A method for detecting alkaline phosphatase activity in soil

Abstract

The invention belongs to the technical field of alkaline phosphatase detection and relates to a method for detecting alkaline phosphatase activity in soil. The method comprises the following steps: step 1. preparing a soil extract; step 2. sequentially adding an L-ascorbic acid-2-phosphate solution and a soil extract into a centrifuge tube, and incubating at 37°C for 30 minutes; and adding a Britton-Robinson buffer to terminate the reaction; step 3. adding a 3,3',5,5'-tetramethylbenzidine solution and a MnBBC dispersion to the reaction solution in step 2, and reacting at a constant temperature of 37±0.1°C for 7 minutes; wherein the MnBBC dispersion is prepared by mixing manganese-rich silicon-modified biochar with water; step 4. measuring the absorbance of a reaction system at 654 nm; observing the chromaticity of the reaction system and monitoring RGB values using a smart phone; and monitoring temperature changes of the reaction system under 808 nm near-infrared laser irradiation. The method comprehensively utilizes multi-dimensional output data such as chromaticity, absorbance, temperature, and RGB values to mutually verify and calibrate the detection results, thereby ensuring the accuracy and credibility of the detection results.

Description

Technical Field

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

[0002] Ginseng is an important traditional Chinese medicinal herb with pharmacological effects such as anti-inflammatory, anti-tumor, and immunity-enhancing properties. China is a major producer of ginseng, but continuous cropping is a major constraint on its production. The impact of soil enzyme activity cannot be ignored. In particular, regulating alkaline phosphatase (ALP) activity plays a positive role in resolving this problem. ALP catalyzes the hydrolysis of organic phosphorus to release available phosphorus for plant uptake and utilization, promoting plant growth. Therefore, ALP activity can serve as an indicator of inorganic phosphorus utilization by plants and microorganisms, playing a crucial role in soil nutrient cycling. Furthermore, literature has reported that ALP is a crucial extracellular enzyme affecting continuous cropping of American ginseng and is closely associated with changes in microbial community structure and composition. Therefore, direct and reliable detection of ALP activity is urgently needed, as it holds important and long-term implications for alleviating this problem.

[0003] So far, several methods for detecting ALP have been reported, such as high-performance liquid chromatography, electrochemical method, radioimmunoassay, etc. Among them, most methods are time-consuming, require complex instruments and tedious operations. In recent years, ultraviolet spectrophotometry and photothermal method have become more attractive for detecting ALP due to their advantages of simplicity and rapidity. For example, the substrate L-ascorbic acid-2-phosphate trisodium salt has been widely used as a traditional method for colorimetric or photothermal ALP sensing. However, false positive results caused by 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 subject to some limitations in practical applications.

[0004] Point-of-care testing (POCT), a key area of in vitro diagnostics, has made tremendous progress in modern analytical chemistry and environmental monitoring. Currently, methods for POCT ALP detection include electrochemical methods, immunoassays, fluorescence methods, and microfluidic chip technology. However, most of these methods require complex equipment and are relatively complicated to operate.

[0005] To improve the sensitivity and stability of point-of-care (POCT) tests, carbon-based materials, as a novel nanomaterial, have recently attracted widespread attention. Carbon-based nanomaterials (such as carbon quantum dots and graphene) have become a hot topic in nanozyme research due to their excellent conductivity, large surface area, and abundant surface functional groups. Specifically, by mimicking the activity of natural enzymes, carbon-based nanomaterials can function as nanozymes, catalyzing the redox reaction of TMB and generating a detectable signal. For example, a one-step-prepared vine biochar was modified into graphene-like molybdenum selenide (MoSe2) with oxidase-like activity, which served as a smart nanozyme sensing platform for voltammetric detection of hesperetin (HP) in orange peel. However, most existing nanozymes require capping and stabilization with protective agents such as polyetherimide (PEI), bovine serum albumin (BSA), and cetyltrimethylammonium bromide (CTAB). This may protect their catalytic sites but reduce their oxidase (OXD) activity.

[0006] Metal-modified biochar, as an emerging carbon-based material, offers significant advantages over traditional carbon-based materials in terms of cost, environmental friendliness, and availability. However, studies on the catalytic activity of metal-modified biochar-based enzymes and their application in the detection of alkaline phosphatase have not been reported. Summary of the Invention

[0007] In view of the above technical problems and defects, the object of the present invention is to provide a method for detecting alkaline phosphatase activity in soil. The method adopts self-designed manganese-rich silicon modified biochar (MnBBC) and realizes 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 assisted application. It has the advantages of high accuracy and credibility of detection results.

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

[0009] A method for detecting alkaline phosphatase activity in soil, the method comprising the following steps:

[0010] Step 1. Prepare soil extract;

[0011] Step 2. Add L-ascorbic acid 2-phosphate solution and soil extract to a centrifuge tube in sequence and incubate at 37°C for 30 min. Add Britton-Robinson buffer to terminate the reaction.

[0012] 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 minutes; wherein the MnBBC dispersion is prepared by mixing manganese-rich silicon modified biochar with water;

[0013] Step 4. Measure the absorbance of the reaction system at 654 nm; observe the color 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 constructed relationship between absorbance change and ALP concentration, the relationship between RGB value and ALP concentration, and the relationship between temperature change and ALP concentration. The multimodal output data of color, absorbance, temperature, and RGB value are mutually verified and corrected to determine the activity of ALP in the soil, where the ALP is alkaline phosphatase.

[0014] As a preferred method of the present invention, the soil extract in step 1 is prepared as follows: 0.5 g of soil sample is placed in a 50 ml centrifuge tube, 20 ml of Britton-Robinson buffer with a pH of 8.0 is added, vortexed for 5 minutes, and centrifuged at 5000 rpm for 10 minutes; the supernatant is filtered through a membrane and diluted for later use.

[0015] As a preferred embodiment of the present invention, 20 uL of 35 mM L-ascorbic acid-2-phosphate solution is added in step 2; the amount of soil extract added is 80 uL;

[0016] In step 3, add 100 μL of 17 mM 3,3′,5,5′-tetramethylbenzidine solution and 100 μL of 0.7 mg / mL MnBBC dispersion.

[0017] 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.

[0018] As a preferred embodiment of the present invention, the manganese-silicon-rich modified biochar is obtained by pyrolysis of a mixture of hawthorn seeds, bentonite and manganese element.

[0019] As a preferred embodiment of the present invention, the manganese-rich silicon modified biochar has 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.

[0020] As a preferred embodiment of the present invention, the linear relationship between the absorbance change and the ALP concentration is: ΔA ALP =0.02524C ALP +0.2624; where ΔA ALPC 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.

[0021] As a preferred embodiment of the present invention, the RGB value of the reaction system was analyzed using a color recognition application of 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.

[0022] As a preferred embodiment of the present invention, a thermal imager is 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 is: ΔT = 0.3913C ALP +3.019;

[0023] 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 = 0.6166°C ALP + 10.27;

[0024] Where 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 exists, that is, the temperature of the reaction system when the soil extract is added, C ALP is the ALP concentration.

[0025] As a further preference of the present invention, the preparation method of the manganese-rich silicon modified biochar is: crushing hawthorn seeds to obtain hawthorn seed powder; weighing hawthorn seed powder and bentonite, mixing them in distilled water, and then adding monohydrated manganese sulfate solution to obtain a suspension; using sodium hydroxide solution to accurately adjust the pH value of the suspension to 10; stirring, ultrasonic treatment, drying, grinding the dried sample, and placing it in a pyrolysis device for pyrolysis; after the pyrolysis is completed, the sample is naturally cooled to room temperature, and the obtained sample is repeatedly rinsed with distilled water to remove impurities; and then dried.

[0026] As a further preferred embodiment of the present invention, the mass ratio of the hawthorn seed powder to the bentonite is 10:1; when pyrolyzed in the pyrolysis device, the temperature is set at 8°C·min -1 The heating rate was increased to 350 °C and maintained for 2 h, with nitrogen continuously introduced during the entire process.

[0027] Advantages and beneficial effects of the present invention:

[0028] (1) The MnBBC provided by the present invention eliminates the need for adding protective agents that could affect OXD activity during the synthesis process, thus perfectly avoiding this disadvantage. Furthermore, unlike nanozymes, it does not require the cumbersome steps and instrumentation required for high-speed centrifugation, significantly reducing time and costs. It also avoids the potential for high-speed centrifugation to cause aggregation, which could affect OXD activity.

