Biological particle screening system, biological particle screening method and biological particle screening chip

Abstract

The purpose of the present invention is to provide a novel technique for efficiently sorting desired biological particles. The invention provides a biological particle sorting system. The biological particle sorting system is configured to sort particles to be collected flowing through a flow channel into a collection flow channel. The biological particle sorting system is configured to be able to perform (as a sorting operation for sorting particles to be collected) a first step for reducing the pressure in the collection flow channel from a reference pressure to direct the particles to be collected into the collection flow channel; a second step for returning the pressure reduced in the first step to the reference pressure; and a third step for mitigating fluctuations in excessive pressure caused by execution of the second step.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a biological particle screening system, a biological particle screening method, and a biological particle screening chip. More specifically, the present disclosure relates to a biological particle screening system configured to screen one or more collection target particles flowing through a flow path into a collection flow path, a biological particle screening method for screening one or more collection target particles flowing through a flow path into a collection flow path, and a biological particle screening chip for performing the screening method. BACKGROUND

[0002] For example, a group of particles such as cells, microorganisms, and liposomes are labeled with a fluorescent dye, and a characteristic of a corresponding particle in the group of particles is measured by emitting a laser beam onto the particle and measuring the intensity and / or pattern of fluorescence generated from the excited fluorescent dye. Examples of a particle analysis device that performs the measurement can include a flow cytometer and a cell sorter.

[0003] For example, a biological particle screening apparatus such as a flow cytometer or a cell sorter analyzes a plurality of particles one by one by emitting a laser beam having a specific wavelength (an excitation beam) onto particles flowing next to each other in a line in a flow path and sensing fluorescence and / or scattered light emitted from each particle, and screens desired biological particles based on the analysis result.

[0004] Several techniques for implementing such biological particle fractionation have been disclosed so far. For example, Patent Literature 1 below discloses a microparticle fractionation device including: a main flow path through which a liquid including microparticles flows; a branch flow path that communicates with the main flow path; an actuator that generates a negative pressure in the branch flow path; and a drive unit that controls a voltage applied to the actuator and generates a pressure change including a staircase waveform component and a kick waveform component in the branch flow path. Patent Literature 2 mentioned below discloses a microparticle fractionation method including a process of collecting microparticles in a liquid flowing through a main flow path into a partial region in which a cross section perpendicular to a flow direction of the liquid in a branch flow path that communicates with the main flow path is formed larger than other portions, by generating a negative pressure in the branch flow path. Patent Literature 3 mentioned below discloses a microparticle collection method performed in a microparticle fractionation mechanism having a flow path structure including a main flow path through which microparticles flow, a collection flow path that collects a collection target particle among the microparticles, a connection flow path that connects the main flow path and the collection flow path, and a liquid supply flow path that is connected to the connection flow path so that the liquid supply flow path can supply a liquid to the connection flow path. The microparticle collection method includes: a flow passage step of causing a first liquid containing microparticles to flow through the main flow path; a determination step of determining whether the microparticles flowing through the main flow path are the collection target microparticles; and a collection step of collecting the collection target microparticles into the collection flow path. In the collection step, the collection target particles are collected into a second liquid that is immiscible with the first liquid in a state in which the collection target particles are included in the first liquid.

[0005] [Citation List]

[0006] [Patent Literature]

[0007] [Patent Literature 1]

[0008] PCT Patent Publication No. WO 2014 / 013802

[0009] [Patent Literature 2]

[0010] Japanese Patent Publication No. 2014-039534

[0011] [Patent Literature 3]

[0012] PCT Patent Publication No. WO 2021 / 084814 SUMMARY

[0013] [Technical Problem]

[0014] Efficient fractionation of target biological particles is required. In particular, fast and stable biological particle fractionation separation is required. For example, fast and stable biological particle fractionation is considered to contribute to further acceleration and improved analysis accuracy.

[0015] In view of this, an object of the present disclosure is to provide a new technology for efficiently screening target biological particles.

[0016] [Solution to the problem]

[0017] The present invention provides a biological particle screening system, which is configured to screen collection target particles flowing through a flow path into a collection flow path, wherein the biological particle screening system is configured to be able to perform, a first step, reducing the pressure in the collection flow path from a reference pressure and guiding the collection target particles into the collection flow path, a second step, restoring the pressure reduced in the first step to the reference pressure; and a third step, reducing excessive pressure changes caused by performing the second step.

[0018] The biological particle fractionation system can be configured to change the pressure in the collection flow path by deforming the collection flow path.

[0019] The biological particle fractionation system may be configured to apply a pulse voltage to the actuator to change the pressure in the collection flow path in one fractionation operation, and the driving waveform of the pulse voltage may be configured to cause the actuator to perform the first step, the second step, and the third step.

[0020] The driving waveform of the pulse voltage may have a first falling edge portion for performing the first step, a rising edge portion for performing the second step, and a second falling edge portion for performing the third step.

[0021] The biological particle screening system may have an information processing unit that performs judgment processing based on the interval between the collection target particle and the subsequent biological particle, and the judgment processing may be a process for determining which screening operation to apply to screen a collection target particle based on whether the interval is within a predetermined numerical range.

[0022] In the determination process, the information processing unit may refer to a step counter value associated with the number of times the screening operation can be performed.

[0023] As a primary screening operation, the biological particle screening system can be configured to be capable of performing a screening operation (hereinafter, also referred to as a "first screening operation"), executing the first step, the second step and the third step, or capable of performing a second screening operation, executing the first step and the second step, and further executing the fourth step of eliminating the deviation of the collection flow path generated by executing the third step when predetermined conditions are met.

[0024] The biological particle fractionation system may have an information processing unit that performs determination processing based on an interval between a collection target particle and a subsequent biological particle, and the information processing unit may select the first fractionation operation or the second fractionation operation based on the interval.

[0025] In the fourth step, the biological particle sieving system can be configured to increase the pressure by one level or to increase the pressure by a plurality of levels.

[0026] In a case where the second sieving operation is performed, the information processing unit can determine whether to perform the fourth step based on an interval between a completion time of the second step in the second sieving operation and a time at which the subsequent biological particle is sensed.

[0027] The biological particle sieving system can be configured to apply a pulse voltage to the actuator to change the pressure in the collection flow path, and a drive waveform of the pulse voltage can be configured such that the actuator performs the first sieving operation or the second sieving operation according to an interval between the collection target particle and the subsequent biological particle.

[0028] The drive waveform for performing the first sieving operation can have a first falling edge portion for performing the first step, a rising edge portion for performing the second step, and a second falling edge portion for performing the third step, and the drive waveform for performing the second sieving operation can have a first falling edge portion for performing the first step and a rising edge portion for performing the second step, and further have a second rising edge portion for performing the fourth step in a case where the fourth step is performed.

[0029] The biological particle sieving system can be configured to increase the pressure by one level in the fourth step, and the second rising edge portion for performing the fourth step can have a single-step rising edge.

[0030] The biological particle sieving system can be configured to be able to increase the pressure by a plurality of levels in the fourth step, and configured to be able to change the number of levels of the increased pressure.

[0031] In the second sieving operation, a fifth step of preventing a particle backflow that can be generated due to the performance of the second step can be further performed.

[0032] The biological particle sieving system can be configured as an emulsion production apparatus, and the emulsion can include emulsion particles having fractionated collection target particles.

[0033] Further, the present disclosure also provides a biological particle sieving method including sieving a collection target particle flowing through a flow path into a collection flow path, wherein the biological particle sieving method includes: performing a first step of decreasing a pressure in the collection flow path from a reference pressure and guiding the collection target particle into the collection flow path, a second step of restoring the pressure decreased at the first step to the reference pressure, and a third step of mitigating an excessive pressure change generated due to the performance of the second step.

[0034] Further, the present disclosure also provides a biological particle sieving chip including a collection flow path in which a collection target particle that flows through the flow path is fractionated, wherein the biological particle sieving chip is used to fractionate the collection target particle by performing a sieving operation including a first step of reducing a pressure in the collection flow path from a reference pressure and guiding the collection target particle into the collection flow path, a second step of restoring the pressure reduced at the first step to the reference pressure, and a third step of mitigating an excessive pressure change due to the performance of the second step. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a diagram depicting a configuration example of a flow path system of a microfluidic chip according to the present disclosure performing a sieving operation.

[0036] Figure 2 is a diagram depicting an enlarged view of a particle sieving unit.

[0037] Figure 3 is a diagram depicting a configuration example of an actuator for generating a negative pressure in a pressure chamber.

[0038] Figure 4 is a diagram depicting an example of a basic drive waveform for sieving a target particle.

[0039] Figure 5 is a diagram schematically depicting states of a suction operation and a discharge operation.

[0040] Figure 6 is a diagram depicting a simulation result of a flow rate in a connection flow path in a case where a pulse drive waveform according to a conventional technique is applied.

[0041] Figure 7 is a diagram for explaining a position of a simulated flow rate.

[0042] Figure 8 is a diagram depicting an example of a drive waveform to be applied to a piezoelectric element to perform a first sieving operation.

[0043] Figure 9 is a diagram representing a simulation result of a flow rate in a connection flow path.

[0044] Figure 10 is a diagram depicting results of an experiment regarding the generation of emulsion particles.

[0045] Figure 11 is a diagram depicting results of an experiment regarding the generation of emulsion particles.

[0046] Figure 12is a graph showing simulation results of flow rates of the connecting flow path in a case where a pulse driving waveform including a falling edge and a waveform other than the pulse driving waveform are used.

[0047] Figure 13 is a graph depicting simulation results of flow rates in a case where the flow path diameter of the connecting flow path is doubled.

[0048] Figure 14 is a graph depicting an example of a driving waveform applied in the second sieving operation.

[0049] Figure 15 is a graph for explaining selection of a sieving operation.

[0050] Figure 16 is an example of a flowchart of a sieving method according to the present disclosure.

[0051] Figure 17 is an example of a flowchart of a waveform selection process according to the present disclosure.

[0052] Figure 18 is a diagram showing an example of a timing chart in a case where the waveform selection process is executed according to the present disclosure.

[0053] Figure 19 is a graph depicting an example of a driving waveform applied in the second sieving operation.

[0054] Figure 20 is a graph for explaining the number of rising operations.

[0055] Figure 21 is a diagram showing an example of a program describing a method executed by a biological particle sieving system according to the present disclosure.

[0056] Figure 22 is a graph depicting an example of a processing program for deciding whether to execute a rising operation.

[0057] Figure 23 is a diagram showing an example of a timing chart in a case where the waveform selection process is executed according to the present disclosure.

[0058] Figure 24 is a graph depicting an example of a driving waveform for executing the second sieving operation including the fifth step.

[0059] Figure 25 is a graph showing simulation results of flow rates in the connecting flow path.

[0060] Figure 26 is a graph showing simulation results of a time change in a position of a particle in a flow direction.

[0061] Figure 27is a view schematically showing the overall configuration of a biological sample analyzer.

[0062] Figure 28A is a block diagram showing a configuration example of an information processing unit.

[0063] Figure 28B is a graph for explaining a waveform of an electrical signal read in by the event sensing circuit 2303.

[0064] Figure 28C is a conceptual diagram for explaining an event data packet.

[0065] Figure 28D is a graph for explaining gating in a histogram and a 2D graph.

[0066] Figure 29 is a view describing an example of an algorithm executed by the information processing unit.

[0067] Figure 30 is a view describing an example of an algorithm executed by the information processing unit.

[0068] Figure 31A is a graph schematically depicting a change in pressure in a collection flow path.

[0069] Figure 31B is a graph schematically depicting a change in pressure in a collection flow path.

[0070] Figure 31C is a graph schematically depicting a change in pressure in a collection flow path.

[0071] Figure 32A is a view describing a configuration example of a biological particle sieving chip according to the present disclosure.

[0072] Figure 32B is an example of a flowchart of a biological particle sieving method according to the present disclosure.

[0073] Figure 33A is a graph depicting measurement results and simulation results of a collection rate.

[0074] Figure 33B is a graph depicting measurement results of purity.

[0075] Figure 33C is a graph depicting a photograph of a fractionated emulsion. DETAILED DESCRIPTION

[0076] Hereinafter, preferred modes for carrying out the present disclosure are explained. Note that the embodiments explained below represent representative embodiments of the present disclosure, and the scope of the present disclosure is not limited only to these embodiments. Note that the present disclosure is explained in the following order.

[0077] 1. First embodiment (biological particle screening system)

[0078] (1) Basic concept

[0079] (2) Configuration example

[0080] (3) Drive waveform

[0081] (4) Generation of emulsion particles

[0082] (5) Usefulness of pulse waveform

[0083] (6) Relationship with flow path cross-sectional area

[0084] (7) Screening process example 1

[0085] (7-1) Example 1 of process program executed by biological particle screening system

[0086] (7-2) Waveform selection process example

[0087] (7-3) Example of timing chart

[0088] (7-4) Configuration example of screening device

[0089] (7-5) Example of information processing unit and example of algorithm executed by information processing unit

[0090] (7-5-1) Example of information processing unit

[0091] (7-5-2) Example of algorithm

[0092] (8) Screening process example 2

[0093] (8-1) Example 1 of process program executed by biological particle screening system

[0094] (8-2) Example of timing chart in case of executing waveform selection process

[0095] (8-3) Example of algorithm

[0096] (9) Screening process example 3

[0097] (10) Configuration example of biological particle screening device

[0098] (11) Embodiment

[0099] 2. Second embodiment (biological particle screening method)

[0100] 3. Third embodiment (biological particle screening chip)

[0101] 1. First embodiment (biological particle screening system)

[0102] (1) Basic Concept

[0103] (Problem)

[0104] For example, a screening device for screening microparticles such as biological particles is configured to selectively screen one or more collection target particles from a large number of particles flowing through a main flow path into a collection flow path. In order to screen the collection target particles into the collection flow path, for example, an operation of sucking the collection target particles into the collection flow path can be performed. As a screening operation for rapidly screening the particles into the collection flow path by such a suction operation, a suction operation can be performed to form a flow from the collection flow path to the main flow path and a discharge operation for restoring a pressure state caused by the suction operation to an initial state before the suction operation. That is, as a screening operation, a set of the suction operation and the discharge operation can be performed. The suction operation can be an operation of forming a flow advancing from the main flow path to the collection flow path, for example, can be performed by lowering the pressure in the collection flow path (i.e., the suction operation). In the collection flow path, a negative pressure is generated. In addition, the discharge operation can be an operation for eliminating or reducing the flow state generated by the suction operation. The discharge operation can be an operation of forming a flow advancing from the collection flow path to the main flow path, but such a flow does not necessarily need to be substantially generated. For example, the discharge operation can be performed by increasing the pressure in the collection flow path.

[0105] Since the discharge operation is an operation of forming a flow advancing from the collection flow path to the main flow path in the screening operation, after the discharge operation is performed, a flow (also referred to as a "discharge flow") advancing from the collection flow path to the main flow path can be generated between the main flow path and the collection flow path. In addition, after the discharge operation is performed, unnecessary flow oscillation is also generated between the main flow path and the collection flow path. The unnecessary oscillation is an oscillation of a flow rate between the main flow path and the collection flow path. For example, particularly in a case where the main flow path and the collection flow path are connected via a connection flow path having a smaller cross-sectional area than the cross-sectional area of the flow path, such a problem can occur.

[0106] Therefore, after a screening operation for screening a specific collection target particle is performed, in order to properly fractionate further lower collection target particles, it is necessary to wait until the discharge flow disappears to perform the screening operation. That is, a predetermined length of time must be allowed between two screening operations. Allowing a longer time between two screening operations hinders rapid screening of the particles, and the particles to be collected can be reduced. Conversely, in a case where no length of time is allowed between two screening operations, the screening process cannot be stably performed.

[0107] In addition, for example, the above-described screening device can produce an emulsion having only desired particles among emulsion particles. That is, the screening device can also be used as an emulsion production device.

[0108] In the case where an emulsion is produced by a screening device, the above-described problems of a discharge flow and unnecessary oscillation can also be problems. Specifically, in a case where screening of particles next to a specific particle flow is performed in a state where a discharge flow produced after classification of a specific particle is not sufficiently reduced or in a state where unnecessary oscillation is produced after classification is performed, emulsion particles having different sizes can be produced. For example, from the viewpoint of screening performance, the smaller the particle size, the more likely screening is to fail, and the larger the particle size, the more likely other particles are to be entrained. Therefore, an emulsion having a variation in emulsion particle size lacks stability in some cases. In addition, from the viewpoint of analysis influence, since a variation in the total amount of a reagent to be input into an emulsion particle is caused by a difference in particle diameter (for example, the larger the particle diameter, the larger the total amount of the reagent), there is a possibility that a variation is produced in an analysis result. Therefore, in some cases, an emulsion having a large variation in emulsion particle size also lacks suitability for analysis using the emulsion.

[0109] (SUMMARY OF THE DISCLOSURE)

[0110] The specific screening operation according to the present disclosure can reduce the above-described discharge flow, and can further reduce unnecessary oscillation. That is, the present disclosure is configured to be able to perform, as a first step of a screening operation for screening a collection target particle, a first step of reducing a pressure in a collection flow path from a reference pressure and guiding one collection target particle into the collection flow path; a second step of restoring the pressure reduced in the first step to the reference pressure; and a third step of reducing an excessive pressure change due to performance of the second step. For example, the pressure in the collection flow path is reduced in the first step, the pressure reduced in the first step is increased in the second step, and then the pressure increased in the second step is reduced again in the third step.

[0111] Note that, in the present specification, the screening operation having the first step, the second step, and the third step is also referred to as a "first screening operation" in order to distinguish the screening operation from other screening operations.

[0112] Further, the screening operation having the first step, the second step, and the third step is preferably performed in a case where an interval between two consecutive flowing collection target particles satisfies a predetermined condition.

[0113] In addition, the collection target particle classified in the one screening operation can be one particle or can be a plurality of particles. That is, the collection target particle refers to one or a plurality of collection target particles.

[0114] In the present specification, the collection target particle can be a biological particle or can be a non-biological particle. As explained below in (7-4), the biological particle can be a cell or a cell-free biological particle, and an example of the non-biological particle can be a bead. Furthermore, the non-biological particle can be a particle configured to be able to collect particles (specifically, biological particles) (hereinafter, also referred to as "particle for collection"). For example, the particle for collection can be a particle that traps a particle on the inside (e.g., inner lumen) of the particle, or can be a particle that traps a particle on the outside (e.g., outer front surface) of the particle. The particle for collection can have a biological component collection unit on the inside or outside thereof. The biological component collection unit can be configured to collect a biological particle and / or a secretion released from a biological particle, and for example, can include a structure that specifically or non-specifically binds to a biological component (e.g., biological component surface marker, etc.) such as an antibody, an enzyme, a protein, a protein fragment, an aptamer, a nucleic acid, or an oligonucleotide. In one embodiment, the collection target particle can be a combination of a non-biological particle and a biological particle collected by the non-biological particle. For example, the collection target particle can be a non-biological particle that has collected a biological particle to be collected inside or outside. For example, the non-biological particle can be a large-diameter particle having a size capable of collecting a biological particle such as a cell inside.

[0115] For example, the size (specifically, the diameter) of the collection target particle can be 1 nm to 1 mm. In several embodiments, for example, the size of the collection target particle can be 50 nm to 500 nm, 50 nm to 300 nm, or 50 nm to 200 nm. The size can apply to a biological particle or can apply to a non-biological particle.

[0116] The sieving operation can be performed by adjusting the pressure in the flow path. The adjustment of the pressure can be performed by deformation of the collection flow path. That is, the biological particle sieving system can be configured to change the pressure in the collection flow path by deforming the collection flow path.

[0117] The biological particle sieving system can be configured to change the pressure in the collection flow path by deforming the collection flow path. Specifically, the collection flow path can be a flow path that collects only the collection target particle from a particle group including the non-collection target particle and the collection target particle.

[0118] In one embodiment, the sieving operation can be performed by an actuator, particularly a piezoelectric actuator, which has been arranged to be able to deform the collection flow path. For example, the actuator can be provided in contact with the front surface of the biological particle sieving chip mentioned later. The collection flow path provided in the chip can be deformed by driving the actuator.

[0119] Further, the deformation of the collection flow path can be performed using pneumatic pressure. For example, the biological particle sieving system can be configured to collect the collection target particles into the collection flow path using pneumatic pressure. For example, the system can include a pneumatic actuator, and the pneumatic actuator can be provided on the chip to perform the deformation of the collection flow path.

[0120] Further, for example, the deformation of the collection flow path can be performed by a device that performs linear motion, particularly a motor such as an electric motor. The biological particle sieving system can have a device provided to perform the deformation of the collection flow path.

[0121] In this way, the biological particle sieving system according to the present disclosure can have a sieving unit including an actuator or a device that deforms the collection flow path as described above.

