Method and system for analyzing nano material spatial distribution effect in nano material-plant interaction mechanism based on micro-fluidic chip

Abstract

The invention belongs to the technical field of nano material evaluation, and provides a method and a system for analyzing a nano material spatial distribution effect in a nano material-plant interaction mechanism based on a micro-fluidic chip. The method comprises the following steps: loading a plant root system into a middle cavity of a micro-fluidic chip (comprising at least three cavities which are communicated with one another), and carrying out plant adaptation; liquid culture media containing nano materials are poured into the cavities on the two sides of the middle cavity respectively, and a plurality of spatial effect models are obtained; the spatial effect model comprises a symmetrical exposure model (the concentration of the nanometer material in the liquid culture medium injected into the cavities in the two sides is the same) and an asymmetric exposure model (the concentration of the nanometer material in the liquid culture medium injected into the cavities in the two sides is different); the total concentrations of nano materials in the liquid culture media filled in the cavities on the two sides in the symmetric exposure model and the asymmetric exposure model are the same; and monitoring and analyzing root growth process data. According to the method, the spatial distribution effect of the nano material in the heterogeneous environment can be accurately analyzed.

Description

Technical Field

[0001] This invention relates to the field of fertilizer evaluation technology, and in particular to a method and system for analyzing the spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism based on microfluidic chips. Background Technology

[0002] Nanomaterials (NMs) are widely used in sustainable agriculture as novel fertilizers or biostimulants, but their environmental effects and biosafety assessments face challenges. Traditional studies often employ the method of homogenizing NMs into soil or culture media to examine their dose-dependent effects. However, the real soil environment exhibits high spatial heterogeneity, leading to non-uniform distribution of NMs in the rhizosphere and the formation of local "hot spots." Therefore, the biological effects of NMs are the result of both dose-dependent and spatial distribution-dependent effects, which traditional methods cannot separate, severely limiting a deeper understanding of the NMs-plant interaction mechanism and guidance for precision agriculture applications.

[0003] In recent years, microfluidic technology has provided a powerful tool for life science research due to its high-precision control of the microenvironment and real-time in-situ observation capabilities. While existing technologies include microfluidic chips for studying plant root-microbe interactions or nutrient gradient responses, there are no reports of systematically applying them to actively manipulate and analyze how the spatial distribution effect of nanometers (NMs), a key environmental variable, independently regulates plant root responses. Therefore, developing a method capable of accurately simulating the spatial heterogeneity of NMs and analyzing their unique biological effects and mechanisms in real-time and in-situ is crucial for advancing basic research and safe applications of environmental nanotechnology. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method and system for analyzing the spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism based on a microfluidic chip. The method of this invention can accurately analyze the spatial distribution effect of nanomaterials in heterogeneous environments.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for analyzing the spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism based on a microfluidic chip, wherein the microfluidic chip includes at least three interconnected cavities; the method includes the following steps: Plant roots were loaded into any central cavity of the microfluidic chip, and after plant adaptation, liquid culture medium containing nanomaterials was infused into the cavities on both sides of the cavity containing the plant roots, resulting in multiple spatial effect models. These models include a symmetrical exposure model and an asymmetrical exposure model. In the asymmetrical exposure model, the concentration of nanomaterials in the liquid culture medium infused into the cavities on both sides is different. In the symmetrical exposure model, the concentration of nanomaterials in the liquid culture medium infused into the cavities on both sides is the same. In both the symmetrical and asymmetrical exposure models, the total concentration of nanomaterials in the liquid culture medium infused into the cavities on both sides is the same. Data on plant root growth processes were monitored in multiple spatial effect models to obtain a database. The spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism was obtained by analyzing the database.

[0006] Preferably, a guide protrusion is provided in the middle cavity of the three cavities, and the cross-sectional shape of the guide protrusion is triangular; the guide protrusion guides the plant roots to grow in a straight line along the cavity.

[0007] Preferably, the three cavities are arranged in parallel, with the two outer cavities symmetrically distributed on both sides of the middle cavity.

[0008] Preferably, the plant includes Arabidopsis thaliana.

[0009] Preferably, the nanomaterial comprises molybdenum disulfide nanosheets.

[0010] Preferably, the concentration of nanomaterials in the liquid culture medium is independently 0~200 mg / L.

[0011] Preferably, during the plant's adaptation and root growth process, the plant's stem is perpendicular to the horizontal plane.

[0012] Preferably, the perfusion flow rate of the liquid culture medium is 0.2~1.0 mL / h.

[0013] Preferably, the root growth process data includes microscopic images, reactive oxygen species, auxin, ferrous ions, and fluorescence images.

[0014] This invention provides a system for analyzing the spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism based on a microfluidic chip, comprising: Microfluidic chips; Growth process monitoring system; The module for processing monitoring data.

[0015] This invention provides a method for analyzing the spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism based on a microfluidic chip. The method involves infusing liquid culture medium containing nanomaterials into two cavities on either side of a cavity containing plant roots within a microfluidic chip, thereby obtaining multiple spatial effect models. These models include a symmetrical exposure model and an asymmetrical exposure model. In the asymmetrical exposure model, the concentration of nanomaterials in the liquid culture medium infused into the two cavities differs. In the symmetrical exposure model, the concentration of nanomaterials in the liquid culture medium injected into the two cavities is the same. In both the symmetrical and asymmetrical exposure models, the total concentration of nanomaterials in the liquid culture medium infused into the two cavities is the same. The liquid culture medium in the two cavities converges in the middle cavity, forming a distinct laminar flow state. This allows the plant roots located in the middle cavity to access either a homogeneous or heterogeneous environment. The symmetrical exposure model eliminates the dose effect, and the spatial distribution effect is reflected in the plant root growth data in the asymmetrical exposure model, thus obtaining the spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism. Specifically, for certain nanomaterials (such as MoS2 nanosheets), changes in their spatial distribution pattern (from symmetric exposure to asymmetric exposure) can trigger unique root responses. This manifests as follows: within a specific concentration window, the asymmetric exposure group significantly promotes root growth compared to the symmetric exposure group (similar to a toxic excitatory effect), while the symmetric exposure group with the same total dose shows inhibition or no significant effect.

