Preparation of a composite nanomotor based on bio-enzyme catalysis-Fenton-like reaction and its application in water treatment

Abstract

This invention belongs to the field of wastewater treatment technology, specifically relating to the preparation of a composite nanomotor based on bio-enzyme catalysis and a Fenton-like reaction, and its application in water treatment. To develop a multi-stage enzyme-driven nanomotor that requires no external fuel and possesses good biocompatibility and high catalytic activity, this invention constructs a three-enzyme cascade nanomotor by loading three cascade reaction enzymes—glucose amylase, glucose oxidase, and lignin peroxidase—on nanoparticles with a magnetic Fe3O4 core, a mesoporous silica shell, and modified with ammoniation. This nanomotor can decompose and remove pollutants in water through enzyme catalysis and a Fenton-like reaction, and can be self-driven by utilizing the concentration gradient and bubbles generated during pollutant decomposition, without relying on external fuel or stimuli (sound, light, magnetism, etc.). It has significant application prospects in the field of wastewater treatment.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment technology, specifically relating to the preparation of a composite nanomotor based on bio-enzyme catalysis-Fenton-like reaction and its application in water treatment. Background Technology

[0002] With the acceleration of industrialization and the increasing demands for water quality, large amounts of industrial wastewater must undergo rigorous treatment to meet discharge standards. Industries such as coking, petrochemicals, garment manufacturing, pharmaceuticals, and food processing generate significant quantities of wastewater containing pollutants such as phenols, azo compounds, and oils. These pollutants, due to their toxicity and reluctance to biodegrade, pose potential threats to the ecological environment and human health, thus necessitating efficient treatment. However, conventional physical, chemical, and biological treatment technologies (such as chemical precipitation, adsorption, chemical oxidation, membrane separation, and activated sludge processes) suffer from high costs, high energy consumption, secondary pollution from residual sludge and chemical agents, and large land area requirements, resulting in less than ideal treatment technologies. Miniaturization, high efficiency, low energy consumption, and the absence of secondary pollution are the goals currently pursued in wastewater treatment technology research and development.

[0003] Nanotechnology offers new solutions to the current challenges of wastewater treatment. Among these, nanomotors are micro- and nanoscale machines, also known as micro- and nanorobots, capable of converting chemical, light, and thermal energy into self-propulsion to perform specific tasks. Nanomotors possess advantages such as small size, large specific surface area, active movement, and the ability to perform precise operations in complex environments, making them promising for applications in biomedicine (e.g., drug delivery, antibacterial agents, treatment of neurodegeneration), biomimicry (e.g., biosensing), and environmental remediation (e.g., wastewater treatment and water quality monitoring). Currently, researchers both domestically and internationally have fabricated various nanomotors for treating different pollutants in wastewater, such as organic compounds, microplastics, hormones, heavy metals, and petroleum. The self-driving and highly catalytic properties of nanomotors have improved the removal efficiency of these pollutants. To date, numerous nanomotors for wastewater treatment have been reported, with most achieving pollutant removal through chemical reactions with added substrates or by external fields (e.g., magnetic fields, light fields, ultrasound). Motors driven by external fields exhibit good directional motion behavior and can operate under confined conditions. However, the demanding environmental requirements of external machines (magnetic fields, infrared light, sound fields, etc.) limit their application. Motors driven by chemical catalysis rely on bubbles or product concentration gradients generated by the catalytic reaction as a power source for self-drive. They possess the ability to degrade pollutants in situ, avoiding dependence on instruments or the external environment. However, they also require external fuel (e.g., H2O2), which can cause secondary pollution. Furthermore, their preparation methods are complex, and they can only degrade a limited range of pollutants. To address the current limitations of micro / nano motors in pollutant degradation, such as the need for external fuel and the limited range of pollutants that can be degraded, it is hoped that using pollutants in water as fuel, and leveraging the concentration gradients and bubbles generated by the pollutants' own catalytic degradation reactions, to drive the micro / nano motors—that is, using pollutant decomposition directly as the driving force—could overcome the need for external fuel and achieve simultaneous nanomotor driving and pollutant removal, providing more favorable conditions for the application of nanomotor technology in wastewater treatment.

