Manganese-silicon-rich modified charcoal, preparation method thereof and application of manganese-silicon-rich modified charcoal as oxidase

Abstract

The invention belongs to the technical field of metal modified biochar, and relates to manganese-rich silicon modified biochar, a preparation method thereof and application of the manganese-rich silicon modified biochar as oxidase. The manganese-silicon-rich modified charcoal is of a porous net structure and comprises elements of Zn, Si, S, P, O, N, Mn, Mg, K, Fe, Ca and Al, the Mn element is evenly distributed in the manganese-silicon-rich modified charcoal with the mass fraction larger than 45% and exists in the form of spinel type Mn3O4, the Ca element exists in the form of CaCO3, and the Si element exists in the form of SiO2. The manganese-silicon-rich modified charcoal has oxidase-like activity, can directly oxidize a specific organic substrate, does not need H2O2 participation, has good chemical stability and catalytic activity, can preserve efficient catalysis in a complex environment, and can be used as oxidase in detection of L-ascorbic acid or alkaline phosphatase.

Description

Technical Field

[0001] The present invention belongs to the technical field of metal-modified biochar, and particularly relates to a manganese- and silicon-rich modified biochar, a preparation method thereof, and an application as an oxidase. Background Art

[0002] Ginseng is an important Chinese herbal medicine with pharmacological effects such as anti-inflammatory, anti-tumor, and immune enhancement. China is a major producer of ginseng. However, an important problem restricting its yield is continuous cropping obstacle. Among them, the influence of soil enzyme activity cannot be ignored. In particular, the regulation of alkaline phosphatase (ALP) activity plays a positive role in solving the continuous cropping obstacle of ginseng. Since ALP can enzymatically hydrolyze organic phosphorus to release available phosphorus for plant absorption and utilization, promoting plant growth. Therefore, the activity of alkaline phosphatase can be used as an index of the inorganic phosphorus utilization rate of plants and microorganisms, and they play a crucial role in the soil nutrient cycle. At the same time, there are literature reports that alkaline phosphatase is a crucial extracellular enzyme affecting the continuous cropping of American ginseng and is closely related to changes in the microbial community structure and composition. Therefore, it is urgent to directly and reliably detect the ALP activity, which has important and long-term significance for alleviating the continuous cropping obstacle of ginseng.

[0003] So far, several detection methods for ALP have been reported, such as high performance liquid chromatography, electrochemistry, radioimmunoassay, etc. Among them, most methods are time-consuming and require complex instruments and cumbersome operations. In recent years, due to the advantages of simplicity and rapidity of ultraviolet spectrophotometry and photothermal method, their use for detecting ALP is more attractive. For example, using the substrate L-ascorbic acid-2-phosphate trisodium salt as a traditional method has been widely used for colorimetric or photothermal ALP sensing. However, false positive results due to light source fluctuations or environmental impacts will directly affect the accuracy and reliability of the designed sensing method, and relying on a single response of color change or different absorbance intensity signals to quantify the ALP activity will be restricted in practical applications.

[0004] Point-of-care testing (POCT), as a key area of in vitro diagnosis, has made great progress in modern analytical chemistry and environmental monitoring. Currently, methods for POCT to detect ALP include electrochemistry, immunoassay, fluorescence, microfluidic chip technology, etc. However, most of these methods require complex equipment and are relatively complex to operate.

[0005] In recent years, carbon-based materials, as a new type of nanomaterials, have received extensive attention in order to improve the sensitivity and stability of POCT detection. Carbon-based nanomaterials (such as carbon quantum dots, graphene, etc.) have become a hot topic in nanozyme research due to their excellent electrical conductivity, large specific surface area, and abundant surface functional groups. Specifically, by mimicking the activity of natural enzymes, carbon-based nanomaterials can act as nanozymes to catalyze the redox reaction of TMB to generate detectable signals. For example, the vine-derived biochar prepared by a one-step method was modified into graphene-like molybdenum diselenide (MoSe2) with peroxidase-like activity as an intelligent nanozyme sensing platform for the voltammetric detection of hesperetin (HP) in orange peel. However, most of the existing nanozymes need to use protectants such as polyetherimide (PEI), bovine serum albumin (BSA), and cetyltrimethylammonium bromide (CTAB) to cover and stabilize them, which may protect their catalytic sites but reduce their oxidase (OXD) activity.

[0006] As an emerging carbon-based material, metal-modified biochar has obvious advantages in terms of cost, environmental friendliness, and easy accessibility compared with traditional carbon-based materials. However, there is no reported literature on the catalytic research of the enzyme-like activity of metal-modified biochar. Summary of the Invention

[0007] In view of the above technical problems and deficiencies, the purpose of the present invention is to provide a manganese- and silicon-rich modified biochar (MnBBC). For the first time, it is detected that the MnBBC carbonyl material has oxidase-like activity, can directly oxidize specific organic substrates without the participation of H2O2, has good chemical stability and catalytic activity, and can maintain high-efficiency catalysis in a complex environment.

[0008] To achieve the above purpose, the present invention adopts the following technical solutions:

[0009] A manganese- and silicon-rich modified biochar, the manganese- and silicon-rich modified biochar has a porous network structure and includes elements such as Zn, Si, S, P, O, N, Mn, Mg, K, Fe, and Ca. Among them, the Mn element is uniformly distributed in the manganese- and silicon-rich modified biochar with a mass fraction of more than 45% and exists in the form of spinel-type Mn3O4, the Ca element exists in the form of CaCO3, and the Si element exists in the form of SiO2.

[0010] As a preference of the present invention, the manganese- and silicon-rich modified biochar is obtained by pyrolyzing a mixture of hawthorn seeds, bentonite, and manganese element.

[0011] The present invention also provides a method for preparing manganese-rich silicon modified biochar, and the preparation method is as follows: crush hawthorn seeds to obtain hawthorn seed powder; weigh the hawthorn seed powder and bentonite, mix them in distilled water, and then add a manganese sulfate monohydrate solution to obtain a suspension; precisely adjust the pH value of the suspension to 10 using a sodium hydroxide solution; stir, perform ultrasonic treatment, dry, grind the dried sample, and place it in a pyrolysis device for pyrolysis; after pyrolysis is completed, naturally cool the sample to room temperature, repeatedly rinse the obtained sample with distilled water to remove impurities; and dry it after rinsing.

[0012] As a further preference of the present invention, the mass ratio of the hawthorn seed powder to the bentonite is 10:1; when pyrolyzing in the pyrolysis device, raise the temperature at a heating rate of 8 °C·min -1 to 350 °C, maintain the reaction for 2 h, and continuously introduce nitrogen throughout the process.

[0013] The present invention first detects that the manganese-rich silicon modified biochar (MnBBC carbonyl material) has oxidase-like activity. Therefore, the manganese-rich silicon modified biochar can be used as an oxidase.

[0014] The manganese-rich silicon modified biochar provided by the present invention can specifically interact with AA (L-ascorbic acid) or ALP in the reaction system by virtue of its oxidase-like activity. Therefore, it can be applied to the detection of L-ascorbic acid or alkaline phosphatase.

[0015] Advantages and beneficial effects of the present invention:

[0016] (1) The MnBBC provided by the present invention does not need to add a protective agent that will affect the OXD activity during the synthesis process, perfectly avoiding this shortcoming. In addition, it does not require cumbersome steps and instrument use such as high-speed centrifugation like nanoenzymes, greatly reducing time and cost, and avoiding the aggregation that may occur to a certain extent during high-speed centrifugation, thereby affecting the OXD activity.

[0017] (2) The MnBBC provided by the present invention has significant peroxidase-like activity. From the development process of biochar, the first-generation biochar was mainly used as a soil conditioner, the second-generation biochar was used as an adsorbent, and MnBBC can be regarded as the third-generation improved biochar. This innovation not only opens up a new direction for the application of biochar but also greatly expands the functional boundaries of biochar. In the field of industrial catalysis, peroxidase-like enzymes have a wide range of application requirements, but traditional peroxidase-like enzymes often have problems such as high cost and poor stability. MnBBC, with its unique peroxidase-like activity, has good chemical stability and catalytic activity, and can maintain high-efficiency catalysis in a complex environment, and is expected to become a very promising alternative material for peroxidase-like enzymes in industry.

[0018] (3) The MnBBC provided by the present invention is different from the traditional peroxidase (POD). POD usually requires hydrogen peroxide (H2O2) as an oxidant to catalyze the oxidation reaction of organic substrates. The use of H2O2 not only increases the cost and operation complexity, but also brings safety hazards. MnBBC does not require the participation of H2O2, has a unique catalytic mechanism, and directly oxidizes specific organic substrates; in addition, in practical applications, the stability of the catalyst is a crucial performance indicator. MnBBC has strong stability. After being placed for a period of time, its catalytic activity does not show a significant decrease, indicating that MnBBC has good storage stability and long-term performance, which provides a strong guarantee for its use in practical industrial production and environmental governance.

[0019] (4) The present invention detects for the first time that the MnBBC carbon-based composite material has oxidase-like activity, which is a special substance that simulates the catalytic function of natural oxidases and has great application potential in many fields such as biosensing and environmental monitoring. MnBBC can specifically react with AA (L-ascorbic acid) or ALP in the reaction system by virtue of its oxidase-like activity, and detect them through a specific signal conversion mechanism. This detection method is expected to provide an efficient, sensitive and convenient way for the quantitative analysis of AA or ALP.

