JP6923181B2 - Fine particle separation device and fine particle separation method - Google Patents

Description

The present invention relates to a fine particle separation device and a fine particle separation method.

Fine particle separation is widely required in medical / biochemical fields, production fields, and the like. For example, in biological research, diagnostic medicine, and regenerative medicine, it is required to separate and collect only specific cells (cancer cells, blood cells, living cells, etc.) from a certain cell population. At present, a centrifugal separation method, a filtration method, a fluorescence activated cell separation method (FACS), a magnetic cell separation method (MACS), and the like are used for separating such biological particles. However, the centrifugal separation method has low accuracy, and the filtration method has a problem that clogging occurs. Further, FACS and MACS require labeling with fluorescence or magnetic beads, require complicated pretreatment, and the apparatus is large and expensive.

In recent years, a fine particle separation method using a microchannel device (Non-Patent Document 1) has been reported as one of the means for solving these problems, and the separation method is roughly classified into the following two types.
(1) Active particle separation method: A method that requires external energy such as electric field, sound field, and magnetic field for separation.
(2) Passive particle separation method: A method that uses only hydraulic action during separation In the case of the active particle separation method, the system is complicated by using external energy, so the passive separation method is expensive. It is desired to realize separation performance. In recent years, as one of the passive separation methods, a case of fine particle separation by the deterministic lateral displacement (DLD) method has been reported (Non-Patent Documents 2 to 4). DLD is a particle separation method that utilizes the flow generated in the fluid by the columns arranged in the flow path, and takes different orbits in the DLD flow path according to the particle characteristics such as particle size, shape, and hardness (Fig. 9). , Particles can be easily separated based on the characteristics of these fine particles. An example (Non-Patent Document 3) has also been reported in which a high separation resolution of 10 nm and a high processing amount of 10 mL / min are used to achieve particle separation.

On the other hand, for the separation of fine particles by DLD, when the fine particle suspension solution is introduced into the DLD flow path, a solution (buffer) in which the fine particles are not suspended is used in order to allow the fine particles to flow in from a specific position, and from both sides. A sheath flow type flow path structure (FIG. 10) that sandwiches the fine particle suspension solution or a flow path structure that sandwiches the fine particle suspension solution between the flow of the buffer and the flow path wall surface (Non-Patent Document 4) has been required. .. Conventionally, if there is no such flow path structure, the fine particles are widely dispersed in the entire DLD flow path (FIG. 11), so that the fine particles cannot be separated by the DLD. However, when such a flow path structure is used, there are problems such as dilution of the analysis sample by a buffer, deterioration of operability due to an increase in the number of liquid feed ports, and complication of the device structure.

D. R. Gossett et al., Anal. Bioanal. Chem., 397, 3249-3267, 2010 L. R. Hung et al., Science, 304, 987-990, 2004. J. McGrath et al., Lab Chip, 14, 4139-4158, 2014. N. Tottori et al., Biomicrofluidics, 10, 0414125, 2016

The present invention solves such a problem, and does not use a flow path structure for sandwiching a fine particle suspension solution using a buffer, and has only a liquid feed flow path for inflowing a fine particle suspension sample as an introduction path. It provides a separation device and a method for separating fine particles using the separation device.

The present invention provides the following invention in order to solve the above problems.
(1) A fine particle separation device for inflowing a liquid in which fine particles are dispersed and separating the fine particles according to their characteristics.
It consists of an inlet and outlet for fine particles, a microchannel for focusing on fine particles, and a microchannel for separating fine particles;
The fine particle separation microchannel is formed in a gap between the arranged columns, and is configured to control the trajectory of the fine particles flowing through the fine particle separation microchannel to separate the fine particles according to the characteristics of the fine particles. The microchannel for focusing on fine particles is provided as an introduction path in front of the microchannel for separating fine particles.
The fine particles in the liquid introduced from the inflow port are configured to be locally arranged in a single or multiple linear lines along the flow in a single flow path by inertial force.
A fine particle separation device, characterized in that the fine particles are configured to flow into a single or a plurality of specific sites of the fine particle separation microchannel in a concentrated and arranged state;

(2) The fine particle separation device according to (1) above, which separates fine particles based on their size, shape or hardness.
(3) The fine particle separation device according to (1) or (2) above, wherein the fine particles are focused by inertial force in a single, two or three linear lines along the flow in the fine particle focusing microchannel. ..
(4) The fine particle separation device according to any one of (1) to (3) above, wherein the fine particles are selected from polymer fine particles, biological fine particles, droplets, metal fine particles, and non-metal particles.

(5) The fine particle separation device according to any one of (1) to (4) above, wherein the liquid in which the fine particles are dispersed is an aqueous suspension.
(6) The fine particle separation device according to any one of (1) to (5) above, wherein the fine particle separation microchannels are formed in a gap between columns having a single separation diameter in which they are arranged.
(7) The fine particle separation device according to any one of (1) to (5) above, wherein the fine particle separation microchannels are formed in gaps between columns having a plurality of separation diameters arranged.

