Microbial culture device based on micro-fluidic chip

Abstract

The invention relates to a microbiological culture device based on a micro-fluidic chip, which is characterized in that two continuous-phase flow channels and a dispersed-phase flow channel are intersected at a cross-shaped channel, and bacterial liquid injected from a dispersed-phase inlet is sheared into first liquid drops; the culture medium inlet is communicated with the sample outlet flow channel of the cross-shaped channel through the culture medium flow channel and is intersected to form a T-shaped channel; the culture medium in the culture medium runner is attracted to move downwards through the adjustable electrode and is fused with the first liquid drop to form a second liquid drop; a splitting channel is arranged at the tail end of a sample outlet flow channel of the cross-shaped channel to form two splitting flow channels, each splitting flow channel is communicated with the corresponding culture chamber, and the splitting channel divides the second liquid drop into two target liquid drops; the culture chamber is used for loading the split target liquid drops and providing a microorganism culture environment. According to the invention, the whole process of generation, splitting, fusion and culture of the liquid drops can be realized, the liquid drop fusion precision is high, the liquid drop production and generation efficiency is high, the large-scale screening requirement can be met, and the culture of microorganisms is facilitated.

Description

Technical Field

[0001] The invention relates to the field of microfluidic technology, in particular to a microorganism cultivation device based on a microfluidic chip. Background Art

[0002] Microfluidic chip technology integrates the basic operating units of biological, chemical, and medical analysis processes, such as sample preparation, reaction, separation, and detection, onto a micrometer-scale chip to complete the entire analysis process. Microfluidic chips for microbial culture are now appearing on the market. Existing microfluidic chips have the following problems: First, they have a single function and only support droplet generation or culture, which makes the entire microbial culture experimental process fragmented and complicated; second, solid electrode materials are easily worn out, and the electric field is unevenly distributed, which will affect the selectivity and accuracy of droplet fusion; third, the droplet generation and manipulation efficiency is insufficient, making it difficult to meet large-scale screening needs. Therefore, existing microfluidic chips are not conducive to the cultivation of microorganisms. Summary of the invention

[0003] Based on this, the purpose of the present invention is to provide a microbial cultivation device based on a microfluidic chip, which adopts an integrated design and can realize the entire process of droplet generation, splitting, fusion, and cultivation. The droplet fusion has high precision and droplet production efficiency, which can meet large-scale screening needs and is conducive to the cultivation of microorganisms.

[0004] The present invention is achieved through the following technical solutions:

[0005] A microorganism culture device based on a microfluidic chip, characterized in that: it comprises a chip body, wherein a continuous phase inlet, a continuous phase flow channel, a dispersed phase inlet, a dispersed phase flow channel, a cross channel, a culture medium inlet, a culture medium flow channel, a T-shaped channel, an adjustable electrode, a split channel, and a culture chamber are arranged in the chip body;

[0006] The continuous phase inlet is connected to the cross channel through two parallel continuous phase flow channels, and the dispersed phase inlet is connected to the cross channel through the dispersed phase flow channel. The two continuous phase flow channels and the dispersed phase flow channel intersect at the cross channel, and the bacterial liquid injected from the dispersed phase inlet is sheared into the first droplet;

[0007] The culture medium inlet is connected to the sample outlet channel of the cross channel through the culture medium flow channel, and they intersect to form the T-shaped channel; the adjustable electrode acts on the T-shaped channel to attract the culture medium in the culture medium flow channel to move downward and merge with the first droplet to form a second droplet;

[0008] The end of the sample outlet channel of the cross channel is provided with the split channel, the split channel is split to form two split channels, each of the split channels is respectively connected to the corresponding culture chamber, and the split channel divides the second droplet into two target droplets;

[0009] The culture chamber is used to load the target droplets that have completed splitting and provide a microorganism culture environment.

