Single cell sorter and single cell sorting method for rare cells or viruses

Abstract

The invention provides a single cell sorter and a single cell sorting method for rare cells or viruses, which are applied to the technical field of biomedical instruments. The single cell sorter comprises: a droplet injection pump for inputting a mixed solution to be sorted; the oil phase injection pump is used for inputting an oil phase; the liquid drop sample inlet is respectively connected with the liquid drop injection pump and the sorting channel; the oil sample inlet is respectively connected with the oil phase injection pump and the sorting channel; the sorting channel is respectively connected with the liquid drop sample inlet and the oil sample inlet and is used for conveying each liquid drop in the plurality of liquid drops; the sorting electrode is used for applying voltage to the current liquid drop for dielectric sorting when receiving the target voltage signal, so that the current liquid drop is sorted into the sorting pool as a target liquid drop; the sorting tank is connected with the sorting channel and is used for receiving the target liquid drops; the waste liquid pool is connected with the sorting channel and used for receiving the waste liquid drops which are not subjected to voltage application by the sorted electrodes, and cell sorting with differential functions can be achieved through the cell sorting device.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical instruments, and in particular to a single cell sorting device and a single cell sorting method for rare cells or viruses. Background Art

[0002] The single cell sorter is an instrument used to separate single cells. Before the instrument performs cell sorting operations, biological samples need to be added to the chip. The single cell sorter can be used to accurately separate single cells in a cell group into a cell receiving device.

[0003] Although the existing single-cell sorters on the market are mature in technology, they generally have problems such as high cost, incomplete functions, low cost performance, slow sorting speed, and low throughput. More importantly, when sorting rare cells or viruses, they often cannot achieve the ideal sorting effect and accuracy, and cannot meet the urgent needs of scientific research and clinical applications.

[0004] In addition, existing technologies also have limitations in sorting different types of cells or microbial mixtures. In particular, when it is necessary to separate specific types of cells or viruses from complex samples, existing single-cell sorters are often unable to perform this task. Summary of the invention

[0005] The present invention provides a single cell sorter and a single cell sorting method for rare cells or viruses, which are used to solve the defect of the prior art that specific types of cells or viruses cannot be separated from complex samples, and realize a single cell sorter with differentiated functions.

[0006] The present invention provides a single-cell sorting instrument and a single-cell sorting method for rare cells or viruses, comprising: a droplet injection pump for inputting a mixed liquid to be sorted, wherein the mixed liquid to be sorted includes a target object, and the target object includes one of the following: rare cells, viruses; an oil phase injection pump for inputting an oil phase; a sorting chip, which is respectively connected to the droplet injection pump and the oil phase injection pump, and the sorting chip comprises: a droplet injection port, an oil injection port, a sorting channel, a sorting electrode, a sorting pool and a waste liquid pool; the droplet injection port is respectively connected to the droplet injection pump and the sorting channel, and is used to receive the mixed liquid to be sorted input by the droplet injection pump, and input the mixed liquid to be sorted into the sorting channel; the oil injection port is respectively connected to the oil phase injection pump and the sorting channel, and is used to receive the The oil phase is input by the oil phase injection pump and input into the sorting channel, the oil phase is used to separate the mixed liquid to be sorted into multiple droplets in the sorting channel; the first end of the sorting channel is respectively connected to the droplet injection port and the oil injection port, the second end of the sorting channel is respectively connected to the sorting pool and the waste liquid pool, the sorting channel is used to transfer each droplet of the multiple droplets; the sorting electrode is connected to the sorting channel, and is used to apply voltage to the current droplet for dielectric sorting when receiving the target voltage signal, so as to sort the current droplet as the target droplet into the sorting pool; the sorting pool is connected to the sorting channel, and is used to receive the target droplet; the waste liquid pool is connected to the sorting channel, and is used to receive the discarded droplets to which the voltage is not applied by the sorting electrode.

[0007] According to a single cell sorter for rare cells or viruses provided by the present invention, the oil phase injection pump includes: a first oil phase injection pump, connected to the oil inlet, used to output the first oil phase at a first frequency, and the first frequency is used to enable the first oil phase to separate the mixed liquid to be sorted into multiple droplets.

[0008] According to a single cell sorter for rare cells or viruses provided by the present invention, the oil phase injection pump also includes: a second oil phase injection pump, connected to the oil inlet, used to output oil phase spaced droplets at a second frequency, the second frequency is used to cut off the continuous water phase to form droplet intervals; a third oil phase injection pump, connected to the oil inlet, used to output the second oil phase at a third frequency, the third frequency is used to make the second oil phase space each of the multiple droplets to a target spacing distance.

[0009] According to a single-cell sorter for rare cells or viruses provided by the present invention, the single-cell sorter for rare cells or viruses also includes: a laser emitter for emitting excitation fluorescence of a target wavelength; a fluorescence microscope optically connected to the laser emitter, for receiving the excitation fluorescence and focusing the excitation fluorescence onto the multiple droplets, so that the droplets containing fluorescent substances produce fluorescence; and determining the fluorescence voltage analog signal corresponding to each droplet; a sorting control module, for outputting a target voltage signal to the sorting electrode when the fluorescence voltage analog signal is in a preset analog signal interval and the droplet width is in a preset droplet width interval.

[0010] According to a single-cell sorter for rare cells or viruses provided by the present invention, the fluorescence microscope includes: a fluorescence detection module, which is used to perform fluorescence detection and image acquisition on each droplet of the multiple droplets to obtain an electrical signal of each droplet and a fluorescence signal of each droplet, wherein the fluorescence signal includes the droplet width; and a signal modulation module, which is used to modulate the fluorescence signal to obtain a fluorescence voltage analog signal.

[0011] According to a single cell sorter for rare cells or viruses provided by the present invention, the fluorescence detection module includes: a dichroic mirror, optically connected to the laser emitter, and used to reflect the excitation fluorescence to the objective lens according to the target angle; the objective lens, optically connected to the dichroic mirror, and used to focus the excitation fluorescence onto the droplet, so that the droplet containing the fluorescent substance produces fluorescence; a beam splitter, optically connected to the objective lens, and used to split the fluorescence in the detection branch to obtain a first fluorescence and a second fluorescence; the first fluorescence is input to a high-speed camera; the second fluorescence is input to the beam splitter; a filter, connected to the beam splitter, and used to The invention is used to filter out stray light of the first fluorescence and the second fluorescence to obtain a first target fluorescence and a second target fluorescence; the high-speed camera is optically connected to the filter, and is used to collect images based on the first target fluorescence to obtain an image signal, and input the image signal to a sorting control module; the spectroscope is optically connected to the laser emitter and the filter, respectively, and is used to split the second target fluorescence to obtain a fluorescence signal, and send the fluorescence signal to a photomultiplier tube; the photomultiplier tube is optically connected to the spectroscope, and is used to receive the fluorescence signal and convert the fluorescence signal into an electrical signal.

[0012] According to a single cell sorter for rare cells or viruses provided by the present invention, the signal modulation module includes: a preamplifier circuit, electrically connected to the photomultiplier tube, for amplifying the electrical signal to obtain an amplified electrical signal; a filter circuit, electrically connected to the preamplifier circuit, for filtering the amplified electrical signal to obtain a filtered electrical signal; an analog-to-digital converter, electrically connected to the filter circuit, for converting the filtered electrical signal into a fluorescent voltage analog signal, and inputting the fluorescent voltage analog signal into a sorting control module.

[0013] According to a single cell sorter for rare cells or viruses provided by the present invention, the sorting control module includes: a computer, electrically connected to a high-speed camera and an analog-to-digital converter, respectively, for receiving an image signal input by the high-speed camera and a fluorescence voltage analog signal input by the analog-to-digital converter, and outputting a target voltage signal when the fluorescence voltage analog signal is in a preset analog signal interval and the droplet width is in a preset droplet width interval; a field programmable gate array, electrically connected to the computer, for outputting a drive signal when the target voltage signal output by the computer is detected; a direct current power supply, electrically connected to the field programmable gate array, for outputting a level signal when the drive signal output by the field programmable gate array is detected; and a high-voltage pulse power supply, electrically connected to the direct current power supply, for amplifying the level signal to obtain a target voltage signal, and outputting the target voltage signal to the sorting electrode.

