Microfluidic device, preparation method thereof and rubber powdery mildew spore collection and detection system

Abstract

The invention provides a microfluidic device and a preparation method thereof, and a rubber powdery mildew spore collection and detection system, the microfluidic device comprises: a pretreatment module provided with an inertial impact channel for separating agglomerated spore particles; the focusing module is connected with the pretreatment module and is provided with a focusing channel, and the focusing channel is used for focusing spore particles in the spore airflow; the separation module is connected with the focusing module, the separation module is provided with a separation channel, and the separation channel is used for separating and obtaining spore particles with preset particle sizes; and the enriching and settling module is connected with the separating module and is provided with a settling and enriching part, and the settling and enriching part is used for settling and collecting spore particles with preset particle sizes. The rubber powdery mildew spore collecting device is simple in structure and convenient to operate, and high-precision and efficient collection of rubber powdery mildew spores can be achieved.

Description

Technical Field

[0001] This application relates to the field of crop detection technology, and more specifically, to microfluidic devices and their preparation methods, and a rubber powdery mildew spore collection and detection system. Background Technology

[0002] Rubber latex, secreted by rubber trees, is a core industrial raw material and the foundation of natural rubber production. Thanks to its excellent insulation, water resistance, and abrasion resistance, rubber materials play an irreplaceable role in strategic fields such as military equipment, medical devices, heavy machinery, and new energy, leading to the widespread application of rubber products. Furthermore, the prevention and control of rubber tree diseases is crucial to both rubber quality and yield.

[0003] Among numerous pests and diseases, powdery mildew on rubber trees poses a particularly prominent threat. This obligate parasite primarily erodes young leaf tissue, initially forming radial, silvery-white hyphae on the leaves, which later evolve into irregular yellow spots. As the disease progresses, a dense powdery layer forms on the underside of the leaves, causing young buds to wither and become deformed, and flowers to develop a powdery covering until they necrotize and fall off. For example, in one year, a large-scale outbreak of powdery mildew occurred in Xishuangbanna: a warm winter prevented older leaves from falling off normally, coupled with prolonged low temperatures and rainy weather in the spring, triggering a major powdery mildew epidemic that resulted in severe defoliation of over 70% of rubber trees, causing significant economic losses. This case reveals that powdery mildew not only reduces latex production by 30%-50% in the current year but also weakens the tree, affecting production capacity for the next 3-5 years. Establishing a scientific prevention and control system has become a core task for ensuring the continuous development of the rubber industry.

[0004] Regarding the collection and detection of rubber powdery mildew, the traditional collection method is to collect samples from glass slides. Subsequently, more efficient spore traps and air samplers were developed, but each has some problems, such as the inability to achieve continuous monitoring, difficulty in carrying them, complex operation, difficulty in reflecting the original state of the collected samples, low concentration, and inability to directly perform detection and analysis. Summary of the Invention

[0005] This application provides a microfluidic device and its preparation method, as well as a rubber powdery mildew spore collection and detection system, which can achieve high-precision and high-efficiency collection and detection of rubber powdery mildew spores.

[0006] In a first aspect, this application provides a microfluidic device for collecting spores of *Pseudomonas aeruginosa*, comprising: a pretreatment module having an inertial impaction channel for separating aggregated spore particles; a focusing module connected to the pretreatment module having a focusing channel with a focusing sheath flow structure at the inlet for focusing spore particles in a spore flow; a separation module connected to the focusing module having a separation channel for separating spore particles of a preset particle size; and an enrichment and sedimentation module connected to the separation module having a sedimentation and enrichment section for sedimenting and collecting spore particles of the preset particle size.

[0007] In some embodiments, the microfluidic device further includes a sample injection module connected to the preprocessing module, and the sample injection module is equipped with a microflow controller.

[0008] In some embodiments, the microfluidic device further includes a transition module that connects the pretreatment module and the focusing module, and the transition module is used to guide and deliver the spore gas flow to the focusing module.

[0009] In some embodiments, the focusing sheath flow structure is configured as a plurality of structures and is symmetrically arranged in the focusing channel.

