A nematode high-throughput drug screening method and system based on nanofriction power generation

Abstract

The application discloses a kind of based on nanometer friction power generation nematode high-throughput drug screening method and system, including nanometer friction power generator and microfluidic chip;The nanometer friction power generator is combined above the microcolumn array in the microfluidic chip correspondingly.Combination of the nanometer friction power generator and microfluidic chip, compared with prior art, realizes miniaturization, low cost, high efficiency collection method, throws away the high observation collection equipment.In addition, the drug dosage of drug screening experiment is controlled within 100 μL, greatly reduce the drug dosage consumed.

Description

Technical Field

[0001] This invention belongs to the field of drug screening technology, specifically relating to a high-throughput drug screening method and system for nematodes based on nano-triboelectric power generation. Background Technology

[0002] In the early stages of modern drug discovery, large-scale screening and evaluation of small biomolecules using high-throughput and low-cost assessment tools has been widely used to identify lead compounds. However, traditional screening methods rely heavily on cell-based biochemical analyses, which makes these strategies challenging to translate into clinical practice. Treatment of complex conditions such as central nervous system disorders and metabolic diseases requires more systematic modulatory therapies. Therefore, the development of in vivo drug screening systems based on small animal models can better evaluate drugs through adsorption, distribution, and toxicity, making it increasingly important for high-throughput screening of compound libraries.

[0003] In addition to conducting extensive experiments using traditional model organisms such as amphibians and mice, researchers have explored novel model organisms like zebrafish, fruit flies, and *C. elegans*, providing rapid solutions for in vivo high-throughput drug screening. Compared to other model organisms, *C. elegans* (or simply nematode), as one of the most thoroughly studied small animal models, has been widely used in large-scale genetic and pharmacological screenings due to its numerous experimental advantages, including small size, short lifespan, transparent body, good genetic characteristics, and low maintenance costs. Furthermore, as a multicellular organism, nematodes have a relatively small number of cells, allowing for exhaustive studies of cell morphology and lineage, as well as genetic analysis. Many *C. elegans* disease models (e.g., pathogen infection) have also seen significant development due to their ability to reproduce unique phenotypes of human diseases.

[0004] However, traditional manual screening methods still present significant challenges in conducting long-term behavioral screening of nematodes while simultaneously achieving rapid, accurate recording and real-time evaluation. Although researchers have developed microfluidic chips or automated microsystems of various sizes for nematode screening, the complexity of external systems limits the applicability of these devices. Currently, almost all platforms rely on high-precision, high-speed cameras for microscopic imaging, and the high cost and low throughput of long-term evaluation limit their use. Therefore, there is an urgent need to develop a practical, economical, and high-throughput in vivo nematode screening system in the fields of biology and pharmacology.

[0005] In recent years, various biomedical sensing systems have successfully demonstrated that nanotriboelectric generators can monitor extremely weak mechanical signals in vivo and in vitro in real time. Their working principle is based on the contact-separation electrostatic effect, that is, obtaining minute amounts of mechanical energy through the contact and separation of two triboelectric layers within the device. By adjusting the material composition, structural design, and scale control of the device, nanotriboelectric generators can be integrated with microfluidic systems to achieve efficient mechanical sensing, thereby monitoring the movement of different organisms in a more economical and effective way, including human motion detection, biosensing of cardiomyocytes, and detection of individual bacterial flagellar movement. Therefore, using nanotriboelectric generators to monitor the behavior of nematodes on a large scale is a novel and very promising method. However, the problem with existing technologies is that traditional methods for long-term, artificially assisted behavioral screening of nematodes, along with rapid, accurate recording and real-time evaluation, require the integration of microfluidic chips or automated microsystems of various sizes. The complex external systems of these devices significantly reduce their applicability. Currently, almost all platforms rely on high-precision, high-speed cameras for microscopic imaging; however, high-precision cameras are expensive, and the high cost and low throughput required for long-term evaluation cannot meet the demands of high-throughput screening. Therefore, developing a practical, convenient, economical, and high-throughput in vivo screening system for nematodes that can collect behavioral data of nematodes under drug influence without the need for expensive equipment is of great significance for subsequent drug screening research. Summary of the Invention

[0006] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a high-throughput drug screening method and system for nematodes based on nano-triboelectric generation. This system, based on a nano-triboelectric generator and a microfluidic chip, realizes a novel in vivo screening strategy. It effectively combines a hierarchical biohybridization triboelectric nanogenerator array (i.e., the chip in the embodiments of this invention) with computational bioinformatics analysis for high-throughput pharmacological evaluation using freely moving *C. elegans*. It effectively solves the problems of conventional animal behavior monitoring methods being laborious, time-consuming, and requiring expensive equipment. Through specially designed micropillars, the behavior of *C. elegans* is effectively converted into triboelectric deformation, generating contact separation motion between two triboelectric layers to produce electrical output. The detection cost is significantly reduced, and it can be combined with a multi-channel recording system or a commercial oscilloscope to record the output signal and extract various biological information for each screened compound. Furthermore, the system has successfully achieved the ability to predict the identity of drugs / compounds by generating information-rich electrical readings from the proposed chip-based platform using a multi-Gaussian kernel-based machine learning method. This makes it easily applicable to various fields, particularly in the exploration of combination drugs with limited samples and resources, and is very useful for accelerating the identification of new therapies with precision drugs.

[0007] In a first aspect, the present invention provides a nano-triboelectric generator, the nano-triboelectric generator comprising a metal core and an outer cladding layer; a gap exists between the metal core and the outer cladding layer.

