JP2006010332A - Method for forming sample solution with small capacity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily separating minor materials such as lymphocytes as a sample solution with a small capacity using a minor flow channel type chip. <P>SOLUTION: This method is to continuously form independent demarcations containing samples being mixed or dissolved into water or hydrophilic samples (target samples) and to form a sample solution with a small capacity. This method includes steps of injecting aqueous solution containing the target samples into a passage 1, injecting oils into a passage 2, forming a passage 3 by joining the passage 1 and the passage 2, forming independent demarcations alternately from the aqueous solution and the oil, and acquiring the independent demarcations of the aqueous solution of which passage 3 contains the target samples. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method of forming a microvolume sample solution. In particular, the present invention relates to a method capable of forming a microvolume sample solution containing a controlled number, eg, one, of biological material such as cells.

  Recently, cell chips are attracting interest as biochips following DNA chips and protein chips. The present inventors used a microarray chip in which hundreds of thousands of microwells with a diameter of 10 μm per chip were arranged in an array, and placed lymphocytes one by one and stimulated antigens to positive cells. We conduct research that leads to the development of efficient and specific antibody drugs by selecting, recovering, and analyzing genes by single unit [Tamiya et al .: Bioindustry, 20 (7), 60-67 (2003)] (Non-Patent Document 1).

In addition, using microfabrication technology used in the semiconductor industry, a minute channel is formed in a chip of several centimeters, where mixing, reaction, detection, etc. are performed, and chemical analysis and synthesis are attempted. It has been. In such a system, a micro-channel type chip is used, and the system itself is called μTAS (micro total analysis systems). For example, this system has been studied to be used for diagnosis such as blood test or as a cell sorter [Takamura: Bioindustry, 20 (12), 52-58 (2003)] ( Non-patent document 2).
Tamiya et al .: Bioindustry, 20 (7), 60-67 (2003) Takamura: Bioindustry, 20 (12), 52-58 (2003)

  A method of selecting and recovering lymphocytes specific for an antigen using a microarray chip is very effective for the development of a specific antibody drug based on one antigen-specific lymphocyte. However, the target is a lymphocyte with a diameter of about 10 μm, the interaction between the microarray chip and the lymphocyte (especially cell adhesion), and the more highly integrated the chip, the more the lymphocytes of other lymphocytes. At present, handling of the microarray chip is not always easy.

  Furthermore, in a microarray chip, it is difficult to perform a reaction as in a cell assay, and it is not easy to take in and out cells. Therefore, it has been desired to develop a flow type chip in place of the microarray chip.

  Accordingly, an object of the present invention is to provide a method capable of easily separating a minute substance such as lymphocytes as a minute volume sample solution using a minute channel type chip.

The present invention for solving the above problems is as follows.
[Claim 1]
A method of forming a microvolume sample solution by continuously forming independent fractions containing a sample mixed with or dissolved in water or a hydrophilic sample (target sample),
Injecting an aqueous solution containing the target sample into the tubular path 1;
Oil is injected into the tubular channel 2;
Path 1 and path 2 join to form a tubular path 3,
In the junction of path 1 and path 2, the aqueous solution and the oil alternately form independent fractions, and the independent fraction of the aqueous solution containing the target sample in path 3 is obtained. Said method.
[Claim 2]
The method according to claim 1, wherein the number of target samples contained in the formed independent fraction is controlled by controlling the concentration of the target sample contained in the aqueous solution and the volume of the independent fraction of the aqueous solution. .
[Claim 3]
The method according to claim 1 or 2, wherein the oil has a viscosity in the range of 10 to 150 cps.
[Claim 4]
The method according to claim 1, wherein the oil is mineral oil, salad oil, or coconut oil.
[Claim 5]
The flow rate of the aqueous solution is in the range of 0.2 to 1.5 μl / min,
The method according to any one of claims 1 to 4, wherein the flow rate of the oil is in the range of 0.2 to 1.5 µl / min.
[Claim 6]
The cross-sectional shape of the paths 1 to 3 is a rectangle,
The width and depth of path 1 are independently in the range of 30-500 μm,
The width and depth of the path 2 are in the range of 30 to 500 μm,
The method according to any one of claims 1 to 5, wherein the width and depth of the path 3 are independently in the range of 30 to 500 µm.
[Claim 7]
In the junction,
The angle between path 1 and path 2 is in the range of 60 ° to 120 °,
The angle formed by path 1 and path 3 is in the range of 120 ° to 150 °,
The method according to any one of claims 1 to 6, wherein an angle formed by the path 2 and the path 3 is in a range of 90 ° to 150 °.
[Claim 8]
The volume of each independent fraction of aqueous solution ranges from 1.0 nl to 1.0 μl;
The method according to any one of claims 1 to 7, wherein the volume of each independent fraction of the oil is in the range of 1.0 nl to 1.0 µl.
[Claim 9]
The method according to any one of claims 1 to 8, wherein the inner walls of the path 1, the path 2, and the path 3 are formed of PDMS, glass, or a combination of PDMS and glass.
[Claim 10]
The method according to claim 1, wherein the target sample is a biological material, an organic material, or an inorganic material.
[Claim 11]
The method according to claim 9, wherein the biological material is a cell, protein, nucleic acid, or cellular tissue.

