JPWO2002095404A1 - New biochip fabrication method - Google Patents

Abstract

Probe molecules arranged in an array on a substrate, i.e., a biochip, can be easily and washed with unbound molecules, and can be denatured by drying the probe molecules during the manufacturing process. Provide a way to make. The method according to the present invention is a method for producing a biochip having a pattern of molecular recognition on a substrate, comprising the following steps: (1) preparing particles of a size that can be handled one by one; (2) Immobilizing a probe for performing molecular recognition on the particles in a test tube; (3) transporting the particles by mechanical means for each type of the probes and disposing the particles at a specific position on the substrate; And (4) fixing the arranged particles at the positions.

Description

TECHNICAL FIELD The present invention relates to a novel biochip manufacturing method.
BACKGROUND ART As a technique for detecting a specific base sequence on DNA, there is a so-called DNA chip. The principle is as follows. 1) Oligonucleotides (DNA having several to about 20 bases) having various sequences are patterned and fixed on a substrate in a two-dimensional array, and 2) A DNA sample with a fluorescent probe is placed on this chip. 3) Under a fluorescence microscope, see where the binding occurs. Since DNA binding occurs by complementarity, if the sample DNA binds to a certain position, a sequence complementary to the sequence fixed at the bonded position must exist on the sample DNA. That is, the sequence of the sample DNA can be detected from the binding position on the pattern. Similarly to this DNA chip, if a protein with molecular recognition such as an antibody is patterned and fixed on a substrate, a protein chip that can detect antigens and the like, and if a sugar chain is patterned and fixed, the It is a chip that detects the molecules that bind.
All of these are based on the principle that a large number of "probes" are systematically fixed at predetermined positions on a substrate, and molecules are identified by detecting the positions where the bonds with foreign molecules occur.
Methods for arranging probes on a substrate include a method of carefully applying a probe solution to a glass substrate one by one using a metal pin, a method of applying using an inkjet method, and a method of selectively performing site-selective chemicals by optical patterning. Methods for performing synthesis have been developed.
The method of applying a solution or the method of applying using an ink jet uses a principle of fixation that probe molecules are adsorbed to a substrate when the applied solution is dried. For example, once a protein is dried, its physiological activity itself may change, and from this point of view, the method of application is not very preferable. In addition, optical patterning also has a problem that molecules are damaged by light irradiation.
On the other hand, in ordinary biochemistry, a microparticle suspension having a diameter of several microns to several tens of microns with a probe fixed on the surface is placed in a 72-well (hole) Petri dish, and the reaction with the suspension is often observed. Done. The problems with this method are that the number of tests that can be performed using a single Petri dish is limited to 72, and that when cleaning particles that do not bind because the fine particles are not fixed to the dish, the fine particles cannot be removed. For example, it may be washed off itself, and it may be difficult to pipette each well with a pipette. There is also an attempt to increase the number of tests per petri dish equivalently by placing microparticles tagged with several types of fluorescent colors and fluorescent intensities in a single well. Intensity measurement requires a complex system.
DISCLOSURE OF THE INVENTION In the present invention, particles having a size that can be handled one by one are prepared, a probe for performing molecular recognition is fixed on the surface of the particles in a test tube, and individual particles are transported by mechanical means. Then, a pattern of the molecular recognition probe is formed on the substrate by arranging and fixing the probe at a fixed position on the substrate for each type of the probe fixed on the surface.
In one embodiment of the present invention, there is provided a method for producing a biochip having a pattern of molecular recognition on a substrate, comprising the following steps:
(1) Prepare particles large enough to handle each one;
(2) immobilizing a probe for performing molecular recognition on the particles in a test tube;
(3) transporting the particles for each type of the probe and disposing them at a specific position on the substrate; and (4) fixing the disposed particles at the position.
The method is provided, comprising:
The probe can be selected from the group consisting of oligonucleotides, sugar chains, and proteins including antibodies.
The diameter of the particles can be between 50 μm and 5 mm.
The particles can have magnetism.
The substrate may have a concavo-convex pattern or a magnetic pattern or both for arranging or arranging the particles.
The fixation of the particles on the substrate can be by adsorption of the magnetic particles by a magnetic pattern on the substrate.
The fixation of the particles can be performed by fitting or entering the particles into concave portions of the concavo-convex pattern on the substrate.
The pattern may be a pattern that is periodically arranged or arranged two-dimensionally.
In another aspect of the present invention, after the particles are introduced into the concave portions of the substrate, the substrate can be covered with a semipermeable membrane or a porous membrane from above and a lid can be placed thereon. Covering in this manner is effective, for example, to prevent drying, wherein the semipermeable or porous membrane includes, but is not limited to, for example, a membrane filter or a nonwoven fabric.
BEST MODE FOR CARRYING OUT THE INVENTION FIG. 1 shows an embodiment of the present invention by taking as an example a case where patterns of 16 types of probes 1-1, 1-2... .
Reference numeral 1 is a test tube containing the probe-immobilized particles 2. In addition to the shape shown in FIG. 1, the test tube has a shape in which particles can be easily transported, a structure in which probes are easily fixed, or a space in which probes are fixed to particles and particles in which probes are fixed. May be exemplified, but is not limited to such an example configuration.
Reference number 2 is a particle having a probe immobilized on its surface. The shape is not limited to the spherical shape as shown in FIG. 1 and may be another shape.
Reference numeral 3 denotes a substrate having a concavo-convex structure having an orderly arrangement, and is formed of silicon, plastic, or the like, and may be formed of a translucent member, but is not limited thereto.
Reference numeral 4 is a marker indicating an address on the substrate. In addition to the symbols shown in FIG. 1, it is only necessary that the particles can be identified and recognized visually or by mechanical, electrical, magnetic, optical, or other various means.
Reference numeral 5 denotes a depression (recess) for fixing the particles, and the depression 5 has a depth of at least unevenness at which the particles on which the probe is fixed can be fixed or a depth of at least the diameter of the particles. In some cases, the size, the shape, and the depth may be sufficient to fix one (or a plurality of) particles in some cases, and are not limited to those shown in FIG.
Reference numeral 6 denotes a magnet for fixing the particles, and the magnet 6 and the depression 5 may be both as shown in FIG.
Reference numeral 7 denotes a transport path of the particles by the transport unit.
Reference numeral 8 is a robot arm used as a particle transport means. The transportation of the particles by the transportation means 8 may be performed approximately one by one (one or more, preferably one by one), and is appropriately adjusted according to the transportation situation. The transport path 7 is also an example, and the particles may be transported from the test tube 1 to the substrate 3 and fixed to a dent or a magnetic force generating part or a predetermined part of a combination thereof, and the transport path may be appropriately adjusted.
First, particles in which magnetic particles are used as particles and all probes are fixed on the surface of all 16 types of probes are prepared in the test tube 1. The magnetic particles include metal particles, plastic particles coated with metal particles, plastic particles containing magnetic powder, and the like.For immobilization of the probe on the particle surface, the probe molecule is attached to the particle surface using a molecular linker. And a method of directly synthesizing on the particle surface. In parallel with this, the depressions (recesses) 5 are arranged in a two-dimensional array on the substrate. A magnet 6 is arranged below each depression 5. The magnet 6 may be a permanent magnet or a magnet that is obtained by temporarily magnetizing a magnetic material with external magnetization. Next, the substrate 3 is filled with the aqueous solution, and the probe-fixed particles 2 are taken out from the 16 kinds of test tubes 1 one by one by the transfer means 8 such as a robot arm, and correspond to the two-dimensional array-shaped depressions 5. Place them one by one at each position (conveyance path 7). The placed particles are fixed by the magnet 6 below the depression 5.
Since the above process and the process can be easily performed in an aqueous solution or under the condition that the particle surface does not dry, the activity of the probe does not decrease due to the drying. In addition, the particles used in the present invention are particles having a diameter of 50 μm or more that can be individually treated. Since the particle size is larger than the latex particles generally used in biochemical assays, the working magnetic force is large and the influence of the flow is relatively large. Therefore, the sample is not washed out by the flow even in the process of introducing or washing the sample after being fixed on the substrate.
According to the biochip manufacturing method of the present invention, a biochip having a high degree of integration can be easily manufactured without drying the probe.
[Brief description of the drawings]
FIG. 1 is a schematic diagram showing the production of a biochip according to the present invention.
List of reference numbers 1 ... Test tube containing probe-immobilized particles 2 ... Particles with probe immobilized on the surface 3 ... Substrate with a regular uneven structure 4 ... Marker indicating address on substrate 5 ... Depression for fixing particles ( Recess)
6 ... A magnet for fixing particles 7 ... A path for transferring particles by a transfer means 8 ... A robot arm used as a particle transfer means

