A thio-pyronine derivative molecular probe, a preparation method and application thereof, and a test paper for detecting biological amine and a preparation method and application thereof

Abstract

The application provides a thio-pyronine derivative molecular probe, a preparation method and application thereof, and test paper for detecting biological amine and a preparation method and application thereof, and relates to the technical field of food safety detection. The thio-pyronine derivative with the structure shown in formula I is used as a molecular probe for detecting biological amine with high sensitivity and selectivity, and has multi-modal response, including olfactory, colorimetric and fluorescent signals, so that the biological amine can be conveniently and real-timely detected.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and in particular to a thiopyronine derivative molecular probe, a preparation method and application thereof, and a test paper for detecting biogenic amines, a preparation method and application thereof. Background Art

[0002] Biogenic amines are primarily indicators of food spoilage caused by fermentation or microbial activity. There is an urgent need to develop sensitive amine detection methods for applications in public health, food safety, environmental monitoring, and other related areas. Current methods for detecting biogenic amines include chromatography, Raman spectroscopy, enzyme-linked immunosorbent assays, molecular imprinting, and sensor technologies. With the continuous advancement of technology and the improvement of testing capabilities, the development of efficient, accurate, convenient, low-cost, and online detection methods will become increasingly important. Detection methods for biogenic amines are rapidly developing, with speed and accuracy constantly improving. New detection methods are constantly emerging, but each method has its own advantages and disadvantages. Due to their toxicity, real-time monitoring of biogenic amine concentrations is crucial for ensuring public health and food safety.

[0003] Molecular probes offer advantages such as simplicity, low cost, high specificity, and high sensitivity, making them a powerful tool for monitoring amines. Numerous papers have been published on molecular probes for the detection of biogenic amines. These probes can be broadly categorized based on their detection principles: nucleophilic addition, aza-Michael addition, dechlorination condensation, Schiff base formation, and esteraminolysis. Although several probes have been used to detect biogenic amines, they still suffer from common challenges. For example, they are limited by the sensitivity of the probe molecules, typically requiring a large amount of biogenic amine to elicit a response, and exhibit long response times. Furthermore, the low nucleophilicity of biogenic amines and their low concentrations in foods and water make real-time, on-site detection of biogenic amines extremely difficult. Therefore, the development of efficient, simple, and field-suitable methods for the detection of biogenic amines is urgent. Summary of the Invention

[0004] The purpose of the present invention is to provide a thiopyronine derivative molecular probe, preparation method and application, and a test paper for detecting biogenic amines, preparation method and application. Compared with existing molecular probes for detecting biogenic amines, the present invention has higher sensitivity and selectivity, and rapidly shows synchronous colorimetric and fluorescence changes and olfactory responses in the presence of biogenic amines.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a thiopyronine derivative molecular probe having a structure shown in Formula I:

[0007]

[0008] The present invention provides a method for preparing the thiopyronine derivative molecular probe described in the above technical solution, comprising the following steps:

[0009] Pyronine thione, iodoethane and an organic solvent are mixed and refluxed to obtain a thiopyronine derivative molecular probe having a structure shown in Formula I.

[0010] The present invention provides the use of the thiopyronine derivative molecular probe described in the above technical solution or the thiopyronine derivative molecular probe prepared by the preparation method described in the above technical solution in detecting biogenic amines.

[0011] The present invention provides a test paper for detecting biogenic amines, comprising a base paper and a thiopyronine derivative molecular probe loaded on the base paper; the thiopyronine derivative molecular probe is the thiopyronine derivative molecular probe described in the above technical solution or the thiopyronine derivative molecular probe prepared by the preparation method described in the above technical solution.

[0012] Preferably, the loading amount of the thiopyronine derivative molecular probe in the test paper for detecting biogenic amines is 1 to 50 nmol / cm 2 .

[0013] The present invention provides a method for preparing a test paper for detecting biogenic amines according to the above technical solution, comprising the following steps:

[0014] Dissolving a thiopyronine derivative molecular probe in an organic solvent to obtain a thiopyronine derivative stock solution; the thiopyronine derivative molecular probe is a thiopyronine derivative molecular probe having a structure shown in Formula I;

[0015] mixing the thiopyronine derivative stock solution and water to obtain an aqueous solution of the thiopyronine derivative;

[0016] The base paper is soaked in the aqueous solution of the thiopyronine derivative to obtain a test paper for detecting biogenic amines.

