WO2015111293A1 - Particle sorter and method for sorting particles - Google Patents

Abstract

A particle sorter and a method for sorting particles are provided with which it is possible to stably form droplets having satisfactory shapes even when the fluid is vibrated at a high frequency. In the particle sorter, particle-containing droplets are ejected from an orifice that produces a fluid stream, and vibrations are applied by a vibrator to a pipeline through which at least a sheath fluid is introduced into a channel that communicates with the orifice.

Description

Particle sorting apparatus and particle sorting method

This technology relates to a particle sorting apparatus and a particle sorting method. More specifically, the present invention relates to a technique for separating and collecting particles based on the result of analysis by an optical method or the like.

Conventionally, an optical measurement method using flow cytometry (flow cytometer) has been used for analysis of living body related microparticles such as cells, microorganisms and liposomes. A flow cytometer is a device that irradiates light on the microparticles that flow through a flow channel formed in a flow cell, a microchip, etc., and detects and analyzes fluorescence and scattered light emitted from the individual microparticles. .

Some flow cytometers have the function of sorting and collecting only particles with specific characteristics based on the analysis results, and the device that specifically sorts cells is called the “cell sorter”. . In this cell sorter, generally, a fluid discharged from the flow path is made into droplets by applying vibration to a flow cell or a microchip by a vibrating element or the like (see Patent Documents 1 and 2 and Non-Patent Document 1).

U.S. Pat. No. 3,826,364 JP 2010-190680 A

Koji Miyazaki, 1 other, “Optimization of droplet generation in high-speed cell sorter”, Transactions of the Japan Society of Mechanical Engineers (B), Japan Society of Mechanical Engineers, December 2003, Volume 69, Number 688, p. 17-22

On the other hand, in the conventional particle sorting apparatus, when high-frequency vibration is applied to the flow cell or microchip in order to increase the droplet formation speed, the shape of the droplet is disturbed, and a well-shaped droplet is stabilized. There is a problem that it cannot be formed. Such disturbance of the shape of the droplet affects the detection accuracy and the sorting accuracy.

Therefore, the main object of the present disclosure is to provide a particle sorting apparatus and a particle sorting method that can stably form a droplet having a good shape even when a fluid is vibrated at a high frequency.

A particle sorting apparatus according to the present disclosure includes a pipe that introduces at least a sheath liquid into a flow path that communicates with an orifice that generates a fluid stream, and a vibrating body that applies vibration to the pipe. A droplet containing particles is ejected.
The channel may be formed in the microchip.
The pipe is, for example, a sheath pipe through which the sheath liquid flows or a merging pipe through which the sheath liquid flows around the sample liquid containing the particles.
The vibrating body is, for example, a piezoelectric element.
In that case, the vibration exciter may be disposed in contact with the pipe.
The vibrating body may be fixed to the pipe via an adhesive.
The vibration exciter may have a through hole, and the pipe may be inserted through the through hole.
In the particle sorting apparatus according to the present disclosure, high-frequency vibration of 1 kHz or more can be applied to the pipe by the vibrating body.
The vibrating body imparts vibration in a direction perpendicular or parallel to the flow direction of the liquid flowing through the pipe, for example.

In the particle sorting method according to the present disclosure, vibration is applied to at least a pipe that introduces sheath liquid into a flow path that communicates with an orifice that generates a fluid stream, and a droplet containing particles is discharged from the orifice. Let

According to the present disclosure, it is possible to stably form a droplet having a good shape even when a fluid is vibrated at a high frequency. Note that the effects described here are not necessarily limited, and may be any of the effects described in the present disclosure.

It is a mimetic diagram showing the composition of the particle sorting device of a 1st embodiment of this indication. FIGS. 2A and 2B are views showing a method of attaching the vibrating body 3 shown in FIG. A and B are figures which show the other attachment method of the vibrating body 3 shown in FIG. 1, A is sectional drawing, B is a top view. A and B are figures which show the other attachment method of the vibrating body 3 shown in FIG. 1, A is sectional drawing, B is a top view. It is a figure which shows the other attachment method of the vibration body 3 shown in FIG. It is sectional drawing which shows typically the structure of the particle sorting apparatus of 2nd Embodiment of this indication. It is a figure which shows the droplet formation state when sheath pressure is 30 psi, A shows the state of the droplet formed with the particle sorter of an Example, B formed with the conventional particle sorter The state of a droplet is shown. It is a figure which shows the droplet formation state when sheath pressure is 35 psi, A shows the state of the droplet formed with the particle sorter of an Example, B formed with the conventional particle sorter The state of a droplet is shown.

