Red soil dry land soil microbial diversity detection method based on multicolor developing test paper

Abstract

The invention discloses a red soil dry land soil microbial diversity detection method based on multicolor developing test paper, and belongs to the technical field of soil environment microbial detection. According to the method, test paper loaded with different color developing agents is designed, a specific color developing reaction is triggered by combining microbial metabolic activity, and color developing characteristics are converted into parameters such as microbial diversity indexes and dominant species proportions by utilizing an image analysis model. The detection device comprises an image acquisition module and a data analysis module, and quantitative association of color development features and diversity is realized through a machine learning model. Compared with the prior art, the method has the advantages that the cost is extremely low, the detection time is shortened to be within 30 minutes, the method is suitable for field real-time monitoring of the red soil dry land, an efficient, low-cost and portable detection tool is provided for soil health management, and the method is particularly suitable for soil health assessment of resource-limited areas.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of soil environmental microorganism detection technology, and particularly relates to a red soil dry land soil microorganism diversity detection method based on a multi-color developing test paper. BACKGROUND

[0002] Red soil dry land microorganism diversity detection has been facing many challenges. Traditional detection methods, such as high-throughput sequencing, PCR amplification and biochemical analysis, can provide relatively accurate microorganism diversity information, but these methods have obvious limitations. On the one hand, the detection equipment is expensive, the operation process is complex, and professional technical personnel are needed to operate and maintain, which makes it difficult to popularize these methods in resource-poor areas or field environments. On the other hand, the laboratory analysis period is relatively long, usually 3-7 days are needed to obtain the results, which cannot meet the real-time monitoring demand. In addition, the threshold of data interpretation is high, and ordinary users are difficult to understand and apply the detection results, which also limits the popularization of microorganism diversity detection in practical applications.

[0003] At the same time, although the existing test paper method is simple to operate and low in cost, it is mainly aimed at a single index (such as pH value, nitrate nitrogen content, etc.), and cannot comprehensively reflect the soil microorganism diversity. More importantly, the existing test paper method lacks a direct correlation model with the richness, evenness and other parameters of the microbial community, and cannot provide quantitative evaluation of the microorganism diversity. With the increasing demand for soil health assessment, especially in resource-limited areas such as red soil dry land, there is an urgent need for a low-cost, portable, rapid and comprehensive microorganism diversity detection method. Such a method should be able to be quickly implemented in the field or resource-poor areas, and provide intuitive and easy-to-understand detection results.

[0004] In the prior art, the water resistance and accelerated aging performance of the multi-color developing test paper have obvious shortcomings. These test papers are prone to color change, blistering, softening and even embrittlement in high humidity environments, resulting in poor fixing effect of the developing agent, which cannot effectively resist water erosion, thereby affecting the accuracy and reliability of the detection results. In addition, under accelerated aging test conditions such as high temperature and light, the developing agent stability of the existing test paper is poor, and the absorbance changes greatly, indicating that it is prone to degradation or deterioration during long-term storage and use, limiting the service life and application range of the test paper. These problems make it difficult for the existing test paper to meet the requirements of stability and durability in practical applications, especially in the field or resource-limited environments, and an improved method is needed to overcome these shortcomings.

[0005] Chinese patent publication No. CN110412239A discloses a gel test paper for rapid detection of microbial CO2 respiration rate and its use method. The test paper is mainly composed of NaOH alkali, acid-base indicator, glycerol and agar, and is divided into high range (1-50mg CO2 respiration rate) and low range (0.1-5mg CO2 respiration rate) two test ranges. By comparison with five color indicators, the respiration intensity and microbial activity level of compost or soil can be quickly judged. Its advantages are high sensitivity, good accuracy, rapid detection (completed within 30 minutes), and no need for expensive instruments. However, the detection index of this invention is single, only for CO2 respiration rate, which cannot comprehensively reflect the soil microbial diversity, and also lacks direct correlation model with microbial community richness, evenness and other parameters, making it difficult to meet the demand for comprehensive detection of microbial diversity in soil health assessment. SUMMARY

[0006] The present application provides a low-cost, portable and rapid (detection time only needs 10-30 minutes) method for detecting microbial diversity in red soil dry land, which overcomes the limitations of single detection index and lack of direct correlation model with microbial community diversity in the prior art, and meets the demand for comprehensive detection of microbial diversity in soil health assessment.

[0007] In order to achieve the above-mentioned application purposes, the present application adopts the following technical solutions: A multi-color developing test paper based on at least three color developing agents responding to microbial metabolic products, extracellular polymers and respiratory activity.

[0008] The color developing agents include bromocresol purple, trypan blue and resazurin.

