Oxidized multi-walled carbon nanotube, and preparation method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGSU UNIV
- Filing Date
- 2024-03-05
- Publication Date
- 2026-06-12
Smart Images
Figure CN118108213B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering and materials preparation technology, specifically relating to an oxidized multi-walled carbon nanotube material, its preparation method, and its application. Background Technology
[0002] Enzymes are common biocatalysts in living organisms, possessing advantages such as high substrate specificity, high catalytic activity, and high catalytic efficiency, leading to their widespread application in production and daily life. However, enzymes still have many shortcomings in practical applications: ① Enzymes are composed of proteins, and their higher conformations are unstable to factors such as heat, strong acids, strong bases, and organic solvents, easily becoming inactive during reactions; ② Pure enzymes are usually expensive; ③ Free enzymes suffer from drawbacks such as high enzyme consumption, difficulty in separation, easy product contamination, and difficulty in reuse, seriously affecting their widespread application in production. Therefore, it is necessary to improve enzyme utilization to enable their large-scale production and application.
[0003] Currently, there are many technologies used to improve enzyme stability and reusability, such as enzyme immobilization, enzyme modification, and protein engineering. Among these, enzyme immobilization technology has become a commonly used method due to its minimal damage to the enzyme. With the rapid development of immobilization materials, various organic and inorganic carriers are used to immobilize enzymes. Compared with free enzymes, immobilized enzymes exhibit higher stability during use, and their larger specific surface area provides a higher binding capacity. Moreover, immobilized enzymes can be separated from the reaction solution through methods such as filtration and centrifugation, which is beneficial for product purification and repeated use of the enzyme. Reaction conditions are easy to control, enabling continuous automated production, reducing costs, and facilitating large-scale industrial production.
[0004] Nanotubes (CNTs) are a class of one-dimensional cylindrical carbon allotropes that have attracted significant research interest due to their exceptional electrical, thermal, and mechanical properties. However, their applications in energy storage, microelectronics, biomedicine, and other industrial fields are limited because pristine CNTs are inert and unreactive, possess a large specific surface area, and exhibit strong intertube van der Waals attraction. This leads to CNT aggregation into large clusters, resulting in poor dispersibility in solution. Furthermore, pristine CNTs are hydrophobic and have low surface energy, making them incompatible with polar solvents. Therefore, it is necessary to introduce surface functional groups and surface defects onto CNTs to address these shortcomings and thus immobilize enzymes. Summary of the Invention
[0005] To address some shortcomings in existing technologies, this invention provides an oxidized multi-walled carbon nanotube material, its preparation method, and its applications. This invention synthesizes oxidized multi-walled carbon nanotubes via an oxidation method. These nanotubes possess a porous three-dimensional structure composed of a sponge-like structure, with a large number of carboxyl and hydroxyl functional groups on their surface. The surface area of the oxidized multi-walled carbon nanotube material is 137-140 m². 2 / g, total pore volume is 0.45-0.50cm³ 3 / g, with a pore size of 13.30-13.40nm; the oxidized multi-walled carbon nanotubes can protect glucoamylase (GLL) from thermal denaturation at 50-60℃ and can be reused multiple times. The oxidized multi-walled carbon nanotube material can efficiently immobilize GLL, with a maximum enzyme loading capacity of 211.28mg / g. The activity of the immobilized GLL enzyme is 4 times higher than that of the free GLL enzyme, which has good practicality.
[0006] To achieve the above-mentioned technical objectives, the present invention employs the following technical means.
[0007] The present invention first provides an oxidized multi-walled carbon nanotube material, which is a porous three-dimensional structure composed of a sponge-like structure, and the surface of the oxidized multi-walled carbon nanotube material has a large number of carboxyl and hydroxyl functional groups.
[0008] Preferably, the surface area of the oxidized multi-walled carbon nanotube material is 137-140 m². 2 / g, total pore volume is 0.45-0.50cm³ 3 / g, with a pore size of 13.30-13.40nm.
[0009] This invention also provides a method for preparing the above-mentioned oxidized multi-walled carbon nanotube material, specifically including the following steps:
[0010] Multi-walled carbon nanotubes were uniformly dispersed in sulfuric acid. Then, an oxidant was added sequentially under stirring, followed by distilled water, to obtain a mixture. The mixture was stirred and heated to react. After the reaction was completed, it was cooled to room temperature to obtain a reaction solution.
