Modified oxidoreductase, preparation method of nanoparticle composition, nanoparticle composition, electrical signal sensor, nanodrug, and nanodrug complex

Abstract

The modified oxidoreductase comprises a dehydrogenated oxidoreductase and a modified chain segment. The dehydrogenated oxidoreductase has at least one dehydrogenated thiol group or at least one dehydrogenated amino group. The dehydrogenated oxidoreductase has the dehydrogenated thiol group, and the modified chain segment has the structure of formula (1) and is bonded to the dehydrogenated thiol group. The dehydrogenated oxidoreductase has the dehydrogenated amino group, and the modified chain segment has the structure of formula (2) and is bonded to the dehydrogenated amino group. In formula (1), A1 is a first chain segment having a first π-conjugated system (i.e., a conjugated double bond system), X is hydrogen, alkyl, carboxyl, amide, or ester group, n1 is an integer from 0 to 20, and m1 is an integer from 0 to 20; in formula (2), A2 is a second chain segment having a second π-conjugated system, n2 is an integer from 0 to 20, and m2 is an integer from 0 to 20.

Description

Technical Field

[0001] This disclosure relates to a modified oxidoreductase and its applications, particularly to a modified oxidoreductase and its applications in the fields of nanoparticle compositions, electrical signal sensors, nanomedicines, and nanomedicine complexes. Prior Technology

[0002] In existing technologies, oxidoreductases have been widely used in various biomedical and industrial fields, such as as functional components of drug delivery systems or as active substances in sensors and catalytic reactions. However, the sensitivity of natural enzymes to environmental conditions such as temperature, pH, organic solvents, or interfacial tension can cause them to lose activity, limiting their stability and reusability in practical applications. To address these issues, researchers have developed various nanoenzymes designed to mimic the structure and function of natural enzymes. Although nanoenzymes can effectively protect the active site of enzymes through their unique nanostructure, thereby improving their stability and catalytic efficiency, they still have some drawbacks. For example, nanoenzymes lack the high specificity of natural enzymes, and some of the constituent materials of nanoenzymes (e.g., metals or metal oxides) may cause biocompatibility problems, increasing the risks associated with their in vivo application. Furthermore, the synthesis of nanoenzymes typically requires harsh conditions such as high temperature and high pressure, which not only complicates the process but also increases production costs, thus posing challenges to large-scale production and clinical translation. Therefore, how to provide a technology that can improve enzyme stability and catalytic efficiency, reduce the risk of enzyme denaturation and inactivation, and combine with drugs to form multifunctional enzyme nanoparticles has become a pressing technical challenge. Summary of the Invention

[0003] According to one or more embodiments of this disclosure, a modified oxidoreductase comprises a dehydrogenated oxidoreductase and a modified segment. The dehydrogenated oxidoreductase has at least one dehydrogenated thiol group or at least one dehydrogenated amino group. The dehydrogenated oxidoreductase has the at least one dehydrogenated thiol group, and the modified segment has the structure of formula (1) and is bonded to the at least one dehydrogenated thiol group. The dehydrogenated oxidoreductase has the at least one dehydrogenated amino group, and the modified segment has the structure of formula (2) and is bonded to the at least one dehydrogenated amino group. Formula (1), where A1 is the first segment having the first π conjugated system (i.e., the conjugated double bond system), X is hydrogen, alkyl, carboxyl, amide or ester, n1 is an integer from 0 to 20, and m1 is an integer from 0 to 20; Equation (2), where A2 is the second chain segment with the second π conjugate system, n2 is an integer from 0 to 20, and m2 is an integer from 0 to 20.

[0004] In one or more embodiments disclosed herein, the first π-conjugated system or the second π-conjugated system is an intra-aromatic ring π-conjugated system.

[0005] In one or more embodiments disclosed herein, the first segment and the second segment are each of equation (3), equation (4), or equation (5): Equation (3); Equation (4); Equation (5).

[0006] In one or more embodiments disclosed herein, the dehydrogenated oxidoreductase is generated by dehydrogenation of glucose oxidase, glucose dehydrogenase, pyruvate oxidase, xanthine oxidase, acetylcholinesterase, lactate oxidase, uricase oxidase, uricase, pyrroloquinoline quinone glucose dehydrogenase-A, pyrroloquinoline quinone glucose dehydrogenase-B, NAD(P)-dependent glutamate dehydrogenase, FAD-dependent glutamate dehydrogenase, cholesterol oxidase, or sulfur-containing enzyme.

[0007] In one or more embodiments disclosed herein, the number of the at least one dehydrogenated thiol group is 1 to 20, and the number of the at least one dehydrogenated amino group is 1 to 20.

[0008] According to one or more embodiments of the present disclosure, a method for preparing a nanoparticle composition includes: placing a plurality of the aforementioned modified oxidoreductases into an aldehyde or aldehyde acid solution with a concentration of 0.1 mM to 0.31 mM.

[0009] In one or more embodiments disclosed herein, the aldehyde or aldehyde acid solution is a monoaldehyde solution, a dialdehyde solution, an aromatic aldehyde solution, an α,β-unsaturated aldehyde solution, a hydroxy aldehyde solution, a ketaldehyde solution, an aldehyde acid solution, or a combination thereof.

[0010] According to one or more embodiments of this disclosure, a nanoparticle composition includes a plurality of enzyme nanoparticles, each enzyme nanoparticle being formed by cross-linking a plurality of modified oxidoreductases and a plurality of aldehyde or aldehyde acid molecules, wherein each modified oxidoreductase includes a dehydrogenated oxidoreductase and a modified segment. The dehydrogenated oxidoreductase has at least one dehydrogenated thiol group or at least one dehydrogenated amino group. The dehydrogenated oxidoreductase has the at least one dehydrogenated thiol group, and the modified segment has the structure of formula (1) and is bonded to the at least one dehydrogenated thiol group. The dehydrogenated oxidoreductase has the at least one dehydrogenated amino group, and the modified segment has the structure of formula (2) and is bonded to the at least one dehydrogenated amino group. Formula (1), where A1 is the first segment having the first π conjugated system (i.e., the conjugated double bond system), X is hydrogen, alkyl, carboxyl, amide or ester, n1 is an integer from 0 to 20, and m1 is an integer from 0 to 20; Equation (2), where A2 is the second chain segment with the second π conjugate system, n2 is an integer from 0 to 20, and m2 is an integer from 0 to 20.

[0011] In one or more embodiments disclosed herein, the average diameter of the enzyme nanoparticles is from 10 nanometers to 5000 nanometers.

[0012] In one or more embodiments disclosed herein, the aldehyde or aldehyde molecule may be a monoaldehyde, dialdehyde, aromatic aldehyde, α,β-unsaturated aldehyde, hydroxy aldehyde, ketaldehyde, aldehyde acid, or a combination thereof.

[0013] In one or more embodiments disclosed herein, the dehydrogenated oxidoreductase system is formed by dehydrogenation of glucose oxidase, and the catalytic efficiency of the nanoparticle composition for glucose, Kcat / KM, is greater than 0.5×104s-1·M-1 and less than 5.0×104s-1·M-1, where Kcat is the catalytic constant and KM is the Michaelis constant.

[0014] According to one or more embodiments of this disclosure, an electrical signal sensor includes an electrode layer and a sensing layer disposed on the surface of the electrode layer. The sensing layer includes the aforementioned nanoparticle composition.

[0015] According to one or more embodiments of this disclosure, a nanomedicine comprises the aforementioned nanoparticle composition.

[0016] According to one or more embodiments of this disclosure, a nano-drug complex includes the aforementioned nanoparticle composition and drug molecules. The drug molecules bind to any one of the modified oxidoreductases in the nanoparticle composition in a releaseable form.

[0017] In one or more embodiments disclosed herein, the drug molecule is an anticancer drug.

[0018] According to the embodiments disclosed above, the modified oxidoreductase exhibits amphiphilic molecular properties by grafting hydrophobic segments, enabling it to be stably dispersed in aqueous solutions and further spontaneously assemble into multifunctional enzyme nanoparticles. This self-assembled structure helps enhance the enzyme's stereostrate stability and functional performance, maintaining high activity and good catalytic efficiency under various pH and temperature conditions, effectively reducing enzyme loss due to denaturation or loss of activity during application. Because these enzyme nanoparticles possess both good stability and biocompatibility, their application potential is broad, covering multiple fields such as biomedicine, environment, food, and bioenergy. Simple Explanation of the Diagram

[0019] To make the above and other objects, features, advantages and embodiments of this disclosure more apparent and understandable, the accompanying drawings are described below: Figures 1A to 1I are scanning electron microscope images of modified glucose oxidase in glutaraldehyde solutions of different concentrations; Figures 2A to 2D are the particle size distribution diagrams of the enzyme nanoparticles shown in Figures 1C to 1F, respectively. Figure 3A shows the average particle size curves of the enzyme nanoparticles at different glutaraldehyde concentrations in Figures 1A to 1H; Figure 3B shows the reaction rate curves of the nanoparticle composition at different glutaraldehyde concentrations. Figure 4 shows the current response curves of the nanoparticle composition at different glutaraldehyde concentrations and different glucose concentrations; Figures 5A and 5B show the UV-Vis absorption spectra of the nanoparticle composition and the modified glucose oxidase, respectively. Figures 6A and 6B are absorbance-time graphs of the nanoparticle composition and the modified glucose oxidase at different glucose concentrations, respectively. Figure 6C shows the absorbance-concentration correction curve for ABTS+. Figures 6D and 6E are Michaelis-Menten curves for the nanoparticle composition and the modified glucose oxidase, respectively. Figures 6F and 6G are Lineweaver-Burk double reciprocal plots of the nanoparticle composition and the modified glucose oxidase, respectively. Figure 7 shows the reaction rate curves of the nanoparticle composition at different pH values; Figure 8A shows the UV-Vis absorption spectra of formula (C) and modified glucose oxidase at different concentrations in dimethyl sulfoxide; Figure 8B shows the concentration-absorbance correction curve for equation (C); Figures 9A and 9B are MALDI-MS images of modified glucose oxidase and glucose oxidase, respectively. Figure 10A shows the current density change curve of the sensor over 7 days in phosphate buffer. Figure 10B is a bar chart showing the change in current density of the sensor after 7 days in phosphate buffer. Figure 10C shows the fluorescence intensity-concentration relationship of the sensor in phosphate buffer. Figure 10D shows the change in the cumulative release efficiency of the sensor over 7 days in phosphate buffer. Figure 11 shows the current changes when different substrates are added to the sensor; and Figure 12 shows the reaction rate curves of the nanoparticle components at different temperatures; Figure 13A is a transmission electron microscope image of a DOX-loaded nanodrug complex. Figure 13B is a partially enlarged view of Figure 13A; and Figure 14 is a side view schematic diagram of an electrical signal sensor according to some embodiments of the present disclosure. Implementation

[0020] The following drawings disclose several embodiments of this disclosure. For clarity, many practical details will be described in the following description. However, it should be understood that these practical details should not be used to limit this disclosure. That is, in some embodiments of this disclosure, these practical details are not essential and therefore should not be used to limit this disclosure.

