Single cell biophysical property detection device and method based on non-compressible channel

Abstract

The present invention provides a single-cell biophysical property detection device and method based on a non-compressible channel, the method comprising: fluorescently staining the cell nucleus of the cell to be tested; injecting the stained cell to be tested into a microfluidic chip, and using a negative pressure driving module to drive the cell to be tested to flow in a non-compressed form in a cell detection channel of the microfluidic chip; using an impedance detection module to record DC resistance data and multiple high-frequency impedance data of the cell to be tested flowing in the cell detection channel, wherein the high frequency range includes: 100kHz-1GHz; using a photoelectric detection module to record the fluorescence intensity data of the cell nucleus of the cell to be tested flowing in the fluorescence detection window; and using a data analysis and processing module to calculate the biophysical properties of the cell to be tested according to the DC resistance data, multiple high-frequency impedance data, and fluorescence intensity data, wherein the biophysical properties of the cell to be tested include cell diameter, cell nuclear diameter, cell membrane specific capacitance, and cytoplasm conductivity.

Description

Technical Field

[0001] The present disclosure belongs to the field of biological detection, and specifically relates to a single cell biophysical property detection device and method based on a non-compressive channel. Background Art

[0002] Cells are the basic structures and units that maintain life activities. The material and energy intake, transformation, storage and utilization of organisms, as well as the storage, processing and expression of biological information are all inseparable from cells. Studying cells is of great significance for exploring the basic laws of life activities of organisms. Early cell research methods were group cell analysis, and the statistical average results obtained could not reflect the differences in biological information between individual cells. Therefore, the importance and necessity of single-cell analysis have gradually been reported in recent years.

[0003] Single-cell analysis usually includes biochemical property detection and biophysical property detection. Compared with the former, the biophysical property detection method has the advantages of no labeling, simple operation and low cost, and is favored by the research in the field of single-cell analysis. The biophysical properties of single cells include parameters such as cell diameter, cell nuclear diameter, cell membrane specific capacitance and cytoplasmic conductivity. Among them, the cell diameter is the size information of the cell; the cell nuclear diameter is the size information of the cell nucleus; the main component of the cell membrane is the phospholipid bilayer. Due to its capacitive reactance, its electrical model can be equivalent to a capacitor, called the cell membrane capacitance, and the cell membrane specific capacitance is the cell membrane capacitance per unit area; the cytoplasm is composed of the cytoplasmic matrix, organelles and other inclusions. Due to the conductive properties of the cytoplasm, its electrical model can be equivalent to a resistor, and the cytoplasmic conductivity is a parameter reflecting the conductivity of the cytoplasm.

[0004] At present, most of the microfluidic impedance cell detectors in the prior art can detect cell size and impedance information, and can achieve a higher detection throughput than traditional fixed cell characterization methods, but they have fewer detection parameters and cannot quantify the inherent characteristics of cells, lacking detection versatility. The microfluidic impedance cell detector based on compression channels has realized the detection of cell diameter, cell membrane specific capacitance, and cytoplasmic conductivity, but the channel cross-sectional area used in this method is small, which can easily lead to blockage of the cell detection channel, a short detection duration, and ignores the biophysical properties of the cell nucleus.

[0005] In summary, current single-cell biophysical detection methods have problems such as few detection parameters and easy channel clogging. Summary of the invention

[0006] In response to the above technical problems, the present disclosure provides a single-cell biophysical property detection device and method based on a non-compressible channel, in order to at least partially solve the above technical problems.

[0007] In order to solve the above technical problems, as one aspect of the present disclosure, a method for detecting biophysical properties of a single cell based on a non-compressive channel is provided, comprising:

[0008] Fluorescently stain the nuclei of cells to be tested;

[0009] Injecting the dyed cells to be tested into the microfluidic chip, and using a negative pressure driving module to drive the cells to be tested to flow in a non-compressed form in a cell detection channel of the microfluidic chip;

[0010] Using an impedance detection module to record the DC resistance data and multiple high-frequency impedance data of the cells to be tested flowing in the cell detection channel, wherein the high frequency range includes: 100kHz-1GHz;

[0011] Using a photoelectric detection module to record the fluorescence intensity data of the cell nucleus of the cell to be tested flowing in the fluorescence detection window; and

[0012] The data analysis and processing module is used to calculate the biophysical characteristics of the cells to be tested based on the DC resistance data, multiple high-frequency impedance data, and fluorescence intensity data, wherein the biophysical characteristics of the cells to be tested include cell diameter, cell nuclear diameter, cell membrane specific capacitance, and cytoplasmic conductivity.

[0013] According to an embodiment of the present disclosure, the cell diameter of the above-mentioned cell to be tested is obtained according to the above-mentioned DC resistance data and Coulter principle;

[0014] According to the cell diameter of the cell to be tested and the multiple high-frequency impedance data, the cell membrane specific capacitance and cytoplasm conductivity of the cell to be tested are obtained based on numerical simulation technology.

