Microfluidic chip and method for manufacturing the same

Abstract

The application discloses a microfluidic chip and a preparation method thereof, and relates to the field of microfluidic chips, and provides a microfluidic chip for detecting high-throughput single-cell intrinsic electrical parameters of adherent cells and a preparation method thereof. The microfluidic chip comprises a first chip and a second chip. The surface of the first chip is provided with a detection electrode. The detection electrode comprises a detection end and an information end. The detection end is formed with a plurality of electrode units. Each electrode unit is used for detecting the intrinsic electrical parameters of a single adherent cell. The information end is used for transmitting the electrical signals of the detection end to the outside. The second chip is attached to the surface of the first chip. The second chip is formed with a microfluidic channel. The microfluidic channel is sequentially provided with a liquid inlet, a cell detection area and a liquid outlet. The liquid inlet is used for introducing cell suspension into the microfluidic channel. A plurality of cell blocking units are formed in the cell detection area, so as to block and retain a single cell respectively. Each cell blocking unit is opposite to each electrode unit in the up-down direction, so as to realize real-time, continuous and high-throughput collection of cell electrical impedance changes through the detection end.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell detection, in particular to a microfluidic chip and a preparation method thereof. BACKGROUND

[0002] Cell impedance sensing technology is that when cells adhere to the base electrode, due to the integrity of the cell membrane, the cells can grow and proliferate on the electrode surface as poor conductors, the local ion environment of the electrode / solution interface changes, and the impedance value increases, so the physiological or pathological state changes of the cells on the electrode surface can be reflected by the changes of the impedance value or the impedance spectrum.

[0003] Except for a few cells such as blood cells, the growth of most types of cells in the human body needs a support surface that can be attached, which is called adherent cells. Compared with detecting cell suspension, detecting cells in adherent state can relatively truly reflect the electrical characteristics of cells in physiological or pathological state. At present, the detection of adherent cell impedance mainly focuses on the cell population level. Due to the heterogeneity between cells, the average electrical characteristic detection results obtained from the cell population cannot reflect the heterogeneity information mixed in the sample, and after detecting the impedance spectrum, the electrical intrinsic parameters of the cells need to be calculated from the cell electrical model. In the case of detecting cell population, the detection results are often affected by factors such as cell density and cell growth geometry on the electrode, and often cannot well meet the simple electrical model.

[0004] In the existing microfluidic chip, although it can be used as a means for capturing single cells, there may be a positional deviation between the single cell array and the electrode array, which leads to the need for multiple repeated operations. Therefore, developing a microfluidic chip for high-throughput single-cell intrinsic electrical parameter detection of adherent cells, which is simple in process, convenient in operation and can realize real-time automatic detection, has very important practical value and innovative significance for accurately and efficiently detecting cell electrical characteristics and analyzing various physiological activities of cells. SUMMARY

[0005] The main purpose of the present application is to provide a microfluidic chip, which aims to solve the problem of difficult accurate and efficient long-time continuous automatic detection of high-throughput single-cell intrinsic electrical parameters of adherent cells.

[0006] To achieve the above-mentioned purpose, the microfluidic chip provided by the present application is used for detecting high-throughput single-cell intrinsic electrical parameters of adherent cells, and the microfluidic chip comprises:

[0007] a first chip, a detection electrode is arranged on the surface of the first chip, the detection electrode comprises a detection end and an information end, a plurality of electrode units are formed on the detection end, each electrode unit is used for detecting the intrinsic electrical parameters of a single adherent cell, and the information end is used for transmitting the electrical signal of the detection end to the outside; and

[0008] The second chip is attached to the surface of the first chip. The second chip has a microfluidic channel. The microfluidic channel is provided with an inlet, a cell detection area and an outlet in sequence. The inlet is used to introduce cell suspension into the microfluidic channel. Multiple cell blocking units are formed in the cell detection area. Each cell blocking unit is used to block and retain a single cell. Each cell blocking unit is opposite to each electrode unit in the vertical direction. The outlet is used to discharge the cell suspension.

