WO2016063389A1

WO2016063389A1 - Microfluidic device, analysis method using same, and analysis device

Info

Publication number: WO2016063389A1
Application number: PCT/JP2014/078160
Authority: WO
Inventors: 峻熊野; 長谷川　英樹; 谷口　伸一; 橋本　雄一郎
Original assignee: 株式会社日立製作所
Priority date: 2014-10-23
Filing date: 2014-10-23
Publication date: 2016-04-28

Abstract

The present invention is an analysis method using a microfluidic device 4 and two magnets arranged on opposite sides of the microfluidic device, the microfluidic device having an introduction channel 3 into which magnetic beads are introduced along with a solution, and an analysis channel 6 and disposal channel 8 that branch off from the introduction channel at a branching section, the device having a dam structure 7 the channel height of which is less than that of the branching section at a connection section with the branching section(s) of the analysis channel and/or disposal channel, and the channel height narrowed by the dam structure being greater than the diameter of the magnetic beads. The method comprises: a step in which magnetic beads inside a microchannel are attracted to one inner wall of the microchannel by magnetic force of one of the magnets; a step in which the magnetic beads are moved to an inner wall of the microchannel on the side opposite the one inner wall by magnetic force of the other of the magnets; and a step in which the other magnet is moved along the microfluidic device, thereby causing the magnetic beads to pass beyond the dam structure. The method realizes highly-robust, high-throughput, and high-sensitivity analysis.

Description

Microfluidic device and analysis method and apparatus using the same

The present invention relates to a method and apparatus for analyzing chemical substances.

The field of micro technology (Micro-Electro-Mechanical Systems: MEMS) using micro-processing technology related to semiconductors has been developed. In particular, Micro-TAS (Micro Total Analytical System), which produces a micro-channel with a width of 1 mm or less on a glass substrate or PDMS (Polydimethylsiloxane) and performs chemical analysis there, is used in various fields such as biotechnology, medicine, and the environment. Has attracted a lot of attention. Micro-TAS integrates and miniaturizes biochemistry and chemical operations (mixing, reaction, separation, detection) that have been performed in the laboratory on a single chip, that is, on a microfluidic device. Compared to conventional analyzers, the amount of samples, reagents, waste liquids, etc. can be reduced. Moreover, since the volume of the reaction solution is small, the reaction efficiency is high and the measurement time can be shortened. In addition, benefits such as cost reduction and portability are shown.

An analysis method in which Micro-TAS is frequently used is an enzyme immunoassay (Enzyme-Linked ImmunoSorbent Assay: ELISA). ELISA is a technique for detecting and quantifying an antigen in a sample solution using an antigen-antibody reaction. In Micro-TAS, there are a case where an antibody is bound to a channel wall surface in a microfluidic device and a case where an antibody is bound to a micro-sized bead surface. In Micro-TAS, since the amount of the reaction solution is small, the contact probability between the antigen and the antibody is increased, and the reaction rate is improved as compared with a normal ELISA.

Patent Document 1 describes a technique for improving the efficiency of ELISA on Micro-TAS. In this document, microbeads to which an antibody is bound are used. Further, a bead flow blocking portion such as a dam structure exists in the flow path of the microfluidic device. In the dam structure, the channel height is narrower than other channels, and the channel height is smaller than the bead diameter. Thus, fluid can pass through the dam structure, but the beads stop at the dam structure. For this reason, beads are concentrated near the dam structure. By generating an antigen-antibody reaction or an enzyme reaction in a concentrated state, the reaction can be efficiently advanced.

There is Patent Document 2 as a technology having a dam structure in a microfluidic device. Patent Document 2 describes a method for discarding beads introduced into a microfluidic device. In the analysis method using beads, it is necessary to refill the beads in order to repeatedly use the microfluidic device. For this reason, after analyzing one sample, it is necessary to first discard the beads introduced into the device. A common disposal method is backwashing. In the backwashing method, the beads are pushed out of the microfluidic device by flowing a washing solution in a direction opposite to that when beads are introduced. However, in order to realize the backwashing method, it is necessary to connect a liquid feed pump to both the inlet side and the outlet side of the microfluidic device in advance, or to replace the pump as necessary, and the operation is complicated. is there. In Patent Document 2, in order to solve this problem, a bead discarding flow path is connected to a dam structure portion that blocks beads. When the beads are introduced into the microfluidic device, the beads are concentrated near the dam structure by closing the valve of the bead disposal flow path and opening the valve at the downstream outlet of the dam structure. After the analysis is completed, the valve at the downstream outlet of the dam structure is closed, the valve of the bead discarding channel is opened, and the cleaning liquid is introduced to push the beads out of the microfluidic device through the discarding channel. In this method, the liquid feeding pump may be installed only on the inlet side of the microfluidic device.

Patent Document 3 also describes a method for discarding beads. This document proposes a microfluidic device having a variable channel cross-sectional area. A valve body that slides in the microfluidic device is installed, and the flow path cross-sectional area is changed by moving the valve body with a motor or the like. In a state in which the cross-sectional area is narrowed, the valve body plays the role of the above-described dam structure portion, and the beads can be stopped. After the analysis, slide the valve body to widen the cross-sectional area and allow the beads to pass through and discard. Also in this method, the liquid feeding pump may be installed only on the inlet side of the microfluidic device.

As in Patent Document 4, magnetic beads may be introduced into the microfluidic device. In this document, magnetic beads to which target molecules are bound are introduced into a microfluidic device and transported to the vicinity of an electrophoresis gel. When magnetic beads are used, the magnetic beads can be trapped by a magnet installed outside the microfluidic device. In Patent Document 4, magnetic beads are concentrated near the dam structure by a magnet. Moreover, it is also possible to move a magnetic bead within a device by moving the magnet outside a microfluidic device like patent document 5. FIG.

WO 03/062823 A1 JP 2007-108075 A JP 2005-851 Gazette JP 2007-47149 A JP 2004-77258 A

In the method of introducing microbeads into a microfluidic device for analysis, a method of trapping and concentrating microbeads and a method of discarding after analysis are important. In Patent Documents 1 to 3, micro beads are trapped using a dam structure. In this case, the height of the dam opening needs to be smaller than the diameter of the bead. Since the manufacture becomes difficult as the height is narrowed, the height of the dam opening is generally about 10 to 20 μm, and the diameter of the beads is 30 μm or more. For example, consider an analysis in which an antibody is bound to the bead surface and reacted with an antigen in a sample. In this case, as the surface area of the beads is increased, the collision probability between the antibody and the antigen is improved, and the reaction rate can be increased. The simplest method for increasing the bead surface area is to make the beads smaller, but there is a problem that the bead diameter is limited by the height of the dam opening as described above.

As in Patent Document 4, magnetic beads can be trapped by the force of a magnet installed outside the microfluidic device. In this case, since the beads can be trapped without a dam structure, the beads can be miniaturized. The diameter of magnetic beads generally used is about 3 μm. The problem with this approach is that the flow rate in the microfluidic device is limited. When the flow of the microchannel of the microfluidic device is assumed to be laminar, magnetic beads oriented parallel to the flow path is the force F _f applied from the fluid expressed by the following equation.

Formula 1

Here, ρ is the density of the fluid, v is the flow velocity, and A is the cross-sectional area of collision of the magnetic beads with the fluid.

