A complex culture system microbial contamination closed-loop monitoring system and method

Abstract

The application discloses a complex culture system microbial contamination closed-loop monitoring system and method, and belongs to the technical field of microfluidic sample pretreatment and process monitoring. The system comprises a gradient spiral zoning module, an online lateral entrapment module, a closed-loop reflux module, an auxiliary pumping and liquid supplementing module and an in-situ monitoring module. Through continuous zoning, cyclic enrichment and lateral entrapment of different scale components in a complex sample, preferential retention and in-situ monitoring of low-abundance microbial contaminants are realized. The application does not need complex labeling and is suitable for contamination early warning and pretreatment of cell culture process liquid, fermentation process liquid, biological process sampling liquid and other complex suspension system samples.

Description

Technical Field

[0001] This application relates to the field of microfluidic sample pretreatment and process monitoring technology, specifically to a closed-loop monitoring system and method for microbial contamination in complex culture systems. Background Technology

[0002] In typical complex suspension culture systems, such as tank-based bio-fermentation in food factories and cell and tissue culture and bioprocess sampling in laboratories, microbial contamination of the culture medium is unavoidable. Early detection and intervention through real-time rapid detection technologies are crucial. Common rapid culture medium detection technologies fall into two categories: one directly monitors the concentration of various substances using electronic sensors in the culture medium, while the other primarily relies on post-processing of the sample solution. The latter inevitably faces challenges such as numerous background particles, low abundance of target microorganisms, high fluid viscosity, and complex composition. Among these non-target background particles are large impurities such as cell debris, cell clusters, and protein flocculants, as well as suspended small particles smaller than the target microorganisms. The presence of these impurities can easily interfere with the identification and observation of target microorganisms and increases the difficulty of sample pretreatment and subsequent monitoring.

[0003] For the detection of microbial contamination in complex sample solutions, current mainstream techniques mainly include post-sample culture in the laboratory environment, staining, microscopic observation, and detection of specific metabolites. However, these methods typically suffer from drawbacks such as numerous processing steps and long detection cycles, easily missing the optimal intervention period. In particular, when the abundance of target microorganisms is low, existing mainstream techniques often suffer from false detection problems, making rapid and stable early monitoring difficult. Therefore, driven by various factors, microfluidic separation, enrichment, and detection technologies are rapidly developing in multiple fields of microbial detection.

[0004] Currently, in the field of microfluidic separation, there are technical approaches such as tortuous channel separation, spiral channel separation, and local microcavity capture. However, most of these methods can only achieve single-pass separation, making it difficult to simultaneously achieve the four processes of "continuous banding, repeated enrichment, online lateral trapping, and in-situ observation" within the same system. Especially for more complex culture system samples, if only a single collection is performed at the end, the target microorganisms are easily under-enriched, resulting in low capture efficiency. If a separate terminal capture cavity is set up, it is easy to miss the moment when the target microbial flow bands are most clearly separated and most stable in a single cycle, leading to inaccurate observation results.

[0005] Therefore, a novel closed-loop microfluidic system is needed to enable target microorganisms in complex culture systems to achieve multi-band distribution within a pre-defined gradient spiral channel, with scale being the primary influencing factor. Furthermore, this system can continuously trap target microorganisms online laterally at the optimal target band location within a single cycle, while simultaneously returning untrapped target band samples to the injection end for repeated banding and enrichment. This improves the pretreatment efficiency of low-abundance microbial contamination and enhances its in-situ monitoring capabilities. Summary of the Invention

[0006] This application discloses a closed-loop monitoring system and method for microbial contamination in complex culture systems, which addresses the problems of low abundance of target microorganisms in complex culture system samples, difficulty in eliminating background interference, limited one-pass separation and enrichment effects, and poor in-situ monitoring capabilities in existing technologies.

[0007] To achieve the above objectives, this application adopts the following technical solution:

[0008] This application provides a closed-loop monitoring system for microbial contamination in complex culture systems, including a gradient spiral banding module, an online lateral trapping module, a closed-loop reflux module, an auxiliary extraction and replenishment module, and an in-situ monitoring module. The gradient spiral banding module includes an inlet, a gradient spiral banding channel, a target side channel outlet, and a non-target side channel outlet. The gradient spiral banding channel is a trapezoidal spiral microchannel with varying curvature. The closed-loop reflux module includes a target side reflux inlet tube, a main reflux tube, and an inlet end reflux return tube connected in sequence. The target side channel outlet is connected to the inlet of the target side reflux inlet tube. The online lateral trapping module is located on one side of the main reflux tube. The inlet end reflux return tube is connected to the inlet or upstream of the inlet. The in-situ monitoring module is located in the area corresponding to the transparent trapping cavity of the online lateral trapping module, and is used for in-situ observation and image acquisition of target microorganisms within the transparent trapping cavity.

