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WO2009110237A1 - Procédé de manipulation de particules et appareil pour le mettre en œuvre - Google Patents

Procédé de manipulation de particules et appareil pour le mettre en œuvre Download PDF

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Publication number
WO2009110237A1
WO2009110237A1 PCT/JP2009/000987 JP2009000987W WO2009110237A1 WO 2009110237 A1 WO2009110237 A1 WO 2009110237A1 JP 2009000987 W JP2009000987 W JP 2009000987W WO 2009110237 A1 WO2009110237 A1 WO 2009110237A1
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Prior art keywords
particles
temperature gradient
solution
target particles
target
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English (en)
Japanese (ja)
Inventor
雅己 佐野
江宏仁
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University of Tokyo NUC
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University of Tokyo NUC
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4022—Concentrating samples by thermal techniques; Phase changes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0454—Moving fluids with specific forces or mechanical means specific forces radiation pressure, optical tweezers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0472—Diffusion

Definitions

  • the present invention relates to a method for manipulating target particles, and in particular, various particles of various sizes and shapes, such as metals, minerals, ceramics, inorganic compounds, organic compounds, fullerenes, carbon nanotubes, polymers, DNA, proteins,
  • the present invention relates to a method that can be used for manipulation and capture of crystals, amorphous, viruses, cells such as E. coli and erythrocytes.
  • Micromanipulation is an effective technique for manipulating small objects such as biomolecules and colloids, and many micromanipulation techniques have been put into practical use so far.
  • electrophoresis, optical tweezers, and magnetic tweezers use electromagnetic force acting on colloidal particles, and exert a local force on the target particle at a microscale or nanoscale size. Can do.
  • Patent Document 1 a technique for separating particles by irradiating moving fine particles with laser light has been developed.
  • thermophoresis phenomenon (sole effect) is known as a means for separating particles in a fluid (Non-patent Document 1).
  • Non-patent Document 2 a thermophoresis phenomenon
  • the thermophoresis phenomenon means that when a temperature gradient exists in the solution, molecules or particles having a positive migration coefficient move to the low temperature region side, and molecules or particles having a negative migration coefficient move to the high temperature region side. This refers to the phenomenon of movement.
  • thermophoresis and the sign of the particle with respect to the applied temperature gradient largely depend on the property of the particle. Therefore, it is an effective means for separating specific colloidal particles, but it is not a universally usable means for manipulating and capturing arbitrary particles.
  • thermophoresis is generally small, and it is difficult to control the direction and magnitude of the force acting on the particles.
  • the present invention has been made in view of such circumstances, and a method for operating / capturing various types of substances including substances such as particles or molecules that have been difficult to operate / capture with conventional micromanipulation.
  • the purpose is to provide.
  • the present inventors obtained new knowledge about utilizing the depletion force / entropy force acting between particles in the development of a new micromanipulation technology, and completed the present invention as a result of earnest research and experiment. It has come to be.
  • the present inventors precisely control the thermophoresis phenomenon of smaller particles (for example, macromolecules) with light, and thereby the direction and magnitude of the depletion force acting on the large particles mixed in the small particle group.
  • I can control. This means that by mixing small particles and larger particles, a desired operation can be performed on large particles regardless of the thermophoretic nature inherent to the substance.
  • the Sore coefficient can be effectively changed by an external operation, and only the relationship between the size and concentration of two or more particles regardless of the type or attribute of the particles, This means that the target particles can be manipulated.
  • the target particles to be manipulated and other particles having a volume smaller than the target particles are present in the solution, and the concentration of the other particles in the solution is higher than the concentration of the target particles. Adjusting the process to (2) causing the target particles to move to a high temperature side of the temperature gradient by generating a temperature gradient in a desired portion of the adjusted solution;
  • a method for manipulating target particles characterized by having:
  • the step (2) preferably generates a temperature gradient in which a temperature difference per unit distance between the high temperature side and the low temperature side is at least 3 ° C./mm or more at a desired portion of the solution. .
  • the step (2) includes irradiating the desired portion with laser light or LED light, It is preferable to generate the temperature gradient.