Further, the force F _m acting on the magnetic beads against the fluid force by arranging the magnet can be expressed by the following equation.

Formula 2

Here, mu is the coefficient of friction with magnetic beads and the microchannel wall, the F _M attraction of the magnet, theta is the angle of the suction force and the wall.

Therefore, the magnetic beads are trapped without being pushed away by the fluid only when F _m > F _f . When considering one magnetic bead, since A is sufficiently small, F _m > F _f is satisfied in many cases. However, when a large number of magnetic beads are trapped, each of them is combined and A becomes large, and the force of the fluid is increased. It becomes easy to lose. The number of magnetic beads to be used affects the reaction rate and the signal at the time of analysis, and the flow rate is a factor that determines the introduction rate of the magnetic beads and affects the throughput of the analysis. Even if it is desired to increase the number of magnetic beads in order to increase the reaction rate, since A becomes large and F _m > F _f does not hold, there may be a problem that the magnetic beads cannot be increased. Further, if the flow rate is increased to increase the throughput, F _f increases, and there is a possibility that the magnetic beads cannot be trapped.

Regarding the bead disposal method, as described above, in Patent Document 1, it is necessary to use the backwash method. In the backwashing method, the beads are pushed out of the microfluidic device by flowing a washing solution in a direction opposite to that when beads are introduced. However, in order to realize the backwashing method, it is necessary to connect a liquid feed pump to both the inlet side and the outlet side of the microfluidic device in advance, or to replace the pump as necessary, and the operation is complicated. .

In Patent Document 2, a flow path for bead disposal is provided in order to avoid the backwashing method. However, the bead discarding channel works as a dead volume. For example, there is a technique in which a target molecule is bound to a bead and then introduced into a microfluidic device, and magnetic beads are trapped and concentrated, and then the target molecule is eluted from the bead and analyzed. In the case of this method, the target molecule diffuses not only to the analysis channel but also to the bead disposal channel, which causes a problem that the concentration of the target molecule in the analysis channel decreases.

Patent Document 3 proposes changing the cross-sectional area of the flow path. For example, when a sliding valve body is used, the degree of sealing of the slide portion is poor, and the probability of liquid leakage increases when the internal pressure inside the microfluidic device increases. On the other hand, the microfluidic device itself can be deformed to change the cross-sectional area of the channel, but it cannot be applied to materials that are difficult to deform, such as glass.

A microfluidic device that uses magnetic beads introduced into a microchannel used in the present invention includes an introduction channel in which magnetic beads are introduced together with a solution, and an analysis channel that branches from the introduction channel at a branching portion. A dam structure that has a waste flow path, and whose flow path height is narrower than the flow path height of the branch part at the connection part between the analysis flow path and the branch part of at least one of the waste flow paths The flow path height narrowed by the dam structure is larger than the diameter of the magnetic beads.

In the present invention, the magnetic beads adsorbing the target molecules are trapped by the force of the dam structure in the microfluidic device and the magnet installed outside. The magnetic beads can get over the dam structure by controlling the movement by magnetic force. Therefore, in one aspect, after the analysis is completed, the magnetic beads are efficiently discarded by controlling the movement of the magnetic beads by the magnetic force and getting over the dam structure. As a result, high-throughput and robust measurement is possible.

According to the present invention, high-throughput and robust measurement can be realized.

Issues, configurations, and effects other than those described above will be clarified by the following description of the embodiments.

The schematic diagram which shows the structural example of a microfluidic device. Schematic which shows an example of the drive sequence in the analysis method using a microfluidic device. The schematic diagram which showed the positional relationship of a microfluidic device and a mass spectrometer. Schematic which shows an example of the analysis system containing a microfluidic device. The schematic diagram which showed the example of the method of driving a magnet in the direction perpendicular | vertical to the microfluidic device surface. The schematic diagram which shows an example of the method of sliding a magnet in the direction parallel to the surface of a microfluidic device. The figure which shows the structural example at the time of using an electromagnet. The schematic diagram which shows the structural example of a magnet. The schematic diagram which shows the example of a dam structure. The schematic diagram which shows the example of the modification method of a magnetic bead. The schematic diagram which shows the manufacturing method of the microfluidic device which has a dam structure. The schematic diagram which shows the manufacturing method of the microfluidic device which has a dam structure. The schematic diagram explaining removal of a microfluidic device. Schematic which shows the other example of the drive sequence in the analysis method using a microfluidic device. The cross-sectional schematic diagram of the microfluidic device with which the ESI chip was connected to the bottom face. The schematic diagram which shows the example which detects a target molecule with a photodetector. The schematic diagram which shows the example which detects a target molecule with a photodetector. The plane schematic diagram which shows the example of the flow-path structure of the microfluidic device which has several flow paths for analysis.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.

Example 1 is an example in which when target molecules are measured by a mass spectrometer, the target molecules are concentrated and ionized using a microfluidic device. In Example 1, magnetic beads having target molecules adsorbed are introduced into a microfluidic device, and the magnetic beads are trapped and concentrated by an external magnet. Subsequently, target molecules are eluted from the magnetic beads and ionized by electrospray ionization (ESI).

FIG. 1 is a schematic diagram showing a configuration example of a microfluidic device of this example, FIG. 1 (a) is a schematic plan view showing a channel structure, and FIG. 1 (b) is a cross-sectional view taken along line BB in FIG. 1 (a). It is a cross-sectional schematic diagram of the device corresponding to a cross section. The material of the microfluidic device 4 is glass, PDMS, or the like. In general, the microfluidic device 4 is manufactured by bonding two plates. Glass has higher adhesion than PDMS, resulting in higher pressure resistance. The size varies depending on the analysis method, but is generally about 30 mm × 70 mm. When the magnetic beads in the microfluidic device 4 are controlled by the magnetic force of the magnet, it is better to bring the magnet as close to the magnetic beads as possible. The thinner the microfluidic device 4 is, the closer the magnet is to the magnetic beads. On the other hand, the strength decreases as the thickness decreases. For this reason, the thickness of the microfluidic device 4 is generally about 1.4 mm. However, it is also possible to bring the magnet closer while maintaining the rigidity of the device by scraping the surface only at the part where the magnet is brought closer. The microfluidic device 4 of the present embodiment has three ports connected to the outside, which are an introduction port 2, a disposal port 1, and an ESI chip connection port 9, respectively. The diameter of the general introduction port 2, the disposal port 1, and the ESI chip connection port 9 is about 100 to 1000 μm, but is not limited to this range. In FIG. 1, the connection piping to the introduction port 2 and the disposal port 1 and the ESI chip are not shown.

