Cmo nanooxidase and cmo-ova-mc-lr probe, kit, preparation method and application thereof

Abstract

The application belongs to the technical field of biological detection, and aims at solving the single color change mode and poor sensitivity of the current ELISA kit, and provides CMO nano-oxidase, a CMO-OVA-MC-LR probe, a kit, a preparation method and application thereof.The CMO nano-oxidase is a raspberry-shaped Co-doped Mn3O4 oxidase, the nano-oxidase is a two-heterojunction CMO of Co3O4 and Mn3O4 synthesized by one-pot hydrothermal method, and the particle size is 110-140 nm.The detection limit of the enzyme-linked immunosorbent kit constructed by the application for microcystin LR (MC-LR) is 0.505 ng / mL, and the quantitative range is 0.195-100 ng / mL.Benefiting from the strong catalytic activity and good biocompatibility of the nano-enzyme, the label and signal response mode open up a new way for the sensitivity improvement of the immunosensor determination, and are conducive to further expanding the application of the immunosensor.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a CMO nano-oxidase and its constructed CMO-OVA-MC-LR probe, kit, preparation method, and application. The nano-oxidase can oxidize TMB to a yellow final product with two electrons, and after binding TMB, it exhibits a colorimetric mode with multiple colors such as green and blue. The probe and kit are used to detect microcystin toxins. Background Technology

[0002] In recent years, eutrophication of water bodies has promoted the rapid growth of algae, leading to algal blooms. Besides worsening the color and odor of water, it also produces toxic metabolites such as algal toxins. Microcystins are a class of cyclic heptapeptide compounds produced by various cyanobacteria, 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, and is the most common and widely distributed category. MC-LR is highly biotoxic because it can inhibit the activity of protein phosphatase and induce apoptosis. Faced with this serious 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). These methods are time-consuming, labor-intensive, and easily affected by sample matrix interference. Enzyme-linked immunosorbent assay (ELISA) is a powerful analytical tool, playing an increasingly important role in biosensors due to its specificity, sensitivity, stability, and reliability. However, due to the inherent characteristics of natural enzymes, such as insufficient sensitivity and catalytic activity, the classic ELISA using horseradish peroxidase (HRP) to catalyze the colorimetric reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) remains limited by relatively poor sensitivity and stability.

[0003] In 2007, Fe3O4 nanoparticles were discovered to possess reaction mechanisms similar to those of biological enzymes, sparking widespread interest and research enthusiasm for nanozymes in the academic community. Compared to natural enzymes, nanozymes offer numerous advantages, including controllable design, superior catalytic activity, robust stability, long-term effectiveness, and large-scale, low-cost production. An increasing number of nanozymes are being discovered, designed, and synthesized, including those made from noble metals, transition metal oxides, metal-organic frameworks (MOFs), metal-doped carbon dots, and emerging single-atom nanozymes. These nanozymes exhibit diverse enzyme-mimicking activities, such as peroxidase (POD), oxidase (OXD), catalase, superoxide dismutase (SOD), and glutathione peroxidase. The utilization of enzyme activity, particularly catalytic oxidation, has broad applications in sensing, sterilization, anticancer activity, 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, developing high-performance nanozymes to improve sensitivity is an urgent task. Manganese (Mn) is one of the most abundant elements on Earth, possessing non-toxicity, low cost, and abundant valence states (Mn). 2+ Mn 3+ Mn 4+ Mn 6+ Mn 7+ Based on the characteristics of [the material], 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 hazards of H2O2 during transportation and storage, and overcoming the inaccuracy of detection caused by the instability of H2O2. To further improve the catalytic activity of nanozymes, many effective strategies have been proposed, including optimizing the raw material ratio, exploring preparation conditions, modifying with organic molecules and doping with other elements to precisely control size, changing morphology and surface potential, modifying surface groups, forming hollow structures, and increasing oxygen vacancies (OVs) and defects to provide high surface energy and abundant active sites. Summary of the Invention

[0005] To address the limitations of current ELISA kits, which rely on a single color-changing mode and have poor sensitivity, this invention provides a CMO nano-oxidase, its constructed CMO-OVA-MC-LR probe, a kit, a preparation method, and its applications.

[0006] The present invention is achieved by the following technical solution: 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 from Co3O4 and Mn3O4 by a one-pot hydrothermal method, with a particle size of 110-140 nm.

