PH and nitrite dual-response fluorescent indicator as well as preparation method and application thereof

Abstract

The invention discloses a pH and nitrite dual-response fluorescent indicator. The pH and nitrite dual-response fluorescent indicator comprises apple pectin and histidine in a mass ratio of 20: 31. The pH and nitrite dual-response fluorescent indicator provided by the invention has the advantages that the preparation method is simple and rapid; the used raw materials are edible, green, environment-friendly and high in safety; the water solubility is good, and the stability is high; the double responsiveness of pH and nitrite is realized. The invention further provides a preparation method and application of the fluorescent indicator. The invention further claims a method for testing pH value and nitrite concentration by using the fluorescent indicator.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, and more specifically, to a fluorescent indicator with dual response to pH and nitrite, a preparation method and application thereof. Background Art

[0002] Although nitrite itself is not toxic or harmful to the human body, when it is ingested into the human body, it will be converted into nitrosamines with strong carcinogenicity, which is harmful to human health. Nitrite has always been one of the factors that pose safety risks to food during food production, processing, and storage. The risk of nitrite is particularly obvious during the planting, processing, and storage of fruits and vegetables. During the storage of fruits and vegetables, the continuous release of carbon dioxide and volatile organic acids will cause the pH value to decrease. Detecting changes in pH during the storage of fruits and vegetables can predict the quality deterioration caused by physiological and biochemical reactions of fruits and vegetables. Therefore, detecting the pH and nitrite content of fruits and vegetables can predict the quality of fruits and vegetables and assess the safety risks of fruits and vegetables.

[0003] Currently, the detection of pH and nitrite in fruits and vegetables is mainly carried out through titration, colorimetry, chromatography, potentiometer and fluorescence methods. In recent years, fluorescence method has become the preferred detection method due to its advantages such as simple operation, rapid response and high sensitivity. The fluorescent nanoprobes used in the existing fluorescence method are all synthetic fluorescent nanoprobes, such as the invention patent with publication number CN 118130434A, which discloses a method for ratiometric fluorescence colorimetric detection of nitrite; another example is the invention patent with publication number CN117003712A, which discloses a fluorescent probe based on phenothiazine for detecting peroxynitrite anions, a synthesis method and its application; and the invention patent with publication number CN117110261A, which discloses a preparation method of a dual-mode MOF sensor for detecting pH based on an alizarin complex indicator. The disclosed fluorescent nanoprobes are all synthetic fluorescent nanoprobes, which generally have the following defects: (1) the selected preparation raw materials either use amine substances, metal ions, or organic solvents, which have low safety; (2) the preparation process is cumbersome and time-consuming; (3) the response time is long and the gradient change is not obvious; (4) it cannot detect pH and nitrite at the same time. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide a fluorescent indicator with dual response to pH and nitrite, the preparation method of which is simple and rapid; the raw materials used are all edible, green, environmentally friendly and safe; it has good water solubility and high stability; and it has dual response to pH and nitrite.

[0006] In order to achieve these objects and other advantages according to the present invention, a fluorescent indicator with dual responses to pH and nitrite is provided, comprising apple pectin and histidine in a mass ratio of 20:31.

[0007] Preferably, the pH response range is 1-13.

[0008] Preferably, the response concentration of nitrite is 0-10 mM.

[0009] The present invention further claims a method for preparing the fluorescent indicator having dual responses to pH and nitrite, comprising: Step 1, preparation of a mixed solution: weigh apple pectin, dissolve it in distilled water and stir for 1-3 hours until the apple pectin is fully dissolved, continue to add histidine and stir until fully mixed to obtain a mixed solution, place the mixed solution in a reactor in an oil bath for reaction for 1-4 hours at a reaction temperature of 120-180°C to obtain a reaction solution; Step 2, separation and purification of the reaction solution: the reaction solution obtained in step 1 is placed in a dialysis bag with a molecular cutoff of 500Da, and dialyzed with distilled water for 32-40 hours, changing the water every 3-5 hours, and collecting the dialysate in the dialysis bag; Step 3: Preparation of fluorescent indicator: Freeze the dialysate obtained in step 2 at -70°C to -90°C for 1 to 3 hours and then freeze dry it in a freeze dryer to obtain the fluorescent indicator. The freeze drying conditions are: cold trap temperature -50 to -70°C ° C, vacuum degree is 4~6Pa, and freeze-drying time is 20~28 h.

