Single-cell impedance detection and sorting system and method

Abstract

The application provides a single-cell impedance detection and sorting system and method. The system comprises a micro-flow sensing module, an electrode substrate and a micro-fluidic chip being stacked, the micro-fluidic chip being provided with a first micro-channel and a second micro-channel in communication with the first micro-channel, the electrode substrate being provided with at least two detection electrode groups arranged at intervals along the extension direction of the first micro-channel, and a verification electrode group corresponding to the second micro-channel; a micro-flow actuating module corresponding to the communication position of the two micro-channels; and a main control processing module for calculating the real-time flow rate of a cell according to the time difference of impedance signals generated by different detection electrode groups on the same cell, determining the delay time of triggering the micro-flow actuating module based on the real-time flow rate of the target cell and outputting a driving signal, and confirming the success of sorting based on the impedance signal feedback of the verification electrode group. The application realizes precise sorting time control through multi-node speed measurement, and verifies the sorting result through an electrical closed loop, thereby significantly improving the sorting precision.

Description

Technical Field

[0001] This application relates to the field of cell sorting, and in particular to a single-cell impedance detection and sorting system and method. Background Technology

[0002] A single-cell impedance detection and sorting system is a miniature device based on microfluidic technology and impedance detection principle for label-free identification and physical sorting of single cells. It is typically equipped with microchannels, detection electrodes, and microactuators. When a single cell flows through the electrode detection area, the cell feature is identified by capturing the change in AC impedance caused by the cell. The target cell is then deflected into the collection channel by the microactuator downstream, thus completing the sorting operation.

[0003] However, when performing cell sorting, existing single-cell impedance detection and sorting systems cannot accurately predict the exact time when a single cell arrives at the sorting position from the detection position due to the inevitable slight fluctuations in the fluid velocity within the microchannel. This results in the microactuator failing to trigger within the optimal time window, leading to missorting or missed sorting of target cells. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this application provides a single-cell impedance detection and sorting system and method.

[0005] On the one hand, this application provides a single-cell impedance detection and sorting system, comprising: A microfluidic sensing module includes a stacked electrode substrate and a microfluidic chip; the microfluidic chip is provided with a first microchannel and a second microchannel communicating with the first microchannel; the electrode substrate is provided with a detection electrode group and a verification electrode group, the number of the detection electrode groups is at least two and they are spaced apart along the extension direction of the first microchannel, the verification electrode groups are provided corresponding to the second microchannel, and both the detection electrode group and the verification electrode group are used to output impedance signals when cells flow through them; A microfluidic actuation module is provided at the connection between the first microchannel and the second microchannel to generate a fluid displacement that drives the target cell to deflect into the second microchannel. The main control processing module is electrically connected to the detection electrode group, the verification electrode group, and the microfluidic actuation module, and is configured to: calculate the real-time flow velocity of the cell based on the time difference between the impedance signals generated by different detection electrode groups on the same cell; when the cell is a target cell, determine the delay time for triggering the microfluidic actuation module based on the real-time flow velocity of the target cell, and output a drive signal; after outputting the drive signal, confirm successful sorting based on the impedance signal fed back by the verification electrode group.

[0006] Furthermore, the microfluidic chip is also provided with a liquid storage cavity communicating with the first microchannel, and the liquid storage cavity and the second microchannel are respectively located on both sides of the first microchannel; the microfluidic actuation module includes a piezoelectric element, the piezoelectric element is electrically connected to the main control processing module, and the piezoelectric element is used to drive the liquid storage cavity to deform under the action of the driving signal, so as to squeeze the liquid in the liquid storage cavity to generate the fluid displacement.

[0007] Furthermore, the piezoelectric element is rigidly fixed to the microfluidic chip by an adhesive layer and covers the opening of the liquid storage cavity.

[0008] Furthermore, the piezoelectric element is a disc-shaped piezoelectric ceramic sheet, and the adhesive layer is an ultraviolet-curable adhesive layer.

[0009] Furthermore, the electrode substrate is a glass substrate, and both the detection electrode group and the verification electrode group are chromium microelectrode arrays deposited on the glass substrate.

[0010] Furthermore, it also includes a circuit board substrate, on which the electrode substrate is stacked. The circuit board substrate has metal pads, which are electrically connected to the pins of the detection electrode group and the pins of the verification electrode group through conductive silver paste.

