Preparation method of antibacterial functional graphene nanocolloid

Abstract

This application discloses a method for preparing antibacterial functional graphene nanocolloids, belonging to the field of graphene technology. Using micron-sized flake graphite with an average lateral dimension of 1-10 μm and a thickness of less than 1 μm as raw material, the raw material undergoes pretreatment with an acidic ammonium sulfate aqueous solution, followed by a three-stage temperature-controlled oxidation process, with hydrogen peroxide added in two steps. After solid-liquid separation, washing, drying, and short-time calcination, a functional graphene oxide intermediate is obtained. The intermediate is dispersed with a biocompatible dispersant and then sequentially subjected to high-shear emulsification, two-stage milling, high-speed mixing under nitrogen protection, and high-pressure microfluidic circulation treatment to obtain a transparent nanocolloid. The product has a lateral dimension D50 of 90-110 nm, 2-3 layers, an oxygen content of 10-15 atomic percent, and excellent antibacterial properties, making it applicable to antibacterial coatings, biomedical equipment, wound dressings, water treatment membranes, and food packaging materials.

Description

Technical Field

[0001] This application relates to the field of graphene technology, and in particular to a method for preparing antibacterial functional graphene nanocolloids. Background Technology

[0002] Graphene is a type of graphene composed of carbon atoms arranged in sp... 2 Two-dimensional carbon nanomaterials with hexagonal honeycomb lattices composed of hybrid orbitals have shown great application potential in materials science, biomedicine, and environmental engineering due to their excellent mechanical, optical, electrical, and chemical stability. Among them, graphene oxide, with its surface rich in oxygen-containing functional groups such as hydroxyl, carboxyl, and epoxy groups, possesses good dispersibility and reactivity, and exhibits significant inhibitory effects on common pathogenic bacteria such as Escherichia coli and Staphylococcus aureus, making it one of the core directions for the research and development of antibacterial functional materials.

[0003] Currently, functionalized graphene, especially few-layer graphene oxide and reduced graphene oxide, shows potential in antibacterial applications due to its ability to induce oxidative stress and membrane damage in microorganisms. However, existing production methods face several core bottlenecks: balancing the degree of oxidation, controlling the number of layers, and difficulties in functionalization. The traditional Hummers process uses strong acids and strong oxidants, leading to over-oxidation, excessively high oxygen content, and damage to the sp. 2 Conjugated structure; structural defects reduce mechanical integrity; residual metal ions are cytotoxic and impair antibacterial activity. Large lateral dimensions and uneven layer distribution: Graphene sheets produced by traditional exfoliation methods have disadvantages such as wide size distribution, inconsistent layer count, and poor transparency. In antibacterial applications, larger sheets tend to encapsulate bacteria rather than penetrate them, while smaller sheets may be internalized without causing membrane damage. Use of toxic or irritating chemicals: hydrazine hydrate or sodium borohydride are used for reduction; N-methylpyrrolidone or dimethylformamide are used for exfoliation and concentrated sulfuric acid; unsuitable for environmentally friendly and biomedical-grade production, especially for antibacterial applications, as residual solvents or heavy metals may lead to false positives in antibacterial results or toxicity to human cells. Summary of the Invention

[0004] To address the problems existing in the prior art, this application proposes a method for preparing antibacterial functional graphene nanocolloids. By using a metal-free, controllable chemical oxidation process tailored to micron-scale sheet structures, combined with a low-temperature calcination process that preserves edge activity, and using micron-sized flake graphite as raw material, and employing only environmentally friendly reagents and mild conditions, antibacterial graphene nanocolloids with controllable lateral dimensions (100 nm), uniform layer number (2-3 layers), and high transparency are prepared. This method solves the technical problems of traditional graphene materials, such as difficulty in precisely controlling the degree of oxidation, uneven distribution of sheet size and layer number, and insufficient antibacterial activity.

[0005] This application provides a method for preparing antibacterial functional graphene nanocolloids, the preparation method comprising:

[0006] (1) Raw material pretreatment: Micron-sized flake graphite is mixed with an acidic aqueous solution containing ammonium persulfate;

[0007] (2) Temperature-controlled oxidation: The first part of hydrogen peroxide is added at low temperature, and the second part of hydrogen peroxide is added at medium temperature. The reaction is carried out at 60°C for 6-10 hours to obtain the reaction solution.

[0008] (3) Intermediate: The reaction solution is subjected to solid-liquid separation, washing, drying, and calcination to obtain a functional graphene oxide intermediate;

[0009] (4) Nanocolloids: The functional graphene oxide intermediate is dispersed in water containing a biocompatible dispersant, and then subjected to high-shear emulsification, two-stage milling, high-speed mixing under nitrogen protection, and high-pressure micro-jet running for 35-45 cycles at 1800-2200 bar; the solid content is adjusted to obtain transparent nanocolloids.

