Droplet microfluidic sorting method, sorting system, electronic device, and storage medium

Abstract

The application discloses a droplet microfluidic sorting method, a sorting system, an electronic device and a storage medium. The droplet microfluidic sorting method comprises the following steps: acquiring a first time point of positive droplet trigger sorting in a microfluidic device; determining a second time point of the positive droplet reaching an outlet of a collection channel according to the first time point and a time length of the positive droplet passing through the collection channel to reach the outlet of the collection channel; the outlet of the collection channel is used for forming an oil droplet; and comparing an oil droplet formation time point closest to the first time point with the second time point to determine a target oil droplet containing the positive droplet. The droplet microfluidic sorting method realizes the automation of sorting a single target droplet, further sorts the target liquid into a droplet containing a single target bacterium or cell, avoids the loss of cells in the demulsification process or the phenomenon that target cells do not exist in part of the well plates or multiple target cells exist in part of the well plates, and thus the efficiency of target sequence acquisition is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic technology, and in particular to a droplet microfluidic sorting method, a sorting system, an electronic device and a storage medium. BACKGROUND

[0002] Microfluidic technology is a technology for processing and manipulating microdroplets of less than 1 picoliter by using microchannels with a scale of tens to hundreds of microns. It involves multiple disciplines, including fluid physics, chemistry, biology and biomedical science, and electronics, and is an emerging interdisciplinary subject. Microfluidic technology has the characteristics of miniaturization, integration, easy preparation and wide application.

[0003] Microfluidic chips, also known as chip laboratories, can integrate microfluidic channel systems in a few square centimeters of chips to perform specific biochemical reactions and achieve micro-total analysis goals. High-throughput microfluidic droplet sorting is a method that wraps cells or bacteria and their reactants in picoliter droplets through micron-sized channels to obtain a target number of micro-reaction systems that are not disturbed by each other. After the contents of the droplets are fully mixed and reacted, the detection of cells or bacteria is completed using an optical system, and the detection results are numerized to obtain comparable electrical peak information. After the comparison of the peak signal and the threshold signal in the software, the results are fed back to the waveform generator and the amplifier to complete the screening of cells or bacteria. Microfluidic droplets only require a small amount of reaction reagent to achieve ultra-high throughput that cannot be achieved by traditional plate screening technology, and thus have important applications in enzyme directed evolution research.

[0004] In a conventional high-throughput droplet microfluidic sorting system, the droplets wrapped with target bacteria or cells are collected in the same sample tube. If the information of light and heavy chains existing in cells secreting antibodies is to be obtained, manual operations such as demulsification recovery and limited dilution distribution need to be performed. The demulsification process may cause loss of cells, and the process of limited dilution distribution may cause Poisson distribution, resulting in the absence of target cells in some well plates or the presence of multiple target cells in some well plates, and thus reducing the efficiency of obtaining target sequences. SUMMARY

[0005] The present application aims to overcome the defects of the prior art that the droplet microfluidic sorting system of the prior art collects droplets of target bacteria or cells together, resulting in the absence of target cells in some well plates or the presence of multiple target cells in some well plates, and provides a droplet microfluidic sorting method, a sorting system, an electronic device and a storage medium.

[0006] The present application solves the above technical problems by the following technical solutions:

[0007] The present application provides a droplet microfluidic sorting method, which comprises:

[0008] acquiring a first time of positive droplet trigger sorting in the microfluidic device;

[0009] determining a second time of the positive droplet reaching the collection channel outlet according to the first time and a time length of the positive droplet passing through the collection channel to reach the collection channel outlet; the collection channel outlet is used to form oil droplets;

[0010] comparing the time of oil droplet formation closest to the first time and the second time to determine a target oil droplet containing the positive droplet.

[0011] Preferably, before the step of determining the second time of the positive droplet reaching the collection channel outlet according to the first time and the time length of the positive droplet passing through the collection channel to reach the collection channel outlet, the droplet microfluidic sorting method further comprises:

[0012] acquiring a first start time and a first end time of oil droplet formation at the collection channel outlet, a second start time and a second end time of oil droplet formation at the disposal channel outlet, a collection channel parameter and an oil phase flow parameter; the disposal channel is used to pass oil droplets not containing the positive droplet;

[0013] determining the time length of the positive droplet passing through the collection channel to reach the collection channel outlet according to the first start time, the first end time, the second start time, the second end time, the collection channel parameter and the oil phase flow parameter.

[0014] Preferably, the collection channel parameter comprises a length and a radius of the collection channel; the oil phase flow parameter comprises a continuous phase oil phase flow and a positive droplet phase oil phase flow;

[0015] The time length of the positive droplet passing through the collection channel to reach the collection channel outlet is determined according to the following formula:

[0016] ;

[0017] ;

[0018] ;

[0019] ;

[0020] wherein, T1 is the first start time of oil droplet formation at the collection channel outlet, T2 is the first end time of oil droplet formation at the collection channel outlet, T is the time interval of oil droplet formation at the collection channel outlet, T is the time interval of oil droplet formation at the disposal channel outlet, a second start time for the oil droplet to be formed at the outlet of the disposal channel, a second end time for the oil droplet to be formed at the outlet of the disposal channel, a flow rate of the oil phase of the continuous phase, a flow rate of the oil phase of the positive droplet phase, a flow rate of the collection channel, a length of the collection channel, a radius of the collection channel, a time length for the positive droplet to pass through the collection channel to reach the outlet of the collection channel.

