A micro-well array for single-cell impedance testing, its preparation method and application

Abstract

The present invention provides a micro-well array for single-cell impedance testing, its preparation method and application, belonging to the technical field of single-cell detection; the micro-well array includes a substrate, on the surface of the substrate, a patterned electrode pair is deposited, and a hydrophobic insulating layer is covered on the electrode pair; micro-wells with upward openings are fixed on the hydrophobic insulating layer; a pair of electrodes is covered on the bottom of each micro-well; the material of the micro-well is a hydrophilic material. After the micro-well array of the present invention is immersed in the cell solution, due to the hydrophobicity of the hydrophobic insulating layer and the hydrophilicity of the micro-well, no liquid droplets remain in the part outside the micro-well. At the same time, because the micro-well is relatively strong and the liquid has surface tension, the micro-droplets containing cells will remain in the micro-well, realizing large-scale sorting of single cells. By connecting the electrodes to an impedance analyzer and performing tests in the present invention, the impedance of a single cell can be obtained. By reasonable wiring, the impedance of the corresponding cell in any certain micro-well can be measured.

Description

Technical Field

[0001] The present invention belongs to the technical field of single-cell detection, and particularly relates to a micro-basin array for single-cell impedance testing, a preparation method thereof, and an application thereof. Background Art

[0002] Due to the heterogeneity among cells, the results obtained from the average effects of population cells in many biological studies cannot provide sufficiently useful information. Therefore, single-cell research has become a hot topic in recent years.

[0003] In the diagnosis of many cancers, it has been found that the "key cells" in diseased tissues have strong proliferation and differentiation abilities, and some can also metastasize, which is one of the important reasons for the deterioration of the disease. Therefore, it is of great significance to study the biological characteristics such as proliferation, differentiation, and migration of cell states at the single-cell level. Some researchers have proved that by using cell impedance technology, the cell state can be monitored and studied at the single-cell level. Due to the non-invasive, non-labeling nature of the technology itself and the ability to monitor the state for a long time, single-cell research using cell impedance technology has unique advantages.

[0004] To achieve single-cell impedance detection, precise manipulation of a single cell is generally required, and a single living cell is efficiently and non-destructively captured on an electrode, allowing the cell to adhere and grow (i.e., attach to the wall) on the electrode, and then the cell impedance is detected to monitor the cell state. Although the prior art has achieved single-cell impedance detection, the detection throughput is small and the detection efficiency is low. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a micro-basin array for single-cell impedance testing, a preparation method thereof, and an application thereof. Based on the micro-basin array of the present invention, high-throughput single-cell impedance detection can be achieved, and the detection efficiency is high.

[0006] The present invention provides a micro-basin array for single-cell impedance testing, including a substrate 1. A patterned plurality of pairs of electrode pairs 2 are deposited on the surface of the substrate 1. A hydrophobic insulating layer 3 is covered on the electrode pairs 2. A plurality of micro-basins 4 with upward openings are fixed on the hydrophobic insulating layer 3. A pair of the electrode pairs 2 is covered on the bottom of each micro-basin 4. The material of the micro-basin is a hydrophilic material. The diameter of the micro-basin 4 is 1.2 to 1.5 times the diameter of the cell.

[0007] Preferably, a number of pairs of electrode pairs 2 distributed in pairs are fixed on the surface of the substrate 1. The electrode pairs 2 are arranged in a matrix and the corresponding micro-basins are selected through row selection lines and column selection lines.

[0008] Preferably, the material of the micro-basin 4 is a hydrophilic material.

[0009] Preferably, the material of the micro-basin 4 includes silicon dioxide, silver or platinum.

[0010] The present invention also provides a method for preparing the micro-basin array described in the above solution, including the following steps:

[0011] 1) Fabricate patterned electrode pairs 2 on the substrate 1;

[0012] 2) Cover the electrode pairs 2 with a hydrophobic material to form a hydrophobic insulating layer 3;

[0013] 3) Fix the micro-basins 4 at positions corresponding to the electrode pairs 2 on the hydrophobic insulating layer 3 to obtain a micro-basin array.

[0014] The present invention also provides a device for single-cell impedance testing, including the micro-basin array described in the above solution or the micro-basin array prepared by the preparation method described above and an impedance analyzer; the impedance analyzer is electrically connected to the electrode pairs 2.

