Seepage-microbial clogging visual experimental device and experimental method based on microfluid

Abstract

The invention relates to the field of biological experiment equipment, in particular to a seepage-microbial clogging visual experiment device based on microfluid and an experiment method.The seepage-microbial clogging visual experiment device comprises an objective table, an experiment component is installed on the objective table, an observation component is arranged below the experiment component, and an image monitoring component is arranged above the experiment component; an optical blockage checking component is also arranged above the experimental component; the experimental component comprises a microfluidic chip sample, the inlet end of the microfluidic chip sample is communicated with an experimental liquid injection system, the outlet end of the microfluidic chip sample is communicated with a waste liquid discharge system, and a pressure monitoring device is arranged on a pipeline through which the microfluidic chip sample is communicated with the experimental liquid injection system; and a flow monitoring device is arranged on a pipeline for communicating the microfluidic chip sample with the waste liquid discharge system. The problem that multi-scale visual observation is difficult to synchronously carry out in the pore medium microorganism blockage experiment process is solved.

Description

Technical Field

[0001] This invention relates to the field of biological experimental equipment, and more specifically to a microfluidic-based visualization experimental device and method for permeation-microbial blockage. Background Technology

[0002] Biofilms are complex biological communities formed by microbial cells and their secreted extracellular polymeric substances (EPS) that adhere tightly to the surface of objects. In real environments, microorganisms often attach to human skin and oral cavity (in the form of dental plaque), food processing equipment, and medical equipment. Their formation often has both advantages and disadvantages. On the one hand, they increase the presence of pathogens, increase the drug resistance of microorganisms, cause pollution and damage to medical devices and implants, and lead to biofilm infection. On the other hand: (1) By using microorganisms to improve the microstructure of soil, soil permeability can be significantly reduced, preventing the transfer of pollutants (bioremediation technology). (2) Biofilms can also remove pollutants and excess nutrients from wastewater (moving bed biofilm reactor). (3) Biofilms can be used to selectively block high-permeability areas, thereby allowing fluids to flow to low-permeability areas to improve oil recovery (microbial enhanced oil recovery technology). Therefore, studying the development of biofilms in porous media and their interaction with fluid flow is of great significance for the application of many engineering technologies. However, most current research on microbial blockage in porous media is based on "black box experiments," inferring the extent of biological blockage within the porous media solely from changes in apparent phenomena such as permeability coefficients and inlet / outlet water quality. This lacks a comprehensive understanding of the entire blockage process and results in a disconnect between microscopic and macroscopic observations. Therefore, developing a multi-scale, real-time dynamic observation platform for a porous media microfluidic model and microbial blockage is of great significance for conducting experiments on microbial-induced porous media blockage. Summary of the Invention

[0003] The purpose of this invention is to provide a microfluidic-based visualization experimental device and method for permeation-microbial blockage, in order to solve the above-mentioned problems and overcome the difficulty of simultaneously conducting multi-scale visualization observations during microbial blockage experiments in porous media.

[0004] To achieve the above objectives, the present invention provides the following solution:

[0005] A microfluidic-based visualization experimental device for seepage-microbial blockage includes a stage on which experimental components are mounted. An observation component is located below the experimental components, an image monitoring component is located above the experimental components, and an optical blockage detection component is located above the experimental components.

[0006] The experimental components include a microfluidic chip sample. The inlet end of the microfluidic chip sample is connected to an experimental liquid injection system, and the outlet end of the microfluidic chip sample is connected to a waste liquid discharge system. A pressure monitoring device is installed on the pipeline connecting the microfluidic chip sample and the experimental liquid injection system, and a flow monitoring device is installed on the pipeline connecting the microfluidic chip sample and the waste liquid discharge system. The image monitoring component, the optical blockage detection component, the pressure monitoring device, and the flow monitoring device are connected to a workstation.

[0007] Preferably, the optical blockage detection component includes a photosensitive receiver, which can be attached to the upper surface of the microfluidic chip sample by moving its position. The photosensitive receiver is signal-connected to an optical signal processing unit, which sends the processed signal to the workstation.

[0008] Preferably, the photosensitive receiver includes a frame in which a plurality of optical fibers are installed. One end of each optical fiber near the microfluidic chip sample is in contact with the top surface of the microfluidic chip sample, and the other end of each optical fiber is connected to the optical signal processing unit. The optical signal processing unit includes a plurality of photosensitive elements, which are connected to a signal processing unit. The signal processing unit sends the signal from the photosensitive elements to the workstation.

