A dynamic biomimetic microfluidic chip, system and method for real-time evaluation of bacterial mucosal penetration

Abstract

This invention discloses a dynamic biomimetic microfluidic chip, system, and method for real-time evaluation of bacterial mucosal penetration ability, belonging to the field of microfluidic chip technology. The chip employs a fully transparent structure bonded to a single layer of PDMS and glass, and features at least two sets of fluid injection channels, a triangular diffusion region with a flexible thin film (FHR membrane), and a laminar flow observation region. A stable bacterial-mucus contact interface layer is formed through laminar diffusion. Controllable lateral pulsating disturbances are generated by the flexible thin film, coupling with the longitudinal flow within the laminar flow observation region to form a dynamic biomimetic flow field simulating the physiological environment of biological mucosa. The accompanying system and method achieve a closed-loop process from biomimetic environment construction and real-time visualization observation to data quantification analysis. This invention is highly versatile and adaptable to various biological mucosal scenarios such as fish mucosa, intestinal mucosa, and respiratory mucosa, solving the technical problems of low biomimeticity, poor versatility, and inability to conduct real-time observation in existing technologies.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chip technology, specifically to a dynamic biomimetic microfluidic chip, system, and method for real-time evaluation of bacterial mucosal penetration ability. It can be applied to the development of fish vaccines, delivery of mucosal vaccines for humans and animals, and research on the mucosal invasion mechanisms of pathogenic bacteria. It is particularly suitable for simulating biological mucus environments with non-Newtonian fluid characteristics to observe the correlation between bacterial movement patterns in mucus and mucosal penetration. Background Technology

[0002] The mucosal barrier is the first line of defense for humans and animals against the invasion of exogenous microorganisms, and it is also the core channel for the in vivo delivery of mucosal vaccines and oral drugs. Whether it is immersion vaccines in aquaculture, oral vaccines for mammals, or intestinal / respiratory targeted drugs, their core function relies on the ability of the carrier bacteria / drug carrier to effectively penetrate the mucus layer of the mucosa and achieve epithelial cell delivery. Therefore, accurately evaluating the mucosal penetration ability of carrier bacteria is a core step in vaccine development, drug screening, and pathogenic mechanism research.

[0003] The mucus layer of biological membranes is essentially a typical non-Newtonian fluid, exhibiting rheological properties such as shear thinning, viscoelasticity, and thixotropy, which are fundamentally different from the hydrodynamic characteristics of conventional Newtonian fluids (such as water and culture media). The biofilm penetration ability of bacteria relies heavily on the oscillation and rotation of their flagella. The resistance to movement, power transmission efficiency, and trajectory in non-Newtonian mucus are drastically different from those in conventional Newtonian water. For example, bacterial flagella typically exhibit uniform helical rotation in Newtonian fluids, but in non-Newtonian mucus, due to the viscoelastic resistance of the mucus, the frequency and amplitude of flagellar oscillation change significantly, and even a switch in movement pattern occurs. This difference directly determines the actual biofilm penetration efficiency of bacteria. Therefore, the core prerequisite for accurately evaluating the biofilm penetration ability of bacteria is to simulate the non-Newtonian fluid characteristics of biological mucus and simultaneously achieve real-time visualization and observation of the bacterial movement process within the mucus.

[0004] Currently, the evaluation methods for bacterial mucosal penetration ability are mainly divided into two categories: in vivo live experiments and in vitro static culture, both of which have obvious technical shortcomings. In vivo experiments: These require the use of laboratory animals for challenge experiments, which are complex, time-consuming, and costly. Furthermore, the variables in the animals are uncontrollable, making it impossible to precisely regulate the non-Newtonian rheological parameters of the mucus, to achieve real-time visualization of the movement of bacteria in the mucus, and to accurately quantify the movement behavior and penetration efficiency of a single bacterium. In vitro static culture: This method, which mainly uses Transwell chambers and plate culture, can only achieve the endpoint detection of penetration rate in a static environment. It cannot simulate the real bacterial-mucus contact interface of biological mucosa, cannot achieve real-time motion visualization observation of bacterial penetration process, and cannot reproduce the dynamic disturbance of the physiological environment of the mucosa (such as water flow disturbance when fish swim, flow field changes during intestinal peristalsis, airflow shearing in the respiratory tract, etc.). This results in a certain deviation between the evaluation results and the real physiological scenario, and cannot provide accurate supporting data for vaccine / drug development.

