A fluorescent probe for detecting nitrite and a preparation method and application thereof

Abstract

The application discloses a nitrite fluorescent probe and a preparation method and application thereof. + The reaction makes the spiro ring open, the conjugated system recovers, the fluorescence emission signal is enhanced, a fluorescence-on detection mode is realized, a boronic acid group is introduced in the probe design, a benzene ring is activated, electrophilic substitution reactivity is improved, the coordination of an amine group and a metal ion is eliminated, the anti-interference ability of the probe to the coordination of metal ions is improved, and rapid, high-selectivity and high-sensitivity detection of nitrite in a complex food matrix is realized. The application also discloses application of the fluorescent probe. The prepared fluorescent probe has the technical effects of good water solubility, high sensitivity, high specificity and strong anti-interference ability.

Description

Technical Field

[0001] The invention belongs to the technical field of nitrite detection, and in particular relates to a fluorescent probe for rapid nitrite detection, and also relates to a preparation method and application of the probe. Background Art

[0002] Nitrite (NO 2- Nitrite is a water-soluble inorganic salt widely used in the food industry as an antioxidant, antibacterial agent, and colorant. Excessive intake of nitrite poses a threat to human health and increases the risk of cancer. In 2008, the World Health Organization established a maximum limit of nitrite of 65 μM. Nitrite detection in food is a crucial aspect of food safety. Developing a convenient, rapid, accurate, and reliable nitrite detection method is crucial for market monitoring of nitrite.

[0003] Fluorescent probes are convenient, sensitive, and rapid, making them ideal for rapid nitrite detection. Based on the diazotization reaction mechanism of nitrite, researchers have constructed various fluorescent nitrite probes. However, the currently reported probes have limitations in practical detection applications, including poor anti-interference capabilities, slow reaction speeds, sensitivity that falls short of the required detection reagents, and the water solubility of the probes, which requires the addition of organic solvents to aid the reaction. Summary of the Invention

[0004] The present invention provides a fluorescent probe for detecting nitrite, a preparation method thereof and an application thereof. Based on the spiro ring-opening reaction of rhodamine, under acidic conditions, the probe molecule reacts with NO + The reaction opens the spiro ring, restores the conjugated system, and enhances the fluorescence emission signal, realizing a fluorescence-on detection mode. At the same time, a boronic acid group is introduced into the probe design to activate the benzene ring, increase the activity of the electrophilic substitution reaction, and eliminate the coordination of the amine group with the metal ion, thereby improving the anti-interference ability of the probe against metal ion coordination, thereby realizing rapid, highly selective, and highly sensitive detection of nitrite in complex food matrices.

[0005] The object of the present invention is achieved in the following manner:

[0006] A fluorescent probe for detecting nitrite, which is a rhodamine B derivative with a spirohydrazide structure, and has the structural formula:

[0007] .

[0008] A method for preparing the above-mentioned fluorescent probe for detecting nitrite uses fluorescein as a precursor and undergoes a chemical reaction to prepare a fluorescent probe having a spirohydrazide structure. The specific preparation steps are as follows:

[0009] Step 1: Preparation of Compound Ⅰ

[0010] Fluorescein solid powder was added to anhydrous dichloromethane and cooled to 0°C in an ice-water bath. Anhydrous pyridine and trifluoromethanesulfonic anhydride solution were added in sequence. The mixture was stirred at 20-30°C for 3-5 hours, diluted with water and extracted. The obtained organic phase was washed with saturated brine, dried, concentrated under reduced pressure, and then gradient eluted with petroleum ether and ethyl acetate as eluents to obtain compound I.

[0011] Step 2: Preparation of Compound II

[0012] Add tris(dibenzylideneacetone)dipalladium, X-Phos, cesium carbonate and thiomorpholine-1,1-dioxide to the sample of Compound I, and finally dissolve it with 1,4-dioxane solution. Heat and reflux at 100-110°C under nitrogen protection for 8-18 hours. After the reaction is complete, add water and dichloromethane for extraction, wash with saturated brine, dry and concentrate under reduced pressure, add 95-105 ml of methanol and slurry for 1.2-2.0 hours, and filter to obtain Compound II.

[0013] Step 3: Preparation of Compound III

[0014] Slowly add a small amount of hydrazine hydrate solution and methanol solution to the compound II sample, heat the reaction system under reflux at 63-70°C for 7-9 hours, cool to 20-30°C, and filter to obtain compound III;

[0015] Step 4: Preparation of fluorescent probes

[0016] A sample of compound III and 2-formylphenylboronic acid were mixed, dichloromethane and anhydrous ethanol were added as solvents, and the mixture was heated under reflux at 78-85°C for 0.5-1.5 hours. After the reaction solution was cooled to 20-30°C, the dichloromethane was removed by rotary evaporation, and the pink solid was filtered and washed with anhydrous ethanol. After drying, a fluorescent probe for detecting nitrite was obtained.

