Microfluidic cell detection device and method

Abstract

The invention provides a microfluidic cell detection device and method, and relates to the technical field of cell detection, the microfluidic cell detection device comprises a microfluidic chip, an impedance detection module, a signal acquisition module and a signal processing module; the micro-fluidic chip is internally provided with a main micro-channel, and a sample inlet and a sheath fluid inlet which are communicated with the inlet end of the main micro-channel; a detection area is further arranged in the micro-fluidic chip, a first reference electrode, a second reference electrode and an exciting electrode are arranged in the detection area, and the first reference electrode, the second reference electrode and the exciting electrode are symmetrically distributed at the bottom of the main micro-channel; the input end of the impedance detection module is electrically connected with the first reference electrode and the second reference electrode, and the output end of the impedance detection module is connected with the input end of the signal acquisition module; the first output end of the signal acquisition module is connected with the excitation electrode, and the second output end of the signal acquisition module is connected with the signal processing module. The reliability of cell detection can be improved.

Description

Technical Field

[0001] This application relates to the field of cell detection, and more specifically, to a microfluidic cell detection device and method. Background Technology

[0002] In biomedical research and clinical testing, high-throughput, label-free, multi-parameter analysis of living cells is of great significance. The electrical and mechanical properties of cells are key indicators of their physiological and pathological states. Microfluidic electrical impedance tomography (IIT) technology, with its advantages of being non-invasive, real-time, and easily integrated, has become an important tool for obtaining this information. This technology applies electrical signals of different frequencies to cells, allowing for the analysis of cell size and membrane properties from the low-frequency response and the acquisition of intracellular electrical information from the mid-frequency response.

[0003] However, the practicality of existing technologies is limited by the cell focusing methods. Currently, focusing mainly relies on inert fluid sheath flow or mechanical structures. The former is prone to causing unstable cell trajectories and signal fluctuations in high-throughput or heterogeneous samples; the latter suffers from channel blockage and difficulty in adapting to cells of different sizes and types. This limits the stability and versatility of the technology in complex samples. Therefore, there is an urgent need to develop a novel detection method and device that can achieve stable, adaptive focusing and simultaneously extract multidimensional electrical and mechanical properties of cells to improve the reliability and breadth of applications of the analysis. Summary of the Invention

[0004] This application provides a microfluidic cell detection device and method, which can improve the reliability of cell detection.

[0005] In a first aspect, embodiments of this application provide a microfluidic cell detection device, comprising: a microfluidic chip, an impedance detection module, a signal acquisition module, and a signal processing module; The microfluidic chip has a main microchannel, and a sample inlet and a sheath fluid inlet connected to the inlet end of the main microchannel. The microfluidic chip also has a detection area, which is equipped with a first reference electrode, a second reference electrode, and an excitation electrode. The first reference electrode, the second reference electrode, and the excitation electrode are symmetrically distributed at the bottom of the main microchannel. The sample inlet is used to inject a cell sample to be tested, and the sheath fluid inlet is used to inject a viscoelastic fluid, so that the viscoelastic fluid and the cell sample to be tested converge in the main microchannel and then flow through the detection area. The input terminal of the impedance detection module is electrically connected to the first reference electrode and the second reference electrode, so that the impedance detection module generates an electrical response signal based on the electrode signals obtained from the first reference electrode and the second reference electrode. The output terminal of the impedance detection module is connected to the input terminal of the signal acquisition module, so that the signal acquisition module generates a detection response signal based on the electrical response signal. The first output terminal of the signal acquisition module is connected to the excitation electrode to apply an excitation signal to the excitation electrode. The second output terminal of the signal acquisition module is connected to the signal processing module so that the signal processing module can obtain the impedance of the cell sample to be tested based on the detection response signal.

[0006] Optionally, the microfluidic chip further includes a collection outlet and a waste liquid outlet, wherein the collection outlet and the waste liquid outlet are connected to the outlet end of the main microchannel, and are used to separate and discharge the detected cell sample and viscoelastic fluid, respectively.

[0007] Optionally, the microfluidic chip includes a substrate and an organosilicon channel structure covering the substrate, wherein the main microchannel, the sample inlet, the sheath fluid inlet, the collection outlet, and the waste liquid outlet are all disposed in the organosilicon channel structure.

[0008] Optionally, the impedance detection module includes a transimpedance amplifier, the two input terminals of which are used to connect the first reference electrode and the second reference electrode, and the output terminal of the transimpedance amplifier is the output terminal of the impedance detection module, which is used to connect to the signal acquisition module.

[0009] Optionally, the signal acquisition module includes: a lock-in amplifier, the input terminal of which is connected to the output terminal of the transimpedance amplifier, the control terminal of which is used to receive an external control signal, and to enable the lock-in amplifier to generate an excitation signal based on the external control signal; the first output terminal of the lock-in amplifier is connected to the excitation electrode, and the second output terminal of the lock-in amplifier is connected to the signal processing module. Optionally, the device further includes: an optical excitation module; the light-emitting surface of the optical excitation module faces the detection area.

[0010] Secondly, embodiments of this application also provide a microfluidic cell detection method, applied to any of the microfluidic cell detection devices described in the first aspect above, the method comprising: Inject the cell sample to be tested into the sample inlet; A viscoelastic fluid is injected into the sheath fluid inlet, so that the viscoelastic fluid and the cell sample to be tested merge in the main microchannel and then flow through the detection area; An excitation signal is applied to the excitation electrode through the signal acquisition module; The electrode signals from the first and second reference electrodes are obtained through the impedance detection module, and the electrical response signal after processing by the impedance detection module is obtained. The electrical response signal is received and processed by the signal acquisition module to obtain the detection response signal; The detection response signal is processed by the signal processing module to obtain the impedance of the cell sample to be tested.

[0011] Optionally, the detection response signal is processed by a signal processing module to obtain the impedance of the cell sample to be tested, including: The impedance of the cell sample to be tested is obtained by the signal processing module based on the real and imaginary components contained in the detection response signal.

[0012] Optionally, the method further includes: The signal acquisition module applies a low-frequency excitation signal and a medium-frequency excitation signal to the excitation electrode to obtain the first impedance generated by the cell sample under the low-frequency excitation signal and the second impedance generated under the medium-frequency excitation signal, respectively. The electrical diameter of the cell sample to be tested is obtained based on the first impedance, and the opacity of the cell sample to be tested is obtained based on the first impedance and the second impedance.

[0013] Optionally, the method further includes: When the cell sample to be tested flows through the detection area, the cells in the detection area are irradiated with light of a preset wavelength through the optical excitation module.

[0014] This application provides a microfluidic cell detection device and method. The device includes a microfluidic chip, an impedance detection module, a signal acquisition module, and a signal processing module. The microfluidic chip has a main microchannel and a sample inlet and a sheath fluid inlet connected to it. A first reference electrode, a second reference electrode, and an excitation electrode are symmetrically arranged in the detection area at the bottom of the main microchannel. The input terminal of the impedance detection module is connected to the two reference electrodes to acquire electrode signals, and its output terminal is connected to the signal acquisition module. The signal acquisition module receives the electrical response signal generated by the impedance detection module based on the electrode signals, and simultaneously provides an excitation signal to the excitation electrode through its first output terminal and a detection response signal obtained based on the electrical response signal to the signal processing module through its second output terminal. The signal processing module finally analyzes the impedance parameters of the cell sample to be tested based on the detection response signal. This device creates a controllable channel environment for the thorough mixing, stable focusing, and directional flow of viscoelastic fluid and the cell sample to be tested through the detection zone. Simultaneously, using the three electrodes in the detection zone, it achieves a complete process of picking up the electrode signals generated when the cells pass through the detection zone, converting them into detection response signals after signal conditioning by the impedance detection module and signal acquisition module, and finally resolving them into impedance parameters by the signal processing module. Structurally, it achieves a high degree of spatial integration of cell focusing, electric field excitation, signal sensing, and data processing, reducing external piping and manual intervention, lowering operational complexity, and improving detection throughput and repeatability. At the same time, it ensures the accuracy and stability of impedance parameters, thus significantly improving the integration, automation, and reliability of cell impedance detection, providing an effective technical platform for high-throughput, label-free cell analysis. Attached Figure Description

[0015] 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.

