WO2015147147A1

WO2015147147A1 - Method for high-speed fixing of biological specimen by using hydrogel and device therefor

Info

Publication number: WO2015147147A1
Application number: PCT/JP2015/059332
Authority: WO
Inventors: 田中　勲; 篠田　晃; 閔姚; 田中　良和
Original assignee: 国立大学法人北海道大学
Priority date: 2014-03-26
Filing date: 2015-03-26
Publication date: 2015-10-01
Also published as: JPWO2015147147A1

Abstract

[Problem] The purpose of the present invention is to provide a novel biological specimen fixing means that is capable of reducing, as much as possible, the amount of time that the biological specimen is exposed to the environment and whereby there is little damage to the biological specimen. [Solution] The present invention provides a method for fixing a biological specimen at high speed by using hydrogel and includes: a step (A) in which a solution (1) and a solution (2) are prepared that form a hydrogel by being mixed; and a step (B) in which droplets of the solution (1) and the solution (2) are brought into contact with the surface of the biological specimen and the biological specimen is fixed at high speed by the hydrogel. As a result of the present invention, a biological specimen can be fixed at high speed, by using a hydrogel having high biocompatibility.

Description

Method and apparatus for fast fixation of biological samples with hydrogel

The present invention relates to a method for fixing protein crystals, cells, biological tissues and other biological samples at high speed with a hydrogel, and an apparatus for performing the method.

Proteins, protein crystals, cells, biological tissues, nucleic acids and other biological samples are generally unstable against drying and high temperatures. Such a biological sample can be stably fixed while maintaining its original function, and simply fixed so as not to affect measurement and analysis at all stages from basic research to applied research. Is an important basic technology, and the need for such technology is still high.

One method for immobilizing a biological sample is to use a hydrogel that causes little physical or chemical damage to the biological sample. For example, Patent Document 1 proposes forming protein crystals in a hydrogel such as agarose. Patent Document 2 proposes a hydrogel substrate for long-term storage and proliferation of cell tissues containing gelatin and polar amino acids. In addition, sodium alginate is widely used as a hydrogel having excellent biocompatibility.

However, hydrogelation usually requires at least minutes time to have the strength required for the fixation of biological samples. Such hydrogelation of minute units can be fixed when it is required to complete fixation in as short a time as possible, for example, when cells are sterically arranged in a living state, or protein crystals are fixed without collapsing. It is disadvantageous in some cases.

Moreover, sodium alginate, which is excellent in biocompatibility, has problems such as high viscosity of the monomer but weak gel strength and high concentration of calcium ions required for gelation. In particular, since calcium ions are ions involved in cell signal transmission, fixation of cells with a hydrogel containing such calcium ions at a high concentration can have an undesirable effect.

On the other hand, it is generally possible to reduce the time required for hydrogelation simply by allowing a high concentration of radicals to exist or heating a mixture of hydrogel monomers. However, most radical and warming treatments can be detrimental to biological samples.

International Publication No. 2009/091053 Special Table 2002-520013

It is an object of the present invention to newly provide a method for immobilizing a biological sample, which can immobilize the biological sample in as short a time as possible and has little damage to the biological sample. Is.

The present inventors appropriately select and combine the monomers that form the hydrogel, contact them in the form of microdroplets, and further allow the hydrogelation accelerator to coexist when the droplets contact, The inventors found that a biological sample can be fixed at high speed with a hydrogel, and completed the following inventions.