The introduction channel 3 communicates with the introduction port 2, and magnetic beads are introduced from the introduction port 2 together with the solution. The end of the analysis channel 6 communicates with the ESI chip connection port 9, and the magnetic beads moved to the analysis channel 6 are detected or processed. In this embodiment, a process for eluting target molecules from the magnetic beads in the analysis flow path 6 is performed. In the discard channel 8, magnetic beads that are communicated with the discard port 1 and discarded are flowed. The introduction flow path 3 branches into the analysis flow path 6 and the waste flow path 8 at the branching portion, and the flow path height is smaller than the flow path height of the branching portion at the connection portion with the branching portion of the waste flow path 8. The dam structure 7 is provided. That is, the dam structure 7 is disposed on the inlet side of a channel having a large conductance during use among the channels branched from the introduction channel 3. In other words, it can also be said that the dam structure 7 is disposed on the waste flow path 8 side of the intersection where the three

micro flow paths

3, 6, and 8 intersect. The dam structure 7 of the embodiment shown in FIG. 1 is configured by a baffle plate that projects to the full width of the flow path from the bottom wall to the top wall of the flow path. The cross section of the flow path is narrowed by the dam structure 7, and in particular, the height direction dimension of the flow path is limited to the dam opening above the dam structure at the position of the dam structure 7. However, the flow path height narrowed by the dam structure 7 is larger than the diameter of the magnetic beads, so that the magnetic beads can pass through the dam opening above the dam structure. The channel has a width of about 100 to 5000 μm and a depth of about 50 to 300 μm. The height of the dam opening of the dam structure 7 must be at least larger than the diameter of the magnetic beads to be used, and is preferably at least twice the diameter of the magnetic beads. A typical dam opening has a height of about 20 μm. Since the diameter of a typical magnetic bead is about 1 to 10 μm, it can pass through a dam opening having a height of 20 μm.

In this embodiment, magnetic beads pass through the dam opening. The height of the dam structure 7 is a value obtained by subtracting the height of the dam opening from the channel depth. Since the typical flow path depth is about 100 μm, the typical height of the dam structure 7 is about 80 μm. The introduced magnetic beads are trapped in the magnetic bead trap part 5 near the dam structure by a magnetic force, and then transported to the analysis channel 6 by applying the magnetic force. It is necessary to introduce all the magnetic beads trapped by the magnetic bead trap section 5 into the analysis flow path 6. This is because if the magnetic beads 13 do not fully enter the analysis flow path 6 and the magnetic beads 13 protrude from the magnetic bead trap section 5, the target molecules eluted from the protruding magnetic beads will also flow to the disposal flow path 8 side due to diffusion. For this reason, it is desirable that the volume of the analysis flow path 6 be equal to or larger than the volume of the trapped magnetic beads, that is, the volume of the magnetic beads introduced into the microfluidic device. The amount of magnetic beads required depends on the amount of target molecules, and the volume of the analysis flow path 6 is designed accordingly. Considering the elution efficiency of the target molecule and the diffusion after the elution, it is desirable to design so that the entire analysis channel 6 is filled with the trapped magnetic beads. The branch part of the flow path described above corresponds to the intersection part of the flow path, and a magnetic force is applied thereto to trap the magnetic beads, so that the branch part of the flow path also overlaps with the magnetic bead trap part 5. That is, the branch part of the flow path, the crossing part of the flow path, and the magnetic bead trap part 5 indicate substantially the same region.

FIG. 2 is a schematic diagram showing an example of a driving sequence in the analysis method using the microfluidic device of this example. In FIG. 2, the magnetic beads 13, the

magnets

11 and 12, and the ESI chip 14 are added to the schematic cross-sectional view of the microfluidic device 4. However, illustration of a microfluidic device holder and a magnet driving mechanism which will be described later is omitted. One

magnet

11, 12 is installed on each of the upper surface side and the bottom surface side of the microfluidic device 4. However, it does not necessarily have to be one by one, and there is no problem even if there is a plurality. Here, of the pair of large planes constituting the outer shape of the microfluidic device 4, the plane on the side where the base of the dam structure 7 is provided is referred to as the bottom surface, and the plane on the opposite side is referred to as the top surface.

As shown in FIG. 2A, at the stage of introducing the magnetic beads 13 into the microfluidic device 4, the lower magnet 12 on the bottom surface side of the microfluidic device 4 is installed near the microfluidic device 4, and the upper magnet on the upper surface side. 11 is installed at a position away from the microfluidic device 4. The lower magnet 12 is closer to the microfluidic device 4 than the upper magnet 11 is. There is no specification for the direction of the pole of the magnet. The magnetic force of the lower magnet 12 acts on the magnetic bead trap portion 5 of the microfluidic device 4.

When the magnetic beads 13 are introduced together with the solution from the introduction port 2 in this magnet installed state, the magnetic beads 13 are attracted to the inner wall of the flow path by the magnetic force of the lower magnet 12 as shown in FIG. Be trapped. The solution introduced from the introduction port 2 flows toward the disposal port 1 and the ESI chip connection port 9 as indicated by arrows. As described above, the diameter of the introduction port 2, the disposal port 1, and the ESI chip connection port 9 is about 100 to 1000 μm, but the tip diameter of the ESI chip 14 is smaller than that. That is, the conductance from the introduction port 2 to the disposal port 1 is larger than the conductance from the introduction port 2 to the tip of the ESI chip. Or it is better to design the device structure to be larger. For example, the conductance from the introduction port 2 to the disposal port 1 is designed to be 100 times the conductance from the introduction port 2 to the tip of the ESI chip. At this time, when the solution is introduced at 100 μL / min, the solution flows at about 100 μL / min on the disposal port side and at about 1 μL / min on the ESI chip connection port side.

In the first embodiment, the dam structure 7 is installed on the waste flow path side where most of the magnetic beads 13 flow due to such a flow rate difference, that is, on the connection portion with the flow path branching portion of the waste flow path 8. When the magnetic beads 13 are introduced, the magnetic beads 13 are attracted to the bottom surface of the flow path by the lower magnet 12. As described above, when the magnetic beads 13 are trapped only by a magnet, they can be trapped only when F _m > F _f . However, in this embodiment, the dam structure 7 is arranged on the waste flow path 8 side of the magnetic bead trap unit 5. Therefore, even if F _f > F _m, the dam structure 7 is present, so that the magnetic beads 13 do not slide due to the force of the fluid. By installing the dam structure 7, it becomes possible to trap the magnetic beads 13 in the magnetic bead trap unit 5 even when the flow rate is increased compared with the case where the dam structure 7 is not installed, and the analysis throughput can be improved. Since the solution flowing in the analysis channel 6 has a low flow rate, the magnetic beads receiving the magnetic attraction force are not pushed toward the analysis channel 6.

After trapping the magnetic beads 13 as shown in FIG. 2A, the lower magnet 12 is slid along the surface of the microfluidic device 4 as shown in FIG. While attracting to the inner wall of the path 6 by magnetic force, it is moved to the vicinity of the ESI chip 14 to be dense. In this state, the eluate is introduced into the microfluidic device 4, the target molecules are eluted from the magnetic beads 13, introduced into the ESI chip 14, and ionized. In order to move the magnetic beads 13 efficiently, the inlet of the analysis flow path 6 is preferably chamfered as shown in FIG.

A general technique for concentrating target molecules using the magnetic beads 13 is to add the magnetic beads 13 to the sample solution containing the target molecules to adsorb the target molecules to the magnetic beads 13, and then to detach the magnetic beads 13. Release. Subsequently, the target molecules are eluted from the magnetic beads 13 by mixing the magnetic beads 13 and the eluate. That is, the target molecule can be concentrated as the amount of the elution solution is smaller than the sample solution. In the technique of adding the magnetic beads 13 to a 1.5 mL microtube or the like and adding the eluate thereto to elute the target molecules, it is difficult to reduce the amount of the eluate due to handling problems. Further, the obtained eluate is usually introduced into a mass spectrometer with a sample injector or the like and analyzed. In the case of this method, the problem arises that the target molecules diffuse in the pipe from the sample injector to the ESI chip 14 and the concentration becomes low.

