Os-Rh nanozyme, preparation method thereof and application thereof in detecting glyphosate

Abstract

The present invention relates to an Os-Rh nanozyme, a preparation method thereof and an application thereof in detecting glyphosate. The present invention provides a novel Os-Rh nanozyme having peroxidase properties. The present invention also provides a method for preparing the Os-Rh nanozyme, which is obtained by reducing an aqueous solution of K2OsCl6, RhCl3·3H2O and glycine under ascorbic acid. The present invention also provides a dual-mode colorimetric platform based on the Os-Rh nanozyme to achieve high-sensitivity detection of glyphosate within a wide linear concentration range. The detection reagent or test paper for detecting glyphosate provided by the present invention can be used in the fields of chemical industry, environment, food and biochemical detection, and has high application value and good market prospects.

Description

Technical Field

[0001] The invention relates to an Os-Rh nanozyme, a preparation method thereof and application thereof in detecting glyphosate, and belongs to the technical field of pesticide residues and food safety detection. Background Art

[0002] Long-term consumption of agricultural products containing pesticide residues can cause great harm to human health, such as stomach pain, vomiting, headaches, etc. In severe cases, it may even lead to Parkinson's disease, cancer, and genetic diseases. For example, glyphosate, as a broad-spectrum, highly effective, non-selective organophosphorus herbicide, has been shown to have certain reproductive toxicity, teratogenicity, and mutagenicity, and can enter the human body through breathing and contact, thus posing a threat to human health. Therefore, in order to reduce the impact of pesticide residues in food on human health, it is necessary to strengthen its detection.

[0003] Traditional methods for detecting pesticides mainly include gas chromatography (GC), high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Although these methods are highly sensitive, they are complex procedures, require large equipment, are costly and require professional operators. In addition, surface-enhanced Raman spectroscopy, fluorescence, and electrochemical methods have also been used for the detection of pesticide residues. However, they also have some disadvantages, such as the need for complex pretreatment processes, poor stability and repeatability, etc. Therefore, it is necessary to develop a simpler and faster alternative method.

[0004] Colorimetry has received increasing attention in recent years because of its simplicity, speed, and easy determination of results, such as the ability to obtain results directly through naked eye observation. Until now, colorimetry is still the national standard method for many food quality inspections. In recent years, nanozymes have become potential substitutes for natural enzymes for pesticide residue detection due to their simple preparation, high stability, excellent catalytic performance, and low cost. However, most of the current nanozyme-based pesticide detection methods mainly focus on forming a cascade reaction system with natural enzymes such as acetylcholinesterase (AChE), choline oxidase (ChOx), and acid phosphatase (ACP). Their detection mechanism is to destroy the cascade reaction process by inhibiting the activity of natural enzymes by pesticides, thereby causing a significant change in the color of the detection system, thereby achieving the purpose of detection. For example, Song and his team prepared a Ce-NC single-atom nanozyme with high peroxidase mimetic activity, and constructed a cascade reaction system with AChE, thereby realizing the portable detection of omethoate, methamidophos, carbofuran, and endosulfan. Zuo et al. successfully designed a colorimetric sensor array composed of Cu-NC nanozymes with excellent peroxidase mimetic activity, AChE and ChOx. In this sensor array, organophosphorus pesticides can reduce the production of H2O2 in the system by inhibiting the activity of AChE, thereby hindering the oxidation of TMB to achieve colorimetric detection of them. For example, Tan and his colleagues constructed a new high-sensitivity colorimetric platform based on the Fe-N / C single-atom nanozyme (with oxidase mimetic activity)-ACP cascade reaction system for the detection of malathion. In this platform, malathion inhibits the activity of ACP, thereby limiting the production of ascorbic acid (AA) in the system, and then restores the oxidative activity of the nanozyme on TMB to achieve sensitive detection of it.

[0005] At present, there are few studies on the direct use of nanozymes to detect pesticides, and only a few reports have been published. For example, Huang et al. synthesized a CuCeTA nanoflower with outstanding peroxidase mimicking activity, and realized direct colorimetric detection of glyphosate by taking advantage of the property that glyphosate can effectively inhibit the peroxidase mimicking activity of CuCeTA. Wu et al. also developed a paper-based platform for rapid detection of glyphosate in agricultural products by taking advantage of the ability of glyphosate to effectively inhibit the peroxidase mimicking activity of Co3O4 nanosheets. Compared with the construction of a cascade catalytic system in combination with natural enzymes, the direct use of nanozymes to detect pesticides has obvious advantages, such as simplicity, convenience, low cost, and high stability (not affected by the stability and activity of natural enzymes). Therefore, it is of great research and practical significance to develop more nanozymes that can be used for direct detection of pesticides, and then develop more direct colorimetric detection methods for pesticides. Summary of the invention

[0006] One purpose of the present invention is to provide a novel Os-Rh nanozyme (osmium-rhodium nanozyme), and another purpose of the present invention is to provide a dual-mode colorimetric detection platform for glyphosate based on Os-Rh nanozyme. The dual-mode detection platform can achieve high-sensitivity detection of glyphosate in a wide linear concentration range (solution platform is 50-1000 μg / L, paper platform is 200-1000 μg / L), the detection limit of the solution platform is 28.37 μg / L, and the detection limit of the paper platform is 400 μg / L (naked eye) / 123.25 μg / L (gray value). When it is used for actual sample detection, it also shows good reliability and accuracy, with a spike recovery rate in the range of 85.47% to 96.51% and an RSD in the range of 0.98% to 4.96%. The glyphosate dual-mode colorimetric detection platform based on Os-Rh nanozymes constructed by this invention can be used in the fields of chemical, environmental, food and biochemical detection, and has high application value and good market prospects.

[0007] The third object of the present invention is to provide the application of the above-mentioned glyphosate dual-mode colorimetric detection platform based on Os-Rh nanozyme in food safety detection.

[0008] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0009] An Os-Rh nanozyme is prepared by mixing aqueous solutions of K2OsCl6, RhCl3·3H2O and glycine, preheating, adding ascorbic acid for reduction incubation, and then dialyzing in deionized water using a dialysis bag to obtain the Os-Rh nanozyme.

[0010] For the Os-Rh nanozyme as described above, preferably, the preheating temperature is 50-70°C, the reduction incubation temperature is 50-70°C, and the molecular weight of the dialysis bag is 500D.

[0011] As described above, in the Os-Rh nanozyme, preferably, the final concentration of K2OsCl6 in the aqueous solution is 2-5 mM, the final concentration of RhCl3·3H2O in the aqueous solution is 1-4 mM, the final concentration of glycine in the aqueous solution is 60-120 mM; and the final concentration of ascorbic acid in the aqueous solution is 0.1-0.3 M.

[0012] More preferably, the final concentration of K2OsCl6 in the aqueous solution is 2 mM, the final concentration of RhCl3·3H2O in the aqueous solution is 1 mM, the final concentration of glycine in the aqueous solution is 81 mM; and the final concentration of ascorbic acid in the aqueous solution is 0.1364 M.