[0021] In this article, the structure of polymers or groups is sometimes represented by a skeleton formula. This representation may omit carbon atoms, hydrogen atoms, and carbon-hydrogen bonds. Of course, if the structural formula explicitly shows atoms or atomic groups, the representation shown by the artist shall prevail.

[0022] Unless otherwise expressly stated, the numerical ranges mentioned in this disclosure shall include their endpoints. For example, expressions such as "A to B", "A~B" or similar phrases mean that the range can be A, B, or any value in between.

[0023] This disclosure provides a modified oxidoreductase, which, after grafting a hydrophobic end, acquires amphiphilic molecular properties, enabling it to exist stably in solution and further self-assemble into multifunctional enzyme nanoparticles. Through this self-assembly structure, the structural stability and functional performance of the enzyme are effectively enhanced, allowing the formed enzyme nanoparticles to maintain high activity and high catalytic efficiency over a wide pH and temperature range, thereby reducing enzyme loss due to denaturation or inactivation. Because these enzyme nanoparticles possess both stability and biocompatibility, they can be widely applied in biomedicine, environmental technology, food industry, and biofuels. Specific applications include drug synthesis, biosensing, and disease diagnosis in the pharmaceutical field; water treatment and pollutant degradation in environmental technology; lactose hydrolysis and starch saccharification in the food industry; and improving enzyme utilization efficiency in biofuel production.

[0024] It should be understood that oxidoreductases (e.g., glucose oxidase) are highly hydrophilic proteins. When placed in aqueous solution, they readily dissolve and disperse in a homogeneous state, lacking sufficient hydrophobic driving force for self-assembly, thus making it difficult to stably form nanoparticles with well-defined interfaces and structures. In response, the modified oxidoreductase disclosed herein is obtained by chemically modifying oxidoreductases to impart amphiphilic characteristics, possessing both hydrophilic and hydrophobic ends, thereby promoting the self-assembly of the modified oxidoreductase in aqueous solution to form stable nanostructures. The modified oxidoreductase disclosed herein can be divided into two types based on structural differences; both types can achieve the effects claimed in this disclosure, as explained below. <Modified Oxidoreductase>

[0025] The first type of modified oxidoreductase is obtained by grafting a hydrophobic modifying segment onto the oxidoreductase using a Michael addition reaction of thiol-maleimide. That is, in this embodiment, the hydrophobic modifying segment is bonded to the oxidoreductase via a thioether bond. Specifically, the modified oxidoreductase includes a dehydrogenated oxidoreductase and a hydrophobic modifying segment, wherein the dehydrogenated oxidoreductase has at least one dehydrogenated thiol group, and the modifying segment is bonded to this dehydrogenated thiol group, wherein the modifying segment has the structure of formula (1): Formula (1), A1 is the first segment having the first π-conjugated system, and X is hydrogen, alkyl (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, isopropyl, isobutyl, sec-butyl, tributyl, isopentyl, neopentyl, sec-pentyl, tripentyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecanyl, octadecyl, nonadecanyl, eicosyl, dodecyl, 3-methylbutyl, 2-methylbutyl), carboxyl, amide, or ester (e.g., formate). Ester, ethyl formate, propyl formate, butyl formate, pentyl formate, hexyl formate, methyl acetate, ethyl acetate, propyl acetate, butyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, octyl acetate, methyl propionate, ethyl propionate, propyl propionate, butyl propionate, methyl butyrate, ethyl butyrate, propyl butyrate, ethyl hexanoate, ethyl heptanoate, ethyl octanoate, ethyl decanoate, methyl benzoate, ethyl benzoate, methyl salicylate, ethyl salicylate, methyl p-hydroxybenzoate), n1 is an integer from 0 to 20, and m1 is an integer from 0 to 20.

[0026] The second type of modified oxidoreductase is obtained by grafting a hydrophobic modifying segment onto the oxidoreductase using a condensation reaction (acetylamine bond formation reaction). In other words, in this embodiment, the hydrophobic modifying segment is bonded to the oxidoreductase via an acetylamine bond. Specifically, the modified oxidoreductase includes a dehydrogenated oxidoreductase and a hydrophobic modifying segment, wherein the dehydrogenated oxidoreductase has at least one dehydrogenated (removed one hydrogen) amino group, and the modifying segment is bonded to the dehydrogenated amino group, wherein the modifying segment has the structure of formula (2): Equation (2), where A2 is the second chain segment with the second π conjugate system, n2 is an integer from 0 to 20, and m2 is an integer from 0 to 20.

[0027] In some embodiments, the first π-conjugated system and the second π-conjugated system may each be an intra-aromatic ring π-conjugated system, that is, the π-conjugated system exists in the conjugated electron cloud of the aromatic ring and is formed by the carbon atoms of the aromatic ring in an extended conjugated structure with alternating single and double bonds. Specifically, the first segment and the second segment may each have the structure of formula (3), formula (4), or formula (5): Equation (3); Equation (4); Equation (5). In a preferred embodiment, the first and second segments can each have the structure of Equation (3). Since the aromatic ring has a stable resonance structure, it can endow the modified segments with good chemical stability, thus reducing their decomposition risk under various environmental conditions. In addition, the hydrophobicity of the aromatic ring helps the modified oxidoreductase form obvious hydrophilic and hydrophobic regions in aqueous solution, effectively promoting its self-assembly behavior, further stabilizing the formation of nanostructures, and improving the overall stability and functional performance of enzyme nanoparticles.

[0028] In some embodiments, the aforementioned dehydrogenating oxidoreductase can be generated via dehydrogenation of glucose oxidase, glucose dehydrogenase, pyruvate oxidase, catalase, xanthine oxidase, acetylcholinesterase, lactate oxidase, uricase oxidase, uricase, pyrroloquinoline quinone glucose dehydrogenase-A, pyrroloquinoline quinone glucose dehydrogenase-B, NAD(P)-dependent glutamate dehydrogenase, FAD-dependent glutamate dehydrogenase, cholesterol oxidase, or sulfur-containing enzymes. In embodiments where the oxidoreductase is glucose oxidase, glucose dehydrogenase, pyrroloquinoline quinone glucose dehydrogenase-A, or pyrroloquinoline quinone glucose dehydrogenase-B, the modified oxidoreductase can perform a redox reaction on glucose. For example, glucose oxidase can catalyze the oxidation of glucose to gluconolactone and hydrogen peroxide (H₂O₂) in the presence of oxygen.

[0029] For the first type of modified oxidoreductase, in some embodiments, the number of dehydrogenated thiol groups is 1 to 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). For the second type of oxidoreductase, the number of dehydrogenated amino groups is 1 to 20 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). In other words, the surface of the dehydrogenated oxidoreductase can be grafted with 1 to 20 hydrophobic modified segments. By grafting hydrophobic modified segments at multiple sites, the ability of oxidoreductases to self-assemble into nanostructures in aqueous solutions can be effectively enhanced, thereby improving their dispersion stability and particle size control. Furthermore, an appropriate number of modified segments can provide structural protection without affecting enzyme activity, improving enzyme stability under different pH and temperature conditions. Moreover, multi-site grafting also helps strengthen the interaction between the enzyme and hydrophobic drugs or carriers, increasing the efficiency of forming drug-complex compounds and enhancing their functional performance in drug delivery, biosensing, and catalytic applications.

[0030] It is worth noting that the modified segments can not only serve as hydrophobic segments, facilitating self-assembly and the formation of stable structures, but also be designed as fluorophores due to their fluorescent properties, endowing the formed enzyme nanoparticles with fluorescent characteristics. This enables the visualization, tracking, and localization of enzymes in vivo or in vitro. Such fluorescence functionality has high application value in the fields of bioimaging, nanoparticle distribution analysis, and site of action identification. It can assist in the real-time observation and evaluation of particle stability, activity retention time, and release dynamics, significantly benefiting the quality control and efficacy optimization of drug delivery systems. <Preparation process of modified oxidoreductase>

[0031] The preparation processes of several embodiments of the first type of modified oxidoreductase and several embodiments of the second type of modified oxidoreductase are described below to demonstrate the feasibility of this disclosure. The entire preparation process can be roughly divided into two steps: first, a hydrophobic modified chain precursor is prepared, and then the hydrophobic modified chain precursor is grafted onto the oxidoreductase. [Preparation of Class I Modified Oxidoreductase] Step 1: Preparation of hydrophobic modified chain segment precursors (list three types) The first type: 4-piperidinyl-1,8-naphthyldimethylimide maleimide

[0032] First, 4-bromo-1,8-naphthalene anhydride (20 mmol) and piperidine (26.2 mmol) were dissolved in 2-methoxyethanol (100 ml) and heated under reflux for 24 hours under nitrogen. After cooling to room temperature, the solvent was removed under reduced pressure, and the residue was recrystallized from ethanol to give orange needle-like crystals of 4-piperidinyl-1,8-naphthalene anhydride, as shown in formula (A). Formula (A). Molecular identification of formula (A): ¹H NMR (300 MHz, CDCl₃, 25°C): δ=1.70–1.80 (m, 2H; CH₂), 1.85–1.95 (m, 4H; 2CH₂), 3.25–3.35 (m, 4H; 2CH₂), 7.20 (d, J=8.4 Hz, 1H; CH), 7.71 (t, J=7.95 Hz, 1H; CH), 8.44 (dd, J= 1, 8.6 Hz, 1H; CH), 8.49 (d, J=8.1 Hz, 1H; CH), 8.57 (dd, J= 1, 7.4 Hz, 1H; CH); MS [ESI⁺]: m / z(%): calcd. 282.1, obsvd. 282.31 [M+H]+

[0033] Next, 20 mmol of 4-piperidinyl-1,8-naphthalene anhydride was dissolved in 150 ml of ethanol, and 6 equivalents of hexaethylenetriamine were added. The reaction mixture was refluxed under nitrogen for 1 hour. After cooling to room temperature, the yellow precipitate was separated by filtration to remove the disubstituted byproduct. The filtrate was concentrated under reduced pressure, and the residue was poured into ice water and extracted with dichloromethane (3 times, 100 ml each time). The organic layer was dried over magnesium sulfate and the solvent was removed under reduced pressure to obtain an orange semi-solid substance (containing the product 4-piperidinyl-1,8-naphthalenedimethimine hexamethyleneamine, as shown in formula (B)). This product was directly used in the next reaction without purification. Formula (B). Molecular identification of formula (B): ¹H NMR (500 MHz, CDCl₃, 25°C): δ = 1.35–1.50 (m, 8H; 3CH₂, NH₂), 1.65–1.75 (m, 4H; 2CH₂), 1.80–1.90 (m, 4H; 2CH₂), 2.60–2.70 (m, 2H; CH₂), 3.15–3.25 (m, 4H; 2CH₂), 4.10–4.20 (m, 2H; CH₂), 7.10–7.20 (m, 1H; CH), 7.60–7.70 (m, 1H; CH), 8.35–8.40 (m, 1H; CH), 8.45–8.50 (m, 1H; CH). 8.55–8.60 (m, 1H; CH)