[0020] (5) In the process of preparing MnBBC, bentonite is introduced to increase the surface roughness of the carbon source, increase the specific surface area, provide more active sites, and facilitate the attachment of metal complexes such as metals and metal oxides to the surface of the carbon source. In addition, it is shown that the microscopic concave-convex structure can enhance the interaction between the metal complex and the carbon source, further improve the stability and uniformity of the attachment (through physical adsorption and chemical bonding), and help to achieve the effective loading of the metal complex on the surface of the carbon source.

[0021] (6) The activity of many traditional catalysts will be significantly affected by changes in pH, resulting in a significant reduction in catalytic efficiency or even loss of activity. However, the MnBBC provided by the present invention has the ability to effectively carry out catalytic work in a relatively wide pH range (the activity is highest at pH 4, and the activity retention rate of MnBBC in the pH 3-5 range is ≥60%), has good tolerance to acidic environments, and is particularly suitable for the detection of alkaline phosphatase activity in ginseng continuous cropping soil. In addition, MnBBC can work effectively at low temperatures (maintaining high catalytic activity at both 4°C and 25°C) and remain stable for a long time, making MnBBC have important application value in cold areas or low-temperature operation scenarios.

[0022] (7) During the process of detecting the alkaline phosphatase (ALP) activity in ginseng continuous cropping soil using the self-designed MnBBC, this invention can effectively avoid the interference of other substances, providing a solid guarantee for obtaining accurate and reliable detection results.

[0023] (8) This invention proposes a comprehensive detection method integrating three detection modes: colorimetry, photothermal, and mobile phone intelligent auxiliary application. Specifically, it integrates visible signals, including the intuitive change of color, absorbance measurement, and quantitative analysis of RGB values (changes in the three color channels of red: R, green: G, blue: B and their superimposed colors), photothermal effect, infrared thermal imaging, and the instant capture and color recognition technology of the smartphone Color Name Recognizer Camera, forming a unique and efficient new detection idea. It provides multi-dimensional information for detection, reflecting the presence and concentration of the target from different angles, greatly improving the accuracy and real-time performance of detection, and overcoming the limitations of a single detection mode in specific environments or complex samples. Moreover, the three-modal detection does not require complex equipment, is easy to operate, and can be completed by following a simple operation process without the need for professional knowledge. The user-friendly operation makes this method suitable for popular point-of-care testing (POCT) applications.

[0024] (9) The three-mode detection method provided by this invention integrating colorimetry, photothermal, and mobile phone intelligence assistance is cost-effective (made from the pyrolysis of agricultural waste) and suitable for large-scale promotion in terms of economy. In terms of detection, it has the characteristics of high-throughput detection, can simultaneously detect multiple samples synchronously, greatly improving the detection efficiency, and has complementary advantages and portability.

[0025] (10) The method provided by this invention comprehensively utilizes multi-dimensional output signals such as chromaticity, absorbance, temperature, and RGB values, and can mutually verify and correct the detection results; when an abnormal signal appears, cross-comparison and analysis can be carried out through other signals to timely discover and correct possible errors, ensuring the accuracy and credibility of the detection results. Brief Description of the Drawings

[0026] Figure 1 Characterization analysis of manganese-rich silicon modified biochar (MnBBC) Figure 1 ; among them, a is the high-resolution scanning electron microscope (HRSEM) image of MnBBC; b - n are the element distribution mapping (Mapping) images of MnBBC; o is the energy dispersive X-ray spectroscopy (EDS) spectrum of MnBBC;

[0027] Figure 2 Characterization analysis of manganese-rich silicon modified biochar (MnBBC) Figure 2; among them, a is the adsorption - desorption curve of manganese - rich silicon - modified biochar; b is the pore size distribution diagram of manganese - rich silicon - modified biochar.

[0028] Figure 3 is the characterization analysis of manganese - rich silicon - modified biochar (MnBBC). Figure 3 ; among them, a - c are the low - to - high - resolution TEM images (500, 100, 5 nm) of manganese - rich silicon - modified biochar; d is the electron paramagnetic resonance spectrum (EPR) of manganese - rich silicon - modified biochar; e is the manganese - rich silicon - modified biochar at 500 - 3500 cm -1 Raman spectroscopy;

[0029] Figure 4 is the peroxidase - like activity analysis of manganese - rich silicon - modified biochar; among them, a is the ultraviolet - visible spectrum and colorimetric diagram of the reaction solution of manganese - rich silicon - modified biochar and 3,3',5,5' - tetramethylbenzidine (TMB) (MnBBC - TMB); b is the observation of the stability of MnBBC - TMB; c is the absorbance of the MnBBC - TMB reaction solution at 4 °C and 25 °C (λ 654 nm); d is the absorbance of the MnBBC - TMB reaction solution at different pH values (λ 654 nm).

[0030] Figure 5 is the steady - state kinetic analysis curve for evaluating the catalytic efficiency of manganese - rich silicon - modified biochar (MnBBC) as a mimetic oxidase, with the substrate being 3,3',5,5' - tetramethylbenzidine (TMB).

[0031] Figure 6 is the absorbance of the reaction solutions of various biochars prepared from different precursors and TMB; among them, a is the absorbance (λ 654 nm) of the reaction systems of hawthorn seed biochar (BC), hawthorn seed - introduced bentonite mixed biochar (BBC), iron - rich modified biochar (FeBBC), zinc - rich modified biochar (ZnBBC), and manganese - rich silicon - modified biochar (MnBBC) with TMB; b is the photo of the reaction solution during the reaction of BC, BBC, FeBBC, ZnBBC, and MnBBC with TMB; c is the absorbance (λ 654 nm) of the reaction systems of manganese - rich silicon - modified biochar from rice husk MnBBC (DK), manganese - rich silicon - modified biochar from sorghum husk MnBBC (GLK), manganese - rich silicon - modified biochar from wild jujube husk MnBBC (SZK), manganese - rich silicon - modified biochar from millet husk MnBBC (XMK), and manganese - rich silicon - modified biochar from hawthorn seed MnBBC (SZZ) with TMB; d is the photo of the reaction solution during the reaction of DK, GLK, SZK, XMK, and SZZ modified biochars with TMB.

[0032] Figure 7Characterization analysis diagrams of various biochars prepared from different precursors: Among them, a - e are high - resolution scanning electron microscope images (HRSEM) of BC, BBC, ZnBBC, FeBBC, and MnBBC, showing the microscopic morphologies of various biochars; f - j are X - ray diffraction (XRD) patterns of BC, BBC, ZnBBC, FeBBC, and MnBBC, analyzing the crystal structures of various biochars;

[0033] Figure 8 X - ray photoelectron spectroscopy spectra (XPS) of biochars BC, BBC, ZnBBC, FeBBC, and MnBBC prepared from different precursors: Among them, a - e are C1s; f - j are O1s; k - o are Si 2p; p - t are S2p;

[0034] Figure 9 Some XPS diagrams and FTIR diagrams: Among them, a is Mn 2p; b is the full XPS spectra of BC, BBC, ZnBBC, FeBBC, and MnBBC; c is the combined Fourier transform infrared spectroscopy (FTIR) spectra of BC, BBC, ZnBBC, FeBBC, and MnBBC;

[0035] Figure 10 Monitoring the concentration - dependent inhibitory effect of AA on the oxidation of TMB catalyzed by MnBBC through ultraviolet - visible absorption spectroscopy and colorimetric imaging: Among them, a is the ultraviolet - visible absorption spectroscopy; b is the colorimetric imaging diagram; c is the standard curve constructed based on the change in absorbance value (ΔA AA ) and the concentration of ascorbic acid (AA);

[0036] Figure 11 Detection of ALP activity based on the MnBBC / AA / ALP cascade signal amplification system: Among them, a is the three - dimensional absorption spectroscopy; b is the colorimetric imaging diagram; c is the standard curve constructed based on the change in absorbance value (ΔA ALP ) and the concentration of ALP;

[0037] Figure 12 Evaluating the selectivity of the alkaline phosphatase (ALP) detection system by ultraviolet spectrophotometry: Among them, a is to investigate the interference effect of structural analogs on the detection system; b is the differential analysis of the signal response of the detection system between the ALP - added group and the non - added group, and the data are expressed as mean ± SD (n = 3);

[0038] Figure 13 Effects of different types of inhibitors on the oxidase - like activity of MnBBC: Among them, a is superoxide dismutase (SOD); b is tryptophan; c is AA; d is catalase; Blank is the control group without added inhibitor;

[0039] Figure 14 is the analysis of the photothermal performance of different systems; among them, a is the reaction system of irradiating MnBBC-TMB-AA with 808 nm laser, and the power increases by 0.5 - 2.2 W / cm 60 uM and its influence on the temperature difference of the system; b is the temperature-time curve of the MnBBC-TMB reaction system, TMB solution and MnBBC dispersion under the irradiation of 808 nm near-infrared laser (2.0 W / cm 2 ); c is the analysis of the synergistic photothermal effect of the MnBBC-TMB composite system relative to single components; 2 ) irradiation; c is the analysis of the synergistic photothermal effect of the MnBBC-TMB composite system relative to single components;

[0040] Figure 15 is the concentration-dependent photothermal response analysis of ALP and AA; among them, a is the temperature change of the MnBBC-TMB-AA system under the irradiation of 808 nm near-infrared laser (2.0 W / cm 2 ); b is the quantitative relationship between the photothermal response signal of the MnBBC-TMB system and the AA concentration within a specific concentration range; c is the temperature change of the reaction solution of the AAP-ALP-MnBBC-TMB multi-stage catalytic system under the irradiation of 808 nm near-infrared laser (2.0 W / cm 2 ); d is the quantitative detection performance of the multi-component synergistic system (the quantitative relationship between the photothermal response signal and the ALP concentration);