[0122] The deformation of the collection flow path of the sieving unit can be controlled by the information processing unit. That is, the biological particle sieving system can have an information processing unit configured to control the deformation of the collection flow path. The information processing unit can drive the sieving unit so that a sieving operation according to the present disclosure is performed. Further, the biological particle sieving system can be configured to be able to perform another sieving operation other than the sieving operation having the first step, the second step, and the third step. The biological particle sieving system can be configured to select a sieving operation for collecting the collection target particles into the collection flow path from among a plurality of sieving operations. The information processing unit can make the selection and then drive the sieving unit so that the selected sieving operation is performed.

[0123] In order to drive the sieving unit, preferably, a drive voltage having a specific drive waveform is applied to the sieving unit. Preferably, the drive voltage is a pulse voltage, and the sieving unit can be driven by a voltage having a pulse waveform.

[0124] The drive waveform of the pulse voltage can be configured so that the sieving unit performs the above-described sieving operation. That is, a pulse voltage having a selected drive waveform is applied to the sieving unit to perform each sieving operation, and the sieving operation corresponding to the drive waveform is performed. The selection of the drive waveform and the configuration of the drive waveform are described later in more detail.

[0125] (Advantages)

[0126] By performing the first step and the second step in one sieving operation, particle sieving is possible, and the problem of the discharge flow and / or unnecessary oscillation can occur as described above. By performing the third step in addition to the first step and the second step, the discharge flow can be mitigated. Further, unnecessary oscillation can also be reduced. Thus, even if the length of time between two sieving operations performed to fractionate two consecutive flows of collection target particles is shorter, the target particles can be fractionated stably. That is, faster and more stable particle sieving is achieved. In this way, the present disclosure can achieve efficient sieving.

[0127] In the device, in order to draw the particles from the main flow path into the collection flow path through the connection flow path in a short time, the suction operation and the discharge operation are continuously performed, for example, by driving the actuator. Since a positive pressure is generated in the pressure chamber by the discharge operation, a large discharge flow is generated between the main flow path and the collection flow path (for example, in the connection flow path) after the discharge operation. Further, unnecessary flow oscillation is also generated after the discharge flow. The present disclosure can suppress generation of the positive pressure generated by the discharge operation and suppress the generation of the discharge flow in the connection flow path. Further, generation of unnecessary oscillation can also be reduced.

[0128] Further, in a case where the suction operation of another collection target particle is performed in a state where the discharge flow is being generated after the sieving of a certain collection target particle, the suction amount decreases. Therefore, in order to appropriately sieve the next collection target particle after the sieving operation of a certain collection target particle, it is necessary to wait until the discharge flow decreases. Since the present disclosure can suppress the generation of the discharge flow in the connection flow path after the sieving operation, the length of time until the discharge flow decreases can be made shorter, and thus the interval of the successive sieving can be made shorter. Therefore, the sieving of the collection target particles arriving at a short time interval becomes possible.

[0129] Further, in a case where unnecessary oscillation is generated after the sieving operation, a variation in the suction amount occurs, and a variation in the size of the emulsion is generated. The present disclosure makes it possible to reduce unnecessary flow oscillation and generate a more uniform emulsion.

[0130] By performing the sieving operation having the first step, the second step, and the third step according to the present disclosure, it becomes possible to suppress the discharge flow generated after the sieving operation and make the interval of the successive sieving shorter, and thus it becomes possible to more efficiently sieve the collection target particles.

[0131] The following describes a configuration example of a flow path system that performs a sieving operation, and the present disclosure is described in more detail with reference to the configuration example.

[0132] (2) Configuration Example

[0133] Figure 1 An example of a flow path system of a microfluidic chip that performs a sieving operation according to the present disclosure is depicted. The flow path system depicted in the figure has a sample liquid flow path 152 and a sheath liquid flow path 154, and these flow paths have the sheath liquid flow path 154 that merges at a merging portion 162. The flow path that merges in the merging portion 162 is referred to as a main flow path 155. A liquid including a plurality of particles is caused to flow through the main flow path.

[0134] The flow path system further includes a collection flow path 159 in which a collection target particle among the plurality of microparticles is collected. That is, the collection target particle among the particles flowing through the main flow path is sieved into the collection flow path.

[0135] A liquid including a plurality of microparticles is caused to flow through the main flow path. In the liquid flowing through the main flow path, the microparticles can be adjacent to each other in a row, and the liquid can form a laminar flow. In the middle of the main flow path, a particle sensing unit 156 (also referred to as a "sensing region" in the present specification) is provided, and light is emitted onto the particle sensing unit. Based on signal information of scattered light and / or fluorescent light generated by the light emission, it is evaluated whether the microparticles are collection target particles.

[0136] Figure 2 An enlarged view of the particle sieving unit is depicted. In the particle sieving unit, the main flow path and the collection flow path are connected by a connection flow path 170, and a liquid supply flow path 161 (also referred to as an "introduction flow path" in the present specification) is connected in the up-down direction, and causes fluid to flow out from the liquid supply flow path. As shown in C in Figure 2 The up-down direction flow is merged in the particle sieving unit (specifically, in the connection flow path), and is branched into the upstream direction and the downstream direction flow. The flow in the upstream direction prevents non-collection target microparticles from flowing into the particle sieving unit.

[0137] On the other hand, in the case of the flow of the collection target particles as shown in B in Figure 2 In the collection flow path, in particular, in the pressure chamber (a region whose volume is changed by an actuator, a cross-sectional area of a plane perpendicular to the advancing direction of the liquid is larger than other parts of the collection flow path) provided in the collection flow path, a negative pressure is generated, and the collection target particles are sucked. In order to generate the negative pressure, the actuator installed outside the pressure chamber is driven, and the volume of the pressure chamber is changed. Specifically, the actuator is driven so that the volume of the pressure chamber increases, and the pressure inside the pressure chamber decreases. Due to the pressure drop, the collection target particles are collected into the collection flow path.

[0138] By using the above-described flow path system, it is also possible to form an emulsion having emulsion particles having only the collection target particles. Specifically, by sieving the collection target particles using a liquid (hereinafter, referred to as a "second liquid") that is immiscible with the liquid (hereinafter, referred to as a "first liquid") flowing through the main flow path as the liquid supplied from the liquid supply flow path, an emulsion is formed. This emulsion is an emulsion in which emulsion particles including the first liquid containing the collection target particles are dispersed in the second liquid. For example, the first liquid can be a hydrophilic liquid (water, an aqueous solution, or an aqueous medium), and the second liquid can be a hydrophobic liquid (an oil-based or an oil-based liquid). Conversely, the first liquid can be a hydrophobic liquid (an oil-based or an oil-based liquid), and the second liquid is a hydrophilic liquid (water, an aqueous solution, or an aqueous medium).

[0139] In Figure 3An example of a configuration of an actuator for generating negative pressure in a pressure chamber is described in FIG. 1. This figure depicts an example of a configuration of a microfluidic chip having a flow path system. The example of the configuration is explained in detail later separately. As depicted in the figure, an actuator 107 is arranged in contact with a collection flow path 159 (specifically, a portion where a pressure chamber is located) on a front surface of the microfluidic chip. The actuator can be, for example, a piezoelectric element such as a piezoelectric element. Collection of collection target particles into the collection flow path is performed by the actuator generating negative pressure in the pressure chamber in the collection flow path to suck the collection target particles into the collection flow path. In order to generate negative pressure, for example, a voltage that induces piezoelectric contraction is applied to the piezoelectric element. By applying the voltage, the pressure chamber is deformed in a direction to increase the volume of the pressure chamber, and negative pressure is generated.

[0140] (3) Drive waveform

[0141] Figure 4 An example of a basic drive waveform applied to the collection target particles for sieving is depicted. This figure depicts a graph of a drive voltage V applied to the actuator with respect to time t. As shown in the figure, the drive waveform has a falling edge portion Wf and a rising edge portion Wr. In addition, there can be a voltage holding portion Wm in which the voltage is held between the falling edge portion Wf and the rising edge portion Wr. The falling edge portion Wf corresponds to a suction operation, and is applied in order to deform the pressure chamber (specifically, to expand the volume of the pressure chamber) to generate negative pressure. The rising edge portion Wr corresponding to a discharge operation is applied to return the deformed pressure chamber to its initial state. At this time, since the application of the rising edge portion is accompanied by a decrease in the volume of the pressure chamber, positive pressure is generated.

[0142] Figure 5 A schematic diagram of the states of the suction operation and the discharge operation is depicted. As shown in A in the figure, by applying the falling edge portion in the drive waveform, negative pressure NP is generated in the collection flow path 159, so that the particles P are collected into the collection flow path (specifically, into the pressure chamber). Thereafter, as shown in B in the figure, by applying the rising edge portion in the drive waveform, positive pressure PP is generated in the collection flow path 159. By this, a force that advances the particles P in the direction of the arrow of B in the figure is generated. However, if the particles P flow downstream from the particle entry port 171 to a certain extent, the particles P will not return to the main flow path again through the connection flow path 170.

[0143] This basic drive waveform is referred to as a pulse drive waveform. In the pulse drive waveform, the suction operation and the discharge operation are performed continuously.

[0144] Figure 6I in FIG. 6 depicts a simulation result of the flow rate in the connection flow path in a case where a pulse drive waveform according to a conventional technique is applied to the piezoelectric element. (A I) in this figure depicts a time change in the drive voltage of the piezoelectric element (vertical axis: drive voltage V, horizontal axis: time t), and (B I) in this figure depicts a time change in the flow rate in the connection flow path (vertical axis: flow rate FV, horizontal axis: time t).

[0145] As shown in Figure 7 , the position of the simulated flow rate is a position indicated by an arrow MP in the connection flow path 170 that connects the main flow path 155 and the collection flow path 159. The cross-sectional average flow rate at this position is the flow rate FV described above. This also applies to other flow rate simulation results.

[0146] Regarding the flow rate FV in (B I), the value "0" in the middle means that the flow rate at this position is 0. The flow rate FV means that, as the value moves upward from "0" to "+", the flow from the main flow path to the collection flow path becomes faster. In addition, the flow rate FV means that, as the value moves downward from "0" to "-", the flow in the direction opposite to the flow from the main flow path to the collection flow path (i.e., the flow from the collection flow path to the main flow path) becomes faster.

[0147] Before the drive waveform is applied, the flow from the collection flow path to the main flow path is generated.

[0148] As shown in Figure 6 , before the drive waveform is applied, the flow from the collection flow path to the main flow path is generated. This can prevent unnecessary particles from traveling to the collection flow path.

[0149] Next, as shown in the region to the right of t1 in (A I) and (B I) in FIG. 6, Figure 6 , immediately after the drive waveform is applied, the suction operation caused by the application of the falling edge makes the fluid rapidly accelerate in the direction of the collection flow path, and then the discharge operation caused by the application of the rising edge makes the fluid rapidly decelerate. By applying the drive waveform, such a suction operation is performed in a short time. However, after the discharge operation, as indicated by an arrow X, a significant flow in the discharge direction is generated in the connection flow path. In addition, after the discharge flow, oscillations of the flow rate (also referred to as "unnecessary oscillations") are generated, although their amplitudes are smaller than the flow rate in the region indicated by the arrow X. That is, the flow rate of the connection flow path is unstable.

[0150] Figure 6II in FIG. 1 depicts conditions assuming that the collection target particles arrive continuously at short time intervals, and simulation results of the flow rate in the connecting flow path in the case where two pulse drive waveforms are applied in succession at short time intervals. (Drive waveform: 15 μs - 10 μs - 15 μs, pulse interval: 100 μs)

[0151] As shown in AII and BII in the figure, due to the short pulse drive interval, the drive voltage application of the second drive is performed at the timing at which the discharge current of the first drive is being generated. In this case, as shown by Y in BII, the flow toward the pressure chamber generated by the application of the second driver is weaker than that of the second driver. If the flow toward the pressure chamber is weakened in this way, there can be a case where the suction amount sufficient to suck in the microparticles cannot be ensured, and the sieving of the microparticles can fail. Therefore, in order to appropriately suck down the microparticles after the sieving operation on the microparticles, a waiting time until the exhaust flow in the connecting flow path decreases and the flow stabilizes is required. This can be an obstacle to achieving fast sieving or efficient sieving.

[0152] In addition, if the pulse drive is performed in a state where the above-described exhaust flow is being generated or in a state where unnecessary vibration is being generated in the case where the device is used as a device for generating an emulsion including emulsion particles having microparticles, the suction amounts obtainable during the first drive and the second drive are different, and thus emulsions having different sizes are generated.

[0153] According to the present disclosure, the above-described first sieving operation is performed. Hereinafter, the first sieving operation will be described with reference to Figure 8 The first sieving operation will be described.

[0154] Figure 8 An example of a drive waveform to be applied to the piezoelectric element to perform the first sieving operation is depicted. As shown in the figure, the pulse drive waveform has a first falling edge portion Wf1, a rising edge portion Wr1, and a second falling edge portion Wf2.

[0155] A hold time Wm1 can be present between the first falling edge portion Wf1 and the rising edge portion Wr1. In addition, a hold time Wm2 can also be present between the rising edge portion Wr1 and the second falling edge portion Wf2.

[0156] In this way, the drive waveform for performing the first sieving operation further has the second falling edge portion after the pulse drive waveform described above with reference to Figure 6 The first sieving operation will be described.

[0157] Note that, in the present specification, the drive waveform having the second falling edge in addition to the first falling edge and the rising edge is also referred to as a "pulse drive waveform including a falling edge".

[0158] Figure 9The simulation results of the flow rate in the connection flow path are depicted in order to depict the advantages of the present disclosure. AI in the figure depicts a pulse driving waveform to be applied including a falling edge according to the present disclosure (driving waveform: fall time-hold time-rise time-hold time-fall time: 15 μs-10 μs-15 μs-10 μs-15 μs, amplitude ratio 0.5). The amplitude ratio is the ratio of the amplitude of the second falling edge portion to the amplitude of the first falling edge portion.

[0159] Thus, by adding the suction operation and the discharge operation after the suction operation, as shown in the flow rate simulation results in BI in the figure, the discharge flow generated after the piezoelectric driving can be suppressed. That is, by Figure 6 the flow rate in the region indicated by the arrow X in BI in Figure 9 is significantly reduced in BI in Figure 6 unnecessary flow oscillation in BI in Figure 9 is also significantly reduced in BI in

[0160] Figure 9 II in the figure shows the simulation results of the flow rate in the connection flow path in the case where the pulse driving waveform to be applied including a falling edge according to the present disclosure is applied twice in succession within a short time interval. AII in the figure depicts a pulse driving waveform to be applied including a falling edge according to the present disclosure (driving waveform: 15 μs-10 μs-15 μs-10 μs-15 μs, amplitude ratio 0.5, pulse interval 100 μs). As shown by the arrow Y in BII in the figure, because the pulse driving waveform including a falling edge used in the present disclosure suppresses the discharge flow generated after the driving waveform is applied, even if the pulse driving waveform including a falling edge is applied with a short driving interval, the flow rate change equal to that during the first driving is obtained during the second driving. That is, the suction amount sufficient to suck in the particles can be ensured even during the second driving. Furthermore, because the waiting time for the discharge flow to decrease becomes unnecessary, or because the waiting time for the suppression of the discharge flow can be reduced, compared to the pulse driving waveform without the second falling edge portion, the particles that arrive in succession with a short time interval can be sieved, and efficient particle sieving becomes possible.

[0161] Furthermore, in the case where an emulsion including emulsion particles having fine particles is produced using the first sieving operation, the suction amounts obtained during the first driving and the second driving are equal. Thus, an emulsion of emulsion particles having uniform sizes can be produced.

[0162] As described above, as a one-time sieving operation for sieving and collecting target particles, it becomes possible to perform effective particle sieving by the first sieving operation in which a first step of reducing the pressure in the collection flow path from the reference pressure and guiding one target particle into the collection flow path, a second step of restoring the pressure reduced in the first step to the reference pressure, and a third step of mitigating excessive pressure variation due to the execution of the second step.

[0163] Referring to Figures 31A to 31C The reference pressure and the excessive pressure variation are explained in more detail.

[0164] Figure 31A is a graph schematically depicting the pressure variation in the collection flow path in the case where the basic drive waveform described in the above-described Figure 4 is applied. The horizontal axis of the graph represents time (t), and the vertical axis of the graph represents pressure (p). The pressure before the application is referred to as the reference pressure PS. At time tl, the pressure in the collection flow path suddenly decreases due to the application of the falling edge portion in the drive waveform. Due to this pressure drop, particles are collected into the collection flow path. Thereafter, the pressure in the collection flow path increases toward the original reference pressure PS due to the rising edge portion. However, for the basic drive waveform, the pressure keeps increasing further after the pressure has reached the reference pressure PS. Thereafter, when a predetermined length of time elapses, the pressure in the collection flow path returns to the reference pressure.

[0165] In this way, in the present specification, the pressure variation exceeding the reference pressure PS due to the application of the rising edge portion is referred to as the excessive pressure variation. In the graph, the area EV1 (area exceeding the reference pressure PS) indicated by the gray color corresponds to the excessive pressure variation.

[0166] By executing the third step according to the present disclosure, the excessive pressure variation is mitigated. Referring to Figure 31B the mitigated pressure variation is explained. This graph is a graph schematically depicting the pressure variation in the collection flow path in the case where the pulse drive waveform including the falling edge depicted in the above-described Figure 8 is applied. In this graph, the horizontal axis of the graph represents time (t), and the vertical axis of the graph represents pressure (p). The pressure before the application is referred to as the reference pressure PS. At time tl, the pressure in the collection flow path suddenly decreases due to the application of the falling edge portion in the drive waveform. Due to this pressure drop, particles are collected into the collection flow path. Thereafter, the pressure in the collection flow path increases toward the original reference pressure PS due to the rising edge portion. According to the present disclosure, the falling edge portion is applied in the third step. In this case, the area EV2 (area exceeding the reference pressure PS) indicated by the gray color in Figure 31A is smaller than EV1. In this way, by executing the third step, it is possible to suppress the excessive pressure variation.

[0167] Note that ideally, it is desirable that in applications such as those Figure 31C After the rising edge portion indicated by the dashed line in EV 0, the pressure change as a result of particle screening does not produce excessive pressure changes. According to the present disclosure, excessive pressure changes can be suppressed and the pressure change in the collection flow path can be made more similar to the ideal pressure change.

[0168] In the present disclosure, the third step is performed so that, for example, the ratio (area value of area EV2) / (area value of area EV1) becomes equal to or lower than 1.0, particularly equal to or lower than 0.9, equal to or lower than 0.8, equal to or lower than 0.7, equal to or lower than 0.6, and even more preferably equal to or lower than 0.5. The area value of area EV1 and the area value of area EV2 are both obtained by integrating "(pressure value that has changed by more than the reference pressure due to application of the rising edge portion) - (reference pressure value)" over the time axis. That is, the third step can be performed so that the overpressure change in the presence of the third step becomes smaller than the overpressure change in the absence of the third step.

[0169] For example, those skilled in the art can appropriately select the waveform applied in the third step based on the first and second steps. In addition, the waveform can also be appropriately changed according to the flow path cross-sectional area or the configuration of the flow path system.

[0170] As described above, in the present disclosure, the biological particle fractionation system can be configured to apply a pulse voltage to the actuator to change the pressure in the collection flow path during a fractionation operation. The driving waveform of the pulse voltage can be configured to cause the actuator to perform the first step, the second step, and the third step.

[0171] The driving waveform of the pulse voltage may have a first falling edge portion configured to perform the first step, a rising edge portion configured to perform the second step, and a second falling edge portion configured to perform the third step.

[0172] In the present disclosure, for example, the amplitude of the first falling edge portion for performing the first step may be equal to or greater than 1 V, preferably equal to or greater than 10 V, and more preferably equal to or greater than 30 V. Furthermore, for example, the amplitude of the first falling edge portion may be equal to or less than 150 V, preferably equal to or less than 100 V, and more preferably equal to or less than 50 V.

[0173] For example, the duration of the first falling edge portion may be equal to or greater than 1 μs, preferably equal to or greater than 5 μs, and more preferably equal to or greater than 15 μs. In addition, for example, the duration of the first falling edge portion may be equal to or shorter than 100 μs, preferably equal to or shorter than 60 μs, and more preferably equal to or shorter than 30 μs.

[0174] For example, the amplitude of the rising portion for performing the second step can be equal to or greater than 1 V, preferably equal to or greater than 10 V, and more preferably equal to or greater than 30 V. In addition, for example, the amplitude of the rising portion can be equal to or less than 150 V, preferably equal to or less than 100 V, and more preferably equal to or less than 50 V.

[0175] For example, the duration of the rising portion can be equal to or greater than 1 μs, preferably equal to or greater than 5 μs, and more preferably equal to or greater than 15 μs. In addition, for example, the duration of the rising portion can be equal to or less than 100 μs, preferably equal to or less than 60 μs, and more preferably equal to or less than 30 μs.

