A Ratiometric Near-Infrared Fluorescent Probe and Its Synthesis Method and Application

Abstract

The present invention discloses a ratiometric near-infrared fluorescent probe, its synthesis method and applications, belonging to the field of organic small molecule ratiometric fluorescent probes. The molecular formula of the near-infrared fluorescent probe is C 32 H 31 N2O3 + , labeled as FS-B1. The maximum absorption wavelength of the near-infrared fluorescent probe in acetonitrile and PBS (30% ACN) is 640 nm, the fluorescence spectrum wavelength is 765 nm, the fluorescence performance is stable, and the response time is fast. In acetonitrile, as the concentration of organic amine increases, and in PBS (30% ACN), as the concentration of NaHSO3 increases, the emission peak of the near-infrared fluorescent probe at 765 nm gradually decreases, the emission peak at 483 nm gradually increases, the absorption peak at 640 nm gradually decreases, the absorption peak at 400 nm gradually increases, and the color of the solution changes significantly and can be distinguished by the naked eye. The near-infrared fluorescent probe realizes the detection of trace amounts of organic amines and NaHSO3 with high sensitivity, and can efficiently and selectively detect trace amounts of biogenic amines produced during food spoilage, showing good application prospects in the field of food safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic small molecule ratiometric fluorescent probes, and particularly relates to a ratiometric near-infrared fluorescent probe, a synthesis method thereof, and an application thereof. Background Art

[0002] With the continuous improvement of living conditions, the public's attention to food safety issues has also increased day by day. The accelerating pace of life has also led to the development of food detection towards the direction of rapidity, accuracy, high efficiency, and safety.

[0003] Meat or protein in food is decomposed due to the influence of factors such as the growth of microorganisms, temperature, and environment, which is called food spoilage. Food spoilage will produce biogenic amines, volatile substances, microorganisms, and their metabolites, etc. These substances all have certain harms to the human body. Therefore, detecting food spoilage is of great significance to food safety and human health. NaHSO3 has the strongest reducibility and bleaching property among sulfite bleaching agents. As a widely used food additive, it is used to inhibit spoilage reactions, oxidation reactions, and microbial reactions caused by bacteria in food and beverages. It is widely used in China for the bleaching of preserved fruits and canned foods, and the maximum usage amount can reach 0.40 g / kg. Although NaHSO3 has advantages such as anti-corrosion, excessive addition is very harmful to the human body. Therefore, detecting the content of NaHSO3 in food is also of great significance to food safety and human health.

[0004] Traditional methods for detecting organic amines and bisulfite (such as chemiluminescence method, chromatography method, electrochemistry method, enzyme method, etc.) have some deficiencies, such as low sensitivity, poor selectivity, slow response speed, and cumbersome sample pretreatment and detection processes. Fluorescent probes have been widely used in the field of food detection in recent years due to their characteristics such as simple operation, rapid response, high sensitivity, good selectivity, and qualitative and quantitative detection. Among them, ratiometric fluorescent probes are an important technology in fluorescence analysis, which can effectively eliminate background errors caused by external factors such as the intensity fluctuation of the excitation light source, photobleaching of the fluorescent reagent, and changes in the concentration of the fluorescent sensing molecule, so as to obtain more accurate results. The present invention designs a ratiometric near-infrared fluorescent probe capable of detecting trace amounts of organic amines and NaHSO3 with high sensitivity under different working conditions, which has practical significance and good application prospects. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a ratiometric near-infrared fluorescent probe capable of regulating the detection of NaHSO3 and organic amines, a synthesis method thereof, and an application thereof, which can efficiently and selectively detect trace amounts of NaHSO3 and biogenic amines generated during food spoilage, and realize the visualization and high-sensitivity detection of trace amounts of NaHSO3 and trace amounts of organic amines.

[0006] The technical solution of the present invention is as follows:

[0007] One of the purposes of the present invention is to provide a ratio-type near-infrared fluorescence probe, and the near-infrared fluorescence probe is labeled as FS-B1, with a molecular formula of C 32 H 31 N2O3 + , and the chemical structural formula is as shown in Ⅰ:

[0008]

[0009] Furthermore, for the fluorescence probe FS-B1 in acetonitrile, its maximum absorption wavelength is at 640 nm, and the fluorescence spectrum wavelength is at 765 nm.

[0010] Another purpose of the present invention is to provide a synthesis method of a ratio-type near-infrared fluorescence probe, including the following steps:

[0011] (1) Mix 3-hydroxy-N,N-dimethylaniline and ethyl benzoylacetate, heat and react, and obtain a yellow solid through separation and purification, which is compound 1. The reaction process is as follows:

[0012]

[0013] (2) Dissolve compound 1 in a tetrahydrofuran solution, cool it to 0 °C, dropwise add methylmagnesium bromide, fully react under normal temperature conditions, quench the obtained solution with an aqueous solution of fluoroboric acid, and extract it into DCM with fluoroboric acid, then dry and separate and purify to obtain a dark red solid, which is compound 2. The reaction process is as follows:

[0014]

[0015] (3) Dissolve the obtained compound 2 and 7-(diethylamino)coumarin-3-carbaldehyde in absolute ethanol, add anhydrous sodium acetate and then heat under reflux. After removing the solvent, a purple compound is obtained. After separation and purification, the near-infrared fluorescence probe FS-B1 is obtained. The reaction process is as follows:

[0016]

[0017] Furthermore, in the step (1), the molar ratio of 3-hydroxy-N,N-dimethylaniline to ethyl benzoylacetate is 1:1.75.

