Hongqi Polysaccharide Functionalized Silver Nanoparticle Colorimetric Sensor, Its Preparation Method and Application in Detecting Methimazole

Abstract

The present invention provides a preparation method of a colorimetric sensor of Hedysarum polybotrys polysaccharide-functionalized silver nanoparticles, and the steps are as follows: Heat a silver nitrate solution to 60-90 °C, adjust the pH to 8-14, and quickly add a Hedysarum polybotrys polysaccharide solution all at once under stirring for reaction until the solution changes from transparent to orange-yellow, then stop heating and cool to obtain the colorimetric sensor of Hedysarum polybotrys polysaccharide-functionalized silver nanoparticles. A syringe paper-based colorimetric detection device for methimazole prepared by using the colorimetric sensor of Hedysarum polybotrys polysaccharide-functionalized silver nanoparticles can be used for quantitative detection of methimazole. The detection device is low in cost, simple to operate, high in sensitivity and strong in specificity, and has been successfully applied to the naked-eye colorimetric detection of methimazole in samples such as human and animal sera and human and animal urine. In a real scenario, the quantitative analysis of the colorimetric detection results can be achieved only by observing with the naked eye or by using a smartphone and computer software, or the concentration of methimazole can be estimated by using a colorimetric card.

Description

Technical Field

[0001] The present invention relates to a colorimetric sensor of Hedysarum polybotrys polysaccharide-functionalized silver nanoparticles, a preparation method thereof, and an application thereof in the detection of methimazole. Background Art

[0002] Methimazole, also known as thiamazole, is a commonly used drug for the treatment of hyperthyroidism, which is the second most common disease in the endocrine system diseases of human beings today. It is worth noting that during the treatment process of this drug, due to the narrow therapeutic window of methimazole, when the blood drug concentration of methimazole is too high, it may cause symptoms such as nausea, dizziness, diarrhea, and even nephritis, lupus erythematosus syndrome, and thrombocytopenia in patients. Therefore, it is particularly crucial to monitor the therapeutic drug concentration during the entire treatment process. In order to monitor the accurate concentration of methimazole, a rapid and sensitive detection method has great practical significance.

[0003] The reported methods for the detection of methimazole include spectrophotometry, liquid chromatography tandem mass spectrometry (LC-MS), enzyme-linked immunosorbent assay technology (EMIT), etc. These methods have satisfactory detection sensitivities. However, they inevitably have defects such as time-consuming, complex operation, and dependence on expensive instruments or equipment. These common disadvantages have hindered their use in real-time monitoring.

[0004] Therefore, it is necessary to establish a method with simple operation, low detection limit, high sensitivity, and good selectivity to achieve the purpose of accurately detecting methimazole in real scenarios. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, based on the characteristic that the specific recognition of methimazole by Hedysarum polybotrys polysaccharide-functionalized silver nanoparticles causes aggregation, the present invention provides a colorimetric sensor of Hedysarum polybotrys polysaccharide-functionalized silver nanoparticles, a preparation method thereof, and a detection device for detecting methimazole with a syringe paper-based structure constructed by a colorimetric sensor of Hedysarum polybotrys polysaccharide-functionalized silver nanoparticles, a syringe, and filter paper. The detection device can simply, quickly, sensitively, real-timely, and quantitatively detect methimazole.

[0006] The principle of the present invention is as follows:

[0007] The surface of Hedysari polysaccharide-modified silver nanoparticles (HPS-AgNPs) has abundant hydroxyl and carbonyl groups of polysaccharides, so it has a negative charge on the surface. The dispersed HPS-AgNPs show orange-yellow color, with a particle size of 5-15 nm. They can all pass through the cellulose acetate filter paper with a pore size of 0.22 μm with almost no color residue. The target ligand methimazole has tertiary amino and primary amino groups and has a positive charge in solution, which will attract the negatively charged nanoparticles to each other, resulting in a shortening of the distance between the nanoparticles and aggregation. After aggregation, the average particle size of the nanoparticles increases significantly and cannot pass through the test filter paper gasket - cellulose acetate filter paper and is intercepted, making the filter paper show a certain color, and the color deepens with the increase of the methimazole concentration. Therefore, the concentration of methimazole in the sample can be inferred by observing and measuring the color depth of the test area of the filter paper.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The present invention provides a preparation method of a Hedysari polysaccharide-functionalized silver nanoparticle colorimetric sensor, and the steps are as follows: Heat the silver nitrate solution to 60-90 °C, adjust the pH to 8-14, and quickly add the Hedysari polysaccharide solution all at once under stirring for reaction until the solution changes from transparent to orange-yellow, then stop heating and cool to obtain the Hedysari polysaccharide-functionalized silver nanoparticle colorimetric sensor.

[0010] Preferably, when the concentration of the Hedysari polysaccharide solution is 1 g / L, the volume ratio of the silver nitrate solution to the Hedysari polysaccharide solution is 20 ml:(50-200) μl; the preferred volume ratio is 20 ml:(75-150) μL; and / or

[0011] The concentration of the silver nitrate solution is 0.1-0.5 mM, preferably 0.2-0.3 mM; and / or

[0012] The heating temperature is 70-80 °C; and / or

[0013] The pH value is adjusted to 9-13.

[0014] When the volume ratio of the silver nitrate solution to the Hedysari polysaccharide solution is lower than 20 ml:50 μL, the product cannot be synthesized.

[0015] Preferably, in the synthesis process, the concentration of silver nitrate is 0.25 mM, the temperature of the reaction system is heated to 70 °C, the pH is 10, and the dosage of Hedysari polysaccharide added is 100 μL; or

[0016] In the synthesis process, the concentration of silver nitrate is 0.25 mM, the temperature of the reaction system is heated to 80 °C, the pH is 10, and the dosage of Hedysari polysaccharide added is 100 μL; or

[0017] During the synthesis process, the concentration of silver nitrate is 0.25 mM, the temperature of the reaction system is heated to 80 °C, the pH is 12, and the dosage of Hedysari Radix polysaccharide added is 100 μL.