[0004] Bioenzymes are environmentally friendly and highly efficient catalysts that generate energy during the catalytic decomposition of pollutants. They possess advantages such as specificity, mild reaction conditions, high catalytic efficiency, and non-toxicity, making them a good choice for driving nanomotors. Therefore, it is possible to consider preparing bioenzymes capable of directly decomposing pollutants in wastewater to drive micro / nanomotors. Simultaneously, enzyme immobilization at the nanoscale can overcome the problems of easy inactivation, poor stability, and low reusability of natural free enzymes. Furthermore, the immobilized enzymes exist in the form of nanomotors, providing power to drive the motor while catalyzing the decomposition of pollutants. This achieves efficient contact between the enzyme and pollutants, improving pollutant removal efficiency and eliminating the need for a power source for the entire motor system, thus saving energy. Currently, the bioenzymes loaded on nanomotors are mostly single enzymes or different types of enzymes to remove different target pollutants, such as using laccase and lipase in combination to decompose bisphenol A and triacetin. However, this would result in limited driving force for nanomotors, and the removal of pollutants would be limited by relying solely on the catalytic action of biological enzymes. If a carrier-loaded cascade enzyme could be used to generate a tiered fuel power source through the combined reaction of biological enzymes and substrates, and the carrier and biological enzyme catalytic intermediates, the driving force of nanomotors would be stronger, the motion effect would be more obvious, and it would be more conducive to achieving stable and efficient pollutant degradation and enhanced nanomotor motion behavior.

[0005] Therefore, it is necessary to develop a multi-stage enzyme-driven nanomotor that requires no external fuel, has good biocompatibility and high catalytic activity to solve the above problems. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, this invention fabricates a three-enzyme cascade nanomotor by immobilizing lignin peroxidase, glucose oxidase, and glucoamylase on nanoparticles with a magnetic Fe3O4 core, a mesoporous silica shell, and modified by amylation. In this nanomotor, the three enzymes—glucosylase, glucose oxidase, and lignin peroxidase—can produce a cascade reaction: glucosylase catalyzes the production of glucose from starch, glucose oxidase utilizes glucose to generate H2O2, and lignin peroxidase utilizes H2O2 to catalyze the decomposition of pollutants. Simultaneously, the Fe3O4 core undergoes a Fenton-like reaction with H2O2 to generate hydroxyl radicals, further decomposing pollutants. The combined effect of these two processes enables the efficient decomposition and removal of pollutants, such as azo dyes. Furthermore, the concentration gradient and bubbles generated during pollutant decomposition provide self-driving capability for the nanomotor.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of this invention provides a method for preparing a three-enzyme cascade nanomotor, which involves first preparing Fe3O4 nanoparticles and modifying them into Fe3O4@SiO2-NH2 nanoparticles, and then loading three cascade reaction enzymes—glucose amylase, glucose oxidase, and lignin peroxidase—on the Fe3O4@SiO2-NH2 nanoparticles to obtain a three-enzyme cascade self-driven nanomotor.

[0009] Preferably, the method for loading three cascade reaction enzymes onto Fe3O4@SiO2-NH2 nanoparticles is as follows: first, Fe3O4@SiO2-NH2 nanoparticles are dispersed in a solvent, then glutaraldehyde is added, the reaction is carried out at room temperature with shaking, and after washing and drying, the nanoparticles are dispersed in PBS buffer solution. Then, glucoamylase, glucose oxidase and lignin peroxidase are added, and after shaking reaction, the three-enzyme cascade nanomotor is obtained.

[0010] Preferably, Fe3O4 nanoparticles are synthesized by a hydrothermal method, and Fe3O4 nanoparticles are modified by tetraethoxysilane (TEOS) / 3-aminopropyltriethoxysilane (APTES) to prepare Fe3O4@SiO2-NH2 nanoparticles.