[0018] Furthermore, in the step (1), the heating reaction time is 22 - 25 h, and the heating reaction temperature is 160 - 200 °C.

[0019] Furthermore, in the step (1), the separation and purification process adopts column chromatography separation, and the chromatography eluent is a mixed solution of dichloromethane / methanol, and the volume ratio of the two is 50:1.

[0020] Furthermore, in the step (2), the molar ratio of compound 1 to methylmagnesium bromide is 1:1.5.

[0021] Furthermore, in the step (2), the concentration of the aqueous solution of fluoboric acid for quenching is 50%, and the concentration of fluoboric acid for extraction is 5%.

[0022] Furthermore, in the separation and purification processes of the steps (2) and (3), column chromatography separation is adopted, and the eluent for chromatography is a mixed solution of dichloromethane / methanol, and the volume ratio of the two is 50:1.

[0023] Furthermore, in the step (3), the molar ratio of compound 2, 7-(diethylamino)coumarin-3-carbaldehyde, and sodium acetate anhydrous is 1:1:0.1.

[0024] Furthermore, in the step (3), during the reflux process, the reaction temperature is 80 - 90 °C, and the reaction time is 15 - 18 h.

[0025] The third object of the present invention is to provide the application of the ratio-type near-infrared fluorescence probe in the highly sensitive detection of trace organic amines and NaHSO3.

[0026] Furthermore, when there are organic amines and NaHSO3 in the environment, the near-infrared fluorescence probe FS-B1 undergoes a nucleophilic addition reaction with the organic amine, resulting in an obvious color change.

[0027] Furthermore, as the concentrations of the organic amine and NaHSO3 increase, the emission peak of the near-infrared fluorescence probe FS-B1 at 765 nm gradually decreases, the emission peak at 483 nm gradually increases, and the emission peak blue-shifts by approximately 165 nm; the absorption peak at 640 nm gradually decreases, the absorption peak at 400 nm gradually increases, the absorption spectrum blue-shifts by approximately 240 nm, and the color of the solution changes from dark blue to light yellow.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. The present invention designs a ratio-type near-infrared fluorescence probe FS-B1 that can regulate the detection of NaHSO3 and organic amines, which is an ideal sensor for rapidly and sensitively detecting organic amines and NaHSO3. This near-infrared fluorescence probe has stable fluorescence performance. Its maximum absorption wavelength in acetonitrile is 640 nm, and the fluorescence spectrum wavelength is 765 nm. It has colorimetric and ratio sensing characteristics with sensitive reaction and obvious color change. When there are organic amines and NaHSO3 in the environment, this near-infrared fluorescence probe can undergo a strong sensing process with rapid response (30 s), high sensitivity, high selectivity, and specificity, realizing the real-time quantitative detection of organic amines and NaHSO3.

[0030] 2. When the near-infrared fluorescent probe FS-B1 synthesized by the present invention can directly detect organic amines and NaHSO3, when reacting with organic amines and NaHSO3, both the ultraviolet-visible absorption spectrum and the fluorescence emission spectrum show a blue shift. The color of the solution changes from the original dark blue to light blue, colorless, and then gradually turns to light yellow. The color change is significant, and it can visually achieve "naked-eye" detection of organic amines and NaHSO3, showing broad application prospects in visual detection and the development of test strips.

[0031] 3. Compared with the existing fluorescent probe technology, the synthesis method of the near-infrared fluorescent probe FS-B1 in the present invention is simple, with easy operation steps, fewer types of raw materials required, and an objective yield. High-energy excitation is not needed during the synthesis process, which is environmentally friendly. The synthesized near-infrared fluorescent probe can be applied to the safety assessment of NaHSO3 and biogenic amines in spoiled food, showing good development prospects in the field of food safety.