[0018] Preferably, the extraction method of Hedysari Radix polysaccharide is as follows: after degreasing Hedysari Radix polysaccharide, it is first enzymatically hydrolyzed with a composite enzyme and then subjected to ultrasonic extraction; the addition amount of the composite enzyme is 1.0% of the mass of Hedysari Radix, the enzymatic hydrolysis temperature is 45 °C, and the enzymatic hydrolysis time is 1 h; the composite enzyme is composed of cellulase and papain, and the mass ratio of the two is 2:1; the ultrasonic temperature is 60 °C, the ultrasonic power is 40 W, the ultrasonic time is 30 min, and the ultrasonic treatment is carried out once.

[0019] The present invention also provides a colorimetric sensor of Hedysari Radix polysaccharide-functionalized silver nanoparticles, which is prepared by the method described above.

[0020] The present invention also provides a syringe paper-based colorimetric detection device for methimazole. The device is composed of a colorimetric sensor of Hedysari Radix polysaccharide-functionalized silver nanoparticles, a syringe, and a filter paper; the pore size of the filter paper is 0.22 μm; preferably, the device further includes a filter holder.

[0021] The present invention also provides an application of a colorimetric sensor of Hedysari Radix polysaccharide-functionalized silver nanoparticles in the preparation of products for detecting methimazole; the detection is not for the purpose of disease diagnosis and treatment.

[0022] Preferably, it is an application of a colorimetric sensor of Hedysari Radix polysaccharide-functionalized silver nanoparticles in the preparation of products for detecting methimazole in human serum and urine, and animal serum and urine.

[0023] The present invention also provides a method for establishing a standard curve for detecting methimazole by a syringe paper-based colorimetric detection device. The detection is not for the purpose of disease diagnosis and treatment. A colorimetric sensor of Hedysari Radix polysaccharide-functionalized silver nanoparticles described above is inhaled into a syringe, and then a series of methimazole solutions with known concentrations are inhaled. After mixing evenly, it is left standing until the reaction is complete. The mixed solution in the syringe is slowly pushed out at a speed of 1 mL / min, so that the mixed solution all passes through the filter paper with a pore size of 0.22 μL. The filter paper is naturally dried, and the gray value of the detection area of the filter paper is measured. A standard curve is established with the methimazole concentration as the abscissa and the ratio of the gray values of the detection areas of the series of standard samples and the blank sample filter papers as the ordinate; the concentration range of the series of methimazole solutions with known concentrations in the mixed solution is 0.1 - 10 μL.

[0024] The present invention also provides a method for detecting methimazole in a sample to be tested, and the detection is not for the purpose of diagnosing and treating diseases. The above-mentioned red astragalus polysaccharide-functionalized silver nanoparticle colorimetric sensor is aspirated into a syringe, and then the sample to be tested is aspirated. After mixing evenly, it is left standing until the reaction is complete. The mixed solution in the syringe is slowly pushed out at a speed of 1 mL / min, so that the mixed solution completely passes through a filter paper with a pore size of 0.22 μL. The filter paper is naturally dried, the gray value of the detection area of the filter paper is measured, the ratio of the gray values of the detection areas of the filter paper of the sample to be tested and the blank sample is calculated, and substituted into the standard curve equation to calculate the concentration of methimazole in the sample to be tested.

[0025] The present invention also provides a colorimetric card for methimazole with a series of concentrations. The above-mentioned red astragalus polysaccharide-functionalized silver nanoparticle colorimetric sensor is aspirated into a syringe, and then a series of methimazole solutions with known concentrations are aspirated. After mixing evenly, it is left standing until the reaction is complete. The mixed solution in the syringe is slowly pushed out at a speed of 1 mL / min, so that the mixed solution completely passes through a filter paper with a pore size of 0.22 μL. The filter paper is naturally dried to obtain a colorimetric card for methimazole with a series of concentrations.

[0026] The present invention also provides a method for detecting methimazole in a sample to be tested, and the detection is not for the purpose of diagnosing and treating diseases, and includes the following steps:

[0027] (1) The above-mentioned red astragalus polysaccharide-functionalized silver nanoparticle colorimetric sensor is aspirated into a syringe, and then a series of methimazole solutions with known concentrations are aspirated. After mixing evenly, it is left standing until the reaction is complete. The mixed solution in the syringe is slowly pushed out at a speed of 1 mL / min, so that the mixed solution completely passes through a filter paper with a pore size of 0.22 μL. The filter paper is naturally dried to obtain a colorimetric card for methimazole with a series of concentrations;

[0028] (2) The above-mentioned red astragalus polysaccharide-functionalized silver nanoparticle colorimetric sensor is aspirated into a syringe, and then the sample to be tested is aspirated. After mixing evenly, it is left standing until the reaction is complete. The mixed solution in the syringe is slowly pushed out at a speed of 1 mL / min, so that the mixed solution completely passes through a filter paper with a pore size of 0.22 μL. The filter paper is naturally dried, and the methimazole concentration range is obtained by observing the color of the filter paper and comparing it with the colorimetric card with the naked eye.

