KR20030018698A - Cell number counting apparatus and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a micro cell counting device including a capillary tube, a storage unit, and a cell detection unit by a MEMS process, and a method of manufacturing the same. The cell counting device of the present invention comprises: a capillary tube formed on a silicon substrate and a passage through which a liquid containing cells to be counted moves; An injection cell storage unit formed at one end of the capillary and storing a liquid including cells injected into the capillary; An outflow cell storage unit formed at the other end of the capillary and storing a liquid including cells flowing out of the capillary; And detecting the number of cells by measuring the electrical resistance of boron formed in the silicon substrate and positioned under a predetermined portion of the capillary and changing according to the amount of transmitted light according to the degree of staining of the cells moving the capillary. A cell detection unit is included, and the cell detection unit is a region formed by doping boron. Thus, the cell counting device according to the present invention can be integrated and miniaturized using a MEMS process to replace expensive existing large cell counters. In addition, by using an electrical resistance detector that responds to light as a cell detector, it is possible to replace the existing complex biochemical differentiation method by providing a function to quickly and accurately discriminate the number of living and dead cells.

Description

Cell counting apparatus and manufacturing method thereof

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a cell counting device and a method of manufacturing the same, and in particular, capillary, reservoir, and cell detection by a microelectromechanical apparatus (MEMS). It relates to an ultra-small cell counting device comprising a part and a method of manufacturing the same.

On the other hand, the present invention relates to a cell counting device and a method for manufacturing the cell counting device that is capable of determining whether the cells are biologically alive or dead while transferring the liquid including the cells, respectively.

On the other hand, the present invention simplifies the manufacturing process and reduces the size by using MEMS technology, as well as the rapid counting results for living and dead cells using the boron electrical resistance detector in response to the irradiated light The present invention relates to a cell counting device and a method of manufacturing the same.

As is well known to those skilled in the art, accurate counting and sorting of cells is essential in many specific fields for medical and biological line experiments, and in particular, it is known that counting and sorting of cells should be done in small amounts for a short time. . Development and research on cell counters and / or sorting devices have been progressed for a long time, but the increase in the size of the device and the increase in the manufacturing cost are obstacles. As a way to solve this problem, researches using MEMS processes have been actively conducted since the 1990s.

On the other hand, the cell counter has a small amount of liquid as a carrier medium, the study of a small amount of liquid carrier medium has been mainly studied for protein separation or DNA separation. Electrophoresis was used as the method of transporting liquids, and the first work using this method began in 1937 by separating the proteins in human serum by Tiselius. On the other hand, electroosmosis, a type of electrophoresis method, is a phenomenon that occurs when capillary tube is used as a carrier of electrophoresis. It was first performed by Hjerten in 1967 and is faster than other carrier media. The advantage is that the cross section of the flow velocity is constant.

1 is a block diagram of a conventional cell counting device.

The cell counter shown in FIG. 1 includes reservoirs 102 and 104, capillary 107, detector 110, signal processor 112, and power supply 114. Cells to be counted are stored in the reservoir 102 at the capillary inlet 106 and counted cells are stored in the reservoir 104 at the capillary outlet 108. When powered by the power supply 114, cells stored in the reservoir 102 by electroosmotic are injected into the capillary inlet 106 and the capillary outlet 108 via the detector 110 through the capillary 107. ) Into the reservoir 104. Cells are counted by the signal processor 112 while they pass through the detector 110. However, in the conventional cell counting device, while the diameter of the capillary tube 107 is very small (several tens of micrometers), the detector 110 and the reservoirs 102 and 104 are relatively large. It is necessary to configure the reservoir as an integrated device.

On the other hand, it is necessary to distinguish between living and dying cells for counting for medical and biological experiments. The differentiation coefficient according to whether the cells are killed or not is used to distinguish the differential responses to the cells 'dyes through a microscope, or to investigate the changes in the labeling proteins exposed to the extracellular membrane or the differential reactions to the chromosomes' dyes. It is done through the method. However, there was no counting method in which the former promptness and the latter accuracy were simultaneously satisfied.

