Preparation of composite nano motor based on bio-enzyme catalysis-Fenton-like reaction and application of composite nano motor in water treatment

Abstract

The invention belongs to the technical field of sewage treatment, and particularly relates to preparation of a composite nano motor based on biological enzyme catalysis-Fenton-like reaction and application of the composite nano motor in water treatment. The invention aims to develop a multistage enzyme self-driven nano motor which does not need additional fuel and has good biocompatibility and high catalytic activity. According to the invention, three cascade reaction enzymes, namely glucamylase, glucose oxidase and lignin peroxidase, are loaded on nano particles which take magnetic Fe3O4 as a core and mesoporous silica as a shell and are subjected to amination modification, so that the three-enzyme cascade nano motor is constructed; the nano motor can decompose and remove pollutants in water through the catalytic action of enzyme and the Fenton-like reaction, can realize self-driving by utilizing the concentration gradient and bubbles generated by decomposing the pollutants, does not need to depend on external fuel and stimulation (sound, light, magnetism and the like), and has an important application prospect in the field of sewage treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of sewage treatment, and specifically relates to the preparation of a composite nanomotor based on bio-enzyme catalysis-Fenton-like reaction and its application in water treatment. Background Art

[0002] With the acceleration of industrialization and the improvement of water environment quality requirements, a large amount of industrial wastewater must be strictly treated to meet the discharge standards. Among them, coking, petrochemical, clothing industry, pharmaceutical and food industries will produce a large amount of wastewater containing pollutants such as phenols, azo and oil, and these pollutants in the wastewater have toxicity, difficult biodegradation and other characteristics, which will bring potential harm to the ecological environment and human health, so it is urgent to carry out efficient treatment. However, conventional physical, chemical and biological treatment technologies (such as chemical precipitation, adsorption, chemical oxidation, membrane separation, activated sludge, etc.) have high cost, high energy consumption, residual sludge and chemicals are prone to secondary pollution, and occupy a large area, resulting in the current treatment technology is not ideal. Miniaturization, high efficiency, low energy consumption and no secondary pollution are the goals pursued by the current technology research and development in the field of sewage treatment.

[0003] Nanotechnology provides a new solution to the current sewage treatment dilemma. Among them, nanomotors are micro-nanoscale machines that can convert other forms of energy such as chemical energy, light energy, and thermal energy into their own propulsion to achieve special tasks, also known as micro-nano robots. Micro-nanomotors have the advantages of small size, large specific surface area, active movement, and fine operation in complex environments, which makes them have good application prospects in biomedical fields (such as drug delivery, antibacterial agents, treatment of neurodegeneration), biomimicry (such as biosensors) and environmental remediation (such as sewage treatment and water quality monitoring). At present, domestic and foreign scholars have prepared a variety of different micro-nanomotors for treating different pollutants in sewage, such as organic compounds, microplastics, hormones, heavy metals, petroleum, etc. Due to the self-driving and high catalytic properties of micro-nanomotors, the removal efficiency of these pollutants is improved. So far, a variety of micro-nanomotors for sewage treatment have been reported, most of which are driven by chemical reactions of external substrates or driven by external fields (such as magnetic fields, light fields, ultrasound, etc.). Among them, the motor driven by external field has good directional motion behavior and can move under restricted conditions. However, since external field driving requires external machines to provide magnetic fields, infrared light, sound fields and other conditions, the requirements for the use environment are relatively harsh, which limits its scope of application. The motor driven by chemical catalytic reaction mainly relies on the bubbles or product concentration gradients generated by chemical catalytic reactions as a power source to achieve self-driving. It has the ability to degrade pollutants in situ, avoiding dependence on instruments and equipment or the external environment during use, but there are also important problems such as the need for external fuel to drive the motor (such as external H2O2), because the presence of external fuel will cause secondary pollution to the environment, the preparation method is relatively complicated, and the type of degraded pollutants is single. In order to solve the current problems of using micro-nano motors to degrade pollutants, such as the need for external fuel and the limited types of degraded pollutants, if the pollutants in the water can be used as fuel, the concentration gradient and bubbles caused by the catalytic degradation reaction of the pollutants themselves can be used to drive the micro-nano motor, that is, the decomposition of pollutants can be directly used as the driving force of the micro-nano motor, it is expected to overcome the problem of the need for external fuel to drive the nanomotor, and realize the simultaneous driving of the nanomotor and the removal of pollutants, providing more favorable conditions for the application of nanomotor technology in sewage treatment.