[0029] In the present invention, silver nitrate is used as a silver precursor, and the polysaccharide extracted from Hedysari Radix is used as a reducing agent, a stabilizer and a modifier to synthesize silver nanoparticles. Under the conditions of heating and stirring and a certain pH value, a water-soluble polysaccharide-functionalized silver nanoparticle (HPS-AgNPs) colorimetric sensor with stable dispersion and a particle size of 5-15 nm is obtained. When the target substance methylmercaptoimidazole is added to the HPS-AgNPs solution, aggregation occurs, and the particle size of the nanoparticles increases significantly. On this basis, the present invention uses HPS-AgNPs as a substrate for colorimetric sensing and constructs a methylmercaptoimidazole syringe paper-based colorimetric detection device together with a syringe, a filter holder and cellulose acetate filter paper. The experimental results show that HPS-AgNPs has the ability to perform sensitive and highly selective colorimetric detection of methylmercaptoimidazole. Correspondingly, the syringe paper-based colorimetric detection device also has good detection performance for methylmercaptoimidazole. The standard curve for detecting methylmercaptoimidazole by this device is calculated as y = 1 + 0.17851x, and the detection limit is as low as 24.6 nM. Using this device in combination with a smartphone and software can achieve qualitative and quantitative analysis of methylmercaptoimidazole. The detection device is inexpensive, easy to operate, highly sensitive and specific, and has been successfully applied to the naked-eye colorimetric detection of methylmercaptoimidazole in complex samples such as human and animal sera and human and animal urine. In a real scenario, quantitative analysis of the colorimetric detection results can be achieved simply by observing with the naked eye or by using a smartphone and computer software, or the concentration of methylmercaptoimidazole can be estimated using a colorimetric card. The sensor has the advantages of being easy to carry, highly sensitive, selective and stable, and has been successfully applied to the detection of methylmercaptoimidazole in complex samples such as human and animal sera and human and animal urine, and has broad prospects and high market value. The above detection of methylmercaptoimidazole in samples is not for the purpose of disease diagnosis and treatment, such as for scientific research analysis. Description of the Drawings

[0030] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention, and do not constitute a limitation to the present invention. In the drawings:

[0031] Figure 1 It is a schematic diagram of a syringe paper-based colorimetric detection device.

[0032] Figure 2 They are photos and ultraviolet absorption spectra of HPS-AgNPs synthesized with different input amounts (50, 100, 150, 250, 500, 1000, 2000 μL) of Hedysari polysaccharide (1 g / L).

[0033] Figure 3 They are photos and ultraviolet absorption spectra of HPS-AgNPs in the presence of different concentrations (0.1, 1, 4, 6, 7.5, 10, 25, 50, 75 μM) of methylmercaptoimidazole in the mixed solution.

[0034] Figure 4 The syringe paper-based colorimetric detection device of the present invention is used for detecting the results photos and standard curves of different concentrations of methimazole.

[0035] Figure 5 The methimazole concentration colorimetric card is made by the syringe paper-based colorimetric detection device according to the detection results.

[0036] Figure 6 The syringe paper-based colorimetric detection device is used for the results photos and standard curves of methimazole detection in human serum samples (A) and urine samples (B).

[0037] Figure 7 The syringe paper-based colorimetric detection device is used for the results photos and standard curves of methimazole detection in rat serum samples (A) and urine samples (B).

[0038] Figure 8 Photos, ultraviolet spectra and bar charts comparing absorbances at a wavelength of 401 nm of HPS-AgNPs in the presence of different substances (methimazole, ampicillin, clarithromycin, erythromycin, paracetamol, starch, glucose, fructose, sucrase, maltose, sorbitol, lactose, 100 μM). Detailed implementation manners

[0039] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available unless otherwise specified.

[0040] Materials / Reagents: Silver nitrate (AgNO3), sodium hydroxide (NaOH) were purchased from Shanghai Sigma-Aldrich Trading Co., Ltd. All glassware required for the experiment was soaked in aqua regia for 12 h, then washed with distilled water and dried for later use.

[0041] The Hedysarum polysaccharide can be prepared by using the common Hedysarum polysaccharide extraction method in the prior art. Preferably, refer to the method in Example 1 of the patent CN201810560882.5, "A method for modifying Hedysarum polysaccharide and applications of the modified Hedysarum polysaccharide", and the preparation method is as follows:

[0042] (1) The Hedysarum medicinal materials were washed, crushed and sieved through an 80-mesh sieve, defatted once with 3 times the amount of ethyl acetate and 85% ethanol respectively, 0.5 h each time, and then dried.

[0043] (2) Weigh 1.0 g of defatted Hedysari Radix dry powder, add 10 mL of tap water, then adjust the pH to 5 with citric acid - sodium citrate buffer solution. Perform enzymatic hydrolysis with a complex enzyme. The addition amount of the complex enzyme is 1.0% of the mass of Hedysari Radix. The enzymatic hydrolysis temperature is 45 °C, and the enzymatic hydrolysis time is 1 h. Inactivation is not required after enzymatic hydrolysis. The complex enzyme is composed of cellulase and papain, and the mass ratio of the two is 2:1.

[0044] (3) After enzymatic hydrolysis with the complex enzyme, perform ultrasonic treatment. The ultrasonic temperature is 60 °C, the ultrasonic power is 40 W, the ultrasonic time is 30 min, and the ultrasonic treatment is carried out once. Then centrifuge, concentrate the filtrate to 1 / 3 of the original volume of the filtrate, add ethanol to make the volume concentration of ethanol reach 70%, let it stand overnight at 4 °C, centrifuge to obtain a precipitate, and vacuum freeze-dry the precipitate to obtain Hedysari Radix polysaccharide. The polysaccharide yield is 16%, and the polysaccharide content is 95%.

[0045] The Hedysari Radix polysaccharide obtained by the above method has relatively high polysaccharide yield and polysaccharide content. Of course, in addition to the above method, Hedysari Radix polysaccharide can also be extracted by the traditional hot water extraction method. The extraction steps are as follows:

[0046] Traditional hot water extraction method: Take 1.0 g of defatted medicinal powder, add 10 mL of tap water, soak at 60 °C for 3 times, each time for 1 h, filter, combine the filtrates, concentrate the filtrate to 1 / 3 of the original volume of the filtrate, add ethanol to make the volume concentration of ethanol 70%, let it stand overnight at 4 °C, centrifuge to obtain a precipitate, and vacuum freeze-dry the precipitate to obtain the Hedysari Radix polysaccharide extracted by hot water. The polysaccharide yield is 6%, and the polysaccharide content is 80%.