[0007] The method for preparing the CMO nano-oxidase includes the following steps: 0.5 g manganese(II) acetate tetrahydrate Mn(CH3COO)2•4H2O, 0.02 g cobalt(II) acetate tetrahydrate Co(CH3COO)2•2H2O and 0.4 g 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 stirred for another 0.5 hours; 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 resulting dark green solution is centrifuged at 9000 rpm for 10 minutes to collect the dark green nano-enzyme product; the dark green nano-enzyme 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] A method for constructing a CMO-labeled chicken ovalbumin-conjugated microcystin LR antigen OVA-MC-LR probe using the CMO nano-oxidase or the CMO nano-oxidase obtained by the method comprises the following steps: 2 μL of 1 mg / mL OVA-MC-LR is added to 1 mL of PBS solution with a concentration of 1 mg / mL CMO, and gently vortexed for 30 minutes at room temperature; then 200 μL of 1% w / v chicken ovalbumin OVA is mixed for 30 minutes to block the active site on the nanozyme surface, which is the antigen probe CMO-OVA-MC-LR, stored at 4°C for later use.

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

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

[0011] Furthermore, the immunosensing kit includes a polystyrene 96-well microplate coated with a murine monoclonal antibody that specifically recognizes microcystin-LR. After incubation, the plate is washed three times, chicken ovalbumin (OVA) is blocked, and after incubation, the plate is washed three times. Standard microcystin solution or target sample solution is added, and after incubation, the plate is washed three times. The CMO-OVA-MC-LR probe is added, and after incubation, the plate is washed six times. TMB substrate solution is added, and the absorbance is measured after 2 minutes of reaction.

[0012] Furthermore, the murine monoclonal antibody specifically recognizing microcystin-LR was coated and incubated overnight at 4°C with a concentration of 0.5 μg / mL. The blocking solution was 0.5% OVA-PBST, and the plate was incubated at 37°C for 45 min. The target was added to the ELISA plate and incubated at 37°C for 30 min. Then, the CMO-OVA-MC-LR probe was added.

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

[0014] The present invention also provides the application of the aforementioned immunosensing kit in the detection of microcystin-LR in lake water, saline lake water, fish, and shrimp.

[0015] The lake water was treated with phosphate buffer to adjust the pH. After the salt lake water was treated at 4°C, the pH of the aqueous solution containing precipitated Na2SO4 was adjusted with phosphate buffer. Fish and shrimp samples were treated with trichloroacetic acid to precipitate proteins and then the pH was adjusted. Finally, the samples were tested using the designed kit.

[0016] Inspired by the synthesis methods of hollow CeO2 and N-doped Mn3O4 nano-oxidases, this invention develops a bayberry-shaped CMO that can form a hydrophobic space to surround TMB and gain two-electron oxidation, exhibiting excellent oxidase activity. Notably, most oxidases and peroxidases catalyze the oxidation of TMB via a single-electron oxidation pathway, producing a blue oxidation product (TMB). ox1 It requires a strong acid to terminate the conversion into a stable yellow oxidation product (TMB). ox2 In contrast, the prepared CMO nanozyme can directly catalyze the oxidation of TMB to a yellow final product (TMB) via a two-electron oxidation pathway. ox2 It exhibits remarkable oxidation capabilities. Furthermore, it is associated with blue TMB. ox1 In comparison, yellow TMB ox2 It exhibits better stability and a larger molar extinction coefficient, facilitating more sensitive analysis and overcoming the cumbersome steps and errors caused by introducing strong acid terminators. Furthermore, unlike the preparation of nanozymes made from precious metals and rare elements, the acquisition of this CMO nanozyme avoids high costs and secondary pollution, and avoids the complex and demanding reaction modes of single-atom nanozymes (SAzymes), demonstrating significant advantages in terms of environmental friendliness and economic benefits.

[0017] A robust CMO was conjugated with OVA-MC-LR to construct an antigen (OVA-MC-LR) probe. The MC-LR target preferentially competes for binding with antibodies (Abs), resulting in a more sensitive and reliable MC-LR detection ELISA kit. The designed analytical platform has been successfully applied to screen real-world samples, including lake water, salt lakes, fish, and shrimp, demonstrating its significant potential in food safety and environmental monitoring.

