A fluorescent probe and its preparation method and application in detecting metabisulfite

Abstract

The present invention provides a fluorescent probe and its preparation method and application in detecting metabisulfite, belonging to the technical field of fluorescent probes. The fluorescent probe CPSP provided by the present invention can undergo Michael addition reaction between the double bond of metabisulfite and has a good response to metabisulfite. After the addition of metabisulfite, it shows obvious proportional fluorescence, and its intensity increases with the addition of S2O5 2‑ The fluorescence emission intensity at 572nm gradually decreases, while that at 481nm gradually increases. 2‑ The concentration changes significantly within the range of 0 to 60 μM and is visible to the naked eye. The fluorescent probe CPSP provided by the present invention can realize the quantitative detection of metabisulfite. 481nm / I 572nm Fluorescence intensity ratio and S2O5 2‑ There is a good linear relationship between the concentrations.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a fluorescent probe and a preparation method thereof, and application thereof in detecting pyrosulfite. Background Art

[0002] Sodium metabisulfite is commonly used as a preservative and color-protecting additive in the dry food and beverage industry. It is a commonly used, inexpensive additive that generates bisulfite in aqueous solution, effectively inhibiting phenolase activity, reducing quinones to phenol, preventing the polymerization of carbonyl compounds, and preserving the original color of the raw material over time. Excessive metabisulfite can cause irreversible damage to cells and tissues, leading to respiratory and cardiovascular diseases, lung cancer, and severe allergic reactions. Consequently, many countries have strict restrictions on the use of metabisulfite in food. To ensure food safety, effective analytical methods must be established for the sensitive and selective detection of metabisulfite in food and biological products.

[0003] To date, many analytical techniques have been developed for the determination of metabisulfite, such as electrochemistry, spectrophotometry, flow injection, and liquid chromatography. However, these methods require expensive instrumentation and complex pretreatment, and are therefore not suitable for the rapid detection of metabisulfite in food.

[0004] Fluorescent probes are a rapid and commonly used analytical technique for imaging and detecting a wide range of biological species due to their simplicity, convenience, high sensitivity, selectivity, and non-invasive nature. However, there are currently no fluorescent probes suitable for detecting metabisulfite in food. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a fluorescent probe and its preparation method and its application in detecting pyrosulfite. The fluorescent probe provided by the present invention is sensitive to S2O5 2- It has fast response speed, low detection limit and good selectivity.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a fluorescent probe having a structure shown in Formula I:

[0008]

[0009] The present invention provides a method for preparing the above fluorescent probe, comprising the following steps:

[0010] Mixing a compound having a structure represented by Formula 1, cyanoacetic acid, piperidine, and anhydrous ethanol solvent to carry out a condensation reaction to obtain a compound having a structure represented by Formula 2;

[0011]

[0012] Mixing the compound having the structure shown in Formula 2, 3-bromo-1-propanol, a condensing agent, and an organic solvent, and performing an esterification reaction to obtain a compound having the structure shown in Formula 3;

[0013]

[0014] The compound having the structure shown in Formula 3, pyridine and ethanol are mixed and subjected to a quaternization reaction to obtain a fluorescent probe having the structure shown in Formula I.

[0015] Preferably, the molar ratio of the compound having the structure shown in Formula 1 to cyanoacetic acid is 1:4-6;

[0016] The molar ratio of the compound having the structure shown in Formula 1 to piperidine is 1:1 to 1.2;

[0017] The condensation reaction temperature is 78-85° C., and the reaction time is 6-10 hours.

[0018] Preferably, the molar ratio of the compound having the structure shown in Formula 2 to 3-bromo-1-propanol is 1.1 to 1.5:1.

[0019] Preferably, the condensing agent includes EDC and HOBt;

[0020] The esterification reaction time is 6 to 10 hours.

[0021] Preferably, the molar ratio of the compound having the structure shown in Formula 3 to pyridine is 1:10-12;

[0022] The temperature of the quaternization reaction is 78-85° C., and the time is 6-10 hours.

[0023] The present invention provides application of the fluorescent probe in detecting pyrosulfite.

