Device for analyzing cells

Abstract

The present invention provides a cell analysis apparatus characterized by comprising: a first substrate formed in a flat shape; a second substrate formed in a flat shape and spaced apart from the first substrate and positioned to face the first substrate; and a plurality of blocking plates formed to protrude in a vertical direction having a preset height on the surface of the first substrate and extending along a first direction from the surface of the first substrate, wherein after loading a sample onto the surface of either the first substrate or the second substrate, the substrate loaded with the sample is slid over another substrate so that the surface of the plurality of blocking plates of the first substrate contacts the second substrate, thereby allowing cells to be analyzed contained in the sample to be placed in the space formed by the plurality of blocking plates, the first substrate, and the second substrate.

Description

Technology Field

[0001] The present invention relates to a cell analysis device, and more specifically, to a cell analysis device that enables accurate counting of cells and observation of cell shape and color by enabling the measurement of cells contained in a sample to be analyzed in single-cell units and by knowing the volume of the sample to be analyzed in advance. Background Technology

[0002] In various research and industrial fields, such as biology, medicine, chemistry, and the environment, it is necessary to analyze particles, materials, or cells. In such cases, a sample containing cells is spread onto a slide, and images acquired by an image acquisition device, such as a microscope, are analyzed.

[0003] For example, in order to diagnose various blood-related diseases at a medical institution, it is important to determine the shape and number of blood cells and components, such as white blood cells, red blood cells, and platelets.

[0004] For this purpose, blood smear methods have traditionally been mainly used, and recently, CBC (complete blood cell count) equipment that automatically counts blood cells by type is being used.

[0005] However, since CBC equipment cannot diagnose diseases by identifying the shape and color of blood cells and has low accuracy in counting, the blood smear method is still widely used in hospitals and other medical institutions along with CBC tests.

[0006] The blood smear method is a method of spreading a thin layer of blood onto a slide glass to create a layer of blood cells in single-cell units, making it easier to observe with a microscope or the like.

[0007] However, this blood smear method has the following problems.

[0008] 1) Smearing is not always consistent (significant variation among users).

[0009] 2) Blood cell counting is more accurate than CBC, but the accuracy is not high.

[0010] 3) Since the area for observing blood cells is limited and the sampling area is also limited, it must be used in conjunction with a CBC.

[0011] 4) The process is complex and requires specialized personnel.

[0012] 5) Non-uniform distribution of blood smeared on the slide glass

[0013] 6) It is difficult to accurately determine the volume of blood when taking a blood smear.

[0014] Meanwhile, automatic blood smear devices have also been proposed, but they have the same problem in that they operate on the same principle as conventional blood smear methods.

[0015] FIG. 1 is a drawing for explaining a conventional blood smear method, showing a top view and a side view of a slide glass smeared with blood.

[0016] As shown in Figure 1, when blood is smeared onto a slide glass, there is a problem in that it is difficult to accurately count blood cells because the exact volume of the smeared blood cannot be known before the blood dries.

[0017] This is because, as shown in the side view of the slide glass, the height (a, a′) of the smeared blood is not constant, and the height is also not constant.

[0018] Figure 2 is a diagram illustrating another problem during blood cell analysis, showing a side view of a slide glass smeared with blood.

[0019] As shown in Fig. 2, in the blood smear method, blood is smeared onto a slide glass substrate, and a microscope objective lens is positioned in the Z-axis direction to acquire an image in the Z-axis direction.

[0020] In this case, if the height (a) in the Z-axis direction is larger than the size of the unit blood cells, two or more blood cells may be arranged in the Z-axis direction. Therefore, as illustrated, when an image is acquired with a microscope, the blood cells appear to overlap, making it difficult to accurately count and analyze the blood cells.

[0021] Figure 3 is a diagram illustrating the conditions for ideal blood cell analysis, showing a side view of a slide glass smeared with blood.

[0022] As described, the height of the slide glass substrate in the Z-axis direction is constant (a=a′), and if the height is known, the exact volume of the area where the image is acquired can be known.

[0023] In addition, when viewed from the side, if the height in the Z-axis direction has a height corresponding to the size of the blood cell so that only one blood cell can be placed, as illustrated, the blood cells will not appear overlapping when the image is acquired, so the blood cells can be observed accurately. Prior art literature

[0024] Republic of Korea Published Patent Application No. 10-2020-0053718 (Published May 19, 2020) The problem to be solved

[0025] The present invention aims to solve the problems described above by providing a cell analysis device that enables accurate counting of cells and observation of cell shape and color by allowing cells contained in a sample to be analyzed to be measured in single-cell units and by knowing the volume of the sample to be analyzed in advance. means of solving the problem

[0026] To solve the problem described above, the present invention provides a cell analysis device characterized by comprising: a first substrate formed in a flat shape; a second substrate formed in a flat shape and spaced apart from the first substrate and positioned to face the first substrate; and a plurality of blocking plates formed to protrude in a vertical direction with a preset height on the surface of the first substrate and extending along a first direction from the surface of the first substrate, wherein after loading a sample onto the surface of either the first substrate or the second substrate, the substrate loaded with the sample is slid over another substrate so that the surface of the plurality of blocking plates of the first substrate contacts the second substrate, thereby allowing cells to be analyzed contained in the sample to be placed in the space formed by the plurality of blocking plates, the first substrate, and the second substrate.

[0027] Here, the height of the plurality of blocking plates can be formed to correspond to the height of a single cell to be analyzed included in the sample.

[0028] Additionally, the plurality of blocking plates may be formed on the surface of the first substrate at predetermined intervals along the first direction and the direction in which the plurality of blocking plates protrude vertically.

[0029] According to another aspect of the present invention, a cell analysis apparatus is provided, comprising: a first substrate formed in a flat shape; a lower substrate having a groove into which the first substrate can be inserted; a plurality of blocking plates formed to protrude in a vertical direction having a preset height on the surface of the first substrate and extending along a first direction on the surface of the first substrate; and an upper substrate disposed on the lower substrate and fixedly coupled to the lower substrate to cover the groove, wherein after loading a sample onto the first substrate, the first substrate is inserted into the groove and slid, thereby allowing cells to be analyzed contained in the sample to be disposed in the space formed by the plurality of blocking plates, the first substrate, and the upper substrate.