[0013] For the Os-Rh nanozyme as described above, preferably, the mixing is performed by vortex mixing, the vortex mixing time is 5 to 20 minutes, the preheating time is 5 to 20 minutes, the incubation time is 1 to 5 hours, and the dialysis time is 8 to 40 hours. More preferably, the vortex mixing time is 10 minutes, the preheating time is 5 minutes, the incubation time is 2 hours, and the dialysis time is 24 hours.

[0014] As mentioned above, Os-Rh nanozyme has peroxidase mimicking activity and can catalyze H2O2 to produce hydroxyl radicals (HO·) to oxidize TMB, and can be used for peroxidase-mimicking applications.

[0015] The use of the Os-Rh nanozyme as described above as or in the preparation of peroxidase-like enzymes.

[0016] The application of the Os-Rh nanozyme as described above in the preparation of a glyphosate detection reagent.

[0017] The detection reagent as described above includes a detection solvent for detecting glyphosate, or a test paper for detecting glyphosate.

[0018] A test paper for detecting glyphosate, wherein an Os-Rh nanozyme solution is coated on the test paper.

[0019] A method for detecting glyphosate based on Os-Rh nanozyme, comprising the following steps:

[0020] S1. After adding the Os-Rh nanozyme solution and glyphosate solutions of different concentrations, i.e., standard products, to a 96-well plate for mixed reaction, the test sample and the Os-Rh nanozyme solution are also added to the 96-well plate, and a blank solution without glyphosate and the Os-Rh nanozyme solution are also added to the 96-well plate as a blank control;

[0021] S2, adding reaction substrate solution to each of the above wells for reaction;

[0022] S3, measuring the ultraviolet absorption spectrum of each well at 650 nm after reaction;

[0023] S4. According to the formula Calculate the inhibition rate of each standard, and draw a standard curve with the standard concentration as the horizontal axis and the inhibition rate of the standard as the vertical axis, where A0 is the absorbance value of the blank control group at 650nm, and A1 is the absorbance value of the standard with glyphosate added at 650nm;

[0024] S5. Substitute the inhibition rate of the sample to be tested into the standard curve to obtain the concentration of glyphosate in the sample to be tested.

[0025] In the method as described above, preferably, in step S1, the mixing time is 15 min; in step S2, the reaction substrate solution is TMB and H2O2 solution, the amount thereof is 200 μL, and the concentrations are 2.0 mM TMB and 10 mM H2O2; and the reaction time is 10 min.

[0026] A paper colorimetric method for detecting glyphosate based on Os-Rh nanozyme comprises the following steps:

[0027] S1. Add different concentrations of glyphosate standard to the test paper containing the Os-Rh nanozyme as described above; at the same time, a solution without glyphosate is used as a blank control, and the test solution is tested at the same time;

[0028] S2. After a period of time, the reaction substrate solution is added dropwise onto the test paper to react;

[0029] S3, take a photo of the test paper after the reaction with a mobile phone, and obtain the gray value of the test paper through software;

[0030] S4. According to the formula Calculate the inhibition rate of each standard, and draw an inhibition standard curve with the concentration of glyphosate in the standard as the horizontal axis and the inhibition rate at the corresponding concentration as the vertical axis; wherein G0 represents the gray value of the blank solution of the test paper when glyphosate and the reaction substrate solution are not added, and G1 represents the gray value of the test paper when glyphosate and the reaction substrate solution are added;

[0031] S5. Substitute the inhibition rate of the sample to be tested into the standard curve to obtain the concentration of glyphosate in the sample to be tested.

[0032] In the paper colorimetry method as described above, preferably, the standing time is 15 minutes, the reaction substrate solution is a solution containing 2.91 mM TMB and 10 mM H2O2; and the reaction time is 8 minutes.

[0033] The paper colorimetric method can be observed with the naked eye. When there is an obvious color change, it can be qualitatively determined that there is glyphosate in the sample to be tested. When the test paper is colorless, it means that the concentration of glyphosate in the sample to be tested is greater than 1200μg / L.

[0034] (III) Beneficial effects

[0035] The beneficial effects of the present invention are:

[0036] The present invention successfully prepared a novel Os-Rh nanozyme with good stability and high catalytic activity for the first time. The nanozyme has high peroxidase mimicking activity and can catalyze H2O2 to produce HO·oxidized TMB. The prepared nanozyme showed a wide linear detection range and excellent detection sensitivity when used for dual-mode colorimetric detection of the pesticide glyphosate. In the solution detection platform, the linear detection range was 50-1000 μg / L, and the detection limit was 28.37 μg / L; in the paper detection platform, the linear detection range was 200-1000 μg / L, the naked eye detection limit was 400 μg / L, and the gray value detection limit was 123.25 μg / L. It also showed satisfactory reliability and accuracy when used for actual sample detection, with the spike recovery rate ranging from 85.47% to 96.51% and the relative standard deviation (RSD) ranging from 0.98% to 4.96%.

[0037] The test paper for detecting glyphosate provided by the present invention can obtain the test result by naked eyes, and does not require professional personnel or professional equipment to obtain the qualitative test result of glyphosate, which is simpler and faster.

[0038] The Os-Rh nanozyme prepared by the present invention has the characteristics of high-performance peroxidase, can be used in the fields of chemical industry, environment, food and biochemical detection, and has high application value and good market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The characterization results of Os-Rh nanozyme, where a is the TEM image, b is the size distribution diagram, c is the EDS spectrum, d is the EDS Mapping spectrum of Os and Rh elements, and e is the XRD spectrum;

[0040] Figure 2 The results of peroxidase-like activity verification of Os-Rh nanozyme are UV-visible absorption spectra measured in the range of 500-800 nm after 10 min of reaction;

[0041] Figure 3 Steady-state kinetic test results of Os-Rh nanozyme on TMB (a and c) and H2O2 (b and d);

[0042] Figure 4The results of the feasibility study of dual-mode direct colorimetric detection of glyphosate based on Os-Rh nanozyme; Figure a shows the feasibility of the solution platform for detecting glyphosate (a: TMB+H2O2+Os-Rh nanozyme, b: TMB+H2O2+glyphosate (700μg / L)+Os-Rh nanozyme, c: TMB+H2O2+glyphosate (1000μg / L)+Os-Rh nanozyme, d: TMB+H2O2+glyphosate (1000μg / L)+Os-Rh nanozyme). 2+glyphosate (700μg / L), e: TMB+H2O2+glyphosate (1000μg / L)); Figure b shows the feasibility of the paper platform to detect glyphosate (a: TMB+H2O2+Os-Rh nanozyme, b: TMB+H2O2+glyphosate (1000μg / L)+Os-Rh nanozyme, c: TMB+H2O2+glyphosate (1000μg / L), d: TMB+H2O2);

[0043] Figure 5 The results of detecting the ability of Os-Rh nanozyme to produce ·OH in the presence or absence of glyphosate, wherein a is the fluorescence spectra of the systems (TA+H2O2+Os-Rh nanozyme) and (TA+H2O2+Os-Rh nanozyme+glyphosate (900 μg / L)) measured at an excitation wavelength of 315 nm; b is the ESR spectra of the systems (DMPO+Os-Rh nanozyme+H2O2) and (DMPO+Os-Rh nanozyme+H2O2+glyphosate (900 μg / L));

