Novel near-infrared aggregation-induced emission dye and probe as well as preparation method and application of novel near-infrared aggregation-induced emission dye and probe

Abstract

The invention belongs to the field of functional dyes in fine chemical engineering, and particularly relates to a novel near-infrared aggregation-induced emission dye and a probe as well as a preparation method and application of the novel near-infrared aggregation-induced emission dye. The near-infrared aggregation-induced emission dye provided by the invention has double-electron donor characteristics of triphenylamine and thiophene, can emit near-infrared aggregation-induced fluorescence with a peak value of 660nm in an aqueous solution, has an excitation wavelength in a visible light region, and has relatively small background fluorescence and scattering signals. The near-infrared aggregation-induced emission probe provided by the invention belongs to a lightening type fluorescent probe, has high specificity and detection speed on 2, 6-dimethylthiophenol, and has practical application value in food samples.

Description

Technical Field

[0001] This invention belongs to the field of fine chemical functional dyes, specifically relating to the preparation of a novel near-infrared aggregation-induced emission dye and probe, and a high-throughput rapid detection method. Background Technology

[0002] Fluorescent probes, due to their high sensitivity, rapid response, good selectivity, and low cost, have become an emerging detection technology and are applied in fields such as food, environment, and biomedicine. Because these fields commonly encounter problems such as complex composition, strong background fluorescence, and severe scattering, developing fluorescent probes with low background and high selectivity is a crucial indicator for achieving practical sample detection. Near-infrared fluorescent probes use near-infrared fluorescent dyes above 650 nm as signal units, avoiding interference from short-wavelength background fluorescence. Simultaneously, the longer wavelength reduces scattering signals from complex samples, and they have been widely used in the detection of biological samples. "Light-up" fluorescent probes, characterized by low initial fluorescence signals, avoid signal interference from their own initial fluorescence. "Light-up" near-infrared fluorescent probes, designed with near-infrared fluorescence in mind, can minimize background fluorescence signals from both the sample and the probe itself, thereby improving the sensitivity and reliability of the detection results.

[0003] 2,6-Dimethylthiophenol, with its low odor threshold and strong sulfurous aroma, is approved for use as a food flavoring and is widely used in baked goods, meat products, beverages, and other food products. Studies have shown that thiophenol compounds are toxic to aquatic organisms and mice (LC50). 50 (The concentration is 0.01-0.4 mM), and long-term exposure may cause health problems such as damage to the central nervous system. Therefore, effective detection of PhSHs content in food is of great significance to the field of food safety science.

[0004] Most existing thiophene fluorescent probes are designed as reactive probes. The nucleophilic reaction of the thiol group of thiophene compounds is the core of the design of this type of fluorescent probe. Therefore, the selectivity of this type of fluorescent probe for different thiophene compounds is not very good. This problem restricts the practical application of this type of probe in complex samples.

[0005] In the food testing industry, a large number of samples need to be tested daily. In existing reports on fluorescent probe technologies, high-throughput detection technology is still uncommon, and cases of its application in food testing are even rarer. Summary of the Invention

[0006] To address the shortcomings of existing methods, this invention provides a novel near-infrared aggregation-induced emission dye and probe, along with their preparation method and applications. The near-infrared aggregation-induced emission dye provided by this invention exhibits two-electron donor characteristics, emitting near-infrared aggregation-induced fluorescence with a peak wavelength of 660 nm in aqueous solution, and its excitation wavelength is located in the visible light region, exhibiting low background fluorescence and scattering signal. The near-infrared aggregation-induced emission probe provided by this invention is a "spot-on" fluorescent probe, demonstrating high specificity and detection speed for 2,6-dimethylthiophenol.

[0007] The technical solution of this invention is a novel near-infrared aggregation-induced emission dye, with the structural formula shown in formula (I): .

[0008] This invention also provides a method for preparing a novel near-infrared aggregation-induced emission dye, as shown in route 1 or route 2, comprising the following steps: (1) Under nitrogen protection and catalysis by a noble metal catalyst, compound M0 and dipinazoboronic acid ester were heated in an organic solvent containing a basic compound to prepare compound M1. (2) Under nitrogen protection and catalysis by a noble metal catalyst, compound M1 and 2,5-dibromothiophene were heated in an organic solvent containing a basic compound to prepare compound M2. (3) Under nitrogen protection and catalysis by a noble metal catalyst, compound M2 reacts with triphenylamine boric acid in an organic solvent containing a basic compound to produce compound I; or; (I) Under nitrogen protection and catalysis by a noble metal catalyst, triphenylamine boric acid and 2,5-dibromothiophene were reacted by heating in an organic solvent containing a basic compound to prepare compound M3; (II) Under nitrogen protection and catalysis by a noble metal catalyst, compound M3 and compound M1 react in an organic solvent containing a basic compound to produce compound I.

[0009] In step (1), the concentration of compound Mo in the organic solvent is 0.08-0.5 mmol / mL, such as 0.09-0.1 mmol / mL in one embodiment; the molar ratio of compound Mo, bis-pinacol boronic acid ester, basic compound and noble metal catalyst is 1:1.5-3:2.5-4:0.04-0.1, such as 1:2:3:0.04-0.05 in one embodiment; the basic compound can be an organic base compound or an inorganic base compound, such as potassium acetate, sodium acetate, potassium carbonate, sodium carbonate, etc. in one embodiment; the noble metal catalyst includes but is not limited to palladium catalyst, ruthenium catalyst, rhodium catalyst, platinum catalyst, etc., such as Pd(dppf)Cl2 in one embodiment; the organic solvent includes but is not limited to dioxane, acetone, ethyl acetate, dimethyl sulfoxide, etc., such as dioxane in one embodiment.