[0009] The preparation method of the multi-color developing test paper is as follows: Step 1, cut cellulose filter paper or nanofiber membrane into strip-shaped test paper; Step 2, Zone 1 (detecting organic acid): the strip-shaped test paper is immersed in 0.5-2 g / L bromocresol purple solution, the bromocresol purple solution is prepared by using an ethanol-water co-solvent system with a volume ratio of 2-4:6-8, containing 0.05wt%-0.2wt% polyvinyl alcohol as a dispersant, and the solution pH is adjusted to 7.0±0.2; Zone 2 (detecting cell membrane): the strip-shaped test paper is further immersed in 0.1-0.5 g / L trypan blue solution, the trypan blue solution is prepared by using a phosphate buffer solution with pH=7.0-8.0, and 0.05wt%-0.1wt% Tween-20 is added as a surfactant; Zone 3 (indicating microbial respiratory activity): finally, the strip-shaped test paper is immersed in 1-3 g / L resazurin solution to uniformly wet the third independent reaction zone of the test paper, the resazurin solution is prepared by using a phosphate buffer solution containing 0.5wt%-2wt% glucose, and it is prepared on demand and stored in the dark; Step 3, the soaked test paper is dried to fix the chromogenic agent on the test paper, and the dried test paper is sealed and stored in the dark.

[0010] Preferably, the preparation method of the multi-color developing test paper is as follows: Step 1, cutting cellulose filter paper or nanofiber membrane into strip-shaped test paper; Step 2, Zone 1 (detecting organic acid): the strip-shaped test paper is immersed in 0.5-2 g / L bromocresol purple solution, the bromocresol purple solution is prepared by using an ethanol-water co-solvent system with a volume ratio of 2-4:6-8, containing 0.05wt%-0.2wt% polyvinyl alcohol as a dispersant, and the solution pH is adjusted to 7.0±0.2; Zone 2 (detecting cell membrane): the strip-shaped test paper is further immersed in 0.1-0.5 g / L trypan blue solution, the trypan blue solution is prepared by using a phosphate buffer solution with pH=7.0-8.0, and 0.05wt%-0.1wt% Tween-20 is added as a surfactant; Zone 3 (indicating microbial respiratory activity): finally, the strip-shaped test paper is immersed in 1-3 g / L resazurin solution to uniformly wet the third independent reaction zone of the test paper, the resazurin solution is prepared by using a phosphate buffer solution containing 0.5wt%-2wt% glucose, and it is prepared on demand and stored in the dark; Step 3, the soaked test paper is dried to fix the chromogenic agent on the test paper, and the dried test paper is sealed and stored in the dark.

[0011] Further preferably, the preparation method of the multi-color developing test paper is as follows: Step 1, cutting cellulose filter paper or nanofiber membrane into strip-shaped test paper; Step 2, Section 1 (Detection of Organic Acids): Immerse the strip test paper in 0.5-2 g / L bromocresol purple solution. The bromocresol purple solution uses an ethanol-water co-solvent system with a volume ratio of 2-4:6-8, containing 0.05wt%-0.2wt% polyvinyl alcohol as a dispersant, 0.3wt%-0.5wt% glutaraldehyde, and 0.2wt%-4wt% carrier material. Adjust the pH of the solution to 7.0±0.2. Section 2 (Detection of Cell Membranes): Immerse the strip test paper in 0.1-0.5 g / L trypan blue solution. The trypan blue solution is prepared using phosphate buffer solution with a pH of 7.0-8.0, and 0.05wt%-0.1wt% is added. Tween-20 as a surfactant, 0.3wt%-0.5wt% glutaraldehyde, and 0.2wt%-4wt% carrier material; Zone 3 (indicating microbial respiratory activity): Finally, immerse the strip test paper in a 1-3 g / L resazurin solution. The resazurin solution is prepared using a phosphate buffer containing 0.5wt%-2wt% glucose, and also contains 0.3wt%-0.5wt% glutaraldehyde and 0.2wt%-4wt% carrier material. Prepare fresh before use and store in the dark. Step 3: Dry the coated test paper to fix the color developer on the test paper. After drying, seal and store it away from light.

[0012] The carrier material is at least one of mesoporous silica, mesoporous alumina, and mesoporous silica-alumina composite oxide materials.

[0013] The preparation method of the mesoporous silicon-aluminum composite oxide material is as follows, in parts by weight: Mix 4-6 parts of tetraisopropyl orthosilicate, 0.4-0.8 parts of aluminum nitrate, 50-70 parts of ethanol, 0.5-2 parts of dodecylamine, and 5-10 parts of water evenly. Add 0.5-2 mol / L sodium hydroxide aqueous solution to adjust the pH to 8-9. Heat to 40-50℃ and stir for 1-5 hours. Then centrifuge, wash and dry, and calcine at 500-600℃ for 1-3 hours to obtain mesoporous silica-alumina composite oxide material.

[0014] Preferably, the method for detecting microbial diversity in red soil dryland based on multicolor colorimetric test paper is as follows: S1. Take a red soil sample, add buffer solution and shake to extract microbial metabolic active components; S2. Add the extract to each reaction zone of the test strip and let it stand for a period of time to allow the color reaction to occur; capture the color development results of the test strip using the image acquisition module; analyze the color development image using the machine learning model in the data analysis module and output the microbial diversity index and diversity level.

[0015] The buffer solution in S1 is phosphate buffer; the shaking time is 2-5 minutes; the standing time in S2 for allowing the colorimetric reaction to occur is 10-30 minutes.

[0016] The microbial diversity indices include the Shannon index and the Inv_Simpson index.