[0010] Preferably, the average transverse dimension of the micron-sized flake graphite in step (1) is 1-10 μm, and the aspect ratio is greater than 10:1.

[0011] Preferably, in step (1), the mass ratio of ammonium persulfate to micron-sized flake graphite is 8:1-12:1.

[0012] Preferably, in step (2), the low temperature is 0-5℃ and the medium temperature is 40℃; the volume ratio of the first part of hydrogen peroxide to the second part of hydrogen peroxide is 2:1.

[0013] Preferably, the calcination conditions in step (3) are 115-125°C and calcination time is 8-12 minutes.

[0014] Preferably, the two-stage grinding in step (4) involves grinding in stages using 0.4-0.6mm zirconia beads and 0.2mm zirconia beads in sequence.

[0015] Preferably, the biocompatible dispersant in step (4) includes polyvinylpyrrolidone, soapberry extract and sodium deoxycholate.

[0016] This application provides, on the one hand, the antibacterial functional graphene nanocolloids prepared by the aforementioned preparation method.

[0017] Preferably, the lateral dimension D50 of the nanocolloid is 90-110 nm, the number of layers is 2-3, the average thickness of AFM is 1.0-1.5 nm, the oxygen content is 10-15 atomic percentage, and the solid content is 0.9-1.1%.

[0018] Another aspect of this application proposes the application of an antibacterial functional graphene nanocolloid in antibacterial coatings, biomedical devices, wound dressings, water treatment membranes, or food packaging materials.

[0019] First, the raw material selected is micron-sized flake graphite with D50=1-10µm, thickness<1µm, carbon purity>99.5%, and ash content<0.5%; this ensures rapid diffusion of the intercalating agent, achieves uniform oxidation of the graphite matrix, and provides a process basis for the final product to reach 100nm-level lateral dimensions.

[0020] Secondly, a staged temperature-controlled oxidation process was specifically optimized for micron-sized flake structures: the timing of hydrogen peroxide addition and the system's reaction kinetics were precisely controlled through three gradient temperature ranges: 0-5℃, 40℃, and 60℃; micron-sized flake graphite possesses a high specific surface area, reaching 2-5m². 2 / g, while large scales are 0.5-1m. 2 / g, therefore, the amount of oxidant needs to be precisely matched to effectively avoid the problems of excessive oxidation and runaway reaction. This process adopts a low-temperature short-time calcination process to remove reaction by-products: treatment in a muffle furnace at 120℃ for 10 minutes can achieve selective decomposition of residual ammonium persulfate. At the same time, this process can effectively remove sulfate by-products coated between micron-sized flake graphite layers, avoiding the re-stacking of graphene sheets during conventional drying, and ensuring the stability and regularity of the product's microstructure.

[0021] Finally, to meet the product's requirements of a 100nm lateral size and a 2-3 layer structure, a multi-stage mechanical exfoliation process was implemented: two stages of sand milling were performed using zirconia beads with particle sizes of 0.4~0.6mm and 0.2mm, followed by online mixing and high-pressure micro-jet (2000bar, 40 cycles). Compared to large flake graphite, using micron-sized flake materials as precursors can reduce the energy input required to achieve the target particle size exfoliation by 60%.

[0022] The beneficial effects of the embodiments in this application are as follows:

[0023] (1) Precise and controllable oxidation degree, stable antibacterial activity: Staged temperature-controlled oxidation and stepwise addition of hydrogen peroxide, combined with low-temperature short-time calcination, precisely control the oxygen content at 10-15 atomic percentages. This retains sufficient edge oxygen-containing functional groups to efficiently generate reactive oxygen species (ROS) to kill bacteria, while avoiding excessive oxidation that damages SP. 2 The structure addresses the issues of uncontrolled oxidation and fluctuating antibacterial properties in traditional processes.

[0024] (2) Uniform sheet size and number of layers, significantly improved antibacterial efficiency: Using micron-sized flake graphite as raw material, combined with multi-level mechanical exfoliation, uniform sheets with a transverse size of 90-110nm and 2-3 layers are obtained. Sheets of about 100nm can be sharply inserted into bacterial cell membranes and induce membrane damage. At the same time, they can flexibly wrap and block nutrient uptake. The three synergistic effects make the antibacterial rate ≥99.99%, which is far superior to traditional graphene materials.

