A flat bottom culture centrifuge bottle which can be repeatedly sterilized

Abstract

The application relates to a repeatable sterilization flat-bottom culture centrifugal bottle, belonging to the technical field of biological experimental equipment, which comprises a bottle body, the bottom of the bottle body is a flat bottom, the upper portion of the bottle body is provided with a bottle mouth, the bottle mouth is screw-connected with a bottle cap, a centrifugal self-locking sealing structure is arranged between the end surface of the bottle mouth and the bottle cap, a sampling valve is arranged on the side wall of the bottle body, and a dissolved oxygen sensing film is arranged on the lower portion of the bottle body. The bottle body is provided with a self-closing sampling valve integrated on the side wall, is matched with the bottle cap provided with the centrifugal self-locking sealing structure and the replaceable air permeable film, can realize 30 sterilization cycles without leakage and multiple sterile sampling, and the air permeable film can be replaced individually without disassembling the cap.

Description

Technical Field

[0001] This invention relates to a re-sterilizable flat-bottomed culture centrifuge bottle, belonging to the field of biological experimental equipment technology. Background Technology

[0002] In biological experiments, centrifuge flasks are commonly used experimental equipment for culturing microorganisms and subsequent centrifugation. Most existing centrifuge flasks have a conical bottom design. Although the conical bottom is beneficial for collecting centrifuged sediment, the liquid is prone to overflow during shake-flask fermentation, and the bottom of the flask is not stable enough, with a high center of gravity, resulting in a spillage rate of >30% during the shaker stage.

[0003] In addition, most existing bottle caps are made of rigid materials and have only rigid threads for pressure retention. They are prone to loosening and leakage at 15,000 g. Furthermore, existing bottle caps do not have good air permeability, which is not conducive to gas exchange during shake flask fermentation. Some bottle caps have a breathable membrane, but the breathable membrane is generally fixed by heat fusion. If contaminated or damaged, the entire bottle cap is scrapped, resulting in high usage costs.

[0004] Moreover, existing culture centrifuge bottles require opening the cap for sampling, resulting in a 12% contamination rate and a 5% loss of bacterial culture. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a re-sterilizable flat-bottomed culture centrifuge bottle that addresses the above-mentioned shortcomings. The bottle has an integrated self-closing sampling valve on its side wall and is equipped with a cap that has a centrifugation self-locking sealing structure and a replaceable breathable membrane. This allows for 30 sterilization cycles without leakage and multiple sterile samplings. The breathable membrane can be replaced separately without removing the cap.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows: a re-sterilizable flat-bottomed culture centrifuge bottle, comprising a bottle body, the bottom of the bottle body being flat, a bottle mouth being provided at the top of the bottle body, a bottle cap being threadedly connected to the bottle mouth, a centrifugal self-locking sealing structure being provided between the end face of the bottle mouth and the bottle cap, a sampling valve being provided on the side wall of the bottle body, and a dissolved oxygen sensing membrane being provided at the bottom of the bottle body.

[0007] As a further improvement to the above technical solution: The centrifugal self-locking sealing structure includes a TPE microring and several rigid locking plates. The TPE microring is disposed on the top inner side of the bottle cap, and the several rigid locking plates are evenly spaced inside the TPE microring.

[0008] The diameter of the TPE microring is slightly larger than the outer diameter of the bottle opening, and the thickness of the rigid locking plate is 0.2 mm.

[0009] The bottle cap inside the TPE microring is provided with a fan-shaped air hole, and a breathable membrane is detachably connected to the fan-shaped air hole.

[0010] The outer periphery of the fan-shaped air hole is provided with a fan-shaped groove, which is an Ω groove, and a pressure ring is engaged in the groove.

[0011] The breathable membrane is a PTFE breathable membrane with an average pore size of 0.22 μm, and the pressure ring is a PTFE-PP ring.

