Water matrix standard substance degerming process based on photocatalysis

Abstract

The invention discloses a water matrix standard substance degerming process based on photocatalysis, and relates to the technical field of water purification, and the process comprises the following steps: introducing a response surface optimization algorithm and a non-dominated sorting genetic algorithm, and constructing a target component retention rate-microbial inhibition rate dual-objective function model; a self-constructed photocatalytic reaction module is adopted to carry out lossless and efficient sterilization treatment on the working solution under a visible light driving condition; carrying out physical and chemical stability adjustment and sterilization effect verification on the water matrix standard substance finished product solution; after the photocatalytic degerming and sterilizing effect verification is completed, sub-packaging into standard containers under the sterile condition; and carrying out principal component structural integrity verification, purity evaluation and principal component content determination on the final finished product, carrying out magnitude assignment by adopting a weighted average method, and finally outputting a quality control analysis report. The method is suitable for sterilization treatment and mass assignment whole-process control of the water-based standard substance.

Description

Technical Field

[0001] This invention relates to the field of water purification technology, and in particular to a photocatalytic water matrix standard sterilization process. Background Technology

[0002] With the increasing demand for water-soluble reference materials in fields such as biomedicine, food safety, and environmental monitoring, the preparation of high-purity, sterile, and structurally stable aqueous matrix reference materials has become a key technical challenge. Aqueous matrix reference materials are typically composed of small organic molecules such as amino acids, sugars, and cyclamate. They require accurate concentrations of the target component in the solution, stable physicochemical properties, and compliance with microbial limit standards to ensure repeatability and accuracy in analytical testing.

[0003] However, existing sterilization methods generally rely on high-temperature and high-pressure sterilization, ultraviolet irradiation, or the addition of chemical disinfectants. While these methods effectively inactivate microorganisms, they often damage the structure of key functional groups in the standard, leading to degradation, oxidation, or isomerization of the main components. This affects the authenticity and traceability of the standard, limiting its application in high-precision fields. Furthermore, many processes lack effective structural verification, component detection, and metrological assignment procedures before and after sterilization, making it difficult to ensure the consistency of the final product's quality and the reliability of its numerical values.

[0004] To address the aforementioned issues, this invention provides a photocatalytic water matrix standard sterilization process. It constructs a thin-layer flow photocatalytic module with controllable light intensity and reaction space, and combines it with real-time microbial detection, principal component structure spectral verification, content determination, and value assignment to form a set of sterilization and quality control methods suitable for water-soluble sensitive components. This ensures sterilization effectiveness while preventing target degradation, achieving comprehensive quality management of standard substances from preparation, sterilization, dispensing to value assignment. Summary of the Invention

[0005] To address the above problems, this invention provides a photocatalytic water matrix standard sterilization process, which solves the problems in the prior art where water matrix standards are easily damaged by high temperature and strong oxidation during sterilization, making it difficult to achieve efficient and non-destructive sterilization and quality traceability.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a photocatalytic water matrix standard sterilization process, comprising the following steps:

[0007] Step S1 involves sterilizing and inhibiting the water matrix standard preparation system throughout the entire process. A response surface optimization algorithm and a non-dominated sorting genetic algorithm are introduced to construct a dual objective function model, obtain the optimal volume fraction of the organic antibacterial solvent, and finally obtain the working solution of the water matrix standard.

[0008] Step S1 also includes the following sub-steps:

[0009] S1-1, glass containers and ultrapure water used for preparing standard solutions are placed in a vertical steam autoclave and treated for 30 minutes at a temperature of 121℃ and a pressure of 0.103MPa. After sterilization, the glass containers are cooled and immediately transferred to a Class 100 laminar flow clean bench and irradiated with a low-pressure ultraviolet lamp with a wavelength of 254nm for 30 minutes for secondary irradiation sterilization.

[0010] S1-2: Transfer the glass container and ultrapure water processed in S1-1 to a pre-sterilized glove box. Weigh the pure component of the water matrix standard and transfer it to a glass container pre-cooled to below 25°C. Add sterile water and stir magnetically at 300 rpm for 15–30 minutes until it is completely dissolved into a homogeneous and transparent solution to obtain the mother liquor of the standard to be sterilized.

[0011] S1-3, a response surface methodology algorithm combined with a non-dominated sorting genetic algorithm is introduced to construct a dual objective function model of "target component retention rate – microbial inhibition rate" to quantitatively calculate the optimal volume fraction of the organic antibacterial solvent. The specific method is as follows:

[0012] Using the volume fraction of the organic antibacterial solvent as the independent variable, and the principal component retention rate R1(v) of the sample after continuous storage at 25℃ for 7 days and the inhibition rate of Escherichia coli and yeast growth at the corresponding concentration R2(v) as dual response variables, experiments were conducted under multiple v-value conditions to establish a response surface regression model. Subsequently, a Pareto front calculation was performed on the above dual-objective response system using a non-dominated sorting genetic algorithm to obtain the volume fraction v* that yields the optimal compromise solution between R1(v) and R2(v). Based on this, the optimal volume fraction of the organic antibacterial solvent was determined, and then the organic antibacterial solvent was slowly added dropwise to the mother liquor of the standard to be sterilized to obtain the working solution of the water matrix standard.

