Flow cytometry sorter, flow cytometry method and sound wave particle control micro-channel chip

Abstract

The invention provides a flow cell sorter, a method and a sound wave particle control micro-channel chip, and relates to the field of flow sorting, a liquid flow system of the flow cell sorter comprises a sound wave particle spacing adjusting unit, the sound wave particle spacing adjusting unit is configured to generate a sound standing wave field in a sample flow channel, the pitch line or the pitch surface of the sound standing wave field is perpendicular to the liquid flow direction, so that the particles move towards the pitch line or the pitch surface under the action of sound radiation force, and the particles are arranged at preset intervals in the liquid flow direction. According to the invention, under the action of the specially designed micro-channel and the piezoelectric acoustic wave device, the spacing of particles in a sample flow is adjusted, so that the particles are uniformly distributed, the probability that the particles are wrapped by the same liquid drop is reduced, and the sorting yield is improved; the particles are focused to the position of a sound wave nodal line in advance by adopting a sound wave focusing technology before sheath fluid focusing, and the width of the sound wave nodal line is relatively small, so that the consistency of the positions of the sample particles and the consistency of the flow velocity at a high sample flow velocity can be ensured, and the sorting purity and the yield are improved.

Description

Technical Field

[0001] This invention relates to the field of flow cytometry technology, and particularly to a flow cytometer, method, and acoustic particle manipulation microchannel chip. Background Technology

[0002] Flow cytometry is an important tool for cell analysis and sorting. Its working principle involves rapidly passing a liquid sample containing cells through the flow cytometer, allowing each cell to continuously pass through a sensing area, thus acquiring the cell's characteristic information. Subsequently, by charging the cell-containing droplets, the charged droplets are deflected under the influence of a subsequent electric field, thereby achieving cell sorting.

[0003] Currently, sheath fluid focusing is used to arrange the cells to be tested in a single file and pass through the laser detection zone. The width of the central sample stream during sheath fluid focusing depends on the flow ratio of the sheath fluid to the sample stream. Because different sample flow rates need to be set during flow cytometry, a wider sample stream distribution occurs at high flow rates, resulting in variations in cell flow rate at different locations. While this doesn't significantly impact the analysis, droplet delay during sorting is closely related to cell flow rate, and differences in cell flow rate will lead to decreased sorting accuracy.

[0004] Furthermore, after the liquid is ejected from the nozzle of the flow cytometer, it is agitated into droplets. These droplets encapsulate cells, and a sorting algorithm determines whether to sort the target droplet. When a droplet contains both target and non-target cells, it may be discarded based on user requirements. This leads to a decrease in the sorting efficiency of target cells, especially when the droplet size is large and the cell concentration is high, the efficiency drop is more significant.

[0005] Therefore, it is necessary to provide a new approach to solve the aforementioned technical problems. Summary of the Invention

[0006] To achieve the above-mentioned objectives and other advantages of the present invention, the first objective of the present invention is to provide a flow cytometer, comprising a fluid flow system, an optical detection system, an electronic system, and a sorting system. The fluid flow system includes an acoustic particle spacing adjustment unit configured to generate an acoustic standing wave field within a sample flow channel. The nodal lines or nodal surfaces of the acoustic standing wave field are perpendicular to the fluid flow direction, and are used to cause particles to move toward the nodal lines or nodal surfaces under the action of acoustic radiation force, thereby achieving a preset spacing arrangement of particles along the fluid flow direction.

[0007] Furthermore, the acoustic particle spacing adjustment unit includes a piezoelectric element acting on a wall perpendicular to the particle flow direction, and the vibration of the piezoelectric element generates an acoustic standing wave field perpendicular to the particle flow direction between the two walls of the sample flow channel.

[0008] Furthermore, the sample flow channel has two opposing walls, and the piezoelectric element is attached to the outer side of at least one of the walls; the driving frequency of the piezoelectric element... satisfy: ,in The velocity of sound in the sample solution. The distance between the two walls is... The number of nodal lines or nodal surfaces in the acoustic standing wave field.