On the other hand, in this embodiment, after concentrating the magnetic beads 13 in the microfluidic device 4, elution of target molecules from the magnetic beads 13 and introduction of the eluate into the ESI chip 14 are performed online. For this reason, the amount of the eluate can be reduced to a high concentration rate. Further, since the target molecules are eluted after transporting the magnetic beads 13 to the vicinity of the ESI chip 14, the eluate does not diffuse and can be ionized while maintaining a high concentration.

As shown in FIG. 2 (c), the target molecules are eluted from the magnetic beads 13 and analyzed, and then the lower magnet 12 is moved to move the magnetic beads 13 to the vicinity of the dam structure 7 along the inner wall of the analysis flow path 6. transport. In order to carry the magnetic beads 13 to the waste channel 8, it is necessary to get over the dam structure 7. Therefore, the lower magnet 12 and the upper magnet 11 are moved as shown in FIG. That is, the lower magnet 12 is separated from the microfluidic device 4 so that the magnetic force of the lower magnet 12 does not reach the magnetic beads 13. On the other hand, by bringing the upper magnet 11 close to the microfluidic device 4 and causing the magnetic force of the upper magnet 11 to act on the magnetic beads 13, the magnetic beads 13 are moved to the inner wall on the upper magnet 11 side.

At this time, since the magnetic beads 13 are stuck to the upper wall of the flow path, when the upper magnet 11 is moved along the microfluidic device 4 as shown in FIGS. The magnetic beads 13 can be transported to the waste flow path 8 over the distance. Furthermore, as shown in FIG. 2G, the magnetic force applied to the magnetic beads is reduced by moving the magnet to a position away from the waste flow path 8, and the magnetic beads 13 are released from the magnetic force. The magnetic beads 13 released from the magnetic force ride on the flow of the solution and are discarded from the disposal port 1 to the outside of the microfluidic device 4.

In the method of this embodiment, the solution flow is flowing from the inlet 2 toward the waste port 1 or the ESI chip connection port 9 even when the magnetic beads 13 are discarded. That is, it is possible to discard the magnetic beads 13 without using the problematic backwashing method.

As a method for discarding the magnetic beads 13, it is conceivable to release the magnetic beads 13 from the magnetic force at the stage of FIG. However, since there is a solution flow from the magnetic bead trap unit 5 to the ESI chip 14 side, a small amount of the magnetic beads 13 flows to the ESI chip 14 side instead of the waste outlet 1 side when the magnetic force is released. Since the tip diameter of the ESI chip 14 is usually small, there is a high possibility that clogging will occur when the magnetic beads 13 flow in. For this reason, when discarding the magnetic beads 13 as in the present embodiment, it is necessary to release the magnetic force after the dam structure 7 is moved over the magnetic beads 13.

There is also a concept of providing a valve on the ESI chip 14 side. If a valve is installed somewhere between the magnetic bead trap section 5 and the ESI chip tip, the magnetic bead will move to the ESI chip 14 side if the magnetic force is released after closing the valve in the stage of FIG. Does not flow and flows only to the disposal port 1 side. However, if a valve is installed between the magnetic bead trap section 5 and the ESI chip tip, the structure becomes very complicated. In addition, when a valve is installed at a portion through which the eluate passes, the portion becomes a dead volume and causes solution diffusion. Thus, also from the viewpoint of the simplicity of the structure and the small dead volume, it can be seen that it is an effective means to get over the dam structure 7 when discarding the magnetic beads 13.

After discarding the magnetic beads 13, the magnet is returned to the position as shown in FIG. 2 (h) and waits for the next measurement. When the magnetic beads 13 are introduced in this state, the state transitions to the state of FIG.

FIG. 3 is a schematic diagram showing the positional relationship between the microfluidic device and the mass spectrometer. The microfluidic device 4 is fixed to the magnet drive mechanism 17 so as to have a predetermined positional relationship. Details of the magnet drive mechanism 17 will be described later.

A DC voltage of about 1 to 5 kV is generally applied to the ESI chip 14 connected to the analysis flow path 6. Charged droplets are sprayed from the ESI chip 14 due to a potential difference from the pore 15 portion of the mass spectrometer 16, and molecules inside the droplets are ionized. The ions are separated and analyzed by the mass-to-charge ratio inside the mass spectrometer 16. If the ESI chip 14 is a conductor, a voltage can be applied by wiring the ESI chip 14 itself. On the other hand, when the ESI chip 14 is a nonconductor such as PEEK (Polyetheretherketone), a method of applying a voltage to the solution is often used. If a part of piping that contacts the solution flowing to the ESI chip 14 is made of metal and a voltage is applied to the pipe, the potential is transmitted to the tip of the ESI chip, and electrospray ionization can be performed.

The assumed risk in this embodiment is that the magnetic beads 13 flow into the tip side of the ESI chip 14 and become clogged with the chip. In order to reduce this risk, as shown in FIG. 3, it is good to arrange | position so that the dam structure 7 side and the waste outlet 1 side may become a lower side with respect to the ESI chip | tip 14 side. In this case, gravity acting on the magnetic beads 13 works in a direction away from the ESI chip 14. For example, even if the magnet is moved at a speed that the magnetic beads 13 cannot follow, the magnetic beads 13 move away from the ESI chip 14 due to gravity if the solution is not flowing or the flow rate of the solution is very slow. To do. However, it is not necessarily limited to the arrangement of FIG. For example, if the risk that all the magnetic beads 13 cannot be transported to the ESI chip 14 at the stage of FIG. 2B is more important than the risk of the magnetic beads 13 flowing into the ESI chip tip side, It is good to install so that the ESI chip | tip 14 side may face down contrary to 3. FIG. Further, the arrangement of the microfluidic device 4 may be limited by the structure around the pore 15 of the mass spectrometer 16.

FIG. 4 is a schematic diagram showing an example of an analysis system including a microfluidic device. The analysis system includes a microfluidic device 4, a magnet drive mechanism 17, a mass spectrometer 16, a sample injector 24, a liquid feed pump 25, a waste liquid valve 23, a high-voltage power supply 20, and a control computer 18. The microchannel, magnetic beads, etc. are not shown. The microfluidic device 4 has been described with reference to FIGS. The magnet drive mechanism 17 includes a magnet, and is configured by a motor or a circuit that moves the magnet. The sample injector 24 and the liquid feeding pump 25 are coupled together, the magnetic beads 13 are introduced together with the solution by the sample injector 24, and the solution is fed by the liquid feeding pump 25. The sample injector 24 and the liquid feed pump 25 are not special special ones, and a general liquid chromatography (LC) system may be used. However, a system capable of delivering the solution at least at about 100 nL / min is desirable. In addition, the system needs to be able to send multiple types of solutions.