[0024] The present invention provides a method for detecting pyrosulfite, comprising the following steps:

[0025] Mixing the sample to be tested with water, performing homogenization and solid-liquid separation to obtain a solution of the sample to be tested;

[0026] The sample solution to be tested is mixed with the fluorescent probe solution. If the fluorescent probe solution changes from pink to light yellow under naked eye observation, the sample to be tested contains metabisulfite;

[0027] Alternatively, the sample solution to be tested is mixed with a fluorescent probe solution. If the fluorescence of the fluorescent probe solution changes from orange-red to bright blue, the sample to be tested contains metabisulfite.

[0028] The present invention provides a method for detecting pyrosulfite, comprising the following steps:

[0029] Mixing the sample to be tested with water, performing homogenization and solid-liquid separation to obtain a solution of the sample to be tested;

[0030] The sample solution to be tested was mixed with the fluorescent probe solution, and the fluorescence intensity of the resulting mixed solution at 572 nm and 481 nm was measured to obtain I 481nm / I 572nm Ratio, according to the I 481nm / I 572nm The content of metabisulfite in the sample to be tested is obtained by using the ratio and a predetermined standard curve;

[0031] The standard curve is I 481nm / I 572nm The linear relationship curve between the ratio and the concentration of metabisulfite solution.

[0032] Preferably, the detection limit of the metabisulfite is 26 nM, and the linear detection range is 26 nM to 60 μM.

[0033] The present invention provides a fluorescent probe having a structure shown in Formula I. The present invention utilizes a coumarin derivative to develop a ratiometric fluorescent probe, designated CPSP. The fluorescent probe CPSP undergoes a Michael addition reaction with the double bond of a pyrosulfite, such as Na2S2O5. The reaction mechanism is shown in Formula A:

[0034]

[0035] The fluorescent probe CPSP has a good response to pyrosulfite and shows obvious proportional fluorescence after the addition of pyrosulfite, and its intensity increases with the increase of S2O5 2- The fluorescence emission intensity at 572 nm changes with the increase of S2O5 2- The fluorescence emission intensity at 481 nm gradually decreases with the increase of S2O5 2- As the concentration increases, the fluorescence of the fluorescent probe CPSP changes from orange-red to bright blue. 2- The concentration changes significantly within the range of 0 to 60 μM and is visible to the naked eye: under naked eye observation, the color of the fluorescent probe CPSP changes from pink to light yellow, which can be used for naked eye identification.

[0036] The fluorescent probe CPSP provided by the present invention can realize the quantitative detection of pyrosulfite. 481nm / I 572nm Fluorescence intensity ratio and S2O5 2- There is a good linear relationship between the concentrations (R2 =0.9969), the detection limit was calculated to be 26 nM, indicating that the CPSP probe is sensitive to S2O5 2- The results of the examples show that other anions do not cause any observable fluorescence changes. 2- When the probe was treated, a significant ratiometric fluorescence response was observed, I 481nm / I 572nm The ratio of S2O5 to CPSP was increased by 439 times. 2- Response time calculation t 1 / 2 . Add 100μM S2O5 2- After that, the fluorescence intensity at 481 nm gradually increases with time and reaches equilibrium. 1 / 2 6s, confirming that the probe and S2O5 2- Quick response between. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the synthetic route of the fluorescent probe CPSP;

[0038] Figure 2 is the H NMR spectrum of the fluorescent probe CPSP of Example 1;

[0039] Figure 3 is the mass spectrum of the fluorescent probe CPSP of Example 1;

[0040] Figure 4 is the UV-visible absorption spectrum of the fluorescent probe CPSP test solution;

[0041] Figure 5 is the fluorescence spectrum of the fluorescent probe CPSP test solution;

[0042] Figure 6 The curve of the fluorescence emission intensity change at 572nm and 481nm of the fluorescent probe CPSP test solution with the increase of Na2S2O5 concentration;

[0043] Figure 7 The fluorescence color of the fluorescent probe CPSP test solution changes with the increase of Na2S2O5 concentration;

[0044] Figure 8 The linear relationship between the fluorescence ratio of the fluorescent probe CPSP at 572 nm and 481 nm and the concentration of Na2S2O5;

