WO2024145763A1

WO2024145763A1 - Micro-droplet screening device and system

Info

Publication number: WO2024145763A1
Application number: PCT/CN2023/070169
Authority: WO
Inventors: 张辉; 周师岳; 栗向; 严向文; 李伟; 徐浩然; 王培毅; 沈梦哲; 郑越; 黄芝淇; 谭亚玲; 莫松劲; 谢庆庆; 董宇亮; 章文蔚
Original assignee: 深圳华大生命科学研究院
Priority date: 2023-01-03
Filing date: 2023-01-03
Publication date: 2024-07-11

Abstract

A micro-droplet screening device, and a system. The micro-droplet screening device comprises an upper computer (1), an optical signal detection apparatus (2), a signal processor (3), and an electric screener (4). The optical signal detection apparatus (2) is used to acquire an optical signal of a droplet to be measured, and convert the optical signal into an electrical signal of the droplet to be measured. The signal processor (3) is electrically connected to the optical signal detection apparatus (2), the signal processor (3) being used to receive and process the electrical signal of the droplet to be measured, and send a screening instruction to the electrical screener (4) according to the strength of the electrical signal. The electric screener (4) is electrically connected to the signal processor (3), the electric screener (4) controlling the droplet to be measured in a microfluidic sorting chip (5) to deflect to the a corresponding flow channel according to the screening instruction, so as to complete screening of the micro-droplet to be measured. The upper computer (1) is electrically connected to the signal processor (3), the upper computer (1) being used to issue a screening instruction and configure a related screening parameter. Provided is a highly integrated and automated micro-droplet screening device, which greatly improves the convenience of microdroplet screening.

Description

Micro-droplet screening equipment and systems

Technical Field

The present invention relates to the field of microfluidic technology, and in particular to a micro-droplet screening device and system.

Background technique

The high-throughput screening technology of microdroplets is to encapsulate single cells and biochemical reaction reagents in monodisperse droplets. After the droplets react under certain conditions, there will be a change in special light signals in the positive droplets containing target cells. This signal change is different from other empty droplets or droplets encapsulating non-target cells. The droplets pass through the microfluidic sorting chip, and the optical signals are collected. By analyzing the optical signals, the sorting threshold is defined. Positive droplets that exceed the threshold will trigger positive sorting electrical pulses when passing through the detection area of the microfluidic chip, and the corresponding positive droplets will be electrically deflected to the microfluidic chip collection channel, thereby realizing the sorting of positive cells.

Microdroplet screening technology can quickly encapsulate single cells in a picoliter volume, and the average daily droplet detection throughput can reach 10 ^7. The tiny reaction systems formed by oil-in-water droplets avoid contamination between different samples, and can achieve high-throughput cell function detection and sorting at the single-cell level. Compared with flow cytometry sorting technology, microdroplet screening technology can detect cell secretions and cell lysis products in addition to intracellular and cell surface signals. It can meet important industrial needs such as monoclonal antibody screening, tumor immunotherapy cell preparation, industrial strain screening, enzyme screening, etc., and has a wide range of commercial application scenarios.

Existing technical means that can be used for monoclonal antibody screening, tumor immunotherapy cell preparation, industrial strain screening, and enzyme directed evolution include: flow cytometry, microplate method, and water-in-oil-in-water tandem flow cytometry. The above technical means usually have defects such as low integration and automation.

Summary of the invention

In order to overcome the above-mentioned defects of the existing screening technology, the present invention provides a micro-droplet screening device and system.

The present invention solves the above technical problems through the following technical solutions:

In a first aspect, a micro-droplet screening device is provided, the micro-droplet screening device comprising a host computer, an optical signal detection device, a signal processor, and an electrical screener;

The optical signal detection device is used to obtain the optical signal of the droplet to be detected, and convert the optical signal into the electrical signal of the droplet to be detected;

The signal processor is electrically connected to the optical signal detection device, and the signal processor is used to receive and process the electrical signal of the droplet to be detected, and send a screening instruction to the electrical filter according to the strength of the electrical signal;

The electrical filter is electrically connected to the signal processor, and the electrical filter controls the droplets to be tested in the microfluidic sorting chip to deflect to the corresponding flow channel according to the screening instruction, so as to complete the screening of the droplets to be tested;

The host computer is electrically connected to the signal processor, and is used to issue the screening instruction and set relevant screening parameters.

Optionally, the optical signal includes at least one of a fluorescence signal, a scattered light signal and an absorbed light signal.

