Living cell sterile workstation based on temperature-controllable micro-fluidic chip
By designing a live cell sterile workstation that integrates microfluidic chips, temperature control devices and sterile operation functions, the problems of complex operation, large equipment dependence and cell infection in the prior art are solved, and efficient and low-cost live cell analysis is achieved.
Patent Information
- Application Number
- CN202421625695.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-10
- Publication Date
- 2025-05-06
- Estimated Expiration
- 2034-07-10
AI Technical Summary
When used for live cell analysis, existing microfluidic chips rely on a variety of instruments and equipment, the operation is complex and the cells are prone to infection. The existing commercial live cell workstations are expensive and have small space, making it difficult to meet user needs.
A live cell sterile workstation based on a controlled temperature microfluidic chip is designed, integrating a microfluidic chip, a temperature control device, an imaging device and a sterile operating device to realize cell capture, perfusion culture and imaging observation, and reduce dependence on external devices.
A high-integration live cell workstation is achieved, which simplifies the operation process, reduces the risk of cell infection, reduces equipment dependence, reduces costs, and provides greater operation space.
Smart Images
Figure CN222834322U_ABST
Abstract
Description
Technical Field
[0001] The utility model belongs to the technical field of microfluidic chip technology and biological experimental equipment, and relates to a living cell sterile workstation based on a temperature-controllable microfluidic chip. Background Art
[0002] Cells are the basic units of structure and function of organisms known so far, except for viruses. Most of the life activities in organisms are completed at the cellular level. In order to reveal the laws of life activities, promote human cognition of life sciences, accelerate the progress of medicine, and ensure human health, it is necessary to conduct in-depth research at the cellular level and explore the life processes such as cell structure, growth and differentiation, metabolism and reproduction, movement and communication, aging and apoptosis, inheritance and evolution. As a cutting-edge technology field in scientific research, microfluidic chips have received extensive attention from researchers in life sciences and cell analysis. Microfluidic chips usually have a microchannel structure comparable to the size of cells. The size and shape of microchannels and functional units can be designed according to actual needs. They have a high degree of controllability and cell manipulation capabilities. They have become a powerful tool for cell analysis and have made significant progress and applications in cell research fields such as cell sorting, gene sequencing, and protein analysis.
[0003] Microfluidic chips are used for live cell analysis, which requires cell capture, culture, and imaging observation. These operations require nutrient solution, pH, and a sterile environment. The culture process requires a temperature of 37°C and a 5% carbon dioxide environment. In order to ensure the survival of cells, when microfluidic chips are used for live cell analysis, cell capture must be performed in a clean bench or biosafety cabinet, and the culture process requires a cell culture box, which relies on many instruments and equipment, increasing the complexity of the operation; imaging observation requires leaving the sterile and culture environment, increasing the probability of cell infection with bacteria and mycoplasma, which may lead to experimental failure. In addition, the existing commercial live cell workstation costs about 500,000 yuan, which is expensive and difficult for general users to purchase; and the small operating space is not conducive to microfluidic chip capture and perfusion culture of cells. Utility Model Content
[0004] The purpose of the utility model is to solve the problems of existing microfluidic chips used for living cell analysis, such as reliance on multiple instruments and equipment, high complexity of operation and susceptibility of cells to infection, to overcome the problems of small space and high price of existing commercial living cell workstations, and to provide a living cell sterile workstation based on a temperature-controllable microfluidic chip. Through this system, it can be used for capturing, perfusion culture and tracking observation of cells in the chip.
[0005] In order to achieve the above technical objectives, the technical solutions adopted by the utility model are as follows:
[0006] A living cell sterile workstation based on a temperature-controllable microfluidic chip comprises a platform, a cell analysis device is placed on the platform, a temperature control device is installed on the cell analysis device, the cell analysis device and the temperature control device outer cover are provided with a sterile sealing cover, an ultraviolet lamp is installed in the sterile sealing cover and the sterile sealing cover is connected to a gas cylinder.
[0007] Preferably, the platform adopts an air-floating vibration-isolated optical platform.
[0008] Preferably, the cell analysis device uses an inverted fluorescence microscope.
[0009] Preferably, the microfluidic chip adopts a PDMS microfluidic chip, the chip cover is provided with a channel for fluid transport; the chip bottom is provided with an array of micropits for capturing single cells.
[0010] Preferably, the temperature control device controls the temperature of the microfluidic chip placed on the ITO glass through the ITO glass.
