Microfluidic perfusion culture integrated chip cartridge for space environment and preparation method thereof

Abstract

The application provides a microfluidic perfusion culture integrated chip cartridge for space environment, which comprises a cartridge frame structure, a microfluidic perfusion culture module and a gas-tight electric connector, the cartridge frame structure is formed into a full-sealing structure by a middle frame plate, an upper cover plate and a lower cover plate, the microfluidic perfusion culture module is enclosed in the cartridge frame structure, and the cartridge frame structure is connected with an external control device through the gas-tight electric connector. The microfluidic perfusion culture integrated chip cartridge is full-sealed, the microorganism in the cartridge is isolated from the outside operator, and is suitable for use in a space environment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical detection, and particularly relates to a microfluidic perfusion culture integrated chip cartridge for space environment and a preparation method thereof. BACKGROUND

[0002] In the direction of space life science and biotechnology, the long-term microgravity, regular magnetic field and rapid alternation of day and night, and special radiation provided by the space station are used to promote the understanding and recognition of the nature of life phenomena and explore scientific laws, carry out researches on the response of living beings to gravity changes, damage under space radiation, and life origin, evolution, development and reproduction, carry out researches on the development and application of biotechnology using space conditions, promote the development of modern life science and biotechnology, and serve human health and social progress.

[0003] The sealing performance of a cell culture device under space environment is required to be higher, and the liquid pipeline connection between the perfusion culture chip and the perfusion equipment in the prior art is needed, which is inconvenient to replace the chip and reconnect the pipeline in the space environment; the perfusion pipeline of the chip is externally connected, which has the risk of liquid leakage if exposed in the equipment, affecting the normal operation of the equipment, and even polluting the space environment; if the chip and the perfusion equipment are integrated, the mechanical and control electrical devices cannot be reused, resulting in waste of load. SUMMARY

[0004] In order to overcome the problems in the prior art, the present application provides a replaceable microfluidic perfusion culture integrated chip cartridge suitable for space environment. The function is to integrate the culture medium, culture chamber, liquid flow path and control valve required for microorganism culture into the microfluidic chip cartridge, to provide a fully closed microorganism culture and detection environment, to reduce the difficulty of manual replacement operation of the microorganism culture chip, and at the same time, to avoid the risk of biological sample leakage in the replacement process.

[0005] The present application provides the following technical solutions:

[0006] A microfluidic perfusion culture integrated chip cartridge for space environment, comprising a cartridge frame structure, a microfluidic perfusion culture module and a gas-tight electrical connector, the cartridge frame structure is formed into a fully sealed structure by a middle frame plate, an upper cover plate and a lower cover plate, the microfluidic perfusion culture module is enclosed inside the cartridge frame structure, and the cartridge frame structure is connected with an external control device through the gas-tight electrical connector.

[0007] Further, the microfluidic perfusion culture module and the gas-tight electrical connector are arranged in the middle frame plate, the two sides of the middle frame plate corresponding to the upper and lower cover plates are provided with annular sealing grooves, sealing rings are arranged in the sealing grooves, and the upper and lower cover plates and the middle frame plate are fixed by bolts.

[0008] Further, the microfluidic perfusion culture module comprises a liquid storage unit, a syringe pump unit, a liquid distribution control unit and a culture detection chamber unit.

[0009] Further, the culture detection chamber unit is fixed on the liquid distribution control unit, and the culture detection chamber unit comprises an upper cover plate and an intermediate hole plate to form a sealed culture chamber, a heating sheet and a temperature sensor are arranged between the upper cover plate and the intermediate hole plate, a microbial filter membrane and a sealing washer are arranged between the intermediate hole plate and the bottom liquid distribution control unit, and the culture chamber is connected to the flow path through the filter membrane.

[0010] Further, the liquid storage bag and the waste liquid bag of the liquid storage unit are fixed in the intermediate frame plate by bolts, the liquid inlet and the waste liquid outlet are respectively connected to the liquid inlet channel and the liquid outlet channel of the liquid distribution control unit, the syringe pump unit is connected to the liquid distribution control unit by a luer connector and communicates with the liquid storage bag, the syringe pump body penetrates through the side wall of the intermediate frame plate, the inner side of the intermediate frame plate is provided with a sealing groove corresponding to the position of the syringe pump body, and an O-ring is arranged in the sealing groove.