[0013] Step S2: Based on the working solution of the water matrix standard, a self-constructed photocatalytic reaction module is used to perform non-destructive and efficient sterilization treatment on the working solution under visible light driving conditions. After filtration through a terminal filter membrane, a high-purity water matrix standard finished solution is obtained.

[0014] Step S2 also includes the following sub-steps:

[0015] S2-1, Constructing a photocatalytic reaction module. The core structure includes an immobilized photocatalytic sheet assembly, a planar reaction chamber, and a wavelength-tunable LED light source array. The photocatalytic sheet assembly uses quartz glass as the substrate material, uniformly coated with PAgT-type nano-TiO2 catalytic slurry, and after natural drying, it is fixed in a special slot using inert, highly transparent silicone adhesive. Subsequently, the photocatalytic sheet is installed on the upper part of the reaction chamber, forming the main channel of the module. The chamber adopts a planar thin-layer design, with a tunable wavelength LED light source array embedded at the top.

[0016] S2-2, the aqueous matrix standard working solution is saturated with dissolved oxygen and then injected into the photocatalytic reaction module at a constant linear velocity. It flows through the reaction chamber equipped with a photocatalytic sheet, forming a thin-layer flow state, and the catalytic surface is irradiated with an LED light source with a wavelength of 365–420 nm. The reaction flow rate and liquid level height are controlled by a voltage stabilizing constant flow device and a liquid level sensing device, and finally a preliminarily sterilized aqueous matrix standard reaction solution is obtained.

[0017] S2-3, an online microbial counting system is installed at the liquid outlet of the photocatalytic module to dynamically monitor the residual microbial activity in the aqueous matrix standard reaction solution. The online microbial counting system uses both fluorescence ATP assay and flow cytometry. The online microbial counting system samples every 5 minutes and compares the results with a preset sterilization threshold. When the detection results show that the number of microorganisms has decreased to 10 compared to the initial load, the system is considered complete. -6 Once the sterilization endpoint is reached, the online microbial counting system automatically shuts off the light and cuts off the input of the water matrix standard reaction solution.

[0018] The water matrix standard reaction solution that has reached the sterilization endpoint is introduced into a filtration device equipped with a PTFE terminal filter membrane with a pore size of 0.22μm. It is then subjected to one-time aseptic filtration through a constant pressure system to remove any possible residual particles of the same size, and finally produce the finished water matrix standard solution.

[0019] Step S3: Perform physicochemical and microbiological tests on the high-purity water matrix standard solution to determine whether the sterilization effect of the high-purity water matrix standard solution meets the standard.

[0020] Step S3 also includes the following sub-steps:

[0021] S3-1, Perform physicochemical property testing on the finished solution of the water matrix standard to check whether its pH value remains within the target range set during the original preparation. Then, based on the set antibacterial solvent replenishment strategy, determine whether antibacterial adjuvants need to be added after sterilization.

[0022] S3-2. Microbial testing is performed on the finished solution of the water matrix standard after the physicochemical properties have been tested. Under aseptic conditions, 1.0 mL of solution sample is collected and inoculated onto the surface of a nutrient agar plate. The plate is incubated at 37°C for 48 hours. After the incubation, observe whether colonies have formed on the plate surface. If colonies are observed during the test, the possible causes need to be analyzed, and the sterilization process parameters need to be adjusted and corrected in the next batch.

[0023] Step S4: After verifying the photocatalytic sterilization and disinfection effects, the high-purity water matrix standard solution is dispensed into standardized containers, sealed and stored by melting and sealing, and the sealing quality and airtightness are checked to obtain the final product.

[0024] Step S4 also includes the following sub-steps:

[0025] S4-1, using chemically stable and high-temperature resistant glass ampoules as finished product containers, the glass ampoules are rinsed multiple times with ultrapure water to remove particles and leachates; after washing, they are dried at a low temperature of 60°C in a clean environment, and then the glass ampoules are placed in a muffle furnace for dry heat sterilization at a temperature of 160°C for 2 hours.

[0026] After microbial testing, the aqueous matrix standard solution is introduced into a sterile operating table. Using a pipette, the aqueous matrix standard solution is injected into each glass ampoule according to the set dosage. If the standard is a homogeneous system, it is directly dispensed in equal volumes. If there are different specific gravities or incompatible components, it is necessary to prepare one ampoule at a time before dispensing, i.e., prepare and seal immediately.

[0027] After dispensing, immediately perform flame sealing. Place the narrow neck of the glass ampoule in an alcohol lamp and heat and rotate it to melt the glass evenly and form a dense and smooth seal. Before sealing, slowly fill the ampoule with nitrogen to replace any residual air. After sealing, use sterile forceps to transfer the glass ampoule to an ampoule holder to cool.

[0028] S4-2, the cooled glass ampoules are sequentially inspected for sealing quality and tightness. Visual inspection is performed to check whether the seal is complete, whether the ampoules are uniform and full, and whether there are cracks, bubbles, or suspicious micropores. Randomly sampled glass ampoules are placed in a vacuum negative pressure device and immersed in a colored indicator liquid to observe whether there is gas leakage and liquid seepage, which serves as the leakage test standard.

[0029] After sampling and testing, standard labels are printed in batches using label printing equipment. The labels include: the name of the standard, batch number, preparation date, concentration parameters, and expiration date. The labels must be affixed to the unsealed part of the glass ampoule to obtain the final product.

