JPWO2014042177A1 - Liquid feeding method, centrifugal separation method, liquid feeding device and centrifugal separator - Google Patents

Abstract

Provided is a centrifugal liquid feeding method that can stabilize a laminar flow in a microchannel provided in a microdevice or the like. By laminating two or more kinds of liquids with different densities in the fine flow path arranged along the rotation direction of the centrifugal rotor, in order from the one with the smallest density, radially outside the centrifugal rotor, the density gradient in the fine flow path Is changed stepwise. In addition to forming a density gradient in the microchannel and matching the direction of density gradient and the direction of centrifugal force, the secondary flow caused by the Coriolis force is suppressed by utilizing the centrifugal force acting on the liquid. Thus, the laminar flow can be stabilized. FIG.

Description

  The present invention relates to a liquid feeding method and the like in a fine channel, and specifically relates to a centrifugal liquid feeding method and the like that can realize stabilization of a laminar flow in a fine channel provided in a microdevice or the like.

In recent years, a few centimeters or so-called micro total analysis system (μTAS) or lab chip (Lab-on-a-chip) has been used as a technology to save time and labor for sample analysis. An ultra-small biochemical analysis device (micro device) of several millimeters has appeared.
A microdevice reproduces a series of processes related to analysis, such as each operation that has been performed manually and sample transfer between operations, on a single substrate. There are merits such as less reaction time, shorter reaction time, and less waste, and it is expected to be used in a wide range of fields such as medical diagnosis, on-site analysis of the environment and food, and production of pharmaceuticals and chemicals.

Usually, a microdevice is formed with various containers such as a reactor and a reservoir, a fine channel connecting them, a valve for controlling the flow of liquid through the fine channel, etc. on a single substrate. It is possible to perform reaction control by heating, cooling, etc. and detection work applying spectroscopic or electrical action.
Examples of liquids used in microdevices include solutions containing blood, proteins, genes, etc., solutions containing solid components such as microorganisms and animal and plant cells, environmental water containing various chemical substances, soil extract water, etc. Examples include various reagents used, buffer solutions, and washing water. Hereinafter, in the present specification, various liquids used in these micro devices are collectively referred to as “liquid”.

By flowing multiple liquids through the microchannel, it is possible to realize a new chemical process and particle separation process that utilizes the phenomenon of substance / object movement at the interface, thus stabilizing the laminar flow in the microchannel. There is a need for a technique for maintaining the interface.
For example, Patent Document 1 discloses a microchip capable of concentrating a sample liquid laminar flow at the center of the flow path and feeding it by partially tapering a confluence flow path between the sample liquid introduction flow path and the sheath liquid introduction flow path. Is disclosed.

  In general, a large and expensive controller or syringe pump is used to control the flow of liquid in the fine flow path, but in addition, a fine flow path is formed on the rotating disk to generate centrifugal force. There is also known a simple liquid pumping method in which a liquid is made to flow. This method has an advantage that a liquid feeding system capable of simultaneously controlling a plurality of liquids can be easily realized because a uniform centrifugal force is obtained radially outward from the center of rotation only by rotating the disk.

  However, Coriolis force acts together with centrifugal force on the liquid flowing in the rotating fine channel. The Coriolis force is proportional to the moving speed of the liquid and the number of rotations of the rotor. The liquid flowing in the center of the fine flow path has a high moving speed, and the liquid flowing near the wall of the fine flow path has a low moving speed. There will be a difference in the Coriolis force acting on the central liquid and the liquid near the wall. As a result, vortex behavior (secondary flow) that twists the liquid in the fine flow path occurs, and the problem that the stability of the laminar flow is lost is known (Patent Documents 2 to 4, Non-Patent Documents). 1 and 2).