  • the temperature gradient may be generated by disposing and facing the heating electrode at the desired location.
  • the step (2) It is desirable that the particles generate a temperature gradient that generates a concentration gradient that increases from the high temperature side to the low temperature side of the temperature gradient. In another embodiment, the step (2) generates a temperature gradient such that the other particles generate a depletion force that acts on the target particle toward the high temperature side of the temperature gradient. It may be allowed.
  • the method further includes the step of (3) moving the predetermined part that generates the temperature gradient and operating the target particle to follow the movement of the predetermined part. This makes it possible to move the target particles freely.
  • the target particles may be manipulated to a predetermined behavior by generating temperature gradients at a plurality of locations.
  • the solution is adjusted so that the volume ratio of the target particles to the other particles is at least 27: 1 or more. Is preferred. More preferably, the step (1) adjusts the solution so that the volume ratio of the target particles to the other particles is at least 8: 1 or more.
  • the temperature gradient and a predetermined portion that generates the temperature gradient are controlled to capture, move, accumulate, concentrate, and rotate the target particle. Any one or a combination of operations is performed.
  • the step (1) is to prepare a plurality of target particles having different sizes in the solution, and the step (2) is performed from a small target particle toward the high temperature side of the temperature gradient.
  • the particles may be accumulated in the order of large target particles, or in the order of large particles to small particles.
  • the target particles are accumulated and crystallized toward the high temperature side of the temperature gradient.
  • the target particle is a particle having a boundary film through which the solution can pass but the other particles cannot pass
  • the step (2) includes the step
  • the boundary film may be moved so as to expand to the low temperature side of the temperature gradient by generating a temperature gradient around the particles.
  • an apparatus for manipulating target particles wherein there are target particles to be manipulated and other particles having a volume smaller than the target particles.
  • a temperature gradient generating unit for moving the target particles.
  • an apparatus capable of performing the method according to the first aspect described above is provided.
  • a kit for use in a method for manipulating a specific target particle including a container, a container held in the container, and having a volume smaller than the specific target particle. And a solution that is adjusted to a concentration that is greater than the concentration of the target particles.
  • a kit capable of performing the method according to the first aspect described above is provided.
  • a solution holding container used in a method for manipulating target particles, wherein the temperature difference per unit distance between the high temperature side and the low temperature side is at least 3 ° C./mm or more.
  • a container is provided that is configured to generate a temperature gradient at a desired location in the solution.
  • this container has a light-absorbing material in at least a part of a surface inscribed with the solution.
  • a container capable of carrying out the method according to the first aspect described above is provided.
  • the method according to the present invention can be used for more general particle manipulation / capturing by using the entropy force induced by the temperature gradient as compared with other conventionally established manipulation techniques. That is, the manipulation method according to the present invention does not adhere to particles of various sizes and shapes, for example, metals, minerals, ceramics, inorganic compounds, organic compounds, fullerenes, carbon nanotubes, polymers such as polystyrene, beads, and the like. It can be used for manipulation and capture of DNA, protein, crystal, amorphous, virus, cells such as E. coli and erythrocytes.
  • FIG. 1 In the solution 1 accommodated in the glass container 5 (liquid holding unit), target particles 2 to be operated and other particles 3 having a smaller volume than the target particles 2 are present, and Adjusting the concentration of the other particles 3 to be higher than the concentration of the target particles 2; (2) By irradiating a desired portion of the adjusted solution 1 with a laser beam L from, for example, a laser light source 4 (temperature gradient generation unit) to generate a local temperature gradient, the high temperature side of the temperature gradient Moving (coagulating, rotating, etc.) the target particles 2 to A method for manipulating the target particle 2, characterized by having: and an apparatus for carrying out the method.
  • a laser beam L for example, a laser light source 4 (temperature gradient generation unit) to generate a local temperature gradient, the high temperature side of the temperature gradient
  • Moving coagulating, rotating, etc.
  • the glass container 5 as the liquid holding unit has a metal film coating 6 deposited on the inner surface thereof, and the laser beam L is condensed in the vicinity of the metal film coating 6.
  • the temperature gradient is generated in the solution 1 through the metal film coating 6.