[0008] In some embodiments of the present invention, the material of the metal core includes: copper, iron, aluminum, and silver.

[0009] In some specific embodiments of the present invention, the material of the metal core is copper.

[0010] In some embodiments of the present invention, the material of the outer coating layer includes: polydimethylsiloxane (PDMS), polyethylene glycol (PEGDA) diacrylate, and methacrylic hydrogel (GelMA).

[0011] In some specific embodiments of the present invention, the material of the outer coating layer is selected as polydimethylsiloxane (PDMS).

[0012] In some embodiments of the present invention, the nano-triboelectric generator may include a plurality of metal cores.

[0013] In some embodiments of the present invention, the plurality of metal cores may be interwoven or combined in any way, including but not limited to being woven into a mesh. In an embodiment of the present invention, the copper mesh has an interlaced hole structure.

[0014] In some specific embodiments of the present invention, the metal core of the nano-triboelectric generator is woven into a mesh structure, more specifically a copper mesh.

[0015] In some embodiments of the present invention, the pore size of the mesh structure is 50-60 μm.

[0016] In some embodiments of the present invention, the pore size of the mesh structure is 60 μm.

[0017] In some embodiments of the present invention, the mesh thickness in the mesh structure is 45–55 μm.

[0018] In some embodiments of the present invention, the mesh thickness in the mesh structure is 55 μm.

[0019] In some embodiments of the present invention, the mesh structure is a grid structure with one layer.

[0020] In this invention, the nano-triboelectric generator is composed of a copper mesh wrapped with a polydimethylsiloxane (PDMS) layer. The size of the tiny gap between the PDMS layer and the copper core is the magnitude of the electrical signal that generates the intensity of the nematode's movement, and the signal can be acquired by combining a self-made multi-channel recording system or a commercial oscilloscope.

[0021] In some embodiments of the present invention, there is a gap of 2 to 5 μm between the metal core and the outer cladding layer.

[0022] In some embodiments of the present invention, there is a 3 μm gap between the metal core and the outer cladding layer.

[0023] In a second aspect, the present invention provides a method for preparing the nano-triboelectric generator described in the first aspect, comprising the following steps: oxidizing a metal core to form an oxide sacrificial layer on its surface, covering the metal core covered with the oxide sacrificial layer with a coating material, and immersing it in an acid solution to remove the oxide sacrificial layer, thereby obtaining the generator.

[0024] In some embodiments of the present invention, the material of the metal core includes: copper, iron, aluminum, and silver.

[0025] In some embodiments of the present invention, the coating material includes: polydimethylsiloxane (PDMS), polyethylene glycol (PEGDA) diacrylate, and methacrylic hydrogel (GelMA).

[0026] In some embodiments of the present invention, the oxidation process includes heating oxidation under air conditions. Specifically, it involves heating oxidation using a box furnace.

[0027] In some embodiments of the present invention, the oxide sacrificial layer is a metal oxide of the metal core.

[0028] In some embodiments of the present invention, the acid solution is hydrochloric acid.

[0029] In some embodiments of the present invention, the gap between the metal core and the cladding material is obtained by etching away the oxide sacrificial layer with acid.

[0030] In some embodiments of the present invention, while the oxide sacrificial layer is etched away, a micro-nano structure similar to the surface of the metal core microstructure is also formed on the inner surface of the coating material. This greatly increases the roughness of the two opposing surfaces of the two friction layers (the metal core and the coating material), significantly increasing the contact area and thus effectively improving the output signal of the nano-triboelectric generator.

[0031] In some embodiments of the present invention, the preparation method of the nano-triboelectric generator is as follows: the metal core is oxidized by heating under air conditions to form an oxide sacrificial layer on its surface, a coating material is spin-coated, and after heating until it is fully cured, the oxide sacrificial layer is removed by immersion in acid solution, and the product is obtained.

[0032] In some embodiments of the present invention, the steps further include washing with water and drying after soaking in acid solution.

[0033] In some embodiments of the present invention, the prepared large-size nano-tribogenerators (thin film form) can be rapidly formed into a large number of nano-tribogenerators with different specific shapes, sizes and mechanical flexibility using high-precision laser cutting technology.

[0034] A third aspect of the present invention provides a microfluidic chip having a cavity, wherein a micropillar array is disposed in the cavity, and a plurality of micropillars are disposed in the micropillar array.

[0035] In some embodiments of the present invention, the diameter of the micropillar is 45–60 μm.

[0036] In some embodiments of the present invention, the diameter of the micropillar is 50 μm.

[0037] In some embodiments of the present invention, the height of the micropillar is 100–150 μm.

[0038] In some embodiments of the present invention, the height of the micropillar is 150 μm.

[0039] In some embodiments of the present invention, the distance between two adjacent micropillars along the edge is 50 to 80 μm.

[0040] In some embodiments of the present invention, the distance between two adjacent micropillars along the edge is 80 μm.

[0041] In some embodiments of the present invention, the micropillars are made of flexible materials and are capable of bending in all directions.

[0042] In some embodiments of the present invention, the flexible material includes: polydimethylsiloxane (PDMS), polyethylene glycol (PEGDA) diacrylate, and methacrylic hydrogel (GelMA).

[0043] In some specific embodiments of the present invention, the flexible material is selected as polydimethylsiloxane (PDMS).

[0044] In this invention, the micropillars transform the horizontal movement of nematodes into deformation and stretching torsion of the PDMS layer on the micropillar nano-triboelectric generator, thereby inducing contact separation motion between the two triboelectric layers and generating electrical output, which plays an important role.