[0030] Here, the lower substrate has a support formed in a flat shape and two side walls formed protruding vertically at both ends of the support, and a plurality of protrusions are formed on the bottom surface of the support by protruding vertically to have a preset height, and the plurality of protrusions are formed extending along a first direction on the surface of the support and may have a height such that the first substrate can be inserted into the groove.

[0031] In addition, by loading a sample onto the first substrate and sliding the first substrate along the first direction while bringing the bottom surface of the first substrate into contact with the surface of the plurality of protrusions, the first substrate is inserted into the groove, thereby allowing the cells to be analyzed contained in the sample to be placed in the space formed by the plurality of blocking plates, the first substrate, and the upper substrate.

[0032] Additionally, an opening is formed in the upper substrate that penetrates in a vertical direction to allow a sample to be loaded, and the first substrate is inserted into the groove and slid so that the first substrate is positioned below the opening, and then a sample can be loaded onto the first substrate through the opening.

[0033] In addition, the height of the plurality of blocking plates can be formed to correspond to the height of a single cell to be analyzed included in the sample.

[0034] Additionally, the plurality of blocking plates may be formed on the surface of the first substrate at predetermined intervals along the first direction and the direction in which the plurality of blocking plates protrude vertically.

[0035] Additionally, the apparatus further includes a support substrate placed on the surface of the lower substrate within the groove, and after loading a sample onto the first substrate, the bottom surface of the first substrate is brought into contact with the surface of the support substrate and is slid along the first direction to be inserted into the groove, thereby allowing the cells to be analyzed contained in the sample to be placed in the space formed by the plurality of blocking plates, the first substrate, and the upper substrate.

[0036] In addition, a plurality of protrusions are formed on the surface of the support substrate by protruding in a vertical direction to have a preset height, and the plurality of protrusions are formed extending along a first direction on the surface of the support substrate and may have a height such that the first substrate can be inserted into the groove.

[0037] In addition, after loading a sample onto the first substrate, the bottom surface of the first substrate is brought into contact with the surface of the plurality of protrusions of the support substrate and is slid along the first direction to be inserted into the groove, thereby allowing the cells to be analyzed contained in the sample to be placed in the space formed by the plurality of blocking plates, the first substrate, and the upper substrate.

[0038] Additionally, the apparatus further includes a support substrate that is positioned below the bottom surface of the lower substrate to support the lower substrate and is fixedly coupled to the upper substrate so as not to vary the gap between the upper substrate and the support substrate, and the lower substrate may be formed of an elastic material.

[0039] In addition, the distance between the surface of the plurality of protrusions and the upper substrate may be smaller than the height of the first substrate.

[0040] Additionally, an opening is formed in the upper substrate that penetrates in a vertical direction to allow a sample to be loaded, and the first substrate is inserted into the groove and slid so that the first substrate is positioned below the opening, and then a sample can be loaded onto the first substrate through the opening.

[0041] In addition, the height of the plurality of blocking plates can be formed to correspond to the height of a single cell to be analyzed included in the sample.

[0042] Additionally, the plurality of blocking plates may be formed on the surface of the first substrate at predetermined intervals along the first direction and the direction in which the plurality of blocking plates protrude vertically.

[0043] In addition, the plurality of protrusions may be formed to extend along a direction perpendicular to the first direction in which the plurality of blocking plates are extended and the direction in which the plurality of blocking plates are vertically protruded.

[0044] In addition, the plurality of protrusions may have an arrangement structure that allows the volume or area of ​​the observation region to be calculated based on the observation pattern observed by overlapping with the plurality of blocking plates when observing a sample by an image acquisition device.

[0045] In addition, a portion of the surface of the first substrate can be formed to protrude higher than other portions. Effects of the invention

[0046] According to the present invention, a cell analysis device can be provided that enables accurate counting of cells and observation of cell shape and color by allowing cells contained in a sample to be analyzed to be measured in single-cell units and by knowing the volume of the sample to be analyzed in advance. Brief explanation of the drawing

[0047] FIG. 1 is a drawing for explaining a conventional blood smear method, showing a top view and a side view of a slide glass smeared with blood. Figure 2 is a diagram illustrating another problem during blood cell analysis, showing a side view of a slide glass smeared with blood. Figure 3 is a diagram illustrating the conditions for ideal blood cell analysis, showing a side view of a slide glass smeared with blood. FIGS. 4 and FIGS. 5 show a cell analysis device (100) according to an embodiment of the present invention, FIG. 4 is an exploded perspective view, and FIG. 5 is a front view of the first substrate (10) and the second substrate (20) combined. FIG. 6 is a drawing for explaining the usage state of a cell analysis device (100), and shows a front view. FIGS. 7 and FIGS. 8 are drawings for explaining a cell analysis device (200) according to another embodiment of the present invention, FIG. 7 is an exploded perspective view, and FIG. 8 is a front view of the entire components combined. FIG. 9 is a plan view of a cell analysis device (200) to explain the process of sample loading and insertion of the first substrate (10) by the opening (61). FIGS. 10 and FIGS. 11 show a cell analysis device (300) according to another embodiment of the present invention, FIG. 10 is an exploded perspective view, and FIG. 11 is a front view of the entire components combined. FIG. 12 is a front view illustrating the operation of a cell analysis device (300) when using a support substrate (80). FIG. 13 is a diagram showing the state of acquiring an image from a sample after the first substrate (10) is inserted into the groove (41). FIG. 14 is a plan view of a cell analysis device (400) according to another embodiment of the present invention. Figure 15 shows an observation pattern according to the extended shape of a plurality of protrusions (50A). FIG. 16 is a front view showing another embodiment of the first substrate (10). FIG. 17 is a front view showing another embodiment of the first substrate (10). FIG. 18 is a drawing showing the close-contact structure of a plurality of blocking plates (30). Figure 19 shows an actual example of an image of observed red blood cells. Figure 20 shows an image of blood loaded into the opening (61). Figure 21 shows an image obtained with a fluorescence microscope. FIG. 22 shows the results of comparing the number of cells in the red blood cell solution and white blood cell solution at each concentration condition with the number of red blood cells and white blood cells counted on the first substrate (10). Figure 23 shows a fluorescence image of HL-60. Specific details for implementing the invention

[0048] Hereinafter, embodiments according to the present invention will be described in detail with reference to the attached drawings.

