Method for detecting activity of alkaline phosphatase in soil

Abstract

The invention belongs to the technical field of alkaline phosphatase detection, and relates to a method for detecting the activity of alkaline phosphatase in soil, which comprises the following steps: step 1, preparing a soil extracting solution; step 2, sequentially adding an L-ascorbic acid-2-phosphate solution and a soil extracting solution into the centrifugal tube, and incubating at 37 DEG C for 30 minutes; a Briton-Robinson buffer solution is added, and the reaction is terminated; step 3, adding a 3, 3 ', 5, 5'-tetramethyl benzidine solution and a MnBBC dispersion liquid into the reaction liquid in the step 2, and reacting at the constant temperature of 37 + / -0.1 DEG C for 7 minutes; wherein the MnBBC dispersion liquid is prepared by mixing manganese-rich silicon modified charcoal with water; step 4, measuring the absorbance of the reaction system at 654 nm; observing the chromaticity of a reaction system, and monitoring an RGB value by using a smart phone; monitoring the temperature change of the reaction system under the irradiation of 808 nm near-infrared laser; according to the method, multi-dimensional output data such as chromaticity, absorbance, temperature and RGB values are comprehensively utilized, detection results can be mutually verified and corrected, and the accuracy and credibility of the detection results are ensured.

Description

Technical Field

[0001] The present invention belongs to the technical field of alkaline phosphatase detection, and particularly relates to a method for detecting the activity of alkaline phosphatase in soil. Background Art

[0002] Ginseng is an important Chinese herbal medicine with pharmacological effects such as anti - inflammation, 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 it plays 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 methods for detecting 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, they are more attractive for detecting ALP. 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 influences 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 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 method, microfluidic chip technology, etc. However, most of these methods require complex equipment and are relatively complicated to operate.

[0005] In recent years, in order to improve the sensitivity and stability of POCT detection, carbon-based materials, as a new type of nanomaterial, have received extensive attention. Carbon-based nanomaterials (such as carbon quantum dots, graphene, etc.) have become a hot spot in nanozyme research due to their excellent electrical conductivity, large specific surface area, and rich 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 biochar prepared by a one-step method is modified into graphene-like molybdenum selenide (MoSe 2 ), which is used as an intelligent nanozyme sensing platform for the voltammetric detection of hesperetin (HP) in orange peel. However, most 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 over traditional carbon-based materials in terms of cost, environmental protection, and easy accessibility. However, there is no report in the existing literature on the catalytic research of the nanozyme activity of metal-modified biochar and its application in the detection of alkaline phosphatase. Summary of the Invention

[0007] In view of the above technical problems and defects, the purpose of the present invention is to provide a method for detecting the activity of alkaline phosphatase in soil. This method uses self-designed manganese-rich silicon-modified biochar (MnBBC) to achieve highly sensitive, anti-interference, and real-time detection of ALP activity in ginseng continuous cropping soil through three detection modes: colorimetry, photothermal, and mobile phone intelligent auxiliary application, with advantages such as high accuracy and credibility of the detection results.

[0008] To achieve the above purpose, the present invention adopts the following technical solutions: A method for detecting the activity of alkaline phosphatase in soil, the method comprising the following steps: Step 1. Prepare a soil extract; 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 to terminate the reaction; Step 3. Add 3,3′,5,5′-tetramethylbenzidine solution and MnBBC dispersion to the reaction solution 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; 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 ALP in the soil, where ALP is alkaline phosphatase.

[0009] As a preference of the present invention, the preparation method of the soil extract in Step 1 is: take 0.5 g of soil sample and place it in a 50 ml centrifuge tube, add 20 ml of Britton-Robinson buffer solution with a pH of 8.0, vortex and oscillate for 5 min, and centrifuge at 5000 rpm for 10 min; take the supernatant and filter it through a membrane, and dilute it for standby.

[0010] As a preference of the present invention, add 20 uL of L-ascorbic acid-2-phosphate solution with a concentration of 35 mM in Step 2; the addition amount of the soil extract is 80 uL; Add 100 uL of 3,3′,5,5′-tetramethylbenzidine solution with a concentration of 17 mM and 100 uL of MnBBC dispersion solution with a concentration of 0.7 mg / mL in Step 3; When irradiating with 808 nm near-infrared laser in Step 4, the constant irradiation power is set to 2.0 W / cm², and the continuous irradiation time is 10 min.

[0011] As a preference of the present invention, the manganese-rich silicon modified biochar is obtained by pyrolysis of a mixture of hawthorn seeds, bentonite, and manganese element.

[0012] As a preference of the present invention, the manganese-rich silicon modified biochar has a porous network structure and includes elements such as Zn, Si, S, P, O, N, Mn, Mg, K, Fe, Ca, Al, among which 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 Mn 3 O 4 and Ca element exists in the form of CaCO 3 and Si element exists in the form of SiO 2 in the form of.

[0013] As a preference of the present invention, the linear relationship between absorbance change and ALP concentration is: ΔA ALP =0.02524C ALP +0.2624; where, ΔA ALPIt is the difference between the absorbance in the presence of ALP and the absorbance value in the absence of ALP, that is, the difference between the absorbance value of the reaction system when adding the soil extract and the absorbance value of the reaction system when not adding the soil extract, C ALP is the ALP concentration.

[0014] As a preference of the present invention, a color recognition application program of a smart phone is used to analyze the RGB value of the reaction system. The linear relationship between the RGB value and the ALP concentration is: B / (R + G + B) = 0.4960 - 0.003073C ALP ; where, R represents the red value, G represents the green value, B represents the blue value, and C ALP is the ALP concentration.

[0015] As a preference of the present invention, an infrared thermal imager is used to monitor the temperature change of the reaction system under the irradiation of an 808 nm near-infrared laser. At this time, the linear relationship between ΔT and the ALP concentration is: ΔT = 0.3913C ALP + 3.019; Or, a high-precision digital display temperature measurement system is used to monitor the temperature change of the reaction system under the irradiation of an 808 nm near-infrared laser. At this time, the linear relationship between ΔT and the ALP concentration is: ΔT = 0.6166C ALP + 10.27; Where, ΔT is the difference between T 0 and T ALP , T 0 represents the temperature of the reaction system in the absence of ALP, that is, the temperature of the reaction system when not adding the soil extract; T ALP represents the temperature of the reaction system in the presence of ALP, that is, the temperature of the reaction system when adding the soil extract, and C ALP is the ALP concentration.

[0016] As a further preference of the present invention, the preparation method of the manganese-rich silicon modified biochar is as follows: the hawthorn seeds are crushed 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; use a sodium hydroxide solution to precisely adjust the pH value of the suspension to 10; stir, perform ultrasonic treatment, dry, grind the dried sample, and place it in a pyrolysis device for pyrolysis; after the pyrolysis is completed, naturally cool the sample to room temperature, repeatedly rinse the obtained sample with distilled water to remove impurities; dry it after rinsing.