[0025] (3) Green and environmentally friendly: No strong acids, heavy metals and toxic organic solvents are used in the whole process. By-products are completely removed by gentle calcination. There are no metal ions and toxic solvent residues. The cell survival rate is >85%, which meets the requirements of high safety scenarios such as biomedical and food contact. It adopts biocompatible composite dispersant and high pressure micro-jet. The colloid is transparent and uniform and has no sedimentation after 30 days. It can be directly used for antibacterial coatings, medical dressings, water treatment membranes and food packaging. It has strong processing adaptability. Detailed Implementation

[0026] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0027] Example 1

[0028] Selection of micron flake graphite

[0029] (1) Selection of graphite type: A systematic comparative experiment was conducted on three graphite morphologies: large flake graphite, irregular powder and micron flake graphite. The comparison results are shown in Table 1. Table 1 shows that the intercalation time is the key parameter that determines the oxidation effect. Ammonium persulfate needs to diffuse fully into the graphite interlayer to ensure the smooth progress of the subsequent oxidation reaction. Micron flake graphite has a thickness of 0.3-0.8µm and has a shorter interlayer diffusion path, which can be fully intercalated within 4-6 hours. Compared with micron flake graphite, the intercalation time of large flake graphite needs to be greater than 24 hours, and the oxidation reaction shows an uneven phenomenon from the edge to the center.

[0030] Table 1 Comparison data of three graphite morphologies

[0031] Large-scale graphite 150 0.6 >100 >24 Irregular powder 20 1.8 45-60 12-18 micron scales 5 3.2 30-40 4-6

[0032] (2) Determination of the temperature for the oxidation chemical reaction of micron-sized scales:

[0033] 1) Hydrolysis process of ammonium persulfate: (NH4)2S2O8 + H2O → (NH4)2SO4 + H2SO4 + 1 / 2O2; This reaction can generate sulfuric acid in situ without the need to add concentrated H2SO4; Micron-sized flake graphite has a high specific surface area, which can significantly accelerate the generation rate of initial acidic substances. The system is prone to local heat accumulation, so setting the temperature to 0-5°C can effectively avoid local overheating and ensure the stability and controllability of the reaction system.

[0034] 2) Co-oxidation of ammonium persulfate and hydrogen peroxide: (NH4)2S2O8 + H2O2 → (NH4)2SO4 + H2SO4 + O2; H2O2 has two functions: a direct oxidant (producing additional oxygen species); and a catalyst for the decomposition of ammonium persulfate, controlling SO4. - • Rate of free radical formation; SO4 - • Free radicals (E°=2.43V) are stronger oxidants than H2O2 (E°=1.78V) and selectively attack the edge sites of graphene. For micron-scale sheets with high edge density, this enables uniform edge functionalization while avoiding excessive damage to the basal surface.

[0035] (3) Mathematical model calculation for the reduction of lateral dimension:

[0036] Starting with micron-sized flake graphite with an initial lateral dimension L0 (µm) and thickness t0 (nm) as the starting material, after oxidation and exfoliation: the final lateral dimension L_f = L0 × (f_oxidation) × (f_exfoliation); where f_oxidation = oxidation factor (0.3-0.5, representing edge erosion during oxidation); f_exfoliation = mechanical reduction factor caused by microjets; for L0 = 5µm (micron-sized flake graphite); L_f = 5 × 0.4 × 0.05 = 0.1µm = 100nm. For L0 = 150µm (large flake graphite), L_f = 150 × 0.4 × 0.05 = 3µm = 3000nm, resulting in an excessively large size. This size control model effectively demonstrates that micron-sized flake graphite is the core key raw material for achieving the target lateral dimension of 100nm in the product.

[0037] Example 2

[0038] Preparation of antibacterial functional graphene nanocolloids

[0039] (1) Preparation of graphite oxide powder:

[0040] 1) Characterization and pretreatment of micron flake graphite: Before oxidation, each batch of micron flake graphite was characterized, and the characterization results are shown in Table 2. Pretreatment: Vacuum drying at 105°C for 2 hours to remove adsorbed moisture and avoid exothermic reaction with ammonium persulfate.