(8) A liquid in which fine particles are dispersed is introduced into a microchannel for focusing on fine particles, and the fine particles in the introduced liquid are linearly arranged along a flow in a single channel by inertial force. Locally arranged in
In a state where the fine particles are concentrated and arranged in a single or a plurality of specific parts of the microchannel for separating fine particles formed in the gap between the arranged columns, the liquid in which the fine particles are dispersed is allowed to flow in.
A method for separating fine particles, which comprises controlling the trajectory of the fine particles flowing through the microchannel for separating fine particles, separating the fine particles according to the characteristics of the fine particles, and causing the fine particles to flow out from the microchannel for separating fine particles.

(9) The method for separating fine particles according to (8) above, which separates fine particles based on their size, shape or hardness.
(10) Separation of the fine particles according to (8) or (9) above, wherein the fine particles are focused by inertial force in a single line, two or three linear lines along the flow in the fine particle focusing microchannel. Method.
(11) The method for separating fine particles according to any one of (8) to (10) above, wherein the fine particles are selected from polymer fine particles, biological fine particles, droplets, metal fine particles, and non-metal particles.

(12) The method for separating fine particles according to any one of (8) to (11) above, wherein the liquid in which the fine particles are dispersed is an aqueous suspension.
(13) The method for separating fine particles according to any one of (8) to (12) above, wherein the microchannels for separating fine particles are formed in a gap between columns having a single separation diameter in which the microchannels are arranged.
(14) The method for separating fine particles according to any one of (8) to (12) above, wherein the microchannels for separating fine particles are formed in gaps between columns having a plurality of separation diameters arranged.

According to the present invention, a fine particle separation device having only a liquid feeding flow path for inflowing a fine particle suspension sample as an introduction path without using a flow path structure for sandwiching the fine particle suspension solution using a buffer, and a fine particle separation device thereof are used. It provides a method for separating fine particles. Furthermore, when the strut is made of a stimulus-responsive polymer, a particle separation device capable of adjusting the separation diameter, which is the particle diameter that is the boundary of the change in the particle trajectory, by changing the flow path geometry by stimulus control and the like. Can provide a method for separating fine particles using.

The figure which shows the relationship between the cross-sectional shape of the microchannel for fine particle focusing by the inertial force of this invention, the focus position of fine particles, and the observation direction. A conceptual diagram of an example of fine particle separation in a fine particle separation microchannel when fine particles are focused in a single line as shown in FIGS. 1 (a) and 1 (e), for example, in the fine particle focusing microchannel. A conceptual diagram of an example of fine particle separation in a fine particle separation microchannel when fine particles are focused in a two-line shape as shown in FIG. 1 (b), for example, in the fine particle focusing microchannel. A conceptual diagram of an example of fine particle separation in a fine particle separation microchannel when fine particles are focused in a three-line shape as shown in FIGS. 1 (c) and 1 (d), for example, in the fine particle focusing microchannel. The schematic which shows one Embodiment of the fine particle separation device of this invention. It is a figure which shows the state of the fine particle focusing by the inertial force in the microchannel for fine particle focusing when the fine particle suspension solution is introduced into the fine particle separation device of this invention. The figure which shows the state of the particle focusing in the particle focusing microchannel under different flow rate conditions. The figure which shows the state of the fine particle separation in the microchannel for fine particle separation when the flow rate is 1 mL / h. The schematic diagram of the DLD flow path and the figure which shows the principle. The figure which shows the DLD fine particle separation device using the sheath flow type flow path. The figure which shows the fine particle trajectory at the DLD flow path inlet when there is no sheath flow type flow path structure.

The fine particle separation device of the present invention is a fine particle separation device for separating inflowing fine particles according to their characteristics. The fine particle separation device comprises an inlet and outlet of a fine particle suspension, a microchannel for focusing on fine particles, and a microchannel for separating fine particles, and the microchannel for separating fine particles is formed in a gap between arranged columns. NS. In the present invention, the trajectory of the fine particles flowing through the microchannel for separating fine particles is controlled so that the fine particles are separated according to the characteristics of the fine particles.

In the fine particle separation device of the present invention, the fine particle focusing microchannel is provided as an introduction path in front of the fine particle separation microchannel, and the fine particles in the liquid introduced from the inflow port are sent into a single flow by inertial force. It is configured to be locally arranged in a single or multiple (eg, 2 or 3) linear lines along the flow along the path to identify single or multiple microchannels for particle separation. It is configured so that the fine particles flow into the site in a concentrated and arranged state.

The struts can preferably be made of materials commonly used in conventional DLDs, such as polymer resins such as polydimethylsiloxane (PDMS), Si, glass and the like. Further, a stimulus-responsive polymer material can be used, and the trajectory of the fine particles flowing through the microchannel is controlled according to the degree of change in the shape of the column due to contraction or swelling in response to the stimulus, and the fine particles are separated according to the characteristics of the fine particles. Being done. The characteristics of the fine particles preferably include size, shape, or hardness.