  • the laser light source 4 is provided with a moving operation unit 8 and is generated by moving the focal position of the laser light L in the direction of the solution 1 or the glass container 5 or in a direction perpendicular thereto. The temperature distribution related to the temperature gradient is changed, and the location where such a temperature gradient is generated can be moved.
  • FIG. 2 (a) to 2 (e) show step-by-step changes in the concentration of other particles (PEG (polyethylene glycol)) mixed in the solution, 0%, 1%, 2%, 3.5%, and 5%.
  • FIG. 3 and FIG. 4 are graphs corresponding to the fluorescence intensity distributions of target particles (fluorescent particles having a diameter of 100 nm) generated when a temperature gradient is formed by irradiating a laser beam to the central portion. is there.
  • the density of the fluorescent particles decreases from the outside (low temperature side) of the thermal gradient toward the center (high temperature side).
  • the PEG concentration is 1%, as shown in FIG. 2 (b)
  • the fluorescent particles are not yet captured, but the degree of depletion near the center is less than that in the case of 0%.
  • the solution having a PEG concentration of 2% as shown in FIG. 2C
  • the capturing of the fluorescent particles is started, and the fluorescence intensity near the center is about twice as high as that of the surroundings.
  • the PEG concentration 3.5% solution and the 5% solution as shown in FIGS.
  • the density at the center increases 10 times and 100 times, respectively, as compared with the periphery.
  • the graph of FIG. 3 shows each state of FIGS. 2A to 2E in relation to the position of the fluorescent particle and the concentration at that position.
  • the slope increases in proportion to the polymer concentration. Therefore, the Sole coefficient changes from a positive value to a negative value as shown in the inset of FIG.
  • thermophoretic coefficient or Sore coefficient
  • the Sore coefficient defined by can be measured using the following relationship.
  • the present inventors obtained the following values for PEG5000 from the slope of the straight line in FIG. 6 (see the Example column).
  • the polymer density is depleted at the center. As described above, the distribution in which the polymer density is depleted at the center behaves like a potential function and enables trapping of target particles.
  • FIGS. 7 (a) and 7 (b) The new manipulation mechanism is illustrated in FIGS. 7 (a) and 7 (b).
  • a layer called a depletion layer always exists on the surface of the particle.
  • the depletion force is generated when two depletion layers overlap, that is, when two particles are close to each other or when a particle and a wall are close to each other (FIG. 7B).
  • FIG. 7 (b) the symmetry of the spatial distribution of the polymer is broken, it is expected that even one isolated particle will generate a depletion force.
  • FIG. 7 (b) it has not been known so far that depletion power appears in such cases.
  • the difference in free energy due to the excluded volume effect of the depleted layer is obtained.
  • the difference in free energy due to the presence of the depleted layer can be estimated as follows using the number of molecules ⁇ n excluded from the volume V dep of the depleted layer.
  • ⁇ is the density of the polymer (in the current situation, the term k B n.DELTA.T is negligible small enough in comparison with the term of k B T ⁇ n). Further, the interaction between the polymers is negligible because the concentration is smaller than the critical concentration (in this case, the following formula) at which the entanglement of the polymer occurs.
  • the magnitude of the depletion force caused by the non-uniformity of the polymer density can be estimated as follows.
  • j T can be written as follows by defining the force f T that causes thermophoresis.
  • thermophoresis 1 / ⁇ is the mobility of the particle, and ⁇ represents the Stokes friction coefficient. Therefore, the force that causes thermophoresis can be expressed as:
  • c 0 is a normalization constant
  • T 0 and ⁇ 0 are the temperature and density at the reference point x 0, respectively (see the example column).
  • the force for trapping particles is proportional to the diameter of the particles. More preferably, when the water repellency at the particle interface is large, it is necessary to consider the case where the fluid boundary condition changes from no slip to slip. In that case, the following relational expression is obtained.
  • l represents the fluid slipperiness on the particle surface in an amount called a slip distance.
  • R g 5.8 nm
  • the inventors of the present application have also found that the new manipulation method described here can be realized using a polymer other than PEG.
