A continuous sample injection microfluidic chip and use method, cell sorting device

Abstract

The application discloses a continuous sample injection micro-fluidic chip and a use method and a cell sorting device, and relates to the technical field of micro-fluidic chips, and comprises a chip, a sample pool arranged on the chip, a sample injection pipeline arranged at one end of the sample pool, a light tweezer sorting channel arranged at the other end of the sample pool, a sheath liquid pipeline arranged on the chip, a sheath liquid inlet arranged at one end of the sheath liquid pipeline, a sheath liquid sorting channel communicated with the other end of the sheath liquid pipeline, and the sheath liquid pipeline and the light tweezer sorting channel are connected, a sample injection interception assembly for cutting off or conducting the sample injection pipeline, in the cell sorting state, the sample injection interception assembly cuts off the sample injection pipeline so that the sample in the sample pool is in a static state, an imaging device is used for observing and distinguishing the target sample in the sample pool, and the target sample is dragged to the end of the light tweezer sorting channel by the light tweezer, so that the target sample is transported to the sheath liquid sorting channel by the sheath liquid in the sheath liquid pipeline, and in the continuous sample injection state, the sample injection interception assembly conducts the sample injection pipeline, and sample injection is carried out in the sample pool through the sample injection pipeline. Compared with the prior art, the application has the beneficial effects that the continuous sample injection and the stable static sorting function can be realized by controlling the interception assembly, and the problems that the single cell sorting cannot continuously inject samples and is easily affected by laminar flow in the sorting process are solved.

Description

Technical Field

[0001] This invention belongs to the field of cell sorting technology, specifically relating to a continuous sample injection microfluidic chip and its usage method, and a cell sorting device. Background Technology

[0002] Microorganisms, as the most diverse species on Earth, play a crucial role in ecosystems, serving as an indispensable link in biomass synthesis, degradation, and cycling. Simultaneously, microorganisms are closely related to human health—the number of microorganisms in the human body is equivalent to ten times that of the body's own cells. However, to date, over 90% of microorganisms cannot be cultured under laboratory conditions. Phenotypic identification, sorting, and genotypic analysis of individual living cells (i.e., "single-cell technology") can avoid the lengthy incubation process of microorganisms, elucidating the deepest levels of heterogeneity and operational mechanisms of living systems. Single-cell technology for microorganisms has long faced this technical challenge: how to isolate individual microbial cells non-destructively and precisely.

[0003] Currently, the main technologies for achieving single-cell isolation of microorganisms include fluorescence flow cytometry (FACS) and micromanipulation techniques (Eppendorf). However, both have their own limitations. For example, FACS requires fluorescent labeling of cells, which often encounters difficulties in labeling or problems affecting cell viability after labeling. In addition, FACS that meets the requirements for microbial isolation is expensive. Micromanipulation techniques involve complex single operations (requiring precise control of the capillary tip position, including needle insertion and withdrawal) and have lower throughput.

[0004] Domestic and international research groups have reported methods for separating microbial single cells using microfluidic techniques or laser ejection strategies. For example, the article "Nondestructive Identification and Accurate Isolation of Single Cells through a Chip with Raman Optical Tweezers" (Anal. Chem.) uses optical tweezers to drag cells from a cell pool to a sorting pool, and then uses a pipette to separate single cells. However, the dragging distance for a single cell is several millimeters, requiring a relatively long manipulation time. Furthermore, the separation channel structure used is quite wide, making it impossible to image the entire channel in the field of view, and easily overlooking the presence of non-target cells.

[0005] Y. Wang, et al., Raman Activarated Cell Ejection for Isolation of Single Cells, Ana l. Chem. This article presents a pulsed laser ejection method. However, the cells need to be naturally dried on the ejection substrate before ejection. In addition, the pulsed laser will generate a strong photothermal phenomenon on the dried substrate, which will seriously affect the physiological activity of the separated cells.

