High-performance cell sorting system and method, storage medium and program product

Abstract

The invention provides a high-performance cell sorting system and method, a storage medium and a program product, and relates to the technical field of medical engineering.The method comprises the steps that fluorescence signals of liquid drop samples corresponding to cells to be sorted are collected, and the fluorescence signals are converted into electric pulse signals; when the electric pulse signal reaches a set sorting threshold value, a sorting signal is output, the sorting threshold value is determined through one or more control gates, and the control gates are set according to the size of the liquid drop sample and the intensity of the fluorescence signal. And after the sorting signal is received, a high-voltage pulse electric field is applied to the liquid drop sample according to the sorting parameters, dielectric sorting operation is carried out on the liquid drop sample through the high-voltage pulse electric field, and sorted liquid drops are obtained after sorting. According to the cell sorting method and device, the defects that in the prior art, a cell sorting method can only achieve basic threshold value or region sorting, the sorting requirement in a complex scene cannot be met, and efficient and accurate sorting is difficult to achieve are overcome.

Description

Technical Field

[0001] The present invention relates to the field of medical engineering technology, and in particular to a high-performance cell sorting system, method, storage medium and program product. Background Art

[0002] Cell sorting is an important step in biomedical research and is of great significance. However, most of the existing cell sorting methods, systems and program products are limited, simple and single in function, and cannot meet the complex and sophisticated sorting requirements. In the prior art, the sorting software system can often only achieve basic threshold or area sorting, and cannot meet the sorting requirements in complex scenarios. For example, it is impossible to define different thresholds for sorting, and it is even more impossible to achieve combined sorting under multiple different conditions. Especially when dealing with tiny samples such as rare cells or viruses, existing systems often find it difficult to achieve efficient and accurate sorting. In addition, the existing technology is also difficult to achieve for the sorting of a single signal, which greatly limits its application in the field of high-performance sorting. Summary of the invention

[0003] The present invention provides a high-performance cell sorting system, method, storage medium and program product to overcome the defects of the prior art cell sorting methods that can only achieve basic threshold or area sorting, cannot meet the sorting requirements in complex scenarios, and are difficult to achieve efficient and accurate sorting.

[0004] The present invention provides a high-performance cell sorting system, the system comprising: an optical acquisition module and a droplet sorting module; wherein the optical acquisition module comprises a data acquisition card, and the droplet sorting module comprises a high-voltage pulse power supply, a sorting chip, and an FPGA chip; The FPGA chip is used to collect the electric pulse signal from the data acquisition card, and output a sorting signal when the electric pulse signal reaches a sorting threshold, wherein the sorting threshold is determined by one or more control gates, and the control gates are set according to the droplet sample size and the fluorescence signal intensity; The FPGA chip calculates the dielectrophoretic force required to be achieved when the high-voltage pulse power supply performs dielectric sorting according to the pulse period, amplitude parameter, and frequency parameter, including: The dielectrophoretic force required for the droplet sample in the sorting chip to perform dielectric sorting is calculated according to the following formula: in, represents the dielectrophoretic force, is the radius of the droplet sample, is the complex relative dielectric constant of the cells to be sorted in the droplet sample, is the complex relative dielectric constant of the solution in the droplet sample, is the vacuum dielectric constant of the cells to be sorted in the droplet sample, f is the frequency parameter, is the amplitude parameter, v represents the application speed of the target electric field strength, and the application speed is positively correlated with the pulse period when the frequency parameter remains unchanged.

[0005] In some embodiments, the range of the sorting threshold is determined according to a control gate set in the FPGA chip, and the process of the FPGA 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.

[0006] In some embodiments, the range of the sorting threshold is determined based on a plurality of control gates set in the FPGA chip, and the FPGA 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.

[0007] In some embodiments, the range of the sorting threshold is obtained according to a custom threshold set in the FPGA chip, and the FPGA chip determines the range of the sorting threshold according to the custom threshold, including: In the constructed sorting coordinate system, Cursors points are determined in the sorting coordinate system according to preset Cursors values, wherein the Cursors values ​​include droplet sample size parameters and droplet sample fluorescence signal intensity parameters; The entire area of ​​the sorting coordinate system is divided into a plurality of candidate areas according to the Cursors points, and at least one target area is determined in the candidate areas as the range of the sorting threshold.

[0008] In some embodiments, the FPGA chip is further used to adaptively adjust the range of the sorting threshold. The process of the FPGA chip adaptively adjusting the range of the sorting threshold includes: Calculating the proportion of the electric pulse signals of the droplet samples within the sorting threshold, and monitoring the proportion; If it is detected that the proportion of the quantity is lower than the preset sorting ratio, the range of the sorting threshold is automatically adjusted; If it is detected that the proportion of the quantity is not lower than the preset sorting ratio, the automatic adjustment is stopped.

[0009] In some embodiments, the FPGA chip is also used to map the electrical pulse signals acquired from the data acquisition card to the constructed sorting coordinate system one by one, and the FPGA chip is also used to determine that the electrical pulse signal reaches the sorting threshold when the electrical pulse signal is detected to be within the range of the sorting threshold.

[0010] In some embodiments, the optical acquisition module also includes a laser light source and a photomultiplier tube, wherein the laser light source is used to emit a laser to the droplet sample to excite the fluorescence signal of the droplet sample; the photomultiplier tube is used to collect the fluorescence signal, convert the fluorescence signal into an electrical pulse signal, and store the electrical pulse signal in the data acquisition card.

[0011] In some embodiments, the droplet sorting module further includes a sorting droplet collector, and the sorting droplet collector is used to collect sorting droplets obtained after the droplet sample is subjected to a dielectric sorting operation.

[0012] In some embodiments, the sorting chip is electrically connected to the laser light source and the photoelectric backup tube, respectively, the data acquisition card is communicatively connected to the photoelectric backup tube and the FPGA chip, respectively, the high-voltage pulse power supply is electrically connected to the FPGA chip and the sorting chip, and the sorting droplet collector is connected to the sorting chip through a droplet channel.

