Visual fluorescent test paper for detecting sulfur dioxide and its derivatives, preparation method and application thereof

Abstract

The application discloses a visual fluorescent test paper for detecting sulfur dioxide and derivatives thereof, a preparation method and application thereof, and comprises a paper base and an SO2 fluorescent probe loaded on the paper base. The fluorescent probe can break the spiro carbon-oxygen atom covalent bond with the assistance of hydroxyl in the paper base, release the reaction site of SO2 and derivatives thereof, and the change of fluorescence can be used for qualitative and quantitative detection of SO2 and derivatives thereof in traditional Chinese medicines. The test paper prepared based on the SO2 fluorescent molecular probe has the advantages of high accuracy, low cost, convenient carrying, simple detection and the like, and can rapidly and effectively detect the content of SO2 in traditional Chinese medicines.

Description

Technical Field

[0001] This invention relates to the field of sulfur dioxide detection technology, and in particular to a visual fluorescent test strip for detecting sulfur dioxide and its derivatives, its preparation method, and its application. Background Technology

[0002] Sulfur dioxide (SO2) and its derivatives are commonly used as preservatives and antioxidants due to their bleaching, anti-enzymatic browning, antibacterial, and antioxidant properties. Sulfur fumigation is often used in the processing of traditional Chinese medicinal herbs such as wolfberry, angelica, astragalus, and codonopsis. Sulfur fumigation can leave SO2 and its derivatives in the herbs. Unscrupulous merchants, in an effort to prolong the preservation of these herbs, may use repeated sulfur fumigation or excessive amounts of sulfur, resulting in dangerously high levels of SO2 and its derivatives. Excessive accumulation of SO2 and its derivatives, such as sulfites and bisulfites, can alter the properties and chemical composition of the herbs, leading to reduced or lost efficacy and adverse reactions such as asthma, lung cancer, stroke, and migraines. Therefore, a convenient and rapid method for detecting SO2 in traditional Chinese medicine is essential.

[0003] Both domestic and international regulations have clearly defined limits for SO2 residues in medicinal herbs. The Chinese Pharmacopoeia sets the SO2 residue limit at 150 mg / kg for common Chinese medicinal herbs and processed medicinal slices, and stipulates a limit of 400 mg / kg for 10 specific herbs, including yam, atractylodes macrocephala, white peony root, and codonopsis pilosula, and their processed medicinal slices. The United States Pharmacopeia limits the addition of bisulfite as a pharmaceutical excipient to 580 mg / kg. Methods for detecting SO2 in Chinese medicine mainly include acid-base titration, gas chromatography, and ion chromatography. However, these methods suffer from drawbacks such as expensive equipment, complex sample pretreatment, and cumbersome detection procedures, making rapid on-site detection of Chinese medicine samples difficult and lacking universality. Currently, rapid SO2 residue detection kits are mainly based on the pararosaniline hydrochloride method, which generates large amounts of toxic substances, including formaldehyde and mercuric tetrachloride. Therefore, developing a highly sensitive, selective, low-cost, and non-toxic fluorescent test strip to achieve rapid on-site qualitative and quantitative determination of SO2 content in Chinese medicine is crucial. Summary of the Invention

[0004] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide a visual fluorescent test strip for detecting sulfur dioxide and its derivatives, its preparation method, and its application. The fluorescent probe contained in the test strip is a pH-activated SO2 fluorescent probe. Under the action of hydrogen bond donors on the paper, the probe undergoes the breaking of the spirocarbon-oxygen covalent bond, releasing reaction sites for SO2 and its derivatives. SO2 and its derivatives alter the conjugated structure of the probe molecule through these reaction sites, thereby causing changes in the probe's color and fluorescence. This can be used for the qualitative and quantitative detection of SO2 and its derivatives.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The first aspect of the present invention provides a visual fluorescent test strip for detecting sulfur dioxide and its derivatives.

