Cell sorting and real-time detection system and method based on combination of microfluidics and uniformly spaced microelectrode arrays

Abstract

The invention belongs to the technical field of cell sorting and cell detection, and relates to a cell sorting and real-time detection system and a cell sorting and real-time detection method combining microfluidics and a uniform-spacing microelectrode array. The system comprises a cell injection chamber, a microfluid channel, a capture trap microstructure array, a cell collection chamber and a microelectrode array, the microelectrode array comprises a hexagonal electrode array and a sinusoidal line electrode array, the side length of the hexagonal electrode array and the cell size meet L = kDc, and the hexagonal electrode array meets d =; the amplitude A of the sine line electrode array is lambda / 4, and the wavelength lambda is two times of the cell diameter. Microfluidic sorting and a microelectrode detection array are combined, two innovative interdigitated electrode structures are adopted, the detection sensitivity is remarkably improved, and the micro-fluidic chip is particularly suitable for cell sorting, cell monitoring, precise diagnosis and biosensing application; an integrated and high-throughput solution is provided for the fields of early diagnosis of cancers, drug screening, personalized medical treatment and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell sorting and cell detection, and particularly relates to a cell sorting and real-time detection system and method combining microfluidics and uniform-pitch microelectrode arrays. The present application provides a microfluidic chip system with the synergistic effect of microfluidic channel magnetic field assisted sorting-microelectrode near-field electrical impedance analysis, and realizes the on-chip microfluidic integrated system of cell sorting and electrical detection through innovative microfluidic structure design and microelectrode array design. The system aims to provide an efficient and accurate cell analysis and screening platform. BACKGROUND

[0002] In the field of cell sorting and detection, traditional technologies usually design sorting modules and detection modules as independent units, that is, first use a separate instrument to sort and enrich cells, and then use a dedicated detection system for targeted cell analysis and detection. The connection between cell sorting and detection is weak, and the sorted cells need to be manually transferred to a culture or analysis device, which can easily lead to sample contamination or loss of activity.

[0003] From the perspective of cell sorting, current technologies mainly use the principles of fluid mechanics and the differences in cell physical properties in microfluidic chips to achieve sorting. For example, filter-based sorting based on cell size differences, and techniques that distinguish based on cell deformation ability. The mainstream magnetic sorting technology uses an external permanent magnet to generate a static magnetic field, which can achieve a sorting efficiency of 80-90%. However, the aggregation of magnetic beads can cause microchannel blockage, and the system usually needs to be manually cleaned after continuous operation for no more than 4 hours. Existing improvement schemes attempt to solve this problem by placing magnetic materials on the side walls of the flow channel, but this causes new problems: the magnetic materials significantly attenuate the signal-to-noise ratio of the detection signal, especially in the impedance detection frequency band (100 kHz -10 MHz), causing about 40% signal loss. Therefore, the corresponding microfluidic structure needs to be improved and precise microfluidic control is required. In terms of fluid control, conventional microfluidic systems use a single pressure source to drive sample flow. This method, although simple in structure, is difficult to accurately control the residence time of cells in the sorting and detection zones. In actual operation, the inlet pressure needs to be adjusted repeatedly, which not only affects the experimental repeatability (flow rate fluctuations can reach ±15%), but also easily damages cells due to excessive shear force (survival rate reduced by 20-30%).

[0004] From the aspect of cell detection analysis, the main principle of using electrodes for cell analysis is to measure the impedance change between electrodes caused by the concentration or growth change of the sample, and then obtain the impedance change relationship corresponding to cell proliferation, drug metabolism or specific modification. This detection method has the advantages of high sensitivity and real-time monitoring. Existing cell detection technologies can be divided into two categories. The invasive detection technology is limited in application due to its time-consuming, large sample size, and damage or impact on the normal function of cells. The non-invasive detection technology has significant advantages, can monitor the natural state of cells in real time and continuously, does not need to destroy or interfere with the natural state of cells, and can maintain the activity and function of cells. Microelectrode has the advantages of high reaction rate and high detection sensitivity. At present, there are schemes for using interdigitated array microelectrodes to complete quantitative measurement and specific detection in the field of biomedicine, which belongs to non-invasive detection technology.