[0017] In the above-mentioned preparation method of the fluorescent probe for detecting nitrite, in step 1, anhydrous pyridine and trifluoromethanesulfonic anhydride solution are added in sequence, stirred at 25° C. for 4 hours, diluted with water, and extracted 2 to 3 times.

[0018] In the preparation method of the fluorescent probe for detecting nitrite, in step 2, heating and reflux at 101° C. for 12 h under nitrogen protection, adding water and dichloromethane for extraction after the reaction is complete, washing with saturated brine, drying and concentrating under reduced pressure, and adding 100 mL of methanol for 1.5 h.

[0019] In the above-mentioned method for preparing the fluorescent probe for detecting nitrite, in step 3, the reaction system is heated under reflux at 65° C. for 8 h, then cooled to 25° C. and filtered.

[0020] The preparation method of the above-mentioned fluorescent probe for detecting nitrite, in the said step 4, heating reflux at 80 ° C for 1 hour, after the reaction solution is cooled to room temperature, rotary evaporation is removed therein of dichloromethane, and suction filtration is carried out. The pink solid is washed with anhydrous ethanol 2-3 times. After the reaction solution is cooled to 25 ° C, rotary evaporation is removed therein of dichloromethane.

[0021] The preparation method of the above-mentioned fluorescent probe for detecting nitrite, in step 1, the mass of fluorescein is 8-15g, the volume of anhydrous dichloromethane is 180-220mL, the volume of anhydrous pyridine solution is 18-23mL, and the volume of trifluoromethanesulfonic anhydride solution is 18-23mL; in step 2, the volume of 1,4-dioxane is 90-120 mL; in step 3, the mass of compound II is 4.5-5.5g, the volume of hydrazine hydrate solution is 0.8-1.2 mL, and the volume of methanol solution is 45-55mL; in step 4, the mass of compound II is 0.5-1.5g, 2-formylphenylboronic acid is 1.3-3.9g; the volume of dichloromethane is 8-12mL, and the volume of anhydrous ethanol is 18-22 mL.

[0022] The preparation method of the above-mentioned fluorescent probe for detecting nitrite, in step 1, the mass of fluorescein is 10 g, the volume of anhydrous dichloromethane is 200 mL, the volume of anhydrous pyridine solution is 20 mL, and the volume of trifluoromethanesulfonic anhydride solution is 20 mL; in step 2, 5.0 g of compound I, 0.8 g of tris(dibenzylideneacetone)dipalladium, 1.2 g of X-Phos, 7.6 g of cesium carbonate and 2.7 g of thiomorpholine-1,1-dioxide, and the volume of 1,4-dioxane are 100 mL; in step 3, the mass of compound II is 3.0 g, the volume of hydrazine hydrate solution is 0.9 mL, and the volume of methanol solution is 50 mL; in step 4, the mass of compound II is 1.0 g, 2-formylphenylboronic acid is 2.6 g, the volume of dichloromethane is 10 mL, and the volume of anhydrous ethanol is 20 mL.

[0023] The above-mentioned fluorescent probe for detecting nitrite is used to prepare a standard probe solution: the fluorescent probe is dissolved in DMSO and diluted with 2 mL of pH = 1.0 HCl solution to a probe solution with a concentration of 6 μM. The sample to be tested is then added and the fluorescence intensity is detected. The nitrite content in the sample is quantitatively calculated based on the linear relationship between the fluorescence intensity and the concentration of the added sample to be tested.

[0024] The above-mentioned fluorescent probe for detecting nitrite is used to make a standard colorimetric card for detecting nitrite: the fluorescent probe is dissolved in DMSO and then diluted with a pH = 1.5 HCl solution to a probe solution with a concentration of 16 μM. Filter paper strips are cut into equal-sized strips and immersed in the 16 μM probe solution. After being taken out and dried, nitrite solutions of different concentrations are added dropwise. The changes in fluorescence intensity are observed to make a standard colorimetric card.

[0025] Compared with the prior art, the present invention has the following technical effects:

[0026] (1) Good water solubility

[0027] The nitrite fluorescent probe of the present invention has good water solubility and can detect and determine the content of nitrite in a pure water system. It is suitable for detecting nitrite in water environment samples (such as groundwater, rivers or lakes, etc.).

[0028] (2) High sensitivity

[0029] The nitrite fluorescent probe of the present invention reacts very sensitively with nitrite and is suitable for detecting nitrite in water environments and foods with a concentration of 0-4.5 μM.

