JP5188767B2 - Cell separator - Google Patents

Description

  The present invention relates to a cell separator for separating a specific component from a fluid containing cells such as animals and plants or cells containing microorganisms.

  For example, when classified into blood cell components (red blood cells, white blood cells, platelets) contained in blood and plasma components (water, plasma proteins), it is generally performed by centrifugation.

  However, as a matter of course, it is necessary to use a centrifuge, and there is a problem that it takes a long time to separate these components. There is also a problem that each operation of the centrifuge is troublesome.

  Moreover, as shown in Patent Document 1, there is a method of separating each component from blood using a filter.

  However, in order to pass through the filter, it is necessary to apply pressure by a syringe pump or the like. Furthermore, although there is a method of separating naturally using gravity, it takes a very long time.

Further, the above separation method has a problem that a large amount of sample is required. This is particularly a problem when using samples that are difficult to collect in large quantities.
Japanese Patent Laid-Open No. 2005-000802

  Accordingly, the present invention has been made to solve the above-mentioned problems all at once, and has a simple configuration, but is capable of instantaneously separating a specific component contained in a cell-containing liquid such as blood. Providing a separator is the main intended task.

That is, the cell separator according to the present invention includes an opening that is in contact with a liquid containing a cell and / or a plant and / or a cell containing microorganisms, and a cell that is provided in communication with the opening and is in contact with the opening. A component separation part that separates a component to be separated, which is a component smaller than a predetermined size, from the liquid by capillarity , and a hydrophobic region is partially formed on the inner surface of the component separation part. and said that you are. Here, the component smaller than the predetermined size means a component having a small predetermined physical size, for example, a component whose diameter is smaller than a predetermined value.

In such a case, since the cell-containing liquid can be introduced into the inside by capillary action simply by bringing the cell-containing liquid into contact with the opening, the cell separator can be used without using external power or piping. The configuration can be simplified. Moreover, since only the component to be separated is introduced into the component separation unit by capillary action, it can be automatically, reliably and instantly separated. Furthermore, since the capillary phenomenon is used, the sample amount can be reduced. In addition, since the hydrophobic region is partially formed on the inner surface of the component separation unit, for example, even when the mechanical dimensional accuracy of the gap of the component separation unit varies, it can be separated with high accuracy. . For example, when only the plasma component is separated from the blood, even if the blood cell component flows into the component separation part due to variation in dimensional accuracy, the blood cell component is adsorbed and collected in the hydrophobic region, and only the plasma component is separated. Will be.

  As a specific embodiment of the component separation unit, it is preferable that the component separation unit has two opposing planes, and the width of the gap between the planes is the predetermined size. In this case, the inflow of the non-separation target component that is a component larger than the predetermined value can be physically prevented only by setting the width of the gap between the planes to a predetermined value. Moreover, the structure of a component separation part can be simplified.

  Specifically, the cell-containing liquid is blood, and the component separation unit separates plasma components from the blood by capillary action. In order to separate plasma components from blood, the width of the gap between the planes is set to 1 μm to 2 μm.

  As a specific embodiment of each region, it is desirable that the hydrophobic region is formed in a pattern.

  According to the present invention configured as described above, it is possible to provide a cell separator capable of instantaneously separating a specific component contained in a cell-containing liquid such as blood with a simple configuration.

  An embodiment of the present invention will be described below with reference to the drawings. 1 is a perspective view of the cell separator 1 according to the present embodiment, FIG. 2 is a cross-sectional view of the cell separator 1, and FIG. 3 shows a pattern P of the hydrophilic region 111a and the hydrophobic region 111b. FIG. 4 is a partially enlarged view, and FIG. 4 is a diagram showing a method for manufacturing the cell separator 1.

  <Device configuration>

  The cell separator 1 according to this embodiment is a so-called plasma separator that separates plasma components from human blood that is a liquid containing cells and the like.