[0017] The method for detecting microbial diversity in red soil dryland based on multicolor colorimetric test paper includes an image acquisition module and a data analysis module; The image acquisition module is used to acquire the colorimetric image of the test strip; the image acquisition module includes an integrated mobile phone camera; The data analysis module maps color features to microbial biodiversity indices using a machine learning model.

[0018] The machine learning model extracts multi-channel color features from the color image using a convolutional neural network and then regresses to predict the Shannon index and the Inv_Simpson index.

[0019] The data analysis module also includes a preset equation for converting the gray values ​​of the red, green and blue channels of the color image into a microbial diversity index. The preset equation is y = -0.0026x + 1.0393, where y is the microbial diversity index and x is the gray value of the red, green and blue channels.

[0020] This invention employs a multi-dimensional colorimetric design, by setting multiple independent reaction zones on the test strip, each loaded with a colorimetric agent that responds to microbial metabolites, extracellular polymeric substances (EPS), and respiratory activity. This design can comprehensively reflect the metabolic characteristics and functional diversity of the microbial community, indirectly characterizing the structural and functional diversity of the microbial community, thus overcoming the limitations of traditional test strip methods that rely on single-index detection.

[0021] This invention also boasts significant advantages in terms of low cost and portability. The test strip is extremely inexpensive to prepare, and the detection device is compatible with smartphones, utilizing the phone's camera for image acquisition and a built-in intelligent analysis module for data analysis. This design makes the detection device lightweight and portable, suitable for rapid field testing without the need for complex laboratory equipment or professional personnel, greatly improving the convenience and accessibility of testing.

[0022] This invention achieves semi-quantitative output of microbial diversity indices through a machine learning model, overcoming the limitations of qualitative detection in traditional test strip methods. During the detection process, an image acquisition module captures the colorimetric image of the test strip, and a machine learning model (such as a convolutional neural network) extracts the multi-channel color features of the colorimetric image. These colorimetric features are then mapped to microbial diversity indices (such as the Shannon index and the Inv_Simpson index) using a pre-defined equation, achieving a semi-quantitative assessment of microbial diversity. The output results are intuitive, allowing users to quickly interpret the results without requiring specialized knowledge.

[0023] The detection method of this invention can complete the detection in a short time, meeting the needs of real-time monitoring. From sample collection to result output, the entire detection process takes only 10-30 minutes, significantly shortening the detection cycle compared to traditional methods. The detection results are displayed in real time via a smartphone app, allowing users to quickly understand the soil microbial diversity and take appropriate measures based on the results.

[0024] This invention not only enables rapid and low-cost detection of soil microbial diversity but also provides a scientific basis for soil health management. Its comprehensive assessment capabilities fully reflect the metabolic activity, community structure, and functional diversity of soil microorganisms, making it suitable for soil health assessment in resource-limited areas such as red soil and dryland, and providing an efficient and convenient detection tool for agricultural production, environmental protection, and ecological restoration.

[0025] The roles of each substance in the further improvements of this invention are as follows: As a crosslinking agent, glutaraldehyde can enhance the adhesion and stability of colorimetric reagents on test paper, effectively resist moisture erosion and degradation under extreme environmental conditions, and significantly improve the water resistance and accelerated aging performance of test paper.

[0026] Mesoporous silica has a high specific surface area and good adsorption properties, which can form a tighter bond with the color developer and cellulose filter paper, thereby enhancing the fixation effect of the color developer on the test paper. At the same time, it effectively blocks the penetration of water, reduces the dissolution and color change of the color developer, and improves the water resistance and stability of the test paper.

[0027] Similar to mesoporous silica, mesoporous alumina also has a high specific surface area and good adsorption properties, which can further enhance the fixation effect of colorimetric agents and improve the chemical stability of test strips under extreme conditions such as high temperature and light. This results in lower absorbance changes in accelerated aging tests and extends the service life of the test strips.

[0028] Mesoporous silica-alumina composite oxide materials, formed through specific preparation processes, not only provide a high specific surface area and good dispersibility similar to nanomaterials, enhancing the binding force between the color developer and cellulose filter paper, but also protect the color developer, effectively resisting the erosion of the color developer by moisture and extreme environmental conditions (such as high temperature and light), thereby significantly improving the stability and preservation performance of the test paper, making it more reliable and durable in practical applications.

[0029] Compared with existing technologies, it has the following advantages: 1) The test strip of this invention has low cost, the device is compatible with smartphones, and it is convenient for field testing. It does not require professional equipment and personnel to operate, which significantly reduces the testing threshold and improves the accessibility of testing.

[0030] 2) The detection time of this invention is only 10-30 minutes, and the results are output in real time through a smartphone APP, which can quickly assess the diversity of soil microorganisms, meet the needs of real-time monitoring, and provide timely guidance for soil management measures.

[0031] 3) This invention uses multi-color colorimetric test strips to comprehensively reflect microbial metabolites, extracellular polymers and respiratory activity. Combined with machine learning models, it achieves semi-quantitative evaluation, breaking through the limitations of traditional test strips in qualitative detection and providing more comprehensive diversity analysis.

