JP2019215202A - Surface observation method and surface observation device of sample in liquid - Google Patents

Abstract

To provide a method and device allowing efficient surface observation of a sample immersed in a liquid.SOLUTION: A surface observation method of a sample in a liquid is provided which includes: a process of changing the height of the liquid level of a preservation liquid in an immersion container; and a process of performing the surface observation of the sample that is stored in the immersion container and is immersed in the preservation liquid in the immersion container. A device used for this method is also provided.SELECTED DRAWING: None

Description

  The present invention relates to a method and an apparatus for observing a surface of a sample in a liquid.

  A capillary array sheet is a type of biochip (DNA chip, etc.) in which probes capable of binding to a target biopolymer in a specimen are immobilized on a large number of spots classified by type on a plane. Has a plurality of through-holes in the thickness direction, and the through-hole is filled with a gel in which a probe is immobilized.

  Capillary array sheet, for example, by drilling a large number of holes in a plurality of stacked sheet-like members or block-like members with a laser or the like, and injecting different probes and gel precursor solutions into the respective holes, gel precursor solution And a method of forming a sheet after polymerizing the fibers (Japanese Patent Application Laid-Open No. 2000-78998), a method in which a fiber aggregate (three-dimensional array) in which hollow fibers are regularly arranged, and a probe in each of the voids of the hollow fibers. And a method of injecting a gel precursor solution, polymerizing the gel precursor solution, and slicing in a direction orthogonal to the fiber axis to form a sheet (Japanese Patent Application Laid-Open Nos. 2000-270877 and 2000-27078). Therefore, it can be manufactured.

  A method for detecting a target biopolymer in a sample using a capillary array sheet is known, and the capillary array sheet is brought into contact with the sample, and the target biopolymer contained in the sample is converted into a capillary array sheet. By binding to the probe contained in the gel of, the obtained conjugate, for example, by detecting with a fluorescent dye or the like applied to the sample, a plurality of types of target biopolymer corresponding to the probe, quantitative or at once It can be examined qualitatively (JP-A-2014-94019, JP-A-2011-133470, and WO2013 / 137247).

  Further, an apparatus and a method for inspecting the presence / absence of a gel in a through-hole of a capillary array sheet have been developed and reported (JP-A-2003-185597, JP-A-2008-107331, and JP-A-2009-107331). 229378, JP-A-2010-112831). These inspection methods determine the presence or absence of a gel in a through-hole by immersing a capillary array sheet to be inspected vertically in a liquid in an immersion tank and measuring the transmitted light transmitted through the gel. .

JP-A-2000-78998 JP 2000-270877 A JP-A-2000-27078 JP 2014-94019 A JP 2011-133470 A WO2013 / 137247 JP 2003-185597 A JP 2008-107331 A JP 2009-229378 A JP 2010-112831 A

  As described above, conventionally, the inspection for the presence or absence of gel in the through-hole of the capillary array sheet has been performed by placing the capillary array sheet in the immersion tank in order to prevent the gel in the capillary array sheet to be inspected from drying. It was immersed in a liquid, and in that state, the transmitted light transmitted through the gel was measured to determine the presence or absence of the gel.

  Conventionally, in a method for detecting a target biopolymer in a specimen using a capillary array sheet, in order to prevent the gel in the capillary array sheet from drying, the capillary array sheet is immersed in a liquid. However, in that state, detection of a fluorescent dye or the like was performed.

  However, air bubbles may be generated and adhere to the inner wall of the immersion container containing the liquid or the surface of the capillary array sheet immersed in the liquid during inspection or detection. In some cases, accurate inspection and detection may not be possible if they overlap with the visual field of the subject.

  Such a problem is not limited to inspection and detection of a capillary array sheet, but is common to all surface observations of a sample housed in an immersion container and immersed in a liquid in the immersion container.

  The present inventors have conducted intensive studies to solve the above problems, and as a result, when immersing the sample in the liquid in the immersion container and observing it, displace the liquid level of the liquid in the immersion container, thereby immersing the sample. It has been found that air bubbles can be prevented from adhering to the inner wall surface of the container and the sample surface, or air bubbles adhering to these surfaces can be removed, and good surface observation of the sample can be achieved.