[0011] More preferably, the effective activity units of the glucoamylase, glucose oxidase, and lignin peroxidase are 2 U / mg, 250 U / mg, and 0.1 U / mg, respectively; and the final concentrations of the glucoamylase, glucose oxidase, and lignin peroxidase in the shaking reaction are 8-12 U / mL, 1-2 U / mL, and 0.4-0.6 U / mL, respectively.

[0012] More preferably, the room temperature shaking reaction takes 5-7 hours, and the second shaking reaction takes 10-15 hours at a temperature of 4°C.

[0013] More preferably, the amount of glutaraldehyde added is 4-6% by volume percentage.

[0014] More preferably, the concentration of the Fe3O4@SiO2-NH2 nanoparticles in the solvent is 0.5-0.7 mg / 1 mL.

[0015] More preferably, the solvent includes (but is not limited to) ethanol.

[0016] The second aspect of the present invention provides a three-enzyme cascade nanomotor prepared using the preparation method described in the first aspect.

[0017] This invention co-immobilizes lignin peroxidase, glucose oxidase, and glucoamylase on nanoparticles with a magnetic Fe3O4 core, a mesoporous silica shell, and an aminated surface. Glucoamylase converts starch in wastewater into glucose, which is then catalyzed by glucose oxidase to decompose into H2O2. Lignin peroxidase then uses H2O2 to decompose azo dyes in the wastewater, generating oxygen. Simultaneously, H2O2 reacts with the Fe3O4 core in a Fenton-like reaction. Throughout the reaction, the oxygen generated by the H2O2 reaction and the concentration gradients from starch and dye degradation act as driving forces, enabling the movement of the enzyme-loaded nanomaterials. This achieves the successful construction of an enzyme cascade self-driven magnetic nanomotor and the efficient removal of pollutants.

[0018] The third aspect of this invention provides the application of the three-enzyme cascade nanomotor described in the second aspect in wastewater treatment, wherein the wastewater contains starch, including (but not limited to) dyeing and printing wastewater and pharmaceutical wastewater containing starch.

[0019] Preferably, the wastewater also contains azo dyes.

[0020] Preferably, the azo dye includes (but is not limited to) Congo red.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] This invention constructs a three-enzyme cascade nanomotor by loading three cascade reaction enzymes—glucose amylase, glucose oxidase, and lignin peroxidase—on nanoparticles with a magnetic Fe3O4 core, a mesoporous silica shell, and modified with ammoniation. This nanomotor can decompose and remove pollutants in water through enzymatic catalysis and a Fenton-like reaction, and can be self-driven by the concentration gradient and bubbles generated during pollutant decomposition, without relying on external fuels or stimuli (sound, light, magnetism, etc.). It has significant application prospects in the field of wastewater treatment. Overall, this invention has the following advantages:

[0023] (1) Glucoamylase, glucose oxidase and lignin peroxidase can produce a cascade reaction. Glucoamylase can catalyze the starch in wastewater to produce glucose. Glucose can be further catalyzed by glucose oxidase to produce H2O2. Then lignin peroxidase will use H2O2 to decompose pollutants in wastewater, such as azo dyes.

[0024] (2) Fe3O4 in the carrier will undergo a Fenton-like reaction with H2O2 produced by glucose oxidase catalyzing glucose, and the resulting hydroxyl radicals can decompose pollutants in mineralized water.

[0025] (3) The synergistic effect of biological enzyme catalysis of lignin peroxidase and chemical oxidation of Fenton-like reaction can achieve efficient removal of pollutants in water. Compared with single biological enzyme catalysis or Fenton-like reaction, the synergistic effect of this system has a higher efficiency in removing pollutants.

[0026] (4) During the reaction, H2O2 produced by the decomposition of pollutants in wastewater can be used to provide substrate H2O2 for lignin peroxidase and Fenton-like reaction without the need for artificial addition of H2O2, which can reduce costs.

