Droplet preparation instrument and antibody screening device

Abstract

The utility model discloses a liquid drop preparation instrument and an antibody screening device, and the liquid drop preparation instrument comprises a chip assembly which comprises a liquid drop generation chip and a liquid drop sorting chip, and the liquid drop sorting chip comprises a sorting electrode; the sample table is used for placing at least one of the liquid drop generating chip and the liquid drop sorting chip; the optical module is optically coupled with the sample table and is used for providing incident light to the sample table and collecting an optical signal generated by the biological sample or the liquid drop in response to the incident light; the liquid path system is in operable conduction with the chip assembly and is used for pumping a solution to the liquid drop generating chip or the liquid drop sorting chip; the control circuit can selectively call a first group of control parameters or a second group of control parameters, the first group of control parameters comprises a first control instruction, and the second group of control parameters comprises a second control instruction; the control circuit comprises a feedback control sub-circuit used for controlling the sorting electrodes. According to the liquid drop preparation instrument, liquid drop preparation can be carried out, and needed liquid drops can be rapidly sorted out.

Description

Technical Field

[0001] This application relates to the technical field of particle sorting, and particularly to a droplet preparation instrument and a device for screening antibodies. Background Art

[0002] Droplet-based microfluidics technology has been widely used in directed evolution (DE) of enzymes in cell-free or cell systems due to its low cost and high throughput. Among them, fluorescence-activated droplet sorting (FADS) is the most commonly used labeling method. It combines microfluidics technology with fluorescence detection principle to sort and analyze cells or molecules in tiny droplets. The significance of this technology lies in that it provides an efficient and sensitive method for high-throughput screening, single-cell analysis, and precise separation of complex biological samples.

[0003] The principle of FADS is based on generating uniform microdroplets in a microfluidic system, with each microdroplet containing a single or a small number of cells. Through fluorescence labeling, cells with specific characteristics can be identified. When these fluorescence-labeled microdroplets flow through the detection area, the system can automatically sort out the target microdroplets based on the intensity of the fluorescence signal. This method has been widely used in many fields such as cell sorting, enzyme activity screening, and antibody discovery.

[0004] With the progress of microfluidics technology and optical detection technology, the application of FADS in biomedical research, drug development, and diagnostic fields is increasing. Especially in single-cell analysis, FADS provides an efficient means to study the heterogeneity between cells, which is of great significance for understanding complex biological processes and disease mechanisms. Summary of the Utility Model

[0005] An object of this application is to propose a droplet preparation instrument and a device for screening antibodies.

[0006] A droplet preparation instrument according to an embodiment of the present application includes: a chip assembly including a droplet generation chip for supporting a biological sample and a droplet sorting chip for supporting droplets to be sorted, the droplet generation chip being configured to generate droplets encapsulating the biological sample, and the droplet sorting chip including sorting electrodes operably electrically connected to the outside; a sample stage for placing at least one of the droplet generation chip and the droplet sorting chip; an optical module optically coupled to the sample stage for providing incident light to the sample stage and collecting optical signals generated by the biological sample or the droplets in response to the incident light; a liquid path system operably connected to the chip assembly for pumping a solution to the droplet generation chip or the droplet sorting chip; a control circuit selectively invoking a first set of control parameters or a second set of control parameters, the first set of control parameters including a first control instruction for driving at least the liquid path system to be coupled to the droplet generation chip, and the second set of control parameters including a second control instruction for driving the liquid path system and the optical module to be coupled to the droplet sorting chip; the control circuit including a feedback control sub-circuit, the feedback control sub-circuit being selectively connected to the sorting electrodes and the optical module based on the second control instruction for controlling the sorting electrodes according to the detection results of the optical module.

[0007] The droplet preparation instrument according to the embodiment of the present application can perform droplet preparation, so as to quickly sort out the required droplets for the next detection, providing a high-throughput, non-destructive, accurate and reliable antibody screening platform, greatly improving the screening efficiency of high-affinity and high-specificity antibodies at the single-cell level, and contributing to the realization of high-throughput, long-length and accurate sequencing.

[0008] In addition, the droplet preparation instrument according to the above embodiment of the present application may further have the following additional technical features:

[0009] In some embodiments, the liquid path system is set under the second set of control parameters to control the ratio of the flow rate of the spacer oil introduced into the droplet sorting chip to the flow rate of the droplets within the range of 7:1 - 14:1.

[0010] In some embodiments, the droplet sorting chip includes a detection flow channel and a target flow channel and a waste liquid flow channel extending in different directions from a sorting point at one end of the detection flow channel, and the sorting electrodes are configured to generate a dielectrophoretic force that guides the fluid at the sorting point to flow towards the target flow channel when electrically connected to an external electrical signal.

[0011] In some embodiments, the optical module includes a light source assembly, a lens assembly, a detector assembly, and an objective lens. The lens assembly is configured to direct the incident light from the light source assembly through the objective lens to the sample stage, and is also configured to transmit the optical signal generated by the biological sample or the droplet in response to the incident light to the detector assembly.

[0012] In some embodiments, the incident light includes incident lasers provided by the light source assembly and having at least two different wavelengths; and / or, the light source assembly includes a first light source, a second light source, and a first dispersion element. The first dispersion element is configured to reflect the incident laser of the first light source and transmit the incident laser of the second light source. The incident lasers of the first light source and the second light source have different wavelengths, and the incident light after coupling the first light source and the second light source through the first dispersion element is directed to the lens assembly.

[0013] In some embodiments, the optical signal includes a fluorescence signal generated by exciting a phosphor in the droplet with the incident laser.

[0014] In some embodiments, the detector assembly includes a first photoelectric sensor and a second photoelectric sensor. The lens assembly further includes a third dispersion element. A part of the fluorescence signal is transmitted through the third dispersion element and transmitted to the first photoelectric sensor, and another part of the fluorescence signal is reflected by the third dispersion element and transmitted to the second photoelectric sensor.

