Method for testing the biostatic properties and durability of a silicone rubber surface

Abstract

The application belongs to the technical field of material surface performance detection, and particularly relates to a detection method for surface anti-biofouling property and durability of silicone rubber, which comprises the following steps: S1, sequentially performing biofilm inhibition rate quantitative detection, anti-bacterial adhesion detection, anti-protein adsorption performance test and surface wettability test on the silicone rubber sample to be detected; S2, performing durability treatment on parallel samples; S3, repeating part or all of the tests in step S1 on the samples treated in step S2; S4, comparing the performance data obtained in step S1 and step S3 to evaluate the anti-biofouling property and durability of the silicone rubber sample. The application organically combines biofilm inhibition rate quantitative detection, anti-bacterial adhesion detection, anti-protein adsorption performance test, surface wettability analysis and durability test, forms a complete evaluation closed loop from molecule adsorption to microbial community and from instant efficiency to long-term stability, and comprehensively covers each key link of biofouling.

Description

Technical Field

[0001] This invention relates to a method for testing the resistance to biofouling and durability of silicone rubber surfaces, belonging to the field of material surface performance testing technology. Background Technology

[0002] Silicone rubber has been widely used in the field of medical devices due to its excellent biocompatibility, good flexibility and outstanding chemical stability. It is used to manufacture various medical devices that come into direct contact with or are implanted in the human body, such as catheters, implants and seals, providing important support for clinical diagnosis and treatment.

[0003] However, the inherent surface properties of silicone rubber pose a significant risk of biocontamination in complex biological environments, both in vivo and in vitro. Specifically, it readily exhibits non-specific protein adsorption and bacterial adhesion. This initially formed protein adsorption layer not only alters the interfacial physicochemical properties of the material but may also trigger unnecessary immune responses in the body. More critically, this adsorption layer provides bridging sites and a nutrient substrate for the adhesion of bacteria and other microorganisms, becoming the initiation point for biofilm formation. Once a complex biofilm forms on the silicone rubber surface, bacterial resistance to antibiotics and the host's immune system will be significantly enhanced, leading to persistent device-related infections. This not only seriously threatens patients' lives and health but also significantly increases the clinical burden, severely limiting the safety and reliability of medical silicone rubber materials.

[0004] To address the aforementioned challenges of resisting biofouling, extensive research has been conducted in related fields, resulting in the development of various silicone rubber surface modification technologies. These include coating with antibacterial coatings, constructing hydrophilic hydration layers, and designing micro- and nano-scale surface topologies. The aim is to enhance the anti-protein adsorption and antibacterial adhesion properties of silicone rubber by altering its surface properties. However, current performance evaluation methods for surface-modified silicone rubber materials have significant limitations, making it difficult to scientifically and comprehensively reflect the actual performance of the modified silicone rubber materials. Specifically, existing evaluation systems rely on simplistic testing methods, with most studies employing only one method (e.g., evaluating antibacterial performance solely through inhibition zone experiments or measuring contact angles to characterize surface hydrophilicity and hydrophobicity). This results in a one-sided performance characterization, failing to form a complete performance evaluation system, hindering systematic analysis of material properties, and failing to comprehensively cover the overall performance required of materials in complex biological environments. Furthermore, significant differences exist in testing methods and experimental conditions used by different laboratories, leading to a lack of comparability in data from various studies and hindering the provision of a unified reference for technological development. In addition, existing evaluation systems generally neglect the systematic examination of silicone rubber durability. The ability of silicone rubber surfaces to maintain their anti-fouling properties after long-term exposure to complex tests such as bodily fluid erosion, enzymatic hydrolysis, and physical friction directly determines the clinical translational value of modified silicone rubber materials, but this core evaluation step is often simplified or even omitted.

[0005] Existing testing methods for the surface modification effects of medical silicone rubber are limited and lack a systematic approach, failing to comprehensively assess the immediate and long-term stability of silicone rubber materials against biofouling. Therefore, developing a standardized, comprehensive testing method that simultaneously covers both immediate performance and long-term stability verification is of paramount practical significance and urgent need for the scientific and accurate evaluation of silicone rubber surface properties. Summary of the Invention

[0006] This invention addresses the shortcomings of existing methods for evaluating the biofouling resistance of silicone rubber surfaces, which are often limited in scope, lack systematicity, and neglect durability assessment. It provides a method for detecting the biofouling resistance and durability of silicone rubber surfaces. This method can quantitatively assess key biofouling processes such as protein adsorption, bacterial adhesion, and biofilm formation, and correlates these with the crucial physical parameter of surface wettability. Finally, aging tests and mechanical friction and wear tests verify the durability of the performance, providing scientific and comprehensive technical support for evaluating the biofouling resistance of silicone rubber materials.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: A method for testing the resistance to biofouling and durability of silicone rubber surfaces, comprising the following steps: S1. Comprehensive test of initial anti-biofouling performance: Using the smooth surface of the silicone rubber to be tested as the control sample and the modified surface of the silicone rubber to be tested as the test sample, the control sample and the test sample of the silicone rubber to be tested were subjected to quantitative detection of biofilm inhibition rate, antibacterial adhesion, anti-protein adsorption performance and surface wettability test in sequence to obtain the initial anti-fouling performance data of the sample. S2. Simulated Environmental Durability Treatment: The samples undergo a durability treatment that simulates the actual use environment. S3. Performance evaluation after durability treatment: For the sample treated in step S2, repeat some or all of the tests in step S1 to obtain the anti-pollution performance data after durability treatment. S4. Comprehensive performance evaluation: Compare the performance data obtained in step S1 and step S3 to evaluate the resistance to biofouling and durability of the silicone rubber sample.

