A bifunctional near-infrared emissive fluorescent probe for simultaneous detection of sulfur dioxide derivatives and volatile amines, and a synthesis method and application thereof

Abstract

A bifunctional near-infrared emission fluorescent probe for the simultaneous detection of sulfur dioxide derivatives and volatile amines, its synthesis method, and its applications are disclosed. The fluorescent probe has the following structural formula: This fluorescent probe features a simple synthesis route, near-infrared emission, fast response speed, and dual-channel response for both colorimetry and fluorescence. This probe can detect HSO3 in real samples such as red wine and white sugar. ‑ And HSO3 in living cells ‑ Fluorescence imaging was performed using triethylamine. Two standard colorimetric cards for fish freshness were prepared using this probe, each divided into a fresh colorimetric zone under sunlight and ultraviolet light, a qualified colorimetric zone under sunlight and ultraviolet light, and a spoilage colorimetric zone under sunlight and ultraviolet light. The sensor tag or sensor film loaded with this probe, combined with the standard colorimetric card, can realize real-time monitoring of fish freshness through colorimetric and fluorescence dual channels without destroying the sample or complicated pretreatment, and the evaluation results are accurate and reliable.

Description

Technical Field

[0001] This invention relates to a bifunctional near-infrared emission fluorescent probe for the simultaneous detection of sulfur dioxide derivatives and volatile amines, its synthesis method, and its applications. Background Technology

[0002] Sulfur dioxide derivatives, bisulfite (HSO3) - ) and sulfites (SO3) 2- Sulfur dioxide, as a food additive, has a long history of use in the food processing industry due to its antibacterial, preservative, bleaching, and antioxidant properties, and is used to extend the shelf life of food. Although sulfur dioxide derivatives have many uses, excessive intake of bisulfites and sulfites can lead to respiratory diseases, allergic reactions, gastrointestinal reactions, and other illnesses. The International Agency for Research on Cancer (IARC) classifies sulfur dioxide derivatives (HSO3) as potentially harmful. - Sulfites are classified as a Group 3 carcinogen. The National Food Safety Standard for the Use of Food Additives (GB 2760—2014) stipulates that the sulfur content (calculated as SO2 content) in white sugar should be less than 0.03 g / kg, and in wine, it is strictly limited to less than 0.25 g / kg. The U.S. Food and Drug Administration (FDA) only permits the addition of no more than 10 ppm of sulfites to food and beverages. Therefore, the development of methods for detecting bisulfites and sulfites in biological systems and food has attracted widespread attention from researchers.

[0003] Amine compounds are widely distributed in the biological world and possess extremely important physiological and biological activities. Proteins, nucleic acids, many hormones, antibiotics, and alkaloids are all derivatives of amines. Most drugs used clinically are also amines or their derivatives. Therefore, understanding the properties, synthesis methods, and detection of amines is fundamental to maintaining human health. Volatile amines are generally produced during meat spoilage, primarily due to enzymatic reactions and microbial metabolism, producing volatile amines such as triethylamine, trimethylamine, cadaverine, putrescine, and spermine. These volatile amines have become important indicators for judging the freshness of meat products. The volatile amines produced by spoiled meat products transform the surrounding environment into an alkaline environment. The freshness of meat products can be monitored by measuring the total amount of volatile amines, i.e., the content of volatile basic nitrogen (TVB-N). However, the national standard method for measuring TVB-N requires expensive equipment, complex pretreatment, damage to the analyte, and is time-consuming. Therefore, developing a simple and convenient method to detect volatile amines and achieve real-time monitoring of meat product freshness has significant application value. Because fish has a short shelf life, it is highly susceptible to spoilage during fishing, transportation, storage, and processing. This not only leads to waste and economic losses, but also poses varying degrees of health risks and foodborne illnesses if ingested. Therefore, monitoring the freshness of fish is crucial for the transportation industry, retailers, and consumers.

[0004] Compared to traditional methods, fluorescence and colorimetric methods have been widely used in the detection of sulfur dioxide derivatives or volatile amines due to their advantages such as simple operation, fast response, and real-time detection. Developing fluorescent probes with dual-channel responses (colorimetric and fluorescence) has become a current research hotspot. To date, many fluorescent probes for detecting sulfur dioxide derivatives or volatile amines individually have been reported. Although these probes have shown good performance, achieving the detection of two targets using a single fluorescent probe remains a challenge. Compared to single-function fluorescent probes, bifunctional fluorescent probes offer "two uses in one probe," resulting in lower application costs, higher molecular utilization, and improved application prospects. Currently, no fluorescent probes capable of simultaneously detecting sulfur dioxide derivatives and volatile amines have been reported. Given the needs of food safety testing, there is an urgent need to develop a bifunctional near-infrared fluorescent probe that can be used for both the detection of sulfur dioxide derivatives in food and the indication of fish freshness. Summary of the Invention

[0005] The technical problem this invention aims to solve is to provide a bifunctional near-infrared emission fluorescent probe for the simultaneous detection of sulfur dioxide derivatives and volatile amines, along with its synthesis method and applications. This fluorescent probe possesses advantages such as a simple synthesis route, near-infrared emission, fast response speed, and dual-channel response for both colorimetry and fluorescence. This probe can detect HSO3 in real food samples such as red wine and white sugar. - And HSO3 in living cells - Fluorescence imaging was performed with triethylamine. Simultaneously, a standard colorimetric card for fish freshness was prepared using this probe. The indicator label combined with the standard colorimetric card enables qualitative analysis of fish freshness without damaging the sample or requiring complex pretreatment. The evaluation results are accurate and reliable, and it can be used for non-destructive, rapid, and real-time detection of salmon freshness.

[0006] The technical solution of this invention is:

[0007] A bifunctional near-infrared fluorescent probe for the simultaneous detection of sulfur dioxide derivatives and volatile amines, its synthesis method, and its application are disclosed. The structural formula of the fluorescent probe is as follows:

[0008] Furthermore, the specific synthesis steps of this fluorescent probe are as follows:

[0009] Using ethanol (5-50 mL) as a solvent, a benzopyran derivative and cycloisopropyl malonate were fed at a molar ratio of 1:(1-2), wherein the benzopyran derivative was 6-(diethylamino)-1,2-dihydrocyclopentano[b]benzopyran-3-carboxaldehyde. Then, acetic acid was added at a mass ratio of benzopyran derivative to acetic acid of 20:1; the mixture was stirred at room temperature for 5 to 8 hours, filtered, and the solid was collected and recrystallized from ethanol to obtain a near-infrared fluorescent probe.