[0009] Preferably, the observation component is an optical microscope, which is located below the experimental component and mounted on the stage.

[0010] Preferably, a light source is provided between the optical microscope and the microfluidic chip sample, and the microfluidic chip sample is placed on the light source.

[0011] Preferably, the experimental liquid injection system includes two first injection pumps and two second injection pumps. A first syringe is mounted on each of the first injection pumps, and a second syringe is mounted on each of the second injection pumps. The first and second injection pumps are signal-connected to the workstation. The outlet of the first syringe is connected to two inlets of a fluid switching valve via a pipeline. The outlet of the second injection pump is connected to the other two inlets of the fluid switching valve via a pipeline. The outlet of the fluid switching valve is connected to the inlet of the microfluidic chip sample via a pipeline. The pressure monitoring device is installed on the pipeline connecting the fluid switching valve and the microfluidic chip sample. The outlet of the microfluidic chip sample is connected to a waste liquid beaker via a pipeline.

[0012] Preferably, a support is mounted on the stage, the light source is mounted on the support, and a temperature sensor is provided on one side of the microfluidic chip sample, the temperature sensor being signal-connected to the workstation.

[0013] A microfluidic permeation-microbial blockage visualization experimental method, the method being implemented based on the experimental apparatus described in the aforementioned scheme, specifically including the following steps:

[0014] Fluid was injected into the microfluidic chip sample using an experimental liquid injection system to expel air from the microfluidic chip sample.

[0015] To prepare the experimental bacterial suspension, the bacterial strain was inoculated into a liquid culture medium and placed in a constant temperature shaker for overnight culture. The bacterial suspension was then re-inoculated into a new liquid culture medium and cultured for a fixed time.

[0016] The experimental apparatus was sterilized using the experimental liquid injection system.

[0017] The injected fluid is converted into a new liquid culture medium using the experimental liquid injection system.

[0018] The cultured bacterial suspension was injected into the microfluidic chip sample and left to stand for a fixed time. After the bacteria successfully attached to the surface of the porous medium, the liquid culture medium was switched and the injection continued.

[0019] The image monitoring component is used to acquire images at regular intervals, and the observation component is used to observe the attachment and growth of microorganisms on the pore surface.

[0020] When the data from the pressure monitoring device and the flow monitoring device are abnormal, the optical blockage detection component is used to check the blockage of the microfluidic chip sample.

[0021] The workstation is used to collect and organize data, and to perform data analysis and calculations.

[0022] The present invention has the following technical effects:

[0023] 1. The monitoring system of this invention employs microscopic imaging technology, combined with fluorescent labeling and high-speed imaging, to achieve real-time, dynamic visualization of the entire process of biofilm growth and blockage at the pore scale. It allows direct observation of how microorganisms attach and proliferate on the pore surface, the secretion of extracellular polymeric substances (EPS) and their gradual filling of pores, and the spatial distribution of the biofilm community, providing a more intuitive perspective for a deeper understanding of bioblockage mechanisms. Simultaneously, it enables macroscopic observation of the porous media, allowing real-time monitoring of the evolution of fluid channels becoming blocked over time.

[0024] 2. Pressure monitoring system: Pressure sensors are installed at the inlet and outlet of the microfluidic chip to record the blockage pressure caused by microbial growth in real time. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a schematic diagram of the structure of the present invention;

[0027] Figure 2 This is a bottom view of the optical sensor receiver of the present invention.

[0028] The components include: 1. Stage; 2. Optical microscope; 3. High-speed camera; 4. Light source; 5. Microfluidic chip sample; 6. Pressure monitoring device; 7. Flow monitoring device; 8. Temperature sensor; 9. First injection pump; 10. Second injection pump; 11. First syringe; 12. Second syringe; 13. Fluid switching valve; 14. Waste liquid beaker; 15. Support; 16. Workstation; 17. Photosensitive receiver; 1701. Frame; 1702. Optical fiber; 18. Optical signal processing unit. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0030] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] Reference Figures 1 to 2 As shown, this embodiment provides a microfluidic-based permeation-microbial blockage visualization experimental device, including a stage 1, an experimental component mounted on the stage 1, an observation component below the experimental component, an image monitoring component above the experimental component, and an optical blockage detection component above the experimental component.

