A protease-containing cleaning composition for electro-spinning cup cleaning and use thereof

Abstract

The application discloses a protease-containing cleaning agent composition for electrotransfection cup cleaning and application thereof, and the protease-containing cleaning agent composition comprises polyethylene glycol p-isooctylphenyl ether, protease K, CaCl2, Tris-HCl and a solvent, wherein the volume concentration percentage of the polyethylene glycol p-isooctylphenyl ether in the cleaning agent composition is 0.1%-2.0%, the concentration of the protease K is 0.05-0.5 mg / mL, the concentration of the Tris-HCl is 10-100 mmol / L, and the concentration of the CaCl2 is 1-10 mmol / L. The protease-containing cleaning agent composition can effectively remove cell fragments, DNA-protein complexes and free nucleic acids remaining in the inner cavity of the electrotransfection cup; the aluminum electrode is not corroded, and the surface of the electrode remains smooth and flat after multiple cycles; there is no cytotoxicity residue, and the cell survival rate and the transfection efficiency of the electrotransfection cup after cleaning used in subsequent electrotransfection experiments have no significant difference with those of a new electrotransfection cup.

Description

Technical Field

[0001] This invention relates to the field of cleaning agents, and more specifically to a protease-containing cleaning agent composition for cleaning electric rotary cups and its application. Background Technology

[0002] The electroporation cup is a core consumable in electroporation transfection experiments. Its inner cavity contains parallel aluminum electrode plates (typically spaced 1 mm, 2 mm, or 4 mm apart). During electroporation, a high-voltage pulse (usually 100-300 V) is applied to the cell suspension, causing reversible micropores to form instantaneously in the cell membrane, allowing exogenous nucleic acids (such as plasmid DNA) to enter the cell. After each electroporation, a large amount of cell debris, DNA-protein complexes, and free nucleic acids remain in the inner cavity of the electroporation cup. If these residues are not thoroughly removed, they will affect the uniformity of the electric field and transfection efficiency in subsequent electroporations, and may cause cross-contamination.

[0003] Currently, manufacturers (such as Bio-Rad and Thermo Fisher) design electroporation cuvettes as disposable consumables and do not recommend reuse (Thermo Fisher's official statement: "washing cannot guarantee removal of all foreign DNA, and cuvettes cannot be easily sterilized"). However, due to the relatively high unit price of electroporation cuvettes (approximately ¥20-50 each), reusing cuvettes in large-scale electroporation experiments is a common cost-control method in laboratories. Several cleaning methods have been reported, summarized by technical approach as follows:

[0004] (1) Sodium hypochlorite (bleach) cleaning method

[0005] After use, the electroporation cup is immersed in a sodium hypochlorite solution containing 0.5%–10% available chlorine for several minutes, followed by rinsing with tap water, deionized water, and 75% ethanol in sequence, and then air-dried in a sterile environment. This protocol is documented in a public protocol published by Chang Bioscience / Science Gateway. Its principle is to utilize the strong oxidizing properties of sodium hypochlorite to degrade nucleic acids and proteins. The disadvantage of this protocol is that residual oxidizing substances (active chlorine) are difficult to completely remove with subsequent ethanol and water rinsing, and residual bleaching agent can cause significant toxicity to cells during subsequent electroporation, leading to a substantial reduction in cell viability (less than 60%).

[0006] (2) Alkaline SDS cleaning method

[0007] The electroporation cup was immersed in a mixed solution containing 0.2 M NaOH and 1% SDS and incubated at room temperature for 15 min, followed by rinsing with pure water, dehydration with ethanol, and air drying. This procedure is a commonly used laboratory method; the alkaline environment denatures proteins and denatures and dissociates DNA, while SDS provides solubilization. However, the strongly alkaline NaOH solution reacts with the aluminum electrode (2Al + 2NaOH + 6H₂O → 2Na[Al(OH)₄] + 3H₂↑), gradually dissolving the electrode surface and causing pitting corrosion. After the electrode surface is roughened, the electric field distribution becomes uneven, making it prone to arcing during electroporation, significantly shortening the effective number of cycles of the electroporation cup, which should not exceed 6 cycles.

[0008] (3) Washing with PBS or pure water

[0009] The electrode is repeatedly rinsed with PBS buffer or ultrapure water, followed by sterilization with 75% ethanol and air-drying. This method is relatively safe, but the cleaning power of PBS / ultrapure water is limited and cannot effectively remove flocculent protein-DNA complexes and membrane lipid residues adhering to the electrode surface.

