Micro-fluidic chip for detecting activity of soil enzyme and application of micro-fluidic chip

Abstract

The invention discloses a micro-fluidic chip for soil enzyme activity detection and application of the micro-fluidic chip, and belongs to the technical field of soil enzyme activity detection. The whole micro-fluidic chip is circular and comprises an upper layer and a lower layer which are bonded, and a plurality of same detection units are distributed around the circle center of the micro-fluidic chip between the upper layer and the lower layer; each set of detection unit comprises a V-shaped soil cavity, a reagent cavity, a reaction cavity and a detection cavity which are sequentially arranged from the circle center to the circumference; except that the tops of two adjacent arms of the two V-shaped soil cavities are respectively provided with a liquid injection hole and an exhaust hole of a soil suspension, the top ends of two adjacent arms of the other V-shaped soil cavities are communicated, so that all the V-shaped soil cavities form a through cavity; the bottom of the V-shaped soil cavity is communicated with the reaction cavity through a micro-channel provided with a capillary valve, and the bottom of the reagent cavity is also communicated with the reaction cavity through a micro-channel provided with a capillary valve; a reagent filling hole is formed in the top of the reagent cavity; the middle lower part of the reaction cavity is communicated with the detection cavity through a siphon valve, the near-circle-center end of the detection cavity is provided with a micro-channel extending towards the circle center direction, and the tail end of the detection cavity is provided with a vent hole. The micro-fluidic chip provided by the invention is used for detecting the enzyme activity of soil and has the advantages of simplicity in operation, high accuracy, large flux and better repeatability.

Description

Technical Field

[0001] The present invention belongs to the technical field of soil enzyme activity detection, and particularly relates to a microfluidic chip for soil enzyme activity detection and its application. Background Art

[0002] Soil enzymes refer to the general term for various enzymes present in the soil medium, which are mainly produced and secreted into the soil by soil microorganisms such as bacteria and fungi, or released into the soil after the decomposition of microbial residues. The main function of soil enzymes is to catalyze the degradation of complex organic monomers or minerals using plant litter, animal remains and excreta, soil organic matter, etc. as substrates, and decompose them into simple small molecule substances for microorganisms and plants to absorb. Soil enzymes play an important role in the ecosystem, mainly reflected in the following four aspects: First, they are the key to the transformation of soil organic matter and the cycling of nutrient elements, promoting the cycling of elements such as carbon, nitrogen, and phosphorus; Second, soil enzymes accelerate the transformation and transfer of energy in the soil ecosystem by catalyzing various biochemical reactions; Third, they are sensitive to environmental changes and can be used as an indicator of the decomposition rate of soil organic matter; Fourth, their activity is affected by soil nutrients, can reflect the health of the ecosystem and soil quality, and helps to maintain soil ecological balance and protect biodiversity. At present, there are many detection methods for soil enzyme activity at home and abroad, including spectrophotometry, fluorescence analysis, radioactive isotope method, and physical methods such as titration method. Spectrophotometry, also known as colorimetry, is based on the principle of mixing an enzyme with a substrate for incubation. The product after incubation can produce a characteristic absorption peak at a specific wavelength. The absorbance values of the standard substance of the product and the sample are measured with a spectrophotometer, and then the activity of the soil enzyme is determined. This method has been applied earlier and more widely, but it has the disadvantages of long time consumption, complex operation, and low sensitivity. In the 1990s, a new method for measuring enzyme activity - fluorescence analysis developed rapidly internationally. It uses a fluorescent group-labeled substrate as a probe to reflect the soil enzyme activity through the change of fluorescence intensity. Although it has the advantages of higher sensitivity and shorter experimental time consumption compared with spectrophotometry, it has the disadvantages of high test cost and difficult dissolution of the substrate. The isotope labeling method is rarely used in actual operation due to its complex operation, high cost, and immature technology for directly extracting enzymes from the soil. The titration method has been gradually phased out due to low accuracy. Currently, the commonly used method for measuring soil enzyme activity is the microplate - fluorescence method, which is an improved technology based on fluorescence analysis. This method places the soil suspension, substrate, and corresponding buffer solution in a 96-well microplate for incubation, and then places it in an enzyme-labeled instrument for detection after the enzyme reaction in the microplate. This method significantly improves the measurement efficiency of soil enzyme activity, but it is more dependent on manual operation. The fineness of the operation directly affects the uniformity of the sample, often resulting in poor parallelism among multiple groups of data for the same soil sample when measuring the same enzyme activity. Moreover, the high cost of the enzyme-labeled instrument limits its popularization and application.

