Droplet microfluidic in-situ test chip and method for research on microbial mineralization crystallization kinetics

Abstract

The invention discloses a droplet microfluidic in-situ test chip and method for microbial mineralization crystallization kinetic research, and relates to the technical field of rock and soil tests. In order to solve the problems that in the prior art, a single crystal crystallization process is difficult to visualize and nucleation sites of crystals are difficult to observe in situ and in real time, the following technical scheme is provided: the device comprises an upper layer channel pattern layer, solid polydimethylsiloxane and cover glass, an oil phase liquid injection port is formed in the top of the upper layer channel pattern layer, and is connected with an annular oil phase pipeline; a first water-phase liquid injection port and a second water-phase liquid injection port are formed in the middle of the area where the annular oil-phase liquid injection pipeline is located, a pipeline connecting the first water-phase liquid injection port and the second water-phase liquid injection port forms a reaction liquid mixing area at the communication position of the water-phase liquid injection pipeline, a liquid drop generation area is formed at the communication position of the oil-phase liquid injection pipeline and the water-phase liquid injection pipeline, and liquid drops form an in-liquid-drop solution mixing area in the snakelike circulation pipeline. And the liquid drops flow out through the liquid outlet. The method has the advantages of high flux, accurate reaction conditions, accurate observation and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of geotechnical tests, and particularly relates to a droplet microfluidic in-situ test chip and method for studying the kinetics of microbial mineralization crystallization. Background Art

[0002] With the development of social economy, traditional construction engineering technologies are facing challenges related to energy conservation, environmental protection, and sustainable development. The construction method integrating ecological concepts has become a new trend in the field of civil engineering. Microbial mineralization is a common natural phenomenon in which microorganisms regulate or induce the deposition of mineral materials through their own metabolic processes. In recent years, it has attracted extensive attention in the fields of civil engineering, environment, materials, etc. By introducing urease-producing bacteria, calcium chloride, and urea into the soil, calcium carbonate can be induced to precipitate between soil particles under suitable conditions, thereby improving the mechanical properties and stability of the soil. Due to its low energy consumption, small disturbance, and good environmental compatibility, this technology has broad application prospects. At present, domestic and foreign scholars have carried out a large number of studies on the reinforcement effect, process, and engineering practice of microbial solidified soil. However, due to the relatively large observation scale of existing testing methods, multiple crystals are usually formed in a large solution system, which makes the crystallization process of single crystals vulnerable to changes in the solution environment. And because the bacteria are small in size and crystal nucleation is random, it is difficult for existing technologies to in-situ and real-time observe the crystal nucleation sites. At present, the nucleation mechanism and crystallization dynamic process of microbial mineralization are still unclear, which greatly limits the application and development of this technology. Summary of the Invention

[0003] The purpose of the present invention is to provide a droplet microfluidic in-situ test chip and method for studying the kinetics of microbial mineralization crystallization, so as to solve the problem that it is difficult for existing technologies to visualize the crystallization process of single crystals and in-situ and real-time observe the crystal nucleation sites.

[0004] The technical solution of the present invention to solve the above technical problems is as follows: A droplet microfluidic in-situ test chip for studying the kinetics of microbial mineralization crystallization, which includes: a solid polydimethylsiloxane upper channel pattern layer, a solid polydimethylsiloxane bottom layer, and a cover glass arranged in sequence from top to bottom. An oil-phase injection port is provided at the top of the upper channel pattern layer, and the oil-phase injection port is connected to the inlet end of an annular oil-phase pipeline. The internal area where the annular oil-phase pipeline is located is provided with a first aqueous-phase injection port and a second aqueous-phase injection port. The first aqueous-phase flow pipeline communicated with the first aqueous-phase injection port and the second aqueous-phase flow pipeline communicated with the second aqueous-phase injection port form a reaction liquid mixing area at the confluence. The annular oil-phase pipeline and the confluence of the first aqueous-phase flow pipeline and the second aqueous-phase flow pipeline form a droplet generation area. The droplets in the droplet generation area form a droplet internal solution mixing area through a serpentine flow pipeline, and finally flow out through an outlet.