[0015] In some embodiments, the optical module further includes an illumination light source and a camera optically coupled to the illumination light source. The camera collects an image of the droplet sorting chip or the droplet generation chip under the illumination of the illumination light source through the objective lens.

[0016] In some embodiments, the lens assembly further includes a beam splitter. The beam splitter is configured to reflect the light emitted by the illumination light source to the camera, and transmit the optical signal in response to the incident laser to the detector assembly.

[0017] In some embodiments, it further includes: a droplet printing module, including a printing channel communicated with the outlet of the target flow channel, and an orifice plate provided corresponding to the printing channel and having a plurality of receiving holes. The droplets flowing out of the target flow channel are discharged through the printing channel into the receiving holes of the orifice plate.

[0018] In some embodiments, it further includes: a motion control circuit. The motion control circuit is connected to a first displacement stage driving the sample stage and a second displacement stage driving the orifice plate, and is configured to control the displacement of the first displacement stage and the second displacement stage.

[0019] Further, the droplet preparation instrument further includes a second camera optically coupled to the end of the printing channel close to the orifice plate, configured to collect an image of the end, and the image of the end includes the number of droplets at the end.

[0020] The present application also provides a device for screening antibodies, including the droplet preparation instrument described above.

[0021] In some embodiments, the device for screening antibodies further includes an incubator for incubating the droplets prepared by the droplet preparation instrument. Description of the Drawings

[0022] Figure 1 is a schematic diagram of a droplet preparation instrument according to an embodiment of the present application.

[0023] Figure 2 is a schematic diagram of the internal structure of a droplet preparation instrument according to an embodiment of the present application.

[0024] Figure 3 is a schematic diagram of an optical module of a droplet preparation instrument according to an embodiment of the present application.

[0025] Figure 4 is a schematic diagram of a droplet printing module of a droplet preparation instrument according to an embodiment of the present application.

[0026] Figure 5 is a control logic block diagram of a droplet preparation instrument according to an embodiment of the present application.

[0027] Figure 6 is a schematic diagram of a droplet sorting chip according to an embodiment of the present application.

[0028] Figure 7 is a schematic diagram of the droplet distribution form in a droplet sorting chip according to an embodiment of the present application.

[0029] Figure 8 is a schematic diagram of a droplet generation chip according to an embodiment of the present application.

[0030] Reference numerals: droplet preparation instrument 100, droplet generation chip 10, first oil phase inlet 11, sample phase inlet 12, confluence port 13, first oil phase flow channel 14, sample phase flow channel 15, introduction channel 101, buffer channel 102, extraction channel 103, confluence flow channel 16, first filter element 171, second filter element 172, droplet sorting chip 20, droplet phase inlet 21, second oil phase inlet 22, detection flow channel 23, target flow channel 24, waste liquid flow channel 25, sorting electrode 26, sample stage 30, optical module 40, first light source 411, second light source 412, camera 421, first photoelectric sensor 422, second photoelectric sensor 423, first filter 431, second filter 432, third filter 433, first dispersion element 441, second dispersion element 442, third dispersion element 443, mirror 451, beam splitter 452, objective lens 453, liquid path system 51, high-voltage drive module 52, feedback control sub-circuit 61, droplet 71, spacer oil 72, control circuit 80, display screen 81, feedback control sub-circuit 82, droplet printing module 83, waste liquid tray 831, second displacement stage 833, servo motor 834, shock absorber 835, motion control circuit 84, illumination light source 85, motion motor drive module 90. Detailed implementation manners

[0031] The embodiments of the present application will be described in detail below. Examples of the embodiments are shown in the drawings, where the same or similar reference numerals represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present application and should not be construed as a limitation of the present application.

[0032] As Figures 1 to 8 , the droplet preparation instrument 100 according to the embodiments of the present application includes: a chip assembly, a sample stage 30, an optical module 40, a liquid path system 51, and a control circuit 80.

[0033] Specifically, the chip assembly includes a droplet generation chip 10 for supporting a biological sample and a droplet sorting chip 20 for supporting the droplets to be sorted. The droplet generation chip is configured to generate droplets encapsulating the biological sample. The droplet sorting chip 20 includes sorting electrodes 26 that are operably electrically connected to the outside. The sample stage 30 is used to place at least one of the droplet generation chip 10 and the droplet sorting chip 20. The optical module 40 is optically coupled to the sample stage 30 for providing incident light to the sample stage and collecting the optical signals generated by the biological sample or the droplets in response to the incident light. The liquid path system 51 is operably connected to the chip assembly for pumping solutions to the droplet generation chip 10 or the droplet sorting chip 20. The control circuit 80 can selectively call a first set of control parameters and a second set of control parameters. The first set of control parameters includes a first control instruction for driving at least the liquid path system to be coupled to the droplet generation chip 10, and the second set of control parameters includes a second control instruction for driving the liquid path system 51 and the optical module to be coupled to the droplet sorting chip 20. The control circuit includes a feedback control sub-circuit 61. The feedback control sub-circuit 61 is selectively connected to the sorting electrodes 26 and the optical module 40 based on the second control instruction for controlling the sorting electrodes 26 according to the detection results of the optical module 40. The above-mentioned first set of control parameters and second set of control parameters respectively correspond to two operation modes of the droplet preparation instrument 100: the droplet generation mode and the droplet sorting mode.

[0034] In the droplet generation mode, the droplet generation chip 10 is correspondingly excited to call the first control instruction, and the liquid path system 51 correspondingly pumps a sample solution (i.e., the continuous phase liquid) and an oil phase solution (i.e., the dispersed phase liquid) into the droplet generation chip 10 to generate droplets in the droplet generation chip 10. Preferably, the liquid path system 51 further collects and stores the droplets from the droplet generation chip 10 in this mode. Further, in some embodiments of the present application, corresponding to the first control instruction, the optical module can be optically coupled to the droplet generation chip 10, project illumination light onto the droplet generation chip 10, and collect real-time images of the droplet generation chip 10 to monitor the droplet generation situation.

