Flexible self-curling microelectrode array system for millimeter-scale whole-soft-bodied animals and method of manufacturing the same

Abstract

This invention discloses a flexible self-rolling microelectrode array system and its fabrication method for electrically stimulating millimeter-sized molluscs, belonging to the field of bioelectrode technology. This flexible self-rolling microelectrode array system is stretchable along the length of the millimeter-sized mollusc and is configured to self-roll around the circumferentially sized mollusc before applying electrical stimulation. Its structure, from bottom to top, includes a base layer, a rolled layer, an intermediate layer, a conductive layer, and an insulating layer. The rolled layer is made of metal, semiconductor, or elastomer, providing the stress required for self-rolling. The conductive layer includes a microelectrode array and pads. The microelectrode array includes multiple electrical stimulation sites, which enable electrical stimulation of multiple segments of the millimeter-sized mollusc. This invention's flexible self-rolling microelectrode array system has a self-rolling structure driven by the residual stress gradient of a multilayer heterogeneous thin film, enabling multimodal compatibility between electrical stimulation intervention and in-situ microscopic observation of millimeter-sized molluscs.

Description

Technical Field

[0001] This invention belongs to the field of bioelectrode technology, specifically relating to a flexible self-rolling microelectrode array system for electrically stimulating millimeter-scale fully mollusc animals and its preparation method. Background Technology

[0002] Millimeter-sized molluscs have wide applications in life sciences and biomedicine, enabling high-throughput screening and rapid toxicological evaluation of targeted drugs. Drosophila larvae, as a typical example of millimeter-sized molluscs, possess advantages such as a semi-transparent body wall and highly quantifiable behavioral patterns (e.g., chemotaxis, escape), making them ideal living vectors for exploring neural circuit development and constructing drug sensitivity screening models. However, millimeter-sized molluscs, including Drosophila larvae, completely lack rigid skeletal support, exhibiting extremely high flexibility, resulting in continuous peristaltic contractions and intense stress deformation during experiments. Existing conventional electrical stimulation methods generally face mechanical mismatch problems when dealing with such highly dynamic soft targets: probe-type invasive electrodes are prone to irreversibly damaging soft tissues, completely depriving larvae of their ability to move freely; conventional two-dimensional planar microelectrode arrays lack three-dimensional topological evolution and dynamic stress release mechanisms, making it impossible to achieve conformal fit with the larvae's near-cylindrical surface in the entire circumference, and the contact points are prone to relative slippage during the larvae's vigorous peristalsis, causing severe contact impedance fluctuations and motion artifacts. These problems severely limit the implementation of precise, non-invasive electrical stimulation interventions with high spatiotemporal resolution and long-term monitoring in millimeter-sized soft-bodied animals such as Drosophila larvae.

[0003] Currently, various electrostimulation electrodes and their supporting equipment technologies have been successfully developed. For example, Chinese patent document CN121153650A discloses an experimental platform for constructing an electrostimulation-behavior mapping relationship, which includes an electrostimulator, an electrostimulation parameter traversal module, and a turtle robot. The turtle robot receives electrostimulation through the electrostimulation electrodes of the electrostimulator, and visual markers are set on its body. This invention solves the problems of low efficiency, strong subjectivity, and difficulty in data correlation associated with manual observation in traditional research. Chinese patent document CN121129206A discloses an organic flexible implantable microelectrode array device and method for bidirectional electrophysiological detection and disease diagnosis of three-dimensional cardiomyocyte spheres. This invention constructs engineered three-dimensional cardiomyocyte spheres, establishes an integrated three-dimensional cardiomyocyte sphere electrophysiological sensing and regulation platform based on an integrated organic flexible implantable microelectrode array device, and proposes a "plug-and-play" implantable detection strategy. This overcomes the spatial detection limitations of planar electrodes and realizes multi-channel electrophysiological signal detection and electrostimulation regulation within the three-dimensional cardiomyocyte spheres. However, the aforementioned electrical stimulation electrodes and their associated equipment technologies are not suitable for millimeter-sized molluscs.