[0041] Figure 16 is the multimodal detection and analysis of the alkaline phosphatase (ALP) activity based on the combination of colorimetry - smartphone - thermal imager; among them, a is the real-time monitoring of the blue change in the MnBBC-TMB-AA-ALP reaction system by the smartphone, and the linear relationship between B / (R + G + B) and the ALP concentration; b is the real-time joint monitoring of the ALP reaction system by the smartphone and the thermal imager under the mapping of 808 nm laser. Within the ALP activity range of 1 - 30 U / L, ΔT shows a significant linear relationship with the ALP activity; c is the photothermal imaging map of the spiked soil; d is the RGB recognition color imaging map of the spiked soil. Specific Embodiments

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the implementation manners of the present invention are not limited thereto. For process parameters not specifically noted, conventional techniques can be referred to. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0043] This embodiment provides a method for detecting the alkaline phosphatase activity in soil, and the method includes the following steps:

[0044] Step 1. Prepare the soil extract;

[0045] Step 2. Sequentially add L-ascorbic acid-2-phosphate solution and soil extract into a centrifuge tube, incubate at 37 °C for 30 min; add Britton-Robinson buffer solution to terminate the reaction;

[0046] Step 3. Add 3,3′,5,5′-tetramethylbenzidine solution and MnBBC dispersion into the reaction solution obtained in Step 2, and react at a constant temperature of 37 ± 0.1 °C for 7 min; wherein, the MnBBC dispersion is prepared by mixing manganese-rich silicon-modified biochar with water; the manganese-rich silicon-modified biochar is self-developed;

[0047] Step 4. Measure the absorbance at 654 nm of the reaction system; observe the chromaticity of the reaction system, and monitor the RGB value using a smartphone; monitor the temperature change of the reaction system under 808 nm near-infrared laser irradiation; calculate the concentration of alkaline phosphatase in the soil based on the established relationships between absorbance change and ALP concentration, RGB value and ALP concentration, and temperature change and ALP concentration respectively, and mutually verify and correct the data through multi-modal output of chromaticity, absorbance, temperature and RGB value, so as to determine the activity of alkaline phosphatase in the soil.

[0048] To enable those skilled in the art to clearly understand how the present invention is implemented, the following is a detailed introduction through specific experiments:

[0049] 1. Experiment:

[0050] 1.1. Materials:

[0051] Hawthorn seeds, rice husks, rice straw, millet husks, sorghum husks, and wild jujube husks were provided by Tianjin YuZhiQuan Trading Co., Ltd. (Tianjin, China); bentonite was purchased from Jilin JinguoHan Technology Co., Ltd. (Jilin, China); chemical reagents zinc sulfate heptahydrate (ZnSO4·7H2O), ferrous sulfate heptahydrate (FeSO4·7H2O), manganese sulfate monohydrate (MnSO4·H2O), sodium hydroxide (NaOH), and biochemical reagents 3,3′,5,5′-tetramethylbenzidine (TMB), L-ascorbic acid (AA), L-ascorbyl-2-phosphate (AAP), superoxide dismutase (SOD), alkaline phosphatase (ALP), catalase (CAT), L-tryptophan (trp), Britton-Robinson buffer (pH 3.0 - pH 8.0), acid phosphatase (ACP), ginsenoside-Rb1 (Rb1), benzoic acid (BA), ginsenoside-Rg1 (Rg1), total ginsenosides (TG), and invertase were all purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).

[0052] 1.2. Preparation of MnBBC biochar:

[0053] The fresh and dry hawthorn seeds (abbreviated as SZZ) were crushed using a crusher and passed through a 60-mesh sieve to obtain hawthorn seed powder. According to the mass ratio of 10:1 (w / w), 10 g of hawthorn seed powder and 1 g of bentonite were weighed and mixed in 100 mL of distilled water, and then 0.2 mol / L manganese sulfate monohydrate (MnSO4·H2O) solution (500 mL) was added. The pH value of the above suspension was precisely adjusted to 10 using 5 M sodium hydroxide (NaOH) solution. The mixture was placed on a magnetic stirrer and stirred at 600 rpm for 2 h, then the mixture was ultrasonically treated for 30 min, transferred to an oven, dried at 80 °C for 48 h, the dried sample was ground, passed through a 60-mesh sieve, and placed in a pyrolysis device at 8 °C·min -1The temperature was raised to 350 °C at a heating rate, and the reaction was maintained for 2 h. Nitrogen was continuously introduced throughout the process to maintain an inert environment and prevent the sample from being oxidized. After pyrolysis was completed, the sample was allowed to cool naturally to room temperature. The obtained sample was repeatedly rinsed with distilled water to remove impurities. The rinsed particles were placed in an oven and dried at 105 °C (±5 °C) for 24 h. The dried sample was stored in a dry, clean, and well-sealed container, and properly labeled for subsequent experiments.

[0054] In this example, the sample obtained by pyrolyzing pure hawthorn seeds was named Biochar (BC), the sample obtained by pyrolyzing the mixture of hawthorn seeds and bentonite was named Bentonite / Biochar (BBC), and the sample obtained by pyrolyzing the mixture of hawthorn seeds, bentonite, and manganese element was named MnBBC, also known as MnBBC (SZZ).

[0055] Referring to the above preparation method, in this example, the preparation of biochar samples with different metals added was also carried out: 0.2 mol / L ZnSO4·7H2O was used to prepare the ZnBBC sample according to the same experimental procedure (1.2) (this sample was obtained by pyrolyzing the mixture of hawthorn seeds, bentonite, and zinc element). FeSO4·7H2O was used to prepare the FeBBC sample according to the same experimental procedure (1.2) (this sample was obtained by pyrolyzing the mixture of hawthorn seeds, bentonite, and iron element).

[0056] In addition, in this example, to compare the uniqueness of MnBBC, the range of raw materials was further expanded. Hawthorn seeds were replaced with rice husks, rice straw, millet husks, sorghum husks, and wild jujube husks respectively, and a series of rice husk manganese-rich silicon modified biochar MnBBC (DK), rice straw manganese-rich silicon modified biochar MnBBC (DC), millet husk manganese-rich silicon modified biochar MnBBC (XMK), sorghum husk manganese-rich silicon modified biochar MnBBC (GLK), and wild jujube husk manganese-rich silicon modified biochar MnBBC (SZK) were prepared according to the above experimental procedure (1.2). These were compared with the hawthorn seed manganese-rich silicon modified biochar MnBBC (SZZ) prepared above to explore the peroxidase-like activity of different biochar raw materials.

[0057] In this example, a total of 10 types of biochars, composite biochars, and metal-modified biochars were synthesized, aiming to comprehensively explore the effects of different preparation methods and raw materials on the properties of biochars to find the biochar with the most peroxidase-like activity.

[0058] It should be noted that in this embodiment, the results of manganese-rich silicon modified biochar from rice husk MnBBC(DK) and manganese-rich silicon modified biochar from rice straw MnBBC(DC) are basically the same. Therefore, only the experimental results of manganese-rich silicon modified biochar from rice husk MnBBC(DK) are provided in this embodiment.

[0059] 1.3. Material characterization:

[0060] The ultraviolet-visible absorption spectrum was recorded using a HITACHI U-2900 ultraviolet-visible spectrophotometer. The multi-scale structure of MnBBC was analyzed using a Hitachi High-Technologies cold field emission scanning electron microscope SU 8600 (equipped with an Oxford Ultim Max 100mm 2 EDS detector), including morphological characterization and elemental analysis. The microstructure was characterized using a JEM-F200 field emission transmission electron microscope (JEOL Ltd.). The nitrogen adsorption-desorption isotherm was measured using a Quantachrome Autosorb-iQ physical adsorption analyzer. The specific surface area was calculated using the BET model, and the mesopore distribution characteristics in the range of 3.5 - 50 nm were analyzed using the BJH theoretical model to obtain the micropore (<2 nm) structure parameters. The phase structure was characterized using a Shimadzu XRD-6100 polycrystalline X-ray diffractometer. The surface chemical state was characterized using a Thermo Fisher Scientific ESCALAB 250Xi X-ray photoelectron spectrometer. The vibration modes of surface functional groups on the biochar were characterized using a Thermo Fisher Nicolet iS50 Fourier transform infrared spectrometer, and the mid-infrared fingerprint region spectrum in the range of 4000 - 400 cm -1 was collected in transmission mode for in-situ surface analysis. The lattice vibration modes were characterized using a HORIBA LabRAM HR Evolution confocal micro-Raman spectrometer. Multi-element quantitative analysis of the samples was performed using a Thermo Scientific iCAP PRO 6300 direct-reading inductively coupled plasma emission spectrometer. A high-precision digital display thermometer (Taizhou Weixing Electric Co., Ltd.), a HIKMICRO thermal imager (Hangzhou Hikvision Microelectronics Sensing Technology Co., Ltd.), an Apple iPhone 13 mobile device (Apple Inc., USA), and an MW-GX808 multimode fiber-coupled laser (Changchun Raishi Optoelectronics Technology Co., Ltd.) were used to jointly build a photo-thermal - smartphone collaborative analysis platform for in-situ monitoring of multiple physical fields.