[0009] Furthermore, the liquid flow system also includes an acoustic particle focusing unit, which is configured to generate an acoustic standing wave field within the sample flow channel to focus particles at a preset position on the liquid flow cross-section.

[0010] Furthermore, the acoustic particle focusing unit includes a piezoelectric element acting on a wall surface parallel to the particle flow direction, and the vibration of the piezoelectric element generates an acoustic pressure nodal line parallel to the particle flow direction at the center of the wall surface of the sample flow channel.

[0011] Furthermore, the sample flow channel has another pair of oppositely arranged walls, and the piezoelectric element is attached to the outer side of at least one of the walls; the driving frequency of the piezoelectric element... satisfy: ,in The velocity of sound in the sample solution. The distance between the two walls is denoted as .

[0012] Furthermore, the fluid flow system also includes: The sheath fluid focusing unit, located downstream of the acoustic particle spacing adjustment unit, is used to encapsulate the acoustically pre-regulated sample flow with sheath fluid to form laminar flow. A flow chamber for carrying the laminar flow and passing it through the optical detection area.

[0013] Furthermore, the sorting system includes: Nozzle, used to form a liquid column from the laminar flow; A sound wave generating device acts on the liquid column or the liquid forming the liquid column to cause the liquid column to break into uniform droplets; A charging device is used to apply a charge to the liquid column at the moment the target droplet breaks, based on the sorting decision of the electronic system, so that the target droplet becomes charged. A deflection device, located downstream of the nozzle, is used to generate a deflection electric field, causing the charged target droplet to deflect its trajectory. A collection device for collecting the deflected target droplets.

[0014] Furthermore, the acoustic wave generating device includes a piezoelectric element fixed on the fluid pool and communicating with the sheath fluid, the vibration direction of which is consistent with the fluid flow direction, and the driving frequency is matched with the desired droplet generation frequency.

[0015] A second objective of this invention is to provide a particle pretreatment method for flow cytometry cell sorting, applied to the aforementioned flow cytometry cell sorter, the method comprising: The sample stream containing particles is allowed to flow through the area affected by the sound waves; An acoustic standing wave field is generated within the area affected by the acoustic wave, and the acoustic radiation force is used to make the particles align at a preset spacing along the direction of liquid flow.

[0016] Furthermore, it also includes: focusing particles to a preset center position of the liquid flow cross section by generating an acoustic standing wave field with nodal lines or nodal surfaces parallel to the flow direction.

[0017] Furthermore, after the step of arranging the particles at a predetermined spacing along the liquid flow direction, the method further includes: Sheath fluid is used to perform hydrodynamic focusing on the sample flow after the spacing has been adjusted, forming a laminar flow with uniform particle velocity and further stretched spacing.

[0018] A third objective of this invention is to provide a cell sorting method, comprising: The cells in the sample stream were pretreated using the method described above; Pretreated cells were subjected to optical detection to obtain cell characteristic signals; Sorting decisions are made based on the cell characteristic signals; The droplets containing the target cells are charged and then deflected in an electric field to achieve sorting.

[0019] A fourth objective of this invention is to provide a microfluidic chip for acoustic particle manipulation, used in the fluid flow system of the aforementioned flow cytometer, wherein the chip integrates: Sample flow inlet and outlet; A microchannel connecting the inlet and the outlet; The first piezoelectric element is positioned corresponding to the opposite wall of the microchannel to generate an acoustic standing wave field. The nodal lines or nodal surfaces of the acoustic standing wave field are perpendicular to the flow direction, so as to arrange the flowing particles at equal intervals in the flow direction.

[0020] Furthermore, it also includes a second piezoelectric element, which is positioned corresponding to another pair of opposing walls of the microchannel, for generating an acoustic standing wave field, wherein the nodal lines or nodal surfaces of the acoustic standing wave field are parallel to the flow direction, so as to focus the flowing particles to the central region of the microchannel.