[0014] According to a single cell sorter for rare cells or viruses provided by the present invention, the mixed liquid to be sorted is a rare virus droplet sample.

[0015] According to a single-cell sorter for rare cells or viruses provided by the present invention, the target object in the rare virus droplet sample is rare virus Contig 1, and the probe sequence of the rare virus Contig 1 is probe B / 56-FAM / rCrUrUrArUrGrGrGrA / ZEN / rGrGrCrArArGrCrArGrCrArArGrUrU / 3IABkFQ / .

[0016] According to a single cell sorter for rare cells or viruses provided by the present invention, the detection tool of the rare virus ContigJo includes: an RNA probe and an RNase H enzyme, wherein the RNA probe includes an RNA oligonucleotide, the 5' end of the RNA oligonucleotide has a fluorescent group coupling, and the 3' end of the RNA oligonucleotide has a quencher; the RNase H enzyme is used to digest the RNA oligonucleotide so that the quencher releases the fluorescent group.

[0017] The present invention also provides a single-cell sorting method for rare cells or viruses, comprising the following steps: inputting a mixed liquid to be sorted through a droplet injection pump, wherein the mixed liquid to be sorted includes a target object, wherein the target object includes one of the following: rare cells, viruses; inputting an oil phase through an oil phase injection pump; receiving the mixed liquid to be sorted input by the droplet injection pump through a droplet inlet of a sorting chip; receiving the oil phase input by the oil phase injection pump through the oil inlet of the sorting chip to separate the mixed liquid to be sorted into multiple droplets; transmitting each of the multiple droplets through a sorting channel of the sorting chip; applying a voltage to a current droplet through a sorting electrode of the sorting chip when receiving a target voltage signal, so as to sort the current droplet as a target droplet into a sorting pool; receiving the target droplet through a sorting pool; receiving discarded droplets to which a voltage is not applied by the sorting electrode through a waste liquid pool.

[0018] The present invention also provides an electronic device, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein when the processor executes the program, the single cell sorting method for rare cells or viruses as described above is implemented.

[0019] The present invention also provides a non-transitory computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the single-cell sorting method for rare cells or viruses as described above.

[0020] The present invention also provides a computer program product, comprising a computer program, wherein when the computer program is executed by a processor, the single cell sorting method for rare cells or viruses as described above is implemented.

[0021] The single cell sorting instrument and single cell sorting method for rare cells or viruses provided by the present invention can accurately input the mixed liquid (including rare cells or viruses) to be sorted and the oil phase into the droplet inlet and oil inlet of the sorting chip through the precise control of the droplet injection pump and the oil phase injection pump. The role of the oil phase is to form a partition in the microfluidic channel, dividing the mixed liquid into a series of tiny droplets, each droplet may contain one or more target objects, thereby improving the accuracy and efficiency of sorting; when the sorting electrode of the sorting chip receives the target voltage signal, it can apply a voltage to the droplets currently flowing through, thereby being able to intelligently identify and separate the target droplets containing the target objects according to preset conditions (such as the specific properties of rare cells or viruses), and transport them to the sorting pool; the sorting pool is used to receive the successfully sorted target droplets, while the unsorted discarded droplets are guided to the waste liquid pool, ensuring the effective use of resources and avoiding unnecessary waste; thereby solving the defect of the related technology that specific types of cells or viruses cannot be separated from complex samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced one by one below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 It is a schematic diagram of the structure of a single cell sorter for rare cells or viruses provided by the present invention.

[0024] Figure 2 It is a schematic diagram of the device structure of the fluorescence detection process provided by the present invention.

[0025] Figure 3 It is a schematic diagram of the device structure of the sorting control process provided by the present invention.

[0026] Figure 4 It is a schematic diagram of the structure of a single cell sorting module for rare cells or viruses provided by the present invention.

[0027] Figure 5 It is a schematic diagram of a gene detection method compatible with MDA conditions provided by the present invention.

[0028] Figure 6 This is one of the schematic diagrams of the detection results of the sample types and probe combinations provided by the present invention.

[0029] Figure 7 This is the second schematic diagram of the detection results of the sample types and probe combinations provided by the present invention.

[0030] Figure 8 This is the third schematic diagram of the detection results of sample types and different probe combinations provided by the present invention.

[0031] Fig. 9 This is the fourth schematic diagram of the detection results of sample types and different probe combinations provided by the present invention.

[0032] Fig.10 This is one of the schematic diagrams of sorting statistical results provided by the present invention.

[0033] Fig.11 This is the second schematic diagram of the sorting statistical results provided by the present invention.

[0034] Fig.12 This is the third schematic diagram of the sorting statistical results provided by the present invention.

[0035] Fig.13 This is the fourth schematic diagram of the sorting statistical results provided by the present invention.

[0036] Fig.14It is a schematic diagram of the process of the single cell sorting method of rare cells or viruses provided by the present invention.

[0037] Fig.15 It is a schematic diagram of the physical structure of the electronic device provided by the present invention. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] The single-cell sorter is an instrument used to separate single cells. Before the instrument performs cell sorting operations, biological samples need to be added to the chip. The single-cell sorter can be used to accurately separate single cells in a cell group into a cell receiving device. The domestic and foreign market situation of single-cell sorters. The domestic market business has just started, with incomplete functions and low cost-performance ratio. There is a lot of market and room for development. The foreign market has high costs, bottlenecks: high cost, low cost-performance ratio, lack of differentiated functions, sorting technology, slow speed, and low throughput. Existing single-cell sorters are not suitable for many application scenarios. For example, they cannot meet the requirements for sorting rare cells / viruses.

[0040] The single cell sorter for rare cells or viruses provided by the present invention can overcome the shortcomings of the prior art, provide a single cell sorter with differentiated functions, realize the sorting of rare cells / viruses, and provide a more convenient means for the analysis of rare cells / viruses. The principle of the single cell sorter for rare cells or viruses is as follows: after the droplet is excited by passing through the fluorescence detection area, a strong fluorescence signal appears in the droplet, the fluorescence signal is collected, and at the same time, according to the intensity output level of the fluorescence signal, the high-voltage pulse power supply output is triggered to complete the dielectric sorting action.

[0041] Figure 1 Schematic diagram of the structure of the single cell sorter for rare cells or viruses provided by the present invention, such as Figure 1 As shown, the above-mentioned single cell sorter for rare cells or viruses includes the following modules: The droplet injection pump 101 is used to input the mixed solution to be sorted, wherein the mixed solution to be sorted includes a target object, and the target object includes one of the following: rare cells, viruses; An oil phase injection pump 102, used for inputting the oil phase; The sorting chip 103 is connected to the droplet injection pump 101 and the oil phase injection pump 102 respectively, and the sorting chip includes: a droplet injection port 1031, an oil injection port 1032, a sorting channel 1033, a sorting electrode 1034, a sorting pool 1035 and a waste liquid pool 1036; The droplet inlet 1031 is connected to the droplet injection pump 101 and the sorting channel 1033 respectively, and is used to receive the mixed liquid to be sorted input by the droplet injection pump 101, and input the mixed liquid to be sorted into the channel; The oil injection port 1032 is connected to the oil phase injection pump 102 and the sorting channel 1033, respectively, and is used to receive the oil phase input by the oil phase injection pump 102 and input the oil phase into the sorting channel. The oil phase is used to separate the mixed liquid to be sorted into multiple droplets in the sorting channel 1033; The first end of the sorting channel 1033 is connected to the droplet injection port 1031 and the oil injection port 1032 respectively, and the second end of the sorting channel 1033 is connected to the sorting pool 1035 and the waste liquid pool 1036 respectively. The sorting channel 1033 is used to transfer each droplet of the multiple droplets; The sorting electrode 1034 is connected to the sorting channel 1033 and is used to apply voltage to the current droplet for dielectric sorting when receiving the target voltage signal, so as to sort the current droplet as the target droplet into the sorting pool 1035; A sorting pool 1035 connected to the sorting channel 1033 for receiving target droplets; The waste liquid pool 1036 is connected to the sorting channel 1033 and is used to receive waste liquid droplets that are not applied with voltage by the sorting electrodes.