[0017] Preferably, the organic solvent comprises acetonitrile or dimethyl sulfoxide.

[0018] Preferably, the concentration of the thiopyronine derivative stock solution is 1 to 10 mmol / L.

[0019] Preferably, the concentration of the aqueous solution of the thiopyronine derivative is 1 to 50 μmol / L.

[0020] The present invention provides the use of the test paper for detecting biogenic amines described in the above technical solution or the test paper for detecting biogenic amines prepared by the preparation method described in the above technical solution in detecting the freshness of drinking water, cosmetics or food.

[0021] The present invention provides a thiopyronine derivative molecular probe. In this invention, the thiopyronine derivative molecular probe undergoes a nucleophilic substitution reaction with a biogenic amine, causing a change in the conjugated structure and simultaneously releasing the easily identifiable olfactory-sensitive molecule ethanethiol. This probe exhibits multimodal responses, including olfactory, colorimetric, and fluorescent signals, and exhibits high sensitivity and selectivity. In particular, it can easily and conveniently identify the presence of biogenic amines through olfaction without the aid of instrumentation.

[0022] The present invention uses a thiopyronine derivative having a structure shown in Formula I as a molecular probe for highly sensitive and selective detection of biogenic amines. It has a multimodal response, including olfactory, colorimetric and fluorescent signals, thereby facilitating real-time detection of biogenic amines.

[0023] The present invention utilizes the thiopyronine derivative molecular probe to prepare test paper for detecting biogenic amines. Test paper loaded with the thiopyronine derivative can achieve multifunctional olfactory, colorimetric, and fluorescent responses to biogenic amines, making qualitative analysis more accurate and reliable. Furthermore, the preparation process is simple and easy to use. By analyzing the RGB color pattern, accurate determination of biogenic amine levels is achieved. The present invention successfully applies the test paper for detecting biogenic amines in real-world scenarios, enabling non-destructive assessment of meat freshness with the help of a smartphone. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The molecular probe PSE synthesized in Example 1 of the present invention 1 H NMR spectrum;

[0025] Figure 2 The molecular probe PSE synthesized in Example 1 of the present invention 13 C NMR spectrum;

[0026] Figure 3 This is the mass spectrum of the molecular probe PSE synthesized in Example 1 of the present invention;

[0027] Figure 4 The absorbance ratio (A) of the molecular probe PSE of the present invention after adding tyramine, tryptamine, cadaverine, spermidine, spermine, benzylamine, histamine, ethanolamine and n-propylamine to react 426nm / A 574nm ) Response plot over time;

[0028] Figure 5 The fluorescence intensity ratio (I 533nm / I 605nm ) Response plot over time;

[0029] Figure 6Figure 1 is the absorption spectrum of the molecular probe PSE of the present invention in aqueous solutions of spermine at different concentrations;

[0030] Figure 7 The fluorescence spectra of the molecular probe PSE of the present invention in aqueous solutions of spermine at different concentrations are shown;

[0031] Figure 8 The colorimetric and fluorescence response diagrams of the molecular probe PSE of the present invention to aqueous solutions of spermine at different concentrations are shown;

[0032] Figure 9 Graph showing the functional relationship between the ratio of the fluorescence intensity of the molecular probe PSE at 533 nm to that at 603 nm and the concentration of spermine;

[0033] Figure 10 This is the response graph of the absorbance change over time after adding spermine to the invented molecular probe PSE;

[0034] Figure 11 Mg is added to the molecular probe PSE of the present invention 2+ 、Zn 2+ 、NO3 - 、SO4 2- 、S 2- , Cl-, tryptamine, tyramine, spermine, cadaverine, spermidine, n-propylamine and histamine;

[0035] Figure 12 The color photographs of the PSE test paper prepared by the present invention when subjected to different concentrations of biogenic amine aqueous solutions under natural light and ultraviolet light;

[0036] Figure 13 The color intensity of the PSE test paper prepared by the present invention changes with the concentration of spermine;

[0037] Figure 14 The following are color photos of PSE test paper under natural light and ultraviolet light as the storage time of beef at 25℃ increases. DETAILED DESCRIPTION

