Preparation and application of a near-infrared ratiometric bisulfite fluorescent probe

Abstract

The application discloses a preparation and application of a near-infrared ratio type bisulfite fluorescent probe, and relates to the technical field of bisulfite fluorescent probes. The probe comprises a near-infrared compound, and a structural formula is as follows: the preparation method and the application of the compound are also provided. The bisulfite ratio fluorescent probe can be reacted with bisulfite to generate a change in a fluorescence spectrum, so that quantitative detection of the bisulfite can be realized. The probe is very sensitive to the reaction with the bisulfite, so that the detection of the bisulfite is facilitated. The probe has good stability, and can be used for a long time. The bisulfite fluorescent probe is a high-selectivity and rapid-response bisulfite fluorescent probe, and is simple to synthesize.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bisulfite fluorescent probe, and particularly to preparation and application of a near-infrared ratio type bisulfite fluorescent probe. BACKGROUND

[0002] Bisulfite has reducing and antibacterial properties, and is often used for cleaning reverse osmosis membranes, removing heavy metal ions and organic matter in water in the field of water treatment. In addition, as a preservative, bisulfite is widely added to food, beverages and pharmaceutical products. However, toxicological studies have shown that high concentrations of bisulfite can induce lung cancer, diarrhea, low blood pressure and many nervous system diseases. Therefore, it is necessary to develop a convenient, rapid, sensitive and highly selective method for detecting and monitoring the content of bisulfite in the environment and organisms.

[0003] So far, some methods for detecting bisulfite have been established, such as chromatography, spectrophotometry, chemiluminescence and electrochemistry. However, most of these methods require expensive equipment and complex procedures, and some of them are destructive and not suitable for biological systems. Fluorescent probes have become a powerful tool for detecting bisulfite in the environment and biological systems due to their non-invasive, high sensitivity, real-time spatial imaging and easy operation. So far, the construction of fluorescent probes for detecting bisulfite has been relatively limited, such as low sensitivity and poor selectivity.

[0004] Therefore, it is an urgent research topic to develop a highly selective and sensitive probe for rapid detection of bisulfite in water and biological systems. SUMMARY

[0005] To solve the above technical problems, the present application discloses a preparation and application of a near-infrared ratio type bisulfite fluorescent probe. The fluorescent probe of the present application has good selectivity, high sensitivity and can quickly respond to bisulfite.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] The present application proposes a near-infrared compound in the first aspect.

[0008] In an optional embodiment, the structure of the near-infrared compound is as follows:

[0009]

[0010] wherein R1, R2, R3, R4, R5 and R6 are independently selected from the group consisting of hydrogen atom, straight-chain or branched alkyl, straight-chain or branched alkoxy, sulfonic acid group, ester group and hydroxyl group; and R1, R2, R3, R4, R5 and R6 can be the same or different.

[0011] Optionally, a near-infrared compound has a structure as follows:

[0012]

[0013] In the structure of the near-infrared compound according to the first aspect of the present application, R1, R2, R3, R4, R5 and R6 are all free hydrogen atoms.

[0014] The second aspect of the present application provides a preparation method of the near-infrared compound.

[0015] In an optional embodiment, the preparation method of the near-infrared compound comprises the following steps:

[0016] (1) dissolving raw material I, cyclohexenone and triethylenediamine in ethanol, wherein the raw material I has a structure as follows: Then, nitrogen is filled, and heating reflux is performed to obtain compound 1,

[0017]

[0018] (2) reacting compound 1 and raw material I in methane sulfonic acid at 80-120℃, then pouring the reaction mixture into ice and stirring for 2-5 min, then adding perchloric acid drop by drop, stirring, filtering, adjusting the pH value of the filtrate to neutral, extracting with dichloromethane, and then performing rotary evaporation under reduced pressure to obtain a product:

[0019]

[0020] Optionally, in step (1), the molar ratio of 4-diethylaminosalicylaldehyde to cyclohexenone is 1:1-1:1.5.

[0021] In step (2), the molar ratio of compound 1 to 4-diethylaminosalicylaldehyde is 1:1-1:1.5.

[0022] Optionally, the reflux time in step (1) is 6-18 h.

[0023] In step (2), the reaction time of compound 1 and 4-diethylaminosalicylaldehyde in methane sulfonic acid at 100℃ is 1-2 h.