[0004] Therefore, there is an urgent need to develop a microelectrode array system for electrical stimulation of millimeter-scale molluscs to solve the problem that existing electrical stimulation technologies cannot achieve precise and non-invasive electrical stimulation intervention and long-term monitoring of millimeter-scale molluscs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a flexible self-rolling microelectrode array system for electrical stimulation of millimeter-scale soft-bodied animals. This system features a self-rolling structure driven by the residual stress gradient of multilayer heterogeneous thin films, enabling multimodal compatibility between millimeter-scale electrical stimulation intervention and in-situ microscopic observation of soft-bodied animals.

[0006] The specific technical solution adopted is as follows: The present invention provides a flexible self-rolling microelectrode array system for electrically stimulating millimeter-sized molluscs. The flexible self-rolling microelectrode array system is stretchable along the length of the millimeter-sized mollusc and is configured to self-roll around the millimeter-sized mollusc in the circumferential direction to perform electrical stimulation. The flexible self-rolling microelectrode array system structure comprises, from bottom to top, a base layer, a rolled layer, an intermediate layer, a conductive layer, and an insulating layer. The base layer material, intermediate layer material, and insulating layer material are all flexible biocompatible materials, including SU-8 series photoresist, polyimide (PI), polydimethylsiloxane (PDMS), or parylene (such as Parylene C). The rolled layer material is a metal, semiconductor, or elastomer, providing the stress required for self-rolling. The conductive layer includes a microelectrode array and pads. The microelectrode array includes multiple electrical stimulation sites, which enable electrical stimulation of multiple segments of a millimeter-scale whole mollusc animal. The pads are connected to the microelectrode array via leads. The insulating layer covers the leads of the conductive layer, exposing the microelectrode array and pads.

[0007] The design principle of the flexible self-rolling microelectrode array system is as follows: the residual stress difference between different thin film layers is used to realize the three-dimensional roll deformation of the device; when the system is released from the support surface, the roll layer releases its internal stress and bends, driving the originally planar system to spontaneously roll inward, and finally forming a three-dimensional tubular structure to wrap the millimeter-sized soft animal; at the same time, the system adopts a stretchable pattern design such as a snake mesh, which enables it to stretch along a specific direction (the length direction of the millimeter-sized soft animal) in terms of physical structure.

[0008] The working process of the flexible self-rolling microelectrode array system is as follows: First, a millimeter-sized mollusc is placed in the working area of ​​the system; then, the system is released and spontaneously rolls up, thus tightly wrapping around the surface of the millimeter-sized mollusc; during electrical stimulation, external electrical signals are transmitted through pads and leads to various electrical stimulation sites exposed at the openings of the insulating layer, thereby precisely stimulating specific segments or multiple segments of the millimeter-sized mollusc; simultaneously, since the basal layer, intermediate layer, and insulating layer are all made of flexible materials with high light transmittance, external optical microscopy equipment can directly pass through the system to perform in-situ fluorescence imaging and behavioral observation of the millimeter-sized mollusc encased inside.

[0009] The substrate layer has a serpentine mesh structure and a thickness of 0.5~1 μm. The substrate layer material is preferably SU-8 2000 series epoxy resin negative photoresist (model 2000.5).

[0010] The described curled layer has a strip array structure perpendicular to the length direction of the millimeter-sized mollusc, with a thickness of 40-80 nm. It provides the stress required for self-curling and encapsulating the millimeter-sized mollusc. The curled layer includes a silver layer and chromium layers located on both sides of the silver layer. By precisely controlling the deposition thickness and rate of the chromium layer, the self-curving radius of curvature can be stably controlled below 500 μm, thereby ensuring that the microelectrode array system can perform omnidirectional, highly conformal encapsulation of millimeter-sized molluscs such as cylindrical fruit fly larvae.