(1) A method in which a biological sample is fast-fixed with a hydrogel, which comprises preparing a solution 1 and a solution 2 that form a hydrogel by mixing, and the solution 1 and the solution 2 Said method comprising the step B of contacting the droplet with the surface of the biological sample and fast fixing the biological sample with hydrogel.
(2) The method according to (1), wherein the step A is a step of preparing a solution 1 containing one of a combination of at least two monomers forming a hydrogel and a solution 2 containing the other.
(3) The method according to (1) or (2), wherein the solution 1 and / or the solution 2 further contains a hydrogelation accelerator.
(4) The method according to (1), wherein the step A is a step of preparing a solution 1 containing one kind of monomer that forms a hydrogel and a solution 2 containing a hydrogelation accelerator for the monomer.
(5) The step A is a step of preparing a solution 1 containing a combination of at least two monomers forming a hydrogel and a solution 2 containing a hydrogelation accelerator for the monomer. Method.
(6) Step B is a step in which droplets of solution 1 and solution 2 discharged from the liquid discharge nozzle are brought into contact with the surface of the biological sample, and the biological sample is fast-fixed with a hydrogel. ) To (5).
(7) The method according to any one of (1) to (6), wherein the biological sample is a protein crystal.
(8) The method according to any one of (1) to (6), wherein the solution 1 and / or the solution 2 is a solution containing a biological sample.
(9) The method according to any one of (2) to (8), wherein the monomer is a multi-branched polyalkylene glycol derivative.
(10) Means for holding the biological sample, at least two for discharging the droplets of the two types of solutions that form a hydrogel by mixing to the biological sample held by the holding means, respectively. An apparatus for rapidly fixing a biological sample with a hydrogel, comprising two discharge nozzles and means for automatically adjusting the position of the holding means and / or the discharge nozzle.
(11) The automatic adjustment means optically detects the position of the biological sample in the holding means, and the detection signal from the detection means is analyzed to contact the droplet on the surface of the biological sample. The apparatus according to (10), further comprising: means for determining the position of the holding means and / or the position of the discharge nozzle for causing to move; and means for moving the holding means and / or the discharge nozzle to the determined position.

According to the present invention, a biological sample can be fixed in a very short time with a hydrogel. Moreover, the three-dimensional molded article containing the biological sample fixed with hydrogel can be prepared by moving continuously, controlling the position which a droplet contacts.

1 is an overall schematic view of an apparatus for rapidly fixing a biological sample with a hydrogel. In the figure, 1 and 2 are inkjet nozzles, 3 is a rotation motor, 4 is a biological sample holder, 5 is a linear stage for moving the position of the inkjet nozzle, and 6 is a holder and a rotation motor. An XY stage for movement, 7 is a linear stage or XY stage drive motor, 8 is a CCD camera, 9 is a pump for sucking and / or discharging a solution, and 10 is a control unit. It is the schematic showing the center part of the apparatus for fixing a biological sample at high speed with hydrogel. In the figure,

reference numerals

1 and 2 denote inkjet nozzles, 3 denotes a rotary motor, 4 denotes a biological sample holder, 5 denotes a linear stage, and 6 denotes an XY stage. It is an enlarged photograph of the lysozyme crystal fixed on Mounted LithoLoops which is a holder. Panels A and B are a copy of the holder and the crystal fixed thereon, from above and from the side.

The present invention is a method for rapidly fixing a biological sample with a hydrogel, which comprises preparing a solution 1 and a solution 2 to form a hydrogel by mixing, and the solution 1 and the solution 2 described above. The method comprises the step B of contacting the biological sample with the surface of the biological sample and fast fixing the biological sample with a hydrogel.

Examples of biological samples to be fixed in the present invention include biological tissues having a size of several mm square or less, fragments or sections thereof (hereinafter referred to as biological tissues, etc.), isolated cells, fungi or bacteria, Examples thereof include intracellular organelles such as mitochondria, endoplasmic reticulum or cell nucleus, and biopolymers such as nucleic acids and proteins.

The preferred subject of the method of the present invention is a cell or protein, and a particularly preferred subject is a protein crystal. Such biological samples include those in a state suspended in a solvent such as water or a suitable buffer as a preferred example. Therefore, the present invention includes a method of fixing a biological sample suspended in such a solvent with a hydrogel.

The high speed referred to in the present invention means at least 10 seconds from the time when the droplets of the solution 1 and the droplets of the solution 2 that are formed by mixing are in contact with each other until the hydrogel is formed. Within 5 seconds, preferably within 5 seconds, more preferably within 3 seconds, and even more preferably within 1 second. In addition, the fixation includes that all of the biological sample is covered and fixed with the hydrogel and a part thereof is covered and fixed with the hydrogel. As an example in which a part is fixed, a case where a biological sample is fixed to the object as a result of formation of a hydrogel between a part of the biological sample and another object can be mentioned. .

