Sterilizing disinfectant and method of preparation

Abstract

The present application relates to the technical field of disinfectant, disclose a kind of sterilization disinfectant and preparation method thereof.The sterilization disinfectant includes by mass percentage: glutaraldehyde 2.0~2.5%, organic-inorganic composite antibacterial agent 0.02~0.15%, antirust agent 0.05~0.30%, complex stabilizer 0.01~0.10%, pH adjusting agent or buffer adjusting system pH to 7.5~8.5, the rest is water.The present application uses glutaraldehyde as main sterilization effective component, uses organic-inorganic composite antibacterial agent as dispersion stability and interface adsorption auxiliary component, improves its local contact efficiency in complex solid-liquid interface scene such as instrument surface, lumen inner wall, biofilm residue and spore pollution, to improve sterilization verification safety margin.The present application is suitable for endoscope, lumen instrument and other heat-intolerant medical instrument high-level disinfection or sterilization treatment.

Description

Technical Field

[0001] This invention relates to the field of disinfectant technology, specifically to a sterilizing disinfectant and its preparation method. Background Technology

[0002] Medical devices are susceptible to contamination by microorganisms such as vegetative bacteria, fungi, viruses, mycobacteria, and bacterial spores during repeated use. For endoscopes, tubular instruments, precision instruments, and some instruments made of polymer materials that cannot be sterilized by high temperature and high pressure, high-level disinfection or sterilization treatment is often required by chemical immersion.

[0003] Glutaraldehyde is a commonly used aldehyde chemical sterilizing agent. It can react with active groups such as amino and thiol groups in microbial proteins, enzymes, nucleic acids, and other biomolecules, thereby disrupting the key structures and metabolic functions of microorganisms. Alkaline glutaraldehyde systems have strong killing capabilities against vegetative bacteria, fungi, viruses, and bacterial spores, and are widely used in the fields of immersion disinfection and sterilization of medical devices.

[0004] However, the glutaraldehyde-only system still has certain limitations. Glutaraldehyde mainly relies on liquid-phase diffusion and chemical cross-linking to exert its bactericidal effect. In areas such as instrument gaps, rough surfaces, luminal walls, and biofilm residues, local contact and penetration efficiency may decrease. Bacterial spores have a dense outer structure and are highly resistant to chemical bactericides, requiring a relatively long reaction time when relying solely on the glutaraldehyde system. The local bactericidal efficiency of glutaraldehyde may also be affected by organic interference or the presence of contaminants on instrument surfaces. Traditional glutaraldehyde formulations primarily focus on liquid-phase bactericidal capabilities, lacking sufficient targeted enhancement for complex solid-liquid interface scenarios such as instrument surfaces, biofilm residues, and luminal walls.

[0005] Quaternary ammonium salt antibacterial agents possess cationic surfactant structures, enabling them to adsorb negatively charged microbial surfaces and disrupt cell membrane structures. However, when used alone, their ability to kill bacterial spores is limited, making them unsuitable as the sole effective component in medical device sterilization agents. Nano-zinc oxide exhibits a high specific surface area and certain antibacterial activity; however, unmodified nano-zinc oxide tends to aggregate and settle in aqueous systems, and its effective contact with microbial surfaces is insufficient.

[0006] Therefore, it is necessary to develop a composite sterilizing agent with glutaraldehyde as the main sterilizing active ingredient and also having interfacial adsorption and dispersion stabilization auxiliary functions, in order to improve its sterilization validation safety margin in complex instrument surfaces and lumen environments. Summary of the Invention

[0007] The purpose of this invention is to provide a glutaraldehyde medical device sterilization and disinfection agent containing an organic-inorganic composite antibacterial agent, its preparation method, and its application in immersion sterilization or high-level disinfection of medical devices.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a glutaraldehyde medical device sterilization and disinfection agent containing an organic-inorganic composite antibacterial agent, comprising the following components by mass percentage: Glutaraldehyde: 2.0%–2.5%; Organic-inorganic composite antibacterial agent: 0.02%~0.15%; Rust inhibitor: 0.05%~0.30%; Complexation stabilizer: 0.01%–0.10%; pH adjuster or buffer: 0.05%–0.50%; Water: Balance; The pH of the sterilizing disinfectant is 7.5 to 8.5.

[0009] Preferably, the mass percentage of glutaraldehyde is 2.1% to 2.4%, more preferably 2.3%.

[0010] Preferably, the organic-inorganic composite antibacterial agent has a mass percentage of 0.05% to 0.12%, more preferably 0.08%.

[0011] Preferably, the rust inhibitor is one or more of sodium nitrite, sodium molybdate, sodium benzoate, borate, and phosphate, and more preferably sodium nitrite.

[0012] Preferably, the complexing stabilizer is one or more of disodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate, sodium citrate, and sodium gluconate, and more preferably disodium ethylenediaminetetraacetate.

[0013] Preferably, the pH adjuster or buffer is one or more of sodium bicarbonate, sodium carbonate, borate buffer, and phosphate buffer.

[0014] The water is preferably purified water, deionized water, or water that meets the requirements for disinfectant production.

