Droplet sorting methods and systems

Abstract

This application discloses a droplet sorting method and system, applied in the field of microfluidics. The method includes: controlling a light source to generate N beams of excitation light with different wavelengths; controlling a first set of optical elements to collimate the N beams of excitation light and transmit them to an objective lens at different angles; controlling the objective lens to focus the N collimated beams, projecting N light spots onto the flow channel of a microfluidic chip, wherein the spacing between adjacent light spots is different from the spacing between adjacent droplets in the flow channel; controlling a second set of optical elements to transmit different fluorescence to different photodetectors, controlling the photodetectors to generate electrical signals based on the fluorescence; if the electrical signal is the fluorescence electrical signal matched by the photodetector, a control signal is generated, and the microfluidic chip is controlled to sort droplets from the flow channel based on the control signal. This application solves the problem in related technologies where multiple light sources with the same optical path simultaneously excite different fluorescent groups in droplets, leading to signal crosstalk between fluorescence channels and affecting droplet sorting results.

Description

Technical Field

[0001] This application relates to the field of microfluidics, and more specifically, to a droplet sorting method and system. Background Technology

[0002] Microfluidics is widely used for directed enzyme evolution in cellular or cell-free systems due to its low cost and high throughput. Among these methods, fluorescence-activated droplet sorting (FADS) is the most commonly used labeling method in microfluidics, used to sort and analyze cells or molecules in tiny droplets. The principle of FADS is as follows: a microfluidic system generates uniform droplets, each containing one or a small number of cells. Cells with specific characteristics are identified through fluorescent labeling. When these fluorescently labeled droplets flow through a detection area, the system automatically sorts the target droplets based on the intensity of the fluorescence signal.

[0003] Because a single fluorescence wavelength is often insufficient for droplet sorting applications, multiple light sources with different spectral center wavelengths are typically needed to excite different fluorophores for more accurate droplet sorting. However, in the FADS system of related technologies, all light sources are present simultaneously with identical optical paths. Different fluorophores in the droplet are excited at the same time, and these different fluorescence signals propagate to the sensor simultaneously, forming a time-synchronized pulse combination. This causes crosstalk of fluorescence signals of other wavelengths into a single fluorescence channel, creating noise that ultimately affects the sorting judgment.

[0004] There is currently no effective solution to the problem in related technologies where multiple light sources with the same optical path simultaneously excite different fluorescent groups in droplets, causing signal crosstalk between fluorescent channels and affecting droplet sorting results. Summary of the Invention

[0005] This application provides a droplet sorting method and system to solve the problem in related technologies where multiple light sources with the same optical path simultaneously excite different fluorescent groups in droplets, causing signal crosstalk between fluorescent channels and affecting the droplet sorting results.

[0006] According to one aspect of this application, a method for sorting droplets is provided. The method includes: providing a microfluidic chip, the microfluidic chip including a detection channel and at least two branch channels extending in different directions from a sorting point at one end of the detection channel; a light source generating N laser beams, wherein the N laser beams have different wavelengths, and N is a positive integer greater than or equal to 2; the N laser beams are combined by an optical guiding element and transmitted in parallel to an objective lens, wherein the N laser beams are transmitted to the objective lens in a non-coaxial manner; the objective lens focuses the N laser beams to form N light spots and projects them onto the detection channel of the microfluidic chip, wherein the spacing between adjacent light spots in the N light spots is different from the spacing between adjacent droplets in the channel; driving each droplet to move along the detection channel and pass through the N light spots sequentially, wherein the N light spots correspond one-to-one to excite droplets carrying a predetermined fluorescent dye to generate fluorescence signals of different wavelengths; N optical detection channels are optically coupled to the channel, and the N optical detection channels sequentially receive fluorescence signals matched with the N light spots; and driving the droplets to sort the droplets to a designated branch channel based on the fluorescence signals detected by at least one optical detection channel.

[0007] Optionally, the light source includes N sub-light sources and N collimating lenses respectively disposed on the optical paths of the sub-light sources. The N laser beams are transmitted to the objective lens in a non-coaxial manner, including: adjusting the positions of the N sub-light sources, and / or adjusting the positions of the N collimating lenses, until the N laser beams are converged by the optical guiding element with different optical axes and transmitted to the objective lens in parallel.

[0008] Optionally, the light guiding element is a dichroic mirror, where at least one laser beam is reflected by the dichroic mirror and transmitted to the objective lens, and at least another laser beam is transmitted through the dichroic mirror and transmitted to the objective lens.

[0009] Optionally, N optical detection channels are optically coupled to the flow channel via an objective lens and at least one dichroic mirror. At least one fluorescence signal is transmitted to one of the optical detection channels via the dichroic mirror, and at least another fluorescence signal is reflected to another optical detection channel via the dichroic mirror. Each optical detection channel includes a photoelectric sensor and a filter located in front of the photoelectric sensor. The filters of different optical detection channels have different transmission wavelengths, and the filters of different optical detection channels transmit fluorescence signals of a specified wavelength to the corresponding photoelectric sensor.

[0010] Optionally, driving the droplets to be sorted to a designated branch channel based on the fluorescence signal detected by at least one optical detection channel includes: generating a dielectrophoretic force at the sorting point that changes the direction of droplet movement based on the fluorescence signal detected by at least one optical detection channel, and sorting the droplets to the designated branch channel.

[0011] Optionally, the method further includes: optically coupling a camera to a microfluidic chip, acquiring images of the detection channel and branch channels in real time, and verifying the droplet sorting results of the microfluidic chip based on the images.

[0012] According to another aspect of this application, a droplet sorting system is provided. The system includes: a microfluidic chip, the microfluidic chip including a detection channel and at least two branch channels extending in different directions from a sorting point at one end of the detection channel; a light source for generating N laser beams, wherein the N laser beams have different wavelengths, and N is a positive integer greater than or equal to 2; a light guiding element for parallel converging of the N laser beams and transmitting them to an objective lens in a non-coaxial manner; and an objective lens for focusing the N laser beams to form N light spots projected onto the detection channel of the microfluidic chip, wherein the spacing between adjacent light spots in the N light spots is proportional to the spacing between adjacent droplets in the channel. The system includes: a power source that drives each droplet to move along the detection channel and pass through N light spots, where the N light spots are used to excite droplets carrying predetermined fluorescent dyes to generate fluorescence signals of different wavelengths; N optical detection channels that are optically coupled to the channel, and the N optical detection channels that receive fluorescence signals matched with the N light spots in sequence; and a sorting driver that is set at a corresponding sorting point and coupled to the N optical detection channels, which drives the droplets to sort to a designated branch channel based on the fluorescence signal detected by at least one optical detection channel.

[0013] Optionally, the system further includes: an illumination source, optically coupled to the sorting point to illuminate the microfluidic chip; and an imaging channel for acquiring images of the sorting point under illumination.

[0014] Optionally, the light source includes N sub-light sources and N collimating lenses respectively disposed in the optical paths of the sub-light sources. The collimating lenses can be moved relative to the sub-light sources to change the propagation path of the sub-light sources.