[0005] Microfluidic chip technology, with its advantages of precise control of microscale flow fields, visualization observation, and biomimetic environment construction, has been gradually applied to fields such as microbial motion observation and cell migration research. However, existing microfluidic chips still have unresolved industry pain points: First, existing laminar flow microfluidic chips are mostly designed for single application scenarios, only able to achieve concentration gradient construction or cell migration detection, and have not been biomimeticly designed for the general physiological structure of biological mucous membranes. They cannot flexibly adapt to the structural characteristics of different types of mucous membranes such as fish mucous membranes, intestinal mucous membranes, respiratory mucous membranes, and oral mucous membranes, resulting in low versatility. Second, existing bacterial motion observation chips are mostly designed with steady-state unidirectional flow channels, which can only simulate static or unidirectional flow. The first problem is that the aquatic environment cannot replicate the dynamic flow field of the physiological environment of the mucosa, which is coupled with "lateral disturbance + longitudinal flow". The biomimetic reproduction degree is low, which can easily lead to the distortion of evaluation results. The second problem is that the existing chips mostly adopt complex structures with multi-layer bonding, which is not only difficult and costly to process, but also cannot achieve a fully transparent design. They cannot meet the real-time observation requirements of conventional optical microscopes and fluorescence microscopes, and the operation threshold is extremely high. The third problem is that there is currently no microfluidic solution that can simultaneously realize the biomimetic construction of non-Newtonian fluid mucus, the simulation of dynamic physiological flow field, the visualization of bacterial movement process, and the accurate quantification of penetration ability. As a result, the industry has always lacked a standardized platform that can accurately evaluate the penetration ability of bacterial mucosa.

[0006] Therefore, developing a microfluidic chip for evaluating bacterial penetration ability that is versatile, highly biomimetic, laminarly stable, convenient for real-time observation, and adaptable to various biological mucosal scenarios is a common technical problem that urgently needs to be solved in many fields such as biomedicine, aquaculture, and microbial research. Summary of the Invention

[0007] To overcome the aforementioned shortcomings of existing technologies, this invention provides a universal biomimetic microfluidic chip, evaluation system, and evaluation method for evaluating microbial mucosal penetration capabilities. It aims to address the technical problems of low biomimeticity (inability to simulate the non-Newtonian fluid characteristics and dynamic physiological flow field of mucosa), poor versatility (inability to adapt to different types of mucosa), and the inability to perform real-time, dynamic, and quantitative analysis of the bacterial penetration process in existing devices and methods for evaluating microbial mucosal penetration capabilities. This invention fills a technological gap in the industry and provides a standardized platform for evaluating bacterial mucosal penetration capabilities. To achieve the above objectives, this invention adopts the following technical solution: In a first aspect, the present invention provides a dynamic biomimetic microfluidic chip for real-time evaluation of bacterial mucosal penetration ability, comprising a fully transparent single-layer substrate structure, wherein the structure is provided with at least two sets of fluid inlet channels, a triangular diffusion region, a laminar flow observation region and a fluid outlet channel; a flexible thin film (FHR membrane) is provided in the triangular diffusion region, and the transverse pulsating disturbance generated by the membrane is coupled with the longitudinal water flow in the laminar flow observation region to form a dynamic biomimetic flow field for simulating the physiological environment of biological mucosa.

[0008] Secondly, the present invention provides a real-time evaluation system for bacterial mucosal penetration ability, including the above-mentioned chip, as well as a fluid drive module, a pulsation control module, a microscopic observation module and a data processing module.

[0009] Thirdly, the present invention provides an evaluation method based on the above-mentioned chip or system, comprising five steps: chip preprocessing, biomimetic flow field construction, laminar interface construction, real-time observation and data acquisition, data quantization and result output.

[0010] Compared with the prior art, the present invention has the following beneficial effects: (1) Dynamic bionics: For the first time, the FHR membrane was transformed into a transverse pulsation disturbance generation component. By coupling the flow field with "transverse pulsation + longitudinal flow", the dynamic physiological environment of biological mucosa was simulated, breaking through the bionic limitations of existing static or unidirectional flow chips.

[0011] (2) Real-time visualization: The fully transparent single-layer structure, combined with the stable bacterial liquid-mucus interface formed by laminar diffusion, can be directly adapted to a microscope to conduct real-time dynamic observation and recording of the bacterial penetration process.