[0005] A testing method for a droplet microfluidic in-situ testing chip for the study of the above-mentioned microbial mineralization crystallization kinetics, comprising the following steps: S1. Prepare a droplet microfluidic chip: Use liquid polydimethylsiloxane to cure and make the upper channel pattern layer, and cover a thin solid polydimethylsiloxane between the upper channel pattern layer and the cover glass of the lower base layer; S2. Set the structure of the droplet microfluidic chip: Set an oil-phase injection port, a first water-phase injection port, a second water-phase injection port and an outlet on the droplet microfluidic chip to construct a reaction liquid mixing area, a droplet generation area and a solution mixing area inside the droplet; S3. Fix the droplet microfluidic chip: Fix the prepared droplet microfluidic chip on the stage of a fluorescence inverted microscope; S4. Prepare solutions and solvents: Use three disposable sterile syringes to suck in microbial mineralization bacterial solution, microbial mineralization cementing solution and continuous phase solution respectively; S5. Connect the syringe pump and the syringe: Connect the three syringes to three tygon hoses through needles respectively, connect the other side of the tygon hoses to steel needles, and fix the syringes on the syringe pump; S6. Connect the droplet microfluidic chip and the syringe: Connect the steel needles to the oil-phase injection port, the first water-phase injection port and the second water-phase injection port of the droplet microfluidic chip respectively, and connect one end of another tygon hose to the outlet of the droplet microfluidic chip through a steel needle; S7. Generate droplets: Control the syringe pump, inject the solutions in the three syringes into the droplet microfluidic chip simultaneously, generate droplets of the required size by adjusting the injection rate of the syringe pump, and observe the generation process under an inverted microscope; S8. Collect droplets: Place a culture dish at one end of the chip outlet on the stage of the fluorescence inverted microscope, and collect the droplets generated in the chip after the droplets are stably generated; S9. Observe the microbial mineralization reaction process: While the solution in the droplet undergoes a crystallization reaction, observe the reaction process in the droplet in-situ and in real-time through an inverted microscope to obtain microbial and crystal information.

[0006] Furthermore, the method for generating droplets in step S7 includes the following steps: S1. Prepare a microbial mineralization bacterial solution: Centrifuge the bacterial solution containing the culture medium obtained after expansion culture, remove the supernatant, and suspend it in sterile physiological saline to obtain a microbial mineralization bacterial solution; S2. Prepare a microbial mineralization cementing solution: Mix the sterile calcium chloride solution and the sterile urea solution evenly to obtain a microbial mineralization cementing solution; S3. Prepare a continuous phase: Mix the surfactant and the oil-based solvent evenly to obtain a continuous phase liquid; S4. Aspirate the microbial mineralization bacterial solution, the microbial mineralization cementing solution, and the continuous phase liquid into disposable syringes respectively. S5. Connect the syringe containing the microbial mineralization bacterial solution to the aqueous phase injection port on the droplet microfluidic chip, and control the injection pump to input the microbial mineralization bacterial solution. S6. Connect the syringe containing the microbial mineralization cementing solution to the aqueous phase injection port on the droplet microfluidic chip, and control the injection pump to input the microbial mineralization cementing solution. S7. Connect the syringe containing the continuous phase to the oil phase injection port on the droplet microfluidic chip, and control the injection pump to input the oil phase. When the continuous phase and the dispersed phase pass through the droplet generation region, "water-in-oil" droplets are obtained. S8. When the generated droplets pass through the droplet mixing region, the solutions inside the droplets are fully mixed evenly.

[0007] Further, the calcium chloride concentration in step S2 is 0.1 - 5 M, and the urea concentration is 0.1 - 5 M; the optical density value of the microbial mineralization bacterial solution in step S1 is OD 600 = 0.2 - 1.8, and the pH is 7.0 - 9.5; the flow rate of the dispersed phase in step S7 is 10 - 150 μL / hr; the flow rate of the continuous phase is 10 - 150 μL / hr. The surfactant in step S3 is any one or more of span80 and Degussa ABIL EM180; the oil solvent is any one or more of liquid paraffin, mineral oil, and dimethyl silicone oil; the content of the surfactant in the continuous phase is 0.1% - 5% (v / v).

[0008] Further, when the research purpose is to verify the crystal nucleation sites, in steps S1 - S3, the following steps are also included: Mix the gelatin solution and the agar solution evenly to obtain a gelatin-agar aqueous solution, mix the microbial mineralization bacterial solution with the gelatin-agar aqueous solution to obtain a microbial-gelatin-agar aqueous solution, mix the microbial mineralization cementing solution with the gelatin-agar aqueous solution to obtain a cementing solution-gelatin-agar aqueous solution, and aspirate the microbial-gelatin-agar aqueous solution, the cementing solution-gelatin-agar aqueous solution, and the continuous phase liquid into disposable syringes respectively. The subsequent steps repeat steps S5 - S8, collect the droplets and cool them to obtain gelatin-agar microgels immobilizing microorganisms, and in-situ and real-time observe the microbial mineralization reaction process in the gelatin-agar microgels through an inverted microscope.