[0044] Figure 6 The results of the optimization of the detection conditions of the solution platform based on Os-Rh nanozyme, where a is the reaction time, b is the concentration of Os-Rh nanozyme, and c is the time for glyphosate (1200 μg / L) to inhibit the activity of Os-Rh nanozyme;

[0045] Figure 7 The optimization results of the paper platform detection conditions based on Os-Rh nanozyme, where a is the reaction time, b is the Os-Rh nanozyme concentration, c is the TMB concentration, and d is the H2O2 concentration;

[0046] Figure 8 The results of the selective exploration of glyphosate detection based on the dual-mode colorimetric platform of Os-Rh nanozyme, where a is the color and absorbance value at 650nm after adding glyphosate and other pesticides to the system (Os-Rh nanozyme + H2O2 + TMB) on the solution platform, and b is the color and grayscale value of the system (Os-Rh nanozyme + H2O2 + TMB) mixed with glyphosate and other pesticides on the paper platform;

[0047] Fig. 9The results of glyphosate detection by the solution platform, where a is the UV-visible absorption spectrum of the system (Os-Rh nanozyme + TMB + H2O2) under different concentrations of glyphosate, b is the inhibition curve of glyphosate on the peroxidase mimicking activity of Os-Rh nanozyme, and c is the linear curve of glyphosate detection by the solution platform;

[0048] Fig.10 The results of glyphosate detection by the paper platform, where a is the color change of the system (Os-Rh nanozyme + TMB + H2O2) under different concentrations of glyphosate, b is the inhibition curve of glyphosate on the peroxidase mimicking activity of Os-Rh nanozyme, and c is the linear curve of glyphosate detection by the paper platform;

[0049] Fig.11 The storage stability results of the paper platform based on Os-Rh nanozyme;

[0050] Fig.12 The results of the dual-mode detection platform and instrument detection method (HPLC-MS) for the detection of real spiked samples, a is the spiked concentration of glyphosate at 500 μg / L and b is the spiked concentration of glyphosate at 900 μg / L;

[0051] Fig.13 are the characterization results of the Os-Rh-(1) nanomaterial in the comparative example, a is the TEM image of the Os-Rh-(1) nanomaterial, b is the peroxidase-like activity verification result of the Os-Rh-(1) nanomaterial, and the UV-visible absorption spectrum measured in the range of 500 to 800 nm after 10 minutes of reaction. DETAILED DESCRIPTION

[0052] Traditional nanozyme-based colorimetric detection of pesticides usually requires the assistance of natural enzymes, and its process and results are easily affected by the stability and activity of natural enzymes. To overcome this limitation, the present invention successfully prepared Os-Rh nanozymes with excellent peroxidase-like activity, and constructed a dual-mode colorimetric platform combining solution colorimetry and paper colorimetry for direct detection of glyphosate. The results showed that glyphosate can sensitively and selectively inhibit the peroxidase-like activity of Os-Rh nanozymes by hindering the decomposition of H2O2 by Os-Rh nanozymes to produce HO·. On this basis, the dual-mode colorimetric platform achieves high-sensitivity detection of glyphosate in a wide linear concentration range (solution platform is 50-1000μg / L, paper platform is 200-1000μg / L), and the detection limit of the solution platform is 28.37μg / L, and the detection limit of the paper platform is 400μg / L (naked eye detection limit) / 123.25μg / L (grayscale detection limit). In addition, the dual-mode colorimetric platform also showed satisfactory reliability and accuracy in practical applications, and has been successfully applied to the detection of actual samples, with a spike recovery rate of 85.47% to 96.51% and a relative standard deviation (RSD) of 0.98% to 4.96%. In short, the dual-mode direct colorimetric detection platform for glyphosate based on Os-Rh nanozymes provided by the present invention has considerable practical application potential. At the same time, it can also provide more inspiration and ideas for the rational design and development of colorimetric sensing methods for rapid detection of pesticides based on nanozymes.

[0053] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0054] Example 1 Preparation and characterization of Os-Rh nanozyme

[0055] 50 μL of 40 mM K2OsCl6 solution, 50 μL of 20 mM RhCl3·3H2O solution and 900 μL of 90 mM glycine solution were added to a 1.5 mL test tube. After vortexing for 10 min, the mixture was preheated at 60 ° C for 5 min. Then, 100 μL of 1.5 M freshly prepared ascorbic acid was added and incubated at 60 ° C for 2 h. Finally, the reaction mixture was dialyzed in deionized water with a 500D dialysis bag for 24 h to obtain the Os-Rh nanozyme stock solution.

[0056] The obtained Os-Rh nanozyme was detected by transmission electron microscopy (TEM), EDS spectrometer, and X-ray diffraction (XRD). Figure 1 As shown. Figure 1 The results of a and b in Figure 2 show that Os-Rh nanozymes are dispersed elliptical particles with an average particle size of about 18.53 nm. EDS elemental analysis results ( Figure 1c) shows that Os-Rh nanozyme contains only Os (osmium) and Rh (rhodium). In addition, as shown in the EDS spectrum results ( Figure 1 d), the two elements are evenly distributed on the Os-Rh nanozyme. In addition, from the XRD results ( Figure 1 e) It can be found that Os-Rh nanozyme has five diffraction peaks at 38.0°, 41.1°, 41.8°, 43.6° and 47.8°, which can correspond to (100), (002) and (101) of fcc Os (JCPDS 06-0662) and (111) and (200) of fcc Rh (JCPDS 05-0658), respectively. The above results indicate that Os-Rh nanozyme was successfully prepared.

[0057] Example 2 Verification of the peroxidase-mimicking activity of Os-Rh nanozyme

[0058] First, the peroxidase mimicking activity of Os-Rh nanozyme was verified by evaluating whether it could catalyze the oxidation of the chromogenic substrate TMB in the presence of H2O2. Briefly, 10 μL of Os-Rh nanozyme solution (20-fold dilution of the stock solution) and 200 μL of reaction substrate solution (containing 2.0 mM TMB and 10 mM H2O2) were added to a 96-well plate and reacted at room temperature for 10 min. The UV-visible absorption spectrum (500-800 nm) of the system was then recorded using an ELISA reader. The group without the addition of Os-Rh nanozyme or H2O2 was set as the control group to observe the effect of H2O2 or Os-Rh nanozyme on the oxidation of TMB. The group without the addition of H2O2 and TMB was set as the blank group, and all results were removed from the blank value.

[0059] The catalytic properties of Os-Rh nanozyme were then further explored by steady-state kinetic analysis. In brief, 10 μL of Os-Rh nanozyme solution (stock solution diluted 20 times) and 200 μL of reaction substrate solution containing 10 mM H2O2 and different concentrations of TMB (2, 1, 0.5, 0.125, 0.0625 mM), or 2.0 mM TMB and different concentrations of H2O2 (10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 mM) were added to a 96-well plate and reacted at room temperature for 10 minutes. The absorbance of the system at a wavelength of 650 nm was then measured by an ELISA instrument. The group without TMB and H2O2 was set as the blank control group, and the blank value was removed from all results. The concentration of oxTMB produced by the above system was calculated as follows:

[0060] A=ξbc(1)

[0061] Where A represents the system absorbance value measured at a wavelength of 650 nm, and ξ represents the molar absorption coefficient of oxTMB (3.9×104 M- 1 cm- 1 ), b represents the thickness of the absorption layer, and c represents the concentration of oxTMB.