[0061] 1.4. Evaluation of the peroxidase-like activity of MnBBC biochar:

[0062] The catalytic oxidation method of 3,3’,5,5’-tetramethylbenzidine (TMB) was adopted to evaluate the peroxidase-like activity of MnBBC. 100 μL of TMB solution (17 mM), 100 μL of MnBBC dispersion (0.7 mg·mL -1 ) and 300 μL of Britton-Robinson buffer (pH 4.0) were successively added into a 1.5 mL polypropylene (PP) centrifuge tube, and the reaction was carried out at room temperature for 7 min. After the reaction, the reaction solution turned blue. The absorbance of the generated blue solution was measured using a UV-visible spectrophotometer at a wavelength of 654 nm, and the measured absorbance data was used for subsequent analysis and evaluation of the peroxidase-like activity of MnBBC.

[0063] To deeply explore the effect of pH on the peroxidase-like activity of MnBBC, the present invention designed to combine MnBBC dispersion (0.7 mg / mL, 100 μL), TMB solution (17 mM, 100 μL) with 300 μL of buffer solutions with different pH values (3, 4, 5, 6, 7 and 8). Each reaction system was continuously reacted at room temperature for 7 min. After the reaction, the absorbance of each reaction system at the characteristic wavelength of 654 nm was immediately measured and recorded using a UV-visible spectrophotometer.

[0064] To deeply explore the effect of temperature on the peroxidase-like activity of MnBBC, the present invention was carried out in the Britton-Robinson buffer (pH 4.0) system (1.4). The specific operation was as follows: the reaction systems were respectively placed under the constant temperature conditions of 4 °C and 25 °C for reaction. After the reaction reached a stable state, the absorbance values at 4 °C and 25 °C of the reaction solution were quickly measured and recorded at the characteristic wavelength of 654 nm using a UV-visible spectrophotometer.

[0065] 1.5. Steady-state kinetic analysis of the peroxidase of MnBBC biochar:

[0066] To study the OXD catalytic kinetic characteristics of MnBBC biochar, in this invention, TMB was used as a substrate to monitor the peroxidase-like kinetic curve of the MnBBC sample. The specific operation was as follows: 100 μL of TMB solutions with different concentration gradients (0 - 25 U / L) were successively added to a 1.5 mL polypropylene centrifuge tube to construct reaction systems under different substrate concentration conditions. Subsequently, 100 μL of 0.7 mg / mL MnBBC dispersion and 300 μL of Britton-Robinson buffer (pH 4.0) were added for reaction. The reaction was carried out at room temperature for 10 min. During the process, the reaction system was monitored in real time using a UV-visible spectrophotometer. The reaction solution was measured every 1 min. Using TMB as the substrate, the steady-state kinetic constants in the MnBBC peroxidase (OXD)-like catalytic system were determined and calculated by UV-visible spectrophotometry (λ = 654 nm). Based on the initial reaction rate data (n = 7 independent repeated experiments), linear fitting was performed through the Lineweaver-Burk double-reciprocal equation:

[0067]

[0068] In the formula, V is the initial reaction rate (mM·min -1 ), V max is the maximum reaction rate (mM·min -1 ), [S] is the substrate concentration (mM), and K m is the Michaelis constant (mM, kinetic constant).

[0069] By performing non-linear regression fitting on the experimental data and substituting it into the above kinetic equation, the steady-state kinetic constants such as V max and K m were accurately calculated, thus providing an important theoretical basis for deeply understanding the action mechanism and catalytic performance of MnBBC in peroxidase-catalyzed reactions.

[0070] 1.6. Research on the colorimetric method and dual combined photothermal detection of AA based on the MnBBC-TMB system:

[0071] The dual-mode sensing analysis of ascorbic acid (AA) based on the MnBBC-TMB system was carried out under the optimized experimental conditions. A reaction mixture with a total volume of 500 μL was accurately prepared as follows: The standard reaction system (17 mM, 100 μL TMB solution, 0.7 mg / mL, 100 μL MnBBC dispersion, 200 μL, pH 4.0 Britton-Robinson buffer solution) and 100 μL AA solutions with different concentration gradients (0 - 90 μM) were successively added to a 1.5 mL polypropylene centrifuge tube. After vortex mixing evenly, the reaction time was accurately controlled for 7 min. After the reaction, 200 μL of the reaction solution was taken and transferred to a 10 mm quartz cuvette, and the absorbance at 654 nm was recorded using a UV-visible spectrophotometer. A dose-absorbance response curve of ΔA and C AA was established. For the combined photothermal detection, an 808 nm laser (power density of 2 W / cm 2 ) was used to irradiate the reaction system, and a high-precision digital display thermometer was used to monitor the temperature field change of the reaction system in real time. By recording the temperature change curve with concentration, the internal relationship between the AA concentration and the temperature change caused by the photothermal effect of the reaction system was analyzed, providing an important basis for in-depth study of the reaction mechanism and development of new detection methods.

[0072] 1.7. Colorimetry - 808 Laser Photothermal Synergistic Strategy for Highly Sensitive Detection of Alkaline Phosphatase (ALP):

[0073] The procedure for determining the ALP activity in this invention is as follows: Accurately pipette 20 μL of 35 mM p-aminophenyl phosphate (AAP) solution and 80 μL of ALP solutions with different concentrations (0 - 30 U / L) into a reaction tube, and incubate in a 37 °C constant temperature water bath for 30 min to allow AAP to react fully under the catalysis of ALP. After incubation, immediately add 200 μL of Britton-Robinson buffer solution with pH 4.0 to terminate the reaction. Then add 100 μL of 17 mM TMB solution and 100 μL of 0.7 mg / mL MnBBC dispersion to the reaction tube in sequence, and vortex mix evenly to form a total reaction solution with a volume of 0.5 mL. Let the above total reaction solution stand for 7 min until the color development is stable, take an appropriate amount of the solution with a micropipette to a quartz cuvette, and measure the absorbance value at 654 nm with a UV-visible spectrophotometer. At the same time, irradiate the reaction system with an 808 nm near-infrared laser (2 W / cm 2 ), and use a high-precision digital display thermometer to monitor the temperature change of the reaction system during laser irradiation in real time. By analyzing the absorbance and temperature change data generated by the change of ALP concentration, the ALP activity was comprehensively analyzed and evaluated.

[0074] 1.8. ALP Colorimetry and Photothermal Multimode Sensing Detection in Smartphone Mode:

[0075] The present invention utilizes the smartphone application Color Name Recognizer Camera (color recognition) to evaluate the color constants of the colorimetric reaction, and combines a thermal imager to monitor the temperature changes generated by the photothermal effect, thereby establishing a photothermal-smartphone-based multi-mode sensing detection method for ALP, aiming to achieve accurate and convenient detection of ALP activity. The operation is the same as that of the above experiment (1.7) until the color development is stable; the "Color Name Recognizer Camera" application of the smartphone is used to immediately capture and recognize the color (RGB) of the reaction solution. This application is an existing program, based on advanced image processing algorithms, which can accurately recognize and quantify color information. At the same time, in order to verify the accuracy of the results, an ultraviolet-visible spectrophotometer is used to measure the absorbance at a wavelength of 654 nm.

[0076] In order to further improve the accurate and stable reading of the temperature rise signal, the present invention designs a portable sensing mode based on a smartphone combined with an infrared thermal imager, that is, during the irradiation with an 808 nm laser (2 W / cm 2 )), the thermal imager is used to continuously monitor the temperature changes (ΔT) generated by the reaction system, and the correlation between the ΔT value and the ALP activity is established. Statistical methods are used to analyze the ΔT value and ALP activity data, and a correlation model between the two is established. The establishment of this model helps to achieve the quantitative analysis of ALP activity by measuring the temperature changes.

[0077] 1.9. Research on the specific recognition of ALP by the MnBBC-based oxidase dual-mode sensing platform and the influence of interfering factors in complex matrices:

[0078] Basis for the selection of interfering factors, structural analogs: ginsenoside Rb1 / Rg1, total ginsenosides; functional analogs: acid phosphatase, sucrase; common matrix components: benzoic acid.

[0079] ALP-free system (blank control group): 100 μL of interfering factor solution (acid phosphatase, sucrase, ginsenoside Rb1, ginsenoside Rg1, total ginsenosides, benzoic acid, with concentrations all being their respective critical concentrations) + standard reaction system (same as 1.6) are sequentially added to a 1.5 mL polypropylene centrifuge tube, vortex-mixed, and reacted at room temperature for 7 min. 200 μL of the reaction solution is taken and transferred to a quartz cuvette, and the absorbance at 654 nm is recorded using an ultraviolet-visible spectrophotometer (the average value of three measurements is taken).

[0080] ALP-containing system (experimental group): Mix 100 μL of interference factor (same as above), 20 μL of 35 mM AAP solution, and 80 μL of 0 - 30 U / L ALP standard solution, and incubate in a constant temperature water bath at 37 °C for 30 min. Add the standard reaction system (same as 1.6), vortex and mix well to prepare a reaction solution with a total volume of 0.5 mL. React at room temperature for 7 min. Transfer 200 μL of the reaction solution to a quartz cuvette and measure the absorbance at 654 nm. Calculate the relative absorbance (A / A0, where A0 is the absorbance without the interference factor) in the presence of each interference factor to evaluate the selectivity of the system.