[0016] Signal transduction mechanism mediating spatial effects: The above biological effects are related to a divalent iron (Fe) band. 2+ This is related to the reactive oxygen species (ROS)-auxin cascade signaling pathway. Specifically, in the asymmetric exposure experimental group, local molybdenum sulfide nanosheet stimulation triggered Fe in the root. 2+ Conduction; Fe 2+ It is necessary for the generation of ROS signals in the root and their transmission to the unexposed side; the ROS signals transmitted to the whole root promote the accumulation of auxin in the root tip; ultimately, this auxin accumulation drives the observed root growth promotion.

[0017] This invention compares and analyzes the differences in various indicators between symmetrical and asymmetrical exposure experimental groups at the same total dose, clarifying the unique biological effects (such as toxic stimulant effects) caused by the variable of "spatial asymmetry," and elucidating the underlying signal transduction network. This invention provides a standardized, reproducible, and mechanistically elucidable research platform for accurately analyzing the spatial distribution effects of biostimulants (such as nanomaterials) in heterogeneous environments. Attached Figure Description

[0018] Figure 1 Schematic diagrams of symmetrical and asymmetrical exposure groups; Figure 2 This is a schematic diagram showing multiple experiments being conducted simultaneously on a microfluidic chip. Figure 3 Image of the object as observed under a microscope; Figure 4 This is a schematic diagram of the observation channel and the direction of liquid flow; Figure 5 along Figure 4 The fluorescence intensity diagram of coumarin was plotted horizontally; Figure 6 Bright-field images of the root systems of the symmetrical and asymmetrical exposure groups at 64 h of growth; Figure 7 The root length during the dynamic growth process of the symmetrical and asymmetrical exposure groups; Figure 8 The relative root elongation of the symmetrical exposure group and the asymmetrical exposure group; Figure 9 The in-situ dynamic propagation and spatial distribution of ROS signals under asymmetric exposure; Figure 10 Fe under asymmetric exposure 2+ In-situ dynamic propagation and spatial distribution of signals; Figure 11 ROS and Fe in roots under unilateral NAC and DFO inhibitor treatment 2+ Changes; Figure 12 Root elongation under NAC and DFO inhibitor treatments; Figure 13 Processing for Sy-25 and Asy-50 DR5:GFP Representative images of fluorescence and comparisons of fluorescence intensity; Figure 14 The effect of auxin synthesis inhibitor treatment on root growth of Asy-50; Figure 15 The auxin inhibitors in Asy-50 affect ROS and Fe 2+ The impact. Detailed Implementation

[0019] This invention provides a method for analyzing the spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism based on a microfluidic chip, wherein the microfluidic chip includes at least three interconnected cavities; the method includes the following steps: Plant roots were loaded into any central cavity of the microfluidic chip, and after plant adaptation, liquid culture medium containing nanomaterials was infused into the cavities on both sides of the cavity containing the plant roots, resulting in multiple spatial effect models. These models include a symmetrical exposure model and an asymmetrical exposure model. In the asymmetrical exposure model, the concentration of nanomaterials in the liquid culture medium infused into the cavities on both sides is different. In the symmetrical exposure model, the concentration of nanomaterials in the liquid culture medium infused into the cavities on both sides is the same. In both the symmetrical and asymmetrical exposure models, the total concentration of nanomaterials in the liquid culture medium infused into the cavities on both sides is the same. Data on plant root growth processes were monitored in multiple spatial effect models to obtain a database. The spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism was obtained by analyzing the database.

[0020] Unless otherwise specified, the raw materials used in this invention are preferably commercially available products.

[0021] The structure of the microfluidic chip is described below.

[0022] In this invention, the microfluidic chip is preferably a structure with a sealed environment formed by sealing an upper chip body and a bottom glass slide. In this invention, the chip body is preferably made of polydimethylsiloxane (PDMS).

[0023] In this invention, the microfluidic chip includes at least three interconnected cavities, which are preferably arranged in parallel, with the two cavities on either side of the central cavity preferably symmetrically distributed on both sides of the central cavity. In this invention, an inlet is preferably located at one end of each of the three cavities, and an outlet is preferably located at the other end. The inlet of the central cavity is used to house plant roots, while the inlets of the two cavities on either side are used to infuse liquid culture medium containing nanomaterials.

[0024] In this invention, of the three cavities, the width of the middle cavity is preferably 300-500 μm, the height is preferably 50-100 μm, and the length is preferably 8000-10000 μm; the width of both side cavities is preferably 100-150 μm, the height is preferably 50-100 μm, and the length is preferably 8000-10000 μm. In a specific embodiment of this invention, the width of the middle cavity is preferably 300 μm, the height is preferably 100 μm, and the length is preferably 8550 μm; the width of both side cavities is preferably 100 μm, the height is preferably 100 μm, and the length is preferably 8200 μm.

[0025] In this invention, a guide protrusion is preferably provided in the middle cavity of the three cavities. The cross-sectional shape of the guide protrusion is preferably triangular, and the triangle is preferably an obtuse triangle. In one specific embodiment of this invention, the three sides of the obtuse triangle have lengths of 235 μm, 220 μm, and 102 μm, respectively. In this invention, the shape of the guide protrusion is preferably a triangular prism, and the height of the triangular prism is preferably 50-100 μm. In this invention, the guide protrusions are preferably arranged symmetrically in two rows, and the distance between the two rows is preferably 130 μm. In one specific embodiment of this invention, the 220 μm × height side of the triangular prism is preferably parallel to the length direction of the middle cavity. This invention does not specifically limit the spacing of each row of guide protrusions, as long as it can guide the plant roots to grow in a straight line along the cavity.