  • a polymer other than PEG For example, it has been confirmed that water-soluble polymers such as polyvinylpyrrolidine (PVP) and chloropolystyrenesulfonic acid (NaPSS) can be realized.
  • PVP polyvinylpyrrolidine
  • NaPSS chloropolystyrenesulfonic acid
  • a promising application in the embodiment of the present invention is the capture of DNA molecules.
  • capturing DNA molecules with laser tweezers is known to be difficult unless DNA is attached to beads or the like
  • several groups have proposed new methods for manipulating DNA.
  • a method combining convection and thermophoresis see Braun D, Libchaber A (2002) Trapping of DNA by thermophoretic depletion and convection. Phys. Rev. Lett. 89: 88103.
  • a high-power laser 100 mW
  • Method of using convection Ichikawa M, Ichikawa H, Yoshikawa K, Kimura Y, (2007) Extension of a DNA molecular by with the heat.
  • the phys. Yoshikawa K (2005) En rapping polymer chain in light well under good solvent condition.J.Phys.Soc.Jpn.74: see 1958-1961), and the like.
  • particles such as DNA can be captured without inducing the flow of the solution fluid. From this, it is clear that the method described here is based on a mechanism completely different from the convection effect.
  • thermophoresis manipulation has been limited so far. This is because the thermal force is generally small.
  • the present inventors have proposed a method for controlling the direction and size of thermophoresis by a simple and clear method. It is based on the osmotic pressure applied to the depleted layer and is due to the fact that the force is amplified because the number of macromolecules is larger than the number of particles to be trapped.
  • the dilute solution has been described for clarifying the mechanism of the present invention.
  • the local concentration of PEG is changed by laser irradiation.
  • the osmotic pressure by PEG can be changed by several atmospheres.
  • the particle size dependence of the trapping force it can be applied to separation by colloid size.
  • an apparatus and method that can facilitate observation by capturing or accumulating cells and particles floating in a solution in an observation field when observing cells and other particles. It is also possible to configure an apparatus for automatically observing suspended cells in culture over time using this mechanism.
  • an apparatus capable of separating, detecting, measuring, or removing target particles by isolating, aspirating, or fixing the target particles captured, moved, accumulated, concentrated, or rotated, and Can provide a method.
  • a DNA sample as a target particle is accumulated near the probe (well, etc.) of the DNA chip, and the probability of contact with the probe is increased, so that the sample and the probe are more efficiently hybridized.
  • a device that can be (that is, fixed) to increase detection or measurement sensitivity is conceivable.
  • an apparatus that moves and accumulates proteins in a protein chip to increase the reaction efficiency between a specimen and a probe (such as an antibody) and to increase the detection sensitivity is also conceivable.
  • the band of target particles (proteins, etc.) after electrophoresis is further concentrated in the gel by the method of the present invention, and then the gel is cut off.
  • the accumulated target particles tend to be arranged in the order of size (from large to small, or from small to large) or oriented along the temperature gradient.
  • a device for measuring the size of target particles (grain, volume, or radius of inertia), sorting or separating according to the size of target particles, and measuring the distribution of the size of target particles in a solution.
  • various methods well known in the technical field of the present invention can be employed for crystallization, selection, and separation.
  • the solution holding unit in the above example, the glass container 5 is used).
  • the moving operation unit 8 is driven to induce the target particles.
  • the temperature gradient may be generated at a plurality of locations, whereby the target particles may be guided more efficiently.
  • the thickness of the container was made to be 2 ⁇ m using a particle spacer.
  • the temperature distribution in the container was performed using 2 ′, 7′Bis (2-carboxyethyl) -5 (6) carbofluorescein (BCECF) (Sigma-Aldrich, Inc. No. 14560) which is a temperature sensitive fluorescent dye ( FIG. 8).
  • BCECF carbofluorescein
  • FIG. 8 a temperature increase of about 1 K was observed every time the laser power was increased by 1 mW.
  • a thick container (20 ⁇ m) was also produced and heated directly using laser absorption of water.
  • the intensity of the image was integrated and averaged in the angular direction around the focus, and the intensity distribution was calculated as a function of radius.
  • x 0 16 ⁇ m where the density asymptotically approaches a constant value was adopted.
