CMO nano oxidase, CMO-OVA-MC-LR probe constructed by CMO nano oxidase, kit, preparation method and application

Abstract

The invention belongs to the technical field of biological detection, and provides a CMO nano oxidase, a CMO-OVA-MC-LR probe constructed by the CMO nano oxidase, a kit, a preparation method and application of the CMO nano oxidase in order to solve the problem that an existing ELISA kit is single in color changing mode and poor in sensitivity. The CMO nano oxidase is waxberry-shaped Co-doped Mn3O4 oxidase, the nano oxidase is an amphiphilic CMO heterojunction synthesized by Co3O4 and Mn3O4 through a one-pot hydrothermal method, and the particle size of the CMO nano oxidase is 110-140 nm. The detection limit of the enzyme linked immunosorbent assay kit constructed by the invention on microcystin LR type (MC-LR) is 0.505 ng / mL, and the quantitative range is 0.195 to 100 ng / mL. Benefited from the strong catalytic activity and good biocompatibility of the nano enzyme, the label and the signal response mode open up a new way for improving the sensitivity of immunosensing determination, and further expansion of the application of an immunosensor is facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a CMO nano-oxidase and a CMO-OVA-MC-LR probe, a kit, a preparation method and an application thereof. The nano-oxidase can oxidize TMB to a yellow end product with two electrons, and presents a colorimetric mode of multiple colors including green and blue after combining with TMB. The probe and the kit are used in detecting microcystin. Background Art

[0002] In recent years, eutrophication of water bodies has promoted the rapid growth of algae, leading to the occurrence of algal blooms. In addition to deteriorating the color and odor of water quality, it also produces toxic metabolites such as algal toxins. Microcystins are a class of cyclic heptapeptide compounds produced by various blue-green algae, composed of seven amino acids. When the two variable amino acids are leucine (L) and arginine (R), it is called microcystin LR (MC-LR), accounting for 46.0-99.8% of the total MC concentration in water, which is the most common and widely distributed category. MC-LR has strong biological toxicity because it can inhibit the activity of protein phosphatases and induce cell apoptosis. Faced with this severe situation, many effective degradation methods have been reported. It is worth mentioning that sensitive and reliable detection is a prerequisite for treatment. Current detection methods mainly include protein phosphatase inhibition, bioassay, and high-performance liquid chromatography (HPLC), which are time-consuming, labor-intensive, and easily interfered by the sample matrix. Enzyme-linked immunosorbent assay (ELISA) is a powerful analytical tool that plays an increasingly important role in biosensors due to its specificity, sensitivity, stability, and reliability. However, due to the inherent properties of natural enzymes, such as insufficient sensitivity and catalytic activity, the classical ELISA using horseradish peroxidase (HRP)-catalyzed 3,3′,5,5′-tetramethylbenzidine (TMB) color development reaction is still limited to relatively poor sensitivity and stability.

[0003] In 2007, Fe3O4 nanoparticles were found to have a reaction mechanism similar to that of biological enzymes, which aroused widespread attention and research enthusiasm for nanozymes in the academic community. Compared with natural enzymes, nanozymes have many advantages, including controllable design, excellent catalytic activity, strong stability, long-term effectiveness, and large-scale low-cost production. More and more nanozymes have been discovered, designed and synthesized, including precious metals, transition metal oxides, metal-organic frameworks (MOFs), metal-doped carbon dots and emerging single-atom nanozymes. These nanozymes exhibit different mimetic enzyme activities, such as peroxidase (POD), oxidase (OXD), catalase, superoxide dismutase (SOD), glutathione peroxidase, etc. The utilization of enzyme activity, especially catalytic oxidation, has a wide range of applications in sensing, sterilization, anticancer and pollutant degradation.

[0004] Currently, various nanozymes have been introduced into ELISA as alternatives to HRP, and their catalytic activity directly affects the sensitivity of the analysis. Therefore, it is an urgent task to develop nanozymes with excellent performance to improve sensitivity. Manganese (Mn) is one of the most abundant elements on earth, with non-toxicity, low cost and abundant valence states (Mn 2+ , Mn 3+ , Mn 4+ , Mn 6+ , Mn 7+ ), its oxide nanomaterials have been found to be promising oxidase nanozymes. Unlike peroxidases, oxidases can promote TMB oxidation in the absence of hydrogen peroxide (H2O2), simplifying the sensing process, avoiding the toxicity and danger of H2O2 during transportation and storage, and overcoming the inaccurate detection caused by the instability of H2O2. In order to further improve the catalytic activity of nanozymes, many effective strategies have been proposed, including optimizing the ratio of raw materials, exploring preparation conditions, modifying with organic molecules and doping with other elements to precisely control the size, change the morphology and surface potential, modifying surface groups, forming hollow structures, increasing oxygen vacancies (OV) and defects to provide high surface energy and abundant active sites. Summary of the invention

[0005] In order to solve the problems of single color change mode and poor sensitivity of current ELISA kits, the present invention provides a CMO nano-oxidase and a CMO-OVA-MC-LR probe constructed therefrom, a kit, a preparation method and an application thereof.