[0021] Preferably, before the step of acquiring the first time at which the positive droplet triggers sorting in the microfluidic device, the droplet microfluidic sorting method further comprises:

[0022] acquiring a fluorescence signal of the droplet in the microfluidic device, and determining the positive droplet according to the fluorescence signal and a preset threshold.

[0023] Preferably, after the step of determining the positive droplet, the droplet microfluidic sorting method further comprises:

[0024] performing deflection processing on the positive droplet, and performing deflection processing on a next positive droplet after a variable deflection cooling time;

[0025] the deflection cooling time is determined according to a deflection time of the positive droplet, an oil droplet formation time closest to the first time, and a time length for the positive droplet to pass through the collection channel to reach the outlet of the collection channel.

[0026] Preferably, after the step of determining the target oil droplet containing the positive droplet, the droplet microfluidic sorting method further comprises:

[0027] moving a collection well of the well plate to the outlet of the collection channel to collect the target oil droplet.

[0028] The present application also provides a droplet microfluidic sorting system, which comprises:

[0029] a positive droplet detection module for acquiring a first time at which a positive droplet triggers sorting in a microfluidic device;

[0030] an oil droplet formation calculation module for determining a second time at which the positive droplet reaches an outlet of a collection channel according to the first time and a time length for the positive droplet to pass through the collection channel to reach the outlet of the collection channel; the outlet of the collection channel is used for forming an oil droplet;

[0031] a target oil droplet determination module for comparing an oil droplet formation time closest to the first time and the second time to determine a target oil droplet containing the positive droplet.

[0032] Preferably, the oil droplet formation calculation module is further configured to obtain a first start time and a first end time of the oil droplet formation at the outlet of the collection channel, a second start time and a second end time of the oil droplet formation at the outlet of the disposal channel, a collection channel parameter and an oil phase flow parameter; the disposal channel is configured to pass the oil droplet not containing the positive droplet;

[0033] The oil droplet formation calculation module is further configured to determine the time length of the positive droplet passing through the collection channel to reach the outlet of the collection channel according to the first start time, the first end time, the second start time, the second end time, the collection channel parameter and the oil phase flow parameter.

[0034] Preferably, the collection channel parameter comprises a length and a radius of the collection channel; the oil phase flow parameter comprises a continuous phase oil phase flow and a positive droplet phase oil phase flow.

[0035] The oil droplet formation calculation module determines the time length of the positive droplet passing through the collection channel to reach the outlet of the collection channel according to the following formula:

[0036] ;

[0037] ;

[0038] ;

[0039] ;

[0040] wherein, T1 is the first start time of the oil droplet formation at the outlet of the collection channel, T2 is the first end time of the oil droplet formation at the outlet of the collection channel, ΔT is the time interval of the oil droplet formation at the outlet of the collection channel, ΔT is the time interval of the oil droplet formation at the outlet of the disposal channel, T3 is the second start time of the oil droplet formation at the outlet of the disposal channel, T4 is the second end time of the oil droplet formation at the outlet of the disposal channel, Qc is the continuous phase oil phase flow, Qd is the positive droplet phase oil phase flow, Q is the flow of the collection channel, L is the length of the collection channel, R is the radius of the collection channel, T is the time length of the positive droplet passing through the collection channel to reach the outlet of the collection channel.

[0041] Preferably, the droplet microfluidic sorting system further comprises:

[0042] A positive droplet determination module is configured to acquire a fluorescence signal of a droplet in the microfluidic device, and determine the positive droplet according to the fluorescence signal and a preset threshold.

[0043] Preferably, the droplet microfluidic sorting system further comprises:

[0044] A droplet deflection module is configured to deflect the positive droplet, and deflect a next positive droplet after a variable deflection cooling time.

[0045] The deflection cooling time is determined according to a deflection time of the positive droplet, a time of forming an oil droplet closest to the first time, and a time length of the positive droplet passing through the collection channel to reach the collection channel outlet.

[0046] Preferably, the droplet microfluidic sorting system further comprises:

[0047] A well plate control module is configured to move a collection well of a well plate to the collection channel outlet to collect the target oil droplet.

[0048] The application further provides an electronic device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor implements the droplet microfluidic sorting method as described above when executing the computer program.

[0049] The application further provides a computer readable storage medium having computer instructions stored thereon, wherein the computer instructions implement the droplet microfluidic sorting method as described above when executed by a processor.

[0050] The positive progress effect of the application is that:

[0051] The droplet microfluidic sorting method provided by the application detects a first time of sorting triggered by a positive droplet, determines a second time of the positive droplet reaching the collection channel outlet according to the first time and a time length of the positive droplet passing through the collection channel to reach the collection channel outlet, compares a time of forming an oil droplet closest to the first time and the second time to determine a target oil droplet containing the positive droplet, sets a deflection cooling time, avoids a single oil droplet containing two positive droplets, realizes the automation of sorting a single target droplet, further sorts the target liquid into a droplet containing a single target bacterium or cell, avoids manual operations such as demulsification recovery, limited dilution method distribution, and the like, for further processing of the target liquid, avoids the loss of cells in the demulsification process, or the phenomenon that some well plates do not contain target cells or some well plates contain multiple target cells in the process of limited dilution method distribution, and thus improves the efficiency of obtaining a target sequence. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1A first flow chart of the droplet microfluidic sorting method in Embodiment 1 of the present application.

