A nanozyme, hydrogel, chromogenic hydrogel kit, detection method, and application

Abstract

This invention belongs to the field of reagent kit technology and discloses a Papain-CuSO4@SiO2 nanozyme. The preparation steps of the nanozyme are as follows: 10 mg / mL of the solution is added while vigorous stirring. ‑1 Papain was added dropwise to a 10 mM CuSO4 solution, stirred, and then nano-SiO2 microspheres were added. The mixture was then sonicated, the pH was adjusted, and the solution was stirred in a water bath. The mixture was then filtered through a membrane to obtain the nanozyme. The nanozyme of this invention is simple to prepare, low in cost, and operates under mild reaction conditions, making it less susceptible to environmental interference and easy to scale up for production. Compared with existing nanozyme synthesis methods, this invention uses inorganic materials such as CuSO4 and SiO2 as precursors, reducing production costs. Furthermore, the nanozyme of this invention is easy to surface modify, enabling rapid and visual detection of solanine content in potatoes.

Description

Technical Field

[0001] This invention belongs to the field of reagent kit technology, and in particular to a nanozyme, hydrogel, chromogenic hydrogel reagent kit, detection method and application. Background Technology

[0002] The potato (Solanum tuberosum L.) is a staple food and economic crop, ranking as the world's fourth largest food crop after rice, corn, and wheat. Potato tubers are rich in the seven essential nutrients for the human body, and approximately one billion people worldwide rely on potatoes as their primary food source. As a member of the nightshade family, the potato plant is rich in solanine, with α-solanine and α-carboxine accounting for about 95% of the total solanine content. A typical mature potato contains 7-100 mg / kg of solanine. -1 While potatoes are generally safe to eat, the solanine content in the green skin and sprouts increases rapidly after long-term storage or exposure to environmental factors such as mechanical damage, high temperatures, and sunlight. When the solanine content exceeds 200 mg / kg... -1 Consuming potatoes at certain times can cause vomiting, convulsions, and even respiratory paralysis leading to death. Therefore, real-time monitoring of solanine in potatoes after harvest is crucial for the safe development of the potato industry.

[0003] Currently, the main methods for detecting solanine in potatoes include colorimetric methods, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), enzyme-linked immunosorbent assay (ELISA), and sensors. However, existing technologies have the following technical problems:

[0004] 1. HPLC and LC-MS are among the most widely used methods for qualitative and quantitative analysis of solanine, both of which offer high recovery rates and sensitivity. However, in practical applications, they require complex sample pretreatment and suffer from problems such as high cost, cumbersome operation, the need for professional personnel, and time consumption, making rapid detection impossible. Furthermore, the bulky equipment prevents on-site testing.

[0005] 2. Colorimetric and thin-layer chromatography methods have low accuracy and are difficult to apply to high-sensitivity quantitative analysis. ELISA has high sensitivity and specificity, but lacks specific antibodies. Sensors have replaced many detection technologies due to their portability and ease of operation; however, sensors suffer from difficulties in construction, signal instability, and poor reproducibility, making the field testing of solanine challenging.

[0006] 3. Existing methods for detecting solanine in potatoes have poor catalytic selectivity and lack specificity. In actual sample testing, complex matrices may interfere with the accuracy of the detection results.

[0007] 4. Currently, nanozyme materials mostly use precious metals such as platinum (Pt), palladium (Pd), gold (Au), and silver (Ag) as raw materials. The synthesis process is time-consuming, expensive, and involves hazardous reaction conditions, limiting the large-scale application of nanozyme materials in solanine detection. Furthermore, most nanozymes lack sufficient catalytic activity and affinity, affecting the timeliness and accuracy of actual detection, thus limiting their practical application. Current methods often involve partial surface modification and coating of nanozymes, which may further hinder their catalytic activity. Therefore, it is urgent to find a method to enhance the peroxidase activity of nanozymes while maintaining their properties and practical applications.

[0008] Through searching, the following patent publications related to this invention patent application were found:

[0009] 1. A method for determining α-solanine and α-carboxine in potatoes and their products (CN110940747A), using acidified methanol, anhydrous sodium sulfate, and anhydrous magnesium sulfate as extraction solvents for α-solanine and α-carboxine, and employing liquid chromatography to determine the content of α-solanine and α-carboxine in potatoes and their products. This method requires specialized and expensive instruments, has a long analysis time, and requires professional operation. This invention uses Cu... 2+ The preparation of nanozymes using this material is simple and low-cost. The hydrogel kit is portable and can be used for rapid detection. During use, smartphones are used to accurately quantify the solanine content in potatoes, solving the problem of bulky instruments and enabling rapid on-site detection and quantification.

[0010] 2. A test strip for detecting solanine and its application (CN114019166A) employs antigen-antibody reaction and competitive inhibition immunochromatographic analysis technology. It determines the residue of solanine in a sample by analyzing the colorimetric reaction of antigen and antibody on the test strip. While this method has high specificity and sensitivity, it cannot determine the solanine content in the sample; it only qualitatively identifies the solanine content and cannot determine whether the sample is edible. This invention uses a smartphone to convert color changes into content information, accurately determining the solanine content and judging the edibility of the sample.

[0011] 3. A method for detecting solanine in potatoes based on the fusion of machine vision and electronic nose technology (CN107748165A) uses computer vision to obtain potato image information and then uses an electronic nose to collect potato odor information, thereby quickly and efficiently determining whether the potato is edible. The machine vision and electronic nose fusion technology used in the experiment requires corresponding equipment and technical support, increasing the detection cost and complexity. Furthermore, deviations in sample acquisition time during the detection process can reduce the accuracy of the detection results. This invention directly extracts solanine from the sample and only requires analysis of color information through a mobile device to achieve quantitative detection, eliminating human visual errors. The measurement results are basically consistent with those obtained by liquid chromatography, achieving accurate, real-time, portable, and simple on-site rapid detection.

