Trichogramma exposure device and preparation method thereof

Abstract

The application discloses an exposure and poisoning device for trichogramma and a preparation method thereof. The exposure and poisoning device for trichogramma comprises a fixed frame with a hole in the middle, and two cover plates which are detachably connected with the fixed frame and used for plugging both sides of the hole. At least one round hole is arranged around the fixed frame, a hollow cylinder pipe is arranged on the round hole, and a gauze is arranged at the outer end of the hollow cylinder pipe. The exposure and poisoning device for trichogramma adopts an integrally-formed PMMA fixed frame, so that the accuracy of the exposure and poisoning test of the trichogramma is effectively avoided from being affected by the use of glue bonding and the like, meanwhile, the problem that the glue is easily peeled off after the device is cleaned after the test is effectively solved, the device is convenient to disassemble and assemble, and is simple and easy to use.

Description

Technical Field

[0001] This invention relates to the technical field of Trichogramma wasp exposure testing equipment, specifically to a Trichogramma wasp exposure and toxicity treatment device and its preparation method. Background Technology

[0002] Red-eyed bee ( Trichogramma spp. As the most widely used oviparous parasitic natural enemy insect globally, *Trichogramma rubescens* possesses highly efficient control capabilities against lepidopteran pests (such as corn borers and cotton bollworms) in agricultural ecosystems, making it a core species in biological control systems. With the accelerated green transformation of agriculture, the ecological risk assessment of chemical pesticides has shifted from single-target pest control to a "non-target biological protection" strategy. GB / T 31270.17-2025, published in 2025 and officially implemented on May 1, 2026, explicitly lists acute toxicity testing of *Trichogramma rubescens* as a legally mandatory requirement for pesticide registration, with the results directly determining whether a pesticide can be approved for registration and field use. This test quantifies the median lethal dose (LR) of pesticides on *Trichogramma rubescens*. 50 The construction of an "ecological safety threshold" provides a scientific basis for pesticide environmental risk classification, green pesticide creation, and integrated pest management (IPM), and is a key technical support for achieving "reduced pesticide use and increased efficiency" and biodiversity conservation.

[0003] Because Trichogramma wasps are extremely small (approximately 0.3 mm), and acute toxicity tests on Trichogramma wasps require extremely high operational precision, current acrylic frames either consist of several panels glued together and then sealed with glue and foam padding. However, Trichogramma wasps are highly sensitive to glue; even after prolonged exposure, the presence of glue during actual testing can cause them to die, affecting the accuracy of the exposure test. Therefore, a user-friendly device is needed that does not affect the accuracy of the Trichogramma exposure test and prevents the wasps from escaping, thus addressing these issues. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the present invention provides a Trichogramma wasp exposure and treatment device and its preparation method.

[0005] The technical solution of the present invention is: a Trichogramma wasp exposure and poisoning device, comprising a fixed frame with a hole in the middle, and two cover plates detachably connected to the fixed frame for sealing both sides of the hole; The fixed frame has at least one round hole around its perimeter, and a hollow cylindrical tube is provided in the round hole, with a mesh at the outer end of the hollow cylindrical tube. The fixed frame is obtained by processing PMMA as the frame matrix using a biomimetic microstructure composite material, then filling the frame matrix with TRNS nanoparticles.

[0006] Note: This invention uses an integrally molded fixed frame and two cover plates to seal the holes. This effectively avoids the impact of using glue or other methods on the accuracy of the Trichogramma exposure test. Because Trichogramma is sensitive to glue, and the device is cleaned after the test, glue can easily come off and is not easy to disassemble. By using biomimetic microstructure composite materials for the manufacture of the fixing frame, the three major characteristics of transparency, odorlessness, and self-sealing can be met simultaneously. The automatic micro-deformation generated by contact with the cover plate can significantly improve the sealing performance of the box and prevent Trichogramma wasps (about 0.3 mm) from escaping through the contact gaps and affecting the accuracy of the test.

[0007] Furthermore, the detachable connection is achieved by fixing the cover plate to the fixed frame with clips, and the side of the fixed frame is provided with a flange for connecting the clips.

[0008] Description: By using clips to fix the cover plate to the fixed frame, disassembly and assembly can be quickly achieved without additional tools. The clip structure achieves one-button operation through the mechanical interlocking of elastic buckles and flanges. No screwdrivers, wrenches, or other tools are needed; the cover plate can be installed and removed with one hand. This feature greatly improves efficiency in operations such as device maintenance. Furthermore, the clips and flanges form a closed-loop mechanical constraint, which, combined with elastic elements, enables dynamic adaptive adjustment of the clamping force. This continuously counteracts the vibration and thermal expansion and contraction stress that may occur in the device, maintaining connection reliability during long-term operation.