[0009] Further, the microbial mineralization bacterial solution is Sarcina pasteurii; the microbial mineralization cementing solution is a mixed solution of a calcium chloride solution and a urea solution, and the concentration ratio of the two is 1:1; the mineral generated by microbial mineralization is calcium carbonate; the droplet size is 20 - 100 μm.

[0010] Furthermore, it also includes the step of nucleic acid staining of the bacteria for microbial mineralization, and the staining agents are SYTO-9 and PI. Subsequent observation of the bacteria is carried out in the fluorescence mode of a fluorescence inverted microscope. The distribution of the bacteria in the droplets is observed through fluorescence imaging, and the mixing condition of the solution in the droplets can also be detected.

[0011] Furthermore, the reaction temperature in the operation steps of S5 - S8 is controlled at 15 - 40 °C.

[0012] Furthermore, in the step of studying the crystal nucleation sites, the temperature during the solution preparation and droplet preparation process is controlled at about 40 °C; the temperature for collecting and cooling the droplets in the step is controlled at 15 - 25 °C.

[0013] The present invention has the following beneficial effects: 1. In order to solve the problem of the lack of an effective experimental method in studying the calcium carbonate crystallization kinetics in microbial mineralization, the present invention proposes a microscale method for in-situ real-time observation of the crystallization process of a single crystal in a microdroplet, which can be used to explore the regulatory effect of environmental conditions on the crystal morphology and growth law, and the method is simple and easy to implement.

[0014] 2. In order to solve the problem that it is difficult to in-situ and real-time observe the crystal nucleation sites in the existing technology of microbial mineralization, the present invention proposes an experimental method in which after generating a microgel to fix bacteria, the localization of the fixed bacteria is clarified under a microscope, and then the nucleation position of the generated crystal is observed in real time. It can be used to explore whether bacterial cells serve as calcium carbonate crystal nucleation sites in microbial mineralization, and the observation result is intuitive and the operation is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a schematic diagram of the pipeline of the droplet microfluidic chip for generating droplets of the present invention; Figure 2 is a three-dimensional schematic diagram of the droplet microfluidic chip for generating droplets of the present invention; Figure 3 is a schematic diagram of the droplets generated in the droplet generation area of the present invention; Figures 1 to 3 The reference numerals shown in are respectively represented as: oil phase injection port 1, first aqueous phase injection port 2, second aqueous phase injection port 3, liquid outlet 4, reaction liquid mixing area 5, droplet generation area 6, solution mixing area 7, upper channel pattern layer 8, cover glass 9, solid polydimethylsiloxane 10, first aqueous phase flow pipeline 11, annular oil phase pipeline 12, serpentine flow pipeline 13, second aqueous phase flow pipeline 14. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0016] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0017] Please refer to Figures 1-2 , the present invention provides a droplet microfluidic in-situ test chip for studying the kinetics of microbial mineralization crystallization and its test method, aiming to solve the problem of the lack of effective experimental methods when studying the calcium carbonate crystallization kinetics in microbial mineralization, and the problem that it is difficult to in-situ and real-time observe the crystal nucleation sites in the prior art of microbial mineralization. The following is a detailed description of the specific implementation manners of the present invention.

[0018] First, prepare a droplet microfluidic chip. The droplet microfluidic chip sequentially includes an upper channel pattern layer 8, a solid polydimethylsiloxane 10, and a cover glass 9 from top to bottom. The upper channel pattern layer 8 is made of cured liquid polydimethylsiloxane, and an oil-phase injection port 1 is provided at its top. The oil-phase injection port 1 is connected to the inlet end of an annular oil-phase pipeline 12. A first aqueous-phase injection port 2 and a second aqueous-phase injection port 3 are provided in the area surrounded by the position of the annular oil-phase pipeline 12. A reaction liquid mixing area 5 is formed at the confluence of the first aqueous-phase flow pipeline 11 communicated with the first aqueous-phase injection port 2 and the second aqueous-phase flow pipeline 14 communicated with the second aqueous-phase injection port 3. A droplet generation area 6 is formed at the intersection of the annular oil-phase pipeline 12 and the confluence of the first aqueous-phase flow pipeline 11 and the second aqueous-phase flow pipeline 14. The droplets in the droplet generation area 6 form a droplet internal solution mixing area 7 in the serpentine flow pipeline 13. The droplets in the serpentine flow pipeline 13 flow out through the liquid outlet 4.