[0030] (3) High specificity and strong anti-interference ability

[0031] The nitrite fluorescent probe of the present invention can selectively react with nitrite specifically, compared with other substances commonly found in food and water environments, including but not limited to aluminum ions (Al 3+ ), magnesium ions (Mg 2+ ), calcium ions (Ca 2 + ), zinc ions (Zn 2+ ), potassium ion (K + ), iron ions (Fe 2+ and Fe 3+ ), fluoride ion (F - ), bromide ion (Br - ), iodide ion (I - ), acetate ion (CH3COO - ), phosphate ions (HPO4 2- ), sulfate ion (SO4 2- ), carbonate ions (CO3 2- ), nitrate ions (NO3 - ), citrate (C5H7O5COO - ), ascorbic acid (AA), cysteine ​​(L-Cys) and glutathione (GSH), etc. The fluorescent probe of the present invention shows high specificity and strong anti-interference ability.

[0032] (4) Dual-mode detection

[0033] The nitrite fluorescent probe of the present invention can also be detected by ultraviolet-visible spectroscopy, and its detection linear range is 0.06~4.0 μM.

[0034] (5) Good stability

[0035] The fluorescence intensity of the nitrite fluorescent probe of the present invention can be kept substantially unchanged for more than 28 minutes in a HCl buffer solution with a pH of 1.0, and the probe has excellent light stability and anti-photobleaching ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 The fluorescence spectrum changes of the probe (6 μM) before and after the addition of nitrite (5 μM).

[0038] Figure 2 The absorption spectrum changes of the probe (6 μM) before and after the addition of nitrite (5 μM).

[0039] Figure 3 The fluorescence signal changes of the probe (6 μM) within 30 min under continuous irradiation at a wavelength of 557 nm.

[0040] Figure 4 This is the fluorescence spectrum of the probe (6 μM) after adding nitrite (0-4.5 μM).

[0041] Figure 5 This is the working curve after the probe (6 μM) was added with nitrite (0~4.5 μM).

[0042] Figure 6 This is the absorption spectrum of the probe (6 μM) after adding nitrite (0~4.5 μM).

[0043] Figure 7 This is the working curve after the probe (6 μM) was added with nitrite (0~4.5 μM).

[0044] Figure 8 It is a test of the ability of the probe (6 μM) to selectively recognize nitrite.

[0045] Figure 9It is a test for nitrite using a probe loaded test strip. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0047] The following Examples 1-6 are examples of preparation methods for detecting nitrite fluorescent probes, and Examples 7-9 are application examples of the fluorescent probes of the present invention.

[0048] Example 1: Synthesis of probe

[0049] The synthetic design route is as follows:

[0050]

[0051] Step 1: Preparation of Compound Ⅰ

[0052] 10.0 g of fluorescein solid powder was weighed into a round-bottom flask, and 200 mL of anhydrous dichloromethane was added. The mixture was cooled to 0°C in an ice-water bath, and then 20 mL of anhydrous pyridine solution and 20 mL of trifluoromethanesulfonic anhydride solution were added in sequence. The mixture was stirred at 25°C for 4 h, diluted with water, and extracted 2 to 3 times. The obtained organic phase was washed with saturated brine, dried, concentrated under reduced pressure, and then gradient eluted with petroleum ether and ethyl acetate as eluents to obtain compound I.

[0053] Step 2: Preparation of Compound II

[0054] Weigh 5.0 g of Compound I, 0.8 g of tris(dibenzylideneacetone)dipalladium, 1.2 g of X-Phos, 7.6 g of cesium carbonate, and 2.7 g of 1,1-thiomorpholine dioxide into a round-bottom flask. Add 100 mL of 1,4-dioxane solution to dissolve the mixture. Heat under reflux at 101°C for 12 hours under nitrogen. After the reaction is complete, extract with water and dichloromethane. Wash with saturated brine, dry, and concentrate under reduced pressure. Add 100 mL of methanol and slurry for 1.5 hours. Filter to obtain Compound II.

[0055] Step 3: Preparation of Compound III

[0056] Weigh 3.0 g of compound II sample into a round-bottom flask, slowly add 0.9 mL of hydrazine hydrate solution and 50 mL of methanol solution, heat the reaction system under reflux at 65 °C for 8 h, cool to 25 °C, and filter to obtain compound III.

[0057] Step 4: Preparation of fluorescent probes

[0058] Weigh 1.0 g of Compound III and 2.6 g of 2-formylphenylboronic acid into a round-bottom flask. Add 10 mL of dichloromethane and 20 mL of anhydrous ethanol as solvents. Heat and reflux at 80°C for 1 hour. After cooling the reaction solution to 25°C, remove the dichloromethane by rotary evaporation. Filter the solution, and wash the pink solid two to three times with anhydrous ethanol. After drying, the fluorescent probe is obtained.