  • ⁇ -DNA was prepared using the cyanine dye YOYO-1 (10 nM) (Molecular Probe, No. Y-3601) in a 10 mM Tris buffer containing 5% PEG 6000 with a 1: 6 substrate to dye. E. Coli. was transferred directly from frozen agar to 10 mM Tris buffer containing 5% PEG6000. Under these conditions, the bacteria do not swim themselves. Red blood cells were provided by one of the inventors (HJ) and were measured at 25% plasma, PEG 6000, 15%.
  • HJ cyanine dye
  • thermophoresis Fluorescent PEG molecules (2%, MW5000, rhodamine-labeled, Nanocs, Inc.) were used to measure thermophoresis of PEG molecules.
  • the PEG concentration was evaluated from the fluorescence intensity as shown in FIGS.
  • the logarithm of the PEG concentration at each point decreases in proportion to the temperature increase at that point.
  • the effect of disappearance of rhodamine-labeled PEG due to heat was measured in another experiment and was confirmed to be ⁇ 1.5% / K. Therefore, a corresponding correction was performed.
  • FIG. 6 shows the corrected data, which is in good agreement with the following equation predicted from the theory of thermophoresis.
  • erythrocytes When erythrocytes were placed in a dilute solution (210 mOS, 5.3 atm) containing PEG 6000, 15% (partial pressure 140 mOS, 3.4 atm) and the vicinity of the erythrocytes was raised by 10 ° C. by laser irradiation, the erythrocytes expanded. This temperature gradient depletes PEG around the red blood cells by 40%, which corresponds to a partial pressure drop of 80 mOS (1.9 atm).
  • the erythrocyte cell membrane is a semi-permeable membrane, and the change in osmotic pressure due to PEG depletion directly affected the shape of erythrocytes.
  • Infrared laser (output: about 4 mW) is focused in a thin container containing the solution, and the temperature rise is small despite a large temperature gradient of 0.1 ° C / ⁇ m at the minimum and 1 ° C / ⁇ m at the maximum.
  • a suppressed (about 4 degrees) temperature distribution was formed (see FIG. 8 and Example column).
  • As a method for forming the temperature gradient two methods were employed, in which a glass film on one side of the container was coated with a light-absorbing film to absorb the light, and a direct light absorption of water molecules. In the configuration of this apparatus, the density distribution of the fine polystyrene particles was measured.
  • FIG. 8 Infrared laser (output: about 4 mW) is focused in a thin container containing the solution, and the temperature rise is small despite a large temperature gradient of 0.1 ° C / ⁇ m at the minimum and 1 ° C / ⁇ m at the maximum.
  • thermophoresis shows the fluorescence intensity distribution of fluorescent polystyrene particles having a diameter of 100 nm. Since polystyrene particles move from a high temperature region to a low temperature region due to the influence of thermophoresis, the fluorescence intensity at the center is weaker than the surroundings. In general, when a molecule or particle has a positive thermophoresis coefficient, it moves to a low temperature region, and when it has a negative thermophoresis coefficient, it moves to a high temperature region. Many objects such as polystyrene particles and DNA molecules are known to have positive thermophoretic coefficients near room temperature.
  • thermophoretic coefficient when a small amount of polymer is added to the solution, polystyrene particles and DNA molecules move to the high temperature side and are trapped regardless of the sign of the thermophoretic coefficient. This can be considered to mean that the sign or size of the thermophoretic coefficient has changed due to the polymer being placed in the temperature gradient.
  • the present inventors verified this phenomenon using various objects.
  • the same polystyrene particles that have escaped from the high temperature in FIG. 9 are placed in a 5% polyethylene glycol (PEG, molecular weight 7500) solution, the particles are actually captured at the focal point of the laser.
  • PEG polyethylene glycol
  • the new manipulation method does not require precise adjustment of the laser beam or special input angle setting. Operation with an obliquely incident laser beam is different from normal laser tweezers. In this manipulation, it is possible to control systematically by changing the polymer concentration, laser power, temperature gradient, and the like. Furthermore, we have confirmed that the same effect occurs with other water-soluble polymers other than PEG. In addition, it was confirmed that the same effect occurred by using polystyrene particles having a particle diameter of 20 nm and PEG having a particle diameter of 6 nm (inertia radius of 3 nm) as target particles.
