JP2012076017A - Microchannel system for elutriator and particle separation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an elutriator apparatus which does not require a connecting mechanism, which is required in a conventional elutriation apparatus, for supplying a liquid to a high speed rotating rotor part using an external pump, and to provide an elutriator system whose apparatus is disposable.SOLUTION: A microchannel system for an elutriator is formed with a channel in a substrate and can rotate around a rotation axis. The channel has an inlet, an outlet, an inlet solution chamber connected to the inlet, and a separation chamber formed between the inlet and the outlet. The distance between the inlet and the rotation axis is shorter than the distance between the outlet and the rotation axis. The distance between the inlet of the separation chamber and the rotation axis is longer than the distance between the outlet of the separation chamber and the rotation axis.

Description

  The present invention continuously separates biological particles such as animal and plant cells, bacteria, viruses, etc., or organelles that are constituents thereof, polymer particles, ceramic particles and metal fine particles according to the particle size and specific gravity. The present invention relates to a microchannel system for carrying out and a centrifugal elutriator apparatus using the same.

  In general, the technology for separating particles such as synthetic polymer fine particles, inorganic powders, and metal powders is not only for basic research, but also for selecting particles of specific sizes in the powder industry, electronics industry, food industry, pharmaceutical industry, etc. And is important in a wide range of fields such as particle size distribution analysis. In addition, a technique for separating and selecting cells having a specific gravity or size from a complex cell population composed of many types of cells is essential in research fields such as regenerative medicine, diagnostic medicine, and biochemistry.

  As a method for separating and classifying fine particles and cells, a continuous centrifugation method called an elutriation method is known, and the apparatus is called an elutriator. The elutriation method is based on the principle that particles gather at the equilibrium position of the flow force opposite to the centrifugal force according to the settling velocity. That is, a gradient layer of particles based on particle size and specific gravity is formed in a chamber installed in the rotor of the centrifuge by centrifugal force acting on the particles and counterflow acting in the opposite direction. By lowering the rotation speed of the rotor or increasing the flow velocity of the counter flow, the equilibrium position of the particles is changed, and the particles are discharged out of the chamber from small particles or particles with small specific gravity. This is a method of collecting this as a fraction and collecting particles having a uniform particle size for each fraction.

  In general, a chamber that gradually expands toward the center of rotation is used. Thereby, the flow direction gradient is provided to the flow velocity of the counter flow. As a result, a gradient of the residence position distribution is formed in the direction of centrifugal (outside) from the center of rotation in the chamber, from small particles to large particles, or from particles with small specific gravity to large particles.

JP-A-7-265742

  However, in the above-described conventional elutriation method, a connection mechanism for feeding liquid using an external pump to the rotor portion rotating at high speed is required. Furthermore, since it is necessary to gradually change the number of revolutions or the amount of liquid delivered by the pump, a mechanism for independently controlling these two parameters is required, and the apparatus itself is very expensive. Therefore, it cannot be a disposable system, and when it is necessary to avoid contamination between samples, it is necessary to clean and sterilize the inside of the flow path for each trial.

  The present invention has been made in view of the above-described problems of the prior art, and the object of the present invention is to eliminate the need for an external liquid feeding device, which is conventionally required, and to dispose the entire device. An object of the present invention is to provide a microchannel system and particle separation method for an Eltriator that can be made inexpensive.

  In order to achieve the above object, the invention according to the first aspect of the present invention is formed of a polymer, glass, or metal material for separating particles having an average particle diameter of R μm (R is 1000 or less). , A micro-channel system for an Eltriator that can be rotated with respect to a specific rotation axis (RA) by an arbitrary rotation device, in which (1) an inlet (DP) and an outlet (DR) A flow path (D) having an inner diameter of 1.5 Rm to 100 mm exists, and (2) the linear distance between the inlet (DP) and the rotation axis (RA) is the straight line between the outlet (DR) and the rotation axis (RA). (3) The flow path (D) is configured so that the flow path (D) is separated from the rotation axis (RA) when the flow path (D) is traced from the inlet (DP) to the outlet (DR). Part where the linear distance gradually decreases (DS) (4) The flow path (D) can hold the liquid inside through the inlet (DP) when the micro flow channel system for the L-tritor is stationary and rotating. (5) The flow path (D) has a structure called a separation chamber (DC) in the part (DS), and (6) a microflow for the eltriator. By rotating the entire path system about the axis of rotation (RA), the particles are introduced into the flow path (D) together with the solution A (composition a), and in the separation chamber (DC), the particles are sized and The particles are separated because they stay at different positions depending on the difference in specific gravity, or at least part of the particles cannot stay. As a result, an excellent effect of eliminating the need for an external liquid delivery device such as a pump, which has been indispensable in the conventional eltriator device, is exhibited.