[0006] 202011027416.4 discloses a microfluidic chip for particle screening and separation, and discloses an oil-coated droplet cell sorting chip. Due to the characteristics of its structure, it cannot achieve secondary sample loading, the chip cannot be reused, resulting in higher costs, and it cannot effectively sort low-abundance samples. Summary of the Invention

[0007] This invention discloses a continuous sample injection microfluidic chip, comprising a chip and further comprising:

[0008] A sample cell is located on the chip, with a sample inlet tube at one end and an optical tweezers sorting channel at the other end;

[0009] A sheath fluid conduit is provided on the chip, with a sheath fluid inlet at one end and a sheath fluid sorting channel at the other end, and the sheath fluid conduit intersects with the end of the optical tweezers sorting channel;

[0010] A sample injection interception assembly is used to cut off or open the sample injection line;

[0011] In the cell sorting state, the sample injection interception component cuts off the sample injection line to keep the sample in the sample pool in a static state. The imaging device is used to observe and identify the target sample in the sample pool, and the target sample is dragged to the end of the optical tweezers sorting channel by optical tweezers so that the target sample can be transported to the sheath fluid sorting channel through the sheath fluid in the sheath fluid line.

[0012] In continuous injection mode, the injection interception component connects the injection pipeline and injects the sample into the sample cell through the injection pipeline.

[0013] In some implementations, it also includes:

[0014] The export component is used to truncate the liquid flow containing the target sample sheath fluid to form droplets containing the target sample.

[0015] In some embodiments, the export component is a gas flow conduit; the gas flow conduit intersects with the sheath fluid sorting channel, and by introducing gas into the gas flow conduit, the liquid flow is sheared by the gas flow to form droplets containing the target sample.

[0016] In some embodiments, the export component is a piezoelectric element; the piezoelectric element is located at the sheath fluid sorting channel, and by applying a rapidly changing voltage signal to the piezoelectric element to deform it, the fluid flow is cut off by vibration to form droplets with the target sample.

[0017] In some embodiments, the injection tubing has a cut-off structure to form a first guide port and a second guide port; the injection retention component is a conductive cavity and communicates with the first guide port and the second guide port, and the outer end of the conductive cavity is an elastic layer; by squeezing or releasing the elastic layer, a cut-off and communication are formed between the first guide port and the second guide port.

[0018] In some embodiments, the sample inlet line, the optical tweezers sorting channel, and the sheath fluid sorting channel are located on the symmetrical central axis of the sample cell.

[0019] In some embodiments, the sheath fluid conduit is provided with two symmetrically distributed on both sides of the sheath fluid sorting channel to form a Y-shaped structure.

[0020] In some embodiments, the airflow pipeline includes a first air passage and a second air passage symmetrically arranged on both sides of the sheath fluid sorting channel; the ends of the first air passage and the second air passage are arranged in a collinear structure with the outlet of the sheath fluid sorting channel and are treated with a superhydrophobic coating.

[0021] This invention discloses a method for using a continuous sample injection microfluidic chip, applied to the continuous sample injection microfluidic chip described in any of the above embodiments. The method for using the continuous sample injection microfluidic chip includes:

[0022] The cell sorting method involves opening the sample inlet tube and injecting the sample into the sample cell. After the sample is injected, the sample inlet tube is cut off and cell sorting is performed in the sample cell. The target sample to be captured is dragged to the intersection of the optical tweezers sorting channel and the sheath fluid tube using optical tweezers. The target sample is then transported to the sheath fluid sorting channel using the sheath fluid in the sheath fluid tube.

[0023] The continuous injection method involves opening the injection line to inject the sample into the sample cell. After the sample injection is completed, the injection line is cut off, and sheath fluid is continuously injected into the sheath fluid line to flush the sheath fluid sorting channel. After flushing, the cells in the sample cell are sorted.

[0024] The target sample extraction method calculates the time it takes for the liquid flow containing the target sample to reach the extraction component, and then sends a signal to the extraction component to cut off the liquid flow containing the target sample, so as to ensure the formation of droplets containing the target sample.

[0025] This invention discloses a cell sorting device, comprising a continuous sample introduction microfluidic chip as described in any of the above embodiments, and further comprising:

[0026] A microfluidic chip placement platform is used to place microfluidic chips and move them horizontally.