[0029] (2) The MnBBC provided by the present invention has significant oxidase-like activity. From the perspective of the development history of biochar, the first generation of biochar was mainly used as a soil conditioner, the second generation of biochar was used as an adsorbent, and MnBBC can be regarded as the third generation of 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, OXD-like substances have a wide range of application needs, but traditional OXD-like substances often have problems such as high cost and poor stability. MnBBC has good chemical stability and catalytic activity due to its unique OXD-like activity, and can maintain efficient catalysis in complex environments. It is expected to become a very promising alternative material to oxidase-like substances in industry.

[0030] (3) The MnBBC provided by the present invention is different from 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 costs and operational complexity, but also poses safety risks. MnBBC does not require the participation of H2O2 and has a unique catalytic mechanism to directly oxidize specific organic substrates. In addition, in practical applications, the stability of the catalyst is a crucial performance indicator. MnBBC is highly stable. 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, providing strong support for its application in actual industrial production and environmental governance.

[0031] (4) This invention first detected that MnBBC carbon-based composite materials have oxidase-like activity. Oxidase-like activity is a unique substance that mimics the catalytic function of natural oxidases and has great application potential in many fields such as biosensing and environmental monitoring. MnBBC, with its oxidase-like activity, can specifically interact with AA (L-ascorbic acid) or ALP in the reaction system, enabling detection of them through a specific signal conversion mechanism. This detection method is expected to provide an efficient, sensitive, and convenient approach for the quantitative analysis of AA or ALP.

[0032] (5) In the present invention, bentonite is introduced into the preparation process of MnBBC to increase the surface roughness of the carbon source, thereby increasing the specific surface area and providing more active sites, which is conducive to 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 improving the stability and uniformity of the attachment (through physical adsorption and chemical bonding), and facilitating the effective loading of the metal complex on the carbon source surface.

[0033] (6) The activity of many traditional catalysts is 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 remains stable for a long time, making MnBBC have important application value in cold areas or low-temperature operation scenarios.

[0034] (7) The present invention uses the self-designed MnBBC to detect the activity of alkaline phosphatase (ALP) in ginseng continuous cropping soil, which can effectively avoid interference from other substances and provide a solid guarantee for obtaining accurate and reliable test results.

[0035] (8) The present invention proposes a comprehensive detection method that integrates 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, and blue: B, and the superimposed colors between them), photothermal effects, 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. In addition, the three-modal detection does not require complex equipment and is easy to operate. It can be completed by following a simple operation process without professional knowledge. The user-friendly operation makes this method suitable for popular point-of-care (POCT) applications.

[0036] (9) The three-mode detection method provided by the present invention, which integrates colorimetry, photothermal analysis, and intelligent mobile phone assistance, is economically low-cost (made by pyrolysis of agricultural waste) and suitable for large-scale promotion. It has high-throughput detection characteristics and can simultaneously detect multiple samples, greatly improving detection efficiency. It also has complementary advantages and is portable.

[0037] (10) The method provided by the present invention comprehensively utilizes multi-dimensional output signals such as chromaticity, absorbance, temperature and RGB values, which can verify and correct the test results. When a certain signal is abnormal, it can be cross-checked and analyzed with other signals to timely discover and correct possible errors, thereby ensuring the accuracy and credibility of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Characterization and analysis of manganese-rich silicon modified biochar (MnBBC) Figure 1 ; Wherein, a is the high-resolution scanning electron microscope (HRSEM) image of MnBBC; bn is the element distribution mapping image of MnBBC; o is the energy dispersive X-ray spectroscopy (EDS) spectrum of MnBBC;

[0039] Figure 2 Characterization and 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 diagram of manganese-rich silicon modified biochar;

[0040] Figure 3 Characterization and analysis of manganese-rich silicon modified biochar (MnBBC) Figure 3 ; ac are low-resolution TEM images (500, 100, 5 nm) of manganese-rich silicon modified biochar; d is the electron paramagnetic resonance (EPR) spectrum of manganese-rich silicon modified biochar; e is the EPR spectrum of manganese-rich silicon modified biochar at 500-3500 cm -1 Raman spectroscopy;

[0041] Figure 4 The following is an analysis of the oxidase activity of manganese-rich silicon modified biochar; a is the UV-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 stability observation of MnBBC-TMB; c is the absorbance (λ) of the MnBBC-TMB reaction solution at 4°C and 25°C. 654 nm); d is the absorbance of MnBBC-TMB reaction solution at different pH values (λ 654 nm);

[0042] Figure 5 This is a steady-state kinetic analysis curve to evaluate the catalytic efficiency of the simulated oxidase of manganese-rich silicon modified biochar (MnBBC), with the substrate being 3,3',5,5'-tetramethylbenzidine (TMB);

[0043] Figure 6 is the absorbance of the reaction solution of various biochars prepared from different precursors and TMB; where 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-silicon-rich modified biochar (MnBBC) and TMB (λ 654 nm); b is a photo of the reaction solution when BC, BBC, FeBBC, ZnBBC, and MnBBC reacted with TMB; c is the absorbance (λ) of the reaction system of rice husk manganese-silicon modified biochar MnBBC (DK), sorghum husk manganese-silicon modified biochar MnBBC (GLK), jujube husk manganese-silicon modified biochar MnBBC (SZK), millet husk manganese-silicon modified biochar MnBBC (XMK), and hawthorn seed manganese-silicon modified biochar MnBBC (SZZ) with TMB 654 nm); d is the photo of the reaction solution when DK, GLK, SZK, XMK, and SZZ modified biochar reacted with TMB;

[0044] Figure 7 These are characterization and analysis diagrams of various biochars prepared from different precursors: ae are high-resolution scanning electron microscope images (HRSEM) of BC, BBC, ZnBBC, FeBBC, and MnBBC, showing the microscopic morphology of various biochars; fj are X-ray diffraction (XRD) patterns of BC, BBC, ZnBBC, FeBBC, and MnBBC, analyzing the crystal structures of various biochars;

[0045] Figure 8 The following are the X-ray photoelectron spectroscopy (XPS) spectra of biochar BC, BBC, ZnBBC, FeBBC, and MnBBC prepared from different precursors: ae is C 1s; fj is O 1s; ko is Si 2p; pt is S 2p;

[0046] Figure 9 Partial XPS and FTIR spectra; a is Mn 2p; b is the full XPS spectrum of BC, BBC, ZnBBC, FeBBC, and MnBBC; c is the combined Fourier transform infrared (FTIR) spectrum of BC, BBC, ZnBBC, FeBBC, and MnBBC;

[0047] Figure 10The concentration-dependent inhibitory effect of AA on TMB oxidation catalyzed by MnBBC was monitored by UV-visible absorption spectroscopy and colorimetric imaging; a is the UV-visible absorption spectrum; b is the colorimetric imaging diagram; c is the absorbance change value (ΔA AA ) and ascorbic acid (AA) concentrations to construct a standard curve;

[0048] Figure 11 It is an ALP activity detection based on the MnBBC / AA / ALP cascade signal amplification system; a is the three-dimensional absorption spectrum; b is the colorimetric imaging diagram; c is the absorbance change value (ΔA ALP ) and ALP concentration to construct a standard curve;

[0049] Figure 12 The selectivity of the alkaline phosphatase (ALP) detection system was evaluated by UV spectrophotometry. (a) investigated the interference of structural analogs on the detection system; (b) analyzed the difference in the signal response of the detection system between the ALP-added group and the group without ALP addition. Data are expressed as mean ± SD (n = 3).

[0050] Figure 13 The figure shows the effects of different types of inhibitors on the activity of MnBBC oxidases; a is superoxide dismutase (SOD); b is tryptophan; c is AA; d is catalase; Blank is the control group without inhibitors.