[0018] Compared with existing technologies, its advantages and positive effects are as follows: Under acidic conditions, this invention can directly oxidize TMB to a yellow final state oxidation product using two electrons, label the antigen to establish a signal probe, and combine the interconversion of TMB oxidation products to achieve multicolor signal immunoassay of microcystin.

[0019] This invention differs from most nanozymes in that they perform single-electron oxidation of TMB, directly oxidizing TMB to its final yellow product using two electrons. The nanozyme forms a hydrophobic cavity, causing hydrophobic TMB to accumulate within the cavity and be oxidized to hydrophilic TMB by reactive oxygen species. ox2 The oxidation product leaves the cavity, thus avoiding combination with TMB to form TMB. ox1 It demonstrates excellent two-electron oxidation capability.

[0020] CMO nanozymes exhibit remarkable catalytic activity (K... m =3.357μM, V max=1.338 μM / s), effectively driving the rapid and direct two-electron oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), converting TMB into stable yellow TMB. ox2 The interconversion between different TMB oxidation products produces a multicolor signal of yellow, green, and blue, allowing for more sensitive detection, without the need for strong acid termination. 50 The half-maximal inhibitory concentration (WMC) was 1.057 ng / mL, representing an 8.4-fold improvement over the classic horseradish peroxidase-based ELISA. The proposed biosensor demonstrated satisfactory performance in real-world environmental and food samples, with recoveries ranging from 80.4% to 112.1%, demonstrating its significant potential for safeguarding public health. Attached Figure Description

[0021] Figure 1 The following is a schematic diagram of the signal material synthesis and sensor principle of the present invention: (A) the preparation principle of CMO and probe; (B) the CMO-driven TMB two-electron oxidation and the UV-Vis absorption peak of the TMB oxidation product; (C) the principle diagram of the multicolor results of the kit for detecting MC-LR.

[0022] Figure 2 Characterization of the signal material CMO for this invention: (A) Transmission electron microscopy image of CMO; (B) Elemental distribution map of CMO; (C) XRD pattern 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;

[0023] Figure 3 Characterization of oxidase activity of CMO, the signaling material of this invention: (A) Citrate buffer containing CMO (0.02 mg / mL) and different concentrations of TMB at 436 nm (TMB ox2 ) and 652nm (TMB) ox1 (A) Photographs and UV-Vis absorbance at (B) TMB, TMB ox1 TMB ox2 TMB ox1 and TMB ox2 (c) UV-Vis absorption spectra of the mixture and CMO; (d) Effect of pH on the oxidase activity of CMO; (e) Kinetic test of CMO-catalyzed TMB oxidation; (f) Kinetic parameters K of various oxidases catalyzing TMB as reported in the literature. m and V max Comparison; Test results of the types of reactive oxygen species generated by (G)CMO; Hydrophobic cavity-mediated TMB two-electron oxidation mechanism of (H)CMO;

[0024] Figure 4 The performance evaluation diagram of the kit of the present invention is as follows: (A) Quantitative analysis of MC-LR by CMO-ELISA; (B) Comparison of the sensitivity of this sensor with reported sensors for detecting MC-LR; (C) Specificity evaluation results of this sensor. Detailed Implementation

[0025] To make the objectives, 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 some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Unless otherwise defined, 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 pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0027] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0029] To address the limitations of single-color signals and low sensitivity in enzyme-linked immunosorbent assay (ELISA) kits, this invention constructs a nanozyme signaling material with superior oxidase activity, capable of directly oxidizing TMB to the final product via double-electrode ablation. To achieve optimal assay performance, an antigen-labeled probe was designed and fabricated, employing a target molecule-priority competition mode to further ensure detection stability. Optimal detection results were achieved through optimization of key parameters. This sensor was ultimately used to detect microcystin-LR, particularly in aquatic environments and aquatic products, demonstrating reliability, sensitivity, and stability.