[0026] In this invention, the three interconnected cavities are referred to as a group, and the microfluidic chip preferably includes multiple groups, more preferably five groups, so as to establish multiple models simultaneously.

[0027] In this invention, the fabrication method of the microfluidic chip preferably includes the following steps: After spin-coating photoresist onto a silicon wafer, photolithography is performed to obtain a master mold; A mixture of monomer and curing agent is poured into the master mold, cured, and then the master mold is peeled off to obtain the chip body. After treating the chip body and the glass substrate with oxygen plasma, the oxygen plasma-treated chip body and the glass substrate are bonded together to obtain the microfluidic chip.

[0028] In this invention, the photoresist is preferably SU-8 2050 photoresist. This invention does not specifically limit the photolithography operation; it can be set according to the cavity to be formed.

[0029] In this invention, the monomer is preferably a PDMS monomer. In this invention, the mass ratio of monomer to curing agent in the monomer-curing agent mixture is preferably 10:1. This invention does not specifically limit the type of curing agent; any commercially available curing agent compatible with the polydimethylsiloxane monomer can be used. In this invention, the curing temperature is preferably 60-70°C, more preferably 65°C; the curing time is preferably 1-3 hours, more preferably 2 hours. In this invention, the curing is preferably carried out in an oven. This invention does not specifically limit the method of peeling off the master mold; any operation well-known to those skilled in the art can be used.

[0030] In this invention, the oxygen plasma treatment is preferably performed in an oxygen plasma cleaner (Harrick Plasma). The power of the oxygen plasma treatment is preferably 80-120W, more preferably 100W; the time is preferably 30-60s, more preferably 40s. This invention does not specifically limit the bonding method, as long as it can form a cavity between the oxygen plasma-treated glass slide substrate and the oxygen plasma-treated chip body. In this invention, the chip body and the glass slide substrate are bonded after oxygen plasma treatment, resulting in irreversible bonding and the fabrication of a complete microfluidic root chip.

[0031] The present invention loads the root system of a plant into any cavity in the middle of the microfluidic chip, and then allows the plant to adapt.

[0032] In this invention, the plant is preferably a seedling. Specifically, the seedling is preferably a seedling with a straight taproot. More preferably, the plant includes Arabidopsis thaliana. Arabidopsis thaliana Columbia ecotype (Col-0) seedlings.

[0033] In this invention, the method for cultivating plant seedlings preferably includes the following steps: Plant seeds are subjected to surface disinfection and vernalization in sequence to obtain vernalized seeds; The vernalized seeds are inoculated onto the tip of a pipette containing a solid culture medium. The tip of the pipette loaded with vernalized seeds is then inserted into the solid culture medium to allow germination, resulting in the plant seedlings.

[0034] In this invention, the disinfectant used for surface disinfection is preferably a sodium hypochlorite solution, and the mass concentration of the sodium hypochlorite solution is preferably 5%; the surface disinfection method is preferably vortex oscillation, and the vortex oscillation time is preferably 3-5 minutes; after surface disinfection, this invention preferably further includes rinsing with double-distilled water, and the number of rinsings with double-distilled water is preferably 3 times. In this invention, the specific process of surface disinfection preferably includes the following steps: soaking plant seeds in a disinfectant and vortexing. In this invention, the vernalization temperature is preferably 4°C, and the time is preferably 2 days. In this invention, the solid culture medium is preferably 1 / 2 Hogrange medium (HM) filled with 0.7% (w / v) plant agar. In this invention, 1 / 2 Hogrange solid culture medium (HM) is chosen because nanomaterials will aggregate in high-concentration nutrient solutions, and the strength of 1 / 2 can reduce nanomaterial aggregation and ensure normal root growth. In this invention, the pipette is preferably a 10µL pipette shortened to a length of about 5mm. In this invention, the tip of the pipette loaded with vernalized seeds is preferably inserted at an angle of 60° to the plane of the solid culture medium. In this invention, the germination temperature is preferably 22°C; the humidity is preferably 50-70%; the germination time is preferably 5-7 days, more preferably 6 days; germination is preferably carried out under intermittent light conditions, preferably 16 hours of light followed by 8 hours of darkness. In this invention, during germination, the pipette is preferably perpendicular to the horizontal plane, that is, the stem of the germinated seedling is perpendicular to the horizontal plane.

[0035] In this invention, the microfluidic chip is preferably sterilized before the plant roots are loaded into any cavity in the middle of the microfluidic chip, and the sterilization time is preferably 15 minutes.

[0036] In this invention, the plant is preferably placed in a pipette. In this invention, the loading angle of the plant's roots is preferably 60° to the plane of the microfluidic chip. In this invention, the specific process of loading the plant's roots into any central cavity of the microfluidic chip preferably includes the following steps: injecting 1 / 2 HM culture medium containing 0.1% (w / v) 2-morpholine ethanesulfonic acid into the inlets of the cavities on both sides of the microfluidic chip; then, selecting seedlings of similar growth in a clean bench, and carefully inserting the pipette tip containing the plant into the root inlet of the central cavity of the microfluidic chip at a 60° angle using fine tweezers, thereby guiding the roots into the central cavity.

[0037] In this invention, the plant adaptation is preferably carried out in a petri dish, and the bottom of the petri dish is preferably lined with moist, lint-free paper to maintain humidity. In this invention, the plant adaptation is preferably carried out under sealed conditions. In this invention, the temperature for plant adaptation is preferably 22°C; the time is preferably 48 hours; the plant adaptation is preferably carried out under intermittent light conditions, preferably 16 hours of light followed by 8 hours of darkness. In this invention, during the plant adaptation process, the plant stem is preferably perpendicular to the horizontal plane; in one specific embodiment of this invention, since the plant is loaded in a pipette, ensuring the pipette is perpendicular to the horizontal plane is sufficient to ensure the plant stem is perpendicular to the horizontal plane; to ensure the pipette is perpendicular to the horizontal plane, the tilt angle of the microfluidic chip is preferably 30°.