[0041] Table 2 Characterization results of micron-sized flake graphite

[0042] D50 horizontal dimensions 1-10µm (preferably 3-7µm) Laser diffraction Scale thickness <1µm (preferably 0.3-0.8µm) SEM cross section carbon purity ≥99.5% Combustion analysis Ash content <0.5% ISO 8004 Specific surface area <![CDATA[2-5m 2 / g]]> <![CDATA[BET (N2 adsorption)]]>

[0043] 2) Initial Oxidation Stage: Add 4L of deionized water (resistivity >18MΩ·cm) to a jacketed glass or PTFE-lined reactor, and start mechanical stirring at 300-400rpm. Slowly add 2kg of ammonium persulfate (ACS grade, purity >98%). Stir at room temperature for 20-30 minutes until completely dissolved. Due to the in-situ generation of H2SO4, the solution pH is approximately 2-3. Subsequently, add 200g of micron-sized flake graphite in batches: Due to the high specific surface area of ​​the micron-sized flakes, the initial wetting process is accompanied by an exothermic effect, so add 10g each time, every 2 minutes, while monitoring the system temperature. When the temperature rises above 35℃, stop feeding and resume operation after the temperature drops below 30℃. The overall feeding time should be controlled at 40-50 minutes. Note: The 10:1 mass ratio of ammonium persulfate to graphite is optimized for micron-sized flakes. Too high a ratio can lead to over-oxidation; too low a ratio can result in incomplete intercalation.

[0044] 3) Low-temperature oxidation stage (0-5℃): Cool the reactor jacket to 0-5℃ using a circulating cooler. Maintain a stirring speed of 400-500 rpm. Wait for the internal temperature to stabilize at ≤5℃ (30-40 minutes); First addition of hydrogen peroxide (400mL 30% w / w H2O2): Use a pressure dropping funnel with a fine tip to ensure uniform distribution, adding at a rate of 10-15mL / min. The reaction is slightly exothermic; maintain the internal temperature at ≤5℃ by adjusting the addition rate. If vigorous bubbling occurs (indicating rapid decomposition of H2O2), further slow down the addition rate. After complete addition, continue stirring at 0-5℃ for 1 hour. The color of the mixture changes from grayish-black to dark brownish-green, indicating the start of graphite intercalation. Technical explanation: Under these temperature conditions, the half-life of H2O2 in the ammonium persulfate system is approximately 4-5 hours, enabling controlled generation of oxidizing active species. The slow reaction process allows the intercalation to occur uniformly throughout the micron-scale structure, effectively avoiding ablation at the scale edges.

[0045] 4) Intermediate-temperature oxidation stage (40°C): The reactor is heated to 40°C at a rate of 0.5°C / min. At 40°C, the half-life of H2O2 decreases to approximately 45 minutes. The second addition of hydrogen peroxide (200 mL 30% w / w H2O2) is then performed at a rate of 5-10 mL / min. This increases the reaction intensity; a small amount of oxygen bubbles will backflow during the process, and the system temperature is maintained at 40±2°C. After the addition is complete, stirring continues for 10 minutes. Since most intercalation has already occurred at 0-5°C, this short stirring time is sufficient to meet the reaction requirements. Technical notes: For micron-scale systems, H2O2 can be added in two stages; while for large-scale systems, it needs to be added three or more times. Due to its high specific surface area, all graphite particles can fully contact the oxidant.

[0046] 5) High-temperature oxidation stage (60°C): Heat to 60°C at a rate of 0.5-1.0°C / min. As peeling begins, the viscosity of the reaction system increases significantly. Maintain the system temperature at 60±2°C and continue the reaction for 8 hours. During this period: 0-2 hours see rapid oxygen release; the system color turns golden brown; 2-6 hours see slow oxygen release, and the system viscosity continues to increase; 6-8 hours see no further gas release; the viscosity of the reaction system reaches its peak. Technical note: Based on the diffusion-controlled oxidation kinetics of the micron-scales, the processing time is determined to be 8 hours. If the processing time is less than 6 hours, incomplete oxidation will occur (product oxygen content <8%); if the processing time exceeds 10 hours, over-oxidation will occur (product oxygen content >18%) and damage the substrate.

[0047] 6) Post-processing:

[0048] Filtration: Cool the reaction mixture to <40°C. Filter using a Buchner funnel or filter press with a 0.45µm PTFE filter membrane, and collect the filtrate (containing (NH4)2SO4, residual H2SO4, and excess H2O2) for neutralization. The filter cake volume is typically 2-3 L.

[0049] Washing: Resuspend the filter cake in 5L of deionized water, stir for 15 minutes, and filter again. Repeat 5-7 times until the pH of the wash water reaches 6.5-7.5 and the conductivity is <50µS / cm. For micron-sized flake products, due to the low ash content of the starting raw material, ionic contamination is reduced, and fewer washing cycles are required (5 times compared to 10-12 times for large flakes).

[0050] Preliminary drying: Transfer the washed filter cake to a glass or PTFE-lined tray. Dry to constant weight in a vacuum oven at 60-80°C (increasing from 60°C to 80°C over 2 hours) and -0.08 MPa vacuum (12-16 hours). Yield at this stage: 180-190 g of dried intermediate from 200 g of starting micron-sized flake graphite (90-95% by mass, accounting for oxygen introduction).