[0034] Subsequently, 20 mmol of 4-piperidinyl-1,8-naphthyldimethylimine hexamethyleneamine was dissolved in 50 ml of dry dichloromethane, 0.35 g of 3-maleimine propionic acid N-hydroxysuccinimine was added, followed by a catalytic amount of pyridine (0.5 ml). The reaction mixture was refluxed under nitrogen for 24 hours. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was washed with brine and extracted with dichloromethane (3 times, 50 ml each time). The organic layer was dried with magnesium sulfate and the solvent was removed under reduced pressure to obtain a semi-solid substance. This semi-solid substance was purified by column chromatography to obtain a yellow solid product, 4-piperidinyl-1,8-naphthyldimethylimine maleimine, which is the hydrophobic modified segment precursor of one embodiment of this invention, as shown in formula (C). Formula (C). Molecular identification using formula (C): ¹H NMR (600 MHz, Chloroform-d) δ 1.36 – 1.44 (m, 4H), 1.45 – 1.52 (m, 2H), 1.67 – 1.77 (m, 4H), 1.86 – 1.94 (m, 4H), 2.54 (t, J = 7.2 Hz, 2H), 3.17 – 3.27 (m, 6H), 3.84 (t, J = 7.2 Hz, 2H), 4.12 – 4.18 (m, 2H), 5.89 (s, 1H), 6.69 (s, 2H), 7.19 (d, J = 8.1 Hz, 1H), 7.68 (dd, J = 8.4, 7.2 Hz, 1H), 8.41 (dd,J= 8.4, 1.2 Hz, 1H), 8.48 (d,J= 8.1 Hz, 1H), 8.55 (dd,J= 7.2, 1.2 Hz, 1H). HRMS [ESI+]: m / z (%): [M+H]+calculated for C30H35N4O5531.2602; found 531.2603 The second type: N-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionic acid)ethyl)-4-(pyrene-1-yl)butyramine

[0035] First, pyrene butyric acid (1.7 mmol) and N-tert-butoxycarbonyl ethylenediamine (1.73 mmol) were dissolved in tetrahydrofuran in an ice bath. Then, N,N-diisopropylethylamine (4.33 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.08 mmol) were added sequentially, and the reaction was stirred for one day. The solvent was removed by rotary condensation, and chloroform was added for resolution. The mixture was extracted with 5% citric acid and saturated brine. The organic layer was collected and dehydrated with anhydrous magnesium sulfate. Finally, the product was purified by column chromatography (mobile phase: methanol:dichloromethane, starting from 2:98). The product was collected, and the solvent was removed by rotary condensation and vacuum was applied to obtain the product (2-(4-(pyrene-1-yl)butyramino)ethyl)carbamate tert-butyl ester, as shown in formula (D). Formula (D). Molecular identification using formula (D): ¹H NMR (600 MHz, DMSO-d⁶) δ 8.38 (d, J = 9.2 Hz, ¹H), 8.29 – 8.25 (m, 2H), 8.22 (dd, J = 8.5, 3.4 Hz, 2H), 8.17 – 8.10 (m, 2H), 8.06 (t, J = 7.6 Hz, ¹H), 7.94 (d, J = 7.8 Hz, 1H), 7.85 (d, J = 5.6 Hz, 1H), 6.77 (s, ¹H), 3.32 – 3.29 (m, 2H), 3.09 (t, J = 6.2 Hz, 2H), 2.99 (d, J = 6.3 Hz, 2H). 2.23 (t, J = 7.3 Hz, 2H), 2.01 (dd, J = 8.7, 6.6 Hz, 2H), 1.33 (s, 9H). HRMS [ESI+]: m / z(%): calcd.453.2149, obsvd.453.2151 [M+Na]+

[0036] Next, tert-butyl (2-(4-(pyrene-1-yl)butyramine)ethyl)carbamate was dissolved in a mixed solution of 30% trifluoroacetic acid and dichloromethane. The mixture was stirred at room temperature for 3 hours to remove the tert-butyloxycarbonyl protecting group. After the reaction was complete, the solvent was completely removed by a nitrogen blowing device. The impurities were then removed by washing with diethyl ether. The solid was collected by filtration and the solid was concentrated by rotary evacuation and the solvent was completely removed by vacuum to obtain the product 2-(4-(pyrene-1-yl)butyramine)ethyl-1-amino, as shown in formula (E). Equation (E). Molecular identification using formula (E): ¹H NMR (600 MHz, DMSO-d⁶) δ 8.37 (d, J = 9.2 Hz, 1H), 8.24 (t, J = 8.3 Hz, 2H), 8.19 (dd, J = 8.6, 4.5 Hz, 2H), 8.14 – 8.07 (m, 3H), 8.03 (t, J = 7.6 Hz, 1H), 7.98 (s, 2H), 7.91 (d, J = 7.8 Hz, 1H), 3.37 – 3.29 (m, 4H), 2.91 (t, J = 6.4 Hz, 2H), 2.29 (t, J = 7.4 Hz, 2H), 2.03 (t, J = 7.7 Hz, 2H)

[0037] Subsequently, 2-(4-(pyrene-1-yl)butano)ethyl-1-amine (1 equivalent) was dissolved in dichloromethane / dimethylformamide and 0.8 ml of pyridine was added. Then, 3-maleiminopropionic acid N-hydroxysuccinimide (1.5 equivalent) was added and the mixture was refluxed until the next day. The product was poured into diethyl ether to precipitate the crude product. The solid was collected and finally purified by column chromatography under vacuum to obtain the product N-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionic acid)ethyl)-4-(pyrene-1-yl)butanamide, which is the hydrophobic modified segment precursor of another embodiment disclosed herein, as shown in formula (F). Formula (F). Molecular identification of formula (F): ¹H NMR (600 MHz, DMSO-d⁶) δ 8.38 (d, J = 9.3 Hz, ¹H), 8.27 (td, J = 7.5, 1.1 Hz, 2H), 8.23 ​​– 8.19 (m, 2H), 8.15 – 8.10 (m, 2H), 8.05 (t, J = 7.6 Hz, 1H), 7.99 – 7.95 (m, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.83 (t, J = 5.3 Hz, 1H), 6.96 (s, 2H), 3.59 – 3.55 (m, 2H), 3.32 – 3.28 (m, 2H), 3.11 – 3.04 (m, 4H), 2.32 – 2.28 (m, 2H), 2.22 (t, J = 7.3 Hz, 2H), 2.04 – 1.98 (m, 2H). HRMS [FD+]: m / z(%): calcd.481.1996, obsvd. 481.1993 [M]. The third type: N-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionic acid)ethyl)-4-(pyrene-1-yl)butyramine

[0038] First, 2-naphthaleneacetic acid (3.61 mmol), N-tert-butoxycarbonyl ethylenediamine (3.34 mmol), 1-hydroxybenzotriazole (3.613 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (4.213 mmol) were dissolved in tetrahydrofuran, and then N,N-diisopropylethylamine (9.05 mmol) was added. After stirring for 17 hours, the solvent was removed by rotary condensation, and ethyl acetate was added to re-dissolve the solvent. The mixture was then extracted sequentially with 1M hydrochloric acid, water, saturated sodium bicarbonate, water, and saturated brine. The organic layer was collected and dehydrated with anhydrous magnesium sulfate. The solvent was then removed by rotary condensation and vacuum was applied to obtain the product (2-(2-(naphthyl-2-yl)acetamino)ethyl)carbamate tert-butyl ester, as shown in formula (G). Formula (G). Molecular identification of formula (G): ¹H NMR (600 MHz, DMSO-d⁶) δ 8.10 (t, J = 5.7 Hz, ¹H), 7.88 – 7.82 (m, 3H), 7.74 (d, J = 1.8 Hz, ¹H), 7.51 – 7.44 (m, 2H), 7.42 (dd, J = 8.4, 1.8 Hz, 1H), 6.79 (t, J = 5.8 Hz, 1H), 3.57 (s, 2H), 3.10 (q, J = 6.4 Hz, 2H), 3.00 (q, J = 6.4 Hz, 2H), 1.37 (s, 9H)

[0039] Next, tert-butyl (2-(2-(naphthyl-2-yl)acetamino)ethyl)carbamate was dissolved in a mixed solution of 30% trifluoroacetic acid and dichloromethane. The mixture was stirred at room temperature for 3 hours to remove the tert-butyloxycarbonyl protecting group. After the reaction was complete, the solvent was completely removed by a nitrogen blowing device. The impurities were then removed by washing with diethyl ether. The solid was collected by filtration and then concentrated by cyclotron and subjected to vacuum to completely remove the solvent, yielding a white solid product 2-(2-(naphthyl-2-yl)acetamino)ethane-1-amine, as shown in formula (H). Formula (H). Molecular identification of formula (H): ¹H NMR (600 MHz, DMSO-d⁶) δ 8.35 (t, J = 5.8 Hz, ¹H), 7.93 (s, ³H), 7.86 (ddd, J = 16.7, 8.2, 2.0 Hz, ³H), 7.76 (d, J = 1.8 Hz, ¹H), 7.52 – 7.42 (m, ³H), 3.62 (s, 2H), 3.31 (q, J = 6.3 Hz, 2H), 2.89 (t, J = 6.5 Hz, 2H)

[0040] Subsequently, 1.84 mmol of 2-(2-(naphth-2-yl)acetamido)ethane-1-amine was dissolved in dichloromethane / dimethylformamide and 0.8 ml of pyridine was added. Then, 2.76 mmol of 3-maleiminopropionic acid N-hydroxysuccinimide was added and the mixture was refluxed until the next day. The product was poured into diethyl ether to precipitate the crude product. The solid was collected and finally purified by column chromatography under vacuum to obtain the product 3-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-N-(2-(2-(naphth-2-yl)acetamido)ethyl)propionamide, which is the hydrophobic modified chain precursor of another embodiment disclosed herein, as shown in formula (I). Formula (I). Molecular identification using formula (I): ¹H NMR (600 MHz, DMSO-d⁶) δ 8.07 (s, ¹H), 7.98 (s, ¹H), 7.88 – 7.82 (m, ³H), 7.74 (t, J = 1.2 Hz, ¹H), 7.51 – 7.44 (m, 2H), 7.42 (dd, J = 8.4, 1.8 Hz, ¹H), 7.00 (s, 2H), 3.61 – 3.56 (m, 4H), 3.07 (td, J = 5.9, 5.1, 2.7 Hz, 4H), 2.32 – 2.28 (m, 2H). HRMS [ESI+]: m / z(%): calcd.402.1424, obsvd.402.1426 [M+Na]+ Step 2: Preparation of modified glucose oxidase

