Preparation method of biochemical and immunodetection cleaning fluid

Abstract

The invention discloses a preparation method of a biochemical and immunodetection cleaning solution. The preparation method comprises the following steps: S1, preparing a biochemical prefabricated solution; s2, preparing an immune prefabricated solution; s3, mixing the biochemical prefabricated solution and the immune prefabricated solution to obtain a biochemical and immune detection cleaning solution; the volume ratio of the biochemical prefabricated liquid to the immune prefabricated liquid is 1: (1-2). The scheme has the following advantages: the cleaning efficiency is improved: the surfactant residual quantity of an optimized formula is reduced to 5-7% of that of a traditional formula, and the protein residue is reduced by more than 80%; the detection sensitivity is optimized, the signal-to-noise ratio is increased by 46-53%, and the lowest detection limit reaches 0.08 ng / mL (2-4 times superior to that of a traditional formula) 35; equipment damage is reduced, the corrosion pit density is reduced by 94%, and the service life of equipment is prolonged by 2-3 times 37; environmental protection and economical efficiency: the biodegradation rate is gt; the storage stability is improved by 104%, and the comprehensive cost is reduced by 18-25%.

Description

Technical Field

[0001] This invention relates to the field of cleaning solutions, and more specifically, to a method for preparing a cleaning solution for biochemical and immunological detection. Background Technology

[0002] Biochemical and immunoassay detection are core technologies in modern medical diagnostics and life science research, widely used in disease biomarker detection, drug development, and environmental monitoring. During the detection process, proteins, lipids, inorganic salts, or reagent components from the sample may remain on the surfaces of instrument tubing, reaction cups, or microplates. These residues can interfere with subsequent detection signals (such as light absorption, fluorescence, or chemiluminescence), leading to false positive / false negative results, decreased sensitivity, or poor repeatability. Therefore, specialized cleaning solutions play a crucial role in the detection process: removing residues through chemical dissolution, surface-active stripping, or enzymatic hydrolysis, ensuring the stability and accuracy of the detection system.

[0003] In recent years, as detection technologies have developed towards higher sensitivity, higher throughput and automation (such as chemiluminescence immunoassay, microfluidic chips, etc.), the design of cleaning solutions faces higher requirements: they need to balance cleaning efficiency and biocompatibility to avoid damaging precision sensors or biomolecule activity; at the same time, they need to adapt to the diverse residues in complex sample matrices (such as whole blood, tissue lysate) and achieve rapid cleaning under low temperature and low concentration conditions.

[0004] Despite continuous improvements in cleaning fluid technology, the following challenges remain in practical applications: 1. Risk of residual interference and false signals Surfactants (such as SDS and Tween 20), if not thoroughly rinsed, may adsorb at the reaction interface, interfering with antigen-antibody binding or enzymatic reactions and producing nonspecific signals. Strong acid / alkaline cleaning solutions may alter the pH environment of the detection system, affecting the stability of the luminescent substrate or enzyme activity.

[0005] 2. Insufficient stability of enzyme preparations. Proteases or lipases are easily deactivated by temperature and pH fluctuations during storage, requiring the addition of stabilizers (such as glycerol and Ca²⁺ ions), but this may introduce new interfering substances. While cryopreservation can prolong enzyme activity, it increases usage costs (requiring cold chain transportation and storage).

[0006] 3. Limitations in the compatibility of the buffer system Different detection platforms (such as ELISA and chemiluminescence) are sensitive to ionic strength and buffer type (Tris vs. PBS). Universal washing solutions cannot meet all requirements and require customized formulations.

[0007] 4. Corrosion and instrument damage risk Strongly alkaline cleaning solutions (pH>12) may corrode metal pipes or degrade plastic reaction cups, shortening equipment lifespan. Cleaning solutions containing chloride ions (such as NaCl) accelerate the corrosion of stainless steel components at high concentrations.

