JP2003207504A - Chip and filter for cytapheresis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To separate constituents of blood from a small amount of blood without clotting or activating the same. <P>SOLUTION: This chip for cytapheresis 10 comprises a first filter base 12 including a mesh filter part 16 of silicon having through micropores arranged like a mesh, and a second filter base 20 including a microporous filter part 21 having through micropores 20b. The first filter base 12 and the second filter base 20 are laminated to be communicated from the mesh filter part 16 to the microporous filter part 12. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a blood cell separation chip and filter, and more particularly to a blood cell separation chip and filter suitable for separating blood cell components from whole blood and collecting plasma or serum samples.

[0002]

2. Description of the Related Art In the fields such as clinical tests using blood and culturing of constituents, plasma or serum obtained by centrifuging unnecessary blood cells, which are unnecessary or interfering substances, from whole blood is usually used as a specimen. Often used as. However, centrifugation requires a large-sized centrifuge and a large amount of blood. In recent years, it has been desired to develop a method for testing with a small amount of blood in order to reduce the burden (pain) on the patient in tests such as chronic diseases and immunological tests.

Therefore, a method of separating blood cells by filtration using a glass fiber filter or the like has been studied. For example, a technique called so-called immunochromatography is disclosed, in which whole blood is dropped on a glass fiber filter, plasma components that have exuded on the opposite side of the dropping surface are collected in a pad, and a test is performed by reacting with a reagent in the pad. ing.

However, this method does not pose a problem for a test completed within the pad, but cannot be applied to a test that needs to be performed using liquid plasma or serum, such as a blood coagulation test. In addition, it is difficult to support inspections that require quantification.

Approximately 100 to 800 μl (microliter) which solves this problem and can be inspected by an automatic analyzer or the like
The blood filtration unit for collecting the liquid plasma or serum is partially put into practical use, and is disclosed in Japanese Patent Application No. 9-196911, Japanese Patent Application Laid-Open No. 10-227788 and the like. In this method, similarly, a glass fiber filter and a microporous membrane are combined and set in a holder, whole blood is dropped, and then suctioned from the opposite side to collect liquid plasma or serum.

[0006]

However, JP-A-10-
Since the blood filtration unit disclosed in JP-A-227788 and the like uses a glass fiber filter, blood is activated by coagulation when it comes into contact with glass, and accurate tests are required for coagulation tests such as PT and APTT. I can't do it. In addition, there is also a problem that blood cell components flow into the gap between the filter and the filter receiver.

Therefore, one of the technical problems to be solved by the present invention is to separate blood cell components from a small amount of blood without coagulation activity.

[0008]

In order to solve the above technical problems, the present invention provides a blood cell separation chip having the following constitution.

The blood cell separation chip is of a type for separating blood cell components. The blood cell separation chip includes first and second filter substrates. The first filter substrate includes a silicon mesh filter portion having penetrating fine holes formed in a mesh shape. The second filter substrate includes a silicon micropore filter portion having micropores penetrating therethrough. The first and second filter substrates are stacked so as to communicate with the mesh filter section and the micropore filter section.

In the above-mentioned structure, silicon is formed into a fine mesh of through holes by, for example, anodization, or
It is possible to form minute holes penetrating therethrough. That is, it is possible to form the mesh filter portion and the micropore filter portion of silicon.

According to the above construction, the small amount of blood is
It is introduced into the mesh filter section. The blood cell component has a relatively low speed of advancing through the fine pores of the mesh filter portion, and the other components are relatively fast, so that the blood component in which most of the blood cell component is separated flows out from the mesh filter portion. The blood component that has passed through the mesh filter section is then introduced into the micropore filter section. The micropores in the micropore filter section are formed to a size that prevents blood cell components from passing through the micropores, so that the blood cell components contained in the blood components that have passed through the mesh filter section are also separated by the micropore filter section. Thus, the blood component that has passed through the micropore filter portion can be prevented from containing a blood cell component.

According to the above construction, the mesh filter portion and the micropore filter portion which are in contact with blood are made of silicon which is inactive to blood, so that blood components are not activated to coagulate.