[0032] The experimental components include a microfluidic chip sample 5, with the inlet end of the microfluidic chip sample 5 connected to an experimental liquid injection system and the outlet end of the microfluidic chip sample 5 connected to a waste liquid discharge system. A pressure monitoring device 6 is installed on the pipeline connecting the microfluidic chip sample 5 and the experimental liquid injection system, and a flow monitoring device 7 is installed on the pipeline connecting the microfluidic chip sample 5 and the waste liquid discharge system. The image monitoring component, the optical blockage detection component, the pressure monitoring device 6, and the flow monitoring device 7 are connected to a workstation 16.

[0033] The scheme is further optimized. The optical blockage inspection component includes a photosensitive receiver 17. The photosensitive receiver 17 can be attached to the upper surface of the microfluidic chip sample 5 by moving its position. The photosensitive receiver 17 is connected to a photoelectric signal processing unit 18, which sends the processed signal to the workstation 16.

[0034] In a further optimized scheme, the photosensitive receiver 17 includes a frame 1701, in which several optical fibers 1702 are installed. One end of each optical fiber 1702 near the microfluidic chip sample 5 is in contact with the top surface of the microfluidic chip sample 5, and the other end of the optical fiber 1702 is connected to the optical signal processing unit 18. The optical signal processing unit 18 includes several photosensitive elements, which are connected to a signal processing unit. The signal processing unit sends the signal from the photosensitive elements to the workstation 16.

[0035] Before the experiment, each optical fiber 1702 is numbered. The arrangement of the optical fibers 1702 corresponds to the pores and channels on the microfluidic chip sample 5. It is used in conjunction with the light source 4. The specific principle is that the light transmittance varies after the colonies grow, resulting in different light intensities entering the optical fiber 1702. A photodiode can be used as the photosensitive element, thus generating different electrical signal intensities. After passing through the signal processing unit, the corresponding numbered light is transmitted to the workstation for recording, allowing determination of which locations are blocked and which are partially blocked. When the pressure monitoring device 6 or the flow monitoring device 7 malfunctions, the state of the microfluidic chip sample 5 can be determined through the photosensitive receiver 17.

[0036] The design was further optimized so that the observation component is an optical microscope 2, which is located below the experimental components and is mounted on the stage 1.

[0037] In a further optimized design, a light source 4 is provided between the optical microscope 2 and the microfluidic chip sample 5, and the microfluidic chip sample 5 is placed on the light source 4.

[0038] Further optimization of the scheme: The experimental liquid injection system includes two first injection pumps 9 and two second injection pumps 10. A first syringe 11 is installed on the first injection pump 9, and a second syringe 12 is installed on the second injection pump 10. The first injection pumps 9 and 10 are connected to the workstation 16 via signal. The liquid outlet of the first syringe 11 is connected to the two inlets of the fluid switching valve 13 via a pipeline. The liquid outlet of the second injection pump 10 is connected to the other two inlets of the fluid switching valve 13 via a pipeline. The liquid outlet of the fluid switching valve 13 is connected to the inlet of the microfluidic chip sample 5 via a pipeline. A pressure monitoring device 6 is installed on the pipeline connecting the fluid switching valve 13 and the microfluidic chip sample 5. The outlet of the microfluidic chip sample 5 is connected to a waste liquid beaker 14 via a pipeline.

[0039] Further optimization of the design involves mounting a support 15 on the stage 1, with the light source 4 mounted on the support 15. A temperature sensor 8 is located on one side of the microfluidic chip sample 5, and the temperature sensor 8 is connected to the workstation 16. The light source 4 is made of a translucent acrylic sheet, with LEDs arranged on one side of the acrylic sheet. This arrangement allows the entire surface of the microfluidic chip sample 5 to be illuminated.

[0040] In this embodiment, the signal connection method of each component can adopt existing technologies such as signal lines and cables, which can realize the mutual transmission of signals, and there are no other special requirements.

[0041] A microfluidic permeation-microbial blockage visualization experimental method, implemented based on the aforementioned experimental setup, specifically includes the following steps:

[0042] Fluid was injected into the microfluidic chip sample 5 using an experimental liquid injection system to expel the air from the microfluidic chip sample 5.

[0043] To prepare the experimental bacterial suspension, the bacterial strain was inoculated into a liquid culture medium and placed in a constant temperature shaker for overnight culture. The bacterial suspension was then re-inoculated into a new liquid culture medium and cultured for a fixed time.

[0044] The experimental apparatus was sterilized using an experimental liquid injection system.

