CN112316993B - Method for acquiring single cell array - Google Patents
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- CN112316993B CN112316993B CN202011190716.4A CN202011190716A CN112316993B CN 112316993 B CN112316993 B CN 112316993B CN 202011190716 A CN202011190716 A CN 202011190716A CN 112316993 B CN112316993 B CN 112316993B
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Abstract
The invention discloses a method for acquiring a single cell array, which specifically comprises the following steps: (1) preparing an ultrathin through hole metal sheet, and cleaning the ultrathin through hole metal sheet for later use; (2) tightly attaching the ultrathin through hole metal sheet to a substrate, and pressing until no bubbles exist; (3) extracting cell suspension, placing the cell suspension in the through hole area of the ultrathin through hole metal sheet, and standing in an incubator; (4) and (3) pumping out the cell suspension, washing the through hole area by using a buffer solution, and then taking down the ultrathin through hole metal sheet to obtain the single cell array. The method does not need bonding or fixture fixing, does not need a sample pool or centrifugation, has simple operation and high efficiency, and provides great help for large-area acquisition of the single cell array; and the raw materials of the ultrathin through hole metal sheet are widely and easily obtained, the processing cost is low, and the ultrathin through hole metal sheet can be subjected to cleaning steps such as ultrasonic cleaning, organic cleaning, boiling cleaning, drying cleaning and the like, and can be repeatedly utilized, so that the cost is reduced.
Description
Technical Field
The invention relates to the technical field of biochips, in particular to a method for acquiring a single cell array.
Background
Data obtained by conventional cell detection methods are based on an average of a large number of cells, and individual cell-specific information obtained by conventional cell detection methods is often considered "erroneous" or "noise" and is ignored or discarded. In modern biomedical research, especially in the study of tumors, conventional cell detection methods often fail to meet the requirements for accurate analysis of cell signals due to cellular heterogeneity.
Single-cell analysis (Single-cell analysis) is an analysis method which focuses on researching the unique response of Single cells to different stimulation signals, overcomes the defects of the traditional cell detection method, and has important significance for the research of Single-cell individuals, the early diagnosis of diseases, the personalized medical treatment and the like.
Single cell research based on hydrodynamic characteristics becomes a current research hotspot due to the characteristics of relative economy, high efficiency, small damage to cells and the like. The existing single cell research based on the hydrodynamic characteristics has the problems of low flux, potential influence on cells (shear stress and the like), high processing precision requirement, high cell loss, short duration (generally less than 24h) and the like. The key factors restricting the development of single cell analysis are the compromise between high throughput and cell capture efficiency, the influence of shear stress on cell activity and other factors.
The through hole chip reported in the past is generally prepared by a micro-nano processing method by using SU8 or PDMS (polydimethylsiloxane) material, and the thickness is generally 20-30 μm, and the through hole chip with the thickness has three problems: firstly, after being attached to a hard substrate, the substrate cannot be tightly attached and can easily slide left and right, so that the substrate is generally required to be bonded or fixed by a clamp; secondly, the thickness is equal to the size of a cell, and single cells cannot enter the hole through free sedimentation due to fluid resistance, so that the cells are driven to enter the deep hole by external force action such as centrifugation for a few minutes; and thirdly, the SU8 through-hole chip is very fragile and is very easy to crack under the action of external force, and secondly, the membrane is difficult to uncover after the SU8 chip is bonded, and PDMS is too soft and is very easy to deform and adhere when being clamped by tweezers.
Therefore, how to provide a method capable of rapidly and accurately acquiring a single cell array is a problem to be solved by those skilled in the art.
Disclosure of Invention
In view of this, the present invention aims to provide a method for obtaining a single cell array, which does not require bonding or fixture fixation, does not require a sample cell, does not require centrifugation, and has the advantages of simple operation, rapid preparation, high efficiency, cost saving, and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for acquiring a single cell array specifically comprises the following steps:
(1) preparing an ultrathin through hole metal sheet, and cleaning the ultrathin through hole metal sheet for later use;
(2) tightly attaching the ultrathin through hole metal sheet to a substrate, and pressing until no bubbles exist;
(3) extracting cell suspension, placing the cell suspension in the through hole area of the ultrathin through hole metal sheet, and standing in an incubator;
(4) and (3) pumping out the cell suspension, washing the through hole area by using a buffer solution, and then taking down the ultrathin through hole metal sheet to obtain the single cell array.
