Method for isolating a hydrophyte virus and use thereof

Abstract

The present application relates to the technical field of aquatic plant virus separation, and discloses a method for separating aquatic plant viruses and application thereof, wherein the method comprises mixing amino-functionalized carbon nanomaterials and aquatic bodies containing plant viruses. The amino-functionalized carbon nanomaterials used in the method can be combined with the plant viruses in the aquatic bodies, thereby improving the adsorption capacity of the amino-functionalized carbon nanomaterials for the plant viruses. The method is simple, efficient and low in cost, and does not produce toxic substances, thereby avoiding secondary pollution possibly caused by chemical disinfection, providing an economic and sustainable solution to the problem of plant virus pollution, and showing a broad application prospect in the field of aquatic plant virus removal.

Description

Technical Field

[0001] This invention relates to the field of carbon nanomaterials technology, specifically to a method for isolating plant viruses in aquatic bodies and its application. Background Technology

[0002] Pepper Mild Mottle Virus (PMMoV) is a common plant virus that primarily infects peppers and other Solanaceae crops. PMMoV exhibits strong environmental stability, surviving for extended periods in water and spreading long distances through irrigation systems. Crops infected with PMMoV show significant growth inhibition, leaf mottling, and fruit deformities, severely impacting agricultural yield and commercial value.

[0003] Currently, the main methods for reducing PMMoV in water bodies include chemical disinfection (including sodium hypochlorite treatment and ozone oxidation), ultraviolet disinfection, and membrane separation. However, these methods all have limitations in practical applications. Dissolved organic matter in the water can competitively react with disinfectants, leading to a decrease in the effective concentration of oxidants, a decline in plant virus inactivation efficiency, and the generation of toxic byproducts. Furthermore, ultraviolet light is easily affected by the optical properties of the water; increased turbidity significantly reduces its penetration, lowering the inactivation efficiency for plant viruses. Although membrane separation technology has a certain retention effect on plant virus particles, the extremely small particle size (nanoscale) of plant viruses makes them difficult to capture or adsorb by the membrane, resulting in less than ideal membrane separation efficiency. Moreover, it faces the serious challenge of membrane fouling in actual operation. This not only increases operating costs but may also affect the lifespan of the membrane material. Therefore, developing efficient, economical, and environmentally friendly new plant virus removal materials is of great significance.

[0004] Carbon nanotubes exhibit significant advantages in pollutant removal due to their unique tubular structure, large specific surface area, and tunable surface chemistry. Therefore, effectively functionalizing carbon nanotubes to achieve efficient separation and removal of plant viruses in aquatic environments has become a pressing issue in agricultural water treatment technology. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of poor removal efficiency, high energy consumption, and environmental pollution (such as the generation of disinfection byproducts and membrane fouling) of existing technologies for plant viruses in water. This invention provides a method for separating plant viruses from water and its application. The amino-functionalized carbon nanomaterials used in this method can bind to plant viruses in water, thereby improving the adsorption capacity of amino-functionalized carbon nanomaterials for plant viruses.

[0006] To achieve the above objectives, the first aspect of the present invention provides a method for isolating plant viruses in aquatic bodies, wherein the method includes mixing amino-functionalized carbon nanomaterials with water containing plant viruses.

[0007] A second aspect of the present invention provides the application of the method described herein in the isolation of aquatic plant viruses.

[0008] Through the above technical solution, the present invention achieves the following technical effects: (1) The amino-functionalized carbon nanotubes used in this invention can bind to plant viruses, thereby improving the adsorption capacity of amino-functionalized carbon nanomaterials for plant viruses in water. In the preferred embodiment, the removal rate of amino-functionalized carbon nanomaterials for plant viruses (especially pepper mild mottle virus, PMMoV) in water is more than 81%. This method shows broad application prospects in the field of water plant virus removal. (2) When the method of the present invention is used to separate and remove plant viruses in water, the plant viruses in the water can be quickly separated by simple filtration or centrifugation. The operation method is simple, efficient and low cost. In addition, the method of the present invention does not produce toxic substances, avoids secondary pollution that may be caused by chemical disinfection, and provides an economical and sustainable solution to the problem of plant virus pollution. (3) The method of the present invention is simple and efficient, requiring no complex equipment throughout the process, and is suitable for large-scale production. Attached Figure Description

[0009] Figure 1 This is a scanning electron microscope (SEM) image of the amino-functionalized carbon nanomaterials prepared in Example 1 of this invention. Detailed Implementation

[0010] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0011] The first aspect of the present invention provides a method for isolating plant viruses in aquatic bodies, wherein the method includes mixing amino-functionalized carbon nanomaterials with water containing plant viruses.