[0010] In some embodiments, the ratio of the focusing airflow rate to the sample airflow rate in the focusing channel is 2:1.

[0011] In some embodiments, the separation channel includes a first separation channel, a second separation channel, and an emission channel. The first separation channel is used to separate impurity particles with a diameter greater than or equal to 40 μm, the second separation channel is used to separate spores with a diameter less than 20 μm, and the emission channel connects the sedimentation enrichment section to the buffer container.

[0012] Secondly, this application also provides a rubber powdery mildew spore collection and detection system, comprising: a sample collection device for collecting suspended spore particles in the air; a gas processing device connected to the sample collection device for drying and flow rate control of the collected spore particle aerosol; a separation and enrichment device including an air pump and a microfluidic device, wherein the air pump is connected to the microfluidic device and the gas processing device, and the microfluidic device is any of the microfluidic devices described above; a light source excitation device including an excitation light source for exciting endogenous flavonoid fluorescent substances inside the spore particles in the sedimentation and enrichment section of the microfluidic device to generate a fluorescence signal, wherein the light source excitation device further includes a first filter, a dichroic mirror, an objective lens, a second filter, and a tube lens disposed on the upper part of the sedimentation and enrichment section to obtain a purified fluorescence signal; and a processing device including a detector and a computer system, wherein the detector is used to acquire the fluorescence signal and transmit it to the computer system, the detector is connected to the computer system, and the computer system is used to identify, count, and analyze the fluorescence signal to obtain disease risk prediction information for rubber plantation powdery mildew.

[0013] In some embodiments, the excitation light source includes an excitation light source module and a first filter, wherein the excitation light source module is an electrically driven broadband light source module and the first filter is a bandpass filter.

[0014] In some embodiments, the receiving surface of the dichroic mirror is disposed facing the first filter, and the angle between the receiving surface of the dichroic mirror and the horizontal plane is 45°.

[0015] Thirdly, this application also provides a method for fabricating a microfluidic device, comprising the following steps: selecting a glass slide as a substrate, sequentially cleaning it with alcohol, wiping it with a lint-free cloth, drying it with nitrogen, and then sealing it with a film; stacking multiple layers of photosensitive dry film under a yellow light environment, pressing the photosensitive dry film at a preset temperature to remove air bubbles from the film layer; precisely aligning the channel mask with the photosensitive layer, exposing it under a photolithography machine to form a microchannel structure, and using a 1:100 ratio of developer solution to remove uncured areas at room temperature; mixing SYLGARD184 base adhesive and curing agent at a mass ratio of 10:1, performing vacuum degassing treatment, and then pouring the mixture to complete chip peeling and bonding; using a concentration gradient of 10³-10 5 The throughput of the spore suspension at CFU / mL was tested to verify the capture efficiency.

[0016] In this embodiment, pretreatment, focusing, separation, and enrichment sedimentation functions are integrated to automate the collection, purification, and harvesting of rubber powdery mildew spores, replacing traditional manual operations. This enables continuous and rapid collection of rubber powdery mildew spores, significantly improving subsequent detection efficiency and accuracy. Furthermore, this microfluidic device is compact, highly integrated, space-saving, easy to install, and convenient to use. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of this application. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 Schematic diagram of a microfluidic device provided in some embodiments of this application;

[0019] Figure 2 Flowcharts illustrating the fabrication method of a microfluidic device provided in some embodiments of this application;

[0020] Figure 3 This is a schematic diagram of a rubber powdery mildew spore collection and detection system provided in some embodiments of this application;

[0021] Figure 4 This is a schematic diagram of the structure of a rubber powdery mildew spore collection and detection system provided in some embodiments of this application;

[0022] Figure 5 This is an internal schematic diagram of a rubber powdery mildew spore collection and detection system provided in some embodiments of this application;

[0023] Figure 6 Schematic diagram of the processing device in a microfluidic device provided in some embodiments of this application;

[0024] Figure 7 This is a finite element simulation diagram of the separation module of a microfluidic device provided in some embodiments of this application;

[0025] Figure 8 Simulation results of flow field velocity, pressure, and spore particle trajectory in the separation module of the microfluidic device provided in the embodiments of this application.