[0035] In the droplet sorting mode, the droplet sorting chip 20 is correspondingly excited to call the second control instruction, and the liquid path system 51 correspondingly pumps a droplet solution containing target droplets and an oil phase liquid (i.e., the spacer oil) into the droplet sorting chip 10 so that the droplets flow through the droplet sorting chip 20 in sequence. In this mode, the optical module projects excitation light onto the droplet sorting chip 20. The target droplets containing the excited substance (such as a fluorescent dye) generate fluorescent signals under the action of the excitation light. After the fluorescent signals are collected by the optical module, the sorting electrodes 26 in the droplet sorting chip 20 are correspondingly driven to collect the target droplets.

[0036] The droplet preparation instrument 100 according to the embodiments of the present application can prepare droplets 71 and quickly sort out the required droplets 71 for the next detection, providing a high-throughput, non-destructive, accurate and reliable antibody screening platform, greatly improving the screening efficiency of high-affinity and high-specificity antibodies at the single-cell level, and contributing to the realization of high-throughput, long-length, and accurate sequencing.

[0037] As Figure 8 , the droplet generation chip 10 of the present application may include: a first oil phase inlet 11, a sample phase inlet 12, a confluence 13, and a flow channel. The first oil phase inlet 11 is used to introduce droplet generation oil, the sample phase inlet 12 is used to introduce a sample, and the flow channel is respectively connected to the first oil phase inlet 11, the sample phase inlet 12, and the confluence 13. The flow channel can be used to connect the first oil phase inlet 11 and the sample phase inlet 12 to the confluence 13. After the droplet generation oil is introduced from the first oil phase inlet 11 and the sample is introduced from the sample phase inlet 12, they can converge at the confluence 13 to generate droplets 71.

[0038] Specifically, in the droplet generation mode, the control circuit 80 calls the second control instruction to couple the liquid path system 51 with the droplet generation chip 10. In this case, the liquid path system 51 can introduce the droplet generation oil from the first oil phase inlet 11 into the flow channel through, for example, a liquid pump and flow towards the confluence 13; introduce the sample from the sample phase inlet 12 into the flow channel through a liquid pump. The introduced droplet generation oil and the sample will converge at the confluence 13, and the droplet generation oil forms a shear force at the confluence to wrap the sample to generate droplets 71. Preferably, the droplets 71 can wrap, for example, a fluorescent dye to generate droplets 71 for screening, which is convenient for subsequent screening of the droplets 71, can improve the screening efficiency and effect, and can control the detection accuracy by controlling the spacing between the droplets 71, thereby realizing high-throughput, non-destructive, accurate and reliable particle screening. Preferably, in the droplet generation mode, the control circuit 80 can also drive the optical module to image, for example, the confluence 13 of the droplet generation chip 10 to monitor the generation situation of the droplets 71 in real time.

[0039] In addition, in some embodiments of the present application, the flow channel may include a first oil phase flow channel 14 and a sample phase flow channel 15. The two ends of the first oil phase flow channel 14 are respectively connected to the first oil phase inlet 11 and the confluence 13, and the two ends of the sample phase flow channel 15 are respectively connected to the sample phase inlet 12 and the confluence 13. By setting the first oil phase flow channel 14 and the sample phase flow channel 15, the sample and the droplet generation oil can be diverted, so that the droplet generation oil and the sample can converge at the confluence 13 to generate droplets 71, and at the same time, buffer and backflow are provided for the droplet generation oil and the sample to avoid impact at the confluence 13, affecting the generation effect of the droplets 71.

[0040] Further, at least one of the first oil phase flow channel 14 and the sample phase flow channel 15 includes an introduction channel 101, a buffer channel 102, and an extraction channel 103. The introduction channel 101, the buffer channel 102, and the extraction channel 103 are connected in sequence, and the extraction channel 103 is connected to the confluence 13. Specifically, the first oil phase flow channel 14 may include the introduction channel 101, the buffer channel 102, and the extraction channel 103. The introduction channel 101 of the first oil phase flow channel 14 is connected to the first oil phase inlet 11, and the extraction channel 103 of the first oil phase flow channel 14 is connected to the confluence 13. The sample phase flow channel 15 may include the introduction channel 101, the buffer channel 102, and the extraction channel 103. The introduction channel 101 of the sample phase flow channel 15 is connected to the sample phase inlet 12, and the extraction channel 103 of the sample phase flow channel 15 is connected to the confluence 13. The buffer channel 102 can provide buffering for the droplet-forming oil or the sample phase, reduce the flow rate of the fluid, and avoid impact, so as to facilitate the generation of stable droplets 71 by the droplet generation chip 10 and facilitate the subsequent sorting and detection of the droplets 71.

[0041] Wherein, the buffer channel 102 can construct a curved flow path. When the fluid (droplet-forming oil or sample) flows in the curved flow channel, the flow direction of the fluid will change, thereby reducing the speed and buffering the fluid, so as to facilitate the combination of the droplet-forming oil and the sample to generate stable droplets 71. In addition, the buffer channel 102 can also be set to include a plurality of curved flow channels connected in series.

[0042] In addition, the width dimension of the extraction channel 103 is smaller than the width dimension of the introduction channel 101. This can buffer the fluid and generate stable droplets 71 at the confluence 13. Combining the foregoing, the first oil phase flow channel 14 may include the introduction channel 101, the buffer channel 102, and the extraction channel 103, and the width dimension of the introduction channel 101 of the first oil phase flow channel 14 is larger than the width dimension of the extraction channel 103; or, the sample phase flow channel 15 may include the introduction channel 101, the buffer channel 102, and the extraction channel 103, and the width dimension of the introduction channel 101 of the sample phase flow channel 15 is larger than the width dimension of the extraction channel 103.