Optionally, the optical signal detection device includes a first light source transmitter, a first light path transmission component, a first light path receiving component, and a first photomultiplier tube;

The first light source transmitter is provided with a first light path transmission component and a first light path receiving component correspondingly;

The first light source emitter excites the optical signal of the droplet to be measured through the first light path transmission component;

The first optical path receiving component is used to obtain the fluorescent signal processed by the filtered light;

Wherein, the first optical path transmission component includes a first beam combiner, a cylindrical lens, and an objective lens, and the first optical path receiving component includes a first dichroic mirror, a first plano-convex lens, and a first filter;

The first photomultiplier tube converts the fluorescence signal into an electrical signal.

Optionally, the optical signal detection device includes a second light source transmitter, a second optical path transmission component, a first optical fiber receiving component, and a second photomultiplier tube;

The second light source emitter is provided with a second light path transmission component correspondingly;

The second light source emitter excites the optical signal of the droplet to be measured through the second light path transmission component;

The first optical fiber receiving component is used to obtain the scattered light signal processed by the filtered light;

Wherein, the second optical path transmission component includes a second beam combiner, a cylindrical lens, and an objective lens, and the first optical fiber receiving component includes an optical fiber receiver, a second plano-convex lens, and a second filter;

The second photomultiplier tube converts the scattered light signal into an electrical signal.

Optionally, the optical signal detection device includes a third light source transmitter, a first optical fiber transmission component, a second optical fiber receiving component, and a third photomultiplier tube;

The first optical fiber transmission component and the second optical fiber receiving component are respectively connected to the first optical fiber interface and the second optical fiber interface distributed on both sides of the flow channel of the microfluidic sorting chip;

The third light source emitter emits light to the first optical fiber interface through the first optical fiber transmission component to stimulate the optical signal of the droplet to be detected;

The second optical fiber receiving component is used to obtain the absorption light signal after the filtered light processing;

Wherein, the second optical fiber receiving assembly comprises an optical fiber receiver, a third plano-convex lens and a third filter;

The third photomultiplier tube converts the absorbed light signal into an electrical signal.

Optionally, the number of the first light source emitter, the second light source emitter and the third light source emitter is multiple;

The wavelength band of the emitted light emitted by each first light source emitter is not completely the same, the wavelength band of the emitted light emitted by each second light source emitter is not completely the same, and the wavelength band of the emitted light emitted by each third light source emitter is not completely the same;

The wavelength band of the emitted light of the first light source emitter and the filtering parameters of the first filter match the fluorescence signal; the wavelength band of the emitted light of the second light source emitter and the filtering parameters of the second filter match the scattered light signal; the wavelength band of the emitted light of the third light source emitter and the filtering parameters of the third filter match the absorbed light signal.

Optionally, the signal processor is used to determine a branch channel that matches the intensity of the electrical signal of the droplet to be detected, and send the screening instruction to the electrical filter.

Optionally, the signal processor is used to send a movement instruction to a host computer when determining that the droplet to be tested is a positive droplet;

The host computer controls the movement of the micro-droplet collecting device according to the movement instruction.

Optionally, the micro-droplet collecting device comprises a plurality of collecting chambers;

The host computer controls the movement of the micro-droplet collecting device when the number of droplets in a collection chamber reaches a preset number. Optionally, the micro-droplet screening device further includes a liquid injection device, which includes a pressure pump, a syringe, and a liquid injection controller;

The liquid injection device is electrically connected to the host computer, and the host computer controls the liquid injection device to inject the dispersed phase and the droplets to be tested into the microfluidic sorting chip;

The syringe is installed in the pressure pump, and the injection controller controls the pressure pump to provide power to the syringe;

An angle is set between the syringe and the horizontal plane.

Optionally, the host computer is used to visually display the optical signal when receiving the optical signal of the droplet to be detected;

The host computer is also used to set parameters of the optical signal detection device, the signal processor and the electrical filter.

Optionally, the micro-droplet screening device further comprises a light-shielding housing;

The optical signal detection device is arranged in the light-shielding housing to block interference from ambient light.

In a second aspect, a micro-droplet screening system is provided, the micro-droplet screening system comprising the microfluidic sorting chip and the micro-droplet screening device described in any one of the above items, the microfluidic sorting chip comprising an optical fiber channel, a sorting component, and at least two branch channels;

The optical fiber channel is connected to the optical fiber interface for transmitting optical signals;

The sorting component is used to sort the droplets to be tested into corresponding branch channels when receiving a deflection instruction.

The beneficial effect of the present invention is that the screening of the droplets to be tested is achieved by setting up a host computer, an optical signal detection device, a signal processor, and an electrical filter. Specifically, the optical signal of the droplets to be tested is obtained by the optical signal detection device, and the optical signal is converted into the electrical signal of the droplets to be tested. The signal processor receives and processes the electrical signal of the droplets to be tested, and sends a screening instruction to the electrical filter according to the strength of the electrical signal. The electrical filter controls the droplets to be tested in the microfluidic sorting chip to deflect to the corresponding flow channel according to the screening instruction, and automatically completes the screening of the droplets to be tested. Thereby, a micro-droplet screening device with high integration and high automation is provided, which greatly improves the convenience of micro-droplet screening.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of a micro-droplet screening device according to an embodiment of the present invention.