[0024] In step (1), triethylenediamine is used as a catalyst, and the main factor affecting the reaction is the feeding ratio of 4-diethylaminosalicylaldehyde to cyclohexenone, so the amount of triethylenediamine is not limited. The purpose of filling nitrogen is to avoid oxidation of the aldehyde group.

[0025] In step (2), the reaction mixture is poured into ice to lower the temperature and dilute methane sulfonic acid, facilitating the subsequent reaction. The magnetic stirrer is added for mixing, and the stirring speed is not limited. The stirring time is 2-5 min. HClO4 is added dropwise to avoid violent reaction. The filtrate is adjusted to neutral pH. If the pH is too low, it will affect the subsequent extraction operation.

[0026] The third aspect of the present application provides a near-infrared ratio type bisulfite fluorescent probe, which comprises a near-infrared compound, and the structural formula is:

[0027]

[0028] The near-infrared wavelength range in the field of fluorescent probes is 650-900 nm, which is not exactly the same as the general expression. The wavelength in this range has strong tissue penetration and is convenient for in vivo imaging.

[0029] The fourth aspect of the present application provides the application of the above-mentioned near-infrared ratio type bisulfite fluorescent probe in detecting the concentration of bisulfite in a sample.

[0030] Optionally, the application comprises the step of contacting the near-infrared ratio type bisulfite fluorescent probe with the sample to be detected.

[0031] Optionally, the sample is water or blood.

[0032] The fifth aspect of the present application provides a detection preparation comprising the near-infrared ratio type bisulfite fluorescent probe provided in the third aspect of the present application.

[0033] The sixth aspect of the present application provides the application of the above-mentioned detection preparation in detecting the concentration of intracellular bisulfite in a sample.

[0034] Optionally, the sample is water or blood.

[0035] The seventh aspect of the present application provides a kit comprising the near-infrared ratio type bisulfite fluorescent probe provided in the third aspect of the present application.

[0036] Optionally, the kit further comprises a buffer for determining the concentration of bisulfite in a sample.

[0037] Optionally, the buffer is a phosphate buffer solution with pH 7.4.

[0038] The eighth aspect of the present application provides the application of the above-mentioned kit in detecting the concentration of intracellular bisulfite in a sample.

[0039] Optionally, the sample is water or blood.

[0040] The present application has the following beneficial effects,

[0041] 1. The application constructs a new near-infrared specific bisulfite ratio fluorescent probe capable of being used for in vivo imaging analysis based on benzopyrylium salt as a recognition receptor. The bisulfite ratio fluorescent probe of the application can react with bisulfite to produce changes in fluorescence spectrum, thereby realizing quantitative detection of bisulfite. Specifically, the bisulfite fluorescent probe of the application reacts with alanine, arginine, isoleucine, aspartic acid, glutamine, serine, glutamic acid, leucine, glycine, histidine, proline, methionine, threonine, cysteine, homocysteine, carbonate ion, potassium ion, zinc ion, magnesium ion, calcium ion, sodium ion, copper ion, ferric ion, ferrous ion, copper ion, potassium superoxide, tert-butyl hydroperoxide, singlet oxygen, hydroxyl radical, peroxynitrite, tert-butyl hydroperoxide radical, hypochlorite, sulfite, hypobromite, nitric oxide, sodium bromide, etc., thereby realizing selective recognition of bisulfite.

[0042] 2. The bisulfite fluorescent probe of the application is very sensitive to bisulfite reaction, thereby being conducive to detection of bisulfite.

[0043] 3. The bisulfite fluorescent probe of the application has good stability, and can be used for long-term storage.

[0044] 4. The bisulfite fluorescent probe of the application is a high-selectivity rapid-response bisulfite fluorescent probe, and is simple to synthesize, which is conducive to commercial popularization and application. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 is the response time of the probe (5 μM) after adding bisulfite (5 μM) according to an embodiment of the application;

[0046] Figure 2 is the fluorescence spectrum of the probe (5 μM) before and after adding bisulfite (0-10 μM) according to an embodiment of the application;

[0047] Figure 3 is the linear relationship of the probe (5 μM) with different concentrations of bisulfite (0-4 μM) according to an embodiment of the application;

[0048] Figure 4 is the influence of different analytes on the fluorescence intensity of the probe (5 μM) according to an embodiment of the application;

[0049] Figure 5 is the fluorescence intensity of the probe (5 μM) after responding with bisulfite (5 μM) in the presence of other analytes according to an embodiment of the application;

[0050] Figure 6is a fluorescence imaging analysis of the probe (10 μM) in HeLa cells for detecting bisulfite according to an embodiment of the present application;

[0051] Figure 7 is a fluorescence imaging analysis of the probe (10 μM) in HeLa cells for reversibly detecting bisulfite according to an embodiment of the present application. DETAILED DESCRIPTION

[0052] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts fall within the scope of the present application.