[0043] In some embodiments of the present application, the width dimension of the lead-out channel 103 is not less than 20 μm and not greater than 50 μm, which further improves the stability of generating the droplets 71. In combination with the foregoing, the first oil-phase flow channel 14 may include an introduction channel 101, a buffer channel 102, and a lead-out channel 103, and the sample-phase flow channel 15 may include an introduction channel 101, a buffer channel 102, and a lead-out channel 103. The width dimension of the lead-out channel 103 of the first oil-phase flow channel 14 is 20 μm to 50 μm, and the width dimension of the lead-out channel 103 of the sample-phase flow channel 15 is 20 μm to 50 μm. In this way, the width dimensions of the lead-out channels 103 of the first oil-phase flow channel 14 and the sample-phase flow channel 15 can be made similar, so as to facilitate the stable generation of droplets 71 at the confluence 13.

[0044] Furthermore, the flow channel may further include a confluence flow channel 16. One end of the confluence flow channel 16 is connected to the confluence 13. Among them, the width of the end of the confluence flow channel 16 connected to the confluence 13 is 20 μm to 50 μm. In this way, the widths of the lead-out channel 103 of the first oil-phase flow channel 14, the lead-out channel 103 of the sample-phase flow channel 15, and the confluence flow channel 16 at the confluence 13 are all similar. Experiments show that more stable droplets 71 can be generated.

[0045] In combination with the foregoing, in some embodiments of the present application, the flow channel further includes a confluence flow channel 16, and the confluence flow channel 16 is connected to the confluence 13 for leading out the droplets 71. Thus, the generated droplets 71 can be stably led out through the confluence flow channel 16, so as to facilitate the collection and storage of the droplets 71.

[0046] In addition, the width dimension of the inlet of the confluence flow channel 16 can be set to be smaller than the width dimension of the outlet of the confluence flow channel 16, so that the droplets 71 generated at the confluence 13 can be stably led out, improving the stability of the droplets 71. The width dimension of the first end of the confluence flow channel 16 can be set to be not less than 20 μm and not greater than 50 μm. In combination with the foregoing, the dimensions of each flow channel at the confluence 13 can be made relatively similar, facilitating the generation of droplets 71.

[0047] In the present application, the droplet-generation oil enters from the first oil-phase inlet 11, and then flows through the buffer channel 102 of the first oil-phase flow channel 14. The buffer channel 102 can stabilize the flow rate of the droplet-generation oil, so that the flow rate of the droplet-generation oil entering the lead-out channel 103 is stable. Moreover, since the width dimension of the lead-out channel 103 in the first oil-phase flow channel 14 is smaller than the width of the introduction channel 101, the amount of the droplet-generation oil can be reduced to a certain extent, thereby realizing the efficient encapsulation of single cells / single particles on the premise of reducing the preparation cost of the droplets 71.

[0048] In addition, the sample-phase flow channels 15 include at least two, and the at least two sample-phase flow channels 15 converge upstream of the confluence 13. Among them, the at least two sample-phase flow channels 15 can be connected to the same sample-phase inlet 12, or can be connected to different sample-phase inlets 12. When the at least two sample-phase flow channels 15 are connected to the same sample-phase inlet 12, the generation efficiency of the droplets 71 can be improved. When the at least two sample-phase flow channels 15 are connected to different sample-phase inlets 12, different samples can be introduced from different sample-phase inlets 12. The different samples can converge upstream of the confluence 13 to complete the combination and be introduced to the confluence 13 to generate the droplets 71, so that different types of droplets 71 can be conveniently generated, and the application range of the droplet generation chip 10 can be improved.

[0049] The two first oil-phase flow channels 14 converge at the confluence 13 to form a shear force, which can conveniently generate the droplets 71 and improve the generation efficiency and stability of the droplets 71. Among them, the at least two first oil-phase flow channels 14 can be connected to the same oil-phase inlet or different oil-phase inlets.

[0050] The first oil-phase inlet 11 is connected with a first filter element 171; or, the sample-phase inlet 12 is connected with a second filter element 172. By filtering out impurities, the stability of the droplet generation chip 10 can be improved, and the flow channel blockage can be avoided.

[0051] The droplet generation chip 10 is configured to be sheet-shaped. It can facilitate loading the droplet generation chip 10 onto the droplet preparation instrument 100 to generate the droplets 71. At the same time, the sheet-shaped droplet generation chip 10 can save the usage of samples and droplet generation oil, reduce costs and be more environmentally friendly.

[0052] In some embodiments of the present application, the liquid path system is set under the second set of control parameters to control the ratio of the flow rate of the spacer oil introduced into the droplet sorting chip to the flow rate of the droplets within the range of 7:1 - 14:1, so as to fully disperse the droplets and avoid the interference of the fluorescence signals between adjacent droplets.

[0053] Such as Figure 1, the sample stage 30 is used to place the droplet sorting chip 20 and the droplet generation chip 10. In particular, different chips can be successively placed on the sample stage 30 to perform different operations, thereby reducing the size of the device. The optical module 40 includes a light source assembly, a lens assembly, a detector assembly, an objective lens 453, etc. The lens assembly is used to guide the incident light of the light source assembly through the objective lens 453 to the sample stage 30, and to transmit the optical signal generated by the biological sample or droplet in response to the incident light to the detector assembly. After the incident light with a specified wavelength is provided by the light source assembly and reaches the sample stage, the specified optical signal filtered from the optical signal returned from the chip assembly is transmitted to the detector assembly through the lens assembly. The liquid path system 51 is used to pump in solutions, such as fluids like the dispersed phase solution or the continuous phase solution, etc. The liquid path system 51 includes, for example, a fluid storage device, a pumping mechanism, a fluid pipeline, etc. The feedback control sub-circuit 61 is activated in the droplet sorting mode to connect the sorting electrode 26 of the droplet sorting chip and the detector assembly of the optical module. In this case, the feedback control sub-circuit 61 controls the sorting electrode 26 according to the detection result of the optical module. The above-mentioned pumping mechanism can be, for example, an industrial injection pump, which is controlled by a PLC. The PLC issues command characters to the injection pump through the RS485 protocol to enable the injection pump to complete functions such as initialization, speed setting, liquid path switching, jog output, etc.