[0053] Figure 2 An internal structure schematic diagram of the microfluidic device in Embodiment 1 of the present application.

[0054] Figure 3 A structure schematic diagram of the droplet microfluidic sorting system in Embodiment 1 of the present application.

[0055] Figure 4 A second flow chart of the droplet microfluidic sorting method in Embodiment 1 of the present application.

[0056] Figure 5 A timing diagram of the positive droplet sorting and collecting channel capillary outlet forming oil droplets in Embodiment 1 of the present application.

[0057] Figure 6 A flow logic diagram of the positive droplet dispensing orifice plate operation in Embodiment 1 of the present application.

[0058] Figure 7 A first structure diagram of the droplet microfluidic sorting system in Embodiment 2 of the present application.

[0059] Figure 8 A second structure diagram of the droplet microfluidic sorting system in Embodiment 2 of the present application.

[0060] Figure 9 A structure schematic diagram of the electronic device in Embodiment 3 of the present application. DETAILED DESCRIPTION

[0061] The present application will be further described below by way of examples, but the present application is not limited in the scope of the examples.

[0062] Embodiment 1

[0063] Please refer to Figure 1 , which is a first flow chart of the droplet microfluidic sorting method in the present embodiment. Specifically, as shown in Figure 1 , the droplet microfluidic sorting method comprises:

[0064] S101, acquiring a first time of positive droplet triggering sorting in the microfluidic device; specifically, Figure 2 An internal structure schematic diagram of the microfluidic device is shown in Figure 2 , Figure 2 1 in the microfluidic device is a position for detection and sorting; when a positive droplet appears in the microfluidic device, the microfluidic device will sort the positive droplet, and the first time of positive droplet triggering sorting in the microfluidic device can be acquired; negative droplets in the microfluidic device will be discarded.

[0065] S102, determining a second time when the positive droplet reaches the collection channel outlet according to the first time and a time length when the positive droplet passes through the collection channel to reach the collection channel outlet; the collection channel outlet is used for forming oil droplets; specifically, Figure 3 The structure diagram of the droplet microfluidic sorting system is shown in FIG. 1. Figure 3 As shown in FIG. 1, Figure 3 2 in FIG. 1 is a microfluidic device, and 3 is a collection channel outlet; the collection channel can include a capillary tube, and the positive droplet passes through the capillary tube to be wrapped by an oil droplet at the collection channel outlet; the second time when the positive droplet reaches the collection channel outlet can be calculated according to the first time when the sorting is triggered by the positive droplet and a time length when the positive droplet passes through the capillary tube to reach the collection channel outlet.

[0066] S103, comparing the first time closest to the oil droplet formation time and the second time to determine a target oil droplet containing the positive droplet; specifically, if the second time is earlier than the first time closest to the oil droplet formation time, the positive droplet will fall along with the oil droplet closest to the first time, and the oil droplet closest to the first time contains the positive droplet; if the second time is later than the first time closest to the oil droplet formation time, the positive droplet will fall along with the oil droplet second closest to the first time, and the oil droplet closest to the first time does not contain the positive droplet, and the oil droplet second closest to the first time contains the positive droplet.

[0067] Please refer to Figure 4 which is a second flowchart of the droplet microfluidic sorting method in the embodiment. Specifically, as shown in FIG. 2, Figure 4 Before step S101 in the embodiment, the droplet microfluidic sorting method further includes:

[0068] S201, acquiring a fluorescence signal of a droplet in the microfluidic device, and determining a positive droplet according to the fluorescence signal and a preset threshold; specifically, the fluorescence of the droplet in the microfluidic device can be detected by using an optical system, and the optical signal is converted into an electrical signal and output to a single-chip microcomputer; the single-chip microcomputer compares the signal of the droplet with the preset threshold to determine the positive droplet, and then judges whether to deflect; if deflection is needed, a deflection signal is sent, which is amplified by an amplifier and then applied to a deflection electrode in the microfluidic device to control the deflection of the target positive droplet.

[0069] In an optional embodiment, before step S102, the droplet microfluidic sorting method further includes:

[0070] S202, acquiring a first starting time and a first ending time of oil droplet formation at the collection channel outlet, a second starting time and a second ending time of oil droplet formation at the discard channel outlet, a collection channel parameter and an oil phase flow parameter; the discard channel is used for discarding oil droplets not containing the positive droplet. Specifically, the negative droplet and the oil droplet not containing the positive droplet are discarded through the discard channel.

[0071] S203, determining the time length of the positive droplet passing through the collection channel to reach the collection channel outlet according to the first starting time, the first ending time, the second starting time, the second ending time, the collection channel parameter and the oil phase flow parameter.

[0072] Specifically, the collection channel parameter includes the length and the radius of the collection channel; the oil phase flow parameter includes the continuous phase oil phase flow and the positive droplet phase oil phase flow;

[0073] The time length of the positive droplet passing through the collection channel to reach the collection channel outlet is determined according to the following formula:

[0074] ;

[0075] ;

[0076] ;

[0077] ;

[0078] wherein, is the first starting time of the oil droplet formed at the collection channel outlet, is the first ending time of the oil droplet formed at the collection channel outlet, is the time interval of the oil droplet formed at the collection channel outlet, is the time interval of the oil droplet formed at the disposal channel outlet, is the second starting time of the oil droplet formed at the disposal channel outlet, is the second ending time of the oil droplet formed at the disposal channel outlet, is the continuous phase oil phase flow, is the positive droplet phase oil phase flow, is the flow of the collection channel, is the length of the collection channel, is the radius of the collection channel, is the time length of the positive droplet passing through the collection channel to reach the collection channel outlet.