[0045] In some embodiments of the present invention, the omnidirectional bending of each micropillar increases the gap between the triboelectric layers.

[0046] In some embodiments of the present invention, the microfluidic chip is prepared using a roll casting method. Of course, those skilled in the art can also prepare it using other methods, including but not limited to the roll casting method, according to the microfluidic chip design requirements of the present invention.

[0047] In some embodiments of the present invention, the design requirements of the microfluidic chip include: having one or more cavities, within which an array of micropillars is disposed. In the present invention, the microfluidic chip has one cavity, with the micropillar array suspended at the top of the cavity. The microfluidic chip structure of the present invention can accommodate dozens of adult nematodes, providing sufficient space for their activity, and can also contain liquid drugs to prevent evaporation. Of course, based on the needs of use, those skilled in the art can appropriately adjust the size of the space to meet specific usage requirements.

[0048] In some embodiments of the present invention, the method for fabricating the microfluidic chip includes: designing a microfluidic chip structure, photolithographically forming a microfluidic chip mold, using a flexible material to cast a film, and punching inlet and outlet holes after curing, thereby obtaining the chip.

[0049] A fourth aspect of the present invention provides a nematode motion detection product, the nematode motion detection product comprising the nano-triboelectric generator described in the first aspect of the present invention and the microfluidic chip described in the third aspect of the present invention; the nano-triboelectric generator is correspondingly coupled above the micropillar array in the microfluidic chip.

[0050] In some embodiments of the present invention, the prepared nano-triboelectric generator and the micro-pillar microfluidic layer (i.e., the microfluidic chip described in the third aspect of the present invention) are tightly bonded after plasma surface treatment, and then a layer of encapsulation material is cast to encapsulate the entire device.

[0051] In some specific embodiments of the present invention, the encapsulation material is selected as polydimethylsiloxane (PDMS).

[0052] In some embodiments of the present invention, as the nematodes crawl and swing in the uniformly distributed column clusters, the drooping columns deform and drag the upper PDMS thin layer, so that the tiny gap between the two triboelectric layers can achieve contact separation, thereby converting the physical movement of the nematodes into an collectable electrical output signal.

[0053] In some embodiments of the present invention, the nematode movement detection product includes a sensor and a chip.

[0054] In some embodiments of the present invention, the sensor includes a front-end sensing component and a sensor element. The chip includes an integrated chip and an in-vivo sensing chip.

[0055] A fifth aspect of the present invention provides a nematode-based detection or screening system, the detection or screening system comprising the nematode movement detection product described in the fourth aspect of the present invention, and external auxiliary equipment.

[0056] In some embodiments of the present invention, the external auxiliary equipment includes a signal collection device and a detection container.

[0057] In some embodiments of the present invention, the signal collection device is used to collect the electrical signals output by the nematode movement detection product.

[0058] In some embodiments of the present invention, the signal collection device includes a digital oscilloscope, an analog oscilloscope, and a virtual oscilloscope.

[0059] A sixth aspect of the present invention provides the application of the nano-triboelectric generator described in the first aspect of the present invention, the microfluidic chip described in the third aspect of the present invention, or the nematode motility detection product described in the fifth aspect of the present invention in drug screening and / or nematode motility detection.

[0060] In some embodiments of the present invention, the specific method of use is as follows: A suspension of *C. elegans* is loaded into a nano-triboelectric nematode chip; the chip is connected to an oscilloscope probe; and the chip is placed in a chip shielding box, thus completing the connection between the chip and the measuring device. When it is necessary to add a trace amount of drug for detection, the drug is added into the nematode chip through the chip's sample inlet. After the drug diffuses into the nematode's body and causes a certain influence on its morphological movement, subsequent observations and data collection can be carried out.

[0061] In this invention, drug screening in nematodes is based on a nanotriboelectric generator. By converting the signals from the horizontal movement of the nematodes into signals from their vertical movement, the unique hierarchical structure of the device itself enables signal transmission and recording, thus breaking through the limitations of traditional single-mode signal acquisition using nanotriboelectric generators. Furthermore, by combining the nanotriboelectric generator with small animal drug screening and employing computational bioinformatics analysis, drug screening functionality using a nanotriboelectric generator has been achieved for the first time. The high sensitivity, high throughput, scalability, and low-cost manufacturing process of the nanotriboelectric generator provide a novel and efficient solution for future drug screening efforts, accelerating drug discovery and research applications in various biomedical and biological fields.

[0062] The beneficial effects of this invention are:

[0063] 1. The nano-triboelectric generator and microfluidic chip in this invention can effectively achieve simple, rapid, and high-throughput drug screening for nematodes.

[0064] 2. The nano-triboelectric generator in this invention can convert the horizontal movement of nematodes in a liquid into vertical contact-separation of the device's hierarchical structure, thereby collecting the movement signals of small animals and providing an efficient, highly sensitive, and low-cost drug screening method.

[0065] 3. This invention combines a nano-triboelectric generator with a microfluidic chip, achieving a miniaturized, low-cost, and high-efficiency energy harvesting method compared to other methods that utilize microfluidic chips for drug screening or nano-triboelectric generators for energy harvesting, thus eliminating the need for expensive observation and harvesting equipment. Furthermore, it controls the amount of drug used in drug screening experiments to within 100 μL, significantly reducing the amount of drug consumed. Attached Figure Description

[0066] Figure 1 This is a schematic diagram of the design of a nano-triboelectric generator in an embodiment of the present invention.

[0067] Figure 2 This is a schematic diagram of the microfluidic chip design in an embodiment of the present invention.