[0042] In an embodiment of the present invention, a droplet injection pump is used to input a mixed solution to be sorted containing target objects such as rare cells and viruses into a sorting chip at a stable flow rate to ensure continuous supply and uniform distribution of the mixed solution.

[0043] The oil phase injection pump is used to inject the oil phase into the sorting chip. The oil phase forms a barrier in the microfluidic channel (i.e., the sorting channel), separating the mixed liquid to be sorted into a series of tiny droplets. Here, the addition of the oil phase makes each droplet an independent microreactor, which is convenient for subsequent screening and separation.

[0044] The sorting chip includes a droplet inlet, an oil inlet, a sorting electrode, a sorting channel, a waste liquid pool and a sorting pool.

[0045] Droplet inlet: receives the mixed liquid to be sorted input by the droplet injection pump.

[0046] Oil inlet: Receives the oil phase input by the oil phase injection pump to separate the mixed liquid to be sorted and obtain multiple droplets.

[0047] Sorting channel: Serves as a channel for droplet transmission, ensuring the orderly flow of droplets within the chip.

[0048] Sorting electrode: When receiving the target voltage signal, it applies voltage to the current droplet and realizes the sorting of droplets through electrical manipulation.

[0049] Waste liquid pool: receives the waste droplets that are not subjected to voltage application by the sorting electrodes.

[0050] Sorting pool: A pool used to receive successfully sorted target droplets.

[0051] In an embodiment of the present invention, when it is detected that the current droplet includes a target object, the sorting electrode receives a target voltage signal and applies voltage to the current droplet for dielectric sorting, so as to sort the current droplet as a target droplet into a sorting pool.

[0052] For example, target detection methods (such as optical detection, electrochemical detection, etc.) are used to determine whether a droplet contains a target object. When a target object is detected, the sorting electrode receives a target voltage signal. After the sorting electrode receives the target voltage signal, a voltage is applied to the current droplet. The magnitude and direction of this voltage are determined based on the difference in dielectric constant between the target object and the surrounding medium. Under the action of the electric field force, droplets containing the target object will have different motion trajectories. Based on the differences in the motion trajectories of the droplets, droplets containing the target object (i.e., target droplets) can be sorted into a sorting pool, thereby achieving droplet sorting.

[0053] According to the single cell sorter for rare cells or viruses provided by the present invention, the oil phase injection pump comprises: The first oil phase injection pump is connected to the oil inlet and is used to output the first oil phase at a first frequency. The first frequency is used to enable the first oil phase to separate the mixed liquid to be sorted into multiple droplets.

[0054] Here, the first frequency can be determined according to the fluid velocity of the mixed liquid to be sorted, and the first oil phase injection pump outputs the first oil phase, which contacts the mixed liquid to be sorted in the microfluidic channel (sorting channel) and cuts the mixed liquid to be sorted based on the flow and shear force of the oil phase. During the cutting process, the mixed liquid to be sorted is divided into multiple tiny droplets, each of which contains a certain number of cells.

[0055] The cutting effect of the first oil phase achieves precise segmentation of the mixed liquid to be sorted, providing samples at the single cell level for subsequent cell analysis; by controlling the flow rate and cutting frequency of the first oil phase, the size of the droplets and the number of cells can be further adjusted to meet the needs of different experiments.

[0056] Through the embodiment of the present invention, by accurately processing the mixed solution to be sorted with the first oil phase, a sample at the single cell level is provided for subsequent cell analysis.

[0057] According to the single cell sorter for rare cells or viruses provided by the present invention, the oil phase injection pump further comprises: A second oil phase injection pump is connected to the oil inlet and is used to output the oil phase at a second frequency to cut off the continuous water phase and form a droplet interval; The third oil phase injection pump is connected to the oil injection port and is used to output the second oil phase at a third frequency. The third frequency is used to make the second oil phase separate each of the multiple droplets into a target separation distance.

[0058] Here, the second frequency and the third frequency may be predetermined and respectively used to space each of the plurality of droplets and control the target spacing distance between different droplets.

[0059] In the embodiment of the present invention, the second oil phase injection pump is connected to the oil inlet and is used to output the oil phase at a second frequency to cut off the continuous water phase and form droplets.

[0060] The third oil phase injection pump is used to output oil phase spacer droplets, which are used to form a physical barrier between multiple droplets, that is, to separate each droplet to prevent them from merging or interfering with each other. The introduction of oil phase spacer droplets increases the stability between droplets and ensures the independence of each droplet during the flow process. The third oil phase injection pump is used to output the second oil phase, which is used to adjust the spacing distance between each droplet in the multiple droplets, that is, to achieve the target distance between the droplets; by precisely controlling the output of the second oil phase, the flow of droplets during the sorting process can be balanced to ensure that each droplet has enough space for subsequent operations and analysis.

[0061] Through the embodiment of the present invention, the second oil phase injection pump and the third oil phase injection pump further enhance the accuracy and stability of droplet generation to ensure that each droplet maintains an appropriate interval during the flow process.

[0062] According to the single cell sorter for rare cells or viruses provided by the present invention, the single cell sorter for rare cells or viruses also includes: A laser transmitter for emitting excitation fluorescence of a target wavelength; A fluorescence microscope, optically connected to the laser emitter, for receiving the excitation fluorescence and focusing the excitation fluorescence onto a plurality of droplets, so that the droplets containing the fluorescent substance generate fluorescence; and determining the fluorescence voltage analog signal and the image signal corresponding to each droplet; The sorting control module is used to output a target voltage signal to the sorting electrode when the fluorescence voltage analog signal is in a preset analog signal interval and the droplet width in the image signal is in a preset droplet width interval.

[0063] In the embodiment of the present invention, the excitation light with a target wavelength of 488 nm is generated by a laser emitter to excite fluorescence, which is propagated along the excitation light path to the dichroic mirror in the fluorescence microscope.

[0064] The fluorescence microscope receives the excitation fluorescence and focuses the excitation fluorescence onto each droplet in the multiple droplets so that the droplets containing the fluorescent substance produce fluorescence, and determines the fluorescence signal of each droplet according to the fluorescence detection module; modulates the fluorescence signal according to the signal modulation module to obtain a fluorescence voltage analog signal, and sends the size of each droplet and the fluorescence voltage analog signal to the computer of the sorting control module.

[0065] The sorting control module detects the image signal and the fluorescence voltage analog signal through a computer, and outputs a target voltage signal to the sorting electrode when the fluorescence voltage analog signal is in a preset analog signal interval and the droplet width is in a preset droplet width interval.

[0066] The single cell sorter and fluorescence microscope for rare cells or viruses provided by the present invention include: A fluorescence detection module, used to perform fluorescence detection and image acquisition on each of the multiple droplets to obtain an electrical signal of each droplet and a fluorescence signal of each droplet, wherein the fluorescence signal includes a droplet width; The signal modulation module is used to modulate the fluorescence signal to obtain a fluorescence voltage analog signal.

[0067] The single cell sorter for rare cells or viruses provided by the present invention and the fluorescence detection module include: a dichroic mirror, optically connected to the laser transmitter, for reflecting the excited fluorescence to the objective lens according to a target angle; an objective lens, optically connected to the dichroic mirror, for focusing the excited fluorescence onto the droplet, so that the droplet containing the fluorescent substance generates fluorescence; The beam splitter is optically connected to the objective lens and is used to split the fluorescence in the detection branch to obtain the first fluorescence and the second fluorescence; the first fluorescence is input to the high-speed camera; the second fluorescence is input to the beam splitter; A filter, connected to the beam splitter, for filtering out stray light from the first fluorescence and the second fluorescence to obtain a first target fluorescence and a second target fluorescence; A high-speed camera, optically connected to the filter, is used to collect images based on the first target fluorescence, obtain image signals, and input the image signals to the sorting control module; A spectroscope, optically connected to the laser emitter and the filter, respectively, for combining the excitation fluorescence and the second target fluorescence to obtain a fluorescence signal, and sending the fluorescence signal to a photomultiplier tube; The photomultiplier tube is optically connected to the spectroscope and is used to receive the fluorescence signal and convert the fluorescence signal into an electrical signal.

