A continuous sample injection digital microfluidic cell sorting system and method based on capacitive detection feedback

Abstract

This invention discloses a continuous injection digital microfluidic cell sorting system and method based on capacitance detection feedback. The system includes: a digital microfluidic chip for providing a droplet manipulation substrate; a capacitance detection module for detecting capacitance signals in a target area; a syringe pump module for replenishing sample fluid into the injection area; a high-pressure drive module for controlling the droplet movement on the digital microfluidic chip; an image acquisition and recognition module for acquiring droplet images from the digital microfluidic chip and identifying cell information within the droplets to obtain cell identification results; and a controller connected to the high-pressure drive module, capacitance detection module, syringe pump module, and image acquisition and recognition module, used to trigger the syringe pump module to quantitatively replenish fluid based on capacitance signals and to control the high-pressure drive module to guide the droplets to the corresponding area based on the cell identification results. This invention improves the continuous operational stability of the digital microfluidic cell sorting process.

Description

Technical Field

[0001] This invention belongs to the fields of digital microfluidics, biochips, droplet manipulation and automated cell sorting technology, specifically relating to a continuous injection digital microfluidic cell sorting system and method based on capacitance detection feedback. Background Technology

[0002] Digital microfluidics, based on the principle of electrowetting, enables the generation, transport, splitting, merging, and routing control of discrete droplets on patterned electrode arrays. It offers advantages such as low sample consumption, programmable flow, and easy integration with microscopic imaging and image recognition systems. Based on these characteristics, digital microfluidic platforms have been used in scenarios such as single-cell manipulation, micro-reactions, and cell sorting. In existing digital microfluidic cell sorting systems, droplet movement is typically controlled by a high-pressure drive sequence, and an image acquisition and recognition module determines whether the droplet contains the target cell, then directs different droplets to different outlets. However, during long-term continuous operation, the liquid coverage in the sample injection area, buffer zone, or droplet generation area can change due to sample consumption, evaporation, pipeline pressure fluctuations, or changes in the chip surface condition. When the liquid volume in the waiting area is insufficient, even if the driving electrode applies a normal voltage, problems such as droplet failure to generate, unstable generation volume, or intermittent droplet generation may occur. Existing solutions mostly rely on manual observation or open-loop liquid supply methods to replenish the sample liquid. Manual fluid replenishment struggles to respond promptly to changes in liquid coverage, while open-loop continuous fluid supply can easily lead to over-injection, droplet size fluctuations, or liquid accumulation within the chip. Furthermore, fluid replenishment control at the supply end is typically independent of downstream droplet identification and sorting routing, lacking a unified scheduling mechanism, making it difficult for the system to stably perform continuous sorting over extended periods. Therefore, there is an urgent need for a continuous injection digital microfluidic cell sorting system capable of online sensing of the liquid coverage status of the waiting area, automatically triggering quantitative fluid replenishment based on detection results, and simultaneously coordinating with droplet generation, image recognition, and sorting routing. Summary of the Invention

[0003] To address the problems of timely detection of liquid shortage in the waiting area, open-loop sample supply, and disconnect between the replenishment process and downstream sorting process in existing digital microfluidic cell sorting systems, this invention provides a continuous injection digital microfluidic cell sorting system and method based on capacitive feedback replenishment. The system collects capacitance signals from at least one of the injection area, buffer zone, and droplet generation area using a capacitance detection module. The controller determines the liquid coverage status based on the capacitance signals. When the capacitance signals meet a preset liquid shortage criterion, the injection pump performs a quantitative replenishment. After replenishment, droplet generation resumes, and droplet sorting is completed using image recognition results.

[0004] To achieve the above objectives, the present invention provides a continuous injection digital microfluidic cell sorting system based on capacitance detection feedback, comprising: A digital microfluidic chip, comprising a sample injection area, a buffer zone, a droplet generation area, a droplet transport area, a central detection area, a sorting and processing area, a target collection area, a waste liquid area, and a post-processing area; The capacitance detection module is connected to a sensing electrode corresponding to at least one of the injection area, buffer zone and droplet generation area, and is used to collect capacitance signals reflecting the liquid coverage state of the corresponding area. The outlet of the syringe pump sample supply module is fluidly connected to the inlet of the sample injection area of ​​the digital microfluidic chip, and is used to replenish the sample liquid to the sample injection area; A high-voltage drive module is connected to the drive electrode array of the digital microfluidic chip and is used to drive droplets to be generated, transported and sorted on the digital microfluidic chip. The image acquisition and recognition module is used to acquire images of droplets within the detection area and identify cell information within the droplets to obtain cell recognition results. The controller is connected to the capacitance detection module, the syringe pump sample supply module, the high-voltage drive module, and the image acquisition and recognition module, respectively. The controller is used to trigger the syringe pump sample supply module to quantitatively replenish fluid based on the capacitance signal, and to control the high-voltage drive module to guide the droplets to the corresponding area based on the cell recognition result.