[0059] Example 2: Synthesis of probe

[0060] The synthetic design route is as follows:

[0061]

[0062] Step 1: Preparation of Compound Ⅰ

[0063] 8.0 g of fluorescein solid powder was weighed into a round-bottom flask, and 180 mL of anhydrous dichloromethane was added. The mixture was cooled to 0°C in an ice-water bath, and then 18 mL of anhydrous pyridine solution and 18 mL of trifluoromethanesulfonic anhydride solution were added in sequence. The mixture was stirred at 20°C for 3 h, diluted with water, and extracted 2 to 3 times. The obtained organic phase was washed with saturated brine, dried, concentrated under reduced pressure, and then gradient eluted with petroleum ether and ethyl acetate as eluents to obtain compound I.

[0064] Step 2: Preparation of Compound II

[0065] Weigh 4.5 g of Compound I, 0.5 g of tris(dibenzylideneacetone)dipalladium, 1.0 g of X-Phos, 7.3 g of cesium carbonate, and 2.4 g of 1,1-thiomorpholine dioxide into a round-bottom flask. Add 90 mL of 1,4-dioxane solution to dissolve the mixture. Heat under reflux at 90°C for 8 hours under nitrogen. After the reaction is complete, extract with water and dichloromethane. Wash with saturated brine, dry, and concentrate under reduced pressure. Add 95 mL of methanol and slurry for 1.2 hours. Filter to obtain Compound II.

[0066] Step 3: Preparation of Compound III

[0067] Weigh 2.5 g of compound II sample into a round-bottom flask, slowly add 0.8 mL of hydrazine hydrate solution and 45 mL of methanol solution, heat the reaction system under reflux at 63 °C for 7 h, cool to 20 °C, and filter to obtain compound III.

[0068] Step 4: Preparation of fluorescent probes

[0069] Weigh 0.5 g of Compound III and 1.3 g of 2-formylphenylboronic acid into a round-bottom flask. Add 8 mL of dichloromethane and 18 mL of anhydrous ethanol as solvents. Heat and reflux at 78°C for 0.5 h. After cooling the reaction solution to 20°C, remove the dichloromethane by rotary evaporation. Filter the solution, and wash the pink solid two to three times with anhydrous ethanol. After drying, the fluorescent probe is obtained.

[0070] Example 3: Synthesis of probe

[0071] The synthetic design route is as follows:

[0072]

[0073] Step 1: Preparation of Compound Ⅰ

[0074] 9.0 g of fluorescein solid powder was weighed into a round-bottom flask, and 190 mL of anhydrous dichloromethane was added. The mixture was cooled to 0°C in an ice-water bath, and then 19 mL of anhydrous pyridine solution and 19 mL of trifluoromethanesulfonic anhydride solution were added in sequence. The mixture was stirred at 22°C for 3.5 h, diluted with water, and extracted 2 to 3 times. The obtained organic phase was washed with saturated brine, dried, concentrated under reduced pressure, and then gradient eluted with petroleum ether and ethyl acetate as eluents to obtain compound I.

[0075] Step 2: Preparation of Compound II

[0076] Weigh 4.8 g of Compound I, 0.6 g of tris(dibenzylideneacetone)dipalladium, 1.2 g of X-Phos, 7.5 g of cesium carbonate, and 2.6 g of 1,1-thiomorpholine dioxide into a round-bottom flask. Add 99 mL of 1,4-dioxane solution to dissolve the mixture. Heat under reflux at 99°C for 11 hours under nitrogen. After the reaction is complete, extract with water and dichloromethane. Wash with saturated brine, dry, and concentrate under reduced pressure. Add 98 mL of methanol and slurry for 1.8 hours. Filter to obtain Compound II.

[0077] Step 3: Preparation of Compound III

[0078] Weigh 2.9 g of compound II sample into a round-bottom flask, slowly add 1.0 mL of hydrazine hydrate solution and 49 mL of methanol solution, heat the reaction system under reflux at 66 ° C for 7.8 h, cool to 24 ° C, and filter to obtain compound III.

[0079] Step 4: Preparation of fluorescent probes

[0080] Weigh 0.9 g of Compound III and 2.5 g of 2-formylphenylboronic acid into a round-bottom flask. Add 9.5 mL of dichloromethane and 21 mL of anhydrous ethanol as solvents. Heat and reflux at 79°C for 1.1 h. After cooling the reaction solution to 24°C, remove the dichloromethane by rotary evaporation. Filter the solution, and wash the pink solid two to three times with anhydrous ethanol. After drying, the fluorescent probe is obtained.

