Femtoliter, picoliter, nanoliter or microliter scale single cell array multi-component precise quantitative reaction system and flexible control method thereof

Abstract

The present application relates to a single-cell on-chip experiment system and a control method thereof, in particular to a system for precise quantitative reaction of a large-scale single-cell array with a femtoliter, picoliter, nanoliter or microliter scale and a flexible control method thereof, wherein the in-chip manipulation units are arranged in a row-column mode, and a transport channel is arranged between the pits in each manipulation unit and the corresponding reaction chamber, and through the action of gravity, buoyancy or fluid driving, the pits can capture a specified number of target objects such as biological particles, droplets or bubbles, and the chip can be rotated to make the target objects in a single column or multiple columns enter the corresponding reaction chamber, each reaction chamber can receive a specified number of various cells, reagents or bubbles to implement storage, mixing, culture or reaction functions, and the target objects arranged in an array can be positioned in two dimensions of row and column by means of dielectric force, electric field force, sound wave, fluid driving or a needle tube type instrument, and can be efficiently discharged, recycled or replaced, and the cell activity is protected during the whole process.

Description

Technical Field

[0001] This invention relates to a microfluidic system and its control method in the fields of biotechnology, life sciences and medical health, and particularly to a large-scale single-cell array multi-component precise quantitative reaction system at the fly-liter, picoliter, nanoliter or microliter level and its flexible control method. Background Technology

[0002] In the fields of biotechnology, life sciences, and healthcare, the precise sorting and array-based reaction analysis of individual bioactive biological particles such as cells, bacteria, and viruses are core tools for drug screening, single-cell analysis, and precision medicine. Droplet microfluidics can generate highly uniform droplets in tiny volumes of femtoliters, picoliters, or nanoliters, and encapsulate single cells in individual droplets or microspaces as independent biochemical reaction containers, making it suitable for high-throughput and high-resolution biochemical analysis. However, existing platforms are mostly based on multi-well plate systems combined with pipetting workstations for control, with reaction volumes typically in the microliter range, resulting in high reagent consumption and limited throughput.

[0003] Technical issues: The existing technology has the following main shortcomings: (1) In terms of reagent handling precision, existing well plates and pipetting systems can usually only reach the microliter or even nanoliter level, making it difficult to achieve accurate measurement and pipetting of liters or picolidoliters of reagents. For applications that are extremely sensitive to reaction volume, such as single-cell analysis and micro-drug efficacy difference analysis, nanoliter or even microliter volumes will lead to excessive dilution of the analyte or waste of reagents.

[0004] (2) In terms of throughput and parallelism, the throughput of traditional well plate systems is limited by physical size and the operating speed of pipetting manipulators, making it difficult to achieve large-scale arrayed parallel reactions and continuous precise concentration gradient distribution.

[0005] (3) In terms of system integration, existing technologies disperse the steps of sorting, adding samples, mixing, and detection to different devices, making the operation process cumbersome and the samples easily damaged or contaminated during the transfer process.

[0006] (4) The micro-volume reaction system leads to huge consumption of precious biological reagents, which seriously restricts the high-throughput screening of scarce samples. Summary of the Invention

[0007] Solution: The present invention provides a large-scale single-cell array multi-component precise quantitative reaction system at the fly-liter, picoliter, nanoliter, or microliter levels, characterized as follows: The target object is a biological particle, droplet, or bubble. The droplet or bubble may or may not contain biological particles. The droplet or bubble may have a shape including but not limited to several of the following: spherical, ellipsoidal, disc-shaped, cylindrical, elongated, cylindrical, olive-shaped, irregular, and other shapes. The concept of droplet includes a liquid segment, and the concept of bubble includes a gas segment.

[0008] The biological particles may be active or inactive, wherein active biological particles include, but are not limited to: whole or part of an organism, whole or part of a microorganism, such as: cells, bacteria, viruses, nematodes, gene fragments, cell clusters, organs (including but not limited to: cell sets with several organ functions), and tissues; inactive biological particles include, but are not limited to: magnetic beads, glue beads, coded beads, granular or blocky substances.

[0009] The volume of the target object, for example but not limited to, the volume of biological particles or the volume of droplets, can occupy a space ranging from the level of femtoliters to picoliters to nanoliters to microliters, that is, it can be in the order of femtoliters, picoliters, nanoliters or microliters.

[0010] The fluid carrier in this invention is an entity capable of containing the flow of liquids, gases or solids or conducting experiments, and includes, but is not limited to, several of the following: microfluidic chips, biochips, gate-like devices, reagent kits, and including, but not limited to, transparent or translucent materials: appliances (especially including but not limited to: experimental equipment), tools or facilities.

[0011] The structure of the fluid carrier includes channels and control units. The types of channels include, but are not limited to, transfer channels and trunk channels. The trunk channel refers to the channel used for fluid flow function, and the types include, but are not limited to, inflow type, outflow type, inflow and outflow type, and single inflow and single outflow type (i.e., no inflow and no outflow, no branching type).

[0012] The control unit, also known as a control unit or functional unit, includes chambers and transport channels. The types of chambers include, but are not limited to, recesses and reaction chambers. The recesses can also be called grooves or limiting grooves. In this invention, recesses are used as a general term. They can stably load a specified number of target materials. The reaction chamber can also be called a reactor, storage chamber, observation chamber, culture chamber, shaking chamber, fusion chamber, or capture chamber. It can have any function of loading, limiting, or fixing target materials and then performing related on-chip experiments, including but not limited to several of the following: positioning, limiting, storage, observation, culture, amplification, reproduction, PCR, reaction, and other experimental steps.

[0013] The volume and precision of the chambers (especially the pits and reaction chambers included in the type), as well as the volume and precision of the droplets or bubbles as the target material type, are all pre-designed and precise: fly-liter, picoliter, nanoliter, or microliter, enabling precise quantitative reactions of multiple components in large-scale single-cell arrays.

[0014] The reaction chamber may include or be connected to a limiting area, or it may not include or be connected to a limiting area.

[0015] The limiting zone, also known as a limiting chamber, positioning zone, locking zone, or restraint zone, takes the form of, but is not limited to, a limiting space or area with complete or partial isolation facilities (examples, but not limited to, channel sidewalls, limiting facilities with holes or gaps, including but not limited to, solid obstacles such as fences, mesh, cylinders, and square pillars), especially including but not limited to, a space or area within the reaction chamber that serves to constrain / limit the position of the target object. The limiting zone refers to a separately defined area with limiting function within the fluid carrier chamber structure, which may or may not have: a curved escape prevention channel. The curved escape prevention channel includes, but is not limited to, arc-shaped, zigzag-shaped, circular, or spiral-shaped channels. The limiting mechanism includes, but is not limited to, only when the fluid carrier's attitude changes at a specific time sequence and matching speed (including but not limited to, rotational speed) (types include, but are not limited to, introductory and extrinsic types, and specific methods include, but are not limited to, continuous rotational angle changes), can the target object enter or leave the limiting zone, or completely leave the specific area (examples, but not limited to, the reaction chamber) including the limiting zone.

[0016] In particular, the limiting zone with the curved escape prevention channel has typical position protection characteristics: for non-introducing fluid carrier attitude changes with specific timing and matching speed, the target object will not enter the limiting zone from the outside, and cannot enter the curved escape prevention channel or can only enter part of the curved escape prevention channel; for non-exiting fluid carrier attitude changes with specific timing and matching speed, the target object will not completely move out of the limiting zone through the curved escape prevention channel, or even if it briefly moves out of the limiting zone to a certain position in the curved escape prevention channel, it will re-enter the original limiting zone in the next stage.

[0017] The above design, employing a specific timing and matching speed mechanism or method, can be used to export a target object from the limiting area or import a new target object.

[0018] In other operations not intended for export (including but not limited to: importing new target substances, for example but not limited to: adding new cells, drugs, reagents or other substances at different times), changes in the attitude of the fluid carrier with specific timing and matching speed for export should be avoided to ensure that the target substance is stably confined within the confinement area or in the curve escape channel near it (in cases where it can return to the confinement area).

[0019] Similarly, in other operations not intended for introduction, changes in the attitude of the fluid carrier with specific timing and matching speed should be avoided to prevent the addition of excess material that could affect the experimental process or results.

[0020] When there are multiple control units, they can be arranged in a matrix (or array) pattern in rows and columns.

[0021] Reactors that are not in the same control unit but are located in the same column / row can share the same transfer channel or have a separate transfer channel.

[0022] In this invention, the concepts of row and column are relative and spatially interchangeable.

[0023] In this invention, regarding space and location, especially between the pit and the reactor, the concept of "correspondence" refers to different chambers or related facilities related to the movement control of the same target object, including but not limited to: the concept of correspondence between the pit and the reaction chamber: pits and reaction chambers that can transfer and deliver target objects to each other according to the user's intention, and they are mutually matched.

[0024] The transfer channel can connect the pit and the corresponding reaction chamber, enabling the movement or transfer of the target object or fluid between the internal cavities of the pit and the corresponding reaction chamber. The volume of the pit cavity is not greater than (i.e., less than or equal to) the volume of the corresponding reaction chamber.

[0025] Number of pits and reaction chambers: Each can be single or multiple. The correspondence between the two can be one-to-one, one-to-many, many-to-one, or many-to-many. The transfer channels of different control units can be independent of each other, or they can be connected, shared, or designed as the same channel.

[0026] The pit and the corresponding reaction chamber are adjacent to each other, and the angle between their opening directions in three-dimensional space is not less than 90 degrees (this angle includes, but is not limited to, a right angle, an obtuse angle, or a 180-degree angle, i.e., the displacement vector of the target object inside the pit and the corresponding reaction chamber after moving out through the opening is 0 or a negative value projected onto the other opening direction, and cannot be a positive value). They are respectively set on different surfaces of the inner wall of the same or connected transfer channel of the same or different control units (the positions include, but are not limited to, the top surface, bottom surface, or side wall of the inner wall of the transfer channel).

[0027] The above mechanism, in particular including but not limited to, optimal design: within the same control unit, the openings of the recess and the corresponding reaction chamber are oriented in opposite directions in three-dimensional space, that is, the angle between the directions of the recess and the openings of the corresponding reaction chamber is 180° in three-dimensional space, the positions are opposite to the channel (including but not limited to: transfer channel), and the line connecting the positions of the recess and the openings of the corresponding reaction chamber is perpendicular to or at an acute angle to the direction of the transfer channel between them, which can achieve the best control effect with the highest efficiency.

[0028] The fluid carrier attitude or structural design may employ, but is not limited to, several of the following different stages (the stage numbers may or may not correspond to the sequence of the control flow): (1) Target acquisition stage: The spatial attitude of the fluid carrier is set as follows: the angle between the direction of the resultant force of gravity and buoyancy on the target object inside the fluid carrier and the direction of the pit opening is not less than 90° (right angle), that is, the angle is a right angle, an obtuse angle or a 180° angle, and the angle between the direction of the resultant force and the direction of the opening of the reaction chamber corresponding to the pit is not greater than 90° (right angle), that is, the angle is a 0° angle, an acute angle or a right angle.

[0029] In the implementation mechanism: when the angle between the direction of the resultant force of gravity and buoyancy on the target object and the direction of the pit opening is a right angle, the target object enters the pit by means of collision, pushing or the turbulence effect of the fluid; when it is not a right angle, the target object can be driven or displaced by the component of the resultant force of gravity and buoyancy on the target object in the direction of the target object's intended movement, so that the target object can enter the pit and be captured.

[0030] In other words, the effectiveness of the fluid carrier's attitude and structural design during the target acquisition process: The target object enters the transfer channel via fluid drive. Under the action of gravity, buoyancy or fluid drive, a specified number of one or more target objects enter the pit, while preventing them from accidentally entering the reaction chamber. Under the action of gravity or buoyancy and the inner wall of the pit, the target object is limited, positioned or captured. The design to prevent accidental entry into the reaction chamber in this step is: the opening direction of the reaction chamber is set to be no more than 90 degrees from the direction of movement of the target object. When the resultant force of gravity and buoyancy is 0, the force is driven by fluid orientation, the inertia of the target object, or other external forces, including but not limited to: electric field force on a pre-charged target object, dielectric force on a neutral target object, magnetic field force on a target object containing magnetic materials, and the force of sound waves, acoustic tweezers, ultrasound, shock waves, optical tweezers, optical traps, etc.

[0031] Based on the different results of comparing the density of the target object with that of the surrounding fluid, the commonly used design or control methods for the above mechanisms include, but are not limited to, several of the following: a) When the density of the target object injected into the fluid carrier is greater than that of the surrounding fluid, the target object inside the fluid carrier is subjected to the resultant force of gravity and buoyancy, with gravity being the dominant force acting in the direction of gravity. The spatial attitude of the fluid carrier is set as follows: The opening direction of the pit forms an angle greater than 90 degrees (i.e., an obtuse angle or a 180-degree angle) with the direction of the buoyancy force on the target object, and an angle less than 90 degrees (i.e., an acute angle or a 0-degree angle) with the direction of the buoyancy force on the target object. The opening direction of the reaction chamber forms an angle no greater than 90 degrees (i.e., a 0-degree angle, an acute angle, or a right angle) with the direction of the gravity force on the target object, and an angle no less than 90 degrees (i.e., a 180-degree angle, an obtuse angle, or a right angle) with the direction of the buoyancy force on the target object. In other words, the angle between the gravity vector of the target object and the opening direction of the pit is greater than a right angle (the angle is obtuse or a 180-degree angle), and the angle between the gravity vector and the opening direction of the reaction chamber is no greater than a right angle (the angle is a right angle, an acute angle, or a 0-degree angle). At the same time, the angle between the fluid flow direction in the transfer channel and the gravity direction is less than 90 degrees (the smaller the angle, the higher the control efficiency), and the angle between the fluid flow direction and the buoyancy direction is greater than 90 degrees (the larger the angle, the higher the control efficiency).

[0032] That is, the gravity vector of the target object has a negative component in the direction of the pit opening and a non-negative component (positive or 0) in the direction of the reaction chamber opening. In particular, including but not limited to: the angle between the direction of the gravity of the target object and the direction of the pit opening is between 100 degrees and 170 degrees, and the angle between the direction of the reaction chamber opening is between 10 degrees and 80 degrees.

