WO2005093416A1

WO2005093416A1 - Substrate for disposing beads and bead disposing method using the same

Info

Publication number: WO2005093416A1
Application number: PCT/JP2005/005339
Authority: WO
Inventors: Takanori Ichiki; Hiroaki Aizawa
Original assignee: Japan Science And Technology Agency
Priority date: 2004-03-26
Filing date: 2005-03-24
Publication date: 2005-10-06
Also published as: JPWO2005093416A1

Abstract

A substrate for disposing beads and a bead disposing method using the substrate enabling the beads capable of efficiently fixing biomolecules to any place in a short time to be stably disposed thereon so that these beads are not peeled off from that place. The substrate (10) for disposing the beads for fixing the magnetic beads (11) in minute reaction chambers (5) comprises a substrate material (1), a magnetic body thin-film (3) disposed on the substrate material (1), and the plurality of minute reaction chambers (5) disposed on the magnetic body thin-film (3). The bead disposing method comprises the steps of disposing a magnet (13) under the substrate (10) for disposing the beads and dripping a dispersion (12), in which the magnetic beads (11) are dispersed, on the substrate (10) to fix the magnetic beads (11) in the minute reaction chambers (5) of the substrate (10) for disposing the beads.

Description

Specification

Bead arranging substrate and bead arranging method using the same

Technical field

The present invention relates to a substrate for arranging beads and a method of arranging beads using the same.

Also, the present invention relates to a bead placing substrate capable of placing beads in a plurality of micro-reaction tanks of the bead placing substrate with high efficiency and achieving stable placement, and a bead placing method using the same.

Background art

[0002] In recent years, the application of micro- and nano-microfabrication technologies cultivated in the semiconductor industry to modern biotechnology has dramatically increased the speed of the reaction processes conventionally performed in test tubes, and the development of technologies for large-scale parallelization has recently been developed. Is progressing rapidly. For example, DNA chips and protein chips that enable large-scale parallel analysis by disposing a large number of DNAs and enzymes with different structures on a glass substrate have already been commercialized and widely used in the field of advanced biotechnology research. I have.

DNA chips are made by directly immobilizing biomolecules on a substrate.

[0003] On the other hand, Non-Patent Document 1 discloses a technique in which a micro semiconductor block is separated from an original substrate, and is dispersed at an arbitrary position on the substrate by spraying the micro semiconductor block on a target substrate in a liquid. Has been disclosed. It is reported that by fixing a biomolecule to a small semiconductor block and using the method described in Non-Patent Document 1, the biomolecule can be arranged at an arbitrary position on a substrate.

Non-Patent Document l: H. Yeh and J. Smith, IEEE Photon. Tech. Lett., Vol. 6, .706, 1994 Disclosure of the invention

Problems to be solved by the invention

[0004] However, according to the conventional method, it is difficult to arrange a large amount of biomolecules on a substrate in a short time by self-assembly by a method of directly immobilizing biomolecules on a substrate. It was also difficult to recover a biomolecule at a desired position from the substrate after the arrangement. In addition, even when the method described in Non-Patent Document 1 is used, the minute semiconductor block is not attracted and fixed on the substrate, the time required for placement is long, and the minute block is placed after placement. Lock release occurred and it was not easy to maintain a stable configuration.

[0005] Therefore, an object of the present invention is to arrange beads capable of immobilizing biomolecules and the like in a short time and with high efficiency at an arbitrary place, and to stably arrange the beads without detachment from an arbitrary place. It is an object of the present invention to provide a substrate for arranging beads, and a method for arranging beads using the same.

Means for solving the problem

The present inventors have conducted intensive studies to solve the above problems, and as a result, have found that the above object can be achieved by combining a magnetic bead with a bead arrangement substrate having a magnetic thin film. Was completed.

[0007] That is, the bead disposing substrate of the present invention is a bead disposing substrate for disposing magnetic beads in a micro-reaction tank, and comprises a substrate material and a magnetic thin film disposed on the substrate material. And a plurality of minute reaction vessels disposed on the magnetic thin film.

