A storage-stable dopamine-containing multifunctional tetra-copolymer, a preparation method thereof and application thereof to the surface of a silicone catheter

Abstract

The application discloses a storage-stable dopamine-containing multifunctional tetra-copolymer and a preparation method and application thereof to a silica gel catheter surface. The copolymer is prepared from dopamine methacrylamide, 2-propenoic acid-2-methoxy ethyl ester, methacryloyloxyethyl trimethyl ammonium chloride and 2-(methacryloyloxy) ethyl N-(3,4-dihydroxyl cyclohexyl) carbamic acid ester through RAFT active radical polymerization under the synergistic effect of a specific pyrazole-based dithio carbamic acid ester chain transfer agent and a Lewis acid. The application effectively inhibits side reactions and realizes accurate control of molecular weight by using a specific structure system, and a storage-stable copolymer solution without gel particles is prepared. Not only the defects of poor bonding force and easy peeling of a traditional coating are effectively overcome, but also the synergistic effect among monomers is utilized to endow the catheter with good lubricity and significantly improve the comprehensive performance of the indwelling catheter in resisting infection and anti-crusting.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials and surface modification technology for medical devices, specifically relating to a storage-stable dopamine-containing multifunctional quaternary copolymer, its preparation method, and its application on the surface of silicone catheters. Background Technology

[0002] Silicone rubber, due to its excellent biocompatibility, chemical stability, and flexibility, is often used to manufacture various interventional medical catheters (such as urinary catheters and drainage tubes). However, the strong hydrophobic and inert surface of silicone makes it prone to non-specific protein adsorption after implantation in the human body, which in turn induces bacterial adhesion, colonization, and the formation of difficult-to-remove biofilms, ultimately leading to serious complications such as catheter-associated urinary tract infections (CAUTI) and crusting of the catheter surface. Compared to other drainage tubes, the urine environment in which urinary catheters are located has high ionic strength, fluctuating pH, and a tendency for supersaturated crystallization, which places extremely stringent and contradictory requirements on the overall performance of the surface coating.

[0003] Existing technologies typically modify silicone surfaces using physical coating or chemical grafting methods. Physical coating with antibiotic or metal ion-containing coatings, while effective in the short term, suffers from drawbacks such as short drug release periods and the potential to induce drug resistance. Furthermore, due to the low surface energy and strong chemical inertness of silicone, ordinary antibacterial polymers struggle to form a stable bond through simple physical adsorption, leading to easy detachment during clinical use. While chemical activation methods such as plasma treatment and UV grafting can enhance adhesion, they rely on expensive equipment and can easily damage the substrate's properties. Inspired by mussel adhesive proteins, catechol-containing copolymers (such as dopamine methacrylamide copolymer, DMA-copolymer) are considered a highly promising technology due to their self-anchoring ability to silicone substrates and the simplicity of a one-step soaking molding process. However, researchers face significant technical challenges in the actual preparation of these dopamine-containing copolymers. The catechol groups in the DMA monomer readily induce oxidative coupling crosslinking and free radical quenching during free radical polymerization. As the degree of polymerization increases, the viscosity of the system rises. Irreversible gelation caused by the coupling of physical entanglement of ultra-high molecular weight chains and chemical cross-linking of catechol will result in the product being insoluble in conventional solvents and losing its coating processability. Although the introduction of living / controlled radical polymerization (such as RAFT) can partially regulate the molecular weight distribution and thus improve solution gelation, the end groups of conventional RAFT reagents (such as dithioesters) are easily degraded and inactivated by nucleophilic attack from catechol or amine groups, and their residual strong color contradicts the regulatory requirements for the colorless and transparent appearance of medical implants.

[0004] Even if the aforementioned challenges of synthetic gelation are overcome through specific methods, resulting in soluble and coatable copolymer solutions, existing technologies still face three fundamental and irreconcilable technical contradictions when dealing with the complex physicochemical environment of long-term indwelling catheters. These contradictions are the root cause of the high failure rate of coatings in catheter scenarios: First, to suppress physical entanglement and gelation during polymerization and storage, existing technologies tend to design coating networks with low crosslinking density and low molecular weight. While this structure ensures the clarity and flowability of the coating solution (meeting processability requirements), it exhibits high swelling rates and low mechanical strength under long-term immersion in urine. The highly swollen coating network not only suffers from drastic deterioration in mechanical properties, but more importantly, it acts as an ionic sponge, absorbing large amounts of Ca from the urine. 2+ Mg 2+ These ions act as heterogeneous nucleation sites within the coating, inducing the crystal growth of calcium phosphate and struvite within the coating body. This generates enormous crystallization pressure, causing the coating to disintegrate and peel off from the silicone surface (losing its service durability).

[0005] Secondly, to reduce mechanical damage to the urethral mucosa caused by silicone drying during catheterization, existing coatings must rely on highly hydrophilic components (such as PEG and PVP) for hydration and lubrication. However, clinical data on urinary catheters show that overly hydrophilic surfaces are a breeding ground for urine crystal deposition. Under conditions where urease-positive bacterial infection leads to an elevated urine pH (>8.0), the hydration layer at highly hydrophilic interfaces preferentially accumulates OH groups. - and PO4 3- This significantly lowers the crystallization nucleation barrier, accelerating the formation of a hard struvite shell. Consequently, the hydrophilic structure designed to alleviate insertion pain ironically becomes a structural contributing factor to severe excision pain and urethral tears in the later stages of indwelling catheterization.

[0006] Third, existing anti-adhesion strategies for coatings are mainly based on the physical repulsion mechanism of the hydration layer. However, *Escherichia coli*, which accounts for more than 75% of the pathogens in catheter CAUTIs, has a specific "lock-and-key" binding mechanism between its FimH protein at the tip of type I pili and the host cell or implant surface. Non-specific hydration layer repulsion cannot effectively shield the FimH protein from its high affinity for mannose residues. If mannose ligands are introduced for specific biological blocking, its polyhydroxy structure will be inactivated by oxidative side reactions in DMA-containing polymerization systems, and glycosylation modification will further aggravate the high viscosity and physical entanglement of the copolymer solution, leading to an exponential increase in the difficulty of gelation control in the upstream synthesis stage.