Hereinafter, modes for carrying out the present disclosure will be described in detail with reference to the accompanying drawings. In addition, this indication is not limited to each embodiment shown below. The description will be given in the following order.

1. First Embodiment (Example of a particle sorting apparatus using a microchip)
2. Second Embodiment (Example of a particle sorting apparatus using a flow cell)

<1. First Embodiment>
First, as a first embodiment of the present disclosure, a particle sorting apparatus that sorts particles using a microchip will be described. FIG. 1 is a schematic diagram illustrating a configuration of a particle sorting apparatus according to a first embodiment of the present disclosure.

[Overall configuration of the device]
The particle sorting apparatus 1 according to the present embodiment sorts and collects particles based on the result of analysis by an optical technique or the like, and as shown in FIG. 1, the microchip 2, the vibrating body 3, and the deflection plate 5a, 5b, recovery containers 6a to 6c, and the like.

[Particles]
The particles analyzed and sorted by the particle sorting apparatus 1 of the present embodiment include biologically related fine particles such as cells, microorganisms and ribosomes, or synthetic particles such as latex particles, gel particles and industrial particles. included.

Biologically relevant microparticles include chromosomes, ribosomes, mitochondria, organelles (cell organelles), etc. that make up various cells. The cells include plant cells, animal cells, blood cells, and the like. Furthermore, microorganisms include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. The biologically relevant microparticles may include biologically relevant polymers such as nucleic acids, proteins, and complexes thereof.

On the other hand, examples of the industrial particles include those formed of an organic polymer material, an inorganic material, or a metal material. As the organic polymer material, polystyrene, styrene / divinylbenzene, polymethyl methacrylate, or the like can be used. Moreover, as an inorganic material, glass, silica, a magnetic material, etc. can be used. As the metal material, for example, gold colloid and aluminum can be used. In addition, although the shape of these fine particles is generally spherical, it may be non-spherical, and the size and mass are not particularly limited.

[Microchip 2]
The microchip 2 includes a sample inlet 22 into which a liquid (sample liquid) containing particles to be sorted is introduced, a sheath inlet 23 into which a sheath liquid is introduced, a suction outlet 24 for eliminating clogging and bubbles, and the like. Is formed. In the microchip 2, the sample liquid is introduced into the sample inlet 22, merged with the sheath liquid introduced into the sheath inlet 23, and sent to the sample flow path, and the orifice 21 provided at the end of the sample flow path. It is discharged from.

In addition, a suction channel communicating with the suction outlet 24 is connected to the sample channel. This suction channel is used to eliminate clogging and air bubbles by creating a negative pressure in the sample channel and temporarily reversing the flow when clogging or air bubbles occur in the sample channel. A negative pressure source such as a vacuum pump is connected to 24.

The microchip 2 can be formed of glass or various plastics (PP, PC, COP, PDMS, etc.). The material of the microchip 1 is desirably a material that is transparent to the measurement light emitted from the light detection unit, has less autofluorescence, and has less optical error due to small wavelength dispersion.

The microchip 2 can be formed by wet etching or dry etching of a glass substrate, or by nanoimprinting, injection molding, or machining of a plastic substrate. The microchip 2 can be formed, for example, by sealing a substrate formed with a sample flow path or the like with a substrate of the same material or a different material.

[Exciter 3]
The vibrating body 3 applies vibration to the liquid flowing through the microchip 2, converts the fluid discharged from the orifice 21 into droplets, and generates a fluid stream (droplet flow) S. For example, it is attached to a sheath pipe 4 for introducing a sheath liquid into the sheath channel. The vibrator 3 is not particularly limited as long as it can vibrate the pipe, but a piezoelectric element is preferable from the viewpoint of controllability and size.