[0010] Preferably, the histidine concentration in the mixed solution in step 1 is 0.5M.

[0011] The present invention further claims a method for testing pH value using a fluorescent indicator having dual responses to pH and nitrite, comprising: A1. Dissolve the fluorescent indicator in distilled water to prepare multiple fluorescent indicator solutions with a concentration of 0.1-10 mg / mL; A2, taking the solution to be tested and the multiple fluorescent indicator solutions obtained in step A1, mixing them thoroughly to obtain multiple pH test solutions; A3. Take the multiple pH test solutions obtained in step A2 and place them in a four-way cuvette, and measure them in a fluorescence spectrophotometer. The fluorescence spectrophotometer conditions were as follows: excitation wavelength 370 nm, slit distance 5 nm, excitation voltage 750 V, scan speed 1200 nm / min, response time 0.08 s; A4. Record the maximum fluorescence intensity I between the emission wavelength of 400-500 nm 400-500 nm Linear fitting was performed with pH value to plot pH-I 400-500 nmFitting curves; A5. Determine the maximum fluorescence intensity I of the object to be tested 400-500 nm , and according to the pH-I obtained in step A4 400-500 nm The pH value of the analyte was calculated by fitting the curve.

[0012] In the above technical solution, based on the method for testing pH value of the fluorescent indicator having dual response to pH and nitrite, the present invention further claims to protect the application of the fluorescent indicator having dual response to pH and nitrite in detecting pH value of food.

[0013] The present invention further claims a method for testing nitrite concentration using the fluorescent indicator having dual responses to pH and nitrite, comprising: B1. Dissolve the fluorescent indicator in distilled water or HCl to prepare multiple fluorescent indicator solutions with a concentration of 0.1-10 mg / mL; B2, taking the solution to be tested and the multiple fluorescent indicator solutions obtained in step B1, and mixing them thoroughly to obtain multiple nitrite test solutions; B3. Place the multiple nitrite test solutions obtained in step B2 in a four-way cuvette and measure them in a fluorescence spectrophotometer. The conditions of the fluorescence spectrophotometer are: excitation wavelength 370 nm, slit distance 5 nm, excitation voltage 750 V, scanning speed 1200 nm / min, response time 0.08 s. B4, respectively set the maximum fluorescence intensity values ​​I at 400-500 nm and 700-800 nm of the fluorescence spectrum 400-500 nm and I 700-800 nm Linear fitting was performed with nitrite concentration C to obtain CI 400-500 nm Fitting curve and CI 700-800 nm Fitting curves; B5. Determine the maximum fluorescence intensity I of the object to be tested 400-500 nm Or the maximum fluorescence intensity I 700-800 nm , and according to the CI obtained in step B4 400-500 nm Fitting curve and CI 700-800 nm The nitrite concentration of the analyte was calculated by fitting the curve.

[0014] In the above technical solution, based on the method for testing nitrite concentration using the fluorescent indicator having dual responses to pH and nitrite, the present invention further claims the use of the fluorescent indicator having dual responses to pH and nitrite in detecting nitrite concentration in food.

[0015] The present invention further claims to protect the use of the fluorescent indicator with dual response to pH and nitrite in the preparation of a fluorescent nanoprobe, wherein the fluorescent probe has fluorescent indicative properties with dual response to pH and nitrite, and the fluorescent nanoprobe also includes a light source, an optical system and a light detector (photomultiplier tube or photodiode).

[0016] The present invention further claims to protect the use of the fluorescent indicator with dual responses to pH and nitrite in the preparation of food packaging films. The food packaging film has fluorescent indicative properties with dual responses to pH and nitrite. The inner side of the food packaging film is coated with the fluorescent indicator with dual responses to pH and nitrite provided by the present invention, or the raw materials for producing the food packaging film are mixed with the fluorescent indicator with dual responses to pH and nitrite provided by the present invention. When the quality of food in the food packaging film changes, the fluorescence intensity of the fluorescent indicator on the food packaging film changes accordingly. According to the change in the fluorescence intensity of the fluorescent indicator on the food packaging film, the quality change state of the food in the food packaging film can be observed with the naked eye, and the quality of the food in the food packaging film can be quickly judged.