The stimulus-responsive polymer material that forms the struts is a hydrogel that responds to physical stimuli such as temperature, light, electric or magnetic fields, or chemical stimuli such as pH, solution composition, and ionic strength. For example, a temperature-responsive polymer material is a hydrogel, which is a polymer having a property of swelling when it contains a large amount of water, and has a function of causing swelling / contraction due to reversible hydration / dehydration due to temperature change. Has. Examples of the temperature-responsive polymer material include poly-N-isopropylacrylamide (PNIPAM), poly-N-vinylalkylamide, polyvinylalkyl ether, etc., but the phase transition temperature is around 30 ° C. Poly-N-isopropylacrylamide is suitable because it can be applied and the shape of the strut can be finely controlled because it exhibits a gentle temperature response. Examples of the photoresponsive polymer material include polyacrylamide hydrogel having an azobenzene-containing crosslinked structure. Examples of the pH-responsive polymer material include a carboxy group-containing polymer having a bulky hydrophobic group in the side chain. Examples of the electroactive polymer materials include polyacrylamide-2-methylpropanesulfonic acid (PAMPS) and the like.

The fine particles to be separated are selected from polymer fine particles such as latex, biological fine particles such as erythrocytes and living cells, droplets, metal fine particles, and non-metal particles such as metal oxide.

The particle size of the fine particles is usually 1 nm to 1 mm, preferably 10 nm to 100 μm.

The microparticles are introduced from the inlet of the microparticle separation device, for example in the form of an aqueous or oily suspension. The aqueous suspension is preferably a solution in which fine particles are suspended in an aqueous solution to which a surfactant is added at a particle concentration of ^{about 10 2} to 10 ^{10 / mL.} The aqueous suspension contains water as a main component and may contain an organic solvent such as alcohols that is compatible with water.

In the fine particle separation device of the present invention, a fine particle focusing microchannel for arranging the arrangement of fine particles introduced from the inflow port by inertial force is provided in front of the fine particle separation microchannel, and the fine particle separation microchannel is specified. It is configured to concentrate and introduce fine particles at the position.

The shape of this microchannel for fine particle focus is
Cross-sectional shape: rectangular, square, semi-circular, triangular;
Aspect ratio (aspect ratio, AR) of the cross-section of the flow path: 0 <AR <1 when the cross-section is a horizontally long rectangle; 1 <AR when the cross-section is a vertically long rectangle; AR = 1 when the cross-section is square.
Channel shape: straight or meandering channel;
Channel length: 1 μm to 1 m, preferably 1 mm to 10 cm;
Channel width: 50 nm to 10 mm;
Channel height: Selected from about 50 nm to 10 mm.
(For references on particle focusing by inertial force, see H. Amini et al.,
Lab Chip, 2014, 2739-2761 and J. Kim et al., Lab Chip, 2016, 16, 992-1001. )

Fig. 1 shows the relationship between the cross-sectional shape of the microchannel for focusing on fine particles by the inertial force of the present invention, the focus position of fine particles, and the observation direction. In FIG. 1, (a) horizontal rectangle 0 <AR <1; (b) vertical rectangle 1 <AR ,; (c) square 1 = AR ,; (d) triangle ,; (e) semicircle, (upper) flow The particle focus position on the cross section of the road, and the (lower) particle focus position when the microchannel is observed from the upper side on the cross section of the channel and the direction perpendicular to the flow (top view).

When the cross-sectional shape of the microchannel for focusing on fine particles is a horizontally long rectangle, the particles are focused on two points in the center of the flow path along the long side on the cross-section of the flow path (Fig. 1a). As shown in the figure, when viewed from above, the particles are observed in a single linearly focused state in the center of the flow path (Fig. 1a). In this case, the particles are introduced into the DLD flow path, which is the fine particle separation part, in a single linearly focused state, and then separated in the DLD flow path (Fig. 2). FIG. 2 shows a conceptual diagram of particle separation in the DLD flow path when the cross-sectional shape of the microchannel for fine particle focus is a horizontally long rectangular (0 <AR <1) or a semicircular cross-sectional shape, and (a) from the separation diameter. Larger particles move to outlet 1 and are collected, particles smaller than the separation diameter are collected from outlet 2, (b) particles larger than the separation diameter move to outlet 3 and are collected, and particles smaller than the separation diameter are collected from outlet 2. Will be recovered. In this case, the aspect ratio (AR) of the rectangular cross section is 0 <AR <1, preferably 0.3 <AR <0.7. The aspect ratio AR is shown by b / a in FIG.

On the other hand, when the cross-sectional shape of the microchannel for fine particle focus is a vertically long rectangle, the particles are focused at two points along the long side on the cross-sectional surface of the channel. When the flow path is viewed from above, the particles are observed in a state where the particles are focused on two places, one near the left and right flow path walls (Fig. 1b). In this case, the particles are introduced into the DLD flow path, which is a micro-channel for separating fine particles, in a state of being focused at two points, and then separated in the DLD flow path by adjusting the geometric shape of the DLD flow path. (Fig. 3). FIG. 3 shows a conceptual diagram of particle separation in the DLD flow path when the cross-sectional shape of the fine particle focusing microchannel is a vertically long rectangle (1 <AR). Particles larger than the separation diameter move to outlet 2, and particles smaller than the separation diameter are collected from outlets 1 and 3. In this case, the aspect ratio (AR) of the rectangular cross section is 1 <AR, preferably 1.4 <AR <3.3.