The term “fixation” as used in the present invention is used in the broad sense of immobilizing an object. Specifically, biological samples such as cells, organelles, and protein crystals retain their biological or biochemical functions. Leaving the desired position with the hydrogel. When preparing biological specimens, in order to protect against degradation due to self-degradation or decay, stopping all biochemical reactions using paraffin or formalin, etc., is also called immobilization. However, the fixation in the present invention is not limited to such a narrow meaning.

One of the preferred embodiments of the present invention is a method for rapidly fixing a biological sample with a hydrogel, which includes a solution 1 containing one of a combination of at least two monomers forming the hydrogel and a solution 2 containing the other. And a step B in which droplets of the solution 1 and the solution 2 are brought into contact with the surface of the biological sample and the biological sample is fast-fixed with a hydrogel.

The combination of at least two types of monomers that form a hydrogel is a combination of monomers in which functional groups that form a cross-linked structure in pairs are introduced separately, and a hydrogel is formed by a cross-linking reaction between the functional groups. It is.

The solution 1 in this embodiment includes one of the above combinations, and the solution 2 includes the other of the above combinations, but each solution may further include another combination of monomers that form a hydrogel. That is, in addition to the form of one solution and one solute containing one monomer in one solution, the form of one solution and multiple solutes containing one or more solutes in one solution may also be used as long as an undesirable phenomenon does not occur in one solution. It can be used as a solution in the invention.

In this embodiment, the solution 1 containing one of the combinations of at least two monomers forming the hydrogel and the solution 2 containing the other are used, which means that only these two types of solutions are used. is not. Therefore, as long as the formation of the hydrogel is not impaired, a solution containing another combination of monomers forming the hydrogel may be additionally used. That is, the combination of the solution may be one or more, and the solution different from the solution 1 and the solution 2 containing the monomer and other substances in a combination different from the combination included in the solution 1 and the solution 2 is used as the solution. Embodiments used with 1 and 2 are also included in this aspect.

Two different types of functional groups that form a cross-linked structure in pairs are combinations of functional groups that cause a so-called click reaction, such as the thiol Michael addition reaction (Devatha et al., Chem. Mater., 2014, Vol. 26, 724-744), for example, thiol groups and maleimide groups, carboxyl groups, vinyl sulfone groups, α, β unsaturated acrylate groups, α, β unsaturated methacrylate groups, α, β unsaturated acrylamide groups or A combination with an α, β unsaturated methacrylamide group, a combination of a carboxyl group and an amino group, and the like can be mentioned.

Monomers that can form a hydrogel include poly (2-hydroxyethyl methacrylate), poly (2-hydroxyethyl acrylate), poly (2-hydroxypropyl acrylate), poly (2-hydroxypropyl methacrylate), poly (2- 2-hydroxybutyl acrylate), poly (2-hydroxybutyl methacrylate) and other hydroxyl group-containing poly (meth) acrylate derivatives with functional groups introduced, poly (methoxypolyethylene glycol acrylate), poly (methoxypolyethylene glycol methacrylate), etc. Phosphoric acid having a phospholipid-like structure such as poly (2-methacryloxyethyl phosphorylcholine), a derivative in which a functional group is introduced into poly (meth) acrylate containing polyethylene glycol chain Derivative functional group is introduced into containing polymethacrylate, and poly such derivatives which functional groups are introduced to the amino group-containing polymers such as (allylamine), a biocompatible polymer monomer is exemplified. In addition, natural polysaccharides such as chondroitin sulfate, heparin, and hyaluronic acid, or derivatives having functional groups introduced into salts or modified products thereof can also be used.

The method of introducing the above functional group into the monomer is known for each functional group. For example, in the case of a carboxyl group and an amine, it was described in Sakai et al. (Macromolecules, 2008, Vol. 41, pages 5379-5384). The method can be used.

One of the combinations of monomers that can be used in the present invention is a biocompatible polymer thiol having a thiol group introduced in hyaluronic acid, chondroitin sulfate or other biocompatible polymer described in JP-T-2010-532396. And a multi-branched polyethylene glycol derivative containing a thiol group-reactive functional group. By adjusting the properties of the solution containing each derivative and adjusting the pH value of the reactive mixture solution by selectively adding an acid-alkaline according to the teaching of JP-T-2010-532396, gelation is performed for 10 seconds or less, In particular, it can be performed in a few seconds.