[0032] Attached drawing reference numerals

[0033] Figure 1 1H NMR spectrum of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention;

[0034] Figure 2 1H NMR spectrum of compound 2 synthesized in Example 1 of the present invention;

[0035] Figure 3 Fluorescence intensity change diagram of the near-infrared fluorescent probe FS-B1 synthesized in Example 2 of the present invention for detecting actual samples of different days;

[0036] Figure 4 Ultraviolet spectral characteristic diagram of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with ethylenediamine in solvents with different polarities and different dielectric constants;

[0037] Figure 5 Ultraviolet spectral characteristic diagram of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with NaHSO3 in organic solvents and non-organic solvents;

[0038] Figure 6 Fluorescence spectral characteristic diagram of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with ethylenediamine in solvents with different polarities and different dielectric constants;

[0039] Figure 7 Fluorescence spectral characteristic diagram of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with NaHSO3 in organic solvents and non-organic solvents;

[0040] Figure 8 UV absorption spectral characteristic diagrams of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with ethylenediamine at different concentrations;

[0041] Figure 9 UV absorption spectral characteristic diagrams of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with hexylamine at different concentrations;

[0042] Figure 10 UV absorption spectral characteristic diagrams of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with diethylenetriamine at different concentrations;

[0043] Figure 11 UV absorption spectral characteristic diagrams of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with 1,5-pentanediamine (cadaverine) at different concentrations;

[0044] Figure 12 UV absorption spectral characteristic diagrams of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with 1,4-butanediamine (putrescine) at different concentrations;

[0045] Figure 13 UV absorption spectral characteristic diagrams of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with spermine at different concentrations;

[0046] Figure 14 UV absorption spectral characteristic diagrams of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with tyramine at different concentrations;

[0047] Figure 15 UV absorption spectral characteristic diagrams of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with histamine at different concentrations;

[0048] Figure 16 UV absorption spectral characteristic diagrams of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with NaHSO3 at different concentrations;

[0049] Figure 17 Fluorescence spectral characteristic diagrams of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with ethylenediamine, where Figure 17 A - B are fluorescence spectra (A: excitation light is 415 nm, B: excitation light is 640 nm), Figure 17 C is the relationship between the fluorescence intensity at 483 nm and the concentration;

[0050] Figure 18 Fluorescence spectral characteristic diagrams of the near-infrared fluorescent probe FS-B1 synthesized in Example 1 of the present invention reacting with hexylamine, whereFigure 18 A-B is the fluorescence spectrum (A: excitation light is 415 nm, B: excitation light is 640 nm), Figure 18 C is the relationship between the fluorescence intensity and concentration at 483 nm;

[0051] Figure 19 Fluorescence spectral characteristic diagram of the near-infrared fluorescence probe FS-B1 synthesized in Example 1 of the present invention reacting with diethylenetriamine, where, Figure 19 A-B is the fluorescence spectrum (A: excitation light is 415 nm, B: excitation light is 640 nm), Figure 19 C is the relationship between the fluorescence intensity and concentration at 483 nm;

[0052] Figure 20 Fluorescence spectral characteristic diagram of the near-infrared fluorescence probe FS-B1 synthesized in Example 1 of the present invention reacting with cadaverine, where, Figure 20 A-B is the fluorescence spectrum (A: excitation light is 415 nm, B: excitation light is 640 nm), Figure 20 C is the relationship between the fluorescence intensity and concentration at 483 nm;

[0053] Figure 21 Fluorescence spectral characteristic diagram of the near-infrared fluorescence probe FS-B1 synthesized in Example 1 of the present invention reacting with putrescine, where, Figure 21 A-B is the fluorescence spectrum (A: excitation light is 415 nm, B: excitation light is 640 nm), Figure 21 C is the relationship between the fluorescence intensity and concentration at 483 nm;

[0054] Figure 22 Fluorescence spectral characteristic diagram of the near-infrared fluorescence probe FS-B1 synthesized in Example 1 of the present invention reacting with spermine, where, Figure 22 A-B is the fluorescence spectrum (A: excitation light is 415 nm, B: excitation light is 640 nm), Figure 22 C is the relationship between the fluorescence intensity and concentration at 483 nm;

[0055] Figure 23 Fluorescence spectral characteristic diagram of the near-infrared fluorescence probe FS-B1 synthesized in Example 1 of the present invention reacting with tyramine, where, Figure 23 A-B is the fluorescence spectrum (A: excitation light is 415 nm, B: excitation light is 640 nm), Figure 23 C is the relationship between the fluorescence intensity and concentration at 483 nm;

[0056] Figure 24 Fluorescence spectral characteristic diagram of the near-infrared fluorescence probe FS-B1 synthesized in Example 1 of the present invention reacting with histamine, where, Figure 24A - B is the fluorescence spectrum (A: excitation light is 415 nm, B: excitation light is 640 nm), Figure 24 C is the relationship between the fluorescence intensity and concentration at 483 nm;

[0057] Figure 25 Fluorescence spectrum characteristics diagram of the near - infrared fluorescence probe FS - B1 synthesized in Example 1 of the present invention when reacting with NaHSO3. Among them, Figure 25 A - B is the fluorescence spectrum (A: excitation light is 415 nm, B: excitation light is 640 nm), Figure 25 C is the relationship between the fluorescence intensity and concentration at 483 nm;

[0058] Figure 26 Fluorescence intensity change diagram of the near - infrared fluorescence probe FS - B1 synthesized in Example 1 of the present invention after reacting with different organic amines in acetonitrile solvent. Detailed implementation mode

[0059] The following further describes the present invention with reference to the attached drawings and preferred embodiments. The given embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention.