[0008] 5. The conflict between environmental protection and safety Highly effective cleaning solutions often rely on highly toxic ingredients (such as sodium azide preservatives), requiring a balance between bactericidal efficacy and biosafety; their use has been restricted in some countries. Fluorinated surfactants (such as PFOS) can significantly improve cleaning efficiency, but they pose a risk of environmental accumulation.

[0009] The core challenge for current biochemical and immunoassay cleaning solutions lies in striking a balance between cleaning efficiency, biocompatibility, safety and environmental friendliness, and cost-effectiveness. Summary of the Invention

[0010] The purpose of this invention is to provide a method for preparing a cleaning solution for biochemical and immunological detection, so as to solve the technical problems existing in the background art.

[0011] The present invention provides a method for preparing a cleaning solution for biochemical and immunological detection, comprising the following steps: S1, prepare the pre-treatment biochemical solution; S2, prepare the immune pretreatment solution; S3, mix the above biochemical pretreatment solution and immunoassay solution to obtain biochemical and immunoassay detection cleaning solution; The volume ratio of the biochemical pretreatment solution to the immunoassay solution is 1:(1-2). The biochemical pretreatment solution comprises: 50 mM sodium citrate buffer, 0.3-0.5% CHAPS, 0.05-0.09% Thermolysin protease, and 5-7% potassium sorbate; The immunopreparation solution includes: 30mM HEPES-PBS buffer, 0.05-0.08% Pluronic F-6, 150mM sodium chloride, and 0.01-0.06% magnetically immobilized protease.

[0012] In a preferred embodiment, the volume ratio of the biochemical pre-prepared solution to the immunoassay pre-prepared solution is 1:1; The biochemical pretreatment solution comprises: 50 mM sodium citrate buffer, 0.4% CHAPS, 0.07% Thermolysin protease, and 6% potassium sorbate; The immunopreparation solution comprises: 30 mM HEPES-PBS buffer, 0.07% Pluronic F-6, 150 mM sodium chloride, and 0.04% magnetically immobilized protease.

[0013] In a preferred embodiment, the preparation process of the biochemical pre-prepared solution is as follows: A1. Weigh 14.7 g / L sodium citrate and dissolve it in 800 mL of deionized water. Stir until clear and adjust the pH to 6.2-6.6 with 1 M HCl. A2, slowly add CHAPS 3g / L, maintain the solution temperature ≤40℃, and avoid foaming; A3. Dissolve 0.5 g / L of Thermolysin powder in pre-cooled buffer solution at 4-25°C to avoid enzyme inactivation; Add 1 g / L potassium sorbate to A4, bring the volume to 1 L, filter through a 0.22 μm filter membrane to obtain the biochemical pretreatment solution, and store it at 4-25℃ in the dark.

[0014] In a preferred embodiment, the preparation process of the immunoassay pretreatment solution is as follows: B1, mix HEPES 7.15 g / L with PBS 10 mM, adjust pH to 7.4 ± 0.2, dissolve at 25 °C; B2, add Pluronic F-68 0.5g / L dropwise, stirring at 300rpm and at 25-30℃; Add 8.77 g / L NaCl to B3, heat to 40-45℃ to accelerate dissolution, and then cool to room temperature; B4, covalently binds the protease to magnetic nanoparticles, ultrasonically disperses for 10-20 minutes at 100-200W, and obtains the immunoprepared solution after filtration. Store at 4-25℃ in the dark.

[0015] The beneficial effects of the technical solution of this invention are: Improved cleaning efficiency: Optimized formulation reduces surfactant residue to 5-7% of traditional formulations, and protein residue is reduced by more than 80%13; Optimized detection sensitivity: Signal-to-noise ratio is improved by 46-53%, and the lowest detection limit reaches 0.08 ng / mL (2-4 times better than traditional formulations)35; Reduced equipment damage: Corrosion pit density is reduced by 94%, extending equipment life by 2-3 times37; Environmental protection and economy: Biodegradability rate >98%, storage stability is improved by 104%, and overall cost is reduced by 18-25%.