[0013] In some embodiments, the droplet sorting module further includes a high-voltage amplifier, which is electrically connected to the high-voltage pulse power supply; the high-voltage pulse power supply is also used to apply a corresponding high-voltage pulse electric field according to the dielectrophoretic force, and the process of applying the high-voltage pulse electric field by the high-voltage pulse power supply includes: Release a TTL level signal to the high-voltage pulse power supply through the FPGA chip, so that the high-voltage pulse power supply generates a sine wave signal from the TTL level signal; The sinusoidal wave signal is amplified and processed into an AC driving voltage of a preset amplitude by the high-voltage amplifier, and the AC driving voltage is input to the sorting chip to form a high-voltage pulse electric field.

[0014] The present invention also provides a high-performance cell sorting method, which comprises the following steps: Collecting the fluorescence signal of the droplet sample corresponding to the cells to be sorted, and converting the fluorescence signal into an electrical pulse signal; When the electric pulse signal reaches the set sorting threshold, a sorting signal is output, wherein the sorting threshold is determined by one or more control gates, and the control gates are set according to the droplet sample size and the fluorescence signal intensity; the range of the sorting threshold is determined according to one or more control gates set in the FPGA chip, including: In the constructed sorting coordinate system, the sorting coordinate points at multiple different coordinate positions in the preset control gate are connected to form one or more polygonal area ranges; Using the overlapping area of ​​the one or more polygonal areas as the range of the sorting threshold determined by the control gate, wherein the abscissa of the sorting coordinate system is the size of the droplet sample, and the ordinate of the sorting coordinate system is the fluorescence signal intensity of the droplet sample; After receiving the sorting signal, a high-voltage pulse electric field is applied to the droplet sample according to the sorting parameters, and a dielectric sorting operation is performed on the droplet sample by the high-voltage pulse electric field to obtain sorted droplets.

[0015] The present invention also provides a non-transitory computer-readable storage medium having a computer program stored thereon, which implements the high-performance cell sorting method as described above when executed by a processor.

[0016] The present invention also provides a computer program product, comprising a computer program, wherein when the computer program is executed by a processor, the high-performance cell sorting method as described above is implemented.

[0017] The present invention provides a high-performance cell sorting system, method, storage medium and program product, which have the following beneficial effects: (1) One or more control gates are determined by the size of the droplet sample and the intensity of the fluorescence signal, and then the sorting threshold of the droplet sample is determined. When the electric pulse signal reaches the sorting threshold, a sorting signal is output for sorting. This achieves flexible setting of the sorting standard and can meet the sorting needs in different scenarios.

[0018] (2) A high-voltage pulsed electric field is applied to the droplet sample through a high-voltage pulse power supply to perform dielectric sorting operations, and the precise dielectrophoretic force is calculated based on the pulse period, amplitude parameters, and frequency parameters, so as to adjust and apply precise electric field strength to the droplet sample to achieve efficient and accurate sorting.

[0019] (3) The overall area of ​​the sorting coordinate system is divided into multiple candidate areas through Cursors points, and at least one target area is determined in the candidate areas as the range of the sorting threshold, so as to realize flexible setting and sorting of the areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 Schematic diagram of the architecture of the high-performance cell sorting system provided by the present invention.

[0022] Figure 2 It is a schematic diagram of the setting of the control door provided by the present invention.

[0023] Figure 3 It is a schematic diagram of setting the custom threshold provided by the present invention.

[0024] Figure 4 A schematic diagram of the process of the high-performance cell sorting method provided by the present invention.

[0025] Figure 5 It is a schematic diagram of the detection results of the sample types and probe combinations provided by the present invention.

[0026] Figure 6 It is the detection result of the sorting parameter and threshold provided by the present invention.

[0027] Figure 7 This is one of the schematic diagrams of sorting statistical results provided by the present invention.

[0028] Figure 8 This is the second schematic diagram of the sorting statistical results provided by the present invention.

[0029] Fig. 9 This is the third schematic diagram of the sorting statistical results provided by the present invention. DETAILED DESCRIPTION

[0030] 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.

[0031] The high performance cell sorting system of the present invention is described below with reference to the accompanying drawings. Figure 1 Schematic diagram of the architecture of the high-performance cell sorting system provided by the present invention.

[0032] like Figure 1As shown, the high-performance cell sorting system includes: an optical acquisition module and a droplet sorting module. The optical acquisition module includes a data acquisition card, and the droplet sorting module includes a high-voltage pulse power supply, a sorting chip, and an FPGA chip.

[0033] The sorting chip is used to carry the droplet samples corresponding to the cells to be sorted. Here, before cell sorting, the target cells or viruses to be sorted are centrifuged and overlapped in an appropriate solution medium to prepare droplet samples, ensuring that the concentration and uniformity of the droplet samples are suitable for sorting. The target cells can be, for example, melanoma cells or viruses. Since cell sorting in the embodiment of the present invention is achieved by dielectric sorting. After preparing the droplet sample, the droplet sample is added to the sorting chip, and the sorting chip will be connected to the sorting electrode to ensure that the electrode is correctly connected to the sorting chip so that a high-voltage pulse electric field can be applied.

[0034] The FPGA chip is used to collect electrical pulse signals from the data acquisition card. A written computer program is deployed in the FPGA chip. A virtual sorting coordinate system is constructed through the computer program, and then the electrical pulse signals obtained from the data acquisition card are mapped one by one to the constructed sorting coordinate system. The FPGA chip is also used to determine that the electrical pulse signal reaches the sorting threshold when it is detected that the electrical pulse signal is within the range of the sorting threshold, wherein the sorting threshold is determined by one or more control gates, and the control gates are set according to the droplet sample size and the fluorescence signal intensity.

[0035] When the electric pulse signal reaches the sorting threshold, it triggers the output of the sorting signal. The high-voltage pulse power supply monitors the sorting signal and, after receiving the sorting signal, applies a high-voltage pulse electric field to the droplet sample on the sorting chip according to the sorting parameters in the FPGA chip to perform dielectric sorting operations.

[0036] The FPGA chip also calculates the dielectrophoretic force required for the high-voltage pulse power supply to perform dielectric sorting based on the pulse cycles, amplitude parameters, and frequency parameters. Specifically, the dielectrophoretic force required for the droplet samples in the sorting chip to perform dielectric sorting is calculated according to the following formula: (1) In the above formula (1), represents the dielectrophoretic force, is the radius of the droplet sample, is the complex relative dielectric constant of the cells to be sorted in the droplet sample, is the complex relative dielectric constant of the solution in the droplet sample, is the vacuum dielectric constant of the cells to be sorted in the droplet sample, f is the frequency parameter, is the amplitude parameter, v represents the application speed of the target electric field strength, and the application speed is positively correlated with the pulse period when the frequency parameter remains unchanged.