[0015] The organic-inorganic composite antibacterial agent of the present invention is prepared by reacting a hydroxyl-containing polyquaternary ammonium salt antibacterial agent with surface epoxy-modified nano zinc oxide, comprising an inorganic nano zinc oxide core and an organic quaternary ammonium salt antibacterial chain segment grafted onto its surface.

[0016] Preferably, the average particle size of the nano-zinc oxide is 20–100 nm.

[0017] Preferably, the organic-inorganic composite antibacterial agent has an average particle size D50 of 80-300 nm and a Zeta potential of +15 mV-+45 mV in the aqueous phase.

[0018] Preferably, the hydroxyl-containing polyquaternary ammonium salt antibacterial agent is a water-soluble or water-dispersible quaternary ammonium salt antibacterial agent containing at least two quaternary ammonium salt groups, at least two hydroxyl groups, and C8-C16 hydrophobic alkyl segments in its molecule.

[0019] The surface-epoxylated zinc oxide nanoparticles are obtained by surface modification of zinc oxide nanoparticles using an epoxy silane coupling agent. Preferably, the silane coupling agent is γ-glycidoxypropyltrimethoxysilane, γ-glycidoxypropyltriethoxysilane, or a combination thereof.

[0020] The preparation method of the organic-inorganic composite antibacterial agent includes the following steps: S1. Disperse nano zinc oxide in an aqueous ethanol solution, adjust the pH to 4.0-6.0, add an epoxy silane coupling agent, and reflux at 50-90℃ for 2-8 hours to obtain surface epoxy-modified nano zinc oxide. S2. Dissolve or disperse the hydroxyl-containing polyquaternary ammonium salt antibacterial agent in water, ethanol, a water-alcohol mixture, or a polar aprotic solvent (such as N,N-dimethylformamide), add the surface-epoxylated nano zinc oxide obtained in step S1, and react at 60-90℃ for 4-12 hours under alkaline catalysis, so that the hydroxyl groups in the hydroxyl-containing polyquaternary ammonium salt antibacterial agent undergo a nucleophilic ring-opening reaction with the surface epoxy groups to form a covalently grafted structure linked by ether bonds; S3. After the reaction is complete, the mixture is centrifuged, washed until neutral, dispersed, or dried to obtain the organic-inorganic composite antibacterial agent.

[0021] In this invention, the organic-inorganic composite antibacterial agent itself possesses certain antibacterial activity. However, its main function in the formulation is not to replace glutaraldehyde as the primary sterilizing agent. Instead, it assists in improving the safety margin of the glutaraldehyde system in harsh scenarios such as instrument surfaces, lumen walls, biofilm residues, and spore contamination by improving the dispersion stability of nano-components, enhancing the adsorption effect at the cation interface, and increasing the probability of local contact at complex solid-liquid interfaces. Glutaraldehyde remains the primary sterilizing agent in the sterilization working solution of this invention.

[0022] Secondly, the present invention provides a method for preparing the above-mentioned sterilizing disinfectant, comprising: A. Preparation of glutaraldehyde base solution; B. Preparation of an activating component containing an organic-inorganic composite antibacterial agent, rust inhibitor, complexing stabilizer, and pH adjuster; C. Before use, mix the glutaraldehyde base solution with the activating components in a certain proportion to obtain a sterilization working solution with a pH of 7.5-8.5 and a glutaraldehyde mass percentage of 2.0%-2.5%.

[0023] Preferably, the activating component includes an organic-inorganic composite antibacterial agent, sodium nitrite, disodium ethylenediaminetetraacetate, and sodium bicarbonate.

[0024] Preferably, the pH of the sterilization working solution after activation is 7.8 to 8.2.

[0025] Thirdly, the present invention provides the application of the above-mentioned sterilizing agent in high-level disinfection or sterilization of medical devices. The sterilizing working solution is suitable for high-level disinfection or sterilization of cleaned medical devices, and the usage conditions are as follows: (1) High-level disinfection: The instrument is completely immersed in the working solution and the lumen is filled. The instrument is treated according to the verified minimum effective treatment time or the time specified in the product instructions. When evaluating the effect of high-level disinfection, the killing test of representative strains of mycobacteria should be added according to the relevant testing requirements of disinfection products. The minimum effective treatment time should be determined by combining suspension test, carrier test and simulated field test. (2) Sterilization treatment: The instrument is completely immersed in the working solution and the lumen is filled. It is treated for the minimum effective treatment time specified in the product instructions or the verified effective sterilization treatment time, preferably 10h; (3) Post-treatment: After the procedure, the instrument surface and lumen should be thoroughly rinsed with sterile water and dried and stored in accordance with the manufacturer’s instructions. (4) Use boundary: When the glutaraldehyde concentration of the working solution is below 2.0%, reaches the upper limit of the validated use period, the pH exceeds the range of 7.5 to 8.5, or obvious sedimentation, stratification, or flocculation appears, it shall not continue to be used as a sterilization working solution; (5) Material suitability: Before use, the applicable scope should be confirmed in accordance with the manufacturer’s instructions and material compatibility verification. Beneficial effects