[0011] Furthermore, it also includes a signal conditioning module, and the main control processing module is electrically connected to the detection electrode group and the verification electrode group through the signal conditioning module; The signal conditioning module includes a lock-in amplifier and a transimpedance amplifier; the transimpedance amplifier is used to convert the current signal output by the detection electrode group and / or the verification electrode group into a voltage signal; the lock-in amplifier is used to provide an excitation signal and demodulate the voltage signal to extract impedance amplitude and phase characteristics.

[0012] On the other hand, this application provides a single-cell impedance detection and sorting method, applied to the single-cell impedance detection and sorting system described in any of the above claims, comprising the following steps: acquiring the impedance signal generated when the cell to be tested passes through at least two of the detection electrode groups in sequence; The real-time flow velocity of the cell is calculated based on the time difference between the impedance signals generated by different detection electrode groups on the same cell. The cell is identified based on the impedance signal generated by the detection electrode group. When the cell is identified as the target cell, the delay time for triggering the microfluidic actuation module is determined based on the real-time flow rate of the target cell. When the delay time is reached, a drive signal is output to the microfluidic actuation module to drive the target cell to deflect into the second microchannel. After outputting the drive signal, the impedance signal fed back by the verification electrode group is obtained to confirm successful sorting.

[0013] Further, the step of identifying the cell based on the impedance signal includes: obtaining the dielectric characteristic parameters of the cell based on the amplitude and / or phase characteristics of the impedance signal; comparing the dielectric characteristic parameters with a preset target cell dielectric characteristic threshold or clustering model; and identifying the cell as the target cell if the comparison result meets the preset conditions.

[0014] Further, determining the delay time for triggering the microfluidic actuation module based on the real-time flow rate of the target cell includes: determining the delay time based on the real-time flow rate and the distance from the downstreammost detection electrode group among the detection electrode groups participating in this real-time flow rate detection to the microfluidic actuation module.

[0015] Further, after outputting the drive signal, the impedance signal fed back by the verification electrode group is acquired to confirm successful sorting, including: Within a preset time window after the drive signal is output, the impedance signal fed back by the verification electrode group is obtained; If the impedance signal fed back by the verification electrode group is successfully obtained within the preset time window, the sorting is confirmed to be successful and the count is updated.

[0016] This application has at least the following beneficial effects: This application employs at least two detection electrode groups spaced apart along the extension direction of the first microchannel. The main control processing module calculates the real-time flow velocity of the cell based on the time difference between the impedance signals generated by different detection electrode groups on the same cell. This real-time flow velocity is then used to determine the delay time for triggering the microfluidic actuation module, ensuring that the microfluidic actuation module is triggered precisely at the moment the target cell reaches the connection point. This overcomes the influence of minute fluctuations in fluid velocity within the microchannel on the sorting timing, significantly improving the accuracy and success rate of single-cell sorting. Simultaneously, a verification electrode group corresponding to the second microchannel confirms successful sorting based on the impedance signal fed back from the verification electrode group after the output drive signal. This achieves closed-loop verification using purely electrical methods, eliminating the need for external optical equipment to confirm sorting results in real time, thus reducing system complexity and cost. Furthermore, the stacked arrangement of the electrode substrate and the microfluidic chip allows for independent manufacturing and assembly of both, facilitating the reuse or replacement of the electrode substrate and effectively reducing the cost per use. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a side view of a single-cell impedance detection and sorting system provided in this embodiment; Figure 2 This is a top view of a single-cell impedance detection and sorting system provided in this embodiment; Figure 3 for Figure 2 A magnified view of a portion of region A in the middle; Figure 4 for Figure 3 A magnified view of a portion of region Q2; Figure 5 This is a top view of the circuit board substrate provided in this embodiment; Figure 6 This is a top view of the electrode substrate provided in this embodiment; Figure 7 This is a top view of the microfluidic chip provided in this embodiment; Figure 8 This embodiment illustrates the driving mechanism of the microfluidic actuator module. Figure 1 ; Figure 9 This embodiment illustrates the driving mechanism of the microfluidic actuator module. Figure 2 ; Figure 10 This is a diagram illustrating the experimental process of a single-cell impedance detection and sorting system provided in this embodiment. Figure 11 This is a schematic diagram of a single-cell impedance detection and sorting system provided in this embodiment; Figure 12 This is a flowchart illustrating a single-cell impedance detection and sorting method provided in this embodiment.

