Electrode-integrated micro-fluidic chip and electrochemical cell migration detection method

Abstract

The invention belongs to the technical field of cell migration detection, and particularly relates to an electrode-integrated micro-fluidic chip, which comprises a cell sowing flow channel, a cell collection flow channel, a series of micro-channels and electrodes, the micro-channels are arranged between the cell sowing flow channel and the cell collecting flow channel in parallel; and two ends of the cell sowing flow channel and the cell collecting flow channel are respectively connected with the electrodes. According to the invention, linear measurement of the number of migrated cells in the microchannel and morphological structure change characterization can be realized, and different types of cells can be distinguished. In addition, the invention also discloses an electrochemical cell migration detection method.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell migration detection, and particularly relates to a microfluidic chip integrated with electrodes and an electrochemical cell migration detection method. Background Art

[0002] Cell migration is a biological process in which cells move directionally through cell body deformation under the guidance of chemical gradients, mechanical stimuli or electrical signals, and plays a key role in physiological and pathological activities such as embryonic development, immune response, tissue repair and tumor metastasis. The in vitro organ-on-a-chip model based on microfluidic technology has become an important experimental platform for studying the mechanism of cell migration because it can accurately simulate the biochemical and mechanical characteristics of the in vivo microenvironment. Existing cell migration detection methods (such as Transwell chambers, scratch assays, etc.) rely on cell fixation and lysis, can only obtain static end-point data and damage sample continuity, have defects such as low time resolution, weak microenvironment regulation ability, and inability to synchronously obtain dynamic information on cell quantity and morphology, and are difficult to reveal the real-time kinetic characteristics of the migration process. The electrochemical detection technology has shown important application value in the study of cell migration behavior due to its non-invasive and real-time monitoring characteristics.

[0003] In the process of implementing the present invention, the inventors found that there are at least the following problems in the prior art:

[0004] There are still some limitations in the existing methods. For example, the system integration degree is insufficient, the detection device and the cell culture device are independent of each other, the operation is complex and it is difficult to achieve in-situ detection. Most of the existing technical methods rely on electrode modification to improve the detection sensitivity of the system, increasing the preparation cost and technical threshold. In addition, the detection dimension of the existing methods is relatively single, mostly analyzing the cell migration path or the migration quantity, and it is difficult to detect the complex interaction between cells and the microenvironment. Summary of the Invention

[0005] The purpose of the present invention is: aiming at the deficiencies of the prior art, on the one hand, to provide a microfluidic chip integrated with electrodes, which can achieve linear measurement of the number of migrating cells in the microchannel and characterization of the morphological structure changes, and realize the differentiation of different types of cells.

[0006] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A microfluidic chip integrated with electrodes, comprising a cell seeding channel, a cell collection channel, a series of microchannels and electrodes; the microchannels are arranged in parallel between the cell seeding channel and the cell collection channel; both ends of the cell seeding channel and the cell collection channel are connected to the electrodes.

[0008] In some possible embodiments, the microfluidic chip template is fabricated by photolithography, and the material of the microfluidic chip is PDMS. The chip is made of PDMS by standard soft lithography process.

[0009] In some possible embodiments, the lengths of the cell seeding channel and the cell collection channel are 1.6 cm, the widths are 1.6 mm, and the heights are 35 μm. The length of the microchannel is 150 μm, the height is 35 μm, and the width is one of 8 μm, 13 μm, 17 μm, and 22 μm, which is used to simulate different degrees of spatial confinement. Each microfluidic chip preferably has microchannels of only one width.

[0010] In some possible embodiments, the cell seeding channel and the cell collection channel have entrances and exits. Through holes are provided at the entrances and exits of the cell seeding channel and the cell collection channel. The through hole at the entrance is used for injecting liquid into the cell channel, and the exit through hole is used for liquid outflow. The entrances and exits are integrated with an electrochemical detection system composed of four electrodes for detecting the electrical properties of cells in the microchannel, which is connected to an electrochemical workstation.

[0011] In some possible embodiments, the diameter of the electrode is 0.2 mm, and the electrode is a platinum wire electrode.

[0012] In some possible embodiments, the electrode is positioned and punched through a 1 mL syringe needle, that is, the 1 mL syringe needle is used for positioning and punching before being integrated into the chip. The electrode is inserted into the microfluidic chip by interference fit for integrating the electrode in the microfluidic chip, that is, then the electrode is inserted into the chip to achieve interference fit, so that the electrode is stably integrated in the microfluidic chip.