[0010] A bifunctional near-infrared emission fluorescent probe for the simultaneous detection of sulfur dioxide derivatives and volatile amines, its synthesis method, and its application are characterized by: detection of HSO3 in a DMSO / PBS (2 / 8, v / v) system. - The test is being conducted for purposes other than disease diagnosis and treatment.

[0011] A bifunctional near-infrared emission fluorescent probe for the simultaneous detection of sulfur dioxide derivatives and volatile amines, its synthesis method, and its applications are characterized by the ability to detect HSO3 within 60 seconds. - The detection is for non-disease diagnosis and non-disease treatment purposes.

[0012] A bifunctional near-infrared emission fluorescent probe for the simultaneous detection of sulfur dioxide derivatives and volatile amines, its synthesis method, and its applications are characterized by their ability to detect HSO3 in red wine and white sugar. - The test is being conducted for purposes other than disease diagnosis and treatment.

[0013] A bifunctional near-infrared fluorescent probe for the simultaneous detection of sulfur dioxide derivatives and volatile amines, its synthesis method, and its applications are characterized by their ability to detect HSO3 in living cells. - Fluorescence imaging with triethylamine is used for non-disease diagnosis and non-disease treatment purposes.

[0014] A bifunctional near-infrared emission fluorescent probe for simultaneously detecting sulfur dioxide derivatives and volatile amines, its synthesis method, and its application are disclosed. The key feature is that the probe-loaded filter paper (Dma / FPS) or the probe-containing polyvinyl alcohol membrane (Dma / PVA) serves as a sensing tag or sensing film to monitor the freshness of salmon flesh. The application is for non-disease diagnosis and non-disease treatment purposes.

[0015] The specific preparation process of the sensor tag or sensor film is as follows:

[0016] Sensor tag DM1 / FPS: Weigh 3.95 mg of fluorescent probe and dissolve it in 10 mL of DMSO. Then, soak a 1.5 cm diameter circular filter paper in the solution overnight. After that, take it out and dry it in an oven to obtain an indicator tag for monitoring the freshness of fish.

[0017] Sensing film DMSO / PVA: 3.2 g of polyvinyl alcohol (PVA) was dissolved in 40 mL of deionized water and magnetically stirred at 90 °C for 2 h to obtain a clear, transparent, viscous solution. Heating was stopped, and the solution was allowed to cool to room temperature. 10 mg of fluorescent probe was dissolved in 1 mL of DMSO and poured into the cooled PVA solution. The solution was quickly stirred until homogeneous, and then ultrasonically degassed to remove air bubbles, yielding a PVA solution containing the fluorescent probe. 15 mL of this solution was poured into a 90 mm diameter glass petri dish and dried in an oven. Finally, the entire PVA film was cut into 1 cm × 1 cm square polyvinyl alcohol films to obtain another indicator label for monitoring the freshness of fish.

[0018] Application of sensor tags or sensor films in determining the freshness of salmon flesh.

[0019] The application of sensor tags or sensor films in determining the freshness of salmon involves placing the sensor tags or sensor films inside the packaging box with the salmon sample, ensuring that the indicator labels do not directly contact the sample. The freshness of the salmon is monitored in real time by comparing the color changes of the sensor tags or sensor films with the corresponding standard color chart, thus identifying the freshness of the salmon.

[0020] Under visible light, the sensor tag DMA / FPS is blue to indicate freshness, purple to indicate compliance, and colorless to indicate non-compliance.

[0021] Under 365nm ultraviolet light, the sensor tag Dma / FPS is fresh when it glows red, qualified when it has no fluorescence, and unqualified when it glows bright green.

[0022] Under visible light, a blue to green color for the Dma / PVA sensing film indicates freshness, a gray color indicates compliance, and an ivory white color indicates non-compliance.

[0023] Under 365nm ultraviolet light irradiation, the Dma / PVA sensing film is considered fresh when it exhibits red fluorescence to no fluorescence, qualified when it exhibits blue fluorescence, and unqualified when it exhibits green fluorescence.

[0024] The beneficial effects of this invention are:

[0025] (1) The fluorescent probe emits at a wavelength of 675 nm, reaching the near-infrared emission region, thus experiencing less interference from scattering and fluorescence background. It can identify HSO3 within 60 seconds in high-water-content systems. - It is responsive to a variety of volatile amines, has a low detection limit, and exhibits a significant color change.

[0026] (2) The sensor tag Dma / FPS or the sensor film Dma / PVA is used as an indicator tag. With the corresponding standard colorimetric card, the freshness of fish meat can be indicated by colorimetry and fluorescence in a dual-channel manner. One probe has two functions, which can save R&D costs and improve the utilization rate of the probe.

[0027] In summary, the fluorescent probe designed and synthesized in this invention can rapidly recognize HSO3 in a DMSO / PBS (2 / 8, v / v) system. - It can also identify various amine solutions in a DMSO / H2O (4 / 6, v / v) system. Furthermore, it can be fabricated into sensor tags or sensor films to identify various volatile amines. The designed sensor tags or sensor films have a simple preparation process and can achieve dual-channel detection of fish freshness using colorimetry and fluorescence, providing accurate and reliable qualitative analysis results. These sensor tags or sensor films enable non-contact, non-destructive, rapid, and real-time monitoring of fish freshness, providing timely and effective freshness information for producers, retailers, and consumers, and have significant practical application value. Attached Figure Description

[0028] Figure 1 It is the fluorescent probe Dma of this invention. 1 H NMR spectrum;

[0029] Figure 2 It is the fluorescent probe Dma of this invention. 13 C NMR spectrum;

[0030] Figure 3 This is the mass spectrum of the fluorescent probe Dma of this invention;

[0031] Figure 4 The present invention is a fluorescent probe Dma and 1:Dma,2:CO3 2- ,3:HCO3 - ,4:C2O4 2- ,5:CH3COO - ,6:PPI,7:HPO4 2- ,8:SO4 2- ,9:S2O3 2- ,10:SCN - ,11:HS - ,12:N3 - ,13:NO2 - ,14:F - ,15:Cl - ,16:Br - ,17:I - ,18:Cys,19:Hcy,20:GSH,21:S 2- ,22:SO3 2- ,23:HSO3- Ultraviolet absorption spectra and changes in sunlight color before and after the treatment;

[0032] Figure 5 The fluorescent probe Dma and CO3 of this invention 2- HCO3 - C2O4 2- CH3COO - PPI, HPO4 2- SO4 2- S2O3 2- SCN - ,HS - N3 - NO2 - ,F - ,Cl - ,Br - ,I - Cys,Hcy,GSH,S 2- SO3 2- HSO3 - Fluorescence emission spectra before and after the interaction with various anions and biothiols;