[0015] Optionally, the sorting driver includes a dielectrophoretic electrode positioned near the sorting point, the dielectrophoretic electrode being coupled to N optical detection channels to generate a dielectrophoretic force at the sorting point that alters the direction of droplet movement based on a fluorescence signal detected by at least one optical detection channel.

[0016] This application employs the following steps: providing a microfluidic chip, the microfluidic chip including a detection channel and at least two branch channels extending in different directions from a sorting point at one end of the detection channel; a light source generating N laser beams, wherein the N laser beams have different wavelengths, and N is a positive integer greater than or equal to 2; the N laser beams are combined by an optical guiding element and transmitted in parallel to an objective lens, wherein the N laser beams are transmitted to the objective lens in a non-coaxial manner; the objective lens focuses the N laser beams to form N light spots and projects them onto the detection channel of the microfluidic chip, wherein the spacing between adjacent light spots in the N light spots is different from the spacing between adjacent droplets in the channel; driving each droplet to move along the detection channel and pass through the N light spots sequentially, wherein the N light spots correspond one-to-one to excite droplets carrying a predetermined fluorescent dye to generate fluorescence signals of different wavelengths; N optical detection channels are optically coupled to the channel, and the N optical detection channels sequentially receive fluorescence signals matched with the N light spots; based on the fluorescence signal detected by at least one optical detection channel, the droplets are driven to sort to a designated branch channel. This invention addresses the problem in related technologies where simultaneous excitation of different fluorescent groups in droplets by multiple identical light sources leads to signal crosstalk between fluorescence channels, affecting droplet sorting results. By controlling the collimated beams of N excitation beams to be transmitted to the objective lens at different angles, and by controlling the spacing between adjacent spots in the focused light spot to differ from the spacing between adjacent droplets in the flow channel, signal crosstalk between fluorescence channels is avoided, thus improving the accuracy of droplet sorting results. Attached Figure Description

[0017] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0018] Figure 1 This is a schematic diagram of a droplet sorting system according to an embodiment of this application;

[0019] Figure 2 This is a schematic diagram of an illumination unit in a droplet sorting system according to an embodiment of this application;

[0020] Figure 3 This is a schematic diagram of an optional droplet sorting system according to an embodiment of this application;

[0021] Figure 4 This is a schematic diagram of the area where the sorting points are located according to an embodiment of this application;

[0022] Figure 5 This is a flowchart of a droplet sorting method according to an embodiment of this application;

[0023] Figure 6 This is a schematic diagram of an electrical signal generated by a photoelectric sensor according to an embodiment of this application;

[0024] Figure 7 This is a schematic diagram of an optional photoelectric sensor generating an electrical signal according to an embodiment of this application. Figure 1 ;

[0025] Figure 8 This is a schematic diagram of an optional photoelectric sensor generating an electrical signal according to an embodiment of this application. Figure 2 ;

[0026] Figure 9 A schematic diagram illustrating the electrical signal generated by a photoelectric sensor in a droplet sorting system according to related technologies;

[0027] Figure 10 This is a schematic diagram of an optional photoelectric sensor generating an electrical signal according to an embodiment of this application. Figure 3 ;

[0028] Figure 11 This is a schematic diagram of an electronic device according to an embodiment of this application. Detailed Implementation

[0029] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0030] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application.

[0031] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this application are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate for the embodiments of this application described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0032] It should be noted that all information (including but not limited to user device information, user personal information, etc.) and data (including but not limited to data used for display, data used for analysis, etc.) involved in this disclosure are information and data authorized by the user or fully authorized by all parties.

[0033] Figure 1 This is a schematic diagram of a droplet sorting system according to an embodiment of this application. Figure 1 As shown, the system includes:

[0034] A microfluidic chip includes a detection channel and at least two branch channels extending in different directions from a sorting point at one end of the detection channel.

[0035] It should be noted that a microfluidic chip is a platform for implementing microfluidic technology. It can control the uniform flow of droplets in the detection channel. When a droplet contains a specific fluorescent dye, it can pull the droplet from the detection channel to a branch channel, thereby achieving the sorting of droplets containing specific fluorescent dyes.

[0036] A light source is used to generate N laser beams, where the N laser beams have different wavelengths and N is a positive integer greater than or equal to 2.

[0037] Optionally, in the droplet sorting system provided in this application embodiment, the light source includes N sub-light sources and N collimating lenses respectively disposed in the optical path of the sub-light sources. The collimating lenses can be moved relative to the sub-light sources to change the propagation path of the sub-light sources.

[0038] Among them, N light sources can be N excitation light sources with different spectral center wavelengths, specifically N lasers with different spectral center wavelengths. The N lasers generate N laser beams with different wavelengths. The lasers are used to excite the fluorescent groups in the droplets in the flow channel of the microfluidic chip. In order to excite different fluorescent groups and thus sort the droplets more accurately, N is a positive integer greater than or equal to 2.

[0039] For example, such as Figure 1 As shown, N can be 2, and the light source can include two excitation light sources L1 and L2. The spectral center wavelengths of the excitation light sources L1 and L2 are selected according to the fluorescent groups contained in the target droplet. For example, if the fluorescent groups are Alexa Flour 532 and Alexa Flour 647, the spectral center wavelengths of the excitation light sources L1 and L2 are 532 nm and 647 nm, respectively.

[0040] The system comprises N collimating lenses, each used to collimate N laser beams to obtain N collimated beams. These collimating lenses can be moved relative to the sub-light sources to alter their propagation paths, thereby controlling the N collimated beams to be transmitted non-coaxially to the objective lens at different angles. Furthermore, in addition to controlling the non-coaxial transmission of the N collimated beams to the objective lens at different angles by adjusting the positions of the collimating lenses, the position of the light source can also be adjusted to control the non-coaxial transmission of the N collimated beams to the objective lens at different angles.

[0041] The optical guiding element is used to combine N laser beams in parallel and transmit them to the objective lens in a non-coaxial manner.

[0042] The light guiding element can be a dispersive element, such as a dichroic mirror. The dispersive element is used to transmit or reflect collimated light of different wavelengths, thus enabling the selection of collimated light of different wavelengths and transmitting N collimated beams to the objective lens in a non-coaxial manner. Non-coaxial means that the optical axes of the N collimated beams are different.

[0043] For example, such as Figure 1 As shown, N can be 2. The light source can include two excitation sources L1 and L2 and two collimating lenses f1 and f2. The collimating lenses f1 and f2 are respectively positioned in the optical paths of the excitation sources L1 and L2 to collimate the two excitation beams generated by the excitation sources L1 and L2, resulting in two collimated beams. The dispersive element can include a dichroic mirror D1, which is positioned at the intersection of the collimated beams from the excitation sources L1 and L2. It is used to reflect the collimated beam from the excitation source L2 and transmit the collimated beam from the excitation source L1, thereby forming a mixed beam. To shorten the optical path and reduce the volume of the liquid-liquid drop system, the first set of optical elements can also include a dichroic mirror D2 and a reflecting mirror M. The mixed beam is reflected by the dichroic mirror D2 to the reflecting mirror M, and the reflected mixed beam is reflected by the reflecting mirror M in a non-coaxial manner to the objective lens.