[0038] The present invention provides a thiopyronine derivative molecular probe having a structure shown in Formula I:

[0039]

[0040] In the present invention, the thiopyronine derivative molecular probe undergoes a nucleophilic substitution reaction with a biogenic amine to generate ethanethiol, resulting in a change in the molecular conjugated structure, thereby causing a change in the colorimetric / fluorescent / olfactory signal. In spoiled food, drinking water, or cosmetics, biogenic amines are not easily identified. However, the ethanethiol generated after reaction with the molecular probe is detectable when the air contains only one 50 billionth of ethanethiol (0.00019 mg / L), that is, when 3 nmol / L of ethanethiol evaporates, its odor can be smelled. This allows for rapid detection of biogenic amines in water, food, and cosmetics without the use of instrumentation. At the same time, the thiopyronine derivative molecular probe is loaded onto a base paper to prepare a test paper, which can achieve qualitative analysis without destroying the sample or complex pretreatment. The results are accurate and reliable, and rapid real-time detection can be achieved.

[0041] The present invention provides a method for preparing the thiopyronine derivative molecular probe described in the above technical solution, comprising the following steps:

[0042] Pyronine thione, iodoethane and an organic solvent are mixed and refluxed to obtain a thiopyronine derivative molecular probe having a structure shown in Formula I.

[0043] In the present invention, the structural formula of the pyroninethione is

[0044] In the present invention, the molar ratio of pyroninethione to iodoethane is preferably 1:5. In the present invention, the ratio of pyroninethione to organic solvent is preferably 0.67-1 mmol:10-15 mL, more preferably 0.67 mmol:10 mL. In the present invention, the organic solvent preferably includes anhydrous acetonitrile or anhydrous dichloromethane.

[0045] In the present invention, the mixing of pyroninethione, ethyl iodide and an organic solvent preferably comprises: dissolving the pyroninethione in an organic solvent and adding ethyl iodide.

[0046] In the present invention, the reflux temperature is preferably 80-85° C., more preferably 82° C.; the reflux time is preferably 2-3 h, more preferably 2 h.

[0047] The present invention preferably cools the resulting system to room temperature after the reflux and then vacuum concentrates to remove the organic solvent to obtain a crude product; the crude product is subjected to column chromatography separation, the components are collected, and the solvent is evaporated to obtain a thiopyronine derivative molecular probe. In the present invention, the eluent used in the column chromatography separation is preferably a mixture of dichloromethane, acetonitrile, and trifluoroacetic acid; the volume ratio of dichloromethane to acetonitrile in the mixture is 50:1, and the volume of trifluoroacetic acid is 0.1% of the total volume of dichloromethane and acetonitrile ((dichloromethane:acetonitrile = 50:1) + 0.1% trifluoroacetic acid).

[0048] The present invention provides the use of the thiopyronine derivative molecular probe described in the above technical solution or the thiopyronine derivative molecular probe prepared by the preparation method described in the above technical solution for detecting biogenic amines. In the present invention, the detection includes qualitative detection or quantitative detection. In the present invention, the use preferably includes: achieving qualitative detection or quantitative detection of biogenic amines in water.

[0049] The present invention provides a test paper for detecting biogenic amines, comprising a base paper and a thiopyronine derivative molecular probe loaded on the base paper; the thiopyronine derivative molecular probe is the thiopyronine derivative molecular probe described in the above technical solution or the thiopyronine derivative molecular probe prepared by the preparation method described in the above technical solution. In the present invention, the loading amount of the thiopyronine derivative molecular probe in the test paper for detecting biogenic amines is 1 to 50 nmol / cm 2 , more preferably 7 to 30 nmol / cm 2 In the present invention, the base paper is preferably filter paper.

[0050] The present invention provides a method for preparing a test paper for detecting biogenic amines according to the above technical solution, comprising the following steps:

[0051] Dissolving a thiopyronine derivative molecular probe in an organic solvent to obtain a thiopyronine derivative stock solution; the thiopyronine derivative molecular probe is a thiopyronine derivative molecular probe having a structure shown in Formula I;

[0052] mixing the thiopyronine derivative stock solution and water to obtain an aqueous solution of the thiopyronine derivative;

[0053] The base paper is soaked in the aqueous solution of the thiopyronine derivative to obtain a test paper for detecting biogenic amines.