[0010] In an optional embodiment, the system further comprises a controller, and a continuous phase driving pump, a dispersed phase driving pump, and a culture medium injection pump electrically connected to the controller; the continuous phase driving pump is used to pump the oil phase substance to the continuous phase inlet, the dispersed phase driving pump is used to pump the bacterial liquid to the dispersed phase inlet, and the culture medium injection pump is used to pump the culture medium to the culture medium inlet; the oil phase substance enters from the continuous phase inlet, flows through the two continuous phase flow channels, and flows to the cross channel, and the bacterial liquid enters from the dispersed phase inlet to the dispersed phase flow channel and flows to the cross channel;

[0011] At the cross channel, the oil phase substances that intersect each other vertically continuously shear the bacterial liquid into first droplets, and the sheared first droplets flow out from the sample outlet channel and merge with the culture medium in the T-shaped channel to form second droplets;

[0012] The second droplet continues to move along the sample outlet channel to reach the splitting channel, and the splitting channel splits the second droplet into two target droplets. The two target droplets enter the corresponding culture chambers through the two splitting channels respectively.

[0013] In an optional embodiment, the controller controls the pressure parameters of the continuous phase drive pump, the dispersed phase drive pump, and the culture medium injection pump to control the concentration and size of the target droplets; and / or,

[0014] The controller controls the operating frequencies of the continuous phase driving pump, the dispersed phase driving pump, and the culture medium injection pump to control the generation rate of target droplets; and / or,

[0015] The controller controls the voltage of the adjustable electrode to control the fusion amount of the culture medium at the T-shaped channel and the concentration of the target droplets.

[0016] In an optional embodiment, a microscope is further included; the microscope is used to take real-time photos of target droplets on the chip body, and obtain images of each target droplet and transmit them to the controller;

[0017] The controller performs image analysis on the target droplets to obtain a target droplet size and a target droplet shape, inputs the target droplet size, the target droplet shape, and control parameters corresponding to the target droplets into a neural network model for training, and continuously corrects the mapping relationship between input parameters and output results in the neural network model, wherein the input parameters are control parameters, and the control parameters include continuous phase drive pump pressure parameters, dispersed phase drive pump pressure parameters, culture medium injection pump pressure parameters, culture medium injection pump working time, and adjustable electrode voltage parameters; the output parameters include target droplet size and target droplet shape; ideal control parameters are determined based on a preset droplet size, a preset droplet shape, and a mapping relationship between input parameters and output results in the neural network model, and the microbial culture device is controlled to operate based on the ideal control parameters.

[0018] In an optional embodiment, the chip body is made of PDMS.

[0019] In an optional embodiment, an incubator is also included, and the culture temperature in the incubator is 36-38°C.

[0020] In an optional embodiment, the adjustable electrode is a liquid metal electrode or a solid nanoelectrode.

[0021] In an optional embodiment, the splitting channel has a conical divider; the conical divider divides the second droplet into two target droplets of the same size.

[0022] The present invention has the following effects: the microbial cultivation device based on the microfluidic chip of the embodiment of the present application adopts an integrated design, which can realize the whole process of droplet generation, splitting, fusion, and cultivation without human intervention, with high droplet fusion accuracy and high droplet production efficiency, which can meet large-scale screening needs and is conducive to the cultivation of microorganisms.

[0023] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the structure of a microfluidic chip in one embodiment of the present invention;

[0025] Figure 2 Schematic diagram of the regulation of the microfluidic chip in one embodiment of the present invention;

[0026] Figure 3 It is a schematic diagram of a state in which a microfluidic chip shears a first droplet in one embodiment of the present invention;

[0027] Figure 4 It is a schematic diagram of a state where a microfluidic chip in an embodiment of the present invention is fused into a second droplet;

[0028] Figure 5 FIG. 4 is a schematic diagram of the state of droplet splitting of a microfluidic chip in an embodiment of the present invention. DETAILED DESCRIPTION

[0029] It should be clear that the described embodiments are only some embodiments of the embodiments of the present application, rather than all embodiments. Based on the embodiments in the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the embodiments of the present application.

[0030] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0031] It should be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may be an intermediate element. When an element is considered to be "connected" or "fixedly connected" to another element, it may be directly connected to the other element or there may be an intermediate element at the same time.