[0017] As a further more preference of the present invention, the mass ratio of the hawthorn seed powder to the bentonite is 10:1; during the pyrolysis in the pyrolysis device, the temperature is raised to 350 °C at a heating rate of 8 °C·min -1 and maintained for 2 h of reaction, and nitrogen is continuously introduced throughout the process.

[0018] Advantages and beneficial effects of the present invention: (1) During the synthesis of the MnBBC provided by the present invention, there is no need to add a protective agent that will affect the OXD activity, thus perfectly avoiding this drawback. In addition, it does not require cumbersome steps and instrument use such as high-speed centrifugation like nanozymes, 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.

[0019] (2) The MnBBC provided by the present invention has significant peroxidase-like activity. From the development history of biochar, the first-generation biochar was mainly used as a soil conditioner, the second-generation biochar as an adsorbent, and the 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 OXD has a wide range of application requirements, but traditional peroxidase-like OXD often has problems such as high cost and poor stability. With its unique peroxidase-like OXD activity, MnBBC has good chemical stability and catalytic activity, 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.

[0020] (3) The MnBBC provided by the present invention is different from traditional peroxidase (POD). POD usually requires hydrogen peroxide (H 2 O 2 ) as an oxidant to catalyze the oxidation reaction of organic substrates, and the use of H 2 O 2 not only increases the cost and operation complexity but also brings safety hazards. MnBBC, on the other hand, does not require H 2 O 2 to participate and has a unique catalytic mechanism, directly oxidizing specific organic substrates; in addition, in practical applications, the stability of the catalyst is a crucial performance indicator. MnBBC has strong stability, and after being placed for a period of time, its catalytic activity has not decreased significantly, indicating that MnBBC has good storage stability and long-term use performance, providing a strong guarantee for it in actual industrial production and environmental governance and other fields.

[0021] (4) The present invention first detected that the MnBBC carbon-based composite material has peroxidase-like activity. Peroxidase-like activity is a characteristic substance that mimics the catalytic function of natural peroxidase and shows great application potential in many fields such as biosensing and environmental monitoring. With its peroxidase-like activity, MnBBC can specifically interact with AA (L-ascorbic acid) or ALP in the reaction system and achieve their detection 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.

[0022] (5) During the preparation of MnBBC of the present invention, bentonite is introduced, which increases the surface roughness of the carbon source, increases the specific surface area, provides more active sites, and is conducive to the attachment of metal complexes such as metals and metal oxides on the surface of the carbon source. In addition, it shows that the microscopic concave-convex structure can enhance the interaction between the metal complex and the carbon source, further improving the stability and uniformity of attachment (through physical adsorption and chemical bonding), and contributing to the effective loading of the metal complex on the surface of the carbon source.

[0023] (6) The activities of many traditional catalysts are significantly affected by changes in pH, resulting in a substantial reduction in catalytic efficiency or even loss of activity. However, the MnBBC provided by the present invention can effectively carry out catalytic work within a relatively wide pH range (with the highest activity at pH 4, and the activity retention rate of MnBBC ≥ 60% within the pH range of 3 - 5), 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 effectively work 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 regions or low-temperature operation scenarios.

[0024] (7) During the detection of alkaline phosphatase (ALP) activity in ginseng continuous cropping soil using the self-designed MnBBC of the present invention, it can effectively avoid the interference of other substances, providing a solid guarantee for obtaining accurate and reliable detection results.

[0025] (8) The present invention proposes a comprehensive detection method integrating three detection modes: colorimetry, photothermal, and mobile phone intelligent auxiliary application. Specifically, it integrates visible signals, including intuitive color changes, 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 instant capture and color recognition technology of the smartphone Color Name Recognizer Camera, forming a unique and efficient detection new idea. It provides multi-dimensional information for detection, reflects the presence and concentration of the target from different angles, greatly improves the accuracy and real-time performance of detection, and overcomes 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 complete the detection simply 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.

[0026] (9) The triple-mode detection method provided by the present invention, which combines colorimetry, photothermal, and mobile phone intelligent assistance, is economically inexpensive (made from the pyrolysis of agricultural waste) and suitable for large-scale promotion. In terms of detection, it has the characteristics of high-throughput detection, can simultaneously detect multiple samples synchronously, greatly improves the detection efficiency, and has complementary advantages and is portable.