[0007] A second aspect of the present invention provides a method for preparing a visual fluorescent test strip for detecting sulfur dioxide and its derivatives.

[0008] A third aspect of the invention proposes an application of a visual fluorescent test strip for detecting sulfur dioxide and its derivatives.

[0009] According to a first aspect of the present invention, a visual fluorescent test strip for detecting sulfur dioxide and its derivatives is provided, comprising a paper substrate and an SO2 fluorescent probe loaded on the paper substrate, the structure of which is shown in Formula I:

[0010]

[0011] In some embodiments of the present invention, the SO2 fluorescent probe undergoes the breaking of the spirocarbon-oxygen covalent bond under the action of the hydrogen bond donor on the paper substrate, releasing the reaction sites of SO2 and its derivatives. SO2 and its derivatives change the conjugated structure of the probe molecule through the reaction sites, thereby causing changes in the probe color and fluorescence.

[0012] In some embodiments of the present invention, the paper substrate is selected from any one of filter paper, sulfuric acid paper, and fiber membrane.

[0013] In some preferred embodiments of the present invention, the paper substrate further includes a substance capable of donating hydrogen bonds.

[0014] In some preferred embodiments of the present invention, the substance having hydrogen-bonding ability is selected from at least one of polyethylene glycol, gelatin, polypropylene glycol, polyglycerol, and polyacrylic acid.

[0015] According to a second aspect of the present invention, a method for preparing the visual fluorescent test strip described in the first aspect is provided, comprising the following steps:

[0016] The paper substrate is immersed in the SO2 fluorescent probe solution and then dried to obtain the final product.

[0017] In some embodiments of the present invention, the soaking time is 5 min to 15 min.

[0018] In some embodiments of the present invention, the drying temperature is 80°C to 95°C and the drying time is 1 hour to 3 hours.

[0019] In some embodiments of the present invention, the solvent of the SO2 fluorescent probe solution is an organic solvent, which is selected from any one of anhydrous ethanol, anhydrous acetonitrile, and anhydrous methanol.

[0020] In some embodiments of the present invention, the concentration of the SO2 fluorescent probe solution is 10 nM to 1 M.

[0021] In some preferred embodiments of the present invention, the preparation method includes: immersing a paper substrate in a solution of a substance capable of donating hydrogen bonds, drying it, then immersing it in the SO2 fluorescent probe solution, and drying it to obtain the final product.

[0022] In some preferred embodiments of the present invention, the paper substrate is immersed in a solution of a substance capable of donating hydrogen bonds for 5 to 15 minutes.

[0023] In some preferred embodiments of the present invention, the mass concentration of the hydrogen-bonding substance in the solution is 10 wt% to 20 wt%.

[0024] According to a third aspect of the present invention, the application of the visual fluorescent test strip described in the first aspect in the detection of sulfur dioxide residues in traditional Chinese medicine is proposed.

[0025] In some embodiments of the present invention, the detection is qualitative or semi-quantitative.

[0026] In some embodiments of the present invention, when qualitative detection is performed, the method of application includes: placing the sample solution to be tested on the visual fluorescent test paper and observing the color of the test paper under natural light or ultraviolet light; if the color of the visual fluorescent test paper is similar to the color of the test paper with a known concentration of SO2 of 0, it indicates that the sample to be tested does not contain SO2; if the color of the visual fluorescent test paper is different from the color of the test paper with a known concentration of SO2 of 0, it indicates that the sample to be tested contains SO2.

[0027] In some embodiments of the present invention, when the detection is semi-quantitative, the method of application includes: taking the sample solution to be tested and placing it on the visualization fluorescent test paper, observing the color of the test paper under natural light or ultraviolet light and comparing it with the standard test paper under the corresponding environment, thereby obtaining the SO2 concentration in the sample to be tested.