  Further, in the invention according to this aspect, although not limited, the flow path (D) is an outlet solution that can hold the liquid inside when stationary and rotating via the outlet (DR). It is preferable to be connected to a chamber (OC). By doing so, it becomes possible to hold the particles separated and discharged from the flow path in the outlet chamber in the apparatus, and it is possible to collect the particles discharged from the flow path after the rotation operation is stopped. It becomes.

  In the invention according to this aspect, although not limited, the separation chamber (DC) has a cross-sectional area S which is gradually or stepwise at least partially as it goes downstream of the flow path (D). Is preferably increased. By doing so, it becomes possible to efficiently form a flow velocity gradient in the separation chamber, so that the difference in the particle retention position due to the difference in size or specific gravity is efficiently formed in the separation chamber. Therefore, the separation accuracy can be improved.

  Further, in the invention according to this aspect, although not limited, the flow path (D) branches at the branch point BP in a section sandwiched between the inlet (DP) and the separation chamber (DC), and at the junction point CP. It is preferable to have at least one branch channel (DB) that rejoins. In other words, by providing a branch flow channel into which particles are not introduced, even if the flow channel is formed in a two-dimensional plane, particles are efficiently introduced not near the wall surface of the branch chamber but near the center. It becomes possible.

  In the invention according to this aspect, although not limited, the inlet solution chamber (IC) preferably has a structure in which a plurality of inlet solution chambers capable of individually introducing solutions are connected in parallel or in series. . In this way, all the introduced cells can be efficiently introduced into the separation chamber, and the composition and physical properties of the solution introduced into the flow path are changed stepwise or gradually. It is also possible.

  Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, the exit solution chamber (OC) is connected with the several exit solution chamber which can collect | recover the solution discharged | emitted from the exit (DR) separately in parallel or in series. It is preferable that it is the structure made. In this way, it is possible to hold the fractions of particles with different properties discharged from the separation chamber over time in different outlet solution chambers for each fraction and collect them individually after the rotation stops. It becomes possible to do.

  Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, it is preferable that the specific gravity of the solution introduce | transduced into a flow path (D) increases in steps or gradually. In this way, it is possible to gradually change the particle retention position without changing the flow velocity in the channel over time, so that efficient particle separation is possible with simpler operations. It becomes.

  Further, in the invention according to this aspect, although not limited, the flow path (D) and the separation chamber (DC) include a groove-like structure processed on the surface of the flat substrate and other flat plates. It is preferable that the substrate is manufactured by bonding. By doing so, for example, it is possible to manufacture an L-triator device at a low cost and in large quantities using a polymer substrate, and thus the device itself can be made disposable.

  In the invention according to this aspect, although not limited, the particles to be separated are preferably biological particles such as cells, viruses, bacteria, and organelles. By separating adult particles, important processes can be realized in research fields such as medicine and biochemistry.

  In addition, a particle separation method according to another aspect of the present invention is to perform particle separation using the microchannel system for an L-triator according to the first aspect. This makes it possible to separate particles and cells with an elutriator apparatus that is simpler and less expensive than conventional methods.

  An eltriator microchannel system according to another aspect of the present invention is an eltriator microchannel system in which a channel is formed on a substrate and is rotatable about a rotation axis. An outlet, an inlet solution chamber connected to the inlet, and a separation chamber formed between the inlet and the outlet, the distance between the inlet and the rotating shaft being shorter than the distance between the outlet and the rotating shaft The distance between the separation chamber inlet and the rotation axis is longer than the distance between the separation chamber outlet and the rotation axis.

  Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, it is preferable to have an exit solution chamber connected to an exit.

  In the invention according to this aspect, although not limited, the cross-sectional area of the separation chamber increases gradually or stepwise from the entrance of the separation chamber toward the exit of the separation chamber. It is preferable.

  In the invention according to this aspect, the flow path is not limited, but the flow path joins the branch point where the flow path branches and the flow path branched by the branch point between the inlet and the separation chamber inlet. It is preferable to have a meeting point.

  Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, it is preferable to have a some inlet solution chamber connected in series or in parallel.

  Moreover, in the invention which concerns on this viewpoint, although it is not necessarily limited, it is preferable to have several exit solution chambers connected in series or in parallel.

  Moreover, in the invention which concerns on this viewpoint, although not necessarily limited, it is preferable to form by bonding together the board | substrate with which the groove | channel corresponding to the flow-path shape was formed, and the board | substrate on a flat plate.

  Since the present invention is configured as described above, a system for supplying liquid from the outside to the flow path portion and the separation chamber becomes unnecessary. Therefore, the particles can be separated and collected only by rotating the device itself. Therefore, for example, by using a flow channel structure formed on an inexpensive polymer substrate, a disposable device can be obtained. Therefore, it is possible to provide an apparatus that does not require processing such as cleaning and sterilization between trials, which has been conventionally required.

  In addition, since the present invention is configured as described above, the apparatus itself can be reduced in size as compared with a conventional apparatus that requires a large centrifuge and a dedicated rotor.