[0003] In summary, given the importance of soil enzymes in soil health assessment indicators, traditional enzyme activity detection methods have deficiencies such as long time consumption, complex operation, low precision, high cost, and over-reliance on manual operation. These methods have limitations in terms of accuracy, analysis efficiency, and cost-effectiveness, restricting their wide application. Therefore, there is an urgent need for a new technology with high detection sensitivity and simple and easy-to-use instruments to improve the detection efficiency and throughput of soil enzyme activity, so as to achieve parallel detection of multiple indicators. Summary of the Invention

[0004] The primary problem to be solved in this application is to provide a microfluidic chip for soil enzyme activity detection, which solves the problems of long time consumption, complex operation, low precision, high cost, and over-reliance on manual operation in traditional enzyme activity detection methods. Another technical problem to be solved in this application is to provide the steps and methods for the specific application of the aforementioned microfluidic chip for soil enzyme activity detection.

[0005] To solve the above problems of the prior art, the technical solution of the present invention is as follows:

[0006] A microfluidic chip for soil enzyme activity detection, the microfluidic chip is circular as a whole, including an upper layer and a lower layer bonded together. Between the upper layer and the lower layer, several sets of identical detection units are distributed around the center of the microfluidic chip; each set of detection units includes a V-shaped soil chamber, a reagent chamber, a reaction chamber, and a detection chamber arranged in sequence from the center to the circumference; the openings of the V-shaped soil chambers in each set of detection units all face the center. Except that a liquid injection hole and an exhaust hole for soil suspension are respectively provided at the top of the adjacent two arms of two V-shaped soil chambers, the adjacent two arms of the remaining V-shaped soil chambers are connected, so that all V-shaped soil chambers form a through chamber; the bottom of the V-shaped soil chamber is connected to the reaction chamber through a microchannel provided with a capillary valve, and the bottom of the reagent chamber is also connected to the reaction chamber through a microchannel provided with a capillary valve, and the capillary valve is the enlarged part in the middle of the microchannel; a reagent injection hole is provided at the top of the reagent chamber; the middle and lower part of the reaction chamber is connected to the detection chamber through a siphon valve, and the siphon valve is a section of microchannel that turns back towards the center of the chip; the detection chamber is cylindrical, and a microchannel extending towards the center is provided at the top of the detection chamber, and a ventilation hole is provided at the end of the microchannel.

[0007] In some embodiments, a redundant chamber is further provided in the middle and lower section of the siphon valve close to the detection chamber.

[0008] In some embodiments, the upper layer of the microfluidic chip is made of an organic material, and the lower layer is made of glass or quartz material.

[0009] In some embodiments, the organic material is polydimethylsiloxane (PDMS) material, polymethyl methacrylate (PMMA), polycarbonate (PC), or polystyrene (PS).

[0010] In some embodiments, the upper layer of the microfluidic chip is made of polydimethylsiloxane (PDMS) material, and the lower layer is made of glass material.

[0011] In some embodiments, the width and height of the microchannel are both 0.5 mm, and the volumes of the reaction chamber, reagent chamber, and detection chamber are not more than 200 μL respectively.

[0012] The preparation method of the microfluidic chip is to recess the detection unit in the upper layer made of organic material by means of mold injection, and then bond the surface of the lower layer after oxygen plasma treatment to the upper layer of the chip to obtain the microfluidic chip.

[0013] Application of the microfluidic chip in the determination of soil enzyme activity.