[0009] Furthermore, the preparation method of the TRNS nanoparticles is as follows: 1) Oil phase preparation: Span 80, Tween 80, and Fe3O4 nanoparticles were sequentially dispersed in 60 mL of n-hexane and magnetically stirred for 30 min; wherein the amount of Span 80 was 3.5~4.8 wt% of n-hexane, the amount of Tween 80 was 1.0~1.5 wt% of n-hexane, and the amount of Fe3O4 nanoparticles was 0.2~0.8 wt% of n-hexane; 2) Aqueous phase preparation: N - Isopropylacrylamide (NIPAM) and sodium 2-acrylamide-2-methylpropanesulfonate (AMPS) monomers were dissolved in deionized water and then added N,N' - Methylenebisacrylamide (MBA), ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) form a uniform aqueous core microemulsion with a particle size distribution (PDI) < 0.15. The aqueous phase volume is 6–10% of the oil phase volume, and the total monomer concentration is 12–18 wt%. The mass ratio of the two monomers is: N- Isopropylacrylamide (NIPAM): Sodium 2-acrylamide-2-methylpropanesulfonate (AMPS) = 80~90: 10~20; N,N' The addition amounts of methylenebisacrylamide (MBA), ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) relative to the total monomers were 0.5–1.0 wt%, 0.2–0.5 wt%, and 0.1–0.2 wt%, respectively. 3) The aqueous phase was slowly added dropwise to the oil phase at a rate of 0.3~0.6 mL / min. The mixture was stirred continuously at 40±1℃ for 60 min (stirring speed of 12000 rpm). Then, high-purity nitrogen was introduced for 10~20 min, and the mixture was kept at a constant temperature of 50±1℃ for 4~5 h under an inert atmosphere to prevent oxidation. The mixture was then cooled to room temperature, centrifuged (12000 rpm, 20 min), and washed several times with n-hexane and ethanol, respectively, to remove unreacted monomers and surfactants, yielding particulate matter. 4) Dialyze the particles in a dialysis bag (MWCO 14000Da) for 42-48 h, changing the deionized water every 6 h to thoroughly remove small molecule impurities. Then, soak the dialyzed particles in 0.1 mM FeCl3 solution at 25℃ for 2-3 h, and then disperse them in PBS buffer at 25℃ and pH 7.4 with stirring for 12-16 h. Add PEG-5000-NHS activated ester, then pre-freeze the resulting particles at -80℃ for 24 h, and freeze-dry them under vacuum for 32-48 h to obtain white powdery TRNS nanoparticles with a particle size of 1-3 μm. Store them in a desiccator, protected from light and moisture, for later use. The amount of PEG-5000-NHS activated ester added relative to the dialyzed particles is 1.5-3.2 wt%.

[0010] Note: This invention is based on N - Isopropylacrylamide (NIPAM) is a temperature-sensitive monomer, and sodium 2-acrylamido-2-methylpropanesulfonate (AMPS) is a hydrophilic comonomer. This allows for precise matching of the metabolic heat-generating temperature of Trichogramma wasps, achieving immediate sealing upon wasp movement, with extremely high reversibility. Compared to existing materials, the TRNS nanoparticles of this invention can maintain a sealing efficiency of >95% after 8000~10000 cycles. Furthermore, Fe3O4 nanoparticles are introduced during the preparation process. On the one hand, the embedded Fe3O4 nanoparticles do not damage the LCST; on the other hand, Fe... 3+ Coordination does not affect thermal response kinetics, giving the prepared TRNS nanoparticles a self-healing function, thereby solving the leakage risk caused by the accumulation of red microcracks during long-term use, and thus significantly improving the sealing effect and durability of TRNS nanoparticle-PMMA composite materials.

[0011] Furthermore, the method for preparing the fixed frame includes the following steps: 1) Preheat the frame mold to 80±2℃ and vacuum dry for 2 hours. Then, gravity-cast the PMMA particles into the frame mold at a casting temperature of 160±2℃ and degas at -0.08 MPa for 12~18 minutes. Subsequently, cool the mold from 160±2℃ to 80±2℃ at a cooling rate of 0.5℃ / min and anneal it at 80℃ for 3~5 hours to obtain the frame matrix. 2) The framework substrate is fabricated using a microcavity array to obtain a microcavity framework; 3) Disperse TRNS nanoparticles in anhydrous ethanol, wherein the amount of TRNS nanoparticles is 0.3~0.8 wt% of anhydrous ethanol, and sonicate for 10~15 min to obtain a dispersion. Then, completely immerse the microcavity frame in the dispersion and adsorb it under a vacuum of -0.09 MPa for 12~18 min. Then, dry and solidify it at 120℃ in a nitrogen atmosphere for 25~40 min to obtain a fixed frame.

[0012] Note: By using food-grade / biological laboratory-grade high-transparency PMMA (such as Evonik Plexiglas® 7N), we ensure that the framework matrix is ​​free of plasticizers and residual monomers. Simultaneously, we composite TRNS nanoparticles with the framework matrix. The internal conditions of the device are dynamically controlled by utilizing the properties of the TRNS nanoparticles, as follows: In a resting state (bees are not active): at a temperature <34℃, TRNS particles shrink, and the gaps in the microcavities are retained, which is conducive to ventilation; During heat triggering (bee activity): the heat generated by bee metabolism raises the local temperature to 35.5℃. The TRNS nanoparticles absorb heat and expand in volume, resulting in a rapid response that creates micro-protrusions of 15~25 μm in height. Furthermore, the expansion of the TRNS nanoparticles and the nanoscale depressions (<5 μm) on the cover plate surface form a lattice-like mechanical interlock, achieving an airtightness of <5×10⁻⁶. -7 Pa·m 3 / s, and when the temperature drops, the sealing interface can automatically release without residual deformation, and it can still maintain a sealing efficiency of >95% after 8000~10000 cycles.