  According to the present invention, it is possible to easily form a microvolume sample solution by changing the flow rate of the fluid without using a moving component. The system used in the method of the present invention is easy to assemble and operate, the compartment formation phenomenon is a continuous and stable process, the separation of specific single cells from the mixed suspension, and the specific single cell genetic material. Can be used for analysis.

  The present invention is a method for forming a microvolume sample solution in which independent fractions containing a sample mixed or dissolved in water or a hydrophilic sample (target sample) are successively formed. Each micro-volume sample solution contains a predetermined amount (number) of the target sample, and by controlling the concentration of the target sample contained in the aqueous solution, the volume of the independent fraction of the aqueous solution, etc. It is also possible to control so that one target sample is included in a minute volume sample solution.

  The method of the present invention can be carried out using a microchannel chip as shown in FIG. The microchannel chip has a tubular path 1, a path 2 and a path 3, respectively, and these three paths merge at the junction. The angle formed by each path in the junction is, for example, an angle formed by the path 1 and the path 2 is in a range of 60 ° to 120 °, and the angle formed by the path 1 and the path 3 is, for example, 120 ° to 150 °. The angle formed by the path 2 and the path 3 is, for example, in the range of 90 ° to 150 °. By setting the angle formed by each path in the merge part to the above range, the aqueous solution and the oil alternately form independent fractions in the merge part of the path 1 and the path 2, and in the path 3 An independent fraction of the aqueous solution containing the target sample is easily obtained.

  The microchannel chip can be formed by bonding a substrate in which grooves corresponding to each path are formed on one main surface and the other substrate so as to form each path having many grooves. Both substrates can be made of resin such as PDMS (polydimethylsiloxane) or glass, and both substrates can be made of resin such as PDMS or glass, or one substrate can be made of resin such as PDMS. It is also possible to form the other substrate by glass. Therefore, the inner walls of the path 1, the path 2 and the path 3 can be formed of a resin such as PDMS, glass, or a combination of a resin such as PDMS and glass. The inner wall of the PDMS path (flow path) can be made hydrophilic by, for example, oxygen plasma ion etching.

  The cross-sectional shape of the paths 1 to 3 is not particularly limited, but may be, for example, a rectangle, a circle, or a semicircle. The cross-sectional area of each path is preferably constant in one path in order to stably form and maintain a fraction in the path 3. Moreover, although it is appropriate that the cross-sectional areas of the paths 1 to 3 are the same, it is not limited to the case where they are the same.

  When the cross-sectional shapes of the paths 1 to 3 are rectangular, the width and depth of the path 1 are independently in the range of, for example, 30 to 500 μm, and the width and depth of the path 2 are standing, for example, 30 to 500 μm. The width and depth of the path 3 can be independently in the range of 30 to 500 μm, for example. Path 1 preferably has a width and depth independently in the range of 30-200 μm, path 2 preferably has a width and depth independently in the range of 30-200 μm, and path 3 preferably The width and depth can independently be in the range of 30-200 μm. When the cross-sectional shapes of the paths 1 to 3 are other than a rectangle, it is appropriate that the cross-sectional area of the rectangle is substantially in the same range. In the case of a circular or semicircular shape, the diameter is suitably in the range of 30 to 500 μm, for example.