[0045] The injected fluid was converted into a new liquid culture medium using an experimental liquid injection system;

[0046] The cultured bacterial suspension was injected into the microfluidic chip sample 5 and left to stand for a fixed time. After the bacteria successfully attached to the surface of the porous medium, the liquid culture medium was switched and the injection continued.

[0047] The image monitoring component is used to acquire images at regular intervals, and the observation component is used to observe the attachment and growth of microorganisms on the pore surface.

[0048] When the data from pressure monitoring device 6 and flow monitoring device 7 are abnormal, the blockage of microfluidic chip sample 5 is checked using an optical blockage inspection component.

[0049] Workstation 16 was used to collect and organize data, and to perform data analysis and calculations.

[0050] This embodiment uses glycerol bacteria as an example; the specific experimental steps are as follows:

[0051] S1: Fabricate microfluidic chip sample 5, according to... Figure 1 The experimental tubing is shown in the diagram. Fluid (here, vacuum-treated deionized water) is injected into the microfluidic chip sample 5 using a silicone tubing, fluid switching valve 13, and a flat-headed steel needle. The pressure monitoring device 6 and flow monitoring device 7 are then connected to the microfluidic chip sample 5. Special attention should be paid to the connection of the pressure monitoring device 6 and flow monitoring device 7. The pressure monitoring device 6 uses a pressure sensor, and the flow monitoring device 7 uses a flow sensor. To ensure the reliability of the measurement data, when connecting the sensors, allow the fluid to slowly enter the sensor cavity, expel the air inside the cavity, and then close the other end of the sensor using a plug. Avoid touching the sensors during the experiment to prevent interference with the data measurement.

[0052] S2: Preparation of experimental bacterial suspension. Frozen glycerol bacteria were inoculated into liquid culture medium at 30℃ and 200 rpm and incubated overnight on a constant-temperature shaker. Before the experiment, the bacterial suspension was re-inoculated into the liquid culture medium at a ratio of 1:1000 and incubated for 3-4 hours until the microorganisms entered the early exponential growth phase.

[0053] S3: Install the experimental setup on the optical platform, place the prepared microfluidic chip sample 5 on the fixing device, turn on the light source and high-speed camera 3, and focus the field of view on the plane of the microfluidic chip sample 5.

[0054] S4: Sterilization of the experimental system. Using a first syringe pump 9 and a first syringe 11, slowly inject 75% alcohol into the chip to sterilize the entire experimental system for 1 hour.

[0055] S5: Start a second injection pump 10 and use the second syringe 12 to switch the injected fluid to a nutrient solution via the fluid switching valve 13, and continue injecting for 3 hours to completely remove the alcohol from the experimental system. It is important to note that the microfluidic chip sample 5 must remain saturated throughout the entire process.

[0056] S6: Turn on another first syringe pump 9 and use another first syringe 11 to inject the bacterial suspension into the microfluidic chip sample 5, and let it stand for 3 hours. After the bacterial cells have successfully attached to the surface of the porous medium, turn on another second syringe pump 10 and use another second syringe 12 to control the switching valve 13 to switch to the culture medium and continue injection. Connect the outlet to the waste liquid beaker 14 to collect the experimental waste liquid, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the concentration of microorganisms in the outflowing waste liquid.

[0057] S7: Turn on the light source 4 and high-speed camera 3 to acquire images at regular intervals and assess the growth and blockage of microorganisms in real time. Simultaneously, the optical microscope 2 can be used to observe in real time the attachment and growth of microorganisms on the pore surface, the secretion of extracellular polymeric substances (EPS), the evolution of cell activity, and the spatial distribution of the biofilm community. When the pressure monitoring device 6 and flow monitoring device 7 show abnormalities, the optical blockage detection component is used to check the blockage of the microfluidic chip sample 5.

[0058] S8: Finally, workstation 16 is used to collect and organize data, and to perform data analysis and calculation.

[0059] In the description of this invention, it should be understood that the terms "longitudinal", "lateral", "up", "down", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this invention, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention.

[0060] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A microfluidic-based visualization experimental device for seepage-microbial blockage, characterized in that, Includes a stage (1), on which an experimental component is mounted, an observation component is provided below the experimental component, an image monitoring component is provided above the experimental component, and an optical blockage detection component is also provided above the experimental component; The experimental components include a microfluidic chip sample (5), the inlet of which is connected to an experimental liquid injection system, and the outlet of which is connected to a waste liquid discharge system. A pressure monitoring device (6) is provided on the pipeline connecting the microfluidic chip sample (5) and the experimental liquid injection system, and a flow monitoring device (7) is provided on the pipeline connecting the microfluidic chip sample (5) and the waste liquid discharge system. The image monitoring component, the optical blockage inspection component, the pressure monitoring device (6), and the flow monitoring device (7) are connected to a workstation (16).