[0005] Microelectrode can significantly improve the mass transfer rate of reactants on the electrode surface due to its small size and large effective surface area. The electrode array structure usually adopts rectangular interdigital cross electrodes and circular linear interdigital cross electrodes, and these two kinds of electrodes have their own shortcomings. From the perspective of electric field distribution characteristics, the electric field of the rectangular interdigital electrode is highly concentrated in the interdigital gap, and the direction anisotropy is obvious, the electric field distribution is uneven, which leads to large measurement error in different directions and reduces the sensing accuracy. And the measurement result is easily affected by the cell adhesion direction, it is difficult to realize isotropic detection. In cell migration or contraction experiments, the direction-dependent electric field distribution will mask the real impedance change. For example, the anisotropic response of cardiomyocyte beating signal may cause the mixing of systolic and diastolic signals. The electric field distribution is different in the length and width directions of the electrode, and strong electric field gradients are easily generated at the sharp corners and edges, which leads to abnormal behavior of cells in these areas and affects the stability of experimental results. Although the electric field distribution of the circular electrode is relatively uniform in the circumferential direction, the electric field strength difference between the center and the edge is significant. The electric field strength of the electrode edge region is usually larger than that of the electrode center region. When cells are mainly concentrated in the electrode edge region, the impedance change in these regions may be more significant, while the impedance change in the center region is relatively small. This uneven distribution will cause large fluctuations in the impedance spectrum data of multiple measurements of the same concentration of cell suspension, affecting the accuracy of impedance measurement, leading to a decrease in impedance detection sensitivity when the cell adhesion position deviates from the center, and cannot meet the needs of precision medicine.

[0006] These technical defects seriously restrict the application effect of microfluidic system and microelectrode cell detection technology in clinical diagnosis, and there is still room for improvement in current research. Research shows that the sensitivity and resolution of the sensor can be improved by further miniaturizing the electrode size and increasing the electrode density. The width and spacing of the electrode have a significant impact on the performance of the sensor. On this basis, simulation software can be used to try to optimize the existing interdigitated array microelectrode structure by comparing the measurement effects of various electrode structures.

[0007] In addition, the traditional integrated electrode mostly uses PCB and surface mounting process, which is relatively complex in design and manufacturing, and the processing precision is limited, so it is difficult to achieve micron-level precision, and therefore there is a problem of limited sensitivity in the detection of micro samples such as cells. The traditional integrated design has fixed electrode shape and connection mode, such as directly integrating the interdigital electrode with the integrated circuit, and connecting the electrode with other circuit elements through the internal wiring and interconnection structure of the chip. This way can achieve high integration and miniaturization, but the manufacturing process is complex and the cost is high, which limits its application in diversified sample detection. SUMMARY

[0008] The purpose of the present application is to solve the above-mentioned problems existing in the prior art, and a cell sorting and real-time detection system and method combining microfluidics and uniform pitch microelectrode array are proposed. The system integrates magnetic field sorting and impedance detection, realizes the organic unity of high-purity sorting and high-precision impedance detection of cells, and is a new microfluidic platform that can realize seamless connection of sorting and detection. The method uses magnetic nanoparticle labeling technology to specifically identify target cells, uses the gradient magnetic field generated by the external magnet to guide the magnetic bead labeled cells into the sorting channel, and uses the magnetic bead capture trap structure designed based on the cross-flow filtration principle to remove free magnetic beads, effectively avoiding the common channel blockage problem in traditional microfluidic sorting; the sorted cells are precisely guided to the detection area at the terminal of the microfluidic channel, and the specially designed metal microelectrode array is used for cell impedance detection, without the need for pre-separation and purification of cell samples, and the mixed sample can be directly sorted on the chip and immediately detected for cell impedance.

[0009] The technical scheme of the present application is:

[0010] The present application provides a cell sorting and real-time detection system combining microfluidics and uniform pitch microelectrode array, which comprises a cell sorting area and a microelectrode detection area, and the cell sorting area comprises:

[0011] A cell injection chamber is used to introduce a cell suspension to be sorted, and the cell suspension comprises free MNPs, target cells labeled with MNPs, and unlabeled cells.

[0012] A microfluidic channel for guiding the flow of a cell suspension in the microfluidic channel to achieve a magnetic field assisted based cell preliminary sorting;

[0013] A capture trap microstructure array for capturing free MNPs to separate the free MNPs from the target cells labeled with MNPs;

[0014] The microelectrode detection area comprises:

[0015] A cell collection chamber for collecting and culturing the sorted cells, including the target cells labeled with MNPs and unlabeled cells;

[0016] A microelectrode array for applying an electric current to the cells to be tested to obtain the impedance change of the electrodes in real time; the microelectrode array comprises two kinds of interdigital electrodes, namely a hexagonal electrode array and a sinusoidal electrode array, the length of the side of the hexagonal electrode array is matched with the cell size according to the formula: L=k·Dc, wherein Dc is the diameter of the target cell, k is a proportional coefficient, and k=0.5-1.0; the pitch of the hexagonal electrode array is equal, and the pitch d and the side length L satisfy the relationship: d= The amplitude A of the sinusoidal electrode array is λ / 4, and the wavelength λ is set to be twice the diameter of the cell.

