Method for detecting nitrite based on nano-enzyme-loaded polymer microneedle patch

Abstract

The invention relates to preparation of a nano-enzyme-loaded porous polymer microneedle patch and application of the nano-enzyme-loaded porous polymer microneedle patch in detection of nitrite, and belongs to the fields of preparation and application of nano-enzymes and food safety detection. The detection method comprises the following steps: S1, preparing a porous polymer microneedle patch; S2, preparing copper and iron doped hydroxyl gallium oxide nano enzyme; and S3, dropwise adding the solution obtained after the TMB is oxidized by the nano-enzyme to the back surface of the patch, and judging the content condition of nitrite according to the color change of the porous polymer microneedle patch. According to the method, a TMB (Tetramethylbenzidine) solution which is oxidized and blue by a hydroxyl gallium oxide nano-enzyme is used as a colorimetric reagent; a specific reaction with nitrite can be carried out; a method for detecting nitrite based on the nano-enzyme is high in specificity, strong in anti-interference capability, high in sensitivity and wide in application range, and pretreatment can be avoided; and the recovery rate is basically more than 90%. The method has a wide application prospect in the field of food detection.

Description

Technical Field

[0001] The invention relates to a preparation method of a porous polymer microneedle patch loaded with nanoenzymes and application thereof in detecting nitrite, and belongs to the field of preparation and application of nanoenzymes and food safety detection. Background Art

[0002] Nitrite is a very common food additive, especially in the processing of meat. Sodium nitrite and potassium nitrite are the two most common types. Sodium nitrite can be used as a coloring agent, color preservative, preservative, etc. Adding sodium nitrite in the meat processing process has the effects of increasing the freshness of meat, making its color bright red, inhibiting the growth of microorganisms, and maintaining the structure and nutritional value of meat products. It is contained in a large amount in pickled meat, marinated meat, smoked meat and other pickled foods. However, sodium nitrite is listed as a Class 2A carcinogen by the World Health Organization. It is a strong oxidant that can combine with hemoglobin after entering the blood, causing tissue hypoxia and poisoning. Excessive intake at one time can even lead to death. Because its appearance is extremely similar to table salt, it is easy to be eaten by mistake. Nitrite is also produced in the food production process. For example, in the pickling process of pickled vegetables, microorganisms such as nitrifying bacteria can reduce nitrate substances to nitrite. Similarly, salted duck eggs and overnight dishes also contain nitrite. Excessive intake of nitrite can cause harm to the human body, so many countries and regions have established corresponding limit standards for nitrite in food.

[0003] In my country's national standards, there are three main methods for detecting nitrite content. The two most common methods are ion chromatography and spectrophotometry. For vegetables and fruits, ultraviolet spectrophotometry can also be used. All three methods require relatively complex sample pretreatment to remove proteins, lipids, pigments or other interfering impurities, and subsequent testing requires additional instruments such as ion chromatographs, spectrophotometers and ultraviolet spectrophotometers. However, all three methods require complex pretreatment processes and require the use of complex instruments and equipment.

[0004] Although there are kits on the market that can detect nitrite content through simple colorimetry, most of the samples they detect are water samples or other liquid samples, which do not require pre-treatment and are directly added to the container and the reagent is added for detection; there are also some kits that can detect solid samples. When detecting solids, this type of kit requires taking a portion of the sample and crushing it, then adding an appropriate amount of pure water to mix it thoroughly, letting it stand for a period of time, and then centrifuging it to take the supernatant and add the colorimetric reagent. Although this method can be used to detect solid substances, it still requires the centrifugation operation, which is not suitable for consumers to conduct on-site testing. In addition, the commonly used reagents for detecting nitrite are naphthylethylenediamine hydrochloride and p-aminobenzenesulfonic acid, which are easily interfered with when detecting nitrite.

[0005] Nanozymes are enzyme mimics that have both the properties of nanomaterials and the catalytic ability of natural enzymes. Compared with natural enzymes, nanozymes have higher stability, can catalyze under different environmental conditions, and are easy to store and transport. At the same time, the preparation process of nanozymes is simple. These advantages make them widely used in the detection field.

