The beneficial effects of the present invention include at least the following:
[0051] 1. The present invention firstly discovered that the reaction system composed of copper nanoclusters, silicon quantum dots and histamine can detect histamine by ratiometric fluorescence spectroscopy, and the detection of histamine has strong selectivity and good specificity.
[0052] 2. The present invention synthesized the copper nanocluster / silicon quantum dot-sodium alginate hydrogel test strip for the first time, which is specific for histamine detection and can realize in-situ visual detection of food samples.
[0053] 3. The present invention provides a ratiometric fluorescence detection method for detecting histamine, which is quick and simple to operate, has high sensitivity, good specificity, low detection limit, and good repeatability; the linear range of the fluorescence method of the present invention is 0.4-7mM, and the detection limit is: 16.6μM (S / N=3). BRIEF DESCRIPTION OF THE DRAWINGS
[0054] Figure 1 Electron microscopy characterization of copper nanoclusters, silicon quantum dots, and hydrogel test strips;
[0055] In the figure: A is a scanning electron microscope image of copper nanoclusters; B is a transmission electron microscope image of silicon quantum dots; C is a scanning electron microscope image of hydrogel.
[0056] Figure 2Spectral graph of the excitation wavelength selected for the detection conditions;
[0057] In the figure: A is the excitation and emission spectra of copper nanoclusters; B is the fluorescence emission spectra of silicon quantum dots under different excitation wavelengths.
[0058] Figure 3 This is a diagram showing the effects of different conditions on histamine detection;
[0059] In the figure: A is the effect of copper nanocluster concentration on histamine detection; B is the effect of reaction time on histamine detection.
[0060] Figure 4 The fluorescence spectra are for detecting different histamine concentrations.
[0061] Figure 5 The standard curve is the ratio of fluorescence intensity to histamine concentration.
[0062] Figure 6 This is a diagram of the selectivity results of the detection method provided by the present invention.
[0063] Figure 7 Photos of hydrogel test strips of fish samples stored at 4°C and 25°C for different periods of time under UV light.
[0064] Figure 8 Photos of hydrogel test strips of shrimp samples stored at 4°C and 25°C for different periods of time under UV light. DETAILED DESCRIPTION
[0065] The present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.
[0066] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.
[0067] All chemical reagents were of analytical grade.
[0068] Experimental Example 1
[0069] Morphological characterization of prepared materials
[0070] Preparation of copper nanoclusters: 0.016 g of copper sulfate and 36 μL of 2,3,5,6-tetrafluorobenzenethiol were mixed in 10 mL of ethanol, stirred at room temperature for 30 min, centrifuged at 5000 rpm for 5 min to collect the yellow precipitate, washed with ethanol three times, and dried at 50° C. to obtain copper nanoclusters. In the copper nanocluster solution described below, ethanol is used as the solvent.
[0071] Preparation of silicon quantum dot solution: Mix 4 mL of 3-aminopropyltrimethoxysilane with 24 mL of deionized water, transfer to a 50 mL polytetrafluoroethylene-lined stainless steel autoclave, and react at 180 ° C for 8 hours. After the reaction is completed, the system is naturally cooled to room temperature. Collect the product, add ethyl acetate 10 times the volume of the product, stir and mix, and let stand until the phases separate. The aqueous phase at the bottom is the purified silicon quantum dot solution. The silicon quantum dot solution used below uses hydrochloric acid to adjust the pH of the silicon quantum dot solution to 7.
[0072] Preparation of hydrogel test strips: Take 1 mL, 2 mg mL -1 The copper nanocluster solution, 0.5 mL of silicon quantum dot solution adjusted to pH 7, and 13.5 mL of sodium alginate solution (1%, w / v, g / mL) were stirred and mixed for 60 min. 0.5 mL of the above mixture and 1 mL of 75 mM calcium chloride solution were added to each hole (2 cm in diameter and 1 cm in height) in the mold, and the mixture was allowed to stand for 60 min. The hydrogel test strips were prepared after washing with deionized water.
[0073] The prepared copper nanoclusters, silicon quantum dots and hydrogel test strips were characterized by scanning and transmission electron microscopy. The experimental results are shown in Figure 1 .like Figure 1 As shown in A, the copper nanocluster has a ribbon-like structure with a width of about 20 nm. Figure 1 Figure B shows that the average diameter of silicon quantum dots is about 3 nm. Figure 1 The results in Figure 3C show that the prepared alginate hydrogel has an obvious porous structure.