[0013] Furthermore, the microcavity array is fabricated as follows: using a femtosecond laser micromachining system (wavelength 800 nm, pulse width 150 fs), 5000 cells / mm are etched within a 0.5 mm wide area on the inner edge of the contact surface between the frame substrate and the cover plate. 2 The biomimetic leaf-shaped microcavity has a diameter of 8-12 μm and a depth of 3-5 μm.

[0014] Explanation: By etching a biomimetic blade pillow microcavity within a 0.5 mm wide area on the inner edge of the contact surface between the frame substrate and the cover plate, the sealing performance can be dynamically controlled by utilizing the characteristics of TRNS nanoparticles. This allows the TRNS nanoparticles to expand and form a lattice-like mechanical interlock with the nanoscale depressions on the cover plate surface. Moreover, when the temperature drops, the sealing interface can automatically release without any residual deformation.

[0015] Furthermore, the microcavity array is fabricated by setting a density of 5~10 cells / mm on the frame substrate. 2 Micro-holes.

[0016] Note: Based on the sealing, we further applied TRNS nanoparticles by incorporating them into the walls of the fixed frame around the holes. This utilizes the properties of TRNS nanoparticles to give the fixed frame a microporous structure similar to an insect eggshell, which can maintain a certain gas exchange function while preventing Trichogramma wasps from escaping from the device. Therefore, it can also be used in experiments such as observing the growth of Trichogramma wasps.

[0017] The present invention also provides a method for preparing a Trichogramma wasp exposure device, comprising the following steps: S1. Wrap the mesh around the outer end of the hollow cylindrical tube, and then insert the outer end of the hollow cylindrical tube into the round hole of the fixed frame to obtain the assembled fixed frame. S2. Place the two cover plates on the upper and lower ends of the assembled fixed frame, respectively, so that the two ends of each cover plate are connected to the fixed frame, thus obtaining the Trichogramma exposed poisoning device.

[0018] Furthermore, an eggshell-like nanofibril array with a height of 200-300 nm and a spacing of 600-800 nm was constructed on the inner wall surface of the frame matrix and the cover plate using anodized aluminum oxide (AAO) templates.

[0019] Explanation: By constructing an eggshell-like nanofibril array on the surface of the frame matrix and the inner wall of the cover plate, a highly uniform nanofibril array is formed, which biomimetically matches the surface structure of the Trichogramma eggshell, forming "mechanical anchor points" to enhance the seal.

[0020] The beneficial effects of this invention are: (1) The Trichogramma exposure and poisoning device of the present invention adopts an integrally molded PMMA fixing frame, which effectively avoids the impact of glue bonding on the accuracy of the Trichogramma exposure and poisoning test. At the same time, it effectively solves the problems of easy detachment of glue when cleaning the device after the test. It is easy to disassemble and assemble, and simple to use.

[0021] (2) By using highly transparent PMMA and TRNS nanoparticles to composite, and by etching biomimetic leaf pillow microcavities on the inner edge of the contact surface between the frame matrix and the cover plate, the sealing performance can be dynamically controlled by utilizing the characteristics of TRNS nanoparticles. This allows the TRNS nanoparticles to expand and form a dot matrix mechanical interlock with the nanoscale depressions on the cover plate surface. Moreover, when the temperature drops, the sealing interface can be automatically released without residual deformation. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the overall structure of the Trichogramma wasp exposure and poisoning device of the present invention; Figure 2 This is an exploded view of the Trichogramma wasp exposure and poisoning device of the present invention; Figure 3 This is a schematic diagram of the internal structure of the Trichogramma wasp exposure and poisoning device of the present invention; Figure 4 This is a schematic diagram of the fixed frame structure of the Trichogramma wasp exposure and poisoning device of the present invention; Among them, 1-fixed frame, 11-hole, 12-round hole, 13-flanged edge, 2-cover plate, 3-hollow cylindrical tube, 4-mesh, 5-clamp. Detailed Implementation

[0023] The present invention will now be described in more detail with reference to specific embodiments, so as to better demonstrate the advantages of the present invention.