Therefore, the blood cell component can be separated from a small amount of blood without coagulation activity.

In the above structure, in the first and second filter substrates, only the mesh filter portion and the micropore filter portion may be formed of silicon, and the other portions may be formed of other than silicon.

Preferably, the periphery of the mesh filter portion and the micropore filter portion is also made of silicon. As a result, it is possible to prevent gaps from being formed around the mesh filter portion and the micropore filter portion, prevent the blood cell component from wrapping around and slip through, and improve the blood cell separation efficiency.

More preferably, the entire first and second filter substrates are made of silicon. Thereby, the mesh filter portion and the micropore filter portion can be easily formed on a part of the first and second filter substrates.
In addition, it is easy to stack filter substrates, and it is possible to form a blood cell separation chip having a small thickness. In addition, a portion (for example, a flow path) other than the mesh filter portion and the micropore filter portion that comes into contact with blood can be made of silicon to prevent blood coagulation activity.

Preferably, the fine pores formed in the mesh filter section have a pore diameter of 5 μm to 50 μm, and the fine pores formed in the fine pore filter section have a pore diameter of 0.5 μm to 5 μm. .

That is, the fine pores of the mesh filter are
In order to separate blood cell components due to the difference in speed, it is necessary to make them larger than the outer shape of blood cell components (5 μm or more). On the other hand, components other than blood cells such as plasma rapidly flow out, the flow path resistance of blood cell components such as red blood cells, white blood cells, and platelets increases, and the time retained in the network structure probabilistically becomes longer and flows out. In order to slow down the mesh size, the fine pores in the mesh filter should not be too large (~
50 μm) is required.

On the other hand, in the micropore filter portion, it may be selected according to the size of the blood cell component to be separated, but in order to be able to separate the blood cell component without leakage, the pore diameter of the micropore is smaller than the outer shape of red blood cells. (~ 5 μm) is required. On the other hand, if the size is too small, it is difficult to process the micropores, the flow path resistance increases, the outflow rate of blood components other than blood cells slows down, and the blood cell separation efficiency decreases. 0.5 μm-) is necessary.

By the way, if silicon is made porous by anodization, fine pores can be formed. According to the anodization, the size and density of the fine pores can be freely controlled, which is suitable for forming the mesh filter portion. When porous silicon is formed by anodization, p-type silicon can form mesh-like fine holes more efficiently and easily than n-type silicon.

[0021] Preferably, the mesh filter portion of the first filter substrate is p-type silicon.

According to the above structure, the mesh filter portion of the first filter substrate can be efficiently formed by making p-type silicon porous by anodizing.

Preferably, the micropore filter portion of the second filter substrate is n-type silicon.

In the above structure, the n-type silicon can form elongated fine pores by anodization even if it has a certain thickness for easy handling.

The present invention also provides a blood cell separating filter having the following constitution.

The blood cell separation filter is a filter for separating blood cell components. The blood cell separation filter includes a filter member made of silicon in which fine holes penetrating are formed in a mesh shape.

In the above structure, the silicon can be made porous by, for example, anodization to form fine pores penetrating therethrough in a mesh form. When the pore size of the micropores formed in the filter member is larger than the size of the blood cell component, the blood cell component can be separated due to the difference in speed between the blood cell component and the other component when passing through the micropore. When the pore size of the micropores formed in the filter member is smaller than the size of the blood cell component and the blood cell component cannot pass through the micropore, only other components can pass through the micropore to separate the blood cell component. .

In the above structure, since the filter member that comes into contact with the blood component is silicon that is coagulation-inactive with respect to blood, the blood component is not coagulation-activated.

Therefore, blood cell components can be separated from a small amount of blood without coagulation activity.

[0030]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a blood cell separation chip according to each embodiment of the present invention will be described with reference to FIGS.

First, the blood cell separation chip 30 according to the first embodiment will be described with reference to FIGS.