[0015] The present invention also provides the application of the micro-basin array described in the above solution, or the micro-basin array prepared by the preparation method described above, or the device described above in single-cell rapid sorting and / or single-cell impedance testing.

[0016] Preferably, the single-cell impedance testing includes the following steps:

[0017] 1) Immerse the micro-basin array in a solution containing cells and then take it out;

[0018] 2) Connect each pair of electrodes to the impedance analyzer and perform testing to obtain the impedance of a single cell.

[0019] The present invention provides a micro-basin array for single-cell impedance testing, including a substrate 1, on the surface of which patterned multiple pairs of electrode pairs 2 are deposited, and a hydrophobic insulating layer 3 is covered on the electrode pairs 2; multiple micro-basins 4 with upward openings are fixed on the hydrophobic insulating layer 3; the bottom of each micro-basin 4 covers a pair of the electrode pairs 2; the material of the micro-basin is a hydrophilic material; the diameter of the micro-basin 4 is 1.2 - 1.5 times the diameter of the cell. After immersing the micro-basin array of the present invention in a cell solution, due to the hydrophobicity of the hydrophobic insulating layer 3 and the hydrophilicity of the micro-basin 4, no liquid droplets remain outside the micro-basin. At the same time, because the liquid has surface tension, the micro-droplets containing cells will remain in the micro-basin, and because the size of the micro-basin 4 matches the cell size, single cells remain in the micro-basin 4, realizing large-scale sorting of single cells. By connecting the electrodes on the micro-basin array to an impedance analyzer and performing testing, the impedance of a single cell can be obtained. Description of the Drawings

[0020] Figure 1The preparation flow chart of the micro-well array of the present invention, where 1 - substrate, 2 - electrode, 3 - hydrophobic insulating layer, 4 - micro-well;

[0021] Figure 2 The flow chart of taking out the micro-well array immersed in a solution containing cells in the present invention;

[0022] Figure 3 The schematic diagram of single-cell impedance measurement in the present invention;

[0023] Figure 4 The schematic diagram of synchronously measuring the cell impedance in the micro-well by row selection and column selection in the present invention;

[0024] Figure 5 The flow schematic diagram of Example 1, where A represents the treatment in Step 1 of Example 1, B represents the treatment in Step 2 of Example 1, C represents the treatment in Step 3 of Example 1, D represents the treatment in Step 4 of Example 1, and E represents the treatment in Step 5 of Example 1;

[0025] Figure 6 The flow schematic diagram of Example 2, where A represents the treatment in Step 1 of Example 2, B represents the treatment in Step 2 of Example 2, C represents the treatment in Step 3 of Example 2, D represents the treatment in Step 4 of Example 2, E represents the treatment in Step 5 of Example 2, and F represents the treatment in Step 6 of Example 2. Detailed implementation manners

[0026] The present invention provides a micro-well array for single-cell impedance measurement, including a substrate 1, on the surface of which patterned multiple pairs of electrode pairs 2 are deposited, and a hydrophobic insulating layer 3 is covered on the electrode pairs 2; multiple micro-wells 4 with upward openings are fixed on the hydrophobic insulating layer 3; the bottom of each micro-well 4 is covered with a pair of the electrode pairs 2; the material of the micro-well is a hydrophilic material; the diameter of the micro-well 4 is 1.2 - 1.5 times the diameter of the cell.

[0027] In the present invention, the substrate 1 preferably includes an insulating sheet; the insulating sheet preferably includes a glass sheet, a quartz sheet or a silicon oxide sheet; the present invention has no special limitation on the thickness of the substrate 1. In the specific implementation process of the present invention, when the substrate 1 is a silicon wafer, the thickness of the substrate 1 is preferably 200 - 500 μm, more preferably 300 - 400 μm; when the substrate 1 is a glass sheet, the thickness of the substrate 1 is preferably 200 - 5000 μm.

[0028] In the present invention, a number of pairwise distributed electrode pairs 2 are fixed on the surface of the substrate 1; the electrode pairs 2 are arranged in a matrix, and the corresponding micro-wells are selected through row selection lines and column selection lines.

[0029] In the present invention, the material of the hydrophobic insulating layer 3 is preferably silicon dioxide; the thickness of the hydrophobic insulating layer 3 is preferably 50 - 150 nm, more preferably 100 nm; the surface of the insulating layer is made hydrophobic by alkylation treatment.