[0006] The present invention is achieved by the following technical scheme: a CMO nano-oxidase, wherein the CMO nano-oxidase is a bayberry-shaped Co-Mn3O4 oxidase; the nano-oxidase is an amphiphilic CMO heterojunction synthesized by a one-pot hydrothermal method of Co3O4 and Mn3O4, and has a particle size of 110-140nm.

[0007] The method for preparing the CMO nano-oxidase comprises the following steps: 0.5 g of tetrahydrated manganese (II) acetate Mn(CH3COO)2•4H2O, 0.02 g of tetrahydrated cobalt (II) acetate Co(CH3COO)2•2H2O and 0.4 g of PVP are dissolved in 15 mL of ethylene glycol; the mixture is stirred vigorously for 1 hour until it is completely dissolved into a transparent orange-red solution; 0.8 mL of 1 mol / L HCl is added to the solution, and the mixture is stirred for another 0.5 hour; the mixture is then heated to 160°C at a heating rate of 20°C / min, and then heated at 160°C for 12 hours; after cooling to room temperature, the obtained dark green solution is centrifuged at 9000 rpm for 10 minutes to collect the dark green nano-oxidase product; the dark green nano-oxidase product is washed three times with distilled water and ethanol respectively, and finally resuspended in 8 mL of distilled water to obtain the CMO nano-oxidase.

[0008] The method for constructing a CMO-labeled chicken ovalbumin-coupled microcystin LR antigen OVA-MC-LR probe using the CMO nanooxidase or the CMO nanooxidase obtained by the method comprises the following steps: 2 μL of 1 mg / mL OVA-MC-LR is added to 1 mL of a PBS solution of CMO at a concentration of 1 mg / mL, and gently vortexed at room temperature for 30 minutes; then 200 μL of 1% w / v chicken ovalbumin OVA is mixed for 30 minutes to block the active sites on the surface of the nanozyme, namely the antigen probe CMO-OVA-MC-LR, which is stored at 4°C for future use.

[0009] The concentration of CMO is 1 mg / mL, the concentration of the antigen OVA-MC-LR is 2 μg / mL, the concentration of the chicken ovalbumin OVA selected for blocking is 0.2%, and the incubation time is 30 minutes.

[0010] The present invention also provides an immunosensor kit prepared by using the prepared CMO-OVA-MC-LR probe as a signal probe.

[0011] Furthermore, the immunosensor kit includes a polystyrene 96-well ELISA plate, which is coated with a mouse monoclonal antibody that specifically recognizes microcystin-LR. The plate is washed three times after incubation, chicken ovalbumin OVA is blocked, the plate is washed three times after incubation, a standard microcystin solution or a target sample solution is added, the plate is washed three times after incubation, a CMO-OVA-MC-LR probe is added, the plate is washed six times after incubation, a TMB substrate solution is added, and the absorbance is measured after reacting for 2 minutes.

[0012] Furthermore, the coated mouse monoclonal antibody that specifically recognized microcystin-LR was incubated at 0.5 μg / mL at 4 °C overnight, the blocking solution was 0.5% OVA-PBST, and the incubation was carried out at 37 °C for 45 min. The target was added to the ELISA plate and incubated at 37 °C for 30 min, and then the CMO-OVA-MC-LR probe was added.

[0013] The target objects are microcystin-LR standards and water environment samples and aquatic product samples containing microcystin-LR.

[0014] The present invention also provides the use of the immunosensor kit in detecting microcystin-LR in lake water, salt lake water, fish and shrimp.

[0015] The lake water is adjusted to pH with phosphate buffer. After the salt lake water is treated at 4°C, the aqueous solution of precipitated Na2SO4 is taken and the pH is adjusted with phosphate buffer. The fish and shrimp samples are treated with trichloroacetic acid to precipitate protein and then the pH is adjusted, and then the designed kit is used for detection.

[0016] Inspired by the synthesis methods of hollow CeO2 and N-doped Mn3O4 nanooxidases, the present invention developed a bayberry-shaped CMO, which can form a hydrophobic space to surround TMB and obtain two-electron oxidation, showing excellent oxidase activity. It is worth mentioning that most oxidases and peroxidases catalyze the oxidation of TMB through a single-electron oxidation pathway to produce a blue oxidation product (TMB ox1 ), which needs to be terminated with a strong acid to be converted into a stable yellow oxidation product (TMB ox2 In contrast, the prepared CMO nanozyme can directly catalyze the oxidation of TMB to a yellow end product (TMB) via a two-electron oxidation pathway. ox2 ), showing extraordinary oxidation ability. In addition, with blue TMB ox1 In comparison, yellow TMB ox2 It has better stability and a larger molar extinction coefficient, which facilitates more sensitive analysis and overcomes the cumbersome steps and errors caused by the introduction of strong acid terminators. In addition, unlike the preparation of precious metal and rare element nanozymes, the acquisition of this CMO nanozyme avoids high costs and secondary pollution, and avoids the complex and demanding reaction mode of single-atom nanozymes (SAzymes), showing significant advantages in environmental friendliness and economic benefits.