[0032] 4) By introducing glutaraldehyde, nanomaterials (such as mesoporous silica and mesoporous alumina) and mesoporous silica-alumina composite oxide materials, this invention significantly improves the water resistance and accelerated aging performance of multicolor colorimetric test paper. Attached Figure Description

[0033] To establish the correlation between the grayscale values ​​of the red, green, and blue channels and the Shannon and Inv-Simpson indices, this invention first conducted a detailed microbial diversity analysis on 35 red soil dryland samples. The Shannon and Inv-Simpson indices of these samples were determined using conventional high-throughput sequencing methods to obtain accurate diversity data. Subsequently, the same group of samples was tested using the multicolor chromogenic test strips of this invention. The colorimetric images of the test strips were acquired through an image acquisition module, and the grayscale values ​​of the red, green, and blue channels were extracted. The grayscale calculation method for the red, green, and blue channels adopted a weighted average method, based on the differences in human eye sensitivity to color, using the formula: Grayscale value = 0.299 × R + 0.587 × G + 0.114 × B, where R, G, and B represent the grayscale value of each channel (red / R, green / G, blue / B), respectively. Statistical analysis was conducted to compare the gray values ​​of the red, green, and blue channels with the Shannon index and Simpson index. The results showed that the gray values ​​of the three channels exhibited significant correlations with both the Shannon and Simpson indices. This correlation indicates that the gray values ​​of the red, green, and blue channels obtained through the colorimetric reaction of the test strips can serve as an effective indicator of microbial diversity, thus providing a new method for rapidly assessing soil microbial diversity.

[0034] To further quantify the relationship between the gray values ​​of the red, green, and blue channels and microbial diversity, a comprehensive analysis of the Shannon index and Simpson index was conducted. Specifically, the microbial diversity index = (single Shannon index / maximum Shannon index + single Simpson index / maximum Simpson index) / 2. Through linear regression analysis, a mathematical model was established between the gray values ​​of the red, green, and blue channels and the microbial diversity index, yielding the equation y = −0.0026x + 1.0393, where y represents the microbial diversity index and x represents the gray values ​​of the red, green, and blue channels. This equation allows for the rapid and accurate estimation of soil microbial diversity through simple measurement of the red, green, and blue channel gray values, thus achieving a quantitative conversion from test strip color development to microbial diversity assessment, providing a rapid and low-cost technical means for soil health assessment.

[0035] Figure 1 The correlation between the gray values ​​of the red, green and blue channels of the test strip in Example 1 and the Shannon index.

[0036] Figure 2 The correlation between the gray values ​​of the red, green and blue channels of the test strip in Example 1 and the Simpson index.

[0037] Figure 3 The correlation between the microbial diversity index and the grayscale values ​​of the three colorimetric channels (red, green, and blue) was tested using the test strip in Example 1. Detailed Implementation

[0038] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Main source of materials: The cellulose filter paper used is slow-speed filter paper for quantitative analysis, with a pore size of 11 μm and a cellulose purity of ≥98%, and is prepared by cutting commercially available chromatography-grade filter paper.

[0040] The phosphate buffer solutions used in the examples were prepared by mixing potassium dihydrogen phosphate (KH2PO4) and disodium hydrogen phosphate (Na2HPO4) in a 1:4 molar ratio, with an ionic strength of 0.01M, and dissolved in ultrapure water.

[0041] Mesoporous silica: Particle size: 5-6 μm, pore size: 5-20 nm, specific surface area 500 m² / g. 2 / g.

[0042] Mesoporous aluminum oxide: particle size ≤ 8 μm, pore volume ≥ 0.8 mL / g, specific surface area ≥ 250 m² 2 / g.

[0043] All other raw materials used in the embodiments and comparative examples of this invention are commercially available products.

[0044] The design concept of this invention is to develop a detection method based on multicolor chromogenic test strips, combined with intelligent analysis technology, to achieve rapid, low-cost, and portable detection of microbial diversity in red soil dryland areas. Specifically, the reaction zone of the multicolor chromogenic test strip is loaded with a chromogenic agent that responds to microbial metabolites, cell membranes, and respiratory activities, indirectly reflecting the structural and functional diversity of the microbial community through a colorimetric reaction. By combining an image acquisition module and a data analysis module, a machine learning model is used to map the features of the chromogenic image to a microbial diversity index, achieving semi-quantitative assessment. This overcomes the limitations of traditional detection methods and meets the soil health assessment needs of resource-constrained areas.

[0045] Example 1 A method for preparing multicolor colorimetric test paper is as follows: Step 1: Use scissors to cut the cellulose filter paper into strips of test paper with a size of 5mm × 20mm; Step 2, Section 1 (Detection of organic acids): Immerse the strip test paper in 1 g / L bromocresol purple solution until evenly moistened. The bromocresol purple solution uses an ethanol-water co-solvent system with a volume ratio of 3:7, containing 0.1 wt% polyvinyl alcohol as a dispersant. Adjust the pH of the solution to 7.0 ± 0.2. Section 2 (Detection of cell membranes): Immerse the strip test paper in 0.3 g / L trypan blue solution until evenly moistened. The trypan blue solution is prepared using phosphate buffer solution with 0.08 wt% Tween-20 added as a surfactant. Section 3 (Indication of microbial respiratory activity): Finally, immerse the strip test paper in 2 g / L resazurin solution until evenly moistened. The resazurin solution is prepared using phosphate buffer solution containing 1 wt% glucose, prepared fresh and stored away from light. Step 3: Vacuum dry the test paper coated with the color developer to ensure that the color developer is completely fixed on the test paper. After drying, seal the test paper in a light-proof container.