[0051] Figure 14 is the photothermal performance analysis of different systems; where a is 808 nm laser irradiation of MnBBC-TMB-AA 60 uM a) The effect of power increase of 0.5-2.2 W / cm² on the temperature difference of the system; b) The temperature-time curves of the MnBBC-TMB reaction system, TMB solution, and MnBBC dispersion under 808 nm near-infrared laser (2.0 W / cm²); c) The synergistic photothermal effect analysis of the MnBBC-TMB composite system relative to a single component;

[0052] Figure 15 Concentration-dependent photothermal response analysis of ALP and AA; where a is the temperature change of the MnBBC-TMB-AA system under 808 nm near-infrared laser irradiation (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 808 nm near-infrared laser irradiation (2.0 W / cm²); d is the quantitative detection performance of the multi-component synergistic system (quantitative relationship between the photothermal response signal and ALP concentration);

[0053] Figure 16 This is a multimodal detection and analysis of alkaline phosphatase (ALP) activity based on a combination of colorimetry, smartphone, and thermal imaging. (a) Real-time monitoring of the blue color change in the MnBBC-TMB-AA-ALP reaction system by a smartphone, showing the linear relationship between B / (R +G + B) and ALP concentration. (b) Real-time monitoring of the ALP reaction system by a smartphone and thermal imaging camera under 808 nm laser mapping. Within the ALP activity range of 1-30 U / L, ΔT shows a significant linear relationship between ALP activity. (c) Photothermal imaging of spiked soil. (d) RGB color recognition imaging of spiked soil. DETAILED DESCRIPTION

[0054] The present invention will be further described below in conjunction with the accompanying drawings and specific examples, but embodiments of the present invention are not limited thereto. For process parameters not particularly noted, reference can be made to conventional techniques. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0055] This embodiment provides a method for detecting alkaline phosphatase activity in soil, which comprises the following steps:

[0056] Step 1. Prepare soil extract;

[0057] Step 2. Add L-ascorbic acid 2-phosphate solution and soil extract to a centrifuge tube in sequence and incubate at 37°C for 30 min. Add Britton-Robinson buffer to terminate the reaction.

[0058] 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; the manganese-rich silicon-modified biochar is self-developed;

[0059] Step 4. Measure the absorbance of the reaction system at 654 nm; observe the color of the reaction system and monitor the RGB values using a smartphone; monitor the temperature changes of the reaction system under 808 nm near-infrared laser irradiation; calculate the concentration of alkaline phosphatase in the soil based on the constructed equations for the relationship between absorbance change and ALP concentration, the relationship between RGB values and ALP concentration, and the relationship between temperature change and ALP concentration. Determine the activity of alkaline phosphatase in the soil by mutual verification and correction of the multimodal output data of color, absorbance, temperature, and RGB values.

[0060] In order to make those skilled in the art clearly understand how the present invention is implemented, the following is a detailed description through specific experiments:

[0061] 1. Experiment:

[0062] 1.1. Materials:

[0063] Hawthorn seeds, rice husks, rice straw, millet husks, sorghum husks, and 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), and 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), and catalase ( 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 purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China).

[0064] 1.2. Preparation of MnBBC biochar

[0065] Freshly dried hawthorn seeds (abbreviated as SZZ) were crushed using a grinder 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, followed by the addition of 0.2 mol / L manganese sulfate monohydrate (MnSO4·H2O) solution (500 mL). The pH value of the suspension was accurately 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. The mixture was then ultrasonically treated for 30 min, transferred to an oven, dried at 80°C for 48 h, ground, passed through a 60-mesh sieve, and placed in a pyrolysis device at 8°C·min -1 The temperature was raised to 350°C at a constant rate and maintained for 2 hours. Nitrogen was continuously introduced throughout the process to maintain an inert environment and prevent sample oxidation. After pyrolysis was complete, the sample was allowed to cool naturally to room temperature. The resulting sample was rinsed repeatedly with distilled water to remove impurities. After rinsing, the pellets were dried in an oven at 105°C (±5°C) for 24 hours. After drying, the sample was stored in a dry, clean, well-sealed container and labeled for subsequent use in experiments.

[0066] In this example, the sample obtained by pyrolysis of hawthorn seeds alone was named Biochar (BC), the sample obtained by pyrolysis of a mixture of hawthorn seeds and bentonite was named Bentonite / Biochar (BBC), and the sample obtained by pyrolysis of a mixture of hawthorn seeds, bentonite, and manganese was named MnBBC, also known as MnBBC (SZZ).

[0067] Referring to the above preparation method, this example also prepared biochar samples with different metal additions: 0.2 mol / L ZnSO4·7H2O was used according to the same experimental process (1.2) to prepare a ZnBBC sample (this sample was obtained by pyrolyzing a mixture of hawthorn seeds, bentonite, and zinc). FeSO4·7H2O was used according to the same experimental process (1.2) to prepare a FeBBC sample (this sample was obtained by pyrolyzing a mixture of hawthorn seeds, bentonite, and iron).

[0068] 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 husk, rice straw, millet husk, sorghum husk, and jujube husk, respectively. According to the above experimental process (1.2), a series of rice husk manganese-silicon modified biochars (MnBBC) (DK), rice straw manganese-silicon modified biochars (MnBBC) (DC), millet husk manganese-silicon modified biochars (MnBBC) (XMK), sorghum husk manganese-silicon modified biochars (MnBBC) (GLK), and jujube husk manganese-silicon modified biochars (MnBBC) (SZK)) were prepared using rice husk (DK), rice straw (DC), millet husk manganese-silicon modified biochars (MnBBC) (XMK), sorghum husk manganese-silicon modified biochars (MnBBC) (GLK), and jujube husk manganese-silicon modified biochars (MnBBC) (SZK) as raw materials. These biochars were then compared with the hawthorn seed manganese-silicon modified biochars (MnBBC) (SZZ) prepared above to explore the oxidase-like activity of different biochar raw materials.

[0069] In this example, a total of 10 different types of biochar, including composite biochar and metal-modified biochar, were synthesized. The purpose was to comprehensively explore the effects of different preparation methods and raw materials on the performance of biochar, in order to find the biochar with the most active enzyme-like effect.

[0070] It should be noted that in this example, the results of the rice husk manganese-rich silicon modified biochar MnBBC (DK) and the rice straw manganese-rich silicon modified biochar MnBBC (DC) are basically the same, so this example only provides the experimental results of the rice husk manganese-rich silicon modified biochar MnBBC (DK).

[0071] 1.3. Material characterization:

[0072] UV-visible absorption spectra were recorded using a HITACHI U-2900 UV-visible spectrophotometer. Multiscale structural elucidation of the MnBBC, including morphological characterization and elemental analysis, was performed using a Hitachi High-Tech cold field emission scanning electron microscope (SU 8600, equipped with an Oxford Ultim Max 100 mm² EDS detector). Microstructural characterization was performed using a JEM-F200 field emission transmission electron microscope (JEOL Ltd.). Nitrogen adsorption-desorption isotherms were measured using a Quantachrome Autosorb-iQ physical adsorption instrument. The specific surface area was calculated using the BET model, and the distribution of mesopores in the 3.5-50 nm range was characterized using the BJH theoretical model. The structural parameters of micropores (<2 nm) were calculated. Phase structure characterization was performed using a Shimadzu XRD-6100 polycrystal X-ray diffractometer. Surface chemical state characterization was performed using a Thermo Fisher Scientific ESCALAB 250Xi X-ray photoelectron spectrometer. The vibrational modes of the biochar surface functional groups were characterized using a Thermo Fisher Nicolet iS50 Fourier transform infrared spectrometer, which collected data in transmission mode at 4000–400 cm-1 Mid-infrared fingerprint spectroscopy enables in situ surface analysis. Lattice vibrational modes were characterized using a HORIBA LabRAM HR Evolution confocal Raman microscope. Multi-element quantitative analysis of the samples was performed using a Thermo Scientific iCAP PRO 6300 full-spectrum direct-reading inductively coupled plasma optical emission spectrometer. A high-precision digital thermometer (Taizhou Weixing Electric Co., Ltd.), a HIKMICRO thermal imager (Hangzhou Hikvision Micro-Image Sensing Technology Co., Ltd.), an Apple iPhone 13 mobile device (Apple Inc.), and an MW-GX808 multimode fiber-coupled laser (Changchun Rays Optoelectronics Technology Co., Ltd.) were used to build a photothermal-smartphone collaborative analysis platform for in situ monitoring of multi-physics fields.

[0073] 1.4. Evaluation of oxidase-like activity of MnBBC biochar:

[0074] The oxidase-like activity of MnBBC was evaluated by catalyzing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB). 100 μL TMB solution (17 mM) and 100 μL MnBBC dispersion (0.7 mg mL -1 ), 300 μL of Britton-Robinson buffer (pH 4.0) were added sequentially to a 1.5 mL polypropylene (PP) centrifuge tube and allowed to react at room temperature for 7 minutes. After the reaction, the reaction solution turned blue. The absorbance of the resulting blue solution was measured using a UV-visible spectrophotometer at a wavelength of 654 nm. The measured absorbance data was used for subsequent analysis and evaluation of MnBBC-type oxidase activity.

