Microfluidic chip for unlabeled or fluorescently labeled single cell sorting and single cell sorting system

Abstract

The utility model provides a micro-fluidic chip for unmarked or fluorescently marked single cell sorting and a single cell sorting system, which do not adopt means for damaging a sorting target, such as high voltage, strong electric field, high-voltage sheath fluid and the like, and can be used for sorting the unmarked or fluorescently marked single cells on the premise of keeping activity. For unlabeled or labeled (such as fluorescence labeled) cells, bacteria, proteins, viruses, genes (fragments or whole segments), cell clusters and other microorganisms, or liquid drops, liquid segments, bubbles or blocky solids containing the biological particles, high-efficiency, accurate and reliable single-individual detection, control and sorting (especially including but not limited to single-cell sorting) are carried out.

Description

Technical Field

[0001] The utility model relates to the fields of biotechnology, life science, and in particular to a technology for flexible and precise cell sorting and reliable manipulation, and relates to a microfluidic chip for single cell sorting and a related single cell sorting system. Background Art

[0002] In the current fields of biotechnology, life sciences, etc., the manipulation, sorting, and screening of single cells are operations commonly used by scientists when conducting scientific research experiments. Traditional methods are mainly manual, using microscopes, pipettes, well plates and other instruments for manual operations, which are inefficient and have poor precision, causing a certain delay in the progress of scientific research. The mature and efficient automated sorting solutions are mainly based on sorting flow cytometers, which use high voltage, high-pressure strong sheath fluid, and direct contact to apply electric charge to cells, bacteria or droplets. The sorting process greatly reduces the activity of biological particles such as cells and bacteria, which is not conducive to experiments in subsequent scientific research links. Summary of the Invention

[0003] In view of the defects and shortcomings of the above technologies, the present invention provides a microfluidic chip for single-cell sorting and a related single-cell sorting system, which does not use means such as high voltage, strong electric field and high-pressure strong sheath fluid to damage the sorting target. While maintaining activity, it can perform high-efficiency, accurate and reliable single-body detection, control and sorting (especially including but not limited to: single-cell sorting) for cells, bacteria, proteins, viruses, genes, cell clusters, other microorganisms, or droplets, liquid segments, bubbles or bulk solids containing the above biological particles.

[0004] The utility model provides a microfluidic chip and a single cell sorting system for single cell sorting, which are characterized by:

[0005] The fluid includes but is not limited to gas, liquid, granular or powdered solid, and mixtures of several types of solids, gases and liquids, and has the property of being flowable because it is composed of flowable substances such as liquids or gases.

[0006] The "flow direction" or "direction" described in this utility model refers to the tangent to the path if the measurement point is non-linear. That is, for example, if the flow direction or path of a channel, fluid flow direction, or fluid flow is non-linear, the direction of such a vector refers to the tangent or parallel line of the curve at the specified point or region.

[0007] In the present invention: the microfluidic chip can be referred to as a chip for short.

[0008] The present invention is directed to fluids containing cells, bacteria or other biological particles, or fluids in the form of droplets, liquid segments or bubbles (partial or complete: the droplets, liquid segments or bubbles contain cells, bacteria or other biological particles). The cells, bacteria, other types of biological particles, droplets, liquid segments or bubbles may be fluorescent or unlabeled.

[0009] The biological particles include active or inactive species, and the types include but are not limited to several of the following: cells, bacteria, proteins, viruses, exosomes, nematodes, genes, tissues, cell clusters, microtissues, microorganisms, and other biological particles.

[0010] The specific biological particles include, but are not limited to: fluorescent cells, fluorescent bacteria, fluorescent droplets (or liquid segments), fluorescent bubbles, unlabeled cells, unlabeled bacteria, unlabeled droplets (or liquid segments), non-fluorescent bubbles, or other types of fluorescent (or unlabeled) biological particles.

[0011] The present invention is directed to: a fluid containing cells, bacteria or other biological particles, or a fluid containing droplets (or liquid segments, or bubbles), wherein the droplets, liquid segments or bubbles contain cells, bacteria or other biological particles.

[0012] Specific biological particles, droplets, liquid segments or bubbles in the above fluid are sorted.

[0013] The fluids manipulated during the sorting process include but are not limited to the following:

[0014] (1) Aqueous liquid containing biological particles such as cells and bacteria, excluding: oil phase or other liquids, gases or solids that are insoluble in water;

[0015] (2) Multiphase fluids containing droplets, wherein some or all of the droplets contain biological particles such as cells and bacteria, such as, but not limited to, oil-in-water droplets (droplets are located in an immiscible oil phase solution), composite droplets (such as, but not limited to, "water-oil-water" type, where the oil-in-water droplets are surrounded by water, i.e., the oil-in-water droplets are surrounded by a layer of water, and all composite droplets are in the water phase solution, or "oil-water-oil" type, where the water-in-oil droplets are surrounded by oil, i.e., the water-in-oil droplets are surrounded by a layer of oil, and all composite droplets are in the oil phase solution);

[0016] (3) Multiphase fluids containing bubbles, especially including but not limited to: multiple droplets or liquid segments separated by bubbles in a pipeline, some or all of which contain biological particles such as cells and bacteria.

[0017] Typically, the present invention targets suspensions containing cells, bacteria or other biological particles, or suspensions in the form of droplets, liquid segments, bubbles, etc. that encapsulate cells, bacteria or other biological particles. The present invention utilizes a microfluidic chip and a single-cell sorting system for single-cell sorting to sort specific biological particles (for example, but not limited to, single unlabeled cells, single fluorescent active bacteria, or a specified number of other types of biological particles) in the suspension from a population and transport them to a specified spatial location.

[0018] In the present invention, a flexible sorting or flexible control method is adopted, that is, the single-cell sorting uses a microfluidic chip and a single-cell sorting system, and all components, parts and other components have the following characteristics: no high-voltage electromagnetic field is used, no ultra-high-pressure sheath fluid similar to flow sorting technology is used, and there is no need to charge the sorting targets (cells or other types of biological particles). Parameters such as the flow rate, flow rate, pressure or pressure of the fluid can be adjusted (or controlled, and can be lowered or increased according to activity requirements), thereby achieving no effect, very little effect, or negligible effect on the activity of sorting targets such as cells and bacteria during the entire operation.

[0019] The single cell sorting system comprises a microfluidic chip for single cell sorting, which has multiple channels that intersect in a common control area through specific channel positions or ports. The common control area is defined as an intersection control area.

[0020] Two or more channels intersect at the intersection control area through ports (or interfaces).

[0021] In particular, it includes: taking the intersection control area as the port (or interface) position and as the flow reference point, then: two or more channels converge at the intersection control area through the port (or interface), among which, taking the intersection control area as the port flow reference point, when one input channel and another output channel are connected through the intersection control area, it can be understood as one channel or as two channels, both of which are applicable scenarios of the present utility model.

[0022] The design of the above-mentioned channel of the present invention includes the case where the intersection control area is the entire channel or a part of the channel, and the confluence points of multiple channels entering the intersection control area can be the same or different. That is, the present invention includes: two or more channels converge at one point, or after multiple channels converge, other channels are set to intersect and connect at a certain distance or after the pipeline is extended. Then all intersection points and the channel parts where each intersection point is connected to each other are defined as the intersection control area.

[0023] The channels can be of input type, output type or bidirectional flow type.

[0024] The categories of the channel ports described in this utility model: "outlet", "inlet" or "inlet and outlet" refer to the main purpose of the channel port or the function intended by the user when in use. The flow direction of the fluid in the port may change in different application scenarios or different internal and external conditions, that is, the flow direction of the fluid in the port does not match the port name. In addition, the directional characteristics (or limitations) of the flow, such as "input" and "output" of the channel, or "unidirectional" and "bidirectional", may also change under different times, conditions or scenarios.

[0025] The single cell sorting system includes several pump and valve structures, devices or facilities that can realize fluid on-off control, and the locations of the pump and valve structures, devices or facilities are not limited to the following:

[0026] (1) Single cell sorting is performed on several channels of a microfluidic chip.

[0027] (2) Channels connected to: catheters or other types of pipes,

[0028] (3) Other locations in the system.

[0029] Preferably, the above-mentioned pump and valve structures, devices or facilities are located between the channel inlet and the intersection control area, or are located on other channels, conduits or other types of pipelines connected to the channel inlet.

[0030] The pump and valve devices or facilities described in the present invention include several of the following: pump and valve devices that can realize controllable on and off of fluids, and their types include but are not limited to: liquid pumps, air pumps, manual valves, electric valves, etc., especially including: solenoid valves, water pumps, constant pressure pumps, negative pressure pumps, and closed container-type devices containing fluids that can provide constant pressure through interfaces. The closed container-type devices especially include: sealed container-type devices or vessels with controllable input and output interfaces, whose cavities have a low air pressure lower than that of the outside or are filled with high-pressure fluids (gas or liquids), for example but not limited to: those filled with gas: high-pressure gas cylinders, constant-pressure gas sealed tanks, or negative-pressure cylinders, vacuum cylinders, other containers containing fluids that can provide constant pressure through interfaces, etc.

[0031] The single cell sorting method uses a plurality of channels of a microfluidic chip, or a plurality of conduits (or other types of pipelines) connected (or linked) via the channels, to have a one-way fluid control link that is "easy to flow in one direction but difficult to flow in the opposite direction" (the other parts of the present invention may be referred to as "one-way control link" for the one-way fluid control link that is "easy to flow in one direction but difficult to flow in the opposite direction"), including structures, components or facilities that achieve the "easy to flow in one direction but difficult to flow in the opposite direction" fluid control effect, including but not limited to the following:

[0032] (1) Tesla one-way valve, including but not limited to: optimized Tesla one-way valve

[0033] (2) Pumps and valves installed on chip channels that can realize fluid on-off control: structures, components, parts, facilities, equipment or instruments,

[0034] (3) Pumps and valves installed on the pipeline connected to the chip channel interface that can realize fluid on-off control: structures, components, parts, facilities, equipment or instruments.