[0010] Step (1): Heat to 110±3℃ and react for 3-5 hours.

[0011] Step (1): The reaction solution obtained from the reaction is cooled to room temperature, water is added to quench the reaction, and the crude product is filtered. The crude product is purified by silica gel column chromatography. The eluent used for silica gel column chromatography is a mixture of ethyl acetate and dichloromethane with a volume ratio of 1:10, which yields compound M1.

[0012] In step (2), the concentration of compound M1 in the organic solvent is 0.05-0.1 mmol / mL, such as 0.06-0.07 mmol / mL in one embodiment; the molar ratio of compound M1, 2,5-dibromothiophene, basic compound and noble metal catalyst is 1:0.8-2:1-2:0.02-0.05, such as 1:0.8-1.5:1.2-1.5:0.02-0.03 in one embodiment; the basic compound can be an organic base compound or an inorganic base compound, such as potassium carbonate, sodium carbonate, potassium bicarbonate, sodium bicarbonate, etc. in one embodiment; the noble metal includes but is not limited to palladium catalyst, ruthenium catalyst, rhodium catalyst, platinum catalyst, etc., such as tetra(triphenylphosphine)palladium in one embodiment; the organic solvent includes but is not limited to tetrahydrofuran, diethyl ether, cyclopentyl methyl ether, etc., such as tetrahydrofuran in one embodiment.

[0013] Step (2): Heat the oil bath to 70±3℃ and react for 5-7 hours.

[0014] Step (2): The organic solvent in the reaction solution is removed by rotary evaporation and then extracted with dichloromethane to obtain compound M2.

[0015] In step (3), the concentration of compound M2 in the organic solvent is 0.01-0.3 mmol / mL, such as 0.02-0.03 mmol / mL in one embodiment; the molar ratio of compound M2, triphenylamine boric acid, basic compound and noble metal catalyst is 1:1-2:2.5-4:0.03-0.08, such as 1:1.3-1.5:2.7-3:0.04-0.045 in one embodiment; the basic compound can be an organic base compound or an inorganic base compound, such as potassium carbonate, sodium carbonate, potassium bicarbonate, sodium bicarbonate, etc. in one embodiment; the noble metal includes but is not limited to palladium catalyst, ruthenium catalyst, rhodium catalyst, platinum catalyst, etc., such as tetrakis(triphenylphosphine)palladium in one embodiment; the organic solvent includes but is not limited to dimethylformamide, dimethyl sulfoxide, ethylene glycol dimethyl ether, etc., such as dimethylformamide in one embodiment.

[0016] Step (3): Heat the oil bath to 130±3℃ and react for 5-6 hours.

[0017] Step (3): The reaction solution obtained from the reaction is cooled to room temperature, water is added to quench the reaction, and the crude product is obtained by column chromatography purification.

[0018] Step (I): The concentration of triphenylamine boric acid in the organic solvent is 0.2-0.6 mmol / mL, such as 0.2 mmol / mL in one embodiment; the molar ratio of triphenylamine boric acid, 2,5-dibromothiophene, basic compound and noble metal catalyst is 1:0.5-2:0.4-1:0.015-0.08, such as 1:0.7-1:0.4-0.6:0.015-0.02 in one embodiment; the basic compound can be an organic base compound or an inorganic base compound, such as potassium carbonate, sodium carbonate, potassium bicarbonate, sodium bicarbonate, etc. in one embodiment; the noble metal includes but is not limited to palladium catalyst, ruthenium catalyst, rhodium catalyst, platinum catalyst, etc., such as tetrakis(triphenylphosphine)palladium in one embodiment; the organic solvent includes but is not limited to xylene, acetone, diethyl ether, etc., such as toluene in one embodiment.

[0019] Step (I): Heat in an oil bath to 100±3℃ and react for 10-12 hours.

[0020] Step (I): The organic solvent in the reaction solution is removed by rotary evaporation. The crude product is purified by silica gel column chromatography with a mesh size of 200-300 to obtain compound M3.

[0021] In step (II), the concentration of compound M3 in the organic solvent is 0.05-0.3 mmol / mL, or 0.07-0.08 mmol / mL in one embodiment; the molar ratio of compound M3, compound M1, basic compound, and noble metal catalyst is 1:1.2-2:0.5-1.5:0.02-0.1, or 1:1.2-1.5:0.7-1:0.025-0.03 in one embodiment; the basic compound can be an organic or inorganic basic compound, such as potassium carbonate, sodium carbonate, potassium bicarbonate, sodium bicarbonate, etc., as one embodiment of the inorganic basic compound; the noble metal includes, but is not limited to, palladium catalyst, ruthenium catalyst, rhodium catalyst, platinum catalyst, etc., such as tetrakis(triphenylphosphine)palladium, as one embodiment of the palladium catalyst; the organic solvent includes, but is not limited to, dimethylformamide, dimethyl sulfoxide, dimethyl ethylene glycol, etc., such as dimethylformamide, as one embodiment of the dimethylformamide.

[0022] This invention also provides a novel near-infrared aggregation-induced emission probe, with the structural formula shown in formula (II): .

[0023] The present invention also provides a method for preparing a novel near-infrared aggregation-induced emission probe, as shown in route 3, the steps of which include: under temperature control, adding triethylamine to an organic solvent containing compound I and 2,4-dinitrobenzenesulfonyl chloride, reacting at room temperature to obtain compound II.

[0024] Furthermore, the ice bath temperature is controlled to 0°C.