  Using the microchannel chip, an aqueous solution containing the target sample is injected into the path 1 and oils are injected into the path 2. It is appropriate to inject the aqueous solution and oil containing the target sample while controlling the flow rate to a predetermined value using a pump such as a micro syringe pump. And in the confluence | merging part of the path | route 1 and the path | route 2, an aqueous solution and oil form an independent fraction by turns, and the independent fraction of the aqueous solution containing the said target sample in the path | route 3 is formed. can get.

  The volume of each independent fraction of the aqueous solution is, for example, in the range of 1.0 nl to 1.0 μl, and the volume of each independent fraction of the oil is, for example, in the range of 1.0 nl to 1.0 μl. The volume of each independent fraction of the aqueous solution and the volume of each independent fraction of the oils can be appropriately controlled by changing the flow rates of the two types of solutions.

  In the present invention, a “sample mixed with or dissolved in water or a hydrophilic sample” (target sample) that is an object for forming an independent fraction is a sample that is mixed or dissolved in water, or is hydrophilic. If it is a sample, it will not restrict | limit in particular. The target sample can be, for example, a biological material, an organic material, or an inorganic material, and further, the biological material can be a cell, a protein, a nucleic acid, a cell tissue, or a fungus body. One of the purposes of the method of the present invention is to form independent fractions in which a single entity (eg, a single particle) is stored as a single fraction, as a substance or collection of substances. For example, when the target sample is a cell according to the present invention, a fraction in which one cell is stored in one independent fraction can be continuously formed.

  Oils preferably have a viscosity in the range of 10 to 150 cps, more preferably in the range of 15 to 100 cps. Examples of oils having a viscosity in this range include mineral oil, silicone oil, and salad oil. And coconut oil. Further, the oils are preferably mineral oil, salad oil, and coconut oil from the viewpoint of relatively low viscosity.

  The flow rate of the aqueous solution containing the target sample is suitably in the range of 0.2 to 1.5 μl / min, for example, and the flow rate of the oils is suitably in the range of 0.2 to 1.5 μl / min. Controlling the flow rate of the aqueous solution and oil containing the target sample in this range is preferable from the viewpoint of forming a cleaner independent fraction (compartment).

  By controlling the concentration of the target sample contained in the aqueous solution containing the target sample and the volume of the independent fraction of the aqueous solution, the number of target samples contained in the formed independent fraction can be controlled. . For example, FIG. 1 shows a state in which the target sample is lymphocytes labeled with fluorescence, and the lymphocytes are stored one by one in an independent fraction of an aqueous solution. The number of lymphocytes stored in the independent fraction of the aqueous solution can be appropriately controlled by controlling the concentration of lymphocytes contained in the aqueous solution containing lymphocytes and the volume of the independent fraction of the aqueous solution. .

  The microvolume sample solution formed by the method of the present invention can be subjected to analysis of the sample contained in each fraction with respect to the independent fraction of each microvolume on the extension of the path 3. For example, Fig. 1 shows that a fraction containing a single lymphocyte (fluorescently labeled) is subjected to an antigen, and the reactivity of the lymphocyte to the antigen is determined by detecting the fluorescence emitted from the lymphocyte. And a method for sorting lymphocytes having reactivity to an antigen. Specifically, this method can be performed as follows. In the flow path in the middle of the path 3, the antigen solution is introduced into the cell solution that has become an independent fraction, and, for example, a fluorescent dye that depends on the intracellular calcium concentration is detected before and after stimulation under a fluorescence microscope. An independent fraction containing lymphocytes with high intensity and responsiveness to the antigen is collected.

  Further, FIG. 1 also shows a method for amplifying the gene for each fraction of lymphocytes whose antigen specificity has been determined by PCR. Specifically, this method can be performed as follows. After detection of antigen-specific lymphocyte cells, a surfactant and a PCR reaction solution are introduced into the flow path through which the cell volume of the independent fraction flows, and cell disruption and PCR reaction are performed. PCR can be performed by controlling the temperature in the flow path using a cartridge heater or the like.