[0005] Optionally, the digital microfluidic chip adopts a parallel plate structure, and the digital microfluidic chip includes an upper cover plate, a lower cover plate, and a spacing control structure; The lower cover plate is provided with a patterned electrode array, a dielectric layer and a hydrophobic layer; The upper cover plate is provided with a grounding conductive layer and a liquid injection hole.

[0006] Optionally, the droplet generation region employs a meniscus-shaped droplet generation electrode, which includes a separation electrode; The end feature dimension of the separation electrode is 0.3mm-0.8mm; The dielectric layer is a SU-8 photoresist layer. When the feature size of the droplet generation electrode is in the range of 0.5mm-0.8mm, the chip gap is 250μm. When the feature size of the droplet generation electrode is in the range of 0.3mm-0.5mm, the chip gap is 60μm.

[0007] Optionally, the capacitance detection module uses a capacitance-to-digital converter chip to communicate with the controller; The communication methods between the capacitor-to-digital converter chip and the controller include: SPI communication, I... 2 C-type communication and serial communication interface; The detection channel of the capacitance detection module includes a reference channel and several sensing channels; the sensing channels are connected to the sensing electrodes corresponding to at least one of the sample injection area, buffer zone and droplet generation area.

[0008] Optionally, the high-voltage drive module includes a 0–300V adjustable high-voltage source and two cascaded HV507 high-voltage drive chips.

[0009] Optionally, the system further includes an isolation switching unit, which is connected between the sensing electrode of the digital microfluidic chip and the high-voltage driving module and the capacitance detection module; Before capacitance sampling, the controller controls the high-voltage drive module to stop outputting, and after a preset discharge time, controls the isolation switching unit to switch the sensing electrode from the high-voltage drive path to the capacitance detection path. After sampling is completed, the capacitance detection path is disconnected and the high-voltage drive path is restored.

[0010] Optionally, the controller triggers the injection pump sample supply module to perform a quantitative fluid replenishment process based on the capacitance signal, including: controlling the injection pump sample supply module to perform a quantitative fluid replenishment when the capacitance signal meets the preset fluid shortage criterion; The preset liquid shortage criterion includes three methods: the capacitance value of a single sampling is lower than the preset threshold, the capacitance value of multiple consecutive samplings is lower than the preset threshold, and the capacitance value after filtering is lower than the preset threshold. The determination of whether the quantitative fluid replenishment is completed includes four methods: the injection pump completes a preset stroke, a preset injection volume is reached, a preset injection time is reached, and the fluid supply corresponding to a preset number of droplets that can be generated is reached.

[0011] Optionally, the image acquisition and recognition module employs trifocal bright-field imaging; The three-focal-plane bright-field imaging includes a central focal plane and two additional focal planes located 5-30 μm above and below the central focal plane, which are used to obtain the spatial distribution information of cells in the droplet and to obtain cell identification results based on the cell physical characteristics to determine the cell information in the droplet. The cell physical characteristics include area, roundness, grayscale distribution, edge contour, and focal plane position features.

[0012] Optionally, when the cell recognition result is a target single-cell droplet, the controller controls the droplet to be guided to the target collection area; When the cell recognition result is an empty droplet or a non-target droplet, the controller guides the droplet to the waste liquid area; When the cell identification result is a multi-cell droplet, the controller directs the droplet to the subsequent processing area.

[0013] This invention also provides a continuous injection digital microfluidic cell sorting method based on capacitance detection feedback, for implementing the aforementioned system, the method comprising the following steps: S1. When the high-voltage drive is off or in a preset sampling window, perform capacitance sampling on at least one of the injection area, buffer area and droplet generation area of ​​the digital microfluidic chip to obtain a capacitance signal reflecting the liquid coverage state. S2. Determine whether the capacitance signal meets the preset liquid shortage criterion; if it does, control the injection pump to perform a quantitative liquid replenishment to the injection area; S3. Stop the infusion when the infusion pump has completed the determination criteria for quantitative fluid refill; S4. Control the high-voltage drive module to generate droplets on the digital microfluidic chip and transport the droplets to the central detection area; S5. Acquire images of droplets in the central detection area, identify the number and type of cells within the droplets, and obtain cell identification results; S6. Based on the cell recognition results, the target single-cell droplet is directed to the target collection area, and the empty droplet or non-target droplet is directed to the waste liquid area. The multi-cell droplet is directed to the subsequent processing area. S7. Repeat S1 to S6 to achieve automated cell sorting under continuous sample injection conditions.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects: First, this invention uses a capacitance detection module to detect the liquid coverage status of at least one of the injection area, buffer zone, and droplet generation area in real time or periodically, and uses the capacitance signal as a trigger signal for the injection pump to replenish liquid, thus changing the injection pump from open-loop liquid supply to on-demand quantitative liquid replenishment, thereby reducing the risk of droplet generation interruption due to liquid shortage. Second, this invention uses a controller to uniformly schedule capacitance detection, injection pump replenishment, high-pressure droplet generation, droplet transport, image recognition, and sorting routing, so that the upstream liquid supply status and the downstream sorting status are coordinated, avoiding the separation of control logic between the liquid supply end and the sorting end. Third, this invention can achieve online detection of the liquid waiting area without significantly increasing the complexity of the chip structure by time-division multiplexing the high-voltage drive path and the capacitance detection path, reducing the interference of the high-voltage drive on the capacitance detection, and improving the stability of the continuous injection process. Attached Figure Description

