Droplet quality control method and device based on laser detection, storage medium and program product

Abstract

The invention discloses a droplet quality control method and equipment based on laser detection, a storage medium and a program product, and the method comprises the steps: obtaining droplet micro-channel signal data collected by a laser device, and monitoring the state of a droplet in a droplet micro-channel based on the droplet micro-channel signal data; wherein the droplet micro-channel comprises single micro-channels and / or a main micro-channel, droplets in the droplet micro-channel comprise droplets in the single micro-channels and / or fused droplets in the main micro-channel, and the droplet micro-channel signal data comprise single micro-channel signal data and / or main micro-channel signal data. After the solution corresponding to each single micro-channel forms liquid drops in each single micro-channel in the corresponding single micro-channel, the liquid drops in each single micro-channel flow to the main micro-channel and are fused in the main micro-channel to form fused liquid drops.

Description

Technical Field

[0001] The present invention relates to the field of gene technology, and in particular to a droplet quality control method, equipment, storage medium and program product based on laser detection. Background Art

[0002] As an important bioanalysis tool, droplet microfluidics has been widely used in single-cell analysis, digital polymerase chain reaction (PCR), immunoassay and other fields. By encapsulating single cells or reaction components in tiny oil droplets, droplet technology provides unprecedented possibilities for high-throughput parallel processing. However, with the deepening of life science research and the increase in application needs, simple droplet encapsulation technology has gradually shown its limitations and is difficult to meet the increasingly complex experimental needs. It is mainly reflected in: (1) The increase in the demand for complex reactions: The development of modern biotechnology requires not only the encapsulation of samples in droplets for simple independent reactions, but also more complex operations, such as fusion, separation, content exchange and multi-step reaction chains between droplets. These operations require precise manipulation inside the droplets to achieve more complex and sophisticated experimental designs. (2) High throughput and diversity: In large-scale biological analysis and drug screening, not only high throughput is required, but also content exchange and mixing between multiple droplets are required to simulate complex biological reaction environments. This demand places higher demands on droplet technology, which needs to be able to process and fuse a large number of droplets with different components in a short period of time and ensure the consistency and reliability of each operation. (3) Multi-omics joint analysis: In single-cell multi-omics analysis, such as the joint analysis of transcriptome, proteome and metabolome, different contents from the same cell source need to be extracted, reacted and analyzed separately. This demand requires droplet technology to flexibly separate and recombine the contents to ensure the integrity and relevance of the data.

[0003] Therefore, the droplet fusion technology in the existing technology faces many challenges: Unstable fusion success rate: Instability in the droplet fusion process is still a major problem. If the fusion efficiency is low or the fusion is incomplete, it will lead to sample loss and data deviation, affecting the reliability of the experimental results. Lack of real-time monitoring means: At present, the success of droplet fusion often depends on post-analysis, and there is a lack of effective real-time monitoring means to ensure the success of the fusion process. In this case, errors in the experiment are difficult to be discovered and corrected in time, increasing the failure rate of the experiment. Complex operating conditions: To achieve efficient droplet fusion, there are precise requirements for operating conditions such as droplet generation frequency, fusion electric field strength, droplet position and size. The control and optimization of these conditions is a major obstacle to the commercialization of droplet fusion technology. Summary of the invention

[0004] In order to solve the existing technical problems, the embodiments of the present invention provide a droplet quality control method based on laser detection, a droplet quality control device based on laser detection, a single-cell sequencing library construction device, a computer-readable storage medium and a computer program product, which can monitor the abnormal status of the entire process of droplet generation, pairing and fusion in real time, so as to facilitate maintenance personnel to promptly discover and eliminate abnormalities.

[0005] In a first aspect, a droplet quality control method based on laser detection is provided, comprising: acquiring droplet microfluidic channel signal data collected by a laser device, and monitoring the state of droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data; wherein the droplet microfluidic channel includes individual single microfluidic channels and / or a main microfluidic channel, and the droplets in the droplet microfluidic channel include droplets in individual single microfluidic channels and / or fused droplets in the main microfluidic channel, and the droplet microfluidic channel signal data includes single microfluidic channel signal data and / or main microfluidic channel signal data, and after the solutions corresponding to individual single microfluidic channels form droplets in individual single microfluidic channels in their respective corresponding single microfluidic channels, the droplets in each single microfluidic channel flow to the main microfluidic channel and merge in the main microfluidic channel to form fused droplets.

[0006] In a second aspect, a droplet quality control device based on laser detection is provided, comprising a memory and a controller, wherein the memory stores a computer program, and when the computer program is executed by the controller, the controller executes the steps of the droplet quality control method based on laser detection provided in an embodiment of the present application.

[0007] In a third aspect, a single-cell sequencing library construction device is provided, including a droplet quality control device based on laser detection provided in an embodiment of the present application.

[0008] In a fourth aspect, a computer-readable storage medium is provided, wherein a computer program is stored on the computer-readable storage medium, and the computer program is used by a controller to execute the steps of a droplet quality control method based on laser detection.

[0009] In a fifth aspect, a computer program product comprises a computer program, which, when executed by a controller, implements the steps of the droplet quality control method based on laser detection as claimed in claim 1.

[0010] The embodiment of the present application uses a laser device to collect real-time droplet microfluidic channel signal data during the droplet monitoring process. The droplet microfluidic channel signal data includes at least one of the following: single microfluidic channel signal data and main microfluidic channel signal data. By analyzing the single microfluidic channel signal data and / or the main microfluidic channel signal data, the droplet conditions of each single microfluidic channel and / or the fused droplets generated in the main microfluidic channel can be monitored, so that the abnormal state of the entire process of droplet generation, pairing, and fusion can be monitored in real time, which is convenient for maintenance personnel to discover abnormalities in time and eliminate abnormalities. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a schematic block diagram of a droplet quality control device based on laser detection in one embodiment;

[0012] Figure 2 Schematic diagram of a microfluidic channel for generating droplets in a droplet quality control device based on laser detection in one embodiment;

[0013] Figure 3 A schematic diagram of droplet fusion in a droplet quality control device based on laser detection in one embodiment;

[0014] Figure 4 is a flow chart of a droplet quality control method based on laser detection in one embodiment;

[0015] Figure 5 is a schematic diagram of single microfluidic channel signal data in one embodiment;

[0016] Figure 6 is a schematic diagram of microfluidic channel signal data corresponding to a standard object in one embodiment;

[0017] Figure 7 is a schematic diagram of a pulse signal of a fused droplet containing a target object in one embodiment;

[0018] Figure 8 is a schematic diagram of a droplet quality control device based on laser detection in one embodiment;

[0019] Fig. 9 Schematic diagram of the structure of a droplet quality control device based on laser detection in one embodiment. DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the technical field of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0022] In the following description, reference is made to “some embodiments”, which describes a subset of all possible embodiments, but it should be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0023] Microfluidics refers to a technology that manipulates fluids in micrometer-scale space. This technology can miniaturize the basic functions of chemistry, biology and other laboratories onto a chip of a few square centimeters, so it is also called a chip laboratory (Lab-on-a-chip). Microfluidic chip systems can manipulate the flow of fluids in tiny channels or components ranging from tens to hundreds of micrometers, and the volume of fluids manipulated can be as small as 10 -9 ~10 -18 L. It integrates the basic operations of biochemical experiments such as sample reaction, preparation, separation, and detection into a very small chip, and uses controllable fluids to form a network of microchannels throughout the microfluidic system. While realizing the functions of conventional biochemical laboratories, it reduces the cost of analysis and detection, speeds up the reaction speed, improves the reaction efficiency, and makes the experiment more controllable.