[0033] b) When the density of the target object injected into the fluid carrier is less than that of the surrounding fluid, the target object inside the fluid carrier is subjected to the resultant force of gravity and buoyancy, with buoyancy being dominant and acting in the direction of buoyancy. The spatial attitude of the fluid carrier is set as follows: The angle between the opening of the pit and the direction of the buoyancy force on the target object is greater than 90 degrees (i.e., an obtuse angle or a 180-degree angle), and the angle between the opening of the pit and the direction of the gravity force on the target object is less than 90 degrees (i.e., an acute angle or a 0-degree angle). The angle between the opening of the reaction chamber and the direction of the buoyancy force on the target object is not greater than 90 degrees (i.e., a right angle, an acute angle, or a 0-degree angle), and the angle between the opening of the reaction chamber and the direction of the gravity force on the target object is not less than 90 degrees (i.e., a right angle, an obtuse angle, or a 180-degree angle). In other words, the angle between the gravity vector on the target object and the opening of the pit is less than a right angle (the angle is an acute angle or a 0-degree angle), and the angle between the gravity vector and the opening of the reaction chamber is not less than a right angle (the angle is a right angle, an obtuse angle, or a 180-degree angle).

[0034] At the same time, the direction of fluid flow in the transfer channel is made such that the angle between the direction of gravity and the direction of gravity is greater than 90 degrees (the larger the angle, the higher the control efficiency), and the angle between the direction of buoyancy and the direction of gravity is less than 90 degrees (the smaller the angle, the higher the control efficiency).

[0035] That is, the gravity vector of the target object has a positive component in the direction of the pit opening and a non-positive component (negative or 0) in the direction of the reaction chamber opening. In particular, including but not limited to: the angle between the direction of gravity of the target object and the direction of the pit opening is between 10 degrees and 80 degrees, and the angle between the direction of gravity and the direction of the reaction chamber opening is between 100 degrees and 170 degrees.

[0036] c) When the density of the target material injected into the fluid carrier is equal to that of the surrounding fluid, the probability of this situation in application is extremely small. It can be achieved by means of fluid directional drive, target inertia, or other external forces (including but not limited to: electric field force on pre-charged target material, dielectric force on neutral target material, magnetic field force on target material containing magnetic material, sound waves, acoustic tweezers, ultrasound, shock waves, optical tweezers, optical traps, etc.).

[0037] The objectives and effects of fluid carrier attitude and structural design in the target acquisition stage: The target object enters the transfer channel via fluid drive. Under the action of gravity, buoyancy, or fluid drive, a specified number of one or more target objects enter the pit, while preventing accidental entry into the reaction chamber (or temporarily allowing entry into the reaction chamber with a subsequent step to remove the target object from the reaction chamber). Under the action of gravity or buoyancy (gravity is the dominant force when the density of the target object in the injected fluid carrier is greater than that of the surrounding fluid; buoyancy is the dominant force when the density of the target object in the injected fluid carrier is less than that of the surrounding fluid), and the action of the inner wall of the pit, the target object is limited, positioned, or captured. The action of the inner wall includes, but is not limited to: the pit opening size is designed to be smaller than its internal cavity (examples, but not limited to: tight opening, narrow opening), or the pit opening is provided with a seal or inner edge smaller than the diameter of its cavity (the shapes of both include, but are not limited to: annular, rectangular). The design to prevent accidental entry into the reaction chamber in this step is: the direction of the reaction chamber opening is set to be no more than 90 degrees from the direction of movement of the target object. (2) Clearing the transfer channel: Once the number of pits capturing a specified number of target objects within the fluid carrier reaches a predetermined value, any excess target objects not captured by the pits in the transfer channel are emptied. In this step, the spatial attitude of the fluid carrier is set as follows: the angle between the direction of the resultant force of gravity and buoyancy on the target object captured by the concave pit and the direction of the opening of the concave pit remains unchanged or, after being changed, the angle is still not less than 90 degrees (i.e., greater than or equal to a right angle), and the angle between the target object captured by the concave pit and the opening of the corresponding reaction chamber remains unchanged or, after being changed, the angle is still not greater than 90 degrees (i.e., less than or equal to a right angle). This allows for the removal of excess target objects in the transfer channel by means of gravity or buoyancy, or by the driving force of the injected fluid, provided that a specified number of target objects that have entered the concave pit and are confined there will not escape from the concave pit, and that excess target objects in the transfer channel will not mistakenly enter the reaction chamber.

[0038] Based on the different results of comparing the density of the target object with that of the surrounding fluid, commonly used design or control methods include, but are not limited to, several of the following: a) When the density of the target object is greater than that of the surrounding fluid, the spatial attitude of the fluid carrier is maintained, or it is set by rotation to make the angle between the opening direction of the pit and the direction of gravity of the target object decrease but not less than 90 degrees (i.e., the angle is ≥90°, which is a right angle, an obtuse angle, or a 180-degree angle), and the angle between the opening direction of the pit and the direction of buoyancy of the target object increase but not greater than 90 degrees (i.e., the angle is ≤90°, which is a right angle, an acute angle, or a 0-degree angle). At the same time, the angle between the direction of fluid flow in the transfer channel and the direction of gravity is less than 90 degrees (the smaller the angle, the higher the control efficiency), and the angle between the direction of buoyancy and the direction of gravity is greater than 90 degrees (the larger the angle, the higher the control efficiency). With the help of gravity (at this time, buoyancy is less than gravity, and the resultant force of gravity and buoyancy is: gravity is the main force), or the driving force of the fluid injected in the transfer channel, the excess target objects that were not captured by the pit in the transfer channel are cleared.

[0039] b) When the density of the target object is less than that of the surrounding fluid, maintain the spatial attitude of the fluid carrier, or rotate it to: increase the angle between the opening direction of the pit and the direction of gravity of the target object but not greater than 90 degrees (i.e., angle ≤ 90° is 0 degrees, acute angle or right angle), and decrease the angle between the opening direction of the pit and the direction of buoyancy of the target object but not less than 90 degrees (i.e., angle ≥ 90° is 180 degrees, obtuse angle or right angle). At the same time, make the direction of fluid flow in the transfer channel make the angle between the direction of gravity and the direction of gravity greater than 90 degrees (the larger the angle, the higher the control efficiency), and the angle between the direction of buoyancy and the direction of buoyancy less than 90 degrees (the smaller the angle, the higher the control efficiency). With the help of buoyancy (at this time, buoyancy is greater than gravity, and the resultant force of gravity and buoyancy is: buoyancy is the main force), or the driving force of the fluid injected in the transfer channel, complete the clearing of the excess target objects in the transfer channel that were not captured by the pit.

[0040] c) When the density of the target object in the injected fluid carrier is equal to that of the surrounding fluid, control is implemented by means of external force. The external force includes, but is not limited to, the driving force of the injected fluid in the transfer channel, dielectric force, acoustic force, and optical tweezers principle force. With the driving force of the injected fluid in the transfer channel, the excess target object that was not captured by the pit in the transfer channel is cleared.

[0041] Through the above a) to c), it is possible to: remove excess target objects in the transfer channel that exceed a specified number, provided that a specified number of target objects that have entered the pit and are confined therein will not escape from the pit, and excess target objects in the transfer channel will not accidentally enter the reaction chamber, by means of gravity or buoyancy, or by the driving force of injecting fluid into the transfer channel. The excess target objects, also referred to as redundant target objects in this invention, include, but are not limited to: target objects that are free in the transfer channel, and target objects that are temporarily at rest at the opening of the pit but have not been stably captured by the pit. The temporary rest at rest means that the target objects have not fully entered the pit. When the fluid flow rate in the transfer channel changes, their force balance is easily broken and they cannot continue to remain at rest.

[0042] During the emptying process of the transfer channel, it is ensured that excess target objects in the transfer channel will not accidentally enter the reaction chamber during the control process of this step. The mechanism includes, but is not limited to: the angle between the opening direction of the reaction chamber and the movement direction of the target object in the transfer channel is not greater than 90 degrees (i.e., 0 degrees, acute angle, or right angle). If there are excess target objects in the transfer channel that have not been captured by the pit, they will be removed from the transfer channel by the aforementioned gravity or buoyancy, or by the fluid drive in the transfer channel, in order to prevent errors in the number of target objects in subsequent operation steps.

[0043] (3) Target transfer stage: When a predetermined number of pits have captured a specified number of target objects, and excess target objects have been removed from the transfer channel where the pit openings are simultaneously connected to the corresponding reaction chamber openings, or when the above control has errors (including but not limited to: missing or exceeding the number of target objects captured by several pits, or excess target objects still existing in the transfer channel) but these errors do not affect the subsequent control effect, are negligible, or are permissible, then: The spatial attitude of the fluid carrier is set as follows: the direction of the resultant force of gravity and buoyancy on the target object captured by the pit is less than 90° (i.e., the angle is less than a right angle) with the direction of the opening of the pit, and the angle with the direction of the opening of the reaction chamber (i.e., the transfer target space) from the position of the target object is less than 90° (i.e., the angle is less than a right angle). At this time, the direction of the resultant force of gravity and buoyancy on the target object is greater than 90° (i.e., the angle is greater than a right angle) with the direction of the opening of the reaction chamber, which can realize the directional movement of the target object in the pit into the interior of the reaction chamber cavity through the opening of the reaction chamber.

[0044] That is, by rotating or shifting, the spatial attitude of the fluid carrier is set so that the resultant force of gravity and buoyancy on the target object in the pit points to the opening of the corresponding reaction chamber. This allows the target object in the pit to be directionally moved into the interior of the reaction chamber through the opening. Since the reaction chamber does not have the spatial limitation of only allowing a specified number of target objects like the pit, the space of the reaction chamber can be equal to or greater than the capacity of the pit. This enables functions such as storage, mixing, reaction, amplification, culture, reproduction, sorting (for example, but not limited to: a portion of the sample is captured through the pit and transferred to the reaction chamber), plate mounting (for example, but not limited to: whether the pit captures the sample or it is subsequently transferred to the corresponding reaction chamber, if the fluid carrier has the function of a culture plate or well plate, then the plate mounting effect is achieved), or selective screening.

[0045] The above design, according to the sequence of steps (1) to (3), can achieve precise quantitative capture and transfer of a large-scale array of a specified number of target objects in a single round of execution through several steps (single step operation or multiple steps continuous operation), or can achieve the collection of multiple components or different target objects (specified number) in multiple rounds of execution. Both execution methods (single round execution and multiple round execution) can achieve functions such as large-scale array storage, mixing, reaction, amplification, culture, reproduction, sorting, or preferential screening of target objects. The target objects include, but are not limited to: single cells, single bacteria, a specified number of droplets, and a specified number of bubbles.

[0046] The mixing, reaction, and manipulation of multiple targets include, but are not limited to: moving different targets from multiple pits to the same target space (including but not limited to: chambers, reaction chambers) in a single round, moving targets from the same pit to the same target space in multiple rounds, and moving different targets loaded in different pits to the same target space in multiple rounds.

[0047] The collection of the above-mentioned different target materials includes, but is not limited to, examples such as: a single reaction chamber can receive a specified number of several types of cells, several types of reagents of specified volumes, and several types of bubbles for reaction or culture observation.

[0048] (4) Selective recovery or discharge of target material: The mechanism for achieving the recycling or discharge process includes, but is not limited to: using external force, especially directional external force, to directly export some or all of the material from any designated number of chambers to the outside of the fluid carrier, or to export it through the chamber opening to the transfer channel, and then export it to a designated space inside or outside the fluid carrier by means of directional external force, especially including but not limited to: directional driving of the fluid in the transfer channel.

[0049] Combining several of the steps (1) to (3) above, it is possible to selectively export or replace all or part of the target objects in several designated chambers (pits, reaction chambers, or other semi-closed or unclosed spaces in the fluid carrier) (the replacement can be implemented in ways including but not limited to: recapture after export), thus realizing the sorting or screening function of the target objects. During the export of the target objects in the chamber, time control can be performed: the export process is stopped when a specified number of partial target objects are exported (the mechanism includes but is not limited to: removing external force, changing the posture of the fluid carrier so that the resultant force of the target object does not point to the opening of the chamber, but to the bottom or side wall), thus realizing the export of partial target objects.

[0050] The selective recovery or discharge of the target material in this invention can be carried out at any time as needed, including but not limited to: directly after a specified number of target materials have been transferred to the reaction chamber, or after several intermediate process steps (including but not limited to: reaction, incubation, reproduction, amplification).

[0051] This process allows for the export or replacement of target materials in any designated chamber (pit, reaction chamber, or other fluid carrier internal space) while remaining unaffected by target materials in other chambers. This method of limiting target materials to chambers also enables selective export or replacement of target materials by detecting their parameters and properties (including but not limited to impedance, fluorescence, imaging, and Raman parameters), thus achieving functions such as target material recovery, sorting, or screening.

[0052] The external forces (including but not limited to: directional external forces, external force action, external force influence) or driving mechanisms (including but not limited to: directional driving, directional control) mentioned in this invention are generated by mechanisms or principles including but not limited to several of the following: sound waves, acoustic tweezers, ultrasound, shock waves, electric field force, dielectric force, magnetic field force, fluid drive (or fluid impact).

[0053] The shape of the external force facility can be a straight line, a curve, or a combination of straight lines and curves.

[0054] The types of external force facilities include, but are not limited to, several of the following: electrodes, magnetic poles (including but not limited to: electrically controlled electromagnets, movable magnetic devices), sound wave generating devices (including but not limited to the following types of wave generating devices: sound waves, acoustic tweezers, ultrasonic waves, shock waves), and fluid flow control devices that can be injected into fluid carriers (especially including but not limited to: fluid flow control devices that can be injected into fluid carrier cavities).

[0055] In this invention, when the external force facility uses electrodes or magnetic poles, the types of electrodes or magnetic poles are divided into: multi-point common type and single-point single-control type. They can also be divided into: movable type and fixed type according to whether they are movable. The shape of the electrodes or magnetic poles can be a straight line, a curve, or a combination of straight lines and curves, including but not limited to: strip, strip, broken line, V-shape, T-shape, arc, wave shape, and shapes with several tips, sharp corners, rounded corners, or tooth-shaped structures, such as: using electric field force to control a charged target object, or using dielectric force to control a target object point by point, row by row, or column by column.