[0015] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings: Figure 1 This is a schematic diagram of the detection process and steps of the continuous injection digital microfluidic cell sorting method according to an embodiment of the present invention; Figure 2 This is a schematic diagram of the control flow and sorting pattern of the continuous injection digital microfluidic cell sorting system according to an embodiment of the present invention. Detailed Implementation

[0016] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0017] It should be noted that the steps shown in the flowchart in the accompanying drawings can be executed in a computer system such as a set of computer-executable instructions, and although a logical order is shown in the flowchart, in some cases the steps shown or described may be executed in a different order than that shown here.

[0018] Example 1 This embodiment provides a continuous injection digital microfluidic cell sorting system and method based on capacitance detection feedback to solve problems in existing technologies such as difficulty in real-time detection of liquid shortage in the injection zone and droplet generation zone, unstable continuous sample supply, and disconnect between upstream replenishment and downstream sorting. This invention introduces capacitance detection control into the digital microfluidic cell sorting system. By sensing the liquid coverage status of the injection zone and buffer zone in real time, the system triggers the injection pump to perform quantitative replenishment when the capacitance value is lower than a preset threshold. After replenishment, droplet generation is automatically restored, thus forming a liquid supply-consumption closed loop, significantly improving the stability of continuous droplet generation and the reliability of sample supply. Simultaneously, this invention unifies upstream liquid supply feedback control with downstream droplet generation, transport, image detection, and droplet sorting within the same state machine scheduling framework, solving the problem of disconnected control logic between the liquid supply end and the sorting end. This enables the system to maintain stable droplet production, high-purity target cell sorting, and efficient automated processing capabilities under long-term continuous operation.

[0019] like Figure 1 As shown, the continuous injection digital microfluidic cell sorting method of the present invention includes the following steps: first, capacitance detection is performed; when insufficient liquid coverage is detected in the target area, a liquid shortage judgment is made and the injection pump is triggered to replenish the sample; after replenishment, droplet generation and transport are performed; then, image acquisition and recognition are performed; finally, the droplets are sorted and guided according to the recognition results, and the above process is repeated to realize automated cell sorting under continuous injection conditions.

[0020] like Figure 2 As shown, the system control flow of this invention includes steps such as syringe pump sample replenishment, capacitance detection, droplet detection, and droplet identification and classification. Specifically, the sample liquid is injected into the digital microfluidic chip via the syringe pump. The capacitance detection unit determines the liquid coverage state, and the image recognition unit identifies cell information within the droplets. Based on the recognition results, the droplets are guided to the target outlet, waste liquid outlet, or subsequent processing area, thereby completing continuous sample injection and automatic sorting.

[0021] As a specific implementation of this embodiment, this embodiment provides a continuous injection digital microfluidic cell sorting system based on capacitance detection feedback. The system includes: a digital microfluidic chip, a high-voltage drive module, a capacitance detection module, an injection pump sample supply module, an image acquisition and recognition module, and a controller.

[0022] The system employs a parallel-plate digital microfluidic chip, measuring 75×74mm, containing 128 independently addressable electrodes. The parallel-plate digital microfluidic chip comprises an upper cover, a lower cover, and a spacing control structure. The lower cover houses a patterned electrode array, a dielectric layer, and a hydrophobic layer, while the upper cover houses a grounded conductive layer and injection holes. The chip is sequentially divided into a sample injection area, a buffer zone, a droplet generation area, a droplet transport area, a central detection area, a sorting and processing area, a target collection area, and a waste liquid area. The storage area measures 8mm×15mm, and the buffer zone measures 3mm×6mm. The droplet generation area utilizes a meniscus-shaped droplet generation electrode, with a feature dimension d at the electrode tip. min The chip spacing is 0.3–0.8 mm, preferably 0.3 mm; the dielectric layer thickness is preferably a 5 μm SU-8 photoresist layer. When the droplet generation electrode feature size is in the range of 0.6–0.8 mm, the chip gap is preferably 250 μm; when the droplet generation electrode feature size is in the range of 0.3–0.5 mm, the chip gap is preferably 60 μm. The system can manipulate a droplet volume of approximately 10 nL per cycle. It should be noted that the chip size, number of electrodes, and area size can be adjusted according to the droplet volume, throughput, and detection requirements, and are not limited to the values ​​mentioned above.

