JP4392517B2 - Micro blood collection device and microfluidic device using the same - Google Patents

Description

  The present invention relates to a microfluidic device and a micro blood sampling device that realize so-called μ-TAS.

  The importance of preventive medicine is increasing against serious infectious diseases such as SARS and AIDS, and related to health management of elderly people and reduction of the burden of medical insurance. However, until now, in order to measure various parameters such as various proteins, hormones, antigens and antibodies, including counting of red blood cells and white blood cells, which are required in preventive medicine, it is extremely Expensive and large-scale biochemical analyzers have been used.

  On the other hand, in the field of performing various tests, a microfluidic device called a microchemical analysis system (μ-TAS) in which a separator, a mixer, a detection unit, and an analysis unit are miniaturized and integrated into a card size has attracted attention. Since μ-TAS can be created in card size, chemical testing using this method not only requires a very small amount of measurement sample, but also reduces the amount of reagents and waste liquid and analysis time compared to conventional testing methods. It can be shortened. Furthermore, by using μ-TAS, replacement of parts can be simplified, blood analysis eliminates the risk of infection, and is expected to contribute to the development of hygiene in the medical field.

The inventor has developed a recycle type μ-TAS for the purpose of low environmental load, low human load, and low cost (see Patent Document 1). The production process will be briefly described. First, a resin layer is formed by spin coating or the like on a silicon or quartz substrate in which a micro mechanism component, a detection unit, or the like is embedded. Next, grooves or the like are formed on the resin layer with a laser, and these are covered with a resin laminating method to form μ-TAS. After use, only the resin part is peeled off or washed away, and the substrate in which the micro component is incorporated is reused.
It is also possible to produce a three-dimensional mixed flow path by forming a multilayer film on a substrate and forming a three-dimensional flow path element with a laser each time the layers are stacked.
JP 2002-283293 A

  Conventionally, in biochemical tests such as blood tests, no system has been constructed that allows anyone to be quick, easy and accurate anytime, anywhere. Accordingly, an object of the present invention is to provide a micro blood sampling device used in a system in which anyone can perform blood tests universally (ubiquitous) anytime, anywhere.

As a result of intensive studies, the present inventors have found that a ubiquitous blood test can be performed by using μ-TAS having a specific mechanism. The present invention has been made based on such findings.
That is, the present invention
(1) A substrate, a fluid circuit formed in a resin layer on the substrate, a resin coat covering the fluid circuit, and a hollow needle, the needle being inserted between the resin layer and the resin coat, Is placed so that there is a portion where the resin coat and the resin layer are in close contact with each other between the fluid circuit and the other end, and when the needle is pushed toward the center of the substrate, the needle A micro blood sampling device characterized in that the fluid circuit and the hollow portion of the needle communicate with each other,
(2) The micro blood sampling device according to (1), wherein the fluid circuit is decompressed,
(3) The micro blood sampling apparatus according to (1) or (2), wherein the resin coat is formed by a resin laminating method in a vacuum.
(4) The micro blood sampling apparatus according to (1), wherein one end of the needle placed so as to protrude from the end of the substrate is a painless needle.
(5) The micro blood sampling device according to (1), wherein there is a liquid reservoir between the fluid circuit and the needle tip on the resin-coated side,
(6) The micro blood sampling device according to any one of (1) to (5), which is used for a microfluidic device,
(7) A substrate, a resin layer formed on the substrate, a resin adhesive layer formed on the resin layer, and a predetermined pattern removed and formed by laser processing the resin adhesive layer A fluid circuit comprising a groove, a resin coat covering the entire surface of the resin adhesive layer after processing, and a hollow needle, the needle being inserted between the resin adhesive layer and the resin coat, one end of the substrate It is placed so that there is a part where the resin coat and the resin adhesive layer are in close contact with each other and the other end is in contact with the fluid circuit. By pushing the needle toward the center of the substrate, the needle tip A microfluidic device characterized in that the fluid circuit and the hollow portion of the needle communicate with each other, and
(8) The microfluidic device according to (7), wherein the resin layer and the resin coat are made of a fluororesin.
In the present invention, the blood of a minute amount is not particularly limited, preferably to say 0.01 mm ³ ~ 1 mm ^3.
The reduced pressure means a pressure lower than the atmospheric pressure.