[0019] Figure label: 100 - Microfluidic sensor module; 110 - Circuit board substrate; 111 - Metal pad; 112 - SMA RF interface; 113 - Observation window; 120 - Electrode substrate; 121 - Detection electrode group; 122 - Verification electrode group; 123 - Excitation electrode; 124 - Differential electrode; 130 - Microfluidic chip; 131 - First microchannel; 132 - Second microchannel; 133 - Liquid reservoir; 1311 - Cell channel; 1312 - Sheath fluid channel; 1313 - Collection channel; 200 - Microfluidic actuation module; 210 - Piezoelectric element; 300 - Main control processing module; 400 - Signal conditioning module; Q1 - Detection area; Q2 - Sorting area; Q3 - Collection area. Detailed Implementation

[0020] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0021] In the description of this application, it should be noted that the terms "vertical", "up", "down", "horizontal", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0022] In the description of this application, it should also be noted that, unless otherwise expressly specified and limited, the terms "set," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0023] The single-cell impedance detection and sorting system and method provided in this embodiment are used to comprehensively solve the technical problem in the prior art that, during cell sorting, due to the unavoidable slight fluctuations in the fluid velocity within the microchannel, the chip cannot accurately predict the specific time when a single cell arrives at the sorting position from the detection position, resulting in the micro-actuator failing to trigger within the optimal time window, thus causing mis-sorting or missed sorting of target cells.

[0024] Example 1 Reference Figures 1 to 11This embodiment provides a single-cell impedance detection and sorting system, including a microfluidic sensing module 100, a microfluidic actuation module 200, a main control processing module 300, and a signal conditioning module 400. The microfluidic sensing module 100 is a multi-layered, nested modular structure used to provide a microenvironment for cell flow, impedance detection, and sorting verification. The microfluidic actuation module 200 generates directional transient fluid displacement at the sorting node, driving the target cells to precisely deflect into the collection branch. The main control processing module 300 is responsible for real-time processing of impedance signals, cell type identification, dynamic flow velocity calculation, determination of actuation pulse delay time, and generation of driving signals. The signal conditioning module 400 performs low-noise amplification and phase-locked demodulation of the weak AC current signals output from the detection electrode group and the verification electrode group.

[0025] Please refer to Figure 1 and Figure 2 In one embodiment, the microfluidic sensing module 100 adopts a multi-layered, nested structure, comprising, from bottom to top, a circuit board substrate 110, an electrode substrate 120, and a microfluidic chip 130. The electrode substrate 120 and the microfluidic chip 130 are stacked. During assembly, after oxygen plasma surface activation treatment, they are precisely aligned and bonded under a microscope using preset alignment marks, thereby forming a completely closed microchannel detection environment that ensures the stability of cell flow and the consistency of impedance detection.

[0026] Please refer to Figure 5 In one embodiment, the circuit board substrate 110 serves as the bottom support and electrical connection board and is a custom-made PCB board. Its upper surface is provided with a plurality of metal pads 111 for forming electrical connections with the electrode group of the electrode substrate 120 above.

[0027] Furthermore, the circuit board substrate 110 integrates multiple standard SMA RF interfaces 112 around its perimeter for connecting to the signal conditioning module 400. Specifically, the SMA RF interface 112 includes interfaces I1 and I3 for input impedance excitation signals, a common ground interface I2, and four pairs of differential electrode output signal interfaces O1-O8 for outputting detection signals to the transimpedance amplifier. The SMA RF interface 112 ensures the integrity and anti-interference capability of the high-frequency AC impedance detection signal during transmission.

[0028] Furthermore, to simultaneously meet the requirements of microscopic optical observation, an observation window 113 is provided in the center of the circuit board substrate 110, which is precisely matched to the outer dimensions of the upper electrode substrate 120. The edge of the observation window has a stepped slot structure, which allows the electrode substrate 120 to be firmly and accurately fitted into the center of the circuit board substrate 110, completely avoiding mechanical slippage during experimental operation and ensuring the reproducibility of the detection area position. At the same time, since the electrode substrate 120 is detachably fitted into the stepped slot of the circuit board substrate 110, when the microfluidic chip 130 needs to be replaced due to biological contamination or wear, only the electrode substrate 120 and the microfluidic chip 130 bonded to it need to be replaced, while the more valuable circuit board substrate 110 and SMA RF interface 112 and other components can be efficiently recycled and reused. The modular nested design of the microfluidic sensor module 100 provided in this embodiment significantly reduces the consumable cost of a single experiment or clinical test to less than 100 yuan, greatly lowering the economic threshold for high-throughput single-cell analysis.

