JP2008292260A - Sample concentration device and sample concentration method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample concentration device concentrating a sample included in fluid, and pouring the sample quantitatively into a microchannel. <P>SOLUTION: This concentration device for concentrating the fluid including the sample has: two channels for injection for injecting the fluid including the sample; a main channel into which the fluid is poured; a sharing part provided on a connection position between the channels for injection and the main channel; a valve for opening/closing a flow of the fluid passing the sharing part from the channels for injection and flowing into the main channel; and a vibration wave generation means for imparting the vibration wave to the fluid held in a part of the channels for injection and the sharing part by the valve. In the sample concentration device, the sample included in the fluid is concentrated by imparting the vibration wave to the held fluid, and the concentrated sample is poured from the sharing part into the main channel. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a sample concentrating device and a sample concentrating method for concentrating a fluid containing a very small amount of sample in a microchannel.

  Analyzing apparatuses having a fine structure have advantages such as improved sensitivity, reduced sample amount, and high-speed analysis compared to a desktop-sized instrument. In addition, the development of a concept called micro total analysis system (μ-TAS) that performs a process from specimen collection to analysis through various processes using a micro space has attracted attention. In analysis of a biological sample, mass spectrometry, electrophoresis, or the like is used, and these techniques can also be made into a microchip.

  However, since analysis in a microfluidic device has a fine structure, a sample amount that is less than the minimum amount that can be handled in a conventional flow injection apparatus is often required. In the conventional electrophoresis chip, when a small amount of the sample 47 in FIG. 3 is poured out, the micro flow channel is formed on the device so that the injection flow channel 44 and the main flow channel 45 intersect in a cross shape, and is shared. A method of dispensing a sample amount corresponding to the volume of the portion 46 has been adopted (see Patent Document 1). At the time of this dispensing, first, a potential gradient is applied in the direction of the injection flow path 44, and the sample 47 is filled to the shared portion 46 at least, and then the applied voltage is adjusted to the direction of the main flow path 45. By switching, a work of dispensing a constant volume of sample 48 is required.

  FIG. 4 shows a method of dispensing after adsorbing the sample in the flow path sharing part of the prior art. First, the sample 54 that has flowed through the injection flow channel 52 is adsorbed by a probe fixed to the flow channel surface in the shared portion 55. Next, when transferring in the direction of the main flow path 53, there is also a method in which the sample 56 is removed by removing the sample 54. In this method, the sample injected into the injection flow channel 52 is concentrated in the shared portion 55 and then poured out toward the main flow channel 53 (see Patent Document 2).

  Further, as a conventional technique using a membrane, as shown in FIG. 5, the sample 64 is concentrated in the shared portion 62 by continuing to transfer the sample 64 from the reservoir 57 toward the membrane 63 in the injection channel 60. There is also a method of pouring a small amount of sample by switching the flow in the direction of the main flow path 61 (see Non-Patent Document 1).

As a conventional technique for concentrating a sample without using a membrane filter, adsorbed molecules, etc., a standing wave 70 is applied using the ultrasonic wave generation sources 67 and 68 in FIG. A method is disclosed in which these are collected in a certain range and poured into a desired flow path 71 (see Patent Document 3).
US Pat. No. 5,900,130 (Section 2, FIGS. 1a and 4b) Japanese Patent Laying-Open No. 2005-274405 (Section 17, FIG. 1) JP-A-9-122480 (Section 7, FIG. 6) Julia Khandurina, Stephen C .; Jacobson, Larry C .; Waters, Robert S. Foote, and Michael Ramsey, “Microfabricated Porous Membrane Structure for Sample Concentration and Electrophoretic Analysis,” Analytical Chemistry19. 71, no. 9, pp 1815-1819, (paragraph 1817, FIG. 3)

  In the method of dispensing a small amount of sample using the cross-shaped channel, a large amount of sample is required for the injection channel 44 from the injection port 41 to the common part 46 at the minimum, but the sample actually extracted in the direction of the main channel 45 48 is a part of the total sample amount determined by the volume of the shared part 46. Therefore, there is a problem that a lot of dead volume is required for extraction. There is also a limitation that the amount of sample to be dispensed is limited to the volume of the shared part of the microchannel.

Further, in the method of adsorbing the sample in the common part, the amount of sample to be dispensed depends on the adsorption efficiency of the sample 54 in the common part 55, so that there is a problem in quantitativeness.
In the method using the membrane, in the liquid feeding method other than the electroosmotic flow, the sample is transferred in the direction of the main flow path 61 before switching the flow in the direction of the main flow path 61, which causes a problem in quantitativeness.

