A method for removing total DNA in wastewater based on graphene oxide combined with hydrogen peroxide

Abstract

This invention belongs to the field of wastewater treatment technology, specifically relating to a method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide. The method involves adding graphene oxide and hydrogen peroxide to wastewater containing DNA, followed by immediate rapid stirring, then slow stirring, and finally allowing the water to settle and separate the purified water. The removal of •OH and •O2 from the graphene oxide-hydrogen peroxide coupling system is achieved through this process. ‑ and 1 The combined effect of O2 oxidation and other processes such as adsorption in the system can effectively remove DNA from wastewater. This method uses readily available raw materials and also provides a new approach for the recycling of waste batteries. It effectively removes total DNA from wastewater, offering a novel and feasible technical method for addressing gene propagation.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment technology, specifically relating to a method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide. It is applicable to the treatment of wastewater containing DNA pollutants, such as genetic engineering wastewater, laboratory wastewater, and fermentation wastewater, and can effectively reduce the total DNA residue in wastewater, ensuring the biosafety of the aquatic environment. Background Technology

[0002] With the rapid development of industries such as biotechnology, genetic engineering, and biomedicine, wastewater discharged from laboratories and production workshops often contains large amounts of total DNA, including recombinant DNA, plasmid DNA, and host cell DNA fragments. Direct discharge of these DNA pollutants could lead to gene diffusion, ecological disturbance, and even biosafety risks. Therefore, the efficient removal of total DNA from wastewater has become a crucial requirement for water environment management.

[0003] Currently, methods for removing DNA from wastewater mainly include physical adsorption, chemical oxidation, and biodegradation. Physical adsorption commonly uses adsorbents such as activated carbon and zeolite, but its adsorption capacity is limited, removal efficiency is low, and it is difficult to completely degrade DNA. Chemical oxidation often uses strong oxidants (such as potassium permanganate and sodium hypochlorite), which can degrade DNA, but easily produce toxic byproducts, causing secondary pollution to the environment. Biodegradation relies on the metabolic activity of microorganisms, has a long treatment cycle, and is greatly affected by environmental conditions (temperature, pH), making it unsuitable for the rapid treatment of high-concentration DNA wastewater. Therefore, developing an efficient, environmentally friendly, and low-cost method for removing total DNA from wastewater to solve the problems of low removal efficiency, secondary pollution, and high treatment costs in existing technologies has become an urgent technical challenge in this field.

[0004] Graphene oxide (GO), an important derivative of graphene, possesses a huge specific surface area and abundant oxygen-containing functional groups (carboxyl, hydroxyl, and epoxy groups), exhibiting excellent adsorption properties. It can adsorb DNA molecules from wastewater through electrostatic interactions and hydrogen bonding. Hydrogen peroxide (H₂O₂), as a mild oxidant, can generate hydroxyl radicals (•OH) under suitable conditions, achieving the oxidative degradation of DNA. Its decomposition products are water and oxygen, with no secondary pollution. Furthermore, GO exhibits an increased band gap due to oxygen atom doping. This doping alters the GO structure, changing its bonding mode from pure sp(s) bonding. 2 Hybridization transforms into sp 2 with sp 3The hybridized state endows GO with oxidative properties. Furthermore, GO possesses advantages such as defects, a wide specific surface area, good hydrophilicity, tunable band gap, large-scale preparation, and environmental friendliness. It not only has the ability to efficiently adsorb organic pollutants but also effectively catalyzes advanced oxidation reactions based on H₂O₂. However, the efficiency of GO in catalytic degradation of organic pollutants is relatively low, failing to meet industrial requirements. Notably, the GO / H₂O₂ system exhibits a synergistic effect, significantly improving catalytic efficiency.