[0053] A near-infrared compound has a general structure as follows:

[0054]

[0055] wherein R1, R2, R3, R4, R5 and R6 are independently selected from the group consisting of a hydrogen atom, a linear or branched alkyl group, a linear or branched alkoxy group, a sulfonic acid group, an ester group and a hydroxyl group; and R1, R2, R3, R4, R5 and R6 can be the same or different.

[0056] A preparation method comprises the following steps:

[0057] (1) dissolving raw material I, cyclohexenone and triethylenediamine in ethanol, wherein the raw material I has a structure as follows: then filling nitrogen, heating and refluxing to obtain compound 1:

[0058]

[0059] (2) reacting compound 1 and raw material I in methane sulfonic acid at 100 ℃, then pouring the reaction mixture into ice and stirring for 2 min, then adding perchloric acid drop by drop, stirring, filtering, adjusting the pH value of the filtrate to neutral, extracting with dichloromethane and rotary evaporating under reduced pressure to obtain a product:

[0060]

[0061] Alternatively, a near-infrared compound has a structure as follows:

[0062]

[0063] that is, R1, R2, R3, R4, R5 and R6 in the structure of the above near-infrared compound are all free hydrogen atoms.

[0064] A preparation method comprises the following steps:

[0065] (1) 4-diethylaminosalicylaldehyde, cyclohexenone and triethylenediamine are dissolved in ethanol, nitrogen is filled, and the reaction is carried out by heating and refluxing to obtain compound 1;

[0066]

[0067] (2) Compound 1 and 4-diethylaminosalicylaldehyde are reacted in methane sulfonic acid at 100℃ to obtain a mixture; the mixture is poured into 300g of ice and stirred for 2min, then 5ml of perchloric acid is added dropwise, and stirred for 5min; after filtration, the filtrate is adjusted to neutral pH, extracted with dichloromethane, and then rotary evaporated under reduced pressure to obtain the product:

[0068]

[0069] It should be noted that adjusting the pH is a routine operation, which will not be described in detail.

[0070] A near-infrared ratio-type bisulfite fluorescent probe, comprising the above-mentioned near-infrared compound, has a structural formula as follows:

[0071]

[0072] A detection preparation comprising the above-mentioned near-infrared ratio-type bisulfite fluorescent probe is used for detecting the concentration of intracellular bisulfite in a sample.

[0073] Optionally, the sample is water or blood.

[0074] A kit comprising the above-mentioned near-infrared ratio-type bisulfite fluorescent probe is used for detecting the concentration of intracellular bisulfite in a sample.

[0075] Optionally, the kit further comprises a buffer for determining the concentration of bisulfite in the sample.

[0076] Optionally, the sample is water or blood.

[0077] Optionally, the detection preparation or kit of the present application further comprises a product instruction manual.

[0078] Example 1

[0079] A near-infrared compound has a structural formula as follows:

[0080]

[0081] The preparation method is as follows:

[0082] (1) 4-diethylaminosalicylaldehyde (3.84 g, 20 mmol), cyclohexenone (2.88 g, 30 mmol) and triethylenediamine (2.24 g, 20 mmol) were dissolved in 25 mL of anhydrous ethanol solution, nitrogen was filled, and heated to reflux under nitrogen for 18 h. After the reaction was completed, the crude product was obtained by rotary evaporation under reduced pressure; the crude product was separated by column chromatography, and a mixture of dichloromethane and petroleum ether (V / V = 15:1) was used as the eluent to obtain 1 108 pure compound 1084 mg, with a yield of 20%;

[0083] (2) Compound 1 (271 mg, 1 mmol) and 4-diethylaminosalicylaldehyde (193 mg, 1 mmol) were added to 5 mL of methane sulfonic acid and reacted at 100°C for 2 h; then, the reaction mixture was poured into 300 g of ice and stirred for 2 min, followed by dropwise addition of 5 mL of perchloric acid and stirring for 5 min; after filtration, the filtrate was adjusted to neutral pH and extracted with dichloromethane; dichloromethane was removed by rotary evaporation under reduced pressure to obtain the crude product; the crude product was separated by column chromatography, and a mixture of dichloromethane and methanol was used as the eluent (V / V = 40:1) to obtain 129 mg of pure product, with a yield of about 30%.