The present invention is based on these findings and includes the following inventions.
[1] a step of displacing the level of the preservation solution in the immersion container;
A step of observing the surface of the sample contained in the immersion container and immersed in the storage solution in the immersion container,
A method for observing a surface of a sample in a liquid, comprising:
[2] The step of displacing the height of the liquid level of the preservation liquid includes, before storing the sample in the immersion container, performing an operation of injecting the preservation liquid to increase the liquid level to a level higher than the observation surface of the sample. The method of [1], which is a step of displacing.
[3] The step of displacing the height of the liquid surface of the preservation liquid includes, after storing the sample in the immersion container, injecting the preservation liquid to change the observation surface of the sample from a height lower than the observation surface of the sample. The method according to [1], which is a step of displacing the height of the liquid surface to a height higher than the height.
[4] The step of displacing the height of the liquid surface of the storage solution includes a step of displacing the height of the liquid surface to a height lower than the observation surface of the sample by an operation of discharging the storage solution, [2]. Or the method of [3].
[5] The method according to any one of [1] to [4], wherein the operation of draining and injecting the storage solution is repeated a plurality of times, thereby repeatedly changing the height of the liquid level of the storage solution a plurality of times.
[6] The method according to any one of [1] to [5], wherein the sample is a biochip.
[7] The method according to [6], wherein the biochip holds a gel.
[8] The method according to [6], wherein the biochip is a capillary array sheet having a plurality of through holes in a thickness direction of the sheet, and holding gel in the through holes.

[9] an immersion container capable of holding a sample,
Liquid level displacement means for displacing the level of the liquid level of the storage solution in the immersion container,
Surface observation means for observing the surface of the sample,
An apparatus for observing a surface of a sample in a liquid, comprising:
[10] The apparatus according to [9], wherein the immersion container includes at least one surface that can transmit light.
[11] The apparatus according to [9] or [10], wherein the liquid level displacing means includes a storage liquid injection device for injecting a storage liquid.
[12] The apparatus according to any one of [9] to [11], including a storage liquid discharging device for discharging a storage liquid as the liquid level displacement unit.
[13] The apparatus according to any one of [9] to [12], wherein the sample is a biochip.
[14] The apparatus according to [13], wherein the biochip holds a gel.
[15] The apparatus according to [13], wherein the biochip is a capillary array sheet having a plurality of through holes in a thickness direction of the sheet, and holding gel in the through holes.

  According to the present invention, it is possible to remove air bubbles attached to the inner wall of a immersion container containing a liquid and the surface of a sample contained in the immersion container and immersed in the liquid by a simple method, Surface observation can be performed efficiently.

FIG. 1 shows a schematic view of the surface of a capillary array sheet. FIG. 2 shows a schematic view of a capillary array sheet housed in a capillary array sheet holder. FIG. 3 is a schematic diagram of a surface observation device for a capillary array sheet. FIG. 4 is a schematic view of a device for observing the surface of a capillary array sheet. FIG. 5 shows that the bubbles (left, arrowhead) attached to the inner wall of the immersion container are displaced by lowering the level of the preservation liquid in the immersion container (lowering (medium, arrow) to raising (immersing)). It is a photograph figure which shows that it removed (right).

  The present invention is used for surface observation of a sample immersed in a liquid. The “sample” is not particularly limited as long as it is subjected to surface observation in a liquid. “Surface observation” includes (but is not limited to) detecting and confirming the shape and condition of a sample or their quality, the presence or absence of substances bound, attached, or contained in a sample, or their condition. included. Surface observation can be performed by sensing and detecting reflected light, scattered light, transmitted light, fluorescence, and the like, which are directly or indirectly derived from the sample or the substance. The “sample” includes, for example, a biochip, and is preferably a capillary array sheet.

  Hereinafter, an example of the present invention using a capillary array sheet as a sample will be described in detail with reference to the drawings.