[0012] The card slot has a depth of 0.5-1.0 mm and a width of 1.0-1.5 mm. The inner wall of the card slot is provided with anti-slip texture with a depth of 0.1-0.3 mm. The card slot and the bottle cap are integrally formed.

[0013] The dissolved oxygen sensing membrane includes a protective membrane, a sensing membrane, and a transition fusion layer co-molded together in sequence. The transition fusion layer is co-injected with the bottle body. The transition fusion layer is a transition region formed by mutual penetration and molecular-level fusion between the molten bottle body and the sensing membrane at the interface during the co-injection process. The sensing membrane is made of Ru(II) complex fluorescent sensitive material, and the protective membrane is made of medical-grade cyclic olefin copolymer.

[0014] The sampling valve is located 10–30 mm from the bottle opening. It is a self-closing sampling valve with a cross-shaped opening in the center. The center of the sampling valve is made of silicone rubber and is integrally molded with the bottle body.

[0015] A method for preparing a re-sterilizable flat-bottomed culture centrifuge flask is as follows: 1. Raw material processing (1) First, dry the medical-grade PC plastic at 115-130℃ for 4 hours to reduce the moisture content to below 0.02% to prevent air bubbles from forming during injection molding; (2) The ruthenium(II) complex powder and COC particles are made into a "masterbatch" together; (3) Vacuum remove air bubbles from medical-grade silicone and cut it into small strips; 2. Use an electric injection molding machine to manufacture the bottle body. (1) Heat the dried PC plastic particles to 280-320℃, extrude them into the mold, and first use an electric injection molding machine to make the bottle shape; (2) Immediately heat the “masterbatch” to 270-300℃, squeeze a small clump on the designated position on the bottle wall to form a dissolved oxygen sensing film with a thickness of 0.1mm; (3) Squeeze out a little more COC transparent plastic to completely wrap the dissolved oxygen sensing membrane to form a protective film; (4) Inspection: The electric injection molding machine is equipped with a laser thickness probe, which measures the dissolved oxygen sensing film thickness every 2 seconds and must be within 0.10±0.01mm; 3. Install the sampling valve – rotating slider mold Transfer the finished bottle to another electric injection molding machine. A small slider extends from the side of the mold. Place the silicone strip on the slider, and the slider closes. After 8 seconds at 170-190℃ and 12MPa, the silicone is vulcanized and fused to the PC bottle wall. The slider is then unscrewed, leaving a cross-shaped slit 0.02-0.07mm wide on the bottle wall. The valve body is then inspected to ensure it is leak-proof. 4. Stress relief Place the bottle in a 130-40℃ hot air oven for 30 minutes to cool slowly. Use a 0.1mm feeler gauge to check the bottom of the bottle to ensure that the flatness is up to standard and that the centrifugation will not cause the bottle to shake. 5. QR code engraving The laser machine burns a Data Matrix QR code onto the bottom of the bottle to a depth of 0.05mm; 6. Two-color injection molding of bottle cap First color: Temperature control: barrel 230-320℃, nozzle 300-320℃, mold 80-110℃; Pressure control: Injection pressure 130-180MPa, holding pressure 40%-60%; Injection speed: 30-100 mm / s; Molding cycle: Inject for 15-30 seconds, cool, rotate 180° to the second color injection station; The groove is made in the bottle cap using a mold; Second color: Immediately inject TPE soft glue and PP hard sheet to make TPE micro rings and rigid locking plates to form a centrifugal self-locking sealing structure; 7. Fabrication of breathable components The pressure ring is made by injection molding medical-grade PC. The PTFE breathable membrane and the pressure ring are bonded together using a hot press at 170-190℃ for 2 seconds and 0.3MPa to form a detachable breathable component.

[0016] The present invention adopts the above technical solution and has the following advantages: 1. A TPE micro-ring and several rigid locking plates are set between the bottle cap and the bottle mouth. When the centrifugal force reaches 5000-15000×g, the rigid locking plates move radially outward under the action of centrifugal force, squeezing the TPE micro-ring to produce elastic deformation, so that the threaded pair gap between the bottle cap and the bottle mouth is further reduced from the initial 0.01-0.05mm, forming dynamic sealing compensation.