SUMMARY OF THE INVENTION The present invention has been made in an effort to provide a micro cell counting apparatus including a capillary tube, a storage unit, and a cell sensing unit by a MEMS process and a method of manufacturing the same, in order to solve the above disadvantages.

Another technical problem to be achieved by the present invention is to provide a cell counting device and a method of manufacturing the same, which are capable of determining whether each cell is biologically alive or dead while transferring a liquid including the cell and counting each.

Another technical problem to be solved by the present invention is to simplify the manufacturing process using MEMS technology and reduce the size thereof, as well as to the living cells and the dead cells by using the electric resistance detector of boron reacting to the irradiated light. The present invention provides a cell counting device and a method of manufacturing the same for providing a fast counting result.

1 is a block diagram of a conventional cell counting device.

2 is a cross-sectional view of a cell counting apparatus according to the present invention.

3A to 3C are plan views of the cell counting apparatus according to the present invention.

Figure 4a to 4k is a vertical cross-sectional view of the process step of the cell counting apparatus according to the present invention.

<Explanation of symbols for the main parts of the drawings>

10 ... boron doped area, 20 ... capillary,

30 ... injection cell storage, 40 ... outflow cell storage,

50 ... glass, 60 ... SU-8,

70 ... oxide, 80 ... silicon substrate

In order to achieve the above technical problem, the present invention provides a cell counting apparatus for staining cells and counting the cells according to the amount of irradiated light, wherein a liquid formed on a silicon substrate and containing the cells to be counted are moved. Capillaries that are channels; An injection cell storage unit formed at one end of the capillary and storing a liquid including cells injected into the capillary; An outflow cell storage unit formed at the other end of the capillary and storing a liquid including cells flowing out of the capillary; And a cell detecting unit formed in the silicon substrate and positioned below a predetermined portion of the capillary and detecting a number of cells by measuring a resistance that is changed according to the amount of transmitted light according to the degree of staining of the cells moving the capillary. It includes, the capillary, injection cell storage, outflow cell storage and cell detection unit provides a cell counting device, characterized in that formed using a MEMS process.

Preferably, the cell detection unit is characterized in that the boron (boron) is doped region formed.

Preferably, the cell staining is characterized in that trypan blue (trypan blue).

Preferably, the living cells and the dead cells are counted by using the difference in cell staining according to whether the cells are killed or not.

Preferably, in order to move the cells by capillary electrophoresis, the capillary further comprises a connecting portion having an electrode function on both sides of the capillary to connect to a power supply provided outside the cell counting device. It is done.

Preferably, the capillary electrophoresis is characterized in that the electroosmosis (electro osmosis).

Preferably, in order to measure the resistance of the cell detection unit, further comprising a connecting portion that functions as an electrode on both sides of the cell detection unit to be connected to the resistance measurement device provided on the outside of the cell counting device.

In order to achieve the above technical problem, the present invention provides a method for manufacturing a cell counting device for staining cells and counting the cells according to the amount of light transmitted, wherein the boron doped region is formed on a predetermined portion of the silicon substrate. First step; Forming a metal layer functioning as an electrode at both ends of the boron doped region to measure the resistance of the boron doped region; A third step of forming a channel for forming a capillary in which a liquid containing cells flows on the boron doped region; And a fourth step of forming a reservoir for storing liquid containing cells at both ends of the channel by drilling a hole in the glass plate and adhering the silicon substrate, and forming a capillary tube between the channel formed in the silicon and the glass plate. It provides a cell counting device manufacturing method characterized in.

Preferably, the method may further include forming an oxide film for allowing light to pass through the boron doped region and protecting the boron doped region after the first step.

Preferably, in the third step, SU-8, which is a negative photoresist, is applied to form a channel.