[0017] Further, the hexagonal electrode array is symmetrically arranged, and the minimum pitch is 8 μm; the minimum pitch of the sinusoidal electrode array is 8 μm.

[0018] In the cell sorting area, the magnetic bead labeled cell sorting is achieved by using an external magnetic field assistance: the initial screening is completed in the inlet area, the accurate sorting is achieved in the middle transition field, and the cell is released without damage at the end. Cooperating with the unique "fishbone type" shunt structure, the capture efficiency of more than 95% can still be maintained under the condition of a flow rate of 1.2 mL / min, and the cell activity loss is controlled within 5%, solving the contradiction between the sorting intensity and the cell survival of the traditional system. The microelectrode detection area adopts the composite design of hexagonal and sinusoidal array microelectrodes, and the sinusoidal electrode can optimize the electric field distribution of the measurement device: by adjusting the wavelength (λ) and amplitude (A), the size of the target cell can be matched, the single sinusoidal period can cover the cell adhesion area, and the effective contact area is maximized. The symmetric arrangement of the hexagonal electrode ensures the uniform distribution of the electric field, reduces the edge effect, and improves the stability of the measurement, and the length (L) and the pitch (d) can also be adjusted to adapt to the cell. Under the same area, the hexagonal electrode contains 30% more electrode fingers than the rectangular electrode, significantly improving the space utilization.

[0019] Further, the materials of the hexagonal electrode and the sinusoidal electrode are gold, and both the electrodes are prepared by micro-nano processing technology.

[0020] This invention employs two interdigitated microelectrode structures: a hexagonal electrode array and a sinusoidal electrode array. Because the spacing between the electrode arrays remains essentially equal, the metal between the arrays is more easily separated during the lift-off process in photolithography, resulting in higher product yield. The uniform electrode spacing also improves the repeatability and consistency of experimental data, especially when comparing multiple experiments or data from different devices. This is crucial for standardized measurements in scientific research and industrial applications.

[0021] The novel planar micro / nano electrodes and microchannel patterns can be fabricated using advanced micro / nano fabrication techniques such as photolithography, while also being compatible with micro / nano fabrication technologies for patterning. Furthermore, in the electrode-channel co-design of this invention, the arrangement is highly flexible, achieved through pre-simulation design and photolithography, allowing for customization according to specific detection needs while reducing manufacturing costs. In addition, users can freely select the cell types to be sorted and the number of detection electrodes according to actual needs, comprehensively improving cell analysis efficiency and detection throughput through the co-design of microelectrodes and microchannels.

[0022] Furthermore, the system is also connected to an analytical instrument for analyzing the quantitative relationship between impedance and the concentration of the tested cells. By comparing the measured unknown concentration of the tested cells with a standard curve, information about the concentration or quantity of the tested cells is obtained, thereby determining the life activities of the cells, including proliferation and differentiation.

[0023] Furthermore, a cell culture plate is also provided in the cell collection chamber, and an electrode structure is provided on the cell culture plate. The cell culture plate is connected to a microfluidic channel and serves as a container for culturing cells.

[0024] This invention also provides a cell sorting and real-time detection method combining microfluidics and a uniformly spaced microelectrode array, comprising the following steps:

[0025] Prepare the cell material to be tested, use MNPs to specifically identify and label the target cells with antibodies, and inject the cell suspension containing multiple cells into the starting chamber, from which it enters the microfluidic channel;

[0026] Unlabeled cells flow along microfluidic channels without an external magnetic field under the influence of microfluidics. Free MNPs and cells labeled with MNPs are attracted by the external magnetic field and deflected towards the side of the microfluidic channel closer to the magnetic field region, separating from the unlabeled cells. When they flow into the magnetic bead trap, the free MNPs are captured, and the cells labeled with MNPs continue to flow into the cell collection chamber. The cells settle and attach to the microelectrode array and are cultured in the cell culture chamber.

[0027] Turn on the instrument, apply an AC signal, and measure and record the electrode impedance in real time.

[0028] Regularly observe the cell status to determine when to end the experiment; at the end of the experiment, analyze the data to assess the cell condition.

[0029] Furthermore, by applying a specific alternating current signal to the microelectrode array and continuously monitoring impedance changes, it is possible to provide real-time feedback on cell proliferation, differentiation, or drug response; the concentration of the cell culture medium can also be obtained from the impedance measured by the electrodes.

[0030] This invention features a flexible structure, enabling measurements of various biological samples under different conditions. It is cost-effective and allows the use of probes instead of testing instruments to read impedance parameters. Furthermore, it can be combined with microfluidic design to flexibly arrange the position and number of sorting channels, and to select the number of electrodes needed when testing mixed samples.