[0016] Figure 1 This application provides a schematic diagram of the structure of a microfluidic cell detection device. Figure 2 A schematic diagram of another microfluidic cell detection device provided in this application; Figure 3 A schematic diagram of another microfluidic cell detection device provided in this application; Figure 4 A schematic diagram of another microfluidic cell detection device provided in this application; Figure 5A schematic diagram of another microfluidic cell detection device provided in this application; Figure 6 A schematic diagram of another microfluidic cell detection device provided in this application; Figure 7 A schematic diagram of another microfluidic cell detection device provided in this application; Figure 8 A schematic flowchart of a microfluidic cell detection method provided in this application; Figure 9 This is a flowchart illustrating another microfluidic cell detection method provided in this application.

[0017] Figure descriptions: 1. Microfluidic chip; 2. Impedance detection module; 3. Signal acquisition module; 4. Signal processing module; 11. Main microchannel; 12. Sample inlet; 13. Sheath fluid inlet; 14. Detection area; 141. First reference electrode; 142. Second reference electrode; 143. Excitation electrode; 15. Collection outlet; 16. Waste liquid outlet; 5. Optical excitation module; 21. Transimpedance amplifier; 31. Lock-in amplifier; 311. Frequency generator; 321. Output superimposed unit; 322. Digital oscillator; 323. Digital processing unit. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, 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 some embodiments of this application, and not all embodiments. The components of the embodiments of this application described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0019] Therefore, the following detailed description of the embodiments of this application provided in the accompanying drawings is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0020] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0021] Before providing a detailed explanation of this application, let's first introduce its application scenarios.

[0022] In cell biology research, clinical in vitro diagnostics, and drug development and screening, rapid, non-invasive, multi-parameter analysis of large numbers of cells is often required to assess their physiological state, identify abnormal cells, or evaluate drug effects. Traditional flow cytometry relies on fluorescent labeling, which suffers from labeling interference, high cost, and limited information dimensions. While electrical impedance tomography (EIT) shows promise, it is often limited by bottlenecks such as poor cell focusing stability, single detection parameters, and difficulty in simultaneously acquiring cellular mechanical and electrical information, thus restricting its widespread application in complex samples and high-precision analyses.

[0023] Based on this, this application provides a microfluidic cell detection device and method. The device includes a microfluidic chip, an impedance detection module, a signal acquisition module, and a signal processing module. The microfluidic chip has a main microchannel and a sample inlet and a sheath fluid inlet connected to it. A first reference electrode, a second reference electrode, and an excitation electrode are symmetrically arranged in the detection area at the bottom of the main microchannel. The input terminal of the impedance detection module is connected to the two reference electrodes to acquire electrode signals, and its output terminal is connected to the signal acquisition module. The signal acquisition module receives the electrical response signal generated by the impedance detection module based on the electrode signals, and simultaneously provides an excitation signal to the excitation electrode through its first output terminal. The signal processing module receives a detection response signal based on the electrical response signal from the signal processing module via its second output terminal. The signal processing module then analyzes the impedance parameters of the cell sample to be tested based on the detection response signal. Structurally, this achieves a high degree of spatial integration of cell focusing, electric field excitation, signal sensing, and data processing, reducing external piping and manual intervention, lowering operational complexity, and improving detection throughput and repeatability. At the same time, it ensures the accuracy and stability of the impedance parameters, thereby significantly improving the integration, automation, and reliability of cell impedance detection, and providing an effective technical platform for high-throughput, label-free cell analysis.

[0024] The following explanation, in conjunction with the accompanying drawings, uses several embodiments to illustrate the concepts.

[0025] Figure 1 This application provides a schematic diagram of the structure of a microfluidic cell detection device, as shown below. Figure 1 As shown, the microfluidic cell detection device includes: a microfluidic chip 1, an impedance detection module 2, a signal acquisition module 3, and a signal processing module 4.

[0026] The microfluidic chip 1 includes a main microchannel 11, and a sample inlet 12 and a sheath fluid inlet 13 connected to the inlet end of the main microchannel 11. Specifically, the sample inlet 12 is used to inject the cell sample to be tested into the main microchannel, and the sheath fluid inlet 13 is used to inject viscoelastic fluid. After the cell sample and viscoelastic fluid meet in the main microchannel 11, the viscoelastic fluid can automatically drive and stably focus the cells to the central axis region of the main microchannel 11 using the normal stress gradient generated during its flow, without the need for external sheath flow or mechanical constraints. This also ensures the stability and consistency of the cells when they subsequently pass through the detection area. This process is accompanied by a controllable, slight, and reversible deformation of the cell sample to be tested. It should be noted that the viscoelastic fluid used in this application consists of a polyethylene oxide (PEO) polymer with a concentration of 0.1% and a molecular weight of 1 MDa. As a high molecular polymer, polyethylene oxide can impart good viscoelasticity to fluids, allowing cells to naturally focus to the center of the channel under normal stress without an external force field, thus enhancing the ability to distinguish the mechanical properties of cells. Furthermore, a certain degree of deformation occurs during the focusing process, which enhances the measurement accuracy of the mechanical properties of cells.

[0027] The microfluidic chip 1 also includes a detection area 14, within which three coplanar electrodes are arranged, including a first reference electrode 141, a second reference electrode 142, and an excitation electrode 143. These electrodes are symmetrically distributed at the bottom of the main microchannel. The excitation electrode is used to receive a sinusoidal excitation signal, while the reference electrode is used to acquire the electrode signal generated when the cell to be tested passes through.

[0028] The input terminal of the impedance detection module 2 is electrically connected to the first reference electrode 141 and the second reference electrode 142. When the focused and deformed test cells pass through the detection area 14 one by one, they will disturb the electric field established by the excitation electrode 143. This disturbance is captured by the two reference electrodes, generating a weak differential signal, i.e., generating an electrode signal. The function of the impedance detection module 2 is to acquire this original electrode signal (usually a micro-current signal) and perform preliminary processing on it to generate an electrical response signal.

[0029] The output terminal of the impedance detection module 2 is connected to the input terminal of the signal acquisition module 3, and the input terminal of the signal acquisition module 3 receives the electrical response signal from the impedance detection module 2.

[0030] The signal acquisition module 3 is connected to the excitation electrode 143 through the first output terminal, and applies an AC excitation signal of a set frequency (e.g., low frequency or medium frequency) to the excitation electrode 143 to establish a stable detection electric field. At the same time, the signal acquisition module 3 further processes the input electrical response signal to generate a detection response signal. Then, the signal acquisition module 3 outputs the detection response signal through its second output terminal.