[0009] Furthermore, the gradient spiral channel is one of a planar spiral channel, a three-dimensional spiral channel, or a spiral-like curved channel, and its cross-section is one of a trapezoidal cross-section, a stepped cross-section, an eccentric cross-section, an asymmetric cross-section, or a gradient cross-section that gradually changes along the flow direction.

[0010] Furthermore, the online lateral trapping module includes a trapping microchannel inlet and a transparent trapping cavity; wherein a plurality of trapping microchannel inlets are sequentially connected to one side of the main reflux pipe; the outlet end of each trapping microchannel inlet is connected to a transparent trapping cavity, which is formed by sequentially arranged first transparent trapping cavities and second transparent trapping cavities connected together; the size of the trapping microchannel inlet is smaller than the accessible scale of large particle background components and larger than or close to the accessible scale of the target microorganism.

[0011] Furthermore, the auxiliary pumping and replenishment module includes a replenishment branch, a storage unit, two microfluidic driven pumps, a pressure regulating branch, and a waste liquid outlet. The replenishment branch is connected to the main return pipe, the pressure regulating branch is connected to the end of the second transparent trapping cavity, the storage unit is connected to the replenishment branch through one of the microfluidic driven pumps, and the pressure regulating branch is connected to the waste liquid outlet through the other of the microfluidic driven pumps.

[0012] Furthermore, a flow-limiting structure is provided between the main return pipe and each of the trapping microchannel inlets, wherein the flow-limiting structure includes one of a slit inlet, a flow-limiting neck, a tapering channel, or a high flow resistance channel, wherein the equivalent hydraulic diameter of each of the trapping microchannel inlets is smaller than the passage scale of non-target large particle background components and larger than or equal to the passage scale of target microorganisms.

[0013] Furthermore, the first transparent trapping cavity and the second transparent trapping cavity are lateral blind cavities or dead-end microcavities, and the two are sequentially connected along the flow direction; the first transparent trapping cavity and the second transparent trapping cavity are preferably formed of transparent resin.

[0014] Furthermore, the in-situ monitoring module includes a microscopic imaging component, an illumination component, an image acquisition component, and an output component, used to monitor the quantity, morphology, aggregation state, or capture growth trend of target microorganisms in the first transparent trapping cavity and the second transparent trapping cavity and output the data through the output component.

[0015] A closed-loop monitoring method for microbial contamination in complex culture systems, comprising the following steps: Step S1: Introduce the complex culture system sample into the gradient spiral distribution channel through the inlet and set the injection flow rate; Step S2: The injection flow rate is used to form a multi-band distribution of the complex culture system sample in the gradient spiral zonal channel, consisting of a large-particle background component band, a target microorganism band, and a small-scale particle component band; Step S3: When the target microbial flow passes through the section with the best single-cycle zoning effect, the target microbial is continuously introduced and retained laterally through the trapping microchannel inlet and pressure regulation branch of the online lateral trapping module; Step S4: The target stream sample that was not trapped by the online lateral trapping module is sent back into the injection section through the closed-loop reflux module for cyclic enrichment; Step S5: Drain the background liquid through the auxiliary pumping and replenishment module, and simultaneously replenish sterile replenishment solution to maintain system volume balance; Step S6: Monitor the contamination status and conduct in-situ observation of the target microorganisms trapped in the first and second transparent trapping cavities using the in-situ monitoring module; Step S7: When the number of newly captured particles, the capture rate, or the particle change trend in the first transparent trapping cavity and the second transparent trapping cavity reach a preset threshold, or when the system reaches a preset running time or a preset number of cycles, stop running and perform final observation or subsequent processing.

[0016] Furthermore, the preset threshold in step S7 includes any one or more of the following: the number of newly added target microorganisms in the first transparent trapping cavity and the second transparent trapping cavity is lower than the threshold, the capture rate per unit time is lower than the threshold, the change in image particles is lower than the threshold, or the particle index of the returned sample drops to a preset range.