[0075] To further investigate the effect of pH on the activity of MnBBC-type oxidases, the present invention combined a MnBBC dispersion (0.7 mg / mL, 100 μL), a 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 incubated at room temperature for 7 minutes. After completion, the absorbance of each reaction system at a characteristic wavelength of 654 nm was immediately measured and recorded using a UV-visible spectrophotometer.

[0076] In order to further explore the effect of temperature on the activity of MnBBC-type oxidase, the present invention was carried out in a Britton-Robinson buffer (pH 4.0) system (1.4). The specific operation was as follows: the reaction system was placed under constant temperature conditions of 4°C and 25°C for reaction, and after the reaction reached a stable state, the reaction solution was quickly measured at a characteristic wavelength of 654 nm using a UV-visible spectrophotometer and the absorbance values at 4°C and 25°C were recorded.

[0077] 1.5. Steady-state kinetic analysis of MnBBC biochar oxidases:

[0078] To investigate the kinetic characteristics of OXD catalysis by MnBBC biochar, the present invention used TMB as a substrate to monitor the oxidase-like kinetic curves of MnBBC samples. Specifically, 100 μL of TMB solutions at varying concentrations (0-25 U / L) were added sequentially to a 1.5 mL polypropylene centrifuge tube to establish a reaction system under varying substrate concentrations. Subsequently, 100 μL of a 0.7 mg / mL MnBBC dispersion and 300 μL of Britton-Robinson buffer (pH 4.0) were added to initiate the reaction. The reaction was conducted at room temperature for 10 minutes, during which the system was monitored in real time using a UV-visible spectrophotometer. The reaction solution was measured every 1 minute. Using TMB as a substrate, UV-visible spectrophotometry (λ = 654 nm) was used to determine and calculate the steady-state kinetic constants of the MnBBC oxidase-like catalytic system. Based on the initial reaction rate data (n = 7 independent replicates), a linear fit was performed using the Lineweaver-Burk double reciprocal equation:

[0079] ;

[0080] 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).

[0081] By performing nonlinear regression fitting on the experimental data and substituting it into the above kinetic equation, V max and K m The steady-state kinetic constants can provide an important theoretical basis for a deeper understanding of the action mechanism and catalytic performance of MnBBC in oxidase-like catalytic reactions.

[0082] 1.6. Study on the combined colorimetric and photothermal detection of AA based on the MnBBC-TMB system:

[0083] The dual-mode sensing analysis of ascorbic acid (AA) based on the MnBBC-TMB system was carried out under optimized experimental conditions. A reaction mixture with a total volume of 500 μL was accurately prepared. The specific operation was 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 solution with different concentration gradients (0-90 μM) were added to a 1.5 mL polypropylene centrifuge tube in sequence. Vortex mix evenly, and the reaction time was precisely controlled to 7 min. After the reaction, 200 ul of the reaction solution was transferred to a 10 mm quartz cuvette, and the absorbance at 654 nm was recorded using a UV-visible spectrophotometer. Establish ΔA and C AA The dose-absorbance response curve of the combined photothermal detection was 808 nm laser (power density 2 W / cm 2 ) irradiated the reaction system and monitored the temperature field in real time using a high-precision digital thermometer. By recording the temperature-concentration curve, the intrinsic relationship between AA concentration and the temperature changes caused by the photothermal effect in the reaction system was analyzed, providing important evidence for in-depth research on the reaction mechanism and the development of new detection methods.

[0084] 1.7. Colorimetric Method - 808 Laser Photothermal Synergy Strategy for Highly Sensitive Detection of Alkaline Phosphatase (ALP):

[0085] The ALP activity assay process is as follows: 20 μL of a 35 mM p-aminophenyl phosphate (AAP) solution and 80 μL of an ALP solution of varying concentrations (0-30 U / L) are precisely pipetted into a reaction tube and mixed. The mixture is then placed in a 37°C water bath and incubated for 30 minutes. AAP is fully reacted under the catalysis of ALP. Upon completion of the incubation, 200 μL of a pH 4.0 Britton-Robinson buffer solution is immediately added to terminate the reaction. 100 μL of a 17 mM TMB solution and 100 μL of a 0.7 mg / mL MnBBC dispersion are then added to the reaction tube, followed by vortex mixing to form a total reaction solution of 0.5 mL. The total reaction solution is allowed to react for 7 minutes until the color develops and stabilizes. An appropriate amount of the solution is then transferred to a quartz cuvette using a micropipette and the absorbance is measured at 654 nm using a UV-visible spectrophotometer. Simultaneously, an 808 nm near-infrared laser (2 W / cm 2) irradiation reaction system, use a high-precision digital thermometer to monitor the temperature change of the reaction system in real time during laser irradiation, and analyze the absorbance and temperature change data generated by ALP concentration changes to comprehensively analyze and evaluate ALP activity.

[0086] 1.8. ALP Colorimetric and Photothermal Multi-Mode Sensing Detection in Smartphone Mode:

[0087] This study utilizes the smartphone application Color Name Recognizer Camera to assess the color constant of the colorimetric reaction and combines it with a thermal imager to monitor temperature changes resulting from the photothermal effect. This method establishes a photothermal-smartphone-based multimodal sensing method for ALP detection, aiming to accurately and conveniently detect ALP activity. The procedure is the same as in the previous experiment (1.7) until the color development stabilizes. The reaction solution is instantly captured and color-recognized (RGB) using the smartphone application "Color Name Recognizer Camera." This existing application uses advanced image processing algorithms to accurately identify and quantify color information. To verify the accuracy of the results, absorbance is measured at a wavelength of 654 nm using a UV-visible spectrophotometer.

[0088] In order to further improve the accuracy and stability of temperature rise signal reading, the present invention designs a portable sensing mode based on a smartphone combined with an infrared thermal imager, that is, the temperature rise signal is measured by an 808 nm laser (2 W / cm 2 During irradiation, a thermal imager was used to monitor the temperature change (ΔT) of the reaction system in real time, and a correlation between ΔT values and ALP activity was established. Statistical methods were used to analyze the ΔT and ALP activity data, and a correlation model was established. This model facilitates quantitative analysis of ALP activity by measuring temperature changes.

[0089] 1.9. Study on the specific recognition of ALP by the MnBBC-like oxidase dual-mode sensing platform and the influence of interfering factors in complex matrices:

[0090] The basis for the selection of interfering factors is: structural analogues: ginsenoside Rb1 / Rg1, total ginsenosides; functional analogues: acid phosphatase, sucrase; common matrix components: benzoic acid.

[0091] ALP-free system (blank control): Add 100 μL of interfering factor solution (acid phosphatase, sucrase, ginsenoside Rb1, ginsenoside Rg1, total ginsenosides, benzoic acid, all at their respective critical concentrations) plus the standard reaction system (same as in 1.6) to a 1.5 mL polypropylene centrifuge tube, vortex to mix, and react at room temperature for 7 min. Transfer 200 μL of the reaction solution to a quartz cuvette and record the absorbance at 654 nm using a UV-visible spectrophotometer (average of three measurements).

[0092] 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. Incubate in a 37°C water bath for 30 min. Add the standard reaction system (same as in 1.6), vortex to mix, and prepare a total reaction volume of 0.5 mL. Incubate 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 in the presence of each interfering factor (A / A0, where A0 is the absorbance in the absence of the interfering factor) to assess system selectivity.

[0093] 1.10. Analysis of the Catalytic Mechanism of MnBBC Oxidase: Reactive Oxygen Species Based on Free Radical Capture:

[0094] MnBBC is a novel catalyst synthesized from agricultural waste. Its oxidase-like (OXD-like) activity 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 elucidate the specific mechanism of its catalytic TMB oxidation, this experiment employed a free radical capture strategy, using specific inhibitors to target and scavenge different ROS, systematically analyzing the key active species and their contributions in the MnBBC catalytic process. The specific experiment is as follows: To the standard reaction system (same as 1.6), free radical scavengers were added: catalase (catalase) - specifically scavenges hydrogen peroxide (H2O2), ascorbic acid (AA) - scavenges hydroxyl radicals (·OH), superoxide dismutase (SOD) - scavenges superoxide anion radicals (O 2 -), tryptophan - scavenges singlet oxygen ( 1 O2), the reaction was carried out at room temperature for 7 min, and then 200 μL of the reaction solution was transferred to a quartz cuvette. The absorbance at 654 nm was recorded using a spectrophotometer (three measurements were averaged) to calculate the relative activity.