[0046] Example 2 A method for preparing multicolor colorimetric test paper is as follows: Step 1: Use scissors to cut the cellulose filter paper into strips of test paper with a size of 5mm × 20mm; Step 2, Section 1 (Detection of organic acids): Immerse the strip test paper in 1 g / L bromocresol purple solution, ensuring even wetting. The bromocresol purple solution uses an ethanol-water co-solvent system with a volume ratio of 3:7, and also contains 0.1 wt% polyvinyl alcohol as a dispersant and 0.4 wt% glutaraldehyde. Adjust the pH of the solution to 7.0 ± 0.2. Section 2 (Detection of cell membranes): Immerse the strip test paper in 0.3 g / L trypan blue solution, ensuring even wetting. The trypan blue solution is prepared using phosphate buffer, with 0.08 wt% Tween-20 added as a surfactant and 0.4 wt% glutaraldehyde. Section 3 (Indication of microbial respiratory activity): Finally, immerse the strip test paper in 2 g / L resazurin solution, ensuring even wetting. The resazurin solution is prepared using phosphate buffer containing 1 wt% glucose and 0.4 wt% glutaraldehyde. Prepare fresh before use and store protected from light. Step 3: Vacuum dry the test paper coated with the color developer to ensure that the color developer is completely fixed on the test paper. After drying, seal the test paper in a light-proof container.

[0047] Example 3 A method for preparing multicolor colorimetric test paper is as follows: Step 1: Use scissors to cut the cellulose filter paper into strips of test paper with a size of 5mm × 20mm; Step 2, Section 1 (Detection of organic acids): Immerse the strip-shaped test paper in a 1 g / L bromocresol purple solution, ensuring even wetting. The bromocresol purple solution uses an ethanol-water co-solvent system with a volume ratio of 3:7, and also contains 0.1 wt% polyvinyl alcohol as a dispersant, 0.4 wt% glutaraldehyde, and 0.3 wt% mesoporous silica. Adjust the pH of the solution to 7.0 ± 0.2. Section 2 (Detection of cell membranes): Immerse the strip-shaped test paper again in a 0.3 g / L trypan blue solution, ensuring even wetting. Trypan blue... The solution was prepared using phosphate buffer, with 0.08 wt% Tween-20 added as a surfactant, 0.4 wt% glutaraldehyde, and 0.3 wt% mesoporous silica added; Zone 3 (indicating microbial respiratory activity): Finally, the strip test paper was immersed in 2 g / L resazurin solution and evenly moistened. The resazurin solution was prepared using phosphate buffer containing 1 wt% glucose, and also contained 0.4 wt% glutaraldehyde and 0.3 wt% mesoporous silica. It was prepared fresh for use and stored in the dark. Step 3: Vacuum dry the test paper coated with the color developer to ensure that the color developer is completely fixed on the test paper. After drying, seal the test paper in a light-proof container.

[0048] Example 4 A method for preparing multicolor colorimetric test paper is as follows: Step 1: Use scissors to cut the cellulose filter paper into strips of test paper with a size of 5mm × 20mm; Step 2, Section 1 (Detection of organic acids): Immerse the strip-shaped test paper in a 1 g / L bromocresol purple solution, ensuring even wetting. The bromocresol purple solution uses an ethanol-water co-solvent system with a volume ratio of 3:7, and also contains 0.1 wt% polyvinyl alcohol as a dispersant, 0.4 wt% glutaraldehyde, and 0.3 wt% mesoporous alumina. Adjust the pH of the solution to 7.0 ± 0.2. Section 2 (Detection of cell membranes): Immerse the strip-shaped test paper again in a 0.3 g / L trypan blue solution, ensuring even wetting. The trypan blue solution... The solution was prepared using phosphate buffer, with 0.08 wt% Tween-20 added as a surfactant, 0.4 wt% glutaraldehyde, and 0.3 wt% mesoporous alumina added; Zone 3 (indicating microbial respiratory activity): Finally, the strip test paper was immersed in 2 g / L resazurin solution and evenly moistened. The resazurin solution was prepared using phosphate buffer containing 1 wt% glucose, and also contained 0.4 wt% glutaraldehyde and 0.3 wt% mesoporous alumina. It was prepared fresh and stored away from light. Step 3: Vacuum dry the test paper coated with the color developer to ensure that the color developer is completely fixed on the test paper. After drying, seal the test paper in a light-proof container.