[0176] For example, the amplitude of the second falling portion for performing the third step can be equal to or greater than 0.1 V, preferably equal to or greater than 1 V, and more preferably equal to or greater than 5 V. In addition, for example, the amplitude of the second falling portion can be equal to or less than 150 V, preferably equal to or less than 100 V, and more preferably equal to or less than 50 V.

[0177] For example, the duration of the second falling portion can be equal to or greater than 1 μs, preferably equal to or greater than 5 μs, and more preferably equal to or greater than 15 μs. In addition, for example, the duration of the second falling portion can be equal to or less than 100 μs, preferably equal to or less than 60 μs, and more preferably equal to or less than 30 μs.

[0178] For example, the hold time between the first falling portion and the rising portion can be equal to or greater than 0 μs. In addition, for example, the hold time can be equal to or less than 150 μs, preferably equal to or less than 50 μs, and more preferably equal to or less than 10 μs. That is, the hold time can be 0 μs, but can be longer than 0 μs.

[0179] For example, the hold time between the rising portion and the second falling portion can be equal to or greater than 0 μs. In addition, for example, the hold time can be equal to or less than 50 μs, preferably equal to or less than 20 μs, and more preferably equal to or less than 10 μs. That is, the hold time can be 0 μs, but can be longer than 0 μs.

[0180] (4) Generation of emulsion particles

[0181] Figure 10Results of experiments on emulsion particle generation are shown in the case where the pulse drive waveform having a falling edge portion and a rising edge portion, but not having a second falling edge portion, is continuously driven twice at an interval of 160-μs or at an interval of 100-μs. In the case where the waveform is continuously driven at an interval of 160-μs, two emulsions are generated with each continuous drive (indicated by two arrows in A in the figure). In contrast, in the case where the waveform is continuously driven at an interval of 100-μs, only one emulsion is generated with each continuous drive (indicated by one arrow in B in the figure). This is because, in the case where the waveform is driven at a short interval of 100-μs, the amount of suction sufficient to generate an emulsion cannot be achieved with the second pulse due to the discharge flow generated after the discharge operation.

[0182] Figure 11 Results of experiments in the case where the pulse drive waveform including a falling edge used in the present disclosure is continuously driven twice at an interval of 160-μs or at an interval of 100-μs are depicted. As shown in the figure, in the case where the pulse drive waveform including a falling edge is used, two emulsions are generated not only in the case where the waveform is continuously driven at an interval of 160-μs, but also in the case where the waveform is continuously driven at an interval of 100-μs (indicated by two arrows in each of A and B in the figure). From these results, it can be known that the present disclosure is also effective for generating emulsions.

[0183] That is, the biological particle screening system of the present disclosure can be used as an emulsion production apparatus. The emulsion production apparatus can produce an emulsion having a fractionated collection target particle in emulsion particles.

[0184] (5) Usefulness of pulse waveform

[0185] Figure 12 Results of simulation of flow rate in a connection flow path in the case where the pulse drive waveform including a falling edge according to the present disclosure and waveforms other than the pulse drive waveform (including a staircase waveform and a staircase waveform including a dip) are used are shown.

[0186] A in the figure depicts the applied drive waveform. a in the figure indicates a pulse waveform including a falling edge, b in the figure indicates a staircase waveform, and c in the figure indicates a staircase waveform including a dip.

[0187] B in the figure indicates results of simulation of flow rate in a connection flow path when the three waveforms are applied. Similar to A in the figure, a indicates results of simulation of a pulse waveform including a falling edge, b indicates results of simulation of a staircase waveform, and c indicates results of simulation of a staircase waveform including a dip.

[0188] As shown by these simulation results, when a step waveform and a step waveform including an undershoot are applied, the duration that the flow velocity in the connecting flow path points in the suction direction is long, and the flow in the discharge direction and unnecessary flow oscillations are also significant. In contrast, when a pulse drive waveform including a falling edge is applied, the duration that the flow velocity in the connecting flow path points in the suction direction is short, and the discharge flow and unnecessary flow oscillations are also small. Therefore, even when compared with the case of applying a step waveform and a step waveform including an undershoot as the piezoelectric drive waveform, the pulse drive waveform including a falling edge achieves suction with the shortest duration and can suppress flow in the discharge direction and unnecessary flow oscillations. Therefore, it can be said that the pulse drive waveform including a falling edge is the most effective drive waveform.

[0189] (6) Relationship with flow path cross-sectional area

[0190] A simulation similar to the simulation described in (3) was performed except that the flow path diameter of the connecting flow path was doubled (the cross-sectional area was doubled). Figure 13 The simulation results depict the flow velocity in the connecting flow path.

[0191] As shown in I in the figure, in the case where the pulse drive waveform of the conventional technology is applied, more significant unnecessary flow oscillation is generated in the connecting flow path due to the increase in the flow path diameter of the connecting flow path.

[0192] In contrast, II in the figure describes the simulation results when the pulse drive waveform including the falling edge according to the present disclosure is applied. This simulation result corresponds to the case where the amplitude ratio kw between the drive amplitude of the second last falling edge portion of the drive waveform and the drive amplitude of the first falling edge portion and the rising edge portion before the second last falling edge portion of the drive waveform is set to 0.5 as in (3) above, and the case where the amplitude ratio kw is set to 1.0.

[0193] When the amplitude ratio kw=0.5, unnecessary oscillation is reduced compared to the case of using a conventional pulse drive waveform as compared to I in the figure. That is, the usefulness of the present invention is shown with various flow path diameters (flow path cross-sectional areas).

[0194] Furthermore, when the amplitude ratio kw = 1.0, which increases the drive amplitude of the second falling edge portion, unnecessary flow oscillations are further reduced compared to the case of an amplitude ratio kw = 0.5. If the flow path diameter of the connecting flow path increases, significant unnecessary oscillations occur, but by changing the amplitude ratio, the advantage of suppressing unnecessary flow oscillations can be improved. Furthermore, even if the flow path diameter of the connecting flow path varies between individual components or a large number of microchips, the advantage of suppressing unnecessary oscillations can be uniformly achieved by adjusting the amplitude ratio between the amplitudes of the first falling edge portion and the rising edge portion and the amplitude of the second falling edge portion.

[0195] In the present disclosure, in order to properly obtain the advantage of the third step, for example, the amplitude ratio can be equal to or greater than 0.01, preferably equal to or greater than 0.05, or more preferably equal to or greater than 0.1, and in particular can be equal to or greater than 0.2, equal to or greater than 0.3, equal to or greater than 0.4, or equal to or greater than 0.5. In addition, for example, the amplitude ratio can be equal to or less than 1.2, equal to or less than 1.1, or equal to or less than 1.0.

[0196] (7) Sieving process example 1

[0197] As described above, the biological particle sieving system according to the present disclosure is configured to be able to perform, as one sieving operation for sieving and collecting a target particle, a first step of reducing the pressure in a collection flow path from a reference pressure and guiding one collection target particle into the collection flow path, a second step of restoring the pressure reduced in the first step to the reference pressure, and a third step of mitigating excessive pressure change due to the performance of the second step. The biological particle sieving system can be configured to perform a sieving operation (also referred to as "first sieving operation") having the first step, the second step, and the third step in a case where a predetermined condition is satisfied. In addition, another sieving operation can be performed in a case where the predetermined condition is not satisfied (or in a case where another predetermined condition is satisfied). The predetermined condition can be a condition related to the relationship (specifically, the temporal relationship or the positional relationship) of the collection target particle to the subsequent biological particle that flows through the collection target particle.

[0198] In one embodiment, in addition to the first sieving operation, the biological particle sieving system can be configured to be able to perform, as the one sieving operation, a second sieving operation to perform the first step and the second step, and to perform a fourth step to further perform, in a case where a predetermined condition is satisfied, cancellation of the offset of the collection flow path generated by the performance of the third step. In this embodiment, depending on the interval (for example, the temporal interval or the positional interval) between the collection target particle and the subsequent biological particle (specifically, the collection target particle), which one of the two sieving operations to be applied can be selected. For example, the interval can be the aforementioned temporal interval ΔTp, can be the interval between the times at which the two particles are sensed, can be the interval between the times at which the two particles reach a certain position (the position at which the two particles are fractionated), or can be the interval (that is, the distance between the positions of the two particles). Depending on such an interval, the sieving operation (specifically, the drive waveform) for the sieving of the collection target particle can be changed.

[0199] In this way, with the configuration capable of performing the second sieving operation in addition to the first sieving operation, the offset of the drive output can be restored to its aforementioned original state. This embodiment, which also includes an algorithm for implementing the sieving method according to the present disclosure, is described in more detail below.

[0200] For example, the biological particle sieving system can determine whether to apply the first sieving operation based on a time interval ΔTp between the collection target particle and the subsequent biological particle (specifically, the collection target particle). That is, the biological particle sieving system can select the first sieving operation or the second sieving operation as the sieving operation for the collection target particle according to the time interval ΔTp.

[0201] Here, the time interval ΔTp can be a time interval between a time point at which a certain collection target particle arrives at the sieving unit (specifically, a position immediately before the connection flow path) and a time point at which a subsequent collection target particle flows immediately after the certain collection target particle arrives at the sieving unit (specifically, a position immediately before the connection flow path).

[0202] Alternatively, the time interval ΔTp can be a time interval between a time point at which a certain collection target particle is sensed and a time point at which a subsequent collection target particle is sensed.

[0203] For example, in a case where the time interval ΔTp is short, the biological particle sieving system can perform the first sieving operation. For example, in order to perform the first sieving operation, as described above with reference to Figure 8 For example, in a case where the time interval ΔTp is short, the biological particle sieving system can perform the first sieving operation. For example, in order to perform the first sieving operation, as described above with reference to

[0204] Further, in a case where the time interval ΔTp is long, the second sieving operation can be performed. In order to perform the second sieving operation, for example, as Figure 14 depicted in FIG. 11, a pulse driving waveform including a rising edge having a second rising edge portion Wr2 in addition to one falling edge portion Wf1 and one rising edge portion Wr1 can be used. In the present specification, a pulse driving waveform further having a second rising edge portion in addition to one falling edge portion and one rising edge portion described in FIG. 10 is also referred to as a "pulse driving waveform including a rising edge". Figure 14 depicted in FIG. 11, a pulse driving waveform including a rising edge having a second rising edge portion Wr2 in addition to one falling edge portion Wf1 and one rising edge portion Wr1 can be used. In the present specification, a pulse driving waveform further having a second rising edge portion in addition to one falling edge portion and one rising edge portion described in FIG. 10 is also referred to as a "pulse driving waveform including a rising edge".

[0205] The following describes advantages of using the two types of sieving operations in this way.

[0206] The continuous sieving interval can be made shorter by the first sieving operation, but if the actuator is driven once by the pulse driving waveform including a falling edge, a shift occurs in the driving output. Therefore, the number of times the actuator can be continuously driven by the pulse driving waveform including a falling edge is limited to a predetermined number.

[0207] On the contrary, since the two discharging operations are successively performed in the second sieving operation, the shift of the drive output can be restored to its initial state, but the interval at which the successive sieving can be performed becomes longer. Based on this, by using the second sieving operation in a case where the time interval ΔTp between the two collection target particles is long, the shift of the drive output generated in the second sieving operation can be restored to its initial state while the collection target particles are appropriately fractionally collected.

[0208] In this way, the drive waveform for performing the second sieving operation can have a first falling edge portion configured to perform the first step and a rising edge portion configured to perform the second step, and can further have a second rising edge portion configured to perform the fourth step in a case where the fourth step is performed.

[0209] The biological particle sieving system can be configured to increase the pressure by one level in the fourth step, and the second rising edge portion for performing the fourth step can have a single-step rising edge. It should be noted that the second rising edge portion can have a multi-step rising edge as described later.

[0210] For example, as shown in FIG. 8, which of the two types of sieving operations to apply can be determined by comparing the time interval ΔTp with a predetermined threshold. Figure 15

[0211] Twf and Twr shown in the figure are as follows, and these can be used as the predetermined threshold.

[0212] Twf: minimum time interval for performing the first sieving operation (minimum time interval for driving the actuator with a pulse drive waveform including a falling edge)

[0213] Twr: minimum time interval for performing the second sieving operation (minimum time interval for driving the actuator with a pulse drive waveform including a rising edge)

[0214] The drive waveform is selected based on the following conditions using these thresholds.

[0215] (a) ΔTp < Twf: The subsequent collection target particle is aborted because the particle interval is too short. Then, based on the time interval between the second subsequent collection target particle and the collection target particle that flows immediately after the subsequent collection target particle, the sieving operation (specifically, the drive waveform) for sieving the collection target particle is determined.

[0216] (b) Twf ≤ ΔTp < Twr: The collection target particle is sieved by the first sieving operation (specifically, sieved with a pulse drive waveform including a falling edge).

[0217] (c) Twr ≤ ΔTp: The collection target particle is sieved by the second sieving operation (specifically, sieved with a pulse drive waveform including a rising edge).​

[0218] Figure 15 Two cases I and II are depicted.

[0219] In case I, events E1 and E2 indicate that light generated by light emission onto the collected target particle is sensed. The position indicated by the middle dotted line of each event is the event sensing time of the event. The time interval ΔTp between the time of sensing event E1 and the time of sensing event E2 is equal to or greater than Twf and shorter than Twr. That is, ΔTp satisfies the condition of (b) described above. Therefore, the first screening operation is selected as the screening operation for screening the collected target particle corresponding to event E1. Then, the collected target particle is collected by the selected first screening operation.

[0220] As for case II, the time interval ΔTp between the time of sensing event E1' and the time of sensing event E2' is equal to or greater than Twf and equal to or greater than Twr. That is, ΔTp satisfies the condition of (c) described above. Therefore, the second screening operation is selected as the screening operation for screening the collected target particle corresponding to event E1'. Then, the collected target particle is collected by the selected second screening operation.

[0221] In this way, the drive waveform applied to screen the collected target particle is selected in accordance with which of the time intervals ΔTp of (a), (b), and (c) corresponds.

[0222] Note that, in case of (a) ΔTp < Twf, it is decided to suspend the subsequent collected target particle (i.e., not to screen the subsequent collected target particle), and the drive waveform to be applied is selected based on the time interval between the second subsequent collected target particle flowing further beside the collected target particle and the collected target particle.

[0223] Further, although in the example described in Figure 15 the time interval ΔTp is the time interval between the times at which two collected target particles are sensed, the time interval ΔTp can be the time interval between the times at which two collected target particles arrive at the particle screening unit (specifically, the position immediately before the connecting flow path).

[0224] For example, these decisions can be executed by an information processing unit of the biological particle screening system. That is, the biological particle screening system can have an information processing unit that performs a decision process based on the interval between the collected target particle and the subsequent biological particle. The decision process can be a process of deciding which screening operation to apply to screen one collected target particle based on whether the interval is within a predetermined numerical range. The configuration of the information processing unit is explained separately in detail.

[0225] Further, the shift of the driving output generated for each driving of the pulse driving waveform including the falling edge can be managed using a step counter. For example, each time the pulse driving waveform including the falling edge is applied, the step counter updates its value (+1). Further, each time the pulse driving waveform including the rising edge is applied, the step counter updates its step counter value by (-1). The step count value in a state where there is no shift of the driving output is defined as 0 (reference value), the step counter value in a state where there is the maximum shift of the driving output is defined as MAX, and the driving waveform is controlled so that the value of the step counter is within a range from 0 to MAX (maximum integer value). That is, the step counter value takes any integer within a range from 0 to MAX.

[0226] For example, in the process of deciding the sieving operation, the information processing unit can refer to the step counter value related to the number of times the sieving operation can be performed. The sieving operation to be applied can be selected based on the step counter value.

[0227] That is, the information processing unit can be configured to select the sieving operation to be applied to the sieving of the collection target particle based on the step counter value and the interval between the collection target particle and the subsequent biological particle.

[0228] The following describes an example of the configuration of the driving waveform for performing the first step, the second step, and the fourth step of the second sieving operation.

[0229] First, the numerical range of the amplitude, the duration of the first falling edge portion for performing the first step and the first rising edge portion for performing the second step, and the hold time can be as described above with respect to the first sieving operation (pulse driving waveform including the falling edge), and the description is also applicable to the second sieving operation.

[0230] Further, for example, the amplitude for the second rising edge portion for performing the fourth step can be equal to or greater than 0.1 V, preferably equal to or greater than 1 V, and more preferably equal to or greater than 5 V. Further, for example, the amplitude of the second rising edge portion can be equal to or less than 150 V, preferably equal to or less than 100 V, and more preferably equal to or less than 50 V.

[0231] For example, the duration of the second rising edge portion can be equal to or longer than 1 μs, preferably equal to or longer than 5 μs, and more preferably equal to or longer than 15 μs. Further, for example, the duration of the second rising edge portion can be equal to or less than 100 μs, preferably equal to or less than 60 μs, and more preferably equal to or less than 30 μs.

[0232] For example, the hold time between the first rising edge portion and the second rising edge portion can be equal to or greater than 0 ps. Also, for example, the hold time can be equal to or less than 100 ps, preferably equal to or less than 50 ps, and more preferably equal to or less than 30 ps.

[0233] (7-1) Example 1 of a processing program executed by a biological particle sorting system

[0234] Referring to Figure 16 and Figure 17 An example of a program of a method executed by a biological particle sorting system according to the present disclosure is described. The former is an example of a flowchart of a sorting processing program executed for each of a plurality of particles flowing through a flow path. The latter is an example of a detailed flowchart of a waveform selection processing in the flowchart.

[0235] As Figure 16 indicated, the biological particle sorting system can be configured to execute an event data acquisition processing S11, a time of arrival calculation processing S12, and a determination processing S13 based on the gate. These processings can be executed for each biological particle flowing in the flow path.

[0236] In step S11, the biological particle sorting system executes an event data acquisition processing. To acquire the event data, the biological particle sorting system acquires data related to light emitted by light emission onto the biological particle flowing through the flow path. For example, the light emission and the light sensing for acquiring the data related to the light can be executed by the light emitting unit and the sensing unit, respectively, which are separately described in the present specification. The data related to the light is transmitted from the sensing unit to the information processing unit mentioned later. In this way, the information processing unit acquires the event data of each biological particle.

[0237] In step S12, the biological particle sorting system (specifically, the information processing unit) calculates the time at which the biological particle arrives at a position immediately before the connection flow path. By performing a sorting operation when the biological particle arrives at the position, the biological particle is collected into the collection flow path. For example, the time can be calculated based on light sensing time data included in the event data.

[0238] The light sensing time data can be the time at which the light is sensed in step S11. The time can be identified based on a technique known to those skilled in the art. For example, the time can be determined based on the distance between the position at which the light emission is performed and the position immediately before the connection flow path and the speed of the biological particle. For example, the distance can be identified in advance based on the structure of the flow path or the structure of the chip. Also, for example, the speed of the biological particle is the speed of the biological particle flowing in the main flow path. For example, the speed can be identified by emitting light onto the biological particle at two different positions. That is, the speed can be determined based on the distance between the two positions and the time between sensing the light generated by the light emission at the two positions.

[0239] In step S13, the biological particle screening system (specifically, the information processing device) performs a determination process based on the gate. In the determination process, the biological particle screening system determines whether the biological particle belongs to the preset gate. More specifically, based on the event data (specifically, based on data related to light sensed by light emitted onto the biological particle), it is determined whether the biological particle belongs to the gate. The gate can be appropriately set by those skilled in the art depending on the type of biological particle that should be screened.

[0240] Step S12 and step S13 can be executed in parallel, or step S13 can be executed after step S12 is executed.

[0241] In step S14, the biological particle screening system (specifically, the information processing unit) determines whether the biological particle is a collection target particle. In the case where it is determined that the biological particle belongs to the gate, the biological particle screening system can determine that the biological particle is a collection target particle.

[0242] Here, in some cases, non-collection target particles exist before or after the biological particle, and in some cases, by performing the screening operation, non-collection target particles are collected into the collection flow path in addition to the biological particle. Therefore, in addition to whether the biological particle belongs to or does not belong to the gate, the biological particle screening system can also determine whether the biological particle is a collection target particle based on whether or not non-collection target particles exist. In the case where non-collection target particles are not allowed to be collected into the collection flow path, a purity priority mode can be employed. In the case where collection of non-collection target particles into the collection flow path is allowed, for example, a throughput priority mode can be employed. In the case where emulsion particles are fractionated, in the case where only emulsion particles having only one collection target particle in the emulsion are collected, a single particle priority mode can be employed. These modes are explained in more detail below.

[0243] In the purity priority mode, the biological particle screening system (specifically, the information processing unit) determines that the biological particle is a collection target particle in the case where it has been determined that the biological particle belongs to the gate and there are no non-collection target particles before and after the biological particle.

[0244] Further, in the case where it has been determined that the biological particle belongs to the gate and there are collection target particles before or after the biological particle, the biological particle screening system determines that the biological particle is a collection target particle.