[0015] According to an embodiment of the present disclosure, the cell membrane specific capacitance and cytoplasm conductivity of the above-mentioned cells to be tested are determined by the following steps:

[0016] Establishing a simulation model, wherein the simulation model includes a rectangular insulating channel having the same size as an actual cell detection channel, and a spherical cell to be detected having a specific conductivity and membrane specific capacitance;

[0017] Simulating the preset cell diameters, cytoplasmic conductivity and cell membrane specific capacitance of the above-mentioned cells to be tested of different sizes, and obtaining a plurality of high-frequency impedance data under the conditions of the determined cell diameters, cytoplasmic conductivity and cell membrane specific capacitance based on the equivalent electrical impedance model of the above-mentioned cells to be tested;

[0018] A lookup table is established between the cell diameter, cytoplasmic conductivity, cell membrane specific capacitance and the multiple high-frequency impedance data. The lookup table is used to determine the cell membrane specific capacitance and cytoplasmic conductivity of the cell to be measured based on the measured cell diameter and the multiple high-frequency impedance data.

[0019] According to the embodiment of the present disclosure, according to the cell diameter D of the above-mentioned cell to be tested c , the length L1 of the cell detection channel, the DC resistance pulse width t1, the interval length L2 of the fluorescence detection window and the fluorescence intensity data pulse width t2, the following formula is used to obtain the cell nucleus diameter D of the cell to be tested. n :

[0020]

[0021] As another aspect of the present disclosure, a single-cell biophysical property detection device based on a non-compressible channel is also provided. The method in the above embodiment is performed by a single-cell biophysical property detection device based on a non-compressible channel. The single-cell biophysical property detection device based on a non-compressible channel includes:

[0022] A microfluidic chip, comprising a cell detection channel, for allowing a single cell to be detected to flow in a non-compressed form;

[0023] An impedance detection module, used to obtain the DC resistance data and multiple high-frequency impedance data of the cells to be tested flowing in the cell detection channel;

[0024] The photoelectric detection module is arranged near the cell detection channel, and is used to provide a fluorescent light source for the fluorescent detection window of the cell detection channel, and to obtain the fluorescence intensity data when the cell nucleus of the cell to be detected passes through the fluorescent detection window;

[0025] A negative pressure driving module, used for driving the cells to be tested to move in the cell detection channel;

[0026] The data analysis and processing module is used to obtain the DC resistance data of the impedance detection module, multiple high-frequency impedance data and the fluorescence intensity data of the photoelectric detection module, and obtain the biophysical characteristics of the cells to be tested based on the DC resistance data, multiple high-frequency impedance data and fluorescence intensity data.

[0027] According to an embodiment of the present disclosure, the above-mentioned microfluidic chip further includes:

[0028] A cell inflow channel, used to inject cells to be tested into the cell detection channel;

[0029] A cell outflow channel, used to allow the cells to be tested to flow out of the cell detection channel;

[0030] Wherein, the cell detection channel is arranged between the cell inflow channel and the cell outflow channel;

[0031] Electrode sheets are respectively arranged in the cell inflow channel and the cell outflow channel and connected to the two ports of the cell detection channel. The electrode sheets are connected to the impedance detection module.

[0032] The shading sheets are arranged at both ends of the cell detection channel and are used to provide fluorescence detection windows for the cell detection channel.

[0033] According to an embodiment of the present disclosure, the cross-sectional area of ​​the cell inflow channel and the cell outflow channel is 3 to 10 times the cross-sectional area of ​​the cell to be tested;

[0034] The cross-sectional area of ​​the cell detection channel is larger than the cross-sectional area of ​​the cell to be detected;

[0035] The length of the cell detection channel is greater than the diameter of the cell to be detected.

[0036] According to an embodiment of the present disclosure, the length and width of the cross-section of the cell inflow channel and the cell outflow channel range from 30 to 60 μm;

[0037] The diameter range of the cells to be tested includes 10 to 20 μm;

[0038] The width and height of the cell detection channel range from 15 to 30 μm;

[0039] The length of the cell detection channel ranges from 20 to 50 μm.

[0040] According to the embodiments of the present disclosure, the diameter length range of the cell nucleus of the above-mentioned cell to be tested includes: 5 to 10 μm;

[0041] The length range of the fluorescence detection window includes: 15 to 30 μm.

[0042] According to an embodiment of the present disclosure, the impedance detection module includes a lock-in amplifier and a data acquisition card, wherein the lock-in amplifier is used to measure the DC resistance data and multiple high-frequency impedance data between the electrode sheets in the microfluidic chip, and the data acquisition card is used to transmit the DC impedance data and multiple high-frequency impedance data output by the lock-in amplifier to the data analysis and processing module;

[0043] The above-mentioned photoelectric detection module includes a microscope and a photomultiplier tube, wherein the above-mentioned microscope is used to provide a fluorescent light source for the above-mentioned fluorescent detection window and receive fluorescence from the above-mentioned fluorescent detection window; the above-mentioned photomultiplier tube is used to measure the fluorescence intensity data output by the above-mentioned microscope.