[0017] The present invention further claims to protect the use of the fluorescent indicator with dual response to pH and nitrite in the preparation of fluorescent test paper with dual response to pH and nitrite, the surface of the fluorescent test paper is coated with the fluorescent indicator with dual response to pH and nitrite provided by the present invention.

[0018] The present invention has at least the following beneficial effects: First, the fluorescent indicator with dual response to pH and nitrite provided by the present invention can simultaneously detect the pH value and nitrite concentration in food, especially fruits and vegetables, that is, the fluorescent indicator with dual response to pH and nitrite provided by the present invention has dual gradient responsiveness to the pH value and nitrite concentration of food (especially fruits and vegetables), wherein the detection range of pH value is 1-13, and the response to pH is sensitive and wide; the detection concentration of nitrite is 0-10mM, the minimum detection limit is 0.79 μM, and it has dual-wavelength linear response at 400-500 nm and 700-800 nm, the reaction time is 1 min, and the response to nitrite is sensitive; Secondly, the fluorescent indicator with dual response of pH and nitrite provided by the present invention is prepared by using edible apple pectin and histidine as raw materials, is green, environmentally friendly and safe, can directly contact food, is biodegradable and renewable, has high salt resistance and light degradation resistance, can stably exert fluorescent properties for a long time, and can be used on food packaging films in addition to conventional testing of food pH and nitrite concentration, thus broadening the application range of fluorescent indicators; Thirdly, the fluorescent indicator with dual response of pH and nitrite provided by the present invention uses distilled water as solvent and can be prepared by hydrothermal method. The preparation method is simple and fast and does not involve organic chemical reagents. Fourthly, the method for detecting pH and nitrite by the fluorescent indicator with dual response to pH and nitrite provided by the present invention is simple and has good detection stability.

[0019] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a scanning electron microscope image of the fluorescent indicator obtained in Example 1 of the present invention; Figure 2 The fluorescent indicator obtained in Example 1 of the present invention 1 H NMR spectra; Figure 3 The fluorescence spectra of the fluorescent indicator obtained in Example 1 of the present invention in response to solutions with different pH values ​​(1-13) are shown; Figure 4 The linear fitting results of the fluorescent indicator obtained in Example 1 of the present invention in response to solutions of different pH values ​​(1-13); Figure 5 The fluorescence spectra of the fluorescent indicator obtained in Example 1 of the present invention in response to different concentrations (0-10 mM) of nitrite; Figure 6 The linear fitting results of the fluorescent indicator obtained in Example 1 of the present invention in response to different concentrations (0-10 mM) of nitrite are shown in FIG. Figure 6 (a) is the linear fitting result of fluorescence intensity and nitrite at the fluorescence emission wavelength of 400-500 nm. Figure 6 (b) The linear fitting results of fluorescence intensity and nitrite at the fluorescence emission wavelength of 700-800 nm Figure 7 The fluorescence spectra of the fluorescent indicators obtained in Examples 1 to 3 of the present invention are shown; Figure 8 The fluorescence spectra of the fluorescent indicators obtained in Examples 1, 4 and 5 of the present invention are shown. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0022] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0023] Example 1 Preparation of fluorescent indicator: Step 1: Weigh 2.5 g of apple pectin and dissolve it in 50 mL of distilled water. Stir magnetically for 2 h at room temperature. Then add histidine to the apple pectin at a concentration of 0.5 M and stir continuously for 5 min to ensure uniform mixing to obtain a mixed solution. Place the obtained mixed solution in a 100 mL reactor and heat it to 160 °C in an oil bath. ° C for 1 h, cooled naturally, and the reaction solution was collected; Step 2: Place the obtained reaction solution in a dialysis bag with a molecular cutoff of 500 Da, dialyze with distilled water for 36 h, change the water every 4 h, and collect the liquid in the dialysis bag to obtain the dialysate; Step 3: Freeze the dialysate obtained in step 2 at -80°C for 2 hours and then freeze-dry it in a freeze dryer to obtain the fluorescent indicator. The scanning electron microscopy results of the fluorescent indicator and 1 The H NMR results are shown in Figure 1 and Figure 2 As shown, the freeze-drying conditions are: cold trap temperature -60°C, vacuum degree 5 Pa, freeze-drying time 24 h. The yield of the fluorescent indicator is 25.6%.