[0005] In existing technologies, GO alone can only achieve physical adsorption of DNA and cannot completely degrade DNA. After adsorption saturation, it is easy to desorb, resulting in unstable treatment effect. When H2O2 is used alone, its oxidation efficiency is low, and a higher amount is required to achieve a certain removal effect, which increases the treatment cost. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide. The method utilizes graphene oxide (not commercially purchased) prepared in-house using a modified Hummers method. By combining the oxidizing effect of hydrogen peroxide with the adsorption effect of graphene oxide, the method achieves efficient and thorough removal of total DNA from wastewater, while reducing treatment costs, avoiding secondary pollution, and overcoming the technical deficiencies of existing methods.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] A method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide includes the following steps: adding graphene oxide to wastewater containing DNA, followed by rapid addition of hydrogen peroxide, immediate and rapid stirring, then slow stirring, and the supernatant separated after settling is the purified water.

[0009] Furthermore, in the above-mentioned method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide, the mass fraction of hydrogen peroxide is 30%.

[0010] Furthermore, in the above-mentioned method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide, the ratio of graphene oxide to hydrogen peroxide is 3 mg / 0.1~1 mL.

[0011] Preferably, the ratio of graphene oxide to hydrogen peroxide is 3 mg / 0.4 mL.

[0012] Furthermore, in the above-mentioned method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide, the dosage of graphene oxide in the wastewater is 30 µg / mL, and the dosage of hydrogen peroxide in the wastewater is 1~10 µL / mL.

[0013] Furthermore, in the above-mentioned method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide, the rapid stirring speed is 600-800 rpm and the rapid stirring time is 1-2 min.

[0014] Furthermore, in the above-mentioned method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide, the slow stirring speed is 100-150 rpm and the slow stirring time is 15-30 min.

[0015] Furthermore, in the above-mentioned method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide, the settling time is 30 minutes.

[0016] Furthermore, in the aforementioned method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide, the graphene oxide is prepared from virgin graphite recovered from waste alkaline batteries, and the specific preparation method includes the following steps:

[0017] 1) Use pliers to pull apart the carbon rods of the waste alkaline battery, wipe the electrodes with paper, wash them with deionized water 3-5 times, place them in a vacuum drying oven at 60℃ and dry for 24 hours. After taking them out, crush them into fine carbon powder. Add 2g of carbon powder to a mixed solution of 37% HCl and 68% HNO3 at a volume ratio of 3:1, treat it in a water bath at 60℃ for 2 hours, then centrifuge it at 13000rpm 1-2 times, wash it with deionized water 3-5 times, and finally dry the treated carbon powder in a vacuum drying oven at 60℃ for 24 hours to obtain raw graphite.

[0018] 2) Take 1g of raw graphite and add it to a mixed solution of 98% H2SO4 and 85% H3PO4 (volume ratio 9:1). Mix well and then add 6g of KMNO4. Heat in a 50℃ water bath for 2.5h, stirring until it turns dark green. Stop the water bath heating and let it cool naturally to room temperature. Then immerse the system in an ice bath for further cooling. Slowly add 400mL of deionized water and 3mL of 30% H2O2, stirring continuously until the reaction stops. Centrifuge at 4℃ at 4000rpm. Take the precipitate and wash it once with deionized water. Remove the supernatant and wash it once with 30% HCl. Wash it again with deionized water and finally with anhydrous ethanol. Place the obtained graphene oxide in a 45℃ vacuum drying oven and dry for 24h to prepare graphene oxide.

[0019] The beneficial effects of this invention are:

[0020] 1. A method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide, wherein the graphene oxide and hydrogen peroxide coupling system contains •OH and •O2. - and 1 O2 can damage DNA through its high oxidizing properties, thereby causing oxidative damage to cell membranes and reducing the total DNA residue in wastewater.

[0021] 2. A method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide. The raw materials are readily available, and the materials can be prepared using graphite recovered from waste batteries, providing a new approach for the recycling of waste batteries. This method is highly efficient in removing DNA from wastewater and can be effectively applied in the field of wastewater treatment, showing promising application prospects. Attached Figure Description

[0022] Figure 1 X-ray diffraction patterns of raw graphite and the prepared graphene oxide.

[0023] Figure 2 The images show the Raman spectra of raw graphite and the prepared graphene oxide.

[0024] Figure 3 This is a comparison chart showing the total DNA removal rate in wastewater under different ratios of graphene oxide to hydrogen peroxide.