[0021] Compared with the prior art, the beneficial effects of the present invention are: This invention employs acoustic wave technology to achieve uniform particle distribution: Due to the nature of fluid flow, the positions of particles in the sample tube are randomly distributed, with some particles spaced far apart and others far apart. This leads to a situation where a single droplet encapsulates multiple particles during downstream droplet encapsulation. Ideally, for sorting, it is desirable for a droplet to encapsulate only one particle, thereby effectively separating the target particle and discarding non-target particles. If a droplet encapsulates both target and non-target particles, the target particle must be discarded to meet high purity requirements, resulting in a decrease in sorting yield. This invention utilizes a specially designed microchannel and piezoelectric acoustic wave devices to adjust the spacing of particles in the sample stream, ensuring a uniform distribution, reducing the probability of particles being encapsulated by the same droplet, and improving the sorting yield.

[0022] This invention employs acoustic focusing technology to achieve particle focusing at specific locations within a liquid flow. Using a single sheath fluid focusing method results in a wider sample flow at high flow rates, leading to a wider particle distribution and varying flow velocities at different locations. Furthermore, the droplet delay used to calculate the distance from the detection point to the sorting point during the sorting process is typically a single, fixed delay, which can cause incorrect timing of charge application to the droplets, reducing sorting purity and yield. This invention utilizes acoustic focusing technology to pre-focus particles at the acoustic nodal line position before sheath fluid focusing. Since the width of the acoustic nodal line is small, it ensures consistency in sample particle position and flow velocity, improving sorting purity and yield.

[0023] This invention is highly efficient and practical: it improves sorting performance without increasing experimental costs or operational difficulty, and is suitable for cell sorting needs in various biomedical fields.

[0024] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0025] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings: Figure 1 A schematic diagram illustrating particle focusing using acoustic wave technology; Figure 2 To illustrate the particle focusing effect achieved using acoustic wave technology; Figure 3 A schematic diagram illustrating the use of acoustic wave technology to achieve equidistant particle arrangement; Figure 4This image illustrates the effect of using acoustic wave technology to achieve an evenly spaced particle arrangement. Figure 5 A schematic diagram illustrating the use of sheath fluid focusing to stretch the interparticle spacing; Figure 6 This is a schematic diagram of the overall system of the flow sorter; Figure 7 This is a flowchart of a particle pretreatment method for flow cytometry cell sorting. Figure 8 This is a flowchart of the cell sorting method. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] In the accompanying drawings, shapes and dimensions may be enlarged for clarity, and the same reference numerals will be used in all figures to indicate the same or similar parts.

[0028] In the following description, terms such as center, thickness, height, length, front, back, rear, left, right, top, bottom, upper, and lower are defined relative to the structure shown in the accompanying drawings. In particular, "height" corresponds to the dimension from top to bottom, "width" corresponds to the dimension from left to right, and "depth" corresponds to the dimension from front to back. These are relative concepts and may vary depending on their location and usage. Therefore, these or other orientations should not be interpreted as restrictive terms.

[0029] Terms involving attachment, connection, etc. (e.g., “connection” and “attachment”) refer to the relationship in which these structures are directly or indirectly fixed or attached to each other through an intermediate structure, as well as movable or rigid attachments or relationships, unless otherwise explicitly stated.

[0030] To address the following technical problems in existing technologies: 1) When using a single sheath fluid focusing method, the sample flow is wider at high flow rates, resulting in a wider particle distribution and different flow rates for particles at different locations. Furthermore, the droplet delay used to calculate the distance from the detection point to the sorting point during the sorting process is usually a single fixed delay, which leads to incorrect timing of applying charge to the droplets, reducing sorting purity and yield.

[0031] 2) After the liquid is ejected from the nozzle of the flow cytometer, it is agitated into droplets. The droplets encapsulate cells, and the sorting algorithm determines whether to sort the target droplet. When a droplet contains both target and non-target cells, it will be discarded according to user needs. This will lead to a decrease in the sorting yield of target cells, especially when the droplet size is large and the cell concentration is high, the yield decrease will be more obvious.

[0032] The acoustic technology of this invention can achieve both uniform particle spacing in the sample tube and centered particle positioning, thereby eliminating the differences in particle position and flow velocity in high-flow-rate samples, as well as the problem of random particle encapsulation caused by random particle distribution, which requires selection in the sorting algorithm. This improves the sorting yield and purity. The specific solution is as follows: Example 1

[0033] A flow cytometer, such as Figure 6 As shown, it includes a fluid flow system, an optical detection system, an electronic system, and a sorting system.