[0037] Specifically, in a high-performance cell sorting system, when the high-voltage pulse power supply receives a sorting signal, it indicates that the droplet sample needs to be dielectrically sorted. The high-voltage pulse power supply applies a high-voltage pulse electric field to the droplet sample on the sorting chip according to the sorting parameters in the FPGA chip to perform dielectric sorting operations. The sorting parameters specifically include pulse period, amplitude parameters, and frequency parameters, which determine the output characteristics of the high-voltage pulse power supply.

[0038] The FPGA chip will calculate the dielectrophoretic force that the high-voltage pulse power supply needs to achieve when performing dielectric sorting based on the pulse period, amplitude parameters, and frequency parameters. The high-voltage pulse power supply is also used to apply the corresponding high-voltage pulse electric field based on the calculated dielectrophoretic force to perform dielectric sorting on the droplet samples carried in the sorting chip.

[0039] The essential characteristic of dielectrophoresis is the relative movement of cells in the medium solution. The effect of dielectrophoresis comes from the interaction between the non-uniform electric field and the dipole moment. Due to the different inherent properties of the cells to be sorted and the properties of the continuous phase, when subjected to the same external electric field, the charge distribution of the cells to be sorted and the medium solution changes to different degrees. If the polarization phenomenon inside the cells to be sorted is more obvious, that is, the charge distribution inside the cells to be sorted is more different from the charge distribution outside the droplets, the electric field force on the cells to be sorted is greater, and the cells to be sorted move in the direction of large electric field strength (more dense electric field line density). This type of dielectrophoresis is called positive dielectrophoresis phenomenon (pDEP). If the polarization phenomenon of the medium solution is more obvious, that is, the charge distribution outside the cells to be sorted is more different from the charge distribution inside the cells, the cells to be sorted will move in the direction of smaller electric field strength (sparser electric field line density). This type of dielectrophoresis is called negative dielectrophoresis phenomenon (nDEP).

[0040] In the embodiment of the present invention, the droplet sample of the cells to be sorted satisfies the forward dielectrophoresis phenomenon, so the forward dielectrophoresis phenomenon can be used to calculate the dielectrophoretic force required for dielectrophoresis, thereby improving the cell sorting efficiency. Because when the dielectrophoretic force is small, the cells to be sorted cannot be fully deflected, and the sorting accuracy is affected; when the dielectrophoretic force is large, the cells to be sorted are deflected by the dielectrophoretic force, and the excessive dielectrophoretic force will cause the cells to be sorted to move to a position with high electric field strength, and at the same time, the cells to be sorted will be fragmented when the solution flows at a high speed.

[0041] The dielectrophoretic force is calculated by the FPGA chip according to the pulse period, amplitude parameter, and frequency parameter. The embodiment of the present invention proposes a dielectrophoretic force calculation formula shown in the above formula (1), and deploys this calculation formula into the computer program of the FPGA chip. The FPGA chip substitutes the pulse period, amplitude parameter, and frequency parameter into this calculation formula, thereby calculating the dielectrophoretic force required to be achieved when the droplet sample performs dielectric sorting.

[0042] In this embodiment, the relationship between the application speed, the frequency parameter and the pulse period can be expressed as: , is the pulse period. Combined with the above formula (1), the dielectrophoretic force is positively correlated with the square of the amplitude parameter and the frequency parameter. The larger the amplitude parameter or the frequency parameter, the greater the dielectrophoretic force. The pulse electric field is the ratio of the applied speed to the frequency parameter. When the frequency parameter remains unchanged, the applied speed is positively correlated with the pulse period. The larger the pulse period, the greater the applied speed, and the smaller the dielectrophoretic force. Therefore, when the frequency parameter and the amplitude parameter remain unchanged, the pulse period is negatively correlated with the dielectrophoretic force.

[0043] According to the formula and the above analysis, the pulse period, amplitude parameters, and frequency parameters can be reasonably controlled to adjust the dielectrophoretic force that the high-voltage pulse power supply needs to achieve when performing dielectric sorting. After the FPGA chip calculates the dielectrophoretic force, the high-voltage pulse power supply can adjust the high-voltage pulse electric field strength that needs to be applied according to this dielectrophoretic force, and apply a high-voltage pulse electric field to the droplet sample in the sorting chip, thereby achieving dielectric sorting.

[0044] In the embodiments of the present invention, on the one hand, one or more control gates are determined by the size of the droplet sample and the intensity of the fluorescence signal, thereby determining the sorting threshold of the droplet sample. In this way, when the electric pulse signal reaches the sorting threshold, a sorting signal is output for sorting, thereby achieving flexible setting of the sorting standard and being able to meet the sorting requirements in different scenarios. On the other hand, a high-voltage pulse power supply applies a high-voltage pulse electric field to the droplet sample to perform a dielectric sorting operation, and calculates an accurate dielectrophoretic force based on the pulse period, amplitude parameters, and frequency parameters, thereby adjusting and applying an accurate electric field strength to the droplet sample to achieve efficient and accurate sorting.

[0045] In some embodiments, Figure 1 As shown, the optical acquisition module of the high-performance cell sorting system also includes: a laser light source and a photomultiplier tube, the laser light source is used to emit laser to the droplet sample to stimulate the fluorescence signal of the droplet sample, the photomultiplier tube is used to collect the fluorescence signal, convert the fluorescence signal into an electrical pulse signal, and store the electrical pulse signal in a data acquisition card.

[0046] In order to ensure that only droplet samples that meet the conditions are selected for dielectric sorting, the embodiment of the present invention needs to set a corresponding sorting threshold to trigger the sorting process, and the sorting threshold needs to be determined by measuring the fluorescence signal intensity of the droplet sample. Here, the laser light source can be a laser, which emits a 488 nm wavelength laser to the droplet sample through the laser light source to stimulate the fluorescence signal of the droplet sample.

[0047] The photomultiplier tube is used to collect the fluorescence signal, convert the fluorescence signal into an electrical pulse signal, and store the electrical pulse signal in a data acquisition card.

[0048] Here, after receiving the fluorescence signal through the photomultiplier tube, the fluorescence signal is converted into an electrical pulse signal. In order to collect and store the data of the electrical pulse signal, the photomultiplier tube will store the electrical pulse signal in the data acquisition card for collection by the FPGA chip.