[0011] Preferably, the thickness of the silver layer in the curled layer is 30~50 nm, and the thickness of the chromium layer in contact with the intermediate layer is 5~20 nm.

[0012] The intermediate layer has a serpentine mesh structure and a thickness of 0.5~1 μm. It partially encapsulates the curled layer to prevent direct connection between the curled layer and the conductive layer. The preferred material for the intermediate layer is SU-8 2000 series epoxy resin negative photoresist (model 2000.5).

[0013] The conductive layer has a serpentine mesh structure and a thickness of 40-100 nm. The conductive layer includes a chromium layer and a gold layer disposed on the chromium layer. Preferably, the thickness of the chromium layer is 3-8 nm.

[0014] The diameter of the electrostimulation site is 80~200 μm; the distance between two adjacent electrostimulation sites is 400~1600 μm; the electrostimulation site is modified with a nanomaterial coating, the nanomaterial including platinum black, poly(3,4-ethylenedioxythiophene) or poly(3,4-ethylenedioxythiophene):polystyrene sulfonate.

[0015] The insulating layer has a serpentine mesh structure and a thickness of 0.5~1 μm. The insulating layer material is preferably SU-8 2000 series epoxy resin negative photoresist (model 2000.5).

[0016] The present invention also provides a method for fabricating the aforementioned flexible self-rolling microelectrode array system, comprising the following steps: (1) Deposit a sacrificial layer on a substrate (silicon dioxide or quartz glass); (2) Using photolithography, a first patterned photoresist is formed on a substrate with a sacrificial layer, and a base layer is formed by baking process; (3) Using photolithography, a second patterned photoresist is formed on the substrate layer, and a curled layer material (preferably resistive vacuum evaporation metal material) is deposited. The photoresist is removed by a stripping process to obtain the curled layer. (4) Using photolithography, a third patterned photoresist is formed on the device obtained in step (3), and an intermediate layer is formed by baking process; (5) Using photolithography, a fourth patterned photoresist is formed on the device obtained in step (4), and a conductive layer material (preferably a resistive vacuum evaporation metal material) is deposited. The photoresist is removed by a stripping process to obtain a conductive layer. The conductive layer includes a microelectrode array and pads. The microelectrode array includes multiple electrical stimulation sites. (6) Using photolithography, a fifth patterned photoresist is formed on the device obtained in step (5), and an insulating layer is formed by baking process; (7) Modify the nanomaterial coating (preferably by electrochemical polymerization) on the electrical stimulation sites of the conductive layer, release the microelectrode array in the copper etching solution, and obtain the flexible self-rolling microelectrode array system.

[0017] This invention also provides a millimeter-scale electrophysiological research method for fully soft animals, utilizing the aforementioned flexible self-rolling microelectrode array system.

[0018] In the existing technologies for electrophysiological research on millimeter-sized soft-bodied animals such as fruit fly larvae, traditional electrical stimulation and recording methods are mainly divided into two categories: invasive microelectrodes and planar microelectrode arrays. However, both have significant technical limitations: (1) Hard probe electrodes are invasive to millimeter-sized soft-bodied animals, causing irreversible damage to the animal's body surface, affecting its natural behavior patterns, and even requiring ex vivo dissection of the animal; (2) Whether it is a hard electrode or a flexible electrode array, there is a "mechanical mismatch" problem in electrical stimulation of millimeter-sized soft-bodied animals. The contact area between the electrode and the millimeter-sized soft-bodied animal is small, making it difficult to achieve all-round wrapping; (3) Planar flexible electrodes lack stress release structures, making it difficult to cope with the stretching and contraction during the movement of fruit fly larvae, and the electrode sites are prone to relative displacement.