[0081] Example 4: Synthesis of probe

[0082] The synthetic design route is as follows:

[0083]

[0084] Step 1: Preparation of Compound Ⅰ

[0085] 15 g of fluorescein solid powder was weighed into a round-bottom flask, and 220 mL of anhydrous dichloromethane was added. The mixture was cooled to 0°C in an ice-water bath, and then 23 mL of anhydrous pyridine solution and 23 mL of trifluoromethanesulfonic anhydride solution were added in sequence. The mixture was stirred at 30°C for 5 h, diluted with water, and extracted 2 to 3 times. The obtained organic phase was washed with saturated brine, dried, concentrated under reduced pressure, and then gradient eluted with petroleum ether and ethyl acetate as eluents to obtain compound I.

[0086] Step 2: Preparation of Compound II

[0087] Weigh 5.1 g of Compound I, 1.0 g of tris(dibenzylideneacetone)dipalladium, 1.4 g of X-Phos, 7.8 g of cesium carbonate, and 3.0 g of 1,1-thiomorpholine dioxide into a round-bottom flask. Add 120 mL of 1,4-dioxane solution to dissolve the mixture. Heat under reflux at 110°C for 18 hours under nitrogen. After the reaction is complete, extract with water and dichloromethane. Wash with saturated brine, dry, and concentrate under reduced pressure. Add 101 mL of methanol and slurry for 2.0 hours. Filter to obtain Compound II.

[0088] Step 3: Preparation of Compound III

[0089] Weigh 3.5 g of compound II sample into a round-bottom flask, slowly add 1.2 mL of hydrazine hydrate solution and 55 mL of methanol solution, heat the reaction system under reflux at 70 °C for 9 h, cool to 30 °C, and filter to obtain compound III.

[0090] Step 4: Preparation of fluorescent probes

[0091] Weigh 1.5 g of Compound III and 3.9 g of 2-formylphenylboronic acid into a round-bottom flask. Add 12 mL of dichloromethane and 22 mL of anhydrous ethanol as solvents. Heat and reflux at 85°C for 1.5 hours. After cooling the reaction solution to 30°C, remove the dichloromethane by rotary evaporation. Filter the solution, and wash the pink solid two to three times with anhydrous ethanol. After drying, the fluorescent probe is obtained.

[0092] Example 5: Synthesis of probe

[0093] The synthetic design route is as follows:

[0094]

[0095] Step 1: Preparation of Compound Ⅰ

[0096] Compound I was prepared by the following steps: Step 1: Preparation of compound I

[0097] Step 2: Preparation of compound II

[0098] Compound II was prepared by the following steps: Step 1: Preparation of compound I

[0099] Step 3: Preparation of compound III

[0100] Compound III was prepared by the following steps: Step 1: Preparation of compound I

[0101] Step 4: Preparation of fluorescent probe

[0102] Compound III was prepared by the following steps: Step 1: Preparation of compound I

[0103] Example 6: Synthesis of probe

[0104] The synthesis design route is as follows:

[0105]

[0106] Step 1: Preparation of compound I

[0107] 14.5 g of fluorescein solid powder was weighed into a round-bottom flask, and 218 mL of anhydrous dichloromethane was added. The mixture was cooled to 0°C in an ice-water bath, and then 21 mL of anhydrous pyridine solution and 22 mL of trifluoromethanesulfonic anhydride solution were added in sequence. The mixture was stirred at 28°C for 4.8 h, diluted with water, and extracted 2 to 3 times. The obtained organic phase was washed with saturated brine, dried, concentrated under reduced pressure, and then gradient eluted with petroleum ether and ethyl acetate as eluents to obtain compound I.

[0108] Step 2: Preparation of Compound II

[0109] Weigh 5.5 g of Compound I, 0.9 g of tris(dibenzylideneacetone)dipalladium, 1.2 g of X-Phos, 7.6 g of cesium carbonate, and 2.8 g of thiomorpholine-1,1-dioxide into a round-bottom flask. Add 118 mL of 1,4-dioxane solution to dissolve the mixture. Heat under reflux at 105°C for 17 hours under nitrogen. After the reaction is complete, extract with water and dichloromethane. Wash with saturated brine, dry, and concentrate under reduced pressure. Add 105 mL of methanol and slurry for 1.7 hours. Filter to obtain Compound II.

[0110] Step 3: Preparation of Compound III

[0111] Weigh 3.4 g of compound II sample into a round-bottom flask, slowly add 1.0 mL of hydrazine hydrate solution and 54 mL of methanol solution, heat the reaction system under reflux at 66 ° C for 8.5 h, cool to 27 ° C, and filter to obtain compound III.

[0112] Step 4: Preparation of fluorescent probes

[0113] Weigh 1.4 g of Compound III and 3.3 g of 2-formylphenylboronic acid into a round-bottom flask. Add 10 mL of dichloromethane and 21 mL of anhydrous ethanol as solvents. Heat and reflux at 84°C for 1.4 hours. After cooling the reaction solution to 29°C, remove the dichloromethane by rotary evaporation. Filter the solution, and wash the pink solid two to three times with anhydrous ethanol. After drying, the fluorescent probe is obtained.