[0011] Further, a hollow sealing screw is arranged on the outer side of the intermediate frame plate to fix the syringe pump body extending out of the intermediate frame plate, and a detachable limiting block is arranged on the outer side of the intermediate frame plate to limit the transverse movement of the push rod of the syringe pump when stored.

[0012] Further, the liquid distribution control unit is provided with a solenoid valve to control the opening and closing of the liquid inlet channel and the liquid outlet channel.

[0013] Further, the length of the card box frame structure is 150-190mm, the width is 80-100mm, the thickness is 20-25mm, the volume of the culture detection chamber is 200-300uL, the volume of the liquid storage bag and the waste liquid bag is 5-10mL, and the volume of the syringe pump is 200-2000uL.

[0014] Further, a handle is arranged on the side of the card box frame structure.

[0015] Further, a gas-tight electrical connector is fixed on the intermediate frame plate, and the gas-tight electrical connector is connected to the heating sheet, the temperature sensor and the solenoid valve, and the external part is plugged into the control device.

[0016] A preparation method of a microfluidic perfusion culture integrated chip card box for space environment, comprising the following steps:

[0017] Step one Assemble the microfluidic perfusion culture module, connect the liquid storage unit, the syringe pump unit and the culture detection chamber unit to the liquid distribution control unit, preinstall microbial dry powder, culture medium and the like in each functional unit according to the requirements, and connect the lead wires of the solenoid valve, the heating sheet and the temperature sensor to the gas-tight electrical connector;

[0018] Step two, fix the microfluidic perfusion culture module on the middle frame, including the fixing of the liquid storage bag, the waste liquid bag and their pipelines;

[0019] Step three, pass the injection pump through the middle frame, put the O ring outside the injection pump body, lock the injection pump on the luer joint, put the O ring into the sealing groove of the middle frame, and fix the hollow sealing screw outside the injection pump body, and the limiting block is connected with the push rod;

[0020] Step four, install the sealing ring in the annular sealing groove on both sides of the middle frame, and connect the upper and lower cover plates with the middle frame by bolts.

[0021] By adopting the above technical scheme, the present application has the following beneficial effects:

[0022] (1) The microfluidic perfusion culture integrated chip card box of the present application is fully closed, and the microorganisms in the interior are isolated from the outside operating personnel, and is suitable for use in space environment.

[0023] (2) The culture medium, perfusion pump, growth detection chamber, constant temperature component and waste liquid storage required for the growth of microorganisms are designed as unit components and integrated in the module.

[0024] (3) Easy to save, the module separates the sterilized culture medium and freeze-dried bacteria powder and is built-in for cold storage. Easy to operate, modular design is adopted, and external control equipment is used. When used, the operating personnel only needs to take out the card box from the package, insert it into the external equipment according to the requirements, and start the program. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a structure schematic view of the microfluidic perfusion culture integrated chip card box of the present application;

[0026] Figure 2 is a structure schematic view of the microfluidic perfusion culture module of the present application;

[0027] Figure 3 is a side view structure schematic view of the culture detection chamber of the present application.

[0028] Wherein: 1-card box frame structure, 2-gas-tight electrical connector, 3-microfluidic perfusion culture module, 4-culture detection chamber unit, 41-upper cover plate, 42-heating sheet, 43-filtration membrane, 44-sealing ring, 5-liquid storage bag, 6-waste liquid bag, 7-injection pump body, 8-hollow sealing screw, 9-limiting block, 10-solenoid valve, 11-handle. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below with reference to the drawings and examples. It should be understood that the structural diagram and specific examples described herein are only used to explain the present application and not used to limit the present application.