[0030] Example 1: Synthesis strategy of CMO and OVA-MC-LR probes as follows Figure 1 As shown in Figure A, the CMO nanozyme was prepared using a one-pot hydrothermal method. The resulting nanomaterial was bound to OVA-MC-LR, and adsorption was blocked by OVA, forming a signal probe. The principle of this CMO for TMB oxidation and multicolor ELISA detection of MC-LR is as follows... Figure 1 B and Figure 1 As shown in C, the specific steps include:

[0031] 1. CMO is prepared by hydrothermal synthesis, such as... Figure 1 A: Dissolve 0.5 g manganese acetate(II) tetrahydrate (Mn(CH3COO)2•4H2O), 0.02 g cobalt acetate(II) tetrahydrate (Co(CH3COO)2•2H2O), and 0.4 g PVP in 15 mL of ethylene glycol. Stir the mixture vigorously for 1 hour until completely dissolved, resulting in a clear orange-red solution. Then, add 0.8 mL of 1 mol / L HCl to the solution and stir for another 0.5 hours. Transfer the mixture to a PTFE-lined stainless steel reactor and heat at 160 °C for 12 hours. After cooling to room temperature, centrifuge the resulting dark green solution at 9000 rpm for 10 minutes to collect the product. Wash the dark green nanozyme product three times with distilled water and ethanol, and finally resuspend it in 8 mL of distilled water.

[0032] 2. Preparation of the CMO-OVA-MC-LR probe, such as... Figure 1 A: Add 2 μL of 1 mg / mL OVA-MC-LR to 1 mL of (1 mg / mL) CMO dissolved in PBS and gently vortex for 30 minutes at room temperature. Then, mix with 200 μL of 1% OVA for 30 minutes to block the active sites on the nanozyme surface and prevent non-specific binding to other substances in the sample. The antigen probe (MC-LR-OVA-CMO) is stored at 4°C for future use.

[0033] 3. This material exhibits oxidase activity of two-electron oxidation of TMB, such as... Figure 1 B: TMB can utilize the reactive oxygen species (ROS) generated by CMO under acidic conditions. 1 O2,•O2 - Direct two-electron oxidation to the yellow final product TMB ox2 In contrast, most oxidases and peroxidases can only oxidize TMB to the blue product (TMB) via a single-electron pathway. ox1 This requires a strong acid to neutralize TMB. ox1 Convert to TMB ox2 The final form is yellow. It's worth noting the blue TMB. ox1 It is an unstable, confusing complex that can decompose into colorless TMB and yellow TMB. ox2 This means that CMO differs from typical oxidases and peroxidases because it has very strong oxidative activity, capable of completely oxidizing TMB to its final yellow state (TMB). ox2 In this system, TMB ox1 and TMBox2 The spectral absorption peaks appear at 652 nm and 436 nm, respectively.

[0034] 4. The principle of multicolor signal presentation in ELISA based on CMO superoxidase is as follows: Figure 1 As shown in C, the detection process specifically includes the following steps:

[0035] Immunoassay Procedure: The ELISA procedure was performed on a clear 96-well microtiter plate according to the following steps. (1) Coating: The wells of the microplate were coated with MC-LR specific antibody (0.5 μg / mL, 100 μL / well in carbonate buffer at pH 9.6) and incubated overnight at 4°C. (2) Blocking: 150 μL / well of 0.5% OVA was added to PBST (pH 7.4, 0.01M PBS containing 0.05% Tween-20) at 37°C to block the remaining binding sites on the plate and incubated for 40 minutes to prevent non-specific adsorption. (3) Addition of Target Analyte: MC-LR standard (0-100 ng / mL, 100 μL) was added to the wells and incubated at 37°C for 30 minutes to be captured by the antibody immobilized in the microplate. After each of the above steps, the plate was washed three times with PBST to remove molecules not adsorbed on the plate and the supernatant. (4) Signal probe introduction: Add 100 μL of CMO-OVA-MC-LR probe and incubate at 37°C for 30 minutes to allow it to be captured by the remaining antibody. To ensure complete removal of probes not adsorbed by the antibodies, wash the plate six times with PBST to avoid false negative results. (5) Substrate colorimetric reaction: Add 100 μL of TMB (20 μg / mL). Due to the strong oxidase activity of CMO, complete colorimetric results can be obtained within 2 minutes without the need for traditional strong acid termination. Measure the absorbance at 436 nm using a microplate reader.

[0036] The CMO-OVA-MC-LR probe was introduced into the ELISA platform for the detection of MC-LR, such as... Figure 1 As shown in Figure C, in a microtiter plate, the target MC-LR preferentially competes for binding with the Abs on the plate, and then the CMO-OVA-MC-LR probe binds to the remaining Abs. When detecting low concentrations of MC-LR, a large number of CMO-OVA-MC-LR probes bind to the micropores, forming hydrophobic cavities and promoting the oxidation of hydrophobic TMB. The resulting oxidation product is 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 This results in a yellow color. At high MC-LR concentrations, the remaining small amount of antibody can only immobilize a small amount of probe, insufficient to form a hydrophobic cavity or encapsulate all TMB, leading to TMB oxidation. ox2 Coupled with the remaining TMB, forming blue TMB.ox1 When there is excess yellow TMB ox2 With blue TMB ox1 When they coexist, the color displayed is green. Therefore, as the target concentration increases, the detection result changes from yellow to green, then to blue, and finally to colorless.

