Micro-corrugated conductive hydrogel based enzyme electrode and manufacturing method thereor and application thereof

Abstract

The present invention relates to a micro-wrinkle conductive hydrogel-based enzyme electrode, a method for manufacturing the same, and an application thereof, comprising: a micro-wrinkle conductive hydrogel having an organic molecule having an amine group infiltrated therein and a metal nanofilm formed on its surface; a conductive host layer having a first stacked structure formed on the conductive hydrogel, wherein a first assembly comprising an organic linker having an amine group and a first metal nanoparticle is repeated; and an enzyme layer having a second stacked structure formed on the conductive host layer, wherein a second assembly comprising an enzyme and a second metal nanoparticle is repeated.

Description

Technology Field

[0001] The present invention relates to a micro-wrinkle conductive hydrogel-based enzyme electrode, a method for manufacturing the same, and an application thereof. Background Technology

[0003] The rapid growth of various portable and medical electronic devices, including microscale and implantable systems, has increased the demand for energy sources, further highlighting the need for renewable and sustainable energy solutions. Among emerging candidates, biocompatible enzyme-based biofuel cells (EBFCs) equipped with enzyme electrodes are receiving significant attention due to their eco-friendly and renewable characteristics. In particular, EBFCs are establishing themselves as an attractive power source for implantable medical devices because they can efficiently generate electricity from biofuels such as glucose and oxygen under physiological conditions. However, despite these advantages, the practical application of EBFCs remains limited due to inherently low output and insufficient operational stability.

[0004] To address these challenges, various strategies have been explored, primarily aiming to increase output by enhancing electron transfer at host electrode / enzyme and enzyme / enzyme interfaces, and to improve operational stability by optimizing enzyme immobilization. Furthermore, significant progress has been made in the development of soft, biocompatible host electrodes with high electrical conductivity to replace conventional carbon-based electrodes, which often exhibit lower conductivity compared to bulk metal-based materials. Achieving high electrical conductivity in the host electrode is essential for efficient electron transfer between the electrode and the enzyme.

[0005] Widely adopted strategies to enhance electron transfer include incorporating conductive fillers, such as carbon nanotubes (CNTs) and metal nanoparticles, into the enzyme layer. For example, glucose oxidase (GOx), commonly used in EBFC anodes, has its redox active site (flavin adenine dinucleotide, FAD) buried 7–15 Å beneath the insulating protein surface, which restricts electron transfer to the host electrode and between adjacent GOx molecules. To address these limitations, integrated conductive fillers act as electron relays to facilitate electron transfer and improve overall power output. Nevertheless, even with the use of such fillers, the achieved power density is often insufficient. Mediated electron transfer (MET)-based EBFCs utilizing electron-hopping redox mediators achieve 1–2 mW cm⁻¹. -2 Although it can provide higher power output in the range, it has significant drawbacks, including biological toxicity, operational instability, complex media synthesis, and a low open-circuit voltage (OCV) of less than 0.80 V. In several recent studies, media-free EBFCs have outputs of several mW / cm² -2 Interest in the development of media-free systems is growing as it is reported that unprecedented power output has been achieved.

[0006] In addition to enhancing the output of electronic relay-based EBFCs, improving enzyme immobilization remains a critical challenge. While various immobilization strategies such as weak adsorption (via van der Waals forces and hydrogen bonding), covalent bonding, crosslinking, and physical capture have been studied, these strategies are still prone to enzyme elution. This is particularly because interfacial interactions at the electrode / enzyme and enzyme / enzyme interfaces are often overlooked when relying on slurry casting. Therefore, devising a strategy that simultaneously promotes efficient electron transfer for high output and ensures stable enzyme immobilization is a key task. Stable enzyme immobilization not only prevents elution but also improves electron transfer efficiency by maintaining close contact between the enzyme and adjacent conductive components.

[0007] Furthermore, the selection of the host electrode is also important in the design of biocompatible and flexible EBFCs. Among various candidate materials, hydrogels are widely known as materials that can generally be safely used in biological systems due to their excellent biocompatibility. To convert these intrinsically insulating hydrogels into electrically conductive hydrogels, various conductive fillers, such as carbon nanotubes (CNTs), metallic components, and conductive polymers, are mixed into the hydrogel matrix through solution blending; this is similar to the strategy used to enhance electron transfer effects in enzyme-based systems. For example, mixing micrometer-sized silver (Ag) flakes into the hydrogel matrix can yield up to 7.3 × 10⁻⁶ in a dry state. 2 S cm -1 Hydrogel composites with high electrical conductivity reaching [value] can be obtained. However, most conductive hydrogels still exhibit relatively low conductivity, generally around 10 -5 ~ 10 1 S cm -1This is the range. These limitations are mainly due to high contact resistance between adjacent conductive fillers or the inherent low conductivity of certain fillers, such as carbon-based materials, and this remains the case even when used in high content. Therefore, very high electrical conductivity (10 4 S cm -1 Obtaining an electrically stable hydrogel host electrode having (excess) remains an important challenge, and is an even more important challenge when the conductive component is not mixed through simple solution blending or slurry casting, but is directly deposited on the surface of a wet insulating hydrogel through mutually complementary interfacial interactions.

[0008] Although various techniques such as printing, hot pressing, and chemical reduction have been used to enhance the electrical conductivity of hydrogel-based host electrodes, these methods often result in insufficient adhesion because they overlook critical interfacial interactions between the conductive composition and the wet hydrogel surface. This weak interfacial bonding can significantly increase contact resistance during mechanical deformation, thereby degrading the overall performance of the electrode. More importantly, these approaches fail to stably immobilize enzymes on the conductive hydrogel surface, which is a critical requirement for EBFC operation. To date, no studies have reported the successful application of these strategies to EBFC electrodes using hydrogel substrates, demonstrating that this remains a major unresolved challenge in the development of high-performance hydrogel-based bioelectrodes. The problem to be solved

[0010] The present invention aims to provide a micro-wrinkle conductive hydrogel-based enzyme electrode, etc., comprising: a micro-wrinkle conductive hydrogel in which an organic molecule having an amine group is infiltrated and a metal nanofilm is formed on the surface; a conductive host layer having a first stacked structure formed on the conductive hydrogel, wherein a first assembly comprising an organic linker having an amine group and a first metal nanoparticle is repeated; and an enzyme layer having a second stacked structure formed on the conductive host layer, wherein a second assembly comprising an enzyme and a second metal nanoparticle is repeated.

[0011] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0013] The present invention provides a micro-wrinkle conductive hydrogel-based enzyme electrode comprising: a micro-wrinkle conductive hydrogel in which an organic molecule having an amine group is infiltrated and a metal nanofilm is formed on the surface; a conductive host layer having a first stacked structure formed on the conductive hydrogel, wherein a first assembly comprising an organic linker having an amine group and a first metal nanoparticle is repeated; and an enzyme layer having a second stacked structure formed on the conductive host layer, wherein a second assembly comprising an enzyme and a second metal nanoparticle is repeated.

[0014] The organic molecule having the above amine group may be one or more selected from the group consisting of polyethyleneimine, tris(2-aminoethyl)amine, and diethylenetriamine.

[0015] The first or second metal nanoparticles may include one or more selected from the group consisting of gold, platinum, nickel, copper, aluminum, gallium, and indium.

[0016] The organic linker having the above amine group may be one or more selected from the group consisting of hydrazine, ethylenediamine, diethylenetriamine, tris(2-aminoethyl)amine, and cysteamine.

[0017] The above enzyme may include one or more selected from the group consisting of glucose oxidase (GOx), catalase (CAT), hemoglobin, and ferritin.

[0018] The number of repetitions of the first or second assembly may be 1 to 20 times.

