CN114602564A - Droplet microfluidic system and control method - Google Patents
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Abstract
The invention discloses a liquid drop microfluidic system and a control method, wherein the system comprises a first oil phase injection pump, a second oil phase injection pump and a microfluidic pipeline; the output port of the first oil phase injection pump is connected to the first input end of the micro-flow pipeline; the output port of the second oil phase injection pump is connected to the second input end of the micro-flow pipeline; the first oil phase injection pump is used for injecting a first oil phase reagent into the microfluidic pipeline to form a first microfluidic liquid drop; the second oil phase injection pump is used for injecting a second oil phase reagent into the first microflow liquid drops to form a plurality of second microflow liquid drops in the first microflow liquid drops, and the second microflow liquid drops are wrapped with sample reagents; the scheme has a simple structure, the operation method is more convenient and fast, the generation process of the liquid drops can be accurately controlled, the pollution and the damage of human factors, an external electric field and a temperature field to the sample are obviously reduced, the loss of the sample is reduced, and the method can be widely applied to the technical field of micro-fluidic.
Description
Technical Field
The invention relates to the technical field of microfluidics, in particular to a droplet microfluidics system and a control method.
Background
Droplet microfluidics is an important branch in the research of microfluidic chips, and is a discontinuous flow microfluidic technology developed on the basis of the traditional continuous flow microfluidic system in recent years, which utilizes two (such as water and oil) or a plurality of liquid phases which are not mutually soluble to generate dispersed micro droplets for experimental operation. With the rapid development of modern high-tech science and technology, micro-droplets in the droplet microfluidic technology have been widely applied to the fields of medicine, biology, power, chemical industry, nuclear energy, petroleum, metallurgy and the like due to the unique hydrodynamic characteristics and micro-scale effect of the micro-droplets. The size, stability and uniformity of droplet generation seriously affect the application effect.
The droplet microfluidic technology mainly comprises droplet generation, splitting and fusion, content analysis and the like. Wherein, the generation of the liquid drop is realized by the combined action of the shearing force and the surface tension of oil-water two phases; the splitting of the liquid drop refers to a liquid drop control technology for splitting one liquid drop into a plurality of liquid drops, and the volume and the concentration of inclusions of the liquid drop can be further regulated and controlled after the liquid drop is generated, so that a high-throughput analysis result is obtained.
In the prior art, a mechanical pump and valve system is generally used for controlling the droplet microfluidic chip. However, in the related systems in the prior art, instruments and equipment are expensive to process, a pump valve system is difficult to obtain flow field stability, and precise control is difficult to realize; and the applied electric field or temperature field is destructive to the liquid drop. Therefore, in the existing research based on the droplet microfluidic technology, a droplet control technology which is simpler, more convenient, mild in operation condition, strong in activity and high in flexibility is urgently needed.
Disclosure of Invention
In view of the above, to at least partially solve one of the above technical problems, an embodiment of the present invention is to provide a droplet microfluidic system and a corresponding control method, so that a channel applied to continuous flow PCR or a cavity of chip-based PCR can meet the requirements of different PCR formats.
The technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides a droplet microfluidic system, which comprises a first oil phase injection pump, a second oil phase injection pump and a microfluidic pipeline; the output port of the first oil phase injection pump is connected to the first input end of the microfluidic pipeline; the output port of the second oil phase injection pump is connected to the second input end of the microfluidic pipeline;
the first oil phase injection pump is used for injecting a first oil phase reagent into the microfluidic pipeline to form a first microfluidic liquid drop; the second oil phase injection pump is used for injecting a second oil phase reagent into the first micro-fluid liquid drop, a plurality of second micro-fluid liquid drops are formed in the first micro-fluid liquid drop, and the second micro-fluid liquid drops are wrapped with sample reagents.
In some optional embodiments, the microfluidic system further comprises at least one sample syringe pump, an output port of the sample syringe being connected to a second input of the microfluidic channel;
the sample injection pump is used for injecting sample reagents into the second microfluidic droplets.
In some optional embodiments, the microfluidic system further comprises sample injection pipes, the number of the sample injection pipes is the same as that of the sample injection pumps, one end of each sample injection pipe is connected to the output port of the sample injection pump, and the other end of each sample injection pipe is connected to the second input end of the microfluidic pipe.