[0013] On the other hand, an electrochemical cell migration detection method is provided, based on the above microfluidic chip integrated with electrodes, including: by establishing a linear relationship between electrochemical parameters and the number of cells, the detection of the number of migrating cells in the microchannel can be realized; according to the fitting result of the number of cells and electrochemical parameters, the sensitivity of cells in the detection can be obtained. Since the detection sensitivities of different types of cells vary greatly, it can be used for the distinction and screening of different cell lines; the electrical properties of migrating cells in microchannels with different widths in the chip are detected. Since cell morphology and migration patterns will significantly affect their electrical properties, this detection method can be used to characterize cell morphology and thus identify changes in cell migration patterns.

[0014] (1) Cell seeding and culture

[0015] Use a pipette to transfer a concentration of 2×10 6 to 3×10 6Inject the cell suspension at a concentration of / mL into the cell seeding flow channel, add an appropriate amount of PBS buffer to the culture dish to maintain humidity, place the culture dish containing the chip in an incubator, and incubate it for 4 hours at 37°C and 5% CO2 to ensure the uniform distribution and growth of cells in the seeding flow channel. After the cells adhere and grow, seal the entrances and exits of the seeding flow channel with 50 μL of culture medium respectively to ensure that the hydrostatic pressure inside the chip is equal everywhere and the culture medium is in a static state. After incubating for 24 h, observe the cell state, and introduce fresh cell culture medium into the cell collection flow channel to provide a migration environment for the cells. Observe and change the medium once every 24 h, and re-seal the entrances and exits of the cell channel with 50 μL of culture medium before putting it back into the incubator. The chip is incubated in the incubator for 3 days to allow a sufficient number of cells to migrate into the microchannel.

[0016] In some possible embodiments, it further includes:

[0017] Use an intermediate frequency for analysis to optimize the extraction of cell electrical properties, and the frequency of the intermediate frequency is 10 3 Hz to 2.5×10 3 Hz.

[0018] (2) Electrochemical cell migration detection

[0019] Use an intermediate frequency of 10 3 Hz to 2.5×10 3 Hz for analysis to optimize the extraction of cell electrical properties. By establishing a linear relationship between electrochemical parameters and the number of cells, the measurement of the number of migrating cells in the microchannel can be achieved. According to the fitting results of the number of cells and electrochemical parameters, the detection sensitivity of the system can be obtained. The detection sensitivities of different types of cells vary greatly, which can be used for the differentiation and screening of different cell lines. Detect the electrical properties of migrating cells in microchannels with different widths in the chip, and characterize the cell morphology based on the correlation between cell morphology and migration mode and electrical properties, and then identify changes in the cell migration mode. Before each measurement, place the chip at room temperature for equilibration to minimize measurement errors caused by temperature changes.

[0020] In some possible embodiments, the electrochemical cell migration detection method based on the microfluidic chip provided by the present invention can be used in combination with other common cell detection means to detect the cells migrating into the microchannel, including immunofluorescence staining method, protein detection method, etc.

[0021] In some possible embodiments, the electrochemical cell migration detection method based on the microfluidic chip provided by the present invention can also be used to study the effects of mechanical cues and biochemical factors on cell migration, and can be used as an experimental platform for further exploring the cell migration mechanism.

[0022] One of the above technical solutions has the following beneficial effects:

[0023] By combining microfluidic technology, the present invention achieves a high degree of simulation of the in-vivo environment for cell migration. The electrodes are integrated outside the cell culture channel to ensure non-destructive and high-precision detection during the cell migration process. Meanwhile, the present invention can monitor the morphological changes and migration patterns of cells in real time, providing more comprehensive data support for studying the cell migration mechanism. In addition, through the fine analysis of electrochemical signals, the present invention can effectively distinguish different types of cells, expanding its application prospects in biomedical research and clinical diagnosis, and having significant advantages and broad application potential in the field of cell migration detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The features, advantages, and technical effects of exemplary embodiments of the present invention will be described below with reference to the accompanying drawings.

[0025] Figure 1 It is a schematic diagram of the overall structure of the microfluidic chip in the present invention.

[0026] Figure 2 It is an equivalent circuit diagram of the electrochemical detection method in the present invention.

[0027] Figure 3 It is a migration map of the cells in the microfluidic chip in the present invention after 3 days of cell incubation.

[0028] Figure 4 It is the impedance value of different numbers of MDA-MB-231 cells in the microchannel, and the fitting curve graph of the number of MDA-MB-231 cells and the impedance value at 1000 Hz.

[0029] Figure 5 It is the impedance value of different numbers of MCF-7 cells in the microchannel, and the fitting curve graph of the number of MCF-7 cells and the impedance value at 1000 Hz.

[0030] Figure 6 It is a comparison graph of the impedance values of equal amounts of MDA-MB-231 cells in a 17-μm-wide microchannel and an 8-μm-wide microchannel.