[0012] (3) High versatility: Through adjustable channel structure, mucus concentration and flow field parameters, it can be adapted to various biological mucosa scenarios such as fish mucosa, intestinal mucosa, and respiratory mucosa.

[0013] (4) Simple process: single-layer PDMS-glass bonded structure, mature processing, controllable cost, compatible with standard capillary tubes and injection pumps, low operation threshold. Attached Figure Description

[0014] Figure 1This is a structural diagram of the mucosal biomimetic microfluidic chip described in this invention. Figure 2 This is a magnified view of a portion of the structure of the FHR membrane and laminar flow region described in this invention. Figure 3 This is a schematic diagram of the bacterial-mucus laminar flow interface and dynamic biomimetic flow field in the laminar flow observation area described in this invention. Figure 4 This is a schematic diagram of the overall architecture of the bacterial mucosal penetration ability evaluation system described in this invention. Figure 5 The following is a flowchart of the evaluation method described in this invention. In the attached image: Figure 1 The chip structure diagram is labeled 1-5. Figure 2 The enlarged view of the local structure is labeled 6-13. Figure 3 The diagram (of laminar interface and biomimetic flow field) is labeled 14-20. Figure 4 (The overall architecture diagram of the evaluation system) is labeled as 21-24, where 1-PDMS layer, 2-glass layer, 3-bacterial liquid inlet, 4-triangular diffusion zone, 5-laminar flow zone, 6-bacterial liquid inlet channel, 7-first biomimetic slime inlet channel, 8-second biomimetic slime inlet channel, 9-laminar flow observation zone, 10-FHR membrane, 11-second biomimetic slime outlet channel, 12-first biomimetic slime outlet channel, 13-bacterial liquid outlet channel, 14-bacterial liquid zone, 15-first biomimetic slime zone, 16-second biomimetic slime zone, 17-deforming FHR membrane, 18-pressure provided by the triangular diffusion zone, 19-elasticity of PDMS material, 20-vertical flow velocity of liquid, 21-microfluidic chip described in this invention, 22-inverted microscope, 23-multichannel syringe pump, 24-bacterial movement trajectory tracking software. Detailed Implementation

[0015] The following embodiments will further illustrate the present invention, but are not intended to limit the invention.

[0016] The core design of this invention is a universal biomimetic microfluidic chip. The following are the optimal implementation methods for three typical scenarios, all based on the core structural design of this invention: Example 1: Dual-channel fish mucosa biomimetic microfluidic chip This embodiment is designed for the evaluation of fish mucosal penetration of immersion vaccines in aquaculture. It is a basic implementation method, and the specific structural parameters and operation methods are as follows: Overall chip structure: It is formed by bonding a single PDMS layer and a glass layer through plasma treatment, and is completely transparent, making it suitable for observation by optical and fluorescence microscopes. The chip has a total length of 50mm, a width of 25mm, and a total height of 1.5mm, with the PDMS layer being 0.5mm thick and the glass layer being 1mm thick.

[0017] Core flow channel parameters: Triangular diffusion region: The inlet width of the narrow channel is 100μm, the triangular diffusion angle is 20°, the total length of the diffusion region is 500μm, the channel transition radius is R=15μm, the width of the wide channel attached to the FHR membrane is 400μm, and the channel depth is 75μm; Laminar flow observation area: The total width is 400μm, of which the width of the bacterial liquid area is 200μm, the width of the biomimetic mucus area is 200μm, the total length of the laminar flow observation area is 8mm, the channel depth is 75μm, and the transition radius at the fluid confluence is R=20μm; Ports and FHR membrane: The inlet / outlet apertures are both 1.0 mm, and the center-to-center distance between adjacent ports is 2.0 mm, which is compatible with standard capillary tubes and syringe pumps; the FHR membrane thickness is 60 μm, which corresponds completely to the bacterial culture zone in the laminar flow observation area.

[0018] Chip fabrication method: A standard soft photolithography process is used. Exemplary steps include: mask fabrication → SU-8 silicon wafer mold fabrication → PDMS casting and curing → drilling and FHR film encapsulation → plasma bonding → hydrophilic modification post-treatment. Those skilled in the art can adjust the specific process parameters of each step according to actual needs, such as the spin coating speed of the SU-8 photoresist, the pre-baking and post-baking times, and the curing temperature and time of the PDMS, which are all conventional technical choices in the field.