As shown in FIG. 8, a blood cell separation chip 1
In No. 0, the first filter substrate 12, the second filter substrate 20, the chip substrate 30, and the glass plate 38 are sequentially laminated and integrated.

The first filter substrate 12 has a mesh filter portion 16. For the mesh filter portion 16, for example, silicon made porous by anodization can be used.

That is, as shown in FIG. 1, a substrate 12 having ap ⁺ diffusion region 14 in a part of n-type silicon is prepared. The p ⁺ diffusion region 14 is formed by, for example, an ion implantation method by using a photoresist as a mask and selectively adding an impurity to a position corresponding to the mesh filter portion 16.

The anodization is performed by using an anodizing jig 1 as shown in FIGS. 2 and 3, for example. Specifically, the O between the pair of holders 2 and 4 made of Teflon (registered trademark)
The substrate 12 is sandwiched via the ring 6 and fixed with bolts 8. Then, a solution of hydrofluoric acid 9 is placed in the spaces inside the holders 2 and 4.
An electric current is caused to flow between the electrodes 3 and 5 which are arranged on both sides of the substrate 12 by filling the a and 9b. As a result, the p ⁺ diffusion region 14 of the substrate 12 is selectively made porous, and as shown in the conceptual diagram of FIG. 4, a large number of fine holes 18 penetrating between the front and back surfaces are formed in a mesh shape. . The hole diameter of the fine holes 18 can be controlled by changing the formation current and the formation time. Since p-type silicon can be made porous more efficiently than n-type silicon, p ⁺ diffusion region 14
Can be efficiently made porous.

As shown in FIG. 8, the second filter substrate 20 has a micropore filter portion 21. The micropore filter portion 21 includes an introduction portion 20a having a V-shaped cross section and a linear micropore 20b extending downward from the apex of the introduction portion 20a.
Consists of. The micropore filter portion 21 can be formed on the second filter substrate 20 in the process shown in FIG. 5, for example.

That is, as shown in FIG. 5A, a (100) plane n-type silicon 20 having a silicon oxide film 22 formed on its surface is prepared. Then, the photoresist is used as a mask by using the photolithography technique, and FIG.
The silicon oxide film 22 is partially removed as indicated by reference numeral 22a in FIG. Next, the silicon oxide film 22
Is used as a mask to anisotropically etch the silicon 20 with KOH. When the (100) plane of the silicon 20 is processed by anisotropic etching, the portion indicated by reference numeral 20a in FIG.
The sidewalls of the etching groove are formed by four (111) planes intersecting at an angle of 7 °.

Silicon 20 having such a shape
In the same manner as described above, anodization is performed using the anodizing jig 1 shown in FIGS. 2 and 3. This allows silicon 2
The electric field is concentrated at the apex of the recessed portion 20a formed by the anisotropic etching of 0, and the reference numeral 20b in FIG.
As shown by, the etching proceeds linearly. When this is advanced, as shown in FIG. 5 (e), the minute holes 20b penetrate.
Also in this case, the diameter of the micropores 20b can be controlled by changing the formation current and the formation time.

Then, as shown in FIG. 5F, the silicon oxide film 22 used as the mask is removed, and the second filter substrate 20 having a large number of minute holes 20b linearly penetrating in the vertical direction is completed. To do.

As shown in FIG. 7, the chip substrate 30 has a filter receiving portion 35 formed on its front surface and a flow path 36 on its rear surface.
Are formed so as to communicate with each other through the through hole 37. The bottom surface of the filter receiving portion 35 has a first groove 35a.
Are radially formed around the through hole 37, and the second groove 35b is formed.
Are formed concentrically around the through hole 37.

The chip substrate 30 can be formed by the process shown in FIG. 6 using silicon, for example.

As shown in FIG. 6A, silicon 30 having silicon oxide films 32 and 34 formed on the front and back surfaces is prepared. For example, a silicon 30 having a thickness of 400 μm, on which silicon oxide films 32 and 34 having a thickness of 1.5 μ are formed on the front and back surfaces, respectively, are used.