[0007] Therefore, a coating technology needs to be developed to resolve the deep contradictions between the processability of the solution (inhibiting gelation), the urine tolerance of the coating (anti-swelling and anti-crystallization), and the biofunctionality of the interface (lubrication and specific anti-infection). Summary of the Invention

[0008] To address the technical bottlenecks in existing technologies where dopamine (catechol) monomers are prone to oxidative crosslinking, uncontrolled molecular weight distribution, and product solution gelation during free radical polymerization, resulting in difficult-to-prepare coating solutions with dark colors and poor storage stability, this invention employs a synergistic strategy of Lewis acid kinetic regulation and specific RAFT reagent activity control to provide a multifunctional copolymer coating with surface self-anchoring function, its preparation method, and its applications.

[0009] The technical solution of this invention is as follows: A storage-stable, dopamine-containing, multifunctional quaternary copolymer. The copolymer is a random copolymer prepared by a reversible addition-fragmentation chain transfer polymerization reaction of four monomers, including monomers A, B, C, and D, in the presence of a chain transfer agent; monomer A is dopamine methacrylamide (DMA), monomer B is 2-methoxyethyl 2-acrylate (MEA), monomer C is methacryloyloxyethyltrimethylammonium chloride (DMC), and monomer D is 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate (CHDU).

[0010] The copolymer has a molecular weight distribution index of 1.1 to 1.5, and the ends of the copolymer molecular chains contain pyrazolyl dithiocarbamate groups derived from the chain transfer agent.

[0011] Preferably, the molar ratio of dopamine methacrylamide (DMA), 2-methoxyethyl 2-acrylate (MEA), methacryloyloxyethyl trimethylammonium chloride (DMC) and 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate (CHDU) is 1 : (5-10) : (0.2-3) : (0.3-5).

[0012] A first aspect of the present invention provides a method for preparing the above-mentioned storage-stable dopamine-containing multifunctional tetromer, characterized by comprising the following steps: Step S1: Obtain dopamine methacrylamide (DMA) monomer; Specifically, this includes reacting dopamine hydrochloride with methacrylic anhydride under alkaline aqueous solution conditions protected by borax decahydrate, followed by extraction and drying to obtain DMA solid; Step S2: RAFT polymerization reaction; The dopamine methacrylamide (DMA), 2-methoxyethyl 2-acrylate (MEA), methacryloyloxyethyltrimethylammonium chloride (DMC), 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate (CHDU), chain transfer agent, initiator, and Lewis acid were dissolved in an organic solvent. After deoxygenation of the reaction system, a constant-temperature polymerization reaction was carried out under sealed conditions. After the reaction was completed, the reaction solution was purified by dialysis and freeze-dried to obtain the quaternary copolymer.

[0013] The core technical improvement of this invention: The chain transfer agent is a pyrazolyl dithiocarbamate compound, preferably: cyanomethyl(3,5-dimethyl-1H-pyrazol)-dithioester; The initiator is azobisisobutyronitrile; The Lewis acid is preferably anhydrous zinc chloride (ZnCl2), which can significantly accelerate the polymerization rate and inhibit gelation; Preferably, the total molar amount of monomer A, monomer B, monomer C and monomer D and the molar ratio of chain transfer agent to initiator are (150-300: 1: (0.1-0.5).

[0014] Preferably, the molar ratio of the Lewis acid to the chain transfer agent is (0.5-2.0):1, and most preferably (1.0-1.2):1.

[0015] In one embodiment, the solvent for the polymerization reaction is selected from one or more of N,N-dimethylformamide (DMF), methanol, or dimethyl sulfoxide (DMSO).

[0016] Preferably, the conditions for the polymerization reaction in step S2 include: The reaction temperature is 60℃~75℃; The reaction time is 6 h to 16 h; The deoxygenation operation employs a freezing-evacuation-thawing cycle or high-purity nitrogen bubbling for more than 30 minutes.

[0017] Preferably, the dialysis bag used in the dialysis has a molecular weight cutoff of 3500 Da, and the dialysis medium is deionized water or acidic aqueous solution to remove unreacted monomers, Lewis acids, and small molecule impurities.

[0018] A second aspect of the present invention provides a medical silicone catheter with a multifunctional coating on its surface.

[0019] The surface of the silicone conduit is coated with a self-anchoring coating formed of the dopamine-containing multifunctional quaternary copolymer described in the first aspect; Preferably, the method for preparing the antibacterial coating includes: The quaternary copolymer is dissolved in a coating solvent to prepare a coating solution, wherein the coating solvent is preferably a mixed solution of isopropanol and Tris-HCl buffer (pH 8.5). The clean silicone tubing is immersed in the coating liquid and deposited by dip-coating for 12 to 24 hours; After removing it, wash and dry it to obtain the product.

[0020] The beneficial effects of this invention are as follows: 1. This invention solves the gelation problem in the synthesis of dopamine-containing polymers, achieving storage stability of the polymer. This invention innovatively introduces a Lewis acid (zinc chloride) and a pyrazole-based RAFT reagent to construct a synergistic system. The core mechanism lies in the fact that, in a non-alkaline polymerization environment, anhydrous zinc chloride can form a monodentate reversible coordination with the catechol group on the dopamine side chain. This electron-withdrawing complexation effect of the Lewis acid acts as an in-situ protecting agent, greatly increasing the dissociation energy of the phenolic hydroxyl group, thereby shielding the free radical quenching activity of catechol and interrupting its oxidation to quinone and subsequent irreversible covalent crosslinking. Combined with the specially selected cyanomethyl (3,5-dimethyl-1H-pyrazole)-dithioester RAFT reagent of this invention, due to its special pyrazole leaving group, it exhibits extremely high chemical stability, perfectly resisting the nucleophilic attack of the amine and phenolic hydroxyl groups in the system without degradation. Meanwhile, in four monomer copolymerization systems with vastly different polarities and reactivity, the RAFT reagent forces all polymer chains to grow synchronously at a uniform rate, effectively suppressing monomer compositional drift and preventing dopamine segments from forming rigid block structures locally.