FIGS. 2 to 5 are diagrams schematically showing how to attach the vibrating body 3. The vibrating body 3 is not particularly limited as long as vibration can be applied to at least a pipe for introducing the sheath liquid into the flow path provided in the microchip 2. For example, when the vibrating body 3 is a piezoelectric element, as shown in FIGS. As shown in FIGS. 4 and 5, for example, by using a piezoelectric element 16 having a cylindrical through hole 16 a and inserting the pipe 14 into the through hole 16 a, the entire circumferential direction of the pipe 14 is extended. It is good also as a structure by which the piezoelectric element 16 is arrange | positioned.

On the other hand, the installation method of the vibrating body 3 is not particularly limited as long as it is installed in a state where vibration can be applied to a predetermined pipe. For example, when the vibrating body 3 is a piezoelectric element, it is disposed so as to contact the pipe 14 as shown in FIGS. In that case, the piezoelectric elements 13 and 16 may be fixed to the pipe 14 via an adhesive as shown in FIGS. 2 and 4, and the fixing members 15a to 15c are used as shown in FIGS. It can also be fixed, and may be fixed in combination.

In the conventional particle sorting apparatus, since a vibrating body is attached to the microchip and the chip guide for holding the microchip, not only the fluid but also peripheral members such as the microchip and the chip guide vibrate. As a result, resonance occurs between the members, and there is a problem in that the droplet shape varies due to the influence. This problem is particularly noticeable when exciting at high frequencies. In contrast, in the particle sorting apparatus 1 of the present embodiment, since vibration is applied to the pipe, resonance between members can be suppressed and the droplet shape can be stabilized.

[Piping]
The pipe for applying vibration by the vibrating body 3 is not limited to the sheath pipe 4, and may be any pipe that introduces at least the sheath liquid into the flow path communicating with the orifice 21. For example, in the case of an apparatus that joins the sheath liquid and the sample liquid in the pipe and introduces them into the microchip 2, the vibrator 3 may be attached to the merge pipe through which the joined liquid flows. Since the sample liquid has a smaller flow rate than the sheath liquid, when only the sample pipe for introducing the sample liquid is vibrated, the fluid discharged from the orifice 21 may not be formed into droplets.

On the other hand, it is preferable that the pipe for applying vibration by the vibrating body 3 is formed of a relatively hard material from the viewpoint of excitation efficiency. Moreover, since the piping may be sterilized and washed with ethanol, hypochlorous acid, or the like, it is preferable that the piping be formed of a material having excellent chemical resistance. Examples of such a piping material include PEEK (polyetheretherketone) resin.

[Charging part]
The charging unit (not shown) applies positive or negative charges to the droplets ejected from the orifice 21, and includes a charge electrode and a voltage source that applies a predetermined voltage to the electrode. ing. The charging electrode is disposed in contact with the sheath liquid and / or sample liquid flowing in the flow path, and applies charge to the sheath liquid and / or sample liquid. For example, the charging electrode is applied to the charged electrode inlet of the microchip 2. Inserted.

[Deflecting plates 5a, 5b]
The deflecting plates 5a and 5b change the traveling direction of each droplet in the fluid stream S by an electric force acting between the electric charges applied to the droplet and guide it to a predetermined recovery container. Yes, with the fluid stream S interposed therebetween. For the deflection plates 5a and 5b, for example, commonly used electrodes can be used. A different positive or negative voltage is applied to each of the deflecting plates 5a and 5b, and when a charged droplet passes through the electric field formed thereby, an electric force (Coulomb force) is generated, and each liquid Drops are attracted in the direction of one of the deflecting plates 5a and 5b.

[Recovery containers 6a to 6c]
The collection containers 6a to 6c collect droplets that have passed between the deflection plates 5a and 5b, and general-purpose plastic tubes or glass tubes can be used for experiments. These collection containers 6a to 6c are preferably arranged so as to be replaceable in the apparatus. Further, a drainage path for the collected droplets may be connected to the collection containers 6a to 6c that receive non-target minute particles.

In addition, the number of the collection containers arranged in the fine particle sorting apparatus 1 is not particularly limited. For example, when more than three collection containers are arranged, each droplet is guided to one of the collection containers depending on the presence / absence of the electric force between the deflecting plates 5a and 5b and the size thereof. It can be recovered.
[Photodetection section]
Furthermore, the particle sorting apparatus 1 of the present embodiment irradiates, for example, light (measurement light) to a predetermined portion of the sample flow path, and emits light (measurement target light) generated from fine particles flowing through the sample flow path. A light detection unit (not shown) for detection is provided. The light detection unit can be configured in the same manner as in conventional flow cytometry. Specifically, a laser light source, an irradiation system consisting of a condensing lens, dichroic mirror, bandpass filter, etc. that collects and irradiates laser light on fine particles, and measurement generated from fine particles by laser light irradiation And a detection system for detecting the target light.