[0014] In some embodiments, the application includes the following steps:

[0015] 1) Inject the soil suspension into the V-shaped soil chamber; inject the fluorescently labeled substrate solution into the reagent chamber;

[0016] 2) Install the microfluidic chip on a centrifugal spin coater;

[0017] 3) Through low-speed centrifugation, the soil suspension and the fluorescently labeled substrate solution in the chip flow into the detection chamber together to form a reaction solution by mixing;

[0018] 4) The reaction solution reacts at a certain temperature for a period of time to cause the soil enzyme in the soil suspension to react with the substrate to generate a fluorescent substance;

[0019] 5) Through high-speed centrifugation, the reaction solution in the reaction chamber flows from the reaction chamber into the detection chamber;

[0020] 6) Use a fluorescence detector to detect the reaction solution in the detection chamber to obtain the fluorescence intensity of the reaction solution, and calculate the activity of the soil enzyme according to the standard curve of the relationship between the concentration of the fluorescent substance prepared in advance by the microfluidic chip and the fluorescence intensity.

[0021] In some embodiments, the soil enzyme is one or more of β-glucosidase, cellobiohydrolase, β-xylosidase, β-galactosidase; N-acetyl-β-glucosaminidase, N-acetyl-β-galactosaminidase, phosphatase, sulfatase.

[0022] Compared with the prior art, the beneficial effects of this application are:

[0023] The microfluidic chip of the present application contains multiple identical detection units, and the sample chambers of the V-shaped structures of all detection units are interconnected. Therefore, multiple detections can be completed with a single sample addition, saving the time for sample addition and reducing the error between samples caused by the sample addition operation. Moreover, when centrifuged, the sample solution in the sample chamber of each V-shaped structure only enters the reaction chamber of its corresponding detection unit, enabling the sample solution injected at one time to be evenly split into each reaction chamber through the sample chamber of the V-shaped structure during centrifugation, ensuring that the volume of the sample solution obtained by each detection unit is equal. Experiments have shown that the microfluidic chip provided by the present application can establish a linear relationship between the fluorescence value for detecting soil-related enzyme activities and the concentration of the standard solution, enabling qualitative and quantitative detection of various enzyme activities in soil. Additionally, the microfluidic chip of the present application for detecting soil enzyme activities has the advantages of simple operation, high accuracy, and good repeatability.

[0024] The present invention fills the gap in the miniaturization, integration, and rapid detection of soil enzyme activities in the microfluidic system. Since the detection of soil enzyme activities is the basis and important research content of soil enzymology and an indispensable analysis index in almost all soil ecosystem and soil microorganism research, the present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the overall structure diagram of the microfluidic chip;

[0026] Figure 2 is the partial enlarged structure diagram of the microfluidic chip;

[0027] Figure 3 is the standard curve graph obtained by measuring the MUF reference material using the microfluidic chip method;

[0028] Figure 4 is the result graph of detecting the BG enzyme activities of different types of soil using the microfluidic chip method;

[0029] Figure 5 is the result graph of detecting the activities of eight enzymes in soil using the microfluidic chip method;

[0030] Figure 6 is the result graph of detecting the activities of eight enzymes in soil using the fluorescence microplate method;

[0031] DESCRIPTION OF THE REFERENCE NUMERALS:

[0032] 1 - V-shaped soil chamber, 2 - reagent chamber, 3 - reaction chamber, 4 - detection chamber, 5 - liquid injection hole, 6 - exhaust hole, 7 - capillary valve, 8 - microchannel, 9 - siphon valve, 10 - reagent filling hole, 11 - redundant chamber, 12 - ventilation hole. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified in detail, the technical means used are all conventional means well-known to those skilled in the art.

[0034] The term

[0035] As used in the present invention, the terms "top", "middle and lower part", and "bottom" are descriptions of different structural parts on the microfluidic chip for soil enzyme activity detection that are arranged from near to far from the center of the circle of the microfluidic chip for soil enzyme activity detection.