  Furthermore, since the present invention is configured as described above, when the number of rotations of the apparatus or the viscosity of the solvent is changed, the flow velocity and centrifugal force balance in the flow path are hardly affected. That is, the accuracy of separation is mainly determined only by the density of the solvent. Therefore, it is difficult to be affected by operating conditions, and even with a very simple system configuration, it is possible to accurately separate and collect cells.

It is the schematic of the microchannel system for eltriators concerning an embodiment, Drawing 1 (a) is a B arrow view in Drawing 1 (b) and Drawing 1 (c), and Drawing 1 (b) is Drawing 1 It is sectional drawing in the A0-A1 line in (a), and FIG.1 (c) is sectional drawing in the A2-A3 line in Fig.1 (a). It is the schematic of the system which has a form different from the system shown in FIG. 1 of the flow-path structure in the micro flow-path system for eltriators which concerns on embodiment. It is the schematic of the system which has a form different from the system shown in FIG. 1 and FIG. 2 of the flow-path structure in the micro flow-path system for eltriators which concerns on embodiment. It is the schematic of the system which has a form different from the system shown in FIG.1, 2, and 3 of the flow-path structure in the microchannel system for eltriators which concerns on embodiment. It is the schematic of the micro flow path system for eltriators used in Example 1, FIG. 5 (a) is a B arrow view in FIG.5 (b), FIG.5 (b) is A0- in FIG.5 (a). FIG. 5C is an enlarged view of the individual flow path (D) and the rotation axis (RA) in FIG. 5A. FIG. 5A and FIG. 5C are almost the actual size. It is a graph which shows the result in Example 1, a horizontal axis shows the fraction number isolate | separated, and the vertical axis | shaft has shown the abundance ratio of each particle | grain. FIG. 3 is a schematic diagram of an L triator microchannel system used in Example 2.

  BEST MODE FOR CARRYING OUT THE INVENTION The best mode of the microchannel system and particle separation method for an L-triator according to the present invention will be described in detail below. However, the present invention can be implemented in many different forms, and is not limited only to the following embodiments and examples.

  FIG. 1 is a schematic view of the microchannel system for an elutriator according to the present embodiment. 1A is a view taken in the direction of arrow B in FIGS. 1B and 1C, and FIG. 1B is a cross-sectional view taken along line A0-A1 in FIG. 1A. 1 (c) is a cross-sectional view taken along line A2-A3 in FIG. 1 (a).

  In FIG. 1A, the flow path (D) is a portion (flow path portion DS) where the linear distance from the rotation axis (RA) becomes short when the flow path is downstream, a separation chamber (DC), It has an inlet (DP) and an outlet (DR), and is connected to the inlet solution chamber (IC) via the inlet (DP). The rotation axis RA is a convenient representation of the position of the center of rotation when the system is rotated by an arbitrary rotating device, and a physical axis for performing a rotational motion is not necessarily attached. It does not mean that

  This micro channel system for an L trilator is formed by bonding two substrates, and the channel structure excluding the inlet solution chamber (IC) is formed on the surface of one of the substrates. The groove structure and the other substrate are planarly configured, and the channel depth is constant, but the channel structure may be formed using three or more substrates, The channel depths may be partially different, and the system may be at least partially connected by circular tubes with the same or different diameters. However, it is more preferable that the flow path structure is at least partially configured in terms of facilitating the production of the flow path structure and enabling more precise production.

  When producing a planar flow path structure, for example, a production technique using a mold such as molding or embossing is preferable in that the flow path structure can be easily produced. Manufacturing techniques such as wet etching, dry etching, laser processing, direct electron beam drawing, and machining can also be used.

  In addition, the material of the microfluidic system when producing a planar flow path structure includes various polymer materials such as PDMS (polydimethylsiloxane) and acrylic, and various metals such as glass, silicon, ceramics, and stainless steel. , Etc., and any two of these materials can be used in combination.

  Furthermore, in the flow channel structure shown in FIG. 1A, the flow channel (D) and the separation chamber (DC) include a flow channel portion linearly extending in the centrifugal direction from the rotation axis (RA), and a rotation axis. Although it is configured by an arc-shaped flow path portion having a center, these structures do not necessarily have to be linear or arc-shaped structures.

  Further, in the flow channel structure shown in FIG. 1 (a), the center of gravity of the micro flow channel system for the eltriator exists on the rotation axis in order to balance the centrifugal force during the rotation operation. If the operation becomes possible, the center of gravity of the system does not necessarily exist on the rotation axis. For example, when the rotation axis is attached to a larger apparatus and the rotation axis is rotated, the rotation axis is located away from the micro flow channel system for the eltriator. There may be.

  In the flow channel structure shown in FIG. 1A, the inlet (DP) of the flow channel (D) is located closer to the rotational axis (RA) than the outlet (DR). On the other hand, when a rotational motion is performed, the solution can be continuously introduced from the inlet to the outlet.

