Single cell mechanical property parallel detection method and device and computer equipment

Abstract

The invention relates to the technical field of cell detection, and discloses a single cell mechanical property parallel detection method and device and computer equipment, and the method comprises the following steps: providing an electric signal for an electrode on an electric deformation chip; acquiring deformation information of cells to be detected in a micro-channel on the electric deformation chip; acquiring and recording an electrical impedance signal when the to-be-detected cell deforms; performing normalization processing on the electrical impedance signal; and enabling the deformation information to correspond to the normalized electrical impedance signal. According to the invention, the mechanical properties of cells are tested based on dielectrophoresis and electrical impedance. An electrical impedance technology is introduced, a deformation signal is converted into an electric signal to be output, the real-time and high-throughput characterization and real-time feedback of the cell mechanical property are realized, and the data extraction and processing process is also simplified by adopting the electric signal to characterize the deformation.

Description

Technical Field

[0001] The present invention relates to the field of cell detection technology, and in particular to a method, device and computer equipment for parallel detection of mechanical properties of single cells. Background Art

[0002] Cancer remains a major public health challenge in my country, and the cancer burden continues to increase. Data show that the five-year overall survival rate for patients with solid malignancies such as esophageal, liver, lung, pancreatic, and gastric cancers has remained below 20% over the past few decades. A key factor contributing to this situation is the high degree of tumor heterogeneity, which exists both within individual tumors and across different tumors. This heterogeneity poses significant challenges to cancer treatment, resulting in existing treatments that can only manage the disease but struggle to achieve a cure. Currently, two main types of detection methods are used to characterize cellular heterogeneity. One approach involves identifying and characterizing several types of biomarkers using traditional biological methods, such as immunofluorescence, RT-PCR, Western blotting, fluorescent chemical probes, and proteomics. These methods enable intuitive and effective detection of specific biomarkers, but they also have numerous drawbacks, such as high cost and the unavoidable need for labeling or invasive cell invasion, which can cause cell damage or even death. Another approach utilizes morphological differences to detect cellular heterogeneity. However, the accuracy of these subjective tests depends largely on the knowledge and experience of the investigator.

[0003] The mechanical phenotype of a cell, a key intrinsic marker of its physiological state, is closely related to important functions such as cell proliferation, differentiation, cell cycle regulation, and malignant transformation. It is associated with human disease and serves as an important biomarker for clinical diagnosis. Since the initial discovery in pleural fluid that metastatic cancer cells exhibit significantly softer properties than benign cells, research on the correlation between cell stiffness and tumor malignancy has continued in depth. Measuring cell mechanics circumvents the need for external labeling (such as fluorescent dyes) and is sensitive to changes in cellular biological composition, making it an attractive non-invasive biomarker. Traditional techniques for measuring single-cell mechanical properties include micropipette placement, optical tweezers, and atomic force microscopy. While these methods offer high spatial resolution, they have low throughput, requiring only skilled personnel using specialized equipment, and measuring single cells requires significant time. Microfluidic methods typically use microscopes and high-speed cameras to record cell deformation. These techniques require expensive imaging equipment and complex image processing algorithms, with throughput limited by the camera's imaging range and subsequent analysis of the imaging data requiring significant time. Not only do existing methods limit the ability to analyze heterogeneous cell populations, the complexity of these technologies also restricts their application in routine clinical analysis or point-of-care testing scenarios, such as rare cell screening and disease diagnosis.

[0004] Therefore, there is an urgent need for a method that can measure the mechanical properties of single cells quickly, with strong scalability, high automation, and high throughput, which is suitable for disease diagnosis, drug screening, and precision medicine. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention provides a method, device and computer equipment for parallel detection of single-cell mechanical properties, which solves the problems of existing cell mechanical properties measurement procedures being cumbersome, requiring high operating experience, taking a long time overall, and having low throughput and automation.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for parallel detection of mechanical properties of single cells, comprising the following steps: providing electrical signals to electrodes on the electrodeformation chip; Obtaining deformation information of the cell to be measured in the microchannel on the electrodeformable chip; Acquire and record the electrical impedance signal of the cell under test when it deforms; Normalizing the electrical impedance signal; The deformation information is matched with the normalized electrical impedance signal.

[0007] Preferably, a microchannel is constructed on the electrodeformation chip; before detection, the electrodeformation chip is calibrated using a control group of cells to be tested that have not been treated in any way to determine a measurement benchmark.

[0008] Preferably, the channel range of the microchannel is controlled within the electrode, and the cells to be tested are in a room temperature environment during detection.