[0017] Compared with the prior art, this application has at least the following beneficial effects:

[0018] 1. This application achieves multi-band distribution in complex culture system samples through a gradient spiral banded flow channel, with the differences between components in different bands mainly reflected in scale. Under continuous flow conditions, this application can distinguish between large background particles, target microbial components, and smaller-scale particle components.

[0019] 2. In this application, the online lateral trapping module is positioned near the target flow zone in the section with the best single-cycle separation effect. Through the synergistic effect of the size-selective trapping microchannel inlet and the pressure regulation branch in the auxiliary pumping and replenishment module, the probability of target microorganisms in the target microbial flow zone entering the transparent trapping cavity is increased and they are kept there, thereby further improving the capture efficiency per unit cycle.

[0020] 3. This application adopts a method that couples online lateral trapping with closed-loop reflux, so that the target microorganisms that are not captured can repeatedly undergo zoning and trapping, thereby achieving more complete continuous enrichment under low abundance conditions.

[0021] 4. This application utilizes the coordinated operation of the pressure regulation branch, waste liquid outlet, and replenishment branch to remove background liquid and smaller-scale impurities to a certain extent during continuous circulation, and maintains the system volume within a stable range, thereby reducing the interference of complex samples on observation results and flow stability.

[0022] 5. This application can complete continuous zoning, online trapping, closed-loop enrichment and in-situ observation within the same system. When dealing with microorganisms in complex culture systems, this application can be used for pollution early warning, pretreatment and visual monitoring. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of a closed-loop monitoring system for microbial contamination in a complex culture system, as described in this application.

[0024] Figure 2This is a structural diagram of a closed-loop monitoring system for microbial contamination in a complex culture system, as described in this application.

[0025] Figure 3 This is a schematic diagram of the multi-band distribution in this application.

[0026] Figure 4 This is a schematic diagram comparing the flow field distribution of representative cross sections of rectangular and trapezoidal sections in this application (where (a) is a schematic diagram of the flow field distribution of the rectangular cross section, and (b) is a schematic diagram of the flow field distribution of the trapezoidal cross section).

[0027] Figure 5 The diagram shows the zonal distribution of particles near the splitting node under different representative injection flow rates (where (a) is the zonal distribution under a flow rate of 1000 μL / min; (b) is the zonal distribution under a flow rate of 1700 μL / min; and (c) is the zonal distribution under a flow rate of 2000 μL / min).

[0028] Figure 6 This is a schematic diagram of the in-situ monitoring module of this application observing, acquiring images, and displaying outputs the corresponding area of ​​the transparent trapping cavity.

[0029] Figure 7 This is a schematic diagram of the closed-loop monitoring method for microbial contamination in a complex culture system according to this application.

[0030] Figure 8 This is a comparison of the number of targets per unit field of view within the transparent trapping cavity over time under closed-loop reflux conditions and single-pass conditions in this application.

[0031] List of reference numerals in the attached diagram:

[0032] Among them, 1-1-Inlet; 1-2-Gradient spiral slurry channel; 1-3-Target side channel outlet; 1-4-Non-target side channel outlet; 1-5-Optimal slurry effect section in a single cycle; 1-6-Large particle background component flow band; 1-7-Target microorganism flow band; 1-8-Small-scale particle component flow band; 2-1-Trap microchannel inlet; 2-2-First transparent trapping chamber; 2-3-Second transparent trapping chamber; 3-1-Target side reflux inlet tube; 3-2-Main reflux tube; 3-3-Inlet end reflux return tube; 4-1-Replenishment branch; 4-2-Storage unit; 4-3-Microfluidic drive pump; 4-4-Pressure regulation branch; 4-5-Waste liquid outlet; 5-In-situ monitoring module. Detailed Implementation

[0033] The present application will be further explained below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the present application. It should be noted that the terms "front", "rear", "left", "right", "up" and "down" used in the following description refer to the directions in the accompanying drawings, and the terms "inner" and "outer" refer to the directions toward or away from the geometric center of a specific component, respectively.

[0034] like Figure 1 and Figure 2 As shown, this embodiment provides a closed-loop monitoring system for microbial contamination in complex culture systems, including a gradient spiral banding module, an online lateral trapping module, a closed-loop reflux module, an auxiliary extraction and replenishment module, and an in-situ monitoring module 5.