[0019] During the preparation process, first pour the liquid polydimethylsiloxane into a mold, and the shape of the mold matches the design of the upper channel pattern layer 8. After the liquid polydimethylsiloxane is cured, the upper channel pattern layer 8 is formed. Then, cover a thin layer of solid polydimethylsiloxane 10 between the upper channel pattern layer 8 and the cover glass 9 of the lower base layer to seal the channels and prevent solution leakage. The thickness of the solid polydimethylsiloxane 10 can be adjusted as needed to ensure the sealing performance and stability of the channels and the focusing accuracy of the microscope.

[0020] Next, the structure of the droplet microfluidic chip is divided. An oil-phase injection port 1, a first aqueous-phase injection port 2, a second aqueous-phase injection port 3, and an outlet port 4 are provided on the droplet microfluidic chip. The oil-phase injection port 1 is used to inject the continuous-phase liquid, and the first aqueous-phase injection port 2 and the second aqueous-phase injection port 3 are respectively used to inject the microbial mineralization bacterial solution and the microbial mineralization cementing solution. The reaction liquid mixing area 5 is located at the connection of the first aqueous-phase injection port 2 and the second aqueous-phase injection port 3 and is used to mix the two aqueous-phase solutions. The droplet generation area 6 is located at the connection of the first aqueous-phase flow pipe 11 and the second aqueous-phase flow pipe 14 with the annular oil-phase pipe 12. When the continuous phase and the aqueous-phase solution meet, droplets are formed under the action of shear force. The serpentine flow pipe 13 forms the solution mixing area 7 inside the droplet, where the droplets are further mixed evenly. Finally, the droplets flow out through the outlet port 4.

[0021] Fix the prepared droplet microfluidic chip on the stage of a fluorescence inverted microscope for subsequent observation and testing. The fluorescence inverted microscope has high resolution and real-time observation ability, and can clearly observe the microbial mineralization reaction process inside the droplets.

[0022] Refer to Figure 3 , and use three disposable sterile syringes to suck in the microbial mineralization bacterial solution, the microbial mineralization cementing solution, and the continuous-phase solution respectively. The microbial mineralization bacterial solution is obtained by centrifuging the bacterial solution containing the culture medium obtained after expansion culture, removing the supernatant, and suspending it in sterile physiological saline. The microbial mineralization cementing solution is obtained by mixing the sterile calcium chloride solution and the sterile urea solution evenly. The concentration range of calcium chloride is 0.1 - 5 M, and the concentration range of urea is 0.1 - 5 M. The optical density value OD 600 of the microbial mineralization bacterial solution is 0.2 - 1.8, and the pH value is 7.0 - 9.5. The continuous-phase solution is obtained by mixing the surfactant and the oil-based solvent evenly. The surfactant can be any one or more of span80 and Degussa ABIL EM180, and the oil-based solvent can be any one or more of liquid paraffin, mineral oil, and dimethyl silicone oil. The content of the surfactant in the continuous phase is 0.1% - 5% (v / v).

[0023] Connect the three syringes to three tygon hoses through needles respectively. The other side of the tygon hoses is connected to steel needles, and the syringes are fixed on the injection pump. The injection pump is used to control the injection rate of the solution to ensure the stable generation of droplets.

[0024] Connect the steel needle connecting the syringe and the hose to the oil-phase injection port 1, the first water-phase injection port 2, and the second water-phase injection port 3 of the droplet microfluidic chip respectively, and connect another section of tygon hose to the liquid outlet 4 of the droplet microfluidic chip through a steel needle. In this way, the solution can be injected into the droplet microfluidic chip through an injection pump, and the generated droplets can be collected and exported through the hose at the liquid outlet.

[0025] The process of generating droplets is as follows: First, control the injection pump to inject the solutions in the three syringes into the droplet microfluidic chip simultaneously. By adjusting the injection rate of the injection pump, the generation rate and size of the droplets can be controlled. Observe the droplet generation process under an inverted microscope. The size of the droplets can be controlled by adjusting the flow rates of the water phase and the oil phase. Generally, the droplet size is 20 - 100 μm. The flow rate of the dispersed phase is 10 - 150 μL / hr, and the flow rate of the continuous phase is also 10 - 150 μL / hr. In this way, the stable generation of droplets can be ensured.