[0019] In one embodiment of the present invention, a method for manufacturing a micro-wrinkle conductive hydrogel-based enzyme electrode is provided, comprising: (a) a step of manufacturing a micro-wrinkle conductive hydrogel by infiltrating an organic molecule having an amine group into a hydrogel and then forming a metal nanofilm on the surface thereof; (b) a step of manufacturing a conductive host layer by forming a first stacked structure in which a first assembly comprising an organic linker having an amine group and a first metal nanoparticle is repeated on the conductive hydrogel; and (c) a step of manufacturing an enzyme layer by forming a second stacked structure in which a second assembly comprising an enzyme and a second metal nanoparticle is repeated on the conductive host layer.

[0020] In another embodiment of the present invention, a biofuel cell comprising the enzyme electrode is provided.

[0021] In another embodiment of the present invention, a biosensor comprising the enzyme electrode is provided. Effects of the invention

[0023] The present invention is characterized by comprising: a micro-wrinkle conductive hydrogel-based enzyme electrode comprising: a micro-wrinkle conductive hydrogel in which an organic molecule having an amine group is infiltrated and a metal nanofilm is formed on the surface; a conductive host layer having a first stacked structure formed on the conductive hydrogel, wherein a first assembly comprising an organic linker having an amine group and a first metal nanoparticle is repeated; and an enzyme layer having a second stacked structure formed on the conductive host layer, wherein a second assembly comprising an enzyme and a second metal nanoparticle is repeated. Accordingly, the enzyme electrode according to the present invention has the advantages of excellent mechanical flexibility, enhanced output, and excellent long-term operational stability.

[0024] Therefore, the present invention can be widely utilized in biofuel cells, biosensors, etc., that require a high surface area, mechanical durability, and long-term operational stability. Brief explanation of the drawing

[0026] FIG. 1 is a schematic diagram of a method for preparing an anode (i.e., 5-GOx-5-MTAH) and a cathode (i.e., Pt-5-MTAH) as enzyme electrodes using a combination of a micro-wrinkle hydrogel and a conductive host layer (i.e., n-MTAH) according to one embodiment of the present invention. Figure 2 shows a schematic diagram of a metal TOA-gold nanoparticle hydrogel (i.e., n-MTAH) using capillary force and ligand exchange-induced metal nanoparticle assembly. Figure 3 shows the characterization of the TOA-gold nanoparticle hydrogel (i.e., TAH). a) Fabrication of the TAH. b) Gradual formation of a porous Au nanoparticle film as the thickness increases from 67 nm (12 hours) to 149 nm (72 hours). c) FE-SEM image of the TAH electrode showing a fine wrinkle surface morphology. d) Capillary-assisted in-situ assembly of the sintered gold thin film on the hydrogel and PEI-driven formation. Figure 4 illustrates the characterization of the TAH. a) Changes in sheet resistance and electrical conductivity over loading time. b) Comparison of electrical conductivity and elasticity between the TAH according to the present invention and a conventional hydrogel-based electrode. c) Change in resistance (R / R0) in the TAH according to bending (blue) and stretching (red) radii (R). d) Change in resistance (R / R0) of the TAH under bending (blue) and stretching (red) for 10,000 cycles at 100% tensile strain, and a photograph (inset). Figure 5 shows a comparison of Au-based hydrogels, such as TAH and citrate Au nanoparticle hydrogel. a) Photograph (left) and planar FE-SEM image of TAH. b) Photograph (left) and planar FE-SEM image of citrate Au nanoparticle hydrogel. c) CV curve of Au-based hydrogel in PBS solution. d) Nyquist plot of Au-based hydrogel in PBS solution. The inset shows an enlarged Nyquist plot of TAH, highlighting significantly lower impedance compared to citrate Au nanoparticle hydrogel. e) Long-term stability of each Au-based hydrogel in PBS solution over 30 days. TAH reached 72% of the initial current density (I0 = 3.0 mA cm⁻¹). -2 , I = 2.1 mA cm -2 It demonstrated excellent long-term stability by maintaining ). In contrast, the citrate Au nanoparticle hydrogel maintained only 18% (I0 = 5.2 × 10⁻⁶). -4 mA cm -2 , I = 9.4 × 10 -5 mA cm -2 ). Figure 6 shows the electrochemical performance of m-GOx-5-MTAH. a) Schematic diagram of 5-GOx-5-MTAH. b) 300 mmol L at 36.5°C. -1 5 mV s in a PBS solution containing glucose -1Cathode current density of m-GOx-5-MTAH as a function of the number of bilayers (m) at the scan rate. c) Nyquist plot of m-GOx-5-MTAH as a function of the number of bilayers. d) As further confirmed by the normalized cathode current density at +0.6 V shown in the inset, glucose concentration (0-300 mmol L⁻¹) -1 Cathode current density of 5-GOx-5-MTAH as ) increases. e) Normalized cathode current density of m-GOx-5-MTAH as a function of the number of double layers. Figure 7 shows the performance of the hydrogel-based EBFC. a) Schematic diagram of the EBFC system. b) 300 mmol L at 36.5°C -1 Power output of a hydrogel-based EBFC in a glucose-containing PBS solution. c) 10 mmol L at 36.5°C -1 Power output of a hydrogel-based EBFC in a glucose-containing PBS solution. d) 300 mmol L at 36.5°C -1 and 10 mmol L -1 Relative power retention rate (P / P0) of hydrogel-based EBFCs in a glucose-containing PBS solution. e) 300 mmol L at 36.5℃ -1 and 10 mmol L -1 Relative OCV retention rate (OCV / OCV0) of hydrogel-based EBFC in a glucose-containing PBS solution. Specific details for implementing the invention

[0027] Biocompatible enzyme-based biofuel cells (EBFCs) equipped with enzyme electrodes demonstrate great potential as power sources for wearable and implantable biomedical devices. However, their practical application has been limited due to low electron transfer efficiency and insufficient operational stability. The present invention aims to provide a mediator-free hydrogel-based EBFC that addresses these challenges by integrating capillary force-assisted assembly and hydrophobic metal nanoparticle deposition. The resulting micro-wrinkled, highly conductive hydrogel electrode provides both enhanced output and long-term stability. To achieve this objective, a hydrogel composed of polyethyleneimine functionalized with amine (-NH2) groups was immersed in a solution of Au nanoparticles (NPs) stabilized with tetraoctylammonium (TOA). This process induces ligand exchange reactions and room-temperature nanoparticle fusion at the interface between the NH2-functionalized hydrogel and the hydrophobic Au NPs, and converts the initial insulating hydrogel into a highly conductive hydrogel with a micro-wrinkled structure through self-assembly by capillary forces. Subsequently, glucose oxidase (GOx) and TOA-gold nanoparticles were deposited on a conductive micro-wrinkle hydrogel to form the anode, and a platinum (Pt) modified hydrogel was used as the cathode. This EBFC exhibited ~3.7 mW cm⁻¹. -2 It achieved high output and maintained ~80% of the initial output even after 30 days of continuous operation, demonstrating significant progress for hydrogel-based EBFCs.

[0029] In other words, the present invention provides a micro-wrinkle hydrogel electrode for a high-performance EBFC, which provides high output and long-term operational stability by in-situ assembling metal NPs onto the wet hydrogel surface and enzyme layer through capillary forces and interfacial interactions (Fig. 1). By combining these strategies, it is possible to form a hydrogel host electrode with electrical conductivity similar to bulk metal, a micro-wrinkle structure, robust mechanical strength, and strong enzyme immobilization through a simple and scalable immersion deposition method. Importantly, the enzyme immobilization used in the present system is based not on weak physical adsorption, but on strong interfacial interactions between the hydrogel surface and the amino acid functional groups of the enzyme, specifically, covalent bonding, thereby ensuring the stability and activity of the immobilized enzyme.