[0044] It should be noted that in the droplet sorting system of the relevant technology, multiple excitation beams are combined into the same optical path. After the combined excitation beam is focused by the objective lens, it forms a single spot at the flow channel, which causes crosstalk of fluorescence signals of other wavelengths into a single fluorescence channel. In this embodiment, N laser beams are combined in parallel and transmitted to the objective lens in a non-coaxial manner, thereby avoiding crosstalk of fluorescence signals between different fluorescence channels.

[0045] The objective lens is used to focus N laser beams to form N light spots that are projected onto the detection channel of the microfluidic chip. The spacing between adjacent light spots in the N light spots is different from the spacing between adjacent droplets in the channel.

[0046] The power source drives each droplet to move along the detection channel and pass through N light spots in sequence. The N light spots are used to excite droplets carrying predetermined fluorescent dyes to generate fluorescent signals of different wavelengths.

[0047] The objective lens consists of multiple lenses used to collect incident light rays and focus them onto a single point to form a light spot. The number of light spots generated by focusing N collimated beams can be adjusted by changing the objective lens parameters, ensuring that the size of these N light spots is similar to the width of the flow channel. Various types of objective lenses can be used; however, to reduce system cost and ensure image clarity, a plan achromatic objective lens is used in this embodiment.

[0048] Because N collimated beams are transmitted to the objective lens at different angles, these angles cause the N collimated beams to form excitation light spots at different positions in the flow channel of the microfluidic chip after being focused by the objective lens. The power source drives each droplet to move along the detection flow channel and pass through the N light spots sequentially. When a droplet in the chip flow channel passes through the excitation light spot, the droplet carrying the fluorescent dye is excited and produces fluorescence. Droplets carrying different fluorescent dyes produce fluorescence signals of different wavelengths when passing through the corresponding light spots. At the same time, by controlling the spacing between adjacent light spots in the N light spots to be different from the spacing between adjacent droplets in the flow channel, crosstalk of fluorescence signals between different fluorescence channels can be avoided. For example, as shown... Figure 1 As shown, N can be 2, and the light source can include two excitation light sources L1 and L2. The excitation light emitted by the two excitation light sources L1 and L2 is focused by the objective lens and generates a first light spot and a second light spot at different positions in the flow channel.

[0049] N optical detection channels are optically coupled to the flow channel, and each of the N optical detection channels receives fluorescence signals that are matched with the N light spots in turn.

[0050] In this method, fluorescence of different wavelengths is transmitted or reflected by a dispersive element, and the fluorescence of different wavelengths is transmitted to different optical detection channels. Each optical detection channel can be equipped with a filter, a focusing lens and a photoelectric sensor. The filter is used to filter out light signals of other wavelengths and leave fluorescence of a preset wavelength. The focusing lens is used to focus the fluorescence of the preset wavelength so as to transmit the fluorescence of the preset wavelength to the corresponding photoelectric sensor.

[0051] For example, such as Figure 1As shown, N can be 2, and the light source can include two excitation light sources L1 and L2. Two different wavelengths of fluorescence are transmitted to two optical detection channels through a dispersive element D3. The first optical detection channel includes a focusing lens f3, a filter F2, and an electrical sensor P1. The second optical detection channel includes a focusing lens f4, a filter F3, and an electrical sensor P2. The dichroic mirror D3 is used to reflect fluorescence of a wavelength matched with the first optical detection channel and transmit fluorescence of a wavelength matched with the second optical detection channel, thereby transmitting different wavelengths of fluorescence to different optical detection channels. The filter F2 on the first optical detection channel further filters the received fluorescence, and the focusing lens f3 focuses the filtered fluorescence onto the corresponding photoelectric sensor P1. The filter F3 on the second optical detection channel further filters the received fluorescence, and the focusing lens f4 focuses the filtered fluorescence onto the corresponding photoelectric sensor P4, so that the photoelectric sensor can perform photoelectric conversion and signal amplification of the corresponding wavelength of fluorescence.

[0052] It should be noted that different photoelectric sensors are used to convert fluorescence of different preset wavelengths into electrical signals and amplify them. A photoelectric sensor can be matched with fluorescence of one preset wavelength or multiple preset wavelengths, depending on the type of fluorescent dye carried by the droplet and the requirements for sorting the droplets. This embodiment does not impose any limitations.

[0053] The photoelectric sensor can be a photomultiplier tube (PMT), which converts weak fluorescence into an electrical signal and amplifies it. If the droplet contains a matching fluorescent dye, the droplet will produce matching fluorescence as it passes through the excitation light spot. The PMT receives the matching fluorescence and outputs a high-level signal. Conversely, if the droplet does not contain a matching fluorescent dye, the PMT will not receive the matching fluorescence and will output a low-level signal. As the droplet continuously flows through the channel, the PMT continuously generates an electrical signal composed of high and low level signals.

[0054] A sorting driver is set at the corresponding sorting point and coupled to N optical detection channels. The sorting driver drives the droplets to be sorted to the designated branch channel based on the fluorescence signal detected by at least one optical detection channel.

[0055] It should be noted that different fluorescent dyes in the droplet emit fluorescence with different center wavelengths when passed through the corresponding excitation light spot. These fluorescences propagate to different photoelectric sensors through different paths. The electrical signals generated by the photoelectric sensors can be analyzed to determine whether the generated electrical signals are the fluorescent electrical signals matched by the current photoelectric sensor. This allows the determination of whether the droplet contains a specific fluorescent dye. If the droplet contains a specific fluorescent dye, the sorting driver drives the droplet to sort the droplet in the detection channel to the designated branch channel based on the detected fluorescence signal, thereby achieving the sorting of droplets containing specific fluorescent dyes.

[0056] The sorting driver includes a dielectrophoretic electrode positioned near the sorting point, which is coupled to N optical detection channels to generate a dielectrophoretic force at the sorting point that changes the direction of droplet motion based on a fluorescence signal detected by at least one optical detection channel.

[0057] Dielectrophoresis electrode, also known as DEP electrode, generates a dielectric force in a non-uniform alternating electric field. This dielectric force pulls the droplets corresponding to the detected fluorescence signal into the branch channel, thereby achieving droplet sorting.

[0058] To achieve more accurate droplet sorting, the droplet sorting system provided in this application embodiment may optionally include: an illumination source, optically coupled to the sorting point to illuminate the microfluidic chip; and an imaging channel for acquiring images of the sorting point under the illumination of the illumination source.