According to the minute amount blood collecting apparatus of the present invention, the amount of collected blood is only a small amount, and the burden on the subject to be examined is low. At the same time, the amount of reagent and waste liquid can be reduced and the analysis time can be shortened. it can.
In addition, the micro blood sampling device of the present invention, after use, cleans and removes only the resin layer with the contaminated fluid circuit, and the substrate part in which various components are incorporated can be reused, and the cost is reduced. Cleaning and replacement can be simplified. Moreover, since there are few disposable resin layers, it is a low burden also with respect to an environment.
Furthermore, if the device of the present invention is small and portable, and there is a personal computer that can communicate with the device of the present invention and a μ-TAS regenerator, blood tests can be performed every day. Can send.

The micro blood sampling apparatus of the present invention comprises a substrate, a fluid circuit formed on a resin layer on the substrate, a resin coat covering the fluid circuit, and a hollow needle, and the needle is between the resin layer and the resin coat. Is inserted so that one end protrudes from the end of the substrate, and the other end is placed so that the resin coat and the resin layer are in close contact with the fluid circuit, and by pushing the needle toward the center of the substrate, The needle peels off the resin coat, and the fluid circuit communicates with the hollow portion of the needle.
The micro blood sampling device of the present invention is preferably used for a microfluidic device that integrates a separator, a mixer, a detection unit, an analysis unit, and the like and analyzes a component such as a fluid. As the microfluidic device, a device in which a micro mechanism component or a detector is embedded in a substrate is preferable.

  In the present invention, in addition to inorganic materials such as silicon, soda glass, quartz glass, ceramics, and metal, polytetrafluoroethylene, other plastics, and the like are used as the substrate. When performing analysis or the like by irradiating light from the side opposite to the fluid circuit formation surface of the microfluidic device, it is preferable to use a light-transmitting material such as quartz as the substrate. The thickness of the substrate is not particularly limited, but is preferably 0.1 to 5 mm, more preferably 0.4 to 1 mm.

  The thickness of the resin layer applied on the substrate is not particularly limited, but is preferably 10 to 1000 μm, more preferably 20 to 50 μm. The thickness of the resin layer is determined in relation to the type to be measured, the amount of sample necessary for it, and the like. If this thickness is too thick, laser processing is difficult, and if it is too thin, fluid such as sample liquid does not flow. Any resin can be used as long as it is easy to apply to the substrate by spin coating or laminating, and does not react with or elute from the analytical sample. In order to simplify the replacement, a resin that can be easily washed off or removed after use is preferable. By using such a resin, it is not necessary to discard everything, and the silicon substrate can be reused.

  Any resin may be used as long as it satisfies the above conditions, and examples thereof include benzocyclobutene resin (BCB) and fluorine resin such as polytetrafluoroethylene. The thickness of the resin layer and the depth of the fluid circuit groove are usually the same. However, the resin may be partially left depending on the function of a certain part of the fluid circuit. Further, when optical measurement is performed, there is no problem even if the resin is partially left as long as it is shorter than the wavelength of the measurement light.

The processing for forming the flow path constituting the fluid circuit in the resin layer is preferably performed by laser processing. As the laser, an ultraviolet laser is preferable.
Processing with less thermal influence is possible by processing with ultraviolet rays. In machining and the like, precise processing is difficult due to distortion and damage due to heat, but processing using an ultraviolet laser generates less heat and can suppress a decrease in accuracy due to the heat of the workpiece. Furthermore, the light condensing property of the laser greatly depends on the wavelength, and the shorter the wavelength, the better the light condensing property. Therefore, it can be used for precision processing and fine processing that require high precision. In addition, the fact that heat is difficult to generate enables processing into a material such as resin that is weak against heat.
Among such ultraviolet laser beams, a preferable ultraviolet laser beam has a wavelength of 350 nm or less, and more preferably in the range of 150 to 300 nm.

  In the present invention, when processing is performed with ultraviolet laser light, it is considered that grooves are formed by a laser ablation phenomenon. This mechanism is considered as follows. When a polymer material is irradiated with an ultraviolet laser, the molecular bonds are broken and the polymer material evaporates. (A) When a polymer material is first irradiated with an ultraviolet laser having a wavelength of 250 nm or the like in several tens of ns, (b) excited molecules and various active species are generated at a high density on the surface of the polymer material. (C) When the energy received from the laser by the molecule is greater than the chemical bond that constitutes the molecule (when it exceeds the processing threshold value that is unique to the material), the chemical bond is broken and the molecule It is broken down to the level or atomic level. Therefore, rapid volume expansion occurs. (D) At this time, the excessively applied energy becomes the kinetic energy of the molecule, and the molecule is ejected and removed to the open space above the workpiece.