[0030] To fabricate a highly conductive hydrogel, a polyacrylic acid-polyacrylamide hydrogel containing polyethyleneimine (PEI) was first synthesized and immersed in a toluene solution containing Au NPs (TOA-gold nanoparticles) stabilized with tetraoctylammonium (TOA). Through this process, vertically and horizontally dense arrays of gold nanoparticles were formed on the hydrogel surface, which was facilitated by continuous capillary transport and ligand exchange reactions (LER) between the NH2-functionalized PEI within the aqueous hydrogel and TOA ligands weakly bound to Au NPs dispersed in a non-polar solvent. This interfacial engineering promoted the room-temperature sintering of the Au NPs to form interconnected Au nanoplates, transforming the initial insulating hydrogel into a highly conductive host electrode. The resulting electrode had a conductivity of approximately 8.4 × 10⁻⁶ 4 S cm -1 It achieved electrical conductivity that surpassed all previously reported hydrogel electrodes, including ion-embedded hydrogels, hydrogels combined with conductive polymers, hydrogels combined with conductive fillers, and hydrogels coated with conductive materials.

[0031] For the EBFC anode, GOx was assembled in a layer-by-layer (LbL) configuration with TOA-gold nanoparticles on a conductive hydrogel through the same LER mechanism in which NH2 groups on the enzyme's amino acid residues interact with TOA ligands. For the cathode, the device structure was completed by depositing Pt, which has excellent oxygen reduction (ORR) activity, onto the conductive hydrogel.

[0032] As a result, the hybrid EBFC composed of a covalently enzyme-linked hydrogel anode and a Pt-modified hydrogel cathode had 300 mmol L -1 Maximum surface power density ~3.7 mW cm⁻² at a glucose concentration of 0.95 V OCV -2 It showed. Physiological glucose conditions (10 mmol L -1 Even in ), the EBFC has an open-circuit voltage (OCV) of approximately 0.92 V and ~2.4 mW cm⁻¹ -2 It maintained an impressive in-plane power density. In addition, even after 30 days of continuous operation, this device retained approximately 80% of its initial output (1.9 mW cm⁻¹). -2 Excellent operational durability was confirmed by maintaining (corresponding to ). The present invention is expected to establish a promising design platform for hydrogel-based EBFCs that provides efficient electron transfer and strong enzyme retention, as well as long-term reliability essential for future bioelectronic and biomedical applications.

[0034] The present invention will be described in detail below.

[0036] Micro-wrinkle conductive hydrogel-based enzyme electrode

[0038] The present invention provides a micro-wrinkle conductive hydrogel-based enzyme electrode comprising: a micro-wrinkle conductive hydrogel in which an organic molecule having an amine group is infiltrated and a metal nanofilm is formed on the surface; a conductive host layer having a first stacked structure formed on the conductive hydrogel, wherein a first assembly comprising an organic linker having an amine group and a first metal nanoparticle is repeated; and an enzyme layer having a second stacked structure formed on the conductive host layer, wherein a second assembly comprising an enzyme and a second metal nanoparticle is repeated.

[0040] First, the micro-wrinkle conductive hydrogel-based enzyme electrode according to the present invention comprises a micro-wrinkle conductive hydrogel in which an organic molecule having an amine group is infiltrated and a metal nanofilm is formed on the surface.

[0041] The above hydrogel may include a polymer containing carboxyl groups. In this case, the carboxyl groups may act as additional crosslinking agents by performing mixed interactions (i.e., hydrogen bonding and electrostatic interactions) with amine groups present in the organic molecule having amine groups. Therefore, there is an advantage in that the mechanical strength of the hydrogel can be improved.

[0042] Specifically, the polymer may be an acrylic acid and acrylamide-based copolymer; an acrylic acid and vinyl alcohol-based copolymer; an acrylic acid homopolymer; or an acrylic acid and acrylonitrile-based copolymer; it is preferably an acrylic acid and acrylamide-based copolymer, and more preferably an acrylic acid and acrylamide-based copolymer (molar ratio 7:3 to 9:1), but is not limited thereto. In this case, the carboxyl group present in the acrylic acid and / or the amine group present in the acrylamide interact with the amine group present in the organic molecule having the amine group, thereby inducing the formation of a polymer network structure with high crosslinking density and increasing the swelling rate of the hydrogel.

[0043] In addition, organic molecules having the amine group are infiltrated into the hydrogel, and as described above, they may interact with functional groups present in the polymer and may be exchanged as ligands for metal nanoparticles described later through capillary transport phenomena.

[0044] The organic molecule having the amine group may be an organic molecule having two or more amine groups, and the weight-average molecular weight of the organic molecule having the amine group is 100 g mol -1 to 5,000 g mol -1 It can be, and 100 g mol -1 Up to 1,000 g mol -1 It is preferable, but not limited thereto. Specifically, the organic molecule having the amine group is preferably one or more selected from the group consisting of polyethyleneimine, tris(2-aminoethyl)amine, and diethylenetriamine, and is more preferably polyethyleneimine, but is not limited thereto.

[0045] In particular, the polyethyleneimine is an organic polymer having numerous amine groups and is characterized by forming a stable network in the horizontal direction within the hydrogel. Accordingly, by uniformly forming the metal nanomembrane described later, it has the advantage of optimizing electrical conductivity and mechanical strength / durability. At this time, the weight-average molecular weight of the polyethyleneimine is 500 g mol -1 to 5,000 g mol -1 It can be, 500 g mol -1 Up to 1,000 g mol -1 It is preferable, but not limited thereto. Meanwhile, when an organic monomer such as tris(2-aminoethyl)amine or diethylenetriamine is applied instead of the polyethyleneimine, such organic monomers penetrate randomly into the hydrogel in various directions. Accordingly, there is a limitation in that the metal nanomembrane described later cannot be uniformly formed.

[0047] Meanwhile, a metal nanomembrane is formed on the surface of the infiltrated hydrogel, and the metal nanomembrane may be a porous nanoplate (nanomembrane) film.

[0048] In order to form the metal nanofilm, it is necessary to deposit a metal nanoparticle-organic ligand (native ligand) on the surface of the infiltrated hydrogel, and a continuous ligand exchange and sintering process is performed as the organic molecule having the amine group causes capillary transport.

[0049] That is, the metal nanomembrane refers to the state after the ligand exchange process, in which the organic ligand that was bound to the metal nanoparticle is partially or completely removed, and the organic molecule having an amine group is bound. At this time, various hydrophobic ligands may be used as the organic ligand, and it is preferable that it be tetraoctylammonium (TOA), but is not limited thereto. The organic molecule having an amine group is a hydrophilic ligand and has superior binding affinity with the metal nanoparticle compared to the organic ligand. Meanwhile, the metal nanomembrane can be bound to the organic linker located in the bottommost layer among the organic linkers having an amine group described later.

[0050] Specifically, the metal nanomembrane may comprise one or more selected from the group consisting of gold, platinum, nickel, copper, aluminum, gallium, and indium, and is preferably gold, but is not limited thereto.

[0052] Meanwhile, the infiltrated hydrogel may have a fine wrinkle structure depending on the repetition of the swelling and deswelling processes.

[0053] In addition, as the time for depositing the metal nanoparticle-organic ligand on the surface of the infiltrated hydrogel increases, the thickness of the metal nanofilm increases, and the thickness is preferably 10 nm to 300 nm, and more preferably 50 nm to 150 nm, but is not limited thereto.

[0055] Next, the micro-wrinkle conductive hydrogel-based enzyme electrode according to the present invention comprises a conductive host layer having a first stacked structure formed on the conductive hydrogel, wherein a first assembly comprising an organic linker having an amine group and a first metal nanoparticle is repeated.

[0056] To further improve the electrical conductivity of the above conductive hydrogel, a first stacked structure can be formed by LbL assembling a first assembly comprising an organic linker having an amine group and a first metal nanoparticle (i.e., a first assembly comprising a first metal nanoparticle through an organic linker having an amine group).

[0057] The organic linker located in the bottom layer among the organic linkers having amine groups can be combined with the first metal nanoparticle through a ligand exchange process while in a state combined with the metal nanomembrane. The organic linker not located in the bottom layer among the organic linkers having amine groups can be combined with the first metal nanoparticles through a ligand exchange process and act as a linker.