Another monomer combination that can be used in the present invention is a multi-branched polyalkylene glycol derivative, preferably a multi-branched polyethylene glycol (PEG) derivative containing a thiol group and a multi-branched PEG derivative containing a thiol-reactive functional group It is a combination.

The combination of such a multi-branched PEG derivative is, for example, a combination of the SUNBRIGHT series SH series (multi-branched PEG derivative having a thiol group) and MA series (multi-branched PEG derivative having a maleimide group) commercially available from NOF Corporation. , PA series (multi-branched PEG derivatives having an amine such as PTE-150PA or HGEO-150PA) and HS series (multi-branched PEG derivatives having a succinimidylcarboxypentyl group), GS series (succinimidyl glutarate) In combination with CS series (multi-branched PEG derivatives with succinimidyl succinate groups), NP series (multi-branched PEG derivatives with p-nitrophenyl carbonate groups, eg HGEO- 200NP) and before Or the like can be mentioned a combination of a PA series.

There are no special restrictions on the number of PEG branches and the types of substituents. Therefore, structures that are not found in commercially available multi-branched PEG derivatives, such as 8-branched PEG derivatives having a maleimide group or 6-branched PEG derivatives having a thiol group, can be used for the synthesis of commercially available multi-branched PEG derivatives. According to the present invention, such a multi-branched PEG derivative can also be used.

In the present invention, it is particularly preferable to select and utilize a hydrogelation accelerator, that is, a substance capable of promoting the formation of a hydrogel, such as a polymerization initiator or a polymerization accelerator, according to the substituent of the monomer. Examples of the hydrogelation accelerator include TEMED, Tris, and disodium hydrogen phosphate when the crosslinking reaction is performed between a thiol group and a maleimide group. In the above embodiment using the solution 1 containing one of the combinations of at least two monomers and the solution 2 containing the other, the hydrogelation accelerator may be contained in one or both solutions.

Another preferred embodiment of the present invention is a method for rapidly fixing a biological sample with a hydrogel, comprising a solution 1 containing one kind of monomer that forms the hydrogel, and a hydrogelation accelerator for the monomer. The method includes the step A of preparing the solution 2 and the step B of bringing the droplets of the solution 1 and the solution 2 into contact with the surface of the biological sample and fast-fixing the biological sample with a hydrogel. .

When using a monomer that can form a hydrogel by itself, prepare a solution 1 containing such a monomer and a solution 2 containing a hydrogelation accelerator such as a polymerization initiator or a polymerization accelerator for the monomer. Alternatively, the biological sample can be fast-fixed with the hydrogel by bringing the droplets of the solution 1 and the solution 2 into contact with the surface of the biological sample. Examples of such monomers include multi-branched PEG derivatives having thiol groups, and examples of hydrogelation accelerators for these monomers include tetramethylethylenediamine (TEMED).

Yet another embodiment of the present invention is a method for rapidly fixing a biological sample with a hydrogel, which comprises a solution 1 containing a combination of at least two monomers forming a hydrogel and a hydrogelation accelerator for the monomer. The method comprising: a step A for preparing a solution 2 containing a solution; and a step B for bringing the droplets of the solution 1 and the solution 2 into contact with the surface of the biological sample and fast-fixing the biological sample with a hydrogel. It is.

When two kinds of monomers that do not form or hardly form a hydrogel simply by mixing are used, a solution 1 containing the two kinds of monomers and a polymerization initiator or polymerization promotion for hydrogel formation by these monomers It is possible to fix a biological sample at high speed with a hydrogel by preparing a solution 2 containing a hydrogelation accelerator such as an agent and bringing the droplets of solution 1 and solution 2 into contact with the surface of the biological sample. it can. Examples of such combinations of two monomers include a combination of a multi-branched PEG derivative having a thiol group and a multi-branched PEG derivative having a maleimide group, and examples of hydrogelation accelerators for these monomers include tetramethylethylenediamine ( TEMED), trishydroxymethylaminomethane (Tris), and disodium hydrogen phosphate. In addition, the solution 1 containing two kinds of monomers contains an inhibitor for hydrogelation at the same time, or the hydrogel is contained in the solution 1 before the contact with the solution 2 that is adjusted to a pH that does not hydrogel. It is preferable to add a condition that does not form.