[0033] Figure 6 The present invention relates to the fluorescent probe Dma recognizing HSO3 when it coexists with 20 anions and biothiols. - Ultraviolet interference detection diagram;

[0034] Figure 7 The present invention relates to the fluorescent probe Dma recognizing HSO3 when it coexists with 20 anions and biothiols. - Fluorescence anti-interference detection image;

[0035] Figure 8 The present invention relates to the fluorescent probe Dma reacting with different concentrations of HSO3. - Changes in fluorescence emission spectra before and after treatment;

[0036] Figure 9 The fluorescent probe Dma of this invention recognizes HSO3. - Detection limit map;

[0037] Figure 10 The fluorescent probe Dma of this invention is added with HSO3 - Response graph showing the change in fluorescence intensity over time;

[0038] Figure 11 The fluorescent probe Dma of this invention is added with HSO3 - Graph showing the changes in fluorescence intensity before and after at different pH levels;

[0039] Figure 12The present invention involves adding HSO3 to the Dma fluorescent probe solution. - The subsequent high-resolution mass spectrum;

[0040] Figure 13 This is a high-resolution mass spectrum of the fluorescent probe Dma solution of the present invention after the addition of n-propylamine;

[0041] Figure 14 The fluorescence intensity of the fluorescent probe Dma in red wine and white sugar samples of this invention is compared with the fluorescence intensity after the addition of HSO3. - Linear relationship graph between concentrations;

[0042] Figure 15 This is a graph showing the changes in cell viability of MCF-7 cells after 24 hours of incubation with different concentrations of the fluorescent probe Dma of this invention.

[0043] Figure 16 The fluorescent probe Dma of this invention is used to detect HSO3 in MCF-7 cells. - Fluorescence imaging; Cells were co-cultured with Dma for 30 min, followed by the addition of a) 0 μM, c) 10 μM, e) 50 μM, g) 100 μM, i) 350 μM HSO3. - Fluorescence images taken in bright field later; b) 0 μM, d) 10 μM, f) 50 μM, h) 100 μM, j) 350 μM HSO3 - Fluorescence image taken later in the dark;

[0044] Figure 17 The UV absorption spectra and color change diagrams under sunlight of the fluorescent probe Dma of this invention before and after the addition of various amines (1. a, 2. aniline, 3. trimethylamine, 4. tyramine, 5. 2-phenylethylamine, 6. diethylamine, 7. tryptamine, 8. 1,2-cyclohexanediamine, 9. n-propylamine, 10. triethylamine, 11. dimethylamine, 12. spermine, 13. putrescine, 14. cadaverine) compounds;

[0045] Figure 18 The fluorescent probe Dma of this invention has fluorescence emission spectra and color change diagrams under ultraviolet light before and after the addition of various amines (1. a, 2. aniline, 3. trimethylamine, 4. tyramine, 5. 2-phenylethylamine, 6. diethylamine, 7. tryptamine, 8. 1,2-cyclohexanediamine, 9. n-propylamine, 10. triethylamine, 11. dimethylamine, 12. spermine, 13. putrescine, 14. cadaverine) compounds;

[0046] Figure 19 These are the fluorescence emission spectra of the fluorescent probe Dma of this invention after adding different concentrations of triethylamine;

[0047] Figure 20 This is a detection limit diagram of the fluorescent probe Dma of this invention for recognizing triethylamine;

[0048] Figure 21 This is a graph showing the change in the ratio of 580 / 675nm fluorescence intensity of the fluorescent probe Dma before and after the addition of triethylamine at different pH values.

[0049] Figure 22 This is a graph showing the change in fluorescence intensity over time after adding triethylamine to the fluorescent probe Dma of this invention.

[0050] Figure 23 These are fluorescence images of the fluorescent probe Dma of this invention in MCF-7 cells after the addition of different concentrations of triethylamine; fluorescence images taken in bright field after cells were co-cultured with Dma for 30 min and then further added with a) 0 μM, d) 10 μM, g) 100 μM, j) 200 μM, m) 500 μM triethylamine; fluorescence images taken in dark field after cells were co-cultured with Dma for 30 min; fluorescence images taken in superimposed field after cells were co-cultured with Dma for 30 min ... bright field after cells were co-cultured with Dma for 30 min; fluorescence images taken in superimposed field after cells were co-cultured with Dma for 30 min; fluorescence images taken in superimposed field after cells were co-cultured with Dma for 30 min; fluorescence images taken in bright field after cells were co-cultured with Dma for 30 min; fluorescence images taken in superimposed field after cells were co-cultured with Dma for 30 min; fluorescence images taken in superimposed field after cells

[0051] Figure 24 These are photographs showing the changes in sunlight color (top) and fluorescence color (bottom) of the sensor tag Dma / FPS prepared in this invention before and after recognizing volatile amines (1. none, 2. ammonia, 3. 1,2-cyclohexanediamine, 4. diethylamine, 5. n-propylamine, 6. triethylamine, 7. spermine, 8. cadaverine, 9. putrescine, 10. 2-phenylethylamine, 11. tyramine, 12. aniline, 13. trimethylamine, 14. dimethylamine, 15. tryptamine).

[0052] Figure 25 These are photographs showing the changes in sunlight color (top) and fluorescence color (bottom) of the Dma / PVA sensing film prepared in this invention before and after recognizing volatile amines (1. none, 2. ammonia, 3. 1,2-cyclohexanediamine, 4. diethylamine, 5. n-propylamine, 6. triethylamine, 7. spermine, 8. cadaverine, 9. putrescine, 10. 2-phenylethylamine, 11. tyramine, 12. aniline, 13. trimethylamine, 14. dimethylamine, 15. tryptamine).

[0053] Figure 26 This is a graph showing the changes in TVB-N content and pH of salmon meat at 4°C over extended storage time.

[0054] Figure 27 This is a graph showing the change in color difference values ​​of the label on salmon meat as the storage time increases at 4°C.

[0055] Figure 28 These are color photographs of salmon flesh at 4°C with the TVB-N content and the corresponding sensor tag Dma / FPS under natural and ultraviolet light as the storage time increases.

[0056] Figure 29 These are photos showing the TVB-N content of salmon meat and the corresponding sensor tag Dma / FPS under natural and ultraviolet light as the storage time of salmon meat in 25℃ smart packaging increases.

[0057] Figure 30 It is a standard colorimetric card made based on the color changes of the TVB-N value of fish meat and the corresponding sensor label Dma / FPS under natural light and ultraviolet light. The upper layer of the standard colorimetric card, from left to right, is the fresh natural light colorimetric area, the qualified natural light colorimetric area, and the spoiled natural light colorimetric area. The lower layer of the standard colorimetric card, from left to right, is the fresh ultraviolet light colorimetric area, the qualified ultraviolet light colorimetric area, and the spoiled ultraviolet light colorimetric area.