[0059] Figure 2 This is a schematic diagram of an illumination unit in a droplet sorting system according to an embodiment of this application, as shown below. Figure 2 As shown, the lighting source can be an LED light source, used to emit a large spot of uniform bright field light above the flow channel. Figure 3 This is a schematic diagram of an optional droplet sorting system according to an embodiment of this application, such as... Figure 3 As shown, the droplet sorting system includes an LED light source, a camera C, a filter F1, a beam splitter S, and a lens f5. The uniform bright-field light emitted by the LED light source is filtered by the filter F1, split by the beam splitter S, and then imaged on the camera after passing through the lens f5. Figure 4 This is a schematic diagram of the area where the sorting point is located according to an embodiment of this application. The camera can display the entire area where the sorting point is located. The image captured by the camera can be mutually verified with the droplet sorting results, thus playing a role in supervising droplet sorting.

[0060] The droplet sorting system of this application utilizes a microfluidic chip, which includes a detection channel and at least two branch channels extending in different directions from a sorting point at one end of the detection channel; a light source for generating N laser beams, wherein the N laser beams have different wavelengths, and N is a positive integer greater than or equal to 2; a light guiding element for parallel converging of the N laser beams and transmitting them to an objective lens in a non-coaxial manner; and an objective lens for focusing the N laser beams to form N light spots that are projected onto the detection channel of the microfluidic chip, wherein the spacing between adjacent light spots in the N light spots is equal to the spacing between adjacent light spots in the channel. The system employs a multi-channel optical system (MCS) with varying droplet spacing. A power source drives each droplet along a detection channel, passing sequentially through N light spots. These N spots are used to excite droplets carrying predetermined fluorescent dyes, generating fluorescence signals of different wavelengths. N optical detection channels, optically coupled to the channel, sequentially receive fluorescence signals matched to the N light spots. A sorting driver, positioned at a corresponding sorting point and coupled to the N optical detection channels, drives the droplets to be sorted into designated branch channels based on the fluorescence signal detected by at least one optical detection channel. This system solves the problem in related technologies where simultaneous excitation of different fluorescent groups from droplets by multiple identical light sources leads to signal crosstalk between fluorescence channels, affecting droplet sorting results. By controlling the collimated beams of N excitation light to be transmitted to the objective lens at different angles, and by controlling the spacing between adjacent light spots in the focused spot to differ from the spacing between adjacent droplets in the channel, signal crosstalk between fluorescence channels is avoided, improving the accuracy of droplet sorting.

[0061] According to an embodiment of this application, a droplet sorting method is provided. The execution entity of this embodiment can be a processor, which stores a program to implement the droplet sorting method of this embodiment.

[0062] It should be noted that before implementing the droplet sorting method of this embodiment through programming, droplets need to be prepared first. Droplets can be prepared using a water-in-oil method, and the size of the prepared droplets is similar to the channel width of the microfluidic chip used for droplet sorting. The droplets contain magnetic beads, smaller than the droplet size, which are enriched with fluorescent dye. After droplet generation, they are pumped into one inlet of the microfluidic chip, and the droplet spacing is controlled by oil at the other inlet. Further, a displacement stage is used to adjust the center of the microfluidic chip's channel to the focal point of the objective lens, and then the droplet sorting method of this embodiment is executed.

[0063] Figure 5 This is a flowchart of a droplet sorting method according to an embodiment of this application. For example... Figure 5 As shown, the method includes the following steps:

[0064] Step S501: Provide a microfluidic chip, which includes a detection channel and at least two branch channels extending in different directions from a sorting point at one end of the detection channel.

[0065] It should be noted that a microfluidic chip is a platform for implementing microfluidic technology. It can control the uniform flow of droplets in the detection channel. When a droplet contains a specific fluorescent dye, it can pull the droplet from the detection channel to a branch channel, thereby achieving the sorting of droplets containing specific fluorescent dyes.

[0066] In step S502, the light source generates N laser beams, wherein the N laser beams have different wavelengths and N is a positive integer greater than or equal to 2.

[0067] The light source can include N excitation sources with different spectral center wavelengths, specifically N lasers with different spectral center wavelengths. These N lasers generate N laser beams with different wavelengths. The lasers are used to excite fluorescent groups in the droplets within the flow channels of the microfluidic chip. To excite different fluorescent groups for more accurate droplet sorting, N is a positive integer greater than or equal to 2. In one embodiment, N is 2, and the light source can include two continuous lasers. The spectral center wavelengths of the lasers are selected based on the fluorescent group; for example, the fluorescent groups are Alexa Flour 532 and Alexa Flour 647, and the spectral center wavelengths of the two lasers are 532 nm and 647 nm, respectively.

[0068] In step S503, the N laser beams are combined by the optical guiding element and then transmitted in parallel to the objective lens, wherein the N laser beams are transmitted to the objective lens in a non-coaxial manner.

[0069] It should be noted that in the droplet sorting system of the related technology, multiple excitation beams are combined into the same optical path. After the combined excitation beam is focused by the objective lens, a single spot is formed at the flow channel, which causes crosstalk of fluorescence signals of other wavelengths into a single fluorescence channel. In this embodiment, the N laser beams are combined in parallel by the optical guiding element and transmitted to the objective lens in a non-coaxial manner, thereby avoiding crosstalk of fluorescence signals between different fluorescence channels.

[0070] In step S504, the objective lens focuses N laser beams to form N light spots and projects them onto the detection channel of the microfluidic chip. The spacing between adjacent light spots in the N light spots is different from the spacing between adjacent droplets in the channel.

[0071] The objective lens consists of multiple transmission elements used to collect incident light and focus it onto a single point to form a light spot. The number of light spots generated by focusing N collimated beams can be adjusted by changing the objective lens parameters, ensuring that the size of these N light spots is similar to the width of the flow channel. Various types of objectives can be used; however, to reduce system cost and ensure image clarity, a plan achromatic objective lens is used in this embodiment.

[0072] Since the N collimated beams are transmitted to the objective lens at different angles, these angles cause the N collimated beams to form excitation light spots at different positions in the flow channel of the microfluidic chip after being focused by the objective lens.

[0073] In step S505, each droplet is driven to move along the detection channel and pass through N light spots in sequence. The N light spots correspond one-to-one to excite the droplets carrying the predetermined fluorescent dye to generate fluorescent signals of different wavelengths.

[0074] Each droplet can be driven by a power source to move along the detection channel and pass through N light spots in sequence. When a droplet in the chip channel passes through the excitation light spot, the droplet carrying the fluorescent dye is excited to produce fluorescence. Droplets carrying different fluorescent dyes pass through the corresponding light spots to produce fluorescence signals of different wavelengths. At the same time, by controlling the spacing between adjacent light spots in the N light spots to be different from the spacing between adjacent droplets in the channel, crosstalk of fluorescence signals between different fluorescence channels can be avoided.

[0075] For example, in this embodiment, the droplets encapsulate biomolecules enriched with different fluorescent groups, such as Alexa Flour 532 and Alexa Flour 647. The light source can include two continuous lasers with spectral center wavelengths of 532 nm and 647 nm, which, after being focused by an objective lens, generate a first and a second light spot at different positions in the flow channel. Droplets carrying Alexa Flour 532 are excited when passing through the first light spot, producing fluorescence at a wavelength of 532 nm, while droplets carrying Alexa Flour 647 are excited when passing through the first light spot, producing fluorescence at a wavelength of 647 nm.