[0026] The attached figures are labeled as follows:

[0027] 100- Rubber powdery mildew spore collection and detection system;

[0028] 1-Sample collection device; 2-Gas processing device; 3-Separation and enrichment device; 4-Light source excitation device; 5-Processing device; 6-Tube;

[0029] 11-Filter plate; 12-Spinning sampling fan; 13-Function funnel; 21-Drying tube; 22-Rotameter; 31-Air pump; 32-Microfluidic device; 33-Chip loading platform; 41-Excitation light source module; 42-First filter; 51-Microscopic observation device; 52-Detector;

[0030] 321-Pretreatment module; 322-Inertial impaction channel; 323-Focusing channel; 324-Focusing sheath flow structure; 325-Transition module; 326-First separation channel; 327-Second separation channel; 328-Emission channel; 329-Sedimentation enrichment section; 511-Dichroic mirror; 512-Objective lens; 513-Second filter; 514-Tube lens. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0032] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used in the description of this application is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. The terms "first," "second," etc., are used to distinguish different objects, not to describe a particular order or hierarchy.

[0033] In this application, the reference to "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that is mutually exclusive with other embodiments.

[0034] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," "linking," and "attachment" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0035] In this application, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. Additionally, in this application, the character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0036] In the embodiments of this application, the same reference numerals denote the same components, and for the sake of brevity, detailed descriptions of the same components are omitted in different embodiments. It should be understood that the thickness, length, width, and other dimensions of various components in the embodiments of this application shown in the accompanying drawings, as well as the overall thickness, length, width, and other dimensions of the integrated device, are merely illustrative and should not constitute any limitation on this application.

[0037] In this application, "multiple" means two or more (including two).

[0038] Please refer to Figure 1 , Figure 1 This is a schematic diagram of a microfluidic device provided in some embodiments of this application.

[0039] This application provides a microfluidic device for the separation and enrichment of *Pseudomonas aeruginosa* spores. The microfluidic device 32 adopts a modular structure, including a pretreatment module 321, a focusing module, a separation module, and an enrichment and sedimentation module. The pretreatment module 321 is equipped with an inertial impaction channel 322, which is used to separate aggregated spore particles. Aggregates and mutually adherent particles are treated and separated through inertial impaction. The sample then enters the focusing module through an airflow. The focusing module is connected to the pretreatment module 321 and is equipped with a focusing channel 323. The inlet of the focusing channel 323 is equipped with a focusing sheath flow structure 324, which focuses the spore particles in the spore airflow into the focusing channel 323. The separation module is connected to the focusing module and is equipped with a separation channel. The separation channel is used to separate spore particles of a preset particle size, resulting in relatively purified *Pseudomonas aeruginosa* spores. An enrichment and sedimentation module is provided at the rear end of the separation module. The enrichment and sedimentation module is connected to the separation module. The enrichment and sedimentation module is provided with a sedimentation and enrichment section 329. The sedimentation and enrichment section 329 is used to sediment and collect spore particles of a preset particle size. The purified rubber powdery mildew spores are enriched in the sedimentation and enrichment section 329.

[0040] The microfluidic device 32 provided in this application embodiment enables the in-situ separation and enrichment of purified rubber powdery mildew spores through a multi-level microfluidic structure.

[0041] In this embodiment, the pretreatment module 321 can be equipped with an aerosol inertial impaction channel 322 for primary separation of spore particles from each other or from other particle clusters. The inertial impaction channel 322 can adopt an impact structure, and the channel adopts a sudden expansion flow channel structure to enhance the inertial separation efficiency. The separation module utilizes the different force conditions of spores and impurity particles under the same separation airflow to achieve directional sorting of target objects, which then flow out of the separation module through the set separation channel. The sedimentation enrichment section 329 can be a gravity sedimentation chamber, which achieves spore enrichment by controlling gravity acceleration and fluid resistance. The impurity outlet is set at the end of the sedimentation enrichment section 329 to discharge non-target particles. The enrichment sedimentation module expands through the channel structure, reduces the flow field velocity, and sediments and enriches the target particles, facilitating detection and observation.