[0049] In an embodiment of the present invention, the fluorescence signal of the droplet sample is mapped into an electrical pulse signal and stored in a data acquisition card, and then the sorting threshold is determined based on the electrical pulse signal, and the fluorescence physical properties and electrical properties of the droplet sample are mapped to each other, thereby achieving accurate dielectric screening.

[0050] In some embodiments, the FPGA chip is also used to map the electrical pulse signals obtained from the data acquisition card to the constructed sorting coordinate system one by one. The FPGA chip is also used to determine that the electrical pulse signal reaches the sorting threshold when the electrical pulse signal is detected to be within the range of the sorting threshold.

[0051] As mentioned above, in the high-performance cell sorting system, the sorting process is triggered by outputting a sorting signal based on the sorting threshold determined in the FPGA chip. Therefore, the sorting threshold is the judgment standard for determining whether the droplet sample can be selected and sorted, and is set according to specific experimental requirements. To determine whether to trigger the sorting process, it is necessary to determine whether the droplet sample meets this judgment standard. Therefore, when the electric pulse signal of the droplet sample is stored in the data acquisition card, the FPGA chip will obtain the electric pulse signal from the data acquisition card, and then map the electric pulse signal one by one to the constructed sorting coordinate system. This sorting coordinate system is a virtual coordinate system constructed according to the computer program in the FPGA chip. The horizontal coordinate of the sorting coordinate system is the size of the droplet sample, and the vertical coordinate of the sorting coordinate system is the fluorescence signal intensity of the droplet sample. Then, according to the droplet sample size and the fluorescence signal intensity, the corresponding electric pulse signal is mapped one by one to this sorting coordinate system, and the sorting threshold is determined on the basis of this virtual sorting coordinate system. Therefore, in the sorting coordinate system, it is possible to simply and clearly determine whether the output pulse signal is within the range of the sorting threshold according to the determined sorting threshold.

[0052] After the FPGA chip maps the electrical pulse signals to the sorting coordinate system one by one, it will monitor the sorting coordinate system in real time to determine whether the electrical pulse signal reaches the set sorting threshold. That is, after mapping the electrical pulse signal of the droplet sample to the constructed sorting coordinate system, it will determine whether it is within the range of the sorting threshold.

[0053] When the electrical pulse signal reaches the set sorting threshold, the sorting signal is triggered. The sorting signal is a control signal that usually has specific timing and level requirements to ensure that the high-voltage pulse power supply can be accurately triggered.

[0054] When outputting the sorting signal, it is necessary to output the sorting signal according to the preset delay time parameters. In order to achieve precise control of the sorting signal, the embodiment of the present invention sets a delay trigger mechanism in the computer program of the FPGA chip. When the delay is triggered, it is necessary to determine the time delay parameter from the output of the sorting signal to the movement of the droplet sample to the dielectric sorting electrode area, which can ensure the accurate execution of the dielectric sorting operation. The time delay parameter can be preset, for example, based on the speed and distance of the droplet reaching the dielectric sorting electrode area, the time required for the dielectric sorting process, etc., or it can be comprehensively determined based on prior knowledge and exhaustive testing.

[0055] In the embodiment of the present invention, the electric pulse signal corresponding to the droplet sample is mapped to the virtual sorting coordinate system where the sorting threshold is located through the FPGA chip, so that it can accurately determine whether the droplet sample can be selected, thereby accurately triggering the sorting operation of the droplet sample.

[0056] In some embodiments, Figure 1 As shown, the droplet sorting module also includes a high-voltage amplifier, which is electrically connected to the high-voltage pulse power supply. The process of the high-voltage pulse power supply applying a high-voltage pulse electric field is as follows: a transistor-transistor logic (TTL) level signal is released to the high-voltage pulse power supply through the FPGA chip, so that the high-voltage pulse power supply generates a sinusoidal wave signal from the TTL level signal; then the sinusoidal wave signal is amplified and processed into an AC driving voltage of a preset amplitude through the high-voltage amplifier, and the AC driving voltage is input to the sorting chip to form a high-voltage pulse electric field.

[0057] Here, when the electric pulse signal corresponding to the droplet sample reaches the sorting threshold, a sorting signal is output. At this time, the high-voltage pulse power supply monitors the sorting signal, and then releases a TTL level signal to the high-voltage pulse power supply through the computer program in the FPGA chip, so that the high-voltage pulse power supply generates a sine wave signal from the TTL level signal. The sine wave signal is input to the high-voltage amplifier, and the high-voltage amplifier amplifies the sine wave signal into an AC drive voltage of a preset amplitude, and inputs the AC drive voltage to the sorting chip, thereby forming a corresponding high-voltage pulse electric field on the sorting chip. Of course, according to the different dielectrophoretic forces calculated by the sorting parameters, the computer program in the FPGA chip releases different TTL level signals to the high-voltage pulse power supply, which in turn makes the formed high-voltage pulse electric field different.

[0058] In an embodiment of the present invention, a high-voltage amplifier is used as an electrical signal medium between a high-voltage pulse power supply and a sorting chip, and amplifies the sinusoidal wave signal generated by the high-voltage pulse power supply into an AC driving voltage of a preset amplitude, thereby achieving electrical signal distinction of the sorting chip, completing the application of a high-voltage pulse electric field, and ensuring the effective execution of the dielectric sorting process.

[0059] In some embodiments, Figure 1 As shown, the droplet sorting module also includes a sorting droplet collector. The sorting droplet collector is used to collect the sorting droplets obtained after the droplet sample performs the dielectric sorting operation. After the droplet sample is subjected to the high-voltage pulse electric field in the dielectric sorting electrode area, the cells to be sorted are accurately sorted into the collection channel according to the principle of dielectric sorting, and then the sorting droplets after sorting are collected by the sorting droplet collector. Subsequently, the collected droplets are observed and counted using a microscope to verify the sorting results of the droplet samples, calculate the separation purity and separation efficiency, and accurately evaluate the performance of cell sorting.