[0021] 2. A synergistic dual defense system of passive anti-fouling and anti-crusting and active sterilization was constructed, which significantly enhanced the antibacterial performance of the coating.

[0022] 3. It achieves stable anchoring of the coating on the inert silicone surface, solving the problem of easy coating peeling.

[0023] 4. The preparation process is mild and simple, with good universality and biocompatibility.

[0024] 5. Based on the specifically introduced D monomer (CHDU), the coating surface of this invention is enriched with cyclic vicinal diol structures. On the one hand, this structure can play a core role in dynamic ion trapping, accurately identifying and chelating locally high concentrations of calcium and magnesium ions, strongly interfering with their collision nucleation pathways, and achieving excellent anti-crusting function; on the other hand, this multi-hydroxyl structure can mimic host cell receptors, competitively binding to the FimH protein active site at the tip of type I fimbriae of pathogenic bacteria (such as Escherichia coli), thereby specifically blocking the initial anchoring of bacteria to the duct surface and cutting off the infection route at the source. Attached Figure Description

[0025] Figure 1 The image shows the hydrogen NMR spectrum of the prepared DMA monomer.

[0026] Figure 2 The image shows the 1H NMR spectrum of the prepared RAFT reagent.

[0027] Figure 3 The 1H NMR spectrum of the terpolymer of Comparative Example 3.

[0028] Figure 4 The zeta potential diagrams are for the quaternary copolymer (B) prepared in Example 1 and the terpolymer (A) of Comparative Example 1.

[0029] Figure 5 The diagram shows the antibacterial effects of S1, D9, D11, and D12.

[0030] Figure 6 SEM images of surfaces S1, D9, D11, and D12 after 3 weeks of in vitro crust formation experiments.

[0031] Figure 7 Cell viability test results for S1 and D12 (a) and fluorescence image of L-929 cell live / dead cell staining test (b). Detailed Implementation

[0032] The technical solution of the present invention will be further explained and described below with reference to specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present invention and should not be construed as limiting the present invention. The scope of protection of the present invention should be determined by the contents of the claims. Modifications and substitutions made by those skilled in the art to the technical solution of the present invention without creative effort all fall within the scope of protection of the present invention. Example 1 1. Synthesis of dopamine methacrylamide (DMA) Weigh 10 g of sodium tetraborate decahydrate and 4 g of sodium bicarbonate, dissolve them in 100 mL of deionized water, and add to a 250 mL three-necked flask. Stir to dissolve. Bubble with nitrogen for 20 min to remove oxygen from the solution. Add 4 g of dopamine hydrochloride and continue stirring until dissolved. Adjust the pH of the solution to above 8 using 1 M NaOH. Dissolve 4 mL of methacrylic anhydride in 40 mL of tetrahydrofuran and slowly add it dropwise to the three-necked flask. React at room temperature with nitrogen purging for 14 h. The resulting product is a white suspension. Post-treatment is as follows: filter to remove excess salt from the reaction product. Then adjust the pH of the solution to 2 with 6 M hydrochloric acid solution. Extract three times with 100 mL of ethyl acetate to obtain a brown organic layer. Dry with anhydrous magnesium sulfate and filter to obtain a clear organic layer. Rotary evaporate to about 50 mL. Slowly add 250 mL of n-hexane with stirring to obtain a gray suspension. Refrigerate at 4 °C to crystallize. Filter the next day and air dry naturally. The product is a gray powder. Collect the product, seal, and refrigerate for later use. Structural identification: 1H NMR spectrum as shown below. Figure 1 As shown.

[0033] 2. Synthesis of cyanomethyl (3,5-dimethyl-1H-pyrazole)-dithioester Step (1): Preparation of intermediate potassium 3,5-dimethyl-1H-pyrazole-1-carbodithioate Potassium hydroxide (6.16 g, 109.75 mmol) was dissolved in tetrahydrofuran (100 mL), and the solution was cooled to 4 °C. 3,5-Dimethylpyrazole (10.00 g, 104.03 mmol) was added to the solution, and the mixture was stirred at 4 °C for 5 minutes. Subsequently, carbon disulfide (8.17 mL, 135.23 mmol) was slowly added dropwise, and the mixture was stirred at 4 °C for 3 minutes, then allowed to cool to room temperature and stirred for another 50 minutes. After the reaction was complete, the mixture was filtered, and the filter cake was washed with diethyl ether and dried to give a light orange solid. This intermediate did not require further purification and was used directly in the next reaction.

[0034] Step (2): Preparation of the target product cyanomethyl (3,5-dimethyl-1H-pyrazole)-dithioester Potassium 3,5-dimethyl-1H-pyrazole-1-carbodithioate (18.60 g, 88.42 mmol) obtained in step (1) was dissolved in deionized water (350 mL) and cooled in an ice-water bath. Chloroacetonitrile (5.92 mL, 92.85 mmol) was added, and the mixture was stirred for 2 minutes in an ice-water bath, then moved to room temperature and reacted for 100 minutes. After the reaction was completed, the mixture was cooled in an ice-water bath for 15 minutes to promote precipitation. The mixture was filtered, and the precipitated yellow solid was collected, washed with ice water, and dried to obtain the first batch of product. The filtrate (mother liquor) was stirred for 2 days, filtered, washed with ice water, and dried to obtain the second batch of product. The two batches of solids were combined to obtain the target product, which is a pale yellow solid. The structure was identified by the 1H NMR spectrum as follows: Figure 2 As shown.