[0035] The "one-way easy, reverse difficult" fluid one-way control link can be composed of one or more "one-way valve structural units", or other forms of fluid one-way control facilities, instruments or functional structures, and the locations include but are not limited to the following: on the channel inside the chip, on the external pipeline (such as a catheter) connected to the channel, and other spatial locations of the system.

[0036] The "one-way easy, difficult reverse" fluid one-way control link described in the present invention includes but is not limited to the following valve body types or their optimized and improved valve body structures: non-controllable, passive realization of the "one-way easy, difficult reverse" function: one-way film valve, Tesla valve (especially including but not limited to several of the following types: optimized Tesla one-way valve, improved Tesla one-way valve, Tesla one-way valve deformation), and with ejector pins, reeds, axial rotating bodies or other structural facilities that can block the fluid in one direction: structure (located inside or outside the chip), valve components, devices or equipment.

[0037] The one-way control link can be inside the microfluidic chip or outside the chip. Preferably, it is arranged between the channel inlet and the intersection control area, or on a conduit (or other type of pipeline) connected to the channel inlet.

[0038] The one-way control link realizes: the fluid can smoothly, easily or fluently reach the intersection control area after passing through the one-way control link. Due to the channel structure or the function and effect of the pump and valve, the flow resistance in this process is small and the flow rate is fast; but if the fluid originating from the intersection control area intends to flow in the opposite direction through the one-way control link, the flow resistance of the reverse flow path is large due to the channel structure or the function and effect of the pump and valve, resulting in the fluid being unable to flow out, difficult to flow out, or able to flow out but with a slow flow rate.

[0039] The above-mentioned channel structure design with a fluid control mechanism of "easy one-way and difficult reverse", or the pump and valve structures, devices or facilities connected (or connected) by the channel (or its connected conduit), can achieve unidirectional flow control of the fluid in the input channel.

[0040] The fluid control of "easy in one direction, difficult in reverse" includes one of the following characteristics:

[0041] (1) The fluid can only flow in the forward direction and cannot flow in the reverse direction;

[0042] (2) The fluid has low flow resistance and fast flow velocity in the forward direction, making it easy to flow. Compared with the forward direction, the fluid can flow in the reverse direction, but the flow resistance is high and the flow velocity is slow, making it relatively difficult to flow.

[0043] Some types of the one-way control links (for example but not limited to: "optimized Tesla one-way valve") include one or more "one-way valve structural units".

[0044] The "one-way valve structural unit" includes at least two types of ports: an outlet and an inlet. The number of each type of port is one or more, preferably, at least one inlet and at least one outlet connected thereto.

[0045] Different ports of the "one-way valve structural unit", especially the interconnected common inlet and corresponding common outlet, are interconnected through at least two channels (at least one straight flow channel and at least one corresponding backflow channel).

[0046] The one-way control link (such as the "optimized Tesla one-way valve") is composed of a plurality of "one-way valve structural units". The structural characteristics of the connection between the channels of adjacent different "one-way valve structural units" connected through the front and back inlets are as follows: along the reverse flow direction of the fluid (the fluid flows from the common outlet to the common inlet through the "one-way valve structural unit"), within the "one-way valve structural unit", when the fluid flows from its common outlet through its internal multiple channels to its common inlet, the fluids in different channels have opposite flow directions or opposite direction components, forming a mutual obstruction effect, and the flow velocity, kinetic energy or momentum offset or weaken each other. If the flow direction after re-convergence is the same as, similar to, collinear, nearly collinear, or the angle between the two is acute, and in the case of an acute angle, the flow direction of the fluid after the above-mentioned reverse convergence, near the common inlet of the outflow, and the angles with the reverse flow channel, straight-through flow channel, etc. at the common outlet of the next "one-way valve structure unit" (along the direction from the common outlet to the common inlet of the "one-way valve structure unit"), are named "return angle" and "straight-through angle" respectively, then the "return angle" shall not be greater than the "straight-through angle".

[0047] In a one-way control link composed of multiple "one-way valve structural units" (such as the "optimized Tesla one-way valve"), when multiple "one-way valve structural units" are connected end to end and used in series, some or all of the "one-way valve structural units" can choose several of the following structures:

[0048] (1) If the straight line, tangent line or other straight line in the direction of fluid flow is used as the axis, the different "one-way valve structure units" in front and behind are alternately mirror-imaged, or with reference to the adjacent ones, the part or all of the "one-way valve structure unit" is modified in several of the following ways: scaling, rotation, shape modification, mirror-image flipping with a straight line in space as the axis, so as to obtain the effect of small forward flow resistance and large reverse flow resistance.

[0049] Typical designs of this utility model include but are not limited to:

[0050] When multiple "one-way valve structure units" are connected end to end and used in series, some or all of the "one-way valve structure units", such as with the straight line, tangent or other spatial straight line (including but not limited to: a spatial straight line with an acute angle to the flow direction) of the fluid flow direction as the axis, the front and rear different "one-way valve structure units" are alternately mirror-flipped, or with reference to the adjacent ones, part or all of the "one-way valve structure unit" is modified in several of the following ways: scaling, rotation, shape modification, mirror flipping with a certain straight line in space as the axis, etc., so as to obtain the effect of small forward flow resistance and large reverse flow resistance of the "optimized Tesla one-way valve".

[0051] Alternatively, the front and rear different "one-way valve structure units" are designed to have the following structural designs: along the direction of fluid flow, with the straight line or tangent of the fluid flow direction as the axis, the front "one-way valve structure unit" is rotated, scaled, deformed, has a simple topological structure change, or is mirror-flipped, and the structure shape of the rear "one-way valve structure unit" is the same, similar, or close. The specific methods of "modification" include but are not limited to: rotation around a point (or line) in space, overall or partial scaling, channel size change, partial or complete change of channel shape, etc.

[0052] The front and rear "check valve structural units" are designed to alternately mirror-flip along the fluid flow direction, with the straight line or tangent of the fluid flow direction as the axis. The mirror-flip changes include mirror-flip, or complex changes such as mirror-flip followed by scaling, rotation at a certain angle, slight changes in size and shape, and simple changes in topology.

[0053] (2) The front and rear adjacent "one-way valve structure units" are rotated at a certain angle with the connection point as the axis. The rotation angle includes but is not limited to: compared with the angle between the return flow channel and the straight flow channel at the common outlet in the "one-way valve structure unit", the angle is the same or similar in size, and the return flow angle is not greater than the straight flow angle.

[0054] In summary, when multiple "one-way valve structural units" are connected end to end, the above-mentioned (1) or (2) measures can be adopted to optimize the structural design and achieve the effect of small forward flow resistance and large reverse flow resistance.

[0055] Among the multiple connecting channels of the "one-way valve structural unit", at least one pair of channels has the same common inlet and common outlet, and the two ends of one channel meet at different intersection points (common inlet and common outlet) from the two ends of the other channel, and the two intersection points have different structural characteristics:

[0056] (a) When fluid flows forward through the plurality of channels having a common inlet and outlet, near the common outlet, the fluids in both channels flow forward and converge at the common outlet. Before the two channels converge near the common outlet, the channels flow in the same direction or form an acute angle with each other.

[0057] The fluids in the two channels flow in the same direction before they converge, or, if the channel into which one of the channels converges is defined as the first channel and the other channel is defined as the second channel, then the projection (vector component) of the vector of the fluid flow direction in the first channel (in the present invention, if such vector corresponds to a non-linear fluid flow path, the tangent of the path at the measurement point or designated area is taken) on the direction of the vector of the fluid flow direction in the second channel that converges with the first channel at a common outlet is in the same direction as the vector of the fluid flow direction in the second channel.

[0058] That is, this structural feature ensures that the two fluids will not hinder each other after converging, and they will flow out smoothly through the common outlet after converging.

[0059] Typically, when a fluid flows forward from a common inlet through two channels to a common outlet, before converging near the common outlet, the flow velocity vectors of the fluids in the two channels are in the same direction or the angle between them is acute, including but not limited to: the fluids in the two channels flow in the same or similar directions, the two fluids do not hinder each other's original flow direction when converging at the common outlet, and the two fluids flow out smoothly through the common outlet after converging;

[0060] (b) When the fluid flows in reverse directions through the multiple channels with a common inlet and outlet, near the common inlet, the fluids in the two channels flow in reverse directions and meet at the common inlet, that is, the directions of the two channels before meeting near the common inlet are opposite or the angles between them are non-acute (obtuse or right angles). Here, if the directions from the common outlet to the common inlet are defined as the reference directions, then: when the internal fluid flows in reverse and converges into the common inlet, the channel whose direction is opposite to the reference direction or whose angles between them are non-acute can be defined as a backflow channel; when the internal fluid flows in reverse and converges into the common inlet, the channel whose direction is the same as the reference direction or whose angles between them are acute can be defined as a straight-through channel.

[0061] The fluids in the two channels flow in opposite directions before they merge. Alternatively, if the channel into which one of the channels merges is defined as the first channel and the other channel is defined as the second channel, then the projection (vector component) of the vector of the fluid flow direction in the first channel on the direction of the vector of the fluid flow direction in the second channel that merges with it at the common inlet is in the same direction as the vector of the fluid flow direction in the second channel.

[0062] That is, in the case of fluid counterflow, this structural feature causes the two fluids to obstruct each other after converging, and the flow rate slows down or becomes difficult to flow out through the common inlet after converging. That is, the projection of the vector of the fluid flow direction in any one channel on the vector direction of the fluid flow direction in the other channel intersecting with it is opposite to the vector of the fluid flow direction in the other channel intersecting with it, that is, the two fluids will obstruct each other after converging, and the total kinetic energy (or absolute value of momentum) of the two fluids will decrease after converging.