[0032] The embodiment of the present application provides a microbial cultivation device based on a microfluidic chip, which can be applied to microbial cultivation and research, and can also be applied to drug screening, metabolic research, environmental monitoring, food industry, synthetic biology, disease research and other fields, and has broad industrial and scientific research value.

[0033] like Figure 1As shown, the microorganism culture device based on the microfluidic chip includes a chip body, and the chip body is provided with a continuous phase inlet A, a continuous phase flow channel, a dispersed phase inlet B, a dispersed phase flow channel, a cross channel 1, a culture medium inlet C, a culture medium flow channel, a T-shaped channel 2, adjustable electrodes (F, G, H, I), a split channel, and a culture chamber (D, E). The continuous phase inlet A is used to pump in oil phase substances, the continuous phase flow channel is used to move the oil phase substances, the dispersed phase inlet B is used to pump in bacterial liquid, and the dispersed phase flow channel is used to move the bacterial liquid. The cross channel 1 is used to shear to form a first droplet, the culture medium inlet C is used to pump in the culture medium, the culture medium flow channel is used to move the culture medium, the T-shaped channel 2 is used to fuse to form a second droplet, the adjustable electrodes (F, G, H, I) are used to adjust the culture medium fusion amount according to the voltage value, the split channel is used to split the second droplet to form a target droplet, and the culture chamber (D, E) is used to load the target droplet that has been split and provide a microorganism culture environment.

[0034] The continuous phase inlet A is connected to the cross channel 1 through two parallel continuous phase flow channels, and the dispersed phase inlet B is connected to the cross channel 1 through the dispersed phase flow channel. The two continuous phase flow channels and the dispersed phase flow channel intersect at the cross channel 1, and the bacterial liquid injected from the dispersed phase inlet B is sheared into the first droplet, and the oil phase material entering through the continuous phase inlet A wraps the bacterial liquid.

[0035] The culture medium inlet C is connected to the sample outlet channel of the cross channel 1 through the culture medium flow channel, and they intersect to form a T-shaped channel 2; the adjustable electrodes (F, G, H, I) act on the T-shaped channel 2 to attract the culture medium in the culture medium flow channel to move downward and merge with the first droplet to form a second droplet; the pressure of the adjustable electrodes (F, G, H, I) can be adjusted to control the downward movement of the culture medium, thereby changing the concentration of the bacterial solution.

[0036] A split channel 3 is provided at the end of the sample outlet channel of the cross channel 1. The split channel 3 splits into two split channels. Each split channel is connected to a corresponding culture chamber (D, E) respectively. The split channel 3 divides the second droplet into two target droplets.

[0037] In this embodiment, the automated cultivation of microorganisms is achieved by manipulating the combination of droplet splitting and fusion in microfluidic technology, which significantly reduces the complexity and time consumption of experimental operations and supports integrated research of the entire process from generation to cultivation.

[0038] Compared with traditional microbial culture, the independent droplet operation unit effectively reduces the risk of cross-contamination, and the miniaturized and highly automated design further saves implementation costs; the use of a cross structure to generate droplets combined with a T-shaped structure can better achieve one-to-one pairing of droplets with high fusion efficiency.

[0039] The microbial cultivation device based on a microfluidic chip in the embodiment of the present application adopts an integrated design and can realize the entire process of droplet generation, splitting, fusion, and cultivation without human intervention. The droplet fusion has high precision and droplet production efficiency is high. The sample is not easy to be cross-contaminated, and it can meet large-scale screening needs and is conducive to the cultivation of microorganisms.

[0040] In an optional embodiment, the microorganism cultivation device based on the microfluidic chip further includes a controller ( Figure 2 The controller includes a computer in the apparatus, and a continuous phase driving pump, a dispersed phase driving pump, and a culture medium injection pump electrically connected to the controller; the continuous phase driving pump is used to pump the oil phase material to the continuous phase inlet, the dispersed phase driving pump is used to pump the bacterial liquid to the dispersed phase inlet, and the culture medium injection pump is used to pump the culture medium to the culture medium inlet. The pressure-driven injection method can adjust the proportion of the injected culture medium to achieve the culture of different proportions of culture medium.