[0019] Example of evaluation operating parameters: In static laminar flow mode, the bacterial solution flow rate is 0.2 μL / min, and the biomimetic mucus flow rate is 0.1 μL / min; the flexible film pulsation frequency is 0.5 Hz, which is used to simulate water disturbance caused by natural water waves and fish swimming; observation is carried out using an inverted fluorescence microscope with a frame rate of 15 fps, and fluorescent labeled bacteria are adapted to an excitation wavelength of 488 nm. Example 2: Three-channel intestinal mucosa biomimetic microfluidic chip The core structure of this embodiment is the same as that of Embodiment 1, with the following differences in parameters: Flow channel structure adjustment: Three sets of fluid injection channels are adopted, namely bacterial liquid injection channel, first biomimetic mucus injection channel (low concentration) and second biomimetic mucus injection channel (high concentration). The total width of the three channels is 400μm, of which the bacterial liquid area is 150μm wide, the dilute mucus area is 125μm wide, and the concentrated mucus area is 125μm wide, forming a gradient mucus interface in the laminar flow observation area to simulate the loose layer and dense layer of intestinal mucus; Flow field parameter adjustment: The transverse pulsation frequency of the FHR membrane was set to 1.5 Hz; the perfusion flow rate was adjusted to 0.3 μL / min for bacterial solution, 0.15 μL / min for dilute mucus, and 0.15 μL / min for concentrated mucus to maintain the stability of the gradient laminar flow interface.

[0020] Example 3: Universal bacterial mucosal penetration ability evaluation system and operation method The evaluation system in this embodiment is built based on the microfluidic chip of any of the above embodiments, and specifically includes: Fluid drive module: It adopts a four-channel commercial syringe pump, which is connected to the chip's inlet and outlet through a PE capillary tube. It supports multi-channel independent flow rate control, with a flow rate adjustment range of 0.01-100μL / min. Pulse control module: It adopts a programmable piezoelectric drive controller (or peristaltic pump) and is connected to the flexible thin film (FHR film) of the chip. The drive frequency adjustment range is 0.1-10Hz, and the drive voltage is adjustable from 0-200V, which can accurately match the disturbance simulation requirements of different mucous membranes. Microscopic observation module: It adopts an inverted fluorescence microscope, equipped with a high-speed CMOS camera, with an adjustable shooting frame rate of 0-100fps, supports bright field optical observation and multi-channel fluorescence observation, and the excitation wavelength is adapted to commonly used fluorescent labels of 405nm, 488nm, 561nm and 640nm. Data processing module: Equipped with a computer system and built-in bacterial movement trajectory tracking software, it can automate the entire process of single bacterial identification, trajectory tracking, penetration rate calculation, and movement parameter statistics, and output core evaluation indicators.

[0021] Based on the general evaluation method of the above system, the specific steps are as follows: Chip pretreatment: The prepared microfluidic chip is passed through deionized water, vacuum degassing is performed to remove air bubbles from the flow channel, and the flow channel is rinsed sequentially with anhydrous ethanol and PBS buffer to complete the hydrophilic modification; the chip is fixed on the stage of an inverted microscope, the microscope field of view is adjusted so that the laminar flow observation area is completely within the imaging field of view, and the matching imaging parameters are set; Biomimetic flow field construction: Based on the type of the target mucosa, prepare the corresponding biomimetic mucus solution, load it into a syringe and connect it to the mucus inlet; load the PBS buffer into the syringe and connect it to the bacterial inlet; set the matching perfusion parameters through the injection pump, and continuously perfuse until a stable longitudinal water flow is formed in the flow channel; start the pulsation control module (such as a piezoelectric drive controller or peristaltic pump), set the matching pulsation frequency, drive the flexible film to generate lateral pulsation disturbance, and cooperate with the longitudinal water flow to form a dynamic biomimetic flow field of the target mucosa; Construction of laminar flow interface: Switch the syringe at the bacterial inlet to the target bacterial suspension (fluorescently labeled engineered bacteria, pathogenic bacteria, vaccine carrier bacteria, etc.), maintain the perfusion parameters and continue perfusion for 3-5 minutes, so that the bacterial suspension and the biomimetic mucus solution can diffuse through laminar flow in the laminar flow observation area to form a stable mucosa-body fluid contact interface layer. Real-time observation and data acquisition: Turn on the fluorescent light source (fluorescence observation mode) or bright field light source corresponding to the excitation wavelength, and continuously photograph the movement and penetration process of bacteria in the laminar flow observation area using a high-speed camera. The acquisition time is 30-120s, and the image sequence data is saved. The changes in the penetration behavior of bacteria under different conditions can be recorded by adjusting the external stimulus parameters (such as light control, drug concentration, etc.). Data quantification and result output: The collected image sequences are imported into the bacterial motion trajectory tracking software to identify and track the motion trajectory of individual bacteria. The core indicators such as the bacterial mucus layer penetration rate, average movement speed, instantaneous speed, directional movement efficiency, and residence time are quantified and calculated. The differences in bacterial penetration ability under different conditions are compared, and the final standardized evaluation results are output.