[0023] The system controller can be an STM32F103ZET6 microcontroller; the high-voltage drive module includes a 0–300V adjustable high-voltage source and two cascaded HV507 high-voltage drive chips to achieve 128 independent channels of drive.

[0024] The capacitance detection module is connected to the sensing electrodes corresponding to the injection area, buffer zone, and / or droplet generation area to detect the capacitance signal of the target area and reflect the liquid coverage status. The capacitance detection module uses a Pcap01 capacitance-to-digital converter chip and communicates with the STM32 controller via an SPI interface. The detection channels include one reference channel and at least seven sensing channels.

[0025] The syringe pump sample supply module is connected to the sample storage container and the sample inlet of the digital microfluidic chip, and is used to replenish sample liquid to the injection area. The controller uses a single threshold-triggered quantitative liquid replenishment control method to control the start and stop of the syringe pump: when the capacitance value is lower than the preset threshold, the syringe pump is started to perform a quantitative liquid replenishment; when the syringe pump completes the preset stroke, preset injection volume, or corresponding preset number of droplets to be generated, the syringe pump is stopped and droplet generation resumes. In this way, the syringe pump is no longer a continuous open-loop liquid supply, but becomes an on-demand triggered quantitative liquid replenishment execution unit.

[0026] The image acquisition and recognition module is used to acquire images of droplets in the detection area and identify the number and / or type of cells within the droplets.

[0027] The controller is connected to the high-voltage drive module, the capacitance detection module, the syringe pump sample supply module, and the image acquisition and recognition module. The controller is configured to: determine whether the injection area, buffer zone, and / or droplet generation area meet the droplet generation conditions based on the capacitance signal output by the capacitance detection module; determine the completion of quantitative fluid replenishment based on any one of the following: the syringe pump completing a preset stroke, reaching a preset injection volume, or reaching a preset supply volume corresponding to the number of droplets that can be generated; control the droplet guidance to the target collection area, waste liquid area, or subsequent processing area based on the recognition results output by the image acquisition and recognition module; and integrate fluid supply feedback control, droplet generation, droplet transport, image detection, and droplet sorting into a unified state machine scheduling framework to achieve automated cell sorting under continuous injection conditions.

[0028] Before system operation, the sensing electrodes can be threshold calibrated. Specifically, the first capacitance reference value C is acquired under conditions of no liquid coverage. air The second capacitance reference value C was collected under the condition of target liquid coverage. liquid According to C air and C liquid The difference between them determines the fluid shortage threshold C. th Preferably, C th Option C is acceptable. air With C liquid The intermediate value between these values, or determined based on the mean and standard deviation of multiple repeated measurements. In actual operation, when the real-time capacitance value is lower than C... th Or, the value of k consecutive samples is lower than C th When insufficient liquid coverage is detected in the corresponding area, the controller employs a single-threshold triggered quantitative replenishment control method to control the syringe pump sample supply module. The buffer capacitance threshold is 80 pF; under air or oil phase conditions, the sensing electrode capacitance is approximately 55 pF; when droplets cover the middle and large electrodes, the capacitance values ​​are approximately 130.4 pF and 167.2 pF, respectively. Each time replenishment is triggered, the syringe pump executes a preset stroke to replenish the sample injection area with the liquid volume required to generate a preset number of droplets.

[0029] In one embodiment, the system includes an isolation switching unit connected between the sensing electrode of the digital microfluidic chip and the high-voltage drive module and capacitance detection module. During the droplet driving phase, the controller connects the sensing electrode to the high-voltage drive path and disconnects the capacitance detection path. During the capacitance sampling phase, the controller first controls the high-voltage drive module to stop output or enter a safe potential state. After a preset discharge time, the controller then controls the isolation switching unit to switch the sensing electrode to the capacitance detection path to collect capacitance signals reflecting the liquid coverage status of the injection area, buffer zone, and / or droplet generation area. After sampling, the controller disconnects the capacitance detection path and restores the high-voltage drive path. The capacitance signal triggers the syringe pump sample supply module to perform quantitative liquid replenishment. After replenishment, the liquid coverage status is confirmed by resampling to determine whether to resume droplet generation and cell sorting processes. When the identification result is a target single-cell droplet, the controller directs the droplet to the target collection area; when the identification result is an empty droplet or a non-target droplet, the controller directs the droplet to the waste liquid area; when the identification result is a multi-cell droplet, the controller directs the droplet to the subsequent processing area.

