JP2006314337A - Method for fixing minute object - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To fix, recover or observe a specific minute object such as a DNA molecule, a cell or a microorganism from a mixture with the other minute objects. <P>SOLUTION: The method for fixing a minute object of the present invention comprises steps of: fixing the minute object together with a gelled medium on a support material by locally gelling the medium in the circumference of the minute object to be fixed in a system comprising the medium capable of reversibly carrying out sol-gel phase transition, the support material and the minute object; adding a photocurable resin to this system; and irradiating the circumference of the gelled medium with light effective for curing the photocurable resin. Furthermore, after fixing the minute object by the method, the gelled medium containing the minute object is collected from the cured photocurable resin or the minute object is collected from the photocurable resin by making the medium in the cured photocurable resin into sol. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for fixing, collecting, or observing a specific minute object from a large number of minute objects, and more specifically, a specific minute object such as a DNA molecule, a cell, or a microorganism is mixed with other minute objects. It relates to a method for fixing, collecting or observing from inside.

  Conventionally, as a method of recovering specific micro objects such as DNA molecules, cells, and microorganisms from other micro objects, a target that has entered a petri dish using a micropipette can be removed while observing under an optical microscope. A method using a cell sorter has been used (Non-patent Document 1). However, in the method using a micropipette, in order to select and collect a minute sample with high purity under a microscope, a technique skilled in the operation is required, and enormous time and cost are required for its implementation. Although the method using the cell sorter has been shown to be effective for an object having a size of about several tens of μm, it is difficult to apply to an object such as a microorganism of about several μm, and the samples are aligned in a row. Since a sequential separation operation is performed, it is impossible to take out an arbitrarily selected target from a randomly dispersed sample at high speed.

In addition, the inventors have devised a high-speed separation system using a non-contact manipulation method in which separation is performed by utilizing laser trap, dielectrophoresis, and microcapillary flow in a microfluidic circuit (Patent Documents 1 to 3). 3). However, since microorganisms are transported directly with a laser trap, there is a risk of damaging the microorganisms. To avoid this, the micro tool is laser trapped and transported indirectly by pushing or pulling the microorganisms. Although a method was devised, there were problems such as difficulty in operation.
In order to solve such a problem, the applicants use a medium having a phase transition temperature that reversibly induces a sol-gel phase transition, and locally gelates this to form a micro object into a gel. A method for recovering a desired minute object was proposed (Japanese Patent Application 2002-131656).

JP 11-346756 A JP2001-095558 JP2001-145478 Proceedings of the Opportunities Society of Japan Vol. 67, No. 635, C, 146-153 (January 2001), Electrophoresis 2001, 22, 283-288

  In the method proposed by the applicants (Japanese Patent Application No. 2002-131656), an attempt was made to separate minute objects such as cells by fixing target cells using a thermal gel aqueous solution that undergoes a sol-gel phase transition with a temperature change. However, if a material that gels by heating is used, it will not be possible to hold minute objects when the temperature drops, and if some gelled materials (such as methylcellulose and poly-N-isopropylacrylamide) are used, the adhesion is weak. Therefore, there is a problem that it cannot be fixed firmly when a large disturbance is applied in the microchannel.

Therefore, by fixing a micro object using a photo-curing resin, and further combining it with a hot gel aqueous solution that undergoes a sol-gel phase transition with a temperature change, it is found that the micro object can be fixed and recovered easily. The present invention has been completed.
The term “fixed” as used herein means that the target minute object cannot be moved spatially, and is not necessarily limited to bonding and fixing the minute object to a support or the like. After fixing in this way, the fixed micro object can be collected or observed as appropriate.

That is, the present invention locally gels a medium around a minute object to be fixed by a system consisting of a medium, a support, and a minute object that reversibly cause a sol-gel phase transition, and Fixing to the support together with the gelled medium (stage A), adding a photocurable resin to the system (stage B), and light effective for curing the photocurable resin around the gelled medium This is a method of fixing a minute object including an irradiation stage.
Still further, in the present invention, after fixing a micro object by this method, a gelled medium containing the micro object or a medium in the cured photo-curing resin is solated from the cured photo-curing resin to be photo-cured. A method of collecting a minute object comprising a step of collecting a minute object from resin.
Still further, the present invention includes a step of removing the medium and minute objects not fixed on the support after the step A, and a step of injecting a medium containing a photocurable resin into the system as the step B. It is the above method to perform.