[0006] Therefore, in view of the current problems of limited sample types for on-site detection and susceptibility to interference in detection, it is of great significance to develop a method for on-site detection that is not restricted by sample types and can specifically detect nitrite. Summary of the invention

[0007] In view of the problems and shortcomings of the prior art, the present invention provides a method for quickly detecting nitrite using a porous polymer microneedle patch loaded with nanoenzymes. The microneedle patch of the present invention can be used as an indicator for detecting the nitrite content in food, without the need for pre-treatment of the sample, and can more simply and quickly detect the nitrite content in commercially available food.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] The first aspect of the present invention provides a method for detecting nitrite based on a porous polymer microneedle patch loaded with nanozymes, characterized in that it comprises steps S1, S2, and S3:

[0010] S1. Preparation of porous polymer microneedle patches:

[0011] S2. Preparation of nanozyme: The nanozyme is a copper-iron doped gallium oxyhydroxide nanomaterial, referred to as nanozyme;

[0012] S3. The porous polymer microneedle patch prepared in step S1 is attached to the surface of the object to be tested, the solution after TMB oxidation by nanozymes is dripped onto the back of the patch, and the nitrite content is determined according to the color change of the porous polymer microneedle patch.

[0013] Further, step S2 specifically includes S21 and S22;

[0014] S21. Mix the gallium salt solution, copper salt solution and iron salt solution and adjust the pH to 9.0-9.5;

[0015] S22. Take the mixed solution obtained in step S21 and perform a hydrothermal synthesis reaction. After the reaction is completed, separate the solid and liquid, wash, and dry to obtain a copper-iron doped gallium oxyhydroxide nanomaterial.

[0016] In some preferred embodiments, in step S21, after the gallium salt solution, the copper salt solution and the iron salt solution are mixed, the step further includes adding CTAB to the mixed solution, and adjusting the pH to 9.0-9.5 after ultrasonic mixing.

[0017] In some embodiments, in step S22, the mixed solution is stirred for 1-1.2 hours.

[0018] In some embodiments, in step S22, the hydrothermal temperature is 80-220° C. and the time is 12-48 hours;

[0019] In some embodiments, in step S22, the washing step is performed with water for 3-5 times and with ethanol for 3-5 times.

[0020] Furthermore, in step S22, the gallium salt solution is Ga 3+ Solution; the copper salt solution is Cu 2+ Solution; the iron salt solution is Fe 3+ Solution.

[0021] In some preferred embodiments, the gallium salt solution is one of Ga(NO3)3 and GaCl3 solutions;

[0022] In some preferred embodiments, the Cu 2+ The salt solution is one of Cu(NO3)2, CuCl2, and CuSO4 solutions;

[0023] In some preferred embodiments, the Fe 3+ The salt solution is one of Fe(NO3)3 and FeCl3 solutions.

[0024] In some preferred embodiments, the amount of CTAB added is 2mmoL Ga 3+ Add 1±0.1mg CTAB,

[0025] In one embodiment, in step S21, the mixed solution: Ga 3+ , Cu 2+ , Fe 3+ The molar ratio is 1:0.6:(0-0.35).

[0026] In some preferred embodiments, in step S21, ammonia water is used to adjust the pH, preferably, ammonia water with a mass concentration of 10%-28% is used.

[0027] In one embodiment, in step S22, the hydrothermal reaction is carried out in a hydrothermal reactor, and the hydrothermal reactor is preferably a stainless steel autoclave lined with polytetrafluoroethylene.

[0028] In one embodiment, in step S22, the gallium oxyhydroxide nanomaterial solid powder is obtained by grinding after drying.

[0029] In a preferred embodiment, the method comprises the following steps: taking a 50 mL beaker, adding 4 mmoL Ga(NO3)3, 2.5 mmoL Cu(NO3)2, and 1 mmoL Fe(NO3)3 respectively thereto, adding 2 mg CTAB to the mixed solution and ultrasonicating for 5 min to mix evenly. The pH of the mixed solution is adjusted to 9.5 with concentrated ammonia water and stirred at room temperature for one hour, and then the solution is transferred to a 25 mL polytetrafluoroethylene-lined autoclave. After reacting in an oven at 120°C for 24 hours, the reactor is moved to a fume hood and cooled to room temperature. The centrifuged precipitate after the reaction is washed 3 to 5 times with water and anhydrous ethanol respectively, and the washed material is placed in a vacuum drying oven at 60°C to dry, and the dried material is ground into powder to obtain copper-iron doped gallium oxyhydroxide nanomaterials.

[0030] In some preferred embodiments, step S3 further includes preparing a colorimetric card based on a linear relationship of color change, and quantitatively determining the nitrite content in the analyte based on the color.