[0063] When fluids flow in opposite directions from a common outlet through two channels to a common inlet, before converging near the common inlet, the fluid velocity vectors in the two channels are in opposite directions or the angle between them is non-acute (obtuse or right angle), including but not limited to: the fluids in the two channels flow in opposite or nearly opposite directions, and when the two fluids intersect at the common outlet, they will hinder the original flow direction of each other, and after the two fluids converge, they will have difficulty flowing out through the common inlet or flow out slowly together.

[0064] In the multiple channels with a common entrance and exit, during the reverse flow of fluid from the common outlet to the common inlet, the direction of the channel when it merges into the common inlet is compared with the direction of the flow channel when it flows in the opposite direction from the common outlet. The channels with the same direction or an acute angle are defined as "straight-through flow channels". The direction of the channel when it merges into the common inlet is compared with the direction of the flow channel when it flows in the opposite direction from the common outlet. The channels with the opposite direction or a non-acute angle (obtuse angle or right angle) are defined as "reverse flow channels".

[0065] From the perspective of spatial geometry, a more specific description is:

[0066] The "one-way valve structural unit" includes two types of ports, namely a common inlet and a common outlet. Each type of interface may have one or more ports, and the two ports are interconnected through at least two channels (at least one straight flow channel and at least one corresponding backflow channel), and have the following characteristics in structure or shape:

[0067] (a) During forward flow, the fluid enters from a common inlet and is divided through at least two channels before converging and flowing out smoothly at or near a common outlet. This process is defined by a spatial vector with the common inlet as the starting point and the common outlet as the end point, which is defined as: the forward flow reference vector. The flow direction below refers to the flow direction of the fluid in the channel. For any section of the straight-through flow channel, the flow direction is the same as the forward flow reference vector or the angle between the two is acute. For the return flow channel, there are several sections at or near the common inlet where the flow direction is opposite to the forward flow reference vector or the angle between the two is non-acute (right angle or obtuse angle). However, for the return flow channel near the common outlet, the flow direction is the same as the forward flow reference vector or the angle between the two is acute.

[0068] (b) In reverse flow, the fluid flows in reverse from the common outlet through at least two channels and then converges at or near the common inlet. Because the velocity vectors of the fluids in at least two channels have opposite or mutually canceling components before the convergence, the sum of the absolute values ​​of the energy or momentum of the two channels after the convergence is lower than that before the convergence, resulting in the inability to flow out, difficulty in flowing out, or even if it can flow out, the flow rate is very slow. The space vector of this process with the common outlet as the starting point and the common inlet as the end point is defined as: the reverse flow reference vector. The following flow path direction refers to the channel direction. The direction of fluid flow at a certain position (or section) in the channel is the same as the direction of the reverse flow reference vector in any section of the straight flow channel, or the angle between the two is acute; while the return flow channel has several sections at or near the common entrance, and the direction of the flow channel is opposite to the direction of the reverse flow reference vector, or the angle between the two is non-acute (right angle or obtuse angle). However, in this case (when the internal fluid flows in the reverse direction), the return flow channel is near the common outlet, and the direction of its flow channel is the same as the direction of the reverse flow reference vector, or the angle between the two is acute.

[0069] Typically, when the fluid flows from the common inlet through different types of flow channels (straight-through flow channel and return flow channel) in the forward direction to the common outlet,

[0070] (1) Near the common outlet, the vector angle between the flow velocities of the fluids in the two channels is 0 degrees or an acute angle (especially including but not limited to: the straight-through flow channel and the return flow channel have the same or similar directions near the common outlet), and the two fluids flow out smoothly through the common outlet after converging;

[0071] (2) Under the same circumstances, near the common inlet, when the fluid is divided from the common inlet and flows into the straight flow channel and the return flow channel respectively, the velocity vector of the internal fluid of at least a part of the channel at the common inlet or other sections of the return flow channel and the velocity vector of the fluid in the two different types of flow channels as described in the previous paragraph (1) when they converge at the common outlet, the angle between the two vectors is: obtuse angle, right angle or 180 degrees,

[0072] (3) Under the same circumstances, near the common inlet, when the fluid is split from the common inlet and flows into the straight-through flow channel and the return flow channel respectively, the angle between the velocity vector of the fluid inside all sections of the straight-through flow channel (the common inlet and all other sections) and the velocity vector of the fluid in the two different types of flow channels as described in the previous paragraph (1) when they converge at the common outlet is: an acute angle or a 0 degree angle.

[0073] The microfluidic chip for single-cell sorting described in the present invention includes a one-way fluid control link that is "easy to flow in one direction but difficult to flow in the reverse direction." The channel structure of the "one-way valve structure unit" is designed such that, if the fluid flows in the reverse direction from the common outlet to the common inlet, during the reverse flow process, the flow resistance of the reverse flow channel is smaller than that of the straight flow channel, or the flow resistance of the reverse flow channel is the same, similar, or close to that of the straight flow channel (including but not limited to: the flow resistance of the reverse flow channel is greater than that of the straight flow channel but the difference is not much).

[0074] The structural design achieves the goal of making the flow resistance of the return flow channel smaller than that of the straight flow channel, including but not limited to the following:

[0075] (a) The cross-sectional area of ​​the reversal passage is larger than that of the straight-through passage, including but not limited to: the cross-sectional area of ​​the reversal passage is wider or deeper than that of the straight-through passage;

[0076] (b) The length of the return flow channel is shorter than that of the straight flow channel;

[0077] (c) The inner wall of the return flow channel is made hydrophobic, oleophobic, or gas-repellent, or the inner wall of the through flow channel is made hydrophilic, oleophobic, or gas-repellent, so that the flow resistance of the fluid when flowing in the return flow channel is smaller than that when flowing in the through flow channel;

[0078] (d) The fluid flow rate in the reflux channel is greater than that in the straight-through channel;

[0079] (e) The fluid velocity in the reflux channel is greater than that in the straight-through channel;

[0080] (f) The absolute value of the fluid momentum in the reversal channel is greater than that in the straight-through channel;

[0081] (g) The effective value of the fluid momentum in the reverse flow channel is greater than that in the straight flow channel.

[0082] The microfluidic chip and single cell sorting system described in the present invention have the following characteristics according to different topological structures and shapes:

[0083] (1) The channel in the chip has a V-shaped topology:

[0084] At least two channels: The microfluidic chip for single cell sorting comprises at least two channels interconnected via an intersection control region. The position where the channels are connected to the intersection control region can be a channel port or any other position of the channel.

[0085] One of the channels contains or is connected (connection method: through the channel inside the chip or the pipeline facilities outside the chip): a one-way fluid control link with "easy one-way, difficult reverse" characteristics. One type of fluid control structure includes: "optimized Tesla one-way valve",

[0086] Other channels, or channels connected to the inlet and outlet: channels, conduits or other types of pipelines, containing pump and valve structures, devices or facilities that can control the flow of fluid in the channels,

[0087] Preferably, the pump and valve structures, devices or facilities are located between the channel entrance and the intersection control area, or on other channels, conduits or other types of pipelines connected to the channel entrance;

[0088] (2) The channels in the chip have a T-shaped topology:

[0089] At least three channels: The microfluidic chip for single-cell sorting uses the intersection control area as the port flow reference point, and includes at least three channels interconnected through the intersection control area: two input channels and one output channel, wherein one input channel and one output channel (such as the driving fluid input channel and the sorting result output channel connected through the intersection control area) have collinear, nearly collinear or acute angle directions (the two channels have collinear, nearly collinear or acute angle directions, which means that the flow vectors of the fluids in the two channels at the port position of the intersection control area are the same or the angle between the two channels in space is an acute angle).

[0090] Preferably: the driving fluid input channel and the sorting result output channel are collinear,

[0091] Another input channel (reagent input channel) includes or is connected (connection method: through an intra-chip channel or an off-chip pipeline facility): a fluid unidirectional control link with "easy one-way flow, difficult reverse flow". One type of fluid control structure includes but is not limited to: "optimized Tesla one-way valve",

[0092] On several channels, or on channels, conduits or other types of pipelines connected to the inlets and outlets of the channels, pump and valve structures, devices or facilities that can realize fluid on-off control are installed.

[0093] Preferably, the pump and valve structures, devices or facilities are located between the channel entrance and the intersection control area, or on other channels, conduits or other types of pipelines connected to the channel entrance.

[0094] The T-type topology "at least three-way channel situation" can be understood as "at least two-way channel situation" if the input channel and an output channel connected to it via the intersection control area and whose channel direction is collinear or nearly collinear with it are understood as one channel.

[0095] (3) The channels in the chip have a "cross" topology:

[0096] At least four channels: The microfluidic chip used for single-cell sorting uses the intersection control area as the port flow reference point, and includes at least four channels interconnected through the intersection control area: two input channels (reagent input channel, driving fluid input channel), and two output channels (sorting result output channel, waste liquid output channel).

[0097] The significant feature of the utility model when using the "cross" topology is that the cross-sectional area of ​​the access port of the waste liquid output channel in the intersection control area (including but not limited to several of the following: port width and depth) is not less than (preferably greater than) the access port of the reagent input channel in the intersection control area, which is used to prevent: the reagent part that is not the sorting target from accidentally entering the driving fluid input channel, the sorting result output channel and other spaces, thereby eliminating experimental errors and enhancing reliability.

[0098] Any input channel and the corresponding output channel have collinear or nearly collinear directions, or the angle between them is acute. The collinear or nearly collinear directions of the two channels refer to the flow vectors of the fluids in the two channels having the same direction or an acute angle between them at the port of the intersection control area. At least one input channel (such as the reagent input channel) contains or is connected (connection method: through the chip channel or the chip external pipeline facilities): a one-way fluid control link with "easy one-way, difficult reverse" characteristics. One type of fluid control structure includes but is not limited to: "optimized Tesla one-way valve",

[0099] Preferably, when the channels in the chip are in a "cross" topology: the driving fluid input channel and the sorting result output channel are collinear or have an acute angle between them; the reagent input channel and the waste liquid output channel are collinear or have an acute angle between them.

[0100] Preferably: the driving fluid input channel and the sorting result output channel are collinear, and the reagent input channel and the waste liquid output channel are collinear.