[0008] Based on the above technical solution, the present invention can also be improved as follows: Furthermore, in step S1, the quantitative detection of the biofilm inhibition rate adopts the biofilm crystal violet staining method. The sample is co-cultured with bacterial suspension to form a biofilm. After crystal violet staining and elution, the absorbance of the dye is measured to quantify the total amount of biofilm and calculate the biofilm inhibition rate.

[0009] Furthermore, in step S1, the antibacterial adhesion detection includes qualitative and quantitative analysis of bacterial adhesion, wherein: The qualitative analysis of bacterial adhesion uses the inhibition zone method, in which the sample is attached to the surface of an agar plate inoculated with bacteria, and after incubation, the size of the sterile area around the sample is observed and measured to qualitatively evaluate the exudation of antibacterial substances on the surface or the contact inhibition ability. The quantitative analysis of bacterial adhesion was performed using the plate colony counting method. After incubating the sample with a bacterial suspension, the bacteria adhering to the surface were washed off and colonies were counted to calculate the antibacterial adhesion rate.

[0010] Furthermore, in step S1, the anti-protein adsorption performance test includes qualitative analysis and quantitative analysis, wherein: The qualitative analysis employed the FITC fluorescent labeling method, incubating the sample with a fluorescein-labeled protein solution, and then observing the distribution of adsorbed proteins on the surface using fluorescence imaging after washing. The quantitative analysis was performed using the BCA protein concentration assay. After incubating the sample with a protein solution and eluting, the total amount of protein adsorbed on the sample surface was determined using a BCA kit, and the anti-protein adsorption rate was calculated.

[0011] Furthermore, in step S1, the surface wettability test uses a contact angle measuring instrument to measure the static water contact angle of the sample.

[0012] Furthermore, in step S2, the simulated environmental durability treatment includes at least one of the following: a. Liquid immersion aging treatment: The sample is immersed in simulated body fluid or phosphate buffer solution and soaked for several days at a constant temperature of 37±1℃. b. Mechanical friction and wear treatment: The sample surface and the standard friction medium are subjected to relative sliding friction under a set normal pressure.

[0013] Furthermore, the standard friction medium is medical gauze, and the positive pressure is achieved by weights placed on the sample, with the weights having a mass of 40-60g.

[0014] Furthermore, in step S4, the antifouling performance is quantified by calculating the biofilm inhibition rate, antibacterial adhesion rate, and antiprotein adsorption rate; among them, a biofilm inhibition rate ≥70%, an antibacterial adhesion rate ≥80%, and an antiprotein adsorption rate ≥80% are evaluated as having good antifouling performance.

[0015] Furthermore, in step S1 or step S3, the comprehensive detection follows the following logic: Quantitative detection of biofilm inhibition rate: if the measured biofilm inhibition rate reaches or exceeds 70%, the predicted antibacterial adhesion rate and antiprotein adsorption rate can reach 80%, respectively. And / or, antibacterial adhesion test: if the measured antibacterial adhesion rate reaches or exceeds 80%, it is predicted that its antiprotein adsorption rate can reach or exceed 80%. Based on the above predictions, subsequent antibacterial adhesion tests and / or antiprotein adsorption performance tests can be selectively simplified.

[0016] The core of this invention lies in the clear correlation between the anti-biofouling performance test indicators, which simplifies the testing process: when performing tests sequentially using the crystal violet staining method, inhibition zone method, plate colony counting method, FITC-BSA fluorescent labeling method, and BCA protein concentration determination method, if the biofilm inhibition rate (i.e., anti-biofilm adhesion rate) reaches 70% or higher, it can be predicted that the antibacterial adhesion rate will likely meet the requirement of 80% or higher; if the antibacterial adhesion rate (i.e., antibacterial rate) reaches 80% or higher, it can be predicted that the anti-protein adsorption rate will likely meet the requirement of 80% or higher. This correlation stems from the progressive mechanism of biofouling—protein adsorption is the basis of bacterial adhesion, and bacterial adhesion is a prerequisite for biofilm formation. High-performance materials need to perform well in each stage. Therefore, excellent results in a later stage (such as biofilm formation) can, in turn, confirm that the performance of the preceding stages (such as bacterial adhesion and protein adsorption) meets the standards, thereby optimizing and simplifying the testing process.

[0017] Furthermore, the bacteria include Gram-negative and Gram-positive bacteria, preferably Escherichia coli and Staphylococcus aureus; the protein is bovine serum albumin.

[0018] The beneficial effects of this invention are as follows: This invention organically combines quantitative detection of biofilm inhibition rate, antibacterial adhesion detection, antiprotein adsorption performance testing, surface wettability analysis, and durability testing to form a complete evaluation closed loop from molecular adsorption to microbial community, from immediate efficacy to long-term stability, comprehensively covering all key aspects of biological pollution.

[0019] This invention provides a variety of methods, from qualitative (such as FITC fluorescent labeling and inhibition zones) to highly quantitative (BCA method, colony counting, crystal violet quantification), with objective and highly comparable results, avoiding the limitations of single methods. By introducing surface wettability (contact angle) testing, macroscopic antifouling performance is correlated with microscopic surface properties (such as hydrophobicity), which helps to deepen the understanding of antifouling mechanisms and provides theoretical guidance for optimizing modification processes.