[0058] Figure 31 It is a standard colorimetric card made based on the TVB-N value of fish meat and the color change of the corresponding sensing film Dma / PVA under natural light and ultraviolet light. The upper layer of the standard colorimetric card, from left to right, is the fresh natural light colorimetric area, the qualified natural light colorimetric area, and the spoiled natural light colorimetric area. The lower layer of the standard colorimetric card, from left to right, is the fresh ultraviolet light colorimetric area, the qualified ultraviolet light colorimetric area, and the spoiled ultraviolet light colorimetric area.

[0059] Figure 32 The present invention provides a method for monitoring the freshness of salmon meat by storing the Dma / FPS sensor tag prepared in this invention with salmon samples at 4°C. The color changes of the tag under sunlight and ultraviolet light are combined with a standard colorimetric card. The salmon meat is considered fresh on day 0 of storage at 4°C, qualified on day 5, and unqualified on day 8.

[0060] Figure 33 The present invention relates to the application of the sensor tag Dma / FPS prepared by this invention to quickly determine the freshness of salmon samples when stored at 25°C. The integrated tag is designed to determine the freshness of the salmon. The salmon is considered fresh when stored at 25°C for 0 hours, qualified when stored for 30 hours, and unqualified when stored for 44 hours.

[0061] Figure 34 The freshness of salmon meat was monitored by storing the Dma / PVA sensing film prepared by this invention with salmon samples at 4°C and observing the color changes of the label under sunlight and ultraviolet light, combined with a standard colorimetric card. The salmon meat was considered fresh on day 0 of storage at 4°C, qualified on day 5, and unqualified on day 8.

[0062] Figure 35This invention relates to the application of an integrated label designed to quickly determine the freshness of salmon samples when the Dma / PVA sensing film prepared in this invention is stored at 25°C. The salmon samples are considered fresh after 0 hours of storage at 25°C, qualified after 30 hours of storage, and unqualified after 44 hours of storage. Detailed Implementation

[0063] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0064] Example 1

[0065] The specific synthesis steps of the fluorescent probe Dma are as follows:

[0066]

[0067] Compound DCA (0.2693 g, 1 mmol), cycloisopropyl malonate (0.2162 g, 1.5 mmol), and acetic acid (10.8 mg) were dissolved in ethanol (10 mL). The mixture was stirred at room temperature for 5 hours, filtered, and the precipitated crude product, a dark green solid, was collected. The product was recrystallized from ethanol to obtain the fluorescent probe Dma with a yield of 79.3%. 1 H NMR spectrum, 13 C NMR spectrum and mass spectrum as follows Figure 1-3 As shown.

[0068] 1 H NMR (400MHz, DMSO-d6) δ8.16(s,1H),7.44(s,1H),7.39(d,J=8.5Hz,1H),6.77(d,J=8.5Hz,1H),6.76(s,1H ),3.47(q,J=6.8Hz,4H),2.93(t,J=6.8Hz,2H),2.82(t,J=6.8Hz,2H),1.64(s,6H),1.13(t,J=6.8Hz,6H).

[0069] 13 C NMR (101MHz, DMSO-d6) δ170.39,155.37,150.95,141.15,131.93,129.63,116. 32,112.43,111.27,102.66,97.74,97.54,44.62,28.11,26.91,25.03,12.86.

[0070] HRMS Calculated for C 23 H 25 NNaO5 + [M+Na]+ calcd:418.1625,found:418.1639.

[0071] Example 2

[0072] Compound DCA (2.693 g, 10 mmol), cycloisopropyl malonate (2.16 g, 15 mmol), and acetic acid (80.7 mg) were dissolved in ethanol (20 mL). The mixture was stirred at room temperature for 6 hours, filtered, and the precipitated crude product, a dark green solid, was collected. Recrystallization from ethanol yielded the fluorescent probe Dma, with a yield of 83.6%. In this example, the fluorescent probe Dma... 1 H NMR spectrum as shown Figure 1 , 13 The C NMR spectrum is as follows Figure 2 High-resolution mass spectrometry, such as Figure 3 As shown.

[0073] Example 3

[0074] Compound DCA (13.465 g, 50 mmol), cycloisopropyl malonate (14.41 g, 100 mmol), and acetic acid (358.6 mg) were dissolved in ethanol (50 mL). The mixture was stirred at room temperature for 8 hours, filtered, and the precipitated crude product, a dark green solid, was collected. This solid was recrystallized from ethanol to obtain the fluorescent probe Dma, with a yield of 72%. In this example, the fluorescent probe Dma... 1 H NMR spectrum as shown Figure 1 , 13 The C NMR spectrum is as follows Figure 2 High-resolution mass spectrometry, such as Figure 3 As shown.

[0075] I. Fluorescent probe Dma recognizes HSO3 - Selective detection:

[0076] A 10 μmol / L fluorescent probe Dma solution was prepared in DMSO / PBS (2 / 8, v / v). 20 μL (50 mmol / L) of 19 anions and 3 biothiols (CO3) were then added to the solution. 2- HCO3 - C2O4 2- CH3COO - PPI, HPO4 2- SO4 2- S2O3 2- SCN - ,HS - N3 - NO2 - ,F - ,Cl - ,Br- ,I - ,S 2- SO3 2- HSO3 - (Cys, Hcy, GSH) was used to detect changes in the absorption spectrum of the solution. From Figure 4 As can be seen, there is a significant absorption peak at 636 nm. When 350 μmol / L HSO3 is added... - When S was added, the absorbance decreased significantly, and the solution color changed from blue to colorless. However, when other analytes were added to the solution, except for S, the absorbance decreased. 2- The solution subsequently turned pale blue with a slight decrease in absorbance. The addition of other anions and biothiols did not significantly alter the absorbance or solution color, indicating that the Dma probe exhibits good UV recognition. Furthermore, the fluorescence emission spectrum of the Dma solution changed after excitation of the fluorescent probe at 580 nm, as shown below. Figure 5 As shown, when using SO3 2- / HSO3 - After treatment, significant fluorescence quenching was observed at 675 nm. When other analytes were added, except for S... 2- The addition of [a specific substance] caused a slight decrease in fluorescence intensity, while other anions had no significant effect on the fluorescence intensity. Therefore, the fluorescent probe Dma recognizes HSO3- via ultraviolet light and fluorescence. - It has good selectivity.