[0042] For example, the focusing channel 323 adopts a focusing sheath flow microchannel structure with a width of 1600 μm and a height of 100 μm. The spores are focused towards the centerline by the compression of the spores in the airflow through the bidirectional sheath flow. The enrichment and sedimentation module is connected to the outlet of the separation module through the sample inlet. The outlet has a width of 500 μm and a height of 100 μm.

[0043] Furthermore, the pretreatment module 321 also includes a sample injection module, which is connected to the pretreatment module 321. Rubber powdery mildew spores are introduced into the chip inlet of the sample injection module. The sample injection module is equipped with a micro-flow controller, which can be, but is not limited to, a glass rotor flow meter 22, to realize automatic flow measurement.

[0044] Furthermore, the microfluidic device 32 also includes a transition module 325, which connects the pretreatment module 321 and the focusing module, and guides and delivers the spore gas flow to the focusing module.

[0045] The focusing module uses a laminar flow-controlled airflow inlet for its focusing airflow inlet. Its internal focusing channel 323 is based on the Dean vortex effect, and the ratio of the focusing airflow rate to the sample airflow rate in the focusing channel is 2:1.

[0046] In one optional embodiment, the focusing sheath flow structure 324 is configured as two or more, and symmetrically arranged in the focusing channel 323. For example, it can be uniformly distributed along the circumference of the focusing channel 323. For example, as shown... Figure 1 As shown, the focusing sheath flow structure 324 adopts a dual sheath flow focusing microchannel, which is symmetrically arranged on the upper and lower sides of the focusing channel 323.

[0047] The separation channel of the separation module can adopt a sorting microchannel structure. Based on the inertial settling of the separated airflow, a stepped, multi-size particle sorting channel structure can be designed, such as... Figure 1 As shown, the sorting microchannel specifically comprises a first separation channel 326, an emission channel 328, and a second separation channel 327. The first separation channel 326 can separate impurity particles of size 40 μm and above, and discharge them from the outlet of the first separation channel 326 into the microfluidic device 32. The emission channel 328 can separate target particles of 20~40 μm, which enter the sedimentation enrichment section 329 for enrichment and sedimentation via the enrichment sedimentation channel. The second separation channel 327 can separate particles smaller than 20 μm, and discharge them from the outlet of the second separation channel 327.

[0048] like Figure 2 As shown. Furthermore, embodiments of this application also provide a method for fabricating the aforementioned microfluidic device, comprising the following steps:

[0049] Step S101: Select a glass sheet as the substrate, and then clean it with alcohol, wipe it with a lint-free cloth, dry it with nitrogen, and then seal it with a film.

[0050] Step S102: Under yellow light environment, multilayer photosensitive dry films are stacked and pressed at a preset temperature to remove air bubbles from the film layer.

[0051] Step S103: The channel mask and the photosensitive layer are precisely aligned, and the microchannel structure is formed by exposure under a photolithography machine. Uncured areas are removed by using a 1:100 ratio developer at room temperature.

[0052] Step S104: Mix SYLGARD184 base adhesive and curing agent at a mass ratio of 10:1, and then pour the mixture after vacuum degassing to complete chip peeling and bonding;

[0053] Step S105, using a concentration gradient of 10³-10 5 The flux of the spore suspension at CFU / mL was tested to verify the capture efficiency and to ensure that the channel transmittance was greater than 92%.

[0054] In step S101, the substrate is first pretreated by selecting a glass sheet with a thickness of 0.4±0.05mm as the substrate. After cleaning with alcohol, it is wiped clean with a lint-free cloth, and its surface is dried by blowing with nitrogen. Then, it is sealed with a film for later use.

[0055] In step S102, the film is applied under yellow light. First, the film layers are stacked: two layers of photosensitive dry film (48μm per layer) are selected and positioned and applied. Then, precision forming is performed: a semi-automatic laminating machine is used in conjunction with a 50℃-70℃ ironing process to achieve bubble-free bonding of the film layers.

[0056] In step S103, the microstructure forming mask is aligned: First, the channel mask is precisely registered with the photosensitive layer; then, exposure is performed for 10 seconds under a photolithography machine to achieve a 100μm aspect ratio structure; then, development is performed: a 1:100 developing solution is prepared and uncured areas are removed at room temperature.