[0035] 3. Synthesis of 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate In a 250 mL round-bottom flask equipped with a magnetic stirrer, 1,2,4-cyclohexanetriol (1.32 g, 10 mmol) was added and dissolved in 50 mL of anhydrous acetone. Then, p-toluenesulfonic acid (0.086 g, 0.5 mmol) was added as a catalyst. The reaction was stirred at room temperature for 12 hours. After the reaction was complete, a small amount of sodium bicarbonate was added for neutralization, the mixture was filtered, and the acetone solvent was removed by rotary evaporation under reduced pressure. The resulting product was then purified by silica gel column chromatography to obtain the hydroxyl-protected intermediate (1,2-O-isopropylidene-1,2,4-cyclohexanetriol). The obtained intermediate (0.86 g, 5 mmol) was dissolved in 20 mL of dichloromethane (DCM). Ethyl methacrylate-2-isocyanate (IEM, 0.77 g, 5 mmol) was slowly added dropwise under nitrogen protection and an ice bath. Subsequently, dibutyltin dilaurate (DBTDL, 0.032 g, 0.05 mmol) was added dropwise as a catalyst, and the reaction was carried out at room temperature in the dark for 24 hours. The DCM solvent was removed by vacuum distillation to obtain a monomer precursor containing a protecting group. The obtained precursor was dissolved in 20 mL of tetrahydrofuran (THF), and 5 mL of 1 M hydrochloric acid aqueous solution was slowly added. The mixture was stirred vigorously at room temperature for 6 hours to remove the ketal protecting group. After the reaction was complete, the system was adjusted to neutral with saturated sodium bicarbonate solution, extracted with ethyl acetate (20 mL × 3 times), the organic phases were combined, dried over anhydrous magnesium sulfate, the solvent was removed by rotary evaporation under reduced pressure, and finally dried at room temperature in a vacuum drying oven to constant weight to obtain 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate.

[0036] 4. Synthesis of tetromers Weigh out dopamine methacrylamide (DMA, 0.45 g, 2 mmol), 2-methoxyethyl 2-acrylate (MEA, 1.9 g, 14 mmol), methacryloyloxyethyltrimethylammonium chloride (DMC, 0.29 g, 1.4 mmol), and 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate (CHDU, 0.29 g, 1 mmol) and dissolve them in 10 mL of N,N-dimethylformamide (DMF). Transfer the solution to a 100 mL dry round-bottom flask. Subsequently, the prepared RAFT reagent cyanomethyl (3,5-dimethyl-1H-pyrazole)-dithioester (17 mg, 0.08 mmol), anhydrous zinc chloride (ZnCl2, 12 mg, 0.09 mmol), and the thermal initiator azobisisobutyronitrile (AIBN, 3 mg, 0.02 mmol) were added to the system. The reaction flask was sealed, and dissolved oxygen was completely removed by a freeze-evacuation-thawing cycle three times. Under nitrogen protection, the reaction flask was placed in a 70°C oil bath for 12 hours. During the reaction, the solution remained clear and transparent with good fluidity, and no gelation occurred. After the reaction, the reaction flask was left open and cooled to room temperature to terminate the polymerization. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and purified by dialyzing in deionized water for 3 days (with the dialyzing water changed every 12 hours) to remove unreacted monomers, solvents, and small molecule impurities. Finally, the solution in the dialysis bag was placed in a freeze dryer for freeze drying to obtain the product. Comparative Example 1 This comparative example is a dopamine-containing terpolymer and its preparation method. The steps are the same as in Example 1, the main difference being that DMC was not used. The steps are as follows: 1-2 are the same as in Example 1.

[0037] 3. Synthesis of terpolymers 0.45 g of dopamine methacrylamide (DMA), 1.9 g of 2-methoxyethyl 2-acrylate (MEA), and 0.29 g of 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate (CHDU) were weighed and dissolved in 10 mL of N,N-dimethylformamide (DMF). Then, the prepared RAFT reagent (17 mg), anhydrous zinc chloride (ZnCl2, 12 mg), and the thermal initiator azobisisobutyronitrile (AIBN, 3 mg) were added to the system. The reaction flask was sealed, and dissolved oxygen was completely removed by a freeze-evacuation-thawing cycle three times. Under nitrogen protection, the reaction flask was placed in a 70°C oil bath for 6 h. During the reaction, the solution remained clear and transparent with good fluidity, and no gelation occurred. After the reaction, the reaction flask was left open and cooled to room temperature to terminate the polymerization. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and purified by dialyzing in deionized water for 3 days (with the dialyzing water changed every 12 hours) to remove unreacted monomers, solvents, and small molecule impurities. Finally, the solution in the dialysis bag was freeze-dried to obtain the product.

[0038] Comparative Example 2 This comparative example demonstrates a method for preparing a terpolymer, with the same steps as in Example 1, the main difference being that DMA was not used. The steps are as follows: 1. Synthesis of cyanomethyl (3,5-dimethyl-1H-pyrazole)-dithioester The synthesis steps are the same as in Example 1.

[0039] 2. Synthesis of 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate The synthesis steps are the same as in Example 1.

[0040] 3. Synthesis of terpolymers Weigh 1.9 g of 2-methoxyethyl 2-acrylate (MEA), 0.29 g of methacryloyloxyethyltrimethylammonium chloride (DMC), and 0.29 g of 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate (CHDU) and dissolve them in 10 mL of N,N-dimethylformamide (DMF). Transfer the solution to a 100 mL dry round-bottom flask. Then, add the prepared RAFT reagent (17 mg), anhydrous zinc chloride (ZnCl2, 12 mg), and the thermal initiator azobisisobutyronitrile (AIBN, 3 mg) to the system. Seal the reaction flask and perform a freeze-evacuation-thawing cycle three times to completely remove dissolved oxygen. Under nitrogen protection, place the reaction flask in a 70 °C oil bath and react for 12 h. During the reaction, the solution remained clear and transparent with good fluidity, and no gelation occurred. After the reaction, leave the reaction flask open and cool to room temperature to terminate the polymerization. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and purified by dialyzing in deionized water for 3 days (with the dialyzing water changed every 12 hours) to remove unreacted monomers, solvents, and small molecule impurities. Finally, the solution in the dialysis bag was freeze-dried to obtain the product.

[0041] Comparative Example 3 This comparative example is a dopamine-containing terpolymer and its preparation method. The steps are the same as in Example 1, the main difference being that CHDU was not used.

[0042] The steps are as follows: 1-2 are the same as in Example 1.