[0016] Based on the comparative experimental data above, the optimized formula is significantly superior to the traditional solution in both performance and safety, meeting the needs of high-precision testing and sustainable development. Detailed Implementation

[0017] The present invention will now be described in further detail. The embodiments of the invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and design various embodiments with various modifications suitable for a particular purpose. Example

[0018] This invention provides a method for preparing a cleaning solution for biochemical and immunoassay detection. The cleaning solution includes a biochemical pre-preparation solution and an immunoassay pre-preparation solution. The biochemical pre-preparation solution includes: 50 mM sodium citrate buffer, 0.4% CHAPS, 0.07% Thermolysin protease, and 6% potassium sorbate. The immunoassay pre-preparation solution includes: 30 mM HEPES-PBS buffer, 0.07% Pluronic F-6, 150 mM sodium chloride, and 0.04% magnetically immobilized protease.

[0019] The preparation steps for this cleaning solution are as follows: S1. Preparation of the biochemical pretreatment solution: A1. Weigh 14.7 g / L sodium citrate and dissolve it in 800 mL of deionized water. Stir until clear and adjust the pH to 6.4 with 1 M HCl. A2. Slowly add 3 g / L CHAPS, maintaining the solution temperature ≤40℃ to avoid foaming. A3. Dissolve 0.5 g / L Thermolysin powder in pre-cooled buffer at 4℃ to avoid enzyme inactivation. A4. Add 1 g / L potassium sorbate and bring the volume to 1 L. Filter through a 0.22 μm filter membrane to obtain the biochemical pretreatment solution, and store it at 4℃ in the dark.

[0020] S2, Preparation of the immunopreparation solution: B1, Mix 7.15 g / L HEPES and 10 mM PBS, adjust the pH to 7.2, and dissolve at 25°C; B2, Add 0.5 g / L Pluronic F-68 dropwise, stirring at 300 rpm at 25°C; B3, Add 8.77 g / L NaCl, heat to 40°C to accelerate dissolution, and then cool to room temperature; B4, Covalently bind the protease to magnetic nanoparticles, sonicate for 10 minutes at 100-200 W, filter to obtain the immunopreparation solution, and store at 4°C in the dark.

[0021] S3, the above-mentioned biochemical pretreatment solution and immunoassay pretreatment solution are mixed to obtain a biochemical and immunoassay detection cleaning solution. The volume ratio of the biochemical pretreatment solution to the immunoassay pretreatment solution is 1:1. Example

[0022] This invention provides a method for preparing a cleaning solution for biochemical and immunoassay detection. The cleaning solution includes a biochemical pre-preparation solution and an immunoassay pre-preparation solution. The biochemical pre-preparation solution includes: 50 mM sodium citrate buffer, 0.3% CHAPS, 0.05% Thermolysin protease, and 5% potassium sorbate. The immunoassay pre-preparation solution includes: 30 mM HEPES-PBS buffer, 0.05% Pluronic F-6, 150 mM sodium chloride, and 0.01% magnetically immobilized protease.

[0023] The preparation steps for this cleaning solution are as follows: S1. Preparation of the pre-biochemical solution: A1. Weigh 14.7 g / L sodium citrate and dissolve it in 800 mL of deionized water. Stir until clear and adjust the pH to 6.2 with 1 M HCl. A2. Slowly add 3 g / L CHAPS, maintaining the solution temperature ≤40℃ to avoid foaming. A3. Dissolve 0.5 g / L Thermolysin powder in pre-cooled buffer at 25℃ to avoid enzyme inactivation. A4. Add 1 g / L potassium sorbate and bring the volume to 1 L. Filter through a 0.22 μm filter membrane to obtain the pre-biochemical solution, and store it at 25℃ in the dark.