[0084] Based on the above pure product, a near-infrared ratio-type bisulfite fluorescent probe was prepared.

[0085] Figure 1 is the response time of the probe (5 μM) after adding bisulfite (5 μM). 25 μL was taken from the probe stock solution and placed in a 5 mL test system, then 5 μM of bisulfite was added to the test system, and the fluorescence intensity change was immediately tested by a fluorescence spectrometer after shaking uniformly. The above determination was carried out in pure water (10 mM PBS, pH 7.4) system, the probe used was the probe prepared in Example 1, and all the spectral tests were measured at 37°C.

[0086] is the fluorescence spectrum of the probe (5 μM) before and after adding bisulfite (0-10 μM). Figure 1 It can be clearly seen that after the addition of bisulfite, the fluorescence intensity at 765 nm reaches a minimum value instantaneously and remains unchanged, which indicates that the probe reacts rapidly with bisulfite and can provide a rapid analysis method for the determination of bisulfite.

[0087] Figure 2 is the fluorescence spectrum of the probe (5 μM) before and after adding bisulfite (0-10 μM).

[0088] A plurality of parallel samples with a probe concentration of 5 μM were prepared in 10 mL cuvettes, and then different concentrations of bisulfite were added to the test system, which was shaken uniformly and then left to stand. The above determination was performed in a pure water (10 mM PBS, pH 7.4) system, the probe used was the probe prepared in Example 1, and all spectral tests were measured at 37 °C. The fluorescence intensity change was tested by a fluorescence spectrometer, and the fluorescence intensity change of the probe (5 μM) after the addition of bisulfite (0-4 μM) was recorded. Figure 2 It can be clearly seen that the fluorescence intensity at 765 nm gradually weakens with the increase of the concentration of bisulfite.

[0089] Figure 3 is a linear relationship diagram of different concentrations of bisulfite (0-4 μM) to the probe (5 μM).

[0090] A plurality of parallel samples with a probe concentration of 5 μM were prepared in 10 mL cuvettes, and then different concentrations of bisulfite were added to the test system, which was shaken uniformly and then left to stand. The above determination was performed in a pure water (10 mM PBS, pH 7.4) system, the probe used was the probe prepared in Example 1, and all spectral tests were measured at 37 °C. The fluorescence intensity change was tested by a fluorescence spectrometer, and the fluorescence intensity change of the probe (5 μM) after the addition of bisulfite (0-4 μM) was recorded. Figure 3 It can be seen that the fluorescence intensity of the fluorescence probe (5 μM) after the addition of bisulfite (0-4 μM) presents a good linear relationship, which proves that the fluorescence probe can be used for quantitative analysis of bisulfite.

[0091] Figure 4 is the influence of different analytes on the fluorescence intensity of the probe (5 μM), and numbers 1-35 are respectively: blank, alanine, arginine, isoleucine, aspartic acid, glutamine, serine, glutamic acid, leucine, glycine, histidine, proline, methionine, threonine, cysteine, homocysteine (50 μM), carbonate ion, potassium ion, zinc ion, magnesium ion, calcium ion, sodium ion, copper ion, potassium superoxide, tert-butyl hydroperoxide, singlet oxygen, hydroxyl radical, peroxynitrite, tert-butyl hydroperoxide radical, hypochlorous acid, sulfite, hypobromous acid (5 μM), nitric oxide, sodium bromide, bisulfite (the concentration of other analytes is 100 μM except for special marking).

[0092] The above determination was performed in a pure water (10 mM PBS, pH 7.4) system, the probe used was the probe prepared in Example 1, and all spectral tests were measured at 37 °C. Specifically, a plurality of parallel samples with a probe concentration of 5 μM were prepared in 10 mL cuvettes, and then a certain amount of analyte was added, which was shaken uniformly, and then the determination was performed after the reaction was completed. The fluorescence intensity change was tested by a fluorescence spectrometer, and the fluorescence intensity change of the probe (5 μM) after the addition of bisulfite (0-4 μM) was recorded. Figure 4 It can be clearly seen that the probe has high selectivity for bisulfite.