Claims (9)

A method for producing a biochip having a pattern of molecular recognition on a substrate, comprising the following steps:
(1) Prepare particles large enough to handle each one;
(2) immobilizing a probe for performing molecular recognition on the particles in a test tube;
(3) transporting the particles for each type of the probe and disposing the particles at a specific position on the substrate; and (4) fixing the disposed particles at the position.
The method comprising: The method according to claim 1, wherein the probe is selected from the group consisting of an oligonucleotide, a sugar chain, and a protein including an antibody. The method of claim 1, wherein the diameter of the particles is between 50 μm and 5 mm. The method of claim 1, wherein the particles are magnetic. The method of claim 1, wherein the substrate has a concavo-convex pattern or a magnetic pattern or both for placing or arranging the particles. The method according to claim 4 or 5, wherein the immobilization of the particles on the substrate is by adsorption of the magnetic particles by a magnetic pattern on the substrate. The method according to claim 1, wherein the fixing of the particles is performed by fitting or entering the particles into recesses of a concavo-convex pattern on the substrate. The method according to claim 1, wherein the pattern is a pattern that is periodically arranged or arranged two-dimensionally. The method according to claim 1, wherein the substrate is covered with a semi-permeable membrane or a porous membrane from above after the particles are introduced into the concave portions of the substrate, and the lid is covered.

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