[0026] This invention uses glutaraldehyde as the main effective sterilization ingredient, ensuring the formulation's basic killing ability against highly resistant microorganisms such as bacterial spores. It introduces an organic-inorganic composite antibacterial agent, enhancing the formulation's adsorption and contact assistance in complex solid-liquid interface environments on top of liquid-phase sterilization. By grafting quaternary ammonium salt antibacterial segments onto the surface of nano-zinc oxide, it improves the dispersion stability of the nano-antibacterial components in the glutaraldehyde aqueous system, reducing aggregation and sedimentation, and increasing the sterilization validation safety margin under stringent evaluation scenarios such as instrument surfaces, lumen walls, biofilm residues, and spore contamination. Detailed Implementation Experimental Example 1:

[0027] Preparation of multiquaternary ammonium salt antibacterial agents containing four quaternary ammonium groups Synthesis principle: (1) The two primary amino groups in 1 molar equivalent of 1,3-propanediamine undergo N-alkylation reaction with 2 molar equivalents of 1-bromohexane under the action of base (K2CO3) to generate intermediate 1 containing two secondary amino groups; (2) The two secondary amine groups of 1 molar equivalent intermediate 1 and 2 molar equivalent 12-bromododecane-1-ol are further N-alkylated under the action of NaOH to generate intermediate 2 containing two tertiary amine groups and two terminal hydroxyl groups; (3) The two tertiary amine groups of 1 molar equivalent intermediate 2 undergo quaternization with 2 molar equivalents of (3-bromopropyl)trimethylammonium bromide. Since (3-bromopropyl)trimethylammonium bromide itself already contains 1 quaternary ammonium group, the reaction simultaneously generates 2 new quaternary ammonium cations. Therefore, the final product molecule contains a total of 4 quaternary ammonium salt groups and 2 hydroxyl groups, and is referred to as a multiquaternary ammonium salt antibacterial agent containing four quaternary ammonium groups.

[0028] Specific preparation method: Preparation of Intermediate 1: 4.0 g K2CO3 and 20 mL n-propanol were added to a three-necked flask and mechanically stirred under nitrogen protection. 0.83 mL (approximately 10 mmol) of 1,3-propanediamine was slowly added dropwise to the flask through a constant-pressure dropping funnel, followed by 2.8 mL (approximately 20 mmol) of 1-bromohexane. The mixture was heated to reflux (approximately 90–95 °C) and reacted for 12 h (appropriate excess alkali and extended reaction time were used to suppress mono-substitution and peralkylation side reactions). After cooling, the inorganic salts were removed by filtration, and the solvent was removed by rotary evaporation. The residue was purified by silica gel column chromatography (dichloromethane:methanol:ammonia = 20:1:0.05, volume ratio), washed three times with toluene / 0.5 wt% NaOH solution, recrystallized three times with anhydrous ethanol, and dried under vacuum at 50 °C for 6 h to obtain a colorless oily / low-melting-point solid intermediate 1, with a yield of approximately 62%.

[0029] Preparation of intermediate 2: 2.4 g (about 10 mmol) of intermediate 1, 5.3 g (about 20 mmol) of 12-bromododecane-1-ol, 1.0 g of NaOH and 40 mL of anhydrous ethanol were added to a three-necked flask and reacted under nitrogen protection at reflux (78 °C) for 10 h. After cooling, NaBr and excess NaOH were removed by suction filtration, and the ethanol was removed by rotary evaporation of the filtrate. The residue was dissolved in dichloromethane, washed three times with water, dried with anhydrous Na2SO4, filtered, and then purified by rotary evaporation. The purified residue was then purified by silica gel column chromatography (dichloromethane:methanol = 10:1, volume ratio) and dried under vacuum at 50 °C for 6 h to obtain a pale yellow waxy solid intermediate 2 with a yield of about 58%.

[0030] Preparation of a multi-quaternary ammonium salt antibacterial agent containing four quaternary ammonium groups: 5.8 g of intermediate 2 and 50 mL of acetonitrile were added to a three-necked flask and stirred at 150 r / min for 10 min under nitrogen protection. 5.2 g (about 20 mmol) of (3-bromopropyl)trimethylammonium bromide was added, and the mixture was refluxed for 10 h. After cooling, the precipitate was poured into acetone, filtered, recrystallized twice by acetonitrile / acetone, and dried under vacuum at 50 °C for 8 h to obtain a white solid target multi-quaternary ammonium salt antibacterial agent with a yield of about 71%.

[0031] Product characterization: 1 HNMR (400MHz, DMSO-D6, δ, ppm): 0.88-0.90 (t, 6H), 1.26-1.38 (m, 36H), 1.41-1.46 (m, 4H), 1.52-1.58 (m, 4H), 1.69-1.7 9(m, 8H), 2.24-2.32(m, 6H), 2.64-2.66(t, 2H), 3.26(s, 18H), 3.38-3.42(t, 8H), 3.54-3.60(m, 8H), 3.72-3.76(m, 8H).