[0027] The imaging module is used for real-time imaging and identification analysis of samples in the sample cell and sheath fluid sorting channel;

[0028] The automatic optical tweezers capture module captures and fixes the identified target sample using optical tweezers; by adjusting the horizontal position of the microfluidic chip placement platform, the target sample is dragged to the intersection of the sheath fluid pipeline and the end of the optical tweezers sorting channel;

[0029] The sheath fluid pipeline pumping device is used to pump sheath fluid into the sheath fluid pipeline, so that the target sample is transported to the sheath fluid sorting channel along with the flow of the sheath fluid.

[0030] The export component control unit calculates the time it takes for the liquid flow containing the target sample to reach the export component position, and then sends a signal to the export component to cut off the liquid flow containing the target sample and form droplets containing the target sample.

[0031] A collection plate placement platform is used to place the collection plate and to receive droplets containing the target sample at the outlet of the sheath fluid sorting channel by displacement;

[0032] The truncation control component is used to control the truncation component to cut off or open the sample inlet line for cell sorting and continuous sample injection.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] This invention suspends cells in an aqueous phase (sheath fluid) and utilizes low-damage near-infrared optical tweezers technology to ensure cell viability during sorting, enabling single-cell culture, sequencing, and other analyses. Thanks to the high resolution of the optical tweezers, precise separation of single cells and particles with dimensions of 0.5 micrometers and above can be achieved. Furthermore, the aqueous phase sorting structure allows for continuous sample introduction and stable static sorting by controlling the retention components, solving the current challenges of continuous sample introduction and susceptibility to laminar flow in single-cell sorting. The continuous sample introduction structure also significantly improves the sorting capabilities for low-abundance samples. Attached Figure Description

[0035] The accompanying drawings, which are provided to further illustrate the invention and constitute a part of this invention, are illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention.

[0036] Figure 1 This is a schematic diagram of the structure of the present invention.

[0037] Figure 2 This is a schematic diagram of the CFD simulation analysis of the sample cell of the present invention.

[0038] 1. Chip; 2. Sample cell; 3. Sheath fluid line; 4. Optical tweezers sorting channel; 5. Sample injection line; 6. Sheath fluid sorting channel. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described and illustrated below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the embodiments provided by this invention without inventive effort are within the scope of protection of this invention.

[0040] Obviously, the accompanying drawings described below are merely some examples or embodiments of the present invention. Those skilled in the art can apply the present invention to other similar scenarios based on these drawings without any inventive effort. Furthermore, it is understood that although the efforts made in this development process may be complex and lengthy, for those skilled in the art related to the content disclosed in this invention, modifications to design, manufacturing, or production based on the technical content disclosed in this invention are merely conventional technical means and should not be construed as insufficient disclosure of the present invention.

[0041] In this invention, the reference to "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it a mutually exclusive, independent, or alternative embodiment. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described in this invention may be combined with other embodiments without conflict.

[0042] This invention discloses a continuous sample injection microfluidic chip, comprising a chip and further comprising:

[0043] The sample cell 2 is located on the chip 1, and one end of it is provided with a sample inlet tube 5, and the other end is provided with an optical tweezers sorting channel 4;

[0044] The sheath fluid conduit 3 is located on the chip 1. One end of the conduit is provided with a sheath fluid inlet, and the other end is connected to the sheath fluid sorting channel 6. The sheath fluid conduit 3 intersects with the end of the optical tweezers sorting channel 4.

[0045] The sample injection interception assembly is used to cut off or open the sample injection line 5. The sample injection interception assembly can have various structural forms to ensure the cutting off or opening of the sample injection line 5. This can be achieved through channel deformation caused by external force, such as through air pressure, mechanical force, magnetic force, temperature-sensitive materials, or biological materials; or by using the vertical movement of a valve or the rotational action of a valve containing an intermediate on / off mechanism to control the opening and closing of the channel; or by controlling the pipe connected to the sample injection line 5 through external force, such as the rapid start and stop of the syringe pump, or the closure of the tubing, to achieve on / off.