[0011] Deionized water and a second oxidant were added to the reaction solution, followed by separation, washing, and drying to obtain the oxidized multi-walled carbon nanotube material.
[0012] Preferably, the ratio of the multi-walled carbon nanotubes, sulfuric acid, oxidant and distilled water is 1g:90mL:4g:50mL.
[0013] Preferably, the oxidant includes one or more of nitrates, permanganates, perchlorates, dichromates, hypochlorites, persulfates, nitrites, and peroxides.
[0014] Preferably, the oxidant is a mixture of sodium nitrate and potassium permanganate; wherein the molar ratio of sodium nitrate to potassium permanganate in the mixture is 1:5-6.
[0015] Preferably, the second oxidant includes one or more of nitrates, permanganates, perchlorates, dichromates, hypochlorites, persulfates, nitrites, and peroxides.
[0016] Preferably, the second oxidant is hydrogen peroxide.
[0017] Preferably, the heating reaction is carried out at 95–100°C for 30–90 minutes.
[0018] Preferably, the volume ratio of deionized water and the second oxidant added to the reaction solution is 5-6 mL:1 mL.
[0019] The present invention also provides the application of the above-mentioned oxidized multi-walled carbon nanotubes in immobilized enzymes.
[0020] Preferably, the immobilized enzyme includes immobilized glucosylamylase.
[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0022] (1) The oxidized multi-walled carbon nanotube material of the present invention is prepared by oxidizing multi-walled carbon nanotubes in a mixture of H2SO4, H2O2, and oxidants (NaNO3 and KMnO4). The strong oxidizing power of the solution increases the disruption of strong covalent bonds and van der Waals attraction between carbon atoms, leading to partial fiber peeling off from the outer wall of the nanotubes and subsequent hydroxylation and carboxylation. In the preparation process of the oxidized multi-walled carbon nanotube material of the present invention, a large number of carboxyl and hydroxyl groups are introduced on the surface of the multi-walled carbon nanotubes through oxidation, forming a sponge-like structure, thereby exhibiting a high enzyme immobilization capacity, with an immobilization load of 211.28 mg / g.
[0023] (2) The hydroxyl and carboxyl groups on the surface of the oxidized multi-walled carbon nanotube material described in this invention are active and form strong covalent bonds with the free amino and carboxyl groups of enzymes, as well as certain active groups, such as the hydroxyl and phenolic groups of threonine and serine, and the imidazole group of histidine. This provides stability for enzymes with controlled active site orientation, thereby improving activity. The activity of the immobilized GLL enzyme is 4 times higher than that of the free GLL.
[0024] (3) Compared with free GLL, the oxidized multi-walled carbon nanotube material oMW-CNTβ immobilized GLL of the present invention exhibits better thermal stability and storage stability. The oxidized multi-walled carbon nanotube material oMW-CNTβ can be reused multiple times, and still maintains the complete sponge-like porous structure after multiple uses, retaining enzyme activity and having good reusability. Attached Figure Description
[0025] Figure 1 This is a schematic diagram of the preparation process of the oxide multi-walled carbon nanotube material (oMW-CNTβ) described in this invention.
[0026] Figure 2 SEM images (A, B, C) and corresponding EDX spectral analysis plots (A1, B1, C1) of pMW-CNT, oMW-CNTα, and oMW-CNTβ.
[0027] Figure 3 FTIR plots of pMW-CNT, oMW-CNTα, and oMW-CNTβ.
[0028] Figure 4 XRD patterns of pMW-CNT, oMW-CNTα, and oMW-CNTβ.
[0029] Figure 5 The diagram shows the oMW-CNTβ nitrogen adsorption-desorption isotherm (A) and its pore size distribution (B).
[0030] Figure 6 Image of GLL labeled with fluorescein isothiocyanate (FITC) fixed on oMW-CNTβ laser confocal scanning microscope.
[0031] Figure 7 The FTIR plots of GLLs were immobilized for pMW-CNT, oMW-CNTα, and oMW-CNTβ, respectively.
[0032] Figure 8 XRD patterns of GLLs immobilized for pMW-CNT, oMW-CNTα, and oMW-CNTβ, respectively.