[0058] Specifically, the organic linker having an amine group is preferably a monomeric linker containing an amine group to perform the role of a linker. This has the advantage of narrowing the spacing between the first metal nanoparticles to strengthen the charge transfer pathway. In particular, the organic linker having an amine group may have a smaller molecular weight compared to the aforementioned organic molecule having an amine group in order to minimize contact resistance. The weight-average molecular weight of the organic linker having an amine group is 30 g mol -1 Up to 500 g mol -1It may be less than 30 g mol -1 Up to 300 g mol -1 It is preferable, but not limited thereto. More specifically, the organic linker having an amine group may be one or more selected from the group consisting of hydrazine, ethylenediamine, diethylenetriamine, tris(2-aminoethyl)amine, and cysteamine. Due to the presence of such an amine group-containing organic linker, a polymer binder is not required, so it may be in a binder-free form.

[0059] Meanwhile, the first metal nanoparticles refer to the state after the ligand exchange process, in which the organic ligand that was bound to the first metal nanoparticles is partially or completely removed, and the linker having an amine group is bound. At this time, various hydrophobic ligands may be used as the organic ligand, and it is preferable that it be tetraoctylammonium (TOA), but is not limited thereto. The linker having an amine group is a hydrophilic ligand and has superior binding affinity with the first metal nanoparticles compared to the organic ligand. Meanwhile, the particle located in the uppermost layer among the first metal nanoparticles may bind to the enzyme located in the lowermost layer among the enzymes described later.

[0060] Specifically, the first metal nanoparticle may include one or more selected from the group consisting of gold, platinum, nickel, copper, aluminum, gallium, and indium, and is preferably gold, but is not limited thereto. Meanwhile, the average size of the first metal nanoparticle may be 1 nm to 100 nm, is preferably 1 nm to 50 nm, and is more preferably 1 nm to 20 nm, but is not limited thereto.

[0061] A first assembly comprising a first metal nanoparticle can form a first stacked structure by LbL assembly through the above-mentioned organic linker having an amine group. At this time, the number of repetitions during LbL assembly may be 1 to 20 times, preferably 2 to 5 times, and more preferably 3 to 5 times, but is not limited thereto. By optimizing the number of repetitions during LbL assembly in this way, electrical conductivity can be improved while simultaneously satisfying manufacturing efficiency and process scalability.

[0063] Next, the micro-wrinkle conductive hydrogel-based enzyme electrode according to the present invention is formed on the conductive host layer and includes an enzyme layer having a second stacked structure in which a second assembly comprising an enzyme and a second metal nanoparticle is repeated.

[0064] A second stacked structure can be formed on the conductive host layer by LbL assembling a second assembly comprising an enzyme and a second metal nanoparticle (i.e., a second assembly comprising a second metal nanoparticle via an enzyme).

[0065] The enzyme located in the lowest layer among the above enzymes can be bound to the second metal nanoparticle through a ligand exchange process while bound to the first metal nanoparticle. The enzyme not located in the lowest layer among the above enzymes can be bound to the second metal nanoparticles through a ligand exchange process and act as a linker. Specifically, the enzyme may include one or more selected from the group consisting of glucose oxidase (GOx), catalase (CAT), hemoglomin, and ferritin, and considering the aspect of maintaining the stability of the enzyme, it is preferable to be glucose oxidase (GOx), but is not limited thereto.

[0066] Meanwhile, the second metal nanoparticle refers to the state after the ligand exchange process, in which the organic ligand that was bound to the second metal nanoparticle is partially or completely removed and the enzyme is bound. At this time, various hydrophobic ligands may be used as the organic ligand, and it is preferable that it be tetraoctylammonium (TOA), but is not limited thereto. Meanwhile, the enzyme is a hydrophilic ligand and has superior binding affinity with the second metal nanoparticle compared to the organic ligand.

[0067] Specifically, the second metal nanoparticle may include one or more selected from the group consisting of gold, platinum, nickel, copper, aluminum, gallium, and indium, and is preferably gold, but is not limited thereto. Meanwhile, the average size of the second metal nanoparticle may be 1 nm to 100 nm, is preferably 1 nm to 50 nm, and is more preferably 1 nm to 20 nm, but is not limited thereto.

[0068] A second assembly containing a second metal nanoparticle can form a second stacked structure by LbL assembly using the enzyme above. At this time, the number of repetitions during LbL assembly may be 1 to 20 times, preferably 2 to 5 times, and more preferably 3 to 5 times, but is not limited thereto. Optimizing the number of repetitions during LbL assembly in this way has the advantage of significantly increasing the current density. If the number of repetitions becomes excessively large, a resistive interface that hinders efficient electron transfer is introduced.

[0070] Method for manufacturing a micro-wrinkle conductive hydrogel-based enzyme electrode

[0072] The present invention provides a method for manufacturing an enzyme electrode based on a micro-wrinkle conductive hydrogel, comprising: (a) a step of manufacturing a micro-wrinkle conductive hydrogel by infiltrating an organic molecule having an amine group into a hydrogel and then forming a metal nanofilm on its surface; (b) a step of manufacturing a conductive host layer by forming a first stacked structure in which a first assembly comprising an organic linker having an amine group and a first metal nanoparticle is repeated on the conductive hydrogel; and (c) a step of manufacturing an enzyme layer by forming a second stacked structure in which a second assembly comprising an enzyme and a second metal nanoparticle is repeated on the conductive host layer.

[0074] First, the method for manufacturing a micro-wrinkle conductive hydrogel-based enzyme electrode according to the present invention includes the step of manufacturing a micro-wrinkle conductive hydrogel by infiltrating an organic molecule having an amine group into the hydrogel and then forming a metal nanofilm on the surface thereof [step (a)].

[0075] As the above hydrogel, the organic molecule having an amine group, and the metal nanomembrane have been described above, a redundant explanation will be omitted.

[0076] In order to infiltrate organic molecules having amine groups into the above hydrogel, it is necessary to immerse the hydrogel in a solution of organic molecules having amine groups.

[0077] In order to form the metal nanomembrane, it is necessary to deposit a metal nanoparticle-organic ligand (native ligand) on the surface of the infiltrated hydrogel, and continuous ligand exchange and sintering processes are performed as the organic molecule having an amine group causes capillary transport. Meanwhile, the infiltrated hydrogel may have a fine wrinkle structure as the swelling and deswelling processes are repeated.

[0079] Next, the method for manufacturing a micro-wrinkle conductive hydrogel-based enzyme electrode according to the present invention comprises the step of manufacturing a conductive host layer by forming a first stacked structure in which a first assembly comprising an organic linker having an amine group and a first metal nanoparticle is repeated on the conductive hydrogel [step (b)].

[0080] As the first metal nanoparticle and the organic linker having an amine group have been described above, a redundant explanation will be omitted.

[0081] The first stacked structure above can be formed by LbL assembling a first assembly comprising an organic linker having an amine group and a first metal nanoparticle (i.e., a first assembly comprising a first metal nanoparticle through an organic linker having an amine group), and the number of repetitions during LbL assembly may be 1 to 20 times, preferably 2 to 5 times, and more preferably 3 to 5 times, but is not limited thereto. By optimizing the number of repetitions during LbL assembly in this way, electrical conductivity can be improved while simultaneously satisfying manufacturing efficiency and process scalability.

[0083] Next, the method for manufacturing a micro-wrinkle conductive hydrogel-based enzyme electrode according to the present invention comprises the step of manufacturing an enzyme layer by forming a second stacked structure in which a second assembly comprising an enzyme and a second metal nanoparticle is repeated on the conductive host layer [step (c)].

[0084] As the above enzyme and the above second metal nanoparticle have been described above, a redundant explanation will be omitted.

[0085] The second stacked structure above can be formed by LbL assembling a second assembly comprising an enzyme and a second metal nanoparticle, and the number of repetitions during LbL assembly may be 1 to 20 times, preferably 2 to 5 times, and more preferably 3 to 5 times, but is not limited thereto. Optimizing the number of repetitions during LbL assembly in this way has the advantage of significantly increasing the current density. If the number of repetitions becomes excessively large, a resistive interface that hinders efficient electron transfer is introduced.