[0056] Methods for attaching charges to a target object include, but are not limited to: carrying positive or negative charges after contact with a positively or negatively charged metal and then leaving the contact point.

[0057] In directional driving, the electric field force is directed at charged target objects, including droplets, bubbles, and biological particles. Droplets or bubbles may or may not contain biological particles.

[0058] In directional driving, the dielectric force targets droplets or bubbles, including but not limited to: water-in-oil droplets, oil-in-water droplets, and gas-in-oil bubbles. The droplets or bubbles may or may not contain: biological particles (including active biological particles and inactive biological particles; examples of active biological particles include, but are not limited to: cells, bacteria, proteins, viruses, and other organisms or microorganisms, in whole or in part; examples of inactive biological particles include, but are not limited to: magnetic beads, fluorescent particles, and inactive: cells, bacteria, proteins, viruses, and other organisms or microorganisms, in whole or in part).

[0059] In directional driving, the magnetic force is directed at a target object containing magnetic material.

[0060] The fluid impact mechanism includes, but is not limited to: an impact-driven method that injects fluid through a channel, and a method that directly sprays fluid through a needle-like device. The latter includes, but is not limited to: tubular or cylindrical mechanisms with cavities. Specific examples, but not limited to, using a needle, syringe, suction tip, or capillary tube to pierce or access the cavity where the target object is located (location or part including, but not limited to, the bottom of the cavity, near the side wall of the cavity, or other spatial locations within the cavity) and spraying fluid to generate a thrust towards the outside of the cavity on the target object.

[0061] In this invention, the types of sound waves include, but are not limited to: surface acoustic waves (SAW), bulk acoustic waves (BAW), acoustic streaming, acoustic tweezers, and bubble-assisted acoustic fluidics.

[0062] The surface acoustic wave types include, but are not limited to: traveling-wave SAW (TSAW), standing-wave SAW (SSAW), and modulated SAW.

[0063] The types of bulk acoustic waves include, but are not limited to: standing bulk acoustic waves (Standing BAW) and traveling bulk acoustic waves (Traveling BAW).

[0064] More detailed description: The selective recovery or discharge of the target material in this invention includes, but is not limited to: employing a directional external force mechanism to drive part or all of the material in any specified number of chambers from their respective chambers into a transfer channel, and then, with the help of external forces such as fluid impact in the transfer channel, to be discharged into a designated space inside or outside the fluid carrier, thereby achieving recovery or discharge. The implementation method and mechanism of the directional external force driving the target material from its respective chamber into the channel include, but are not limited to, several of the following: a) External force facilities are installed outside the fluid carrier for contact-type control of the fluid carrier. The external force facility can controllably emit or generate external force mechanisms including, but not limited to, several of the following: sound waves, acoustic tweezers, ultrasound, shock waves, electric field force, dielectric force, magnetic field force, positive pressure fluid drive, and negative pressure suction. The external force device provides contact-based control of the fluid carrier: it approaches, penetrates, or connects to a specific location on the fluid carrier, generating the required directional external force to drive a specific target object. The approach methods include, but are not limited to, approaching the specific location or target object through pre-set holes or openings. The access methods include, but are not limited to: contacting or connecting to: a force-applying device pre-embedded or pre-set inside the fluid carrier; the access methods include, but are not limited to: through an interface or exposed electrodes. In the aforementioned piercing control method, the internal space of the fluid carrier may or may not be pierced. The piercing method can be achieved through the aforementioned external force mechanism, including but not limited to: inserting a needle-like device into the cavity of the fluid carrier to drive the fluid jet, or using negative pressure suction (attraction or absorption). Another method of piercing without penetrating (the internal space of the fluid carrier) employs external force mechanisms other than positive pressure driving and negative pressure suction, which can prevent leakage of liquid or gas.

[0065] Especially when the control units or targets inside the fluid carrier are arranged in a row and column array, a two-dimensional spatial positioning method is adopted to locate several specific targets (one or more) and apply external force for directional control.

[0066] b) External force facilities are installed on the outside of the fluid carrier for non-contact control of the fluid carrier: The external force facility can controllably emit or generate external force mechanisms including but not limited to several of the following: sound waves, acoustic tweezers, ultrasound, shock waves, electric field force, dielectric force, magnetic field force, and non-contact control of the fluid carrier: applying directional external force to a target object at a specific location in a specific direction outside the fluid carrier, especially when the control unit or target object inside the fluid carrier is arranged in a row and column array, using a two-dimensional spatial positioning method to achieve positioning of several specific targets (one or more) and apply directional control of external force.

[0067] c) Non-electrode-type external force facilities are installed inside the fluid carrier to implement fixed-point control of the target object: The non-electrode type external force facility is set at a specific position in each control unit. The controllable external force mechanism that can be emitted or generated includes, but is not limited to, several of the following: sound waves, acoustic tweezers, ultrasound, shock waves, magnetic field force. If the external force is activated at any specified point, a directional external force will be applied to the target object at the specific position. d) Electrode-type external force devices are installed inside the fluid carrier to implement point-by-point control of the target object: The electrode-type external force facilities can be set at specific locations in each control unit. The controllable external force mechanism that can be emitted or generated includes, but is not limited to, several of the following: electric field force, dielectric force. When the power is turned on, the external force is generated, or the external force is enabled at any specified point, a directional external force can be applied to the target object at several specific locations. The point-to-point control described in d) above specifically includes: implementing matrix-based fixed-point control on the target object. Array-type (also referred to as row-column type in this invention) control: The laying direction of electrode-type external force facilities includes, but is not limited to: the angle between the electrode and the direction of the transfer channel is acute or right, wherein the effect is best when the angle is right; The fluid carrier is configured such that the target object within the chamber, under the influence of gravity or buoyancy and confined by the chamber walls, achieves force equilibrium. The set of several (one or more) control units (whose pits or reaction chambers are directly connected to the transport channel) that flow through each transport channel (located including but not limited to: between the pit and the corresponding reaction chamber) is defined as a column / row. Then, the set of single-row control units that are perpendicular to it and controlled or affected by the same on / off logic command through several (one or more) electrodes is defined as a row / column. The two are different in spatial dimensions, and the control units (or specifically: pits or reaction chambers) are positioned and controlled from two different dimensions, row and column. For example, the transfer channel between the pit and the corresponding reaction chamber, and the set of pits or reaction chambers affected by it, if defined as a column, then the set of pits or reaction chambers affected by one or more electrodes (controlled by the same command) is defined as a row; For example, the transfer channel between the pit and the corresponding reaction chamber, and the set of pits or reaction chambers affected by it, if defined as a row, then the set of pits or reaction chambers affected by one or more electrodes (controlled by the same command) is defined as a column. For any manipulator chamber within the fluid carrier (including but not limited to: pits, reaction chambers), unique row and column-dimensional positioning can be achieved using several of the following external forces for displacement control: sound waves, acoustic tweezers, ultrasound, shock waves, electric field force, dielectric force, magnetic field force, fluid impact, and negative pressure suction. In this example, electric field force or dielectric force is used: Electrodes are energized (using either electric field or dielectric principles; in the case of electric field force, the electrodes drive and control a charged target object; if the voltage applied to the electrodes has the same polarity as the charge on the target object, a repulsive force is used; if the voltage applied to the electrodes has different polarities from the charge on the target object, an attractive force is used). (Form) This method enables the removal of all or part of the target object from a selected number of rows (or columns) of chambers within a cavity, driving (sucking out or pushing) it from the chamber to an external transfer channel near its opening. Simultaneously, fluid is injected into the transfer channels of the selected columns (or rows). (Note: During this process, the rows with charged electrodes are combined with the columns with injected fluid, or the columns with charged electrodes are combined with the rows with injected fluid. The spatial dimensions of the rows and columns cannot be the same, but the spatial positions can be interchanged.) This allows for the direct removal of some or all of the target objects from the chambers of any selected number of control units distributed in a matrix, or their export to a designated space via the transfer channels driven by the injected fluid.

[0068] In the above four items a) to d), the shape of the external force facility (including but not limited to: electrode) can be a straight line, a curve, or a combination of straight lines and curves. When the external force facility uses electrodes or magnetic poles, it can be divided into: multi-point common type and single-point single-control type. It can also be divided into: movable type and fixed type according to its mobility attribute.

[0069] When using dielectric electric fields, the electrode shapes include, but are not limited to, those that can generate a non-uniform strong electric field: broken lines, straight lines or curves with V-shaped structures, and straight lines or curves with arc structures.

[0070] In this invention, the method of applying charge to the target object (e.g., but not limited to: cells, droplets) includes, but is not limited to: the target object carrying a positive charge after contacting a positively charged metal and then leaving it; the target object carrying a negative charge after contacting a negatively charged metal and then leaving it.

[0071] The selective recovery or discharge of the target object in this invention uses electric field force and dielectric force as examples to illustrate the array (or row and column, matrix) control mechanism. Other external force methods are similar or analogous, and other mechanism principles can also achieve the same effect: they all serve as external forces other than gravity and buoyancy to manipulate droplets, directly removing the target object from the chamber, or first moving it to the external channel of the chamber, and then realizing the recovery, discharge (or export) of the target object through some external force or fluid drive. The fluid carrier's attitude and structural design are as follows: The fluid carrier is equipped with several electrodes (the number can be one or more), and the laying or setting method includes, but is not limited to: setting them at a certain angle (especially including, but not limited to: right angle) to the direction of the transport channel. The angle between the two (the angle between scalar line segments: take the smaller of the angles formed by their intersection) ranges from 0 to 90 degrees, especially including: 30 to 90 degrees, with 90 degrees being the optimal or commonly used angle. The number of electrodes can be one or more, and the number of cavities in the control unit controlled or affected by each electrode can be one or more.

[0072] The fluid carrier is configured such that the resultant force of gravity and buoyancy on the target object within the cavity (pit, reaction chamber, or other space within the fluid carrier) is greater than or equal to 0 in the direction from the target object's location to the bottom of the cavity, and less than or equal to 0 in the direction from the target object's location to the opening or inlet / outlet of the cavity (except in the following cases: the cavity has a tight opening structure, the opening of the cavity is narrowed, or the opening of the cavity has an annular or mesh-like limiting mechanism). That is, the direction of the resultant force of gravity and buoyancy on the target object is from the target object's location to the bottom or side wall of the cavity, but cannot be towards the opening of the reaction chamber. This allows the target object to achieve force balance under the action of gravity or buoyancy and the limitation of the inner wall of the cavity (including but not limited to: pit, reaction chamber), and thus be limited or positioned.

[0073] Taking the recovery or discharge of target substances within the reaction chamber as an example, specific settings include, but are not limited to: When the density of the target object is greater than that of the surrounding fluid, the attitude of the fluid carrier is set such that the angle between the direction of the reaction chamber opening and the direction of gravity is not less than 90 degrees (i.e., a right angle, an obtuse angle, or a 180-degree angle, but not an acute angle or a 0-degree angle). When the density of the target object is less than that of the surrounding fluid, the attitude of the fluid carrier is set so that the angle between the direction of the reaction chamber opening and the direction of gravity is no greater than 90 degrees (i.e., 0 degrees, acute angle, or right angle, but not obtuse angle or 180 degrees). The selective recovery or discharge process of the target material in this invention includes, but is not limited to: mobile recovery control, methods for exporting, removing, or replacing the target material, and may be selected from, but not limited to: The device employs movable directional external force apparatus (including but not limited to: pointed, strip-shaped, or rod-shaped heads), in single or multiple (multiple arrangements can be arrayed or non-arrayed), and in specific types including but not limited to several of the following: electrodes, magnetic poles, and acoustic wave, acoustic tweezers, ultrasonic, or shock wave generators. This movable directional external force apparatus can move in one-dimensional, two-dimensional, or three-dimensional space and can approach a specific location on the target object from outside the fluid carrier, or penetrate a specific location on the fluid carrier to approach the target object. The penetration is designed to prevent leakage of liquid or gas, without penetrating the internal space of the fluid carrier, allowing the directional external force apparatus to approach the target object. The recovery or discharge control methods include but are not limited to several of the following: Several directional external force devices outside the fluid carrier, by moving, approach the target object from the outside of the fluid carrier or penetrating the inside of the fluid carrier (but without penetrating the internal space of the fluid carrier to prevent negative phenomena such as leakage of liquid or gas) and exert controllable external force, the methods including but not limited to several of the following mechanisms: (1) The electrodes are charged to form a non-uniform electric field, which drives the target in the fluid in a directional manner through dielectric force; (2) The electrodes are charged to form an electric field (either uniform or non-uniform), and the electric field force is used to attract or push the charged target in the fluid: (if the electrodes and the target have opposite polarities, they attract each other and produce a pulling effect; if the electrodes and the target have the same polarity, they repel each other and produce a pushing effect), thereby realizing the directional driving of the target in the fluid. (3) The magnetic force generated by the magnetic poles attracts or pushes the target object containing magnetic material in the fluid (the magnetic poles close to the target object and the magnetic poles closer to the magnetic material in the target object have opposite magnetic properties and attract each other to produce a pulling effect, while the magnetic poles closer to the magnetic material in the target object have the same magnetic properties and repel each other to produce a pushing effect). For cases where the magnetic material in the target object is easy to deflect or move: the method of attracting each other with opposite magnetic properties to produce a pulling effect is adopted. (4) By using a sound wave, acoustic tweezers, ultrasonic wave or shock wave generating device, the energy of the wave is used to pull the target object closer or push it away, thereby realizing the directional driving of the target object in the fluid.

[0074] All of the above mechanisms achieve directional movement of the target object along a specified direction in a non-contact manner with the target object or fluid carrier, thereby realizing recovery or other displacement control. Alternatively, a contact manner with the fluid carrier can be selected to achieve the same control purpose for the target object.

[0075] In the above-mentioned methods of contacting the fluid carrier, the directional external force facility pierces but does not penetrate the fluid carrier. The piercing position includes, but is not limited to, several of the following: the side of the channel, the top of the channel, the bottom of the channel, and the island position set in the middle of the channel. The piercing position is not connected to the channel and attracts or pushes the target object in the nearby chamber (including but not limited to: pit, reaction chamber) to leave its original position and enter the channel. The target object is then driven to the designated space by the driving action of injecting fluid into the channel.