[0031] Furthermore, step S1 includes S11, S12, and S13:

[0032] S11. Prepare the pore-forming stock solution: dissolve the pore-forming agent PEG in a 2-methoxyethanol solution to obtain a solution A;

[0033] S12 Preparation of monomer solution: Glycidyl methacrylate, trimethylolpropane trimethacrylate and triethylene glycol dimethacrylate three solutions were mixed to obtain solution B, stored at -15 ℃ - 20 ℃, heated to room temperature before use;

[0034] S13. Curing and molding: After mixing the solution A and the solution B, add a photoinitiator and mix well. Cast the obtained porous polymer solution on a mold. After degassing under negative pressure, cure it under ultraviolet light under oxygen-proof conditions. After molding, take the patch out of the mold, heat it in a mixture of ethanol and water, remove the porogen and dry it to obtain a porous polymer microneedle patch.

[0035] Porous polymer microneedle patches are a large category of microneedle patches. Porous polymer microneedle patches have a large number of randomly distributed pores, which are conducive to the rapid wetting of the microneedle tips and can use capillary action to quickly extract tissue fluid from the sample. They have good biocompatibility, high mechanical strength and are non-toxic.

[0036] In one embodiment, in step S11, the solution preparation condition is to heat the solution to 50-60° C.; and the amount of the porogen PEG added is 0.6 g / 3 g to 0.7 g / 3.5 g.

[0037] In a preferred embodiment, in step S11, the temperature is heated to 50°C to dissolve the porogen PEG.

[0038] In a preferred embodiment, in step S12, the added amounts of reagents are: 1 mL of glycidyl methacrylate, 0.523 mL of trimethylolpropane trimethacrylate, and 1.57 mL of triethylene glycol dimethacrylate.

[0039] In a preferred embodiment, in step S13, the photoinitiator is 1-hydroxycyclohexyl phenone, and the amount added is 0.1 g ± 0.01 g; in step 3, the ultraviolet light wavelength is 365 nm, and the irradiation time is 0.75 to 1 hour. The preferred irradiation time is 1 hour.

[0040] In one embodiment, in step S13, the ratio of the mixed solution of ethanol and water is 1:1 by volume, the heating temperature is 50-60°C, and this step needs to be repeated 3-4 times. Preferably, heating is performed at 60°C for 1 hour.

[0041] In one embodiment, in step S13, a proper amount of porous polymer microneedle solution is cast into the microneedle mold, and the amount of the porous polymer microneedle solution added is 400-500 μL.

[0042] In one embodiment, the mold used is a PDMS mold, with a needle tip height of 1500 μm, a bottom diameter of 550 μm, a needle tip distance of 1200 μm, an array of 8*8 particles, a patch size of 12*12 mm, and a groove depth of 2 mm.

[0043] In one embodiment, in step S13, the specific operation of negative pressure degassing is: using a vacuum drying oven at a temperature of room temperature to 37° C., evacuating to 0.8 to 1.0 MPa, maintaining the pressure for 3 minutes, and repeating the operation 3 to 5 times.

[0044] In one embodiment, in step S13, the condition for isolating oxygen is to place the mold in an environment filled with nitrogen.

[0045] In a preferred embodiment, the method for preparing the porous polymer microneedle patch comprises the following steps:

[0046] Preparation of pore-forming stock solution: Under heating conditions of 60°C, 0.6g of pore-forming agent PEG is dissolved in 3g of 2-methoxyethanol solution to obtain solution A. Preparation of monomer stock solution: 1mL of glycidyl methacrylate, 0.523mL of trimethylolpropane trimethacrylate and 1.57mL of triethylene glycol dimethacrylate are mixed to obtain solution B. Preparation of porous polymer microneedle patch: The solution A and the solution B are mixed, and after mixing, 0.1g of photoinitiator is added, and mixed to obtain a porous polymer microneedle patch solution. 400-500μL of the solution is cast on the mold, and then it is vacuumed to 1.0MPa at room temperature, and the pressure is maintained for 3 minutes. The operation is repeated 3-5 times, and negative pressure degassing is performed three to four times. Under nitrogen-filled conditions, ultraviolet light is irradiated at a wavelength of 365nm for 1 hour for curing. After molding, the patch was taken out of the mold, placed in a solution of ethanol and water in a volume ratio of 1:1 and heated at 60°C for 1 hour. This operation was repeated three times to remove the porogen, and a porous polymer microneedle patch was obtained after drying.