[0068] In the embodiment of the present invention, the fluorescence detection module includes a variety of photoelectric devices to convert the fluorescence signal into an electrical signal and output it to the photomultiplier tube, and the high-speed camera captures the motion state of the droplet passing through the objective lens. Here, the fluorescence microscope is equipped with a high-speed camera and a photomultiplier tube for fluorescence detection. When in use, the photomultiplier tube is inserted into the eyepiece, and the high-speed camera is inserted into the side light path exit.

[0069] refer to Figure 2 , Figure 2 It is a schematic diagram of the device structure of the fluorescence detection process provided by the present invention, which includes: a power supply, an amplifier, a fluorescence microscope, a laser with a wavelength of 488 nm (i.e., a laser transmitter), an objective lens, a chip (i.e., a sorting chip), a dichroic mirror, a beam splitter, a high-speed camera, a filter, a photomultiplier tube (PMT), and an FPGA (i.e., a field programmable gate array).

[0070] In an embodiment of the present invention, a laser emitter emits an excitation fluorescence with a target wavelength of 488 nm. The excitation fluorescence propagates along the excitation optical path and is reflected by a dichroic mirror before entering the objective lens. After being focused by the objective lens, it is emitted onto a droplet on the sorting chip. If the droplet contains a fluorescent substance, the droplet will be excited to produce fluorescence. The fluorescence enters the detection optical path from the objective lens and is divided into two by a beam splitter on the detection optical path. One part of the light is detected by a high-speed camera after stray light is filtered out by a filter and converted into an image signal and sent to a computer. Before entering the photomultiplier tube, the other part of the light is filtered out by a corresponding bandpass filter (i.e., the second target fluorescence) after stray light is filtered out. A beam splitter is used to respectively combine the excitation light (i.e., the excitation fluorescence) and the distinguished emission light (i.e., the second target fluorescence), and then is focused by a focusing lens into the photomultiplier tube for conversion, so as to convert the fluorescence signal into an electrical signal output, and the electrical signal emitted by the photomultiplier tube is recorded by a signal acquisition card.

[0071] According to the single cell sorter for rare cells or viruses provided by the present invention, the signal modulation module comprises: A preamplifier circuit is electrically connected to the photomultiplier tube and is used to amplify the electrical signal to obtain an amplified electrical signal; A filter circuit is electrically connected to the preamplifier circuit and is used to filter the noise of the amplified electrical signal to obtain a filtered electrical signal; The analog-to-digital converter is electrically connected to the filter circuit and is used for converting the filtered electrical signal into a fluorescence voltage analog signal and inputting the fluorescence voltage analog signal into the sorting control module.

[0072] Continue to refer Figure 2After the photomultiplier tube converts the fluorescence signal into an electrical signal, it needs to be amplified, filtered, and processed before it can be sent to the AD module (i.e. A / D) to be converted into a digital signal. The signal processing chip (i.e. field programmable gate array) performs subsequent processing on the signal.

[0073] According to the single cell sorter for rare cells or viruses provided by the present invention, the sorting control module comprises: A computer is electrically connected to the high-speed camera and the analog-to-digital converter, respectively, and is used to receive the image signal input by the high-speed camera and the fluorescence voltage analog signal input by the analog-to-digital converter, and output a target voltage signal when the fluorescence voltage analog signal is in a preset analog signal interval and the droplet width is in a preset droplet width interval; A field programmable gate array, electrically connected to the computer, and configured to output a driving signal when a target voltage signal output by the computer is detected; A DC power supply, electrically connected to the field programmable gate array, and configured to output a level signal when detecting a driving signal output by the field programmable gate array; The high-voltage pulse power supply is electrically connected to the DC power supply, and is used to amplify the level signal to obtain a target voltage signal, and output the target voltage signal to the sorting electrode.

[0074] refer to Figure 3 , Figure 3 It is a schematic diagram of the device structure of the sorting control process provided by the present invention, which includes: a computer, a USB, a field programmable gate array (FPGA), a TTL level signal (output by a DC power supply), a high-voltage power supply (i.e., a high-voltage pulse power supply), and an electrode (i.e., a sorting electrode).

[0075] like Figure 3 As shown, the chip electrode is connected to a high-frequency high-voltage power supply (high-voltage pulse power supply), and is connected to a computer through a field programmable gate array, wherein the field programmable gate array includes a control box USB isolated digital quantity I / O unit, which drives the DC power supply on and off. The control signal for generating the high-voltage pulse voltage is output by program control, and then outputs a level signal through the digital output interface of the field programmable gate array control box USB isolated digital quantity I / O unit, triggering the high-voltage pulse power supply output to complete the dielectric sorting action.

[0076] That is, the field programmable gate array (FPGA) is used to analyze in real time whether there is a target voltage signal in the signal. If the target voltage signal is detected, the DC power supply output of the target voltage is controlled, and the high-voltage power supply (i.e., high-voltage pulse power supply) is driven through the control box to trigger sorting.

[0077] The DC power supply is a square wave signal of a certain frequency, and the voltage is amplified by a high-voltage amplifier and output. The sorting control module includes a microcontroller and a signal processing circuit, which can generate a TTL signal according to the fluorescence intensity of the droplets. The TTL signal can further trigger the high-voltage pulse power supply to generate a square wave pulse voltage, which is amplified by the voltage amplifier and transmitted to the sorting chip. The resulting dielectrophoresis effect sorts the target droplets into the collection channel.

[0078] refer to Figure 4 , Figure 4 It is a schematic diagram of the structure of a single-cell sorting module for rare cells or viruses provided by the present invention, which includes: a fluorescence detection module, a signal modulation module, a control module, a power supply, a voltage amplification module and a microfluidic chip.

[0079] In an embodiment of the present invention, a fluorescence detection module is used to detect the (analog) fluorescence signal of each droplet in a plurality of droplets, and then the fluorescence signal is adjusted by a signal modulation module and input into a control module. The control module drives a signal source according to the fluorescence signal so that a power supply drives a voltage source, and then amplifies the voltage (for driving the droplets) by a voltage amplification module, thereby generating a dielectrophoresis effect to sort the target droplets in the microfluidic chip (sorting chip) into a collection channel.

[0080] Through the embodiments of the present invention, by simultaneously limiting the fluorescence signal and the droplet width, samples whose fluorescence signals meet the preset interval but whose droplet morphology does not meet the requirements can be more effectively excluded, thereby avoiding misjudgment. The stability of the droplet width has an important influence on the stability of the fluorescence signal. By limiting the droplet width, it is possible to ensure that the droplet morphology is consistent each time it is detected, thereby ensuring the stability and reliability of the fluorescence signal.

[0081] In order to realize rare cell sorting, the present invention needs to independently write software to realize: based on the Labview software development environment, develop the Sorter sorting program; develop the signal acquisition and processing program, the USB digital I / O module output sorting signal program, and the signal source generation software. Connect the software to the sorting instrument, set the parameters of the sorting instrument to realize real-time signal acquisition of different functions; analyze and determine whether the droplet signal is the target voltage signal, and then output the sorting signal through the FPGA (i.e., field programmable gate array) to realize rare cell sorting.

[0082] The following describes an example of a single-cell sorter for rare cells or viruses provided by the present invention in a practical application scenario.

[0083] In an embodiment of the present invention, the sorting chip (control system) is placed on the stage of an inverted fluorescence microscope for operation, and a high-speed camera is used to observe the movement of microdroplets. For example, a 1 mL syringe is driven by an injection pump to inject the separation oil into the chip. Image acquisition is completed by an inverted fluorescence microscope and a high-speed camera. The design platform of the inverted fluorescence microscope is very firm and has no jitter. The high-power LED lamp provides bright light to the field of view, which can meet the high requirements and clarity of image brightness in the experiment. The high-speed camera uses a 1280 x 1024 CMOS sensor, which has a higher frame rate, frame synchronization and exposure progress.