[0012] 4. A nanozyme, probe, and test strip for rapid on-site detection of solanine in potatoes, and its application (CN115851678A). This invention describes an immunoassay test strip for rapid on-site detection of solanine in potatoes, in which a nanozyme combined with a monoclonal antibody is mounted as a probe on the test strip. The solanine content in potatoes can be detected via enzyme-linked immunosorbent assay (ELISA). While this method has high sensitivity and accuracy, the test strip may be affected by external environmental interference during use, reducing its stability and thus affecting the accuracy of the detection results. Furthermore, the test strip has high environmental requirements in practical applications and may not be suitable for high-temperature, high-humidity environments. This invention utilizes hydrogel to encapsulate the nanozyme. The hydrogel effectively protects the nanozyme from external interference, improving the stability of the kit and enabling its application in different detection environments. The microenvironment provided by the hydrogel enhances the nanozyme's reactivity, increasing the reaction rate and color intensity during the detection process.

[0013] By comparison, the present invention patent application is fundamentally different from the aforementioned patent publications. Summary of the Invention

[0014] The purpose of this invention is to overcome the shortcomings of the prior art and provide a nanozyme, hydrogel, colorimetric hydrogel kit, detection method and application.

[0015] The technical solution adopted by this invention to solve its technical problem is:

[0016] A Papain-CuSO4@SiO2 nanozyme, the preparation steps of which are as follows:

[0017] With vigorous stirring at 950 rpm, 10 mg mL -1Papain was added dropwise to 10 mM CuSO4 solution, followed by stirring at 35 °C for 15 min. Then, nano-SiO2 microspheres were added and sonicated for 10 min. The pH of the system was adjusted to 3.5 with 3 M NaOH solution, and the mixture was stirred in a water bath at 37 °C for 2 h. The resulting solution was filtered through a 0.22 μm aqueous filter membrane to obtain pure Papain-CuSO4@SiO2 nanozyme, which was in liquid form. The nanozyme solution was stored in a sealed container at 4 °C.

[0018] The volume ratio of papain to CuSO4 solution (mL:mL) is 5:1, and the mass ratio of papain to nano-SiO2 microspheres (mg:mg) is 5:3.

[0019] The Papain-CuSO4@SiO2 hydrogel, which utilizes the Papain-CuSO4@SiO2 nanozyme as described above, is prepared by the following method:

[0020] Acrylamide monomer, 0.1M N-methylenebisacrylamide (MBAA), 0.1M ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) were added to the system. Water was added to adjust the total mass of the system. The solution was then quickly poured into a mold and placed in an oven for 4 hours to obtain acrylamide hydrogel.

[0021] Acrylamide hydrogel was soaked in Papain-CuSO4@SiO2 nanozyme for 24 hours to obtain Papain-CuSO4@SiO2 hydrogel;

[0022] The acrylamide monomer accounts for 12% of the total mass of the system, the molar ratio of MBAA to APS is 1:5, the mass ratio of acrylamide monomer to MBAA to TEMED is 1500:1:4, and the oven heating temperature is 50℃.

[0023] A kit for the quantitative detection of solanine in potatoes was prepared using the Papain-CuSO4@SiO2 hydrogel as described above. The preparation method of the kit is as follows:

[0024] 20 μL of solanine aqueous solution was mixed with 20 μL of acetylcholinesterase (ACHE) solution and incubated at 37 °C for 2 h. Solanine inhibited the activity of ACHE. Subsequently, 20 μL of choline oxidase (CHO) solution, acetylcholine chloride (ATCH) solution, and 0.1 mM pH 7.5 Tris-HCl were added to the above mixture in sequence and incubated at 37 °C for 30 min. Finally, the reaction solution was added to Papain-CuSO4@SiO2 hydrogel to obtain a solanine detection hydrogel kit.

[0025] The concentration of ACHE was 2.5 U / mL. -1 The concentration of CHO was 6 U / mL. -1 The concentration of ATCH was 100 mM.

[0026] The method for detecting solanine content in potatoes using the kit described above includes the following steps:

[0027] Add 80 μL of 0.2 mM pH 4.0 acetate-sodium acetate buffer (HAc-NaAc) and 200 μL of 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric reagent to the obtained solanine detection hydrogel kit. The colorimetric reaction of the kit is triggered, and the gel color can be observed to lighten from blue as the solanine content increases, showing a gradient change. The color change is recorded using ColorPicker software, and the color information is converted into R values. The R value of the hydrogel when the solanine content is 0 is selected as R0, and the relationship curve between R / R0 and solanine concentration is established to obtain a real-time quantitative detection platform for solanine. Then, by measuring the R / R0 value of the reaction of solanine in potato samples under the kit and comparing it with the standard curve, the solanine content in the actual samples is determined.

[0028] The concentration of ACHE was 2.5 U / mL. -1 The concentration of CHO was 6 U / mL. -1 The concentration of ATCH was 100 mM.

[0029] The advantages and positive effects of this invention are as follows:

[0030] 1. Compared with traditional peroxidase preparation methods, the nanozymes of this invention are simple to prepare, low in cost, and operate under mild reaction conditions, are less susceptible to interference from environmental factors, and are easy to scale up for production. Compared with existing nanozyme synthesis methods, this invention uses inorganic materials such as CuSO4 and SiO2 as precursors, reducing production costs. Furthermore, the nanozymes of this invention are easy to surface modify, enabling rapid and visual detection of solanine content in potatoes.

[0031] 2. This invention utilizes the natural enzyme papain and Cu 2+ Nanomaterials (Papain-CuSO4@SiO2) were prepared using SiO2 as a raw material. The introduction of SiO2 increased the specific surface area of ​​the synthesized nanozyme, which is beneficial for its binding with the catalytic site and improves the catalytic effect.

[0032] 3. This invention utilizes a smartphone to collect color information from the hydrogel, converting color changes into RGB information. The relationship between the amount of H2O2 inhibited by solanine and the R / R0 ratio is analyzed. The solanine content in potatoes is accurately determined using RGB detection mode, achieving on-site quantitative detection. Furthermore, the application of smartphones eliminates errors in visual judgment, and the results from the kit are consistent with those from liquid chromatography.