Next, the step of dry etching the silicon oxide film 32 is performed twice on the surface by using the photolithography technique. That is, the silicon oxide film 32
Photoresist is applied to, and a predetermined mask pattern is exposed and developed. Then, after the silicon oxide film 32 is dry-etched, the photoresist is removed. In the first step, as shown by reference numeral 32a in FIG. 6B, the silicon oxide film 32 is removed corresponding to the portions to be the first groove 35a and the second groove 35b of the filter receiving portion 35. In the second process, reference numeral 3 in FIG.
As shown by 2b, the silicon oxide film 32 is removed corresponding to the entire filter receiving portion 35 to provide a step.

Also on the back surface, the silicon oxide film 34 is similarly removed by two steps. In the first step, as shown by reference numeral 34a in FIG. 6D, the silicon oxide film 34 is removed corresponding to the portion to be the through hole 37. In the second step, as shown by reference numeral 34b in FIG. 6E, the portion corresponding to the flow path 36 is removed and a step is provided.

Next, ICP (Inductively)
With a Coupled Plasma etching apparatus, dry etching of the silicon 30 using the silicon oxide films 32 and 34 having steps as a mask and dry etching of the silicon oxide films 32 and 34 are alternately repeated.

That is, the silicon 30 is dry-etched on the surface, and as shown in FIG.
From the silicon 30, the first groove 35 of the filter receiving portion 35
The portion 30a corresponding to a and the second groove 35b is removed. Next, the silicon oxide film 32 is dry-etched to remove the portion 32c corresponding to the entire filter receiving portion 35, as shown in FIG. Next, silicon 30
6D, the portion 30b corresponding to the entire filter receiving portion 35 is removed from the silicon 30 as shown in FIG. 6 (h).

Similarly, on the back surface, dry etching of the silicon 30 is performed, and as shown in FIG.
The portion 30c corresponding to the through hole 37 is removed from the silicon 30. Next, the silicon oxide film 34 is dry-etched to remove the silicon oxide film 34 in the portion 34c corresponding to the flow path 36, as shown in FIG. Next, dry etching of the silicon 30 is performed, and FIG.
As shown in (k), from the silicon 30, a portion 30d corresponding to the flow path 36 and a portion 30 corresponding to the through hole 37.
e and are removed.

Finally, the remaining silicon oxide films 32 and 34
Dip silicon in hydrofluoric acid and use wet etching to remove silicon 30
To remove from.

As shown in FIG. 8, a glass plate 38 having suction holes 39 is bonded to the chip substrate 30 formed in a desired shape by anodic bonding. Glass plate 38
Is subjected to coagulation inactivation treatment on blood.

The first filter substrate 12, the second filter substrate 20, and the chip substrate 30 are attached and integrated by direct bonding (diffusion bonding).

The blood cell separation chip 10 thus formed is set in the holder 40 shown in FIG. 9, for example.

As shown in FIG. 9, the holder 40 is generally placed in the recess 45 of the frame 44 in the blood cell separation chip 10.
And blood receiver 43 are inserted, and cover 42 is fixed with screw 48. Thereby, the suction hole 3 of the blood cell separation chip 10
The suction pad 46 is pressure-bonded to the periphery of 9.

Whole blood is dropped into the through hole 43a of the blood receiver 43 and is sucked by a suction pump or the like (not shown) connected to the tube insertion interface 47 via a tube. The suction pump may be, but is not limited to, a syringe pump or an ironing pump.
In the whole blood, in the blood cell separation chip 10, blood cell components are separated by the mesh filter portion 16 and the micropore filter portion 21, and blood components including plasma and serum are blood receiving portion 3
5, it flows into the flow path 36 from the first groove 35a or the second groove 35b through the through hole 37.

Specifically, most of the blood cell components are separated by the mesh filter portion 16, and the blood cell components that have passed through the mesh filter portion 16 without separation are separated by the micropore filter portion 21.