[0009] A parallel detection device for the mechanical properties of a single cell includes an electric deformation chip and an electrical impedance signal processing module. The electric deformation chip includes a microdevice and a probe station for an external circuit. The microdevice is made by bonding a microelectrode array and a microchannel. The electrical impedance signal processing module is used to collect and process the cell deformation signals on the microelectrode array in real time, and convert the electrical impedance signals into corresponding cell deformation information.

[0010] Preferably, the microchannel is made of dimethylsiloxane material, and the microelectrode array uses indium tin oxide as the electrode material.

[0011] Preferably, the microelectrode array is distributed and a heat spreader compatible with the microdevice is fabricated to control the cells in the microdevice to be in a room temperature environment.

[0012] Preferably, the inlet and outlet of the microchannel are respectively connected to a programmable syringe pump. When a round of testing is completed, the tested cell suspension is pushed out and a new cell suspension is automatically pushed into the channel to continue testing.

[0013] Preferably, each electrode in the microelectrode array has a pointed triangular structure at one end close to the microchannel.

[0014] Preferably, the electrical impedance signal processing module includes a hardware circuit system, which includes a main control and storage module, a signal generating module, an impedance detection module, a programmable scanning module and a power supply module. The signal generating module provides an electrical signal for cell deformation, the impedance detection module obtains cell deformation information, and the programmable scanning module realizes rapid scanning of the impedance signals of each electrode; the electrical impedance signal processing module also includes a software part, which executes the following workflow: the main control and storage module outputs a serial microelectrode selection signal; one microelectrode is connected to the impedance detection module for testing; the signal is digitally filtered and Fourier transformed in the main control and storage module; after completing a test, the data set is transmitted to the computer host; in the computer host, the electrical impedance signal is quickly analyzed and pattern recognized to realize the corresponding conversion between the impedance signal and the cell deformation information.

[0015] A computer device comprising a memory and a processor, wherein the memory is used to store a computer program, and the processor is used to execute the computer program and implement the parallel detection method for single-cell mechanical properties as described in the above scheme when executing the computer program.

[0016] The present invention has the following beneficial effects: The single-cell mechanical properties parallel detection method, device and computer equipment have high sensitivity and can quickly detect the deformation ability of a single cell and identify the deformation ability of a small number of abnormal cells in a normal cell sample.

[0017] Compared with traditional biological methods, this single-cell mechanical properties parallel detection method, device and computer equipment realizes a label-free and non-destructive high-throughput cell mechanical properties testing platform, uses dielectrophoresis to detect cell mechanical properties, uses electrical impedance to extract cell parameters, couples mechanical and electrical signals to analyze cell map characteristics from multiple angles, and quickly and accurately analyzes cell heterogeneity while avoiding the use of expensive high-speed cameras.

[0018] (3) The method, device and computer equipment for parallel detection of mechanical properties of single cells are highly flexible and can realize dynamic measurement of mechanical properties of cells. The method includes a device, circuit and storage device, which not only does not require complex technology and expensive equipment, but also has high scalability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the detection system architecture of the present invention; Figure 2 Schematic diagram of the microdevice structure of the present invention; Figure 3 Schematic diagram of the impedance detection module system architecture of the present invention.

[0020] In the figure: 1. Electrodeformable chip; 2. Microdevice; 3. Programmable scanning module; 4. Main control and storage module; 5. Impedance detection module; 6. Signal generation module; 7. Microelectrode; 8. Microchannel; 9. Programmable injection pump. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention. Example 1

[0022] See also Figure 1 The present invention provides a technical solution: a method for parallel detection of single cell mechanical properties, comprising the following steps: Providing electrical signals to the electrodes on the electrodeformation chip 1; Obtaining deformation information of the cell to be measured in the microchannel 8 on the electrodeformable chip 1; Acquire and record the electrical impedance signal of the cell under test when it deforms; Normalizing the electrical impedance signal; The deformation information is matched with the normalized electrical impedance signal.

[0023] This method tests the mechanical properties of cells based on dielectrophoresis and electrical impedance. The introduction of electrical impedance technology converts deformation signals into electrical signal output, enabling real-time and high-throughput characterization of cell mechanical properties and real-time feedback. The use of electrical signals to characterize deformation also simplifies the process of data extraction and processing. It has the following advantages: (1) High sensitivity, rapid detection of the deformation ability of individual cells, and identification of the deformation ability of a small number of abnormal cells in normal cell samples; (2) Compared with traditional biological methods, it realizes a label-free and non-destructive high-throughput cell mechanical properties testing platform. Dielectrophoresis is used to detect cell mechanical properties, and electrical impedance is used to extract cell parameters. Mechanical and electrical signals are coupled to analyze cell atlas characteristics from multiple angles, allowing for rapid and accurate analysis of cell heterogeneity while avoiding the use of expensive high-speed cameras; (3) This method is highly flexible and enables dynamic measurement of cell mechanical properties, including devices, circuits, and storage devices. It does not require complex technology and expensive equipment and is highly scalable.