[0019] This invention presents a flexible self-rolling microelectrode array system with a self-rolling structure driven by the residual stress gradient of multilayer heterogeneous thin films. By depositing a rolled layer (chromium / silver / chromium trimetallic stress layer) with specific stress mismatch characteristics on the surface of a flexible substrate, the device achieves spontaneous evolution from a two-dimensional planar structure to a three-dimensional tubular structure, enabling omnidirectional conformal encapsulation of millimeter-sized soft-bodied animals. Furthermore, the main body of this flexible self-rolling microelectrode array system adopts a serpentine stretchable mesh topology. This design utilizes the geometrical nonlinear deformation of the serpentine lines as an effective stress release unit, transforming the macroscopic tensile or contractile strain generated by the peristalsis of millimeter-sized soft-bodied animals such as fruit fly larvae into microscopic torsion within the lines. This achieves mechanical decoupling between the electrode array and soft tissue, minimizing the interference of physical constraints on the larvae's natural behavioral patterns. In addition, this open mesh structure provides ample space for physiological metabolism and gas exchange within the organism. Combined with a transparent material component with high optical transmittance, it achieves multimodal compatibility between electrical stimulation intervention and in-situ microscopic observation, improving the physiological activity and monitoring accuracy of long-term biological experiments.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The flexible self-rolling microelectrode array system of the present invention adopts a specific serpentine stretchable mesh structure design, and achieves extremely low equivalent Young's modulus and elastic recovery capability within a large strain range through geometric topology optimization. This structure can highly adapt to the surface deformation of millimeter-sized soft-bodied animals such as fruit fly larvae during vigorous movement behaviors such as peristalsis and contraction, avoiding relative slippage between the electrodes and the skin, thereby ensuring accurate spatial positioning of the electrical stimulation site during dynamic processes.

[0021] (2) The flexible self-rolling microelectrode array system of the present invention is based on a stress-driven self-rolling mechanism. By precisely controlling the residual stress between the multilayer thin films, the microelectrode array spontaneously evolves from a two-dimensional plane to a three-dimensional tubular structure. The radius of curvature of this self-rolling structure can be precisely adjusted for the physical characteristics of millimeter-sized molluscs at different ages, thereby achieving a seamless conformal integration of the microelectrode array with the cylindrical worm surface. Compared with traditional planar electrode contact, the flexible self-rolling microelectrode array system of the present invention greatly increases the effective contact area and significantly reduces the interfacial contact impedance, realizing omnidirectional electrical stimulation of millimeter-sized molluscs such as fruit fly larvae.

[0022] (3) The flexible self-rolling microelectrode array system of this invention uses a flexible biocompatible material with high optical transmittance and exhibits colorless and transparent properties across the entire wavelength range. This feature enables the flexible self-rolling microelectrode array system of this invention to be seamlessly integrated with various advanced biological observation methods such as calcium ion imaging, fluorescence microscopy, and optogenetics. Researchers can achieve real-time monitoring of millimeter-scale whole-mollusc muscle tissue and neuronal activity while applying precise electrical stimulation, providing multi-dimensional experimental data for exploring the biological regulatory mechanisms under electrical intervention.

[0023] (4) This invention integrates multiple micron-sized electrical stimulation sites on a flexible substrate using micro-nano fabrication technology. The arrangement of these electrical stimulation sites closely matches the segmental structure of millimeter-sized whole molluscs, providing extremely high spatial resolution. Based on this, this invention can not only achieve precise local stimulation within a single segment, but also flexibly configure synergistic stimulation schemes within the same segment or across segments according to experimental needs, providing a powerful tool for studying long-range conduction and system-level behavioral responses in the nervous system of millimeter-sized whole molluscs. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the structure of a flexible self-rolling microelectrode array system.

[0025] Figure 2 This is a schematic diagram of the functional partitions of a flexible self-rolling microelectrode array system.

[0026] Figure 3 This is a flowchart of the fabrication process for a flexible self-rolling microelectrode array system, where af represents different fabrication stages.

[0027] Figure 4 This is a schematic diagram of a flexible self-rolling microelectrode array system combined with a fruit fly larva.

[0028] Figure 5 This is a physical image of a flexible self-rolling microelectrode array system combined with a fruit fly larva.

[0029] Figure 6 The structure-property relationship between the curvature of the flexible self-rolling microelectrode array system and the parameters of the rolled layer is presented.