[0028] In some embodiments of the present invention, the preparation method of the standard test strip includes: preparing a series of NaHSO3 aqueous solutions of different concentrations, wherein the concentration of SO2 is calculated as NaHSO3, placing them on the surface of the visual fluorescent test strip respectively, and drying them to obtain the test strip.

[0029] In some embodiments of the present invention, the series of concentrations are 0 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, and 1 M. As the NaHSO3 concentration increases, the color of the test paper changes from blue to light blue, then turns pale yellow at 1 mM, and becomes yellow again as the concentration increases further. The fluorescence color of the test paper gradually weakens from red to pale blue fluorescence, and then changes to a gradient of increasing blue fluorescence as the concentration increases.

[0030] The beneficial effects of this invention are:

[0031] 1. The visual fluorescent test strip provided by this invention can perform qualitative and semi-quantitative detection of SO2 content in traditional Chinese medicine: In the absence of SO2, the test strip shows a distinct blue color; in the presence of SO2, the color reaction of the test strip is affected. As the SO2 concentration increases, the blue color of the test strip gradually weakens, then turns pale yellow at 1 mM, and the yellow color gradually strengthens with increasing SO2 content. Under ultraviolet light irradiation, in the absence of SO2, the test strip shows red fluorescence. With increasing SO2 concentration, the red fluorescence weakens and exhibits a gradient change, reaching blue fluorescence at an SO2 concentration of 10 μM. Furthermore, as the SO2 concentration continues to increase, the blue fluorescence deepens, showing a clear gradient change. The standard colorimetric card prepared according to this invention can be used for qualitative and semi-quantitative analysis of SO2 content in traditional Chinese medicine.

[0032] 2. The visualization fluorescent test strip for detecting SO2 in traditional Chinese medicine provided by this invention has a simple preparation process, readily available raw materials, and high yield, making it suitable for industrial application; it is also portable, convenient to use, and can quickly and effectively detect the SO2 content in traditional Chinese medicine. Attached Figure Description

[0033] Figure 1 For compound I 1 H NMR spectrum;

[0034] Figure 2 For compound I 13 C NMR spectrum;

[0035] Figure 3 Compound I and different concentrations of HSO3 - UV spectrum of the reaction;

[0036] Figure 4 Compound I and different concentrations of HSO3 - Fluorescence emission spectrum of the reaction (blue fluorescence, Em = 420 nm);

[0037] Figure 5 Compound I and different concentrations of HSO3 -Fluorescence emission spectrum of the reaction (red fluorescence, Em = 570 nm);

[0038] Figure 6 The visualization fluorescent test strip prepared in Example 1 under natural light;

[0039] Figure 7 The visualization fluorescent test strip prepared in Example 1 is shown under ultraviolet light;

[0040] Figure 8 The visualization fluorescent test strip prepared in Example 2 is shown under natural light;

[0041] Figure 9 The visualization fluorescent test strip prepared in Example 2 is shown under ultraviolet light;

[0042] Figure 10 The visualization fluorescent test strip prepared in Example 3 is shown under natural light;

[0043] Figure 11 The visualization fluorescent test strip prepared in Example 3 is shown under ultraviolet light;

[0044] Figure 12 The standard colorimetric card for the visualization fluorescent test strip prepared in Example 1;

[0045] Figure 13 The visualization fluorescent test strip prepared in Example 1 was used to detect SO2 residues in traditional Chinese medicine. Detailed Implementation

[0046] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0047] Example 1

[0048] 2 mmol of 1,1,2-trimethyl-1H-benzo[e]indole (compound II) and 2.4 mmol of 2-bromoethanol were heated in 5 mL of anhydrous acetonitrile for 10 h at 82 °C. The reaction system was placed at room temperature, and the polarity was adjusted with ethyl acetate until a large amount of solid precipitated. The mixture was filtered under reduced pressure, and the filter cake was washed 2-3 times with ethyl acetate and dried in a dryer at 65 °C for 3 h to obtain 3-(2-hydroxyethyl)-1,1,2-trimethyl-1H-benzo[e]indole bromide (compound III).