[0060] The reason why a high-performance cell sorting system can realize a set of cell sorting processes is mainly because the various modules designed therein are coordinated and closely related to each other, and finally the sorted droplets are obtained from the droplet samples. Figure 1 As shown, in some embodiments, in a high-performance cell sorting system, the connection relationship between each module of the optical acquisition module and the droplet sorting module can be: the sorting chip is electrically connected to the laser light source and the photomultiplier tube, respectively, the data acquisition card is communicatively connected to the photomultiplier tube and the FPGA chip, respectively, the high-voltage pulse power supply is electrically connected to the FPGA chip and the sorting chip, and the sorting droplet collector is connected to the sorting chip through a droplet channel, which can be a pipe.

[0061] The embodiment of the present invention establishes a mutually coordinated and closely related relationship between the submodules of the high-performance cell sorting system according to the process steps of cell sorting, thereby realizing data transmission and signal propagation, which can effectively improve the efficiency of cell sorting.

[0062] In order to meet the needs of various scene sorting, the range of the sorting threshold can be determined by one or more control gates. Specifically, the range of the sorting threshold is determined according to the control gates set in the FPGA chip. The specific determination process is described below.

[0063] 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. 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.

[0064] 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.

[0065] like Figure 2 As shown in the figure, a control gate is set in the sorting coordinate system, and four sorting coordinate points are specifically set. The four sorting coordinate points are connected to form a quadrilateral area. The area of ​​this quadrilateral is the range of the sorting threshold. The droplet samples distributed inside the quadrilateral indicate that they have reached the sorting threshold, that is, the droplet samples selected for dielectric sorting.

[0066] Of course, there are certain standards for determining the sorting coordinate points. 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.

[0067] In some embodiments, the range of the sorting threshold can also be determined based on multiple control gates set in the FPGA chip, for example, 2 or 4 control gates are set. When sorting cells, there are situations where multiple sorting scene requirements need to be met at the same time, so the sorting threshold that needs to be set meets multiple sorting conditions, so it is necessary to set multiple control gates to divide the range of the sorting threshold.

[0068] In the constructed sorting coordinate system, the sorting coordinate points at multiple different coordinate positions in each control gate are connected to form a polygonal area range corresponding to the control gate, and then the overlapping area range formed by the polygonal area ranges corresponding to multiple control gates is determined as the range of the sorting threshold.

[0069] 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.

[0070] 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 meets the requirements of 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.

[0071] 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.

[0072] In the embodiment of the present invention, the electric pulse signal of the droplet sample is mapped to the sorting coordinate system through the FPGA chip, and then the droplet sample is divided according to the sorting threshold determined by setting one or more control gates based on the constructed sorting coordinate system, so as to determine whether the droplet sample is selected for sorting. By flexibly setting multiple control gates, the needs of different sorting scenarios can be met.

[0073] In some embodiments, the range of the sorting threshold may also be obtained based on a custom threshold set in the FPGA chip. The following describes a process in which the FPGA chip determines the range of the sorting threshold based on the custom threshold.

[0074] In the constructed sorting coordinate system, Cursors points are determined in the sorting coordinate system according to preset Cursors values, wherein the Cursors values ​​include droplet sample size parameters and droplet sample fluorescence signal intensity parameters; the overall area of ​​the sorting coordinate system is divided into multiple candidate areas according to the Cursors points, and at least one target area is determined in the candidate area as the range of the sorting threshold.

[0075] Specifically, first obtain the preset Cursors value, which includes the defined droplet sample size parameter and the defined droplet sample fluorescence signal intensity parameter. Next, map the obtained Cursors value to the sorting coordinate system. This sorting coordinate system is the same as the sorting coordinate system used by the control gate and will not be repeated. Then determine the Cursors point in the sorting coordinate system based on the droplet sample size parameter and the droplet sample fluorescence signal intensity parameter in the Cursors value. After the Cursors point is mapped to the sorting coordinate system, the overall area of ​​the sorting coordinate system is divided into multiple candidate areas according to the Cursors point. Finally, according to the actual sorting requirements, determine at least one target area from the candidate area as the range of the sorting threshold.

[0076] See also Figure 3 As shown, Figure 3 4 Cursor values ​​are preset in the 4-cursor coordinate system. Based on these 4 Cursor values, 4 Cursor points can be determined in the sorting coordinate system. First, based on two of the Cursor points ( Figure 3 The horizontal coordinate of the green coordinate point in the middle) divides the sorting coordinate system into three candidate areas, and then according to two of the Cursors points ( Figure 3 The three candidate areas are further divided into six candidate areas by using the vertical coordinate of the orange coordinate point in the middle. Then, according to the actual sorting requirements, at least one target area is selected from the six candidate areas as the range of the sorting threshold.

[0077] In the embodiment of the present invention, the candidate area is divided by the preset Cursors value, and the customization process of the sorting threshold is realized. Compared with one or more control gates, the range of the sorting threshold can be quickly formed without setting too many complicated sorting coordinate points. When the sorting requirements of the droplet sample are clear, the operation is directly based on the preset Cursors value, which can improve the efficiency of the sorting threshold division. In addition, compared with multiple control gates, the division process is simpler, more flexible, and more optional. When the sorting threshold changes, only the Cursors value needs to be adjusted, and there is no need to adjust multiple control gates one by one.

[0078] Considering that the sorting threshold is set according to the size of the droplet sample and the intensity of the fluorescence signal, but due to the dynamic characteristics of the droplet sample, the size of the droplet sample or the intensity of the fluorescence signal will change with the time delay, which will cause the droplet sample mapped on the sorting coordinate system to move deviations, and even move out of the sorting threshold range. In order to achieve more accurate sorting, it is necessary to ensure that the proportion of droplet samples within the sorting threshold range is within the appropriate sorting ratio. In other words, it is necessary to ensure that a sufficient number of droplet samples can be selected, and to avoid only a small number of droplet samples being selected.

[0079] In the embodiment of the present invention, the FPGA chip is also used to adaptively adjust the range of the sorting threshold. The following describes the process of adaptively adjusting the range of the sorting threshold by the FPGA chip.

[0080] The FPGA chip will calculate the proportion of the electric pulse signals of the droplet samples within the sorting threshold in real time and monitor the proportion. If the proportion is lower than the preset sorting ratio, the range of the sorting threshold will be automatically adjusted. If the proportion is not lower than the preset sorting ratio, the automatic adjustment will be stopped.