[0025] Figure 4 Figure 1 shows a comparison of reactive oxygen species quenching experiments under different graphene oxide to hydrogen peroxide ratios. Figure 2a shows the total DNA removal rate of each quenching system, and Figure 3b shows the contribution of various reactive oxygen species to total DNA degradation. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] Example 1

[0028] This embodiment uses effluent from a laboratory-operated sequencing batch reactor (SBR) to simulate wastewater from a real wastewater treatment plant, specifically including the following steps:

[0029] 1) Laboratory operation of SBR microbial community and water total DNA enrichment stage:

[0030] The raw water for the experiment was taken from a stably operating sequencing batch reactor (SBR), and the inoculum sludge was taken from the aeration tank of a municipal wastewater treatment plant. Artificial nutrient water was prepared, and a certain concentration of erythromycin thiocyanate was added for microbial acclimation and cultivation, promoting the enrichment of microbial cells and total DNA in the water. Specific influent components and concentrations are shown in Table 1.

[0031] Table 1 SBR Influent Formulation

[0032]

[0033] 2) Start-up phase of SBR operation in the laboratory:

[0034] 1.5L of activated sludge and 2.5L of water were mixed and added to the SBR. The SBR cycle was set to 6 hours, including 0.05 hours of influent, 3.6 hours of aerobic operation, 1.8 hours of anoxic operation, 0.5 hours of sedimentation, and 0.05 hours of effluent, with four cycles per day. After stable operation, the total DNA content in the water was sampled periodically. Once the system stabilized, the supernatant after sedimentation in the sequencing batch reactor was taken as the wastewater to be treated.

[0035] 3) The stage of self-preparation of graphene oxide using the existing modified Hummers method with raw graphite recovered from waste alkaline batteries:

[0036] Graphene oxide can be obtained through commercial purchase or laboratory preparation. The graphene oxide used in this embodiment was prepared using the conventional modified Hummers method, rather than being commercially purchased, to ensure that the material properties are stable and controllable, thus providing a guarantee for the effect of subsequent joint treatment.

[0037] S1: Use pliers to pull apart the carbon rod of the waste alkaline battery, wipe the electrode with paper, wash it 4 times with deionized water, place it in a vacuum drying oven at 60℃ and dry it for 24 hours. After taking it out, crush it into fine carbon powder. Add 2g of carbon powder to a mixed solution of 30mL of 37% HCl and 10mL of 68% HNO3 (volume ratio 3:1), treat it in a water bath at 60℃ for 2 hours, then centrifuge it at 13000rpm 1-2 times, wash it 4 times with deionized water, and then dry the treated carbon powder in a vacuum drying oven at 60℃ for 24 hours to obtain the original graphite G(R).

[0038] S2: Take 1g of the raw graphite obtained in step S1) and add it to 120mL of a mixed solution of 98% H2SO4 and 14mL of 85% H3PO4 (volume ratio 9:1). Mix well and then add 6g of KMNO4. Heat in a water bath at 50℃ for 2.5h, stirring until it turns dark green. Stop the water bath heating and let it cool naturally to room temperature. Then immerse the system in an ice bath for further cooling. Slowly add 400mL of deionized water and 3mL of 30% H2O2, stirring continuously until the reaction stops. Centrifuge at 4℃ at 4000rpm. Take the precipitate and wash it once with deionized water. Remove the supernatant and wash it once with 30% HCl. Wash it again with deionized water and finally wash it once with ethanol. Place the obtained graphene oxide in a vacuum drying oven at 45℃ and dry it for 24h to prepare graphene oxide GO.