[0008] In addition, in the method for arranging beads according to the present invention, when arranging the magnetic beads in the micro-reaction tank of the substrate for arranging beads, a magnet is arranged below the substrate for arranging beads, And a step of dropping a dispersion liquid in which the magnetic beads are dispersed.

In the present invention, the magnetic beads are attracted into the micro-reaction tank by the action of the magnetic force of the magnetic beads and the magnetic thin film, so that the magnetic beads can be easily arranged and a stable arrangement can be obtained. In addition, since the magnetization of the magnetic thin film remains after the magnet is removed, the magnetic beads can maintain a stable arrangement.

The invention's effect

According to the present invention, for example, even a magnetic bead on which a biomolecule is immobilized can be controlled by an external magnetic field and can be easily and reliably filled in a microreactor. It becomes possible to form a large number of micro reaction vessels.

[0011] Furthermore, by coating a magnetic thin film on a substrate and utilizing the magnetization phenomenon of the magnetic thin film, a large amount of magnetic beads can be simultaneously filled in a plurality of minute reaction vessels on a magnetic bead disposing substrate. Power S can. In this way, magnetic beads can be efficiently filled into multiple micro reaction vessels Thereby, simplification of filling and speeding up can be realized as compared with the conventional method. In addition, due to the residual magnetization of the magnetic thin film, the holding power of the magnetic beads on the substrate surface is increased, and the detachment of the magnetic beads due to running water or the like can be prevented.

Brief Description of Drawings

FIG. 1 is a schematic view showing a process for producing a substrate for fixing beads according to an embodiment of the present invention.

FIG. 2 is an enlarged photograph of a micro-reaction tank formed on a substrate for arranging beads.

FIG. 3 is a schematic view of an apparatus used for observing an array of magnetic beads using an optical microscope.

FIG. 4 is a schematic view showing a step of producing a substrate for fixing beads according to another embodiment of the present invention.

FIG. 5 is (a) a microphotograph of magnetic beads under irradiation of white light and (b) a microphotograph of magnetic beads under irradiation of green light in Example 4.

Explanation of symbols

[0013] 1 Substrate material

2 Adhesive layer

3 Magnetic thin film

4 Negative photoresist SU— 8

5 Micro reaction tank

10 substrate

11 Magnetic beads

12 Dispersion

13 magnet

14 Optical microscope

15 Arithmetic processing unit

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be specifically described.

The substrate material that can be used for the substrate for arranging beads of the present invention is preferably a transparent glass or a plastic material, more preferably a material that does not emit fluorescence. Transparent board By using the material, optical observation of the biopolymer arbitrarily adsorbed on the surface of the magnetic beads becomes possible. In addition, by using a material that does not emit fluorescence for the substrate material, the accuracy of the optical measurement can be improved.

Next, by magnetizing the magnetic thin film laminated on the substrate material, the magnetic beads can be fixed in the minute reaction tank. As the material of the magnetic thin film, metals such as nickel, nickel alloys, iron and iron alloys can be suitably used. In the present invention, it is preferable to use a magnetic material having a large remanent magnetization. . The thickness of the magnetic thin film is preferably 0.2 to 2 x m for the purpose.

In the present invention, an adhesive layer of chromium or the like can be provided between the substrate material and the magnetic thin film provided thereon to enhance the adhesiveness between the two.

[0017] The plurality of micro-reaction tanks provided on the magnetic thin film can be formed by using a known photolithography technique, which is a fine processing technique cultivated in the semiconductor industry. As a material for forming the micro-reaction tank, a transparent photoresist, polydimethylsiloxane (PDMS), or the like can be suitably used.

[0018] The filling rate of the magnetic beads into the reaction vessel depends on the diameter of the reaction vessel, and the larger the diameter of the reaction vessel is slightly larger than the diameter of the magnetic beads, the higher the filling rate is. Is 112 times the diameter of the magnetic beads. Further, in filling one magnetic bead into one microreactor, the depth of the microreactor is preferably 112 times the diameter of the magnetic beads.