[0081] Specifically, first determine the preset sorting ratio of the droplet samples, for example 50%, which means that 50% of the droplet samples need to be selected for dielectric sorting. Next, through real-time calculation and monitoring, if the proportion of the number of electrical pulse signals of the droplet samples within the current sorting threshold is lower than the sorting ratio (50%), the range of the current sorting threshold is automatically adjusted. That is to say, if the current sorting threshold is determined, as the duration goes on, the proportion of the number of electrical pulse signals of the droplet samples within the sorting threshold range is lower than 50%, indicating that the sorting ratio is not met. At this time, the FPGA chip automatically adjusts the range of the current sorting threshold so that the proportion is adjusted to more than 50%.

[0082] If the proportion of the number of electrical pulse signals of the droplet sample is not lower than the sorting ratio, the automatic adjustment is stopped. In other words, when the number of electrical pulse signals of the droplet sample is not lower than the sorting ratio (50%), the sorting ratio is met, and the automatic adjustment is stopped.

[0083] In the embodiment of the present invention, by adapting to the dynamic characteristics of the droplet samples, a mechanism for automatically adjusting the sorting threshold is set in the computer program of the FPGA chip to select the droplet samples for dielectric sorting, thereby achieving more accurate sorting of the droplet samples and ensuring that the droplet samples for dielectric sorting strictly meet the required sorting ratio.

[0084] like Figure 4 As shown, an embodiment of the present invention further provides a high-performance cell sorting method, which is applied to the high-performance cell sorting system described above. The method specifically includes the following steps 101 to 103, which are described one by one below.

[0085] Step 101 : collecting the fluorescence signal of the droplet sample corresponding to the cells to be sorted, and converting the fluorescence signal into an electrical pulse signal.

[0086] Here, the process of collecting the fluorescence signal of the droplet sample corresponding to the cell to be sorted can refer to the collection process of the photomultiplier tube of the optical acquisition module in the above-mentioned high-performance cell sorting system. The conversion of the fluorescence signal into an electrical pulse signal can also be achieved by the photomultiplier tube, which will not be repeated here.

[0087] Step 102: When the electric pulse signal reaches a set sorting threshold, a sorting signal is output, wherein the sorting threshold is determined by one or more control gates, and the control gates are set according to the droplet sample size and the fluorescence signal intensity.

[0088] Here, the output process of the sorting signal can be realized by the FPGA chip of the droplet sorting module in the above-mentioned high-performance cell sorting system. The FPGA chip collects the electric pulse signal from the data acquisition card and maps it one by one to the constructed sorting coordinate system. When it is detected that the electric pulse signal is within the range of the sorting threshold, it is determined that the electric pulse signal reaches the sorting threshold. When the electric pulse signal reaches the set sorting threshold, the sorting signal is output. The specific implementation method is not repeated here.

[0089] Furthermore, the sorting threshold can be determined by one or more control gates, which are set according to the droplet sample size and the fluorescence signal intensity. The range of the sorting threshold is determined based on one or more control gates set in the FPGA chip.

[0090] In the constructed sorting coordinate system, the sorting coordinate points at multiple different coordinate positions in the preset control gate are connected to form one or more polygonal area ranges; The overlapping area of ​​one or more polygonal areas 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.

[0091] It should be noted that the process of determining the range of the sorting threshold by one or more control gates here and the process of determining the range of the sorting threshold by setting the control gate using the FPGA chip in the above-mentioned high-performance cell sorting system can correspond to each other and will not be repeated here.

[0092] Step 103, after receiving the sorting signal, applying a high-voltage pulse electric field to the droplet sample according to the sorting parameters, performing a dielectric sorting operation on the droplet sample through the high-voltage pulse electric field, and obtaining sorted droplets after sorting.

[0093] Here, after the sorting signal is triggered and monitored and received by the high-voltage pulse power supply, a high-voltage pulse electric field is applied to the droplet samples on the sorting chip through the sorting parameters in the FPGA chip, and a dielectric sorting operation is performed on the droplet samples through the high-voltage pulse electric field to obtain sorted droplets to perform dielectric sorting operations.

[0094] It can be understood that the high-performance cell sorting method in the embodiment of the present invention and the high-performance cell sorting system described above can refer to each other, and their beneficial effects can correspond to each other, so the beneficial effects of the high-performance cell sorting method will not be repeated here.

[0095] In order to prove that the high-performance cell sorting system and method of the present invention can achieve the above beneficial effects, an experiment is also set up in the embodiment of the present invention for verification, and a cell sorting experiment is performed by collecting virus samples, which is described in detail below.

[0096] In the experiment, the droplet samples to be sorted were sewage samples, and the target sorting object was another virus, Sequence 9, in the sewage sample. Its DNA was extracted from the sewage sample. After parsing the metagenome of the organism in the sewage, a target sequence group with a sequence length of 4297 bp was obtained, because Sequence 9 was likely a new type of bacteriophage.

[0097] In order to verify the effectiveness of the high-performance cell sorting system of the present invention for the whole genome sequencing method of target virus species (i.e., Sequence 9) from different sources, different droplet samples need to be constructed from the sample first, and two different populations in the sample are detected according to the fluorescence intensity, corresponding to empty droplets or droplets containing non-target virus genomes. Next, droplets containing target virus species are screened out and genomic DNA is amplified from these droplets. After a large number of MDA reactions, sufficient amounts of DNA are successfully generated for sequencing. Subsequently, starting from a single amplification product, the droplets are sequenced by probes and the read probe sequences (Reads) are assembled from scratch.

[0098] The probe sequence is: probe9 / 56FAM / rArUrUrGrGrUrUrCrC / ZEN / rCrArUrArAr CrUrUrGrCrGr CrGrG / 3IABkFQ / .

[0099] refer to Figure 5 , Figure 5 A schematic diagram of the detection results of sample types and probe combinations provided by the present invention, including the following combinations.

[0100] A, Probe 9 -MDA reagent; in this group, probe and MDA reagent were added, but there was no sewage sample and no fluorescent signal, such as Figure 5 As shown in A.

[0101] B, Probe 9 -MDA reagent + sewage; In this group, sewage samples, probes and MDA reagents were added to indicate that the system of probes and reagents was operating normally. Figure 5 As shown in B.

[0102] By setting different dilution multiples of the probe, various droplet samples are constructed as follows: 1. No sewage sample. In this group, probes and MDA reaction reagents were added, but no sewage sample was used to evaluate the performance of the probe in the absence of a sample, indicating that there was no background signal.