[0031] Signal processing module 4 (e.g., a computer and dedicated analysis software) is connected to the second output terminal of signal acquisition module 3 and is used to detect the response signal. Signal processing module 4 has a built-in preset analysis algorithm that can process the detected response signal and finally extract and output key impedance parameters characterizing the physical properties of the cell, i.e., the impedance of the cell sample under test, such as the electrical diameter reflecting cell size and membrane properties, and the opacity reflecting the electrical heterogeneity inside the cell. At the same time, since the cell under test undergoes deformation related to its own rigidity during the previous viscoelastic fluid focusing process, this deformation causes a systematic and regular change in the measured values ​​of its "electrical diameter" and "opaqueness", which directly affects the electrode signal captured by the reference electrode. Therefore, by analyzing the impedance parameters (electrical diameter, opacity, etc.) of the cell sample under test, this device can indirectly and synchronously infer the rigidity information of the cell under test.

[0032] In practical setup, the various modules in the microfluidic cell detection device are electrically and fluidly connected to form a complete detection system. The microfluidic chip 1 guides the cell sample and viscoelastic fluid to the main microchannel 11 through the sample inlet 12 and sheath fluid inlet 13, and performs impedance measurement through the detection area 14. The impedance detection module 2 is connected to two reference electrodes, and the signal acquisition module 3 receives the electrical response signal, processes it, and transmits it to the signal processing module 4. Finally, the signal processing module 4 analyzes the impedance characteristics of the cells, completing the cell detection and analysis.

[0033] In this embodiment, a microfluidic cell detection device composed of the aforementioned microfluidic chip, impedance detection module, signal acquisition module, and signal processing module creates a controllable channel environment for the thorough mixing, stable focusing, and directional flow of viscoelastic fluid and the cell sample to be tested through the detection zone. Simultaneously, the three electrodes in the detection zone enable the complete process of picking up the electrode signals generated when the cells pass through the detection zone, converting them into detection response signals after signal conditioning by the impedance detection module and signal acquisition module, and finally resolving them into impedance parameters by the signal processing module. Structurally, this achieves a high degree of spatial integration of cell focusing, electric field excitation, signal sensing, and data processing, reducing external piping and manual intervention, lowering operational complexity, and improving detection throughput and repeatability. It also ensures the accuracy and stability of impedance parameters, thereby significantly improving the integration, automation, and reliability of cell impedance detection, providing an effective technical platform for high-throughput, label-free cell analysis.

[0034] exist Figure 1 Based on the microfluidic cell detection device provided in the corresponding embodiments, in one possible implementation, this application also provides an implementation example of a microfluidic cell detection device. Figure 2This is a schematic diagram of another microfluidic cell detection device provided in this application. Figure 2 As shown, the microfluidic chip 1 also includes a collection outlet 15 and a waste liquid outlet 16. The collection outlet 15 and the waste liquid outlet 16 are connected to the outlet end of the main microchannel 11 and are used to separate and discharge the detected cell sample and viscoelastic fluid, respectively.

[0035] The collection outlet 15 guides cell samples that have passed through the detection zone and undergone impedance testing to a designated collection path for further use in subsequent cell analysis, culture, optical observation, or downstream experiments. The waste outlet 16 guides the viscoelastic fluid and background fluid (excluding cells) out of the system, preventing fluid buildup at the main microchannel 11 outlet and ensuring stable flow throughout the device. By setting the collection outlet 15 and waste outlet 16 as independent fluid branches, not only can the targeted separation of detected cells be achieved, but the circulation path of the viscoelastic fluid within the device is also guaranteed. This maintains a stable flow state within the main microchannel 11 during long-term, continuous detection, further improving detection repeatability and operational stability of the microfluidic cell detection device.

[0036] In one possible implementation, Figure 1 Based on the provided microfluidic cell detection device, to ensure the stability of the aforementioned diversion process, a specific channel ratio structure is typically designed at the outlet end of the main microchannel 11. This ensures that different fluids are automatically diverted to different outlet ends based on flow resistance. Through this structure, when the detected cell sample and viscoelastic fluid continue flowing downstream after the detection zone 14, the cells, due to their hydrodynamic characteristics, preferentially enter the collection outlet 15, while the viscoelastic fluid and background liquid mainly flow to the waste liquid outlet 16, ensuring the integrity and purity of cell collection.

[0037] In this embodiment, after the detection is completed, the cell sample and viscoelastic fluid can be effectively separated, thereby enabling controllable output of the sample after detection in multiple experimental scenarios and improving the overall system's flexibility, functional scalability, and usability.

[0038] exist Figure 2 Based on the microfluidic cell detection device provided in the corresponding embodiment, optionally, the microfluidic chip 1 includes a substrate and an organosilicon channel structure covering the substrate, wherein the main microchannel 11, sample inlet 12, sheath fluid inlet 13, collection outlet 15 and waste liquid outlet 16 are all disposed in the organosilicon channel structure.

[0039] Specifically, the substrate is preferably a glass substrate, on which a first reference electrode 141, a second reference electrode 142, and an excitation electrode 143 are fabricated using photolithography and metal evaporation processes. The silicone channel structure is preferably a polydimethylsiloxane (PDMS) channel layer. PDMS is a widely used silicone elastomer with good biocompatibility, gas permeability, optical transparency, and ease of molding. By replicating the flow channel pattern on the mold using soft photolithography, a three-dimensional structure including a main microchannel 11, a sample inlet 12, a sheath fluid inlet 13, a collection outlet 15, and a waste outlet 16 can be formed. During fabrication, the cured PDMS channel layer is tightly bonded to the glass substrate with the detection area 14, thereby forming a complete and closed microfluidic chip 1. This PDMS-glass composite structure not only has good optical transparency for easy observation, but its elastic PDMS material is also easy to connect to external tubing, and it has excellent biocompatibility, making it suitable for long-term detection of live cells.

[0040] The following provides a specific implementation of the microfluidic chip 1 in this embodiment. First, a channel master mold is prepared on a silicon wafer using SU-8 photoresist through photolithography, wherein the width of the main microchannel 11 is set to 50 μm and the height to 30 μm. Then, PDMS prepolymer and curing agent are mixed at a mass ratio of 10:1, vacuum degassed, and poured onto the master mold, and baked at 65°C for 4 hours to obtain the PDMS channel structure. Simultaneously, a Ti / Au (20nm / 200nm) coplanar three-electrode structure (detection area 14) is fabricated on a glass substrate using photolithography and metal evaporation processes. The electrode width and spacing are both 20 μm, and they are symmetrically distributed in the detection area 14. Finally, the PDMS channel structure and the glass substrate are aligned and bonded after oxygen plasma treatment for 2 minutes to form a complete microfluidic chip 1 structure.

[0041] To clearly demonstrate the microfluidic cell detection device provided in this embodiment, this application also provides implementation examples of the microfluidic cell detection device. Figure 3 This is a schematic diagram of another microfluidic cell detection device provided in this application. Figure 3 As shown, the substrate of the microfluidic chip 1 is glass, and the detection region 14 structure is pre-fabricated on its surface; while covering the glass substrate is a PDMS channel layer (i.e., PDMS channel layer replicated by soft photolithography) Figure 3 (Showing all channels covering the base).

[0042] In this embodiment, by employing a microfluidic chip comprising a substrate and an organosilicon channel structure covering it, the main microchannel, each inlet and outlet can be integrated in a high-precision, integrated manner within a flexible, transparent organosilicon material. This facilitates cell observation and long-term experiments, enhancing the practicality and ease of operation of the device. Thus, while maintaining detection performance, the device's integration, applicability, and scalability are further optimized.