[0245] In addition, the biological particle screening system determines that the biological particle is not a collection target particle in the case where it has been determined that the biological particle belongs to the gate but there are non-collection target particles before or after the biological particle.

[0246] Further, the biological particle screening system determines that the biological particle is not the collection target particle, in a case where it has been determined that the biological particle does not belong to the gate, independently of whether or not there is a non-collection target particle before or after the biological particle.

[0247] In the acquisition rate priority mode, the biological particle screening separation system (specifically, the information processing unit) determines that the biological particle is the collection target particle, in a case where it has been determined that the biological particle belongs to the gate and there is no non-collection target particle before and after the biological particle.

[0248] Further, the biological particle screening system determines that the biological particle is the collection target particle, in a case where it has been determined that the biological particle belongs to the gate but there is a non-collection target particle before or after the biological particle.

[0249] Further, the biological particle screening system determines that the biological particle is not the collection target particle, in a case where it has been determined that the biological particle does not belong to the gate, independently of whether or not there is a non-collection target particle before or after the biological particle.

[0250] In the single particle priority mode, the biological particle screening system (specifically, the information processing unit) determines that the biological particle is the collection target particle, in a case where it has been determined that the biological particle belongs to the gate and there is no particle before or after the biological particle.

[0251] Further, the biological particle screening system determines that the biological particle is not the collection target particle, in a case where it has been determined that the biological particle belongs to the gate but there is a particle before or after the biological particle. That is, the biological particle screening system determines that the biological particle is not the collection target particle, in a case where it has been determined that the biological particle belongs to the gate but there is a collection target particle before or after the biological particle, unlike in the case of the purity priority mode.

[0252] Further, the biological particle screening system determines that the biological particle is not the collection target particle, in a case where it has been determined that the biological particle does not belong to the gate, independently of whether or not there is a particle before or after the biological particle.

[0253] In the purity priority mode described above, in a case where one collection target particle and a subsequent collection target particle flowing after the one collection target particle approach each other, it is decided to screen the one collection target particle. That is, in some cases, in one screening operation for screening one collection target particle, a subsequent collection target particle is also screened together with the one collection target particle. Thereby, the purity of the object particle can be improved. In contrast, in the single particle priority mode, in a case where one collection target particle and a subsequent particle flowing after the one collection target particle approach each other, it is decided not to screen the one collection target particle, independently of whether the subsequent particle is a collection target particle or a non-collection target particle. Thereby, in one screening operation, an emulsion particle including only one collection target particle is formed, and formation of an emulsion particle including two particles can be prevented.

[0254] In step S15, the biological particle screening system (specifically, the information processing apparatus) selects a drive waveform to be applied to the particle determined as a collection target particle in step S14. The drive waveform selection processing is performed using the time interval ΔTp calculated in step S12 with respect to the arrival time of the collection target particle and the arrival time of the collection target particle (also referred to as "subsequent collection target particle") flowing immediately after the collection target particle calculated in step S12. The drive waveform selection processing will be described later with reference to Figure 17 A more specific example of the selection processing will be described.

[0255] In step S16, the biological particle screening system performs a screening operation using the drive waveform selected in step S15. By the screening operation, the collection target particle is collected into the collection flow path. The screening operation can be performed by a screening unit to be described later. For example, the screening unit can be implemented using an actuator, more specifically a piezoelectric element, which is provided to be able to change the volume in the collection flow path.

[0256] (7-2) Waveform selection processing example

[0257] The following will be described with reference to Figure 17 A more specific example of the waveform selection processing in step S15 described above will be described. The biological particle screening system (specifically, the information processing unit included in the system) can be configured to perform each step in the waveform selection processing.

[0258] In step S20, the information processing unit starts the drive waveform selection processing.

[0259] In step S21, the information processing unit calculates a time interval ΔTp between a time at which the collection target particle reaches a position immediately before the connecting flow path and a time at which the subsequent collection target particle flowing immediately after the collection target particle reaches the position immediately before the connecting flow path. Both of the times of arrival can be the times calculated in step S12 described above. The biological particle sorting system can utilize a difference between the two times of arrival as the time interval ΔTp.

[0260] In step S22, the information processing unit can compare the time interval ΔTp calculated in step S21 with a predetermined threshold value (also referred to as a "first threshold value" in the present specification). The prescribed first threshold value can also be the threshold value Twf described above. The first threshold value can be preset by a person skilled in the art, and can be preset, for example, based on a region of the liquid collected into the collection flow path in the case where the pulse driving waveform to be applied is applied.

[0261] In step S22, in the case where the time interval ΔTp is equal to or greater than the first threshold value (for example, in the case where Twf ≤ ΔTp), the information processing unit advances the process to step S23.

[0262] In step S22, in the case where the time interval ΔTp is not equal to or greater than the first threshold value (for example, in the case where ΔTp < Twf), the information processing unit advances the process to step S29.

[0263] In step S23, the information processing unit can compare the time interval ΔTp and a predetermined threshold value (also referred to as a "second threshold value" in the present specification). The prescribed second threshold value can be the threshold value Twr described above. The second threshold value can be preset by a person skilled in the art, and can be preset, for example, based on a time until an ejection flow generated in the case where the sorting operation is performed disappears, a time until an amplitude of unnecessary oscillation generated in the case where the sorting operation is performed becomes equal to or smaller than a predetermined value, or the like.

[0264] In step S23, in the case where the time interval ΔTp is shorter than the second threshold value (for example, in the case where ΔTp < Twr), the information processing unit advances the process to step S24.

[0265] In step S23, in the case where the time interval ΔTp is not smaller than the second threshold value (for example, in the case where Twr ≤ ΔTp), the information processing unit advances the process to step S27.

[0266] In step S24, the information processing unit determines whether the step counter value is smaller than the maximum value. The step counter value can be a value of the step counter related to the number of times of execution of the first sieving operation. For example, the step counter value can take any integer value, and in a case where the value has reached a predetermined value, this can mean that the first sieving operation cannot be executed. For example, in a case where the step counter value is the maximum value, this can mean that the pressure in the collection flow path cannot be further reduced.

[0267] In step S24, in a case where the step counter value is smaller than the maximum value, the information processing unit advances the process to step S25. In a case where the step counter value is smaller than the maximum value, the first sieving operation can be executed, and by executing the first sieving operation, more efficient sieving becomes possible.

[0268] In step S24, in a case where the step counter value is not smaller than the maximum value (for example, in a case where the step counter value is the maximum value), the information processing unit advances the process to step S26.

[0269] In step S25, the information processing unit selects a pulse drive waveform for executing the first sieving operation. Specifically, a pulse drive waveform including a falling edge is selected.

[0270] In step S25, because there is a subsequent collection target particle in a position relatively close to the position of the collection target particle (for example, a position satisfying Twf≤ΔTp

[0271] For example, in step S25, the timing of the pulse drive waveform (specifically, the pulse drive waveform including a falling edge) to which the first sieving operation is applied can be written to the memory.

[0272] Further, after the waveform selection of step S25 (for example, after executing the sieving operation using the selected pulse drive waveform), the biological particle sieving system can execute a process of incrementing the step counter value. Because the number of times of continuous execution of the first sieving operation is limited to a predetermined number of times by the offset as described above, the number of times of continuous execution of the first sieving operation can be managed by increasing the step counter value, and the subsequent sieving operation can be appropriately selected.

[0273] In step S26, the information processing unit suspends the subsequent collection target particle. In step S36, since the step counter value is the maximum value, the first sieving operation cannot be performed. Therefore, the subsequent collection target particle is not regarded as a collection target. Then, the biological particle sieving system performs the waveform selection process based on the relationship (specifically, the time interval ΔTp) between the second subsequent collection target particle flowing immediately after the subsequent collection target particle and the collection target particle.

[0274] In step S27, the information processing unit determines whether the step counter value is greater than 0. The step counter value can be the value of the step counter referred to in step S24.

[0275] In step S27, in a case where the step counter value is greater than 0 (for example, in a case where the step counter value is any integer greater than 0 and equal to or less than the maximum value), the information processing unit advances the process to step S28. The step counter value greater than 0 means that the above-described offset has been generated. In view of this, the offset can be eliminated by advancing the process to step S28 and performing the second sieving operation. Therefore, the number of times the first sieving operation can be continuously performed can be increased. This contributes to efficient sieving of particles.

[0276] In step S27, in a case where the step counter value is not greater than 0 (for example, in a case where the step counter value is 0), the information processing unit advances the process to step S25. The step counter value being 0 means that the above-described offset has not been generated. Therefore, it is not necessary to perform the second sieving operation.

[0277] In step S28, the information processing unit selects a pulse drive waveform for performing the second sieving operation.

[0278] In step S28, since there is a subsequent collection target particle at a position relatively far from the collection target particle (for example, a position satisfying Twr≤ ΔTp), even if the second sieving operation is performed, the sieving of the subsequent collection target particle is not affected by the exhaust flow and / or unnecessary oscillation, or, even if affected, the effect is within an allowable range for stable sieving. Furthermore, the above-described offset can be eliminated by performing the second sieving operation. Therefore, the number of times the first sieving operation can be continuously performed can be increased. This contributes to efficient sieving of particles.

[0279] For example, in step S28, the timing of the pulse drive waveform (specifically, the pulse drive waveform including a rising edge) to which the second sieving operation is applied can be written to the memory.

[0280] Further, after the waveform selection in step S28 (for example, after performing the sieving operation using the selected pulse drive waveform), the information processing unit can perform a process of decreasing the step counter value. Because the number of times of continuously performing the first sieving operation is limited to a predetermined number of times by the offset as described above, the number of times of continuously performing the first sieving operation has increased by decreasing the step counter value, and the subsequent sieving operation can be appropriately selected.

[0281] In step S29, the information processing unit suspends the subsequent collection target particle. The suspension can mean that the decision that the subsequent collection target particle has been decided as a collection target is changed to the decision that the subsequent collection target particle is a non-collection target. That is, the suspension makes the subsequent collection target particle a non-collection target.

[0282] In step S22 before step S29, it is determined that the interval between the subsequent collection target particle and the collection target particle is too short (ATp < Twf), and if the sieving operation of the subsequent collection target particle as a collection target is performed in this case, the subsequent collection target particle cannot be appropriately fractionated. Further, if the sieving operation for producing emulsion particles is performed in this case, the size of the produced emulsion particles becomes significantly different from those of other emulsion particles with high probability. Therefore, by the suspension process, the biological particle sieving separation can be stably performed.

[0283] In the case where the suspension process is performed, the information processing unit returns the process to step S20, and can perform the waveform selection process illustrated in the figure based on the interval between the collection target particle and the second subsequent collection target particle that flows immediately after the subsequent collection target particle. Thereby, efficient particle sieving becomes possible.

[0284] By the waveform selection process as described above, an appropriate drive waveform can be selected according to the interval between the collection target particle and the subsequent collection target particle. Further, the first sieving operation can be performed at an appropriate timing, and this contributes to achieving efficient particle sieving. Further, the second sieving operation can be performed at an appropriate timing, and the pressure in the collection flow path can be appropriately controlled.

[0285] In this way, the biological particle sieving system according to the present disclosure can be configured to be able to perform the first sieving operation or the second sieving operation as a one-time sieving operation. Further, the biological particle sieving system can have an information processing unit that performs a decision process based on the interval between one collection target particle and a subsequent biological particle, and the information processing unit can select the first sieving operation or the second sieving operation based on the interval.

[0286] In one embodiment, the biological particle sieving system can be configured to apply a pulsed voltage to the actuator to change the pressure in the collection flow path. Then, the drive waveform of the pulsed voltage can be configured such that the actuator performs the first sieving operation or the second sieving operation according to the interval.

[0287] (7-3) Example of a timing chart

[0288] Referring to Figure 18 An example of a timing chart that explains the execution of the above-described waveform selection process is described. This chart is an example of a timing chart, and this timing chart depicts events E11 to E19 in which a collection target particle is sensed. A in the chart indicates an event sensing time, B in the chart indicates an application timing of a pulse drive waveform including a falling edge, C in the chart indicates an application timing of a pulse drive waveform including a rising edge, and D indicates a drive signal.

[0289] The waveform selection process performed for each event is described below.

[0290] With respect to the collection target particle of event E11, the information processing unit calculates ΔTp at step S21. More specifically, the information processing unit calculates the time interval ΔTp between the collection target particle of event E11 and the collection target particle of event E12, which is a subsequent collection target particle that flows immediately after the collection target particle of event E11.

[0291] As shown in the chart, the calculated ΔTp is longer than Twf and shorter than Twr. Thus, as a result of the determination process in steps S22 and S23, the information processing unit advances the process to step S24.

[0292] In step S24, the information processing unit refers to the step counter value. The step counter value is 0. Thus, the information processing unit advances the process to step S25. In step S25, the information processing unit selects the first sieving operation as the sieving operation to be applied to the sieving of the collection target particle. That is, the pulse drive waveform including the falling edge is selected.

[0293] In response to the selection of the first sieving operation, the information processing unit increases the step counter value by 1, that is, changes the step counter value from “0” to “1”, as shown in the chart. By this, the case where a shift has occurred in the collection flow path (or the drive voltage) is managed.

[0294] With respect to the collection target particle of event E12, the information processing unit calculates ΔTp at step S21. More specifically, the information processing unit calculates the time interval ΔTp between the collection target particle of event E12 and the collection target particle of event E13, which is a subsequent collection target particle that flows immediately after the collection target particle of event E12.

[0295] As shown in the figure, the calculated ΔTp is longer than Twf, and furthermore, longer than Twr. Thus, as a result of the determination processing in step S22 and step S23, the information processing unit advances the processing to step S27.

[0296] In step S27, the information processing unit refers to the step counter value. The step counter value is 1. Thus, the information processing unit advances the processing to step S28. In step S28, the information processing unit selects the second sieving operation as the sieving operation to be applied to the collection target particle. That is, the pulse drive waveform including the rising edge is selected.

[0297] In response to the selection of the second sieving operation, as shown in the figure, the information processing unit decrements the step counter value by 1, that is, changes the step counter value from "1" to "0". Thereby, the management is made in a case where the offset in the collection flow path (or the drive voltage) has been eliminated.

[0298] As for the collection target particle of event E13, the same processing as that of the collection target particle of event El is executed, and the information processing unit selects the first sieving operation as the sieving operation to be applied to the collection target particle. That is, the pulse drive waveform including the falling edge is selected.

[0299] As for events E14 and E15 as well, similar processing is executed, and the information processing unit selects the first sieving operation as the sieving operation to be applied to the collection target particle. That is, the pulse drive waveform including the falling edge is selected. Note that as a result of the successive execution of events E13, E14, and E15, the step counter value is gradually changed from "0" to "3 (maximum value)".

[0300] As for the collection target particle of event E16, in step S21, the information processing unit calculates ΔTp. More specifically, the information processing unit calculates the time interval ΔTp between the collection target particle of event E16 and the collection target particle of event E17 (the subsequent collection target particle flowing after the collection target particle of event E16).

[0301] As shown in the figure, the calculated ΔTp is longer than Twf, and furthermore, longer than Twr. Thus, as a result of the determination processing in step S22 and step S23, the information processing unit advances the processing to step S27.

[0302] In step S27, the information processing unit refers to the step counter value. The step counter value is the maximum value, which is 3. Thus, the information processing unit advances the processing to step S28. In step S28, the information processing unit selects the second sieving operation as the sieving operation to be applied to the collection target particle. That is, the pulse drive waveform including the rising edge is selected.

[0303] In response to the selection of the second sieving operation, the information processing unit decrements the step counter value by 1, i.e., changes the step counter value from "3" to "2", as shown in the figure.

[0304] As for the collection target particles of event E17 and event E18, similar processing is also performed, and the information processing unit selects the second sieving operation as the sieving operation to be applied to the sieving of the collection target particles. That is, the pulse drive waveform including the rising edge is selected. Note that as a result of the successive execution of events E16, E17, and E18, the step counter value is changed from "3 (the maximum value)" to "0" step by step.

[0305] As for the collection target particles of event E19, the same processing as that of the collection target particles of event E11 is performed, and the information processing unit selects the first sieving operation as the sieving operation to be applied to the sieving of the collection target particles. That is, the pulse drive waveform including the falling edge is selected.

[0306] By performing the drive waveform selection processing as described above, the first sieving operation is applied in the case where the interval between two successive collection target particles is short, so that it is possible to improve the sieving efficiency. Furthermore, the offset generated by the execution of the first sieving operation is eliminated at an appropriate timing.

[0307] (7-4) Configuration example of sieving apparatus

[0308] For example, the biological particle sieving system according to the present disclosure can have a light emitting unit, a sensing unit, an information processing unit, and a sieving unit as explained below. The light emitting unit, the sensing unit, the information processing unit, and the sieving unit can be as explained below with respect to the biological sample analyzer apparatus, and the explanation is also applicable to the biological particle sieving of the present disclosure. The light emitting unit, the sensing unit, the information processing unit, and the sieving unit can be present in one apparatus, or can be installed distributively in multiple apparatuses.

[0309] Figure 27 An exemplary configuration of a biological sample analyzer of the present disclosure is shown. Figure 27 The biological sample analyzer 6100 shown in FIG. 11 includes a light irradiation unit 6101 that irradiates a biological sample S flowing in a flow path C with light, a detection unit 6102 that detects light generated by irradiating the biological sample S with light, and an information processing unit 6103 that processes information about the light detected by the detection unit. The biological sample analyzer 6100 is, for example, a flow cytometer or an imaging cytometer. The biological sample analyzer 6100 can include a sorting unit 6104 that sorts out a specific biological particle P in the biological sample. The biological sample analyzer 6100 including the sorting unit is, for example, a cell sorter.

[0310] (Biological sample)

[0311] The biological sample S can be a liquid sample containing biological particles. The biological particles are, for example, cells or non-cellular biological particles. The cells can be living cells, and more specific examples thereof include blood cells (such as red blood cells and white blood cells) and reproductive cells (such as sperm and zygotes). Furthermore, the cells can be those collected directly from a sample such as whole blood, or can be cultured cells obtained after culturing. The non-cellular biological particles are, for example, extracellular vesicles, or particularly exosomes and microvesicles. The biological particles can be labeled with one or more labeling substances such as dyes (particularly fluorescent dyes) and fluorescent dye-labeled antibodies. Note that particles other than biological particles can be analyzed by the biological sample analyzer of the present disclosure, and beads or the like can be analyzed for calibration or the like.

[0312] (flow path)

[0313] The flow path C is designed so as to form a flow of the biological sample S. Specifically, the flow path C can be designed so as to form a flow in which the biological particles contained in the biological sample are aligned substantially in a line. The flow path structure including the flow path C can be designed so as to form a laminar flow. Specifically, the flow path structure is designed so as to form a laminar flow in which a flow of the biological sample (sample flow) is surrounded by a flow of sheath liquid. The design of the flow path structure can be appropriately selected by those skilled in the art, or a known design can be employed. The flow path C can be formed as a flow path structure such as a microchip (a chip having a flow path of micrometer order) or a flow cell. The width of the flow path C is 1 mm or less, and specifically, can be 10 pm or more and 1 mm or less. The flow path C and the flow path structure including the flow path C can be made of a material such as plastic or glass.

[0314] The biological sample analyzer of the present disclosure is designed so as to irradiate the biological sample flowing in the flow path C, or specifically the biological particles in the biological sample, with light from the light irradiation unit 6101. The biological sample analyzer of the present disclosure can be designed so that the irradiation point of light on the biological sample is located in the flow path structure in which the flow path C is formed, or can be designed so that the irradiation point is located outside the flow path structure. An example of the former case can be a configuration in which light is emitted onto the flow path C in a microchip or a flow cell. In the latter case, the biological particles after exiting the flow path structure (specifically, a nozzle portion thereof) can be irradiated with light, and for example, an air jet type flow cytometer can be employed.

[0315] (light irradiation unit)

[0316] The light irradiation unit 6101 includes a light source unit that emits light and a light guide optical system that guides the light to an irradiation point. The light source unit includes one or more light sources. The type of the light source is, for example, a laser light source or an LED. The wavelength of the light emitted from each light source can be any wavelength of ultraviolet light, visible light, and infrared light. The light guide optical system includes, for example, optical components such as a beam splitter, a mirror, or an optical fiber. The light guide optical system can also include a lens group for light condensation, and include, for example, an objective lens. There can be one or more irradiation points at which the biological sample and the light intersect. The light irradiation unit 6101 can be designed to collect light emitted onto one irradiation point from one light source or different light sources.