[0095] 1.11. Detection of ALP in real soil samples using a MnBBC-type oxidase system:

[0096] Spike recovery testing is of great scientific significance in soil alkaline phosphatase (ALP) activity determination. Its core purpose is to evaluate assay accuracy, matrix interference, and method reliability by spiking known concentrations of the target substance (i.e., ALP enzyme). Because soil is a complex, heterogeneous system composed of minerals, organic matter, microorganisms, and metabolites, its physicochemical properties (such as pH, ionic strength, and humic acid content) and coexisting substances (such as heavy metals, enzyme inhibitors, and adsorbent colloids) can significantly interfere with ALP activity determination. For example, inhibition / activation effects: Humic acid can bind to the enzyme through hydrophobic interactions, altering its conformation; heavy metal ions (such as Cu²⁺ and Cd²⁺) can competitively bind to the enzyme's active site. Adsorption loss: The negative surface charge of soil clay minerals (such as montmorillonite and kaolinite) can adsorb the positively charged ALP enzyme, leading to an underestimation of the free enzyme concentration. Optical interference: Pigments or suspended particles in soil extracts can interfere with the signal measured by spectrophotometry or fluorescence. Spike-in-situ analysis, by adding exogenous ALP standards to soil samples, can quantify the combined impact of these interfering factors on test results. If the recovery deviates from the theoretical value (e.g., <90% or >110%), it indicates that the extraction buffer needs to be optimized (e.g., by adding the chelating agent EDTA to desorb metal ions) or the test conditions need to be improved (e.g., by centrifugation to remove turbidity).

[0097] Parallel experimental design for spiked detection: Soil samples were divided into an unspiked group (to measure background ALP activity) and a spiked group (to add known amounts of ALP enzyme 5 / 10 / 15 U / L). The recovery rate was calculated by comparing the activity difference between the two groups.

[0098] This study validated the detection performance of MnBBC-type oxidases for ALP in soil using spiked soil samples. Five typical soil samples were selected: a soil sample with no ginseng cultivation history (CK), a soil sample from a five-year continuous ginseng crop, and soil samples from the rhizosphere of ginseng plants from seven, 15, and 30 years of cultivation (5Y, 7Y, 15Y, and 30Y). The samples were air-dried. 0.5 g of each soil sample was placed in a 50 ml centrifuge tube. 20 ml of Britton-Robinson buffer (pH 8.0) was added, vortexed for 5 minutes, and centrifuged at 5000 rpm for 10 minutes. The supernatant was filtered through a membrane and serially diluted 100-fold for later use.

[0099] The concentration settings were unspiked (0 U / L), low spiked (5 U / L), medium spiked (10 U / L), and high spiked (15 U / L). The reaction system was as follows: 20 μL of AAP solution (35 mM) and 80 μL of ALP standard solutions (5 U / L, 10 U / L, and 15 U / L, pH 8.0) were added to a centrifuge tube and incubated at 37°C for 30 min. The reaction was immediately terminated by adding 200 μL of Britton-Robinson (pH 4.0). The reaction solution was then added with 100 μL of TMB solution (17 mM) and 100 μL of MnBBC dispersion (0.7 mg / mL). The reaction was incubated at 37 ± 0.1°C for 7 min, and the absorbance at 654 nm was immediately measured (the average of three measurements was taken). Ginseng soil sample testing: Replace the ALP standard solution with 80 μL of soil extract to control the total volume to 0.5 mL. Follow the same steps as above. Calculate the ALP activity based on the standard curve and express the results as ALP activity per unit mass of soil (U / L).

[0100] The recovery rate was calculated as follows: recovery rate = (measured value - control group) / added value × 100%.

[0101] 2. Results and Discussion

[0102] 2.1. Characterization of MnBBC Biochar

[0103] Figure 1 and 2 This is a characterization analysis diagram 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). Combined nitrogen adsorption-desorption isotherm ( Figure 2 a) and BJH pore size distribution ( Figure 2 b), indicating that the material exhibits a pore system dominated by mesopores (average pore diameter of 9.781 Å), with a specific surface area ( S BET ) is 35.128 m 2 / g, pore volume 0.085 m 2 / g, and its type IV(a) isotherm and H3 hysteresis loop further confirmed that the mesoporous voids were consistent with the HRSEM morphology, which was beneficial to the diffusion of reactants and the exposure of active sites, thereby enhancing the catalytic performance.

[0104] Elemental Mapping and Energy Dispersive X-ray Spectroscopy (EDS) were used to analyze the elemental composition of MnBBC. Figure 1b-1o). Results show that Mn, with a mass fraction of 46.14 wt.%, is uniformly distributed on the surface of the material, primarily in the Mn(II) / Mn(III) oxidation states (as confirmed by XPS analysis). This provides abundant active centers for its oxidase-like activity. Furthermore, trace metal elements (Fe: 0.19 wt.%, Zn: 0.83 wt.%) and non-metallic elements (S: 8.03 wt.%) were detected in the material, potentially further regulating 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.%).

[0105] In the present invention, ICP-OES (inductively coupled plasma optical emission spectrometer) analysis results show that the Si element content in MnBBC is 13580 ppm, the Mn element content is 186670 ppm, and the S element content is 7880 ppm.

[0106] Figure 3 This is a characterization analysis diagram 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), this microstructure is highly consistent with the three-dimensional interconnected void structure observed by HRSEM. Further high-resolution HRTEM was used to analyze MnBBC in depth, and clear lattice fringes were observed ( Figure 3 c). After precise measurement, the lattice spacings were 0.288 nm, 0.284 nm, and 0.335 nm, respectively. By comparing with JCPDS, they corresponded 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 MnBBC forms a multiphase composite structure during the modification process. In order 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, the MnBBC system showed a clear characteristic response of hydroxyl radical (•OH), while this signal was not observed in the blank control group. This comparison strongly confirmed that the generation of •OH originated from the oxidase-like activity of MnBBC. Raman spectroscopy ( Figure 3 e) Provide information for studying the carbon matrix and chemical bond state of MnBBC. The results show that the carbon skeleton of MnBBC has a typical sp 2 Hybrid characteristics, including D peak (1331cm -1 , A1g symmetry) reflects the defects or edge structures in the carbon structure, and the G peak (1575cm -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 and reorganization of the carbon matrix. The increase in the degree of graphitization has a significant impact on the performance of the material, the most direct manifestation of which is the improvement of the material's electronic conductivity. In addition, in the Raman spectrum, the 2210 cm -1 The Mn-O vibration peak at 1924 cm -1 The Si-O stretching vibration peaks at and respectively confirm the loading of the manganese oxide phase and the retention of the SiO2 skeleton derived from the bentonite template. The two synergistically optimize the dispersion of the active sites and the stability of the structure of the material, further providing strong support for the high efficiency performance of MnBBC in catalytic reactions.

[0107] 2.2. Oxidase-like activity of MnBBC:

[0108] The present invention comprehensively evaluates the oxidase-like activity performance of MnBBC. Figure 4 This is an analysis of the oxidase activity of manganese-silicon modified biochar. First, in the comparative experiment, when MnBBC and TMB exist alone, the solution does not change color. When MnBBC and TMB are mixed, the solution turns blue. By UV-visible absorption spectroscopy analysis ( Figure 4 a) A strong absorption peak was observed at 654 nm. This phenomenon indicates that TMB was oxidized to oxTMB, thus proving that MnBBC possesses OXD activity.

[0109] Further study on the effect of temperature and pH on its activity showed that ( Figure 4 c) MnBBC maintains high catalytic activity at both 4°C and 25°C, confirming its low-temperature adaptability advantage. The catalytic activity of MnBBC shows a significant pH dependence ( Figure 4d): The activity was highest at pH 4 (ΔA = 1.02), 90% activity was retained at pH 3 (ΔA = 0.959), and activity gradually decreased with increasing pH in the pH range of 5-8, consistent with the proton-coupled electron transfer mechanism of typical Mn-based nanozymes. Notably, the MnBBC maintained ≥60% activity in the pH range of 3-5 (vs. pH 4), demonstrating its good tolerance to acidic environments (such as ginseng continuous cropping soil), surpassing most metal-organic framework (MOF) nanozymes (which typically experience activity decay >50%).

[0110] Long-term storage stability: The catalytic activity of MnBBC did not significantly decrease after storage at room temperature for 50 days (ΔA=0.98±0.008, Figure 4 b) Outperforming easily inactivated enzymes (e.g., HRP activity decreases by >30% after 7 days of storage). Combined with its wide pH response (3-5), low-temperature tolerance (4-25°C), and long-term stability, MnBBC demonstrates outstanding potential for environmental sensing and point-of-care testing (POCT).