Further, the present invention irradiates a medium around a micro object to be fixed in a system composed of a photo-curing resin, a support, and a micro object with light effective for curing the photo-curing resin. Fixing to the support together with the medium (stage C), adding to the system a medium that reversibly induces a sol-gel phase transition (stage D), and locally gelling the medium around the micro object This is a method for fixing a minute object comprising the steps of:
Furthermore, the present invention performs a step of removing the medium and minute objects not fixed on the support after the step C, and a medium that reversibly causes a sol-gel phase transition as the step D. The method of performing the step of injecting into the above.

  Conventionally, characterization of cells such as microorganisms has been performed using population cell experiments because it has been difficult to observe active biomolecules while directly observing individual cells. In the proposed method, it is possible to randomly select a target from dispersed micro objects and fix it alive, and to perform characteristic investigation while directly observing individual cells. In addition, since information on the fixed position is stored, observation of a plurality of cells can be automated by controlling the electric stage.

Examples of the photocurable resin used in the method of the present invention include photopolymerization systems (unsaturated polyester systems, acrylates, nylon systems (photopolymerization oligomers + polymers), En addition reaction systems, cationic polymerization systems, etc.), photocrosslinking types (metal ion heavy). Chromic acid type photosensitive resin (bichromate + polymer), photodimerization type photosensitive resin (cinnamate polymer), etc.) and photodegradable type (photodegradable crosslinked type (azide compound + polymer), photodegradable insoluble type ( Diazo compounds + polymers) and the like, and prepolymers containing polyethylene glycol or polypropylene glycol (for example, ENTG-3800 (Kansai Paint)) are preferred.
Although the wavelength of light for curing such a resin depends on the resin, it is preferably 290 nm to 800 nm, more preferably 320 nm to 800 nm. Particularly, the light of 320 nm to 400 nm is hardly damaged by DNA, and in addition to visible light. It is preferable to select one that does not harden because observation with visible light is possible.
Examples of the light irradiation means include a mercury lamp, a chemical lamp (wavelength 300 to 400 nm), a He-Cd laser (325 nm), and a Nd: YAG laser triple wave (355 nm). A filter for wavelength control, a lens for condensing light, and the like may be appropriately put on these.
In this case, the medium may be only a photo-curing resin, or may be one in which this is dissolved in an appropriate solvent or the like.

Since the support is for fixing the target and fixing the target when removing the unnecessary objects, there is no particular limitation as long as it can serve such a purpose. Usually, it is a container in which a minute object and a medium are placed, but a support body having a bar shape, a plate shape, a net shape, or a lattice shape may be used separately from the container. There is no restriction | limiting in particular in the material of a support body. However, if the support is transparent, it is possible to shine light through the support or to observe the target, and furthermore, since a microscope of the type observed by the transmission illumination method can be used, preferable.
The medium causes a sol-gel phase transition depending on the temperature, and it is convenient if the medium can be heated. For example, if the support is a transparent electrode (ITO), it can be irradiated with an infrared laser beam to be locally heated, or the whole or part can be heated by energization, which is particularly preferable.
As the micro object that is the object of the method of the present invention, any object can be used as long as it is incorporated into a gelled medium, and in particular, the size is several nm to about 200 μm, preferably several nm to about 30 μm. Is suitable. The method of the present invention is particularly suitable for separating and recovering micro objects such as DNA molecules, cells or microorganisms. Such a minute object can usually be identified visually or through a microscope, such as the shape, size, color, fluorescence reaction, etc. of the sample. May be. In the present invention, such selection does not necessarily need to be made visually, and may be automated using an automatic identification device using a CCD camera or the like, a device that automatically senses a fluorescent reaction, or the like.

The medium that reversibly causes the sol-gel phase transition may be any medium that reversibly causes the sol-gel phase transition. Among them, it is easy to use one that causes such a phase transition depending on the temperature. That is, this medium preferably has a sol-gel phase transition temperature, is gelled by being heated above the phase transition temperature, and is solified by being cooled below the phase transition temperature. This phase transition temperature is preferably from room temperature to 90 ° C., more preferably from 20 to 85 ° C., and even more preferably from 30 to 60 ° C., although it depends on the properties of the micro object that is the subject of the present invention. In particular, in order to separate and recover microorganisms, the phase transition temperature is preferably 30 to 50 ° C. In this case, the medium is preferably a water-soluble cellulose derivative or a solution thereof. As the cellulose derivative, methyl cellulose is more preferable, and examples thereof include Metrows SM, SH, and SE manufactured by Shin-Etsu Chemical Co., Ltd. Furthermore, a polymer of poly N isopropyl acrylamide and a polyethylene glycol derivative or the like may be used as the medium. This polymer is commercially available, for example, as meviol gel (registered trademark, Rika Ikeda, transition temperature 22 ° C.), but the conversion temperature can be set to 30 to 32 ° C. by molecular design.
For example, methyl cellulose (Metroze SM) is usually clouded when heated to about 55 ° C. with a 2% solution, and becomes sol again by cooling. This change is reversible with respect to the temperature change, and methylcellulose is a fiber and is harmless to the living body and human body. Furthermore, gelation temperature falls by mixing NaCl, NaOH, etc. For example, adding 5% NaCl to this 2% solution reduces the gelation temperature to about 40 ° C.