[0025] Furthermore, the concentration of compound I in the organic solvent is 10-60 μmol / mL, and in one embodiment, it is 30 μmol / mL; the molar ratio of compound I, 2,4-dinitrobenzenesulfonyl chloride, and triethylamine is 1:1.5-2:2.4-3, and in one embodiment, it is 1:1.5:2.4-2.5; the organic solvent includes, but is not limited to, dichloromethane, toluene, tetrahydrofuran, etc., such as dichloromethane in one embodiment.

[0026] Further, the organic solvent in the reaction solution was removed by rotary evaporation, and the crude product was purified by silica gel column chromatography with a mesh size of 200-300 to obtain compound II.

[0027] The novel near-infrared aggregation-induced emission dye (compound of formula (I)) provided by the present invention can be used to prepare fluorescent products, including but not limited to fluorescent materials, fluorescent devices, fluorescent apparatuses or fluorescent equipment; it can also be used to prepare fluorescent products for detection, including but not limited to fluorescent reagents, fluorescent devices, fluorescent apparatuses or fluorescent equipment.

[0028] This invention provides a product containing a novel near-infrared aggregation-induced emission dye (compound of formula (I)) provided above.

[0029] The novel near-infrared aggregation-induced emission probe (compound of formula (II)) provided by the present invention can be used to detect 2,6-dimethylthiophenol, or to prepare detection products for 2,6-dimethylthiophenol, including but not limited to reagents, materials, devices, apparatus or equipment.

[0030] This invention provides a product for the detection of 2,6-dimethylthiophenol, containing a novel near-infrared aggregation-induced emission probe (compound of formula (II)) provided above by this invention.

[0031] The present invention also provides a method for detecting 2,6-dimethylthiophenol, comprising the steps of: adding the sample solution to be tested into a reaction system containing a novel near-infrared aggregation-induced emission probe (formula (II)) provided by the present invention, and performing qualitative and / or quantitative determination of 2,6-dimethylthiophenol in the sample solution by standard addition method, external standard method, or internal standard method.

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects: The fluorescent dye (I) of this invention is a near-infrared naphthalimide-based AIE dye constructed using two donors (triphenylamine and thiophene), exhibiting a significantly improved quantum efficiency compared to existing naphthalimide-based near-infrared AIE dyes. Based on this, a fluorescent probe is designed to achieve specific detection of 2,6-dimethylbenzylthiophenol by exploiting the difference in reaction kinetics between benzenesulfonate esters and benzylthiophenol compounds with different nucleophilic properties, demonstrating good selectivity and anti-interference performance. Furthermore, a high-throughput detection scheme is proposed for the quantitative detection of 2,6-dimethylbenzylthiophenol, possessing practical application value in food samples. Attached Figure Description

[0033] Figure 1 This is the 1H NMR spectrum of the fluorescent dye (I) described in this invention.

[0034] Figure 2 This is the hydrogen NMR spectrum of the fluorescent probe (II) described in this invention.

[0035] Figure 3The absorption spectra of fluorescent dye (I) and probe (II) in pure water are shown. It can be seen that the absorption spectra of the two are not significantly different, with absorption peaks in the visible light region ranging from 400 to 600 nm, indicating effective absorption in this region, which can be used to excite the fluorescence emission of the compound. Meanwhile, the absorption peaks of the two are located at 450 nm and 470 nm, respectively, with little difference, indicating that both have extremely high absorption efficiency for excitation light in the 450–470 nm range. Therefore, the preferred excitation wavelength is determined to be 450–470 nm.

[0036] Figure 4 The fluorescence spectra of fluorescent dye (I) and probe (II) in pure water are shown, with an excitation wavelength of 450 nm. It can be seen that the fluorescence spectrum of dye (I) covers the region above 600–850 nm, with an emission peak at 700 nm, classifying it as a near-infrared aggregation-induced emission dye. Probe (II) exhibits a dark fluorescence state under the same testing conditions, with no obvious fluorescence signal in its spectrum, consistent with the characteristics of a photoinduced electron transfer fluorescent probe.

[0037] Figure 5 The probe (II) of this invention exhibits thermokinetic responses to 2,6-dimethylthiophenol at 20°C and 37°C. The excitation wavelength is 450 nm, the detection wavelength is 660 nm, and the detection time is 0–3000 s. It can be seen that the reaction rate and conversion rate increase with increasing temperature. At 20°C, there is a turning point at 180 s, after which the reaction rate decreases significantly, making it suitable for rapid detection of 2,6-dimethylthiophenol. Therefore, the preferred detection temperature is 20°C and the detection time is 3 minutes.

[0038] Figure 6 A flowchart illustrating a high-throughput detection scheme using the probe of this invention.

[0039] Figure 7 The probe (II) of this invention was used with the above-described high-throughput detection scheme. Fluorescence intensity-concentration relationships of different thiols were plotted at preferred detection temperatures and times. The excitation wavelength was 450 nm, the detection wavelength was 660 nm, the detection time was 3 minutes, and the detection temperature was 20°C. It can be seen that under the above detection conditions, among the three food flavoring thiophenols, 2,6-dimethylthiophenol exhibited significantly better fluorescence intensity signals than the other two thiophenols in the concentration range of 30-100 μM.

[0040] Figure 8 The graph shows the stability of the probe (compound II) of this invention under different pH conditions and its response to 2,6-dimethylthiophenol.