[0029] Please refer to Figure 1 , Figure 2 and Figure 6 In one embodiment, the electrode substrate 120 is preferably a glass substrate on which a chromium (Cr) microelectrode array is deposited using micro-nano fabrication processes to form a detection electrode group 121 and a verification electrode group 122. This embodiment uses chromium as the microelectrode material, which is far less expensive than precious metals such as gold and platinum used in traditional solutions, while also possessing excellent conductivity, fully meeting the requirements for establishing a high-frequency electric field and sensing weak impedance disturbances within the microchannel.

[0030] Each detection electrode group 121 and each verification electrode group 122 includes an excitation electrode 123 and a differential electrode 124, used to form a local electric field sensitive region within the microchannel on the microfluidic chip 130, and output an impedance signal when cells flow through it. The number of detection electrode groups 121 is at least two. In this embodiment, four detection electrode groups 121 (referred to as E1, E2, E3, and E4 in sequence along the fluid direction) are used as an example. They are arranged at intervals along the extension direction of the first microchannel 131, with a known and precisely controllable spacing (e.g., the spacing between E1 and E2 is set to 2000 μm), thereby outputting multiple impedance pulses when cells flow through them sequentially, used to measure the flow velocity of a single cell in real time. The number of verification electrode groups 122 is at least one, in this embodiment one group (referred to as E5), located at the entrance of the second microchannel 132, used to sense target cells deflected into the second microchannel 132, and output an impedance signal as the cells flow through it, serving as a verification signal for successful sorting.

[0031] In one embodiment, the lead-out pins of each detection electrode group 121 and verification electrode group 122 on the electrode substrate 120 are electrically connected to the corresponding metal pads 111 on the circuit board substrate 110 via epoxy conductive silver paste under normal temperature or low temperature (e.g., 60°C baking for accelerated curing) conditions. This completely eliminates the traditional process of high-temperature soldering of glass substrate pins to the PCB. This embodiment, by employing a flexible encapsulation method using epoxy conductive silver paste at normal / low temperature, eliminates the problem of thermal stress concentration caused by differences in thermal expansion coefficients during high-temperature soldering, thereby preventing the generation of microcracks in the glass substrate and significantly improving the overall shock resistance of the microfluidic chip structure, the long-term sealing reliability of the flow channels, and the yield rate.

[0032] Please refer to Figure 2 , Figure 3 and Figure 7 In one embodiment, the microfluidic chip 130 is preferably made of polydimethylsiloxane (PDMS), which has good biocompatibility and elasticity, and its interior is pre-formed with a microchannel network using an SU-8 photolithography molding process. This microchannel network includes at least one first microchannel 131, one second microchannel 132, and a reservoir 133. The first microchannel 131 includes a cell channel 1311, a sheath fluid channel 1312, and a collection channel 1313, which converge to form the collection channel 1313. The second microchannel 132 serves as a collection pathway for target cells, forming a Y-shaped connection with the collection channel 1313. A detection area Q1, a sorting area Q2, and a collection area Q3 are sequentially formed along the flow direction on the first microchannel 131 and the second microchannel 132. The liquid storage chamber 133 is also connected to the first microchannel 131 in the sorting area Q2, and is respectively set on both sides of the first microchannel 131 with the second microchannel 132, forming a push-pull sorting structure.

[0033] Furthermore, each detection electrode group 121 is arranged at intervals along the extension direction of the cell channel 1311 and / or the collection channel 1313.

[0034] Furthermore, since the SMA RF interface 112 includes a common ground interface I2, the piezoelectric drive circuit and the detection circuit share the same reference potential, preventing leakage current from entering the measurement circuit and effectively preventing leakage current from interfering with the impedance measurement results in the liquid storage chamber 133.

[0035] Please refer to Figure 2 , Figure 3 and Figure 7 In one embodiment, the microfluidic actuation module 200 is set at the connection point (hereinafter referred to as the sorting fork) between the first microchannel 131 and the second microchannel 132, and is used to generate transient fluid displacement that drives the target cell to deflect into the second microchannel 132 within a millisecond time window.