[0087] Applications of Micro-wrinkle Conductive Hydrogel-Based Enzyme Electrodes

[0089] The present invention provides a biofuel cell or biosensor comprising the above-described micro-wrinkle conductive hydrogel-based enzyme electrode.

[0091] The biofuel cell or biosensor according to the present invention includes the micro-wrinkle conductive hydrogel-based enzyme electrode, and since the micro-wrinkle conductive hydrogel-based enzyme electrode has been described above, a redundant description will be omitted.

[0092] In the above biofuel cell or biosensor, the enzyme electrode can act as an anode.

[0093] Meanwhile, a micro-wrinkled conductive hydrogel having an organic molecule having an amine group infiltrated inside and a metal nanofilm formed on its surface can be used as a cathode applicable to the above-mentioned biofuel cell or biosensor; a conductive host layer having a first stacked structure formed on the conductive hydrogel, wherein a first assembly comprising an organic linker having an amine group and a first metal nanoparticle is repeated; and a Pt layer formed on the conductive host layer may be used. In this case, the Pt layer may be deposited by a known method such as sputtering.

[0095] As described above, the present invention is characterized as an enzyme electrode based on a micro-wrinkle conductive hydrogel comprising: a micro-wrinkle conductive hydrogel in which an organic molecule having an amine group is infiltrated and a metal nanofilm is formed on the surface; a conductive host layer having a first stacked structure formed on the conductive hydrogel, wherein a first assembly comprising an organic linker having an amine group and a first metal nanoparticle is repeated; and an enzyme layer having a second stacked structure formed on the conductive host layer, wherein a second assembly comprising an enzyme and a second metal nanoparticle is repeated. Accordingly, the enzyme electrode according to the present invention has the advantages of excellent mechanical flexibility, enhanced output, and excellent long-term operational stability.

[0096] Therefore, the present invention can be widely utilized in biofuel cells, biosensors, etc., that require a high surface area, mechanical durability, and long-term operational stability.

[0098] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0100] [Example]

[0101] Example 1: Design and fabrication of a hydrogel electrode

[0102] To prepare a conductive hydrogel host electrode (Fig. 3a), a copolymerized polyacrylic acid-polyacrylamide (PAA-co-PAAm) hydrogel template was first synthesized. This template was prepared at pH 11 and 20 mg / mL -1 NH2-functionalized PEI(M at the concentration wPEI was bound to the hydrogel by immersion in a solution of ~800. At this alkaline pH, a significant portion of the carboxylic acid (COOH) groups in the PAA component of the hydrogel (pKa of PAA ~4.5, i.e., the pH at which 50% of the functional groups are ionized) were deprotonated to form carboxylate ions (COO₃). - While ) formed, PEI component (pK a ~10.5) maintained a partially protonated state. The same negative charge COO - Due to electrostatic repulsion during the period, the hydrogel swelled to more than six times its original volume.

[0103] Based on these results, the swollen PEI-hydrogel was immersed in a toluene solution containing TOA-gold nanoparticles with a diameter of approximately 8 nm. This treatment resulted in the formation of a high-density layer of Au NPs on the surface of the PEI-hydrogel. Simultaneously, the hydrogel underwent significant deswelling, and the final volume of the Au nanoparticle-assembled PEI-hydrogel decreased to approximately 31% of the initial swollen state. Furthermore, as the deposition time of the Au NPs increased, the initially adsorbed Au nanoparticles gradually transformed into a porous Au nanoplate film composed of numerous Au nanoparticles. This transformation caused a noticeable increase in film thickness from ~67 nm after 12 hours to ~149 nm after 72 hours (Fig. 3b). It has been reported that for Ag or Au nanoparticles with low cohesive energy, strong metallic bonding can occur between adjacent metal nanoparticles when the interparticle spacing is less than 5-7 Å.

[0104] In addition, the TOA-gold nanoparticle-based hydrogel electrode (abbreviated as TAH electrode) exhibited a unique micro-wrinkle surface morphology, as confirmed by FE-SEM (field emission scanning electron microscope) images (Fig. 3c).

[0105] This increase in thickness and unique morphological evolution of the Au nanoplates are primarily attributed to the NH2 groups of PEI contained in TAH, which exhibited a stronger binding affinity for the Au nanoparticle surface than the ammonium groups of the natural TOA ligand. Specifically, loosely bound TOA ligands on the Au NP surface were replaced by the NH2 groups of PEI at the water / toluene interface through a continuous ligand exchange reaction. Furthermore, this ligand exchange process was sustained as PEI continuously migrated via capillaries from within the hydrogel to the outer TOA-gold nanoparticle layer, thereby promoting the growth of thick, interconnected Au nanoplate films. In contrast, for spin-coated TOA-gold nanoparticle films without ligand exchange, the interparticle interface between adjacent TOA-gold nanoparticles consisted of two bulky TOA ligand layers (M of TOA). w It consisted of ~546) which hindered close contact. As a result, these films did not undergo fusion at room temperature, and the sheet resistance was ~10 7 Ω sq -1 It was expected to exhibit electrical insulation characteristics exceeding [value].

[0106] These observations were further supported by experiments conducted using a two-phase liquid system consisting of an aqueous PEI solution (water) and a toluene-based TOA-gold nanoparticle dispersion (toluene). As shown in Fig. 3d, the dispersed TOA-gold nanoparticles gradually assembled into an Au nanoplate layer at the water / toluene interface. This transformation was driven by LER at the NP surface, where hydrophobic TOA ligands were replaced by hydrophilic PEI molecules. Consequently, the surface properties of the Au NPs transitioned from hydrophobic to hydrophilic, significantly enhancing the migration of capillary-driven PEI to the upper, initially hydrophobic TOA-gold nanoparticle layer. This capillary action, very similar to the mechanism observed in hydrogel systems, enabled continuous sintering through the self-assembly of Au NPs, forming a porous Au nanoplate structure at the interface. As a result, as the ligand exchange and sintering processes continued, both the vertical and lateral dimensions of the resulting Au NP film gradually increased.

[0107] Unlike previously reported nanoparticle assembly methods (typically low-density monolayers or electrostatic interaction-based multilayers formed through hundreds of multiple deposition steps), the capillary force-induced metal nanoparticle assembly strategy according to the present invention provided distinct advantages in terms of electrical and mechanical properties by directly and robustly forming conductive components on a wet hydrogel substrate. Conductive hydrogels prepared using this approach exhibited very high electrical conductivity (~8.4 × 10⁻⁶) after 72 hours of immersion. 4 S cm -1 ) and low sheet resistance (0.8 Ω sq -1 ) was shown (Fig. 4a).

[0108] For comparison, NH3 in PEI + COO of gold nanoparticles stabilized with citrate ions dispersed in water at pH 4 (i.e., anionic gold nanoparticles) -Hydrogel host electrodes were fabricated through electrostatic interactions between groups. However, this method failed to form a high-density gold nanoparticle array on the hydrogel template due to electrostatic repulsion between homocharged gold nanoparticles, resulting in significantly degraded electrical performance. Specifically, the citrate gold nanoparticle-based hydrogel electrode exhibited very low conductivity (~2.0 × 10⁻⁶). -3 S cm -1 ) and high sheet resistance (~1.2 × 10⁻⁶ 6 Ω sq -1 In addition, these hydrogel electrodes exhibited a relatively flat and smooth surface morphology due to the very low packing density (< 30%) of the Au nanoparticle arrays induced by electrostatic interactions, which stood in stark contrast to TAH, which had a fine wrinkled surface morphology induced by swelling-deswelling.

[0109] The TAH electrode possessed excellent electrical properties and exhibited outstanding mechanical flexibility. It maintained conductivity under both bending and tensile conditions with a bending radius (R) of approximately 1 mm. In particular, the TAH electrode maintained its electrical performance for 10,000 cycles of 100% uniaxial tension, demonstrating excellent durability even under repetitive mechanical deformation (Fig. 4b, c). Furthermore, the TAH electrode withstood various deformations, including bending, crumpling, twisting, and tension, without compromising structural integrity. These results clearly demonstrated that the TAH assembled with high-density TOA-gold nanoparticles achieved excellent electrical conductivity, exhibiting superior performance compared to citrate gold nanoparticle-based hydrogels as well as most previously reported conductive hydrogels (Fig. 4d). Specifically, the TAH maintained high conductivity even under deformation, proving excellent mechanical-electrical stability suitable for flexible bioelectronic applications.