[0004] Biological enzyme is an environmentally friendly and efficient catalyst. It can generate energy in the process of catalytic decomposition of pollutants. It has the advantages of specificity, mild reaction conditions, high catalytic efficiency, and non-toxicity. It is a good choice for driving nano motors. Therefore, it is possible to consider preparing biological enzymes that can directly decompose pollutants in sewage and wastewater to drive micro-nano motors. At the same time, the nano-immobilization of enzymes can be achieved, overcoming the problems of easy inactivation of natural free enzymes, poor stability, and low reuse rate. The immobilized enzyme exists in the form of a nano motor. While the biological enzyme catalyzes the decomposition of pollutants, it can also provide power to make the motor move, thereby achieving efficient contact between the enzyme and the pollutant, improving the removal efficiency of the pollutant, and making the entire motor system do not need to provide a power source, saving energy consumption. At present, the biological enzymes loaded by nano motors are mostly single enzymes, or enzymes of different types, to remove different target pollutants, such as using laccase and lipase to decompose bisphenol A and triacetin. However, this will result in limited driving force of the nanomotor, and the removal of pollutants will only rely on the catalytic action of biological enzymes, which will limit the removal efficiency. If a carrier-loaded cascade enzyme can be used to generate a stepped fuel power source by utilizing the combined reaction of biological enzymes and substrates, and carriers and biological enzyme-catalyzed intermediates, the driving force of the nanomotor can be made stronger, the movement effect more obvious, and it will be more conducive to achieving stable and efficient pollutant degradation and enhanced nanomotor movement behavior.

[0005] In view of this, it is necessary to develop a multi-stage enzyme self-driven nanomotor that does not require external fuel and has good biocompatibility and high catalytic activity to solve the above problems. Summary of the invention

[0006] In order to overcome the deficiencies of the above-mentioned prior art, the present invention prepares a three-enzyme cascade nanomotor by co-fixing lignin peroxidase, glucose oxidase and glucoamylase on nanoparticles with magnetic Fe3O4 as the core, mesoporous silica as the shell and amino-modified. The three biological enzymes of glucoamylase, glucose oxidase and lignin peroxidase in the nanomotor can produce a cascade reaction, glucoamylase catalyzes starch to produce glucose, glucose oxidase uses glucose to produce H2O2, lignin peroxidase can use H2O2 to catalyze the decomposition of pollutants, and at the same time, the inner core Fe3O4 can react with H2O2 to produce hydroxyl free radicals to decompose pollutants. The combined action of the two can achieve efficient decomposition and removal of pollutants, such as azo dyes, and at the same time, the concentration gradient and bubbles generated by the decomposition of pollutants can realize the self-driving of the nanomotor.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a method for preparing a three-enzyme cascade nanomotor, namely, first preparing Fe3O4 nanoparticles and modifying them to form Fe3O4@SiO2-NH2 nanoparticles, and then loading three cascade reaction enzymes, namely 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 on Fe3O4@SiO2-NH2 nanoparticles is as follows: first disperse the Fe3O4@SiO2-NH2 nanoparticles in a solvent, then add glutaraldehyde, shake react at room temperature, wash and dry, then disperse in PBS buffer solution, then add glucose amylase, glucose oxidase and lignin peroxidase, and then shake react to obtain a three-enzyme cascade nanomotor.

[0010] Preferably, Fe3O4 nanoparticles are synthesized by a hydrothermal method, and the 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 2U / mg, 250U / mg and 0.1U / mg respectively; the final concentrations of the glucoamylase, glucose oxidase and lignin peroxidase in the shaking reaction are 8-12U / mL, 1-2U / mL and 0.4-0.6U / mL respectively.

