WO2024012029A1

WO2024012029A1 - Microfluidic perfusion culture integrated chip cartridge for space environment and preparation method therefor

Info

Publication number: WO2024012029A1
Application number: PCT/CN2023/093003
Authority: WO
Inventors: 邓玉林; 徐建栋; 张莉明; 任培
Original assignee: 北京理工亘舒科技有限公司
Priority date: 2022-07-11
Filing date: 2023-05-09
Publication date: 2024-01-18
Also published as: CN115011473A

Abstract

A microfluidic perfusion culture integrated chip cartridge for the space environment, comprising a cartridge frame structural body, a microfluidic perfusion culture module and an airtight electric connector. The cartridge frame structural body is of a full-sealing structure formed by an intermediate frame plate, an upper cover plate and a lower cover plate, and the microfluidic perfusion culture module is sealed in the cartridge frame structural body. The cartridge frame structural body is connected to an external control device by means of the airtight electric connector. The microfluidic perfusion culture integrated chip cartridge is fully sealed and microorganisms therein are isolated from external operators, so that the cartridge is suitable for being used in a space environment.

Description

A microfluidic perfusion culture integrated chip cartridge for space environment and its preparation method

Cross-references to related applications

This application requires priority for the Chinese patent application with application number 202210808260.6 and titled "A microfluidic perfusion culture integrated chip cartridge for space environment and its preparation method" submitted to the China State Intellectual Property Office on July 11, 2022. rights, the entire contents of which are incorporated herein by reference.

Technical field

The present disclosure belongs to the field of biomedical detection, and specifically relates to a microfluidic perfusion culture integrated chip cartridge for space environment and a preparation method thereof.

Background technique

In the direction of space life science and biotechnology, the long-term microgravity, regular magnetic field, rapid alternation of day and night, and special radiation provided by the space station are used to promote the understanding and understanding of the nature of life phenomena and explore scientific laws. The goal is to carry out research on the induction of gravity changes by living bodies, damage under space radiation, as well as the origin, evolution, development and reproduction of life, carry out research on the development and application of biotechnology using space conditions, and promote the development of modern life sciences and biotechnology. Serve human health and social progress.

In space environments, higher sealing performance requirements for cell culture devices are required. The perfusion culture chip and perfusion equipment in the related art require liquid pipeline connection, and it is inconvenient to replace the chip and reconnect the pipeline in a space environment. If the chip is connected to an external perfusion pipeline and is exposed inside the device, there is a risk of liquid leakage, which will affect the normal operation of the device and even pollute the space environment. If the chip and perfusion equipment are integrated, the mechanical and control electrical components cannot be reused, resulting in a waste of load.

Contents of the invention

Some embodiments of the present disclosure provide a replaceable microfluidic perfusion culture integrated chip cartridge suitable for space environments. The microfluidic perfusion culture integrated chip cartridge provided by some embodiments of the present disclosure integrates the culture medium, culture chamber, liquid flow path, control valve and other components required for microbial culture into the microfluidic chip cartridge, providing a comprehensive The closed microbial culture and detection environment reduces the difficulty of manual replacement of microbial culture chips and at the same time avoids the risk of biological sample leakage during the replacement process.

Some embodiments of the present disclosure provide an integrated chip cartridge for microfluidic perfusion culture in space environments, which includes a cartridge frame structure, a microfluidic perfusion culture module and an airtight electrical connector. The cartridge frame structure is composed of a middle frame plate , the upper and lower covers form a fully sealed structure, the microfluidic perfusion culture module is enclosed inside the cartridge frame structure, and the cartridge frame structure is connected to external control equipment through an airtight electrical connector.

In some optional embodiments, the microfluidic perfusion culture module and the air-tight electrical connector are provided in the middle frame plate, and annular sealing grooves are provided on both sides of the middle frame plate corresponding to the upper and lower cover plates. There is a sealing ring, and the upper and lower cover plates and the middle frame plate are fixed with bolts.

In some optional embodiments, the microfluidic perfusion culture module includes a liquid storage unit, a syringe pump unit, a liquid dispensing control unit and a culture detection chamber unit.