[0043] 3. Synthesis of terpolymers 0.45 g of dopamine methacrylamide (DMA), 1.9 g of 2-methoxyethyl 2-acrylate (MEA), and 0.29 g of methacryloyloxyethyltrimethylammonium chloride (DMC) were weighed and dissolved in 10 mL of N,N-dimethylformamide (DMF). Then, the prepared RAFT reagent (17 mg), anhydrous zinc chloride (ZnCl2, 12 mg), and the thermal initiator azobisisobutyronitrile (AIBN, 3 mg) were added to the system. The reaction flask was sealed, and dissolved oxygen was completely removed by a freeze-evacuation-thawing cycle three times. Under nitrogen protection, the reaction flask was placed in a 70°C oil bath for 6 hours. During the reaction, the solution remained clear and transparent with good fluidity, and no gelation occurred. After the reaction, the reaction flask was left open and cooled to room temperature to terminate the polymerization. The reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and purified by dialyzing in deionized water for 3 days (with the dialyzing water changed every 12 hours) to remove unreacted monomers, solvents, and small molecule impurities. Finally, the solution in the dialysis bag was freeze-dried to obtain the product. The structure was identified by a 1H NMR spectrum as follows: Figure 3 As shown.

[0044] Comparative Example 4 This comparative example is a dopamine-containing quaternary copolymer and its preparation method. The steps are the same as in Example 1, except that cyanomethyl (3,5-dimethyl-1H-pyrazole)-dithioester and anhydrous zinc chloride were not used.

[0045] 1. Synthesis of dopamine methacrylamide (DMA) The synthesis steps are the same as in Example 1.

[0046] 2. Synthesis of 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate The synthesis steps are the same as in Example 1.

[0047] 3. Synthesis of tetromers Weigh out 0.45 g of dopamine methacrylamide (DMA), 1.9 g of 2-methoxyethyl 2-acrylate (MEA), 0.29 g of methacryloyloxyethyltrimethylammonium chloride (DMC), and 0.29 g of 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate (CHDU) and dissolve them in 10 mL of N,N-dimethylformamide (DMF). Transfer the solution to a 100 mL dry round-bottom flask. Then, add the thermal initiator azobisisobutyronitrile (AIBN, 3 mg) to the system. Seal the reaction flask and perform a freeze-evacuation-thawing cycle three times to completely remove dissolved oxygen. Under nitrogen protection, place the reaction flask in a 70°C oil bath for constant temperature reaction. During the polymerization process, an extremely long induction period was observed in the reaction solution, indicating that the catechol groups of dopamine exhibit strong free radical polymerization inhibition activity in the absence of in-situ coordination protection by anhydrous zinc chloride. As the reaction proceeded, the solution color rapidly deepened to a dark reddish-brown, the viscosity of the system increased sharply, and finally, a large amount of insoluble hard lumps precipitated at the bottom of the reaction flask. Attempts to dissolve the resulting mixture for GPC testing revealed that most of the product could not be reconstituted in conventional solvents. Therefore, the comparative product could not be used for subsequent dialysis purification and coating preparation steps.

[0048] Comparative Example 5 This comparative example is a dopamine-containing quaternary copolymer and its preparation method. The steps are the same as in Example 1, except that cyanomethyl (3,5-dimethyl-1H-pyrazole)-dithioester is not used.

[0049] 1. Synthesis of dopamine methacrylamide (DMA) The synthesis steps are the same as in Example 1.

[0050] 2. Synthesis of 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate The synthesis steps are the same as in Example 1. 3. Synthesis of tetromers 0.45 g of dopamine methacrylamide (DMA), 1.9 g of 2-methoxyethyl 2-acrylate (MEA), 0.29 g of methacryloyloxyethyltrimethylammonium chloride (DMC), and 0.29 g of 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate (CHDU) were weighed and dissolved in 10 mL of N,N-dimethylformamide (DMF), and transferred to a 100 mL dry round-bottom flask. Anhydrous zinc chloride (ZnCl2, 12 mg) and the thermal initiator azobisisobutyronitrile (AIBN, 3 mg) were then added to the system. The reaction flask was sealed, and the dissolved oxygen was completely removed by a freeze-evacuation-thawing cycle three times. Under nitrogen protection, the reaction flask was placed in a 70°C oil bath and reacted for 12 h. After the reaction, the system macroscopically lost its fluidity. This is not due to chemical cross-linking, but rather because the system lacks a RAFT reagent for activity regulation, causing the reaction to degenerate into uncontrolled free radical polymerization. Uncontrolled chain growth leads to high molecular weight and severe compositional drift, resulting in the local enrichment of dopamine monomers on the polymer chains, forming microblocks. The severe physical entanglement between polymer chains, coupled with extremely strong intermolecular hydrogen bonds and π-π stacking interactions between the enriched dopamine segments, causes the system to exhibit extremely viscous gelation on a macroscopic scale. Therefore, the comparative product cannot be subjected to subsequent dialysis purification and coating preparation steps.

[0051] Comparative Example 6 This comparative example is a dopamine-containing quaternary copolymer and its preparation method. The steps are the same as in Example 1, the main difference being that anhydrous zinc chloride was not used.

[0052] 1-3 are the same as in Example 1.

[0053] 4. Synthesis of tetromers Weigh 0.45 g of dopamine methacrylamide (DMA), 1.9 g of 2-methoxyethyl 2-acrylate (MEA), 0.29 g of methacryloyloxyethyltrimethylammonium chloride (DMC), and 0.29 g of 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate (CHDU) and dissolve them in 10 mL of N,N-dimethylformamide (DMF). Transfer the solution to a 100 mL dry round-bottom flask. Then, add the prepared RAFT reagent cyanomethyl (3,5-dimethyl-1H-pyrazole)-dithioester (17 mg) and the thermal initiator azobisisobutyronitrile (AIBN, 3 mg) to the system. Seal the reaction flask and perform a freeze-evacuation-thawing cycle three times to completely remove dissolved oxygen. Under nitrogen protection, place the reaction flask in a 70 °C oil bath and react for 12 h. After the reaction was completed, a large amount of insoluble white turbidity appeared at the bottom of the reaction flask. Although the upper liquid had a certain viscosity, its flowability was poor. Experiments confirmed that in the absence of Lewis acid (zinc chloride) activation, the polymerization kinetics could not compete with dopamine for polymerization inhibition and self-crosslinking side reactions, leading to uncontrolled product structure and gelation. Therefore, the comparative product could not be subjected to subsequent dialysis purification and silicone tube coating preparation steps.