Hereinafter, the present invention will be described in more detail with reference to examples.
PDMS chips were fabricated using photolithography technology (Whitesides et al., 2001). The first step in PDMS microchannel chip fabrication is the fabrication of a PDMS device template. This mold was cut from a 4 cm diameter silicon wafer, cut 4 cm, washed in water for 10 min, then in boiling acetone for about 10 min, and dried with a nitrogen blower.

2 ml of a photoresist, SU-8 50 (Microchem, USA), was placed on a silicon substrate and coated with a spin coater at 2000 rpm for 10 seconds. The solvent was removed from the photoresist by soft baking at 95 ^° C. for 20 min. Photo using the channel 1 and the contact angle between the flow path 2 having a 60, 90 or 120 ^o Y- shaped in design photomask (50 [mu] m x 50 [mu] m or 100 [mu] m x 100 [mu] m (width and depth)) Lithography was performed. Photolithography was performed by irradiating with UV rays at about 380 mJ / cm ² for 30 seconds using a mask aligner, and then removing unpolymerized photoresist using a developer to form a template for SU-8.

Next, the PDMS monomer and its cross-linking agent were mixed thoroughly at a volume ratio of 10: 1, and the air bubbles generated during the mixing were removed under vacuum, and the mixture was poured into a SU-8 mold, 80 ^o Cured at C for 60 min. The PDMS tip is peeled off from the SU-8 mold, the two inlets and outlets are formed in needle shape, the tube is attached, sealed with PDMS mixture (10: 1) and again cured at 120 ^° C for 10 min. It was. The surface of the obtained PDMS microchannel chip was made hydrophilic by oxygen plasma ion etching and adhered to a glass substrate. Contact angles between channels 1 and 2 are 60, 90 and 120 ^o , and dimensions (width and depth) are 50 μm x 50 μm And PDMS / glass chip devices with 100 μm × 100 μm Y-shaped microchannels were used for fluorescent single particle separation and single B-cell analysis and sorting (FIG. 1).
.

  The microchannel chip device was placed in a fluorescence microscope. Image production (imaging) was performed with a CCD camera. This was easy for real-time imaging and was controlled by a computer. Aqueous and non-aqueous mineral oil solutions were pumped using 100 μl microsyringes.

Visualization of compartment formation of aqueous (dissolved neutral red) and non-aqueous solutions, such as mineral oil and commercial salad oil and coconut oil, using 50 μm × 50 μm and 100 μm × 100 μm microchannel channels The measurement was carried out with a fluorescence stereographic microscope equipped with a CCD camera at a flow rate in the range of 0.2-1.5 μl / min. A neutral red 0.1% (w / v) aqueous solution was prepared with distilled water, and undissolved neutral red particles were removed by a filter. Mineral oil was used as it was. They were pumped to two different inlets of a device with a contact angle of 60 ^° between the flow channels at various flow rates, and the neutral red aqueous solution and mineral oil were visualized by light microscopy. 0.1% (w / v) FITC solution as the aqueous phase, mineral oil as the non-aqueous phase, at a flow rate of 0.2-2.0 μl / min, in a PDMS / glass microchannel device at an angle of 60, 90 or 120 ^o A similar test was conducted.

A system using a 60 ^° angle PDMS / glass microchannel device is shown in FIGS. A in FIG. 2 is a photograph of the whole microchannel device, and B shows a state in which is independently fractionated. FIG. 5 is a system using coconut oil and eosin aqueous solution, A is an image observed using an optical microscope, and B is an image observed using a fluorescence microscope.

A system using a 90 ^° angle PDMS / glass microchannel device is shown in FIG. A is a photograph of the entire microchannel device, and B shows a state in which an aqueous FITC solution and mineral oil are separated into independent fractions.