The invention has the advantages that the invention utilizes the special flexibility and rigidity characteristics of the ultrathin through hole metal sheet, can be directly and tightly attached to a substrate (flat substrate) for adherent cell growth like a 'film', and then realizes the adherent cell entrance by utilizing the natural sedimentation of the cells, thereby efficiently finishing the preparation of the high-flux single cell array.
The invention does not need a clamp, a sample pool, bonding and centrifugation, and the acquisition method has simple operation and high efficiency, and provides great help for large-area acquisition of the single cell array; and the raw materials of the ultrathin through hole metal sheet are widely and easily obtained, the processing cost is low, and the ultrathin through hole metal sheet can be subjected to cleaning steps such as ultrasonic cleaning, organic cleaning, boiling cleaning, drying cleaning and the like, and can be repeatedly utilized, so that the cost is reduced.
Further, in the step (2), a buffer layer is firstly paved on the substrate, and then the ultrathin through hole metal sheet is tightly attached to the buffer layer and is tightly pressed until no bubbles exist.
Further, in the step (2), a coating layer is formed on the substrate by using a coating substance, and then the ultrathin through hole metal sheet is tightly attached to the coating layer and is compressed until no bubbles exist.
Further, in the step (2), a buffer layer is firstly paved on the substrate, a coating layer is formed on the buffer layer by adopting a coating substance, and finally the ultrathin through hole metal sheet is tightly attached to the coating layer and is tightly pressed until no air bubbles exist.
Furthermore, the buffer layer is made of Polydimethylsiloxane (PDMS) or hydrogel.
The further technical scheme has the beneficial effects that materials such as polydimethylsiloxane, hydrogel and the like can be tightly attached to the surfaces of substrates such as glass and the like, so that the cell culture is facilitated, and the method is mainly suitable for other plane substrates which are not suitable for directly carrying out cell culture, such as substrates with flat surfaces, such as common glass sheets, metal, plastic and the like.
Further, the coating material is any one of adhesion promoting substances such as Fibronectin (FN), MSC adherence promoting agent, Polylysine (PLL), collagen, and aminopropyltriethoxysilane.
The substrate or the buffer layer which needs to obtain the single cell array is coated and incubated by adopting the cell adhesion promoting substance, different assembly modes and high adsorbability of the coating substance are effectively combined, so that the single cells can be more quickly adsorbed by the coating substance, the adherence time of the cells is greatly shortened (shortened from 1-2h to 20-30min), and the success rate of the single cell array is improved to a certain extent.
Further, in the step (1), the preparation method of the ultrathin through hole metal sheet is laser drilling or micromachining.
The laser drilling method adopted by the further technical scheme has the advantages of high drilling speed, high efficiency, good economic benefit and the like, the prepared ultrathin through hole metal sheet has smaller depth-diameter ratio and high flux, parameters such as metal sheet patterns, hole spacing, aperture density and the like can be adjusted, the automation degree is high, the factory operation is simple and easy to realize, and the acquisition quality of the single cell array is fundamentally ensured.
Furthermore, the thickness of the ultrathin through hole metal sheet is less than or equal to 10 μm, the aperture is 10-25 μm (the diameter of 1 cell is less than the aperture of 2 cells), the hole spacing is 80-100 μm (the hole spacing is greater than 2 hole diameters), and the shape is arbitrary (such as rectangle); preferably, the ultra-thin via metal sheet has a thickness of 5 μm, a pore diameter of 25 μm, and a pore pitch of 100 μm. In addition, when the aperture of the ultrathin through hole metal sheet is 25-50 μm, the multi-cell array can be obtained.
The ultrathin through hole metal sheet has the advantages that the ultrathin through hole metal sheet is simple in structure and accurate in size, cells can be ensured to fall in each through hole individually, and the area of a single cell array is greatly increased; more importantly, the thickness of the ultrathin through hole metal sheet is far smaller than the size of a single cell, and the fluid resistance is extremely low, so that cells can naturally settle into the hole without being driven into the hole by external forces such as centrifugation and the like.
Further, in the step (1), the specific operation of cleaning is as follows: firstly, placing the ultrathin through hole metal sheet in absolute ethyl alcohol for ultrasonic treatment, then sequentially using absolute ethyl alcohol and deionized water for cleaning, finally using nitrogen for blow-drying, and carrying out high-pressure or ultraviolet sterilization; wherein the power of ultrasonic treatment is 0.3-0.5W/cm2The temperature is 20-100 deg.C, and the time is at least 5 min.