[0076] Step S506: N optical detection channels are optically coupled to the flow channel, and the N optical detection channels successively receive fluorescence signals that match the N light spots.

[0077] In this method, fluorescence of different wavelengths is transmitted or reflected by a dispersive element, and the fluorescence of different wavelengths is transmitted to different optical detection channels. Each optical detection channel can be equipped with a filter, a focusing lens and a photoelectric sensor. The filter is used to filter out light signals of other wavelengths and leave fluorescence of a preset wavelength. The focusing lens is used to focus the fluorescence of the preset wavelength so as to transmit the fluorescence of the preset wavelength to the corresponding photoelectric sensor.

[0078] Different photoelectric sensors are used to convert fluorescence of different preset wavelengths into electrical signals and amplify them. For example, when the first photoelectric sensor is matched with fluorescence of a first preset wavelength and the second photoelectric sensor is matched with fluorescence of a second preset wavelength, if fluorescence of the first preset wavelength is generated, it is transmitted to the first photoelectric sensor through a dispersive element and a focusing lens. The first photoelectric sensor then converts the fluorescence of the first preset wavelength into an electrical signal and amplifies it. If fluorescence of the second preset wavelength is generated, it is transmitted to the second photoelectric sensor through a dispersive element and a focusing lens. The second photoelectric sensor then converts the fluorescence of the second preset wavelength into an electrical signal and amplifies it. Furthermore, it should be noted that a photoelectric sensor can be matched with one preset wavelength of fluorescence or multiple preset wavelengths of fluorescence, depending on the type of fluorescent dye carried by the droplet and the requirements for droplet sorting. This embodiment does not impose such a limitation.

[0079] The photoelectric sensor can be a photomultiplier tube, and the power supply voltage can be 0.4V. The photomultiplier tube can convert weak fluorescence into an electrical signal and amplify it. Optionally, in the droplet sorting method provided in the embodiments of this application, each photoelectric sensor generates a first level signal when it receives matching fluorescence and generates a second level signal when it does not receive matching fluorescence. The first level signal and / or the second level signal form an electrical signal.

[0080] It should be noted that the first level signal can be a high level signal and the second level signal can be a low level signal. If the droplet contains a matching fluorescent dye, the droplet will generate matching fluorescence after passing through the corresponding excitation light spot. The photomultiplier tube will receive the matching fluorescence and output a high level signal. Conversely, if the droplet does not contain a matching fluorescent dye, the photomultiplier tube will not receive the matching fluorescence and will output a low level signal. Since the droplet in the flow channel is constantly flowing, the photomultiplier tube will continuously generate an electrical signal composed of high and low level signals.

[0081] Step S507: Based on the fluorescence signal detected by at least one optical detection channel, the droplets are driven to sort the droplets to the designated branch channel.

[0082] It should be noted that different fluorescent dyes emit fluorescence with different center wavelengths when exposed to the corresponding excitation light spots. These fluorescences propagate to different photoelectric sensors through different paths. The electrical signals generated by the photoelectric sensors can be analyzed to determine whether the generated electrical signals are the fluorescent electrical signals matched by the current photoelectric sensor. This allows for the determination of whether the droplet contains a specific fluorescent dye. If the droplet contains a specific fluorescent dye, the detected fluorescence signal can be used to drive the droplet to sort the droplets in the detection channel to a designated branch channel, thereby achieving the sorting of droplets containing specific fluorescent dyes.

[0083] The droplet sorting method provided in this application embodiment includes a microfluidic chip comprising a detection channel and at least two branch channels extending in different directions from a sorting point at one end of the detection channel; a light source generates N laser beams, wherein the N laser beams have different wavelengths and N is a positive integer greater than or equal to 2; the N laser beams are combined by a light guiding element and transmitted in parallel to an objective lens, wherein the N laser beams are transmitted to the objective lens in a non-coaxial manner; the objective lens focuses the N laser beams to form N light spots and projects them onto the detection channel of the microfluidic chip, wherein the spacing between adjacent light spots is different from the spacing between adjacent droplets in the channel; each droplet is driven to move along the detection channel and pass through the N light spots sequentially, wherein each of the N light spots corresponds to a droplet carrying a predetermined fluorescent dye to generate a fluorescence signal of different wavelengths; N optical detection channels are optically coupled to the channel, and the N optical detection channels sequentially receive fluorescence signals matched with the N light spots; based on the fluorescence signal detected by at least one optical detection channel, the droplets are driven to sort to a designated branch channel. This invention addresses the problem in related technologies where simultaneous excitation of different fluorescent groups in droplets by multiple identical light sources leads to signal crosstalk between fluorescence channels, affecting droplet sorting results. By controlling the collimated beams of N excitation beams to be transmitted to the objective lens at different angles, and by controlling the spacing between adjacent spots in the focused light spot to differ from the spacing between adjacent droplets in the flow channel, signal crosstalk between fluorescence channels is avoided, thus improving the accuracy of droplet sorting results.

[0084] There are various ways to control N collimated beams to be transmitted to the objective lens at different angles. Optionally, in the droplet sorting method provided in the embodiments of this application, the light source includes N sub-light sources and N collimating lenses respectively disposed on the optical paths of the sub-light sources. The N laser beams are transmitted to the objective lens in a non-coaxial manner, including: adjusting the positions of the N sub-light sources, and / or adjusting the positions of the N collimating lenses, until the N laser beams are converged by the light guiding element at different optical axes and transmitted to the objective lens in parallel.

[0085] The N sub-sources can be N excitation sources with different spectral center wavelengths, specifically N lasers with different spectral center wavelengths. N collimating lenses are used to collimate the N laser beams, resulting in N collimated beams. For lasers with fine-tuning mechanisms, adjusting the fine-tuning knobs at the clamping ends of the N lasers can offset the optical axis of the laser from the center of the lens. This causes the collimated beams corresponding to different excitation beams to propagate at a small angle when reaching the objective lens, resulting in different collimated beams being transmitted to the objective lens in a non-coaxial manner. After focusing by the objective lens, the resulting light spots are located at different positions in the flow channel; for example, adjacent light spots can be separated by tens of micrometers.

[0086] In addition to adjusting the laser, the position of the lens can also be adjusted to offset the optical axis of the laser from the center of the lens. This can also cause the collimated light corresponding to different excitation beams to form a small angle when it is transmitted to the objective lens, so that different collimated beams are transmitted to the objective lens in a non-coaxial manner, and the light spot formed after being focused by the objective lens is located at different positions in the flow channel.

[0087] Furthermore, when processing and installing the optical components of the droplet sorting system, the positions of the light source and lens can be set so that the optical axes of the collimated light corresponding to different excitation lights do not overlap when they are transmitted to the objective lens. This allows different collimated lights to be transmitted to the objective lens in a non-coaxial manner, and the light spots formed after being focused by the objective lens are located at different positions in the flow channel.