[0011] More preferably, the temperature control device includes a PID temperature controller, a transformer, and a K-type thermocouple, wherein the PID temperature controller is used to set the temperature of the ITO glass; the transformer is used to convert the AC 220V voltage into a 12V DC voltage and apply it to the ITO glass, so that the ITO glass generates heat; the K-type thermocouple is used to feed back the temperature of the ITO glass to the PID temperature controller.
[0012] Preferably, it further comprises a sample injection device, and the microfluidic chip is connected to the sample injection device through an external pipeline.
[0013] Preferably, an operation port is provided on the front side of the sterile sealing cover.
[0014] Preferably, the gas cylinder is filled with 5% CO2.
[0015] Preferably, the ultraviolet lamp is installed on the top of the sterile sealing cover.
[0016] The beneficial effects of the utility model are:
[0017] (1) High integration. The living cell workstation integrates microfluidic chips, temperature control devices, imaging devices, sterile operation devices, etc., so that the process of microfluidic chip capture, observation and cell culture can be completed in the workstation.
[0018] (2) Less reliance on equipment. No clean bench or cell culture box is required, and the use of too many instruments is avoided.
[0019] (3) Easy to operate. The operation process is simple and easy for operators to master, which facilitates the promotion and use of the device.
[0020] (4) Large operating space. The sterile sealed cover has a large space and can be designed according to actual needs. The microfluidic chip captures and cultures cells, and the perfusion equipment used can be placed in the sterile sealed cover without affecting the operation process.
[0021] (5) Low risk of cell infection. The entire process is completed in situ in the living cell workstation, without the need to move to other equipment for operation, reducing the risk of cell infection during movement.
[0022] (6) Low cost. The workstation devices are all designed and built by ourselves, using low-priced raw materials and high user acceptance.
[0023] (7) Wide range of applications. This platform can be used for all imaging and analysis studies related to living cells or tissues, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 It is a schematic diagram of the structure of the utility model based on the temperature-controllable living cell sterile workstation.
[0025] Figure 2 It is a structural schematic diagram of the temperature control device in the utility model.
[0026] Figure 3 The following are mask design drawings. (a, b) chip cover mask design drawings; (c, d) chip bottom mask design drawings; (e) chip bottom micro-pit array mask design drawings.
[0027] In the figure:
[0028] 1. Air-floating vibration-isolated optical platform; 2. Inverted fluorescence microscope; 3. Temperature control device; 31. Transformer; 32. PID temperature controller; 33. K-type thermocouple; 4. Microfluidic chip; 5. Sterile sealing cover; 6. Gas cylinder. DETAILED DESCRIPTION
[0029] The present invention is described in detail below in conjunction with specific implementation methods. The following specific embodiments are helpful for those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be pointed out that, without departing from the concept of the present invention, the device can be subjected to a number of deformations or modifications. These all fall within the scope of protection of the present invention.
[0030] In the description of the present utility model, it should be noted that the terms "above", "below", etc. indicate directions or positional relationships based on the directions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present utility model and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific direction, be constructed and operated in a specific direction, and therefore cannot be understood as a limitation on the present utility model. In addition, unless otherwise clearly specified and limited, the terms "installation", "connection", etc. should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be directly connected, it can be indirectly connected through an intermediate medium, or it can be internal communication between two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present utility model can be understood according to the specific circumstances.
[0031] In addition, unless otherwise specified, the components used in the following embodiments are all existing components, and their corresponding connection methods can also be achieved through conventional technical means, which will not be described one by one in this application.
[0032] Example
[0033] Combination Figure 1 The present embodiment provides a living cell sterile workstation based on a temperature-controllable microfluidic chip, comprising an air-floating vibration-isolating optical platform 1, an inverted fluorescence microscope 2 is placed on the air-floating vibration-isolating optical platform 1, a temperature control device 3 is installed on the inverted fluorescence microscope 2, the temperature control device 3 controls the temperature of a microfluidic chip 4 placed on the ITO glass through the ITO glass, a sterile sealing cover 5 is provided on the outer cover of the inverted fluorescence microscope 2 and the temperature control device 3, an operation port may be opened on the front side of the sterile sealing cover 5 for easy operation, an ultraviolet lamp (conventional functional component, not shown in the figure, usually preferably installed on the top of the sterile sealing cover 5) is installed in the sterile sealing cover 5, the sterile sealing cover 5 is connected to a gas cylinder 6, and the gas cylinder 6 may be filled with 5% CO2.
[0034] Preferably, the microfluidic chip 4 is a PDMS microfluidic chip, the chip cover is provided with channels for fluid transport, and the chip bottom is provided with array micropits for capturing single cells.