The detection system includes, for example, a PMT (Photo Multiplier Tube), an area image sensor such as a CCD or a CMOS element. The irradiation system and the detection system may be configured by the same optical path or may be configured by separate optical paths. In addition, the measurement target light detected by the detection system of the light detection unit is light generated from the microparticles by irradiation of the measurement light, such as forward scattered light, side scattered light, Rayleigh scattering, and Mie scattering. It can be scattered light or fluorescence. These measurement target lights are converted into electrical signals, and the optical characteristics of the microparticles are detected based on the electrical signals.

[Operation]
Next, the operation of the particle sorting apparatus 1 of the present embodiment will be described. When the particles are separated by the particle sorting apparatus 1 of the present embodiment, the sample liquid containing the particles to be sorted is introduced from the sample pipe connected to the sample inlet 22 and connected to the sheath inlet 23. A sheath liquid is introduced from the sheath pipe 4.

At this time, the sheath pipe 4 is vibrated by the vibrating body 3 to impart vibration to the sheath liquid introduced into the microchip 2. The direction of applying vibration by the vibration exciter 3 may be either a vertical direction or a parallel direction with respect to the flow direction of the liquid flowing in the pipe. Is preferred.

For example, in the case where the pipe 14 is inserted into the through hole 16a of the piezoelectric element 16 shown in FIGS. 4 and 5, the piezoelectric element 16 expands and contracts in the longitudinal direction of the pipe 14, and therefore parallel to the liquid flow direction. Vibration is applied to the. On the other hand, in the case where the plurality of piezoelectric elements 13 are arranged opposite to each other with the piping 14 shown in FIGS. 2 and 3, the piezoelectric elements 13 expand and contract in the direction perpendicular to the piping 14, so Thus, vibration is applied in the vertical direction. In this case, the excitation efficiency by the piezoelectric element is higher in the radial contraction than in the longitudinal expansion and contraction.

Thereafter, for example, the detection of the optical characteristics of the particles and the detection of the flow rate (flow velocity) of the particles and the interval of the particles are performed by the light detection unit, for example. The detected optical characteristics, flow velocity, interval, and the like of the detected particles are converted into electrical signals and output to the overall control unit (not shown) of the apparatus. On the other hand, the laminar flow of the sample liquid and the sheath liquid that has passed through the light irradiation part of the sample flow path is discharged from the orifice 21 to the space outside the microchip 2. At that time, at least the sheath liquid is vibrated by the vibrating body 3, so that the fluid discharged from the orifice 21 is turned into droplets.

Then, each droplet charged in the sample channel is changed in its traveling direction by the deflecting plates 5a and 5b based on the detection result in the light detection unit, and is guided to the predetermined collection containers 6a to 6c to be collected. Is done.

Since the particle sorting apparatus of this embodiment vibrates the piping, it can form droplets without receiving the influence of resonance between the members and the shape of the microchip and its peripheral members. Thereby, even when a high frequency vibration of 1 kHz or more is applied, a droplet having a good shape can be stably formed. The particle sorting apparatus of the present embodiment is suitable when vibrating in the range of 1 kHz to 200 kHz, and particularly preferably in the range of 10 kHz to 100 kHz.

In addition, when a vibrating body is attached to the microchip, it was difficult to make the chip disposable from the viewpoint of cost, but in the particle sorting apparatus of this embodiment, the vibrating body is attached to the pipe. Therefore, a disposable chip can be realized.

<2. Second Embodiment>
Next, a particle sorting apparatus that sorts particles using a flow cell will be described as a second embodiment of the present disclosure. FIG. 6 is a schematic diagram illustrating a configuration of a particle sorting apparatus according to the second embodiment of the present disclosure. In FIG. 6, the same components as those of the particle sorting apparatus 1 of the first embodiment shown in FIG.