[0024] The generation of droplets on a microfluidic chip is a process in which one phase of fluid is dispersed in another phase of immiscible or partially miscible fluid. One of the two immiscible liquids is used as the continuous phase and the other as the dispersed phase. The dispersed phase is dispersed in a tiny volume (10 -15 ~10 -9 The L) unit is dispersed in the continuous phase to form droplets. At present, the methods for forming droplets can be divided into two categories: passive method and active method. The passive method refers to controlling the generation of droplets by controlling the microchannel structure and the two-phase flow rate ratio, while the active method generally drives and controls the generation of droplets by external force.

[0025] After droplet generation, the cells or nuclei in each droplet release their nucleic acids (DNA or RNA) in preparation for amplification, which is performed using specific amplification techniques such as multiple displacement amplification (MDA) or PCR amplification by droplets.

[0026] See also Figure 1, is a schematic block diagram of a droplet quality control device based on laser detection in one embodiment. The droplet quality control device based on laser detection includes but is not limited to the following devices or components: a controller 10, a gas generating device 11, a regulating device 12, a laser device 13, a pressure detecting device 14, a gas impurity removal device 15, a pressure output device 16 and a display device 17. The controller 10 is connected to each of the other devices respectively. As the core of the droplet quality control device based on laser detection, the controller is responsible for coordinating and managing the functions of each component. The controller receives data from each device, processes and analyzes the data of each device, and generates different commands to control each device. The gas generating device 11 is used to provide stable and adjustable air pressure for use in the entire experimental process. The air pressure generating device 11 is a key device for maintaining the pressure environment required for the experiment to ensure that the experimental conditions meet the preset standards. The gas impurity removal device 15 is used to remove moisture from the gas to prevent water vapor from affecting the experimental accuracy and equipment performance. Dry gas helps maintain the stability of the system and extend the service life of the equipment. The air pressure generated by the air pressure generating device 11 is output to each single microchannel through the pressure output device 16 to maintain the pressure in each single microchannel. The pressure detection device 14 is used to detect the pressure level of each single microchannel in real time. The pressure detection device 14 can accurately measure and record the slight changes in the pressure in each single microchannel, providing key data for the experiment. The regulating device 12 is used to control the rate and amount of gas flowing into each single microchannel to ensure the uniformity and stability of the experimental conditions. The regulation of the regulating device 12 can be automatically adjusted according to the pressure data fed back by the pressure detection device 14 to respond to the changes in demand during the experiment. The regulating device 12 is connected to the air pressure generating device 11. The laser device 13 is used to project a laser signal to the detection area of ​​the droplet microchannel. The laser signal encounters the droplet microchannel to form a detection signal, thereby obtaining the droplet microchannel signal data, wherein the detection signal includes but is not limited to the signal formed by the reflection and / or refraction of the laser signal encountering the droplet microchannel. The detection signal during the droplet monitoring process can be captured by the laser device 13, so as to facilitate the analysis of whether there is a fault in the droplet monitoring process or whether the generated droplets are qualified according to the signal during the droplet monitoring process. The display device 17 may be a display component integrated in the droplet quality control device based on laser detection, or an external display device connected to the droplet quality control device based on laser detection, etc. The display device 17 is used to display the display data sent by the controller 10 for intuitive viewing by the user.

[0027] In an optional implementation, at least one laser device 13 can be configured for each single microchannel, so that the microchannel signal data corresponding to each single microchannel can be collected. For the main microchannel, at least one laser device 13 can also be set to collect the main microchannel signal data corresponding to the main microchannel. It can be understood that the laser device 13 used for the single microchannel and the laser device 13 used in the main microchannel can be configured at the same time, or only one of them can be configured separately. Each laser device 13 corresponds to a laser detection area, and when the droplet passes through the laser detection area, the signal data in the droplet microchannel at this time can be collected.

[0028] In some embodiments, the droplet quality control device based on laser detection may also include an image acquisition device, which is used to acquire real-time droplet microfluidic channel images during the droplet monitoring process, and transmit the captured images and / or videos to the display device 17 in real time. The image acquisition device 13 can capture detailed images of each moment in the droplet monitoring process, so as to facilitate the analysis of whether there is a fault in the droplet monitoring process or whether the generated droplets are qualified according to the images in the droplet monitoring process. The image acquisition device may include but is not limited to the following components: a high frame rate camera, a collimating lens, a fill light, etc., wherein the high frame rate camera has high definition and high-speed image capture capabilities to capture rapidly changing experimental conditions. The collimating lens is used to focus and guide light to accurately irradiate the photosensitive element to ensure image quality. The fill light is used to provide illumination in a weak light environment, so that a clear image can be obtained in a weak environment. The image acquisition device may be one or more. The adjustment device 12 may be one or more proportional valves.

[0029] In some embodiments, the droplet quality control equipment based on laser detection may also include a fluorescence detection device, the position of which corresponds to the detection area in the main microfluidic channel, and is used to detect whether a target substance exists in the melt droplets in the main microfluidic channel. The target substance is a pre-fluorescently labeled substance, such as nucleic acid, etc.

[0030] Figure 2 is a schematic diagram of a microfluidic channel for generating droplets in a droplet quality control device based on laser detection in one embodiment, and Figure 3 Schematic diagram of droplet fusion in a droplet quality control device based on laser detection in an embodiment. The schematic diagram shown in this embodiment includes two single microchannels, a first single microchannel and a second single microchannel. The air pressure output by the pressure output device 16 maintains the pressure of the two single microchannels. Under the action of pressure, different solutions in each single microchannel flow in their respective channels and form droplets in their respective single microchannels. The droplets in each single microchannel flow to the main microchannel and merge with each other in the main microchannel to form fused droplets. The fused droplets are as shown in FIG. Figure 3 shown.

[0031] See also Figure 4 , is a flow chart of a droplet quality control method based on laser detection provided in an embodiment of the present application. The droplet quality control method based on laser detection is applied to a gene sequencer, and the droplet quality control method based on laser detection includes the following steps:

[0032] S11, acquiring droplet microfluidic channel signal data collected by a laser device.

[0033] In this embodiment, the laser device can be used to detect a single microchannel or a main microchannel, so the droplet microchannel includes each single microchannel and / or the main microchannel. The laser device includes a transmitting device and a receiving device, the transmitting device is used to send a laser signal to the laser detection area, and the receiving device receives the signal detected in the laser detection area. Since there are droplets flowing in the droplet microchannel, the size, spacing, flow rate and other data of the droplets will affect the received detection signal. For the laser device used for a single microchannel, the signal collected is the single microchannel signal data; for the laser device used for the main microchannel, the signal collected is the main microchannel signal data. The droplet microchannel signal data includes the single microchannel signal data and / or the main microchannel signal data. If the droplet microchannel includes a single microchannel, the droplets in the droplet microchannel include the droplets in the single microchannel; if the droplet microchannel includes a main microchannel, the droplets in the droplet microchannel include the fused droplets in the single microchannel. The droplet microfluidic channel signal data may be signal data collected over a period of time, or may be signal data collected once.