  FIG. 1 is a schematic plan view showing the configuration of the “capillary array sheet”. The capillary array sheet (1) has a flat sheet member (2) that does not transmit light. The sheet member (2) is provided with a plurality of through holes (4) so as to penetrate between both surfaces of the sheet member (2), and the through holes are filled with the gel (3). A probe capable of binding to a target biopolymer in a sample is fixed to the gel (3). The shape of the through-hole in the surface of the capillary array sheet (1) is not particularly limited, but preferably has, for example, a circular cross section or a rectangular cross section.

  Although the material of the sheet member (2) is not particularly limited, for example, a material in which carbon black is kneaded into a resin such as acryl, polycarbonate, urethane, etc., which hardly reflects light and hardly transmits light is preferable.

  Although the gel is not particularly limited, for example, acrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, N-acryloylaminoethoxyethanol, N-acryloylaminopropanol, N-methylolacrylamide, N-vinylpyrrolidone, hydroxyethyl methacrylate, A gel obtained by copolymerizing at least one or more monomers such as (meth) acrylic acid and allyldextrin and a polyfunctional monomer such as methylenebis (meth) acrylamide and polyethylene glycol di (meth) acrylate is exemplified. In addition, examples of gels that can be used for the capillary array sheet include gels of agarose, alginic acid, dextran, polyvinyl alcohol, polyethylene glycol, and the like, and gels obtained by crosslinking these.

  Probes immobilized on the gel include nucleic acids such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), and oxypeptide nucleic acid (OPNA) that can bind to a target biopolymer in a sample, and proteins ( Antibodies or fragments thereof, antigens or fragments thereof, lectins, etc.), polysaccharides, and derivatives thereof.

  The capillary array sheet slices a fiber assembly (three-dimensional array) in which a plurality of biopolymer-immobilized gel-retaining hollow fibers on which different probes are immobilized are regularly arranged in a direction orthogonal to the fiber axis. It can be manufactured by doing.

  However, the present invention provides a capillary array sheet manufactured by another method, for example, forming a plurality of holes with a laser beam or the like in a plurality of stacked sheet-like members or block-like members, and forming different nucleic acids in these holes. It is manufactured by injecting a biopolymer probe such as a biopolymer probe and a gel precursor solution, polymerizing the gel precursor solution in the hole, and then cutting this at a desired thickness in a direction orthogonal to the direction in which the hole extends. Capillary array sheets can also be used.

The capillary array sheet (1) may be used in the present form, or may be used in order to facilitate protection and operation (for example, accommodation in and recovery from the immersion container (12) described below). 2, it may be used in a form accommodated in a capillary array sheet holder (5). The capillary array sheet holder (5) can be made of an arbitrary member (for example, the same material as the sheet member (2)) as long as the observation operation is not hindered. Further, the shape can be any shape as long as the observation work is not hindered.
Hereinafter, the surface observation device (6) of the present invention will be described.

1. Surface Observation Apparatus The surface observation apparatus (6) is an apparatus for observing the surface of a sample in a liquid, a surface observation means for observing the surface of the sample, an immersion container capable of accommodating the sample, and And a liquid level displacing means for displacing the height of the liquid level of the storage liquid.
FIG. 3 is a schematic diagram illustrating an example of a surface observation device for a capillary array sheet.
The surface observation device (6) includes a light source (7) as a surface observation unit, and an imaging unit (10) having a condenser lens (8) and a detection unit (9). ) And a immersion container (12) capable of accommodating the capillary array sheet (1), and a filling / discharging device for injecting / discharging a preservation solution into / from the immersion container as a liquid level displacing means. (13) can be provided. The light source (7), the immersion container (12), and the imaging unit (10) are arranged on the optical axis of the condenser lens (8) in this order.

(1-1) Light source The light source (7) does not need to be used as long as sufficient brightness for observing the surface of the capillary array sheet (1) is obtained depending on the use environment of the present apparatus. A light source can be used depending on the purpose. Examples of the light source (7) include a halogen lamp, a mercury lamp, an LED, a laser, and the like. By irradiating the surface of the capillary array sheet (1) with excitation light, fluorescence observation of the capillary array sheet is performed. be able to. In this case, the user may change the wavelength, intensity, irradiation angle, and the like of the light each time according to the observation state. In FIG. 3, the light source (7) is shown as being a transmitted illumination, but it is also possible to irradiate the light from the condenser lens side.
In addition, the light source (7) can be used together with an emission unit adjusting means (not shown) such as a field stop, if necessary.