[0030] In this specific implementation, the controller first shuts off the high-voltage drive in each scheduling cycle and switches to the capacitance detection path via a relay to sample the sensing electrodes in the buffer and storage areas. Experimental calibration shows that the sensing capacitance value is approximately 55 pF under air or oil phase conditions; when droplets cover the middle and large electrodes, the capacitance values ​​are approximately 130.4 pF and 167.2 pF, respectively. Therefore, this embodiment sets the buffer insufficient liquid volume judgment threshold to 80 pF and the storage area insufficient liquid volume judgment threshold to 90 pF. When the real-time capacitance value of the buffer is lower than 80 pF, the controller starts the injection pump to replenish the sample injection area; the controller triggers the injection pump to perform a quantitative liquid replenishment; under a preferred operating condition, the single quantitative liquid replenishment volume is 0.5-15 μL, or corresponds to the continuous generation of a preset number of standard microdroplets; after the liquid replenishment is completed, the controller resumes the cell sorting process.

[0031] During continuous sorting, the system uses approximately 10 nL droplets as the basic control unit. The drive distance step time Δt is set to 0.50 s, the arrival interval step number Narr is set to 4, and the corresponding droplet generation frequency G is 0.5 Hz. The target sorting quantity Ntarget is set to 200. The controller's operating logic is as follows: first, capacitance sampling is performed to determine whether generation is allowed; then, position updates and routing processing are performed on the existing droplets on the chip; when droplets are present in the detection area, image acquisition and recognition are performed; finally, new droplet generation is performed only when the liquid supply status meets the requirements, the generation area is idle, and the downstream congestion is below the threshold. Through the above logic, the system balances the continuous upstream liquid supply with the limited downstream processing capacity.

[0032] In this embodiment, image acquisition employs trifocal bright-field imaging, including a central focal plane and two additional focal planes located 15 μm above and below the central focal plane, used to acquire spatial distribution information of cells within the droplet. The image recognition module extracts the area and roundness features of the cells to determine whether the droplet is a cell-free droplet, a single-cell droplet, or a multi-cell droplet, and further distinguishes between target cells and non-target cells. Preferably, the processing time for a single frame image is less than 100 ms, and the total decision time for a single droplet is less than 500 ms. For algal samples, target cells may include at least one of Haematococcus pluvialis, Chlorella vulgaris, and Synechococcus. When the recognition result is a target single-cell droplet, the controller guides the droplet to the target collection area; when the recognition result is an empty droplet or a non-target droplet, it guides it to the waste liquid area; when the recognition result is a multi-cell droplet, it guides it to the subsequent processing area. Under preferred operating conditions, the system can achieve a sorting purity of 97.9%, a recovery rate of over 98.1%, and an output capacity of 20 target droplets / min. The image acquisition and recognition module can employ bright-field imaging, fluorescence imaging, multi-focal-plane bright-field imaging, or a combination thereof. In a preferred embodiment, trifocal-plane bright-field imaging is used, including a central focal plane and two additional focal planes located 15 μm above and below the central focal plane. Recognition features include, but are not limited to, cell area, roundness, grayscale intensity, edge contour, color information, fluorescence intensity, and focal plane sharpness.

[0033] This invention extends capacitance detection from simply determining the presence of liquid to providing a continuous injection control signal, enabling the syringe pump to switch from open-loop sample supply to on-demand liquid replenishment, which significantly improves the stability of continuous droplet formation. Existing experiments have shown that after introducing capacitance detection, the number of droplets formed under culture medium conditions increased from (37.0±3.4) to (59.2±0.8), and under water conditions, it increased from (38.2±2.3) to (59.8±0.4), with the coefficients of variation (CV) decreasing from 9.17% and 5.97% to 1.41% and 0.75%, respectively.

[0034] This invention unifies the upstream liquid supply feedback closed loop and the downstream visual sorting closed loop into the same controller and the same state machine scheduling framework, solving the problem of the separation in traditional systems where "liquid supply relies on manual labor and sorting relies on automation".

[0035] This invention introduces continuous sample injection control on the basis of existing cell digital microfluidic sorting platforms, which can operate continuously with approximately 10 nL droplets, a step size of 0.50 s, and a generation frequency of 0.5 Hz, while maintaining 97.9% purity, a recovery rate of over 98.1%, and an output capacity of 20 target droplets / min.