[0043] exist Figure 1 Based on the microfluidic cell detection device provided in the corresponding embodiments, in one possible implementation, this application also provides an implementation example of a microfluidic cell detection device. Figure 4 This is a schematic diagram of another microfluidic cell detection device provided in this application. Figure 4 As shown, the impedance detection module 2 includes a transimpedance amplifier 21. The two input terminals of the transimpedance amplifier 21 are used to connect the first reference electrode 141 and the second reference electrode 142. The output terminal of the transimpedance amplifier 21 is the output terminal of the impedance detection module 2, which is used to connect the signal acquisition module 3.

[0044] When the cell sample to be tested is stably focused and passes through the detection area 14 under the drive of viscoelastic fluid, it will disturb the alternating electric field established by the excitation electrode 143, thereby generating a differential current signal between the two reference electrodes.

[0045] Since the first reference electrode 141 and the second reference electrode 142 are symmetrically arranged on both sides of the detection area 14, the two input terminals of the transimpedance amplifier 21 are connected to the first reference electrode 141 and the second reference electrode 142, thereby receiving the differential current signal generated by these two reference electrode paths. This differential current signal is the electrode signal received by the impedance detection module 2. The transimpedance amplifier 21 simultaneously acquires and amplifies the signals from the two reference electrodes through its differential structure, converting the weak current signal into a stable voltage signal to improve the signal-to-noise ratio and resolvability of the subsequent signal acquisition module 3. This voltage signal is the electrical response signal generated by the impedance detection module 2 and is output to the signal acquisition module 3.

[0046] Specifically, refer to Figure 4The transimpedance amplifier 21 has a differential amplification unit inside, each with a fixed input gain (e.g., a gain of G=1K), used to convert the electrode signal at the input terminal into a current-to-voltage signal. At the same time, the internal AC coupling capacitor AC filters out low-frequency noise and DC drift, keeping the output signal stable and suitable for impedance measurement under intermediate frequency excitation conditions. The transimpedance amplifier 21 is further configured with a subsequent buffer amplification unit, whose gain is generally set to unity gain (e.g., G=1), used to provide the differentially converted AC voltage signal, i.e., the electrical response signal, to the signal acquisition module 3 to ensure the signal amplitude is stable, thus facilitating subsequent processing.

[0047] In this embodiment, the transimpedance amplifier 21 is set as the core component of the impedance detection module 2, which can accurately capture the electrode signal even when the cell under test generates only a very small impedance disturbance, while maintaining good amplitude consistency and phase accuracy, thus improving the reliability and stability of the device.

[0048] exist Figure 4 Based on the microfluidic cell detection device provided in the corresponding embodiments, in one possible implementation, this application also provides an implementation example of a microfluidic cell detection device. Figure 5 This is a schematic diagram of another microfluidic cell detection device provided in this application. Figure 5 As shown, the signal acquisition module 3 includes: a lock-in amplifier 31, the input terminal of which is connected to the output terminal of the transimpedance amplifier 21, and the control terminal of the lock-in amplifier 31 is used to receive an external control signal, which enables the lock-in amplifier 31 to generate an excitation signal based on the external control signal; the first output terminal of the lock-in amplifier 31 is connected to the excitation electrode 143, and the second output terminal of the lock-in amplifier 31 is connected to the signal processing module 4. Specifically, the input terminal of the lock-in amplifier 31 is used to receive the electrical response signal processed by the transimpedance amplifier 21. The external control signal received by the control terminal of the lock-in amplifier 31 is usually provided by the host computer connected to it, and is used to set the frequency, amplitude, and waveform parameters of the excitation signal. Optionally, the lock-in amplifier 31 can be an HF2LI dual-channel lock-in amplifier.

[0049] Reference Figure 5 The lock-in amplifier 31 integrates an independent frequency generator 311, which includes an output superimposed unit 321 and a digital oscillator 322. The output terminal of the output superimposed unit 321 serves as the first output terminal of the lock-in amplifier 31 and is connected to the excitation electrode 143. The input terminal of the digital oscillator 322 serves as the control terminal of the lock-in amplifier 31 and is used to receive external control signals.

[0050] The digital oscillator 322 is used to directly generate a frequency-stable reference signal internally. It can generate excitation waveforms with different frequency ranges according to the settings of external control signals. Its internal numerically controlled oscillation architecture makes the output frequency precisely adjustable, with low phase noise and high spectral purity. The output superimposed unit 321 is used to perform online superposition and amplitude shaping of the frequency signals generated by the digital oscillator 322. In one possible implementation, the lock-in amplifier 31 can generate excitation signals of different frequencies simultaneously within a single hardware structure, realizing a dual-frequency or multi-frequency coexistence excitation mode, so as to establish a multi-band electric field in the detection area 14 according to different measurement requirements.

[0051] Specifically, the digital oscillator 322 generates two sinusoidal digital waveforms based on external control signals (e.g., 1MHz and 5MHz). The output superimposed unit 321 receives these two signals, performs linear superposition, and dynamically adjusts and shapes the overall amplitude of the superimposed composite waveform to ensure that the final output excitation signal maintains the set relative amplitude and phase relationship at each frequency component. This composite excitation signal is applied to the excitation electrode 143 to achieve synchronous multi-frequency impedance excitation of the cell, thereby exciting the electrode signals at different frequencies through a single cell passage event. Subsequently, the lock-in amplifier 31 uses the electrical response signal generated based on the composite excitation signal to decouple and extract the detection response signals of the cell at 1 MHz and 5 MHz, respectively. Finally, the signal processing module 4 models and analyzes the detection response signal to obtain the impedance parameters of the cell sample to be tested. This enables the simultaneous acquisition of the electrical diameter (mainly based on 1 MHz) reflecting cell size and the cytoplasmic conductivity and membrane permeability (mainly based on 5 MHz) reflecting internal structure and membrane state in a single measurement, thereby accurately capturing the multidimensional electrical characteristics of the cell (such as electrical diameter, cytoplasmic conductivity, and membrane permeability). In addition, the lock-in amplifier 31 is internally equipped with a digital processing unit 323 for signal demodulation. The two input terminals of the digital processing unit 323, the input signal + and the input signal -, serve as the input terminals of the lock-in amplifier 31 to receive the output terminal of the transimpedance amplifier 21. The output terminal of the digital processing unit 323 serves as the second output terminal of the lock-in amplifier 31 and is connected to the signal processing module 4.

[0052] The digital processing unit 323 may include modules for cross-correlation calculation, amplitude-phase demodulation, or quadrature component extraction. It uses a reference signal (in phase and frequency with the excitation signal) generated by the numerical oscillator 322 to separate the amplitude and phase of the input electrical response signal, thereby obtaining the real and imaginary components of the electrical response signal. These demodulated signals are transmitted as detection response signals through the second output terminal to the signal processing module 4 for further processing, ultimately obtaining the impedance of the cell sample to be tested.

[0053] In this application, the signal processing module 4 can be a dedicated hardware processor or implemented by a computer. One possible approach is that when the signal processing module 4 is implemented by a computer, it not only receives the detection response signal from the lock-in amplifier 31, but also communicates with the control terminal of the lock-in amplifier 31 to send external control signals (setting the excitation frequency, amplitude, switching the operating mode, etc.), enabling the lock-in amplifier 31 to adjust its operating state according to the set frequency, amplitude, and strategy, thereby achieving intelligent detection and high repeatability measurement throughout the entire process.

[0054] In this embodiment, the lock-in amplifier 31 is set as the core component of the signal acquisition module 3, so that the functions of excitation signal generation, synchronous demodulation and signal output are realized in the same hardware module. No external frequency source or independent demodulation circuit is required, which reduces the number of system components, avoids phase drift caused by asynchronous external frequency sources, and improves the stability, noise resistance and repeatability of detection results.