Preferably, in order to move the cells by capillary electrophoresis during the formation of the metal layer of the second step, a metal layer serving as an electrode is formed on both sides of the capillary.

Preferably, the capillary electrophoresis is characterized in that the electroosmotic.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description of the present invention, if it is determined that detailed descriptions of related well-known technologies or configurations may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted. In addition, terms to be described below are terms defined in consideration of functions in the present invention, which may vary according to the intention or custom of a user or an operator. Therefore, the definition should be made based on the contents throughout the specification.

2 is a cross-sectional view of a cell counting apparatus according to the present invention.

Referring to FIG. 2, the cell counting device according to the present invention is formed using a MEMS process, and includes a boron doped region 10, a capillary tube 20, an injection cell storage unit 30, and a cell detector. Outflow cell storage unit 40 is included. The capillary tube 20 is a passage through which the liquid containing cells to be counted moves, and the injection cell storage unit 30 formed at one end of the capillary tube 20 stores the liquid containing the cells injected into the capillary tube 20. The outflow cell storage unit 40 formed at the other end of the capillary tube 30 is a portion for storing the liquid containing the cells outflowing from the capillary tube 20. The boron doped region 10 is formed in the silicon substrate 80 and is positioned below a predetermined portion of the capillary 20 and is changed according to the amount of transmitted light depending on the degree of staining of cells moving the capillary 20. It is a cell detector that detects the number of cells by measuring the electrical resistance of boron.

On the other hand, in order to move the liquid containing the cells by electroosmosis, which is a kind of capillary electrophoresis (electro osmosis) is further provided with a connecting portion (32, 42) to function as electrodes on both sides of the capillary tube 20 It is connected to a power supply (not shown) provided on the outside of the device. Further, in order to measure the resistance of the boron doped region 10, a storage measuring device (not shown) provided on the outside of the cell counting device further includes connection parts 12 and 14 serving as electrodes on both sides of the boron doped region 10. )).

In addition, in order to allow light to pass through the boron doped region 10 and to protect the boron doped region 10, an oxide film 70 is formed, and a SU-8 (60), which is a negative photoresist, is formed on the oxide film 70. Is applied and exposed with an ultraviolet mask to create a channel through which the liquid containing the cells moves. In addition, the channel covers the glass film 50 to form the capillary tube 20.

Looking at the counting action of the cell counting device according to the present invention. The cells to be counted are stained with trypan blue and injected into the capillary 20 in the injection cell reservoir 30. For cell transfer by electroosmotic, power is applied to the electrodes 32 and 42 installed at the reservoirs 30 and 40 at both ends of the capillary tube, and the resistance change is detected from the electrodes 12 and 14 in the boron doped region 10. do. The light irradiated through the glass plate 50 changes the intensity of the light reaching the boron doped region 10 according to the degree of staining of the cells moving the capillary tube 20, and the light irradiated to the boron doped region 10. Since the electric resistance of the boron doped region 10 varies according to the intensity, the number of cells moving through the capillary tube 20 can be counted. On the other hand, resistance change is divided into three stages, based on the difference in staining according to whether cells die or die. That is, trypan blue strongly stains only dying cells, so when cells are not moving, they show the lowest resistance, when living cells are moving, and intermediate cells are moving, and when dead cells are moving. The resistance value of the highest stage is shown. In order to increase the accuracy of the cell count, the measurement is performed at a constant temperature.

3A, 3B and 3C are plan views of the cell counting apparatus according to the present invention, where FIG. 3A shows a glass plate 50 or a glass lid, FIG. 3B shows an electrode arrangement on an oxide film, and FIG. 3C shows an arrangement Shows a channel made of SU-8 on the electrode.

The cell counting apparatus described above with reference to Figs. 3A, 3B and 3C will be further briefly described. The glass plate 50 shown in FIG. 3A serves as a cover for forming the capillary tube 20 (FIG. 2) and allows light to pass through the boron doped region 10 (FIG. 2). In addition, a hole is formed in the glass plate 50 to form the injection cell storage unit 30 and the outflow cell storage unit 40.