  • a container according to an embodiment of the present invention is a solution holding container used in a method for manipulating target particles, and a temperature difference per unit distance between a high temperature side and a low temperature side is at least 3 ° C./mm or more. The temperature gradient is generated at a desired position of the solution.
  • a container is prepared by optimizing the shape, shape, capacity, volume, size, weight, etc. of the container.
  • this container can obtain what can produce the said temperature difference efficiently by providing a light absorptive material in at least one part of the surface inscribed in the said solution.
  • the light absorbing material include chromium and ITO (indium tin oxide) film.
  • ITO is a compound obtained by adding several percent of tin oxide (SnO 2 ) to indium oxide (In 2 O 3 ).
  • a laser beam, infrared light, or the like is condensed on the light-absorbing material to generate a temperature gradient at a desired location of the solution held in the container.
  • ITO film Infrared Phys. Technol. 36 (1995) 779-784 is known to absorb infrared rays.
  • ITO converts light energy into heat energy and is held in the container by the infrared absorption action of ITO as described above. It is possible to generate a temperature gradient in the solution. Further, by applying this ITO to a desired location of the container, it is possible to indirectly generate a temperature gradient at a desired location of the solution held in the container.
  • infrared rays it is preferable to use infrared rays as the type of light to be collected, but is not limited to infrared rays, and depending on the type of light-absorbing material applied to the container, The type can be changed as appropriate. In other words, any combination of light-absorbing material and light may be used as long as the condensed light can be converted into heat by condensing a specific type of light on the light-absorbing material. .
  • the container is configured so that the above-described temperature gradient is generated at a desired position in the solution by irradiation with a long wavelength laser beam, a change in focal depth, or the like.
  • a kit according to an embodiment of the present invention is adjusted to a concentration at which a container and other particles having a smaller volume than the specific target particle are larger than the concentration of the target particle. And a solution. That is, in order to make it possible to prepare a solution and a container applicable to the method for manipulating target particles according to the present invention by mixing target particles to be manipulated, the kit according to an embodiment of the present invention includes a container. And a solution in which other particles having a smaller volume than the target particles are adjusted to a concentration that is higher than the concentration of the target particles.
  • Examples of the other particles according to this embodiment include polymers such as polyethylene glycol (PEG), polyvinyl pyrrolidine (PVP), and chloropolystyrene sulfonic acid (NaPSS), as described later. If it can achieve a structure, it will not specifically limit.
  • PEG polyethylene glycol
  • PVP polyvinyl pyrrolidine
  • NaPSS chloropolystyrene sulfonic acid
  • the local temperature gradient is generated by condensing the laser beam.
  • the present invention is not limited to this method. The same effect can be obtained even if a gradient is generated.
  • FIG. 11 shows an example in which a temperature gradient is generated around the endothermic member by arranging the endothermic member in the particles and irradiating the endothermic member with uniform light.
  • the target particle can be manipulated and trapped by biasing the temperature gradient around the target particle.
  • FIG. 11B it is possible to rotate the target particles in a specific operation, for example, by arranging a heat absorbing member having a specific shape.
  • other particles used in the present specification refers to all substances that can exist in a solution.
  • specific examples include polymers such as polyethylene glycol (PEG), polyvinylpyrrolidine (PVP), and chloropolystyrene sulfonic acid (NaPSS).
  • PEG polyethylene glycol
  • PVP polyvinylpyrrolidine
  • NaPSS chloropolystyrene sulfonic acid
  • the shape is spherical, rod-like, porous or irregular, and the particle size is the first item (absolute value) in exp in equation (6) in comparison with the target particle. Any particle size that satisfies the condition of being larger than the item (absolute value) may be used. That is, when the particle size ratio is small, it is possible to satisfy the condition by increasing the density of “other particles”.
  • the particle size is not limited to these, but it is sufficient that the particle size is at least smaller than the target particle, preferably 1/2 or less of the target particle, more preferably 1/7 or less of the target particle. More preferably, it is 1/10 or 1/30 or less of the target particle.