[0114] Example 7: Application of probe

[0115] a. Changes in fluorescence and absorption spectra of the test probe before and after nitrite recognition

[0116] Implementation plan: Add 6 μL of 2 mM probe stock solution to 2 mL of pH = 1.0 HCl solution, mix well, and record the UV-visible absorption and fluorescence spectrum signals respectively. Then add 5 μL of 2 mM sodium nitrite stock solution to the above mixture, mix well and react fully, and record the UV-visible absorption and fluorescence spectrum signals respectively. The results are as follows Figure 1 and Figure 2 shown.

[0117] Depend on Figure 1 It can be clearly seen that when nitrite is added, the fluorescence intensity at 557nm changes significantly at the same excitation wavelength (533nm). Figure 2 It can be seen that the absorption intensity value at 532nm increases significantly, and the color change before and after addition can be used for naked eye identification: the solution is colorless before the addition of nitrite, and the color becomes orange-red after the addition.

[0118] b. Test the stability of fluorescent probes

[0119] Implementation plan: Add 6 μL of 2 mM probe stock solution to 2 mL of pH = 1 HCl solution, mix well, and record the changes in the fluorescence signal of the probe solution at 557 nm within 30 minutes under continuous irradiation with 533 nm wavelength light. Figure 3 shown.

[0120] Depend on Figure 3 It can be seen that when nitrite is added, the fluorescent probe reacts rapidly with nitrite, the fluorescence intensity reaches a maximum after 118 seconds, and the fluorescence intensity remains basically unchanged within 30 minutes, indicating that the fluorescent probe of the present invention reacts relatively quickly with nitrite, and the probe molecule has relatively excellent photostability and resistance to photobleaching, which can provide a fast and accurate analytical method for the measurement or detection of nitrite.

[0121] c. Test the working concentration of fluorescent probe for nitrite

[0122] Implementation plan: Add 0~4.5μM NO2 to 2mL of pH=1.0 HCl solution containing 6μM probe - The solution was mixed thoroughly and reacted at room temperature for 9 minutes. The changes in the fluorescence signal and absorbance signal of each solution were recorded using a fluorescence spectrometer and a UV-visible spectrophotometer, and the changes in the color of the solution under sunlight and 365nm light were recorded. The results are as follows: Figure 4-Figure 7 shown.

[0123] Depend on Figure 4 It can be seen that with the increase of nitrite concentration, the fluorescence intensity at 557nm gradually increased; Figure 5 It can be seen that at 557 nm, the probe exhibits a good linear relationship with nitrite concentrations ranging from 0.03 to 2.0 μM and from 2.0 to 4.5 μM. Figure 6 It can be seen that with the increase of nitrite concentration, the absorption intensity at 532nm gradually increases; Figure 7As can be seen, at 532 nm, the probe exhibits a good linear relationship with nitrite concentrations between 0.06 and 4.0 μM. These data demonstrate that the probe of the present invention can respond sensitively to trace amounts of nitrite and can be used to determine nitrite using both fluorescence spectroscopy and UV-visible spectroscopy.

[0124] d. Testing the specificity of the fluorescent probe for nitrite

[0125] Implementation: Add 3.0 μM NO2 to 2 mL of pH 1.0 HCl solution containing 6 μM probe. - solution and 150 μM aluminum ions (Al 3+ ), magnesium ions (Mg 2+ ), calcium ions (Ca 2+ ), zinc ions (Zn 2+ ), potassium ion (K + ), iron ions (Fe 2+ and Fe 3+ ), fluoride ion (F - ), bromide ion (Br - ), iodide ion (I - ), acetate ion (CH3COO - ), phosphate ions (HPO4 2- ), sulfate ion (SO4 2- ), carbonate ions (CO3 2- ), nitrate ions (NO3 - ), citrate (C5H7O5COO - ), ascorbic acid (AA), cysteine ​​(L-Cys) and glutathione (GSH) solution, reacted at room temperature for 9 minutes, and recorded the changes in the fluorescence signal of the solution at 557nm. The results are as follows Figure 8 shown.

[0126] Depend on Figure 8 It can be seen that the probe of the present invention has a high selectivity for nitrite and can react specifically with nitrite. The fluorescence intensity changes significantly before and after the reaction, while the fluorescence intensity of other common analytes does not change significantly after the probe reacts with them.