[0060] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels without special instructions;

[0061] In the following quantitative tests, three repeated experiments are set, and the results are averaged;

[0062] The experimental methods in the following embodiments are all conventional methods without special instructions;

[0063] The dry solvents used in the reaction are dried using molecular sieve 4A (sodium - A type molecular sieve) or molecular sieve 3A (potassium - A type molecular sieve);

[0064] The inert atmosphere used in the reaction selects argon as the protective gas;

[0065] 1 1H NMR spectra are recorded on a JEOL ECZ600S (600 MHz) spectrometer using CDCl3 or CD3OD as the solvent;

[0066] According to the internal TMS (trimethylsilane) reference data, report the chemical shift in parts per million of the front field;

[0067] Coupling constants (J) are expressed in hertz (Hz), and spin multiplicities are represented by s (singlet), d (doublet), t (triplet), and m (multiplet);

[0068] Column chromatography used in the reaction employed a thick-walled glass column and silica gel (300 - 400 mesh); thin-layer chromatography (TLC) was carried out using a commercially available 0.25 mm silica gel plate and visualized under an ultraviolet lamp;

[0069] The ultraviolet absorption spectrum in solution was obtained using a Shimadzu UV-1900 ultraviolet-visible-near-infrared spectrophotometer;

[0070] The fluorescence spectrum was measured using a Spectrofluorometer FS5 fluorescence spectrometer; the mass spectrum was recorded on a ThermoFisher high-performance liquid chromatography-mass spectrometry (HPLC-MS) instrument.

[0071] Example 1

[0072] This example provides a ratiometric near-infrared fluorescence probe, and its synthesis method includes the following steps:

[0073] S1. Mix 3-hydroxy-N,N-dimethylaniline and ethyl benzoylacetate, heat and react at 180 °C for 24 h, and separate and purify using a dichloromethane / methanol mixed solution with a volume ratio of 50:1 to obtain a yellow solid, which is Compound 1. Perform nuclear magnetic resonance hydrogen spectrum analysis on it, and the spectrum is as shown in the appendix. The preparation process is as follows: Figure 2 as shown, and the preparation process is as follows:

[0074]

[0075] S2. Dissolve Compound 1 in a tetrahydrofuran solution, cool to 0 °C, dropwise add methylmagnesium bromide, and react fully at room temperature for 12 h. Quench the resulting solution with 50% aqueous fluoboric acid solution, extract it with 5% fluoboric acid into DCM, dry it, and separate and purify using a dichloromethane / methanol mixed solution with a volume ratio of 50:1 to obtain a dark red solid, which is Compound 2, with a yield of 29%. The preparation process is as follows:

[0076]

[0077] S3. Dissolve the obtained Compound 2 and 7-(diethylamino)coumarin-3-carbaldehyde in absolute ethanol, add anhydrous sodium acetate, and heat under reflux at 85 °C for 16 h. After removing the solvent, a purple compound is obtained. Separate and purify it using a dichloromethane / methanol mixed solution with a volume ratio of 50:1 to obtain the near-infrared fluorescence probe FS-B1, with a yield of 50%. Perform nuclear magnetic resonance hydrogen spectrum analysis on it, and the spectrum is as shown in the appendix. The synthesis process is as follows: Figure 1 as shown, and the synthesis process is as follows:

[0078]

[0079] Among them, this synthesis method can also adjust S1 to react by heating at 160 °C for 25 h or react by heating at 200 °C for 22 h according to the actual operation conditions, and adjust S3 to reflux by heating at 80 °C for 18 h or reflux by heating at 90 °C for 15 h.

[0080] Example 2

[0081] In this example, the ratio-type near-infrared fluorescent probe in Example 1 was used to detect biogenic amines in the actual sample of shrimp, including the following steps:

[0082] S1. Select fresh live shrimp as the sample, cook the shrimp and place it at room temperature, and take the pieces on the first day, the third day, and the fifth day for dilution;

[0083] S2. Under the excitation lights of 415 nm and 640 nm, measure the fluorescence spectra of the near-infrared fluorescent probe buffer solution FS-B1 (10 μM) added with different volumes (0 - 60 μL) of cooked shrimp juice, record the maximum fluorescence intensity at the peak, and draw a line graph according to the obtained data.

[0084] As shown in the appendix Figure 3 As shown, with the change of time, under the action of microorganisms, the amine production of the shrimp meat increases, the concentration of biogenic amines in the sample rises, the fluorescence value at 483 nm increases after the near-infrared fluorescent probe binds to the amine, and the amine production reaches the peak on the third day and then the amount of biogenic amines decreases, and the fluorescence value also decreases. To sum up, through the binding of the near-infrared fluorescent probe and biogenic amines, the trend of food spoilage can be reflected indirectly, so the fluorescent probe FS-B1 can be used to evaluate the degree of food spoilage.