[0031] The beneficial effects of this invention are:

[0032] (1) This invention innovatively combines a microelectrode structure with a microfluidic sorting system. The sorting, detection, and culture processes do not require cell transfer, maintaining the cells in their original state. It is suitable for fragile samples such as stem cells and primary cells, avoiding contamination or cell damage caused by sample transfer in traditional techniques. The modular design supports multi-channel parallel processing, and the magnetic beads can be magnetically recycled and reused, reducing the cost per detection. The system can be adapted to various magnetic bead labeling strategies (such as immunomagnetic beads and functionalized magnetic beads) to meet diverse needs such as circulating tumor cell detection and immune cell sorting.

[0033] (2) In terms of detection, this invention employs two innovative interdigitated electrode structures. The sinusoidal microelectrode increases the contact area by changing the wavelength and amplitude to allow the electrode curvature to naturally conform to the cell shape. The high-density arrangement significantly increases the effective electrode area ratio, achieving optimal morphological matching with cells of different sizes. Experimental results show that it can increase the contact area by more than 40% for HeLa cells with a diameter of 20 μm, significantly improving detection sensitivity. The hexagonal microelectrode array eliminates the anisotropy problem of traditional rectangular electrodes through symmetrical arrangement design, improving the uniformity of electric field distribution by up to 35%. Both electrodes can be fabricated using mature micro-nano fabrication technology, with a minimum spacing of 8 μm. Under the same area, it can accommodate 30% more electrode fingers than conventional electrodes, significantly improving space utilization and detection sensitivity. By applying a small alternating current signal, the electrode can measure the impedance change in the cell culture medium in real time, thereby obtaining a standard curve between cell number and impedance, accurately quantifying the cell number.

[0034] (3) This invention combines microfluidic technology, improving the real-time detection effect and biocompatibility of microelectrodes. It is expected to capture specific biomolecules (such as antibodies, proteins, or DNA) and convert them into analyzable electrical signals through corresponding biochemical reactions, further enhancing the biochemical analysis function of this technology. This invention overcomes the limitations of existing cell sorting and detection equipment, such as large size, complex operation, and low efficiency, and provides a convenient, highly sensitive, highly stable, and low-cost microfluidic sorting and microelectrode detection array solution. It is particularly suitable for cell sorting, cell monitoring, precision diagnosis, and biosensing applications, providing an integrated, high-throughput solution for early cancer diagnosis, drug screening, personalized medicine, and other fields. It has significant academic value and broad application prospects. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the overall system of the present invention;

[0036] Figure 2 A schematic diagram of the structure and logic of the cell sorting region;

[0037] Figure 3 A schematic diagram of a microstructure array for magnetic bead trapping;

[0038] Figure 4 This is a diagram of a hexagonal electrode structure.

[0039] Figure 5 This is a diagram of a sinusoidal electrode structure.

[0040] Figure 6 HeLa cells and cardiomyocytes were added to the design of the sinusoidal electrode wavelength and amplitude parameters for comparison.

[0041] Figure 7 Schematic diagram of cell-attached electrodes;

[0042] Figure 8 Comparison of potential distribution diagrams for three different electrode types: hexagonal, sinusoidal, and circular electrodes;

[0043] Figure 9 The hexagonal, sinusoidal, and circular electrodes are spaced 110 mm apart at their centers. The curve showing the change of electric field intensity with the x-axis;

[0044] Figure 10 These are the operational steps of the method of the present invention. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] To further understand the present invention, it will be further described in conjunction with the accompanying drawings and embodiments.

[0047] Example 1

[0048] This invention provides a cell sorting and real-time detection system combining microfluidics and a uniformly spaced microelectrode array, such as... Figure 1 As shown, the entire system is fabricated on a silicon wafer or a high-purity quartz substrate, but PET or other flexible substrates can also be used. The system is divided into two main areas: a cell sorting area and a microelectrode detection area, which work together to achieve efficient cell sorting and analysis of electrophysiological characteristics.

[0049] The system consists of three main parts: a PDMS-silicon-based composite microfluidic structure, a magnetic field-assisted sorting module, and a microelectrode impedance analysis unit. The core area of ​​the chip adopts a unique "sorting-detection" dual-segment layout, which effectively solves the problems of separation of sorting and detection links and lack of time synchronization in traditional solutions.

[0050] In the cell sorting region, this system combines magnetic nanoparticle (MNP) labeling technology, magnetic field-assisted sorting principle, physical screening of micropillar array structure, and microfluidic manipulation technology to form an efficient and reliable cell sorting mechanism. The main body of the microfluidic structure is made of polydimethylsiloxane (PDMS) material.