[0041] The production process of microbial droplets is as follows: the oil phase material enters from the continuous phase inlet A, flows through two continuous phase flow channels, and flows to the cross channel 1, and the bacterial liquid enters the dispersed phase flow channel from the dispersed phase inlet B and flows to the cross channel 1; at the cross channel 1, the oil phase materials that intersect each other vertically continuously shear the bacterial liquid into the first droplet, and the sheared first droplet flows out from the sample outlet flow channel and merges with the culture medium in the T-shaped channel 2 to form a second droplet; the second droplet continues to move along the sample outlet flow channel to reach the splitting channel 3, and the splitting channel 3 splits the second droplet into two target droplets, and the two target droplets enter the corresponding culture chambers (D and E) through the two splitting flow channels respectively.

[0042] In an optional embodiment, the controller controls the pressure parameters of the continuous phase drive pump, the dispersed phase drive pump, and the medium injection pump to control the concentration and size of the target droplets. Figure 2 As shown, in the flow control part, the flow control of each pump can be achieved by controlling the pressure parameters of each pump through a computer.

[0043] The controller controls the operating frequencies of the continuous phase driving pump, the dispersed phase driving pump, and the medium injection pump to control the generation rate of the target droplets.

[0044] The controller controls the voltage of the adjustable electrode to control the fusion amount of the culture medium at the T-shaped channel and the concentration of the target droplets. Figure 2 As shown, during electric field control, the power supply transmits the electrode voltage to the adjustable electrode of the microfluidic chip through the amplifier. The adjustable electrode acts on the T-shaped channel of the microfluidic chip to attract the culture medium to move downward. Different electrode voltages can attract culture medium with different flow rates to move downward, thereby controlling the formation of microdroplets with different proportions. At the same time, the amplifier transmits the electrode voltage acting on the microfluidic chip to the oscilloscope for display.

[0045] Furthermore, the device includes a microscope; the microscope is used to take real-time photos of the target droplets on the chip body, obtain images of each target droplet, and transmit them to the controller, which displays the droplet images on the display screen and presents them to the experimenter in a visual image format, so as to facilitate accurate control of the experiment and the formation of research reports. Figure 2 As shown, the electron microscope is connected to a computer, and the droplet image is collected through the electron microscope, transmitted to the computer, and displayed on the computer. Optionally, when the experiment is completed, the waste liquid on the microfluidic chip can be recycled.

[0046] In order to predict and adjust the size and shape of the target droplets, this embodiment also adjusts the pressure parameters of each pump based on the current target droplet size and target droplet shape to prepare droplets of various forms to better meet the experimental needs. Specifically, the controller performs image analysis on the target droplets to obtain the target droplet size and target droplet shape, inputs the target droplet size, target droplet shape, and control parameters corresponding to the target droplets into the neural network model for training, and continuously corrects the mapping relationship between the input parameters and the output results in the neural network model, wherein the input parameters are control parameters, and the control parameters include the continuous phase drive pump pressure parameters, the dispersed phase drive pump pressure parameters, the culture medium injection pump pressure parameters, the working time of the culture medium injection pump, and the voltage parameters of the adjustable electrode, and the output parameters include the target droplet size and target droplet shape. According to the preset droplet size, the preset droplet shape, and the mapping relationship between the input parameters and the output results in the neural network model, the ideal control parameters are determined, and the microbial culture device is controlled to work according to the ideal control parameters.

[0047] On the basis of the above embodiments, the chip body is made of PDMS. The microorganism culture device also includes an incubator, and the culture temperature in the incubator is 36-38°C, specifically 37°C. During culture, the entire microfluidic chip can be placed in the incubator and maintained at a constant temperature for a preset culture time. Specifically, a heating unit is provided under the culture chamber. The heating unit can be an electrically controlled heating wire or an electrically controlled heating plate. In this way, the temperature in the culture chamber reaches the optimum temperature for microbial culture by heating and heating the heating unit to control the temperature conditions for microbial culture. Since the microbial droplets are mainly deposited at the bottom of the culture chamber, the heating unit located at the bottom of the culture chamber can heat the culture chamber evenly.