In the present invention, the means for reversibly causing the phase transition between the gel and the sol may be appropriately selected depending on the property of the substance that causes the phase transition between the gel and the sol, but the phase transition between the gel and the sol is reversible depending on the temperature. It is convenient to use a substance that causes it.
Although there is no restriction | limiting in particular as the method of local heating, A microelectrode may be formed by microfabrication and resistance wire heating may be performed or it may heat locally with an infrared irradiation apparatus.
When resistance wire heating of a heating electrode or the like is performed, and the object is a microorganism or the like, it is preferable that the resistance wire is covered with an insulator so that they are not killed by energization. Examples of the insulator include silicon oxide, silicon nitride, and polyimide resin.
As this local heating method, it is particularly preferable to heat the medium to the phase transition temperature or higher by infrared irradiation and to cool the medium to the phase transition temperature or lower by stopping the irradiation. A known infrared irradiation device can be used, but an infrared laser is preferably used. The laser may be irradiated to the object as it is, or may be irradiated so as to be focused on the object using a lens or the like. Any infrared laser such as a YAG laser (Nd-YAG laser), Nd: YVO ₄ laser, CO ₂ laser, or ruby laser may be used, but it is convenient to use a YAG laser or Nd: YVO ₄ laser. It is.
Moreover, you may pour hot water from an injection | pouring port as a heating method.

Hereinafter, although embodiment etc. of this invention are shown, it does not intend limiting this invention.
First, a method for fixing a target object together with a photo-curing resin and separating, collecting or observing the target object will be described.
A microchip having a fine flow path as shown in FIG. 1 is manufactured and set on a microscope stage. Various other microchips are conceivable, but the one shown in FIG. 2 is actually easy to use.
The microchannel is manufactured by micromachining. For example, the microfluidic chip may be manufactured by molding with PDMS (polydimethylsiloxane), or may be manufactured using glass. The opening and closing of the valve may be performed by deforming a tube connected to the microfluidic chip or by making a micro small valve in the microchip and controlling it.
A liquid containing sample cells, microorganisms, etc. is injected from the sample input port. The separation unit supplements target cells to be selected based on heating and cooling of optical tweezers or thermal gel. An aqueous solution containing a photocurable resin is charged from the photocurable resin charging port. At this time, the valve of the reagent introduction port is closed.
The point after the confluence of the two microchannels is observed with an inverted microscope, and the target is selected based on the shape, size, color, fluorescence reaction, etc. of the sample. The target is fixed by locally irradiating light having a wavelength that cures around the target to cure the photocurable resin.
FIG. 3 shows an example in which a target is fixed in the photocurable resin. In this example, there is a merit that the position of the target can be accurately fixed. However, since the target is fixed comprehensively, there is a possibility that the movement of the target and the movement of the substrate when the reagent is flowed may be hindered.

Next, a method of separating, collecting or observing the target by curing the photo-curing resin around the target will be described.
Use the same equipment and procedure as above. The curing procedure is shown in FIGS.
In this example, the target is not irradiated with light having a wavelength to be cured, but is irradiated so as to surround the periphery of the target, and the target is fixed in a space formed by curing the photocurable resin in a pocket or an annular shape.
Figure 4 shows an example in which the periphery of the target is cured by half. However, the position of the target cannot be precisely fixed because it is fixed in space, but there is an advantage that the movement of the target and the movement of the substrate are difficult to hinder. is there.
The above procedure is repeated to fix the required number of targets, and then a reagent such as a culture solution or a fluorescent reagent is flowed from the reagent charging port to remove impurities at the same time. After the reagent is charged, for example, all the valves can be closed and the reaction can be observed in a closed environment, or the reagent can be observed periodically or continuously circulated.
FIG. 5 further shows an example in which the periphery of the target is completely cured in a cylindrical shape. In this example, since the target is not fixed, the target can be easily taken out from the upper part.
Furthermore, the target can be surrounded by a photo-curing resin, and a hole through which cells can enter and exit can be made. An example is shown in FIG. In FIG. 6, the target is surrounded by a cylindrical photo-curing resin, and the upper opening is opened or closed by controlling the temperature of the thermal gel. Since the thermal gel causes a sol-gel phase transition by temperature control, the opening and closing of the entrance can be controlled.