In addition, when the cross-sectional shape of the microchannel for fine particle focus is square, the particles are focused at four points along each side on the cross-section of the flow path (Fig. 1c). When the flow path is viewed from above, the particles are observed in a state where the particles are focused on three places, one near the wall surfaces of the left and right flow paths and one in the center of the flow path (Fig. 1c). In this case, the particles are introduced into the DLD flow path, which is the particle separation part, in a focused state at three points, and then separated in the DLD flow path (Fig. 4). FIG. 4 is a conceptual diagram of fine particle separation in the DLD flow path when the cross-sectional shape of the fine particle focusing microchannel is square (AR = 1) or triangular. (a, b) Particles larger than the separation diameter move to outlets 2 and 4 and are collected, and particles smaller than the separation diameter are collected from outlets 1, 3 and 5. The aspect ratio (AR) of the cross section in this case is AR = 1.

Further, even if the cross-sectional shape of the microchannel for fine particle focusing is other than the above-mentioned shape, the particles can be focused at a specific position on the cross-sectional shape of the flow path. For example, when the cross section is triangular as shown in FIG. 1 (d), the particles can be focused on three points on the cross section, and the particles are focused on three linear lines from the observation direction in the figure. The condition is observed. Further, when the cross section is semicircular as shown in FIG. 1 (e), the particles can be focused on two places on the cross section, and the particles are focused on one line from the observation direction in the figure. The state is observed.

Further, even if the cross-sectional shape of the microparticle focusing microchannel is the same, the number of focuses when viewed from above can be changed by changing the direction thereof. For example, the flow path of the square cross section of FIG. 1 (c) is rotated by 45 degrees, or the flow path of the triangular cross section of FIG. 1 (d) is rotated by 30 degrees, or the flow path of the semicircular cross section shown in FIG. 1 (e). By rotating the flow path by 90 degrees, it is possible to have two focus positions when viewed from above.

The flow of the microchannel for fine particle focusing is preferably a Reynolds number Re of 0.01 to 2400 or a laminar flow state.

The convergence width when the fine particles are introduced from the fine particle focusing microchannel due to inertial force to the fine particle separation microchannel having a column arrangement structure is the gap between the columns arranged in the fine particle separation microchannel (d). It is preferable that the convergence width is equivalent to that of. For example, a microchannel device is a polydimethylsiloxane (polydimethylsiloxane) for sealing a DLD channel formed between strut sequences made of the temperature-responsive polymer poly-N-isopropylacrylamide (PNIPAM) and the channel. It consists of a polydimethylsulfonic: PDMS) flow path. A DLD strut by PNIPAM is _{produced on a glass substrate by photolithography using, for example, a film mask having a strut diameter (D p} ) of 20 μm, a strut gap (d) of 30 μm, and a strut arrangement inclination (θ) of 0.05 rad. Here, the inclination (θ) of the column arrangement is the inclination in the arrangement direction of the columns in the plan view. The PDMS flow path for sealing the DLD flow path transfers a pattern from a mold prepared using a negative photoresist "SU-8" based on the epoxy resin "EPON SU-8" to PDMS on a Si substrate. It is produced by. After aligning the DLD strut made on the glass substrate with the PDMS flow path, they are bonded together to form a device.

In one embodiment of the present invention, in the case of a flow path using PDMS as a support, a flow path mold is made of epoxy resin “EPON SU-8” or Si, and then the flow path shape pattern is transferred to PDMS. In the case of a channel using Si, it is preferable to etch Si before use.

The shape of the support column is not limited to a cylinder, and may be any columnar body. For the arrangement of the columns, the shape of the columns, the diameter of the columns, the gap between the columns (d), and the inclination (θ) of the arrangement of the columns are preferably set according to the characteristics of the particles such as the size, shape, and hardness of the fine particles to be separated. Can be done by.

For example, in the case of separation according to the size of the fine particles, the strut diameter is set from 10 nm to 10 mm, the gap between the strut is 1 nm to 10 mm, larger than the diameter of the separated fine particles, and the inclination of the strut arrangement is about 0.01 to 0.5 rad. The array of struts is configured to have a single separation diameter (ie, the particle diameter that borders the changes in the particle trajectory due to the geometry of the flow path), or to have multiple separation diameters. You may be.

In the method for separating fine particles of the present invention, a liquid in which fine particles are dispersed is introduced into a microchannel for focusing on fine particles, and the fine particles in the introduced liquid are simply introduced along a flow in a single flow path by an inertial force. Locally arranged in one or more linear lines; in a state where fine particles are concentrated and arranged in a single or multiple specific sites of a microchannel for separating fine particles formed in a gap between arranged columns. , The liquid in which the fine particles are dispersed is flowed in; the orbit of the fine particles flowing through the fine particle separation microchannel is controlled to separate the fine particles according to the characteristics of the fine particles, and the fine particles are discharged from the fine particle separation microchannel.