[0077] II. Fluorescent probe Dma recognizes HSO3 - Anti-interference detection:

[0078] 10 μmol / L fluorescent probe Dma was dissolved in DMSO / PBS (2 / 8, v / v), and 20 μL (50 mmol / L) of different analytes (CO3) were added to each solution. 2- HCO3 - C2O4 2- CH3COO - PPI, HPO4 2- SO4 2- S2O3 2- SCN - ,HS - N3 - NO2 - ,F - ,Cl - ,Br - ,I - ,S 2- HSO3 -(Cys, Hcy, GSH) was used to test the UV absorption and fluorescence emission spectra of the solution. Then, 350 μmol / L HSO3 was added to each solution containing the respective anion. - The ultraviolet absorption and fluorescence emission spectra of the solution were tested. The results are as follows: Figure 6 and Figure 7 As shown, when HSO3 is added - When different analytes were added, the absorbance at 636 nm and the fluorescence intensity at 675 nm showed almost no significant change (green square). Further addition of HSO3 to the above solution... - At (350 μmol / L), both absorbance and fluorescence intensity decreased significantly (red square), indicating that probe Dma can still recognize HSO3 even in the presence of other anions and biothiols. - It has good anti-interference capabilities.

[0079] III. Fluorescent probe Dma against HSO3 - Titration test:

[0080] 10 μmol / L fluorescent probe Dma in DMSO / PBS (2 / 8, v / v) solution, with 0–350 μmol / L HSO3 added respectively. - The changes in the fluorescence emission spectrum of the test solution, such as Figure 8 As shown. With HSO3 - As the concentration increases, the emission peak intensity at 675 nm gradually decreases. This decrease is observed when 350 μmol / L HSO3 is added. - When the emission peak intensity no longer changes, it indicates that saturation has been reached.

[0081] IV. Fluorescent probe Dma for HSO3 - Detection limit test:

[0082] In a DMSO / PBS (2 / 8, v / v) solution containing probe Dma, the fluorescence intensity of at least 11 parallel samples was measured, according to the formula ∑(X i -X) 2 =(X1-X) 2 +(X2-X) 2 +……+(X n -X) 2 Find the sum of the differences of squares (X) i For each measurement of the receptor's fluorescence intensity, X is the average fluorescence intensity, and n is the number of tests (n≥11). Then, according to the formula: S=[∑(X i -X) 2 / (n-1)] 0.5Calculate the sensitivity S, and then use the detection limit formula: Detection limit = 3S / K, where K is the slope of the selected linear segment (Note: the linear segment is based on a titration plot, with the x-axis representing ion concentration and the y-axis representing fluorescence intensity). The detection limit is calculated to be 3.993 μmol / L (see...). Figure 9 This indicates that the probe has a low detection limit and has certain practical application value.

[0083] V. Fluorescent probe Dma for HSO3 - Response time test:

[0084] The fluorescence intensity of the probe Dma in DMSO / PBS (2 / 8, v / v) solution changed over time, and the results are as follows: Figure 10 As shown, when 350 μmol / L HSO3 is added... - Subsequently, the fluorescence intensity of the probe at 675 nm decreased significantly and stabilized within 60 seconds, indicating that the probe Dma can rapidly detect HSO3. - .

[0085] VI. Fluorescent probe Dma for HSO3 - pH response test:

[0086] To demonstrate the practicality of Dma, we examined the effects of different pH values. For example... Figure 11 As shown, the probe Dma only exhibited strong fluorescence emission at pH 3-11. Subsequently, HSO3 was added to the probe Dma. - The fluorescence intensity decreased significantly only at pH 6-11, indicating that Dma can detect HSO3 within the pH range of 6-11. - .

[0087] VII. The fluorescent probe of the present invention for HSO3 - The recognition mechanism for n-propylamine is as follows:

[0088]

[0089] To determine whether probe Dma recognizes HSO3 - The mechanism, Dma+HSO3 - High-resolution mass spectrometry was tested. For example... Figure 12 As shown, Dma + HSO3 - A new peak appears at m / z = 500.1353, which is the probe [M+HSO3] - +Na] + The corresponding peak (theoretical value 500.1350) indicates that HSO3 -A nucleophilic addition reaction occurred with Dma, disrupting the conjugated system and hindering intramolecular charge transfer of Dma, leading to fluorescence quenching and a fading of the solution color from blue to colorless, thus enabling "naked-eye" and fluorescent recognition of HSO3. - After reacting n-propylamine with the fluorescent probe Dma, the resulting solution was analyzed by high-resolution mass spectrometry. Figure 13 As shown, a new peak appears at m / z = 438.0566 in Dma+CH3CH2CH2NH2, which corresponds to the peak of the product generated after the ester group in the probe Dma is aminolyzed (theoretical value is 438.0551). This indicates that after Dma recognizes n-propylamine, the entire conjugated system is not destroyed, the electron-withdrawing ability of the carbonyl group in the generated product is changed, and the intramolecular charge transfer of Dma still exists, resulting in a decrease in fluorescence intensity at long wavelengths and an increase in fluorescence intensity at short wavelengths, as well as a significant change in color.

[0090] VIII. Application of fluorescent probe Dma in red wine and white sugar:

[0091] Dilute 1 mL of red wine to 100-fold with PBS (pH = 7.4) buffer solution to prepare a corresponding DMSO / PBS (2 / 8, v / v) solution containing red wine. Use this solution to dilute 250 μL of probe Dma into a 25 mL volumetric flask to obtain the required test solution. Dilute 0.5 g of white sugar with DMSO / PBS (2 / 8, v / v) solution, add 250 μL of probe Dma solution, and dilute into a 25 mL volumetric flask to obtain the required test solution. Subsequently, add different concentrations of HSO3 to Dma (10 μmol / L) solutions containing different amounts of red wine and white sugar. - The fluorescence intensity change at 675 nm was recorded. For example... Figure 14 As shown, when HSO3 is added - A good linear relationship was observed at concentrations of 5-35 μM. Recovery rates were calculated (Table 1): the recovery rate of red wine sample 1 was between 97.29% and 100.41%, the recovery rate of red wine sample 2 was between 97.27% and 98.16%, the recovery rate of white sugar sample 1 was between 97.42% and 101.09%, and the recovery rate of white sugar sample 2 was between 99.43% and 101.85%. This working method was compared with traditional methods for detecting HSO3 in food samples. - Compared with titration methods for the concentration of HSO3, the error was small (Table 2). Good recovery rates and data compared with traditional titration methods indicate that the probe Dma can accurately detect HSO3 in actual samples of red wine and white sugar. - content.

[0092] Table 1 shows the detection of HSO3 by the fluorescent probe Dma of this invention in red wine and white sugar samples. - Recovery rate and standard deviation

[0093]

[0094] The experiment was repeated three times.