[0010] (4) Dilute hydrochloric acid cleaning method

[0011] The method involves immersing the electroporation vessel in dilute HCl to degrade DNA and proteins. However, due to the vigorous reaction between aluminum and hydrochloric acid (2Al + 6HCl → 2AlCl3 + 3H2↑), this method is highly corrosive to aluminum electrodes and is explicitly prohibited in publicly available laboratory guidelines.

[0012] (5) Long-term immersion method in 75% ethanol

[0013] After pouring 95% or 75% ethanol into the electroporation vessel and letting it stand for 24 hours, it is then vacuum dried or air-dried. This method only serves to disinfect and dehydrate, and its effectiveness in removing cell debris, protein, and nucleic acid residues is limited. The performance of the electroporation vessel is not fully restored after cleaning.

[0014] In summary, existing methods cannot simultaneously achieve both "efficient removal of protein / nucleic acid residues" and "protection of aluminum electrodes from corrosion / residue of cytotoxic substances." Strong cleaning agents (alkalis, oxidants, acids) inevitably lead to electrode corrosion or cytotoxicity; weak cleaning agents (PBS, ethanol) cannot completely remove contaminants. Therefore, there is an urgent need for a cleaning solution that can effectively remove protein and nucleic acid residues from the inner cavity of the electroporation cup while being non-corrosive and non-cytotoxic to the aluminum electrode. Summary of the Invention

[0015] The purpose of this invention is to provide a protease-containing cleaning agent composition for cleaning electroporation cups and its application. This composition effectively removes residual cell debris, DNA-protein complexes, and free nucleic acids from the inner cavity of the electroporation cup; it does not corrode aluminum electrodes, and the electrode surface remains smooth and flat after multiple cycles of use; it leaves no cytotoxic residue; when the cleaned electroporation cup is used in subsequent electroporation experiments, the cell viability and transfection efficiency are not significantly different from those of a new electroporation cup; it allows the electroporation cup to be stably reused more than 10 times, and the transfection efficiency (EGFP positivity rate) remains above 95% after each cleaning.

[0016] In one aspect of the invention, a protease-containing cleaning composition for cleaning electroporation cups is provided. According to embodiments of the invention, the composition comprises polyethylene glycol p-isooctylphenyl ether (Triton X-100), proteinase K, CaCl2, Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride, and a solvent. In the cleaning composition, the volume concentration percentage of polyethylene glycol p-isooctylphenyl ether is 0.1%-2.0%; the concentration of proteinase K is 0.05-0.5 mg / mL, with a specific activity ≥30 U / mg; the concentration of Tris-HCl is 10-100 mmol / L; and the concentration of CaCl2 is 1-10 mmol / L.

[0017] Triton X-100 molecules contain a p-tert-octylphenyl hydrophobic head and a hydrophilic polyoxyethylene chain composed of approximately 9–10 ethoxy units. It is a nonionic surfactant with a critical micelle concentration (CMC) of approximately 0.015% (w / v). At a working concentration of 0.5%, well above the CMC, Triton X-100 efficiently solubilizes phospholipids and membrane proteins in the cell membrane phospholipid bilayer, gently exfoliating cell membrane debris from the electrode surface and dispersing it in solution. Compared to anionic surfactants (such as SDS), Triton X-100 is uncharged and does not cause significant protein denaturation through strong charge interactions. Therefore, it has no inhibitory effect on the catalytic activity of proteinase K, which is beneficial for maintaining high enzyme activity in the washing system. Furthermore, the low-foaming nature of Triton X-100 allows for a more thorough rinsing step after washing, reducing the risk of cytotoxicity caused by surfactant residue. The preferred concentration range for Triton X-100 is 0.1%–2.0% (v / v). When the concentration is too low (< 0.1%), the ability to solubilize membrane fragments is insufficient; when the concentration is too high (> 2.0%), it increases the burden of subsequent rinsing and the marginal benefit decreases.

[0018] Proteinase K is a serine endopeptide isolated from *Tritirachium album*, with a relative molecular weight of approximately 28.9 kDa and an optimal pH of 7.5–12.0. It specifically hydrolyzes peptide bonds in proteins, degrading large proteins and protein components in DNA-protein complexes into smaller peptides or amino acids, thus eliminating their adhesiveness. Proteinase K exhibits broad substrate specificity and maintains high activity in the presence of nonionic surfactants, making it particularly suitable for co-processing with Triton X-100. Compared to anionic surfactants (such as SDS), proteinase K retains or even slightly enhances its catalytic activity in the presence of the nonionic surfactant Triton X-100, making it an ideal enzyme for co-processing with Triton X-100.