[0049] FIGS. 4 and FIGS. 5 show a cell analysis device (100) according to an embodiment of the present invention, FIG. 4 is an exploded perspective view, and FIG. 5 is a front view of the first substrate (10) and the second substrate (20) combined.

[0050] Referring to FIGS. 4 and 5, the cell analysis device (100) includes a first substrate (10), a second substrate (20), and a plurality of blocking plates (30).

[0051] The first substrate (10) and the second substrate (20) are formed in a flat shape, and the second substrate (20) is spaced apart from the first substrate (10) and arranged to face the first substrate (10).

[0052] Hereinafter, the mutually opposing surfaces of the first substrate (10) and the second substrate (20) are referred to as surfaces.

[0053] In FIGS. 4 and 5, the first substrate (10) and the second substrate (20) are formed in a rectangular shape, but this is exemplary and they may have other shapes such as a circle or a triangle.

[0054] The first substrate (10) and the second substrate (20) can be formed from materials such as polymer, glass, metal, paper, etc., and can be formed from transparent, translucent, or opaque materials.

[0055] A plurality of blocking plates (30) are formed on the surface of the first substrate (10) and are formed to protrude in a vertical direction to have a preset height (h).

[0056] Additionally, a plurality of blocking plates (30) are formed extending along a first direction on the surface of the first substrate (10).

[0057] Here, the first direction may be the Y-axis direction in the XYZ coordinate system as illustrated. In this case, the direction in which the plurality of blocking plates (30) protrude vertically becomes the Z-axis direction, and since the second substrate (20) is arranged to face the first substrate (10), the plurality of blocking plates (30) are formed to protrude in the direction of the second substrate (10).

[0058] Here, the height (h) of the plurality of blocking plates (30) is formed to correspond to the height of a single cell to be analyzed included in the sample.

[0059] For example, when the sample is blood and blood cells such as red blood cells or white blood cells are analyzed, the multiple blocking plates (30) can be formed to a height of about 10 to 20 μm corresponding to the height of one unit blood cell.

[0060] In addition, when analyzing platelets, the height of the multiple blocking plates (30) can be formed to be about 3 to 5 μm, corresponding to the height of a single unit platelet cell.

[0061] Additionally, as shown in FIG. 5, a plurality of blocking plates (30) are formed on the surface of the first substrate (20) at a predetermined interval (d) along the first direction and the direction in which the plurality of blocking plates (30) protrude vertically.

[0062] The spacing (d) between the multiple blocking plates (30) can also be set based on the type of cell to be analyzed included in the sample.

[0063] The spacing (d) between the multiple blocking plates (30) is preferably constant, but some may be formed to have different spacing (d).

[0064] After loading a sample onto the surface of either the first substrate (10) or the second substrate (20), the substrate loaded with the sample can be slid over another substrate so that the second substrate (20) comes into contact with the surface of the plurality of blocking plates (30) of the first substrate (10) as shown in FIG. 5, thereby allowing the cells to be analyzed contained in the sample to be placed in the space formed by the plurality of blocking plates (30), the first substrate (10), and the second substrate (20).

[0065] FIG. 6 is a drawing for explaining the usage state of a cell analysis device (100), and shows a front view.

[0066] Figure 6 shows an example where the sample is blood and the blood cells contained in the blood are analyzed.

[0067] First, as shown in (A) of FIG. 6, blood is loaded onto the first substrate (10).

[0068] Next, as shown in (B) and (C), when the plurality of blocking plates (30) are slid while the surface of the second substrate (20) is brought into contact with the surface of the plurality of blocking plates (30) formed on the first substrate (10), a space is formed by the plurality of blocking plates (30), the first substrate (10), and the second substrate (20), and blood cells contained in the blood are placed in this space.

[0069] At this time, as described above, since the height (h) of the plurality of blocking plates (30) is formed to correspond to the height of one blood cell to be analyzed, the blood cells can be arranged to be evenly distributed in the XY plane so as not to overlap each other in the Z-axis direction, as shown in (C) of FIG. 6.

[0070] In this state, if the objective lens of the microscope is positioned below the first substrate (10) or above the second substrate (20) to acquire an image, an image can be obtained in which the blood cells do not overlap each other. Therefore, the number, shape, color, etc. of the blood cells can be accurately observed.

[0071] In addition, since the height (h) and spacing (d) of the plurality of blocking plates (30) are predetermined values, the volume of the space formed by the plurality of blocking plates (30), the first substrate (10), and the second substrate (20), as shown in FIG. 6, is also a value that can be known in advance. Therefore, it is possible to count blood cells in a predetermined volume for each space.

[0072] Meanwhile, FIG. 6 illustrates an example in which blood is loaded onto the first substrate (10), but it is also possible to place the second substrate (20) at the bottom and then slide the first substrate (10) after loading the blood.

[0073] In addition, in FIG. 6, the second substrate (20) is shown sliding along the X-axis direction, but this is also exemplary and it may be slid along the Y-axis direction or on the XY plane.

[0074] FIGS. 7 and FIGS. 8 are drawings for explaining a cell analysis device (200) according to another embodiment of the present invention, FIG. 7 is an exploded perspective view, and FIG. 8 is a front view of the entire components combined.

[0075] The cell analysis device (200) of FIGS. 7 and 8 has the same basic principle as the cell analysis device (100) of FIGS. 4 to 6, but differs in that it includes a housing structure that uses a lower substrate (40) and an upper substrate (60) instead of a second substrate (20).