[0030] Example 1

[0031] As shown in Figure 1 , 2 , the present application provides a microfluidic perfusion culture integrated chip cartridge for space environment, which comprises a cartridge frame structure 1, a microfluidic perfusion culture module 3 and a gas-tight electrical connector 2. The cartridge frame structure is formed into a full-sealed structure by a middle frame plate, an upper cover plate and a lower cover plate. The microfluidic perfusion culture module is enclosed inside the cartridge frame structure. The cartridge frame structure is connected with an external control device through the gas-tight electrical connector.

[0032] The microfluidic perfusion culture module and the gas-tight electrical connector are arranged in the middle frame plate. The middle frame plate is provided with annular sealing grooves on the two sides corresponding to the upper and lower cover plates. Sealing rings are arranged in the sealing grooves. The upper and lower cover plates are bolted with the middle frame plate. The microfluidic perfusion culture module is used for culture and detection of biological samples. The gas-tight electrical connector is connected with an external power supply and a controller to control the electrical elements inside the integrated chip cartridge. Through a set perfusion culture program, the perfusion culture cartridge is controlled to achieve the functions of constant culture temperature, resuscitation of microorganisms to be cultured, provision of timed and quantitative culture medium perfusion, detection of OD value and fluorescence value data of microorganisms, etc.

[0033] The length of the cartridge frame structure is 150-190 mm, the width is 80-100 mm, and the thickness is 20-25 mm. Preferably, the length of the cartridge frame structure is 155 mm, the width is 85 mm, and the thickness is 24 mm. A handle is arranged on the side of the cartridge frame structure. The operator only contacts the outer surface of the perfusion culture cartridge, avoiding the pollution caused by the operator to cause the failure of microbial culture. At the same time, the microorganisms inside the cartridge will not leak into the environment where the operator is located, avoiding pollution to the environment.

[0034] Example 2

[0035] As shown in Figure 3 , the microfluidic perfusion culture module comprises a liquid storage unit, a syringe pump unit, a liquid distribution control unit and a culture and detection chamber unit. The culture and detection chamber unit 4 is fixed on the liquid distribution control unit. The culture and detection chamber unit is composed of an upper cover plate 41 and a middle aperture plate to form a sealed culture chamber. A heating sheet 42 and a temperature sensor are arranged between the upper cover plate and the middle aperture plate. A microbial filter membrane 43 and a sealing washer 44 are arranged between the middle aperture plate and the bottom liquid distribution control unit. The culture chamber is connected with the flow path through the filter membrane. The volume of the culture and detection chamber can be 200-300 uL, preferably 300 uL.

[0036] The storage bag 5 and the waste liquid bag 6 of the storage unit are fixed in the middle frame plate by bolts, and the storage port and the waste liquid port are connected to the liquid inlet channel and the liquid outlet channel of the distribution control unit respectively, and the volume of the storage bag and the waste liquid bag can be 5-10 mL, preferably 6 mL.

[0037] The syringe pump body 7 is placed in the middle frame plate and fixed on the distribution control unit by a luer joint, in communication with the liquid channel of the storage bag, and the volume of the syringe pump can be 1-2 mL, preferably 1 mL. The syringe pump body penetrates the side wall of the middle frame plate, and the inner side of the middle frame plate is provided with a sealing groove corresponding to the position of the syringe pump body, and an O-ring is arranged in the sealing groove.

[0038] A hollow sealing screw 8 is arranged on the outer side of the middle frame plate to fix the syringe pump body extending out of the middle frame plate, and a detachable limiting block 9 is arranged on the outer side of the middle frame plate to limit the transverse movement of the push rod of the syringe pump when stored.

[0039] The distribution control unit is provided with a solenoid valve to control the opening and closing of the liquid inlet channel and the liquid outlet channel. The number of solenoid valves is adapted to the number of culture chambers. A gas-tight electrical connector is fixed on the middle frame plate, and the gas-tight electrical connector is connected to the heating film, the temperature sensor and the solenoid valve respectively, and is plugged with the external control device. The control device provides switch control of two solenoid valves, real-time temperature reading of the temperature sensor, power supply of the heating film, etc.; at the same time, the external control device also provides the push-pull power of the syringe pump.