[0020] This invention innovatively incorporates durability testing into the evaluation system, which can effectively predict the performance retention rate of modified surfaces under simulated real-world usage environments (immersion in body fluids, mechanical friction), and has important guiding value for the clinical application prospects of materials.

[0021] Within the systematic testing framework of this invention, there is an inherent logical correlation and statistical trend among different levels of anti-fouling performance indicators (biofilm inhibition rate > antibacterial adhesion rate > antiprotein adsorption rate). Biofilm formation represents the most mature stage of pollution and is the most difficult to inhibit. Therefore, if a sample exhibits excellent anti-biofilm performance (e.g., inhibition rate ≥ 70%), it is highly likely to exhibit equal or better performance in upstream stages (antibacterial and antiprotein adsorption). This principle allows for reasonable prediction of lower-level indicators (e.g., antibacterial rate, antiprotein adsorption rate) based on the results of higher-level indicators (e.g., biofilm inhibition rate) when using this method for routine screening or quality control. This enables selective simplification of the testing process and improvement of evaluation efficiency while ensuring the reliability of the assessment.

[0022] The method of this invention has clear steps, and the reagents and instruments used are all common laboratory equipment, which is easy to standardize and promote in different laboratories, providing a reliable tool for the research and development, quality control and product evaluation of medical silicone rubber materials. Attached Figure Description

[0023] Figure 1 This is a schematic diagram illustrating the operation process of the crystal violet staining method for biological membranes in a specific embodiment; Figure 2 This is a schematic diagram illustrating the operation process of the inhibition zone method in a specific implementation embodiment; Figure 3 This is a schematic diagram illustrating the operation process of the plate colony counting method in a specific implementation embodiment; Figure 4 This is a schematic diagram illustrating the principle of the FITC fluorescent labeling method in a specific implementation method; Figure 5 This is a schematic diagram illustrating the principle of the BCA protein concentration determination method in a specific implementation embodiment; Figure 6 This is a schematic diagram illustrating the operation process of the body fluid immersion aging treatment in a specific embodiment; Figure 7 This is a schematic diagram of the operation process of mechanical friction and wear treatment in a specific embodiment; Figure 8 This is a schematic diagram illustrating the specific operation process of the detection method of the present invention; Figure 9 The results are observed using the FITC fluorescent labeling method in a specific implementation. Detailed Implementation

[0024] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0025] Example 1 I. Experimental Preparation 1. Experimental Samples: GA-2760A / B silicone rubber was selected, and its main components are shown in Table 1 below. The surface of the silicone rubber samples was processed using a femtosecond laser with a laser power of 5W, a frequency of 200 kHz, a scanning speed of 60 mm / s, and a scanning interval of 0.1 mm. The processed structures were a grid, grooves, a biomimetic lotus leaf, and a biomimetic fish scale, designated as the test samples. A smooth silicone rubber surface was used as a control sample.

[0026] Table 1. Composition of Silicone Rubber

[0027] 2. Experimental strains: Escherichia coli (EPEC, enteropathogenic Escherichia coli) and Staphylococcus aureus (ATCC25923), both of which are common pathogens associated with medical device-related infections and are commercially available.

[0028] 3. Main reagents and instruments: FITC-BSA (fluorescein-labeled bovine serum albumin), BCA protein detection kit, crystal violet staining solution, simulated body fluid, BHI culture medium and other biological and chemical reagents were all from Shanghai Maclean Biotechnology Co., Ltd.; ELISA reader (SpectraMax Paradigm, Molecular Devices, USA), confocal laser scanning microscope (CLSM, Zeiss LSM 800, Germany), contact angle meter (OCA 15EC, Dataphysics, Germany), CO2 incubator (Nu-5800, NuAire, USA), biosafety cabinet (NU-425-400S, NuAire, USA), etc.

[0029] 4. Core evaluation indicators: anti-protein adsorption rate, anti-bacterial adhesion rate (bacteriostatic rate) and biofilm inhibition rate. The evaluation criteria for each indicator are shown in Table 2 below.

[0030] Table 2 Evaluation Criteria for Core Indicators

[0031] II. Specific Operating Procedures for Testing See Figure 8 The method for testing the resistance to biofouling and durability of silicone rubber surfaces proposed in this invention specifically includes the following steps: Step 1: Quantitative detection of biomembrane inhibition rate – Crystal violet staining method for biomembranes See Figure 1 After sterilization, each sample was placed in a 24-well plate, and 100 μL of bacterial culture with a concentration of 1×10⁻⁶ was taken from each well. 6 CFU / mL *E. coli* and *Staphylococcus aureus* cultures were added to the corresponding wells and incubated at 37°C for 48 hours. After incubation, the sample surface was rinsed three times with PBS to remove unattached airborne impurities. Then, 100 μL of 0.1% methanol was added to the wells for 15 min to denature and fix the proteins in the biofilm onto the sample surface. Subsequently, 100 μL of 0.1% crystal violet staining solution was added, ensuring complete coverage of the sample surface, and staining was performed for 15 min. After removing the staining solution, the sample surface was rinsed with PBS until the rinse solution was free of purple color to remove unbound free crystal violet. The samples were air-dried in a fume hood for 30 min, and then eluted with 200 μL of 33% glacial acetic acid for 30 min to dissolve the crystal violet dye. Finally, the absorbance at 590 nm was measured using a microplate reader. Each sample group was prepared in triplicate to ensure data reliability. The biofilm inhibition rate was calculated based on the biofilm absorbance of each sample using the following formula: ; Where A represents the absorbance of the biofilm in each sample.