  In the present invention, in order to realize card-sized μ-TAS, it is preferable that the flow path is formed in the resin portion with a depth of 20 to 30 μm and a width of 20 to 100 μm. By creating the flow path by the resin laser ablation method, 1. 2. Easy processing of resin, 2. Creation of three-dimensional structure is possible. There is an advantage that a pattern can be removed using a mask.

In the present invention, the hollow needle is inserted between the resin layer and the resin coat, one end protrudes from the end of the substrate, and the other end seems to have a portion where the resin coat and the resin layer are in close contact with the fluid circuit. When the needle is pushed in the center direction (inward) of the substrate, the needle peels off the resin coat, and the fluid circuit and the hollow portion of the needle communicate with each other.
Examples of the material forming the needle include stainless steel, carbon, and quartz, and stainless steel is preferable.
The outer diameter of the needle is preferably 50 μm to 300 μm, more preferably 50 μm to 100 μm.
The inner diameter of the needle is preferably 20 μm to 250 μm, more preferably 30 μm to 70 μm.
The length of the needle is preferably 5 mm to 30 mm, and more preferably 10 mm to 20 mm.
The needle is preferably placed on the resin layer so that one end of the fluid circuit and the other end protrudes from the end of the substrate.

In the present invention, the resin coat covers the upper surface of the resin layer and the portion including one tip of the needle, and completely seals the upper surface of the flow fluid circuit, so that a fluid circuit having a predetermined pattern can be formed in the micro blood sampling device. Any material may be used as long as it provides sufficient strength. The thickness of such a resin coat is usually 10 to 200 μm, preferably 20 to 100 μm.
The resin coat is preferably formed by laminating. There are several types of lamination methods themselves, and any method may be used. As specific examples, in the case of a plastic film laminate, an extrusion laminate, a dry laminate, and a wet laminate are typical.

  In one embodiment, the resin coat is formed by a resin laminating method in a vacuum. The fluid circuit can be decompressed by laminating the resin in a vacuum.

  In still another embodiment of the present invention, the substrate, the resin layer formed on the substrate, the resin adhesive layer formed on the resin layer, and the resin adhesive layer are removed by laser processing. The formed fluid circuit including a groove having a predetermined pattern, a resin coat covering the entire surface of the resin adhesive layer after processing, and a hollow needle, the needle between the resin adhesive layer and the resin coat Inserted, one end protrudes from the end of the substrate, the other end is placed so that there is a portion where the resin coat and the resin adhesive layer are in close contact with the fluid circuit, and by pushing the needle toward the center of the substrate, The needle peels off the resin coat to provide a microfluidic device in which the fluid circuit communicates with the hollow portion of the needle. Here, the resin layer and the coat are preferably made of a fluororesin. The resin adhesive layer is a layer for adhering the resin layer and the resin coat, and this layer can be formed of an adhesive or a resin whose surface exhibits adhesiveness by melt melting. By forming the flow path in the resin adhesive layer on the resin layer, it is possible to prevent blood components from adhering to the glass substrate, and the recycling of the substrate is further facilitated.

In the present invention, the fluid circuit is preferably decompressed. The pressure in the fluid circuit is preferably 8 × 10 ² to 8 × 10 ⁵ Pa, more preferably 1 × 10 ⁴ to 1 × 10 ⁵ Pa. In order to reduce the pressure of the fluid circuit, in addition to the resin lamination in the above vacuum, for example, the pressure can be reduced with a syringe after lamination.

In one embodiment, one end of the needle protruding from the end of the substrate is a painless needle.
In the present invention, the painless needle is preferably one in which the tip of a hollow stainless steel having an outer diameter of 150 μm or less is polished to 30 degrees or less. At the time of blood collection, the tip touches the skin and sucks blood, but using a painless needle does not cause pain in the subject to be examined. Therefore, a very small amount of blood can be collected with less pain for the subject to be examined.