[0038] In this invention, the plant adaptation enables the plant's root system to adapt to the environment of the microfluidic chip and extend its growth along the central cavity.

[0039] After the plants adapt, the present invention injects liquid culture medium containing nanomaterials into the two cavities on both sides of the cavity containing the plant roots to obtain multiple spatial effect models.

[0040] In this invention, the spatial effect model includes a symmetric exposure model and an asymmetric exposure model.

[0041] In this invention, the asymmetric exposure model refers to the different concentrations of nanomaterials in the liquid culture medium injected into the two cavities.

[0042] In this invention, the symmetrical exposure model is that the concentration of nanomaterials in the liquid culture medium injected into both cavities is the same.

[0043] In this invention, the total concentration of nanomaterials in the liquid culture medium injected into the cavities on both sides of the symmetrical and asymmetric exposure models is the same.

[0044] In this invention, the nanomaterial preferably comprises molybdenum disulfide nanosheets. This invention uses molybdenum disulfide nanosheets as the nanomaterial for research, and it has already been applied to agricultural nano-fertilization, while also possessing the ability to regulate root redox homeostasis.

[0045] In this invention, the preparation method of the liquid culture medium containing nanomaterials preferably includes the following steps: dispersing the nanomaterials in a liquid culture medium to form a mother liquor; then diluting the mother liquor with the liquid culture medium to obtain the liquid culture medium containing nanomaterials. In this invention, the dispersion is preferably carried out under ice-water bath and ultrasonic conditions, with the ultrasonic power preferably being 300W; the ultrasonication is preferably carried out under intermittent conditions, preferably 2 seconds of ultrasonication followed by a 2-second pause; the ultrasonication is preferably carried out using an ultrasonic cell disruptor. In this invention, the total dispersion time is preferably 30 minutes.

[0046] In this invention, the liquid culture medium is preferably 1 / 4 Hoagland's liquid culture medium. In this invention, the concentration of nanomaterials in the liquid culture medium is preferably 0-200 mg / L, specifically preferably 0 mg / L, 12.5 mg / L, 25 mg / L, 50 mg / L, 100 mg / L, or 200 mg / L.

[0047] In this invention, the preferred flow rate of the liquid culture medium is 0.2~1.0 mL / h, specifically preferably 0.2 mL / h, 0.3 mL / h, 0.4 mL / h, 0.5 mL / h, 0.6 mL / h, 0.7 mL / h, 0.8 mL / h, 0.9 mL / h, or 1 mL / h. In this invention, the flow rate of the liquid culture medium in the two cavities is preferably the same. Setting the flow rate of the liquid culture medium to 0.2~1.0 mL / h in this invention maintains a stable chemical gradient while avoiding mechanical stress on the roots.

[0048] In this invention, the liquid culture medium injected into the two side cavities converges in the middle cavity in a cross-flow manner, so that the plant roots placed in the middle cavity can grow in the liquid culture medium injected into the two side cavities. By adjusting the concentration of nanomaterials in the liquid culture medium injected into the two side cavities, the homogeneous and heterogeneous environments can be controlled, so as to obtain and analyze the spatial distribution effect of nanomaterials.

[0049] In this invention, the total concentration of nanomaterials in the liquid culture medium perfused in both cavities of the symmetrical and asymmetric exposure models is the same to achieve iso-dose comparison. For example, comparing the "symmetrical exposure experimental group 25 mg / L (one-sided)" with the "asymmetric exposure experimental group 50 mg / L (one-sided)". The method of this invention utilizes the laminar flow characteristics and spatial controllability of microfluidic chips to construct symmetrical and asymmetric exposure experimental groups, thereby treating "spatial distribution" as an independent experimental variable and analyzing the effect of nanomaterial spatial distribution on plant root growth. In this invention, the mechanism by which asymmetric exposure promotes growth includes an Fe-dependent process triggered by the exposure. 2+The transduction of ROS signals from the exposed side to the unexposed side, and the accumulation of auxin in the root tip mediated by this ROS signal.

[0050] After obtaining multiple spatial effect models, this invention monitors the data of plant root growth processes in multiple spatial effect models to obtain a database.

[0051] In this invention, during the growth of the plant root system, the plant stem is preferably perpendicular to the horizontal plane.

[0052] In this invention, the data on the plant root growth process preferably include microscope images, fluorescence images, reactive oxygen species, auxins, and ferrous ions.

[0053] In this invention, during the monitoring of microscopic images, the preferred acquisition time interval is 2-4 hours during the rapid root growth period (e.g., 40-80 hours after exposure); and 6-8 hours during the growth plateau period. This invention allows for the acquisition of root length and relative growth rate through microscopic image monitoring. In this invention, the root length is preferably measured using ImageJ software and its plugins (such as "Manual Tracking" or "Root-Angel"), or through semi-automated analysis to improve data objectivity and throughput.

[0054] In this invention, the monitoring of reactive oxygen species (ROS) preferably includes ROS detection and ROS scavenging. In this invention, the ROS detection preferably uses an H2DCF-DA probe, the working concentration of which is preferably 5-20 μM, and the incubation time in the dark is preferably 10-20 min. In this invention, the ROS scavenging preferably uses N-acetylcysteine ​​(NAC), the working concentration of which is preferably 0.1-1.0 mM.

[0055] In this invention, the monitoring of auxin preferably includes auxin distribution and auxin synthesis inhibition. In this invention, the auxin distribution is preferably monitored using transgenic Arabidopsis expressing the DR5:GFP reporter gene. In this invention, the auxin synthesis inhibition is preferably monitored using L-kynurenine (Kyn), with a working concentration of 0.5–2.0 μM. The inhibitor L-kynurenine (Kyn) is preferably pretreated for 1–3 h before chemical exposure, and a "control group only" must be included to exclude the influence of the inhibitor itself on basal growth.