[0009] Optionally, the microfluidic channel includes a drainage channel segment located between the inlet and the cell detection area, wherein the drainage channel segment has a drainage structure on the side adjacent to the cell detection area, the drainage structure being used to guide cells into each of the cell barrier units; and / or,

[0010] The first chip surface is further provided with a counter electrode to form a circuit with the detection electrode, and the distance between the counter electrode and the detection end is [missing information]. ,in, .

[0011] Optionally, the height of the microfluidic channel in the vertical direction is The maximum cell size is ,in, ; and / or,

[0012] The thickness of the detection electrode is ,in, .

[0013] Optionally, the cell blocking unit includes two protruding blocks, with one end of the two blocks adjacent to each other and their opposite side portions inclined away from each other to form a V-groove between the two blocks.

[0014] Optionally, the distance between the adjacent ends of the two stops is , .

[0015] Optionally, the surface of the first chip is formed with etched grooves for mounting the detection electrodes.

[0016] Optionally, the surface of the first chip is provided with an adhesion layer to adhere the detection electrode to the etching groove, wherein the thickness of the adhesion layer is [missing information]. , ; and / or,

[0017] The depth of the etching groove is ,in, .

[0018] Optionally, the surface of the first chip is provided with an insulating layer, which covers the detection electrode and exposes the detection end and the information end.

[0019] Optionally, the thickness of the insulating layer is ,in, .

[0020] The present invention also proposes a method for fabricating the above-mentioned microfluidic chip, the method comprising the following steps:

[0021] The substrate surface of the first chip is patterned with a metal or alloy coating.

[0022] An etch groove with a first depth is etched on the substrate surface;

[0023] The adhesive layer and electrode material are sputtered sequentially;

[0024] The electrode material is patterned with an insulating layer to block the detection end and information end of the detection electrode;

[0025] Insulating layers were prepared by plasma-enhanced chemical vapor deposition;

[0026] The photoresist at the detection end and information end of the detection electrode is stripped to form the first chip;

[0027] The inlet and outlet of the liquid are mechanically etched on the second chip substrate, and microfluidic channels and barrier structures are etched to form the second chip;

[0028] The first chip and the second chip are bonded together to form a microfluidic chip.

[0029] In the technical solution of this invention, after the cell suspension is input from the inlet of the second chip, multiple single cells in the microfluidic channel are blocked by the cell blocking unit in the cell detection area, so that the single cells in the original suspension become adherent cells, and part of the cell-blocked suspension is discharged from the outlet. The second chip is attached to the surface of the first chip, and each of the cell blocking units and each of the electrode units are opposite to each other. The parameter changes of each adherent cell can be recorded by each of the electrode units, and the electrical signal is transmitted to the external impedance detection system through the information terminal of the detection electrode. Cell impedance changes can be collected in real time, continuously and with high throughput, so as to further analyze the physiological changes of cells and realize long-term continuous automated monitoring of single cells. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0031] Figure 1 This is a schematic diagram of the structure of an embodiment of the microfluidic chip provided by the present invention;

[0032] Figure 2 for Figure 1 A magnified view of a portion of point A in the middle.

[0033] Explanation of icon numbers:

[0034]

[0035] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] It should be noted that all directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of the present invention are only used to explain the relative positional relationship and movement of each component in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indication will also change accordingly.

[0038] Furthermore, the use of terms such as "first" and "second" in this invention is for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of that feature. Additionally, the technical solutions of the various embodiments can be combined with each other, but only on the basis of being achievable by those skilled in the art. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed by this invention.

[0039] Cell impedance sensing technology is based on the fact that when cells adhere to a base electrode, due to the integrity of the cell membrane, the cells can act as poor conductors and grow and proliferate on the electrode surface. The local ionic environment at the electrode / solution interface changes, and the impedance value increases. Therefore, physiological or pathological changes in the cells on the electrode surface can be reflected by changes in impedance value or impedance spectrum.