[0111] 2.3. Steady-state kinetic analysis of MnBBC biochar oxidases:

[0112] The present invention studied the enzymatic reaction characteristics of TMB oxidation catalyzed by MnBBC using the Michaelis-Menten kinetic model. Figure 5 As shown in the figure, with the increase of substrate concentration (0-25 mM), the initial reaction velocity (V) shows a typical hyperbolic saturation trend, which is consistent with the Michaelis-Menten kinetic law. The key parameters obtained by nonlinear fitting are: the maximum reaction rate V max = (0.9263±0.023)×10 -8 M·s -1 , Michaelis constant K m = 0.5260±0.15 mmol / L. To further verify the kinetic model, linear regression analysis was performed using the Lineweaver-Burk double reciprocal plot (1 / V vs 1 / [S]). max With K m The values were consistent with the nonlinear fitting results (relative error < 5%).

[0113] K m The smaller the size, the stronger the affinity, and the lower the K m The values indicate that MnBBC has a high affinity for TMB (K m = 0.5260mmol / L), which is better than most biomimetic oxidase materials (such as Fe3O4 nanozyme K m ≈8-12 mM). V max The value is 0.9263×10 -8 M·s -1The catalytic efficiency is comparable to that of natural enzymes (e.g., horseradish peroxidase HRP catalyzes the conversion of TMB to V max ≈1.2×10 -8 M·s -1 The above parameters indicate that MnBBC can achieve a catalytic turnover rate comparable to that of biological enzymes while maintaining high substrate binding capacity.

[0114] 2.4. Oxidase-like activity of biochar prepared from different precursors:

[0115] The present invention studies the oxidase-like activity (catalytic activity) of various biochars prepared from different precursors. Figure 6 As shown in Figure 2, by comparing the TMB oxidation ability (λ=654 nm) of different metal-modified biochars, it was found that only manganese-rich silicon-modified biochar (MnBBC) showed significant advantages. Figure 6 As shown in a, the absorbance of the MnBBC system (A=1.086±0.12) 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 verified that only the MnBBC-TMB system showed a characteristic dark blue color ( Figure 6 b), indicating its ability to efficiently catalyze the oxidation of TMB to generate oxTMB. In addition, by comparing the synthesis of manganese-rich silicon-modified biochars using different precursors, it was found that the MnBBC type OXD synthesized using hawthorn seeds as precursors was the highest, with significant differences ( P <0.01).

[0116] 2.5. Comparative analysis of biochar prepared from different precursors:

[0117] The present invention characterizes and analyzes the biochar prepared from different precursors. Figure 7 The biochar materials prepared from different precursors were characterized by high-resolution scanning electron microscopy (HRSEM), including hawthorn seed biochar (BC), hawthorn seed mixed with bentonite (BBC), iron-rich modified biochar (FeBBC), zinc-rich modified biochar (ZnBBC), and manganese-silicon-rich modified biochar (MnBBC). Among them, BC and BBC showed dense block morphology, smooth surface and low porosity ( Figure 7 a-7b), the introduction of bentonite on the surface did not significantly change its macroscopic morphology. In the single metal modified samples, ZnBBC ( Figure 7 c) FeBBC Figure 7 d) presents a granular structure, while the MnBBC particles before pyrolysis are evenly dispersed, and the MnBBC after pyrolysis ( Figure 7e) It presents an interlaced porous network structure, indicating that pyrolysis treatment has a particularly significant effect on the morphology of MnBBC, providing an ideal carrier for substrate transport and active site exposure.

[0118] Combined with X-ray diffraction (XRD) analysis ( Figure 7 f-7j) spectrum, BC is at 20°-30° (2 θ ), indicating that it is mainly composed of amorphous carbon with low crystallinity, resulting in limited active site density ( Figure 7 f). BBC adds the (012) and (104) crystal diffraction peaks of calcium carbonate (CaCO3) and the (003) and (110) peaks of bentonite. However, the inherent catalytic activity of the newly added crystalline phase is low, which together with the compactness of the SEM morphology leads to limited catalytic performance and fails to significantly improve the overall performance ( Figure 7 g). XRD pattern of MnBBC ( Figure 7 j) shows the characteristic peaks of (101), (112), and (211) crystal planes belonging to Mn3O4, confirming its spinel structure. Scherrer formula calculation shows that the Mn3O4 grain size is 15.3 nm, and its high-index crystal planes, such as (112), account for 42%, which is beneficial to the active sites (Mn 3+ -O-Mn 2+ ) is exposed, which can effectively adsorb substrates and carry out catalytic reactions. Compared with the (104) and (113) peaks of α-Fe2O3 in FeBBC and the (100) and (002) peaks of ZnO in ZnBBC, which correspond to corundum and wurtzite structures respectively, their grain sizes are larger (Fe2O3: 28 nm; ZnO: 35 nm), and the surface active crystal planes such as Fe2O3 (001) and ZnO (101) account for less than 20%, resulting in a significantly lower active site density than that of MnBBC ( Figure 7 h-7i).

[0119] Figure 8 、 Figure 9 The XPS spectra of biochar 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)、S 2p( Figure 8p-8t), Mn 2p (9a); from the X-ray photoelectron spectroscopy (XPS) full spectrum analysis of BC, BBC, ZnBBC, FeBBC, and MnBBC (9b), the C 1s spectra of BC and BBC showed C–C (284.5 eV), C–O–C (286.3 eV), and O—C=O (288.8 eV) functional groups. The C–O (532.5 eV) and C=O (530.8 eV) peaks in the O 1s spectrum indicated the presence of hydroxyl groups, ether bonds, and carboxyl groups on their surfaces. The O—C=O content in BBC increased (from 8% in BC to 15%), but the lack of metal active sites resulted in no significant improvement in catalytic activity. The 102.5 eV peak (SiO2) in the Si 2p spectrum and the weak S2p signal (<0.5 at.%) confirmed that the introduction of bentonite did not introduce sulfur impurities.

[0120] Metal-loaded sample: The C 1s and O 1s spectra of MnBBC retain the carbon and oxygen functional groups of BC, with the addition of Mn–O bonds (529.8 eV). The Mn 2p3 / 2 peak shows that Mn 2+ (641.2 eV) and Mn 3+ (643.5 eV), indicating the formation of multivalent Mn3O4. FeBBC and ZnBBC detected Fe–O (530.2 eV) and Zn–O (530.5 eV) bonds corresponding to α-Fe2O3 and ZnO, respectively, but its single oxidation state (Fe 3+ 、Zn 2+ ) and high oxygen vacancy formation energy (Fe2O3: 2.1 eV; ZnO: 2.5 eV) limit the generation rate of active oxygen species (•O2⁻, •OH), resulting in significantly lower catalytic activity than MnBBC. Mn 3+ / Mn 2+ Multivalent state cycle (TOF = 3.2 s -1 ) is significantly better than Fe 3+ / Fe 2+ (TOF = 0.8 s -1 ) and Zn 2+ a single oxidation state.

[0121] The Fourier transform infrared spectrum (FTIR) of the above corresponding biochar is as follows Figure 9 As shown in c, the FTIR combined image analysis shows that all samples have a peak at 3445 cm -1 (OH), 1625 cm -1 (C=O), 1122 cm -1 The characteristic peaks at (CO) indicate that its surface is rich in hydroxyl, carbonyl, and ether bonds, but lacks metal oxide active sites, which limits its catalytic performance, such as BC. BBC retains the characteristic peaks of the carbon skeleton of BC, with a new peak at 1025 cm-1 (Si–O antisymmetric stretching), 876 cm -1 、617 cm -1 (Si-O symmetrical stretching), confirming the introduction of bentonite. The ν3 vibration peak of calcium carbonate (CaCO3) (1430 cm -1 ) did not significantly change the surface catalytic activity, indicating that the mechanical stability was improved but the active sites were not increased. FeBBC at 561 cm -1 The Fe–O characteristic vibration peak (A1g mode of α-Fe2O3) appears at the 3+ It exists in the form of iron oxide. Its limited oxidase activity (TOF = 0.8 s -1 ) is attributed to Fe 3+ / Fe 2+ Insufficient single electron transfer capability (Hubbard U =4.5 eV). 474 cm -1 The Zn–O vibration peak at (E2 mode of ZnO) matches the XPS Zn 2p3 / 2 (1021.5 eV), indicating that Zn 2+ In the form of zinc oxide. Although ZnO has photocatalytic potential, its wide band gap (3.3 eV) limits the carrier concentration (n=10 15 cm −3 ), resulting in weak TMB oxidation activity (TOF = 0.6 s -1 MnBBC 621 cm -1 (Mn–O stretching vibration) and 531 cm -1 The characteristic peak of (Mn–O–Mn bending vibration) is consistent with the vibration mode of spinel Mn3O4. Hydroxyl (3423 cm -1 ) and carbonyl (1595 cm -1 ) enhances the substrate adsorption capacity, combined with Mn 3+ / Mn 2+ Multivalent state cycling (TOF = 3.2 s -1 ), significantly improving the TMB oxidation performance.