[0045] Figure 9 The selectivity of the fluorescent probe CPSP to different ions;

[0046] Figure 10is the response time of the fluorescent probe CPSP to Na2S2O5;

[0047] Figure 11 The fluorescence intensity of the probe test solution when the sample solution is added in an amount of 0 to 45 μL. DETAILED DESCRIPTION

[0048] The present invention provides a fluorescent probe having a structure shown in Formula I:

[0049]

[0050] In the present invention, the method for preparing the fluorescent probe comprises the following steps:

[0051] Mixing a compound having a structure represented by Formula 1, cyanoacetic acid, piperidine, and anhydrous ethanol solvent to carry out a condensation reaction to obtain a compound having a structure represented by Formula 2;

[0052]

[0053] Mixing the compound having the structure shown in Formula 2, 3-bromo-1-propanol, a condensing agent, and an organic solvent, and performing an esterification reaction to obtain a compound having the structure shown in Formula 3;

[0054]

[0055] The compound having the structure shown in Formula 3, pyridine and ethanol are mixed and subjected to a quaternization reaction to obtain a fluorescent probe having the structure shown in Formula I.

[0056] The present invention mixes a compound having a structure shown in Formula 1, cyanoacetic acid, piperidine and anhydrous ethanol solvent, and performs a condensation reaction to obtain a compound having a structure shown in Formula 2. In the present invention, the source of the compound having the structure shown in Formula 1 is commercially available or self-prepared. As a specific embodiment of the present invention, when the source of the compound having the structure shown in Formula 1 is commercially available, the CAS number of the compound having the structure shown in Formula 1 is: 57597-64-5, and the purchase manufacturer is preferably Anaiji Chemical. When the source of the compound having the structure shown in Formula 1 is self-prepared, the preparation method preferably includes the following steps:

[0057] mixing 4-(diethylamino)-salicylaldehyde, diethyl malonate, piperidine and ethanol to carry out a first condensation reaction to obtain a first condensation reaction product;

[0058] The first condensation reaction product is mixed with N,N-dimethylformamide containing phosphorus oxychloride to carry out a formylation reaction to obtain a compound having a structure shown in Formula 1.

[0059] The present invention mixes 4-(diethylamino)-salicylaldehyde, diethyl malonate, piperidine and ethanol to perform a first condensation reaction to obtain a first condensation reaction product. In the present invention, the molar ratio of the 4-(diethylamino)-salicylaldehyde to the diethyl malonate is preferably 1:1, and the molar ratio of the 4-(diethylamino)-salicylaldehyde to the piperidine is preferably 1:1. In the present invention, the ethanol is preferably anhydrous ethanol. In the present invention, the temperature of the first condensation reaction is preferably reflux temperature, and the time is preferably 6 hours.

[0060] After the first condensation reaction, the present invention preferably performs post-treatment on the obtained first condensation reaction product, and the post-treatment preferably includes the following steps:

[0061] The first condensation reaction product is cooled and spin-dried, concentrated hydrochloric acid and glacial acetic acid are added and refluxed, the pH value of the resulting system is adjusted to weak acidity with NaOH solution to precipitate a solid, and the resulting solid-liquid mixture is filtered, dried and separated by column chromatography in sequence to obtain a condensation reaction product.

[0062] In the present invention, the reflux time is preferably 6 h, and the concentration of the NaOH solution is preferably 0.1 M. In the present invention, the pH value of the weak acid is specifically 5.

[0063] In the present invention, the first condensation reaction product is mixed with N,N-dimethylformamide containing phosphorus oxychloride, and a formylation reaction is performed to obtain a compound having a structure shown in Formula 1. In the present invention, the method for preparing N,N-dimethylformamide containing phosphorus oxychloride preferably comprises the following steps: phosphorus oxychloride is added dropwise to N,N-dimethylformamide in an ice bath under nitrogen protection for activation for 2 hours until the solution turns light pink or light yellow.

[0064] In the present invention, the temperature of the formylation reaction is preferably reflux temperature, and the time is preferably overnight.