[0054] The present invention dissolves a thiopyronine derivative in an organic solvent to obtain a thiopyronine derivative stock solution. In the present invention, the organic solvent preferably comprises acetonitrile or dimethyl sulfoxide (DMSO). In the present invention, the concentration of the thiopyronine derivative stock solution is preferably 1 to 10 mmol / L.

[0055] After obtaining the thiopyronine derivative stock solution, the present invention mixes the thiopyronine derivative stock solution with water to obtain an aqueous solution of the thiopyronine derivative. In the present invention, the water is preferably deionized water.

[0056] In the present invention, the concentration of the aqueous solution of the thiopyronine derivative is preferably 1 to 50 μmol / L, more preferably 10 μmol / L.

[0057] After obtaining an aqueous solution of a thiopyronine derivative, the present invention soaks a base paper in the aqueous solution of the thiopyronine derivative to obtain a test paper for detecting biogenic amines. In the present invention, the base paper is preferably filter paper. In the present invention, the soaking temperature is preferably room temperature, and the soaking time is preferably 8 to 12 hours.

[0058] In the present invention, after the soaking, the solvent in the test paper is preferably removed to obtain a test paper for detecting biogenic amines. In the present invention, the method for removing the solvent in the test paper preferably includes: volatilizing in air to dryness.

[0059] The present invention provides the use of the biogenic amine test paper described in the above technical solution for testing the freshness of drinking water, cosmetics, or food. The method is preferably used to test the freshness of meat, and more preferably to test the freshness of beef. In the present invention, the method preferably includes placing the biogenic amine test paper and the meat to be tested in a culture dish, with the test paper not in direct contact with the meat to be tested. Under ultraviolet light, the meat freshness is monitored in real time based on the color change of the test paper to identify the meat's freshness. In the present invention, the linear distance between the biogenic amine test paper and the meat to be tested is preferably 0.1 to 5 centimeters, more preferably 0.1 centimeter. In the present invention, the biogenic amine test paper is preferably placed directly above the meat to be tested. In the present invention, the wavelength of the ultraviolet light is preferably 365 nm. In the present invention, when the biogenic amine test paper emits red fluorescence, the meat to be tested is fresh; when the biogenic amine test paper emits yellow fluorescence, the meat to be tested is acceptable; and when the biogenic amine test paper emits green fluorescence, the meat to be tested is unacceptable.

[0060] The present invention provides a thiopyronine derivative molecular probe for detecting biogenic amines with multiple functional responses, including olfactory, colorimetric, and fluorescent color changes. The thiopyronine derivative molecular probe exhibits rapid response and high selectivity for biogenic amines, with a low detection limit of 0.03 μmol / L. Furthermore, the present invention provides a portable and cost-effective test paper for detecting biogenic amines, providing a practical and non-destructive method for assessing meat freshness. This paper-based method outperforms traditional instruments in terms of low cost, portability, time efficiency, and ease of use.

[0061] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0062] Example 1

[0063] The specific synthesis steps of the molecular probe PSE are as follows:

[0064] 0.6 mmol of 4-(dimethylamino)salicylaldehyde and 0.6 mmol of 3-dimethylaminophenol were dissolved in 5 mL of 95 wt% concentrated phosphoric acid, heated to 160°C for reaction, and the reaction was stopped after 5 h. The reaction mixture was cooled to room temperature, and the reaction system was poured into 30 mL of water. A 30 wt% NaOH solution was added to adjust the pH of the solution to 8, and then 6 mmol of ammonium hexafluorophosphate was added. A solid precipitated, which was filtered, washed three times with water, and vacuum dried (at 120°C for 2 h) to obtain a pyronine solid.

[0065] 3.73 mmol of sodium sulfide was dissolved in 15 mL of N,N-dimethylformamide (DMF), and then 0.373 mmol of the pyronine solid was added. The mixture was heated to 140° C. and reacted for 12 h. The heating was stopped, and the mixture was cooled to room temperature and concentrated in vacuo to remove DMF. The mixture was then separated by column chromatography (eluent: dichloromethane: petroleum ether = 1:1 (v / v)). The collected fractions were spin-dried to obtain a yellow solid pyroninethione.