[0122] 2.6. Ascorbic acid (AA) detection based on MnBBC-TMB system:

[0123] The present invention uses UV-visible absorption spectroscopy and colorimetric imaging to detect the dependence of AA on the inhibition of MnBBC-catalyzed TMB oxidation at different concentrations. The results are as follows Figure 10As shown. In this system, oxidized TMB (oxTMB) is a water-soluble blue chemical whose color change can serve as a reliable indicator for detecting certain reducing agents (such as AA). Based on this characteristic, it is expected that a sensor for detecting AA can be developed by detecting the color change caused by the oxTMB / TMB ratio affected by the AA concentration. Figure 10 As shown in a, the AA concentration increases from 0 to 90 μM (0, 15, 30, 45, 60, 75, 90 μM, from top to bottom), showing a gradual attenuation trend of the characteristic absorption peak at 654 nm. At the same time, the colorimetric imaging shows that the blue color intensity also decreases synchronously ( Figure 10 b). The underlying mechanism of this phenomenon is that MnBBC catalyzes the oxidation of TMB to produce oxTMB, while the addition of AA, acting as a reducing agent, reduces the resulting oxTMB. Visual observation revealed that the blue color of the solution gradually faded with increasing AA concentration, consistent with the colorimetric imaging results. In the UV-visible absorption spectrum, the absorbance at 654 nm gradually decreased as the AA concentration increased from 0 to 90 μM. This result is consistent with the color change trend observed by colorimetric imaging, further confirming the concentration-dependent inhibitory effect of AA on the MnBBC-catalyzed TMB oxidation reaction.

[0124] The present invention is based on the absorbance change value (ΔA AA ) and ascorbic acid (AA) concentration C AA A standard curve was constructed and the results were as follows Figure 10 c; where ΔA AA is the absorbance value in the presence of ascorbic acid (A AA ) and the absorbance value (A0) when ascorbic acid is not present, that is, (ΔA AA = A0−A AA ). After linear regression analysis, the linear regression equation is ΔA AA =0.006546C AA −0.04322 (R 2 =0.9937), which showed that in the range of AA concentration from 15 to 90 μM, ΔA AA With AA concentration C AA There is a significant linear relationship.

[0125] 2.7. ALP activity detection based on the MnBBC / AA / ALP cascade signal amplification system (AAP-ALP-MnBBC-TMB):

[0126] The present invention demonstrates ALP activity detection based on the MnBBC / AA / ALP cascade signal amplification system. Figure 11 a is 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) decayed regularly, which was consistent with 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 decreases with increasing ALP activity. ALP concentration ≥30 U·L -1 When , the absorbance signal is completely quenched, defining the upper detection limit of the system.

[0127] The present invention also shows the absorbance change value (ΔA ALP ) and ALP concentration C ALP The standard curve between Figure 11 c; where ΔA ALP is the absorbance in the presence of ALP (A ALP ) and the absorbance value (A0) when ALP is not present, (ΔA ALP =A0−A ALP At 654 nm, the absorbance change (ΔA ALP ) showed 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 was 1-30 U / L, and the limit of detection (LOD) was 0.3 U / L, demonstrating its highly sensitive quantitative detection of ALP activity. Therefore, the cascade signal amplification system constructed in this invention based on MnBBC-type oxidases, ascorbic acid (AA), and alkaline phosphatase (ALP) achieves highly sensitive detection of ALP activity.

[0128] 2.8. Specific recognition of alkaline phosphatase (ALP) by MnBBC oxidases:

[0129] The present invention evaluates the selectivity of the alkaline phosphatase (ALP) detection system by ultraviolet spectrophotometry, and investigates the interference effects of structural analogs (ginsenoside Rb1, Rg1, and total ginsenosides), functional analogs (acid phosphatase, sucrase), and a common matrix component (benzoic acid) on the detection system (the critical concentrations are ginsenoside Rb1, Rg1, total ginsenoside TG, acid phosphatase ACP, sucrase, and benzoic acid BA); Figure 12 a is the result of interference with the analysis, the test concentrations of ginsenoside Rb1 (1.7 mM), Rg1 (0.51 mM), total ginsenosides (5 mg / ml), acid phosphatase (100 U / L), sucrase (105 U / L), benzoic acid (1 mM) at 654 nm, the results show that the system has high tolerance to structural / functional analogs and matrix interference. In addition, the present invention analyzed the difference in the signal response of the detection system between the ALP addition group and the blank control group. The results are as follows Figure 12 As shown in b, in the comparison of the signals between the ALP-added group (30 U / L) and the blank control group (mean ± SD, n = 3), 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 distractor group ( p >0.05). This is likely due to the specific catalysis of TMB oxidation by Mn3O4 on the MnBBC surface, the strict substrate selectivity of the AAP→AA pathway mediated by ALP for phosphatases, and the hydrophobic structure of ginsenosides and organic acids that restricts their π-π interactions with TMB / AA. These results demonstrate that the developed colorimetric biosensor exhibits high selectivity and satisfactory specificity for ALP.

[0130] 2.9. Catalytic Mechanism of MnBBC Oxidase

[0131] The present invention studies the reaction mechanism of the MnBBC catalytic system. Figure 13 shows the effects of different inhibitors on the MnBBC catalytic system, revealing the specificity of the reaction mechanism. The inhibitor types used are: (a) superoxide dismutase (SOD), (b) tryptophan (Trptophan), (d) ascorbic acid (AA), and (e) catalase (Catalase). The experimental results show that after treatment with catalase, SOD, or Trptophan, the oxidation level of TMB remains relatively stable. However, when AA is added, the blue color becomes lighter. AA, as a strong reducing agent, directly scavenges ·OH (·OH + AA → oxidized AA + H2O), blocking the TMB oxidation pathway and reducing the formation of oxTMB. SOD, Trptophan, and Catalase had no significant effect. SOD (scavenges O2· - ): indicates O2· - It is not a key intermediate; Trptophan (scavenges ¹O2): excludes the participation of singlet oxygen; Catalase (decomposes H2O2): confirms that H2O2 is a non-essential intermediate. OH is the only active species directly involved in TMB oxidation. The catalytic activity of MnBBC is derived from the manganese species on its surface: the multivalent manganese (Mn 2+ → Mn 3+) can activate oxygen (O2) through single electron transfer to produce reactive oxygen species ROS. Therefore, the catalytic oxidation mechanism of manganese-silicon modified biochar may be that MnBBC directly generates ·OH by activating O2, which acts as the main oxidant to attack TMB to generate oxTMB, and AA inhibits the reaction by scavenging ·OH. Other ROS (O2· - , ¹O₂, and H₂O₂) do not participate in the dominant pathway. This mechanism differs from traditional nanozymes in that it does not require H₂O₂, thus avoiding their limitations. Furthermore, the inert carbonyl matrix of biochar reduces nonspecific adsorption, improving detection selectivity and enhancing interference resistance. This provides new insights into the design of H₂O₂-independent nanozyme sensors and also reveals the synergistic catalytic effect of the biochar-metal manganese-silicon composite.