[0055] exist Figure 1 Based on the microfluidic cell detection device provided in the corresponding embodiments, in one possible implementation, this application also provides an implementation example of a microfluidic cell detection device. Figure 6 This is a schematic diagram of another microfluidic cell detection device provided in this application. Figure 6 As shown, the microfluidic cell detection device also includes: an optical excitation module 5; the light-emitting surface of the optical excitation module 5 faces the detection area 14.

[0056] By aligning the light-emitting surface of the optical excitation module with the detection area, cells flowing through the detection area 14 can undergo optical excitation under specific light intensity and spectral conditions while simultaneously receiving impedance measurement. By achieving spatial overlap between photoexcitation and impedance detection in the detection area 14, simultaneous measurement of the functional state of cell membrane molecules can be realized.

[0057] Specifically, the optical excitation module 5 is used to apply light stimulation of a preset wavelength to the cells passing through the detection area 14 to excite or regulate the functional state of their membrane proteins or ion channels. The optical excitation module 5 may include a combination structure of an inverted fluorescence microscope and an sCMOS camera. The inverted fluorescence microscope has a built-in narrowband filter module in its optical path, such as an excitation filter BP460–495, a dichroic mirror DM505, and an emission filter BA510–550. This type of narrowband filter system can provide stable illumination conditions within a specific wavelength range, allowing blue light to continuously illuminate the detection area, thereby achieving effective excitation of photosensitive proteins (such as rsChRmine) expressed on the cell membrane. The sCMOS camera can simultaneously acquire changes in optical signals after excitation to analyze the behavior of the cell membrane during the photoexcitation process. For example, in a typical application scenario, the light stimulation is a blue light pulse with a wavelength of 450 nm. This wavelength can specifically activate the light-sensitive proteins expressed on the cell membrane, thereby inducing a transient change in transmembrane ion current or membrane potential. The pulse width, frequency and intensity of the light stimulation can be designed according to experimental requirements to achieve controllable excitation of the functional state of the cell membrane and synchronous impedance monitoring.

[0058] It should be noted that the optical excitation module 5 can not only provide single-band light stimulation, but also, in some implementations, be configured with multiple excitation channels with separated spectra. Each channel is equipped with a corresponding excitation filter, dichroic mirror, and emission filter, enabling it to independently or superimpose excitation of different types of photosensitive proteins or membrane proteins, thus achieving membrane functional state analysis under combined light stimulation conditions. When cells are stimulated by light in the detection area 14, their membrane electrophysiological activity may change rapidly. These changes can be captured in real time by impedance detection, allowing the optical excitation module 5 and the impedance detection module 2 to work together to achieve electrical monitoring of changes in the functional state of cell membrane molecules.

[0059] In this embodiment, by adding an optical excitation module 5 to the microfluidic cell detection device, the device can not only acquire the electrophysical parameters of cells based on viscoelastic focusing and impedance measurement, but also simultaneously collect changes in cell membrane function under light stimulation. This combines optical stimulation with electrical response, enabling multimodal joint detection of cell deformation, impedance measurement, and optical excitation within the same device. It can be used in various cell function analysis fields such as screening photosensitive proteins, drug intervention detection, and membrane electrical activity monitoring, thus improving the device's ability to perform complex functional analysis.

[0060] Based on the above embodiments, in one possible implementation, this application also provides an example of a microfluidic cell detection device. Figure 7 This is a schematic diagram of another microfluidic cell detection device provided in this application. Figure 7As shown, the microfluidic cell detection device includes: a microfluidic chip 1, a transimpedance amplifier 21, a lock-in amplifier 31, a signal processing module 4, and an optical excitation module 5.

[0061] The microfluidic chip 1 includes a main microchannel 11, a sample inlet 12, a sheath fluid inlet 13, a detection zone 14, a collection outlet 15, and a waste liquid outlet 16. Transimpedance amplifier 21 is a component of impedance detection module 2. Its two input terminals are used to connect to the first reference electrode 141 and the second reference electrode 142 to receive electrode signals and generate an electrical response signal based on them. Lock-in amplifier 31 is a component of signal acquisition module 3. Lock-in amplifier 31 integrates an independent frequency generator 311, which includes an output superimposed unit 321 and a digital oscillator 322. The output terminal of the output superimposed unit 321 serves as the first output terminal of lock-in amplifier 31 and is connected to the excitation electrode 143. The input terminal of the digital oscillator 322 serves as the control terminal of lock-in amplifier 31 to receive external control signals. Lock-in amplifier 31 also has a digital processing unit 323. The two input terminals of digital processing unit 323, the + and - input signals, serve as the input terminals of lock-in amplifier 31 to receive the output terminal of transimpedance amplifier 21 and generate a detection response signal based on the electrical response signal input to transimpedance amplifier 21. The output terminal of digital processing unit 323 serves as the second output terminal of lock-in amplifier 31 and is connected to signal processing module 4. The signal processing module 4 is a computer, which is connected not only to the output of the digital processing unit 323 to process the detection response signal to obtain the impedance of the cell sample to be detected, but also to the input of the digital oscillator 322 to send external control signals to it. Meanwhile, the light-emitting surface of the optical excitation module 5 faces the detection area 14.

[0062] Based on this embodiment, the microfluidic cell detection device achieves full-process integration and closed-loop control from cell sample introduction, focusing, excitation, signal acquisition to data processing and optical excitation. Specifically, the cell sample and viscoelastic fluid are fed into the main microchannel 11 through the sample inlet 12 and sheath fluid inlet 13, respectively. Under viscoelastic action, they automatically focus and deform before flowing through the detection zone 14. The digital oscillator 322 in the lock-in amplifier 31 generates a target frequency waveform according to the control signal issued by the signal processing module 4. The waveform is then synthesized by the output superimposed unit 321 and applied to the excitation electrode 143 to establish a multi-frequency electric field in the detection zone. When the cell sample passes through the detection zone 14, the electrode signal generated by the disturbance electric field is picked up by the first reference electrode 141 and the second reference electrode 142 and converted into an electrical response signal by the transimpedance amplifier 21. This electrical response signal is input to the digital processing unit 323 of the lock-in amplifier 31 for synchronous demodulation and outputs a detection response signal to the signal processing module 4 for impedance parameter extraction. At the same time, the signal processing module 4 can dynamically adjust the excitation frequency and mode through the control signal to achieve adaptive detection. The optical excitation module 5 provides light stimulation to the detection zone 14, enabling the device to simultaneously acquire the electrical impedance response and membrane function changes under light excitation in a single cell passage event. Subsequently, the tested cell samples and viscoelastic fluid are separated and discharged through the downstream collection outlet 15 and waste liquid outlet 16. The cells that have completed the test can be recycled or discarded according to experimental needs, ensuring the continuity and controllability of the test process.

[0063] In this embodiment, by achieving spatial synchronization and signal closed-loop of electrical, optical and fluid technologies, parallel, highly sensitive and programmable detection of cellular multi-physical field responses is realized, thereby improving the device's multimodal analysis capabilities and automation level.

[0064] Based on the microfluidic cell detection device provided in any of the above embodiments, this application also provides a microfluidic cell detection method. Figure 8 This is a schematic flowchart of a microfluidic cell detection method provided in this application. Figure 8 As shown, this microfluidic cell detection method includes: S101, inject the cell sample to be tested into the sample inlet.