[0047] The second aspect of the present invention provides an application of the method for detecting nitrite in food detection, wherein the food is food containing tissue fluid; the food includes but is not limited to fresh meat, pre-prepared dishes, and vegetables.

[0048] In some embodiments of the present invention, for detecting fresh meat, the steps include: taking 720 μL of PBS buffer solution with pH 3.5, adding 200 μL of 200 mM hydrogen peroxide solution, 60 μL of 3 mg / mL nanozyme suspension and 20 μL of 5 mM TMB solution, and fully reacting for 8 to 10 minutes. Insert the prepared porous polymer microneedle patch into the fresh meat to be tested, draw 200 μL of the blue solution that reacts and drips it on the back of the microneedle patch, react for 10 minutes, observe that the solution changes from blue to green, take a photo with a mobile phone to record the color and use software to analyze its color RGB, so as to obtain the content of nitrite in the sample to be tested.

[0049] The third aspect of the present invention provides a rapid test kit for nitrite in food based on a porous polymer microneedle patch loaded with nanozymes; the kit comprises: a polymer microneedle patch, a solution after TMB is oxidized by nanozymes, and a colorimetric card; the rapid test kit is used to perform detection according to the detection method.

[0050] Furthermore, the color of the colorimetric card is prepared according to the color-concentration change relationship; and the nitrite content in the analyte is quantitatively determined according to the color.

[0051] Furthermore, the method for preparing the solution after nanozyme oxidizes TMB is as follows: PBS buffer solution, hydrogen peroxide solution, and nanozyme suspension are mixed, and then TMB solution is added for oxidation.

[0052] Furthermore, in the kit, the PBS buffer solution, hydrogen peroxide solution, and nanozyme suspension are mixed and then packaged; the TMB solution is packaged separately; and when in use, the two are mixed and oxidized before use.

[0053] The beneficial effects produced by the present invention include at least:

[0054] 1. The colorimetric reagent of the present invention is TMB solution which is oxidized to blue by gallium oxyhydroxide nanozyme; the oxidized TMB can react specifically with nitrite, thereby avoiding interference from other ions, enhancing the anti-interference ability of the detection, and making the detection result more accurate;

[0055] The method for detecting nitrite based on the nanozyme has high detection sensitivity, strong anti-interference, wide application range, and is easy to use. It has high detection accuracy and sensitivity below 250 mg / kg; the recovery rate is basically above 90%;

[0056] 2. The nanomaterial preparation process of the present invention is simple, efficient, and low-cost. In addition, nanozymes are easier to store and transport than natural enzymes and are relatively relaxed in terms of reaction environment conditions.

[0057] 3. The present invention uses a mobile phone to collect color information on the back of the patch, and can convert the color information into RGB. By establishing a linear relationship between nitrite content and G / B, an intelligent method can be used to avoid pre-processing and conveniently detect whether fresh meat contains nitrite and its content;

[0058] 4. The porous needle patch of the present invention is made of a variety of polymers, and the needle tip has good mechanical properties, which is sufficient to penetrate into the tissue of the object to be tested; the capillary action of the porous structure is used to quickly extract tissue fluid in meat; and the porous polymer microneedle patch is easy to prepare, the preparation process is pollution-free, and has a wide range of applications and can be used for industrial production.

[0059] Of course, any product implementing the present invention does not necessarily need to achieve all of the above-mentioned technical effects at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0061] Figure 1 This is an appearance morphology picture of the porous polymer microneedle patch prepared in Example 1;

[0062] Figure 2 This is a morphology image of the needle tip portion of the porous polymer microneedle patch prepared in Example 1;

[0063] Figure 3 This is a scanning electron microscope image of the needle tip portion of the porous polymer microneedle patch prepared in Example 1;

[0064] Figure 4 This is a graph showing the relationship between the texture force and displacement of the porous polymer microneedle patch prepared in Example 1;

[0065] Figure 5 This is a SEM image of the copper-iron doped gallium oxyhydroxide nanomaterial prepared in Example 2;

[0066] Figure 6 This is an X-ray diffraction pattern of the copper-iron doped gallium oxyhydroxide nanomaterial prepared in Example 2;

[0067] Figure 7 The linear relationship diagram between the sodium nitrite content and the G / B value obtained in Example 3;

[0068] Figure 8 Colorimetric detection of the color change of nitrite in pork by adding the copper-iron-doped gallium oxyhydroxide nanomaterial prepared in Example 2 to the microneedle patch prepared in Example 1;

[0069] Fig. 9 This is a graph showing the specificity experimental results of Example 5;

[0070] Fig.10 This is the absorbance value diagram of the solution after the reaction in Example 6 at 652nm. DETAILED DESCRIPTION

[0071] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0072] Unless otherwise specified, the sources of the reagents used in the embodiments of the present invention can be purchased from the market.