[0035] The gradient spiral sizing module includes an inlet 1-1, a gradient spiral sizing channel 1-2, a target-side channel outlet 1-3, and a non-target-side channel outlet 1-4. The gradient spiral sizing channel 1-2 is a trapezoidal spiral microchannel with varying curvature, used for continuous sizing of samples from complex culture systems. The gradient spiral sizing channel 1-2 can be one of a planar spiral channel, a three-dimensional spiral channel, or a spiral-like curved channel, and its cross-section can be one of a trapezoidal cross-section, a stepped cross-section, an eccentric cross-section, an asymmetric cross-section, or a gradient cross-section that gradually changes along the flow direction. This embodiment uses a trapezoidal cross-section as an example.

[0036] The closed-loop reflux module includes a target-side reflux inlet pipe 3-1, a main reflux pipe 3-2, and an inlet-side reflux return pipe 3-3 connected in sequence. The target-side flow channel outlet 1-3 is connected to the inlet of the target-side reflux inlet pipe 3-1, and is used to introduce the separated target-side sample into the closed-loop circuit. The inlet-side reflux return pipe 3-3 is connected to the inlet 1-1 or its upstream, and is used to return untrapped target-side samples to the system inlet, achieving cyclic enrichment.

[0037] The online lateral trapping module is located on one side of the main reflux pipe 3-2 and is connected to the main reflux pipe 3-2 through the trapping microchannel inlet 2-1, used for the lateral introduction and retention of target microorganisms during the separation process. Figure 2As shown, the online lateral trapping module includes trapping microchannel inlets 2-1 and transparent trapping chambers. Several trapping microchannel inlets 2-1 are sequentially connected to one side of the main return pipe 3-2. The outlet end of each trapping microchannel inlet 2-1 is connected to the transparent trapping chamber. This transparent trapping chamber is formed by sequentially connecting a first transparent trapping chamber 2-2 and a second transparent trapping chamber 2-3. The size of the trapping microchannel inlets 2-1 is smaller than the accessible size of large background particles and larger than or close to the accessible size of the target microorganisms, thereby achieving size-selective retention of the target microorganisms. A flow-limiting structure is provided between the main return pipe 3-2 and each trapping microchannel inlet 2-1, which can be one of a slit inlet, a flow-limiting neck, a converging channel, or a high-resistance channel. The equivalent hydraulic diameter of each trapping microchannel inlet 2-1 is smaller than the passage size of non-target large background particles and larger than or equal to the passage size of the target microorganisms.

[0038] The first transparent trapping cavity 2-2 and the second transparent trapping cavity 2-3 are preferably two-stage tandem lateral blind cavity structures connected in sequence, formed of transparent resin. After the target microorganism enters the first transparent trapping cavity 2-2 laterally through the trapping microchannel inlet 2-1, some of the target microorganism continues to migrate to and remain in the second transparent trapping cavity 2-3, thereby forming a step-by-step capture and in-situ observation area.

[0039] The auxiliary drainage and replenishment module is connected to the end of the main reflux pipe 3-2 and the online lateral trapping module, respectively, to drain part of the background liquid and small-scale impurities, and simultaneously replenish sterile liquid to maintain system volume balance. Figure 2 As shown, the auxiliary suction and replenishment module includes a replenishment branch 4-1, a storage unit 4-2, two microfluidic pumps 4-3, a pressure regulating branch 4-4, and a waste outlet 4-5. The replenishment branch 4-1 is connected to the main return pipe 3-2 and is used to replenish the system with sterile liquid. The pressure regulating branch 4-4 is connected to the end of the second transparent trapping chamber 2-3. The storage unit 4-2 is connected to the replenishment branch 4-1 through one of the microfluidic pumps 4-3; the pressure regulating branch 4-4 is connected to the waste outlet 4-5 through the other microfluidic pump 4-3. This pressure regulating branch 4-4 is used to create continuous suction under the action of the microfluidic pump 4-3, thereby enhancing the tendency of target microorganisms to migrate into the transparent trapping chamber and discharging excess waste liquid.

[0040] The in-situ monitoring module 5 is located in the corresponding area of ​​the transparent trapping cavity of the online lateral trapping module, and is used for in-situ observation and image acquisition of target microorganisms within the transparent trapping cavity. The in-situ monitoring module 5 includes a microscopic imaging component, an illumination component, an image acquisition component, and an output component. Figure 6As shown, this module is used to continuously monitor the quantity, morphology, aggregation state or capture growth trend of target microorganisms in the first transparent trapping cavity 2-2 and the second transparent trapping cavity 2-3, and output the results through output components such as a display.