[0036] In this embodiment, the microfluidic actuation module 200 includes a piezoelectric element 210, which is electrically connected to the main control processing module 300. The piezoelectric element 210 is used to drive the liquid storage chamber 133 to deform under the action of the driving signal, so as to squeeze the liquid in the liquid storage chamber 133 to generate fluid displacement.

[0037] Specifically, the piezoelectric element 210 is a disc-shaped piezoelectric ceramic sheet (PZT, lead zirconate titanate), the diameter of which matches the width of the reservoir 133.

[0038] Specifically, the piezoelectric element 210 is directly bonded and fixed to the top surface of the microfluidic chip 130 body at the opening of the liquid storage cavity 133 through an adhesive layer formed by ultraviolet curing adhesive (UV adhesive).

[0039] Unlike traditional methods that involve drilling holes in the chip and allowing the piezoelectric element to directly contact the fluid, this embodiment directly bonds and fixes the piezoelectric element 210 to the top surface of the microfluidic chip 130 at the opening of the liquid reservoir 133. Since the microfluidic chip 130 is made of PDMS, which inherently possesses elasticity, this embodiment can efficiently transmit mechanical deformation by utilizing the inherent elasticity of the PDMS body, while eliminating the complexity of the process required by traditional methods that necessitate the bonding of separate thin films. Furthermore, the integrated, sealed structure completely prevents leakage problems that may occur during the operation of the piezoelectric element 210. In addition, this embodiment fully utilizes the insulating properties of the PDMS body to form a fully bonded coupling structure, completely eliminating air gaps that may exist between the piezoelectric element and the chip in traditional assembly methods.

[0040] The piezoelectric element 210 is electrically connected to the main control processing module 300 via a wire. Please refer to... Figure 8 , Figure 9 and Figure 10During operation, when the main control processing module 300 determines that the target cell is about to reach the sorting fork, it outputs a high-voltage drive pulse (e.g., a half-wave sine or square wave pulse with an amplitude of 40V and a pulse width of 20ms) to the piezoelectric element 210 based on the dynamically calculated delay time. Upon receiving the pulse, the piezoelectric element 210 undergoes instantaneous longitudinal bending deformation based on the inverse piezoelectric effect. This deformation directly and efficiently drives the deformation of the liquid storage chamber 133 through the rigid adhesive layer, squeezing the incompressible liquid within the chamber. The squeezed liquid is instantly ejected from the outlet of the liquid storage chamber 133 into the first microchannel 131, forming a concentrated and directional transient fluid displacement that precisely pushes the target single cell, which happens to be flowing through the sorting fork, laterally into the opposite second microchannel 132. This mechanical coupling structure fundamentally improves the electromechanical conversion efficiency. Actual tests show that the response time from receiving the driving pulse to generating effective fluid displacement is only 1.5ms, with extremely high mechanical deformation transmission efficiency. This ensures that the precise deflection of a single cell is completed within the optimal execution window at the millisecond level, greatly improving the sorting accuracy and purity of high-speed flowing particles.

[0041] Please refer to Figures 1 to 11 In one embodiment, the main control processing module 300 may be implemented by a field-programmable gate array (FPGA) and / or a microcontroller to ensure extremely low signal processing and control latency (total processing latency is controlled within 40 μs). The main control processing module 300 is electrically connected to the detection electrode group 121 and the verification electrode group 122 through the signal conditioning module 400, and is also electrically connected to the microfluidic actuation module 200.

[0042] The signal conditioning module 400 mainly includes a lock-in amplifier and a transimpedance amplifier (TIA). The transimpedance amplifier is used to convert the weak current signals output by the detection electrode group 121 and the verification electrode group 122 into voltage signals with low noise. The lock-in amplifier outputs an AC excitation signal (such as a frequency of 500kHz and an amplitude of 500mV) to the excitation electrode, and demodulates the voltage signal output by the transimpedance amplifier to extract the impedance amplitude and phase characteristics reflecting the dielectric characteristics of the cell from the noise background, and transmits them to the main control processing module 300 in real time.

[0043] The main control processing module 300 is configured as follows: As the cell flows through the detection zone, the real-time flow velocity of the cell is calculated based on the time difference between the impedance signals generated by different detection electrode groups 121 on the same cell. Specifically, when the same cell passes through electrode groups I1 and I2 with a known spacing in sequence, two impedance spikes are generated, and the main control processing module 300 records the time difference between the two spikes. The real-time flow rate is (where L is the distance between I1 and I2).