  The solution A, which is a particle suspension solution, is dropped into the inlet solution chamber (IC) with respect to this flow channel structure, and then the entire micro flow channel system for the L trilator is rotated with respect to the rotation axis (RA) by an arbitrary rotating device. When the rotation operation is performed, the solution A is introduced from the inlet (DR) by the effect of centrifugal force.

  In addition, it is possible to prevent scattering or dissipation of the solution A and particles due to the rotation operation by covering at least partly the opening of the inlet solution chamber (IC) after dropping the solution. The same effect can be achieved by narrowing the opening of the (IC) or by inclining the wall surface of the solution chamber with respect to the rotation axis direction.

  In this case, the flow path (D) and the separation chamber (DC) are preferably filled with a solution B that is a solution not containing particles in advance, and the composition of the solution B is the same as the composition of the solution A. Or close.

  With respect to the particles introduced into the channel D, in the channel part (DS), centrifugal force is applied from the rotation axis to the centrifugal side, and the flow is applied in the direction of the rotation axis. -Particles having specific gravity and shape can be retained at a specific position in the separation chamber. On the other hand, particles that are sufficiently small or sufficiently small in specific gravity are discharged from the outlet (DR). In the present embodiment, a solution chamber can be incorporated in the L-triator device, and liquid feeding can be performed using centrifugal force generated by rotating the device itself.

  In addition, after rotating for an appropriate time, the solution in the inlet solution chamber (IC) is replaced with the solution B, and the rotation operation is performed again, thereby efficiently separating the particles in the inlet solution chamber (IC). (DC) or exit (DR) can be introduced.

  In addition, after rotating for an appropriate time, the solution in the inlet solution chamber (IC) is replaced with the solution C, which is a solution having a higher specific gravity than the solution A, and the rotation operation is performed again, so that the solution enters the separation chamber (IC). The staying particles can be discharged from the outlet (DR) step by step, and separation is achieved.

  In addition, the number of rotations in the rotation operation is preferably in the range of 100 to 30000 rpm, more preferably in the range of 500 to 10000 rpm, but may be in a range that does not adversely affect particles such as cells to be introduced. .

  The viscosity of Solution A, Solution B, and Solution C to be used is not limited as long as it can be stably operated. However, if the viscosity is too high, the flow rate is lowered, so that it is preferably 100 cP or less. .

  The flow rate in the flow path is determined by the rotational speed, the viscosity of the solution, and the size (width, length, etc.) of the flow path. These values are determined in the separation chamber (DC) by the flow path. From the viewpoint that it is necessary to maintain a state where the flow velocity is uniformly decelerated with expansion, a stable laminar flow is maintained in the flow path, specifically, the Reynolds number is 1000 or less. If it is in range. In the case where the flow path has a width or depth of 1 mm or less and a centrifugal force-driven liquid feeding is performed, it is easy to achieve this condition.

  FIG. 2 is a schematic view of another example of the flow path (D) in the micro flow path system for an L trilator.

  The flow path (D) shown in FIG. 2 has a branch flow path (DB) that branches at the branch point (BP) and merges at the merge point (CP). At the branch point (BP), almost 50% of the flow rate introduced from the inlet is distributed to the branch flow path (DB), and the particles are on the opposite side of the rotating shaft by centrifugal force until reaching the rejoining point. In other words, the particles pass along the wall surface of the channel, that is, the particles are not introduced into the branch flow path (DB), so that the particles pass near the center of the cross section of the flow path at the rejoining point. Therefore, even in the separation chamber (DC), the particles can flow near the center, and the accumulation of particles on the wall surface can be suppressed, so that the accuracy of separation can be improved.

  In the flow channel structure shown in FIG. 2, the branching / re-merging portion (DB) is constituted by a branching structure into two. However, as long as the effects described above are obtained, the number of the branching / rejoining parts (DB) is not necessarily two. Further, the position is not necessarily the position shown in FIG. However, it is preferable that the rejoining portion is close to the separation chamber (DC) as shown in FIG. In addition, the flow rate introduced into the branch channel (DB) may not be 50% as long as the position where the particles flow is near the center in the separation chamber (DC).

  Further, in the flow channel structure in FIG. 2, the separation chamber (DC) has a portion that increases in width as it goes downstream (that is, as it goes from the inlet to the outlet of the separation chamber), that is, the cross-sectional area S increases. The design is such that the flow rate gradually decreases. This makes it possible to increase the size or specific gravity range of the particles staying in the separation chamber (DC).

  In addition, the flow channel structure in FIG. 2 has an outlet solution chamber (OC) capable of holding the solution inside the micro flow channel system for the L-triator, so that it stays in the separation chamber (DC). It is possible to individually collect particles discharged in stages from the separation chamber (DC).

  In addition, the flow path structure in FIG. 2 has a portion that becomes wider as it goes downstream, that is, has a portion where the cross-sectional area S increases, and is designed so that the flow velocity gradually decreases. As a result, it is possible to widen the size or specific gravity range of the particles staying in the separation chamber (DC).