[0065] Before the detection begins, the operator or an automated sampler injects the cell sample to be tested into the main microchannel 11 through the sample inlet 12 of the microfluidic chip 1 using a syringe pump, pressure controller, or gravity drive. The cell sample to be tested is typically a population of live cells suspended in a buffer solution. After entering the main microchannel 11 through the sample inlet 12, it moves downstream along the channel direction, providing an initial sample flow for cell detection.

[0066] S102, viscoelastic fluid is injected into the sheath fluid inlet, so that the viscoelastic fluid and the cell sample to be tested merge in the main microchannel and then flow through the detection area.

[0067] Specifically, a pre-prepared viscoelastic fluid (such as a 0.1% PEO solution with a molecular weight of 1 MDa) is injected into the main microchannel 11 at a stable flow rate through the sheath fluid inlet 13. After the viscoelastic fluid and the test cell sample merge in the main microchannel 11, the viscoelastic fluid can automatically drive and stably focus the cells to the central axis region of the main microchannel 11 without the need for external sheath flow or mechanical constraints, utilizing the normal stress gradient generated during its flow. This process is accompanied by a controllable, slight, and reversible deformation of the test cell sample, the degree of which is related to the rigidity of the cells and is reflected in a distinguishable change in the electrical signal in subsequent impedance measurements. The focused cells, carried by the viscoelastic fluid, flow sequentially and stably through the detection area 14 located downstream of the channel.

[0068] S103 applies an excitation signal to the excitation electrode through the signal acquisition module.

[0069] Specifically, the signal acquisition module 3 generates an AC excitation signal with a specific frequency, amplitude, and waveform (e.g., a sine wave with an amplitude of 5 Vpp and a frequency of 1 MHz and / or 5 MHz) according to preset parameters. This excitation signal is applied to the excitation electrode 143 arranged in the detection area 14 of the microfluidic chip 1, thereby establishing a stable and uniform AC detection electric field environment in the main microchannel 11 of the detection section, providing an excitation source for subsequent impedance detection.

[0070] S104: The electrode signals from the first reference electrode and the second reference electrode are obtained through the impedance detection module, and the electrical response signal after processing by the impedance detection module is obtained.

[0071] Specifically, when a focused and deformed cell passes through the detection area 14, its presence disturbs the original electric field distribution, thereby inducing a weak current signal (i.e., electrode signal) on the first reference electrode 141 and the second reference electrode 142 symmetrically arranged on both sides of the excitation electrode 143. The two input terminals of the impedance detection module 2 are electrically connected to these two reference electrodes respectively, for real-time acquisition of the electrode signal and preliminary processing, thereby obtaining the electrical response signal generated by the impedance detection module 2.

[0072] S105 receives and processes the electrical response signal through the signal acquisition module to obtain the detection response signal.

[0073] Specifically, the signal acquisition module 3 receives the electrical response signal from the impedance detection module 2 and processes it further. This processing includes, but is not limited to, signal amplification, filtering and noise reduction, and response extraction at characteristic frequencies. The signal acquisition module 3 converts the electrical response signal into a standardized signal, i.e., the detection response signal, which is convenient for subsequent analysis. This process significantly improves the signal-to-noise ratio and stability of the signal, providing a high-quality signal foundation for subsequent feature parameter extraction.

[0074] S106 processes the detection response signal through the signal processing module to obtain the impedance of the cell sample to be tested.

[0075] Specifically, the detection response signal from the signal acquisition module 3 is received by the signal processing module 4. The detection response signal is processed and analyzed using algorithms built into the signal processing module 4 (such as peak detection, waveform analysis, and equivalent circuit model fitting), ultimately extracting and outputting key impedance parameters characterizing the cell's physical properties, i.e., the impedance of the cell sample under test. Examples include the electrical diameter reflecting cell size (mainly based on low-frequency excitation signals) and the opacity reflecting internal electrical heterogeneity of the cell (mainly based on mid-frequency excitation signals). By analyzing these impedance parameters and their combinations, cell type, state, and physical properties can be identified and distinguished under label-free conditions. Simultaneously, while measuring the impedance of the cell sample under test, cell rigidity information can also be indirectly inferred.

[0076] In this embodiment, the cell sample to be tested and the viscoelastic fluid are injected into the microfluidic chip and then combined. Viscoelasticity is used to automatically focus and controllably deform the cells. An excitation signal is applied to the excitation electrode in the detection area to establish a detection electric field. The electrode signals of the two reference electrodes are acquired by the impedance detection module and an electrical response signal is generated. The signal acquisition module then processes the signal to obtain the detection response signal. Finally, the signal processing module analyzes and extracts the impedance parameters of the cells to be tested. The entire detection process is highly automated. No additional adjustment or multi-device cooperation is required from the time the cells enter the channel to the completion of impedance acquisition. It features high speed, continuity, and non-contact operation, which can meet the needs of high-throughput detection. While maintaining detection accuracy, it significantly improves detection efficiency, providing an efficient, sensitive, and stable solution for label-free cell analysis.

[0077] The above Figure 8 Based on the provided microfluidic cell detection method, optionally, in step S106 above, the detection response signal is processed by a signal processing module to obtain the impedance of the cell sample to be tested, which includes: S610 obtains the impedance of the cell sample to be tested based on the real and imaginary components contained in the detection response signal through the signal processing module.

[0078] Specifically, the signal processing module 4 can perform frequency domain feature separation and complex impedance analysis on the detection response signal from the signal acquisition module 3. Specifically, since the detection response signal is an AC signal induced by the cell perturbation electric field at the excitation frequency, it inherently contains both in-phase (real) and orthogonal (imaginary) information that varies with the electric field. The signal processing module 4 can extract the real component in phase with the excitation signal and the imaginary component orthogonal to the excitation signal from the detection response signal using built-in digital signal processing algorithms (such as phase reference decomposition and Fourier component extraction). After obtaining the real and imaginary components, the signal processing module 4 can further combine the two parts of information according to the calculation relationship of complex impedance to obtain the impedance value of the cell sample under test at the excitation frequency, providing a data basis for cell feature analysis.

[0079] In one possible implementation, the signal acquisition module 3 is a lock-in amplifier (LCAP). Since the LCAP internally has a reference signal path synchronized with the excitation signal and can perform multiplication and low-pass filtering on the input electrical response signal, it directly separates and outputs the real component in phase with the reference signal and the orthogonal imaginary component in the electrical response signal. The signal processing module 4 receives the real and imaginary components from the LCAP and calculates the impedance of the cell sample under test accordingly. This eliminates the need for complex frequency domain decomposition calculations, improving the stability and sensitivity of impedance calculation and further enhancing the accuracy of cell electrical characteristic identification.

[0080] In this embodiment, the impedance of the cell sample to be tested is calculated by the signal processing module based on the real and imaginary components contained in the detection response signal. This enables the acquisition of complete impedance information of the cell under specific excitation conditions, enhances the ability to distinguish subtle differences in physical properties between cells, and achieves more refined analysis of cell electrical properties.

[0081] In the above Figure 8 Based on the provided microfluidic cell detection method, this application also provides an implementation example of the microfluidic cell detection method. Figure 9 This is a schematic flowchart of another microfluidic cell detection method provided in this application. Figure 9 As shown, based on the above S101-S106, this microfluidic cell detection method further includes: S710 applies low-frequency and medium-frequency excitation signals to the excitation electrode through the signal acquisition module, and obtains the first impedance generated by the cell sample under the low-frequency excitation signal and the second impedance generated under the medium-frequency excitation signal, respectively.