[0047] I. Preparation of the functionalized silver nanoparticles colorimetric sensor of Hedysari Radix polysaccharide (HPS-AgNPs colorimetric sensor) of the present invention is as follows:

[0048] Add 20 mL of silver nitrate solution to a 200 mL round-bottom flask, stir and heat it to 60 - 90 °C with a magnetic stirring heating jacket. Add sodium hydroxide solution to adjust the pH of the solution to 8 - 14. Under magnetic stirring, quickly add 50 - 200 μL of Hedysari Radix polysaccharide solution (1 g / L) to the mixed solution at one time. After constant temperature heating and stirring for 1 h, the color of the solution gradually changes from transparent to orange-yellow. Stop heating and let the product solution cool at room temperature to obtain an orange-yellow solution, which is the HPS-AgNPs colorimetric sensor. Pour the obtained solution into a triangular flask or beaker and store it in a 4 °C refrigerator for standby.

[0049] Among them, the concentration of the silver nitrate solution is 0.1 mM to 0.5 mM, preferably in the range of 0.2 - 0.3 mM.

[0050] The dosage of silver precursor is a key parameter in the synthesis process of nanoparticles and has an important influence on the particle size and morphology of the obtained nanoparticles. To optimize the reactant dosage, while maintaining the temperature of the reaction system at 70 °C and the pH at 10, 100 μL of the dosage of Hedysari polysaccharide was added, and the HPS-AgNPs synthesized using different concentrations of AgNO3 were compared. It was found that when the concentration of AgNO3 was between 0.1 mM and 0.5 mM, the color of the HPS-AgNPs solution deepened with the increase of the AgNO3 concentration, the characteristic ultraviolet absorption intensity increased, the concentration and particle size of the nanoparticle solution both increased. When the AgNO3 concentration was in the range of 0.2 - 0.3 mM, the color and particle size of the nanoparticle solution were moderate. When the AgNO3 concentration was 0.25 mM, the particle size and color depth of the product were the most suitable.

[0051] The heating temperature of the reaction system is 60 - 90 °C, and the preferred heating temperature is 70 - 80 °C.

[0052] The reaction temperature is an important factor affecting the experimental rate. Keeping other experimental conditions unchanged, with the concentration of silver nitrate being 0.25 mM, the pH being 10, and 100 μL of the dosage of Hedysari polysaccharide added, the reaction conditions and the product HPS-AgNPs under different temperature conditions were compared. It was found that when the temperature was below 50 °C, the color of the reaction mixture hardly changed within one hour, indicating that the synthesis of nanoparticles could not be promoted below 50 °C. When the temperature was above 60 °C, the color change rate of the reaction mixture accelerated and turned orange-yellow within a few minutes. However, when the temperature was above 90 °C, the reaction became too violent, and the evaporation of water caused the product to concentrate and the color to be too deep. The color and particle size of the nanoparticle product produced under the condition of 80 °C were the most suitable. Therefore, 60 °C - 90 °C is the preferred reaction temperature, and 70 °C - 80 °C is selected as the optimal reaction temperature.

[0053] The pH value of the reaction system is 8 - 14, and the pH value is preferably adjusted to 9 - 13.

[0054] During the synthesis of HPS-AgNPs, the pH value of the reaction solution has a great influence on the progress of the reaction and the morphological characteristics of the products. Keeping other experimental conditions unchanged, the temperature of the reaction system was 80 °C, the concentration of silver nitrate was 0.25 mM, and the dosage of Hedysarum polybotrys polysaccharide added was 100 μL. The effect of acidity on the synthesis was studied in the pH range of 2-14. When the pH value was in the range of 2-6, the color of the mixed solution hardly changed after 1 h of reaction, and no characteristic absorption peak appeared in the ultraviolet spectrum. The synthesis of HPS-AgNPs was not suitable under acidic conditions. Under alkaline conditions with a pH value in the range of 8-14, the color of the reaction solution gradually changed to orange-yellow within a few minutes. With the increase of alkalinity, the ultraviolet absorption peak of the solution showed a certain degree of red shift. It can be seen that alkaline conditions are more suitable for the synthesis of HPS-AgNPs, and with the increase of alkalinity, the particle size of the nanoparticles increases slightly, and the product properties of the nanoparticles produced at pH 12 are the best. The reason may be that under alkaline conditions, AgNO3 is converted into silver oxide insoluble in water. Silver oxide can slowly release Ag + , which is beneficial to the uniform growth of nanoparticles and improves their monodispersity. However, when the concentration of silver oxide is too high, the reaction rate exceeds the nucleation rate, and large-sized HPS-AgNPs are produced accordingly. According to the above experimental results, pH 8-14 was selected as the preferred pH value range, and pH 9-13 was selected as the best acid-base condition to obtain HPS-AgNPs with a high concentration and uniform particle size.

[0055] The following are several optimal preferred examples of this application. Under the following conditions, HPS-AgNPs with a high concentration, moderate and uniform color and particle size of the nanoparticle solution can be obtained:

[0056] Preferably, the concentration of silver nitrate in the synthesis process is 0.25 mM, the temperature of the reaction system is heated to 70 °C, the pH is 10, and the dosage of Hedysarum polybotrys polysaccharide added is 100 μL.

[0057] Preferably, the concentration of silver nitrate in the synthesis process is 0.25 mM, the temperature of the reaction system is heated to 80 °C, the pH is 10, and the dosage of Hedysarum polybotrys polysaccharide added is 100 μL.

[0058] Preferably, the concentration of silver nitrate in the synthesis process is 0.25 mM, the temperature of the reaction system is heated to 80 °C, the pH is 12, and the dosage of Hedysarum polybotrys polysaccharide added is 100 μL.