[0074] Experimental Example 2
[0075] Selection of the optimal excitation wavelength for detection conditions
[0076] The experimental method is: using fluorescence spectroscopy, record the excitation spectrum of copper nanoclusters in ethanol and the emission spectrum at the optimal excitation wavelength; record the emission spectrum of silicon quantum dots at different excitation wavelengths. The experimental results are shown in Figure 2 ,Depend on Figure 2 As shown in Figure A, the excitation spectrum of copper nanoclusters has two maximum peaks at 330 and 430 nm. At excitation wavelengths of 330 and 430 nm, copper nanoclusters exhibit strong luminescence, with the emission peak located at 615 nm. At an excitation wavelength of 330 nm, its spectral peak shape is less complete, while at 430 nm, its peak shape is complete but the emission intensity is relatively low. Figure 2As shown in Figure B, when the excitation wavelength is 320-390 nm, the emission wavelength of silicon quantum dots slightly red-shifts from 415-450 nm, indicating that it has the characteristic of wavelength excitation dependence. The inventors found that selecting an excitation wavelength of 380 nm as the optimal excitation wavelength for the detection system can ensure the spectrum integrity of copper nanoclusters at this wavelength, have a certain fluorescence emission intensity, and ensure that silicon quantum dots have good luminescence performance.
[0077] Selection of the Optimal Experimental Conditions for Histamine Detection
[0078] The experimental method is: fix the silicon quantum dot concentration and reaction time, and change the copper nanocluster concentration; fix the silicon quantum dot concentration and copper nanocluster concentration, and change the reaction time. Mix the copper nanocluster solution, the silicon quantum dot solution with a neutral pH value (pH value of 7) and the histamine solution, and dilute to 1.5mL with deionized water. After the reaction is allowed to stand, a mixed solution of each system is obtained, and the histamine concentration in the detection system is 7mM. Detect and record the fluorescence emission spectrum at an excitation wavelength of 380nm.
[0079] The experimental results are shown in Figure 3 , Figure 3 A shows that 100 μL of copper nanocluster solutions of different concentrations (0.25, 0.5, 1, 2, 4 mg·mL) were added to the above system (1.5 mL after constant volume). -1 ) The corresponding test result diagram, the test results of different concentrations and different reaction times are shown in Figure 3 From A and B in the figure, we can see that the concentration of copper nanoclusters is 2 mg·mL -1 (The final concentration in the system is 133.3 μg mL -1 ), room temperature reaction time 60min is the best experimental condition for histamine detection. Figure 3 As shown in A, with the addition of copper nanoclusters, △F (F 615 / F 437 The ratio difference) increased and reached a stable state, and the copper nanocluster concentration was selected to be 2 mg·mL -1 (The final concentration in the system is 133.3 μg mL -1 ). Reaction time Figure 3 As shown in B. F 615 / F 437 It is stable after 60 min, so 60 min is selected as the optimal reaction time for histamine detection.
[0080] Experimental Example 3
[0081] Linear range and detection limit of ratiometric fluorescence detection
[0082] The experimental conditions were: 100 μL, 2 mg mL -1The copper nanocluster solution, 50 μL of silicon quantum dot solution adjusted to a neutral pH value, and histamine solutions of different concentrations were mixed and fixed to 1.5 mL with deionized water. After the reaction was allowed to stand for 60 min, the mixed solutions of each system were obtained. The fluorescence emission spectra of each system at an excitation wavelength of 380 nm were detected ( Figure 4 ). The ratio of the fluorescence intensity at 615nm and 437nm is used as F 615 / F 437 The standard curve was prepared by using the concentration of histamine solution (0.4, 1, 2, 3.5 and 7 mM) as the ordinate and the concentration of histamine solution (0.4, 1, 2, 3.5 and 7 mM) as the abscissa. Figure 5 ), used to detect the concentration of histamine in the sample to be tested.
[0083] The experimental results are shown in Figure 4 , Figure 5 , from which it can be seen that the linear range of this method is: 0.4-7mM and the detection limit is: 16.6μM (S / N=3).