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

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

[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 by the preparation method described in the first aspect.

[0017] The present invention co-immobilizes lignin peroxidase, glucose oxidase and glucoamylase on nanoparticles with magnetic Fe3O4 as core, mesoporous silica as shell and amino modification, and uses glucoamylase to convert starch in wastewater into glucose, which is then catalytically decomposed into H2O2 by glucose oxidase, and then lignin peroxidase uses H2O2 to decompose azo dyes in wastewater and generate oxygen. At the same time, H2O2 can produce a Fenton-like reaction with the core Fe3O4. During the entire reaction process, the oxygen generated by the H2O2 reaction and the concentration gradient generated by the degradation of starch and dye can all play the role of driving force, so that the nanomaterial loaded with enzymes moves, thereby realizing the successful construction of enzyme cascade self-driven magnetic nanomotor and the efficient removal of pollutants.

[0018] The third aspect of the present invention provides the use of the three-enzyme cascade nanomotor described in the second aspect in wastewater treatment, wherein the wastewater contains starch, including (but not limited to) printing and dyeing 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 present invention has the following beneficial effects:

[0022] The present invention constructs a three-enzyme cascade nanomotor by loading three cascade reaction enzymes, namely glucoamylase, glucose oxidase and lignin peroxidase, on nanoparticles with magnetic Fe3O4 as the core, mesoporous silica as the shell and amino-modified. The nanomotor can decompose and remove pollutants in water through the catalytic action of enzymes and Fenton-like reactions, and can use the concentration gradient and bubbles generated by the decomposition of pollutants to achieve self-driving without relying on external fuel and stimulation (sound, light, magnetism, etc.), which has important application prospects in the field of sewage treatment. In general, the present invention has the following advantages:

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

[0024] (2) The Fe3O4 in the carrier will undergo a Fenton-like reaction with the H2O2 produced by glucose catalyzed by glucose oxidase. The generated hydroxyl free radicals can decompose pollutants in the mineralized water.

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

[0026] (4) During the reaction process, the H2O2 produced by the decomposition of pollutant starch in the sewage can be used to provide substrate H2O2 for lignin peroxidase and Fenton-like reactions, without the need to artificially add H2O2, which can reduce costs.

[0027] (5) The power that drives the nanomotor comes from the concentration gradient generated by the decomposition of pollutants through enzyme catalysis and Fenton-like reactions, as well as the oxygen bubbles produced when lignin peroxidase utilizes H2O2, without the need for external fuel or external environmental stimulation. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0030] Figure 3 CLSM photos of the three-enzyme cascade nanomotor (A. bright field; B. blue - glucose oxidase; C. red - glucoamylase; D. green - lignin peroxidase; E. three fluorescence superposition);

[0031] Figure 4 The degradation effect of three-enzyme cascade nanomotor, two-enzyme (glucoamylase and glucose oxidase) cascade nanoparticles, free three enzymes, inactivated immobilized three enzymes, and modified ferrosoferric oxide nanoparticles on Congo red;

[0032] Figure 5 The three-enzyme cascade nanomotor and the two-enzyme (glucoamylase and glucose oxidase) cascade nanoparticles are in Congo red and starch solution, and DMPO is used as a free radical scavenger to capture the spin of OH in the reaction system by an electron paramagnetic resonance (EPR) spectrometer at a temperature of 30°C and pH = 6.0.

[0033] Figure 6 This is a diagram of the movement behavior of the three-enzyme cascade nanomotor observed in starch and Congo red solution using a laser confocal microscope (LSM800with Airyscan). DETAILED DESCRIPTION

[0034] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in each embodiment of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0036] Example: Preparation of a three-enzyme (glucoamylase, glucose oxidase and lignin peroxidase) cascade nanomotor

[0037] (1) Fe3O4 nanoparticles were first synthesized by a hydrothermal method, and then modified by TEOS / APTES (for specific methods, please refer to "Preparation of laccase-driven nanomotors and their application in pollutant degradation. Huang Yongwei. Master's thesis of Harbin Institute of Technology. 2021.").