[0039] Figure 1 The X-ray diffraction patterns are shown for pristine graphite G(R) and the prepared graphene oxide GO. The prepared graphene oxide material is black in appearance. When observed under an X-ray diffractometer, the material appears as follows... Figure 1 As shown, the original graphite exhibits a significant (002) peak at 26.54°, and the interlayer spacing is calculated to be 0.3356 nm according to Bragg's formula. After oxidation, the characteristic peaks of graphite disappear significantly, and GO shows a new diffraction peak at 11.10°, exhibiting a sharp peak corresponding to the (001) crystal plane, with an interlayer spacing of 0.7965 nm, indicating the presence of functional groups and defects in the GO structure. The interlayer spacing of GO is much larger than that of the original graphite, which is because the introduction of oxygen functional groups on the GO layers leads to an increase in the interlayer spacing, indicating that the oxidation degree of GO is relatively high. GO also shows a (100) peak at 42.42°, revealing the vortex layer bands of the disordered carbon material, indicating the existence of a vortex layered disordered structure. The above results show that GO was successfully oxidized, proving the successful synthesis of GO.

[0040] Figure 2 The images show the Raman spectra of pristine graphite G(R) and the prepared graphene oxide (GO). The Raman spectra of pristine graphite and GO are shown below. Figure 2 As shown, the Raman spectrum of pristine graphite is at 1353 cm⁻¹. -1 and 1582cm -1 This corresponds to the D and G bands. GO is at approximately 1358cm. -1 and 1581cm -1 The area exhibits D and G peaks, respectively. The D band is typically formed by defects or sps on the GO surface. 3 The presence of carbon atoms is the cause, therefore the D band peak corresponds to sp. 3 Structural features include carbon atoms and vacancy defects, grain boundary formation, etc., while the appearance of G-bands originates from sp. 2 The relative vibrations within the carbon atom plane, with a prominent G peak characteristic, indicate that the sample has a crystal structure. The ratio of D-band to G-band peak intensity (ID / IG) is a commonly used indicator to characterize the degree of GO defects; a high ID / IG ratio indicates that its sp... 3 The increase in structural domains implies an increase in defect density, and these defects may be sites for the generation of reactive oxygen species (ROS). Raman spectroscopy data showed an ID / IG ratio of 0.9386, indicating a higher defect density in the GO structure, attributed to the increased interlayer spacing caused by the introduction of oxygen functional groups. This result is consistent with XRD analysis.

[0041] 4) Reaction stage:

[0042] The experimental setup consisted of a series of 1L beakers. 100mL of the wastewater to be treated (supernatant from the sequencing batch reactor after sedimentation in step 2) was added to each beaker. Then, an equal amount of 3mg graphene oxide was added to each beaker, followed immediately by 0.1mL, 0.4mL, and 0.7mL of 30% (w / w) H₂O₂. The graphene oxide to hydrogen peroxide ratios were 3mg / 0.1mL, 3mg / 0.4mL, and 3mg / 0.7mL, respectively. The mixture was immediately stirred vigorously at 600rpm for 2 minutes, then slowly stirred at 100rpm for 20 minutes. The beakers were then allowed to settle naturally for 30 minutes.

[0043] Meanwhile, graphene oxide was replaced with an equal amount of original graphite and H2O2 with a mass fraction of 30% from the same batch as a comparison. Graphene oxide / graphite without the addition of 30% H2O2 was also compared with graphene oxide / graphite without the addition of 30% H2O2.

[0044] DNA extraction was performed using a bacterial genomic DNA extraction kit from China Plant Gene Biotechnology Co., Ltd. DNA concentration was determined spectrophotometrically. DNA purity was determined by calculating the absorbance ratio (A260 / A280) at 260 nm and 280 nm. In this example, the DNA purity extracted from the raw wastewater after sedimentation in the SBR reactor was 1.83, which meets the purity requirements for aquatic microbial genomic DNA detection and can be used for subsequent quantitative detection.

[0045] The total genomic DNA content detected is shown in Table 2.

[0046] Table 2 Total genomic DNA removal rate in wastewater

[0047]

[0048] The removal of total genomic DNA under different treatment conditions (G(R), GO, H2O2, G(R) / H2O2 coupling system, GO / H2O2 coupling system) is as follows: Figure 3 As shown in the figure, the DNA removal effect reached 88.4% when 3 mg of graphene oxide and 0.4 mL of 30% H2O2 were added, compared with the control group.