[0036] This embodiment also provides a continuous injection digital microfluidic cell sorting method based on capacitance detection feedback, including the following steps: S1, Capacitive sampling is performed on the injection area and / or droplet generation area to obtain a capacitance signal reflecting the liquid coverage state; S2, when the capacitance value is lower than the preset threshold, it is determined that the corresponding area is not adequately covered by liquid, and the injection pump is started to perform a quantitative liquid replenishment; S3: When the syringe pump completes the liquid supply corresponding to the preset stroke, preset liquid volume, or preset number of droplets that can be generated, stop the liquid replenishment and resume droplet generation; S4, the generated droplets are transported to the detection area for image acquisition and recognition, and the results of cell number and / or cell type recognition are obtained in the droplets; S5, based on the identification results, controls the droplet to be directed to the target collection area, waste liquid area or subsequent treatment area; S6. Repeat steps S1 to S5 to achieve automated cell sorting under continuous injection conditions.

[0037] When the sample is algal cells, the target cells include at least one of Haematococcus pluvialis, Chlorella vulgaris, and Synechococcus, and the identification features include cell area and roundness.

[0038] Example 2 A continuous injection digital microfluidic cell sorting system based on capacitance detection feedback includes: a digital microfluidic chip, comprising an injection zone, a buffer zone, a droplet generation zone, a droplet transport zone, a central detection zone, a sorting and processing zone, a target collection zone, a waste liquid zone, and a post-processing zone; a capacitance detection module connected to a sensing electrode corresponding to at least one of the injection zone, buffer zone, and droplet generation zone, for acquiring capacitance signals reflecting the liquid coverage state of the corresponding zone; an injection pump module, the outlet of which is fluidly connected to the injection inlet of the digital microfluidic chip, for replenishing sample liquid to the injection zone; and a high-pressure drive module connected to the drive electrode array of the digital microfluidic chip, for driving the generation, transport, and sorting of droplets on the digital microfluidic chip. The system includes a sorting module; an image acquisition and recognition module for acquiring droplet images within the detection area and identifying the number and / or type of cells within the droplets to obtain cell identification results; and a controller connected to the capacitance detection module, the syringe pump sample supply module, the high-voltage drive module, and the image acquisition and recognition module. The controller is configured to: acquire capacitance signals within a preset sampling window after the high-voltage drive module stops outputting or enters a safe potential; control the syringe pump sample supply module to perform a quantitative fluid replenishment when the capacitance signal meets a preset fluid shortage criterion; resume droplet generation after quantitative fluid replenishment is completed and the liquid coverage condition is confirmed to meet the droplet generation conditions; and control the high-voltage drive module to guide the droplets to the target collection area, waste liquid area, or subsequent processing area based on the cell identification results.

[0039] The digital microfluidic chip adopts a parallel plate structure, which includes an upper cover plate, a lower cover plate, and a spacing control structure. The lower cover plate is provided with a patterned electrode array, a dielectric layer, and a hydrophobic layer. The upper cover plate is provided with a grounded conductive layer and a liquid injection hole. The digital microfluidic chip is divided into a sample injection area, a buffer zone, a droplet generation area, a droplet transport area, a central detection area, a sorting and processing area, a target collection area, and a waste liquid area.

[0040] The droplet generation region employs a meniscus-shaped droplet generation electrode, which includes a separation electrode. The end feature size of the separation electrode is 0.3 mm to 0.8 mm. The dielectric layer is a SU-8 photoresist layer. When the feature size of the droplet generation electrode is in the range of 0.5 mm to 0.8 mm, the chip gap is 250 μm. When the feature size of the droplet generation electrode is in the range of 0.3 mm to 0.5 mm, the chip gap is 60 μm.

[0041] The capacitance detection module uses a capacitance-to-digital converter chip and communicates with the controller via an SPI interface. The detection channel of the capacitance detection module includes a reference channel and several sensing channels. The sensing channels are connected to the sensing electrodes corresponding to at least one of the sample injection area, buffer zone, and droplet generation area.

[0042] The high-voltage drive module includes a 0–300V adjustable high-voltage source and two cascaded HV507 high-voltage drive chips.

[0043] The capacitance detection module includes a capacitance-to-digital converter chip, which communicates via SPI and I / O. 2 It communicates with the controller via a C or serial communication interface.

[0044] The system also includes an isolation switching unit connected between the sensing electrode of the digital microfluidic chip and the high-voltage drive module and capacitance detection module. Before capacitance sampling, the controller controls the high-voltage drive module to stop output or enter a safe potential state, and after a preset discharge time, controls the isolation switching unit to switch the sensing electrode from the high-voltage drive path to the capacitance detection path. After capacitance sampling is completed, the controller controls the isolation switching unit to disconnect the capacitance detection path and restore the high-voltage drive path. The isolation switching unit includes at least one of a relay, a solid-state relay, a high-voltage analog switch, an optocoupler disconnect switch, or a switch matrix.