[0032] The structure of a multi-quaternary ammonium salt antibacterial agent containing four quaternary ammonium groups can be described as follows: 1,3-propanediamine serves as the central linking unit, with its two nitrogen atoms respectively attached to a C6 alkyl chain and a C12 alkyl chain containing terminal hydroxyl groups. Further, it undergoes a quaternization reaction with (3-bromopropyl)trimethylammonium bromide to form a multi-quaternary ammonium salt antibacterial agent containing four quaternary ammonium salt groups and two terminal hydroxyl groups. Its specific structural formula is as follows: . Experimental Example 2:

[0033] Preparation of surface-epoxylated nano zinc oxide Synthesis principle: The trimethoxy group of γ-glycidoxypropyltrimethoxysilane (KH560) hydrolyzes to generate Si-OH, which condenses with the hydroxyl groups on the surface of nano-zinc oxide to form a stable covalent bond, introducing epoxy groups into the surface of nano-zinc oxide. The mass ratio of KH560 to nano-zinc oxide is 0.04 to 0.10:1.

[0034] Specific preparation method: 95 mL of anhydrous ethanol and 5 mL of deionized water were added to a 250 mL three-necked flask equipped with a mechanical stirrer, reflux condenser, and thermometer. The mixture was stirred at 500 rpm for 30 min. 2 g of nano-zinc oxide (average particle size 30 nm) was added, and the pH was adjusted to 4.5–5.5 with dilute acetic acid. The mixture was stirred for 2 h to ensure complete dispersion. 0.12 g of KH560 was slowly added, and the temperature was raised to 70 °C. The mixture was stirred and kept under reflux condensation for 4 h. After the reaction, the suspension was separated by high-speed centrifugation and washed successively with acetone, ethanol, and deionized water to remove unreacted silane coupling agent and small molecule byproducts. The resulting solid was vacuum dried at 60 °C for 8 h to obtain surface-epoxylated nano-zinc oxide. If necessary, solid-liquid separation can also be performed using a 0.05–0.22 μm filter membrane or ultrafiltration. Experiment Example 3:

[0035] Preparation of organic-inorganic composite antibacterial agents Synthesis Principle: Under NaOH catalysis, the epoxy groups on the surface of surface-epoxylated zinc oxide nanoparticles undergo a nucleophilic ring-opening etherification reaction with the hydroxyl groups in a multi-quaternary ammonium salt antibacterial agent containing four quaternary ammonium groups, forming a stable covalent structure linked by -O-. The mass ratio of the multi-quaternary ammonium salt antibacterial agent to surface-epoxylated zinc oxide nanoparticles is 0.3–1:1.

[0036] Specific preparation method: 1g of surface-epoxylated nano zinc oxide, 60mL of N,N-dimethylformamide (DMF) and 20mL of deionized water were added to a three-necked flask and stirred at 200r / min for 1h under nitrogen protection; the temperature was raised to 90℃, 0.5g of a multi-quaternary ammonium salt antibacterial agent containing four quaternary ammonium groups and 0.1g of sodium hydroxide were added, and the reaction was kept at this temperature for 5h; the temperature was cooled to room temperature, centrifuged, washed with deionized water until neutral, and dried under vacuum at 60℃ for 8h to obtain an organic-inorganic composite antibacterial agent.

[0037] Uniform characterization sample numbering Sample A: Unmodified nano zinc oxide; Sample B: Surface-epoxylated nano-zinc oxide; Sample C: A polyquaternary ammonium salt antibacterial agent containing four quaternary ammonium groups (pure); Sample D: Physical mixture of surface-epoxylated nano zinc oxide and multi-quaternary ammonium salt (without NaOH, before washing); Sample E: The product of sample D after centrifugation and washing 5 times; Sample F: Organic-inorganic composite antibacterial agent of the present invention (before washing); Sample G: The product of sample F after centrifugation and washing 5 times.

[0038] (a) Thermogravimetric analysis (TGA) Test conditions: N2 atmosphere, 30–800℃, heating rate 10℃ / min. Sample A was unmodified nano-zinc oxide, sample B was surface-epoxylated nano-zinc oxide, sample D was the physical mixture of surface-epoxylated nano-zinc oxide and multi-quaternary ammonium salt antibacterial agent before washing, sample E was the product of sample D after 5 centrifugal washings, sample F was the organic-inorganic composite antibacterial agent of this invention before washing, and sample G was the product of sample F after 5 centrifugal washings. The results are shown in Table 1.

[0039] Table 1. Thermogravimetric analysis results of different samples

[0040] As shown in Table 1, the increased organic weight loss of sample B compared to sample A indicates that the epoxy silane coupling agent has been introduced onto the surface of the nano-zinc oxide. Physically mixed sample D exhibited a high apparent loading of quaternary ammonium salts before washing, but after five centrifugal washes, the apparent loading of quaternary ammonium salts in sample E decreased to 0.6 wt%, with a retention rate of approximately 4.9%. In contrast, sample G, obtained after five centrifugal washes of the covalently grafted sample F of this invention, still maintained an apparent loading of 10.4 wt% of quaternary ammonium salts, with a retention rate of approximately 89.7%. These results demonstrate that the multi-quaternary ammonium salt antibacterial segments do not primarily exist on the surface of nano-zinc oxide through physical adsorption, but rather form a stable grafted structure through an epoxy ring-opening etherification reaction.

[0041] Note: Sample C is a purified multi-quaternary ammonium salt antibacterial agent containing four quaternary ammonium groups, which can be used for structural characterization by infrared spectroscopy, nuclear magnetic resonance, elemental analysis, etc., but is not included in the calculation of nano zinc oxide surface loading and washing retention rate in Table 1.