The beneficial effect of adopting above-mentioned further technical scheme lies in, ultra-thin through-hole sheetmetal can get rid of pollutants such as particulate matter, greasy dirt on the sheetmetal surface after the clearance, and further high pressure or ultraviolet sterilization are favorable to the aseptic culture of cell. In addition, after the steps of ultrasonic cleaning and sterilization, the ultrathin through hole metal sheet can be repeatedly utilized, so that the experiment repetition is facilitated, and the cost is further reduced.
Further, in the step (2), the ultra-thin metal sheet with through holes may be fixed by a magnet, a jig, an adhesive layer, or other means, instead of being naturally pressed.
Further, in the step (3), the standing time is 20-120min, preferably 20-60min, and more preferably 30 min; the temperature in the cell culture box is 36-38, preferably 37 ℃; the concentration of carbon dioxide is 4% -6%, preferably 5%.
The further technical scheme has the beneficial effects that after the incubator is kept still for a period of time, the cells can naturally settle into the holes under the action of gravity and further adhere to the culture medium. For the substrate without coating, the standing time of the cells generally needs 60-120min to ensure the effective adhesion of the cells and the substrate; for the coated substrate, the standing time of the cells is generally 20-30min, and the effective adhesion of the cells and the substrate can be ensured. Conversely, too long a time may result in stacking adhesion between cells.
In addition, in the step (3), the cell suspension can be combined with a high-speed centrifugal device except for adopting a natural sedimentation (standing) mode; alternatively, the single cell array may be accessed by replacing other devices, such as by using a vacuum pressurizing device.
Further, in the step (4), the buffer solution is any one of Phosphate Buffered Saline (PBS), Hankel's Balanced Salt Solution (HBSS) and Dui's Phosphate Buffered Saline (DPBS); the number of washes was 2-3.
The technical scheme has the beneficial effects that after the ultrathin through hole metal sheet is kept stand for a certain time, the buffer solution is used for slightly washing the upper layer of the ultrathin through hole metal sheet, so that the cells which are not adhered to the substrate on the upper layer are washed away, and the interference of the cells on the single cell array after the sheet is taken off in the next step can be reduced. It is noted that excessive washing may also wash out cells in the wells, reducing the efficiency of single cell preparation.
According to the technical scheme, compared with the prior art, the invention has the following beneficial effects:
1. the ultrathin through hole metal sheet adopted by the invention has flexibility and rigidity, can be clamped by tweezers without deformation, can be tightly attached to the substrate without sliding, has strong pressure resistance, and can not damage and deform under the action of the clamp; and can carry on washing steps such as supersound, organic, boiling, oven drying, therefore can reuse;
2. the surface of the ultrathin through hole metal sheet adopted by the invention has certain hydrophobicity, the contact angle is larger, and the cell suspension can keep a liquid drop shape after being dripped into the through hole area, so that the cell suspension cannot flow away from the through hole area; in addition, a centrifugation step is not needed, so that the invention does not need to additionally increase a sample loading area of a sample cell for limiting the sample, namely, a clamp is not needed, bonding is not needed, centrifugation is not needed, and the sample cell is not needed;
3. because of the limitation of the thickness of the metal sheet of the ultrathin through hole, the fluid resistance is very small, and cells can be easily deposited into the through hole to adhere to the wall without external forces such as centrifugation and the like when going into the through hole;
4. the invention effectively combines different assembly modes with high adsorbability of the coating material, so that the single cells can be more quickly adsorbed by the coating material, thereby greatly increasing the area of the single cell array and improving the experimental efficiency;
5. the ultrathin through hole metal sheet selected by the invention has the advantages of wide and easily available raw materials and low processing cost, can be subjected to cleaning steps such as ultrasonic cleaning, organic cleaning, boiling cleaning, drying cleaning and the like, and can be recycled, so that the cost is reduced.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a side view of an assembled chip device (substrate + ultra-thin via metal sheet) according to example 1 of the present invention;
FIG. 2 is a side view of an assembled chip device (substrate + buffer layer + ultra-thin via metal sheet) according to example 2 of the present invention;
FIG. 3 is a side view of an assembled chip device (substrate + coating layer + ultra-thin via metal sheet) according to example 3 of the present invention;
FIG. 4 is a side view of an assembled chip device (substrate + buffer layer + coating layer + ultra-thin via metal sheet) according to example 4 of the present invention;
FIG. 5 is a schematic structural diagram of an ultra-thin via metal sheet according to example 4 of the present invention;
FIG. 6 is a diagram illustrating the capturing effect of the single cell array in example 4 of the present invention.