[0044] Based on the above technical solutions, it can be seen that the single-cell biophysical property detection device and method based on the non-compressed channel disclosed in the present invention has at least one of the following beneficial effects:

[0045] (1) In the embodiments of the present disclosure, the cells to be tested are stained to facilitate observation of the nucleus of a single cell to be tested; the cells to be tested are introduced into the cell detection channel of the microfluidic chip and flow in a non-compressed form, which, on the one hand, makes the detection duration longer, the operation easier, and improves the detection flux; on the other hand, it does not deform the cells to be tested, nor does it damage the cells to be tested. The DC resistance data, multiple high-frequency impedance data, and fluorescence intensity data of the cells to be tested can be obtained by using the impedance detection module and the photoelectric detection module, and the cell biophysical characteristic parameters of a single cell to be tested, such as cell diameter, cell nuclear diameter, cell membrane specific capacitance, and cytoplasmic conductivity, can be obtained by using the data analysis and processing module.

[0046] (2) The detection method disclosed in the present invention can obtain multiple biophysical characteristic parameters of a single cell to be tested, making it more practical in the cell field. In addition, the non-compressed cell detection channel used in the present invention overcomes the problem that traditional cell detection channels are easily blocked. Cells flow in the cell detection channel in a non-compressed form, the detection duration is longer, and the operation procedure is relatively simple.

[0047] (3) The electrodes are respectively arranged in the cell inflow channel and the cell outflow channel to collect the cell impedance of the cells to be tested when entering and flowing out of the cell detection channel. The impedance data can be combined with the Coulter principle to obtain the diameter of the cells to be tested, and combined with numerical simulation technology to obtain the cell membrane specific capacitance and cytoplasm conductivity of the cells to be tested.

[0048] (4) The shading sheets are arranged at both ends of the cell detection channel to provide a fluorescence detection window for the cell detection channel, so as to obtain the fluorescence intensity data of the cell nucleus of the cell to be detected and realize the cell nucleus detection of the cell to be detected. In addition, the shading sheet can be used to cover part of the microfluidic chip outside the cell detection channel, so as to ensure that the photoelectric detection module only detects within the fluorescence detection window without being disturbed by the rest of the part. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a schematic diagram of the process of the single cell biophysical property detection method based on non-compressible channels disclosed in the present invention;

[0050] Figure 2 is a schematic diagram of the structure of the microfluidic chip in an embodiment of the present disclosure;

[0051] Figure 3 is a process flow chart for preparing a microfluidic chip in an embodiment of the present disclosure;

[0052] Figure 4 is a schematic diagram of a single cell biophysical property detection device based on a non-compressible channel in an embodiment of the present disclosure;

[0053] Figure 5 is a dynamic schematic diagram of the cells to be detected entering and flowing out of the cell detection channel in the embodiment of the present disclosure;

[0054] Figure 6 is a data graph of the DC resistance and multiple high-frequency impedances of the cells to be tested when flowing in the cell detection channel in the embodiment of the present disclosure;

[0055] Figure 7 It is a graph of the fluorescence intensity change of a single cell to be tested when it flows in the cell detection channel in the embodiment of the present disclosure.

[0056] [Description of Reference Numerals]

[0057] 1-cell inflow channel; 2-cell detection channel; 3-shading sheet; 4-electrode sheet; 5-cell to be tested; 6-cell nucleus; 7-cell outflow channel; 101-photomultiplier tube; 102-microscope; 103-photoelectric detection module; 104-microfluidic chip; 105-negative pressure drive module; 106-data analysis and processing module; 107-data acquisition card; 108-phase-locked amplifier; 109-impedance detection module DETAILED DESCRIPTION

[0058] In order to make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0059] Based on the problems of few detection parameters, easy clogging of cell detection channels and short detection duration in current single-cell biophysical detection methods, it is necessary to propose a microfluidic chip with multiple detection parameters, high throughput and not easy to clog, and a single-cell biophysical property detection device and method based on non-compressive channels contained therein, to detect parameters such as the diameter of a single cell to be tested, the diameter of the cell nucleus, the specific capacitance of the cell membrane, and the cytoplasmic conductivity.

[0060] Figure 1 It is a schematic diagram of the process of the single-cell biophysical property detection method based on non-compressive channels disclosed in the present invention.

[0061] like Figure 1 As shown, the single-cell biophysical property detection method based on the non-compressive channel disclosed in the present invention includes steps S101 to S105.

[0062] Step S101: Fluorescence staining of the nuclei of the cells to be tested.

[0063] Step S102: injecting the dyed cells to be tested into the microfluidic chip, and using a negative pressure driving module to drive the cells to be tested to flow in a cell detection channel of the microfluidic chip in a non-compressed state.