[0084] like Figure 1 As shown, the schematic diagram of the sorting chip specifically includes: a droplet inlet, an oil inlet, a sorting electrode, a sorting channel, a waste liquid pool, and a sorting pool; the droplet enters the sorting chip through the inlet and reaches the detection light site. The laser irradiates the droplet, and the droplet generates a fluorescence signal. The fluorescence signal of the droplet is detected. According to the detected fluorescence signal, it is determined whether the droplet is a target droplet, and then a voltage is applied; the droplet entering the fluorescence detection part is detected, and the droplet without the target voltage signal is not applied with voltage and flows into the waste liquid port. The fluorescence signal is collected by the sorting instrument, and the computer compares the fluorescence with the database to identify the type of signal. If it contains the target voltage signal, a voltage is applied to complete the dielectric sorting.

[0085] According to the single cell sorter for rare cells or viruses provided by the present invention, the mixed liquid to be sorted is a rare virus droplet sample.

[0086] In some embodiments, the target object of the mixed liquid to be sorted is virus, and the details can be referred to the following embodiments.

[0087] Example 1

[0088] Example 1 of the present application provides a method for sorting rare cells and viruses, comprising the following steps: Step 1: dilute the cell mixture containing rare cells or viruses, and encapsulate the rare cells or viruses in droplets; In this embodiment, the virus to be sorted is a virus Contig 1 in a sewage sample, and its DNA is extracted from the sewage sample. After the metagenome of the organism in the sewage is analyzed, a target sequence group with a sequence length of 4808 bp is obtained; In some embodiments, the sequence group includes two open reading frames (ORFs), one encoding a protein consisting of 1088 amino acids and the other encoding a protein consisting of 168 amino acids. These protein sequences do not match any sequences in existing protein sequence databases, further illustrating the uniqueness of the series. In order to gain a deeper understanding of the structure and function of these proteins, the present invention used the AlphaFold tool to generate their predicted protein structures, and then searched the protein structure database, showing a match. The smaller protein folds into a structure similar to the anti-CRISPR protein. The larger protein contains two domains, one of which matches a lysozyme inhibitor and the other matches a class of RNA-dependent RNA polymerases. These RNA polymerases are typically involved in the production of small interfering siRNAs.

[0089] Step 2: performing a specific reaction in the droplet, obtaining a corresponding fluorescence signal interval under specific reaction conditions, and obtaining a droplet size interval; The present invention first dilutes the sewage sample and encapsulates the viral genome into droplets according to the statistical principle of Poisson distribution, ensuring that most droplets contain only one viral genome. In order to amplify the cloned copy of each encapsulated genome, the present invention performs an MDA reaction in the droplets. The polymerase and other MDA reagents are co-encapsulated with the genome in the droplets, and then the whole genome amplification is initiated. The amplification reaction starts after the droplets are formed, ensuring the efficiency and specificity of the amplification reaction and obtaining the corresponding fluorescence signal interval.

[0090] Step 3: Enrichment or sorting of rare cells or viruses in the cell mixture is achieved based on the fluorescence signal interval combined with the droplet size interval.

[0091] The present invention screens out droplets containing genomes and performs fluorescence-activated droplet sorting in a microfluidic sorter according to the fluorescence signal interval. This step can accurately separate droplets containing the target genome, providing high-quality samples for subsequent analysis.

[0092] According to the single cell sorter for rare cells or viruses provided by the present invention, the target object in the rare virus droplet sample is rare virus Contig 1, and the probe sequence of rare virus Contig 1 is probe B / 56-FAM / rCrUrUrArUrGrGrGrA / ZEN / rGrGrCrArArGrCrArGrCrArArGrUrU / 3IABkFQ / (SEQ ID NO.1).

[0093] After the enrichment of the virus is completed, the classified droplets are collected in a test tube, and a demulsifier is added to destroy the emulsion structure to release the target genome from the droplets. The released genome copies are then used as templates for a second batch of MDA amplification to produce sufficient DNA for subsequent sequencing analysis. Finally, the present invention processes the amplified products, constructs a sequencing library, and performs high-throughput sequencing. In order to assemble the sequencing reads into the final genome sequence, the present invention filters the original sequencing reads, eliminates low-quality reads and human contamination, and the remaining reads are assembled into Contigs. The obtained genome sequence is matched with the genome in the NCBI database, thereby achieving accurate identification and analysis of the viral genome.

[0094] In order to verify the effectiveness of the whole genome sequencing method for target virus species from different sources, the present invention plans to re-sort from the peak samples. Two different populations were detected based on the fluorescence intensity. The main population corresponds to empty droplets or droplets containing non-target virus genomes, while the bright population represents droplets containing targets. Next, the present invention screens out bright droplets and amplifies genomic DNA from these droplets. Through a large number of MDA reactions, the present invention successfully produces sufficient amounts of DNA for sequencing. Subsequently, the present invention performs sorted droplet sequencing from a single amplification product and assembles read sequences from scratch. The probe sequence is probe B / 56-FAM / rCrUrUrArUrGrGrGrA / ZEN / rGrGrCrArArGrCrArGrCrArArGrUrU / 3IABkFQ / (SEQ ID NO.1).

[0095] refer to Figure 5 , Figure 5 It is a schematic diagram of a gene detection method compatible with MDA conditions provided by the present invention, which includes: a target genome, a sequence-specific RNA probe, ribonuclease H, and incubation; According to the single cell sorter for rare cells or viruses provided by the present invention, the detection tool for rare virus Contig 1 includes: an RNA probe and an RNase H enzyme, wherein the RNA probe includes an RNA oligonucleotide, the 5' end of the RNA oligonucleotide has a fluorescent group coupling, and the 3' end of the RNA oligonucleotide has a quencher; the RNase H enzyme is used to digest the RNA oligonucleotide to release the fluorescent group from the quencher.

[0096] In order to successfully isolate the probe sequence of unknown target genome Contig 1, such as Figure 5As shown in Figure 2, a gene detection method compatible with MDA conditions was designed. The detection uses a designed RNA probe and RNase H enzyme. The RNA probe is a special RNA oligonucleotide with a fluorescent group coupled to its 5' end and a quencher at its 3' end. The oligonucleotide sequence is designed to target the contigs of the target viral species. In order to ensure that the probe can bind accurately and stably to the target gene, we selected a probe that is not easy to form a stable secondary structure and has a melting temperature above 30 RNA sequence. For common fluorophore-quencher pairs, the optimal length of the oligonucleotide is 18-24 bp. We used 5'FAM (fluorescein) / 3'IBFQ ( ) and selected an oligonucleotide sequence of 22 bp.

[0097] RNase H is an endonuclease that digests RNA only when the RNA chain is bound to its complementary DNA chain. During the MDA process, if a droplet contains a target genome, the RNA probe will actively approach the complementary sequence in the genome to form a DNA-RNA complex. Once RNase H binds to these specific complexes, it precisely digests the RNA oligonucleotide, thereby releasing the fluorophore from the quencher. Therefore, when the droplet containing the target genome is excited, a high fluorescence signal is presented. In contrast, in droplets containing non-target genomes or no genomes at all, the RNA probe is not digested by RNase H and the fluorophore remains in a quenched state.

[0098] refer to Figure 6 , Figure 6 It is one of the schematic diagrams of the detection results of the sample types and probe combinations provided by the present invention, which includes the following groups.

[0099] A. No sewage sample + probe D. In this group, no sewage sample (ie, No sewage sample) was used, but only probe D (probe D / 56-FAM / rGrGrGrUrGrUrCrCrU / ZEN / rGrUrUrGrGrArUrArCrUrCrCrGrUrG / 3IABkFQ / (SEQ ID NO.2)) was used. As a control experiment, it was used to evaluate the performance of probe D in the absence of sample or background signal (A1 is the original figure, A2 is the image after the experiment).

[0100] B. Sewage sample + crAssphage probe. In this group, a sewage sample (i.e., Sewage sample) and a crAssphage probe (i.e., crAssphage probe) were used. crAssphage is a bacteriophage commonly found in human fecal samples. This group was used to detect the presence or concentration of crAssphage in sewage and verify the operation of the droplet and probe system (B1 is the original image, and B2 is the image after the experiment).