Systems using a 120 ^° angle PDMS / glass microchannel device are shown in FIGS. FIG. 3 is a system using mineral oil and FITC aqueous solution, A is a photograph of the whole micro-channel type device, and B shows a state where FITC aqueous solution and mineral oil are separated into independent fractions. FIG. 4 shows a system using salad oil and an eosin aqueous solution. A is an image observed using an optical microscope, and B is an image observed using a fluorescence microscope. FIG. 6 shows a system in which fluorescent beads are separated using mineral oil. A is an image observed using an optical microscope, and B is an image observed using a fluorescence microscope.

  Table 1 below summarizes the experimental conditions in the system using each microchannel device of FIGS.

  Waste liquid was collected from the outlet of the device. The flow rate of the liquid solution could be controlled by a micro syringe pump. The compartment volume can also be controlled by controlling the flow rate.

A microchannel device using a two-phase liquid was used to separate single particulates into each compartment. This is an example of a single cell analysis. The size of the used polystyrene beads (diameter 4.5 μm) was selected to be slightly smaller than the target B-cell (6-8 μm diameter). Prepare a fluorescent-labeled polystyrene bead solution (10 ⁵ beads per ml) in distilled water to make an aqueous phase, and use a 100 μl microsyringe at the inlet of the microchannel device as the opposite phase with mineral oil. Feeds were 0.2, 0.5 and 1.0 μl / min. A separation of single particulates into each aqueous compartment was observed.

Figure 2 shows an experiment conducted using a neutral red aqueous solution and mineral oil and a chip designed with a contact angle of 60 ^° between the channels. Neutral red aqueous solution and mineral oil enter the wells of the independent channels, transport along the microchannels, mix with each other at the intersection of the Y-shaped microchannels, spontaneous formation of mineral oil and neutral red compartments Was observed. The formation of stable and uniform dimensions of nanoliter capacity providing compartments for neutral red aqueous and non-aqueous (mineral oil) phases in the device flow path was only visualized by pressure flow from the microsyringe pump (Figure 2).

  The formation of compartments with nanoliter and sub-nanoliter capacities was affected by the contact angle between the device channels and the shape (design) of the channels. Compartment formation was relatively easy in all three devices with different contact angles examined. It also showed stable aqueous and mineral oil solution compartments, and the two-phase compartment formation was equally distributed.

  Compartment formation is highly dependent on the viscosity of the liquid used in the microchannel device. Instead of mineral oil (viscosity 20 cps), coconut oil (viscosity 31 cps) and salad oil (canola oil) (viscosity 60 cps) were used to examine the effect of the viscosity of the liquid on the compartment formation. As a result, when either coconut oil or canola oil was used, a compartment was formed as in the case of using mineral oil.

  These results indicated that the viscosity of the non-aqueous liquid played a major role in forming the liquid compartment in the microchannel. For the formation of compartments, mineral oil (viscosity 20 cps) is most suitable as the non-aqueous liquid among oils. The design of the channel shape, particularly the contact angle between the channels, the liquid flow rate, the liquid surface tension, the surface characteristics of the channel surface and the viscosity of the liquid, are the key factors in the formation of non-aqueous and aqueous solution compartments in the microchannel. It is a determinant. A stable, uniformly sized compartment that provides nanoliter volumes of aqueous and non-aqueous (mineral oil) solutions allows for rapid single cell separation from mixed cell suspensions and allows for single target specific targets. Allows detection of cells.

It was also observed that single particles in each compartment move quickly in the microchannel. Particles appear to be linear due to insufficient speed of the CCD camera used. The separation of single particulates or single cells in each compartment was highly dependent on the channel geometry. This is because a channel having a dimension of 50 × 50 μm ² was more suitable than a device having a dimension of 100 × 100 μm ² . This appears to be due to the higher surface tension that occurs in the 50 × 50 μm ² channel. The separation of single particulates in each compartment is somewhat different from the formation of compartments with aqueous and non-aqueous liquids alone. Single particulate separation into each aqueous compartment can be done with low flow mineral oil (0.2 μl / min), at high flow rates it is difficult to observe the single particle state and the formation of aqueous and non-aqueous compartments is disrupted There was also. Although identifying important parameters is difficult, one or more parameters appear to play a critical role in compartment formation and particle or cell separation. From the above results, the selection of important parameters varies from system to system and may also require consideration of particles or cells and sometimes cell types.