[0081] 1.10. Analysis of the catalytic mechanism of MnBBC-like oxidase: Reactive oxygen species based on radical trapping:

[0082] As a novel catalyst synthesized from agricultural waste, the oxidase (OXD-like) activity of MnBBC stems from the synergistic effect between the transition metal Mn active site and the carbon matrix. Its catalytic mechanism may be closely related to the reactive oxygen species (ROS) generated during the reaction. To clarify the specific mechanism of its catalyzing the oxidation of TMB, this experiment adopted a radical trapping strategy to specifically target and scavenge different ROS with specific inhibitors, and systematically analyzed the key active species and their contribution degrees during the catalytic process of MnBBC. The specific experiment is as follows: In the standard reaction system (same as 1.6), add radical scavengers respectively: Catalase - specifically scavenges hydrogen peroxide (H2O2), ascorbic acid (AA) - scavenges hydroxyl radical (·OH), superoxide dismutase (SOD) - scavenges superoxide anion radical (O 2 -), tryptophan (Trptophan) - scavenges singlet oxygen ( 1 O2), react at room temperature for 7 min, then transfer 200 μL of the reaction solution to a quartz cuvette, and use a spectrophotometer to record the absorbance at 654 nm (average value after three measurements), and calculate the relative activity.

[0083] 1.11. Spike test of ALP in real soil samples based on the MnBBC-like oxidase system:

[0084] The spike recovery test is of great scientific significance in the determination of soil alkaline phosphatase (ALP) activity. Its core purpose is to evaluate the accuracy of the detection method, matrix interference effect and method reliability by adding a target substance with a known concentration (i.e., ALP enzyme). Since soil is a complex heterogeneous system composed of minerals, organic matter, microorganisms and metabolites, its physical and chemical properties (such as pH, ionic strength, humic acid content) and coexisting substances (such as heavy metals, enzyme inhibitors, adsorptive colloids) may significantly interfere with the detection of ALP activity. For example: Inhibition / activation effect: Humic acid can bind to the enzyme through hydrophobic interaction and change its conformation; heavy metal ions (such as Cu 2+ , Cd 2+ ) may competitively bind to the enzyme active site. Adsorption loss: The negative charge on the surface of soil clay minerals (such as montmorillonite, kaolinite) will adsorb the positively charged ALP enzyme, resulting in an underestimation of the free enzyme concentration. Optical interference: Pigments or suspended particles in the soil extract may interfere with the signal determination of spectrophotometry or fluorescence method. The spike recovery test can quantify the comprehensive impact of the above interference factors on the detection results by adding an exogenous ALP standard to the soil sample. If the recovery rate deviates from the theoretical value (such as <90% or >110%), it indicates that the extraction buffer needs to be optimized (such as adding a chelating agent EDTA to desorb metal ions) or the detection conditions need to be improved (such as centrifugal filtration to remove turbidity).

[0085] Parallel experimental design of the spike recovery test: The soil samples are divided into an unspiked group (to measure the background ALP activity) and a spiked group (adding known amounts of ALP enzyme 5 / 10 / 15 U / L), and the recovery rate is calculated by comparing the activity difference between the two groups.

[0086] In this invention, the detection performance of MnBBC-like oxidase for ALP in soil is verified through a spike experiment. Five typical soil samples are selected: a soil sample without ginseng planting history (CK), a continuous cropping soil with 5-year ginseng planting, and soil samples around the rhizosphere of ginseng planted for 7 years, 15 years, and 30 years (5Y / 7Y / 15Y / 30Y), which are air-dried. 0.5 g of each soil sample is placed in a 50 ml centrifuge tube, 20 ml of Britton-Robinson buffer (pH 8.0) is added, and it is vortexed for 5 min and centrifuged at 5000 rpm for 10 min; the supernatant is filtered through a membrane and diluted 100 times step by step for standby.

[0087] The concentration settings were without spiking (0 U / L), low-concentration spiking (5 U / L), medium-concentration spiking (10 U / L), and high-concentration spiking (15 U / L). The reaction system was as follows: In a centrifuge tube, add successively: 20 μL AAP solution (35 mM), 80 μL ALP standard solutions at different concentrations (5 U / L, 10 U / L, 15 U / L, pH 8.0), incubate at 37 °C for 30 min; immediately add 200 μL Britton-Robinson (pH 4.0) to terminate the reaction, add 100 μL TMB solution (17 mM) and 100 μL MnBBC dispersion (0.7 mg / mL) to the reaction solution, carry out a constant-temperature reaction at 37 ± 0.1 °C for 7 min, and immediately measure the absorbance at 654 nm (take the average of three measurements). Detection of ginseng soil samples: Take 80 μL of soil extract to replace the ALP standard solution, control the total volume to 0.5 mL, and the other steps are the same as above. Calculate the ALP activity according to the standard curve, and the result is expressed as the ALP activity (U / L) per unit mass of soil.

[0088] The formula for the recovery rate is: Recovery Rate = (measured value - control group) / added value × 100%.

[0089] 2. Results and Discussion:

[0090] 2.1. Characterization of MnBBC Biochar:

[0091] Figure 1 and 2 are the characterization analysis diagrams of manganese-rich silicon-modified biochar (MnBBC). Through high-resolution scanning electron microscopy (HRSEM) diagrams, the microscopic morphology of manganese-rich silicon-modified biochar (MnBBC) was characterized, and it was found that it has a three-dimensional interconnected porous network structure ( Figure 1 a). Combining the nitrogen adsorption-desorption isotherm ( Figure 2 a) and the BJH pore size distribution ( Figure 2 b), it shows that this material exhibits a mesoporous-dominated pore system (average pore size is ), the specific surface area (S BET ) is 35.128 m 2 / g, the pore volume is 0.085 m 2 / g. Its type IV(a) isotherm and H3-type hysteresis loop further confirm that the mesoporous voids are consistent with the HRSEM morphology, which is conducive to the diffusion of reactants and the exposure of active sites, thus enhancing the catalytic performance.

[0092] Furthermore, elemental mapping and energy-dispersive X-ray spectroscopy (EDS) were used to analyze the elemental composition of MnBBC ( Figure 1b-1o). The results show that Mn element is uniformly distributed on the material surface with a mass fraction of 46.14 wt.%, and mainly exists in the Mn(II) / Mn(III) oxidation state (verified by XPS), which provides abundant active centers for its peroxidase-like activity. In addition, trace metal elements (Fe: 0.19 wt.%, Zn: 0.83 wt.%) and non-metal element (S: 8.03 wt.%) are detected in the material, which may further regulate the catalytic performance through electron transfer or synergistic effect. The contents of each element are as follows: Zn: 0.83 wt.%, Si: 0.23 wt.%, S: 8.03 wt.%, P: 0.01 wt.%, O: 0.23 wt.%, N: 0.01 wt.%, Mn: 46.14 wt.%, Mg: 0.06 wt.%, K: 0.04 wt.%, Fe: 0.19 wt.%, Ca: 0.50 wt.%, Al: 0.17 wt.% (error range ±0.05 wt.%).

[0093] In the present invention, the analysis results of ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer) show that the content of Si element in MnBBC is 13580 ppm, the content of Mn element is 186670 ppm, and the content of S element is 7880 ppm.

[0094] Figure 3 Figure for the characterization analysis of manganese-rich silicon modified biochar (MnBBC). The structure of MnBBC was characterized by transmission electron microscopy (TEM), and the results showed a typical porous network morphology ( Figure 3 a, 3b), which is highly consistent with the three-dimensional interconnected void structure observed by HRSEM. Further in-depth analysis of MnBBC was carried out by high-resolution HRTEM, and clear lattice fringes were observed ( Figure 3 c). After precise measurement, the lattice spacings are 0.288 nm, 0.284 nm, and 0.335 nm respectively. By comparing with JCPDS, they correspond to the crystal planes of manganese oxide (Mn3O4, JCPDS No. 18-0803), calcium carbonate (CaCO3, JCPDS No. 88-1807), and silicon dioxide (SiO2, JCPDS No. 46-4015) respectively. This result clearly shows that a multiphase composite structure is formed in MnBBC during the modification process. To further explore the catalytic mechanism of MnBBC, in-situ electron paramagnetic resonance (EPR) spectroscopy was used for research ( Figure 3 d). In the presence of MnBBC, a characteristic response of obvious hydroxyl radicals (·OH) appears in the MnBBC system, while this signal is not observed in the blank control group. This comparative structure strongly confirms that the generation of ·OH is due to the peroxidase-like activity of MnBBC. Raman spectroscopy ( Figure 3e) Provide information for studying the carbon matrix and chemical bond states of MnBBC. The results show that the carbon skeleton of MnBBC has typical sp 2 hybridization characteristics, where the D peak (1331 cm -1 , A1g symmetry) reflects the defects or edge structures in the carbon structure, and the G peak (1575 cm -1 , E2g symmetry) corresponds to the in-plane stretching vibration of graphitized carbon. The intensity of MnBBC (ID / IG = 0.56) is relatively low, indicating that the modification process promotes the graphitization reorganization of the carbon matrix. The improvement of the graphitization degree has an important impact on the performance of the material. The most direct manifestation is the improvement of the electron conduction ability of the material. In addition, the Mn-O vibration peak at 2210 cm -1 and the Si-O stretching vibration peak at 1924 cm -1 in the Raman spectrum respectively confirm the loading of the manganese oxide phase and the retention of the SiO2 skeleton derived from the bentonite template. The two cooperate to optimize the dispersion of the active sites of the material and the stability of the structure, further providing strong support for the high performance of MnBBC in catalytic reactions.