[0057] In step S104, functional packaging is performed. First, SYLGARD184 base adhesive and curing agent are mixed at a mass ratio of 10:1 and then poured after vacuum degassing (pressure -0.08MPa, time 15min). Then, the chip is peeled off and bonded.

[0058] In step S105, the performance of the microfluidic device 32 is verified, and a three-level detection system is constructed: First, the active focusing module adopts a fluid dynamic focusing structure to achieve spore pre-arrangement through focused airflow; then, the capture is verified by using a concentration gradient spore suspension (103~105 CFU / mL) for throughput testing to ensure that the capture efficiency reaches the ideal result; finally, optical compatibility is ensured, with a channel transmittance greater than 92%, making it compatible with a microscopic imaging system.

[0059] The microfluidic device fabrication method provided in this application enhances process robustness while maintaining core technical indicators by optimizing the tolerance range of process parameters (such as temperature fluctuation ±5℃ and time deviation ±5s).

[0060] like Figure 3 As shown. This application also provides a rubber powdery mildew spore collection and detection system 100 for collecting and detecting rubber powdery mildew spores. The system includes: a sample collection device 1, a gas processing device 2, a separation and enrichment device 3, a light source excitation device 4, and an image processing device 5. The devices and components within the devices are connected by flexible hoses 6.

[0061] The sample collection device 1 is located on the outermost side of the system and can actively capture and collect spore particles suspended in the air, and supply the collected spore particle samples to the gas processing device 2.

[0062] like Figure 4 and Figure 5 As shown. Exemplarily, the sample collection device 1 includes a filter plate 11, a spin sampling fan 12, and a funnel 13. The filter plate 11 is located on the outermost side of the device housing, while the spin sampling fan 12 and funnel 13 are located inside the device housing. Powered by the spin sampling fan 12, suspended spore particles in the air are drawn into the device housing. They first undergo primary filtration through the filter plate 11 to separate impurities. Then, the aerosol enters the funnel 13 and exits from the top outlet of the funnel 13. The top outlet is connected to a rotor flow meter 22 via a hose 6. After passing through the rotor flow meter 22, the sample enters the next unit.

[0063] The gas treatment device 2 includes a drying tube 21 and a rotor flow meter 22. The drying tube 21 is connected to a funnel 13 to dry the aerosol containing enriched spore particles collected in the funnel 13, ensuring that the aerosol remains dry. The rotor flow meter 22 is located at the rear end of the drying tube 21 and at the front end of the separation and enrichment device 3. The rotor flow meter 22 is connected to the microfluidic device 32 of the rotor flow meter 22 via a flexible hose 6 to control the flow rate of the gas entering the microfluidic device 32. Excess gas is introduced into an external buffer container through the emission channel 328 to achieve harmless emission.

[0064] The rotor flowmeter 22 can employ a rotor precision flow regulator, which, based on predetermined fluid dynamics simulation parameters, stably controls the inlet flow rate within the range of 15-20 mL / min.

[0065] Furthermore, the gas processing device 2 also includes a filter assembly located at the rear end of the drying tube 21, and the two are connected by a hose 6. The filter assembly can remove other particulate impurities from the filtered air.

[0066] The separation and enrichment device 3 includes an air pump 31 and a microfluidic device 32. The air pump 31 can be a miniature air pump 31, which provides power. The microfluidic device 32 is fixed on the chip loading platform 33. The chip outlet is connected to the negative pressure generating device through a silicone tube 6. The microfluidic device 32 uses a channel structure to amplify the sedimentation enrichment section 329 of gravity sedimentation that changes the flow field velocity. The air pump 31 draws air into the inlet of the focusing sheath flow structure 324 on the side of the microfluidic device 32 to focus the sample in the internal channel of the microfluidic device 32, and then separates and obtains purified rubber powdery mildew fungal spores.

[0067] After the powdery mildew spore suspension is collected by the sample collection device 1 and processed by the gas processing device 2, it is then screened by the microfluidic device 32 of the separation and enrichment device 3 to obtain purified rubber powdery mildew fungal spores.