[0033] 4. The polyacrylamide hydrogel selected in this invention has a large specific surface area and high hydrophilicity, enabling rapid uptake of potato tissue fluid and accelerating the reaction rate of nanozymes and chromogenic agents with solanine in potatoes. Confining the nanozyme within a highly specific hydrogel further enhances its reactivity through the confinement effect, optimizing the colorimetric effect and improving detection accuracy. The hydrogel effectively protects the nanozyme from external interference, improving the stability of the kit.

[0034] 5. This invention is the first to develop and successfully apply a colorimetric hydrogel kit for the quantitative detection of solanine in potatoes, which has high specificity and sensitivity.

[0035] Solanine specifically binds to the active site of ACHE, inhibiting ACHE's degradation of ATCH and thus weakening the color reaction. The Papain-CuSO4@SiO2 nanozyme-ACHE-CHO cascade system used in this invention has reaction selectivity and signal amplification effects, ensuring that only solanine can initiate the cascade reaction during detection. Signal amplification allows this invention to detect low concentrations of solanine, enhancing the sensitivity and specificity of the kit. The hydrogel kit of this invention is suitable for detecting concentrations of 0.0335-2 mg / mL. -1 The presence of solanine, a major interfering substance in potatoes, did not significantly affect the detection system.

[0036] 6. This invention employs a Papain-CuSO4@SiO2 nanoenzyme-ACHE-CHO cascade system, which enhances the anti-interference ability of the hydrogel. Even in the presence of various bioactive substances commonly found in potatoes, the detection of solanine can still maintain high accuracy, effectively avoiding interference from other coexisting substances in potatoes.

[0037] 7. This invention is the first to develop and successfully apply a colorimetric hydrogel kit for the quantitative detection of solanine in potatoes, which has the advantages of low cost, portability, and rapid color development.

[0038] The nanozyme of this invention uses inorganic materials such as CuSO4 and SiO2 as precursors, resulting in low production costs. Data can be read within 10 minutes of sample addition, and the instantaneous reaction makes the nanozyme of this invention convenient for on-site detection. The hydrogel in the kit is small in volume (80×10×4mm), making it easy to carry. During the detection process, the combination of smartphone to collect and analyze color information eliminates the error of visual judgment, realizing the rapid on-site handheld detection of solanine.

[0039] 8. The nanozymes of the present invention have a small size. The introduction of SiO2 increases the specific surface area of ​​the nanozymes and improves their catalytic efficiency. Furthermore, confining the nanozymes in the hydrogel enhances the stability of the kit and the accuracy of the detection results through the confinement effect.

[0040] The nanozyme of this invention is a near-spherical particle with an average diameter of 35.83 nm. Its small size and large specific surface area improve catalytic efficiency. The microenvironment provided by the hydrogel enhances the catalytic activity of the nanozyme through the confinement effect and expands the colorimetric signal. The actual detection results have a good correlation with the liquid chromatography measurement results (r = 0.991). Moreover, the hydrogel protects the nanozyme from external interference. The color change of the hydrogel is small after 3 hours of reaction, which effectively improves the stability of the kit. Attached Figure Description

[0041] Figure 1 This is a schematic diagram illustrating the synthesis and assay principles of the reagent kit in this invention;

[0042] Figure 2 Figure 1 shows the synthesis principle and characterization diagram of Papain-CuSO4@SiO2 nanozyme in this invention; Figure 2a is a schematic diagram of the synthesis principle of Papain-CuSO4@SiO2 nanozyme; Figures 3b and 4c are scanning electron microscope (SEM) images of Papain-CuSO4@SiO2 nanozyme at 300 nm and transmission electron microscope (TEM) images at 100 nm; Figure 4d is the FT-IR image of Papain-CuSO4@SiO2 nanozyme; Figure 5e is the XRD pattern of Papain-CuSO4@SiO2 nanozyme; Figure 6f is the XPS pattern of Papain-CuSO4@SiO2 nanozyme; and Figure 7g is a graph showing the determination of the kinetic constant of Papain-CuSO4@SiO2 nanozyme.

[0043] Figure 3Figure 1 shows the reaction connection diagram of Papain-CuSO4@SiO2 nanozyme in this invention and the optimization diagram of the experimental conditions for the catalytic reaction. Among them, Figure 2a is the color reaction diagram of Papain-CuSO4@SiO2 nanozyme with added TMB and H2O2; Figures 3bg are the effects of different CuSO4:papain volume ratios (1:5-5:1), different pH (3-5.5), different SiO2 addition amounts (0-35mg), different temperatures (40℃-60℃), different catalytic times (0-30min), and different concentrations of TMB (1.2-2.2mM) on the color development of the nanozyme.

[0044] Figure 4 Figure 1 shows the reaction connection diagram of the hydrogel kit in this invention and the optimization diagram of the experimental conditions for the catalytic reaction; wherein, Figure 2a is the colorimetric reaction diagram of the hydrogel kit with added TMB and H2O2; Figures 3b and 4d are the reaction diagrams of different concentrations of ACHE (1.5-4.5 U / mL) respectively. -1 CHO (0-10U mL) -1 The effect of ) and ATCH (80-200mM) on the color development of the hydrogel kit;

[0045] Figure 5 These are characterization images of the Papain-CuSO4@SiO2 hydrogel in this invention; Figures a and c are scanning electron microscope (SEM) images of the polyacrylamide hydrogel at 100 nm and 200 nm, respectively; Figures b and d are SEM images of the Papain-CuSO4@SiO2 hydrogel at 100 nm and 200 nm, respectively; Figure e is the FT-IR image of the Papain-CuSO4@SiO2 hydrogel; and Figure f is the XRD pattern of the Papain-CuSO4@SiO2 hydrogel.

[0046] Figure 6 Figures show a comparison of the catalytic activity of the hydrogel kit for solanine detection in this invention, and a standard curve for solanine standards. Figures ab and b show a comparison of the color development effects of the hydrogel kit and the Papain-CuSO4@SiO2 nanozyme solution; Figure cd shows the stability of the color development of the Papain-CuSO4@SiO2 nanozyme solution; Figure ef shows the stability of the color development of the hydrogel kit; Figure g shows the standard curve of the hydrogel kit against solanine standards; and Figure h shows the specificity detection of the hydrogel kit.