[0036] A microfluidic chip for soil enzyme activity detection (hereinafter referred to as the microfluidic chip) has a structure as Figure 1 and Figure 2 shown. It is generally circular with a radius of about 50 mm and is formed by bonding a PDMS layer and a glass substrate layer. Nine sets of identical detection units are evenly distributed between the two layers around the center of the circle. Each set of detection units includes a V-shaped soil chamber 1, a reagent chamber 2, a reaction chamber 3, and a detection chamber 4 arranged in sequence from the center of the circle of the microfluidic chip to the circumference. Among them, the openings of the V-shaped soil chambers 1 in each set of detection units all face the center of the circle, that is, the bottom of the V-shaped soil chamber 1 is the far center end, and the tops of the two arms of the V-shaped soil chamber 1 are the near center ends. Except that liquid injection holes 5 and exhaust holes 6 for soil suspension are respectively provided at the tops of the adjacent two arms of two V-shaped soil chambers 1, the tops of the adjacent two arms of the remaining V-shaped soil chambers 1 are connected, so that all the V-shaped soil chambers 1 form a through chamber with a total volume of 900 ± 5 μL. In each set of detection units, the bottom of the V-shaped soil chamber 1 is connected to the reaction chamber 3 through a microchannel 8 provided with a capillary valve 7. The bottom of each reagent chamber 2 is also connected to the reaction chamber 3 through a microchannel 8 provided with a capillary valve 7. The capillary valve 7 is the enlarged part in the middle of the microchannel 8 and is a cylindrical cavity with a bottom circle diameter of 1 ± 0.2 mm and a height of 1 mm. The width and height of all the microchannels 8 in the microfluidic chip are both 0.5 ± 0.1 mm. The volume of the reagent chamber is 50 ± 2 μL, and a reagent injection hole 10 is also provided at the top for injecting buffer solution or reaction substrate solution. The middle and lower part of the reaction chamber 3 is connected to the detection chamber 4 through a siphon valve 9. The siphon valve 9 is a section of microchannel 8 that turns towards the center of the circle, and a redundant chamber 11 is provided in the middle and lower section close to the detection chamber 4. The detection chamber is a cylinder with a radius of 3 mm and a volume of 100 ± 5 μL. A long strip-shaped microchannel 8 is provided at the near center end of the detection chamber 4, and an air vent 12 is provided at the end of the microchannel.

[0037] It should be noted that except for the V-shaped soil chamber, the shapes of the reagent chamber, reaction chamber, and detection chamber are not limited as long as they are conducive to detection. In this embodiment, the reagent chamber and the reaction chamber are trapezoids with arc-shaped upper and lower bottoms, while the detection chamber is a cylinder. In addition, the middle and lower part of the reaction chamber 3 is not the bottom. Soil will be deposited at the bottom. The distance from the middle and lower part to the bottom is H, and the size of H can be adjusted according to actual needs.

[0038] The method for preparing the microfluidic chip in this embodiment is as follows: all the chambers and channel structures of the chip are integrally cast and formed through a mold, and are recessed in the PDMS layer. Then, the flat glass substrate layer is treated by oxygen plasma. Finally, the treated glass substrate layer is bonded to the PDMS layer to form a complete detection unit chamber structure, and a microfluidic chip is obtained. The specific steps of bonding are: the PDMS layer (with the structural surface facing up) and the glass substrate layer are simultaneously placed in a plasma cleaner for surface activation treatment. The key parameters for cleaning are vacuum pumping for 1 minute, oxygen plasma treatment for 50 seconds, the treatment voltage is 720V, and the current is 0.230A. The activated PDMS layer and the glass substrate layer are quickly taken out, and their activated surfaces are closely attached together.