[0046] During cell sorting, the sample injection retention component cuts off the sample injection line 5 so that the sample in the sample pool 2 is in a static state. The imaging device is used to observe and identify the target sample in the sample pool. The target sample is dragged to the end of the optical tweezers sorting channel 4 by optical tweezers so that the target sample can be transported to the sheath fluid sorting channel 6 through the sheath fluid in the sheath fluid line 3.

[0047] In continuous injection mode, the injection interception component connects the injection pipeline 5, and the sample is injected into the sample cell 2 through the injection pipeline 5.

[0048] In some implementations, it also includes:

[0049] The export component is used to truncate the liquid flow containing the target sample sheath fluid to form droplets containing the target sample.

[0050] Specifically, the truncation method can be achieved using either air-jet droplet printing or piezoelectric droplet printing.

[0051] Air-jet droplet printing: Rapid rise and fall of air pressure can cause droplet formation and ejection. By controlling the change of air pressure, the size and release speed of the droplets can be adjusted. When air pressure acts on the liquid inside the chip, the liquid will be ejected from the nozzle to form droplets. The size of the droplets can be controlled by adjusting the magnitude of the air pressure and the duration of action.

[0052] In some embodiments, the output component is an airflow conduit; the airflow conduit intersects with the sheath fluid sorting channel, and by introducing gas into the airflow conduit, the liquid flow is sheared by the airflow to form droplets with the target sample.

[0053] Piezoelectric droplet printing: By applying a rapidly changing voltage signal to the piezoelectric element, the piezoelectric element will expand and contract rapidly, generating vibration; the vibration of the piezoelectric element will cause pressure fluctuations in the liquid inside the chip, thereby forming droplets at the nozzle; by precisely controlling the frequency and amplitude of the voltage signal, the size of the droplets and the jetting speed can be precisely controlled.

[0054] In some embodiments, the deriving component is a piezoelectric element; the piezoelectric element is disposed at the sheath fluid sorting channel 6, and the piezoelectric element is deformed by applying a rapidly changing voltage signal to it, thereby truncates the liquid flow by vibration to form droplets with the target sample.

[0055] In some embodiments, the sample injection interception assembly adopts an elastic squeeze valve structure. The sample injection line has a split, cut-off structure, with a first guide port and a second guide port formed on opposite sides of the split. The sample injection interception assembly is a conductive cavity that communicates with the first and second guide ports, and the outer end of the conductive cavity is an elastic layer. By squeezing or releasing the elastic layer, a cut-off and conductive connection is formed between the first and second guide ports.

[0056] In some embodiments, the sample inlet line 5, the optical tweezers sorting channel 4, and the sheath fluid sorting channel 6 are located on the symmetrical central axis of the sample cell 2; the sheath fluid line 3 is provided with two symmetrically distributed on both sides of the sheath fluid sorting channel 6 to form a Y-shaped structure.

[0057] A microfluidic chip for particle screening and separation disclosed in patent 202011027416.4 requires a channel width of approximately 30 micrometers in its T-shaped structure to function as a static flow field for cell storage. Therefore, it can only be manufactured using soft lithography, which is demanding and costly. Furthermore, double-sided adhesive etching cannot provide the micrometer-level precision of traditional PDMS chip soft lithography, resulting in channel widths of at least 100 micrometers. This invention, however, optimizes the sheath fluid channel structure and chip composition, achieving the same flow field stability even with a channel width of 200 micrometers. Coupled with existing microfluidic chip double-sided adhesive technology, it enables large-scale mass production, reducing costs. The invention is validated through fluid simulation CFD analysis to ensure that the flow velocity within the sorting cell is not affected by external channels. Additionally, the sorting cell has only one outlet connected to the optical tweezers sorting channel 4, with the other end forming a blind end through a retaining component, further ensuring channel stability during sorting.

[0058] In some embodiments, the airflow conduit includes a first air path and a second air path symmetrically arranged on both sides of the sheath fluid sorting channel; the ends of the first and second air paths are collinearly arranged with the outlet of the sheath fluid sorting channel. This collinear arrangement ensures the stability of droplet fragmentation during air pressure-induced droplet breakage.