[0022] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the core design principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A dynamic biomimetic microfluidic chip for real-time evaluation of bacterial mucosal penetration ability, characterized in that, It includes a single-layer substrate structure, which is formed by bonding a PDMS layer and a glass layer and is completely transparent. The single-layer substrate structure is provided with: At least two sets of fluid injection channels, including a bacterial liquid injection channel and a biomimetic mucus injection channel, with the ends of the two sets of fluid injection channels converging; The laminar flow observation area has its inlet connected to the junction of the two sets of fluid inlet channels; The fluid sampling channel is connected to the outlet of the laminar flow observation area; The triangular diffusion zone has only one fluid port that communicates with the outside. The end of the triangular diffusion zone without a port is separated from the sidewall of the laminar flow observation zone by a flexible film (FHR film). The width of the effective deformation zone of the flexible film matches the width of the laminar flow observation zone. The fluid port is used to connect an external fluid drive device to drive the flexible film to generate reciprocating deformation, thereby providing lateral pulsating disturbance to the fluid in the laminar flow observation area; After the bacterial liquid injection channel and the biomimetic mucus injection channel are filled with fluid, a stable bacterial liquid-mucus contact interface layer is formed in the laminar flow observation area through laminar flow diffusion. The transverse pulsating disturbance provided by the FHR membrane, together with the longitudinal water flow in the laminar flow observation area, forms a dynamic biomimetic flow field that simulates the physiological environment of the biological mucus. The fully transparent single-layer substrate structure is adapted for use with microscopes to observe in real time the process of bacteria penetrating the interface layer.

2. The microfluidic chip according to claim 1, characterized in that, The thickness of the PDMS layer is 0.5 mm, and the thickness of the glass layer is 1 mm.

3. The microfluidic chip according to claim 1, characterized in that, The inlet width of the fine channel in the triangular diffusion region is 100 μm, the triangular diffusion angle is 20°, the total length of the diffusion region is 500 μm, the channel transition radius is R = 15 μm, the width of the wide channel attached to the FHR membrane is 400 μm, and the channel depth is 75 μm.

4. The microfluidic chip according to claim 1, characterized in that, The total width of the laminar flow observation area is 400 μm, of which the width of the bacterial liquid area is 150 μm, the width of the first biomimetic slime area is 125 μm, the width of the second biomimetic slime area is 125 μm, the total length of the laminar flow observation area is 8 mm, the channel depth is 75 μm, and the transition radius at the fluid confluence is R=20 μm.

5. The microfluidic chip according to claim 1, characterized in that, The port diameter of both the fluid inlet channel and the fluid outlet channel is 1.0 mm, and the center-to-center distance between adjacent ports is 2.0 mm. The ports are used to adapt to the connection of standard capillary tubes and injection pumps.

6. The microfluidic chip according to claim 1, characterized in that, The FHR membrane has a thickness of 60 μm and corresponds perfectly to the bacterial culture area in the laminar flow observation zone, ensuring that lateral disturbances uniformly cover the entire observation interface.

7. The microfluidic chip according to claim 1, characterized in that, The PDMS layer and the glass layer are bonded by plasma treatment. The inner surface of the PDMS layer is provided with a hydrophilic modified coating. The hydrophilic modified coating is used to prevent bacteria from non-specifically adhering in the flow channel, and at the same time adapts to the observation requirements of fluorescence microscopy.

8. The microfluidic chip according to claim 1, characterized in that, The fluid injection channel is provided in three sets: a bacterial liquid injection channel, a first biomimetic slime injection channel, and a second biomimetic slime injection channel. The first and second biomimetic slime injection channels are used to introduce biomimetic slime of different concentrations to form a gradient slime interface layer in the laminar flow observation area.

9. The microfluidic chip according to claim 1, characterized in that, The width ratio of the bacterial liquid zone and the biomimetic mucus zone in the laminar flow observation area can be adjusted to match the biomimetic requirements of mucus layer thickness for different types of biological mucosa.