[0037] Example 2: Synthesis and characterization results of CMO nanozymes are as follows Figure 2 As shown: First, the microstructure of CMO was observed using an electron microscope. (As shown...) Figure 2 As shown in Figure A, the TEM image clearly reveals the size and morphology of CMO, which is uniform in size, ranging from approximately 140 to 200 nm, and has a regular bayberry-shaped structure. The corresponding X-ray energy-dispersive spectroscopy (EDS) image of CMO is also shown. Figure 2 B) shows that Mn, Co, and O elements are uniformly distributed in the nanomaterial, indicating the successful synthesis of CMO. To explore the crystal structure of CMO, powder X-ray diffraction (XRD) was used, and the results are as follows: Figure 2 As shown in Figure C, CMO exhibits 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) crystal planes of Mn3O4 (JCPDS No. 24-0734). Five characteristic peaks at 2θ values ​​of 31.3°, 36.9°, 45.0°, 59.4°, and 65.2° are attributed to the (220), (311), (400), (511), and (440) crystal planes of Co3O4 (JCPDS No. 43-1003). These results indicate that CMO, with similar diffraction peaks to Mn3O4 and Co3O4, is a heterojunction combining the crystal structures of Mn3O4 and Co3O4. Simultaneously, X-ray photoelectron spectroscopy (XPS) was used to investigate the surface composition, elemental valence states, and chemical forms of Mn, Co, and O in CMO. The investigation revealed that CMO is composed of Mn, Co, O, N, and C, with distinct peaks at Mn 2p, Mn 3p, Co 2p, O 1s, N 1s, and C 1s. Figure 2 D). For example Figure 2 As shown in Figure E, the Mn 2p peak indicates that Mn 2p1 / 2 and Mn 2p3 / 2 are located at BE values ​​of 652.95 and 641.15 eV, respectively, and the 11.8 eV distance indicates the oxidation state of Mn. The peak with a binding energy of 643.6 eV can be attributed to high-spin Mn.4+ State. High proportion of Mn 3+ This indicates the presence of more oxygen vacancies, which promotes higher mimicry enzyme activity in the prepared nanozyme. 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, while distinct oscillating satellite peaks at 786.0 eV and 802.2 eV indicate that the Co 2p spectrum reveals Co 2+ and Co 3+ Coexistence. For Figure 2 The 1s spectrum of O in G, after fine fitting and deconvolution, shows three peaks at 531.8, 531.2, and 529.65 eV. These peaks are attributed to surface-adsorbed molecules (such as PVP or H2O) surrounding the oxygen vacancy (OV), adsorbed oxygen species, and typical metal-oxygen bonds (Mn-O, Co-O), or lattice oxygen, respectively. The surface-adsorbed oxygen is an oxide defect and is the reason for its high reactivity. The FT-IR spectra of PVP, Co3O4, Mn3O4, and CMO are shown below. Figure 2 As shown in H. These four materials are in the range of 3300-3700 cm. -1 The broadband at this point is attributed to the OH stretching vibrations of the absorbed water molecules. For PVP, 2954 cm⁻¹ -1 The characteristic absorption band at 1663 cm⁻¹ is designated as the CH stretching vibration. -1 The absorption band at this point also originates from the coincidence of the C=O stretching vibration and the OH bending vibration. 127 cm -1 The strong peak is an absorption peak caused by the bending vibration of the methylene group, while the 1287 cm⁻¹ peak is an absorption peak caused by the bending vibration of the methylene group. -1 The peaks are caused by CN stretching vibrations. In Co3O4, they are located at 836 and 727 cm⁻¹. -1 The two strong absorption peaks are attributed to the vibrational absorption of the Co-O bond. Similarly, the 605 cm⁻¹ peak was derived from the Mn-O stretching vibration of Mn₃O₄. -1 and 494 cm -1 Two characteristic absorption bands at 626 and 512 cm⁻¹. In the CMO spectrum, these are the two characteristic absorption bands at 626 and 512 cm⁻¹. -1 The peak is attributed to the stretching vibration of the MO bond and is slightly shifted compared to Mn3O4, which is influenced by Co doping. It is located at 1635 cm⁻¹. -1 The short peaks originate from the C=O stretching vibration and OH bending vibration of the PVP binding, consistent with the XPS results. These characterization spectra verify the successful preparation of CMO.