[0101] The "at least four-channel situation" of the "cross" topology can be understood as "at least two-channel situation" if the input channel and an output channel that is connected to it through the intersection control area and whose channel directions are collinear or nearly collinear with it are understood as one channel.

[0102] Pump and valve structures, devices or facilities that can realize fluid on-off control are set on several channels, or on channels, conduits or other types of pipelines connected to the outlets and inlets of the channels.

[0103] Preferably, the above-mentioned pump and valve structures, devices or facilities are located between the channel inlet and the intersection control area, or on the channel, conduit or other type of pipeline connected to the inlet.

[0104] The present invention has the following notable features when using a "cross" topology: the cross-sectional area (including but not limited to: port width, depth) of the access port of the waste liquid output channel in the intersection control area is not less than (especially but not limited to: preferably greater than) the access port of the reagent input channel in the intersection control area, which is used to prevent: the reagent part that is not the sorting target from accidentally entering the driving fluid input channel, the sorting result output channel and other spaces, thereby eliminating experimental errors and enhancing reliability.

[0105] The detection link of the single cell sorting system described in the present invention includes sensors and other types of structures, especially including but not limited to photoelectric sensors and light sensors.

[0106] The types of photoelectric sensors particularly include photosensitive elements, such as, but not limited to, photodiodes and PMTs that can convert light signals into electrical signals, which are used to identify marked targets (including but not limited to fluorescently marked targets, such as fluorescently marked cells, bacteria, etc.). When excited by a light source (the excitation light source includes but is not limited to lasers, mercury lamps, halogen lamps, and LED lamps), the marked targets generate fluorescence, which is converted into electrical signals and recognized by the control system, which is then used as a basis for implementing sorting operations.

[0107] In addition to the above solutions, the detection link of the single-cell sorting system may also include: light sensors that can identify reflected light, refracted light or scattered light, or sensing devices, facilities, instruments or components that can be used to identify the outline, color, shape of unmarked targets (including but not limited to cells, bacteria or other unmarked biological particles), or can identify the shadows formed by unmarked targets blocking light (or identifying the phenomenon that causes the transmitted light to weaken). Devices, instruments or facilities with recording functions may also be selected to achieve detection and identification of targets through image recognition functions.

[0108] The single cell sorting system includes a light sensor that can identify reflected light, refracted light or scattered light, or includes a recording device-type component that can be used to identify reflected light, refracted light or scattered light, or for identifying the outline, color or shape of unmarked targets. The recording device can also identify the target through its image recognition function.

[0109] The "one-way easy, difficult to reverse" fluid one-way control link includes but is not limited to the following valve body types or their optimized and improved valve body structures: non-controllable, passive "one-way easy, difficult to reverse" functions: one-way diaphragm valves, Tesla valves, and structures with ejector pins, reeds, axial rotating bodies or other moving parts that can block fluid in one direction: structures, valve components, devices or equipment.

[0110] The input channel adopts a Tesla valve structure to realize the reagent or other fluid containing biological particles such as cells, bacteria, cell clusters, etc. Under the fluid one-way control link mechanism of "easy one-way, difficult reverse", through the one-way control link such as optimizing the Tesla valve structure, when a specified number of biological particles enter the intersection control area, several channels (including but not limited to: driving fluid input channels) connected to the same intersection control area can be controlled to open and inject the driving fluid, especially including the preferred case of high-speed injection of the driving fluid, the driving fluid generates in all directions when it reaches the intersection control area The pressure or pressure can produce a blocking effect: it prevents the "reagent (or fluid) part manipulated in the next round" from continuing to enter the intersection control area. The "reagent (or fluid) part manipulated in the next round" refers to: the reagent or fluid part that enters the intersection control area for sorting and control in the next round or next stage in the channel between the intersection control area and the one-way control link, or in the channel of the one-way control link itself, that is, after the fluid sorting control in the current intersection control area is completed, the reagent or fluid part to be sorted enters the intersection control area in the next sorting control stage or round.

[0111] The above-mentioned blocking effect is achieved by: the controlled "next round manipulation reagent (or fluid) part" stops flowing, or flows in the direction of its source (ie, the opposite side of the intersection control area of ​​its channel).

[0112] The driving fluid can push part or all of the fluid in the intersection control area containing a specified number of biological particles through several intended low-resistance channels connected to the target position (the mechanism for achieving low flow resistance includes but is not limited to several of the following: other channels have a large flow resistance due to their own structural characteristics, or due to the setting or connection of other channels: pump valve structures, facilities or instruments, the channel flow resistance is adjustable, or the channel can be closed and cut off, and the flow resistance is large), out of the intersection control area, and move to a specified space or target position, including but not limited to: several holes on a porous plate, test tubes, collectors, or other receivable vessels, to achieve the sorting of the specified number of biological particles.

[0113] The single-cell sorting system of the present invention may include a well plate, a test tube, an EP tube, a centrifuge tube, a culture dish, a syringe or other types of instruments with several storage cavities, as a collection device for a specified number of sorting targets (such as cells, bacteria, and other types of biological particles) after sorting, and especially includes a multi-cavity (subspace) collection device, such as a well plate (including but not limited to: 6-well plate, 12-well plate, 24-well plate, 48-well plate, 96-well plate, 384-well plate, 1536-well plate, and well plates with other numbers of holes). At the end of each round of sorting, different wells in the well plate are switched one by one as the sorting destination, so as to realize a multi-unit space collection device, in which a specified number of sorting targets are sorted into each unit space. For example, if the sorting target is a cell and the specified number is 1, continuous single-cell flexible sorting operation for the well plate can be realized, and the single-cell well plate planting operation is automated.

[0114] If a test tube or other single-cavity device is used (including but not limited to several of the following: syringes, culture dishes, EP tubes, centrifuge tubes, watch glasses, reagent bottles, culture bottles, liquid storage bottles, etc.), the sorted target objects can be output to the device with a single storage cavity without switching the sorting destination.

[0115] In the above process, reagents containing biological particles or other fluids, under the action of a one-way fluid control link such as an optimized Tesla valve, which is "easy to flow in one direction but difficult to flow in the reverse direction", can only flow in one direction into the intersection control area and cannot flow back in the reverse direction. Or, although it can flow back in the reverse direction, the flow rate is relatively low (compared to the case without a one-way fluid control link). That is, it is relatively easy for the fluid to enter the intersection control area through the one-way control link, but it is relatively difficult or impossible to flow back in the reverse direction or flow back in the intersection control area, thereby achieving some of the following goals, but not limited to:

[0116] (1) In the intersection control area, the reagent or other fluid portion containing a specified number of biological particles will not flow back from the intersection control area to its source channel, or even if it can flow back, the flow rate is low, and the reflux portion does not contain biological particles or contains but is negligible;

[0117] (2) At or near the location of the one-way control link of the fluid where “one-way flow is easy but reverse flow is difficult”, other biological particles in the channels connected to this link that have not entered the intersection control area will not mistakenly enter the intersection control area when receiving fluid pressure from other channels. Because of its one-way control mechanism, it will not flow away from the intersection control area in the reverse direction. Or, even if it flows away from the intersection control area in the reverse direction, the flow displacement is shorter than when there is no one-way control mechanism, which is conducive to faster injection into the intersection control area during the next round (or stage) of sorting operations;

[0118] (3) Due to the setting of different flow resistances of different outflow channels, all or most of the reagents or fluids that are the sorting targets in the intersection control area enter the low-resistance outflow channel connected to the intended target position, while other non-intended outflow channels with higher flow resistance cannot flow out, flow out less, or the outflow amount is negligible, which does not affect the sorting results or has a small impact. The mechanism includes but is not limited to: setting the flow resistance of the target outflow channel smaller than that of other outflow channels, thereby achieving the goal of all or most of the fluid with a specified number of biological particles in the intersection control area to flow out through the intended or designated channel with small flow resistance; or, through pump and valve structures, devices or facilities, achieving the goal of closing or increasing the flow resistance of other channels except the designated channel connected to the intended target space;

[0119] (4) The intended outflow channel is located at or near the junction of the intersection control area, and its direction is collinear with the flow direction of the driving fluid in the intersection control area or at an acute angle, thereby utilizing the fluid inertia to achieve: the driving fluid causes the reagent or fluid portion containing a specified number of biological particles in the intersection control area to flow out through the intended (designated) channel with low flow resistance that is connected to the target space, and move to the designated space or target position.

[0120] (5) Unintended outflow channels, which prevent or reduce the outflow of fluid from the intersection control area, include some of the following:

[0121] (a) Unintended outflow channels can be partially or completely closed,

[0122] (b) The flow rate of the non-intentional outflow channel can be adjusted.

[0123] (c) Taking the flow direction of the driving fluid at or just entering the intersection control area as the reference direction, the angle between the direction of any non-intentional outflow channel at the point where it connects with the intersection control area and the reference direction among all outflow channels shall not be less than the angle between the direction of the intended outflow channel at the point where it connects with the intersection control area and the reference direction. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1 This is a structural diagram of the "one-way valve structural unit" in the "one-way easy, reverse difficult" fluid one-way control link of the utility model;

[0125] Figure 2 This is a schematic diagram of the structural principle of the "one-way valve structural unit" described in the present utility model when the internal fluid flows in the forward and reverse directions;

[0126] Figure 3 This is a schematic diagram of the structure principle of the microfluidic chip for single cell sorting in a V-shaped topology in the single cell sorting system of the present invention;

[0127] Figure 4This is a schematic diagram of the structure principle of the microfluidic chip for single cell sorting in the T-type topology in the single cell sorting system of the present invention;

[0128] Figure 5 This is a schematic diagram of the structural principle of the microfluidic chip used for single cell sorting in a cross-topology in the single cell sorting system of the present invention. DETAILED DESCRIPTION

[0129] The following embodiment of the present invention is only one of the specific forms of the present invention, and is provided for the purpose of describing the present invention in more detail, rather than limiting the scope of the present invention or the application form of the present invention.