[0034] S12. Monitoring the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data.

[0035] In this embodiment, the laser device 13 can collect real-time droplet microfluidic channel signal data in the droplet monitoring process in real time, and can analyze the collected droplet microfluidic channel signal data to monitor the droplet state. The droplet state includes at least one of the following: the droplet conditions in each single microfluidic channel, the fused droplet conditions in the main microfluidic channel, and the generation frequency ratio of each single microfluidic channel. The droplet conditions include but are not limited to droplet size data, droplet spacing, and droplet flow rate. The fused droplet conditions include but are not limited to any one of the following: droplet fusion rate, fused droplet size data, fused droplet spacing, fused droplet flow rate, fusion rate of fused droplets, fusion droplet encapsulation rate, whether the fused droplets have target substances, and the successful fusion rate of fused droplets. Whether there are abnormal problems in the monitoring droplet monitoring process is determined by whether the droplet conditions of each single microfluidic channel and / or the fused droplet conditions generated in the main microfluidic channel meet the preset conditions.

[0036] In some embodiments, when the droplet conditions passing through each single microchannel and / or the fused droplet conditions generated in the main microchannel do not meet the preset conditions, it is determined that the state of the droplets in the droplet microchannel is abnormal, and an early warning prompt is executed. In an optional implementation, the method of executing the early warning prompt includes but is not limited to one or more of the following combinations: executing an abnormal prompt on the user interface, sending an abnormal prompt to the terminal device of the relevant personnel. When executing the abnormal prompt on the user interface, an image, pressure data, etc. corresponding to the abnormal situation can be displayed.

[0037] In the above embodiment, the real-time droplet microfluidic channel signal data in the droplet monitoring process is collected in real time by a laser device, and the droplet microfluidic channel signal data includes at least one of the following: single microfluidic channel signal data, main microfluidic channel signal data. By analyzing the single microfluidic channel signal data and / or the main microfluidic channel signal data, the droplet conditions of each single microfluidic channel and / or the fusion droplet conditions generated in the main microfluidic channel can be monitored, so that the abnormal state of the entire process of droplet generation, pairing, and fusion can be monitored in real time, which is convenient for maintenance personnel to discover abnormalities in time and eliminate abnormalities.

[0038] In some embodiments, the state of the droplets in the droplet microfluidic channel includes at least one or more of the following combinations: the generation frequency of droplets in each single microfluidic channel, the relationship between the current droplet generation frequencies corresponding to all single microfluidic channels, the size data of droplets in the droplet microfluidic channel, the droplet spacing in the droplet microfluidic channel, the flow rate of droplets in the droplet microfluidic channel, the fusion rate of droplets in the main microfluidic channel, whether there are target objects in the fused droplets in the main microfluidic channel, the wrapping rate of the fused droplets in the main microfluidic channel, whether the fused droplets in the main microfluidic channel are successfully fused droplets, the successful fusion rate of fused droplets in the main microfluidic channel, and the content data of the target substance in the successfully fused droplets.

[0039] In this embodiment, the current droplet generation frequency relationship can be expressed by the ratio of the generation frequencies of all single microchannels, where the generation frequency indicates the generation speed of the droplets; the fusion rate indicates the proportion of droplets in each single microchannel that successfully fuse together to form fused droplets; the encapsulation rate indicates the proportion of fused droplets containing the target object in the fused droplets; the successfully fused droplets indicate the presence of fluorescently labeled target substances in the fused droplets; and the successful fusion rate indicates the proportion of successfully fused droplets in the fused droplets. The target can be a specified substance or a carrier carrying the specified substance. The specified substance includes, but is not limited to, one or more of the following combinations of nucleic acids, proteins, carbohydrates, lipids, or other organic or inorganic solvents. For example, the target is a gel ball with a specified DNA tag.

[0040] In the above embodiment, the process of droplet generation, pairing, and fusion is monitored from multiple angles, and abnormalities in the monitoring process are discovered in time to ensure the success rate of droplet fusion.

[0041] In some embodiments, monitoring the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data includes:

[0042] Based on the signal data of each single microchannel, the pulse signal interval corresponding to each single microchannel is obtained, and based on the pulse signal interval corresponding to each single microchannel, the generation frequency of droplets in each single microchannel is calculated;

[0043] According to the generation frequency of droplets in each single microfluidic channel, the current droplet generation frequency relationship corresponding to all single microfluidic channels is calculated;

[0044] Based on the current droplet generation frequency relationship, the pressure data of each single microfluidic channel is controlled.

[0045] In this embodiment, for a single microchannel, in the laser detection area of ​​the laser device, when a droplet passes through the single microchannel, a pulse signal will exist in the collected single microchannel signal data. One droplet corresponds to one pulse signal, such as Figure 5 As shown, Figure 5 FIG. 1 is a schematic diagram of single microchannel signal data in an embodiment. In a single microchannel, when two adjacent droplets pass through the laser detection area, single microchannel signal data is formed. Each droplet corresponds to a pulse signal, such as Figure 5 The T shown is a pulse signal interval, and the pulse signal interval indicates the time interval between two adjacent pulse signals. In an optional implementation, for a single microchannel, multiple pulse signal intervals are obtained, and the average value of the pulse signal interval is calculated based on the multiple pulse signal intervals, and the average value of the pulse signal interval is used as the pulse signal interval corresponding to the single microchannel. The generation frequency of droplets in a single microchannel = 1 / pulse signal interval corresponding to a single microchannel. For other single microchannels, the above method can also be used to calculate, so as to obtain the generation frequency of droplets in each single microchannel. The generation frequency indicates the generation speed of droplets.

[0046] The current droplet generation frequency relationship represents the ratio of the generation frequencies of all single microchannels. For example, in the case of two single microchannels, the current droplet generation frequency relationship is the ratio of the generation frequency of the first single microchannel to the generation frequency of the second single microchannel. The current droplet generation frequency relationship is related to the droplet fusion rate. If the droplet generation frequency of a single microchannel is faster or slower, it will cause waste of reagents and reduce the droplet fusion rate. Therefore, the current droplet generation frequency relationship is compared with the preset generation frequency ratio to determine whether the current droplet generation frequency relationship meets the preset generation frequency conditions, thereby controlling the pressure data of each single microchannel.

[0047] In an optional implementation, the controlling of the pressure data of each single microchannel based on the current droplet generation frequency relationship includes at least one of the following:

[0048] Compare the current droplet generation frequency relationship with a preset generation frequency ratio, obtain a single microchannel with a generation frequency lower than a first generation frequency value, and increase the pressure data of the single microchannel with a generation frequency lower than the first generation frequency value;

[0049] The current droplet generation frequency relationship is compared with a preset generation frequency ratio, a single microchannel having a generation frequency higher than a second generation frequency value is obtained, and pressure data of the single microchannel having a generation frequency higher than the second generation frequency value is reduced.

