WO2015147147A1 - Procédé de fixation haute vitesse d'échantillon biologique à l'aide d'hydrogel et son dispositif - Google Patents

Procédé de fixation haute vitesse d'échantillon biologique à l'aide d'hydrogel et son dispositif Download PDF

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WO2015147147A1
WO2015147147A1 PCT/JP2015/059332 JP2015059332W WO2015147147A1 WO 2015147147 A1 WO2015147147 A1 WO 2015147147A1 JP 2015059332 W JP2015059332 W JP 2015059332W WO 2015147147 A1 WO2015147147 A1 WO 2015147147A1
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solution
biological sample
hydrogel
monomer
contact
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PCT/JP2015/059332
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English (en)
Japanese (ja)
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田中　勲
篠田　晃
閔 姚
田中　良和
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国立大学法人北海道大学
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Priority to JP2016510474A priority Critical patent/JPWO2015147147A1/ja
Publication of WO2015147147A1 publication Critical patent/WO2015147147A1/fr

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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
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N1/31—Apparatus therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027—General features of the devices
    • G01N2035/1034—Transferring microquantities of liquid
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G01N2035/1027—General features of the devices
    • G01N2035/1034—Transferring microquantities of liquid
    • G01N2035/1037—Using surface tension, e.g. pins or wires

Definitions

  • 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.
  • 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.
  • sodium alginate is widely used as a hydrogel having excellent biocompatibility.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the solution 1 and / or the solution 2 further contains a hydrogelation accelerator.
  • 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.
  • 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.
  • the biological sample is a protein crystal.
  • 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.
  • a biological sample can be fixed in a very short time with a hydrogel.
  • 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.
  • 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
  • 6 is a holder and a rotation motor.
  • 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
  • 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.
  • reference numerals 1 and 2 denote inkjet nozzles
  • 3 denotes a rotary motor
  • 4 denotes a biological sample holder
  • 5 denotes a linear stage
  • 6 denotes an XY stage.
  • 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.
  • 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.
  • 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.
  • 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.
  • a part 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. .
  • 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.
  • 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, ⁇ , ⁇ unsaturated acrylate groups, ⁇ , ⁇ unsaturated methacrylate groups, ⁇ , ⁇ unsaturated acrylamide groups or A combination with an ⁇ , ⁇ 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.
  • 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.
  • 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.
  • 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.
  • PEG polyethylene glycol
  • 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
  • HS series multi-branched PEG derivatives having a succinimidylcarboxypentyl group
  • GS series succinimidyl glutarate
  • CS series multi-branched PEG derivatives with succinimidyl succinate groups
  • NP series multi-branched PEG derivatives with p-nitrophenyl carbonate groups, eg HGEO- 200NP
  • 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.
  • the hydrogelation accelerator include TEMED, Tris, and disodium hydrogen phosphate when the crosslinking reaction is performed between a thiol group and a maleimide group.
  • 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. .
  • 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.
  • a hydrogelation accelerator such as a polymerization initiator or a polymerization accelerator for the monomer.
  • 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.
  • 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.
  • a hydrogelation accelerator such as an agent
  • 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.
  • 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.
  • 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.
  • a hydrogelation accelerator is used.
  • 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.
  • the pH when the solutions are in contact with each other is preferably 7 or more.
  • 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.
  • the lower limit may be 3% by weight or more, or 1% by weight or more.
  • 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.
  • 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 ⁇ L.
  • a suitable liquid ejection nozzle 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.
  • the volume per droplet discharged may be larger.
  • 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.
  • a nozzle for discharging a droplet (volume) in the range of several tens of picoliters (pL) to 1 ⁇ L 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.
  • 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.
  • the droplets of the solution 1 and the solution 2 and the droplet of the biological sample are ejected toward a single point substantially simultaneously or successively.
  • the biological sample is fast-fixed with a hydrogel by contacting these solutions.
  • 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.
  • 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.
  • 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.
  • 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.
  • a biological sample is fast-fixed with a hydrogel by contact.
  • 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.
  • the hydrogels are controlled at the positions where the different biological samples are controlled.
  • 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.
  • the solution 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.
  • a suitable solvent such as PBS (phosphate buffered saline).
  • PBS phosphate buffered saline
  • 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.
  • 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.
  • 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.
  • FIGS. 1 and 2 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
  • 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.
  • the inkjet nozzles 1 and 2 are attached to a linear stage 5 that operates in the Z-axis direction.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 0.4 ⁇ ), 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.
  • 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 ⁇ m.
  • the optical detection device is preferably capable of transmitting captured image data as a detection signal to a spatial position determination unit described later.
  • the optical detection unit is preferably a CCD camera.
  • 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.
  • 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.
  • 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.
  • 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.
  • the object to be moved by the driving means may be either the holding means, the discharge nozzle, or both of them.
  • 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.
  • Lysozyme crystals 100 ⁇ 100 ⁇ 30 ⁇ m 3 cubes
  • 0.1 M sodium acetate buffer solution pH 4.6
  • Protein Wave's Mounted Litho Loops mesh diameter 0.4 ⁇
  • 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.
  • 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.

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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention vise à proposer un nouveau moyen de fixation d'échantillon biologique qui est capable de réduire, autant que possible, la quantité de temps pendant laquelle l'échantillon biologique est exposé à l'environnement et ce par quoi il y a un faible endommagement à l'échantillon biologique. À cet effet, la présente invention porte sur un procédé de fixation d'échantillon biologique à haute vitesse à l'aide d'hydrogel et comprend : une étape (A) dans laquelle une solution (1) et une solution (2) sont préparées, lesquelles forment un hydrogel en étant mélangées ; et une étape (B) dans laquelle des gouttelettes de la solution (1) et de la solution (2) sont mises en contact avec la surface de l'échantillon biologique et l'échantillon biologique est fixé à haute vitesse par l'hydrogel. Par conséquence de la présente invention, un échantillon biologique peut être fixé à haute vitesse, à l'aide d'un hydrogel ayant une biocompatibilité élevée.
PCT/JP2015/059332 2014-03-26 2015-03-26 Procédé de fixation haute vitesse d'échantillon biologique à l'aide d'hydrogel et son dispositif WO2015147147A1 (fr)

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