JP 2011-179945 A Japanese Patent No. 4836270 Japanese Patent No. 4247390 JP 2011-218348 A

Jens Ducree et al., Multilamination of flows in planar networks ofrotating microchannels, Microfluidics and Nanofluidics, 2, 78-84, (2006). Jens Ducree, et al., Patterning of flow and mixing in rotatingradial microchannels, Microfluidics and Nanofluidics, 2, 97-105, (2006).

In Patent Documents 2 to 4 (see, for example, [0006] of Patent Document 2), mention is made of the effect of a continuous density gradient suppressing the disturbance of the liquid phase in a centrifugal tube in general centrifugation. However, it is not always sufficient as a means for suppressing the secondary flow generated by the Coriolis force acting on the liquid accompanied by the flow.
In addition, all the inventions described in Patent Documents 2 to 4 perform fine particle classification (centrifugation) by the sedimentation velocity method after suppressing disturbance in the liquid phase to some extent, and fine particle classification by the sedimentation equilibrium method. At present, there is no technology that realizes this.

  In view of such problems, the present invention provides a centrifugal liquid feeding method, a centrifugal separation method, a liquid feeding device, and a centrifugal separator that can realize stabilization of a laminar flow in a microchannel provided in a microdevice or the like. For the purpose.

In the liquid feeding method of the present invention, two or more kinds of liquids having different densities are stacked on the radially outer side of the centrifugal rotor in order from the smaller density in the fine flow path arranged along the rotation direction of the centrifugal rotor. The density gradient in the fine channel is changed stepwise instead of continuously.
Further, the secondary flow caused by the Coriolis force acting toward the inside or outside in the radial direction with respect to the liquid is weakened by a centrifugal force.
Moreover, it acts on each liquid by making the gradient direction (direction from low density to high density) of the density of each of the two or more types of liquids changed stepwise coincide with the direction of action of the centrifugal force. It is characterized by stabilizing the laminar flow by weakening the secondary flow caused by the Coriolis force with a centrifugal force.
Further, the width of the fine channel is set to 10 μm or more and 1 mm or less.
Further, the density of each liquid is in the range of 0.9 g / cc to 1.2 g / cc, and the difference in density between the two kinds of laminated liquid is 0.01 g / cc or more.

In addition, the centrifugal method of the present invention is characterized in that a fine particle sample is contained in at least one of the plurality of liquids, and the fine particle sample is separated by a density gradient centrifugal method.
The fine particles may be at least one of cells, microorganisms, liposomes and other bio-related fine particles, latex particles, gel particles, industrial particles and other synthetic particles.

The liquid feeding device of the present invention includes a fine flow path arranged along the rotation direction of the centrifugal rotor, and the two or more types of liquids having different densities are placed in the fine flow path in order from the smaller density. The density gradient in the fine channel is changed not in a continuous manner but in a staircase pattern by being laminated on the outside in the radial direction.
Further, the secondary flow caused by the Coriolis force acting on the liquid inward or outward in the radial direction is weakened by centrifugal force.
Moreover, it acts on each liquid by making the gradient direction (direction from low density to high density) of the density of each of the two or more types of liquids changed stepwise coincide with the direction of action of the centrifugal force. It is characterized by stabilizing the laminar flow by weakening the secondary flow caused by the Coriolis force with a centrifugal force.
Further, the width of the fine channel is set to 10 μm or more and 1 mm or less.
Further, the density of each liquid is in the range of 0.9 g / cc to 1.2 g / cc, and the difference in density between the two kinds of laminated liquid is 0.01 g / cc or more.

A centrifugal separator according to the present invention uses the above-described liquid feeding device, and a fine particle sample is contained in at least one of the plurality of liquids, and the fine particle sample is separated by a density gradient centrifugal separation method. It is characterized by doing.
The fine particles may be at least one of cells, microorganisms, liposomes and other bio-related fine particles, latex particles, gel particles, industrial particles and other synthetic particles.