[0017] Robust CMO was coupled to OVA-MC-LR to construct an antigen (OVA-MC-LR) probe, and the MC-LR target preferentially competed with the binding of antibodies (Abs), establishing a more sensitive and reliable MC-LR detection ELISA kit. The designed analytical platform has been successfully applied to screen real samples, including lake water, salt lakes, fish and shrimp, demonstrating its great potential in food safety and environmental monitoring.

[0018] Compared with the prior art, the advantages and effects of the present invention are as follows: the present invention can directly double-electron oxidize TMB to a yellow final oxidation product under acidic conditions, label the antigen to establish a signal probe, combine the mutual conversion of the TMB oxidation product to establish a multi-color signal immunoassay of microcystin.

[0019] The present invention is different from the single-electron oxidation of TMB by most nanozymes, and can directly oxidize TMB to a yellow product in the final state with two electrons. The nanozyme forms a hydrophobic cavity, so that the hydrophobic TMB accumulates in the cavity and is oxidized to hydrophilic TMB by active oxygen free radicals. ox2 The oxidation product leaves the cavity and avoids combining with TMB to form TMB ox1 , demonstrating excellent two-electron oxidation ability.

[0020] CMO nanozymes exhibit extraordinary catalytic ability (K m =3.357μM, V max=1.338 μM / s), effectively driving the rapid direct two-electron oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) to convert TMB into stable yellow TMB ox2 The interconversion between different TMB oxidation products produces a yellow-green-blue multicolor signal, allowing for more sensitive detection, IC 50 The half-maximal inhibitory concentration (HAIC) was 1.057 ng / mL, which was 8.4 times higher than that of the classic horseradish peroxidase-based ELISA. The proposed biosensor showed satisfactory performance in real environmental and food samples with recoveries ranging from 80.4% to 112.1%, demonstrating its great potential in safeguarding public health. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the synthesis of the signal material and the sensor principle of the present invention: (A) Preparation principle of CMO and probe; (B) CMO-driven two-electron oxidation of TMB and UV-visible spectrum absorption peak of TMB oxidation product; (C) Schematic diagram of the multi-color results of the kit for detecting MC-LR; Figure 2 Characterization of the signal material CMO of the present invention: (A) transmission electron microscope image of CMO; (B) element distribution diagram of CMO; (C) XRD spectrum of CMO; (D) full range scanning XPS spectrum of CMO; (E) Mn 2p spectrum of CMO; (F) Co 2p spectrum of CMO; (G) O 1s spectrum of CMO; (H) FTIR spectra of PVP, Co3O4, Mn3O4 and CMO; Figure 3 The oxidase activity characterization diagram of the signal material CMO of the present invention: (A) Citrate buffer containing CMO (0.02 mg / mL) and different concentrations of TMB at 436 nm (TMB ox2 ) and 652nm (TMB ox1 ) and UV-vis absorbance; (B) TMB, TMB ox1 、TMB ox2 、TMB ox1 and TMB ox2 The UV-visible absorption spectrum of the mixture and CMO; (C) The effect of pH on the oxidase activity of CMO; (DE) Kinetic test of TMB oxidation catalyzed by CMO; (F) Kinetic parameters K of various oxidases catalyzed by TMB reported in the literature m and V max Comparison; (G) Test results of reactive oxygen free radical species generated by CMO; (H) Mechanism of two-electron oxidation of TMB mediated by the hydrophobic cavity of CMO; Figure 4The performance evaluation diagrams of the kit of the present invention are as follows: (A) quantitative analysis of MC-LR by CMO-ELISA; (B) sensitivity comparison between the sensor and the reported sensor for detecting MC-LR; (C) specificity evaluation results of the sensor. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0024] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0025] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0026] In order to solve the problem of single color signal and low sensitivity of enzyme-linked immunosorbent assay (ELISA), the present invention constructs a nanoenzyme signal with super strong oxidase activity, which can directly double-electrically self-oxidize TMB into the final product. In order to obtain the best measurement performance, a probe of antigen-labeled signal is designed and prepared, and the target molecule priority competition mode is adopted to further ensure the stability of the detection. By optimizing important parameters, the best detection effect is obtained. Finally, the sensor is used to detect microcystin-LR, especially for the detection of water environment and aquatic products, and has the characteristics of reliability, sensitivity and stability.

[0027] Example 1: Synthesis strategy of CMO and OVA-MC-LR probes Figure 1 As shown in A, CMO nanozymes were prepared by a one-pot hydrothermal method. The resulting nanomaterial was combined with OVA-MC-LR and blocked by OVA through adsorption to form a signal probe. The principle of the oxidation of TMB by this CMO and the detection of MC-LR by multicolor ELISA is shown in Figure 1 B and Figure 1 C, specifically including the following steps: 1. Preparation of CMO by hydrothermal synthesis, such as Figure 1 A: 0.5 g of manganese (II) acetate tetrahydrate (Mn(CH3COO)2•4H2O), 0.02 g of cobalt (II) acetate tetrahydrate (Co(CH3COO)2•2H2O), and 0.4 g of PVP were dissolved in 15 mL of ethylene glycol. The mixture was vigorously stirred for 1 hour until it was completely dissolved and a transparent orange-red solution was obtained. After that, 0.8 mL of 1 mol / L HCl was added to the solution and stirred for another 0.5 hour. The mixture was then transferred to a polytetrafluoroethylene (PTFE) liner in a stainless steel reactor and heated at 160 °C for 12 hours. After cooling to room temperature, the obtained dark green solution was centrifuged at 9000 rpm for 10 minutes to collect the product. The dark green nanozyme product was washed three times with distilled water and ethanol, respectively, and finally resuspended in 8 mL of distilled water.