[0085] Performance Test

[0086] 1. UV spectral test of the ratio-type near-infrared fluorescent probe FS-B1 in Example 1 with 10 eq of ethylenediamine in solvents with different polarities and different dielectric constants:

[0087] S1. Select four solvents with different polarities and different dielectric constants through two conditions of solvent polarity and dielectric constant: petroleum ether, tetrahydrofuran, water, N,N-dimethylformamide, and acetonitrile.

[0088] S2. Dissolve the probe FS-B1 obtained in Example 1 in dimethyl sulfoxide, place it in four solvents of petroleum ether, tetrahydrofuran, water, N,N-dimethylformamide, and acetonitrile, and measure the ultraviolet absorption spectrum after reacting with 10 eq of ethylenediamine.

[0089] As shown in the appendix Figure 4As shown, the near-infrared fluorescent probe FS-B1 in the ultraviolet spectrum shows a maximum absorption value at 640 nm and exhibits the same colorimetric sensing characteristics as ethylenediamine in four solvents: petroleum ether, tetrahydrofuran, water, N,N-dimethylformamide, and acetonitrile.

[0090] 2. Fluorescence spectrum test of the ratiometric near-infrared fluorescent probe FS-B1 in Example 1 with 10 eq of ethylenediamine in solvents with different polarities and different dielectric constants:

[0091] S1. Four solvents with different polarities and different dielectric constants are screened out through two conditions of solvent polarity and dielectric constant: petroleum ether, tetrahydrofuran, water, N,N-dimethylformamide, and acetonitrile.

[0092] S2. The near-infrared fluorescent probe FS-B1 obtained in Example 1 is dissolved in dimethyl sulfoxide and placed in four solvents: petroleum ether, tetrahydrofuran, water, N,N-dimethylformamide, and acetonitrile. The fluorescence spectrum after the reaction with 10 eq of ethylenediamine is measured, and a bar chart based on the highest fluorescence value at 483 nm is made. The conditions of the fluorescence spectrometer are: λex = 640 nm / 415 nm, Step: 1.00 nm, Dwell: 0.1 s, Repeats: 2, Bdw = 2.

[0093] As shown in Figure 6 A-C, when excited at 415 nm in the fluorescence spectrum, the near-infrared fluorescent probe FS-B1 has a maximum fluorescence value at 483 nm, and when excited at 640 nm, the near-infrared fluorescent probe FS-B1 has a maximum fluorescence value at 765 nm; when 10 eq of ethylenediamine is added, the fluorescence intensity at 765 nm gradually decreases, while the fluorescence intensity at 483 nm gradually increases. The near-infrared fluorescent probe FS-B1 and ethylenediamine exhibit the same fluorescence change characteristics in four solvents: petroleum ether, tetrahydrofuran, water, N,N-dimethylformamide, and acetonitrile. However, in the acetonitrile solution, the near-infrared fluorescent probe FS-B1 has a higher fluorescence intensity at 483 nm and the highest fluorescence intensity at 765 nm. After adding 10 eq of ethylenediamine, the change degree is obvious. Therefore, acetonitrile solvent can be selected as a good medium for detecting organic amines by the near-infrared fluorescent probe FS-B1.

[0094] 3. Ultraviolet spectrum test of the ratiometric near-infrared fluorescent probe FS-B1 in Example 1 with 10 eq of NaHSO3 in organic solvents and non-organic solvents:

[0095] S1. Four buffer solvents are screened: PBS, PBS(30% ACN), acetonitrile, and tetrahydrofuran.

[0096] S2. Dissolve the probe FS-B1 obtained in Example 1 in dimethyl sulfoxide, place it in four solvents: PBS, PBS (30% ACN), acetonitrile, and tetrahydrofuran, and measure the ultraviolet absorption spectrum after reacting with 10 eq of NaHSO3.

[0097] As shown in the appendix Figure 5 As shown, in the ultraviolet spectrum, the near-infrared fluorescent probe FS-B1 shows the maximum absorption value at 640 nm, presenting the same colorimetric sensing characteristics as NaHSO3 in four solvents: PBS, PBS (30% ACN), acetonitrile, and tetrahydrofuran.

[0098] 4. Fluorescence spectrum test of the ratio-type near-infrared fluorescent probe FS-B1 in Example 1 with 10 eq of NaHSO3 in organic solvents and non-organic solvents:

[0099] S1. Screen four buffer solvents: PBS, PBS (30% ACN), acetonitrile, and tetrahydrofuran.

[0100] S2. Dissolve the near-infrared fluorescent probe FS-B1 obtained in Example 1 in dimethyl sulfoxide, place it in four solvents: PBS, PBS (30% ACN), acetonitrile, and tetrahydrofuran, and measure the fluorescence spectrum after reacting with 10 eq of NaHSO3 and the column chart based on the highest fluorescence value at 483 nm. The conditions of the fluorescence spectrometer are: λex = 640 nm / 415 nm, Step: 1.00 nm, Dwell: 0.1 s, Repeats: 2, Bdw = 2.