[0024] In this embodiment, a microchannel 8 is constructed on the electrodeformable chip 1 . Before detection, the electrodeformable chip 1 is calibrated using a control group of cells to be tested that have not been treated in any way to determine a measurement benchmark.

[0025] In this embodiment, the channel range of the microchannel 8 is controlled within the electrode, and the cells to be tested are in a room temperature environment during detection. In order to achieve the effect of one electrode adsorbing one cell, in addition to controlling the cell concentration, the channel range can be limited and controlled as much as possible within the electrode. The shaded part represents the microelectrode 7. For cancer cell experiments, the distance between the electrodes is set to 40um. If other cells are used, the electrode distance is adjusted according to the actual situation. 52 pairs of microelectrodes 7 are drawn here, which is only a schematic diagram. According to experimental requirements, the number of electrodes can be expanded to hundreds or thousands to increase the number of cells measured in parallel. The microelectrodes 7 are distributed in an array and in order to avoid the influence of temperature on cell deformability, a heat spreader compatible with the device is made to control the cells in the microdevice 2 to be in a room temperature environment. Example 2

[0026] Reference Figure 1 As shown, a device for parallel detection of single-cell mechanical properties includes an electrodeformation chip 1 and an impedance signal processing module. The electrodeformation chip 1 includes a microdevice 2 and a probe station with an external circuit. The microdevice 2 is made of a bonded array of microelectrodes 7 and microchannels 8. The impedance signal processing module is used to collect and process cell deformation signals from the microelectrode array in real time, converting the impedance signals into corresponding cell deformation information. The device mainly includes the electrodeformation chip 1 and the impedance signal processing module. The electrodeformation chip 1 module includes a microdevice 2 and a probe station with an external circuit. The microdevice 2 is made of a bonded array of microelectrodes 7 and microchannels 8, which are used to detect cell deformation and receive and output electrodeformation signals. The impedance signal processing module consists of both hardware and software. The hardware component not only provides electrical signals for cell deformation and impedance measurement, but also rapidly collects and processes cell deformation signals from the electrode array in real time. The software component is used to implement automated signal processing, rapid analysis and pattern recognition, and convert the impedance signals into corresponding cell deformation information.

[0027] In this embodiment, the microchannels 8 are made of dimethylsiloxane, and the microelectrode array 7 uses indium tin oxide as its electrode material. Polydimethylsiloxane was chosen as the material for the microchannels 8 due to its excellent gas permeability, non-toxicity, ease of manufacture, and low cost. Compared to gold and platinum, the electrode shape, color, and transparency do not affect cell observation. After calibration of the electrodeformable chip 1 is complete, other electrode materials may be used.

[0028] In this embodiment, the microelectrodes 7 are distributed in an array and a heat spreader compatible with the microdevice 2 is fabricated to control the cells in the microdevice 2 to be kept at room temperature.

[0029] In this embodiment, the inlet and outlet of microchannel 8 are each connected to a programmable syringe pump 9. When a test cycle is completed, the tested cell suspension is ejected, and a new cell suspension is automatically pushed into the channel to continue testing. To increase test throughput and reduce manual operation, a programmable syringe pump 9 can be connected to the inlet and outlet of the channel. When a test cycle is completed, the tested cell suspension is ejected, and a new cell suspension is automatically pushed into the channel to continue testing.

[0030] In this embodiment, each electrode in the microelectrode array 7 has a pointed triangular structure at one end near the microchannel 8. The "pointed-to-flat" electrode has the best cell deformation effect, and the triangular structure enables targeted cell capture, ensuring that one "pointed" electrode attracts one cell.

[0031] In this embodiment, the impedance signal processing module includes a hardware circuit system, which includes a main control and storage module 4, a signal generating module 6, an impedance detection module 5, a program-controlled scanning module 3 and a power supply module. The signal generating module 6 provides an electrical signal for cell deformation, the impedance detection module 5 obtains cell deformation information, and the program-controlled scanning module 3 realizes a rapid scan of the impedance signal of each electrode; the impedance signal processing module also includes a software part, which executes the following workflow: the main control and storage module 4 outputs a serial microelectrode 7 selection signal; a microelectrode 7 is connected to the impedance detection module 5 for testing; the signal is digitally filtered and Fourier transformed in the main control and storage module 4; after completing a test, the data set is transmitted to the computer host; in the computer host, the impedance signal is quickly analyzed and pattern recognized to realize the conversion of impedance signal and cell deformation information. The hardware system interface is designed to be made into a probe station form, using a retractable spring probe to be placed on the ITO electrode to control signal transmission. The middle electrode is grounded, and the surrounding electrode array is connected to the signal positive pole. When the electrical signal is output, the cell is adsorbed on the surrounding electrodes. Next, the function and circuit composition of each module are introduced in detail.