[0049]

[0050] 2 mmol of 7-diethylaminocoumarin (compound IV) was dissolved in 5 mL of N,N-dimethylformamide (DMF), and 200 μL of phosphorus oxychloride (POCl3) was used as a catalyst. The mixture was stirred at 65 °C for 2 h, cooled to room temperature, and the reaction was quenched with ice water. Then, 20 wt% NaOH was added dropwise to adjust the pH until a large amount of precipitate was formed. The pH was measured to be 7 using pH paper. The mixture was filtered under reduced pressure and dried under vacuum to obtain the crude product. The crude product was then recrystallized from ethyl acetate. The recrystallized precipitate was dried in a blower dryer at 65 °C for 2 h to obtain 7-diethylamino-3-aldehyde-coumarin (compound V).

[0051]

[0052] 1 mmol of compound III and 1.1 mmol of compound V were reacted in 5 mL of anhydrous ethanol (EtOH) at 78.9 °C for 10 h. After cooling to room temperature, the solvent was evaporated, and the mixture was recrystallized with ethyl acetate. The mixture was filtered under reduced pressure, dried under vacuum, washed 2-3 times with ethyl acetate, and dried under vacuum to give (E)-2-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)-3-(2-hydroxyethyl)-1,1-dimethyl-1H-benzoindole-3-onium (compound VI).

[0053]

[0054] At room temperature, 1 mmol of compound VI was mixed with 5 mL of saturated sodium carbonate (Na₂CO₃) and 5 mL of ethyl acetate (EA) and stirred for 1 h. After the reaction, the solution was separated by a separatory funnel. The supernatant was evaporated to dryness using a rotary evaporator, and then recrystallized with ethyl acetate. The recrystallized precipitate was dried in a 65°C dryer for 2 h to obtain compound I. 1 H NMR spectrum see Figure 1 , 13 CNMR spectrum see Figure 2 As shown.

[0055]

[0056] Compound I was prepared at a concentration of 1.0 × 10⁻⁶. -5 Prepare a 1 mM sodium bisulfite (NaHSO3) solution by adding 50 mL of a mol / L solution (15 mL of anhydrous ethanol and 35 mL of water). Take 3 mL of the compound I solution into a cuvette, and add 0 μL, 3 μL, 12 μL, 30 μL, 45 μL, and 60 μL of the prepared NaHSO3 solution to the cuvette, respectively, until the NaHSO3 solution is dissolved. - The concentration ratio with compound I was 0–2 equiv. The absorbance of the six solutions was scanned at 230 nm–800 nm using a UV-spectrophotometer, and the results are shown below. Figure 3The absorbance in the near-infrared region at 589 nm increases with the absorption of bisulfite (HSO3). - The absorbance in the blue light region at 400 nm decreases with increasing concentration of bisulfite (HSO3). - The fluorescence intensity increased with increasing concentration. This indicates that the SO2 derivative reacted with compound I, terminating molecular conjugation and weakening the original red fluorescence of the molecule, resulting in the blue fluorescence of coumarin. Six solutions were excited with 400 nm excitation light using a fluorescence-spectrophotometer, and the emission spectra ranged from 420 nm to 750 nm. The results are as follows: Figure 4 The fluorescence is strongest at 473 nm, and the fluorescence increases with HSO3. - The emission intensity increases with increasing concentration. Six groups of solutions were excited using a 550 nm excitation light under a fluorescence spectrophotometer; the emission spectra ranged from 570 nm to 850 nm. The results are as follows: Figure 5 The fluorescence is strongest at 670 nm, and the fluorescence increases with HSO3. - The effect weakens as the concentration increases. Compound I is sensitive to SO2 and its derivatives, exhibiting a significant color change; therefore, using this molecule to prepare test paper is highly feasible.