  In addition, it is possible to control the range of the particle size staying in the separation chamber (DC) by designing an appropriate flow path. The flow path design described here is a value such as the shortest distance between the rotation axis (RA) and the inlet (DP) and the outlet (DR), the position and shape of the separation chamber (DC), or the diameter of the flow path (D). Is set according to the particles to be separated.

  In order to achieve the separation of specific particles, the flow channel network is considered as an analog of the resistance circuit, that is, based on the idea that the flow distribution is proportional to the pressure and inversely proportional to the flow resistance. It is desirable to go through a step of calculating or pre-calculating the distribution ratio of the flow rate at each point by calculating and considering the pressure at each point in the flow channel and the resistance value of the flow channel structure. In the calculation of resistance in that case, the Hagen-Poiseuille equation or the like can be used.

  FIG. 3 is a schematic diagram of a microchannel system for an elutriator having multiple inlet / outlet solution chambers.

  The microchannel system for an L-triator shown in FIG. 3 has a structure in which three inlet solution chambers (IC1, IC2, IC3) that can introduce solutions independently are connected in series by a channel, It has a structure in which three outlet solution chambers (OC1, OC2, OC3) capable of collecting a solution independently are connected in series by flow paths.

  In the micro channel system for an L-triator shown in FIG. 3, after the channel (D) is filled with the solution B, the solution A that is a particle suspension in OC3, the solution B that does not contain particles in OC2, and the solution A in OC1 Since each solution C having a different composition from each other is introduced and rotated, and these solutions are sequentially introduced into the flow path (D), the automatic operation of solutions having different compositions can be performed without stopping the rotating operation. Introduction is possible. It is desirable to put a solution having a higher specific gravity in the inlet solution chamber located closer to the rotation axis, and it is desirable to introduce the particle suspension solution into the inlet solution chamber located far from the rotation axis. However, if the separation purpose can be achieved, solutions having different densities and compositions may be introduced, and a larger number of inlet solution chambers may exist.

  In addition, since there are a plurality of outlet solution chambers (OC) in the L channel microchannel system shown in FIG. 3, the solution discharged from the channel (D) can be changed into outlet solution chambers (OC) that are different over time. It is possible to hold it. That is, in this case, since the discharged solutions are held in the outlet solution chambers of OC3, OC2, and OC1 in the order of discharge, they can be recovered independently after stopping the rotation. These outlet solution chambers (OC) can be used in the number or volume according to the purpose.

  FIG. 4 is a schematic diagram of another example of a microchannel system for an ertriator having multiple inlet / outlet solution chambers.

  4 has a structure in which three outlet solution chambers (OC1, OC2, OC3) capable of collecting solutions independently are connected in series by flow paths. ing. By providing each outlet solution chamber with a mechanism that does not introduce a solution of a certain amount or more, the solution discharged from the flow path (D) is allowed to flow from the upstream outlet solution chamber (OC1) to the downstream outlet solution chamber (OC3). Since the discharged solutions are held in the outlet solution chambers of OC1, OC2, and OC3 in the discharged order, it is possible to collect them independently after stopping the rotation. .

  As the particles to be separated using the above microchannel system, depending on the purpose, polymer particles such as polystyrene, metal fine particles, ceramic particles, or physical or chemical treatment of the surface thereof may be performed. In addition to the applied particles, biological particles such as animal and plant cells, bacteria, viruses, and organelles that are constituents thereof can be used.

  In addition, various solutions can be used as the particle suspension depending on the purpose. For example, when polymer particles or metal particles are used as the particles, in addition to aqueous solutions containing various chemical substances, organic solvents, ionic A fluid or the like can be used. Furthermore, when biological particles such as cells are used as the particles, it is preferable to use an aqueous solution that is isotonic with cells, such as a cell culture solution or a buffer solution. However, in the case of cells that are resistant to relatively hypotonic or hypertonic solutions, such as bacteria and plant cells, it is not always necessary to be isotonic. For the convenience of operation, a system in which the difference between the specific gravity of the solution and the specific gravity of the particles is not extremely large is more preferable, and the particle group to be separated includes particles having a specific gravity smaller than that of the solution A. It is desirable that

  In addition, as a means for rotating the micro flow channel system for the Eltriator, a normal centrifugal separator, an electric motor, or the like can be used, but any device that can maintain a constant rotational speed for a certain period of time may be used.

  By the system configuration and operation described above, it is possible to accurately and simply separate and collect only certain particles and cells according to the purpose from the mixture of particles and cells.

  In addition, since the microchannel system for an L trilator according to the present embodiment can be made disposable by forming a solution chamber, a channel, and a separation chamber structure using an inexpensive material, for example, a polymer, This is a system that makes it possible to eliminate the process of cleaning and sterilizing the inside of the flow path, which is necessary when avoiding contamination.

  Hereinafter, the effect of the present invention was confirmed by actually producing a microchannel system for an L-triator according to the above embodiment and performing a particle separation method. This will be described below.