FIG. 2 is a schematic diagram of a signal processing feedback control board according to an embodiment of the present invention.

FIG. 3 is a schematic diagram of a microfluidic sorting chip according to an embodiment of the present invention.

FIG. 4 is a schematic diagram of an optical signal detection device according to an embodiment of the present invention.

FIG. 5 is a schematic diagram of a liquid injection device according to an embodiment of the present invention.

FIG. 6 is a schematic diagram of the device system software according to an embodiment of the present invention.

FIG. 7 is a schematic diagram of the overall structure of a micro-droplet screening device according to an embodiment of the present invention.

FIG. 8 is a scatter plot of droplet data according to an embodiment of the present invention.

Detailed ways

The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

FIG1 is a schematic diagram of a micro-droplet screening device provided by an exemplary embodiment of the present invention, and the micro-droplet screening device includes a host computer 1, an optical signal detection device 2, a signal processor 3, and an electrical filter 4. The optical signal detection device 2 is used to obtain the optical signal of the droplet to be tested and convert the optical signal into the electrical signal of the droplet to be tested. The signal processor 3 is electrically connected to the optical signal detection device 2, and the signal processor 3 is used to receive and process the electrical signal of the droplet to be tested, and send a screening instruction to the electrical filter 4 according to the intensity of the electrical signal. The electrical filter 4 is electrically connected to the signal processor 3, and the electrical filter 4 controls the droplet to be tested in the microfluidic sorting chip 5 to deflect to the corresponding flow channel according to the screening instruction to complete the screening of the droplet to be tested. The host computer 1 is electrically connected to the signal processor 3, and is used to issue a screening instruction and set relevant screening parameters. Among them, the relevant screening parameters include: droplet judgment conditions, electrical pulse amplitude, frequency and duration of electrical deflection, etc.

Wherein, referring to FIG. 2 , the signal processor 3 may be a signal processing feedback control board, including signal input ports 2121 to 2128, a signal processing center 22, a deflection electric pulse 231 and a droplet distribution electric pulse 232. When in use, the electric signal of the droplet to be tested is input from the signal input ports 2121 to 2128. The signal processing center 22 includes a droplet signal extraction module, a droplet signal feature calculation module, a droplet judgment module and an electric pulse trigger module, which are used to process the electric signal input from the control board signal input ports 2121 to 2128, and through the above modules, the required droplet to be tested is judged. After the judgment, a screening instruction is sent to the electric filter 4, and the screening instruction may be a deflection electric pulse 231. The signal processing feedback control board also includes an ADC module composed of multiple ADCs (Analog-to-Digital Converters), and the ADC module can sample the first photomultiplier tube, the second photomultiplier tube and the third photomultiplier tube simultaneously and continuously. The ADC module stores the digital signal into the memory for data analysis and processing. The signal processing feedback control board can also complete background signal filtering, and simultaneously collect characteristic values of multiple signals (different optical signals, or parameters of different bands of the same optical signal), including but not limited to peak value, average value, signal width, and perform signal calculations within and between channels to screen the droplets to be tested.

3, the micro-droplet screening device needs to use a microfluidic sorting chip 5 when performing droplet screening. The microfluidic sorting chip 5 includes at least one droplet inlet, at least one oil phase inlet, at least two outlets (collection port and waste liquid port), a screening electrode, a shielding electrode, and at least one optical fiber insertion port. For example, FIG3 shows a microfluidic sorting chip 5 suitable for a micro-droplet screening device, including a sheath liquid inlet (511, 512), a droplet phase inlet (513), a positive droplet outlet (521), a negative droplet outlet (522), a low-melting-point metal infusion port (531, 533, 541, 544), an atmospheric pressure connection port (532, 534), a wire insertion port (542, 543), an optical fiber insertion port (551, 552, 553), a PDMS infusion port (561, 562, 563), a main flow path (57), a branch flow path (518, 519), a pressure relief channel (520), shielding electrodes (5211, 5212), and a screening electrode (54).

The micro droplets to be tested flow into the microfluidic sorting chip 5 from the droplet phase inlet (513). When the micro droplets to be tested pass through the detection area of the microfluidic sorting chip 5, the optical signal of each micro droplet to be tested is detected by the optical signal detection device 2, and the optical signal is converted into an electrical signal (such as a voltage signal) of the droplet to be tested. The signal processor determines whether to send a screening instruction through the intensity of the electrical signal (such as the magnitude of the voltage signal). If the intensity of the electrical signal meets the preset sorting threshold, the screening instruction will be sent to the electrical filter 4. The electrical filter 4 will trigger the deflection electric pulse, which can realize the sorting of positive droplets into the corresponding branch channel, such as the branch flow path (518, 519). The positive droplets are the micro droplets to be tested that need to be screened out.