[0073] In an embodiment of the present invention, a porous polymer microneedle patch loaded with nanozymes is provided for detecting nitrite in fresh meat, and the method comprises the following steps:

[0074] Step 1, prepare a solution for nanozyme oxidation of TMB: add hydrogen peroxide solution, TMB solution (3,3',5,5'-tetramethylphenylenediaminoacetate) and nanozyme material to PBS buffer to obtain a solution in which TMB is oxidized and turns blue; Step 2, detect the content of nitrite in fresh meat: insert a porous microneedle patch into fresh meat, add the TMB solution that turns blue after oxidation to the back of the patch, observe the degree of color change of the solution, and determine the content of nitrite in the sample.

[0075] In one embodiment, in step 1, the pH of the PBS buffer is 3.5, the concentration of the hydrogen peroxide solution is 200 mM, the concentration of the TMB solution is 5 mM, the solvent is a DMSO solution, and the concentration of the nanozyme material is 3 mg / mL.

[0076] In one embodiment, the volume ratio of each reaction solution is: PBS buffer: hydrogen peroxide solution: TMB solution: nanozyme material suspension = 72:20:2:6.

[0077] In one embodiment, in step 1, the time for TMB to oxidize to blue is 8 to 10 minutes.

[0078] In one embodiment, in step 2, the amount of oxidized TMB solution added to the back of the patch is 150-200 μL.

[0079] In one embodiment, in step 2, the solution change reaction time is 6 to 10 minutes.

[0080] Example 1 Preparation of porous polymer microneedle patch:

[0081] (1) Preparation of porogen solution: Dissolve 0.6 g of porogen PEG in 3 g of 2-methoxyethanol solution at 60°C to obtain solution A;

[0082] (2) preparing a monomer solution: mixing 1 mL of glycidyl methacrylate, 0.523 mL of trimethylolpropane trimethacrylate, and 1.57 mL of triethylene glycol dimethacrylate to obtain solution B;

[0083] (3) Preparation of porous polymer microneedle patch: Mix the solution A and the solution B, add 0.1g of photoinitiator after mixing, mix well, and prepare a porous polymer microneedle patch solution. Cast 400-500μL of the solution on the mold, and then evacuate it to 0.8-1.0MPa at room temperature, maintain the pressure for 3 minutes, repeat this operation 3-5 times, perform negative pressure degassing several times, and suck out the excess solution. In a nitrogen environment, cure it under ultraviolet light at a wavelength of 365nm for 1 hour. After molding, take the patch out of the mold, place it in a solution made of ethanol and water in a volume ratio of 1:1 and heat it at 60 degrees for 1 hour, repeat three times to remove the porogen, and obtain a porous polymer microneedle patch after drying.

[0084] Test of the porous polymer microneedle patch prepared in Example 1:

[0085] The porous polymer microneedle patch prepared in Example 1 was photographed for its appearance. The testing method was as follows: the microneedle patch was clamped with tweezers, and photographed with a mobile phone under appropriate light and background. The appearance of the porous polymer microneedle patch prepared in Example 1 is shown in FIG. Figure 1 shown.

[0086] The porous polymer microneedle patch prepared in Example 1 was photographed for needle tip morphology. The testing method was as follows: using a stereo microscope to photograph the needle tip morphology, setting the magnification on the microscope, placing the microneedle patch under appropriate light and angle, and photographing it. Figure 2 This is a partial needle tip morphology image of the porous polymer microneedle patch prepared in Example 1. Figure 3 This is a scanning electron microscope image of the needle tip portion of the porous polymer microneedle patch prepared in Example 1; the porous structure of the microneedle patch is conducive to the upward transmission of liquid through capillary action.