[0027] (5) The driving force for the movement of the nanomotor comes from the concentration gradient generated by the decomposition of pollutants by bio-enzyme catalysis and Fenton-like reaction, as well as the oxygen bubbles generated when lignin peroxidase uses H2O2, without the need for external fuel or external environmental stimulation. Attached Figure Description

[0028] Figure 1 The morphology of the three-enzyme cascade nanomotor particles dispersed in aqueous solution;

[0029] Figure 2 Fourier transform infrared spectrum of a three-enzyme cascade nanomotor;

[0030] Figure 3 CLSM images of a three-enzyme cascade nanomotor (A. Bright field; B. Blue - glucose oxidase; C. Red - glucosyl amylase; D. Green - lignin peroxidase; E. Superimposed of three fluorescence types);

[0031] Figure 4 The degradation effects of Congo red on three-enzyme cascade nanomotors, two-enzyme (glucosyl amylase and glucose oxidase) cascade nanoparticles, free three enzymes, inactivated immobilized three enzymes, and modified iron oxide nanoparticles are shown in the figure.

[0032] Figure 5 The spin capture EPR signal of ·OH in Congo red and starch solutions using DMPO as a free radical scavenger was obtained by electron paramagnetic resonance (EPR) spectroscopy at 30 °C and pH 6.0 for a three-enzyme cascade nanomotor and a two-enzyme (glucosylase and glucose oxidase) cascade nanoparticles.

[0033] Figure 6 The image shows the motion behavior of a three-enzyme cascade nanomotor observed in starch and Congo red solution using a laser confocal microscope (LSM800 with Airyscan). Detailed Implementation

[0034] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0036] Example: Preparation of a cascaded nanomotor of three enzymes (glucosylamylase, glucose oxidase, and lignin peroxidase)

[0037] (1) Fe3O4 nanoparticles were first synthesized by hydrothermal method, and then modified by TEOS / APTES (for specific methods, refer to "Preparation of Laccase-Driven Nanomotors and Their Application in Pollutant Degradation. Huang Yongwei. Master's Thesis, Harbin Institute of Technology. 2021.").

[0038] (2) 0.6 mg of modified nanoparticles (Fe3O4@SiO2-NH2) were dispersed in 1 mL of anhydrous ethanol, and 0.05 mL of 5% glutaraldehyde was added. The mixture was shaken for 6 hours at room temperature. After the reaction was complete, the nanoparticles were washed 6 times with anhydrous ethanol and dried under vacuum at 60 °C for 4 hours. The nanoparticles were then dispersed in 1 mL of PBS buffer solution (pH = 6.0), and 10 U / mL of glucoamylase, 1 U / mL of glucose oxidase, and 0.5 U / mL of lignin peroxidase were added (the effective activity units of glucoamylase, glucose oxidase, and lignin peroxidase were 2 U / mg, 250 U / mg, and 0.1 U / mg, respectively). The mixture was then shaken at 4 °C for 12 hours to obtain a three-enzyme cascade nanomotor. The morphology of the three-enzyme cascade nanomotor particles dispersed in aqueous solution is shown in the figure. Figure 1 As shown.

[0039] pass Figure 2 The Fourier transform infrared spectra show that a value of 597 cm⁻¹ was observed in the Fe₃O₄ and Fe₃O₄@SiO₂ curves. -1 and 1093cm -1 The peaks at these points represent the Fe-O-Fe vibration and the Si-O-Si reverse stretching vibration, respectively, indicating that Fe3O4 was successfully synthesized and coated with SiO2. A new peak at 1634 cm⁻¹ appears in the Fe3O4@SiO2-NH2 curve. -1 (NH bending vibration peak) and 3358cm -1The (NH stretching vibration peak) indicates that amino functionalization of the nanoparticles has been achieved. Furthermore, the 1413 cm⁻¹ peak in the Fe₃O₄@SiO₂-NH₂-enzymes curve demonstrates this. -1 The CN stretching vibration peak indicates that the enzyme was successfully immobilized on the surface of the nanoparticles. The presence of these functional groups indicates that the enzyme-driven nanomotor was successfully synthesized. Simultaneously, through... Figure 3 The CLSM image of the nanomotors (AD) shows that the green fluorescence is lignin peroxidase stained with FTIC, the blue fluorescence is glucose oxidase stained with Cy-5, and the red fluorescence is glucosyl amylase stained with Rhodamine B. The nanoparticles are clearly encapsulated by these three enzymes through CLSM. Figure 3 (E) shows the effect after the three fluorescences are superimposed. It can be seen that the distribution of the three enzymes in the same part of the nanoparticle is relatively consistent, which shows that the three enzymes are uniformly fixed on the nanoparticle.