[0132] 2.10. Study on the photothermal performance of MnBBC-type oxidases during catalysis:

[0133] In the present invention, the oxidation of TMB (ox-TMB) not only produces a significant color development reaction, but also exhibits a strong photothermal effect in the near-infrared range. Figure 14 The power dependence and synergistic effect of photothermal performance are demonstrated. This unique photothermal conversion characteristic can be used to establish a direct / indirect proportional relationship between temperature and target analyte concentration, which is called "photothermal detection". Therefore, under 808 nm near-infrared laser irradiation, MnBBC-TMB-AA 60 uM The composite reaction system was subjected to a systematic power gradient experiment (0.5-2.2 W / cm²). The experimental data showed that when the irradiation power was 2 W / cm² for 10 min, the system temperature difference ΔT reached the maximum value (ΔT max ), so it was determined as the optimized condition for subsequent experiments ( Figure 14 a). Because excessively high power density and prolonged exposure may cause photobleaching during the experiment, thus affecting the detection results, under 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 a good temperature-time linear relationship ( Figure 14 b), which shows that the system has stable photothermal response characteristics. In addition, the present invention establishes a synergistic effect analysis model ( Figure 14 c) It was found that the improvement of the photothermal performance of the composite system MnBBC-TMB comes from the synergistic effect of multiple components; Figure 14 c, the synergistic temperature difference ΔT is the temperature of the composite system (T MnBBC - TMB ) and T0 (ΔT=T MnBBC - TMB −T0) and TMB monomer solution temperature (T TMB ) and T0 (ΔT=TTMB −T0), 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 induces efficient near-infrared photothermal conversion properties, which provides a direct method for constructing colorimetric and photothermal dual-mode detection.

[0134] The present invention found through experiments that the concentrations of alkaline phosphatase (ALP) and ascorbic acid (AA) were dependent on the photothermal response. Figure 15 As shown, Figure 15 a shows the temperature changes 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 show that as the AA concentration gradient increases (0-90 μM), the equilibrium temperature of the system shows a concentration-dependent downward 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 (T0-T AA ), T AA and T0 are the system temperatures when AA exists and does not exist, respectively. The experimental results show that ΔT is related to the AA concentration C AA It showed a good linear relationship in the range of 15-90 μM, and the linear regression equation was: 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 photothermal curve was recorded and obtained in real time using a high-precision digital temperature measurement system. The experimental results show that as 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, and the temperature difference ΔT is (T0-T ALP ), T ALP and T0 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 ALP concentration C ALP It shows a good linear relationship, and the linear equation is ΔT = 0.6166C ALP + 10.27, R² = 0.9939, and the detection limit (LOD) of this detection system is 0.3 U / L. Therefore, within a specific range, the MnBBC system can achieve rapid real-time detection of ALP through photothermal detection.

[0135] 2.11. Multi-mode sensing detection:

[0136] This invention is based on a multimodal detection of alkaline phosphatase (ALP) activity using a colorimetric, smartphone, and thermal imaging camera. To enable 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 a selected specific area into RGB values. Specifically, a color recognition application on the smartphone is used to analyze the red (R), green (G), and blue (B) values, known as RGB values. The results show that within a 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, such as Figure 16 As shown in a.

[0137] In addition, the present invention used a thermal imager to monitor the ALP reaction system in real time under 808 nm laser mapping. The results are shown in Figure 16b. In the ALP activity range of 1-30 U / L, ΔT and ALP activity showed a significant linear relationship, and the linear equation was ΔT = 0.3913C ALP +3.019, R² = 0.9951; the detection limit of this detection system is 0.29 U / L. This shows that the sensing method of the present invention has the ability to output multimodal signals such as colorimetry, absorbance, temperature, and RGB values, which can effectively avoid the errors that may be caused by a single detection method and significantly improve the reliability of ALP activity detection results.

[0138] In order to demonstrate the advantages of the ALP detection method provided by the present invention, it is compared with other reported colorimetric and fluorescence methods for ALP detection, as follows:

[0139] Table 1 Comparison of detection limits of analytical methods and synthetic materials in ALP biosensors

[0140] method Material <![CDATA[Detection limit (U L -1 )]]> Remark blood glucose meter 8.9 Fluorescence assay TPEPy-pY 6.6 Fluorescence assay rGQDs / CS 7.8 Fluorescence assay 3D DNA 50 Fluorescence assay <![CDATA[CsPbBr3@PMMA]]> 4.85 Colorimetry AgNPrs 5 Colorimetry <![CDATA[TiO2]]> 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

[0141] Table 2 Comparison of detection ranges of analytical methods and synthetic materials in ALP biosensors

[0142] method Material <![CDATA[Detection range (U L -1 )]]> Remark Fluorescence assay <![CDATA[CD / SiO2]]> 0.12-15 Fluorescence assay <![CDATA[NaYF4]]> 0.06-12 Fluorescence assay UCNPS 0.15-8 Fluorescence assay Fe-N800CS 0.2-10 Fluorescence assay PB 0.25-6 Fluorescence assay <![CDATA[NH2-MIL-101(Fe)]]> 0.2-20 Fluorescence assay <![CDATA[(LaF3: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

[0143] The results show 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 be adapted to the detection of soil samples with a wider range of concentrations.

[0144] The results of the spiked determination (ultraviolet) of ALP in soil by the present invention are shown in Table 3, the results of the spiked determination (photothermal) of ALP in soil by the above method are shown in Table 4, and the photothermal imaging is shown in Table 5. Figure 16 c shows the RGB results of the spiked determination of ALP in soil. Figure 16 As shown in d.

[0145] Table 3. Determination of ALP in soil by spiked ionization (UV)

[0146]

[0147] Table 4 ALP in soil by spiked determination (photothermal) and thermal imaging

[0148]

[0149] The above results prove that the determination results of the method provided by the present invention are accurate, reliable and have good reproducibility.

[0150] The above are specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection 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. Prepare soil extract; Step 2. Add L-ascorbic acid 2-phosphate solution and soil extract to a centrifuge tube in sequence and 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 minutes; wherein the MnBBC dispersion is prepared by mixing manganese-rich silicon modified biochar with water; The manganese-rich silicon modified biochar is obtained by pyrolysis of a mixture of hawthorn seeds, bentonite and manganese; the manganese-rich silicon modified biochar has a porous network structure and includes 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; Step 4. Determine the absorbance of the reaction system at 654 nm; observe the colorimetry of the reaction system and monitor the RGB values 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 constructed equations for the relationship between absorbance change and ALP concentration, the relationship between RGB values and ALP concentration, and the relationship between temperature change and ALP concentration, and determine the activity of ALP in the soil by mutual verification and correction of the multimodal output data of colorimetry, absorbance, temperature, and RGB values, wherein the ALP is alkaline phosphatase.

2. The method for detecting alkaline phosphatase activity in soil according to claim 1, wherein: 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 (pH 8.0) was added, the tube was vortexed for 5 minutes, and centrifuged at 5000 rpm for 10 minutes; 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, wherein: In step 2, add 20 μL of 35 mM L-ascorbic acid 2-phosphate solution; the amount of soil extract added is 80 μL; In step 3, add 100 μL of 17 mM 3,3′,5,5′-tetramethylbenzidine solution and 100 μL of 0.7 mg / mL MnBBC dispersion; During the 808 nm near-infrared laser irradiation in step 4, the constant irradiation power was set to 2.0 W / cm 2 , the continuous irradiation time is 10min.

4. The method for detecting alkaline phosphatase activity in soil according to claim 1, wherein: 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.

5. The method for detecting alkaline phosphatase activity in soil according to claim 1, wherein: The RGB values of the reaction system were analyzed using a color recognition application on a smartphone. The linear relationship between the RGB values 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.

6. The method for detecting alkaline phosphatase activity in soil according to claim 1, wherein: The temperature change of the reaction system under 808nm near-infrared laser irradiation was monitored using a thermal imager. The linear relationship between ΔT and ALP concentration was: ΔT = 0.3913°C. ALP +3.019; Alternatively, a high-precision digital temperature measurement system was used to monitor the temperature change of the reaction system under 808nm near-infrared laser irradiation. At this time, the linear relationship between ΔT and ALP concentration was: ΔT = 0.6166°C ALP +10.27; Where 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 exists, that is, the temperature of the reaction system when the soil extract is added, C ALP is the ALP concentration.

7. The method for detecting alkaline phosphatase activity in soil according to claim 1, wherein: The preparation method of the manganese-rich silicon modified biochar comprises the following steps: crushing hawthorn seeds to obtain hawthorn seed powder; weighing the hawthorn seed powder and bentonite, mixing them in distilled water, and then adding a monohydrated manganese sulfate solution to obtain a suspension; The pH value of the suspension is precisely adjusted to 10 using sodium hydroxide solution; the suspension is stirred, ultrasonically treated, dried, and the dried sample is ground and placed in a pyrolysis device for pyrolysis; after the pyrolysis is completed, the sample is naturally cooled to room temperature, and the obtained sample is repeatedly rinsed with distilled water to remove impurities; and then dried after rinsing.

8. The method for detecting alkaline phosphatase activity in soil according to claim 7, wherein: The mass ratio of the hawthorn seed powder to the bentonite is 10:1; when pyrolyzed 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 continuously introduced during the entire process.

Images