[0040] The recovery and culture control process of the microfluidic perfusion culture integrated chip card box:

[0041] 1) Insert the chip card box into the device and start the control program;

[0042] 2) First, control the heating film to start heating, and at the same time, monitor the temperature of the culture detection chamber unit by the temperature sensor, and feedback adjust the heating film to make the temperature of the chamber reach and constant at 37℃;

[0043] 3) Microbial recovery stage:

[0044] a. The device pulls the syringe pump push rod of the chip card box to suck 800uL of culture medium in the storage bag;

[0045] b. Open the solenoid valve one, connect the syringe pump and the culture chamber one flow path, and push the syringe pump to perfuse 400uL of culture medium into the culture chamber one;

[0046] c. Keep valve one open, open valve two, switch the syringe pump flow path to culture chamber two, continue to push the syringe pump to perfuse 400uL of culture medium, and complete the liquid perfusion of the two chambers;

[0047] d. Close valve one and valve two, keep 1h at constant temperature 37℃, let the microbial dry powder completely recover.

[0048] 4) Continuous perfusion culture and detection phase:

[0049] a. According to the research needs, detect the OD value or fluorescence value of the microorganism through the transparent window of the culture detection chamber unit;

[0050] b. The device pulls the injection pump push rod of the chip card box to suck 800uL of culture medium in the storage liquid bag;

[0051] c. Open electromagnetic valve one, connect the injection pump and the culture chamber one flow path, and push the injection pump to perfuse 15uL of culture medium to the culture chamber one;

[0052] d. Keep valve one open, open valve two, switch the injection pump flow path to culture chamber two, continue to push the injection pump to perfuse 15uL of culture medium, complete the liquid perfusion of the two chambers;

[0053] e. Close valve one and valve two, keep static culture for 10min, then repeat steps c and d to complete intermittent continuous perfusion;

[0054] f. According to the needs, detection can be completed before each perfusion, or continuous detection can be performed;

[0055] g. According to the experimental needs, the whole perfusion culture process can last for 8-12h, and the liquid amount of the injection pump can be repeated in step b to continue to suck from the storage liquid bag.

[0056] 5) End and close phase:

[0057] a. After the culture is completed, the device will automatically return the injection pump to zero position, stop heating, and close all electromagnetic valves;

[0058] b. Take out the perfusion culture integrated chip card box and discard it after packaging.

[0059] Example 3

[0060] The present application provides a preparation method of a microfluidic perfusion culture integrated chip card box for space environment, comprising the following steps:

[0061] Step one Assemble the microfluidic perfusion culture module, connect the storage liquid unit, injection pump unit, and culture detection chamber unit on the liquid distribution control unit, and preinstall microbial dry powder, culture medium, etc. in each functional unit according to the needs. The leads of the electromagnetic valve, heating sheet, and temperature sensor are connected with the airtight electrical connector.

[0062] Step two Fix the microfluidic perfusion culture module on the middle frame plate, including the fixation of the storage liquid bag, waste liquid bag, and their pipelines.

[0063] Step three: the injection pump passes through the middle frame plate, the O-ring is sleeved outside the injection pump body, the injection pump is locked and fixed on the luer joint, the O-ring is sent into the sealing groove of the middle frame plate, the hollow sealing screw is sleeved and fixed outside the injection pump body, and the limiting block is clamped with the push rod;

[0064] Step four: the sealing ring is installed in the annular sealing groove on the inner and outer sides of the middle frame plate, and the upper and lower cover plates and the middle frame plate are connected by bolts.

[0065] Through experimental tests, the card box as a whole can provide overall sealing, and when the internal pressure of the card box reaches 1.2 atmospheres, the contents of the card box can still be guaranteed not to leak. It can be guaranteed that in a vacuum environment or in the extreme case that the pressure outside the card box is completely lost, the card box will not have a risk of leakage.