[0049] The reaction equation for the graphene oxide-hydrogen peroxide coupling system is as follows:

[0050]

[0051] HOO• (hydroperoxygen radical), also known as the all-hydroxyl radical, is a superoxide radical (O2).- The protonated form of HOO•. In aquatic environments with physiological pH levels, HOO• mainly exists in the deprotonated form•O2. - It exists; however, in acidic pH environments (such as lysosomes), the dominant species is HOO•.

[0052] In this invention, the graphene oxide / hydrogen peroxide coupling system not only utilizes the oxidizing effect of hydrogen peroxide, but also efficiently activates the •OH and •O2 generated by hydrogen peroxide. - and 1 Strong oxidizing agents such as O2 can penetrate deep into the bacterial cell membrane and directly oxidize and break the phosphodiester bonds and base structures of intracellular DNA, thereby achieving efficient degradation of total DNA in wastewater.

[0053] The generation of active substances in the graphene oxide / hydrogen peroxide coupling system requires the participation of hydrogen peroxide, as increasing the amount of hydrogen peroxide added promotes the generation of active substances, thereby accelerating DNA degradation. To achieve the best treatment effect, the concentration ratio of graphene oxide to hydrogen peroxide needs to be controlled. In this invention, the optimal dosage ratio of graphene oxide to 30% (w / w) hydrogen peroxide is 3 mg / 0.4 mL.

[0054] Figure 3 This chart compares the total DNA removal rates in wastewater under different graphene oxide to hydrogen peroxide ratios. After treatment with a mixture of graphene oxide and hydrogen peroxide, the total DNA removal rate in the wastewater reached 88.4%.

[0055] The significant reduction in total DNA concentration in wastewater under the graphene oxide and hydrogen peroxide coupling system indicates that the removal of total DNA from wastewater is not merely a matter of physical adsorption. The graphene oxide and hydrogen peroxide coupling system can directly destroy the DNA molecular structure through the oxidation of active free radicals, thereby reducing the risk of genetic pollution to the environment from the treated effluent.

[0056] Figure 4 Figure 1 shows a comparison of reactive oxygen species quenching experiments under different graphene oxide to hydrogen peroxide ratios. Figure 2a shows the total DNA removal rate of each quenching system, and Figure 3b shows the contribution of various reactive oxygen species to total DNA degradation.

[0057] Because standalone graphite systems, standalone graphene oxide systems, and standalone hydrogen peroxide systems do not produce substances containing •OH and •O2 under any external conditions. - and 1This study only investigated reactive oxygen species, including O2, in the coexisting system of graphene oxide and hydrogen peroxide. The reactive oxygen species in the graphene oxide and hydrogen peroxide coupled system primarily react with the target pollutant, total DNA. The added TBA, p-BQ, and L-histidine act as •OH and •O2 ions. - and 1 The O2 quenchers TBA, p-BQ, and L-histidine mainly compete with reactive free radicals in the system, consuming reactive species and thus inhibiting DNA removal. After adding 2 mM TBA, the total DNA removal rate in the system at different graphene oxide / hydrogen peroxide ratios decreased from 44.1% to 40.5% and then back to 40.1%. After adding 2.59 mM p-BQ, the total DNA removal rate in the system at different graphene oxide / hydrogen peroxide ratios increased from 29.1% to 46.7% and then back to 53.5%. After adding 6.44 mM L-histidine, the total DNA removal rate in the system at different graphene oxide / hydrogen peroxide ratios decreased from 35.1% to 26.8% and then increased to 37.3%. These results indicate that in the GO / H2O2 system, at low graphene oxide / hydrogen peroxide ratios, O2 plays a major role in total DNA removal. - Calculation yields •O2 - The contribution of α is approximately 42%, and the contribution of •OH is approximately 12%. 1 O2 contributes approximately 30% to the removal efficiency, while the remaining 15% is attributed to the adsorption of graphene oxide and other oxidation processes. The graphene oxide / hydrogen peroxide ratio plays a major role in total DNA removal. 1 O2, calculated to obtain 1 O2 contributes approximately 49%, and •OH contributes approximately 23%. - The contribution of graphene oxide to total DNA removal is approximately 11%, with the remaining removal efficiency contributed by the adsorption of graphene oxide and other oxidation processes, accounting for approximately 18%. At high graphene oxide / hydrogen peroxide ratios, the main factor contributing to total DNA removal is also... 1 O2, calculations show that ¹O2 contributes approximately 35%, and •OH contributes approximately 30%, •O2 - The contribution of graphene oxide is about 6%, while the remaining removal effect is contributed by the adsorption of graphene oxide and other oxidation effects, accounting for about 29%.