[0024] exist Figure 1 The fluorescent indicator prepared in Example 1 showed clusters in the full field of view of the electron microscope. Further magnification and analysis showed that the fluorescent indicator prepared in Example 1 showed a pectin-like network structure, which indicates that after histidine was incorporated into apple pectin, it combined with apple pectin molecules to form a stable binding state. Figure 2 In the embodiment 1, the fluorescent indicator prepared 1 H NMR results showed that the peaks at 4.99, 4.33, 3.90 and 1.21 ppm were attributed to H-1, H-4, H-3 and CH3(Rha) of pectin, respectively. The main chain and side chain structure of pectin still existed in histidine-apple pectin. The chemical shifts at 1.30, 1.56, 2.83 and 3.32 ppm were caused by the hydrogen protons on the imidazole ring of histidine, but 1 Some new peaks appeared on the H NMR, which were attributed to the hydrogen in histidine combining with apple pectin to form new hydrogen types, further proving that histidine was incorporated into apple powder and combined with apple pectin molecules to form a stable binding state under the reaction temperature and reaction time provided in the present application.

[0025] The pH response effect of the fluorescent indicator obtained in Example 1 was tested: A series of solutions with a pH of 1-13 were prepared using 0.2 M HCl and 0.2 M NaOH. The prepared fluorescent indicator was dissolved in distilled water at a concentration of 10 mg / mL. During the test, 0.2 mL of 10 mg / mL fluorescent indicator aqueous solution was added to 1.8 mL of pH 1-13 solution and mixed. 2 mL of the mixed solution was added to a four-way cuvette and measured in a fluorescence spectrophotometer. The conditions of the fluorescence spectrophotometer are: excitation wavelength 370 nm, slit distance 5 nm, excitation voltage 750 V, scanning speed 1200 nm / min, response time 0.08 s. The maximum fluorescence intensity I400-500nm between the emission wavelength of 400-500 nm was recorded and linearly fitted with the pH value (X). The response spectrum results of the fluorescent indicator obtained in Example 1 to solutions with different pH values ​​are shown as follows. Figure 3 The fitting results are shown in Figure 4 As shown, the results show that the fitting equation is I 400-500 nm =-224.84X+4513.9, R 2 =0.9816.

[0026] The response of the fluorescent indicator obtained in Example 1 to nitrite was tested: The prepared fluorescent indicator was dissolved in 0.2 M HCl solution, and the concentration of the fluorescent indicator after dissolution was 10 mg / mL. Take 0.2 mL of the fluorescent indicator hydrochloric acid aqueous solution, add 1.8 mL of sodium nitrite solution of different concentrations (0-0.1 mM), mix and react for 1 min, and measure the fluorescence spectrum with a fluorescence spectrophotometer. The response spectrum of the fluorescent indicator obtained in Example 1 to different concentrations (0~10 mM) of nitrite is as follows: Figure 5 The fluorescence spectrophotometer conditions were as follows: excitation wavelength 370 nm, slit distance 5 nm, excitation voltage 750 V, scanning speed 1200 nm / min, response time 0.08 s. The maximum fluorescence intensity values ​​I at 400-500 nm and 700-800 nm of the fluorescence spectrum were recorded respectively. 400-500 nm and I 700-800 nm A linear fit was performed with the nitrite concentration C. When the nitrite concentration was 0.1-0.001 mM, I 400-500 nm It is linearly related to the nitrite concentration C. The fitting results are as follows Figure 6 (a) shows that the fitting equation is I 400-500 nm =-14020C+6258.4,R 2 =0.9685;I 700-800 nm It is linearly related to the nitrite concentration C. The fitting results are as follows Figure 6 (b) shows that the fitting equation is I 700-800 nm =-3893.3C+1857.5,R2 =0.9793.

[0027] The detection limit was calculated by LOD (limit of detection) = 3σbi / m (where m represents the slope of the curve in the graph of the relationship between the response ion concentration and the fluorescence intensity change, and σbi represents the standard deviation of the blank experimental data). After calculation, the detection limit of the fluorescent nanoprobe for nitrite was 0.78 μM.