As an embodiment of the present invention, a method of separating, collecting or observing a target by fixing the target with a temperature-sensitive gel once and curing a photo-curing resin around the target is shown.
In this example, a photocurable resin and a temperature sensitive sol / gel are used in combination. An example is shown in FIG.
First, a solution in which a minute object is dispersed in a solution in which a temperature sensitive gel is dissolved is introduced into a minute channel from an introduction port. At this time, the valves of the photocurable resin charging port and the reagent charging port are closed. The target object is selected based on the shape, size, color, fluorescence reaction, etc. of the sample while observing with an inverted microscope in the region after the confluence with the photocurable resin. The selected target is irradiated with a laser having a wavelength that is absorbed by the solution and locally heated to be gelled (a).
Next, a photocurable resin is introduced into the microchannel (b). At this time, the valves of the charging port and the reagent charging port are closed. A light having a wavelength at which the photocurable resin is cured is locally irradiated to a place where the target is fixed by gelation, and is cured so as to cover the gelled part (c). The target object is fixed in the space on the dome formed by the photocurable resin cured by releasing the gelation after curing (d).
This procedure is repeated to fix the required number of targets, and then the reagent is introduced into the microchannel from the reagent introduction port and the reaction is observed.
In the example of FIG. 4 described above, the photocurable resin remains in the fixed space. In particular, when a hydrophobic photocurable resin is used, there is a possibility that droplets are generated in water and the observation is excluded. However, in this example, the space is fixed in advance with a temperature-sensitive gel, and the surrounding area is covered with a photocurable resin. Therefore, even with a hydrophobic photocurable resin, droplets are not generated in the space, and the observation is excluded. There is no merit. As in the other examples, the opening and closing of the valve may be performed by deforming a tube connected to the microfluidic chip or by making a micro-small valve in the microchip and controlling it.

It is a figure (plan view) showing an example of a microchip having a flow path. It is a figure which shows another example of a microchip. It is a figure (sectional drawing) which shows the example which fixes a target object in photocurable resin. It is a figure which shows the procedure which hardens | cures the photocurable resin around the target object (half). The upper figure is a plan view and the lower figure is a sectional view. It is a figure which shows the procedure which hardens | cures the photocurable resin around the target object (the remaining half). The upper figure is a plan view and the lower figure is a sectional view. It is a figure which shows the example which hardens | cures all the circumference | surroundings of a target object in a cylinder shape. The upper figure is a plan view and the lower figure is a sectional view. It is a figure which shows the example which used together photocurable resin and temperature sensitive sol / gel. It is all sectional drawing.

Claims (5)

In a system consisting of a medium, a support, and a micro object reversibly causing a sol-gel phase transition, the medium around the micro object to be fixed is gelled locally and the micro object is combined with the gelled medium. A step comprising fixing to the support (step A), adding a photocurable resin to the system (step B), and irradiating the periphery of the gelated medium with light effective for curing the photocurable resin. How to fix an object. After fixing the micro object by the method according to claim 1, the gelled medium containing the micro object is recovered from the cured photo-curing resin, or the medium in the cured photo-curing resin is solated. A method for collecting a micro object comprising a step of recovering a micro object from a photo-curing resin. 3. The method according to claim 1, wherein after the step A, a step of removing a medium and minute objects that are not fixed on a support is performed, and as the step B, a step of injecting a medium containing a photocurable resin into the system is performed. The method described. In a system composed of a photo-curing resin, a support, and a micro object, a medium around the micro object to be fixed is irradiated with light effective for curing the photo-curing resin, and the micro object is applied to the support together with the medium. A micro object comprising a fixing step (C step), a step of adding a medium that reversibly causes a sol-gel phase transition to the system (D step), and a step of locally gelling the medium around the micro object. Fixing method. After the step C, a step of removing the medium and fine objects not fixed on the support is performed, and as the step D, a step of injecting a medium that reversibly causes a sol-gel phase transition into the system is performed. The method of claim 4.