In some optional embodiments, the culture detection chamber unit is fixed on the liquid dispensing control unit. The culture detection chamber unit consists of an upper cover plate and a middle well plate. The upper cover plate and the middle well plate form a sealed culture chamber. There are heating plates and temperature sensors between the orifice plates. There are microbial filter membranes and sealing gaskets between the middle orifice plate and the bottom liquid separation control unit. The culture chamber and the flow path are connected through the filter membrane.

In some optional embodiments, the liquid storage bag and waste liquid bag of the liquid storage unit are fixed in the middle frame plate through bolts, and the liquid storage port and waste liquid port are respectively connected to the liquid inlet channel of the liquid dispensing control unit. On the liquid outlet channel, the syringe pump unit is connected to the dispensing control unit through a Luer connector and is connected to the liquid path of the liquid storage bag. The syringe pump body passes through the side wall of the middle frame plate, and the inside of the middle frame plate corresponds to the syringe pump body. There is a sealing groove, and an O-ring is provided in the sealing groove.

In some optional embodiments, hollow sealing screws are provided on the outside of the middle frame plate to fix the syringe pump body extending out of the middle frame plate, and removable limit blocks are provided on the outside of the middle frame plate for limiting storage. The plunger of the syringe pump moves laterally.

In some optional embodiments, the liquid separation control unit is provided with a solenoid valve to control the opening and closing of the liquid inlet channel and the liquid outlet channel.

In some optional embodiments, the length of the cartridge frame structure is 150-190mm, the width is 80-100mm, and the thickness is 20-25mm. The volume of the culture detection chamber is 200-300uL, and the liquid storage bag, waste The volume of the liquid bag is 5-10mL, and the volume of the syringe pump is 200-2000uL.

In some optional embodiments, the length of the cartridge frame structure may be 155 mm, the width of the cartridge frame structure may be 85 mm, and the thickness of the cartridge frame structure may be 24 mm.

In some optional embodiments, the volume of the culture detection chamber may be 300 uL.

In some optional embodiments, the volume of the liquid storage bag of the liquid storage unit may be 6 mL, and the volume of the waste liquid bag of the liquid storage unit may be 6 mL.

In some optional embodiments, the volume of the syringe pump of the syringe pump unit may be 1 mL.

In some optional embodiments, a handle is provided on the side of the cartridge frame structure.

In some optional embodiments, the air-tight electrical connector is fixed on the middle frame plate, the air-tight electrical connector is connected to the heating plate, the temperature sensor and the solenoid valve respectively, and is plugged into the control device externally.

In some optional embodiments, the airtight electrical connector can be connected to an external controller to control the electrical components inside the microfluidic perfusion culture integrated chip cartridge.

In some optional embodiments, the airtight electrical connector can be connected to an external power source.

Some embodiments of the present disclosure also provide a method for preparing an integrated chip cartridge for microfluidic perfusion culture in a space environment, including the following steps:

Step 1: Assemble the microfluidic perfusion culture module and connect the liquid storage unit, syringe pump unit, and culture detection chamber unit On the liquid separation control unit, each functional unit is pre-installed with microbial dry powder, culture medium, etc. as required, and the leads of the solenoid valve, heating film and temperature sensor are connected to an airtight electrical connector;

Step 2: Fixedly connect the microfluidic perfusion culture module to the middle frame plate, including the fixation of the liquid storage bag, waste liquid bag and their pipelines;

Step 3: The syringe pump passes through the middle frame plate, and the O-ring is placed outside the syringe pump body. The syringe pump is locked and fixed on the Luer connector. The O-ring is fed into the sealing groove of the middle frame plate. The hollow sealing screw set is fixed on the syringe pump. Outside the body, the limit block snaps into the push rod;

Step 4: Install sealing rings in the annular sealing grooves on both sides of the middle frame plate, and connect the upper and lower cover plates to the middle frame plate with bolts.

Using the above technical solutions, some embodiments of the present disclosure have at least the following beneficial effects:

(1) The microfluidic perfusion culture integrated chip cartridge is fully enclosed, and the microorganisms inside are isolated from outside operators, making it suitable for use in space environments.

(2) Fully integrated. The culture medium, perfusion pump, growth detection chamber, constant temperature components and waste liquid storage required for microbial growth are all designed as unit components and integrated in the module.