[0054] The droplet preparation instrument further includes a first displacement stage coupled to the sample stage 30, and a droplet printing module 83 connected to the sample stage to receive the sorted target droplets. The droplet printing module 83 has a second displacement stage, which is used to support the target container, and the target droplets are collected in the target container after being guided by the printing module 83. The above-mentioned displacement stage is preferably driven by a motor. Correspondingly, the droplet preparation instrument includes a motion motor drive module 90. The first displacement stage is used to drive the chip assembly to position the chip assembly, and the second displacement stage drives the target container to generate displacement. Correspondingly, the control circuit further includes a motion control circuit 84, which is used to receive displacement instructions matching the first set of control parameters or the second set of control parameters, drive the motion motor drive module 90, and then precisely control the displacement of the first displacement stage and / or the second displacement stage.

[0055] As Figure 3As shown, the optical module includes a bright-field imaging optical path and a fluorescence detection optical path. Among them, the bright-field imaging optical path includes: a surface light source 85 that projects bright-field illumination light onto the corresponding chip on the sample stage 30, and the optical signal generated by the chip in response to the bright-field light source is collected by the objective lens 453 and then collected by the camera to generate an image of the droplet sorting chip or the droplet generation chip under the illumination of the illumination light source. Specifically, the illumination light source 85 outputs a collimated bright-field illumination (for example, infrared wavelength) beam, and cooperates with the camera 421 of the optical module 40 to form an image of the chip flow channel. The illumination light source 85 of the present application is composed of an LED and a lens. The wavelength of the LED does not overlap with the fluorescence wavelength. The illumination light source 85 adopts an epi-illumination method. The illumination light emitted by the LED is collimated by the lens and then irradiates the chip, and the transmitted light enters the subsequent optical path through the objective lens 453. The camera 421 is associated with the display screen 81, and the image of the chip flow channel is displayed on the display screen 81, which is used to monitor whether the sample of the droplet sorting chip 20 is working properly, whether the flow channel is blocked, and preliminarily judge the sorting accuracy of the droplets 71. Subsequently, this image can be used for the AI machine vision algorithm to assist the FADS system in intelligent sorting. The droplet printing module 83 discharges the sorted single droplet 71 through a fluid manner from the pipeline and falls into the target container (such as a microplate). In this case, the second displacement stage can be driven by the drive motor drive module 90 to distribute the droplets 71 into different holes in the microplate.

[0056] As Figure 6, the droplet sorting chip 20 of the present application may include a droplet phase inlet 21, a second oil phase inlet 22, a detection flow channel 23, a target flow channel 24, and a waste liquid flow channel 25. The droplet phase inlet 21 and the second oil phase inlet 22 are connected to the detection flow channel 23. The droplet phase inlet 21 is used to introduce droplets 71, and the second oil phase inlet 22 is used to introduce spacer oil 72. The detection flow channel 23 is connected to the target flow channel 24 and the waste liquid flow channel 25. Specifically, during the sorting process by the droplet preparation instrument 100, the droplets 71 (which can be the droplets 71 generated by the aforementioned droplet generation chip or other droplets 71) can enter the droplet sorting chip 20 through the droplet phase inlet 21. In addition, the spacer oil 72 can enter the droplet sorting chip 20 through the second oil phase inlet 22 to separate the droplets 71 introduced through the droplet phase inlet 21. The distance between the droplets 71 can be adjusted by controlling the flow rates of the droplets 71 and the spacer oil 72. After the droplets 71 enter the droplet sorting chip 20 at intervals through the spacer oil 72, they flow towards the detection flow channel 23. Due to the action of the spacer oil 72, the droplets 71 are separated and can pass through the detection flow channel 23 intermittently in sequence. The droplets 71 passing through the detection flow channel 23 will be detected by the droplet preparation instrument 100 to determine whether the droplets 71 in the detection flow channel 23 are the required droplets 71. By judging whether the droplets 71 passing through the detection flow channel 23 are the required droplets 71 through the droplet preparation instrument 100, when the droplets 71 in the detection flow channel 23 are the required droplets 71, the droplet preparation instrument 100 can control the droplets 71 to flow towards the target flow channel 24. When the droplets 71 in the detection flow channel 23 are not the required droplets 71, the droplet preparation instrument 100 can control the droplets 71 to flow towards the waste liquid flow channel 25. Of course, the droplet sorting chip 20 can be set for other purposes. For example, it can detect the number or percentage of droplets 71 that meet the requirements in the droplets 71 to be sorted, etc.

[0057] The droplet sorting chip 20 according to the embodiment of the present application can achieve the sorting of droplets 71, so as to quickly sort out the required droplets 71 for the next step of detection, providing a high-throughput, non-destructive, accurate and reliable antibody screening platform, greatly improving the screening efficiency of high-affinity and high-specificity antibodies at the single-cell level, and contributing to the realization of high-throughput, long-length, and accurate sequencing.

[0058] The droplet preparation instrument 100 provided by the present application, when calling the second control instruction for droplet 71 sorting, includes the following steps:

[0059] 1. Place the droplet sorting chip 20 in the sample stage 30. Set the sorting parameters of each module (which can be done through the display screen 81). The sorting parameters can include the wavelength and power of the incident light (such as the excitation light) of the light source assembly; the light intensity, gain, and exposure duration of the illumination light of the illumination light source; the specifications of the sample loading device of the liquid path system 51, the flow rate of the droplets 71, and the flow rate of the spacer oil 72; the high-voltage driving voltage, frequency, signal holding duration; the sorting threshold of the droplets 71, etc.