[0051] 7) Removal of ammonium persulfate (short-time low-temperature calcination):

[0052] Place the dried intermediate (180-190g) into an alumina or quartz crucible. The material bed depth should not exceed 2cm to ensure uniform heating. Place the crucible in a muffle furnace preheated to 120°C, close the furnace door, and calcine for 10 minutes. During this period: 0-2 minutes: residual moisture escapes; 2-6 minutes: (NH4)2S2O8 decomposes: (NH4)2S2O8 → 2NH3 + H2S2O7 (or SO3 + H2O), then further decomposes into SO2 and O2; 6-10 minutes: decomposition complete; white smoke (NH3, SO2) stops. Remove the crucible and cool to room temperature on a heat-resistant surface in a fume hood. The final product is functional graphene oxide powder with an oxygen content of 12-15% (measured by XPS), and ammonium persulfate is undetectable by ion chromatography (detection limit: 10ppm).

[0053] The reason for choosing 120°C for 10 minutes is as follows: Thermogravimetric analysis (TGA) of the drying intermediate showed: 80-110°C: 5% mass loss (adsorbed water); 110-125°C: 12% mass loss (decomposition of ammonium persulfate); >125°C: gradual mass loss (decomposition of oxygen-containing functional groups on graphene); a duration of 10 minutes at 120°C completely removes ammonium persulfate while retaining >95% of the target oxygen-containing functional groups. Longer times (>20 minutes) or higher temperatures (>130°C) will begin to decrease the oxygen content.

[0054] (2) Preparation of transparent nanocolloids (100nm, 2-3 layers):

[0055] An antibacterial functional graphene nanocolloid was produced with a lateral dimension D50 of 100nm ± 10nm, 2-3 layers, a solid content of 1% ± 0.1%, an oxygen content of 10-15%, and a transparent appearance (UV-Vis transmittance of 0.1% diluted solution at 660nm > 70%).

[0056] 1) Preparation of colloidal suspensions:

[0057] Use 3.8L of deionized water. Dispersant selection: For antibacterial applications, the dispersant must be non-toxic, biocompatible, and preferably possess intrinsic antibacterial synergistic effects. Recommended dispersants include: polyvinylpyrrolidone (PVP K30): 0.2-1.0% w / w, providing steric stabilization and auxiliary antibacterial properties; soapberry thorn extract: 0.3-1.0% w / w, with natural broad-spectrum antibacterial activity; sodium deoxycholate (bile salt): 0.01-0.05% w / w, providing strong electrostatic stabilization and natural antibacterial properties. Add the dispersant to the water and stir at 300-500 rpm for 10-20 minutes until completely dissolved.

[0058] Add functional graphene oxide powder: Slowly add 160g of powder from step (1). Adding rate: 5-10g per minute, stirring continuously. The powder will initially float, then slowly hydrate and disperse.

[0059] Premixing: Continue stirring at 500-800 rpm for 30-60 minutes. The mixture will darken in color and form a viscous dark brown suspension. At this point, the average particle size is usually 5-20 µm (aggregates).

[0060] 2) Shear emulsification pretreatment:

[0061] Equipment: High-shear rotor-stator emulsifier, equipped with a 20-30mm diameter generator; settings: 8000-12000rpm, linear speed 15-25m / s.

[0062] Process: Pass the colloidal suspension through an emulsifier. For batch operations, circulate the entire volume for 20-40 minutes; for continuous operations, pass through once and maintain a residence time of 10-20 minutes.

[0063] Temperature control: Use a jacketed container or in-line heat exchanger to maintain the temperature ≤30°C. High shear will generate heat (>50°C), causing graphene to aggregate.

[0064] Endpoint: After shear emulsification, the particle size was reduced to D50 = 500-1000 nm as measured by dynamic light scattering (DLS). The viscosity of the suspension was significantly reduced, and the color was more uniform.

[0065] 3) Two-stage grinding (micron-level wet grinding):

[0066] Equipment: Horizontal pin-type sand mill with zirconia chamber lining; Two-stage operating parameters: Grinding chamber pressure: 0.2-0.4MPa; Circulation flow rate: 10-20L / h; Grinding speed: 2000-3000rpm; Cooling water temperature: 15℃; Feed suspension: Output of step 2), diluted if necessary to maintain pumpability.

[0067] a. Perform the first grinding using 0.4-0.6mm zirconia beads:

[0068] Bead filling amount: 70-80% of the chamber volume; Duration: 3 hours; Sampling plan: 5 mL samples are taken at 0, 1, 2, and 3 hours for DLS measurement. Expected particle size after 3 hours: D50 = 200-300 nm. Appearance: Dark brown, opaque.