[0017] Specific performance characteristics: Sealing reliability: Under centrifugation conditions of 15000×g and 10 minutes, there was no leakage of liquid (including water, cell culture medium, fermentation broth, etc.) inside the bottle, and all sealing parts such as bottle mouth, sampling valve, and vent membrane remained intact; Pressure resistance: It can withstand an internal air pressure of 0.4MPa for 48 hours without the generation of bubbles, demonstrating excellent sealing performance; Repeatability: After 30 autoclave cycles, the sealing performance remains stable without any degradation; 2. It adopts a snap-on replaceable breathable membrane design. The breathable components include a slot, a breathable membrane and a pressure ring. The pressure ring and the slot are interference fit. The disassembly force is controlled within the range of 3-10N, which ensures both the firmness during use and the ease of quick replacement.

[0018] Specific performance characteristics: Replacement time: The entire process, from removing the old film to installing the new film, can be completed within 5-15 seconds, with an average of about 10 seconds; Reusability: The breathable membrane assembly can be repeatedly assembled and disassembled 20-50 times without significant decrease in disassembly force; Sealing reliability: After replacement, there is no leakage between the breathable membrane and the bottle cap, and the breathability is stable; 3. The sampling valve is made of liquid silicone rubber (LSR) through secondary injection molding. The valve body has a cross-shaped slit in the center (2-5mm in length and 0.01-0.10mm in width). When the sampling needle is punctured, the slit expands to allow liquid to pass through; after the needle is removed, the elastic recovery of the silicone rubber closes the slit, achieving self-sealing.

[0019] Specific performance characteristics: Dead volume control: The internal channel design of the sampling valve is optimized, and the dead volume is controlled to ≤50μL, which is far lower than the loss caused by traditional open-cap sampling; Liquid surface stability: The sampling valve is located on the side wall of the bottle, 10-50 mm from the bottle mouth. It will not disturb the liquid surface vortex during sampling, thus maintaining the stability of the shaker culture. Sampling efficiency: The amount of each sample can be controlled between 0.1-5 mL to meet different experimental needs; Sterility guaranteed: The sampling process does not require opening the cap, avoiding approximately 12% of the risk of contamination; 4. The bottle body is made of medical-grade PC or PPCO material, and undergoes annealing treatment at 130-140℃ for 25-35 minutes to eliminate internal stress. The dissolved oxygen sensing membrane is formed using a co-injection molding process, creating a five-layer sandwich structure of PC / PPCO-sensing membrane-COC, with a strong interface bond.

[0020] Specific performance characteristics: Sterilization tolerance: After 30 cycles of high-pressure steam sterilization at 121-125℃ for 20-40 minutes, the change in yellowness index (ΔYI) of the bottle is <4.0, and the light transmittance retention rate is ≥85%; Functional stability: Fluorescence signal loss in the dissolved oxygen sensing window <5%, sensitivity retention ≥90%; Mechanical properties: The flatness of the bottle bottom remains ≤0.10mm, the self-closing force of the sampling valve is ≥0.15N, and the disassembly force of the breathable membrane shows no significant decrease; Sealing performance: All sealing components, including centrifugal seal, sampling valve seal, and breathable membrane seal, remain intact and leak-free.

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the structure of a re-sterilizable flat-bottomed culture centrifuge bottle according to an embodiment of the present invention; Figure 2 This is a schematic diagram of the internal structure of the bottle cap in an embodiment of the present invention; Figure 3 This is a cross-sectional view of the card slot in an embodiment of the present invention; Figure 4 This is a cross-sectional view of the pressure ring being placed into the slot in an embodiment of the present invention; Figure 5 This is a cross-sectional view of the dissolved oxygen sensing membrane and the bottle body being co-injected together in an embodiment of the present invention.