[0032] (1) Main control and storage module 4: The function of this module is to coordinate and control the workflow of the other modules and support the signal processing algorithm to process the collected electrical impedance signals. This module uses the Artix-7 series chip XC7A35T-2FGG484 launched by Xilinx as the main control chip. The chip has 250 user IO pins, which is sufficient to connect and control the other modules. At the same time, it has rich computing and storage resources to meet the resource requirements of high-speed signal processing algorithms. The main components of this module also include a DDR3 memory chip (MT41K128M16JT) that can store the time series of electrical impedance signals. A USB2.0 high-speed transmission chip (CY7C68013) is used to achieve high-speed data transmission between the circuit and the host computer. The chip has a complete USB protocol parsing and data transceiver engine with a transmission speed of up to 40 MB / s.

[0033] (2) Signal Generator Module 6: This module superimposes two sinusoidal alternating signals with different frequencies and amplitudes generated by the signal generator. One signal is high-frequency and high-voltage, generating the dielectrophoretic force, while the other is low-frequency and low-voltage, detecting the electrical impedance signal of cell deformation. A CA3140 high-speed operational amplifier is used to form an adjustable-gain, in-phase summing amplifier to sum the two signals and generate a superimposed signal. The superimposed signal is connected in parallel to each pair of electrodes.

[0034] (3) Programmable scanning module 3: It is composed of multiple shift registers working in series. The main control and storage module 4 outputs a serial electrode selection signal, which is converted into a parallel signal output by the shift register. The parallel signal is connected to the base of the electrode selection transistor, while the collector is connected to the electrode and the emitter is connected to the impedance detection module 5. In this way, the main control and storage module 4 can control the electrical impedance signal scanning detection on each electrode.

[0035] (4) Impedance Detection Module 5: Impedance detection module 5 is the most important functional module of the hardware circuit. The programmable scanning module 3 can select a certain electrode to be connected to one arm of the Xilin bridge. It is planned to connect the bridge to the non-inverting input and inverting input of a differential amplifier, perform differential operation on the output electrical signal and amplify it. Two AD630 chips are used to form an orthogonal phase-locked amplifier to perform phase-locked amplification on the differential signal. After analog-to-digital conversion, the amplified signal is transmitted to the DDR3 memory chip of the main control module for storage and subsequent signal processing.

[0036] (5) Power supply module: Four 18650 rechargeable lithium batteries are connected in series and divided into two paths. One path is reduced to 5 V by a three-terminal regulator to power the components; the other path is converted into two independent 9 V outputs by a DC-DC component. The two paths are connected in series and output ±9 V after being stabilized by a three-terminal regulator to power the operational amplifier.

[0037] After designing the circuit in modules, make the PCB circuit board and solder the components for testing.

[0038] Next, the high-speed impedance signal processing software component is required. First, the firmware for the main control module must be developed using Verilog HDL and the Zynq development tool. The workflows for each module are written using Verilog HDL and the Zynq development tool. The main control module outputs a serial microelectrode 7 selection signal, and one microelectrode 7 is connected to the impedance detection module 5 for testing. The signal undergoes digital filtering and Fourier transform processing in the main control module. After a test is completed, the data set is transferred to a host computer via a USB interface. Within the host computer, a convolutional neural network-based fast impedance signal analysis and pattern recognition algorithm is developed using the Matlab platform (the "Electrical Impedance Imaging Algorithm Based on Two-Dimensional Convolutional Neural Network, by Zhao Shaofeng and Li Jing (School of Engineering Science, University of Science and Technology of China, Hefei, Anhui)" can also be used for identification and analysis, enabling the conversion of impedance signals into cell deformation information.