[0068] The microfluidic device 32 in this application embodiment can be the same microfluidic device 32 used for the separation and collection of rubber powdery mildew spores in the above embodiments.

[0069] The sampling, separation, and enrichment principles in this embodiment are illustrated below: Powered by a spinning sampling fan 12, suspended particles in the air are drawn into the device. The aerosol containing enriched suspended particles first undergoes primary impurity separation via a filter plate 11. Then, the aerosol enters a funnel 13 and exits from its top outlet. This top outlet is connected to the inlet of a drying tube 21 via a hose 6. After the drying tube 21 absorbs and dries the sample, the sample enters a rotor flowmeter 22 from the outlet of the drying tube 21. The rotor flowmeter 22 controls the flow rate, and the sample then enters the sedimentation enrichment section 329 of the microfluidic device 32 through the hose 6 and the inlet of the microfluidic device 32. The sample moves within the channels of the microfluidic device 32. Spore particles with a diameter of 40 μm or larger are discharged through the outlet of the first separation channel 326, while spore particles with a diameter of 20 μm or smaller are discharged through the outlet of the second separation channel 327. Target spore particles with a diameter between 20 μm and 40 μm enter the sedimentation enrichment section 329 for sedimentation and enrichment. The outlet of the microfluidic device 32 is connected to the air pump 31, and the redundant gas at the back end is discharged from the outlet of the air pump 31 from the microfluidic device 32.

[0070] The rubber powdery mildew spore collection and detection system 100 also includes a light source excitation device 4, a microscopic observation device 51, and a processing device 5. The microscopic observation device 51 includes an excitation light source, a first filter 42, a dichroic mirror 511, an objective lens 512, a second filter 513, and a tube lens 514. The excitation light source excites the endogenous flavonoid fluorescent substances inside the spore particles in the sedimentation enrichment section 329 of the microfluidic device 32 to generate a fluorescent signal. The upper part of the sedimentation enrichment section 329 is sequentially provided with a dichroic mirror 511, an objective lens 512, a second filter 513, and a tube lens 514 to obtain a purified fluorescent signal.

[0071] In this embodiment, the light source excitation device 4 forms two optical paths: an excitation optical path and an emission optical path. The excitation optical path is the optical path formed by the excitation light source generating light and the rubber powdery mildew spores inside the sedimentation and enrichment section 329. The emission optical path is the optical path formed from the rubber powdery mildew spores to the treatment device 5.

[0072] For example, the excitation device emits 375nm wavelength excitation light. This excitation light passes through a 375 / 20nm excitation first filter 42, which filters for specific wavelengths of excitation light, limiting stray light interference. The purified 375nm excitation light reaches a dichroic mirror 511, whose cutoff wavelength is set at approximately 400nm. This mirror reflects short-wavelength light below 400nm and transmits long-wavelength light above 400nm. Therefore, the 375nm excitation light is reflected by the dichroic mirror 511, with the reflected light pointing vertically downwards. The downward-propagating excitation light converges to a focal point, precisely illuminating the rubber powdery mildew spores enriched inside the sedimentation enrichment section 329 of the microfluidic device 32.

[0073] like Figure 6 As shown. In an optional embodiment, the excitation light source includes an excitation light source module 41 and a first filter 42. The excitation light source module 41 is an electrically driven broadband light source module, and the first filter 42 is a bandpass filter.

[0074] Specifically, the excitation light source module 41 is equipped with an electrically driven broadband light source (e.g., 300-700 nm) and connected to a bandpass excitation first filter 42 (e.g., 360-370 nm). A dichroic mirror 511, mounted at an angle, is configured to reflect the monochromatic excitation light output from the excitation filter to the sample region and transmit the long-wavelength emitted light generated by the sample. A second emission filter 513 is equipped with a bandpass filter (e.g., 420-460 nm) to block excitation light leakage. The sample region receives spore samples output from the sedimentation enrichment section 329. NAD(P)H within the spores is stimulated to produce 460 nm fluorescence, and senescent spores exhibit enhanced fluorescence intensity.