[0047] Figure 7 This is a graph showing the detection results of solanine content in actual potato samples using the hydrogel reagent kit of this invention. Detailed Implementation

[0048] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0049] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0050] A Papain-CuSO4@SiO2 nanozyme, the preparation steps of which are as follows:

[0051] With vigorous stirring at 950 rpm, 10 mg mL -1 Papain was added dropwise to 10 mM CuSO4 solution, followed by stirring at 35 °C for 15 min. Then, nano-SiO2 microspheres were added and sonicated for 10 min. The pH of the system was adjusted to 3.5 with 3 M NaOH solution, and the mixture was stirred in a water bath at 37 °C for 2 h. The resulting solution was filtered through a 0.22 μm aqueous filter membrane to obtain pure Papain-CuSO4@SiO2 nanozyme, which was in liquid form. The nanozyme solution was stored in a sealed container at 4 °C.

[0052] The volume ratio of papain to CuSO4 solution (mL:mL) is 5:1, and the mass ratio of papain to nano-SiO2 microspheres (mg:mg) is 5:3.

[0053] The Papain-CuSO4@SiO2 hydrogel, which utilizes the Papain-CuSO4@SiO2 nanozyme as described above, is prepared by the following method:

[0054] Acrylamide monomer, 0.1M N-methylenebisacrylamide (MBAA), 0.1M ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) were added to the system. Water was added to adjust the total mass of the system. The solution was then quickly poured into a mold and placed in an oven for 4 hours to obtain acrylamide hydrogel.

[0055] Acrylamide hydrogel was soaked in Papain-CuSO4@SiO2 nanozyme for 24 hours to obtain Papain-CuSO4@SiO2 hydrogel;

[0056] The acrylamide monomer accounts for 12% of the total mass of the system, the molar ratio of MBAA to APS is 1:5, the mass ratio of acrylamide monomer to MBAA to TEMED is 1500:1:4, and the oven heating temperature is 50℃.

[0057] A kit for the quantitative detection of solanine in potatoes was prepared using the Papain-CuSO4@SiO2 hydrogel as described above. The preparation method of the kit is as follows:

[0058] 20 μL of solanine aqueous solution was mixed with 20 μL of acetylcholinesterase (ACHE) solution and incubated at 37 °C for 2 h. Solanine inhibited the activity of ACHE. Subsequently, 20 μL of choline oxidase (CHO) solution, acetylcholine chloride (ATCH) solution, and 0.1 mM pH 7.5 Tris-HCl were added to the above mixture in sequence and incubated at 37 °C for 30 min. Finally, the reaction solution was added to Papain-CuSO4@SiO2 hydrogel to obtain a solanine detection hydrogel kit.

[0059] The concentration of ACHE was 2.5 U / mL. -1 The concentration of CHO was 6 U / mL. -1 The concentration of ATCH was 100 mM.

[0060] The method for detecting solanine content in potatoes using the kit described above includes the following steps:

[0061] Add 80 μL of 0.2 mM pH 4.0 acetate-sodium acetate buffer (HAc-NaAc) and 200 μL of 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric reagent to the obtained solanine detection hydrogel kit. The colorimetric reaction of the kit is triggered, and the gel color can be observed to lighten from blue as the solanine content increases, showing a gradient change. The color change is recorded using ColorPicker software, and the color information is converted into R values. The R value of the hydrogel when the solanine content is 0 is selected as R0, and the relationship curve between R / R0 and solanine concentration is established to obtain a real-time quantitative detection platform for solanine. Then, by measuring the R / R0 value of the reaction of solanine in potato samples under the kit and comparing it with the standard curve, the solanine content in the actual samples is determined.

[0062] The concentration of ACHE was 2.5 U / mL. -1 The concentration of CHO was 6 U / mL. -1 The concentration of ATCH was 100 mM.

[0063] Specifically, the relevant preparation and testing methods are as follows:

[0064] The mechanism of this invention is as follows:

[0065] like Figure 1As shown, the carrier of this portable kit is a hydrogel material with a target response, which possesses Papain-CuSO4@SiO2 nanozyme with peroxidase-like activity. In the presence of H2O2, it catalyzes the oxidation of colorless TMB to blue oxTMB. Simultaneously, ATCH is hydrolyzed by ACHE to thiocholine, which is then oxidized by CHO and O2 to generate H2O2. Solanine, as an ACHE inhibitor, inhibits ACHE-catalyzed ATCH to H2O2, thereby suppressing the color reaction of the kit. In practical applications, Color Picker software is used to convert the color change of the kit into digital information. The obtained R-value data is substituted into the previously obtained linear relationship curve of "R / R0 - solanine concentration" to calculate the concentration of solanine in the sample, achieving rapid on-site detection of solanine.

[0066] The relevant preparation and testing methods of this invention are as follows:

[0067] Example 1: Preparation, optimization, and characterization of a Papain-CuSO4@SiO2 nanozyme

[0068] 1. Preparation of Papain-CuSO4@SiO2 nanozymes

[0069] 10 mg mL was added with vigorous stirring (950 rpm). -1 Papain was added dropwise to 10 mL of CuSO4 solution to obtain Papain-CuSO4. The mixture was then stirred at 35 °C for 15 min, followed by the addition of nano-SiO2 microspheres and sonication for 10 min. The pH of the system was adjusted to 3.5 with 3 M NaOH solution, and the mixture was stirred in a water bath at 37 °C for 2 h. The resulting solution was filtered through a 0.22 μm aqueous filter membrane to obtain pure Papain-CuSO4@SiO2 nanozyme. The nanozyme solution (the obtained product is liquid) was stored sealed at 4 °C.

[0070] The volume ratio of Papain to CuSO4 solution is 5:1 (mL:mL), and the mass ratio of Papain to nano-SiO2 microspheres is 5:3 (mg:mg).