The cell structure comprises a substrate 1, an ultrathin through hole metal sheet 2, a cell 3, a buffer layer 4 and a coating layer 5.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following examples, the preparation method of the buffer layer was: weighing 100g of Dow Corning A in a paper cup by using an electronic balance, sucking the Dow Corning B by using a disposable dropper, dripping 10g of Dow Corning B, uniformly stirring, slowly pouring the Dow Corning B onto a glass dish, waiting until the Dow Corning B is completely spread and has no bubbles, horizontally putting the glass dish into an oven at 80 ℃ for baking, taking out the glass dish after one hour, and cutting the polydimethylsiloxane without bubbles for later use.
Example 1
The method for acquiring the single cell array (the substrate and the ultrathin through hole metal sheet) specifically comprises the following steps:
(1) preparing an ultrathin through hole metal sheet with the thickness of 5 microns, the aperture of 10 microns and the hole spacing of 80 microns by adopting laser drilling, and cleaning the through hole area for later use; wherein, the specific operation of clearance is: firstly, the ultra-thin through hole metal sheet is placed in absolute ethyl alcohol for ultrasonic treatment, and the power is 0.3W/cm2Sequentially cleaning with anhydrous ethanol and deionized water at 20 deg.C for 5min, blow-drying with nitrogen gas, autoclaving, and observing whether the via hole region is completely cleaned with microscope, or cleaning according to the above steps;
(2) tightly attaching the ultrathin through hole metal sheet to a culture dish, and pressing until no bubbles exist;
(3) extracting 400 mu L of cell suspension prepared in advance by using a pipette, placing the cell suspension in the through hole area of the ultrathin through hole metal sheet to enable the cell suspension to be level with the upper surface of the ultrathin through hole metal sheet, and placing the cell suspension in an incubator for standing for 2 hours at the temperature of 36 ℃ and the carbon dioxide concentration of 4%;
(4) taking out, removing cell suspension, washing the through hole region with Hankel's balanced salt solution for 2 times, marking the position of the through hole region at the bottom of the culture dish for observation under a microscope, and finally slowly taking down the ultrathin through hole metal sheet by using a pair of tweezers to obtain the single cell array.
Example 2
The method for acquiring the single cell array (the substrate, the buffer layer and the ultrathin through hole metal sheet) specifically comprises the following steps:
(1) preparing an ultrathin through hole metal sheet with the thickness of 5 microns, the aperture of 25 microns and the hole spacing of 80 microns by adopting micromachining, and cleaning a through hole area for later use; wherein, the specific operation of clearance is: firstly, the ultra-thin through hole metal sheet is placed in absolute ethyl alcohol for ultrasonic treatment, and the power is 0.3W/cm2Sequentially cleaning with anhydrous ethanol and deionized water at 30 deg.C for 8min, blow-drying with nitrogen gas, autoclaving, and observing whether the via hole region is completely cleaned with microscope, or cleaning according to the above steps;
(2) firstly, paving a polydimethylsiloxane buffer layer on a culture dish, tightly attaching an ultrathin through hole metal sheet to the polydimethylsiloxane buffer layer, and pressing until no air bubbles exist;
(3) extracting 400 mu L of cell suspension prepared in advance by using a pipette, placing the cell suspension in the through hole area of the ultrathin through hole metal sheet to enable the cell suspension to be level with the upper surface of the ultrathin through hole metal sheet, and placing the cell suspension in an incubator for standing for 1h at the temperature of 36 ℃ and the carbon dioxide concentration of 6%;
(4) taking out, removing cell suspension, washing the through hole region for 3 times by using a Du's phosphate buffer solution, marking the position of the through hole region at the bottom of the culture dish to facilitate observation under a microscope, and finally, slowly taking down the ultrathin through hole metal sheet and the polydimethylsiloxane buffer layer by using tweezers in sequence to obtain the single cell array.