[0039] After the system is set up and before use, the device is calibrated. The electrodeformable chip 1 is calibrated with a control group of untreated cancer cells to determine the measurement baseline. The first step is sample preparation. Before measurement, cells are digested with 0.25% trypsin, counted, and resuspended in dielectrophoresis working buffer solution. The cell density is about 10 4 cells / mL. The dielectrophoresis buffer working solution is prepared with deionized water and consists of a 0.3 M inositol solution with an osmotic pressure of approximately 290 mOsm / kg. The pH of the solution is adjusted to 7.4 using NaOH / HCl. The conductivity is then adjusted by titrating the D-PBS solution with the dielectrophoresis buffer working solution. After the preparations are completed, the cell suspension is slowly introduced into the channel, and an electrical signal is applied to control cell stretching and relaxation. After the cells are stabilized on the device, voltages of varying magnitudes are applied to the microelectrodes 7. Cell calibration is divided into two parts. One part uses a high-speed camera to record the corresponding cell deformation information. The microdevice 2 is placed on the stage of an inverted microscope, and the high-speed camera transmits the cell deformation information back to the computer, where the recorded position is observed and adjusted in real time on the computer screen. Image morphology technology is used to rapidly extract cell image information. The other part uses the impedance detection module 5 to acquire and record the electrical signals during cell deformation. Ultimately, the deformation information obtained through image processing is matched to the normalized impedance signal.

[0040] This solution is not only applicable to the detection of cancer cells, but can also be used to test other cells, such as red blood cells and immune cells, by adjusting the shape and spacing of the microelectrodes 7. By acquiring cell deformation and electrical information, cell heterogeneity can be analyzed in multiple dimensions. Example 3

[0041] A computer device includes a memory and a processor, the memory is used to store a computer program, the processor is used to execute the computer program and implement the single cell mechanical property parallel detection method as described in Example 1 when executing the computer program.

[0042] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "include," "comprise," or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations. The phrase "includes an element defined by..." does not exclude the presence of other identical elements in the process, method, article, or device that includes the element.

[0043] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for parallel detection of single cell mechanical properties, characterized in that: The following steps are involved: Providing electrical signals to the electrodes on the electrodeformable chip: The electrodeformable chip includes a microdevice and a probe station connected to an external circuit. The microdevice is made by bonding a microelectrode array and a microchannel. The individual electrodes in the microelectrode array have a pointed triangular structure at one end near the microchannel. The channel range of the microchannel is controlled within the electrode. During the detection, the cells to be tested are at room temperature. Obtaining deformation information of the cell to be measured in the microchannel on the electrodeformable chip; Acquire and record the electrical impedance signal of the cell under test when it deforms; Normalizing the electrical impedance signal; The deformation information is matched with the normalized electrical impedance signal.

2. The method for parallel detection of single-cell mechanical properties according to claim 1, characterized in that: A microchannel is constructed on the electrodeformation chip. Before detection, the electrodeformation chip is calibrated using a control group of cells to be tested that have not been treated in any way to determine a measurement benchmark.

3. A device for parallel detection of single-cell mechanical properties, characterized by: The device comprises the electric deformation chip and the electrical impedance signal processing module as described in claim 1 or 2, wherein the electrical impedance signal processing module is used to collect and process the cell deformation signal on the microelectrode array in real time, and convert the electrical impedance signal into corresponding cell deformation information.

4. The device for parallel detection of single-cell mechanical properties according to claim 3, characterized in that: The microchannel is made of dimethylsiloxane material, and the microelectrode array uses indium tin oxide as electrode material.

5. The device for parallel detection of single-cell mechanical properties according to claim 4, characterized in that: The microelectrode array is distributed and a heat spreader compatible with the microdevice is made to control the cells in the microdevice to be in a room temperature environment.

6. A single cell mechanical properties parallel detection device according to claim 4 or 5, characterized in that: The inlet and outlet of the microchannel are respectively connected to a programmable syringe pump. When a round of testing is completed, the tested cell suspension is pushed out and a new cell suspension is automatically pushed into the channel to continue the test.

7. The device for parallel detection of single-cell mechanical properties according to claim 3, characterized in that: The electrical impedance signal processing module includes a hardware circuit system, which includes a main control and storage module, a signal generation module, an impedance detection module, a programmable scanning module and a power supply module. The signal generation module provides an electrical signal for cell deformation, the impedance detection module obtains cell deformation information, and the programmable scanning module realizes rapid scanning of the impedance signals of each electrode; the electrical impedance signal processing module also includes a software part, which executes the following workflow: the main control and storage module outputs a serial microelectrode selection signal; one microelectrode is connected to the impedance detection module for testing; the signal is digitally filtered and Fourier transformed in the main control and storage module; after completing a test, the data set is transmitted to a computer host; in the computer host, the electrical impedance signal is quickly analyzed and pattern recognized to realize the corresponding conversion between the impedance signal and the cell deformation information.

8. A computer device, characterized in that: The computer device includes a memory and a processor, the memory is used to store a computer program, and the processor is used to execute the computer program and implement the single-cell mechanical property parallel detection method according to any one of claims 1 to 2 when executing the computer program.

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