[0028] 2. Preparation of CMO-OVA-MC-LR probe, such as Figure 1 A: 2 μL of 1 mg / mL OVA-MC-LR was added to 1 mL (1 mg / mL) CMO dissolved in PBS and gently vortexed for 30 minutes at room temperature. Then, 200 μL of 1% OVA was mixed for 30 minutes to block the active sites on the surface of the nanozyme and prevent nonspecific binding with other substances in the sample. The antigen probe (MC-LR-OVA-CMO) was stored at 4°C for future use.

[0029] 3. The material exhibits oxidase activity for two-electron oxidation of TMB, e.g. Figure 1 B: TMB can generate reactive oxygen species (ROS) under acidic conditions through CMO ( 1 O2, •O2 - ) is directly oxidized with two electrons to the yellow final product TMB ox2 In contrast, most oxidases and peroxidases can only oxidize TMB to a blue product (TMB) via a single-electron pathway. ox1 ), which requires strong acid to convert TMB ox1 Transformed into TMB ox2 The yellow final form. It is worth noting that the blue TMB ox1 It is an unstable complex that can be decomposed into colorless TMB and yellow TMB ox2 This means that CMO is different from typical oxidases and peroxidases in that it has a very strong oxidative activity and is able to completely oxidize TMB to its final yellow state (TMB ox2 ). In this system, TMB ox1 and TMB ox2 The spectral absorption peaks appear at 652 nm and 436 nm, respectively.

[0030] 4. The principle of multicolor signal presentation by ELISA based on CMO superoxidase is as follows Figure 1 As shown in C, the detection process specifically includes the following steps: Immunoassay procedure: The ELISA procedure was performed in a transparent 96-well microtiter plate according to the following steps. (1) Coating: The wells of the microtiter plate were coated with MC-LR-specific antibodies (0.5 μg / mL in carbonate buffer solution at pH 9.6, 100 μL / well) and incubated overnight at 4°C. (2) Blocking: 150 μL / well of 0.5% OVA was added to PBST (pH 7.4, 0.01 M PBS containing 0.05% Tween-20) at 37°C to block the remaining binding sites on the plate and incubate for 40 minutes to prevent nonspecific adsorption. (3) Target analyte addition: MC-LR standards (0-100 ng / mL, 100 μL) were added to the wells and incubated at 37°C for 30 minutes to be captured by the antibodies immobilized in the microtiter plate. After each of the above steps, the plate was washed three times with PBST to remove molecules that were not adsorbed to the plate and the supernatant. (4) Signal probe introduction: Add 100 μL CMO-OVA-MC-LR probe and incubate at 37°C for 30 minutes to allow it to be captured by the remaining antibodies. To ensure complete removal of probes that are not adsorbed to the plate by Abs, wash the plate six times with PBST to avoid false negative results. (5) Substrate color development reaction: Add 100 μL TMB (20 μg / mL). Due to the strong oxidase activity of CMO, a complete colorimetric result can be obtained within 2 minutes without the need for traditional strong acid termination. The absorbance is measured at 436 nm using a microplate reader.

[0031] The CMO-OVA-MC-LR probe was introduced into the ELISA platform for the detection of MC-LR, such as Figure 1 C. In a microtiter plate, the target MC-LR preferentially competes with the Abs on the plate, and then the CMO-OVA-MC-LR probe binds to the remaining Abs. When low concentrations of MC-LR are detected, a large number of CMO-OVA-MC-LR probes bind to the microwells, forming a hydrophobic cavity that promotes the oxidation of hydrophobic TMB. The resulting oxidation product TMB ox2 Due to its hydrophilicity, it then leaves the hydrophobic space and enters the aqueous phase, separating from TMB and preventing coupling to form TMB ox1 , resulting in a yellow color. At high MC-LR concentrations, the small amount of antibody remaining can only fix a small amount of probe, which is insufficient to form a hydrophobic cavity or encapsulate all TMB, resulting in oxidized TMB ox2 Coupled with the remaining TMB to form blue TMB ox1 When excess yellow TMB ox2 With blue TMB ox1When coexisting, the color appears green. Therefore, as the target concentration increases, the detection result changes from yellow to green, then to blue, and finally to colorless.