The magnetic beads 13 introduced from the sample injector 24 pass through the introduction pipe 21 and are introduced into the microfluidic device 4. After completing the mass analysis in the drive sequence as shown in FIG. 2, the magnetic beads 13 are discarded from the waste flow path 8 to the waste liquid tank 26 through the waste pipe 22 and the waste liquid valve 23. Any type of mass spectrometer 16 may be used, such as a triple quadrupole mass spectrometer, an ion trap, and a time-of-flight mass spectrometer. Further, in this embodiment, since the target molecule is ionized by ESI, it is not necessary to be a mass spectrometer as long as it is an apparatus for analyzing ions. For example, it is possible to analyze with an ion mobility meter.

As a premise of the present embodiment, the surface of the magnetic bead 13 is modified in some way so that target molecules can be adsorbed. The target molecules are adsorbed in a solution containing a large amount of H ₂ O under, for example, serum or phosphate buffered saline (PBS). On the other hand, when the magnetic beads 13 are added to the organic solvent, the target molecules are eluted into the organic solvent. Based on this premise and FIGS. 1 to 4, an example of the drive sequence of this embodiment will be described.

(1) The magnetic beads 13 on which the target molecules are adsorbed are introduced into the microfluidic device 4 through the sample injector 24 together with the aqueous solution. The flow rate at that time is, for example, 100 μL / min. The aqueous solution is, for example, PBS. At this time, the waste liquid valve 23 connected to the waste flow path 8 is in an open state, and the solution introduced from the introduction port 2 flows out of the microfluidic device 4 through the waste port 1 and the ESI chip 14. To do.

(2) The introduced magnetic beads 13 are trapped in the vicinity of the dam structure 7, that is, in the magnetic bead trap section 5 by the magnetic force of the lower magnet 12.

(3) The introduction of the soot solution is stopped, and the magnetic beads 13 are concentrated in the vicinity of the ESI chip connection port 9 by moving the lower magnet 12 to the ESI chip 14 side without the flow of the solution.

(4) An organic solvent is introduced from the feed pump 25 into the microfluidic device 4 as an eluent. For example, methanol. The organic solvent need not necessarily be 100%, and may be a 50% aqueous methanol solution. The waste liquid valve 23 may be introduced in an open state or may be introduced in a closed state. Alternatively, the waste liquid valve 23 may be closed after a certain amount of organic solvent is introduced. When the conductance on the waste outlet 1 side is sufficiently larger than that on the ESI chip 14 side, the waste flow path 8 side is filled with the organic solvent before the organic solvent is introduced into the entire analysis flow path 6. It is desirable to close the waste liquid valve 23 when the waste flow path 8 side is filled with the organic solvent. However, the introduction flow rate is adjusted so that the flow rate flowing to the ESI chip 14 side is about 1 μL / min. When the waste liquid valve 23 is closed, the liquid feed amount of the liquid feed pump 25 is set to about 1 μL / min, and when the target molecule is eluted while the waste liquid valve 23 is open, the waste outlet 1 side and the ESI chip The flow rate is determined based on the conductance ratio on the 14th side.

(5) The target molecules adsorbed on the magnetic beads 13 are eluted by the contact between the organic solvent and the magnetic beads 13. The eluted molecules are introduced into the ESI chip 14 and ionized by ESI.

(6) The ionized molecules are analyzed by the mass spectrometer 16.

(7) After elution is completed, stop sending the organic solvent. By moving the magnet, the magnetic beads 13 are transported to the magnetic bead trap unit 5.

(8) The upper magnet 11 and the lower magnet 12 are moved so that the magnetic beads 13 get over the dam structure 7 and the magnetic beads are introduced into the waste flow path 8.

(9) The magnetic beads 13 are released from the magnetic force by moving the upper magnet 11 to a position away from the waste flow path 8.

(10) The waste liquid valve 23 is opened and the cleaning solution is introduced into the microfluidic device 4 to push the magnetic beads 13 out of the microfluidic device 4 from the waste outlet 1.

(11) Return the saddle magnet to its original position and enter the standby state for the next measurement.

Here, ESI is applied as an ionization method, but this embodiment is not limited to ESI. Although similar to ESI, a thermospray ionization method using no voltage may be used. Alternatively, the eluate may be vaporized and then ionized. In that case, atmospheric pressure chemical ionization (APCI), photoionization (PI), barrier discharge ionization (DBDI), secondary electrospray ionization (SESI), etc. It is also possible to use. Alternatively, after dropping the eluate onto a plate or the like, desorption electrospray ionization (DESI) or matrix-assisted laser desorption / ionization (Matrix Assisted Desorption / Ionization, MALDI) may be used.

FIG. 5 is a schematic diagram showing an example of a method for driving a magnet in a direction perpendicular to the surface of the microfluidic device. The upper magnet 11 and the lower magnet 12 held by the magnet drive mechanism are coupled via a magnet holder 27 and are driven by a rack and pinion mechanism 29 in synchronization with a direction perpendicular to the device surface. That is, the two

magnets

11 and 12 arranged with the microfluidic device 4 interposed therebetween are driven so that when one magnet approaches the microfluidic device 4, the other magnet moves away. In other words, the magnet drive mechanism drives the two

magnets

11 and 12 so that when one magnet acts on the microchannel of the microfluidic device 4, the other magnet stops acting. In this embodiment, the rotational motion by the DC motor is converted into the vertical motion of the magnet. It is desirable to monitor the position of the magnet with an optical sensor or the like and control the rotation of the DC motor.

The distance between the

magnets

11 and 12 should be changed according to the magnitude of the magnetic force. When the distance between the magnets is short, both magnetic forces act on the magnetic beads 13. When the distance between the two magnets is equal to the magnetic bead 13, the magnetic bead 13 cannot be brought to one side of the flow path as shown in FIG. For this reason, when one magnet is approaching the microfluidic device 4, the other magnet is sufficiently separated from the microfluidic device 4 and the magnetic force from the one magnet is mainly acting on the magnetic beads 13. Need to produce. However, the fact that the distance between the magnets increases means that the driving distance of the magnets increases, and a problem arises that the size of the entire apparatus increases. For example, when a magnet having a strength of about 100 to 300 mT is used, the distance between the magnets may be about 10 to 30 mm. However, the range is not limited to this range, and the distance between the magnets is not limited as long as the magnetic beads 13 can be moved as in the sequence of FIG.

Further, it is not always necessary to connect the upper magnet 11 and the lower magnet 12. You may attach a drive mechanism to each and move independently. The moving method of the magnet is not limited to the rack and pinion mechanism 29, and any method can be used as long as it can be controlled by computer control, such as a stepping motor. If the control system becomes uncontrollable due to a software error or the like, the magnet may collide with the microfluidic device 4. For this reason, it is desirable to prepare a physical stopper so that the magnet does not collide with the microfluidic device 4 even when control becomes impossible.

FIG. 5 also shows the microfluidic device holder 28. The microfluidic device holder 28 is divided into upper and lower parts, one has a concave structure into which the microfluidic device 4 can be inserted and is fixed to the magnet drive mechanism 17, and the other is a pressing plate for fixing the microfluidic device 4. It is. In a state where the microfluidic device 4 is inserted into the recess of the microfluidic device holder, the microfluidic device 4 is fixed by, for example, screwing the holding plate. Although the microfluidic device holder 28 is omitted in other drawings, it is usually used together with the microfluidic device 4. If the entire microfluidic device 4 is covered with a holder, the magnet cannot be brought closer to the microfluidic device 4 by the thickness of the holder. In view of this, the holder of the present embodiment is scraped according to the shape of the magnet only at the portion where the magnet approaches, and has a structure in which the magnet is as close to the microfluidic device 4 as possible. Although it is possible to cut a large area without necessarily matching the shape of the magnet, there is a case where a decrease in rigidity becomes a problem.