[0030] Step S5 involves verifying the structural integrity of the main components, assessing their purity, and determining their content in the final product ampoules. The weighted average method is used to assign values, and a quality control analysis report is output.

[0031] Step S5 also includes the following sub-steps:

[0032] S5-1, the integrity of the main component structure of the final product is verified. Fourier transform infrared spectroscopy is used to identify its characteristic absorption peaks, confirm that the key functional groups have not undergone structural changes, and compare it with the reference standard sample spectrum before sterilization. Then, gas chromatography-mass spectrometry is used to detect the fragmentation characteristic peaks of the main component, match the mass spectrometry database, and determine whether its molecular skeleton is intact.

[0033] S5-2, Purity assessment and main component content determination: Under the premise of confirming that the structure has not changed, quantitative analysis of the standard components is performed to construct the content information of the main components. The purity of the main components is determined by the "impurity deduction method". After the calculation is completed, the impurities are deducted to obtain the true purity of the main components. The mass of the main components in each final product is calculated in combination with the dispensing volume of the glass ampoule.

[0034] S5-3, Randomly select 3-5 final products from the same batch for parallel testing, determine the concentration of the main component, and calculate the relative standard deviation.

[0035] Subsequently, based on the content data obtained by the analytical method calibrated by metrological traceability, the weighted average method was used to assign the values; at the same time, the expanded uncertainty of the standard was calculated according to the impurity deduction ratio and the error range of the analytical instrument; after the assignment was completed, a quality control analysis report was generated, including the main component name, content value, purity, expanded uncertainty, analytical method and metrological traceability path information.

[0036] Compared with the prior art, the beneficial effects of the present invention are:

[0037] This invention constructs a photocatalytic reaction module with thin-layer flow characteristics and visible light response capability, replacing traditional sterilization methods under high temperature, high pressure, or strong oxidation environments. This achieves non-destructive and efficient inactivation of microorganisms in water-based standard materials. In terms of physical structure, the module adopts a combination of a ≤2mm thin-layer reaction chamber, an immobilized nano-TiO2 catalyst sheet, and a wavelength-tunable LED light source. This not only improves the light energy utilization rate of the catalytic interface, but also significantly enhances the uniformity and repeatability of the sterilization process by precisely controlling the flow rate, illuminance, and dissolved oxygen conditions. It avoids common problems such as bubble blockage and overexposure, ensuring that the target components are not structurally damaged during the sterilization process.

[0038] This invention adds a real-time online microbial detection and endpoint determination module after sterilization. Based on the dual technologies of ATP bioluminescence and flow cytometry, it determines the sterilization endpoint in a coordinated manner, which has high sensitivity and high throughput. This allows the sterilization degree of each batch of standard materials to be quantified and controlled. It also has automatic interception and terminal filter membrane safety protection measures to effectively prevent secondary contamination and improve the intelligence and closed-loop control level of the sterilization process.

[0039] This invention establishes a complete process for principal component structure verification, content determination, and titration assignment. Infrared spectroscopy, mass spectrometry, and nuclear magnetic resonance are used to confirm that the principal component structure remains unchanged. Impurities are screened using HPLC quantitative analysis, moisture titration, and ICP-MS multidimensional detection. The purity of the principal component is calculated using the mass balance method, enabling accurate titration of standard substances and traceability of the SI system. This solves problems such as opaque titration values ​​and large batch-to-batch variations in existing processes, significantly improving the product's application value in quality control, regulations, and standard setting. Attached Figure Description

[0040] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0041] Figure 1 This is a process flow diagram of the present invention. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but is merely a selection of embodiments of the present invention.

[0043] Please refer to Figure 1 , Figure 1 This is a flow chart of a photocatalytic water matrix standard sterilization process provided by an embodiment of the present invention, including the following steps:

[0044] Step S1 involves sterilizing and inhibiting the water matrix standard preparation system throughout the entire process. Response surface methodology and non-dominated sorting genetic algorithm are introduced to construct a dual objective function model of "target component retention rate – microbial inhibition rate" to obtain the optimal volume fraction of the organic antibacterial solvent.

[0045] S1-1, Sterilization of raw materials, solvents, and glassware: To minimize the initial microbial contamination level during preparation, a combined sterilization strategy is implemented for the containers and solvents used to prepare standard solutions. Glass containers, namely volumetric flasks, graduated pipettes, and magnetic stir rods, are placed in a vertical autoclave and treated for 30 minutes at a temperature of 121°C and a pressure of 0.103 MPa to achieve thorough moist heat sterilization. After sterilization, the glass containers are cooled and immediately transferred to a Class 100 laminar flow clean bench, and then irradiated with a low-pressure ultraviolet lamp at a wavelength of 254 nm for 30 minutes for secondary irradiation sterilization to eliminate any possible residual heat-resistant spore-forming microorganisms.

[0046] Meanwhile, the ultrapure water used for preparation, namely high-purity deionized water with a resistivity ≥18.2MΩ·cm, is treated under the same high temperature and high pressure conditions, and then subjected to ultraviolet irradiation for 30 minutes before use to ensure that the ultrapure water itself does not carry any active bacteria.