[0101] C. RS1-1 sewage sample + probe D. In this group, RS1-1 sewage sample (i.e. RS1-1sewage sample) and probe D (i.e. probe D) were used to detect the presence or concentration of a certain substance in this type of sewage sample (C1 is the original image, and C2 is the image after the experiment).

[0102] D. RS1-2 sewage sample + probe D. RS1-2 sewage sample and probe D were used in this group. RS1-2 represents a specific source or classification of sewage samples, which is used to detect the presence or concentration of target substances in RS1-2 sewage samples (D1 is the original image, and D2 is the image after the experiment).

[0103] E. Alcosan sample + probe D. Alcosan sample and probe D were used in this group to evaluate the presence or concentration of target substances in the Alcosan sample (E1 is the original image, and E2 is the image after the experiment).

[0104] refer to Figure 7 , Figure 7 This is the second schematic diagram of the detection results of the sample types and probe combinations provided by the present invention, which includes the following groups.

[0105] A. Probe D and In this group, Probe D and As a control experiment, it is used to evaluate the performance of probe D in the absence of sample or background signal (A1 is the original image, A2 is the image after the experiment).

[0106] B. Probe D, template and In this group, probe D and template DNA were used, but RNase H was not used to verify the effect of the system (B1 is the original image, and B2 is the image after the experiment).

[0107] C. Probe D, template and In this group, probe D, template DNA and , verify the function of the system. In this case, the system operates normally (C1 is the original image, and C2 is the image after the experiment).

[0108] refer to Figure 8 , Figure 8 This is the third schematic diagram of the detection results of sample types and different probe combinations provided by the present invention, wherein: Figure 8 The upper figures (upper part) in the figure are all bright field. Figure 8 The lower rows of graphs (lower panels) in the figure all represent fluorescence fields.

[0109] There was no fluorescence signal in the sewage sample and probe D (probe D / 56-FAM / rGrGrGrUrGrUrCrCrU / ZEN / rGrUrUrGrGrArUrArCrUrCrCrGrUrG / 3IABkFQ / (SEQ ID NO.2)), indicating that the effect of probe D was not good.

[0110] Positive control, adding positive-crAssphage probe, there is a fluorescent signal, indicating that the whole system is operating normally.

[0111] In the sewage sample and probe B ( / 56-FAM / rCrUrUrArUrGrGrGrA / ZEN / rGrGrCrArArGrCrArGrCrArArGrUrU / 3IABkFQ / (SEQ ID NO.1)), some droplets had strong fluorescence signals, indicating that probe B was a suitable probe for Contig 1.

[0112] Sewage sample and probe M ( / 56-FAM / rUrCrGrCrUrArCrArG / ZEN / rCrArArArCrArUrCrGrUrGrCrUrGrA / 3IABkFQ / (SEQ ID NO.3)), some droplets have fluorescence signals, indicating that probe M is a suitable probe for Contig 1.

[0113] refer to Fig. 9 , Fig. 9 4 is a schematic diagram of the detection results of sample types and different probe combinations provided by the present invention, wherein: Fig. 9 The middle and upper rows (upper part) are all bright field. Fig. 9 The middle and lower rows (lower panels) all represent fluorescent fields.

[0114] Negative-no sewage sample, added with negative-probe B, no fluorescence signal, which is the negative control.

[0115] Negative-no sewage sample, added with negative-probe M, no fluorescence signal, which is the negative control.

[0116] Positive-sewage sample, added with positive-crAssphage probe, has fluorescent signal, which is the positive control.

[0117] Probe B, when probe B is added to the sewage sample, there is a fluorescence signal, and the fluorescence effect of probe B is better than that of probe M, indicating that probe B is the best probe.

[0118] Probe M. When probe M is added to the sewage sample, there is a fluorescence signal, but the fluorescence intensity is significantly lower than that of probe B.

[0119] In some embodiments, the sewage is infinitely diluted to ensure that each droplet contains only one virus. The droplets containing the probe, MDA reagent and sewage sample are injected into the sorting chip and the sorting experiment is performed in the sorting instrument. At PMT 1 0.48, , Burst cycles 25, Amplitude 6.9 V, Frequency 25000 Hz, and under the sorting conditions of Gate 1 and Gate 2, droplets containing rare viruses were sorted.

[0120] The range of the sorting threshold is determined by the control gate set in the chip. The process of the chip determining the sorting threshold through the control gate includes: In the constructed sorting coordinate system, the sorting coordinate points at multiple different coordinate positions in the preset control gate are connected to form a polygonal area range; The polygonal area range is used as the range of the sorting threshold determined by the control gate, wherein the abscissa of the sorting coordinate system is the droplet sample size, and the ordinate of the sorting coordinate system is the fluorescence signal intensity of the droplet sample.

[0121] Specifically, in the constructed sorting coordinate system, according to the preset control gate, a plurality of sorting coordinate points at different coordinate positions are connected, and the polygonal area range formed after the connection can be used as the range of the sorting threshold determined by the control gate.

[0122] The sorting coordinate points are connected to form a polygonal area. For example, three sorting coordinate points form a triangular area, and four sorting coordinate points form a quadrilateral area. The electrical pulse signal of the droplet sample may be mapped within the range of this polygonal area. The range of the polygonal area determined by the control gate is the range of the sorting threshold. The droplet sample whose electrical pulse signal is within the polygonal area indicates that it has reached the sorting threshold and is selected as the droplet sample for dielectric sorting.

[0123] It should be noted that the determination of the sorting coordinate points also has certain standards. For example, in some sorting scenarios, the size of the droplet sample must reach a certain size range, or the fluorescence signal intensity must reach a certain intensity range before it can be sorted. Different sorting scenarios set different size ranges and intensity ranges.

[0124] Furthermore, the range of the sorting threshold is determined based on a plurality of control gates set in the chip. The chip determines the range of the sorting threshold through the plurality of control gates, including: In the constructed sorting coordinate system, multiple sorting coordinate points at different coordinate positions in each control gate are connected to form a polygonal area range corresponding to the control gate; The overlapping area range formed by the polygonal area ranges corresponding to the multiple control gates is determined as the range of the sorting threshold.

[0125] When the range of the sorting threshold is determined by multiple control gates, in the constructed sorting coordinate system, multiple sorting coordinate points at different coordinate positions set in each control gate are connected to form a polygonal area range corresponding to the control gate.

[0126] When setting multiple control gates, the setting processes between the control gates do not interfere with each other and are independent of each other. In the constructed sorting coordinate system, multiple sorting coordinate points with different coordinate positions can be set for each control gate one by one, and then the sorting coordinate points of the corresponding control gates can be connected. In this way, the sorting coordinate points of each control gate form a polygonal area range, and multiple polygonal area ranges will be formed. Finally, the overlapping area range formed by the polygonal area ranges of multiple control gates is determined as the range of the sorting threshold. The overlapping area is the area that satisfies multiple control gates, and the range of this area can be determined as the range of the sorting threshold, thereby realizing the flexible control of the sorting threshold by multiple control gates.

[0127] For example, in a cell sorting scenario, two control gates are needed to determine the range of the sorting threshold. Therefore, four sorting coordinate points and three sorting coordinate points are set in the sorting coordinate system. The four sorting coordinate points of the first control gate are connected to form a quadrilateral area range, and the three sorting coordinate points of the second control gate are connected to form a triangular area range. Then the overlapping area range of the quadrilateral area range and the triangular area range can be determined as the range of the sorting threshold.

[0128] refer to Fig.10 , Fig.10 This is one of the schematic diagrams of the sorting statistical results provided by the present invention.

[0129] Under the same sorting parameter conditions, 24338 fluorescent droplets were sorted out from 2467090 droplets (i.e., 0.9865063698527415% before sorting).

[0130] refer to Fig.11 , Fig.11 This is the second schematic diagram of the sorting statistical results provided by the present invention.

[0131] Under the same sorting parameter conditions, 8246 fluorescent droplets were sorted out from 323242 droplets (i.e., 0.25509730573192446% before sorting).