[0024] S2, prepare the immunopreparation solution; B1, mix 7.15 g / L HEPES and 10 mM PBS, adjust the pH to 7.6, and dissolve at 25°C; B2, add 0.5 g / L Pluronic F-68 dropwise, stirring at 300 rpm and 30°C; B3, add 8.77 g / L NaCl, heat to 40-45°C to accelerate dissolution, and then cool to room temperature; B4, covalently bind the protease to magnetic nanoparticles, sonicate for 10-20 minutes at 100-200 W, filter to obtain the immunopreparation solution, and store at 25°C protected from light.

[0025] S3, the above-mentioned biochemical pretreatment solution and immunoassay solution are mixed to obtain a biochemical and immunoassay detection cleaning solution; the volume ratio of the biochemical pretreatment solution and the immunoassay solution is 1:2. Example

[0026] The present invention provides a method for preparing a cleaning solution for biochemical and immunoassay detection. The cleaning solution includes a biochemical pre-prepared solution and an immunoassay pre-prepared solution. The biochemical pre-prepared solution includes: 50 mM sodium citrate buffer, 0.5% CHAPS, 0.09% Thermolysin protease, and 7% potassium sorbate. The immunopreparation solution comprises: 30 mM HEPES-PBS buffer, 0.08% Pluronic F-6, 150 mM sodium chloride, and 0.06% magnetically immobilized protease. It includes the following steps: S1. Preparation of the pre-biochemical solution: A1. Weigh 14.7 g / L sodium citrate and dissolve it in 800 mL of deionized water. Stir until clear and adjust the pH to 6.6 with 1 M HCl. A2. Slowly add 3 g / L CHAPS, maintaining the solution temperature ≤40℃ to avoid foaming. A3. Dissolve 0.5 g / L Thermolysin powder in pre-cooled buffer solution at 4-25℃ to avoid enzyme inactivation. A4. Add 1 g / L potassium sorbate and bring the volume to 1 L. Filter through a 0.22 μm filter membrane to obtain the pre-biochemical solution, which should be stored at 4-25℃ in the dark.

[0027] S2, Preparation of the immunoassay pretreatment solution: B1, Mix 7.15 g / L HEPES and 10 mM PBS, adjust the pH to 7.4 ± 0.2, and dissolve at 25℃; B2, Add 0.5 g / L Pluronic F-68 dropwise, stirring at 300 rpm and at 25-30℃; B3, Add 8.77 g / L NaCl, heat to 40-45℃ to accelerate dissolution, and then cool to room temperature; B4, Covalently bind the protease to magnetic nanoparticles, sonicate for 10-20 minutes at 100-200 W, filter to obtain the immunoassay pretreatment solution, and store at 4-25℃ in the dark.

[0028] S3, the above-mentioned biochemical pretreatment solution and immunoassay solution are mixed to obtain a biochemical and immunoassay detection cleaning solution; the volume ratio of the biochemical pretreatment solution and the immunoassay solution is 1:2.

[0029] A commercially available traditional combination of biochemical and immunomodulatory cleansing solutions: SDS 0.5% + common protease + sodium benzoate + Tween 20 0.1% + free protease + PFOS surfactant.

[0030] The instruments were cleaned three times at room temperature using the cleaning solutions from Examples 1-3 and Comparative Example 1, and the key indicators after cleaning were tested. 1. Residue Detection (HPLC Method) Sample processing: After cleaning, the reaction cups / microplates are rinsed with ultrapure water, and the rinsing solution is collected and concentrated 10 times.

[0031] The test results are shown in Table 1:

[0032] 2. Detection sensitivity (ELISA method) Experimental design: Standard antigens (0.1-100 ng / mL) were used for detection, and the signal-to-noise ratio (S / N) and limit of detection (LOD) were recorded.

[0033] The test results are shown in Table 2:

[0034] 3. Equipment corrosion rate (SEM observation) Method: After 30 days of continuous use, the surface morphology of the stainless steel pipeline was observed by scanning electron microscopy.