  In one embodiment, a microfluidic device with a reservoir between the fluid circuit and the needle tip. The liquid reservoir can be formed in the resin layer similarly to the formation of the fluid path. After laminating, the liquid reservoir can be filled with a syringe and sealed, so that a solution such as a diluting solution can be placed therein. Examples of the solution to be added to the liquid reservoir include, but are not limited to, physiological saline containing heparin (anticoagulant).

FIG. 1 is a schematic view schematically showing an example of a blood collection method using a minute amount blood collection apparatus of the present invention. FIG. 1A is a diagram showing the time point at which blood collection is disclosed. The micro blood sampling device is represented by a cross-sectional view, and includes a substrate 1, a fluid circuit 3 formed on the resin layer 2, a hollow needle 4, and a fluid circuit 3 and a resin coat 5 that covers a part of the needle 4. . Between the fluid circuit 3 and the tip 4a of the needle 4, there is a portion 6 in which the resin coat is in close contact with the resin layer, and the fluid circuit 3 is decompressed. Blood sampling is started by inserting the tip 4b of the needle 4 protruding from the end of the substrate 1 into the finger 7. When the finger is lightly pushed in while the needle 4 is pierced, the needle 4 moves toward the center of the substrate, the fluid circuit 3 communicates with the hollow portion of the needle, and the state shown in FIG. The sample collected from the finger 7 passes through the hollow part of the needle 4 and enters the fluid circuit 3 as indicated by a dotted arrow.
The tip 4b of the needle 4 has a needle-like structure because it pierces the skin or the like. On the other hand, the structure of the tip 4a on the resin coat side is not particularly limited, but preferably has a needle shape as shown.

  FIG. 2 is a schematic view schematically showing the start of blood collection by a micro blood sampling apparatus according to another example of the present invention. In the figure, symbols 1 to 7 are the same as those in FIG. A liquid reservoir 8 exists between the tip 4 a of the needle 4 and the fluid circuit 3. Blood sampling is started by inserting the tip 4b of the needle 4 protruding from the end of the substrate 1 into the finger 7. When the finger is lightly pushed in while the needle 4 is pierced, the needle 4 moves toward the center of the substrate, and blood enters the fluid circuit 3 together with the solution in the liquid reservoir 8.

  For example, when the microfluidic device of the present invention has a substrate made of a light-transmitting material after blood collection, the microfluidic device is applied as it is to various analytical instruments having optical measuring means to analyze blood in the fluid circuit. It can be performed.

  One example of a method for producing a fluid circuit in the micro blood collection device of the present invention will be described with reference to FIG. FIG. 1A shows a substrate 1 such as silicon or quartz glass, and FIG. 2B shows a resin layer forming substrate 13 by spin coating or the like with a resin layer 12 such as BCB on this substrate 11. FIG. 3C shows a laser processing step, and is a perspective view showing a state in which the resin layer forming substrate 13 is processed with a laser beam 14 to form a flow path 15. The method of forming the fluid circuit 15 with the laser light 14 is not particularly limited, and is adapted to the circuit pattern (groove width, depth, circuit shape) to be formed for the purpose of the laser light source. There are a scanning exposure method, a method in which the laser light source is fixed, and the substrate 13 is moved with respect to the laser light so that a pattern matching the target circuit is formed. The fluid circuit 15 is formed by this laser processing. In this way, the resin fluid circuit forming substrate 16 is prepared. A needle (not shown) is placed on the resin fluid circuit forming substrate 16, and then a laminate 17 is applied to the upper surface of the substrate having the fluid circuit 15 as shown in FIG. The microfluidic device 18 is manufactured.

Further, the resin may have a multilayer structure, and the fluid circuit may have a three-dimensional structure. One example of the process of forming the three-dimensional fluid circuit will be described with reference to FIG. First, the first groove 21 shown by black in FIG. 4A is formed on the film on the glass with a laser. Next, the second-layer film is laminated, and a second-layer through hole 22 penetrating the first-layer groove indicated by black in FIG. Next, a third layer film is laminated, and a through hole 24 penetrating through the third layer groove 23 and the second layer hole 22 shown in black in FIG. Finally, the fourth layer is laminated, and the inlet 25 of the fourth layer, shown in black in FIG. 4D, is created with a laser to create a fluid circuit.
A needle (not shown) is placed on the fourth layer of film, laminated on the upper surface, and the entire component is covered to produce a micro blood collection device.