[0012] According to some embodiments of the present invention, the mass ratio of the amino-functionalized carbon nanomaterial to the water containing plant viruses is 1:(100-500).

[0013] In this invention, the process of mixing amino-functionalized carbon nanomaterials with water containing plant viruses is the process of adsorbing plant viruses by amino-functionalized carbon nanomaterials. The entire adsorption process is carried out at room temperature, such as around 25°C.

[0014] To improve the adsorption efficiency of amino-functionalized carbon nanomaterials for plant viruses, the mixing conditions preferably include: a stirring speed of 200-300 rpm and a mixing time of 30-60 min.

[0015] In this invention, the method further includes solid-liquid separation of the mixture after mixing the amino-functionalized carbon nanomaterials and the water containing plant viruses, thereby achieving rapid separation and removal of plant viruses from the water. Preferably, the solid-liquid separation method includes filtration or centrifugation, more preferably filtration.

[0016] Preferably, the pore size of the filter membrane is 0.45-0.8 μm.

[0017] In this invention, the amino-functionalized carbon nanomaterial refers to the carboxylation modification of multi-walled carbon nanotubes by using a strong oxidizing acid (such as sulfonic acid or a mixture of concentrated sulfuric acid and concentrated nitric acid) (introducing carboxyl groups on the surface of carbon nanotubes) to obtain carboxylated multi-walled carbon nanotubes; then, the carboxylated multi-walled carbon nanotubes are chemically reacted with nitrogen-containing functional groups (amide reaction) to introduce amino groups on the surface of carbon nanotubes in the form of amide bonds to obtain amino-modified carbon nanomaterials.

[0018] According to some embodiments of the present invention, the preparation method of the amino-functionalized carbon nanomaterial includes the following steps: (1) Carboxylated multi-walled carbon nanotubes and water were mixed and sonicated to obtain a suspension of carboxylated multi-walled carbon nanotubes; (2) The carboxylated multi-walled carbon nanotube suspension and amine compounds were mixed and subjected to amide reaction to obtain amino-functionalized carbon nanomaterials.

[0019] In this invention, there is no particular limitation on the source of carboxylated multi-walled carbon nanotubes; they can be prepared in-house or obtained commercially. In the specific embodiments provided in this invention, the carboxylated multi-walled carbon nanotubes are obtained commercially, for example, from Shanghai Maclean Biochemical Technology Co., Ltd., product model 308068-56-6. The purity of the carboxylated multi-walled carbon nanotubes is >95%, the inner diameter (ID) is 2-5 nm, the outer diameter (OD) is <8 nm, the length is 0.5-2 μm, and the carboxyl content is approximately 3.9 wt%.

[0020] The inventors of this invention have discovered that the amino-functionalized carbon nanomaterials prepared by the method of this invention have abundant amino groups on their surface and carry a certain amount of charge (potential value meets a specific range), which enables the amino-functionalized carbon nanomaterials to bind efficiently with plant viruses, thereby improving the adsorption capacity of amino-functionalized carbon nanomaterials for plant viruses in water.

[0021] According to some embodiments of the present invention, the plant virus is selected from at least one of pepper mild mottle virus (PMMoV), tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and potato virus Y (PVY virus).

[0022] Preferably, the plant virus is pepper mild mottle virus.

[0023] According to some embodiments of the present invention, the zeta potential value of the amino-functionalized carbon nanomaterial is 12-25 mV, preferably 17-24 mV, and more preferably 17.5-21 mV.

[0024] In this invention, the Zeta potential value of the amino-functionalized carbon nanomaterial is determined by a Zeta potential and particle size analyzer (the instrument was purchased from Malvern, product model Zetasizer Nano ZS).

[0025] In this invention, the pore size of the amino-functionalized carbon nanomaterials was measured using an electron microscope scanner (the electron microscope scanner was purchased from Nippon Electron Ltd., instrument model JSM-7800F).

[0026] According to some embodiments of the present invention, in step (1), based on the mass of 1g of the carboxylated multi-walled carbon nanotubes, the amount of water used is 100-1000mL, preferably 200-500mL.