[0027] (10) The method provided by the present 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, so as to timely discover and correct possible errors and ensure the accuracy and reliability of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the 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; Figure 2 is the characterization analysis of manganese-rich silicon modified biochar (MnBBC); Figure 2 among them, a is the adsorption and desorption curve of manganese-rich silicon modified biochar; b is the pore size distribution map of manganese-rich silicon modified biochar; Figure 3 is the characterization analysis of manganese-rich silicon modified biochar (MnBBC); Figure 3 among them, a-c are the low-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; 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); Figure 5It is the steady-state kinetic analysis curve for evaluating the catalytic efficiency of manganese-rich silicon-modified biochar (MnBBC) mimicking oxidase, with 3,3',5,5'-tetramethylbenzidine (TMB) as the substrate; Figure 6 They are the absorbances of reaction solutions of various biochars prepared from different precursors and TMB; among them, a is the absorbance of the reaction system of hawthorn seed biochar (BC), hawthorn seed-introduced bentonite mixed biochar (BBC), iron-rich modified biochar (FeBBC), zinc-rich modified biochar (ZnBBC), manganese-rich silicon-modified biochar (MnBBC) and TMB (λ 654 nm); b is the photo of the reaction solution during the reaction of BC, BBC, FeBBC, ZnBBC, MnBBC and TMB; c is the absorbance of the reaction system of manganese-rich silicon-modified biochar MnBBC from rice husk (DK), manganese-rich silicon-modified biochar MnBBC from sorghum husk (GLK), manganese-rich silicon-modified biochar MnBBC from wild jujube husk (SZK), manganese-rich silicon-modified biochar MnBBC from millet husk (XMK), manganese-rich silicon-modified biochar MnBBC from hawthorn seed (SZZ) and TMB (λ 654 nm); d is the photo of the reaction solution during the reaction of DK, GLK, SZK, XMK, SZZ modified biochar and TMB; Figure 7 They are the characterization analysis diagrams of various biochars prepared from different precursors: among them, a-e are the high-resolution scanning electron microscope images (HRSEM) of BC, BBC, ZnBBC, FeBBC, MnBBC, showing the microscopic morphologies of various biochars; f-j are the X-ray diffraction (XRD) patterns of BC, BBC, ZnBBC, FeBBC, MnBBC, analyzing the crystal structures of various biochars; Figure 8 They are the X-ray photoelectron spectroscopy spectra (XPS) of biochars BC, BBC, ZnBBC, FeBBC, MnBBC prepared from different precursors: among them, a-e are C 1s; f-j are O 1s; k-o are Si 2p; p-t are S 2p; Figure 9 They are partial XPS diagrams and FTIR diagrams: among them, a is Mn 2p; b is the full XPS spectrum of BC, BBC, ZnBBC, FeBBC, MnBBC; c is the combined Fourier transform infrared spectroscopy (FTIR) spectrum of BC, BBC, ZnBBC, FeBBC, MnBBC; Figure 10 It is to monitor the concentration-dependent inhibition of AA on the oxidation of TMB by MnBBC through ultraviolet-visible absorption spectroscopy and colorimetric imaging; among them, a is the ultraviolet-visible absorption spectrum; b is the colorimetric imaging diagram; c is the standard curve constructed based on the absorbance change value (ΔA AA ) and the concentration of ascorbic acid (AA); Figure 11 is the detection of ALP activity based on the MnBBC / AA / ALP cascade signal amplification system; where, a is the three-dimensional absorption spectrum; b is the colorimetric imaging map; c is the standard curve constructed based on the absorbance change value (ΔA ALP ) and the ALP concentration; Figure 12 is the evaluation of the selectivity of the alkaline phosphatase (ALP) detection system by ultraviolet spectrophotometry; where, 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 ALP-added group and the non-added group to the detection system, and the data are expressed as mean ± SD (n = 3); Figure 13 is the effect of different types of inhibitors on the activity of MnBBC-like oxidase; where, a is superoxide dismutase (SOD); b is tryptophan; c is AA; d is catalase; Blank is the control group without added inhibitor; Figure 14 is the analysis of the photothermal performance of different systems; where, a is the reaction system of MnBBC-TMB-AA irradiated by an 808 nm laser, and the influence on the temperature difference of the system when the power increases from 0.5 - 2.2 W / cm²; b is the temperature-time curve of the MnBBC-TMB reaction system, TMB solution and MnBBC dispersion under irradiation by an 808 nm near-infrared laser (2.0 W / cm²); c is the analysis of the synergistic photothermal effect of the MnBBC-TMB composite system relative to single components; 60 uM is the concentration-dependent photothermal response analysis of ALP and AA; where, a is the temperature change of the MnBBC-TMB-AA system under irradiation by an 808 nm near-infrared laser (2.0 W / cm²); 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 irradiation by an 808 nm near-infrared laser (2.0 W / cm²); d is the quantitative detection performance of the multi-component synergistic system (the quantitative relationship between the photothermal response signal and the ALP concentration); Figure 15 Figure 16 ​Multimodal detection and analysis of alkaline phosphatase (ALP) activity based on 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 monitoring of the ALP reaction system jointly by the smartphone and the thermal imager under the mapping of 808 nm laser. In the range of ALP activity from 1 to 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 implementation mode

[0029] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the implementation modes 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.

[0030] This embodiment provides a method for detecting the activity of alkaline phosphatase in soil, and the method includes the following steps: Step 1. Prepare a soil extract. Step 2. Add L-ascorbic acid-2-phosphate solution and soil extract into a centrifuge tube in sequence, incubate at 37 °C for 30 min; add Britton-Robinson buffer to terminate the reaction. Step 3. Add 3,3′,5,5′-tetramethylbenzidine solution and MnBBC dispersion to the reaction solution 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. Step 4. Measure the absorbance at 654 nm of the reaction system; observe the chromaticity of the reaction system, and use a smartphone to monitor the RGB value; monitor the temperature change of the reaction system under the irradiation of 808 nm near-infrared laser; 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 output multimodally by chromaticity, absorbance, temperature and RGB value, so as to determine the activity of alkaline phosphatase in the soil.

[0031] To enable those skilled in the art to clearly understand how the present invention is implemented, the following will be introduced in detail through specific experiments: 1. Experiment: 1.1. Materials: 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 (ZnSO 4 ·7H 2 O), ferrous sulfate heptahydrate (FeSO 4 ·7H 2 O), manganese sulfate monohydrate (MnSO 4 ·H 2 O), 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).

[0032] 1.2. Preparation of MnBBC biochar: The fresh and dry hawthorn seeds (abbreviated as SZZ) were crushed using a pulverizer and passed through a 60-mesh sieve to obtain hawthorn seed powder. According to a 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. Subsequently, 0.2 mol / L manganese sulfate monohydrate (MnSO 4 ·H 2O) solution (500 mL). 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. After that, 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, and heated to 350 °C at a heating rate of 8 °C·min -1 and maintained at this temperature for 2 h of reaction. 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, labeled, and prepared for subsequent experiments.

[0033] 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).

[0034] 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 ZnSO 4 ·7H 2 O 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). FeSO 4 ·7H 2 O 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).

[0035] In addition, in this example, in order 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 manganese-rich silicon-modified biochars MnBBC (DK), MnBBC (DC), MnBBC (XMK), MnBBC (GLK), and MnBBC (SZK) with rice husks (DK), rice straw (DC), millet husks (XMK), sorghum husks (GLK), and wild jujube husks (SZK) as raw materials were prepared according to the above experimental procedure (1.2). These were compared with the manganese-rich silicon-modified biochar MnBBC (SZZ) prepared from hawthorn seeds above to explore the laccase-like activity of different biochar raw materials.

[0036] In this example, a total of 10 types of different types of biochar, composite biochar, and metal-modified biochar were synthesized, aiming to comprehensively explore the effects of different preparation methods and raw materials on the properties of biochar in order to find the biochar with the most enzyme-like activity.

[0037] It should be noted that in this example, the results of manganese-rich silicon modified biochar MnBBC(DK) from rice husk and manganese-rich silicon modified biochar MnBBC(DC) from rice straw are basically the same. Therefore, only the experimental results of manganese-rich silicon modified biochar MnBBC(DK) from rice husk are provided in this example.

[0038] 1.3. Material characterization: The ultraviolet-visible absorption spectrum was recorded using a HITACHI U-2900 type 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 100 mm² EDS detector), including morphology 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 type physical adsorption instrument. 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 type polycrystalline X-ray diffractometer. The surface chemical state was characterized using a Thermo Fisher Scientific ESCALAB 250Xi type X-ray photoelectron spectrometer. The vibration modes of the surface functional groups of the biochar were characterized using a Thermo Fisher Nicolet iS50 Fourier transform infrared spectrometer. The mid-infrared fingerprint region spectrum was collected in transmission mode at 4000 - 400 cm -1 to achieve in-situ surface analysis. The lattice vibration modes were characterized using a HORIBA LabRAM HR Evolution type confocal micro-Raman spectrometer. The multi-element quantitative analysis of the sample was performed using a Thermo Scientific iCAP PRO 6300 full-spectrum direct-reading inductively coupled plasma emission spectrometer. A high-precision digital display thermometer (Taizhou Weixing Electric Co., Ltd.), a HIKMICRO thermal imager (Hangzhou Hikvision Microimaging 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 to achieve in-situ monitoring of multiple physical fields.