[0050] In this embodiment, the first generation frequency value and the second generation frequency value are calculated based on a preset generation frequency ratio, and a single microfluidic channel with a generation frequency lower than the first generation frequency value or a single microfluidic channel with a generation frequency higher than the second generation frequency value is obtained, and the pressure data of these single microfluidic channels are adjusted, so that the generation frequency of these single microfluidic channels can be adjusted, so that the current droplet generation frequency relationship is within the error range of the preset generation frequency ratio, thereby ensuring the efficiency of droplet fusion.

[0051] In the above embodiment, the pulse signal interval corresponding to each single microchannel is obtained, and based on the pulse signal interval corresponding to each single microchannel, the generation frequency of droplets in each single microchannel is calculated, and the current droplet generation frequency relationship corresponding to all single microchannels is calculated, and the current droplet generation frequency relationship is compared with the preset generation frequency ratio. When the current droplet generation frequency relationship does not meet the preset generation frequency condition, a single microchannel with a generation frequency lower than the first generation frequency value is obtained or a single microchannel with a generation frequency higher than the second generation frequency value is obtained, and the pressure data of these single microchannels are adjusted, so that the generation frequency of these single microchannels can be adjusted so that the current droplet generation frequency relationship is within the error range of the preset generation frequency ratio. This can ensure the success rate of droplet fusion and reduce the waste of reagents.

[0052] In some embodiments, before monitoring the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data, the method further includes:

[0053] A standard pulse width is obtained, where the standard pulse width is obtained by placing a standard object with a preset diameter in the droplet microchannel and flowing the standard pulse width according to a received signal of the laser device.

[0054] In this embodiment, the standard object may be a standard rubber ball, which is placed in the droplet microfluidic channel to flow, so that when the standard object passes through the laser detection area of ​​the laser device, the corresponding microfluidic channel signal data can be obtained. Figure 6 As shown, Figure 6Schematic diagram of microfluidic signal data corresponding to a standard object in an embodiment, a pulse signal corresponding to a standard rubber ball collected twice in a row, a pulse width is represented by T4. In an optional implementation, multiple pulse signals can be extracted from the microfluidic signal data, and the pulse width of each pulse signal can be calculated, and the average value of the pulse width of each pulse signal is accumulated as the standard pulse width. Since the standard object has a preset diameter, the interval between droplets, size data, etc. can be estimated based on the standard pulse width. The standard pulse width indicates the average width of the pulse signal corresponding to the standard object.

[0055] In the above embodiment, by acquiring the standard pulse width corresponding to the standard object with a preset diameter, the relevant data of the droplets can be accurately estimated subsequently, so as to accurately monitor the status of the droplets during monitoring and detect abnormalities in time.

[0056] In some embodiments, monitoring the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data includes:

[0057] Based on the droplet microfluidic channel signal data, acquiring at least one pulse width corresponding to the droplet microfluidic channel;

[0058] Calculating size data of a droplet in the droplet microfluidic channel based on the plurality of pulse widths, the preset diameter and the standard pulse width;

[0059] Based on the droplet size data, abnormal warning operations are performed.

[0060] In this embodiment, this step can be used to calculate the size data of droplets in a single microchannel, and can also be used to calculate the size data of droplets after fusion in the main microchannel. For a single microchannel or a main microchannel, at least one pulse width can be obtained from the single microchannel signal data or the main microchannel signal data. For each pulse width, based on the preset diameter and the standard pulse width, a droplet size can be obtained, that is, the size data of the droplet. If a pulse width is T1, the preset diameter is D, and the standard pulse width is T5, then a droplet size d = D*T1 / T5, so there is a droplet size corresponding to each pulse width. Based on the droplet size data, determine whether the droplets in the single microchannel and / or the main microchannel meet the preset conditions. When it is determined that the droplets in the single microchannel and / or the main microchannel do not meet the preset conditions based on the droplet size data, perform an abnormal warning operation.

[0061] In an optional implementation, performing an abnormal warning operation based on the size data of the droplets includes:

[0062] For a single microchannel, obtaining size data of multiple droplets; calculating an average droplet size based on the size data of the multiple droplets, and performing an abnormal warning operation when the average droplet size does not meet a preset average droplet size; or

[0063] For the main microfluidic channel, unfused target droplets are screened according to the size data of the droplets, and the number of target droplets is calculated. Based on the number of target droplets, an abnormal warning operation is performed.

[0064] In the above embodiment, during the monitoring process of droplets, the size data of droplets in a single microchannel and / or a main microchannel can be monitored. Based on the size data of the droplets, when it is determined that the droplets in the single microchannel and / or the main microchannel do not meet the preset conditions, an abnormal warning operation is performed, thereby accurately monitoring the abnormalities in the droplet monitoring process, improving the success rate of droplet fusion, and reducing the waste of reagent resources.

[0065] In some embodiments, monitoring the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data includes:

[0066] Based on the droplet microfluidic channel signal data, obtaining a pulse signal interval corresponding to the droplet microfluidic channel;

[0067] Calculating the droplet interval in the droplet microchannel based on the pulse signal interval corresponding to the droplet microchannel, the preset diameter and the standard pulse width;

[0068] Based on the droplet interval in the droplet micro-channel, an abnormality warning operation is performed.

[0069] In this embodiment, this step can be used to calculate the size data of droplets in a single microchannel, and can also be used to calculate the size data of droplets after fusion in the main microchannel. For a single microchannel or a main microchannel, the pulse signal interval corresponding to the single microchannel or the main microchannel is calculated, which is similar to the method for obtaining the pulse signal interval corresponding to the single microchannel described in the above embodiment, and will not be repeated here. If the pulse signal interval corresponding to the single microchannel or the main microchannel is T2, the preset diameter is D, and the standard pulse width is T5, then the droplet interval d3 in the single microchannel or the main microchannel is D*T2 / T5. In an optional implementation, the pulse signal interval corresponding to the droplet microchannel may not be the average pulse signal interval, but represents multiple pulse signal intervals corresponding to the droplet microchannel. For a pulse signal interval, the above formula can be used to calculate the droplet interval corresponding to the pulse signal interval, thereby obtaining the droplet interval corresponding to multiple pulse signal intervals, and the average droplet interval can be obtained based on the droplet interval corresponding to multiple pulse signal intervals. In an optional implementation, based on the droplet spacing in the droplet microchannel, performing an abnormal warning operation includes at least one of the following: performing an abnormal warning operation when the average droplet spacing in a single microchannel or a main microchannel is not within the error range of a preset droplet spacing; or performing an abnormal warning operation when the number of droplet spacings that are not within the error range of a preset droplet spacing in a single microchannel or a main microchannel exceeds a preset number. When the droplet spacing in a single microchannel or a main microchannel is uneven, it indicates that the pressure in the single microchannel or the main microchannel is unstable, and the cause needs to be checked in time.