[0076] The selective recovery or discharge process of the target material in this invention, within the chambers inside the fluid carrier (including but not limited to: pits, reaction chambers, and other spaces within the fluid carrier), includes methods for the extraction, removal, recovery, or replacement of the target material, as well as methods for: The external directional force device used on the fluid carrier is a needle-like instrument (i.e., a needle-like or tube-like device, object, or tool with a cavity, including but not limited to: a device or instrument with a hollow needle-like, tubular, or cylindrical structure, which may or may not have a tip, for example but not limited to: needles, capillaries, pipettes, suction tips, especially including but not limited to: rigid hollow pointed pipettes or needles), which pierces and penetrates the channel or cavity, and the mechanism includes but is not limited to several of the following: a) Utilizing the suction effect of negative pressure: In needle-like devices, the pressure inside the cavity is lower than the pressure at the insertion point of the fluid carrier, allowing the target object near the insertion point to be drawn into the cavity of the needle-like device, and then removed to any space. During this process, if there is fluid in the external channel connected to the chamber and it can be injected into the chamber, it can fill the gap caused by the target object and fluid being sucked away by the needle-like device: the loss of material and the reduction of pressure in the local space. This can ensure that during the above-mentioned operation, the chamber space where the target object is located and other spaces of the connected fluid carrier have no fluid flow or a small flow, thus minimizing the disturbance and impact on other chambers or space targets. b) Utilizing the driving effect of positive pressure: In the cavity of a needle-like device, the fluid pressure is greater than the pressure in the space near the insertion point of the fluid carrier. The needle-like device ejects fluid at the insertion point of the fluid carrier, increasing the fluid pressure in the cavity and making it greater than the pressure in the external channel connected to the cavity. This allows the fluid to drive the target object out of the cavity and into the external channel, where it is then moved to a designated space (including but not limited to: being exported to any space through the fluid carrier interface). During this process, if the pressure in the external channel connected to the chamber is not greater than the pressure in the controlled chamber, the target can be absorbed from the chamber. This can compensate for the increase in the amount of material and the increase in pressure in the local space caused by the fluid ejected from the syringe-type device. This can ensure that the disturbance and impact on the target in other chambers or fluid spaces are minimized during the above control process. c) Utilizing the simultaneous "blowing" and "suction" action of positive and negative pressure: Needle-like devices that pierce a fluid carrier and penetrate its channel or cavity, including two types: Type 1: The fluid pressure inside the cavity is greater than the pressure inside the fluid carrier, causing fluid to be ejected at the puncture point, using positive pressure to drive the target away from its original position. Type 2: The fluid pressure inside the cavity is less than the pressure inside the fluid carrier, using negative pressure to absorb the target at the puncture point. Both types of needle-like devices are inserted near the target (especially including but not limited to: puncture...). (Since the positions are both located on both sides of the target object in three-dimensional space), the target object can be efficiently moved and enter the second type of needle-like device with lower pressure under the "blowing" and "suction" action of the two types of needle-like devices. By controlling the two types of needle-like devices with different flow directions, and at the same time, the fluid volumes of "blowing" and "suction" are the same or similar, it can be ensured that during the above operation, the chamber space where the controlled target object is located and other spaces of the connected fluid carrier have no fluid flow or a small flow volume, so as to minimize the disturbance and influence on the target object in other chambers or spaces.

[0077] The control method for the large-scale single-cell array multi-component precise quantitative reaction system of the present invention (flux, picoliter, nanoliter, or microliter) is characterized by: a method for returning escaped target material from the chamber that has not been exported but has been driven or escaped (including in any stage of the present invention, the target material escapes, is removed, or is discharged from the chamber such as the pit or reaction chamber) to the transport channel, including but not limited to: The method disables external force facilities (i.e., eliminates the external force exerted on the target object by the external force facilities, which is the opposite of the word "enable," for example, but not limited to: previously charged electrodes being de-energized or grounded, magnetic external force facilities being demagnetized or moved away from the control point), and maintains or resets the fluid carrier attitude so that the target object previously driven (sucked out or pushed) into the transfer channel in the control unit chamber has a component of gravity and buoyancy greater than 0 in the direction from its current position to the chamber it was previously removed from (specifically, but not limited to: the opening, interior, or bottom of the chamber). The preferred method is that the resultant force of gravity and buoyancy points towards the opening or interior of the reaction chamber (including but not limited to: the bottom of the chamber) from which the target object was previously located (the source location before removal), so that the target object that was not removed from the transfer channel returns to the chamber from which it originated (the previously removed location, the source location) (for example, but not limited to: a pit, a reaction chamber). In this process, no external force other than gravity and buoyancy is used, and the impact on the activity of the target object is minimal. This is a flexible control method of the present invention.

[0078] In the selective recovery or discharge process of the target objects of this invention, the external force mechanisms of electric field force and dielectric force are facilitated by the pre-laying of electrodes on and off row by row or column by column, thus enabling the use of a matrix-type two-dimensional fixed-point control mechanism. As for the action mechanisms of sound waves, acoustic tweezers, ultrasound, shock waves, magnetic field force, fluid impact, etc., the external force facilities are more likely to achieve single-point or multi-point directional control. They can be used to directionally drive only a few selected target objects, causing them to leave the chamber and enter the external channel of the chamber. Then, with the help of external forces such as fluid impact, the target objects are recovered, sorted, screened, or moved to any designated space.

[0079] In this invention, the electrode type is: a type with multiple control points shared (abbreviated as: multi-point simultaneous control type or multi-point common type) or a type with a single control point single control (abbreviated as: single-point single control). According to whether it is movable, it is divided into: movable type and fixed type. The electrode shape is: straight line, curve, or a combination or combination of the two (straight line and curve). Examples include, but are not limited to: broken line, line with a tip or protrusion, especially including but not limited to: in the case of pre-laid, the electrode shape is designed with a V-shaped tip or an arc protrusion.

[0080] The present invention employs an electric field force to carry a charge on a target object such as a droplet, including but not limited to: setting an exposed electrode connected to an electrical signal in a reaction chamber or other chamber, the target object coming into contact with the charge, and the positive or negative charge being selectable by the positive or negative voltage of the electrical signal connected to the electrode.

[0081] In each stage of this invention, the driving force of the fluid injected into the transfer channel or the inertia of the target object can be used simultaneously to improve control efficiency. The angle between the direction of the fluid flow in the transfer channel and the direction of the resultant force of gravity and buoyancy on the target object (when centrifugal force or other external force is used for control, the resultant force may or may not be replaced by centrifugal force or other external force) is less than 90°, that is, the angle can be 0 degrees, acute angle or right angle. In particular, in the target object capture stage, the angle between the direction of the fluid driving the target object in the transfer channel and the direction of the pit opening is not less than 90 degrees (right angle, obtuse angle or 180 degrees angle), and the angle between the opening direction of the reaction chamber corresponding to the pit and the direction of the target object's movement in the transfer channel is not greater than 90 degrees (right angle, acute angle or 0 degrees angle). More importantly, the large-scale single-cell array multi-component precise quantitative reaction system of the present invention, which can be implemented at capacities of 1000 liters, 1000 liters, 1000 liters, or 1000 liters, can achieve effects including but not limited to: directional driving of target analytes (for example, but not limited to: reagents or contrast agents of different volumes) in multiple columns / rows of pits to the same reaction chamber, the specific design, mechanism or method of which is described below: The angle between the fluid flow direction in the transfer channel within the fluid carrier and the direction of the resultant force of gravity and buoyancy on the controlled target is 0 degrees, an acute angle, or a right angle. The 0-degree angle means that the fluid flow direction and the direction of the resultant force are in the same direction.

[0082] Within the same control unit, the openings of several (i.e. one or more) columns / rows of recesses all face or are near the openings of reaction chambers (corresponding to, the same or different ones within the same control unit), including but not limited to: if the direction of the transfer channel is defined as a column, then the same column of control units, the same column of recesses or the same column of reaction chambers share the same transfer channel.

[0083] Within the same control unit, to ensure that any target object captured in a pit can be moved into the corresponding reaction chamber under external force, other pits along the path from the pit opening to the corresponding reaction chamber or its opening, or near that path, that might obstruct or affect the target object's directional movement into the corresponding reaction chamber, are all provided with bottom holes through which the target object can pass. (That is, to ensure that if there is an intermediate pit between any target object captured in a pit and the corresponding reaction chamber opening, then each intermediate pit has a bottom hole, and the target object can move through the bottom hole and opening of the intermediate pit sequentially under external force to the reaction chamber.) The corresponding reaction chamber opening and enters the reaction chamber. To ensure the smooth progress of this movement process: the size of the target object is not larger than the size of the bottom hole and opening of the intermediate recess through which it passes, or although the size of the target object is larger than the size of the bottom hole of the intermediate recess through which it passes, it can pass through the bottom hole and opening of the intermediate recess through short-term deformation or rotation and under the action of external force; within the same control unit, on the movement path from the recess opening to the corresponding reaction chamber of the same control unit, or near the path, if there is a solid structure of the fluid carrier (including but not limited to: channel sidewall) forming an obstruction, then the solid structure has a through hole through which the target object can pass.

[0084] Specifically, to ensure that the target object captured in any pit can be smoothly moved into the target reaction chamber, if there is a channel wall or solid obstacle between its position and the opening of the reaction chamber corresponding to the pit, the channel wall or solid obstacle shall have a through hole. The target object can pass through the through hole under the action of external force, move to the opening of the target reaction chamber corresponding to the pit from which it originated, and enter the reaction chamber. To ensure that the movement process proceeds smoothly: the size of the target object is not larger than the size of the through hole through the intermediate transfer channel wall, or although the size of the target object is larger than the size of the through hole through the intermediate transfer channel wall, it can pass through the through hole of the intermediate transfer channel wall by means of short-term deformation or rotation under the action of external force.

[0085] Within the same control unit, different pits whose moving targets are the same reaction chamber and whose moving paths are collinear have cavity volumes and minimum bottom hole sizes that are inversely proportional to the path length from the corresponding reaction chamber opening, and can be arranged linearly or nonlinearly. Furthermore, the target objects captured by the pit with the bottom hole are all larger than the size of the bottom hole, which not only limits the captured target objects but also prevents them from escaping from the bottom hole, thus achieving reliable capture, limiting or positioning of the target objects. Using the reaction chamber as a location reference, along the movement path from the opening of the distant pit to the opening of the corresponding reaction chamber, or near that path, if there are other nearby pits that act as obstructions, these pits are provided with bottom holes. If there is a solid structure that acts as an obstruction for the fluid carrier, this solid structure has through holes. In this case, the largest target object that the distant pit can capture can enter the corresponding reaction chamber under external force through the bottom hole of the nearby pit or the through hole of the solid structure. Measures include, but are not limited to, ensuring that the diameter of the largest target object that the pit can capture is not less than its distance from the opening of the distant pit to the opening of the corresponding reaction chamber. The size of the bottom hole or through hole of the solid structure on or near the movement path, or by relying on the change in the properties of the target object, including but not limited to deformation, compression or slimming, and driven by external force (e.g., but not limited to: centrifugal force, electric field force, magnetic field force, dielectric force, sound wave, acoustic tweezers, ultrasound, shock wave, fluid drive, etc.), enables the target object captured in the cavity of the pit at a distance to pass through the bottom hole of the pit (moving from the bottom hole of the pit through the pit opening) or through hole on or near the movement path, and enter the corresponding reaction chamber through the corresponding reaction chamber opening.

[0086] In this invention, the bottom hole of the recess, in addition to ensuring that the target object matched with the recess will not leak out from the bottom hole, is designed to allow distant target objects that are farther from the reaction chamber and whose travel path coincides with the position of the bottom hole of the recess to pass more smoothly. The bottom hole of the recess is designed to be wide-mouthed, that is, the cross-sectional area of ​​the bottom hole cavity on the path of the distant target object moving towards the target reaction chamber is proportional to the distance from the reaction chamber. In other words, when the distant target object moves towards the target reaction chamber and passes through the bottom hole of the recess on the path of travel, the bottom hole has a gradually widening structure that narrows along the travel path, and the narrowest area of ​​the bottom hole can also allow the distant target object to pass through. This makes it easier for the distant target object to enter the bottom hole. Even if the travel path of the distant target object deviates, it can be eliminated or corrected, thereby improving the reliability of the system.

[0087] The collection of the above-mentioned different targets, for example but not limited to: the same reaction chamber can receive a specified number of several types of cells, different types of reagents of specified volumes, and several types of bubbles for mixing, reaction, or culture observation. In particular, including but not limited to: the pit volumes in different control units are designed in a gradient manner, which can realize precise gradient experiments. Measures include but are not limited to: aligning the same row of reaction chambers with different rows of pits arranged in a gradient manner. Here, the design is that the cavity capacity of the pits in different rows is different, which facilitates the capture of droplets or bubbles of the same volume in the same row. The sample can be injected in one go, which improves efficiency. However, for connected or same transfer channels, different pits on the same side can have the same or different designed capacities (or volumes). The different internal volumes of the pit cavities include, but are not limited to, gradient, descending, or ascending arrangements. The corresponding control mechanism is as follows: Droplets or bubbles are injected into the pits at different times, in different batches, or in different rounds, with the injection order being: larger objects first, then smaller. This is because larger objects cannot enter smaller cavities, and once a large object enters a pit that can only accommodate it, that pit cannot accept other objects (including smaller objects). The method of injecting smaller objects first, then larger ones, in batches is prohibited because this method may cause smaller objects to enter larger pits that are not their target locations. This not only enters the wrong location but also hinders or prevents subsequent larger objects from entering the corresponding larger pits, causing control errors or mistakes.

[0088] To achieve precise positioning and displacement at the nanometer and micrometer levels, accurately locate the chamber or target position of the control unit, or a specific position within the fluid carrier, position marking is performed. The methods include, but are not limited to, several of the following: light marking, color marking, specific shape mechanism marking, and character marking (including but not limited to: serial number, letter, and symbol). The types of light marking include, but are not limited to: fluorescence, self-luminescence, refracted light, transmitted light, and reflected light.