[0039] 1.4. Evaluation of the peroxidase-like activity of MnBBC biochar: The peroxidase-like activity of MnBBC was evaluated by the method of catalyzing the oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB). 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 to 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 blue solution generated by the reaction was measured using a UV-visible spectrophotometer, and the measurement wavelength was set at 654 nm. The measured absorbance data were used for subsequent analysis and evaluation of the peroxidase-like activity of MnBBC.

[0040] To further explore the effect of pH on the peroxidase-like activity of MnBBC, the present invention designed a combination of MnBBC dispersion (0.7 mg / mL, 100 μL), TMB solution (17 mM, 100 μL), and 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.

[0041] To further 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 in a constant temperature condition of 4°C and 25°C for reaction. After the reaction reached a stable state, the absorbance values of the reaction solution at 4°C and 25°C were quickly measured and recorded at the characteristic wavelength of 654 nm using a UV-visible spectrophotometer.

[0042] 1.5. Steady-state kinetic analysis of the peroxidase of MnBBC biochar: 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 a reaction system 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 the 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: ; where 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), K m is the Michaelis constant (mM, kinetic constant).

[0043] 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.

[0044] 1.6. Research on the colorimetric method and combined photothermal detection of AA based on the MnBBC-TMB system: 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 precisely 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 of 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 precisely 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 (with a 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 change curve of temperature with concentration, the internal relationship between the AA concentration and the temperature change generated 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.

[0045] 1.7. Colorimetry - 808 Laser Photothermal Synergistic Strategy for Highly Sensitive Detection of Alkaline Phosphatase (ALP): The ALP activity determination process of the present invention is as follows: 20 μL of 35 mM p-aminophenyl phosphate (AAP) solution and 80 μL of ALP solutions with different concentrations (0 - 30 U / L) were precisely pipetted and mixed in a reaction tube, and incubated in a 37 °C constant temperature water bath for 30 min to allow AAP to react fully under the catalysis of ALP. After the incubation, 200 μL of Britton-Robinson buffer solution with pH 4.0 was immediately added to terminate the reaction. Then, 100 μL of 17 mM TMB solution and 100 μL of 0.7 mg / mL MnBBC dispersion were successively added to the reaction tube, and vortex mixed evenly to form a total reaction solution with a volume of 0.5 mL. The above total reaction solution was allowed to stand and react for 7 min until the color development was stable. An appropriate amount of the solution was taken with a micropipette and transferred to a quartz cuvette, and the absorbance value was measured at 654 nm using a UV-visible spectrophotometer. At the same time, an 808 nm near-infrared laser (2W / cm 2 ) was used to irradiate the reaction system, and a high-precision digital display thermometer was used to monitor the temperature change of the reaction system during the 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.

[0046] 1.8. Colorimetric and photothermal multimode sensing detection under smartphone mode: The present invention uses the smartphone application Color Name Recognizer Camera (color recognition) to evaluate the color constants of colorimetric reactions, and combines a thermal imager to monitor the temperature changes generated by the photothermal effect, establishing a photothermal-smartphone-based ALP multimode sensing detection method, aiming to achieve accurate and convenient detection of ALP activity. The operation is the same as the above experiment (1.7) until the color development is stable; use the "Color Name Recognizer Camera" application of the smartphone to instantaneously capture and recognize the color (RGB) of the reaction solution. This application is an existing program, based on advanced image processing algorithms, accurately recognizes and quantifies color information. At the same time, in order to verify the accuracy of the results, the absorbance is measured at a wavelength of 654 nm using a UV-visible spectrophotometer.

[0047] 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 change (ΔT) generated by the reaction system, and establish the correlation between the ΔT value and the ALP activity. Statistical methods are used to analyze the ΔT value and ALP activity data, and establish a correlation model between the two. The establishment of this model helps to achieve the quantitative analysis of ALP activity by measuring the temperature change.

[0048] 1.9. Research on the specific recognition of ALP by the MnBBC-like oxidase dual-mode sensing platform and the influence of interfering factors in complex matrices: Basis for the selection of interfering factors, structural analogs: ginsenoside Rb1 / Rg1, total ginsenosides; functional analogs: acid phosphatase, sucrase; common matrix components: benzoic acid.

[0049] ALP-free system (blank control group): Add 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) to a 1.5 mL polypropylene centrifuge tube in sequence, vortex mix, and react at room temperature for 7 min. Take 200 μL of the reaction solution and transfer it to a quartz cuvette, and use a UV-visible spectrophotometer to record the absorbance at 654 nm (take the average of three measurements).

[0050] ALP-containing system (experimental group): Mix 100 μL of the interfering 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 / A 0 , A 0 is the absorbance without the interfering factor), and evaluate the selectivity of the system.

[0051] 1.10. Analysis of the catalytic mechanism of MnBBC-like oxidase: Reactive oxygen species based on radical trapping: As a new catalyst synthesized from agricultural waste, the oxidase (OXD-like) activity of MnBBC stems from the synergistic effect of 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 process. To clarify the specific mechanism of its catalysis of TMB oxidation, this experiment adopted a radical trapping strategy, specifically targeting and scavenging different ROS with specific inhibitors to systematically analyze 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 scavenging hydrogen peroxide (H 2 O 2 ), ascorbic acid (AA) - scavenging hydroxyl radicals (·OH), superoxide dismutase (SOD) - scavenging superoxide anions (O 2 -), tryptophan - scavenging singlet oxygen ( 1 O 2 ). The reaction is carried out at room temperature for 7 min, and then 200 uL of the reaction solution is transferred to a quartz cuvette, and the absorbance at 654 nm is recorded using a spectrophotometer (average value after three measurements), and the relative activity is calculated.

[0052] 1.11. Spiked detection of ALP in real soil samples based on the MnBBC-like oxidase system: The spike recovery test has important 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 ALP activity detection. For example: Inhibition / activation effect: Humic acid can bind to the enzyme through hydrophobic interaction, changing its conformation; heavy metal ions (such as Cu²⁺, Cd²⁺) 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 underestimated 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 it is necessary to optimize the extraction buffer (such as adding a chelating agent EDTA to desorb metal ions) or improve the detection conditions (such as centrifugal filtration to remove turbidity).

[0053] Parallel experimental design of the spike recovery test: The soil samples are divided into an unspiked group (measuring 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.