2. The microfluidic-based permeation-microbial blockage visualization experimental device according to claim 1, characterized in that, The optical blockage detection component includes a photosensitive receiver (17), which can be attached to the upper surface of the microfluidic chip sample (5) by moving its position. The photosensitive receiver (17) is signal connected to an optical signal processing unit (18), which sends the processed signal to the workstation (16).

3. The microfluidic-based permeation-microbial blockage visualization experimental device according to claim 2, characterized in that, The photosensitive receiver (17) includes a frame (1701), in which a plurality of optical fibers (1702) are installed. One end of the plurality of optical fibers (1702) near the microfluidic chip sample (5) is in contact with the top surface of the microfluidic chip sample (5), and the other end of the optical fibers (1702) is connected to the optical signal processing unit (18). The optical signal processing unit (18) includes a plurality of photosensitive elements, which are connected to a signal processing unit. The signal processing unit sends the signal of the photosensitive elements to the workstation (16).

4. The microfluidic-based permeation-microbial blockage visualization experimental device according to claim 1, characterized in that, The observation component is an optical microscope (2), which is located below the experimental component and is mounted on the stage (1).

5. The microfluidic-microbial blockage visualization experimental device according to claim 1, wherein the image monitoring component is a high-speed camera (3), the high-speed camera (3) is signal connected to the optical signal processing component (18), the high-speed camera (3) is mounted on the movable arm, and the movable arm is mounted on the stage (1).

6. The microfluidic-based permeation-microbial blockage visualization experimental device according to claim 4, characterized in that, A light source (4) is provided between the optical microscope (2) and the microfluidic chip sample (5), and the microfluidic chip sample (5) is placed on the light source (4).

7. The microfluidic-based permeation-microbial blockage visualization experimental device according to claim 1, characterized in that, The experimental liquid injection system includes two first injection pumps (9) and two second injection pumps (10). A first syringe (11) is installed on the first injection pump (9), and a second syringe (12) is installed on the second injection pump (10). The first injection pump (9) and the second injection pump (10) are connected to the workstation (16) via signal. The liquid outlet of the first syringe (11) is connected to the two liquid inlets of the fluid switching valve (13) via a pipeline. The liquid outlet of the second injection pump (10) is connected to the other two liquid inlets of the fluid switching valve (13) via a pipeline. The liquid outlet of the fluid switching valve (13) is connected to the inlet of the microfluidic chip sample (5) via a pipeline. The pressure monitoring device (6) is installed on the pipeline connecting the fluid switching valve (13) and the microfluidic chip sample (5). The outlet of the microfluidic chip sample (5) is connected to a waste liquid beaker (14) via a pipeline.

8. The microfluidic-based permeation-microbial blockage visualization experimental device according to claim 6, characterized in that, A support bracket (15) is installed on the stage (1), and the light source (4) is installed on the support bracket (15). A temperature sensor (8) is provided on one side of the microfluidic chip sample (5), and the temperature sensor (8) is connected to the workstation (16) via signal.

9. A microfluidic permeation-microbial blockage visualization experimental method, characterized in that, The method is implemented based on the experimental apparatus according to any one of claims 1-8, and specifically includes the following steps: Fluid is injected into the microfluidic chip sample (5) through an experimental liquid injection system to expel the air inside the microfluidic chip sample (5); To prepare the experimental bacterial suspension, the bacterial strain was inoculated into a liquid culture medium and placed in a constant temperature shaker for overnight culture. The bacterial suspension was then re-inoculated into a new liquid culture medium and cultured for a fixed time. The experimental apparatus was sterilized using the experimental liquid injection system. The injected fluid is converted into a new liquid culture medium using the experimental liquid injection system. The cultured bacterial suspension was injected into the microfluidic chip sample (5), and left to stand for a fixed time. After the bacteria successfully attached to the surface of the pore medium, the liquid culture medium was switched and the injection continued. The image monitoring component is used to acquire images at regular intervals, and the observation component is used to observe the attachment and growth of microorganisms on the pore surface. When the data from the pressure monitoring device (6) and the flow monitoring device (7) are abnormal, the optical blockage detection component is used to check the blockage of the microfluidic chip sample (5). The workstation (16) is used to collect and organize data, and to perform data analysis and calculation.

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