[0054] Comparative Example 7 This comparative example is a dopamine-containing quaternary copolymer and its preparation method. The steps are the same as in Example 1, the main difference being that a commercially available RAFT reagent of dithiobenzoate (CPDB) is used, but anhydrous zinc chloride is added to the polymerization system.

[0055] 1. Synthesis of dopamine methacrylamide (DMA) The synthesis steps are the same as in Example 1.

[0056] 2. Synthesis of 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate The synthesis steps are the same as in Example 1.

[0057] 3. Synthesis of tetromers Weigh 0.45 g of dopamine methacrylamide (DMA), 1.9 g of 2-methoxyethyl 2-acrylate (MEA), 0.29 g of methacryloyloxyethyltrimethylammonium chloride (DMC), and 0.29 g of 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate (CHDU) and dissolve them in 10 mL of N,N-dimethylformamide (DMF). Transfer the solution to a 100 mL dry round-bottom flask. Then, add commercially available RAFT reagent 2-cyano-2-propyl dithiobenzoate (CPDB, 18 mg) and anhydrous zinc chloride (ZnCl2, 12 mg) to the system. Finally, add the thermal initiator azobisisobutyronitrile (AIBN, 3 mg). Seal the reaction flask and perform a freeze-evacuation-thawing cycle three times to completely remove dissolved oxygen from the system. The post-reaction phenomena were the same as in Comparative Example 5, with the reaction solution exhibiting gelation. This may be due to the poor chemical stability of commercially available conventional dithiobenzoic acid ester RAFT reagents (CPDB). In the complex system of this invention, CPDB is highly susceptible to attack by monomer side-chain amine groups or free phenolic hydroxyl groups, resulting in irreversible ammonolysis or degradation. The destruction of the active end groups of the RAFT reagent leads to the loss of chain transfer control in the system, causing the reaction to degenerate into uncontrolled free radical polymerization, which in turn triggers the physical entanglement and gelation of ultra-long chains. Therefore, the product of this comparative example could not be subjected to subsequent dialysis purification and coating preparation steps.

[0058] Comparative Example 8 This comparative example is a dopamine-containing quaternary copolymer and its preparation method. The steps are the same as in Example 1, the main difference being that a commercially available RAFT reagent of dithiobenzoate (CPDB) is used, and anhydrous zinc chloride is not added to the polymerization system.

[0059] 1. Synthesis of dopamine methacrylamide (DMA) The synthesis steps are the same as in Example 1.

[0060] 2. Synthesis of 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate The synthesis steps are the same as in Example 1.

[0061] 3. Synthesis of tetromers Weigh 0.45 g of dopamine methacrylamide (DMA), 1.9 g of 2-methoxyethyl 2-acrylate (MEA), 0.29 g of methacryloyloxyethyltrimethylammonium chloride (DMC), and 0.29 g of 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate (CHDU) and dissolve them in 10 mL of N,N-dimethylformamide (DMF). Transfer the solution to a 100 mL dry round-bottom flask. Then, add the commercially available RAFT reagent 2-cyano-2-propyl dithiobenzoate (CPDB, 18 mg) and the thermal initiator azobisisobutyronitrile (AIBN, 3 mg) to the system. Seal the reaction flask and perform a freeze-evacuation-thawing cycle three times to completely remove dissolved oxygen. Under nitrogen protection, place the reaction flask in a 70°C oil bath for constant temperature reaction. The post-reaction phenomena were the same as those in Comparative Example 5. The system exhibited extremely high viscosity gelation on a macroscopic scale, therefore the product of this comparative example could not be subjected to subsequent dialysis purification and coating preparation steps.

[0062] Example 2 Preparation of a multifunctional coating on the surface of silicone tubes: 1. Silicone tubing pretreatment: Cut an appropriate amount of medical silicone catheter (made of PDMS), first ultrasonically clean it with ethanol for 10 minutes to remove surface oil, then ultrasonically clean it with deionized water for 10 minutes, and then dry it with nitrogen gas for later use.

[0063] 2. Preparation of coating solution: Prepare a 10 mM Tris-HCl buffer solution, then mix the Tris-HCl buffer solution and isopropanol at a 1:1 ratio to obtain a mixed solution, and adjust the pH to 8.5 with sodium hydroxide.

[0064] A certain amount of the quaternary copolymer prepared in Example 1 was weighed and dissolved in the above mixed solution to prepare a modified coating solution with a polymer concentration of 20 mg / mL.

[0065] 3. Immersion coating: The pretreated silicone tube was completely immersed in the modified coating solution. Under room temperature (25°C) and open, ventilated conditions, the beaker was placed on a shaker and shaken at low speed for 24 hours.

[0066] 4. Post-processing: After coating, the silicone tube was removed and its surface was repeatedly rinsed with plenty of deionized water to remove any unadsorbed or poorly bonded polymer layers. Finally, the silicone tube was dried in a 50°C vacuum oven to constant weight, resulting in a silicone tube with a stable self-anchoring multifunctional coating, labeled as S1 - Example 1 conduit.

[0067] Comparative Example 9 This comparative example demonstrates a method for preparing a multifunctional coating on the surface of a silicone tube. The steps are the same as in Example 2, with the main difference being that the effective component in the coating solution comes from Comparative Example 1. The resulting silicone tube is named D9-Comparative Example 1 conduit.

[0068] Comparative Example 10 This comparative example demonstrates a method for preparing a multifunctional coating on the surface of a silicone tube. The steps are the same as in Example 2, with the main difference being that the effective component in the coating solution comes from Comparative Example 2. The resulting silicone tube is named D10-Comparative Example 2 conduit.

[0069] Since the polymer coating cannot be effectively anchored to the surface of the silicone tube and suffers from severe peeling when exposed to water, no further experiments were conducted.