[0066] 0.67 mmol of pyroninethione was added to a round-bottom flask and dissolved in 10 mL of anhydrous acetonitrile. Then 3.35 mmol of iodoethane was added, and the mixture was heated to 82°C and refluxed under condensation for 2 hours. After cooling to room temperature, the anhydrous acetonitrile was removed by vacuum concentration to obtain a crude product. The product was then separated by column chromatography (eluent: (dichloromethane:acetonitrile = 50:1) + 0.1% trifluoroacetic acid), and the collected components were spin-dried to obtain a thiopyronine derivative molecular probe (PSE) having the structure shown in Formula I with a yield of 91% and a purity of 95%.

[0067] Figure 1 The molecular probe PSE synthesized in Example 1 of the present invention 1 H NMR spectrum; Figure 2 The molecular probe PSE synthesized in Example 1 of the present invention 13 C NMR spectrum; Figure 3 This is the mass spectrum of the molecular probe PSE synthesized in Example 1 of the present invention.

[0068] 1 H NMR (600MHz, CD3CN) δ8.29 (d, J=9.6Hz, 2H), 7.14 (dd, J=9.6, 9.6Hz, 2H), 6.74 (d, J=2.4Hz, 2H), 3.29 (s, 12H), 3.21 (q, J=7.2Hz, 2H), 1.27 (t, J=7.2Hz, 3H). 13C NMR (151MHz, CD3CN) δ159.88, 158.52, 157.63, 132.07, 117.53, 115.47, 96.97, 41.37, 34.43, 15.92. ESI-MS:calcd for[M+H] + :327.1526.Found:m / z 327.1524.

[0069] Test Example 1

[0070] The detection principle of PSE for biogenic amines is as follows:

[0071]

[0072] The thiopyronine derivative molecular probe prepared by the present invention has good sensitivity and selectivity and can react with different biogenic amines. The reaction principle of this molecular probe is based on the low electron cloud density of the carbon atom at the meso position of the thiopyronine derivative, which easily undergoes a nucleophilic substitution reaction with biogenic amines to produce an amino-substituted pyronine and a byproduct, ethanethiol. This in turn causes changes in colorimetry, fluorescence, and odor, thereby effectively identifying biogenic amines.

[0073] Test Example 2

[0074] Detection of PSE recognition of amines: To evaluate the sensing ability of PSE to biogenic amines, the present invention monitored the reactions of PSE with typical biogenic amines, including tyramine, tryptamine, cadaverine, spermidine, spermine, benzylamine, histamine, ethanolamine, and n-propylamine.

[0075] The PSE prepared in Example 1 was dissolved in 3.05 mL of acetonitrile to obtain a PSE stock solution with a concentration of 1 mmol / L, which was then diluted with deionized water to obtain a PSE aqueous solution with a concentration of 10 μmol / L. Different types of biogenic amines were weighed and dissolved in deionized water to prepare different types of biogenic amine aqueous solutions with a concentration of 10 mmol / L. The different types of biogenic amine aqueous solutions with a concentration of 10 mmol / L were added to the PSE aqueous solution with a concentration of 10 μmol / L to obtain mixed solutions. The concentration of biogenic amine in each mixed solution was 100 μmol / L. Each mixed solution contained 1% acetonitrile (v / v). Each mixed solution was placed in a 3 mL cuvette for detection.

[0076] Figure 4 The absorbance ratio (A) of the molecular probe PSE of the present invention after adding tyramine, tryptamine, cadaverine, spermidine, spermine, benzylamine, histamine, ethanolamine and n-propylamine to react 426nm / A 574nm ) over time, given by Figure 4It can be seen that with the change of time, the absorption peak at 574nm in the UV-visible spectrum disappears, and a new absorption peak appears at 426nm.

[0077] Figure 5 The fluorescence intensity ratio (I 533nm / I 605nm ) over time, given by Figure 5 As can be seen, over time, the PSE emission peak at 605 nm disappears, while a new peak appears at 533 nm. The fluorescence changes from red to green. Simultaneously, the characteristic odor of ethanethiol can be clearly detected during the measurement. Therefore, PSE exhibits universal sensitivity to typical biogenic amines.