[0062] The oxTMB concentration produced per unit time (V0) is then calculated according to the following formula:

[0063]

[0064] Wherein, t is the reaction time, which is 10 min in the present invention.

[0065] Then, the reciprocal of the oxTMB concentration generated per unit time (1 / V0) and the reciprocal of the substrate TMB (or H2O2) concentration (1 / [S]) were plotted as the ordinate and abscissa, respectively. Finally, the Michaelis constant (K) was calculated according to the following formula: m ) and maximum reaction speed (V max ):

[0066]

[0067] The results are as follows Figure 2 As shown, it shows that Os-Rh nanomaterials cannot directly catalyze the oxidation of TMB, but in the presence of H2O2, TMB can be catalyzed and oxidized into a blue product with the strongest absorption near 650nm, which indicates that Os-Rh nanomaterials have peroxidase-like activity.

[0068] In steady-state kinetic studies, the Michaelis constant (K m ) value reflects the affinity of the enzyme to the corresponding substrate. The smaller the value, the stronger the affinity. Maximum initial velocity (V max ) represents the maximum speed of the catalytic reaction. The larger the value, the faster the reaction speed. Figure 3 a and 3b are typical Michaelis-Menten curves established with TMB and H2O2 as substrates, respectively. Based on these results, the corresponding Lineweaver-Burk plots were obtained ( Figure 3 c and 3d). After the final calculation, the K of Os-Rh nanozyme to TMB m and V max 0.26 mM and 76.64 × 10 -8 M / s, K for H2O2 m and V max 0.38mM and 48.39×10 -8 M / s. Table 1 lists the K values ​​of HRP and other reported nanozymes with peroxidase-like activity. m and V max .

[0069] Table 1. Comparison of K of Os-Rh nanozyme and other catalysts m and V max

[0070]

[0071] From the above, it can be found that the affinity and maximum reaction rate of Os-Rh nanozyme to TMB and H2O2 are higher than those of HRP and other nanozymes, which indicates that the Os-Rh nanozyme obtained in the present invention has good peroxidase-like activity and is expected to become a candidate material for nanoperoxidase.

[0072] Example 3 Feasibility study of dual-mode direct colorimetric detection of glyphosate based on Os-Rh nanozyme

[0073] By evaluating the inhibitory ability of high concentration of glyphosate on the peroxidase-like activity of Os-Rh nanozyme on solution platform and paper platform respectively, the feasibility of direct detection of glyphosate by dual-mode colorimetric platform based on Os-Rh nanozyme was explored.

[0074] In brief, on the solution platform, 10 μL of Os-Rh nanozyme solution (stock solution diluted 20 times and 10 μL of glyphosate solution (700 μg / L or 1000 μg / L) were first added to a 96-well plate. After mixing at room temperature for 15 minutes, 200 μL of reaction substrate solution containing 2.0 mM TMB and 10 mM H2O2 was added, and the reaction continued for 10 minutes at room temperature. Subsequently, an iPhone 14 pro was used to take pictures, and a microplate reader was used to obtain the ultraviolet absorption spectrum of the reaction system at 500-800 nm. In addition, the group without glyphosate or Os-Rh nanozyme was set as the control group.

[0075] For the paper platform, first add 10 μL of Os-Rh nanozyme solution (the stock solution was diluted 30 times) to a clean test paper with a diameter of 8 mm and dry it naturally at room temperature to prepare a glyphosate detection test paper. Then, add 10 μL of glyphosate solution (1000 μg / L) to the test paper and leave it at room temperature for 15 minutes. Then, add 20 μL of reaction substrate solution (containing 2.91 mMTMB and 10 mM H2O2) to the test paper. After 8 minutes of reaction at room temperature, the color of the test paper was recorded with an iPhone 14pro, and the grayscale (G) value of the test paper was obtained using Image J software. In addition, the group without glyphosate and Os-Rh nanozyme was set as the control group, and the group containing glyphosate and Os-Rh nanozyme was set as the blank control group. △G is the difference between the G value of the blank group and the G value of the test group or the control group.

[0076] The results are as follows Figure 4 As shown, Figure 4a shows that after adding different concentrations of glyphosate to the system (TMB+H2O2+Os-Rh nanozyme), the color of the solution (tubes b and c) changes from the original blue to light blue or even colorless, corresponding to a significant decrease in the absorption peak at 650nm. This indicates that glyphosate can effectively inhibit the peroxidase-like activity of Os-Rh nanozyme, and the inhibition increases with the increase in glyphosate concentration. In addition, test tubes d and e are mixtures of substrate solution (TMB+H2O2) and glyphosate of different concentrations, both of which are colorless and have no characteristic absorption peak at a wavelength of 650nm, indicating that glyphosate has no effect on the oxidation of TMB in the system. The above results show that the solution platform for direct colorimetric detection of glyphosate using Os-Rh nanozyme is feasible.

[0077] Likewise, from Figure 4 As can be seen in b, when the substrate solution (TMB+H2O2) was dripped onto the paper (containing Os-Rh nanozyme), the paper turned blue-green (paper a), corresponding to a higher △G value (G value of the blank group - G value of the sample group), which can be attributed to the peroxidase-like activity of the Os-Rh nanozyme. When a certain amount of high concentration (1000 μg / L) of glyphosate was dripped before the substrate solution was added, the paper could not change color (paper b), indicating that glyphosate can also effectively inhibit the peroxidase-like activity of the Os-Rh nanozyme on the paper platform, and can therefore be used to design a new paper platform for direct colorimetric detection of glyphosate.

[0078] In summary, Os-Rh nanozyme can be used to develop a dual-mode colorimetric detection platform (solution platform and paper platform) for direct detection of glyphosate.

[0079] Example 4 Study on the direct colorimetric detection mechanism of glyphosate based on Os-Rh nanozyme

[0080] A large number of studies have shown that the essence of nanozyme peroxidase mimicking activity is that nanozymes catalyze the decomposition of H2O2 to produce HO·, which is then further oxidized to chromogenic substrates (such as TMB, ABTS, etc.). Therefore, it is reasonable to speculate that glyphosate inhibits its peroxidase-like activity by hindering the HO· produced by the decomposition of H2O2 catalyzed by Os-Rh nanozymes. In order to verify this speculation, the present invention uses terephthalic acid (TA) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as HO· capture agents to explore the ability of Os-Rh nanozymes to catalyze H2O2 to produce HO· in the presence or absence of glyphosate, as follows:

[0081] After the systems (TA (70 μL, 5 mM) + H2O2 (600 μL, 10 mM) + Os-Rh nanozyme (30 μL, the stock solution was diluted 20 times)) and (TA (70 μL, 5 mM) + H2O2 (600 μL, 10 mM) + Os-Rh nanozyme (30 μL, the stock solution was diluted 20 times) + glyphosate (900 μg / L)) were reacted at 45 °C for 30 min, the fluorescence spectra of the systems were recorded using a fluorescence spectrophotometer with an excitation wavelength of 315 nm. In addition, after the systems (DMPO (100 μL, 50 mM) + Os-Rh nanozyme (30 μL, 20-fold dilution of the stock solution) + H2O2 (600 μL, 10 mM)) and (DMPO (100 μL, 50 mM) + Os-Rh nanozyme (30 μL, 20-fold dilution of the stock solution) + H2O2 (600 μL, 10 mM) + glyphosate (900 μg / L)) were reacted at pH 6 for 5 min, the electron spin resonance (ESR) spectra of the systems were monitored using a Bruker A300 spectrometer.