[0110] The micro-wrinkle structure of TAH is expected to provide superior electrochemical performance as a host electrode compared to conventional electrodes with flat and smooth structures. First, as confirmed by FE-SEM and optical microscopy, TAH exhibited a unique wrinkle structure in stark contrast to the flat surface of the citrate gold nanoparticle-based hydrogel prepared via electrostatic interactions (Fig. 5a, b). This unique structure was formed by swelling-deswelling kinetics following the assembly of TOA-gold nanoparticles via capillaries onto the PEI hydrogel. In particular, the wrinkle structure of TAH was able to improve the electrochemical performance of the electrode by increasing the effective surface area of ​​the electrode-electrolyte interface.

[0111] To verify this possibility, the electrochemical performance of TAH was compared with that of a citrate Au nanoparticle-based hydrogel. TAH measured 3.0 mA cm⁻¹ at +0.6 V. -2 It showed a significantly high surface current density of 5.2 × 10 -4 mA cm -2 It significantly outperformed the citrate Au NP-based hydrogel shown in Man (Fig. 5c). Furthermore, electrochemical impedance spectroscopy (EIS) revealed additional advantages of TAH. In particular, the ~4.2 × 10⁻⁶ of the citrate Au nanoparticle-based hydrogel 4 1.1 × 10 compared to Ω 2 It exhibited a much lower equivalent series resistance (ESR) of Ω (Fig. 5d). Additionally, TAH showed a steeper Warburg slope in the low-frequency region, indicating enhanced ion diffusion and charge transport behavior. In a long-term stability test conducted in phosphate-buffered saline (PBS) for 30 days, TAH maintained 72% of its initial current density, whereas the citrate Au nanoparticle-based hydrogel maintained only ~18% (Fig. 5e), further demonstrating the superior operational durability of TAH.

[0112] To further evaluate the structural advantages of the present invention, a control electrode (i.e., an Au sputtered hydrogel) was fabricated by directly sputtering a thin Au layer onto a hydrogel. This control electrode exhibited high electrical conductivity (~2.2 × 10⁻⁶). 2 S cm -1 ) and low sheet resistance (0.2 Ω sq -1 It showed ), but the electrochemical performance was poor. At this time, the surface current density was 7.5 × 10 -2 mA cm -2 It was merely, and the ESR was ~4.3 × 10 2 The Ω value was relatively high. Furthermore, severe surface delamination and degradation were observed during electrochemical operation, significantly reducing performance and stability. Due to these structural defects, Au sputtered hydrogels were unsuitable for use as reliable host electrodes in electrochemical applications.

[0113] The structural durability of TAH was evaluated through a rigorous 100-day cycle test involving repeated air drying and immersion in PBS. TAH demonstrated excellent resilience, maintaining 81% of its initial conductivity (6.8 × 10⁻⁶) even after 30 days. 4 S cm -1 ), even after 100 days, 63%(5.3 × 10 4 S cm -1 It maintained its structure. Upon re-immersion in PBS, partial re-swelling occurred, resulting in a slight decrease in electrical conductivity, but the overall corrugated structure was completely preserved. This reversible structural response highlighted the robustness and adaptability of the hydrogel under long-term environmental stress. Furthermore, the excellent electrical performance of TAH was attributed primarily to the interconnected and finely corrugated Au nanoplates. Due to the unique integration of high electrical conductivity, mechanical integrity, and long-term stability, TAH has been positioned as a highly promising electrode platform for high-performance bioelectrochemical systems and other flexible energy devices.

[0114] Based on these results, we aimed to further enhance the electrical conductivity of conductive TAH. To this end, TOA-gold nanoparticles were linked with an NH2-functionalized monomer linker, such as tris-(2-aminoethyl)amine (TREN) (M w It was additionally assembled on TAH via an LbL assembly process using ~ 146). In this case, bulky TOA groups loosely bound to the surface of Au NPs were replaced by NH2 groups of TREN via LER. This is similar to existing systems containing PEI-hydrogel and TOA-gold nanoparticles, which was confirmed by Fourier Transform Infrared (FTIR) spectroscopy. This additional LbL assembly of TREN and TOA-gold nanoparticles promoted the substitution of terminal TOA ligands, significantly enhancing the electrical conductivity of TAH, thereby further strengthening interparticle contact and reducing interfacial resistance. Furthermore, the (TOA-gold nanoparticle / TREN) of TAH n When the number of multilayer stacking layers (n) is increased from 1 to 5, the generated (TOA-gold nanoparticles / TREN) n The electrical conductivity of the / TAH electrode has been significantly improved, with a value of ~1.1 × 10 5 S cm -1 From ~1.5 × 10 5 S cm -1 The conductivity increased to the level of bulk metal, which approached the conductivity of the bulk metal. This highly conductive hydrogel electrode is hereinafter referred to as n-MTAH (Metallic TOA-Gold Nanoparticle Hydrogel), where n represents the number of TOA-Gold Nanoparticle / TREN stacks. Although increasing the number of stacks can further improve electrical conductivity, the maximum number of stacks was limited to 5 to consider manufacturing efficiency and process scalability. The electrode with 5 stacks (n = 5) is hereinafter referred to as 5-MTAH.

[0115] Based on these results, the mechanical properties were further evaluated by analyzing the stress-strain profiles of the PEI-hydrogel, TAH, and high-conductivity 5-MTAH electrodes. First, the PEI-hydrogel exhibited excellent ductility and elasticity, displaying a Young's modulus of approximately 65 kPa and an elongation at break of approximately 289%. Additionally, TAH showed a Young's modulus of approximately 181 kPa and an elongation at break of approximately 222%. On the other hand, the 5-MTAH electrode exhibited a slightly lower Young's modulus of approximately 182 kPa and a reduced elongation of approximately 222%, which was likely due to rigid and fused Au nanoplates. The inclusion of these domains significantly improved electrical conductivity but somewhat reduced the elasticity of the material. Nevertheless, the n-MTAH electrode maintained a good level of mechanical flexibility while preserving high electrical performance. These results highlight the multifunctional properties of hydrogel-based electrodes, namely high electrical conductivity, mechanical durability, and a large electroactive surface area, which can position them as a promising platform for next-generation wearable energy systems, particularly highly flexible and stretchable EBFCs.

[0117] Example 2: Preparation and Performance of Hydrogel Anode

[0118] Based on these results, GOx (GOx concentration 10 mg mL) on a 5-MTAH host electrode -1 , 0.5 mol L -1 A hydrogel-based anode for an EBFC was fabricated by performing repeated LbL combinations of a PBS solution containing a concentration of NaCl and a TOA-gold nanoparticle solution (in toluene). In this configuration, the TOA-gold nanoparticles acted as conductive linkers connecting vertically adjacent GOx layers, facilitating efficient electron transfer throughout the multilayer structure (Figs. 2 and 6a).

[0119] First, under ambient conditions, PBS (20 mmol L -1 5 mV s at pH 7.4) -1The electrochemical performance of n-MTAH host electrodes with various stacking iterations (n ​​= 1 to 5) was evaluated using cyclic voltammetry (CV) at a scan rate. (TOA-gold nanoparticles / TREN) n As the number of stacking layers (n) of the multilayer increases, the cathodic current density at +0.6 V is 3.2 mA cm⁻¹ -2 3.8 mA cm at (n = 1) -2 It gradually increased to (n = 5), which could be attributed to the increased electroactive surface area provided by the additional Au nanoparticle layer. EIS analysis supported this observation, showing that the ESR consistently decreased from approximately 98 Ω (n = 1) to 60 Ω (n = 5). These results collectively suggested that the electron transfer rate improved and the total impedance decreased as the number of stacking layers increased. Based on these results, 5-MTAH was selected as the optimal host electrode for the fabrication of an anode through GOx immobilization.