[0069] b. Perform a second grinding using 0.2mm zirconia beads:

[0070] Replace with 0.2mm diameter yttrium-stabilized zirconia beads; duration: 3 hours. Sampling plan: Sampling at 3, 4, 5, and 6 hours cumulatively, with an expected particle size of D50 of 100-120nm after a cumulative 6 hours (3+3). Appearance: Dark translucent brown.

[0071] Two-stage grinding technology explained: First, large beads (0.4-0.6mm) are used to effectively break down agglomerates from 500-1000nm to 200-300nm. Then, small beads (0.2mm) are used to achieve the final target of 100nm. Using only small beads from the beginning is inefficient (long grinding time, high wear), while using only large beads cannot achieve <150nm due to the minimum gap between the beads.

[0072] 4) High-speed mixing (nano-wetting / surface conditioning):

[0073] Equipment: High-speed disperser, equipped with a serrated impeller or rotor-stator mixer, capable of reaching 8000 rpm in 5-10L containers.

[0074] Procedure: Transfer the milled suspension to a mixing container. Set the rotation speed to 8000 rpm.

[0075] Nitrogen purging: Continuously purge the suspension with nitrogen at a rate of 0.5-1.0 L / min. Nitrogen purging serves three purposes: removing dissolved oxygen that may lead to oxidative degradation; preventing re-agglomeration by maintaining an inert atmosphere; and helping to dissipate the heat generated by high-speed mixing. Duration: 3 hours. During this period, the suspension temperature should be maintained at 25-35°C (using a cooling jacket if necessary).

[0076] What happens in this step: High shear and nitrogen purging promote the embedding of water molecules between the graphene layers, further separating any remaining stacked sheets. Dispersant molecules also rearrange on the graphene surface to maximize steric stabilization.

[0077] 5) High-pressure microjets:

[0078] Equipment: High-pressure homogenizer / microjet machine, equipped with diamond or ceramic interactive chamber; Pressure: 2000 bar (200 MPa, approximately 29,000 psi); Number of cycles: 40; Feed rate per cycle: 5-10 L / h.

[0079] Cooling: The microjets generate a large amount of heat due to cavitation. A plate heat exchanger is used after the interaction chamber to cool the product back to 25-35°C before recirculation.

[0080] Sampling plan: Particle size (DLS) is measured every 10 cycles. After 10 cycles: D50 = 120-140 nm; after 20 cycles: D50 = 105-115 nm; after 30 cycles: D50 = 95-105 nm; after 40 cycles: D50 = 90-100 nm. The reason for 40 cycles is that for graphene oxide produced from micron-sized flake graphite, the relationship between the number of microjets (N) and the transverse size (D) follows a power law: D(N) = D_initial × N - 0.3. Starting from D_initial = 120 nm after milling, if the relationship continues indefinitely, 40 cycles yield D = 120 × 40 - 0.3 ≈ 120 × 0.3² ≈ 38 nm. However, a plateau is reached at 90-100 nm because further size reduction requires spp fracture. 2 The required energy exceeds what the microfluidic generator can provide. 40 cycles is the optimal point: fewer cycles result in particles >100nm, while more cycles lead to diminishing returns and increase energy consumption and potential contamination from chamber wear.

[0081] 6) Final Adjustment and Quality Control

[0082] The solids content is measured by drying 5g of the suspension at 105°C for 24 hours, with a target of 1% ± 0.1%. If the solids content is higher than 1.1%, dilute with deionized water containing the same concentration of dispersant as used in the preparation of the colloidal suspension. If the solids content is lower than 0.9%, concentrate by gentle rotary evaporation under reduced pressure at 40°C; the final product is a transparent to translucent dark brown nanocolloid. When diluted with water at a ratio of 1:100, the solution should be light brown and clear (without visible particles). Filter through a 200µm nylon mesh to remove any large agglomerates (this rarely occurs if the procedure is performed correctly).