[0030] Figure 7 This is a comparison of the electrochemical performance of a flexible self-rolling microelectrode array system before and after nanomaterial modification.

[0031] Figure 8 This image shows the fluorescence imaging results of the neuromuscular junction of fruit fly larvae under different electrical stimulation voltages using a flexible self-rolling microelectrode array system.

[0032] Figure reference numerals: 1. Substrate layer, 2. Curved layer, 3. Intermediate layer, 4. Conductive layer, 5. Insulating layer, 6. Microelectrode array, 7. Lead, 8. Pad. Detailed Implementation

[0033] To make the objectives, features, and advantages of this invention more apparent and understandable, a detailed description is provided below through specific embodiments. Many specific details are set forth in the following description to provide a thorough understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the invention can be combined appropriately without mutual conflict.

[0034] Unless otherwise specified, the operating methods in the following examples are generally performed under conventional conditions or as recommended by the manufacturer. Contents not described in detail in this specification are prior art known to those skilled in the art. Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0035] Example 1 To address the technical challenge of providing stable and precise electrical stimulation to millimeter-sized molluscs such as fruit fly larvae in existing technologies, this invention provides a flexible self-rolling microelectrode array system for electrical stimulation of millimeter-sized molluscs. Utilizing this system and its unique self-rolling and stretchable structure, precise electrical stimulation intervention with high spatiotemporal resolution is achieved even when fruit fly larvae are in a free-moving state.

[0036] Figure 1 This is a schematic diagram of the flexible self-rolling microelectrode array system of the present invention. Figure 1As shown, its structure includes a base layer 1, a curled layer 2, an intermediate layer 3, a conductive layer 4, and an insulating layer 5. The base layer 1 is a biocompatible flexible material with a serpentine, stretchable mesh structure, preferably SU-8 2000 series epoxy resin negative photoresist (model 2000.5), and the thickness of the base layer is 0.5~1 μm. The curled layer 2 is formed on the base layer 1, specifically as a horizontal (perpendicular to the length direction of the millimeter-scale mollusc) strip array, providing the stress required for self-curling. The curled layer includes a silver layer and chromium layers located on both sides of the silver layer, and the thickness of the curled layer is 40~80 nm, of which the thickness of the silver layer is 30~50 nm, and the thickness of the chromium layer in contact with the intermediate layer is 5~20 nm. The intermediate layer 3 is a biocompatible flexible material with a serpentine, stretchable mesh structure, preferably SU-8 The 2000 series epoxy resin negative photoresist (model 2000.5) has an intermediate layer 3 formed on the curled layer 2 and the base layer 1, partially encapsulating the curled layer 2 to prevent direct connection between the curled layer 2 and the conductive layer 4. The thickness of the intermediate layer is 0.5~1 μm. The conductive layer 4 is formed on the intermediate layer 3 and includes a microelectrode array 6, leads 7, and pads 8. The microelectrode array 6 includes multiple electrical stimulation sites, which are longitudinally staggered and span multiple segments of the fruit fly larva. The pads 8 are connected to the microelectrode array 6 via leads 7. The conductive layer 4 consists of a chromium layer and a gold layer disposed on the chromium layer, with a thickness of 40~100 nm, of which the thickness of the chromium layer is 3~8 nm. The insulating layer 5 is a biocompatible flexible material with a serpentine stretchable mesh structure, preferably SU-8. 2000 series epoxy resin negative photoresist (model 2000.5); insulating layer 5 is formed on conductive layer 4; insulating layer 5 covers lead 7 and exposes microelectrode array 6 and pad 8; the thickness of insulating layer is 0.5~1 μm.

[0037] Specifically, the diameter of the electrical stimulation site is 80–200 μm, and the distance between two adjacent electrical stimulation sites is 400–1600 μm. Electrical stimulation can be performed between segments of the same body and between segments of the Drosophila larva. Furthermore, the diameter of the electrical stimulation site and the distance between two adjacent electrical stimulation sites can be adjusted according to the location of the electrical stimulation.