The inventors of the present application have elucidated through experiments and simulations what size, direction and position the Coriolis force is acting on the liquid flowing in the fine channel, and obtained knowledge Based on this, the present invention has been completed which can suppress the secondary flow caused by the Coriolis force and can stabilize the liquid flowing in the fine flow path.
According to the liquid feeding method of the present invention, two or more kinds of liquids having different densities are stacked on the radially outer side of the centrifugal rotor in order from the smaller density, thereby changing the density gradient in the fine flow path in a stepped manner. .
For example, when two types of liquids are used, as shown in the flow velocity distribution of FIG. 1A, the liquid flowing through the center P1 (the center in the width direction and the vertical direction) of the fine channel has the fastest moving speed, The liquid flowing in the vicinity of the wall surfaces P2 to P5 (the wall surface in the width direction and the wall surface in the vertical direction) has a relatively low moving speed. Since the Coriolis force is proportional to the flow velocity of the liquid, as shown in FIG. 1B, when the longitudinal section of the microchannel (the section along the radial direction of the centrifugal rotor) is viewed, the center P1 of the microchannel Thus, a large Coriolis force is applied outward from the wall, and a relatively small Coriolis force is applied in the vicinity of the vertical wall surfaces P4 and P5. Therefore, the radially inner liquid (liquid 1) of the centrifugal rotor enters the outer liquid (liquid 2) by a large Coriolis force in the vicinity of the point P1, and the liquid 2 has a relatively weak Coriolis force. The liquid is pushed in the direction of the liquid 1 from the vicinity of the points P4 and P5. As a result, a vortex behavior (secondary flow) that twists the flow of the liquid 1 and the liquid 2 occurs in the cross section of the flow path, and the stability of the laminar flow is lost in the fine flow path.

Therefore, as shown in FIG. 1C, the density of the liquid 2 is made relatively larger than the density of the liquid 1 as in the present invention to form a step-like density gradient in the fine channel. The centrifugal force acting on the liquid 2 is made relatively larger than the centrifugal force acting on the liquid 1. In this case, when the vertical cross section of the fine channel is viewed, a larger centrifugal force than the liquid 1 acts on the liquid 2 uniformly in the vertical direction, and the liquid 2 is outside the fine channel by the centrifugal force. Will be pressed against the wall surface W1.
As a result, the force that pushes the liquid 2 due to the Coriolis force as described above from the vicinity of the points P4 and P5 to the liquid 1 side is weakened by the centrifugal force acting on the liquid 2, and the vortex behavior ( (Secondary flow) is less likely to occur, so that the laminar flow can be stabilized.
That is, the present invention dares to form a density gradient in the fine flow path, and makes the centrifugal direction acting on the liquid by matching the direction of density gradient (direction from low density to high density) and the direction of action of centrifugal force. The secondary flow due to the Coriolis force is suppressed by using the force to stabilize the laminar flow.
In this specification, the density gradient changes in a “stepped shape” means that the density change in the direction along the radial direction of the centrifugal rotor is not spatially continuous with respect to the liquid in the fine flow path. Shall point to.

Further, as shown in FIG. 2, the direction in which the Coriolis force acts on the liquid changes depending on the rotation direction of the centrifugal rotor when the direction in which the liquid flows is constant. That is, as shown in FIG. 2A, when both the liquid flowing direction and the rotation direction of the centrifugal rotor are counterclockwise, the Coriolis force acts outward in the radial direction of the centrifugal rotor. On the other hand, as shown in FIG. 2B, when the liquid flow direction is counterclockwise and the rotation direction of the centrifugal rotor is clockwise, the Coriolis force acts inward of the centrifugal rotor in the radial direction. In the present invention, regardless of whether the Coriolis force acts inward or outward in the radial direction of the centrifugal rotor, the secondary flow caused by the Coriolis force is weakened by the centrifugal force and laminar flow is caused. Can be stabilized.
In the liquid feeding method of the present invention, the stability of the interface can be maintained even when the rotational speed and flow rate of the centrifugal rotor are increased as compared with the conventional method in which the density of a plurality of liquids flowing through the fine flow path is the same. For example, if the flow rate is about 100 times faster than the conventional method, the processing time can be greatly reduced.