[0317] (detection unit)

[0318] The detection unit 6102 includes at least one photodetector that detects light generated by irradiating light onto a biological particle. For example, the light to be detected can be fluorescent light or scattered light (such as one or more of forward scattered light, backward scattered light, and side scattered light). Each photodetector includes, for example, one or more light receiving elements, and has an array of light receiving elements. Each photodetector can include one or more photomultiplier tubes (PMTs) and / or photodiodes such as APDs and MPPCs as light receiving elements. The photodetector includes, for example, a PMT array in which a plurality of PMTs are arranged in a one-dimensional direction. The detection unit 6102 can also include an image sensor such as a CCD or a CMOS. With the image sensor, the detection unit 6102 can acquire an image (for example, such as a bright field image, a dark field image, or a fluorescent image) of a biological particle.

[0319] The detection unit 6102 includes a detection optical system that causes light of a predetermined detection wavelength to reach a corresponding photodetector. The detection optical system includes a light splitting unit such as a prism or a diffraction grating, or a wavelength separation unit such as a dichroic mirror or a filter. For example, the detection optical system is designed to disperse light generated by light irradiation to a biological particle, and detect the dispersed light using more light detectors than the number of fluorescent dyes that label the biological particle. A flow cytometer including such a detection optical system is called a spectral flow cytometer. Further, for example, the detection optical system is designed to separate light of a fluorescent wavelength band corresponding to a specific fluorescent dye from light generated by light irradiation to a biological particle, and cause a corresponding photodetector to detect the separated light.

[0320] The detection unit 6102 can further include a signal processing unit that converts an electric signal obtained by the light detector into a digital signal. The signal processing unit can include an A / D converter as a device that performs the conversion. The digital signal obtained by the conversion performed by the signal processing unit can be sent to the information processing unit 6103. The digital signal processing unit 6103 can process the digital signal into light-related data (hereinafter, also referred to as "light data"). For example, the light data can be light data including fluorescence data. More specifically, the light data can be data of light intensity, and the light intensity can be light intensity data of light including fluorescence (the light intensity data can include characteristic amounts such as area, height, and width).

[0321] (Information processing unit)

[0322] For example, the information processing unit 6103 includes a processing unit that performs processing of various types of data (e.g., light data) and a storage unit that stores various types of data. In a case where the processing unit acquires light data corresponding to a fluorescent dye from the detection unit 6102, the processing unit can perform fluorescence leakage correction (compensation processing) on the light intensity data. In the case of a spectral flow cytometer, the processing unit also performs fluorescence separation processing on the light data, and acquires light intensity data corresponding to a fluorescent dye. For example, the fluorescence separation processing can be performed by the unmixing method disclosed in JP 2011-232259 A. In a case where the detection unit 6102 includes an image sensor, the processing unit can acquire morphological information on a biological particle based on an image acquired by the image sensor. The storage unit can be designed to be able to store the acquired light data. The storage unit can be designed to be able to further store spectral reference data to be used in unmixing processing.

[0323] In a case where the biological sample analyzer 6100 includes a sorting unit 6104 described later, the information processing unit 6103 can determine whether or not to sort a biological particle based on the light data and / or the morphological information. Then, the information processing unit 6103 controls the sorting unit 6104 based on the determination result, and can sort the biological particle by the sorting unit 6104.

[0324] The information processing unit 6103 can be designed to be able to output various types of data (e.g., such as light data and images). For example, the information processing unit 6103 can output various data (e.g., such as a two-dimensional curve or a spectral curve) generated based on the light data. For example, the information processing unit 6103 can also be designed to be able to accept input of various types of data, and accept user's gate processing of a plot. The information processing unit 6103 can include an output unit (e.g., such as a display) or an input unit (e.g., such as a keyboard) for performing the output or the input.

[0325] The information processing unit 6103 can be designed as a general-purpose computer, and can be designed as an information processing device including, for example, a CPU, a RAM, and a ROM. The information processing unit 6103 can be included in a housing including the light irradiation unit 6101 and the detection unit 6102, or can be located outside the housing. Furthermore, various processes or functions to be executed by the information processing unit 6103 can be implemented by a server computer or a cloud connected via a network.

[0326] (Sorting unit)

[0327] The sorting unit 6104 sorts biological particles according to the determination result by the information processing unit 6103. The sorting method can be a method of generating a droplet containing a biological particle by vibration, applying an electric charge to the droplet to be sorted, and controlling the moving direction of the droplet by an electrode. The sorting method can be a method for sorting by controlling the traveling direction of a biological particle in a flow path structure. For example, the flow path structure has a control mechanism based on pressure (injection or suction) or electric charge. An example of the flow path structure can be a chip having a flow path structure in which a flow path C is branched into a recovery flow path and a waste liquid flow path on the downstream side, and a specific biological particle is collected in the recovery flow path (for example, a chip disclosed in JP 2020-76736 A).

[0328] (7-5) Examples of information processing unit and examples of algorithm executed by information processing unit

[0329] (7-5-1) Example of information processing unit

[0330] The following describes a more specific configuration example of the information processing unit and an example of the algorithm of the sieving process executed by the information processing unit.

[0331] [Overall configuration of information processing unit]

[0332] Figure 28A is a block diagram illustrating a configuration example of an information processing unit. The information processing unit 23 illustrated in the figure has a plurality of circuits 2302 to 2309 each connected to a bus 2301. Note that the information processing unit can also be referred to as a drive unit or a control unit.

[0333] Specifically, the circuit denoted by reference numeral 2302 in the figure is an analog-to-digital conversion circuit 2302. In addition, the circuit denoted by reference numeral 2303 is an event sensing circuit 2303. In addition, the circuit denoted by reference numeral 2304 is an arrival time calculation circuit 2304. Further, the circuit denoted by reference numeral 2305 is a selection circuit 2305. In addition, the circuit denoted by reference numeral 2306 is an output queue circuit 2306. In addition, the circuit denoted by reference numeral 2307 is an output timing generation circuit 2307. In addition, the circuit denoted by reference numeral 2308 is an output signal generation circuit 2308. In addition, the circuit denoted by reference numeral 2309 is an MPU (micro processing unit) 2309. Note that the analog-to-digital conversion circuit 2302 is written as "A / D" in the figure.

[0334] Furthermore, as shown in the figure, the information processing unit 23 has a clock counter 2310. The clock counter 2310 is connected to the event sensing circuit 2303, the arrival time calculation circuit 2304, the gating circuit 2305, the output queue circuit 2306, the output timing generation circuit 2307, and the output signal generation circuit 2308.

[0335] Furthermore, as shown in the figure, the information processing unit 23 has a PC I / O unit (input / output interface circuit for personal computer connection) 2311 connected to the MPU 2309 and a control PC 2312 connected to the PCI / O unit 2311 .

[0336] Furthermore, as shown in the figure, the information processing unit 23 has a digital-analog conversion circuit 2313 connected to the output signal generating circuit 2308. Note that the digital-analog conversion circuit 2313 is written as "D / A" in the figure.

[0337] [Details of the information processing unit]

[0338] [Analog-to-digital conversion circuit]

[0339] The analog-to-digital conversion circuit 2302 is a circuit located downstream (on the output side) of the detection unit 6102 (see Figure 27 ) and is connected to the detection unit 6102. In addition, a plurality of analog-to-digital conversion circuits 2302 are arranged. Here, the analog-to-digital conversion circuits 2302 can be arranged in the same number as the number of channels of the detection unit 6102 to correspond to the plurality of lights (wavelength regions) detected by the detection unit 6102. Alternatively, the analog-to-digital conversion circuits 2302 can be arranged in the same number as the number of sensors of the detection unit 2302.

[0340] Each analog-digital conversion circuit 2302 receives input of an electric signal corresponding to the analog-digital conversion circuit 2302, which is output from the detection unit 6102. The electric signal is an analog signal obtained by photoelectric conversion of light (fluorescent light and scattered light) detected by the detection unit 6102 by the detection unit 6102. Then, each analog-digital conversion circuit 2302 converts each input electric signal from an analog signal to a digital signal. Further, each analog-digital conversion circuit 2302 outputs the electric signal converted to a digital signal to the downstream.

[0341] [Event sensing circuit]

[0342] The event sensing circuit 2303 is a circuit located downstream of each analog-digital conversion circuit 2302, and is connected to each analog-digital conversion circuit 2302.

[0343] The event sensing circuit 2303 receives input of an electric signal output from each analog-digital conversion circuit 2302. Then, the event sensing circuit 2303 uses a specific signal in the corresponding input electric signal as a trigger signal for identifying a microparticle. That is, in a case where a value of the trigger signal satisfies a predetermined condition, the event sensing circuit 2303 identifies that an electric signal has been detected from a microparticle. In addition, the trigger signal can also be an electric signal of light having the highest intensity among the plurality of lights detected by the detection unit 6102 (for example, forward scattered light), but is not limited thereto.

[0344] In addition, as depicted in Figure 28B , the event sensing circuit 2303 reads in a waveform of each input electric signal, and calculates a width, a height, and an area of the read-in waveform. Further, as depicted in Figure 28C , using the corresponding calculated values and the like of the waveform, the event sensing circuit 2303 creates an event data packet in which each electric signal is associated with one microparticle corresponding to the corresponding calculated values and the like. The event data packet is an instance of measurement data of one microparticle. Then, the event sensing circuit 2303 outputs the created event data packet to the downstream.

[0345] Here, the event data packet includes an item (hereinafter, referred to as a first item) in which data recording is completed at the time of creation of the packet. Further, the event data packet includes an item (hereinafter, referred to as a second item) which is updated as a process related to the electric signal corresponding to the packet is performed after the creation of the packet.

[0346] For example, the first item includes the following items.

[0347] - width, height, and area of the waveform of the electric signal

[0348] - number of identified microparticles (event number)

[0349] - number of electric signals used as trigger signals

[0350] - trigger signal sensing time

[0351] Note that the number of electric signals used as the trigger signal can be the channel number. Further, a signal input from the clock counter 2310 can be used to record the trigger signal sensing time. This signal can represent the number of clock signals input to the clock counter 2310 from a clock generation circuit (not shown) and counted by the clock counter 2310.

[0352] In contrast, for example, the second item includes the following items.

[0353] - time at which the microparticle should be collected

[0354] - first flag indicating whether or not to collect the microparticle

[0355] - second flag indicating whether or not to collect the microparticle

[0356] Note that the first flag is a flag set by the gating circuit 2305. In contrast, the second flag is a flag set by the output queue circuit 2306. The first flag and the second flag can be substantially set to 1 or 0 and used to determine whether or not to collect the corresponding particle. Further details of each flag are mentioned later.

[0357] [Arrival time calculation circuit]

[0358] As shown in the figure, the arrival time calculation circuit 2304 is a circuit located downstream of the event sensing circuit 2303 and connected to the event sensing circuit 2303.

[0359] The arrival time calculation circuit 2304 receives input of the event data packet output from the event sensing circuit 2303. Then, based on the input event data packet, the arrival time calculation circuit 2304 calculates the arrival time of the microparticle (target particle) to the communication port (the inlet of the connection flow path on the main flow path side) as the time at which the microparticle should be collected in the second item. In the following description, the "time at which the microparticle should be collected" is instead referred to as the "arrival time". The arrival time calculation circuit 2304 records the arrival time calculated by the calculation in the event data packet and outputs the event data packet after recording downstream.

[0360] The arrival time can be calculated by adding the required time (delay time) for the target particle in the sensing region to arrive at the communication port to the trigger signal sensing time included in the second item. Further, the clock counter value can be used to calculate the arrival time.

[0361] [Gating circuit]

[0362] The gating circuit 2305 is a circuit located downstream of the event sensing circuit 2303 and connected to the event sensing circuit 2303.

[0363] The gating circuit 2305 receives an input of the event data packet output from the event sensing circuit 2303. Then, the gating circuit 2305 sets the first flag in the input event data packet. Further, the gating circuit 2305 outputs the event data packet after setting the first flag downstream.

[0364] The setting of the first flag can be performed based on a threshold preset for a parameter of each electric signal included in the event data packet. In this case, the threshold can be related to at least one of a width, a height, and an area of the waveform. Then, in a case where the parameter satisfies the threshold, a value of the first flag can be set to a value (e.g., “1”) indicating that the particle is collected. In contrast, in a case where the parameter does not satisfy the threshold, a value of the first flag can be set to a value (e.g., “0”) indicating that the particle is not collected.

[0365] In addition, the threshold can be a range designated by gating in advance. Here, the gating is a process of designating a range corresponding to a target particle by surrounding the range on a distribution map representing a characteristic distribution of the particles in the particle group. This gating is performed before the target particle collection operation starts. Note that the distribution map can be created by a GUI (Graphical User Interface) on the PC 2312. Further, the gating can be performed by the gating circuit 2305.

[0366] Here, Figure 28D A on the left side depicts a result of gating a histogram as an example of the distribution map. The horizontal axis of the histogram represents a parameter, and the vertical axis represents a particle number. However, the parameter in the figure is an area of a waveform of an electric signal corresponding to channel number 1 (Ch1), and a parameter other than this can be used. Then, the rectangular frame in the figure is a gate that designates a range corresponding to a target particle, and this range can be used as a threshold for the setting of the first flag.

[0367] In contrast, Figure 28D B on the middle right side depicts a result of gating a 2D (two-dimensional) map as another example of the distribution map. In this 2D map, mutually different parameters are assigned to the horizontal axis and the vertical axis. The parameter of the horizontal axis in the figure is an area of a waveform of an electric signal corresponding to channel number 2 (Ch2), and the parameter of the vertical axis in the figure is an area of a waveform of an electric signal corresponding to channel number 3 (Ch3). However, a parameter other than the area of the waveform of the electric signal can be used. Then, the rectangular frame in the figure is a gate that designates a range corresponding to a target particle, and this range can be used as a threshold for the setting of the first flag.

[0368] [Output queue circuit]

[0369] Returning to Figure 28AThe output queue circuit 2306 is a circuit located downstream of the arrival time calculation circuit 2304 and the gating circuit 2305, and is connected to the arrival time calculation circuit 2304 and the gating circuit 2305.

[0370] The output queue circuit 2306 receives the input of the event data packet output from the arrival time calculation circuit 2304 and the event data packet output from the gating circuit 2305. Then, the output queue circuit 2306 integrates (synthesizes) the event data packets included in the input event data packets and representing the same particle (i.e., having the same event number) into one event data packet. The integrated event data packet includes the delay time and the first flag written therein. Note that the integration of the event data packets can be performed by either of the arrival time calculation circuit 2304 and the gating circuit 2305 through communication between the circuits 2304 and 2305. Further, the arrival time calculation circuit 2304 and the gating circuit 2305 can be connected in series.

[0371] Further, the output queue circuit 2306 arranges the serially input event data packets of mutually different particles in descending order of the arrival times included in the event data packets. Here, the event data packet that has been input to the output queue circuit 2306 and waits for the output of the drive waveform for collecting the corresponding particle is defined as an "output queue". The output queue is updated according to the input of a new event data packet to the output queue circuit 2306.

[0372] Further, the output queue circuit 2306 determines whether or not to collect the particle corresponding to each event data packet, depending on whether the screening operation mode is the purity priority mode, the acquisition rate priority mode, or the single particle priority mode. Note that the purity priority mode, the acquisition rate priority mode, and the single particle priority mode are operation modes of the information processing unit 23 that are selectively set in advance before the start of the particle collection operation. The mode setting can be performed by the control PC 2312 via various types of user interfaces.

[0373] Here, the purity priority mode is a mode in which, in a case where a target particle and a non-target particle flow close to each other and the possibility that the two particles are collected together is high, the target particle is intentionally treated as a "non-target particle (not acquired)", and the purity of the collected particles is enhanced. That is, in a case where the purity priority mode is set, a target particle close to a non-target particle is not collected, but is discarded.

[0374] In contrast, the acquisition rate priority mode is a mode in which, in a case where a target particle and a non-target particle flow close to each other and the possibility that the two particles are collected together is high, the two particles are acquired together, and even if the purity of the collected particles decreases, the number of acquired particles increases.

[0375] The single-particle priority mode is a screening mode, which is useful in a case where a target particle is collected as, for example, an emulsion particle. The single-particle priority mode is a mode in which, in a case where a certain target particle and another particle (a target particle or a non-target particle) flow close to each other, it is likely that the two particles are collected together, and multiple particles are contained in one emulsion particle, a specific target particle is intentionally considered as a "non-target particle (not to be acquired)" and is not screened. That is, the single-particle priority mode can also be referred to as a mode for improving the collection rate of a single-particle emulsion. That is, in a case where the single-particle priority mode is set, a target particle close to a particle is not collected, but is discarded.

[0376] Then, the output queue circuit 2306 sets a second flag on the basis of a result of determining whether to collect the microparticle in accordance with the set mode. At this time, in a case where it is determined to collect the microparticle, the output queue circuit 2306 can set the second flag to "1". In contrast, in a case where it is determined not to collect the microparticle, the output queue circuit 2306 can set the second flag to "0". Note that such a flag setting mode can not be the only example.

[0377] Further, the output queue circuit 2306 writes an application timing of a drive waveform that should be applied to the actuator into a memory. The memory can be a RAM (Random Access Memory). In addition, the memory can be connected to the bus 2301, or can be built in the circuit of the information processing unit 23 or the control PC 2312. Furthermore, when the application timing is written, the set values of the first flag and the second flag can be referred to. In this case, in a case where both flags have been set to values indicating the execution of collection, such an application timing of collecting the corresponding microparticle can be written. In addition, the drive waveform can be a drive voltage.

[0378] Here, the output queue circuit writes an application timing of a drive waveform to be used in the memory. In a case where a pulse drive waveform is applied as the drive waveform, as described above Figure 4 in the above description, the pulse waveform includes a falling edge portion and a rising edge portion. The falling edge portion forms a front portion of the pulse waveform, and the rising edge portion forms a rear portion of the pulse waveform. The height (amplitude) of the falling edge portion and the height of the rising edge portion are made equal to each other.

[0379] The falling edge portion is applied for deforming the pressure chamber in a direction of increasing the volume of the pressure chamber and generating a pressure change in a negative direction immediately after the application of the falling edge. The falling edge portion can generate a force to weaken the pressing pressure so as to increase the volume in the pressure chamber. In a case where the actuator is a piezoelectric element, the falling edge portion can have a waveform to decrease a drive voltage applied to the stretched piezoelectric element and cause the piezoelectric element to contract.

[0380] In contrast, an up-slope drive waveform is applied for deforming the pressure chamber so as to reduce the volume of the pressure chamber and generate a pressure change in a positive direction immediately after the application of the drive waveform. The up-slope drive waveform can generate a force to reinforce the pressing pressure so as to reduce the volume in the pressure chamber. In the case where the actuator is a piezoelectric element, the up-slope drive waveform can be a waveform that increases the drive voltage applied to the contracted piezoelectric element and extends the piezoelectric element.

[0381] In the case where the output queue circuit writes the application timing of the pulse drive waveform including the above-described down-slope portion and up-slope portion to the memory, the output queue circuit can write the arrival time included in the event data packet as the application timing of the pulse drive waveform.

[0382] [Output timing generation circuit]

[0383] As Figure 28A The output timing generation circuit 2307 is connected to the output queue circuit 2306. The output timing generation circuit 2307 reads out the arrival time of the event data packet arranged at the start of the output queue written to the RAM by the output queue circuit 2306 from the RAM. Then, the output timing generation circuit 2307 compares the read arrival time with the value of the signal from the clock counter 2310 and generates an output timing signal at the arrival time. Here, the output timing signal is a signal for assigning the output timing of the drive waveform. The output timing generation circuit 2307 outputs the generated output timing signal downstream. Further, the output queue circuit 2307 can transmit a completion signal to the output queue circuit 2306 after outputting the output timing signal and prompt the update of the output queue.

[0384] In the case where the drive waveform is a pulse waveform, the output timing generation circuit 2307 refers to the memory in which the application timing of the down-slope portion and up-slope portion is written by the output queue circuit 2306. Then, the output timing generation circuit 2307 compares the application timing of the down-slope portion and up-slope portion in the memory with the value of the signal from the clock counter 2310 and generates an output timing signal for each waveform portion. Further, the output timing generation circuit 2307 outputs the generated output timing signal downstream.

[0385] [Output signal generation circuit]

[0386] The output signal generation circuit 2308 is a circuit located downstream of the output timing generation circuit 2307 and connected to the output timing generation circuit 2307.

[0387] The output signal generation circuit 2308 receives input of the output timing signal output from the output timing generation circuit 2307. Then, the output signal generation circuit 2308 generates a drive waveform (output signal) corresponding to the input output timing signal, and outputs the drive waveform downstream. Further, after outputting the drive waveform, the output signal generation circuit 2308 updates the step counter and the output state signal. Note that the output state signal is a signal indicating a state in which the waveform is being deactivated / being output (can be output (enabled) / cannot be output (disabled)).

[0388] Here, the step counter indicates a stepwise output level of the drive waveform. That is, the step counter indicates a deviation value of the drive waveform from a reference value that changes in steps as the number of applications of the drive waveform increases. The output difference of each level difference corresponding to one level of the step counter is constant. The step counter and the output state signal can be input to the output queue circuit 2306 or the output timing generation circuit 2307, and used for processing performed by each circuit 2306 and 2307.