[0038] Preferably, the electrostimulation sites are modified with nanoparticles. The nanoparticle materials include platinum black, poly(3,4-ethylenedioxythiophene), and poly(3,4-ethylenedioxythiophene):polystyrene sulfonate, etc. In electrostimulation detection, the nanoparticles reduce interfacial impedance and phase shift, increase charge injection capacity, and improve mechanical compliance at the interface with the fruit fly larvae.

[0039] Figure 2This is a functional partition diagram of the flexible self-coiling microelectrode array system. Along the length of the millimeter-scale soft-bodied animal, it can be divided into a fixed zone, a stretching zone, a larval extension zone, a coiling zone, an electrical stimulation electrode zone, and a device flipping zone. These multiple functional partitions work together to integrate the flexible self-coiling microelectrode array system with the fruit fly larva and facilitate subsequent electrical stimulation experiments.

[0040] Figure 3 The fabrication process flow diagram for the flexible self-rolling microelectrode array system includes the following steps: Step 1: A 5nm / 100nm thick chromium / copper metal film is deposited on the surface of a silicon dioxide wafer after surface cleaning using resistive vacuum evaporation deposition to form a sacrificial layer, such as... Figure 3 As shown in Figure a.

[0041] Step 2: Spin-coat SU-8 adhesion promoter onto the surface of the oxygen plasma-activated sacrificial layer. After baking at 95 ℃, spin-coat SU-8 2000.5 photoresist with a thickness of 1000 nm. After pre-baking at 95 ℃ for 3 min, exposure with a mask, post-baking at 95 ℃ for 3 min, development, and curing at 180 ℃ for 20 min, a base layer is formed. Figure 3 As shown in b.

[0042] Step 3: Spin-coat AZ5214 photoresist to a thickness of 2 μm on the oxygen plasma-activated substrate surface. After pre-baking at 120℃ for 3 min, exposure with a mask, and development, a curled layer inversion pattern is formed. A 1.5 nm / 40 nm / 20 nm thick chromium / silver / chromium metal film is deposited using resistive vacuum evaporation deposition. The photoresist is then removed using a lift-off process to form the curled layer, as shown below. Figure 3 As shown in c.

[0043] Step 4: Spin-coat SU-8 2000.5 photoresist to a thickness of 500 nm onto the surface of the oxygen plasma-activated rolled layer. After pre-baking at 95 °C for 3 min, exposure using a mask, post-baking at 95 °C for 3 min, development, and curing at 180 °C for 20 min, a flexible intermediate layer is formed. Figure 3 As shown in d.

[0044] Step 5: Spin-coat AZ5214 photoresist to a thickness of 2 μm onto the surface of the oxygen plasma-activated flexible intermediate layer. After pre-baking at 120 °C for 3 min, exposure using a mask, and development, a conductive layer structure is formed. A 5 nm / 80 nm thick chromium / gold metal layer is then deposited using resistive vacuum evaporation deposition. The photoresist is removed via a lift-off process to form the conductive layer, which consists of pads, a serpentine mesh of leads, and a microelectrode array. Figure 3 As shown in e.

[0045] Step 6: Spin-coat SU-8 2000.5 photoresist with a thickness of 500nm onto the surface of the conductive layer activated by oxygen plasma. After pre-baking at 95℃ for 3 min, exposure with a mask, post-baking at 95℃ for 3 min, development, and curing at 180℃ for 20 min, an insulating layer is formed.