[0038] Example 3: Verification of the oxidase-like (OXD) performance and kinetic tests of the prepared CMO, the results are as follows: Figure 3 As shown.

[0039] 1. Catalytic steps and results of CMO oxidation of TMB: The OXD-like activity of CMO was studied by presenting a colored product with TMB: 10 μL of 0.2 mg / mL CMO was introduced into 100 μL of 2-200 μg / mL TMB dispersed in a pH 3.6 citrate buffer solution. Figure 3 As shown in Figure A, with increasing TMB substrate concentration, three types of oxidation products appeared, with the color changing from yellow to green and finally to blue. This change is attributed to the limited amount of CMO binding TMB, leading to saturation of its oxidation capacity. Initially, CMO can catalyze some TMB to form the yellow product TMB. ox2 As TMB concentration increases, some TMB... ox2 It combines with TMB to form the blue product TMB. ox 1. With TMB ox2 They appear green when coexisting. As TMB concentration continues to increase, all yellow TMB... ox2 Both combine with TMB to form the blue product TMB. ox1 The ultraviolet-visible absorption spectra of these signal materials are as follows: Figure 3 As shown in B, TMB exhibits the strongest absorption at 286 nm, while the blue TMB... ox1 Three absorption peaks were observed at 286 nm, 370 nm, and 652 nm, respectively. Yellow TMB catalyzed by CMO. ox2 An absorption peak is observed at 436 nm, which is pure TMB. ox2 The absorption peak was red-shifted at 420 nm and was set as the standard wavelength for recording absorbance. TMBo x1 and TMB ox2 The green mixture has these four peaks.

[0040] 2. Screening for optimal pH and determination of kinetics: such as Figure 3 As shown in Figure C, the CMO exhibits oxidase activity at pH 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. 100 μL of TMB stock solution (2 mg / mL dissolved in ethanol) was added to a final volume of 10 mL of citrate buffer (0.02 M, optimal pH) to prepare the substrate solution. In a 96-well ELISA plate, 5–200 μL of the substrate solution was added to wells containing 275–80 μL of acetate buffer. After adding 20 μL of the synthesized CMO solution, the color development time (5–20 s) and color development status (yellow indicates TMB) were recorded for each well. ox2 Green indicates TMB ox1 and TMB ox2 The mixed state; blue indicates TMB. oxlThe absorbance (at 436 nm or 652 nm) and concentration of the oxidation product were measured. The concentration of the oxidation product was calculated using the Lambert-Beer law. The oxidation rate was obtained by combining the reaction time. A regression curve corresponding to the reaction rate at the TMB concentration was established, and the Michaelis constant (K) was obtained by fitting it with the Michaelis-Menton equation. m ) and maximum speed (V max ).

[0041]

[0042] In the formula: A is absorbance; ε represents the molar extinction coefficient; b is the length of the light source passing through the solution; c is the concentration of the solution; V represents the reaction rate, which increases with the increase of the substrate TMB concentration [S] until it reaches a constant V. max K m It is the Michaelis constant, which represents the reaction rate as V. max A specific substrate concentration at half the concentration.

[0043] The kinetic measurement results are as follows Figure 3 As shown in D and E, at a fixed oxidase concentration, the reaction rate gradually accelerates to a constant level with increasing substrate TMB concentration. By fitting the data to the Michaelis-Menton equation (enzyme kinetic function), K was obtained. m 3.357 μM, V max The value is 1.338 μM / s. Compared to HRP used in classic ELISA (TMB K), m and V max The Ki of the prepared CMO was 730 μM and 0.147 μM / s, respectively. m It decreased by nearly three orders of magnitude, while V max This represents an 8.1-fold increase. Furthermore, these parameters were compared with those of previously reported nano-oxidases (see...). Figure 3 (F and Table 1) indicate that CMO oxidase has a stronger affinity for the substrate TMB (K m Smaller size, faster catalytic reaction rate.