In this embodiment, the host computer 1, the optical signal detection device 2, the signal processor 3, and the electrical filter 4 are integrated to realize the screening of the droplets to be tested, thereby providing a micro-droplet screening device with high integration and high automation, which greatly improves the convenience of micro-droplet screening.

Optionally, the optical signal includes at least one of a fluorescent signal, a scattered light signal, and an absorbed light signal. Wherein, at least one of the fluorescent signal, the scattered light signal, and the absorbed light signal is selected for screening according to different characteristics of the micro-droplets to be detected. For example, the first micro-droplet to be detected needs to be judged as a positive micro-droplet by the optical signal intensity of both the fluorescent signal and the scattered light signal, and the second micro-droplet to be detected needs to be judged as a positive micro-droplet by the optical signal intensity of the fluorescent signal, the scattered light signal, and the absorbed light signal.

Optionally, referring to FIG4 , the optical signal detection device 2 includes a first light source emitter (at least one first light source emitter is selected arbitrarily from 212 to 215), a first light path transmission component, a first light path receiving component, and a first photomultiplier tube (a first photomultiplier tube corresponding to the first light source emitter is selected from 281 to 284). The first light source emitter is provided with a first light path transmission component and a first light path receiving component. The first light source emitter excites the optical signal of the droplet to be measured through the first light path transmission component. The first light path receiving component is used to obtain the fluorescent signal processed by the filtered light. The first optical path transmission component includes a first beam combiner (a first beam combiner corresponding to the first light source emitter is selected from 221 to 224), a cylindrical lens 23, and an objective lens 293. The first optical path receiving component includes a first dichroic mirror (a first dichroic mirror corresponding to the first light source emitter is selected from 241 and 241 to 246), a first plano-convex lens (a plano-convex lens corresponding to the first light source emitter is selected from 261 to 268 as the first plano-convex lens), and a first filter (a filter corresponding to the first light source emitter is selected from 271 to 278 as the first filter). The first photomultiplier tube converts the fluorescence signal into an electrical signal.

In addition to the above-mentioned devices, the optical signal detection device 2 also includes an illumination light source 295, a stage 294, a tube lens 291, and a high-speed camera 292. Before detecting the fluorescence signal, the microfluidic sorting chip 5 is placed on the stage. When the high-speed camera observes that the droplets are arranged in a single row in the flow channel of the microfluidic sorting chip 5 and the spacing passes stably, it means that the fluorescence signal can be measured. The first light source emitter is used to emit light sources such as lasers and infrared light. The type of light emitted by the first emission light source is not limited here. The first optical path transmission component includes a first beam combining mirror, a cylindrical lens, and an objective lens. The first optical path transmission component is used to transmit the light path emitted by the first emission light source to the stage. The first optical path receiving component includes a first dichroic mirror, a first plano-convex lens, and a first filter, which is used to obtain the fluorescence signal after filtering. After receiving the fluorescence signal, the first photomultiplier tube converts the fluorescence signal into an electrical signal, which can be a voltage value. When different fluorescence signals of the same droplet to be measured need to be measured, multiple first light source emitters can be provided, and the number of first light path transmission components, first light path receiving components and first photomultiplier tubes corresponds to each other. According to the measurement requirements, the wavelength band of the light of the first light source emitter can be adjusted, and the filtering parameters of the first filter are in a corresponding relationship with the wavelength band of the light of the first light source emitter.

Optionally, referring to FIG. 4 , the optical signal detection device 2 includes a second light source transmitter (at least one second light source transmitter is selected arbitrarily from 212 to 215), a second optical path transmission component, a first optical fiber receiving component, and a second photomultiplier tube (at least one photomultiplier tube is selected arbitrarily from 285 to 288). The second light source transmitter is provided with a second optical path transmission component. The second light source transmitter excites the optical signal of the droplet to be detected through the second optical path transmission component. The first optical fiber receiving component is used to obtain the scattered light signal processed by the filtered light. Among them, the second transmission component includes a second beam combining mirror (a second beam combining mirror corresponding to the second light source transmitter is selected from 221 to 224), a cylindrical lens 23, and an objective lens 293. The first optical fiber receiving component includes a first optical fiber receiver (at least one first optical fiber receiver is selected arbitrarily from 252 to 255), a second plano-convex lens (a plano-convex lens corresponding to the first optical fiber receiver is selected from 265 to 268 as a second plano-convex lens), and a second filter (a filter corresponding to the first optical fiber receiver is selected from 275 to 278 as a second filter). The second photomultiplier tube converts the scattered light signal into an electrical signal.