Example 1
FIG. 5 is a microchannel system diagram according to the present embodiment. 5A is a view taken in the direction of arrow B in FIG. 5B, FIG. 5B is a cross-sectional view taken along line A0-A1 in FIG. 5A, and FIG. 5C is in FIG. It is an enlarged view of an individual flow path (D) and a rotating shaft (RA).

  This microchannel system for an L-trilator is bonded vertically to a flat polymer substrate (PDMS (polydimethylsiloxane)) having a fine groove structure and a flat polymer substrate (PDMS) not having a groove structure. It is formed by.

  A channel structure is formed on the lower surface of the upper polymer substrate, and its depth is about 20 μm. With regard to this value, it is possible to adopt a flow channel structure having an arbitrary value up to 100 mm depending on the size of the particles to be separated, and the same processing is applied to the upper surface of the lower substrate. The flow path structure may be partially different in depth.

  In addition, this micro flow channel system for an L trilator includes four micro flow channel structures having the same function. This number may be one per system or plural. Moreover, each flow path structure may be different from the structure having the same function.

  The inlet solution chamber (IC) and the outlet solution chamber (OC) are each constituted by a through hole formed in the upper substrate and a lower substrate. In addition, each is partially blocked by a removable inlet seal structure (IS) and outlet seal structure (OS), and when the system is not rotating, the solution can be injected or recovered from the outside using a pipette or the like. In addition, when the system is rotated, the solution inside the chamber is held without splashing outside. As the sealing structure, various materials or shapes can be used as long as they are in close contact with the upper substrate, such as a commercially available adhesive tape or a flat polymer substrate. In addition to the structure shown in FIG. 5, if the solution can be exchanged with the outside, and the scattering of the solution during rotation can be prevented, the chamber structure inclined in the centrifugal direction or the depth direction In addition, a chamber structure having a different cross-sectional area may be employed.

  In this micro flow channel system for an L trilator, the inlet solution chamber (IC) and the outlet solution chamber (OC) are connected by a flow channel (D), and the inlet solution chamber (IC) and the outlet solution chamber (OC) flow. It is not defined as a part of the path (D).

  When the flow path (D) traces the flow path from the inlet to the outlet, the flow path once turns to the outside and then turns so as to approach the rotation axis. And a separation chamber (DC) in which the width gradually expands and converges downstream thereof.

  The width of the flow path (D) is 100 μm in portions other than the separation chamber (DC), and the maximum value in the separation chamber (DC) is 5 mm. Similarly to the channel depth, these values can adopt a channel structure having an arbitrary value up to 100 mm depending on the properties of the particles to be separated and the maximum processing amount of the particles.

  In addition, in the microchannel system for an Eltriator proposed in the present invention, the particle size of particles staying in the separation chamber (DC) can be changed by changing the width, depth, and length of the separation chamber (DC). The range of specific gravity can be changed. Therefore, it is desirable to adopt flow path structures with different designs according to the properties of the particles to be separated.

  Further, the theoretical value of the distribution ratio of the flow rate at the branch point BP is 50%, that is, the flow rate is designed so that the flow rates are equally divided and merge at the junction (CP). This flow rate distribution ratio can be calculated and set based on the Hagen-Poiseuille equation that expresses the relationship between flow rate, pipe diameter, and pressure loss. It is possible to set.

  In the above configuration, the separation of polystyrene particles having different particle diameters performed as an example of the separation of particles using the above-described micro flow channel system for an L triator will be described.

  First, 0.5% Tween 80 aqueous solution was used as the particle suspension solution (solution A), and polystyrene particles having diameters of 1.0 μm, 3.0 μm, and 5.0 μm were suspended.

  After filling the channel (D) with solution B (0.5% Tween 80 aqueous solution), the solution in the inlet solution chamber (IC) is replaced with the particle suspension, and then the inlet solution chamber (IC) and the outlet solution chamber. The inlet seal structure (IS) and the outlet seal structure (OC) were respectively attached to (OC), and the system was rotated at 1500 rpm using a rotating device (spin coater). As the inlet and outlet seal structures, PDMS thin layer sheets (thickness of about 0.5 mm) were used.

  Then, the rotation was stopped every 300 seconds or 900 seconds, and 10 μL of a solution having the same or different composition was dropped into the inlet chamber (IC), and the solution held in the outlet solution chamber (OC) was recovered and recovered. The solutions were named fraction 1 to fraction 7, respectively. Here, fraction 1 is the number of the solution collected after the first rotation operation, and the solution and rotation time in each rotation operation 1 to 7 are as follows. Rotation operation 1: 0.5% Tween 80 aqueous solution (specific gravity 1.00 g / mL), 300 seconds, rotation operation 2: 0.5% Tween 80 aqueous solution, 900 seconds, rotation operation 3: 0.5% Tween 80 aqueous solution, 900 seconds, Rotating operation 4: 0.5% Tween 80 aqueous solution, 300 seconds, rotating operation 5: 0.5% Tween 80 + 2.7% CsCl aqueous solution (specific gravity 1.02 g / mL), 300 seconds, rotating operation 6: 0.5% Tween 80 + 2. 7% CsCl aqueous solution, 300 seconds, rotating operation 7: 0.5% Tween 80 + 5.4% CsCl aqueous solution (specific gravity 1.04 g / mL), 900 seconds.