[0078] Test Example 3

[0079] PSE titration test for amines:

[0080] A titration test of spermine was conducted in aqueous solution. The PSE prepared in Example 1 was weighed and dissolved in 3.05 mL of acetonitrile to obtain a 1 mmol / L PSE stock solution, which was then diluted with deionized water to obtain a 10 μmol / L PSE aqueous solution. Spermine was weighed and dissolved in deionized water to prepare a 10 mmol / L spermine aqueous solution. The spermine aqueous solution was gradually added to the 10 μmol / L PSE aqueous solution to prepare solutions with varying spermine concentrations. UV absorption and fluorescence spectra were measured and recorded. Each solution contained 1% acetonitrile (v / v) and was placed in a 3 mL cuvette for testing.

[0081] Figure 6 The absorption spectra of the molecular probe PSE of the present invention in aqueous solutions of spermine at different concentrations are shown in FIG. Figure 6 It can be seen that with the gradual addition of spermine concentration, the absorption peak of PSE at 574 nm gradually decreased, and at the same time a new absorption peak appeared at 426 nm that gradually increased.

[0082] Figure 7 The fluorescence spectra of the molecular probe PSE of the present invention in aqueous solutions of spermine at different concentrations are shown in FIG. Figure 7 It can be seen that the fluorescence emission peak of PSE at 603nm gradually decreases, and the fluorescence emission peak at 533nm gradually increases. This transition leads to a significant change in fluorescence color from red to green ( Figure 8 Importantly, the fluorescence intensity ratio of PSE (I 533nm / I 603nm ) showed a good linear correlation with spermine concentration (see Figure 9Furthermore, the detection limit of spermine was determined to be 0.03 μmol / L. These results indicate that PSE can be used for the quantitative determination of biogenic amines in solution.

[0083] Test Example 4

[0084] PSE response time test to amines:

[0085] The PSE prepared in Example 1 was weighed and dissolved in 3.05 mL of acetonitrile to obtain a PSE stock solution with a concentration of 1 mmol / L, which was diluted with deionized water to obtain a PSE aqueous solution with a concentration of 10 μmol / L; spermine was weighed and dissolved in deionized water to prepare a spermine aqueous solution with a concentration of 10 mmol / L; the spermine aqueous solution was added to the PSE aqueous solution with a concentration of 10 μmol / L to obtain a mixed solution, wherein the concentration of spermine in the mixed solution was 100 μmol / L, and the mixed solution contained 1% acetonitrile (v / v). The solution was placed in a 3 mL cuvette for detection, and the ultraviolet absorption and fluorescence spectra were measured and recorded to obtain the change in fluorescence intensity of the aqueous solution of the molecular probe PSE over time. The results are shown as follows: Figure 10 As shown, when spermine (100 μmol / L) was added to the PSE solution (10 μmol / L), the reaction between PSE and spermine was completed within 30 minutes.

[0086] The present invention further verifies the selectivity of PSE for biogenic amines. A series of biogenic amine solutions with ion or molecular concentrations of 10 mmol / L were prepared using deionized water. Mg and Mg were added to the 10 μmol / L PSE aqueous solution. 2+ 、Zn 2+ 、NO3 - 、SO4 2- 、S 2- , Cl- and biogenic amine aqueous solutions, and test the changes in the fluorescence intensity of the molecular probe PSE aqueous solution at 533 nm. Figure 11 As shown, when Mg is added 2+ 、Zn 2+ 、NO3 - 、SO4 2- 、S 2- After addition of chlorine and Cl-, the fluorescence spectrum of PSE showed negligible changes. Significant fluorescence emission was observed only after the addition of biogenic amines. These data indicate that PSE can selectively detect biogenic amines even in the presence of other substances.

[0087] Application Example 1

[0088] 1 mg of the PSE prepared in Example 1 was weighed and dissolved in 3.05 mL of acetonitrile to obtain a PSE stock solution, which was then diluted in deionized water to obtain a PSE aqueous solution with a PSE concentration of 10 μmol / L;

[0089] A 1 cm×1 cm square filter paper was soaked in the PSE aqueous solution overnight, and then evaporated to dryness in the air to obtain a test paper for detecting biogenic amines (PSE test paper).

[0090] The test paper for detecting biogenic amines prepared in this application example consists of filter paper and PSE loaded on the filter paper; the loading amount of PSE in the test paper for detecting biogenic amines is 7.21 nmol / cm 2 .