[0082] Specifically, in actual operation, the signal acquisition module 3 applies a preset low-frequency excitation signal (e.g., a sine wave with a frequency of 1 MHz and an amplitude of 5 Vpp) to the excitation electrode 143 of the detection area 14. When the cell under test passes through the detection area 14, the impedance detection module 2 and the signal acquisition module 3 work together to complete a complete signal acquisition and processing flow (corresponding to S104-S106), thereby obtaining the first impedance generated by the cell under test under the low-frequency excitation signal.

[0083] Subsequently, during the passage of the same cell through the detection zone 14 or before the next cell enters the detection zone 14, the signal acquisition module 3 switches the excitation signal parameters, applying a preset intermediate frequency excitation signal (e.g., a sine wave with a frequency of 5 MHz and an amplitude of 5 Vpp) to the excitation electrode 143. Similarly, signal acquisition and processing are performed to obtain the second impedance generated by the cell under test under this intermediate frequency excitation signal. Through this switching method, the impedance response of the same cell or cell population at two different characteristic frequencies can be obtained.

[0084] S720, the electrical diameter of the cell sample to be tested is obtained based on the first impedance, and the opacity of the cell sample to be tested is obtained based on the first impedance and the second impedance.

[0085] Specifically, after the signal processing module 4 processes the signal to obtain the first impedance and the second impedance based on the low-frequency excitation signal and the medium-frequency excitation signal, the electrical diameter of the cell sample to be tested can be calculated based on the first impedance according to the preset analytical algorithm built into the signal processing module 4.

[0086] Furthermore, the first impedance and the second impedance are jointly analyzed by the signal processing module 4. Since the intermediate frequency excitation signal is more sensitive to changes in the dielectric properties inside the cell, by calculating the ratio or difference of the impedance response at the two frequencies (e.g., comparing their amplitude or phase), a composite parameter reflecting the electrical heterogeneity inside the cell sample under test, namely the opacity, can be obtained. The opacity is closely related to the cell's conductivity, membrane permeability, and internal structure.

[0087] In one possible implementation, the signal acquisition module 3 integrates a frequency generator, which comprises a digital oscillator and an output superimposed unit. The digital oscillator generates preset low-frequency and mid-frequency digital waveforms, while the output superimposed unit performs real-time superposition, amplitude shaping, and amplitude adjustment on the frequency signals from the digital oscillator. When needed, two or more frequency signals can be linearly superimposed into a composite excitation signal, ensuring that the final composite excitation signal output to the excitation electrode 143 maintains a set relative amplitude and phase relationship among multiple frequency components. Through this frequency generator structure, the signal acquisition module 3 can not only sequentially output low-frequency and mid-frequency excitation signals but also select to operate in single-frequency excitation mode or composite multi-frequency excitation mode according to detection requirements. In the composite excitation mode, the digital oscillator simultaneously generates two sinusoidal signals of different frequencies (e.g., 1 MHz and 5 MHz). After being superimposed by the output superimposed unit, a composite excitation signal containing both frequency components is formed and applied to the excitation electrode 143. This allows each cell passing through the detection area 14 to simultaneously receive electric field excitation at both frequencies in the same passage event, thereby enabling the impedance detection module 2 to acquire a single electrical response signal containing multi-frequency information. After receiving the electrical response signal, the signal acquisition module 3 separates, filters, and extracts features from different frequency channels through its internal synchronous demodulation structure, obtaining two sets of detection response signals corresponding to low and medium frequencies. These two sets of detection response signals are then output to the signal processing module 4. The signal processing module 4 further processes the two sets of detection response signals to obtain the corresponding first impedance and second impedance. This allows the low-frequency impedance parameter reflecting cell electrical properties and the medium-frequency impedance parameter reflecting cell cytoplasmic conductivity and membrane permeability to be obtained simultaneously in a single cell event. Based on the comparison of the difference between the first and second impedances, the opacity of the cell sample under test can be obtained.

[0088] Compared to sequentially applying excitation signals, using composite excitation signals can reduce the waiting time for electric field establishment and switching, improve the utilization rate of single-cell events, and thus maintain multi-frequency impedance detection capability under conditions of higher flow rates and higher detection throughput. It also significantly improves the calculation accuracy and stability of electrical diameter and opacity, enhances the ability to distinguish cell size, membrane properties, cytoplasmic conductivity, membrane permeability and internal heterogeneity, and achieves simultaneous acquisition of multi-dimensional electrical characteristics.

[0089] In this embodiment, by applying low-frequency and medium-frequency excitation signals, and simultaneously acquiring the two parameters of cell electrical diameter and cell opacity, the ability to identify cell characteristics is enhanced, making it more applicable.

[0090] The above Figure 8 Based on the provided microfluidic cell detection method, optionally, in addition to the above S101-S106, the method further includes: S810: When the cell sample to be tested flows through the detection area, the cells in the detection area are irradiated with light of a preset wavelength through the optical excitation module.

[0091] In one possible implementation, the microfluidic cell detection device further includes an optical excitation module. Specifically, during the process of the cell sample entering the detection zone 14 after being focused by a viscoelastic fluid, the optical excitation module projects excitation light of a preset spectral range, such as a blue light pulse with a wavelength of 450 nm, into the detection zone 14, so that the cells flowing through the detection zone 14 receive optical stimulation while impedance measurement is being performed. This illumination is used to induce responsive changes in cell membrane proteins or membrane electrical activity, such as changes in membrane potential, changes in ion channel permeability, or activation responses of photosensitive proteins. The electrical changes generated by the cells under light stimulation are immediately reflected in the impedance signal, and the transient response generated by light stimulation can be captured by the impedance detection module 2.

[0092] In this example, by combining photoexcitation with impedance detection, the electrophysical changes of cells under photostimulation conditions can be obtained simultaneously in the same detection, enabling higher-dimensional measurement of cell function.

[0093] To describe this application more clearly, specific embodiments are described below.

[0094] Example 1: Detection Method Flow Based on Microfluidic Cell Detection Device. This example provides a basic detection method using a microfluidic cell detection device, demonstrating the complete operational steps from cell inflow into the microchannel to output impedance parameters. First, a sample suspension containing the cells to be tested is prepared, and a specific viscoelastic fluid (such as a 0.1% PEO solution with a molecular weight of 1 MDa) is used as the sheath fluid. The fluid is then loaded into a syringe and injected into the sample inlet and sheath fluid inlet of the microfluidic chip, respectively. Subsequently, the control signal acquisition module simultaneously outputs low-frequency (1 MHz) and medium-frequency (5 MHz) AC excitation signals to the excitation electrodes. Cells focus, deform, and pass through the detection area one by one under the action of the viscoelastic fluid. The impedance detection module acquires the electrode signals in real time and performs preliminary processing, converting them into electrical response signals that are transmitted to the signal acquisition module. The signal acquisition module performs lock-in amplification and demodulation on the electrical response signals to obtain the real (X) and imaginary (Y) data of each cell passage event at 1 MHz and 5 MHz, forming a detection response signal, which is then transmitted to the signal processing module. Finally, the signal processing module analyzes the detection response signal to obtain impedance parameters characterizing the cell's physical properties, such as electrical diameter and opacity, enabling label-free detection of cell size, internal structure, and membrane properties. The data from thousands of cells are ultimately distributed in a characteristic pattern on a two-dimensional scatter plot of electrical diameter and opacity.