[0041] Figure 4 (a) is a schematic diagram of the flow field distribution in a rectangular cross-section. Figure 4 (b) is a schematic diagram of the flow field distribution across the trapezoidal cross-section, where A and B are orientation marks of the cross-sectional profile. Figure 4 It is evident that the flow field distribution under rectangular cross-section conditions is relatively symmetrical, while the flow field distribution under trapezoidal cross-section conditions exhibits spatial offset characteristics along the cross-sectional direction. Based on this cross-sectional difference, this application employs a trapezoidal cross-section gradient spiral zoning channel to enable components of different scales to form more easily distinguishable positional distributions within the cross-section, and to provide a structural basis for downstream target-side diversion and subsequent trapping.

[0042] After the complex culture system sample enters the flow channel, under the combined effects of fluid inertial lift, Dean secondary flow drag force, and size differences between different particles, the components of different sizes in the sample can form a distinguishable multi-flow zone distribution in the optimal zoning effect section 1-5 of a single cycle. Figure 2 In the diagram, AB represents the selected representative cross-sectional location, used for subsequent comparative analysis of the flow field distribution across the cross-section. Specifically, as shown... Figure 3 As shown, in the trapezoidal cross-section of this embodiment, small-scale particle component flow bands 1-8, target microorganism flow bands 1-7, and large-particle background component flow bands 1-6 are sequentially formed from side B to side A. The large-particle background component flow bands 1-6 occupy a relatively wide area laterally, approximately half the width of the cross-section; while the target microorganism flow bands 1-7 and 1-8 each occupy roughly half of the remaining lateral area. The target microorganism flow bands 1-7 are located between the small-scale particle component flow bands 1-8 and the large-particle background component flow bands 1-6, and maintain a distinguishable interface with the adjacent flow bands. These flow bands gradually stabilize downstream of the channel and form a target-side sample flow at the target-side channel outlet 1-3, which is beneficial for subsequent continuous trapping.

[0043] To illustrate the influence of injection flow rate on the gradient spiral zonation state and the zonal distribution of particles near the splitting node, three representative injection flow rate conditions were selected for observation, such as... Figure 5 As shown. Among them, Figure 5 (a) corresponds to an injection flow rate of 1000 μL / min. Figure 5 (b) corresponds to an injection flow rate of 1700 μL / min. Figure 5 (c) corresponds to an injection flow rate of 2000 μL / min. (From...) Figure 5It is evident that under different injection flow rates, the lateral focusing position of particle components near the splitting node, the clarity of the banded interface, and the relative spacing between bands all change. When the injection flow rate is low, although the particle bands show a certain stratification trend, the banded interface is relatively unclear, and the stability of the banded distribution near the splitting node is relatively average; when the injection flow rate is moderate... Figure 5 As shown in (b), the zonal boundaries of each component are relatively clear, and the main band position is relatively stable, which is more conducive to the formation of target side sample flows that can be used for subsequent splitting and online lateral trapping. When the injection flow rate is further increased, the zonal distribution near the splitting node can still be maintained, but its interface clarity and local stability change compared to the more suitable injection flow rate conditions. Therefore, by reasonably setting the injection flow rate to 1700 μL / min, a more clearly defined and more stable particle zonal distribution can be formed near the splitting node, thus providing more favorable conditions for the subsequent splitting, lateral introduction, and closed-loop reflux enrichment of target components. Figure 5 The spatial offset features shown indicate that the pre-set target side path can correspond to the subsequent branch path on the side closer to the target microbial flow zone and connect with the online lateral trapping module, so that the target component can enter the subsequent trapping area while maintaining a high local concentration and a relatively stable lateral position.

[0044] The method and process of this application are as follows: Figure 7 As shown, it includes the following steps: Step S1: Liquid is replenished to the system from the liquid storage unit 4-2 via the liquid replenishment branch 4-1, the number of cycles is set, and the system initialization is completed.

[0045] Step S2: Start the microfluidic drive pump 4-3, pre-rinse and defoam the microfluidic channel, then introduce the complex culture system sample into the gradient spiral zoning channel 1-2 through the inlet 1-1, and set the injection flow rate.

[0046] Step S3: The injection flow rate is used to create a multi-band distribution in the gradient spiral zonal channel 1-2, consisting of large-particle background component bands 1-6, target microorganism bands 1-7, and small-scale particle component bands 1-8. For example... Figure 5 As shown, by setting the injection flow rate to 1700 μL / min appropriately, a more clearly defined and more stable particle band distribution can be formed near the splitting node.