[0049] Example 5 A method for preparing multicolor colorimetric test paper is as follows: Step 1: Use scissors to cut the cellulose filter paper into strips of test paper with a size of 5mm × 20mm; Step 2, Section 1 (Detection of organic acids): Immerse the strip-shaped test paper in a 1 g / L bromocresol purple solution, ensuring even wetting. The bromocresol purple solution uses an ethanol-water co-solvent system with a volume ratio of 3:7, and also contains 0.1 wt% polyvinyl alcohol as a dispersant, 0.4 wt% glutaraldehyde, 0.15 wt% mesoporous silica, and 0.15 wt% mesoporous alumina. Adjust the pH of the solution to 7.0 ± 0.2. Section 2 (Detection of cell membranes): Immerse the strip-shaped test paper again in a 0.3 g / L trypan blue solution, ensuring even wetting. The trypan blue solution is prepared using phosphate buffer with 0.08 wt% added. Tween-20 as a surfactant, 0.4 wt% glutaraldehyde, 0.15 wt% mesoporous silica, and 0.15 wt% mesoporous alumina; Zone 3 (indicating microbial respiratory activity): Finally, immerse the strip-shaped test paper in a 2 g / L resazurin solution, ensuring even wetting. The resazurin solution is prepared using a phosphate buffer solution containing 1 wt% glucose, and also contains 0.4 wt% glutaraldehyde, 0.15 wt% mesoporous silica, and 0.15 wt% mesoporous alumina. Prepare fresh before use and store protected from light. Step 3: Vacuum dry the test paper coated with the color developer to ensure that the color developer is completely fixed on the test paper. After drying, seal the test paper in a light-proof container.

[0050] Example 6 A method for preparing multicolor colorimetric test paper is as follows: Step 1: Use scissors to cut the cellulose filter paper into strips of test paper with a size of 5mm × 20mm; Step 2, Section 1 (Detection of organic acids): Immerse the strip-shaped test paper in a 1 g / L bromocresol purple solution, ensuring even wetting. The bromocresol purple solution uses an ethanol-water co-solvent system with a volume ratio of 3:7, and also contains 0.1 wt% polyvinyl alcohol as a dispersant, 0.4 wt% glutaraldehyde, and 0.3 wt% mesoporous silica-alumina composite oxide material. Adjust the pH of the solution to 7.0 ± 0.2. Section 2 (Detection of cell membranes): Immerse the strip-shaped test paper again in a 0.3 g / L trypan blue solution, ensuring even wetting. The trypan blue solution is prepared using phosphate buffer with 0.08 wt% added. Tween-20 as a surfactant, 0.4wt% glutaraldehyde, and 0.3wt% mesoporous silica-alumina composite oxide material; Zone 3 (indicating microbial respiratory activity): Finally, immerse the strip-shaped test paper in a 2g / L resazurin solution and wet it evenly. The resazurin solution is prepared with phosphate buffer containing 1wt% glucose, and also contains 0.4wt% glutaraldehyde and 0.3wt% mesoporous silica-alumina composite oxide material. It should be prepared fresh and stored away from light. Step 3: Vacuum dry the test paper coated with the color developer to ensure that the color developer is completely fixed on the test paper. After drying, seal the test paper in a light-proof container.

[0051] The preparation method of the mesoporous silicon-aluminum composite oxide material is as follows, in parts by weight: Five parts of tetraisopropyl orthosilicate, 0.6 parts of aluminum nitrate, 60 parts of ethanol, 1 part of dodecylamine and 8 parts of water were mixed evenly. A 1 mol / L sodium hydroxide aqueous solution was added to adjust the pH to 8.5. The mixture was heated to 45°C and stirred for 3 hours. After centrifugation, the mixture was washed and dried, and then calcined at 550°C for 2 hours to obtain a mesoporous silica-alumina composite oxide material.

[0052] Example 7 A method for detecting soil microbial diversity in red soil dryland based on multicolor chromogenic test strips is as follows: S1. Collect 1g of soil sample from red soil dryland, put 1g of soil sample into centrifuge tube, add 5mL of phosphate buffer, use a vortex shaker to shake the sample for 2 minutes to fully dissolve the microbial metabolites into the buffer, let stand for a while, and after the soil particles settle, take the supernatant for subsequent detection. S2. Add 100 μL of supernatant evenly to the test strip and let it stand for 15 minutes to allow the colorimetric reaction to proceed fully. During this process, microbial metabolites, extracellular polymers, and respiratory activity will react with the chromogenic agent on the test strip, causing the test strip color to change. Use an image acquisition device to capture the colorimetric results of the test strip, ensuring sufficient and uniform lighting during the capture to avoid shadows or reflections affecting image quality. After capturing the image, save it and upload it to the accompanying APP. The APP will automatically read the grayscale values ​​(intensity values ​​of the red, green, and blue channels) of the test strip image. The machine learning model built into the APP will analyze the colorimetric features according to the preset equation, and the model will output the following results: microbial diversity index (such as Shannon index, Inv_Simpson index), dominant species ratio, and diversity level (high, medium, low).

[0053] Interpretation of Results: Microbial biodiversity index: Shannon index: Measures the diversity and evenness of a microbial community. A higher value indicates a richer variety of microorganisms and a more even distribution.

[0054] Inv_Simpson index: measures the diversity of a microbial community; the higher the value, the richer the microbial community.

[0055] Dominant species percentage: This reflects the relative proportion of a specific microbial species in a community. A high percentage may indicate that certain microorganisms dominate the community.

[0056] Based on the output diversity index and level, determine the status of soil microbial diversity: High diversity level: The soil microbial community is rich and uniform, with good ecological functions.