[0068] Figure 3 This is a schematic diagram of the fabrication process of the nano-triboelectric generator in an embodiment of the present invention.

[0069] Figure 4 This is a schematic diagram of the micropillar nano-triboelectric generator nematode chip structure in an embodiment of the present invention.

[0070] Figure 5 This is a physical image of the micropillar nano-triboelectric generator nematode chip in an embodiment of the present invention.

[0071] Figure 6 The diagram shows the internal structure of the microfluidic chip in the embodiment of the present invention, where a is a micropillar array (scale bar 100 μm); b is a single micropillar (scale bar 20 μm); and c is a representative microscopic image of a crawling Caenorhabditis elegans worm in the device (scale bar 100 μm).

[0072] Figure 7 This is a schematic diagram illustrating the coupling principle of contact triboelectric effect and electrostatic induction in the nano-triboelectric generator of this invention.

[0073] Figure 8 This is a simulation diagram of the nano-triboelectric generator in an embodiment of the present invention.

[0074] Figure 9The stability characterization results of the micropillar nano-triboelectric generator in the embodiments of the present invention are shown below. Among them, a is the output voltage signal result of the micropillar nano-triboelectric generator after 10,000 cycles of mechanical motor contact; b is the waveform comparison of the voltage output results of the nano-triboelectric generator after the first contact and the 10,000th contact; c is the numerical comparison of the voltage output results of the nano-triboelectric generator after the first contact and the 10,000th contact, and there is no significant difference between the two output results.

[0075] Figure 10 This is a photograph of the micropillar nano-triboelectric generator energy storage lamp in an embodiment of the present invention.

[0076] Figure 11 The nanoindentation instrument in this embodiment of the invention measures the sensitivity of the micropillar nano-triboelectric generator.

[0077] Figure 12 The results show the control group and experimental group, as well as the comparison of output electrical signals at different caffeine concentrations.

[0078] Figure 13 The figures show the time-domain and frequency-domain characteristics of the micropillar nano-triboelectric generator in this embodiment of the invention. Specifically, a) is a comparison of the time-domain signal waveforms of 37 drugs tested in the experiment; b) is a comparison of the peak distribution histograms of the time-domain signals of 37 drugs tested in the experiment; and c) is a comparison of the fast Fourier transform heatmaps of the frequency-domain signals of 37 drugs tested in the experiment.

[0079] Figure 14 To utilize cluster analysis of the similarity results of 37 drugs, where a represents the 8 cluster results formed by blind identification cluster analysis; and b represents the similarity network distribution of the experimental results of the 37 drugs.

[0080] Figure 15 This is a schematic diagram illustrating the structural differences between the microcolumn microfluidic nano-triboelectric generator in this embodiment of the invention and a traditional nano-triboelectric generator.

[0081] Figure 16 This invention provides a performance comparison between the microcolumn microfluidic nano-triboelectric generator in this embodiment and a traditional nano-triboelectric generator. Detailed Implementation

[0082] To make the objectives, technical solutions, and effects of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described in this specification are merely for explaining the invention and are not intended to limit the invention.

[0083] Unless otherwise specified, all experimental materials and reagents used are commercially available consumables and reagents.

[0084] High-throughput drug screening system for nematodes based on nano-triboelectric power generation

[0085] In this invention, the core device (nanotriboelectric generator sensor) of the nematode high-throughput drug screening system based on nanotriboelectric power generation mainly consists of two parts: one part is a nanotriboelectric generator used for the detection and transmission of nematode movement signals, and the other part is a microfluidic chip used for drug loading and providing space for the movement of model organisms.

[0086] The nano-triboelectric generator of this invention consists of a copper mesh coated with a polydimethylsiloxane (PDMS) layer. For example... Figure 1 As shown: the size of the tiny gap between the PDMS layer and the copper core is the magnitude of the electrical signal that generates the intensity of the nematode's movement, and the signal can be acquired by combining a homemade multi-channel recording system or a commercial oscilloscope.

[0087] The key to the microfluidic chip in this invention lies in its internal structure, which is composed of an array of micropillars. For example... Figure 2 As shown: Each micropillar inside the chip can bend in all directions, which increases the gap between the triboelectric layers.

[0088] When nematodes added to the micropillar-based nano-triboelectric generator (NTG) nematode chip respond to drug stimulation, exhibiting changes such as altered body oscillation frequency, body bending degree, or movement speed, these phenotypes cause the nematodes to continuously collide with the micropillars. Therefore, the micropillars dynamically amplify the electrical signals generated by the movement of *C. elegans* during drug stimulation. The key to the nano-triboelectric generator lies in the "contact-separation" electrical signal generation formed by its hierarchical structure. Combining these two components into a high-throughput drug screening system for nematodes allows for drug screening with only an extremely small amount of drug filling the microfluidic chip. This successfully demonstrates that the informative electrical signal data generated by the proposed nano-triboelectric generator-based microfluidic platform is sufficient to predict the properties and efficacy of drugs / compounds.

[0089] The fabrication of the nano-tribogen sensor in this invention mainly involves combining a micro-nano pillar microfluidic chip mold fabricated using soft photolithography technology with a microfluidic chip fabricated by replicating microfluidic hollow channels from the mold using PDMS, thus completing the fabrication of the micro-nano pillar nano-tribogen sensor.