In the present invention, the monomer is used in the form of a solution, particularly an aqueous solution. The pH of the solution is desirably adjusted with an appropriate acid, alkali, or a buffering agent so that the pH when the solutions come into contact with each other is a pH suitable for the gelation reaction of the monomer. The same applies when a hydrogelation accelerator is used. In the case of a combination of a multi-branched PEG derivative having a thiol group and a multi-branched PEG derivative having a maleimide group, the pH at which the solutions come into contact with each other is in the range of 5 to 8, preferably 6.5 to 7.5. It is preferable to adjust so that it may exist in this range. Particularly in the case of the thiol Michael addition reaction, the pH when the solutions are in contact with each other is preferably 7 or more. Furthermore, the solution may contain arbitrary components such as sodium chloride and other salts and sugars for isotonicity as long as high-speed gelation is not hindered.

The monomer concentration in the solution must be higher than the concentration at which high-speed gelation is possible in the combination used, but in any combination, the lower limit may be 5% by weight or more. When using an accelerator, the lower limit may be 3% by weight or more, or 1% by weight or more. On the other hand, the upper limit of the concentration depends on the volume of the ejected droplet, but in the present invention, it may be 30% by weight or less, preferably 20% by weight or less.

The present invention includes a step B in which the droplets of the solution 1 and the solution 2 are brought into contact with the surface of the biological sample and the biological sample is fixed at high speed with a hydrogel.

One of the preferred embodiments of the step B is to bring the droplet of the solution 1 and the droplet of the solution 2 into contact with the biological sample supported by an appropriate holder on the surface of the biological sample. In this method, all or part of a biological sample is fixed with a hydrogel.

The solution is preferably ejected from a suitable liquid ejection nozzle as a droplet having a volume (volume) ranging from several tens of picoliters (pL) to 1 μL. For example, when a portion of a protein crystal placed on a suitable holding member is fast-fixed with a hydrogel so that the protein crystal is fixed to the holding member, a liquid having a volume per drop of 50 to 200 pL. It is preferable to eject the droplet toward the protein crystal. Such a small amount of droplets can be mixed in a short time after contact, and a hydrogel can be formed in a shorter time. However, when fixing a large biological sample or in the case of a droplet containing cells, the volume per droplet discharged may be larger.

As the liquid discharge nozzle, any liquid discharge nozzle of plunger type, air pressure type, screw valve type or the like can be used as long as it can discharge the above-mentioned droplets. Microinjectors and low-capacity automatic pipetters can also be used.

A particularly preferred liquid discharge nozzle for the purpose of the present invention is an inkjet nozzle. As a nozzle for discharging a droplet (volume) in the range of several tens of picoliters (pL) to 1 μL, an inkjet nozzle having a piezo element or a heating type inkjet can be used. The use of an ink jet nozzle having elements is more preferred.

Also, the droplets of the two solutions are ejected while adjusting the direction and the distance between the ejection nozzle and the contact position so that the droplets come into contact with each other on or close to the surface of the biological sample.

In another preferred embodiment of the step B, the droplets of the solution 1 and the solution 2 and the droplet of the biological sample, for example, the solution 3 containing cells, are ejected toward a single point substantially simultaneously or successively. In this method, the biological sample is fast-fixed with a hydrogel by contacting these solutions.

In this embodiment, a molded product in which a biological sample is fixed with a hydrogel can be produced by continuously moving the solution in contact with the solution. In addition, multiple solutions containing different biological samples are prepared, and the order in which they are discharged or the position where they are brought into contact is appropriately controlled, so that different biological samples are fixed with hydrogel at controlled positions. It is also possible to produce a three-dimensional molded product.

The application of 3-D printer technology to biological samples is described in Chemistry and Biology, 2013, Volume 512, for example, Nakamura et al. (Vol. 30-34), Furukawa et al. (Vol. 36- 37 pages) and various other proposals have been made. The method of the present invention can be easily performed by utilizing such additive manufacturing type 3-D printer technology. Specifically, ABS resin, photo-curing resin, and other three-dimensional molding materials used in 3-D printer technology are replaced with a solution of the monomer according to the present invention, preferably a biocompatible monomer, to obtain a desired biological material. By controlling the discharge together with the solution containing the sample, a three-dimensional molded product in which the biological sample is fixed with a hydrogel can be produced.