10. A real-time evaluation system for bacterial mucosal penetration ability, characterized in that, The system includes the dynamic biomimetic microfluidic chip as described in any one of claims 1-9, as well as a fluid drive module, a pulsation control module, a microscopic observation module, and a data processing module. The fluid drive module is connected to the inlet and outlet of the microfluidic chip via a capillary tube and is used to control the perfusion rate and perfusion mode of the fluid. The pulsation control module is connected to the FHR membrane of the microfluidic chip and is used to adjust the frequency and amplitude of the transverse pulsation disturbance. The microscopic observation module is aligned with the fully transparent microfluidic chip and is used to acquire image data of the movement and penetration process of bacteria in the laminar flow observation zone in real time. The data processing module is communicatively connected to the microscopic observation module and is used to perform quantitative analysis on the acquired image data and output the evaluation results of the bacteria's mucosal penetration ability.

11. The evaluation system according to claim 10, characterized in that, The fluid drive module is an injection pump, which supports multi-channel independent flow rate control to adjust the perfusion flow rate of bacterial solution and biomimetic mucus of different concentrations.

12. The evaluation system according to claim 10, characterized in that, The microscopic observation module is an optical microscope or a fluorescence microscope, and the excitation wavelength of the fluorescence microscope is adapted to the observation needs of engineered bacteria expressing fluorescent proteins.

13. The evaluation system according to claim 10, characterized in that, The data processing module is equipped with bacterial movement trajectory tracking software, which is used to quantitatively analyze the core evaluation indicators of bacterial penetration rate, instantaneous movement speed, average movement speed, and directional movement efficiency.

14. The evaluation system according to claim 10, characterized in that, The pulsation control module is a peristaltic pump or a piezoelectric drive controller, used to drive the flexible film to generate transverse pulsations at a set frequency to match the physiological environmental disturbance simulation requirements of different biological mucosa.

15. A method for real-time evaluation of bacterial mucosal penetration ability, characterized in that, Based on the dynamic biomimetic microfluidic chip according to any one of claims 1-9, or the real-time evaluation system according to any one of claims 10-14, the system comprises the following steps: S1 – Chip pretreatment: hydrophilic modification and venting treatment of the flow channel of the microfluidic chip; fixing the chip on the microscope stage; and aligning and adjusting the field of view of the microscopic observation module to the laminar flow observation area; S2 – Biomimetic flow field construction: introducing a biomimetic mucus solution matching the target mucus into the biomimetic mucus injection channel through the fluid driving module, introducing a buffer solution into the bacterial liquid injection channel, and setting the perfusion parameters to form a stable longitudinal water flow; activating the pulsation control module to drive the FHR membrane to generate transverse pulsation disturbance, which, in conjunction with the longitudinal water flow, forms a simulated target. Dynamic biomimetic flow field of mucosal physiological environment; S3 – Laminar flow interface construction: The bacterial suspension is switched to the bacterial inlet channel by the fluid drive module, and the set perfusion parameters are maintained so that the bacterial suspension and biomimetic mucus solution form a stable bacterial-mucus contact interface layer in the laminar flow observation area through laminar diffusion; S4 – Real-time observation and data acquisition: The movement and penetration process of bacteria in the laminar flow observation area are continuously and in real time captured by the microscopic observation module, and image sequence data is acquired; S5 – Data quantification and result output: The acquired image sequence data is processed by the data processing module, the movement trajectory of bacteria is tracked, the penetration ability related indicators are quantified and calculated, and the evaluation results of the bacteria's penetration ability against the target mucosa are output.

16. The evaluation method according to claim 15, characterized in that, In step S2, the transverse pulsation frequency can be adjusted within the range of 0.1-5Hz, where 0.5Hz is used to simulate the natural water wave disturbance of the mucosa in an aquatic environment, and the 1-2 Hz frequency is used to simulate the mucosal environment disturbance of mammalian intestinal peristalsis.

17. The evaluation method according to claim 15, characterized in that, In step S4, the shooting frame rate of the microscopic observation module is not less than 10fps, and the continuous shooting time is not less than 30s.

18. The evaluation method according to claim 15, characterized in that, In step S5, by adjusting the perfusion flow rate, pulsation frequency, laminar flow zone width ratio, or biomimetic mucus concentration, the penetration ability evaluation under different bacterial species or different types of biological mucosa scenarios can be completed.

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