  Specifically, as shown in FIG. 1 and FIG. 2, this is provided in contact with the blood in contact with the contact opening 1 </ b> A and the contact opening 1 </ b> A. A component separation unit 1B for introducing a plasma component into the inside by capillary action and separating the blood cell component and the plasma component; and a collection opening 1C for taking out the plasma component separated by the component separation unit 1B. ing.

  The plasma component is a component containing water, plasma protein, and the like, and is a component to be separated that is smaller than a predetermined size (1 μm to 2 μm in the present embodiment). The blood cell component is a component containing red blood cells, white blood cells, and platelets, and is a non-separation target component larger than the predetermined size (1 μm to 2 μm).

  The component separation unit 1B physically separates components having a physical size equal to or less than a predetermined value, and has surfaces 1111 and 12a facing each other substantially in parallel (see FIG. 2). The width of the gap between the opposing surfaces 1111 and 12a of the present embodiment is set smaller than the size of blood cell components (red blood cells, white blood cells, and platelets) that are non-separation target components, and the plasma components that are separation target components are physically It is set to a separable size.

  Specifically, the width of the gap between the opposing surfaces 1111 and 12a is set to 1 μm to 2 μm, which is smaller than the physical size (diameter) of the blood cell component.

  The component separating portion 1B is formed from a main body member 11 in which a rectangular recess 111 is formed in a plan view, and a cover member 12 that is joined on the main body member 11 and covers the middle part of the groove recess 111. Has been.

  Then, by joining the main body member 11 and the cover member 12, one of the openings formed at both ends of the recess 111 is used as the contact opening 1A. This contact opening 1A is a long hole having the same width as the width of the recess 111, and when blood plasma components flow into the component separation part 1B by capillary action, blood cell components in the blood are part of the opening 1A. Even if clogged, it does not interfere with the inflow of plasma components.

  Since one of the openings formed at both ends of the recess 111 is used as the contact opening 1A in this way, it is not necessary to provide a hole constituting the opening 1A in the main body member 11 and the cover member 12, and the manufacturing is simplified. Can do.

  Further, in the state in which the main body member 11 and the cover member 12 are joined, the width of the gap between the bottom surface 1111 of the recess 111 of the main body member 11 and the flat surface 12a of the cover member 12 facing the bottom surface 1111 is 1 μm to 2 μm.

  The main body member 11 of this embodiment is a rectangular silicon substrate, and the cover member 12 is a rectangular glass substrate.

  Further, as shown in FIGS. 1 and 2, a pattern P including a hydrophilic region 111 a and a hydrophobic region 111 b is formed on the bottom surface 1111 of the recess 111.

  Specifically, as shown in FIG. 3, the strip-shaped hydrophilic region 111 a and the strip-shaped hydrophobic region 111 b so that the plasma component is almost perpendicular to the direction in which the plasma component proceeds by capillary action (the arrow direction in FIG. 3). Are alternately formed so as to be equally spaced.

  Specifically, the hydrophilic region 111a is formed with a width of 100 μm, and the hydrophobic region 111b is formed with a width of 50 μm so as to be sandwiched between the hydrophilic regions 111a. Further, the hydrophilic region 111a and the hydrophobic region 111b are formed in the entire width direction of the component separating portion 1B (direction perpendicular to the traveling direction due to capillary action). That is, the regions 111 a and 111 b are formed over the entire width direction of the bottom surface 1111 of the recess 111.

  Thus, since the hydrophobic region 111b is formed substantially perpendicular to the traveling direction due to the capillary phenomenon, the liquid that has flowed in due to the capillary phenomenon always passes over the hydrophobic region 111b, and the blood cell component is temporarily separated into the component separation unit 1B. Even if it flows in, the blood cell component is adsorbed and collected by the hydrophobic region 111b during the inflow process, so that only the plasma component can be reliably separated.