[0041] In this step, the hydrophobic modified precursor obtained in step one is grafted onto the surface of glucose oxidase (GOx). Specifically, glucose oxidase (0.05 g) is dissolved in 14 mL of phosphate buffer, stirred continuously at room temperature, and placed in a 100 mL round-bottom flask. Next, the hydrophobic modified precursor (approximately 10 moles of GOx) is dissolved in 7 mL of dimethyl sulfoxide, and then added in portions (1 mL every 5 minutes) to the stirred GOx solution. The entire reaction is carried out at room temperature in the dark for 48 hours. After the reaction, the solution is transferred to a dialysis membrane with a molecular weight cutoff (MWCO) of 12.4 kDa for dialysis to remove excess hydrophobic modified precursor and dimethyl sulfoxide. Fresh deionized water is then replaced every 12 hours, and dialysis is performed for a total of three days. Next, the solvent was removed using a freeze dryer to obtain GOx with a hydrophobic modified segment (i.e., modified glucose oxidase, represented by (J) below, where GOx' is GOx lacking an SH group), which is a yellow solid (approximately 21 mg). The modified glucose oxidase should be protected from light and stored at 4°C. Formula (J). [Preparation of Type II Modified Oxidoreductase] Step 1: Preparation of hydrophobic modified chain segment precursors (list two examples) The first type: 6-(1,3-dione-6-(piperidin-1-yl)-1H-benzo[de]isoquinoline-2(3H)-yl)-2-octylaminohexanoic acid

[0042] First, 4-bromo-1,8-naphthalic anhydride (5 mmol) and piperidine (10 mmol) were mixed and dissolved in 50 mL of chlorobenzene. The mixture was stirred under reflux for 8 hours, and the reaction was monitored by thin-layer chromatography. After cooling to room temperature, the mixture was extracted with dichloromethane and brine, and dehydrated with anhydrous magnesium sulfate. The solvent was then removed using a rotary vacuum concentrator, and methanol was added for recrystallization to obtain a yellow, long needle-like solid product, 4-piperidinyl-naphthalic anhydride, as shown in formula (K). Formula (K). Molecular identification of formula (K): 1H NMR (300 MHz, [D6] DMSO): δ = 1.65–1.80 (m, 2H, CH2), 1.8–1.95 (m, 4H, CH2), 3.30 (t, J = 5.3 Hz, 4H, CH2), 4.75 (s, 2H, CH2), 7.37 (dd, J = 7.5, 7.2 Hz, 1H, CH), 7.88 (dd, J = 8.0 Hz, 1H, CH), 8.43 (d, J = 8.1 Hz, CH), 8.45–8.60 (m, 2H, CH)

[0043] Next, 1 g of 2-chlorotriphenylmethyl chloro resin (hereinafter referred to as resin) was swelled in anhydrous dichloromethane for 40 minutes. Then, Nα-fluorenemethoxycarbonyl-Nε-tert-butoxycarbonyl-L-lysine (Fmoc-lysine (Boc)) (2.00 mmol) and N,N-diisopropylethylamine (6.8 mmol) were mixed, dissolved in an appropriate amount of anhydrous dimethylformamide, and added to a special apparatus to react with the resin for one hour to allow the amino acids to bind with the resin. The mixture was washed three times with anhydrous dimethylformamide (at least 2 minutes each time). Then, anhydrous dichloromethane (8 ml), methanol (1.5 ml), and N,N-diisopropylethylamine (0.5 ml) were mixed and added to the mixture to react for 30 minutes. The mixture was washed three times with anhydrous dimethylformamide (at least 2 minutes each time). Finally, 20 ml of anhydrous dichloromethane was added to the mixture. The Nα-fluorenemethoxycarbonyl protecting group was removed by reacting a mixed solution of piperidine and dimethylformamide for 30 minutes, followed by two repeated washings (2 minutes each). Finally, octanoic acid (3.4 mmol), benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate (2.00 mmol), and N,N-diisopropylethylamine (6 mmol) were dissolved in an appropriate amount of anhydrous dimethylformamide, and this solution was added to the apparatus to react with the resin and couple it with the amino acids. After reacting overnight, the completed white solid product (peptide derivative) octanoyllysine was cleaved from the resin using a mixed solution of 90% trifluoroacetic acid and 10% deionized water, as shown in formula (L). Formula (L). Molecular identification of formula (L): ¹H NMR (500 MHz, D₂O) δ 4.36 (dd, J = 8.8, 4.9 Hz, ¹H), 3.00 (t, J = 7.5 Hz, 2H), 2.30 (t, J = 7.1 Hz, 2H), 1.97 – 1.88 (m, ¹H), 1.82 – 1.75 (m, ¹H), 1.73 – 1.66 (m, 2H), 1.61 (d, J = 5.9 Hz, 2H), 1.47 (dd, J = 15.0, 7.2 Hz, 2H), 1.28 (d, J = 10.9 Hz, 10H), 0.85 (d, J = 6.6 Hz, 3H); HRMS [ESI-]: m / z (%): Theoretical value 272.2100, Actual value 272.2110 [MH-]

[0044] Subsequently, 4-piperidinyl-1,8-naphthalene anhydride (400 mg) and octyl lysine (200 mg) were dissolved in 20 ml of triethylamine in ethanol solution and stirred under reflux for 6 hours. The reaction was monitored by thin-layer chromatography every 2 hours. After the reaction was complete, the solvent was removed by rotary evaporator. Finally, the compound was purified by column chromatography. 4-piperidinyl-1,8-naphthalene anhydride was first removed with dichloromethane, and then purified with an extract of dichloromethane:methanol = 9:1 to obtain the yellow solid product 6-(1,3-dione-6-(piperidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)-2-octanamidohexanoic acid (6-(1,3-dioxo-6-(piperidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)-2-octanamidohexanoic acid). acid), namely the hydrophobic modified chain segment precursor of one embodiment of the present disclosure, as shown in formula (M). As shown in formula (M). Molecular identification of formula (M): ¹H NMR (500 MHz, CDCl₃) δ 8.54 (d, J = 6.7 Hz, ¹H), 8.47 (d, J = 7.3 Hz, ¹H), 8.38 (d, J = 8.3 Hz, ¹H), 7.66 (s, ¹H), 7.16 (d, J = 7.9 Hz, ¹H), 6.63 (s, ¹H), 4.45 (s, ¹H), 4.18 (d, J = 21.9 Hz, 2H), 3.23 (s, 4H), 2.27 (dd, J = 15.3, 7.6 Hz, 2H), 2.01 (s, 2H), 1.88 (s, 4H), 1.72 (s, 2H). 1.61 (s, 2H), 1.47 (d,J= 7.3 Hz, 2H), 1.32 – 1.08 (m, 10H), 0.83 (d,J= 6.9 Hz, 3H).13C NMR (125 MHz, DMSO-d 6) δ 172.17, 163.54, 163.00, 156.68, 132.23, 130.54, 129.16, 125.79, 125.45, 122.52, 115.02, 114.90, 53.95, 51.71, 45.55, 40.02, 39.85, 39.69, 39.52, 39.35, 39.19, 39.02, 35.02, 31.15, 30.82, 28.46, 28.34, 27.26, 25.70, 25.61, 25.22, 23.83, 23.04, 22.01, 13.91, 8.80. HRMS [ESI-]: m / z (%): Theoretical value 534.2968, Actual value 534.2998 [MH-] The second type: 6-(1,3-dione-6-(piperidin-1-yl)-1H-benzo[de]isoquinoline-2(3H)-yl)-2-palmitoylaminohexanoic acid

[0045] First, the aforementioned 4-piperidinyl-1,8-naphthalene anhydride (i.e., formula (K)) was prepared.

[0046] Next, 1 g of 2-chlorotriphenylmethyl chloro resin (hereinafter referred to as resin) was swelled in anhydrous dichloromethane for 40 minutes. Then, Nα-fluorenemethoxycarbonyl-Nε-tert-butoxycarbonyl-L-lysine (Fmoc-lysine (Boc)) (2.00 mmol) and N,N-diisopropylethylamine (6.8 mmol) were mixed, dissolved in an appropriate amount of anhydrous dimethylformamide, and added to a special apparatus to react with the resin for one hour to allow the amino acids to bind with the resin. The mixture was washed three times with anhydrous dimethylformamide (at least 2 minutes each time). Then, anhydrous dichloromethane (8 ml), methanol (1.5 ml), and N,N-diisopropylethylamine (0.5 ml) were mixed and added to the mixture to react for 30 minutes. The mixture was washed three times with anhydrous dimethylformamide (at least 2 minutes each time). Finally, 20 ml of anhydrous dichloromethane was added to the mixture. The Nα-fluorenemethoxycarbonyl protecting group was removed by reacting a mixture of piperidine and dimethylformamide for 30 minutes, followed by two repeated washings (2 minutes each). Finally, palmitic acid (3.4 mmol), benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate (2.00 mmol), and N,N-diisopropylethylamine (6 mmol) were dissolved in an appropriate amount of anhydrous dimethylformamide, and this solution was added to the apparatus to react with the resin and couple it with the amino acids. After reacting overnight, the completed white solid product (peptide derivative) palmitoyllysine (as shown in formula (N)) was cleaved from the resin using a mixture of 90% trifluoroacetic acid and 10% deionized water. Formula (N). Molecular identification of formula (N): ¹H NMR (500 MHz, DMSO) δ 12.48 (s, ¹H), 7.99 (d, J = 7.7 Hz, ¹H), 7.70 (s, 2H), 4.15 (s, ¹H), 2.76 (s, 2H), 2.10 (t, J = 7.0 Hz, 2H), 1.69 (s, ¹H), 1.60 – 1.44 (m, 5H), 1.29 (d, J = 51.0 Hz, 26H), 0.85 (d, J = 6.4 Hz, 3H). ¹³C NMR (125 MHz, DMSO-d⁶) δ 173.71, 172.30, 51.39, 38.61, 35.04, 31.26, 30.47, 29.02, 28.78, 28.67, 28.61, 26.51, 25.22, 22.40, 22.06, 13.92; HRMS [ESI-]: m / z (%): theoretical value 384.3352, actual value 384.3357 [MH-].