Here, the stanchion may preferably be made of a material commonly used in conventional DLDs, such as a polymer resin such as polydimethylsiloxane (PDMS), Si, glass or the like. Furthermore, using a stimulus-responsive polymer material, the trajectory of the fine particles flowing through the microchannel is controlled according to the degree of change in the shape of the strut due to contraction or swelling in response to the stimulus, and the fine particles are separated according to the characteristics of the fine particles. You may.

The characteristics of the fine particles include their size, shape or hardness, and the fine particles will be separated based on their size, their shape, or their hardness.

The stimulus-responsive polymer material that forms the struts is a hydrogel that responds to physical stimuli such as temperature, light, electric or magnetic fields, or chemical stimuli such as pH, solution composition, and ionic strength. For example, a temperature-responsive polymer material is a hydrogel, which is a polymer having a property of swelling when it contains a large amount of water, and has a function of causing swelling / contraction due to reversible hydration / dehydration due to temperature change. Has.

The fine particles to be separated are selected from polymer fine particles such as latex, biological fine particles such as erythrocytes and living cells, metal fine particles, and non-metal particles such as metal oxide.

The particle size of the fine particles is usually 1 nm to 1 mm, preferably 10 nm to 100 μm.

The microparticles are introduced from the inlet of the microparticle separation device, for example in the form of an aqueous or oily suspension. The aqueous suspension is preferably a solution in which fine particles are suspended in an aqueous solution to which a surfactant is added at a particle concentration of ^{about 10 2} to 10 ^{10 / mL.} The aqueous suspension contains water as a main component and may contain an organic solvent such as alcohols that is compatible with water.

In the fine particle separation method of the present invention, a fine particle focus microchannel for arranging the arrangement of fine particles introduced from the inflow port by inertial force is provided in front of the fine particle separation microchannel, and the fine particle separation microchannel is provided. It is configured to concentrate fine particles at a specific position.

The shape of this microchannel for fine particle focus is
Cross-sectional shape: rectangular, square, semi-circular, triangular;
Aspect ratio (aspect ratio, AR) of the cross-section of the flow path: 0 <AR <1 when the cross-section is a horizontally long rectangle; 1 <AR when the cross-section is a vertically long rectangle; AR = 1 when the cross-section is square.
Channel shape: straight or meandering channel;
Channel length: 1 μm to 1 m, preferably 1 mm to 10 cm;
Channel width: 50 nm to 10 mm;
Channel height: Selected from about 50 nm to 10 mm.

Fig. 1 shows the relationship between the cross-sectional shape of the microchannel for focusing on fine particles by the inertial force of the present invention, the focus position of fine particles, and the observation direction. In FIG. 1, (a) horizontal rectangle 0 <AR <1; (b) vertical rectangle 1 <AR ,; (c) square 1 = AR ,; (d) triangle ,; (e) semicircle, (upper) flow The particle focus position on the road cross section, and the particle focus position when the (lower) microchannel is observed from the upper side of the flow path cross section and the direction perpendicular to the flow (top view).

When the cross-sectional shape of the microchannel is a horizontally long rectangle, the particles are focused at two points in the center of the channel along the long side on the cross section of the channel (Fig. 1a). As shown in the figure, when viewed from above, the particles are observed in a single linearly focused state in the center of the flow path (Fig. 1a). In this case, the particles are introduced into the DLD flow path, which is the fine particle separation part, in a single linearly focused state, and then separated in the DLD flow path (Fig. 2). FIG. 2 shows a conceptual diagram of particle separation in the DLD flow path when the cross-sectional shape of the microchannel for fine particle focus is a horizontally long rectangular (0 <AR <1) or a semicircular cross-sectional shape, and (a) from the separation diameter. Larger particles move to outlet 1 and are collected, particles smaller than the separation diameter are collected from outlet 2, (b) particles larger than the separation diameter move to outlet 3 and are collected, and particles smaller than the separation diameter are collected from outlet 2. Will be recovered. In this case, the aspect ratio (AR) of the rectangular cross section is 0 <AR <1, preferably 0.3 <AR <0.7. The aspect ratio AR is shown by b / a in FIG.

On the other hand, when the cross-sectional shape of the microchannel for fine particle focus is a vertically long rectangle, the particles are focused at two points along the long side on the cross-sectional surface of the channel. When the flow path is viewed from above, the particles are observed in a state where the particles are focused on two places, one near the left and right flow path walls (Fig. 1b). In this case, the particles are introduced into the DLD flow path, which is a micro-channel for separating fine particles, in a state of being focused at two points, and then separated in the DLD flow path by adjusting the geometric shape of the DLD flow path. (Fig. 3). FIG. 3 shows a conceptual diagram of particle separation in the DLD flow path when the cross-sectional shape of the fine particle focusing microchannel is a vertically long rectangle (1 <AR). Particles larger than the separation diameter move to outlet 2, and particles smaller than the separation diameter are collected from outlets 1 and 3. In this case, the aspect ratio (AR) of the rectangular cross section is 1 <AR, preferably 1.4 <AR <3.3.