[0084] Experimental Example 4
[0085] Selectivity analysis of analytical methods
[0086] The experimental conditions were as follows: copper nanocluster solution (100 μL, 2 mg mL -1 ), a silicon quantum dot solution (50 μL) adjusted to a neutral pH, and a histamine solution (750 μL, 20 mM) (or a blank water sample, interferences of the same concentration, including phenylethylamine, tryptamine, putrescine, and tyramine) were mixed. The volume was made up to 1.5 mL with deionized water, and the reaction was allowed to stand at room temperature for 60 minutes. The fluorescence emission spectrum was detected at an excitation wavelength of 380 nm, and the ratio of the fluorescence intensity at 615 nm and 437 nm was used as the fluorescence intensity ratio F 615 / F 437 A bar graph is drawn with the value as the vertical axis and histamine and other interferents as the horizontal axis to determine the specificity of the method for histamine response.
[0087] Results Figure 6 , it can be seen that this method has strong selectivity and good detection specificity for histamine.
[0088] Example 1
[0089] Spiked testing of histamine in fish
[0090] The copper nanoclusters and silicon quantum dot solutions prepared in Experimental Example 1 were used.
[0091] Construction of fluorescence analysis method and histamine spike detection: 1.0 g of fish sample was mixed with 10 mL of 5% trichloroacetic acid, ultrasonically treated for 20 min, and the supernatant was collected by centrifugation. 20 mL of n-hexane was added to remove fat, and the bottom aqueous phase was collected by phase separation. 100 μL of 2 mg mL -1Copper nanocluster solution, 50 μL silicon quantum dot solution (pH 7) and sample solution (histamine solution was added to a final concentration of 3.00 mM and 6.00 mM, respectively) were mixed, and the volume was adjusted to 1.5 mL with deionized water. After standing for 60 minutes at room temperature, the fluorescence emission spectrum was detected at an excitation wavelength of 380 nm. The corresponding standard curve was prepared using the histamine solution, and the relevant spiked experimental results calculated using the prepared standard curve are shown in Table 1. The spiked recovery of the fish solution was 101.3% to 102.3%, and the relative standard deviation (RSD) was 3.03 to 3.95%, indicating that this method has good practicality and repeatability.
[0092] Table 1. Analysis results of histamine in fish meat (n=3)
[0093]
[0094] Example 2
[0095] Spiked test of histamine in shrimp meat
[0096] The copper nanoclusters and silicon quantum dot solutions prepared in Experimental Example 1 were used.
[0097] Construction of fluorescence analysis method and histamine spike detection: 1.0 g shrimp meat sample was mixed with 10 mL 5% trichloroacetic acid, ultrasonically treated for 20 min, and the supernatant was collected by centrifugation. 20 mL of n-hexane was added to remove fat, and the bottom aqueous phase was collected by phase separation. 100 μL 2 mg mL -1 Copper nanocluster solution, 50 μL silicon quantum dot solution (pH 7) and sample solution (histamine solution was added to a final concentration of 3.00 mM and 6.00 mM, respectively) were mixed, and the volume was adjusted to 1.5 mL with deionized water. After standing for 60 minutes at room temperature, the fluorescence emission spectrum was detected at an excitation wavelength of 380 nm. The corresponding standard curve was prepared using the histamine solution, and the results of the relevant spiked experiments calculated using the prepared standard curve are shown in Table 2. The recovery rate of the shrimp meat solution spiked was 111.0% to 112.3%, and the relative standard deviation (RSD) was 1.72 to 7.13%, indicating that this method has good practicality and repeatability, and has the potential to detect histamine in food samples.
[0098] Table 2. Analysis results of histamine in shrimp meat (n=3)
[0099]
[0100] Example 3
[0101] Fluorescence Visualization Detection of Histamine in Fish Meat
[0102] The copper nanoclusters and silicon quantum dot solutions prepared in Experimental Example 1 were used.
[0103] Preparation of hydrogel test strips and visual detection of histamine in fish samples: The hydrogel test strips prepared in Experimental Example 1 were placed in sealed centrifuge tubes together with 2.0 g of fish meat, and placed at 4°C and 25°C respectively. After different reaction times, photos of the test strips were taken with a smartphone (or camera) under 365 nm ultraviolet light (ultraviolet lamp). The experimental results are shown in Figure 7 , reflecting the changes in histamine content in fish samples at different temperatures over time. When stored at 25°C, the histamine level increased significantly on the second day, and the fluorescent image of the test strip changed from light red to blue, indicating that histamine was released rapidly at high temperatures. When stored at 4°C, the fluorescent image of the test strip remained light red after 4 days, indicating that the histamine content was low and did not change much.