[0032] The results are shown in Tables 3 and 4: Table 3. Data on inhibition rate of Escherichia coli biofilm

[0033] Table 4. Data on inhibition rate of Staphylococcus aureus biofilm

[0034] In the unaged state, the biofilm content on microstructured surfaces is significantly lower than that on smooth surfaces. The biofilm inhibition rates of the mesh surface against *Escherichia coli* and *Staphylococcus aureus* reached 70.14% and 73.56%, respectively. The biofilm inhibition experiment measured the total amount of biofilm (including viable bacteria, dead bacteria, and extracellular polymers). Compared to the plate colony counting experiment (which only involves viable bacteria), inhibiting biofilm is more difficult, resulting in a lower inhibition rate. These data indicate that the mesh surface exhibits the highest inhibition efficacy for both strains, followed by lotus leaf, grooved, and fish scale surfaces, showing a consistent overall trend. This suggests that the surface micro / nanostructures play a crucial regulatory role in bacterial adhesion and biofilm formation.

[0035] Step 2: Antibacterial adhesion behavior test 2.1 Inhibition zone method: See Figure 2 Take 100 μL of bacterial culture with a concentration of 1×10 6 CFU / mL *E. coli* bacterial suspension and *Staphylococcus aureus* suspension were evenly spread onto the surface of BHI solid agar medium using a sterile spreader. The samples were then sterilized, and each sample surface was attached to the agar surface inoculated with bacterial suspension. After incubating the plates at 37°C for 24 hours, the presence of a transparent inhibition zone around the sample was observed to qualitatively determine the exudation of antibacterial substances.

[0036] After the inhibition zone incubation was completed, no visible transparent inhibition zones appeared around any of the five samples, indicating that no soluble antibacterial substances with significant inhibitory effects on colony growth diffused or released from the sample surface into the surrounding agar medium. This result first rules out the dominant role of chemical sterilization mechanisms, thus highlighting the core contribution of surface physical structure to the subsequently observed antibacterial effect. Furthermore, from a biosafety perspective, this characteristic suggests that these materials are highly safe as potential biomedical materials, avoiding biocompatibility risks associated with the release of ions or molecules.

[0037] 2.2 Plate colony counting method: See Figure 3 After sterilization, each sample was placed in a 24-well plate, and 100 μL of bacterial culture with a concentration of 1×10⁻⁶ was taken from each well.6 CFU / mL *E. coli* and *Staphylococcus aureus* bacterial suspensions were added to the corresponding wells and incubated at 37°C for 6 hours. After incubation, the sample surface was rinsed three times with PBS to remove unattached airborne bacteria. The sample was then transferred to a sterile centrifuge tube containing 5 mL PBS and vortexed vigorously for 2 minutes to ensure thorough elution of surface-adhered bacteria into the PBS. The eluent was diluted tenfold, and 100 μL of the bacterial suspension was inoculated onto BHI solid agar plates using the spread plating method. The inoculated plates were incubated at 37°C for 24 hours, after which the number of surface colonies was counted, and the inhibition rate was quantitatively calculated.

[0038] ; Where C represents the number of colonies in each sample.

[0039] The results are shown in Tables 5 and 6.

[0040] Table 5. Antibacterial data against Escherichia coli

[0041] Table 6. Antibacterial data against Staphylococcus aureus

[0042] Quantitative analysis was performed using a standardized plate count method, with a smooth surface as a control. The colony counts were as high as 793±57 CFU (Escherichia coli) and 695 CFU±72 (Staphylococcus aureus), providing a clear baseline for evaluating the antibacterial effect. The experimental data clearly show that all four treated sample surfaces exhibited highly significant antibacterial capabilities, but their antibacterial efficacy showed a clear gradient due to differences in surface microstructure. Specifically, for Escherichia coli, the grid surface showed the most outstanding antibacterial rate, reaching 92.69% (58±6 CFU), followed by the lotus leaf structure (91.17%, 70±9 CFU), grooved structure (89.53%, 83±8 CFU), and fish scale structure (82.98%, 135±12 CFU). The experimental results against Staphylococcus aureus showed a highly consistent trend: the grid structure performed best (inhibition rate 90.65%, colony count 65±8 CFU), followed by the lotus leaf structure (89.50%, 73±11 CFU), the groove structure (87.34%, 88±13 CFU), and the fish scale structure (85.47%, 101±15 CFU). In summary, the antibacterial adhesion effect was: grid > biomimetic lotus leaf > groove > biomimetic fish scale.

[0043] Step 3: Anti-protein adsorption performance test 3.1 FITC fluorescent labeling method: See Figure 4 First, the samples were equilibrated in phosphate-buffered saline (PBS, pH 7.4) for 2 hours. Then, they were immersed in a 1.0 mg / mL FITC-BSA phosphate-buffered saline solution and incubated at 37°C for 2 hours to ensure adsorption equilibrium. After incubation, the sample surface was gently rinsed three times with PBS solution to remove physically adsorbed protein molecules. The fluorescence signals on different surfaces were imaged using a laser scanning confocal microscope to indirectly observe protein adsorption. The results are shown in [link to results]. Figure 9 The results showed that the surface of ordinary silicone rubber exhibited high-intensity, uniformly distributed green fluorescence, indicating that bovine serum albumin (BSA) was densely and extensively adsorbed on its surface. In stark contrast, the fluorescence signal on the surface of superhydrophobic silicone rubber was extremely weak, with an intensity comparable to the background noise level, making it almost visually indistinguishable.