[0040] Except for a few cells such as blood cells, the growth of most types of cells in the human body requires a supportive surface for adhesion, hence the term adherent cells. Compared to detecting cell suspensions, detecting cells in an adherent state can more accurately reflect the electrical properties of cells under physiological or pathological conditions. Currently, the detection of impedance in adherent cells mainly focuses on the cell population level. Due to the heterogeneity between cells, the average electrical properties obtained from the cell population cannot reflect the heterogeneity information mixed in the sample. After obtaining the impedance spectrum, the intrinsic electrical parameters of the cells need to be calculated from the cell electrical model. However, when detecting cell populations, the detection results are affected by factors such as cell density and the geometry of cell growth on the electrode, and often do not conform well to a simple electrical model.

[0041] While existing microfluidic chips can be used to capture single cells, positional misalignment between the single-cell array and the electrode array can occur, necessitating repeated operations. Therefore, developing a high-throughput microfluidic chip for detecting the intrinsic electrical parameters of adherent cells—one that is simple to fabricate, easy to operate, and capable of real-time automated detection—is of significant practical value and innovative importance for accurately and efficiently detecting cellular electrical properties and analyzing various cellular physiological activities.

[0042] To address the above problems, this invention proposes a microfluidic chip. Figures 1 to 2 This is one embodiment of the present invention.

[0043] In this embodiment of the invention (hereinafter referred to as this embodiment), the microfluidic chip 1000 is as follows: Figures 1 to 2As shown, a high-throughput single-cell intrinsic electrical parameters detection method for adherent cells is described. The microfluidic chip 1000 includes a first chip 1 and a second chip 2. The surface of the first chip 1 is provided with a detection electrode 11, which includes a detection end 111 and an information end 112. The detection end 111 forms multiple electrode units 1111, each of which is used to detect the intrinsic electrical parameters of a single adherent cell. The information end 112 is used to transmit the electrical signal from the detection end 111 to the outside. The second chip 2 is attached to the surface of the first chip 1. On the other side, the second chip 2 forms a microfluidic channel 21, on which an inlet 211, a cell detection area 212, and an outlet 213 are sequentially provided. The inlet 211 is used to introduce cell suspension into the microfluidic channel 21. Multiple cell blocking units 2121 are formed in the cell detection area 212. Each cell blocking unit 2121 is used to block and retain a single cell, and each cell blocking unit 2121 is opposite to each electrode unit 1111 in the vertical direction. The outlet 213 is used to discharge the cell suspension.

[0044] In the technical solution of the present invention, after the cell suspension is input from the inlet 211 of the second chip 2, the cell blocking unit 2121 in the cell detection area 212 blocks multiple single cells in the microfluidic channel 21, so that the single cells in the original suspension become adherent cells, and the suspension after partial cell blocking is discharged from the outlet 213. The second chip 2 is attached to the surface of the first chip 1, and each of the cell blocking units 2121 and each of the electrode units 1111 are opposite to each other. The parameter changes of each adherent cell can be recorded by each of the electrode units 1111, and the electrical signal is transmitted to the external impedance detection system through the information terminal 112 of the detection electrode 11. The changes in cell impedance can be collected in real time, continuously and with high throughput, so as to further analyze the physiological changes of the cell and realize long-term continuous automated monitoring of single cells.

[0045] To divert cells within the suspension so that each cell blocking unit 2121 can block single cells, the microfluidic channel 21 includes a drainage channel section between the inlet 211 and the cell detection area 212. A drainage structure 214 is provided on the side of the drainage channel section adjacent to the cell detection area 212. The drainage structure 214 is used to divert the suspension and guide cells into each cell blocking unit 2121 when the cell suspension enters the drainage channel section, so that single cells can be blocked and retained by the cell blocking unit 2121. The remaining cells and the suspension are discharged from the outlet 213 after passing through the cell blocking unit 2121. The drainage structure 214 consists of multiple spaced protrusions. The protrusions connect the upper and lower sidewalls of the drainage channel section in the thickness direction of the second chip 2 to form a drainage port between two adjacent protrusions. When the suspension flows through the drainage port, the multiple drainage ports divide the suspension into multiple streams, each stream corresponding to each cell blocking unit 2121, so as to guide the cells into each cell blocking unit 2121.