[0059] Example 1

[0060] I. The preparation of the Hedysarum polybotrys polysaccharide-functionalized silver nanoparticle colorimetric sensor (HPS-AgNPs colorimetric sensor) of the present invention is as follows:

[0061] Add 20 mL of 0.25 mM silver nitrate solution to a 200 mL round-bottom flask, stir and heat it to 70 °C with a magnetic stirring heating mantle, add sodium hydroxide solution to adjust the pH of the solution to 12, and quickly add 100 μL of Hedysarum polybotrys polysaccharide solution (1 g / L) to the mixed solution at one time under magnetic stirring. After heating and stirring for 1 h, the color of the solution gradually changes from transparent to orange-yellow. Stop heating and let it stand at room temperature until the product solution cools down to obtain an orange-yellow solution, which is the HPS-AgNPs colorimetric sensor. Pour the obtained solution into an Erlenmeyer flask or beaker and store it in a 4 °C refrigerator for later use.

[0062] II. Construction of the syringe paper-based detection device:

[0063] Materials / Reagents: The HPS-AgNPs colorimetric sensor prepared in Step 1; cellulose acetate filter paper with a pore size of 0.22 μm was purchased from Shanghai Xingya Materials Co., Ltd.; a 2.5 mL syringe was purchased from Jiangsu Zhiyu Medical Equipment Co., Ltd.; a filter holder was purchased from Ruixing Experimental Equipment Co., Ltd.

[0064] Construction method: As Figure 1 shown, the device for detecting methimazole consists of three parts: a 2.5 mL syringe, a reusable filter holder, and a circular cellulose acetate filter paper. Aspirate the prepared HPS-AgNPs solution (450 μL) as the colorimetric substrate into the 2.5 mL syringe, cut the whole cellulose acetate filter paper into a circle with a diameter of 10 mm, place the circular filter paper into the filter holder, fix and tighten it with a washer, and finally connect the filter holder with the filter paper to the syringe needle.

[0065] Figure 1 It is a schematic diagram of the syringe paper-based colorimetric detection device.

[0066] III. Compatibility of products obtained with different amounts of Hedysarum polybotrys polysaccharide input in the colorimetric detection equipment

[0067] Since the amount of Hedysarum polybotrys polysaccharide input will affect the particle size and morphology of the synthesized HPS-AgNPs product, only nanoparticles with a smaller particle size can pass through the filter paper with a pore size of 0.22 μm without leaving a trace and having no impact on the experiment. Therefore, it is necessary to investigate the compatibility of products obtained with different amounts of Hedysarum polybotrys polysaccharide input in the colorimetric detection equipment.

[0068] Materials / Reagents: According to the synthesis method of the HPS-AgNPs colorimetric sensor in Step 1, while keeping other conditions unchanged, change the input amount of Hedysarum polybotrys polysaccharide (1 g / L) (50, 100, 150, 250, 500, 1000, 2000 μL) to obtain different HPS-AgNPs colorimetric sensors.

[0069] Method: Use a syringe to inhale 450 μL of HPS-AgNPs solution synthesized with different input amounts of Hedysari Radix Polysaccharide, then inhale 50 μL of the blank sample. Connect the needle to a filter, and slowly push out the liquid in the syringe at a speed of 1 mL / min. All the liquid passes through the filter paper in the filter. Take out the filter paper and let it dry, and observe the color of the filter paper with the naked eye.

[0070] Results: From Figure 2 It can be seen that when the input amount of Hedysari Radix Polysaccharide is 50 μL or 100 μL, there is no color residue on the filter paper, and it almost remains the original pure white. When the input amount of Hedysari Radix Polysaccharide is greater than or equal to 150 μL, the filter paper shows orange to dark brown, indicating that a large amount of silver nanoparticles remain on the filter paper. The darker color will cause great interference to the detection experiment. Therefore, an input amount of Hedysari Radix Polysaccharide below 100 μL is more appropriate.

[0071] Figure 2 Photos and ultraviolet absorption spectra of HPS-AgNPs synthesized with different input amounts (50, 100, 150, 250, 500, 1000, 2000 μL) of Hedysari Radix Polysaccharide (1 g / L).

[0072] IV. Response of HPS-AgNPs to Different Concentrations of Methimazole

[0073] To confirm the ability of HPS-AgNPs to detect methimazole, it is necessary to investigate the response of HPS-AgNPs to different concentrations of methimazole.

[0074] Materials / Reagents: HPS-AgNPs; Methimazole was purchased from Shanghai Sigma-Aldrich Reagent Co., Ltd.

[0075] Method: Take 10 ampoules, add 450 μL of the HPS-AgNPs prepared in the first step to each ampoule, and then add different concentrations (1, 10, 40, 60, 75, 100, 250, 500, 750 μM), 50 μL of methimazole aqueous solution to obtain a mixed solution. After vortexing and mixing the mixed solution, let it stand for 1 min until the color no longer changes, and detect the sample solution with a UV spectrophotometer.

[0076] Results: From Figure 3It can be seen that when the concentration range of methimazole in the mixed solution is 0 - 10 μM, the color gradually changes from orange-yellow to brown, and the color depth deepens with the increase of methimazole concentration. When the concentration of methimazole in the mixed solution is higher than 10 μM, the color of the solution remains dark brown and does not change any more, indicating that the detection limit has been reached. From the results of ultraviolet spectrophotometry, it can be seen that the characteristic absorption peak of HPS-AgNPs with the addition of blank samples appears at about 401 nm. The absorbance at 401 nm gradually decreases with the increase of methimazole concentration, and the peak shape changes from sharp to smooth. This is due to the addition of methimazole target substances causing the aggregation of HPS-AgNPs, resulting in changes in their particle size, morphology and absorbance characteristics. After the methimazole concentration is greater than 10 μM, the absorption intensity of HPS-AgNPs no longer changes with the concentration. It shows that the absorbance of HPS-AgNPs at 401 nm is correlated with the concentration of methimazole (0.1 - 10 μM) in the sample solution.

[0077] Figure 3 Photographs and ultraviolet absorption spectra of HPS-AgNPs in the presence of different concentrations (0.1, 1, 4, 6, 7.5, 10, 25, 50, 75 μM) of methimazole in the mixed solution.