[0044] Table 1. Comparison of Michaelis constants (K0) for the catalytic oxidation of TMB by different nano-oxidases. m ) and maximum reaction rate (V max )

[0045]

[0046] 3. Investigation into the mechanism of catalytic oxidation of CMO: The types of reactive oxygen species (ROS) generated were determined through free radical scavenging experiments. Isopropanol (IPA), furfuryl alcohol (FFA), p-benzoquinone (BQ), and Na2-EDTA can scavenge hydroxyl radicals (•OH) and quench singlet oxygen, respectively. 1 O2), consumes superoxide anion free radicals (·O 2- ), neutralizing holes (h + At the optimal pH (100 μL), 10 μL of 50 mM IPA, FFA, BQ, and Na2-EDTA were added to 20 μL of CMO solution, respectively. 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 as follows. Figure 3 As shown in G, the addition of FFA and BQ to the catalytic system significantly reduced the colorimetric reaction of TMB oxidation, because they respectively captured [the catalytic reaction]. 1 O2 and •O 2- This indicates that CMO can catalyze the production of O2 in an acidic environment. 1 O2 and •O2 - This oxidizes TMB.

[0047] We hypothesized the mechanism by which CMO acts as an oxidase to trigger the two-electron oxidation of TMB, such as... Figure 3 As shown in Figure H, a large amount of precursor PVP was used in the preparation of CMO. Due to its viscosity, even after washing three times with water and ethanol, some undissociated PVP remained firmly bound to the surface of the synthesized material. Therefore, the binding of PVP endows the nanomaterial with amphiphilicity, i.e., hydrophilicity and hydrophobicity. In the aqueous phase, CMO can form hydrophobic microcavities, which facilitates the encapsulation of hydrophobic TMB. After being oxidized by free radicals, the resulting 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 TMB. ox2 The coupling between them leads to unusual two-electron oxidation. If excessive TMB is introduced into the system, the hydrophobic cavity will be insufficient to encapsulate all the TMB, resulting in TMB... ox2 Combined with TMB to form blue TMB ox1 When TMB ox1 and TMB ox2 When coexisting, the solution appears green; when TMB... ox2 It combines completely with excess TMB to form TMB ox1 At that time, the system turns blue.

[0048] Example 4: Evaluation of the analytical performance of the CMO-based kit for microcystin-LR, the results are as follows: Figure 4 As shown.

[0049] 1. Sensitivity Definition: A four-parameter logistic regression equation is used to fit the relationship between absorbance change and target substance concentration. According to the fitting formula, the limit of detection (LOD) is calculated based on the x-value corresponding to the y-value obtained by subtracting three times the standard deviation from the blank control group. The detection range is from 10% to 90% of the inhibition concentration (IC50). 10 -IC 90 ).

[0050]

[0051] In the formula: 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.

[0052] Sensitivity results are as follows Figure 4 As shown in Figure A, with increasing MC-LR concentration, the yellow and blue signals gradually decreased until they became colorless, which is consistent with the competition mode. A good four-parameter logistic regression (R0.05) relationship was established in the MC-LR range of 0.195 to 100 ng / mL by plotting the absorbance at 436 nm versus MC-LR concentration. 2 =0.994). Based on the fitted equation, the limit of detection (LOD) of 0.505 ng / mL MC-LR was calculated by subtracting three times the standard deviation from the blank value. This LOD is lower than the World Health Organization's safe drinking water value (1 ng / mL) and the tolerable daily intake (TDI) of 0.04 μg / kg body weight. Similarly, the quantitative analysis range of the classic ELISA is 1.22–102.7 ng / mL, with a LOD of 1.01 ng / mL. Therefore, compared to the traditional ELISA's 8.878 ng / mL, this IC based CMO sensor... 50 The target concentration (the half-maximal competitive inhibition rate, representing the sensitivity of the competitive immunoassay) was 1.057 ng / mL, an improvement of 8.4 times.

[0053] 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. Results are as follows: Figure 4 As shown in Figure 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 elicited a significant signal reduction response. These findings demonstrate that the CMO biosensor possesses satisfactory sensitivity and specificity in detecting MC-LR.