[0041] Figure 9The bar chart shows the selectivity and anti-interference performance of the probe (II) of the present invention for various anions and cations at the preferred detection temperature and time. The excitation wavelength was 450 nm, the detection wavelength was 660 nm, the detection time was 3 minutes, and the detection temperature was 20 °C. It can be seen that the probe does not have a fluorescent response to common anions and cations, but a significant fluorescence enhancement occurs after the addition of 2,6-dimethylthiophenol. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Example 1: Probe Synthesis Method Compound M0 (500 mg, 1.36 mmol), bis-pinacolborate (690 mg, 2.72 mmol), Pd(dppf)Cl2 (40 mg, 54.67 μmol), and potassium acetate (400 mg, 4.08 mmol) were dissolved in dioxane (14 mL) and stirred at 110 °C for 3 h under nitrogen protection. After cooling the reaction solution to room temperature, the reaction was quenched with water, and the crude product was obtained by filtration. The reaction mixture was purified by silica gel (ethyl acetate / dichloromethane = 1 / 10) column chromatography to give a pale yellow solid M1 (340 mg, 60% yield). 1 H NMR (400 MHz, CDCl3) δ ppm 1.48 (s, 12 H), 6.75 (t, J = 2.08 Hz, 1 H), 6.85 - 6.93 (m, 2 H), 7.40 (t, J = 8.07 Hz, 1 H), 7.83 (dd, J = 8.44, 7.34 Hz, 1H), 8.34 (d, J = 7.21 Hz, 1 H), 8.61 - 8.68 (m, 2 H), 9.19 (dd, J = 8.50, 1.04Hz, 1 H).

[0044] Weigh 100 mg (0.41 mmol) of 2,5-dibromothiophene, 206 mg (0.5 mmol) of M1, and 12 mg (10.33 μmol) of tetra(triphenylphosphine)palladium into a 20 mL two-necked flask. Dissolve the M1 in 8 mL of THF, and under nitrogen protection, inject 0.3 mL of potassium carbonate aqueous solution (2 M). Stir the mixture in an oil bath at 70 °C for 5 h. After the reaction is complete, cool the mixture. Remove tetrahydrofuran by rotary evaporation, and then extract the crude product three times with 20 mL of dichloromethane. The crude product is then subjected to silica gel column chromatography (200-300 mesh) (eluent: DCM:EA = 20:1) to obtain intermediate M2. 1 H NMR (400 MHz, CDCl3) δ ppm 6.78 -6.82 (m,2 H), 6.86 - 6.89 (m,1 H), 7.32 (t, J = 8.19 Hz, 1 H), 7.42 (d, J =391 Hz, 1H), 7.49 (d, J =3.91 Hz,1H), 7.94-7.99 (m, 2 H), 8.52 (d, J = 7.58Hz, 1H), 8.58 (d, J =7.21 Hz, 1 H), 8.68 (d, J =8.56 Hz,1 H), 9.68 (s,1 H).

[0045] Compound M2 (100 mg, 0.22 mmol), triphenylamine boric acid (84 mg, 0.29 mmol), and Pd(PPh3)4 (10 mg, 8.65 μmol) were placed in a 25 mL two-necked flask, dissolved in 10 mL of DMF, and the mixture was placed under nitrogen protection. 0.3 mL of potassium carbonate aqueous solution (2 M) was then added, and the mixture was stirred in an oil bath at 130 °C for 5 h. After the reaction was complete, the mixture was cooled to room temperature, quenched with water, and filtered to obtain the crude product. The crude product was then purified by column chromatography using PE:EA = 20:1 (V / V) as the eluent to give a red solid (I) (36 mg, 26% yield). 1 H NMR (400 MHz, CDCl3) δ ppm 6.80 (t, J = 2.08 Hz, 1 H), 6.89 (dd, J =7.76, 0.92 Hz, 1 H), 6.95 (dd, J = 8.19, 1.83 Hz, 1 H), 7.07 - 7.11 (m, 2 H), 7.13 (d,J = 8.68 Hz, 2 H), 7.15 - 7.19 (m, 4 H), 7.29 - 7.34 (m, 4 H), 7.34 -7.39 (m, 2 H), 7.42 (t, J = 8.07 Hz, 1 H), 7.54 - 7.59 (m, 2 H), 7.82 (dd, J =8.50, 7.40 Hz, 1 H), 7.90 (d, J = 7.70 Hz, 1 H), 8.66 (d, J = 7.58 Hz, 1 H), 8.71(dd, J = 1.00 Hz, 1 H), 8.81 (dd, J = 8.56, 0.86 Hz, 1 H).

[0046] Another route: Weigh 500 mg (2.07 mmol) of 2,5-dibromothiophene, 896 mg (3.10 mmol) of triphenylamine boric acid, and 60 mg (51.92 μmol) of tetra(triphenylphosphine)palladium into a 50 mL two-necked flask. Dissolve the mixture in 15 mL of toluene, and under nitrogen protection, inject 0.8 mL of potassium carbonate aqueous solution (2M). Stir the mixture in an oil bath at 100 °C for 10 h. After the reaction is complete, cool to room temperature and remove the toluene by rotary evaporation at 50 °C to obtain the crude product. Finally, purify the crude product by silica gel column chromatography (eluent: PE) using a 200-300 mesh silica gel screen to obtain a white solid (385 mg, yield 46%). 1 H NMR (400 MHz, CDCl3) δ ppm 6.97 (d, J = 3.91 Hz, 1 H), 7.02 (d, J = 3.91Hz, 1 H), 7.05 - 7.10 (m, 4 H), 7.12 - 7.16 (m, 4 H), 7.27 - 7.28 (m, 1 H), 7.29 - 7.32 (m, 3 H), 7.39 (d, J = 8.56 Hz, 2 H).