[0038] (2) 0.6 mg of the 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 at room temperature for 6 hours on an oscillator. After the shaking reaction, the mixture was washed 6 times with anhydrous ethanol and dried in a vacuum at 60°C for 4 hours. The mixture was then dispersed in 1 mL of PBS buffer solution (pH = 6.0), and a final concentration of 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 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 FIG. Figure 1 shown.

[0039] pass Figure 2 It can be seen from the Fourier transform infrared spectra that the 597 cm -1 and 1093cm -1 The peaks at 1634 cm-1 represent the Fe-O-Fe vibration peak and the Si-O-Si anti-stretching vibration, respectively, indicating that Fe3O4 was successfully synthesized and coated by SiO2. -1 (NH bending vibration peak) and 3358cm -1(NH stretching vibration peak) indicates that the amino functionalization of the nanoparticles has been achieved. In addition, the peak at 1413 cm -1 The CN stretching vibration peak at indicates that the enzyme is successfully fixed to the surface of the nanoparticles. The appearance of these functional groups can be used to determine that the enzyme drives the successful synthesis of the nanomotor. Figure 3 From the CLSM photos of the nanomotor (AD), we can see that the green fluorescence is the lignin peroxidase stained with FTIC, the blue fluorescence is the glucose oxidase stained with Cy-5, and the red fluorescence is the glucoamylase stained with rhodamine B. Through CLSM, it can be clearly observed that the nanoparticles are wrapped by these three enzymes. Figure 3 (E) is 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 nanoparticles is relatively consistent, which shows that the three enzymes are evenly fixed on the nanoparticles.

[0040] Comparative Example: Preparation of Dual Enzyme (Glucoamylase and Glucose Oxidase) Cascade Nanomotors

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

[0042] 0.6 mg of the three-enzyme cascade nanomotor of the embodiment was added to a solution with a starch concentration of 1 wt% and a Congo red concentration of 350 μM to 450 μM, and the pH was adjusted to 6.0, and then placed under the condition of T = 30°C to treat Congo red. At the same time, the degradation effects of the dual enzyme (glucoamylase and glucose oxidase) cascade nanomotor, free three enzymes, inactivated immobilized three enzymes (three enzyme cascade motors sterilized by high pressure steam at 1.01 MPa, 121°C, 20 min), and modified ferrosoferric oxide nanoparticles (Fe3O4@SiO2-NH2) on Congo red were compared.

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

[0044] like Figure 5 As shown, DMPO was used as a free radical scavenger to measure the three-enzyme cascade nanomotor + starch ( Figure 5 A), three-enzyme cascade nanomotor + starch + Congo red ( Figure 5 B), dual enzyme (glucoamylase-glucose oxidase) cascade nanomotor + starch ( Figure 5 C), and dual enzyme (glucoamylase-glucose oxidase) cascade nanomotor + starch-Congo red ( Figure 5 D) The presence of free radicals in the system (reaction system volume 600 μL, Congo red concentration 350-450 μM, starch 1wt%, nanomotor 0.6 mg). The spin capture EPR signal diagram of ·OH in the reaction system at 30°C and pH 6.0 by an electron paramagnetic resonance EPR spectrometer can be determined. It can be determined that ·OH signals appear in all four systems. It is judged that the cascade reaction of glucoamylase and glucose oxidase produces H2O2, and then H2O2 and Fe3O4 produce a Fenton-like reaction to generate ·OH, and the strong oxidizing property of ·OH can achieve efficient removal of Congo red.