[0047] Step S4: When the target microbial flow 1-7 flows through the section 1-5 where the single-cycle zoning effect is optimal, the target microbial is continuously introduced and retained laterally through the trapping microchannel inlet 2-1 and pressure regulating branch 4-4 of the online lateral trapping module.

[0048] Step S5: The target stream sample that was not trapped by the online lateral trapping module is sent back into the injection section through the closed-loop reflux module for cyclic enrichment.

[0049] Step S6: Drain the background liquid and smaller particle components through the auxiliary pumping and replenishment module, and simultaneously replenish sterile replenishment solution to maintain system volume balance.

[0050] Step S7: Monitor the contamination status and conduct in-situ observation of the target microorganisms trapped in the first transparent trapping cavity 2-2 and the second transparent trapping cavity 2-3 through the in-situ monitoring module 5.

[0051] Step S8: If the number of cycles does not reach the preset value, return to step S3 to continue the enrichment process; when the number of cycles reaches the preset value, stop the microfluidic drive and perform final observation and result output through the in-situ monitoring module 5. The preset threshold includes any one or more of the following: the number of newly added target microorganisms in the first transparent trapping cavity 2-2 and the second transparent trapping cavity 2-3 is lower than the threshold, the capture rate per unit time is lower than the threshold, the change in image particles is lower than the threshold, or the particle index of the returned sample decreases to a preset range. These thresholds are used to automatically determine whether the enrichment process has achieved the expected effect.

[0052] To verify the effectiveness of this method, a closed-loop reflux and single-pass comparison experiment was set up under the same conditions. Figure 8 As shown, within the same observation period, using the closed-loop reflux method of this application, the overall number of target microorganisms in the target area with a preset number of cycles of 5 was higher than that under the single-pass condition, and continued to increase over time; in contrast, the number of targets under the single-pass condition increased more slowly and showed a slowing trend in the later stages. These results indicate that the closed-loop reflux module can enable target components that were not immediately trapped to re-participate in the zoning and subsequent trapping processes, significantly improving the accumulation efficiency of target components.

[0053] Furthermore, two points should be noted regarding the applicable samples in this embodiment. First, the complex culture system in this application can be cell culture medium, fermentation process broth, bioprocess sampling broth, suspension culture system, process sample containing cell debris and protein flocculents, industrial culture system sample containing high background particles, or other similar complex suspension system sample. Second, this application can be used independently as a pretreatment enrichment module for microbial contamination of complex culture systems, or as an in-situ visual monitoring unit in a pollution early warning system.

[0054] The technical means disclosed in this application are not limited to the technical means disclosed in the above embodiments, but also include technical solutions composed of any combination of the above technical features.

Claims

1. A closed-loop monitoring system for microbial contamination in complex culture systems, characterized in that, It includes a gradient spiral zoning module, an online lateral trapping module, a closed-loop reflux module, an auxiliary pumping and replenishment module, and an in-situ monitoring module (5); The gradient spiral slicing module includes an inlet (1-1), a gradient spiral slicing channel (1-2), a target-side channel outlet (1-3), and a non-target-side channel outlet (1-4); wherein the gradient spiral slicing channel (1-2) is a trapezoidal spiral microchannel with varying curvature. The closed-loop reflux module includes a target-side reflux inlet tube (3-1), a main reflux tube (3-2), and an injection-side reflux return tube (3-3) connected in sequence; the target-side flow channel outlet (1-3) is connected to the inlet of the target-side reflux inlet tube (3-1); the injection-side reflux return tube (3-3) is connected to the injection port (1-1) or the upstream of the injection port (1-1); The online lateral trapping module is located on one side of the main reflux pipe (3-2) and includes a trapping microchannel inlet (2-1) and a transparent trapping cavity. A plurality of trapping microchannel inlets (2-1) are sequentially connected to one side of the main reflux pipe (3-2). The outlet end of each trapping microchannel inlet (2-1) is connected to the transparent trapping cavity, which is formed by sequentially connecting a first transparent trapping cavity (2-2) and a second transparent trapping cavity (2-3). The size of the trapping microchannel inlet (2-1) is smaller than the accessible scale of large background particles and larger than or close to the accessible scale of the target microorganism. The auxiliary pumping and replenishment module includes a replenishment branch (4-1), a storage unit (4-2), two microfluidic pumps (4-3), a pressure regulating branch (4-4), and a waste liquid outlet (4-5). The replenishment branch (4-1) is connected to the main return pipe (3-2), the pressure regulating branch (4-4) is connected to the end of the second transparent trapping chamber (2-3), the storage unit (4-2) is connected to the replenishment branch (4-1) through one of the microfluidic pumps (4-3), and the pressure regulating branch (4-4) is connected to the waste liquid outlet (4-5) through the other microfluidic pump (4-3). The in-situ monitoring module (5) is located in the area corresponding to the transparent trapping cavity of the online lateral trapping module, and is used for in-situ observation and image acquisition of target microorganisms in the transparent trapping cavity.