[0024] Example 1: As Figure 1-Figure 4 As shown, a Trichogramma wasp exposure and venom-contamination device includes a fixed frame 1 with a hole 11 in the center, and two cover plates 2 detachably connected to the fixed frame 1 for sealing both sides of the hole 11; the detachable connection is achieved by clamps 5 to fix the cover plates 2 to the fixed frame 1, and the side of the fixed frame 1 is provided with flanges 13 for connecting the clamps 5. The fixing frame 1 has at least one circular hole 12 around its perimeter, and a hollow cylindrical tube 3 is provided on the circular hole 12. A mesh 4 is provided at the outer end of the hollow cylindrical tube 3. The fixing frame 1 has dimensions of 100 mm × 80 mm × 30 mm (length × width × height), and the hole 11 has dimensions of 60 mm × 60 mm × 30 mm. The cover plate 2 is a commercially available glass plate with dimensions of 100 mm × 80 mm × 30 mm (length × width × height). The clip 5 is a commercially available clip. The mesh 4 is a commercially available mesh with a mesh size <0.3 mm. The hollow cylindrical tube 3 is made of food-grade / biological experimental grade high-transparency PMMA (e.g., ...). Evonik Plexiglas® 7N Made of 15mm in length and 16mm in diameter; the groove depth of the two flanges 13 is 80mm×10mm×20mm (length×width×height). The preparation method of the above-mentioned Trichogramma wasp exposure and venom-contamination device includes the following steps: S1. Wrap the mesh 4 around the outer end of the hollow cylindrical tube 3, and then insert the outer end of the hollow cylindrical tube 3 into the round hole 12 of the fixed frame 1 to obtain the assembled fixed frame 1. S2. Place the two cover plates 2 on the upper and lower ends of the assembled fixed frame 1 respectively, so that the two ends of each cover plate 2 are connected to the fixed frame 1, thus obtaining the Trichogramma exposed poisoning device.

[0025] The fixed frame 1 is obtained by fabricating a micro-cavity array of a biomimetic microstructure composite material with PMMA as the frame matrix, followed by filling the frame matrix with TRNS nanoparticles. The preparation method of the fixed frame 1 includes the following steps: S1. Preheat the frame mold to 80±2℃ and vacuum dry for 2 hours. Gravity cast PMMA particles into the frame mold at a casting temperature of 160±2℃. Degas under vacuum at -0.08 MPa for 15 minutes, then cool from 160±2℃ to 80±2℃ at a cooling rate of 0.5℃ / min. Anneal at 80℃ for 4 hours to obtain the frame matrix. The PMMA particles have a purity ≥99.9% and a molecular weight Mw = 120000±5000. This is achieved using food-grade / biological laboratory-grade high-transparency PMMA (such as...). Evonik Plexiglas® 7N Ensure no plasticizers or residual monomers are present; S2. Microcavity array fabrication is performed on the frame substrate to obtain a microcavity frame; the microcavity array fabrication is as follows: using a femtosecond laser micromachining system (wavelength 800 nm, pulse width 150 fs), 5000 cavities / mm are etched in a 0.5 mm wide area on the inner edge of the contact surface between the frame substrate and the cover plate. 2 The biomimetic leaf-shaped microcavities have a diameter of 8-12 μm and a depth of 3-5 μm; and are arranged on the frame substrate at a density of 8 microcavities / mm². 2 micropores S3. Disperse TRNS nanoparticles in 180 mL of anhydrous ethanol, wherein the amount of TRNS nanoparticles is 0.6 wt% of anhydrous ethanol. Sonicate for 12 min to obtain a dispersion. Then, completely immerse the microcavity frame in the dispersion and adsorb under a vacuum of -0.09 MPa for 15 min. Then, dry and solidify at 120 °C for 35 min under a nitrogen atmosphere to obtain the fixed frame 1.

[0026] The preparation method of the TRNS nanoparticles is as follows: 1) Oil phase preparation: Span 80, Tween 80, and Fe3O4 nanoparticles were sequentially dispersed in 60 mL of n-hexane and magnetically stirred for 30 min to form a homogeneous oil phase, ensuring emulsification stability; wherein, the amount of Span 80 used was 4.0 wt% of n-hexane, the amount of Tween 80 used was 1.2 wt% of n-hexane, and the amount of Fe3O4 nanoparticles used was 0.6 wt% of n-hexane; 2) Aqueous phase preparation: N - Isopropylacrylamide (NIPAM) and sodium 2-acrylamide-2-methylpropanesulfonate (AMPS) monomers were dissolved in deionized water and then added N,N' Methylenebisacrylamide (MBA), ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) form a uniform aqueous core microemulsion with a particle size distribution (PDI) < 0.15. The aqueous phase volume is 6–10% of the oil phase volume, and the total monomer concentration is 15 wt%. The mass ratio of the two monomers is: N - Isopropylacrylamide (NIPAM): Sodium 2-acrylamide-2-methylpropanesulfonate (AMPS) = 86:14; N,N' The amounts of methylenebisacrylamide (MBA), ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) added relative to the total monomers were 0.83 wt%, 0.35 wt%, and 0.18 wt%, respectively. 3) The aqueous phase was slowly added dropwise to the oil phase at a rate of 0.5 mL / min. The mixture was stirred continuously at 40±1℃ for 60 min (stirring speed of 12000 rpm). Then, high-purity nitrogen was introduced for 15 min, and the mixture was kept at a constant temperature of 50±1℃ for 4.5 h under an inert atmosphere to prevent oxidation. The mixture was then cooled to room temperature, centrifuged (12000 rpm, 20 min), and washed three times each with n-hexane and ethanol to remove unreacted monomers and surfactants, yielding particulate matter. 4) The particles were dialyzed in a dialysis bag (MWCO 14000Da) for 48 h, with deionized water replaced every 6 h to thoroughly remove small molecule impurities. The dialyzed particles were then soaked in 0.1 mM FeCl3 solution at 25°C for 2.5 h, and then dispersed in PBS buffer at 25°C and pH 7.4 for 15 h with stirring. PEG-5000-NHS activated ester was added, and the resulting particles were pre-frozen at -80°C for 24 h and then freeze-dried under vacuum for 48 h to obtain white powdery TRNS nanoparticles with a particle size of 1~3 μm. These were stored in a desiccator, protected from light and moisture, for later use. The amount of PEG-5000-NHS activated ester added relative to the dialyzed particles was 2.8 wt%.