[0090] Specifically, the preparation method of the above-mentioned nano-triboelectric generator is as follows:

[0091] The nano-triboelectric generator of this invention consists of a copper core and a double triboelectric layer covering the outer surface of the copper core and a PDMS layer. A certain gap exists between the copper core and the PDMS layer, which is achieved by eliminating the CuO generated during the oxidation of the copper mesh under air heating conditions. The preparation process is as follows: Figure 3As shown, a copper mesh was heated and oxidized in a box furnace at 500℃ for 4 hours to form a CuO layer on its surface. The CuO layer, acting as a sacrificial layer, was removed during the subsequent removal process. After the CuO-coated copper mesh cooled, PDMS was spin-coated onto the mesh surface at 2500 rpm for 15 seconds. Once the PDMS layer stabilized, it was transferred to an 80℃ oven for complete curing, forming a solid PDMS layer. The PDMS-coated copper mesh was then immersed in a 1M hydrochloric acid solution until the CuO layer on the surface was removed, creating gaps of approximately 3 μm. Simultaneously with the etching away of the CuO layer, a micro / nano structure similar to the copper mesh microstructure was formed on the inner surface of the PDMS layer. The two roughened surfaces of the two friction layers (the copper core and the PDMS layer surrounding it) significantly increased the contact area, effectively improving the output signal of the nano-triboelectric generator. After washing away residual hydrochloric acid with deionized water, the prepared device was completely air-dried with an air gun, and electrodes were attached to the surface for further integration with a micropillar microfluidic chip.

[0092] Design and fabrication of the above-mentioned microfluidic chip:

[0093] The microfluidic chip structure was designed using SolidWorks 3D modeling software. The micropillar array microfluidic layer designed in this invention is a key component of the nematode high-throughput drug screening system. For example... Figure 2 As shown, it consists of a cavity with a micropillar array suspended at the top. This structure can accommodate dozens of adult nematodes, providing sufficient space for their movement, and also holds the liquid medicine to prevent evaporation. Specifically, in this microfluidic chip, the diameter of each micropillar in the micropillar array is 50 μm, the height is 150 μm, and the distance between adjacent micropillars along their edges is 80 μm.

[0094] After designing the structure using modeling software, a micro-nanopillar microfluidic chip mold (TENG) was fabricated on a double-sided polished single silicon wafer using soft photolithography. Before fabrication, the single silicon wafer was immersed in anhydrous ethanol and ultrapure water at a volume ratio of 1:3 and ultrasonically cleaned for 10 minutes. The wafer was then removed and placed on a 35°C heating stage to dry the surface moisture. It was then dried with an air gun and cleaned with lint-free paper to remove any remaining water. Negative photoresist (SU8-2050, MicroChem) was poured onto the cleaned single-crystal silicon wafer and placed in a spin coater. The spin coater speed was set in stages: first at 500 rpm / min to evenly distribute the photoresist, and second at 1700 rpm / min to spread the photoresist to a height of 150 μm. After spin coating, the wafer was allowed to stand for 15 minutes before being placed on a heating stage for two-step pre-baking: first at 65°C for 5 minutes, then at 95°C for 15 minutes. Afterward, the wafer was allowed to cool to room temperature. A photomask with the designed pattern was pressed onto a silicon wafer coated with SU-8. The microfluidic structure was transferred to the surface of the SU-8 coated single-crystal silicon wafer by standard UV exposure for 48 seconds. After removing the mask, the silicon wafer underwent a distribution and baking process: first, heating at 65°C for 4 minutes, then at 95°C for 10 minutes. The wafer was then allowed to cool to room temperature. The wafer was then developed in a developer solution for 10 minutes. After development, the photoresist on the silicon wafer surface was rinsed alternately with isopropanol and ultrapure water. Finally, the SU-8 micro / nanopillar microfluidic chip mold was hardened at 150°C for 10 minutes and then cooled to room temperature, completing the fabrication of the micro / nanopillar microfluidic chip mold. The prepared mold was then treated with trifluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane in a vacuum dryer to prevent PDMS adhesion during the molding process.

[0095] In preparing the PDMS layer for the microfluidic chip, the vacuum-degassed PDMS and its corresponding crosslinking agent (PDMS purchased from Guangzhou Chensheng Biotechnology Co., Ltd., consisting of prepolymer A (PDMS) and crosslinking agent B) were mixed at a mass ratio of 10:1, thoroughly stirred, and poured into a mold. The mixture was then partially cured in an 85°C oven for 5 minutes. After the PDMS layer hardened, the prepared TENG was pressed tightly onto its surface, and the entire device was coated with the same proportion of PDMS. After completely removing air bubbles and degassing in a vacuum chamber, the mold with the PDMS layer was heated in an 80°C oven for 2-3 hours. The fully cured PDMS layer was removed, and holes were punched at both ends to form inlet and outlet ports. Finally, after plasma treatment, the nano-triboelectric generator with the micropillar microfluidic chip was firmly bonded to a glass slide / printed circuit board (PCB), forming a... Figure 4 and Figure 5 The micropillar nano-triboelectric generator (chip device) shown is the final product used.

[0096] The prepared nano-triboelectric generator and micro-pillar microfluidic layer were tightly bonded after plasma surface treatment, and then a layer of PDMS was cast to encapsulate the entire device, forming a micro / nano-pillar nano-triboelectric generator sensor, which was used to obtain a nematode high-throughput drug screening system. As the nematodes crawl and swing in the uniformly distributed column clusters, the drooping columns deform, dragging the thin PDMS layer above, and the tiny gap between the two triboelectric layers achieves contact separation, thereby converting the nematode's physical motion into a collectable electrical output signal.

[0097] Specifically, the signal analysis included recording a 200-second sequence of drug signals and processing it against its corresponding control group signals. The processing involved first normalizing each signal sequence to a range of 0 to 1, then dividing it into 10 segments for further investigation. Next, the envelope of each signal segment was extracted at intervals of 50 data points. Finally, a Fast Fourier Transform (FFT) was performed to obtain spectral information at a frequency of 40 Hz.