[0031] Among them, the description of the reference numerals is as follows:

[0032] 1 - glass substrate; 2 - microfluidic chip; 3 - electrode. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0033] As used in the specification and claims, certain terms are used to refer to specific components. Those skilled in the art should understand that hardware manufacturers may use different terms to refer to the same component. The specification and claims do not distinguish components by the difference in names, but by the difference in their functions. As used throughout the specification and claims, "comprising" is an open-ended term and should be interpreted as "comprising but not limited to". "Substantially" means within an acceptable error range, and those skilled in the art can solve technical problems within a certain error range and basically achieve the technical effect.

[0034] The present invention will be further described in detail below in conjunction with the accompanying drawings, but this is not intended to limit the present invention.

[0035] A microfluidic chip with integrated electrodes, comprising a cell seeding channel, a cell collection channel, a plurality of microchannels and electrodes; the microchannels are arranged in parallel between the cell seeding channel and the cell collection channel; both ends of the cell seeding channel and the cell collection channel are respectively connected to the electrodes. See Figure 1 As shown, the left channel is the cell inoculation channel, the right channel is the cell collection channel, a glass substrate 1 is provided at the bottom of the microfluidic chip, and the electrodes are led out from both ends of the microfluidic chip.

[0036] In the microfluidic chip with integrated electrodes according to the present invention, the microfluidic chip template is manufactured by photolithography, and the material of the microfluidic chip is PDMS. The chip is made of PDMS by standard soft lithography.

[0037] In the microfluidic chip with integrated electrodes according to the present invention, the lengths of the cell seeding channel and the cell collection channel are 1.6 cm, the widths are 1.6 mm, and the heights are 35 μm. The length of the microchannel is 150 μm, the height is 35 μm, and the width is one of 8 μm, 13 μm, 17 μm and 22 μm, which is used to simulate different degrees of space limitation. Each microfluidic chip preferably has only one width of microchannel.

[0038] In the microfluidic chip with integrated electrodes according to the present invention, the cell seeding channel and the cell collection channel have inlets and outlets. Through holes are provided at the inlets and outlets of the cell seeding channel and the cell collection channel. The through hole at the inlet is used to inject liquid into the chip, and the outlet through hole is used for liquid to flow out. An electrochemical detection system composed of four electrodes is integrated at the inlets and outlets, which is used for detecting the electrical characteristics of cells in the microchannel and is connected to an electrochemical workstation.

[0039] In the microfluidic chip with an integrated electrode according to the present invention, the diameter of the electrode is 0.2 mm, and the electrode is a platinum wire electrode. The electrode is positioned and punched through a 1 mL syringe needle, that is, before being integrated into the chip, a 1 mL syringe needle is used for positioning and punching. The electrode is inserted into the microfluidic chip through interference fit for integrating the electrode in the microfluidic chip, that is, then the electrode is inserted into the chip to achieve interference fit, so that the electrode is stably integrated in the microfluidic chip.

[0040] Electrochemical cell migration detection method:

[0041] It includes detecting the number of cells during migration by establishing a linear relationship between electrochemical parameters and the number of cells; obtaining the sensitivity of cells in the detection according to the fitting result of the number of cells and electrochemical parameters for the differentiation and screening of different cell lines; detecting the electrical characteristics of migrating cells in microchannels with different widths in the chip to characterize cell morphology, and further identifying changes in the cell migration pattern.

[0042] Preferably, it further includes: using intermediate frequency for analysis to optimize the extraction of cell electrical characteristics, and the frequency of the intermediate frequency is 10 3 Hz to 2.5×10 3 Hz.

[0043] Preferably, the electrochemical cell migration detection method based on the microfluidic chip provided by the present invention can be used in combination with other common cell detection means to detect the cells migrating into the microchannel, including immunofluorescence staining method, protein detection method, etc.

[0044] Preferably, the electrochemical cell migration detection method based on the microfluidic chip provided by the present invention can also be used to study the effects of mechanical cues and biochemical factors on cell migration, and can be used as an experimental platform for further exploring the cell migration mechanism.

[0045] Example 1

[0046] Manufacture of the microfluidic chip

[0047] A mold is made through a photolithography mask plate for casting. Using soft lithography, the PDMS prepolymer and curing agent are mixed at a ratio of 5:1, stirred for 2 min to be fully mixed, then poured into the mold, and after being evacuated by a vacuum machine, it is dried and cured, and finally demolded to obtain the chip. To facilitate seeding, liquid replacement, and subsequent detection, a punch with a diameter of 1.5 mm is used to punch the inlets and outlets of the cell seeding channel and the collection channel. The chip is ultrasonically cleaned successively with absolute ethanol and deionized water to remove the influence of surface impurities and organic matter residues on the experiment. After being dried, it is bonded to the glass slide through oxygen plasma to seal the microchannel. After bonding, it is baked again to make the bonding tighter.