[0054] Example 5: Real-world Sample Analysis: Environmental and food samples susceptible to MC-LR contamination were tested to evaluate the practicality of the proposed ELISA kit. Representative samples included Lingde Lake from Shanxi University, Yuncheng Salt Lake, fish, and shrimp. Water samples from Lingde Lake were directly tested after pH adjustment with 0.1 M pH 7.4 PBS (original sample:PBS = 9:1, v / v). Given the high concentration of Na₂SO₄ in Yuncheng Salt Lake, whose solubility decreases with low temperatures, the samples were refrigerated at 4°C to precipitate salt, thus reducing interference with Ag-Ab recognition. The supernatant was collected and then added to PBS to adjust the pH as described above. 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 precipitate. The supernatant was adjusted to pH 7.4 with NaOH. The obtained sample solutions were tested using the proposed ELISA.

[0055] The results are shown in Table 2. Except for a concentration of 1.85 ng / mL detected in the brown salt pond water of Yuncheng Salt Lake, MC-LR was not detected in the green and blue salt pond water of Lingde Lake, Yuncheng Salt Lake, or in fish or shrimp. The accuracy of the analysis was evaluated through standard spiking and recovery experiments. The results showed that the recoveries of MC-LR in these environmental water samples and aquatic products ranged from 80.4% to 112.1%, with all RSDs below 10%, indicating that the designed detection platform has satisfactory reliability and broad application potential.

[0056] Table 2: Detection of microcystin in real samples using CMO-based kits (n=3)

[0057]

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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.

[0059] References:

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Claims

1. A CMO nano-oxidase, characterized in that: The CMO nano-oxidase is a bayberry-shaped Co-Mn3O4 oxidase; the TEM detection particle size of this nano-oxidase is 140-200 nm; the specific preparation method includes the following steps: 0.5 g manganese acetate (II) tetrahydrate Mn(CH3COO)2•4H2O, 0.02 g cobalt acetate (II) tetrahydrate Co(CH3COO)2•2H2O and 0.4 g PVP are dissolved in 15 mL of ethylene glycol; Stir vigorously for 1 hour until completely dissolved into a transparent orange-red solution; add 0.8 mL of 1 mol / L HCl to the solution and stir for another 0.5 hours; 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 9000 rpm for 10 minutes to collect the dark green nanozyme product; wash the dark green nanozyme product three times with distilled water and ethanol respectively, and finally resuspend it in 8 mL of distilled water, which is CMO nano oxidase.

2. A method for constructing a CMO-labeled chicken ovalbumin-conjugated microcystin LR antigen OVA-MC-LR probe using the CMO nano-oxidase described in claim 1, characterized in that: The procedure includes the following steps: 2 μL of 1 mg / mL OVA-MC-LR is added to 1 ml of PBS solution containing 1 mg / mL CMO, and the mixture is gently vortexed at room temperature for 30 minutes; then 200 μL of 1% w / v chicken ovalbumin OVA is mixed and mixed for 30 minutes to block the active site on the nanozyme surface, which is the antigen probe MC-LR-OVA-CMO, stored at 4°C for later use; The concentration of CMO was 1 mg / mL, the concentration of antigen OVA-MC-LR was 2 μg / mL, the concentration of OVA used for blocking was 0.2%, and the incubation time was 30 min.

3. An immunosensing kit prepared using the CMO-OVA-MC-LR probe prepared according to claim 2 as a signal probe.

4. The immunosensing kit according to claim 3, characterized in that: The immunosensing kit includes a polystyrene 96-well microplate coated with a murine monoclonal antibody that specifically recognizes microcystin-LR. After incubation, the plate is washed three times, then chicken ovalbumin (OVA) is blocked, followed by three washes. Standard microcystin solution or target sample solution is added, followed by three washes. The plate is then incubated and washed three times. The CMO-OVA-MC-LR probe is added, followed by six washes. TMB substrate solution is added, and the absorbance is measured after 2 minutes of reaction.

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

6. The immunosensing kit according to claim 4, characterized in that: The target substances are microcystin-LR standard and water environment samples and aquatic product samples containing microcystin-LR.

7. The application of the immunosensing kit according to claim 3 in the detection of microcystin-LR in lake water, saline lake water, fish, and shrimp.

8. The application according to claim 7, characterized in that: The lake water was treated with phosphate buffer to adjust the pH. After the salt lake water was treated at 4°C, the pH of the aqueous solution containing precipitated Na2SO4 was adjusted with phosphate buffer. Fish and shrimp samples were treated with trichloroacetic acid to precipitate proteins and then the pH was adjusted. Finally, the samples were tested using the designed kit.

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