[0019] In the cleaning system of this invention, the protein components in the residue from the electroporation process specifically hydrolyzed by proteinase K are short peptides, which disintegrate the protein-dependent DNA-protein complexes "anchored" to the electrode surface. The free DNA, now free of protein binding, is effectively removed in the subsequent water rinsing step.

[0020] The preferred concentration range for proteinase K is 0.05–0.5 mg / mL. Too low an enzyme concentration results in insufficient protein hydrolysis; too high a concentration increases costs without significantly improving cleaning effectiveness.

[0021] Tris-HCl serves as a buffer system to maintain the optimal pH range for proteinase K; CaCl2 contains Ca... 2+ It is an essential cofactor for the activity of proteinase K, through interaction with Ca in the enzyme molecule. 2+ Binding sites (Ca1 and Ca2) maintain the enzyme's thermal stability and conformational integrity.

[0022] The synergistic effect of Triton X-100 and proteinase K: Triton X-100 first gently solubilizes membrane debris, assisting in the exposure of proteins and DNA-protein complexes, without inhibiting proteinase K activity; proteinase K efficiently hydrolyzes protein components, causing the DNA-protein complexes to disintegrate, and the DNA, having lost its protein "anchor," is eluted along with the washing buffer. The entire system is carried out in mild Tris-HCl buffer (pH 8.0), supplemented with Ca2+. 2+ As a cofactor for proteinase K activity, it does not chemically corrode aluminum electrodes, and the residues are not cytotoxic.

[0023] In addition, a protease-containing cleaning agent composition for cleaning electroporation cups according to the above embodiments of the present invention may also have the following additional technical features:

[0024] In some embodiments of the present invention, the solvent is ultrapure water.

[0025] In some embodiments of the present invention, the pH of the Tris-HCl is 7.5-9.0. This pH is within the optimal activity range of proteinase K (pH 7.5-12.0) and is chemically inert to the aluminum electrode.

[0026] In another aspect of the present invention, the present invention provides a method for preparing a protease-containing cleaning agent composition for cleaning electroporation cups. According to an embodiment of the present invention, the method includes the following steps: dissolving Triton X-100 in ultrapure water by stirring; adding Tris-HCl and CaCl2, stirring evenly, finally adding proteinase K, adjusting the volume with solvent, then filtering with a filter membrane for sterilization, and storing in the dark at 2-6°C.

[0027] In another aspect, the present invention provides a method for cleaning an electrostatic precipitator. According to an embodiment of the present invention, the method includes the following steps:

[0028] (1) Inject the protease-containing cleaning agent composition for cleaning electric rotating cup into the electric rotating cup to be cleaned, and after repeated blowing and agitation, incubate at 25-45°C for 10-30 min;

[0029] (2) Discard the cleaning solution and rinse repeatedly with ultrapure water 3-7 times;

[0030] (3) After disinfection by soaking in 60%-75% (volume percentage) ethanol, rinse with ultrapure water and air dry.

[0031] In addition, the cleaning method for an electric rotary cup according to the above embodiments of the present invention may also have the following additional technical features:

[0032] In some embodiments of the present invention, the electric transfer cup after drying in step (3) is sterilized by ultraviolet irradiation.

[0033] In some embodiments of the present invention, the electro-spinning cup is an aluminum embedded electrode electro-spinning cup.

[0034] Compared with the prior art, the beneficial effects of the present invention are:

[0035] (1) High cleaning efficiency and stable transfection efficiency

[0036] After being cleaned with the cleaning agent of this invention, the fluorescence positivity rate of the electroporation cuvettes containing EGFP plasmids after electroporation into mouse embryonic stem cells (mESCs) remained above 95% (mean 95.2%–96.8%) in 10 consecutive "contamination-cleaning-electroplation" cycles, showing no significant difference from that of fresh electroporation cuvettes (which typically have a positivity rate of approximately 96%–98%). In contrast, the fluorescence rate of the PBS-washed group dropped below 80% in the first round.

[0037] (2) It does not corrode aluminum electrodes, significantly extending the service life of the electric rotary cup.