[0389] In the case where the drive waveform is a pulse waveform, the output signal generation circuit 2308 generates and outputs a falling edge portion and a rising edge portion, respectively.

[0390] [Digital-analog conversion circuit]

[0391] The digital-analog conversion circuit 2313 receives input of the drive waveform output from the output signal generation circuit 2308. Then, the digital-analog conversion circuit 2313 converts the input drive waveform from a digital signal to an analog signal, and outputs the analog signal to the drive circuit of the actuator 31.

[0392] Note that the information processing unit 23 in the above-described Figure 28A may be applied to apply a drive waveform other than a pulse waveform.

[0393] (7-5-2) Example of Algorithm

[0394] Examples of the algorithm executed by the information processing unit are described in Figure 29 . In the flowchart shown in the figure, the following first to third processes are executed separately and in parallel.

[0395] [First process]

[0396] In the first process, first, in step S151-1, it is determined whether there is an input of an event (event data packet). In this determination, the event data packet input from the upstream circuit (for example, the arrival time calculation circuit 2304 and the gating circuit 2305) is used. Then, in the case where the determination result of step S151-1 is affirmative, the process proceeds to step S151-2, and in the case where the determination result of step S151-1 is negative, step S151-1 is repeated.

[0397] Next, in step S151-2, the output queue is updated by adding a new event corresponding to the determination of "there is an input" in step S151-1 to the output queue.

[0398] Finally, in step S151-3, based on the output queue updated in step S151-2, it is reevaluated whether to collect (acquire / not acquire) the particle, and the process returns to step S151-1. Here, the reason why the expression "reevaluate" is used is because the evaluation in this step corresponds to the process of reevaluating the event already in the queue (the event that has been evaluated and for which the second flag has already been set). In the case of purity priority, since the event appended later to the queue can be close to the event appended earlier, the process of this step effectively functions.

[0399] [Second Process]

[0400] In the second process, in step S152, with respect to the event to be acquired next (the collection target particle), it is selected whether to acquire the event by applying a pulse drive waveform including a falling edge or to acquire the event by applying a pulse drive waveform including a rising edge, and the arrival time recorded in the event data packet is written to the memory. The drive waveform selection process can be performed as explained in (7-2) and (7-3) above. Alternatively, the drive waveform selection process can also be performed as explained in (8) or (9) below. The pulse drive waveform selected by the selection process is written to the memory.

[0401] [Third Process]

[0402] In the third process, in the case where the application of the pulse driving waveform including the falling edge is selected in the second process, first, in step S153-1, the application timing of the falling edge portion of the event that should be acquired by the application of the pulse driving waveform including the falling edge is written to the memory. The written application timing is referred to by the output timing generation circuit. Next, in step S153-2, it is determined whether the trigger output of the pulse driving waveform including the falling edge has been completed. The falling edge trigger output is the output of the pulse driving waveform containing the falling edge generated by the output timing generation circuit to the output signal generation circuit. Finally, in step S153-3, the output queue is updated by deleting the event that has completed the trigger output of the pulse waveform containing the falling edge from the output queue, and the process returns to step S153-1.

[0403] [Fourth process]

[0404] In the fourth process, in the case where the application of the pulse driving waveform including the rising edge is selected in the second process, first, in step S154-1, the application timing of the event that should be acquired by the application of the pulse driving waveform including the rising edge is written to the memory. The written application timing is referred to by the output timing generation circuit. Next, in step S154-2, it is determined whether the trigger output of the pulse driving waveform including the rising edge has been completed. The pulse waveform trigger output containing the rising edge is the output of the rising edge portion generated by the output timing generation circuit to the output signal generation circuit. Finally, in step S154-3, the output queue is updated by deleting the event that has completed the trigger output of the pulse waveform including the rising edge from the output queue, and the process returns to step S154-1.

[0405] (8)Screening process example 2

[0406] In the second screening operation explained in the above (7), the second rising edge portion is configured to raise one level of pressure. That is, single-step offset elimination is performed. To eliminate the offset, the second rising edge portion has a single-step rising edge.

[0407] In one embodiment of the present disclosure, the pressure increase at the fourth step is not limited to a single-step pressure increase, and can be a multi-step pressure increase. That is, at the fourth step, offset elimination with one or more levels can be performed. To eliminate the offset, the second rising edge portion can have a rising edge with one or more steps.

[0408] Further, in the present embodiment, the number of levels for eliminating the offset can be changed. For example, the number can be changed according to the interval between the completion time of the second step and the time at which the subsequent biological particle (e.g., a collection target particle) is sensed.

[0409] To increase the number of levels of pressure in this way in the fourth step, the second rising edge portion for performing the fourth step can have a multi-step rising edge. Thereby, the step counter value can be effectively returned. This contributes to reducing the frequency of the step counter value becoming the maximum value, and furthermore, can enhance the sieving efficiency.

[0410] In addition, in some cases, it is not necessary to perform the offset cancellation. For example, in some cases, it is not necessary to return the step counter value. Therefore, in the second sieving operation, the fourth step can not be performed. In view of this, the biological particle sieving system can be configured to perform the fourth step in a case where a predetermined condition is satisfied. For example, the information processing unit can decide whether to perform the fourth step based on an interval between a completion time of the second step in the second sieving operation and a time at which a subsequent biological particle is sensed.

[0411] The embodiment will be described in more detail below.

[0412] As described above with reference to (7), when the step counter value is the maximum value, it is not possible to perform sieving using the pulse drive waveform including the falling edge. Therefore, a subsequent collection target particle that satisfies the condition that the time interval between the collection target particles is short (ΔTp < Twr) is aborted. Thus, the sieving efficiency is improved by reducing the frequency at which the step counter reaches the maximum value.

[0413] In the present embodiment, as Figure 19 depicted in (8), in a case where the time interval ΔTp between two collection target particles is long, a pulse drive waveform including a multi-step rising edge added with a multi-step rising edge portion is used. The pulse drive waveform including a multi-step rising edge shown in the figure has a first falling edge portion Wf1 and a first rising edge portion Wr1, and further has a second rising edge portion Wr2. The second rising edge portion Wr2 is a rising edge portion including three rising edge portions Wr21, Wr22, and Wr23, that is, multi-step offset cancellation is performed.

[0414] Note that each of the hold times Wm1 to Wm4 is set between adjacent two waveform portions.

[0415] In this way, in a case where ΔTp is long, a larger number of rising operations can be performed. By referring to ΔTp, an appropriate number of rising operations can be selected.

[0416] In addition, in order to decide whether it is possible or not possible to perform a rising operation and decide the number of rising operations, another index relating to the time interval between the collection target particle and the subsequent particle can be referred to. ΔTr(n) is described below, but another time interval can be referred to.

[0417] For example, the number of times of performing the rising operation can be selected based on the rising edge application completion time and the arrival time of the subsequent collection target particle.

[0418] Figure 20 The relationship between the time interval ΔTr(n) between the particles and the pulse driving waveform including the multi-step rising edge is described. The parameters in the figure have the following meanings.

[0419] ΔTr(n): Time interval between the rising edge application completion time and the arrival time of the subsequent collection target particle (or the falling edge application start time for the collection of the subsequent collection target particle (n: number of times of application of the continuous rising edge. n≥0.)

[0420] Tmr: Minimum time interval allowing the next application of the rising edge

[0421] In this embodiment, for example, the time interval ΔTr(n) is calculated each time the rising edge is applied.

[0422] For example, with respect to Figure 20 In the case where the first rising edge portion Wr1 is applied (in the case where n=0), the time interval ΔTr(0) between the rising edge portion Wr1 application completion time and the arrival time of the subsequent collection target particle is calculated, this time interval is compared with the minimum time interval Tmr, and then in the case where ΔTr(0)>Tmr, the next first rising edge portion Wr21 is applied.

[0423] Next, in the case where the second rising edge portion Wr21 is applied, the time interval ΔTr(1) between the rising edge portion Wr21 application completion time and the arrival time of the subsequent collection target particle is calculated, this time interval is compared with the minimum time interval Tmr, and then in the case where ΔTr(1)>Tmr, the next second rising edge portion Wr22 is applied.

[0424] Next, in the case where the second rising edge portion Wr22 is applied, the time interval ΔTr(2) between the rising edge portion Wr22 application completion time and the arrival time of the subsequent collection target particle is calculated, this time interval is compared with the minimum time interval Tmr, and then in the case where ΔTr(2)>Tmr, the next third rising edge portion Wr23 is applied.

[0425] In this way, each time the time interval ΔTr(n) is calculated, it is compared with the minimum time interval Tmr, and then, in the case of ΔTr(n) > Tmr, the next rising edge portion is applied. Then, after the next rising edge portion is applied, the time interval ΔTr(n+1) is further calculated, it is compared with the minimum time interval Tmr, and then, in the case of ΔTr(n+1) > Tmr, another next rising edge portion is applied.

[0426] In this way, in the embodiment, it can be further decided whether to apply the rising edge portion based on the time interval ΔTr(n).

[0427] Further, in the present embodiment, in the case of ΔTr(n) ≤ Tmr as a result of the comparison, it is determined that the rising edge portion is not applied. This is because, if the rising edge portion is applied in this case, it can affect the subsequent particle sieving.

[0428] Further, in the present embodiment, in the case of the step counter value being 0, it is determined that the rising edge portion is not applied. This is because, in this case, the rising edge portion does not need to be applied.

[0429] For example, by applying the rising edge portion Wr2 shown in (2) of FIG. 6, the step counter value becomes 0. Thereby, the pressure increasing process ends. Figure 20

[0430] That is, the comparison and application of the rising edge portion can be repeated until ΔTr(n) < Tmr or the step counter value becomes 0.

[0431] In this way, in this embodiment, the biological particle sieving system can repeatedly perform the pressure increase in the collection flow path by applying the rising edge portion until the time interval ΔTr(n) calculated each time the rising edge portion is applied becomes shorter than the predetermined value Tmr.

[0432] As described above, in the present embodiment, it can be decided whether to increase the pressure in the collection flow path based on the time interval ΔTr(n). In addition, in this decision, the step counter value can be referred to.

[0433] Even in the case where the rising operation is performed multiple times in the fourth step of the second sieving operation, each waveform portion can be configured similarly to the case where the rising operation is performed once as mentioned in (7) above.

[0434] The amplitude and duration of the first falling edge portion for performing the first step and the first rising edge portion for performing the second step, and the numerical range of the hold time can be as explained above with respect to the first sieving operation (pulse drive waveform including falling edge), and the explanation is also applicable to the second sieving operation in the present example. ​

[0435] For example, the amplitude of each of the plurality of rising edge portions corresponding to the fourth step may be equal to or greater than 0.1 V, preferably equal to or greater than 1 V, and more preferably equal to or greater than 5 V. Furthermore, for example, the amplitude of each of the plurality of rising edge portions may be equal to or less than 150 V, preferably equal to or less than 100 V, and more preferably equal to or less than 50 V.

[0436] For example, the duration of each of the plurality of rising edge portions corresponding to the fourth step may be equal to or longer than 1 μs, preferably equal to or longer than 5 μs, and more preferably equal to or longer than 15 μs. In addition, for example, the duration of each of the plurality of rising edge portions may be equal to or less than 100 μs, preferably equal to or less than 60 μs, and more preferably equal to or less than 30 μs.

[0437] Furthermore, for example, the hold time between two consecutive rising edge portions may be equal to or greater than 0 μs. Furthermore, for example, the hold time may be equal to or less than 100 μs, preferably equal to or less than 50 μs, and more preferably equal to or less than 30 μs. That is, the hold time may be 0 μs, but may be longer than 0 μs.

[0438] (8-1) Example 1 of the processing procedure performed by the biological particle screening system

[0439] Reference Figure 21 An example of the procedure of the method performed by the biological particle screening system according to the present disclosure is described. Figure 16 The flowchart in FIG. 1 is identical to the flowchart in FIG. 2 , except that step S30 (the step surrounded by a dashed line) is added. In step S30, a process is performed to determine whether to execute the fourth step and, if so, to determine the level of pressure increase. These decisions are made based on the time interval ΔTr(n). Because this process is related to further pressure increase, it is also referred to as "additional pressure increase processing" in this specification. By executing step S30, it is possible to determine whether to eliminate offset, and further, if offset elimination is desired, offset elimination can be performed at an appropriate level.

[0440] In the case where the second screening operation is selected in the waveform selection process (ie, the second screening operation is selected in the waveform selection process), step S30 is executed. In the case where step S28 is executed). More specifically, in Figure 16 In step S15, the second screening operation has been selected, and then in a case where the second screening operation has been performed in step S16, step S30 is performed.

[0441] In a case where the first sieving operation has been selected at step S15, and then the first sieving operation has been executed at step S16, step S30 can not be executed, and the process can proceed to step 17.

[0442] In this way, step S30 can be executed only in a case where the second sieving operation has been selected.

[0443] A more specific example of step S30 will be described below with reference to Figure 22 A more specific example of step S30 will be described below with reference to

[0444] At step S31, the biological particle sieving system starts the additional pressure increase process.

[0445] At step S32, the biological particle sieving system calculates a time interval ΔTr(n). To calculate ΔTr(n), an up-ramp application completion time in the second sieving operation executed at step S16 is acquired. Then, the time interval ΔTr(n) is calculated based on the up-ramp application completion time and a subsequent arrival time of the target particle to be collected.

[0446] At step S33, the biological particle sieving system determines whether the step counter value is greater than 0. In a case where the step counter value is greater than 0, the biological particle sieving system causes the process to proceed to step S34. In a case where the step counter value is not greater than 0 (i.e., in a case where the step counter value is 0), the biological particle sieving system causes the process to proceed to step S36.

[0447] At step S34, the biological particle sieving system determines whether the time interval ΔTr(n) is longer than Tmr. In a case where ΔTr(n) is longer than Tmr, the biological particle sieving system causes the process to proceed to step S35. In a case where ΔTr(n) is not longer than Tmr (i.e., in a case where ΔTr(n) is equal to or smaller than Tmr), the biological particle sieving system causes the process to proceed to step S35.

[0448] At step S35, the biological particle sieving system decides to further increase the pressure in the collection flow path. According to the decision result, for example, the biological particle sieving system applies an up-ramp portion, and eliminates the offset of the collection flow path. By applying the up-ramp portion, the collection flow path is deformed, and the volume of the collection flow path is reduced. Thus, the pressure in the collection flow path can be further increased. After the determination, the biological particle sieving system changes n to n+1, and returns the process to step S32, and calculates ΔTr(n+1). In this way, steps S32 to S35 are repeated until the step counter value becomes 0 or ΔTr(n) ≤ Tmr.

[0449] At step S36, the biological particle sieving system ends the additional pressure increase process.

[0450] As described above, the biological particle sieving system can be configured to decide whether or not to execute the fourth step. Further, in a case where the fourth step is to be executed, the biological particle sieving system can be configured to be able to perform the offset elimination process a plurality of times in the fourth step. Further, the biological particle sieving system can be configured to be able to change the number of times of the offset elimination process to be executed in the fourth step. The number of times of the offset elimination process to be executed can be determined based on the interval calculated by the predetermined scheme (specifically, the time interval such as ΔTr(n)). These decisions can be performed by the information processing unit described above. This decision method contributes to improving or speeding up the efficiency of the particle sieving.

[0451] Further, the offset elimination process can increase the pressure in the collection flow path, as described above. That is, in a case where the fourth step is executed, the biological particle sieving system can be configured to increase the pressure at one level or to increase the pressure at a plurality of levels. The levels can be decided based on the interval calculated by the predetermined scheme. These decisions can be performed by the information processing unit. According to the decision result, the information processing unit drives the sieving unit and causes the sieving unit to execute the selected sieving operation.

[0452] (8-2) Example of timing chart in a case where the waveform selection process is executed

[0453] Reference Figure 23 An example of a timing chart explaining the execution of the above-described waveform selection process is described. This chart is an example of a timing chart, and this timing chart depicts events E21 to E29 in which a collection target particle is sensed. A in the chart indicates the event sensing time, B in the chart indicates the application timing of the pulse drive waveform including the falling edge, C in the chart indicates the application timing of the pulse drive waveform (the pulse drive waveform not including the rising edge or the pulse drive waveform including the rising edge), and D indicates the drive signal. E indicates the application time of the rising edge portion related to the additional pressure increase process.

[0454] The waveform selection process performed for each event is described below.

[0455] The waveform selection process related to the collection target particle of the event E21 is the same as that of the event E11 described in (7-3) above. Thus, the description of the event E21 is omitted.

[0456] With respect to the collection target particle of the event E22, the waveform selection process is performed similarly to the event E12 described in (7-3) above, and by the waveform selection process, the second sieving operation is selected as the sieving operation to be applied to the sieving of the collection target particle, that is, the pulse drive waveform including the rising edge is selected. Then, in step S16, the second sieving operation is executed, so that the step counter value is 0.

[0457] Next, as a result of selecting the second sieving operation, the biological particle sieving system executes step S30 as explained above in (8-1). An example of the details of step S30 can be one described in (8-1) above. Figure 22

[0458] In step S31 in the figure, the biological particle sieving system starts the additional pressure increase processing.

[0459] The biological particle sieving system acquires ΔTr(n) in step S32. Next, in step S33, the biological particle sieving system refers to the step counter value. Since the step counter value is 0, the processing goes to step S36, the pressure is not increased, and the processing related to the pressure increase ends.

[0460] Events E23 to E25 are the same as events E13 to E15 explained above in (7-3). Thus, the explanation of these events is omitted.

[0461] As for the collection target particle of event E26, the waveform selection processing is performed the same as event E16 explained above in (7-3), by which the second sieving operation is selected as the sieving operation employed for the sieving of the collection target particle, that is, the pulse drive waveform including the rising edge is selected. Then, in step S16, the second sieving operation is executed.

[0462] Next, as a result of selecting the second sieving operation, the biological particle sieving system executes step S30 as explained above in (8-1).

[0463] In step S31 in (8-1) above, the biological particle sieving system starts the additional pressure increase processing. Figure 22

[0464] In step S32, the biological particle sieving system acquires ΔTr(n).

[0465] Next, in step S33, the biological particle sieving system refers to the step counter value. Since the step counter value is greater than 0 (i.e., 3 (the maximum value)), the processing goes to step S34.

[0466] In step S34, the biological particle sieving system compares ΔTr(n) and Tmr. Here, n is 1. Since ΔTr(1) > Tmr, the biological particle sieving system advances the processing to step S35.

[0467] In step S35, the biological particle sieving system decides to increase the pressure. Thus, the biological particle sieving system applies the rising edge portion, changes n from 1 to 2, and returns the processing to step S32.

[0468] ​​In step S32, the bioparticle separation system obtains ΔTr(2) and then executes steps S33 to S35. In step S35, the bioparticle separation system decides to increase the pressure. Therefore, the bioparticle separation system applies the rising edge portion, changes n from 2 to 3, and returns the process to step S32.

[0469] The biological particle fractionation system acquires ΔTr(3) in step S32, but since the step counter value is 0 in step S33, the process proceeds to step S36. Thus, the additional pressurization process ends.

[0470] Regarding the collection target particles of events E27 and E28, a waveform selection process is performed similarly to event E16 described above in (7-3), and through the waveform selection process, the second screening operation is selected as the screening operation to be applied to the screening of the collection target particles. As a result of selecting the second screening operation, the biological particle screening system executes step S30.

[0471] exist Figure 22 In step S31 , the bioparticle separation system starts another pressure increase process.

[0472] In step S32, the biological particle classification system obtains ΔTr(n).

[0473] Next, in step S33, the biological particle classification system refers to the step counter value. Since the step counter value is 0, the process proceeds to step S36. Thus, the additional pressurization process ends.

[0474] For the collection target particles of event E29, the same processing as event E21 is performed.

[0475] (8-3) Example of the algorithm

[0476] use Figure 30 The flowchart depicted in FIG. 1 illustrates an example of an algorithm embodying particle classification according to a modified example of the present disclosure. Figure 30 Only the Figure 29The changed part. The difference from the previous embodiment example is the processing content of the fourth processing performed by the output queue circuit. After the output of the pulse drive waveform in S200-1 to S200-3 and the update of the output queue, the processing in S200-4 to S200-6 is performed to output a multi-step rising edge. First, in S200-4, it is determined whether a rising edge is further applied after the output of the pulse drive waveform. In the case where the application of the rising edge is to be continuously applied as a result of the decision, in S200-5, the application timing of the rising edge is written to the memory. Subsequently, in S200-6, it is determined whether the trigger output of the rising edge has been completed. After the trigger output of the rising edge is completed, the processing returns to S200-4, and it is again determined whether the rising edge is applied. While the result of the decision on whether to apply the rising edge remains positive, the output of the falling edge is repeated. In the case where the result of the determination is negative, the output of the multi-step falling edge is completed.