[0056] In this invention, the fluorescence imaging monitoring preferably uses a fixed microscope with all parameters such as exposure time and gain to ensure the comparability of fluorescence intensity between different samples.

[0057] In this invention, the monitoring of ferrous ions preferably includes ferrous ion detection and ferrous ion chelation. In this invention, the ferrous ion (Fe... 2+ The detection preferably uses a RhoNox-1 probe, with a preferred working concentration of 2-10 μM and a preferred incubation time of 15-25 minutes in the dark. Before using the RhoNox-1 probe, the chip channels are preferably cleaned with phosphate buffer (pH 6.5) to remove interference from the culture medium. In this invention, the ferrous ion chelation preferably uses deferoxamine (DFO), with a preferred working concentration of 0.1-1.0 mM.

[0058] After obtaining the database, the present invention analyzes the database to obtain the spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism.

[0059] The present invention does not specifically limit the operation of the analysis, and any operation known to those skilled in the art can be used.

[0060] Figure 1 The diagram shows the symmetrical and asymmetrical exposure groups. Symmetry means that the concentration of nanomaterials in the liquid culture medium poured into the two cavities is the same, while asymmetry means that the concentration of nanomaterials in the liquid culture medium poured into the two cavities is different. Specifically, the concentration of nanomaterials in the liquid culture medium poured into one cavity is 0, while the concentration of nanomaterials in the liquid culture medium poured into the other cavity is the total concentration of nanomaterials in the symmetrical group. Figure 2 This is a schematic diagram showing multiple experiments being conducted simultaneously on a microfluidic chip. Figure 3 Microscopic observation of the actual object.

[0061] The method of this invention can decouple the dose effect and spatial distribution effect of nanomaterials, and reveal the unique biological functions and molecular mechanisms of the spatial distribution effect. This method provides a novel research paradigm and technical support for evaluating the real-world behavior of nanomaterials in complex soil environments and designing novel nano-agrochemicals.

[0062] This invention also provides a system for analyzing the spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism based on a microfluidic chip, comprising: Microfluidic chips; Growth process monitoring system; The module for processing monitoring data.

[0063] The system provided by this invention includes a microfluidic chip. In this invention, the structure of the microfluidic chip is preferably the same as the above-described technical solution and will not be repeated here.

[0064] The system provided by this invention includes a growth process monitoring system. In this invention, the growth process monitoring system preferably includes an inverted microscope, which is preferably equipped with an environmental control unit, which preferably includes a temperature control unit and a CO2 control unit. In this invention, the environmental control unit is configured to maintain the physiological stability of live samples.

[0065] In this invention, the growth process monitoring system preferably includes a reactive oxygen species (ROS) monitoring system, which preferably includes an ROS detection system and an ROS scavenging system.

[0066] In this invention, the growth process monitoring system preferably includes an auxin monitoring system, and the auxin monitoring system preferably includes an auxin distribution monitoring system and an auxin synthesis inhibition monitoring system.

[0067] In this invention, the growth process monitoring system preferably includes a fluorescence imaging monitoring system.

[0068] In this invention, the growth process monitoring system preferably includes a ferrous ion monitoring system, which preferably includes a ferrous ion detection system and a ferrous ion chelation monitoring system.

[0069] The system provided by this invention includes a monitoring data processing module. In this invention, the monitoring data processing module preferably includes ImageJ software and its plugins (such as "Manual Tracking" or "Root-Angel"), custom Python / MATLAB scripts, and professional image processing software. The following detailed description, in conjunction with embodiments, illustrates the method and system for analyzing the spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism based on microfluidic chips, but these should not be construed as limiting the scope of protection of this invention.

[0070] Example 1 Experimental Content and Objectives: This embodiment aims to construct and verify the feasibility and stability of the microfluidic chip—the fundamental experimental device for this study. The core objective is to confirm that the platform can successfully support the long-term healthy growth of Arabidopsis thaliana seedling roots and achieve independent and stable fluid supply on both sides, laying the foundation for subsequent space exposure experiments.

[0071] (1) Microfluidic chip fabrication using standard soft lithography: First, SU-8 2050 photoresist is spin-coated on a silicon wafer and photolithography is performed to form a master mold containing a central cavity, a central groove (width 300μm, height 100μm, length 8550μm) and a first side cavity and a second side cavity on both sides, a first side groove and a second side groove (width 100μm, height 100μm, length 8200μm), and photolithography guides the protrusions in the central groove. Polydimethylsiloxane (PDMS) monomers and curing agents were mixed at a mass ratio of 10:1, degassed, and poured onto a master mold. The mixture was cured in a 65°C oven for 2 hours. The silicon wafer was then peeled off to obtain a PDMS chip body containing a groove structure. A 1mm diameter biopsy needle was used to create a plant inlet at one end of the central groove and a liquid outlet at the other end. Liquid outlets were also created at the ends of the first and second side grooves, and liquid inlets at the other ends. The liquid outlets of the central groove, the first side groove, and the second side groove were located at the same end. The PDMS chip body and a cleaned glass slide were placed in an oxygen plasma cleaner (Harrick Plasma) and treated at 100W power for 40 seconds. Immediately after treatment, they were bonded together to form an irreversible bond, resulting in a complete microfluidic chip. The guide protrusions in the central cavity are as follows: Figure 1 As shown, in the central cavity with a length of 8550μm, two rows of guide protrusions are uniformly arranged at intervals of 130μm. The height of the guide protrusions is 100μm, and the dimensions of the cross-sectional triangle are 235μm×220μm×102μm.