[0028] Example 2 Preparation of fluorescent indicator: Step 1: Weigh 2.5 g of apple pectin and dissolve it in 50 mL of distilled water. Stir it magnetically for 2 h at room temperature. Then add histidine to the apple pectin at a concentration of 0.5 M and stir it continuously for 5 min to ensure uniform mixing to obtain a mixed solution. Place the obtained mixed solution in a 100 mL reactor and heat it to 120°C in an oil bath for 1 h. Cool it naturally and collect the reaction solution. Step 2: Place the obtained reaction solution in a dialysis bag with a molecular cutoff of 500 Da, dialyze with distilled water for 36 h, change the water every 4 h, and collect the liquid in the dialysis bag to obtain the dialysate; Step 3: Freeze the dialysate obtained in step 2 at -80°C for 2 hours and then freeze-dry it in a freeze dryer to obtain the fluorescent indicator. The freeze-drying conditions are: cold trap temperature -60°C, vacuum degree 5 Pa, and freeze-drying time 24 hours.

[0029] Example 3 Preparation of fluorescent indicator: Step 1: Weigh 2.5 g of apple pectin and dissolve it in 50 mL of distilled water. Stir magnetically for 2 h at room temperature. Then add histidine to the apple pectin at a concentration of 0.5 M and stir continuously for 5 min to ensure uniform mixing to obtain a mixed solution. Place the obtained mixed solution in a 100 mL reactor and heat it to 180°C in an oil bath. ° C for 1 h, cooled naturally, and the reaction solution was collected; Step 2: Place the obtained reaction solution in a dialysis bag with a molecular cutoff of 500 Da, dialyze with distilled water for 36 h, change the water every 4 h, and collect the liquid in the dialysis bag to obtain the dialysate; Step 3: Freeze the dialysate obtained in step 2 at -80°C for 2h and then freeze-dry it in a freeze dryer to obtain the fluorescent indicator. The freeze-drying conditions are: the cold trap temperature is -60 ° C, vacuum degree was 5 Pa, and freeze-drying time was 24 h.

[0030] Example 4 Preparation of fluorescent indicator: Step 1: Weigh 2.5 g of apple pectin and dissolve it in 50 mL of distilled water. Stir it magnetically for 2 h at room temperature. Then add histidine to the apple pectin at a concentration of 0.5 M and stir it continuously for 5 min to ensure uniform mixing to obtain a mixed solution. Place the obtained mixed solution in a 100 mL reactor and heat it to 160°C in an oil bath for 2 h. Cool it naturally and collect the reaction solution. Step 2: Place the obtained reaction solution in a dialysis bag with a molecular cutoff of 500 Da, dialyze with distilled water for 36 h, change the water every 4 h, and collect the liquid in the dialysis bag to obtain the dialysate; Step 3: Freeze the dialysate obtained in step 2 at -80°C for 2h and then freeze-dry it in a freeze dryer to obtain the fluorescent indicator. The freeze-drying conditions are: the cold trap temperature is -60 ° C, vacuum degree was 5 Pa, and freeze-drying time was 24 h.

[0031] Example 5 Preparation of fluorescent indicator: Step 1: Weigh 2.5 g of apple pectin and dissolve it in 50 mL of distilled water. Stir it magnetically for 2 h at room temperature. Then add histidine to the apple pectin at a concentration of 0.5 M and stir it continuously for 5 min to ensure uniform mixing to obtain a mixed solution. Place the obtained mixed solution in a 100 mL reactor and heat it to 160°C in an oil bath for 4 h. Cool it naturally and collect the reaction solution. Step 2: Place the obtained reaction solution in a dialysis bag with a molecular cutoff of 500 Da, dialyze with distilled water for 36 h, change the water every 4 h, and collect the liquid in the dialysis bag to obtain the dialysate; Step 3: Freeze the dialysate obtained in step 2 at -80°C for 2h and then freeze-dry it in a freeze dryer to obtain the fluorescent indicator. The freeze-drying conditions are: the cold trap temperature is -60 ° C, vacuum degree was 5 Pa, and freeze-drying time was 24 h.