In addition to the above-mentioned devices, the optical signal detection device 2 also includes an illumination light source 295, a stage 294, a tube lens 291, and a high-speed camera 292. Before detecting the scattered light signal, the microfluidic sorting chip 5 needs to be placed on the stage. When the high-speed camera observes that the droplets are arranged in a single row in the flow channel of the microfluidic sorting chip 5 and the spacing passes stably, it means that the scattered light signal can be measured. The second light source transmitter is used to emit light sources such as lasers and infrared light. The type of light emitted by the second light source is not limited here. The second optical path transmission component includes a second beam combiner, a cylindrical lens, and an objective lens. The first optical fiber receiving component includes an optical fiber receiver, a second plano-convex lens, and a second filter. The optical fiber receiver is connected to the interface of any optical fiber insertion port of the microfluidic sorting chip 5. The optical fiber receiving component is used to obtain the scattered light signal processed by the filtered light. After receiving the scattered light signal, the photomultiplier tube converts the scattered light signal into an electrical signal, which can be a voltage value. When different scattered light signals of the same droplet to be measured need to be measured, multiple second light source transmitters can be provided, and the number of second optical path transmission components, first optical fiber receiving components and photomultiplier tubes corresponds to each other. According to the measurement requirements, the wavelength band of the light of the second light source transmitter can be adjusted, and the filtering parameters of the second filter are in a corresponding relationship with the wavelength band of the light of the second light source transmitter.

Optionally, referring to FIG. 4 , the optical signal detection device 2 includes a third light source transmitter 211 and its optical fiber interface 251, a first optical fiber transmission component, a second optical fiber receiving component, and a third photomultiplier tube (at least one photomultiplier tube is selected arbitrarily from 285 to 288). The first optical fiber transmission component and the second optical fiber receiving component are respectively connected to the first optical fiber interface and the second optical fiber interface distributed on both sides of the flow channel of the microfluidic sorting chip 5. The third light source transmitter 211 emits light to the first optical fiber interface through the first optical fiber transmission component to excite the optical signal of the droplet to be measured. The second optical fiber receiving component is used to obtain the absorption light signal processed by the filtered light. Among them, the second optical fiber receiving component includes a second optical fiber receiver (at least one second optical fiber receiver is selected arbitrarily from 252 to 255), a third plano-convex lens (a plano-convex lens corresponding to the second optical fiber receiver is selected from 265 to 268 as the third plano-convex lens) and a third filter (a filter corresponding to the second optical fiber receiver is selected from 275 to 278 as the third filter), and the third photomultiplier tube converts the absorption light signal into an electrical signal.

In addition to the above-mentioned devices, the optical signal detection device 2 also includes an illumination light source 295, a stage 294, a tube mirror 291, and a high-speed camera 292. Before detecting the absorption light signal, the microfluidic sorting chip 5 needs to be placed on the stage. When the high-speed camera observes that the droplets are arranged in a single row in the flow channel of the microfluidic sorting chip 5 and pass through with a stable spacing, it means that the absorption light signal can be measured. The third light source transmitter is used to emit light sources such as lasers and infrared light. The type of light emitted by the third light source is not limited here. The first optical fiber transmission component and the second optical fiber receiving component are respectively connected to the first optical fiber interface and the second optical fiber interface distributed on both sides of the flow channel of the microfluidic sorting chip 5. The third light source transmitter emits light to the first optical fiber interface through the first optical fiber transmission component to excite the optical signal of the droplet to be measured. The second optical fiber receiving component is used to obtain the absorption light signal processed by filtered light.

Optionally, there are multiple first light source emitters, second light source emitters, and third light source emitters. The wavelength band of the emitted light emitted by each first light source emitter is not completely the same, the wavelength band of the emitted light emitted by each second light source emitter is not completely the same, and the wavelength band of the emitted light emitted by each third light source emitter is not completely the same. The wavelength band of the emitted light of the first light source emitter and the filtering parameters of the first filter match the fluorescent signal, the wavelength band of the emitted light of the second light source emitter and the filtering parameters of the second filter match the scattered light signal, and the wavelength band of the emitted light of the third light source emitter and the filtering parameters of the third filter match the absorbed light signal.