[0076] Referring to FIGS. 7 and 8, the cell analysis device (200) includes a first substrate (10), a plurality of blocking plates (30), a lower substrate (40), and an upper substrate (60).

[0077] The first substrate (10) is the same as described in FIGS. 4 to 6, and the plurality of blocking plates (30) are also the same, so a detailed description of them is omitted.

[0078] The lower substrate (40) has a groove (41) into which the first substrate (10) can be inserted.

[0079] The lower substrate (40) has a support (42) formed in a flat shape, and two side walls (43, 44) that are formed protruding vertically at both ends of the support (42) when the lower substrate (40) is viewed from the front as shown in FIG. 8.

[0080] Two side walls (43, 44) have a preset thickness and height, and a groove (41) is formed by the surface of these two side walls (43, 44) and the support (42).

[0081] The upper part of the groove (41) may be open, and an upper substrate (60) is positioned over the groove (41) to cover the groove (41) as described below.

[0082] Meanwhile, on the bottom surface of the support (42), a plurality of protrusions (50) may be formed, although they are not essential.

[0083] A plurality of protrusions (50) are formed by protruding vertically to have a preset height on the bottom surface of the support (42), and can be formed extending along a first direction on the surface of the support (42).

[0084] Here, the first direction is the same direction as the direction in which the plurality of blocking plates (30) described above are extended. That is, as described above, the plurality of protrusions (50) are formed by extending along the Y-axis direction in the XYZ coordinate system, and the direction in which the plurality of protrusions (50) protrude vertically is the Z-axis direction.

[0085] Here, the height of the plurality of protrusions (50) is such that the first substrate (10) can be inserted into the groove (41).

[0086] That is, the height of the plurality of protrusions (50) is formed such that the distance between the upper substrate (60) arranged to cover the groove (41) from the surface of the plurality of protrusions (50) corresponds to the height of the first substrate (10).

[0087] In addition, in FIGS. 7 and 8, the multiple protrusions (30) are shown in the shape of a rectangular prism, but this is exemplary and a cylinder or triangular prism shape may also be used.

[0088] The upper substrate (60) is placed on the lower substrate (40) and fixedly coupled to the lower substrate (40) to cover the groove (41).

[0089] Both ends of the upper substrate (60) can be fixed to the lower substrate (40) by being in close contact with the surfaces of the two side walls (43, 44) of the lower substrate (40).

[0090] The cell analysis device (200) of FIGS. 7 to 8 operates as follows.

[0091] First, if a plurality of protrusions (50) are not formed on the lower substrate (40), after loading a sample onto the first substrate (10), the first substrate (10) can be inserted into the groove (41) and slid along the first direction.

[0092] In this case, the bottom surface of the first substrate (10) can slide while in contact with the surface of the lower substrate (40). Once insertion is complete, cells to be analyzed contained in the sample can be placed in the space formed by the plurality of blocking plates (30), the first substrate (10), and the upper substrate (60).

[0093] In addition, when a plurality of protrusions (50) are formed on the lower substrate (40), when a sample is loaded onto the first substrate (10) and the first substrate (10) is inserted into the groove (41) of the lower substrate (40), the bottom surface of the first substrate (10) is brought into contact with the surface of the plurality of protrusions (50) and the first substrate (41) is inserted into the groove (41) while sliding along the first direction, i.e., the Y-axis direction.

[0094] At this time, since only the multiple protrusions (50) are in contact with the bottom surface of the first substrate (10), the contact area can be reduced compared to the case where there are no multiple protrusions (50), so the first substrate (10) can be inserted more smoothly.

[0095] Meanwhile, an opening (61) that penetrates in the vertical direction can be formed in the upper substrate (60) to allow loading of a sample.

[0096] FIG. 9 is a plan view of a cell analysis device (200) to explain the process of sample loading and insertion of the first substrate (10) by the opening (61).

[0097] Referring to FIG. 9, as shown in (A), the first substrate (10) is slid along the first direction, i.e., the Y-axis direction, and about half of the first substrate (41) is inserted into the groove (41) so that the first substrate (41) is positioned below the opening (61).

[0098] In this state, a sample is loaded onto the first substrate (10) through the opening (61). The area where the sample is loaded on the first substrate (10) is indicated by a red dotted line.

[0099] Next, when the first substrate (10) loaded with the sample is continuously slid along the first direction again, as shown in (B), the first substrate (10) is completely inserted into the groove (41), and at this time, it can be seen that the sample loading area also moved along with the movement of the first substrate (10).

[0100] FIGS. 10 and FIGS. 11 show a cell analysis device (300) according to another embodiment of the present invention, FIG. 10 is an exploded perspective view, and FIG. 11 is a front view of the entire components combined.

[0101] The cell analysis device (300) of FIGS. 10 and 11 has the same basic principle as the cell analysis device (200) of FIGS. 7 and 8, but differs in that it further includes a support substrate (70).

[0102] Referring to FIGS. 10 and 11, the cell analysis device (300) includes a first substrate (10), a lower substrate (40A), an upper substrate (60), a base substrate (70), and a support substrate (80).

[0103] The first substrate (10) and the upper substrate (60) are the same as those described in FIGS. 7 and FIGS. 8, so a detailed description is omitted.

[0104] The lower substrate (40A) is identical to the lower substrate (40) described in FIGS. 7 and 8, except that it is not formed with a plurality of protrusions (50), so a detailed description of this is also omitted.

[0105] The support substrate (70) is placed on the surface of the lower substrate (40A) within the groove (41).

[0106] A plurality of protrusions (50A) as described in FIGS. 7 and FIGS. 8 may be formed on the surface of the base substrate (70).

[0107] A plurality of protrusions (50A) are formed by protruding vertically on the surface of the support substrate (70) to have a preset height, and are formed extending along a first direction on the surface of the support substrate (70).

[0108] Here, the first direction is the same as the direction in which the multiple blocking plates (30) are extended, as previously explained.

[0109] In addition, the height of the plurality of protrusions (50A) is such that, as previously described, the first substrate (10) can be inserted into the groove (41).