[Overall configuration of the device]
As shown in FIG. 6, the particle sorting device 11 of the present embodiment sorts and collects particles based on the result of analysis by an optical method or the like, and uses a flow cell 12 instead of a microchip. Other than the above, the particle sorting apparatus of the first embodiment is the same as that described above. In this particle sorting apparatus 11 as well, a vibrating body 3 for applying vibration is attached to at least a pipe for introducing a sheath liquid into a flow path communicating with an orifice 31 provided in the flow cell 12.

[Flow cell 12]
The flow cell 12 includes a sample inlet 32 into which a liquid (sample liquid) containing particles to be sorted is introduced, a sheath inlet 33 into which a sheath liquid is introduced, and the like. In the flow cell 12, the sample liquid is introduced into the sample inlet 32, merged with the sheath liquid introduced into the sheath inlet 33, and then discharged from the orifice 31.
[Operation]
When the particles are sorted by the particle sorting device 11 of the present embodiment, a sample liquid containing the particles to be sorted is introduced from the sample pipe 7 connected to the sample inlet 32 and connected to the sheath inlet 33. The sheath liquid is introduced from the sheath pipe 4. At this time, the sheath pipe 4 is vibrated by the vibrating body 3 to impart vibration to the sheath liquid introduced into the flow cell 12.

Thereafter, for example, the detection of the optical characteristics of the particles and the detection of the flow rate (flow velocity) of the particles and the interval of the particles are performed by the light detection unit, for example. The detected optical characteristics, flow velocity, interval, and the like of the detected particles are converted into electrical signals and output to the overall control unit (not shown) of the apparatus. On the other hand, the laminar flow of the sample liquid and the sheath liquid that has passed through the light irradiation part of the sample flow path is discharged from the orifice 31 to the space outside the flow cell 12. At that time, at least the sheath liquid is vibrated by the vibrating body 3, so that the fluid discharged from the orifice 31 is turned into droplets.

Each droplet charged in the sample channel is changed in its traveling direction by the deflecting plates 5a and 5b based on the detection result in the light detection unit, and is guided to the predetermined collection containers 6a to 6c and collected. .

In the conventional particle sorting apparatus, since the vibrating body is attached to the flow cell, not only the fluid but also the flow cell and its peripheral members vibrate. For this reason, resonance occurs between the members, and the droplet shape is likely to vary due to the influence. However, in the particle sorting apparatus 1 of the present embodiment, the vibration is applied to the pipe, so the resonance between the members. Can be suppressed, and the droplet shape can be stabilized. The configuration and effects other than those described above in the microparticle sorting apparatus of the present embodiment are the same as those of the first embodiment described above.

However, the flow cell is more expensive than the microchip, and if the nozzle is clogged, the flow cell needs to be replaced greatly. In addition, it is extremely difficult to completely sterilize an apparatus using a flow cell because it requires long-time washing in order to eliminate contamination with particles used in the previous measurement. For this reason, from the viewpoint of operability and cost, it is preferable to use a microchip rather than a flow cell, and it is preferable to use an apparatus using a microchip, particularly when it is necessary to perform measurement under aseptic conditions.

In addition, the present disclosure can take the following configurations.
(1)
A pipe for introducing at least sheath fluid into a flow path communicating with an orifice for generating a fluid stream;
A vibrating body for applying vibration to the pipe,
A particle sorting device for discharging droplets containing particles from the orifice.
(2)
The particle sorting apparatus according to (1), wherein the flow path is formed in a microchip.
(3)
The particle sorting device according to (1) or (2), wherein the pipe is a sheath pipe through which a sheath liquid flows or a joining pipe through which a sheath liquid flows around a sample liquid containing the particles.
(4)
The particle sorting apparatus according to any one of (1) to (3), wherein the vibrating body is a piezoelectric element.
(5)
The particle sorting apparatus according to any one of (1) to (4), wherein the vibrating body is disposed in contact with the pipe.
(6)
The particle sorting apparatus according to any one of (1) to (5), wherein the vibrating body is fixed to the pipe via an adhesive.
(7)
The particle sorting apparatus according to any one of (1) to (6), wherein the vibrating body has a through hole, and the pipe is inserted through the through hole.
(8)
The particle sorting device according to any one of (1) to (7), wherein the vibration exciter imparts high-frequency vibration of 1 kHz or more to the pipe.
(9)
The particle sorting apparatus according to any one of (1) to (8), wherein the vibration exciter imparts vibration in a direction perpendicular to a flow direction of the liquid flowing through the pipe.
(10)
The particle sorting device according to any one of (1) to (8), wherein the vibration exciter imparts vibration in a direction parallel to a flow direction of the liquid flowing through the pipe.
(11)
A particle sorting method in which vibration is applied to at least a pipe for introducing sheath liquid into a flow path communicating with an orifice that generates a fluid stream by a vibrating body, and droplets containing particles are discharged from the orifice.