[0095] 2.2. Peroxidase-like activity of MnBBC:

[0096] The present invention comprehensively evaluates the peroxidase-like activity performance of MnBBC Figure 4 for the analysis of the peroxidase-like activity of manganese-rich silicon modified biochar. First, in the comparative experiment, when MnBBC and TMB exist alone, the solution has no color change. When MnBBC and TMB are mixed, the solution turns blue. Through ultraviolet-visible absorption spectroscopy analysis ( Figure 4 a), a strong absorption peak appears at 654 nm. This phenomenon indicates that TMB is oxidized to the oxidized form of oxTMB, thus proving that MnBBC has OXD activity.

[0097] Further study the effects of temperature and pH on its activity. The temperature experiment shows ( Figure 4 c) that MnBBC maintains high catalytic activity at 4 °C and 25 °C, confirming its advantage of low-temperature adaptability. The catalytic activity of MnBBC shows significant pH dependence ( Figure 4 d): The activity is the highest at pH 4 (ΔA = 1.02), 90% of the activity is retained at pH 3 (ΔA = 0.959), and the activity gradually decreases with the increase of pH in the range of pH 5-8, which is consistent with the proton-coupled electron transfer mechanism of typical Mn-based nanozymes. It is worth noting that the activity retention rate of MnBBC is ≥60% in the pH range of 3-5 (vs. pH 4), indicating its good tolerance to acidic environments (such as ginseng continuous cropping soil, etc.), which is better than most metal-organic framework (MOFs) nanozymes (usually the activity decay >50%).

[0098] Long-term storage stability: The catalytic activity of MnBBC did not show significant decay after 50 days of storage at room temperature (ΔA = 0.98 ± 0.008, Figure 4 b), which is better than that of the easily inactivated bioenzymes (such as the activity of HRP decreased by > 30% after 7 days of storage). Combining with the wide pH response (3 - 5), low temperature tolerance (4 - 25 °C) and long-term stability, MnBBC shows outstanding application potential in the fields of environmental sensing and point-of-care testing (POCT).

[0099] 2.3. Steady-state kinetic analysis of MnBBC biochar-based oxidase:

[0100] In this invention, the enzymatic reaction characteristics of MnBBC catalyzing the oxidation of TMB were studied through the Michaelis kinetics model, and the results are as Figure 5 shown. With the increase of substrate concentration (0 - 25 mM), the initial reaction velocity (V) showed a typical hyperbolic saturation trend, conforming to the Michaelis-Menten kinetics law. The key parameters were obtained through non-linear fitting: the maximum reaction rate V max = (0.9263 ± 0.023) × 10 -8 M·s -1 , and the Michaelis constant K m = 0.5260 ± 0.15 mmol / L. To further verify the kinetic model, linear regression analysis was carried out through the Lineweaver-Burk double reciprocal plot (1 / V vs 1 / [S]), and the obtained V max and K m values were consistent with the results of non-linear fitting (relative error < 5%).

[0101] The smaller the K m , the stronger the affinity. The lower K m value indicates that MnBBC has a high affinity for TMB (K m =

[0102] 0.5260 mmol / L), which is better than most biomimetic oxidase materials (such as Fe3O4 nanozyme K m ≈ 8 - 12 mM). The V max value reaches the order of 0.9263 × 10 -8 M·s -1 , confirming that its catalytic efficiency reaches the level of natural enzymes (such as the V max ≈ 1.2 × 10 -8 M·s -1 of horseradish peroxidase HRP catalyzing TMB). The above parameters indicate that while maintaining a high substrate binding ability, MnBBC can achieve a catalytic turnover rate comparable to that of bioenzymes.

[0103] 2.4. Oxidase-like activities of biochars prepared from different precursors:

[0104] The present invention studies the peroxidase-like activity (catalytic activity) of various biochars prepared from different precursors. The results are as Figure 6 shown. By comparing the TMB oxidation ability (λ = 654 nm) of different metal-modified biochars, it is found that only manganese-rich silicon-modified biochar (MnBBC) shows significant advantages. As Figure 6 shown in a, the absorbance (A = 1.086 ± 0.12) of the MnBBC system is 6.0 times and 8.1 times that of iron-rich (FeBBC: 0.181 ± 0.03) and zinc-rich (ZnBBC: 0.133 ± 0.06) modified materials, respectively. The colorimetric results further verify that only the MnBBC-TMB system presents a characteristic dark blue Figure 6 (b), indicating its ability to efficiently catalyze the oxidation of TMB to generate oxTMB. In addition, by comparing the manganese-rich silicon-modified biochars synthesized from different precursors, it is found that the MnBBC synthesized from hawthorn seeds as the precursor has the highest OXD-like activity, with a significant difference (P < 0.01).

[0105] 2.5. Comparative analysis of biochars prepared from different precursors:

[0106] The present invention conducts a characterization analysis of biochars prepared from different precursors. Figure 7 Characterizations of biochar materials prepared from different precursors by high-resolution scanning electron microscopy (HRSEM) are shown, including hawthorn seed biochar (BC), hawthorn seed-incorporated bentonite hybrid biochar (BBC), iron-rich modified biochar (FeBBC), zinc-rich modified biochar (ZnBBC), and manganese-rich silicon-modified biochar (MnBBC); among them, both BC and BBC exhibit a dense blocky morphology with a flat surface and low porosity Figure 7 (a-7b), and the introduction of surface bentonite does not significantly change their macroscopic morphology. Among the single-metal modified samples, ZnBBC Figure 7 (c) and FeBBC Figure 7 (d) present a granular structure, while the MnBBC particles before pyrolysis are uniformly dispersed, and the pyrolyzed MnBBC Figure 7 (e) shows an interlaced porous network structure, indicating that the pyrolysis treatment has a particularly significant effect on the morphology regulation of MnBBC, providing an ideal carrier for substrate transport and active site exposure.

[0107] Combined with the X-ray diffraction XRD analysis Figure 7 (f-7j) patterns, it can be seen that the broad diffraction peak of BC between 20° - 30° (2θ) indicates that it is mainly composed of amorphous carbon with low crystallinity, resulting in a limited density of active sites Figure 7f). The diffraction peaks of (012) and (104) crystal planes of newly added calcium carbonate (CaCO3) and the (003) and (110) peaks of bentonite appear in BBC. However, the inherent catalytic activity of the newly added crystalline phase is relatively low. Coupled with the compactness of the SEM morphology, the catalytic performance is limited and the overall performance is not significantly improved. Figure 7 g). The XRD pattern of MnBBC Figure 7 j) shows the characteristic peaks of (101), (112), and (211) crystal planes attributed to Mn3O4, confirming its spinel structure. Calculation by the Scherrer formula shows that the grain size of Mn3O4 is 15.3 nm. The proportion of its high-index crystal planes, such as (112), reaches 42%, which is beneficial to the exposure of active sites (Mn 3+ -O-Mn 2+ ), and can effectively adsorb substrates and carry out catalytic reactions. In contrast, in FeBBC, the (104) and (113) peaks of α-Fe2O3 and the (100) and (002) peaks of ZnO in ZnBBC correspond to the corundum and wurtzite structures respectively. Their grain sizes are larger (Fe2O3: 28 nm; ZnO: 35 nm), and the proportion of surface active crystal planes, such as Fe2O3(001) and ZnO(101), is less than 20%, resulting in a significantly lower density of active sites than that of MnBBC. Figure 7 h-7i).

[0108] Figure 8 、 Figure 9 The XPS spectra of biochars BC, BBC, ZnBBC, FeBBC, and MnBBC prepared from different precursors are shown: C1s( Figure 8 a-8e), O 1s( Figure 8 f-8j), Si 2p( Figure 8 k-8o), S2p( Figure 8 p-8t), Mn 2p(9a); From the analysis of the full X-ray photoelectron spectroscopy (XPS) spectra (9b) of BC, BBC, ZnBBC, FeBBC, and MnBBC: The C1s spectra of BC and BBC show functional groups of C-C (284.5 eV), C-O-C (286.3 eV), and O—C=O (288.8 eV). The peaks of C-O (532.5 eV) and C=O (530.8 eV) in the O 1s spectrum indicate that their surfaces contain hydroxyl groups, ether bonds, and carboxyl groups. The content of O—C=O in BBC increases (from 8% in BC to 15%), but the lack of metal active sites results in no significant improvement in catalytic activity. The peak of 102.5 eV (SiO2) in the Si2p spectrum and the weak S2p signal (<0.5 at.%) confirm that the introduction of bentonite does not introduce sulfur impurities.

[0109] Metal-loaded samples: The C1s and O1s spectra of MnBBC retain the carbon-oxygen functional groups of BC, and a new Mn-O bond (529.8 eV) appears. The deconvolution of Mn 2p3 / 2 shows the coexistence of Mn 2+ (641.2 eV) and Mn 3+ (643.5 eV), indicating the formation of multivalent Mn3O4. The Fe-O (530.2 eV) and Zn-O (530.5 eV) bonds are detected in FeBBC and ZnBBC, corresponding to α-Fe2O3 and ZnO respectively. However, their single oxidation states (Fe 3+ , Zn 2+ ) and high oxygen vacancy formation energies (Fe2O3: 2.1 eV;

[0110] ZnO: 2.5 eV) limit the generation rate of reactive oxygen species (·O2−, ·OH), resulting in significantly lower catalytic activity than MnBBC. The Mn 3+ / Mn 2+ multivalent cycle (TOF = 3.2 s -1 ) is significantly superior to that of Fe 3+ / Fe 2+ (TOF = 0.8 s -1 ) and the single oxidation state of Zn 2+ .