[0047] Subsequently, 4-piperidinyl-1,8-naphthalene anhydride (400 mg) and palmityl lysine (200 mg) were dissolved in 20 mL of ethanol solution and stirred under reflux for 6 hours. The reaction was monitored by thin-layer chromatography every 2 hours. After the reaction was complete, the solvent was removed by rotary evaporator. Finally, the compound was purified by column chromatography. 4-piperidinyl-1,8-naphthalene anhydride was first removed with dichloromethane, and then purified with an extract of dichloromethane:methanol = 9:1 to obtain the yellow solid product 6-(1,3-dione-6-(piperidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)-2-palmitamidohexanoic acid (6-(1,3-dioxo-6-(piperidin-1-yl)-1H-benzo[de]isoquinolin-2(3H)-yl)-2-palmitamidohexanoic acid). acid), which is the hydrophobic modified segment precursor of another embodiment disclosed herein, as shown in formula (O). Formula (O). Identification of formula (O) molecule: ¹H NMR (500 MHz, CDCl₃) δ 8.53 (d, J = 6.8 Hz, ¹H), 8.45 (d, J = 8.1 Hz, ¹H), 8.36 (d, J = 7.9 Hz, ¹H), 7.65 (s, ¹H), 7.15 (d, J = 7.9 Hz, ¹H), 6.62 (s, ¹H), 4.44 (s, ¹H), 4.21 – 4.10 (m, 2H), 3.22 (s, 4H), 2.31 – 2.19 (m, 3H), 1.99 (s, 2H), 1.87 (d, J = 4.7 Hz, 5H), 1.72 (s, 3H), 1.59 (d,J= 7.2 Hz, 3H), 1.47 (d,J= 7.2 Hz, 3H), 1.33 – 1.07 (m, 26H), 0.87 (dd,J= 8.4, 5.5 Hz, 3H).13C NMR (125 MHz, DMSO-d 6) δ=172.15, 163.53, 162.99, 156.68, 132.22, 130.54, 129.17, 125.78, 125.46, 122.52, 115.03, 114.89, 53.96, 51.65, 45.56, 40.02, 39.85, 39.69, 39.52, 39.35, 39.19, 39.02, 35.01, 31.24, 30.81, 29.02, 28.97, 28.68, 28.65, 28.47, 27.24, 25.70, 25.22, 23.83, 23.04, 22.04, 13.90, 8.81. HRMS [ESI-]: m / z (%): Theoretical value 646.4220, Actual value 646.4225 [MH-] Step 2: Preparation of modified glucose oxidase

[0048] In this step, the hydrophobic modified precursor obtained in step one is grafted onto the surface of glucose oxidase (abbreviated GOx). Specifically, the hydrophobic modified precursor (formula (M): 30 mg / formula (O): 36 g)) and sodium bicarbonate (6 mg) are taken, and the mixture is dissolved in 2 ml of water and stirred at room temperature for 10 minutes. Then, phosphate buffer solution (pH = 7.54, 4 ml) is added, followed by N,N-diisopropylcarbodiimide (12 mg) and hydroxybenzotriazole (8.6 mg) in sequence, and stirred at room temperature for 1.5 hours. Then, glucose oxidase (50 mg) is added and stirred at room temperature for 24 hours. Finally, the mixture is dialyzed for 2 days to remove impurities, and finally lyophilized to obtain the solid product modified glucose oxidase (represented by (P) below, where GOx' is GOx lacking an NH2 group). Formula (P). <Nano Particle Composition>

[0049] The modified oxidoreductase disclosed herein possesses amphiphilic molecular properties due to the grafting of hydrophobic ends, which facilitates its self-assembly into multifunctional enzyme nanoparticles, thereby forming the nanoparticle composition disclosed herein. Specifically, the nanoparticle composition comprises multiple enzyme nanoparticles, and each enzyme nanoparticle is formed by cross-linking multiple modified oxidoreductases with multiple aldehyde or aldehyde-acid molecules. In detail, aldehyde or aldehyde-acid molecules can undergo a Schiff-base reaction with the exposed amino groups on the surface of the modified oxidoreductase to form Schiff-base linkages, thereby causing multiple modified oxidoreductases to cross-link and stably aggregate to form enzyme nanoparticles. These enzyme nanoparticles possess high catalytic stability and activity, can maintain function for extended periods under various environmental conditions, and significantly improve enzyme utilization efficiency, making them suitable for a variety of biomedical and industrial applications.

[0050] In some embodiments, the modified oxidoreductase can be placed in an aldehyde or aldehyde acid solution with a concentration of 0.1 mM to 0.31 mM (e.g., 0.11 mM, 0.13 mM, 0.15 mM, 0.17 mM, 0.19 mM, 0.21 mM, 0.23 mM, 0.25 mM, 0.27 mM, 0.29 mM) for a cross-linking reaction to form enzyme nanoparticles. During the cross-linking process, the concentration of aldehyde or aldehyde acid molecules in the aldehyde or aldehyde acid solution can control the cross-linking density and size of the resulting enzyme nanoparticles, thereby affecting their structural stability and catalytic function. Specifically, when the concentration of aldehyde or aldehyde acid molecules is low, the degree of cross-linking of the modified oxidoreductase is limited, the resulting enzyme nanoparticles are larger in size, have a looser structure, and relatively lower catalytic activity. As the concentration of aldehydes or aldehyde acids increases, the cross-linking reaction intensifies, causing the modified oxidoreductases to aggregate more tightly. The resulting enzyme nanoparticles gradually shrink in size, thus improving catalytic activity and stability. However, when the concentration of aldehydes or aldehyde acids exceeds a certain threshold, excessive cross-linking can lead to excessive aggregation of enzyme nanoparticles, causing the particle size to increase again, thereby affecting catalytic efficiency and stability.

[0051] In some embodiments, the aldehyde or aldehyde acid solution may be a monocarbonyl aldehyde solution, a dialdehyde solution, an aromatic aldehyde solution, an α,β-unsaturated aldehyde solution, a hydroxy aldehyde solution, a keto aldehyde solution, an aldehyde acid solution, or a combination thereof. In other words, the aldehyde or aldehyde acid molecules in the aldehyde or aldehyde acid solution may be monocarbonyl aldehydes, dialdehydes, aromatic aldehydes, α,β-unsaturated aldehydes, hydroxy aldehydes, keto aldehydes, aldehyde acids, or combinations thereof. In some embodiments, the aldehyde or aldehyde acid solution may contain... or or or or or or or Aldehydes or aldehyde acid molecules, or combinations thereof.

[0052] In some embodiments, the average diameter of the enzyme nanoparticles can be from 10 nanometers to 5000 nanometers (e.g., 50 nanometers, 100 nanometers, 150 nanometers, 200 nanometers, 250 nanometers, 300 nanometers, 350 nanometers, 400 nanometers, 450 nanometers, 500 nanometers, 550 nanometers, 600 nanometers, 650 nanometers, 700 nanometers, 750 nanometers, 800 nanometers, 850 nanometers, 900 nanometers, 1000 nanometers, 1100 nanometers, 1200 nanometers, 1300 nanometers, 1400 nanometers, 1500 nanometers, 1600 nanometers, 1700 nanometers, 1800 nanometers, 1900 nanometers, 2000 nanometers, 2500 nanometers, 3000 nanometers, 3500 nanometers, 4000 nanometers, 4500 nanometers). The wide size range of these enzyme nanoparticles gives them excellent biocompatibility and tunability, allowing for customization to meet specific application needs. Smaller enzyme nanoparticles offer higher specific surface area, thereby enhancing catalytic efficiency and reaction rates; while larger enzyme nanoparticles improve stability and durability, making them suitable for long-term enzyme reactions. Furthermore, the controllability of particle size helps optimize enzyme stability in different environments, further expanding their applications in drug synthesis, biosensing, and environmental technologies.

[0053] In some embodiments, when glucose oxidase is used as an oxidoreductase to prepare a modified cycloprotease (i.e., when the dehydrogenated oxidoreductase system is dehydrogenated by glucose oxidase), the catalytic efficiency (Kcat / KM) of the nanoparticle composition for glucose is greater than 0.5 × 10⁴ s⁻¹·M⁻¹ and less than 5 × 10⁴ s⁻¹·M⁻¹, where Kcat is the catalytic constant and KM is the Michaelis constant. For example, the catalytic efficiency of the nanoparticle composition for glucose can be 10⁴ s⁻¹·M⁻¹, 1.5 × 10⁴ s⁻¹·M⁻¹, 2 × 10⁴ s⁻¹·M⁻¹, 2.5 × 10⁴ s⁻¹·M⁻¹, 3 × 10⁴ s⁻¹·M⁻¹, 3.5 × 10⁴ s⁻¹·M⁻¹, 4 × 10⁴ s⁻¹·M⁻¹, or 4.5 × 10⁴ s⁻¹·M⁻¹. In detail, Kcat represents the number of substrate molecules that each enzyme active site can catalyze per unit time, and is a core indicator for evaluating enzyme catalytic ability; KM represents the enzyme's affinity for the substrate, and the smaller the value, the easier it is for the enzyme to form a complex with the substrate; the ratio of the two, Kcat / KM, is an important indicator for evaluating enzyme performance at low substrate concentrations, and the larger the value, the higher the efficiency and selectivity of the enzyme. Therefore, enzyme nanoparticles not only maintain the high activity of natural enzymes, but also possess modified stability and high substrate affinity, making them particularly suitable for applications in fields such as bioconversion, biosensing, and medical diagnostics where reaction rates and selectivity are extremely demanding.

[0054] In some embodiments, when glucose oxidase is used as an oxidoreductase to prepare modified cycloprotozymes (i.e., when the dehydrogenated oxidoreductase system is dehydrogenated by glucose oxidase), the nanoparticle composition is stably viable under conditions of pH 5 to 9 (e.g., 6, 7, 8) and temperature 15°C to 60°C (e.g., 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, or 55°C), and within this pH and temperature range, The catalytic efficiency Kcat / KM of the nanoparticle composition for glucose can be greater than 0.5×10⁴ s⁻¹·M⁻¹ and less than 5×10⁴ s⁻¹·M⁻¹ (e.g., 10⁴ s⁻¹·M⁻¹, 1.5×10⁴ s⁻¹·M⁻¹, 2×10⁴ s⁻¹·M⁻¹, 2.5×10⁴ s⁻¹·M⁻¹, 3×10⁴ s⁻¹·M⁻¹, 3.5×10⁴ s⁻¹·M⁻¹, 4×10⁴ s⁻¹·M⁻¹, 4.5×10⁴ s⁻¹·M⁻¹). <Preparation process of nanoparticle components>

[0055] The preparation processes of several embodiments of the nanoparticle composition are described below to demonstrate the feasibility of this disclosure. [Preparation of nanoparticle components using a type-1 modified oxidoreductase]

[0056] The first-generation modified glucose oxidase (2 mg) was dissolved in deionized water (200 μl). Then, ethanol was gradually added (5 μl at a time) until the solution became turbid. Next, glutaraldehyde was added, and the mixture was stirred at room temperature for 1.5 hours. After the reaction, the formed nanoparticles were collected by centrifugation (12000 g, 15 min). The collected precipitate was resuspended in a mixture of ethanol and deionized water and centrifuged again under the same conditions. Subsequently, the nanoparticles were washed twice with ultrapure water under the same conditions. The final precipitate was resuspended in deionized water and stored at 4°C until use. [Preparation of nanoparticle components using a type II modified oxidoreductase]

[0057] Take 10 mg of modified glucose oxidase (Type II), and slowly add 2 mL of isopropanol (200 μL at a time, stirring thoroughly after each addition). Once the solution becomes turbid, add 20 μL of 8% glutaraldehyde to crosslink the modified oxidoreductase and stir at room temperature for 12 hours. Then centrifuge (12000 g, 15 min). Next, collect the clear supernatant, add the same solvent (isopropanol), and centrifuge again. Repeat this step twice. Then, resuspend the resulting precipitate in isopropanol using an ultrasonic bath and store at 4°C until use. <Nano Drugs>