In addition, when the cross-sectional shape of the microchannel for fine particle focus is square, the particles are focused at four points along each side on the cross-section of the flow path (Fig. 1c). When the flow path is viewed from above, the particles are observed in a state where the particles are focused on three places, one near the wall surfaces of the left and right flow paths and one in the center of the flow path (Fig. 1c). In this case, the particles are introduced into the DLD flow path, which is the particle separation part, in a focused state at three points, and then separated in the DLD flow path (Fig. 4). FIG. 4 is a conceptual diagram of fine particle separation in the DLD flow path when the cross-sectional shape of the fine particle focusing microchannel is square (AR = 1) or triangular. (a, b) Particles larger than the separation diameter move to outlets 2 and 4 and are collected, and particles smaller than the separation diameter are collected from outlets 1, 3 and 5. The aspect ratio (AR) of the cross section in this case is AR = 1.

Further, even if the cross-sectional shape of the microchannel for fine particle focusing is other than the above-mentioned shape, the particles can be focused at a specific position on the cross-sectional shape of the flow path. For example, when the cross section is triangular as shown in FIG. 1 (d), the particles can be focused on three points on the cross section, and the particles are focused on three linear lines from the observation direction in the figure. The condition is observed. Further, when the cross section is semicircular as shown in FIG. 1 (e), the particles can be focused on two places on the cross section, and the particles are focused on one line from the observation direction in the figure. The state is observed.

Further, even if the cross-sectional shape of the microparticle focusing microchannel is the same, the number of focuses when viewed from above can be changed by changing the direction thereof. For example, the flow path of the square cross section of FIG. 1 (c) is rotated by 45 degrees, or the flow path of the triangular cross section of FIG. 1 (d) is rotated by 30 degrees, or the flow path of the semicircular cross section shown in FIG. 1 (e). By rotating the flow path by 90 degrees, it is possible to have two focus positions when viewed from above.

The flow of the microchannel for fine particle focusing is preferably a Reynolds number Re of 0.01 to 2400 or a laminar flow state.

The convergence width when the fine particles are introduced from the fine particle focusing microchannel due to inertial force to the fine particle separation microchannel having a column arrangement structure is the gap between the columns arranged in the fine particle separation microchannel (d). It is preferable that the convergence width is equivalent to that of. For example, a microchannel device is a polydimethylsiloxane (polydimethylsiloxane) for sealing a DLD channel formed between strut sequences made of the temperature-responsive polymer poly-N-isopropylacrylamide (PNIPAM) and the channel. It consists of a polydimethylsulfonic: PDMS) flow path. A DLD strut by PNIPAM is _{produced on a glass substrate by photolithography using, for example, a film mask having a strut diameter (D p} ) of 20 μm, a strut gap (d) of 30 μm, and a strut arrangement inclination (θ) of 0.05 rad. Here, the inclination (θ) of the column arrangement is the inclination in the arrangement direction of the columns in the plan view. The PDMS flow path for sealing the DLD flow path transfers a pattern from a mold prepared using a negative photoresist "SU-8" based on the epoxy resin "EPON SU-8" to PDMS on a Si substrate. It is produced by. After aligning the DLD strut made on the glass substrate with the PDMS flow path, they are bonded together to form a device.

In one embodiment of the present invention, in the case of a flow path using PDMS as a support, a flow path mold is made of epoxy resin “EPON SU-8” or Si, and then the flow path shape pattern is transferred to PDMS. In the case of a channel using Si, it is preferable to etch Si before use.

The shape of the support column is not limited to a cylinder, and may be any columnar body. For the arrangement of the columns, the shape of the columns, the diameter of the columns, the gap between the columns (d), and the inclination (θ) of the arrangement of the columns are preferably set according to the characteristics of the particles such as the size, shape, and hardness of the fine particles to be separated. Can be done by.

For example, in the case of separation according to the size of the fine particles, the strut diameter is set from 10 nm to 10 mm, the gap between the strut is 1 nm to 10 mm, larger than the diameter of the separated fine particles, and the inclination of the strut arrangement is about 0.01 to 0.5 rad. The array of struts is configured to have a single separation diameter (ie, the particle diameter that borders the changes in the particle trajectory due to the geometry of the flow path), or to have multiple separation diameters. You may be.

In the method for separating fine particles of the present invention, a liquid in which fine particles are dispersed is introduced into a microchannel for focusing on fine particles, and the fine particles in the introduced liquid are simply introduced along a flow in a single flow path by an inertial force. Locally arranged in one or more linear lines; in a state where fine particles are concentrated and arranged in a single or multiple specific sites of a microchannel for separating fine particles formed in a gap between arranged columns. , The liquid in which the fine particles are dispersed is flowed in; the orbit of the fine particles flowing through the fine particle separation microchannel is controlled to separate the fine particles according to the characteristics of the fine particles, and the fine particles are discharged from the fine particle separation microchannel.