[0034] The fluid flow system includes a sample tube, a sheath fluid pool, an acoustic particle spacing adjustment unit, an acoustic particle focusing unit, a flow chamber, and a nozzle. The acoustic particle spacing adjustment unit is the core improvement. This unit is configured to generate an acoustic standing wave field within the sample flow channel. The nodal lines or nodal surfaces of the acoustic standing wave field are perpendicular to the fluid flow direction, causing particles to move towards these nodal lines or nodal surfaces under the influence of acoustic radiation force, thereby achieving a preset spacing arrangement of particles along the fluid flow direction. The acoustic particle focusing unit is configured to generate an acoustic standing wave field within the sample flow channel before or after the sample flow enters the acoustic particle spacing adjustment unit, focusing the particles to preset positions on the fluid flow cross-section.

[0035] This embodiment employs acoustic wave technology to achieve particle focusing and uniform arrangement: a wall parallel to the particle flow direction is constructed within the sample loading pipe, and piezoelectric elements are attached to the outer wall of the pipe. The vibration of the piezoelectric elements drives the wall to vibrate, forming a standing wave. An acoustic pressure nodal line parallel to the particle flow direction is generated at the center of the wall. Under the action of acoustic radiation force, the particles move towards the standing wave nodal line, forming a single-row arrangement.

[0036] A wall perpendicular to the particle flow direction is constructed within the sample loading pipe. Piezoelectric elements are attached to the outer wall of the pipe. The vibration of the piezoelectric elements causes the wall to vibrate, forming a standing wave. This generates multiple acoustic pressure nodal lines perpendicular to the particle flow direction between the two walls. Under the influence of acoustic radiation, the particles move towards the standing wave nodal lines, forming an evenly spaced arrangement.

[0037] In some embodiments, the acoustic particle spacing adjustment unit includes a first piezoelectric element acting on a wall perpendicular to the particle flow direction, and the vibration of the first piezoelectric element generates an acoustic standing wave field perpendicular to the particle flow direction between the two walls of the sample flow channel.

[0038] The sample flow channel has two opposing walls, and the first piezoelectric element is attached to the outer side of at least one of the walls; the driving frequency of the first piezoelectric element... satisfy: (1), where The velocity of sound in the sample solution. The distance between the two opposing walls is defined as follows: The number of nodal lines or nodal surfaces in the acoustic standing wave field.

[0039] In other embodiments, the acoustic particle focusing unit includes a second piezoelectric element acting on a wall surface parallel to the particle flow direction, and the vibration of the second piezoelectric element generates an acoustic pressure nodal line parallel to the particle flow direction at the center of the wall surface of the sample flow channel.

[0040] The sample flow channel has two opposing walls, and the second piezoelectric element is attached to the outer side of at least one of the walls; the driving frequency of the second piezoelectric element... satisfy: (2), of which The velocity of sound in the sample solution. The distance between the two opposing walls is the second type of wall.

[0041] The fluid flow system in the flow cytometer provided by this invention arranges cells at uniform intervals and forms a single-row formation that passes sequentially through the detection zone. Acoustic wave technology is used for the equidistant and single-row arrangement of cells. A second piezoelectric element acts on a wall parallel to the particle flow direction. The vibration of the piezoelectric element drives the wall to vibrate, forming a standing wave, which generates an acoustic pressure nodal line parallel to the particle flow direction at the center of the wall. Under the action of acoustic radiation force, the particles move towards the standing wave nodal line, forming a single-row arrangement. Specifically, the piezoelectric element is bonded to the outer surface of the channel, and the driving frequency matches the above formula (2).

[0042] The first piezoelectric element acts on a wall perpendicular to the particle flow direction. The vibration of the piezoelectric element drives the wall to vibrate, forming a standing wave. Multiple acoustic pressure nodal lines perpendicular to the particle flow direction are generated between the two walls. Under the action of acoustic radiation force, the particles move towards the standing wave nodal lines and form an evenly spaced arrangement. Specifically, the piezoelectric element is bonded to the outer surface of the channel, and the driving frequency is consistent with the above formula (1).