[0051] like Figure 1 As shown, the cell sorting area includes a cell injection chamber for introducing the cell suspension to be sorted; a microfluidic channel for guiding the flow of the cell suspension inside the chip; a trap microstructure array for capturing excess MNPs, achieving separation of free MNPs from target cells labeled with MNPs; an outlet collection chamber; and a microelectrode detection area for collecting and fixing the cells to be tested at the microelectrode array structure in subsequent detection, thereby achieving better near-field cell impedance detection.

[0052] A magnetic bead injection channel is set at the inlet of the microchannel, such as... Figure 2 As shown, the surface of the magnetic beads is modified with specific antibodies. After mixing with the magnetic bead sample, the target cells are labeled by the magnetic beads through antigen-antibody binding. Then, they flow through the magnetic field pre-sorting zone and are enriched into the sorting channel under the action of the magnetic field. Non-target cells are discharged from the non-magnetic field zone outlet, thereby achieving preliminary cell sorting based on the magnetic field.

[0053] However, not only cells labeled with magnetic beads can flow into the microchannels near the magnetic field region, but also unlabeled blank magnetic beads will flow into the microchannels near the magnetic field along with the labeled cells under the influence of the magnetic field. This inevitably leads to the consumption and waste of a large number of blank magnetic beads. Moreover, since the magnetism exhibited by magnetic beads is positively correlated with their quantity, the presence of magnetic beads can prevent cells from attaching smoothly to the microelectrodes during near-field sensing of cells, affecting the detection effect. Furthermore, the presence of magnetism can cause severe drift and distortion of the impedance spectrum of the cells being analyzed. Therefore, to improve the accuracy of cell analysis, it is necessary to separate blank magnetic beads from labeled cells as much as possible.

[0054] like Figure 3 As shown, by designing a trap microstructure array that only allows small-sized magnetic beads to enter, blank magnetic beads are captured and fixed within the trap structure as much as possible, thereby achieving further sorting of blank magnetic beads and labeled cells. The trap microstructure array designed in this way is parallel to the liquid flow direction, belonging to the cross-flow filtration method. Furthermore, it filters out small-sized magnetic beads while allowing large-sized cells to flow through the microstructure, effectively avoiding the problem of microchannel clogging. The trap microstructure array incorporates a gradient filtration structure, where each section of the filtration structure is a filtration point. Each stage filters the mixed cells as they pass through the filtration structure. After multiple stages of filtration, the number of free magnetic beads in the microchannel is significantly reduced, achieving near-complete filtration. The principle and process of this specific method are as follows: When cells labeled with MNPs flow together with free MNPs in a microchannel near a magnetic field, the magnetic beads can smoothly enter the magnetic bead trap and be captured because the diameter of the magnetic beads is much smaller than that of the labeled cells. Larger cells, on the other hand, cannot enter the trap and continue to flow along the microchannel. They will then enter the cell collection area, where some cells will be confined to the microelectrode array detection area for impedance spectroscopy analysis.

[0055] like Figure 1 As shown, the microelectrode detection area includes a cell collection chamber and a microelectrode array disposed within the cell collection chamber.

[0056] In the cell collection chamber, sorted cells settle and adhere to the microelectrode array, thus reducing the impact of fluid disturbance on detection. The fixed cells are then cultured directly in the detection chamber with appropriate culture medium added and temperature and humidity controlled. A specific alternating current signal is applied to the microelectrode array, and impedance changes are continuously monitored to provide real-time feedback on cell proliferation, differentiation, or drug response (e.g., a decrease in impedance after anticancer drug treatment reflects apoptosis).

[0057] The electrode portion of the interdigitated electrode serves as the platform and electron transfer channel during electrochemical detection. The size and structure of its electrode array are the most important factors affecting the detection results, such as the uniformity of the electrode electric field distribution and the severity of anisotropy. Based on common interdigitated electrode structures, this invention further optimizes the electrode design, proposing two novel interdigitated electrode microstructures: a hexagonal array electrode structure and a sinusoidal array electrode structure. Their basic structures are as follows: Figure 4 and Figure 5 As shown, in these two structural designs, the surface area occupied by the electrode array is maximized, while the spacing between each array is kept equal by alternating their arrangement, which ensures the uniformity of the electric field distribution, reduces the error of the detection results, and improves repeatability and reliability. Figure 4 The design of the hexagonal electrode balances electrode density and edge electric field distribution, making it better suited for cell detection. Figure 5 The electrode geometry parameters are optimized through mathematical models (such as amplitude A=λ / 4, wavelength λ matching cell size) to adapt to different cell types (such as 10-50 μm cells). The periodic waveform structure of the sinusoidal microelectrode can generate a local uniform electric field (compared to circular electrodes), reduce edge effects, and improve the sensitivity and consistency of impedance detection.