[0032] Example 2: Synthesis and characterization results of CMO nanozymes Figure 2 As shown: First, the microstructure of CMO was observed by electron microscopy. Figure 2 As shown in A, the TEM image clearly shows the size and morphology of CMO, which is uniform in size ranging from about 140 to 200 nm with a regular bayberry-shaped structure. The corresponding X-ray energy dispersive spectrum (EDS) image of CMO ( Figure 2 B) shows that Mn, Co and O elements are evenly distributed in the nanomaterial, indicating that the synthesis of CMO is successful. In order to explore the crystal structure of CMO, powder X-ray diffraction (XRD) measurement technology was used, and the results are shown in Figure 2 C. CMO shows 14 typical diffraction peaks at 2θ = 17.8°, 28.5°, 32.0°, 35.8°, 37.8°, 44.0°, 50.6°, 53.3°, 55.6°, 59.6°, 64.4°, 69.6° and 73.3°, corresponding to the (101), (112), (103), (211), (004), (220), (105), (312), (303), (321), (224), (400), (305) and (413) planes of Mn3O4, respectively (JCPDS No. 24-0734). The five characteristic peaks at 2θ values ​​of 31.3°, 36.9°, 45.0°, 59.4° and 65.2° were attributed to the (220), (311), (400), (511) and (440) crystal planes of Co3O4, respectively (JCPDS No.43-1003). These results indicate that CMO, which has similar diffraction peaks of Mn3O4 and Co3O4, is a heterojunction combining the crystal structures of Mn3O4 and Co3O4. At the same time, the surface composition, elemental valence state and chemical form of Mn, Co and O in CMO were studied using X-ray photoelectron spectroscopy (XPS) measurements. The investigation showed that CMO is composed of Mn, Co, O, N and C, with obvious peaks for Mn 2p, Mn 3p, Co 2p, O 1s, N 1s and C 1s ( Figure 2 D). Figure 2 As shown in Figure E, the Mn 2p peaks indicate that Mn 2p1 / 2 and Mn 2p3 / 2 are located at BE values ​​of 652.95 and 641.15 eV, respectively, and the distance of 11.8 eV indicates the oxidation state of the Mn element. The peak with a binding energy value of 643.6 eV can be attributed to the high spin Mn 4+ High proportion of Mn 3+This indicates that there are more oxygen vacancies, which promotes higher mimetic enzyme activity of the prepared nanozymes. Similarly, the Co 2p spectrum ( Figure 2 F) The two main peaks at 795.8 and 780.4 eV are attributed to Co 2p1 / 2 and Co 2p3 / 2, respectively, and there are obvious oscillating satellite peaks at 786.0 eV and 802.2 eV, indicating that the Co 2p spectrum reveals the Co 2+ and Co 3+ coexistence. Figure 2 The O 1s spectrum in G, after fine fitting and deconvolution, has three peaks at 531.8, 531.2 and 529.65 eV, which are respectively attributed to the surface adsorbed molecules (such as PVP or H2O) around the oxygen vacancies (OV), adsorbed oxygen species and typical metal oxygen bonds (Mn-O, Co-O) or lattice oxygen. The surface adsorbed oxygen belongs to oxide defects and is the reason for the high efficiency. The FT-IR spectra of PVP, Co3O4, Mn3O4 and CMO are shown in Figure 2. Figure 2 H. These four materials have a wavelength of 3300-3700 cm -1 The broad band at 2954 cm is attributed to the OH stretching vibration of absorbed water molecules. -1 The characteristic absorption band at 1663 cm-1 was assigned to CH stretching vibration. -1 The absorption band at 127 cm also comes from the overlap of C=O stretching vibration and OH bending vibration. -1 The strong peak at 1287 cm is caused by the bending vibration of methylene, while the -1 The peaks are caused by CN stretching vibration. In Co3O4, the peaks are located at 836 and 727 cm -1 The two strong absorption peaks are attributed to the vibration absorption of Co-O bonds. Similarly, the 605 cm absorption peak is derived from the Mn-O stretching vibration of Mn3O4. -1 and 494 cm -1 There are two characteristic absorption bands at 626 and 512 cm in the CMO spectrum. -1 The peak at 1635 cm-1 is attributed to the stretching vibration of the MO bond and is slightly shifted compared to Mn3O4, which is affected by Co doping. -1 The short peaks of α-PbO originated from the C=O stretching vibration and OH bending vibration of PVP, which is consistent with the XPS results. These characterization spectra verified the successful preparation of CMO.

[0033] Example 3: Performance verification and kinetic test of the prepared CMO-like oxidase (OXD). The results are as follows Figure 3 shown.

[0034] 1. CMO oxidation catalytic steps and results of TMB: TMB presents colored products to study the OXD-like activity of CMO: 10 μL of 0.2 mg / mL CMO was introduced into 100 μL of 2-200 μg / mL TMB dispersed in pH 3.6 citric acid buffer solution. Figure 3 As shown in A, as the concentration of TMB substrate increases, three types of oxidation products appear, and the color changes from yellow to green and finally to blue. This change is attributed to the limited amount of CMO that binds to TMB, resulting in its saturated oxidation capacity. CMO can initially catalyze some TMB to form the yellow product TMB ox2 As TMB concentrations increased, some ox2 Combines with TMB to form blue product TMB ox 1. TMB ox2 When TMB coexists, it is green. As the concentration of TMB continues to increase, all yellow TMB ox2 Both combine with TMB to form a blue product TMB ox1 The UV-visible absorption spectra of these signal materials are as follows Figure 3 B. TMB shows the strongest absorption at 286 nm, while blue TMB ox1 There are three absorption peaks at 286nm, 370nm and 652nm. Yellow TMB catalyzed by CMO ox2 The absorption peak is at 436 nm, which is pure TMB. ox2 The absorption peak of TMBo is red-shifted at 420 nm and is set as the standard wavelength for recording absorbance. x1 and TMB ox2 The green mixture has these four peaks.