[0120] Subsequently, GOx was immobilized on a 5-MTAH electrode through additional LbL assembly with TOA-gold nanoparticles (GOx / TOA-gold nanoparticles) m A multilayer structure was formed. The resulting hydrogel-based anode is hereinafter referred to as m-GOx-5-MTAH, where m represents the number of (GOx / TOA-gold nanoparticle) LbL stacking layers. This assembly was performed in PBS at room temperature. In particular, the NH2 portion of the amino acid residues in GOx exhibited a strong affinity for the surface of Au nanoparticles, promoting a Ligand Exchange Reaction (LER) between GOx and loosely bound TOA ligands on the nanoparticles, which was confirmed through UV-vis spectroscopy and FTIR spectroscopy.

[0121] Importantly, the method according to the present invention enabled direct interfacial contact between conductive Au nanoparticles and GOx without relying on insulating organic linkers (e.g., bulky ligands bonded to the surface of Au NPs) or polymerizable binders. While electrostatically stabilized metal nanoparticles in aqueous solutions often experience strong electrostatic repulsion, hydrophobic TOA-gold nanoparticles dispersed in non-polar solvents such as toluene exhibited minimized interparticle repulsion. Consequently, they could be assembled densely and uniformly on the GOx layer. Furthermore, the TOA-gold nanoparticle layer used to form the amphiphilic LbL multilayer composed of hydrophilic GOx and hydrophobic TOA-gold nanoparticles was identical to that applied to 5-MTAH, characterized by the fact that GOx nanoblends with Au nanoparticles in both vertical and horizontal directions (Fig. 6a). This unique structural integration suggested that electron transfer between adjacent GOx molecules, as well as between GOx and the underlying 5-MTAH, could be significantly enhanced compared to conventional slurry-cast GOx films.

[0122] To further verify this possibility, the electrochemical behavior of m-GOx-5-MTAH was investigated (Figs. 6b, c). As the number of stacking layers (m) of m-GOx-5-MTAH increased from 1 to 7, the cathodic current density showed a significant increase up to m = 5 and then gradually decreased (Fig. 6b). At the same time, the ESR of m-GOx-5-MTAH was 300 mmol L⁻¹. -1 There was a slight increase from approximately 59 Ω (m = 0) to approximately 127 Ω (m = 7), suggesting that excessive multilayer formation introduces resistive interfaces that hinder efficient electron transfer (Fig. 6c). The electrocatalytic reaction of 5-GOx-5-MTAH was performed in PBS (pH 7.4) at glucose concentrations (0–300 mmol L⁻¹). -1It was further evaluated as a function of ). As the glucose concentration increased, the cathodic current density gradually increased (Fig. 6d), indicating that the catalytic oxidation of glucose by GOx increased. This correlation was further supported by the normalized current density values ​​obtained after subtracting the background signal measured at +0.6 V in glucose-free PBS. At the same time, within the same glucose concentration range, the ESR values ​​were as follows: glucose concentrations from 0 to 300 mmol L -1 It was found to increase slightly as it increased. This increase was attributed to electron transfer associated with the glucose oxidation reaction.

[0123] In addition, to elucidate the effect of the number of layers on catalytic performance, normalized cathodic current densities were systematically investigated from m = 1 to 7 (Fig. 6e). At m = 1, 1.3 mA cm⁻¹ -2 From m = 5, 9.3 mA cm -2 A consistent increase was observed with a peak, indicating that gradual LbL assembly significantly enhances GOx activity. Notably, the 5-GOx-5-MTAH electrode showed 17.9 mA cm⁻¹ -2 It exhibited the maximum face current density, confirming that the observed enhancement is primarily attributed to glucose oxidation catalyzed by GOx. However, when the number of layers was further increased to m = 7, the face current density dropped to 16.5 mA cm⁻¹ due to mass transfer limitation, increased steric hindrance, or increased interfacial resistance within the excessively thick multilayer structure. -2 It was reduced to . These research results suggested that the number of stacking layers m = 5 provides an optimal configuration for maximizing electron transfer efficiency and catalytic performance, and highlighted the important role of LbL engineering in fine-tuning enzyme electrode structures.

[0124] To further verify the electron transfer kinetics of the optimized 5-GOx-5-MTAH, the Laviron model was applied to the apparent heterogeneity electron transfer rate constant (k s ) was estimated. CV was performed at various scan rates (ν), and as a result, the cathode and anode peak current densities increased linearly with scan rate, indicating a surface-controlled quasi-reversible redox process. As the scan rate increased, the inter-peak potential separation (ΔE p = E pa - E pc ) also increased. (E - E 0 Through the linear relationship between ) and log(ν), k s Values ​​could be extracted, and for 5-GOx-5-MTAH, 3.7±0.2 s -1 We obtained , which demonstrated efficient catalytic kinetics at the electrode-enzyme interface. This high rate constant was 100 mV s⁻¹. -1 A relatively small ΔE of 83 mV at a scan rate p It is supported by carbon nanomaterials (2.1 s -1 ), conductive polymer (2.2 s -1 ) and metal-organic complexes (1.1 s -1 It surpassed values ​​reported for other electrode systems such as ). In surface-controlled electrochemical systems, ΔE of less than 200 mV p generally reflected a rapid heterogeneous electron transfer process. Therefore, the observed small ΔE p Effective enzyme immobilization and rapid electron exchange were confirmed at the bioelectrode interface.

[0125] To evaluate the effect of multilayer assembly on electron transfer rate, for both 1-GOx-5-MTAH electrodes, k s The values ​​were additionally measured. This comparison is additional (GOx / TOA-gold nanoparticles) m The beneficial effect of multilayer structures on increasing interfacial electron transfer rates was highlighted.

[0126] The operational stability of the 5-GOx-5-MTAH anode was further evaluated through individual time-current measurements. After 48 hours of continuous operation, the anode was approximately 87% (15.6 mA cm⁻¹) of the initial current density. -2 This high cathodic current density and long-term operational stability were mainly attributed to the synergistic effect of the (GOx / TOA-gold nanoparticle)m multilayer assembly and the micro-wrinkle structure of the hydrogel-based electrode, which comprehensively improved electron transfer efficiency and mechanical robustness.

[0128] Example 3: Preparation and Performance of a Hydrogel Cathode

[0129] The 5-MTAH platform effectively functions as a cathode host in EBFCs, where ORR significantly influences both electrochemical efficiency and long-term stability. Therefore, enhancing ORR activity is crucial for improving overall device performance. In this context, the insertion of Au NPs within the fine wrinkle structure of 5-MTAH was able to provide a synergistic effect. Specifically, the large electroactive surface area and unique catalytic properties of Au NPs promoted efficient ORR reaction rates, while the wrinkle morphology increased the density of accessible active sites, thereby enhancing the reaction rate.

[0130] To further enhance ORR performance, Pt, a benchmark ORR catalyst, was introduced as a co-catalyst. This was achieved by immersing 5-MTAH in a Pt precursor solution and then performing a chemical reduction reaction using sodium borohydride (NaBH4). As a result, the Pt-modified structure (Pt-5-MTAH) exhibited uniform Pt deposition without damaging the underlying structure.

[0131] Based on these results, ORR performance was evaluated using PBS under nitrogen saturation, oxygen saturation, and ambient conditions. At -0.6 V (relative to Ag / AgCl), the surface current density of Pt-5-MTAH was -17.6 mA cm⁻¹.-2 (Nitrogen), -34.0 mA cm -2 (Ambient), -53.3 mA cm -2 It was (oxygen). When normalized to the nitrogen (N2) baseline, the cathodic current density under ambient and oxygen conditions was -16.4 mA cm⁻¹, respectively. -2 and -35.7 mA cm -2 was. In comparison, pure 5-MTAH (no Pt) exhibited a significantly lower current density under the same conditions: -1.7 mA cm⁻¹. -2 (Nitrogen), -3.1 mA cm -2 (Ambient), -3.9 mA cm -2 (Oxygen). This confirmed the excellent catalytic activity of Pt-5-MTAH.