[0058] The above phenomena demonstrate that the graphene oxide / hydrogen peroxide coupling system can significantly remove total DNA from wastewater. When the graphene oxide / hydrogen peroxide ratio is 3 mg / 0.4 mL, the total DNA removal rate in wastewater can reach 88.4%. The graphene oxide / hydrogen peroxide coupling system achieves DNA removal through multiple synergistic pathways: on the one hand, GO catalyzes the generation of •OH and •O2 from H2O2.- and 1 Reactive oxygen species, including O2, directly oxidize and damage the molecular structure of DNA. On the other hand, GO, with its oxygen-containing functional groups and large specific surface area, can enrich DNA in water through adsorption, further improving the removal effect and effectively reducing the risk of genetic pollution in water bodies.

Claims

1. A method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide, characterized in that, The process includes the following steps: adding graphene oxide to wastewater containing DNA, followed by rapid addition of hydrogen peroxide, immediate and rapid stirring, then slow stirring, and finally allowing the supernatant to settle and separate, which is the purified water.

2. The method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide according to claim 1, characterized in that, The mass fraction of the hydrogen peroxide is 30%.

3. The method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide according to claim 2, characterized in that, The ratio of graphene oxide to hydrogen peroxide is 3 mg / 0.1~1 mL.

4. The method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide according to claim 3, characterized in that, The ratio of graphene oxide to hydrogen peroxide is 3 mg / 0.4 mL.

5. The method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide according to claim 3, characterized in that, The amount of graphene oxide added to the wastewater is 30 µg / mL, and the amount of hydrogen peroxide added to the wastewater is 1~10 µL / mL.

6. The method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide according to claim 1, characterized in that, The rapid stirring speed is 600-800 rpm, and the rapid stirring time is 1-2 minutes.

7. The method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide according to claim 1, characterized in that, The slow stirring speed is 100-150 rpm, and the slow stirring time is 15-30 min.

8. The method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide according to claim 1, characterized in that, The settling time is 30 minutes.

9. The method for removing total DNA from wastewater based on graphene oxide combined with hydrogen peroxide according to claim 1, characterized in that, The graphene oxide is prepared from raw graphite recovered from waste alkaline batteries, and the specific preparation method includes the following steps: 1) Use pliers to pull apart the carbon rods of the waste alkaline battery, wipe the electrodes with paper, wash them with deionized water 3-5 times, place them in a vacuum drying oven at 60℃ and dry for 24 hours. After taking them out, crush them into fine carbon powder. Add 2g of carbon powder to a mixed solution of 37% HCl and 68% HNO3 at a volume ratio of 3:1, treat it in a water bath at 60℃ for 2 hours, then centrifuge it at 13000rpm 1-2 times, wash it with deionized water 3-5 times, and finally dry the treated carbon powder in a vacuum drying oven at 60℃ for 24 hours to obtain raw graphite. 2) Take 1g of raw graphite and add it to a mixed solution of 98% H2SO4 and 85% H3PO4 (volume ratio 9:1). Mix well and then add 6g of KMNO4. Heat in a 50℃ water bath for 2.5h, stirring until it turns dark green. Stop the water bath heating and let it cool naturally to room temperature. Then immerse the system in an ice bath for further cooling. Slowly add 400mL of deionized water and 3mL of 30% H2O2, stirring continuously until the reaction stops. Centrifuge at 4℃ at 4000rpm. Take the precipitate and wash it once with deionized water. Remove the supernatant and wash it once with 30% HCl. Wash it again with deionized water and finally with anhydrous ethanol. Place the obtained graphene oxide in a 45℃ vacuum drying oven and dry for 24h to prepare graphene oxide.

Images