FIG. 3B shows the electrode arrangement on the oxide film 70. The reservoirs 30, 40 on both sides of the capillary tube 20 (FIG. 2) are moved to allow the liquid containing cells to move by electroosmotic, which is a type of capillary electrophoresis. 2; terminals 34 and 44 having electrodes 32 and 42 at positions, and for connecting the electrodes 32 and 42 to a power supply (not shown) provided outside of the cell counter; Means 36, 46 for connecting the terminals 34, 44 and the electrodes 32, 42 are provided. In addition, in order to measure the resistance of the boron doped region 10, electrodes 12 and 14 are provided on both sides of the boron doped region 10, and the electrodes 12 and 14 are stored and stored outside the cell counter. Terminals 16, 18 for connecting to an apparatus (not shown) and means 17, 19 for connecting the terminals 16, 18 and the electrodes 12, 14.

FIG. 3C shows a channel made of SU-8 over the disposed electrodes, a negative photoresist on the oxide film 70 formed to allow light to pass through the boron doped region 10 and to protect the boron doped region 10. A photoresist) SU-8 (60) is applied and exposed with an ultraviolet mask to create a channel 62 through which liquid, including cells, moves. The channel 62 also covers the glass film 50 (FIG. 2) to form a capillary tube 20 (FIG. 2).

Figure 4a to 4k is a vertical cross-sectional view of the cell counting device according to the process step according to the present invention, the cell counting device manufacturing method according to the present invention will be described with reference to Figures 4a to 4k.

Referring to FIG. 4A, as a step of washing the silicon substrate 80, the silicon substrate 80 having a thickness of 400 μm, in a <100> direction, and the N-type silicon substrate 80 are sulfuric acid (H ₂ SO ₄ ) and hydrogen peroxide (H ₂ O ₂ ). Clean for 5 minutes in a 1: 2 dilution to remove contaminants, metals and organics.

Referring to FIG. 4B, as a step of forming oxide films 72 and 74 on the silicon substrate 80, the silicon substrate 80 is placed in an oxidation furnace and oxidized with DI (deionized) water at 1000 ° C. for 6 hours. Oxide films 72 and 74 (SiO ₂ -Silicon Oxide) of about 1.2 mu m are formed.

Referring to FIG. 4C, as a step of removing the oxide layer 72 of the boron doped region, the HMDS is coated on the top surface with a 0.3 μm rotary coating (4 seconds at 250 rpm and 35 seconds at 5000 rpm) for soft baking at 90 ° C. for 100 seconds. After soft baking, the AZ 5214 photoresist was coated with a rotary coating of about 1.3 μm (4 seconds at 250 rpm, 35 seconds at 5000 rpm) and soft baked at 90 ° C. for 50 seconds. An ultraviolet (UV) mask having an area to be doped with boron is exposed to ultraviolet light for 10 seconds at a 16 kW / cm 2 intensity. After developing for 1 minute in AZ 300MIF developer, it is hard baked at 120 ° C. for 100 seconds in an oven or hot plate. The oxide layer 72 is etched for 13 minutes in a buffered HF (BHF) solution to remove the oxide layer in the boron doped region. The etched substrate is washed for 3 minutes in a TCE solution, 3 minutes in an acetone solution and 3 minutes in a methyl alcohol solution to remove the organic photoresist, and then dried with nitrogen (N ₂ ).

Referring to FIG. 4D, as a step of generating the boron doped region 10, a boron diffusion process is performed to generate a resistance pattern of the resistance detector. Pre-deposition is performed at a depth of about 0.2 μm using a boron tribromide (BBr ₃ ) liquid source at 1000 ° C. for 1 hour.

Referring to FIG. 4E, as an oxide film is removed, the oxide film is completely removed from the HF solution for 13 minutes.