[0087] The polymer microneedle patch prepared in Example 1 was subjected to a mechanical property test, and the test method was as follows: using a texture analyzer and selecting a P / 25 probe; parameter settings: the measurement mode and option were TPA, the pre-measurement rate was 1 mm / s, and the force measurement began when the sensor touched the tip; the mid-measurement rate was 0.2 mm / s, and the force measurement ended when 50% of the strain occurred. The experiment was repeated, and the experimental results were converted into force results, and it was found that the average bearing force of the porous polymer microneedle patch tip was 41.81 N, which was sufficient to penetrate meat tissue. Figure 4 This is a force-displacement relationship diagram of the porous microneedle patch prepared in Example 1.

[0088] Example 2 Preparation of copper-iron doped gallium oxyhydroxide nanomaterials

[0089] (1) Preparation of Ga(NO3)3 solution: Take Ga(NO3)3 reagent and mix it with ultrapure water until the material is completely dissolved to obtain a 0.5 M Ga(NO3)3 solution.

[0090] (2) Preparation of Cu(NO3)2 solution: Take the Cu(NO3)2 reagent and mix it thoroughly with ultrapure water until the material is completely dissolved to obtain a 0.5 M CuNO3)2 solution.

[0091] (3) Preparation of Fe(NO3)3 solution: Take Fe(NO3)3 reagent and mix it with ultrapure water until the material is completely dissolved to obtain a 0.5M Fe(NO3)3 solution.

[0092] (4) Take 4 mL of 0.5 M Ga(NO3)3 solution, 2.5 mL of 0.5 M CuNO3)2 solution and 1 mL of 0.5 M Fe(NO3)3 solution, mix them evenly, add 2 mg of CTAB reagent, and ultrasonicate for 5 min. The mixed homogeneous solution is adjusted to pH 9.5 with 28% concentrated ammonia water and stirred at room temperature for 1 hour.

[0093] (5) The mixed solution obtained in (4) was transferred to a high-pressure stainless steel reactor lined with polytetrafluoroethylene and reacted in an oven at 120° C. for 24 hours.

[0094] (6) The reaction mixture of (5) was placed in a fume hood to cool to room temperature, centrifuged, the supernatant was discarded, and the precipitate was washed three times with ultrapure water and anhydrous ethanol respectively. The precipitate obtained after washing was placed in a vacuum drying oven at 60° C. and dried. The dried solid was ground into powder to obtain copper-iron doped gallium oxyhydroxide nanomaterials.

[0095] Figure 5 This is a scanning electron microscope image of the copper-iron doped gallium oxyhydroxide nanomaterial prepared in Example 2; the synthesized nanomaterial is rod-shaped with uniform size, smooth surface, few impurities and high purity; the size is nanoscale, with a diameter of 20-40nm and a length of 200-300nm.

[0096] Figure 6 This is an X-ray diffraction pattern of the copper-iron doped gallium oxyhydroxide nanomaterial prepared in Example 2. The diffraction peaks at 2θ=18.1°, 21.5°, 26.7°, and 37.2° show the synthesis of gallium oxyhydroxide, and the diffraction peak at 37.46° proves the successful doping of copper and iron.

[0097] Example 3 Establishment of the linear relationship between nitrite content and color G / B value detected by porous polymer microneedle patch loaded with nanozymes

[0098] The porous polymer microneedle patch prepared in Example 1 and the copper-iron-doped gallium oxyhydroxide nanozyme prepared in Example 2 were used to establish a linear relationship between the sodium nitrite content and the colorimetric G / B value. 1-1.1g of gelatin was added to 19-20mL of sodium nitrite solution of different concentrations at 50-60°C and heated until completely dissolved, and poured into a glass container to cool and solidify. The patch was inserted into the solidified gelatin, the solution was fully absorbed for 3-5 minutes, 720μL of PBS buffer solution with pH 3.5 was taken, 200μL of 200mM hydrogen peroxide solution, 60μL of 3mg / mL nanozyme suspension and 20μL of 5mM TMB solution were added, and the solution was fully reacted for 8-10 minutes until the solution turned blue, and 200μL of the blue solution was absorbed to the back of the patch, and the reaction was 8-10 minutes. The color of the solution was observed to change from blue to green. Take a picture with a mobile phone and analyze the color difference RGB value according to the software.

[0099] Establish a linear relationship between sodium nitrite concentration and color G / B as shown in Figure 7 .