Further, the surface of the substrate for arranging beads and the inner wall of the microreactor of the present invention may be coated with a blocking agent for preventing nonspecific adsorption of biomolecules, for example, polyethylene glycol (PEG) or 2-methacryloyloxetyl phosphorylcholine (MPC). ) Can be suitably coated. By coating with a strong blocking agent, non-specific adsorption of biomolecules on the substrate surface or the inner wall of the microreactor can be suppressed.

[0020] Further, it is preferable that the micro reaction tank is hydrophilized. By subjecting the microreactor to hydrophilic treatment by irradiation with oxygen plasma or the like, the liquid (magnetic beads dispersed liquid) is easily filled into the reaction tank, and the filling rate is improved.

[0021] Furthermore, the magnetic thin film at the bottom of the microreactor may be removed by a known method such as wet etching. Observation becomes possible. For example, it becomes possible to determine the expression of cells and proteins by fluorescence.

Next, in the bead placement method of the present invention, when placing the magnetic beads in the microreaction tank of the above-described bead placement substrate, a magnet is placed below the bead placement substrate, and the magnet is placed on the substrate. Then, a dispersion liquid in which magnetic beads are dispersed is dropped. Thereby, the magnetic beads are attracted to the micro-reaction tank by the attraction of the magnet. Furthermore, the magnetic beads are dispersed by appropriately moving the magnet in a direction parallel to the substrate, and the filling rate in the reaction tank is improved. The strength of the magnetic field applied to the bead placement substrate by the magnet is preferably 100 to 10,000 gauss in order to obtain a desired effect.

[0023] As described above, the filling of the inside of the reaction tank with the liquid is facilitated by making the micro reaction tank hydrophilic, but the same effect can be obtained by using a surfactant.

Further, in the present invention, the magnetic thin film is magnetized by the magnet disposed under the substrate, and the magnetic beads remain in the magnetic thin film even after the magnetic beads are attracted to the reaction tank and the magnet is removed. It is possible to keep it fixed.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

Example 1

First, as shown in FIG. 1 (a), a chromium film is formed as an adhesive layer 2 on a transparent glass substrate material 1 of 20 mm × 20 mm, and nickel film, which is a magnetic material, has a film thickness of 0. Thus, a magnetic thin film 3 was obtained. Thereafter, a negative photoresist SU-8 indicated by reference numeral 4 in the figure was applied so as to have a thickness of 3 × m (FIG. L (b)). Further, the pattern of 10,000 micro-reactors with a diameter of 3 μm was transferred by contact exposure, and the circular pattern was removed with a developer to form a micro-reactor 5 (FIG. 1 (c)). Finally, the surface was subjected to hydrophilization by irradiating the surface with oxygen plasma at 100 mTorr, 200 W for 5 minutes using a flat-plate inductively coupled plasma generator, and a substrate for bead placement was fabricated (Fig. 1 (d)). ). Fig. 2 shows an enlarged photograph of the prepared bead placement substrate.

Next, as schematically shown in FIG. 3, a magnetic beam having a diameter of 2.8 / m A dispersion liquid 12 in which the particles 11 were dispersed was dropped. At this time, a magnet 13 having a magnetic flux density of 2000 Gauss was arranged on the back surface of the substrate 10 in order to guide the magnetic beads 11 into the micro reaction vessel 5. Further, by appropriately moving the magnet 13 in a direction parallel to the substrate 10, the induction of the magnetic beads 11 into the minute reaction tank 5 was promoted.

After the surface of the substrate 10 is washed with pure water while the magnet 13 is fixed to the back surface of the substrate 10, the arrangement of the magnetic beads 11 is observed with an optical microscope 14 connected to an arithmetic processing unit 15. did. As a result of observation with the optical microscope 14, it was confirmed that the magnetic beads 11 were filled one by one into each micro reaction vessel 5 with a high probability of 95% or more.

Comparative Example 1

A substrate 10 was prepared in the same manner as in Example 1 except that nickel was not formed as the magnetic thin film 3, and the same test was performed.As a result, the filling rate in the microreactor 5 was 10% or less. Lower and lower values were shown. From this, it was confirmed that the filling rate was improved by the effect of the nickele film.