[0079] Figure 5 is the timing diagram of the positive droplet sorting in the microfluidic device and the oil droplet formed at the capillary outlet of the collection channel. As shown in Figure 5 , the time of the oil droplet formed at the collection channel outlet is acquired by the photoelectric sensor (the starting and ending time of the collection channel outlet is and ), according to the time interval of the oil droplet formed at the collection outlet , the time of the target droplet flowing from the “detection-sorting” position of the microfluidic device to the collection channel outlet and the time of the positive droplet triggering sorting to judge whether to move the displacement platform. The standardization well plate is used to receive the positive droplet, and the well plate position of the positive droplet is received. When the positive droplet is sorted, the flow rate of the collection channel is , the positive droplet collection outlet is formed at a time interval to form an oil droplet and fall into the well plate, and the falling timing is not affected by whether the positive droplet is deflected. The formation time of the previous oil droplet is , and the formation time of the current oil droplet is , wherein , at the time , the positive droplet exceeding the set threshold triggers sorting, the deflection electrode applies a deflection voltage, the target droplet flows into the droplet collection channel under the action of the electrophoretic force, and flows to the droplet collection capillary outlet after , at this time, the time is , and the software judges the time when the well plate collects the positive droplet by comparing the relationship between the two times and .

[0080] In another optional embodiment, after step S201, the droplet microfluidic sorting method further comprises:

[0081] S204, deflection processing is performed on the positive droplet, and after a variable deflection cooling time, deflection processing is performed on the next positive droplet. The deflection cooling time is determined according to the deflection time of the positive droplet, the formation time of the oil droplet closest to the first time, and the time length of the positive droplet passing through the collection channel to reach the collection channel outlet. Specifically, in order to avoid the problem that multiple positive droplets trigger deflection at a short time interval (less than ), a variable deflection cooling time is needed. The positive droplets generated within the cooling start time will not trigger deflection, is calculated by using , and , and is . The end time of the cooling is calculated by and , that is, . The positive droplets between the times and no longer trigger sorting.

[0082] In addition, after step S103, the droplet microfluidic sorting method further comprises:

[0083] S205, moving the collection well of the well plate to the collection channel outlet to collect the target oil droplet.

[0084] The following example illustrates the procedure for sorting positive droplets. Figure 6 The flowchart illustrates the operation of a positive droplet distribution plate. Step 1: Power on the device, but do not activate the sorting function. Input the flow rate of the droplet sample to be sorted into the software. and the flow rate of the continuous oil phase After the biochemical sample droplets to be sorted and the dispersed oil phase begin to flow stably, the software uses two sets of photoelectric sensors to obtain the exact moment when oil droplets are generated in the collection channel. and And the exact moment when oil droplets are generated in the discarded channel. and And calculate the time interval for oil droplet formation. as well as Calculate the flow rate allocated to the collection channel. and by collecting the capillary length of the channel ,radius and the traffic allocated to the collection channel. Calculate the time required for the target droplet to flow from the "detection-sorting" position in the microfluidic chip to the collection channel outlet. The second step is to enable the sorting function and refresh the initial time of oil droplet generation. The termination time of oil droplet formation was calculated. When a positive droplet is detected, the software obtains the moment when the positive droplet triggers sorting. The time when the positive droplet flows to the outlet of the collection channel is calculated. The third step is for the software to determine the termination time of oil droplet formation. The moment when the positive droplet flows to the collection channel outlet The order of events, if time Prior to time As positive droplets fall with the current oil droplet, the software controls the displacement platform to move the collection hole of the orifice plate to the collection channel outlet to catch the oil droplet containing the positive droplet, and calculates the cooling time to prevent two adjacent positive droplets from entering the same orifice plate collection hole. Then calculate the time when the cooldown ends. ,exist arrive Positive droplets between time points will no longer undergo deflection; if time points... After the time Positive droplets will fall with the oil droplets following the current one. The software controls the displacement platform to move the disposal hole on the orifice plate to the collection channel outlet to collect oil droplets not coated with positive droplets. After the current oil droplet is disposed of, the displacement platform is again controlled to move the collection hole on the orifice plate to the collection channel outlet to collect the next oil droplet coated with a positive droplet. The time when this oil droplet falls is... and calculate the cooling time to avoid the two adjacent positive droplets before and after entering the same well of the well plate and calculate the time when the cooling time ends , the positive droplet is no longer deflected between the time to .

[0085] The droplet microfluidic sorting method provided by the embodiment first determines the time length of the positive droplet passing through the collection channel to reach the outlet of the collection channel through the droplet generation information of the collection channel, the collection channel parameters and the oil phase flow parameter, then detects the first time when the positive droplet triggers sorting, and determines the second time when the positive droplet reaches the outlet of the collection channel according to the first time and the time length of the positive droplet passing through the collection channel to reach the outlet of the collection channel, compares the time when the oil droplet is formed closest to the first time and the second time to determine the target oil droplet containing the positive droplet, and sets a variable deflection cooling time to avoid the same oil droplet wrapping multiple positive droplets, realizes the automation of sorting a single target droplet, and further sorts the target liquid into a droplet containing a single target bacteria or cell, without the need to manually process the target liquid through demulsification recovery, limited dilution method distribution and the like, avoids the loss of cells in the demulsification process, or the phenomenon that there is no target cell in part of the well plate or multiple target cells exist in part of the well plate in the process of limited dilution method distribution, thereby improving the efficiency of obtaining the target sequence.