In another preferred embodiment of the step B, one or both of the solution 1 and the solution 2 contains a biological sample, for example, a cell, and the droplets of the solution 1 and the solution 2 are ejected toward one point. In this method, a biological sample is fast-fixed with a hydrogel by contact.

Also in this embodiment, it is possible to produce a molded article in which a biological sample is fixed with a hydrogel by continuously moving while controlling the position where the solution is brought into contact. In addition, by preparing multiple solutions each containing different biological samples and appropriately controlling the order in which the droplets are ejected or the position where they are brought into contact with each other, the hydrogels are controlled at the positions where the different biological samples are controlled. It is also possible to produce a three-dimensional molded product fixed in (1). Such a molded product can also be easily manufactured by utilizing the 3-D printer technology as described above.

In the case of discharging droplets of a solution containing a biological sample, the solution must be a suitable solvent that can keep the biological sample stable, as long as it does not interfere with high-speed hydrogelation with monomers. Typically, it is desirable to prepare based on a highly biocompatible buffer such as PBS (phosphate buffered saline). In addition, optional components such as stabilizers and preservatives may be added as necessary.

The present invention provides a means for holding a biological sample, at least for discharging droplets of two kinds of solutions that form a hydrogel by mixing to the biological sample held by the holding means, respectively. There is provided an apparatus for rapidly fixing a biological sample with a hydrogel, comprising two discharge nozzles and means for automatically adjusting the position of the holding means and / or the discharge nozzle. In particular, the automatic adjustment means optically detects the position of the biological sample in the holding means, the detection signal from the detection means is analyzed, and the droplet is brought into contact with the surface of the biological sample. A biological sample is fast-fixed with a hydrogel, comprising means for determining the position of the holding means and / or the position of the discharge nozzle for moving and means for moving the holding means and / or the discharge nozzle to the determined position An apparatus is provided.

An example of the configuration of the apparatus is shown in FIGS. 1 and 2 as an example. FIG. 1 is a schematic view of the entire apparatus, and FIG. 2 is a schematic view of a central portion of the apparatus. The apparatus shown in FIG. 1 is an example of an apparatus intended to immobilize protein crystals.

Inkjet nozzles

1 and 2, rotation motor 3, holder 4, linear stage 5, XY stage 6, drive motor 7, includes a CCD camera 8, a pump 9 and a control unit 10.

In this embodiment, the

inkjet nozzles

1 and 2 are attached to a linear stage 5 that operates in the Z-axis direction. Further, the rotary motor 3 in which the holder 4 is coupled to the rotary shaft is attached to an XY stage 6 that operates in the X-axis and Y-axis directions. Each of the linear stage 5 and the XY stage 6 includes a drive motor 7 that moves each stage according to a command from the control unit 10.

The solution is sent to the

inkjet nozzles

1 and 2 from the two containers filled with the

solutions

1 and 2 by the pump 9, and the droplet of the solution 1 from the inkjet nozzle 1 and the droplet of the solution 2 from the inkjet nozzle 2. Each is discharged. It is desirable that the tip of the inkjet nozzle and the liquid level in the container are arranged at the same height in the vertical direction.

The CCD camera 8 is arranged so as to photograph the tip of the holder 4 connected to the rotation shaft of the rotary motor 3. When the rotary motor 3 is driven, the holder 4 is rotated, and the position of the biological sample is photographed by the CCD camera 8 from a plurality of angles. Image data captured by the CCD camera 8 is sent to the control unit 10.

The control unit 10 analyzes detailed positional information of the biological sample sent from the CCD camera 8 as image data under program control, and performs inkjet recording so that the droplets ejected on the surface of the biological sample come into contact with each other. Signals for moving the

nozzles

1 and 2 and the rotary motor 3 are transmitted to the drive motor 7 of the linear stage 5 and the XY stage 6. In addition, a signal for adjusting the direction of the sample is transmitted to the rotary motor 3, and a signal for discharging the solution is transmitted to the

inkjet nozzles

1 and 2, respectively.