FIG. 6 is a schematic diagram showing an example of a method of sliding a magnet in a direction parallel to the surface of the microfluidic device. The ESI chip 14 and the magnetic beads 13 are not shown. The magnet drive mechanism 17 causes the two

magnets

11 and 12 to perform a synchronized slide and independent slide along the surface of the microfluidic device 4.

One magnet 12 is coupled to a drive stage 30 driven by a stepping motor. As shown in FIG. 5, since the upper magnet 11 and the lower magnet 12 are coupled, when one magnet slides with the drive stage 30, the other magnet also slides in synchronization. However, as shown in FIG. 2B, when the magnetic beads 13 are transported to the ESI chip 14 side, only the lower magnet 12 slides and the upper magnet 11 does not slide. This is because if the upper magnet 11 is also slid, the upper magnet 11 collides with the ESI chip 14. In the example shown in FIG. 6, the slider 31, the conston spring 32, and the stopper 45 are installed so that the upper magnet 11 does not follow the lower magnet 12. A stopper 45 exists on the ESI chip side, and the stopper 45 and the magnet 11 collide so that the magnet cannot advance ahead of the stopper 45. The slide in the reverse direction is restrained by the conston spring 32. The method for adjusting the position of the magnet is not limited to the method described in FIG. 6, and any method may be used as long as the movement as shown in FIG. 2 is possible.

The magnet used in this embodiment may be anything as long as it can apply a magnetic force to the magnetic beads 13. Among permanent magnets, neodymium magnets are strong and easy to use. On the other hand, an electromagnet may be used as shown in FIG. It is a general electromagnet to wind a coil 34 around a bar having a high magnetic permeability such as an iron core 33. If an electromagnet is used in contrast to the case where a permanent magnet is used, a drive mechanism as shown in FIG. 5 is not necessary. Since an electromagnet generates a magnetic force only when an electric current is flowing, it is only necessary to pass an electric current through the coil 34 of the upper magnet 11 and the lower magnet 12 to be used. For example, if a current is passed through the lower magnet 12 in the stages of FIGS. 2A to 2C and the upper magnet 11 in the stages of FIGS. 2D to 2F, this can be achieved without the drive mechanism shown in FIG. The control method of the magnetic beads 13 described up to here can be realized. However, it is difficult for an electromagnet to generate a magnetic force equivalent to that of a neodymium magnet. A large current is required and heat generation becomes a major issue. In addition, if the magnetic field is changed by arranging electromagnets and passing a current only through the necessary electromagnets, the position of the magnetic beads can be controlled without sliding the magnets. When an electromagnet is used as the magnet, the magnet drive mechanism 17 stops energizing the electromagnet on the other side when energizing the electromagnet on the other side, that is, with respect to the microchannel of the microfluidic device. When the electromagnet on one side applies a magnetic force, the two electromagnets are driven so that the electromagnet on the other side stops the action.

The shape of the magnet is not necessarily a spherical shape as shown in FIG. FIG. 8 is a schematic diagram showing an example of the structure of a magnet. Only the tip as shown in FIG. 8A may have a spherical shape, a rectangular parallelepiped as shown in FIG. 8B, or a triangular pyramid as shown in FIG. 8C. The magnetic beads gather at the portion where the magnetic force is the strongest. However, when there are a plurality of corners such as a rectangular parallelepiped, there arises a problem that the locations where the magnetic beads 13 are concentrated are dispersed.

FIG. 9 is a schematic sectional view showing an example of a dam structure. FIGS. 9A to 9E are schematic partial cross-sectional views showing only the portion corresponding to the dam structure 7 of FIG. 1B in an enlarged manner. This shows a state of being trapped in a branch portion of the magnetic beads, that is, a magnetic bead trap portion.

As shown in FIGS. 9A to 9E, the dam structure 7 can have various shapes. FIG. 9A shows the dam structure 7 shown so far, and has a rectangular cross section. FIG. 9B shows a dam structure having an inverted triangular cross section in which the width of the dam bottom is narrower than the top. The magnetic beads 13 inserted in the gaps at the bottom do not move even if the flow rate increases. That is, the trap efficiency is increased. However, there are problems in that the manufacturing is difficult and the efficiency of disposal may be reduced. The dam structure 7 shown in FIG. 9C has a trapezoidal cross section. Even if the structure shown in FIG. 9A is to be manufactured, the structure shown in FIG. In the case of the dam structure 7 having a trapezoidal cross section, the magnetic beads 13 may slide up the dam structure 7 when the flow velocity increases. Also, as shown in FIG. 9 (d), there is a structure in which a recess is provided in the microchannel, and the wall of the recess constitutes a barrier that must be overcome for the magnetic beads entering the recess to escape from the recess. This is the category of the dam structure 7 in the present invention. The portion of the vertical wall on the left side of the recess illustrated in FIG. 9D corresponds to the connection portion between the branch portion of the micro flow channel and the waste flow channel, and the location of the vertical wall on the right side of the recess is the micro flow channel. Corresponds to the connection between the branch and the analysis flow path. According to this dam structure, since the position of the magnetic beads 13 can be shifted downward from the flow of the fluid, the probability of being washed away by the fluid decreases. As shown in FIG. 9E, the structure in which the flow path height is changed stepwise also acts as the dam structure 7 of the present invention to prevent the magnetic beads from being washed away by the fluid. The portion of the vertical wall on the left side of the depression shown in FIG. 9 (e) corresponds to the connection portion between the branch portion of the microchannel and the waste channel.

In the present embodiment, a NanoESI chip is assumed as the ESI chip 14. NanoESI chips generally have an outer diameter of about 350 μm and a tip inner diameter of about 10 to 100 μm. When the NanoESI chip is used, the flow rate to flow to the chip side is preferably 5 μL / min or less. In addition to a hollow chip inside, a chip in which an LC column is included may be used. In addition to the LC column packed with particles, a chip containing a monolithic column may be used. When the LC column is included, LC separation can be performed at that portion. Moreover, when the eluate for eluting the target molecules from the magnetic beads is not suitable for ionization, the solution can be exchanged by using an LC column.

There are various methods for adsorbing magnetic beads and target molecules. The adsorption method is not particularly limited as long as it can be eluted on the microfluidic device 4. For example, it is conceivable to modify the polymer 36 around the magnetic beads 13 as shown in FIG. This acts as a general solid phase extraction agent. For example, it is modified with C18. When the magnetic beads 13 are introduced into a solution rich in H ₂ O, the target molecules move into the polymer 36. Thereafter, when the magnetic beads 13 are introduced into the organic solvent, the target molecules are eluted. This is a method utilizing the polarity of molecules.