[0075] The dichroic mirror 511 in this embodiment operates on the following principle: it reflects short wavelengths (excitation light, such as 360 nm) and transmits long wavelengths (emission light, such as 460 nm). After the sample enters the enrichment region, the excitation light source module 41 receives input, and the electrical energy drives the mercury lamp to output a broadband light of 300-700 nm, providing excitation energy. The first filter 42 is a bandpass filter, selectively transmitting through multiple layers of media to block non-target wavelengths and output purified monochromatic excitation light of 360-370 nm.

[0076] The endogenous flavonoid fluorescent substances in the spores of *Pseudomonas aeruginosa* generate a characteristic emission spectrum (e.g., 460 nm) under photoexcitation. The 460 nm emission light emitted by the spores (along with some scattered excitation light and other stray light) propagates upwards and reaches the dichroic mirror 511 again. Due to the characteristics of the dichroic mirror 511, it can transmit light longer than 400 nm, allowing the 460 nm emission light to pass smoothly through it. Any 375 nm excitation light scattered back by the sample is reflected back to the light source by the dichroic mirror 511, thus being effectively separated. The transmitted light contains the target emission light at 460 nm, but may also contain weak fluorescence or noise of other wavelengths. The beam then passes through the objective lens 512 and is filtered by a second filter 513. This second filter 513 only allows light with a center wavelength of 460 nm and a bandwidth of 25 nm to pass through, achieving secondary filtering and optimization of the characteristic emission spectrum, significantly improving the signal-to-noise ratio. Finally, the purified 460nm fluorescence is guided to the processing device 5 through the tube lens 514.

[0077] In order to improve the receiving efficiency of the excitation light source, the receiving surface of the dichroic mirror 511 is set towards the first filter 42, and the angle between the receiving surface of the dichroic mirror 511 and the horizontal plane is 45°.

[0078] The processing device 5 includes a detector 52 and a computer system. The detector 52 is used to acquire fluorescence signals and transmit them to the computer system. The detector 52 uses a scientific-grade CCD sensor to capture fluorescence signals and transmits them to the computer system in real time through an interface. The computer system identifies, counts and analyzes the fluorescence signals to obtain disease risk prediction information for rubber plantation powdery mildew.

[0079] The computer system uses a deep learning-based computer vision model to extract spore morphological features and automatically count them. The count data is uploaded to a cloud analysis platform via an interface. Combined with historical meteorological data and field parameters, a risk level prediction model for powdery mildew (PDB) in rubber plantations is constructed using a spore density-disease incidence regression analysis algorithm.

[0080] For example, the computer system performs finite element simulation on the separated parts of the chip, sets relevant simulation parameters, and verifies the particle collection efficiency, such as... Figure 7 As shown, particles ranging in size from 10 to 40 μm were selected, and the collection quantity and efficiency were statistically analyzed. The results are as follows. Figure 8 As shown, the collection efficiency for target particles with a size of 20-40 μm is over 95%, achieving the ideal collection effect.

[0081] Therefore, the rubber powdery mildew spore collection and detection system 100 provided in this application embodiment is simple to operate and highly accurate, avoids mechanical repetitive work, improves detection efficiency, and can quickly detect the number of powdery mildew spores and make predictions on the incidence of rubber tree powdery mildew based on experimental models.

[0082] This application addresses the challenges of detecting latent spores and the delayed prediction of disease outbreaks in rubber powdery mildew by proposing an integrated rubber powdery mildew spore collection and detection system. Based on a multi-level microfluidic structure, it achieves a functional chain for in-situ spore separation, enrichment, and quantitative analysis, enabling latent spore capture, high-precision optical detection, and disease prediction, forming a closed loop from spore concentration detection to disease risk level output.

[0083] The microfluidic device and its preparation method, as well as the rubber powdery mildew spore collection and detection system provided in this application, have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. It should be noted that those skilled in the art can make several improvements and modifications to this application without departing from the principles of this application, and these improvements and modifications also fall within the protection scope of this application.