(3) Easy to store, the module separately builds the sterilized culture medium and freeze-dried bacterial powder and stores them in a cold storage. Easy to operate, adopts modular design and works with external control equipment. When using it, the operator only needs to take the card box out of the package, insert the external device as required, and start the program.

Description of drawings

Figure 1 is a schematic structural diagram of a microfluidic perfusion culture integrated chip cartridge provided by some embodiments of the present disclosure;

Figure 2 is a schematic structural diagram of a microfluidic perfusion culture module provided by some embodiments of the present disclosure;

Figure 3 is a schematic side structural view of a culture detection chamber provided by some embodiments of the present disclosure.

Among them: 1-cassette frame structure, 2-airtight electrical connector, 3-microfluidic perfusion culture module, 4-culture detection chamber unit, 41-upper cover, 42-heating plate, 43-filtration Membrane, 44-sealing ring, 5-liquid storage bag, 6-waste liquid bag, 7-syringe pump body, 8-hollow sealing screw, 9-limit block, 10-solenoid valve, 11-handle.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present disclosure more clear, the present disclosure will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the structural diagrams and specific embodiments described here are only used to explain the present disclosure and are not used to limit the present disclosure.

As shown in Figures 1 and 2, some embodiments of the present disclosure provide an integrated chip cartridge for microfluidic perfusion culture in a space environment. The microfluidic perfusion culture integrated chip cartridge includes a cartridge frame structure 1, a microfluidic perfusion culture module 3 and an airtight electrical connector 2. The cartridge frame structure can be formed into a fully sealed structure by the middle frame plate, the upper cover plate and the lower cover plate, and the microfluidic perfusion culture module 3 can be enclosed inside the cartridge frame structure 1 . The cartridge frame structure 1 can Connect to external control equipment through airtight electrical connector 2.

The microfluidic perfusion culture module 3 and the airtight electrical connector 2 can be arranged in the middle frame plate, and the middle frame plate can be provided with annular sealing grooves on both sides corresponding to the upper and lower cover plates. A sealing ring can be provided in the annular sealing groove, and the upper and lower cover plates and the middle frame plate can be fixed by bolts. The microfluidic perfusion culture module 3 can be used to culture and detect biological samples, and the airtight electrical connector 2 can be used to connect to an external power supply and controller to control the electrical components inside the integrated chip card box. Through the set perfusion culture program, the perfusion culture cartridge is controlled to achieve a constant culture temperature, resuscitate the microorganisms to be cultured, provide regular and quantitative culture medium perfusion, and detect the OD value and fluorescence value data of the microorganisms.

In some optional embodiments, the length of the cartridge frame structure may be 150-190 mm, the width may be 80-100 mm, and the thickness may be 20-25 mm. In other optional embodiments, the length of the cartridge frame structure may be 155 mm, the width may be 85 mm, and the thickness may be 24 mm. A handle can be provided on the side of the card box frame structure. The operator only touches the outer surface of the perfusion culture cartridge to avoid microbial culture failure due to contamination introduced by personnel during operation; at the same time, the microorganisms inside the cartridge will not leak into the environment where the operator is located to avoid contamination of the environment.

As shown in Figure 3, in some embodiments of the present disclosure, the microfluidic perfusion culture module may include a liquid storage unit, a syringe pump unit, a liquid dispensing control unit and a culture detection chamber unit. The culture detection chamber unit 4 can be fixed on the liquid dispensing control unit. The culture detection chamber unit may be composed of an upper cover plate 41 and a middle well plate to form a sealed culture chamber. A heating plate 42 and a temperature sensor may be provided between the upper cover plate and the middle orifice plate. A microbial filter membrane 43 and a sealing gasket 44 are provided between the middle orifice plate and the liquid separation control unit located below the middle orifice plate, and the culture chamber is connected to the flow path through the microbial filter membrane. The volume of the culture detection chamber can be 200~300uL. In some optional embodiments, the volume of the culture detection chamber is 300uL.