[0058] The nanoparticle composition disclosed herein can be applied to the development of nanomedicines. Specifically, nanomedicines can include the aforementioned nanoparticle composition, which can actively exert enzymatic activity in vivo, catalyzing reactions against specific substrates. In some embodiments, when glucose oxidase is used as an oxidoreductase to prepare modified cycloprotease, the resulting enzyme nanoparticles can catalyze the decomposition of glucose in the tumor microenvironment, producing gluconic acid and hydrogen peroxide (H2O2), effectively cutting off the glucose supply to tumor cells and thus inhibiting their growth. Since glucose is a key nutrient source for tumor cell metabolism, the metabolic deprivation effect achieved through glucose consumption can achieve the anti-cancer effect of starvation therapy, selectively destroying tumor cells and inhibiting their proliferation. Notably, the H2O2 produced by glucose decomposition can also generate highly reactive hydroxyl radicals (.OH), causing oxidative damage to tumor cells. Based on the above, the nanomedicine disclosed herein can be combined with various treatment methods such as phototherapy, gas therapy, chemotherapy, catalytic therapy, and immunotherapy to achieve synergistic effects across multiple modalities. <Nano-drug complex>

[0059] This disclosure also provides a nano-drug complex comprising the aforementioned nanoparticle composition and drug molecules, wherein the drug molecules bind to the modified oxidoreductase in the nanoparticle composition in a releaseable form. By actively catalyzing substrate reactions and altering the tumor microenvironment at the target site through the nanoparticle composition, and releasing drug molecules in the process, the drug delivery effect can be achieved while inhibiting the nutrient supply to tumor cells, further realizing the dual therapeutic benefits of drug therapy and enzyme therapy. In some embodiments, the drug molecule can be an anticancer drug and can bind to the nanoparticle composition through suitable methods such as covalent bonding, electrostatic adsorption, hydrophobic interaction, or π-π stacking. For example, a hydrophilic drug can be encapsulated in the aqueous core of the enzyme nanoparticles, while a hydrophobic drug can be embedded in the hydrophobic region of the enzyme nanoparticles. This design can achieve stable encapsulation and controlled release of drug molecules, and increase the circulation time and accumulation of drugs in the body and tumor tissue, thereby enhancing therapeutic effects and reducing side effects. <Preparation Process of Nanopharmaceutical Complexes>

[0060] In some embodiments, during the preparation of the nano-drug complex, drug molecules can be added first to a solution of modified oxidoreductase that has not yet self-assembled, and then aldehydes or aldehyde acid molecules can be added to promote the self-assembly behavior of the modified oxidoreductase. Through this method of co-self-assembling the modified oxidoreductase and drug molecules, drug molecules can be stably encapsulated within the enzyme nanoparticles during enzyme self-assembly, thereby forming a stable nano-drug complex.

[0061] The preparation processes of several embodiments of the nano-drug complex are described below to demonstrate the feasibility of this disclosure. [Nano-drug complex loaded with doxorubicin (DOX)]

[0062] The aforementioned modified glucose oxidase (2 mg) was dissolved in deionized water (200 μL). Then, 0.1 mg of DOX hydrochloride (dissolved in 20 μL of deionized water) was added, and the mixture was stirred continuously. Ethanol was then gradually added (20 μL each time) to the modified glucose oxidase solution until the solution became cloudy. At this point, the ethanol to water ratio was approximately 3:2. Glutaraldehyde was then added, and the mixture was stirred at room temperature for 8 hours. The DOX-loaded enzyme nanoparticles were then collected by centrifugation (12000 g, 15 min). The precipitate was resuspended in an ethanol-deionized water mixture and centrifuged again under the same conditions. The mixture was then washed twice with ultrapure water under the same conditions. Finally, the DOX-loaded nanodrug complex was collected and stored at 4°C. Please refer to Figures 13A-13B, where Figure 13A is a transmission electron microscopy (TEM) image (15000X magnification) of the DOX-loaded nano-drug complex 200, and Figure 13B is a magnified view of a portion of Figure 13A. The TEM images show that the nano-drug complex 200 is a complete and clearly defined granular (spherical) structure with a measured particle size of 30.46 ± 6.71 nanometers, confirming that drug molecules can be stably encapsulated within enzyme nanoparticles during enzyme self-assembly, thereby forming a stable nano-drug complex. [Nano-drug complex loaded with paclitaxel (PTX)]

[0063] The aforementioned modified glucose oxidase (2 mg) was dissolved in deionized water (200 μL). Then, 0.1 mg of paclitaxel (dissolved in 20 μL ethanol) was added, and the mixture was stirred continuously. Ethanol was then gradually added (20 μL each time) to the modified glucose oxidase solution until the solution became cloudy. At this point, the ethanol to water ratio was approximately 1:2. Glutaraldehyde was then added, and the mixture was stirred at room temperature for 8 hours. The PTX-loaded enzyme nanoparticles were then collected by centrifugation (12000 g, 15 min). The precipitate was resuspended in an ethanol-deionized water mixture and centrifuged again under the same conditions. The mixture was then washed twice with ultrapure water under identical conditions. Finally, the PTX-loaded nano-drug complex was collected and stored at 4°C. <Signal Sensor>

[0064] The modified oxidoreductase and nanoparticle composition disclosed herein can also be applied to the development of electrical signal sensors. Specifically, please refer to Figure 14, which is a side view of an electrical signal sensor 10 according to some embodiments of this disclosure. The electrical signal sensor 10 may include an electrode layer 11 and a sensing layer 12 disposed on the surface 11a of the electrode layer 11, wherein the sensing layer 12 contains the aforementioned modified oxidoreductase or nanoparticle composition. Such a sensing layer can generate an electrochemical signal, such as the generation of hydrogen peroxide, through a specific catalytic reaction between the modified oxidoreductase and a target substrate (e.g., glucose), thereby triggering a current change detectable by the electrode. Based on this, the sensor can effectively convert changes in substrate concentration into corresponding electrical signals, thereby achieving highly sensitive and selective electrochemical detection. In some embodiments, the sensor can be applied to blood glucose monitoring.

[0065] Because the nanoparticle system is formed through a self-assembly mechanism, its high specific surface area and uniform dispersion provide the sensing layer with more reactive sites, thereby effectively improving the contact probability and reaction efficiency between the enzyme and the substrate. This structural advantage significantly enhances the intensity of the catalytically generated electrical signal and reduces the detection limit of the overall sensing system, enabling it to detect extremely low concentrations of target molecules. Furthermore, the self-assembled nanoparticles also help expand the sensor's operating concentration range, covering target detection needs from trace amounts to high concentrations, and improving overall sensitivity and reproducibility, while also exhibiting a high degree of discernibility for subtle concentration changes. Since the modified oxidoreductase disclosed in this study possesses excellent stability and enzymatic activity, and its self-assembly as a nanoparticle carrier further enhances its stability and resistance to degradation in the sensing environment, it helps extend the lifespan of the sensing layer and reduce background noise. In summary, this sensing design not only possesses highly sensitive and stable electrochemical sensing performance but is also suitable for portable or wearable biomonitoring devices, with the potential to achieve long-term, continuous detection.

[0066] In the following description, the effectiveness of this disclosure will be verified through several comparative examples and several embodiments. It should be understood that this disclosure should not be interpreted as limiting by the embodiments described below. <Experimental Example 1: Particle Size Analysis of Enzyme Nanoparticles>

[0067] In this experimental example, scanning electron microscopy (SEM) was used to measure the particle size of the enzyme nanoparticles in the nanoparticle composition. Specifically, modified glucose oxidase was prepared using the method described above for [preparing a first-class modified oxidoreductase] (note: using a first hydrophobic modified chain precursor), and the modified glucose oxidase was prepared into a nanoparticle composition using the method described above for [preparing a nanoparticle composition using a first-class modified oxidoreductase]. The correlation between glutaraldehyde concentration and the particle size of the enzyme nanoparticles in the nanoparticle composition was analyzed.

[0068] Please refer to Figures 1A to 1I for the experimental results, which are scanning electron microscope images of modified glucose oxidase in glutaraldehyde solutions of different concentrations. More specifically, the glutaraldehyde concentrations in Figures 1A to 1I are 0 mM (code I), 0.10 mM (code II), 0.1 mM (code III), 0.2 mM (code IV), 0.21 mM (code V), 0.22 mM (code VI), 0.23 mM (code VII), 0.31 mM (code VIII), and 0.35 mM (code IX), respectively, and the scale bar (white line in the lower right corner) in each figure represents 1.0 micrometer. The SEM images show that, in the initial state, as the glutaraldehyde concentration increases, the particle size and aggregation degree of the enzyme nanoparticles 100 (white particles in Figures 1A to 1I) self-assembled by the modified glucose oxidase gradually decrease. However, when the concentration exceeds 0.21 mM, the particle size and aggregation degree actually increase. The results above show that the concentration of glutaraldehyde has a significant effect on the self-assembly behavior of modified glucose oxidase, and there is an optimal concentration range (e.g., 0.2 mM to 0.23 mM) that can form enzyme nanoparticles with small particle size and uniform dispersion.

[0069] To further verify this, dynamic light scattering (DLS) was used to analyze the particle size of enzyme nanoparticles 100 in Figures 1A to 1I. The experimental results are presented in Table 1, and some of the results are further presented in Figures 2A to 2D, where Figures 2A to 2D are the particle size distribution maps F1 of enzyme nanoparticles 100 in Figures 1C to 1F, respectively. The experimental results show that the average particle size of enzyme nanoparticles 100 generally decreases with increasing glutaraldehyde concentration, reaching a minimum at a glutaraldehyde concentration of 0.21 mM, consistent with the results observed in the SEM images.

[0070] Table 1 Glutaraldehyde concentration (mM) Average particle size of enzyme nanoparticles (nm) 0 463.28±23.47 0.19 324.97±21.05 0.10 389.36±15.61 0.20 267.54±27.30 0.21 183.19±40.45 0.22 296.31±45.95 0.23 326.33±20.71 0.31 583.54±35.24 0.35 860.25±73.93

[0071] Subsequently, the results from Table 2 were plotted as Figure 3A, which is the average particle size curve of enzyme nanoparticles 100 at different glutaraldehyde concentrations (Figure F2) from Figures 1A to 1H. (Note: Concentrations exceeding 0.35 mM were not further investigated due to significant precipitation in Figure 1I). The symbols (I) to (IX) in Figure 3A correspond to symbols I to IX in Figures 1A to 1H. The results show a non-linear V-shaped relationship between glutaraldehyde concentration and enzyme nanoparticle size. Specifically, the average particle size reaches its minimum at a concentration of 0.21 mM, decreasing from approximately 475 nm to approximately 150 nm. This indicates that at lower concentrations, the enzyme nanoparticles remain relatively large due to limited cross-linking; as the glutaraldehyde concentration increases, the cross-linking reaction intensifies, the particle structure becomes more compact, leading to a reduction in size; when the concentration exceeds... At 0.21 mM, excessive cross-linking leads to particle aggregation, ultimately resulting in an increase in particle size. Combined SEM and DLS analyses show that high concentrations of glutaraldehyde can promote the formation of enzyme nanoparticles, but excessive cross-linking agents cause aggregation, leading to larger particles. This explains the key role of glutaraldehyde in the interaction between enzyme nanoparticles and glutaraldehyde.