[0086] Embodiment 2

[0087] Please refer to Figure 7 , which is the first structural schematic diagram of the droplet microfluidic sorting system in the embodiment. Specifically, as shown in Figure 7 , the droplet microfluidic sorting system comprises:

[0088] The positive droplet detection module 21 is configured to obtain the first time when the positive droplet triggers sorting in the microfluidic device; specifically, Figure 2 is a schematic diagram of the internal structure of the microfluidic device, as shown in Figure 2 , Figure 2 1 in the above is the position of the microfluidic device for detection and sorting; when the positive droplet appears in the microfluidic device, the microfluidic device will sort the positive droplet, and the first time when the positive droplet triggers sorting in the microfluidic device can be obtained; the negative droplet in the microfluidic device will be discarded.

[0089] The oil droplet formation calculation module 22 is configured to determine the second time when the positive droplet reaches the outlet of the collection channel according to the first time and the time length of the positive droplet passing through the collection channel to reach the outlet of the collection channel; the outlet of the collection channel is configured to form an oil droplet; specifically, Figure 3 is a structural schematic diagram of the droplet microfluidic sorting system, as shown in Figure 3As shown, Figure 3 2 is a microfluidic device, and 3 is a collection channel outlet; the collection channel can include a capillary tube, and the positive droplet passes through the capillary tube and is wrapped by an oil droplet at the collection channel outlet; the first time of sorting triggered by the positive droplet and the time length of the positive droplet passing through the capillary tube to reach the collection channel outlet can be used to calculate the second time of the positive droplet reaching the collection channel outlet.

[0090] The target oil droplet determination module 23 is configured to compare the first time closest to the oil droplet formation time and the second time to determine the target oil droplet containing the positive droplet. Specifically, if the second time is earlier than the oil droplet formation time closest to the first time, the positive droplet will fall with the oil droplet closest to the first time, and the oil droplet closest to the first time contains the positive droplet; if the second time is later than the oil droplet formation time closest to the first time, the positive droplet will fall with the oil droplet next to the first time, and the oil droplet closest to the first time does not contain the positive droplet, and the oil droplet next to the first time contains the positive droplet

[0091] Please refer to Figure 8 which is a second structural schematic diagram of the droplet microfluidic sorting system in the embodiment. Specifically, as shown in Figure 8 In the embodiment, the droplet microfluidic sorting system further includes:

[0092] The positive droplet determination module 24 is configured to acquire the fluorescence signal of the droplet in the microfluidic device, and determine the positive droplet according to the fluorescence signal and a preset threshold. Specifically, the fluorescence of the droplet in the microfluidic device can be detected by using an optical system, and the optical signal is converted into an electrical signal and output to a single-chip microcomputer. The single-chip microcomputer compares the signal of the droplet with a preset threshold to determine the positive droplet, and then makes a judgment on whether to deflect. If deflection occurs, a deflection signal is sent, which is amplified by an amplifier and then applied to the deflection electrode in the microfluidic device to control the deflection of the target positive droplet.

[0093] In an optional embodiment, the oil droplet formation calculation module 22 is further configured to acquire the first starting time and the first ending time of the oil droplet formed at the collection channel outlet, the second starting time and the second ending time of the oil droplet formed at the discard channel outlet, the collection channel parameter, and the oil phase flow parameter; the discard channel is used to pass through the oil droplet not containing the positive droplet;

[0094] The oil droplet formation calculation module 22 is further configured to determine the time length of the positive droplet passing through the collection channel to reach the collection channel outlet according to the first starting time, the first ending time, the second starting time, the second ending time, the collection channel parameter, and the oil phase flow parameter.

[0095] Specifically, the collection channel parameter includes the length and the radius of the collection channel; and the oil phase flow parameter includes the continuous phase oil phase flow and the positive droplet phase oil phase flow.

[0096] The oil droplet formation calculation module determines the time it takes for a positive droplet to travel through the collection channel and reach the collection channel outlet using the following formula:

[0097] ;

[0098] ;

[0099] ;

[0100] ;

[0101] in, The initial moment at which oil droplets form at the collection channel outlet. The first termination moment for the formation of oil droplets at the collection channel outlet. To collect the time interval for oil droplets to form at the channel outlet, The time interval for oil droplets to form at the outlet of the disposal channel. This marks the second initiation point at which oil droplets form at the outlet of the disposal channel. This is the second termination moment when oil droplets form at the outlet of the disposal channel. For continuous phase oil phase flow rate, The flow rate of the positive droplet phase oil phase is... To collect channel traffic, To collect the length of the channel, To collect the radius of the channel, The time it takes for the positive droplet to travel from the collection channel to the collection channel outlet.