[0082] Figure 5 The results in a show that the system (TA+H2O2+Os-Rh nanozyme) has a strong fluorescence emission near 435nm. This is because Os-Rh nanozyme catalyzes H2O2 to produce a large amount of HO·, which can be captured by TA to produce fluorescent TA-OH. However, when glyphosate (900μg / L) was added, the fluorescence intensity of the system decreased significantly, indicating that glyphosate can inhibit Os-Rh nanozyme from catalyzing H2O2 to produce HO·. In addition, from Figure 5 b It can be found that the system (DMPO+Os-Rh nanozyme+H2O2) can effectively generate a high signal with a ratio of 1:2:2:1, corresponding to the ESR signal of HO·. However, the system (DMPO+Os-Rh nanozyme+H2O2+glyphosate) hardly generates such a signal, which further indicates that glyphosate has the ability to inhibit the catalysis of H2O2 by Os-Rh nanozyme to produce HO·.

[0083] In summary, glyphosate inhibits the peroxidase-like activity of Os-Rh nanozyme by hindering the HO· produced by the decomposition of H2O2 catalyzed by Os-Rh nanozyme, thereby achieving direct colorimetric detection of glyphosate.

[0084] Example 5 Optimization of conditions for detecting glyphosate using a dual-mode colorimetric platform based on Os-Rh nanozymes

[0085] To achieve the best sensing performance for glyphosate detection, the sensing conditions of the solution platform and paper platform were optimized, respectively.

[0086] For the solution platform, the optimized conditions included the concentration of Os-Rh nanozyme (expressed as the dilution multiple of the original solution: 10, 20, 40, 80, 160), the enzymatic reaction time (2, 5, 10, 15, 20, 25 min) and the time for glyphosate to inhibit the activity of Os-Rh nanozyme (5, 10, 15, 20, 25, 30 min). In brief, the optimization of Os-Rh nanozyme concentration and enzymatic reaction time was as follows: 10 μL of Os-Rh nanozyme solution diluted a certain number of times and 200 μL of reaction substrate solution (containing 2.0 mM TMB and 10 mM H2O2) were added to a 96-well plate, and after a certain reaction time at room temperature, the absorbance value of the system at 650 nm was measured. The optimization steps for glyphosate to inhibit the activity time of Os-Rh nanozyme are as follows: first, add 10 μL of Os-Rh nanozyme solution (the stock solution was diluted 20 times) and 10 μL of 1200 μg / L glyphosate solution to a 96-well plate. After mixing for a period of time, add 200 μL of reaction substrate solution (containing 2.0 mMTMB and 10 mM H2O2), and continue to react at room temperature for 10 minutes. Then, measure the absorbance value of the system at 650 nm. The group without adding the reaction substrate solution was set as the blank control group, and the final absorbance values ​​of all groups were the absorbance values ​​after removing the absorbance value of the blank control group.

[0087] For the paper platform, the optimized conditions include enzymatic reaction time (2, 4, 6, 8, 10, 12, 14min), Os-Rh nanozyme concentration (expressed by the dilution multiple of the stock solution, 0, 10, 20, 30, 40, 50), TMB concentration (0.42, 1.25, 2.08, 2.91, 3.74mM) and H2O2 concentration (0.63, 1.25, 2.5, 5, 10, 20mM). The optimization procedure is as follows: 10μL of Os-Rh nanozyme solution diluted a certain multiple and 20μL of reaction substrate solution containing a certain concentration of TMB and H2O2 are added dropwise to the prepared glyphosate detection test paper. After reacting for a certain period of time at room temperature, the color of the test paper was recorded by taking a photo with an iPhone 14pro, and the grayscale (G) value of the test paper was obtained using Image J software. The test paper without the addition of the reaction substrate solution was set as the blank group, and the △G value of the test paper was the difference between the G values ​​of the blank group and the test group.

[0088] Figure 6 a shows that as the reaction time increases, the absorbance value of the system gradually increases, and then stabilizes after 10 minutes, indicating that TMB in the system can be completely oxidized within 10 minutes. Therefore, 10 minutes is selected as the optimal reaction time. Figure 6As shown in Figure b, as the dilution factor of Os-Rh nanozyme increases (concentration decreases), the absorbance value of the reaction system hardly changes at first. When the dilution factor is greater than 20, the absorbance value gradually decreases. This indicates that when the dilution factor of Os-Rh nanozyme is less than or equal to 20, TMB in the system can be completely oxidized within 10 minutes, so the final dilution factor is set to 20. Figure 6 c It can be seen that as the inhibition time of glyphosate on Os-Rh nanozyme increases, the absorbance value of the system gradually decreases and is almost zero after 15 minutes, which means that glyphosate (1200 μg / L) can completely inhibit the activity of Os-Rh nanozyme after 15 minutes of action, so 15 minutes is selected as the optimal inhibition time. In short, the optimal detection conditions of the solution platform are: the reaction time is 10 minutes, the concentration of Os-Rh nanozyme is 20 times the original solution, and the inhibition time of glyphosate on Os-Rh nanozyme is 15 minutes.

[0089] Figure 7 a shows that as the reaction time increases, the △G value of the paper first gradually increases (2 to 8 minutes) and then tends to be stable (8 to 14 minutes), indicating that TMB can be completely oxidized and colored on the paper within 8 minutes, so 8 minutes is selected as the optimal reaction time. Figure 7 b shows that as the dilution factor increases (concentration decreases), the △G value of the paper first gradually increases (this is because the excessive concentration of Os-Rh nanozyme causes TMB peroxidation, making the paper appear yellow-green, which leads to a decrease in the △G value), and then gradually decreases. When the dilution factor is 30, the △G value of the paper is the largest, so the final dilution factor is set to 30. Figure 7 The results of c show that as the TMB concentration increases, the △G value of the paper gradually increases and then stops changing (after 2.91 mM), so 2.91 mM is selected as the appropriate TMB concentration. In addition, Figure 7 d shows that when the concentration of H2O2 is greater than 10mM, the △G value of the paper hardly changes, so 10mM is selected as the appropriate concentration of H2O2. In general, the optimal detection conditions of the paper platform are: reaction time of 8min, Os-Rh nanozyme concentration diluted 30 times of the stock solution, and TMB and H2O2 concentrations of 2.91mM and 10mM, respectively.