[0045] The preset liquid shortage criterion includes any of the following: the capacitance value of a single sampling is lower than the preset threshold; the capacitance value of multiple consecutive samplings is lower than the preset threshold; or the capacitance value after filtering is lower than the preset threshold.

[0046] The criteria for determining whether quantitative fluid replenishment is completed include: the injection pump completing a preset stroke, reaching a preset injection volume, reaching a preset injection time, or reaching a supply volume corresponding to a preset number of droplets that can be generated.

[0047] The controller performs capacitance detection, liquid shortage judgment, quantitative liquid replenishment, droplet generation, droplet transport, image recognition, and droplet sorting according to the state machine. Furthermore, the controller only controls the high-voltage drive module to generate new droplets when the liquid supply status meets the requirements, the droplet generation area is idle, and the downstream droplet congestion status is below a preset threshold.

[0048] The image acquisition and recognition module adopts trifocal bright-field imaging; trifocal bright-field imaging includes a central focal plane and two additional focal planes located 5-30 μm above and below the central focal plane, which are used to acquire spatial distribution information of cells in the droplet, and identify the number of cells and / or cell type in the droplet based on cell area, roundness, gray scale distribution, edge contour and / or focal plane position features.

[0049] When the cell identification result is a target single-cell droplet, the controller directs the droplet to the target collection area; when the cell identification result is an empty droplet or a non-target droplet, the controller directs the droplet to the waste liquid area; when the cell identification result is a multi-cell droplet, the controller directs the droplet to the subsequent processing area.

[0050] A continuous injection digital microfluidic cell sorting method based on capacitance detection feedback includes the following steps: S1, when the high-voltage drive is off or in a preset sampling window, capacitance sampling is performed on at least one region of the injection area, buffer zone, and droplet generation area of ​​the digital microfluidic chip to obtain a capacitance signal reflecting the liquid coverage state; S2, it is determined whether the capacitance signal meets the preset liquid shortage criterion; if it does, the injection pump is controlled to perform a quantitative liquid replenishment to the injection area; S3, when the injection pump completes the preset stroke, reaches the preset injection volume, reaches the preset injection time, or reaches the preset injection time, the cell sorting method is further refined. When the preset supply volume corresponding to the number of droplets that can be generated is reached, the replenishment is stopped; S4, the high-pressure drive module is controlled to generate droplets on the digital microfluidic chip and transport the droplets to the detection area; S5, droplet images in the detection area are acquired, the number of cells and / or cell type in the droplets are identified, and cell identification results are obtained; S6, based on the cell identification results, the target single-cell droplets are guided to the target collection area, the empty droplets or non-target droplets are guided to the waste liquid area, and the multi-cell droplets are guided to the subsequent processing area; S7, S1 to S6 are executed in a loop to realize automated cell sorting under continuous sample injection conditions.

[0051] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A continuous injection digital microfluidic cell sorting system based on capacitance detection feedback, characterized in that, include: A digital microfluidic chip, comprising a sample injection area, a buffer zone, a droplet generation area, a droplet transport area, a central detection area, a sorting and processing area, a target collection area, a waste liquid area, and a post-processing area; The capacitance detection module is connected to a sensing electrode corresponding to at least one of the injection area, buffer zone and droplet generation area, and is used to collect capacitance signals reflecting the liquid coverage state of the corresponding area. The outlet of the syringe pump sample supply module is fluidly connected to the inlet of the sample injection area of ​​the digital microfluidic chip, and is used to replenish the sample liquid to the sample injection area; A high-voltage drive module is connected to the drive electrode array of the digital microfluidic chip and is used to drive droplets to be generated, transported and sorted on the digital microfluidic chip. The image acquisition and recognition module is used to acquire images of droplets within the detection area and identify cell information within the droplets to obtain cell recognition results. The controller is connected to the capacitance detection module, the syringe pump sample supply module, the high-voltage drive module, and the image acquisition and recognition module, respectively. The controller is used to trigger the syringe pump sample supply module to quantitatively replenish fluid based on the capacitance signal, and to control the high-voltage drive module to guide the droplets to the corresponding area based on the cell recognition result.

2. The continuous injection digital microfluidic cell sorting system based on capacitance detection feedback according to claim 1, characterized in that, The digital microfluidic chip adopts a parallel plate structure and includes an upper cover plate, a lower cover plate, and a spacing control structure. The lower cover plate is provided with a patterned electrode array, a dielectric layer and a hydrophobic layer; The upper cover plate is provided with a grounding conductive layer and a liquid injection hole.