[0044] 3.2 BCA Protein Concentration Assay: First, the sample was equilibrated in phosphate buffer (pH 7.4) for 2 hours. Then, it was immersed in a 4.5 mg / mL bovine serum albumin solution and incubated at 37°C for 2 hours to simulate the physiological adsorption process. Afterward, it was gently rinsed with PBS to remove loosely bound proteins. Finally, it was immersed in a 2% (w / w) sodium dodecyl sulfate (SDS) aqueous solution, sonicated for 10 minutes, and shaken for 2 hours to completely elute the proteins firmly adsorbed on the sample surface. The protein concentration in the eluent was accurately determined using a Micro BCA protein assay kit. The absorbance at 562 nm was measured using a microplate reader, and the protein adsorption amount and anti-protein adsorption rate were calculated using a standard curve (see [link to standard curve]). Figure 5 ).

[0045] ; Where Q represents the amount of protein adsorbed in each sample.

[0046] The results are shown in Table 7.

[0047] Table 7 Anti-protein adsorption rate data

[0048] Table 7 shows that the smooth surface exhibits the strongest protein adsorption capacity, with an adsorption amount as high as 113.40 ± 6.37 µg / mL. In stark contrast, the protein adsorption amounts of the four biomimetic superhydrophobic surfaces are significantly reduced, and the order of anti-protein adsorption performance from best to worst is: mesh > biomimetic lotus leaf > groove > biomimetic fish scale, which is highly consistent with the results of antibacterial adhesion behavior. Quantitative results show that all four biomimetic superhydrophobic surfaces exhibit excellent anti-protein adsorption performance. Compared with the smooth surface, the protein adsorption amounts of the mesh, lotus leaf, groove, and fish scale surfaces are significantly reduced by 85.70%, 83.21%, 80.60%, and 78.90%, respectively. In other words, their anti-protein adsorption performance is improved by 7.0 times, 6.0 times, 5.2 times, and 4.7 times, respectively. The formula for calculating the improvement factor is: Where Q represents the amount of protein adsorbed in each sample.

[0049] Step 4: Correlation Analysis of Surface Wettability and Antifouling Performance – Surface Contact Angle Measurement Method The material surface was ultrasonically cleaned for 10 minutes each with acetone, anhydrous ethanol, and deionized water to remove surface impurities. Then, the static water contact angle of the surface was measured using a contact angle meter. Five different points were selected for testing each sample, and the average value was taken as a quantitative indicator of surface wettability. Correlation analysis was performed between the contact angle data and the antifouling performance indicators obtained in steps 1-3 to verify the intrinsic relationship between surface hydrophilicity / hydrophobicity and antifouling performance. The results are shown in Table 8.

[0050] Table 8 Surface Wettability (Static Water Contact Angle) Data

[0051] Further analysis of the contact angle data of different surfaces can reveal their wettability characteristics and adaptability to complex environments in greater depth. Table 8 shows that smooth surfaces exhibit the worst hydrophobicity, while the contact angles of all four superhydrophobic surfaces remain above 150°, generally maintaining the performance ranking of "mesh > lotus leaf > groove > fish scale". This ranking is completely consistent with its performance ranking in the anti-fouling test, further confirming the core role of surface micro / nanostructure design in ensuring superhydrophobic stability.

[0052] Step 5: Durability Testing and Performance Retesting 5.1 Body fluid immersion aging test: See Figure 6To simulate the chemical erosion and aging effects of the biological environment on the material surface, samples were immersed in simulated body fluid at a constant temperature of 37°C for 12 days. Throughout the aging cycle, samples were removed, gently rinsed, and dried every 48 hours, and the contact angle test in step 4 and the core biofouling resistance tests in steps 1-3 were repeated, with performance changes recorded. By comparing the experimental data before and after the 12-day aging treatment in simulated body fluid, the evolution of surface wettability and biofouling resistance over time and their correlation can be clearly observed. The results are shown in Tables 9-12.

[0053] Table 9 Comparison of wettability (static water contact angle) of various surfaces

[0054] After aging treatment, the static water contact angle of all sample surfaces decreased to varying degrees. The contact angle of the smooth surface decreased significantly from 110.2° to 95.0°, indicating enhanced hydrophilicity. The contact angles of the four superhydrophobic surfaces also decreased, but remained at a relatively high level (146.7°-151.1°), with the performance ranking consistent with that before aging (mesh > biomimetic lotus leaf > groove > biomimetic fish scale).

[0055] Table 10 Comparison of anti-protein adsorption properties of various surfaces

[0056] After aging treatment, the protein adsorption capacity of the smooth surface increased from 113.40±6.37 μg / mL to 162.37±5.38 μg / mL, while the adsorption capacity of the four superhydrophobic surfaces also increased by 1.5-2 times. Nevertheless, the anti-protein adsorption rate of the superhydrophobic surfaces only decreased slightly, and the ranking of performance was exactly the same as before aging.