[0047] S1-2: The standard substance solution is prepared artificially in a low-oxygen sterile environment to avoid structural denaturation of the target compound caused by bacteria or oxidizing gases in the air. The glass container and ultrapure water processed in S1-1 are transferred to a pre-sterilized glove box. The glove box is maintained with a nitrogen atmosphere, and the oxygen volume fraction is controlled at ≤1% and the relative humidity is <10%. Under this environment, the pure component of the water matrix standard (i.e., the target small organic molecule compound) is weighed to an accurate value of 0.1 mg and transferred to a glass container pre-cooled to below 25°C. Then, sterile water is added dropwise to the predetermined volume, and the mixture is magnetically stirred at 300 rpm for 15–30 min until it is completely dissolved into a homogeneous and transparent solution.

[0048] For pH-sensitive substances, such as those containing functional groups like amines or carboxyls, 0.01–0.05 mol·L⁻¹ of phosphate buffer or acetate buffer can be added beforehand to adjust the overall pH value to the target range. After dissolution, the prepared solution should be immediately sealed and stored in a glove box for later use to prevent aerosol particles or other microorganisms from entering the system during temporary storage, thereby obtaining a physicochemically stable and microbial risk-controlled stock solution of the sterilization standard.

[0049] S1-3, optimization of organic antibacterial solvent volume fraction and pH stability control: To improve the resistance of aqueous matrix standards to microbial invasion during storage and transportation, an antibacterial strategy is introduced without affecting the chemical structure of the target components.

[0050] Traditional methods typically rely on experience to set fixed volume fractions of organic antibacterial solvents, but they do not fully consider the structural retention behavior of the target analyte in the presence of the solvent, failing to achieve the optimal balance between different solvent concentrations and their inhibitory effects on microbial growth. Therefore, this invention introduces a response surface methodology (RSM) combined with a non-dominated sorting genetic algorithm (NSGA-II) to construct a dual objective function model of "target component retention rate – microbial inhibition rate," used to quantitatively calculate the optimal volume fraction of the organic antibacterial solvent. The specific method is as follows:

[0051] The volume fraction (v) of the organic antibacterial solvent was used as the independent variable, with a range of [0.1%, 10%]. The retention rate of the principal components after continuous storage at 25℃ for 7 days and the inhibition rate against the growth of *E. coli* and yeast at the corresponding concentrations were used as dual response variables. Experiments were conducted under multiple v-value conditions to establish a response surface regression model. The specific fitting form is as follows:

[0052]

[0053] Where R1(v) represents the retention rate of the principal component, R2(v) represents the antibacterial rate, and v represents the volume fraction of the organic antibacterial solvent. These are the constant term, first-order coefficient, and second-order coefficient of the fitted curve, respectively, which are determined by fitting experimental data.

[0054] Subsequently, the Pareto front calculation was performed on the above dual-objective response system using the non-dominated sorting genetic algorithm (NSGA-II) to obtain the volume fraction v* that achieves the optimal compromise solution between R1(v) and R2(v), that is, the proportion of organic antibacterial solvent added to obtain sufficient antibacterial efficacy without significantly damaging the principal component structure.

[0055] In practice, the optimal volume fraction of the organic antibacterial solvent (methanol or acetonitrile) is determined by the calculated v* as the standard. Then, the organic antibacterial solvent, which has been sterilized by a 0.22μm filter membrane, is slowly added to the mother liquor of the standard to be sterilized. The mixture is then magnetically stirred for 5 minutes to ensure uniform mixing and to obtain the working solution of the water matrix standard.

[0056] If the addition of an organic antibacterial solvent causes the solution pH to deviate from the target range, or if the target substance itself is pH sensitive, then 0.1 mol·L⁻¹ hydrochloric acid or sodium hydroxide should be used for slow titration to adjust the pH value back to the range of 6.0–8.0, further suppressing microbial growth and maintaining solution stability.

[0057] It should be noted that water-based standards refer to standard solutions obtained by artificially dissolving selected water-soluble small organic molecule compounds (such as amino acids, sugars, cyclamate, etc.) in high-purity water at a quantitative concentration. They are suitable as calibration standards or quality control references in analytical testing. They are characterized by structural sensitivity and susceptibility to contamination, and the stability of the components must be strictly ensured during the sterilization process.

[0058] Step S2: Based on the water-based standard working solution, a self-constructed photocatalytic reaction module is used to perform non-destructive and efficient sterilization treatment on the working solution under visible light-driven conditions, ensuring that the microbial contaminants in the solution are completely inactivated and maintaining the structural and concentration stability of the original components to the greatest extent.

[0059] S2-1, a photocatalytic reaction module specifically designed for the sterilization treatment of water-based standards was constructed. Its core structure includes an immobilized photocatalytic sheet assembly, a planar reaction chamber, and a wavelength-tunable LED light source array. The specific construction process is as follows:

[0060] First, a photocatalyst sheet was prepared, using quartz glass as the substrate material, at a density of 0.05 g·cm⁻¹. -2 The PAgT-type nano-TiO2 catalyst slurry is uniformly coated with a mass loading ratio and then naturally dried. It is then fixed in a special slot with inert, highly transparent silicone adhesive to ensure that the thickness of the catalyst sheet does not exceed 1.0 mm, so as to avoid obstructing the liquid flow channel of the reaction chamber.