[0072] The laminar flow properties of the microfluidic chip were verified: a 10 mM coumarin solution was used as a tracer. A dye-containing fluid and a separate dye-free solution were simultaneously and stably injected into the first and second cavities of the microfluidic chip using a syringe pump. The observation area of ​​the microfluidic chip was then imaged using an inverted fluorescence microscope. After stable laminar flow was established, clear fluorescence distribution images were acquired laterally from the untreated side to the treated side. The results are shown below. Figure 4 As shown in the figure. Finally, the acquired fluorescence images were quantitatively analyzed using professional image processing software. By extracting the fluorescence intensity distribution data perpendicular to the flow direction, a curve of fluorescence intensity changing with channel position was plotted, and the results are shown in the figure. Figure 5 .from Figure 4 It can be seen that the liquid flows in the microfluidic chip, and a laminar flow effect is formed within the microfluidic chip. From... Figure 5 It can be seen that the fluorescence intensity increases significantly only on the side where coumarin is applied, indicating that the microfluidic chip can achieve a stable laminar flow effect and can be controlled independently.

[0073] (2) Plant culture: Arabidopsis seeds were soaked in 5% sodium hypochlorite solution, vortexed for 4 minutes, and then rinsed 3 times with sterile double-distilled water to obtain surface-sterilized Arabidopsis seeds. The seeds were vernalized at 4℃ for 2 days to obtain vernalized seeds. The vernalized seeds were then inoculated onto 10µL pipette tips (trunked to about 5mm in length) filled with solid culture medium (1 / 2 HM medium of 0.7% (w / v) plant agar). The pipette tips containing vernalized seeds were inserted at a 60° angle into the solid culture medium (1 / 2 HM medium of 0.7% (w / v) plant agar) for germination for 6 days. During germination, the solid culture medium was placed at a 30° angle in a light incubator. All experiments were conducted under long-day conditions (16 hours of light, 22℃, and 60% relative humidity).

[0074] (3) Plant loading and adaptation culture: First, sterilize the processed chip under ultraviolet light for 15 min. Then, inject 1 / 2 HM medium containing 0.1% (w / v) 2-morpholine ethanesulfonic acid into the inlets of the cavities on both sides of the microfluidic chip. Next, select seedlings of similar growth in a clean bench and carefully insert the pipette tip loaded with the plant into the root inlet of the middle cavity of the microfluidic chip at a 60° angle with fine tweezers to guide the roots into the middle cavity. Place the loaded microfluidic chip into a sterile 150 mm petri dish with a moist lint-free paper at the bottom to maintain humidity. After sealing the petri dish, place it in a light incubator (22℃, 16 hours light / 8 hours dark) at a 30° angle under the same conditions as plate culture for 48 h to allow the roots to adapt to the chip environment and extend along the channels.

[0075] Example 2 Discovery and characterization of the spatial distribution effect of MoS2 nanosheets The purpose of this embodiment is to investigate the effects of different spatial distribution patterns of molybdenum disulfide (MoS2) nanosheets (symmetric and asymmetric exposure groups) on Arabidopsis root growth using the platform established in Example 1. The aim is to directly compare whether and how spatial distribution independently alters the phenotypic response of root growth under the same total exposure dose.

[0076] (1) Preparation of liquid culture medium containing nanomaterials: Weigh MoS2 nanosheet powder and add it to 1 / 4 strength Hoagland liquid medium (1 / 4 HM). Use an ultrasonic cell disruptor (300W power, 2s on, 2s off) and sonicate in an ice-water bath for 30 min to obtain a uniformly dispersed stock solution with a concentration of 1000 mg / L. Then dilute the stock solution with 1 / 4 HM to the required concentrations (12.5 mg / L, 25 mg / L, 50 mg / L, 100 mg / L, 200 mg / L).

[0077] (2) Experimental grouping: Seedlings adapted to culture within the microfluidic chip were randomly divided into different treatment groups (each group had at least 5 independent root systems); the following groups were set up by connecting the inlet through an injection pump and a polyethylene tubing: Control group: both cavities were perfused with blank 1 / 4 HM at a flow rate of 0.5 mL / h; Symmetrical exposure experimental group (Sy): both cavities were perfused with the same concentration of MoS2 nanosheets / 1 / 4 HM suspension (12.5 mg / L (Sy-12.5), 25 mg / L (Sy-25), 50 mg / L (Sy-50), 100 mg / L (Sy-100)); Asymmetric exposure experimental group (Asy): one cavity was perfused with MoS2 nanosheets / 1 / 4 HM suspension (25 mg / L (Asy-25), 50 mg / L (Asy-50), 100 mg / L (Asy-100), 200 mg / L (Asy-200)), with a blank 1 / 4 HM perfused in the other cavity. For example, the total MoS2 supply was the same for the Asy-50 group and the Sy-25 group.

[0078] (3) Dynamic acquisition of phenotypic data: After the start of exposure (recorded as 0 hours), the microfluidic chip was placed on the stage of an inverted microscope (such as Olympus IX73). Using the microscope's automatic imaging function, one bright-field image of the root tip region was taken every 4 hours for 120 hours. The results are as follows: Figure 6 As shown, from Figure 6 It can be seen that under symmetrical exposure conditions, as the concentration of MoS2 nanosheets increases (12.5~100 mg·L⁻¹), the effect of the nanosheets on the overall health of the product increases. -1 ), root length was reduced compared to the control group; under asymmetric exposure conditions, low concentration MoS2 nanosheets (25 mg·L⁻¹) showed a greater reduction compared to the control. -1 No significant changes were observed in the medium concentration of MoS2 nanosheets (50 mg·L⁻¹). -1 High concentrations of MoS2 nanosheets (100 and 200 mg·L⁻¹) significantly promoted root growth. -1 It significantly inhibits root growth.