[0042] (ii) Fourier transform infrared spectroscopy (FTIR) Test results show that sample A is at 3400cm. -1 (Surface-OH) and 430-500cm -1 (Zn-O) exhibits characteristic absorption. Sample B shows an additional 2925 / 2855 cm⁻¹ compared to sample A. -1 (-CH2-), 1100 / 1040cm -1 (Si-O-Si / Si-O-Zn) and 910cm -1 Characteristic peaks (of epoxy groups) confirm successful coupling of KH560. Sample F relative to sample B: 910 cm⁻¹ -1 The characteristic peak of the epoxy group was significantly weakened (epoxy group consumption), and a new peak at 1480 cm⁻¹ was added. -1 N + (CH3)3 characteristic absorption, 2925 / 2855 cm⁻¹ -1 The intensity of the alkyl peak increased to 3400 cm⁻¹. -1 The hydroxyl peak position shifts slightly towards lower wavenumbers (due to the formation of new -OH and -O- environments after ring opening). These characteristics simultaneously support two key pieces of evidence: the consumption of epoxy groups and the introduction of quaternary ammonium salt structures.

[0043] (iii) X-ray photoelectron spectroscopy (XPS) The test results showed that: Sample A only detected Zn2p, O1s, and trace C1s (adsorbed carbon); Sample B, compared to Sample A, showed Si2p (approximately 102.5 eV, attributed to Si-O-Zn / Si-O-Si) and a significantly enhanced C1s peak; Sample F, compared to Sample B, further showed an N1s peak (approximately 402.3 eV, attributed to N⁺ quaternary ammonium nitrogen), and the percentage of N atoms increased from 0.1% (B) to 3.2% (F). XPS results verified the loading of quaternary ammonium salt structures on the nano-zinc oxide surface.

[0044] (iv) Antibacterial performance test The shaking contact method (GB / T21510) was used. Tested bacterial species: *Escherichia coli* ATCC25922 and *Staphylococcus aureus* ATCC6538. Sample concentration: 100 mg / L; initial bacterial concentration: approximately 1 × 10⁻⁶. 6 CFU / mL, contacted at 37℃ with shaking for 2 hours. Results are shown in Table 2.

[0045] Table 2. Antibacterial performance test results of different samples

[0046] The antibacterial rate of sample G (after washing) was still ≥98.3%, proving that the quaternary ammonium salt structure is stable and the antibacterial durability is significantly better than that of the physical mixing system (sample E). Example 1:

[0047] A sterilizing disinfectant, wherein the sterilizing working solution comprises the following components by weight percentage: Glutaraldehyde: 2.30%; Organic-inorganic composite antibacterial agent: 0.08%; Sodium nitrite: 0.10%; Disodium ethylenediaminetetraacetate: 0.05%; Sodium bicarbonate: appropriate amount; Water: Balance; The pH of the sterilization working solution is 8.05; Its preparation method is as follows: Glutaraldehyde is added to water and stirred evenly to obtain glutaraldehyde base solution; sodium nitrite, disodium ethylenediaminetetraacetate, sodium bicarbonate and organic-inorganic composite antibacterial agent are added to water and stirred evenly to obtain activation component; before use, glutaraldehyde base solution and activation component are mixed and stirred evenly to obtain the sterilization working solution. Example 2:

[0048] A sterilizing disinfectant, wherein the sterilizing working solution comprises the following components by weight percentage: Glutaraldehyde: 2.10%; Organic-inorganic composite antibacterial agent: 0.05%; Sodium nitrite: 0.08%; Disodium ethylenediaminetetraacetate: 0.03%; Sodium bicarbonate: appropriate amount; Water: Balance; The pH of the sterilization working solution is 7.85; The preparation method is the same as in Example 1. Example 3:

[0049] A sterilizing disinfectant, wherein the sterilizing working solution comprises the following components by weight percentage: Glutaraldehyde: 2.40%; Organic-inorganic composite antibacterial agent: 0.12%; Sodium nitrite: 0.15%; Disodium ethylenediaminetetraacetate: 0.08%; Sodium carbonate and sodium bicarbonate: appropriate amount; Water: Balance; The pH of the sterilization working solution is 8.20; The preparation method is the same as in Example 1. Comparative Example 1:

[0050] The organic-inorganic composite antibacterial agent in Example 1 was replaced with an equal amount of "unmodified nano zinc oxide + a physical mixture of multi-quaternary ammonium salt antibacterial agents containing four quaternary ammonium groups", and the rest was the same as in Example 1. Comparative Example 2:

[0051] The organic-inorganic composite antibacterial agent in Example 1 was replaced with an equal amount of surface-epoxylated nano zinc oxide, and the rest was the same as in Example 1. Comparative Example 3:

[0052] The organic-inorganic composite antibacterial agent in Example 1 was replaced with an equal amount of "physical adsorption system obtained by mixing surface-epoxylated nano zinc oxide and a multi-quaternary ammonium salt antibacterial agent containing four quaternary ammonium groups without adding NaOH", and the rest was the same as in Example 1. Comparative Example 4:

[0053] The glutaraldehyde-free quaternary ammonium salt composite disinfection system includes 0.08% organic-inorganic composite antibacterial agent, 0.10% sodium nitrite, 0.05% disodium ethylenediaminetetraacetate, appropriate amount of sodium bicarbonate, and water as the balance, with a pH of 8.05. Performance testing:

[0054] Unless otherwise specified, the test strains include *Escherichia coli* ATCC 25922, *Staphylococcus aureus* ATCC 6538, *Pseudomonas aeruginosa* ATCC 15442, *Candida albicans* ATCC 10231, and *Bacillus subtilis* var. *niger* spores ATCC 9372. For high-level disinfection efficacy evaluation, a bactericidal test of representative mycobacterial strains should be added according to the relevant disinfection product testing requirements. The minimum effective contact time should be determined by combining suspension tests, carrier tests, and simulated field tests. The inoculum concentration is an initial bacterial suspension concentration of 1×10^7~5×10^7 CFU / mL; the carrier test yields a recovered bacterial count of 1×10^6~5×10^6 CFU per tablet. The neutralizing agent was a complex neutralization system consisting of 3.0 g / L lecithin, 30.0 g / L Tween 80, 5.0 g / L glycine, 5.0 g / L sodium thiosulfate, and phosphate buffer. Before use, the neutralizing agent was tested according to disinfectant testing requirements to confirm its effective neutralizing ability against the test samples and its lack of adverse effects on the test bacteria. The culture conditions were: bacteria cultured at 37℃ for 48 h, Candida albicans cultured at 28℃ for 72 h, and spores cultured at 37℃ for 7 days for observation. For the suspension method, a log-kill value ≥5.00 was used as the qualification criterion for bacterial vegetative cells, a log-kill value ≥4.00 was used as the qualification criterion for fungi, and a log-kill value ≥3.00 was used as the preliminary evaluation criterion for spores. For the carrier method, sterility of the entire carrier was used as the basis for sterilization determination. I. Dispersion Stability Test:

[0055] The samples were placed at 25℃ in the dark, and the particle size D50, PDI, Zeta potential, appearance, and sedimentation rate were measured at 0h and 14d. The results are shown in Table 3.

[0056] Table 3. Dispersion stability test results II. Quantitative Killing Experiment with Suspension:

[0057] The test was conducted at 20℃ for a specified time, and the results are shown in Table 4.

[0058] Table 4 Results of quantitative killing test of suspension (log kill value) III. Quantitative Killing Experiment of Carrier:

[0059] Bacillus subtilis var. niger spores were contaminated on the surfaces of stainless steel sheets, PTFE sheets, and silica sheets. After drying, the sheets were placed in working solution and treated at 20°C for 3 h and 5 h. The results are shown in Table 5.

[0060] Table 5. Results of the quantitative killing test on the carrier (log kill value) IV. Simulated on-site sterilization experiment of medical devices:

[0061] Stainless steel hinged instruments, PTFE lumens, and silicone lumens were selected. Bacillus subtilis var. niger spores were contaminated in key areas, dried, and then immersed in the working solution for treatment. The minimum effective sterilization time specified in the product manual of this invention is 10 hours; the 5-hour data in this experiment is for stringent condition evaluation (used to compare the sterilization safety margin of each formulation) and is not considered a recommended usage time. The results are shown in Table 6.

[0062] Table 6 Results of Simulated On-Site Sterilization Tests for Medical Devices

[0063] As shown in Table 6, under the 10-hour sterilization time specified in the product instructions, no positive carriers were detected in Example 1 and Comparative Examples 1, 2, and 3 (containing glutaraldehyde), meeting the sterilization criteria under the experimental conditions. Comparative Example 4 (without glutaraldehyde) still showed positive carriers under the 10-hour condition, failing to meet the sterilization criteria. Furthermore, in the stringent condition evaluation with a 5-hour shortened reaction time, Example 1 had 0 / 100 positive carriers, while Comparative Examples 1, 2, and 3 showed different numbers of positive carriers, indicating that the formulation of this invention has a higher sterilization safety margin in complex instrument surfaces and lumen simulation scenarios. The 5-hour data is only used for comparing stringent conditions between different formulations and is not considered a recommended usage time; the recommended minimum effective reaction time for sterilization treatment in this invention remains 10 hours or an effective reaction time determined through product validation. V. Anti-biofilm auxiliary performance test:

[0064] A 24-hour biofilm model of *Pseudomonas aeruginosa* was established on the inner wall of a stainless steel sheet and a PTFE tube. The samples were then immersed in the test samples for 30 minutes (to reduce viable cell count) and 60 minutes (to determine residual biofilm using the crystal violet method). The results are shown in Table 7.

[0065] Table 7 Results of Anti-biofilm Auxiliary Performance Tests VI. Effects of Organic Interfering Substances on Experiments:

[0066] According to the "Disinfection Technical Specifications", three conditions were set to conduct a spore-killing test on stainless steel sheets (3h). The results are shown in Table 8.

[0067] Table 8. Results of the experiment on the effects of organic interfering substances (log kill value) VII. Continuous Use Stability Test:

[0068] The activated working solution from Example 1 was placed in a covered sterilization tank and used continuously at room temperature to simulate continuous use. Various indicators were tested, and the results are shown in Table 9.