[0095] Table 2 shows the detection of HSO3 by the fluorescent probe Dma of this invention in red wine and white sugar samples. - Comparison with traditional titration methods

[0096]

[0097] The experiment was repeated three times.

[0098] IX. Cytotoxicity assay of fluorescent probe Dma and its effect on HSO3 - Cell imaging:

[0099] To investigate the biological applications of the probe Dma, its toxicity was first tested using a CCK-8 assay kit. For example... Figure 15 As shown, when Dma concentrations were 0-50 μmol / L, cell viability was greater than 85% after 24 hours, indicating that Dma had low cytotoxicity and good biocompatibility. MCF-7 cells were co-incubated with 10 μmol / L probe Dma at 37°C for 30 min, exhibiting significant red fluorescence in the dark. Cells were then washed three times with PBS, and different concentrations of HSO3 were added. - (10, 50, 100 and 350 μmol / L) and incubated for 30 min, with HSO3 - As the concentration increases, the red fluorescence gradually weakens until it disappears. Figure 16 This indicates that Dma can inhibit HSO3 in living cells. - Perform fluorescence imaging.

[0100] 10. Selectivity of fluorescent probe Dma for amine compounds

[0101] A 10 μmol / L solution of fluorescent probe Dma was prepared by adding 2 mL of DMSO / H2O (4 / 6, v / v) solution. Then, 20 μL and 50 mmol / L solutions of various amines (aniline, trimethylamine, tyramine, 2-phenylethylamine, diethylamine, tryptamine, 1,2-cyclohexanediamine, n-propylamine, triethylamine, dimethylamine, spermine, putrescine, and cadaverine) were added respectively, and the changes in UV and fluorescence were observed. Figure 17 The ultraviolet spectroscopy revealed that, except for aniline, the solutions of the other 12 amine compounds all caused varying degrees of decrease and blue shift in the maximum absorption peak at 640 nm of the Dma, and the solution color changed from blue to other colors. Figure 18Fluorescence spectroscopy revealed that, except for aniline, the solutions of the other 12 amine compounds all caused a decrease in the fluorescence emission of Dma at 675 nm, while fluorescence appeared and significantly increased at 580 nm, changing from red to orange fluorescence. These results indicate that Dma can identify a variety of amine compounds by colorimetry and fluorescence in DMSO / H2O (4 / 6, v / v) solution, with a wide recognition range.

[0102] XI. Recognition performance of fluorescent probe Dma for triethylamine, a representative amine compound

[0103] First, the fluorescence titration of amine compounds with Dma was tested. Taking triethylamine as an example, the relationship between the fluorescence intensity of 10 μmol / L Dma in DMSO / H2O (4 / 6, v / v) solution and the concentration of triethylamine solution was tested. Figure 19 As shown, as the concentration of added triethylamine gradually increases, the fluorescence intensity at 675 nm gradually decreases, while the fluorescence intensity at 580 nm gradually increases. When a 400 μmol / L triethylamine solution is added, the fluorescence intensity no longer changes, indicating that saturation has been reached. Other amine compounds, except for aniline, exhibit similar changes.

[0104] Secondly, the detection limit of DMA for triethylamine was calculated. Based on the fluorescence titration data, the abscissa X = log[guest] was calculated, where [guest] is the ion concentration, i.e., the triethylamine concentration. The ordinate was calculated as (I max -I) / (I max -I min ), where I max The fluorescence maximum value at 675 nm, I min Let I be the fluorescence minimum at 675 nm, and I be the variable at 675 nm. Plot a dot plot. According to Y = AX + B (where A is the slope of the selected straight line and B is the intercept), when Y = 0, the calculated value is the detection limit. The detection limit is calculated using the formula 10... -A / B The detection limit was determined to be 80.7 μmol / L (see...). Figure 20 This indicates that the probe has good sensitivity in detecting triethylamine in aqueous solutions and has the potential for practical application.

[0105] Furthermore, the effect of pH on the detection of amine compounds was investigated. Taking triethylamine as an example, the ratio of fluorescence intensity at 580 nm to 675 nm was plotted on the ordinate. The fluorescence intensity ratio of Dma under different pH conditions is shown below. Figure 21As shown, the probe Dma exhibits strong stability within a pH range of 2-11. The addition of triethylamine increases the fluorescence intensity ratio, and changes in the fluorescence intensity ratio are observed at pH 5-11. Fluorescence at 580 nm is enhanced, while fluorescence at 675 nm is weakened, indicating significant changes before and after recognition. We can conclude that within a pH range of 5-11, the fluorescent probe Dma demonstrates good recognition efficacy for triethylamine.

[0106] For rapid detection, response time is a key factor. After adding 400 μmol / L triethylamine solution to a solution containing Dma, the change in fluorescence intensity at 675 nm over time was monitored. Figure 22 As shown, the fluorescence intensity of the Dma solution decreased significantly and then leveled off within 2 hours. This rapid response laid the foundation for the subsequent preparation of indicator tags to monitor the freshness of fish in real time.

[0107] Finally, the cell imaging effect of Dma on triethylamine was tested. MCF-7 cells were co-incubated with 10 μmol / L probe Dma at 37°C for 30 min, as shown in the figure. Figure 23 As shown, there is obvious red fluorescence in the dark field. Then, after washing three times with PBS, different concentrations of triethylamine (0, 10, 100, 200, 500 μmol / L) were added and incubated for 30 min. As the concentration of triethylamine gradually increased, the red fluorescence gradually weakened, indicating that the probe Dma can perform fluorescence imaging of triethylamine in live cells.

[0108] 12. The effectiveness of the indicator label in identifying volatile amines in a simulated environment (cylindrical glass bottle, 57mm high, 18mm in diameter).

[0109] The label preparation process is as follows:

[0110] Weigh 3.95 mg of fluorescent probe and dissolve it in 10 mL of DMSO. Soak a 1.5 cm diameter circular filter paper in the solution overnight, then remove it and dry it in an oven to obtain the sensor tag Dma / FPS for monitoring the freshness of fish.

[0111] 3.2 g of polyvinyl alcohol (PVA) was dissolved in 40 mL of deionized water and magnetically stirred at 90 °C for 2 h to obtain a clear, transparent, viscous solution. Heating was stopped, and the solution was allowed to cool to room temperature. 10 mg of fluorescent probe was dissolved in 1 mL of DMSO and poured into the cooled PVA solution. The solution was quickly stirred until homogeneous, and then ultrasonically degassed to remove air bubbles, yielding a PVA solution containing the fluorescent probe. 15 mL of this solution was poured into a 90 mm diameter glass petri dish and dried in an oven. Finally, the entire PVA film was cut into 1 cm × 1 cm square polyvinyl alcohol films to obtain the Dma / PVA sensing film for monitoring the freshness of fish.