[0057] Table 11 Comparison of antibacterial rates of various surfaces

[0058] Before aging, samples with microstructured surfaces showed significantly higher antibacterial rates against *Escherichia coli* and *Staphylococcus aureus* than smooth surfaces, with the mesh surface achieving antibacterial rates of 92.69% (*E. coli*) and 90.65% (*Staphylococcus aureus*), respectively. After aging, the antibacterial rates of all surfaces decreased to varying degrees. The mesh surface's antibacterial rate against *E. coli* decreased from 92.69% to 80.98%, and against *Staphylococcus aureus* from 90.65% to 80.97%. Samples with microstructured surfaces showed a smaller decrease in antibacterial rate. The mesh surface maintained antibacterial rates of 80.98% and 80.97% against both strains, respectively. Although the antibacterial rates of the biomimetic lotus leaf, groove, and biomimetic fish scale surfaces decreased sequentially, they were still significantly higher than those of the smooth surface for the same strains. This indicates that microstructured surfaces can retain some superhydrophobic properties, reducing the effective contact between live bacteria and the surface, thus maintaining relatively excellent antibacterial performance even after aging.

[0059] Table 12 Comparison of biofilm inhibition rates on various surfaces

[0060] After aging, the biofilm content on all surfaces increased, while the inhibition rate decreased simultaneously. However, the anti-biofilm advantage of samples with microstructured surfaces remained clear. Specifically, the biofilm inhibition rate of the mesh surface against the two strains decreased to 66.58% and 68.45%, respectively, with a decrease of only 3.56-5.11 percentage points. Although the biofilm inhibition rates of the biomimetic lotus leaf, groove, and biomimetic fish scale surfaces decreased sequentially, their biofilm absorbance values ​​were still higher than those of the same strains on smooth surfaces.

[0061] 5.2 Mechanical friction and wear test: Design a quantitative tribology and wear assessment device to evaluate the mechanical stability of a surface. See also Figure 7 The sample was inverted so that it was in direct contact with standard medical gauze. A 50g weight was placed on top of the material to simulate slight positive pressure. The silicone rubber sample was pulled at a constant speed of 2 cm / s to induce relative sliding between it and the gauze. After each 20 cm friction stroke, the sample was cleaned, and the contact angle test in step 4 and the core anti-biofouling performance test in steps 1-3 were immediately repeated to evaluate the performance degradation under mechanical action. The results are shown in Tables 13-16.

[0062] Table 13 Comparison of wettability (static water contact angle) of various surfaces

[0063] As shown in the table above, friction and wear caused a decrease in the static water contact angle of all tested surfaces. Specifically, the contact angle of the smooth surface decreased from 110.2° to 99.3°, indicating further enhancement of hydrophilicity. The contact angles of the four superhydrophobic surfaces also decreased, but the final values ​​remained at a high level (147.3°-150.1°), and the performance ranking remained exactly the same as before wear. The mesh surface exhibited the best durability, indicating that its microstructure has a relatively good ability to maintain integrity under mechanical stress.

[0064] Table 14 Comparison of anti-protein adsorption properties of various surfaces

[0065] After abrasion treatment, the protein adsorption capacity on smooth surfaces increased from 113.40±6.37 μg / mL to 201.28±8.22 μg / mL, while the adsorption capacity on superhydrophobic surfaces also increased by 1-2 times (e.g., from 16.21 μg / mL to 32.83 μg / mL on the mesh surface). Correspondingly, the anti-protein adsorption rates decreased; for example, the inhibition rate on the mesh surface decreased from 85.70% to 83.69%. Nevertheless, the inhibition rate of the superhydrophobic surface after abrasion was still significantly higher than that of the smooth surface, and the ranking of performance among the surfaces remained unchanged, demonstrating that the superhydrophobic surface possesses good mechanical stability.

[0066] Table 15 Comparison of antibacterial properties of various surfaces against Escherichia coli

[0067] After the abrasion treatment, the antibacterial rate of each sample decreased to varying degrees, while the ranking of the performance of each surface remained unchanged.

[0068] Table 16 Comparison of the inhibitory performance of various surfaces against Staphylococcus aureus biofilm

[0069] Wear treatment also led to an increase in biofilm biomass on all surfaces. For Staphylococcus aureus, the absorbance value of the mesh surface increased from 0.0524±0.0069 before wear to 0.0853±0.0128 after wear, while its biofilm inhibition rate decreased from 73.56% to 67.44%. Although the biofilm inhibition rate of each superhydrophobic surface decreased after wear, it was still much higher than that of the smooth surface, and the performance ranking remained unchanged.

[0070] The systematic method described in this invention not only clearly distinguishes the advantages and disadvantages of the immediate anti-fouling performance of silicone rubber surfaces with different microstructures, but more importantly, it quantitatively reveals the performance degradation law under simulated usage conditions, proving the optimality of the mesh structure in terms of comprehensive performance and durability. This provides a comprehensive and reliable experimental basis for material selection and modification direction.

[0071] Example 2 This embodiment aims to verify whether the antibacterial and antiprotein adsorption properties of a sample can be effectively predicted when its anti-biofilm properties are known. *Escherichia coli* (EPEC) was used as the experimental strain in this embodiment.

[0072] 1. Sample preparation: Experimental Samples: Five groups of samples were designed using silicone rubber of model KE-2092-40, and the following surface treatments were performed on each sample: Sample A: The superhydrophobic mesh surface was processed using laser technology.

[0073] Sample B: The grooved superhydrophobic surface was processed using laser technology.

[0074] Sample C: Smooth silicone rubber surface without any modification.

[0075] Sample D: The surface is coated with an antibacterial coating that slowly releases silver ions, but the coating surface is relatively rough and easily traps bacteria.

[0076] Sample E: The surface is grafted with a highly hydrophilic PEG layer (polyethylene glycol), which has excellent resistance to protein adsorption. However, the PEG layer has poor physical stability in humid environments and is easily damaged, leading to bacterial adhesion.