[0132] refer to Fig.12 , Fig.12 This is the third schematic diagram of the sorting statistical results provided by the present invention.

[0133] Under the same sorting parameter conditions, 4606 fluorescent droplets were sorted out from 1865286 droplets (i.e., 0.2469326419648247% before sorting).

[0134] refer to Fig.13 , Fig.13 This is the fourth schematic diagram of the sorting statistical results provided by the present invention.

[0135] Under the same sorting parameter conditions, 886 fluorescent droplets were sorted out from 1,688,937 droplets (i.e., 0.05245903192363007% before sorting).

[0136] The single cell sorting method for rare cells or viruses provided by the present invention is described below. The single cell sorting method for rare cells or viruses described below and the single cell sorting instrument for rare cells or viruses described above can be referenced to each other.

[0137] refer to Fig.14 , Fig.14 It is a schematic diagram of the process of the single cell sorting method of rare cells or viruses provided by the present invention.

[0138] Step 1401, inputting a mixed solution to be sorted through a droplet injection pump, wherein the mixed solution to be sorted includes a target object, and the target object includes one of the following: rare cells, viruses; Step 1402, inputting the oil phase through the oil phase injection pump; Step 1403, receiving the mixed liquid to be sorted inputted by the droplet injection pump through the droplet injection port of the sorting chip, and inputting the mixed liquid to be sorted into the sorting channel; Step 1404, receiving the oil phase inputted by the oil phase injection pump through the oil injection port of the sorting chip, and inputting the oil phase into the sorting channel, where the oil phase is used to separate the mixed liquid to be sorted into a plurality of droplets in the sorting channel; Step 1405, transferring each of the plurality of droplets through a sorting channel of a sorting chip; Step 1406, when receiving the target voltage signal, the sorting electrode of the sorting chip applies voltage to the current droplet for dielectric sorting, so as to sort the current droplet as a target droplet into a sorting pool; Step 1407, receiving target droplets through a sorting pool; Step 1408, receiving the waste liquid droplets to which the voltage is not applied by the sorting electrode through the waste liquid pool.

[0139] Specifically, the single-cell sorting method for rare cells or viruses provided by the present invention can implement all the method steps implemented by the single-cell sorting instrument embodiment for rare cells or viruses, and can achieve the same technical effects. The parts and beneficial effects that are the same as those in the method embodiment will not be described in detail herein.

[0140] Fig.15 is a schematic diagram of the physical structure of the electronic device provided by the present invention, such as Fig.15As shown, the electronic device may include: a processor 1510, a communications interface 1520, a memory 1530 and a communications bus 1540, wherein the processor 1510, the communications interface 1520 and the memory 1530 communicate with each other via the communications bus 1540. The processor 1510 may call the logic instructions in the memory 1530 to execute a single cell sorting method for rare cells or viruses, the method comprising: inputting a mixed liquid to be sorted through a droplet injection pump, wherein the mixed liquid to be sorted includes a target object, and the target object includes one of the following: rare cells, viruses; inputting an oil phase through an oil phase injection pump; receiving the mixed liquid to be sorted input by the droplet injection pump through the droplet injection port of the sorting chip, and inputting the mixed liquid to be sorted into the sorting channel; and inputting the mixed liquid to be sorted into the sorting channel through the oil injection port of the sorting chip. The port receives the oil phase input by the oil phase injection pump, and inputs the oil phase into the sorting channel, where the oil phase is used to separate the mixed liquid to be sorted into multiple droplets in the sorting channel; each of the multiple droplets is transmitted through the sorting channel of the sorting chip; when the sorting electrode of the sorting chip receives the target voltage signal, a voltage is applied to the current droplet for dielectric sorting, so as to sort the current droplet as a target droplet into the sorting pool; the target droplet is received through the sorting pool; and the waste droplets to which the voltage is not applied by the sorting electrode are received through the waste liquid pool.

[0141] In addition, the logic instructions in the above-mentioned memory 1530 can be implemented in the form of a software functional unit and can be stored in a computer-readable storage medium when it is sold or used as an independent product. Based on such an understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art or the part of the technical solution, can be embodied in the form of a software product, and the computer software product is stored in a storage medium, including several instructions for a computer device (which can be a personal computer, a server, or a network device, etc.) to perform all or part of the steps of the method described in each embodiment of the present invention. The aforementioned storage medium includes: U disk, mobile hard disk, read-only memory (ROM, Read-Only Memory), random access memory (RAM, Random Access Memory), disk or optical disk, etc. Various media that can store program codes.

[0142] On the other hand, the present invention also provides a computer program product, which includes a computer program, which can be stored on a non-transitory computer-readable storage medium. When the computer program is executed by a processor, the computer can execute the single-cell sorting method for rare cells or viruses provided by the above methods, the method comprising: inputting a mixed liquid to be sorted through a droplet injection pump, wherein the mixed liquid to be sorted includes a target object, and the target object includes one of the following: rare cells, viruses; inputting an oil phase through an oil phase injection pump; receiving the mixed liquid to be sorted input by the droplet injection pump through the droplet inlet of the sorting chip; Sorting the mixed liquid, and inputting the mixed liquid to be sorted into the sorting channel; receiving the oil phase input by the oil phase injection pump through the oil inlet of the sorting chip, and inputting the oil phase into the sorting channel, the oil phase is used to separate the mixed liquid to be sorted into multiple droplets in the sorting channel; transmitting each droplet of the multiple droplets through the sorting channel of the sorting chip; applying voltage to the current droplet for dielectric sorting when receiving the target voltage signal through the sorting electrode of the sorting chip, so as to sort the current droplet as the target droplet into the sorting pool; receiving the target droplet through the sorting pool; receiving the discarded droplets to which the sorting electrode has not applied voltage through the waste liquid pool.

[0143] On the other hand, the present invention also provides a non-transitory computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, is implemented to execute the single-cell sorting method for rare cells or viruses provided by the above-mentioned methods, the method comprising: inputting a mixed liquid to be sorted through a droplet injection pump, wherein the mixed liquid to be sorted includes a target object, and the target object includes one of the following: rare cells, viruses; inputting an oil phase through an oil phase injection pump; receiving the mixed liquid to be sorted input by the droplet injection pump through the droplet inlet of the sorting chip, and injecting the mixed liquid to be sorted into the droplet injection pump; Input into the sorting channel; receive the oil phase input by the oil phase injection pump through the oil inlet of the sorting chip, and input the oil phase into the sorting channel, the oil phase is used to separate the mixed liquid to be sorted into multiple droplets in the sorting channel; transmit each of the multiple droplets through the sorting channel of the sorting chip; when the sorting electrode of the sorting chip receives the target voltage signal, the voltage is applied to the current droplet for dielectric sorting, so as to sort the current droplet as the target droplet into the sorting pool; receive the target droplet through the sorting pool; receive the discarded droplets to which the voltage is not applied by the sorting electrode through the waste liquid pool.

[0144] The device embodiments described above are merely illustrative, wherein the units described as separate components may or may not be physically separated, and the components displayed as units may or may not be physical units, that is, they may be located in one place, or they may be distributed on multiple network units. Some or all of the modules may be selected according to actual needs to achieve the purpose of the scheme of this embodiment. Ordinary technicians in this field can understand and implement it without paying creative labor.

[0145] Through the description of the above implementation methods, those skilled in the art can clearly understand that each implementation method can be implemented by means of software plus a necessary general hardware platform, and of course, can also be implemented by hardware. Based on this understanding, the above technical solution is essentially or the part that contributes to the prior art can be embodied in the form of a software product, and the computer software product can be stored in a computer-readable storage medium, such as ROM / RAM, a disk, an optical disk, etc., including a number of instructions for a computer device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in each embodiment or some parts of the embodiments.