(1-2) Imaging Unit The imaging unit (10) includes a condenser lens (8) and a detection unit (9). As the condenser lens (8), for example, an objective lens for a microscope can be used. As the detection means (9), for example, a CCD sensor, a CMOS sensor, a line sensor, or the like can be used. Further, the photographing section (10) may be fixed to a movable table (not shown). The moving table is indicated by a focus adjusting means (not shown) for adjusting the distance in the optical axis direction between the condenser lens (8) and the detecting means (9) and the capillary array sheet (1). This allows movement on the optical axis of the condenser lens (8).

(1-3) Immersion Container The immersion container (12) preferably includes at least one surface that can transmit light so that surface observation of the capillary array sheet (1) is not hindered. Such a surface may be made of a material having a high light transmittance. As such a material, for example, glass, quartz, polypropylene, polyethylene, polyester, acrylic, polycarbonate, polysulfone, or the like can be used.

  The immersion container (12) is connected to an opening (sample insertion part (14)) for inserting the capillary array sheet (1) and an injection / discharge device (13) to be described later, as necessary. A preservation solution inlet / outlet can be provided. The preservation liquid injection and discharge port may be used for both injection and discharge, or may be separate ones used for injection and discharge respectively. It is preferable that at least the outlet of the preservative solution is provided at a position lower than the observation surface of the capillary array sheet (1).

  Furthermore, the immersion container (12) may be fixed to a stage (not shown) that is relatively movable in a direction orthogonal to the optical axis of the condenser lens (8). The moving direction and the moving amount of the stage can be instructed by a detection position adjusting means (not shown), so that all the through holes of the capillary array sheet (1) are within the field of view of the imaging unit (10). It is possible to fit.

  The preservation solution (11) is for preventing the sample from drying, and in the case of the capillary array sheet (1), is for preventing the gel held in the through hole from drying. The preservation liquid (11) may be any liquid that prevents drying of the sample and does not contaminate or degrade the sample, and preferably has high light transmittance. Further, a liquid having a low viscosity, in which the liquid level is easily displaced by the injection / discharge device described above, is more preferable. Examples of such a liquid include water, salt solution, and the like.

(1-4) Injection / Discharge Device Injection and discharge of the storage solution (11) can be performed by an injection / discharge device (13).
The injection / discharge device (13) may be a single device having a pump capable of switching operation between injection and discharge, or may be an injection device connected to an injection pump and a discharge device connected to a discharge pump. It may be a device in combination with a discharge device.
As an example of the configuration of the injection / discharge device (13), the configuration illustrated in FIG. 3 will be described.

  The injecting / discharging device (13) shown in FIG. 3 includes a storage liquid tank (17), an injecting line (15), and a discharging line (16). Each of the devices has a device, and is connected to a storage solution injection / discharge port provided in a water tank portion of the immersion container (12). When injecting the preservation solution into the water tank part of the immersion container (12), the preservation solution is sent via the infusion line (15). Conversely, when the preservation solution is discharged, the discharge line (16) is connected. The liquid is sent via the hopper.

FIG. 4 shows another example of the filling / discharging device.
The injecting / discharging device (13) includes a storage solution tank (17) and an injecting line (15 '), and a waste liquid tank (18) and a discharging line (16'). Each line is an injecting device (not shown). And a discharge device.
When injecting the preservation solution into the water tank part of the immersion container (12), the injection line (15 ') can be inserted into the immersion container (12) (for example, from the sample insertion part (14)). The liquid is sent via the. Conversely, when draining the preservation solution, insert the end of the drain line until it is lower than the observation surface of the capillary array sheet (for example, from the sample insertion part (14)), and pass through the drain line. The liquid is sent.

2. Surface observation method The surface observation method of the present invention is a method for observing the surface of the sample in a liquid, a step of displacing the height of the liquid level of the storage solution in the immersion container, and housed in the immersion container, Observing the surface of the sample immersed in the storage solution in the immersion container.