[0027] According to some embodiments of the present invention, in step (2), based on the mass of 1g of the carboxylated multi-walled carbon nanotubes, the amount of the amine compound is 15-100g, preferably 20-80g, and more preferably 20-40g.

[0028] According to some embodiments of the present invention, the amine compound is selected from at least one of polyethyleneimine, ethylenediamine and hexamethylenediamine, preferably polyethyleneimine.

[0029] In this invention, polyethyleneimine is obtained commercially, for example, from Shanghai E. En Chemical Technology Co., Ltd., product model number 9002-98-6.

[0030] According to some embodiments of the present invention, in step (1), the conditions for ultrasound include: ultrasound frequency of 40-80kHz and ultrasound time of 30-60min.

[0031] According to some embodiments of the present invention, in step (2), the conditions for the amide reaction include: a temperature of 50-60°C and a time of 12-15h.

[0032] According to some embodiments of the present invention, the method further includes pretreatment of the carboxylated multi-walled carbon nanotubes.

[0033] Preferably, the pretreatment method includes washing the carboxylated multi-walled carbon nanotubes with organic alcohol and water, respectively. The number of washes is not particularly limited and can be adjusted according to actual conditions, such as 1-3 times.

[0034] More preferably, the organic alcohol is selected from at least one of methanol, ethanol and propanol.

[0035] According to some embodiments of the present invention, the method further includes washing the amino-functionalized carbon nanomaterials with water and drying the solid particles obtained from solid-liquid separation. Preferably, the washing is performed 5-7 times.

[0036] Preferably, the solid-liquid separation method is selected from filtration and / or centrifugation, and more preferably filtration.

[0037] Preferably, the pore size of the filter membrane is 0.45-0.8 μm.

[0038] Preferably, the drying conditions include a temperature of 50-60°C and a time of 12-15 hours.

[0039] In this invention, the water used is ultrapure water; the ethanol refers to anhydrous ethanol reagent.

[0040] According to a particularly preferred embodiment of the present invention, a method for isolating plant viruses in aquatic bodies is provided, the method comprising the following steps: (1) Wash the carboxylated multi-walled carbon nanotubes with ethanol and ultrapure water 3-5 times respectively. Add the washed carboxylated multi-walled carbon nanotubes to ultrapure water and sonicate for 45-60 min at an ultrasonic frequency of 65-80 kHz to obtain a carboxylated multi-walled carbon nanotube suspension. Based on the mass of 1g of the carboxylated multi-walled carbon nanotubes, the amount of water used is 100-200mL; The mass ratio of the amino-functionalized carbon nanomaterial to the water containing plant viruses is 1:(100-200). (2) Add polyethyleneimine to the above suspension, heat and stir at 55-60℃ for 13-15h, wash the synthesized material with ultrapure water 5-7 times after the reaction, filter (filter membrane pore size is 0.45μm), and dry the solid particles obtained at 55-60℃ for 13-15h to prepare amino-functionalized carbon nanomaterials (the zeta potential value of amino-functionalized carbon nanomaterials is 17.5-20mV). Based on the mass of 1g of the carboxylated multi-walled carbon nanotubes, the amount of polyethyleneimine used is 20-30g. (3) The prepared amino-functionalized carbon nanomaterials were mixed with water containing plant viruses and stirred at room temperature (25℃) for 30-60 min at a stirring speed of 200-300 rpm. The mixture was filtered (the filter membrane pore size was 0.45-0.8 μm), and the supernatant was then taken into a centrifuge tube to determine the content of pepper mild mottle virus in the water. The mass ratio of the amino-functionalized carbon nanomaterial to the water containing plant viruses is 1:(100-500).

[0041] The present invention will be described in detail below through embodiments.

[0042] Carboxylated multi-walled carbon nanotubes were purchased from Shanghai Maclean Biotechnology Co., Ltd., product model number 308068-56-6; among them, the purity of carboxylated multi-walled carbon nanotubes is >95%, inner diameter (ID) is 2-5nm, outer diameter (OD) is <8nm, length is 0.5-2μm, and carboxyl content is about 3.9wt%.

[0043] The zeta potential of amino-functionalized carbon nanomaterials was determined using a zeta potential and particle size analyzer (Malvern, Zetasizer Nano ZS).

[0044] The pore size of the amino-functionalized carbon nanomaterials was measured using an electron microscope scanner (JSM-7800F, purchased from NEC Corporation, Japan).

[0045] Polyethyleneimine was purchased from Shanghai E. En Chemical Technology Co., Ltd.