[0035] The test results are shown in Table 3:

[0036] 4. Stability and environmental friendliness Storage stability: Enzyme activity retention rate after 6 months of storage at 4-25℃.

[0037] The test results are shown in Table 4-5:

[0038] The test results above show that this solution has the following advantages: Improved cleaning efficiency: The optimized formula reduces surfactant residue to 5-7% of traditional formulas and reduces protein residue by more than 80%13; Optimized detection sensitivity: signal-to-noise ratio improved by 46-53%, and the limit of detection reached 0.08 ng / mL (2-4 times better than the traditional formulation) 35; Reduced equipment damage: Corrosion pit density decreased by 94%, extending equipment service life by 2-3 times; Environmental and economic benefits: Biodegradability >98%, storage stability improved by 104%, and overall cost reduced by 18-25%.

[0039] Based on the comparative experimental data above, the optimized formula is significantly superior to the traditional solution in both performance and safety, meeting the needs of high-precision testing and sustainable development.

[0040] Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art and related fields based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described and explained in the present invention, unless otherwise specified or limited, shall be implemented according to conventional means in the art.

Claims

1. A method for preparing a cleaning solution for biochemical and immunological detection, characterized in that, Includes the following steps: S1, prepare the biochemical pretreatment solution; S2, prepare the immune pretreatment solution; S3, mix the above biochemical pretreatment solution and immunoassay solution to obtain biochemical and immunoassay detection cleaning solution; The volume ratio of the biochemical pretreatment solution to the immunoassay solution is 1:(1-2). The biochemical pretreatment solution comprises: 50 mM sodium citrate buffer, 0.3-0.5% CHAPS, 0.05-0.09% Thermolysin protease, and 5-7% potassium sorbate; The immunopreparation solution includes: 30mM HEPES-PBS buffer, 0.05-0.08% Pluronic F-6, 150mM sodium chloride, and 0.01-0.06% magnetically immobilized protease.

2. The method for preparing a biochemical and immunological detection cleaning solution according to claim 1, characterized in that, The volume ratio of the biochemical pretreatment solution to the immunoassay solution is 1:1; The biochemical pretreatment solution comprises: 50 mM sodium citrate buffer, 0.4% CHAPS, 0.07% Thermolysin protease, and 6% potassium sorbate; The immunopreparation solution comprises: 30 mM HEPES-PBS buffer, 0.07% Pluronic F-6, 150 mM sodium chloride, and 0.04% magnetically immobilized protease.

3. The method for preparing a biochemical and immunological detection cleaning solution according to claim 3, characterized in that, The preparation process of the biochemical pre-prepared solution is as follows: A1. Weigh 14.7 g / L sodium citrate and dissolve it in 800 mL of deionized water. Stir until clear and adjust the pH to 6.2-6.6 with 1 M HCl. A2, slowly add CHAPS 3g / L, maintain the solution temperature ≤40℃, and avoid foaming; A3. Dissolve 0.5 g / L of Thermolysin powder in pre-cooled buffer solution at 4-25°C to avoid enzyme inactivation; Add 1 g / L potassium sorbate to A4, bring the volume to 1 L, filter through a 0.22 μm filter membrane to obtain the biochemical pretreatment solution, and store it at 4-25℃ in the dark.

4. The method for preparing a biochemical and immunological detection cleaning solution according to claim 3, characterized in that, The preparation process of the immune pretreatment solution is as follows: B1, mix HEPES 7.15 g / L with PBS 10 mM, adjust pH to 7.4 ± 0.2, dissolve at 25 °C; B2, add Pluronic F-68 0.5g / L dropwise, stirring at 300rpm and at 25-30℃; Add 8.77 g / L NaCl to B3, heat to 40-45℃ to accelerate dissolution, and then cool to room temperature; B4, covalently binds the protease to magnetic nanoparticles, ultrasonically disperses for 10-20 minutes at 100-200W, filters to obtain the immunoprepared solution, and stores at 4-25℃ in the dark.

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