[0110] That is, the distance between the upper substrate (60) positioned to cover the groove (41) from the surface of the plurality of protrusions (50A) corresponds to the height of the first substrate (10).

[0111] The support substrate (80) is positioned below the bottom surface of the lower substrate (40A) and performs the function of supporting the lower substrate (40A). The support substrate (80) is not essential and may be omitted as needed. An embodiment using the support substrate (80) will be described in detail below with reference to FIG. 12.

[0112] When the support substrate (80) is not used, the cell analysis device (300) of FIGS. 10 and FIGS. 11 operates as follows.

[0113] First, a sample is loaded onto the first substrate (10), and the first substrate (10) is inserted into the groove (41) of the lower substrate (40). At this time, the first substrate (41) is inserted into the groove (41) while sliding along the first direction, with the bottom surface of the first substrate (10) in contact with the surface of the plurality of protrusions (50A) of the support substrate (70).

[0114] At this time, as previously explained, since only the multiple protrusions (50A) come into contact with the bottom surface of the first substrate (10), the contact area can be reduced so that the first substrate (10) can slide smoothly.

[0115] When the first substrate (10) is fully inserted into the groove (41), a space is formed by the first substrate (10), a plurality of blocking plates (30) and the upper substrate (60), and blood cells contained in the blood are placed in this space.

[0116] In this case, as described in FIG. 9, a sample may be loaded through the opening (61). That is, as described above, the first substrate (10) is inserted into the groove (41) so that a part of the first substrate (41) is positioned below the opening (61), and a sample can be loaded onto the first substrate (10) through the opening (61). Afterward, the first substrate (10) loaded with the sample can be fully inserted into the groove (41).

[0117] Meanwhile, if a plurality of protrusions (50A) are not formed on the support substrate (70), after loading a sample onto the first substrate (10), the first substrate (10) can be inserted into the groove (41) of the lower substrate (40) and slid. In this case, the bottom surface of the first substrate (10) can be slid while in contact with the surface of the support substrate (70). Once insertion is complete, cells to be analyzed contained in the sample can be placed in the space formed by the plurality of blocking plates (30), the first substrate (10), and the upper substrate (60).

[0118] Next, the operation of the cell analysis device (300) when using a support substrate (80) is described.

[0119] FIG. 12 is a front view illustrating the operation of a cell analysis device (300) when using a support substrate (80).

[0120] Referring to FIG. 12, the support substrate (80) is positioned below the bottom surface of the lower substrate (40A) as described above and performs the function of supporting the lower substrate (40A).

[0121] The support substrate (80) is fixedly coupled to the upper substrate (60) so that the gap between the upper substrate (60) and the support substrate (80) does not change.

[0122] In the embodiment of FIG. 12, the lower substrate (40A) is formed of an elastic material that can contract or expand due to pressure or external force, and the support substrate (80) is fixedly coupled to the upper substrate (60) and performs the function of causing the lower substrate (40A) to contract, expand, and deform in the vertical direction due to elasticity. This is to allow the first substrate (10) to be inserted more closely into the groove (41).

[0123] In this case, as shown in the enlarged view at the bottom of FIG. 12, the distance (a′) between the surface of the plurality of protrusions (50A) and the upper substrate (60) can be formed to be smaller than the height (a) of the first substrate (10).

[0124] In this state, when the first substrate (10) is inserted into the groove (41) in the manner described above, the bottom surface of the first substrate (10) is brought into contact with the surface of the plurality of protrusions (50A) and a slight downward force is applied while inserting it into the groove (41), and thus downward pressure is applied by the support substrate (70), and thus the lower substrate (40A) formed of an elastic material is pressed downward, and deformation occurs in the part indicated by "T" in FIG. 12.

[0125] That is, as the lower substrate (40A) is pressed downward, the gap between the upper substrate (60) and the lower substrate (40A) at the "T" portion widens, and as a result, the support substrate (70) also moves downward (indicated by the dotted line). Accordingly, the bottom surface of the first substrate (10) can be slid and inserted into the groove (41) as described above while coming into contact with the surface of the plurality of protrusions (50A).

[0126] According to this configuration, the first substrate (10) and the upper substrate (60) can be inserted into the groove (41) while being in close contact.

[0127] At this time, as described above, since only the surface of the plurality of protrusions (50) and the bottom surface of the first substrate (10) are in contact, the contact area can be minimized, so that the first substrate (10) can be inserted smoothly while being in close contact with the upper substrate (60).

[0128] FIG. 13 is a diagram showing the state of acquiring an image from a sample after the first substrate (10) is inserted into the groove (41).

[0129] FIG. 13 shows the state in which the first substrate (10) is inserted into the groove (41) through the process described above, and it can be seen that a sample shown in red is loaded in the space formed by the first substrate (10), a plurality of blocking plates (30), and the upper substrate (60).

[0130] In this state, as illustrated, if the objective lens of the microscope is positioned on the upper substrate (60) to acquire an image, an image can be obtained in which the blood cells do not overlap each other.

[0131] FIG. 14 is a plan view of a cell analysis device (400) according to another embodiment of the present invention.

[0132] The cell analysis device (400) of FIG. 14 is identical to the cell analysis device (300) described above, but differs in that a plurality of protrusions (50A) formed on the support substrate (70) are formed extending along the X-axis direction, that is, in a direction perpendicular to the first direction in which a plurality of blocking plates (30) are extended and the direction in which a plurality of blocking plates (30) are vertically protruded.

[0133] At this time, if the upper substrate (60) is formed from a transparent material, and the objective lens of the microscope is positioned on the upper substrate (60) as described in FIG. 13 to obtain an image of the sample, then as shown in the lower part of FIG. 14, a plurality of protrusions (50A) and a plurality of blocking plates (30) are observed overlapping, so an observation pattern showing a grid-like pattern can be observed.

[0134] Since the area or volume of the observation region can be determined by this grid-shaped observation pattern, there is an advantage in that the number of target cells contained in the sample per unit volume can be accurately counted or measured.