[0477] (9) Screening processing example 3

[0478] The fourth step of the second screening operation explained in the above (7) is increased by one level. Further, the fourth step of the second screening operation explained in the above (8) is performed with a pressurization method having one or a plurality of levels.

[0479] In the present disclosure, in the second screening operation, a fifth step of preventing backflow of particles that can be generated due to the execution of the second step can be further executed before the fourth step. For example, in the fifth step, the pressure increased in the second step, for example, can be lowered again and thereafter raised again. Then, the fourth step is executed after the fifth step.

[0480] Referring to Figure 24 The second screening operation of executing the fifth step is explained in more detail. The figure depicts an example of a pulse drive waveform to be used for executing the second screening operation of executing the fifth step. As shown in the figure, the pulse drive waveform for executing the second screening operation in which the fifth step is executed has a second falling edge portion Wf2, a second rising edge portion Wr2, and a third rising edge portion Wr3 (including a rising edge portion Wr31, a rising edge portion Wr32, and a rising edge portion Wr33) in addition to the first falling edge portion Wf1 and the first rising edge portion Wr1.

[0481] As shown in the figure, the second falling edge portion Wf2 and the second rising edge portion Wr2 exist after the first falling edge portion Wf1 and the first rising edge portion Wr1. The second falling edge portion Wf2 and the second rising edge portion Wr2 can suppress the discharge flow, and can also suppress unnecessary oscillation.

[0482] Further, as shown in the drawing, there is a third rising portion Wr3 after the second rising portion Wr2. The configuration of the third rising portion can be appropriately changed according to the step counter mentioned later. Further, the third rising portion Wr3 can not be provided. Although the third rising portion Wr3 is a multi-step (three-step) rising edge having three rising portions (rising portion Wr31, rising portion Wr32, and rising portion Wr33), in the drawing, the number of steps can be changed. For example, the rising portion Wr33 can be omitted so that the number of steps is 2, or further, the rising portion Wr31 and the rising portion Wr32 can be omitted so that the number of steps is 1. Alternatively, a rising portion can be further added, and the third rising portion Wr3 can be configured as a rising portion having four steps or more.

[0483] In the present specification, a pulse drive waveform having a second falling portion and a second rising portion in addition to a first falling portion and a first rising portion is also referred to as a "pulse drive waveform including a falling edge / rising edge".

[0484] In the present disclosure, in a case where the interval ΔTp between one collection target particle and a subsequent collection target particle flowing immediately after the one collection target particle is long, the second sorting operation including the fifth step described above can be used as a sorting operation for sorting one collection target particle. Therefore, generation of a discharge flow can be suppressed, and stable particle sorting can be performed. From the viewpoint of suppressing a discharge flow in the second sorting operation described above, the second sorting operation including the fifth step is excellent.

[0485] Referring to Figure 25 The second sorting operation including the fifth step is described below.

[0486] The drawing depicts a simulation result (BI) of a flow rate in a connection flow path in a case where an actuator is driven by a pulse drive waveform including a rising edge (AI) to perform a second sorting operation not including the fifth step, and a simulation result (BII) of a flow rate in a connection flow path in a case where an actuator is driven by a pulse drive waveform including a falling edge / rising edge (AII) to perform a second sorting operation including the fifth step.

[0487] As shown in the figure, a significant discharge current is generated immediately after driving by the pulse driving waveform including the rising edge. As shown in the graph BI, the discharge current is generated after a time twl has passed from the start of applying the driving waveform. In contrast, the generation of the discharge current is delayed by adding the second falling edge portion in the pulse driving waveform including the falling edge / rising edge. As shown in the graph BII, the discharge current is generated after a time tw2 has passed from the start of applying the driving waveform, and the discharge current is generated after twl. The delay in generating the discharge current allows the particles collected in the collection flow path to further flow downstream of the collection flow path. Therefore, even if the discharge current is generated, if the generation of the discharge current is delayed, it is possible to prevent the collected particles from flowing back from the collection flow path (specifically, to the outside of the collection flow path) through the connection flow path.

[0488] Further, Figure 26 Analog results (I) of the time variation of the position of the collected particles in the flow direction in the case where the actuator is driven with the pulse driving waveform including the rising edge to perform the second sorting operation not including the fifth step, and analog results (II) of the time variation of the position of the collected particles in the flow direction in the case where the actuator is driven with the pulse driving waveform including the falling edge / rising edge to perform the second sorting operation including the fifth step are depicted.

[0489] In the figure, the vertical axis represents the position of the sorted particles in the flow direction. Moving toward “+” means moving toward a position further downstream in the collection flow path, and moving toward “-” means moving closer to the main flow path. “C” is the center position of the liquid supply flow path. “E” is the front end position of the connection flow path, i.e., the position of the inlet of the connection flow path on the main flow path side.

[0490] Each simulation is performed under two conditions in which the amplitude of the driving waveform is different (driving amplitude: medium, driving amplitude: small). In the figure, Am represents the analog results in the case where the driving amplitude is medium, and As represents the analog results in the case where the driving amplitude is small.

[0491] In order to collect the particles to be sorted in the collection flow path, the particles to be sorted need to advance toward the collection flow path from the position C of the liquid supply flow path connected in the connection flow path.

[0492] In the case where the driving amplitude is “medium”, the particles are transported toward the collection flow path past the position C in the connection flow path by both the pulse driving waveform including the rising edge and the pulse driving waveform including the falling edge / including the rising edge, and the sorting is successful.

[0493] In addition, it is known that in the case where fractionation is performed with the pulse driving waveform including the rising edge, a large amount of discharge current is generated immediately after driving, so the fractionated particles stay at a specific position during the period in which the discharge current is generated.

[0494] In contrast, in the case of fractionation using a pulse drive waveform including a falling edge / rising edge, the generation of the discharge flow is delayed, and thus the particles to be fractionated are already transported further downstream at the same time.

[0495] In the condition of a small drive amplitude, in the case of fractionation using a pulse drive waveform including a rising edge, stagnation due to the discharge flow is generated before the position C in the liquid supply flow path, and fractionation fails.

[0496] In contrast, in the case of fractionation using a pulse drive waveform including a falling edge / rising edge, there is a significant time difference between the drive and the generation of the discharge flow, and thus the particles to be fractionated can be transported further downstream, and the collection of the particles to be fractionated succeeds.

[0497] According to these results, compared with the screening using a pulse drive waveform including a rising edge, the screening using a pulse drive waveform including a falling edge / rising edge can be performed even with a smaller drive amplitude, and can be more stable particle screening.

[0498] As described above, in the second screening operation, a fifth step of preventing backflow of particles that can be generated due to the execution of the second step can be further performed. The fifth step can be provided between the second step and the fourth step on the time axis of one screening operation.

[0499] The waveform applied for performing the fifth step can have a falling edge portion and a rising edge portion (and optionally a hold time portion between these two waveform segments).

[0500] For example, the amplitude of the falling edge portion for performing the fifth step can be equal to or greater than 0.1 V, preferably equal to or greater than 1 V, and more preferably equal to or greater than 5 V. Further, for example, the amplitude of the falling edge portion can be equal to or less than 150 V, preferably equal to or less than 100 V, and more preferably equal to or less than 50 V.

[0501] For example, the duration of the falling edge portion for performing the fifth step can be equal to or greater than 1 µs, preferably equal to or greater than 5 µs, and more preferably equal to or greater than 15 µs. Further, for example, the duration of the falling edge portion can be equal to or shorter than 100 µs, preferably equal to or shorter than 60 µs, and more preferably equal to or shorter than 30 µs.

[0502] For example, the amplitude of the rising edge portion for performing the fifth step can be equal to or greater than 0.1 V, preferably equal to or greater than 1 V, and more preferably equal to or greater than 5 V. Further, for example, the amplitude of the rising edge portion can be equal to or less than 150 V, preferably equal to or less than 100 V, and more preferably equal to or less than 50 V.

[0503] For example, the duration of the rising edge portion for executing the fifth step may be equal to or greater than 1 μs, preferably equal to or greater than 5 μs, and more preferably equal to or greater than 15 μs. In addition, for example, the duration of the rising edge portion may be equal to or less than 100 μs, preferably equal to or less than 60 μs, and more preferably equal to or less than 30 μs.

[0504] For example, the hold time of the fifth step may be equal to or greater than 0 μs, and may preferably be equal to or greater than 10 μs. Furthermore, for example, the hold time may be equal to or less than 100 μs, preferably equal to or less than 60 μs, and more preferably equal to or less than 40 μs. That is, the hold time may be 0 μs, but may be longer than 0 μs.

[0505] (10) Configuration example of biological particle screening equipment

[0506] In one embodiment, the biological particle screening system of the present disclosure can be configured as a biological particle screening device, for example, a cell screening device. The biological particle screening device can be a device for analyzing and / or screening biological particles in a microchip without forming droplets. The biological particle screening device can be configured to perform the above-mentioned inspection process. Figure 32A and Figure 32B This embodiment will be described.

[0507] Figure 32A A schematic diagram depicts a configuration example of a bioparticle fractionation microchip and a configuration example of a bioparticle analysis device including the microchip. Figure 32B An example of a flow chart of a bioparticle screening operation performed by a bioparticle analysis apparatus is depicted.

[0508] Figure 32A The biological particle separation microchip 150 described in has a sample liquid flow path 152 and a sheath liquid flow path 154 that merges with the sample liquid flow path 152 at a junction 162. The biological particle separation microchip 150 is further provided with a sample liquid inlet 151 and a sheath liquid inlet 153.

[0509] Note that the portion of the sheath fluid path 154 is composed of Figure 32A The portion indicated by the dotted line is at a position lower than the sample liquid flow path 152 indicated by the solid line (a position offset in the optical axis direction mentioned later), and at a position where the flow path indicated by the dotted line and the flow path indicated by the solid line intersect, these flow paths are not connected to each other. Figure 32A, the sample liquid flow path 152 is depicted as making two turns between the sample liquid inlet 151 and the confluence portion 162, and this is to make it easier to distinguish the sample liquid flow path 152 from the sheath liquid flow path 154. The sample liquid flow path 152 may be configured linearly between the sample liquid inlet 151 and the confluence portion 162 without making turns in this manner.

[0510] In the biological particle screening operation, a sample liquid including biological particles is introduced into the sample liquid flow path 152 from the sample liquid inlet 151 , and a sheath liquid not including biological particles is introduced into the sheath liquid flow path 154 from the sheath liquid inlet 153 .

[0511] The biological particle fractionation microchip 150 includes a merging flow channel 155 having a merging portion 162 at one end thereof.

[0512] The sample liquid and the sheath liquid merge at the confluence 162 and then flow toward the particle screening unit 157 in the merging flow path 155. Specifically, the sample liquid and the sheath liquid merge at the confluence 162 and form, for example, a laminar flow in which the sample liquid is surrounded by the sheath liquid. Preferably, the biological particles are adjacent to each other in approximately a line in the laminar flow. The flow path structure including the sample liquid flow path 152 and the two sheath liquid flow paths 154 merging at the confluence 162, and the merging flow path 155 having the confluence 162 at one end thereof forms a laminar flow including biological particles flowing adjacent to each other in approximately a line. Thus, in the luminescence of the sensing area 156 described below, it is easy to distinguish between light generated by luminescence toward one biological particle and light generated by luminescence toward another biological particle.

[0513] The biological particle fractionation microchip 150 further includes a particle fractionation unit 157 at the other end of the merging flow channel 155 . Figure 12 Depicts an enlarged view of the particle screening unit 157. Figure 12 As shown in FIG. 1A , at the other end, the merging flow path 155 is connected to the bioparticle collecting flow path 159 via the connecting flow path 170. Figure 2 As depicted in FIG. 1A , the merging flow path 155 , the connecting flow path 170 , and the biological particle collecting flow path 159 may be coaxial.

[0514] In the case of collecting the target particles and flowing them to the particle screening unit 157, as shown in FIG. Figure 2 As shown in FIG. 1B , a flow is formed that flows from the merging flow path 155 through the connecting flow path 170 into the biological particle collecting flow path 159, and the collection target particles are collected in the biological particle collecting flow path 159. In this way, the collection target particles flow into the biological particle collecting flow path 159 through the connecting flow path 170.

[0515] In a case where the flow of the biological particles that are not the target particles to the particle fractionation unit 157, the flow of the biological particles that are not the target particles to the branch flow path 158, as shown in FIG. 18B. In this case, the flow to the biological particle collection flow path 159 is not formed. Figure 2 In this case, the flow to the biological particle collection flow path 159 is not formed.

[0516] As shown in FIG. 18C, the biological particle collection flow path 159 is formed to linearly extend from the particle fractionation unit 157, make a U-turn, and then reach the same surface as the surface formed by the sample liquid inlet 151 and the sheath liquid inlet 153. The liquid flowing through the biological particle collection flow path 159 is discharged from the collection flow path terminal 163 to the outside of the chip. Figure 32A As shown in FIG. 18D, the two branch flow paths 158 are also formed to linearly extend from the particle fractionation unit 157, make a U-turn, and then reach the same surface as the surface formed by the sample liquid inlet 151 and the sheath liquid inlet 153. The liquid flowing in the branch flow paths 158 is discharged from the branch flow path terminals 166 to the outside of the chip.

[0517] Figure 32A In In FIG. 18E, the representation method of the biological particle collection flow path 159 is changed from a solid line to a broken line at the portion where the biological particle collection flow path 159 makes a U-turn. This change indicates that the position in the optical axis direction is changed in the middle. By changing the position in the optical axis direction in this way, the biological particle collection flow path 159 and the branch flow paths 158 are not communicated at the portion where the biological particle collection flow path 159 and the branch flow paths 158 cross.

[0518] Figure 32A The collection flow path terminal 163 and the two branch flow path terminals 166 are each formed on the surface on which the sample liquid inlet 151 and the sheath liquid inlet 153 are formed. In addition, an introduction flow path inlet 164 for introducing a liquid into the introduction flow path 161 is also formed on the surface. In this way, in the biological particle fractionation microchip 150, all the inlets from which a liquid is introduced and the outlets from which a liquid is discharged are formed on one surface. Therefore, it becomes easier to attach the chip to the biological particle analysis device 100. For example, connection between the flow path provided to the biological particle analysis device 100 and the flow path of the biological particle fractionation microchip 150 becomes easier than in a case where the inlets and / or the outlets are formed on two or more surfaces.

[0519] As shown in FIG. 18F, the biological particle fractionation microchip 150 has an introduction flow path 161 for introducing a liquid into a connection flow path 170.

[0520] As shown in FIG. 18G and FIG. 18H, the biological particle fractionation microchip 150 has an introduction flow path 161 for introducing a liquid into a connection flow path 170. Figure 32A Figure 2

[0521] ​​​The connection flow path 170 is filled with the liquid by introducing the liquid from the introduction flow path 161 into the connection flow path 170. Thus, it is possible to prevent non-target biological particle from entering the biological particle collection flow path 159.

[0522] The biological particle sieving microchip 150 has two branch flow paths 158 connected to the merging flow path 155 at the other end of the merging flow path 155. In this way, in the biological particle sieving microchip used in the present technology, the merging flow path can branch into a connection flow path and at least one branch flow path.

[0523] The biological particle other than the target particle flows to either of the two branch flow paths 158 without entering the biological particle collection flow path 159.

[0524] Further, as Figure 32A indicated, the biological particle sieving microchip 150 is included as a part of the biological particle analysis device 100 including a light emitting unit 101, a detection unit 102, and a control unit 103 in addition to the microchip. The light emitting unit 101, the detection unit 102, and the control unit 103 correspond to the light irradiation unit 6101, the detection unit 6102, and the information processing unit 6103 mentioned in (2) above, respectively, and the description thereof also applies to the present structural example. As Figure 13 indicated, the control unit 103 of the biological particle analysis device 100 can include a signal processing unit 104, a determination unit 105, and a hierarchical control unit 106.

[0525] As Figure 32B indicated, the biological particle sieving operation using the above-described biological particle sieving microchip 150 includes a flow-through step S101 of causing a liquid containing biological particles to flow through the merging flow path 155, a determination step S102 of determining whether the biological particles flowing through the merging flow path 155 are the collection target particles, and a collection step S103 of collecting the collection target particles to the biological particle collection flow path 159. Each step will be described below.

[0526] (10-1) Flow-through step

[0527] In the flow-through step S101, a sample liquid including biological particles and a sheath liquid not including biological particles are introduced from the sample liquid inlet 151 and the sheath liquid inlet 153, and are introduced into the sample liquid flow path 152 and the sheath liquid flow path 154, respectively.

[0528] The sample liquid and the sheath liquid are merged at the merging portion 162, and for example, a laminar flow in which the sample liquid is surrounded by the sheath liquid is formed. Preferably, the biological particles are approximately in line with each other in the laminar flow. That is, at the flow-through step S101, a laminar flow including biological particles flowing in approximately one row next to each other can be formed.

[0529] In this manner, in the flow step S101 , the liquid containing the biological particles is caused to flow particularly as a laminar flow in the merging flow path 155 . The liquid flows from the merging portion 162 to the particle fractionating unit 157 in the merging flow path 155 .

[0530] (10-2) Determine the steps

[0531] In the determination step S102, it is determined whether the biological particle flowing through the merging flow path 155 is a collection target particle. The determination unit 105 can make this determination. The determination unit 105 can make the determination based on the light emitted by the light emitting unit 101 onto the biological particle.

[0532] The signal processing unit 104 included in the control unit 103 can process the waveform of the digital electrical signal obtained by the detection unit 102 and generate information (data) regarding the characteristics of the light to be determined by the determination unit 105. As information related to the characteristics of the light, for example, the signal processing unit 104 can obtain one, two, or three of the width, height, and area of ​​the waveform from the waveform of the digital electrical signal. In addition, for example, the information regarding the characteristics of the light may include the time at which the light was sensed. In particular, in embodiments in which scattered light and / or fluorescence are sensed, the processing performed by the signal processing unit 104 described above can be performed.

[0533] The determination unit 105 included in the control unit 103 determines whether the biological particles passing through the flow path are flowing collection target particles based on light generated by light emission onto the biological particles.

[0534] (10-3) Collection steps

[0535] In the collecting step S103, the biological particles determined as the collection target particles in the determining step S102 are collected in the biological particle collecting flow path 159. The collecting step S103 is performed in the particle fractionation unit 157 in the microchip 150. In the particle fractionation unit 157, the laminar flow flowing through the merging flow path 155 is branched into two branching flow paths 158. Figure 10 The particle screening unit 157 shown in FIG has two branch flow paths 158, but the number of branch flow paths is not limited to two. For example, the particle screening unit 157 may be provided with one or more (e.g., two, three, or four, etc.) branch flow paths. The branch flow paths may be configured as follows: Figure 10 The structure may be configured to branch in a Y-shape on one plane, or may be configured to branch in three dimensions.

[0536] In the collecting step S103, a pressure change in the biological particle collecting flow path 159 causes the collection target particles to be collected into the biological particle collecting flow path through the connection flow path. The collection can be performed by generating a negative pressure in the biological particle collecting flow path 159, for example, as described above. The negative pressure can be generated by deforming the wall defining the biological particle collecting flow path 159 by the actuator 107 (specifically, a piezoelectric actuator) attached outside the microchip 150, for example. The negative pressure can form a flow into the biological particle collecting flow path 159. To generate the negative pressure, the actuator 107 can be attached outside the microchip 150, for example, so that the wall of the biological particle collecting flow path 159 can be deformed. The deformation of the wall can change the inner space of the biological particle collecting flow path 159 and generate the negative pressure. The actuator 107 can be a piezoelectric actuator, for example. When the collection target particles are sucked into the biological particle collecting flow path 159, the sample liquid included in the laminar flow or the sample liquid and the sheath liquid included in the laminar flow can also flow into the biological particle collecting flow path 159. In this way, the collection target particles are sieved in the particle sieving unit 157 and collected into the biological particle collecting flow path 159.

[0537] To prevent the biological particles other than the collection target particles from entering the biological particle collecting flow path 159 through the connection flow path 170, the connection flow path 170 is provided with the introduction flow path 161. The liquid is introduced from the introduction flow path 161 into the connection flow path 170. Due to the introduction of the liquid, the connection flow path 170 is filled with the liquid. Further, a part of the liquid forms a flow from the connection flow path 170 toward the merging flow path 155, and this prevents the biological particles other than the collection target particles from entering the biological particle collecting flow path 159. The liquid that forms the flow from the connection flow path 170 toward the merging flow path 155 does not flow in the merging flow path 155 due to the flow of the liquid flowing through the merging flow path 155 and toward the branch flow path 158, and flows through the branch flow path 158 similarly to the liquid.

[0538] It should be noted that the remaining part of the liquid introduced into the connection flow path 170 flows to the biological particle collecting flow path 159. Therefore, the biological particle collecting flow path 159 can be filled with the liquid.