[0039] The flow of liquid on the microfluidic chip provided in this application is driven by centrifugal force. In the static state, due to the surface tension between the liquid and the contact surface of the microfluidic chip, as well as the influence of the structures and dimensions of capillary valves, microchannels, etc., the liquid in each chamber cannot flow in or out by gravity. Under low-speed centrifugation, the through V-shaped soil chamber can evenly divide the soil suspension of the same sample into nine parts and distribute it to the reaction chambers in nine detection units. At the same time, the substrate solution in the reagent chamber also flows into the reaction chamber and mixes with the soil suspension to form a reaction solution. Due to the existence of the swirling siphon valve, the reaction solution in the reaction chamber can only further flow into the detection chamber under high-speed centrifugation and wait for fluorescence detection.

[0040] The chip provided in this application can detect (but not limited to) eight key enzymes in soil: β-glucosidase (BG), cellobiohydrolase (CBH), β-xylosidase (BX), β-galactosidase (GAL); N-acetyl-β-glucosaminidase (NAG), N-acetyl-β-galactosaminidase (NAGA), phosphatase (AP), sulfatase (SF). The fluorescence-labeled substrate is cleaved under the catalytic action of soil extracellular enzymes to release a fluorescent group. By measuring the fluorescence intensity, the size of enzyme activity can be reflected, and the accurate enzyme activity can be further calculated. The enzyme activity result is expressed as the amount of fluorescent substance produced per unit mass of soil per unit time, and the unit is nmol g -1 h -1 , where the standard substance is 4-methylumbelliferone (MUF). As shown in Table 1, after the soil enzyme to be measured reacts with the corresponding substrate in the solution, MUF will be released. By detecting the fluorescence intensity generated by the reaction to produce MUF, according to the standard curve of the concentration of MUF prepared in advance using the microfluidic chip and the fluorescence intensity, the activity sizes of the eight key enzymes in the soil can be calculated.

[0041] Table 1 Soil Enzymes and Substrates

[0042] Number Soil enzyme to be measured Fluorescently labeled substrate 1 β-glucosidase (BG) 4-MUF-β-D-glucopyranoside 2 N-acetyl-β-glucosaminidase (NAG) 4-MUF-N-acetyl-β-D-glucosaminide 3 Phosphatase (AP) 4-MUF-phosphate 4 Sulfatase (SF) 4-MUF-sulfate 5 β-xylosidase (BX) 4-MUF-β-D-xylopyranoside 6 Cellobiohydrolase (CBH) 4-MUF-β-cellobioside 7 β-galactosidase (GAL) 4-MUF-β-D-galactopyranoside 8 N-acetyl-β-galactosaminidase (NAGA) 4-MUF-N-acetyl-β-D-galactosaminide

[0043] Method for using the microfluidic chip provided by this application:

[0044] I. Reagent loading: Mix the buffer solution (25 μL) and substrate (25 μL) required for the reaction and add the mixture to the reagent chamber. Reserve one to three reagent chambers and add 50 μL of buffer solution as a blank control. For glycosidase, phosphatase, and arylsulfatase, use 0.1 M MES buffer solution and adjust the pH to 6.1. Different types of enzymes correspond to different reaction substrates, and the substrate concentration is controlled at 400 μM. After the addition is completed, seal the filling port with materials such as transparent tape. This step is usually pre-prepared during mass production to avoid the influence of experiments in different batches on the detection results and ensure the consistency and repeatability of the experiments.

[0045] II. Soil suspension filling: Prepare a soil suspension according to a fixed ratio, adding 1 g of soil to 10 mL of water, vortexing to evenly disperse the soil particles into the liquid to prepare the soil suspension, and then immediately take 900 μL of the suspension and inject it into the soil chamber, sealing the liquid injection hole.

[0046] III. Install the centrifugal microfluidic chip on a centrifugal spin coater and fix it by means of snap fixation.

[0047] IV. Centrifugal mixing and light-shielding cultivation: Use the centrifugal spin coater to centrifuge the microfluidic chip at a low speed of 1500 - 2500 r / min for 6 s. The soil suspension and the reagent are thrown into the reaction chamber together to achieve a mixing reaction. Subsequently, under the condition of (30 ± 3 °C), perform constant-temperature light-shielding cultivation for 0.5 h.