[0048] Example 2

[0049] Migration detection of MDA-MB-231 cells and MCF-7 cells

[0050] DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin was used as the cell culture medium. Using the microfluidic chip and method of the present invention, MDA-MB-231 cells and MCF-7 cells were cultured and detected respectively. On the 3rd day of chip culture, the cell status in each region of the chip is shown in Figure 3 As shown, it can be found that the cells in both flow channels and microchannels showed good growth states, indicating that the constructed microfluidic chip can be used for subsequent cell migration and detection experiments. The detection results indicate that there is a positive correlation between the cell number and the total impedance value. The fitting results of the cell number and the electrical property parameters are shown in Figure 4 and Figure 5 As shown, it shows that the detection system can achieve linear measurement of the number of migrating cells in the microchannel, and the detection sensitivity of the chip seeded with MDA-MB-231 cells is 136.204 Ω / cell, and the detection sensitivity of the chip seeded with MCF-7 cells is 448.273 Ω / cell. The difference in system sensitivity can be attributed to the differences in cell size and migration patterns between the two cell lines. Therefore, based on the differences in their electrical properties, the present invention can be used as a feasible tool for distinguishing different types of cells.

[0051] Example 3

[0052] Using the same cell culture method as in Example 2, MDA-MB-231 cells were cultured in the microfluidic chip of the present invention (the widths of the microchannels are 17 μm and 8 μm). The number of cells migrating in the microchannels was monitored by a microscope, and two groups of chips with the same number of migrating cells were obtained. Among them, the cells on the 17-μm group chips adopted a mesenchymal migration pattern, and the cells on the 8-μm group chips partly adopted an amoeboid migration pattern. Using the method of the present invention to detect the above two groups of chips, it was found that the electrochemistry parameters of the 17-μm group cells were slightly lower than those of the 8-μm group. Repeating the above experiment, statistical analysis was performed on the detection results to evaluate the correlation between the proportion of migration patterns in the two chips and the average value of cell electrochemistry parameters, as shown in Figure 6 As shown, according to the experimental results, it can be concluded that the average value of electrochemistry parameters increases with the increase in the proportion of amoeboid migration pattern. This indicates that the present invention can be used to characterize cell morphology and further identify changes in cell migration patterns.

[0053] Based on the disclosure and teachings of the above specification, those skilled in the art to which the present invention pertains are also able to make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments described above. Any obvious improvements, substitutions, or variations made by those skilled in the art based on the present invention fall within the protection scope of the present invention. In addition, although some specific terms are used in this specification, these terms are only for convenience of description and do not constitute any limitation to the present invention.

Claims

1. A microfluidic chip integrated with an electrode, characterized in that: It includes a cell seeding channel, a cell collection channel, multiple microchannels and electrodes; The microchannels are arranged in parallel between the cell seeding channel and the cell collection channel; Both ends of the cell seeding channel and the cell collection channel are respectively connected to the electrodes.

2. The microfluidic chip with an integrated electrode according to claim 1, characterized in that: The microfluidic chip is fabricated by soft lithography, and the material of the microfluidic chip is PDMS.

3. The microfluidic chip with an integrated electrode according to claim 1, characterized in that: The lengths of the cell seeding channel and the cell collection channel are 1.6 cm, the widths are 1.6 mm, and the heights are 35 μm.

4. The microfluidic chip with an integrated electrode according to claim 1, characterized in that: The lengths of the microchannels are 150 μm, the heights are 35 μm, and the widths are one of 8 μm, 13 μm, 17 μm and 22 μm.

5. The microfluidic chip with an integrated electrode according to claim 4, wherein: The cell seeding channel and the cell collection channel have inlets and outlets, and an electrochemical detection system composed of four electrodes is integrated at the inlets and outlets for detecting the electrical properties of cells in the microchannels.

6. The microfluidic chip with an integrated electrode according to claim 1, characterized in that: The diameters of the electrodes are 0.2 mm, and the electrodes are platinum wire electrodes.

7. The microfluidic chip with an integrated electrode according to claim 6, characterized in that: The electrodes are positioned and punched through syringe needles, and the electrodes are inserted into the microfluidic chip by interference fit for integrating the electrodes in the microfluidic chip.

8. An electrochemical cell migration detection method, characterized in that: The microfluidic chip with integrated electrodes according to claim 1, comprising: Detecting the number of cells during migration by establishing a linear relationship between electrochemical parameters and the number of cells; Obtaining the sensitivity of cells in the detection according to the fitting result of the number of cells and electrochemical parameters for distinguishing and screening different cell lines; Detecting the electrical properties of migrating cells in microchannels with different widths in the chip to characterize cell morphology and further identify changes in cell migration patterns.

9. The electrochemical cell migration detection method according to claim 8, characterized in that, It further includes: Using intermediate frequency for analysis to optimize the extraction of cell electrical properties.

10. The electrochemical cell migration detection method according to claim 9, characterized in that: The frequency of the intermediate frequency is 10 3 Hz to 2.5×10 3 Hz.

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