[0070] In the above embodiment, during the droplet monitoring process, the droplet spacing in a single microchannel and / or a main microchannel can be monitored. Based on the droplet spacing, when it is determined that the droplet spacing in the single microchannel and / or the main microchannel does not meet the preset droplet spacing conditions, an abnormal warning operation is performed, thereby accurately monitoring abnormalities in the droplet monitoring process, improving the success rate of droplet fusion, and reducing the waste of reagent resources.

[0071] In some embodiments, monitoring the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data includes:

[0072] Calculating the flow rate of the droplet in the droplet microchannel according to the preset diameter and the standard pulse width;

[0073] When the flow rate of the droplets in the droplet microchannel does not meet the preset flow rate condition, an abnormal warning operation is performed.

[0074] In this embodiment, since the various parameter conditions of the standard object in the droplet microfluidic channel and the droplet monitoring process are the same, the flow rate of the standard object can be used to represent the flow rate of the droplet. Droplet flow rate S = preset diameter / standard pulse width. The droplet flow rate cannot be too fast or too slow. Too fast or too slow will affect the fusion rate of the droplets in the main microfluidic channel. In an optional implementation, when the flow rate of the droplets in the droplet microfluidic channel does not meet the preset flow rate conditions, performing an abnormal warning operation includes one of the following: when the flow rate of the droplets is lower than the first flow rate preset value, performing an abnormal warning operation to prompt that the flow rate is too slow; when the flow rate of the droplets is higher than the second flow rate preset value, performing an abnormal warning operation to prompt that the flow rate is too fast; wherein the second flow rate preset value is greater than the first flow rate preset value.

[0075] In the above embodiment, during the monitoring process of droplets, the droplet flow rate in the droplet microfluidic channel can be monitored. Based on the droplet flow rate, when it is determined that the droplet flow rate in the droplet microfluidic channel does not meet the preset flow rate condition, an abnormal warning operation is performed, thereby accurately monitoring the abnormalities in the droplet monitoring process, improving the success rate of droplet fusion, and reducing the waste of reagent resources.

[0076] In some embodiments, monitoring the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data includes:

[0077] Obtaining the total amount of droplets flowing in the target single microfluidic channel within a target time period, and obtaining target droplets whose droplet sizes in the main microfluidic channel and the droplet sizes in the target single microfluidic channel are within a preset range within a monitoring time period corresponding to the target time period, and calculating the number of target droplets;

[0078] Calculating a fusion rate based on the target number of droplets and the total amount of droplets;

[0079] When the fusion rate is lower than the preset fusion rate, an abnormal warning operation is performed.

[0080] In this embodiment, the target single microfluidic channel can be any single microfluidic channel. The monitoring time period is used for the time period in the main microfluidic channel. Since the droplets in the single microfluidic channel need to flow into the main microfluidic channel first and then fuse the droplets, the monitoring time period will be later than the target time period. The monitoring time period is used to monitor the time period of the main microfluidic channel, and the target time period is used to monitor the time period of the target single microfluidic channel. In an optional implementation, the total amount of droplets in the target time period can be determined by the number of pulse signals in the signal data of the target single microfluidic channel in the target time period. Generally, the size data of the fused droplets is larger than the size data of the droplets in the single microfluidic channel. Therefore, the target droplets whose droplet size is within the preset range and the droplet size in the target single microfluidic channel can be obtained from the fused channel in the main microfluidic channel. The target droplets are unfused droplets. For example, if the total amount of droplets in the target time period is 100 and the number of target droplets in the monitoring time period is 20, the number of droplets involved in the fusion is 80, and the fusion rate is 80 / 100. That is, the fusion rate is (total number of droplets - target number of droplets) / total number of droplets. The fusion rate refers to the proportion of droplets in each single microchannel that successfully fuse together to form fused droplets.

[0081] In the above embodiment, during the monitoring process of droplets, the fusion rate of droplets in the main microchannel can be monitored. When the fusion rate is lower than the preset fusion rate, an abnormal warning operation is performed, thereby accurately monitoring the abnormalities in the droplet monitoring process, improving the success rate of droplet fusion, and reducing the waste of reagent resources.

[0082] In some embodiments, monitoring the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data includes:

[0083] Acquiring main microchannel signal data, and extracting pulse signals corresponding to each droplet in the main microchannel from the main microchannel signal data;

[0084] A droplet whose signal attribute of the corresponding pulse signal in the main microchannel meets the preset signal attribute conditions is determined to be a fused droplet containing the target object; wherein the signal attribute includes at least one of the following: there is an opposite trough between two peak positions in the pulse signal, and the pulse width is greater than the pulse width of the pulse signal corresponding to the droplet in the single microchannel.

[0085] In this embodiment, the signal data corresponding to the fused droplets can be collected through the laser detection area corresponding to the main microfluidic channel. Since there may be droplets that have been fused, but there are no DNA label colloid balls wrapped in the droplets, if there are no DNA label colloid balls wrapped in the droplets, the gene data cannot be accurately detected later. Therefore, it is necessary to monitor whether the fused droplets in the main microfluidic channel are droplets wrapped with labels. Figure 7 As shown, Figure 7 FIG. 1 is a schematic diagram of a pulse signal of a fused droplet containing a target object in an embodiment. The reverse trough is as shown in FIG. Figure 7 As shown in Figure 5 As shown, an inverted peak appears in the middle of the pulse waveform of the fused droplet wrapped by the label, and the waveform becomes wider. Moreover, the peak of the fused droplet wrapped by the label is higher than the peak of the pulse signal corresponding to the droplet in the single microfluidic channel.

[0086] In an optional embodiment, monitoring the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data further includes:

[0087] Obtaining the total number of fused droplets in the main microfluidic channel and the number of fused droplets containing target objects;

[0088] Calculating the encapsulation rate according to the total number of fused droplets in the main microfluidic channel and the number of fused droplets containing the target object;

[0089] When the package rate is lower than a preset package value, an abnormal warning operation is performed.

[0090] In this embodiment, the total number of fused droplets in the main microfluidic channel within a preset time period can be monitored. The main microfluidic channel signal data within a preset time period can be obtained, and the size data of each droplet in the main microfluidic channel can be obtained according to the main microfluidic channel signal data. The fused droplets are determined according to the size data of each droplet in the main microfluidic channel. For example, the droplets with size data higher than the preset drop size data are determined as fused droplets, and then the total number of fused droplets is calculated. The encapsulation rate is the number of fused droplets with target objects / the total number of fused droplets. The encapsulation rate indicates the proportion of fused droplets with target objects in the fused droplets.

[0091] In the above embodiment, during the monitoring process of droplets, the encapsulation rate of droplets in the main microchannel can be monitored. When the fusion rate is lower than the preset encapsulation value, an abnormal warning operation is performed, thereby accurately monitoring the abnormalities in the droplet monitoring process, improving the success rate of droplet fusion, and reducing the waste of reagent resources.