[0046] In the following examples, ethanol refers to anhydrous ethanol.

[0047] Example 1 (1) The carboxylated multi-walled carbon nanotubes (100 mg) were washed three times with ethanol and ultrapure water respectively. The washed carboxylated multi-walled carbon nanotubes were added to 100 mL of ultrapure water and sonicated for 60 min at an ultrasonic frequency of 80 kHz to obtain a carboxylated multi-walled carbon nanotube suspension (mass fraction of 1 mg / mL). (2) Add 2g of polyethyleneimine to the above suspension, heat and stir at 60°C for 15h, and after the reaction is completed, wash the synthesized material with ultrapure water 7 times, filter (the filter membrane pore size is 0.45μm), and dry the solid particles obtained at 60°C for 15h to prepare amino-functionalized carbon nanomaterials (the zeta potential value of amino-functionalized carbon nanomaterials is 17.8mV). The prepared amino-functionalized carbon nanomaterials were subjected to transmission electron microscopy (results are shown below). Figure 1 As shown). By Figure 1 It can be seen that the amino-functionalized carbon nanomaterials prepared in Example 1 form a network-like structure inside, which increases their specific surface area and is beneficial for the adsorption of plant viruses.

[0048] Example 2 (1) The carboxylated multi-walled carbon nanotubes (100 mg) were washed twice with ethanol and ultrapure water respectively. The washed carboxylated multi-walled carbon nanotubes were added to 100 mL of ultrapure water and sonicated for 40 min at an ultrasonic frequency of 60 kHz to obtain a carboxylated multi-walled carbon nanotube suspension (mass fraction of 1 mg / mL). (2) Add 4g of polyethyleneimine to the above suspension, heat and stir at 50°C for 12h. After the reaction is complete, wash the synthesized material with ultrapure water 5 times, filter (filter membrane pore size is 0.8μm), and dry the solid particles obtained at 50°C for 12h to prepare amino-functionalized carbon nanomaterials (the zeta potential value of amino-functionalized carbon nanomaterials is 20.8mV).

[0049] Example 3 The method of Example 1 was followed, except that in step (2), the amount of polyethyleneimine used was 8g, and the other steps and conditions were the same as in Example 1, and amino-functionalized carbon nanomaterials (the Zeta potential value of the amino-functionalized carbon nanomaterials was 24mV) were prepared.

[0050] Example 4 Following the method of Example 1, except that the polyethyleneimine in step (2) was replaced with ethylenediamine, and the other steps and conditions were the same as in Example 1, amino-functionalized carbon nanomaterials were prepared (the zeta potential value of the amino-functionalized carbon nanomaterials was 12.3 mV).

[0051] Example 5 The method of Example 1 is different in that the amount of ultrapure water used in step (1) is 600 mL, and the other steps and conditions are the same as in Example 1, and amino-functionalized carbon nanomaterials (the Zeta potential value of amino-functionalized carbon nanomaterials is 9.2 mV) are prepared.

[0052] Example 6 The method of Example 1 is different in that the ultrasonic frequency in step (1) is 20 kHz, and the other steps and conditions are the same as in Example 1, and amino-functionalized carbon nanomaterials (the zeta potential value of amino-functionalized carbon nanomaterials is 11.5 mV) are prepared.

[0053] Example 7 The method of Example 1 is the same as in Example 1, except that in step (2), the temperature of the amide reaction is 40°C and the time is 4h. Other steps and conditions are the same as in Example 1, and amino-functionalized carbon nanomaterials are prepared (the zeta potential value of the amino-functionalized carbon nanomaterials is 8.4mV).

[0054] Example 8 The method of Example 1 was followed, except that in step (2), the amount of polyethyleneimine used was 0.5g, and the other steps and conditions were the same as in Example 1, and amino-functionalized carbon nanomaterials (the zeta potential value of the amino-functionalized carbon nanomaterials was 9.7mV) were prepared.

[0055] Example 9 The method of Example 1 was followed, except that in step (2), the amount of polyethyleneimine used was 1g, and the other steps and conditions were the same as in Example 1, and amino-functionalized carbon nanomaterials were prepared (the zeta potential value of the amino-functionalized carbon nanomaterials was 11.8mV).

[0056] Example 10 The method of Example 1 was followed, except that in step (2), the amount of polyethyleneimine used was 16g, and the other steps and conditions were the same as in Example 1, and amino-functionalized carbon nanomaterials were prepared (the zeta potential value of the amino-functionalized carbon nanomaterials was 50.7mV).