In some optional embodiments, the output port of the second oil phase injection pump is provided with an injection needle, and the second oil phase injection pump is connected to the second input end through the injection needle; the other end of the sample injection pipeline is arranged in the injection needle, and a gap is reserved between the inner wall of the injection needle and the sample injection pipeline.
In some alternative embodiments, the sample injection conduit is a polyetheretherketone tube.
In some alternative embodiments, the microfluidic conduit is a silicone conduit.
In some optional embodiments, the microfluidic system further comprises a stretching module for stretching the sample injection conduit.
In some optional embodiments, the microfluidic system further comprises a flow rate control module for controlling a flow rate of a sample reagent in the sample injection conduit.
In a second aspect, the present invention provides a droplet flow control method, comprising the steps of:
injecting a first oil phase reagent into the micro-flow pipeline to form first oil phase liquid drops;
injecting a sample reagent into a second oil phase reagent to form a second oil phase droplet, and injecting the second oil phase droplet into the first oil phase droplet to form a target sample droplet, wherein the target sample droplet comprises a plurality of second oil phase droplets.
In some optional embodiments, the method further comprises at least one of:
stretching the sample injection pipeline of the sample reagent by a stretching module so as to improve the flow rate of the sample reagent in the sample injection pipeline;
and controlling the flow rate of the sample reagent in the sample injection pipeline through a flow rate control module.
Advantages and benefits of the present invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention:
according to the technical scheme of droplet microfluidics, a first oil phase reagent can be injected into a micro-flow pipeline through a first oil phase injection pump to form a first microfluidic droplet; and then injecting the second oil phase liquid drop wrapped with the sample reagent into the first oil phase liquid drop through a second oil phase injection pump to form a sample liquid drop.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic view of a droplet microfluidic system according to the present invention;
FIG. 2 is a schematic illustration of the formation of an oil bath droplet containing a plurality of sample droplets in an example of the invention;
FIG. 3 is a schematic view of a liquid drop injected through a needle in an embodiment of the present invention;
FIG. 4 is a schematic diagram of another droplet microfluidic system of the present invention;
FIG. 5 is a flow chart of the steps of a droplet flow control method according to the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
In the description of the present invention, it is to be understood that the terms "length," "upper," "lower," "front," "rear," "left," "right," "top," "inner," "outer," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the figures, which are based on the orientation or positional relationship shown in the figures, and are used for convenience in describing the invention and to simplify the description, but are not intended to indicate or imply that the device or element so referred to must have a particular orientation, be constructed in a particular orientation, and be operated in a particular orientation, and thus are not to be construed as limiting the invention. Furthermore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless otherwise specified.
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
Single-cell RNA-sequencing (scRNA-seq) is a new technology that has been developed in recent years, and the technology can obtain a full transcriptome expression profile from a single-cell level, amplify and perform high-throughput sequencing, thereby efficiently detecting the gene expression level in a single cell, and has important application values in the fields of diagnosis and treatment of tumors, design of targeted drugs, development and differentiation of stem cells, and the like.
Single cell transcriptome sequencing can sequence hundreds of thousands of cells simultaneously because each cell is tagged with a different tag, and each mRNA is similarly assigned a unique molecular tag in order to distinguish each mRNA in each cell. In the prior art, a micro-fluidic system or platform for single cell sequencing mainly places single cells and single microspheres with molecular tags in a micropore environment, after the cells are cracked, different cells can carry specific cell tags, and different mRNAs of the same cell are marked by different molecular tags through reverse transcription. The method can track the cell source of each gene, can quantify mRNA, obviously improves sequencing flux, reduces sequencing cost and lightens the influence of amplification preference.
However, the latest single cell sequencing based on the droplet microfluidic technology exists at present, a plurality of complicated steps can be integrated on one microfluidic chip, the traditional microspheres with molecular labels have the problems of high manufacturing cost, difficult preparation, low preparation success rate, unstable effects of different batches and the like, and the most serious is that the droplet microfluidic technology is difficult to form single package on the traditional microspheres, so that the stability and the accuracy of a single cell sequencing result are influenced; therefore, in the existing research based on the droplet microfluidic technology, a droplet control technology which is simpler and more convenient, mild in operation condition, strong in initiative and high in flexibility is urgently needed.
In a first aspect, as shown in fig. 1, the droplet microfluidic system provided by the present invention mainly includes a first oil phase injection pump 101, a second oil phase injection pump 102, and a microfluidic pipeline 103;
wherein, the output port of the first oil phase injection pump 101 is connected to the first input end of the microfluidic pipeline 103; the output port of the second oil phase injection pump 102 is connected to a second input of the microfluidic conduit 103.