[0065] In the present invention, after the formylation reaction, the present invention preferably performs post-treatment on the obtained formylation reaction liquid, and the post-treatment preferably comprises the following steps:

[0066] The formylation reaction solution is poured into ice water while hot to precipitate a solid, which is then filtered, dried, and recrystallized to obtain a pure compound having the structure shown in Formula 1.

[0067] In the present invention, the solvent used in the recrystallization is preferably anhydrous ethanol.

[0068] The present invention mixes a compound having a structure shown in Formula 1, cyanoacetic acid, piperidine and anhydrous ethanol solvent, and performs a condensation reaction to obtain a compound having a structure shown in Formula 2.

[0069] In the present invention, the molar ratio of the compound having the structure represented by Formula 1 to cyanoacetic acid is preferably 1:4-6, more preferably 1.3:5.87; the molar ratio of the compound having the structure represented by Formula 1 to piperidine is preferably 1:1-1.2, more preferably 1:1.

[0070] In the present invention, the ethanol is preferably anhydrous ethanol. In the present invention, the temperature of the condensation reaction is preferably 78 to 85°C, more preferably 80°C; and the time is preferably 6 to 10 hours, more preferably 8 hours.

[0071] After the condensation reaction, the present invention preferably performs post-treatment on the obtained condensation reaction solution, and the post-treatment preferably includes the following steps:

[0072] The condensation reaction liquid is filtered, and the obtained solid is washed and recrystallized to obtain a pure compound having the structure shown in Formula 2.

[0073] In the present invention, the washing is preferably rinsing, and the detergent used in the washing is preferably n-hexane. In the present invention, the solvent used in the recrystallization is preferably anhydrous ethanol.

[0074] In the present invention, the compound having the structure represented by Formula 2, 3-bromo-1-propanol, a condensing agent, and an organic solvent are mixed and subjected to an esterification reaction to obtain a compound having the structure represented by Formula 3. In the present invention, the molar ratio of the compound having the structure represented by Formula 2 to 3-bromo-1-propanol is preferably 1.1 to 1.5:1, more preferably 1.2 to 1.25:1.

[0075] In the present invention, the condensing agent preferably includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-hydroxybenzotriazole (HOBt). In the present invention, the molar ratio of the compound having the structure shown in Formula 2 to EDC is preferably 1:1, and the molar ratio of the compound having the structure shown in Formula 2 to HOBt is preferably 1:1.

[0076] In the present invention, the organic solvent is preferably dichloromethane. In the present invention, the mixing method is preferably: firstly mix the compound having the structure shown in Formula 2, the condensing agent and the organic solvent, stir at room temperature for 30 minutes, and then add 3-bromo-1-propanol.

[0077] In the present invention, the esterification reaction is preferably carried out under stirring; the temperature of the esterification reaction is preferably room temperature, and the time is preferably 6 to 10 hours, more preferably 7 to 8 hours.

[0078] After the esterification reaction, the present invention preferably performs post-treatment on the obtained esterification reaction liquid, and the post-treatment preferably includes the following steps:

[0079] The esterification reaction liquid was quenched with water, and the obtained quenched liquid was extracted with dichloromethane. The organic phases were combined and purified by silica gel column chromatography to obtain a pure compound having the structure shown in Formula 3.

[0080] After obtaining the compound having the structure represented by Formula 3, the present invention mixes the compound having the structure represented by Formula 3, pyridine, and ethanol, and performs a quaternization reaction to obtain a fluorescent probe having the structure represented by Formula I. In the present invention, the molar ratio of the compound having the structure represented by Formula 3 to pyridine is preferably 1:10-12, more preferably 1:10-11.

[0081] In the present invention, the temperature of the quaternization reaction is preferably reflux temperature, and the time is overnight.

[0082] After the ion exchange, the present invention preferably filters the obtained quaternization reaction solution and washes the obtained solid to obtain a pure fluorescent probe having the structure shown in Formula I. In the present invention, the detergent used in the washing is preferably ethanol.

[0083] The present invention provides the use of the fluorescent probe in detecting metabisulfite. In the present invention, the metabisulfite is preferably sodium metabisulfite.