[0146] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A single cell sorter for rare cells or viruses, characterized in that: include: A droplet injection pump, used for inputting a mixed solution to be sorted, wherein the mixed solution to be sorted includes a target object, and the target object includes one of the following: rare cells, viruses; Oil phase injection pump, used for inputting oil phase; A sorting chip is connected to the droplet injection pump and the oil phase injection pump respectively, and the sorting chip comprises: a droplet injection port, an oil injection port, a sorting channel, a sorting electrode, a sorting pool and a waste liquid pool; The droplet inlet is connected to the droplet injection pump and the sorting channel respectively, and is used to receive the mixed liquid to be sorted input by the droplet injection pump, and input the mixed liquid to be sorted into the sorting channel; The oil injection port is connected to the oil phase injection pump and the sorting channel respectively, and is used to receive the oil phase input by the oil phase injection pump and input the oil phase into the sorting channel. The oil phase is used to separate the mixed liquid to be sorted into a plurality of droplets in the sorting channel; The first end of the sorting channel is connected to the droplet injection port and the oil injection port respectively, the second end of the sorting channel is connected to the sorting pool and the waste liquid pool respectively, and the sorting channel is used to transfer each droplet of the plurality of droplets; The sorting electrode is connected to the sorting channel and is used to apply voltage to the current droplet for dielectric sorting when receiving the target voltage signal, so as to sort the current droplet as a target droplet into the sorting pool; The sorting pool is connected to the sorting channel and is used to receive the target droplets; A waste liquid pool, connected to the sorting channel, for receiving waste liquid droplets to which voltage is not applied by the sorting electrode; The oil phase injection pump comprises: A first oil phase injection pump connected to the oil inlet and used to output the first oil phase at a first frequency, wherein the first frequency is used to enable the first oil phase to separate the mixed liquid to be sorted into a plurality of droplets; The oil phase injection pump also includes: A second oil phase injection pump, connected to the oil inlet, for outputting oil phase interval droplets at a second frequency, wherein the second frequency is used to cut off the continuous water phase to form droplet intervals; A third oil phase injection pump is connected to the oil injection port and is used to output the second oil phase at a third frequency. The third frequency is used to make the second oil phase space each of the multiple droplets to a target spacing distance.

2. The single cell sorter for rare cells or viruses according to claim 1, characterized in that: The single cell sorter for rare cells or viruses also includes: A laser transmitter for emitting excitation fluorescence of a target wavelength; A fluorescence microscope, optically connected to the laser emitter, for receiving the excitation fluorescence and focusing the excitation fluorescence onto the plurality of droplets, so that the droplets containing the fluorescent substance generate fluorescence; and determining a fluorescence voltage analog signal corresponding to each droplet; The sorting control module is used to output a target voltage signal to the sorting electrode when the fluorescence voltage analog signal is in a preset analog signal interval and the droplet width is in a preset droplet width interval.

3. The single cell sorter for rare cells or viruses according to claim 2, characterized in that: The fluorescence microscope comprises: A fluorescence detection module, used to perform fluorescence detection and image acquisition on each of the plurality of droplets to obtain an electrical signal of each droplet and a fluorescence signal of each droplet, wherein the fluorescence signal includes a droplet width; The signal modulation module is used to modulate the electrical signal to obtain a fluorescence voltage analog signal.

4. The single cell sorter for rare cells or viruses according to claim 3, characterized in that: The fluorescence detection module comprises: a dichroic mirror, optically connected to the laser emitter, for reflecting the excitation fluorescence to the objective lens according to a target angle; The objective lens is optically connected to the dichroic mirror and is used to focus the excitation fluorescence onto the droplet so that the droplet containing the fluorescent substance produces fluorescence; A beam splitter, optically connected to the objective lens, for splitting the fluorescence in a detection branch to obtain a first fluorescence and a second fluorescence; the first fluorescence is input to a high-speed camera; the second fluorescence is input to a beam splitter; a filter, connected to the beam splitter, for filtering out stray light of the first fluorescence and the second fluorescence to obtain a first target fluorescence and a second target fluorescence; The high-speed camera is optically connected to the filter, and is used to collect images based on the first target fluorescence, obtain image signals, and input the image signals into the sorting control module; The spectroscope is optically connected to the laser emitter and the filter, respectively, and is used to combine the excitation fluorescence and the second target fluorescence to obtain a fluorescence signal, and send the fluorescence signal to a photomultiplier tube; The photomultiplier tube is optically connected to the spectroscope and is used to receive the fluorescence signal and convert the fluorescence signal into an electrical signal.

5. The single cell sorter for rare cells or viruses according to claim 3, characterized in that: The signal modulation module comprises: A preamplifier circuit, electrically connected to the photomultiplier tube, for amplifying the electrical signal to obtain an amplified electrical signal; A filter circuit, electrically connected to the preamplifier circuit, for filtering the amplified electrical signal to obtain a filtered electrical signal; The analog-to-digital converter is electrically connected to the filter circuit and is used for converting the filtered electrical signal into a fluorescence voltage analog signal and inputting the fluorescence voltage analog signal into a sorting control module.

6. The single cell sorter for rare cells or viruses according to claim 2, characterized in that: The sorting control module comprises: A computer, electrically connected to the high-speed camera and the analog-to-digital converter, respectively, for receiving the image signal input by the high-speed camera and the fluorescence voltage analog signal input by the analog-to-digital converter, and outputting a target voltage signal when the fluorescence voltage analog signal is in a preset analog signal interval and the droplet width is in a preset droplet width interval; a field programmable gate array, electrically connected to the computer, and configured to output a driving signal when detecting the target voltage signal output by the computer; A direct current power supply, electrically connected to the field programmable gate array, and configured to output a level signal when detecting the driving signal output by the field programmable gate array; A high-voltage pulse power supply is electrically connected to the DC power supply, and is used to amplify the level signal to obtain a target voltage signal, and output the target voltage signal to the sorting electrode.

7. The single cell sorter for rare cells or viruses according to claim 1, characterized in that: The mixed liquid to be sorted is a rare virus droplet sample.

8. The single cell sorter for rare cells or viruses according to claim 7, characterized in that: The target object in the rare virus droplet sample is rare virus Contig 1, and the probe sequence of the rare virus Contig 1 is probeB / 56-FAM / rCrUrUrArUrGrGrGrA / ZEN / rGrGrCrArArGrCrArGrCrArArGrUrU / 3IABkFQ / .

9. The single cell sorter for rare cells or viruses according to claim 8, characterized in that: The detection tool for the rare virus Contig 1 includes: an RNA probe and an RNase H enzyme, wherein the RNA probe includes an RNA oligonucleotide, the 5' end of the RNA oligonucleotide has a fluorescent group coupling, and the 3' end of the RNA oligonucleotide has a quencher; the RNase H enzyme is used to digest the RNA oligonucleotide so that the quencher releases the fluorescent group.

10. A single cell sorting method for rare cells or viruses, characterized in that: include: Inputting a mixed solution to be sorted through a droplet injection pump, wherein the mixed solution to be sorted includes a target object, and the target object includes one of the following: rare cells, viruses; The oil phase is introduced through an oil phase injection pump; The mixed liquid to be sorted is received by the droplet injection pump through the droplet injection port of the sorting chip, and the mixed liquid to be sorted is input into the sorting channel; The oil phase input by the oil phase injection pump is received through the oil injection port of the sorting chip, and the oil phase is input into the sorting channel, wherein the oil phase is used to separate the mixed liquid to be sorted into a plurality of droplets in the sorting channel; passing each of the plurality of droplets through a sorting channel of the sorting chip; When receiving the target voltage signal, the sorting electrode of the sorting chip applies voltage to the current droplet for dielectric sorting, so as to sort the current droplet as a target droplet into a sorting pool; receiving the target droplet through the sorting cell; receiving, through a waste liquid pool, discarded liquid droplets to which voltage is not applied by the sorting electrode; Outputting the first oil phase at a first frequency through a first oil phase injection pump, wherein the first frequency is used to enable the first oil phase to separate the mixed liquid to be sorted into a plurality of droplets; Outputting oil phase interval droplets at a second frequency through a second oil phase injection pump, wherein the second frequency is used to cut off the continuous water phase to form droplet intervals; The second oil phase is outputted at a third frequency through a third oil phase injection pump, and the third frequency is used to enable the second oil phase to space each of the plurality of droplets to a target spacing distance.

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