[0102] Figure 5 This is a timing diagram showing the separation of positive droplets and the formation of oil droplets at the capillary outlet of the collection channel within a microfluidic device. Figure 5 As shown, the moment when oil droplets form at the outlet of the oil droplet collection channel is obtained by a photoelectric sensor (the time of the oil droplet outlet of the collection channel is...). and ), based on the time interval of oil droplets formed at the collection outlet The time it takes for the target droplet to flow from the "detection-sorting" position in the microfluidic device to the collection channel outlet. and the timing of positive droplet-triggered sorting To determine whether the displacement platform has moved, a standardized orifice plate is used to collect positive droplets, and the position of the orifice plate is also determined. During positive droplet sorting, the flow rate of the collection channel is... Positive droplet collection outlet at time intervals Oil droplets form and fall into the orifice plate, and the timing of their fall is unaffected by the presence or absence of positive droplet deflection. The formation time of the previous oil droplet is... The formation time of this oil droplet is wherein at , the positive droplet exceeding the set threshold triggers sorting, the deflection electrode applies a deflection voltage, the target droplet flows into the droplet collection channel under the action of electrophoretic force, and after , the target droplet flows to the droplet collection capillary outlet, at which time , the software determines the time when the positive droplet is collected by the orifice plate by comparing the relationship between and .

[0103] In another alternative embodiment, the droplet microfluidic sorting system further comprises:

[0104] a droplet deflection module 25 for deflection processing of the positive droplet, and deflection processing of the next positive droplet after a variable deflection cooling time; the deflection cooling time is determined according to the deflection time of the positive droplet, the oil droplet formation time closest to the first time, and the time length of the positive droplet passing through the collection channel to reach the collection channel outlet. Specifically, in order to avoid the problem that multiple positive droplets trigger deflection at a short time interval (less than ), a variable deflection cooling time is added, during which time the positive droplets generated will not trigger deflection, using , and to calculate , and then using and to calculate the end time of cooling , that is , the positive droplets between and no longer trigger sorting.

[0105] In addition, the droplet microfluidic sorting system further comprises:

[0106] an orifice plate control module 26 for moving the collection orifice of the orifice plate to the collection channel outlet to collect the target oil droplet.

[0107] The operation process of sorting the positive droplet is described by way of example as follows. Figure 6 The flowchart of the orifice plate operation for the positive droplet is as follows. First, the device is powered on, the sorting function is not started, and the flow rate and the flow rate of the continuous phase oil phase are input into the software. After the biochemical sample droplet to be sorted and the dispersed phase oil phase begin to flow stably, the software obtains the start time of the collection channel generated oil droplet through two sets of photoelectric sensors and and the time of the generation of the oil droplet and and the time interval of the generation of the oil droplet is calculated and the flow rate distributed to the collection channel is calculated and the capillary length of the collection channel , the radius and the flow rate distributed to the collection channel the time required for the target droplet to flow from the "detection-sorting" position in the microfluidic chip to the outlet of the collection channel is calculated . The second step, the sorting function is turned on, and the initial time of the generation of the oil droplet is refreshed the end time of the generation of the oil droplet is calculated when the positive droplet is detected, the software obtains the time of the positive droplet triggering the sorting the time of the positive droplet flowing to the outlet of the collection channel is calculated . The third step, the software judges the precedence of the end time of the generation of the oil droplet and the time of the positive droplet flowing to the outlet of the collection channel , if the time is prior to the time , the positive droplet will fall with the current oil droplet, then the software controls the displacement platform to move the collection well of the well plate to the outlet of the collection channel to receive the oil droplet wrapping the positive droplet, and calculates the cooling time to avoid the entry of the adjacent positive droplets into the same collection well of the well plate , and the time of the end of the cooling time is calculated , the positive droplet is not subjected to the deflection operation between the time and the time ; if the time is posterior to the time , the positive droplet will fall with the oil droplet after the current oil droplet, then the software controls the displacement platform to move the disposal well of the well plate to the outlet of the collection channel to receive the oil droplet not wrapping the positive droplet, and controls the displacement platform to move the collection well of the well plate to the outlet of the collection channel to receive the next oil droplet wrapping the positive droplet after the disposal of the current oil droplet, the time of the falling of the oil droplet is , and the cooling time to avoid the entry of the adjacent positive droplets into the same collection well of the well plate is calculated , and the time of the end of the cooling time is calculated , the positive droplet is not subjected to the deflection operation between the time and the time .

[0108] The droplet microfluidic sorting system provided by the embodiment determines the time length of the positive droplet passing through the collection channel to reach the outlet of the collection channel through the droplet generation information of the collection channel, the collection channel parameters and the oil phase flow parameter, then detects the first time of triggering sorting of the positive droplet, determines the second time of the positive droplet reaching the outlet of the collection channel according to the first time and the time length of the positive droplet passing through the collection channel to reach the outlet of the collection channel, compares the time of forming the oil droplet closest to the first time and the second time to determine the target oil droplet containing the positive droplet, and sets a variable deflection cooling time to avoid the same oil droplet wrapping multiple positive droplets, so as to realize the automation of sorting a single target droplet, further sort the target liquid into a droplet containing a single target bacteria or cell, and avoid the manual operation of the target liquid for further processing, such as demulsification recovery, limited dilution method distribution, cell loss in the demulsification process, or the phenomenon that there is no target cell in part of the well plate or multiple target cells exist in part of the well plate in the process of limited dilution method distribution, thereby improving the efficiency of obtaining the target sequence.