Specifically, in an embodiment, a first oil phase injection pump is used for injecting a first oil phase reagent into a microfluidic pipeline to form a first microfluidic droplet; the second oil phase injection pump is used for injecting a second oil phase reagent into the first microflow liquid drops, a plurality of second microflow liquid drops are formed in the first microflow liquid drops, and the second microflow liquid drops wrap the sample reagent. In an exemplary embodiment, the process for preparing the encapsulated multi-sample reagent droplets by the droplet microfluidic system is as follows: firstly, inserting one or a plurality of polyether ether ketone (PEEK) pipelines into a second oil phase injection pump, and injecting a specific sample reagent into each PEEK pipeline so that a sample liquid drop is wrapped in the oil phase reagent of the second injection pump to form a primary sample liquid drop; meanwhile, injecting a first oil phase reagent, namely an oil bath reagent, into the micro-flow pipeline through a first oil phase injection pump, then injecting the preliminary sample liquid drop into the oil bath reagent in the micro-flow pipeline, and finally forming an oil bath liquid drop containing a plurality of sample liquid drops by controlling the flow rate of the sample reagent in the PEEK pipeline and the flow rate of the oil bath reagent in the micro-flow pipeline as shown in FIG. 2; for example, in one embodiment, the prepolymer solution is an internal phase, the oil phase material is an external phase, micro droplets are prepared, and then the micro droplets are polymerized on line at 30-65 ℃ for 10-14 hours to obtain the soluble hydrogel microspheres. It should be noted that in the system of the embodiment, the first oil phase reagent and the second oil phase reagent may be the same oil phase reagent, so that the second syringe pump has better compatibility with the oil bath reagent in the microfluidic pipeline when injecting the sample liquid drop; in addition, the injection pump in the embodiment system can also inject the reagent in an electric control mode, so that the reagent injection process is easier to control, and the flow rate and the flow velocity of the reagent injection are also easier to control. The syringe pump in the example system may also be replaced with a syringe for the sake of simplifying the control flow.
The present embodiment system differs from a continuous flow system in that droplet-based microfluidics allows independent control of individual droplets, so that droplets and bubbles can be transported, mixed and analyzed separately as microreactors. Droplets produced in example systems vary in diameter from a few microns to hundreds of microns, resulting in volumes varying from femto liters to nano liters. In such small volumes, reagent consumption is even reduced and mixing efficiency is further improved, which facilitates rapid reactions and examinations in chemical synthesis and biochemical analysis; furthermore, precise control and manipulation of fluids in example systems is geometrically limited to a small range, typically greater than 1 micron but less than 1 millimeter.
In some alternative embodiments, the microfluidic system further comprises at least one sample syringe pump, an output port of the sample syringe being connected to the second input end of the microfluidic channel;
in particular, the sample injection pump in the system is used for injecting the sample reagent into the second microfluidic droplets, so that the metering of the sample reagent injected into the oil bath reagent and the flow rate during the injection process are easier to control, and finally the reagent droplets with the target volume are obtained.
In some optional embodiments, the microfluidic system further comprises sample injection pipes, wherein the number of the sample injection pipes is consistent with the number of the sample injection pumps; one end of the sample injection pipeline is connected to the output port of the sample injection pump, and the other end of the sample injection pipeline is connected to the second input end of the microfluidic pipeline;
in particular, the sample injection tube in the embodiment may include, but is not limited to, a Polyetheretherketone (PEEK) tube, a teflon tube, and the like capillary tube; and the sample injection pipe can be made more slender by the stretched or stretched module in the embodiment. In the embodiment, the container side wall of the second oil phase injection pump is perforated, and the sample injection pipeline penetrates through the side wall of the injection pump, enters the inner space of the injection pump and is fixed at the position of the output port of the injection pump.
In some alternative embodiments, the output port of the second oil phase injection pump in the system is provided with an injection needle, wherein the second oil phase injection pump is connected to the second input end through the injection needle; the other end of the sample injection pipeline is arranged in the injection needle, and a gap is reserved between the inner wall of the injection needle and the sample injection pipeline.