[0092] In some embodiments, monitoring the state of a droplet in the droplet microfluidic channel based on the droplet microfluidic channel signal data includes at least one of:

[0093] Acquire signals corresponding to each fused droplet in the main microfluidic channel by means of a fluorescence detection device, determine the fused droplet corresponding to the signal with the fluorescence signal as a successfully fused droplet, acquire the total number of the successfully fused droplets and the number of fused droplets in the main microfluidic channel, calculate the successful fusion rate according to the total number of successfully fused droplets and the number of fused droplets in the main microfluidic channel, and perform an abnormal warning operation when the successful fusion rate is lower than a preset successful fusion rate; or

[0094] According to the fluorescence signal intensity of the successfully fused droplet, the content data of the target substance labeled with the fluorescent dye in the successfully fused droplet is determined.

[0095] In this embodiment, after the droplets in each single microchannel in the main microchannel are fused, fused droplets are obtained, but there may be some droplets that are fused but not successfully fused. Fluorescent markers can be added to certain substances in advance, and a fluorescent detection device can be used to detect whether there is a fluorescent signal in the signal of the fused droplet. If there is a fluorescent signal, it indicates a successfully fused droplet. Therefore, the fused droplets can be first screened out by the size data of the droplets, and then the signal corresponding to the fused droplet is detected to see if there is a fluorescent signal. Specifically, a fluorescent detection device emits light of a certain wavelength to the fluorescent detection area of ​​the main microchannel, and the fused droplets with fluorescent markers are stimulated to emit a fluorescent signal of a specific wavelength. If a fluorescent signal of a specific wavelength is detected, it indicates that there is a fluorescent signal in the signal of the fused droplet, that is, the droplets are successfully fused. The successful fusion rate represents the proportion of successfully fused droplets in the fused droplets. In some embodiments, the content data of the target substance in the successfully fused droplet can also be determined based on the intensity value of the fluorescent signal, where the content data includes but is not limited to concentration, quantity, proportion, etc.

[0096] In the above embodiment, during the monitoring process of droplets, the successful fusion rate of droplets in the main microchannel can be monitored. When the fusion rate is lower than the preset successful fusion rate, an abnormal warning operation is performed. The content of target substances in the successfully fused droplets can also be monitored to ensure that the subsequent droplets are reliable droplets.

[0097] In some embodiments, the method further comprises:

[0098] Acquiring droplet microfluidic channel image information, wherein the droplet microfluidic channel image information includes single microfluidic channel image information and / or main microfluidic channel image information;

[0099] Based on the droplet microfluidic channel image information, the state of the droplet in the droplet microfluidic channel is monitored.

[0100] In this embodiment, the single microchannel image information and / or the main microchannel image information can be analyzed to monitor the size data, spacing, morphology, etc. of the droplets in the single microchannel, and combined with one or more of the above embodiments, the state of the droplets in the droplet microchannel can be monitored. When the state of the droplets monitored based on the droplet microchannel signal data is inconsistent with the state of the droplets monitored based on the droplet microchannel image information, an abnormal warning operation can be performed, and the acquired droplet microchannel image can be sent to the terminal device for final confirmation by the user.

[0101] In the above embodiments, real-time image analysis and laser detection are combined to monitor the processes of droplet generation, pairing, fusion, etc. in real time from different angles, thereby improving the accuracy and success rate of the experiment.

[0102] On the other hand, the present application provides a computer program product, including a computer program, which, when executed by a processor, implements the droplet quality control method based on laser detection described in any embodiment of the present application.

[0103] Among them, in the computer program product, the optional implementation form of the program module architecture of the computer program that implements each step of the method can be a droplet quality control device based on laser detection. Figure 8 An embodiment of the present application provides a droplet quality control device based on laser detection, including: an acquisition module 81, used to acquire droplet microfluidic channel signal data collected by a laser device, and a monitoring module 82, used to monitor the state of droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data; wherein the droplet microfluidic channel includes each single microfluidic channel and / or a main microfluidic channel, and the droplets in the droplet microfluidic channel include droplets in each single microfluidic channel and / or fused droplets in the main microfluidic channel, and the droplet microfluidic channel signal data includes single microfluidic channel signal data and / or main microfluidic channel signal data, and after the solutions corresponding to each single microfluidic channel form droplets in each single microfluidic channel in their respective corresponding single microfluidic channels, the droplets in each single microfluidic channel flow to the main microfluidic channel and merge in the main microfluidic channel to form fused droplets.

[0104] Optionally, the state of the droplets in the droplet microfluidic channel includes at least one or more of the following combinations: the generation frequency of droplets in each single microfluidic channel, the relationship between the current droplet generation frequencies corresponding to all single microfluidic channels, the size data of droplets in the droplet microfluidic channel, the droplet spacing in the droplet microfluidic channel, the flow rate of droplets in the droplet microfluidic channel, the fusion rate of droplets in the main microfluidic channel, whether there are target objects in the fused droplets in the main microfluidic channel, the wrapping rate of the fused droplets in the main microfluidic channel, whether the fused droplets in the main microfluidic channel are successfully fused droplets, the successful fusion rate of fused droplets in the main microfluidic channel, and the content data of the target substance in the successfully fused droplets.

[0105] Optionally, the monitoring module 82 is further used for:

[0106] Based on the signal data of each single microchannel, the pulse signal interval corresponding to each single microchannel is obtained, and based on the pulse signal interval corresponding to each single microchannel, the generation frequency of droplets in each single microchannel is calculated;

[0107] According to the generation frequency of droplets in each single microfluidic channel, the current droplet generation frequency relationship corresponding to all single microfluidic channels is calculated;

[0108] Based on the current droplet generation frequency relationship, the pressure data of each single microfluidic channel is controlled.

[0109] Optionally, the monitoring module 82 is further used for:

[0110] Compare the current droplet generation frequency relationship with a preset generation frequency ratio, obtain a single microchannel with a generation frequency lower than a first generation frequency value, and increase the pressure data of the single microchannel with a generation frequency lower than the first generation frequency value;

[0111] The current droplet generation frequency relationship is compared with a preset generation frequency ratio, a single microchannel having a generation frequency higher than a second generation frequency value is obtained, and pressure data of the single microchannel having a generation frequency higher than the second generation frequency value is reduced.

[0112] Optionally, the monitoring module 82 is further used for:

[0113] A standard pulse width is obtained, where the standard pulse width is obtained by placing a standard object with a preset diameter in the droplet microchannel and flowing the standard pulse width according to a received signal of the laser device.

[0114] Optionally, the monitoring module 82 is further used for:

[0115] Based on the droplet microfluidic channel signal data, acquiring at least one pulse width corresponding to the droplet microfluidic channel;

[0116] Calculating size data of a droplet in the droplet microfluidic channel based on the plurality of pulse widths, the preset diameter and the standard pulse width;

[0117] Based on the droplet size data, abnormal warning operations are performed.

[0118] Optionally, the monitoring module 82 is further used for:

[0119] Based on the droplet microfluidic channel signal data, obtaining a pulse signal interval corresponding to the droplet microfluidic channel;

[0120] Calculating the droplet interval in the droplet microchannel based on the pulse signal interval corresponding to the droplet microchannel, the preset diameter and the standard pulse width;

[0121] Based on the droplet interval in the droplet micro-channel, an abnormality warning operation is performed.