[0070] Comparative Example 11 This comparative example describes a method for preparing an antibacterial coating on the surface of a silicone tube. The steps are the same as in Example 2, with the main difference being that the effective component in the coating solution comes from Comparative Example 3. The resulting silicone tube is named D11-Comparative Example 3 conduit.

[0071] Comparative Example 12 Clean silicone tubing D12 - blank catheter without any treatment.

[0072] Test case 1. Zeta potential test The quaternary copolymer obtained in Example 1 (S1 - polymer B corresponding to the catheter in Example 1) and the ternary copolymer obtained in Comparative Example 1 (D9 - polymer A corresponding to the catheter in Comparative Example 1) were respectively prepared into solutions for Zeta potential testing. Aqueous dispersions of the test samples with a mass concentration of 0.5 mg / mL were subjected to three consecutive measurement cycles at room temperature. The results are as follows: Figure 4 As shown, the terpolymer (A) without added DMC monomer exhibits a weak positive charge (approximately +1.0 mV); while the quaternary copolymer (B) of this invention, due to the successful introduction of the quaternary ammonium salt monomer (DMC), exhibits a strong positive potential as high as approximately +20.8 mV. This not only demonstrates the successful copolymerization of the DMC monomer but also provides a crucial charge basis for the subsequent active contact film-breaking sterilization of the coating. 2. Antibacterial test.

[0073] (1) Preparation of liquid culture medium Weigh 1g of tryptone, 0.5g of yeast extract, and 1g of sodium chloride separately. Transfer the weighed powders to an Erlenmeyer flask, add 100mL of deionized water, and stir continuously until the mixed solution is uniform and transparent. Ensure that all solutes are completely dissolved, then place the Erlenmeyer flask in an autoclave and sterilize at 121℃ and 101kPa for 30 minutes. After sterilization, remove the flask, cool it to room temperature, and store it in a refrigerator at 4℃ for later use.

[0074] (2) Preparation of TSA agar medium First, weigh out 4.5g of tryptone, 2.25g of yeast extract, 4.5g of sodium chloride, and 6.75g of agar powder, and add these powders sequentially to a 500mL Erlenmeyer flask. Then add 450mL of deionized water and stir thoroughly with a glass rod until all solids are completely dissolved. Place the flask in an autoclave and sterilize at 121℃ and 101kPa for 120 minutes. After sterilization, remove the container and allow it to cool to approximately 50℃, before the agar solidifies. Before the agar solidifies, pour the culture medium into sterile petri dishes, adding approximately 15-20mL of medium to each dish. After pouring, gently shake the petri dishes to distribute the medium evenly, and then allow them to solidify.

[0075] (3) Bacterial culture The target strain was inoculated into sterile culture medium and cultured in a shaker at 37°C for 24 h. The bacterial culture was then diluted with fresh culture medium to approximately 10⁻⁶. 6 Prepare a concentration of CFU / mL for later use.

[0076] (4) Antibacterial test Sterilized D12-blank catheter, D11-comparative example 3 catheter, D9-comparative example 1 catheter, and S1-Example 1 catheter were cut into 2 cm long segments, with at least 3 replicates for each sample. The four types of catheters were then placed in 5 mL of diluted bacterial solution and incubated for 24 h. After incubation, the catheters were removed. The surfaces of the catheters were cleaned with sterile PBS buffer to remove any bacteria. Each catheter was then placed in 3 mL of sterile PBS buffer and sonicated for 5 min to remove any bacteria adhering to the surface. 100 μL of bacterial PBS solution was then added to agar plates in a clean bench and spread evenly using a disposable spreader. The inoculated plates were placed in a 37°C incubator and incubated for 24 h. The colony counts were then determined. The experimental results are as follows: Figure 5 As shown in the figure. In vitro antibacterial plate count test results showed that the surface of the unmodified silicone tube (D12 - blank catheter) contained a large number of viable bacteria, lacking antibacterial defense capabilities; the ternary copolymer-coated silicone tube (D9 - Comparative Example 1 catheter) without the bactericidal monomer DMC only showed a weak antibacterial effect, indicating that although the hydrophilic hydration layer alone can produce a passive anti-adhesion physical barrier, it cannot fundamentally inactivate pathogens. In contrast, the ternary copolymer coating without CHDU monomer (D11 - Comparative Example 3 catheter) and the quaternary copolymer coating of the present invention (S1 - Example 1 catheter) both exhibited extremely strong bactericidal efficacy, with viable bacteria being almost completely eliminated. This fully confirms that the antibacterial function originates from the active contact membrane-breaking bactericidal mechanism brought about by the positive charge of the DMC quaternary ammonium salt, and that the introduction of CHDU does not interfere with its bactericidal performance.

[0077] 3. Anti-crust test Both ends of the same length of catheter segments—D12 (blank), D11 (comparative example 3), D9 (comparative example 1), and S1 (Example 1)—were completely sealed to eliminate interference from crystal deposition on the inner wall. Three parallel samples were prepared for each group. Each group of catheters was suspended in an in vitro simulated bladder device (conical flask) using a 0.5 mm diameter guidewire. Then, a solution containing 1 × 10⁻⁶... 4 Artificial urine containing CFU / mL of Proteus mirabilis was prepared. Fresh artificial urine was continuously pumped into the device at a rate of 0.5 mL / min using a peristaltic pump, while a thermostatic magnetic stirrer (37℃, 60 rpm) was activated to simulate the microfluidic environment of the human bladder. The pH value of the artificial urine in the in vitro bladder was measured every 24 hours. After three weeks of operation, the catheter was removed, and the loose crystals on the surface of the catheter were gently washed away with ultrapure water before being dried in an oven at 55℃ for 12 hours. Subsequently, the sample was fixed on the sample stage with conductive adhesive, sputtered with gold, and characterized by SEM. The results are as follows: Figure 6 As shown, the strongly hydrophobic interface of the unmodified pure silicone tube (D12 - blank tube) readily induces non-specific heterogeneous nucleation, resulting in a visible, rough, white, hard-shell-like deposit on its surface after 21 days of testing. In contrast, the control tube D11 - Comparative Example 3, which does not contain D monomer (CHDU), exhibits a highly hydrated hydrophilic surface that provides numerous effective heterogeneous nucleation sites for calcium phosphate and struvite crystals, with densely packed, layered crystal clusters visible under SEM. In stark contrast to D11 - Comparative Example 3, although D9 - Comparative Example 1 tube (without DMC) and S1 - Example 1 tube (quaternary copolymer) differ in composition, both contain D monomer (CHDU), resulting in extremely smooth surfaces with only a very thin, transparent film-like deposit under the same experimental conditions.