[0044] Based on the impedance amplitude and / or phase characteristics extracted by the lock-in amplifier, the cell is determined to be a target cell (such as a circulating tumor cell) by means of a preset threshold comparison or clustering algorithm.

[0045] When the target cell is identified, the calculated real-time flow rate is used as a basis. And the distance from the downstreammost detector electrode in the real-time flow velocity detection group (i.e., the one closest to the microfluidic actuator module 200, for example, I2 when using I1 and I2 to calculate the flow velocity) to the center of the sorting fork. Subtracting the inherent processing delay of the system Dynamically determine the delay time for triggering the microfluidic actuator module 200. And accurately output the drive signal when the delay time is reached.

[0046] After the output drive signal, the sorting is further confirmed based on the impedance signal fed back by the verification electrode group 122 within a preset time window. If the impedance signal is successfully obtained within the preset time window, the sorting is confirmed to be successful and the target cell count is updated.

[0047] In this embodiment, at least two detection electrode groups 121 are spaced apart along the extension direction of the first microchannel 131. The main control processing module 300 calculates the real-time flow velocity of the cell based on the time difference between the impedance signals generated by different detection electrode groups 121 on the same cell. Then, based on the real-time flow velocity, the delay time for triggering the microfluidic actuation module 200 is determined, so that the microfluidic actuation module 200 can be triggered at the precise moment when the target cell reaches the connection point. This overcomes the influence of small fluctuations in fluid flow velocity within the microchannel on the sorting timing, significantly improving the accuracy and success rate of single-cell sorting. At the same time, through the verification electrode group 122 corresponding to the second microchannel 132, the successful sorting is confirmed based on the impedance signal fed back by the verification electrode group after the output drive signal. This achieves closed-loop verification based on pure electrical means, which can complete the real-time confirmation of sorting results without relying on external optical equipment, reducing system complexity and cost.

[0048] Example 2 Reference Figures 1 to 12 This embodiment provides a single-cell impedance detection and sorting method, which can be applied to the single-cell impedance detection and sorting system described in Embodiment 1 above. The method includes the following steps: Step S1: Start the injection pump to inject the suspension containing the test cells and the sheath fluid into the first microchannel 131 of the microfluidic chip 130 at a set flow rate (e.g., 100 μL / min), forming a stable single-cell focused flow. Simultaneously, the lock-in amplifier in the control signal conditioning module 400 of the main control processing module 300 outputs an AC excitation signal (e.g., frequency 500 kHz, amplitude 500 mV) to the excitation electrode of the detection electrode group 121, establishing a stable high-frequency non-uniform electric field in the detection area, preparing for subsequent impedance detection.

[0049] Step S2: Under fluid force, a single cell sequentially passes through multiple detection electrode groups 121 spaced along the first microchannel 131. Taking the cell sequentially passing through the first detection electrode group I1 and the second detection electrode group I2 as an example, when the cell enters the electrode sensitive area of ​​I1, it causes a change in the impedance between the electrodes, which, after processing by a transimpedance amplifier and a lock-in amplifier, generates a first impedance spike; when the cell continues to flow into the sensitive area of ​​I2, a second impedance spike is generated. The main control processing module 300 accurately records the time difference between the appearance of these two impedance spikes. Given the design spacing L between I1 and I2 (e.g., L = 2000 μm), the real-time flow velocity of the cell can be calculated. By setting up multi-node velocity measurement electrode groups (such as I1-I4), the operator can flexibly combine the four detection electrode groups 121 in pairs according to actual needs, select the optimal velocity measurement baseline, and further improve the robustness and accuracy of flow velocity detection.

[0050] Simultaneously, the main control processing module 300 also obtains the dielectric characteristic parameters of the cell (such as cell volume, membrane capacitance, cytoplasmic conductivity, etc.) based on the impedance amplitude and phase characteristics extracted by the lock-in amplifier. These dielectric characteristic parameters are then compared with pre-calibrated and stored target cell dielectric characteristic thresholds or clustering models; if the comparison result meets preset conditions, the cell is identified as the target cell. This feature extraction method based on impedance amplitude and phase characteristics enables label-free, non-invasive, and accurate identification of live single cells.