[0122] Optionally, the monitoring module 82 is further used for:

[0123] Calculating the flow rate of the droplet in the droplet microchannel according to the preset diameter and the standard pulse width;

[0124] When the flow rate of the droplets in the droplet microchannel does not meet the preset flow rate condition, an abnormal warning operation is performed.

[0125] Optionally, the monitoring module 82 is further used for:

[0126] Obtaining the total amount of droplets flowing in the target single microfluidic channel within a target time period, and obtaining target droplets whose droplet sizes in the main microfluidic channel and the droplet sizes in the target single microfluidic channel are within a preset range within a monitoring time period corresponding to the target time period, and calculating the number of target droplets;

[0127] Calculating a fusion rate based on the target number of droplets and the total amount of droplets;

[0128] When the fusion rate is lower than the preset fusion rate, an abnormal warning operation is performed.

[0129] Optionally, the monitoring module 82 is further used for:

[0130] Acquiring main microchannel signal data, and extracting pulse signals corresponding to each droplet in the main microchannel from the main microchannel signal data;

[0131] A droplet whose signal attribute of the corresponding pulse signal in the main microchannel meets the preset signal attribute conditions is determined to be a fused droplet containing the target object; wherein the signal attribute includes at least one of the following: there is an opposite trough between two peak positions in the pulse signal, and the pulse width is greater than the pulse width of the pulse signal corresponding to the droplet in the single microchannel.

[0132] Optionally, the monitoring module 82 is further used for:

[0133] Obtaining the total number of fused droplets in the main microfluidic channel and the number of fused droplets containing target objects;

[0134] Calculating the encapsulation rate according to the total number of fused droplets in the main microfluidic channel and the number of fused droplets containing the target object;

[0135] When the package rate is lower than a preset package value, an abnormal warning operation is performed.

[0136] Optionally, the monitoring module 82 is further used for:

[0137] Acquire signals corresponding to each fused droplet in the main microfluidic channel by means of a fluorescence detection device, determine the fused droplet corresponding to the signal with the fluorescence signal as a successfully fused droplet, acquire the total number of the successfully fused droplets and the number of fused droplets in the main microfluidic channel, calculate the successful fusion rate according to the total number of successfully fused droplets and the number of fused droplets in the main microfluidic channel, and perform an abnormal warning operation when the successful fusion rate is lower than a preset successful fusion rate; or

[0138] According to the fluorescence signal intensity of the successfully fused droplet, the content data of the target substance labeled with the fluorescent dye in the successfully fused droplet is determined.

[0139] Optionally, the monitoring module 82 is further used for:

[0140] Acquiring droplet microfluidic channel image information, wherein the droplet microfluidic channel image information includes single microfluidic channel image information and / or main microfluidic channel image information;

[0141] Based on the droplet microfluidic channel signal data and the droplet microfluidic channel image information, the state of the droplets in the droplet microfluidic channel is monitored.

[0142] It can be understood by those skilled in the art that Figure 8 The structure of the droplet quality control device based on laser detection does not constitute a limitation on the droplet quality control device based on laser detection, and each module can be implemented in whole or in part by software, hardware, and a combination thereof. The above modules can be embedded in or independent of the controller in the computer device in the form of hardware, or can be stored in the memory in the computer device in the form of software, so that the controller can call and execute the operations corresponding to the above modules. In other embodiments, the droplet quality control device based on laser detection may include more or fewer modules than those shown in the figure.

[0143] See also Fig. 9 On the other hand, an embodiment of the present application further provides a droplet quality control device 200 based on laser detection, including a controller 10 and a memory 18, wherein the memory 18 stores a computer program. When the computer program is executed by the controller, the controller 10 executes the steps of the droplet quality control method based on laser detection provided in any of the above embodiments of the present application.

[0144] The controller 10 is a control center, which uses various interfaces and lines to connect various parts of the entire computer device, and executes various functions of the computer device and processes data by running or executing software programs and / or modules stored in the memory 18, and calling data stored in the memory 18. Optionally, the controller 10 may include one or more processing cores; preferably, the controller 10 may integrate an application controller and a modem controller, wherein the application controller mainly processes the operating system, user pages and application programs, etc., and the modem controller mainly processes wireless communications. It is understandable that the above-mentioned modem controller may not be integrated into the controller 10.

[0145] The memory 18 can be used to store software programs and modules. The controller 10 executes various functional applications and data processing by running the software programs and modules stored in the memory 18. The memory 18 can mainly include a program storage area and a data storage area, wherein the program storage area can store an operating system, an application required for at least one function (such as a sound playback function, an image playback function, etc.), etc.; the data storage area can store data created according to the use of the computer device, etc. In addition, the memory 18 can include a high-speed random access memory, and can also include a non-volatile memory, such as at least one disk storage device, a flash memory device, or other volatile solid-state storage devices. Accordingly, the memory 18 can also include a memory controller to provide the controller 10 with access to the memory 18.

[0146] On the other hand, an embodiment of the present application further provides a storage medium storing a computer program. When the computer program is executed by a controller, the controller executes the steps of the droplet quality control method based on laser detection provided in any of the above embodiments of the present application.

[0147] On the other hand, an embodiment of the present application further provides a single-cell sequencing device, including a droplet quality control device based on laser detection provided by any of the above embodiments of the present application.

[0148] On the other hand, an embodiment of the present application further provides a single-cell sequencing library construction device, including a droplet quality control device based on laser detection provided by any of the above embodiments of the present application.

[0149] Those skilled in the art can understand that all or part of the processes in the methods provided in the above embodiments can be completed by instructing the relevant hardware through a computer program, and the program can be stored in a non-volatile computer-readable storage medium. When the program is executed, it can include the processes of the embodiments of the above methods. Among them, any reference to memory, storage, database or other media used in the embodiments provided in the present application can include non-volatile and / or volatile memory. Non-volatile memory may include read-only memory (ROM), programmable ROM (PROM), electrically programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM) or flash memory. Volatile memory may include random access memory (RAM) or external cache memory. As an illustration and not limitation, RAM is available in many forms, such as static RAM (SRAM), dynamic RAM (DRAM), synchronous DRAM (SDRAM), double data rate SDRAM (DDRSDRAM), enhanced SDRAM (ESDRAM), synchronous link (Synchlink) DRAM (SLDRAM), memory bus (Rambus) direct RAM (RDRAM), direct memory bus dynamic RAM (DRDRAM), and memory bus dynamic RAM (RDRAM).

[0150] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. The protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A droplet quality control method based on laser detection, characterized in that: include: Acquire the droplet microfluidic signal data collected by the laser device, Based on the droplet microfluidic channel signal data, the state of droplets in the droplet microfluidic channel is monitored; wherein the droplet microfluidic channel includes each single microfluidic channel and / or a main microfluidic channel, the droplets in the droplet microfluidic channel include droplets in each single microfluidic channel and / or fused droplets in the main microfluidic channel, the droplet microfluidic channel signal data includes single microfluidic channel signal data and / or main microfluidic channel signal data, and after the solutions corresponding to each single microfluidic channel form droplets in each single microfluidic channel in their respective corresponding single microfluidic channels, the droplets in each single microfluidic channel flow to the main microfluidic channel and merge in the main microfluidic channel to form fused droplets.