From the

inkjet nozzles

1 and 2 arranged at predetermined positions, droplets of the

solutions

1 and 2 are ejected toward the biological sample, respectively, and contact with the surface of the biological sample starts formation of a hydrogel. Is done. In addition, by appropriately driving the drive motor 7 and / or the rotary motor 3 of the linear stage 5 and the XY stage 6 together with the discharge, the position where the droplet contacts on the surface of the biological sample can be finely adjusted. .

The means for holding the biological sample may be any member that can hold the biological sample by other methods such as pinching, scooping, puncturing, hanging, and placing. When the biological sample is a protein crystal, a preferable example of the means is a protein crystal holder, Protein Wave, which has a mesh structure, Mounted LithoLoops (mesh diameter 0.4Φ), and a product made by Hampton Research, which has a loop structure. Examples include Mounted Cryo Loop. The holding means may be an independent member so that the operator can work manually, or may be formed integrally with another member, for example, the rotation shaft of the rotary motor 3. .

The discharge nozzle is preferably an ink jet nozzle, and may be appropriately selected from commercially available ink jet nozzles according to the amount of liquid droplets to be discharged. Special materials and shapes are not particularly required. Preferred examples include IJHD series (IJHD-100, IJHD-300, etc.) manufactured by Microjet.

The means for automatically adjusting the position of the holding means and / or the discharge nozzle is based on the positional information of the holding means and / or the discharge nozzle, and the droplet of the solution 1 and the droplet of the solution 2 on the surface of the biological sample. Is a means for automatically adjusting the position of the holding means and / or the discharge nozzle so as to come into contact with each other. One aspect of the automatic adjustment means is a means for optically detecting the position of the biological sample in the holding means; a detection signal from the detection means is analyzed, and a droplet is brought into contact with the surface of the biological sample. Automatic adjusting means including means for determining the position of the holding means and / or the position of the discharge nozzle for causing the nozzle to move, and means for moving the holding means and / or the discharge nozzle to the determined position.

The detection means may be an optical detection device having a resolution and sensitivity sufficient to measure or identify the position of the biological sample held by the holder at least on the order of mm, preferably on the order of μm. The optical detection device is preferably capable of transmitting captured image data as a detection signal to a spatial position determination unit described later. Typically, the optical detection unit is preferably a CCD camera. However, the present invention is not limited to this, and an infrared camera or other optical detection device may be used.

The means for analyzing the detection signal from the detection means and determining the position of the holding means and / or the discharge nozzle for discharging the droplet onto the biological sample is typically a computer having a high speed processor. Control unit. The control unit, which is the determination means, includes a process for calculating a position for discharging a droplet to the biological sample of the holding means and / or the discharge nozzle based on a detection signal transmitted from the detection means, that is, image data. A program for executing a process for transmitting a command for moving the holding means and / or the discharge nozzle toward the position to the moving means described below is incorporated.

The means for moving the holding means and / or the discharge nozzle is a driving means that can move the holding means and / or the discharge nozzle in a three-dimensional space in accordance with a command from the determining means. Examples of such driving means include an electric motor or other electrically driven one, a hydraulic or hydraulic pressure driven one, a compressed air driven one or the like. In the present invention, an electrical drive means that can finely adjust the spatial position, that is, the X-axis direction, the Y-axis direction, and the Z-axis direction, is easy to assemble the device, and is easy to reduce the size of the entire device. It is preferable to use a linear stage, an automatic stage, a servo motor, or a stepping motor. Further, the object to be moved by the driving means may be either the holding means, the discharge nozzle, or both of them.

The following examples further illustrate the present invention.

<Example 1>
1) Manufacture of high-speed fixing device CCD camera (Watec) combining two inkjet nozzles (both IJHD-100 from Microjet) connected to two liquid holding containers and zoom lens TV-ZH manufactured by Chuo Seiki WAT-231S2), Rotating motor (OMRON), Protein Wave Mounted Loops that can be connected to the tip of the rotating shaft of the rotating motor, XY stage for adjusting the position of the rotating motor in the X-axis direction and Y-axis direction, 2 A high-speed fixing device including a linear stage that adjusts the position of two inkjet nozzles in the Z-axis direction and a computer unit in which a position control program for both stages is incorporated was manufactured. The position control program was self-made.