[0130] Figure 1 The figure shows a schematic structural diagram of a "one-way valve structural unit" in the one-way fluid control link of the present invention, which is "easy to one-way and difficult to reverse". This structure is a component of the following multiple embodiments.

[0131] Figure 2 This is a schematic diagram of the structural principle of the "one-way valve structural unit" described in the present invention when the internal fluid flows forward and reverse, wherein the common inlet is shown as label A, the common outlet is shown as label D, and the forward flow direction of the fluid in the channel refers to: from label A, through label B or C, to label D; the reverse flow direction refers to: from label D, through label B or C, to label A.

[0132] Figure 1 The "check valve structure unit" shown in the figure includes two ports: a common inlet (indicated by label A) and a common outlet (indicated by label D). The two ports are interconnected through two channels (a straight-through flow channel indicated by labels A→B→D, and a return flow channel indicated by labels A→C→D). The structure or shape has the following characteristics:

[0133] (a) In forward flow, if Figure 2 As shown in part (a), after the fluid labeled A enters the common inlet, it is divided into two channels, labeled A→B→D and A→C→D, and then converges at the common outlet labeled D and can flow out smoothly. The spatial vector with the common inlet (labeled A) as the starting point and the common outlet (labeled D) as the end point is defined as the forward flow reference vector. The flow direction of any section of the straight flow channel (labeled A→B→D) is the same as the direction of the forward flow reference vector, or the angle between the two is acute. The return flow channel (labeled A→C→D) has several sections at the common inlet (labeled A) (the front section of the labeled A→C section) whose flow direction is opposite to the direction of the forward flow reference vector, or the angle between the two is non-acute. However, near the common outlet (labeled D) (the labeled C→D section), the return flow channel has the same direction as the forward flow reference vector, or the angle between the two is acute.

[0134] (b) In reverse flow, if Figure 2 As shown in part (b), the fluid flows from the common outlet (as shown by label D) through two channels (the reverse straight-through flow channel as shown by label D→B→A, and the reverse return flow channel as shown by label D→C→A), diverges in the opposite direction, and then converges at or near the common inlet (as shown by label A). Because the fluid velocity vectors in at least two channels have opposite or mutually canceling components before confluence (such as the reverse straight-through flow channel section as shown by label B→A, and the reverse return flow channel section as shown by label C→A), the sum of the absolute values ​​of the energy or momentum of the two after confluence is lower than that before confluence, resulting in the inability to flow out, difficulty in flowing out, or the ability to flow out but at a very slow flow rate after confluence. This process starts with the common outlet (as shown by label D) and ends with the common inlet (as shown by label A). The spatial vector of a point is defined as: the reverse flow reference vector. The following flow channel direction refers to the flow direction of the fluid in the channel. The flow channel direction of any section of the reverse straight flow channel (shown by label D→B→A) is the same as the direction of the reverse flow reference vector or the angle between the two is an acute angle; and the reverse return flow channel (shown by label D→C→A) has several sections at the common entrance (shown by label A) or near it (such as the rear section of the section labeled C→A), and the flow channel direction is opposite to the direction of the reverse flow reference vector or the angle between the two is not an acute angle (right angle or obtuse angle). However, in this case, the return flow channel is near the common outlet (shown by label D), and its flow channel direction (shown by label D→C) is the same as the direction of the reverse flow reference vector or the angle between the two is an acute angle.

[0135] All embodiments of the present invention adopt a flexible sorting or control method: no high-voltage electromagnetic field is used, no ultra-high-pressure sheath fluid similar to flow sorting technology is used, and there is no need to charge the sorting targets (cells or other types of biological particles). Parameters such as the flow rate, flow rate, pressure or pressure of the fluid can be adjusted (or controlled, and can be lowered or increased according to activity requirements). Then, during the entire operation, the activity of the sorting targets such as cells and bacteria is not affected, the effect is very small, or even if there is an effect, it can be ignored.

[0136] Example 1 - The channel in the chip has a V-shaped topology:

[0137] Figure 3 This is a schematic diagram of the structural principle of the microfluidic chip used for single cell sorting in the V-shaped topology of the single cell sorting system of the present invention.

[0138] The microfluidic chip used for single cell sorting includes two channels shown by labels E→Q and F→Q, which are interconnected through an intersection control area (shown by label Q). The position where the channel is connected to the intersection control area is a channel port.

[0139] The reagent input channel indicated by label E→Q includes a fluid one-way control link with the characteristics of “easy one-way flow and difficult reverse flow”: “optimized Tesla one-way valve”, such as Figure 3 As shown, by several Figure 1 The multiple "one-way valve structural units" shown are connected end to end, and adjacent units are designed in a mirror-flipped shape along the horizontal direction of the figure. The functional part of the entire "optimized Tesla one-way valve" is composed of N (N is a positive integer) one-way valve structural units, which are connected end to end in an alternating mirror-flipped form. This method reduces the forward flow resistance as much as possible while maximizing the reverse flow resistance.

[0140] A solenoid valve (such as K1) that can control the flow of fluid in the channel can be set on the driving fluid input channel (as shown by label F→Q) or on the conduit connected to the other side of the channel connected to the intersection control area (the channel is at the entrance shown by label F).

[0141] The reagent input channel (indicated by the label E→Q) for injecting the suspension of biological particles to be sorted is connected to a catheter for injecting reagents at its inlet (label E). The injection power includes but is not limited to several of the following: pumps (pressure pumps, constant pressure pumps), high-pressure gas cylinders, and other fluids driven by pumps and valves. In addition, the control link may include (or not include): controllable valves, device functional structures, facilities or instruments and equipment, in conjunction with the corresponding sorting process, to control the flow rate or start and stop status of the reagent input in this link.

[0142] The implementation process of this embodiment is as follows:

[0143] The biological reagent (types include but are not limited to: suspension containing cells, bacteria or other biological particles, or fluid in the form of droplets, bubbles, etc. that encapsulate cells, bacteria or other biological particles) is driven through the pump valve structure or functional device to enter the channel (labeled E→Q) through the input port indicated by label E.

[0144] At the same time, when the driving fluid in the other input channel (labeled F→Q) is not found to have a specified number of biological particles entering the intersection control area through the solenoid valve K1 and the catheter, the solenoid valve K1 is closed. At this time, the driving fluid cannot enter the intersection control area, and the fluid in the intersection control area cannot or is difficult to: flow in the opposite direction from label Q to label F.

[0145] When the cell (or bacteria, or other biological particle) suspension reagent to be sorted flows from inlet E through the optimized Tesla one-way valve composed of N one-way valve structural units on the reagent input channel (labeled E→Q), due to its relatively low flow resistance, it smoothly reaches the intersection control zone (indicated by label Q) in the forward direction. Real-time detection in the intersection control zone indicates that a specified number (e.g., a single) cell has entered the intersection control zone. The solenoid valve K1 is immediately opened. Driven by the powerful pressure of an external pressure pump (or syringe pump), the driving fluid rapidly reaches the intersection control zone (indicated by label Q) through the driving fluid input channel (indicated by label F) at a higher flux rate than the suspension reagent entering the intersection control zone. The driving fluid then pushes the reagent portion containing the specified number of biological particles previously at the intersection control zone through the sorting result output channel (indicated by label Q→G), either directly or via a conduit to a specific well in the orifice plate, or a designated target space. The solenoid valve K1 on the driving fluid input channel or on the conduit connected to the driving fluid input channel inlet is closed, completing the sorting of the specified number of cells at the designated location in the current round.

[0146] During the above process, after the solenoid valve K1, which controls the on / off switching of the driving fluid input channel, is opened, the driving fluid, characterized by high flow rate, high pressure, or high energy, rapidly reaches the intersection control region indicated by Q through the driving fluid input channel. This exerts a reverse thrust on the cell suspension reagent (i.e., the reagent used for sorting in subsequent rounds) in the reagent input channel indicated by E→Q, which is connected to but does not include the intersection control region. This prevents the reagent from entering the sorting result output channel (labeled Q→G) or the designated sorting target location such as the orifice plate through the intersection control region, thereby avoiding sorting errors and mistakes. Furthermore, because the reverse flow resistance of the optimized Tesla one-way valve in the reagent input channel indicated by E→Q is relatively high, the reagent used for sorting in the subsequent round cannot or has difficulty flowing backward through the reagent input channel to its inlet in the direction of E. As a result, the reagent stays in place or flows in reverse for a short distance, facilitating the rapid entry of the cell suspension reagent for the next round into the intersection control region, thereby efficiently carrying out the next round of sorting.

[0147] The above process is repeated repeatedly, and different wells in the well plate (including but not limited to: 96-well plate or 384-well plate) are continuously switched as the sorting destination, so as to realize continuous single-cell flexible sorting operation for the well plate - if the specified number is 1, the single-cell well plate plating operation is realized.

[0148] If a test tube or other single-cavity device (such as a syringe, culture dish, EP tube) is used, the sorted target object can be output to the device with a single storage cavity without switching the sorting destination.

[0149] The driving fluid flows into the input channel indicated by the label F→Q. A solenoid valve is provided on the conduit connected to its inlet (at the label F). The other end of the solenoid valve is connected to a driving fluid with high pressure or high speed. The methods for achieving high pressure include but are not limited to: injection of high-pressure fluid (gas or liquid), drive by pumps (constant pressure pumps, pressure pumps or other types of pumps and valves), and drive by other power methods.

[0150] Example 2 - The channel in the chip has a T-shaped topology:

[0151] Figure 4 This is a schematic diagram of the structural principle of the microfluidic chip used for single cell sorting in the T-type topology of the single cell sorting system of the present invention.

[0152] The T-type topology is a situation where three channels are connected in the intersection control area: two input channels and one output channel. The driving fluid input channel F→Q and the sorting result output channel Q→G are collinear. This structural design can drive the fluid from the inlet marked F through the intersection control area marked Q to the outlet indicated by G. In this process, the flow resistance is lower and the fluid flow speed is faster.