[0058] The curved shape of a sinusoidal electrode determines the unique characteristics of its potential distribution. The periodicity of the sinusoidal curve causes the potential to change periodically on the electrode. This periodic potential distribution can provide a more stable measurement signal within a specific frequency range. The sinusoidal electrode of this invention employs wavelength (λ) and amplitude (A) parameterization design, enabling the electrode to match the size of the target cell (e.g., 20 μm HeLa cells), thereby maximizing the contact area between the electrode surface and the cell and improving sensitivity. Through the periodic electric field distribution of the sinusoidal electrode, a more stable and sensitive signal can be provided within a specific frequency range, improving the response to cell behavior. Dc: Target cell diameter (e.g., approximately 20 μm for HeLa cells, approximately 15 μm for cardiomyocytes). λ: Sine wave wavelength, set to twice the cell diameter to ensure that a single peak-trough cycle covers a cell attachment area. A: Amplitude, set to λ / 4 to match the electrode curvature with the cell membrane curvature, maximizing the contact area. When λ / A=4, the electric field gradient (∇E) on the surface of the sinusoidal electrode is significantly reduced compared to that of the straight electrode, and the current density concentration phenomenon caused by the edge effect is significantly improved, specifically as follows: Figure 6 As shown.

[0059] like Figure 7 As shown, gold with good biocompatibility is used. In the design, the two electrodes can be placed as close as possible. When using the test, after the cell suspension is added to the well plate, it is left to stand for 10-15 minutes (avoid shaking) to promote the natural sedimentation of the cells to the electrode surface, so that the cells can adhere to the electrodes well.

[0060] From the perspective of electric field distribution, the arrangement of hexagonal electrodes results in a more uniform potential distribution. The formula for matching the side length (L) to cell size is: L = k⋅Dc, where Dc is the target cell diameter (e.g., Dc = 20 μm for HeLa cells), and k is a scaling factor (k = 0.5 − 1.0, adapting to the cell attachment area). For HeLa cells (Dc = 20 μm), L = 15 μm is used to ensure that the hexagonal side length covers 50%-75% of the cell attachment area. The spacing (d) and side length (L) satisfy d = The relationship between the hexagonal electrodes ensures a uniform electric field distribution. The relatively consistent angles and distances between adjacent hexagonal electrodes facilitate a more regular propagation of the potential within the electrode array. Compared to circular electrodes, hexagonal electrodes exhibit less edge effect, resulting in a smoother change in potential across the entire electrode region. This allows for more accurate potential difference data when measuring impedance, thereby improving the precision of impedance measurements.

[0061] The electrode design of this invention is compatible with existing microscope systems (such as the Olympus IX83), facilitating the observation of the physiological state of cells on a microelectrode array. It is also designed for standard interfaces, allowing seamless integration into existing cell impedance devices and reducing equipment upgrade costs. Due to its standardized design, laboratories can freely choose the number, arrangement, and well plate configuration of electrodes to meet the needs of experiments of varying scales.

[0062] This electrode device achieves the function of measuring the analyte using impedance spectroscopy. It includes a cell culture device with the aforementioned electrodes, which can be designed as a well plate structure on a glass or PET substrate containing one or more wells. Each well is a cell culture area with the aforementioned gold microelectrode. An impedance detection device containing several cell culture devices can measure the resistance in each well of each cell culture device in real time. The integrated well plate design can be placed in a cell viability maintenance instrument, featuring temperature control. A constant carbon dioxide concentration ensures the viability of the biological sample. Furthermore, due to the high sensitivity of the medium and electronic components to humidity, the measured temperature and humidity can be well maintained. Figure 10 This is the main step in experimental measurement using microelectrodes.

[0063] Furthermore, the two electrode designs mentioned in this invention can be used in combination. Different electrode holes can employ various electrode designs; for example, a hexagonal electrode array can be used above, and a sinusoidal electrode array below. The number and arrangement of electrode holes can be flexibly set according to the detection target, without limitation. For example, 2, 4, 6, or 8 holes are all possible. The electrodes within each electrode hole can also be replaced as needed, suitable for various scenarios and requirements. Using the above device to load cells and applying millivolt-level AC signals at the pads (Pads represent circuit leads used to provide electrical connections) at both ends of the electrodes generates an electric field that can pass through the analyte. When the concentration of the analyte changes, the dielectric constant between the electrodes also changes, resulting in a change in the real-time measured sensor impedance. The impedance data is recorded on a connected PC, and the quantitative relationship between the sensor impedance and the analyte concentration is analyzed. When measuring unknown concentrations of the analyte, the data is compared with a standard curve to obtain information about the concentration or quantity of the sample, thereby determining cell proliferation, differentiation, and other life activities.