[0077] 2. Testing Method: First, the five groups of samples were tested using the "crystal violet staining method" to obtain their biofilm inhibition rate (the amount of biofilm in sample C with a smooth surface was taken as the baseline, i.e., 0% inhibition rate).

[0078] If a sample has a biofilm inhibition rate ≥ 70%, its antibacterial rate ≥ 80% and its anti-protein adsorption rate ≥ 80% can be predicted. Therefore, the sample is considered to meet the requirements, and the testing procedure can be simplified. If a sample has a biofilm inhibition rate < 70%, its antibacterial rate < 80% and its anti-protein adsorption rate < 80% can be predicted. Therefore, the sample is considered to potentially not meet the requirements, and the testing procedure should not be simplified.

[0079] 3. Experimental Results: Table 17 Data on the resistance to biofouling of samples from Example 2

[0080] 4. Conclusion: In the evaluation system of materials' resistance to biofouling, anti-protein adsorption rate, anti-bacterial adhesion rate, and biofilm inhibition rate are selected as core indicators. These are based on the dynamic, phased characteristics of the biofouling formation process, aiming to systematically characterize the antifouling mechanism of materials from the molecular level to the community level. Anti-protein adsorption rate targets the initial stage of pollution. Given that the preferential adsorption of proteins on the material surface can form a "conditional interfacial film," altering the physicochemical properties of the interface and mediating microbial colonization, inhibiting this process can block the biofouling chain at its source. Anti-bacterial adhesion rate focuses on the early colonization stage. Bacterial surface adhesion, whether reversible or irreversible, is a prerequisite for successful microbial colonization. This indicator can effectively assess the material's ability to inhibit initial microbial attachment under static or dynamic environments. Biofilm inhibition rate targets the mature stage of pollution. Biofilms form a three-dimensional network structure through the secretion of extracellular polymeric substances (EPS), exhibiting strong environmental resistance but easily causing practical problems such as infection, corrosion, and increased fluid resistance. Therefore, this indicator reflects the comprehensive inhibition efficacy of materials against the long-term formation and development of pollutant communities. The three indicators mentioned above correspond sequentially to the key stages of the biocontamination chain, from initiation to development to maturity, covering the entire time-series process from protein adsorption and bacterial adhesion to biofilm formation. This provides a scientific and systematic evaluation basis for the research and development and application of materials in the medical device field. This embodiment experimentally verifies the rationality and value of the prediction logic proposed in this invention: For high-performance samples (such as sample A), once the "anti-biofilm test" yields a positive result (≥70% inhibition rate), strong confidence in its comprehensive anti-fouling performance can be established. Based on this, subsequent detailed antibacterial and anti-protein adsorption quantitative tests can be simplified, significantly improving screening efficiency.

[0081] For samples with medium to low performance (such as samples B and C) and samples with special surfaces (such as samples D and E), the anti-biofilm test results did not reach the threshold (<70% inhibition rate). In such cases, optimistic predictions should not be made, and subsequent complete quantitative tests must be conducted to comprehensively assess their performance shortcomings. The experimental data fully confirm this judgment, and samples D and E reveal why biofilm indicators are more reliable initial screening indicators, as they reflect the ability to resist the final and most complex stages of contamination.

[0082] Example 3 Based on the conclusions of Example 2, this example demonstrates a simplified evaluation process using the predictive logic of the present invention. This example uses *Escherichia coli* (EPEC) as the experimental strain.

[0083] This embodiment aims to quickly screen out candidate materials with potentially satisfactory comprehensive antifouling performance (biofilm inhibition ≥70%, antibacterial ≥80%, antiprotein ≥80%) from six different types of silicone rubber with the same mesh structure surface (referred to as samples 1-6).

[0084] Among them, the six different types of silicone rubber are KE-2092-40 (sample 1), Silbione 4717 A / B (sample 2), Silastic 7-6860 Biomedical Grade LSR (sample 3), LIM 6050 (sample 4), SH745U (sample 5), and Elastosil LR 3040 / 40 (sample 6).

[0085] Traditional methods require quantitative tests on all six samples, including biofilm, colony count, and BCA protein adsorption, totaling 18 sets of experiments.

[0086] Using the method of the present invention: Step 1 (Initial Screening): All 6 samples were tested using the "Biomembrane Crystal Violet Staining Method" (6 sets of experiments). The results are shown in Table 18.

[0087] Step 2 (Analysis and Prediction): The test results show that the biofilm inhibition rate of samples 1, 3 and 6 is ≥70%, while the other samples are <70%.

[0088] Step 3 (Simplified Testing): Based on the prediction logic, only samples 1, 3, and 6 were subsequently verified using the "plate colony counting method" and "BCA protein concentration determination method" (3+3 groups of experiments). The remaining samples failed the high-level screening and were deemed to have substandard overall performance, or were to be retested later as needed. In this embodiment, to verify the rationality of the prediction results of the present invention, all 6 groups of samples underwent subsequent testing.

[0089] Efficiency improvement: The total number of tests was reduced from 18 to (6+3+3) = 12, reducing the testing workload by nearly 33%, while allowing resources to be concentrated on the most promising candidate materials.