[0153] Another input channel: reagent input channel, including a fluid one-way control link with "easy one-way, difficult reverse": "optimized Tesla one-way valve"; such as Figure 4 As shown, including several Figure 1 The multiple "one-way valve structural units" shown are connected end to end, and adjacent units are connected end to end in a ring shape, but the adjacent units are not mirror-reversed in structure. The functional part of the entire "optimized Tesla one-way valve" is composed of N (N is a positive integer) one-way valve structural units, which do not need to be alternately mirror-reversed and adopt a ring or curved end-to-end connection form. This method, like other embodiments, can reduce the forward flow resistance as much as possible while maximizing the reverse flow resistance.

[0154] A solenoid valve (such as K1) that can control the flow of fluid in the channel can be installed on the driving fluid input channel (as shown by the label F→Q) or on the conduit connected to the other side of the channel connecting the control area (at the entrance of the channel shown by the label F).

[0155] The reagent input channel (indicated by the label E→Q) for injecting the biological particle suspension to be sorted includes a partial or complete: annular or curved channel form.

[0156] At the entrance of the reagent input channel (labeled E), a conduit for injecting reagents is connected. The injection power includes but is not limited to several of the following: pumps (pressure pumps, constant pressure pumps), high-pressure gas cylinders, and other fluids driven by pumps and valves. In addition, the control link may include (or not include): controllable valves, device functional structures, facilities or instruments and equipment, in conjunction with the corresponding sorting process, to control the flow rate or start and stop status of the reagent input in this link.

[0157] The implementation process of this embodiment is as follows:

[0158] The biological reagent (types include but are not limited to: suspension containing cells, bacteria or other biological particles, or fluid in the form of droplets, bubbles, etc. that encapsulate cells, bacteria or other biological particles) is driven through the pump valve structure or functional device to enter the channel (E→Q) through the reagent input port indicated by the label E.

[0159] At the same time, when the driving fluid input channel (F→Q) is connected to the driving fluid via the solenoid valve K1 and the catheter and no specified number of biological particles are found to enter the intersection control area, the solenoid valve K1 is closed. At this time, the driving fluid cannot enter the intersection control area, and the fluid in the intersection control area cannot or is difficult to flow in the opposite direction from label Q to label F.

[0160] When the cell (or bacteria, or other biological particle) suspension reagent to be sorted flows from inlet E through the optimized Tesla one-way valve composed of N one-way valve structural units on the reagent input channel (labeled E→Q), due to its relatively low flow resistance, it smoothly reaches the intersection control area (indicated by label Q) in the forward direction. Real-time detection in the intersection control area indicates that a specified number (e.g., a single) cell has entered the intersection control area. The solenoid valve K1 is immediately opened. Driven by the strong pressure of an external pressure pump (or syringe pump), the driving fluid quickly enters the intersection control area from the driving fluid input channel through label F at a higher flux rate than the suspension reagent entering the intersection control area. The driving fluid then pushes the reagent portion containing the specified number of biological particles at the intersection control area through the sorting result output channel (indicated by label Q→G), directly or through a conduit to a specific well in the orifice plate, or other designated target space. The solenoid valve K1 on the driving fluid input channel or on the conduit connected to the driving fluid input channel inlet is closed, completing the sorting of the specified number of cells at the designated location in the current round.

[0161] During the above process, after the solenoid valve K1, which controls the on / off switching of the driving fluid input channel, is opened, the driving fluid, characterized by high flow rate, high pressure, or high energy, rapidly reaches the intersection control region indicated by Q through the driving fluid input channel. This exerts a reverse thrust on the cell suspension reagent (i.e., the reagent used for sorting in subsequent rounds) in the reagent input channel indicated by E→Q, which is connected to the intersection control region but does not include the intersection control region, preventing the reagent from entering the sorting result output channel (labeled Q→G) or the designated sorting target location such as the orifice plate through the intersection control region, thereby avoiding sorting errors and mistakes. In addition, because the reverse flow resistance of the optimized Tesla one-way valve in the reagent input channel indicated by E→Q is relatively high, the reagent used for sorting in the subsequent round cannot or has difficulty flowing backward through the reagent input channel to its inlet in the direction of E. As a result, the reagent stays in place or flows in reverse for a short distance, facilitating the rapid entry of the cell suspension reagent for the next round into the intersection control region, thereby efficiently carrying out the next round of sorting.

[0162] The above process is repeated repeatedly, and different wells in the well plate (including but not limited to: 96-well plate or 384-well plate) are continuously switched as the sorting destination, so as to realize continuous single-cell flexible sorting operation for the well plate - if the specified number is 1, the single-cell well plate plating operation is realized.

[0163] If a test tube or other single-cavity device (such as a syringe, culture dish, EP tube) is used, the sorted target object can be output to the device with a single storage cavity without switching the sorting destination.

[0164] The driving fluid flows into the input channel indicated by the label F→Q, and a solenoid valve is provided on the conduit connected to its inlet F. The other end of the solenoid valve is connected to a driving fluid with high pressure or high speed. The methods for achieving high pressure include but are not limited to: injection of high-pressure fluid (gas or liquid), drive by pumps (constant pressure pumps, pressure pumps or other types of pumps and valves), and drive by other power methods.

[0165] In the T-type topology three-way channel situation described in this embodiment, if the driving fluid input channel (F→Q) and the sorting result output channel (Q→G) connected to it through the intersection control area and with the channel direction collinear with it are understood as one channel, the above-mentioned "three-way channel situation" can be understood as a "two-way channel situation".

[0166] Example 3 - The channels in the chip are in a "cross" topology:

[0167] The "cross" topology is a situation where at least four channels are included: the microfluidic chip used for single-cell sorting uses the intersection control area as the port flow reference point, and includes at least four channels interconnected through the intersection control area: two input channels and two output channels, any input channel and a corresponding output channel (the reagent input channel corresponds to the waste liquid output channel; the driving fluid input channel and the sorting result output channel), the channel directions are collinear, nearly collinear (the directions or directions of the two channels are collinear or nearly collinear, which means that the flow vector directions of the fluids in the two channels at the port position of the intersection control area are the same or the angle between the two is an acute angle) or the angle is an acute angle; wherein at least one input channel (reagent input channel) includes or is connected (connection method: through the channel inside the chip or the pipeline facility outside the chip): a fluid unidirectional control link with "easy one-way and difficult reverse", and one of the types of fluid control structures includes: "optimized Tesla one-way valve".

[0168] Figure 5 This is a schematic diagram of the structural principle of the microfluidic chip used for single cell sorting in a cross-topology in the single cell sorting system of the present invention.

[0169] The "cross" topology of this embodiment is a situation in which four channels are connected in the intersection control area: two input channels and two output channels, wherein the input channel E→Q and the output channel Q→H are collinear; the input channel F→Q and the output channel Q→G are collinear. Such a structural design can realize the driving fluid from the inlet at label F through the intersection control area at label Q to the outlet indicated by label G. In this process, the flow resistance is lower and the fluid flow speed is faster. Similarly, after the reagent enters through label E, it enters from the outlet of the "optimized Tesla one-way valve" through the intersection control area at label Q to the outlet indicated by label G. In this process, the flow resistance is lower and the fluid flow speed is faster.

[0170] Note: The significant feature of the present invention when using the "cross" topology is that the cross-sectional area of ​​the access port of the waste liquid output channel in the intersection control area (including but not limited to several of the following: port width, depth) is not less than (preferably greater than) the access port of the reagent input channel in the intersection control area, which is used to prevent: the reagent part that is not the sorting target from accidentally entering the driving fluid input channel, the sorting result output channel and other spaces, thereby eliminating experimental errors and enhancing reliability.

[0171] The reagent input channel (indicated by the label E→Q) contains a fluid one-way control link with the characteristics of "easy one-way and difficult reverse": the "optimized Tesla one-way valve"; Figure 5 As shown, including several Figure 1The multiple "one-way valve structural units" shown are connected end to end, and adjacent units are designed in a mirror-flipped shape along the horizontal direction of the figure. The functional part of the entire "optimized Tesla one-way valve" is composed of N (N is a positive integer) one-way valve structural units, which are connected end to end in an alternating mirror-flipped form. This method reduces the forward flow resistance as much as possible while maximizing the reverse flow resistance.

[0172] The following positions can be set up with solenoid valves that can control the flow of fluid in the channel:

[0173] A solenoid valve K1 may be provided on the driving fluid input channel (as indicated by the label F→Q), or on the conduit connected to the other side of the channel that connects to the intersection control area (at the inlet of the channel indicated by the label F);

[0174] A solenoid valve K2 can be installed on the sorting result output channel (as shown by the label Q→G), or on the conduit connecting the channel to the other side of the intersection control area (at the outlet of the channel as shown by the label G);

[0175] A solenoid valve K3 can be provided on the waste liquid output channel (as indicated by the label Q→H), or on the conduit connected to the other side of the channel connected to the intersection control area (at the outlet of the channel indicated by the label H).

[0176] The reagent input channel E→Q for injecting the biological particle suspension to be sorted is connected to a conduit for injecting reagents at its inlet (labeled E). The injection power includes but is not limited to several of the following: pumps (pressure pumps, constant pressure pumps), high-pressure gas cylinders, and other fluids driven by pumps and valves. In addition, the control link may include (or not include): controllable valves, device functional structures, facilities or instruments and equipment, in conjunction with the corresponding sorting process, to control the flow rate or start and stop status of the reagent input in this link.

[0177] The implementation process of this embodiment is as follows:

[0178] Before the sorting is started, or during the target capture phase when no specified number of biological particles are found to have entered the intersection control zone, the solenoid valves K1 and K2 are closed, and the solenoid valve K3 is opened.

[0179] Through an external or internal pump-valve structure or pump-valve functional device, the biological reagent (types include but are not limited to: suspensions containing cells, bacteria or other biological particles, or fluids in the form of droplets, bubbles, etc. that encapsulate cells, bacteria or other biological particles) is driven to enter the reagent input channel (labeled E→Q) through the input port indicated by label E, and through the optimized Tesla one-way valve composed of N one-way valve structural units of the reagent input channel (labeled E→Q), it can reach the intersection control area (indicated by label Q) smoothly and quickly in the forward direction.