Here, the stanchion may preferably be made of a material commonly used in conventional DLDs, such as a polymer resin such as polydimethylsiloxane (PDMS), Si, glass or the like. Furthermore, using a stimulus-responsive polymer material, the trajectory of the fine particles flowing through the microchannel is controlled according to the degree of change in the shape of the strut due to contraction or swelling in response to the stimulus, and the fine particles are separated according to the characteristics of the fine particles. You may.

The characteristics of the fine particles include their size, shape or hardness, and the fine particles will be separated based on their size, their shape, or their hardness.

The stimulus-responsive polymer material that forms the struts is a hydrogel that responds to physical stimuli such as temperature, light, electric or magnetic fields, or chemical stimuli such as pH, solution composition, and ionic strength. For example, a temperature-responsive polymer material is a hydrogel, which is a polymer having a property of swelling when it contains a large amount of water, and has a function of causing swelling / contraction due to reversible hydration / dehydration due to temperature change. Has.

The fine particles to be separated are selected from polymer fine particles such as latex, biological fine particles such as erythrocytes and living cells, metal fine particles, and non-metal particles such as metal oxide.

The particle size of the fine particles is usually 1 nm to 1 mm, preferably 10 nm to 100 μm.

The microparticles are introduced from the inlet of the microparticle separation device, for example in the form of an aqueous or oily suspension. The aqueous suspension is preferably a solution in which fine particles are suspended in an aqueous solution to which a surfactant is added at a particle concentration of ^{about 10 2} to 10 ^{10 / mL.} The aqueous suspension contains water as a main component and may contain an organic solvent such as alcohols that is compatible with water.

Next, in the present invention, a case where separation by particle shape is performed will be described. For example, in the case of disk-shaped particles such as red blood cells (thickness 2 μm, diameter 6 μm), the apparent diameter of the particles in the DLD flow path changes depending on the flow posture of the particles, resulting in separation behavior (Displacement mode, Zigzag mode). It is known to have an impact. Therefore, we used a separation method (JP Beech et al., Lab Chip, 12, 1048-1051, 2012) in which the posture of erythrocytes (diameter direction and thickness direction of the disk) was controlled with respect to the support of the DLD, and the rotation of erythrocytes. Since separation methods (KK Zeming et al., Nat. Commun., 4, 1625, 2013) have been reported so far, it is possible to perform separation by particle shape by applying the separation method of the present invention to these. can.

Further, in the present invention, a case where separation is performed based on the hardness of the particles will be described. Since shear stress acts on the particles in the DLD flow path, the degree of deformation of the particles differs according to the difference in hardness of the particles. For example, even if the particles have the same diameter in the stationary state, the particles that are more easily deformed have a smaller apparent diameter in the DLD flow path than the particles that do not deform. That is, even if the particles have the same size, it is possible to separate the particles by utilizing the difference in the degree of deformation of the particles. For example, in the case of erythrocytes, since the hardness of normal erythrocytes and that of malaria-infected erythrocytes are different, it is expected that disease can be detected by separating erythrocytes using the hardness as an index (T. Krueger et al., Biomicrofluidics, 8, 054114, 2014). By applying the separation method of the present invention to these, separation can be performed according to the hardness of the particles.

Example 1
FIG. 5 is a schematic view showing one embodiment of the fine particle separation device of the present invention. It is composed of a microchannel (introduction path) for focusing fine particles by inertial force and a microchannel for separating fine particles having a deterministic lateral displacement (DLD) strut arrangement. The microchannel for focusing on fine particles, which is the introduction path, is a linear channel (width 50 μm, height 25 μm, length 2.5 cm) having a rectangular cross section (aspect ratio 0.5), and is a downstream DLD channel (microparticle separation micro). The parameters of the flow path) were a flow path length of 23 mm, a flow path width of 3 mm, a column diameter (D _p ) of 30 μm, a distance between columns (d) of 20 μm, and an inclination of the column arrangement of 0.05 rad. The device was made by transferring a pattern from a template made using a negative photoresist "SU-8" based on the epoxy resin "EPON SU-8" to polydimethylsiloxane (PDMS) on a Si substrate. It was formed by adhering the prepared PDMS flow path and a glass substrate after oxygen plasma treatment.

The introduction specimen diameter 13μm and 7μm polystyrene beads (Thermo Fisher Co., Bangs Laboratories Inc.) 0.1 v / v% of the surfactant Tween (R) 20 (Sigma) particle concentration 1.0 × 10 ⁵ in an aqueous solution A solution suspended at / mL was prepared. The particle suspension solution was sent by a syringe pump (KD Scientific, KDS200).