[0043] In one specific design, the acoustic particle spacing adjustment unit includes two microchannels, one at the front and one at the back. The first microchannel (focusing function): as shown... Figure 1 , Figure 2 As shown, this microchannel has a pair of wide walls parallel to the flow direction. Piezoelectric ceramic sheets (PZTs) are bonded to the outer side of one or both wide walls. When an alternating current signal of a specific frequency is applied, the PZT vibration excites the wall vibration, forming an acoustic standing wave field distributed along the width of the channel in the liquid within the channel. According to the principle of acoustic radiation force, particles such as cells will move towards the acoustic pressure nodes (nodal lines). The channel width is designed... and drive frequency ( , (At the speed of sound), a stable sound pressure nodal line can be formed in the center of the flow channel. Cells flowing through this section will be efficiently focused onto this central nodal line, achieving a single-row arrangement.

[0044] Second microchannel (gap adjustment function): such as Figure 3 , Figure 4 As shown, this microchannel has a pair of walls perpendicular to the flow direction. Another PZT is bonded to the outside of these walls. By applying a frequency of... drive signal ( This is the length of the flow channel. To achieve the desired number of nodal lines (e.g., 3), multiple acoustic pressure nodal surfaces distributed along the flow direction can be formed within the flow channel. Cells will be trapped at these equally spaced nodal surfaces under the influence of acoustic radiation, thus forming a roughly uniformly spaced queue upon outflow.

[0045] In this embodiment, sheath fluid is used to further increase the spacing between particles and increase the particle velocity: at the outlet of the sample loading tube after the action of sound waves, sheath fluid is added to the fluid pool for focusing. The sheath fluid wraps the sample to form a laminar flow, which passes through the laser detection point.

[0046] In some embodiments, the fluid flow system further includes: The sheath fluid focusing unit, located downstream of the acoustic particle spacing adjustment unit, is used to encapsulate the acoustically pre-regulated sample flow with sheath fluid to form laminar flow. A flow chamber for carrying the laminar flow and passing it through the optical detection area.

[0047] The sample stream, pre-conditioned by acoustic waves, then merges with the sheath fluid from the sheath fluid reservoir and enters the flow chamber. For example... Figure 5 As shown, the sheath fluid focuses and further encapsulates and stretches the sample flow, increasing the longitudinal distance between cells and accelerating the sample flow to a stable laminar flow state.

[0048] The optical detection system includes an excitation module and an optical path collection module. The excitation module consists of multiple lasers of different wavelengths and beam-shaping optical elements. The lasers provide monochromatic light of specific wavelengths, which are shaped into fixed shapes and sizes. These beams illuminate the flow chamber, forming elliptical beams at the detection points. When cells carrying fluorescent markers pass through the laser detection points, they are excited to generate forward scattered light (FSC), side scattered light (SSC), and various fluorescence (FL). The optical path collection module includes a collection lens, a series of dichroic mirrors, and a bandpass filter, guiding light signals of different wavelengths to corresponding photodetectors (e.g., photodiodes for FSC, photomultiplier tubes for SSC and fluorescence). Specifically, these signals are guided and filtered by a series of lenses, beam splitters, and filters, ultimately being distributed to different detection channels. The detectors are connected to photodetectors at their ends. Photomultiplier tubes detect side scattered light and fluorescence, while photodiodes are typically used to detect stronger forward scattered light.

[0049] The electronic system converts optical signals into analyzable digital information and includes a signal amplifier, an analog-to-digital converter, and an embedded processor. The analog electrical signal output from the detector is pre-amplified, converted into a digital signal by the analog-to-digital converter, and then analyzed in real time by the embedded processor. Based on a preset gating strategy, the characteristics of each cell are determined in real time, and a sorting decision is made.

[0050] This embodiment employs acoustic wave technology to achieve the breakup of the liquid column into droplets: a piezoelectric element is installed in the fluid pool, and its vibration direction is consistent with the fluid flow direction. This vibration acts on the sheath fluid or the confluence of the sheath fluid and the sample. The confluence passes through a nozzle with micropores and is injected into the air. Under the action of the applied acoustic wave, the continuous liquid column breaks into uniformly distributed droplets.