[0027] Example 2: This example differs from Example 1 in that the preparation process of the frame matrix is ​​different. Specifically, the frame mold is preheated to 80±2℃ and vacuum dried for 2 hours. PMMA particles are gravity-cast into the frame mold at a casting temperature of 160±2℃. The mold is then degassed under vacuum at -0.08 MPa for 12 minutes. Subsequently, the temperature is reduced from 160±2℃ to 80±2℃ at a cooling rate of 0.5℃ / min. Finally, the mold is annealed at 80℃ for 3 hours to obtain the frame matrix.

[0028] Example 3: This example differs from Example 1 in that the preparation process of the frame matrix is ​​different. Specifically, the frame mold is preheated to 80±2℃ and vacuum dried for 2 hours. PMMA particles are gravity-cast into the frame mold at a casting temperature of 160±2℃. The mold is then degassed under vacuum at -0.08 MPa for 18 minutes. Subsequently, the temperature is reduced from 160±2℃ to 80±2℃ at a cooling rate of 0.5℃ / min. Finally, the mold is annealed at 80℃ for 5 hours to obtain the frame matrix.

[0029] Example 4: This example differs from Example 1 in that the preparation process of TRNS nanoparticles loaded onto the framework matrix is ​​different. Specifically, TRNS nanoparticles are dispersed in 180 mL of anhydrous ethanol, with the amount of TRNS nanoparticles being 0.3 wt% of the anhydrous ethanol. The mixture is ultrasonically treated for 10 min to obtain a dispersion. Subsequently, the microcavity framework is completely immersed in the dispersion and adsorbed under a vacuum of -0.09 MPa for 12 min. Then, it is dried and cured at 120°C under a nitrogen atmosphere for 25 min to obtain the fixed framework 1.

[0030] Example 5: This example differs from Example 1 in that the preparation process of TRNS nanoparticles loaded onto the framework matrix is ​​different. Specifically, TRNS nanoparticles are dispersed in 180 mL of anhydrous ethanol, with the amount of TRNS nanoparticles being 0.8 wt% of the anhydrous ethanol. The mixture is ultrasonically treated for 15 min to obtain a dispersion. Subsequently, the microcavity framework is completely immersed in the dispersion and adsorbed under a vacuum of -0.09 MPa for 18 min. Then, it is dried and cured at 120°C under a nitrogen atmosphere for 40 min to obtain the fixed framework 1.

[0031] Example 6: This example differs from Example 1 in that the oil phase preparation process in the preparation of TRNS nanoparticles is different. Specifically, the amount of Span 80 is 3.5 wt% of n-hexane, the amount of Tween 80 is 1.0 wt% of n-hexane, and the amount of Fe3O4 nanoparticles is 0.2 wt% of n-hexane.

[0032] Example 7: This example differs from Example 1 in that the oil phase preparation process in the preparation of TRNS nanoparticles is different. Specifically, the amount of Span 80 is 4.8 wt% of n-hexane, the amount of Tween 80 is 1.5 wt% of n-hexane, and the amount of Fe3O4 nanoparticles is 0.8 wt% of n-hexane.

[0033] Example 8: This example differs from Example 1 in that the aqueous phase preparation process in the preparation of TRNS nanoparticles is different. Specifically, the aqueous phase preparation process is different. N - Isopropylacrylamide (NIPAM) and sodium 2-acrylamide-2-methylpropanesulfonate (AMPS) monomers were dissolved in deionized water and then added N,N' Methylenebisacrylamide (MBA), ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) form a uniform aqueous core microemulsion with a particle size distribution (PDI) < 0.15. The aqueous phase volume is 6% of the oil phase volume, and the total monomer concentration is 12 wt%. The mass ratio of the two monomers is: N - Isopropylacrylamide (NIPAM): Sodium 2-acrylamide-2-methylpropanesulfonate (AMPS) = 80:20; N,N' The amounts of methylenebisacrylamide (MBA), ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) added relative to the total monomers were 0.5 wt%, 0.2 wt%, and 0.1 wt%, respectively.