[0026] Place the culture dish at one end of the liquid outlet of the chip on the stage of the fluorescence inverted microscope. After the droplets are stably generated, collect the droplets generated in the chip and introduce them into the culture dish for subsequent observation.

[0027] Observe the process of microbial mineralization reaction. While the crystallization reaction occurs in the solution inside the droplet, observe the reaction process in the droplet in situ and in real time through an inverted microscope. Real-time images and information of microorganisms and crystals can be obtained, so as to analyze the kinetic parameters of microbial mineralization crystallization. During the observation process, the magnification and focal length of the microscope can be adjusted to obtain clearer images and more accurate data.

[0028] Specifically, in order to improve the accuracy and reliability of the observation, nucleic acid staining can also be performed on the microbial mineralization bacteria. Fluorescent dyes such as SYTO-9 and PI can be selected as the staining agents. Inject the stained microbial mineralization bacterial solution into the droplet microfluidic chip, and then observe it in the fluorescence mode of the fluorescence inverted microscope. In this way, the position and morphology of the bacteria can be identified more clearly, and thus the nucleation position of the crystals can be judged more accurately.

[0029] During the observation process, the reaction conditions can also be regulated as needed. For example, parameters such as the pH value, temperature, ion concentration, and type of calcium source of the reaction solution can be adjusted to explore the effects of different environmental conditions on the crystal morphology and growth law. The reaction temperature is controlled within the range of 15 - 40 °C to ensure the activity of microorganisms and the normal growth of crystals.

[0030] In addition, in order to further solve the problem that it is difficult to in-situ and real-time observe the crystal nucleation sites in the existing technology of microbial mineralization, the present invention also proposes a method for generating microgels to immobilize bacteria and then observing the crystal nucleation positions. The specific steps are as follows: First, mix the gelatin solution and the agar solution evenly to obtain a gelatin-agar aqueous solution. Then, mix the microbial mineralization bacterial solution with the gelatin-agar aqueous solution to obtain a microbe-gelatin-agar aqueous solution, and mix the microbial mineralization cementing solution with the gelatin-agar aqueous solution to obtain a cementing solution-gelatin-agar aqueous solution. Aspirate the microbe-gelatin-agar aqueous solution, the cementing solution-gelatin-agar aqueous solution, and the continuous phase into disposable syringes respectively. Repeat the above droplet generation process for the subsequent steps, collect the droplets and cool them to 15-25 °C to obtain gelatin-agar microgels immobilizing the microbes. In-situ and real-time observe the microbial mineralization reaction process in the gelatin-agar microgels through an inverted microscope. Since the gelatin-agar microgels have the function of immobilizing bacteria, the positional relationship between the bacteria and the calcium carbonate nucleation can be clearly observed, thus solving the controversial problem of "whether calcium carbonate takes bacteria as the nucleation site" in microbial mineralization.

[0031] In the specific implementation process, the microbial mineralization bacterial solution can select bacteria with mineralization ability such as Sporosarcina pasteurii. The microbial mineralization cementing solution is a mixed solution of other calcium ion solutions such as calcium chloride and a urea solution, and the concentration ratio of the two can be adjusted to 1:1 or other appropriate ratios. The mineral generated by microbial mineralization is calcium carbonate or other minerals with research value.

[0032] Through the droplet microfluidics in-situ testing method of the present invention, in-depth research on the crystallization kinetics of microbial mineralization can be achieved. This method has the advantages of high throughput, precise reaction conditions, accurate observation, etc., and can quickly obtain the crystallization parameters and kinetic information of microbial mineralization. At the same time, by introducing hydrogel materials to immobilize bacteria and observing the positional relationship between the bacteria and the calcium carbonate nucleation, the nucleation mechanism of microbial mineralization can be explored and the hypothesis that bacteria serve as nucleation sites can be verified.

[0033] In the specific implementation process, further optimization and adjustment can also be carried out according to experimental needs. For example, the size, shape, and quantity of the droplets can be adjusted to meet different experimental requirements; the composition and concentration of the reaction solution can be optimized to obtain a more ideal crystallization effect; other advanced characterization techniques and instruments such as in-situ spectroscopy, in-situ electrochemistry, and in-situ thermology can also be combined to expand the research scope.