[0180] At this time, the driving fluid cannot enter the intersection control area because the solenoid valve K1 is closed, and the reagent in the intersection control area cannot or is difficult to flow in the opposite direction from label Q to label F through the driving fluid input channel. At the same time, because the solenoid valve K2 is closed, the reagent in the intersection control area cannot or is difficult to flow from label Q to label G through the sorting result output channel.

[0181] When the biological reagents arrive at the intersection control area through the reagent input channel, the system uses its detection part (including but not limited to: strong light induced fluorescence detection for fluorescently stained targets, scattered light, reflected light, transmitted light, shadow area and other light detection for unlabeled targets, and graphic visual detection of the outline, color, and properties of any target) to detect in real time whether the sorting targets in the intersection control area have reached the specified number:

[0182] Real-time detection is performed in the intersection control area. If no specified number of cells (such as a single cell, or other specified number) are found to enter the intersection control area, the reagent will enter the waste liquid output channel (labeled Q→H) through the intersection control area. At this time, the solenoid valve K3 is open, and the solenoid valves K1 and K2 are closed. The driving fluid input channel and the sorting result output channel are blocked, and the reagent can only be exported to the external designated space through the waste liquid output channel.

[0183] After real-time detection in the intersection control area, if a specified number of cells are found to have entered the intersection control area, the solenoid valve K3 is immediately closed, and the solenoid valves K2 and K1 are opened in sequence. Driven by the strong pressure of the external pressure pump (or syringe pump), the driving fluid enters the intersection control area at a flux and speed higher than the reagent (cell, bacteria or other biological particle suspension) in the reagent input channel, and quickly reaches the intersection control area indicated by label F. Afterwards, the driving fluid pushes the reagent part containing the specified number of biological particles in the intersection control area, through the sorting result output channel (indicated by label Q→G), directly or through the catheter to the specified target space such as the specific hole in the 96-well plate. After the process is completed, the solenoid valves K1 and K2 are immediately closed, and the solenoid valve K3 is opened, completing the sorting of the specified number of cells at the specified location in the current round, and re-entering the target capture stage.

[0184] During the above process, after solenoid valve K3 is closed and solenoid valves K2 and K1 are opened, the driving fluid with characteristics such as high flow rate, high pressure, or high energy quickly reaches the intersection control area indicated by label Q. This exerts a reverse thrust on the cell suspension reagent (i.e., the reagent used for sorting in subsequent rounds) in the reagent input channel (indicated by label E→Q) connected to the intersection control area but not including the intersection control area, preventing the reagent from entering the designated sorting target position such as the orifice plate through the intersection control area, nor entering the driving fluid input channel or the sorting result output channel, thereby avoiding sorting errors and mistakes. In addition, because the reverse flow resistance of the optimized Tesla one-way valve in the reagent input channel indicated by label E→Q is relatively high, the reagent used for sorting in the subsequent round cannot or has difficulty in reverse flow to its inlet in the direction of label E, and stays in place or flows in reverse for a short distance, which facilitates the rapid entry of the cell suspension reagent of the next round into the intersection control area, thereby efficiently carrying out the next round of sorting.

[0185] The above process is repeated repeatedly, and different wells in the well plate are constantly switched as sorting destinations, so that continuous single-cell flexible sorting operations can be achieved for the well plate - if the specified number is 1, a single-cell well plate transplanting operation is achieved.

[0186] If a test tube or other single-cavity device (such as a syringe, culture dish, EP tube) is used, the sorted target object can be output to the device with a single storage cavity without switching the sorting destination.

[0187] In this embodiment, a solenoid valve may be provided on the driving fluid input channel indicated by the label F→Q through which the driving fluid flows, or on a conduit connected to its inlet F. The other end of the solenoid valve is connected to a driving fluid with high pressure or high speed. Methods for achieving high pressure include, but are not limited to, injection of high-pressure fluid (gas or liquid), drive by a pump (constant pressure pump, pressure pump, or other type of pump or valve), and drive by other power methods.

[0188] In the "cross" topology described in this embodiment, in the case of four channels, if an input channel is connected to an output channel via an intersection control area and the channel directions are collinear or at an acute angle (such as nearly collinear) with the output channel, it is understood as one channel.

[0189] For example, in this embodiment, if the driving fluid input channel (labeled F→Q) and the collinear sorting result output channel (labeled Q→G) are combined or understood as one channel, and the reagent input channel (labeled E→Q) and the collinear waste liquid output channel (labeled Q→G) are combined or understood as one channel,

[0190] The above-mentioned "four-way channel situation" can be understood as a "two-way channel situation".

Claims

1. A microfluidic chip and single-cell sorting system for label-free or fluorescently labeled single-cell sorting, characterized by: The single cell sorting system includes: a microfluidic chip for single cell sorting, and pump and valve structures, devices or facilities, The microfluidic chip for single cell sorting comprises a plurality of channels interconnected through a junction control area, and also comprises an inlet and an outlet, both of which are connected to the channels respectively. The single cell separation is performed by using a one-way control link on a plurality of channels of a microfluidic chip, or on a conduit or non-conduit pipe connected to the channels, which is easy to flow in one direction but difficult to flow in the reverse direction. The one-way control link is referred to as a one-way control link. The one-way control link is composed of a plurality of one-way valve structural units. The one-way control link is located inside or outside the microfluidic chip. Some types of one-way control links, such as "optimized Tesla one-way valve", include one or more "one-way valve structural units". The "one-way valve structural unit" includes two types of ports: a common inlet and a common outlet. The two ports are interconnected through at least one straight-through flow channel and at least one corresponding backflow channel, and have the following structural or morphological characteristics: (a) During forward flow, the fluid enters from a common inlet and is divided through at least two channels before converging and flowing out smoothly at or near a common outlet. This process is defined as a spatial vector with the common inlet as the starting point and the common outlet as the end point, which is defined as: a forward flow reference vector. The flow path direction mentioned below refers to the flow direction of the fluid in the channel. For any section of the straight-through flow channel, the flow path direction is the same as the forward flow reference vector or the angle between the two is acute. The return flow channel has several sections at or near the common inlet, whose flow path direction is opposite to the forward flow reference vector or the angle between the two is non-acute. The non-acute angle includes: a right angle or an obtuse angle. However, the return flow channel is near the common outlet, and its flow path direction is the same as the forward flow reference vector or the angle between the two is acute. (b) During reverse flow, the fluid flows in reverse from the common outlet through at least two channels and then converges at or near the common inlet. The spatial vector of this process, which takes the common outlet as the starting point and the common inlet as the end point, is defined as: the reverse flow reference vector. The flow direction mentioned below refers to the flow direction of the fluid in the channel. Then, the flow direction of any section of the straight flow channel is the same as the direction of the reverse flow reference vector or the angle between the two is acute. The return flow channel has several sections at or near the common inlet, and the flow direction is opposite to the direction of the reverse flow reference vector or the angle between the two is non-acute. The non-acute angle includes: a right angle or an obtuse angle. In this case, the return flow channel is near the common outlet, and its flow direction is the same as the direction of the reverse flow reference vector or the angle between the two is acute.

2. The microfluidic chip and single cell sorting system for single cell sorting according to claim 1, characterized in that: The one-way fluid control link with the feature of "easy one-way flow but difficult reverse flow" includes the following valve body types: Non-controllable, passive "one-way easy, reverse difficult" function: one-way membrane valve, Tesla valve, Or with ejector pins, reeds, axial rotating bodies, or structural facilities that can block fluid movement in one direction: structures, valve components, devices or equipment, "One-way valve structural unit" channel: When the fluid flows in the reverse direction from the common outlet to the common inlet, in order to achieve a structural design in which the flow resistance of the reverse flow channel is smaller than that of the straight flow channel during the reverse flow process, the following design is adopted: (a) The cross-sectional area of ​​the reversal channel is larger than that of the straight-through channel, including: the cross-sectional area of ​​the reversal channel is wider than that of the straight-through channel, or the cross-sectional area of ​​the reversal channel is deeper than that of the straight-through channel; Or: (b) the length of the reversal channel is shorter than the straight-through channel; Or: (c) the inner wall of the return flow channel is made hydrophobic, oleophobic, or gas-repellent, or the inner wall of the through flow channel is made hydrophilic, oleophobic, or gas-repellent, so that the flow resistance of the fluid when flowing in the return flow channel is smaller than that when flowing in the through flow channel; or: (d) channel setting or connection: pump valve type structure, device, facility or apparatus; Alternatively: (e) the channel flow resistance can be adjusted; Alternatively: (f) the channel can be closed and cut off.

3. The microfluidic chip and single cell sorting system for single cell sorting according to claim 1, characterized in that: "One-way valve structural unit", different "one-way valve structural units" connected through the inlet and outlet, and adjacent to each other, have the following structural features at the channel connection: In the reverse flow direction of the fluid, in the "one-way valve structure unit", when the fluid flows from its common outlet through its internal multiple channels to its common inlet, if the flow direction after re-convergence is the same as the flow direction of the return flow channel at the common outlet of the next "one-way valve structure unit" to flow through, or the two are at an acute angle, In the case of acute angles, the direction of the fluid flow after the reverse confluence, near the common inlet where it flows out, and the direction of the blocking flow channel and the straight flow channel at the common outlet of the next "one-way valve structure unit" are respectively named: "blocking angle" and "straight-through angle". The "blocking angle" is not greater than the "straight-through angle". The one-way control link, such as the "optimized Tesla one-way valve", is composed of multiple "one-way valve structural units". When the multiple "one-way valve structural units" are connected end to end and used in series, some or all of the "one-way valve structural units" have the following structural design (1) or (2): (1) For example, with the straight line, tangent line, or straight line in space as the axis, the different "one-way valve structure units" in front and behind are alternately mirror-reversed, or with reference to the adjacent ones, part or all of the "one-way valve structure unit" is modified in the following ways: scaling, rotation, shape modification, or mirror-reversal with a straight line in space as the axis, so as to obtain the effect of small forward flow resistance and large reverse flow resistance; (2) The front and rear adjacent "one-way valve structure units" rotate at a certain angle with the connection point as the axis.