[0090] Example 6 Selective study of glyphosate detection based on dual-mode colorimetric platform of Os-Rh nanozyme

[0091] In order to evaluate the selectivity of Os-Rh nanozyme for dual-mode direct detection of glyphosate, the inhibitory ability of other eight common pesticides (methamidophos, paraquat, dimethoate, profenofos, chlorpyrifos, phoxim, phoxim and phoxim) on the peroxidase-like activity of Os-Rh nanozyme under optimal conditions was investigated on the solution platform and paper platform, respectively. The test concentration of each pesticide was 1200 μg / L.

[0092] On the solution platform, 10 μL of Os-Rh nanozyme solution (stock solution diluted 20 times) and 10 μL of 1200 μg / L pesticide solution were mixed in a 96-well plate for 15 minutes. Then 200 μL of reaction substrate solution (containing 2.0 mM TMB and 10 mM H2O2) was added and reacted at room temperature for 10 minutes. Finally, the absorbance value of the reaction system at 650 nm was measured using an ELISA instrument. The group without pesticide addition was set as the blank control group.

[0093] On the paper platform, first add 10 μL of Os-Rh nanozyme solution (the stock solution was diluted 30 times) to a clean test paper with a diameter of 8 mm, dry it naturally, and obtain a glyphosate detection test paper. Next, add 10 μL of 1200 μg / L pesticide solution to the test paper and leave it at room temperature for 15 minutes. Subsequently, add 20 μL of reaction substrate solution (containing 2.91 mM TMB and 10 mM H2O2) and react at room temperature for 8 minutes. Then take a picture with an iPhone 14pro, and use Image J software to obtain the grayscale value (G) of the test paper. △G is the difference between the G value of the blank control group (without pesticides and reaction substrates) and the G value of the test group.

[0094] like Figure 8 As shown in a, under the same conditions, the sample wells with glyphosate added were colorless, and the absorbance at 650nm was very small. In contrast, the blank control wells and the sample wells with other pesticides added were blue, and the absorbance at 650nm was also similar. The above results show that, except for glyphosate, other pesticides cannot inhibit the peroxidase-like activity of Os-Rh nanozymes, that is, other pesticides do not interfere with the glyphosate direct colorimetric detection solution platform developed based on Os-Rh nanozymes.

[0095] Likewise, from Figure 8 b It can be seen that under the same conditions, only the sample paper with glyphosate added did not show color (the corresponding △G value was very low), while the blank control paper and the sample paper with other pesticides added showed blue-green color, and the △G value of the sample paper with other pesticides added was similar to that of the blank control paper. This indicates that other pesticides will not interfere with the development of the glyphosate direct colorimetric detection paper platform based on Os-Rh nanozyme.

[0096] In conclusion, the dual-mode colorimetric platform based on Os-Rh nanozyme has high selectivity for the direct detection of glyphosate.

[0097] Example 7 Dual-mode colorimetric platform based on Os-Rh nanozyme pair for glyphosate detection

[0098] On the solution colorimetric platform, 10 μL of Os-Rh nanozyme solution (the stock solution was diluted 20 times) and 10 μL of glyphosate solution of different concentrations (50-1200 μg / L) were mixed in a 96-well plate for 15 minutes. Then, 200 μL of reaction substrate solution (containing 2.0 mM TMB and 10 mM H2O2) was added. After reacting at room temperature for 10 minutes, the ultraviolet absorption spectrum of the reaction system in the range of 450-800 nm was measured using an ELISA instrument. The group without glyphosate was set as the blank control group. The inhibition rate of glyphosate on the peroxidase-like activity of Os-Rh nanozyme was calculated according to the following formula (4):

[0099]

[0100] Where A0 is the absorbance value of the blank control group at 650nm, and A1 is the absorbance value of the glyphosate-added group at 650nm. The inhibition curve was drawn with glyphosate concentration as the horizontal axis and inhibition rate as the vertical axis. In addition, a linear fit was performed between inhibition rate and glyphosate concentration in the concentration range of 50 to 1000 μg / L. When the signal-to-noise ratio (S / N) was 3, the detection limit (LOD) of the glyphosate solution platform was calculated according to formula (5).

[0101] LOD = 3 × SD / m (5)

[0102] Wherein, SD is the standard deviation of the multiple measurement signals of the blank control group, and m is the slope of the standard curve.

[0103] On the paper colorimetric platform, 10 μL of Os-Rh nanozyme solution (the stock solution was diluted 30 times) was dripped onto a clean test paper with a diameter of 8 mm and dried naturally at room temperature to prepare a glyphosate detection test paper. Then 10 μL of glyphosate solution of different concentrations (50-1200 μg / L) was dripped onto the prepared glyphosate detection test paper. After standing at room temperature for 15 minutes, 20 μL of reaction substrate solution (containing 2.91 mM TMB and 10 mM H2O2) was added. The test paper was then reacted at room temperature for 8 minutes, and then photographed with an iPhone 14pro, and the grayscale value (G) of the test paper was obtained by Image J software. The inhibition rate of glyphosate on the peroxidase-like activity of Os-Rh nanozyme is calculated by formula (6):

[0104]

[0105] Wherein, G0 represents the G value of the test paper when no glyphosate and reaction substrate solution are added, and G1 represents the G value of the test paper when glyphosate and reaction substrate solution are added. The following procedures are consistent with those of the above solution colorimetric platform. Fig. 9 As shown in a, with the increase of glyphosate concentration, the solution color of the system (Os-Rh nanozyme + TMB + H2O2 + glyphosate) gradually changes from the original blue to colorless, which corresponds to the gradual weakening of their absorption peak intensity at 650nm. Fig. 9 The results of a can be converted to obtain the relationship between glyphosate concentration and its inhibitory effect on Os-Rh nanozyme activity ( Fig. 9 b and 9c). Fig. 9 b is a scatter plot of 50-1200 μg / L, which shows that the inhibitory effect of glyphosate on the activity of Os-Rh nanozyme gradually increases with the increase of glyphosate concentration and eventually reaches equilibrium, which is consistent with the results of the optical photograph. In addition, Fig. 9 c is a linear fitting diagram of the data points from 50 to 1000 μg / L in Figure 9b. Fig. 9 c, it can be found that the inhibitory effect of glyphosate on the activity of Os-Rh nanozyme in the concentration range of 50-1000 μg / L showed a good linear relationship, and the linear correlation coefficient (R 2 ) is 0.998. After calculation, the detection limit (LOD) of this solution platform for glyphosate is 28.37 μg / L. Compared with other glyphosate detection methods (Table 2), the detection limit of this solution platform is lower, indicating that this method has good sensitivity.