[0101] As shown in the appendix Figure 7 As shown in A - C, in the fluorescence spectrum, when excited at 415 nm, the near-infrared fluorescent probe FS-B1 has a maximum fluorescence value at 483 nm, and when excited at 640 nm, the near-infrared fluorescent probe FS-B1 has a maximum fluorescence value at 765 nm; when 10 eq of NaHSO3 is added, the fluorescence intensity at 765 nm gradually decreases, while the fluorescence intensity at 483 nm gradually increases. The near-infrared fluorescent probe FS-B1 and NaHSO3 present the same fluorescence change characteristics in four solvents: PBS, PBS (30% ACN), acetonitrile, and tetrahydrofuran. However, in the PBS (30% ACN) solution, the near-infrared fluorescent probe FS-B1 has a higher fluorescence intensity at 483 nm and the highest fluorescence intensity at 765 nm, and the change degree is obvious after adding 10 eq of NaHSO3. Therefore, the PBS (30% ACN) solvent can be selected as a good medium for detecting NaHSO3 by the near-infrared fluorescent probe FS-B1.

[0102] 5. UV absorption spectrum test of the ratiometric near-infrared fluorescent probe FS-B1 in Example 1 for different concentrations of ethylenediamine, n-hexylamine, diethylenetriamine, 1,5-pentanediamine, 1,4-butanediamine, spermine, tyramine, and histamine:

[0103] S1. Dissolve the near-infrared fluorescent probe FS-B1 obtained in Example 1 in dimethyl sulfoxide and dilute it with an acetonitrile buffer solution to a 10 μM probe buffer solution.

[0104] S2. Take several portions of the above solution and add ethylenediamine, n-hexylamine, diethylenetriamine, 1,5-pentanediamine, 1,4-butanediamine, spermine, tyramine, and histamine solutions respectively, so that their concentrations are: 0 - 1000 μM, 0 - 1000 μM, 0 - 500 μM, 0 - 700 μM, 0 - 400 μM, 0 - 100 μM, 0 - 1500 μM, 0 - 700 μM. Then measure the UV absorption spectra of the near-infrared fluorescent probe FS-B1 after reacting with different concentrations of amines.

[0105] As shown Figures 8 - 15 in the figure, in the UV spectrum, the near-infrared fluorescent probe FS-B1 shows the maximum absorption value at 640 nm. After reacting with ethylenediamine, n-hexylamine, diethylenetriamine, 1,5-pentanediamine, 1,4-butanediamine, spermine, tyramine, and histamine respectively, it shows the same colorimetric sensing characteristics. As the concentrations of ethylenediamine, n-hexylamine, diethylenetriamine, 1,5-pentanediamine, 1,4-butanediamine, spermine, tyramine, and histamine increase, the absorption peak of the near-infrared fluorescent probe FS-B1 at 640 nm gradually decreases, while the absorption peak at 400 nm gradually increases. During the process of concentration increase, the color of the solution changes from the original dark blue to light blue, colorless, and finally yellow, indicating that the near-infrared fluorescent probe FS-B1 can visually achieve "naked-eye" detection of organic amines.

[0106] 6. UV absorption spectrum test of the ratiometric near-infrared fluorescent probe FS-B1 in Example 1 for different concentrations of NaHSO3:

[0107] S1. Dissolve the near-infrared fluorescent probe FS-B1 obtained in Example 1 in dimethyl sulfoxide and dilute it with a PBS (30% ACN) buffer solution to a 10 μM probe buffer solution.

[0108] S2. Take the above solution and add NaHSO3 so that its concentration is: 0 - 300 μM. Then measure the UV absorption spectra of the near-infrared fluorescent probe FS-B1 after reacting with different concentrations of NaHSO3.

[0109] As shown Figure 16As shown, in the ultraviolet spectrum, the near-infrared fluorescent probe FS-B1 shows a maximum absorption value at 640 nm. With the increase in the concentration of NaHSO3, the absorption peak of the near-infrared fluorescent probe FS-B1 at 640 nm gradually decreases, while the absorption peak at 400 nm gradually increases. During the process of concentration increase, the color of the solution changes from the original dark blue to light blue, colorless, and finally yellow, indicating that the near-infrared fluorescent probe FS-B1 can visually achieve "naked-eye" detection of NaHSO3.