[0034] In summary, the present invention provides a droplet microfluidic in-situ test chip for the study of microbial mineralization crystallization kinetics and its test method. This method has the advantages of high throughput, precise reaction conditions, accurate observation, etc., and can achieve in-depth research on microbial mineralization crystallization kinetics. At the same time, by introducing a hydrogel material to immobilize bacteria and observing the positional relationship between bacteria and calcium carbonate nucleation, the microbial mineralization nucleation mechanism can be further explored and related hypotheses can be verified. The technical solution of the present invention provides new ideas and methods for the research in the field of microbial mineralization, and has important academic value and practical value.

[0035] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A droplet microfluidic in-situ test chip for the study of microbial mineralization crystallization kinetics, characterized in that, Comprising: An upper channel pattern layer (8), a solid polydimethylsiloxane (10), and a cover glass (9) arranged in sequence from top to bottom. An oil phase injection port (1) is provided at the top of the upper channel pattern layer (8). The oil phase injection port (1) is connected to the inlet end of an annular oil phase pipeline (12). Inside the area of the annular oil phase injection pipeline (12), a first aqueous phase injection port (2) and a second aqueous phase injection port (3) are provided. A first aqueous phase flow pipeline (11) communicated with the first aqueous phase injection port (2) and a second aqueous phase flow pipeline (14) communicated with the second aqueous phase injection port (3) form a reaction liquid mixing area (5) at the confluence. The pipeline formed after the annular oil phase pipeline (12) intersects with the first aqueous phase flow pipeline (11) and the second aqueous phase flow pipeline (14) forms a droplet generation area (6). Droplets in the droplet generation area (6) form a solution mixing area (7) inside the droplet in a serpentine flow pipeline (13). The droplets in the serpentine flow pipeline (13) flow out through an outlet (4).

2. The testing method of the droplet microfluidic in-situ testing chip for microbial mineralization crystallization kinetics research according to claim 1, characterized in that, Including the following steps: S1. Prepare a droplet microfluidic chip: Use liquid polydimethylsiloxane to solidify to make an upper channel pattern layer (8), and cover a thin layer of solid polydimethylsiloxane (10) between the upper channel pattern layer (8) and the cover glass (9) of the lower substrate layer; S2. Set the structure of the droplet microfluidic chip: Set an oil phase injection port (1), a first aqueous phase injection port (2), a second aqueous phase injection port (3), and an outlet (4) on the droplet microfluidic chip, and construct a reaction liquid mixing area (5), a droplet generation area (6), and a solution mixing area (7) inside the droplet; S3. Fix the droplet microfluidic chip: Fix the prepared droplet microfluidic chip on the stage of a fluorescence inverted microscope; S4. Prepare solutions and solvents: Use three disposable sterile syringes to respectively suck in microbial mineralization bacterial solution, microbial mineralization cementation solution, and continuous phase liquid; S5. Connect the syringe pump and the syringe: Connect the three syringes to three tygon hoses respectively through needles. The other side of the tygon hoses is connected to steel needles, and fix the syringes on the syringe pump; S6. Connect the droplet microfluidic chip and the syringe: Connect the steel needles in the step S5 to the oil phase injection port (1), the first aqueous phase injection port (2), and the second aqueous phase injection port (3) of the droplet microfluidic chip respectively, and connect one end of another section of tygon hose to the outlet of the droplet microfluidic chip through a steel needle; S7. Generate droplets: Control the syringe pump, inject the solutions in the three syringes into the droplet microfluidic chip simultaneously, generate droplets of the required size by adjusting the injection rate of the syringe pump, and observe the generation process under an inverted microscope; S8. Collect droplets: Place a culture dish at one end of the chip outlet on the stage of the fluorescence inverted microscope. After the droplets are stably generated, collect the droplets generated in the chip; S9. Observe the microbial mineralization reaction process: While the solution in the droplet undergoes a crystallization reaction, in-situ and real-time observe the reaction process in the droplet through an inverted microscope to obtain microbial and crystal information.