[0098] Internal structure observation of nano-triboelectric generator

[0099] Structural analysis of the nano-triboelectric generator in the above embodiments revealed that it possesses a multi-layered mesh structure. This characteristic allows for the simple, rapid, and low-cost fabrication of the nano-triboelectric generator. Specifically, the nano-triboelectric generator structure in the above embodiments uses a copper mesh as a substrate, with a mesh aperture of approximately 60 μm and a mesh thickness of approximately 55 μm. Under controlled heating oxidation time, heating temperature, and hydrochloric acid corrosion, a micro-gap of approximately 3 μm is formed between the two triboelectric dielectric layers. The micro / nano-scale topological structure formed on the surface of the copper core after acid corrosion of CuO also significantly increases the contact area between the two triboelectric layers, giving the nano-triboelectric generator structure higher output characteristics and detection sensitivity. Furthermore, using high-precision laser cutting technology, large-scale nano-triboelectric generators (thin-film form) can be rapidly mass-produced to achieve specific shapes, sizes, and mechanical flexibility.

[0100] Internal structure observation of micropillar microfluidic chip

[0101] Structural analysis of the micropillar microfluidic chip in the above embodiments revealed that each micropillar has a diameter of 50 μm and a height of 150 μm, with an 80 μm distance between adjacent micropillars along their edges. These micropillars play a crucial role in converting the horizontal movement of the nematode into deformation and torsion of the PDMS layer on the micropillar nano-triboelectric generator, thereby inducing contact separation between the two triboelectric layers and generating electrical output.

[0102] Relevant characterization diagrams are as follows Figure 6 As shown.

[0103] Performance characterization of micropillar nano-triboelectric generator

[0104] (1) Feasibility:

[0105] Unlike traditional nano-triboelectric generators that detect motion deformation measured in millimeters per centimeter due to human behavior, the displacement produced by nematodes during movement is measured in micrometers. The micropillar nano-triboelectric generator in this embodiment operates on the principle of coupling between the contact triboelectric effect and electrostatic induction. Figure 7 As shown, when an external force is applied, surface charge transfer occurs in the contact area due to the triboelectric effect during the cyclic contact and separation process of the two triboelectric layers. Electrons transfer from the copper core surface to the PDMS layer, resulting in a negative charge on the PDMS surface and a positive charge on the upper copper mesh surface. When the external force is released, electrons in the upper PDMS layer and the copper core are driven to flow back and forth in the external circuit to balance the potential difference formed on the copper core and the PDMS layer, thereby generating a continuous electrical signal.

[0106] (2) Mechanical analysis:

[0107] like Figure 8 As shown, based on the principles of structural mechanics, after performing a three-dimensional finite element analysis on the micropillar nanotriboelectric generator in this embodiment of the invention, it was found that when the micropillar nanotriboelectric generator is working, as the nematode crawls sinusoidally through the cavity of the microfluidic chip, the nematode squeezes the micropillar and causes deflection. The bending horizontal force caused by the squeezing is decomposed into vertical and horizontal forces. In the simulation, the range of this total bending force is set to be from a few microNewtons to tens of millinewtons. When a horizontal force acts on a specific micropillar, it will cause vertical reflection on the upper part of the flexible PDMS layer. When the horizontal force is less than 200 μN, this vertical deformation can still easily cause contact and friction between the copper mesh surface and the PDMS layer.

[0108] (3) Stability:

[0109] After further stability characterization of the micropillar nano-triboelectric generator in the embodiments of the present invention, it was found that when using a linear motor with a force range of 10-50 mN or a nanoindenter with an indentation force range of 200-800 μN to scrape the surface of the micropillar nano-triboelectric generator, the device structure can maintain its integrity and function stability even after the motor applies a force of 50 mN and reciprocates 10,000 times. Figure 9 (As shown). When a micropillar nano-triboelectric generator is combined with an energy storage circuit, it is possible to store an output voltage of approximately 2V to light up an LED (such as...). Figure 10 (As shown).

[0110] (4) Sensitivity:

[0111] Sensitivity characterization was performed on the micropillar nanotriboelectric generator in this embodiment of the invention. A microNewton force was applied to a single micropillar using a nanoindenter. Figure 11 As shown, even under a very small force of 200 μN, a potential of 0.25 ± 0.02 mV can be achieved, indicating that the layered micropillar nano-triboelectric generator has good sensitivity to small deformations / forces.

[0112] Further testing was conducted by adding *Caenorhabditis elegans*, the nematode species required for the experiment, to the apparatus. The results were as follows: Figure 12 As shown, compared to an empty device without animals, pulse potential outputs of various sizes and irregular polarities can be clearly observed. Furthermore, it was found that the micropillar nanotriboelectric generator in this embodiment can not only detect the electrical signal output generated by nematode behavior stimulated by different concentrations of drugs (caffeine was shown as the drug in this embodiment) at relatively low doses from 10 μM to 50 μM, but also clearly detect the differences in electrical signal output at different concentrations. The results indicate that the micropillar nanotriboelectric generator device used in this invention can be used for the detection of minute deformations in nematodes and is suitable for evaluating nematode behavior under drug influence.