3. The continuous injection digital microfluidic cell sorting system based on capacitance detection feedback according to claim 2, characterized in that, The droplet generation region employs a meniscus-shaped droplet generation electrode, which includes a separation electrode; The end feature dimension of the separation electrode is 0.3mm-0.8mm; The dielectric layer is a SU-8 photoresist layer. When the feature size of the droplet generation electrode is in the range of 0.5mm-0.8mm, the chip gap is 250μm. When the feature size of the droplet generation electrode is in the range of 0.3mm-0.5mm, the chip gap is 60μm.

4. The continuous injection digital microfluidic cell sorting system based on capacitance detection feedback according to claim 2, characterized in that, The capacitance detection module uses a capacitance-to-digital converter chip to communicate with the controller. The communication methods between the capacitor-to-digital converter chip and the controller include: SPI communication, I... 2 C-type communication and serial communication interface; The detection channel of the capacitance detection module includes a reference channel and several sensing channels; the sensing channels are connected to the sensing electrodes corresponding to at least one of the sample injection area, buffer zone and droplet generation area.

5. The continuous injection digital microfluidic cell sorting system based on capacitance detection feedback according to claim 1, characterized in that, The high-voltage drive module includes a 0–300 V adjustable high-voltage source and two cascaded HV507 high-voltage drive chips.

6. The continuous injection digital microfluidic cell sorting system based on capacitance detection feedback according to claim 1, characterized in that, The system also includes an isolation switching unit, which is connected between the sensing electrode of the digital microfluidic chip and the high-voltage driving module and the capacitance detection module. Before capacitance sampling, the controller controls the high-voltage drive module to stop outputting, and after a preset discharge time, controls the isolation switching unit to switch the sensing electrode from the high-voltage drive path to the capacitance detection path. After sampling is completed, the capacitance detection path is disconnected and the high-voltage drive path is restored.

7. The continuous injection digital microfluidic cell sorting system based on capacitance detection feedback according to claim 1, characterized in that, The controller, based on the capacitance signal, triggers the injection pump sampling module to perform a quantitative fluid replenishment process, including: when the capacitance signal meets the preset fluid shortage criterion, controlling the injection pump sampling module to perform a quantitative fluid replenishment; The preset liquid shortage criterion includes three methods: the capacitance value of a single sampling is lower than the preset threshold, the capacitance value of multiple consecutive samplings is lower than the preset threshold, and the capacitance value after filtering is lower than the preset threshold. The determination of whether the quantitative fluid replenishment is completed includes four methods: the injection pump completes a preset stroke, a preset injection volume is reached, a preset injection time is reached, and the fluid supply corresponding to a preset number of droplets that can be generated is reached.

8. The continuous injection digital microfluidic cell sorting system based on capacitance detection feedback according to claim 1, characterized in that, The image acquisition and recognition module uses trifocal bright-field imaging; The three-focal-plane bright-field imaging includes a central focal plane and two additional focal planes located 5-30 μm above and below the central focal plane, which are used to obtain the spatial distribution information of cells in the droplet and to obtain cell identification results based on the cell physical characteristics to determine the cell information in the droplet. The cell physical characteristics include area, roundness, grayscale distribution, edge contour, and focal plane position features.

9. The continuous injection digital microfluidic cell sorting system based on capacitance detection feedback according to claim 1, characterized in that, When the cell recognition result is a target single-cell droplet, the controller guides the droplet to the target collection area; When the cell recognition result is an empty droplet or a non-target droplet, the controller guides the droplet to the waste liquid area; When the cell identification result is a multi-cell droplet, the controller directs the droplet to the subsequent processing area.

10. A continuous injection digital microfluidic cell sorting method based on capacitance detection feedback, characterized in that, For implementing the system as described in claim 1, the method includes the following steps: S1. When the high-voltage drive is off or in a preset sampling window, perform capacitance sampling on at least one of the injection area, buffer area and droplet generation area of ​​the digital microfluidic chip to obtain a capacitance signal reflecting the liquid coverage state. S2. Determine whether the capacitance signal meets the preset liquid shortage criterion; if it does, control the injection pump to perform a quantitative liquid replenishment to the injection area; S3. When the infusion pump completes the quantitative fluid replenishment, the decision to stop fluid replenishment is based on the completion of the procedure. S4. Control the high-voltage drive module to generate droplets on the digital microfluidic chip and transport the droplets to the central detection area; S5. Acquire images of droplets in the central detection area, identify the number and type of cells within the droplets, and obtain cell identification results; S6. Based on the cell recognition results, the target single-cell droplet is directed to the target collection area, and the empty droplet or non-target droplet is directed to the waste liquid area. The multi-cell droplet is directed to the subsequent processing area. S7. Repeat S1 to S6 to achieve automated cell sorting under continuous sample injection conditions.

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