[0038] The alkaline SDS method results in electrode pitting corrosion in the 3rd-5th rounds due to the chemical reaction between NaOH and aluminum, and the arc discharge frequency becomes unusable around the 6th round. The cleaning agent system of this invention has a slightly alkaline pH (pH 8.0) and its components are chemically inert to aluminum. After accelerated corrosion experiments (20-day continuous immersion), no obvious corrosion was observed on the electrode surface by scanning electron microscopy, and the corrosion current density measured by electrochemical Tafel testing showed no significant difference compared to the pure water control group.

[0039] (3) No cytotoxicity

[0040] The sodium hypochlorite method results in a significant decrease in cell viability (< 60%) after electroporation due to residual oxidizing chlorine. The Triton X-100 and proteinase K in the cleaning agent of this invention are standard reagents used in biochemistry laboratories, and no toxic residues were observed after five rinses with pure water and immersion in ethanol. In the cyclic experiments, the cell viability remained > 85%, showing no significant difference compared to the control group with the new electroporation cup.

[0041] (4) The cleaning mechanism is clear, and the two components have a synergistic effect.

[0042] Unlike existing methods that rely solely on a single chemical action (oxidation, acid-base, dissolution), this invention utilizes the synergistic effect of the mild physical solubilization of nonionic surfactants and the specific catalytic hydrolysis of biological enzymes: Triton X-100 gently solubilizes membrane fragments without inhibiting proteinase K activity; proteinase K efficiently hydrolyzes proteins, causing DNA-protein complexes to disintegrate. These two mechanisms are mutually dependent and synergistic, achieving a cleaning effect that is "1+1>2".

[0043] (5) Easy to operate and low cost

[0044] All components of the cleaning agent are commonly used reagents in molecular biology laboratories, making preparation simple and the cost per cleaning cycle extremely low (approximately ¥0.2 / cycle). The entire cleaning process takes about 1 hour (including incubation and drying) and requires no special equipment. Attached Figure Description

[0045] Figure 1 This is a cell state diagram of day 1 after the first round of electroporation in Application Example 1 of this invention;

[0046] Figure 2 This is a cell state diagram of day 1 after the fifth round of electroporation in Application Example 1 of this invention;

[0047] Figure 3 This is a cell state diagram of day 1 after the tenth round of electroporation in Application Example 1 of this invention;

[0048] Figure 4 This is a diagram of the cell state on day 1 after the first round of electroporation in Comparative Example 4 of this invention;

[0049] Figure 5 This is a diagram of the cell state on day 1 after the fifth round of electroporation in Comparative Example 4 of this invention;

[0050] Figure 6 This is a diagram of the cell state on day 1 after the tenth round of electroporation in Comparative Example 4 of this invention;

[0051] Figure 7 This is a diagram of the cell state on day 1 after the third round of electroporation in Comparative Example 2 of this invention;

[0052] Figure 8 This is a diagram of the state of day1 cells after the first round of electroporation in Comparative Example 3 of this invention. Detailed Implementation

[0053] 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, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0054] Example 1

[0055] A protease-containing cleaning agent composition for cleaning electroporation cups comprises Triton X-100, proteinase K, Tris-HCl (pH 8.0), and CaCl2, wherein the final concentration of Triton X-100 is 0.5% (v / v), the final concentration of proteinase K is 0.1 mg / mL, the final concentration of CaCl2 is 5 mM, and then the volume is adjusted to 100 mL with ultrapure water.

[0056] A method for preparing a protease-containing cleaning agent composition for electroporation cup cleaning includes the following steps:

[0057] Dissolve 0.5 mL of Triton X-100 in 80 mL of ultrapure water by stirring; add 5 mL of 1 M Tris-HCl (pH 8.0) and 0.5 mL of 1 M CaCl2, and stir until homogeneous; add 10 mg of proteinase K, and bring the volume to 100 mL with ultrapure water; filter through a 0.22 μm filter membrane for sterilization; store at 4°C protected from light.

[0058] Example 2

[0059] A protease-containing cleaning agent composition for cleaning electroporation cups comprises Triton X-100, proteinase K, Tris-HCl (pH 8.0), and CaCl2. The final concentration of Triton X-100 is 0.1% (v / v), the final concentration of proteinase K is 0.05 mg / mL, the final concentration of Tris-HCl (pH 8.0) is 50 mM, and the final concentration of CaCl2 is 5 mM. The mixture is then brought to a final volume of 100 mL using ultrapure water.