[0109] Embodiment 3

[0110] Figure 9 The electronic device provided by the embodiment 4 of the present application is shown in a structural schematic diagram. The electronic device includes a memory, a processor, and a computer program stored in the memory and executable on the processor, and the processor implements the droplet microfluidic sorting method of the embodiment 1 when executing the program. Figure 9 The electronic device 30 shown is merely an example and should not impose any limitation on the function and use range of the embodiment of the present application.

[0111] As shown in Figure 9 The electronic device 30 can be in the form of a general computing device, for example, it can be a server device. The components of the electronic device 30 can include but are not limited to the above-mentioned at least one processor 31, the above-mentioned at least one memory 32, and a bus 33 connecting different system components including the memory 32 and the processor 31.

[0112] The bus 33 includes a data bus, an address bus and a control bus.

[0113] The memory 32 can include a volatile memory, such as a random access memory (RAM) 321 and / or a cache memory 322, and can further include a read-only memory (ROM) 323.

[0114] The memory 32 can further include a program / utility 325 having a set of the program modules 324, such as an operating system, one or more application programs, other program modules, and program data, and each of these examples, or some combination thereof, can include implementation of a network environment.

[0115] The processor 31 performs various function applications and data processing by running the computer programs stored in the memory 32, such as the droplet microfluidic sorting method of embodiment 1 of the present application.

[0116] The electronic device 30 can also communicate with one or more external devices 34 such as a keyboard, a pointing device, etc. through an input / output (I / O) interface 35. Further, the model generating device 30 can communicate with one or more networks such as a local area network (LAN), a wide area network (WAN), and / or a public network such as the Internet, through a network adapter 36. As depicted, the network adapter 36 communicates with the other modules of the model generating device 30 through the bus 33. It should be appreciated that other hardware and / or software modules can be used in conjunction with the model generating device 30, such as a microcode, device drivers, redundant processing units, external disk drive arrays, RAID (Redundant Array of Independent Disks) systems, tape drives, and data backup storage systems, etc., although not depicted.

[0117] It should be noted that although several units / modules or sub-units / modules of the electronic device are mentioned in the foregoing detailed description, such a division is merely exemplary and not mandatory. Indeed, according to an embodiment of the application, the features and functions of two or more units / modules described above can be embodied in one unit / module. Conversely, the features and functions of one unit / module described above can be further divided into units / modules embodied by several units / modules.

[0118] Embodiment 4

[0119] The present embodiment provides a computer readable storage medium having stored thereon a computer program, the program being executed by a processor to implement the droplet microfluidic sorting method of embodiment 1.

[0120] More specifically, the readable storage medium can include, but is not limited to, a portable disc, a hard disk, a random access memory, a read-only memory, an erasable programmable read-only memory, an optical storage device, a magnetic storage device, or any suitable combination of the above.

[0121] In a possible implementation, the present application can also be implemented in the form of a program product, which includes program codes for causing a terminal device to perform the droplet microfluidic sorting method of embodiment 1 when the program product is run on the terminal device.

[0122] programmable logic arrays, field programmable gate arrays, programmable logic devices, microcode, etc. In this manner, the program code can implement a virtual machine that operates in response to execution of the program code by the processor or microprocessor-based computer system.

[0123] Although the present application has been described in connection with the embodiments thereof, it will be understood that the application is capable of further modifications. This application is intended to cover any variations, uses or adaptations of the application other than those shown in the specification.

Claims

1. A method of droplet microfluidic sorting, the method comprising: The droplet microfluidic sorting method comprises: acquiring a first time of positive droplet triggered sorting in a microfluidic device; determining a second time of the positive droplet reaching an outlet of a collection channel according to the first time and a time length of the positive droplet passing through the collection channel to reach the outlet of the collection channel; the outlet of the collection channel is used to form an oil droplet; comparing the second time with an oil droplet formation time closest to the first time to determine a target oil droplet containing the positive droplet; the step of comparing the second time with the oil droplet formation time closest to the first time to determine the target oil droplet containing the positive droplet comprises: if the second time is earlier than the oil droplet formation time closest to the first time, the positive droplet falls with the oil droplet closest to the first time, and the oil droplet closest to the first time contains the positive droplet.

2. The droplet microfluidic sorting method of claim 1, wherein, Before the step of determining the second time of the positive droplet reaching the outlet of the collection channel according to the first time and the time length of the positive droplet passing through the collection channel to reach the outlet of the collection channel, the droplet microfluidic sorting method further comprises: acquiring a first start time and a first end time of the outlet of the collection channel forming an oil droplet, a second start time and a second end time of the outlet of a discard channel forming an oil droplet, a collection channel parameter and an oil phase flow parameter; the discard channel is used to pass through an oil droplet not containing the positive droplet; determining the time length of the positive droplet passing through the collection channel to reach the outlet of the collection channel according to the first start time, the first end time, the second start time, the second end time, the collection channel parameter and the oil phase flow parameter.