[0030] In the present invention, the diameter of the micro-basin 4 is 1.2 - 1.5 times the diameter of the cell; the inner diameter of the micro-basin 4 is preferably 8 - 12 μm, more preferably 10 μm. In the present invention, the material of the micro-basin 4 is a hydrophilic material; the material of the micro-basin 4 includes silicon dioxide, silver or platinum.

[0031] In the present invention, there is no special limitation on the spacing between two adjacent micro-basins 4, preferably 15 - 25 μm, more preferably 20 μm.

[0032] The present invention also provides a method for preparing the micro-basin array described in the above solution, including the following steps:

[0033] 1) Fabricate a patterned electrode pair 2 on the substrate 1;

[0034] 2) Cover the electrode pair 2 with a hydrophobic material to form a hydrophobic insulating layer 3;

[0035] 3) Fix the micro-basins 4 at the positions corresponding to the electrode pair 2 on the hydrophobic insulating layer 3 to obtain a micro-basin array.

[0036] For the preparation flow chart of the micro-basin array of the present invention, see Figure 1 .

[0037] The present invention first fabricates a patterned electrode pair 2 on the substrate 1. The present invention has no special limitation on the method of fabricating the patterned electrode pair 2, and the conventional methods of photolithography, evaporation and stripping in the art can be used.

[0038] After forming the electrode pair 2, the present invention covers the electrode pair 2 with a hydrophobic material to form a hydrophobic insulating layer 3. The present invention has no special limitation on the method of covering the hydrophobic material, and the conventional methods of covering the hydrophobic material in the art can be used.

[0039] After obtaining the hydrophobic insulating layer 3, the present invention fixes the micro-basins 4 at the positions corresponding to the electrode pair 2 on the hydrophobic insulating layer 3 to obtain a micro-basin array. The present invention has no special limitation on the method of fixing the micro-basins, and the conventional methods of photolithographic development, sputtering and stripping in the art can be used.

[0040] The present invention also provides a device for single-cell impedance testing, including the micro-basin array described in the above solution or the micro-basin array prepared by the preparation method described above and an impedance analyzer; the impedance analyzer is electrically connected to the electrode pair 2.

[0041] The present invention also provides an application of the micro-well array described in the above solution, or the micro-well array prepared by the preparation method, or the device in single-cell rapid sorting and / or single-cell impedance testing.

[0042] In the present invention, the single-cell impedance testing includes the following steps:

[0043] 1) Immerse the micro-well array in a solution containing cells and then take it out;

[0044] 2) Connect each pair of electrodes to an impedance analyzer and perform a test to obtain the impedance of a single cell.

[0045] For the flowchart of immersing the micro-well array in a solution containing cells and then taking it out in the present invention, see Figure 2.

[0046] In the present invention, first immerse the micro-well array in a solution containing cells and then take it out; there is no special limitation on the immersion depth in the present invention, and it is sufficient that the micro-wells are fully immersed.

[0047] Connect the electrodes to an impedance analyzer and perform a test to obtain the impedance of a single cell; the test includes synchronous testing or single testing. In the present invention, the impedance of cells in the micro-wells is synchronously tested by row selection and column selection. For a schematic diagram, see Figure 4 , taking Figure 4 as an example, the present invention can select any one micro-well in the array by selecting one row from rows 1 to 4 and one column from columns A to D, and measure the impedance of the cells therein.

[0048] For a schematic diagram of single-cell impedance testing in the present invention, see Figure 3 .

[0049] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention.

[0050] Example 1

[0051] 1. Take a quartz wafer with a length of 2 cm and a width of 1 cm, ultrasonically clean it with acetone and isopropanol for 5 minutes respectively, and dry it with nitrogen. For a schematic diagram, see A in Figure 5 .

[0052] 2. Fabricate patterned electrodes on the quartz wafer by photolithography, sputtering 10 nm of chromium and 60 nm of gold, and stripping. For a schematic diagram, see B in Figure 5 .

[0053] 3. Cover a hydrophobic layer on the electrodes by photolithography, sputtering 10 nm thick silicon oxide, and stripping. For a schematic diagram, see C in Figure 5 .