[0127] Example 8: Testing the detection of nitrite in actual samples using fluorescent probes

[0128] Implementation 1: Take appropriate amounts of bottled water and lake water, filter them through a 0.22μm filter membrane, and set aside. Take 1.0mL of the bottled water sample and 1.1mL of the lake water sample, respectively, and add 6μL of the 2mM probe stock solution to a pH 1.0 system composed of 0.2mol / L HCl. Then, add 0.0μL, 0.4μL, 1.5μL, and 3.5μL of the 2mM sodium nitrite stock solution, respectively. After reacting at room temperature for 9 minutes, record the changes in the fluorescence signal at 557nm, substitute the test results into the plotted standard curve, and calculate the spike recovery. The results are shown in Table 1.

[0129] Implementation Plan 2: Weigh 5.0 g of ham sausage and pickled vegetable homogenate respectively into a 500 mL beaker, add 12.5 mL of saturated sodium borate solution first, then add 150 mL of 70°C hot water, boil in a boiling water bath for 15 minutes, cool to room temperature, add 5.0 mL of 106 g / L potassium ferrocyanide solution, stir evenly, then add 5.0 mL of 220 g / L zinc acetate solution and 27.5 mL of ultrapure water, stir evenly, let it stand for 30 minutes to precipitate protein, and then use a desktop high-speed centrifuge to centrifuge at 10,000 r / min for 5 minutes, and take the supernatant for subsequent testing. To a pH 1 system consisting of 0.4 mL of ham sausage extract and 0.4 mL of pickle extract, along with 0.2 mol / L HCl, 6 μL of the 2 mM probe stock solution was added. Then, 0.0 μL, 0.4 μL, 1.5 μL, and 3.5 μL of the 2 mM sodium nitrite stock solution were added. After reacting at room temperature for 9 minutes, the changes in the fluorescence signal at 557 nm were recorded and the results were substituted into the prepared standard curve to calculate the spike recovery. The results are shown in Table 1.

[0130] Table 1 shows that lake water, ham, and pickles contain a certain amount of nitrite. Calculated concentrations are 0.0385 mg / kg, 2.62 mg / kg, and 2.06 mg / kg, respectively, all within the permitted limits in my country. The actual nitrite levels detected in the samples were highly consistent with the theoretically expected nitrite levels, with recoveries ranging from 91.7% to 106.0% and RSDs less than 4.60% (N = 3). These results demonstrate that the fluorescent probe of the present invention can accurately determine nitrite content in complex food matrices and demonstrates its applicability.

[0131] Table 1. Results of nitrite content determination in environmental water, bottled water, ham sausage and pickled vegetable samples by probe

[0132] .

[0133] Example 9: Preparation of probe-loaded test strips

[0134] Implementation plan: Cut filter paper strips of 0.7×0.8 cm in size and soak them in 1 mL of HCl solution with a pH of 1.5 and a probe concentration of 16 μM. After soaking for 15 minutes, take them out and let them air dry to obtain portable nitrite detection test strips. 15 μL of NO2 at concentrations of 0 μM, 20 μM, 50 μM, 100 μM, 150 μM, and 250 μM was added to the prepared test strips. - After 9 minutes of reaction, observe the color change of the test strip under a 365nm UV lamp and make a standard colorimetric card. Figure 9 shown.

[0135] Depend on Figure 9 It can be seen that as the NO2 in the solution added to the test strip - As the concentration increases from 0μM to 250μM, under the irradiation of 365nm ultraviolet light, it can be clearly observed that the yellow fluorescence of the test strip gradually deepens, and when the concentration of nitrite is 20μM, the test strip has a significant color change.

[0136] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0137] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention to be protected, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

Claims

1. A fluorescent probe for detecting nitrite, characterized in that: The fluorescent probe is a rhodamine B derivative with a spirohydrazide structure, and its structural formula is: 。 2. A method for preparing a fluorescent probe for detecting nitrite according to claim 1, characterized in that: Using fluorescein as a precursor, a fluorescent probe with a spirohydrazide structure was prepared through a chemical reaction. The specific preparation steps are as follows: Step 1: Preparation of Compound Ⅰ Fluorescein solid powder was added to anhydrous dichloromethane and cooled to 0°C in an ice-water bath. Anhydrous pyridine and trifluoromethanesulfonic anhydride solution were added in sequence. The mixture was stirred at 20-30°C for 3-5 hours, diluted with water and extracted. The obtained organic phase was washed with saturated brine, dried, concentrated under reduced pressure, and then gradient eluted with petroleum ether and ethyl acetate as eluents to obtain compound I. Step 2: Preparation of Compound II Compound I was added to tris(dibenzylideneacetone)dipalladium, X-Phos, cesium carbonate, and thiomorpholine-1,1-dioxide, and finally dissolved with 1,4-dioxane solution. The mixture was heated under reflux at 100-110°C for 8-18 hours under nitrogen protection. After the reaction was complete, water and dichloromethane were added for extraction. The mixture was washed with saturated brine, dried, and concentrated under reduced pressure. 95-105 ml of methanol was added for slurrying for 1.2-2.0 hours, and filtered to obtain compound II. Step 3: Preparation of Compound III Slowly add a small amount of hydrazine hydrate solution and methanol solution to the compound II sample, heat the reaction system under reflux at 63-70°C for 7-9 hours, cool to 20-30°C, and filter to obtain compound III; Step 4: Preparation of fluorescent probes A sample of compound III and 2-formylphenylboronic acid were mixed, dichloromethane and anhydrous ethanol were added as solvents, and the mixture was heated under reflux at 78-85°C for 0.5-1.5 hours. After the reaction solution was cooled to 20-30°C, the dichloromethane was removed by rotary evaporation, and the pink solid was filtered and washed with anhydrous ethanol. After drying, a fluorescent probe for detecting nitrite was obtained.