[0023] In the diagram, 1-bottle body, 2-bottle cap, 3-sampling valve, 4-dissolved oxygen sensing membrane, 5-QR code, 6-TPE micro-ring, 7-fan-shaped pores, 8-toothed protrusion, 9-cross opening, 10-rigid locking plate, 11-slot, 12-pressure ring, 13-protective film, 14-sensing film, 15-transition fusion layer. Detailed Implementation

[0024] 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 some embodiments of the present invention, and not all embodiments. 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.

[0025] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," and "counterclockwise," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention. Example 1

[0026] like Figure 1-5 As shown, a re-sterilizable flat-bottomed culture centrifuge bottle includes a bottle body 1, which is injection molded from medical-grade PC or PPCO in one piece. The bottom of the bottle body is flat, and the bottle body 1 has a bottle mouth at the top. The bottle mouth is provided with an M44 internal thread, and a bottle cap 2 is threadedly connected to the bottle mouth. The bottle cap 2 has several tooth-like protrusions 8 on its outer periphery.

[0027] A centrifugal self-locking sealing structure is provided between the bottle mouth end face and the bottle cap 2. The centrifugal self-locking sealing structure includes a TPE micro ring 6 and several rigid locking plates 10. The TPE micro ring 6 is located on the top inner side of the bottle cap 2. The diameter of the TPE micro ring 6 is slightly larger than the outer diameter of the bottle mouth. Several rigid locking plates 10 are evenly spaced inside the TPE micro ring 6. The thickness of the rigid locking plates 10 is 0.2mm. When the bottle cap 2 is screwed onto the bottle mouth, the inner side of the TPE micro ring 6 contacts the outer edge of the bottle mouth. During centrifugation, when the centrifugal force is ≥15000×g, several rigid locking plates 10 move outward to squeeze the TPE micro ring 6, so that the threaded pair gap is ≤0.02mm.

[0028] The bottle cap 2 inside the TPE microring 6 has a fan-shaped air hole 7. A breathable membrane is detachably connected to the fan-shaped air hole 7. The breathable membrane is a PTFE breathable membrane with an average pore diameter of 0.22μm. The outer periphery of the fan-shaped air hole 7 has a fan-shaped groove 11. The groove 11 is an Ω groove with a depth of 0.5-1.0mm and a width of 1.0-1.5mm. The inner wall of the groove 11 has anti-slip texture with a depth of 0.1-0.3mm. The groove 11 is integrally formed with the bottle cap 2.

[0029] A pressure ring 12 is engaged in the slot 11. The pressure ring 12 is a PTFE-PP ring. The pressure ring 12 presses the breathable membrane into the slot 11. When the breathable membrane needs to be replaced, simply remove the pressure ring 12, remove the breathable membrane and replace it with a new one. The operation is simple and convenient, and it can be reused ≥30 times.

[0030] A sampling valve 3 is provided on the side wall of the bottle body 10–30 mm from the bottle mouth. The sampling valve 3 is a self-closing sampling valve. The sampling valve 3 has a cross-shaped opening 9 in the center. The material of the center of the sampling valve 3 is silicone rubber. The sampling valve 3 is integrally formed with the bottle body 1. There is no leakage after 48 hours. The rebound force after 100 punctures is ≥0.2N.

[0031] The lower part of the bottle body 1 is provided with a dissolved oxygen sensing membrane 4. The bottom of the dissolved oxygen sensing membrane 4 is 3.5cm away from the bottom of the bottle. The width of the dissolved oxygen sensing membrane 4 is 0.8cm and the height of the dissolved oxygen sensing membrane 4 is 1.2cm. The dissolved oxygen sensing membrane 4 and the bottle body 1 are injection molded together. The dissolved oxygen sensing membrane 4 includes a protective film 13, a sensing film 14 and a transition fusion layer 15 that are sequentially co-molded together. The transition fusion layer 15 and the bottle body 1 are co-molded together.