[0035] 2. Screening of optimal pH and kinetic determination: Figure 3 As shown in C, the CMO has oxidase activity when the pH is less than 6. Through comprehensive comparison, pH 3.6 was selected as the optimal catalytic environment for the two-electron oxidation of TMB by CMO. Add 100 μL of TMB stock solution (2 mg / mL dissolved in ethanol) to a final volume of 10 mL of citric acid buffer (0.02 M, optimal pH value) to prepare the substrate solution. In a 96-well ELISA plate, add 5-200 μL of substrate solution to the wells containing 275-80 μL of acetic acid buffer solution. After adding 20 μL of the synthesized CMO solution, record the color development time (5-20 s), color development state (yellow indicates TMB) of each well ox2 ; Green indicates TMB ox1 and TMB ox2 Mixed state; blue represents TMB oxl) and absorbance (at 436 nm or 652 nm). The concentration of the oxidation product was calculated using the Lambert-Beer law. Combined with the reaction time, the rate of the oxidation reaction was obtained. A regression curve of the reaction rate corresponding to the TMB concentration was established, and the Michaelis constant (K) was obtained after fitting with the Michaelis-Menton equation. m ) and maximum speed (V max ).

[0036] Where: A is the absorbance; ε is the molar extinction coefficient; b is the length of the light source through the solution; c is the concentration of the solution; V is the reaction rate, which increases with the increase of the substrate TMB concentration [S] until it reaches a constant V max , K m is the Michaelis constant, which indicates the reaction rate V max The specific substrate concentration at half the concentration of

[0037] The results of kinetic assays were Figure 3 As shown in D and E, at a fixed oxidase concentration, as the concentration of substrate TMB increases, the reaction rate gradually accelerates to a constant level. By fitting the data to the Michaelis-Menton equation (enzyme kinetic function), the K m 3.357μM, V max is 1.338 μM / s. Compared with HRP used in classic ELISA (TMB’s K m and V max 730 μM and 0.147 μM / s, respectively). m decreased by nearly 3 orders of magnitude, while V max In addition, these parameters were compared with those of reported nanooxidases (see Figure 3 F and Table 1 ), indicating that CMO oxidase has a stronger affinity for the substrate TMB (K m smaller), and the catalytic reaction rate is faster.

[0038] Table 1 Comparison of the Michaelis constants (K) of different nanooxidases for TMB oxidation m ) and the maximum reaction rate (V max ) 3. Mechanism of catalytic oxidation of CMO: Through free radical capture experiments, the types of reactive oxygen species (ROS) generated were determined. Isopropyl alcohol (IPA), furfuryl alcohol (FFA), p-benzoquinone (BQ) and Na2-EDTA can scavenge hydroxyl radicals (•OH), quench singlet oxygen ( 1O2), consumes superoxide anion radicals (·O 2- ), neutralization holes (h + ). At the optimal pH value (100 μL), 10 μL of 50 mM IPA, FFA, BQ, and Na2-EDTA were added to 20 μL of CMO solution. After incubation for 10 minutes, 50 μL of 0.02 mg / mL TMB was introduced and the absorbance was measured at 436 nm. The results are shown in Figure 3 As shown in G, the addition of FFA and BQ to the catalytic system significantly reduced the color reaction of TMB oxidation, because they captured 1 O2 and •O 2- , indicating that CMO can catalyze the production of O2 in an acidic environment 1 O2 and •O2 - Thereby oxidizing TMB.

[0039] We speculated that the mechanism by which CMO acts as an oxidase to trigger the two-electron oxidation of TMB, such as Figure 3 H. A large amount of precursor PVP was used in the preparation of CMO. Due to its viscosity, even after three washes with water and ethanol, some undissociated PVP was firmly bound to the surface of the synthetic material. Therefore, the binding of PVP gave the nanomaterial amphiphilicity, i.e., hydrophilic and hydrophobic properties. In the aqueous phase, CMO can form hydrophobic microcavities, which facilitates the encapsulation of hydrophobic TMB. After being oxidized by free radicals, the generated TMB ox2 Due to its hydrophilicity, it leaves the hydrophobic cavity and enters the aqueous phase, avoiding binding with TMB to form a complex TMB ox1 Therefore, these hydrophobic cavities effectively block TMB and ox2 If an excess of TMB is introduced into the system, the hydrophobic cavity will not be sufficient to encapsulate all the TMB, resulting in the unusual two-electron oxidation of TMB. ox2 Combines with TMB to form blue TMB ox1 When TMB ox1 and TMB ox2 When TMB ox2 Completely combines with excess TMB to form TMB ox1 , the system turns blue.

[0040] Example 4: Evaluation of the analytical performance of the CMO-based kit for microcystin-LR. The results are as follows: Figure 4 shown.

[0041] 1. Sensitivity definition: A four-parameter logistic regression equation was used to fit the relationship between the absorbance change and the concentration of the target substance. According to the fitting formula, the detection limit (LOD) was calculated based on the x value corresponding to the y value obtained by subtracting three times the standard deviation from the blank group. The detection range was 10% to 90% inhibition concentration (IC 10 -IC 90 ).