[0111] The Fourier transform infrared spectra (FTIR) of the corresponding biochars are shown in Figure 9 c. It can be seen from the analysis of the FTIR composite diagram that all samples show characteristic peaks at 3445 cm -1 (O-H), 1625 cm -1 (C=O), and 1122 cm -1 (C-O), indicating that their surfaces are rich in hydroxyl, carbonyl, and ether bonds, but lack metal oxide active sites, resulting in limited catalytic performance, such as BC. BBC retains the characteristic peaks of the carbon skeleton of BC and adds new peaks at 1025 cm -1 (asymmetric stretching of Si-O), 876 cm -1 , and 617 cm -1 (symmetric stretching of Si-O), confirming the introduction of bentonite. The ν3 vibration peak of calcium carbonate (CaCO3) (1430 cm -1 ) does not significantly change the surface catalytic activity, indicating that the mechanical stability is improved but the active sites are not increased. An Fe-O characteristic vibration peak appears at 561 cm -1 in FeBBC (A1g mode of α-Fe2O3). Combining with XPS Fe 2p3 / 2 (710.8 eV) and Fe-O bond (530.2 eV), it is confirmed that Fe 3+ exists in the form of iron oxide. Its limited oxidase activity (TOF = 0.8 s -1 ) is attributed to Fe3+ / Fe 2+ Insufficient single-electron transfer ability (Hubbard U = 4.5 eV). The Zn-O vibration peak (E2 mode of ZnO) at 474 cm -1 of ZnBBC matches with XPS Zn 2p3 / 2 (1021.5 eV), indicating that Zn 2+ exists in the form of zinc oxide. Although ZnO has photocatalytic potential, its wide bandgap (3.3 eV) limits the carrier concentration (n = 10 15 cm -3 ), resulting in weak oxidation activity of TMB (TOF = 0.6 s -1 ). The characteristic peaks at 621 cm -1 (Mn…O stretching vibration) and 531 cm -1 (Mn…O…Mn bending vibration) of MnBBC are consistent with the vibration modes of spinel-type Mn3O4. The synergistic effect of hydroxyl groups (3423 cm -1 ) and carbonyl groups (1595 cm -1 ) enhances the substrate adsorption ability. Combining with the Mn 3+ / Mn 2+ multivalent state cycle (TOF = 3.2 s -1 ), the oxidation performance of TMB is significantly improved.

[0112] 2.6. Detection of ascorbic acid (AA) based on the MnBBC-TMB system:

[0113] The present invention detects the dependence of AA on inhibiting the oxidation of TMB by MnBBC at different concentrations through ultraviolet-visible absorption spectroscopy and colorimetric imaging, and the results are as Figure 10 shown. In this system, oxidized TMB (oxTMB) is a water-soluble blue chemical substance, and its color change can be used as a reliable indicator for detecting certain reducing agents (such as AA). Based on this characteristic, it is expected to develop a sensor for detecting AA by detecting the color change caused by the oxTMB / TMB ratio affected by the AA concentration. As Figure 10 shown in a, as the AA concentration increases sequentially from 0 - 90 μM (0, 15, 30, 45, 60, 75, 90 μM, from top to bottom), the characteristic absorption peak at 654 nm shows a gradually decreasing trend. At the same time, colorimetric imaging shows that the blue color development intensity also weakens synchronously ( Figure 10b). The internal mechanism of this phenomenon is as follows: MnBBC can catalyze the oxidation of TMB to generate oxTMB, and the added AA, as a reducing agent, can cause the generated oxTMB to undergo a reduction reaction. Through direct visual observation, as the concentration of AA increases continuously, the blue color of the solution gradually fades, which is consistent with the colorimetric imaging result. In the ultraviolet-visible absorption spectrum, during the process of the AA concentration ranging from 0 to 90 μM, the absorbance at 654 nm gradually decreases, and this result is consistent with the color change trend observed by colorimetric imaging, further confirming that AA has a concentration-dependent inhibitory effect on the MnBBC-catalyzed oxidation reaction of TMB.

[0114] Based on the change value of absorbance (ΔA AA ) and the concentration C of ascorbic acid (AA), AA a standard curve was constructed, and the results are shown in Figure 10 c; where ΔA AA is the difference between the absorbance value (A AA ) in the presence of ascorbic acid and the absorbance value (A0) in the absence of ascorbic acid, that is, (ΔA AA = A0 - A AA ). Through linear regression analysis, the linear regression equation is ΔA AA = 0.006546C AA - 0.04322 (R 2 = 0.9937). This result shows that in the range of AA concentration from 15 to 90 μM, ΔA AA has a significant linear relationship with the AA concentration C AA .

[0115] 2.7. Detection of ALP activity based on the MnBBC / AA / ALP cascade signal amplification system (AAP-ALP-MnBBC-TMB):

[0116] This invention demonstrates the detection of ALP activity based on the MnBBC / AA / ALP cascade signal amplification system. Figure 11 a shows the ALP concentration-dependent response: as the ALP concentration gradient increases (0 - 30 U·L -1 ), the intensity of the characteristic absorption peak (654 nm) of oxTMB decays regularly, conforming to the pseudo-first-order kinetic model (R 2 = 0.9935). Figure 11 b shows the visual colorimetric analysis of the MnBBC / AA / ALP system: the blue intensity of the solution (oxTMB → TMB) gradually weakens as the ALP activity increases. When the ALP concentration ≥ 30 U·L -1 , the absorbance signal is completely quenched, defining the detection upper limit of the system.

[0117] The present invention also shows the absorbance change value (ΔA ALP ) of the MnBBC / AA / ALP system and the standard curve between it and the ALP concentration C ALP , as shown in Figure 11 c; wherein, ΔA ALP is the difference between the absorbance (A ALP ) in the presence of ALP and the absorbance value (A0) in the absence of ALP, (ΔA ALP = A0 - A ALP ). At 654 nm, the absorbance change (ΔA ALP ) and the ALP activity show a significant linear relationship in the range of 1 - 30 U / L (linear regression equation: ΔA ALP = 0.02524C ALP + 0.2624, R 2＝ 0.9935). The limit of quantification (LOQ) of this method is 1 - 30 U / L, and the limit of detection (LOD) is 0.3 U / L, indicating its high-sensitivity quantitative detection ability for ALP activity. Therefore, the cascade signal amplification system based on MnBBC-like oxidase, ascorbic acid (AA) and alkaline phosphatase (ALP) constructed by the present invention realizes the high-sensitivity detection of ALP activity.

[0118] 2.8. Specific recognition of alkaline phosphatase (ALP) by MnBBC-like oxidase:

[0119] The present invention evaluates the selectivity of the alkaline phosphatase (ALP) detection system by ultraviolet spectrophotometry, and respectively examines the interference effects of structural analogs (ginsenoside Rb1, Rg1, total ginsenosides), functional analogs (acid phosphatase, sucrase) and common matrix components (benzoic acid) on the detection system (the critical concentrations are ginsenoside Rb1, Rg1, total ginsenosides TG, acid phosphatase ACP, sucrase, benzoic acid BA); Figure 12 a shows the analysis results affected by interfering substances. The absorbances at 654 nm of the test concentrations of ginsenoside Rb1 (1.7 mM), Rg1 (0.51 mM), total ginsenosides (5 mg / ml), acid phosphatase (100 U / L), sucrase (10 5 U / L), and benzoic acid (1 mM) are shown. The results show that this system has high tolerance to structural / functional analogs and matrix interference. In addition, the present invention analyzes the difference in the signal response of the ALP addition group and the blank control group to the detection system. The results are shown in Figure 12As shown in Fig. b, in the signal comparison between the ALP addition group (30 U / L) and the blank control group (mean ± SD, n = 3), only the ALP or ALP + interferent group showed a significant decrease in absorbance (***p < 0.001), while there was no statistical difference in the single interferent group (p > 0.05). This may be because Mn3O4 on the surface of MnBBC specifically catalyzes the oxidation of TMB, the ALP-mediated AAP→AA pathway has strict substrate selectivity for phosphatases, and the hydrophobic structures of ginsenosides and organic acids limit their π-π interactions with TMB / AA. The above results indicate that the developed colorimetric biosensing method has high selectivity and satisfactory specificity for ALP.

[0120] 2.9. Catalytic mechanism of MnBBC-like oxidase:

[0121] This invention studies the reaction mechanism of the MnBBC catalytic system. Figure 13 For the effects of different inhibitors on the MnBBC catalytic system, the specificity of the reaction mechanism is revealed; the types of inhibitors used are: (a) superoxide dismutase (SOD), (b) tryptophan, (d) ascorbic acid (AA), (e) catalase. It can be seen from the experimental results that after treatment with catalase, SOD or tryptophan, the oxidation level of TMB remains in a relatively stable state. However, when AA is added, the blue color becomes lighter. As a strong reducing agent, AA directly scavenges ·OH (·OH + AA → oxidized AA + H2O), blocking the TMB oxidation pathway and resulting in a decrease in the generation of oxTMB. SOD, tryptophan, and catalase have no significant effect. SOD (scavenging O2· - ): indicating that O2· - is not a key intermediate; tryptophan (scavenging 1 O2): excluding the participation of singlet oxygen; catalase (decomposing H2O2): confirming that H2O2 is a non-essential intermediate. ·OH is the only active species directly involved in the oxidation of TMB. The catalytic activity of MnBBC stems from the manganese species loaded on its surface: the multivalent state of manganese (Mn 2+ →Mn 3+ ) can activate oxygen (O2) through single electron transfer to generate reactive oxygen species ROS. Therefore, the catalytic oxidation mechanism of manganese-rich silicon-modified biochar may be that MnBBC directly generates ·OH by activating O2, and the latter attacks TMB as the main oxidant to generate oxTMB, and AA inhibits the reaction by scavenging ·OH, and other ROS (O2· - , 1O2, H2O2) do not participate in the dominant path. The difference between this mechanism and traditional nanozymes is that it does not require H2O2, avoiding its limitations. Moreover, the inert carbonyl groups of biochar reduce non-specific adsorption, improve detection selectivity, and have strong anti-interference ability. It provides a new idea for designing H2O2-independent nanozyme sensors and also reveals the synergistic catalytic effect of biochar-metal manganese silicate composites.