[0046] Step 7: Connect the device obtained in Step 6 to the electrochemical workstation and modify the nanomaterials on the electrostimulation site using an electrochemical polymerization method. The specific process is as follows: Prepare a 14 mM 1x phosphate-buffered saline (PBS) precursor solution of 3,4-ethylenedioxythiophene (EDOT) and drop it onto the surface of the device; perform electrochemical deposition using a standard three-electrode system, with a platinum wire as the counter electrode, an Ag / AgCl electrode as the reference electrode, and the device as the working electrode; perform electrochemical polymerization using chronoamperometry, setting the parameters of the electrochemical workstation as follows: after a 2-second rest period, step the working potential positively to a constant 1.4 V and continue the reaction for 180 seconds, thereby polymerizing and generating a poly(3,4-ethylenedioxythiophene) (PEDOT) nanocoating on the surface of the electrostimulation site.

[0047] This flexible self-rolling microelectrode array system is stretchable along the length of a millimeter-sized mollusc and is configured to self-roll around the millimeter-sized mollusc before applying electrical stimulation. Figure 4 This is a schematic diagram illustrating the integration of a flexible, self-rolling microelectrode array system with a fruit fly larva. When released from the support surface, this flexible, self-rolling microelectrode array system can bind to the fruit fly larvae and modulate their behavior through electrical stimulation.

[0048] Figure 5 The image shows a real-world photograph of a flexible self-curling microelectrode array system combined with a fruit fly larva. It can be seen that when the system is released from the support surface, the curling layer releases its internal stress and bends, driving the originally planar system to spontaneously curl inward, ultimately forming a three-dimensional tubular structure that wraps around the fruit fly larva. The fruit fly larva and the biocompatible flexible material SU8 are well bonded together, forming a tight wrapping.

[0049] Figure 6This paper presents the relationship between the curl curvature and the parameters of the curled layer in a flexible self-curving microelectrode array system. Experimental results show that the self-curving driving force of the flexible self-curving microelectrode array system is positively correlated with the thickness of the outermost chromium (Cr) layer in the curled layer. The thickness of the first chromium layer is 1.5 nm, and the thickness of the second silver layer is 40 nm. Increasing the thickness of the outermost chromium layer (the chromium layer in contact with the middle layer) within a preset range increases the residual stress gradient between the three metal layers, thereby enhancing the device's curl deformation capability. Through process optimization, the curl curvature radius of this flexible microelectrode array can be precisely controlled to below 500 μm. Since the average diameter of third-instar fruit fly larvae is typically between 0.5 mm and 1 mm, the curl curvature achieved in this invention ensures that the electrode array forms a tight circumferential wrap and highly conformal contact with the fruit fly larvae's body surface.

[0050] Figure 7 This study compares the electrochemical performance of the electrostimulation sites in a flexible, self-rolling microelectrode array system before and after modification with nanomaterial coatings. Electrochemical impedance spectroscopy (EIS) results show that by electrodepositing a poly(3,4-ethylenedioxythiophene) (PEDOT) nanoparticle modification layer on the surface of the electrostimulation sites, the interfacial impedance of the electrodes at the characteristic frequency of 1 kHz bioelectrophysiological signals significantly decreased from an initial 899 kΩ to 130 kΩ. This dramatic decrease in impedance effectively reduces energy loss and thermal interference during electrostimulation, improving charge injection efficiency while ensuring signal stability and biointerfacial safety of the microelectrode array in long-duration, high-frequency electrostimulation tasks.

[0051] Figure 8 This study demonstrates the electrophysiological results of simultaneous fluorescence imaging of the neuromuscular junction (NMJ) in Drosophila larvae using a flexible self-coiling microelectrode array system under different stimulation voltage conditions. The experiment used a square wave electrical stimulation lasting 1 s as the stimulation signal, triggered at t=5 s. Observations revealed that when the applied stimulation voltage crossed a critical threshold (approximately 4 V between adjacent stimulation sites in this embodiment), the fluorescence intensity at the neuromuscular junction of the Drosophila larvae significantly increased. This phenomenon quantitatively verifies that the multi-electrode array provided by this invention can effectively couple bioelectrical signals and achieve precise regulation of the neuromuscular conduction pathway across tissue impedance. Furthermore, these experimental results clarify the effective operating voltage range of the flexible self-coiling microelectrode array system in in vivo applications, providing crucial parameter basis for subsequent high spatiotemporal resolution precision electro-intervention and drug screening models.