[0084] The present invention provides a method for detecting pyrosulfite, comprising the following steps:

[0085] Mixing the sample to be tested with water, performing homogenization and solid-liquid separation to obtain a solution of the sample to be tested;

[0086] The sample solution to be tested is mixed with the fluorescent probe solution. If the fluorescent probe solution changes from pink to light yellow under naked eye observation, the sample to be tested contains metabisulfite;

[0087] Alternatively, the sample solution to be tested is mixed with a fluorescent probe solution. If the fluorescence of the fluorescent probe solution changes from orange-red to bright blue, the sample to be tested contains metabisulfite.

[0088] The present invention mixes a sample to be tested with water, performs homogenization, and performs solid-liquid separation to obtain a sample solution to be tested. In the present invention, the sample to be tested is preferably a food or beverage. In the present invention, the mass ratio of the sample to be tested to water is preferably 1:10 to 100, more preferably 1:10 to 50.

[0089] In the present invention, the homogenization method is preferably homogenization; and the solid-liquid separation method is preferably filtration.

[0090] In the present invention, the fluorescent probe solution is preferably an aqueous solution or a PBS solution. In the present invention, the concentration of the fluorescent probe solution is preferably 10 to 30 μM, more preferably 10 to 20 μM; the volume ratio of the sample solution to the fluorescent probe solution is preferably 1:100 to 1000, more preferably 1:100 to 500.

[0091] The present invention mixes the sample solution to be tested with the fluorescent probe solution. In the present invention, the mixing method is preferably to add the sample solution to be tested into the fluorescent probe solution.

[0092] In the present invention, if the fluorescent probe solution changes from pink to light yellow under naked eye observation, the sample to be tested contains metabisulfite. Alternatively, if the fluorescence of the fluorescent probe solution changes from orange-red to bright blue, the sample to be tested contains metabisulfite.

[0093] The present invention provides a method for detecting pyrosulfite, comprising the following steps:

[0094] Mixing the sample to be tested with water, performing homogenization and solid-liquid separation to obtain a solution of the sample to be tested;

[0095] The sample solution to be tested was mixed with the fluorescent probe solution, and the fluorescence intensity of the resulting mixed solution at 572 nm and 481 nm was measured to obtain I 481 nm / I 572nm Ratio, according to the I 481nm / I 572nm The content of metabisulfite in the sample to be tested is obtained by using the ratio and a predetermined standard curve;

[0096] The standard curve is I 481nm / I 572nm The linear relationship curve between the ratio and the concentration of metabisulfite solution.

[0097] In the present invention, the sample to be tested is preferably food or beverage. In the present invention, the mass ratio of the sample to be tested to water is preferably 1:10 to 100, more preferably 1:10 to 50.

[0098] In the present invention, the homogenization method is preferably homogenization; and the solid-liquid separation method is preferably filtration.

[0099] In the present invention, the fluorescent probe solution is preferably an aqueous solution or a PBS solution. In the present invention, the concentration of the fluorescent probe solution is preferably 10 to 30 μM, more preferably 10 to 20 μM; the volume ratio of the sample solution to the fluorescent probe solution is preferably 1:100 to 1000, more preferably 1:100 to 500.

[0100] In the present invention, the excitation wavelength for testing the fluorescence intensity at 572 nm and 481 nm is 443 nm.

[0101] In the present invention, the method for obtaining the standard curve preferably comprises the following steps:

[0102] Provide multiple gradient metabisulfite standard solutions with known concentrations;

[0103] The pyrosulfite standard solution of known concentration is used as the sample solution to be tested, and the pyrosulfite standard solution is mixed with the fluorescent probe solution to obtain the corresponding I of the pyrosulfite standard solution with different concentrations. 481nm / I 572nm Ratio, with the concentration of metabisulfite as the horizontal axis and I 481nm / I 572nm The ratio is used as the vertical axis to draw the standard curve.

[0104] As a specific embodiment of the present invention, the standard curve is y=0.14306x+0.03662; wherein y is the fluorescence intensity ratio of the fluorescent probe at 572nm and 481nm. 481nm / I 572nm , x is the concentration of metabisulfite.