[0104] Example 4
[0105] Fluorescence Visualization Detection of Histamine in Shrimp Meat
[0106] Preparation of hydrogel test strips and visual detection of histamine in shrimp meat samples: The hydrogel test strips prepared in Experimental Example 1 were placed in sealed centrifuge tubes together with 2.0 g of shrimp meat, and placed at 4°C and 25°C respectively. After different reaction times, photos of the test strips were taken with a smartphone (or camera) under 365 nm ultraviolet light (ultraviolet lamp). The experimental results are shown in Figure 8 , reflecting the changes in histamine content in shrimp samples at different temperatures over time. When stored at 25°C, the histamine level increased significantly on the second day, and the fluorescence image of the test strip changed from light red to blue, indicating that histamine was released rapidly at high temperatures. When stored at 4°C, the fluorescence image of the test strip remained light red after 4 days, indicating that the histamine content was low and did not change much. The results show that the developed hydrogel test strip has great application potential in the visualization monitoring of food spoilage and can be used to evaluate the freshness of food.
Claims
1. A fluorescence ratio method for detecting histamine, characterized in that: The steps include: The copper nanocluster solution, silicon quantum dot solution and histamine solution of different concentrations were mixed and fixed to the same volume. After the reaction was allowed to stand, the mixed solutions of each system were obtained. The fluorescence emission spectra of each system under an excitation wavelength of 380 nm were detected. The ratio of the fluorescence intensity at wavelengths of 615 nm and 437 nm was used as F. 615 / F 437 A standard curve is prepared with the concentration of histamine solution as the vertical axis and the concentration of histamine solution as the horizontal axis, which is used to detect the concentration of histamine in the sample to be tested.
2. The fluorescence ratio method according to claim 1, wherein: The volume was adjusted to the same with deionized water, and the mixture was allowed to stand at room temperature for more than 60 minutes to obtain a mixed solution of each system; Preferably, the mixture is diluted to the same volume with deionized water, and the mixture is allowed to react at room temperature for 60 minutes to obtain a mixed solution of each system.
3. The fluorescence ratio method according to claim 1, wherein: The copper nanocluster solution, silicon quantum dot solution and histamine solution of different concentrations were mixed and fixed to the same volume. The final concentration of copper nanocluster added to each system was 17-267 μg mL -1 ; Preferably, the final concentration of copper nanoclusters added to each system is 130-140 μg·mL -1 ; Preferably, the final concentration of copper nanoclusters added to each system is 133.3 μg·mL -1 ; Preferably, in the fluorescence ratio method, the volumes of silicon quantum dot solutions added are equal; Preferably, the volume ratio of the added silicon quantum dot solution to the fixed volume is 1:(290-310); Preferably, the ratio of the volume of the silicon quantum dot solution added to the volume after constant volume is 1:
300.
4. The fluorescence ratio method according to claim 1, wherein: When preparing the standard curve, the final concentration of histamine was 0.4-7 mM; Preferably, in the fluorescence ratio method, the concentration of histamine in the sample to be tested is detected by a standard curve method; Preferably, the copper nanocluster solution, silicon quantum dot solution and sample solution to be tested are mixed by the same method as that for preparing the standard curve, and the volume is fixed to the same volume with deionized water. After the reaction is allowed to stand, a mixed solution of each system is obtained, and the fluorescence emission spectrum at an excitation wavelength of 380 nm is detected and recorded. The ratio of the fluorescence intensity at wavelengths of 615 nm and 437 nm is used as the value F 615 / F 437 , using the prepared standard curve, calculate the histamine content in the sample to be tested.
5. The fluorescence ratio method according to claim 1, wherein: The following steps are involved: 100 μL, 2 mg mL -1 The copper nanocluster solution, 50 μL silicon quantum dots and histamine solutions of different concentrations were mixed and fixed to 1.5 mL with deionized water. After the reaction was allowed to stand for 60 min, the mixed solutions of each system were obtained. The fluorescence emission spectra of each system were detected at an excitation wavelength of 380 nm. The ratio of the fluorescence intensity at wavelengths of 615 nm and 437 nm was used as the fluorescence intensity ratio F. 615 / F 437 A standard curve is prepared with the concentration of histamine solution as the ordinate and the concentration of histamine solution as the abscissa, which is used to detect the concentration of histamine in the sample to be tested; Preferably, the linear range of the detection method is 0.4-7 mM, and the detection limit is 16.6 μM.
6. A hydrogel, characterized in that The invention comprises the following components: copper nanoclusters, silicon quantum dot solution, sodium alginate and calcium chloride.