[0051] Step S3: When the target cell is identified in step S2, the main control processing module 300 immediately calculates the real-time flow rate of the cell based on step S2. And the precise distance from the downstreammost electrode in the detection electrode group participating in this real-time flow velocity detection (i.e., the one closest to the microfluidic actuator module 200, for example, I4 if I3 and I4 are used to calculate the flow velocity) to the center of the sorting fork. The delay time required to trigger the microfluidic actuator module 200 is calculated in real time. ,in This is the total delay inherent in the system's signal processing and logic calculations. The main control processing module 300 uses a high-precision hardware timer to manage this delay. Then, a preset high-voltage drive pulse (e.g., a half-wave sine or square wave pulse with an amplitude of 40V and a pulse width of 20ms) is output to the piezoelectric element 210. This dynamic delay calculation mechanism enables the system to perform personalized time compensation for the instantaneous flow rate of each single cell, thereby overcoming the influence of fluid flow rate fluctuations within the microchannel on the sorting timing in principle.

[0052] Step S4: After receiving the driving pulse, the piezoelectric element 210 (disc-type piezoelectric ceramic sheet) undergoes instantaneous longitudinal bending deformation based on the inverse piezoelectric effect. This deformation is transmitted to the liquid storage chamber 133 through the UV adhesive layer, squeezing the liquid inside the chamber and instantly generating a directional transient fluid displacement. This fluid displacement laterally crosses the first microchannel 131, precisely pushing the target cell that is flowing through the sorting fork away from its original main flow path, causing it to deflect into the second microchannel 132. Non-target cells and other fluids continue to flow along the first microchannel 131 to the downstream waste outlet, thereby completing high-precision single-cell physical sorting.

[0053] Step S5: After the drive signal is output in step S3, the main control processing module 300 opens a preset verification time window (e.g., 50ms from the end of the actuation pulse) and continuously monitors the impedance signal output by the verification electrode group 122 (I5) set at the entrance of the second microchannel 132. If the impedance signal fed back by the verification electrode group 122 is successfully obtained within the preset time window, the main control processing module 300 confirms that the sorting is successful and updates the target cell count (e.g., increments the target cell counter by 1). If the impedance signal fed back by the verification electrode group 122 is not obtained within the preset time window, the sorting is considered to have failed, and the system can record this abnormal event. The verification and counting results can be uploaded to the host computer in real time for online plotting, statistical analysis, and visualization using scripts such as Python or MATLAB.

[0054] This method, through multi-node impedance velocimetry and dynamic delay compensation mechanisms, completely overcomes the adverse effects of minute fluctuations in fluid velocity within microchannels on sorting timing, controlling the triggering time error of piezoelectric actuation to the millisecond level, effectively avoiding mis-sorting and missed sorting. Simultaneously, based on a purely electrical closed-loop verification design using the verification electrode assembly, it completely eliminates the dependence on bulky external high-speed imaging equipment, reducing the total closed-loop verification delay of the system to the millisecond level. Combining the above designs, the overall single-cell detection and sorting throughput of the system can exceed 8 cells / s, providing core components and methodological support for portable, high-throughput, and low-cost point-of-care testing (POCT) devices.

[0055] Those skilled in the art will understand that the modules or steps described above in this application can be implemented using general-purpose computing devices. They can be centralized on a single computing device or distributed across a network of multiple computing devices. Optionally, they can be implemented using computer-executable program code, thereby allowing them to be stored in a storage device for execution by a computing device, or they can be fabricated as separate integrated circuit modules, or multiple modules or steps can be fabricated as a single integrated circuit module. Thus, this application is not limited to any particular combination of hardware and software.

[0056] Note that the above description is merely a preferred embodiment and the technical principles employed in this application. Those skilled in the art will understand that this application is not limited to the specific embodiments described herein, and various obvious changes, readjustments, and substitutions can be made without departing from the scope of protection of this application. Therefore, although this application has been described in detail through the above embodiments, this application is not limited to the above embodiments. Many other equivalent embodiments may be included without departing from the concept of this application, and the scope of this application is determined by the scope of the appended claims.