[0071] 2. Characterization of Papain-CuSO4@SiO2 nanozymes

[0072] The morphology of nanozymes was obtained using transmission electron microscopy (TEM) and scanning electron microscopy (SEM), and their unique physicochemical properties were studied using characterization techniques such as particle size, zeta potential, XPS, and XRD. The results are as follows: Figure 2 As shown in the SEM and TEM images, Papain-CuSO4@SiO2 consists of nearly spherical particles with an average diameter of approximately 35.83 nm. Figure 2 (b, c) Due to the adsorption of SiO2, the Zeta potential of Papain-CuSO4@SiO2 (2.49 mV) is lower than that of Papain-CuSO4 (1.82 mV). Peaks of SiO2 can be observed in both XRD and FT-IR spectra, indicating that SiO2 was successfully adsorbed onto the Papain-CuSO4 nanozyme. Figure 2 d, e). XPS results showed that C-Si and C-SO were generated during the synthesis process. x The chemical bond with SH demonstrates the successful combination of Papain, CuSO4, and SiO2. Figure 2 f).

[0073] 3. Peroxidase-like activity of Papain-CuSO4@SiO2 nanozymes

[0074] (1) Peroxidase-like activity of Papain-CuSO4@SiO2 nanozymes

[0075] TMB and H2O2 solutions with a volume ratio of 1:1 (μL:μL) were added to the prepared Papain-CuSO4@SiO2 nanozyme solution and Papain-CuSO4@SiO2 hydrogel, respectively, and the catalytic reaction was carried out at room temperature and in the dark. The results are as follows. Figure 3 As shown in a and 4a, the experiments demonstrate that the prepared Papain-CuSO4@SiO2 nanozyme is colorless and transparent, and has little impact on the subsequent colorimetric reaction. After the addition of the nanozyme, the originally colorless TMB and H2O2 solutions rapidly turned blue, indicating that the prepared Papain-CuSO4@SiO2 nanozyme possesses good peroxidase-like activity.

[0076] TMB is a 2.2 mM dimethyl sulfoxide solution, and H2O2 is a 0.1% aqueous solution.

[0077] (2) Optimization of preparation conditions for Papain-CuSO4@SiO2 nanozymes

[0078] Four key factors in the nanozyme preparation process—the volume ratio of CuSO4 to Papain, the amount of nano-SiO2 microspheres added, pH, and temperature—were optimized to obtain the best color development effect. First, different volume ratios (1:5-5:1) of 10 mM CuSO4 and 10 mg / mL... -1 Papain prepared Papain-CuSO4@SiO2 precursor, followed by the addition of nano-SiO2 microspheres ( Papain-CuSO4@SiO2 nanozymes were prepared at pH 4.5 and 50℃ (i.e., by mass ratio); secondly, 10 mL of CuSO4 was mixed with 10 mg of CuSO4. -1 Papain was mixed at a volume ratio of 5:1, and then different masses of nano-SiO2 microspheres were added. Papain-CuSO4@SiO2 nanozymes were prepared at pH 4.5 and 50℃ (i.e., by mass ratio); next, 10 mM CuSO4 was mixed with 10 mg / mL... -1 Papain was mixed at a volume ratio of 5:1, and then nano-SiO2 microspheres were added. Papain-CuSO4@SiO2 nanozymes were prepared in different pH (3-5.5) and 50℃ environments (i.e., by mass ratio); finally, 10 mM CuSO4 was mixed with 10 mg / mL... -1 Papain was mixed at a volume ratio of 5:1, and then nano-SiO2 microspheres were added. Papain-CuSO4@SiO2 nanozymes were prepared in environments with pH 3.5 and different temperatures (40℃-60℃) using a mass ratio of 1:1:1 (μL:μL:μL). The obtained nanozymes were then mixed thoroughly with 1 mM TMB and 0.1% H2O2 in a volume ratio of 1:1:1 (μL:μL:μL) in test tubes. After reacting for 3 min, the absorbance of the reaction solution at 652 nm was measured using a microplate reader.

[0079] The results are as follows Figure 3 As shown in the figure, the experiment indicates that the volume ratio of CuSO4 to Papain is 5:1. The optimal mass ratio (i.e., 5:1) for nanozyme catalysis and color development was observed, with the best color development effect observed in a PBS buffer system prepared at 50℃ and pH 3.5. As the preparation temperature increased from 40℃ to 50℃, the OD value of the nanozyme gradually increased, but at 55℃, the OD value plummeted from 1.77 to 0.60. The nanozyme prepared at 50℃ exhibited the strongest peroxidase activity. Papain showed the highest catalytic oxidation rate at pH 3.5, and the weakly acidic environment was conducive to maintaining H2O2 stability. Furthermore, with increasing amounts of nano-SiO2 microspheres, the OD value showed a trend of first increasing and then decreasing. A small amount of nano-SiO2 microspheres can provide more adsorption sites on the nanozyme surface, but excessive nano-SiO2 microspheres may shield the active sites on the nanozyme surface or block electron pathways. It can be seen that the optimal volume ratio of CuSO4 to Papain is 5:1. There is a synergistic effect between the (i.e., mass ratio) and the nanozymes prepared can be improved in a synergistic way.