Example 3
The method for acquiring the single cell array (substrate + coating layer + ultrathin through hole metal sheet) specifically comprises the following steps:
(1) preparing an ultrathin through hole metal sheet with the thickness of 5 microns, the aperture of 25 microns and the hole spacing of 100 microns by adopting laser drilling, and cleaning the through hole area for later use; wherein, the specific operation of clearance is: firstly, the ultra-thin through hole metal sheet is placed in absolute ethyl alcohol for ultrasonic treatment, and the power is 0.4W/cm2At 40 deg.C for 10min, sequentially cleaning with anhydrous ethanol and deionized water, blow-drying with nitrogen gas, ultraviolet sterilizing, and observing with microscopeInspecting whether the through hole area is completely cleaned or not, and otherwise, continuously cleaning according to the steps;
(2) using a pipette to transfer the melted adhesive protein into a culture dish and completely cover the same, then pumping out the residual adhesive protein, forming a coating layer on the culture dish, tightly attaching the ultrathin through hole metal sheet to the coating layer, and pressing until no bubbles exist;
(3) extracting 400 μ L of prepared cell suspension with a pipette, placing in the through hole region of the ultrathin through hole metal sheet to make the cell suspension level with the upper surface of the ultrathin through hole metal sheet, and standing in an incubator at 38 deg.C for 30min with carbon dioxide concentration of 4%;
(4) taking out, removing cell suspension, washing the through hole region for 3 times by using a Du's phosphate buffer solution, marking the position of the through hole region at the bottom of the culture dish to facilitate observation under a microscope, and finally slowly taking down the ultrathin through hole metal sheet by using a pair of tweezers to obtain the single cell array.
Example 4
The method for acquiring the single cell array (substrate + buffer layer + coating layer + ultrathin through hole metal sheet) specifically comprises the following steps:
(1) preparing an ultrathin through hole metal sheet with the thickness of 5 microns, the aperture of 25 microns and the hole spacing of 100 microns by adopting laser drilling, and cleaning the through hole area for later use; wherein, the specific operation of clearance is: firstly, the ultra-thin through hole metal sheet is placed in absolute ethyl alcohol for ultrasonic treatment, and the power is 0.5W/cm2Sequentially cleaning with anhydrous ethanol and deionized water at 100 deg.C for 15min, blow-drying with nitrogen gas, ultraviolet sterilizing, and observing whether the via hole region is completely cleaned with microscope, or cleaning according to the above steps;
(2) firstly, a polydimethylsiloxane buffer layer is paved on a substrate, then a liquid-moving gun is used for moving the melted adhesive protein on the polydimethylsiloxane buffer layer and completely covering the polydimethylsiloxane buffer layer, then the rest adhesive protein is extracted, a coating layer is formed on the polydimethylsiloxane buffer layer, and finally the ultrathin through hole metal sheet is tightly attached to the coating layer and is tightly pressed until no bubbles exist;
(3) extracting 400 μ L of prepared cell suspension with a pipette, placing in the through hole region of the ultrathin through hole metal sheet to make the cell suspension level with the upper surface of the ultrathin through hole metal sheet, and standing in an incubator at 37 deg.C for 20min with carbon dioxide concentration of 5%;
(4) taking out, removing the cell suspension, washing the through hole region for 3 times by using a phosphate buffer solution, marking the position of the through hole region at the bottom of the culture dish to facilitate observation under a microscope, and finally, slowly taking down the ultrathin through hole metal sheet and the polydimethylsiloxane buffer layer by using tweezers in sequence to obtain the single cell array.
As can be seen from FIGS. 5 and 6, the high-throughput single-cell array is effectively obtained on the substrate, and the array pattern is equivalent to the pattern of the original ultrathin through-hole metal chip.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. A method for acquiring a single cell array is characterized by comprising the following steps:
(1) preparing an ultrathin through hole metal sheet, and cleaning the ultrathin through hole metal sheet for later use;
the preparation method of the ultrathin through hole metal sheet is laser drilling or micromachining, the thickness of the ultrathin through hole metal sheet is less than or equal to 10 microns, and the aperture is 10-25 microns;
(2) tightly attaching the ultrathin through hole metal sheet to a substrate, and pressing until no bubbles exist;
(3) extracting cell suspension, placing the cell suspension in the through hole area of the ultrathin through hole metal sheet, and standing in an incubator;
(4) and (3) pumping out the cell suspension, washing the through hole area by using a buffer solution, and then taking down the ultrathin through hole metal sheet to obtain the single cell array.
2. The method for obtaining a single cell array as claimed in claim 1, wherein in step (2), a buffer layer is first laid on the substrate, and then the ultra-thin via metal sheet is tightly attached to the buffer layer and compressed until no air bubbles are formed.
3. The method for obtaining a single cell array as claimed in claim 1, wherein in step (2), a coating layer is formed on the substrate by using a coating material, and then the ultra-thin via metal sheet is tightly attached to the coating layer and pressed until no bubble is formed.