[0106] Based on the high sensitivity of Os-Rh nanozyme in detecting glyphosate in solution, a colorimetric paper detection platform was established to make the detection more portable and convenient. Fig.10 As shown. Fig.10 It can be clearly seen in a that as the glyphosate concentration increases, the blue-green color of the test paper becomes lighter and lighter until it disappears. Especially when the concentration exceeds 400 μg / L, the color of the test paper is significantly different from that of the blank control paper, so this concentration can be regarded as the naked eye detection limit of the glyphosate paper platform. The color of the paper was converted into the corresponding grayscale value using Image J software, and finally the grayscale value was converted into the inhibition rate of glyphosate on the activity of Os-Rh nanozyme, thereby obtaining a relationship diagram between glyphosate concentration and its inhibitory effect on the activity of Os-Rh nanozyme ( Fig.10 b and c). Similar to the solution platform, the inhibitory effect of glyphosate on the activity of Os-Rh nanozymes gradually increased with the increase of glyphosate concentration and finally reached equilibrium ( Fig.10 b) and showed a good linear relationship in the concentration range of 200-1000 μg / L, R 2 is 0.996( Fig.10c). The detection limit of glyphosate by the colorimetric paper platform was calculated to be 123.25 μg / L, which is lower than many other methods. Therefore, the colorimetric paper platform provides a convenient and sensitive detection platform for glyphosate.

[0107] Table 2. Comparison of glyphosate detection methods based on nanozymes

[0108]

[0109] Example 8 Study on the room temperature storage stability of the paper colorimetric platform based on Os-Rh nanozyme

[0110] Considering the convenience of future storage, we explored the stability of Os-Rh nanozyme colorimetric test paper at room temperature by measuring the catalytic reaction activity at 0, 10, 20, 30, 40, 50 and 60 days. Simply put, 20 μL of reaction substrate solution (containing 2.91 mM TMB and 10 mM H2O2) was added to the colorimetric test paper for different storage days. After reacting at room temperature for 8 minutes, the color of the test paper was recorded with an iPhone 14pro, and the color of the test paper was converted into a grayscale value (G value) using Image J software. The G value on day 0 was set as a relative activity of 100%.

[0111] like Fig.11 As shown, the detection activity of the colorimetric paper platform did not decrease significantly within 60 days, indicating that the prepared colorimetric test paper has excellent room temperature storage stability. Compared with other pesticide rapid detection products (mostly stored at 4°C to maintain the catalytic activity of natural enzymes), the colorimetric paper platform developed by the present invention is easier to store and is convenient for promotion and application in actual glyphosate detection.

[0112] Example 9 Detection of glyphosate in real spiked samples using a dual-mode colorimetric platform based on Os-Rh nanozymes

[0113] In order to verify the feasibility of the dual-mode colorimetric detection platform developed based on Os-Rh nanozymes in actual sample detection, apples, oranges, cucumbers and green beans were selected as actual samples for glyphosate residue analysis. First, these samples were washed with distilled water and air-dried for use. Then, 1000 μL of a certain concentration (2500 μg / L or 4500 μg / L) of glyphosate solution was evenly sprayed on their surfaces. Subsequently, these samples were air-dried in a fume hood for 24 hours to obtain spiked samples. Then, the surface of the spiked sample was repeatedly sprayed with 5 mL of distilled water, the sprayed solution was collected and filtered with a 0.22 μm filter membrane to obtain the solution to be tested. Finally, the solution colorimetric platform and paper colorimetric platform constructed in this study and the HPLC-MS method were used to detect the concentration of glyphosate in the solution to be tested.

[0114] The solution colorimetric method for detecting the concentration of glyphosate includes the following steps:

[0115] 1) Add 10 μL of Os-Rh nanozyme solution (the stock solution was diluted 20 times) and 10 μL of standard glyphosate solution of different concentrations (50-1200 μg / L) into a 96-well plate;

[0116] 2) mixing the mixed solution obtained in step 1) at room temperature for 15 minutes;

[0117] 3) Add 200 μL of reaction substrate solution (containing 2.0 mM TMB and 10 mM H2O2) to the solution in step 2);

[0118] 4) reacting the mixed solution obtained in step 3) at room temperature for 10 min;

[0119] 5) using a microplate reader to measure the ultraviolet absorption spectrum of the solution obtained in step 4) at 650 nm, and setting the group without adding glyphosate as a blank control group;

[0120] 6) The inhibition rate of glyphosate on the peroxidase-like activity of Os-Rh nanozyme was calculated according to the following formula:

[0121]

[0122] In the formula, A0 is the absorbance value of the blank control group at 650nm, and A1 is the absorbance value of the glyphosate-added group at 650nm. The inhibition standard curve is drawn with the concentration of glyphosate as the horizontal axis and the inhibition rate at the corresponding concentration as the vertical axis.

[0123] The solution of the sample to be tested is substituted for the above standard glyphosate solution for testing, and the inhibition rate of the sample to be tested is measured. The glyphosate concentration in the sample to be tested can be obtained by substituting the inhibition rate into the inhibition standard curve.

[0124] The paper colorimetric method detects the concentration of glyphosate, and the steps include:

[0125] 1) Add 10 μL of Os-Rh nanozyme solution (the stock solution was diluted 30 times) onto a clean circular test paper with a diameter of 8 mm;

[0126] 2) drying the test paper obtained in step 1) at room temperature to prepare a glyphosate detection test paper;

[0127] 3) adding 10 μL of standard glyphosate solutions of different concentrations (50-1200 μg / L) to the glyphosate test paper obtained in step 2);

[0128] 4) placing the test papers containing different concentrations of glyphosate obtained in step 3) at room temperature for 15 minutes;

[0129] 5) Add 20 μL of reaction substrate solution (containing 2.91 mM TMB and 10 mM H2O2) to the test paper obtained in step 4) and react at room temperature for 8 min;

[0130] 6) Use an iPhone 14pro to take a photo of the test paper color obtained in step 5), and use Image J software to obtain the grayscale value (G) of the test paper;

[0131] 7) The inhibition rate of glyphosate on the peroxidase-like activity of Os-Rh nanozyme was calculated by formula (6):

[0132]

[0133] In the formula, G0 represents the G value of the blank solution of the test paper when no glyphosate and reaction substrate solution are added, and G1 represents the G value of the test paper when glyphosate and reaction substrate solution are added. The inhibition standard curve is drawn with the concentration of glyphosate as the horizontal axis and the inhibition rate at the corresponding concentration as the vertical axis.

[0134] The solution of the sample to be tested is substituted for the above standard glyphosate solution for testing, and the inhibition rate of the sample to be tested is measured. The glyphosate concentration in the sample to be tested can be obtained by substituting the inhibition rate into the inhibition standard curve.

[0135] The recovery rate and relative standard deviation of glyphosate added to the actual sample measured and calculated are shown in Table 3, and the glyphosate concentrations measured by the three methods are used as the vertical axis to draw a bar graph. Fig.12 .

[0136] Table 3. Recoveries and relative standard deviations of glyphosate spiked into real samples

[0137]

[0138]

[0139] a indicates that apples, tomatoes, oranges and eggplants were purchased from the local market. Each sample was measured three times (n=3).