[0059] Specifically, the sample cell 2 can be in the form of a rhombus structure. The sample inlet pipe 5 and the optical tweezers sorting channel 4 are located at the two ends on both sides of it. In the design, the sheath fluid pipe and the air flow pipe are located on both sides of the sample cell 2 from the inside to the outside, and both are symmetrical structural designs. The inlet of the sheath fluid pipe and the air flow pipe is a single port located on the central axis of symmetry of the sample cell 2.

[0060] This invention also discloses a method for using a continuous injection microfluidic chip, applicable to any of the continuous injection microfluidic chips described in the above embodiments. The method for using the continuous injection microfluidic chip includes:

[0061] The cell sorting method involves opening the sample inlet tube and injecting the sample into the sample cell. After sample injection, the sample inlet tube is cut off, and sheath fluid is continuously injected into the sheath fluid tube. The sample in the sample cell is kept stable by the intersection of the optical tweezers sorting channel and the sheath fluid tube. The target sample to be captured is dragged to the intersection of the optical tweezers sorting channel and the sheath fluid tube by optical tweezers. The target sample is then transported to the end of the sheath fluid sorting channel by the sheath fluid in the sheath fluid tube.

[0062] The continuous injection method involves opening the injection line to inject the sample into the sample cell. After the sample injection is completed, the injection line is cut off, and sheath fluid is continuously injected into the sheath fluid line to flush the sheath fluid sorting channel. After flushing, the cells in the sample cell are ready to be sorted.

[0063] The target sample extraction method calculates the time it takes for the liquid flow containing the target sample to reach the extraction component, and then sends a signal to the extraction component to cut off the liquid flow containing the target sample, so as to ensure the formation of droplets containing the target sample.

[0064] The present invention also discloses a cell sorting device, comprising a continuous sample introduction microfluidic chip according to any of the above embodiments, and further comprising:

[0065] A microfluidic chip placement platform is used to place microfluidic chips and can be adjusted and moved in the horizontal direction; it can be designed as a two-axis or three-axis transport platform as needed.

[0066] The imaging module is used for real-time bright-field / fluorescence imaging of samples in the sample cell and sheath fluid sorting channel;

[0067] The automatic optical tweezers capture module is used to analyze and identify samples in the sample cell and capture and fix the identified target samples using optical tweezers; by adjusting the horizontal position of the microfluidic chip placement platform, the target sample is dragged to the intersection of the sheath fluid pipeline and the end of the optical tweezers sorting channel;

[0068] The sheath fluid pipeline pumping device is used to pump sheath fluid into the sheath fluid pipeline, thereby transporting the target sample through the sheath fluid to the sheath fluid sorting channel;

[0069] The export component control unit calculates the time it takes for the liquid flow containing the target sample to reach the export component position, and then sends a signal to the export component to cut off the liquid flow containing the target sample and form droplets containing the target sample.

[0070] A collection plate placement platform is used to place the collection plate and to receive the target sample from the outlet of the sheath fluid sorting channel by displacement;

[0071] The interception control component is used to control the interception component to achieve the cut-off or opening of the injection line.

[0072] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A continuous sample injection microfluidic chip, comprising a chip, characterized in that, Also includes: A sample cell is located on the chip, with a sample inlet tube at one end and an optical tweezers sorting channel at the other end; A sheath fluid conduit is provided on the chip, with a sheath fluid inlet at one end and a sheath fluid sorting channel at the other end, and the sheath fluid conduit intersects with the end of the optical tweezers sorting channel; The sample injection interception component is used to cut off the sample injection line in the sorting state and to open the sample injection line in the continuous sample injection state. The export component, through its control unit, calculates the time it takes for the liquid flow containing the target sample to reach the position of the export component, and applies an active cutoff action to the liquid flow in the sheath liquid sorting channel to form droplets containing the target sample; In the cell sorting state, the sample injection retention component cuts off the sample injection line to keep the sample in the sample cell in a static state. The imaging device is used to observe and identify the target sample in the sample cell, and the optical tweezers are used to drag the target sample to the end of the optical tweezers sorting channel so that the target sample can be transported to the sheath fluid sorting channel through the sheath fluid in the sheath fluid line. The output component cuts off the liquid flow containing the target sample to form droplets. In continuous injection mode, the injection interception component connects the injection pipeline and injects the sample into the sample cell through the injection pipeline.

