JP4374230B2 - Biopolymer detection method and biochip - Google Patents

Description

  The present invention relates to a method for detecting biopolymers such as DNA and RNA (RNA is a transcription product from DNA, ie, mRNA, rRNA, tRNA, or small RNA), and a protein, and a biochip used therefor. is there.

  Conventionally, a technique for decoding a biopolymer (hereinafter, DNA is taken as an example) using a microarray chip is well known (see, for example, Patent Document 1). And this kind of DNA microarray chip is usually formed as follows, and can decode DNA.

  On a glass (or plastic) substrate, probe DNA having a sequence complementary to the target mRNA (cDNA) is spotted and fixed in an array. On top of that, a target mRNA (cDNA) with a fluorescent label is dropped. At this time, the probe and target of complementary sequences hybridize and bind, but those that do not bind do not.

  After the hybridization has progressed sufficiently, the substrate is washed with a washing buffer solution, and the unhybridized target is washed away. Next, the presence and amount of mRNA (cDNA) as a target can be measured by optically reading the position and light amount of the fluorescent label with a reader (see, for example, Patent Document 2).

Japanese Unexamined Patent Publication No. 2000-131237 (second page, FIG. 1-3) Japanese Unexamined Patent Publication No. 2000-235035 (second page, FIG. 7-9)

However, the conventional DNA microarray can obtain the target data by the series of protocols as described above, but actually has various problems on the protocol at each stage. As a result, the obtained data has many problems such as certainty, reproducibility, repetitive characteristics, sensitivity, etc. Therefore, standardization of experimental data does not progress, and in addition to the problems in terms of content, it is a clinical site. However, DNA microarrays have not yet become widespread.
Among the various problems, items that are particularly problematic are S / N ratio, detection sensitivity, detection time, accuracy, and reproducibility.

  The present invention solves the above-mentioned problems, and provides a biopolymer detection method using an antigen-antibody reaction that improves the S / N ratio, improves the detection sensitivity, and shortens the detection time, and the biochip used in the method It aims at realizing.

In order to achieve such a problem, in the present invention,
A biopolymer detection method for detecting a target biopolymer by capturing the target biopolymer on the substrate side,
A target biopolymer labeled with fluorescence and a bead having a probe biopolymer and a polyclonal antibody for addressing ID recognition of beads or an address probe peptide or a biopolymer address linker immobilized on a surface are placed in a solution, and the target biopolymer After hybridizing the polymer and the probe biopolymer, the address linker is antigen-antibody reaction with the address probe peptide or biopolymer or polyclonal antibody that has an antigen / antibody relationship with the address linker immobilized on the substrate. It is characterized by capturing.

Since beads are used in the present invention, the surface area of the beads is much larger than the surface area of conventional DNA chips, and many probe biopolymers can be immobilized on the surface of the beads. Opportunities between the probe biopolymer and the target biopolymer in the solution are greatly increased, and the target biopolymer can be captured with extremely high sensitivity. Generally, the sensitivity is about 1000 times or more that of a DNA array.
An address linker for ID recognition is further fixed to the beads. As the address linker, a polyclonal antibody, an address probe peptide, or a biopolymer is used.

On the other hand, the site on the substrate contains an address probe peptide, biopolymer, or polyclonal antibody that has a relationship between the address linker and the antigen / antibody (having a much stronger binding force than DNA-DNA hybridization). It is fixed. When the beads are poured onto the substrate, the address linker is captured with a strong binding force to the address probe peptide, the biopolymer or the polyclonal antibody by the antigen-antibody reaction.
In this way, the target biopolymer can be captured on the substrate with a high S / N ratio and high detection sensitivity.

  In this case, as in claim 2, when the target biopolymer and beads are placed in a container together with a buffer solution and stirred by physical, electrical, or chemical means, the conventional method simply uses a Brownian motion to provide a complementary probe. Compared to searching, the present invention searches for complementary probes by obtaining energy from other sources, which greatly increases the chance of hesitation, resulting in faster hybridization between the probe biopolymer and the target biopolymer. can do.

  As the beads, magnetic beads as described in claim 3 or beads using metal or plastic are used. The target biopolymer is RNA (mRNA or rRNA or tRNA or small RNA), cDNA, or protein that is a transcription product from DNA as in claim 4.

As is apparent from the above description, the present invention has the following effects.
(1) Since the beads have a large surface area, they can bind a large amount of probe DNA. Therefore, a very small amount of target biopolymer in the solution can be easily captured with extremely high sensitivity (sensitivity about 1000 times that of a general DNA array). Can do.
(2) Since the target DNA can be hybridized and bound to many probe DNAs bound to one bead, the S / N ratio can be easily increased.

(3) Since many probe DNAs are bound to one bead and the solution is stirred, the chance of wrinkling between the target DNA and the probe DNA increases, and the detection time (mainly the time required for hybridization) The target DNA and the probe DNA can be hybridized with extremely high sensitivity.