[0057] Example 11 The method of Example 1 was followed, except that in step (2), the amount of polyethyleneimine used was 32g, and the other steps and conditions were the same as in Example 1, and amino-functionalized carbon nanomaterials were prepared (the zeta potential value of the amino-functionalized carbon nanomaterials was 57.7mV).

[0058] Comparative Example 1 The method of Example 1 was followed, except that polyethyleneimine in step (2) was replaced with ascorbic acid, and the other steps and conditions were the same as in Example 1, to prepare hydroxyl-functionalized carbon nanomaterials (the zeta potential of the hydroxyl-functionalized carbon nanomaterials was -12mV).

[0059] Test case The amino-functionalized carbon nanomaterials prepared in the above examples and comparative examples were added to water containing plant viruses (self-made), and the removal rate of pepper mild mottle virus (PMMoV) in the water was measured.

[0060] Methods for preparing water containing plant viruses include: (1) Take 300g of diseased leaves infected with pepper mild mottle virus, grind them with liquid nitrogen, add an equal amount of 0.02mol / LPB (pH=7.4) buffer, homogenize, filter with 3 layers of gauze or centrifuge at low speed for 5min, and take the supernatant. (2) Add 8% (w / v) n-butanol to the supernatant, stir magnetically for 20 min, clarify, centrifuge at 4000 rpm and 4℃ for 20 min, and take the supernatant; (3) Add NaCl and polyethylene glycol 6000 to the supernatant to a final concentration of 5.8% and 4% (w / v), respectively. Stir at 4°C until fully dissolved, let stand overnight at 4°C, centrifuge at 4000 rpm for 30 min at 4°C, and collect the precipitate. (4) The precipitate was fully suspended in 50 mL of 0.01 mol / L PB (pH=7.4) buffer solution, stirred at 4°C until fully dissolved, centrifuged at 4000 rpm for 20 min at 4°C, and the supernatant was collected. (5) Add 20% (w / v) sucrose pad, centrifuge at 36000 rpm at 4℃ for 1.5 h, and take the precipitate; suspend the precipitate in 4 mL of 0.01 mol / L PB (pH=7.4) buffer; (6) Centrifuge at 25000 rpm and 4℃ for 2 hours using a 30% (w / v) sucrose linear gradient column; place 1 mL into a centrifuge tube and measure OD260 / 280 using a UV spectrophotometer. (7) Collect the virus-rich portion, concentrate at 36,000 rpm, centrifuge at 4°C for 2 hours, and collect the precipitate; (8) Dissolve the precipitate in 0.01 mol / L PB (pH=7.4) buffer, centrifuge at 10000 rpm for 20 min at 4℃, take the supernatant (which is the purified solution of PMMoV), and store at -20℃ for later use.

[0061] The preparation method of the sucrose linear gradient centrifuge column includes: preparing 30%, 20%, and 10% sucrose solutions (w / v) using 0.02 mol / L Pb (pH=7.4). Then, using a syringe, 10 mL of the 10% (w / v) sucrose solution is drawn and injected along the wall of the centrifuge tube. Next, 10 mL of the 20% (w / v) sucrose solution is drawn and gently injected along the wall of the centrifuge tube under the 10% (w / v) sucrose pad. Similarly, 10 mL of the 30% (w / v) sucrose solution is drawn and gently injected under the 20% (w / v) sucrose pad. The column is then incubated at 4°C for 24 hours to equilibrate the sugar solution, forming a natural sucrose gradient column from bottom to top, ranging from 30% to 10% (w / v).

[0062] Method for determining the removal rate of pepper mild mottle virus in water: 100 mg of amino-functionalized carbon nanomaterials were added to 50 mL of water containing plant virus (the content of pepper mild mottle virus was 41 ng / L). The mixture was stirred at room temperature (25℃) (stirring speed was 200 rpm). After stirring for 30 min and 60 min, the mixture was filtered (filter membrane pore size was 0.8 μm). Then, 1 mL of supernatant was taken into a centrifuge tube, and the content of pepper mild mottle virus in the water was determined according to the detection method below. The removal rate of pepper mild mottle virus in the water was calculated according to the formula below. Each treatment was set up with 3 replicates. The results are shown in Table 1.

[0063] The content of pepper mild mottle virus in water was determined by enzyme-linked immunosorbent assay (ELISA).