The mesh filter section 16 separates blood cell components by the difference in speed of blood components passing through the fine holes 18. That is, when the blood component flows through the fine holes 18 of the mesh filter portion 16, small-sized components such as plasma other than blood cells flow out relatively quickly without resistance. on the other hand,
Large-sized blood cell components such as red blood cells, white blood cells, and platelets have a large flow channel resistance, and stochastically hold for a long time in the micropores 18 to slow outflow. Figure 4
As shown in, the fine holes 18 are formed in a mesh shape and have a complicated shape such as branching and joining like a maze,
Blood cell components follow complex pathways. Therefore, it takes time for the blood cell component to flow out from the micropores 18. Therefore,
From the mesh filter unit 16, the blood component in which most of the blood cell components have been separated flows out.

The micropore filter section 21 has a micropore 20b.
Is smaller than the size of the blood cell component to prevent passage of the blood cell component, thereby separating the blood cell component.

The mesh filter portion 16 alone cannot completely separate the blood cell components, which may cause separation leakage of the blood cell components. With the micropore filter section 21 alone,
The micropores 20b may be clogged with blood cell components, which may reduce the blood cell separation efficiency. By combining both, blood cell components can be efficiently separated from whole blood.

Next, a specific example of the blood cell separation chip 10 will be described.

Production Example 1 The first filter substrate 12 was made of silicon having a thickness of 200 μm. Anodic formation was carried out using a solution in which 47% hydrofluoric acid and ethanol were mixed in a ratio of 1: 1 under conditions of a current density of 20 mA / cm ² and a formation time of 30 minutes, and micropores 18 having a pore diameter of about 20 μm were formed. The mesh filter part 16 of porous silicon was formed. Second filter substrate 20
Was 100 μm thick n-type silicon with a 1.5 μm silicon oxide film formed on the front and back surfaces. After anisotropic etching, 47% hydrofluoric acid and ethanol were mixed at a ratio of 1: 1 and irradiated with light from the back surface of the substrate under conditions of current density of 20 mA / cm ² and formation time of 100 minutes. Anodization was performed to form micropores 20b having a pore diameter of about 1 μm in the micropore filter portion 21.

[0060]Example 1 The blood cell separation chip 10 produced in Production Example 1 and shown in FIG.
Blood cells were separated using the holder 40 prepared above. Well
First, 200 μl (microliter) of whole blood from a healthy person
Collect blood and use a pipettor in the through hole 43a of the blood receiver 43.
And dripped. The hematocrit value of this blood was measured
However, it was 42.9%. Simultaneously with the dropping of this whole blood
From the blood cell separation chip 10 via a tube.
Suction was started with the syringe pump provided. Suction speed
It was set to 100 μl per minute. This gives 40 μl of blood
Plasma can be obtained and the obtained plasma volume (V₁) Is used
Total blood volume (V₀) Divided by (V ₁/ V₀) Is plasma
The collection efficiency was 20%. Also obtained this way
Plasma was subjected to a complex factor T coagulation test and was centrifuged.
The value obtained was the same as that of plasma obtained in
It was confirmed that blood cells could be separated without sex.

As described above, in order to prevent coagulation activity due to contact of blood with glass, instead of the glass fiber filter, porous silicon having a network structure having fine pores and a large number of linearly penetrating longitudinally are used. Blood cells can be separated by combining silicon with holes. As a result, coagulation activity due to glass does not occur, and since the filter made of silicon and the chip body can be joined together without a gap, the blood cell component does not slip through the filter and the filter receiver.

Therefore, a blood cell component can be efficiently separated from a small amount of blood without causing coagulation activity.

Next, a blood cell separating apparatus using a blood cell separating filter according to a second embodiment of the present invention will be described with reference to FIG.
Will be described with reference to.

As shown in the schematic view of FIG. 10, in the blood cell separating apparatus, a filter holding plate 58 holding a blood cell separating filter 59 is placed on the base plate 50. The blood cell separation filter 59 is porous silicon that is formed into an appropriate shape after anodizing p-type silicon as in the mesh filter portion 16 of the first embodiment. The blood cell separation filter 59 may be configured to be detachable from the filter holding plate 58, or may be integrated with the filter holding plate 58.