  When the particles were introduced into the separation chamber (DC) by rotating, the particles were not flowed into the branch flow path (DB) when using the micro flow path system for the Eltriator shown in FIG. In (CP), since the particles flowed near the center of the flow path, it was confirmed that the particles were introduced into the central portion of the separation chamber (DC). In the case of the flow path (D) that does not have the branch flow path (DB), the particles are introduced into the separation chamber (DC), but the concentration thereof is non-uniform, and particles deposited near the wall surface are also confirmed. It was.

  When the solution in the channel (D) is a 0.5% Tween 80 aqueous solution and the rotation speed is 1500 rpm, 1.0 μm particles do not stay in the separation chamber (DC), and 3.0 μm particles. Was confirmed to stay mainly in the downstream part of the separation chamber (DC), and particles of 5.0 μm mainly stayed in the upstream part of the separation chamber (DC).

  FIG. 6 is a graph showing the abundance ratio of particles separated by the above rotation operation for each fraction. It was confirmed that particles having a diameter of 1.0 μm, 3.0 μm, and 5.0 μm were efficiently separated.

(Example 2)
FIG. 7 is a diagram of the microchannel system of the present embodiment. The micro channel system and particle separation method for an L-triator shown in FIG. 7 are designed and manufactured for blood cell separation. Like the channel system shown in FIG. 5, four micro-channel systems (D) are provided. The outlet solution chamber (OC) is located on the more centrifugal side than the flow path system shown in FIG. The channel depth, width, and shape of the separation chamber (DC) are the same as the channel structure shown in FIG.

  As the particles to be separated, human blood cells (including red blood cells and white blood cells) were used. As a solution, a blood diluted solution (solution A) in which PBS 1 (phosphate buffered saline) 9 containing 0.1% BSA was mixed with blood 1 was used.

  First, the channel (D) was filled with PBS containing 0.1% BSA (solution B), and then the solution B remaining in the inlet solution chamber (IC) was replaced with the blood dilution solution (solution A). .

  When the system was rotated at 1500 rpm for 300 seconds, it was confirmed that both red blood cells and white blood cells were introduced into the separation chamber (DC), so solution B was further dropped into the inlet solution chamber (IC), and the rotation operation was performed at 1500 rpm for 900 seconds. As a result, it was confirmed that only leukocytes were selectively retained in the separation chamber (DC), and separation of erythrocytes and leukocytes was achieved. Furthermore, in the separation chamber (DC), it was also observed that small white blood cells stay on the downstream side and large white blood cells stay on the upstream side. It was also possible to discharge the accumulated white blood cells into the outlet solution chamber (OC) by increasing the specific gravity of the solution stepwise.

  In addition, in the separation of polystyrene fine particles using the microchannel system for eltriator shown in FIG. 5 and the blood cell separation using the microchannel system for eltriator shown in FIG. As a result of the experiment, there was almost no change in the position where the particles stayed in the separation chamber (DC) in the range of 500 to 5000 rpm. This is considered to be because the flow velocity in the separation chamber (DC) and the moving speed of the particles due to centrifugal force are both proportional to the number of rotations of the system, so that their effects have canceled each other. In other words, the particle separation method using the micro channel system for an L-triator according to the present invention is not easily affected by the number of rotations, so that it is considered that a highly reliable separation operation can be easily performed.

  Further, in the separation of the polystyrene fine particles using the microchannel system for eltriator shown in FIG. 5 and the blood cell separation using the microchannel system for eltriator shown in FIG. As a result, when the viscosity of the solution was in the range of 1 cP to 20 cP, there was almost no change in the position where the particles stayed in the separation chamber (DC). This is considered to be because the flow velocity in the separation chamber (DC) and the moving speed of the particles due to centrifugal force are both inversely proportional to the viscosity of the solution, so that these effects cancel each other. In other words, the particle separation method using the microchannel system for an L trilator according to the present invention is not easily affected by the viscosity of the solution, and the behavior of the particles is almost determined by the specific gravity of the solution. It is thought to be possible easily.

  Since the present invention is configured as described above, it is a complicated process for continuously feeding liquid to the rotating chamber portion by using an external pump or the like, which is necessary in the existing Eltriator system. The equipment is unnecessary, and it is thought that it can contribute to a significant reduction in the equipment price.

  In addition, since the present invention is configured as described above, the entire system can be formed of an inexpensive polymer material or the like, and a disposable apparatus can be provided. Therefore, it is considered that it is possible to provide a useful cell separation device that can be easily used in research fields such as diagnostic medicine, regenerative medicine, biochemistry and cell biology. By providing a disposable apparatus, it is not necessary to perform a washing operation of the apparatus, which is essential in a normal elutriator apparatus, and it is possible to prevent cross contamination between samples.