[0090] Table 18 Data on the resistance to biofouling of samples from Example 3

[0091] This simplified evaluation process fully demonstrates the significant advantages of this invention in the initial screening stage of materials' anti-biofouling performance. By using biofilm inhibition rate as the key initial screening threshold (≥70%), candidate materials with potential comprehensive anti-fouling capabilities can be accurately identified, effectively avoiding the cumbersome and costly subsequent quantitative tests on antibacterial and anti-protein adsorption for a large number of obviously substandard samples. Experimental results show that for six different types of mesh-structured silicone rubber samples, the workload of testing was reduced from the traditional 18 groups to 12 groups after adopting the method of this invention, a reduction of nearly 33%. At the same time, samples 1, 3, and 6 were successfully screened as qualified materials with biofilm inhibition rate ≥70% and measured antibacterial and anti-protein adsorption rates ≥80%, and the predicted results were completely consistent with the actual test results. This not only greatly improves the efficiency of material screening and saves manpower, material resources, and time costs, but more importantly, it ensures the reliability and accuracy of the screening results, providing an efficient and scientific evaluation tool for the rapid research and development and application of silicone rubber materials in the field of anti-biofouling.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for testing the resistance to biofouling and durability of silicone rubber surfaces, characterized in that, Includes the following steps: S1. Comprehensive test of initial anti-biofouling performance: Using the smooth surface of the silicone rubber to be tested as the control sample and the modified surface of the silicone rubber to be tested as the test sample, the control sample and the test sample of the silicone rubber to be tested were subjected to quantitative detection of biofilm inhibition rate, antibacterial adhesion, anti-protein adsorption performance and surface wettability test in sequence to obtain the initial anti-fouling performance data of the sample. S2. Simulated Environment Durability Treatment: The samples undergo a durability treatment simulating actual use environments. This simulated environment durability treatment includes: a. Liquid immersion aging treatment: The sample is immersed in simulated body fluid or phosphate buffer solution and soaked for several days at a constant temperature of 37±1℃. b. Mechanical friction and wear treatment: The sample surface is subjected to relative sliding friction with a standard friction medium under a set normal pressure; S3. Performance evaluation after durability treatment: Repeat some or all of the tests in step S1 on the test samples treated in step S2 to obtain the anti-pollution performance data after durability treatment. S4. Comprehensive performance evaluation: Compare the performance data obtained in steps S1 and S3 to evaluate the anti-biofouling and durability of the silicone rubber sample. The anti-fouling performance is quantified by calculating the biofilm inhibition rate, antibacterial adhesion rate, and anti-protein adsorption rate. Among them, the biofilm inhibition rate ≥70%, antibacterial adhesion rate ≥80%, and anti-protein adsorption rate ≥80% are evaluated as having good anti-fouling performance. In step S1 or step S3, the comprehensive detection follows the following logic: Quantitative detection of biofilm inhibition rate: if the measured biofilm inhibition rate reaches or exceeds 70%, the predicted antibacterial adhesion rate and antiprotein adsorption rate can reach 80%, respectively. And / or, antibacterial adhesion test: if the measured antibacterial adhesion rate reaches or exceeds 80%, it is predicted that its antiprotein adsorption rate can reach or exceed 80%. Based on the above predictions, subsequent antibacterial adhesion tests and / or antiprotein adsorption performance tests can be selectively simplified.

2. The method for testing the resistance to biofouling and durability of silicone rubber surfaces according to claim 1, characterized in that, In step S1, the quantitative detection of biofilm inhibition rate adopts the biofilm crystal violet staining method. The sample is co-cultured with bacterial suspension to form a biofilm. After crystal violet staining and elution, the absorbance of the dye is measured to quantify the total amount of biofilm and calculate the biofilm inhibition rate.

3. The method for testing the resistance to biofouling and durability of silicone rubber surfaces according to claim 1, characterized in that, In step S1, the antibacterial adhesion test includes qualitative and quantitative analysis of bacterial adhesion, wherein: The qualitative analysis of bacterial adhesion uses the inhibition zone method, in which the sample is attached to the surface of an agar plate inoculated with bacteria, and after incubation, the size of the sterile area around the sample is observed and measured to qualitatively evaluate the exudation of antibacterial substances on the surface or the contact inhibition ability. The quantitative analysis of bacterial adhesion was performed using the plate colony counting method. After incubating the sample with a bacterial suspension, the bacteria adhering to the surface were washed off and colonies were counted to calculate the antibacterial adhesion rate.

4. The method for testing the resistance to biofouling and durability of silicone rubber surfaces according to claim 1, characterized in that, In step S1, the anti-protein adsorption performance test includes qualitative analysis and quantitative analysis, wherein: The qualitative analysis employed the FITC fluorescent labeling method, incubating the sample with a fluorescein-labeled protein solution, and then observing the distribution of adsorbed proteins on the surface using fluorescence imaging after washing. The quantitative analysis was performed using the BCA protein concentration assay. After incubating the sample with a protein solution and eluting, the total amount of protein adsorbed on the sample surface was determined using a BCA kit, and the anti-protein adsorption rate was calculated.

5. The method for testing the resistance to biofouling and durability of silicone rubber surfaces according to claim 1, characterized in that, In step S1, the surface wettability test uses a contact angle meter to measure the static water contact angle of the sample.

6. The method for testing the resistance to biofouling and durability of silicone rubber surface according to claim 1, characterized in that, In step S2, the standard friction medium is medical gauze, and the positive pressure is achieved by weights placed on the sample, with the weights having a mass of 40-60g.

7. The method for testing the resistance to biofouling and durability of silicone rubber surfaces according to claim 1, characterized in that, The bacteria include Gram-negative and Gram-positive bacteria; the protein is bovine serum albumin.

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