[0080] (3) Optimization of the catalytic environment of Papain-CuSO4@SiO2 nanozymes

[0081] The effects of different concentrations of TMB and catalytic duration on nanozyme catalysis were investigated to determine the optimal catalytic conditions for Papain-CuSO4@SiO2. First, 10 mM CuSO4 was mixed with 10 mg / mL... -1 Papain was mixed at a volume ratio of 5:1, and then nano-SiO2 microspheres were added. Papain-CuSO4@SiO2 nanozymes were prepared at pH 3.5 and 50℃ (mass ratio), and then catalyzed with 0.1% H2O2. Different concentrations of TMB (1.2 mM-2.2 mM) were used for color development. The volume ratio of nanozyme to H2O2 and TMB was 1:1:1 (μL:μL:μL). After 3 min of reaction, the absorbance of the reaction solution at 652 nm was measured using a microplate reader. Next, 10 mM CuSO4 was mixed with 10 mg / mL... -1 Papain was mixed at a volume ratio of 5:1, and then nano-SiO2 microspheres were added. Papain-CuSO4@SiO2 nanozymes were prepared at pH 3.5 and 50℃ (mass ratio), and then reacted with 0.1% H2O2 and 2.2mM TMB at a volume ratio of 1:1:1 (μL:μL:μL). After different catalytic times (0-30 min), the absorbance of the reaction solution at 652 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0082] The results are as follows Figure 3 As shown in fg, experiments indicate that the nanozyme exhibits the best colorimetric effect in a system with a TMB concentration of 2.2 mM, catalyzed for 10 min. With increasing TMB concentration, the OD value shows an increasing trend, reaching 2.03 at a TMB concentration of 2.2 mM, indicating that the synthesized nanozyme at this concentration possesses strong peroxidase activity. Furthermore, with prolonged catalytic reaction time, the OD value shows a rapid increase, then tends to stabilize after 10 min. Therefore, the nanozyme synthesized in this invention, while demonstrating significant colorimetric effect, greatly shortens the detection time. It can be seen that the TMB concentration of 2.2 mM and the catalytic time of 10 min have a synergistic effect, collectively improving the relevant performance of the prepared nanozyme.

[0083] (4) Steady-state kinetics experiment of Papain-CuSO4@SiO2 nanozyme

[0084] During the experiment, the steady-state kinetic parameters of the Papain-CuSO4@SiO2 nanozyme were calculated by measuring the absorbance at 652 nm of the reaction solution using an ELISA reader under optimal conditions, with the H2O2 concentration fixed and the TMB concentration varied (1-15 mM), or the TMB concentration fixed and the H2O2 concentration varied (10-50 mM).

[0085] like Figure 2 As shown in g, experiments indicate that when TMB and H2O2 are used as substrates, the Kc of Papain-CuSO4@SiO2 is... m The values ​​were 0.056 mM (TMB) and 0.089 mM (H2O2), respectively, both higher than those of Papain-CuSO4 nanozyme. The Vt value of Papain-CuSO4@SiO2 for TMB was... max Value (3.04×10) -6 Ms -1 (Higher than Papain-CuSO4 (2.15×10)) -6 Ms -1 The results indicate that the nanozyme exhibits higher catalytic efficiency after the addition of SiO2. It can be seen that there is a synergistic effect between the substrate TMB and H2O2, which can synergistically improve the relevant performance of the prepared nanozyme.

[0086] Example 2: Preparation and Performance Verification of a Hydrogel Reagent Kit for Solanine Detection

[0087] 1. Preparation of a hydrogel kit for solanine detection

[0088] (1) Optimization of colorimetric conditions for the Papain-CuSO4@SiO2-ACHE-CHO cascade system

[0089] To achieve the optimal colorimetric effect for solanine detection, the effects of ACHE, CHO, and ATCH concentrations on the catalytic effect of the prepared Papain-CuSO4@SiO2 nanozyme solution were investigated. First, different concentrations of ACHE (1.5-4.5 U / mL) were... -1 ), 1U mL -1CHO, 120 mMATCH and 0.1 mM pH 7.5 Tris-HCl were incubated at 37 °C for 30 min in a volume ratio of 1:1:1:1 (μL:μL:μL:μL) to obtain reagent A. Subsequently, 2.2 mM TMB, 0.2 mM pH 4.0 acetate-sodium acetate buffer (HAc-NaAc) and the prepared Papain-CuSO4@SiO2 nanozyme solution were added sequentially. The volume ratio of reagent A to the above three solutions was 2:5:2:19 (μL:μL:μL:μL). After reacting at room temperature and in the dark for 1 h, the absorbance of the reaction solution at 652 nm was measured using an ELISA reader.

[0090] Secondly, 2.5 U mL -1 ACHE, different concentrations of CHO (0-10 U / mL) -1 120 mM ATCH and 0.1 mM pH 7.5 Tris-HCl were incubated at 37 °C for 30 min in a volume ratio of 1:1:1:1 (μL:μL:μL:μL) to obtain reagent A. Subsequently, 2.2 mM TMB, 0.2 mM pH 4.0 HAc-NaAc and the prepared Papain-CuSO4@SiO2 nanozyme solution were added sequentially. The volume ratio of reagent A to the above three solutions was 2:5:2:19 (μL:μL:μL:μL). After reacting at room temperature and in the dark for 1 h, the absorbance of the reaction solution at 652 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0091] Finally, 2.5 U mL -1 ACHE, 6U mL -1 CHO (0-10U mL) -1 Different concentrations of ATCH (80-200 mM) and 0.1 mM pH 7.5 Tris-HCl were incubated at 37°C for 30 min in a volume ratio of 1:1:1:1 (μL:μL:μL:μL) to obtain reagent A. Subsequently, 2.2 mM TMB, 0.2 mM pH 4.0 HAc-NaAc, and the prepared Papain-CuSO4@SiO2 nanozyme solution were added sequentially, with the volume ratio of reagent A to the above three solutions being 2:5:2:19 (μL:μL:μL:μL). After reacting for 1 h at room temperature and in the dark, the absorbance of the reaction solution at 652 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader.

[0092] like Figure 4 As shown in Figure bd, the experiment indicates that the nanozyme is effective at an ACHE concentration of 2.5 U / mL. -1 The concentration of CHO is 6 U / mL. -1 The best colorimetric effect was observed in a system with an ATCH concentration of 100 mM. It can be seen that an ACHE concentration of 2.5 U / mL... -1The concentration of CHO is 6 U / mL. -1 Furthermore, when the ATCH concentration is 100 mM, the three components have a synergistic effect, which can synergistically improve the relevant properties of the prepared nanozyme.