[0091] Test Example 5

[0092] PSE test paper test for biogenic amines:

[0093] Different amounts of biogenic amines were weighed and dissolved in deionized water to prepare a 10 mmol / L biogenic amine stock solution. This amine stock solution was then diluted in the deionized water solution to obtain aqueous solutions of biogenic amines with concentrations ranging from 0 to 600 μmol / L. The PSE test paper prepared in Application Example 1 was immersed in aqueous solutions of biogenic amines (including spermine, cadaverine, tyramine, spermidine, and histamine) at different concentrations. The color changes under sunlight and the fluorescence changes under 365 nm illumination were then recorded using a smartphone. The biogenic amines were visually measured by observing the color and fluorescence changes.

[0094] Figure 12 The following graph shows the color (CM mode) and fluorescence intensity (FL mode) of the PSE test paper prepared according to the present invention as a function of biogenic amine concentration. The PSE test paper appears deep red and turns pink after exposure to a 50 μmol / L biogenic amine solution. As the biogenic amine concentration increases, the color of the PSE test paper gradually changes to yellow, and the fluorescence color of the PSE test paper gradually changes from deep red to yellow-green and finally to green. This demonstrates its potential as an indicator of the levels of various biogenic amines.

[0095] Monitoring the content of biogenic amines can help consumers accurately assess the freshness of meat. To achieve this goal, the present invention uses a smartphone to capture an image of a PSE test strip in daylight and then uses a color picker application to obtain RGB color and color intensity data. By analyzing the color intensity values, the present invention found a linear correlation between the color intensity of the PSE test strip and the spermine concentration ( Figure 13 ), the linear regression equation is y=1.3216x+22.5963(R 2 =0.99). This indicates that the level of biogenic amines can be determined by RGB analysis of PSE test paper, and therefore, PSE test paper is expected to become a portable tool for on-site detection of biogenic amines.

[0096] Table 1 RGB values ​​and color intensities of PSE test paper when detecting different concentrations of spermine

[0097]

[0098]

[0099] Test Example 6

[0100] The recognition effect of PSE test paper on amines in the simulated environment of drinking water and cosmetics:

[0101] According to the National Standard of the People's Republic of China GB / T 21970-2008, the biogenic amine content of putrescine, cadaverine, spermidine, spermine, and histamine in drinking water must be controlled between 2.0 mg / L and 40.0 mg / L. To verify the responsiveness of the PSE test paper prepared in the present invention to amines, the PSE test paper prepared in Application Example 1 was placed in a spermine aqueous solution (200 μmol / L) for 30 seconds. After the test paper was removed, a distinct odor of ethyl mercaptan was detected, simulating the test paper's response to amines in an environment where the amine content in drinking water exceeds the standard.

[0102] The 2015 edition of the "Technical Specifications for Safety of Cosmetics" stipulates that the content of dialkanolamines in monoalkanolamines must not exceed 0.5%, while both ethanolamine and diethanolamine fall within this range. To verify the responsiveness of the PSE test paper prepared in this invention to amines, 10 μL of an 8.2 mmol / L ethanolamine aqueous solution was accurately pipetted and applied to the PSE test paper prepared in Application Example 1. A distinct odor from the volatilization of ethanethiol was detected, simulating the test paper's response to amines under conditions where the amine content in cosmetics exceeds the standard. This method can detect biogenic amines at the nanomolar level, which is of great significance for the safety testing of drinking water and cosmetics.

[0103] Test Example 7

[0104] Practical application of PSE test paper:

[0105] The present invention conducted a preliminary investigation using PSE test paper to detect the biogenic amines released at different storage times at 25°C ( Figure 14 Fresh beef samples were placed in a Petri dish, covered with plastic wrap, and the PSE test paper prepared in Application Example 1 was placed on the plastic wrap. A handheld UV lamp (365 nm) and a smartphone camera were used to capture photos of the scene. Images and R / G data from the PSE test paper showed that the initial red fluorescence of the PSE test paper turned pink after storage for 8 hours, and then turned green after 16 hours, indicating increasing levels of volatile biogenic amines.