Specifically, as shown in fig. 3, in the embodiment of the injection needle, one or more PEEK tubes are inserted, each PEEK tube is injected with a specific sample, and oil is passed through the gap between the PEEK tubes in the needle. Inserting the needle head into the silica gel pipeline, and finally forming an oil bath liquid drop containing a plurality of sample liquid drops by controlling the flow rate of the liquid drops in each PEEK pipe and the flow rate of the oil bath in the second injector; in addition, it should be noted that in the embodiment, the injection needle can also play a role in fixing, and in addition, the injection needle can keep concentric with the microfluidic pipeline, so that the liquid drop flow is normally carried out.
In some alternative embodiments, the sample injection conduit is a polyetheretherketone tube; specifically, the Polyetheretherketone (PEEK) plastic raw material is an aromatic crystal type thermoplastic polymer material, and has the advantages of high mechanical strength, high temperature resistance, impact resistance, flame retardance, acid and alkali resistance, hydrolysis resistance, wear resistance, irradiation resistance and good electrical property.
In some alternative embodiments, the microfluidic conduit is a silicone conduit; specifically, the silicone tube is telescopic, so that the syringe can be sleeved to play a role in wrapping. The injection needle of the injection pump is inserted into the silicone tube, and the flexibility of the silicone tube plays a role in wrapping.
In some alternative embodiments, the microfluidic system further comprises a stretching module for stretching the sample injection conduit; in particular, embodiments stretch and elongate the sample injection conduit by a stretching module to provide a more elongated conduit having a shorter cross-sectional radius to form a smaller volume sample droplet. In some necessary application scenarios, the embodiment may also heat the sample injection pipe for better stretching effect.
In some alternative embodiments, the microfluidic system further comprises a flow rate control module for controlling a flow rate of the sample reagent in the sample injection conduit.
In some alternative embodiments, as shown in fig. 4, the embodiment directly inserts a plurality of fine pipes into the needle of a syringe, then further inserts the needle into a plastic pipe with a larger radius, completely glues the gap between the needle and the fine pipe with glue, and respectively flows different fluids such as water into the plurality of fine pipes to realize the formation of a plurality of droplets.
In a second aspect, as shown in fig. 5, the present application further provides a droplet flow control method, including steps S100-S200:
s100, injecting a first oil phase reagent into the micro-flow pipeline to form first oil phase liquid drops;
s200, injecting a sample reagent into the second oil phase reagent to form a second oil phase liquid drop, and injecting the second oil phase liquid drop into the first oil phase liquid drop to form a target sample liquid drop, wherein the target sample liquid drop comprises a plurality of second oil phase liquid drops.
Specifically, in the embodiment, one or a plurality of polyether ether ketone (PEEK) pipelines are inserted into the second oil phase injection pump, and a specific sample reagent is injected into each PEEK pipeline, so that the sample liquid drop is wrapped in the oil phase reagent of the second injection pump to form a primary sample liquid drop; meanwhile, a first oil phase reagent, namely an oil bath reagent, is injected into the microfluidic pipeline through a first oil phase injection pump, then the preliminary sample liquid drop is injected into the oil bath reagent in the microfluidic pipeline, and by controlling the flow rate of the sample reagent in the PEEK pipeline and the flow rate of the oil bath reagent in the microfluidic pipeline, as shown in fig. 2, the oil bath liquid drop containing a plurality of sample liquid drops is finally formed.
In some optional embodiments, the method further comprises at least one of steps S300 and S400:
s300, stretching the sample injection pipeline of the sample reagent through a stretching module to improve the flow rate of the sample reagent in the sample injection pipeline;
and S400, controlling the flow rate of the sample reagent in the sample injection pipeline through the flow rate control module.
From the above specific implementation process, it can be concluded that the technical solution provided by the present invention has the following advantages or advantages compared to the prior art:
1. the scheme of the invention is simple to operate, and the distribution proportion of the micro-droplet sample can be controlled;
2. the scheme of the invention realizes the sampling function of the tiny sample through sample distribution and sampling operation with higher proportion, realizes the sampling function of the tiny sample in the aspect of rapid detection of rare viruses in the field of biomedical detection, obviously reduces the consumption of the sample and has outstanding advantages;
3. according to the scheme, the operation of the micro-droplets is performed in the micro-fluidic chip, so that the pollution of human factors to the sample is obviously reduced;
4. the scheme of the invention can generate a large number of micro-reactors in a short time, each liquid drop can be used as an independent micro-reactor, the volume can be as small as picoliter or femtoliter, the consumption of samples and reagents is greatly reduced, and the reaction time is shortened.