[0033] Figure 9 TGA plots of oMW-CNTβ and oMW-CNTβ immobilized GLLs.
[0034] Figure 10 Figures showing the load amounts of the fixed load GLLs for pMW-CNT, oMW-CNTα, and oMW-CNTβ.
[0035] Figure 11 Graphs showing the optimal pH (A) and temperature (B) for free and immobilized GLL.
[0036] Figure 12 The graph shows the relative enzyme activities of free GLLs and immobilized GLLs with pMW-CNT, oMW-CNTα, and oMW-CNTβ.
[0037] Figure 13 The thermal stability (A) and storage stability (B) of free and oMW-CNTβ immobilized GLL.
[0038] Figure 14 Figure showing the activity of oMW-CNTβ-immobilized GLL after repeated use. Detailed Implementation
[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.
[0040] Example 1: Preparation of oxidized multi-walled carbon nanotube materials
[0041] This embodiment follows Figure 1 The schematic diagram shown illustrates the fabrication process of oxide multi-walled carbon nanotube material (oMW-CNTβ). The specific steps for preparing oxide multi-walled carbon nanotube material are as follows:
[0042] 0.5 g of multi-walled carbon nanotubes were ultrasonically treated for 10 minutes to uniformly disperse them in 45 mL of sulfuric acid. Then, 0.2 g of sodium nitrate and 2.0 g of potassium permanganate were added sequentially under stirring, followed by the gradual addition of 50 mL of distilled water to obtain a mixture. The mixture was stirred and heated to 100 °C for 60 minutes to enhance oxidation. After the reaction, the mixture was cooled to room temperature to obtain a reaction solution. 150 mL of deionized water was gradually added, followed by 25 mL of hydrogen peroxide (H₂O₂). The solution was centrifuged at 8000 rpm to separate the precipitate. The recovered precipitate was intermittently washed three times with 0.1 M NaOH and distilled water for 10 minutes each time, followed by centrifugation at 8000 rpm to remove residual acid or salt. The precipitate was then freeze-dried to obtain a sponge-like powder, yielding the final sponge-like oxidized multi-walled carbon nanotube material oMW-CNTβ.
[0043] To investigate the effects of oxidized multi-walled carbon nanotubes (MWCNTs) with different oxidation methods on immobilized enzymes, this embodiment also prepared oxidized MWCNTs with other oxidation treatments:
[0044] 0.1 g of pMW-CNT was added to a piranha solution containing 30 mL of sulfuric acid and 20 mL of H₂O₂. The mixture was dispersed by sonication for 10 minutes and stirred in an ice bath for 6 hours. 200 mL of distilled water was gradually added to the mixture, and the mixture was separated by vacuum filtration. The mixture was then washed three times with 0.1 M NaOH to reach a neutral pH, and then freeze-dried. The resulting oxidized multi-walled carbon nanotube product was named oMW-CNTα.
[0045] The microstructure, morphology, and elemental composition of the material were investigated using FE-SEM and built-in energy dispersive X-ray (EDX) spectroscopy, such as... Figure 2 As shown. From Figure 2It can be seen that the morphology of the original pMW-CNTs and the MW-CNTs treated with piranha solution (solution α) showed almost no structural change compared to those treated with a mixture of H2SO4, H2O2, NaNO3, and KMnO4 (solution β), both exhibiting a sponge-like porous structure. EDX quantification of the amounts of carbon and oxygen present in the MW-CNTs revealed that the oxidation of the sample treated with solution β (oMW-CNTβ) containing 7.59% oxygen was significantly improved compared to treatment with solution α (oMW-CNTα) containing 2.04% oxygen (pMW-CNT) and untreated MW-CNTs (pMW-CNT) containing 0.73% oxygen.
[0046] The Fourier transform infrared (FT-IR) spectrum of the material was recorded using a spectrometer, and the results are as follows: Figure 3 As shown. Figure 3 This indicates that no obvious peak was observed in pMW-CNT, but oMW-CNTβ was observed at 3478. -1 and 1631cm -1 A deliberate peak appeared at the frequency. At 3478 cm⁻¹ -1 The broad spectrum with a peak at 1631 cm⁻¹ represents the stretching vibration of the OH group, while the peak at 1631 cm⁻¹ represents the stretching vibration of the OH group. -1 The peak value is related to the stretching vibration of the carbonyl or carboxyl (C=O) groups located at the edge of oMW-CNTβ.