[0040] Comparative Example: Preparation of a dual-enzyme (glucosylamylase and glucose oxidase) cascade nanomotor

[0041] Fe3O4 nanoparticles were first synthesized via a hydrothermal method and modified using TEOS / APTES. Then, using the modified nanoparticles (Fe3O4@SiO2-NH2) as the substrate material, 0.6 g of the nanoparticles were dispersed in 1 mL of anhydrous ethanol, followed by the addition of 0.05 mL of 5% glutaraldehyde. The mixture was shaken at room temperature for 6 hours. After the reaction was complete, the nanoparticles were washed six times with anhydrous ethanol and then vacuum-dried at 60 °C for 4 hours. The resulting nanoparticles were then dispersed in PBS buffer solution, and glucoamylase and glucose oxidase were added to a final concentration of 10 U / mL. The mixture was then shaken at 4 °C for 12 hours to obtain a dual-enzyme cascade nanomotor. Experimental example: Degradation performance of the nanomotor on pollutants.

[0042] 0.6 mg of the three-enzyme cascade nanomotor from the example was added to a solution with a starch concentration of 1 wt% and a Congo red concentration of 350 μM–450 μM, and the pH was adjusted to 6.0. The solution was then placed at T = 30 °C to treat the Congo red. The degradation effects of the two-enzyme (glucosylase and glucose oxidase) cascade nanomotor, free three enzymes, inactivated immobilized three enzymes (three-enzyme cascade motor sterilized by autoclaving at 1.01 MPa, 121 °C, and 20 min), and modified iron oxide nanoparticles (Fe3O4@SiO2-NH2) on Congo red were compared.

[0043] like Figure 4As shown, when the substrate (Congo red) concentration is 350 μM to 450 μM, the removal rate of Congo red by the three-enzyme cascade nanomotor is over 90%, which is higher than that of other materials. This indicates that in addition to the weak adsorption effect of the nanomaterial (Fe3O4@SiO2-NH2, inactivated three-enzyme cascade nanomotor) and the simple decomposition effect of the enzyme (free three enzymes), other major reactions for removing Congo red also occur, and the reactivity of the three-enzyme cascade nanomotor is significantly higher than that of the two-enzyme cascade nanomaterial.

[0044] like Figure 5 As shown, DMPO was used as a free radical scavenger to determine the free radical content of the three-enzyme cascade nanomotor + starch (…). Figure 5 A), a three-enzyme cascade nanomotor + starch + Congo red ( Figure 5 B), a dual-enzyme (glucosylamylase-glucose oxidase) cascade nanomotor + starch ( Figure 5 C), and a dual-enzyme (glucosylamylase-glucose oxidase) cascade nanomotor + starch-Congo red ( Figure 5 D) The presence of free radicals in the system (reaction volume 600 μL, Congo red concentration 350-450 μM, starch 1 wt%, nanomotor 0.6 mg). Spin capture EPR signal maps of ·OH in the reaction system were obtained using electron paramagnetic resonance (EPR) spectroscopy at 30℃ and pH 6.0. The presence of ·OH signals in all four systems indicates that the reaction is a cascade reaction of glucoamylase and glucose oxidase producing H₂O₂, which then reacts with Fe₃O₄ in a Fenton-like reaction to generate ·OH. The strong oxidizing properties of ·OH enable efficient removal of Congo red.