[0064] Step S103: using an impedance detection module to record the DC resistance data and multiple high-frequency impedance data of the cells to be tested flowing in the cell detection channel, wherein the high frequency range includes: 100kHz-1GHz.

[0065] Step S104: using a photoelectric detection module to record the fluorescence intensity data of the cell nucleus of the cell to be detected flowing in the fluorescence detection window.

[0066] Step S105: using a data analysis and processing module to calculate the biophysical properties of the cells to be tested based on the DC resistance data, multiple high-frequency impedance data, and fluorescence intensity data, wherein the biophysical properties of the cells to be tested include cell diameter, cell nuclear diameter, cell membrane specific capacitance, and cytoplasmic conductivity.

[0067] Through the embodiments of the present disclosure, the nuclei of the cells to be tested are stained to detect the nuclei of a single cell to be tested; the cells to be tested are introduced into the cell detection channel of the microfluidic chip and flow in a non-compressed form, which, on the one hand, makes the detection duration longer, the operation easier, and improves the detection flux; on the other hand, it does not cause the cells to be tested to deform, nor does it cause damage to the cells. The DC resistance data, multiple high-frequency impedance data, and cell fluorescence intensity data of the cells to be tested can be obtained by using the impedance detection module and the photoelectric detection module, and the biophysical characteristic parameters of a single cell to be tested, such as cell diameter, cell nuclear diameter, cell membrane specific capacitance, and cytoplasmic conductivity, can be obtained by the data analysis and processing module. The detection method of the present disclosure can obtain multiple biophysical characteristic parameters of a single cell to be tested, making it more practical in the field of cells. Moreover, the non-compressed cell detection channel used in the present disclosure overcomes the problem that the channel is easily blocked in the microfluidic impedance cell detector based on the compression channel, and the detection duration is longer and the operation is easier.

[0068] According to an embodiment of the present disclosure, in step S101, an appropriate amount of fluorescent dye is added to the cell suspension to be tested, and then cultured at an appropriate cell culture temperature for 20-30 minutes, and then washed with PBS buffer solution (phosphate buffered saline solution) or culture fluid. The purpose is to make the cell nucleus obviously colored while the cytoplasm is not obviously colored, so that the fluorescence pulse signal changes caused by the cell nucleus can be detected under a photomultiplier tube.

[0069] According to an embodiment of the present disclosure, before injecting the stained cells to be tested into the microfluidic chip, all channels in the microfluidic chip need to be filled with cell culture fluid to avoid the generation of bubbles in the channels due to negative pressure during operation, thereby affecting the cell fluidity and impedance measurement accuracy.

[0070] According to an embodiment of the present disclosure, in step S102, a negative pressure driving module is used to apply negative pressure at the cell outflow channel to drive the cells to be tested to enter the cell detection channel, flow in the cell detection channel, and drive the cells to flow out of the cell detection channel.

[0071] According to an embodiment of the present disclosure, in step S103, the cell diameter of the cell to be tested is obtained based on the DC resistance data and the Coulter principle; based on the cell diameter of the cell to be tested and impedance data at multiple high frequencies, the cell membrane specific capacitance and cytoplasm conductivity of the cell to be tested are obtained based on numerical simulation technology.

[0072] According to the embodiment of the present disclosure, in step S104 and step S105, the data analysis and processing module is used to process the data tested by the impedance detection module and the photoelectric detection module, and the conversion relationship obtained by numerical simulation based on the DC resistance data and the Coulter principle can be obtained to obtain the diameter data of the cell to be tested. According to the cell diameter of the cell to be tested and the impedance data under multiple high frequencies, the cell membrane specific capacitance and cytoplasm conductivity of the cell to be tested can be obtained based on the numerical simulation technology. According to the cell diameter D of the cell to be tested c , the length L1 of the cell detection channel, the DC resistance pulse width t1, the interval length L2 of the fluorescence detection window and the fluorescence intensity data pulse width t2, the following formula is used to obtain the cell nucleus diameter D of the cell to be tested. n :

[0073]

[0074] According to the embodiments of the present disclosure, before performing single-cell biophysical property detection of a non-compressible channel, a cell biophysical property detection device for a non-compressible channel needs to be built first, and the working mechanism of the device includes: first connecting a microfluidic chip, an impedance detection module, a photoelectric detection module, a negative pressure drive module and a data analysis and processing module, wherein the measuring ends of the phase-locked amplifier of the impedance detection module are respectively connected to two electrode sheets in the microfluidic chip, and the output end of the phase-locked amplifier is connected to the data analysis and processing module through a data acquisition card; the microscope of the photoelectric detection module is arranged near the cell detection channel in the microfluidic chip, the acquisition end of the photomultiplier tube is connected to the light port of the microscope, and the output end of the photomultiplier tube is connected to the data analysis and processing module; the output end of the negative pressure drive module is connected to the cell outflow channel of the microfluidic chip module through a closed catheter.