4. The microfluidic chip and single cell sorting system for single cell sorting according to claim 1, characterized in that: The single cell sorting system includes not only microfluidic chips, pump and valve structures, devices or facilities for single cell sorting, but also detection parts and control systems. Pump and valve structures, devices or facilities are installed on several channels of the microfluidic chip for single cell separation, or on the conduits or pipes connected to the channels. Detection part: Real-time detection of whether the sorting target objects in the intersection control area have reached the specified number, Control system: The electrical signal is recognized by the control system and used as the basis for implementing sorting operations.

5. The microfluidic chip and single cell sorting system for single cell sorting according to claim 2, characterized in that: The fluid one-way control link includes: a Tesla valve structure with "easy one-way and difficult reverse", the Tesla valve structure includes an optimized Tesla one-way valve, Through the one-way fluid control link, when a specified number of biological particles enter the intersection control area, By controlling the opening of the driving fluid input channel connected to the same intersection control area and injecting the driving fluid at high speed, the driving fluid generates pressure or pressure in all directions when it reaches the intersection control area, which can produce a blocking effect: making it impossible for the "next round of manipulation reagents or fluid parts" to continue entering the intersection control area. The driving fluid can push part or all of the fluid in the intersection control area containing a specified number of biological particles to flow out of the intersection control area through the intended low-resistance channel connected to the target location and move to a specified space or target location, including: several wells on a multi-well plate, test tubes, collectors, or receivable containers, to achieve the sorting of the specified number of biological particles. The intended outflow channel is at or near the junction of the intersection control area, and its direction is collinear with the flow direction of the driving fluid in the intersection control area or at an acute angle. The mechanism of the unintended outflow channel to prevent or reduce the outflow of fluid from the intersection control area includes: taking the flow direction of the driving fluid at or just entering the intersection control area as the reference direction, the angle between the direction of the unintended outflow channel at the connection with the intersection control area and the reference direction of all outflow channels is not less than the angle between the direction of the intended outflow channel at the connection with the intersection control area and the reference direction, The single cell sorting system includes an apparatus with a storage cavity, such as a well plate, a test tube, an EP tube, a centrifuge tube, a culture dish, a syringe, a well plate, as a designated number of sorting targets, and a collection device after sorting. Among them, well plate sorting is adopted. At the end of each round, different wells in the well plate are switched one by one as sorting destinations. If the sorting target is a cell, the specified number is 1, and continuous single-cell flexible sorting operation for the well plate can be realized. The automated operation of the single-cell well plate is connected to: a collection device of the single-cell sorting system; or an instrument with a single storage cavity, such as a test tube, a syringe, a culture dish, or an EP tube, which can output the sorted target to an instrument with a single storage cavity, serving as a collection device of the single-cell sorting system after sorting.

6. The microfluidic chip and single cell sorting system for single cell sorting according to claim 1, characterized in that: Single cell separation uses a microfluidic chip, and multiple channels are interconnected through the intersection control area. Channel structures include: V-type topology: It contains at least two channels that are interconnected through the intersection control area. The location where the channel is connected to the intersection control area can be the channel port or any location of the channel. One of the channels includes, or is connected via an intra-chip channel or an off-chip piping facility: a fluid one-way control link that is "easy to flow in one direction but difficult to flow in the reverse direction," and the type of the fluid one-way control link includes: an "optimized Tesla one-way valve"; On several channels, or channels connected to the inlet and outlet: channels, conduits or pipelines, containing pump and valve structures, devices or facilities that can control the flow of fluid in the channels, The above-mentioned pump and valve structures, devices or facilities are installed in the following locations: between the channel entrance and the intersection control area, or on the channel, conduit or pipeline connected to the channel entrance.

7. The microfluidic chip and single cell sorting system for single cell sorting according to claim 1, characterized in that: Single cell separation uses a microfluidic chip, and multiple channels are interconnected through the intersection control area. Channel structures include: T-type topology: Taking the intersection control area as the port flow reference point, it includes at least three channels interconnected through the intersection control area, two input channels and one output channel, wherein the driving fluid input channel and the sorting result output channel have collinear directions or acute angles; the other input channel includes, or is connected through the chip channel or chip external pipeline facilities: a fluid one-way control link with "easy one-way and difficult reverse", the types of which include: "optimized Tesla one-way valve", This type of fluid control structure also includes: the driving fluid input channel and the sorting result output channel are collinear, On several channels, or on the channels, conduits or pipelines connected to the inlets and outlets of the channels, pump valve structures, devices or facilities that can realize fluid on-off control are installed. The above-mentioned pump and valve structures, devices or facilities are located between the channel entrance and the intersection control area, or in the channel, conduit or pipeline connected to the channel entrance. The T-type topology "at least three-way channel situation" becomes "at least two-way channel situation" if the input channel is connected to an output channel via an intersection control area, and the channel direction is collinear or nearly collinear with the output channel, that is, one channel.

8. The microfluidic chip and single cell sorting system for single cell sorting according to claim 1, characterized in that: Single cell separation uses a microfluidic chip, and multiple channels are interconnected through the intersection control area. The channel structure includes: "cross" topology: Taking the intersection control area as the port flow reference point, it contains at least four channels interconnected through the intersection control area: two input channels and two output channels, any input channel corresponds to an output channel, and the reagent input channel corresponds to the waste liquid output channel; the driving fluid input channel and the sorting result output channel, the channel directions are collinear or the angle is acute; at least one of the input channels contains, or is connected through the chip channel or chip external pipeline facilities: a fluid one-way control link with "easy one-way and difficult reverse", the types of which include: "optimized Tesla one-way valve", The fluid control structure type includes: the driving fluid input channel and the sorting result output channel are collinear, the reagent input channel and the waste liquid output channel are collinear, This type of fluid control structure includes: a waste liquid output channel that is collinear with or at an acute angle to the reagent input channel; the cross-sectional area of ​​the access port in the intersection control area is set to include: the port width or depth is not less than the access port of the reagent input channel in the intersection control area, and is used to prevent: the reagent part that is not the sorting target from accidentally entering the driving fluid input channel and the sorting result output channel, thereby eliminating experimental errors and enhancing reliability. The "at least four-way channel situation" of the "cross" topology can be understood as "at least two-way channel situation" if the input channel and an output channel connected to it via the intersection control area and whose channel direction is collinear or nearly collinear with it are understood as one channel. On several channels, or on the channels, conduits or pipelines connected to the inlets and outlets of the channels, pump valve structures, devices or facilities that can realize fluid on-off control are installed. The above-mentioned pump and valve structures, devices or facilities are installed in the following locations: between the channel inlet and the intersection control area, or on the channel, conduit or pipeline connected to the inlet.

9. The single cell sorting system according to claim 1, wherein: The pump and valve structures, devices or facilities are located in the following locations: (1) or (2): (1) Single cell sorting is performed on several channels of a microfluidic chip. (2) Channel connected to: catheter or pipe, The pump and valve structures, devices or facilities include (1) or (2) of the following: (1) Pump and valve devices that can realize controllable on and off of fluids, including: liquid pumps, air pumps, manual valves, electric valves, Especially including: solenoid valves, water pumps, constant pressure pumps, negative pressure pumps; (2) A closed container device containing fluid that can provide a constant pressure through an interface, such as: a high-pressure gas cylinder, a constant-pressure gas sealed tank, or a negative-pressure cylinder, a vacuum cylinder, The single-cell sorting system is constructed such that: a high-voltage electromagnetic field is not used, an ultra-high-pressure sheath fluid of flow sorting technology is not used, and there is no need to charge the sorting target; alternatively, the flow rate, flow rate, pressure or pressure of the fluid are adjustable or controllable parameters, which can be lowered or raised according to activity requirements, thereby achieving flexible sorting or flexible control methods.

10. The microfluidic chip and single cell sorting system for single cell sorting according to claim 1, characterized in that: The one-way control link is located inside or outside the microfluidic chip, and is arranged between the channel inlet and the intersection control area, or on a conduit or pipeline connected to the channel inlet; The detection process includes sensors, which can be selected from the following (1), (2), (3) or (4): (1) Photoelectric sensors, including photosensitive elements, such as photodiodes and PMTs that can convert light signals into electrical signals, are used to identify marked targets, including fluorescently labeled targets, such as fluorescently labeled cells and bacteria. When excited by a light source, the marked target produces fluorescence, which is converted into an electrical signal and then recognized by the control system, which is then used as a basis for sorting operations. The excitation light sources include lasers, mercury lamps, halogen lamps, and LED lamps. (2) Light sensors that can identify reflected, refracted, or scattered light, or can be used to identify the outline, color, or shape of unmarked targets, such as cells, bacteria, or unmarked biological particles. (3) Sensor devices, facilities, instruments or components that can identify the shadows formed by unmarked objects blocking light or the phenomenon that causes the transmitted light to weaken. (4) Devices, instruments or facilities with recording functions that detect and identify targets through image recognition functions; The one-way control link, such as the "optimized Tesla one-way valve", is composed of multiple "one-way valve structural units". When the multiple "one-way valve structural units" are connected end to end and used in series, some or all of the "one-way valve structural units" have the following structural design (1) or (2): (1) If the straight line, tangent line or spatial straight line in the direction of fluid flow is used as the axis, the different "one-way valve structure units" before and after are alternately mirror-reversed, or with reference to the adjacent ones, part or all of the "one-way valve structure unit" is modified as follows: scaling, rotation, shape modification, or mirror-reversal with a certain straight line in space as the axis, so as to obtain the effect of small forward flow resistance and large reverse flow resistance. (2) The front and rear adjacent "one-way valve structure units" rotate at a certain angle with the connection point as the axis.

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