[0078] V. Establishment of the standard curve for the detection of methimazole by the syringe paper-based colorimetric detection device

[0079] Materials / Reagents: Syringe paper-based colorimetric sensor; HPS-AgNPs; Methimazole was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; Human serum and urine samples were collected from healthy volunteers in the School of Pharmacy of Lanzhou University, China, and animal serum and urine samples were collected from healthy rats.

[0080] Method: According to the synthesis method of the HPS-AgNPs colorimetric sensor in Step 1, use a syringe to aspirate 450 μL of the HPS-AgNPs prepared in Step 1 respectively, and then aspirate 50 μL of methimazole sample solutions with concentrations of 0, 1, 10, 40, 60, 75, 100 μM respectively. After shaking and mixing evenly, let it stand for 1 min until the reaction is complete. Then connect the needle to the filter implanted with cellulose acetate filter paper, and slowly push out the liquid in the syringe at a speed of 1 mL / min to make the sample solution all pass through the filter paper in the filter. Use tweezers to take out the filter paper from the filter and let it dry naturally. Observe the color of the filter paper with the naked eye, then take a photo with a smartphone or camera and process the resulting picture with the professional image processing software ImageJ to obtain the gray value of the detection area of the filter paper. Establish a standard curve of the ratio of the gray value of the detection sample to the gray value of the blank sample and the concentration of methimazole, and quantify the color depth of the detection area by measuring the gray intensity of the detection area of the filter paper. Establish a colorimetric card of the color of the paper-based colorimetric area and the concentration of methimazole according to the experimental results.

[0081] Results: As Figure 4 shown, a standard curve was established with the concentration of methimazole as the abscissa and the ratio of the gray value of the detection area of the test sample to that of the blank sample filter paper as the ordinate. It can be seen from the figure that there is a linear relationship in the range of 0.1 μM - 10 μM of methimazole concentration in the sample solution. The fitted linear equation is y = 1 + 0.17851x, and the correlation coefficient R 2 is 0.97913. The detection limit was calculated to be 24.6 nM, which can be used for the quantitative detection of methimazole.

[0082] Figure 4 This is the result photo and standard curve of the syringe-based colorimetric detection device of the present invention for detecting different concentrations of methimazole.

[0083] Figure 5 The methimazole concentration colorimetric card was established according to the experimental results. In the actual scenario, the experimenter can obtain the methimazole concentration range by comparing the color of the filter paper with the colorimetric card, or first establish a standard curve through a smartphone or camera and ImageJ software to obtain a more accurate methimazole concentration value.

[0084] Figure 5 This is the methimazole concentration colorimetric card made by the syringe-based colorimetric detection device according to the detection results.

[0085] VI. Detection of actual samples

[0086] Human serum samples and human urine samples were collected from healthy volunteers in the School of Pharmacy of Lanzhou University, China. Animal serum samples and animal urine samples were collected from rats. Various samples were pretreated as follows: an appropriate amount of acetonitrile solution was added to the urine samples, centrifuged to remove proteins, and diluted 10-fold. The blood samples were allowed to stand for several hours to separate the serum. 5% (v:v) perchloric acid solution was added to 0.5 mL of serum samples for protein precipitation. After centrifugation at 8000 r / min for 10 min, the supernatant was collected, and a certain volume of NaOH (0.1 M) was added to adjust the sample to neutral. Before use, the pretreated serum solution was diluted 5-fold and filtered through a 0.22 μm filter membrane.

[0087] After pretreatment, human serum, human urine samples, rat serum, and rat urine samples were added with different concentrations of methimazole and detected using the above method.

[0088] Results: As Figure 6 can be seen, a standard curve was established with the concentration of methimazole as the abscissa and the ratio of the gray value of the detection area of the test sample to that of the blank sample filter paper as the ordinate. For the detection of human serum samples, it can be seen from the figure that there is a linear relationship in the range of 0.1 μM - 10 μM of methimazole concentration, which conforms to the linear regression equation: y = 1 + 0.27942x, R2 = 0.977, the naked-eye detection limit is 100 nM, and the LOD is 65.3 nM. Similarly, for urine samples, the regression equation was calculated as y = 1 + 0.25066x, and the correlation coefficient R 2 was 0.989, the naked-eye detection limit was 100 nM, and the LOD value was 52.6 nM. For rat serum samples, the regression equation was calculated as y = 1 + 0.27942x, and the correlation coefficient R 2 was 0.97733. For rat urine samples, the regression equation was calculated as y = 1 + 0.25066x, and the correlation coefficient R 2 was 0.98952( Figure 7 ).

[0089] Figure 6 are the photos of the detection results and the standard curves of methimazole in human serum samples (A) and urine samples (B) by the syringe paper-based colorimetric detection device.

[0090] Figure 7 are the photos of the detection results and the standard curves of methimazole in rat serum samples (A) and urine samples (B) by the syringe paper-based colorimetric detection device.

[0091] VII. Verification of the experimental system:

[0092] Materials / Reagents: Syringe paper-based colorimetric detection device; HPS-AgNPs; Methimazole, Ampicillin, Clarithromycin, Erythromycin, Paracetamol were purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; Starch, Glucose, Fructose, Sucrase, Maltose, Sorbitol, Lactose were purchased from Tianjin Baishi Chemical Co., Ltd.

[0093] Selectivity experiment

[0094] Take 12 ampoules, add 450 μL of HPS-AgNPs to each vial, and then add 50 μL of interfering substances such as drugs and biomolecules that may be present in biological samples (Ampicillin, Clarithromycin, Erythromycin, Paracetamol, Starch, Glucose, Fructose, Sucrase, Maltose, Sorbitol, Lactose, 100 μM), as well as 50 μL of methimazole at a concentration of 10 μM. Shake well and let stand for 1 min for detection, and measure its ultraviolet absorption at wavelengths from 300 nm to 800 nm. Calculate the difference in absorbance values at 401 nm between the above detection samples and the blank control, and establish a bar chart for comparison.