[0054] The present invention verifies the detection performance of MnBBC-like oxidase for ALP in soil through a spike experiment. Five typical soil samples are selected: a soil sample without ginseng planting history (CK), a continuous cropping soil planted with ginseng for 5 years, 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. Take 0.5 g of each soil sample and place it in a 50 ml centrifuge tube, add 20 ml of Britton-Robinson buffer (pH 8.0), vortex for 5 min, and centrifuge at 5000 rpm for 10 min; take the supernatant and filter it through a membrane, and dilute it 100 times step by step for standby.

[0055] The concentration settings were unspiked (0 U / L), low-concentration spiked (5 U / L), medium-concentration spiked (10 U / L), and high-concentration spiked (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 at 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, react at a constant temperature of 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 remaining steps are the same as above. Calculate the ALP activity according to the standard curve, and the results are expressed as the ALP activity (U / L) per unit mass of soil.

[0056] The formula for the recovery rate is: Recovery Rate = (measured value - control group) / added value × 100%.

[0057] 2. Results and discussion: 2.1. Characterization of MnBBC biochar: Figure 1 and 2 are the characterization analysis diagrams of manganese-rich silicon-modified biochar (MnBBC). The microscopic morphology of manganese-rich silicon-modified biochar (MnBBC) was characterized by high-resolution scanning electron microscopy (HRSEM), 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 9.781 Å), the specific surface area ( S BET ) is 35.128 m 2 / g, and 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 beneficial to the diffusion of reactants and the exposure of active sites, thus enhancing the catalytic performance.

[0058] 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 the 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 elements (S: 8.03 wt.%) are detected in the material, which may further regulate the catalytic performance through electron transfer or synergistic effects. The content of each element is 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.%).

[0059] 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.

[0060] 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 (Mn 3 O 4 , JCPDS No.18-0803), calcium carbonate (CaCO 3 , JCPDS No.88-1807), and silicon dioxide (SiO 2 , 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 3d). In the presence of MnBBC, a distinct characteristic response of 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 stems from the peroxidase-like activity of MnBBC. Raman spectroscopy ( Figure 3 e) provides information on the carbon matrix and chemical bond states of MnBBC. The results show that the carbon skeleton of MnBBC exhibits typical sp 2 hybridization characteristics, where the D peak (1331 cm -1 , A 1 g symmetry) reflects the defects or edge structures in the carbon structure, and the G peak (1575 cm -1 , E 2 g 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 enhancement of the material's electron conduction ability. 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 SiO 2 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.

[0061] 2.2. Peroxidase-like activity of MnBBC: 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 color of the solution does not change. When MnBBC and TMB are mixed, the solution turns blue. Through ultraviolet-visible absorption spectroscopy ( Figure 4 a), a strong absorption peak appears at 654 nm. This phenomenon indicates that TMB is oxidized to the oxidized oxTMB, thus proving that MnBBC has OXD activity.

[0062] Further study on the effects of temperature and pH on its activity shows that the temperature experiment ( Figure 4 c) shows that MnBBC maintains high catalytic activity at both 4 °C and 25 °C, confirming its advantage in low-temperature adaptability. The catalytic activity of MnBBC shows significant pH dependence ( Figure 4d): It has the highest activity at pH 4 (ΔA = 1.02), retains 90% of its activity at pH 3 (ΔA = 0.959), and its 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 MnBBC has an activity retention rate ≥ 60% in the pH range of 3 - 5 (vs. pH 4), indicating its good tolerance to acidic environments (such as continuous ginseng cropping soil), which is superior to most metal-organic framework (MOF) nanozymes (usually with an activity decay > 50%).

[0063] Long-term storage stability: The catalytic activity of MnBBC shows no significant decay after 50 days of storage at room temperature (ΔA = 0.98 ± 0.008, Figure 4 b), which is superior to the easily inactivated biocatalysts (such as HRP with an activity decline > 30% after 7 days of storage). Combining the broad 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).

[0064] 2.3. Steady-state kinetic analysis of MnBBC biochar-like oxidase: In this invention, the enzymatic reaction characteristics of MnBBC catalyzing the oxidation of TMB were studied through the Michaelis kinetic model, and the results are as Figure 5 shown. With the increase of substrate concentration (0 - 25 mM), the initial reaction velocity (V) shows a typical hyperbolic saturation trend, conforming to the Michaelis-Menten kinetic 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 are consistent with the non-linear fitting results (relative error < 5%).

[0065] K m The smaller the value, the stronger the affinity. The lower K m value indicates that MnBBC has a higher affinity for TMB (K m = 0.5260 mmol / L), which is superior to most biomimetic oxidase materials (such as Fe 3 O 4 nanozyme K m ≈8 - 12 mM). The V max value reaches 0.9263×10 -8 M·s-1 Magnitude, confirming that its catalytic efficiency reaches the level of natural enzymes (such as the V of horseradish peroxidase HRP catalyzing TMB max ≈1.2×10 -8 M·s -1 ). The above parameters indicate that while maintaining a high substrate binding ability, MnBBC can achieve a catalytic turnover rate comparable to that of bioenzymes.

[0066] 2.4. Oxidase-like activity of biochars prepared from different precursors: The present invention studies the oxidase-like activity (catalytic activity) of various biochars prepared from different precursors, and 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 the 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 the 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 prepared from hawthorn seeds as the precursor has the highest OXD, with a significant difference ( P <0.01).

[0067] 2.5. Comparative analysis of biochars prepared from different precursors: The present invention characterizes and analyzes the biochars prepared from different precursors, Figure 7 as shown by the characterization of biochar materials prepared from different precursors by high-resolution scanning electron microscopy (HRSEM), including 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); 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), FeBBC ( Figure 7 d) present a granular structure, while the MnBBC particles before pyrolysis are evenly 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.

[0068] Combined with X-ray diffraction (XRD) analysis ( Figure 7 f-7j), it can be seen from the spectrum that BC has a broad diffraction peak between 20° and 30° (2 θ ), indicating that it is mainly composed of amorphous carbon with low crystallinity, resulting in a limited density of active sites ( Figure 7 f). The diffraction peaks of the (012) and (104) crystal planes of newly added calcium carbonate (CaCO 3 ) in BBC and the (003) and (110) peaks of bentonite, but the inherent catalytic activity of the newly added crystalline phase is relatively low. Together with the compactness of the SEM morphology, it leads to limited catalytic performance and fails to significantly improve the overall performance ( Figure 7 g). The XRD pattern of MnBBC ( Figure 7 j) shows the characteristic peaks of the (101), (112), and (211) crystal planes attributed to Mn 3 O 4 , confirming its spinel structure. Calculation by the Scherrer formula shows that the grain size of Mn 3 O 4 is 15.3 nm, and 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. Comparing with the (104) and (113) peaks of α-Fe 2 O 3 in FeBBC and the (100) and (002) peaks of ZnO in ZnBBC, which correspond to the corundum and wurtzite structures respectively, their grain sizes are larger (Fe 2 O 3 : 28 nm; ZnO: 35 nm), and the proportion of surface active crystal planes, such as Fe 2 O 3 (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).