[0032] Comparative Example 1 Preparation of fluorescent indicator: Step 1: Weigh 2.5 g of apple pectin and dissolve it in 50 mL of distilled water. Stir it magnetically for 2 h at room temperature. Then add lysine to the apple pectin at a concentration of 0.1 M and stir it continuously for 5 min to ensure uniform mixing to obtain a mixed solution. Place the obtained mixed solution in a 100 mL reactor and heat it in an oil bath to 160 ° C for 1 h, cooled naturally, and the reaction solution was collected, wherein the amount of lysine added was the preferred amount; Step 2: Place the obtained reaction solution in a dialysis bag with a molecular cutoff of 500 Da, dialyze with distilled water for 36 h, change the water every 4 h, and collect the liquid in the dialysis bag to obtain the dialysate; Step 3: Freeze the dialysate obtained in step 2 at -80°C for 2h and then freeze-dry it in a freeze dryer to obtain the fluorescent indicator. The freeze-drying conditions are: the cold trap temperature is -60 ° C, vacuum degree of 5 Pa, freeze-drying time of 24 h. The yield of the fluorescent indicator is 22.6%.

[0033] The pH response effect of the fluorescent indicator obtained in Comparative Example 1 was tested: A solution with a pH of 1-13 was prepared by 0.2 M HCl and 0.2 M NaOH. The fluorescent indicator prepared in Comparative Example 1 was dissolved in distilled water at a concentration of 10 mg / mL. During the test, 1.8 mL of pH 1-13 solution was added with 0.2 mL of 10 mg / mL fluorescent indicator aqueous solution and mixed. 2 mL of the mixed solution was added to a four-way cuvette and measured in a fluorescence spectrophotometer. The conditions of the fluorescence spectrophotometer were: excitation wavelength 370 nm, slit distance 5 nm, excitation voltage 750 V, scanning speed 1200 nm / min, and response time 0.08 s. The maximum fluorescence intensity I between the emission wavelength 400-500 nm was recorded. 400-500 nm A linear fit was performed with pH value (X), and the result showed that the equation was I 400-500 nm =-136.23X+6964.9, R 2 =0.2353, the degree of linear relationship is low.

[0034] The response results of the fluorescent indicator obtained in Comparative Example 1 to nitrite were tested: The fluorescent indicator prepared in Comparative Example 1 was dissolved in 0.2 M HCl solution. The concentration of the fluorescent indicator after dissolution was 10 mg / mL. Take 0.2 mL of the fluorescent indicator hydrochloric acid aqueous solution, add 1.8 mL of sodium nitrite solution of different concentrations (0-0.1 mM), mix and react for 1 min, and measure the fluorescence spectrum in a fluorescence spectrophotometer. The conditions of the fluorescence spectrophotometer are: excitation wavelength 370 nm, slit distance 5 nm, excitation voltage 750 V, scanning speed 1200 nm / min, response time 0.08 s. Record the maximum fluorescence intensity values ​​I at 400-500 nm and 700-800 nm of the fluorescence spectrum respectively. 400-500 nm and I 700-800 nm A linear fit was performed with the nitrite concentration C. The results showed that the maximum fluorescence of the fluorescent probe at 400-500 nm and 700-800 nm had no concentration-dependent response to the nitrate concentration.

[0035] Comparative Example 2 Preparation of fluorescent indicator: Step 1: Weigh 2.5 g of apple pectin and dissolve it in 50 mL of distilled water. Stir it magnetically for 2 h at room temperature. Then add arginine to the apple pectin at a concentration of 0.8 M and stir it continuously for 5 min to ensure uniform mixing to obtain a mixed solution. Place the obtained mixed solution in a 100 mL reactor and heat it in an oil bath to 160 ° C for 1 h, cooled naturally, and the reaction solution was collected, wherein the amount of arginine added was the preferred amount; Step 2: Place the obtained reaction solution in a dialysis bag with a molecular cutoff of 500 Da, dialyze with distilled water for 36 h, change the water every 4 h, and collect the liquid in the dialysis bag to obtain the dialysate; Step 3: Freeze the dialysate obtained in step 2 at -80°C for 2h and then freeze-dry it in a freeze dryer to obtain the fluorescent indicator. The freeze-drying conditions are: the cold trap temperature is -60 ° C, vacuum degree of 5 Pa, freeze-drying time of 24 h. The yield of the fluorescent indicator is 20.3%.