3. The droplet microfluidic in-situ testing method for microbial mineralization crystallization kinetics according to claim 2, characterized in that The method for generating droplets in the step S7 includes the following steps: S1. Preparation of microbial mineralization bacterial liquid: Centrifuge the bacterial liquid containing the culture medium obtained after amplification culture, remove the supernatant, and suspend it in sterile physiological saline to obtain the microbial mineralization bacterial liquid; S2. Preparation of microbial mineralization cementing liquid: Mix the sterile calcium chloride solution and the sterile urea solution evenly to obtain the microbial mineralization cementing liquid; S3. Preparation of continuous phase: Mix the surfactant and the oil-based solvent evenly to obtain the continuous phase liquid; S4. Aspirate the microbial mineralization bacterial liquid, the microbial mineralization cementing liquid, and the continuous phase liquid into disposable syringes respectively; S5. Connect the syringe containing the microbial mineralization bacterial liquid to the aqueous phase injection port (2) on the droplet microfluidic chip, and control the injection pump to input the microbial mineralization bacterial liquid; S6. Connect the syringe containing the microbial mineralization cementing liquid to the aqueous phase injection port (3) on the droplet microfluidic chip, and control the injection pump to input the microbial mineralization cementing liquid; S7. Connect the syringe containing the continuous phase liquid to the oil phase injection port (1) on the droplet microfluidic chip, and control the injection pump to input the oil phase. When the continuous phase and the dispersed phase pass through the droplet generation region (6), "water-in-oil" droplets are obtained; S8. When the generated droplets pass through the droplet generation region (7), the solutions in the droplets are fully mixed evenly.

4. The droplet microfluidic in-situ testing method for microbial mineralization crystallization kinetics according to claim 3, characterized in that, The calcium chloride concentration in step S2 is 0.1 - 5 M, and the urea concentration is 0.1 - 5 M; the optical density value of the microbial mineralization bacterial solution in step S1 is OD 600 = 0.2 - 1.8, and the pH is 7.0 - 9.5; the flow rates of the microbial mineralization bacterial solution in step S5 and the microbial mineralization cementing solution in step S6 are 10 - 150 μL / hr; the flow rate of the continuous phase is 10 - 150 μL / hr; The surfactant in step S3 is any one or two of span80 and Degussa ABIL EM180; the oil-based solvent is any one or more of liquid paraffin, mineral oil, and dimethyl silicone oil; the content of the surfactant in the continuous phase is 0.1%-5% (v / v).

5. The droplet microfluidic in-situ testing method for microbial mineralization crystallization kinetics research according to claim 3, characterized in that In steps S1-S3, the following steps are further included: Mix the gelatin solution and the agar solution evenly to obtain the gelatin-agar aqueous solution, mix the microbial mineralization bacterial liquid with the gelatin-agar aqueous solution to obtain the microbial-gelatin-agar aqueous solution, mix the microbial mineralization cementing liquid with the gelatin-agar aqueous solution to obtain the cementing liquid-gelatin-agar aqueous solution, and aspirate the microbial-gelatin-agar aqueous solution, the cementing liquid-gelatin-agar aqueous solution, and the continuous phase liquid into disposable syringes respectively. The subsequent steps repeat steps S5-S8, collect the droplets and cool them to obtain the gelatin-agar microgel immobilizing microorganisms, and in-situ and real-time observe the microbial mineralization reaction process in the gelatin-agar microgel through an inverted microscope.

6. The droplet microfluidic in-situ testing method for microbial mineralization crystallization kinetics research according to any one of claims 2-5, characterized in that The microbial mineralization bacterial liquid is Sporosarcina pasteurii; the microbial mineralization cementing liquid is a mixed solution of calcium chloride solution and urea solution, and the concentration ratio of the two is 1:1; the mineral generated by microbial mineralization is calcium carbonate; the droplet size is 20-100 μm.

7. The droplet microfluidic in-situ testing method for microbial mineralization crystallization kinetics according to any one of claims 2-5, characterized in that, It also includes the step of nucleic acid staining of the microbial mineralization bacteria, and the staining agents are SYTO-9 and PI. The subsequent observation of the bacteria is carried out in the fluorescence mode of a fluorescence inverted microscope.

8. The droplet microfluidic in-situ testing method for microbial mineralization crystallization kinetics according to claim 3, wherein The reaction temperature of the operation steps in steps S5-S8 is controlled at 15-40°C.

9. The droplet microfluidic in-situ testing method for microbial mineralization crystallization kinetics according to claim 5, characterized in that, The temperature during the solution preparation and droplet preparation process in the above steps is controlled at 40°C; the temperature during the collection and cooling of the droplets in the above steps is controlled at 15-25°C.

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