[0069] Figure 8 , Figure 9 shows the XPS spectra of biochars BC, BBC, ZnBBC, FeBBC, and MnBBC prepared from different precursors: C1s ( Figure 8 a-8e), O 1s ( Figure 8 f-8j), Si 2p ( Figure 8 k-8o), S 2p ( Figure 8p-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 C 1s spectra of BC and BBC show C–C (284.5 eV), C–O–C (286.3 eV), and O—C=O (288.8 eV) functional groups. The peaks of C–O (532.5 eV) and C=O (530.8 eV) in the O 1s spectrum indicate that its surface contains hydroxyl, ether, and carboxyl groups. The increased content of O—C=O in BBC (from 8% in BC to 15%) but the lack of metal active sites result in no significant improvement in catalytic activity. The peak at 102.5 eV in the Si 2p spectrum (SiO 2 ) and the weak S2p signal (<0.5 at.%) confirm that the introduction of bentonite does not introduce sulfur impurities.

[0070] Metal-loaded samples: The C 1s and O 1s spectra of MnBBC retain the carbon-oxygen functional groups of BC, and a new Mn–O bond (529.8 eV) appears. The Mn 2p 3 / 2 peak fitting shows the coexistence of Mn 2+ (641.2 eV) and Mn 3+ (643.5 eV), indicating the formation of multivalent Mn 3 O 4 . For FeBBC and ZnBBC, Fe–O (530.2 eV) and Zn–O (530.5 eV) bonds are detected, corresponding to α-Fe 2 O 3 and ZnO respectively. However, their single oxidation states (Fe 3+ , Zn 2+ ) and high oxygen vacancy formation energies (Fe 2 O 3 : 2.1 eV; ZnO: 2.5 eV) limit the generation rate of reactive oxygen species (•O 2 ⁻, •OH), resulting in significantly lower catalytic activity than MnBBC. The Mn 3+ / Mn 2+ multivalent cycle (TOF = 3.2 s -1 ) is significantly better than that of Fe 3+ / Fe 2+ (TOF = 0.8 s -1 ) and the single oxidation state of Zn 2+ .

[0071] The corresponding Fourier transform infrared spectroscopy (FTIR) of the above biochars is shown in Figure 9 c. It can be seen from the analysis of the FTIR combined diagram that all samples show a peak at 3445 cm -1(O-H), 1625 cm -1 (C=O), 1122 cm -1 (C-O) shows characteristic peaks, indicating that its surface is rich in hydroxyl, carbonyl, and ether bonds, but lacks 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 1025 cm -1 (Si–O antisymmetric stretching), 876 cm -1 , 617 cm -1 (Si-O symmetric stretching), confirming the introduction of bentonite. The ν 3 vibration peak of calcium carbonate (CaCO 3 ) (1430 cm -1 ) does not significantly change the surface catalytic activity, indicating improved mechanical stability but no increase in active sites. FeBBC shows a characteristic vibration peak of Fe–O (A -1 mode of α-Fe 2 O 3 ) at 561 cm 1 . Combining with XPS Fe 2p 3 / 2 (710.8 eV) and the 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 the insufficient single-electron transfer ability of Fe 3+ / Fe 2+ (Hubbard U = 4.5 eV). The Zn–O vibration peak (E -1 mode of ZnO) of ZnBBC at 474 cm 2 matches with XPS Zn 2p 3 / 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 TMB oxidation activity (TOF = 0.6 s -1 ). The characteristic peaks of MnBBC at 621 cm -1 (Mn–O stretching vibration) and 531 cm -1 (Mn–O–Mn bending vibration) are consistent with the vibration modes of spinel-type Mn 3 O 4 . Hydroxyl (3423 cm -1 ) and carbonyl (1595 cm -1The synergistic effect enhances the substrate adsorption ability and combines with Mn 3+ / Mn 2+ The multivalent state cycle (TOF = 3.2 s -1 ), significantly improving the oxidation performance of TMB.

[0072] 2.6. Detection of ascorbic acid (AA) based on the MnBBC-TMB system: 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. 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), a gradual attenuation trend of the characteristic absorption peak at 654 nm is shown. At the same time, colorimetric imaging shows that the blue color intensity also weakens synchronously ( Figure 10 b). The internal mechanism of this phenomenon is that 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 AA concentration continuously increases, the blue color of the solution gradually fades, which is consistent with the colorimetric imaging results. In the ultraviolet-visible absorption spectrum, during the process of AA concentration from 0 - 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 the concentration-dependent inhibitory effect of AA on the oxidation reaction of MnBBC catalyzing TMB.

[0073] The present invention constructs a standard curve based on the absorbance change value (ΔA AA ) and the ascorbic acid (AA) concentration C AA . The results are as Figure 10 shown in c; among them, ΔA AA is the difference between the absorbance value (A AA ) in the presence of ascorbic acid and the absorbance value (A 0 ) in the absence of ascorbic acid, that is, (ΔA AA = A 0 −A AA ). Through linear regression analysis, the linear regression equation is obtained as ΔA AA =0.006546C AA −0.04322 (R 2= 0.9937), and the result indicates that within the range of AA concentration from 15 - 90 μM, ΔA AA has a significant linear relationship with AA concentration C AA .

[0074] 2.7. Detection of ALP activity based on the MnBBC / AA / ALP cascade signal amplification system (AAP - ALP - MnBBC - TMB): 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 of oxTMB (654 nm) 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 with the increase of ALP activity. When the ALP concentration ≥ 30 U·L -1 , the absorbance signal is completely quenched, defining the upper detection limit of the system

[0075] This invention also shows the standard curve between the change value of absorbance (ΔA ALP ) of the MnBBC / AA / ALP system and ALP concentration C ALP , as shown in Figure 11 c; where ΔA ALP is the difference between the absorbance (A ALP ) in the presence of ALP and the absorbance value (A 0 ) in the absence of ALP, (ΔA ALP = A 0 − A ALP ). At 654 nm, the change in absorbance (ΔA ALP ) has a significant linear relationship with ALP activity 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 in this invention realizes the high - sensitivity detection of ALP activity

[0076] 2.8. Specific recognition of alkaline phosphatase (ALP) by MnBBC-like oxidase: In this invention, the selectivity of the alkaline phosphatase (ALP) detection system was evaluated by ultraviolet spectrophotometry. 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 were investigated respectively (the critical concentrations were ginsenoside Rb1, Rg1, total ginsenosides TG, acid phosphatase ACP, sucrase, benzoic acid BA). Figure 12 a shows the influence of interfering substances on the analysis results. 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) were measured. The results showed that the system had high tolerance to structural / functional analogs and matrix interference. In addition, the differences in the signal responses of the ALP addition group and the blank control group to the detection system were analyzed in this invention. The results are as Figure 12 shown in b. In the signal comparison (mean ± SD, n = 3) between the ALP addition group (30 U / L) and the blank control group, only the ALP or ALP + interfering substance group showed a significant decrease in absorbance (*** p <0.001), while there was no statistical difference in the single interfering substance group ( p >0.05). This may be because Mn 3 O 4 on the surface of MnBBC specifically catalyzes the oxidation of TMB. The ALP-mediated AAP→AA pathway has strict substrate selectivity for phosphatases. 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.