[0048] V. Result detection: Use the centrifugal spin coater to centrifuge the microfluidic chip at a high speed of 3500 - 4500 r / min for 30 s. Under the action of centrifugal force, the soil particles remain at the bottom of the reaction chamber, and the reaction reagent enters the detection chamber through the siphon valve, separating the two and terminating the reaction. Finally, perform fluorescence detection on the detection chamber at an excitation wavelength of 355 nm and an emission wavelength of 460 nm to obtain the fluorescence value.

[0049] VI. Enzyme activity calculation: Calculate the enzyme activity according to the MUF standard curve prepared in advance using the microfluidic chip.

[0050] Example 1: Determination of the MUF standard curve based on the microfluidic chip

[0051] Take 1 g of soil sample, add 10 mL of sterile deionized water, mix using a vortex oscillator, and prepare a soil suspension. After that, take 900 μL and inject it into the soil chamber on the microfluidic chip. Inject 45 μM MUF standard solution into the reagent chamber of the microfluidic chip, and add an appropriate amount of MES buffer to control the total volume of a single reaction unit on the chip to be 150 μL to obtain standard curves with final concentrations of 0 μmol / L, 3 μmol / L, 6 μmol / L, 9 μmol / L, and 12 μmol / L. The concentration of the standard curve can be adjusted according to the sample. Perform fluorescence detection at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. Perform linear fitting between the fluorescence values measured on the chip and the concentrations of the standard solutions, as Figure 2 shown. The linear equation of the standard curve for MUF determination is y = 697.57x + 129.84, and the correlation coefficient is 0.9905; there is a significant positive linear correlation (p < 0.05) between the fluorescence values measured on the chip and the concentrations of the standard solutions.

[0052] Example 2: Influence of Different Types of Soils on the Stability of the Detection Results of the Microfluidic Chip

[0053] Inject 25 μL of MES buffer and 25 μL of substrate solution into the reagent chamber of the microfluidic chip respectively, and seal the filling holes. Take 1 g of red soil, black soil, and fluvo-aquic soil respectively, add 10 mL of sterile deionized water, mix using a vortex oscillator, and prepare a soil suspension. After that, take 900 μL and inject it into the soil chamber on the microfluidic chip. Centrifuge and mix at 2000 r / s, incubate in the dark at a constant temperature of (30 ± 3) °C for 0.5 h, and then perform fluorescence detection at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. Taking β-glucosidase as an example, set seven parallels for each soil type and calculate the within-group coefficient of variation (CV). The results are as Figure 3 shown. The CV values of red soil, black soil, and fluvo-aquic soil are 10.6%, 14.0%, and 11.4% respectively, indicating that the microfluidic chip method can obtain relatively stable enzyme activity results for different soil types.

[0054] Comparative Example 1: Comparative Analysis of the Microfluidic Chip Method and the Traditional Fluorescent Microplate Method for Determining Eight Enzyme Activities in Soils

[0055] Taking fluvo-aquic soil with long-term chemical fertilizer application as the soil sample, the activities of eight enzymes, namely β-glucosidase (BG), cellobiohydrolase (CBH), β-xylosidase (BX), β-galactosidase (GAL), N-acetyl-β-glucosaminidase (NAG), N-acetyl-β-galactosaminidase (NAGA), sulfatase (SF), and phosphatase (AP), were detected using a microfluidic chip, and a comparison was made using the traditional fluorescence microplate method outside the chip. Three replicates were set for each enzyme. The average value and standard deviation of each enzyme activity were calculated for the measured fluorescence values, and the results are as shown in Figure 5 , 6 . The error bars in the figure represent the standard deviation of each group of data, reflecting the degree of data dispersion. It can be seen that while simplifying the operation process, the measurement accuracy of the microfluidic chip is comparable to that of the traditional method, and the error of some enzyme activity detections is smaller. Therefore, it has advantages over the traditional microplate method.

[0056] The above description is illustrative rather than restrictive for the present invention. Those of ordinary skill in the art understand that many modifications, variations, or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present invention.