[0048] In traditional microfluidic chips, solid electrode materials are easily worn out and the electric field is unevenly distributed, which affects the selectivity and accuracy of droplet fusion. To solve the above problems, in this embodiment, the adjustable electrode is a liquid metal electrode or a solid nanoelectrode to enhance the uniformity and controllability of the electric field, while reducing the complexity of chip manufacturing, improving the accuracy of droplet fusion, reducing experimental costs, and extending the service life of the chip. At the same time, the active method of electric fusion is adopted, which greatly improves the efficiency compared to the passive fusion method and can meet the needs of high-throughput experiments.

[0049] In addition, if the fusion failure rate is high, the electric field strength (voltage) and frequency are dynamically adjusted to improve the fusion success rate; if the adjustable electrode overheats or the droplet ruptures, the electric field strength is reduced to protect the experimental conditions.

[0050] The splitting channel has a conical divider; the splitting flow channels on both sides of the conical divider have the same diameter, and the conical divider divides the second droplet into two target droplets of the same size. Adding a conical divider greatly improves the splitting efficiency and assists in achieving high-throughput experimental requirements.

[0051] like Figures 3 to 5 As shown, at the cross channel 1, the oil phase material shears the bacterial liquid into the first droplet. At the T-shaped channel 2, a certain voltage is applied to the adjustable electrode to attract the culture medium to move downward and merge with the first droplet to form a second droplet. The second droplet reaches the splitting channel 3 and is divided into two target droplets by the conical splitting channel. The target droplets enter different culture chambers for culture.

[0052] Specifically, during the research process, the inventors were able to obtain at least the following morphological images of microbial droplets for observation.

[0053] Set the continuous phase driving pump pressure to 1.0psi, the dispersed phase driving pump pressure to 1.0psi, the injection pressure of the culture medium injection pump to 1.0psi, the injected liquid volume to about 20nl, and the adjustable electrode voltage to 300v to prepare spherical droplets with a size of 100um.

[0054] Set the continuous phase driving pump pressure to 1.0psi, the dispersed phase driving pump pressure to 1.1psi, the injection pressure of the culture medium injection pump to 1.0psi, the injected liquid volume to about 20nl, and the voltage of the adjustable electrode to 300v, then kidney-shaped droplets can be prepared with a size of 100um.

[0055] The continuous phase driving pump pressure is set to 1.0psi, the dispersed phase driving pump pressure is set to 1.1psi, the injection pressure of the culture medium injection pump is set to 1.0psi, the injected liquid volume is about 20nl, and the voltage of the adjustable electrode is set to 300v. Spherical droplets can be prepared with a spherical droplet size of 130um.

[0056] The continuous phase driving pump pressure is set to 1.0psi, the dispersed phase driving pump pressure is set to 1.1psi, the injection pressure of the culture medium injection pump is set to 1.2psi, the injected liquid volume is about 30nl, and the voltage of the adjustable electrode is set to 300v. Spherical droplets can be prepared with a spherical droplet size of 130um.

[0057] Set the continuous phase driving pump pressure to 1.0psi, the dispersed phase driving pump pressure to 1.1psi, the injection pressure of the culture medium injection pump to 1.2psi, the injected liquid volume to about 30nl, and the voltage of the adjustable electrode to 270v, then kidney-shaped droplets can be prepared with a size of 130um.

[0058] Compared with the prior art, the microbial cultivation device based on a microfluidic chip in the embodiment of the present application adopts an integrated design, which can realize the entire process of droplet generation, splitting, fusion, and cultivation without human intervention. The droplet fusion has high precision and droplet production efficiency, which can meet large-scale screening needs and is conducive to the cultivation of microorganisms.