[0100] Example 4: Detection of nitrite in fresh meat using porous polymer microneedle patch loaded with nanozymes

[0101] The nitrite content in fresh meat was detected using the porous polymer microneedle patch capable of loading nanozymes prepared in Example 1. Pig 1 and Pig 2 are the experimental results of pork from different parts of pork, and 5 groups were parallelly tested for each concentration.

[0102] Pork was spiked with the spike method to make the theoretical nitrite concentrations of 50, 100, 150, 200, and 250 mg / kg, respectively. The patch was attached to the surface of the treated fresh meat. 720 μL of PBS buffer solution with pH 3.5 was taken, 200 μL of 200 mM hydrogen peroxide solution, 60 μL of 3 mg / mL nanozyme suspension, and 20 μL of 5 mM TMB solution were added, and the solution was fully reacted for 8 to 10 minutes until the solution turned blue. 200 μL of the blue solution was drawn to the back of the patch and reacted for 8 to 10 minutes. The color of the solution was observed to change from blue to green. Take a photo with a mobile phone, and the nitrite content can be calculated by analyzing the color difference RGB value according to the software.

[0103] Table 1 is the data of nitrite content in pork detected in Example 4

[0104]

[0105] As shown in Table 1, the detection method of the present application has high detection sensitivity and a wide range of application. It has high detection accuracy and sensitivity below 250 mg / kg; and the recovery rate is basically above 90%.

[0106] Example 5 Specificity Test

[0107] The blue mixed solution after TMB is oxidized has the highest ultraviolet absorption peak at 652nm, and after the oxidized TMB reacts with nitrite, the solution changes from blue to green, the ultraviolet absorption peak at 652nm decreases, and the ultraviolet absorption peak at 445nm increases. Therefore, the specificity of the nanozyme prepared in Example 2 to nitrite is verified by adding / not adding interfering ions at 445nm and 652nm.

[0108] Take 700 μL of PBS buffer solution at pH 3.5, add 200 μL of 200 mM hydrogen peroxide solution, 60 μL of 3 mg / mL nanozyme suspension and 20 μL of 5 mM TMB solution, react for 8-10 minutes, and add 20 μL of 5 mM Fe 3+ Mg 2+ , Ca 2+ , K + 、Na + 、SO4 2- , glucose, urea, isoleucine, lysine, and set up a blank group at the same time. After reacting for 8-10 minutes, measure its UV absorption; take 680μL of pH 3.5 PBS buffer solution, keep other conditions unchanged, add other ions and 20μL of 5mM NaNO2 solution, keep other reaction conditions unchanged, and measure its UV absorption in the same way. The results are as follows Fig. 9 As shown in (experimental results of specific selective reaction). After reacting with nitrite, the solution changes from blue to green, and the ratio of A652 / A445 decreases. (Fe 3+ The solution itself is yellow, and the yellow mixed with the blue solution after the reaction will turn green, which has some interference, but due to NO 2- The difference in data when present / absent is not large, which may also indicate that there is no further reaction with nitrite and the color changes to green).

[0109] Example 6 Specificity Test

[0110] Take 700 μL of PBS buffer solution at pH 3.5, add 200 μL of 200 mM hydrogen peroxide solution, 60 μL of 3 mg / mL nanozyme suspension and 20 μL of 5 mM TMB solution, react for 8-10 minutes, and add 20 μL of 5 mM Fe 3+ Mg 2+ , Ca 2+ , K + 、Na + 、SO4 2- 、NO2 - , glucose, urea, isoleucine, lysine, and a blank group were set up at the same time to measure their ultraviolet absorption values ​​at 652nm. The results are as follows Fig.10 .

[0111] Fig.10 Since nitrite reacts with oxidized TMB, the solution changes from blue to green, so the absorbance at 652nm decreases. Other ions do not react with oxidized TMB, and the solution remains blue. 2- The absorbance value of the solution after the reaction at 652nm is significantly lower than that of the others.

[0112] The data of Examples 5-6 show that the nanozyme prepared in Example 2 of the present application has good specificity for detecting nitrite.

[0113] In summary, the present invention discloses an application of a porous polymer microneedle patch and copper-iron doped gallium oxyhydroxide in the detection of nitrite content; a TMB solution oxidized to blue by gallium oxyhydroxide nanozyme is used as a colorimetric reagent; a specific reaction can occur with nitrite, thereby avoiding interference from other ions and enhancing the anti-interference of the detection; a method for detecting nitrite based on the nanozyme has high detection sensitivity and a wide range of applications, and can also use intelligent methods to avoid pre-treatment, and conveniently detect whether fresh meat contains nitrite and its content; it has high detection accuracy and sensitivity below 250 mg / kg; and a recovery rate of more than 90%. The preparation process of the nanomaterial is simple, efficient, and low-cost, and the nanozyme is easier to store and transport than the natural enzyme, and is relatively loose to the reaction environment conditions.