[0032] The transition fusion layer 15 is a transition region formed by the mutual penetration and molecular-level fusion of the molten bottle body 1 and the sensing membrane 14 at the interface during the co-injection molding process. The thickness of the transition fusion layer 15 is 0.1 mm.

[0033] The sensing membrane 14 is made of Ru(II) complex (ruthenium complex) fluorescent sensitive material, which usually exists in the form of solid solution or polymer film. The thickness of the sensing membrane 14 is 0.1 mm.

[0034] The protective film 13 is made of medical-grade cyclic olefin copolymer (COC), which has extremely high light transmittance (greater than 91%), extremely low fluorescence background, excellent bioinertness, and very low protein adsorption. It effectively protects the sensing membrane from interference from culture medium components, and its hydrophobic surface is easy to clean. Its processing temperature is close to that of PC, facilitating a strong bond through co-injection molding. The thickness of the protective film 13 is 0.05 mm.

[0035] A QR code 5 is also provided at the bottom of the bottle body 1. Example 2

[0036] A method for preparing a re-sterilizable flat-bottomed culture centrifuge flask is as follows: 1. Raw material processing (1) First, dry the medical-grade PC plastic at 115-130℃ for 4 hours to reduce the moisture content to below 0.02% to prevent air bubbles from forming during injection molding; (2) The ruthenium(II) complex powder and COC particles are made into a "masterbatch" together; (3) Vacuum remove air bubbles from medical-grade silicone and cut it into small strips.

[0037] 2. Use an electric injection molding machine to manufacture the bottle body. (1) Heat the dried PC plastic particles to 280-320℃, extrude them into the mold, and first use an electric injection molding machine to make the bottle shape; (2) Immediately heat the “masterbatch” to 270-300℃, squeeze a small clump on the designated position on the bottle wall to form a dissolved oxygen sensing film 4 with a thickness of 0.1mm; (3) Squeeze out a little more COC transparent plastic to completely wrap the dissolved oxygen sensing membrane 4 to form a protective film 13; (4) Inspection: The electric injection molding machine is equipped with a laser thickness probe. The thickness of the dissolved oxygen sensing film is measured every 2 seconds and must be within 0.10±0.01mm.

[0038] 3. Install the sampling valve – rotating slider mold Transfer the finished bottle to another electric injection molding machine. A small slider extends from the side of the mold. Place the silicone strip on the slider, and the slider closes. After 8 seconds at 170-190℃ and 12MPa, the silicone is vulcanized and fused to the PC bottle wall. The slider is then unscrewed, leaving a cross-shaped slit 0.02-0.07 mm wide on the bottle wall. The valve body is then inspected to ensure it is leak-proof.

[0039] 4. Stress relief Place the bottle in a 130-40℃ hot air oven for 30 minutes to cool slowly. Use a 0.1mm feeler gauge to check the bottom of the bottle to ensure that the flatness is up to standard and that the centrifugation will not cause it to shake.

[0040] 5. QR code engraving The laser machine burns a Data Matrix QR code on the bottom of the bottle, with a depth of 0.05mm. The code contains the serial number and the material batch number.

[0041] 6. Two-color injection molding of bottle cap First color: Temperature control: barrel 230-320℃, nozzle 300-320℃, mold 80-110℃; Pressure control: Injection pressure 130-180MPa, holding pressure 40%-60%; Injection speed: 30-100 mm / s; Molding cycle: Inject for 15-30 seconds, cool, rotate 180° to the second color injection station; Create a slot 11 in the bottle cap using a mold.

[0042] Second color: Immediately inject TPE soft glue and PP hard sheet to make TPE micro rings 6 and rigid locking plates 10 to form a centrifugal self-locking sealing structure.