Among them, when the number of the first light source emitter, the second light source emitter and the third light source emitter is multiple, the fluorescence signal, scattered light signal and absorption light signal of the same droplet to be tested, or any one of the three, can be detected simultaneously by the optical signal detection device 2. By setting the first light source emitter, the second light source emitter and the third light source emitter and the corresponding required components, fluorescence signals of different bands, scattered light signals and absorption light signals of different bands can also be detected simultaneously. The power of the first light source emitter, the second light source emitter and the third light source emitter can be adjusted, usually between 0 milliwatts (mW) and 100 milliwatts (mW). The first light source emitter, the second light source emitter and the third light source emitter can be synthesized into one optical path through a beam combiner and irradiated into the flow channel of the microfluidic sorting chip 5 through an objective lens. The droplet passes through the laser position of the flow channel to generate an excited optical signal.

In this embodiment, the fluorescence signal, scattered light signal and absorbed light signal of the droplet to be tested are detected simultaneously, which meets the requirement of detecting multiple optical signals of the droplet to be tested in one experiment, and provides a micro-droplet screening device with high integration and high automation, which greatly improves the convenience and diversity of micro-droplet screening.

Optionally, the signal processor 3 is used to send a movement instruction to the host computer 1 when determining that the droplet to be tested is a positive droplet, and the host computer 1 controls the movement of the micro-droplet collecting device according to the movement instruction.

1 and 2 , the signal processor 3 may be a signal processing feedback control board. When the droplet to be tested is determined to be a positive droplet through analysis, a movement instruction is sent to the host computer 1. The host computer 1 controls the movement of the micro-droplet collecting device 6 according to the movement instruction. The movement instruction may emit a deflection electric pulse 231 for the droplet.

Optionally, the micro-droplet collecting device 6 includes a plurality of collecting chambers, and the host computer 1 controls the micro-droplet collecting device to move when the number of droplets in a collecting chamber reaches a preset number.

Wherein, referring to Fig. 1, the micro-droplet collecting device 6 includes an oil droplet detection sensor 601, and when the oil droplet detection sensor 601 detects droplets flowing out of the hose, a trigger signal will be generated. The host computer 1 can control the electric stage, on which the micro-droplet collecting device 6 can be placed, and the micro-droplet collecting device 6 can be a 96-well plate or a 384-well plate (or a well plate with other numbers of holes). Whether to move is determined by the result of the optical signal analysis, the deflection instruction (for example, the deflection electric pulse 231) and the trigger signal of the oil droplet detection sensor 601. In the actual detection process, it can also be set according to actual needs to move when each well plate collects a preset number of droplets. The waste liquid tank 602 is used to collect unscreened waste droplets.

In this embodiment, automatic collection of droplets is achieved, providing a micro-droplet screening device with high integration and high automation, which greatly improves the convenience and diversity of micro-droplet screening.

Optionally, referring to FIG5 , the micro-droplet screening device further comprises a liquid injection device 7, which comprises a pressure pump, a syringe 2111 to 2113, and a liquid injection controller 24. The liquid injection device is electrically connected to the host computer 1, and the host computer 1 controls the liquid injection device to inject the dispersed phase and the droplets to be tested into the microfluidic sorting chip 5. The syringe is installed in the pressure pump, and the liquid injection controller controls the pressure pump to provide power to the syringe. An angle is set between the syringe and the horizontal plane.

Among them, the injection device includes multiple precision pressure pumps and injection controllers. The pressure pump can be driven by a stepper motor. According to the test needs, different types of syringes of 0.5mL-100mL can be installed. The injection speed can be controlled, and the speed range is 1nL/min-200mL/min. It also has both push and pull functions to meet the droplet preparation and screening needs of different samples. In actual use, the sample to be tested can be infused into a disposable syringe to avoid mutual contamination of different batches of experimental samples. The sample in the syringe is connected to the microfluidic chip through a hose, and the installed syringe forms an angle with the horizontal plane.

In this embodiment, the injection device is controlled by the host computer 1, and the screening requirements of different samples are met, providing a micro-droplet screening device with high integration and high automation, which greatly improves the convenience and diversity of micro-droplet screening.

Optionally, the host computer 1 is used to visually display the optical signal when receiving the optical signal of the droplet to be detected. The host computer 1 is also used to set parameters for the optical signal detection device 2, the signal processor 3 and the electrical filter 4.

6 shows the device system software applicable to the host computer 1, including a microfluidic sorting chip stage movement parameter setting module 111 (used to control the movement of the stage in the three directions of XYZ), a light source transmitter power setting module 112, a pressure pump operation parameter setting module 113, a signal source selection/signal source gain setting module 114, a sorting threshold setting module 115, a sorting electric pulse amplitude/duration setting module 116, and a single droplet dispensing stage movement parameter setting module 117. The required parameters are read in real time through a high-speed camera monitoring interface 121 and an optical visualization display interface 122 (visualization display of single-channel and multi-channel optical signals).