The base plate 50 has micropores 52 similar to the micropores 21b of the micropore filter portion 21 of the first embodiment.
And a flow path 56 are formed so as to communicate with each other through the communication hole 54. At the end of the flow path 56, the tube 6
8 is inserted and sucked by the syringe pump 60. The syringe pump 60 suctions by moving the piston rod 64 of the syringe 62 with the stepping motor 66, for example.

The blood cell separator is a filter 5 for separating blood cells.
The whole blood dropped on 9 is sucked by the syringe pump 60, and the blood cell component is separated by the blood cell separation filter 59 and the micropores 52.

By using the silicon blood cell separation filter 59, the blood cell component can be separated from a small amount of blood while preventing the coagulation activity.

The present invention is not limited to the above embodiments, but can be implemented in various modes.

For example, the blood cell separation chip may be manufactured by a method different from the method described in the first embodiment. Further, in the first and second filter substrates, a material other than silicon, such as ceramic or resin, may be used for the portion other than the mesh filter portion and the micropore filter portion.
Further, the mesh filter portion and the micropore filter portion may not be opposed to each other, but may be arranged at a shifted position. Further, the micropores of the micropore filter section may be curved instead of being linear.

The blood cell separation filter may be used by incorporating it into an apparatus or device other than the chip.

The blood cell separation may be performed without suction.

[Brief description of drawings]

FIG. 1 is a cross-sectional view of anodized silicon.

FIG. 2 is an exploded perspective view of an anodizing jig.

FIG. 3 is an explanatory diagram of anodization.

FIG. 4 is a conceptual diagram of porous silicon.

FIG. 5 is an explanatory diagram of a processing step of the second filter substrate.

FIG. 6 is an explanatory diagram of a chip substrate processing step.

FIG. 7 is a top view and a bottom view of the chip substrate.

FIG. 8 is a cross-sectional view of the blood cell separation chip according to the first embodiment of the present invention.

9 is an explanatory diagram of a method of using the blood cell separation chip of FIG.

FIG. 10 is a conceptual diagram of a blood cell separation device incorporating a blood cell separation filter according to a second embodiment of the present invention.

[Explanation of symbols]

10 Blood cell separation chip 12 First filter substrate 16 mesh filter 18 fine holes 20 Second filter substrate 20b micropore 21 Micropore filter part 59 Blood Cell Separation Filter

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Toshimitsu Fujiwara             2-3-3 Azuchi-cho, Chuo-ku, Osaka-shi, Osaka Prefecture             Osaka International Building Minolta Co., Ltd. (72) Inventor Shigeo Yamashita             2-3-3 Azuchi-cho, Chuo-ku, Osaka-shi, Osaka Prefecture             Osaka International Building Minolta Co., Ltd. F term (reference) 2G045 BA08 BB04 CA25 HB02 HB03                       HB05 JA07                 2G052 AA30 AD29 CA03 CA18 CA19                       EA03 EA13 JA01 JA16                 4D019 AA03 BA01 BB07 BB09 BB10                       BD01 BD03 CA10 CB03 CB06

Claims (5)

[Claims] 1. A blood cell separation chip for separating blood cell components, comprising: a first filter substrate including a silicon mesh filter portion having penetrating micropores formed in a mesh shape; and penetrating micropores. And a second filter substrate including a silicon micropore filter portion, wherein the first filter substrate and the second filter substrate are laminated so as to communicate from the mesh filter portion to the micropore filter portion. A blood cell separation chip characterized in that 2. The micropores formed in the mesh filter section have a pore size of 5 μm to 50 μm, and the micropores formed in the micropore filter section have a pore size of 0.
The blood cell separation chip according to claim 1, which has a diameter of 5 μm to 5 μm. 3. The mesh filter portion of the first filter substrate is p-type silicon,
The blood cell separation chip according to claim 1. 4. The blood cell separation chip according to claim 1, wherein the micropore filter portion of the second filter substrate is n-type silicon. 5. A blood cell separation filter for separating blood cell components, comprising a filter member made of silicon with penetrating micropores formed in a mesh shape.