[0057] Cut the filter paper into 1.5cm × 3cm pieces and dry them in an oven at 80–95℃ for 10 minutes. Then soak them in an aqueous solution for 5–15 minutes, followed by drying them in a forced-air dryer at 80–95℃ for 1–3 hours. Finally, place the dried filter paper in a petri dish and add 50mL of a solution with a concentration of 1.0 × 10⁻⁶. -5 Immerse the sample in a mol / L solution of compound I (prepared with anhydrous ethanol as solvent) for 5–15 min, then remove and dry in an oven at 80–95 °C for 1–3 h to obtain fluorescent test paper.

[0058] The test strip prepared in this embodiment is blue under natural light, and turns slightly bluer after adding water. Adding one drop of 10μM HSO3... - The test strip then turned yellow, see Figure 6 As shown; under ultraviolet light, the test strip itself exhibits red fluorescence, which becomes even redder when water is added, and when one drop of 10μM HSO3 is added... - The test strip then turned blue, see Figure 7 As shown. Figure 6 and Figure 7 The numbers from left to right correspond to blank, water added, and HSO3 added, respectively. - The situation.

[0059] Example 2

[0060] Compound I was prepared according to Example 1. Filter paper was cut into 1.5cm × 3cm pieces and dried in an oven at 80–95°C for 10 min. Then, it was immersed in 15wt% polyethylene glycol for 5–15 min, followed by drying in a forced-air dryer at 80–95°C for 1–3 h. The dried filter paper was then placed in a petri dish, and 50 mL of 1.0 × 10⁻⁶ wt% polyethylene glycol solution was added. -5 Immerse the sample in a mol / L solution of compound I (prepared with anhydrous ethanol as solvent) for 5–15 min, then remove and dry in an oven at 80–95 °C for 1–3 h to obtain fluorescent test paper.

[0061] The test strip prepared in this embodiment is colorless under natural light, turns light blue after adding water, and turns light blue after adding one drop of 10μM HSO3. - The test strip then turned yellow, see Figure 8 As shown; the test strip itself fluoresces red under ultraviolet light, and also fluoresces red when water is added. Adding one drop of 10μM HSO3... - The test strip then turned blue, see Figure 9 As shown. Figure 8 and Figure 9 The numbers from left to right correspond to blank, water added, and HSO3 added, respectively. - The situation.

[0062] Example 3

[0063] Compound I was prepared according to Example 1. Filter paper was cut into 1.5cm × 3cm pieces and dried in an oven at 80–95°C for 10 min. Then, it was soaked in a 20wt% gelatin solution for 5–15 min, followed by drying in a forced-air dryer at 80–95°C for 1–3 h. The dried filter paper was then placed in a petri dish, and 50 mL of a 1.0 × 10⁻⁶ solution was added. -5 Immerse the sample in a mol / L solution of compound I (prepared with anhydrous ethanol as solvent) for 5–15 min, then remove and dry in an oven at 80–95 °C for 1–3 h to obtain fluorescent test paper.

[0064] The test strip prepared in this embodiment is blue under natural light and shows no significant change after adding water. Adding one drop of 10μM HSO3... - The test strip then turned yellow, see Figure 10 As shown; the test strip itself fluoresces red under ultraviolet light, and also fluoresces red when water is added. Adding one drop of 10μM HSO3... - The test strip then turned a very bright blue. Figure 11 As shown. The test paper prepared in this example is thicker than the test papers prepared in Examples 1 and 2. It does not change significantly after being dripped with water. The test paper is more stable, and the color contrast is more obvious and the fluorescence is brighter after reacting with SO2 (and its derivatives). Figure 10 and Figure 11The numbers from left to right correspond to blank, water added, and HSO3 added, respectively. - The situation.

[0065] Test case

[0066] The fluorescent test strips prepared in Example 1 were reacted with different concentrations of SO2, namely 0 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, and 1 M, respectively. Colorimetric cards were then prepared based on these concentrations, and the results are as follows: Figure 12 As shown: the test strip shows a clear color change and a sensitive response.