2) High-speed fixation of protein crystals In the liquid holding container 1 of the high-speed fixation apparatus of 1) above, a SUBRIGHT (registered trademark) PTE-100MA solution (10% by weight, hydrogelation), a high purity activated PEG derivative from NOF Corporation The liquid holding container 2 was filled with 150 μL each of the company's SUNBRIGHT (registered trademark) PTE-100SH solution (10 wt%).

Using Lysozyme crystals (100 × 100 × 30 μm ³ cubes) in 0.1 M sodium acetate buffer solution (pH 4.6) containing 1.25 M sodium chloride using Protein Wave's Mounted Litho Loops (mesh diameter 0.4Φ) Then, about 1 μL of the buffer solution was scooped up and connected to the rotary shaft of the rotary motor of the high-speed fixing device of 1). The high-speed fixing device is operated, and droplets of solution 1 and solution 2 (200 pL / droplet) are ejected from the inkjet nozzles toward the biological sample on the holder, and are brought into contact with the surface of the lysozyme crystal. A hydrogel was formed. This discharge was performed while continuously moving the position of the holder to fix the lysozyme crystal to the holder. The time required from discharging the droplet to fixing was about 3 seconds. FIG. 3 shows an enlarged photograph of lysozyme crystals fixed with hydrogel on Mounted LithoLoops as a holder. The transparent cube on the left side of each panel is a lysozyme crystal fixed with hydrogel together with the buffer solution.

The holder was removed from the shaft of the rotary motor, and the fixed lysozyme crystal was soaked in a 0.1 M sodium acetate buffer solution (pH 4.6) containing 1.25 M sodium chloride. It was kept stable without collapsing, and could be used for searching for inhibitors using subsequent X-ray crystal structure analysis.

The present invention can fix a biological sample in a shorter time, and is used for, for example, protein crystal analysis, drug development using protein crystals (search for inhibitors), functional analysis of cells or biological tissues, and the like. be able to. It can also be used for the production of three-dimensional molded products containing biological samples.

1 Inkjet nozzle 1
2 Inkjet nozzle 2
3 Rotating motor 4 Holder 5 Linear stage 6 XY stage 7 Drive motor 8 CCD camera 9 Pump 10 Control unit

Claims

A method of fast-fixing a biological sample with a hydrogel, comprising preparing a solution 1 and a solution 2 that form a hydrogel by mixing, and a droplet of the solution 1 and the solution 2 Said method comprising the step B of bringing the biological sample into contact with the surface of the biological sample and fast fixing the biological sample with a hydrogel.
The method according to claim 1, wherein step A is a step of preparing a solution 1 containing one of a combination of at least two monomers forming a hydrogel and a solution 2 containing the other.
The method according to claim 1 or 2, wherein the solution 1 and / or the solution 2 further contains a hydrogelation accelerator.
The method according to claim 1, wherein the step A is a step of preparing a solution 1 containing one kind of monomer forming a hydrogel and a solution 2 containing a hydrogelation accelerator for the monomer.
The method according to claim 1, wherein the step A is a step of preparing a solution 1 containing a combination of at least two monomers forming a hydrogel and a solution 2 containing a hydrogelation accelerator for the monomer.
Step B is a step in which droplets of solution 1 and solution 2 discharged from the liquid discharge nozzle are brought into contact with the surface of the biological sample and the biological sample is fast-fixed with a hydrogel. The method in any one of.
The method according to any one of claims 1 to 6, wherein the biological sample is a protein crystal.
The method according to any one of claims 1 to 6, wherein the solution 1 and / or the solution 2 is a solution containing a biological sample.
The method according to any one of claims 2 to 8, wherein the monomer is a multi-branched polyalkylene glycol derivative.
Means for holding a biological sample, at least two discharge nozzles for discharging droplets of two kinds of solutions that form a hydrogel by mixing to the biological sample held by the holding means, respectively And a device for fast fixing a biological sample with a hydrogel, comprising means for automatically adjusting the position of the holding means and / or the discharge nozzle.
A means for optically detecting the position of the biological sample in the holding means; and a means for analyzing a detection signal from the detection means to bring the droplet into contact with the surface of the biological sample. 11. The apparatus according to claim 10, comprising means for determining the position of the holding means and / or the position of the discharge nozzle, and means for moving the holding means and / or the discharge nozzle to the determined position.