[0088] This embodiment adjusts the position of the light source and the collimating lens so that the collimated light corresponding to different excitation beams is focused by the objective lens and the spot position at the flow channel of the microfluidic chip is different. This avoids the problem of crosstalk between fluorescence signals between different fluorescence channels caused by multiple excitation beams being combined and focused by the objective lens to form a single spot. This lays the foundation for improving the accuracy of droplet sorting results.

[0089] To avoid crosstalk between fluorescence signals from different fluorescence channels, in addition to controlling the positions of different light spots in the flow channel to prevent overlap, it is also necessary to adjust the spacing between adjacent light spots to be different from the spacing between adjacent droplets in the flow channel. Optionally, in the droplet sorting method provided in this application embodiment, the spacing between adjacent light spots in N light spots is controlled to be different from the spacing between adjacent droplets in the flow channel by adjusting the parameters of the objective lens and / or adjusting the angle at which N collimated beams are transmitted to the objective lens until the spacing between adjacent light spots in N light spots is different from the spacing between adjacent droplets in the flow channel.

[0090] Specifically, after the droplets are generated, they are pushed into one inlet of the flow channel of the microfluidic chip by a pump, and the spacing between adjacent droplets is controlled by oil at the other inlet. The spacing between adjacent droplets can be equal. After the spacing between adjacent droplets is fixed, the spacing between adjacent light spots is further controlled so that the spacing between adjacent light spots is different from the spacing between adjacent droplets in the flow channel.

[0091] One way to control the spacing between adjacent light spots is to adjust the parameters of the objective lens. These parameters can include magnification, numerical aperture, etc. By adjusting the objective lens parameters, the size of the light spot can be adjusted, which indirectly adjusts the spacing between adjacent light spots, thus making the spacing between adjacent light spots different from the spacing between adjacent droplets in the flow channel.

[0092] Another method for controlling the spacing between adjacent light spots is to adjust the angle at which the N collimated beams are transmitted to the objective lens, thereby directly adjusting the spacing between adjacent light spots so that the spacing between adjacent light spots differs from the spacing between adjacent droplets in the flow channel. For example, adjusting the angle at which the N collimated beams are transmitted to the objective lens may include adjusting the position of the light source or adjusting the position of the collimating lens corresponding to the light source.

[0093] It should be noted that in the multi-wavelength droplet sorting systems of related technologies, crosstalk exists between channels in the fluorescence excited by excitation light of different wavelengths. This makes it impossible to accurately identify whether the photoelectric sensor collects the true signal or crosstalk noise from other channels. However, in this application, the spacing between adjacent light spots is controlled to be different from the spacing between adjacent droplets in the flow channel. When droplets flow sequentially through different excited light spots in the flow channel of the microfluidic chip, the fluorescence signals received by channels of different wavelengths are separated in the time domain. The crosstalk signals between different channels cannot be superimposed on other channels. Figure 6 This is a schematic diagram of an electrical signal generated by a photoelectric sensor according to an embodiment of this application, as shown below. Figure 6 As shown, when N is 2, the signal containing two channels can be clearly distinguished from the real signal and crosstalk noise on the time waveform diagram, avoiding incorrect sorting of droplets and thus improving the accuracy of droplet sorting.

[0094] In one optional implementation, the distance between adjacent droplets is d2, and the distance between the two excitation spots is d1. To avoid crosstalk between fluorescence signals from different channels, the following condition must be satisfied: d2 ≠ d1. The droplets flow uniformly within the channel at a velocity of v. Fluorescence 1 is generated at time t1, fluorescence 2 at time t2, and the distance between the two excitation spots is d1 = v(t2 - t1), which must satisfy v(t2 - t1) ≠ d1. Furthermore, it should be noted that the droplet diameter is d, and the distance between the two excitation spots is d1. To further avoid signal crosstalk, the following condition must be satisfied: d < d1.

[0095] Figure 7This is a schematic diagram of an optional photoelectric sensor generating an electrical signal according to an embodiment of this application. Figure 1 .like Figure 7 As shown, the fluorescence at different wavelengths occurs at different times. To avoid crosstalk between the fluorescence signals of the droplets, only one droplet generates a single fluorescence signal at a single time point. The fluorescence 1 is generated at time t1, the fluorescence 2 is generated at time t2, the droplet diameter is d, the distance between the two excitation spots is d1, the droplet diameter is smaller than the distance between the two excitation spots d1, and the distance the droplet moves between t1 and t2 is d2. If d1 = d2, crosstalk will still occur between the fluorescence signals of the two channels.

[0096] Figure 8 This is a schematic diagram of an optional photoelectric sensor generating an electrical signal according to an embodiment of this application. Figure 2 .like Figure 8 As shown, two fluorescence signals are generated at a single time point. The distance between the two excitation spots is d1, the droplet diameter is d which is smaller than the distance between the two excitation spots d1, and the distance between the two droplets is d2. If d1 ≠ d2, the fluorescence signals of the two channels will not cause crosstalk.

[0097] The first set of optical elements is selected according to the wavelength of the light source. Optionally, in the droplet sorting method provided in the embodiments of this application, the light guiding element is a dichroic mirror. At least one laser beam is reflected by the dichroic mirror and transmitted to the objective lens, and at least another laser beam is transmitted through the dichroic mirror and transmitted to the objective lens.

[0098] In this system, N sub-light sources can be N lasers with different spectral center wavelengths. N collimating lenses are respectively positioned in the optical path of each laser to collimate the N excitation beams, resulting in N collimated beams. The optical guiding element can include multiple dichroic mirrors, which select the desired wavelength of light. When the N-1 lasers are arranged approximately perpendicular to the first laser in their optical paths, the first dichroic mirror is positioned at the intersection of the collimated beams of the first and second lasers. This first dichroic mirror reflects the collimated beam of the second laser and transmits the collimated beam of the first laser, forming a mixed beam. Each dichroic mirror other than the first one is positioned at the intersection of the mixed beam of the current collimated beam and the collimated beam before it. Each mirror reflects the current collimated beam and transmits the mixed beam before it, resulting in a further mixed beam. The last dichroic mirror outputs the resulting mixed beam to the objective lens. It should be noted that the N collimated beams in the mixed beam are transmitted to the objective lens at different angles.

[0099] To shorten the optical path and thus reduce the volume of the liquid-liquid separation system, a dichroic mirror can be placed on the optical path of the mixed beam formed by the N collimated beams, and a reflecting mirror parallel to the dichroic mirror can be placed. The mixed beam of the N collimated beams is reflected by the dichroic mirror, and the reflected mixed beam is reflected again to the objective lens by the reflecting mirror.

[0100] In this embodiment, N collimating lenses, each arranged in the optical path of a different sub-light source, and a set of dispersive elements are used to transmit the mixed beam formed by N collimated beams to the objective lens at different angles, laying the foundation for the objective lens to focus and obtain different light spots.