[0054] 4. Fix the micro-basins on the hydrophobic layer through photolithography, sputtering, and lift-off. See the schematic diagram in Figure 5 D in

[0055] 5. Perform wire bonding on the electrode sheet. See the schematic diagram in Figure 5 E in

[0056] Among them, the specific process of photolithography is as follows: Spin-coat AR-P5350 photoresist on the substrate using a spin coater CEEAPOGEE X-PRO II, with specific parameters of 500 rpm for 10 s and 4000 rpm for 60 s. Bake on a hot plate at 105 °C for 5 min. Use an ultraviolet lithography machine SUSS MA6 to expose through a mask for 2.5 s. Develop in a developer solution of AR300-26: H 2 O = 1:7 for 30 s. Dry with nitrogen.

[0057] The specific process of sputtering is as follows: Use a sputtering device DENTON DISCOVERY-635 to coat the sample, with specific parameters of chromium: DC 100 W for 60 s to 10 nm; gold: DC 200 W for 60 s to 60 nm; silicon oxide: RF 300 W for 720 s to 100 nm; platinum: DC 200 W for 200 s to 100 nm; silver: DC 40 W for 278 s to 100 nm.

[0058] The specific process of lift-off is as follows: Use acetone to wash off the photoresist on the substrate sputtered with materials, leaving the patterned materials.

[0059] For the specific process of fixing the micro-basins, see (Tian F, Li M, Pu D, et al. A Versatile and Environmentally Friendly Microfabrication Process for Producing Micro-Basin Array for Single Cell Analysis[C] / / 2021 IEEE 34th International Conference on Micro Electro Mechanical Systems (MEMS). IEEE, 2021).

[0060] Example 2

[0061] 1. Take a quartz sheet with a length of 2 cm and a width of 2 cm, ultrasonically clean it with acetone and isopropyl alcohol for 5 min respectively, and dry it with nitrogen. See the schematic diagram in Figure 6 A in

[0062] 2. Fabricate the patterned electrodes on the quartz wafer by photolithography, sputtering 10 nm of chromium and 60 nm of gold, and lift-off. See the schematic diagram in Figure 6 B in

[0063] 3. Define the shape of the isolation part of silicon oxide by photolithography. See the schematic diagram in Figure 6 C in

[0064] 4. Define the electrode shape by photolithography. See the schematic diagram in Figure 6 D in

[0065] 5. Hydrophobic layer + protective electrode, photolithography, sputter silicon oxide. See the schematic diagram in Figure 6 E in

[0066] 6. Fix the micro-basin on the hydrophobic layer by photolithography development, sputtering, and lift-off. See the schematic diagram in Figure 6 F in

[0067] 7. Perform wire bonding on the electrodes.

[0068] The specific processes of photolithography, sputtering, lift-off, and micro-basin fixation are the same as those in Embodiment 1.

[0069] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention rather than all of them. People can also obtain other embodiments according to this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a micro-well array for single-cell impedance testing or a device for single-cell impedance testing in single-cell rapid sorting and / or single-cell impedance testing; The single-cell impedance testing comprises the following steps: 1) Immerse the micro-well array in a solution containing cells and then take it out; 2) Connect each pair of electrodes to an impedance analyzer and conduct a test to obtain the impedance of a single cell; The micro-well array includes a substrate (1), on the surface of which patterned multiple pairs of electrode pairs (2) are deposited, and a hydrophobic insulating layer (3) is covered on the electrode pairs (2); a plurality of micro-wells (4) with upward openings are fixed on the hydrophobic insulating layer (3), and the bottom of each micro-well (4) is covered with a pair of the electrode pairs (2); the material of the micro-well is a hydrophilic material; the diameter of the micro-well (4) is 1.2 - 1.5 times the diameter of the cell; The device for single-cell impedance testing includes the micro-well array and an impedance analyzer; the impedance analyzer is electrically connected to the electrode pairs (2).

2. The application according to claim 1, characterized in that the electrode pairs (2) are arranged in a matrix, and corresponding micro-wells are selected through row selection lines and column selection lines.

3. The application according to claim 1, characterized in that the material of the micro-well (4) includes silicon dioxide, silver or platinum.

4. The application according to claim 1, characterized in that the preparation method of the micro-well array comprises the following steps: 1) Fabricate patterned electrode pairs (2) on the substrate (1); 2) Cover a hydrophobic material on the electrode pairs (2) to form a hydrophobic insulating layer (3); 3) Fix micro-wells (4) at positions corresponding to the electrode pairs (2) on the hydrophobic insulating layer (3) to obtain a micro-well array.

Images