[0077] 2.9. Catalytic mechanism of MnBBC-like oxidase: In this invention, the reaction mechanism of the MnBBC catalytic system was studied. Figure 13 shows the effects of different inhibitors on the MnBBC catalytic system, revealing the specificity of the reaction mechanism. The types of inhibitors used were: (a) superoxide dismutase (SOD), (b) tryptophan, (d) ascorbic acid (AA), and (e) catalase. It can be seen from the experimental results that after treatment with catalase, SOD, or tryptophan, the oxidation level of TMB remained in a relatively stable state. However, when AA was added, the blue color became lighter. As a strong reducing agent, AA directly scavenged ·OH (·OH + AA → oxidized AA + H 2O), blocking the oxidation pathway of TMB and resulting in a decrease in the generation of oxTMB. SOD, Trptophan, and Catalase have no significant effect. SOD (scavenging O 2 · - ): indicating that O 2 · - is not a key intermediate; Trptophan (scavenging ¹O 2 ): excluding the participation of singlet oxygen; Catalase (decomposing H 2 O 2 ): confirming that H 2 O 2 is a non-essential intermediate product. ·OH is the only active species directly participating in the oxidation of TMB. The catalytic activity of MnBBC stems from the manganese species loaded on its surface: the multivalent states of manganese (Mn 2+ → Mn 3+ ) can activate oxygen (O 2 ) 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 O 2 , and the latter attacks TMB as the main oxidant to generate oxTMB. AA inhibits the reaction by scavenging ·OH, and other ROS (O 2 · - , ¹O 2 , H 2 O 2 ) do not participate in the dominant pathway. The difference between this mechanism and traditional nanozymes is that it does not require H 2 O 2 , 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 H 2 O 2 -independent nanozyme sensors and also reveals the synergistic catalytic effect of biochar-metal manganese silicon composites.

[0078] 2.10. Study on the photothermal performance during the oxidase-like catalysis of MnBBC: In the present invention, the oxidation of TMB (ox-TMB) not only produces an obvious color reaction but also exhibits a strong photothermal effect in the near-infrared range. Figure 14 The power dependence and synergistic effect study of the photothermal performance are shown. 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, for MnBBC-TMB-AA 60 uMA systematic power gradient experiment (0.5 - 2.2 W / cm²) was carried out on the composite reaction system. The experimental data showed that when irradiated continuously at a power of 2 W / cm² for 10 min, the temperature difference ΔT of the system reached the maximum value (ΔT max ), so it was determined as the optimized condition for the subsequent experiment ( Figure 14 a). Since too high power density and long-time exposure may lead to photobleaching during the experiment, thus affecting the detection results. Therefore, at a constant irradiation power of 2.0 W / cm² and a reaction time of 10 minutes, the MnBBC-TMB composite system, TMB solution and MnBBC dispersion all showed good temperature-time linear relationships ( Figure 14 b), which indicated that the system had stable photothermal response characteristics. In addition, by establishing a synergistic effect analysis model ( Figure 14 c), the present invention found that the improvement of the photothermal performance of the composite system MnBBC-TMB originated from the synergistic effect of multiple components; Figure 14 in c, the synergistic temperature difference ΔT was the difference between the temperature of the composite system (T MnBBC - TMB ) and T 0 (ΔT = T MnBBC - TMB −T 0 ) and the difference between the temperature of the TMB monomer solution (T TMB ) and T 0 (ΔT = T TMB −T 0 ), and T 0 was the reference temperature of the MnBBC dispersion (T 0 = T MnBBC ). The experimental results showed that within a specific range, only the catalytic reaction of MnBBC catalyzing TMB produced blue oxTMB (λmax = 654 nm), and caused efficient near-infrared photothermal conversion characteristics, which provided a direct method for constructing colorimetric and photothermal dual-mode detection.

[0079] The present invention found through experiments that the concentrations of alkaline phosphatase (ALP) and ascorbic acid (AA) were dependent on the photothermal response. The results were as Figure 15 shown. Figure 15 a shows the temperature change of the MnBBC-TMB-AA system under 808 nm irradiation (2.0 W / cm²) (recorded in real time by a digital temperature monitoring system). The results showed that with the increase of the AA concentration gradient (0 - 90 μM), the equilibrium temperature of the system showed a concentration-related 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 (T 0 -T AA ), and T AAand T 0 are the system temperatures in the presence and absence of 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, R² = 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 / cm²). The high - precision digital display temperature measurement system is used to record and obtain the photothermal curve in real time. 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 (T 0 - T ALP ), where T ALP and T 0 are the system temperatures in the presence and absence of ALP, respectively; Figure 15 d shows the quantitative detection performance of the multi - component synergistic system: in 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, R² = 0.9939. The detection limit (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.

[0080] 2.11. Multi - mode sensing detection: The present invention is based on the combined colorimetric - smartphone - thermal imager for multi - modal detection of alkaline phosphatase (ALP) activity. To achieve on - site monitoring of AA, a smartphone analysis platform is established using the MnBBC system. The smartphone monitors the blue 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 the B / change value (R + G + B) and the ALP concentration: B / (R + G + B)=0.4960 - 0.003073C ALP , R 2 = 0.9913, as Figure 16 shown in a.

[0081] In addition, under the 808 nm laser mapping, the present invention uses a thermal imager to monitor the ALP reaction system in real time. As shown in 16b, within the ALP activity range of 1 - 30 U / L, ΔT and ALP activity show a significant linear relationship, and the linear equation is ΔT = 0.3913C ALP +3.019, R² = 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 multi-modal signal output functions such as chromaticity, absorbance, temperature, and RGB value, can effectively avoid the errors that may occur in a single detection method, and significantly improves the reliability of the ALP activity detection result.