[0075] Figure 2 It is a schematic diagram of the structure of the microfluidic chip in the embodiment of the present disclosure.

[0076] like Figure 2As shown, the present disclosure provides a microfluidic chip, including: a cell inflow channel 1, used to inject a cell to be tested 5 into a cell detection channel 2; a cell outflow channel 7, used to make the cell to be tested 5 flow out of the cell detection channel 2; the cell detection channel 2 is arranged between the cell inflow channel 1 and the cell outflow channel 7, and is used to make a single cell to be tested 5 flow in a non-compressed form; an electrode sheet 4, which is respectively arranged in the cell inflow channel 1 and the cell outflow channel 7, and is connected to the two ports of the cell detection channel 2, and the electrode sheet is connected to the impedance detection module 109, and is used to detect the impedance of the cell to be tested 5 entering and flowing out of the cell detection channel 2; a light shielding sheet 3, which is arranged at both ends of the cell detection channel 2, and is used to provide a fluorescence detection window for the cell detection channel 2.

[0077] Through the embodiments of the present disclosure, the cell detection channel is designed to allow a single cell to be tested to flow in a non-compressed form, which can ensure that the cell to be tested does not block the detection channel, the detection duration is longer, the operation is simpler, the detection flux is improved, and the shape of the cell to be tested is not affected. The electrode sheets are distributed on both sides of the cell detection channel and connected to the two ports of the cell detection channel, which can ensure that the impedance detection module measures the impedance data in the cell detection channel, wherein the impedance data includes DC resistance data and multiple high-frequency impedance data (the high-frequency range is generally 100kHz-1GHz). The shading sheet is set at both ends of the cell detection channel to cover the microfluidic chip outside the cell detection channel, which can ensure that the photoelectric detection module only detects the cell detection channel without being interfered by the rest of the parts, and also provides a fluorescence detection window for the cell detection channel.

[0078] According to an embodiment of the present disclosure, the cross-sectional areas of the cell inflow channel 1 and the cell outflow channel 7 are 3 to 10 times the cross-sectional area of ​​the cell to be tested 5; the cross-sectional area of ​​the cell detection channel 2 is larger than the cross-sectional area of ​​the cell to be tested 5; and the length of the cell detection channel 2 is larger than the diameter of the cell to be tested 5.

[0079] According to the embodiments of the present disclosure, the cross-sectional length and width range of the cell inflow channel and the cell outflow channel includes 30 to 60 μm; the diameter range of the cells to be tested includes 10 to 20 μm; the width and height range of the cell detection channel includes 15 to 30 μm; and the length range of the cell detection channel includes 20 to 50 μm.

[0080] According to the embodiments of the present disclosure, the sizes of the cell inflow channel, the cell outflow channel and the cell detection channel in the microfluidic chip can be adjusted according to the size of the cells to be detected.

[0081] Through the embodiments of the present disclosure, the cross-sectional area of ​​the cell detection channel is set to be larger than the cross-sectional area of ​​the cells to be detected, so that the cells to be detected can flow individually in the cell detection channel in a non-compressed form. The length of the cell detection channel is designed to be larger than the length of the cells to be detected, so that the cells to be detected can fully flow in the cell detection channel and the detection duration is longer.

[0082] According to an embodiment of the present disclosure, the diameter length range of the cell nucleus of the cell to be detected includes: 5 to 10 μm; the length range of the fluorescence detection window includes: 15 to 30 μm.

[0083] According to an embodiment of the present disclosure, the length of the fluorescence detection window can be adjusted according to the diameter of the cell nucleus of the cell to be detected.

[0084] According to the embodiments of the present disclosure, the length of the fluorescence detection window is set to be greater than the length of the nucleus of the cell to be detected, so as to more accurately obtain the size information of the nucleus of the cell to be detected.

[0085] Figure 3 It is a process flow chart for preparing the microfluidic chip in the embodiment of the present disclosure.

[0086] exist Figure 3 In a, the first step is to spin-coat a layer of SU8-5 photoresist on a glass sheet, and then perform pre-baking, exposure, and post-baking in sequence without developing to form a positive mold of a cell detection channel.

[0087] exist Figure 3 In b, the second step is to spin-coat a layer of SU8-25 photoresist on the existing SU8-5 photoresist, and then perform pre-bake, exposure, post-bake and other processes in sequence.

[0088] exist Figure 3 In c, the third step is to develop and harden the two layers of photoresist, thereby producing positive molds of the cell inflow channel and the cell outflow channel.

[0089] exist Figure 3 In d, the fourth step is to pour the mixture of PDMS and its curing agent on the mold made in the first three steps.

[0090] exist Figure 3 In e, the fifth step is to demold the PDMS prepared in the fourth step after solidification, so as to obtain the PDMS as the microfluidic chip structure and punch holes at corresponding positions.

[0091] exist Figure 3 In FIG. 5 , the sixth step is to spin-coat a layer of AZ 1500 photoresist on another cleaned glass sheet, and then perform pre-baking and exposure treatments in sequence.