[0051] Cell sorting is achieved by applying an electric charge to the droplets: a voltage is applied to the sheath fluid in the fluid cell the instant the target droplet leaves the liquid column, and the voltage is returned to zero after the target droplet leaves the liquid column. A high-voltage electrode is placed below the fluid cell, generating a high-voltage electrostatic field. When a charged droplet passes through the high-voltage electric field, it is deflected. The position of the charged droplet changes compared to other droplets, and the target charged droplet is collected through a collection tube.

[0052] In some embodiments, the sorting system includes: Nozzle, used to form a liquid column from the laminar flow; A sound wave generating device acts on the liquid column or the liquid forming the liquid column to cause the liquid column to break into uniform droplets; A charging device is used to apply a charge to the liquid column at the moment the target droplet breaks, based on the sorting decision of the electronic system, so that the target droplet becomes charged. A deflection device, located downstream of the nozzle, is used to generate a deflection electric field, causing the charged target droplet to deflect its trajectory. A collection device for collecting the deflected target droplets.

[0053] The acoustic wave generating device includes a third piezoelectric element fixed on the fluid pool and communicating with the sheath fluid. Its vibration direction is consistent with the fluid flow direction, and its driving frequency matches the desired droplet generation frequency.

[0054] The continuous jet of liquid ejected from the nozzle breaks into uniform droplets under the vibration of a third piezoelectric element in the acoustic wave generator, which is synchronized with the droplet generation frequency. Based on sorting decisions and precisely calculated droplet delay, the electronic system controls a charging device to apply a momentary voltage pulse to the liquid jet just before the target droplet breaks, thus charging the droplet. The charged droplets fall together with the uncharged droplets, passing through a pair of deflectors carrying several kilovolts of DC high voltage. Depending on the sign and amount of charge, the charged droplets are deflected at a specific angle in the electrostatic field, ultimately falling into a designated collection device, achieving sorting. Non-target droplets fall in a straight line into the waste liquid tank.

[0055] The third piezoelectric element provided by this invention acts on the sheath fluid. The sheath fluid, passing through a nozzle, breaks into uniform droplets, preparing for charge separation. The driving frequency of the third piezoelectric element corresponds to the number of droplets generated per second. Specifically, the third piezoelectric element is fixed to a fluid pool, and its end face communicates with the sheath fluid through a seal. Within the fluid pool, sheath fluid focusing further widens the particle spacing and simultaneously increases the particle velocity.

[0056] This invention provides a flow cytometer based on acoustic particle manipulation technology, primarily used in flow cytometers and related sorting equipment to improve cell sorting yield and purity. For example, in medical diagnostic and therapeutic applications, this technology can be used to efficiently sort and separate specific cell types, such as tumor cells and immune cells, thereby providing high-quality cell samples for disease diagnosis and personalized treatment. Through acoustic particle manipulation technology, this system can significantly improve sorting yield and purity, enhance sorting reliability, and is suitable for various cell sorting applications. Example 2

[0057] A microfluidic chip for controlling acoustic particles is provided for use in the fluidization system of the aforementioned flow cytometer / fluidization system. For a detailed description of the flow cytometer / fluidization system, please refer to the corresponding description in the aforementioned flow cytometer embodiments; it will not be repeated here. The chip integrates: Sample flow inlet and outlet; A microchannel connecting the inlet and the outlet; The first piezoelectric element is positioned corresponding to the opposite wall of the microchannel to generate an acoustic standing wave field. The nodal lines or nodal surfaces of the acoustic standing wave field are perpendicular to the flow direction, so as to arrange the flowing particles at equal intervals in the flow direction.

[0058] Furthermore, it also includes a second piezoelectric element, which is positioned corresponding to another pair of opposing walls of the microchannel, for generating an acoustic standing wave field, wherein the nodal lines or nodal surfaces of the acoustic standing wave field are parallel to the flow direction, so as to focus the flowing particles to the central region of the microchannel.