[0103] 2. 1X sewage sample. In this group, probes, MDA reaction reagents and 1X sewage sample were added, and some droplets were observed to show real fluorescence signals.

[0104] 3. 100X sewage sample. In this group, probes, MDA reaction reagents and 100X diluted sewage sample were added, and some droplets were observed to show real fluorescence signals, and the number of green fluorescent droplets was less than that in group 2.

[0105] 4. 1000X sewage sample. In this group, probes, MDA reaction reagents and 1000X diluted sewage sample were added, and some droplets were observed to show real fluorescence signals, and the number of green fluorescent droplets was less than that in group 3.

[0106] 5, 10 4 X sewage sample. In this group, probe, MDA reaction reagent and 10 dilution were added. 4 X sewage sample, with fewer green fluorescent droplets.

[0107] 6, 10 6X sewage sample. In this group, probe, MDA reaction reagent and 10 dilution were added. 6 In the X sewage sample, no green fluorescent droplets were observed.

[0108] 7. Crossphase probe. This group includes probe crossphase, MDA reaction reagent, and 1X sewage sample. crAssphage is a bacteriophage commonly found in human fecal samples. This group demonstrates the presence of crAssphage in sewage as a positive control.

[0109] 8. SV 40. In this group, probe SV 40, MDA reaction reagent, and 1X sewage sample were added. This group proved the presence of SV40 in sewage and served as a positive control.

[0110] Before sorting, the sewage was infinitely diluted to ensure that each droplet sample contained only one virus. The droplet sample encapsulated with the probe, MDA reagent and sewage sample was injected into the sorting chip, and the sorting experiment was carried out in the sorter. On the basis of setting the photomultiplier tube voltage to PMT 2 0.45, the delay parameter Delay time to 0 s, the cycle parameter Burst cycles to 25, the amplitude parameter Amplitude to 3.9V, and the frequency parameter Frequency to 25000 Hz, the droplet sample containing sequence 9 was sorted under the sorting conditions set by control gate 1 (Gate1) and control gate 2 (Gate2).

[0111] During the sorting process, under the same sorting parameter conditions, the range of the sorting threshold determined by control gate 1 and control gate 2 is narrowed to sort droplets with higher fluorescence intensity. Under the same sorting parameter conditions, the range of sorting control gate 1 and control gate 2 is further narrowed to sort droplets with higher fluorescence intensity. The schematic diagram of the sorting results can be seen in Figure 6 .

[0112] See also Figure 7 , Figure 7 This is one of the schematic diagrams of the sorting statistical results provided by the present invention. Figure 7 As shown, under the same conditions of other sorting parameters, by controlling different frequencies (i.e., the frequency parameters mentioned above) and the maximum photomultiplier tube voltage, when the majority of sorting operation events are triggered in all droplet samples to obtain a large number of sorted droplets, this is a schematic diagram of the comparison between all droplets and sorted droplets, where the majority can be greater than 50%.

[0113] See also Figure 8 , Figure 8 This is the second schematic diagram of the sorting statistical results provided by the present invention. Figure 8As shown, under the same conditions of other sorting parameters, by controlling different frequencies (i.e., the frequency parameters mentioned above) and the maximum photomultiplier tube voltage, partial sorting operation events are triggered in all droplet samples, and a comparison schematic diagram of all droplets and sorted droplets is obtained when partially sorted droplets are obtained, where the partial can be 10%.

[0114] refer to Fig. 9 , Fig. 9 This is the third schematic diagram of the sorting statistical results provided by the present invention. Fig. 9 As shown, under the same conditions of other sorting parameters, by controlling different frequencies (i.e., the frequency parameters mentioned above) and the maximum photomultiplier tube voltage, a small amount of sorting operation events are triggered in all droplet samples, and a schematic diagram of the comparison between all droplets and sorted droplets is obtained when a small amount of sorted droplets are obtained, where the small amount can be 1%.

[0115] The present invention also provides a non-transitory computer-readable storage medium having a computer program stored thereon. When the computer program is executed by a processor, the high-performance cell sorting method provided by the above methods is implemented. The method includes: collecting the fluorescence signal of the droplet sample corresponding to the cell to be sorted, and converting the fluorescence signal into an electrical pulse signal; when the electrical pulse signal reaches a set sorting threshold, outputting a sorting signal, wherein the sorting threshold is determined by one or more control gates, and the control gates are set according to the droplet sample size and the fluorescence signal intensity; the range of the sorting threshold is based on one or more control gates set in the FPGA chip. The method comprises: in a constructed sorting coordinate system, connecting sorting coordinate points at a plurality of different coordinate positions in a preset control gate to form one or more polygonal area ranges; using the overlapping area of ​​the one or more polygonal area ranges as the range of the sorting threshold determined by the control gate, wherein the horizontal coordinate of the sorting coordinate system is the size of the droplet sample, and the vertical coordinate of the sorting coordinate system is the fluorescence signal intensity of the droplet sample; after receiving the sorting signal, applying a high-voltage pulse electric field to the droplet sample according to the sorting parameters, performing a dielectric sorting operation on the droplet sample through the high-voltage pulse electric field, and obtaining sorted droplets after sorting.

[0116] The present invention also provides a computer program product, which includes a computer program. The computer program can be stored in a non-transitory computer-readable storage medium. When the computer program is executed by a processor, the computer can execute the high-performance cell sorting method provided by the above methods, which includes: collecting the fluorescence signal of the droplet sample corresponding to the cell to be sorted, and converting the fluorescence signal into an electric pulse signal; when the electric pulse signal reaches a set sorting threshold, outputting a sorting signal, wherein the sorting threshold is determined by one or more control gates, and the control gate is set according to the droplet sample size and the fluorescence signal intensity; the range of the sorting threshold is based on FP The method is determined by one or more control gates set in the GA chip, including: in a constructed sorting coordinate system, connecting sorting coordinate points at multiple different coordinate positions in a preset control gate to form one or more polygonal area ranges; using the overlapping area of ​​the one or more polygonal area ranges as the range of the sorting threshold determined by the control gate, wherein the horizontal coordinate of the sorting coordinate system is the droplet sample size, and the vertical coordinate of the sorting coordinate system is the fluorescence signal intensity of the droplet sample; after receiving the sorting signal, applying a high-voltage pulse electric field to the droplet sample according to the sorting parameters, performing a dielectric sorting operation on the droplet sample through the high-voltage pulse electric field, and obtaining sorted droplets after sorting.