  Furthermore, in the extraction method using ultrasonic waves, the collection of the sample 69 into the flow path 71 does not take quantitativeness into account, and there is a possibility that a part of the sample 69 does not fit in the desired flow path. There is a problem that there is. Furthermore, since the sample 71 is physically inserted into the channel and the sample 69 is accommodated in the capillary 71, the sample 69 is sufficiently collected to move the capillary instead of the micro channel. There is a problem that it can be applied only to those having a large size.

  The present invention has been made in view of such background art, and reduces the dead volume in the flow channel for injection, concentrates a small amount of sample contained in the fluid, and quantitatively transfers the sample to the micro flow channel. The present invention provides a sample concentrating device and a sample concentrating method that can be dispensed into a container.

  A micro sample concentration device that solves the above problem is a concentration device that concentrates a fluid containing a sample, and includes at least two injection channels for injecting the fluid containing the sample, and a main channel for dispensing the fluid. A shared portion provided at a position where the injection flow path and the main flow path are connected; a valve that opens and closes a flow of fluid that flows from the injection flow path to the main flow path through the common flow section; and injection by the valve And a vibration wave generating means for applying a vibration wave to the fluid held in the shared portion and concentrating the sample contained in the fluid by applying the vibration wave to the held fluid. The concentrated sample is poured into the main channel from the common part.

  A micro sample concentration method that solves the above problem is a concentration method for concentrating a fluid containing a sample, and includes at least two injection channels for injecting a fluid containing a sample, and a main channel for dispensing the fluid. A shared portion provided at a position where the injection flow path and the main flow path are connected; a valve that opens and closes a flow of fluid that flows from the injection flow path to the main flow path through the common flow section; and injection by the valve The sample contained in the fluid is concentrated by applying a vibration wave to the held fluid using a vibration wave generating means for applying a vibration wave to a part of the flow path and the fluid held in the common part. The concentrated sample is poured into the main channel from the common part.

The present invention can provide a sample concentrating apparatus and a sample concentrating method that can concentrate a sample contained in a fluid and quantitatively dispense the sample into a microchannel.
In the sample concentration apparatus and method of the present invention, the total amount of sample in the space closed by the valve is obtained by concentrating the sample contained in the fluid existing in the injection channel, which is a microchannel, in the common part. Can be efficiently poured into the main channel.

Further, since the volume of the sample to be dispensed is not limited only to the volume of the shared portion, there is an effect that the degree of freedom of the amount of sample to be dispensed increases.
Further, by determining the volume of the injection channel with a valve, it is possible to quantitatively dispense the concentrated sample into the main channel.

Hereinafter, the present invention will be described in detail.
A micro sample concentration device according to the present invention is a concentration device for concentrating a fluid containing a sample, and includes at least two injection channels for injecting a fluid containing a sample, a main channel for dispensing the fluid, A common portion provided at a position where the injection flow channel and the main flow channel are connected; a valve that opens and closes the flow of fluid that flows from the injection flow channel to the main flow channel through the common flow channel; A vibration wave generating means for applying a vibration wave to the fluid held in a part of the path and the shared portion, and concentrating a sample contained in the fluid by applying the vibration wave to the held fluid; It is characterized by pouring the prepared sample into the main channel from the common part.

  The vibration wave generating means is preferably a means for generating an ultrasonic wave vibration wave, but is not limited thereto. As a specific vibration wave generating means, it is preferable that the ultrasonic wave generating source and the reflection plate or two ultrasonic wave generating sources are used, and a node or a belly of an ultrasonic standing wave is applied to the shared part.

The sample is preferably a cell, a particle, or a molecule adsorbed on the particle, and the molecule may be a biomolecule containing DNA or protein.
It is preferable that at least one of the width and the depth of the cross-sectional dimension of the channel is 0.1 μm or more and 1000 μm or less.

  The micro sample concentration method according to the present invention is a concentration method for concentrating a fluid containing a sample, wherein at least two injection channels for injecting a fluid containing a sample, a main channel for dispensing the fluid, A common portion provided at a position where the injection flow channel and the main flow channel are connected; a valve that opens and closes the flow of fluid that flows from the injection flow channel to the main flow channel through the common flow channel; Using a vibration wave generating means for applying a vibration wave to the fluid held in a part of the path and the shared part, the sample contained in the fluid is concentrated by applying the vibration wave to the held fluid, and concentrated. It is characterized by pouring the prepared sample into the main channel from the common part.