The present invention can be applied to various processes that could not be used because a plurality of liquids are mixed in the fine channel by the Coriolis force.
For example, when the density of a liquid (dissolution solution) intended to dissolve the cell membrane is adjusted to the midpoint between the density of the cell and the cell nucleus, it flows along with the cell suspension so that it flows on the outer periphery of the microchannel. During the centrifugation, the cells are pressed against the lysate, and a reaction in which nuclei are removed at the interface proceeds. Nuclei removed from the cells are taken into the lysate, but cells that have not been lysed do not enter the lysate, so only the nuclei can be selected and recovered in the lysate.
In addition, when a water-soluble substance (ion or the like) in an organic solvent is extracted into an aqueous system, the diffusion distance of the substance in the fine channel is short by contacting these liquids in the fine channel. A process such as liquid-liquid extraction of a water-soluble substance from an organic solvent can be realized with high efficiency. Conversely, it is also possible to extract a hydrophobic substance from an aqueous system into an organic solvent or the like.

The width of the fine channel is preferably 10 μm or more and 1 mm or less. In general separation processing, the centrifugal rotor is often rotated at 3,000 rpm. If the width of the fine channel is designed within the above range under these conditions, the Reynolds number will be approximately 100 or less, and the viscosity and flow rate of the liquid will be comprehensive. The highest resolution can be achieved by taking into account the above.
According to the centrifugation method of the present invention, the density gradient centrifugation method that has been conventionally performed in a test tube size can be made into a microdevice, so that the separation apparatus can be configured at low cost, and a small amount of liquid (sample) can be used and the sample can be prepared. There is also an advantage that a tank for storage can be mounted on the centrifugal rotor. Furthermore, since the centrifuge operation can be automated if the continuous method is used, manual operations such as forming a density gradient in the centrifuge tube and collecting fractions concentrated at the density medium interface as in the past. There is an advantage that reproducibility and separation accuracy are improved.
The centrifugal method of the present invention can also be used as a method for producing various fine particles such as monodisperse silica fine particles, monodisperse polymer fine particles, magnetic recording media, toners, pigments, and silicon nanoparticles.

In the present specification, when fine particles are contained in the liquid, examples of the fine particles include bio-related fine particles such as cells, microorganisms, and liposomes, and synthetic particles such as latex particles, gel particles, and industrial particles.
Biologically related microparticles include chromosomes, liposomes, mitochondria, organelles (organelles) that constitute various cells. The cells include animal cells (such as blood cells) and plant cells. Microorganisms include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Furthermore, biologically relevant microparticles include biologically relevant polymers such as nucleic acids, proteins, and complexes thereof.
The industrial particles may be, for example, an organic or inorganic polymer material, a metal, or the like.
Organic polymer materials include polystyrene, styrene / divinylbenzene, polymethyl methacrylate, and the like. Inorganic polymer materials include glass, silica, magnetic materials, and the like. Metals include gold colloid, aluminum and the like. The shape of these fine particles is generally spherical, but may be non-spherical, and the size and mass are not particularly limited.

Schematic diagrams (a) to (c) showing Coriolis force and centrifugal force acting on the liquid in the fine channel. Figures (a) and (b) for explaining the direction in which the Coriolis force acts on the liquid Schematics for explaining the liquid feeding method (a) to (d) The figure which shows the state in the microchannel in Example 1 (a) and (b) The figure which shows the structure of the liquid feeding apparatus in Example 2, and a centrifuge. The figure which shows the result of having compared the pattern formed when water and Percoll are used as a liquid, changing Percoll density and flow velocity. The figure which shows the mixed state of each liquid collect | recovered by sample collection port L2 and R2. The figure which shows the result at the time of changing the rotation speed of a centrifugal rotor. The figure which shows the result of having isolate | separated the sample which suspended the microparticles | fine-particles in the blood by density gradient centrifugation. The figure which shows the result of the separation experiment of the blood and resin microparticles | fine-particles performed using the structure which branched the flow-path exit into three.