[0093] Figure 5The fluorescence intensity of the probe (5 μM) in the presence of other analytes after response to bisulfite (5 μM), No. 1-34 are: blank, alanine, arginine, isoleucine, aspartic acid, glutamine, serine, glutamic acid, leucine, glycine, histidine, proline, methionine, threonine, cysteine, homocysteine (50 μM), carbonate ion, potassium ion, zinc ion, magnesium ion, calcium ion, sodium ion, copper ion, potassium superoxide, tert-butyl hydroperoxide, singlet oxygen, hydroxyl radical, peroxynitrite, tert-butyl hydroperoxide radical, hypochlorite, sulfite, hypobromite (5 μM), nitric oxide, sodium bromide (other analyte concentrations are 100 μM unless otherwise specified).

[0094] By Figure 5 It can be seen that the response of the probe to carbon monoxide is not interfered by other detection substances.

[0095] Figure 6 is the fluorescence imaging of the probe (10 μM) in cells.

[0096] Figure 6 is the fluorescence imaging analysis of the probe (10 μM) in HeLa cells for detecting bisulfite. First, image the HeLa cells without adding the probe (Figure A), then image the cells after incubating the probe (10 μM) for 20 min (Figure B); image another three groups of cells after incubating the probe (10 μM) for 20 min and then adding bisulfite (10 μM, 20 μM, 50 μM) for 20 min (Figures C-E). The fluorescence imaging effect of the probe in living cells for reversibly detecting bisulfite is analyzed by calculating the ratio of the fluorescence intensity of the red channel to the green channel. Figure 6 It can be seen that the probe has a good fluorescence imaging effect for detecting bisulfite in cells.

[0097] Figure 7 is the fluorescence imaging analysis of the probe (10 μM) in HeLa cells for reversibly detecting bisulfite. First, image the HeLa cells without adding the probe (Figure A), then image the cells after incubating the probe (10 μM) for 20 min (Figure B); image another group of cells after incubating the probe (10 μM) for 20 min, adding bisulfite (50 μM) for 20 min, and then adding H2O2 (20 μM) for 20 min (Figure D). The fluorescence imaging effect of the probe in living cells for reversibly detecting bisulfite is analyzed by calculating the ratio of the fluorescence intensity of the red channel to the green channel. Figure 7 It can be seen that the probe can reversibly detect bisulfite in cells.

[0098] Example 2

[0099] (1) 4-diethylaminosalicylaldehyde (3.84 g, 20 mmol), cyclohexenone (2.88 g, 30 mmol) and triethylenediamine (2.24 g, 20 mmol) were dissolved in 25 mL of anhydrous ethanol solution, nitrogen was filled, heated to reflux under nitrogen for 12 h, after the reaction was completed, the crude product was obtained by rotary evaporation under reduced pressure; the crude product was separated by column chromatography, a mixture of dichloromethane and petroleum ether (V / V = 15:1) was used as eluent, and 1542 mg of pure compound 1 was obtained, with a yield of 10%;

[0100] (2) Compound 1 (271 mg, 1 mmol) and 4-diethylaminosalicylaldehyde (289 mg, 1.5 mmol) were added to 5 mL of methane sulfonic acid and reacted at 100°C for 2 h; then, the reaction mixture was poured into 300 g of ice and stirred for 2 min, and then 5 mL of perchloric acid was added dropwise and stirred for 5 min; after filtration, the filtrate was adjusted to neutral pH and extracted with dichloromethane; dichloromethane was removed by rotary evaporation under reduced pressure to obtain the crude product; the crude product was separated by column chromatography, a mixture of dichloromethane and methanol was used as eluent (V / V = 40:1), and 171 mg of pure product was obtained, with a yield of about 40%.