The molecular template polymer (Molecular imprinted polymer: MIP) selects molecules to be adsorbed rather than the solid phase extractant. When the polymer 36 is synthesized using the target molecule as a template, and then the target molecule is removed, the shape of the polymer 36 becomes a structure that can specifically capture the target molecule. This MIP may be modified on the surface of the magnetic beads 13. When MIP is used, the target molecule can be eluted with an organic solvent as in the case of the solid phase extraction agent.

Antibody 37 can adsorb molecules more specifically than MIP. The antibody 37 may be bound to the surface of the magnetic bead 13 as shown in FIG. In order to bind the antibody 37 to the magnetic bead 13, first, a molecule capable of binding to the antibody 37 such as Protein A or Protein G is bound to the surface of the magnetic bead 13. Alternatively, streptavidin is bound to the surface of the magnetic beads 13, and the biotinylated antibody 37 is bound thereto. When the antibody 37 and the target molecule are bound, the target molecule is generally eluted by denaturing the antibody 37 with an acidic solution. When elution is performed with an acidic solution, the acidic solution is not suitable for ESI, and thus the solution needs to be replaced before ESI. For this purpose, it is preferable to use the ESI chip 14 including the LC column as described above. The target molecule dissolved in the acidic solution is trapped with an LC column and eluted with an organic solvent.

There are two main methods for manufacturing the microfluidic device 4 having the dam structure 7. FIG. 11 and FIG. 12 are schematic views showing respective manufacturing methods. Basically, the microfluidic device 4 is manufactured by laminating two sheets. In the method of FIG. 11, both the upper plate 38 and the lower plate 39 are processed and bonded together. In this case, it is necessary to attach the upper plate 38 and the lower plate 39 with high accuracy. On the other hand, in the method shown in FIG. 12, the upper plate 38 is not processed, and only the lower plate 39 is processed. In order to make the dam structure 7, it is necessary to cut the depth of the lower plate 39 in two stages. For this reason, processing becomes complicated and takes time.

FIG. 13 is a schematic diagram for explaining the removal of the microfluidic device. The analysis method of this embodiment can perform a plurality of analyzes without replacing the microfluidic device 4. That is, the introduction, analysis, and disposal of the magnetic beads 13 are repeated. However, when a large amount of analysis is performed and the microfluidic device 4 has deteriorated, it is preferable to replace the device. In this case, it is desirable that only the microfluidic device 4 and the ESI chip 14 are replaced as shown in FIG. 13, and the magnet and the magnet drive mechanism 17 are not replaced. Of course, when the drive stage 30 or the DC motor deteriorates, the magnet drive mechanism 17 also needs to be replaced, but the usable period is longer than that of the microfluidic device 4 and the ESI chip 14. The microfluidic device 4 and the ESI chip 14 are not necessarily replaced at the same time, and may be replaced independently.

Although omitted in FIG. 13, the microfluidic device 4 is fixed to the microfluidic device holder 28, and when it is desired to replace the microfluidic device 4, the microfluidic device holder 28 is generally replaced. is there. In this case, it is desirable that the magnet driving mechanism 17 and the microfluidic device holder 28 are not fixed by screws, but the microfluidic device holder 28 can be attached and detached from the magnet driving mechanism 17 with one touch.

As shown in FIG. 13, the magnet drive mechanism 17 is fixed to a magnet drive mechanism fixing portion 48 via a fixing scaffold 49. The fixing scaffold 49 is fixed to the mass spectrometer 16 or integrated with the mass spectrometer 16.

The feature of the present embodiment is that the trap efficiency of the magnetic beads 13 is increased by utilizing the dam structure 7 and the magnetic force, thereby enabling high-throughput analysis. The high concentration target molecules can be ionized by transporting and concentrating the magnetic beads 13 to the vicinity of the ESI chip 14. Furthermore, by controlling the magnetic force applied to the magnetic bead 13, the dam structure 7 is moved over the magnetic bead 13 and efficient destruction of the magnetic bead 13 is realized, so that the robustness is high.

FIG. 14 is a schematic diagram showing another example of a driving sequence in an analysis method using a microfluidic device. The microfluidic device 4 of the present embodiment has a structure similar to that of the first embodiment. However, the difference from the first embodiment is that the dam structure 7 is not only the side of the waste flow path 8 of the magnetic bead trap part but also the flow path for analysis. It is also a point arranged on the 6 side. From the conductance ratio of the flow path on the waste flow path 8 side and the flow path on the ESI chip 14 side, most of the solution flows to the waste flow path 8 side. For this reason, there is a high probability that the magnetic beads also flow to the waste flow path 8 side. However, a small amount of the magnetic beads 13 receives the force of the fluid toward the analysis channel 6 side. In order to reduce the possibility that the magnetic beads 13 may flow to the analysis flow channel 6 side without being trapped, the microfluidic device 4 of the present embodiment has a cross section (branch) where the three flow channels intersect the dam structure 7. The magnetic bead trap section 5 set in the section) is installed on the analysis flow path 6 side as well as the waste flow path 8 side.

In this case, the driving sequence is partly more complicated than in the first embodiment. In the case of Example 1, the lower magnet 12 was merely slid along the microfluidic device 4 when the magnetic beads 13 trapped in the magnetic bead trap unit 5 were transported to the analysis flow path 6 side. However, in the case of Example 2, as shown in FIGS. 14B to 14E, the magnetic beads 13 are first moved to the upper magnet 11 side to get over the dam structure 7 and then again to the lower magnet 12 side. After bringing the magnetic beads 13 together, they are transported to the vicinity of the ESI chip connection port 9. After the target molecule is eluted and analyzed, as shown in FIGS. 14 (f) to (h), the magnetic beads 13 are moved from the lower magnet 12 side to the upper magnet 11 side and then moved to the waste flow path 8. To transport.

Although the sequence is more complicated than in Example 1, the risk of the magnetic beads 13 flowing into the analysis channel 6 side during trapping can be reduced. There is no difference between the first and second embodiments except that one dam structure 7 is added and the driving sequence becomes complicated as a result.

In addition, when the ESI chip 14 is installed downward as shown in FIG. 15, when the magnetic beads 13 are transported to the vicinity of the ESI chip connection port 9, after passing the dam, the magnetic beads 13 are moved to the lower magnet 12 side. There is no need to attract. In a state where the magnetic beads 13 are brought close to the upper magnet 11 side, the upper magnet is slid to the vicinity of the ESI chip to elute the target molecules. After the target molecule is eluted and analyzed, the upper magnet 11 is slid along the microfluidic device 4 as it is, and the magnetic beads 13 are transported to the disposal channel 8 and discarded.

Example 3 differs from Examples 1 and 2 in that the target molecule is detected not by mass spectrometry but by the photodetector 42. The structure of the microfluidic device 4 is the same as in the first or second embodiment. Similarly to the first and second embodiments, target molecules are adsorbed on the magnetic beads 13 and introduced into the microfluidic device 4. The introduced magnetic beads 13 are trapped in the magnetic bead trap unit 5 by the magnetic force and the dam structure 7.

In the example shown in FIG. 16, target molecules eluted from the magnetic beads 13 are detected. For detection, a laser light 41 is generated by a laser light source 40 and a color reaction or absorbance is used. Alternatively, the concentration can be analyzed using the thermal lens effect. As shown in FIG. 17, there is also a method in which excitation light 43 is irradiated from a laser light source 40 onto magnetic beads on which target molecules are adsorbed, and fluorescence 44 is observed with a photodetector 42. This is a general ELISA.