[0135] Meanwhile, the principle described in the example of the embodiment of FIG. 14, which is applied to a cell analysis device (300), can, of course, also be applied to a cell analysis device (200). In this case, the only difference is that the plurality of protrusions (50) are formed on the lower substrate (40) rather than on the support substrate (70).

[0136] In addition, other observation patterns other than the grid-shaped pattern shown in Fig. 14 may also be used.

[0137] Figure 15 shows an observation pattern according to the extended shape of a plurality of protrusions (50A).

[0138] Figure 15 (A) shows a configuration in which a plurality of protrusions (50A) are not extended along one direction but are arranged to appear as a two-dimensional array when viewed from a plane, and Figure 15 (B) shows a configuration in which a plurality of protrusions (50A) are extended in the same direction as described in Figure 14 but are not extended continuously but are arranged separated from each other with spacing.

[0139] In addition to the form shown in FIG. 15, the plurality of protrusions (50A) may have various other arrangement structures that allow the volume or area of ​​the observation region to be calculated by the observation pattern observed by overlapping with the plurality of blocking plates (30) when observing the sample by an image acquisition device.

[0140] FIG. 16 is a front view showing another embodiment of the first substrate (10).

[0141] The first substrate (10) of FIG. 16 differs from the embodiments described above in that the volume of the space formed by the first substrate (10), a plurality of blocking plates (30), and the second substrate (20) or the upper substrate (60) is not all the same.

[0142] To this end, a portion (S2, S3) of the surface of the first substrate (10) is formed to protrude higher than another portion (S1), so that the distances (a, b, c) between the surface of the first substrate (10) and the second substrate (20) or the upper substrate (60) can be made different from each other.

[0143] As shown in the illustration, the distance between the second substrate (20) or the upper substrate (60) from surface S1 of the first substrate (10) is c, and the distance between surfaces S2 and S3 from the second substrate (20) or the upper substrate (60) is a and b, respectively.

[0144] According to this structure, when the sample contains multiple types of cells to be analyzed and their sizes differ, there is an advantage in that multiple target cells can be analyzed simultaneously in a single device.

[0145] For example, when analyzing blood, c can be formed to a value of about 12 to 16 μm so that single white blood cells are placed in the space formed by the surface (S1) of the first substrate (10), b can be formed to a value of about 3 to 5 μm so that single red blood cells are placed in the space formed by the surface (S2), and a can be formed to a value of 2 μm or less so that single platelet cells are placed in the space formed by the surface (S3).

[0146] In this state, blood samples are loaded, and the first substrate (10) is inserted into the groove (41) as described above, so that blood cells such as red blood cells and white blood cells, and platelets can be separated and arranged in each space. In this state, the cells contained in the sample can be analyzed by an image acquisition device as described above.

[0147] FIG. 17 is a front view showing another embodiment of the first substrate (10).

[0148] FIG. 17 is basically the same as the first substrate (10) of FIG. 16, wherein A) shows a symmetrical arrangement structure of a plurality of blocking plates (30) of the first substrate (10), and B) shows a case where each space is separated from each other by a plurality of blocking plates (30).

[0149] In this way, multiple blocking plates (30) of various types can be used.

[0150] Next, specific experimental examples of the cell analysis device (300) of FIGS. 10 and FIGS. 11 will be described.

[0151] In the cell analysis device (300) of FIGS. 10 and 11, the thickness of the first substrate (10) is 500 μm, and the height of the plurality of blocking plates (30) is formed to be 15 μm for white blood cells and 4 μm for red blood cells. In addition, the width of one of the plurality of blocking plates (30) is 50 μm, and the distance between the plurality of blocking plates (30) is formed to be 400 μm.

[0152] The first substrate (10) was formed of PDMS (polydimethylsiloxane).

[0153] The height of the multiple protrusions (50A) of the support substrate (70) was formed to be 150 μm and the thickness to be 100 μm, and the spacing between them was set to 2 mm. The support substrate (70) was formed of a transparent urethane material.

[0154] The upper substrate (60) and the support substrate (80) are formed from transparent acrylic plates, and the thickness is 1 mm.

[0155] In addition, the height of the two side walls (43, 44) of the lower substrate (40A) was set to 3.4 mm, and the lower substrate (40A) was formed of PDMS (polydimethylsiloxane).

[0156] In the case where the first substrate (10) is formed of PDMS, PDMS is a silicone-based material and has adhesive properties, so it may be difficult to slide when inserted into the groove (41) as described above. Therefore, the support substrate (70) was formed of urethane, a material that allows for good sliding.

[0157] In addition, as described above, the adhesion or friction between the first substrate (10) and the support substrate (70) can be minimized by the plurality of protrusions (50A) formed on the support substrate (70), so that the first substrate (10) can be smoothly inserted into the groove (41) and slide.

[0158] FIG. 18 is a drawing showing the close-contact structure of a plurality of blocking plates (30).

[0159] The left side of FIG. 18 shows the state of inserting the first substrate (10) into the groove (41) after loading a sample through the opening (61) of the cell analysis device (300) as described above.

[0160] Here, 50 μL of water mixed with food coloring at an arbitrary concentration was used as a sample for the experiment.

[0161] In this state, when the first substrate (10) was examined through an optical microscope, it was confirmed that the area where the sample was loaded did not move to another channel by the plurality of blocking plates (30), as shown on the right side of FIG. 18.

[0162] Meanwhile, to verify the operation of the cell analysis device (300) according to the present invention, a red blood cell concentrate provided by the Ulsan Blood Center of the Korean Red Cross was mixed with PBS (phosphate buffered saline, pH 7.4) in a ratio of red blood cells:PBS = 4.5:5.5 to prepare a composition similar to blood, and this was used as a sample for the experiment.

[0163] Figure 19 shows an actual example of an image of observed red blood cells.