7. The method for preparing the hydrogel according to claim 6, characterized in that: The steps include: The copper nanocluster solution, the silicon quantum dot solution and the sodium alginate solution were stirred and mixed, a calcium chloride solution was added and allowed to stand, and the solution was washed with deionized water to prepare a hydrogel; Preferably, the method for preparing the hydrogel comprises the following steps: Take 2 mg mL -1 The copper nanocluster solution, the silicon quantum dot solution and the sodium alginate solution with a mass volume ratio of 1% are mixed in a volume ratio of 1: (0.3-0.8): (10-15), and the mixture is stirred for 55-65 minutes to obtain a mixture, and the mixture is mixed with a 75mM calcium chloride solution in a volume ratio of 1: (1.8-2.2), and the mixture is allowed to stand for more than 60 minutes, and washed with deionized water to prepare a hydrogel; Preferably, the method for preparing the hydrogel comprises the following steps: Take 2 mg mL -1 The copper nanocluster solution, the silicon quantum dot solution and the sodium alginate solution with a mass volume ratio of 1% were mixed in a volume ratio of 1:0.5:13.5, and stirred for 60 minutes to obtain a mixture, and the mixture was mixed with a 75mM calcium chloride solution in a volume ratio of 1:2, and allowed to stand for more than 60 minutes, and washed with deionized water to prepare a hydrogel; Preferably, the hydrogel is prepared into strips or sheets using a mold.
8. Use of the hydrogel according to claim 6 or the hydrogel prepared by the method according to claim 7 in detecting histamine in a sample; Preferably, the hydrogel according to claim 6 or the hydrogel prepared by the method according to claim 7 is used to evaluate the freshness of a sample by detecting histamine in the sample; Preferably, the sample is food; Preferably, the food comprises fish, shrimp and dairy products.
9. A method for visual detection of histamine, characterized in that: The steps include: Placing the hydrogel according to claim 6 or the hydrogel prepared by the method according to claim 7 in contact with a sample to be tested or immersing the hydrogel in the sample to be tested, irradiating the hydrogel with ultraviolet light, and judging the change of histamine in the sample to be tested according to the color change; Preferably, in the histamine visualization detection method, the wavelength of ultraviolet light irradiation is 365 nm; Preferably, in the histamine visualization detection method, as the histamine content in the sample to be tested increases, the color of the hydrogel gradually changes from red to blue.
10. In the fluorescence ratio method for detecting histamine according to claim 1, the hydrogel according to claim 6, or the method for preparing the hydrogel according to claim 7, the solvent of the copper nanocluster solution is ethanol; Preferably, the pH value of the silicon quantum dot solution is adjusted to 7.0-7.4; Preferably, the method for preparing copper nanoclusters comprises the following steps: Copper sulfate, 2,3,5,6-tetrafluorothiophenol and ethanol are stirred and mixed, and the precipitate is collected after centrifugation, washed with ethanol, and dried to obtain copper nanoclusters; Preferably, the method for preparing the copper nanoclusters comprises the following steps: Copper sulfate and 2,3,5,6-tetrafluorothiophenol were mixed in ethanol at a mass volume ratio of 1:(1-3) g / mL, stirred at room temperature for more than 30 min, centrifuged to collect the precipitate, washed with ethanol for more than 3 times, and dried to obtain copper nanoclusters; Preferably, the method for preparing the copper nanoclusters comprises the following steps: Copper sulfate and 2,3,5,6-tetrafluorothiophenol were mixed in ethanol at a mass volume ratio of 1:2.25 g / mL, stirred at room temperature for more than 30 min, centrifuged to collect the precipitate, washed with ethanol for more than 3 times, and dried to obtain copper nanoclusters; Preferably, the method for preparing the silicon quantum dot solution comprises the following steps: 3-aminopropyltrimethoxysilane was mixed with deionized water and transferred into an autoclave for reaction. After the reaction was completed, the mixture was cooled to room temperature, the product was collected, ethyl acetate was added, the mixture was mixed and allowed to stand until the phases were separated, and the aqueous phase at the bottom was the silicon quantum dot solution. Preferably, the method for preparing the silicon quantum dot solution comprises the following steps: Mix 3-aminopropyltrimethoxysilane and deionized water evenly, with a volume ratio of 3-aminopropyltrimethoxysilane to deionized water of 1:(5-6); transfer to an autoclave for reaction at 180-200°C for more than 8 hours, cool to room temperature, and collect the product; add ethyl acetate with a volume 5-10 times that of the product, mix and let stand until the phases are separated, and the water phase at the bottom is the silicon quantum dot solution.