[0046] To ensure the integrity of the circuit of the detection electrode 11, a counter electrode 12 is also provided on the surface of the first chip 1. The counter electrode 12, also known as an auxiliary electrode, is used to form an electrical circuit with the detection electrode 11. To ensure the stability of the electrical circuit, the distance between the counter electrode 12 and the detection end 111 is... It should be within the range of 50μm to 2000μm.

[0047] To ensure that the cells in the suspension have suitable flow space, the microfluidic channel 21 is located vertically, that is, at the height of the second chip 2 in the thickness direction. With the largest size of the cell Should meet This ensures smooth cell flow without being too large to hinder or obstruct cell flow.

[0048] In this embodiment, the thickness of the detection electrode 11 is 20 nm to 100 nm, and the material used is gold, platinum or indium tin oxide.

[0049] To specifically achieve the cell-blocking effect of the cell-blocking unit 2121, in this embodiment, as follows: Figure 2As shown, the cell blocking unit 2121 includes two protruding blocks 2121a. One end of each block 2121a is adjacent to the other, and their opposite sides are inclined away from each other, thus forming a V-shaped groove between them. The opening of the V-shaped groove faces the drainage channel section to block and retain cells within it. The bottom of the V-shaped groove is open to allow for the flow of the suspension. Further, the distance between the adjacent ends of the two blocks 2121a is... ,in, Specifically, the width of the through portion at the bottom of the V-shaped groove is between 2 μm and 5 μm. Each electrode unit 1111 corresponds to a V-shaped groove, ensuring that when cells are retained within the V-shaped groove, the electrode unit 1111 is covered, preventing it from being exposed in the suspension and improving the accuracy of cell impedance data detection. Furthermore, the motor unit is circular or square in shape, with a diameter or side length between 10 μm and 100 μm.

[0050] To facilitate the accurate placement of the detection electrode 11, an etching groove is formed on the surface of the first chip 1 to sputter the detection electrode 11 within the etching groove, thus facilitating the fabrication and shaping of the detection electrode 11. The depth of the etching groove is [details to be inserted here]. , This is to accommodate the sputtering of the adhesive layer and the detection electrode 11 during subsequent processes.

[0051] To specifically achieve the placement of the detection electrode 11 on the substrate surface of the first chip 1, an adhesion layer is provided on the surface of the substrate of the first chip 1. The adhesion layer is used to adhere the detection electrode 11 to the etching groove, thereby ensuring that the detection electrode 11 is stably formed within the etching groove. The thickness of the adhesion layer is [not specified in the original text]. , .

[0052] The surface of the first chip 1 is provided with an insulating layer, which covers the detection electrode 11 and exposes the detection end 111 and the information end 112. This prevents the detection electrode 11 from interfering with signal transmission at the connection point between the detection end 111 and the information end 112 during electrical signal transmission, exposing only the detection end 111 and the information end 112. This facilitates the acquisition of relevant cell parameters and connection to external devices. The thickness of the insulating layer is [not specified in the original text]. The range is .