  In addition, although the width | variety of the hydrophilic region 111a of this embodiment is 100 micrometers, and the width | variety of the hydrophobic area | region 111b is 50 micrometers, it is not restricted to this. However, since the size of red blood cells among blood cell components is about 8 μm, it is effective that the width of the hydrophobic region 111b has a width larger than that. If this width is excessively widened, it is considered that the effect of the capillary phenomenon decreases and the separation time becomes long. In the worst case, the capillary phenomenon may not occur.

  <Manufacturing method>

  Next, the manufacturing method of the cell separator 1 (plasma separator) of this embodiment is demonstrated with reference to FIG.

  For example, a photoresist is applied to a silicon substrate 11 having a thickness of 500 μm, and patterning is performed using photolithography (FIG. 4A).

  Next, using the photoresist as a mask, the silicon substrate 11 is etched using a dry etching apparatus to form a recess 111 (FIG. 4B). At this time, the depth of the recess 111 is 1 μm to 2 μm.

Then, the photoresist is removed, and the silicon substrate 11 is thermally oxidized to form a silicon oxide film (SiO ₂ film) on the surface of the silicon substrate 11 (FIG. 4C). The thickness of this silicon oxide film is about 50 nm.

  Next, in order to remove the silicon oxide film formed in the recess 111, a photoresist is applied and patterning is performed using photolithography (FIG. 4D).

  Next, using the photoresist as a mask, the silicon oxide film is etched using a dry etching apparatus (FIG. 4E), and the photoresist is removed. Thereby, the pattern P of the hydrophilic region 111a and the hydrophobic region 111b is formed on the bottom surface 1111 of the recess 111.

  The glass substrate 12 is bonded to the silicon substrate 11 subjected to the above processing by, for example, anodic bonding (FIG. 4F).

  <Plasma separation method>

  Next, a method for separating plasma components from blood using the cell separator 1 of the present embodiment will be described.

  First, blood is dropped into the contact opening 1 </ b> A of the cell separator 1.

  Then, blood is automatically sucked into the component separation unit 1B from the contact opening 1A by capillary action and flows into the component separation unit 1B.

  At this time, blood cell components (red blood cells, white blood cells, and platelets) in the blood do not flow into the component separation unit 1B having a gap of 1 μm to 2 μm because the diameter is about 2 μm or more. Therefore, only the plasma component in the blood flows into the component separation unit 1B. In this way, only the plasma component can be separated from the blood.

  In addition, due to the processing accuracy of the recess 111 to the main body member 11 and the bonding accuracy of the main body member 11 and the cover member 12, the gap of the component separation unit 1B is 2 μm or more, and the blood cell component enters the component separation unit 1B The blood cell component is adsorbed and collected in the hydrophobic region 111b provided on the bottom surface 1111 of the recess 111. Thereby, plasma components can be accurately separated from blood even if there is some variation in processing accuracy.

  The separated plasma can be used for biochemical analysis of liver function test items such as GOT, GTP, and γ-GTP, and diabetes test items such as blood glucose and hemoglobin A1c.

  As a specific analysis method of the plasma thus separated, the plasma is optically analyzed by sucking and collecting from the collection opening 1C with a pump, a syringe, etc., and then mixing with a measurement reagent and detecting the absorbance. It is possible to do.

  It is also conceivable to apply a measurement sample to one or both of the opposing surfaces 1111 and 12a. Then, a light source for irradiating the inspection light is provided on one side of the cell separator 1 (the main body member 11 side), and a light detector is provided on the other side (the cover member 12 side) to detect light transmitted through the cell separator 1. Then, it is conceivable to analyze the plasma by calculating the absorbance of the plasma component by processing the detection signal of the photodetector. Note that a plurality of sets of light sources and light detection units may be provided in pairs. It is also conceivable to analyze by introducing the cell separator 1 into an analyzer equipped with such a light source and a photodetector.

  <Effect of this embodiment>

  According to the cell separator 1 according to the present embodiment configured as described above, the blood can be introduced into the inside by capillary action simply by bringing the blood into contact with the opening 1A. The configuration of the cell separator 1 can be simplified without using it. In addition, since only the plasma component is introduced into the component separation unit 1B by capillary action, the plasma component can be separated automatically, reliably and instantaneously. In addition, since the capillary phenomenon is used, the amount of blood sample can be reduced.