[0089] Specific locations on the fluid carrier, such as but not limited to: control units, channels, pits, or reaction chambers, are marked by fluorescence. The fluid carrier is provided with several channels specifically for location marking, and only at each identification point that needs to be identified, a difference is set in shape, size, or feature, including but not limited to: protrusions, bends, forks, branches, slots, blind holes, and through holes. The above-described method for position marking using fluorescence involves: a separate, interconnected channel is provided for the position to be marked on the fluid carrier, or multiple positions to be marked are connected in series or parallel via a common channel. Fluorescent material is injected through the channel or opening. In the case of multiple positions injected through a common channel, the common channel between each position can be blocked before use to prevent excitation light from illuminating it (preventing fluorescence) or to block the space between it and the observation instrument (so that although fluorescence is produced, it cannot be observed). This forms a discrete number of fluorescently marked positions. When illuminated by excitation light, the observation instrument can determine the specific position of the fluid carrier based on the fluorescence position, including but not limited to several of the following: control unit, channel, pit, or reaction chamber. In each step of this invention, the detection methods for the target object include, but are not limited to, several of the following: fluorescence or self-luminescence detection, impedance or admittance detection, Raman signal detection, voltage or current signal detection, image information detection (including but not limited to: computer vision detection), size detection (including but not limited to: particle size detection), color detection, etc.

[0090] In this invention, in the aforementioned cases where gravity or buoyancy is used as the primary force for control, centrifugal force can be used instead of gravity or buoyancy by rotating or moving the fluid carrier. The corresponding control methods are the same or similar. The so-called primary force refers to one or more external forces acting on the target object with the largest absolute value. When the target object in the fluid is only subjected to gravity and buoyancy, the primary force is the one with the larger absolute value between gravity and buoyancy. Specific mechanisms and methods for using centrifugal force include, but are not limited to, several of the following steps: (1) In the capture stage, centrifugal force is used as the main force, so that the angle between the direction of centrifugal force and the direction of the pit opening is greater than 90 degrees (obtuse angle or 180° angle). The target object enters the pit that can only accommodate a specified number of target objects under the drive of centrifugal force, thus achieving the capture of the specified number of target objects; when the target object is taken out of the specified pit, external force is used. (2) The transfer channel is cleared so that the angle between the direction of the centrifugal force and the direction of the pit opening is not less than 90 degrees (right angle, obtuse angle or 180° angle), the target object in the pit is limited, and the excess target object in the transfer channel is removed. (3) In the process of transferring, recovering or discharging the target object, external force (including but not limited to several of the following: sound wave, acoustic tweezers, ultrasonic wave, shock wave, electric field force, dielectric force, magnetic field force) or centrifugal force is used to remove the target object from the designated pit or reaction chamber for recovery, discharge or transfer to other spaces.

[0091] In cases where centrifugal force is used: the angle between the opening direction of the pit or reaction chamber and the direction of centrifugal force is less than 90 degrees (acute angle or 0° angle). Specific methods include, but are not limited to: rotating the opening direction of the pit or reaction chamber relative to the direction of centrifugal force by 90 to 270 degrees, so that the angle between the opening direction of the pit or reaction chamber and the direction of centrifugal force is 0 degrees or acute angle, without needing to seal the opening of the pit or reaction chamber where the target object is located or setting up obstacles to restrict the escape of the target object; When it is necessary to transfer the target object to the reaction chamber or other space, simply position the target object so that the angle between the direction of the opening (or entrance / exit) of the reaction chamber or other space to which it is to be transferred and the direction of the external force or centrifugal force is greater than 90 degrees (obtuse angle or 180-degree angle).

[0092] When the density of the target object is less than that of the surrounding fluid, the opening direction of the cavity such as the pit or reaction chamber is reversed in the above-mentioned specific mechanism and method of centrifugal force, so that the similar control effect of each link (1) to (3) can be achieved in a similar way.

[0093] In this invention, the use of gravity, buoyancy, or a weak external force as a driving method can significantly improve the activity of the target object, all of which fall within the scope of the flexible control method of this invention.

[0094] The control mechanism for positioning or limiting the pit or reaction chamber of the present invention is very suitable for Raman sorting applications based on Raman analyzers or Raman sensors.

[0095] Regarding the aforementioned "technical problem," the present invention offers numerous solutions, including, but not limited to, typical examples: A large-scale array of control units is incorporated within a fluid carrier (e.g., microfluidic chips, biochips, gates). The openings of the recesses within each control unit and their corresponding reaction chambers are at a 180° angle and are positioned opposite each other via transport channels. Through gravity, buoyancy, or fluid actuation, a specified number of biological particles (such as cells, bacteria, or viruses), reagent droplets, or bubbles are captured through the recesses. The chip is then rotated, allowing the captured targets in one or more rows of recesses to enter their corresponding reaction chambers without obstruction. Each reaction chamber can receive a specified number of cells, various reagents, or bubbles for storage, mixing, culture, or reaction. Furthermore, the array of targets can be positioned along both rows and columns using dielectric forces, electric fields, acoustic waves, acoustic tweezers, fluid actuation, or needle-like instruments, enabling efficient discharge, recovery, or replacement. Cell viability is protected throughout the entire process.

[0096] Beneficial effects: The present invention has the following significant beneficial effects: (1) It can achieve single-cell sorting while ensuring cell viability, and accurately control the liquid volume through several (one or more) reagents with liter or picoliter precision, and combine experiments with single cells to achieve a precision improvement and volume reduction of 2 to 5 orders of magnitude compared with traditional microliter and nanoliter systems.

[0097] (2) Significantly reduces reagent consumption and experimental costs. The pixel-level reaction volume reduces the amount of precious biological reagents used by N orders of magnitude (N≥2) compared to the traditional well plate system, and the consumption of screening reagents can be reduced to one part per million.

[0098] (3) Achieve ultra-high throughput large-scale arrayed parallel reaction, and a single batch can accommodate tens of thousands or even millions of independent reaction units for parallel experiments, with throughput increased by 3 to 5 orders of magnitude compared to the traditional well plate system.

[0099] (4) The flexible sorting method, which is characterized by low hydraulic pressure, low voltage, no high temperature, and no aerosol, significantly protects the viability of fragile cells. At the same time, the entire chain of sorting, sample addition, mixing, culture, and detection is integrated into a single chip, avoiding damage and contamination caused by sample transfer.

[0100] (5) Traditional methods use micro-enlargement reaction systems through well plates or test tubes, which is similar to studying a single goldfish in a swimming pool. The micro-enlargement reaction system of this invention is similar to studying a single goldfish in a fish tank, which effectively enriches the target analyte. Compared with traditional methods, the detection sensitivity is significantly improved, and the error is low, the efficiency is high, and it is more economical. Attached Figure Description

[0101] Figure 1 This is a schematic diagram of the structure of the two different control units of the present invention, which are respectively configured into a 2×2 matrix.

[0102] Figure 2 This is a schematic diagram of the multi-row recessed control unit structure within the fluid carrier of the present invention.

[0103] Figure 3 This is a schematic diagram illustrating the principle and structure of the array-type single-cell sheet capture, transfer, culture, and recovery system of the present invention, which combines a transverse electrode with two-dimensional control of longitudinal fluid flow.

[0104] Figure 4 This is a schematic diagram illustrating the principle and process of a control unit with a limiting area in the reaction chamber for capturing, transferring, injecting or adding drugs, culturing, recovering and resetting 3×2 array single-cell slices, as described in Embodiment 1 of the present invention.

[0105] Figure 5 This is a schematic diagram illustrating the principle and process of a single-space reaction chamber control unit for capturing, transferring, culturing, recovering, and resetting 3×4 array-type single-cell slices, as shown in Embodiment 2 of the present invention.

[0106] Figure 6 This is a schematic diagram illustrating the principle and process of a control unit with multiple rows of pits corresponding to a single row of reaction chambers to achieve multiple capture, co-directional transfer, and mixed culture on a 3×1 array of single-cell sheets, as described in Embodiment 3 of the present invention.

[0107] In the above figures: i, i+1, i+2, i+3, etc., indicate the row number of the control unit array; j, j+1, j+2, j+3, etc., indicate the column number of the control unit array.

[0108] Figure 1 This is a schematic diagram showing the structure of two different single-row recessed control units of the present invention, each forming a 2×2 matrix. It includes the two basic control unit types shown in sub-figures (a) and (b).

[0109] Figure 1Neutron diagram (a) shows a 2×2 array structure composed of control units with a single spatial reaction chamber. The dashed box Qij indicates a single control unit. The structure includes: a pit indicated by label AKij, a reaction chamber indicated by label FYSij corresponding to the pit, and a transfer channel indicated by label ZTDij that is connected and shared by the two. The opening of the pit AKij and the corresponding reaction chamber FYSij are opposite to the transfer channel ZTDij. The solid black arrow indicated by label FLj indicates the flow direction of cell suspension or other liquid reagents in the transfer channel ZTDij. This direction is perpendicular to the opening direction of the pit and makes an angle of 45° with the opening direction of the reaction chamber FYSij. The two controllable fluids indicated by labels FLj and FLj+1 converge through the two transfer channels.

[0110] Figure 1 The neutron diagram (b) shows a 2×2 array structure composed of control units with limiting areas in the reaction chamber. Its structure, part numbers and design features are similar to those in sub-diagram (a). The difference is that in the scheme of sub-diagram (b), a limiting area marked XWij is added to the reaction chamber of the control unit. This limiting area shares the arc-shaped bottom of the reaction chamber and has the characteristics of being easy to enter but difficult to enter. The characteristic of the easy entry and difficult exit is as follows: In sub-figure (b), when the direction of the resultant force of gravity and buoyancy on the target object such as cells or droplets originating from the pit indicated by label AKij is horizontally pointing to the left side of the figure, the target object automatically moves to the bottom of the reaction chamber through the reaction chamber inlet indicated by label FYSij, along the downward sloping side wall of the reaction chamber. Especially with the help of the arc-shaped design at the bottom of the reaction chamber, under the action of the resultant force of gravity and buoyancy horizontally to the left, it converges at the limiting area indicated by label XWij at the center of the bottom of the reaction chamber. Under the premise that the posture of the entire fluid carrier (such as microfluidic chip or gate device) remains unchanged and the gravity and buoyancy remain unchanged, the target object such as cells or droplets is difficult to leave the limiting area, thus achieving locking or limiting.

[0111] Figure 2 This is a schematic diagram of a multi-row recessed control unit structure in a fluid carrier for use in multi-component collection experiments according to the present invention. It includes two control units indicated by labels Qi and Qi+1, which represent a high-order application design of the present invention. The two control units share three transfer channels indicated by labels ZTDa, ZTDb, and ZTDc.

[0112] Figure 2The control unit indicated by the symbol Qi includes a reaction chamber indicated by FYSi. Corresponding to this reaction chamber are three recesses indicated by the symbols AKia, AKib, and AKic, respectively. The maximum volume of the target object that the three recesses can capture is inversely proportional to the distance from the recess to the entrance of the corresponding reaction chamber: that is, the closer the recess is to the corresponding reaction chamber, the larger its capacity. Furthermore, the center position of the opening of the three recesses is spatially linear or nearly linear with a point (including but not limited to: the center point) of the opening of the corresponding reaction chamber. Among the recesses with a linear spatial relationship between their opening positions, the middle recess located between the corresponding reaction chamber and the rightmost recess has a hole at its bottom. The hole is sized to allow the largest target object captured by the right-hand recess to pass through and enter the left-hand reaction chamber. In this way, the control unit can capture three different target objects at once through three rows of recesses, and then directly or through the bottom hole of the middle recess, enter the reaction chamber corresponding to the left-hand recess under the action of external force. This enables the collection, mixing, reaction, culture, or other uses of droplets containing cells, reagent A, and reagent B in aqueous phases at the petroliter, nanoliter, or microliter levels. The external force implementation or design mechanism includes, but is not limited to, setting the attitude of the fluid carrier so that the resultant force of gravity and buoyancy on the target object points towards the entrance of the left-hand reaction chamber.

[0113] Figure 2The control unit indicated by label Qi+1 includes a reaction chamber indicated by FYS(i+1), and the corresponding pits include two groups of six pits, AK(i+1)a1, AK(i+1)b1, AK(i+1)c1 and AK(i+1)a3, AK(i+1)b2, AK(i+1)c2, respectively. Their operating mechanism and principle are the same as the control unit indicated by label Qi. Additionally, connected to the transfer channel indicated by label ZTDa, there is a small-capacity pit indicated by label AK(i+1)a2. Its volume differs from the other pits connected to the same transfer channel, meaning that injecting a target object of the same size all at once will cause an error: the smaller target object will occupy the cavity of the large pit in the non-target space, causing an error itself and preventing the larger target object from entering the target space of the large pit cavity. The corresponding mechanism and method is: in the transfer channel indicated by label ZTDa, injecting objects in a large-to-small order... For the same batch of target materials, first, the three large pits AKia, AK(i+1)a1, and AK(i+1)a3 capture a batch of target materials of the same size. Because large-sized target materials cannot enter the small pits whose cavity capacity is smaller than their size, after the transfer channel is emptied, small-sized target materials matching the cavity capacity of the small-capacity pit indicated by AK(i+1)a2 are placed in the same way. Since the large pits connected by the same transfer channel have been filled with a specified number of large-sized target materials in the previous round of operation, there will be no spare space to put the small-sized target materials injected into the transfer channel in subsequent rounds. In this way, target materials of different capacities can be captured separately through the same connected transfer channel in different rounds according to the principle of large to small, and then they can be merged into the same reaction chamber. This can achieve the mixing of target materials of different doses and sizes (such as reagent droplets or bubbles), especially including but not limited to: gradient experiments, such as Figure 2 In the middle, if AK(i+1)a3 is changed to a through hole with a cavity smaller than AK(i+1)a1, and AK(i+1)a2 is deleted (i.e., the pit is blocked), then Figure 2 The reaction chamber with two control units can obtain single captured cells from AKia and AK(i+1)a1 respectively. At the same time, it can also obtain droplet reagents with gradient characteristics from AKib, AK(i+1)b1, AK(i+1)b2, etc. (FYSi obtains 1 droplet, FYS(i+1) obtains 2 droplets, and by expanding the control units in this way, it is easy to achieve: FYS(i+2) obtains 3 droplets, FYS(i+3) obtains 4 droplets, or: FYS(i+2) obtains 4 droplets, FYS(i+3) obtains 8 droplets. The number of control units and the number of rows of pits can be increased and expanded to realize single cell + gradient drug mixing experiments), realizing the diversity and flexibility of experimental methods.