It is preferable that the amount of sample dispensed is smaller, equal or larger than the volume of the common part.
In the present invention, a sample contained in a fluid is concentrated using a vibration wave, and the principle is that an acoustic reflection wave generated from a vibration wave transmission source is used. The force F received by a particle existing at a distance x from a node or a node constituting a standing wave is

It is expressed. Where p ₀ is the sound pressure amplitude, V _c is the volume of the particle, λ is the sound wave wavelength, k is the wave number vector, φ, β, and ρ are

Ρ _c is the particle density, ρ _w is the solvent density, β _c is the particle compressibility, and β _w is the solvent compressibility. Here, φ is called a φ factor, and in the positive value, the particles are accumulated in the nodes of the standing wave, and in the negative value, the particles are accumulated in the antinodes of the standing wave.

The present invention uses the above principle to concentrate the sample existing in the injection channel into a common part constituted by the injection channel and the main channel, and then pours it into the main channel.
FIG. 1 is a conceptual diagram showing an embodiment of a sample concentrator of the present invention. Hereinafter, it demonstrates in detail using FIG.

  The sample concentrator of the present invention comprises two injection channels 4 and 5 for injecting a fluid containing a sample, a main channel 6 for dispensing the fluid, and the injection channels 4 and 5 and the main channel 6. A shared portion 7 provided at a connection position, valves 10, 11, 12, and 13 that open and close the flow of fluid that flows from the injection flow path to the main flow path through the shared portion 7, and for injection by the valve Ultrasonic wave generation means 80 for applying ultrasonic waves to the fluid held in a part of the flow path and the shared part is included.

  The injection channels 4 and 5 and the main channel 6 are micro channels. As a cross-sectional dimension of the microchannel preferable in the present invention, the cross-sectional dimension of at least one of the width and the depth is 0.1 μm or more and 1000 μm or less. The cross-sectional dimensions of both the width and depth may be different or the same. A flow path from a contact point between the injection flow path 4 and the flow path 6 to a contact point between the injection flow path 5 and the main flow path 6 is defined as a shared portion 7. Further, the configuration of the micro flow path may be a cross-shaped flow path structure in which the injection flow path 5 exists in the extending direction of the injection flow path 4 and the main flow path 6. Further, the main channel 6 does not need to be used only for the purpose of concentration and analysis, and may pass through any process after sample extraction.

  The material of the sample concentrator of the present invention need not be particularly limited, such as glass, ceramic, metal, plastic, or a hybrid thereof, but is preferably a member that hardly absorbs ultrasonic waves as much as possible.

  The valves 10, 11, 12, and 13 do not need to have any particular restrictions on the form, but a form that can be opened and closed multiple times, such as a form that blocks the flow path with a diaphragm, is preferable. The valve 10 is positioned immediately before the injection flow path 4 bends in the direction of the shared portion 7, and the valve 11 is positioned at the boundary with the shared portion 7 in the direction of the reservoir 2 in the main flow path 6. The valve 12 is disposed immediately after bending in the direction away from the shared portion 7 in the injection flow path 5, and the valve 13 is disposed at the boundary with the shared section 7 on the opposite side of the main flow path 6 from the reservoir 2.

  In addition, the distance from reservoir 1 to valve 10 in injection channel 4 and the distance from valve 12 to reservoir 3 in injection channel 5 are preferably as short as possible in order to reduce the amount of sample required. .

  The ultrasonic wave generation means 80 includes an ultrasonic wave generation source 8 and a reflector or an ultrasonic wave generation source 9. The ultrasonic wave generation source 8 has at least a length of the common part 7 in the direction of the main flow path 6 and oscillates ultrasonic waves with a width that covers the entire space closed by the valves 10, 11, 12, and 13. It is installed at a position as close to the injection channel 4 as possible. The reflector or the ultrasonic wave generation source 9 installed as close as possible to the injection flow path 5 in FIG. 1 is another ultrasonic wave generation source having the same size as the ultrasonic wave generation source 8 or an ultrasonic wave source. A reflection plate that reflects sound waves from the sound wave generation source 8 may be used, and it is only necessary that the standing wave can be configured in combination with the ultrasonic wave generation source 8.

  Examples of the sample solution 15 include a bead, a cell, or a molecule immobilized on the bead, which is diluted in a solvent at an appropriate concentration.