[0069] Table 9 Results of continuous use stability test

[0070] Note: The 5-hour stringent condition evaluation results were defined as follows: after immersion in a simulated carrier stainless steel sheet contaminated with Bacillus subtilis spores for 5 hours, it was transferred to a culture medium and incubated at 37°C for 7 days. No viable bacteria growth was observed on any of the carriers, and the result was considered sterile. This evaluation is used to compare the performance changes of the working solution during continuous use and is not intended as a recommended sterilization time. Based on the data in Table 9, the effective usage period of the sterilization working solution of this invention is limited to within 10 days after activation. 8. Metal corrosion resistance and material compatibility testing:

[0071] Each material was immersed in the working solution of Example 1 for 72 hours, and the results are shown in Table 10.

[0072] Table 10 Material compatibility test results IX. Rinsing Residue Test:

[0073] After each carrier was immersed in the working solution of Example 1, it was rinsed 3 and 5 times with sterile water, and the residue on the instrument surface was tested. The results are shown in Table 11.

[0074] Table 11 Results of rinsing residue test.

[0075]

[0076] With increasing flushing cycles, both glutaraldehyde and zinc residues showed a decreasing trend. For luminal instruments, it is recommended to flush thoroughly at least 5 times to significantly reduce residues. Application example:

[0077] (1) High-level disinfection: Immerse the thoroughly cleaned medical device completely in the sterilization working solution obtained in Example 1; for tubular devices, ensure the working solution completely fills the lumen and removes all air bubbles. Allow the device to act at room temperature for the validated minimum effective time before removing it. The high-level disinfection time should be based on product validation results and the instructions for use; for example, it may be validated to be 45–60 minutes.

[0078] (2) Sterilization treatment: Immerse the thoroughly cleaned instruments completely in the sterilization working solution; for tubular instruments, the lumen should be completely filled with the working solution. Remove the instruments after 10 hours at room temperature or after the effective sterilization time determined by product validation.

[0079] (3) Post-treatment: After high-level disinfection or sterilization, the instrument surface and lumen should be thoroughly rinsed with sterile water. For lumen instruments, it is recommended to rinse at least 5 times to reduce glutaraldehyde and nano-component residues. After rinsing, dry and store according to the instrument manufacturer's instructions.

[0080] (4) Boundaries of use: When the glutaraldehyde concentration of the working solution is below 2.0%, the activated solution has been used for more than 10 days, the pH exceeds the range of 7.5 to 8.5, or obvious sedimentation, stratification, or flocculation appears, it shall not continue to be used as a sterilization working solution.

[0081] (5) Material compatibility: Before use, the scope of application should be confirmed in accordance with the instructions of the medical device manufacturer and in conjunction with material compatibility verification; for devices containing aluminum, copper, silicone, polycarbonate or other sensitive materials, a special compatibility verification should be performed before use. in conclusion:

[0082] This invention provides a glutaraldehyde sterilizing agent for medical devices containing an organic-inorganic composite antibacterial agent. The system uses glutaraldehyde as the main effective sterilizing component and the organic-inorganic composite antibacterial agent as a dispersion stabilizing and interfacial adsorption auxiliary component. Epoxy groups are introduced onto the surface of nano-zinc oxide via an epoxy-silane coupling agent, and further, a nucleophilic ring-opening etherification reaction occurs with the hydroxyl-containing polyquaternary ammonium salt antibacterial agent, allowing the polyquaternary ammonium salt antibacterial segments to be stably grafted onto the surface of the nano-zinc oxide, thereby improving the dispersion stability and wash retention of the nano-component in the glutaraldehyde aqueous system.

[0083] Experimental results show that, compared with physically mixed systems, the organic-inorganic composite antibacterial agent of this invention maintains a high quaternary ammonium salt structure retention rate after multiple washes and exhibits a higher safety margin for killing bacteria under stringent evaluation scenarios such as complex instrument surfaces, lumen inner walls, biofilm residues, and spore contamination. This sterilizing agent can be used for high-level disinfection or sterilization of endoscopes, lumen instruments, precision instruments, and other heat-sensitive medical devices. In practical use, the validated minimum effective contact time, effective shelf life, minimum effective concentration of glutaraldehyde, and material compatibility results should be used as the boundaries of application.

Claims

1. A sterilizing disinfectant, characterized in that, By mass percentage, it includes the following components: Glutaraldehyde 2.0%–2.5%; Organic-inorganic composite antibacterial agent: 0.02%–0.15%; Rust inhibitor 0.05%~0.30%; Complexing stabilizer 0.01%–0.10%; pH adjuster or buffer 0.05%–0.50%; The remainder is water; The pH of the sterilizing disinfectant is 7.5–8.5; The organic-inorganic composite antibacterial agent comprises an inorganic nano-zinc oxide core and an organic quaternary ammonium salt antibacterial chain segment grafted onto the surface of the nano-zinc oxide via covalent bonds. The covalent bonds are formed by a nucleophilic ring-opening etherification reaction between the hydroxyl groups in the hydroxyl-containing polyquaternary ammonium salt antibacterial agent and the epoxy groups on the surface of the epoxy-oxidized nano-zinc oxide.

2. The sterilizing disinfectant according to claim 1, characterized in that, The hydroxyl-containing polyquaternary ammonium salt antibacterial agent is a water-soluble or water-dispersible quaternary ammonium salt antibacterial agent containing at least two quaternary ammonium salt groups, at least two hydroxyl groups, and C8-C16 hydrophobic alkyl segments in its molecule.