  Moreover, since it is not the structure using pores, such as a filter, clogging can be prevented. In addition, the cell separator 1 can be made compact.

  <Other modified embodiments>

  The present invention is not limited to the above embodiment. In the following description, the same reference numerals are given to members corresponding to the above-described embodiment.

  In the above embodiment, the liquid containing cells is human blood, and the plasma component is separated from the blood. In addition, for example, the specific component from the cell-containing liquid containing cells of animals and plants other than humans is used. May be used, or a characteristic component may be separated from a microorganism-containing liquid containing microorganisms.

  In addition, the pattern P of the hydrophilic region 111a and the hydrophobic region 111b of the above embodiment is provided perpendicular to the traveling direction due to capillary phenomenon, but as shown in FIG. It may be provided with a predetermined angle (30 degrees in FIG. 5). In this embodiment as well, as shown in FIG. 3, the liquid that has flowed in due to capillary action always passes over the hydrophobic region 111b, and even if the blood cell component flows into the component separation portion 1B, In this process, the blood cell component is adsorbed and collected by the hydrophobic region 111b, so that only the plasma component can be reliably separated. Furthermore, as the hydrophobic region 111b is inclined, the liquid flows faster in the component separation unit 1B, and the time required for separation can be shortened. Further, as shown in FIG. 6, the strip-like hydrophilic regions 111a and the strip-like hydrophobic regions 111b may be alternately formed at equal intervals so as to be substantially parallel to the traveling direction by capillary action. . In this case, the flow of the liquid in the component separation unit 1B becomes faster, and the separation time can be shortened.

  In the above-described embodiment, the hydrophobic region 111b has a band shape. However, the hydrophobic region 111b may be formed by arranging strips in the longitudinal direction, or having a curved or bent shape. There may be.

  Furthermore, as shown in FIG. 7, a plurality of circular hydrophobic regions 111b may be formed apart from each other. In this case, the hydrophobic region 111b is not limited to a circular shape, and may have other shapes such as a polygonal shape such as a triangular shape or a rectangular shape, or an elliptical shape. In this case, the liquid can easily come into contact with the hydrophobic region 111b, and the non-separation target component such as a blood cell component can be easily adsorbed, so that the liquid can be reliably separated.

  Further, as shown in FIG. 8, the distance between the two hydrophobic regions 111 b that form a pair may be narrowed toward the downstream in the traveling direction due to capillary action. In FIG. 8, each hydrophobic region 111b has a strip shape, but may have other shapes. In this case, as the sample goes downstream, the sample can easily come into contact with the upstream corner of the hydrophobic region 111b, and can easily adsorb a non-separation target component such as a blood cell component, so that the sample can be reliably separated. it can.

  In addition, the hydrophobic regions 111b may be formed in a lattice shape or a staggered lattice shape, and the hydrophobic regions 111b may be densely provided on the contact opening 1A side in the component separation portion 1B. May be.

  The pattern P may be formed on the cover member 12 or may be formed on both the main body member 11 and the cover member 12.

  In addition, in the embodiment, the contact opening 1A and the sampling opening 1C are openings formed by the main body member 11 and the cover member 12, but as shown in FIG. It may be a through hole provided in the member 12. The through hole in FIG. 9 is a long hole so that even if blood cell components in the blood clog the entrance of the component separation unit 1B, the capillary phenomenon is not easily affected.

  Furthermore, in the said embodiment, after forming the recessed part 111 in the main body member 11, the cover member 12 is joined to the main body member 11, and the component separation part 1B is formed, but in addition, the main body member 11, the cover member 12, It is also conceivable that a spacer is formed between them.