[0095] Example 2: Cell size detection method based on microfluidic cell detection device. Human umbilical vein endothelial cells (EA.hy926) and cervical cancer cells (HeLa) were selected as test samples, prepared into suspensions, and injected into the sample inlet of the microfluidic chip. After the viscoelastic fluid was injected through the sheath fluid inlet, both types of cells were automatically focused to the center of the main microchannel and flowed sequentially through the detection area. An excitation signal was applied through the signal acquisition module, and impedance acquisition and signal processing were completed sequentially to obtain the multi-frequency impedance parameters of each cell. After mapping the obtained impedance data to a two-dimensional impedance feature space composed of electrical diameter and opacity, it can be observed that EA.hy926 and HeLa cells form independent distribution areas in this two-dimensional space, showing a clear separation trend. Thus, the impedance characteristics can be used to distinguish cells of different types and physical properties.

[0096] Example 3: Detection of cell rigidity using a microfluidic cell detection device. Human erythrocytes were used as the test sample and treated with different concentrations of glutaraldehyde (a cross-linking agent that hardens cells) and diamide (an oxidizing agent that cross-links and hardens the cytoskeleton). By applying an excitation signal and acquiring the impedance response, the electrical diameter and opacity of erythrocytes under each treatment condition were obtained. The results showed that glutaraldehyde treatment caused the electrical diameter to gradually shift to the right with increasing concentration, reflecting a decrease in cell deformability; diamide treatment caused the distribution of cells in the electrical characteristic space to tend to be concentrated, reflecting a decrease in heterogeneity caused by hardening. As the drug concentration increased, the distribution of the electrical diameter of erythrocytes shifted towards the larger direction, and the opacity increased significantly, thereby capturing the changes in cell rigidity induced by the drug.

[0097] Example 4: Detection of drug blocking agent effects using a microfluidic cell detection device. HEK293T cells stably expressing photosensitive proteins were selected as samples and injected into a microfluidic chip along with a viscoelastic fluid. When the cells flowed through the detection area, a 450nm blue light pulse was applied to the detection area via an optical excitation module, exciting the photosensitive proteins and generating changes in membrane current and membrane potential. This process was completed simultaneously with impedance measurement; therefore, the transient membrane property changes caused by photoexcitation were directly reflected in the impedance response. By comparing the changes in the cell impedance response before and after light irradiation, the activity of membrane channels could be evaluated. Furthermore, the experiment was repeated after pretreating cells with different concentrations (e.g., 5μM–200μM) of calcium channel blockers (e.g., verapamil) for different durations (e.g., 30 minutes, 2 hours, 4 hours). The collected impedance trajectories showed a consistent inhibitory trend in impedance parameters under different drug concentrations and time conditions, and the degree of inhibition increased with increasing treatment time, thus allowing for the evaluation of the potential of membrane protein function and drug effects.

[0098] Optionally, this application also provides a program product, such as a computer-readable storage medium, including a program that, when executed by a processor, performs the above-described method embodiments.

[0099] The integrated units implemented as software functional units described above can be stored in a computer-readable storage medium. These software functional units, stored in a storage medium, include several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) or processor to execute some steps of the methods of the various embodiments of this application. The aforementioned storage medium includes various media capable of storing program code, such as USB flash drives, portable hard drives, read-only memory (ROM), random access memory (RAM), magnetic disks, or optical disks.

[0100] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0101] The above description is merely a specific embodiment 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 technical scope 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 microfluidic cell detection device, characterized in that, include: Microfluidic chip, impedance detection module, signal acquisition module, and signal processing module; The microfluidic chip has a main microchannel, and a sample inlet and a sheath fluid inlet connected to the inlet end of the main microchannel. The microfluidic chip also has a detection area, which is equipped with a first reference electrode, a second reference electrode, and an excitation electrode. The first reference electrode, the second reference electrode, and the excitation electrode are symmetrically distributed at the bottom of the main microchannel. The sample inlet is used to inject a cell sample to be tested, and the sheath fluid inlet is used to inject a viscoelastic fluid, so that the viscoelastic fluid and the cell sample to be tested converge in the main microchannel and then flow through the detection area. The input terminal of the impedance detection module is electrically connected to the first reference electrode and the second reference electrode, so that the impedance detection module generates an electrical response signal based on the electrode signals obtained from the first reference electrode and the second reference electrode. The output terminal of the impedance detection module is connected to the input terminal of the signal acquisition module, so that the signal acquisition module generates a detection response signal based on the electrical response signal. The first output terminal of the signal acquisition module is connected to the excitation electrode to apply an excitation signal to the excitation electrode. The second output terminal of the signal acquisition module is connected to the signal processing module so that the signal processing module can obtain the impedance of the cell sample to be tested based on the detection response signal.

2. The apparatus according to claim 1, characterized in that, The microfluidic chip is further provided with a collection outlet and a waste liquid outlet, which are connected to the outlet end of the main microchannel and are used to divert and discharge the detected cell sample and viscoelastic fluid, respectively.

3. The apparatus according to claim 2, characterized in that, The microfluidic chip includes a substrate and an organosilicon channel structure covering the substrate. The main microchannel, the sample inlet, the sheath fluid inlet, the collection outlet, and the waste liquid outlet are all disposed in the organosilicon channel structure.

4. The apparatus according to claim 1, characterized in that, The impedance detection module includes a transimpedance amplifier, the two input terminals of which are used to connect the first reference electrode and the second reference electrode, and the output terminal of the transimpedance amplifier is the output terminal of the impedance detection module, which is used to connect to the signal acquisition module.

5. The apparatus according to claim 4, characterized in that, The signal acquisition module includes: a lock-in amplifier, the input terminal of which is connected to the output terminal of the transimpedance amplifier, the control terminal of which is used to receive an external control signal, and to enable the lock-in amplifier to generate an excitation signal based on the external control signal; the first output terminal of the lock-in amplifier is connected to the excitation electrode, and the second output terminal of the lock-in amplifier is connected to the signal processing module.

6. The apparatus according to claim 1, characterized in that, The device further includes an optical excitation module; the light-emitting surface of the optical excitation module faces the detection area.

7. A microfluidic cell detection method, characterized in that, The method, applied to the microfluidic cell detection device according to any one of claims 1-6, comprises: Inject the cell sample to be tested into the sample inlet; A viscoelastic fluid is injected into the sheath fluid inlet, so that the viscoelastic fluid and the cell sample to be tested merge in the main microchannel and then flow through the detection area; An excitation signal is applied to the excitation electrode through the signal acquisition module; The electrode signals from the first and second reference electrodes are obtained through the impedance detection module, and the electrical response signal after processing by the impedance detection module is obtained. The electrical response signal is received and processed by the signal acquisition module to obtain the detection response signal; The detection response signal is processed by the signal processing module to obtain the impedance of the cell sample to be tested.

8. The method according to claim 7, characterized in that, The detection response signal is processed by a signal processing module to obtain the impedance of the cell sample to be tested, including: The impedance of the cell sample to be tested is obtained by the signal processing module based on the real and imaginary components contained in the detection response signal.

9. The method according to claim 7, characterized in that, The method further includes: The signal acquisition module applies a low-frequency excitation signal and a medium-frequency excitation signal to the excitation electrode to obtain the first impedance generated by the cell sample under the low-frequency excitation signal and the second impedance generated under the medium-frequency excitation signal, respectively. The electrical diameter of the cell sample to be tested is obtained based on the first impedance, and the opacity of the cell sample to be tested is obtained based on the first impedance and the second impedance.

10. The method according to claim 7, characterized in that, The method further includes: When the cell sample to be tested flows through the detection area, the cells in the detection area are irradiated with light of a preset wavelength through the optical excitation module.

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