[0092] exist Figure 3In g, the seventh step is to develop the photoresist layer already formed in the sixth step to remove the photoresist at the electrode position.

[0093] exist Figure 3 h, the eighth step is to sputter metal Cr / Au based on the seventh step.

[0094] exist Figure 3 In step i, the ninth step is to perform a stripping operation on the metal Cr / Au sputtered on the photoresist to obtain the desired electrode sheet.

[0095] exist Figure 3 In step j, the tenth step is to spin-coat another layer of AZ 1500 on the electrode sheet of the ninth step, and then perform pre-baking, exposure and other treatments in sequence.

[0096] exist Figure 3 In k, the eleventh step is to develop the existing layer of photoresist on the basis of the tenth step to remove the photoresist at the position of the light shielding sheet.

[0097] exist Figure 3 1, the twelfth step is to sputter the ZnO thin film based on the eleventh step.

[0098] exist Figure 3 In step m, the thirteenth step is to strip off the ZnO film sputtered on the photoresist in the twelfth step to obtain the desired light shielding film.

[0099] exist Figure 3 n, the fourteenth step is to align and bond the PDMS prepared in the fifth step and the glass sheet containing electrodes and light shielding film in the thirteenth step to form a complete microfluidic chip.

[0100] Figure 4 It is a schematic diagram of a single-cell biophysical property detection device based on a non-compressible channel in an embodiment of the present disclosure.

[0101] like Figure 4 As shown, the single-cell biophysical property detection device based on non-compressive channels provided by the present disclosure includes: a microfluidic chip 104, an impedance detection module 109, a photoelectric detection module 103, a negative pressure driving module 105, and a data analysis and processing module 106.

[0102] According to the embodiment of the present disclosure, the impedance detection module 109 includes a phase-locked amplifier 108 and a data acquisition card 107, wherein the phase-locked amplifier 108 is used to obtain the DC resistance data of the cells to be tested in the cell detection channel under DC conditions and the impedance data of whether the cells to be tested pass through the cell detection channel under at least two high-frequency (high-frequency range 100kHz-1GHz) conditions, and the data acquisition card 107 is used to transmit the DC resistance data and multiple high-frequency impedance data output by the phase-locked amplifier 108 to the data analysis and processing module 106. The measuring ends of the impedance detection module 109 in the embodiment of the present disclosure are respectively connected to the electrode sheets 4 in the microfluidic chip 104, and are used to obtain the DC resistance data and multiple high-frequency impedance data of the cells to be tested flowing in the cell detection channel.

[0103] According to an embodiment of the present disclosure, the photoelectric detection module 103 includes a microscope 102 and a photomultiplier tube 101, wherein the microscope 102 is arranged near the cell detection channel to provide a fluorescent light source for the fluorescence detection window; the collection end of the photomultiplier tube 101 is arranged at the light port of the microscope to obtain the fluorescence intensity data caused by the cell nucleus passing through the fluorescence detection window.

[0104] According to an embodiment of the present disclosure, the negative pressure driving module 105 is connected to the microfluidic chip 104 through a closed catheter, and the negative pressure driving module 105 includes a pressure calibrator, wherein the pressure calibrator is used to generate and control the output negative pressure to drive cell movement.

[0105] According to an embodiment of the present disclosure, the data analysis and processing module 106 is used to obtain the DC resistance data of the impedance detection module 109, multiple high-frequency impedance data and the fluorescence intensity data of the photoelectric detection module 103, to obtain the cell diameter based on the DC resistance data, to obtain the cell membrane specific capacitance and the cytoplasm conductivity based on the cell diameter and the multiple high-frequency impedance data, and to obtain the cell nucleus diameter of the cell to be tested based on the cell diameter, the DC resistance data and the fluorescence intensity data.

[0106] Through the embodiments of the present disclosure, the microfluidic chip is the core part of the single-cell biophysical property detection device based on the non-compressed channel, and the cell-related biophysical property data is obtained by the movement of the cell to be tested in the cell detection channel. When the detection device is working, the impedance detection module is connected to the electrode sheet in the microfluidic chip, the impedance data is collected, and transmitted to the data analysis and processing module, wherein the impedance data includes DC resistance data and impedance data under multiple high frequencies. The microscope in the photoelectric detection module is set near the cell detection channel in the microfluidic chip to provide a fluorescent light source for the fluorescence detection window, wherein the photomultiplier tube collection end is connected to the light port of the microscope to detect the fluorescence intensity data when the cell nucleus passes through, and the fluorescence intensity data is converted into an electrical signal, which is transmitted to the data analysis and processing module through the output end. The output end of the negative pressure drive module is connected to the cell outflow channel of the microfluidic chip through a closed conduit to control the movement of the cell to be tested in the microfluidic chip. The data analysis and processing module processes and analyzes the signals received from the impedance detection module and the photoelectric detection module, and can obtain the cell diameter, cell nucleus diameter, cell membrane specific capacitance, and cytoplasmic conductivity of a single cell to be tested.