The liquid storage bag 5 and waste liquid bag 6 of the liquid storage unit can be fixed in the middle frame plate through bolts. The liquid storage port of the liquid storage bag 5 and the waste liquid port of the waste liquid bag 6 are respectively connected to the liquid inlet channel and the liquid outlet channel of the liquid dispensing control unit. The volumes of the liquid storage bag and waste liquid bag can be 5 to 10 mL respectively. In some optional embodiments, the volume of the liquid storage bag is 6 mL. In some optional embodiments, the volume of the waste liquid bag is 6 mL.

The syringe pump body 7 can be placed in the middle frame plate, fixed on the liquid dispensing control unit through a Luer connector, and connected to the liquid path of the liquid storage bag. The volume of the syringe pump can be 1-2mL. In some optional embodiments, the volume of the syringe pump is 1 mL. The syringe pump body can pass through the side wall of the middle frame plate. The inner side of the middle frame plate is provided with a sealing groove at a position corresponding to the syringe pump body, and an O-ring is provided in the sealing groove.

Hollow sealing screws 8 can be provided on the outside of the middle frame plate to fix the syringe pump body extending out of the middle frame plate. A removable limiting block 9 can be provided on the outside of the middle frame plate to limit the lateral movement of the push rod of the syringe pump during storage.

The liquid separation control unit may be 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 can be adapted to the number of culture chambers. The air-tight electrical connector can be fixed on the middle frame plate. The air-tight electrical connector Heating plates, temperature sensors and/or solenoid valves can be connected separately and plugged into external control devices. The control device can provide switch control of the two-way solenoid valve, real-time temperature reading of the temperature sensor and/or power supply of the heating film, etc. In addition, the external control device also provides the push and pull power of the syringe pump.

The microfluidic perfusion culture integrated chip cartridge recovery and culture control process provided by some embodiments of the present disclosure can be:

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

2) First, control the heating film to start heating, and at the same time, the temperature sensor monitors the temperature of the culture detection chamber unit, and feedback adjusts the heating film so that the temperature of the chamber reaches and remains constant at 37°C;

3) Microbial recovery stage:

a. The device pulls the syringe pump push rod of the chip cartridge and absorbs 800uL of culture medium in the liquid storage bag;

b. Open the solenoid valve 1, connect the flow path between the syringe pump and culture chamber 1, and push the syringe pump to infuse 400uL culture medium into culture chamber 1;

c. Keep valve 1 open, open valve 2, switch the syringe pump flow path to culture chamber 2, continue to push the syringe pump to infuse 400uL culture medium, and complete the liquid perfusion of the two chambers;

d. Close valve 1 and valve 2 and keep it at a constant temperature of 37°C for 1 hour to allow the dry microbial powder to completely recover.

4) Continuous perfusion culture and detection stage:

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

b. The device pulls the syringe pump push rod of the chip cartridge and absorbs 800uL of culture medium in the liquid storage bag;

c. Open the solenoid valve 1, connect the flow path between the syringe pump and culture chamber 1, and push the syringe pump to infuse 15uL culture medium into culture chamber 1;

d. Keep valve 1 open, open valve 2, switch the syringe pump flow path to culture chamber 2, continue to push the syringe pump to infuse 15uL culture medium, and complete the liquid perfusion of the two chambers;

e. Close valve 1 and valve 2, maintain static culture for 10 minutes, and then repeat steps c and d to complete intermittent continuous perfusion;

f. If necessary, the test can be completed before each perfusion, or continuous testing can be performed;

g. The entire perfusion culture process can last 8 to 12 hours according to experimental needs. If the amount of liquid in the syringe pump is insufficient during the process, you can repeat step b and continue to draw from the liquid storage bag.

5) Ending and closing stage:

a. After the culture is completed, the equipment will automatically return the syringe pump to zero, stop heating, and close all solenoid valves;

b. Take out the perfusion culture integrated chip cartridge, package it and discard it.

Step 1: Assemble the microfluidic perfusion culture module. The assembly process includes connecting the liquid storage unit, syringe pump unit, and culture detection chamber unit to the liquid separation control unit. Each functional unit is pre-installed with microbial dry powder, culture medium, etc. as needed. , in which the leads of the solenoid valve, heating film and temperature sensor are connected to an airtight electrical connector;

After experimental testing, the cartridge provided by some embodiments of the present disclosure can provide overall sealing, and even when the internal pressure of the cartridge reaches 1.2 atmospheres, it can still ensure that the contents of the cartridge will not leak. The cartridge according to some embodiments of the present disclosure can ensure that there is no risk of leakage in a vacuum environment or in extreme situations when the external pressure of the cartridge is completely lost.