2. The closed-loop monitoring system for microbial contamination in a complex culture system according to claim 1, characterized in that, The gradient spiral channel (1-2) is one of a planar spiral channel, a three-dimensional spiral channel, or a spiral-like curved channel, and its cross-section is a trapezoidal cross-section.

3. The closed-loop monitoring system for microbial contamination in a complex culture system according to claim 1, characterized in that, A flow-limiting structure is provided between the main return pipe (3-2) and each of the trapping microchannel inlets (2-1). The flow-limiting structure includes one of a slit inlet, a flow-limiting neck, a converging channel, or a high flow resistance channel. The equivalent hydraulic diameter of each trapping microchannel inlet (2-1) is smaller than the passage scale of non-target large particle background components and larger than or equal to the passage scale of target microorganisms.

4. The closed-loop monitoring system for microbial contamination in a complex culture system according to claim 1, characterized in that, The first transparent trapping cavity (2-2) and the second transparent trapping cavity (2-3) are lateral blind cavities or dead-end microcavities, and the two are connected in sequence along the flow direction; the first transparent trapping cavity (2-2) and the second transparent trapping cavity (2-3) are formed of transparent resin.

5. The closed-loop monitoring system for microbial contamination in a complex culture system according to claim 1, characterized in that, The in-situ monitoring module (5) includes a microscopic imaging component, an illumination component, an image acquisition component, and an output component, which are used to monitor the quantity, morphology, aggregation state, or capture growth trend of target microorganisms in the first transparent trapping cavity (2-2) and the second transparent trapping cavity (2-3) and output the data through the output component.

6. A closed-loop monitoring method for microbial contamination in complex culture systems, characterized in that, The closed-loop monitoring system for microbial contamination in a complex culture system as described in any one of claims 1-5 specifically includes the following steps: Step S1: Introduce the complex culture system sample into the gradient spiral banding channel (1-2) through the inlet (1-1) and set the injection flow rate; Step S2: The sample from the complex culture system is made to form a multi-band distribution in the gradient spiral zonal channel (1-2) by the injection flow rate, consisting of a large-particle background component band (1-6), a target microorganism band (1-7), and a small-scale particle component band (1-8). Step S3: When the target microbial flow (1-7) flows through the section (1-5) with the best single-cycle zoning effect, the target microbial is continuously introduced and retained laterally through the trapping microchannel inlet (2-1) and pressure regulating branch (4-4) of the online lateral trapping module; Step S4: The target stream sample that was not trapped by the online lateral trapping module is sent back into the injection section through the closed-loop reflux module for cyclic enrichment; Step S5: Drain the background liquid through the auxiliary pumping and replenishment module, and simultaneously replenish sterile replenishment solution to maintain system volume balance; Step S6: Monitor the contamination status and conduct in-situ observation of the target microorganisms trapped in the first transparent trapping cavity (2-2) and the second transparent trapping cavity (2-3) using the in-situ monitoring module (5); Step S7: When the number of new captures, the capture rate or the particle change trend in the first transparent trapping cavity (2-2) and the second transparent trapping cavity (2-3) reach a preset threshold, or when the system reaches a preset running time or a preset number of cycles, stop running and perform final observation or subsequent processing.

7. The closed-loop monitoring method for microbial contamination in a complex culture system according to claim 6, characterized in that, The preset threshold in step S7 includes any one or more of the following: the number of newly added target microorganisms in the first transparent trapping cavity (2-2) and the second transparent trapping cavity (2-3) is lower than the threshold, the capture rate per unit time is lower than the threshold, the change in image particles is lower than the threshold, or the particle index of the returned sample drops to a preset range.

Images