[0047] Next, compound M3 (1.2 g, 2.95 mmol), compound M1 from the original route (1.47 g, 3.54 mmol), and Pd(PPh3)4 (85 mg, 73.56 μmol) were weighed into a 100 mL two-necked flask, dissolved in 40 mL of DMF, and under nitrogen protection, 1 mL of potassium carbonate aqueous solution (2 M) was injected. The mixture was stirred in an oil bath at 130 °C for 8 h. After the reaction was completed, the mixture was cooled to ambient temperature, the reaction was quenched with water, and the crude product was obtained by filtration. The crude product was then purified by column chromatography using PE:EA = 20:1 (V / V) as the eluent to give a red solid (I) (1.4 g, 77% yield). 1 H NMR (400 MHz, DMSO-d6) δ ppm 6.74 - 6.82(m, 2 H),6.87 (d, J =7.46 Hz, 1 H), 7.01 (d, J =8.56 Hz, 2 H) , 7.05 - 7.14 (m,6 H) , 7.30 - 7.38 (m, 17 H) , 7.56 (d, J =3.55 Hz, 1 H), 7.62 (d, J =3.79 Hz, 1H), 7.68 (d, J =8.56 Hz, 2 H) , 8.47 - 8.54 (m, 1 H) , 8.56 (d, J =7.21 Hz, 1 H), 8.80 (d, J =8.56 Hz, 1 H), 9.77 (s, 1 H).

[0048] Finally, compound (I) (100 mg, 150 μmol) and 2,4-dinitrobenzenesulfonyl chloride (60 mg, 225 μmol) were added to the reaction tube in a molar ratio of 1:1.5. After dissolving in 5 mL of dichloromethane, the mixture was cooled to 0 °C, and 50 μL of triethylamine was added dropwise under ice bath conditions. After returning to room temperature, the mixture was stirred for 2 hours, and then the dichloromethane was removed by rotary evaporation to obtain the crude product. The target product was purified by silica gel column chromatography (200-300 mesh) (eluent PE:DCM = 1:4 (V / V)) to obtain the dark red target product (II). 1 H NMR (400 MHz, DMSO-d6) δ ppm 7.01 (d, J=8.68 Hz, 2 H), 7.05 -7.15 (m, 6 H), 7.29 (d, J =9.17 Hz, 1 H), 7.32 - 7.39 (m, 4 H), 7.39 - 7.45 (m, 3H), 7.54 - 7.64 (m, 2 H), 7.65 - 7.74 (m, 3 H), 7.93 - 7.98 (m, 1 H), 8.00 (d, J =7.70 Hz, 1 H), 8.48 - 8.61 (m, 3 H), 8.82 (d, J =8.80 Hz, 1 H), 8.90 (d, J =2.81Hz, 1 H). Example 2: A further photophysical performance testing scheme for fluorescent dye (compound I) and fluorescent molecular probe (compound II) is provided, the steps of which are as follows: 1) Add the prepared fluorescent probe (compound I) to a 5 mL volumetric flask, then add DMSO to dissolve and dilute to volume to prepare a 1 mM stock solution. Add the prepared fluorescent probe (compound II) to a 5 mL volumetric flask, then add DMSO to dissolve and dilute to volume to prepare a 1 mM stock solution.

[0049] 2) Add 1980 μL of deionized water to a 3.5 mL four-sided transparent quartz cuvette using a pipette. Place the cuvette in a UV-Vis analyzer, set the test range to 280 nm to 600 nm, and click "baseline" to test the baseline of the pure water solvent.

[0050] 3) Using a pipette, add 20 μL of a 1 mM fluorescent dye stock solution to the cuvette from step 2), mix thoroughly, and obtain a 10 μM fluorescent dye test solution. Place the cuvette in a UV-Vis analyzer and scan the range from 280 nm to 600 nm to obtain the UV-Vis absorption spectrum of the fluorescent dye (compound I). Then place the cuvette in a fluorescence spectrometer, set the excitation wavelength to 470 nm, and scan the range from 500 nm to 850 nm to obtain the fluorescence spectrum of the dye (compound I).

[0051] 4) Clean and dry the cuvette. Add 1980 μL of deionized water using a pipette, followed by 10 μL of 1 mM fluorescent molecular probe stock solution. Mix thoroughly to obtain a 10 μM probe test solution. Place the cuvette in a UV-Vis analyzer and scan the range from 280 nm to 600 nm to obtain the UV-Vis absorption spectrum of the fluorescent molecular probe (compound II). Place the test solution in the sample chamber of the fluorescence spectrometer, set the excitation wavelength to 470 nm, and scan the range from 500 nm to 850 nm to obtain the fluorescence spectrum of the probe (compound II).

[0052] 5) Process the spectral data obtained in steps 3) and 4), and merge them to obtain a comparative UV-Vis absorption spectrum. Figure 3 ) and fluorescence spectrum ( Figure 4 ).

[0053] Example 3: A high-throughput performance testing scheme for the detection of 2-methoxythiophenol, 2-ethylthiophenol, and 2,6-dimethylthiophenol using a fluorescent molecular probe (compound II) is further provided. The procedure is as follows: Figure 6 As shown, the steps are as follows: 1) Preparation of probe stock solution (1 mM): Accurately weigh 4.2 mg (5 μmol) of fluorescent probe (compound II), place it in a 5 mL volumetric flask, dissolve it with DMSO and dilute to the mark, shake well to obtain a 1 mM fluorescent probe (compound II) stock solution. Store the solution at 4 °C protected from light.