In this embodiment, through the parameter setting and visual display of the equipment system software, it is convenient to observe the display interface and adjust the parameters at any time during the detection process, providing a highly integrated and highly automated micro-droplet screening device, which greatly improves the convenience and diversity of micro-droplet screening.

Optionally, the micro-droplet screening device further includes a light-shielding housing, and the optical signal detection device 2 is disposed in the light-shielding housing to block interference from ambient light.

7 shows a schematic diagram of the overall structure of the micro-droplet screening device, including a light-shielding housing 81 and a frame 82, and the micro-droplet screening device is placed on the frame 82. When in use, the light-shielding housing 81 can block the interference of external ambient light, ensuring that the micro-droplet screening device is not disturbed by ambient light in a non-darkroom working environment.

In this embodiment, a light-shielding shell is provided to reduce interference with fluorescence signals, scattered light signals and absorbed light signals in actual measurements, thereby providing a micro-droplet screening device with high integration and automation, which greatly improves the convenience and diversity of micro-droplet screening.

Optionally, the micro-droplet screening device also includes a power supply system, which provides power for all sub-devices that require power, such as the high-speed camera, the first light source emitter, the second light source emitter, the third light source emitter, the pressure pump, the electric stage, etc., and provides adjustable gain voltage output for multiple photomultiplier tubes.

In the example of screening E. coli droplets:

(1) Screening preparation stage: The plasmid with the red fluorescent protein gene was transformed into E. coli. After overnight culture on a plate, a single clone was picked and the seed solution was cultured in liquid LB at 37°C and 220 rpm. The seed solution was transferred to LB medium at a ratio of 1%, and shaken at 37°C and 220 rpm until OD600 = 0.6. The bacteria were collected and centrifuged at 1000g for 5 minutes. The bacteria were washed three times with autoinduction medium, and then respun with autoinduction medium and diluted to OD600 = 0.1. The suspended bacterial solution was wrapped by the oil phase to form microdroplets with a droplet size of 20 μm. At this concentration, the proportion of droplets without E. coli was 74%. After the E. coli was wrapped in the droplets, the droplets were cultured at 37°C, and the E. coli multiplied in the droplets and expressed red fluorescent protein.

(2) Adjustment stage: Place the microfluidic sorting chip on the stage, adjust the movement of the stage so that the chip detection and sorting area is within the field of view of the high-speed camera, install the syringe containing droplets and oil phase on the pressure pump, connect the syringe outlet to the microfluidic sorting chip through a hose, adjust the droplet phase flow rate to 15μL/h, and the two sheath liquid phase flow rates are both 300uL/h. When the high-speed camera observes that the droplets are arranged in a single row in the flow channel and the spacing is stable, start the first light source emitter and the second light source emitter, adjust the photomultiplier tube gain value, and collect the scattered light signal and red fluorescent protein signal data of the bacteria in the droplets.

(3) Analysis phase: See FIG8 for a scatter plot of the collected droplet data. The horizontal axis is the voltage parameter of the droplet scattering signal, and the vertical axis is the voltage parameter of the red fluorescent protein signal in the droplet. The box in the figure delimits the sorting threshold range. After the sorting threshold is determined, when the detected droplet signal value is within the threshold range, it will be judged as a positive droplet. The signal processor generates a screening instruction, and the positive droplet is deflected under the dielectrophoretic force and enters the corresponding branch channel. At the same time, the droplet distribution electric pulse controls the movement of the electric stage to collect the positive droplets into the micro-droplet collection device, thereby realizing the collection of positive droplets.

Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that these are only examples, and various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

Claims

A micro-droplet screening device, characterized in that the micro-droplet screening device comprises a host computer, an optical signal detection device, a signal processor, and an electrical screener;

The optical signal detection device is used to obtain the optical signal of the droplet to be detected, and convert the optical signal into the electrical signal of the droplet to be detected;

The signal processor is electrically connected to the optical signal detection device, and the signal processor is used to receive and process the electrical signal of the droplet to be detected, and send a screening instruction to the electrical filter according to the intensity of the electrical signal;

The electrical filter is electrically connected to the signal processor, and the electrical filter controls the droplets to be tested in the microfluidic sorting chip to deflect to the corresponding flow channel according to the screening instruction, so as to complete the screening of the droplets to be tested;

The host computer is electrically connected to the signal processor, and is used to issue the screening instruction and set relevant screening parameters.
The micro-droplet screening device as described in claim 1 is characterized in that the optical signal includes at least one of a fluorescent signal, a scattered light signal and an absorbed light signal.
The micro-droplet screening device according to claim 2, characterized in that the optical signal detection device includes a first light source emitter, a first light path transmission component, a first light path receiving component, and a first photomultiplier tube;

The first light source transmitter is provided with a first light path transmission component and a first light path receiving component correspondingly;