[0035] Combination Figure 2 The temperature control device 3 includes a PID temperature controller 32, a transformer 31, and a K-type thermocouple 33. The PID temperature controller 32 is used to set the temperature of the ITO glass; the transformer 31 is used to convert the AC 220V voltage into a 12V DC voltage and apply it to the ITO glass to make the ITO glass generate heat; the K-type thermocouple 33 is used to feed back the temperature of the ITO glass to the PID temperature controller 32.
[0036] Preferably, it further comprises a sample injection device, and the microfluidic chip is connected to the sample injection device via an external pipeline.
[0037] A method for constructing the above-mentioned living cell sterile workstation is now described in detail using the PDMS microfluidic chip as an example. The specific implementation steps are as follows:
[0038] S1. Design and preparation of silicon wafer mold: Use CAD software to design the mask, such as Figure 3 As shown. After the mask design and processing is completed, a 4-inch single-polished silicon wafer is used to make a mold using photolithography. After the silicon wafer is cleaned, dried, and modified, a layer of SU-8 photoresist is applied to the single-polished surface of the silicon wafer. According to the parameters of the photoresist, the photoresist is spun with a coating machine to obtain the desired photoresist thickness (the photoresist thickness at this time is the depth of the chip channel produced later). After pre-baking, exposure, post-baking, development, hardening and other processes, the finished silicon wafer mold is prepared.
[0039] S2. Preparation of PDMS chip: After the silicon wafer mold is made, mix PDMS prepolymers A and B in a ratio of 10:1, stir evenly, and use a vacuum dryer to remove bubbles. The silicon wafer mold is silanized, and the degassed prepolymer is poured on the silicon wafer (pour on the balance to grasp the mass of the poured PDMS prepolymer, and the amount poured at this time will determine the thickness of the chip), and the silicon wafer and prepolymer are cured in a vacuum oven at 85°C for 30-60 minutes. After curing, take it out, separate the PDMS from the mold, and cut and punch the PDMS. After cleaning the PDMS, use plasma to bond the cover and bottom film of the PDMS to prepare the finished chip.
[0040] S3. Homemade temperature control device, the main preparation steps are as follows: The control output mode selected by the PID temperature controller (XMT612) is 1: relay J1 alarm output, relay J2 contact PID control output, SSR output is invalid, AH2, AL2 settings are invalid. This control mode is mainly used for constant temperature control mode, SV is the temperature setting value. Ports 1 and 2 of the PID temperature controller terminal are connected to the positive and negative poles of the 220V power supply respectively; ports 9 and 10 are connected to the positive and negative poles of the thermocouple respectively, and the sensor head of the thermocouple is attached to the ITO layer of the chip to realize real-time temperature monitoring; J2 (ports 13 and 14) is used as a switch, port 13 is connected to the copper foil of the chip, port 14 is connected to the positive pole of the 12V voltage, and the negative pole of the 12V voltage is connected to the copper foil at the other end of the chip to realize power supply and heating of the chip.
[0041] S4. After the wiring is completed, set the parameters of the temperature controller. Connect the power supply of the PID temperature controller and the power supply of the transformer respectively, and supply power to the PID temperature controller and the transformer. Then set the initial function parameters of the PID temperature controller according to the operating instructions of the PID temperature controller.
[0042] S5. After the parameter setting is completed, perform the instrument self-tuning. Set the temperature to 50℃ and perform a self-tuning to let the instrument calculate the appropriate PID parameters. Since the constant temperature selected in this experiment is 37℃, after the above self-tuning is completed, set the temperature to 37℃ and perform self-tuning again to obtain the best PID adjustment parameters. After completing the self-tuning, the temperature control operation can be carried out normally.
[0043] S6. Construction of a living cell workstation. Place an inverted fluorescence microscope on a suspended vibration-isolated optical platform. Design a sterile sealed cover that matches the inverted fluorescence microscope, with a UV lamp on the top of the sterile sealed cover, and place the inverted fluorescence microscope in the sterile sealed cover. Use a polyvinyl chloride (PVC) sealing strip to seal the gap between the sterile sealed cover and the inverted fluorescence microscope. Insulate the inverted fluorescence microscope workbench with 3M tape and set the temperature control device on the microscope workbench. Finally, connect a gas cylinder containing 5% CO2 to the sterile sealed cover to obtain a living cell workstation.
[0044] The present application can also be used to culture cells in microfluidic chips or culture dishes made of other materials and perform imaging analysis. Depending on the chip material or container for culturing cells, the preparation method of the chip or container needs to be appropriately adjusted; the other steps remain basically unchanged.