[0045] In addition, three enzymes (glucosidase, glucose oxidase, and lignin peroxidase) were stained with Cy-5, filtered, dialyzed, and then immobilized on nanoparticles according to the method described in the example. The three-enzyme cascade nanomotor was then added to a 1 wt% starch and 450 μM Congo red solution. After stabilization, it was observed under a 100× objective lens in a laser confocal dish. Figure 6 As shown in the figure, the large particles are the substrate starch. Within a short time, the three-enzyme cascade motor, stained blue by Cy-5, quickly approaches the substrate starch (from left to right, downwards in the figure) and reacts. This indicates that the driving force of this three-enzyme cascade nanomotor comes from the concentration gradient generated by the bio-enzyme catalysis and Fenton-like reaction decomposing pollutants, as well as the oxygen bubbles generated when lignin peroxidase utilizes H2O2, without requiring external fuel or external environmental stimulation.

[0046] In summary, the nanomotors constructed using the method of this invention can decompose and remove starch and azo dyes from water through enzymatic catalysis and Fenton-like reactions. On the one hand, the removal of azo dyes in the reaction system is the result of the combined action of biological enzyme catalysis and Fenton-like reactions, resulting in higher efficiency in removing azo dyes. On the other hand, the concentration gradient and bubbles generated by the decomposition of pollutants using a three-enzyme cascade reaction in the reaction system can drive the movement of nanomaterials, enabling rapid movement of micro- and nanomotors without external fuel or stimuli (sound, light, magnetism, etc.).

[0047] The above describes the preparation of nanomotors and their process for removing pollutants. However, this invention is not limited to the hydrothermal synthesis of iron oxide nanoparticles; iron oxide nanoparticles synthesized by other precipitation methods can also be used as carrier materials. The treated solutions are not limited to Congo red solutions; any wastewater containing starch and lignin peroxidase substrates can be used, such as textile dyeing wastewater and pharmaceutical wastewater.

[0048] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. An application of a three-enzyme cascade nanomotor in wastewater treatment, characterized in that, The wastewater contains starch and azo dyes; The preparation of the three-enzyme cascade nanomotor involves first preparing Fe3O4 nanoparticles and modifying them into Fe3O4@SiO2-NH2 nanoparticles, and then loading three cascade reaction enzymes, namely glucose amylase, glucose oxidase, and lignin peroxidase, onto the Fe3O4@SiO2-NH2 nanoparticles to obtain the three-enzyme cascade self-driven nanomotor. The method for loading three cascade enzymes onto Fe3O4@SiO2-NH2 nanoparticles is as follows: First, Fe3O4@SiO2-NH2 nanoparticles are dispersed in a solvent, then glutaraldehyde is added, the mixture is shaken at room temperature, washed, and dried, and then dispersed in PBS buffer solution. Next, glucoamylase, glucose oxidase, and lignin peroxidase are added, and after shaking, a three-enzyme cascade nanomotor is obtained. The effective activity units of the glucoamylase, glucose oxidase, and lignin peroxidase are 2 U / mg, 250 U / mg, and 0.1 U / mg, respectively. The final concentrations of the glucoamylase, glucose oxidase, and lignin peroxidase during the shaking reaction are 8-12 U / mL, 1-2 U / mL, and 0.4-0.6 U / mL, respectively.

2. The application according to claim 1, characterized in that, The room temperature shaking reaction lasted for 5-7 hours, and the second shaking reaction lasted for 10-15 hours at 4°C.

3. The application according to claim 1, characterized in that, The amount of glutaraldehyde added is 4-6% by volume percentage.

4. The application according to claim 1, characterized in that, The concentration of the Fe3O4@SiO2-NH2 nanoparticles in the solvent is 0.5-0.7 mg / 1 mL.

5. The application according to claim 1, characterized in that, The azo dyes include Congo red.

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