[0057] Medium diversity level: The microbial community diversity is moderate, the soil ecosystem is relatively healthy, but there may be some ecological pressure.

[0058] Low diversity level: Soil microbial communities may be disturbed, resulting in poor ecological function, and appropriate soil improvement measures may be required.

[0059] Test Example 1 Water resistance test: The multicolor test strips prepared in Examples 1-6 of this invention were cut into several test pieces of the same size and placed in a humidity control chamber, ensuring that the sample surface was exposed to the air. The relative humidity in the humidity control chamber was adjusted to 100%RH and maintained at 30℃. The color change, bubbling, softening, and brittleness of the test strips were observed over 24 hours. The color change and physical state of the test strips under high humidity were recorded. Test strips with no significant color change and good physical state showed good water resistance.

[0060] Table 1

[0061] Test Example 2 Accelerated aging test: Accelerated aging testing is a method to rapidly assess the stability of chromogenic agents during long-term storage by simulating extreme conditions. The specific procedure is as follows: The multicolor chromogenic test strips prepared in Examples 1-6 of this invention are stored at 45°C and under ultraviolet light, respectively. Samples are taken every week, and the absorbance is measured at the maximum absorption wavelength of the chromogenic agent using an ultraviolet spectrophotometer. The stability of the chromogenic agent is assessed by recording the changes in absorbance; the smaller the change in absorbance, the better the stability of the chromogenic agent.

[0062] Specific standards for absorbance changes: Absorbance change less than 5%: The colorimetric reagent is very stable under the test conditions and is suitable for long-term storage.

[0063] Absorbance variation is between 5% and 10%: The colorimetric reagent has moderate stability and may need to be stored under specific conditions (such as low temperature and protection from light).

[0064] Absorbance change exceeding 10% indicates poor stability of the colorimetric agent.

[0065] Table 2

[0066] As can be seen from test examples 1-2, the multicolor colorimetric test paper prepared in Example 6 has the best water resistance and accelerated aging performance.

[0067] Example 2 demonstrates that the addition of 0.4 wt% glutaraldehyde significantly improved the moisture resistance and accelerated aging performance of multicolor colorimetric test paper. Glutaraldehyde, as a cross-linking agent, enhances the adhesion and stability of the colorimetric agent on the test paper. In the water resistance test, the test paper with added glutaraldehyde exhibited less color change and physical damage, indicating better fixation of the colorimetric agent and effective resistance to moisture erosion. In the accelerated aging test, the absorbance of the test paper with added glutaraldehyde showed a significant decrease, indicating that the colorimetric agent maintains high stability under extreme conditions such as high temperature and light exposure, and is not prone to degradation or deterioration.

[0068] Examples 3, 4, and 5, by adding mesoporous silica, mesoporous alumina, or a mixture of both, significantly improved the moisture resistance and accelerated aging performance of multicolor colorimetric test papers. Mesoporous silica and mesoporous alumina possess high specific surface area and excellent adsorption properties, enabling them to form a tighter bond with the colorimetric agent and cellulose filter paper, thereby enhancing the fixation effect of the colorimetric agent on the test paper. In the moisture resistance test, these nanomaterials effectively blocked moisture penetration, reducing the dissolution of the colorimetric agent and color changes. In the accelerated aging test, the addition of nanomaterials improved the chemical stability of the colorimetric agent, allowing it to maintain low absorbance changes even under high temperature and light conditions, exhibiting better resistance to degradation.

[0069] The mesoporous silica-alumina composite oxide material used in Example 6 exhibits superior moisture resistance and accelerated aging performance compared to the mesoporous silica or mesoporous alumina used in other examples. This mesoporous silica-alumina composite oxide material, prepared through a specific process, possesses a unique structure and properties. It not only provides a high specific surface area and good dispersibility similar to nanomaterials, enhancing the binding force between the colorimetric agent and the cellulose filter paper, but also protects the colorimetric agent, effectively resisting the erosion of the colorimetric agent by moisture and extreme environmental conditions (such as high temperature and light). This improves the stability and preservation performance of the test paper.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multi-color colorimetric test strip, characterized in that, The container is loaded with at least three chromogenic agents that respond to microbial metabolites, extracellular polymers, and respiratory activity; the chromogenic agents include bromocresol purple, trypan blue, and resaazine.

2. A method for preparing the multicolor colorimetric test paper as described in claim 1, characterized in that, The method is as follows: Step 1: Cut cellulose filter paper or nanofiber membrane into strip-shaped test strips; Step 2: Prepare bromocresol purple solution with a concentration of 0.5-2 g / L, trypan blue solution with a concentration of 0.2-0.8 g / L, and resazurin solution with a concentration of 1-3 g / L, respectively. Immerse the strip-shaped test paper in bromocresol purple solution, then immerse it in trypan blue solution, and finally immerse it in resazurin solution. Step 3: Dry the soaked test paper to fix the color developer on the test paper. After drying, seal and store it away from light.