[0112] During fish spoilage, some volatile metabolic byproducts are produced, such as triethylamine, diethylamine, and n-propylamine. Detecting these volatile metabolic byproducts can monitor the freshness of the fish. To verify whether the indicator tag is selective for volatile amines, we placed the indicator tag in the top space of a 0.5% volatile amine aqueous solution to simulate the fish spoilage environment and observe whether the sensor tag Dma / FPS and the sensor film Dma / PVA respond to volatile amines.

[0113] First, 1 mL of a 50 mmol / L volatile amine was added to 1 mL of distilled water to prepare a 25 mmol / L volatile amine solution. Fourteen amines were selected as experimental groups: ammonia, 1,2-cyclohexanediamine, diethylamine, n-propylamine, triethylamine, spermine, cadaverine, putrescine, 2-phenylethylamine, tyramine, aniline, trimethylamine, dimethylamine, and tryptamine. An empty bottle with an indicator label served as a blank control group. Figure 24 As shown, the Dma / FPS sensor tag is blue in sunlight and red in fluorescence. When exposed to different amines, except for tryptophan, which showed no significant change in sunlight color, the other 13 amines turned purple or colorless under sunlight, and their fluorescence color changed to bright green or non-fluorescent. Both sunlight and fluorescence colors showed significant changes, indicating that the Dma / FPS sensor tag is sensitive to volatile amines and has good dual colorimetric and fluorescence responses. This also suggests that the Dma / FPS sensor tag has the potential to indicate the freshness of fish. Figure 25 As shown, the Dma / PVA sensing film is blue in sunlight and red in fluorescence. When exposed to different amines, except for tryptamine which shows no significant change in sunlight color, the other 13 amines turn purple, green, gray, etc. in sunlight, and their fluorescence color changes to purple, blue, or no fluorescence. Both sunlight and fluorescence colors show significant changes, indicating that the Dma / PVA sensing film is sensitive to volatile amines and has good colorimetric and fluorescence dual responses. This also shows that the Dma / PVA sensing film has the potential to indicate the freshness of fish.

[0114] XIII. Preparation of Standard Colorimetric Cards for Labels

[0115] To prepare a standard colorimetric chart, we need to monitor the color changes of the sensor tag or sensing film under natural light and fluorescent light during the process of fish meat from fresh to spoilage. We selected salmon meat as the experimental subject, removed the skin from the back meat, and divided it into several small pieces of 5-6g each. These pieces were placed in disposable 90mm diameter petri dishes. The sensor tag or sensing film was placed in the opening above the lid of each petri dish, the lids were sealed, and the dishes were stored at 4℃. The TVB-N content, pH value, and color difference of the salmon meat were measured every 24 hours (see [link to relevant documentation]). Figure 26 and Figure 27 ), and collected natural light and fluorescence photos of the sensor tag or sensing film, and obtained data such as Figure 28 and Figure 29 As shown, the TVB-N content of fresh salmon meat shows an overall upward trend starting from 2.33 ± 0.06 mg / 100 g. The TVB-N content reaches 15.87 ± 0.06 mg / 100 g on the 5th day and 30.33 ± 0.06 mg / 100 g on the 8th day, while the acceptable limit value of TVB-N is 30 mg / 100 g. Therefore, the 8th day of storage of salmon meat at 4°C can be regarded as the critical value of storage deterioration. Under natural light, the sensing label Dma / FPS changes from the initial blue to purple and finally to colorless; under 365 nm ultraviolet light, the indicator label changes from the initial red fluorescence to non-fluorescent and finally to bright green fluorescence. Under natural light, the sensing film Dma / PVA changes from the initial blue to gray and finally to ivory white; under 365 nm ultraviolet light, the indicator label changes from the initial red fluorescence to blue fluorescence and finally to green fluorescence.

[0116] According to the provisions of the national standard of the People's Republic of China GB / T 18108-2019 (General Rules for Fresh Seawater Fish), the maximum limit of TVB-N content in seawater fish is 30 mg / 100 g. When the TVB-N value ≤ 15 mg / 100 g, the sample is of excellent grade; when 15 mg / 100 g < TVB-N value ≤ 30 mg / 100 g, the sample is qualified; when the TVB-N value > 30 mg / 100 g, the sample is unqualified at this time.

[0117] Based on the measured TVB-N values and the corresponding photos of the natural light color and ultraviolet light color of the sensing label Dma / FPS, we selected the sensing label Dma / FPS corresponding to the TVB-N values of 2.33 mg / 100 g and 13.53 mg / 100 g, which show dark blue and light blue respectively under natural light and red fluorescence and magenta fluorescence under ultraviolet light irradiation, as the reference standards for the fresh part in the standard color comparison card. When the sensing label Dma / FPS shows one of the above color states, it can be judged that this sample is a fresh product (see Figure 30 the fresh area in). We selected the sensing label Dma / FPS corresponding to the TVB-N values of 15.87 mg / 100 g and 25.67 mg / 100 g, which show purple and light purple respectively under natural light and almost no fluorescence under ultraviolet light irradiation, as the reference standards for the qualified part in the standard color comparison card. When the sensing label Dma / FPS shows one of the above color states, it can be judged that this sample is a qualified product (see Figure 30(Medium-compliant area). The sensor labels Dma / FPS corresponding to TVB-N values ​​of 30.33 mg / 100g and 32.67 mg / 100g are selected. These labels are colorless under natural light and exhibit bright green fluorescence under ultraviolet light. These are used as reference standards for non-compliant portions in the standard colorimetric card. When the sensor label Dma / FPS displays one of these color states, the sample can be judged as a non-compliant product (see...). Figure 30 (Corruption zone in China).

[0118] Based on the measured TVB-N values ​​and the corresponding natural and ultraviolet light colors of the sensing film Dma / PVA, we selected Dma / PVA with TVB-N values ​​of 2.33 mg / 100g and 13.53 mg / 100g as reference standards for the fresh portion in the standard colorimetric card. When the sensing film Dma / PVA displays one of these color states, the sample can be considered a fresh product (see...). Figure 31 (Mid-fresh area). The sensor film Dma / PVA with TVB-N values ​​of 15.87 mg / 100g and 25.67 mg / 100g, which appears gray under natural light and exhibits blue fluorescence under ultraviolet light, were selected as the reference standard for the qualified portion in the standard colorimetric card. When the sensor film Dma / PVA displays one of the above color states, the sample can be judged as a qualified product (see...). Figure 31 (Medium-Qualified Zone). The sensor film Dma / PVA with TVB-N values ​​of 30.33 mg / 100g and 32.67 mg / 100g, which appears ivory white under natural light and green fluorescence under ultraviolet light, was selected as the reference standard for the non-compliant portion in the standard colorimetric card. When the sensor film Dma / PVA displays one of the above color states, the sample can be judged as a non-compliant product (see...). Figure 31 (Corruption zone in China).