(2-1) Storage of sample in immersion container The storage of the capillary array sheet (1) in the immersion container (12) is performed by inserting the capillary array sheet (1) into the opening of the immersion container (12). The sample is inserted vertically from the sample insertion section (14)) to a predetermined position where the range to be observed on the capillary array sheet (1) falls within the field of view of the imaging section (10).

  "Insert vertically" means that the capillary array sheet (1) is moved, inserted into the immersion container (12), and held and fixed at a predetermined position in the immersion container (12), Alternatively, the capillary array sheet (1) may be gripped and fixed, and the immersion container (12) may be moved so that the capillary array sheet (1) is inserted. The sample can be stored in the immersion container at any timing, whether the storage solution (11) is empty in the immersion container (12) or the state where the storage solution (11) has already been injected.

(2-2) Step of Displacing the Level of the Preservation Liquid in the Immersion Vessel The height of the preservation liquid (11) in the immersion vessel (12) can be adjusted by injecting / discharging a liquid level displacing means. Using the device (13), the displacement can be effected by injecting and / or draining the preservative solution (11) in the immersion vessel (12).

  The preservation liquid (11) can be injected before or after housing the capillary array sheet (1) in the immersion container (12), but preferably, the immersion container (12) is used. It is injected after the capillary array sheet (1) is accommodated therein. The preservation solution (11) gradually raises the liquid surface from a position lower than the observation surface of the capillary array sheet (1) to a position higher than the observation surface, so that the inner wall of the immersion vessel (12) and the capillary array sheet (1). Air bubbles generated and adhered to the surface of the object can be removed, and obstruction of observation by the air bubbles can be avoided.

  The preservation liquid (11) can be discharged before or after the capillary array sheet (1) is accommodated in the immersion container (12). The preservation liquid (11) may be discharged at least to a position lower than the observation surface of the capillary array sheet (1) until the preservation liquid (11) drops.

  The speed at which the liquid level of the preservation liquid (11) is changed depends on the shape of the immersion container (12), the shape of the preservation liquid injection port, the composition of the preservation liquid, etc. Just fine.

  The operation of injecting and / or discharging the preservation solution (11) can be repeated a plurality of times (for example, 2, 3, 4, 5, or more times).

(2-3) Step of Observing Surface of Sample Dipped in Preservation Solution The surface of the capillary array sheet (1) immersed in the preservation solution (11) in the immersion container (12) is observed.
In the surface observation, if necessary, the surface of the capillary array sheet (1) is focused by the focus adjusting means provided in the photographing unit (10), and the observation surface of the capillary array sheet (1) is adjusted. When the whole does not fit in the visual field range, observation is performed by the imaging unit (10) while shifting the observation surface of the capillary array sheet (1) each time by the detection position adjusting means provided in the immersion container (12). Can be.

  If the observation operation is hindered by bubbles generated during surface observation, as described in step (2-2), gradually raise the liquid level of the storage solution (11) from a position lower than the observation surface to a position higher than the observation surface. Thus, air bubbles generated and attached to the inner wall of the immersion container (12) and the surface of the capillary array sheet (1) can be removed. That is, once the storage liquid in the immersion container (12) is discharged to a position lower than the observation surface of the capillary array sheet (1) by the injection / discharge device (13), and then the storage liquid (11) is injected again. Thus, bubbles can be removed.

  Examples of the present invention will be described below.

Example 1 Production of Capillary Array Sheet A 0.12 mm thick perforated plate in which 19 × 12, 228 holes having a diameter of 0.32 mm are arranged in a grid pattern at intervals of 0.42 mm in the center of the plate. Using two sheets, a hollow fiber made of polycarbonate (outer diameter 0.28 mm, inner diameter 0.18 mm, length 500 mm) colored with carbon black passes through all the holes of the perforated plate, and passes through these two fibers. The separated perforated plate is separated by 50 mm, and between the separated perforated plates, a polyurethane resin colored with carbon black is filled. A hollow fiber array was obtained.

  The hollow fiber in the obtained hollow fiber array is filled with a gel precursor solution and a 40-based nucleic acid probe of a different type, respectively, and the gel precursor solution is polymerized, and then, in a microtome, in a direction perpendicular to the fiber axis. A capillary array sheet was obtained by cutting a thin piece having a thickness of 0.25 mm.