In alternative embodiments, the functions/acts noted in the block diagrams may occur out of the order noted in the operational illustrations. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality/acts involved. Furthermore, the embodiments presented and described in the flow charts of the present invention are provided by way of example in order to provide a more thorough understanding of the technology. The disclosed methods are not limited to the operations and logic flows presented herein. Alternative embodiments are contemplated in which the order of various operations is changed and in which sub-operations described as part of larger operations are performed independently.
Furthermore, although the present invention is described in the context of functional modules, it should be understood that, unless otherwise stated to the contrary, one or more of the functions and/or features may be integrated in a single physical device and/or software module, or one or more of the functions and/or features may be implemented in a separate physical device or software module. It will also be appreciated that a detailed discussion of the actual implementation of each module is not necessary for an understanding of the present invention. Rather, the actual implementation of the various functional modules in the apparatus disclosed herein will be understood within the ordinary skill of an engineer, given the nature, function, and internal relationship of the modules. Accordingly, those skilled in the art can, using ordinary skill, practice the invention as set forth in the claims without undue experimentation. It is also to be understood that the specific concepts disclosed are merely illustrative of and not intended to limit the scope of the invention, which is defined by the appended claims and their full scope of equivalents.
The logic and/or steps represented in the flowcharts or otherwise described herein, e.g., an ordered listing of executable instructions that can be considered to implement logical functions, can be embodied in any computer-readable medium for use by or in connection with an instruction execution system, apparatus, or device, such as a computer-based system, processor-containing system, or other system that can fetch the instructions from the instruction execution system, apparatus, or device and execute the instructions.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
While the preferred embodiments of the present invention have been illustrated and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A liquid drop microfluidic system is characterized by comprising a first oil phase injection pump, a second oil phase injection pump and a microfluidic pipeline; the output port of the first oil phase injection pump is connected to the first input end of the microfluidic pipeline; the output port of the second oil phase injection pump is connected to the second input end of the microfluidic pipeline;
the first oil phase injection pump is used for injecting a first oil phase reagent into the microfluidic pipeline to form a first microfluidic liquid drop; the second oil phase injection pump is used for injecting a second oil phase reagent into the first micro-fluid liquid drop, a plurality of second micro-fluid liquid drops are formed in the first micro-fluid liquid drop, and the second micro-fluid liquid drops are wrapped with sample reagents.
2. The droplet microfluidic system according to claim 1, further comprising at least one sample syringe pump, an output port of said sample syringe being connected to a second input of said microfluidic channel;
the sample injection pump is used for injecting sample reagents into the second microfluidic droplets.
3. The droplet microfluidic system according to claim 2, further comprising sample injection pipes, wherein the number of the sample injection pipes is the same as that of the sample injection pumps, one end of each sample injection pipe is connected to an output port of the sample injection pump, and the other end of each sample injection pipe is connected to a second input end of the microfluidic pipe.
4. The droplet microfluidic system according to claim 3, wherein an output port of the second oil phase injection pump is provided with an injection needle, and the second oil phase injection pump is connected to the second input end through the injection needle; the other end of the sample injection pipeline is arranged in the injection needle, and a gap is reserved between the inner wall of the injection needle and the sample injection pipeline.
5. A droplet microfluidic system according to claim 3, wherein said sample injection conduit is a polyetheretherketone tube.
6. The droplet microfluidic system of claim 1, wherein the microfluidic channel is a silicone channel.
7. A droplet microfluidic system according to any of claims 3-5, further comprising a stretching module for stretching the sample injection conduit.
8. A droplet microfluidic system according to any of claims 3-5, further comprising a flow rate control module for controlling the flow rate of sample reagent in the sample injection conduit.
9. A droplet microfluidic control method applied to a droplet microfluidic system as claimed in claim 1, the method comprising the steps of:
injecting a first oil-phase reagent into the micro-flow pipeline to form first oil-phase liquid drops;
injecting a sample reagent into a second oil phase reagent to form a second oil phase droplet, and injecting the second oil phase droplet into the first oil phase droplet to form a target sample droplet, wherein the target sample droplet comprises a plurality of second oil phase droplets.
10. A droplet flow control method according to claim 9, further comprising at least one of:
stretching a sample injection pipeline of the sample reagent by a stretching module so as to improve the flow rate of the sample reagent in the sample injection pipeline;
and controlling the flow rate of the sample reagent in the sample injection pipeline through a flow rate control module.
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| 李建国: "《高等仪器分析》", 苏州大学出版社, pages: 148 * |
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