[0083] 7) The characterization results of micron-scale derivative products are shown in Table 3;

[0084] Table 3 Characterization results of micron-scale derivative products

[0085] Lateral dimension (D50) 100nm±10nm 98±6nm Malvern Zetasizer, TEM image analysis D90 (Maximum Size) <150nm 132±12nm Laser diffraction number of floors 2-3 floors 2-3 layers (TEM average value) <![CDATA[TEM (count ≥ 100 pieces), Raman I2D / IG ratio > 1.2]]> thickness 1.0-1.5nm 1.2±0.3nm AFM (Mica Tape Mode) Oxygen content (atomic %) 10-15% 12.8±1.2% XPS (C1s and O1s peaks) Carbon / Oxygen Ratio (C / O) 5.7-9.0 6.8±0.8 XPS C=O / CO ratio (functional group distribution) 0.4-0.6 0.52 XPS C1s peak fitting Solid content 1.0%±0.1% 1.02±0.04% Gravimetric method (105°C, 24h) pH value (stock solution) 6.0-7.5 6.8±0.3 pH meter electrical conductivity <500µS / cm 320±50µS / cm conductivity meter Zeta potential -40 to -60mV -48±5mV Electrophoretic light scattering UV-Vis transmittance (0.01% dilution, 660nm) >70% 76±4% UV-Vis spectrophotometer Settlement stability (30 days, room temperature) No visible sediment No precipitation, D50 increase <10% Visual inspection + DLS Antibacterial activity (Escherichia coli, 4h, 50µg / mL) ≥99.9% reduction 99.997% (4.5 logarithmic decrease) Plate counting method Antibacterial activity (Staphylococcus aureus, 4h, 50µg / mL) ≥99.9% reduction 99.99% (4.0 logarithmic decrease) Plate counting method Cytotoxicity (L929 fibroblasts, 72h, 100µg / mL) >80% survival rate 88±5% survival rate MTT method Residual ammonium persulfate <10ppm <2ppm Ion chromatography Residual metal ions (Mn, Fe, Cu, Cr, Ni) <1ppm per Below detection limit (ICP-MS) ICP-MS

[0086] Comparative Example 1

[0087] Large flake graphite (>50µm)

[0088] (1) Follow the same oxidation and exfoliation conditions as in Example 2, but use 150µm large flake graphite as the starting material.

[0089] (2) Results: After oxidation: D50 = 80µm (limited size reduction); After complete peeling: D50 = 2.8µm (2800nm), not reaching 100nm; Number of layers: 8-12 layers; Antibacterial activity: logarithmic reduction of 1.2 at 50µg / mL (94% kill rate); Conclusion: Under the same process conditions, large scales cannot be reduced to 100nm.

[0090] Comparative Example 2

[0091] Irregular graphite powder (D50=20µm, aspect ratio<5:1)

[0092] (1) Following the same oxidation and exfoliation conditions as in Example 2, irregular graphite powder was selected as the starting material.

[0093] (2) Results: After oxidation: uneven color (some particles were over-oxidized, and some were under-oxidized); After peeling: wide size distribution (D50=350nm, D90=1200nm), number of layers: 4-7 layers, with great variation, yield of 2-3 layer products: <20%. Conclusion: Non-flake morphology leads to uneven oxidation and peeling.

[0094] Comparative Example 3

[0095] Hummers method for micron-sized flake graphite

[0096] (1) Using the same micron-sized graphite as in Example 2 as the starting material, the Hummers method was used for the experiment.

[0097] (2) Results: Oxidation was severe; even with an ice bath, the temperature still soared to >80°C. After washing: Metal ion residue (Mn: 450 ppm, determined by ICP-MS). After stripping: D50 = 180 nm, oxygen content 22% (over-oxidation). Antibacterial activity: Logarithmic reduction of 2.8, but due to the residual Mn, it was cytotoxic. Conclusion: Even with the use of micron-sized scales, the Hummers method introduces metal contamination and over-oxidation.

[0098] Test case

[0099] Antibacterial performance test

[0100] (1) The antibacterial properties of Escherichia coli were determined under the conditions of diluting the sample concentration of Example 2, Comparative Example 1 and Comparative Example 3 to 50 µg / mL and antibacterial action time of 4 h, as shown in Table 4.

[0101] Table 4 Comparison of antibacterial activities

[0102] Comparative Example 1 (Large Scales) 1.2 Comparative Example 3 (Hummers) 2.1 Example 2 4.5(≥99.997%)

[0103] (2) According to Table 4, the antibacterial log reduction value of the product of this application is 4.5, while that of large flake graphite is only 1.2, and that of conventional Hummers process product is 2.1.

[0104] The antibacterial mechanism of 100nm, 2-3 layer graphene is as follows: The antibacterial activity of the prepared nanocolloids originates from three synergistic mechanisms: Cell membrane disruption through edge insertion: The sharp edges of the 100nm sheets insert into the bacterial lipid bilayer; Molecular dynamics simulations show that sheets with lengths of 50-150nm have the optimal edge curvature to achieve membrane penetration; Larger sheets (>200nm) are in a flat state, while smaller sheets (<50nm) are ingested by bacteria without lysis. Induced oxidative stress: Oxygen-containing functional groups (XPS measured at 10-15%, mainly edge-based -COOH and -OH) generate reactive oxygen species (ROS), including superoxide anions (O2). - • and hydroxyl radicals (•OH); micron-scale-derived graphene has a high edge density, maximizing ROS generation per unit mass. Physical encapsulation and isolation: 2-3 layers of flakes (approximately 1-1.5 nm thick) are flexible enough to adapt to the bacterial surface, yet rigid enough to maintain sharp edges, encapsulating bacteria and blocking nutrient uptake.