  • the volume is not limited to these, but it is sufficient that the volume is at least smaller than the target particle, preferably 1/8 or less of the target particle, more preferably 1/147 or less of the target particle. More preferably, it is desirable that it is 1/1000 or 1/27000 or less of the target particle.
  • the particle diameter and volume used in the present specification can be calculated by regarding the range of the inertia radius as a spherical particle.
  • the particle size and volume of the particle are calculated based on the correlation length of the entanglement network. It is possible.
  • the shape of the “target particle” used in the present specification includes a spherical shape, a rod shape, a porous body, or an irregular shape, and the particle size is not limited to these, but preferably at least It is desirable that it is 10 nm or more. Further, the volume is not limited to these, but it is preferable that the volume is at least 1000 nm 3 or more.
  • solution used in the present specification is a liquid or gel mixture composed of two or more substances having an arbitrary solvent and solute.
  • thermophoretic used in the specification of the present application means that particles existing in a region having a temperature gradient are caused by the thermal motion of the particles or the interaction between the molecules and water molecules. It is a phenomenon that moves from the high temperature side to the low temperature side or from the low temperature side to the high temperature side. In general, when a molecule or particle has a positive thermophoretic coefficient, it moves to the low temperature side, and when it has a negative thermophoretic coefficient, it moves to the high temperature side. Many objects such as polystyrene particles and DNA molecules are known to have a positive thermophoretic coefficient near room temperature.
  • the “depletion force” used in the specification of the present application refers to a force generated when the distance between any two particles approaches so that other polymers in the solution cannot enter.
  • concentration used in the present specification means not a weight concentration but a number density.
  • a laser light source that irradiates laser light has been described as an example of a temperature gradient generating unit that generates a temperature gradient in a solution.
  • a temperature gradient may be generated by irradiating LED light. good.
  • a temperature gradient may be generated by disposing the heating electrode at a desired position of the solution and facing the heating electrode.
  • local heating may be realized by using a long wavelength laser beam or changing the focal depth of the laser beam without using a metal film coating, and a temperature gradient may be generated.
  • the concentration of other particles may be any as long as it generates a temperature gradient that causes a concentration gradient that increases from the high temperature side to the low temperature side of the temperature gradient.
  • the gradient may be about 3 ° C./mm, and various means other than the above-described examples are conceivable.
  • the temperature gradient generating unit generates a temperature gradient such that the other particles generate a depletion force that acts on the target particle toward the high temperature side of the temperature gradient. I just need it.
  • the movement operation unit that moves the predetermined part that causes the temperature gradient and operates the target particle so as to follow the movement of the predetermined part is not limited to the above example, and various kinds of units can be used. It is possible to adopt. For example, instead of moving the light source, a method of moving the glass container side may be used.
  • FIG. 1 is a schematic diagram for forming a temperature gradient at a desired location in a solution according to the present invention.
  • FIG. 2 is a diagram showing the density distribution of 100 nm diameter particles at various polymer concentrations.
  • (A)-(e) are intensity distributions of fluorescent particles in a temperature gradient in PEG 6000 molecules at concentrations of 0%, 1%, 2%, 3.5%, and 5%. The laser is focused in the center. The scale bar is 10 ⁇ m.
  • FIG. 3 is a diagram showing the density distribution of 100 nm diameter particles at various polymer concentrations. The radial density distribution of particles at each polymer concentration is shown. From bottom to top, data for 0%, 1%, 2%, 3.5%, and 5% PEG solutions, respectively.
  • FIG. 4 is a diagram showing the relationship between the temperature increase and the particle density increase at each point in FIG. The inset shows the relationship between the effective Sore coefficient and the PEG concentration.
  • FIG. 5 is a diagram showing a concentration distribution of a polymer in a temperature gradient. The spatial distribution of fluorescent PEG molecules in the vicinity of the laser spot is shown (using rhodamine-labeled PEG 5000).
  • FIG. 6 is a graph showing the relationship between the logarithm of the concentration of fluorescent PEG molecules and the local temperature rise. The solid line is a regression line and its slope gives the sore coefficient.