2. The droplet quality control method based on laser detection according to claim 1, characterized in that: The state of the droplets in the droplet microfluidic channel includes at least one or more of the following combinations: the generation frequency of droplets in each single microfluidic channel, the relationship between the current droplet generation frequencies corresponding to all single microfluidic channels, the size data of the droplets in the droplet microfluidic channel, the droplet spacing in the droplet microfluidic channel, the flow rate of the droplets in the droplet microfluidic channel, the fusion rate of the droplets in the main microfluidic channel, whether there is a target object in the fused droplets in the main microfluidic channel, the encapsulation rate of the fused droplets in the main microfluidic channel, whether the fused droplets in the main microfluidic channel are successfully fused droplets, the successful fusion rate of the fused droplets in the main microfluidic channel, and the content data of the target substance in the successfully fused droplets.

3. The droplet quality control method based on laser detection according to claim 1, characterized in that: The monitoring of the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data comprises: Based on the signal data of each single microchannel, the pulse signal interval corresponding to each single microchannel is obtained, and based on the pulse signal interval corresponding to each single microchannel, the generation frequency of droplets in each single microchannel is calculated; According to the generation frequency of droplets in each single microfluidic channel, the current droplet generation frequency relationship corresponding to all single microfluidic channels is calculated; Based on the current droplet generation frequency relationship, the pressure data of each single microfluidic channel is controlled.

4. The droplet quality control method based on laser detection according to claim 3, characterized in that: The controlling of the pressure data of each single microchannel based on the current droplet generation frequency relationship includes at least one of the following: Compare the current droplet generation frequency relationship with a preset generation frequency ratio, obtain a single microchannel with a generation frequency lower than a first generation frequency value, and increase the pressure data of the single microchannel with a generation frequency lower than the first generation frequency value; The current droplet generation frequency relationship is compared with a preset generation frequency ratio, a single microchannel having a generation frequency higher than a second generation frequency value is obtained, and pressure data of the single microchannel having a generation frequency higher than the second generation frequency value is reduced.

5. The droplet quality control method based on laser detection according to claim 2, characterized in that: Before monitoring the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data, the method further includes: A standard pulse width is obtained, where the standard pulse width is obtained by placing a standard object with a preset diameter in the droplet microchannel and flowing the object according to a received signal of the laser device.

6. The droplet quality control method based on laser detection according to claim 5, characterized in that: The monitoring of the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data comprises: Based on the droplet microfluidic channel signal data, acquiring at least one pulse width corresponding to the droplet microfluidic channel; Calculating size data of a droplet in the droplet microfluidic channel based on the plurality of pulse widths, the preset diameter and the standard pulse width; Based on the droplet size data, abnormal warning operations are performed.

7. The droplet quality control method based on laser detection according to claim 5, characterized in that: The monitoring of the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data comprises: Based on the droplet microfluidic channel signal data, obtaining a pulse signal interval corresponding to the droplet microfluidic channel; Calculating the droplet interval in the droplet microchannel based on the pulse signal interval corresponding to the droplet microchannel, the preset diameter and the standard pulse width; Based on the droplet interval in the droplet micro-channel, an abnormality warning operation is performed.

8. The droplet quality control method based on laser detection according to claim 5, characterized in that: The monitoring of the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data comprises: Calculating the flow rate of the droplet in the droplet microchannel according to the preset diameter and the standard pulse width; When the flow rate of the droplets in the droplet microchannel does not meet the preset flow rate condition, an abnormal warning operation is performed.

9. The droplet quality control method based on laser detection according to claim 2, characterized in that: The monitoring of the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data comprises: Obtaining the total amount of droplets flowing in the target single microfluidic channel within a target time period, and obtaining target droplets whose droplet sizes in the main microfluidic channel and the droplet sizes in the target single microfluidic channel are within a preset range within a monitoring time period corresponding to the target time period, and calculating the number of target droplets; Calculating a fusion rate based on the target number of droplets and the total amount of droplets; When the fusion rate is lower than the preset fusion rate, an abnormal warning operation is performed.

10. The droplet quality control method based on laser detection according to claim 2, characterized in that: The monitoring of the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data comprises: Acquiring main microchannel signal data, and extracting pulse signals corresponding to each droplet in the main microchannel from the main microchannel signal data; A droplet whose signal attribute of the corresponding pulse signal in the main microchannel meets the preset signal attribute conditions is determined to be a fused droplet containing the target object; wherein the signal attribute includes at least one of the following: there is an opposite trough between two peak positions in the pulse signal, and the pulse width is greater than the pulse width of the pulse signal corresponding to the droplet in the single microchannel.

11. The droplet quality control method based on laser detection according to claim 10, characterized in that: The monitoring of the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data further comprises: Obtaining the total number of fused droplets in the main microfluidic channel and the number of fused droplets containing target objects; Calculating the encapsulation rate according to the total number of fused droplets in the main microfluidic channel and the number of fused droplets containing the target object; When the package rate is lower than a preset package value, an abnormal warning operation is performed.

12. The droplet quality control method based on laser detection according to claim 2, characterized in that: The monitoring of the state of the droplets in the droplet microfluidic channel based on the droplet microfluidic channel signal data includes at least one of: Acquire signals corresponding to each fused droplet in the main microfluidic channel by means of a fluorescence detection device, determine the fused droplet corresponding to the signal with the fluorescence signal as a successfully fused droplet, acquire the total number of the successfully fused droplets and the number of fused droplets in the main microfluidic channel, calculate the successful fusion rate according to the total number of the successfully fused droplets and the number of fused droplets in the main microfluidic channel, and perform an abnormal warning operation when the successful fusion rate is lower than a preset successful fusion rate; or According to the fluorescence signal intensity of the successfully fused droplet, the content data of the target substance labeled with the fluorescent dye in the successfully fused droplet is determined.

13. The droplet quality control method based on laser detection according to any one of claims 1 to 12, characterized in that: The method further comprises: Acquiring droplet microfluidic channel image information, wherein the droplet microfluidic channel image information includes single microfluidic channel image information and / or main microfluidic channel image information; Based on the droplet microfluidic channel signal data and the droplet microfluidic channel image information, the state of the droplets in the droplet microfluidic channel is monitored.

14. A droplet quality control device based on laser detection, characterized in that: The method comprises a memory and a controller, wherein the memory stores a computer program, and when the computer program is executed by the controller, the controller executes the steps of the method according to any one of claims 1 to 13.

15. A single-cell sequencing library construction device, characterized in that: Including a droplet quality control device based on laser detection as described in claim 14.

16. A computer-readable storage medium, characterized in that: The computer-readable storage medium stores a computer program, and when the computer program is executed by the controller, the droplet quality control method based on laser detection as described in any one of claims 1 to 13 is implemented.

17. A computer program product comprising a computer program, characterized in that When the computer program is executed by the controller, the droplet quality control method based on laser detection as described in any one of claims 1 to 13 is implemented.

Images