[0079] (4) Growth Data Analysis: Import the time series images into ImageJ software. Use the "Segmented Line" tool to manually trace the path from the root-stem junction to the root tip. The software automatically calculates the root length (in pixels, which can be converted to micrometers using a scale). Export the root length data for all time points, use GraphPad Prism software to plot the root growth curve, and calculate the absolute growth rate within a specific time interval. The results are as follows: Figure 7 and Figure 8 As shown, from Figure 7 and Figure 8It can be seen that the root growth of all treatment groups exhibits an S-shaped curve with the exposure time of the nanomaterials, which can be divided into three stages: initial growth delay (0-40h), exponential growth (40-80h), and asymptotic growth (80-120h). Comparison revealed that the difference in the intermediate growth stage is the main factor determining the final growth difference. Specifically, at 64h, the root length of the Asy-50 treatment group was significantly higher than that of the control group, the Sy-25 group, and the Sy-50 group by 17.0%, 18.3%, and 26.9%, respectively.

[0080] Example 3: ROS and Fe in the root system 2+ In-situ imaging of signal spatial distribution Based on the discovery that Asy-50 treatment promotes root growth, this experiment aims to investigate its potential early signaling events. The objective is to utilize fluorescent probes to visualize in situ, in real-time, the interaction of reactive oxygen species (ROS) and ferrous ions (Fe2+) under asymmetric exposure. 2+ The location, intensity, and spatial propagation dynamics of these two key signaling molecules in the root system.

[0081] (1) Real-time ROS imaging: The Asy-50 treatment group's chip was selected. After approximately 60 hours of exposure (the period when the growth-promoting effect was apparent), perfusion was briefly stopped, and the channel was gently rinsed three times with pre-warmed PBS buffer (pH 7.4). Subsequently, the culture medium was replaced with PBS solution containing 10 μM H2DCF-DA (ROS probe), and incubated in the dark for 15 min. After washing away excess probe with PBS, the chip was returned to the microscope. Simultaneously with restarting the unilateral perfusion of MoS2 suspension, time-series imaging of the fluorescence channel (excitation light 488 nm, emission light collection band 500-550 nm) was initiated, continuously recording at a rate of 30 seconds / frame for 15 min. The dynamic process of the ROS fluorescence signal starting from the treated side and spreading to the untreated side was recorded. The results are as follows: Figure 9 As shown, the left image is a real-time image of the distribution of the ROS fluorescent probe H2DCFA in the Asy-50 root system, and the right image is the change of the ROS fluorescent probe H2DCFA (relative to the initial state) in the Asy-50 root system with exposure time and distance from the exposed root side. Figure 9 It can be seen that ROS initially increases on the treated side of the root and then translocates to the untreated side within 15 minutes. This signal transduction-like ROS translocation mechanism resulted in an overall 1.5-fold increase in root ROS levels at 15 minutes compared to the initial level, confirming the ability of MoS2 nanosheets to influence root redox homeostasis.

[0082] (2) Fe 2+ Distribution detection: An additional Asy-50 and control chip were used. After 64 hours of exposure, the chips were washed with PBS and then perfused with 5 μM RhoNox-1 (Fe2+).2+ The probe was incubated in PBS solution for 20 min in the dark. After washing away excess probe with PBS, the chip was placed back into the microscope. Simultaneously, while restarting the unilateral perfusion of MoS2 suspension, time-series imaging was initiated using the fluorescence channel (excitation wavelength 540 nm, emission and collection wavelength 560-620 nm), continuously recording images at a rate of 30 seconds per frame for 15 minutes to record Fe. 2+ The dynamic process of fluorescence signal initiation from the treated side and diffusion towards the untreated side, as shown in the following figures. Figure 10 As shown, the left figure shows the Fe root system of Asy-50. 2+ Real-time imaging of the distribution of the fluorescent probe RhoNox-1; the right image shows the Fe2+ root system of Asy-50. 2+ The variation of the fluorescent probe RhoNox-1 (relative to its initial state) with exposure time and distance from the exposed root side. From Figure 10 It can be seen that: Fe 2+ Initially, the increase was observed on the treated side of the root, followed by transduction to the untreated side within 15 minutes. This signal transduction-like effect of Fe... 2+ The conduction mechanism allows for the presence of Fe in the roots at 15 minutes. 2+ The overall level increased by 2.0 times compared to the initial level, confirming the ability of MoS2 nanosheets to affect the redox homeostasis of the root system.

[0083] Example 4: Chemogenetic verification of the necessity of key signaling pathways This experiment aims to verify the ROS and Fe observed in Example 3. 2+ Is the signal change a necessary causal link in the Asy-50 treatment's promotion of root growth? The aim is to establish a causal chain of "spatial stimulation producing a specific signal that affects growth phenotype" by using a specific pharmacological inhibitor to block a specific signal while applying spatial stimulation.

[0084] (1) Inhibitor intervention experimental design: Based on the Asy-50 treatment in Example 2, the following inhibitor co-treatment groups were added: Asy-50+NAC group: 0.5mM N-acetylcysteine ​​(NAC, broad-spectrum ROS scavenger) was added for 3h before MoS2 suspension injection; Asy-50+DFO group: 0.5mM deferoxamine (DFO, iron ion chelator) was added for 12h before MoS2 suspension injection; The Sy-25 treatment group with the same exposure dose as the Asy-50 treatment group was used as the control. Figure 11 ROS and Fe in roots under unilateral NAC and DFO inhibitor treatment 2+ Changes. From Figure 11 It can be seen that the inhibitor has a significant inhibitory effect.

[0085] (2) Dual detection: First, signal detection was performed. Following the procedure in Example 3, the roots of the corresponding groups were stained with H2DCF-DA or RhoNox-1 to observe the effect of the inhibitor on ROS or Fe. 2+ The blocking effect of signal spatial patterns was then assessed. Phenotypic detection was then performed, and the root length of each group was measured and compared with that of the Asy-50 group (without inhibitor) following the procedure in Example 2. Figure 12 The graph shows root elongation under inhibitor treatments. The left image represents representative root growth images under ROS inhibitor (NAC) and iron chelating agent (DFO) treatments, while the right image shows the relative root elongation rate of the corresponding treatment groups. Results are as follows: Figure 12 As shown, under unilateral exposure of MoS2 nanosheets, the treatment of lateral root ROS and Fe 2+ The root growth-promoting effect disappears after inhibition.