[0113] Micro-column nano-triboelectric generator for micro-drug screening in nematodes

[0114] The micro-drug screening relies primarily on the micro-nano-pillar triboelectric generator sensor, composed of the nano-tribogenerator and the micro-pillar microfluidic chip described in the above embodiments. This sensor forms the basis for measuring the vigorous movement of nematodes under drug stimulation. In addition to the crucial micro-nano-pillar triboelectric generator sensor (or micro-nano-pillar triboelectric generator chip, referred to as "chip" in this invention), the high-throughput drug screening system for nematodes requires the use of other external equipment. This external equipment mainly includes an oscilloscope (SDS5054X, SIGLENT) and a metal shielding box for housing the chip. Furthermore, depending on data recording requirements, the external equipment configured with this nematode chip triboelectric generator also needs to include external storage devices. The oscilloscope test probe connects to the electrode wires of the chip to receive signals and transmit them in real time to the machine for waveform display. The metal shielding box is used to shield against environmental noise interference.

[0115] In use, a suspension of adult *C. elegans* (YA) worms is loaded into the nano-triboelectric generator nematode chip. Specifically, the *C. elegans* (YA) worm suspension is prepared by washing adult *C. elegans* worms off solid culture medium (NGM) with M9 solution and then using a pipette to thoroughly mix the prepared nematode suspension. 50 μL of the suspension is then added to the nematode chip through the chip's sample inlet.

[0116] The microfluidic chip has a liquid inlet and outlet at each end. After the *C. elegans* enters the microfluidic chip channel, the chip is connected to the oscilloscope probe and placed inside the chip shielding box, completing the connection between the chip and the measurement device. When it is necessary to add a trace amount of drug for detection, 50 μL of the prepared drug is pipetted and added to the nematode chip through the chip's inlet. After the drug diffuses into the nematode and causes a certain effect on its morphology, subsequent observations and data collection can be carried out.

[0117] The practical effect of using micro / nano pillar nano-triboelectric generator chips for drug screening of nematodes

[0118] Without any physical / chemical stimulation, nematodes propel themselves forward or backward on agarose medium primarily through muscle contraction and relaxation in a sinusoidal pattern. In this invention, the micro-nano-pillar triboelectric generator chip, constructed based on a micropillar nano-triboelectric generator and a microfluidic chip, contains a microfluidic chamber with micropillars, enabling nematodes on the chip to exhibit a similar gait to those on culture medium. This allows for the study of nematode motility and chemotaxis under different drug conditions.

[0119] In this embodiment, a micro-nano pillar nano-triboelectric generator chip was used to evaluate and screen 37 randomly selected chemical drugs, each with a concentration of 100 μM. The acute changes in the behavior of the drugs induced in animals (nematodes) were observed, and different but repeatable electrical signal outputs generated by different drugs were acquired using an oscilloscope.

[0120] The specific operation is the same as in the above embodiment: First, fill the micropillar nanotriboelectric generator chip with the M9 solution used in the experiment and remove air bubbles from the chip. Then, use a pipette to inject 50 μL of a uniform nematode suspension into the micropillar nanotriboelectric generator chip, test and record the signal as a control group. Then, inject 50 μL of different chemical drugs into the micropillar nanotriboelectric generator chip, wait for 3 minutes to allow the nematodes to be completely immersed in the chemical drug environment, and record the electrical signal output for 200 seconds at a sampling rate of 2500 data points / s and save it for further analysis. After each experiment, the micropillar nanotriboelectric generator chip is washed with ultrapure water and dried at 30°C to evaporate the moisture on the chip. Before each drug measurement experiment, a blank control experiment is performed first to eliminate surrounding interference.

[0121] Meanwhile, to further analyze the nematode response to different drug treatments, the raw electrical signals recorded by the micropillar nano-triboelectric generator chip were analyzed in terms of amplitude and frequency. Some drug information in this embodiment is shown in Table 1.

[0122] Table 1. Drug Selection and Effects

[0123] Drug Name use Effect Amitriptyline Serotonin and norepinephrine neurotransmitter reuptake inhibitors Clinically used as a tricyclic antidepressant Clozapine Antipsychotic drugs It can cause tachycardia during use. Barmatine Isoquinoline alkaloids of the protoberberine class Slowing down the behavior of nematodes

[0124] Essentially, while changes in animal behavior are multidimensional and quantitative, the micropillar nano-triboelectric generator chip of this invention provides an in vivo fingerprint for each screened compound. Visually revealing some distinct differences in the electrical signals of specific drugs through temporal (signal envelope) and frequency (fast Fourier transform) characteristics of the acquired electrical signals demonstrates the variability between different drugs.

[0125] like Figure 13 As shown, amitriptyline induced the highest signal amplitude compared to other compounds; the time-domain electrical signal envelope curve of clozapine also clearly showed a significant increase in the frequency of the electrical peak. However, under the influence of bamatine, the amplitude of the electrical signal output of nematode behavior decreased sharply.

[0126] To further extract the intrinsic consistency between biometric fingerprints containing rich information, fast Fourier transform pattern fingerprints were collected for each test drug, and a multi-type dataset was formed by combining the time-domain signal envelope. For example... Figure 13 As shown, by analyzing the heatmap of the Fast Fourier Transform (FFT), drugs that promote nematode behavior exhibit very high amplitudes at specific frequencies in the FFT heatmap, while drugs that slow down nematode behavior show no significant amplitude at the characteristic frequencies of the heatmap.