[0043] 7. Fabrication of breathable components The pressure ring 12 is made by injection molding medical-grade PC. The PTFE breathable membrane and the pressure ring 12 are bonded together using a hot press at 170-190 ℃ for 2 seconds and 0.3MPa to form a detachable breathable component.

[0044] 8. Assembly and Testing Install the breathable component on the bottle cap. Example 3

[0045] The performance of a re-sterilizable flat-bottomed culture centrifuge bottle of the present invention was tested.

[0046] I. Sealing performance test 1. Centrifugal sealing test Test conditions: Centrifuge at 15000×g for 10 minutes, then inject 500mL of water or cell culture medium into the bottle; Judgment criteria: No leakage, and all sealing parts such as bottle opening, sampling valve, and vent membrane are intact; Test results: After 30 high-pressure sterilization cycles, the sealing performance remained stable without any degradation.

[0047] 2. Pressure tolerance test Test conditions: Apply 0.4 MPa air pressure internally and maintain for 48 hours; Judgment criteria: No bubbles are generated, and the pressure drop rate is ≤5%; Test results: All samples passed the test, demonstrating excellent sealing reliability.

[0048] II. Test of breathable membrane performance 1. Ease of replacement test Test conditions: Record the operation time from removing the old membrane to installing the new membrane; Judgment criteria: Replacement time ≤ 10 seconds, disassembly force controlled within the range of 3-10N; Test results: The average replacement time is about 8 seconds, the disassembly force is about 6N, and it can be reassembled and disassembled 20-50 times.

[0049] 2. Breathability test Test conditions: Using a PTFE breathable membrane, the oxygen permeability was measured; Judgment criteria: Oxygen permeability ≥ 5000 cm³ / m²·24h·atm; Test results: The air permeability is stable and meets the requirements for high oxygen tension culture.

[0050] III. Sampling Valve Performance Testing 1. Dead volume test Test conditions: Measure the internal channel volume of the sampling valve; Judgment criteria: Dead volume ≤ 50 μL; Test results: The actual dead volume is approximately 45 μL, which does not interfere with the liquid surface vortex.

[0051] 2. Puncture durability test Test conditions: 100 punctures with a 22G needle, and the force of needle withdrawal on the 100th puncture is measured; Judgment criterion: Self-closing force ≥ 0.15N; Test results: The force required to remove the needle on the 100th attempt was approximately 0.25 N, and the sealing performance remained good.

[0052] 3. Sealing performance test Test conditions: After 100 punctures, apply a pressure of 0.4 MPa and maintain it for 48 hours; Judgment criteria: No bubbles are produced; Test results: All samples passed the test and there was no leakage.

[0053] IV. Dissolved Oxygen Sensing Membrane Performance Testing 1. Light transmittance test Test conditions: Transmittance at 460nm wavelength was measured using a spectrophotometer; Judgment criteria: Initial light transmittance ≥ 90%; Test results: Initial transmittance was approximately 92%, and fluorescence signal loss was <5%.

[0054] 2. Sensitivity Test Test conditions: Two-point calibration was performed using standard gases with oxygen concentrations of 0% and 21%. Judgment criterion: Linearity R² ≥ 0.995; Test results: The calibration curve showed good linearity, and the sensitivity retention rate was ≥90%.

[0055] V. Verification of Repeatable Sterilization Performance 1. Sterilization tolerance test Test conditions: 30 cycles of high-pressure steam sterilization at 121-125℃ for 20-40 minutes; Judgment criteria: Yellowness index change (ΔYI) < 4.0, transmittance retention ≥ 85%; Test results: ΔYI=2.1, transmittance retention rate is approximately 89%.

[0056] 2. Functional stability test Test conditions: After 30 sterilization cycles, all performance indicators were retested; Judgment criteria: All performance indicators still meet the usage requirements; Test results: All sealing parts, including centrifugal seal, sampling valve seal, and breathable membrane seal, remained intact. The dissolved oxygen sensing membrane functioned stably, and the QR code readability was 100%.