[0059] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and the present invention is also intended to include these modifications and modifications.

Claims

1. A microorganism cultivation device based on a microfluidic chip, characterized in that: The chip body comprises a continuous phase inlet, a continuous phase flow channel, a dispersed phase inlet, a dispersed phase flow channel, a cross channel, a culture medium inlet, a culture medium flow channel, a T-shaped channel, an adjustable electrode, a splitting channel, and a culture chamber; The continuous phase inlet is connected to the cross channel through two parallel continuous phase flow channels, and the dispersed phase inlet is connected to the cross channel through the dispersed phase flow channel. The two continuous phase flow channels and the dispersed phase flow channel intersect at the cross channel, and the bacterial liquid injected from the dispersed phase inlet is sheared into the first droplet; The culture medium inlet is connected with the sample outlet flow channel of the cross channel through the culture medium flow channel, and they intersect to form the T-shaped channel; The adjustable electrode acts on the T-shaped channel to attract the culture medium in the culture medium flow channel to move downward and merge with the first droplet to form a second droplet; The end of the sample outlet channel of the cross channel is provided with the split channel, the split channel is split to form two split channels, each of the split channels is respectively connected to the corresponding culture chamber, and the split channel divides the second droplet into two target droplets; The culture chamber is used to load the target droplets that have completed splitting and provide a microorganism culture environment.

2. The microorganism cultivation device based on a microfluidic chip according to claim 1, characterized in that: Also includes a controller, and a continuous phase driving pump, a dispersed phase driving pump, and a culture medium injection pump electrically connected to the controller; The continuous phase driving pump is used to pump the oil phase material to the continuous phase inlet, the dispersed phase driving pump is used to pump the bacterial liquid to the dispersed phase inlet, and the culture medium injection pump is used to pump the culture medium to the culture medium inlet.

3. The microorganism cultivation device based on a microfluidic chip according to claim 2, characterized in that: The controller controls the pressure parameters of the continuous phase drive pump, the dispersed phase drive pump, and the culture medium injection pump to control the concentration and size of the target droplets; and / or, The controller controls the operating frequencies of the continuous phase driving pump, the dispersed phase driving pump, and the culture medium injection pump to control the generation rate of target droplets; and / or, The controller controls the voltage of the adjustable electrode to control the fusion amount of the culture medium at the T-shaped channel and the concentration of the target droplets.

4. The microorganism cultivation device based on a microfluidic chip according to claim 3, characterized in that: It also includes a microscope; the microscope is used to take real-time photos of target droplets on the chip body, and obtain images of each target droplet and transmit them to the controller; The controller performs image analysis on the target droplets to obtain a target droplet size and a target droplet shape, inputs the target droplet size, the target droplet shape, and control parameters corresponding to the target droplets into a neural network model for training, and continuously corrects the mapping relationship between input parameters and output results in the neural network model, wherein the input parameters are control parameters, and the control parameters include continuous phase drive pump pressure parameters, dispersed phase drive pump pressure parameters, culture medium injection pump pressure parameters, culture medium injection pump working time, and adjustable electrode voltage parameters; the output parameters include target droplet size and target droplet shape; ideal control parameters are determined based on a preset droplet size, a preset droplet shape, and a mapping relationship between input parameters and output results in the neural network model, and the microbial culture device is controlled to operate based on the ideal control parameters.

5. The microorganism cultivation device based on a microfluidic chip according to claim 1, characterized in that: The chip body is made of PDMS.

6. The microorganism cultivation device based on a microfluidic chip according to claim 1, characterized in that: The method also includes an incubator, wherein the culture temperature in the incubator is 36 to 38°C.

7. The microorganism cultivation device based on a microfluidic chip according to claim 1, characterized in that: The adjustable electrode is a liquid metal electrode or a solid nanoelectrode.

8. The microorganism cultivation device based on a microfluidic chip according to claim 1, characterized in that: The splitting channel has a conical divider; the conical divider divides the second liquid droplet into two target liquid droplets of the same size.

Images