[0047] The crystal structures of pMW-CNT, oMW-CNTα, and oMW-CNTβ were studied using X-ray diffraction, and the structures are as follows: Figure 4 As shown. From Figure 4 As can be seen, all samples exhibit two characteristic peaks at approximately 26.08° and 43.26°θ, corresponding to the (002) and (100) planes of the graphite structure, respectively (JCPDF No. 75-1621). However, the intensity of the diffraction peaks decreases with increasing oxidation, with oMW-CNTβ showing the lowest intensity. Comparison with EDX spectra suggests that oxidation treatment results in a higher concentration of oxygen groups on the MW-CNT surface, which may affect the crystal structure by increasing oxidation and reducing the intensity peaks of the samples.
[0048] The surface area and porosity of the support module used for enzyme immobilization are important parameters because they allow for easy enzyme penetration or provide numerous active binding sites on its surface to improve enzyme attachment. The surface area and porosity of oMW-CNTβ were examined using the BET and BJH models, respectively, and the results are as follows: Figure 5 As shown in Figures A and 5B, the surface area of oMW-CNTβ was found to be 137.52 m². 2 / g, total pore volume is 0.457cm³ 3 / g, pore size 13.30nm. Specific surface area compared to the original pMW-CNT (140m²). 2 Compared to g), the surface area after oxidation is slightly reduced. Due to the large surface area and porosity of MW-CNT, in order to limit enzyme loss, it is necessary to increase the number of surface carboxyl and hydroxyl functional groups to enhance the intermolecular attraction with the enzyme and prevent leakage or loss after fixation.
[0049] Example 2: oMW-CNTβ immobilized enzyme
[0050] This embodiment uses GLL as an example to study the ability of materials to immobilize GLL. To determine the immobilization ability of the materials, this embodiment studies the oMW-CNTβ immobilized GLL prepared in Example 1 by fluorescent labeling, confocal laser scanning microscopy, FT-IR spectroscopy, and X-ray diffraction, and performs thermogravimetric (TGA) analysis on oMW-CNTβ and oMW-CNT-β@GLL.
[0051] (1) Fluorescent labeling and confocal laser scanning microscopy were used to investigate the ability of oMW-CNTβ to immobilize GLLs:
[0052] To determine whether GLL was successfully immobilized on oMW-CNT, the enzyme was labeled with FITC fluorescent dye and then immobilized on the material. The green fluorescence image of the immobilized FITC-labeled GLL excited at 488 nm is shown under a confocal laser scanning microscope. The fluorescence characteristics of the immobilized FITC probe GLL are as follows: Figure 6 As shown in the figure, a strong green fluorescence signal can be seen, indicating that the FITC-labeled GLL was successfully immobilized on oMW-CNTβ.
[0053] (2) FT-IR spectrum and crystal structure of oMW-CNTβ immobilized GLL
[0054] The FT-IR spectra of oMW-CNTβ-immobilized GLLs were recorded using a spectrometer. Figure 7 This indicates that after oMW-CNTβ immobilization of GLL, the additional peak is attributed to the tensile vibrations of OH and NH (3332 cm⁻¹). -1 ) and amide I (1665 and 1614 cm) of adsorbed protein molecules. -1 ) and amide II (1441cm) -1 The in-plane stretching mode characteristics of GLL further indicate that GLL is fixed to oMW-CNTβ.
[0055] The crystal structure of oMW-CNTβ immobilized with GLL was studied using X-ray diffraction. No changes in the crystal structure were observed, indicating that the low crystallinity of GLL did not affect the general crystallinity of oMW-CNTβ. Figure 8 ).