  In the present invention, the advantages of beads and the advantages of DNA arrays are combined. The good thing about beads is that they have a large surface area that allows you to bind a lot of probe DNA (much more than binding probe DNA to a flat site) and move freely through the solution. As a result, the chance to clash with the target biopolymer in the solution is dramatically improved, and as a result, a very small amount of the target DNA in the solution can be captured with extremely high sensitivity (generally about 1000 times the DNA array That is the point.

  However, on the other hand, there is a drawback that it is not known which DNA is bound to which bead ID, that is, which bead. In order to recognize the ID of this bead, various attempts have been made such as using color beads or identifying with two color light sources, but the problem is that the number that can be identified is small, and the device is complicated and expensive. There is a problem that the size, size, and handling become difficult. In the present invention, the problem can be solved well by distinguishing the protein on the beads and the array by the antigen-antibody reaction.

Hereinafter, the present invention will be described in detail with reference to the drawings. 1 to 3 are diagrams for explaining the principle of an example of the biopolymer detection method according to the present invention. Here, the case where the biopolymer is DNA will be described.
1 to 3 are diagrams for explaining the principle of an example of the biopolymer detection method according to the present invention. Here, the case where the biopolymer is DNA will be described.

As shown in FIG. 1, probe DNA 2 is immobilized on the surface of beads 1. As the beads, magnetic beads, beads using metal or plastic, and the like can be used.
In addition, an address linker 3 for recognizing bead identification information (ID) is fixed on the surface of the bead 1. As the address linker 3, an antigen for address determination or an antibody for address determination (monoclonal or polyclonal) is used.
On the other hand, a fluorescent tag 5 is added (labeled) to the target RNA, cDNA or protein (hereinafter referred to as RNA) 4.

  The beads 1, the target RNA 4, and the buffer solution 6 prepared as described above are put together in a container 7, and stirred by physical, electrical, or chemical means as necessary. As a result, the target DNA 4 in a complementary relationship with the probe DNA 2 on the surface of the bead 1 is bound.

Next, the bound beads are poured onto the arrayed sites 11 of the biochip 10 shown in FIG. 2A is a side view, and FIG. 2B is a plan view.
On the site 11, an address probe protein 12 that captures the ID recognition address linker 3 immobilized on the surface of the bead 1 by an antigen-antibody reaction is immobilized in advance. 3 is an enlarged view of a circled portion A in FIG.

The address linker 3 and the address probe protein 12 are bound by an antigen / antibody reaction. The fluorescent label 5 can recognize which probe site 11 the bead 1 is bound to the address probe protein 12. The fluorescent label can be easily detected by a fluorescent reader (not shown).
In this way, the presence and amount of the target RNA 4 can be measured efficiently.

  The above description merely shows a specific preferred embodiment for the purpose of explaining and illustrating the present invention. Therefore, the present invention is not limited to the above-described embodiments, and includes many changes and modifications without departing from the essence thereof.

  For example, as an address linker, an address probe peptide or a biopolymer can be used in addition to a polyclonal antibody. In this case, either an address probe peptide, a biopolymer or a polyclonal antibody having an antigen / antibody relationship with the address linker is immobilized on the substrate correspondingly.

It is a figure explaining the principle of an example of the biopolymer detection method which concerns on this invention. It is another explanatory drawing explaining the principle of an example of the biopolymer detection method which concerns on this invention. It is another explanatory drawing explaining the principle of an example of the biopolymer detection method which concerns on this invention.

Explanation of symbols

1 Bead 2 Probe protein 3 Address linker 4 Target RNA
5 Fluorescent tag 6 Solution 7 Container 10 Substrate 11 Site 12 Address probe protein

Claims (4)

A biopolymer detection method for detecting a target biopolymer by capturing the target biopolymer on the substrate side,
A target biopolymer labeled with fluorescence and a probe biopolymer and a bead having a polyclonal antibody for address ID recognition of beads or an address probe peptide or a biopolymer address linker immobilized on the surface are placed in a solution, and the target biopolymer After hybridizing the polymer and the probe biopolymer, the address linker is antigen-antibody reaction with the address probe peptide or biopolymer or polyclonal antibody that has an antigen / antibody relationship with the address linker immobilized on the substrate. The biopolymer detection method characterized by capturing by this.   The biopolymer detection method according to claim 1, wherein the target biopolymer and beads are placed in a container together with a buffer solution and stirred by physical, electrical, or chemical means.   3. The biopolymer detection method according to claim 1, wherein magnetic beads or beads using metal or plastic are used as the beads.   The biopolymer detection method according to any one of claims 1 to 3, wherein the target biopolymer is RNA, cDNA, or protein that is a transcription product from DNA.

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