[0106] In order to test the freshness of beef, the total volatile base nitrogen (TVBN) content of beef samples was measured. The assessment criteria for beef freshness are as follows: a TVBN value below 15 mg / 100 g indicates freshness, a TVBN value below 25 mg / 100 g indicates staleness but still edible, and a TVBN value exceeding 25 mg / 100 g indicates spoilage and inedible. The present invention further compares the TVBN analysis data with the results of the PSE test paper. Figure 14 As shown, the PSE test paper still emits red fluorescence within 8 hours of storage, the green / red (G / R) ratio is less than 1.0, and the corresponding TVBN value is less than 15.0 mg / 100 g, indicating that the beef remains fresh. When the storage time is extended to 12 hours, the TVBN content increases to 16.24 mg / 100 g, the fluorescence of the PSE test paper turns yellow, and the green / red (G / R) ratio is greater than 1.0, indicating that there is slight deterioration. When the storage time is extended to 16 hours, the PSE test paper becomes yellow-green, and the TVBN level reaches 19.6 mg / 100 g, indicating that the beef is no longer fresh. After storage at 25°C for 24 hours, the fluorescence of the PSE test paper turns green, and the TVBN value exceeds 25.0 mg / 100 g, indicating obvious deterioration and the beef cannot be eaten (see Figure 14 These observations indicate that the PSE test strips provide consistent results with those of the TVBN assay, while offering significant advantages in convenience, speed, cost-effectiveness, and portability.

[0107] To test the immediacy of the test paper's response to biogenic amines, the present invention applied PSE test paper to beef samples. The test paper exhibited distinct color changes, indicating different TVBN values ​​for beef samples stored at different times. Therefore, PSE test paper represents a highly advantageous method for rapid, non-destructive, and visual assessment of meat freshness.

[0108] The PSE test paper of the present invention is simple to prepare and can provide multifunctional responses to biogenic amines using olfactory, colorimetric, and fluorescent methods, making qualitative analysis more accurate and reliable. The PSE test paper can rapidly and non-destructively monitor biogenic amine content in real time, making it suitable for drinking water and cosmetics safety testing and promising for food freshness testing. In underdeveloped regions, the PSE test paper of the present invention can provide timely and effective freshness information through rapid detection of biogenic amines, demonstrating its practical application.

[0109] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A use of a thiopyronine derivative molecular probe in detecting biogenic amines for non-diagnostic and non-therapeutic purposes, characterized in that: The thiopyronine derivative molecular probe has a structure shown in Formula I:

2. The use of the thiopyronine derivative molecular probe according to claim 1 in detecting biogenic amines for non-diagnostic and non-therapeutic purposes, wherein the preparation method of the thiopyronine derivative molecular probe comprises the following steps: Pyronine thione, iodoethane and an organic solvent are mixed and refluxed to obtain a thiopyronine derivative molecular probe having a structure shown in Formula I.

3. A test paper for detecting biogenic amines, comprising a base paper and a thiopyronine derivative molecular probe loaded on the base paper; the thiopyronine derivative molecular probe has a structure shown in Formula I:

4. The test paper for detecting biogenic amines according to claim 3, characterized in that The loading amount of the thiopyronine derivative molecular probe in the test paper for detecting biogenic amines is 1 to 50 nmol / cm 2 .

5. The method for preparing the test paper for detecting biogenic amines according to any one of claims 3 to 4, comprising the following steps: Dissolving a thiopyronine derivative molecular probe in an organic solvent to obtain a thiopyronine derivative stock solution; the thiopyronine derivative molecular probe is a thiopyronine derivative molecular probe having a structure shown in Formula I; mixing the thiopyronine derivative stock solution and water to obtain an aqueous solution of the thiopyronine derivative; The base paper is soaked in the aqueous solution of the thiopyronine derivative to obtain a test paper for detecting biogenic amines.

6. The preparation method according to claim 5, characterized in that The organic solvent includes acetonitrile or dimethyl sulfoxide.

7. The preparation method according to claim 5, characterized in that The concentration of the thiopyronine derivative stock solution is 1 to 10 mmol / L.

8. The preparation method according to claim 5, characterized in that The concentration of the aqueous solution of the thiopyronine derivative is 1 to 50 μmol / L.

9. Use of the test paper for detecting biogenic amines according to any one of claims 3 to 4 or the test paper for detecting biogenic amines prepared by the preparation method according to any one of claims 5 to 8 in detecting the freshness of drinking water, cosmetics or food.

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