Hereinafter, the present invention will be described more specifically with reference to examples.
Example 1
Example 1 will be described with reference to FIG.

  First, the valves 10 and 12 are opened, and a sample solution 15 containing a sample whose concentration is adjusted in advance in the direction of the reservoir 3 is injected from the reservoir 1 through the injection channel 4, the shared portion 7, and the injection channel 5. To do. Valves 11 and 13 are closed. At this time, the sample solution is transferred by a method using a capillary phenomenon in a micro flow path, a pressure applied to the reservoir 1 by an external pump, or a pressure flow caused by a decompression in the reservoir 3, and electricity between the reservoir 1 and the reservoir 3. There is no particular limitation on the method such as osmotic flow or dielectrophoresis. After the flow of the sample solution 15 passes through the valve 12, the valve is closed in the order of the valve 12 and the valve 10.

  Next, the ultrasonic wave generation source 8 is operated to oscillate a sound wave in the direction of the injection channel. If 9 is a reflector in 9 installed near the injection flow path 5 in FIG. 1, a standing wave node or belly is placed on the shared portion 7 by frequency control of only the ultrasonic wave generation source 8. Can be configured. If 9 is another ultrasonic wave generation source, a superposition of a wave of standing waves in the sharing unit 7 by superposition of a sound wave oscillated from the ultrasonic wave generation source 8 and a wave oscillated from the ultrasonic wave generation source 9. The belly can be configured. In addition, a node or an abdomen can be constructed by superimposing sound waves also at a position other than the shared part 7, and in this case, the sample solution 15 is transferred to the shared part 7 while being trapped by the ultrasonic node or the abdomen. Also good.

  One ultrasonic node or antinode is formed in the injection channels 4 and 5, that is, the distance between the ultrasonic wave generation source 8 and the reflector 9 (or the ultrasonic wave generation source 9) as indicated by the standing wave 14 is set. The frequency is adjusted so as to be a half wavelength, and the nodes or antinodes are arranged in the shared portion 7. Whether to form a node or a belly depends on the φ factor of the sample to be concentrated. At this time, in the entire space closed by the valves 10 and 12 in the injection channels 4 and 5 and the valves 11 and 13 in the main channel 6, the sample solution 15 is accumulated in the directions 16 and 17 in which the sample solution 15 is accumulated in the common unit 7. The force works toward the shared part 7. In FIG. 1, the standing wave 14 is illustrated as constituting a node in the shared portion 7, but it may be configured as an abdomen, and the sample can be concentrated as in the present embodiment. is there.

  After the sample solution 15 is concentrated to the common part 7, the valves 11 and 13 are opened, and the sample is poured into the main channel 6 by applying pressure from the reservoir 2 connected to the main channel 6. Further, the sample may be poured out into the main channel 6 by a method such as electroosmotic flow or dielectrophoresis, as well as the pressure flow by pressurization, and the method for transferring the sample solution 15 is not particularly limited.

Example 2
As Example 2, application of the present invention in a plurality of main channels will be described with reference to FIG. FIG. 2 is a conceptual diagram showing another embodiment of the sample concentrator of the present invention.

  The injection channel 22 has reservoirs 18 and 21 at both ends, and immediately before the injection channel 22 bends from the reservoir 18 toward the shared portion 25, the injection channel 19 extends from the shared portion 26 toward the reservoir 21. Immediately after bending, the valve 32 is installed. Valves 30 and 33 are arranged at the boundary between the shared portion 25 and the main flow path 23, and valves 31 and 34 are arranged at the respective boundaries between the shared portion 26 and the main flow path 24.

  The shared portion 25 refers to a region where the injection channel 22 and the main channel 23 intersect. Similarly, the sharing unit 26 indicates a region where the injection channel 22 and the main channel 24 intersect. Further, in the present embodiment, the injection flow path is illustrated by a single straight line, but it may be in contact with each other without crossing as in the first embodiment.

  The sample solution 36 is injected into the injection flow path 22 by opening the valves 29 and 32, and after the sample solution proceeds from the valve 32 toward the reservoir 21, the valves 32 and 29 are closed in this order. A standing wave 35 is oscillated between the ultrasonic wave generation source 27 and the reflector 28 or the ultrasonic wave generation source 28 so as to have a frequency such that nodes or antinodes exist in the shared portions 25 and 26. When there are two main flow paths in FIG. 2, the oscillated ultrasonic wave corresponds to exactly one wavelength.