[0064] The cell culture plate substrate is made of glass or PET, which are stable and relatively inexpensive. Gold is used to fabricate the interdigitated electrodes, as it has good biocompatibility and conductivity. The electrode structure is manufactured using micro-nano fabrication techniques such as photolithography. The specific steps are as follows: First, a layer of photoresist is spin-coated onto the substrate. After the mask with the electrode structure is etched on it, the pattern on the mask is transferred to the substrate by photolithography. Then, a gold layer is evaporated or sputtered using electron beam evaporation. Chromium or titanium can be used as an adhesion layer between the substrate and the gold. Afterward, the photoresist is peeled off to obtain the fabricated interdigitated array microelectrodes. Microfluidic sorting channels with corresponding numbers of pores are fabricated on the obtained culture plate. The microfluidic channel material at the corresponding position of each electrode structure needs to have a certain height to serve as a container for culturing cells. After the microfluidic channel structure is fabricated, the complete cell culture plate structure is obtained.

[0065] Example 2

[0066] This invention provides a cell sorting and real-time detection method combining microfluidics and a uniformly spaced microelectrode array, comprising the following steps:

[0067] Prepare the cell material to be tested (including the cells to be tested and other cells), add MNPs, use MNPs to specifically identify and label the target cells with antibodies, and inject a cell suspension containing multiple cells (including free MNPs, cells labeled with MNPs, and unlabeled cells) into the starting chamber and enter the microfluidic channel from the chamber.

[0068] A magnet is added to one side of the microfluidic channel to form a magnetic field. Unlabeled cells flow along the microfluidic channel without an external magnetic field under the action of the microfluidic fluid. Free MNPs and cells labeled with MNPs are attracted by the external magnetic field and deflected to the side of the microfluidic channel closer to the magnetic field area. In this way, the cells to be tested are separated from other unlabeled cell samples.

[0069] When free MNPs and cells labeled with MNPs flow into the magnetic bead trap, the free MNPs are captured, and the cells labeled with MNPs continue to flow into the cell collection chamber. The cells settle and attach to the microelectrode array. A suitable culture medium is added to the cell culture chamber and the temperature and humidity are controlled for cell culture.

[0070] Turn on the instrument, apply a specific AC signal to the microelectrode array, and measure the electrode impedance in real time and record the data;

[0071] The cell state is observed regularly using equipment such as microscopes to determine when the experiment should be ended; at the end of the experiment, the data is analyzed to determine the condition of the cells.

[0072] Based on the recorded changes in electrode impedance, it is possible to provide real-time feedback on cell proliferation, differentiation, or drug response. For example, a decrease in impedance after treatment with anticancer drugs reflects cell apoptosis. The concentration of cell culture medium can also be obtained from the impedance measured by the electrodes.

[0073] Comparative Example 1

[0074] Figure 8 The diagrams compare the potential distribution of three different electrode types: hexagonal, sinusoidal, and circular electrodes. It can be seen that because hexagonal and sinusoidal electrodes have relatively regular edges and the spacing between adjacent electrodes is equal everywhere, the electric field distribution is more uniform compared to that of circular electrodes. Figure 9 The three electrodes are spaced 110 mm apart at the center. The curve showing the change of electric field intensity with the X-axis shows that, with the same electrode spacing, the circular electrode has the smallest electric field intensity, the sinusoidal electrode has the largest electric field intensity, and the hexagonal electrode is in between. Therefore, the sinusoidal electrode and the hexagonal electrode are superior to the circular electrode in terms of sensitivity. Thus, the design of the new electrode can improve the stability and sensitivity of impedance measurement.

[0075] The hexagonal electrode array, through its symmetrical arrangement, ensures a uniform electric field distribution, reducing edge effects and electric field gradient problems inherent in traditional rectangular electrodes, thus making the measurement of cell behavior more stable and reliable. This ensures a uniform electric field distribution between adjacent electrodes (isotropic error <5%). Compared to rectangular interdigitated electrodes, hexagonal interdigitated electrodes are more densely packed on a plane. Within the same area, hexagonal electrodes can accommodate more electrode fingers, improving space utilization and the effective contact area of ​​the electrodes, thus enhancing detection sensitivity and efficiency. Hexagonal electrodes can accommodate 30% more electrode fingers than rectangular electrodes within the same area, resulting in a significant improvement in space utilization.