The first light source emitter excites the optical signal of the droplet to be measured through the first light path transmission component;

The first optical path receiving component is used to obtain the fluorescent signal processed by the filtered light;

Wherein, the first optical path transmission component includes a first beam combiner, a cylindrical lens, and an objective lens, and the first optical path receiving component includes a first dichroic mirror, a first plano-convex lens, and a first filter;

The first photomultiplier tube converts the fluorescence signal into an electrical signal.
The micro-droplet screening device according to claim 2, characterized in that the optical signal detection device 2 comprises a second light source transmitter, a second optical path transmission component, a first optical fiber receiving component, and a second photomultiplier tube;

The second light source emitter is provided with a second light path transmission component correspondingly;

The second light source emitter excites the optical signal of the droplet to be measured through the second light path transmission component;

The first optical fiber receiving component is used to obtain the scattered light signal processed by the filtered light;

Wherein, the second optical path transmission component includes a second beam combiner, a cylindrical lens, and an objective lens, and the first optical fiber receiving component includes an optical fiber receiver, a second plano-convex lens, and a second filter;

The second photomultiplier tube converts the scattered light signal into an electrical signal.
The micro-droplet screening device according to claim 2, characterized in that the optical signal detection device includes a third light source transmitter, a first optical fiber transmission component, a second optical fiber receiving component, and a third photomultiplier tube;

The first optical fiber transmission component and the second optical fiber receiving component are respectively connected to the first optical fiber interface and the second optical fiber interface distributed on both sides of the flow channel of the microfluidic sorting chip;

The third light source emitter emits light to the first optical fiber interface through the first optical fiber transmission component to stimulate the optical signal of the droplet to be detected;

The second optical fiber receiving component is used to obtain the absorption light signal after the filtered light processing;

Wherein, the second optical fiber receiving assembly comprises an optical fiber receiver, a third plano-convex lens and a third filter;

The third photomultiplier tube converts the absorbed light signal into an electrical signal.
The optical signal detection device according to any one of claims 3 to 5, characterized in that the number of the first light source emitter, the second light source emitter and the third light source emitter is multiple;

The wavelength band of the emitted light emitted by each first light source emitter is not completely the same, the wavelength band of the emitted light emitted by each second light source emitter is not completely the same, and the wavelength band of the emitted light emitted by each third light source emitter is not completely the same;

The wavelength band of the emitted light of the first light source emitter and the filtering parameters of the first filter match the fluorescence signal; the wavelength band of the emitted light of the second light source emitter and the filtering parameters of the second filter match the scattered light signal; the wavelength band of the emitted light of the third light source emitter and the filtering parameters of the third filter match the absorbed light signal.
The micro-droplet screening device as described in claim 1 is characterized in that the signal processor is used to determine the branch channel that matches the intensity of the electrical signal of the droplet to be tested, and send the screening instruction to the electrical filter.
The micro-droplet screening device according to claim 7, characterized in that the signal processor is used to send a movement instruction to the host computer when determining that the droplet to be tested is a positive droplet;

The host computer controls the movement of the micro-droplet collecting device according to the movement instruction.
The micro-droplet screening device according to claim 8, characterized in that the micro-droplet collecting device includes a plurality of collecting chambers;

The host computer controls the micro-droplet collecting device to move when the number of droplets in a collection chamber reaches a preset number.
The micro-droplet screening device according to claim 1, characterized in that the micro-droplet screening device also includes a liquid injection device, and the liquid injection device includes a pressure pump, a syringe, and a liquid injection controller;

The liquid injection device is electrically connected to the host computer, and the host computer controls the liquid injection device to inject the dispersed phase and the droplets to be tested into the microfluidic sorting chip;

The syringe is installed in the pressure pump, and the injection controller controls the pressure pump to provide power to the syringe;

An angle is set between the syringe and the horizontal plane.
The micro-droplet screening device according to claim 1, characterized in that the host computer is used to visually display the optical signal when receiving the optical signal of the droplet to be tested;

The host computer is also used to set parameters of the optical signal detection device, the signal processor and the electrical filter.
The micro-droplet screening device according to claim 7, characterized in that the micro-droplet screening device further comprises a light-shielding housing;

The optical signal detection device is arranged in the light-shielding housing to block interference from ambient light.
A micro-droplet screening system, characterized in that the micro-droplet screening system comprises a microfluidic sorting chip and a micro-droplet screening device according to any one of claims 1 to 12; the microfluidic sorting chip comprises an optical fiber channel, a sorting component, and at least two branch channels;

The optical fiber channel is connected to the optical fiber interface for transmitting optical signals;

The sorting component is used to sort the droplets to be tested into corresponding branch channels when receiving a deflection instruction.