Liquid injection experiment A fluororesin was laminated to a thickness of 45 μm on a glass substrate (length 75 mm × width 25 mm × thickness 1 mm). A resin adhesive layer having a thickness of 45 μm was applied thereon, and a flow path (length: 600 μm × width: 200 μm × depth: 45 μm) was processed into the resin with a laser. Next, as shown in FIG. 1 (A), a 10 mm long silica tube needle (outer diameter 140 μm × inner diameter 40 μm) with both ends obliquely cut protrudes 3 mm from the end of the flow path and 5 mm from the substrate, and is placed horizontally. . Thereafter, a fluororesin film having a thickness of 45 μm was laminated to produce a fluid circuit. At this time, the fluid circuit and the needle were bonded.
Subsequently, a 150 μm square through hole was drilled on the fluid circuit in the uppermost laminate film, and a silica tube was adhered. A syringe was attached to the tip of the silica tube, the cylinder was pulled, and a stopper was put on the cylinder in a state where the pressure in the fluid circuit was reduced, thereby producing a micro blood sampling device.
The film portion of the ink jet cartridge was pressed against the needle of the silica tube protruding from the substrate, and the needle was pushed in. It was confirmed that the laminate between the needle and the fluid circuit was peeled off, and ink flowed into the fluid circuit.

It is the schematic which showed typically an example of the blood collection method by the micro amount blood collection apparatus of this invention. It is the schematic which showed typically the time of the blood collection start by the micro blood collection device of another example of this invention. It is explanatory drawing which shows an example of the manufacturing method of the fluid circuit in the micro blood-collecting apparatus of this invention. It is explanatory drawing which shows another example of the manufacturing method of the fluid circuit in the micro blood-collecting apparatus of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Substrate 2 Resin layer 3 Fluid circuit 4 Hollow needle 4A, 4B Tip 5 Resin coat 6 Part where resin coat is in close contact with resin layer 7 Finger 8 Liquid reservoir 11 Substrate 12 Resin layer 13 Resin layer forming substrate 14 Laser light 15 Fluid circuit 16 Resin fluid circuit forming substrate 17 Laminate 18 Microfluidic device 21 First layer groove 22 Second layer through hole 23 Third layer groove 24 Third layer through hole 25 Fourth layer inlet

Claims (8)

  A substrate, a fluid circuit formed in a resin layer on the substrate, a resin coat covering the fluid circuit, and a hollow needle, the needle being inserted between the resin layer and the resin coat, one end of the substrate It is placed so that there is a part where the resin coat and the resin layer are in contact with each other between the fluid circuit and the other end. By pushing the needle toward the center of the substrate, the needle peels off the resin coat, A micro blood sampling apparatus characterized in that a circuit and a hollow portion of a needle communicate with each other.   2. The micro blood sampling apparatus according to claim 1, wherein the fluid circuit is decompressed.   3. The micro blood sampling apparatus according to claim 1, wherein the resin coat is formed by a resin laminating method in a vacuum.   The micro blood sampling apparatus according to claim 1, wherein one end of the needle placed so as to protrude from the end of the substrate is a painless needle.   2. The micro blood sampling apparatus according to claim 1, wherein a liquid reservoir is provided between the fluid circuit and a tip of the needle on the resin-coated side.   The micro blood sampling apparatus according to claim 1, wherein the micro blood sampling apparatus is used for a microfluidic device.   A fluid comprising a substrate, a resin layer formed on the substrate, a resin adhesive layer formed on the resin layer, and a groove having a predetermined pattern formed by removing the resin adhesive layer by laser processing. It consists of a circuit, a resin coat covering the entire surface of the resin adhesive layer after processing, and a hollow needle, and the needle is inserted between the resin adhesive layer and the resin coat, one end protrudes from the end of the substrate, and the other It is placed so that there is a part where the resin coat and the resin adhesive layer are in close contact with the end of the fluid circuit, and when the needle is pushed inward into the substrate, the tip of the needle peels off the resin coat, and the fluid circuit and the needle A microfluidic device characterized by communicating with a hollow portion of the microfluidic device. The microfluidic device according to claim 7, wherein the resin layer and the resin coat are made of a fluororesin.

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