  • FIG. 7 shows a mechanism of manipulation by entropy force driven by thermophoresis.
  • FIG. 8 is a diagram showing local temperature distribution and thermophoresis of fluorescent polystyrene particles. The temperature distribution in a container measured with the temperature sensitive fluorescent dye BCECF is shown. The inset shown in the upper right shows that the experimental container and the upper surface of the container are coated with a chromium thin film, the laser is focused, and the temperature is increased by absorption.
  • FIG. 8 is a diagram showing local temperature distribution and thermophoresis of fluorescent polystyrene particles. The temperature distribution in a container measured with the temperature sensitive fluorescent dye BCECF is shown. The inset shown in the upper right shows that the experimental container and the upper surface of the container are coated with a chromium thin film, the laser is focused, and the temperature is increased by absorption.
  • FIG. 9 is a diagram showing the density distribution of fluorescent polystyrene particles having a diameter of 100 nm during a temperature gradient.
  • the scale bar is 10 ⁇ m.
  • FIG. 10 is a diagram showing various objects captured in the polymer solution.
  • A fluorescent polystyrene particles having a diameter of 100 nm
  • polystyrene particles having a diameter of 500 nm are present.
  • a three-dimensional colloidal crystal formed by heating a solution (d) phage DNA, (e) E. coli E. coli. coli.
  • F Red blood cells.
  • FIG. 11 is a schematic view of rotating the target particles using the present invention.
  • FIG. 12 is a diagram showing changes in the shape of red blood cells before and after laser irradiation.
  • FIG. 13 is a diagram showing changes in the shape of red blood cells before and after laser irradiation.

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Abstract

L'invention concerne un procédé de manipulation/piégeage de divers types de substances, notamment des substances telles que des particules ou des molécules, qui sont difficiles à manipuler ou à piéger par micromanipulation conventionnelle. Le procédé a pour objectif la manipulation de particules cibles, et se caractérise en ce qu'il comprend (1) une étape dans laquelle les particules cibles à manipuler et d'autres particules de volume inférieur à celui des particules cibles sont introduites dans une solution, la concentration des autres particules dans la solution étant ajustée de façon à être plus élevée que la concentration des particules cibles, et (2) une étape dans laquelle un gradient de températures est établi dans la solution dans une position désirée de façon à déplacer les particules cibles vers le côté des températures plus élevées du gradient de température.
PCT/JP2009/000987 2008-03-05 2009-03-04 Procédé de manipulation de particules et appareil pour le mettre en œuvre Ceased WO2009110237A1 (fr)

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JP2016031722A (ja) * 2014-07-30 2016-03-07 株式会社デンソー 熱泳動条件の決定方法及び決定装置
JP2017202446A (ja) * 2016-05-11 2017-11-16 公立大学法人大阪府立大学 微小物体の捕集装置および捕集キットならびに微小物体の捕集方法
CN113188959A (zh) * 2021-04-29 2021-07-30 国家纳米科学中心 微纳生物颗粒热泳检测装置及方法
CN114843002A (zh) * 2022-04-26 2022-08-02 深圳大学 一种基于光热扩散泳的光镊装置及微粒的操控方法
CN116046512A (zh) * 2022-11-30 2023-05-02 江苏省农业科学院 基于热泳富集技术对微塑料的高效富集和检测方法

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JP2016031722A (ja) * 2014-07-30 2016-03-07 株式会社デンソー 熱泳動条件の決定方法及び決定装置
JP2017202446A (ja) * 2016-05-11 2017-11-16 公立大学法人大阪府立大学 微小物体の捕集装置および捕集キットならびに微小物体の捕集方法
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CN113188959A (zh) * 2021-04-29 2021-07-30 国家纳米科学中心 微纳生物颗粒热泳检测装置及方法
CN114843002A (zh) * 2022-04-26 2022-08-02 深圳大学 一种基于光热扩散泳的光镊装置及微粒的操控方法
CN116046512A (zh) * 2022-11-30 2023-05-02 江苏省农业科学院 基于热泳富集技术对微塑料的高效富集和检测方法

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