[0059] This embodiment provides a standalone chip product that implements the aforementioned acoustic pre-conditioning function. The chip is made of glass or silicon and has continuous microchannels etched internally. A first PZT and a second PZT are mounted at specific locations on the chip's exterior. The second PZT corresponds to the wide-area wall of the channel used for focusing, and the first PZT corresponds to the end face of the channel used for spacing adjustment. The chip has a standard fluid interface, allowing for easy connection to the fluid path of a flow cytometer as an upstream pretreatment module. Example 3

[0060] A particle pretreatment method for flow cytometry cell sorting is provided, applied to the aforementioned flow cytometry cell sorter. For a detailed description of the flow cytometry cell sorter, please refer to the corresponding description in the above-described flow cytometry cell sorter embodiments; it will not be repeated here. Figure 7 As shown, the method includes: S1. Allow the sample stream containing particles to flow through the area affected by the sound waves; S2. An acoustic standing wave field is generated within the acoustic wave action area, and the acoustic radiation force is used to make the particles align at a preset spacing along the liquid flow direction.

[0061] Preferably, it further includes: S3, focusing the particles to a preset center position of the liquid flow cross section by generating an acoustic standing wave field with nodal lines or nodal surfaces parallel to the flow direction.

[0062] To further increase the particle spacing and simultaneously improve particle velocity, after the step of arranging the particles at a preset spacing along the liquid flow direction, the method further includes: S4. Use sheath fluid to perform hydrodynamic focusing on the sample flow after the spacing arrangement has been adjusted, forming a laminar flow with uniform particle velocity and further stretched spacing.

[0063] After being focused by the sheath fluid, single-row, equidistant, and centrally located cell queues pass through the laser detection point in sequence, generating optical signals. The system then analyzes the signals, identifies the target cells, and accurately calculates the time it takes for them to form droplets. At the correct time, the droplets containing the target cells are charged, and an electrostatic field is used to sort them into designated collection tubes. Example 4

[0064] A cell sorting method, such as Figure 8 As shown, it includes: S100. The cells in the sample stream are pretreated using the particle pretreatment method described above. For a detailed description of the particle pretreatment method, please refer to the corresponding description in the above particle pretreatment method embodiment, which will not be repeated here.

[0065] S200: Perform optical detection on the pretreated cells to obtain cell characteristic signals; S300, Make a sorting decision based on the cell characteristic signals; S400: Charge the droplet containing the target cell and deflect the charged droplet in the electric field to achieve sorting.

[0066] Through the above embodiments, the present invention effectively solves the problems mentioned in the background art. Acoustic pre-focusing ensures consistency in detection and sorting timing, while acoustic spacing adjustment reduces the multi-cell encapsulation rate. The synergistic effect of these two methods significantly improves the sorting yield without sacrificing purity, achieving high-performance cell sorting.

[0067] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A flow cytometer for cell sorting, comprising a fluid flow system, an optical detection system, an electronic system, and a sorting system, characterized in that, The liquid flow system includes an acoustic particle spacing adjustment unit, which is configured to generate an acoustic standing wave field in the sample flow channel. The nodal lines or nodal surfaces of the acoustic standing wave field are perpendicular to the liquid flow direction, so as to make the particles move towards the nodal lines or nodal surfaces under the action of acoustic radiation force, thereby realizing the arrangement of particles at a preset spacing along the liquid flow direction.

2. The flow cytometer as described in claim 1, characterized in that, The acoustic particle spacing adjustment unit includes a piezoelectric element that acts on a wall perpendicular to the particle flow direction. The vibration of the piezoelectric element generates an acoustic standing wave field perpendicular to the particle flow direction between the two walls of the sample flow channel.

3. A flow cytometer as described in claim 2, characterized in that, The sample flow channel has two opposing walls, and the piezoelectric element is attached to the outer side of at least one of the walls; the driving frequency of the piezoelectric element... satisfy: ,in The velocity of sound in the sample solution. The distance between the two walls is... The number of nodal lines or nodal surfaces in the acoustic standing wave field.

4. A flow cytometer as described in claim 1, characterized in that, The liquid flow system also includes an acoustic particle focusing unit, which is configured to generate an acoustic standing wave field in the sample flow channel and focus the particles to a preset position on the liquid flow cross section.