[0127] Furthermore, a learning method incorporating multiple Gaussian kernels was used to learn a similarity measure between each pair of fingerprints in the data. Based on this method, a block structure was adopted in the resulting sample-to-sample similarity matrix, which was then used for further hierarchical clustering. The clustering analysis resulted in the blind identification of eight clusters, revealing multiple lines of evidence indicating that compounds with related fingerprints often have the same therapeutic indications. Figure 14As shown in the similarity network, the identified clusters are relatively far apart from each other. Furthermore, the tightness of intra-cluster connectivity displayed by the similarity network strongly suggests considerable consistency within each individual fingerprint cluster of drugs. Moreover, some structurally similar compounds can be successfully distinguished by the fingerprints identified by this system. For example, matrine, isomamatrine, and oxymatrine were grouped into groups 7, 5, and 2, respectively. Although these alkaloids have similar chemical structures, their spatial configurations differ, which corresponds to the significantly different cardiotetracycline-induced patterns reported in practice (existing studies have shown that the anti-damage effects induced by matrine and isomamatrine are mediated through different molecular targets, including mucosa and kappa-opioid receptors). Silymarin and baicalin, two flavonoids used in different treatment strategies for metabolic syndrome and liver-related diseases, exhibit different characteristics compared to other compounds and clustered into group 4 in this embodiment. However, another isoquinoline alkaloid, palmatine, which is also widely used in the treatment of metabolic syndrome and liver-related diseases, was identified in group 8. The pharmacokinetic profile of palmatine is more complex than that of silymarin and baicalin. Palmatine has a much wider range of clinical applications, such as in the treatment of central nervous system disorders. Furthermore, most drugs in group 6 have been found to have antidepressant / antidepressant-like effects. These analytical results indicate that the micropillar microfluidic nanotriboelectric generator of this invention can be used for high-throughput in vivo screening of drugs with different therapeutic functions.

[0128] Performance Comparison of Micro-column Microfluidic Nano-Triboelectric Generator and Traditional Nano-Triboelectric Generator

[0129] The micropillar microfluidic nanotriboelectric generator described in this invention is compared with a traditional nanotriboelectric generator. The operation method of the micropillar microfluidic nanotriboelectric generator is the same as that in the above embodiments. The traditional nanotriboelectric generator only has two dielectric layers in contact and separation, and does not have a microfluidic chip or nanopillar structure (such as...). Figure 15 (As shown), the operation method is as described in the instruction manual. The test is conducted on the premise that all other external conditions are completely consistent.

[0130] The results are as follows Figure 16 As shown, when a force of 50 mN is applied, the maximum output voltage of the pillarless microfluidic nanotriboelectric nanogenerator is only about 1.5 mV. Compared with non-pillar devices (i.e., conventional nanotriboelectric nanogenerators), the pillar-based microfluidic nanotriboelectric nanogenerator in this embodiment of the invention exhibits nearly twice the signal enhancement conversion, making this layered bio-hybrid triboelectric nanogenerator array highly sensitive. Furthermore, it was found that if the copper mesh surface lacks nanostructures, the electrical signal output amplitude of the nanotriboelectric nanogenerator will also be significantly reduced.

[0131] Comparison of microcolumn microfluidic nano-triboelectric generators with traditional nematode drug screening techniques

[0132] The micropillar microfluidic nanotriboelectric generator in this invention requires minimal external support for drug screening, has a simple overall structure, and can be easily integrated with microfluidic systems to achieve efficient mechanical sensing. It also has the potential to monitor nematode behavior on a large scale in a cost-effective manner. Currently, almost all existing platforms rely on high-precision, high-speed cameras for microscopic imaging. Camera imaging suffers from high long-term evaluation costs and low throughput, limiting the use of existing platforms. The novel in vivo screening strategy based on the micropillar microfluidic nanotriboelectric generator screening system in this invention combines a hierarchical nanotriboelectric generator with a microfluidic chip and biological components. With computational bioinformatics analysis to assist in high-throughput pharmacological evaluation, drug screening results can be obtained more efficiently, conveniently, and quickly. This allows for high-throughput microscopic screening of nematodes, significantly reducing waste and loss of human and material resources, and offering significant application advantages.

[0133] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A nematode movement detection product, characterized in that, The nematode movement detection product includes a nano-triboelectric generator and a microfluidic chip; The nano-triboelectric generator is correspondingly attached to the micropillar array in the microfluidic chip; The nano-triboelectric generator is composed of a metal inner core and an outer cladding layer; there is a gap between the metal inner core and the outer cladding layer, and the gap is 2~5μm. The metal core of the nano-triboelectric generator is woven into a mesh structure. In the mesh structure, the pore size is 50~60μm; The microfluidic chip has a cavity, and a micropillar array is disposed in the cavity, and a plurality of micropillars are disposed in the micropillar array; The micropillars have a diameter of 45-60 μm, a height of 100-150 μm, and a distance of 50-80 μm between adjacent micropillars along their edges. The micropillars are made of flexible materials and can be bent in all directions.

2. The nematode movement detection product according to claim 1, characterized in that, The method for preparing the nano-triboelectric generator includes the following steps: oxidizing the metal core to form an oxide sacrificial layer on its surface, covering the metal core with the oxide sacrificial layer with a coating material, and immersing it in acid to remove the oxide sacrificial layer, thus obtaining the generator.

3. The nematode movement detection product according to claim 1, characterized in that, The nematode movement detection product includes sensors and chips.

4. A nematode-based detection or screening system, characterized in that, The detection or screening system includes the nematode movement detection product according to any one of claims 1-3, and external auxiliary equipment.

5. The detection or screening system according to claim 4, characterized in that, The external auxiliary equipment includes a signal collection device and a detection container; the signal collection device is used to collect the electrical signals output by the nematode movement detection product.

6. The use of the nematode motility detection product according to any one of claims 1-3 in drug screening and / or nematode motility detection.

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