[0060] A method for preparing a protease-containing cleaning agent composition for cleaning electric rotary cups is the same as the preparation method in Example 1, except that the amount of raw materials is different.

[0061] Example 3

[0062] A protease-containing cleaning agent composition for cleaning electroporation cups comprises Triton X-100, proteinase K, Tris-HCl (pH 8.0), and CaCl2. The final concentration of Triton X-100 is 0.5% (v / v), the final concentration of proteinase K is 0.2 mg / mL, the final concentration of Tris-HCl (pH 8.0) is 50 mM, and the final concentration of CaCl2 is 5 mM. The mixture is then brought to a final volume of 100 mL using ultrapure water.

[0063] A method for preparing a protease-containing cleaning agent composition for cleaning electric rotary cups is the same as the preparation method in Example 1, except that the amount of raw materials is different.

[0064] Example 4

[0065] A protease-containing cleaning agent composition for cleaning electroporation cups comprises Triton X-100, proteinase K, Tris-HCl (pH 8.0), and CaCl2. The final concentration of Triton X-100 is 2% (v / v), the final concentration of proteinase K is 0.5 mg / mL, the final concentration of Tris-HCl (pH 8.0) is 50 mM, and the final concentration of CaCl2 is 5 mM. The mixture is then brought to a final volume of 100 mL using ultrapure water.

[0066] A method for preparing a protease-containing cleaning agent composition for cleaning electric rotary cups is the same as the preparation method in Example 1, except that the amount of raw materials is different.

[0067] Application Example 1

[0068] A method for cleaning an electric rotary cup includes the following steps:

[0069] 1. Experimental materials

[0070] Electro-spinning cup: Fisherbrand FB102, 2 mm gap, aluminum embedded electrode.

[0071] mESC cell suspension: C57BL / 6N mouse embryonic stem cells (mESC), 1 × 10⁻⁶ 7 cells / mL.

[0072] Plasmid: pMAX-EGFP.

[0073] Electroporation apparatus: Bio-Rad Gene Pulser Xcell (parameters: 250 V / 500 μF).

[0074] Detection: Flow cytometry (FITC channel, EGFP positivity rate detected 48 h after electroporation) was performed using a BD FACSCanto / Beckman CytoFLEX flow cytometer.

[0075] 2. Establishment of Standard Pollution Model

[0076] Take a brand new electroporation cuvette, add 400 μL of mESC cell suspension to each cuvette, and perform electroporation according to the parameters mentioned above. After electroporation, aspirate the cell suspension; visible flocculent cell debris remains in the inner cavity of the cuvette. Allow to air dry at room temperature for 30 min to obtain a standardized contaminated electroporation cuvette.

[0077] 3. Cleaning process

[0078] (1) The protease-containing cleaning agent composition for cleaning the electroporation cup prepared in Example 1 was injected into the electroporation cup and mixed by blowing, and then incubated at 37°C for 15 min;

[0079] (2) Discard the cleaning agent composition in the electric rotating cup, and then repeatedly blow the electric rotating cup with ultrapure water 5 times;

[0080] (3) Soak the electric rotary cup in 75% ethanol for 30 minutes, discard the ethanol, and then rinse with pure water;

[0081] (4) After the electric transfer cup is placed in a fume hood to dry, it is sterilized by ultraviolet irradiation to ensure that the electric transfer cup is in a sterile and dry state before the next use.

[0082] 4. Cyclic Experiment Procedure

[0083] Take 6 brand new electroporation cuvettes (labeled R01-R06), establish a standard contamination model according to the above method → ​​clean according to the standard cleaning procedure in step (3) → electroporate EGFP plasmid into mESC → detect the EGFP positivity rate by flow cytometry 48 h after electroporation → establish the contamination model again → repeat the above steps for a total of 10 cycles. Record the mean ± standard deviation of the fluorescence positivity rate of the 6 electroporation cuvettes in each cycle.

[0084] The results of the cyclic experiment are as follows:

[0085] Table 1 Results of Cyclic Experiment

[0086] Loop count EGFP positivity rate (Mean ± SD, %) 1 96.8 ± 1.2 2 96.5 ± 1.5 3 96.2 ± 1.8 4 96.0 ± 1.6 5 95.8 ± 1.4 6 95.5 ± 1.7 7 95.3 ± 1.5 8 95.1 ± 1.9 9 95.6 ± 1.3 10 95.2 ± 1.6

[0087] As shown in Table 1, the EGFP positivity rate remained above 95% after 10 cycles, showing no significant difference from the new electroporation cup.