5. A flow cytometer as described in claim 4, characterized in that, The acoustic particle focusing unit includes a piezoelectric element that acts on a wall surface parallel to the particle flow direction. The vibration of the piezoelectric element generates an acoustic pressure nodal line parallel to the particle flow direction at the center of the wall surface of the sample flow channel.

6. A flow cytometer as described in claim 5, characterized in that, The sample flow channel has another pair of oppositely arranged walls, and the piezoelectric element is attached to the outer side of at least one of the walls; the driving frequency of the piezoelectric element... satisfy: ,in The velocity of sound in the sample solution. The distance between the two walls is denoted as .

7. A flow cytometer as described in any one of claims 1 to 6, characterized in that, The fluid flow system also includes: The sheath fluid focusing unit, located downstream of the acoustic particle spacing adjustment unit, is used to encapsulate the acoustically pre-regulated sample flow with sheath fluid to form laminar flow. A flow chamber for carrying the laminar flow and passing it through the optical detection area.

8. A flow cytometer as described in claim 7, characterized in that, The sorting system includes: Nozzle, used to form a liquid column from the laminar flow; A sound wave generating device acts on the liquid column or the liquid forming the liquid column to cause the liquid column to break into uniform droplets; A charging device is used to apply a charge to the liquid column at the moment the target droplet breaks, based on the sorting decision of the electronic system, so that the target droplet becomes charged. A deflection device, located downstream of the nozzle, is used to generate a deflection electric field, causing the charged target droplet to deflect its trajectory. A collection device for collecting the deflected target droplets.

9. A flow cytometer as described in claim 8, characterized in that, The acoustic wave generating device includes a piezoelectric element fixed on the fluid pool and communicating with the sheath fluid. Its vibration direction is consistent with the fluid flow direction, and its driving frequency matches the desired droplet generation frequency.

10. A particle pretreatment method for flow cytometry cell sorting, applied to a flow cytometry cell sorter as described in any one of claims 1 to 9, characterized in that, The method includes: The sample stream containing particles is allowed to flow through the area affected by the sound waves; An acoustic standing wave field is generated within the area affected by the acoustic wave, and the acoustic radiation force is used to make the particles align at a preset spacing along the direction of liquid flow.

11. A particle pretreatment method for flow cytometry cell sorting as described in claim 10, characterized in that, Also includes: By generating an acoustic standing wave field with nodal lines or nodal surfaces parallel to the flow direction, particles are focused to a predetermined center position of the liquid flow cross section.

12. The particle pretreatment method for flow cytometry cell sorting as described in claim 10, characterized in that, After the step of arranging the particles at a predetermined spacing along the liquid flow direction, the method further includes: Sheath fluid is used to perform hydrodynamic focusing on the sample flow after the spacing has been adjusted, forming a laminar flow with uniform particle velocity and further stretched spacing.

13. A cell sorting method, characterized in that, include: The cells in the sample stream are pretreated using the method described in any one of claims 10 to 12; Pretreated cells were subjected to optical detection to obtain cell characteristic signals; Sorting decisions are made based on the cell characteristic signals; The droplets containing the target cells are charged and then deflected in an electric field to achieve sorting.

14. A microfluidic chip for controlling acoustic particles, used in the fluid flow system of a flow cytometer as described in any one of claims 1 to 9, characterized in that, The chip integrates: Sample flow inlet and outlet; A microchannel connecting the inlet and the outlet; The first piezoelectric element is positioned corresponding to the opposite wall of the microchannel to generate an acoustic standing wave field. The nodal lines or nodal surfaces of the acoustic standing wave field are perpendicular to the flow direction, so as to arrange the flowing particles at equal intervals in the flow direction.

15. The acoustic particle-controlled microfluidic chip as described in claim 14, characterized in that, It also includes a second piezoelectric element, which is positioned to correspond to another pair of opposing walls of the microchannel, for generating an acoustic standing wave field, wherein the nodal lines or nodal surfaces of the acoustic standing wave field are parallel to the flow direction, so as to focus the flowing particles to the central region of the microchannel.

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