[0095] Results: As Figure 8As shown, only in the presence of methimazole did the HPS-AgNPs exhibit an obvious color change and spectral shape change observable by the naked eye. The difference in absorbance at a wavelength of 401 nm from the blank sample was much greater than that of other samples, indicating that these substances would not interfere with the experimental results for the detection of methimazole.

[0096] Figure 8 Photographs of HPS-AgNPs, UV spectra, and a bar graph comparing absorbances at a wavelength of 401 nm in the presence of different substances (methimazole, ampicillin, clarithromycin, erythromycin, paracetamol, starch, glucose, fructose, sucrase, maltose, sorbitol, lactose, 100 μM).

[0097] Example 2

[0098] The preparation of the Hedysari polysaccharide-functionalized silver nanoparticle colorimetric sensor (HPS-AgNPs colorimetric sensor) of the present invention is as follows:

[0099] Add 20 ml of 0.25 mM silver nitrate solution to a 200 mL round-bottom flask, stir and heat with a magnetic stirring heating mantle to 70 °C, add sodium hydroxide solution to adjust the pH of the solution to 10, and quickly add 100 μL of Hedysari polysaccharide solution (1 g / L) to the mixed solution at one time under magnetic stirring. After heating and stirring for 1 h, the color of the solution gradually changes from transparent to orange-yellow. Stop heating and let the product solution cool at room temperature to obtain an orange-yellow solution, which is the HPS-AgNPs colorimetric sensor. Pour the obtained solution into an Erlenmeyer flask or beaker and store it in a 4 °C refrigerator for later use.

[0100] Example 3

[0101] The difference between the present invention and Example 2 is that the temperature of the reaction system is heated to 80 °C, and the remaining steps and parameters are the same as those in Example 2.

[0102] Example 4

[0103] The difference between the present invention and Example 2 is that the temperature of the reaction system is heated to 80 °C and the pH is 12, and the remaining steps and parameters are the same as those in Example 2.

[0104] Example 5

[0105] The preparation of the Hedysari polysaccharide-functionalized silver nanoparticle colorimetric sensor (HPS-AgNPs colorimetric sensor) of the present invention is as follows:

[0106] Add 20 ml of 0.1 mM silver nitrate solution to a 200 mL round-bottom flask, stir and heat it to 60 °C with a magnetic stirring heating mantle. Add sodium hydroxide solution to the solution to adjust the pH of the solution to 9. Under magnetic stirring, quickly add 100 μL of Hedysarum polysaccharide solution (1 g / L) to the mixed solution at one time. After heating and stirring for 1 h, the color of the solution gradually changes from transparent to orange-yellow. Stop heating and let it stand at room temperature until the product solution cools down to obtain an orange-yellow solution, which is the HPS-AgNPs colorimetric sensor. Pour the obtained solution into an Erlenmeyer flask or a beaker and store it in a 4 °C refrigerator for later use.

[0107] Example 6

[0108] The preparation of the Hedysarum polysaccharide-functionalized silver nanoparticle colorimetric sensor (HPS-AgNPs colorimetric sensor) of the present invention is as follows:

[0109] Add 20 ml of 0.5 mM silver nitrate solution to a 200 mL round-bottom flask, stir and heat it to 90 °C with a magnetic stirring heating mantle. Add sodium hydroxide solution to the solution to adjust the pH of the solution to 14. Under magnetic stirring, quickly add 50 μL of Hedysarum polysaccharide solution (1 g / L) to the mixed solution at one time. After heating and stirring for 1 h, the color of the solution gradually changes from transparent to orange-yellow. Stop heating and let it stand at room temperature until the product solution cools down to obtain an orange-yellow solution, which is the HPS-AgNPs colorimetric sensor. Pour the obtained solution into an Erlenmeyer flask or a beaker and store it in a 4 °C refrigerator for later use.

[0110] Example 7

[0111] The preparation of the Hedysarum polysaccharide-functionalized silver nanoparticle colorimetric sensor (HPS-AgNPs colorimetric sensor) of the present invention is as follows:

[0112] Add 20 ml of 0.2 mM silver nitrate solution to a 200 mL round-bottom flask, stir and heat it to 80 °C with a magnetic stirring heating mantle. Add sodium hydroxide solution to the solution to adjust the pH of the solution to 11. Under magnetic stirring, quickly add 60 μL of Hedysarum polysaccharide solution (1 g / L) to the mixed solution at one time. After heating and stirring for 1 h, the color of the solution gradually changes from transparent to orange-yellow. Stop heating and let it stand at room temperature until the product solution cools down to obtain an orange-yellow solution, which is the HPS-AgNPs colorimetric sensor. Pour the obtained solution into an Erlenmeyer flask or a beaker and store it in a 4 °C refrigerator for later use.

[0113] Example 8

[0114] The preparation of the Hedysarum polysaccharide-functionalized silver nanoparticle colorimetric sensor (HPS-AgNPs colorimetric sensor) of the present invention is as follows:

[0115] Add 20 ml of 0.3 mM silver nitrate solution to a 200 mL round-bottom flask, stir and heat it to 70 °C with a magnetic stirring heating mantle. Add sodium hydroxide solution to adjust the pH of the solution to 13. Under magnetic stirring, quickly add 50 μL of Hedysarum polysaccharide solution (1 g / L) to the mixed solution at one time. After heating and stirring for 1 h, the color of the solution gradually changes from transparent to orange-yellow. Stop heating and let the product solution cool at room temperature to obtain an orange-yellow solution, which is the HPS-AgNPs colorimetric sensor. Pour the obtained solution into an Erlenmeyer flask or beaker and store it in a refrigerator at 4 °C for standby.