[0105] In the present invention, the detection limit of the metabisulfite is 26 nM, and the linear detection range is 26 nM to 60 μM.

[0106] The fluorescent probe provided by the present invention, its preparation method and its application in detecting metabisulfite are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0107] Example 1

[0108] according to Figure 1 The synthetic route for preparing the fluorescent probe CPSP is as follows:

[0109] (1) Preparation of Compound 1: 4-(Diethylamino)-salicylaldehyde (1.93 g, 10 mmol) was dissolved in anhydrous ethanol (30 mL), and diethyl malonate (3.2 g, 10 mmol) and piperidine (1 ml, 10 mmol) were added and refluxed for 6 h. The reaction was monitored by thin-layer chromatography. After the reaction was completed, the mixture was cooled and the solvent was dried by spin drying. Concentrated hydrochloric acid (20 mL) and glacial acetic acid (20 mL) were directly added and refluxed for 6 h. The reaction system was adjusted to weak acidity with 0.1 M NaOH to precipitate a large amount of light yellow solid, which was filtered, dried, and purified by column chromatography. Formylation: Add phosphorus oxychloride dropwise to purified N,N-dimethylformamide in an ice bath under nitrogen protection and activate for 2 h. The reaction turns light pink or light yellow. Dissolve the product from the previous step in purified N,N-dimethylformamide and add it to the reaction system. Reflux overnight. Pour it into 50 mL of ice water while hot. A large amount of red solid will precipitate. After stirring for 30 min, filter, dry, and recrystallize from anhydrous ethanol to obtain red crystals with a yield of 70%.

[0110] (2) Preparation of compound 2: Compound 1 (320 mg, 1.3 mmol) and cyanoacetic acid (500 mg, 5.87 mmol) were dissolved in anhydrous ethanol, piperidine (130 μL, 1.3 mmol) was added, and the mixture was heated under reflux overnight. The precipitated red solid was filtered, washed with n-hexane, and the solid was recrystallized from anhydrous ethanol. The yield was 83%.

[0111] (3) Preparation of Compound 3: Compound 2 (350 mg, 1.12 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (214.7 mg, 1.12 mmol), and 1-hydroxybenzotriazole (151.3 mg, 1.12 mmol) were dissolved in dichloromethane and stirred at room temperature for 30 min. 3-Bromo-1-propanol (68.2 mg, 0.93 mmol) was added, stirred overnight, and quenched with water. The aqueous solution was extracted with dichloromethane, and the combined organic phases were dried over magnesium sulfate and purified by silica gel column chromatography to obtain 411.2 mg of a red solid in a yield of 73%.

[0112] (4) Preparation of compound CPSP: Compound 3 was dissolved in anhydrous ethanol, and 10 equivalents of pyridine were added. The mixture was heated under reflux overnight. The resulting solid was filtered and washed with ethanol to obtain a red solid, which is the fluorescent probe CPSP.

[0113] Figure 2 This is the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe CPSP of Example 1. 1HNMR (400MHz, DMSO-d6)δ9.15(d,J=5.6Hz,2H),8.76(s,1H),8.61(t,J=7.8Hz,1H),8.17(m,3H),7.64(d,J=9.2Hz,1H),6.87(dd,J=9.1,2.2Hz,1H),6.68(s,1H),4.76(t,J=6.8Hz,2H),4.38(t,J=5.8Hz,2H),3.55(dd,J=13.8,6.9Hz,2H),2.48-2.38(m,2H),1.17(t,J=7.0Hz,6H), proving that the target product was obtained.

[0114] Figure 3 This is the mass spectrum of the fluorescent probe CPSP of Example 1.