Referring to FIG. 4F, as a step of forming an oxide film 70 to protect the boron doped region 10, the applied boron layer is 1.6 μm deep for 2 hours at 1050 ° C. by a drive-in. Spread. Then, after the diffusion process is completed to allow light to pass through the boron doped region 10, which serves as a resistance detector, and to protect the boron doped region, an oxide film 70 having a height of 2000 kPa for 30 minutes by flowing water vapor at the same temperature of 1050 캜. , Silicon oxide; SiO ₂ ).

Referring to FIG. 4G, the contact holes 11 and 13 are formed to measure the resistance of the boron doped region 10, and the contact holes 11 and 13 are formed at both ends of the boron doped region 10. On the top surface of the substrate. The HMDS is coated on the top surface with a 0.3 μm rotary coating (4 seconds at 250 rpm, 35 seconds at 5000 rpm) and soft baked at 90 ° C. for 50 seconds. An ultraviolet (UV) mask having an electrode region is exposed to ultraviolet light for 10 seconds at 16 mA / cm 2 intensity. After developing for 1 minute in AZ 300MIF developer, hard bake at 120 ° C. for 100 seconds in an oven or hot plate. The oxide film 70 is etched for 150 seconds in a buffered HF (BHF) solution to remove the oxide film in the electrode region. The etched substrate is washed for 3 minutes in a TCE solution, 3 minutes in an acetone solution and 3 minutes in a methyl alcohol solution to remove organic photoresist, and then dried with nitrogen (N ₂ ).

Referring to FIG. 4H, electrodes 32 and 42 for electroosmosis and electrodes 12 and 14 for resistance change measurement are generated, and electrodes 32 and 42 for electroosmosis on the upper surface of the substrate 80. Plating and patterning to produce electrodes 12 and 14 for resistance change measurement. A layer of chromium / gold (Cr / Au) metal is deposited using a thermal evaporator in turn to form a plate base. Chromium (91, 92, 93, 94) has a total deposition rate of 2.5 to 3 mA / s for about 2 minutes between 55 A and 60 A current to improve adhesion between the substrate and gold (12, 14, 32, 42). 350 kV is deposited. Gold deposits a total of 1200 mA at a deposition rate of 10 mA / s for about 2 minutes between currents 55 A to 60 A. The metal layer deposited on the front surface of the substrate was subjected to soft baking at 90 ° C. for 100 seconds using HMDS (approximately 4 microseconds at 250 rpm and 35 seconds at 5000 rpm). (4 seconds at 250 rpm, 35 seconds at 5000 rpm) and soft bake at 90 ° C. for 50 seconds. An ultraviolet (UV) mask for excluding the plating layer except for the electrode region is exposed to ultraviolet light for 10 seconds at 16 ㎽ / cm 2 intensity. After developing for 1 minute in AZ 300MIF developer, it is hard baked in oven or hot plate for 120 seconds for 100 seconds. The etching solution capable of etching gold and chromium, respectively, is mixed in a 3: 1 ratio of HCl: H ₂ O ₂ , immersed for 1 minute, and then rinsed. Again, the substrate is washed for 3 minutes in a TCE solution, 3 minutes in an acetone solution and 3 minutes in a methyl alcohol solution to remove the organic photoresist and dried with nitrogen (N ₂ ).

Referring to Figure 4i, as a step of rotating the SU-8 (60), SU-8 (70wt% EPON, 30wt% GBL) to rotate the coating (200rpm) to make a channel that the passage of the solution containing the cells move 4 seconds at 1000 rpm for 35 seconds). It is then prebaked at 95 ° C. for 15 minutes.

Referring to FIG. 4J, as a step of forming a channel in the SU-8 60, an ultraviolet mask for making a channel in the SU-8, which is a negative photoresist, is exposed at around 365 nm at an intensity of 300 to 400 mJ / cm 2. do. Post bake at 95 ° C. for 15 minutes and develop and wash for 15 minutes in propyleneglycol monomethylether acetate (PGMEA). Hard bake at 200 ° C. for 30 minutes. Boil NMP (1-methyl-2-pyrrolidinone) or O ₂ ashing is used for complete removal of SU-8.