[0116] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of a Hedysari polysaccharide-functionalized silver nanoparticle colorimetric sensor in a product for detecting methimazole, characterized in that: A detection device based on methimazole, which consists of a colorimetric sensor of Hedysarum polysaccharide-functionalized silver nanoparticles, a syringe and a filter paper; the pore size of the filter paper is 0.22 μm; The preparation method of the colorimetric sensor of Hedysarum polysaccharide-functionalized silver nanoparticles is as follows: heat the silver nitrate solution to 60-90 °C, adjust the pH to 8-14, and quickly add the Hedysarum polysaccharide solution all at once under stirring for reaction until the solution changes from transparent to orange-yellow, then stop heating and cool to obtain the colorimetric sensor of Hedysarum polysaccharide-functionalized silver nanoparticles; when the concentration of the Hedysarum polysaccharide solution is 1 g / L, the volume ratio of the silver nitrate solution to the Hedysarum polysaccharide solution is 20 ml:(50-200) μl.

2. The application according to claim 1, wherein: When the concentration of the Hedysarum polysaccharide solution is 1 g / L, the volume ratio of the silver nitrate solution to the Hedysarum polysaccharide solution is 20 ml:(75-150) μL.

3. The application according to claim 1, wherein: The concentration of the silver nitrate solution is 0.1-0.5 mM; and / or The heating temperature is 70-80 °C; and / or The pH is adjusted to 9-13.

4. The application according to claim 3, wherein: The concentration of the silver nitrate solution is 0.2-0.3 mM.

5. The application according to claim 4, characterized in that: The concentration of silver nitrate is 0.25 mM, the temperature of the reaction system is heated to 70 °C, the pH is 10, and the dosage of Hedysarum polysaccharide added is 100 μL; or The concentration of silver nitrate is 0.25 mM, the temperature of the reaction system is heated to 80 °C, the pH is 10, and the dosage of Hedysarum polysaccharide added is 100 μL; or The concentration of silver nitrate is 0.25 mM, the temperature of the reaction system is heated to 80 °C, the pH is 12, and the dosage of Hedysarum polysaccharide added is 100 μL.

6. The application according to any one of claims 1-5, characterized in that: The extraction method of Hedysarum polysaccharide is as follows: after defatting Hedysarum polysaccharide, first carry out enzymatic hydrolysis with a composite enzyme, and then carry out ultrasonic extraction; the addition amount of the composite enzyme is 1.0% of the mass of Hedysarum, the enzymatic hydrolysis temperature is 45 °C, and the enzymatic hydrolysis time is 1 h; the composite enzyme consists of cellulase and papain, and the mass ratio of the two is 2:1; the ultrasonic temperature is 60 °C, the ultrasonic power is 40 W, the ultrasonic time is 30 min, and the ultrasonic treatment is carried out once.

7. The application according to claim 1, characterized in that: The device consists of a colorimetric sensor of Hedysarum polysaccharide-functionalized silver nanoparticles, a syringe, a filter paper and a filter holder.

8. The application according to claim 1, wherein: The application of the device in detecting human serum and urine, animal serum and urine.

9. A method for establishing a standard curve of methimazole, characterized in that: Draw the colorimetric sensor of Hedysarum polysaccharide-functionalized silver nanoparticles described in any one of claims 1-6 into a syringe, then draw in a series of methimazole solutions with known concentrations, mix well and let stand until the reaction is complete, slowly push out the mixed solution in the syringe at a speed of 1 mL / min so that the mixed solution all passes through the filter paper with a pore size of 0.22 μm, air-dry the filter paper naturally, measure the gray value of the detection area of the filter paper, use the methimazole concentration as the abscissa, and use the ratio of the gray values of the detection areas of the series of standard samples and the blank sample filter papers as the ordinate to establish a standard curve; the concentration range of the series of methimazole solutions with known concentrations in the mixed solution is 0.1-10 μM.

10. A method for detecting methimazole in a sample to be tested, characterized in that: A colorimetric sensor of Hedysarum polybotrys polysaccharide-functionalized silver nanoparticles described in any one of claims 1-6 is aspirated into a syringe, and then the sample to be tested is aspirated. After mixing evenly, it is left standing until the reaction is complete. The mixed solution in the syringe is slowly pushed out at a speed of 1 mL / min, so that the mixed solution completely passes through a filter paper with a pore size of 0.22 μm. The filter paper is naturally air-dried, the gray value of the detection area of the filter paper is measured, the ratio of the gray values of the detection areas of the filter paper of the sample to be tested and the blank sample is calculated, and it is substituted into the standard curve equation in claim 9 to calculate the concentration of methimazole in the sample to be tested.

11. Series concentration methimazole color comparison card, characterized in that: A colorimetric sensor of Hedysarum polybotrys polysaccharide-functionalized silver nanoparticles described in any one of claims 1-6 is aspirated into a syringe, and then a series of methimazole solutions with known concentrations are aspirated. After mixing evenly, it is left standing until the reaction is complete. The mixed solution in the syringe is slowly pushed out at a speed of 1 mL / min, so that the mixed solution completely passes through a filter paper with a pore size of 0.22 μm. The filter paper is naturally air-dried to obtain a colorimetric card for methimazole with a series of concentrations.

12. A method for detecting methimazole in a sample to be tested, characterized in that: Comprising the following steps: (1) A colorimetric sensor of Hedysarum polybotrys polysaccharide-functionalized silver nanoparticles described in any one of claims 1-6 is aspirated into a syringe, and then a series of methimazole solutions with known concentrations are aspirated. After mixing evenly, it is left standing until the reaction is complete. The mixed solution in the syringe is slowly pushed out at a speed of 1 mL / min, so that the mixed solution completely passes through a filter paper with a pore size of 0.22 μm. The filter paper is naturally air-dried to obtain a colorimetric card for methimazole with a series of concentrations; (2) A colorimetric sensor of Hedysarum polybotrys polysaccharide-functionalized silver nanoparticles described in any one of claims 1-6 is aspirated into a syringe, and then the sample to be tested is aspirated. After mixing evenly, it is left standing until the reaction is complete. The mixed solution in the syringe is slowly pushed out at a speed of 1 mL / min, so that the mixed solution completely passes through a filter paper with a pore size of 0.22 μm. The filter paper is naturally air-dried, and the concentration range of methimazole is obtained by observing the color of the filter paper through the colorimetric card with the naked eye.

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