[0115] Example 2 Spectral response of fluorescent probe CPSP to Na2S2O5

[0116] Prepare a 1mM stock solution of CPSP. Accurately measure 30μL of the probe stock solution into 2.97mL of PBS to prepare a 10μM probe test solution. Weigh Na2S2O5 and dissolve it in water to prepare a 10mM Na2S2O5 stock solution. Add 3μL of the Na2S2O5 stock solution at a time to obtain test sample solutions of varying concentrations. After incubation for 5 minutes, measure the absorption and emission spectra of the system. Figure 4 and Figure 5 They are the changes of UV-visible absorption spectrum and fluorescence spectrum of fluorescent probe CPSP test solution, Figure 4 and Figure 5 The illustrations show the color changes of the test solutions without and with the addition of Na2S2O5.

[0117] Depend on Figure 4 It can be seen that the probe has the maximum absorption peak at 520nm. 2- With the increase of concentration (0-60 μM), the absorption peak gradually decreases; a new absorption peak is generated at 410 nm. Figure 4 The insets show the presence and absence of S2O5 2- The colorimetric image of the solution changes from pink to pale yellow and can be identified by the naked eye.

[0118] also, Figure 5 It was also observed that S2O5 2- It has a significant ratiometric fluorescence response. 2- After that, the fluorescence emission intensity at 572 nm gradually decreased, and the fluorescence emission intensity at 481 nm gradually increased. Figure 5 The inset shows that the fluorescence of CPSP changes from orange-red to bright blue.

[0119] Figure 6 The fluorescence emission intensity change curves of the fluorescent probe CPSP test solution at 572nm and 481nm as the Na2S2O5 concentration increases; Figure 7 The fluorescence color of the fluorescent probe CPSP test solution changes with the increase of Na2S2O5 concentration.

[0120] Figure 8 The linear relationship between the fluorescence ratio of the fluorescent probe CPSP at 572nm and 481nm and the concentration of Na2S2O5 is shown in the regression curve equation: y = 0.14306x + 0.03662; where y is the fluorescence intensity ratio of the fluorescent probe at 572nm and 481nm. 481nm / I 572nm , x is the concentration of Na2S2O5.

[0121] The sensitivity of the fluorescent probe CPSP for Na₂S₂O₅ was as follows: limit of detection (LOD) = 3σbi / m, where σbi is the standard deviation of fluorescence from 10 scans of the probe test solution, and m is the slope of the fit between emission intensity and probe concentration. The signal-to-noise ratio (S / N) was 3. The LOD was 26 nM, demonstrating the high sensitivity of the fluorescent probe CPSP for Na₂S₂O₅.

[0122] Example 3 Selectivity of fluorescent probe CPSP for different ions

[0123] Prepare a probe test solution with a concentration of 10 μM for later use. 2- ), potassium thiocyanate (SCN - ), sodium thiosulfate (S2O3 2- ), potassium persulfate (S2O8 2- ), sodium sulfate (SO4 2- ), barium sulfide (S 2- ), sodium chloride (Cl - ), sodium carbonate (CO3 2- ), calcium nitrate (NO3 - ) and potassium dihydrogen phosphate (H2PO4 - ) solution. The concentrations of Na2S2O5 and Na2SO3 solutions were 10 mM and 20 mM, respectively, and the other compounds were prepared as 100 mM solutions. 3 μL of each solution was added to the 10 μM CSP test solution described in Example 2, and the fluorescence intensity at 572 nm and 481 nm was measured. Figure 9 The selectivity of the fluorescent probe CPSP to different ions is Figure 9 Samples 1 to 10 correspond to sodium metabisulfite (S2O5 2- ), potassium thiocyanate (SCN -), sodium thiosulfate (S2O3 2- ), potassium persulfate (S2O8 2- ), sodium sulfate (SO4 2- ), barium sulfide (S 2- ), sodium chloride (Cl - ), sodium carbonate (CO3 2- ), calcium nitrate (NO3 - ) and potassium dihydrogen phosphate (H2PO4 - ).like Figure 9 It can be seen that the probe only has 2- Fluorescence will only occur in the presence of .

[0124] Example 4 Response time of fluorescent probe CPSP to Na2S2O5

[0125] 3 μL of the Na2S2O5 solution (concentration of 10 mM) described in Example 2 was added to the 10 μM probe test solution, and the fluorescence change trend over time was observed at 481 nm. Figure 10 After fitting the image, we can get t 1 / 2 =6s, which shows that the fluorescent probe CPSP of the present invention can quickly detect Na2S2O5.