[0052] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A flexible, self-rolling microelectrode array system for electrically stimulating millimeter-scale fully mollusc animals, characterized in that, The flexible self-rolling microelectrode array system is stretchable along the length of a millimeter-sized mollusc and is configured to self-roll around the millimeter-sized mollusc before applying electrical stimulation. The flexible self-rolling microelectrode array system structure comprises, from bottom to top, a base layer, a rolled layer, an intermediate layer, a conductive layer, and an insulating layer. The base layer material, intermediate layer material, and insulating layer material are all flexible biocompatible materials, including SU-8 series photoresist, polyimide, polydimethylsiloxane, or parylene. The rolled layer material is a metal, semiconductor, or elastomer, providing the stress required for self-rolling. The conductive layer includes a microelectrode array and pads. The microelectrode array includes multiple electrical stimulation sites, which enable electrical stimulation of multiple segments of a millimeter-scale whole mollusc animal. The pads are connected to the microelectrode array via leads. The insulating layer covers the leads of the conductive layer, exposing the microelectrode array and pads.

2. The flexible, self-scrolling microelectrode array system of claim 1, wherein, The substrate layer has a serpentine mesh structure and a thickness of 0.5~1 μm.

3. The flexible, self-scrolling microelectrode array system of claim 1, wherein, The curled layer has a strip array structure perpendicular to the length direction of the millimeter-scale whole mollusc, with a thickness of 40~80 nm. The curled layer includes a silver layer and chromium layers located on both sides of the silver layer.

4. The flexible, self-scrolling microelectrode array system of claim 1, wherein, The intermediate layer has a serpentine mesh structure and a thickness of 0.5~1 μm, which is used to prevent the curled layer from being directly connected to the conductive layer.

5. The flexible, self-scrolling microelectrode array system of claim 1, wherein, The conductive layer has a serpentine mesh structure and a thickness of 40~100 nm. The conductive layer includes a chromium layer and a gold layer disposed on the chromium layer.

6. The flexible, self-scrolling microelectrode array system of claim 1, wherein, The diameter of the electrostimulation site is 80~200 μm; the distance between two adjacent electrostimulation sites is 400~1600 μm; the electrostimulation site is modified with a nanomaterial coating, the nanomaterial including platinum black, poly(3,4-ethylenedioxythiophene) or poly(3,4-ethylenedioxythiophene):polystyrene sulfonate.

7. The flexible self-rolling microelectrode array system according to claim 1, characterized in that, The insulating layer has a serpentine mesh structure and a thickness of 0.5~1 μm.

8. The method for fabricating a flexible self-rolling microelectrode array system according to any one of claims 1-7, characterized in that, Includes the following steps: (1) Deposit a sacrificial layer on the substrate; (2) Using photolithography, a first patterned photoresist is formed on a substrate with a sacrificial layer, and a base layer is formed by baking process; (3) Using photolithography, a second patterned photoresist is formed on the substrate layer, and a curled layer material is deposited. The photoresist is removed by a stripping process to obtain the curled layer. (4) Using photolithography, a third patterned photoresist is formed on the device obtained in step (3), and an intermediate layer is formed by baking process; (5) Using photolithography, a fourth patterned photoresist is formed on the device obtained in step (4), a conductive layer material is deposited, and the photoresist is removed by a stripping process to obtain a conductive layer; the conductive layer includes a microelectrode array and pads, and the microelectrode array includes multiple electrical stimulation sites; (6) Using photolithography, a fifth patterned photoresist is formed on the device obtained in step (5), and an insulating layer is formed by baking process; (7) Modify the nanomaterial coating on the electrical stimulation sites of the conductive layer to obtain the flexible self-rolling microelectrode array system.

9. A millimeter-scale electrophysiological research method for whole molluscs animals, characterized in that, The flexible self-rolling microelectrode array system according to any one of claims 1-7 is utilized.

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