It should be noted that the effects described in the present specification are merely examples and are not limited, and other effects may be obtained.

Hereinafter, the effects of the present disclosure will be described in detail with reference to examples and comparative examples. In the present embodiment, the shape of the droplets to be formed, etc., using the particle sorting device shown in FIG. 2 and the conventional particle sorting device attached to the chip guide on the piezoelectric element and vibrating the microchip, etc. Compared.

At that time, two piezoelectric elements (20 m type manufactured by NEC TOKIN Corporation) were used as the vibrator, and a microchip having an orifice diameter of 85 μm was used. The driving voltage of the piezoelectric element was 3.6 V, the driving frequency was about 40 kHz, and the frequency at which the droplet generated from the tip of the chip (Break off length: BOL) was the shortest was selected. The measurement was performed under two conditions: sheath pressure 30 psi (sheath fluid flow rate: about 15 m / second) and sheath pressure 35 psi (sheath fluid flow rate: about 17 m / second).

7A and 7B are results when the sheath pressure is 30 psi, and FIGS. 8A and 8B are results when the sheath pressure is 35 psi. FIGS. 7A and 8A are views showing the state of droplets formed by the particle sorting apparatus of the embodiment, and FIGS. 7B and 8B are the states of droplets formed by the conventional particle sorting apparatus. FIG. Although the droplets D formed by the conventional particle sorting apparatus shown in FIGS. 7B and 8B vary in shape, the particle sorting apparatus of the embodiment in which the sheath pipe shown in FIGS. 7A and 8A is vibrated. The droplet D formed by the above was excellent in shape stability.

From the above results, according to the present disclosure, it was confirmed that even when the fluid was vibrated at a high frequency, it was possible to stably form a droplet having a good shape.

DESCRIPTION OF SYMBOLS 1,11 Particle fractionation device 2 Microchip 3 Excitation body 4 Sheath piping 5a, 5b Deflection plate 6a-6c Recovery container 7 Sample piping 12 Flow cell 13, 16 Piezoelectric element 14 Piping 15a-15c Fixing member 16a Through-hole 21, 31 Orifice 22, 32 Sample inlet 23, 33 Sheath inlet 24 Suction outlet

Claims (11)

A pipe for introducing at least sheath fluid into a flow path communicating with an orifice for generating a fluid stream;
A vibrating body for applying vibration to the pipe,
A particle sorting device for discharging droplets containing particles from the orifice. The particle sorting apparatus according to claim 1, wherein the flow path is formed in a microchip. 2. The particle sorting apparatus according to claim 1, wherein the pipe is a sheath pipe through which a sheath liquid flows or a merge pipe through which a sheath liquid flows around a sample liquid containing the particles. The particle sorting apparatus according to claim 1, wherein the vibrating body is a piezoelectric element. The particle sorting device according to claim 4, wherein the vibrating body is disposed in contact with the pipe. The particle sorting device according to claim 4, wherein the vibrating body is fixed to the pipe via an adhesive. The particle sorting device according to claim 4, wherein the vibrating body has a through hole, and the pipe is inserted through the through hole. The particle sorting apparatus according to claim 1, wherein the vibration exciter imparts high-frequency vibration of 1 kHz or more to the pipe. The particle sorting device according to claim 1, wherein the vibrating body imparts vibration in a direction perpendicular to a flow direction of the liquid flowing through the pipe. The particle sorting device according to claim 1, wherein the vibrating body imparts vibration in a direction parallel to a flow direction of the liquid flowing through the pipe. A particle sorting method in which vibration is applied to at least a pipe for introducing a sheath liquid into a flow path communicating with an orifice that generates a fluid stream by a vibrating body, and droplets containing particles are discharged from the orifice.

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