3. The sterilizing disinfectant according to claim 1, characterized in that, The surface-epoxylated nano-zinc oxide is obtained by surface modification of nano-zinc oxide with an epoxy-silane coupling agent; the epoxy-silane coupling agent is selected from one or two of γ-glycidyl etheroxypropyltrimethoxysilane and γ-glycidyl etheroxypropyltriethoxysilane; the mass ratio of the epoxy-silane coupling agent to nano-zinc oxide is 0.04 to 0.10:

1.

4. The sterilizing disinfectant according to claim 1, characterized in that, The average particle size of the nano zinc oxide is 20-100 nm; the average particle size D50 of the organic-inorganic composite antibacterial agent measured at 25°C, with a dispersion concentration of 0.1 wt% in deionized water and a pH of 7.0-8.5 is 80-300 nm, and the Zeta potential is +15 mV to +45 mV.

5. The sterilizing disinfectant according to claim 1, characterized in that: The mass percentage of glutaraldehyde is 2.1% to 2.4%; The organic-inorganic composite antibacterial agent has a mass percentage of 0.05% to 0.12%. The rust inhibitor is selected from one or more of sodium nitrite, sodium molybdate, sodium benzoate, borate, and phosphate. The complexing stabilizer is selected from one or more of disodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate, sodium citrate, and sodium gluconate. The pH adjuster or buffer is selected from one or more of sodium bicarbonate, sodium carbonate, borate buffer, and phosphate buffer.

6. The sterilizing disinfectant according to claim 1, characterized in that, The hydroxyl-containing polyquaternary ammonium salt antibacterial agent is a polyquaternary ammonium salt antibacterial agent containing four quaternary ammonium groups. Its structure is as follows: with 1,3-propanediamine as the central linking unit, its two nitrogen atoms are respectively linked to a C6 alkyl chain and a C12 alkyl chain containing terminal hydroxyl groups, and further undergoes a quaternization reaction with (3-bromopropyl)trimethylammonium bromide to form a polyquaternary ammonium salt antibacterial agent containing 4 quaternary ammonium salt groups and 2 terminal hydroxyl groups in the molecule.

7. The method for preparing the sterilizing disinfectant according to any one of claims 1 to 6, characterized in that, include: A. Prepare a glutaraldehyde base solution containing glutaraldehyde and water; B. Prepare an activating component containing an organic-inorganic composite antibacterial agent, rust inhibitor, complexing stabilizer, and pH adjuster or buffer; C. Before use, mix the glutaraldehyde base solution with the activating components in a certain proportion to obtain a sterilization working solution with a pH of 7.5-8.5 and a glutaraldehyde mass percentage of 2.0%-2.5%.

8. The preparation method according to claim 7, characterized in that, The preparation steps of the organic-inorganic composite antibacterial agent are as follows: S1. Disperse nano zinc oxide in an aqueous ethanol solution, adjust the pH to 4.0-6.0, add an epoxy silane coupling agent, reflux at 50-90℃ for 2-8 hours, separate, wash, and dry to obtain surface epoxy-modified nano zinc oxide; S2. Dissolve or disperse the hydroxyl-containing polyquaternary ammonium salt antibacterial agent in a solvent, add the surface-epoxylated nano-zinc oxide obtained in step S1, and react at 60-90°C for 4-12 hours under alkaline catalysis, so that the hydroxyl groups in the hydroxyl-containing polyquaternary ammonium salt antibacterial agent undergo a nucleophilic ring-opening etherification reaction with the epoxy groups on the surface of the surface-epoxylated nano-zinc oxide; the mass ratio of the hydroxyl-containing polyquaternary ammonium salt antibacterial agent to the surface-epoxylated nano-zinc oxide is 0.3-1:1; S3. After the reaction is complete, the mixture is centrifuged, washed until neutral, dispersed or dried to obtain an organic-inorganic composite antibacterial agent.

9. The application of the sterilizing disinfectant according to any one of claims 1 to 6 in high-level disinfection or sterilization of medical devices, characterized in that, The medical devices mentioned are heat-sensitive medical devices, endoscopic devices, tubular devices, precision devices, or devices made of polymer materials; When in use, the instrument is completely immersed in the sterilizing working solution, and the lumen is filled with the sterilizing working solution; High-level disinfection action time shall be performed in accordance with the minimum effective action time verified or the time specified in the product instructions, and sterilization action time shall be 10 hours or the verified effective sterilization action time; When the glutaraldehyde concentration in the working solution is below 2.0%, or when it has been used for more than 10 days after activation, or when obvious sedimentation or stratification appears, it must not be used as a sterilization working solution.

10. The application of the sterilizing disinfectant according to any one of claims 1 to 6 in treating solid-liquid interface contamination of medical devices, characterized in that, The solid-liquid interface contamination includes Pseudomonas aeruginosa biofilm contamination, bacterial spore contamination, or a combination thereof. The application involves immersing a cleaned, contaminated instrument carrier in the sterilization working solution to reduce the level of microbial contamination on the instrument surface, the inner wall of the lumen, or complex structural parts.

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