[0061] The photocatalyst sheet is then installed on the upper part of the reaction chamber, forming the main channel of the module. The chamber adopts a flat, thin-layer design, with the height of each layer controlled to ≤2mm, ensuring that the working liquid forms a uniform thin-layer flow on the surface of the photocatalyst sheet, improving light transmission efficiency and catalytic reaction interface contact rate. A 365–420nm adjustable wavelength LED light source array is embedded at the top, with a luminous flux controlled to ≤10mW·cm². -2 To meet the light requirements under different component stability conditions.

[0062] It should be noted that, to ensure safety and efficiency, the module adopts a fully sealed structure, and all seals are made of inert materials to prevent the migration of organic components or interference from light-induced side reactions. It is also equipped with a reflector to improve light energy utilization and is equipped with leakage testing and light uniformity detection procedures to ensure stable operation of the reaction device and consistent illumination.

[0063] S2-2, Continuous sterilization under visible light-driven operation is performed within the constructed reaction module, as detailed below:

[0064] First, the working solution of the aqueous matrix standard to be treated was saturated with dissolved oxygen. Pure oxygen was injected into the pretreatment chamber, and the dissolved oxygen concentration was increased to ≥6 mg·L using microbubble aeration. -1This ensures the continuous generation of hydroxyl radicals (·OH) and superoxide anions (·O2) during the catalytic reaction. - It contains reactive oxygen species (ROS) to enhance sterilization capabilities.

[0065] Subsequently, the oxygen-saturated working solution was injected into the photocatalytic reaction module at a constant linear velocity, flowing through the reaction chamber equipped with photocatalysts. The flux within the photocatalytic reaction module was controlled at 1-5 mL / min. -1 To ensure the liquid forms a thin-layer flow state within the reaction layer ≤2mm thick, turbulence interference is avoided; and the catalytic surface is irradiated with an LED light source of 365–420nm wavelength, with a light intensity not exceeding 10mW·cm². -2 To simulate the actual sterilization efficiency under visible light conditions;

[0066] To prevent reaction dead zones and catalytic blind spots, the system is equipped with a pressure-stabilizing constant flow unit and a liquid level sensing device to strictly control the reaction flow rate and liquid level. Air bubbles must be prevented from entering during the entire sterilization process to avoid local light obstruction. The total sterilization time is adjusted according to the initial load, solution properties and catalytic efficiency, and is generally controlled within the range of 15–60 minutes. No additional chemical sterilizing agents or heating and pressurization treatment is required, making it particularly suitable for temperature-sensitive or oxidation-sensitive small molecule component systems. After the above treatment, a preliminarily sterilized water matrix standard reaction solution is obtained.

[0067] S2-3, Real-time Sterilization Monitoring and Endpoint Determination: To ensure the photocatalytic sterilization effect meets the standards and avoids damage to the component structure caused by excessive light exposure, an online microbial counting system is installed at the liquid outlet of the photocatalytic module to dynamically monitor the activity of residual microorganisms in the reaction solution. The system comprehensively uses two methods: fluorescence ATP method and flow cytometry. The ATP method is based on the light signal generated by the release of ATP catalyzed by luciferase, and the number of viable bacteria in a unit volume of reaction solution is calculated by converting the signal intensity. Flow cytometry combines staining agents to distinguish between dead and live cells and outputs the percentage of viable bacteria in real time.

[0068] The online microbial counting system samples every 5 minutes and compares the results with a preset sterilization threshold. The system detects microorganisms when the count has decreased to 10 compared to the initial load. -6 Once the sterilization endpoint is reached, the system automatically shuts off the light and cuts off the liquid input to ensure that the components are not damaged by over-exposure.

[0069] Finally, the reaction solution is introduced into a filtration device equipped with a PTFE terminal filter membrane with a pore size of 0.22μm. The solution is then subjected to sterile filtration through a constant pressure system to remove any possible residual particles. If the system operating pressure difference increases, an automatic warning will be issued for filter membrane replacement or channel blockage to ensure the safe operation of the sterilization system. The final product is a water matrix standard solution with stable structure and concentration and no residual active microorganisms.

[0070] S3, Photocatalytic Post-processing and Effect Verification: The physicochemical properties and microbiological properties of the finished solution of the water matrix standard obtained after filtration through the terminal filter membrane are tested to ensure that it meets the requirements for long-term preservation and subsequent analysis.

[0071] S3-1, Physicochemical property verification and stability adjustment: First, the physicochemical properties of the aqueous matrix standard solution are tested to check whether its pH value is maintained within the target range set during the original preparation (e.g., pH 6.0–8.0). If the pH value is slightly off due to by-product reactions or free radical-mediated reactions in the photocatalytic reaction, it should be slowly adjusted by titration with sterilized 0.1 mol·L⁻¹ hydrochloric acid or sodium hydroxide to restore it to the appropriate buffer range, so as to avoid degradation of standard components or changes in biological activity due to pH abnormalities.

[0072] Subsequently, based on the antibacterial solvent replenishment strategy set in steps S1-3, it is determined whether antibacterial adjuvants need to be added after sterilization. If no antibacterial organic solvent was added during the initial preparation, the antibacterial solvent that has been sterilized by a 0.22μm filter membrane should be slowly added dropwise to the liquid according to the volume fraction v* obtained by the response optimization algorithm after sterilization is ensured, and magnetic stirring should be maintained for 5 minutes to ensure that it is fully mixed with the standard substance.