[0078] 4. Cytotoxicity test 4.1 The in vitro cytotoxicity of the D12-blank duct and the S1-Example 1 duct was evaluated using the mouse fibroblast cell line L-929. Three groups were set up: a blank group (containing only complete culture medium and MTT reagent, without cells), a control group (containing normally growing cells, complete culture medium, and MTT reagent), and an experimental group (containing cells, duct sample extract, and MTT reagent). The extract was prepared as follows: the D12-blank duct and the S1-Example 1 duct were soaked in physiological saline for 24 h to obtain the extract, which was then filtered through a 0.22 μm needle filter.

[0079] L-929 cells were routinely cultured in complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. After digestion and counting, the cells were seeded into 96-well plates at a density of approximately 5000 cells per well and then incubated at 37°C with 5% CO2 for 24 hours to allow the cells to adhere and grow. The original medium was then discarded, and the cells were cultured again in extract medium containing a specific concentration of the sample. The experimental groups used extract medium from D12-blank catheters and S1-Example 1 catheters, respectively, while the positive control group used physiological saline. After culturing the cells for 24 hours, 20 μL of MTT reagent was added to each well of the seeded plates, and the plates were incubated in the dark for 4 hours. After purple formazan crystals formed, an appropriate amount of dissolving solution was added to completely dissolve the crystals, and the optical density at 570 nm was measured using a microplate reader. The results are as follows: Figure 7 As shown in Figure a, during consecutive test cycles of 24 h, 48 h, and 72 h, the relative cell viability of the S1-Example 1 catheter group was significantly higher than the 70% cytotoxicity threshold specified in ISO 10993-5.

[0080] 4.2 Live / Dead Cell Staining Assay To further observe the effect of the coating on cytotoxicity, L-929 cells were seeded in laser confocal culture dishes and incubated for 48 h. After cell adhesion, 100 µL of extracts from D12-blank catheters and S1-Example 1 catheters were co-incubated with L-929 cells for 24 h. L-929 cells were characterized using the Calcein-AM / PI live / dead cell staining method. Results are as follows: Figure 7 As shown in b.

[0081] Visual observation using a laser confocal microscope revealed that all cells within the field of view exhibited green fluorescence, indicating that the cells were alive; almost no red fluorescence signals representing cell death or apoptosis were observed, further demonstrating the high biocompatibility of the constructed coating.

[0082] To limit the scope of this invention, those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of this invention should be included within the protection scope of this invention.

Claims

1. A storage-stable dopamine-containing multifunctional tetra-copolymer, characterized in that, The copolymer is a random copolymer prepared by a reversible addition-fragmentation chain transfer polymerization reaction of four monomers, including monomers A, B, C, and D, in the presence of a chain transfer agent and a Lewis acid; monomer A is dopamine methacrylamide, monomer B is 2-methoxyethyl 2-acrylate, monomer C is methacryloyloxyethyltrimethylammonium chloride, and monomer D is 2-(methacryloyloxy)ethyl N-(3,4-dihydroxycyclohexyl)carbamate; The molar ratio of monomers A, B, C and D is 1 : (5-10) : (0.2-3) : (0.3-5); The chain transfer agent is cyanomethyl (3,5-dimethyl-1H-pyrazole)-dithioester, and the Lewis acid is anhydrous zinc chloride.

2. The storage-stable dopamine-containing multi-functional tetra-copolymer according to claim 1, wherein: The copolymer has a molecular weight distribution index of 1.1 to 1.5, and the ends of the copolymer molecular chains contain pyrazolyl dithiocarbamate groups derived from the chain transfer agent.

3. The method for preparing the storage-stable dopamine-containing multifunctional tetra- copolymer according to any one of claims 1-2, characterized in that, The method is as follows: the monomers A, B, C, and D, the chain transfer agent, the initiator, and the Lewis acid are dissolved in an organic solvent, and after deoxygenation, a constant-temperature polymerization reaction is carried out under sealed conditions. After the reaction is completed, the reaction solution is purified by dialysis and freeze-dried to obtain the quaternary copolymer; the initiator is azobisisobutyronitrile.

4. The method for preparing the storage-stable dopamine-containing multifunctional tetromer according to claim 3, characterized in that, The total molar amount of monomers A, B, C and D and the molar ratio of chain transfer agent to initiator are (150-300): 1: (0.1-0.5).

5. The method for preparing the storage-stable dopamine-containing multifunctional tetropolymer according to claim 3, characterized in that, The molar ratio of the Lewis acid to the chain transfer agent is (0.5–2.0):

1.

6. The method for preparing the storage-stable dopamine-containing multifunctional tetromer according to claim 3, characterized in that, The polymerization reaction is carried out at a temperature of 60℃ to 75℃ for a reaction time of 6h to 16h.

7. A surface-modified silicone tube, characterized in that, The surface of the silicone tube is covered with a self-anchoring coating formed by the storage-stable dopamine-containing multifunctional quaternary copolymer of any one of claims 1-2 or the storage-stable dopamine-containing quaternary copolymer prepared by the method of any one of claims 3-6; the coating is formed by dissolving the copolymer in a mixed solvent to prepare a coating solution, and immersing the silicone tube in the coating solution for in-situ oxidative crosslinking assembly.

8. The surface-modified silicone tube according to claim 7, characterized in that, The copolymer was dissolved in a mixed solvent with a pH of 8.0–9.0 to prepare a coating solution.

9. The surface-modified silicone tube according to claim 7, characterized in that: The mixed solvent is isopropanol and Tris-HCl buffer at a volume ratio of 1:(0.5-2).

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