[0110] 7. Fluorescence spectrum test of the ratiometric near-infrared fluorescent probe FS-B1 in Example 1 for different concentrations of ethylenediamine, n-hexylamine, diethylenetriamine, 1,5-pentanediamine, 1,4-butanediamine, spermine, tyramine, and histamine:

[0111] S1. Take several buffer solutions of the near-infrared fluorescent probe FS-B1, and add ethylenediamine, n-hexylamine, diethylenetriamine, 1,5-pentanediamine, 1,4-butanediamine, spermine, tyramine, and histamine solutions respectively, so that their concentrations are: 0 - 70 μM, 0 - 100 μM, 0 - 60 μM, 0 - 200 μM, 0 - 400 μM, 0 - 100 μM, 0 - 1000 μM, 0 - 700 μM. Under the conditions of excitation light at 415 nm and 640 nm, measure the fluorescence spectra of the near-infrared fluorescent probe FS-B1 after reacting with different concentrations of organic amines respectively. Among them, the conditions of the fluorescence spectrometer are: λex = 640 nm / 415 nm, Step: 1.00 nm, Dwell: 0.1 s, Repeats: 2, Bdw = 2.

[0112] As Figure 17 A - B ~ Figure 24 As shown in A - B, in the fluorescence spectrum, the behavior of the near-infrared fluorescent probe FS-B1 is consistent with that in the ultraviolet spectrum, showing similar sensitivity and selectivity. When the excitation light is 640 nm, with the addition of ethylenediamine, n-hexylamine, diethylenetriamine, 1,5-pentanediamine, 1,4-butanediamine, spermine, tyramine, and histamine, the fluorescence of the near-infrared fluorescent probe FS-B1 at 765 nm gradually weakens; when the excitation light is 415 nm, with the addition of ethylenediamine, n-hexylamine, diethylenetriamine, 1,5-pentanediamine, 1,4-butanediamine, spermine, tyramine, and histamine, the fluorescence intensity of the near-infrared fluorescent probe FS-B1 at 765 nm gradually decreases, while the fluorescence intensity at 483 nm gradually increases, and an isosbestic point is formed at about 630 nm.

[0113] From Figure 17 C ~ Figure 24As can be seen from Figure C, there is a good linear relationship between the fluorescence intensity of the near-infrared fluorescent probe FS-B1 and the concentrations of ethylenediamine, n-hexylamine, diethylenetriamine, 1,5-pentanediamine, 1,4-butanediamine, spermine, tyramine, and histamine (linear correlation coefficient R 2 = 0.9956, R 2 = 0.9927, R 2 = 0.9788, R 2 = 0.9978, R 2 = 0.9992, R 2 = 0.9902, R 2 = 0.9989, R 2 = 0.9947). Therefore, the near-infrared fluorescent probe FS-B1 can quantitatively detect organic amines, and the detection limits of the near-infrared fluorescent probe FS-B1 for ethylenediamine, n-hexylamine, diethylenetriamine, 1,5-pentanediamine, 1,4-butanediamine, spermine, tyramine, and histamine can be calculated using the formula, which are 1.14 μM, 0.13 μM, 0.27 μM, 0.81 μM, 0.34 μM, 1.87 μM, 1.64 μM, and 2.73 μM, respectively.

[0114] As shown in the appendix Figure 26 The near-infrared fluorescent probe FS-B1 has a certain selectivity for organic amines. Comparing the fluorescence values before the reaction, the fluorescence intensity increased by 145 times after reacting with ethylenediamine and 54 times after reacting with tyramine. It can be seen that the selectivity of the near-infrared fluorescent probe FS-B1 for organic amines is related to the structure of the organic amines, being more sensitive to aliphatic organic amines and less sensitive to aromatic organic amines.

[0115] 8. Fluorescence spectrum test of the ratiometric near-infrared fluorescent probe FS-B1 in Example 1 for different concentrations of NaHSO3:

[0116] S1. Take the buffer solution of the near-infrared fluorescent probe FS-B1 and add the NaHSO3 solution to make its concentration 0 - 300 μM. Under the conditions of excitation light at 415 nm and 640 nm, measure the fluorescence spectra of the near-infrared fluorescent probe FS-B1 after reacting with different concentrations of NaHSO3. Among them, the conditions of the fluorescence spectrometer are: λex = 640 nm / 415 nm, Step: 1.00 nm, Dwell: 0.1 s, Repeats: 2, Bdw = 2.

[0117] As Figure 25As shown in A - B, in the fluorescence spectrum, the behavior of the near - infrared fluorescent probe FS - B1 is consistent with that in the ultraviolet spectrum, showing similar sensitivity and selectivity. When the excitation light is 640 nm, with the addition of NaHSO3, the fluorescence of the near - infrared fluorescent probe FS - B1 at 765 nm gradually weakens; when the excitation light is 415 nm, with the addition of NaHSO3, the fluorescence intensity of the near - infrared fluorescent probe FS - B1 at 765 nm gradually decreases, while the fluorescence intensity at 483 nm gradually increases, and an isosbestic point is formed at about 630 nm.

[0118] As can be seen from Figure 25 C, the fluorescence intensity of the near - infrared fluorescent probe FS - B1 has a good linear relationship with the concentration of NaHSO3 (linear correlation coefficient R 2 = 0.9968). Therefore, the near - infrared fluorescent probe FS - B1 can quantitatively detect NaHSO3, and the detection limit of the near - infrared fluorescent probe FS - B1 for NaHSO3 can be calculated to be 0.16 μM using the formula.