  Ultrasonic force is generated, and the sample solution 36 existing in the injection channel 22 receives the force in the direction of the arrows 37, 38, 39, or 40 at the position where the injection channel 22 is present, or the shared portion 25 or shared. Concentrated in part 26. Thereafter, the valves 30 and 33 are opened to pour the sample into the main flow path 23, and the valves 31 and 34 are opened to pour the sample into the main flow path 24. The method of transferring the sample solution 36 in the direction of the main channels 23 and 24 is not particularly limited, and the dispensing time need not be synchronized.

  Example 2 described the method of pouring a sample into two main channels. However, even in three or more main channels, if each main channel is installed at half-wavelength intervals, the frequency of the ultrasonic transmission source is adjusted. A node or an abdomen is formed in each common part, and the sample extraction method of the present invention can be used.

  The sample concentrator of the present invention can concentrate a sample contained in a fluid and quantitatively pour the sample into a microchannel, so that it contains cells and biomolecules using microchannels and capillaries. It can be used for microflow injection to be used for separation and purification of samples.

It is a conceptual diagram which shows one embodiment of the sample concentration apparatus of this invention. It is a conceptual diagram which shows the other embodiment of the sample concentration apparatus of this invention. It is a conceptual diagram which shows the sample extraction method using the cross-shaped flow path of a prior art. It is a conceptual diagram which shows the sample extraction method using the sample concentration method of a prior art. It is a conceptual diagram which shows the sample extraction method using the filter of a prior art. It is a conceptual diagram which shows the sample concentration method using the ultrasonic wave of a prior art.

Explanation of symbols

1, 2, 3 Reservoir 4 Injection channel 5 Injection channel 6 Main channel 7 Shared portion 8 Ultrasonic source 9 Reflector or ultrasonic source 10 Valve 11 Valve 12 Valve 13 Valve 14 Standing wave 15 Sample solution 18, 19, 20, 21 Reservoir 22 Injection channel 23, 24 Main channel 25, 26 Shared portion 27 Ultrasonic source 28 Reflector or ultrasonic source 29, 30, 31, 32, 33, 34 Valve 35 Fixed Standing wave 36 Sample solution 44 Injection channel 45 Main channel 46 Shared portion 47 Sample 48 Sample 52 Injection channel 53 Main channel 54 Sample 55 Shared unit 56 Sample 57 Reservoir 60 Injection channel 61 Main channel 62 Shared unit 64 Sample 66 Flow path 67, 68 Ultrasonic wave generation source 69 Sample 70 Standing wave 71 Flow path 80 Ultrasonic wave generation means

Claims (6)

  A concentration device for concentrating a fluid containing a sample, wherein at least two injection channels for injecting a fluid containing a sample, a main channel for discharging the fluid, and the injection channel and the main channel are connected to each other. A shared portion provided at a position, a valve that opens and closes the flow of fluid that flows from the injection flow path to the main flow path through the shared portion, and a portion of the injection flow path that is held by the shared portion by the valve. Vibration wave generating means for applying a vibration wave to the fluid, and concentrating the sample contained in the fluid by applying the vibration wave to the retained fluid, and pouring the concentrated sample from the common part into the main channel. A sample concentrating device characterized in that it is dispensed.   2. The vibration wave generating means includes an ultrasonic wave generation source and a reflection plate, or two ultrasonic wave generation sources, and imparts a node or an antinode of an ultrasonic standing wave to the shared part. The sample concentrator described in 1.   The sample concentration apparatus according to claim 1, wherein the sample is a cell, a particle, or a molecule adsorbed on the particle.   4. The sample concentrator according to claim 1, wherein at least one of a width and a depth of a cross-sectional dimension of the flow path is 0.1 μm or more and 1000 μm or less.   A concentration method for concentrating a fluid containing a sample, wherein at least two injection channels for injecting a fluid containing a sample, a main channel for discharging the fluid, and the injection channel and the main channel are connected to each other. A shared portion provided at a position, a valve that opens and closes the flow of fluid that flows from the injection flow path to the main flow path through the shared portion, and a portion of the injection flow path that is held by the shared portion by the valve. The sample contained in the fluid is concentrated by applying the vibration wave to the retained fluid using the vibration wave generating means for applying the vibration wave to the fluid, and the concentrated sample is poured into the main channel from the common part. A sample concentration method characterized by comprising:   The sample concentration method according to claim 5, wherein the introduction amount of the sample is smaller than, equal to, or larger than the volume of the common part.

Images