The above-described embodiments only represent some implementations of the present disclosure, and their descriptions are relatively specific and detailed, but should not be construed as limiting the patent scope of the present disclosure. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present disclosure, and these all fall within the protection scope of the present disclosure. Therefore, the protection scope of the patent disclosed shall be determined by the appended claims.

Industrial applicability

Some embodiments of the present disclosure provide a microfluidic perfusion culture integrated chip cartridge for space environment and a preparation method thereof. The cartridge includes a cartridge frame structure, a microfluidic perfusion culture module and an airtight electrical connector. The cartridge frame structure is composed of a middle frame plate, an upper cover plate and a lower cover plate to form a fully sealed structure. The microfluidic perfusion culture module The module is enclosed within the cartridge frame structure, and the cartridge frame structure is connected to external control equipment through an airtight electrical connector. The microfluidic perfusion culture integrated chip cartridge provided by some embodiments of the present disclosure is fully enclosed, and the microorganisms inside are isolated from external operators, and is suitable for use in space environments. The microfluidic perfusion culture integrated chip cartridge provided by some embodiments of the present disclosure is fully integrated. The culture medium, perfusion pump, growth detection chamber, constant temperature components and waste liquid storage required for microbial growth are all designed as unit components and integrated in in the module. The microfluidic perfusion culture integrated chip cartridge provided by some embodiments of the present disclosure is easy to store. The module separately builds the sterilized culture medium and freeze-dried bacterial powder and stores them in cold storage. The microfluidic perfusion culture integrated chip cartridge provided by some embodiments of the present disclosure is easy to operate, adopts a modular design, and works with external control equipment. When using it, the operator only needs to take the card box out of the package, insert the external device as required, and start the program.

In addition, it can be understood that the microfluidic perfusion culture integrated chip cartridge for space environment of the present application is reproducible and can be used in a variety of industrial applications. For example, the microfluidic perfusion culture integrated chip cartridge of the present application for space environment can be used in any device that requires bacterial culture.