FIG. 6 shows the focusing of the fine particles due to the inertial force when the particle suspension solution is introduced into the device at a flow rate (Q) of 1 mL / h. It was confirmed that the introduced fine particles flowed in a state where the beads were dispersed in the entire flow path in the upstream portion of the microchannel for focusing on the fine particles (FIG. 6a). Beads with a diameter of 13 μm moved to the center of the flow path in the middle part of the flow path for fine particle focus (Fig. 6b), and beads with diameters of 13 μm and 7 μm both moved to the center of the flow path in the downstream part of the flow path for fine particle focus (Fig. 6b). FIG. 6c) The situation was confirmed. This is because the flow path length required for the fine particles to move to the center of the flow path is inversely proportional to the cube of the particle diameter. On the other hand, when the flow rate was set to low (Q = 0.5 mL / h, 0.1 mL / h), the fine particles did not move sufficiently to the center of the flow path, and at Q = 1 mL / h, the diameters were 13 μm and 7 μm. It was confirmed that the beads sufficiently moved to the center of the flow path (FIGS. 7a to 7c; FIG. 7 is a diagram showing a state of particle focusing in the particle focusing microchannel under different flow rate conditions). It is considered that this is because the flow path length required for the particles to move to the center of the flow path is inversely proportional to the flow velocity.

FIG. 8 shows a state of fine particle separation in the fine particle separation microchannel (DLD flow path) when the flow rate is set to 1 mL / h. After the fine particles are introduced in a focused state at a specific position in the DLD flow path (Fig. 8a), the beads with a diameter of 13 μm take a displacement mode trajectory that follows the inclination of the DLD strut arrangement and have a diameter. It was confirmed that the 7 μm beads take a zigzag mode orbit in the same direction as the flow (Fig. 8b). In the downstream part of the DLD flow path, it was confirmed that beads having diameters of 13 μm and 7 μm were separated and then collected from outlet 1 and outlet 2, respectively (FIG. 8c). That is, fine particle separation was realized by a fine particle separation device without using a microchannel structure in which a fine particle suspension solution is sandwiched by a buffer, such as a sheath flow type.

According to the present invention, a fine particle separation device having only a liquid feeding flow path for inflowing a fine particle suspension sample as an introduction path without using a flow path structure for sandwiching the fine particle suspension solution using a buffer, and a fine particle separation device thereof are used. A method for separating fine particles can be provided.

Claims (14)

It is a fine particle separation device for inflowing a liquid in which fine particles are dispersed and separating the fine particles according to their characteristics.
It consists of an inlet and outlet for fine particles, a microchannel for focusing on fine particles, and a microchannel for separating fine particles;
The fine particle separation microchannel is formed in a gap between the arranged columns, and is configured to control the trajectory of the fine particles flowing through the fine particle separation microchannel to separate the fine particles according to the characteristics of the fine particles. The microchannel for focusing on fine particles is provided as an introduction path in front of the microchannel for separating fine particles.
The fine particles in the liquid introduced from the inflow port are configured to be locally arranged in a single or multiple linear lines along the flow in a single flow path by inertial force.
A fine particle separation device, characterized in that the fine particles are configured to flow into a single or a plurality of specific sites of the fine particle separation microchannel in a concentrated and arranged state; The fine particle separation device according to claim 1, wherein the fine particles are separated based on their size, shape or hardness. The fine particle separation device according to claim 1 or 2, wherein in the microchannel for focusing fine particles, fine particles are focused by inertial force in a single line, two or three linear lines along a flow. The fine particle separation device according to any one of claims 1 to 3, wherein the fine particles are selected from polymer fine particles, biological fine particles, droplets, metal fine particles, and non-metal particles. The fine particle separation device according to any one of claims 1 to 4, wherein the liquid in which the fine particles are dispersed is an aqueous suspension. The fine particle separation device according to any one of claims 1 to 5, wherein the fine particle separation microchannel is formed in a gap between columns having a single separation diameter. The fine particle separation device according to any one of claims 1 to 5, wherein the fine particle separation microchannel is formed in a gap between columns having a plurality of separation diameters. The liquid in which the fine particles are dispersed is introduced into the microchannel for focusing on the fine particles, and the fine particles in the introduced liquid are locally localized in a single line or a plurality of lines along the flow in the single channel by inertial force. Arranged in;
In a state where the fine particles are concentrated and arranged in a single or a plurality of specific parts of the microchannel for separating fine particles formed in the gap between the arranged columns, the liquid in which the fine particles are dispersed is allowed to flow in.
A method for separating fine particles, which comprises controlling the trajectory of the fine particles flowing through the microchannel for separating fine particles, separating the fine particles according to the characteristics of the fine particles, and causing the fine particles to flow out from the microchannel for separating fine particles. The method for separating fine particles according to claim 8, wherein the fine particles are separated based on their size, shape or hardness. The method for separating fine particles according to claim 8 or 9, wherein the fine particles are focused by inertial force in a single line, two or three linear lines along the flow in the fine particle focusing microchannel. The method for separating fine particles according to any one of claims 8 to 10, wherein the fine particles are selected from polymer fine particles, biological fine particles, droplets, metal fine particles, and non-metal particles. The method for separating fine particles according to any one of claims 8 to 11, wherein the liquid in which the fine particles are dispersed is an aqueous suspension. The method for separating fine particles according to any one of claims 8 to 12, wherein the microchannels for separating fine particles are formed in a gap between columns having a single separation diameter in which they are arranged. The method for separating fine particles according to any one of claims 8 to 12, wherein the microchannels for separating fine particles are formed in gaps between columns having a plurality of separation diameters in which the microchannels are arranged.

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