An embodiment of the liquid feeding method of the present invention will be described.
As shown in FIG. 3A, in this embodiment, two types of Percoll 10, 20 (first Percoll 10 (low density) and second Percoll 20 (high density)) and fine particles having different densities as liquids are used. Blood 30 is used as a liquid containing a sample, and plasma 30a, nucleated red blood cells (NRBC) 30b, and red blood cells 30c contained in blood 30 are separated and collected. The density of the first percoll 10 is adjusted to be larger than the floating density of the plasma 30a and smaller than the floating density of the nucleated red blood cell 30b, and the density of the second percoll 20 is larger than the floating density of the nucleated red blood cell 30b, And it is adjusted so that it may become smaller than the floating density of the red blood cells 30c.

The flow paths 12, 22, and 32 extend from the containers 11, 21, and 31 containing the first percoll 10, the second percoll 20, and the blood 30 in the radial direction of the centrifugal rotor. Are connected to the upstream end of one microchannel 40 arranged along the rotation direction. Further, the downstream end of the fine channel 40 is branched into three, and the plasma 30a, the nucleated red blood cell 30b, and the red blood cell 30c are collected at each branch portion.
As shown in FIG. 3B, in the fine channel 40, the first Percoll 10 having a lower density of the two types of Percoll is arranged closer to the rotation center of the centrifugal rotor, and the second Percoll 20 having a higher density. Are stacked on the outside to make the density gradient stepwise. Further, the blood 30 is arranged closer to the center of rotation than the first percoll 10.

When two types of Percoll 10, 20 and blood 30 are caused to flow in the fine flow path 40 while rotating the centrifugal rotor, the plasma 30a moves to the side closest to the center of rotation in the fine flow path 40 due to the centrifugal force, causing nucleation. The red blood cell 30b moves between the first percoll 10 and the second percoll 20, and the red blood cell 30c moves outside the second percoll 20.
As shown in FIGS. 3C and 3D, only the plasma 30a is recovered at the first branch portion, only the nucleated red blood cell 30b is recovered at the second branch portion, and finally the red blood cell 30c is recovered. Is done.

  Generally, there are a batch method and a continuous method for collecting fine particles. The batch method can perform only a small amount of processing, and the conditions vary slightly for each operation from the start of rotation to acceleration, constant rotation, rotation deceleration, and stop, so the collection accuracy varies from batch to batch. There is a problem such as coming out. On the other hand, in the continuous method, the sample throughput can be increased compared to the batch method, and the collection accuracy can be made uniform. The present invention can be applied to both systems, but is preferably applied to a continuous system.

  The material of the fine channel is not particularly limited, and examples of the organic material include silicone resin, acrylic resin, polystyrene, polyolefin, polyester, polycarbonate, fluororesin, elastomer such as silicone rubber and fluororubber, Examples of inorganic materials include glass, quartz, alumina, zirconia, and the like. Moreover, the process for adjusting physical properties, such as wettability, may be given to each container and the inner wall of a flow path.

Further, by adjusting the material, length, shape, etc. of each flow path 12, 22, 32 extending in the radial direction of the centrifugal rotor from each container containing the first percoll, the second percoll, and blood, a fine flow can be obtained. The flow rate of each liquid in the channel can be adjusted. For example, the flow rate can be suppressed by making the flow path meander in an S-shape so that each liquid does not flow easily in the flow path.
In this embodiment, three types of liquids (first percoll, second percoll, and blood) having different densities are allowed to flow through the fine flow path. However, four or more types of liquids may be allowed to flow. As a result, a multi-stage density gradient can be formed, and the resolution can be made more accurate.
In addition, by providing a structure (filter, column, etc.) that obstructs the flow in a part of the fine channel, unnecessary substances contained in the liquid can be removed, or a plurality of liquids can be added to the predetermined channel in the fine channel. A function of stirring at a position may be provided. Alternatively, a plurality of fine channels may be arranged along the rotation direction of the centrifugal rotor.