[0088] Comparative Example 1

[0089] A method for cleaning an electric rotary cup includes the following steps:

[0090] (1) Prepare a cleaning agent by mixing NaOH and SDS (sodium dodecyl sulfate), wherein the final concentration of NaOH is 0.2 mol / L and the final concentration (mass / volume percentage (w / v)) of SDS is 1%;

[0091] (2) Soak the contaminated electric rotary cup from Application Example 1 in the cleaning agent for 15 minutes, and then rinse it 5 times with pure water;

[0092] (4) Soak the electric rotary cup in 75% ethanol for 30 minutes, discard the ethanol, and then rinse with pure water.

[0093] (5) After the electric transfer cup is placed in a fume hood to dry, it is sterilized by ultraviolet irradiation to ensure that the electric transfer cup is in a sterile and dry state before the next use.

[0094] Comparative Example 2

[0095] A method for cleaning an electric rotary cup includes the following steps:

[0096] (1) Use 0.5% (mass percentage) available chlorine NaClO solution as cleaning agent;

[0097] (2) Soak the contaminated electric rotary cup from Application Example 1 in the cleaning agent for 15 minutes, and then rinse it 5 times with pure water;

[0098] (4) Soak the electric rotary cup in 75% ethanol for 30 minutes, discard the ethanol, and then rinse with pure water.

[0099] (5) After the electric transfer cup is placed in a fume hood to dry, it is sterilized by ultraviolet irradiation to ensure that the electric transfer cup is in a sterile and dry state before the next use.

[0100] Comparative Example 3

[0101] A method for cleaning an electric rotary cup includes the following steps:

[0102] (1) 1×PBS (phosphate-buffered saline, pH 7.4) solution was used as the cleaning agent;

[0103] (2) Use the cleaning agent to repeatedly blow on the contaminated electric rotary cup in Case 1 10 times, and then rinse it 5 times with pure water;

[0104] (4) Soak the electric rotary cup in 75% ethanol for 30 minutes, discard the ethanol, and then rinse with pure water.

[0105] (5) After the electric transfer cup is placed in a fume hood to dry, it is sterilized by ultraviolet irradiation to ensure that the electric transfer cup is in a sterile and dry state before the next use.

[0106] Comparative Example 4

[0107] Subsequent experiments were conducted using brand new, unused electric rotary cups without adding any cleaning agents.

[0108] Application Example 1 and Comparative Examples 1-4 were subjected to a "contamination-cleaning-electroplating" cyclic experiment, with 6 repeats per group (n = 6). After each cycle, the following two methods were used for testing:

[0109] (1) Flow cytometry detection of EGFP positivity: 48 h after electroporation, cells from each group were collected, digested with trypsin, washed with PBS, and then analyzed for EGFP fluorescence positivity using a flow cytometer (BD FACSCanto, FITC channel). 10,000 cells were analyzed per group per test. A negative control gate was established using untransfected cells. EGFP positivity rate = number of fluorescently positive cells / total number of cells × 100%.

[0110] (2) Cell viability counting using a hemocytometer: 24 h after electroporation, cell suspensions from each group were collected, stained with trypan blue, and the number of live and dead cells were counted using a hemocytometer. Cell viability = number of live cells / (number of live cells + number of dead cells) × 100%. Each group had 3 technical replicates, and the mean value was taken.

[0111] The results of application example 1 are shown in the table below:

[0112] Table 2. Results of the cyclic experiment in Application Example 1

[0113] Cycle round EGFP positivity rate (Mean ± SD, %) Cell viability (Mean ± SD, %) Electrode surface state 1 96.8 ± 1.2 89.2 ± 3.1 The electrode surface is smooth and free of visible corrosion. 3 96.2 ± 1.8 88.5 ± 2.9 The electrode surface is smooth and free of visible corrosion. 5 95.8 ± 1.4 87.5 ± 2.8 The electrode surface is smooth and free of visible corrosion. 7 95.3 ± 1.5 87.1 ± 3.2 The electrode surface is smooth and free of visible corrosion. 10 95.2 ± 1.6 86.8 ± 3.5 The electrode surface is smooth and free of visible corrosion.