2. The continuous sample injection microfluidic chip according to claim 1, characterized in that, The export component is an airflow conduit; the airflow conduit intersects with the sheath fluid sorting channel, and by inputting gas into the airflow conduit, the liquid flow is sheared by the airflow to form droplets containing the target sample.

3. The continuous sample injection microfluidic chip according to claim 1, characterized in that, The export component is a piezoelectric element; the piezoelectric element is located at the sheath fluid sorting channel, and by applying a rapidly changing voltage signal to the piezoelectric element to deform the piezoelectric element, the liquid flow is cut off by vibration to form droplets with the target sample.

4. The continuous sample injection microfluidic chip according to claim 1, characterized in that, The sample inlet tubing has a cut-off structure to form a first guide port and a second guide port; the sample inlet retention component is a conductive cavity and is connected to the first guide port and the second guide port, and the outer end of the conductive cavity is an elastic layer; By squeezing or releasing the elastic layer, a cutoff and connection are formed between the first guide and the second guide.

5. The continuous sample injection microfluidic chip according to claim 1, characterized in that, The sample inlet line, the optical tweezers sorting channel, and the sheath fluid sorting channel are located on the symmetrical central axis of the sample cell.

6. The continuous sample injection microfluidic chip according to claim 1, characterized in that, The sheath fluid pipeline has two symmetrically distributed on both sides of the sheath fluid sorting channel to form a Y-shaped structure.

7. The continuous sample injection microfluidic chip according to claim 2, characterized in that, The airflow pipeline includes a first air path and a second air path symmetrically arranged on both sides of the sheath fluid sorting channel; the ends of the first air path and the second air path are arranged in a collinear structure with the outlet of the sheath fluid sorting channel and are treated with a superhydrophobic coating.

8. A method of using a continuous sample injection microfluidic chip, characterized in that, The method of using the continuous injection microfluidic chip, applied to any one of claims 1-7, comprises: The cell sorting method involves opening the sample inlet tube and injecting the sample into the sample cell. After the sample is injected, the sample inlet tube is cut off and cell sorting is performed in the sample cell. The target sample to be captured is dragged to the intersection of the optical tweezers sorting channel and the sheath fluid tube using optical tweezers. The target sample is then transported to the sheath fluid sorting channel using the sheath fluid in the sheath fluid tube. The continuous injection method involves opening the injection line to inject the sample into the sample cell. After the sample injection is completed, the injection line is cut off, and sheath fluid is continuously injected into the sheath fluid line to flush the sheath fluid sorting channel. After flushing, the cells in the sample cell are sorted. The target sample extraction method calculates the time it takes for the liquid flow containing the target sample to reach the extraction component, and then sends a signal to the extraction component to cut off the liquid flow containing the target sample, so as to ensure the formation of droplets containing the target sample.

9. A cell sorting device, characterized in that, The continuous injection microfluidic chip as described in any one of claims 1-7 further includes: A microfluidic chip placement platform is used to place microfluidic chips and move them horizontally. The imaging module is used for real-time imaging and identification analysis of samples in the sample cell and sheath fluid sorting channel; The automatic optical tweezers capture module captures and fixes the identified target sample using optical tweezers; by adjusting the horizontal position of the microfluidic chip placement platform, the target sample is dragged to the intersection of the sheath fluid pipeline and the end of the optical tweezers sorting channel; The sheath fluid pipeline pumping device is used to pump sheath fluid into the sheath fluid pipeline, so that the target sample is transported to the sheath fluid sorting channel along with the flow of the sheath fluid. The export component control unit calculates the time it takes for the liquid flow containing the target sample to reach the export component position, and then sends a signal to the export component to cut off the liquid flow containing the target sample and form droplets containing the target sample. A collection plate placement platform is used to place the collection plate and to receive droplets containing the target sample at the outlet of the sheath fluid sorting channel by displacement; The truncation control component is used to control the truncation component to cut off or open the sample inlet line for cell sorting and continuous sample injection.

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