[0066] The above-described embodiments only express the implementation of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent of the present application. It should be pointed out that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A microfluidic perfusion culture integrated chip cartridge for space environment, comprising a cartridge frame structure, a microfluidic perfusion culture module, and a hermetically sealed electrical connector, characterized in that, The card box frame structure is formed by a middle frame plate, upper and lower cover plates into a full sealing structure, the microfluidic perfusion culture module is enclosed in the card box frame structure, the card box frame structure is connected with the external control device through the air-tight electric connector; the microfluidic perfusion culture module comprises a liquid storage unit, a syringe pump unit, a liquid distribution control unit and a culture detection chamber unit, the liquid storage bag and the waste liquid bag of the liquid storage unit are fixed in the middle frame plate through bolts, the liquid inlet and the waste liquid outlet are respectively connected to the liquid inlet channel and the liquid outlet channel of the liquid distribution control unit, the syringe pump unit is connected to the liquid distribution control unit through a luer connector and communicates with the liquid storage bag, the syringe pump body penetrates through the side wall of the middle frame plate, the inner side of the middle frame plate is provided with a sealing groove at the corresponding position of the syringe pump body, and an O-ring is arranged in the sealing groove.

2. The microfluidic perfusion culture integrated chip cartridge of claim 1, wherein, The microfluidic perfusion culture module and the air-tight electric connector are arranged in the middle frame plate, the two sides of the middle frame plate corresponding to the upper and lower cover plates are provided with annular sealing grooves, and sealing rings are arranged in the sealing grooves; the upper and lower cover plates and the middle frame plate are fixed through bolts.

3. The microfluidic perfusion culture integrated chip cartridge of claim 2, wherein, The culture detection chamber unit is fixed on the liquid distribution control unit, the culture detection chamber unit is composed of an upper cover plate and a middle hole plate into a sealed culture chamber, a heating sheet and a temperature sensor are arranged between the upper cover plate and the middle hole plate, a microbial filter membrane and a sealing washer are arranged between the middle hole plate and the bottom liquid distribution control unit, and the culture chamber and the flow path are connected through the filter membrane.

4. The microfluidic perfusion culture integrated chip cartridge of claim 3, wherein, A hollow sealing screw is arranged on the outer side of the middle frame plate to fix the syringe pump body extending out of the middle frame plate, and a detachable limiting block is arranged on the outer side of the middle frame plate to limit the transverse movement of the push rod of the syringe pump when stored.

5. The microfluidic perfusion culture integrated chip cartridge of claim 4, wherein, The liquid distribution control unit is provided with an electromagnetic valve to control the opening and closing of the liquid inlet channel and the liquid outlet channel.

6. The microfluidic perfusion culture integrated chip cartridge of claim 2, wherein, The length of the card box frame structure is 150-190 mm, the width is 80-100 mm, the thickness is 20-25 mm, the volume of the culture detection chamber is 200-300 uL, the volume of the liquid storage bag and the waste liquid bag is 5-10 mL, and the volume of the syringe pump is 200-2000 uL.

7. The microfluidic perfusion culture integrated chip cartridge of claim 2, wherein, A handle is arranged on the side of the card box frame structure.

8. The microfluidic perfusion culture integrated chip cartridge of claim 1, wherein, The air-tight electric connector is fixed on the middle frame plate, the air-tight electric connector is connected with the heating sheet, the temperature sensor and the electromagnetic valve respectively, and the external control device is plugged in.

9. A preparation method of the microfluidic perfusion culture integrated chip card box for space environment according to claim 5, characterized in that comprising the following steps: Step one: assemble the microfluidic perfusion culture module, connect the liquid storage unit, the syringe pump unit and the culture detection chamber unit to the liquid distribution control unit, and connect the lead wires of the electromagnetic valve, the heating sheet and the temperature sensor to the air-tight electric connector; Step two: fix the microfluidic perfusion culture module on the middle frame plate, including the fixation of the liquid storage bag, the waste liquid bag and the pipelines thereof; Step three: the syringe pump penetrates through the middle frame plate, the O-ring is sleeved outside the syringe pump body, the syringe pump is locked and fixed on the luer connector, the O-ring is sent into the sealing groove of the middle frame plate, the hollow sealing screw is sleeved and fixed outside the syringe pump body, and the limiting block is clamped to the push rod. Step four In the annular sealing groove of the inner and outer sides of the middle frame plate, install the sealing ring, and connect the upper and lower cover plates with the middle frame plate by bolts.

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