[0073] S3-2, Terminal verification of sterilization effect: To verify the microbial inactivation efficiency of the photocatalytic sterilization process, the finished solution of the water matrix standard after the physicochemical properties have been tested should be subjected to microbial testing. The specific operation is as follows: Under aseptic conditions, 1.0 mL of the sterilized solution sample is collected and inoculated onto the surface of the nutrient agar plate. The plate is incubated at 37℃ for 48 h. After the incubation, observe whether there are colonies forming on the plate surface.

[0074] If no colonies grow on the plate, it means that the treatment process has reduced the number of microorganisms to 10% of the initial value. -6 The following results indicate that the sterilization effect of the batch of standard solution meets the 6log sterilization standard. If colonies are observed during testing, possible causes need to be analyzed, such as insufficient reaction time, low catalyst loading, or air bubbles introducing and obscuring the reaction surface. At the same time, the sterilization process parameters should be adjusted, including extending the light exposure time, increasing the catalyst surface area, optimizing the flow rate and dissolved oxygen concentration, etc., and corrected in the next batch to ensure the adaptability and consistency of the sterilization process under different standard material backgrounds.

[0075] Step S4: After verifying the photocatalytic sterilization and disinfection effects, the high-purity water matrix standard solution is dispensed into standardized containers under aseptic conditions and sealed for airtight preservation.

[0076] S4-1, using chemically stable and high-temperature resistant glass ampoules as finished product containers, the glass ampoules are rinsed multiple times with ultrapure water to remove particles and leachates; after washing, they are dried at a low temperature of 60°C in a clean environment, and then the glass ampoules are placed in a muffle furnace for dry heat sterilization at a temperature of 160°C for 2 hours.

[0077] After microbial testing, the finished solution of the aqueous matrix standard is introduced into a sterile operating table. Using a sterile pipette, the solution is injected into each glass ampoule according to the set dosage, ensuring that the volume of each ampoule is controlled within ±2%. If the standard is a homogeneous system, it can be directly dispensed in equal volumes. If there are different specific gravities or incompatible components, each ampoule must be prepared and sealed immediately before dispensing to ensure that the components are uniform.

[0078] After dispensing, immediately perform flame sealing. Place the narrow neck of the glass ampoule in an alcohol lamp and heat and rotate it to ensure the glass is heated evenly and melted, forming a dense and smooth seal. Before sealing, slowly fill the ampoule with nitrogen to replace any residual air inside. After sealing, use sterile forceps to transfer the glass ampoule to an ampoule rack for cooling to avoid uneven cooling that could damage the ampoule.

[0079] S4-2, Sealing quality inspection and information label recording: The cooled glass ampoules are sequentially inspected for sealing quality and tightness. First, visually inspect whether the seal is complete, whether the ampoules are uniform and full, and whether there are any cracks, bubbles or suspicious micropores. Randomly sample the glass ampoules and place the samples in a vacuum negative pressure device. Immerse them in a colored indicator liquid to observe whether there is gas leakage or liquid seepage, which serves as the leakage test standard.

[0080] After sampling and testing, standard labels are printed in batches using label printing equipment. The labels include information such as the name of the standard, batch number, preparation date, concentration parameters, and expiration date. The labels must be affixed to the non-sealed parts of the glass ampoules to ensure resistance to high humidity and prevent them from falling off. All ampoules are entered into the registration system with batch data simultaneously for tracking and management to obtain the final product.

[0081] Step S5: To ensure that the composition and structure of the standard substances in the final product are not destroyed and that their purity, concentration and uniformity meet the requirements for value transfer, the final product must undergo systematic component confirmation, content determination and uniformity testing, and value assignment for this batch of standard substances must be carried out based on traceability data.

[0082] S5-1, Principal component structure integrity verification: Randomly select samples from the final product and conduct spectral confirmation tests on the principal component structure. Specifically:

[0083] First, Fourier transform infrared spectroscopy (FT-IR) was used to identify characteristic absorption peaks, confirming that key functional groups had not undergone structural changes, and the spectrum was compared with that of a reference standard sample before sterilization to ensure spectral consistency. Then, gas chromatography-mass spectrometry (GC-MS) was used to detect the fragmentation characteristic peaks of the main component, and the results were matched with a mass spectrometry database to determine the integrity of its molecular skeleton. If necessary, further analysis was conducted... 1 H-NMR and 13 C-NMR analysis of the hydrogen and carbon nuclei of the main component confirmed that its chemical structure had not undergone isomerization or degradation.

[0084] If all spectral comparison results show consistency with the original standard and no abnormal signal peaks appear, it can be determined that no impurities were introduced during the photocatalytic sterilization process, nor was the molecular structure of the main component destroyed.

[0085] S5-2, Purity Assessment and Main Component Content Determination: Under the premise of confirming no structural changes, quantitative analysis of the standard components is performed to construct the main component content information. The purity of the main component is determined using the "impurity deduction method," specifically including the following detection dimensions:

[0086] 1. Use high performance liquid chromatography (HPLC) to obtain the peak area of ​​the main component, calculate its concentration quantitatively based on the pre-constructed calibration curve, and check for interference from other impurity peaks;

[0087] 2. The moisture content was determined at the ppm level using Karl Fischer coulometric titration to ensure that no moisture contamination was introduced during the photocatalysis and encapsulation process;

[0088] 3. Analyze possible metallic element impurities in the solution using inductively coupled plasma mass spectrometry (ICP-MS), especially detecting catalyst residues such as titanium and silver, as well as leaching from glass containers.