[0114] Figure 2 The reference numerals DK(i+1)a1, DK(i+1)a3, DK(i+1)b1, and DK(i+1)b2 are the bottom holes of the pit. The bottom holes of the pit can be designed to gradually narrow and tighten along the direction of the target object's movement, or they can be designed with equal width, gradually widening, gradually widening and then gradually narrowing, or gradually narrowing and then gradually widening along the direction of the target object's movement. All of the above different designs of the bottom holes of the pit must ensure that the target object passes through smoothly and that the target object captured by the pit does not leak out from its bottom hole.

[0115] Figure 3 The symbols Qij, Q(i+1), and (j+1) indicate two control units, whose design and control mechanisms are the same. Figure 1 Neutron diagram (a) scheme, Figure 3 The design incorporates four electrodes (which can be replaced with other external force devices based on different principles, including but not limited to magnetic poles and ultrasonic generators) labeled HVi, HVi+1, HVi+2, and HVi+3. Each electrode has a force application point protruding towards the target chamber at the location where the control unit needs to apply force. This not only enhances the non-uniformity of the electric field or strengthens the effects of external forces such as electric field and dielectric force, but also prevents excessively strong electric field and dielectric force from being generated at other locations on the electrode besides the force application point, thus preventing misoperation. For example, labels Qij and Q(i+3),j indicate two different shaped force application points. Each control unit has one or more of these force application points. Of course, in practical applications, the above-mentioned force application points can be retained or removed. However, when using a dielectric force control scheme, the above-mentioned force application points help to realize or enhance the required non-uniform electric field, achieving a better control effect.

[0116] The force application points of the four electrodes are all closer to the reaction chamber in the control unit than other parts of the electrodes. To explain the diversity of the electrodes, the positions and shapes of the four electrodes are different. In practical applications, one or more of them can be used. The shape types of the electrode force application points are as follows: the electrode indicated by HVi is a protruding needle-shaped T-shape, and the electrode protrusion or protrusion part is indicated by Vij; the electrode indicated by HVi+1 is a protruding solid triangle; the electrode indicated by HVi+2 is a protruding V-shape; the electrode indicated by HVi+3 is a protruding arc shape, and the electrode protrusion or protrusion parts are indicated by V(i+3),(j+1). The above electrode shapes can be selected according to their advantages in processing technologies such as potting, pre-embedding, electroplating, and screen printing.

[0117] The location of the electrode force application point can be as follows: Figure 3 As shown in the diagram, columns j and j+3 are positioned at the bottom or top layer of the transfer channel near the target control path node (including but not limited to: fluid channels). Figure 3The two-dimensional graphic cross-section shown is parallel to the bottom or top layer. The electrode may or may not penetrate the internal space of the transfer channel. The penetration method (including the case of exposure) requires measures such as preventing electrode electrolysis, leakage, and liquid leakage. Non-penetration is a more reliable solution. Alternatively, it can be as follows: Figure 3 As shown in columns j+1 and j+2, they are positioned on the side of the transfer channel closer to the target control path node, which is more convenient for applying force (including but not limited to: the location of non-chamber, non-channel solid parts in the fluid carrier). Control mechanisms employing electric field force or dielectric force, including but not limited to: for row i+1 (i.e. Figure 3 When the control unit (second row from the top) is in control, the upper HVi+1 row is powered on (connecting the electrical signal shown by the label HV, the voltage of which is DC or AC, including but not limited to: sine wave, triangle wave, square wave, such as: pulse electrical signal with a value greater than 1V and a frequency higher than 100Hz), and the lower HVi+2 row is grounded (shown by the label GND), working together with the HVi+1 row to achieve the desired better electric field effect.

[0118] Figure 3 The electrodes are arranged in rows, which can affect all control units in the row. This makes it convenient to combine the fluid impact or other longitudinal (i.e., column direction) external forces in the transfer channel to implement array-type layout and row-column fixed-point control. In practical applications, electrodes can also be set point by point for each control unit. The electrode is only used for target object control within the control unit. This allows for the direct selection of any one or more of the control units in the array-type layout to implement precise drive or control of the target object at a specific point or in a chamber. Detailed implementation method: The preferred embodiment of the present invention:

[0119] Example 1: As in the present invention Figure 4 As shown, the control unit with a limiting area in the reaction chamber realizes the principle and process of capturing, transferring, injecting or adding drugs, culturing, recovering and resetting on a 3×2 array of single-cell sheets. In this embodiment, the fluid carrier adopts a microfluidic chip (hereinafter referred to as chip). The figure includes 9 sub-figures (a) to (i) according to the sequential operation. At the top of each sub-figure: the left arrow of label G1 indicates the direction of gravity followed when the chip attitude is set for the control situation where the density of the target object is greater than that of the driving fluid. At this time, the resultant force of gravity and buoyancy on the target object in the driving fluid is expressed as the direction of gravity. The right arrow of label G2 indicates the direction of gravity followed when the chip attitude is set for the control situation where the density of the target object is less than that of the driving fluid. At this time, the resultant force of gravity and buoyancy on the target object in the driving fluid is expressed as the direction of buoyancy.

[0120] The following describes a scenario where single-cell droplets (shown as black spheres in the image) and reagent droplets (shown as white spheres in the image) are used as target objects, both with densities less than the oil-like fluid (referred to as oil) driving their movement. Control is implemented based on buoyancy, using the direction of gravity indicated by the arrow to the right of label G2 to set the chip's position and attitude. The specific steps, methods, and principles of this embodiment are explained. If the target object being manipulated in the application has a density greater than the fluid driving its movement, then... Figure 4 The arrow to the left of G1 indicates the direction of gravity, setting the chip's position and orientation; the control principle is similar.

[0121] This embodiment Figure 4 In each sub-diagram, the controllable oil, via the black arrows near labels FLj and FLj+1, flows along the two transport channels j and j+1 respectively, passing through three control units: a reaction chamber with a limiting area on the left and a recess on the right. It then converges into the inflow channel above the sub-diagram and, driven by the oil injected on the left, flows through the outlet of the right channel to a designated space inside or outside the chip. The gray diamond-shaped icons between the recesses and the corresponding reaction chambers in each transport channel indicate the insertion points where electrodes or other force-applying devices can be inserted. These points are insertion points or pre-fabricated blind holes that are not connected to the channels or chambers within the chip. Electrodes are inserted along these points but do not penetrate the channels or chambers within the chip, approaching and controlling the target object near that point.

[0122] The electrode insertion method includes, but is not limited to: the insertion point on the side of the chip where the electrode is inserted is a soft material into which a hard electrode can be inserted (for example, but not limited to: PDMS, soft rubber); a better solution is a pre-fabricated blind hole, which is not connected to the channels or chambers inside the chip, so that the electrode can be easily inserted and approached after positioning.

[0123] Figure 4 As shown in neutron diagram (a), the preparation step involves evacuating air from each chamber inside the chip to the outside through channels or interfaces to remove all or part of the gas inside the chip, preventing negative effects such as the generation of bubbles later. This step can be ignored if the chip has already been immersed in liquid or if this step is not required.

[0124] Figure 4As shown in neutron diagram (b), in the capture stage: the chip orientation is set according to the gravity direction indicated by the arrow on the right side of G2. The black spheres in the transport channel represent active single-cell droplets. Under the action of buoyancy and oil drive, they move from bottom to top in the direction indicated by the arrows labeled FLj and FLj+1. The angle between this direction of movement and the direction of the reaction chamber opening on the left side of the transport channel is an acute angle (including but not limited to: 45°). The angle between the buoyancy direction and the direction of the reaction chamber opening is an acute angle or 0°. Therefore, it can prevent single-cell droplets from accidentally entering the reaction chamber in this step. At the same time, this transport... The direction of movement is perpendicular to the direction of the opening of the pit on the right side of the transport channel through which the droplet flows, and the direction of the buoyancy force on the single-cell droplet is at an obtuse angle (including but not limited to: 135°) to the direction of the pit opening. When the single-cell droplet flows through the pit opening under the action of buoyancy, it enters the pit. The pit can only allow a specified number of target objects (Note: the specified number is 1 in this embodiment, but it can be multiple in other applications, and the principle is similar). Other single-cell droplets flowing through the pit cannot enter it again, thus achieving pit capture of a specified number of single-cell droplets of 1.

[0125] Figure 4 As shown in neutron diagram (c), the clearing process is as follows: In the previous step, after a predetermined number (partial or complete) of single-cell droplets have been captured in the pits, the chip orientation remains unchanged, or the gravity direction indicated by the right arrow of G2 is set for better results. Oil without droplets is injected from bottom to top into the two transport channels along the arrow directions indicated by labels FLj and FLj+1. At this time, the single-cell droplets in the pits are limited by buoyancy and the inner wall of the pits and are difficult to move out of the pits. The excess or redundant single-cell droplets in the transport channels that have not been captured by the pits are driven by the oil and discharged from the transport channels through the upper channel. In this process, because the angle between the buoyancy direction and the reaction chamber opening direction is an acute angle or 0 degrees, the single-cell droplets will not accidentally enter the reaction chamber.

[0126] Figure 4 As shown in neutron diagram (d), the transfer process involves rotating the chip clockwise (or, more preferably, rotating the chip so that the direction of buoyancy of the single-cell droplet is perpendicular to the chip, and then maintaining an orientation perpendicular to the fluid flow direction in the transfer channel) until the orientation is set according to the gravity direction indicated by the arrow on the right side of G2, achieving an angle of 0° or an acute angle between the buoyancy direction and the opening direction of the pit. The single-cell droplet in the pit enters the corresponding confinement area in the reaction chamber without error through the transfer channel, allowing for observation, storage, culture, amplification, PCR, and other operations.

[0127] Figure 4As shown in neutron diagrams (e) to (f), in the drug addition process: if other reagents need to be injected into / participated in cell-related experiments in addition to the single-cell droplets that have already entered the reaction chamber, as shown in sub-diagram (e), the reagent droplets shown in the white spheres above can be injected into the target reaction chamber once or multiple times using the same method as in sub-diagrams (b) to (d). During this process, the buoyancy direction of the single-cell droplets that have entered the confinement zone is prevented from pointing towards the entrance and exit of the confinement zone to prevent escape. In this step of the embodiment, the chip orientation direction that may cause the target to escape incorrectly is eliminated.

[0128] As shown in sub-figure (f), the added reagent droplets converge, fuse, mix, react, or interact with the previous single-cell droplets. The reagent droplets include, but are not limited to, nutrient solutions, lysis buffers, and graded drug reagents. Sub-figures (e) and (f) are optional and can be used individually or multiple times as needed. The fusion methods include, but are not limited to, using specialized reagents (including, but not limited to, demulsifiers) or using an electric field to destroy or weaken the surface activity of the droplets. The mixing methods include, but are not limited to, shaking to mix them uniformly.

[0129] Figure 4 As shown in neutron diagrams (g) to (h), in the recovery phase: the chip orientation is set according to the gravity direction indicated by the arrow on the right side of G2 in sub-diagram (g). Within each reaction zone, small spheres (half black and half white represent droplets fused with subsequently added reagent droplets; if the above-mentioned drug addition step is not performed, these droplets can be understood as equivalent to the black spheres representing single-cell droplets in previous steps) escape from the limiting area of ​​the reaction chamber under buoyancy, but do not leave the reaction chamber. At this point, by arbitrarily selecting a force application point within a control unit, for example, the control unit represented by point (i, j+1) in the control unit array shown in the diagram, force is applied using electrodes, etc. The device is inserted into the force application point near the center of the transfer channel outside the reaction chamber of the control unit. The electrode is inserted but not penetrated through. After the electrode is energized, the droplet can be driven by dielectric force or by electric field force to move out of the reaction chamber and into the transfer channel. At this time, the oil in the direction indicated by the arrow of column j+1 is selected and injected from bottom to top, driving the droplet that has moved out of the reaction chamber and into the transfer channel to move out to any designated space inside or outside the chip through the upper merging channel, realizing the export, discharge or recovery of the target object in any reaction chamber of any designated array control unit.

[0130] Figure 4 As shown in neutron diagram (i), in the reset step: the chip orientation is set according to the gravity direction indicated by the arrow on the right side of G2 in sub-diagram (i). Droplets that have escaped from the limiting area but have not been exported, discharged, or recovered by the electrode can re-enter the limiting area under the action of buoyancy and the inner wall of the reaction chamber in this step, and wait for subsequent processing.

[0131] The above-mentioned array-type control unit can be selected for node recovery by using needle-like instruments for blowing and suction control (or positive and negative pressure difference method), including but not limited to several of the following: (1) using needle-like instruments to directly pierce the space where the target object is located, and using negative pressure or suction action to directly adsorb and move it to the designated destination space; (2) using needle-like instruments to pierce the space where the target object is located, and using positive pressure or injection fluid to drive the target object to the transfer channel, and then injecting fluid into the transfer channel to drive it to the designated destination space; (3) using a multi-cavity needle-like instrument with positive and negative pressure combination (the flow direction in the two types of needle-like cavities is opposite), piercing the space where any selected target object is located in the fluid carrier, with several needles in the multi-cavity needle-like instrument spraying fluid and several other needles absorbing fluid, so that the selected target object can be directly sucked away and moved to the designated destination space. In this invention, "several" refers to one or more.