[0140] from Fig.12It can be seen that the glyphosate spike recovery results measured by the dual-mode colorimetric detection platform based on Os-Rh nanozymes are very similar to those measured by the HPLC-MS method. In addition, it can be seen from Table 3 that the glyphosate spike recovery rates of the colorimetric solution platform and colorimetric paper platform based on Os-Rh nanozymes are 86.27% to 96.51% and 85.47% to 95.64%, respectively, and the RSDs are 1.38% to 3.87% and 0.98% to 4.96%, respectively. Compared with the glyphosate spike recovery rate (86.31% to 97.86%) and RSD (1.83% to 3.37%) detected by the HPLC-MS method, the deviation is very small. These results show that the established dual-mode colorimetric detection platform based on Os-Rh nanozymes has good accuracy and reliability in practical applications, and has considerable practical application potential in the field of rapid glyphosate detection.

[0141] Comparative Example 1

[0142] In a 1.5 mL test tube, 50 μL of 160 mM K2OsCl6 solution, 50 μL of 100 mM RhCl3·3H2O solution, and 900 μL of 50 mM glycine solution were added. After vortexing for 10 min, the mixture was preheated at 50 °C for 5 min. Then, 100 μL of 0.55 M freshly prepared ascorbic acid was added and incubated at 50 °C for 1 h. Finally, the reaction mixture was dialyzed in deionized water using a 500D dialysis bag for 8 h to obtain the Os-Rh-(1) nanomaterial stock solution.

[0143] The results are as follows Fig.13 .(a) TEM image of Os-Rh-(1) nanomaterials, (b) verification results of the peroxidase-like activity of Os-Rh-(1) nanomaterials, the UV-visible absorption spectrum measured in the range of 500-800 nm after 10 min of reaction showed that the Os-Rh-(1) nanomaterials prepared under this condition did not have a regular shape, and the color reaction results ( Fig.13 b) It shows that the Os-Rh-(1) nanomaterial prepared under these conditions does not have peroxidase mimicking activity.

[0144] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any person skilled in the art can use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. An application of Os-Rh nanozyme in the preparation of a glyphosate detection reagent, characterized in that: The preparation method of the Os-Rh nanozyme is to mix K2OsCl6, RhCl3·3H2O and an aqueous solution of glycine, preheat, add ascorbic acid for reduction incubation, and then dialyze in deionized water using a dialysis bag to obtain the Os-Rh nanozyme; The preheating temperature is 50-70°C, the reduction incubation temperature is 50-70°C, and the molecular weight of the dialysis bag is 500D; The final concentration of K2OsCl6 in the aqueous solution is 2-5 mM, the final concentration of RhCl3·3H2O in the aqueous solution is 1-4 mM, the final concentration of glycine in the aqueous solution is 60-120 mM; and the final concentration of ascorbic acid in the aqueous solution is 0.1-0.3 M.

2. The use according to claim 1, characterized in that The mixing is performed by vortex mixing, the vortex mixing time is 5 to 20 minutes, the preheating time is 5 to 20 minutes, the incubation time is 1 to 5 hours, and the dialysis time is 8 to 40 hours.

3. The use according to claim 1, characterized in that The detection reagent includes a detection solvent for detecting glyphosate or a test paper for detecting glyphosate.

4. A test paper for detecting glyphosate, wherein an Os-Rh nanozyme solution is coated on the test paper, wherein the preparation method of the Os-Rh nanozyme is to mix K2OsCl6, RhCl3·3H2O and an aqueous solution of glycine, preheat, add ascorbic acid for reduction incubation, and then dialyze in deionized water using a dialysis bag to obtain the Os-Rh nanozyme; The preheating temperature is 50-70°C, the reduction incubation temperature is 50-70°C, and the molecular weight of the dialysis bag is 500D; The final concentration of K2OsCl6 in the aqueous solution is 2-5 mM, the final concentration of RhCl3·3H2O in the aqueous solution is 1-4 mM, the final concentration of glycine in the aqueous solution is 60-120 mM; and the final concentration of ascorbic acid in the aqueous solution is 0.1-0.3 M.

5. A method for detecting glyphosate based on Os-Rh nanozyme, comprising the following steps: S1. After adding the Os-Rh nanozyme solution and glyphosate solutions of different concentrations, i.e., standard products, to a 96-well plate for mixed reaction, the sample to be tested and the Os-Rh nanozyme solution are also added to the 96-well plate, and a blank solution without glyphosate and the Os-Rh nanozyme solution are also added to the 96-well plate as a blank control; the preparation method of the Os-Rh nanozyme is to mix K2OsCl6, RhCl3·3H2O and an aqueous solution of glycine, preheat, add ascorbic acid for reduction incubation, and then dialyze in deionized water using a dialysis bag to obtain the Os-Rh nanozyme; The preheating temperature is 50-70°C, the reduction incubation temperature is 50-70°C, and the molecular weight of the dialysis bag is 500D; The final concentration of K2OsCl6 in the aqueous solution is 2-5 mM, the final concentration of RhCl3·3H2O in the aqueous solution is 1-4 mM, the final concentration of glycine in the aqueous solution is 60-120 mM; the final concentration of ascorbic acid in the aqueous solution is 0.1-0.3 M; S2, adding reaction substrate solution to each of the above wells for reaction; S3, measuring the ultraviolet absorption spectrum of each well at 650 nm after reaction; S4. According to the formula Calculate the inhibition rate of each standard, and draw a standard curve with the standard concentration as the horizontal axis and the inhibition rate of the standard as the vertical axis, where A0 is the absorbance value of the blank control group at 650nm, and A1 is the absorbance value of the standard with glyphosate added at 650nm; S5. Substitute the inhibition rate of the sample to be tested into the standard curve to obtain the concentration of glyphosate in the sample to be tested.

6. A paper colorimetric method for detecting glyphosate based on Os-Rh nanozyme, comprising the following steps: S1. Add different concentrations of glyphosate standard to the test paper containing Os-Rh nanozyme; use the solution without glyphosate as blank control, and test the solution at the same time; wherein, The preparation method of the Os-Rh nanozyme is to mix K2OsCl6, RhCl3·3H2O and an aqueous solution of glycine, preheat, add ascorbic acid for reduction incubation, and then dialyze in deionized water using a dialysis bag to obtain the Os-Rh nanozyme; The preheating temperature is 50-70°C, the reduction incubation temperature is 50-70°C, and the molecular weight of the dialysis bag is 500D; The final concentration of K2OsCl6 in the aqueous solution is 2-5 mM, the final concentration of RhCl3·3H2O in the aqueous solution is 1-4 mM, the final concentration of glycine in the aqueous solution is 60-120 mM; the final concentration of ascorbic acid in the aqueous solution is 0.1-0.3 M; S2. After a period of time, the reaction substrate solution is added dropwise onto the test paper to react; S3, take a photo of the test paper after the reaction with a mobile phone, and obtain the gray value of the test paper through software; S4. According to the formula Calculate the inhibition rate of each standard, and draw an inhibition standard curve with the concentration of glyphosate in the standard as the horizontal axis and the inhibition rate at the corresponding concentration as the vertical axis; wherein G0 represents the gray value of the blank solution of the test paper when glyphosate and the reaction substrate solution are not added, and G1 represents the gray value of the test paper when glyphosate and the reaction substrate solution are added; S5. Substitute the inhibition rate of the sample to be tested into the standard curve to obtain the concentration of glyphosate in the sample to be tested.

Images