[0036] The pH response effect of the fluorescent indicator obtained in Comparative Example 2 was tested: A solution with a pH of 1-13 was prepared using 0.2 M HCl and 0.2 M NaOH. The prepared fluorescent indicator was dissolved in distilled water at a concentration of 10 mg / mL. During the test, 1.8 mL of pH 1-13 solution was added with 0.2 mL of 10 mg / mL fluorescent indicator aqueous solution and mixed. 2 mL of the mixed solution was added to a four-way cuvette and measured in a fluorescence spectrophotometer. The conditions of the fluorescence spectrophotometer were: excitation wavelength 370 nm, slit distance 5 nm, excitation voltage 750 V, scanning speed 1200 nm / min, and response time 0.08 s. The maximum fluorescence intensity I400-500 nm between the emission wavelength of 400-500 nm was recorded and linearly fitted with the pH value (X). The results showed that the equation was I 400-500 nm =68.464X+4656.6, R 2 =0.0778, the degree of linear relationship is low.

[0037] The response results of the fluorescent indicator obtained in Comparative Example 2 to nitrite were tested: The prepared fluorescent indicator was dissolved in 0.2 M HCl solution, and the concentration of the fluorescent indicator after dissolution was 10 mg / mL. Take 0.2 mL of the fluorescent indicator hydrochloric acid aqueous solution, add 1.8 mL of sodium nitrite solution of different concentrations (0-0.1 mM), mix and react for 1 min, and measure the fluorescence spectrum in a fluorescence spectrophotometer. The conditions of the fluorescence spectrophotometer are: excitation wavelength 370 nm, slit distance 5 nm, excitation voltage 750 V, scanning speed 1200 nm / min, response time 0.08 s. Record the maximum fluorescence intensity values ​​I at 400-500 nm and 700-800 nm of the fluorescence spectrum respectively. 400-500 nm and I 700-800 nm A linear fit was performed with the nitrite concentration C. The results showed that the maximum fluorescence of the fluorescent probe at 400-500 nm and 700-800 nm had no concentration-dependent response to the nitrate concentration.

[0038] In Examples 1 to 3, the reaction temperature of histidine-apple pectin was changed to prepare different histidine-apple pectin fluorescent indicators. The fluorescence spectra of each fluorescent indicator are as follows: Figure 7 In Example 1, Example 4 and Example 5, the reaction time of histidine-apple pectin was changed to prepare different histidine-apple pectin fluorescent indicators. The fluorescence spectra of each fluorescent indicator are shown in Figure 8 As shown. Figure 7 It can be seen that the histidine-apple pectin fluorescence indicator prepared at three reaction temperatures has a detection wave at a wavelength of 400-500 nm, which has the property of indicating pH value and nitrite concentration. Among them, the histidine-apple pectin fluorescence indicator prepared at a reaction temperature of 160°C has the largest detection wave intensity at a wavelength of 400-500 nm. Figure 8 It can be seen that the histidine-apple pectin fluorescent indicator prepared under three reaction times has a detection wave at a wavelength of 400-500 nm, and has the property of indicating pH value and nitrite concentration. Among them, the histidine-apple pectin fluorescent indicator prepared under a reaction time of 1 h has the largest detection wave intensity at a wavelength of 400-500 nm.

[0039] Comparative Examples 1 and 2 changed the types of amino acids in the fluorescent indicator. The lysine-apple pectin fluorescent indicator prepared in Comparative Example 1 had poor responsiveness to pH at a wavelength of 400-500 nm, and had no response to nitrite concentration at wavelengths of 400-500 nm and 700-800 nm. The arginine-apple pectin fluorescent indicator prepared in Comparative Example 2 had poor responsiveness to pH at a wavelength of 400-500 nm, and had no response to nitrite concentration at wavelengths of 400-500 nm and 700-800 nm.

[0040] The number of equipment and processing scales described here are used to simplify the description of the present invention. The application, modification and variation of the fluorescent indicator with dual response to pH and nitrite and the preparation method of the present invention are obvious to those skilled in the art.

[0041] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A fluorescent indicator with dual response to pH and nitrite, characterized in that: It comprises apple pectin and histidine in a mass ratio of 20:

31.

2. The fluorescent indicator with dual response of pH and nitrite as claimed in claim 1, characterized in that: The pH response range is 1-13.

3. The fluorescent indicator with dual response of pH and nitrite as claimed in claim 1, characterized in that: The response concentration of the nitrite is 0-10 mM.