[0132] The aforementioned clearing process, namely the clearing process of the transfer channel, includes mechanisms including but not limited to: When the density of the target object is greater than that of the surrounding fluid, the spatial attitude of the fluid carrier is set by rotation or displacement to reduce the angle between the opening direction of the pit and the direction of gravity acting on the target object (i.e., the opening direction of the pit rotates to the horizontal direction (i.e., the direction perpendicular to the direction of gravity) but is not parallel to the horizontal direction), but the angle is not less than a right angle (i.e., a right angle or an obtuse angle). Under the premise that the specified number of target objects that have entered the pit and are confined will not escape from the pit, and that excess target objects in the transfer channel will not accidentally enter the reaction chamber, the excess target objects in the transfer channel (more than a specified number of target objects) are removed by means of gravity (at this time, buoyancy is less than gravity, and the resultant force of gravity and buoyancy is: gravity is the main force) or by the driving force of the fluid injected into the transfer channel (the types of excess target objects include but are not limited to: those that are free in the transfer channel, those that are temporarily positioned at the opening of the pit due to force balance, and those that have not been stably captured by the pit). When the density of the target object is less than that of the surrounding fluid, the spatial attitude of the fluid carrier is set by rotation or displacement to increase the angle between the opening direction of the pit and the direction of gravity of the target object (i.e., the opening direction of the pit rotates to the horizontal direction (i.e., the direction perpendicular to the direction of gravity) but does not coincide with the horizontal direction), but the angle is not greater than a right angle (i.e., a right angle or an acute angle). Under the premise that the specified number of target objects that have entered the pit and are confined will not escape from the pit, and that excess target objects in the transfer channel will not accidentally enter the reaction chamber, the excess target objects in the transfer channel (more than a specified number of target objects) are removed by means of buoyancy (at this time, buoyancy is greater than gravity, and the resultant force of gravity and buoyancy is: buoyancy is the main force) or by the driving force of the fluid injected into the transfer channel. The types of excess target objects include but are not limited to: those that are free in the transfer channel, those that are temporarily positioned at the opening of the pit due to force balance, and those that have not been stably captured by the pit). Other embodiments of the present invention:

[0133] Example 2: As in the present invention Figure 5 The diagram shows the principle and process of capturing, transferring, culturing, recovering and resetting a 3×4 array of single-cell slices using a single-space reaction chamber control unit.

[0134] As mentioned above Figure 3 Similar to related designs, the electrodes in this embodiment are protruding needle-shaped T-shaped and pre-embedded under the bottom surface of the channel inside the microfluidic chip, but do not pierce or expose the chip's internal cavity or channel. The target object is selected as a single-cell droplet, whose density is greater than the light oil (hereinafter referred to as oil) that drives its movement. The four electrodes Ei to Ei+3 in the microfluidic chip are arranged in rows, that is, each electrode can affect the target object in the control unit of a row after being powered on.

[0135] First, the preparation step: vacuuming and venting the chip's internal chamber. This step is optional and can be omitted if needed.

[0136] Figure 5 As shown in neutron diagram (a), in the capture stage: the chip orientation is set according to the gravity direction indicated by the left arrow of G1. The single-cell droplet, represented by the black ball in the transport channel, moves directionally through the four transport channels (from bottom to top) indicated by the four arrows (FLj to FLj+3) under the action of gravity and the drive of light oil. The angle between this direction of movement and the direction of the reaction chamber opening on the left side of the transport channel diagram is 45°, and the angle between the direction of gravity and the direction of the reaction chamber opening is 0°. Therefore, it can prevent the single-cell droplet from accidentally entering the reaction chamber in this step. At the same time, the direction of movement is perpendicular to the direction of the pit opening on the right side of the transport channel diagram. The angle between the direction of gravity on the single-cell droplet and the direction of the pit opening is 135°. Thus, the single-cell droplet enters the pit under the action of gravity. The pit can only accommodate one cell (Note: This is Example 1; in other applications, the number can be specified as multiple, and the principle is similar). Other cells flowing through the pit cannot enter it again, thus realizing the pit capture of single cells.

[0137] Figure 5As shown in neutron diagram (b), the clearing process is as follows: In the previous step, after single-cell droplets have been captured in a predetermined number of pits, the chip orientation remains unchanged, or rotates clockwise at an angle not greater than 45 degrees, and is set according to the gravity direction indicated by the left arrow of G1 (at this time, the angle between the pit opening direction and the gravity direction is about 100°). The light oil that does not contain cells is injected into the four transport channels along the four arrow directions indicated by labels FLj to FLj+3. At this time, the single-cell droplets in the pits are limited by gravity and the restriction effect of the pit inner wall. The excess free single-cell droplets or other droplets in the transport channels that have not been captured by the pits are driven by the light oil and discharged from the transport channels through the upper converging channel. In this process, because the angle between the gravity direction and the reaction chamber opening direction is an acute angle, the free single-cell droplets or other droplets will not accidentally enter the reaction chamber.

[0138] Figure 5 As shown in neutron diagram (c), the transfer process involves stabilizing and rapidly rotating the chip to the direction of gravity as indicated by the left arrow of G1, achieving a 0° angle between the direction of gravity and the opening direction of the pit. Single-cell droplets in the pit can then be transferred through the transfer channel into the corresponding reaction chamber, enabling observation, storage, culture, amplification, PCR, and other operations.

[0139] Figure 5 As shown in neutron diagrams (d) to (e), in the recovery process: the chip orientation is set according to the gravity direction indicated by the left arrow of G1 in sub-diagram (d). Under the influence of gravity, all droplets inside the chip are located at the bottom of the reaction chamber. At this time, by taking any electrode corresponding to a row of control units, such as Ei+1, powering on the electrical signal indicated by the label HV, while keeping the electrodes corresponding to other rows of control units grounded, the single-cell droplets in the reaction chambers of the four control units in row i+1 are respectively drawn out into different transport channels. Afterwards, according to... Figure 5 As shown in neutron diagram (e), any transport channel can be selected. In this embodiment, the transport channel in column j+1 is selected. Light oil without cells is injected along the direction of the black arrow indicated by label FLj+1. This allows the single-cell droplets in the culture chamber of the selected control unit in row 2 and column 2 to be exported or recovered through the transport channel and the confluence channel.

[0140] Figure 5 As shown in neutron diagram (f), in the reset step: the chip orientation is set according to the gravity direction indicated by the left arrow of G1 in sub-diagram (f). Droplets that have escaped from the reaction chamber into the transfer channel but have not been selected for export, discharge or recycling can re-enter the reaction chamber under the action of gravity in this step and wait for subsequent processing.

[0141] Example 3: As in the present invention Figure 6 The diagram shows the principle and process of how the control unit of multiple rows of pits corresponding to single rows of reaction chambers realizes multiple capture, co-directional transfer, and mixed culture on a 3×1 array of single-cell sheets.

[0142] This embodiment reveals a high-end application of microfluidic chips or gate-like devices: a single transfer can bring together multiple groups of target materials of different sizes (flipper, picoliter, nanoliter, or microliter) into the same reaction chamber, enabling more efficient and precise microscopic experimental operations at the single-cell and single-molecule levels.

[0143] The foregoing Figure 2 The relevant design and principles are suitable for this embodiment. Based on a similar structure, this embodiment uses a 3×1 array control unit to achieve precise control of the target object in a single reaction chamber corresponding to multiple rows of pits.

[0144] Figure 6 The control principle, mechanism, and process of this embodiment are explained through six sub-figures (a) to (f) arranged in chronological order. In each sub-figure, the left arrow of label G1 indicates the direction of gravity followed when setting the chip attitude for the control case where the density of the target object is greater than that of the fluid driving it; the right arrow of label G2 indicates the direction of gravity followed when setting the chip attitude for the control case where the density of the target object is less than that of the fluid driving it. In this embodiment, the density of all target objects is less than that of the heavy oil driving its movement, so the latter baseline scheme is adopted: the attitude of the microfluidic chip is set according to the direction of gravity shown by the right arrow of label G2.

[0145] This embodiment features a 3×1 array-type microfluidic chip structure, such as... Figure 6 Neutron diagram (b) shows a microfluidic chip, including three control units: Qi, Qi+1, and Qi+2. The three units share three transport channels indicated by black arrows near labels FLj, FLj+1, and FLj+2. In the transport channel indicated by black arrows near label FLj, there are two different types of pits on the right side of the diagram: six large pits and one small pit. The pits that are directly connected in other transport channels are all of the same type, and their cavity sizes are also the same or similar. In the same control unit, the pits that move toward the same reaction chamber, have collinear paths, and are located in different transfer channels have the following characteristics, including but not limited to: (1) their cavity volume is inversely proportional to the distance from the target reaction chamber; (2) except for the column farthest from the reaction chamber, the bottom of the other pits is provided with bottom holes; (3) the bottom holes provided in the pits can accommodate the target objects in all columns on the right side of the figure: the collinear pits.

[0146] First, the preparation step: vacuuming and venting the chip's internal chamber. This step is optional and can be omitted if needed.

[0147] Figure 6As shown in neutron diagram (a), in the capture stage: the chip orientation is set according to the gravity direction indicated by the arrow on the right side of G2. A large white sphere representing a single-cell droplet is injected into the transport channel (i.e., column j) pointed to by label FLj. A medium black sphere representing reagent A and a small black sphere representing reagent B are injected into the two transport channels (columns j+1 and j+2, respectively). In this way, the capture of the matching target objects in the large pit in column j, the medium pit in column j+1, and the small pit in column j+2 is completed in one go (matching means that the pit cavity can capture only a specified number of target objects, and the relationship between the two is matching). Only the third small pit from the top (sequence number) in column j is still empty, and the first round of capture stage is completed.

[0148] Figure 6 As shown in neutron diagram (b), during the emptying process: the chip attitude remains unchanged, and heavy oil that does not contain cells is injected into the three transport channels in the directions of the three arrows marked FLj to FLj+2. At this time, the target objects in the pit are limited by buoyancy and the inner wall of the pit. The excess free target objects in the transport channels that are not captured by the pit are all driven by the heavy oil and discharged from the transport channels through the channel shown below. In addition, because the angle between the buoyancy direction and the reaction chamber opening direction is an acute angle, the free target objects will not accidentally enter the reaction chamber.

[0149] Figure 6 As shown in neutron diagram (c), the second round of capture is the same as the mechanism shown in sub-diagram (a). A small ball representing reagent D is injected into the j-th column transport channel to complete the capture of the target object by matching the third small pit from the top to the bottom in the j-th column transport channel.

[0150] Figure 6 As shown in sub-diagram (d), the second round of emptying is the same mechanism as shown in sub-diagram (b). Heavy oil that does not contain the target is injected into the transfer channel in column j along the direction of the arrow indicated by label FLj to remove the free target that was not captured by the pit.

[0151] Figure 6 As shown in neutron diagrams (e) to (f), the transfer process involves stabilizing and rapidly rotating the chip to a position where the direction of gravity is set according to the arrow on the right side of G2, achieving a 0° angle between the buoyancy direction and the pit opening direction. Targets in the three rows of pits can directly enter the corresponding reaction chamber in the control unit, either directly or through the bottom holes of other pits, without any obstacles blocking their path. Observation, storage, culture, amplification, PCR, and other operations can then be performed. Subsequent recovery, discharge, sorting, screening, and resetting processes can employ mechanisms and methods similar to those in Example 1 or Example 2.

[0152] In this invention, the bottom hole of the recess, in addition to ensuring that the target object matched with the recess will not leak out from the bottom hole, is also designed to be gradually widened. This design allows the target object to move along the path of the recess towards the target reaction chamber. The bottom hole on the path of the movement is widened and then narrowed, and the narrowest area of ​​the bottom hole can also allow the target object to pass through. This makes it easier for the target object to enter the bottom hole. Even if the target object deviates from its path, it can be eliminated or corrected, thus improving the reliability of the system.

[0153] In this way, this embodiment, with the help of the three rows of recesses in the control unit, and the design of recess cavities and bottom holes of various sizes, can capture three different target substances at once, or capture more different target substances in multiple rounds. Then, directly or through the bottom hole of the middle recess, under the action of buoyancy, gravity or other external forces, the target substances are drawn into the corresponding target reaction chamber, realizing the mixing of target substances of different dosages and sizes (such as droplets containing active cells, reagent droplets or bubbles). It can perform experimental functions such as storage, collection, fusion, mixing, reaction, culture, lysis, sorting, and screening, and has the diversity, advancement and efficiency of experimental methods for single-cell or molecular-level reaction systems.

[0154] Industrial applicability: This invention has clear industrial applicability. The microfluidic chip can be mass-produced using standard soft lithography processes with mature materials such as PDMS, and is highly compatible with existing MEMS fabrication platforms. The system can seamlessly interface with peripheral equipment such as automated control, high-resolution imaging, and fluorescence detection to achieve fully automated operation. This invention can be widely applied in fields such as antibody screening, synthetic biology, single-cell sequencing, drug dosage response studies, and circulating tumor cell detection, and has broad industrialization prospects and market value.

[0155] Free content of the sequence list: None.

Claims

1. A large-scale single-cell array multi-component precise quantitative reaction system at the liter, picoliter, nanoliter, or microliter level, characterized in that: The target object is a biological particle, droplet, or bubble. The biological particle may be active or inactive. Active biological particles include whole or part of a biological or microbial organism. The droplet or bubble may or may not contain biological particles. The fluid carriers included are entities that can contain the flow of liquids, gases or solids or be used for experiments, including: biochips, microfluidic chips, gate-like devices, reagent kits, and instruments containing transparent or translucent materials. The fluid carrier includes a channel and a control unit, and the channel types include transfer channels and main channel; The control unit includes a chamber and a transfer channel, and the chamber types include: recesses and reaction chambers; The recess can stably hold a specified number of target objects and is connected to the corresponding reaction chamber via a transfer channel. The transfer channels of different control units can be independent of each other, or they can be connected, shared, or designed as the same channel. The pit and the corresponding reaction chamber are adjacent in opening position, with the opening direction angle not less than 90 degrees, and are respectively set on different sides of the inner wall of the transfer channel. The volume of the pit cavity is not greater than the volume of the corresponding reaction chamber. The fluid carrier attitude or structural design, in chronological order, is as follows: (1) Target capture stage: The direction of the resultant force of gravity and buoyancy on the target object in the fluid carrier is at an angle of not less than 90 degrees with the direction of the pit opening and at an angle of not more than 90 degrees with the direction of the reaction chamber opening. In this stage, the target object enters the transfer channel by fluid drive. Under the action of gravity, buoyancy or fluid drive, a specified number of one or more target objects enter the pit while preventing them from accidentally entering the reaction chamber. Under the action of gravity or buoyancy and the inner wall of the pit, the target object is limited, positioned or captured. (2) Emptying the transfer channel: The target object captured by the concave pit in the fluid carrier is subjected to the resultant force of gravity and buoyancy. The angle between the direction of the resultant force and the opening direction of the concave pit remains unchanged or is not less than 90 degrees after change. The angle between the target object and the opening direction of the corresponding reaction chamber of the concave pit remains unchanged or is not greater than 90 degrees after change. It can be achieved by using gravity or buoyancy, or by driving the injected fluid, to remove excess target objects in the transfer channel, provided that a specified number of target objects that have entered the concave pit and are limited will not escape from the concave pit, and excess target objects in the transfer channel will not accidentally enter the reaction chamber. (3) Target transfer stage: The spatial attitude of the fluid carrier is set as follows: the target captured by the pit is subjected to the resultant force of gravity and buoyancy, and the angle between the direction of the pit opening is less than 90 degrees, and the angle between the direction of the target from the position of the target to the opening of the corresponding reaction chamber of the pit is less than 90 degrees, so that the target in the pit can be directionally moved into the interior of the reaction chamber through the opening of the reaction chamber. By operating continuously according to the above sequence, a single round can be executed to accurately capture and transfer a specified number of target substances, or multiple rounds can be executed to collect multiple components or different target substances. Both execution methods can realize the storage, mixing, reaction, amplification, culture, reproduction, sorting, plate loading, or selective screening of target substances.