Claims

1. A microfluidic device for collecting of Oidium neolycopersici spores, characterized in that, The microfluidic device comprises: a pretreatment module (321) provided with an inertial impact channel (322) for separating agglomerated spore particles; a focusing module connected to the pretreatment module (321) and provided with a focusing channel (323) having an inlet provided with a focusing sheath flow structure (324) for focusing spore particles in a spore gas flow; a separation module connected to the focusing module and provided with a separation channel for separating spore particles of a preset particle size; an enrichment and sedimentation module connected to the separation module and provided with a sedimentation enrichment part (329) for sedimentation and collection of spore particles of a preset particle size.

2. The microfluidic device of claim 1, wherein, The microfluidic device further comprises a sample injection module connected to the pretreatment module (321) and provided with a micro-flow controller.

3. The microfluidic device of claim 2, wherein, The microfluidic device further comprises a transition module (325) connected to the pretreatment module (321) and the focusing module, and used for guiding and transporting the spore gas flow to the focusing module.

4. The microfluidic device of any one of claims 1-3, wherein, The focusing sheath flow structure (324) is provided in multiple and symmetrically arranged in the focusing channel (323).

5. The microfluidic device of claim 3, wherein, The ratio of the flow rate of the focusing gas flow in the focusing channel (323) to the flow rate of the sample gas flow is 2:

1.

6. The microfluidic device of claim 1, wherein, The separation channel comprises a first separation channel (326) for separating impurity particles with a particle size greater than or equal to 40 μm, a second separation channel (327) for separating spores with a particle size less than 20 μm, and a discharge channel (328) connected to the sedimentation enrichment part (329) and a buffer container.

7. An Oidium neolycopersici spore collection detection system characterized by, The microfluidic device comprises: a sample collection device for collecting suspended spore particles in the air; a gas treatment device (2) connected to the sample collection device for drying and flow rate control of the collected spore particle aerosol; a separation and enrichment device (3) comprising a gas pump (31) and a microfluidic device (32), wherein the gas pump (31) is connected to the microfluidic device (32) and the gas treatment device (2), and the microfluidic device (32) is the microfluidic device (32) of any one of claims 1-6; a light source excitation device (4) comprising an excitation light source for exciting endogenous flavonoid fluorescent substances inside spore particles in the sedimentation enrichment part (329) of the microfluidic device (32) to generate a fluorescent signal, and further comprising a first filter (42), a dichroic mirror (511), an objective lens (512), a second filter (513), and a cylindrical lens (514) arranged on the upper part of the sedimentation enrichment part (329) to obtain a purified fluorescent signal; The processing device (5) comprises a detector (52) for acquiring the fluorescent signal and transmitting to a computer system, the detector (52) is connected to the computer system, and the computer system is used for identifying, counting and analyzing the fluorescent signal to obtain the disease risk prediction information of the rubber forest powdery mildew.

8. The system for detecting the collection of Microsphaera nashi spores according to claim 7, wherein The excitation light source comprises an excitation light source module (41) and a first filter (42), the excitation light source module (41) is an electric energy driven wide spectrum light source module, and the first filter (42) is a band-pass filter.

9. The system for detecting the collection of Microsphaera nashi spores according to claim 8, wherein The receiving surface of the dichroic mirror (511) is arranged towards the first filter (42), and the angle between the receiving surface of the dichroic mirror (511) and the horizontal plane is 45°.

10. A method for producing a microfluidic device, for producing a microfluidic device according to any one of claims 1 to 6, characterized in that The method comprises the following steps: Glass slices are selected as substrates, and are sequentially cleaned with alcohol, wiped with a dust-free cloth, dried with nitrogen blowing, and then sealed with a film; In a yellow light environment, a plurality of layers of photosensitive dry films are stacked, the photosensitive dry films are pressed at a preset temperature to discharge bubbles in the film layers; The channel mask and the photosensitive layer are accurately aligned, exposed under a photoetching machine to form a micro-channel structure, and the un-solidified area is removed using a 1:100 ratio developing solution under room temperature conditions; SYLGARD184 base glue and a curing agent are mixed at a mass ratio of 10:1, vacuum degassing treatment is performed, and then pouring is performed to complete chip peeling and bonding; A spore suspension with a concentration gradient of 10 3 -10 5 CFU / mL was used for flux tests to verify the capture efficiency.

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