4. The method for preparing the fluorescent indicator with dual response of pH and nitrite as claimed in claim 1, characterized in that: include: Step 1, preparation of a mixed solution: weigh apple pectin, dissolve it in distilled water and stir for 1-3 hours until the apple pectin is fully dissolved, continue to add histidine and stir until fully mixed to obtain a mixed solution, place the mixed solution in a reactor in an oil bath for reaction for 1-4 hours at a reaction temperature of 120-180°C to obtain a reaction solution; Step 2, separation and purification of the reaction solution: the reaction solution obtained in step 1 is placed in a dialysis bag with a molecular cutoff of 500Da, and dialyzed with distilled water for 32-40 hours, changing the water every 3-5 hours, and collecting the dialysate in the dialysis bag; Step 3: Preparation of fluorescent indicator: Freeze the dialysate obtained in step 2 at -70°C to -90°C for 1 to 3 hours and then freeze dry it in a freeze dryer to obtain the fluorescent indicator. The freeze drying conditions are: cold trap temperature -50 to -70°C ° C, vacuum degree is 4~6 Pa, and freeze-drying time is 20~28 h.

5. The method for preparing the fluorescent indicator with dual response of pH and nitrite as claimed in claim 4, characterized in that: In step 1, the histidine concentration in the mixed solution is 0.5 M.

6. The method for testing pH value by a fluorescent indicator with dual response of pH and nitrite as claimed in claim 1, characterized in that: include: A1. Dissolve the fluorescent indicator in distilled water to prepare multiple fluorescent indicator solutions with a concentration of 0.1-10 mg / mL; A2, taking the solution to be tested and the multiple fluorescent indicator solutions obtained in step A1, mixing them thoroughly to obtain multiple pH test solutions; A3. Take the multiple pH test solutions obtained in step A2 and place them in a four-way cuvette, and measure them in a fluorescence spectrophotometer. The fluorescence spectrophotometer conditions were as follows: excitation wavelength 370 nm, slit distance 5 nm, excitation voltage 750 V, scan speed 1200 nm / min, response time 0.08 s; A4. Record the maximum fluorescence intensity I between the emission wavelength of 400-500 nm 400-500 nm Linear fitting was performed with pH value to plot pH-I 400-500 nm Fitting curves; A5. Determine the maximum fluorescence intensity I of the object to be tested 400-500 nm , and according to the pH-I obtained in step A4 400-500 nm The pH value of the analyte was calculated by fitting the curve.

7. The method for testing nitrite concentration by a fluorescent indicator with dual response of pH and nitrite as claimed in claim 1, characterized in that: include: B1. Dissolve the fluorescent indicator in distilled water or HCl solution to prepare multiple fluorescent indicator solutions with a concentration of 0.1-10 mg / mL; B2, taking the solution to be tested and the multiple fluorescent indicator solutions obtained in step B1, and mixing them thoroughly to obtain multiple nitrite test solutions; B3. Place the multiple nitrite test solutions obtained in step B2 in a four-way cuvette and measure them in a fluorescence spectrophotometer. The conditions of the fluorescence spectrophotometer are: excitation wavelength 370 nm, slit distance 5 nm, excitation voltage 750 V, scanning speed 1200 nm / min, response time 0.08 s. B4, respectively set the maximum fluorescence intensity values ​​I at 400-500 nm and 700-800 nm of the fluorescence spectrum 400-500 nm and I 700-800 nm Linear fitting was performed with nitrite concentration C to obtain CI 400-500 nm Fitting curve and CI 700-800 nm Fitting curves; B5. Determine the maximum fluorescence intensity I of the object to be tested 400-500 nm Or the maximum fluorescence intensity I 700-800 nm , and according to the CI obtained in step B4 400-500 nm Fitting curve and CI 700-800 nm The fitting curve corresponds to the calculated nitrite concentration of the analyte.

8. Use of the fluorescent indicator with dual response to pH and nitrite as claimed in claim 1 in the preparation of fluorescent nanoprobes, characterized in that: The fluorescent probe has fluorescence indication with dual response to pH and nitrite.

9. Use of the fluorescent indicator with dual response of pH and nitrite as claimed in claim 1 in preparing food packaging film, characterized in that: The food packaging film has fluorescence indication with dual response of pH and nitrite.

10. Application of the fluorescent indicator with dual response of pH and nitrite as claimed in claim 1 in preparing fluorescent test paper with dual response of pH and nitrite.

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