[0093] (2) Preparation of Papain-CuSO4@SiO2 hydrogel

[0094] Acrylamide monomer, 0.1M N,N-methylenebisacrylamide (MBAA), 0.1M ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) were added to the system. Water was added to adjust the total mass of the system, and the solution was quickly poured into a mold and placed in an oven for 4 hours to obtain an acrylamide hydrogel. The acrylamide hydrogel was then immersed in a Papain-CuSO4@SiO2 nanozyme solution for 24 hours to obtain a Papain-CuSO4@SiO2 hydrogel.

[0095] The acrylamide monomer accounts for 12% of the total mass of the system (g:g), the molar ratio of MBAA to APS is 1:5 (M:M), the mass ratio of acrylamide monomer to MBAA to TEMED is 1500:1:4 (mg:mg:mg), and the oven heating temperature is 50℃.

[0096] (3) Characterization of Papain-CuSO4@SiO2 hydrogel

[0097] The morphology of the hydrogel was obtained using SEM, and its unique physicochemical properties were studied by XRD, FT-IR, and other characterization methods. The results are as follows: Figure 5 As shown in the image, SEM images reveal the three-dimensional fibrous structure of the hydrogel, indicating that the Papain-CuSO4@SiO2 nanozyme was successfully loaded onto the hydrogel. The shifts and changes in the spectral peaks in the FT-IR and XRD images further confirm the attachment of the Papain-CuSO4@SiO2 nanozyme to the hydrogel. Figure 5 e, f).

[0098] (4) Preparation of a hydrogel kit for the detection of solanine

[0099] Mix solanine aqueous solution with 2.5 U mL -1 After mixing with ACHE, the mixture was incubated at 37°C for 2 hours to inhibit the activity of ACHE by solanine; subsequently, 6 U / mL of the mixture was added sequentially. -1The reaction solution consisted of CHO, 100 mM ATCH, and 0.1 mM pH 7.5 Tris-HCl, with a volume ratio of solanine, ACHE, CHO, ATCH, and Tris-HCl of 1:1:1:1:1 (μL:μL:μL:μL). After incubation at 37°C for 30 min, the reaction solution was added to the Papain-CuSO4@SiO2 hydrogel to obtain the solanine detection hydrogel kit.

[0100] Adding 0.2 mM pH 4.0 HAc-NaAc and 2.2 mM TMB at a volume ratio of 2:5 (μL:μL) to the obtained solanine detection hydrogel kit triggered the colorimetric reaction of the kit. The gel color was observed to lighten with increasing solanine content, becoming a lighter blue. Color changes were recorded using Color Picker software, and the color information was converted into R values. The R value of the hydrogel with a solanine content of 0 was selected as R0, and a curve showing the relationship between R / R0 and solanine concentration was established, thus obtaining the solanine quantitative real-time detection platform described in this invention. Furthermore, by measuring the R / R0 value of the solanine reaction in potato samples under the kit's conditions and comparing it with the standard curve, the solanine content in actual samples was determined.

[0101] 2. Comparison of catalytic activity between the solanine detection hydrogel kit and Papain-CuSO4@SiO2 solution

[0102] Papain-CuSO4@SiO2 nanozyme solution and Papain-CuSO4@SiO2 hydrogel were incubated with ACHE, CHO and ATCH at a volume ratio of 1:1:1:1 (μL:μL:μL:μL) for 1 h at room temperature and in the dark. The absorbance and R / R0 value of the reaction solution at 652 nm were measured, and the stability of the colorimetric reaction of nanozyme solution and hydrogel in 1-3 h was determined.

[0103] The results are as follows Figure 6 As shown in Figure af, with increasing H2O2 concentration, the color development effect of Papain-CuSO4@SiO2 hydrogel was significantly higher than that of Papain-CuSO4@SiO2 nanozyme solution. This may be due to the larger specific surface area of ​​the hydrogel and its higher affinity for the reactants. Compared with the solution, using hydrogel as a carrier showed a more significant color development effect and greater potential for practical application. Furthermore, after 3 hours of reaction, the color development of Papain-CuSO4@SiO2 nanozyme solution degraded rapidly due to instability, while the color change of Papain-CuSO4@SiO2 hydrogel, analyzed by Color Picker software, showed less color change, indicating a more stable state.

[0104] 3. Specificity test of the solanine detection hydrogel kit

[0105] Choose a concentration of 1 mg / mL -1 Starch, bovine serum albumin, glutamic acid, lysine, glucose, and sucrose standards were used as interfering agents in a 0.1 mg / mL solution. -1 Under the condition of the presence of solanine, the above-mentioned interfering agent was added to the reaction system (the same as the solanine detection hydrogel kit reaction system in step 1 (4) of Example 2), wherein the volume ratio of the interfering agent to the solanine solution was 1:1 (μL:μL). Figure 6 As shown in h, the experiment shows that the coexistence of the main interfering substances in the potato has no significant effect on the detection system, and the Papain-CuSO4@SiO2 hydrogel kit has high selectivity.

[0106] Example 3: Solanine Detection Hydrogel Kit for the Detection of Solanine Standards

[0107] Different concentrations of solanine standard solutions (0-2 mg / mL) were used. -1 ) and 2.5U mL -1 ACHE was mixed at a volume ratio of 1:1 (μL:μL) and incubated at 37°C for 2 hours. Then, 6 U mL of a solution with a volume ratio of 1:1:1 (μL:μL:μL) was added sequentially. -1 CHO, 100 mMATCH, and 0.1 mM pH 7.5 Tris-HCl were incubated at 37 °C for 30 min. Finally, a 2:5 (μL:μL) mixture of 0.2 mM pH 4.0 HAc-NaAc and 2.2 mM TMB was added to the Papain-CuSO4@SiO2 hydrogel. After reacting at room temperature and in the dark for 1 h, the hydrogel color was analyzed using Color Picker software. A series of color changes in the hydrogel kit could be observed visually under sunlight. The detection results are shown below. Figure 6 As shown in g, the color intensity of the hydrogel decreases with increasing solanine concentration, and there is a good linear relationship between R / R0 and solanine concentration (R 2 =0.9953), linear fit of calibration data, R / R0 = 0.72 × C 茄碱 +1.0. The standard curve plotted is suitable for detecting 0.0335–2 mg / mL. -1 A wide range of solanine molecules are detected with high sensitivity. The hydrogel kit, combined with ColorPicker software, enables rapid quantitative monitoring of solanine in potatoes.