[0101] The second set of optical elements is selected according to the wavelength of the light source. Optionally, in the droplet sorting method provided in this application embodiment, N optical detection channels are optically coupled to the flow channel via an objective lens and at least one dichroic mirror. At least one fluorescence signal is transmitted to one of the optical detection channels via the dichroic mirror, and at least another fluorescence signal is reflected to another optical detection channel via the dichroic mirror. A single optical detection channel includes a photoelectric sensor and a filter located in front of the photoelectric sensor. The filters of different optical detection channels have different transmission bands, and the filters of different optical detection channels transmit fluorescence signals of a specified wavelength to the corresponding photoelectric sensor.

[0102] The beam-splitter selects the desired wavelength of light, transmitting fluorescence of different wavelengths to different optical detection channels. Each optical detection channel can be equipped with a filter, a focusing lens, and a photoelectric sensor. When N-1 optical detection channels are arranged approximately perpendicular to the first optical detection channel, each beam-splitter is positioned at the intersection of the output light path of the objective lens and the receiving path of the current optical detection channel. It reflects fluorescence of the wavelength matched to the current optical detection channel and transmits fluorescence of other wavelengths, thus transmitting fluorescence of different wavelengths to different optical detection channels. The filter in the optical detection channel further filters the received fluorescence, and the focusing lens in the optical detection channel focuses the filtered fluorescence onto the corresponding photoelectric sensor, enabling the photoelectric sensor to perform photoelectric conversion and signal amplification of the corresponding wavelength of fluorescence.

[0103] In this embodiment, a dichroic mirror, N focusing lenses respectively set in the optical paths of different photoelectric sensors, and N filters respectively set in the optical paths of different focusing lenses transmit fluorescence of different wavelengths to the receiving paths of different photoelectric sensors, laying the foundation for the photoelectric sensors to generate electrical signals and perform droplet sorting judgment based on the electrical signals.

[0104] Dielectrophoresis can be used for droplet sorting. Optionally, in the droplet sorting method provided in the embodiments of this application, the droplets are driven to be sorted to a designated branch channel based on the fluorescence signal detected by at least one optical detection channel. This includes: generating a dielectrophoretic force at the sorting point that changes the direction of droplet movement based on the fluorescence signal detected by at least one optical detection channel, and sorting the droplets to the designated branch channel.

[0105] In this process, different excitation light spots are pre-adjusted to the center of the flow channel of the microfluidic chip and close to the DEP (Dielectrophoresis) electrode. The DEP includes a ground electrode and a charged electrode. The DEP electrode generates a dielectric force in a non-uniform alternating electric field. The dielectric force is used to pull the target droplet into the sorting channel, thereby realizing droplet sorting. Figure 4 This is a schematic diagram of the area where the sorting points are located according to an embodiment of this application, and is a screenshot of the area where the sorting points are located under a microscope, such as... Figure 4 As shown, the electrode generates a high-voltage alternating current signal, which pulls the positive droplet to the upper branch channel, while the cathode droplet flows away along the detection channel, thus achieving droplet sorting.

[0106] To further ensure the effectiveness of droplet sorting, the droplet sorting method provided in this application embodiment may optionally include: optically coupling a camera to a microfluidic chip, acquiring images of the detection channel and branch channels in real time, and verifying the droplet sorting results of the microfluidic chip based on the images.

[0107] The droplet sorting system includes an LED light source that emits a large, uniform bright field light. This light, after passing through an objective lens and lens system, is imaged on a camera, displaying the entire sorting area. The camera-acquired image can be cross-validated with the droplet sorting results, thus monitoring the sorting process. Furthermore, the camera and illumination source assist in observing the position of the microfluidic chip moved by the displacement stage, aligning the center of the microfluidic chip's channel with the focal point of the objective lens, thereby ensuring effective droplet sorting.

[0108] It should be noted that multi-wavelength fluorescent droplet sorting systems in related technologies typically combine light beams of different wavelengths into the same path, then focus them onto a point in the center of the microfluidic chip after passing through an objective lens, thereby exciting the fluorescent signal in the droplet and transmitting the signal to the sensor. Figure 9 This is a schematic diagram illustrating the electrical signal generated by the photoelectric sensor in a droplet sorting system based on related technologies. It shows the signal collected when the droplet contains only a single-colored fluorescent group. Figure 9As shown, in addition to the channel corresponding to the sample fluorescence emission wavelength having a signal, due to the existence of crosstalk between channels, other fluorescence channels that should not have signals also exhibited considerable crosstalk noise. The signal waveforms of other fluorescence channels that should not have signals are consistent with the signal waveforms of the fluorescence emission wavelength channels, which can be inferred to be crosstalk noise. This crosstalk noise will cause the monochromatic fluorescent droplet to be judged as a dual-color fluorescent droplet, resulting in incorrect droplet sorting.

[0109] In this application, multiple excitation light sources form different light spots on the flow channel through the objective lens. When the droplet passes through different light spots in the flow channel of the microfluidic chip in sequence, the droplet generates fluorescence under the excitation light and is received by the photomultiplier tube. Figure 10 This is a schematic diagram of an optional photoelectric sensor generating an electrical signal according to an embodiment of this application. Figure 3 ,like Figure 10 As shown, when there are two excitation light sources, forming two different light spots on the flow channel through the objective lens, and two photomultiplier tubes are used as photodetectors, the droplet itself contains unenriched dye. When the droplet flows through the light spot, there is a fluorescent signal substrate. When the droplet contains magnetic beads, the magnetic beads enrich the fluorescent dye. Because the size of the magnetic beads is smaller than the droplet, the time it takes for the magnetic beads to pass through the light spot is shorter than the time it takes for the droplet to pass through the light spot, resulting in stronger fluorescence than the unenriched dye dispersed in the droplet. In a series of fluorescence signals, some signals will have a higher peak fluorescence signal above the background fluorescence signal, such as... Figure 10 At the far left, because the two excitation light spots are located at different positions in the flow channel, the droplets flow through the light spots for different times, and the peak signal received by the photomultiplier tube differs in the time domain. Compared with related technologies, this application enables the originally overlapping crosstalk noise and the real signal to be accurately distinguished, ensuring the accuracy of the sorting criteria.

[0110] It should be noted that the steps shown in the flowchart in the accompanying drawings can be executed in a computer system such as a set of computer-executable instructions, and although a logical order is shown in the flowchart, in some cases the steps shown or described may be executed in a different order than that shown here.

[0111] This application also provides a computer storage medium for storing a program, wherein the program, when running, controls the device containing the non-volatile storage medium to perform a droplet sorting method.

[0112] This application also provides an electronic device. Figure 11 This is a schematic diagram of an electronic device according to an embodiment of this application, such as... Figure 11As shown, the electronic device 110 includes a processor and a memory; the memory stores computer-readable instructions, and the processor executes the computer-readable instructions, wherein the computer-readable instructions, when executed, perform a droplet sorting method. The electronic device in this document can be a server, PC, PAD, mobile phone, etc.