Example 2

As in Example 1, first, a chromium film was formed as an adhesive layer 2 on a transparent glass substrate material 1 of 20 mm × 20 mm, and nickel was used as a magnetic material so that the film thickness became 0.5 μΐη. (Fig. L (a)). A negative photoresist SU-8 indicated by reference numeral 4 in the figure was applied to the magnetic thin film 3 of the nickele so as to have a thickness of 3-10 x m (FIG. 1 (b)). Next, the pattern of the circular microreactor 5 having a diameter of 3, 4, 5, 6, 7, and 10 zm was transferred by contact exposure, and the circular pattern was removed with a developer to form the microreactor 5 ( Figure 1 (c)). Finally, using a flat plate type inductively coupled plasma generator, the surface was subjected to hydrophilization treatment by irradiating the surface with oxygen plasma at 100 mTorr and 200 W for 5 minutes to prepare a substrate for bead placement (Fig. 1 (d) )).

Next, as schematically shown in FIG. 3, a dispersion liquid 12 in which 4 × 10 ⁶ magnetic beads 11 having a diameter of 2.8 _μm are dispersed on the manufactured substrate 10 is dropped. A magnet 13 was fixed to the back surface of the substrate 10 to guide the magnetic beads 11 into the reaction tank 5. Further, by moving the magnet 13, the induction of the magnetic beads 11 into the microreactor 5 was promoted.

After cleaning the surface with pure water with the magnet 13 fixed to the back surface of the substrate 10, the same as in Example 1 was performed. The state of the arrangement of the magnetic beads 11 was observed with the optical microscope 14 connected to the arithmetic processing unit 15 as described above. Observation with an optical microscope 14 revealed that the filling rate of the magnetic beads 11 increased in proportion to the diameter of the microreactor 5. In addition, a micro-reaction tank having a large diameter was filled with a plurality of magnetic beads 11.

[0032] When the depth of the microreactor 5 was 6 µm or more, a plurality of magnetic beads 11 were mixed in the longitudinal direction. When the diameter of the microreactor 5 was 6 μm or less, one magnetic bead 11 was filled in one microreactor 5. In addition, in the micro-reaction tank 5 having a large diameter, the magnetic beads 11 are detached from the micro-reaction tank 5 having a diameter substantially the same size as the force of the magnetic beads 11 detached during washing. Not observed. From the above, it was clarified that the depth and diameter of the microreactor 5 were suitably 6 μm or less.

Example 3

As in the case of Examples 1 and 2, first, as shown in FIG. 4 (a), on a transparent glass substrate material 1 of 20 mm × 20 mm, a nickel material, which is a magnetic material, was formed to a thickness of 0.5 μη. Then, a magnetic thin film 3 was obtained. Thereafter, a negative photoresist SU-8 indicated by reference numeral 4 in the figure was applied to a thickness of 3 / im (FIG. 4 (b)). Further, the patterns of 10,000 circular microreactors 5 having a diameter of 3 / im were transferred by contact exposure, and the circular patterns were removed with a developer to form microreactors 5 (FIG. 4 (c)). In order to observe the filling of the magnetic beads with an inverted microscope, the magnetic thin film 3 (nickel film) at the bottom of the microreactor 5 was removed by wet etching (Fig. 4 (d)). Finally, the surface was subjected to a hydrophilic treatment by irradiating the surface with oxygen plasma at 100 mTorr and 200 W for 5 minutes using a flat-plate inductively coupled plasma generator, and a substrate for bead placement was fabricated (Fig. 4 (e)). ).

Next, an aqueous dispersion in which 4 × 10 ⁶ magnetic beads of 2.8 μm in diameter were dispersed was dropped on the prepared substrate, and the arrangement of the magnetic beads was observed from the bottom of the substrate with an optical microscope. Observed. As a result of observation with an inverted optical microscope, it was possible to observe the state in which the magnetic beads were filled in the micro reaction tank.