[0101] Example 3

[0102] (1) 4-diethylaminosalicylaldehyde (3.84 g, 20 mmol), cyclohexenone (1.92 g, 20 mmol) and triethylenediamine (2.24 g, 20 mmol) were dissolved in 25 mL of anhydrous ethanol solution, nitrogen was filled, heated to reflux under nitrogen for 12 h, after the reaction was completed, the crude product was obtained by rotary evaporation under reduced pressure; the crude product was separated by column chromatography, a mixture of dichloromethane and petroleum ether (V / V = 15:1) was used as eluent, and 1542 mg of pure compound 1 was obtained, with a yield of 10%;

[0103] (2) Compound 1 (271 mg, 1 mmol) and 4-diethylaminosalicylaldehyde (289 mg, 1.5 mmol) were added to 5 mL of methane sulfonic acid and reacted at 100°C for 2 h; then, the reaction mixture was poured into 300 g of ice and stirred for 2 min, and then 5 mL of perchloric acid was added dropwise and stirred for 5 min; after filtration, the filtrate was adjusted to neutral pH and extracted with dichloromethane; dichloromethane was removed by rotary evaporation under reduced pressure to obtain the crude product; the crude product was separated by column chromatography, a mixture of dichloromethane and methanol was used as eluent (V / V = 40:1), and 171 mg of pure product was obtained, with a yield of about 40%.

[0104] Example 4

[0105] (1) 4-diethylaminosalicylaldehyde (3.84 g, 20 mmol), cyclohexenone (1.92 g, 20 mmol) and triethylenediamine (2.24 g, 20 mmol) were dissolved in 25 mL of anhydrous ethanol solution, nitrogen was filled, and heated to reflux under nitrogen for 18 h. After the reaction was completed, the crude product was obtained by rotary evaporation under reduced pressure; the crude product was separated by a chromatographic column, and a mixture of dichloromethane and petroleum ether (V / V = 15:1) was used as an eluent to obtain 1650 mg of pure compound 1, with a yield of 12%;

[0106] (2) Compound 1 (271 mg, 1 mmol) and 4-diethylaminosalicylaldehyde (193 mg, 1 mmol) were added to 5 mL of methane sulfonic acid and reacted at 100°C for 2 h; then, the reaction mixture was poured into 300 g of ice and stirred for 2 min, and then 5 mL of perchloric acid was added dropwise and stirred for 5 min; after filtration, the filtrate was adjusted to neutral pH and extracted with dichloromethane; dichloromethane was removed by rotary evaporation under reduced pressure to obtain the crude product; the crude product was separated by a chromatographic column, and a mixture of dichloromethane and methanol was used as an eluent (V / V = 40:1) to obtain 107 mg of pure product, with a yield of about 25%.

[0107] The bisulfite fluorescent probe of the present application has the remarkable feature of being able to highly selectively and ultra-sensitively recognize bisulfite and accurately quantitatively analyze bisulfite in the presence of other substances in the human body.

[0108] Of course, the above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present application should also be within the scope of protection of the present application.

Claims

1. A near-infrared compound, characterized in that, The general structural formula is: ， Among them, R1, R2, R3, R4, R5, and R6 are all hydrogen atoms.

2. A method for preparing a near-infrared compound as described in claim 1, characterized in that, Includes the following steps: (1) Dissolve raw material I, cyclohexenone, and triethylenediamine in anhydrous ethanol. The structural formula of raw material I is: Then, nitrogen gas is introduced, and the mixture is heated under reflux at 75-80 degrees Celsius to obtain compound 1. The structural formula of compound 1 is as follows: , ； (2) Compound 1 and raw material I were reacted in methanesulfonic acid at 100°C for 2 hours. Then, the reaction mixture was poured into ice and stirred for 2-5 minutes. Perchloric acid was then added dropwise, stirred, filtered, and the pH of the filtrate was adjusted to neutral. After extraction with dichloromethane, the filtrate was evaporated under reduced pressure to obtain the near-infrared compound of claim 1. 。 3. The method for preparing a near-infrared compound as described in claim 2, characterized in that, In step (1), the molar ratio of 4-diethylaminosalicylic acid aldehyde and cyclohexenone is 1:1 to 1:1.5; In step (2), the molar ratio of compound 1 and 4-diethylaminosalicylaldehyde is 1:1 to 1:1.

5.

4. The method for preparing a near-infrared compound as described in claim 2, characterized in that, The reflux time in step (1) is 6-18 h.

5. The application of a near-infrared compound as described in claim 1 in the preparation of a near-infrared ratiometric bisulfite fluorescent probe.

6. A reagent kit, characterized in that, Including the near-infrared compounds as described in claim 1.

7. A reagent kit as described in claim 6, characterized in that, It also includes a buffer for determining the concentration of bisulfite in the sample.

8. The reagent kit as described in claim 7, characterized in that, The buffer is a phosphate buffer solution.

Images