  The system is easy to assemble and operate, and can be easily controlled by changing the fluid flow rate without using moving components. The compartment formation phenomenon is a continuous and stable process that can be used to separate specific single cells from a mixed suspension and to analyze specific single cell genetic material.

  According to the present invention, for example, it is possible to screen for antigen-specific lymphocyte cells and to produce monoclonal antibodies having high affinity from these cells. Therefore, it is considered that it can be a new system (new device) for producing an antibody drug. In addition to performing various bioassays in each compartment, a variety of assays can be performed in a single experimental operation in a single platform without cross-contamination by the non-aqueous phase. It enables numerous assay formats using trace liquid reagents and can be an attractive μTAS (micro total analysis system).

Explanatory drawing of the microchannel type | mold chip | tip used with the method of this invention. The state of an experiment conducted using a neutral red aqueous solution and mineral oil and a chip designed with a contact angle of 60 ^° between the channels is shown. The state of the experiment carried out in a system using PDMS / glass micro-channel type device and mineral oil with a contact angle of 120 ^° channel is shown. The state of an experiment conducted in a system using a PDMS / glass micro-channel type device and a salad oil with a contact angle of 120 ^° channel is shown. An experiment conducted in a system using a PDMS / glass micro-channel type device and a coconut oil having a contact angle of 60 ^° between the channels is shown. The state of the experiment which isolate | separated the fluorescent bead using PDMS / glass microchannel type | mold device and the mineral oil of the contact angle of 120 ^o channel | paths is shown. System using system Shows how the experiments performed by the system using the PDMS / glass microchannel type device contact angle of 90 ^o passages comrades.

Claims (11)

A method of forming a microvolume sample solution by continuously forming independent fractions containing a sample mixed with or dissolved in water or a hydrophilic sample (hereinafter referred to as a target sample),
Injecting an aqueous solution containing the target sample into the tubular path 1;
Oil is injected into the tubular channel 2;
Path 1 and path 2 merge to form a tubular path 3;
In the junction of path 1 and path 2, the aqueous solution and the oil alternately form independent fractions, and the independent fraction of the aqueous solution containing the target sample in path 3 is obtained. Said method. The method according to claim 1, wherein the number of target samples contained in the formed independent fraction is controlled by controlling the concentration of the target sample contained in the aqueous solution and the volume of the independent fraction of the aqueous solution. . The method according to claim 1 or 2, wherein the oil has a viscosity in the range of 10 to 150 cps. The method according to any one of claims 1 to 3, wherein the oils are mineral oil, salad oil, or coconut oil. The flow rate of the aqueous solution is in the range of 0.2 to 1.5 μl / min,
The method according to any one of claims 1 to 4, wherein the flow rate of the oil is in the range of 0.2 to 1.5 µl / min. The cross-sectional shape of the paths 1 to 3 is a rectangle,
The width and depth of path 1 are independently in the range of 30-500 μm,
The width and depth of the path 2 are in the range of 30 to 500 μm,
The method according to any one of claims 1 to 5, wherein the width and depth of the path 3 are independently in the range of 30 to 500 µm. In the junction,
The angle between path 1 and path 2 is in the range of 60 ° to 120 °,
The angle formed by path 1 and path 3 is in the range of 120 ° to 150 °,
The method according to any one of claims 1 to 6, wherein an angle formed by the path 2 and the path 3 is in a range of 90 ° to 150 °. The volume of each independent fraction of aqueous solution ranges from 1.0 nl to 1.0 μl;
8. The method according to any one of claims 1 to 7, wherein the volume of each independent fraction of oil is in the range of 1.0 nl to 1.0 [mu] l. The method according to any one of claims 1 to 8, wherein the inner walls of the path 1, the path 2, and the path 3 are formed of PDMS, glass, or a combination of PDMS and glass. The method according to claim 1, wherein the target sample is a biological material, an organic material, or an inorganic material. The method according to claim 9, wherein the biological material is a cell, protein, nucleic acid, cell tissue, or fungus body.