[0126] Example 5 Detection of Na2S2O5 Content in Preserved Fruits Using Fluorescent Probe CPSP

[0127] Accurately weigh 5g of preserved fruit (apricot) and add it to 10mL of ultrapure water. Homogenize and filter to obtain the sample solution. Add 5μL of sample solution to the 10μM probe test solution at a time and measure the fluorescence intensity at the excitation wavelength of 443nm to obtain I 481nm / I 572nm The ratio was substituted into the regression curve equation in Example 2 to calculate the content of Na2S2O5 in the sample. The content of Na2S2O5 in preserved fruit under different sample solution addition amounts is shown in Table 1. The fluorescence intensity of the probe test solution under 0-45 μL sample solution addition amount is shown in Table 1. Figure 11 The final calculated content of Na2S2O5 in preserved fruit was 216.9±6.5mg / kg.

[0128] Table 1 Content of Na2S2O5 in preserved fruit at different sample solution addition amounts

[0129]

[0130] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A fluorescent probe having the structure shown in Formula I:

2. The method for preparing the fluorescent probe according to claim 1, comprising the following steps: Mixing a compound having a structure represented by Formula 1, cyanoacetic acid, piperidine, and anhydrous ethanol solvent to carry out a condensation reaction to obtain a compound having a structure represented by Formula 2; Mixing the compound having the structure shown in Formula 2, 3-bromo-1-propanol, a condensing agent, and an organic solvent, and performing an esterification reaction to obtain a compound having the structure shown in Formula 3; The compound having the structure shown in Formula 3, pyridine and ethanol are mixed and subjected to a quaternization reaction to obtain a fluorescent probe having the structure shown in Formula I.

3. The preparation method according to claim 2, characterized in that The molar ratio of the compound having the structure shown in Formula 1 to cyanoacetic acid is 1:4-6; The molar ratio of the compound having the structure shown in Formula 1 to piperidine is 1:1 to 1.2; The condensation reaction temperature is 78-85° C., and the reaction time is 6-10 hours.

4. The preparation method according to claim 2, characterized in that The molar ratio of the compound having the structure shown in Formula 2 to 3-bromo-1-propanol is 1.1 to 1.5:

1.

5. The preparation method according to claim 2 or 4, characterized in that The condensing agent includes EDC and HOBt; The esterification reaction time is 6 to 10 hours.

6. The preparation method according to claim 2, characterized in that The molar ratio of the compound having the structure shown in Formula 3 to pyridine is 1:10-12; The temperature of the quaternization reaction is 78-85° C., and the time is 6-10 hours.

7. Use of the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 6 in the detection of metabisulfite.

8. A method for detecting metabisulfite, comprising the following steps: Mixing the sample to be tested with water, performing homogenization and solid-liquid separation to obtain a solution of the sample to be tested; The sample solution to be tested is mixed with the fluorescent probe solution. If the fluorescent probe solution changes from pink to light yellow under naked eye observation, the sample to be tested contains metabisulfite; Alternatively, the sample solution to be tested is mixed with a fluorescent probe solution. If the fluorescence of the fluorescent probe solution changes from orange-red to bright blue, the sample to be tested contains metabisulfite. The fluorescent probe is the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 6.

9. A method for detecting metabisulfite, comprising the following steps: Mixing the sample to be tested with water, performing homogenization and solid-liquid separation to obtain a solution of the sample to be tested; The sample solution to be tested was mixed with the fluorescent probe solution, and the fluorescence intensity of the resulting mixed solution at 572 nm and 481 nm was measured to obtain I 481nm / I 572nm Ratio, according to the I 481nm / I 572nm The content of metabisulfite in the sample to be tested is obtained by using the ratio and a predetermined standard curve; The standard curve is I 481nm / I 572nm The linear relationship curve between the ratio and the concentration of metabisulfite solution; The fluorescent probe is the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to any one of claims 2 to 6.

10. The detection method according to claim 9, characterized in that: The detection limit of the metabisulfite is 26 nM, and the linear detection range is 26 nM to 60 μM.

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