[0107] In order to make the purpose, technical solution and advantages of the present disclosure clearer, the technical solution and testing principle of the present disclosure are further explained below through specific embodiments combined with the accompanying drawings. It should be noted that the following specific embodiments are only used as examples, and the protection scope of the present disclosure is not limited thereto.

[0108] Figure 5 is a dynamic schematic diagram of the cells to be detected entering and flowing out of the cell detection channel in the embodiment of the present disclosure; Figure 6 It is a data graph of the DC resistance and multiple high-frequency impedances of the cells to be tested when flowing in the cell detection channel in the embodiment of the present disclosure.

[0109] Will Figure 5 and Figure 6 In combination, when the stained cells to be tested flow through the electrode sheet from the cell inflow channel into the cell detection channel ( Figure 5 a-5b) will produce a rising peak of a pulse ( Figure 6 ab segment); when the cells to be tested flow in the cell detection channel ( Figure 5 b-5c), a peak platform will be generated due to the lack of resistance change ( Figure 6 When the cells to be tested flow out of the cell detection channel and enter the cell outflow channel, a pulse falling peak will be generated because the cells to be tested pass through the electrode sheet again ( Figure 6cd segment in the middle). According to the amplitude of the DC resistance pulse and the Coulter principle, the diameter of the cell can be obtained. When the cell diameter of the cell to be tested is larger, the resistance value in the cell detection channel caused by the increase is larger. The specific conversion relationship from the DC resistance data to the diameter of the cell to be tested is obtained according to the numerical simulation. According to the measured diameter of the cell to be tested and multiple high-frequency impedance data, a table comparison is performed in a lookup table constructed according to the numerical simulation, and the values ​​of the cell membrane specific capacitance and cytoplasm resistance of the cell to be tested can be obtained, wherein the lookup table is obtained by numerical simulation of multiple high impedance data and DC resistance data of cells of different diameters with different cell membrane specific capacitance and cytoplasm conductivity, and stored in the data analysis and processing module.

[0110] It should be noted that the present disclosure adopts a finite element analysis method based on Comsol software in the numerical simulation process. First, a rectangular insulating channel (i.e., a cell detection channel) of the same size as the actual detection channel is defined, and the cells to be tested are defined as spheres with specific conductivity and membrane specific capacitance. In the numerical simulation, the cell diameter, cytoplasmic conductivity, and cell membrane specific capacitance of cells to be tested of different sizes are simulated to obtain a plurality of high-frequency impedance data under the conditions of a determined cell diameter, cytoplasmic conductivity, and cell membrane specific capacitance, and a lookup table is established. According to the Coulter principle, the cell diameter and the DC resistance directly correspond to each other, and the cell diameter can be obtained; according to the equivalent electrical impedance model of the cell, under a determined cell diameter, the cell membrane specific capacitance and the cytoplasmic conductivity correspond to a plurality of high-frequency impedance data.

[0111] Figure 7 It is a graph of the fluorescence intensity change of a single cell to be tested when it flows in the cell detection channel in the embodiment of the present disclosure.

[0112] Will Figure 5 and Figure 7 In combination, assuming that the cell to be tested moves at a constant speed in the cell detection channel, when the nucleus of the cell to be tested passes through the light shielding plates at both ends of the cell detection channel and moves in the fluorescence detection window area, there is a change in fluorescence intensity, wherein the movement of the cell to be tested in the detection channel may have the following relationship:

[0113]

[0114] The moving distance corresponding to the fluorescence intensity pulse width t2 is the length of the light shielding sheet interval L2 plus the cell nucleus diameter D n ; Based on the known cell diameter D c , the length L1 of the cell detection channel, the DC resistance pulse width t1, the interval length L2 of the fluorescence detection window and the fluorescence intensity data pulse width t2.

[0115] According to the proportional relationship in formula 1, the diameter D of the nucleus of the cell to be tested can be calculated. n .