[0060] 2. Start droplet 71 sorting. The pumping mechanism of the liquid path system 51 can include a dispersed-phase microfluidic pump and a continuous-phase microfluidic pump. The dispersed-phase microfluidic pump can introduce the droplets 71 into the droplet sorting chip 20 from the droplet-phase inlet 21; at the same time, the continuous-phase microfluidic pump introduces the spacer oil 72 into the droplet sorting chip 20 from the second oil-phase inlet 22. When the droplets 71 and the spacer oil 72 meet at the intersection of the dispersed-phase main channel and the continuous-phase channel, the spacer oil 72 separates the droplets 71 in the dispersed phase, so that the droplets 71 move forward in the channel in an orderly manner with a certain interval along with the spacer oil 72.

[0061] 3. In addition, to achieve accurate sorting, in addition to maintaining the uniformity of the droplets 71, controlling the oil speed of the oil phase so that the droplets 71 move forward in the channel in an orderly manner with a certain interval in the droplet sorting chip 20 is another important means to ensure accurate sorting. In this application, the size of the droplets 71 can be set to 20 - 50 pL, preferably in the range of 30 - 45 pL; through the liquid path system 51, the flow rate of the spacer oil 72 and the flow rate of the droplets 71 can be maintained at 7:1 - 14:1. Or rather, the liquid path system is set under the second set of control parameters to control the ratio of the flow rate of the spacer oil introduced into the droplet sorting chip 20 to the flow rate of the droplets within the range of 7:1 - 14:1. The interval distribution between the droplets 71 is as Figure 7As shown. In some embodiments of the present application, when the speed of the spacer oil 72 is 600 μL / h and the rate of the droplet 71 is 45 μL / h, the sorting accuracy of the droplet 71 is about 100%. As the rate of the droplet 71 gradually increases, when the rate of the droplet 71 is 85 μL / h, the sorting accuracy of the droplet 71 is about 95% at this time. Thus, by controlling the uniformity of the droplet 71 size and the flow rate ratio of the oil phase to the droplet 71, the interval between the droplets 71 can be better controlled, so as to ensure that there is a uniform interval between the droplets 71 and they pass through the fluorescence detection area in sequence and orderly, further improving the sorting accuracy of the droplet 71. In the present application, the sorting accuracy of the droplet 71 is not less than 95%. Therefore, the size of the droplet 71 in the present application is not less than 20 pL and not greater than 50 pL; or, the liquid path system 51 is configured to control the ratio of the flow rate of the spacer oil 72 to the flow rate of the droplet 71 within the range of 7:1 - 14:1; or, the liquid path system 51 is configured to control the flow rate of the spacer oil 72 to be 600 μL / h and the flow rate of the droplet 71 is not greater than 85 μL / h; or, the liquid path system 51 is configured to control the flow rate of the spacer oil 72 to be 600 μL / h and the flow rate of the droplet 71 is not less than 45 μL / h and not greater than 85 μL / h.

[0062] 4. When the droplet 71 reaches the excitation area, the incident light provided by the optical module 40 scans each passing droplet 71, and the generated fluorescence signal is received by the detector assembly. When the intensity of the received optical signal (fluorescence signal) meets the set sorting parameters, it is considered as the target droplet 71. After the signal is converted into an electrical signal by the detector assembly, it is amplified and transmitted to the feedback control sub - circuit 61. Optionally, the excitation area can be configured as circular, elliptical or linear.

[0063] Optionally, as Figure 6 shown, the droplet sorting chip 20 includes a detection flow channel 23 and a target flow channel 24 and a waste liquid flow channel 25 extending from a sorting point P at one end of the detection flow channel 23 in different directions. The sorting electrode 26 is configured to generate a dielectrophoretic force that guides the fluid at the sorting point P to flow towards the target flow channel 24 when it is electrically connected to an external electrical signal. The feedback control sub - circuit 61 determines whether it is a target droplet 71 by receiving the electrical signal. If it is a target droplet 71, it outputs an instruction to the high - voltage driving module 52 to activate the sorting electrode 26, generating a non - uniform electric field. Under the action of the non - uniform electric field, a dielectrophoretic force acting on the droplet 71 is generated to deflect the droplet 71 to flow towards the target channel 24; if the fluorescence signal does not reach the set threshold of the sorting parameters, it is determined as a non - target droplet 71. At this time, the feedback control sub - control circuit 61 does not send a high - voltage driving instruction, and the non - target droplet 71 flows towards the waste liquid flow channel 25.

[0064] As Figure 3, the incident light includes incident lasers with at least two different wavelengths provided by a light source assembly. Optionally, the light source assembly includes a first light source 411, a second light source 412, and a first dispersion element 441. The first dispersion element 441 is configured to reflect the incident laser of the first light source 411 and transmit the incident laser of the second light source 412. The incident lasers of the first light source and the second light source have different wavelengths. The incident light after the first light source 411 and the second light source 412 are coupled by the first dispersion element 441 is directed towards the lens assembly. Specifically, the light source assembly includes at least two excitation light sources with different spectral center wavelengths (such as 525 nm and 638 nm). The incident lights of the first light source 411 and the second light source 412 respectively become collimated lights after passing through a collimating lens. The incident lights of multiple light sources pass through the first dispersion element 441 (the dispersion element can be a dichroic mirror, a grating, or a prism, etc.), adjust the incident angle, and then combine the incident lasers of the first light source 411 and the second light source 412 and further propagate along the optical path.

[0065] Furthermore, the detector assembly includes a first photoelectric sensor 422 and a second photoelectric sensor 423. The lens assembly further includes a third dispersion element 443. A part of the fluorescence signal is transmitted through the third dispersion element 443 and transmitted to the first photoelectric sensor 422, and another part of the fluorescence signal is reflected by the third dispersion element 443 and transmitted to the second photoelectric sensor 423. The first photoelectric sensor 422 and the second photoelectric sensor 423 respectively detect the fluorescence signals generated by the incident lasers of different wavelengths.

[0066] As shown in the figure, the above-mentioned incident laser and the fluorescence signal in response to the incident laser are confocal.