Claims

1. A microfluidic chip for detecting soil enzyme activity, characterized in that, The overall shape of the microfluidic chip is circular, including an upper layer and a lower layer bonded together. Between the upper layer and the lower layer, several sets of identical detection units are distributed around the center of the microfluidic chip; each set of detection units includes a V-shaped soil chamber, a reagent chamber, a reaction chamber, and a detection chamber arranged in sequence from the center to the circumference; the openings of the V-shaped soil chambers of each set of detection units all face the center. Except that a liquid injection hole and an exhaust hole for soil suspension are respectively provided at the top of the adjacent two arms of two V-shaped soil chambers, the adjacent two arms of the remaining V-shaped soil chambers are connected, so that all V-shaped soil chambers form a through chamber; the bottom of the V-shaped soil chamber is communicated with the reaction chamber through a microchannel provided with a capillary valve, and the bottom of the reagent chamber is also communicated with the reaction chamber through a microchannel provided with a capillary valve. The capillary valve is a part where the middle of the microchannel bulges; a reagent injection hole is provided at the top of the reagent chamber; the middle and lower part of the reaction chamber is communicated with the detection chamber through a siphon valve, and the siphon valve is a section of microchannel that turns back towards the center; the detection chamber is cylindrical, and a microchannel extending towards the center is provided at its top, and a ventilation hole is provided at the end of the microchannel.

2. The microfluidic chip for detecting soil enzyme activity according to claim 1, characterized in that, A redundant chamber is also provided in the middle and lower section of the siphon valve close to the detection chamber.

3. The microfluidic chip for detecting soil enzyme activity according to claim 1, wherein The upper layer of the microfluidic chip is made of an organic material, and the lower layer is made of glass or quartz.

4. The microfluidic chip for detecting soil enzyme activity according to claim 3, wherein The organic material is polydimethylsiloxane, polymethyl methacrylate, polycarbonate or polystyrene.

5. The microfluidic chip for detecting soil enzyme activity according to claim 4, wherein, The upper layer of the microfluidic chip is made of polydimethylsiloxane, and the lower layer is made of glass.

6. The microfluidic chip for detecting soil enzyme activity according to claim 5, characterized in that The width and height of the microchannel are both 0.5 mm, and the volumes of the reaction chamber, reagent chamber, and detection chamber are respectively not more than 200 μL.

7. The preparation method of the microfluidic chip for soil enzyme activity detection according to any one of claims 3-6, characterized in that, By means of mold injection, the detection units are recessed in the upper layer prepared from the organic material, and then the surface of the lower layer is treated by oxygen plasma and bonded to the upper layer of the chip to obtain the microfluidic chip.

8. Application of the microfluidic chip for detecting soil enzyme activity according to any one of claims 1-6 in the determination of soil enzyme activity.

9. The application according to claim 8, wherein Including the following steps: 1) Inject the soil suspension into the V-shaped soil chamber; inject the fluorescently labeled substrate solution into the reagent chamber; 2) Install the microfluidic chip on a centrifugal spinner; 3) Through low-speed centrifugation, the soil suspension and the fluorescently labeled substrate solution in the chip flow into the detection chamber together to form a reaction solution by mixing; 4) The reaction solution reacts at a certain temperature for a period of time, so that the soil enzyme in the soil suspension reacts with the substrate to generate a fluorescent substance; 5) Through high-speed centrifugation, the reaction solution in the reaction chamber flows from the reaction chamber into the detection chamber; 6) Use a fluorescence detector to detect the reaction solution in the detection chamber, obtain the fluorescence intensity of the reaction solution, and calculate the activity of the soil enzyme according to the standard curve of the relationship between the concentration of the fluorescent substance prepared in advance by the microfluidic chip and the fluorescence intensity.

10. The application according to claim 8 or 9, characterized in that, The soil enzyme is one or several of β-glucosidase, cellobiohydrolase, β-xylosidase, β-galactosidase; N-acetyl-β-glucosaminidase, N-acetyl-β-galactosaminidase, phosphatase, sulfatase.

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