[0122] 2.10. Study on the photothermal performance during the catalytic process of MnBBC-like oxidase:

[0123] In the present invention, the oxidation of TMB (ox-TMB) not only produces an obvious color reaction but also exhibits a powerful photothermal effect in the near-infrared range. Figure 14 It demonstrates the power dependence and synergistic effect study of the photothermal performance. This unique photothermal conversion characteristic can be used to establish a direct / indirect proportional relationship between temperature and the concentration of the target analyte, called "photothermal detection". Therefore, under the irradiation of an 808 nm near-infrared laser, a systematic power gradient experiment (0.5 - 2.2 W / cm 60uM ) was carried out on the MnBBC-TMB-AA 2 composite reaction system. The experimental data show that when irradiated continuously at a power of 2 W / cm 2 for 10 min, the temperature difference ΔT of the system reaches the maximum value (ΔT max ), so it is defined as the optimized condition for the subsequent experiment ( Figure 14 a). Since too high a power density and long-term exposure may cause photobleaching during the experiment, thus affecting the detection results, therefore, at a constant irradiation power of 2.0 W / cm 2 , and reacting for 10 minutes, the MnBBC-TMB composite system, TMB solution, and MnBBC dispersion all show good temperature-time linear relationships ( Figure 14 b), which indicates that the system has stable photothermal response characteristics. In addition, in the present invention, by establishing a synergistic effect analysis model ( Figure 14 c), it is found that the improvement of the photothermal performance of the MnBBC-TMB composite system comes from the synergistic effect of multiple components; Figure 14 In c, the synergistic temperature difference ΔT is the difference between the temperature of the composite system (T MnBBC-TMB ) and T0 (ΔT = T MnBBC-TMB - T0) and the difference between the temperature of the TMB monomer solution (T TMB ) and T0 (ΔT = T TMB - T0), where T0 is the reference temperature of the MnBBC dispersion (T0 = T MnBBC)。The experimental results show that within a specific range, only MnBBC catalyzes the catalytic reaction of TMB to produce blue oxTMB (λmax = 654 nm), and exhibits efficient near-infrared photothermal conversion characteristics, which provides a direct method for constructing colorimetric and photothermal dual-mode detection.

[0124] Through experiments, the present invention discovers that the concentrations of alkaline phosphatase (ALP) and ascorbic acid (AA) are dependent on the photothermal response. The results are as Figure 15 shown Figure 15 a shows the temperature change of the MnBBC-TMB-AA system under 808 nm irradiation (2.0 W / cm 2 ²) (recorded in real time by a digital temperature monitoring system). The results show that as the AA concentration gradient increases (0 - 90 μM), the equilibrium temperature of the system shows a concentration-dependent decreasing trend; Figure 15 b shows the quantitative relationship between the photothermal response signal of the MnBBC-TMB system and the AA concentration within a specific concentration range; where ΔT is the difference between (T0 - T AA ), T AA and T0 are the system temperatures with and without AA respectively. The experimental results show that ΔT and the AA concentration C AA show a good linear relationship in the range of 15 - 90 μM. The linear regression equation is: ΔT = 0.08235C AA + 5.834, R 2 = 0.9910. Figure 15 c shows the temperature change of the reaction solution of the ALP-AAP-TMB-MnBBC multi-stage catalytic system under 808 nm near-infrared laser irradiation (2.0 W / cm 2 ²). The high-precision digital temperature measurement system is used to record in real time and obtain the photothermal curve. The experimental results show that when the ALP concentration gradient increases (1 - 30 U / L), the equilibrium temperature of the system shows a significant concentration-dependent decrease, from 53.5 °C to 25.5 °C. The temperature difference ΔT is the difference between (T0 - T ALP ), T ALP and T0 are the system temperatures with and without ALP respectively; Figure 15 d shows the quantitative detection performance of the multi-component synergistic system: within the range of 1 - 30 U / L ALP concentration, ΔT and the ALP concentration C ALP show a good linear relationship. The linear equation is ΔT = 0.6166C ALP + 10.27, R 2 = 0.9939. The limit of detection (LOD) of this detection system is 0.3 U / L. Therefore, within a specific range, the MnBBC system can achieve rapid and real-time detection of ALP through photothermal detection.

[0125] 2.11. Multimode sensing detection:

[0126] The present invention is based on the combined colorimetric-smartphone-thermal imager for multimodal detection of alkaline phosphatase (ALP) activity. To achieve on-site monitoring of AA, a smartphone analysis platform was established using the MnBBC system. The smartphone monitors the blue color change in the MnBBC reaction system in real time, and then converts the color intensity of the selected specific area into RGB values; specifically, the color recognition application of the smartphone is used to analyze the values of red (R), green (G), and blue (B), namely the so-called RGB values; as a result, in the concentration range of 1-30 μM, the LOD is 0.24 U / L, and there is a good linear relationship between B / change value (R+G+B) and ALP concentration: B / (R+G+B)=0.4960 - 0.003073C ALP ,R 2 =0.9913, as Figure 16 shown in a.

[0127] In addition, under the 808 nm laser mapping, the present invention uses a thermal imager to monitor the ALP reaction system in real time. The results are shown in 16b. In the ALP activity range of 1-30 U / L, there is a significant linear relationship between ΔT and ALP activity, and the linear equation is ΔT = 0.3913C ALP +3.019, R 2 =0.9951; the detection limit of this detection system is 0.29 U / L; thus, it can be seen that this sensing method of the present invention has multimodal signal output functions such as chromaticity, absorbance, temperature, and RGB values, can effectively avoid the errors that may be generated by a single detection method, and significantly improves the reliability of the ALP activity detection results.

[0128] To prove the advantages of the ALP detection method provided by the present invention, it was compared with the reported colorimetric method and fluorescence method for ALP detection. The specific situation is as follows:

[0129] Table 1 Comparison of detection limits of analysis methods and synthetic materials in ALP biosensing

[0130]

[0131]

[0132] Table 2 Comparison of detection ranges of analysis methods and synthetic materials in ALP biosensing

[0133]

[0134] The above results prove that the detection method provided by the present invention has the advantages of wide detection range, low detection limit, high sensitivity, simplicity, low cost, self-calibration, and portability, and can adapt to the detection of soil samples with a wider concentration range.

[0135] The results of the spike recovery determination (UV) of ALP in soil by the above method of the present invention are shown in Table 3, and the results of the spike recovery determination (photo-thermal) of ALP in soil are shown in Table 4. The photo-thermal imaging diagram is as Figure 16 shown in c, and the RGB results of the spike recovery determination of ALP in soil are as Figure 16 shown in d.

[0136] Table 3 Spike recovery determination (UV) of ALP in soil

[0137]

[0138]

[0139] Table 4 Spike recovery determination (photo-thermal) of ALP in soil, thermal imaging detection

[0140]

[0141] The above are the specific implementation manners of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A manganese-rich silicon-modified biochar, characterized in that, The manganese-rich silicon-modified biochar has a porous network structure and contains elements such as Zn, Si, S, P, O, N, Mn, Mg, K, Fe, and Ca. Among them, the Mn element is uniformly distributed in the manganese-rich silicon-modified biochar with a mass fraction greater than 45% and exists in the form of spinel-type Mn3O4, the Ca element exists in the form of CaCO3, and the Si element exists in the form of SiO2.

2. The modified biochar rich in manganese and silicon according to claim 1, characterized in that, The manganese-rich silicon-modified biochar is obtained by pyrolyzing a mixture of hawthorn seeds, bentonite, and manganese element.

3. The preparation method of the manganese-rich silicon modified biochar according to claim 1, characterized in that, The preparation method is as follows: The hawthorn seeds are crushed to obtain hawthorn seed powder; a certain amount of hawthorn seed powder and bentonite are weighed and mixed in distilled water, and then a manganese sulfate monohydrate solution is added to obtain a suspension. The pH value of the suspension is precisely adjusted to 10 using a sodium hydroxide solution; it is stirred, ultrasonically treated, dried, the dried sample is ground, and placed in a pyrolysis device for pyrolysis; after pyrolysis is completed, the sample is naturally cooled to room temperature, and the obtained sample is repeatedly rinsed with distilled water to remove impurities; after rinsing, it is dried.

4. The preparation method of the manganese-rich silicon modified biochar according to claim 3, characterized in that, The mass ratio of the hawthorn seed powder to the bentonite is 10:1; during pyrolysis in the pyrolysis device, it is heated to 350 °C at a heating rate of 8 °C·min -1 -1 and kept reacting for 2 h, and nitrogen gas is continuously introduced throughout the process.

5. Application of the manganese-rich silicon-modified biochar prepared in claim 3 or 4 as a peroxidase-like enzyme.

6. Application of the manganese-rich silicon-modified biochar prepared in claim 3 or 4 in detecting L-ascorbic acid or alkaline phosphatase.

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