[0057] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A single-cell impedance detection and sorting system, characterized in that, include: A microfluidic sensing module includes an electrode substrate and a microfluidic chip stacked together; the microfluidic chip is provided with a first microchannel and a second microchannel communicating with the first microchannel; The electrode substrate is provided with a detection electrode group and a verification electrode group. There are at least two detection electrode groups, which are spaced apart along the extension direction of the first microchannel. The verification electrode group is provided corresponding to the second microchannel. Both the detection electrode group and the verification electrode group are used to output impedance signals when cells flow through them. A microfluidic actuation module is provided at the connection between the first microchannel and the second microchannel to generate a fluid displacement that drives the target cell to deflect into the second microchannel. The main control processing module is electrically connected to the detection electrode group, the verification electrode group, and the microfluidic actuation module, and is configured to: calculate the real-time flow velocity of the cell based on the time difference between the impedance signals generated by different detection electrode groups on the same cell; when the cell is a target cell, determine the delay time for triggering the microfluidic actuation module based on the real-time flow velocity of the target cell, and output a drive signal; after outputting the drive signal, confirm successful sorting based on the impedance signal fed back by the verification electrode group.

2. The single-cell impedance detection and sorting system according to claim 1, characterized in that, The microfluidic chip is also provided with a liquid storage cavity communicating with the first microchannel, and the liquid storage cavity and the second microchannel are respectively located on both sides of the first microchannel; The microfluidic actuation module includes a piezoelectric element, which is electrically connected to the main control processing module. The piezoelectric element is used to drive the liquid storage cavity to deform under the action of the driving signal, so as to squeeze the liquid in the liquid storage cavity to generate the fluid displacement.

3. The single-cell impedance detection and sorting system according to claim 2, characterized in that, The piezoelectric element is rigidly fixed to the microfluidic chip by an adhesive layer and covers the opening of the liquid storage cavity.

4. The single-cell impedance detection and sorting system according to claim 3, characterized in that, The piezoelectric element is a disc-shaped piezoelectric ceramic sheet, and the adhesive layer is an ultraviolet-cured adhesive layer.

5. The single-cell impedance detection and sorting system according to claim 1, characterized in that, The electrode substrate is a glass substrate, and both the detection electrode group and the verification electrode group are chromium microelectrode arrays deposited on the glass substrate.

6. The single-cell impedance detection and sorting system according to claim 1, characterized in that, The microcurrent sensing module also includes a circuit board substrate, on which the electrode substrate is stacked. The circuit board substrate has metal pads, which are electrically connected to the pins of the detection electrode group and the pins of the verification electrode group through conductive silver paste.

7. The single-cell impedance detection and sorting system according to claim 1, characterized in that, It also includes a signal conditioning module, and the main control processing module is electrically connected to the detection electrode group and the verification electrode group through the signal conditioning module; The signal conditioning module includes a lock-in amplifier and a transimpedance amplifier; the transimpedance amplifier is used to convert the current signal output by the detection electrode group and / or the verification electrode group into a voltage signal; the lock-in amplifier is used to provide an excitation signal and demodulate the voltage signal to extract impedance amplitude and phase characteristics.

8. A single-cell impedance detection and sorting method, applied to the single-cell impedance detection and sorting system according to any one of claims 1 to 7, characterized in that, include: Acquire the impedance signal generated when the cell to be tested passes through at least two of the detection electrode groups in sequence; The real-time flow velocity of the cell is calculated based on the time difference between the impedance signals generated by different detection electrode groups on the same cell. The cell is identified based on the impedance signal generated by the detection electrode group. When the cell is identified as the target cell, the delay time for triggering the microfluidic actuation module is determined based on the real-time flow rate of the target cell. When the delay time is reached, a drive signal is output to the microfluidic actuation module to drive the target cell to deflect into the second microchannel. After outputting the drive signal, the impedance signal fed back by the verification electrode group is obtained to confirm successful sorting.

9. The single-cell impedance detection and sorting method according to claim 8, characterized in that, Determining the delay time for triggering the microfluidic actuation module based on the real-time flow velocity of the target cell includes: The delay time is determined based on the real-time flow rate and the distance from the downstreammost detection electrode group to the microfluidic actuator module among the detection electrode groups participating in this real-time flow rate detection.

10. The single-cell impedance detection and sorting method according to claim 9, characterized in that, After outputting the drive signal, the impedance signal fed back by the verification electrode group is obtained to confirm successful sorting, including: Within a preset time window after the drive signal is output, the impedance signal fed back by the verification electrode group is obtained; If the impedance signal fed back by the verification electrode group is successfully obtained within the preset time window, the sorting is confirmed to be successful and the count is updated.

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