[0067] Furthermore, the lens assembly further includes a beam splitter 452. The beam splitter 452 is configured to reflect the light emitted by the illumination light source to the camera 421 and transmit the fluorescence signal in response to the above to the detector assembly. The lens assembly includes a mirror, a second dispersion element 442, a first filter 431, a beam splitter 452, and a third dispersion element 443 arranged in sequence along a straight line direction. The incident laser is directed towards the objective lens 453 through the second dispersion element 442 and the mirror. The light emitted by the illumination light source is directed towards the camera 421 through the mirror, the second dispersion element 442, the first filter 431, and the beam splitter 452. The above-mentioned fluorescence signal is directed towards the detector assembly through the mirror, the second dispersion element 442, the first filter 431, the beam splitter 452, and the third dispersion element 443.

[0068] Specifically, the incident laser after the combination of the light source components is incident on the second dispersion element 442, reflected by the second dispersion element 442 to the objective lens 453, and focused to form a single light spot in the detection area of the droplet sorting chip 20 (such as the detection flow channel 23 of the droplet sorting chip 20). By controlling the parameters of the components (such as the objective lens 453) in the optical module 40, the size of the incident light spot is controlled to be close to the width of the chip flow channel. When the sample droplet 71 containing fluorescence flows through this area, the fluorescent particles in the droplet 71 generate fluorescence signals under the excitation of the incident light. The fluorescence signal, the residual incident laser reflected by the interface, and the mixed light formed by the illumination light pass through the objective lens 453 and the second dispersion element 442 again. Since the second dispersion element 442 does not reflect fluorescence, the fluorescence and the residual excitation light are initially separated. Subsequently, the mixed light passes through the first filter 431. The first filter 431 has a high reflectivity to the excitation light, and most of the residual excitation light in the mixed light is removed after passing through. The mixed light then passes through a beam splitter 452. The beam splitter 452 separates the mixed light in a certain ratio (such as 1:9). The reflected part passes through the tube lens and then enters the camera 421 of the image detection module for imaging, and the image information is displayed on the display screen 81. The transmitted mixed light passes through the third dispersion element 443. The third dispersion element 443 reflects the fluorescence and excitation light corresponding to the wavelength to the third filter 433. After being focused by the lens, it is detected by the first photoelectric sensor 422 (such as a photomultiplier tube). The remaining transmitted mixed light passes through the second filter 432, and only the fluorescence signal of the specified wavelength (such as 680 nm) remaining in the mixed light is focused by the lens and detected by the second photoelectric sensor 423 (photomultiplier tube).

[0069] The foregoing optical module 40 faces the sample stage 30, and the generated light spot area covers the width range of the detection flow channel 23. The shape of the light spot can be circular, elliptical or linear, etc., to ensure that the incident light spot can excite the maximum fluorescence intensity of the excited substance (such as a fluorescent dye) in the droplet 71 when scanning each droplet 71, thereby optimizing the detection efficiency and detection sensitivity of the photomultiplier tube.

[0070] Optionally, the display screen 81 displays the image information captured by the camera 421 in real time and further feeds back the operation status of the droplet 71 sorting process in the droplet sorting mode, including monitoring the distance between droplets 71 in the microfluidic flow channel, the blockage condition of the flow channel, etc. It should be understood that the present application can also combine AI machine vision algorithms to assist in counting the sorting throughput and sorting accuracy of droplets 71 and display them on the display screen 81.

[0071] In some embodiments of the present application, the droplet printing module 83 includes a printing channel communicated with the outlet of the target flow channel 24, and an orifice plate provided with a plurality of receiving holes corresponding to the printing channel. The droplets flowing out of the target flow channel are discharged through the printing channel into the receiving holes of the orifice plate. The principle structure of the droplet printing module 83 in the present application is asFigure 4 As shown. After the target droplet 71 is sorted out, it needs to be automatically placed at different positions on the microplate according to requirements. The two-dimensional second displacement stage 833 drives the target container (microplate) to move relative to the printing channel so that the droplet 71 can fall onto different positions on the microplate. To ensure that each droplet 71 dropping into the wells on the microplate is single, therefore, in some embodiments of the present application, the droplet sorter 100 further includes a second camera optically coupled to the printing channel. The second camera images the printing channel to monitor the number of droplets in the printing channel in real time and determines whether there is only one droplet 71 within the specified position of the printing channel. When the number of droplets 71 does not meet the actual requirements, the movable waste liquid tray 831 is moved below the printing channel to prevent the droplets that do not meet the quantity requirements from dropping into the microplate. To make the second displacement stage 833 lightweight, the entire second displacement stage 833 is preferably made of aluminum. Further, since the entire second displacement stage 833 has components with fast response movement, in order to reduce the impact of vibration on the device, shock-absorbing blocks 835 are provided at the joints to reduce the impact of the second displacement stage 833 on the device during movement.

[0072] In some embodiments of the present application, the motion control circuit 84 that controls the displacement of the first displacement stage and the second displacement stage can be controlled by a PLC. The motion control circuit 84 can control the motion control of the X / Y / Z axes of the first displacement stage, the motion control of the second displacement stage 833 driving the target container (such as a microplate), etc. The first displacement stage is preferably driven by a stepper motor, where the X-axis and Y-axis use pulse control and the Z-axis is controlled by a bus protocol (EntherCAT). The second displacement stage is optionally driven by a servo motor 834 and is all controlled by the bus protocol (EntherCAT).

[0073] The present application also provides a device for screening antibodies, including the aforementioned droplet preparation instrument 100.

[0074] In addition, the device for screening antibodies further includes an incubator for incubating the droplets 71 generated by the droplet preparation instrument 100. After the droplet preparation instrument 100 generates the droplets 71, the droplets 71 are first collected in the incubator, and an incubator for culturing cells is used for cell culture for a period of time. After the culture is completed, it is sent to the droplet preparation instrument 100 for steps such as optical fiber irradiation and sorting.