4. The method for obtaining a single cell array as claimed in claim 1, wherein in the step (2), a buffer layer is first laid on the substrate, a coating layer is formed on the buffer layer by using a coating material, and finally the ultra-thin via metal sheet is tightly attached to the coating layer and is compressed until no bubbles are formed.
5. The method for obtaining a single cell array according to claim 2 or 4, wherein the buffer layer is polydimethylsiloxane or hydrogel.
6. The method of claim 3 or 4, wherein the coating material is any one of fibronectin, MSC adherence promoting agent, polylysine, collagen and aminopropyltriethoxysilane.
7. The method for acquiring a single cell array according to any one of claims 1 to 4, wherein in step (1), the specific operation of cleaning is: firstly, placing the ultrathin through hole metal sheet in absolute ethyl alcohol for ultrasonic treatment, then sequentially using absolute ethyl alcohol and deionized water for cleaning, finally using nitrogen for blow-drying, and carrying out high-pressure or ultraviolet sterilization; wherein the power of ultrasonic treatment is 0.3-0.5W/cm2, the temperature is 20-100 deg.C, and the time is at least 5 min.
8. The method for acquiring the single cell array according to any one of claims 1 to 4, wherein in the step (3), the standing time is 20 to 120min, the temperature is 36 to 38 ℃, and the concentration of carbon dioxide is 4 to 6 percent.
9. The method for obtaining a single cell array according to any one of claims 1 to 4, wherein in step (4), the buffer solution is any one of phosphate buffer, Hankel's balanced salt solution and Du's phosphate buffer.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2851418A1 (en) * | 2012-05-14 | 2015-03-25 | Hitachi Chemical Company, Ltd. | Cancer cell-trapping metal filter, cancer cell-trapping metal filter sheet, cancer cell-trapping device, and manufacturing methods therefor |
| CN107118938A (en) * | 2017-04-07 | 2017-09-01 | 中北大学 | The unicellular arrangement of fluid enhancing dielectrophoresis and control chip and preparation method thereof |
| CN109852530A (en) * | 2019-03-29 | 2019-06-07 | 中国科学院上海微系统与信息技术研究所 | A kind of micro-fluidic chip and its device and method integrating circulating tumor cell capture, cracking and detection of nucleic acids |
| CN110951613A (en) * | 2019-12-30 | 2020-04-03 | 北京大学 | Single cell array chip and method for screening ion channel medicine |
| CN111019829A (en) * | 2019-12-30 | 2020-04-17 | 北京大学 | Single cell array chip and its preparation method and use |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2138844B1 (en) * | 2008-06-26 | 2012-04-25 | SpheroTec GmbH | Method for testing the response of cells to exposures with therapeutics |
| EP2536819A4 (en) * | 2010-02-16 | 2017-01-25 | The University of North Carolina At Chapel Hill | Array of micromolded structures for sorting adherent cells |
| JP6409988B2 (en) * | 2016-02-01 | 2018-10-24 | 日立化成株式会社 | Cell capture filter, cell capture device, cell capture method, cell observation method, and cell culture method |
| CN107389642A (en) * | 2017-08-01 | 2017-11-24 | 北京科技大学 | Unicellular efficient capture, the imaging of high intension and full transcriptome analysis apparatus and method |
| CN111774110A (en) * | 2020-06-28 | 2020-10-16 | 中国科学院大连化学物理研究所 | Biological analysis chip capable of realizing cell capture and fixation |
-
2020
- 2020-10-30 CN CN202011190716.4A patent/CN112316993B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2851418A1 (en) * | 2012-05-14 | 2015-03-25 | Hitachi Chemical Company, Ltd. | Cancer cell-trapping metal filter, cancer cell-trapping metal filter sheet, cancer cell-trapping device, and manufacturing methods therefor |
| CN107118938A (en) * | 2017-04-07 | 2017-09-01 | 中北大学 | The unicellular arrangement of fluid enhancing dielectrophoresis and control chip and preparation method thereof |
| CN109852530A (en) * | 2019-03-29 | 2019-06-07 | 中国科学院上海微系统与信息技术研究所 | A kind of micro-fluidic chip and its device and method integrating circulating tumor cell capture, cracking and detection of nucleic acids |
| CN110951613A (en) * | 2019-12-30 | 2020-04-03 | 北京大学 | Single cell array chip and method for screening ion channel medicine |
| CN111019829A (en) * | 2019-12-30 | 2020-04-17 | 北京大学 | Single cell array chip and its preparation method and use |
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