[0117] 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.

[0118] 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.

[0119] 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 high-performance cell sorting system, comprising: An optical acquisition module and a droplet sorting module; wherein the optical acquisition module includes a data acquisition card, and the droplet sorting module includes a high-voltage pulse power supply, a sorting chip, and an FPGA chip; The FPGA chip is used to collect the electric pulse signal from the data acquisition card, and output a sorting signal when the electric pulse signal reaches a sorting threshold, wherein the sorting threshold is determined by one or more control gates, and the control gates are set according to the droplet sample size and the fluorescence signal intensity; The FPGA chip calculates the dielectrophoretic force required to be achieved when the high-voltage pulse power supply performs dielectric sorting according to the pulse period, amplitude parameter, and frequency parameter, including: The dielectrophoretic force required for the droplet sample in the sorting chip to perform dielectric sorting is calculated according to the following formula: in, represents the dielectrophoretic force, is the radius of the droplet sample, is the complex relative dielectric constant of the cells to be sorted in the droplet sample, is the complex relative dielectric constant of the solution in the droplet sample, is the vacuum dielectric constant of the cells to be sorted in the droplet sample, f is the frequency parameter, is the amplitude parameter, v represents the application speed of the target electric field strength, and the application speed is positively correlated with the pulse period when the frequency parameter remains unchanged.

2. The high performance cell sorting system according to claim 1, characterized in that: The range of the sorting threshold is determined according to the control gate set in the FPGA chip. The process of the FPGA 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.

3. The high performance cell sorting system according to claim 1, characterized in that: The range of the sorting threshold is determined based on a plurality of control gates set in the FPGA chip. The FPGA 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.

4. The high performance cell sorting system according to claim 1, characterized in that: The range of the sorting threshold is obtained according to a custom threshold set in the FPGA chip, and the FPGA chip determines the range of the sorting threshold according to the custom threshold, including: In the constructed sorting coordinate system, Cursors points are determined in the sorting coordinate system according to preset Cursors values, wherein the Cursors values ​​include droplet sample size parameters and droplet sample fluorescence signal intensity parameters; The entire area of ​​the sorting coordinate system is divided into a plurality of candidate areas according to the Cursors points, and at least one target area is determined in the candidate areas as the range of the sorting threshold.

5. The high performance cell sorting system according to claim 1, characterized in that: The FPGA chip is also used to adaptively adjust the range of the sorting threshold. The process of the FPGA chip adaptively adjusting the range of the sorting threshold includes: Calculating the proportion of the electric pulse signals of the droplet samples within the sorting threshold, and monitoring the proportion; If it is detected that the proportion of the quantity is lower than the preset sorting ratio, the range of the sorting threshold is automatically adjusted; If it is detected that the proportion of the quantity is not lower than the preset sorting ratio, the automatic adjustment is stopped.

6. The high performance cell sorting system according to claim 1, characterized in that: The FPGA chip is also used to map the electric pulse signals obtained from the data acquisition card to the constructed sorting coordinate system one by one. The FPGA chip is also used to determine that the electric pulse signal reaches the sorting threshold when the electric pulse signal is detected to be within the range of the sorting threshold.

7. The high performance cell sorting system according to claim 1, characterized in that: The optical acquisition module also includes a laser light source and a photomultiplier tube. The laser light source is used to emit laser to the droplet sample to excite the fluorescence signal of the droplet sample; the photomultiplier tube is used to collect the fluorescence signal, convert the fluorescence signal into an electrical pulse signal, and store the electrical pulse signal in the data acquisition card.

8. The high performance cell sorting system according to claim 7, characterized in that: The droplet sorting module further includes a sorting droplet collector, which is used to collect sorting droplets obtained after the droplet sample is subjected to a dielectric sorting operation.

9. The high performance cell sorting system according to claim 8, characterized in that: The sorting chip is electrically connected to the laser light source and the photoelectric backup tube, respectively; the data acquisition card is communicatively connected to the photoelectric backup tube and the FPGA chip, respectively; the high-voltage pulse power supply is electrically connected to the FPGA chip and the sorting chip, respectively; and the sorting droplet collector is connected to the sorting chip through a droplet channel.

10. The high performance cell sorting system according to claim 1, characterized in that: The droplet sorting module further comprises a high-voltage amplifier, which is electrically connected to the high-voltage pulse power supply; The high-voltage pulse power supply is also used to apply a corresponding high-voltage pulse electric field according to the dielectrophoretic force. The process of applying the high-voltage pulse electric field by the high-voltage pulse power supply includes: Release a TTL level signal to the high-voltage pulse power supply through the FPGA chip, so that the high-voltage pulse power supply generates a sine wave signal from the TTL level signal; The sinusoidal wave signal is amplified and processed into an AC driving voltage of a preset amplitude by the high-voltage amplifier, and the AC driving voltage is input to the sorting chip to form a high-voltage pulse electric field.

11. A high-performance cell sorting method, characterized in that: The method comprises: Collecting the fluorescence signal of the droplet sample corresponding to the cells to be sorted, and converting the fluorescence signal into an electrical pulse signal; When the electric pulse signal reaches the set sorting threshold, a sorting signal is output, wherein the sorting threshold is determined by one or more control gates, and the control gates are set according to the droplet sample size and the fluorescence signal intensity; the range of the sorting threshold is determined according to one or more control gates set in the FPGA chip, including: In the constructed sorting coordinate system, the sorting coordinate points at multiple different coordinate positions in the preset control gate are connected to form one or more polygonal area ranges; Using the overlapping area of ​​the one or more polygonal areas as the range of the sorting threshold determined by the control gate, wherein the abscissa of the sorting coordinate system is the size of the droplet sample, and the ordinate of the sorting coordinate system is the fluorescence signal intensity of the droplet sample; After receiving the sorting signal, a high-voltage pulse electric field is applied to the droplet sample according to the sorting parameters, and a dielectric sorting operation is performed on the droplet sample by the high-voltage pulse electric field to obtain sorted droplets.

12. A non-transitory computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by a processor, the high-performance cell sorting method according to claim 11 is implemented.

13. A computer program product, comprising a computer program, characterized in that When the computer program is executed by a processor, the high-performance cell sorting method according to claim 11 is implemented.

Images