[0067] The SO2 content in different traditional Chinese medicines was detected using the fluorescent test strip prepared in Example 1: 1g each of tremella, wolfberry, astragalus, ginger slices, licorice, chrysanthemum, ophiopogon japonicus, and angelica sinensis were weighed and soaked in 5mL of water for 5 minutes. Then, one drop of each was placed on the fluorescent test strip, and the color change was observed. The results are as follows: Figure 13 As shown, from left to right, the ingredients are: Tremella fuciformis, Lycium barbarum, Astragalus membranaceus, ginger slices, licorice root, chrysanthemum, Ophiopogon japonicus, Angelica sinensis, and a blank control. It was found that Lycium barbarum and Astragalus membranaceus had high SO2 content. The colorimetric card showed that the SO2 concentration in their aqueous solutions was 1 mM, which translates to 530 mg / kg of SO2 in the tested samples, exceeding the residue limits for traditional Chinese medicine stipulated in the Chinese Pharmacopoeia. In contrast, Tremella fuciformis and Angelica sinensis had low SO2 content. The colorimetric card showed that the SO2 concentrations in their aqueous solutions were 100 nM and 10 nM, respectively, meaning the SO2 content in the tested samples was 0.53 mg / kg for Tremella fuciformis and 0.053 mg / kg for Angelica sinensis, both meeting the standards stipulated in the Chinese Pharmacopoeia.

[0068] The test strips prepared in Examples 2 and 3 can achieve similar or better results than those in Example 1, which will not be elaborated here.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A visual fluorescent test strip for detecting sulfur dioxide and its derivatives, characterized in that, The invention includes a paper substrate and an SO2 fluorescent probe loaded on the paper substrate, the structure of which is shown in Formula I: ； The paper substrate also includes a substance capable of donating hydrogen bonds, which is selected from at least one of polyethylene glycol, gelatin, polypropylene glycol, polyglycerol, and polyacrylic acid.

2. A method for preparing the visual fluorescent test strip according to claim 1, characterized in that, The process includes the following steps: immersing the paper substrate in a solution of a substance capable of donating hydrogen bonds, drying it, then immersing it in the SO2 fluorescent probe solution, and drying it to obtain the final product.

3. The preparation method according to claim 2, characterized in that, The concentration of the SO2 fluorescent probe solution is 10 nM to 1 M.

4. The preparation method according to claim 2, characterized in that, The soaking time is 5 to 15 minutes.

5. The preparation method according to claim 2, characterized in that, The drying temperature is 80-95℃, and the time is 1-3 hours.

6. The application of the visual fluorescent test strip according to claim 1 in the detection of sulfur dioxide residues in traditional Chinese medicine, characterized in that, The detection is qualitative or semi-quantitative.

7. The application according to claim 6, characterized in that, The detection method includes: placing the sample solution to be tested on the visual fluorescent test paper, observing the color of the test paper under natural light or ultraviolet light; if the color of the visual fluorescent test paper is similar to the color of the test paper with a known concentration of SO2 of 0, it indicates that the sample to be tested does not contain SO2; if the color of the visual fluorescent test paper is different from the color of the test paper with a known concentration of SO2 of 0, it indicates that the sample to be tested contains SO2.

8. The application according to claim 6, characterized in that, The detection method includes: placing the sample solution to be tested on the visual fluorescent test paper, observing the color of the test paper under natural light or ultraviolet light and comparing it with the standard test paper under the corresponding environment to obtain the SO2 concentration in the sample to be tested.

9. The application according to claim 8, characterized in that, The preparation method of the standard test strip includes: preparing a series of NaHSO3 solutions of different concentrations, wherein the concentration of SO2 is calculated as NaHSO3, placing them on the surface of the visual fluorescent test strip respectively, and drying them to obtain the test strip.

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