[0054] 2) Preparation of standard stock solutions (50 mM): Using a microsyringe, accurately measure 30.5 μL (0.25 mmol, density 1.15 g / mL) of 2-methoxythiophenol, 33 μL (0.25 mmol, density 1.05 g / mL) of 2-ethylthiophenol, and 32.5 μL (0.25 mmol, density 1.06 g / mL) of 2,6-dimethylthiophenol, and place them in separate 5 mL volumetric flasks. Dissolve them in DMSO and dilute to the mark. Shake well to obtain 50 mM stock solutions of 2-methoxythiophenol, 2-ethylthiophenol, and 2,6-dimethylthiophenol.

[0055] 3) Preparation of probe working solution A (50 μM): Use a pipette to measure 2.5 mL of the 1 mM probe stock solution from step 1), dilute it with HEPES buffer (10 mM, pH 7.4) and bring the volume to 50 mL to obtain a probe working solution with a probe concentration of 50 μM.

[0056] 4) Preparation of standard working solution B (200 μM): Use a pipette to measure 200 μL of the 50 mM standard stock solution from step 2) above, dilute with HEPES buffer (10 mM, pH 7.4) and bring the volume to 50 mL to obtain a working solution of 2-methoxythiophenol, 2-ethylthiophenol, and 2,6-dimethylthiophenol with a detection concentration of 200 μM.

[0057] 5) Using an 8-channel adjustable pipette, add the corresponding volumes of the three liquids to each column according to Table 1 below, ensuring that the liquids in each column are added simultaneously.

[0058] 6) After all the liquids in step 5) have been added, gently shake the 96-well plate to ensure complete mixing. Then wait 1 hour to ensure complete reaction in each well.

[0059] 7) Place the 96-well plate into the sample chamber of the ELISA reader. Set the ELISA reader parameters: excitation wavelength: 470 nm; scan emission wavelength: 660 nm.

[0060] 8) Record the fluorescence intensity values ​​of 8 parallel wells at each concentration point (a total of 12 concentrations).

[0061] 9) Calculate the average value and standard deviation of fluorescence intensity at each concentration point.

[0062] 10) Plot a standard working curve with the final concentrations (μM) of 2-methoxythiophenol, 2-ethylthiophenol, and 2,6-dimethylthiophenol on the x-axis and the corresponding average fluorescence intensity on the y-axis. Collect the fluorescence spectrum of the sample and plot the fluorescence intensity-concentration curve at 660 nm. Figure 7 ).

[0063] Table 1. Reagent Dosage for High-Throughput Testing Channels Example 4: A kinetic assay protocol for the detection of 2,6-dimethylthiophenol using probe molecule (compound II) is further provided. The steps are as follows: 1) Add the prepared fluorescent probe (compound II, 4.2 mg) to a 5 mL volumetric flask, then dissolve and dilute to volume with DMSO to prepare a 1 mM stock solution. Accurately measure 32.5 μL (0.25 mmol, density 1.06 g / mL) of 2,6-dimethylthiophenol using a microsyringe, place it in a 5 mL volumetric flask, dissolve and dilute to the mark with DMSO, and shake well to obtain a 50 mM 2,6-dimethylthiophenol stock solution. 2) Add 1990 μL of HEPES buffer solution to a 3.5 mL four-sided transparent quartz cuvette using a pipette, then add 10 μL of probe stock solution using a pipette, mix well, and obtain a probe test solution with a concentration of 5 μM. 3) Place the micro stirring magnet into the quartz cuvette containing the test solution prepared in step 2), place it in the sample chamber of the fluorescence spectrometer, start stirring, and set the stirring speed to 3; turn on the sample cell temperature control and control the reaction temperature to 20℃; 4) Open the Kenetic test mode, set the excitation wavelength to 470 nm, and the scanning emission wavelength to 660 nm; the scanning time is 3600 s, and the emission intensity is scanned once every 10 s; 5) After 30 seconds after the scan begins, use a microsyringe to add 4 μL of 50 mM 2,6-dimethylthiophenol stock solution to the test liquid in the quartz cuvette through the well plate. At this time, the concentration of 2,6-dimethylthiophenol in the cuvette is 100 μM. 6) After completing the above reaction, the kinetic curve of the probe reaction with 2,6-dimethylthiophenol was obtained with the fluorescence intensity at 660 nm as the ordinate, the time of addition of 2,6-dimethylthiophenol stock solution as the abscissa, and the reaction temperature as 20℃. 7) Adjust the reaction temperature of the sample cell to 37℃ and repeat the above steps to obtain the reaction kinetic curve of the probe on 2,6-dimethylthiophenol at 37℃. 8) Combine the reaction kinetic curves obtained at the two temperatures to obtain... Figure 5 .