[0034] Example 9: This example differs from Example 1 in that the aqueous phase preparation process in the preparation of TRNS nanoparticles is different. Specifically, the aqueous phase preparation process is different. N - Isopropylacrylamide (NIPAM) and sodium 2-acrylamide-2-methylpropanesulfonate (AMPS) monomers were dissolved in deionized water and then added N,N' Methylenebisacrylamide (MBA), ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) form a uniform aqueous core microemulsion with a particle size distribution (PDI) < 0.15. The aqueous phase volume is 10% of the oil phase volume, and the total monomer concentration is 18 wt%. The mass ratio of the two monomers is: N - Isopropylacrylamide (NIPAM): Sodium 2-acrylamide-2-methylpropanesulfonate (AMPS) = 90:10; N,N' The amounts of methylenebisacrylamide (MBA), ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) added relative to the total monomers were 1.0 wt%, 0.5 wt%, and 0.2 wt%, respectively.

[0035] Example 10: The difference between this example and Example 1 is that the polymerization process in the preparation of TRNS nanoparticles is different. Specifically, the aqueous phase is slowly added dropwise to the oil phase at a dropping rate of 0.3 mL / min, and the mixture is continuously stirred at 40±1℃ for 60 min (stirring speed of 12000 rpm). Then, high-purity nitrogen is introduced for 10 min, and the reaction is carried out at a constant temperature of 50±1℃ for 4 h.

[0036] Example 11: The difference between this example and Example 1 is that the polymerization process in the preparation of TRNS nanoparticles is different. Specifically, the aqueous phase is slowly added dropwise to the oil phase at a dropping rate of 0.6 mL / min, and the mixture is continuously stirred at 40±1℃ for 60 min (stirring speed of 12000 rpm). Then, high-purity nitrogen gas is introduced for 20 min, and the reaction is carried out at a constant temperature of 50±1℃ for 5 h.

[0037] Example 12: This example differs from Example 1 in that the post-processing of TRNS nanoparticles is different. Specifically, the particles were dialyzed in a dialysis bag (MWCO 14000Da) for 42 h, with deionized water replaced every 6 h to thoroughly remove small molecule impurities. Then, the dialyzed particles were soaked in a 0.1 mM FeCl3 solution at 25°C for 2 h, and then dispersed in PBS buffer at 25°C and pH 7.4 with stirring for 12 h. PEG-5000-NHS activated ester was added, and the resulting particles were pre-frozen at -80°C for 24 h and then freeze-dried under vacuum for 32 h to obtain white powdery TRNS nanoparticles with a particle size of 1~3 μm. The amount of PEG-5000-NHS activated ester added relative to the dialyzed particles was 1.5 wt%.

[0038] Example 13: This example differs from Example 1 in that the post-processing of TRNS nanoparticles is different. Specifically, the particles were dialyzed in a dialysis bag (MWCO 14000Da) for 48 h, with deionized water replaced every 6 h to thoroughly remove small molecule impurities. Then, the dialyzed particles were soaked in a 0.1 mM FeCl3 solution at 25°C for 3 h, and then dispersed in PBS buffer at 25°C and pH 7.4 with stirring for 16 h. PEG-5000-NHS activated ester was added, and the resulting particles were pre-frozen at -80°C for 24 h and then freeze-dried under vacuum for 48 h to obtain white powdery TRNS nanoparticles with a particle size of 1~3 μm. The amount of PEG-5000-NHS activated ester added relative to the dialyzed particles was 3.2 wt%.

[0039] Example 14: This example differs from Example 1 in that the microcavity array fabrication method is different. Based on Example 2, a density of 5 cells / mm is set on the frame substrate. 2Micro-holes.

[0040] Example 15: This example differs from Example 1 in that the microcavity array fabrication method is different. Based on Example 2, a density of 10 cells / mm is set on the frame substrate. 2 Micro-holes.

[0041] Example 16: The difference between this example and Example 1 is that an anodic aluminum oxide (AAO) template is used to construct an eggshell-like nanofibril array with a height of 200-300 nm and a spacing of 600-800 nm on the inner wall surface of the frame matrix and the cover plate.

[0042] Experimental example: I. Experimental Objective: To verify the self-sealing performance of the fixed frame prepared by the novel biomimetic microstructure composite material, and to evaluate its advantages over traditional glue-bonded containers in reducing volatile organic compound (VOC) interference, ensuring the accuracy and biorelevance of toxicity test results.

[0043] II. Experimental Principle: 1) A combined box body consisting of a fixed frame made of biomimetic microstructure composite material and a glass plate as a cover; 2) An interference control group was set up for the glue. It has been proven that the release of VOCs such as formaldehyde, benzene, and acetone by epoxy resin or cyanoacrylate glue during the curing process can significantly interfere with the olfactory localization and behavioral activity of Trichogramma wasps, leading to an abnormally high mortality rate and constituting a false positive interference in the experiment.

[0044] 3) The effectiveness of the device for quantifying the gas leakage rate and 24-hour mortality rate under the same bee colony was compared with that of the test box.

[0045] III. Experimental Design:

[0046] Note: According to GB / T 31270.17-2025, the experiment must meet the following conditions to be valid: the mortality rate of the solvent control group is ≤5%, and the mortality rate of the experimental group shows a dose-response relationship.