Example 2: Surface observation Injection of the preservation solution The injection line for injecting the preservation solution and the discharge line for discharging the preservation solution are directly connected to the preservation solution injection / discharge port provided in the glass-like immersion vessel, respectively. The preservation solution was injected into the immersion container through the production line.

2. Capillary array sheet storage Insert the capillary array sheet vertically from the sample insertion section provided at the top of the immersion container, immerse it in the storage solution in the immersion container, fix it, and adjust the detection position The observation position and the focus position were adjusted using the device and the focus adjusting device.

3. Surface observation A surface observation image was acquired with a CCD sensor (BS-41L manufactured by Bitrun) in the imaging unit in a state where transmitted light (white LED) was irradiated from the back of the capillary array sheet.
When the captured surface observation image was confirmed, air bubbles were attached to the observation surface of the capillary array sheet (FIG. 5, left, arrowhead).

4. Removal of air bubbles The preservation solution in the immersion vessel is discharged once through the discharge line until the liquid level is lower than the observation surface of the capillary array sheet (Fig. 5, middle, arrow), and then injected. The preservation solution was re-injected via the application line until it reached a position higher than the observation surface of the capillary array sheet (FIG. 5, right).
When the surface observation image of the capillary array sheet was acquired again by the CCD sensor of the photographing unit, it was confirmed that bubbles were removed.

1 Capillary array sheet 2 Sheet member 3 Gel 4 Through hole 5 Capillary array sheet holder 6 Surface observation device 7 ... Light source 8... Condensing lens 9... Detecting means 10. Sample insertion unit 15 Injection line 16 Discharge line 15 'Injection line 16' Discharge line 17 Preservation liquid tank 18 Waste liquid tank

Claims (15)

A step of displacing the level of the preservation liquid in the immersion container,
A step of observing the surface of the sample contained in the immersion container and immersed in the storage solution in the immersion container,
A method for observing a surface of a sample in a liquid, comprising:   The step of displacing the height of the liquid level of the preservation solution is a step of displacing the height of the liquid level to a level higher than the observation surface of the sample by an operation of injecting the preservation solution before storing the sample in the immersion container. The method of claim 1, wherein   The step of displacing the height of the liquid level of the preservation solution, after storing the sample in the immersion container, by the operation of injecting the preservation solution, from a height lower than the observation surface of the sample to a height exceeding the observation surface of the sample. The method according to claim 1, wherein the step of displacing the level of the liquid surface is performed.   4. The method according to claim 2, wherein the step of displacing the height of the liquid level of the storage liquid includes a step of displacing the liquid level to a height lower than the observation surface of the sample by an operation of discharging the storage liquid. The described method.   The method according to any one of claims 1 to 4, wherein the height of the liquid level of the storage solution is repeatedly changed a plurality of times by repeating the operation of discharging and injecting the storage solution a plurality of times.   The method according to any one of claims 1 to 5, wherein the sample is a biochip.   7. The method according to claim 6, wherein the biochip holds a gel.   The method according to claim 6, wherein the biochip is a capillary array sheet having a plurality of through holes in a thickness direction of the sheet, and holding gel in the through holes. An immersion container capable of holding a sample,
Liquid level displacement means for displacing the level of the liquid level of the storage solution in the immersion container,
Surface observation means for observing the surface of the sample,
An apparatus for observing a surface of a sample in a liquid, comprising:   The apparatus according to claim 9, wherein the immersion container includes at least one surface that can transmit light.   The apparatus according to claim 9, further comprising a storage liquid injection device configured to inject a storage liquid as the liquid level displacement unit.   The apparatus according to any one of claims 9 to 11, further comprising a storage liquid discharging device configured to discharge a storage liquid as the liquid level displacement unit.   The device according to any one of claims 9 to 12, wherein the sample is a biochip.   14. The device according to claim 13, wherein the biochip holds a gel.   14. The apparatus according to claim 13, wherein the biochip is a capillary array sheet having a plurality of through holes in a thickness direction of the sheet, and holding gel in the through holes.