[0105] In summary, this application addresses the key bottlenecks of traditional graphene antibacterial materials, such as excessive oxidation, uneven size, inconsistent number of layers, residual toxicity, and low antibacterial efficiency. It proposes an integrated preparation route using micron-sized flake graphite as raw material and employing a metal-free, environmentally friendly stepwise temperature-controlled oxidation combined with low-temperature short-time calcination and multi-stage mechanical precise exfoliation. This approach successfully prepared highly transparent antibacterial graphene nanocolloids with a lateral size D50 of 90-110 nm, 2-3 layers, and an oxygen content of 10-15 atomic percentage.

[0106] Through three-stage temperature control and stepwise addition of hydrogen peroxide, the oxidation degree is precisely controllable. Utilizing two-stage milling and high-pressure microfluidics, uniform sheet size and layer count are ensured. The entire process avoids the use of strong acids, heavy metals, and toxic solvents, resulting in thorough removal of byproducts and excellent biocompatibility. The prepared colloid exhibits potent antibacterial activity against common pathogens through a synergistic mechanism of membrane puncture damage, reactive oxygen species oxidative stress, and physical encapsulation. It can be widely applied in antibacterial coatings, medical devices, wound dressings, water treatment membranes, and food packaging, providing a stable and feasible technical solution for the large-scale and green preparation of high-performance, safe graphene antibacterial materials.

[0107] Although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. A method for preparing antibacterial functional graphene nanocolloids, characterized in that, The preparation method includes: (1) Raw material pretreatment: Micron-sized flake graphite is mixed with an acidic aqueous solution containing ammonium persulfate; (2) Staged temperature-controlled oxidation: The first part of hydrogen peroxide is added at low temperature, the second part of hydrogen peroxide is added at medium temperature, and the reaction is carried out at 60°C to obtain the reaction solution. (3) Intermediate: The reaction solution is subjected to solid-liquid separation, washing, drying, and calcination to obtain a functional graphene oxide intermediate; (4) Nanocolloids: The functional graphene oxide intermediate is dispersed in water containing a biocompatible dispersant, and then subjected to high-shear emulsification, two-stage milling, high-speed mixing under nitrogen protection, and high-pressure microjet; the solid content is adjusted to obtain transparent nanocolloids.

2. The method for preparing an antibacterial functional graphene nanocolloid as described in claim 1, characterized in that, In step (1), the average transverse dimension of the micron-scale graphite flakes is 1-10 μm, and the aspect ratio is greater than 10:

1.

3. The method for preparing an antibacterial functional graphene nanocolloid as described in claim 1, characterized in that, In step (1), the mass ratio of ammonium persulfate to micron-sized flake graphite is 8:1-12:

1.

4. The method for preparing an antibacterial functional graphene nanocolloid as described in claim 1, characterized in that, In step (2), the low temperature is 0-5℃ and the medium temperature is 40℃; the volume ratio of the first part of hydrogen peroxide to the second part of hydrogen peroxide is 2:

1.

5. The method for preparing an antibacterial functional graphene nanocolloid as described in claim 1, characterized in that, The calcination conditions in step (3) are 115-125°C for 8-12 minutes.

6. The method for preparing an antibacterial functional graphene nanocolloid as described in claim 1, characterized in that, The two-stage grinding in step (4) involves grinding in stages using 0.4-0.6mm zirconia beads and 0.2mm zirconia beads in sequence.

7. The method for preparing an antibacterial functional graphene nanocolloid as described in claim 1, characterized in that, The biocompatible dispersant in step (4) includes polyvinylpyrrolidone, soapberry extract and sodium deoxycholate.

8. An antibacterial functional graphene nanocolloid prepared by any of the preparation methods described in claims 1-7.

9. The antibacterial functional graphene nanocolloid as described in claim 8, characterized in that, The nanocolloid has a lateral dimension D50 of 90-110 nm, 2-3 layers, an average AFM thickness of 1.0-1.5 nm, an oxygen content of 10-15 atomic percent, and a solid content of 0.9-1.1%.

10. The application of the antibacterial functional graphene nanocolloid as described in claim 8 or 9 in antibacterial coatings, biomedical devices, wound dressings, water treatment membranes, or food packaging materials.