[0047] IV. Experimental Results: According to the standard GB / T 31270.17-2025 "Test Guidelines for Environmental Safety Evaluation of Chemical Pesticides Part 17: Acute Toxicity Test of Natural Enemy Trichogramma wasps", a solvent control method was used. The mortality rate of the solvent control group should be controlled at ≤5%, while the mortality rate of the glue-bonded group, due to VOCs interference, can show an abnormal increase of >15% as clearly indicated in the literature. Three replicates were taken for each group, and the average value was calculated. The results are shown in Table 1 below. Table 1 Comparison of mortality data for Trichogramma wasps

[0048] As can be seen from the results in Table 1 above, the mortality rate of the test device using the biomimetic microstructure composite material + glass plate (Example 1) of the present invention is significantly lower than that of the control group. This shows that by using the biomimetic microstructure composite material + glass plate, other toxic interferences can be significantly reduced, effectively improving the accuracy of the acute toxicity test of Trichogramma wasps.

[0049] In order to investigate the effects of different biomimetic microstructure composite materials on the experimental setup, the following investigation was conducted: Investigation 1: Differences between biomimetic microstructure composite materials and PMMA materials Using Example 1 (bionic microstructure composite material + glass plate) and an experimental setup composed of PMMA + glass plate as examples, the only difference between the two groups is the fixing frame material. The solvent control method described above was used, with three replicates for each group and the average value taken. The results are shown in Table 2 below: Table 2 Comparison of mortality data for Trichogramma wasps

[0050] As can be seen from the results in Table 2 above, the mortality rate of the solvent control using the fixation frame made of PMMA, compared with the fixation frame made of biomimetic microstructure composite material, both meet the standards of "GB / T 31270.17-2025 Environmental Safety Evaluation Test Guidelines for Chemical Pesticides Part 17: Acute Toxicity Test of Natural Enemy Trichogramma". However, the fixation frame made of biomimetic microstructure composite material can reduce the interference of other factors on the accuracy of the Trichogramma test to a greater extent.

[0051] Inquiry 2: Differences in the preparation processes of biomimetic microstructure composite materials The different preparation processes of TRNS nanoparticles loaded onto the framework matrix in Examples 4 and 5 are denoted as L1 and L2; the different oil phase preparation processes in the preparation of TRNS nanoparticles in Examples 6 and 7 are denoted as L3 and L4; the different aqueous phase preparation processes in the preparation of TRNS nanoparticles in Examples 8 and 9 are denoted as L5 and L6; the different post-treatment processes in the preparation of TRNS nanoparticles in Examples 12 and 13 are denoted as L7 and L8; and the different microcavity array fabrication processes in the framework matrix in Examples 14 and 15 are denoted as L9 and L10. Comparative experiments were conducted on the biomimetic microstructure composite materials prepared above, with a control group set up as follows: Control: The difference from Example 1 is that Fe3O4 nanoparticles were not added in the oil phase preparation, and the dialyzed particles were not immersed in FeCl3 solution in the post-treatment. The rest of the preparation process was the same as in Example 1. Each group was repeated three times, and the average value was taken. The results are shown in Table 3 below: Table 3 Comparison of mortality data for Trichogramma wasps

[0052] As can be seen from the results in Table 3 above, the mortality rate of the fixed frames prepared by each preparation process meets the standard of "GB / T 31270.17-2025 Environmental Safety Evaluation Test Guidelines for Chemical Pesticides Part 17: Acute Toxicity Test of Natural Enemy Trichogramma wasps" when compared with the solvent control. However, after adjusting the preparation process of the biomimetic microstructure composite material, except for L2 and L10, the mortality rate of Trichogramma wasps increased to varying degrees when the other biomimetic microstructure composite materials were applied to the test device compared with the preparation process of Example 1. Therefore, the biomimetic microstructure composite materials prepared in Examples 1, 5, and 15 can reduce the interference of other factors on the accuracy of the Trichogramma wasp test to a greater extent when applied to the fixed frame. The specific process parameters can be selected as needed according to the actual production conditions.

Claims

1. A device for exposing and contaminizing Trichogramma wasps, characterized in that, It includes a fixed frame (1) with a hole (11) in the middle, and two cover plates (2) detachably connected to the fixed frame (1) for sealing the two sides of the hole (11). The fixed frame (1) has at least one round hole (12) around its perimeter, and a hollow cylindrical tube (3) is provided on the round hole (12), with a mesh (4) provided at the outer end of the hollow cylindrical tube (3). The fixed frame (1) is obtained by processing the frame matrix with PMMA as the frame matrix using a biomimetic microstructure composite material, and then filling the frame matrix with TRNS nanoparticles.

2. The Trichogramma wasp exposure and venom-contamination device according to claim 1, characterized in that, The cover plate (2) is detachably connected to the fixed frame (1). The detachable connection is achieved by using a clip (5) to fix the cover plate (2) to the fixed frame (1). The side of the fixed frame (1) is provided with a flange (13) for connecting the clip (5).