[0164] As shown in FIG. 19, after loading a sample onto the first substrate (10) of the cell analysis device (300) and inserting it into the groove (41), an image was acquired and verified using an optical microscope. It was confirmed that the red blood cells were spread out well on the first substrate (10) in single-cell units without any burst or cut red blood cell shapes, and it was observed that no red blood cells were visible at the part where the first substrate (10) meets the upper substrate (60), thus confirming that the adhesion between them was well achieved.

[0165] Figure 20 shows an image of blood loaded into the opening (61).

[0166] As described above, the prepared red blood cell solution was dropped at the location indicated in FIG. 20 to check whether red blood cells are injected by capillary action even on the first substrate (10) with a height of 5 μm. As a result, it was confirmed that not a single red blood cell is injected by itself because the height of the internal channel between the multiple blocking plates (30) is too low, as shown in FIG. 20. In other words, it was confirmed that when the first substrate (10) is inserted into the groove (41) and slid as in the present invention, red blood cells can be injected into the channel with a height of 5 μm, allowing for observation on a one-cell basis.

[0167] In addition, using a first substrate (10) having multiple blocking plates (30) with a height of 15 μm, it was confirmed whether leukocytes are also smeared in single-cell units and whether observation is easy.

[0168] To this end, 5×10 HL-60 cells stained with Hoechst (used as leukocyte substitutes for experimental verification) were added to the red blood cell solution prepared as described above. 6 / mL, 1×10 7 After mixing / mL, loading it onto the first substrate (10), inserting and sliding it into the groove (41), and observing it using a fluorescence microscope (DAPI filter).

[0169] Figure 21 shows an image obtained with a fluorescence microscope, and as shown in Figure 21, it was confirmed that identification and counting of HL-60 cells are possible at the single-cell level.

[0170] Next, the results of an experiment to accurately count red blood cells using the cell analysis device (300) of the present invention will be explained.

[0171] To verify this, three types of red blood cell solutions (2.5 × 10⁻⁶) 9 cell / mL, 3.5×10 9 cell / mL, 4.5×10 9Prepare cell / mL in PBS, and to confirm the possibility of leukocyte counting, stain HL-60 with Hoechst and mix with red blood cell solution to obtain a mixture (red blood cell count: 4.5×10⁶ 9 The following three HL-60 conditions with cell / mL fixed: 5×10 6 cell / mL, 1×10 7 cell / mL, 2×10 7 A mixture of cell / mL in PBS was prepared.

[0172] A. Red blood cell solution (mix the following number of red blood cells in PBS)

[0173] i. 2.5×10 9 cell / mL

[0174] ii. 3.5×10 9 cell / mL

[0175] iii. 4.5×10 9 cell / mL

[0176] B. Leukocyte solution (PBS-based)

[0177] i. Red blood cells 4.5×10 9 cell / mL + HL-60 5×10 6 cell / mL

[0178] ii. Red blood cells 4.5 × 10⁶ 9 cell / mL + HL-60 1×10 7 cell / mL

[0179] iii. Red blood cells 4.5×10 9 cell / mL + HL-60 2×10 7 cell / mL

[0180] To prepare red blood cell solutions, concentrated red blood cells (provided by the Ulsan Blood Center of the Korean Red Cross) were counted using a disposable hemocytometer (C-Chip, NanoEntek) and prepared by diluting them in PBS according to three different concentration conditions.

[0181] In addition, to prepare the leukocyte solution (a solution mixed with red blood cells and HL-60), the number of red blood cells was fixed using the method described above, and the HL-60 prepared by Hoechst staining (culture medium composition: RPMI1640 + fetal bovine serum 10% + 1% penicillin / streptomycin) was counted on a C-Chip under three conditions (5×10⁻⁶). 6 cell / mL, 1×10 7 cell / mL, 2×10 7 It was prepared by diluting it in PBS according to the recipe.

[0182] A red blood cell solution was loaded onto a first substrate (10) having multiple blocking plates (30) of 5 μm height, and a white blood cell solution was loaded onto a first substrate (10) having multiple blocking plates (30) of 15 μm height, and then inserted (slid) into a groove (41), and then an image was taken at 400× magnification using an optical microscope (Olympus CKX53) (in the case of the white blood cell solution, fluorescence imaging was performed using a DAPI fluorescence filter, Long pass filter: excitation= 361~389 nm; emission= ≤435 nm;) and the data was analyzed.

[0183] A. Red blood cells: For each red blood cell concentration condition, three random areas of the first substrate (10) were photographed, and the total number of red blood cells in the three images was added to form one set, for a total of three sets (n=3). The red blood cell concentration was calculated by determining the volume of the area where red blood cells were counted, and for each red blood cell concentration condition (2.5×10 9 cell / mL, 3.5×10 9 cell / mL, 4.5×10 9 It was compared with cell / mL, and the one-sample t-test was used for statistics.

[0184] B. White blood cells: For each white blood cell concentration condition, 40 to 50 random areas of the first substrate (10) were photographed, and the total number of white blood cells was added to form one set, resulting in a total of 3 sets (n=3). The white blood cell concentration was calculated by determining the volume of the area where white blood cells were counted, and for each white blood cell concentration condition (5×10 6 cell / mL, 1×10 7 cell / mL, 2×10 7 It was compared with cell / mL. The single-sample t-test was used for statistics.

[0185] The number of cells in the red blood cell solution and white blood cell solution at each concentration condition and the number of red blood cells and white blood cells counted on the first substrate (10) were compared using a single-sample t-test.

[0186] FIG. 22 shows the results of comparing the number of cells in the red blood cell solution and white blood cell solution at each concentration condition with the number of red blood cells and white blood cells counted on the first substrate (10).

[0187] As shown in FIG. 22, under all conditions P It was confirmed that the -value was 0.05 or higher, and was not significantly different from the set number of cells. This means that when red blood cells and white blood cells are counted using the cell analysis device (300) of the present invention, they can be counted accurately.

[0188] Next, the results of verifying the possibility of cell staining using a staining sample applied in advance to the first substrate (10) are explained.