[0119] XIV. Practical Application of Indicator Labels

[0120] The freshness of salmon was monitored in practice using sensor tags Dma / FPS and sensor films Dma / PVA, respectively, and the salmon sample processing was the same as described above. Figure 32 and Figure 33The color changes of the sensing label Dma / FPS and the sensing film Dma / PVA of salmon during storage in a 4°C refrigerator with increasing storage time are shown. In newly purchased salmon, the sensing label Dma / FPS appears blue under natural light and emits red fluorescence under ultraviolet light, corresponding to the fresh part in the standard color comparison card; when the storage time is 5 days, the natural light color of the indicating label is purple and there is no fluorescence under ultraviolet light, indicating that it is still a qualified product at this time. When the storage time is further increased to 8 days, the natural light color of the indicating label is colorless and there is strong bright green fluorescence under ultraviolet light, indicating that the salmon has deteriorated at this time and the unqualified product is inedible.

[0121] In newly purchased salmon, the sensing film Dma / PVA appears blue under natural light and emits red fluorescence under ultraviolet light, corresponding to the fresh part in the standard color comparison card; when the storage time is 5 days, the natural light color of the indicating label is gray and there is weak blue fluorescence under ultraviolet light, indicating that it is still a qualified product at this time. When the storage time is further increased to 8 days, the natural light color of the indicating label is ivory white and there is medium green fluorescence under ultraviolet light, indicating that the salmon has deteriorated at this time.

[0122] In addition, we integrated the sensing label Dma / FPS or the sensing film Dma / PVA with the standard color comparison card to design an integrated label, and investigated the real-time monitoring of the freshness of salmon fish at room temperature, and then realized the application of quickly judging the freshness of fish. Figure 34 and Figure 35 The color changes of the sensing label Dma / FPS and the sensing film Dma / PVA of salmon with increasing storage time at 25°C room temperature are shown. In the newly purchased salmon fish, the sensing label Dma / FPS appears blue under natural light and emits red fluorescence under ultraviolet light, corresponding to the fresh part in the standard color comparison card. The actually measured TVB-N content is 6.53, indicating that the salmon fish is indeed fresh at this time, indicating that the sensing label can accurately evaluate the fresh level of the salmon fish through the colorimetric and fluorescence dual channels. When the salmon is stored at 25°C for 30 h, the color of the sensing label Dma / FPS changes from blue to purple, and the red fluorescence changes to weak fluorescence or even no fluorescence, indicating that the salmon is slightly decomposed and reaches sub-fresh, and is a qualified product at this time; the actually measured TVB-N content is 15.4, indicating that the salmon fish is indeed qualified at this time, indicating that the sensing label can accurately evaluate the qualified level of the salmon fish through the colorimetric and fluorescence dual channels. Until 44 h later, the label is colorless and emits bright green fluorescence, indicating that the salmon has deteriorated and is inedible. The actually measured TVB-N content is 30.33, indicating that the salmon fish has indeed deteriorated at this time, indicating that the sensing label can accurately evaluate the unqualified level of the salmon fish through the colorimetric and fluorescence dual channels.

[0123] Newly purchased salmon from the same batch was also placed in smart packaging containing the Dma / PVA sensor film. The sensor film appeared blue under natural light and emitted red fluorescence under ultraviolet light, corresponding to the fresh portion on the standard colorimetric card, with an initial TVB-N content of 6.53. Similarly, after 30 hours of storage at room temperature, the Dma / PVA smart label changed from blue to gray, and the fluorescence color changed from red to blue, indicating a change in quality from fresh to acceptable, with an actual measured TVB-N content of 15.4. After 44 hours of storage at room temperature, the Dma / PVA sensor film appeared ivory white with strong green fluorescence, indicating spoilage, and the actual measured TVB-N content reached 30.33. These results demonstrate that the Dma / PVA sensor film constructed using the probe Dma can accurately evaluate the freshness level of salmon flesh through both colorimetric and fluorescence dual-channel methods.

[0124] Both the Dma / FPS sensor tag and the Dma / PVA sensor film indicator tag can accurately determine the actual freshness of salmon meat by comparing it with a standard colorimetric card under dual-mode signal changes at room temperature of 4℃ and 25℃. This helps consumers purchase qualified salmon products and helps retailers sell near-expiry products in a timely manner, which has positive guiding significance for ensuring food safety.

[0125] In summary, our prepared multifunctional near-infrared fluorescent probe Dma can be used to detect sulfur dioxide derivatives and volatile amines, exhibiting both colorimetric and fluorescence dual-mode responses. In DMSO / PBS (2 / 8, v / v) solution, Dma can rapidly detect HSO3. - It exhibits high sensitivity and a wide applicable pH range. Furthermore, DMA has been successfully applied to the detection of HSO3 in food samples such as red wine and white sugar. - It can also affect HSO3 in living cells. - Fluorescence imaging with triethylamine has biological application value. Furthermore, the two indicator tags of this invention—the Dma / FPS sensor tag and the Dma / PVA sensor film—are simple to prepare and can both achieve dual-channel indication of fish freshness through colorimetry and fluorescence, providing accurate and reliable qualitative analysis results. These indicator tags enable rapid, real-time, non-contact, and non-destructive monitoring of fish freshness, providing timely and effective freshness information for producers, retailers, and consumers, and have significant practical application value.

Claims

1. A bifunctional near-infrared emission fluorescent probe for the simultaneous detection of sulfur dioxide derivatives and volatile amines, characterized in that: The fluorescent probe has the following structural formula:

2. The application of the dual-functional near-infrared emission fluorescent probe for simultaneous detection of sulfur dioxide derivatives and volatile amines according to claim 1, characterized in that: HSO3 in a DMSO / PBS system with a volume ratio of 2 / 8 - The test is being conducted for purposes other than disease diagnosis and treatment.

3. The application of the dual-functional near-infrared emission fluorescent probe for simultaneous detection of sulfur dioxide derivatives and volatile amines according to claim 1, characterized in that: HSO3 in red wine and white sugar - The test is being conducted for purposes other than disease diagnosis and treatment.

4. The application of the dual-functional near-infrared emission fluorescent probe for simultaneous detection of sulfur dioxide derivatives and volatile amines according to claim 1, characterized in that: HSO3 in living cells - Fluorescence imaging with triethylamine is used for non-disease diagnosis and non-disease treatment purposes.

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