[0064] Example 5: Further, a testing protocol is provided to assess the stability of the probe molecule (compound II) under different pH conditions and its response to 2,6-dimethylthiophenol. The steps are as follows: 1) Add 1990 μL of solution with pH values ​​ranging from 2 to 12 to a four-sided transparent quartz cuvette using a pipette. Then, add 10 μL of 1 mM probe stock solution using a pipette and mix thoroughly to obtain a 5 μM probe test solution. After standing for 30 minutes, place the solution in the sample cell of a fluorescence spectrometer, set the excitation wavelength to 470 nm, and scan the fluorescence emission intensity from 500 to 850 nm to obtain the fluorescence spectra of the probe under 11 pH conditions. 2) Take the fluorescence intensity at 660 nm in the fluorescence spectrum data obtained in step 1) as the ordinate and pH as the abscissa to plot a fluorescence intensity line graph to evaluate the stability of the probe under different pH conditions. 3) Further, add 4 μL of 50 mM 2,6-dimethylthiophenol stock solution to the solutions at each pH condition in step 1) using a microsyringe. The total amount of 2,6-dimethylthiophenol added to the quartz cuvette is then 100 μM. After shaking and letting stand for 30 minutes, place the cuvette in the sample cell of the fluorescence spectrometer, set the excitation wavelength to 470 nm, and scan the fluorescence emission intensity from 500 to 850 nm to obtain the fluorescence spectra of the probe's response to 2,6-dimethylthiophenol under 11 pH conditions. 4) Take the fluorescence intensity at 660 nm in step 3) as the ordinate and pH as the abscissa to plot a fluorescence intensity line graph to evaluate the stability of the probe's response to 2,6-dimethylthiophenol under different pH conditions. 5) Combine the two line graphs drawn in steps 1) and 2) to obtain the stability of the probe molecule under different pH conditions and its response stability to 2,6-dimethylthiophenol, as shown below. Figure 8 As shown.

[0065] Example 6: A further testing scheme for the selectivity and anti-interference ability of the fluorescent molecular probe (compound II) for the detection of 2,6-dimethylthiophenol is provided. The steps are as follows: 1) To test the selectivity and anti-interference performance of the probe (compound II) for different ions, the selected targets included: Na + K + Cu + Zn 2+ Mg 2+ Fe 3+ Al 3+ Ca 2+ F - Cl - ,Br - I - SO4 2- SO3 2- CO3 2- ,ClO - NO2 - ; 2) Weigh out the corresponding water-soluble substances for each ion in the appropriate stoichiometric ratio (sodium sulfite 12.61 mg, sodium carbonate 5.3 mg, sodium sulfate 71 mg, sodium nitrite 3.45 mg, potassium chloride 3.73 mg, potassium bromide 5.95 mg, calcium chloride 5.55 mg, sodium fluoride 2.1 mg, sodium iodide 7.49 mg) and place them in a 5 mL volumetric flask. Add deionized water to dissolve and dilute to the mark on the volumetric flask, so that the concentration of each ion is 10 mM. Shake well and place in the refrigerator (4°C). o C) Store in a dark place; 3) Add 1990 μL of deionized water to a four-sided transparent quartz cuvette using a pipette, then add 10 μL of probe stock solution using a pipette, mix thoroughly to obtain a 5 μM probe test solution. Add 20 μL of 10 mM sodium sulfite stock solution to the above test solution, then Na... + The ion concentration was 100 μM. After shaking, the mixture was placed in a fluorescence spectrometer with an excitation wavelength of 470 nm and a scanning range of 500 nm to 850 nm. The probe molecule (compound II) was then used to detect the reaction of Na+ with Na+. + Selective response spectrum; 4) Add the 100 μM Na to the solution in step 3). + In the probe test solution, 4 μL of 2,6-dimethylbenzylthiophenol stock solution was added using a microsyringe, resulting in a 2,6-dimethylbenzylthiophenol concentration of 100 μM in the test solution. After shaking and incubation for 30 minutes, the solution was tested. The excitation wavelength was set to 470 nm, and the scanning range was 500 nm to 850 nm. The probe molecule (compound II) was then analyzed in the presence of Na. + Effect of interference on the response of 2,6-dimethylthiophenol; 5) For all Na ions + K + Cu + Zn 2+ Mg 2+ Fe 3+ Al 3+ Ca 2+ F - Cl - ,Br - I - SO4 2- SO3 2- CO3 2- ,ClO - NO2 - Repeat steps 3) and 4) to obtain the fluorescence spectrum of the probe molecule after the addition of ions at a concentration of 100 μM, and the response fluorescence spectrum of 2,6-dimethylthiophenol under the interference of ions at a concentration of 100 μM. 6) Process the above two sets of data, and plot a bar chart of the selectivity and anti-interference ability of the probe for the detection of 2,6-dimethylthiophenol with fluorescence intensity as the ordinate and corresponding to various ions (e.g., Figure 9 ).