[0042] Where: A1 and A2 represent the maximum and minimum asymptotes of the curve, respectively. P is the slope parameter of the curve, and x0 is the x-position parameter corresponding to the midpoint between the two asymptotes.

[0043] Sensitivity results are as follows Figure 4 As shown in A, as the concentration of MC-LR increased, the yellow and blue signals gradually decreased until they became colorless, which is consistent with the competition model. By plotting the absorbance at 436 nm versus MC-LR concentration, a good four-parameter logistic regression relationship (R 2 =0.994). According to the fitting equation, the detection limit of 0.505 ng / mL MC-LR was calculated by subtracting three times the standard deviation from the blank value, which is lower than the safe drinking water value (1 ng / mL) and the tolerable daily intake (TDI) of 0.04 μg / kg body weight set by the World Health Organization. Similarly, the quantitative analysis range of the classic ELISA is 1.22-102.7 ng / mL, and the detection limit is 1.01 ng / mL. It can be seen that the IC of this CMO-based sensor is much higher than that of 8.878 ng / mL of the traditional ELISA. 50 (the concentration of the target substance corresponding to the half-maximum competitive inhibition rate, representing the sensitivity of the competitive immunoassay) was 1.057 ng / mL, an increase of 8.4 times.

[0044] 2. Specificity identification: The specificity of the CMO-based ELISA was investigated by detecting other amino acid-containing substances and organic contaminants, including glutathione, histamine, doxorubicin, tetracycline, oxytetracycline, and dibutyl phthalate. These drugs were dissolved in PBS at a concentration of 1 μg / mL and tested, with PBS serving as a negative control. The results are shown in Figure 2. Figure 4 As shown in B, high concentrations (1 μg / mL) of other substances produced the same detection results as the negative control, while only 20 ng / mL of MC-LR caused a significant signal decrease reaction. These findings demonstrated that the CMO biosensor has satisfactory sensitivity and specificity in detecting MC-LR.

[0045] Example 5: Actual sample analysis: Environmental samples and food samples susceptible to MC-LR contamination were tested to evaluate the practicality of the proposed ELISA kit. Representative samples selected included Lingde Lake of Shanxi University, Yuncheng Salt Lake, fish and shrimp. Water samples from Lingde Lake of Shanxi University were directly tested after adjusting the pH value with 0.1 M pH 7.4 PBS (original sample: PBS = 9:1, v / v). Given that Yuncheng Salt Lake contains a high concentration of Na2SO4, whose solubility decreases with low temperature, the samples were refrigerated at 4 °C to precipitate the salt, thereby reducing its interference with Ag-Ab recognition. The supernatant was taken and then added to PBS to adjust its pH value according to the above method. For fish and shrimp samples, 1 g of chopped sample was dispersed in 2 mL of 3% (v / v) trichloroacetic acid, shaken for 10 minutes, and centrifuged to remove the precipitate. The supernatant was adjusted to pH 7.4 with NaOH. The obtained sample solution was tested using the proposed ELISA.

[0046] The results are shown in Table 2. Except for the concentration of 1.85 ng / mL detected in the brown salt pool water of Yuncheng Salt Lake, MC-LR was not detected in the green and blue salt pool water of Lingde Lake, Yuncheng Salt Lake, or in fish or shrimp. The accuracy of the analysis was evaluated by standard addition and recovery experiments, and the results showed that the recoveries of MC-LR in these environmental water samples and aquatic products were 80.4-112.1%, and all RSDs were less than 10%, indicating that the designed detection platform has satisfactory reliability and wide application potential.

[0047] Table 2: Detection of microcystins in real samples by CMO-based kit (n=3) Finally, 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0048] References: [1] J. Xi, R. Zhang, L. Wang, W. Xu, Q. Liang, J. Li, et al., ANanozyme-Based Artificial Peroxisome Ameliorates Hyperuricemia and IschemicStroke, Adv. Funct. Mater. 31 (2020) 2007130. [2] Y. Wu, L. Jiao, X. Luo, W. Xu, X. Wei, H. Wang, H. Yan, W. Gu, B.Xu, D. Du, Y. Lin, C. Zhu, Oxidase-Like Fe-N-C Single-Atom Nanozymes for theDetection of Acetylcholinesterase Activity, Small 15 (2019) e1903108. [3] Y. Wang, Z. Zhang, G. Jia, L. Zheng, J. Zhao, X. Cui, Elucidatingthe mechanism of the structure-dependent enzymatic activity of Fe-N / C oxidasemimics, Chem. Commun. 55 (2019) 5271-5274. [4] V. Baldim, N. Yadav, N. Bia, A. Graillot, C. Loubat, S. Singh, A.Karakoti, J. Berret, Polymer-Coated Cerium Oxide Nanoparticles asOxidoreductase-like Catalysts, ACS Appl. Mater. Interfaces 12 (2020) 42056-42066. [5] C. J. Yu, T. H. Chen, J. Y. Jiang, W. L. Tseng, Lysozyme-directedsynthesis of platinum nanoclusters as a mimic oxidase, Nanoscale 6 (2014)9618-9624. [6] A. Asati, S. Santra, C. Kaittanis, S. Nath, J. M. Perez, Oxidase-Like Activity of Polymer-Coated Cerium Oxide Nanoparticles, Angew. Chem. Int.Ed. Engl. 48 (2009) 2308-2312. [7] W. Zhang, X. Niu, S. Meng, X. Li, Y. He, J. Pan, F. Qiu, H. Zhao,M. Lan, Histidine-mediated tunable peroxidase-like activity of nanosized Pdforphotometric sensing of Ag + , Sensor Actuat B-Chem. 273 (2018) 400-407. [8] H. Cheng, S. Lin, F. Muhammad, Y. Lin, H. Wei, RationallyModulate the Oxidase-like Activity of Nanoceria for Self-Regulated Bioassays,ACS Sens. 1 (2016) 1336-1343. [9] T. Dong, X. Zhang, J. Yuan, Z. Lin, P. Yin, H. Yu, M. Wang, A.Liu, Sensitive Lateral Flow Immunoassay Based on Specific Peptide andSuperior Oxidase Mimics with a Universal Dual-Mode Significant SignalAmplification, Anal. Chem. 95 (2023) 12532−12540.