[0064] The removal rate (%) of chili pepper mild mottle virus in water body = 100% × (content of chili pepper mild mottle virus in original water body - content of chili pepper mild mottle virus in treated water body) / (content of chili pepper mild mottle virus in original water body).

[0065] Table 1

[0066] A comparison of Examples 1-4 with Examples 5-11 shows that the preferred method of the present invention can further improve the adsorption capacity of amino-functionalized carbon nanomaterials for chili pepper mottle virus.

[0067] Compared with Example 1, Comparative Example 1 replaced polyethyleneimine with ascorbic acid, and the potential value of the prepared hydroxyl-functionalized carbon nanomaterial dropped sharply, resulting in a significant reduction in the adsorption capacity of the hydroxyl-functionalized carbon nanomaterial for pepper mottle virus.

[0068] In summary, the amino-functionalized carbon nanomaterials prepared using the embodiments of the present invention have a high removal effect on pepper mottle virus in water.

[0069] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for isolating plant viruses from aquatic bodies, characterized in that, The method includes mixing amino-functionalized carbon nanomaterials with water containing plant viruses; The plant virus is selected from at least one of pepper mild mottle virus (PMMoV), tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and potato virus Y (PVY virus); The zeta potential of the amino-functionalized carbon nanomaterial is 12-25 mV. The preparation method of the amino-functionalized carbon nanomaterials includes the following steps: (1) Carboxylated multi-walled carbon nanotubes and water were mixed and sonicated to obtain a suspension of carboxylated multi-walled carbon nanotubes; (2) A carboxylated multi-walled carbon nanotube suspension and amine compounds were mixed and subjected to an amide reaction to obtain amino-functionalized carbon nanomaterials; The amine compound is selected from at least one of polyethyleneimine, ethylenediamine, and hexamethylenediamine.

2. The method according to claim 1, wherein, The plant virus in question is pepper mild mottle virus; And / or, the zeta potential of the amino-functionalized carbon nanomaterial is 17-24 mV.

3. The method according to claim 2, wherein, The zeta potential of the amino-functionalized carbon nanomaterial is 17.5-21 mV.

4. The method according to claim 1, wherein, The mass ratio of the amino-functionalized carbon nanomaterial to the water containing plant viruses is 1:(100-500).

5. The method according to claim 1, wherein, The mixing conditions include: a stirring speed of 200-300 rpm and a mixing time of 30-60 min.

6. The method according to claim 1, wherein, In step (1), based on the mass of 1g of the carboxylated multi-walled carbon nanotubes, the amount of water used is 100-1000mL; And / or, in step (2), the amount of the amine compound used is 15-100g, based on the mass of 1g of the carboxylated multi-walled carbon nanotubes.

7. The method according to claim 6, wherein, In step (1), based on the mass of 1g of the carboxylated multi-walled carbon nanotubes, the amount of water used is 200-500mL; And / or, in step (2), the amount of the amine compound used is 20-80g, based on the mass of 1g of the carboxylated multi-walled carbon nanotubes.

8. The method according to claim 7, wherein, In step (2), based on the mass of 1g of the carboxylated multi-walled carbon nanotubes, the amount of the amine compound used is 20-40g.

9. The method according to claim 1, wherein, The amine compound is polyethyleneimine.

10. The method according to any one of claims 1-9, wherein, In step (1), the conditions for ultrasound include: ultrasound frequency of 40-80kHz and ultrasound time of 30-60min. And / or, in step (2), the conditions for the amide reaction include: a temperature of 50-60°C and a time of 12-15h.

11. The method according to any one of claims 1-9, wherein, The method further includes pretreatment of the carboxylated multi-walled carbon nanotubes.

12. The method according to claim 11, wherein, The pretreatment method includes washing the carboxylated multi-walled carbon nanotubes with organic alcohol and water, respectively.

13. The method according to claim 12, wherein, The organic alcohol is selected from at least one of methanol, ethanol and propanol.

14. The method according to any one of claims 1-9, wherein, The method further includes washing the amino-functionalized carbon nanomaterials with water and drying the solid particles obtained from solid-liquid separation.

15. The method according to claim 14, wherein, The solid-liquid separation method is selected from filtration and / or centrifugation.

16. The method of claim 14, wherein, The drying conditions include a temperature of 50-60℃ and a time of 12-15 hours.

17. The application of the method according to any one of claims 1-16 in the isolation of aquatic plant viruses.

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