[0076] The shape of sinusoidal interdigitated electrodes allows for more efficient use of area within a given space, enabling a greater electrode distribution. This makes them more suitable for applications requiring numerous electrodes for multi-point measurements or high-resolution detection, such as large-scale biochip detection or electrical monitoring of cell arrays. While circular interdigitated electrodes offer relatively good isotropy, hexagonal and sinusoidal interdigitated electrodes exhibit more consistent electric field distribution and electrical performance across multiple directions, reducing measurement differences caused by variations in orientation. They are superior in studies of the isotropic properties of samples or applications requiring uniform electrical information from multiple directions, such as researching the three-dimensional electrical behavior of biomolecules or cells in solution or changes in the isotropic electrical properties of biological tissues.

[0077] The electrode design of this invention offers high flexibility, allowing for adjustments to the arrangement and number of electrodes to meet diverse sample detection requirements based on varying experimental needs. The electrodes feature uniform spacing and a simple structure, making them suitable for manufacturing using advanced micro-nano fabrication processes. Compared to traditional integrated electrodes, this invention offers significant advantages in terms of cost and production efficiency.

[0078] By optimizing the electrode structure and manufacturing process, this invention effectively reduces manufacturing costs and provides higher sorting throughput and more stable test data. In various application scenarios, this flexible design and cost advantage make this invention promising for future development.

[0079] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, alterations, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cell sorting and real-time detection system combining microfluidics and a uniformly spaced microelectrode array, characterized in that, The system includes a cell sorting area and a microelectrode detection area, wherein the cell sorting area includes: A cell injection chamber is used to introduce a cell suspension to be sorted, the cell suspension comprising free MNPs, target cells labeled with MNPs, and unlabeled cells; Microfluidic channels are used to guide cell suspensions to flow within the microfluidic channels, enabling preliminary cell sorting based on magnetic field assistance. A trap microstructure array is used to capture free MNPs, enabling the separation of free MNPs from target cells labeled with MNPs; The microelectrode detection area includes: The cell collection chamber is used to collect and culture sorted cells, including target cells labeled with MNPs and unlabeled cells. A microelectrode array is used to apply current to the target cell and acquire the impedance change of the electrode in real time. The microelectrode array includes two types of interdigitated electrodes: a hexagonal electrode array and a sinusoidal electrode array. The side length of the hexagonal electrode array is adapted to the cell size using the formula: L = k⋅Dc, where Dc is the target cell diameter and k is a scaling factor, k = 0.5-1.

0. The hexagonal electrode array has equal spacing, and the spacing d satisfies d = ... The relationship is as follows: the amplitude A of the sinusoidal electrode array is A=λ / 4, and the wavelength λ is set to twice the cell diameter.

2. The system according to claim 1, characterized in that, The hexagonal electrode array is symmetrically arranged with a minimum spacing of 8 μm, and the sinusoidal electrode array has a minimum spacing of 8 μm.

3. The system according to claim 1, characterized in that, Both the hexagonal and sinusoidal electrodes are made of gold and are fabricated using micro-nano fabrication techniques.

4. The system according to claim 1, characterized in that, The system is also connected to an analytical instrument for analyzing the quantitative relationship between impedance and the concentration of the tested cells. By comparing the measured unknown concentration of the tested cells with a standard curve, information about the concentration or number of the tested cells can be obtained, thereby determining the life activities of the cells, including proliferation and differentiation.

5. The system according to claim 1, characterized in that, The cell collection chamber is also equipped with a cell culture plate, which has an electrode structure and is connected to a microfluidic channel, serving as a container for culturing cells.

6. A cell sorting and real-time detection method combining microfluidics and a uniformly spaced microelectrode array, characterized in that, Includes the following steps: Prepare the cell material to be tested, use MNPs to specifically identify and label the target cells with antibodies, and inject the cell suspension containing multiple cells into the starting chamber, from which it enters the microfluidic channel; Unlabeled cells flow along microfluidic channels without an external magnetic field under the influence of microfluidics. Free MNPs and cells labeled with MNPs are attracted by the external magnetic field and deflected towards the side of the microfluidic channel closer to the magnetic field region, separating from the unlabeled cells. When they flow into the magnetic bead trap, the free MNPs are captured, and the cells labeled with MNPs continue to flow into the cell collection chamber. The cells settle and attach to the microelectrode array and are cultured in the cell culture chamber. Turn on the instrument, apply an AC signal, and measure and record the electrode impedance in real time. Regularly observe the cell status to determine when to end the experiment; at the end of the experiment, analyze the data to assess the cell condition.

7. The method according to claim 6, characterized in that, By applying a specific alternating current signal to the microelectrode array and continuously monitoring impedance changes, it is possible to provide real-time feedback on cell proliferation, differentiation, or drug response; the concentration of the cell culture medium can also be obtained from the impedance measured by the electrodes.

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