[0114] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for detecting nitrite based on a porous polymer microneedle patch loaded with nanozymes, characterized in that: The steps include: S1. Preparation of porous polymer microneedle patches: S2. Preparation of nanozyme: The nanozyme is a copper-iron doped gallium oxyhydroxide nanomaterial, referred to as nanozyme; S3. The porous polymer microneedle patch prepared in step S1 is attached to the surface of the object to be tested, the solution after the nanozyme oxidizes TMB is dripped on the back of the patch, and the nitrite content is determined according to the color change of the porous polymer microneedle patch; The nanozyme has uniform size, smooth surface, a diameter of 20-40 nm, and a length of 200-300 nm.

2. The method for detecting nitrite based on a porous polymer microneedle patch loaded with nanozymes according to claim 1, characterized in that: Step S2 includes: S21. Mix the gallium salt solution, copper salt solution and iron salt solution and adjust the pH to 9.0-9.5; S22. Take the mixed solution obtained in step S21 and perform a hydrothermal synthesis reaction. After the reaction is completed, separate the solid and liquid, wash, and dry to obtain a copper-iron doped gallium oxyhydroxide nanomaterial.

3. The method for detecting nitrite based on a porous polymer microneedle patch loaded with nanozymes according to claim 1, characterized in that: Step S1 includes: S11. Prepare the pore-forming stock solution: dissolve the pore-forming agent PEG in a 2-methoxyethanol solution to obtain a solution A; S12 Preparation of monomer solution: Glycidyl methacrylate, trimethylolpropane trimethacrylate and triethylene glycol dimethacrylate three solutions were mixed to obtain solution B, stored at -15 ℃ - 20 ℃, heated to room temperature before use; S13. Curing and molding: After mixing the solution A and the solution B, add a photoinitiator and mix well. Cast the obtained porous polymer solution on a mold. After degassing under negative pressure, cure it under ultraviolet light under oxygen-free conditions. After molding, take the patch out of the mold, remove the porogen, and dry it to obtain a porous polymer microneedle patch.

4. The method for detecting nitrite based on a porous polymer microneedle patch loaded with nanozymes according to claim 2, characterized in that: In step S21, after the gallium salt solution, the copper salt solution and the iron salt solution are mixed, CTAB is added to the mixed solution, and the pH is adjusted to 9.0-9.5 after ultrasonic mixing.

5. The method for detecting nitrite based on a porous polymer microneedle patch loaded with nanozymes according to claim 2, characterized in that: In step S22, the mixed solution is stirred for 1-1.2 hours. and / or, in step S22, the hydrothermal temperature is 80-220° C. and the time is 12-48 hours; And / or, in step S22, the washing step is performed with water for 3-5 times and with ethanol for 3-5 times.

6. The method for detecting nitrite based on a porous polymer microneedle patch loaded with nanozymes according to claim 2, characterized in that: In step S22, the gallium salt solution is Ga 3+ Solution; the copper salt solution is Cu 2+ Solution; the iron salt solution is Fe 3+ Solution.

7. The method for detecting nitrite based on a porous polymer microneedle patch loaded with nanozymes according to claim 4, characterized in that: The amount of CTAB added is 2mmoL Ga 3+ Add 1±0.1mg CTAB, The concentration of the ammonia water is 10%-28%.

8. The method for detecting nitrite based on a porous polymer microneedle patch loaded with nanozymes according to claim 1, characterized in that: Step S3 also includes establishing a linear relationship between the sodium nitrite content and the colorimetric G / B value to quantitatively determine the nitrite content in the sample to be tested.

9. Application of the method according to any one of claims 1 to 8 in food testing, characterized in that: The food is food containing tissue fluid; the food includes but is not limited to raw meat, pre-prepared dishes, and vegetables.

10. A rapid test kit for nitrite in food based on a porous polymer microneedle patch loaded with nanozymes, characterized in that: include: Polymer microneedle patch, nanoenzyme oxidized TMB solution, colorimetric card; The rapid test kit is used to perform detection according to the detection method described in any one of claims 1 to 8.

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