3. The droplet microfluidic sorting method of claim 2, wherein, The collection channel parameter comprises a length and a radius of the collection channel; the oil phase flow parameter comprises a continuous phase oil phase flow and a positive droplet phase oil phase flow; The time length of the positive droplet passing through the collection channel to reach the outlet of the collection channel is determined according to the following formula: ； ； ； ； wherein, is a first start time at which oil droplets are formed at the outlet of the collection channel, is a first end time at which oil droplets are formed at the outlet of the collection channel, is a time interval during which oil droplets are formed at the outlet of the collection channel, is a time interval during which oil droplets are formed at the outlet of the disposal channel, is a second start time at which oil droplets are formed at the outlet of the disposal channel, is a second end time at which oil droplets are formed at the outlet of the disposal channel, is a flow rate of the continuous phase oil phase, is a flow rate of the positive droplet phase oil phase, is a flow rate of the collection channel, is a length of the collection channel, is a radius of the collection channel, is a time duration for the positive droplet to travel through the collection channel to reach the outlet of the collection channel.

4. The droplet microfluidic sorting method of claim 1, wherein, Before the step of acquiring the first time of positive droplet triggered sorting in a microfluidic device, the droplet microfluidic sorting method further comprises: acquiring a fluorescence signal of a droplet in the microfluidic device, and determining the positive droplet according to the fluorescence signal and a preset threshold.

5. The droplet microfluidic sorting method of claim 4, wherein, After the step of determining the positive droplet, the droplet microfluidic sorting method further comprises: performing deflection processing on the positive droplet, and performing deflection processing on a next positive droplet after a variable deflection cooling time; The deflection cooling time is determined according to a deflection time of the positive droplet, an oil droplet formation time closest to the first time and a time length of the positive droplet passing through the collection channel to reach the outlet of the collection channel.

6. The droplet microfluidic sorting method of claim 1, wherein, After the step of determining the target oil droplet containing the positive droplet, the droplet microfluidic sorting method further comprises: moving a collection well of a well plate to the outlet of the collection channel to collect the target oil droplet.

7. A droplet microfluidic sorting system, characterized in that, The droplet microfluidic sorting system comprises: a positive droplet detection module configured to acquire a first time of positive droplet triggered sorting in a microfluidic device; The oil droplet formation calculation module is used to determine the second time when the positive droplet arrives at the collection channel outlet based on the first time and the time it takes for the positive droplet to reach the collection channel outlet through the collection channel; the collection channel outlet is used to form oil droplets. The target oil droplet determination module is used to compare the oil droplet formation time closest to the first time with the second time to determine the target oil droplet containing the positive droplet; The target oil droplet determination module is used to determine that if the second time is earlier than the oil droplet formation time closest to the first time, the positive droplet falls with the oil droplet closest to the first time, and the oil droplet closest to the first time includes the positive droplet.

8. The droplet microfluidic sorting system of claim 7, wherein, The oil droplet formation calculation module is also used to obtain the first start time and the first end time of oil droplet formation at the collection channel outlet, the second start time and the second end time of oil droplet formation at the disposal channel outlet, collection channel parameters, and oil phase flow parameters; the disposal channel is used to pass oil droplets that do not contain the positive droplets; The oil droplet formation calculation module is also used to determine the time it takes for the positive droplet to reach the collection channel outlet through the collection channel based on the first start time, the first end time, the second start time, the second end time, the collection channel parameters, and the oil phase flow rate parameters.

9. The droplet microfluidic sorting system of claim 8, wherein, The collection channel parameters include the length and radius of the collection channel; the oil phase flow rate parameters include the continuous phase oil phase flow rate and the positive droplet phase oil phase flow rate. The oil droplet formation calculation module determines the time it takes for the positive droplet to reach the collection channel outlet through the collection channel according to the following formula: ； ； ； ； wherein, is a first start time at which oil droplets are formed at the outlet of the collection channel, is a first end time at which oil droplets are formed at the outlet of the collection channel, is a time interval during which oil droplets are formed at the outlet of the collection channel, is a time interval during which oil droplets are formed at the outlet of the disposal channel, is a second start time at which oil droplets are formed at the outlet of the disposal channel, is a second end time at which oil droplets are formed at the outlet of the disposal channel, is a flow rate of the continuous phase oil phase, is a flow rate of the positive droplet phase oil phase, is a flow rate of the collection channel, is a length of the collection channel, is a radius of the collection channel, is a time duration for the positive droplet to travel through the collection channel to reach the outlet of the collection channel.

10. The droplet microfluidic sorting system of claim 7, wherein, The droplet microfluidic sorting system also includes: A positive droplet determination module is used to acquire the fluorescence signal of the droplet in the microfluidic device and determine the positive droplet based on the fluorescence signal and a preset threshold.

11. The droplet microfluidic sorting system of claim 10, wherein, The droplet microfluidic sorting system also includes: A droplet deflection module is used to deflect the positive droplet and, after a variable deflection cooling time, deflect the next positive droplet. The deflection cooling time is determined based on the deflection time of the positive droplet, the formation time of the droplet closest to the first time, and the time it takes for the positive droplet to reach the outlet of the collection channel.

12. The droplet microfluidic sorting system of claim 7, wherein, The droplet microfluidic sorting system also includes: An orifice plate control module is used to move the collection holes of the orifice plate to the outlet of the collection channel to collect the target oil droplets.

13. An electronic device comprising a memory, a processor, and a computer program stored on the memory and executable on the processor, characterized in that, When the processor executes the computer program, it implements the droplet microfluidic sorting method as described in any one of claims 1 to 6.

14. A computer readable storage medium having stored thereon computer instructions, wherein, The computer instructions, when executed by the processor, implement the droplet microfluidic sorting method as described in any one of claims 1 to 6.

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