[0072] Furthermore, this study further analyzed the steady-state kinetics of the nanoparticle composition at different glutaraldehyde concentrations using the Michaelis-Menten and Lineweaver-Burk models. The results are shown in Figure 3B, which is a graph F3 depicting the reaction rate curves of the nanoparticle composition at different glutaraldehyde concentrations. The symbols (I) to (IX) in Figure 3B correspond to symbols I to IX in Figures 1A to 1H. The experimental results show that the glutaraldehyde concentration and the catalytic efficiency of the nanoparticle composition exhibit a non-linear, inverted V-shaped relationship. When the glutaraldehyde concentration is 0.21 mM, the enzyme nanoparticles have the smallest particle size, and the active surface region of the enzyme is most exposed, thus reaching a peak reaction rate. Conversely, at lower concentrations, due to incomplete cross-linking, the particle size is larger and the structure is loose, limiting enzyme stability and activity, resulting in a lower reaction rate. As the glutaraldehyde concentration continues to increase, excessive cross-linking causes the enzyme nanoparticles to aggregate, hindering reaction sites, and the reaction rate decreases again. This shows that reducing particle size helps improve catalytic efficiency, but excessive condensation and aggregation can reduce catalytic efficiency, highlighting the importance of controlling the degree of crosslinking in enzyme nanoparticle design. <Experimental Example 2: Electrochemical Analysis of Enzyme Nanoparticles>

[0073] In this experimental example, modified glucose oxidase was prepared using the method described above for [Preparation of Type I Modified Oxidoreductase] (Note: using the first hydrophobic modified chain precursor), and the modified glucose oxidase was prepared into nanoparticles using the method described above for [Preparation of Nanoparticles Using Type I Modified Oxidoreductase]. The current response of the nanoparticles at different glutaraldehyde concentrations and different glucose concentrations (2 mM, 10 mM, and 24 mM) was measured by amperometric analysis at a fixed voltage of 0.65 V.

[0074] Please refer to Figure 4 for the experimental results, which shows the current response curves of the nanoparticle composition at different glutaraldehyde and glucose concentrations (F4). Additionally, the experimental data are summarized in Table 2. The results show that at glucose injection concentrations of 2 mM, 10 mM, and 24 mM, the current intensity increases with increasing glutaraldehyde concentration, reaching a peak at 0.21 mM. At this optimal concentration (0.21 mM), the corresponding current intensities are 4.3 μA, 18.0 μA, and 28.3 μA, respectively. When the glutaraldehyde concentration exceeds 0.21 mM, the current intensity decreases sharply with further increases in the crosslinking agent concentration. This trend is inversely related to the particle size changes observed in DLS analysis, indicating that smaller nanoparticles correspond to higher current intensities. This phenomenon is due to the increased surface area of ​​the nanoparticles, enabling them to more effectively immobilize the enzyme and interact with glucose, improving the sensor's sensitivity and signal output, further demonstrating the advantages of the nanoparticle composition in electrochemical glucose detection.

[0075] Table 2 Glutaraldehyde concentration (mM) Glucose concentration (mM) twenty four 10 2 Average current (μA) Standard deviation Average current (μA) Standard deviation Average current (μA) Standard deviation 0 16.82 0.05 10.31 0.47 2.53 0.19 0.06 17.89 0.75 10.72 0.59 2.54 0.13 0.10 18.69 0.40 11.50 0.22 2.52 0.08 0.15 20.71 0.41 13.83 0.18 3.45 0.11 0.19 22.18 0.44 14.11 0.45 3.45 0.33 0.20 25.59 0.38 17.18 0.60 3.85 0.32 0.21 28.26 1.35 17.96 0.64 4.32 0.14 0.22 19.05 0.58 11.48 0.65 3.27 0.23 0.23 16.18 0.12 9.83 0.08 2.40 0.04 0.27 14.80 0.41 9.15 0.12 2.29 0.10 0.31 12.81 0.32 7.63 0.04 1.77 0.18 0.35 11.05 0.28 5.98 0.44 1.43 0.13 <Experimental Example 3: Kinetic Analysis of Enzyme Nanoparticles>

[0076] In this experimental example, modified glucose oxidase was prepared using the method described above for [Preparation of Type I Modified Oxidoreductase] (Note: using the first hydrophobic modified chain precursor). The modified glucose oxidase was then converted into a nanoparticle composition using the method described above for [Preparation of Nanoparticle Composition Using Type I Modified Oxidoreductase]. The UV-Vis absorption spectra of the modified glucose oxidase and the nanoparticle composition catalyzing 10 mM glucose were measured under equal enzyme concentration (2 μg / ml). The experimental results are shown in Figures 5A and 5B, which are the UV-Vis absorption spectra of the nanoparticle composition and the modified glucose oxidase, respectively. The absorption peak appears at 250 nm, corresponding to the UV-Vis absorption peak of H2O2 reported in the literature. Furthermore, the nanoparticle composition produced a higher concentration of H2O2 from glucose, indicating that its catalytic performance was superior to that of the unnanomodified modified oxidoreductase.

[0077] Next, the catalytic efficiency of the nanoparticle composition was investigated using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. Specifically, in a cascade reaction involving horseradish peroxidase (HRP) and ABTS, the modified glucose oxidase and the nanoparticle composition catalyzed the oxidation of glucose to gluconolactone and H₂O₂, respectively, in the presence of oxygen. HRP then reacted with H₂O₂ to promote the oxidation of colorless ABTS to green ABTS radical cations (ABTS⁺). Colorimetric analysis of enzyme activity was then performed. The deepening of the glucose solution color confirmed that, under the same glucose, HRP, and ABTS concentrations and reaction times, the catalytic efficiency of the nanoparticle composition was significantly higher.

[0078] For further analysis, enzyme kinetics were then analyzed using Michaelis-Menten curves and Lineweaver-Burk double reciprocal plots. Specifically, the time-dependent absorbance of ABTS+ at different glucose concentrations at 740 nm was measured (Figures 6A and 6B), and the absorbance data were converted to ABTS+ concentration (Figure 6C). Michaelis-Menten curves for the nanoparticle composition and modified glucose oxidase were plotted (Figures 6D and 6E), and Lineweaver-Burk double reciprocal plots for the nanoparticle composition and modified glucose oxidase were then plotted using double reciprocal conversion (Figures 6F and 6G). Specifically, Figures 6A and 6B are absorbance-time plots (F6) of the nanoparticle composition and modified glucose oxidase at different glucose concentrations, respectively; Figure 6C is the absorbance-concentration correction curve (F7) of ABTS+; Figures 6D and 6E are Michaelis-Menten curves (F8) of the nanoparticle composition and modified glucose oxidase, respectively; and Figures 6F and 6G are Lineweaver-Burk double reciprocal plots (F9) of the nanoparticle composition and modified glucose oxidase, respectively. The enzyme kinetic parameters (VMax, KM, Kcat, and Kcat / KM) of the nanoparticle composition and modified glucose oxidase obtained from the above enzyme kinetic analysis are summarized in Table 3. VMax represents the maximum reaction rate at saturated glucose concentration, KM is the glucose concentration at which the reaction rate reaches 1 / 2VMax, and the meanings of Kcat and Kcat / KM are explained above and will not be repeated here.

[0079] Table 3 VMax (μMs-1) KM (mM) Kcat (s-1) Kcat / KM (M-1s-1) Modified glucose oxidase 0.2411 25.49 388.81 1.49 * 104 Nanoparticles 0.2646 9.33 421.87 4.52 * 10⁴

[0080] Experimental results show that the catalytic efficiency (Kcat / KM) of the nanoparticle composition is more than 300% higher than that of the modified glucose oxidase, highlighting its enhanced catalytic ability. Notably, the catalytic efficiency of the nanoparticle composition disclosed in this study is competitive with that of glucose oxidase reported in the literature, and is approximately 66 times higher than the catalytic efficiency of glucose oxidase nanoparticles (676 M⁻¹s⁻¹). <Experimental Example 4: Analysis of the Effect of pH on the Stability of Enzyme Nanoparticles>

[0081] In this experimental example, modified glucose oxidase was prepared using the method described above for [Preparation of Type I Modified Oxidoreductase] (Note: using the first hydrophobic modified chain precursor), and the modified glucose oxidase was then prepared into nanoparticles using the method described above for [Preparation of Nanoparticles Using Type I Modified Oxidoreductase]. Next, deionized aqueous solutions with different pH values ​​were prepared, and peroxidase (HRP, concentration 0.4 μg / mL) and ABTS (concentration 2.5 mM) were added respectively. The nanoparticles (concentration 0.1 μg / mL) were then added to the above mixture, and 1 mL of this mixture was placed in a UV-Vis spectrometer (model V670, JASCO), with the detection wavelength set to 740 nm (the characteristic absorption wavelength of ABTS+). Subsequently, glucose was added, and the absorbance change over time was measured. The ABTS+ concentration was then converted to time using the standard calibration curve of ABTS+ and the slope of the reaction rate. Various enzyme kinetic parameters were calculated using the Michaelis-Menten kinetic model, supplemented by the Lineweaver-Burk model for further analysis, to explore the steady-state enzyme kinetic behavior of the nanoparticle composition at different pH values. The experimental results are shown in Figure 7, which is the reaction rate curve of the nanoparticle composition at different pH values ​​(F10). Figure 7 shows that the catalytic efficiency of the nanoparticle composition varies with a bell-shaped curve at different pH values, reaching its highest value at pH=7, with Kcat / KM approximately 4.8 × 10⁴ s⁻¹·M⁻¹, indicating that it has optimal enzyme activity in a neutral environment. Although the catalytic efficiency of the nanoparticle composition decreased slightly in slightly acidic (pH=5, Kcat / KM is about 0.5×104s-1·M-1) and slightly alkaline (pH=9, Kcat / KM is about 104s-1·M-1) environments, it maintained stable enzyme activity in the overall range of pH=5 to pH=9, indicating that the nanoparticle composition has good pH tolerance and wide application flexibility. [] <Experimental Example 5: Determination of the number of hydrophobic modified segments in modified oxidoreductase>

[0082] In this experimental example, modified glucose oxidase was prepared using the method described above for [Preparation of the first type of modified oxidoreductase] (Note: using the first hydrophobic modified segment precursor), and the number of hydrophobic modified segments on the modified glucose oxidase was measured using a UV-Vis spectrometer. Specifically, the structure of formula (C) in the first hydrophobic modified segment precursor was dissolved in dimethyl sulfoxide at various concentrations, and its absorbance at a wavelength of 410 nm was measured (see Figure 8A, which is the UV-Vis absorption spectrum F11 of formula (C) and modified glucose oxidase at different concentrations in dimethyl sulfoxide, where curves L2~L6 represent concentrations of 0.188 mM, 0.24 mM, 0.47 mM, 0.94 mM, and 1.88 mM), and a calibration curve was plotted based on this (see Figure 8B, which is the concentration-absorbance calibration curve F12 of formula (C)). Next, the modified glucose oxidase at a concentration of 6.25 μM was dissolved in dimethyl sulfoxide, and its absorbance at a wavelength of 410 nm was measured (see curve L1 in Figure 8A). The concentration of the hydrophobic modified segment formed by formula (C) in the modified glucose oxidase was calculated to be 21.56 μM via the calibration curve, which is 3.45 times the concentration of the modified glucose oxidase, indicating that there are approximately 3 hydrophobic modified segments in the modified glucose oxidase.