3. The method for preparing a fluorescent probe for detecting nitrite according to claim 2, wherein: In the step 1, anhydrous pyridine and trifluoromethanesulfonic anhydride solution are added in sequence, stirred at 25° C. for 4 hours, diluted with water, and extracted 2 to 3 times.

4. The method for preparing a fluorescent probe for detecting nitrite according to claim 2, wherein: In the step 2, heating and reflux at 101° C. for 12 h under nitrogen protection, adding water and dichloromethane for extraction after the reaction is complete, washing with saturated brine, drying and concentrating under reduced pressure, and adding 100 mL of methanol for slurrying for 1.5 h.

5. The method for preparing a fluorescent probe for detecting nitrite according to claim 2, wherein: In the step 3, the reaction system was heated under reflux at 65° C. for 8 h, cooled to 25° C., and filtered.

6. The method for preparing a fluorescent probe for detecting nitrite according to claim 2, wherein: In step 4, the reaction mixture is heated under reflux at 80° C. for 1 h. After the reaction solution is cooled to room temperature, dichloromethane is removed by rotary evaporation, and the mixture is filtered. The pink solid is washed 2 to 3 times with anhydrous ethanol and dried to obtain a fluorescent probe for detecting nitrite.

7. The method for preparing a fluorescent probe for detecting nitrite according to claim 2, wherein: In step 1, the mass of fluorescein is 8-15 g, the volume of anhydrous dichloromethane is 180-220 mL, the volume of anhydrous pyridine solution is 18-23 mL, and the volume of trifluoromethanesulfonic anhydride solution is 18-23 mL; in step 2, the volume of 1,4-dioxane is 90-120 mL; in step 3, the mass of compound II is 4.5-5.5 g, the volume of hydrazine hydrate solution is 0.8-1.2 mL, and the volume of methanol solution is 45-55 mL; in step 4, the mass of compound III is 0.5-1.5 g, 2-formylphenylboronic acid is 1.3-3.9 g; the volume of dichloromethane is 8-12 mL, and the volume of anhydrous ethanol is 18-22 mL.

8. The method for preparing a fluorescent probe for detecting nitrite according to claim 2, wherein: In the step 1, the mass of fluorescein is 10 g, the volume of anhydrous dichloromethane is 200 mL, the volume of anhydrous pyridine solution is 20 mL, and the volume of trifluoromethanesulfonic anhydride solution is 20 mL; in the step 2, 5.0 g of compound I, 0.8 g of tris(dibenzylideneacetone)dipalladium, 1.2 g of X-Phos, 7.6 g of cesium carbonate, and 2.7 g of thiomorpholine-1,1-dioxide, and the volume of 1,4-dioxane are 100 mL; in the step 3, the mass of compound II is 3.0 g, the volume of hydrazine hydrate solution is 0.9 mL, and the volume of methanol solution is 50 mL; in the step 4, the mass of compound III is 1.0 g, 2-formylphenylboronic acid is 2.6 g, the volume of dichloromethane is 10 mL, and the volume of anhydrous ethanol is 20 mL.

9. An application of the fluorescent probe for detecting nitrite according to claim 1, characterized in that: Prepare a standard probe solution: Dissolve the fluorescent probe in DMSO and dilute it with 2 mL of pH 1.0 HCl solution to a probe solution with a concentration of 6 μM. Then add the sample to be tested and detect the fluorescence intensity. Based on the linear relationship between the fluorescence intensity and the concentration of the sample to be tested, quantitatively calculate the nitrite content in the sample.

10. The use of the fluorescent probe for detecting nitrite according to claim 9, characterized in that: A standard colorimetric card for detecting nitrite was made: the fluorescent probe was dissolved in DMSO, and then diluted with a pH = 1.5 HCl solution to a probe solution with a concentration of 16 μM. The filter paper was cut into strips of the same size, and the strips were immersed in the probe solution with a concentration of 16 μM. After being taken out and dried, nitrite solutions of different concentrations were added dropwise, and the changes in fluorescence intensity were observed to make a standard colorimetric card.

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