[0189] First, to check whether white blood cells can be stained when blood is loaded onto the first substrate (10) and inserted into the groove (41), Hoechst diluted in PBS to 20 μg / ml was loaded in the form of a droplet onto the first substrate (10) having a plurality of block plates (30) with a height of 15 μm and dried in a 65°C oven for 4 hours.

[0190] And, HL-60 cells 1×10 6cell / mL, red blood cells 4.5×10 9 After loading 20 μL of the mixture of cell / mL mixed in PBS onto the first substrate dried by Hoechst, the result was observed by inserting it into the groove (41).

[0191] Figure 23 shows a fluorescence image of HL-60.

[0192] As shown in Fig. 23, after 0 to 5 minutes, the first substrate (10) was observed with a fluorescence microscope using a DAPI filter, and it was confirmed that the nuclei of the HL-60 cells were stained well.

[0193] Although the present invention has been described above with reference to preferred embodiments, the invention is not limited to the above embodiments, and it is understood that various modifications and variations are possible within the scope of the invention as understood by the appended claims and drawings.

[0194] For example, although the above examples mainly described cases where the sample to be analyzed is blood, they are not limited thereto and can, of course, be applied to all other types of cells as well. Explanation of the symbols

[0195] 100,200,300,400... Cell analysis device 10...1st substrate 20...2nd board 30...multiple barrier plates 40...lower circuit board 50...multiple protrusions 60...Upper board 70...support circuit board 80...support board

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 A cell analysis device comprising: a first substrate formed in a flat shape; a lower substrate having a groove into which the first substrate can be inserted; a plurality of blocking plates formed to protrude in a vertical direction to have a preset height on the surface of the first substrate and extending along a first direction on the surface of the first substrate; and an upper substrate disposed on the lower substrate and fixedly coupled to the lower substrate to cover the groove, wherein after loading a sample onto the first substrate, the first substrate is inserted into the groove and slid, thereby allowing cells to be analyzed contained in the sample to be disposed in the space formed by the plurality of blocking plates, the first substrate, and the upper substrate. Claim 5 A cell analysis device according to claim 4, wherein the lower substrate has a support formed in a flat shape and two side walls formed protruding vertically at both ends of the support, and a plurality of protrusions are formed on the bottom surface of the support by protruding vertically to have a preset height, and the plurality of protrusions are formed extending along a first direction on the surface of the support and have a height such that the first substrate can be inserted into the groove. Claim 6 A cell analysis device according to claim 5, characterized in that a sample is loaded onto the first substrate, and the first substrate is inserted into the groove by sliding it along the first direction while bringing the bottom surface of the first substrate into contact with the surface of the plurality of protrusions, thereby allowing cells to be analyzed contained in the sample to be placed in the space formed by the plurality of blocking plates, the first substrate, and the upper substrate. Claim 7 A cell analysis device according to claim 4, wherein the upper substrate has an opening formed vertically through it to load a sample, and the first substrate is inserted into the groove and slid so that the first substrate is positioned below the opening, and then a sample is loaded onto the first substrate through the opening. Claim 8 A cell analysis device according to claim 4, characterized in that the height of the plurality of blocking plates is formed to correspond to the height of a single cell to be analyzed included in the sample. Claim 9 A cell analysis device according to claim 4, wherein the plurality of blocking plates are formed on the surface of the first substrate at predetermined intervals along the first direction and the direction in which the plurality of blocking plates protrude vertically. Claim 10 A cell analysis device according to claim 4, further comprising a support substrate disposed on the surface of the lower substrate within the groove, and wherein, after loading a sample onto the first substrate, the bottom surface of the first substrate is brought into contact with the surface of the support substrate and is inserted into the groove by sliding it along the first direction, thereby allowing cells to be analyzed contained in the sample to be disposed in the space formed by the plurality of blocking plates, the first substrate, and the upper substrate. Claim 11 A cell analysis device according to claim 10, wherein a plurality of protrusions are formed on the surface of the support substrate by protruding in a vertical direction to have a preset height, and the plurality of protrusions are formed extending along a first direction on the surface of the support substrate and have a height such that the first substrate can be inserted into the groove. Claim 12 A cell analysis device according to claim 11, characterized in that after loading a sample onto the first substrate, the bottom surface of the first substrate is brought into contact with the surface of the plurality of protrusions of the support substrate and is slid along the first direction to be inserted into the groove, thereby allowing the cells to be analyzed contained in the sample to be placed in the space formed by the plurality of blocking plates, the first substrate, and the upper substrate. Claim 13 A cell analysis device according to claim 10, further comprising a support substrate disposed below the bottom surface of the lower substrate to support the lower substrate and fixedly coupled to the upper substrate so as not to vary the gap with the upper substrate, wherein the lower substrate is formed of an elastic material. Claim 14 A cell analysis device according to claim 13, characterized in that the distance between the surface of the plurality of protrusions and the upper substrate is smaller than the height of the first substrate. Claim 15 A cell analysis device according to claim 10, wherein the upper substrate has an opening formed vertically through it to load a sample, and the first substrate is inserted into the groove and slid so that the first substrate is positioned below the opening, and then the sample is loaded onto the first substrate through the opening. Claim 16 A cell analysis device according to claim 10, characterized in that the height of the plurality of blocking plates is formed to correspond to the height of a single cell to be analyzed included in the sample. Claim 17 A cell analysis device according to claim 10, wherein the plurality of blocking plates are formed on the surface of the first substrate at predetermined intervals along the first direction and the direction in which the plurality of blocking plates protrude vertically. Claim 18 A cell analysis device according to claim 5 or claim 11, wherein the plurality of protrusions are formed to extend along a first direction in which the plurality of blocking plates are extended and a direction perpendicular to the direction in which the plurality of blocking plates are vertically protruded. Claim 19 A cell analysis device according to claim 5 or claim 11, wherein the plurality of protrusions have an arrangement structure that allows the volume or area of ​​an observation region to be calculated by an observation pattern observed overlapping with the plurality of blocking plates when observing a sample by an image acquisition device. Claim 20 A cell analysis device according to claim 4, characterized in that a portion of the surface of the first substrate is formed to protrude higher than other portions.

Images