As a solution, the laminar flow stabilization effect of phosphate buffer (PBS, density 1.006 g / cc) and percoll was verified. Place a micro flow path with a radius of curvature of 55 mm on the concentric circle of a compact disk (centrifugal rotor), and set the compact disk from two adjacent entrances so that PBS flows on the inner circumference side and percoll flows on the outer circumference side. The solution was fed by centrifugal force by rotating. The rotation speed at the time of liquid feeding is 2000 rpm, and the flow in the microchannel at this time is approximately 40 mm / sec (1.5 ul / sec). When the density of Percoll was gradually brought closer to that of PBS from 1.075 g / cc, the influence of the secondary flow did not appear until the density of 1.02 g / cc (see FIG. 4A).
In addition, when the same experimental system is replaced with a liquid of the same density (colored water), it is known that 40% of the flow is mixed due to the influence of the secondary flow. Therefore, it can be seen that if there is a density difference of 0.014 g / cc as a density difference and a density difference of about 1.5% as a ratio, there is an effect of stabilizing the laminar flow.
In addition, when polystyrene latex particles (density 1.075 g / cc or less) are suspended in the PBS and the density of Percoll is 1.075 g / cc in the experimental system, the particles are PBS immediately after the merge of PBS and Percoll. It was confirmed that the particles were converged at the interface by centrifugal force and maintained at the interface between Percoll and PBS at a few centimeters after the merging, as opposed to being dispersed inside (Fig. 4 (b)). )reference).

As shown in FIG. 5, a liquid feeding device provided with a flow path resistance adjusting unit that can adjust the ease of liquid flow by meandering the flow path in the middle from the sample inlets L <b> 1 and R <b> 1 to the periphery.
The present invention is achieved by injecting blue-colored percoll (Percoll, high density) into the sample inlet L1 and red-colored water (low density) into the sample inlet R1, and rotating the centrifugal rotor counterclockwise. The laminar flow stabilization effect was verified.
FIG. 6 shows the result of comparing patterns formed by flowing water and Percoll at the same time while changing the Percoll density and flow rate.
Below each photograph, the density difference between Percoll and water (Percoll density-water density) is shown. The leftmost photograph shows a case where both are water, and a stripe pattern cannot be formed, but the density of Percoll increases and the stripe pattern becomes clear as the density difference increases. The upper and lower stages have the same number of rotations and different flow rates.
Comparing these, in order to clarify the fringe pattern at a higher flow rate, that is, to suppress the secondary flow and stabilize the laminar flow, it is necessary to have a large density difference between the two liquids. I understand.

FIG. 7 shows the mixed state obtained by spectroscopic analysis of the liquids collected at the sample collection ports L2 and R2. The flow path is branched into two just before the sample recovery port, and when the interface between the two liquids is clear, the unmixed dye is recovered.
As shown in FIG. 7, it can be seen that by increasing the density of the blue Percoll, the sample is less mixed and a clearer interface is formed. In addition, when there is no density difference, it is known that a clear interface cannot be formed unless the flow rate is suppressed to about 0.1 μl / s. However, by applying the stepwise density gradient of the present invention, It can be seen that the interface is maintained even under the condition of 11.8 μl / s, which is about 100 times higher.
FIG. 8 shows the results when the rotational speed of the centrifugal rotor is changed under the same flow rate condition. In this case, there is no significant dependence on the rotational speed, and it can be said that the effect is stably exhibited against variations in the rotational speed.