Example 4

The following experiment was performed as a model experiment of biomolecule screening by affinity in an aqueous solution. First, a substrate for arranging beads was prepared by the method shown in Example 1. Base Quartz glass, which does not emit fluorescence in the visible light region with high UV transmittance, was used as the substrate material of the plate. In addition, a PEG (polyethylene glycol) film was coated on the substrate surface to prevent nonspecific adsorption of proteins.

Next, the obtained bead placement substrate was filled with magnetic beads whose surface was modified with biotin using PEG, and an aqueous solution in which streptavidin serving as a target molecule was dispersed was poured. To facilitate optical observation, streptavidin used was fluorescently labeled with Texas Red. Finally, the aqueous solution in which streptavidin was dispersed in pure water was washed.

[0037] The washed magnetic beads were observed with a fluorescence microscope. Figure 5 shows the results. Figure 5 (a) is a micrograph of magnetic beads under white light irradiation, and (b) is a micrograph of magnetic beads under green light irradiation. Strong red fluorescence was observed from the magnetic beads, confirming that streptavidin, a target molecule, was placed on the magnetic beads fixed in the microreaction tank.

Industrial applicability

The present invention provides a substrate structure and arrangement suitable for immobilizing biomolecules on a magnetic bead surface and then disposing the biomolecules on a substrate instead of directly immobilizing the biomolecules on a substrate like a DNA chip. Provide a way. By using this method, a large amount of biomolecules can be arranged on a substrate by self-assembly in a short time, and it is also easy to collect a biomolecule at a desired position from the substrate after the arrangement. . This technology can be applied to large-scale parallel screening that is required for evolutionary molecular engineering that rapidly evolves biopolymers to create useful molecules. Therefore, the present invention is expected to contribute to a wide range of industrial fields, such as new drug development, the environment, bioprocesses, and foods, as basic elemental technologies of advanced molecular reactors that realize the theory of advanced molecular engineering.

Claims

The scope of the claims

[I] A bead arrangement substrate for disposing magnetic beads in a micro-reaction tank, comprising: a substrate material; a magnetic thin film provided on the substrate material; and a magnetic thin film provided on the magnetic thin film. And a plurality of micro reaction tanks.

2. The substrate for arranging beads according to claim 1, further comprising an adhesive layer between the substrate material and the magnetic thin film.

3. The substrate for arranging beads according to claim 1, wherein the substrate material is a transparent glass or plastic material, and is a material that does not emit fluorescence.

[4] The substrate for arranging beads according to claim 1, wherein the diameter of the minute reaction tank is 112 times the diameter of the magnetic beads.

5. The substrate for arranging beads according to claim 1, wherein the depth of the micro-reaction tank is 112 times the diameter of the magnetic beads.

6. The bead placement substrate according to claim 1, wherein the micro-reaction tank is formed of a transparent photoresist or polydimethylsiloxane (PDMS).

7. The bead placement substrate according to claim 1, wherein the magnetic thin film is a magnetic material selected from the group consisting of nickel, a nickel alloy, iron, and an iron alloy.

[8] The substrate for arranging beads according to claim 1, wherein the substrate surface and the inner wall of the microreactor are coated with a blocking agent for preventing nonspecific adsorption of biomolecules.

9. The substrate for arranging beads according to claim 1, wherein the micro-reaction tank is hydrophilized.

10. The bead placing substrate according to claim 1, wherein the magnetic thin film at the bottom of the minute reaction tank is removed.

[II] In arranging the magnetic beads in the micro-reaction tank of the bead arrangement substrate according to claim 1, a magnet is arranged below the bead arrangement substrate, and the magnetic beads are dispersed on the substrate. A method for arranging beads, comprising a step of dropping the dispersion liquid.

12. The bead arrangement method according to claim 11, wherein the magnet is appropriately moved in a direction parallel to the substrate to guide the magnetic beads to the micro reaction vessel.

13. The bead placing method according to claim 11, wherein the strength of the magnetic field applied to the bead placing substrate by the magnet is 100 to 10,000 Gauss.