2. The large-scale single-cell array multi-component precise quantitative reaction system of claim 1 (flux, picoliter, nanoliter, or microliter scale) is characterized in that: In several of the aforementioned steps, the driving force of the fluid injected into the transfer channel or the inertia of the target object is used to improve control efficiency. The angle between the fluid flow direction in the transfer channel within the fluid carrier and the direction of the resultant force of gravity and buoyancy on the controlled target object is 0 degrees, an acute angle, or a right angle. In particular, in the target object capture step, the angle between the flow direction of the fluid driving the target object in the transfer channel and the opening direction of the pit is not less than 90 degrees, and the angle between the opening direction of the reaction chamber corresponding to the pit and the movement direction of the target object in the transfer channel is not greater than 90 degrees. Including the selective recovery or discharge of target materials, combined with the recapture, emptying, or transfer of target materials, it enables the selective export or replacement of target materials within the chambers, thus achieving the sorting or screening function of target materials. The mechanism for this step includes: through the application of directional external force, some or all of the material from any designated number of chambers is directly exported to the outside of the fluid carrier, or exported from the chamber to the transfer channel through the chamber opening, and then exported to a designated space inside or outside the fluid carrier by the directional drive of the fluid in the transfer channel or the application of directional external force, thereby achieving recovery, discharge, or recapture and replacement. The directional external force or directional drive mechanism or principle includes several of the following: sound waves, ultrasound, shock waves, acoustic tweezers, electric field force, dielectric force, magnetic field force, and fluid impact. The electric field force is directed at the charged target object. The dielectric force is directed at droplets or bubbles, which may or may not contain: biological particles. The magnetic force is directed at the target object containing magnetic material. The fluid impact mechanism involves inserting or connecting a needle-like device into the cavity containing the target object and ejecting fluid, thereby generating a thrust towards the outside of the cavity on the target object.

3. The large-scale single-cell array multi-component precise quantitative reaction system of claim 2 (flux, picoliter, nanoliter, or microliter scale) is characterized in that: In the selective recovery or discharge of the target object, the directional external force drives the target object from its chamber into the channel, and then, with the help of fluid impact or external force in the specific channel, achieves recovery, discharge, or recapture for replacement. The implementation method and mechanism of the directional external force include several of the following: a) External force facilities are installed on the outside of the fluid carrier for contact-type control of the fluid carrier. The mechanisms include several of the following: sound waves, ultrasound, shock waves, electric field force, dielectric force, and magnetic field force. These external force facilities control the fluid carrier by approaching, penetrating, or connecting to specific locations on the fluid carrier. Methods include: through pre-set holes or openings; contacting or connecting to: a force-applying device pre-embedded or pre-installed inside the fluid carrier. When the control units or target objects inside the fluid carrier are arranged in a row-and-column array, a two-dimensional spatial positioning method is used to locate the specific target and apply directional external force control. In the penetrating control method, the fluid carrier's internal space may or may not be penetrated. Penetrating without penetrating prevents leakage of liquid or gas. b) External force facilities are installed outside the fluid carrier for non-contact control of the fluid carrier: The mechanism includes several of the following: sound waves, ultrasound, shock waves, electric field force, dielectric force, and magnetic field force. Non-contact control of the fluid carrier involves applying a directional external force to a target object at a specific location outside the fluid carrier, along a specific direction. When the control units or target objects inside the fluid carrier are arranged in a row-and-column array, a two-dimensional spatial positioning method is used to locate the specific target and apply directional external force control. c) Non-electrode-type external force facilities are installed inside the fluid carrier to implement targeted control of the target object: The non-electrode-type external force facilities are set at specific locations in each control unit, and the controllable external force mechanisms that can be emitted or generated include several of the following: sound waves, ultrasonic waves, shock waves, and magnetic fields. By activating the external force at any designated point, a directional external force can be applied to the target object at that specific location. d) Electrode-type external force facilities are installed inside the fluid carrier to implement point-by-point control of the target object: The electrode-type external force facilities can be set at specific positions of each control unit. The controllable external force mechanism includes several of the following: electric field force, dielectric force. When power is applied or an external force is generated at any specified point, a directional external force is applied to the target object at the specific position. The point-by-point control includes, in particular, row-and-column fixed-point control of the target object: If the set of several control units that flow through each transfer channel is defined as a column / row, then the set of single-row control units controlled or affected by several electrodes perpendicular to it is defined as a row / column. Then, by energizing the electrodes, electric field force or dielectric force is used to drive the selected rows / columns of target objects from the chamber to the external transfer channel. At the same time, fluid is selectively injected into the transfer channels of several columns / rows. Then, by row and column positioning, the target objects in the chambers of any selected several control units are exported to the specified space through the transfer channels under the drive of the injected fluid. In the above serial numbers a) to d), the shape of the external force facility can be a straight line, a curve, or a combination of straight lines and curves. When the external force facility uses electrodes, the electrode type is: multi-point common type or single-point single-control type, movable type or fixed type, and the electrode shape is: straight line, curve, or a combination of both.

4. The large-scale single-cell array multi-component precise quantitative reaction system of claim 3 (flux, picoliter, nanoliter, or microliter scale) is characterized in that: The selective recovery or discharge of the target object employs a mobile, directional external force facility, which may be single or multiple and includes several types of the following: electrodes, magnetic poles, and sound wave, ultrasonic wave, or shock wave generating devices. This mobile, directional external force facility can move in one-dimensional, two-dimensional, or three-dimensional space and can be located outside the fluid carrier or penetrate but not through the fluid carrier to approach the target object and apply a controllable external force. The mechanism includes several of the following: (1) The electrodes are charged to form a non-uniform electric field, which drives the target object in the fluid in a directional manner through dielectric force. (2) The electrodes are charged to form an electric field, which attracts or drives the charged target in the fluid by means of the electric field force: the electrodes and the target have opposite polarities and attract each other, and have the same polarity and repel each other, thereby realizing the directional driving of the target in the fluid. (3) The magnetic force generated by the magnetic poles attracts or pushes the target object containing magnetic material in the fluid: the magnetic poles closer to the target object and the magnetic poles closer to the magnetic material in the target object attract each other if their magnetic properties are opposite, and repel each other if their magnetic properties are the same. (4) By using a sound wave, ultrasonic wave, or shock wave generator, the energy of the wave is used to attract or push the target object closer to or further away, thereby achieving directional driving of the target object in the fluid. All of the above mechanisms achieve the directional movement of the target object along a specified direction in a non-contact manner or method, thereby enabling the target object to be recovered, discharged, recaptured and replaced, or otherwise controlled for displacement.

5. The large-scale single-cell array multi-component precise quantitative reaction system of claim 1 (flux, picoliter, nanoliter, or microliter scale), characterized in that: Including the selective recovery or removal of the target object, within the internal chambers of the fluid carrier, the methods for exporting, expelling, recovering, removing, or replacing the target object include: using a directional external force device outside the fluid carrier, such as a needle-like instrument, to pierce and penetrate the channel or chamber, with mechanisms including several of the following: a) Utilizing the suction effect of negative pressure: In needle-like devices, the pressure inside the cavity is lower than the pressure at the insertion point of the fluid carrier, so that the target near the insertion point is sucked into the cavity of the needle-like device and can then be moved out to any space. b) Utilizing the driving effect of positive pressure: In the cavity of the needle-like device, the fluid pressure is greater than the pressure of the space near the insertion point in the fluid carrier. The needle-like device ejects fluid at the insertion point of the fluid carrier, increasing the fluid pressure in the cavity. This allows the fluid to drive the target object out of the cavity and into the connected external channel. Then, through the directional driving of the fluid in the external channel, it moves to the designated space. c) Utilizing the simultaneous "blowing" and "suction" action of positive and negative pressure: Needle-like instruments that pierce the fluid carrier and penetrate its channels or cavities, including two types: The first type has a fluid pressure in the cavity that is greater than the pressure of the space inside the pierced fluid carrier, and fluid is ejected at the piercing point of the fluid carrier, using positive pressure to drive the target object away from its original position; the second type has a fluid pressure in the cavity that is less than the pressure of the space inside the pierced fluid carrier, and negative pressure is used to absorb the target object at the piercing point of the fluid carrier. Both types of needle-like instruments are inserted near the target object, so the target object is efficiently moved and enters the lower-pressure second type of needle-like instrument under the "blowing" and "suction" action of the two types of needle-like instruments. By controlling the two types of needle-like instruments with different flow directions, and simultaneously ensuring that the fluid volumes "blown" and "suctioned" are the same or similar, it can be ensured that during the operation, the space of the cavity where the controlled target object is located and other spaces of the connected fluid carrier have no fluid flow or very little flow, minimizing the disturbance and impact on other cavities or spaces of the target object.

6. The large-scale single-cell array multi-component precise quantitative reaction system according to any one of claims 1 to 5, characterized in that: In each of the aforementioned steps, the detection methods for the target object include several of the following: fluorescence or self-luminescence detection, impedance or admittance detection, Raman signal detection, voltage or current signal detection, image information detection, size detection, and color detection.

7. The large-scale single-cell array multi-component precise quantitative reaction system according to any one of claims 1 to 5, characterized in that: Within the same control unit, several columns / rows of recessed openings all face or are near the reaction chamber opening. Within the same control unit, to ensure that all targets captured in the pits can be moved into the corresponding reaction chambers under external force, other pits along or near the path from the pit opening to the corresponding reaction chamber opening, which may obstruct or affect the movement of the targets, are provided with bottom holes through which the targets can pass. Within the same control unit, if a solid structure of a fluid carrier forms an obstruction along or near the movement path from the pit opening to the corresponding reaction chamber, such solid structure has a through-hole through which the targets can pass. Within the same control unit, different pits whose moving targets are the same reaction chamber and whose paths are collinear have cavity volumes and minimum bottom hole sizes that are inversely proportional to the path length from the corresponding reaction chamber opening, and can be arranged linearly or nonlinearly. The size of the target object captured by the pit with the bottom hole is larger than the size of the bottom hole, which not only limits the captured target object but also prevents it from escaping from the bottom hole.

8. The large-scale single-cell array multi-component precise quantitative reaction system according to any one of claims 1 to 5, characterized in that: The fluid carrier and reaction chamber include or are connected to a limiting area with a limiting function. The limiting mechanism includes: only when the fluid carrier's attitude changes at a specific time sequence and matching speed, can the target object enter or move out of the limiting zone, or completely leave from a specific range including the limiting zone; For fluid carrier attitude changes that are not specific in timing and matching speed, the target object will not enter the limiting zone from the outside. For fluid carrier attitude changes that are not specific in timing and matching speed, the target object will not completely move out of the limiting zone, or even if it moves out of the limiting zone briefly, it will re-enter the original limiting zone in the next stage.

9. The large-scale single-cell array multi-component precise quantitative reaction system according to any one of claims 1 to 5, characterized in that: To achieve micron-level precise positioning or displacement, accurately locate the position of the control unit chamber or target object, or a specific location within the fluid carrier, position marking is performed. This can be achieved through several of the following methods: optical marking, color marking, specific shape mechanism marking, and character marking. Specific locations within the fluid carrier can be categorized as: control units, channels, recesses, and reaction chambers. One method of marking by fluorescence involves setting several channels on the fluid carrier specifically for location marking. Only at each identification point that needs to be identified, a different shape, size, or feature is set, including several of the following: protrusion, bend, fork, branch, slot, blind hole, through hole. The method of using fluorescence for position marking includes: setting a separate connected channel for the position to be marked, or connecting multiple positions to be marked in series or parallel through a common channel, injecting fluorescent material through the channel or opening; in the case of multiple positions injected through a common channel, the common channel between each position can be blocked before use so that the excitation light cannot shine on it, or the space between it and the observation instrument can be blocked, thus forming a number of discrete fluorescent marked positions. When the excitation light shines on it, the observation instrument can determine the specific position of the relevant fluid carrier based on the fluorescent position.

10. A flexible control method for a large-scale single-cell array multi-component precise quantitative reaction system of any one of claims 1 to 5, characterized in that: Return methods for target objects that were not extracted from the chamber but were driven or escaped into the transport channel: To disable the external force facility, eliminate its external force action on the target object, and maintain or reset the fluid carrier attitude as follows: the resultant force of gravity and buoyancy on the target object that was previously driven into the transfer channel from the cavity of the control unit is greater than 0 in the direction from its current position to the cavity from which it was previously moved out, so that the target object that was not exported from the transfer channel returns to the cavity from which it originated. By using electric field force or dielectric force control, the electrode is set at the position where the control unit needs to apply force control, with the electrode force application point protruding towards the cavity of the control target. In addition to strengthening the non-uniformity of the electric field or enhancing the effect of external forces such as electric field and dielectric force, it can also prevent excessively strong electric field, dielectric force or other external forces from being generated at other positions on the electrode except for the electrode force application point, thus preventing misoperation. The volume and precision of the chamber, as well as the volume and precision of the droplets or bubbles, are all pre-designed and precise: fly-liter, picoliter, nanoliter, or microliter, enabling large-scale single-cell array multi-component precise quantitative reactions.

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