[0057] 3. Mechanical performance testing Test conditions: After 30 sterilization cycles, measure the flatness of the bottle bottom and the self-closing force of the sampling valve; Judgment criteria: Bottle bottom flatness ≤ 0.10 mm, sampling valve self-closing force ≥ 0.15 N; Test results: The flatness of the bottle bottom is approximately 0.08 mm, and the self-closing force of the sampling valve is approximately 0.25 N.

[0058] VI. Sterility Assurance Test 1. Aseptic validation Test conditions: After sterilization by 25kGy electron beam irradiation, sterility test was performed; Judgment criteria: Complies with USP <71> Aseptic testing requirements; Test results: All samples passed sterility verification.

[0059] 2. Endotoxin testing Test conditions: Use 17.2 mL of ultrapure water to rinse, and collect the rinsing solution as the test solution; Judgment criteria: Endotoxin <0.25 EU / mL; Test results: Endotoxin content is approximately 0.15 EU / mL.

[0060] VII. Comprehensive Performance Evaluation After comprehensive performance testing, the re-sterilizable flat-bottomed culture centrifuge flask of this invention performs excellently in the following aspects: (1) Sealing reliability: Zero leakage under centrifugal conditions of 15000×g, and no leakage for 48 hours under pressure of 0.4MPa; (2) Ease of operation: The breathable membrane can be replaced within 10 seconds, and the dead volume of the sampling valve is ≤50μL; (3) Functional stability: After 30 sterilization cycles, all performance indicators still meet the usage requirements; (4) Aseptic assurance: meets the requirements of USP aseptic testing method and endotoxin standard.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A re-sterilizable flat-bottomed culture centrifuge flask, characterized in that: The bottle includes a bottle body (1), the bottom of which is flat, and the bottle mouth is located at the top of the bottle body (1). The bottle mouth is threadedly connected to a bottle cap (2). A centrifugal self-locking sealing structure is provided between the end face of the bottle mouth and the bottle cap (2). A sampling valve (3) is provided on the side wall of the bottle body, and a dissolved oxygen sensing membrane (4) is provided at the bottom of the bottle body (1).

2. The re-sterilizable flat-bottomed culture centrifuge bottle according to claim 1, characterized in that: The centrifugal self-locking sealing structure includes a TPE microring (6) and several rigid locking plates (10). The TPE microring (6) is disposed on the top inner side of the bottle cap (2), and several rigid locking plates (10) are evenly spaced inside the TPE microring (6).

3. A re-sterilizable flat-bottomed culture centrifuge flask according to claim 2, characterized in that: The diameter of the TPE microring (6) is slightly larger than the outer diameter of the bottle mouth, and the thickness of the rigid locking piece (10) is 0.2 mm.

4. A re-sterilizable flat-bottomed culture centrifuge flask according to claim 3, characterized in that: The bottle cap (2) inside the TPE microring (6) is provided with a fan-shaped air hole (7), and a breathable membrane is detachably connected to the fan-shaped air hole (7).

5. A re-sterilizable flat-bottomed culture centrifuge flask according to claim 4, characterized in that: The outer periphery of the fan-shaped air hole (7) is provided with a fan-shaped groove (11), the groove (11) is an Ω groove, and a pressure ring (12) is engaged in the groove (11).

6. A re-sterilizable flat-bottomed culture centrifuge flask according to claim 5, characterized in that: The breathable membrane is a PTFE breathable membrane with an average pore size of 0.22 μm, and the pressure ring (12) is a PTFE-PP ring.

7. A re-sterilizable flat-bottomed culture centrifuge flask according to claim 6, characterized in that: The depth of the slot (11) is 0.5-1.0 mm and the width is 1.0-1.5 mm. The inner wall of the slot (11) is provided with anti-slip texture, the depth of which is 0.1-0.3 mm. The slot (11) and the bottle cap (2) are integrally formed.