[0108] Example 4: Solanine Detection Hydrogel Kit for the Detection of Actual Potato Samples at Different Stages

[0109] Accurately weigh 1g of freeze-dried potato pulp and add 5mL of extraction solution (V甲醇 :V 甲酸 =500:1 (volume ratio), and ultrasonic-assisted extraction was performed for 30 min, followed by shaking and overnight incubation at 25℃. The extract was centrifuged at 8000 rpm for 5 min, and the supernatant was passed through a 0.22 μm aqueous filter membrane. Before testing, the potato sample solution needed to be concentrated to near dryness using a nitrogen blower, and then diluted to volume with deionized water at a volume ratio of 1:1 (mL:mL) to obtain the solanine test solution. The test solution was then mixed with 2.5 U mL of... -1 ACHE was mixed at a volume ratio of 1:1 (μL:μL) and incubated at 37°C for 2 hours. Subsequently, 6 U mL of a solution with a volume ratio of 1:1:1 (μL:μL:μL) was added to the above mixture. -1 CHO, 100 mMATCH, and 0.1 mM pH 7.5 Tris-HCl were incubated at 37 °C for 30 min. Finally, a 2:5 (μL:μL) mixture of 0.2 mM pH 4.0 HAc-NaAc and 2.2 mM TMB was added. This reaction solution was then added to a Papain-CuSO4@SiO2 hydrogel. After reacting for 1 h at room temperature and in the dark, color changes were recorded using ColorPicker software, the R value was measured, and the solanine content in the potato test solution was calculated based on the standard curve. The detection results are shown in Table 1. Figure 7 As shown, the LOD value of the hydrogel kit is 0.0335 mg / mL. -1 The kit is highly sensitive to changes in solanine content in actual potato samples, with a relative standard deviation of less than 0.33. It also shows good correlation with the values ​​determined by liquid chromatography (r = 0.991), indicating that the two detection methods are consistent and that the kit has potential applicability and reliability in actual samples.

[0110] Table 1. Reagent kit and liquid chromatograph of the present invention for the detection of solanine in actual potato samples.

[0111]

[0112]

[0113] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A kit for quantitative detection of solanigrin in potato prepared by using papain-CuSO4@SiO2 hydrogel of papain-CuSO4@SiO2 nanozyme, characterized in that: The preparation method of the kit is as follows: After 20 μL of the solubility solution of solamum and 20 μL of the acetylcholinesterase (ACHE) solution are mixed and incubated at 37 ℃ for 2 h, the solamum inhibits the activity of ACHE, then 20 μL of the choline oxidase (CHO) solution, 20 μL of the acetylcholine chloride (ATCH) solution and 0.1 mM pH 7.5 Tris-HCl are sequentially added to the above mixture, and the mixture is incubated at 37 ℃ for 30 min, and finally the reaction solution is added to the Papain-CuSO4@SiO2 hydrogel to obtain a solamum detection hydrogel kit; wherein the concentration of ACHE is 2.5 U mL -1 , the concentration of CHO is 6 U mL -1 , and the concentration of ATCH is 100 mM; The preparation steps of the Papain-CuSO4@SiO2 nanenzyme are as follows: With vigorous stirring, i.e. 950 rpm, 10 mg mL -1 Papain was added dropwise into 10 mM CuSO4 solution, followed by continued stirring at 35 ℃ for 15 min, then nano-SiO2 microspheres were added and ultrasonicated for 10 min, the pH of the system was adjusted to 3.5 with 3 M NaOH solution, and the resulting solution was stirred at 37 ℃ for 2 h. The obtained pure Papain-CuSO4@SiO2 nanoszyme solution was filtered with a 0.22 μm water filter membrane and stored in a refrigerator at 4 ℃. The volume ratio mL:mL of the papain:CuSO4 solution is 5:1, and the mass ratio mg:mg of the papain:nano-SiO2 microspheres is 5:3; The preparation method of the Papain-CuSO4@SiO2 hydrogel is as follows: The acrylamide monomer, 0.1 M N,N-methylenebisacrylamide (MBAA), 0.1 M ammonium persulfate (APS) and tetramethyl ethylenediamine (TEMED) are added to the system, water is added to adjust the total mass of the system, and then the solution is quickly poured into a mold and placed in an oven for 4 h to obtain an acrylamide hydrogel; The acrylamide hydrogel is soaked in the Papain-CuSO4@SiO2 nanenzyme for 24 h to obtain a Papain-CuSO4@SiO2 hydrogel. The mass of the acrylamide monomer accounts for 12% of the total mass of the system, the molar ratio of MBAA:APS is 1:5, the mass ratio of the acrylamide monomer:MBAA:TEMED is 1500:1:4, and the heating temperature of the oven is 50 ℃.

2. A method for detecting the content of solanin in potatoes by using the kit as claimed in claim 1, characterized in that: The following steps are included: 80 μL of 0.2 mM pH 4.0 acetic acid-sodium acetate buffer HAc-NaAc and 200 μL of 3,3',5,5'-tetramethylbenzidine color developing agent are added to the obtained solamum detection hydrogel kit, the color developing reaction of the kit is triggered, the color of the gel can be observed to become lighter blue and present a gradient change with the increase of the solamum content, Color Picker software is used to record the color change, the color information is converted into R value, the R value of the hydrogel when the solamum content is 0 is selected as R0, a relationship curve of R / R0 and the solamum concentration is established, a solamum quantitative real-time detection platform is obtained, and the determination of the solamum content in the actual sample is completed by determining the R / R0 value of the reaction of the solamum in the potato sample in the kit and comparing with the standard curve. wherein the concentration of ACHE is 2.5 U mL -1 , the concentration of CHO is 6 U mL -1 , and the concentration of ATCH is 100 mM.

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