[0066] Example 7: A further detection scheme for the content of 2,6-dimethylthiophenol in actual food samples using near-infrared fluorescent molecular probes is provided. The steps are as follows: 1) Add the prepared fluorescent probe (compound II, 4.2 mg) to a 5 mL volumetric flask, then add DMSO to dissolve and dilute to volume to prepare a 1 mM stock solution; 2) Add 3.3 μL of 2,6-dimethylthiophenol (25 μmol) (density 1.06 g / mL) to a 5 mL volumetric flask using a microsyringe, then add DMSO to dissolve and dilute to the mark to prepare a 5 mM stock solution; 3) Add 1990 μL of deionized water to a four-sided transparent quartz cuvette using a pipette, then add 10 μL of 1 mM probe stock solution using a pipette, mix well, and obtain a 5 μM probe test solution. 4) After shaking well, test on the instrument. Set the excitation wavelength to 470 nm and the scanning range to 500 nm~850 nm to obtain the fluorescence intensity spectrum of the probe molecule with a concentration of 5 μM. 5) Add 1 μL of 5 mM 2,6-dimethylthiophenol stock solution to a quartz cuvette using a microsyringe to obtain a test solution with a 2.5 μM 2,6-dimethylthiophenol concentration. Shake well and let stand for 30 minutes, then test using the same parameters to obtain the fluorescence intensity of the probe molecule in deionized water in response to a 2.5 μM 2,6-dimethylthiophenol concentration. 6) Repeat the above steps four times, changing the amount of 2,6-dimethylthiophenol stock solution added in step 5), adding 2 μL, 4 μL, 8 μL, and 16 μL respectively, to obtain the fluorescence intensity spectra of the probe molecule in deionized water in response to 2,6-dimethylthiophenol added at concentrations of 5 μM, 10 μM, 20 μM, and 40 μM respectively. 7) Process the fluorescence intensity spectra of the probe molecule in deionized water for 0, 2.5 μM, 5 μM, 10 μM, 20 μM and 40 μM concentrations of 2,6-dimethylthiophenol. Take the fluorescence intensity at 660 nm as the ordinate and the corresponding concentration of 2,6-dimethylthiophenol as the abscissa to plot the standard curve of the probe response to the concentration of 2,6-dimethylthiophenol. 8) Food Sample Pretreatment: Weigh 2.000 g each of air-dried meat jerky, biscuits, and potato chips, freeze-dry them under vacuum at low temperature for 4 hours, carefully grind the solid samples and sieve them (300 mesh), weigh 1.000 g of the powder, add 2 mL of DMSO solvent to soak the sample, and sonicate for 10 minutes. Centrifuge the soaked samples (8000 rpm, 5 minutes), remove the centrifuge tube, transfer the supernatant to a 5 mL volumetric flask, and dilute to 5 mL with DMSO to prepare the test solutions for the three samples of meat jerky, biscuits, and potato chips; 9) Add 1990 μL of deionized water to a four-sided transparent quartz cuvette using a pipette, then add 10 μL of 1 mM probe stock solution using a pipette, mix well to obtain a 5 μM probe test solution. Add the above actual sample test solution to the test solution containing the probe, set the excitation wavelength to 470 nm and the scanning range to 500 nm~850 nm, and immediately perform fluorescence spectroscopy. Wait half an hour to ensure complete reaction, and then perform a fluorescence spectroscopy test again using the same parameters to obtain the fluorescence spectra before and after the probe reaction. 10) Take the fluorescence spectrum obtained in step 9) and calculate the difference in fluorescence emission at 660 nm wavelength before and after the probe is added to the food sample test solution, that is, the fluorescence change before and after the reaction to remove background fluorescence; 11) Substitute the fluorescence change after removing background fluorescence into the standard curve calculated in step (1), and the content of 2,6-dimethylthiophenol in the actual sample can be obtained by conversion, as shown in Table 2. It can be seen that the probe detection scheme is suitable for the detection of 2,6-dimethylthiophenol in the concentration range of food additives.

[0067] Table 2 shows the thiophenol content in the samples calculated from actual sample testing.

Claims

1. A novel near-infrared aggregation-induced emission dye, characterized in that, The structural formula is shown in equation (I): 。 2. The method for preparing a novel near-infrared aggregation-induced emission dye as described in claim 1, characterized in that, As shown in Route 1 or Route 2, the steps include: (1) Under nitrogen protection and catalysis by a noble metal catalyst, compound M0 and dipinazoboronic acid ester were heated in an organic solvent containing a basic compound to prepare compound M1. (2) Under nitrogen protection and catalysis by a noble metal catalyst, compound M1 and 2,5-dibromothiophene were heated in an organic solvent containing a basic compound to prepare compound M2. (3) Under nitrogen protection and catalysis by a noble metal catalyst, compound M2 reacts with triphenylamine boric acid in an organic solvent containing a basic compound to produce compound I; or; (I) Under nitrogen protection and catalysis by a noble metal catalyst, triphenylamine boric acid and 2,5-dibromothiophene were reacted by heating in an organic solvent containing a basic compound to prepare compound M3; (II) Under nitrogen protection and catalysis by a noble metal catalyst, compound M3 and compound M1 react in an organic solvent containing a basic compound to produce compound I.

3. A novel near-infrared aggregation-induced emission probe, characterized in that, The structural formula is shown in equation (II): 。 4. The method for preparing a novel near-infrared aggregation-induced emission probe as described in claim 3, characterized in that, As shown in Route 3, the steps include: Under controlled temperature conditions, triethylamine was added to an organic solvent containing compound I and 2,4-dinitrobenzenesulfonyl chloride, and the reaction was carried out at room temperature to prepare compound II.

5. A novel near-infrared aggregation-induced emission dye prepared by the method of claim 1 or claim 2 is used to prepare fluorescent products or fluorescent products for detection.

6. The application according to claim 5, characterized in that, The fluorescent products include reagents, materials, devices, apparatuses, or equipment.

7. A novel near-infrared aggregation-induced emission probe according to claim 3 or a novel near-infrared aggregation-induced emission probe prepared by the preparation method according to claim 4, used for the detection of 2,6-dimethylthiophenol, or used to prepare a detection product for 2,6-dimethylthiophenol.

8. The application according to claim 7, characterized in that, The products used for testing include reagents, materials, devices, apparatus, or equipment.

9. A product characterized in that, The invention comprises a novel near-infrared aggregation-induced emission dye as described in claim 1 or prepared by the preparation method of claim 2, or a novel near-infrared aggregation-induced emission probe as described in claim 3 or prepared by the preparation method of claim 4.

10. A method for detecting 2,6-dimethylthiophenol, characterized in that, The steps include: The sample solution to be tested is added to a reaction system containing a novel near-infrared aggregation-induced emission probe as described in claim 3 or a novel near-infrared aggregation-induced emission probe prepared by the preparation method described in claim 4. The qualitative and / or quantitative determination of 2,6-dimethylthiophenol in the sample solution to be tested is performed by the standard addition method, the external standard method, or the internal standard method.

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