8. A re-sterilizable flat-bottomed culture centrifuge flask according to claim 7, characterized in that: The dissolved oxygen sensing membrane (4) includes a protective membrane (13), a sensing membrane (14) and a transition fusion layer (15) co-molded together in sequence. The transition fusion layer (15) is co-injected together with the bottle body (1). The transition fusion layer (15) is a transition region formed by mutual penetration and molecular-level fusion of the molten bottle body (1) and the sensing membrane (14) at the interface during the co-injection process. The sensing membrane (14) is made of Ru(II) complex fluorescent sensitive material, and the protective membrane (13) is made of medical grade cyclic olefin copolymer.

9. A re-sterilizable flat-bottomed culture centrifuge flask according to claim 8, characterized in that: The sampling valve (3) is located 10–30 mm from the bottle opening. The sampling valve (3) is a self-closing sampling valve. The sampling valve (3) has a cross-shaped opening (9) in the center. The material of the center of the sampling valve (3) is silicone rubber. The sampling valve (3) is integrally formed with the bottle body (1).

10. A re-sterilizable flat-bottomed culture centrifuge flask according to any one of claims 1-9, characterized in that: The preparation method is as follows:

1. Raw material processing (1) First, dry the medical-grade PC plastic at 115-130℃ for 4 hours to reduce the moisture content to below 0.02% to prevent air bubbles from forming during injection molding; (2) The ruthenium(II) complex powder and COC particles are made into a "masterbatch" together; (3) Vacuum remove air bubbles from medical-grade silicone and cut it into small strips; 2. Use an electric injection molding machine to manufacture the bottle body. (1) Heat the dried PC plastic particles to 280-320℃, extrude them into the mold, and first use an electric injection molding machine to make the bottle shape; (2) Immediately heat the "masterbatch" to 270-300℃, squeeze a small clump on the designated position on the bottle wall to form a dissolved oxygen sensing film with a thickness of 0.1mm (4). (3) Squeeze out a little more COC transparent plastic to completely wrap the dissolved oxygen sensing membrane (4) to form a protective film (13). (4) Inspection: The electric injection molding machine is equipped with a laser thickness probe, which measures the thickness of the dissolved oxygen sensing membrane (4) every 2 seconds. The thickness must be within 0.10±0.01mm.

3. Install the sampling valve – rotating slider mold Transfer the finished bottle to another electric injection molding machine. A small slider extends from the side of the mold. Place the silicone strip on the slider, and the slider closes. After 8 seconds at 170-190℃ and 12MPa, the silicone is vulcanized and fused to the PC bottle wall. The slider is then unscrewed, leaving a cross-shaped slit 0.02-0.07mm wide on the bottle wall. The valve body is then inspected to ensure it is leak-proof.

4. Stress relief Place the bottle in a 130-40℃ hot air oven for 30 minutes to cool slowly. Use a 0.1mm feeler gauge to check the bottom of the bottle to ensure that the flatness is up to standard and that the centrifugation will not cause the bottle to shake.

5. QR code engraving The laser machine burns a Data Matrix QR code onto the bottom of the bottle to a depth of 0.05mm; 6. Two-color injection molding of bottle cap First color: Temperature control: barrel 230-320℃, nozzle 300-320℃, mold 80-110℃; Pressure control: Injection pressure 130-180MPa, holding pressure 40%-60%; Injection speed: 30-100 mm / s; Molding cycle: Inject for 15-30 seconds, cool, rotate 180° to the second color injection station; (11) A slot is made in the bottle cap using a mold. Second color: Immediately inject TPE soft glue and PP hard sheet to make TPE micro rings (6) and rigid locking sheet (10) to form a centrifugal self-locking sealing structure; 7. Fabrication of breathable components The pressure ring (12) is made by injection molding of medical grade PC. The PTFE breathable membrane and the pressure ring (12) are bonded together using a hot press at 170-190℃ for 2 seconds and 0.3MPa to form a detachable breathable component.

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