[0114] As shown in Table 2, in Application Example 1, during 10 cycles of "contamination-washing-electroporation," the EGFP positivity rate consistently remained between 95.2% and 96.8%, showing no significant difference compared to Comparative Example 4 (P > 0.05, t-test). Cell viability consistently remained between 86.8% and 89.2%. Figure 1 , Figure 2 and Figure 3The results show the cell survival of day 1 after the first, fifth, and tenth rounds of electroporation in Example 1, compared with Comparative Example 4. Figure 4 , 5 6) No significant difference. After 10 cycles, the electrode surface remained smooth and flat to the naked eye and under magnification, without pitting, discoloration or roughening.

[0115] The results of Comparative Example 1 are shown in the table below:

[0116] Table 3 Results of the cyclic test in Comparative Example 1

[0117] Cycle round EGFP positivity rate (Mean ± SD, %) Cell viability (Mean ± SD, %) Electrode surface state 1 94.5 ± 2.1 85.0 ± 3.2 The electrode surface is smooth and free of visible corrosion. 3 88.3 ± 3.5 82.1 ± 3.8 Slight pitting appeared on the electrode surface 5 79.6 ± 4.2 78.5 ± 4.5 Increased pitting and surface roughening 6 — — Frequent arcing led to the termination of the experiment.

[0118] As shown in Table 3, the EGFP positivity rate of Comparative Example 1 was close to that of Application Example 1 (94.5%) in the first round. However, with the increase of the number of cycles, the corrosion of the aluminum electrode by NaOH gradually accumulated. From the third round onwards, visible pitting appeared on the electrode surface, the electric field uniformity decreased, and the EGFP positivity rate dropped to 88.3%. In the fifth round, the pitting further intensified, the electrode surface became rough, and the EGFP positivity rate dropped to 79.6%. In the sixth round of electro-rotation, the arcing frequency increased significantly, the voltage became unstable, and the experiment was terminated.

[0119] The results of Comparative Example 2 are shown in the table below:

[0120] Table 4. Results of the cyclic test in Comparative Example 2

[0121] Cycle round EGFP positivity rate (Mean ± SD, %) Cell viability (Mean ± SD, %) Electrode surface state 1 93.2 ± 2.8 72.5 ± 5.1 Smooth electrode surface 2 90.1 ± 3.5 52.3 ± 4.8 Smooth electrode surface 3 — — The experiment was terminated due to the massive death of cells.

[0122] As shown in Table 4, the positive rate of EGFP in the comparative group was maintained in the first and second rounds (93.2% and 90.1%, respectively), but the cell survival rate was significantly lower than that of other groups from the first round (72.5%), and further plummeted to 52.3% in the second round. In the third round, a large number of cells died, such as... Figure 7 As shown, the number of surviving cells was significantly lower than in other groups. This is because sodium hypochlorite is a strong oxidizing agent; despite rinsing with pure water and soaking in ethanol, active chlorine remained in the micropores of the electrode surface, which was released during subsequent electroporation and became toxic to the cells. In the third round, the cell viability was below 40%, with a large number of cells dying, making it impossible to obtain effective transfection data, and the experiment was terminated.

[0123] The results of Comparative Example 3 are shown in the table below:

[0124] Table 5. Results of the cyclic test in Comparative Example 3

[0125] Cycle round EGFP positivity rate (Mean ± SD, %) Cell viability (Mean ± SD, %) Electrode surface state 1 78.5 ± 4.3 86.0 ± 2.5 Flocculent residues are visible on the electrode surface 2 65.2 ± 5.1 84.8 ± 3.0 Increased residue 3 48.7 ± 6.3 83.5 ± 3.2 Obvious residue, unstable voltage 4 — — arcing terminates

[0126] As shown in Table 5, Comparative Example 3, despite repeated washing with PBS, failed to effectively remove the protein-DNA complex adhering to the electrode surface. The EGFP positivity rate dropped to 78.5% in the first round, significantly lower than that of Application Example 1 (96.8%). With increasing cycle count, residues accumulated, resulting in noticeable flocculent residues on the electrode surface and severe interference with the electric field distribution. The EGFP positivity rate decreased to 65.2% in the second round and further to 48.7% in the third round, with unstable voltage and increased arcing frequency during electroporation. The experiment was terminated in the fourth round due to the inability to complete electroporation via arcing. Notably, the cell viability rate of Comparative Example 3 (83.5%–86.0%) was similar to that of Application Example 1. Figure 8 As shown, this indicates that PBS itself is not cytotoxic; its defect lies purely in the electric field interference caused by insufficient washing ability.