[0082] To prove the advantages of the ALP detection method provided by the present invention, it is compared with the reported colorimetric method and fluorescence method for ALP detection. The specific situation is as follows: Table 1 Comparison of detection limits of analytical methods and synthetic materials in ALP biosensing Method Material <![CDATA[Detection limit (U L -1 )]]> Remark Blood glucose meter 8.9 Fluorometry TPEPy-pY 6.6 Fluorometry rGQDs / CS 7.8 Fluorometry 3D DNA 50 Fluorometry <![CDATA[CsPbBr 3 @PMMA]]> 4.85 Colorimetry AgNPrs 5 Colorimetry <![CDATA[TiO 2 > 3 Colorimetry protein@3D DNA 19.7 Colorimetry Cu-TpBpy-COF) 7.17 Colorimetry MnBBC 0.3 This work Photothermal effect MnBBC 0.3 This work Smartphone RGB MnBBC 0.24 This work Table 2 Comparison of detection ranges of analytical methods and synthetic materials in ALP biosensing Method Material <![CDATA[Detection range (U L -1 )]]> Remark Fluorometry <![CDATA[CD / SiO 2 > 0.12-15 Fluorometry <![CDATA[NaYF 4 > 0.06-12 Fluorometry UCNPS 0.15-8 Fluorometry Fe-N800CS 0.2-10 Fluorometry PB 0.25-6 Fluorometry <![CDATA[NH 2 -MIL-101(Fe)]]> 0.2-20 Fluorometry <![CDATA[(LaF 3 :Eu / PEI / Ag)]]> 2-16 Colorimetry AuNPs 3-18 Colorimetry Fe / C NS 0.05-6 Colorimetry / Photothermal method / Smartphone RGB MnBBC 1-30 This work The 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.

[0083] The results of the spiked determination (ultraviolet) of ALP in soil by the present invention in the above manner are shown in Table 3, and the results of the spiked 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 spiked determination of ALP in soil are as Figure 16 shown in d.

[0084] Table 3 Spiked determination (ultraviolet) of ALP in soil

[0085] Table 4 Spiked determination (photo-thermal) of ALP in soil, thermal imaging detection

[0086] The above results prove that the method provided by the present invention has accurate and reliable determination results and good reproducibility.

[0087] The above are the specific embodiments 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 within 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 method for detecting alkaline phosphatase activity in soil, characterized in that: The method comprises the following steps: Step 1. preparing soil extract; Step 2. Add L-ascorbic acid-2-phosphate solution and soil extract solution to the centrifuge tube in sequence, incubate at 37°C for 30 min; add Britton-Robinson buffer to terminate the reaction; Step 3. Add 3,3′,5,5′-tetramethylbenzidine solution and MnBBC dispersion to the reaction solution of step 2, and react at 37±0.1°C for 7 min; wherein the MnBBC dispersion is prepared by mixing manganese-rich silicon modified biochar with water; Step 4. Determine the absorbance of the reaction system at 654 nm; observe the chromaticity of the reaction system, and use a smart phone to monitor the RGB value; 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 constructed relationship between absorbance change and ALP concentration, RGB value and ALP concentration, and temperature change and ALP concentration, and verify and correct each other through multimodal output data of chromaticity, absorbance, temperature and RGB value, so as to determine the activity of ALP in the soil, wherein the ALP is alkaline phosphatase.

2. The method for detecting alkaline phosphatase activity in soil according to claim 1, characterized in that: The soil extract in step 1 was prepared as follows: 0.5 g of soil sample was placed in a 50 ml centrifuge tube, 20 ml of Britton-Robinson buffer with a pH of 8.0 was added, vortexed for 5 min, and centrifuged at 5000 rpm for 10 min; the supernatant was filtered through a membrane and diluted for later use.

3. The method for detecting alkaline phosphatase activity in soil according to claim 1, characterized in that: In step 2, add 20 uL of 35 mM L-ascorbic acid-2-phosphate solution; the amount of soil extract added is 80 uL; In step 3, add 100 uL of 17 mM 3,3′,5,5′-tetramethylbenzidine solution and 100 uL of 0.7 mg / mL MnBBC dispersion; During 808 nm near-infrared laser irradiation in step 4, the constant irradiation power was set to 2.0 W / cm², and the continuous irradiation time was 10 min.

4. The method for detecting alkaline phosphatase activity in soil according to claim 1, characterized in that: The manganese-silicon-rich modified biochar is obtained by pyrolyzing a mixture of hawthorn seeds, bentonite and manganese elements.

5. The method for detecting alkaline phosphatase activity in soil according to claim 1, characterized in that: The manganese-rich silicon modified biochar is a porous network structure, including Zn, Si, S, P, O, N, Mn, Mg, K, Fe, Ca, and Al elements, wherein 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.

6. The method for detecting alkaline phosphatase activity in soil according to claim 1, characterized in that: The linear relationship between absorbance change and ALP concentration is: ΔA ALP =0.02524C ALP +0.2624; where ΔA ALP C is the difference between the absorbance when ALP is present and when ALP is not present, that is, the difference between the absorbance of the reaction system when soil extract is added and when no soil extract is added. ALP is the ALP concentration.

7. The method for detecting alkaline phosphatase activity in soil according to claim 1, characterized in that: The RGB value of the reaction system was analyzed using a color recognition application on a smartphone. The linear relationship between the RGB value and the ALP concentration was: B / (R+G+B)=0.4960-0.003073C ALP ; Among them, R represents the red value, G represents the green value, B represents the blue value, C ALP is the ALP concentration.

8. The method for detecting alkaline phosphatase activity in soil according to claim 1, characterized in that: The temperature change of the reaction system under 808 nm near-infrared laser irradiation was monitored by a thermal imager. The linear relationship between ΔT and ALP concentration was: ΔT = 0.3913C ALP +3.019; Alternatively, a high-precision digital temperature measurement system was used to monitor the temperature change of the reaction system under 808 nm near-infrared laser irradiation. At this time, the linear relationship between ΔT and ALP concentration was: ΔT = 0.6166C ALP + 10.27; Among them, ΔT is the difference between T0 and T ALP T0 represents the temperature of the reaction system when ALP does not exist, that is, the temperature of the reaction system when no soil extract is added; T ALP represents the temperature of the reaction system when ALP is present, that is, the temperature of the reaction system when the soil extract is added, C ALP is the ALP concentration.

9. The method for detecting alkaline phosphatase activity in soil according to claim 4, characterized in that: The preparation method of the manganese-rich silicon modified biochar is as follows: crushing hawthorn seeds to obtain hawthorn seed powder; weighing the hawthorn seed powder and bentonite, mixing them in distilled water, and then adding monohydrated manganese sulfate solution to obtain a suspension; Use sodium hydroxide solution to accurately adjust the pH value of the suspension to 10; stir, ultrasonicate, dry, grind the dried sample, and place it in a pyrolysis device for pyrolysis; after the 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.

10. The method for detecting alkaline phosphatase activity in soil according to claim 9, characterized in that: The mass ratio of the hawthorn seed powder to the bentonite is 10:1; during pyrolysis in the pyrolysis device, the temperature is 8°C·min -1 The heating rate was raised to 350 °C and maintained for 2 h, with nitrogen being continuously introduced during the whole process.

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