[0119] 9. Spike recovery test of the ratiometric near - infrared fluorescent probe FS - B1 for the detection of biogenic amines in actual samples in Example 1:

[0120] S1. After cooking fresh shrimp meat and leaving it for three days, take crushed pieces for dilution. Under excitation lights of 415 nm and 640 nm, measure the fluorescence spectra of the near - infrared fluorescent probe FS - B1 buffer solution (10 μM) added with different volumes (0 - 60 μL) of the supernatant of cooked shrimp meat crushed pieces and different concentrations of biogenic amines (putrescine, cadaverine, spermine, tyramine).

[0121] S2. Obtain the fluorescence values from the fluorescence spectra in S1, substitute them into the standard curves of different biogenic amines to calculate the content of amines, and then calculate the spike recovery rate according to the spike recovery formula. The spike recovery formula is as follows:

[0122] P (recovery rate)=(spiked sample amount - sample measured amount) / spiked amount;

[0123] The calculation results are shown in Table 1:

[0124] Table 1 Spike recovery rates of the near - infrared fluorescent probe FS - B1 for the detection of biogenic amines in actual samples

[0125]

[0126] As can be seen from Table 1, the average recovery rates of different biogenic amines at different concentrations in the sample are all within a good range, indicating that the use of the near - infrared fluorescent probe FS - B1 for the detection of biogenic amines in spoiled food is effective.

[0127] The above are only embodiments of the present invention, and thus do not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, shall similarly be included within the patent protection scope of the present invention.

Claims

1. A ratiometric near-infrared fluorescence probe, characterized in that, The near-infrared fluorescent probe is labeled as FS-B1, and its molecular formula is C 32 H 31 N2O3 + , and its chemical structural formula is as shown in Ⅰ:

2. A method for synthesizing the ratio-type near-infrared fluorescence probe according to claim 1, characterized in that, The following steps are adopted: (1) 3-Hydroxy-N,N-dimethylaniline and ethyl benzoylacetate are mixed and heated for reaction. After separation and purification, a yellow solid, which is Compound 1, is obtained. Its structural formula is as shown in II: (2) Compound 1 is dissolved in a tetrahydrofuran solution and cooled to 0 °C. Methylmagnesium bromide is added dropwise. After fully reacting at room temperature, the obtained solution is quenched with an aqueous solution of fluoroboric acid and extracted into DCM with fluoroboric acid. After drying and separation and purification, a dark red solid, which is Compound 2, is obtained. Its structural formula is as shown in III: (3) The obtained Compound 2 and 7-(diethylamino)coumarin-3-carbaldehyde are dissolved in absolute ethanol. After adding anhydrous sodium acetate, the mixture is heated under reflux. After removing the solvent, a purple compound is obtained. After separation and purification, a near-infrared fluorescent probe FS-B1 is obtained. Its structural formula is as shown in IV:

3. The synthesis method of a ratio-type near-infrared fluorescent probe according to claim 2, characterized in that, In the step (1), the molar ratio of 3-hydroxy-N,N-dimethylaniline to ethyl benzoylacetate is 1:1.

75.

4. The synthesis method of a ratio-type near-infrared fluorescent probe according to claim 2, characterized in that, In the separation and purification process in the step (1), column chromatography separation is adopted. The chromatography eluent is a mixed solution of dichloromethane / methanol, and the volume ratio of the two is 50:

1.

5. The synthesis method of a ratio-type near-infrared fluorescent probe according to claim 2, characterized in that, In the step (2), the molar ratio of Compound 1 to methylmagnesium bromide is 1:1.

5.

6. The synthesis method of a ratio-type near-infrared fluorescent probe according to claim 2, characterized in that, In the step (2), the concentration of the aqueous solution of fluoroboric acid used for quenching is 50%, and the concentration of fluoroboric acid used for extraction is 5%.

7. The synthesis method of a ratio-type near-infrared fluorescent probe according to claim 2, characterized in that, In the separation and purification processes in the steps (2) and (3), column chromatography separation is adopted. The chromatography eluent is a mixed solution of dichloromethane / methanol, and the volume ratio of the two is 50:

1.

8. The synthesis method of a ratio-type near-infrared fluorescent probe according to claim 2, characterized in that, In the step (3), the molar ratio of Compound 2, 7-(diethylamino)coumarin-3-carbaldehyde, and anhydrous sodium acetate is 1:1:0.

1.

9. Application of a ratiometric near-infrared fluorescent probe as described in Claim 1 in the preparation of a highly sensitive detection reagent for detecting trace organic amines and NaHSO3.

10. Use of the ratio-type near-infrared fluorescent probe as described in claim 9 in the preparation of a highly sensitive detection reagent for detecting trace organic amines and NaHSO3, characterized in that, When organic amines and NaHSO3 are present in the environment, the near-infrared fluorescent probe FS-B1 undergoes a nucleophilic addition reaction with organic amines and NaHSO3, resulting in an obvious color change.

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