Claims

An integrated chip cartridge for microfluidic perfusion culture in space environment, including a cartridge frame structure, a microfluidic perfusion culture module and an airtight electrical connector. It is characterized in that the cartridge frame structure consists of a middle frame plate, an upper The cover plate and the lower cover plate form a fully sealed structure. The microfluidic perfusion culture module is enclosed inside the cartridge frame structure. The cartridge frame structure is connected to external control equipment through an airtight electrical connector.
The microfluidic perfusion culture integrated chip cartridge according to claim 1, characterized in that the microfluidic perfusion culture module and the air-tight electrical connector are arranged in a middle frame plate, and the middle frame plate is in contact with the An annular sealing groove is provided on the corresponding two sides of the upper cover plate and the lower cover plate, and a sealing ring is provided in the annular sealing groove. The upper cover plate and the lower cover plate are respectively fixed with the middle frame plate through bolts. .
The microfluidic perfusion culture integrated chip cartridge according to claim 1 or 2, characterized in that the microfluidic perfusion culture module includes a liquid storage unit, a syringe pump unit, a liquid dispensing control unit and a culture detection chamber unit .
The microfluidic perfusion culture integrated chip cartridge according to claim 3, characterized in that the culture detection chamber unit is fixed on the liquid separation control unit, and the culture detection chamber unit consists of an upper cover plate and a middle hole. The plates form a sealed culture chamber. A heating plate and a temperature sensor are provided between the upper cover plate and the middle orifice plate of the culture detection chamber unit. The middle orifice plate and the liquid separation control located below the middle orifice plate A microbial filter membrane and a sealing gasket are provided between the units, and the culture chamber is connected to the flow path through the microbial filter membrane.
The microfluidic perfusion culture integrated chip cartridge according to claim 3 or 4, characterized in that the liquid storage bag and waste liquid bag of the liquid storage unit are fixed in the middle frame plate through bolts, and the liquid storage bag The liquid storage port and the waste liquid port of the waste liquid bag are respectively connected to the liquid inlet channel and the liquid outlet channel of the liquid dispensing control unit, and the syringe pump unit is connected to the liquid dispensing control unit through a Luer connector. It is connected to the liquid path of the liquid storage bag. The syringe pump body passes through the side wall of the middle frame plate. There is a sealing groove on the inside of the middle frame plate at a position corresponding to the syringe pump body. There is an O-ring in the sealing groove. .
The microfluidic perfusion culture integrated chip cartridge according to claim 5, wherein a hollow sealing screw is provided on the outside of the middle frame plate to fix the syringe pump body extending out of the middle frame plate. A removable limit block is provided on the outside of the middle frame plate to limit the lateral movement of the push rod of the syringe pump during storage.
The microfluidic perfusion culture integrated chip cartridge according to any one of claims 3 to 5, characterized in that the liquid separation control unit is provided with a solenoid valve to control the liquid separation control unit. Opening and closing of the liquid inlet channel and liquid outlet channel.
The microfluidic perfusion culture integrated chip cartridge according to any one of claims 3 to 7, characterized in that the length of the cartridge frame structure is 150-190mm, the width is 80-100mm, and the thickness is 20-25mm, the volume of the culture detection chamber is 200-300uL, the volume of the liquid storage bag of the liquid storage unit is 5-10mL, the volume of the waste liquid bag of the liquid storage unit is 5-10mL, so The volume of the syringe pump of the syringe pump unit is 200-2000uL.
The microfluidic perfusion culture integrated chip cartridge according to any one of claims 3 to 7, characterized in that the length of the cartridge frame structure is 155 mm, and the width of the cartridge frame structure is 85mm, and the thickness of the cartridge frame structure is 24mm.
The microfluidic perfusion culture integrated chip cartridge according to any one of claims 3 to 7, wherein the volume of the culture detection chamber is 300 uL.
The microfluidic perfusion culture integrated chip cartridge according to any one of claims 3 to 7, characterized in that the volume of the liquid storage bag of the liquid storage unit is 6 mL, and the waste of the liquid storage unit The volume of the liquid bag is 6mL.
The microfluidic perfusion culture integrated chip cartridge according to any one of claims 3 to 7, wherein the volume of the syringe pump of the syringe pump unit is 1 mL.
The microfluidic perfusion culture integrated chip cartridge according to claim 2, characterized in that a handle is provided on the side of the cartridge frame structure.
The microfluidic perfusion culture integrated chip cartridge according to claim 4 or 7, characterized in that an airtight electrical connector is fixed on the middle frame plate, and the airtight electrical connector is connected to the The heating plate, the temperature sensor included in the culture detection chamber unit and/or the solenoid valve included in the shunt control unit are connected and plugged into the external control device.
The microfluidic perfusion culture integrated chip cartridge according to any one of claims 1 to 14, characterized in that the airtight electrical connector is connected to an external controller to control the microfluidic perfusion. The electrical components inside the culture integrated chip cartridge are controlled.
The microfluidic perfusion culture integrated chip cartridge according to any one of claims 1 to 14, wherein the airtight electrical connector is connected to an external power supply.
A method for preparing an integrated chip cartridge for microfluidic perfusion culture in a space environment, which is characterized in that the preparation method includes the following steps:

Step 1: Assemble the microfluidic perfusion culture module of claim 4, connect the liquid storage unit, syringe pump unit, and culture detection chamber unit to the liquid dispensing control unit, and connect the leads of the solenoid valve, heating film, and temperature sensor to Airtight electrical connector connection;

Step 2: Fixedly connect the microfluidic perfusion culture module to the middle frame plate, including the fixation of the liquid storage bag, waste liquid bag and their pipelines;

Step 3: The syringe pump passes through the middle frame plate, and the O-ring is placed outside the syringe pump body. The syringe pump is locked and fixed on the Luer connector. The O-ring is fed into the sealing groove of the middle frame plate. The hollow sealing screw set is fixed on the syringe pump. Outside the body, the limit block snaps into the push rod;

Step 4: Install sealing rings in the annular sealing grooves on both sides of the middle frame plate, and connect the upper and lower cover plates to the middle frame plate with bolts.