[0053] This invention also proposes a method for fabricating the microfluidic chip 1000, the method comprising the following steps: performing a metal or alloy coating patterning treatment on the substrate surface of the first chip 1; then etching an etching trench with a first depth, the first depth ranging from 500 nm to 3000 nm, on the surface; then sequentially sputtering an adhesion layer and electrode material to form the detection electrode 11 and attaching the detection electrode 11 to the etching trench; subsequently, patterning an insulating layer on the electrode 12 material to block the detection end 111 and information end 112 of the detection electrode 11, preventing the detection end 111 and the information end 112 from being mistakenly covered during subsequent covering of the insulating layer, further improving the flow rate. An insulating layer is coated on the detection electrode 11 by plasma-enhanced chemical vapor deposition. The photoresist at the detection end 111 and the information end 112 is stripped by gas etching to expose the detection end 111 and the information end 112, facilitating the acquisition of cell-related data and connection to external devices, thus forming the first chip 1. The substrate of the second chip 2 is photolithographically or mechanically etched to form the inlet and outlet 213 and the microfluidic channel 21, wherein the unetched portion of the microfluidic channel 21 forms the barrier structure, thus forming the second chip 2. The first chip 1 and the second chip 2 are then bonded together to prepare the microfluidic chip 1000.

[0054] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention's specification and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A microfluidic chip for detecting high-throughput intrinsic electrical parameters of adherent single cells, characterized in that, The microfluidic chip includes: A first chip has detection electrodes on its surface. Each detection electrode includes a detection end and an information end. The detection end has multiple electrode units, each used to detect the intrinsic electrical parameters of a single adherent cell. The information end is used to transmit the electrical signal from the detection end to the outside. The second chip is attached to the surface of the first chip. The second chip has a microfluidic channel. The microfluidic channel is provided with an inlet, a cell detection area and an outlet in sequence. The inlet is used to introduce cell suspension into the microfluidic channel. Multiple cell blocking units are formed in the cell detection area. Each cell blocking unit is used to block and retain a single cell. Each cell blocking unit is opposite to each electrode unit in the vertical direction. The outlet is used to discharge the cell suspension.

2. The microfluidic chip as described in claim 1, characterized in that, The microfluidic channel includes a drainage channel segment located between the inlet and the cell detection zone. The drainage channel segment has a drainage structure on the side adjacent to the cell detection zone, the drainage structure being used to guide cells into each of the cell barrier units; and / or The surface of the first chip is further provided with a counter electrode to form a circuit with the detection electrode, and the distance between the counter electrode and the detection end is [missing information]. ,in, .

3. The microfluidic chip as described in claim 1, characterized in that, The microfluidic channel has a vertical height of [missing information]. The maximum cell size is ,in, ; and / or, The thickness of the detection electrode is ,in, .

4. The microfluidic chip as described in claim 1, characterized in that, The cell blocking unit includes two protruding blocks, with one end of the two blocks adjacent to each other and their opposite side portions inclined away from each other to form a V-groove between the two blocks.

5. The microfluidic chip as described in claim 4, characterized in that, The distance between the nearest ends of the two stops is , .

6. The microfluidic chip as described in claim 1, characterized in that, The surface of the first chip has etched grooves for mounting the detection electrodes.

7. The microfluidic chip as described in claim 6, characterized in that, The surface of the first chip is provided with an adhesion layer to adhere the detection electrode to the etching groove, wherein the thickness of the adhesion layer is [missing information]. , ; and / or, The depth of the etching groove is ,in, .

8. The microfluidic chip as described in claim 1, characterized in that, The surface of the first chip is provided with an insulating layer, which covers the detection electrode and exposes the detection end and the information end.

9. The microfluidic chip as described in claim 8, characterized in that, The thickness of the insulating layer is ,in, .

10. A method for fabricating a microfluidic chip as described in any one of claims 1 to 9, characterized in that, Includes the following steps: The substrate surface of the first chip is patterned with a metal or alloy coating. An etching groove with a first depth is etched on the substrate surface; The adhesive layer and electrode material are sputtered sequentially; The electrode material is patterned with an insulating layer to block the detection end and information end of the detection electrode; Insulating layers were prepared by plasma-enhanced chemical vapor deposition; The photoresist at the detection end and information end of the detection electrode is stripped to form the first chip; The inlet and outlet of the liquid are mechanically etched on the substrate of the second chip, and microfluidic channels and barrier structures are etched to form the second chip; The first chip and the second chip are bonded together to form a microfluidic chip.

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