[0045] It is understandable that the material of the microfluidic chip is not limited to PDMS; the container for culturing cells is not limited to microfluidic chips; the objects of capture, culture and imaging analysis are not limited to single cells; the preparation method of the microfluidic chip is not limited to photolithography, and the internal design of the chip is not limited to micro-pits; the temperature control device is not limited to PID type temperature control equipment; the platform used for placing the sterile workstation is not limited to the air flotation vibration isolation optical platform; the cell analysis device used in the sterile workstation is not limited to an inverted fluorescence microscope; the size of the sterile sealing cover is not limited and can be adjusted according to actual needs; the culture method of cells in the chip is not limited to perfusion culture.
[0046] The working process of using the above-constructed living cell sterile workstation for the capture, cultivation and imaging observation of in situ animal cells is as follows:
[0047] S1. Connect the bonded closed PDMS chip to the external pipe, and then connect the other end of the external pipe to the manual syringe;
[0048] S2. Place the chip and perfusion device in a living cell sterile workstation, place the chip on the ITO glass, spray with 75% ethanol and sterilize with UV light;
[0049] S3. Place the chip under a microscope, adjust the syringe flow rate, and observe the cell capture status under the microscope;
[0050] S4. First, use 75% ethanol and PBS solution to rinse the inside of the chip respectively;
[0051] S5. Then adjust the flow rate of the syringe to slowly inject a certain concentration of cell suspension into the chip, then stop the injection and observe the cell capture status under a microscope. After a period of time, observe that the cells have settled into the micropits, then use the culture medium to slowly flush away the cells outside the micropits, and repeat this step 2-3 times;
[0052] S6. Finally, the culture medium is continuously and slowly injected into the chip by perfusion to culture single cells and observe the status and survival time of single cells.
[0053] Those skilled in the art can understand that the method of the above embodiment has the advantages of high integration, simple operation, strong controllability, low cost, etc., and can be easily mastered by the operator.
[0054] In this embodiment, specific examples are used to illustrate the implementation methods and obtained products of the present application. The description of the above embodiments is only used to help understand the method and core idea of the utility model; at the same time, for general technicians in this field, according to the idea of the utility model, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on the utility model.
Claims
1. A living cell sterile workstation based on a temperature-controllable microfluidic chip, characterized in that: The invention comprises a platform, on which a cell analysis device is placed, on which a temperature control device is installed, and the outer covers of the cell analysis device and the temperature control device are provided with a sterile sealing cover, in which an ultraviolet lamp is installed, and the sterile sealing cover is connected with a gas cylinder.
2. A living cell sterile workstation based on a temperature-controllable microfluidic chip according to claim 1, characterized in that: The platform adopts an air-floating vibration-isolating optical platform.
3. A living cell sterile workstation based on a temperature-controllable microfluidic chip according to claim 1 or 2, characterized in that: The cell analysis device uses an inverted fluorescence microscope.
4. A living cell sterile workstation based on a temperature-controllable microfluidic chip according to claim 1, characterized in that: The microfluidic chip adopts a PDMS microfluidic chip, a chip cover is provided with a channel for fluid transport, and an array of micropits is provided on the chip bottom film for capturing single cells.
5. A living cell sterile workstation based on a temperature-controllable microfluidic chip according to claim 1, characterized in that: The temperature control device controls the temperature of the microfluidic chip placed on the ITO glass through the ITO glass.
6. A living cell sterile workstation based on a temperature-controllable microfluidic chip according to claim 5, characterized in that: The temperature control device includes a PID temperature controller, a transformer, and a K-type thermocouple. The PID temperature controller is used to set the temperature of the ITO glass; the transformer is used to convert an AC 220V voltage into a 12V DC voltage and apply it to the ITO glass to make the ITO glass generate heat; the K-type thermocouple is used to feed back the temperature of the ITO glass to the PID temperature controller.
7. A living cell sterile workstation based on a temperature-controllable microfluidic chip according to claim 1 or 4, characterized in that: It also includes a sample injection device, and the microfluidic chip is connected to the sample injection device through an external pipeline.
8. The living cell sterile workstation based on a temperature-controllable microfluidic chip according to claim 1, characterized in that: An operation port is provided on the front side of the sterile sealing cover.
9. A living cell sterile workstation based on a temperature-controllable microfluidic chip according to claim 1, characterized in that: The gas cylinder is filled with 5% CO2.
10. A living cell sterile workstation based on a temperature-controllable microfluidic chip according to claim 1 or 8, characterized in that: The ultraviolet lamp is installed on the top of the sterile sealing cover.