[0056] (3) Thermogravimetric (TGA) analysis of oMW-CNTβ and oMW-CNT-β@GLL
[0057] The thermal stability of oMW-CNTβ and oMW-CNT-β@GLL was studied by thermogravimetric analysis, such as Figure 9 As shown, both oMW-CNTβ and oMW-CNTβ@GLL exhibited single-step weight loss, with significant weight loss beginning at 158 °C. The TGA curves of oMW-CNTβ@GLL show that oMW-CNTβ@GLL experienced a larger weight loss compared to oMW-CNTβ, which may be attributed to the loss of moisture content and the decomposition of adsorbed protein molecules. The weight reduction of oMW-CNTβ can be attributed to the decomposition of carboxyl and hydroxyl groups attached to the MW-CNT surface. The similarity in weight loss patterns indicates the structural stability of pMW-CNT, due to the strong covalent and van der Waals attraction within its well-defined graphite framework. After heating from 25 °C to 800 °C, oMW-CNTβ lost a total of 54.08% of its initial weight, while the immobilized GLL oMW-CNT-β@GLL lost 69.43%. This suggests that immobilization of GLL did not improve the thermal stability of oMW-CNT, as protein molecules readily decompose with increasing temperature.
[0058] Example 3: Study on GLL Fixation Capacity
[0059] The immobilization capacity of GLLs in the study materials (pMW-CNT, oMW-CNTα, and oMW-CNTβ) was determined. Enzyme immobilization was performed by adding 0.6 mg / mL GLL (1 mL) to 1 mg of material. The amount of enzyme in the supernatant was quantified at specific time points over 12 hours at 25°C. Figure 10 As shown, immobilization increased over time, with pMW-CNT reaching its maximum loading capacity of 146.67 mg / g at 6 hours, and oMW-CNTα and oMW-CNTβ reaching their maximum immobilization capacities of 175.13 mg / g and 211.28 mg / g, respectively, at 9 hours.
[0060] The higher loading capacity of oMW-CNTβ is likely attributed to the large number of carboxyl and hydroxyl groups introduced onto the surface during oxidation. Hydroxyl and carboxyl groups are reactive and can form strong covalent bonds with the free amino and carboxyl groups of the enzyme, as well as certain active groups, such as the hydroxyl and phenolic groups of threonine and serine, and the imidazole group of histidine. This provides stability to the enzyme with controlled active site orientation, thereby enhancing activity. The lower loading capacity of pMW-CNT can be understood as being due to insufficient functional groups required for enzyme interaction. Therefore, at equilibrium, low ionic interactions and the lack of oxygen-containing groups that promote stable binding lead to the detachment of GLLs from the support, which may explain the decrease in enzyme adsorption after 6 hours.
[0061] Example 4: Optimization of GLL Immobilization Conditions
[0062] Because oMW-CNTβ possesses high immobilization capacity and enhances enzyme activity, the effects of parameters such as temperature and pH on the activity of GLL immobilized on oMW-CNTβ were further investigated. During the investigation, the activity at the optimal pH and temperature was defined as 100%, and the relative activities under different pH and temperature conditions were calculated. The results are as follows: Figure 11 As shown.
[0063] The optimal pH and temperature for maximizing GLL activity were explored in 50 mM sodium phosphate buffer (supplemented with 50 mM NaCl and 3 mM KCl) at pH 6.0–9.0 and temperatures 20–70 °C. NaCl and KCl, as neutral salts, completely ionize in aqueous medium to form Na+. + K + and Cl - Its interaction with counterions on the protein surface provides maximum stability and solubility. For example... Figure 11 As shown in Figure A, free GLL exhibits the highest enzyme activity at pH 7. oMW-CNTβ@GLL maintains relatively high activity at pH 8.0–9.0, indicating that oMW-CNTβ@GLL has better pH tolerance compared to free GLL.
[0064] The effect of temperature on immobilized enzymes was investigated by incubating at pH 7 at 20, 30, 30, 40, 50, 60, and 70 °C for 6 h. The results are as follows: Figure 11 As shown in Figure B, the results indicate that the immobilization efficiency is high at higher temperatures; however, above 40°C, the activities of both free and immobilized enzymes begin to decrease, so the enzyme activity is highest at 40°C.
[0065] Therefore, the optimal conditions for GLL immobilization are pH=7 and temperature=40℃.
[0066] Enzyme activity was compared between free GLLs and GLLs immobilized with pMW-CNT, oMW-CNTα, and oMW-CNTβ under optimal conditions (pH 7 and 40°C). Figure 12 It was observed that the enzyme activities of GLLs immobilized with pMW-CNT, oMW-CNTα, and oMW-CNT-β increased by approximately 1.7, 2.23, and 4.04 times, respectively, compared to free GLLs. This indicates that oMW-CNTβ, with the highest surface oxidation capacity, exhibits the highest activity due to its strong interaction with GLL enzymes.