[0113] Those skilled in the art will understand that embodiments of this application can be provided as methods, systems, or computer program products. Therefore, this application can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, this application can take the form of a computer program product embodied on one or more computer-usable storage media (including but not limited to disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0114] This application is described with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of this application. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, special-purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, generate instructions for implementing the flowchart... Figure 1 One or more processes and / or boxes Figure 1 A device that provides the functions specified in one or more boxes.

[0115] These computer program instructions may also be stored in a computer-readable storage medium that can direct a computer or other programmable data processing device to function in a particular manner, such that the instructions stored in the computer-readable storage medium produce an article of manufacture including instruction means, which are implemented in a process Figure 1 One or more processes and / or boxes Figure 1 The function specified in one or more boxes.

[0116] These computer program instructions may also be loaded onto a computer or other programmable data processing equipment to cause a series of operational steps to be performed on the computer or other programmable equipment to produce a computer-implemented process, thereby providing instructions that execute on the computer or other programmable equipment for implementing the process. Figure 1 One or more processes and / or boxes Figure 1 The steps of the function specified in one or more boxes.

[0117] In a typical configuration, a computing device includes one or more processors (CPU), input / output interfaces, network interfaces, and memory.

[0118] Memory may include non-persistent memory in computer-readable media, such as random access memory (RAM) and / or non-volatile memory, like read-only memory (ROM) or flash RAM. Memory is an example of computer-readable media.

[0119] Computer-readable media includes both permanent and non-permanent, removable and non-removable media that can store information using any method or technology. Information can be computer-readable instructions, data structures, modules of programs, or other data. Examples of computer storage media include, but are not limited to, phase-change memory (PRAM), static random access memory (SRAM), dynamic random access memory (DRAM), other types of random access memory (RAM), read-only memory (ROM), electrically erasable programmable read-only memory (EEPROM), flash memory or other memory technologies, CD-ROM, digital versatile optical disc (DVD) or other optical storage, magnetic tape, magnetic magnetic disk storage or other magnetic storage devices, or any other non-transferable medium that can be used to store information accessible by a computing device. As defined herein, computer-readable media does not include transient computer-readable media, such as modulated data signals and carrier waves.

[0120] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0121] The above are merely embodiments of this application and are not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A method for sorting droplets, characterized in that, include: A microfluidic chip is provided, the microfluidic chip including a detection channel and at least two branch channels extending in different directions from a sorting point at one end of the detection channel; The light source generates N laser beams, wherein the N laser beams have different wavelengths, and N is a positive integer greater than or equal to 2; The N laser beams are combined by an optical guiding element and then transmitted in parallel to the objective lens, wherein the N laser beams are transmitted to the objective lens in a non-coaxial manner; The objective lens focuses the N laser beams to form N light spots and projects them onto the detection channel of the microfluidic chip. The spacing between adjacent light spots in the N light spots is different from the spacing between adjacent droplets in the channel. The droplets are driven to move along the detection channel and pass sequentially through the N light spots, wherein... N light spots correspond one-to-one to excite the droplets carrying predetermined fluorescent dyes to generate fluorescent signals of different wavelengths; N optical detection channels are optically coupled to the flow channel, and the N optical detection channels sequentially receive fluorescence signals that match the N light spots; Based on the fluorescence signal detected by at least one of the optical detection channels, the droplets are driven to sort the droplets to the designated branch channels.

2. The method according to claim 1, characterized in that, The light source includes N sub-light sources and N collimating lenses respectively disposed in the optical paths of the sub-light sources. The N laser beams are transmitted to the objective lens in a non-coaxial manner, including: Adjust the positions of the N sub-light sources and / or adjust the positions of the N collimating lenses until the N laser beams converge through the light guiding element with different optical axes and are transmitted in parallel to the objective lens.

3. The method according to claim 1, characterized in that, The light guiding element is a dichroic mirror. At least one of the laser beams is reflected by the dichroic mirror and transmitted to the objective lens, and at least another laser beam is transmitted through the dichroic mirror and transmitted to the objective lens.

4. The method according to claim 1, characterized in that, The N optical detection channels are optically coupled to the flow channel via the objective lens and at least one dichroic mirror. At least one fluorescence signal is transmitted through the dichroic mirror to one of the optical detection channels, and at least another fluorescence signal is reflected through the dichroic mirror to another optical detection channel. Each optical detection channel includes a photoelectric sensor and a filter located in front of the photoelectric sensor. The filters of different optical detection channels have different transmission bands, and the filters of different optical detection channels transmit the fluorescence signal of a specified wavelength to the corresponding photoelectric sensor.

5. The method according to claim 1, characterized in that, The step of driving the droplet to sort the droplet to the designated branch channel based on the fluorescence signal detected by at least one of the optical detection channels includes: generating a dielectrophoretic force at the sorting point that changes the direction of the droplet's movement based on the fluorescence signal detected by at least one of the optical detection channels, and sorting the droplet to the designated branch channel.

6. The method according to claim 1, characterized in that, The method further includes: optically coupling a camera to the microfluidic chip, acquiring images of the detection channel and the branch channel in real time, and verifying the droplet sorting results of the microfluidic chip based on the images.

7. A droplet sorting system, characterized in that, include: A microfluidic chip, the microfluidic chip including a detection channel and at least two branch channels extending in different directions from a sorting point at one end of the detection channel; A light source is used to generate N laser beams, wherein the N laser beams have different wavelengths and N is a positive integer greater than or equal to 2; a light guiding element is used to combine the N laser beams in parallel and transmit them to the objective lens in a non-coaxial manner. The objective lens is used to focus the N laser beams to form N light spots that are projected onto the detection channel of the microfluidic chip. The spacing between adjacent light spots in the N light spots is different from the spacing between adjacent droplets in the channel. A power source drives each droplet to move along the detection channel and pass through the N light spots in sequence, wherein the N light spots are used to excite the droplets carrying predetermined fluorescent dyes to generate fluorescent signals of different wavelengths. N optical detection channels optically coupled to the flow channel, the N optical detection channels sequentially receiving fluorescence signals matched with the N light spots; and a sorting driver set corresponding to the sorting point and coupled to the N optical detection channels, the sorting driver driving the droplet to sort the droplet to the designated branch flow channel based on the fluorescence signal detected by at least one of the optical detection channels.

8. The droplet sorting system according to claim 7, characterized in that, The system also includes: An illumination source, optically coupled to the sorting point, is used to illuminate the microfluidic chip; and An imaging channel is used to acquire images of the sorting points generated under the illumination of the light source.

9. The droplet sorting system according to claim 7, characterized in that, The light source includes N sub-light sources and N collimating lenses respectively disposed in the optical paths of the sub-light sources. The collimating lenses can be moved relative to the sub-light sources to change the propagation path of the sub-light sources.

10. The droplet sorting system according to claim 7, characterized in that, The sorting driver includes a dielectrophoretic electrode disposed near the sorting point, the dielectrophoretic electrode being coupled to the N optical detection channels to generate a dielectrophoretic force at the sorting point that changes the direction of droplet movement based on a fluorescence signal detected by at least one of the optical detection channels.

Images