[0127] The results of Comparative Example 4 are shown in the table below:

[0128] Table 6. Results of the cyclic experiment in Comparative Example 4

[0129] Cycle round EGFP positivity rate (Mean ± SD, %) Cell viability (Mean ± SD, %) Electrode surface state 1 97.5 ± 1.1 92.0 ± 1.8 Brand new electro-rotor, with smooth electrodes. 5 96.8 ± 1.3 91.5 ± 2.0 Brand new electro-rotor, with smooth electrodes. 10 97.2 ± 1.0 91.8 ± 1.9 Brand new electro-rotor, with smooth electrodes.

[0130] As shown in Table 6, in Comparative Example 4, brand new electroporation cups were used in each round without a washing step, serving as a positive control. The EGFP positivity rate remained between 96.8% and 97.5%, and the cell viability remained between 91.5% and 92.0%, serving as the evaluation benchmark for the washing effect of the cleaning agent of this invention. In Application Example 1, there were no significant differences in EGFP positivity rate and cell viability between each round and Group E (P > 0.05), indicating that the electroporation cups washed with the cleaning agent of this invention were comparable to the new electroporation cups in terms of transfection performance and cell safety.

[0131] The following table compares the test results of Comparative Examples 1-4 and Application Example 1:

[0132] Table 7. Comparison of test results between Comparative Examples 1-4 and Application Example 1

[0133] Comparison Dimensions Application Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Transfection efficiency after 10 cycles > 95% Round 6 terminated Poor cell condition, with a large number of cells dying. < 80% (First Round) > 96% (benchmark) Cell viability > 85% medium < 40% > 85% > 90% (benchmark) Number of times to reuse ≥ 10 ≤ 6 ≤ 2 (Valid) ≤ 3 1 Single cost ~¥0.2 ~¥0.1 ~¥0.05 ~¥0.02 ¥20–50 Cleaning mechanism Mild solubilization + enzymatic hydrolysis synergistic effect Strong base denaturation + solubilization Strong oxidative degradation Physical rinsing —

[0134] Based on the above experimental results, the cleaning agent of the present invention (Application Example 1) is significantly superior to existing technologies (Comparative Examples 1-3) in terms of EGFP positivity rate, cell viability, and electrode surface integrity, and shows no significant difference compared to the new electroporation cup control group (Comparative Example 4). Hemocytometer counting results show that the cell viability rate in Application Example 1 remained above 85% in each round, with no cytotoxic residue.

[0135] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the present invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A protease-containing cleaning agent composition for cleaning electroporation cups, characterized in that: The cleaning agent composition includes polyethylene glycol p-isooctylphenyl ether, proteinase K, CaCl2, Tris-HCl, and a solvent. In this composition, the volume concentration percentage of polyethylene glycol p-isooctylphenyl ether is 0.1%-2.0%, the concentration of proteinase K is 0.05-0.5 mg / mL, the concentration of Tris-HCl is 10-100 mmol / L, and the concentration of CaCl2 is 1-10 mmol / L.

2. The protease-containing cleaning agent composition for cleaning electroporation cups according to claim 1, characterized in that: The solvent is ultrapure water.

3. The protease-containing cleaning agent composition for cleaning electroporation cups according to claim 1, characterized in that: The pH of the Tris-HCl is 7.5-9.

0.

4. A method for preparing a protease-containing cleaning agent composition for electroporation cup cleaning according to any one of claims 1-3, characterized in that, Includes the following steps: Dissolve Triton X-100 in ultrapure water by stirring; add Tris-HCl and CaCl2, stir until homogeneous, then add proteinase K, bring the solvent to a final volume, filter through a membrane for sterilization, and store at 2-6°C in the dark.

5. A method for cleaning an electric rotary cup, characterized in that, Includes the following steps: (1) Inject the protease-containing cleaning agent composition for cleaning electric rotating cups according to any one of claims 1-3 into the electric rotating cup to be cleaned, and incubate at 25-45°C for 10-30 min after repeated blowing and agitation. (2) Discard the cleaning solution and rinse repeatedly with ultrapure water 3-7 times; (3) After disinfection by soaking in 60%-75% ethanol, rinse with ultrapure water and air dry.

6. The cleaning method for an electro-rotating cup according to claim 5, characterized in that: After drying in step (3), the electric rotary cup is sterilized by ultraviolet irradiation.

7. The cleaning method for an electro-rotating cup according to claim 5, characterized in that: The electro-rotating cup is an aluminum embedded electrode electro-rotating cup.

Images