  In addition, in the above-described embodiment, the component separation unit 1B has the opposed planes 1111 and 12a, and the width of the gap is 1 μm to 2 μm. Any cross-sectional area having a size that allows only the components to pass therethrough may be used. For example, the component separation part 1B may be a passage having a cross-sectional diameter of 1 μm to 2 μm.

  Further, as shown in FIG. 10, the main body member 11 may be provided with a concave groove 112 and the main body member 11 and the cover member 12 may be joined. In the cell separator 1, the openings of the concave grooves 112 formed on both side ends serve as the contact opening 1 </ b> A and the collection opening 1 </ b> C.

  In the said embodiment, although the component separation part 1B is formed from the silicon substrate 11 and the glass substrate 12, it is not limited to this. For example, the main body member 11 and the cover member 12 may be flat plates made of a resin material such as acrylic resin. In this case, the recess 111 is manufactured using injection molding or imprint molding. The pattern P of the hydrophilic region 111a and the hydrophobic region 111b is formed by applying hydrophilic coating or adding hydrophilicity by plasma treatment because the resin itself has hydrophobicity. As the hydrophilic coating, it is conceivable to coat polysaccharides such as heparin, nucleotides such as DNA, or compounds such as surfactants. Further, for the joining of the main body member 11 and the cover member 12, heat fusion or ultrasonic bonding or the like is used.

  In addition, as a modification for performing separation and analysis at the same time, a plasma or measurement reagent is reacted by applying a powdery or solid measurement reagent in advance to an area where plasma is separated, and the reaction is optically performed. Alternatively, it is conceivable to analyze plasma by electrochemical measurement.

  In addition, as shown in FIG. 11, the measurement sensor 3 may be built in the component separation unit 1B in advance, and measurement may be performed when plasma flows. Note that reference numeral 4 in FIG. 11 denotes an external connection terminal that is connected to the sensor 3 and guides a signal from the sensor 3 to the outside. Specifically, a method in which the measurement sensor 3 is an enzyme electrode or the like and performs electrochemical measurement is conceivable.

  In addition, some or all of the above-described embodiments and modified embodiments may be combined as appropriate, and the present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the spirit of the present invention. .

The perspective view of the cell separator which concerns on one Embodiment of this invention. Sectional drawing of the cell separator in the same embodiment. The elements on larger scale which show the pattern of a hydrophobic region and a hydrophilic region. The figure which shows the manufacturing method of the cell separator in the embodiment. The elements on larger scale which show the pattern of another hydrophobic area | region and a hydrophilic region. The elements on larger scale which show the pattern of another hydrophobic area | region and a hydrophilic region. The elements on larger scale which show the pattern of another hydrophobic area | region and a hydrophilic region. The elements on larger scale which show the pattern of another hydrophobic area | region and a hydrophilic region. The perspective view of the cell separator which concerns on a 1st modification. The perspective view of the cell separator which concerns on a 2nd modification. The top view of the cell separator which concerns on a 3rd modification.

DESCRIPTION OF SYMBOLS 1 ... Cell separator 1A ... Contact opening part 1B ... Component separation part 1C ... Collection opening part 11 ... Main body member 1111 ... The surface (recessed part) which forms the component separation part of a main body member Bottom)
12 ... Cover member 12a ... The surface which forms the component separation part of a cover member

Claims (4)

An opening that comes into contact with a liquid such as cells containing animals and plants and / or cells containing microorganisms;
A component separation unit that is provided in communication with the opening and that separates a component to be separated, which is a component smaller than a predetermined size, from the liquid containing cells and the like in contact with the opening by capillarity and separates the component to be separated; And
A cell separator in which a hydrophobic region is partially formed on the inner surface of the component separation part .   The cell separator according to claim 1, wherein the component separation unit has two opposing planes, and a width of a gap between the planes is the predetermined size. The cell-containing liquid is blood,
The cell separator according to claim 1 or 2, wherein the component separator separates a plasma component from the blood by capillary action. The cell separator according to claim 1, 2, or 3, wherein the hydrophobic region is formed by patterning.