3. A method for preparing the multicolor colorimetric test paper as described in claim 1, characterized in that, The preparation method of the multicolor colorimetric test paper is as follows: Step 1: Cut cellulose filter paper or nanofiber membrane into strip-shaped test strips; Step 2, Zone 1 (Detection of organic acids): Immerse the strip test paper in 0.5-2 g / L bromocresol purple solution; The bromocresol purple solution uses an ethanol-water co-solvent system with a volume ratio of 2-4:6-8, containing 0.05wt%-0.2wt% polyvinyl alcohol as a dispersant and 0.3wt%-0.5wt% glutaraldehyde. The pH of the solution is adjusted to 7.0±0.

2. Zone 2 (cell membrane detection): The strip test paper is then immersed in 0.1-0.5 g / L trypan blue solution, prepared using Tris-HCl buffer solution at pH 7.5-8.5, with 0.03wt%-0.08wt% Triton X-100 added as a surfactant and 0.3wt%-0.5wt% glutaraldehyde. Zone 3 (indicating microbial respiratory activity): Finally, the strip test paper is immersed in 1-3 g / L resazurin solution, prepared using phosphate buffer solution containing 0.5wt%-2wt% glucose and 0.3wt%-0.5wt% glutaraldehyde. The solution should be prepared fresh and stored away from light. Step 3: Dry the soaked test paper to fix the color developer on the test paper. After drying, seal and store it away from light.

4. A method for preparing the multicolor colorimetric test paper as described in claim 1, characterized in that, The preparation method is as follows: Step 1: Cut cellulose filter paper or nanofiber membrane into strip-shaped test strips; Step 2, Section 1 (Detection of Organic Acids): Immerse the strip test paper in 0.5-2 g / L bromocresol purple solution. The bromocresol purple solution uses an ethanol-water co-solvent system with a volume ratio of 2-4:6-8, containing 0.05wt%-0.2wt% polyvinyl alcohol as a dispersant, 0.3wt%-0.5wt% glutaraldehyde, and 0.2wt%-4wt% carrier material. Adjust the pH of the solution to 7.0±0.

2. Section 2 (Detection of Cell Membranes): Immerse the strip test paper in 0.1-0.5 g / L trypan blue solution. The trypan blue solution is prepared using phosphate buffer solution with a pH of 7.0-8.0, and 0.05wt%-0.1wt% is added. Tween-20 as a surfactant, 0.3wt%-0.5wt% glutaraldehyde, and 0.2wt%-4wt% carrier material; Zone 3 (indicating microbial respiratory activity): Finally, immerse the strip-shaped test paper in 1-3g / L resazurin solution to evenly wet the third independent reaction zone of the test paper. The resazurin solution is prepared with phosphate buffer containing 0.5wt%-2wt% glucose, and also contains 0.3wt%-0.5wt% glutaraldehyde and 0.2wt%-4wt% carrier material. It should be prepared fresh and stored away from light. Step 3: Dry the coated test paper to fix the color developer on the test paper. After drying, seal and store it away from light. The carrier material is at least one of mesoporous silica, mesoporous alumina, and mesoporous silica-alumina composite oxide materials. The preparation method of the mesoporous silicon-aluminum composite oxide material is as follows, in parts by weight: Mix 4-6 parts of tetraisopropyl orthosilicate, 0.4-0.8 parts of aluminum nitrate, 50-70 parts of ethanol, 0.5-2 parts of dodecylamine, and 5-10 parts of water evenly. Add 0.5-2 mol / L sodium hydroxide aqueous solution to adjust the pH to 8-9. Heat to 40-50℃ and stir for 1-5 hours. Then centrifuge, wash and dry, and calcine at 500-600℃ for 1-3 hours to obtain mesoporous silica-alumina composite oxide material.

5. A method for detecting microbial diversity in red soil dryland based on multicolor colorimetric test paper as described in claim 1, characterized in that, The detection method includes an image acquisition module and a data analysis module; The image acquisition module is used to acquire the colorimetric image of the test strip; The data analysis module maps color features to microbial biodiversity indices using a machine learning model.

6. The detection method as described in claim 5, characterized in that, The machine learning model extracts multi-channel color features from the color image using a convolutional neural network and then regresses to predict the Shannon index and the Inv_Simpson index.

7. The detection method as described in claim 5, characterized in that, The data analysis module also includes a preset equation for converting the gray values ​​of the red, green and blue channels of the color image into a microbial diversity index. The preset equation is y = -0.0026x + 1.0393, where y is the microbial diversity index and x is the gray value of the red, green and blue channels.

8. The detection method as described in claim 5, characterized in that, The method for detecting soil microbial diversity in red soil dryland based on multicolor colorimetric test paper is as follows: S1. Take a red soil sample, add buffer solution and shake to extract microbial metabolic active components; S2. Add the extract to each reaction zone of the test strip and let it stand for a period of time to allow the color reaction to occur; capture the color development results of the test strip using the image acquisition module; analyze the color development image using the machine learning model in the data analysis module and output the microbial diversity index and diversity level.

9. The detection method as described in claim 8, characterized in that, The buffer solution in S1 is phosphate buffer; the shaking time is 2-5 minutes; the standing time in S2 for allowing the colorimetric reaction to occur is 10-30 minutes.

10. The detection method as described in claim 8, characterized in that, The microbial diversity indices include the Shannon index and the Inv_Simpson index.

Images