[0132] In addition, the long-term operational stability of Pt-5-MTAH was evaluated under ambient PBS conditions. The Pt-modified electrode maintained approximately 82% of its initial current density after 30 days, whereas pure 5-MTAH maintained only about 71% during the same period. Collectively, these results demonstrated that Pt-5-MTAH exhibits significantly enhanced ORR activity, improved charge transfer efficiency, and stable long-term operation through the synergistic effect of the highly active Pt catalyst and the robust electroactive 5-MTAH host electrode.

[0134] Example 4: Output and Efficiency of Hydrogel-Based EBFC

[0135] To evaluate the actual performance of hydrogel-based EBFCs, devices were assembled by combining a 5-GOx-5-MTAH anode with a Pt-5-MTAH or pure 5-MTAH cathode. All tests were conducted at 300 or 10 mmol L under ambient conditions. -1 It was performed in PBS containing glucose (Fig. 7a). To minimize parasitic current, normal output measurements were performed using external resistances in the range of 1 kΩ to 10 MΩ. 300 mmol L -1In glucose, a complete EBFC (5-GOx-5-MTAH / Pt-5-MTAH) has a maximum power density of ~3.7 mW cm⁻¹. -2 It achieved ~1.6 mW cm -2 It was approximately 2.3 times higher than the Pt-free device (5-GOx-5-MTAH / 5-MTAH) reached (Fig. 7b). Similarly, 10 mmol L -1 In glucose, a complete EBFC is ~2.4 mW cm⁻¹ -2 By delivering the output of the Pt-free system (~0.7 mW cm²) -2 It exhibited approximately 3.4 times better performance than (Fig. 7c). These results clearly demonstrated the superior power performance of the full EBFC at both low and high glucose concentrations. This improvement was attributed to the synergistic pairing of the highly enzymatic 5-GOx-5-MTAH anode and the Pt-MTAH cathode, in which ORR activity was optimized through NaBH4 reduction. Further supporting this is that the full EBFC at +0.95 V (300 mmol L⁻¹) -1 ) and +0.92 V(10 mmol L -1 It showed a high OCV, which was significantly higher than the Pt-free system (+0.76 V and +0.62 V, respectively) under ambient conditions.

[0136] In addition, the hydrogel-based EBFC demonstrated excellent long-term operational stability. 300 mmol L -1 Under glucose-containing ambient conditions, a full EBFC has an initial power density of approximately 2.9 mW cm⁻¹ after 30 days of continuous operation. -2 Maintained (~78%) (Fig. 7d). 10 mmol L -1 At glucose, ~1.9 mW cm -2 While maintaining (~80%), Pt-free EBFC was ~0.5 mW cm during the same period. -2 It maintained (~77%). Voltage maintenance measurements showed a similar trend (Fig. 7e). The full EBFC was 300 and 10 mmol L.-1 In glucose, ~84% (+0.80 V) and ~86% (+0.79 V) of the initial OCV were maintained, respectively, whereas Pt-free EBFCs maintained ~81% (+0.62 V) and ~85% (+0.53 V) under the same conditions. This remarkable stability was attributed primarily to the hierarchical electrode design incorporating various interfacial and structural strategies: (i) the integration of capillary forces and ligand exchange drives of metal nanoparticles between PEI and TOA-gold nanoparticles, (ii) covalent bonding between TREN and TOA-gold nanoparticles, and (iii) amphiphilic coassembly of GOx and TOA-gold nanoparticles.

[0138] In this invention, the development of a hydrogel-based EBFC with high flexibility and biocompatibility, high electron transfer efficiency, increased areal power density, and long-term operational stability was demonstrated. These characteristics were achieved through the assembly of metal nanoparticles induced by capillary forces and ligand exchange. In particular, PEI infiltrated into the hydrogel performed multiple functions: acting as a crosslinking agent to control the mechanical properties of the hydrogel and as a strong binding linker between the hydrogel and Au nanoparticles, as well as serving as a modifying additive for the formation of Au nanoplatelets. This process was approximately 1.5 × 10⁻⁶ 5 S cm -1 We derived highly conductive 5-MTAH with a micro-wrinkle surface morphology through adsorption during repetitive swelling / deswelling cycles, possessing excellent electrical conductivity, and confirmed that it maintains very stable electrical properties under various mechanical stresses. Additional (GOx / TOA-gold nanoparticles) on 5-MTAH as the anode and Pt-5-MTAH as the cathode m When the multilayer was LbL-assembled, the resulting hydrogel-based EBFC was ~3.7 mW cm⁻¹ -2It exhibited excellent output and maintained an efficiency of approximately 80% of the initial area power density even after 30 days of continuous operation. Given that the strategy according to the present invention is based on interfacial assembly to improve both electron transfer rate and enzyme immobilization, this can provide extensive potential for hydrogel-based electrodes not only in EBFCs but also in various electrochemical systems requiring high surface area, mechanical durability, and long-term operational stability.

[0140] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A micro-wrinkle conductive hydrogel-based enzyme electrode comprising: a micro-wrinkle conductive hydrogel formed by infiltrating an organic molecule having an amine group inside, comprising a hydrogel containing a carboxyl group-containing polymer, and a metal nanofilm formed by depositing metal nanoparticles on the surface of said hydrogel; a conductive host layer having a first stacked structure formed on said conductive hydrogel, wherein a first assembly comprising an organic linker having an amine group and a first metal nanoparticle is repeated; and an enzyme layer having a second stacked structure formed on said conductive host layer, wherein a second assembly comprising an enzyme and a second metal nanoparticle is repeated, wherein said organic molecule having an amine group interacts with said carboxyl group-containing polymer and is bound as a hydrophilic ligand of said metal nanoparticle through capillary transport phenomena. Claim 2 A micro-wrinkle conductive hydrogel-based enzyme electrode according to claim 1, characterized in that the organic molecule having an amine group is one or more selected from the group consisting of polyethyleneimine, tris(2-aminoethyl)amine, and diethylenetriamine. Claim 3 A micro-wrinkle conductive hydrogel-based enzyme electrode according to claim 1, characterized in that the first or second metal nanoparticle comprises one or more selected from the group consisting of gold, platinum, nickel, copper, aluminum, gallium, and indium. Claim 4 A micro-wrinkle conductive hydrogel-based enzyme electrode according to claim 1, characterized in that the organic linker having an amine group is one or more selected from the group consisting of hydrazine, ethylenediamine, diethylenetriamine, tris(2-aminoethyl)amine, and cysteamine. Claim 5 A micro-wrinkle conductive hydrogel-based enzyme electrode according to claim 1, characterized in that the enzyme comprises one or more selected from the group consisting of glucose oxidase (GOx), catalase (CAT), hemoglobin, and ferritin. Claim 6 A micro-wrinkle conductive hydrogel-based enzyme electrode according to claim 1, characterized in that the number of repetitions of the first or second assembly is 1 to 20. Claim 7 (a) a step of preparing a micro-wrinkle conductive hydrogel by infiltrating an organic molecule having an amine group into a hydrogel containing a carboxyl group-containing polymer and then depositing metal nanoparticles on the surface to form a metal nanofilm; (b) a step of preparing a conductive host layer by forming a first stacked structure in which a first assembly comprising an organic linker having an amine group and a first metal nanoparticle is repeated on the conductive hydrogel; and (c) a step of preparing an enzyme layer by forming a second stacked structure in which a second assembly comprising an enzyme and a second metal nanoparticle is repeated on the conductive host layer, wherein in step (a), the organic molecule having an amine group interacts with the carboxyl group-containing polymer and is bound as a hydrophilic ligand of the metal nanoparticle through capillary transport. Claim 8 A biofuel cell comprising an enzyme electrode according to any one of claims 1 to 6. Claim 9 A biosensor comprising an enzyme electrode according to any one of claims 1 to 6.

Images