[0075] ​In the description of the present application, it should be understood that the orientation or positional relationship indicated by the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation to the present application.

[0076] In addition, the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one of such features. In the description of the present application, the meaning of "a plurality" is at least two, such as two, three, etc., unless otherwise specifically and clearly defined.

[0077] In the present application, unless otherwise clearly specified and defined, the terms "mounted", "connected", "coupled", "fixed", etc. shall be construed in a broad sense. For example, it may be a fixed connection, a detachable connection, or integrated; it may be a mechanical connection or an electrical connection; it may be directly connected or indirectly connected through an intermediate medium, and it may be the internal communication of two elements or the interaction relationship between two elements, unless otherwise clearly defined. For those of ordinary skill in the art, the specific meanings of the above terms in the present application can be understood according to specific circumstances.

[0078] In the present application, unless otherwise clearly specified and defined, the first feature being "on" or "under" the second feature may be that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature being "above", "over" and "on top of" the second feature may be that the first feature is directly above or obliquely above the second feature, or merely indicates that the first feature has a higher horizontal height than the second feature. The first feature being "under", "beneath" and "underneath" the second feature may be that the first feature is directly below or obliquely below the second feature, or merely indicates that the first feature has a lower horizontal height than the second feature.

[0079] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0080] Although the embodiments of this application have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limitations on this application. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of this application.

Claims

1. A droplet preparation instrument, characterized in that, Comprising: A chip component, including a droplet generation chip for supporting a biological sample and a droplet sorting chip for supporting the droplets to be sorted. The droplet generation chip is configured to generate droplets encapsulating the biological sample, and the droplet sorting chip includes sorting electrodes operably electrically connected to the outside; A sample stage for placing at least one of the droplet generation chip and the droplet sorting chip; An optical module optically coupled to the sample stage, configured to provide incident light to the sample stage and collect optical signals generated by the biological sample or the droplets in response to the incident light; A liquid path system operably connected to the chip component for pumping a solution to the droplet generation chip or the droplet sorting chip; A control circuit selectively invoking a first set of control parameters or a second set of control parameters. The first set of control parameters includes a first control instruction for driving at least the liquid path system to be coupled with the droplet generation chip, and the second set of control parameters includes a second control instruction for driving the liquid path system and the optical module to be coupled with the droplet sorting chip; The control circuit includes a feedback control sub-circuit, which is selectively connected to the sorting electrodes and the optical module based on the second control instruction, and is configured to control the sorting electrodes according to the detection results of the optical module.

2. The droplet preparation instrument according to claim 1, characterized in that, The liquid path system is set under the second set of control parameters to control the ratio of the flow rate of the spacer oil flowing into the droplet sorting chip to the flow rate of the droplets within the range of 7:1 - 14:1; And / or, the droplet sorting chip includes a detection flow channel, a target flow channel and a waste liquid flow channel extending from a sorting point at one end of the detection flow channel in different directions. The sorting electrodes are configured to generate a dielectrophoretic force that guides the fluid at the sorting point to flow towards the target flow channel when electrically connected to an external electrical signal.

3. The droplet preparation instrument according to claim 1, wherein The optical module includes a light source assembly, a lens assembly, a detector assembly and an objective lens. The lens assembly is configured to guide the incident light of the light source assembly through the objective lens to the sample stage, and to transmit the optical signals generated by the biological sample or the droplets in response to the incident light to the detector assembly.

4. The droplet preparation instrument according to claim 3, characterized in that, The incident light includes incident lasers provided by the light source assembly having at least two different wavelengths; and / or, the light source assembly includes a first light source, a second light source and a first dispersion element. The first dispersion element is configured to reflect the incident laser of the first light source and transmit the incident laser of the second light source. The incident lasers of the first light source and the second light source have different wavelengths, and the incident light after being coupled by the first light source, the second light source and the first dispersion element is directed towards the lens assembly.

5. The droplet preparation instrument according to claim 4, characterized in that, The optical signal includes a fluorescence signal generated by the phosphor in the droplets being excited by the incident laser.

6. The droplet preparation instrument according to claim 5, characterized in that, The detector assembly includes a first photoelectric sensor and a second photoelectric sensor. The lens assembly further includes a third dispersion element. A part of the fluorescence signal is transmitted through the third dispersion element and transmitted to the first photoelectric sensor, and another part of the fluorescence signal is reflected by the third dispersion element and transmitted to the second photoelectric sensor.

7. The droplet preparation instrument according to claim 4, wherein The optical module further includes an illumination light source and a camera optically coupled to the illumination light source. The camera collects an image of the droplet sorting chip or the droplet generation chip under the illumination of the illumination light source through the objective lens.

8. The droplet preparation instrument according to claim 7, characterized in that, The lens assembly further includes a beam splitter configured to reflect the light emitted by the illumination light source to the camera and transmit the optical signal in response to the incident laser to the detector assembly.

9. The droplet preparation instrument according to claim 2, characterized in that, Further included is: A droplet printing module, including a printing channel communicated with the outlet of the target flow channel and an orifice plate provided corresponding to the printing channel and having a plurality of receiving holes. Droplets flowing out of the target flow channel are discharged through the printing channel into the receiving holes of the orifice plate.

10. The droplet preparation instrument according to claim 9, wherein, Further included is: A motion control circuit, which is connected to a first displacement stage driving the sample stage and a second displacement stage driving the orifice plate, and is configured to control the displacement of the first displacement stage and the second displacement stage.

11. The droplet preparation instrument according to claim 9, characterized in that, The droplet preparation instrument further includes a second camera optically coupled to the printing channel, configured to collect an image of the printing channel, and the image of the printing channel includes the quantity information of the droplets.

12. An apparatus for screening antibodies, characterized in that, Including the droplet preparation instrument according to any one of claims 1-11.

13. The device according to claim 12, characterized in that, The device for screening antibodies further includes an incubator for incubating the droplets prepared by the droplet preparation instrument.

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