FIG. 9 shows the result of separating a sample in which fine particles (fluorescence) are suspended in blood (non-fluorescence) by density gradient centrifugation (precipitation equilibrium method). Since the density of blood cells is greater than that of Percoll, blood cells settle through the interface between the sample solvent and Percoll. On the other hand, as can be seen from the fluorescence image, the density of the fine particles is smaller than the density of Percoll, so that the fine particles do not pass through the interface.
FIG. 10 shows an experiment for separating blood and resin fine particles using a structure in which the outlet of the flow path is branched into three. As shown in each graph, the sample flowing in the lower stage (see Fig. 9) does not contain beads, and it is possible to obtain a sample in which only blood cells are separated, and particles can be separated by the sedimentation equilibrium method. I understand that

  A liquid feeding method by a centrifugal method that can realize stabilization of a laminar flow in a fine channel provided in a microdevice or the like, and has industrial applicability.

10 First Percoll 20 Second Percoll 30 Blood 30a Plasma 30b Nucleated Red Blood Cell 30c Red Blood Cell 11 Container 21 Container 31 Container 12 Channel 22 Channel 32 Channel 40 Channel

Claims (14)

  By laminating two or more kinds of liquids with different densities in the fine flow path arranged along the rotation direction of the centrifugal rotor, in order from the one with the smallest density, radially outside the centrifugal rotor, the density gradient in the fine flow path A liquid feeding method characterized in that the step is changed stepwise instead of continuously.   2. The liquid feeding method according to claim 1, wherein the secondary flow caused by the Coriolis force acting on the liquid inward or outward in the radial direction is weakened by centrifugal force.   By aligning the gradient direction of the density of each of the two or more liquids changed in a stepped manner (the direction from low density to high density) and the direction of action of the centrifugal force, Coriolis acting on each liquid The liquid feeding method according to claim 1, wherein the laminar flow is stabilized by weakening the secondary flow caused by force with a centrifugal force.   The liquid feeding method according to any one of claims 1 to 3, wherein a width of the fine channel is set to 10 µm or more and 1 mm or less.   The density of each liquid is in the range of 0.9 g / cc to 1.2 g / cc, and the density difference between the two kinds of laminated liquid is 0.01 g / cc or more. The liquid feeding method according to claim 1.   The fine particle sample is contained in at least one liquid among the plurality of liquids, and the fine particle sample is separated by a density gradient centrifugal separation method. Centrifugation method using liquid feeding method.   The centrifugal separation method according to claim 6, wherein the fine particles are at least one of cells, microorganisms, liposomes and other bio-related fine particles, latex particles, gel particles, industrial particles and other synthetic particles.   A fine flow path arranged along the rotation direction of the centrifugal rotor is provided, and two or more types of liquids having different densities are stacked in the fine flow path in the radial direction outside the centrifugal rotor in order from the smaller density. The liquid feeding device is characterized in that the density gradient in the fine channel is changed stepwise instead of continuously.   The liquid feeding device according to claim 8, wherein a secondary flow caused by a Coriolis force acting toward the inner side or the outer side in the radial direction with respect to each liquid is weakened by a centrifugal force.   By aligning the gradient direction of the density of each of the two or more liquids changed in a stepped manner (the direction from low density to high density) and the direction of action of the centrifugal force, Coriolis acting on each liquid The liquid feeding device according to claim 8 or 9, wherein a secondary flow caused by force is weakened by centrifugal force to stabilize a laminar flow.   The liquid feeding device according to any one of claims 8 to 10, wherein a width of the fine channel is set to 10 µm or more and 1 mm or less.   The density of each liquid is in the range of 0.9 g / cc to 1.2 g / cc, and the density difference between the two kinds of laminated liquid is 0.01 g / cc or more. The liquid feeding device according to claim 1.   The fine particle sample is contained in at least one liquid among the plurality of liquids, and the fine particle sample is separated by a density gradient centrifugal separation method. Centrifugal separator using a liquid delivery device. 14. The centrifugal separator according to claim 13, wherein the fine particles are at least one of cells, microorganisms, liposomes and other bio-related fine particles, latex particles, gel particles, industrial particles and other synthetic particles.

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