Referring to FIG. 4K, as a step of covering the glass plate to form the storage parts 30 and 40 and the capillary tube 20, a hole is drilled in a 500 mm thick glass plate 50 with a 1 mm drill and the AZ 5214 glass surface for adhesion. And adhere to SU-8 (60). Hold for 10 minutes on the bond for a firm bond. By covering the glass plate, a capillary is formed between the glass plate 50 and the oxide film 70 on the silicon substrate 80, and the holes in the glass plate 50 and the silicon substrate 80 form the storage portions 30 and 40. By doing so, a cell counting device according to one embodiment of the present invention is completed.

As such, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. Therefore, the above-described embodiments are to be understood as illustrative in all respects and not as restrictive. The scope of the present invention is shown by the following claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention. do.

As described above, the cell counting device according to the present invention can be integrated and miniaturized using a MEMS process to replace expensive existing large cell counters. In addition, by using an electrical resistance detector that responds to light as a cell detector, it is possible to replace the existing complex biochemical differentiation method by providing a function to quickly and accurately discriminate the number of living and dead cells.

Claims (12)

In the cell counting device for staining the cells and counting the cells in accordance with the amount of light transmitted, A capillary tube formed on a silicon substrate and a passage through which a liquid containing cells to be counted moves; An injection cell storage unit formed at one end of the capillary and storing a liquid including cells injected into the capillary; An outflow cell storage unit formed at the other end of the capillary and storing a liquid including cells flowing out of the capillary; And A cell detection unit formed in the silicon substrate and positioned below a predetermined portion of the capillary and detecting a number of cells by measuring a resistance that is changed according to the amount of transmitted light according to the degree of staining of the cells moving the capillary; And the capillary, injection cell storage, outflow cell storage, and cell detection unit are formed using a Micro Electro Mechanical System (MEMS) process. The cell counting device of claim 1, wherein the cell detection unit is a region formed by doping with boron. The cell counting device according to claim 1 or 2, wherein the cell staining is trypan blue. 4. The cell counting device according to claim 3, wherein the living and the dead cells are counted by using the difference in cell staining according to whether cells die or die. The power supply device of claim 1 or 2, further comprising a connecting portion that functions as an electrode on both sides of the capillary tube to move the cells by capillary electrophoresis. Cell counting device, characterized in that to connect to. 6. The cell counting device of claim 5, wherein the capillary electrophoresis is electro osmosis. According to claim 1 or 2, In order to measure the resistance of the cell detection unit further comprises a connecting portion that functions as an electrode on both sides of the cell detection unit to connect to the resistance measurement device provided on the outside of the cell counting device Cell counting device, characterized in that. In the method of manufacturing a cell counting device for staining the cells and counting the cells in accordance with the amount of light transmitted, Generating a boron doped region in a predetermined portion of the silicon substrate; Forming a metal layer functioning as an electrode at both ends of the boron doped region to measure the resistance of the boron doped region; A third step of forming a channel for forming a capillary in which a liquid containing cells flows on the boron doped region; And And a fourth step of forming a reservoir for puncturing a glass plate and adhering it onto the silicon substrate to store liquid containing cells at both ends of the channel, and forming a capillary between the channel formed in the silicon and the glass plate. Cell counting device manufacturing method. The method of claim 8, further comprising: forming an oxide film for allowing light to pass through the boron doped region and protecting the boron doped region after the first step. The method according to claim 8, wherein in the third step, SU-8, which is a negative photoresist, is applied to form a channel. The method of claim 8, further comprising forming metal layers that function as electrodes on both sides of the capillary tube to move the cells by capillary electrophoresis when the metal layer is formed in the second step. The method of claim 11, wherein the capillary electrophoresis is electroosmotic.