[0083] Furthermore, the modified glucose oxidase and glucose oxidase were analyzed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-MS). The results are shown in Figures 9A and 9B, which are the MALDI-MS spectra of the modified glucose oxidase and glucose oxidase, respectively. In Figure 9A, the signal of the modified glucose oxidase appears at 6434.790 (where the molecular weight of the fragment of formula (C) is 530 g / mol), and in Figure 9B, the signal of the glucose oxidase appears at 6360.983 (where each glucose oxidase consists of two identical subunits, and the molecular weight of each subunit is approximately 74200 Da), indicating that the average molar ratio of formula (C) to glucose oxidase is approximately 1.6, that is, approximately 1.6 fragments of formula (C) are grafted onto one glucose oxidase molecule. Based on the combined results of UV-Vis and MALDI-MS, the coupling ratio between formula (C) and glucose oxidase is approximately 1 to 3, indicating that the hydrophobic modified chain segment formed by formula (C) is successfully coupled with glucose oxidase. <Experiment Example 6: Sensor Stability>

[0084] In this experimental example, modified glucose oxidase was prepared using the method described above for [Preparation of the first type of modified oxidoreductase] (Note: using the first hydrophobic modified chain precursor). The modified glucose oxidase was then prepared into nanoparticles using the method described above for [Preparation of nanoparticles using the first type of modified oxidoreductase]. These nanoparticles and the modified glucose oxidase were then used to fabricate sensors E1 and E2, respectively. The stacked structure of sensor E1 is: platinum electrode / poly(3,4-ethylenedioxythiophene (PEDOT) layer / a complex of modified glucose oxidase, bovine serum albumin, and glutaraldehyde / Nafion™ film. The stacked structure of sensor E2 is the same as that of sensor E1, except that the complex of modified glucose oxidase, bovine serum albumin, and glutaraldehyde in sensor E1 is replaced with modified glucose oxidase. Next, stability tests were performed on sensors E1 to E2.

[0085] The results are shown in Figures 10A to 10D. Figure 10A is a graph showing the current density changes of sensors E1-E2 in phosphate buffer after 7 days, and Figure 10B is a bar graph showing the current density changes of sensors E1-E2 in phosphate buffer after 7 days. The results show that the activity loss of sensor E1 is significantly lower. Figure 10C is a graph showing the fluorescence intensity-concentration relationship of sensors E1-E2 in phosphate buffer. The results show that the modified glucose oxidase in sensor E1 has lower solubility than the glucose oxidase in sensor E2, and therefore has higher stability. Figure 10D is a graph showing the cumulative release efficiency of sensors E1-E2 in phosphate buffer after 7 days. The results show that the modified glucose oxidase in sensor E1 has stability due to its nanostructure, and is not easily lost in large quantities in a short period of time. This effectively reduces the risk of enzyme inactivation, thereby maintaining stable enzyme activity over a long period of time and improving the reliability and accuracy of the sensor under long-term operation. <Experimental Example 7: Specificity of Modified Oxidoreductase>

[0086] In this experimental example, the enzyme specificity of the aforementioned sensor E1 was tested. Please refer to Figure 11, which shows the current changes (voltage 0.65V) when different substrates were added to sensor E1. At time points (a) to (f), glucose (2mM), ascorbic acid (0.34mM), acetaminophen (1.3mM), fructose (2mM), galactose (3.3mM), and glucose (2mM) were added sequentially. The results show that the current signal significantly increased upon the addition of glucose, indicating that the sensor has good selectivity and specificity for glucose. However, there was no significant current response to common interfering substances such as ascorbic acid, acetaminophen, fructose, and galactose, demonstrating that the sensor possesses high selectivity and can effectively avoid non-specific signal interference, improving its reliability and accuracy in detecting glucose in actual biological fluids. <Experimental Example 8: Analysis of the Influence of Temperature on the Stability of Enzyme Nanoparticles>

[0087] In this experimental example, modified glucose oxidase was prepared using the method described above for [Preparation of Type I Modified Oxidoreductase] (Note: using the first hydrophobic modified chain precursor), and the modified glucose oxidase was then prepared into a nanoparticle composition using the method described above for [Preparation of Nanoparticle Composition Using Type I Modified Oxidoreductase]. Next, an enzyme reaction solution containing the nanoparticle composition (concentration 0.1 μg / mL), HRP (concentration 0.4 μg / mL), and ABTS (concentration 2.5 mM) was prepared. 400 μL of the reaction solution was placed in a J-815 circular dichroism spectrometer for subsequent measurements, and the circulating thermostatic water bath was turned on to adjust the temperature to the desired set value. Once the reaction system stabilized at the target temperature, the measurement wavelength was set to 740 nm (the characteristic absorption wavelength of ABTS+), and glucose was added as the reaction substrate. Subsequently, the change in absorbance over time was recorded. By using the standard curve and absorbance change rate of ABTS+, the corresponding ABTS+ concentration change can be calculated. Then, based on the Michaelis-Menten kinetic model, various enzyme kinetic parameters can be calculated to evaluate the catalytic efficiency and stability of nanoparticle components under different temperature conditions.

[0088] The experimental results are shown in Figure 12, which is the reaction rate curve of the nanoparticle composition at different temperatures (F14). Figure 12 shows that the nanoparticle composition exhibits enzymatic activity at temperatures ranging from 15°C to 60°C, with the best catalytic efficiency at 35°C, where Kcat / KM is approximately 6.0 × 10⁴ s⁻¹·M⁻¹. Furthermore, it maintains high reactivity at room temperature (25°C), demonstrating that the nanoparticle composition exhibits good stability and catalytic ability within the common operating temperature range. Overall, the catalytic efficiency of the nanoparticle composition for glucose at temperatures ranging from 15°C to 60°C is greater than 0.5 × 10⁴ s⁻¹·M⁻¹ and less than 6.5 × 10⁴ s⁻¹·M⁻¹ (e.g., 10⁴ s⁻¹·M⁻¹, 1.5 × 10⁴ s⁻¹·M⁻¹, 2 × 10⁴ s⁻¹·M⁻¹, 2.5 × 10⁴ s⁻¹·M⁻¹, 3 × 10⁴ s⁻¹·M⁻¹, 3.5 × 10⁴ s⁻¹·M⁻¹, 4 × 10⁴ s⁻¹·M⁻¹, 4.5 × 10⁴ s⁻¹·M⁻¹, 5.5 × 10⁴ s⁻¹·M⁻¹, 6 × 10⁴ s⁻¹·M⁻¹), demonstrating that the nanoparticle composition possesses good temperature tolerance and broad application flexibility.

[0089] Although the present disclosure has been described above with reference to embodiments, it is not intended to limit the present disclosure. Anyone skilled in the art may make various modifications and refinements without departing from the spirit and scope of the present disclosure. Therefore, the scope of protection of the present disclosure shall be determined by the appended claims.

[0090] I,II,III,IV,V,VI,VII,VIII,IX,(I),(II),(III),(IV),(V),(VI),(VII),(VIII),(IX): code number 10: Signal Sensor 11: Electrode layer 11a: Surface 12: Sensing layer 100: Enzyme nanoparticles 200: Nano-drug complex F1: Particle size distribution diagram F2: Average Particle Size Curve F3: Reaction rate curve F4: Current Response Curve F5: UV-Vis absorption spectrum F6: Absorbance-Time Plot F7: Absorbance-Concentration Correction Curve F8: Michaelis-Menten curve F9: Lineweaver-Burk Double Countdown Chart F10: Reaction rate curve F11: UV-Vis absorption spectrum F12: Concentration-Absorbance Correction Curve F13: MALDI-MS chart F14: Reaction Rate Curve L1~L6: Curves E1~E2: Sensors (a),(b),(c),(d),(e),(f): Time points

[0091] Domestic storage information (please note in order of storage institution, date, and number) none Overseas storage information (please note in the order of storage country, institution, date, and number) none

Claims

1. A modified oxidoreductase, comprising: A dehydrogenated oxidoreductase having at least one dehydrogenated thiol group, wherein the number of the at least one dehydrogenated thiol group is 1 to 20; and a modified segment having the structure of formula (1) and bonded to the at least one dehydrogenated thiol group: Formula (1), wherein A1 is a first segment having a first π-conjugation system, the first π-conjugation system being an aromatic ring π-conjugation system, X being hydrogen, alkyl, carboxyl, amide or ester, n1 being an integer from 0 to 20, and m1 being an integer from 0 to 20.

2. The modified oxidoreductase as claimed in claim 1, wherein the first segment is of formula (3), formula (4) or formula (5): Formula (3); Formula (4); Formula (5).

3. The modified oxidoreductase as claimed in claim 1, wherein the dehydrogenated oxidoreductase is generated by dehydrogenation of glucose oxidase, glucose dehydrogenase, pyruvate oxidase, xanthine oxidase, acetylcholinesterase, lactate oxidase, uricase oxidase, uricase, pyrroloquinolinequinone glucose dehydrogenase-A, pyrroloquinolinequinone glucose dehydrogenase-B, NAD(P)-dependent glutamate dehydrogenase, FAD-dependent glutamate dehydrogenase, cholesterol oxidase, or a sulfur-containing enzyme.

4. A method for preparing a nanoparticle composition, comprising: A plurality of modified oxidoreductases as described in claim 1 are placed in a dialdehyde solution with a concentration of 0.1 mM to 0.31 mM.

5. A nanoparticle composition, comprising: A plurality of enzyme nanoparticles, each of which is cross-linked from a plurality of modified redox enzymes as described in claim 1 and a plurality of dialdehyde molecules.

6. The nanoparticle composition as claimed in claim 5, wherein the average diameter of the enzyme nanoparticles is from 10 nanometers to 5000 nanometers.

7. The nanoparticle composition as claimed in claim 5, wherein the dehydrogenated oxidoreductase is formed by dehydrogenation of glucose oxidase, and the catalytic efficiency of the nanoparticle composition for glucose, Kcat / KM, is greater than 0.5 × 10⁴ s⁻¹·M⁻¹ and less than 5.0 × 10⁴ s⁻¹·M⁻¹, where Kcat is the catalytic constant and KM is the Michaelis constant.

8. An electrical signal sensor, comprising: One electrode layer; And a sensing layer disposed on the surface of the electrode layer, wherein the sensing layer comprises nanoparticles as described in claim 5.

9. A nano-drug comprising: The nanoparticle composition as described in claim 5.

10. A nano-drug complex, comprising: The nanoparticle composition as described in claim 5; And a drug molecule, in a releasable form, binds to any of the modified oxidoreductases in the nanoparticle composition.

11. The nano-drug complex as claimed in claim 10, wherein the drug molecule is an anticancer drug.

Images