[0116] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present disclosure. It should be understood that the above description is only a specific embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. A method for detecting the biophysical properties of a single cell based on a non-compressive channel, characterized in that: The method comprises: Fluorescently stain the nuclei of cells to be tested; Injecting the stained cells to be tested into the microfluidic chip, and using a negative pressure driving module to drive the cells to be tested to flow in a cell detection channel of the microfluidic chip in a non-compressed form; Using an impedance detection module to record the DC resistance data and multiple high-frequency impedance data of the cells to be tested flowing in the cell detection channel, wherein the high frequency range includes: 100 kHz-1 GHz; Using a photoelectric detection module to record the fluorescence intensity data of the cell nucleus of the cell to be tested flowing in the fluorescence detection window; and Using a data analysis and processing module, according to the DC resistance data, the multiple high-frequency impedance data, and the fluorescence intensity data, the biophysical characteristics of the cells to be tested are calculated, wherein the biophysical characteristics of the cells to be tested include cell diameter, cell nuclear diameter, cell membrane specific capacitance, and cytoplasmic conductivity; Wherein, the cell membrane specific capacitance and cytoplasm conductivity of the cell to be tested are determined by the following steps: Establishing a simulation model, wherein the simulation model includes a rectangular insulating channel having the same size as an actual cell detection channel, and a spherical cell to be detected having a specific conductivity and membrane specific capacitance; Simulating the preset cell diameters, cytoplasmic conductivity and cell membrane specific capacitance of the cells to be tested of different sizes, and obtaining a plurality of high-frequency impedance data under the conditions of the determined cell diameters, cytoplasmic conductivity and cell membrane specific capacitance based on the equivalent electrical impedance model of the cells to be tested; Establishing a lookup table between the cell diameter, cytoplasmic conductivity, cell membrane specific capacitance and the multiple high-frequency impedance data, wherein the lookup table is used to determine the cell membrane specific capacitance and cytoplasmic conductivity of the cell to be measured according to the measured cell diameter and the multiple high-frequency impedance data; According to the cell diameter of the cell to be measured , length of cell detection channel , DC impedance pulse width , the interval length of the fluorescence detection window and the pulse width in the fluorescence intensity data , the following formula is used to obtain the nuclear diameter of the cell to be tested : (Formula 1).

2. The method according to claim 1, characterized in that According to the DC resistance data and the Coulter principle, the cell diameter of the cell to be tested is obtained; According to the cell diameter of the cell to be measured and the multiple high-frequency impedance data, the cell membrane specific capacitance and cytoplasm conductivity of the cell to be measured are obtained based on numerical simulation technology.

3. A single cell biophysical property detection device based on a non-compressible channel, characterized in that: The method according to any one of claims 1 to 2 is performed by a single-cell biophysical property detection device based on a non-compressible channel, and the single-cell biophysical property detection device based on a non-compressible channel comprises: A microfluidic chip, comprising a cell detection channel, for allowing a single cell to be detected to flow in a non-compressed form; An impedance detection module, used to obtain DC resistance data and multiple high-frequency impedance data of the cells to be tested flowing in the cell detection channel; A photoelectric detection module is arranged near the cell detection channel, and is used to provide a fluorescent light source for the fluorescent detection window of the cell detection channel, and to obtain the fluorescence intensity data when the cell nucleus of the cell to be detected passes through the fluorescent detection window; A negative pressure driving module, used for driving the cells to be tested to move in the cell detection channel; The data analysis and processing module is used to obtain the DC resistance data, multiple high-frequency impedance data and fluorescence intensity data of the impedance detection module, and obtain the biophysical characteristics of the cells to be tested based on the DC resistance data, multiple high-frequency impedance data and fluorescence intensity data.

4. The device according to claim 3, characterized in that The microfluidic chip also includes: A cell inflow channel, used to inject cells to be tested into the cell detection channel; A cell outflow channel, used to allow the cells to be tested to flow out of the cell detection channel; Wherein, the cell detection channel is arranged between the cell inflow channel and the cell outflow channel; Electrode sheets, respectively arranged in the cell inflow channel and the cell outflow channel and connected to two ports of the cell detection channel, and the electrode sheets are connected to the impedance detection module; The light shielding sheets are arranged at two ends of the cell detection channel and are used to provide fluorescence detection windows for the cell detection channel.

5. The device according to claim 4, characterized in that The cross-sectional areas of the cell inflow channel and the cell outflow channel are 3 to 10 times the cross-sectional areas of the cells to be tested; The cross-sectional area of ​​the cell detection channel is larger than the cross-sectional area of ​​the cell to be detected; The length of the cell detection channel is greater than the diameter of the cell to be detected.

6. The device according to claim 5, characterized in that The length and width of the cross-sections of the cell inflow channel and the cell outflow channel range from 30 to 60 μm; The diameter range of the cells to be tested includes 10-20 μm; The width and height of the cell detection channel range from 15 to 30 μm; The length of the cell detection channel ranges from 20 to 50 μm.

7. The device according to claim 3, characterized in that The diameter length range of the cell nucleus of the cell to be tested includes: 5-10 μm; The length range of the fluorescence detection window includes: 15~30 μm.

8. The device according to claim 3, characterized in that The impedance detection module includes a lock-in amplifier and a data acquisition card, wherein the lock-in amplifier is used to measure the DC resistance data and multiple high-frequency impedance data between the electrode sheets in the microfluidic chip, and the data acquisition card is used to transmit the DC impedance data and multiple high-frequency impedance data output by the lock-in amplifier to the data analysis and processing module; The photoelectric detection module includes a microscope and a photomultiplier tube, wherein the microscope is used to provide a fluorescent light source for the fluorescent detection window and receive fluorescence from the fluorescent detection window; the photomultiplier tube is used to measure the fluorescence intensity data output by the microscope.

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