[0089] After the above test data are calculated for mass balance, the impurities are deducted to obtain the true purity of the main component. The mass or concentration of the main component of each final product is then calculated in combination with the dispensing volume of the glass ampoule.

[0090] S5-3, Homogeneity test and determination of values: Parallel tests are performed on 3-5 randomly selected final products from the same batch. The concentration values ​​of the main components are measured and the relative standard deviation (RSD) is calculated. If the fluctuation range is controlled within the specified uncertainty, the batch of standard material is judged to have good homogeneity of contents.

[0091] Subsequently, based on the content data obtained by the analytical method calibrated by metrological traceability, the weighted average method was used to assign the value; at the same time, the expanded uncertainty of the standard was calculated according to the impurity deduction ratio and the error range of the analytical instrument; if the concentration of the principal component has been cross-validated by multiple methods, the average result can be selected as the recommended value, and the analytical method and uncertainty calculation method should be noted.

[0092] After the assignment is completed, a quality control analysis report is generated, which includes information such as the main component name, content value, purity, expanded uncertainty, analytical method, and metrological traceability path, and serves as the technical documentation accompanying the standard.

[0093] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations will be apparent to those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A photocatalytic water matrix standard sterilization process, characterized in that, Includes the following steps: Step S1 involves sterilizing and inhibiting the water matrix standard preparation system throughout the entire process. A response surface optimization algorithm and a non-dominated sorting genetic algorithm are introduced to construct a dual objective function model, obtain the optimal volume fraction of the organic antibacterial solvent, and finally obtain the working solution of the water matrix standard. Step S2: Based on the working solution of the water matrix standard, a self-constructed photocatalytic reaction module is used to perform non-destructive and efficient sterilization treatment on the working solution under visible light driving conditions. After filtration through a terminal filter membrane, a high-purity water matrix standard finished solution is obtained. Step S3: Perform physicochemical and microbiological tests on the high-purity water matrix standard solution to determine whether the sterilization effect of the high-purity water matrix standard solution meets the standard. Step S4: After verifying the photocatalytic sterilization and disinfection effects, the high-purity water matrix standard solution is dispensed into standardized containers, sealed and stored by melting and sealing, and the sealing quality and airtightness are checked to obtain the final product. Step S5 involves verifying the structural integrity of the main components, assessing their purity, and determining their content in the final product ampoules. The weighted average method is used to assign values, and a quality control analysis report is output.

2. The photocatalytic water matrix standard sterilization process according to claim 1, characterized in that: Step S1 also includes the following sub-steps: S1-1, glass containers and ultrapure water used for preparing standard solutions are placed in a vertical steam autoclave and treated for 30 minutes at a temperature of 121℃ and a pressure of 0.103MPa. After sterilization, the glass containers are cooled and immediately transferred to a Class 100 laminar flow clean bench and irradiated with a low-pressure ultraviolet lamp with a wavelength of 254nm for 30 minutes for secondary irradiation sterilization. S1-2: Transfer the glass container and ultrapure water processed in S1-1 to a pre-sterilized glove box. Weigh the pure component of the water matrix standard and transfer it to a glass container pre-cooled to below 25°C. Add sterile water and stir magnetically at 300 rpm for 15–30 minutes until it is completely dissolved into a homogeneous and transparent solution to obtain the mother liquor of the standard to be sterilized. S1-3, a response surface methodology combined with a non-dominated sorting genetic algorithm is introduced to construct a dual objective function model of "target component retention rate – microbial inhibition rate" to quantitatively calculate the optimal volume fraction of the organic antibacterial solvent. The specific method is as follows: Using the volume fraction of organic antibacterial solvent as the independent variable, and the retention rate of principal component R1(v) and the inhibition rate of Escherichia coli and yeast growth at the corresponding concentrations after the sample was stored at 25℃ for 7 consecutive days as dual response variables, experimental measurements were carried out under multiple v values ​​to establish a response surface regression model. Subsequently, a non-dominated sorting genetic algorithm was used to perform Pareto front calculations on the above dual-objective response system to obtain the volume fraction v* that yields the optimal compromise solution between R1(v) and R2(v); and based on this, the optimal volume fraction of the organic antibacterial solvent was determined. Then, the organic antibacterial solvent was slowly added dropwise to the mother liquor of the standard to be sterilized to obtain the working solution of the water matrix standard.