[0045] In addition, three enzymes (glucoamylase, glucose oxidase and lignin peroxidase) were stained with Cy-5, filtered and dialyzed, and then the three enzymes were fixed on the nanoparticles according to the method of the embodiment. Then, the three-enzyme cascade nanomotor was added to 1wt% starch and 450μM Congo red solution, and after stabilization, it was placed in a laser confocal dish under a 100× objective lens for observation. Figure 6 As shown in the figure, the large particles are substrate starch. In a short period of time, it can be seen that the three-enzyme cascade motor stained with blue fluorescence by Cy-5 quickly approaches the substrate starch (from left to right, downward in the figure) and reacts. This shows that the power of the three-enzyme cascade nanomotor comes from the concentration gradient generated by the decomposition of pollutants by biological enzyme catalysis and Fenton-like reaction, as well as the oxygen bubbles generated when lignin peroxidase uses H2O2, without the need for external fuel and external environmental stimulation.

[0046] In summary, the nanomotor constructed by the method of the present invention can decompose and remove starch and azo dyes in water through enzyme catalysis and Fenton-like reaction. 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 reaction, and the efficiency of removing azo dyes is higher; on the other hand, the concentration gradient and bubbles generated by the three-enzyme cascade reaction in the reaction system to decompose pollutants can drive the movement of nanomaterials, realizing the rapid movement of micro-nano motors without external fuel and stimulation (sound, light, magnetism, etc.).

[0047] The above is the process of preparing nanomotors and removing pollutants, but the present invention is not limited to the hydrothermal synthesis of ferroferric oxide nanoparticles. Ferroferric oxide nanoparticles synthesized by other precipitation methods can also be used as carrier materials. The treated solution is not limited to Congo red solution, as long as it is wastewater containing starch and lignin peroxidase substrates, such as textile printing and dyeing wastewater, pharmaceutical wastewater, etc.

[0048] The embodiments of the present invention are 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 of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A method for preparing a three-enzyme cascade nanomotor, characterized in that: First, Fe3O4 nanoparticles were prepared and modified to make Fe3O4@SiO2-NH2 nanoparticles. Then, the three cascade reaction enzymes, namely glucose amylase, glucose oxidase, and lignin peroxidase, were loaded on the Fe3O4@SiO2-NH2 nanoparticles to obtain a three-enzyme cascade self-driven nanomotor.

2. The method for preparing a three-enzyme cascade nanomotor according to claim 1, characterized in that: The method for loading three cascade reaction enzymes on Fe3O4@SiO2-NH2 nanoparticles is as follows: first disperse the Fe3O4@SiO2-NH2 nanoparticles in a solvent, then add glutaraldehyde, shake the reaction at room temperature, wash and dry, then disperse in PBS buffer solution, then add glucose amylase, glucose oxidase and lignin peroxidase, and then shake the reaction to obtain a three-enzyme cascade nanomotor.

3. The method for preparing a three-enzyme cascade nanomotor according to claim 2, characterized in that: The effective activity units of the glucoamylase, glucose oxidase and lignin peroxidase are 2U / mg, 250U / mg and 0.1U / mg respectively; the final concentrations of the glucoamylase, glucose oxidase and lignin peroxidase in the shaking reaction are 8-12U / mL, 1-2U / mL and 0.4-0.6U / mL respectively.

4. The method for preparing a three-enzyme cascade nanomotor according to claim 2, characterized in that: The room temperature shaking reaction time is 5-7 hours, and the second shaking reaction temperature is 4° C. and the time is 10-15 hours.

5. The method for preparing a three-enzyme cascade nanomotor according to claim 2, characterized in that: Calculated by volume percentage, the added amount of glutaraldehyde is 4-6%.

6. The method for preparing a three-enzyme cascade nanomotor according to claim 2, characterized in that: The concentration of the Fe3O4@SiO2-NH2 nanoparticles in the solvent is 0.5-0.7 mg / 1 mL.

7. A three-enzyme cascade nanomotor prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the three-enzyme cascade nanomotor according to claim 7 in wastewater treatment, characterized in that: The wastewater contains starch and azo dyes.

9. The use according to claim 8, characterized in that: The wastewater also contains azo dyes.

10. The use according to claim 9, characterized in that: The azo dyes include Congo red.

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