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Claims

1. A CMO nanooxidase, characterized in that: The CMO nano-oxidase is a bayberry-shaped Co-Mn3O4 oxidase; the nano-oxidase is an amphiphilic CMO heterojunction synthesized by a one-pot hydrothermal method using Co3O4 and Mn3O4, and has a particle size of 110-140 nm.

2. A method for preparing the CMO nanooxidase according to claim 1, characterized in that: The method comprises the following steps: 0.5 g of tetrahydrated manganese (II) acetate Mn(CH3COO)2·4H2O, 0.02 g of tetrahydrated cobalt (II) acetate Co(CH3COO)2·2H2O and 0.4 g of PVP are dissolved in 15 mL of ethylene glycol; Stir vigorously for 1 hour until it is completely dissolved into a transparent orange-red solution; add 0.8mL 1mol / L HCl to the solution and stir for another 0.5 hour; then heat the mixture to 160°C at a heating rate of 20°C / min, and then heat at 160°C for 12 hours; after cooling to room temperature, centrifuge the obtained dark green solution at 9000rpm for 10 minutes to collect the dark green nanozyme product; wash the dark green nanozyme product with distilled water and ethanol three times respectively, and finally resuspend it in 8mL distilled water, which is the CMO nanooxidase.

3. A method for constructing a CMO-labeled chicken ovalbumin-coupled microcystin LR antigen OVA-MC-LR probe using the CMO nanooxidase according to claim 1 or the CMO nanooxidase obtained by the method according to claim 2, characterized in that: The method comprises the following steps: 2 μL of 1 mg / mL OVA-MC-LR is added to 1 ml of PBS solution of CMO at a concentration of 1 mg / mL, and the mixture is gently vortexed for 30 minutes at room temperature; 200 μL of 1% w / v chicken egg albumin OVA is then mixed for 30 minutes to block the active sites on the surface of the nanozyme, i.e., the antigen probe MC-LR-OVA-CMO, which is stored at 4°C for future use; The CMO concentration is 1 mg / mL, the antigen OVA-MC-LR concentration is 2 μg / mL, the OVA concentration selected for blocking is 0.2%, and the incubation time is 30 minutes.

4. An immunosensor kit prepared using the CMO-OVA-MC-LR probe prepared according to claim 3 as a signal probe.

5. The immunosensor kit according to claim 4, characterized in that: The immunosensor kit comprises a polystyrene 96-well ELISA plate, which is coated with a mouse monoclonal antibody that specifically recognizes microcystin-LR, and the plate is washed three times after incubation, chicken ovalbumin OVA is blocked, and the plate is washed three times after incubation, a standard microcystin solution or a target sample solution is added, and the plate is washed three times after incubation, a CMO-OVA-MC-LR probe is added, and the plate is washed six times after incubation, and a TMB substrate solution is added, and the absorbance is measured after the reaction for 2 minutes.

6. The immunosensor kit according to claim 5, characterized in that: The coated mouse monoclonal antibody that specifically recognizes microcystin-LR was incubated at a concentration of 0.5 μg / mL at 4°C overnight, the blocking solution was 0.5% OVA-PBST, and the incubation was at 37°C for 45 min. The target was added to the ELISA plate and incubated at 37°C for 30 min, and then the CMO-OVA-MC-LR probe was added.

7. The immunosensor kit according to claim 5, characterized in that: The target objects are microcystin-LR standards and water environment samples and aquatic product samples containing microcystin-LR.

8. Use of the immunosensor kit according to claim 4 in detecting microcystin-LR in lake water, salt lake water, fish and shrimp.

9. The use according to claim 8, characterized in that: The lake water is adjusted to pH with phosphate buffer. After the salt lake water is treated at 4°C, the aqueous solution of precipitated Na2SO4 is taken and the pH is adjusted with phosphate buffer. The fish and shrimp samples are treated with trichloroacetic acid to precipitate protein and then the pH is adjusted, and then the designed kit is used for detection.

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