3. The Trichogramma wasp exposure and venom-contamination device according to claim 1, characterized in that, The preparation method of the TRNS nanoparticles is as follows: 1) Oil phase preparation: Span 80, Tween 80, and Fe3O4 nanoparticles were sequentially dispersed in 60 mL of n-hexane and magnetically stirred for 30 min; wherein the amount of Span 80 was 3.5~4.8 wt% of n-hexane, the amount of Tween 80 was 1.0~1.5 wt% of n-hexane, and the amount of Fe3O4 nanoparticles was 0.2~0.8 wt% of n-hexane; 2) Aqueous phase preparation: N -Isopropylacrylamide and sodium 2-acrylamide-2-methylpropanesulfonate, two monomers, are dissolved in deionized water and then added... N,N' - Methylenebisacrylamide, ammonium persulfate, tetramethylethylenediamine, wherein the volume of the aqueous phase is 6-10% of the volume of the oil phase, and the total monomer concentration is 12-18 wt%; the mass ratio of the two monomers is: N -Isopropylacrylamide:Sodium 2-acrylamide-2-methylpropanesulfonate = 80~90:10~20; N,N' The addition amounts of methylenebisacrylamide, ammonium persulfate, and tetramethylethylenediamine relative to the total monomers are 0.5–1.0 wt%, 0.2–0.5 wt%, and 0.1–0.2 wt%, respectively. 3) The aqueous phase was slowly added dropwise to the oil phase at a rate of 0.3-0.6 mL / min. The mixture was stirred continuously at 40±1℃ for 60 min, followed by the introduction of high-purity nitrogen gas for 10-20 min. The mixture was then reacted at a constant temperature of 50±1℃ for 4-5 h. After cooling to room temperature, the mixture was centrifuged and washed several times with hexane and ethanol, respectively, to obtain particulate matter. 4) Dialyze the particles in a dialysis bag for 42-48 h, changing the deionized water every 6 h. Then, soak the dialyzed particles in 0.1 mM FeCl3 solution at 25℃ for 2-3 h, and then disperse them in PBS buffer at 25℃ and pH 7.4 with stirring for 12-16 h. Add PEG-5000-NHS activated ester, then pre-freeze the resulting particles at -80℃ for 24 h, and freeze-dry them under vacuum for 32-48 h to obtain white powdery TRNS nanoparticles with a particle size of 1-3 μm. The amount of PEG-5000-NHS activated ester added relative to the dialyzed particles is 1.5-3.2 wt%.

4. The Trichogramma wasp exposure and venom-contamination device according to claim 3, characterized in that, The method for preparing the fixed frame (1) includes the following steps: 1) Preheat the frame mold to 80±2℃ and vacuum dry for 2 hours. Then, gravity-cast the PMMA particles into the frame mold at a casting temperature of 160±2℃ and degas at -0.08 MPa for 12~18 minutes. Subsequently, cool the mold from 160±2℃ to 80±2℃ at a cooling rate of 0.5℃ / min and anneal it at 80℃ for 3~5 hours to obtain the frame matrix. 2) The framework substrate is fabricated using a microcavity array to obtain a microcavity framework; 3) Disperse TRNS nanoparticles in anhydrous ethanol, wherein the amount of TRNS nanoparticles is 0.3~0.8 wt% of anhydrous ethanol, sonicate for 10~15 min to obtain a dispersion, then completely immerse the microcavity frame in the dispersion, adsorb under vacuum negative pressure of -0.09 MPa for 12~18 min, and then dry and solidify at 120℃ in a nitrogen atmosphere for 25~40 min to obtain a fixed frame (1).

5. The Trichogramma wasp exposure and venom-contamination device according to claim 4, characterized in that, The microcavity array is fabricated by etching a density of 5000 cells / mm within a 0.5 mm wide area on the inner edge of the contact surface between the frame substrate and the cover plate. 2 The biomimetic leaf pillow microcavity has a diameter of 8~12 μm and a depth of 3~5 μm.

6. The Trichogramma wasp exposure and venom-contamination device according to claim 4, characterized in that, The microcavity array is fabricated by setting a density of 5-10 cells / mm on a frame substrate. 2 Micro-holes.

7. A method for preparing a Trichogramma wasp exposure and venom-contamination device according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1. Wrap the mesh (4) around the outer end of the hollow cylindrical tube (3), and then insert the outer end of the hollow cylindrical tube (3) into the round hole (12) of the fixed frame (1) to obtain the assembled fixed frame (1). S2. Place the two cover plates (2) on the upper and lower ends of the assembled fixed frame (1) respectively, so that the two ends of each cover plate (2) are connected to the fixed frame (1) respectively, to obtain the Trichogramma exposure and poisoning device.

8. The method for preparing a Trichogramma wasp exposure and venom-contamination device according to claim 7, characterized in that, An eggshell-like nanofibril array with a height of 200~300 nm and a spacing of 600~800 nm was constructed on the inner wall surface of the fixed frame (1) and the cover plate (2) using anodized aluminum oxide (AAO) template.

Images