3. The photocatalytic water matrix standard sterilization process according to claim 1, characterized in that: Step S2 also includes the following sub-steps: S2-1, Constructing a photocatalytic reaction module. The core structure includes an immobilized photocatalytic sheet assembly, a planar reaction chamber, and a wavelength-tunable LED light source array. The photocatalytic sheet assembly uses quartz glass as the substrate material, uniformly coated with PAgT-type nano-TiO2 catalytic slurry, and after natural drying, it is fixed in a special slot using inert, highly transparent silicone adhesive. Subsequently, the photocatalytic sheet is installed on the upper part of the reaction chamber, forming the main channel of the module. The chamber adopts a planar thin-layer design, with a tunable wavelength LED light source array embedded at the top. S2-2, the aqueous matrix standard working solution is saturated with dissolved oxygen and then injected into the photocatalytic reaction module at a constant linear velocity. It flows through the reaction chamber equipped with a photocatalytic sheet, forming a thin-layer flow state, and the catalytic surface is irradiated with an LED light source with a wavelength of 365–420 nm. The reaction flow rate and liquid level height are controlled by a voltage stabilizing constant flow device and a liquid level sensing device, and finally a preliminarily sterilized aqueous matrix standard reaction solution is obtained. S2-3, an online microbial counting system is installed at the liquid outlet of the photocatalytic module to dynamically monitor the residual microbial activity in the aqueous matrix standard reaction solution. The online microbial counting system uses both fluorescence ATP assay and flow cytometry. The online microbial counting system samples every 5 minutes and compares the results with a preset sterilization threshold. When the detection results show that the number of microorganisms has decreased to 10 compared to the initial load, the system is considered complete. -6 Once the sterilization endpoint is reached, the online microbial counting system automatically shuts off the light and cuts off the input of the water matrix standard reaction solution. The water matrix standard reaction solution that has reached the sterilization endpoint is introduced into a filtration device equipped with a PTFE terminal filter membrane with a pore size of 0.22μm. It is then subjected to one-time aseptic filtration through a constant pressure system to remove any possible residual particles of the same size, and finally produce the finished water matrix standard solution.

4. The photocatalytic water matrix standard sterilization process according to claim 1, characterized in that: Step S3 also includes the following sub-steps: S3-1, Perform physicochemical property testing on the finished solution of the water matrix standard to check whether its pH value remains within the target range set during the original preparation. Then, based on the set antibacterial solvent replenishment strategy, determine whether antibacterial adjuvants need to be added after sterilization. S3-2. Microbial testing is performed on the finished solution of the water matrix standard after the physicochemical properties have been tested. Under aseptic conditions, 1.0 mL of solution sample is collected and inoculated onto the surface of a nutrient agar plate. The plate is incubated at 37°C for 48 hours. After the incubation, observe whether colonies have formed on the plate surface. If colonies are observed during the test, the possible causes need to be analyzed, and the sterilization process parameters need to be adjusted and corrected in the next batch.

5. The photocatalytic water matrix standard sterilization process according to claim 1, characterized in that: Step S4 also includes the following sub-steps: S4-1, using chemically stable and high-temperature resistant glass ampoules as finished product containers, the glass ampoules are rinsed multiple times with ultrapure water to remove particles and leachates; after washing, they are dried at a low temperature of 60°C in a clean environment, and then the glass ampoules are placed in a muffle furnace for dry heat sterilization at a temperature of 160°C for 2 hours. After microbial testing, the aqueous matrix standard solution is introduced into a sterile operating table. Using a pipette, the aqueous matrix standard solution is injected into each glass ampoule in sequence according to the set dosage. If the standard composition is a homogeneous system, it is directly dispensed in equal volumes. If there are different specific gravities or incompatible components, it is necessary to prepare one ampoule at a time before dispensing, that is, prepare and seal it immediately. After dispensing, immediately perform flame sealing. Place the narrow neck of the glass ampoule in an alcohol lamp and heat and rotate it to melt the glass evenly and form a dense and smooth seal. Before sealing, slowly fill the ampoule with nitrogen to replace any residual air. After sealing, use sterile forceps to transfer the glass ampoule to an ampoule holder to cool. S4-2, the cooled glass ampoules are sequentially inspected for sealing quality and tightness. Visual inspection is performed to check whether the seal is complete, whether the ampoules are uniform and full, and whether there are cracks, bubbles, or suspicious micropores. Randomly sampled glass ampoules are placed in a vacuum negative pressure device and immersed in a colored indicator liquid to observe whether there is gas leakage and liquid seepage, which serves as the leakage test standard. After sampling and testing, standard labels are printed in batches using label printing equipment. The labels include: the name of the standard, batch number, preparation date, concentration parameters, and expiration date. The labels must be affixed to the unsealed part of the glass ampoule to obtain the final product.

6. The photocatalytic water matrix standard sterilization process according to claim 1, characterized in that: Step S5 also includes the following sub-steps: S5-1 verifies the structural integrity of the principal components of the final product by using Fourier transform infrared spectroscopy to identify its characteristic absorption peaks. We confirmed that the key functional groups had not undergone structural changes and compared them with the chromatograms of the reference standard sample before sterilization. Then, we used gas chromatography-mass spectrometry to detect the fragmentation characteristic peaks of the main component, matched them with the mass spectrometry database, and determined whether its molecular skeleton was intact. S5-2, Purity assessment and determination of main component content: Under the premise of confirming that the structure has not changed, quantitative analysis of the components of the standard is performed to construct the content information of the main component, and the purity of the main component is determined by the "impurity deduction method". After the calculation is completed, the true purity of the main component is obtained by deducting the impurities, and the mass of the main component in each final product is calculated in combination with the dispensing volume of the glass ampoule. S5-3, Randomly select 3-5 final products from the same batch for parallel testing, determine the concentration of the main component, and calculate the relative standard deviation. Subsequently, based on the content data obtained by the analytical method calibrated by metrological traceability, the weighted average method was used to assign the values; at the same time, the expanded uncertainty of the standard was calculated according to the impurity deduction ratio and the error range of the analytical instrument; after the assignment was completed, a quality control analysis report was generated, including the main component name, content value, purity, expanded uncertainty, analytical method and metrological traceability path information.

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