[0067] Example 5: Study on the thermal stability of immobilized enzymes
[0068] This study investigated the stability of immobilized enzymes at different temperatures. The thermostability of free and immobilized GLL was assessed by incubation at 4, 40, 45, 50, 55, 60, and 70°C for 60 minutes. Figure 13 As shown in Figure A. Residual activity was then assessed by setting the sample incubated at 4°C as the reference point (100%). The results showed that free GLL completely lost its activity after 60 minutes at 55°C and 60°C, while immobilized GLL@oMW-CNT retained 58.1% and 19.5% of its GLL activity, respectively, after 60 minutes at 55°C and 60°C.
[0069] This embodiment also investigated the storage stability of the enzyme, such as Figure 13 As shown in Figure B, free GLLs without any preservatives tend to lose activity over time within 30 days, especially when stored at room temperature. By day 20, the activity of free GLLs decreased by approximately 17%, and by day 30, by approximately 38%. GLLs immobilized with oMW-CNTβ retained almost all of their activity (98.3%) by day 30. The structural rigidity provided by oMW-CNTβ prevents GLL denaturation or the formation of ionic interactions that could lead to denaturation.
[0070] Therefore, compared to free GLL, the oMW-CNTβ described in this invention has better thermal stability.
[0071] Example 6: Study on the recyclability of immobilized GLL
[0072] Recyclability and reusability are among the decisive factors in the synthesis and application of enzyme immobilization materials. For practical application, the recyclability of immobilized GLLs was also investigated. oMW-CNTβ@GLLs could be easily recovered from the reaction system for reuse by centrifugation. The enzyme activity of the first reaction was defined as the initial activity (100%), and subsequent activities were compared to it. Figure 14As shown, after five consecutive repeated uses, the GLL enzyme activities of GLL@oMW-CNTβ immobilized were 89%, 81%, 66%, 61%, and 56%, respectively.
[0073] In summary, the oMW CNTβ material with a sponge-like structure described in this invention can immobilize GLL. This invention features simple processing, strong enzyme binding capacity, increased enzyme activity, improved thermal and storage stability, convenient recovery, and reusability, overcoming many shortcomings of free enzymes in practical applications mentioned in the background art, thereby greatly promoting the utilization of enzymes in large-scale production.
[0074] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.
Claims
1. A method for preparing an oxidized multi-walled carbon nanotube material, characterized by, The preparation method includes: Multi-walled carbon nanotubes were uniformly dispersed in sulfuric acid. Then, an oxidant was added sequentially under stirring, followed by distilled water, to obtain a mixture. The mixture was stirred and heated to react. After the reaction was completed, it was cooled to room temperature to obtain a reaction solution. Deionized water and a second oxidant were added to the reaction solution, followed by separation, washing, and drying to obtain the oxidized multi-walled carbon nanotube material. The ratio of the multi-walled carbon nanotubes, sulfuric acid, oxidant and distilled water is 1 g: 90 mL: 4 g: 50 mL; The oxidant is a mixture of sodium nitrate and potassium permanganate; in the mixture, the molar concentration ratio of sodium nitrate to potassium permanganate is 1:5~6; The second oxidant is hydrogen peroxide; The conditions for the heating reaction are to react at 95~100℃ for 30-90 minutes; In the reaction solution, the volume ratio of deionized water to the second oxidant is 5-6 mL: 1 mL. The oxidized multi-walled carbon nanotube material is a porous three-dimensional structure composed of a sponge-like structure, and has a large number of carboxyl and hydroxyl functional groups on the surface of the oxidized multi-walled carbon nanotube material; the surface area of the oxidized multi-walled carbon nanotube material is 137-140 m 2 / g, the total pore volume is 0.45-0.50 cm 3 / g, and the pore size is 13.30-13.40 nm.
2. The oxidized multi-walled carbon nanotubes prepared by the method of claim 1.
3. The application of the oxidized multi-walled carbon nanotubes according to claim 2 in immobilized enzymes.
4. Use according to claim 3, characterized in that, The immobilized enzyme includes immobilized glucosylamylase.