  Furthermore, since the present invention is configured as described above, even if the amount of the sample (the number of particles or the volume of the particle suspension) is small, the particles can be efficiently collected without losing the sample. Since it becomes possible to separate and collect, it is very efficient in separating particles such as precious cells.

Further, the present invention is configured as described above, and can automatically change the composition of the solution by a single rotation operation, so that it is possible to provide a system that is very easy to automate apparatuses and operations. It is expected to exert an excellent effect of.

Claims (17)

It is made of polymer, glass, or metal material for separating particles having an average particle size of R μm (R is 1000 or less), and is rotated about a specific rotation axis (RA) by an arbitrary rotating device. An eltriator microchannel system that can be
(1) There is a flow path (D) having an inner diameter of 1.5 Rm to 100 mm connecting the inlet (DP) and the outlet (DR),
(2) The linear distance between the inlet (DP) and the rotation axis (RA) is configured to be shorter than the linear distance between the outlet (DR) and the rotation axis (RA).
(3) When the flow path (D) is traced from the inlet (DP) to the outlet (DR), the linear distance from the rotational axis (RA) gradually decreases. Have at least one part (DS),
(4) The flow path (D) is an inlet solution chamber (IC) that can hold the liquid therein via the inlet (DP) when the micro flow channel system for an L-triator is stationary and rotating. Connected with
(5) The flow path (D) has a separation chamber (DC) in a part (DS),
(6) By rotating the entire microchannel system for an L-retriator with respect to the rotation axis (RA), the particles are introduced into the channel (D) together with the solution A (composition a), and the separation chamber (DC) ), The particles stay at different positions depending on the difference in size and specific gravity, or at least a part of the particles cannot stay, so that the particles are separated. system.   The elt according to claim 1, wherein the flow path (D) is connected via the outlet (DR) to an outlet solution chamber (OC) capable of holding a liquid therein when stationary and rotating. Micro flow system for rieta.   The L triator according to claim 1 or 2, wherein the separation chamber (DC) has its cross-sectional area S increasing gradually or stepwise at least partially as it goes downstream of the flow path (D). Microchannel system for use.   The flow path (D) has at least one branch flow path (DB) that branches at a branch point BP and rejoins at a merge point CP in a section sandwiched between the inlet (DP) and the separation chamber (DC). The microchannel system for an L-triator according to any one of claims 1 to 3.   5. The microfluidic flow for an eltriator according to claim 1, wherein the inlet solution chamber (IC) has a structure in which a plurality of inlet solution chambers capable of individually introducing solutions are connected in parallel or in series. Road system.   6. The outlet solution chamber (OC) has a structure in which a plurality of the outlet solution chambers capable of individually collecting solutions discharged from the outlet (DR) are connected in parallel or in series. The micro flow channel system for an L-triator according to Item.   The micro-channel system for an eltriator according to any one of claims 1 to 6, wherein the specific gravity of the solution introduced into the channel (D) increases stepwise or gradually.   The said flow path (D) and the said separation chamber (DC) are produced by bonding together the groove-shaped structure processed on the surface of the flat substrate, and another flat substrate. The micro flow channel system for an Eltriator according to any one of the above.   9. The microchannel system for an eltriator according to claim 1, wherein the particles to be separated are biological particles such as cells, viruses, bacteria, and organelles.   A particle separation method using the microchannel system for an L-triator according to any one of claims 1 to 9. A micro-channel system for an elutriator having a channel formed in a substrate and capable of rotating about a rotation axis,
The flow path has an inlet, an outlet, an inlet solution chamber connected to the inlet, and a separation chamber formed between the inlet and the outlet;
The distance between the inlet and the rotating shaft is shorter than the distance between the outlet and the rotating shaft, and the distance between the inlet of the separation chamber and the rotating shaft is the distance between the outlet of the separation chamber and the rotating shaft. A microchannel system for an Eltriator that is longer than the distance between the shafts.   The microchannel system for an ertlator according to claim 11, further comprising an outlet solution chamber connected to the outlet.   12. The microchannel system for an eltriator according to claim 11, wherein a cross-sectional area of the separation chamber increases gradually or stepwise from an inlet of the separation chamber toward an outlet of the separation chamber.   12. The micro flow for an Eltriator according to claim 11, wherein the flow path has a branch point where the flow path branches and a merge point where the flow path branched by the branch point joins between the inlet and the separation chamber inlet. Road system.   12. The microchannel system for an eltriator according to claim 11, comprising a plurality of inlet solution chambers connected in series or in parallel.   12. The microchannel system for an eltriator according to claim 11, comprising a plurality of outlet solution chambers connected in series or in parallel.   12. The microchannel system for an eltriator according to claim 11, wherein the microchannel system is formed by bonding a substrate on which a groove corresponding to the channel shape is formed and a substrate on a flat plate.