Even when the analysis is performed using the photodetector 42, the disposal method of the magnetic beads 13 is the same as in the first and second embodiments. As shown in FIG. 2 and FIG. 15, by controlling the magnetic force acting on the magnetic beads 13, the magnetic beads 13 are moved over the dam structure 7 and moved to the disposal flow path 8. The method of this embodiment improves the trap efficiency of the magnetic beads 13 by using the dam structure 7 and the magnetic force at the same time, and enables high-throughput analysis. Further, by controlling the magnetic force acting on the magnetic beads 13, it is possible to make the magnetic beads 13 get over the dam structure 7, thereby realizing efficient disposal of the magnetic beads 13.

FIG. 18 is a schematic plan view showing an example of the channel structure of the microfluidic device of this example having a plurality of analysis channels. As shown in FIG. 18, in the microfluidic device 4 of this embodiment, in addition to the analysis flow path 6, a second analysis flow path 46 and a second ESI chip connection port 47 are arranged. Other points are the same as in the first and second embodiments. In this embodiment, by preparing three or more magnets, a new magnetic bead 13 is trapped by the magnetic bead trap unit 5 while the target molecule is being eluted from the magnetic bead 13 near the ESI chip connection port 9. It is also possible to transport to the second analysis channel 46. Alternatively, the second analysis channel 46 side can be used for the ELISA analysis as shown in the third embodiment.

18, the dam structure 7 is installed only on the disposal flow path 8 side, but may be installed on the analysis flow path 6 side or the second analysis flow path 46 side. Whether or not a dam structure is necessary depends on the flow velocity of the liquid flowing in the flow path. As the flow rate is increased, the measurement throughput can be increased, but the force of the fluid acting on the magnetic beads 13 is increased. When the magnetic beads 13 cannot be trapped only by the magnetic force, it is necessary to install the dam structure 7. In this embodiment, two analysis channels are arranged, but three or more may be arranged if necessary.

In addition, this invention is not limited to an above-described Example, Various modifications are included. For example, the above-described embodiments have been described in detail for easy understanding of the present invention, and are not necessarily limited to those having all the configurations described. Further, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment. Further, it is possible to add, delete, and replace other configurations for a part of the configuration of each embodiment.

DESCRIPTION OF SYMBOLS 1 ... Waste port, 2 ... Inlet port, 3 ... Introduction channel, 4 ... Microfluidic device, 5 ... Magnetic bead trap part, 6 ... Analytical channel, 7 ... Dam structure, 8 ... Waste channel, 9 ... ESI Chip connection port, 11 ... upper magnet, 12 ... lower magnet, 13 ... magnetic bead, 14 ... ESI chip, 15 ... pore, 16 ... mass spectrometer, 17 ... magnet drive mechanism, 23 ... waste liquid valve, 24 ... sample injector , 25 ... liquid feed pump, 28 ... microfluidic device holder, 30 ... drive stage, 31 ... slider, 32 ... conston spring, 40 ... laser light source, 42 ... photodetector, 44 ... fluorescence, 45 ... stopper

Claims

A microfluidic device in which magnetic beads are introduced into a microchannel and used, wherein the magnetic beads are introduced together with a solution, an analysis channel that branches from the introduction channel at a branching portion, and a waste stream A dam having a flow path height lower than a flow path height of the branch portion at a connection portion of at least one of the analysis flow channel and the waste flow channel with the branch portion. An analysis method using a microfluidic device having a structure and a flow path height narrowed by the dam structure being larger than a diameter of a magnetic bead, and two magnets arranged with the microfluidic device interposed therebetween,
Attracting magnetic beads in the microchannel to one inner wall of the microchannel by the magnetic force of one magnet;
Moving the magnetic beads to the inner wall opposite to the inner wall by the magnetic force of the other magnet;
Moving the other magnet along the microfluidic device to cause the magnetic beads to cross the dam structure;
The analysis method characterized by having.
The analysis method according to claim 1,
Introducing magnetic beads into the introduction flow path;
Trapping magnetic beads by magnetic force in the vicinity of the dam structure;
Moving the magnetic beads to the flow path for analysis by moving a magnet;
A step of eluting the target molecules adsorbed on the magnetic beads by introducing the eluate into the analysis channel;
Moving the magnetic beads to the vicinity of the dam structure by moving a magnet after analysis of the target molecule;
The analysis method characterized by having.
The analysis method according to claim 1,
Moving the magnetic beads moved to the vicinity of the dam structure over the dam structure to the waste channel;
Reducing the magnetic force applied to the magnetic beads by moving the magnet to a position away from the waste flow path;
The analysis method characterized by having.
The analysis method according to claim 2,
An analysis method characterized by closing a valve connected to the waste flow path when eluting the target molecule from a magnetic bead.
The analysis method according to claim 2,
The analysis method further comprising the step of ionizing the eluted target molecule.
The analysis method according to claim 2,
The analysis method further comprising the step of ionizing the eluted target molecule by electrospray ionization.
The microfluidic device of claim 1, wherein
The analysis method characterized in that the volume of the flow path for analysis is equal to or greater than the volume of magnetic beads introduced into the microfluidic device.
A microfluidic device that uses and introduces magnetic beads in a microchannel,
An introduction flow path through which the magnetic beads are introduced together with the solution, an analysis flow path branched from the introduction flow path at the branching section, and a disposal flow path,
The connection portion of the flow path for analysis and the waste flow path to the branch portion of at least one flow path has a dam structure in which the flow path height is smaller than the flow path height of the branch portion, A microfluidic device characterized in that the flow path height narrowed by the dam structure is larger than the diameter of the magnetic beads.
The microfluidic device of claim 8, wherein
The microfluidic device, wherein the dam structure is provided at a connection portion with the branch portion of the disposal channel.
The microfluidic device of claim 8, wherein
A microfluidic device, wherein an ion source is connected to the analysis flow path.
The microfluidic device of claim 8, wherein
The microfluidic device, wherein the waste channel is connected to a valve.
A microfluidic device according to claim 8;
Two magnets disposed across the microfluidic device;
A magnet drive mechanism that holds and drives the two magnets;
The analysis apparatus characterized in that the magnet driving mechanism drives the two magnets such that when one magnet acts on the microchannel of the microfluidic device, the other magnet stops acting. .
The analyzer according to claim 12, wherein
The analysis apparatus characterized in that the magnet driving mechanism drives the two magnets such that when one magnet approaches the microfluidic device, the other magnet moves away.
The analyzer according to claim 12, wherein
The analysis apparatus characterized in that the magnet drive mechanism causes the two magnets to perform a synchronized slide and independent slide along the surface of the microfluidic device.
The analyzer according to claim 14, wherein
When the magnetic beads are introduced into the introduction flow path, the magnet drive mechanism causes a magnetic force of a magnet disposed on the base side of the dam structure to act on the branch portion of the micro flow path so that the magnetic beads are moved to the branch portion. When the magnetic beads are discarded, the magnetic force of the magnet arranged on the flow path side narrowed by the dam structure acts on the magnetic beads, and the magnet is slid to cause the magnetic beads to cross the dam structure. Analysis equipment.