[0086] Example 5: Confirmation of downstream auxin response and pathway endpoint This experiment aims to elucidate the downstream pathways of the final signaling pathways in the spatial stimulus signaling pathway. The goal is to confirm whether asymmetric MoS2 exposure, through the aforementioned signaling cascade, ultimately leads to the local accumulation of auxin, a key hormone regulating growth, in the root tip, and to verify the necessity of this accumulation for growth promotion.

[0087] (1) Auxin reporter gene imaging: Transgenic Arabidopsis thaliana expressing DR5:GFP (auxin response reporter gene) was used, and the blank, Asy-50 and Sy-25 treatments in Example 2 were repeated. After 64 hours of exposure, the GFP fluorescence intensity of the root tip was observed and photographed directly under a fluorescence microscope (GFP channel: excitation light 488nm, emission light collection band 500-550nm) to compare the differences in auxin reporter activity between the two groups.

[0088] (2) Auxin synthesis inhibition experiment: In wild-type Arabidopsis thaliana, an Asy-50+Kyn treatment group was set up, i.e., 1 μM L-kynurenine (Kyn, a competitive inhibitor of the auxin synthesis inhibitor TAA1 / TAR) was added before the addition of MoS2 suspension for 24 h. After 64 h, the root length of the Asy-50+Kyn group was measured and compared with the Asy-50 group (without inhibitor) and the Sy-25 group. Kyn treatment could specifically eliminate the growth-promoting effect of Asy-50, indicating that auxin biosynthesis is the final step necessary for this spatial effect.

[0089] Reverse verification of ROS and Fe 2+ The signal is located upstream of auxin accumulation: ROS and Fe were measured in the Asy-50+Kyn treatment group after 64 h. 2+The relative fluorescence intensity was measured. The relative fluorescence intensity of DR5:GFP (auxin-responsive reporter gene) expressed in the Asy-50+NAC and Asy-50+DFO treatment groups was measured after 64 h.

[0090] The results are as follows Figures 13-15 As shown, where, Figure 13 Processing for Sy-25 and Asy-50 DR5:GFP Representative fluorescence images and fluorescence intensity comparisons are shown. The left image shows representative images of DR5:GFP fluorescence in the root tips of each treatment group, and the right image shows the change in DR5:GFP fluorescence intensity in the root tips relative to the control group. Figure 14 The effects of auxin synthesis inhibitor treatment on the root growth of Asy-50 are shown in the image. The left image represents the root growth of Asy-50 after Kyn treatment, and the right image shows the root elongation rate of the corresponding treatment group. Figure 15 The effects of auxin inhibitors in Asy-50 on root ROS and Fe 2+ The left figure shows the effect of auxin inhibitors in Asy-50 on the root ROS fluorescent probes H2DCFA and Fe. 2+ Imaging of the fluorescent probe RhoNox-1; the right image shows the change in fluorescence intensity in the roots under the corresponding probe. From Figures 13-15 It can be seen that the fluorescence intensity of DR5:GFP in the root tips of the Asy-50 treatment group was significantly enhanced compared with the control group and the Sy-25 treatment group. Under iron deficiency or in the presence of ROS scavengers, DR5:GFP gene expression was significantly downregulated. Conversely, inhibiting auxin biosynthesis with Kyn did not significantly alter ROS and Fe levels in the roots. 2+ The overall content. These results indicate that auxin biosynthesis is downstream of redox signaling.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for analyzing the spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism based on a microfluidic chip, characterized in that, The microfluidic chip includes at least three interconnected cavities; the method includes the following steps: Plant roots were loaded into any central cavity of the microfluidic chip, and after plant adaptation, liquid culture medium containing nanomaterials was infused into the cavities on both sides of the cavity containing the plant roots, resulting in multiple spatial effect models. These models include a symmetrical exposure model and an asymmetrical exposure model. In the asymmetrical exposure model, the concentration of nanomaterials in the liquid culture medium infused into the cavities on both sides is different. In the symmetrical exposure model, the concentration of nanomaterials in the liquid culture medium infused into the cavities on both sides is the same. In both the symmetrical and asymmetrical exposure models, the total concentration of nanomaterials in the liquid culture medium infused into the cavities on both sides is the same. Data on plant root growth processes were monitored in multiple spatial effect models to obtain a database. The spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism was obtained by analyzing the database.

2. The method according to claim 1, characterized in that, A guide protrusion is provided in the middle cavity of the three cavities. The cross-sectional shape of the guide protrusion is triangular. The guide protrusion guides the plant roots to grow in a straight line along the cavity.

3. The method according to claim 1, characterized in that, The three cavities are arranged in parallel, with the two outer cavities symmetrically distributed on both sides of the middle cavity.

4. The method according to claim 1, characterized in that, The plants mentioned include Arabidopsis thaliana.

5. The method according to claim 1, characterized in that, The nanomaterials include molybdenum disulfide nanosheets.

6. The method according to claim 1 or 5, characterized in that, The concentration of nanomaterials in the liquid culture medium is independently 0~200 mg / L.

7. The method according to claim 1, characterized in that, During the process of plant adaptation and root growth, the plant stem is perpendicular to the horizontal plane.

8. The method according to claim 1, characterized in that, The perfusion flow rate of the liquid culture medium is 0.2~1.0 mL / h.

9. The method according to claim 1, characterized in that, The root growth process data includes microscopic images, reactive oxygen species, auxin, ferrous ions, and fluorescence images.

10. A system for analyzing the spatial distribution effect of nanomaterials in the nanomaterial-plant interaction mechanism based on a microfluidic chip, characterized in that, include: Microfluidic chips; Growth process monitoring system; The module for processing monitoring data.

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