[0539] The flow that has flowed to the branch flow path 158 can be discharged to the outside of the microchip through the branch flow path terminal 160. Further, the collection target particles collected into the biological particle collecting flow path 159 can be discharged to the outside of the microchip through the collection flow path terminal 163. The collection flow path terminal 163 can be connected with a container via a flow path such as a tube. The collection target particles can be collected in the container.

[0540] (11) Embodiment

[0541] The driving waveform and the sorting process explained in the sorting process example 3 above (9) are implemented in the biological particle sorting system according to the present disclosure, and the sorting process is performed using the driving waveform. The sorting process is performed in the single particle priority mode explained in (7-1) and (7-5) above, and the sorting process is performed to form an emulsion containing the collection target particles. That is, in the present implementation example, the biological particle sorting system is configured as an emulsion production apparatus, and the advantage of the present disclosure is examined by evaluating the sorting performance of the emulsion production apparatus.

[0542] To evaluate the sorting performance, a mixed bead sample including two types of fluorescent beads having mutually different fluorescent characteristics is prepared as a sample including the collection target particles and the non-collection target particles. The mixed bead sample is introduced into the emulsion production apparatus, and the sorting process is performed under each condition of various target concentrations and event rates. Three concentrations of 96.9%, 48.7%, and 18.2% are used as the various target concentrations.

[0543] Further, to verify the experimental results, a simulation is also performed assuming that the collection rate follows a Poisson distribution in the case where the particles arrive at the classification unit. The simulation is performed for each of the case where the driving waveform explained in the sorting process example 3 above (9) is applied and the case where the pulse waveform of AI in (I, upper side) is applied. Figure 6

[0544] An emulsion is formed by the sorting method. Further, the collection rate and the purity of the collection target particles are evaluated under each condition. The collection rate, the purity, and the evaluation results are explained below.

[0545] The collection rate is calculated by counting the log file of the emulsion production apparatus and the collected emulsion particles. Figure 33A The measurement results of the collection rate are plotted. The graph plots the collection rate measured for each case of the three target concentrations. The collection rates in the cases of 96.9%, 48.7%, and 18.2% are indicated by black circular marks, dark gray triangular marks, and light gray square marks, respectively.

[0546] Further, the simulation results in the case where the driving waveform explained in the sorting process example 3 above (9) is applied are also plotted in (I) of Figure 33A , and the example simulation in (I) of Figure 33A , and the example simulation in (I) of Figure 6 ​The simulation results in the case of the above-described pulse waveform of the AI in the present disclosure. In the figure, the black dotted line indicates the simulation results in the case of applying the present disclosure in the case of the target concentration 96.9%, and the black dot-dashed line indicates the simulation results in the case of applying the conventional technique in the case of the same target concentration. The dark gray dotted line indicates the simulation results in the case of applying the present disclosure in the case of the target concentration 48.7%, and the dark gray dot-dashed line indicates the simulation results in the case of applying the conventional technique in the case of the same target concentration. The light gray dotted line indicates the simulation results in the case of applying the present disclosure in the case of the target concentration 18.2%, and the light gray dot-dashed line indicates the simulation results in the case of applying the conventional technique in the case of the same target concentration.

[0547] From the comparison of the collection rate of the experimental results and the simulation results of the implementation examples, it can be seen that the collection rate of the experimental results closely matches the simulation results.

[0548] In addition, the comparison between the results of the collection rate in the case of applying the driving waveform according to the present disclosure and the results of the collection rate of the conventional technique indicates that, in all conditions of the three target concentrations, the value of the results of the former case is higher than the value of the results of the latter case. For example, in the condition that the target concentration is 48.7% and the event rate is 6000 eps, the experimental results and the simulation results in the case of applying the driving waveform according to the present disclosure are about 66%, while the simulation results of the conventional technique are about 57%. In this way, it can be seen that the present disclosure can improve the collection rate of the target particles.

[0549] In addition, as the event rate increases, the difference between the collection rate in the case of applying the driving waveform according to the present disclosure and the collection rate in the case of applying the conventional technique increases. This is considered to be because, as the event rate increases, the rate of collecting target particles arriving at short time intervals increases, and therefore the target particles that cannot be collected increase as the conventional technique in which the time interval of the continuous screening is long. That is, it is also known that the present disclosure particularly significantly improves the collection rate in the case where the event rate has increased.

[0550] Figure 33B The measurement results of the purity are depicted. The purity is defined as the ratio of the emulsion particles including only the single collection target beads to the number of the emulsion particles including beads (regardless of the bead type and the number) in the collected emulsion. Figure 33C A portion of the image obtained by imaging the collected emulsion with a fluorescence microscope is depicted. Figure 33C A on the left side is an image imaged in the condition that only the collection target beads produce fluorescence, and the white dots (indicating the fluorescence of the collection target beads) in each emulsion particle are the collection target beads. Figure 33CB on the right side is an image imaged under the condition that only non-collection target beads generate fluorescence. From these photographs, it can be seen that each of the collected emulsion particles includes only one collection target bead. Furthermore, the experimental results of purity show that under all measurement conditions, emulsions including emulsion particles containing a single collection target particle were formed at a high purity of equal to or greater than about 98%. In this way, it has been revealed that the present application makes it possible to fractionate emulsion particles having collection target particles at a high purity.

[0551] These results confirm that, compared to conventional techniques, the present disclosure makes it possible to produce emulsions including emulsion particles containing fractionation target particles at a high purity, while achieving a high collection rate.

[0552] 2. Second embodiment (biological particle fractionation method)

[0553] The present disclosure also provides a biological particle fractionation separation method. The method includes performing the fractionation process explained in "1. First embodiment". More specifically, the method includes performing the fractionation process explained in "(7) Fractionation process example 1", "(8) Fractionation process example 2", or "(9) Fractionation process example 3" in "1. First embodiment". The above "1. First embodiment" relating to these fractionation processes also applies to the biological particle fractionation method of the present disclosure.

[0554] Specifically, the fractionation operation is the first fractionation operation explained in "1. First embodiment". That is, in one embodiment, the present disclosure provides a biological particle fractionation method including fractionating collection target particles flowing through a flow path into a collection flow path. The method can include, as one fractionation operation for fractionating the collection target particles, performing a first step to reduce the pressure in the collection flow path from a reference pressure and to guide the collection target particles into the collection flow path, a second step to restore the pressure reduced at the first step to the reference pressure, and a third step to mitigate an excessive pressure change due to performing the second step.

[0555] Furthermore, in addition to performing the first fractionation operation, the biological particle fractionation method can include performing the second fractionation operation explained in "1. First embodiment". That is, the biological particle fractionation method can include performing the first fractionation operation or the second fractionation operation as one fractionation operation for fractionating the collection target particles.

[0556] The biological particle fractionation method (specifically, the first fractionation operation and the second fractionation operation) can be performed by the biological particle fractionation system mentioned in "1. First embodiment", and specifically, can be performed by the fractionation unit.

[0557] Further, the biological particle screening method can include a decision process based on the interval between the collected target particle and the subsequent biological particle explained in "1. First Embodiment". For example, the decision process is a process of deciding which screening operation to apply to screen one collected target particle based on whether the interval is within a predetermined numerical range.

[0558] The decision process can be executed by the biological particle screening system mentioned in "1. First Embodiment". And specifically, it can be executed by the information processing unit mentioned in "1. First Embodiment".

[0559] Further, for example, the biological particle screening method can be executed as an emulsion production method. That is, the present disclosure also provides an emulsion production method including executing the first screening operation. In the emulsion production method, the first screening operation can be executed as one screening operation for screening the collected target particle. Further, in one embodiment, the first screening operation or the second screening operation can be executed as one screening operation for screening the collected target particle. Through these screening operations, emulsion particles containing one biological particle are produced. Further, in the emulsion production method, a process of deciding which screening operation to apply to screen one collected target particle can be executed.

[0560] Further, in the emulsion production method, preferably, the single particle priority mode mentioned in "1. First Embodiment" above can be adopted. By executing the decision process in the single particle priority mode, it is possible to prevent two or more biological particles from being included in each emulsion particle in the emulsion to be manufactured.

[0561] Further, the present disclosure also provides a program for causing a biological particle screening system (specifically, a biological particle screening apparatus or an emulsion production apparatus, for example, a flow cytometer) to execute a biological particle screening method. For example, the program can be stored on the biological particle screening system (specifically, an information processing device, and more specifically, a storage unit), or can be stored on an information storage medium. For example, the information storage medium can be an SD card, a micro SD card, a CD, a DVD, a flash memory, or a magnetic recording medium.

[0562] 3. Third Embodiment (Biological Particle Screening Chip)

[0563] The present disclosure also provides a biological particle sieving chip for performing the biological particle sieving method (specifically, the sieving process) mentioned in the above "1. First Embodiment" and "2. Second Embodiment". That is, the description in the above "1. First Embodiment" and "2. Second Embodiment" also applies to the biological particle sieving chip of the present disclosure. For example, the chip can be a chip having the flow path system described in the above "1. First Embodiment". For example, the chip can be the chip described in (10) in the above "1. First Embodiment", but is not limited to this chip.

[0564] That is, in one embodiment, the present disclosure also provides a biological particle sieving chip having a collection flow path into which a collection target particle flowing through a flow path is sieved, and the biological particle sieving chip is used to perform a biological particle sieving method according to the present disclosure. For example, the biological particle sieving method can include performing a sieving operation as one sieving operation for sieving the collection target particle, the sieving operation including: a first step of reducing a pressure in the collection flow path from a reference pressure and guiding the collection target particle into the collection flow path; a second step of restoring the pressure reduced in the first step to the reference pressure; and a third step of mitigating an excessive pressure change due to the performance of the second step. By performing the sieving operation, the collection target particle is sieved.

[0565] Further, in addition to performing the first sieving operation, the biological particle sieving method can include performing the second sieving operation described in the above "1. First Embodiment". That is, the biological particle sieving method can include performing the first sieving operation or the second sieving operation as one sieving operation for sieving the collection target particle.

[0566] Further, the present disclosure also provides a biological particle sieving chip for performing an emulsion production method according to the present disclosure. The method of preparing an emulsion is as described in the above "1. First Embodiment" and "2. Second Embodiment". The chip is particularly suitable for producing an emulsion including an emulsion particle, the emulsion particle including a biological particle.

[0567] Note that the present disclosure can also employ the following configurations. [1]

[0569] A biological particle sieving system configured to sieve a collection target particle flowing through a flow path into a collection flow path, wherein

[0570] As one sieving operation for sieving the collection target particle, the biological particle sieving system is configured to be able to perform:

[0571] a first step of reducing a pressure in the collection flow path from a reference pressure and guiding the collection target particle into the collection flow path,

[0572] a second step of restoring the pressure reduced in the first step to the reference pressure, and

[0573] a third step of mitigating an excessive pressure change due to the execution of the second step. [2]

[0575] The biological particle sieving system according to [1], wherein

[0576] The biological particle sieving system is configured to change the pressure in the collection flow path by deforming the collection flow path. [3]

[0578] The biological particle sieving system according to [1] or [2], wherein

[0579] The biological particle sieving system is configured to apply a pulse voltage to the actuator to change the pressure in the collection flow path in the one sieving operation, and

[0580] The drive waveform of the pulse voltage is configured so that the actuator performs the first step, the second step, and the third step. [4]

[0582] The biological particle sieving system according to [3], wherein

[0583] The drive waveform of the pulse voltage has:

[0584] a first falling edge portion for performing the first step,

[0585] a rising edge portion for performing the second step, and

[0586] a second falling edge portion for performing the third step. [5]

[0588] The biological particle sieving system according to any one of [1] to [4], comprising:

[0589] an information processing unit that performs a determination process based on an interval between a collection target particle and a subsequent biological particle, wherein

[0590] The determination process is a process of determining which sieving operation to apply to the one collection target particle for sieving based on whether the interval is within a predetermined numerical range. [6]

[0592] The biological particle sieving system according to [5], wherein

[0593] In the determination process, the information processing unit refers to a step counter value related to the number of times the sieving operation can be performed. [7]

[0595] The biological particle sieving system according to any one of [1] to [6], wherein

[0596] As a one-time sieving operation, the biological particle sieving system is configured to be able to perform:

[0597] a sieving operation (hereinafter, also referred to as "first sieving operation") that performs the first step, the second step, and the third step, or

[0598] a second sieving operation that performs the first step and the second step, and further performs a fourth step that eliminates a shift of the collection flow path generated by performing the third step, in a case where a predetermined condition is satisfied. [8]

[0600] The biological particle sieving system according to [7], comprising:

[0601] an information processing unit that performs determination processing based on an interval between the collection target particle and the subsequent biological particle, wherein

[0602] the information processing unit selects the first sieving operation or the second sieving operation based on the interval. [9]

[0604] The biological particle sieving system according to [7] or [8], wherein

[0605] In the fourth step, the biological particle sieving system is configured to increase the pressure by one level or to increase the pressure by a plurality of levels.

[10]

[0607] The biological particle sieving system according to [8] or [9], wherein

[0608] In a case where the second sieving operation is performed, the information processing unit determines whether to perform the fourth step based on an interval between a completion time of the second step in the second sieving operation and a time at which the subsequent biological particle is sensed.

[11]

[0610] The biological particle sieving system according to any one of [7] to

[10] , wherein

[0611] the biological particle sieving system is configured to apply a pulse voltage to the actuator to change the pressure in the collection flow path, and

[0612] The drive waveform of the pulse voltage is configured so that the actuator performs the first or second sieving operation in accordance with the interval between the collected target particles and the subsequent biological particles.

[12]

[0614] The biological particle sieving system according to

[11] , wherein

[0615] The drive waveform for performing the first sieving operation has a first falling edge portion for performing the first step, a rising edge portion for performing the second step, and a second falling edge portion for performing the third step, and

[0616] The drive waveform for performing the second sieving operation has a first falling edge portion for performing the first step and a rising edge portion for performing the second step, and further has a second rising edge portion for performing the fourth step in the case where the fourth step is performed.

[13]

[0618] The biological particle sieving system according to

[12] , wherein

[0619] The biological particle sieving system is configured to increase the pressure by one level in the fourth step, and

[0620] The second rising edge portion for performing the fourth step has a single-step rising edge.

[14]

[0622] The biological particle sieving system according to

[12] or

[13] , wherein

[0623] The biological particle sieving system is configured to be able to increase the pressure at a plurality of levels in the fourth step, and is configured to be able to change the number of levels at which the pressure is increased.

[15]

[0625] The biological particle sieving system according to any one of [7] to

[14] , wherein

[0626] In the second sieving operation, a fifth step of preventing backflow of particles that can occur due to the performance of the second step is further performed.

[16]

[0628] The biological particle sieving system according to any one of [1] to

[15] , wherein

[0629] The biological particle sieving system is configured as an emulsion production apparatus, and

[0630] The emulsion includes emulsion particles having the sieved collected target particles.

[17]

[0632] A biological particle fractionation method, comprising:

[0633] fractionating collection target particles flowing through a flow path into a collection flow path, wherein

[0634] as one fractionation operation for fractionating the collection target particles, the biological particle fractionation method includes performing:

[0635] a first step of decreasing a pressure in the collection flow path from a reference pressure and guiding the collection target particles into the collection flow path,

[0636] a second step of restoring the pressure decreased in the first step to the reference pressure, and

[0637] a third step for mitigating an excessive pressure change due to performing the second step.

[18]

[0639] A biological particle fractionation chip, comprising:

[0640] a collection flow path in which collection target particles flowing through a flow path are fractionated;

[0641] the biological particle fractionation chip fractionates the collection target particles by performing a fractionation operation, as one fractionation operation for fractionating the collection target particles, the fractionation operation includes:

[0642] a first step of decreasing a pressure in the collection flow path from a reference pressure and guiding the collection target particles into the collection flow path,

[0643] a second step of restoring the pressure decreased in the first step to the reference pressure, and

[0644] a third step for mitigating an excessive pressure change due to performing the second step.

[0645] [Reference numeral list]

[0646] 155: main flow path

[0647] 156: particle sensing unit

[0648] 159: collection flow path

[0649] 161: liquid supply flow path

[0650] 170: connection flow path

[0651] 107: actuator

Claims

1. A biological particle screening system, configured to screen target particles flowing through a flow path into a collection flow path, wherein: As a primary screening operation for screening the collected target particles, the biological particle screening system is configured to be able to perform: In the first step, the pressure in the collection flow path is reduced from a reference pressure, and the collection target particles are guided into the collection flow path. a second step of restoring the pressure reduced in the first step to the reference pressure, and The third step is to alleviate the excessive pressure change caused by performing the second step.

2. The biological particle screening system according to claim 1, wherein: The biological particle fractionation system is configured to change the pressure in the collection flow path by deforming the collection flow path.

3. The biological particle screening system according to claim 1, wherein: The biological particle screening system is configured to apply a pulse voltage to the actuator to change the pressure in the collection flow path during the primary screening operation, and The driving waveform of the pulse voltage is configured to cause the actuator to perform the first step, the second step, and the third step.

4. The biological particle screening system according to claim 3, wherein: The driving waveform of the pulse voltage has: The first falling edge portion is used to execute the first step, The rising edge portion is used to perform the second step, and The second falling edge portion is used to execute the third step.

5. The biological particle screening system according to claim 1, comprising: An information processing unit that performs determination processing based on an interval between a collection target particle and a subsequent biological particle, wherein The determination process is a process of determining which screening operation to apply to screen one of the collection target particles based on whether the interval is within a predetermined numerical range.

6. The biological particle screening system according to claim 5, wherein: In the determination process, the information processing unit refers to a step counter value associated with the number of times the screening operation can be performed.

7. The biological particle screening system according to claim 1, wherein: As a screening operation, the biological particle screening system is configured to be able to perform: A screening operation (hereinafter, also referred to as "first screening operation"), performing the first step, the second step and the third step, or The second screening operation is to execute the first step and the second step, and further execute a fourth step of eliminating the deviation of the collection flow path generated by executing the third step when a predetermined condition is satisfied.

8. The biological particle screening system according to claim 7, comprising: An information processing unit that performs a determination process based on an interval between a collection target particle and a subsequent biological particle, wherein The information processing unit selects the first screening operation or the second screening operation based on the interval.

9. The biological particle screening system according to claim 7, wherein: In a fourth step, the biological particle fractionation system is configured to increase the pressure by one level or to increase the pressure by multiple levels.

10. The biological particle screening system according to claim 8, wherein: In the case where the second screening operation is performed, the information processing unit determines whether to perform the fourth step based on an interval between a completion time of the second step in the second screening operation and a time when the subsequent biological particle is sensed.

11. The biological particle screening system according to claim 7, wherein The biological particle separation system is configured to apply a pulse voltage to the actuator to change the pressure in the collection flow path, and The driving waveform of the pulse voltage is configured to cause the actuator to perform the first screening operation or the second screening operation according to an interval between a collection target particle and a subsequent biological particle.

12. The biological particle screening system according to claim 11, wherein: The driving waveform for performing the first screening operation has a first falling edge portion for performing the first step, a rising edge portion for performing the second step, and a second falling edge portion for performing the third step, and The driving waveform for executing the second screening operation has a first falling edge portion for executing the first step and a rising edge portion for executing the second step, and further has a second rising edge portion for executing the fourth step when executing the fourth step.

13. The biological particle screening system according to claim 12, wherein: The biological particle screening system is configured to increase the pressure by one level in the fourth step, and The second rising edge portion for executing the fourth step has a single-step rising edge.

14. The biological particle screening system according to claim 12, wherein: The biological particle fractionation system is configured to be capable of increasing the pressure at a plurality of levels in the fourth step, and is configured to be capable of changing the number of levels to which the pressure is increased.

15. The biological particle screening system according to claim 7, wherein: In the second screening operation, a fifth step of preventing backflow of particles that may be generated by the execution of the second step is further performed.

16. The biological particle screening system according to claim 1, wherein: The bioparticle screening system is configured as an emulsion production device, and The emulsion includes emulsion particles with sieved collection target particles.

17. A method for screening biological particles, comprising: The target particles flowing through the flow path are screened into the collection flow path, wherein As a primary screening operation for screening the collected target particles, the biological particle screening method includes performing: In the first step, the pressure in the collection flow path is reduced from a reference pressure, and the collection target particles are guided into the collection flow path. a second step of restoring the pressure reduced in the first step to the reference pressure, and The third step is for alleviating excessive pressure changes caused by executing the second step.

18. A bioparticle screening chip, comprising: a collection flow path, wherein the collection target particles flowing through the flow path are screened; The bio-particle screening chip screens the collected target particles by performing a screening operation, as a screening operation for screening the collected target particles, the screening operation includes: In the first step, the pressure in the collection channel is reduced from a reference pressure, and the collection target particles are guided into the collection channel. a second step of restoring the pressure reduced in the first step to the reference pressure, and The third step is for alleviating excessive pressure changes caused by executing the second step.

Images