JP4288982B2 - Surface for detecting intermolecular interactions - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a detection surface for detecting hybridization and other intermolecular interactions. More specifically, the metal thin film portion to which the detection molecule is fixed is formed at an interval, and the detection molecule is fixed at an appropriate density, thereby causing steric hindrance during the intermolecular interaction. The present invention relates to an intermolecular interaction detection surface devised to prevent a reduction in reaction efficiency.
[0002]
[Prior art]
Currently, integrated substrates for bioassays called DNA chips or DNA microarrays (hereinafter collectively referred to as “DNA chips”), in which predetermined DNA is finely arranged by microarray technology, are used for gene mutation analysis, SNPs (single nucleotides). Polymorphism) analysis, gene expression frequency analysis, etc., and has begun to be widely used in drug discovery, clinical diagnosis, pharmacogenomics, forensic medicine and other fields.
[0003]
Since this DNA chip has a large number of DNA oligo chains and cDNA (complementary DNA) integrated on a glass substrate or silicon substrate, comprehensive analysis of intermolecular interactions such as hybridization becomes possible. It is characterized by dots.
[0004]
Here, Patent Document 1 discloses a solid phase immobilization in which avidin molecules are fixed to a single layer only on a specific site where probe biomolecules on the surface of a substrate including a metal substrate are to be attached using photolithography technology and etching technology. A surface-treated substrate having a film formed thereon is disclosed.
[0005]
Further, in Patent Document 2, the probe used for detecting the inspection target substance is captured in advance in fine particles, the fine particles are fixed to a portion partitioned in a lattice pattern on the surface of the substrate, and the probe is captured in each partition. A technique for finely fixing fine particles in a single layer is disclosed.
Further, as shown in FIG. 7 schematically showing the attached prior art, a metal thin film 101 such as gold is formed on the entire surface of the substrate 100 made of synthetic resin, etc. Generally, a detection molecule to be chemically bound is immobilized.
[0006]
[Patent Document 1]
JP 2002-153272 A [Patent Document 2]
Japanese Patent Laid-Open No. 2002-253232
[Problems to be solved by the invention]
However, in the above-described conventional DNA chip technology, a nucleotide chain for detection such as a DNA probe is immobilized at a detection surface site (spot site) by being tangled or rounded in a random coil shape, and has a high integration density. Thus, steric hindrance occurs during hybridization with the target molecule or other interaction.
[0008]
For this reason, there is a problem that the efficiency of the interaction is poor, the reaction takes a long time, and there is a possibility of showing false positive or false negative. In particular, in the technique of immobilizing detection molecules on the entire detection surface (see FIG. 7), the above problem is significant because of the high integration density of the detection molecules.
[0009]
Therefore, the present invention forms a metal thin film portion on which the detection molecules are fixed at intervals on the detection surface, and fixes the detection molecules at an appropriate density, thereby preventing the interaction between molecules. The main purpose is to prevent a reduction in reaction efficiency due to steric hindrance.
[0010]
[Means for Solving the Problems]
In order to solve the above technical problem, in the present application, first, an intermolecular interaction between a detection molecule having a mercapto group (—SH) or a disulfide group (—S—S—) and a target molecule. A detection surface on a substrate facing a reaction region providing a field, wherein a metal thin film portion is provided on the substrate at an interval, and the molecule for detection is connected to the metal thin film portion via the mercapto group or disulfide group. Provided are an intermolecular interaction detection surface devised so as to immobilize and an intermolecular interaction detection substrate provided with the detection surface.
[0011]
That is, the intermolecular interaction detection surface according to the present invention does not employ a configuration in which the detection molecule is immobilized on the entire detection surface, but is a point like an island on the detection surface. As shown, the detection molecules are devised so as to be immobilized only on (the surface of) the thin metal film portion formed at intervals.
[0012]
As a result, since the density of detection molecules (immobilized) on the detection surface can be suppressed to a low level, it is added later to detection molecules that are entangled or rounded in a random coil shape. Alternatively, a reaction field environment in which a target molecule to be sent is easily accessible is formed. That is, since an environment in which the influence of steric hindrance is small is formed in the reaction between molecules, hybridization and other interaction between the detection molecule and the target molecule proceed efficiently.
[0013]
The intermolecular interaction detection surface of the present invention is fixed on the detection surface by adjusting the area per metal thin film portion formed on the detection surface and the number of metal thin film portions formed per unit area of the detection surface. The integration density of chemical detection molecules can be freely adjusted.
[0014]
What is necessary is just to form the said metal thin film part in a thin film form with the metal material which reacts with a mercapto group (-SH) or a disulfide group (-S-S-), for example, can be formed from a gold thin film. A metal such as gold has a function of reacting with a mercapto group (—SH) or a disulfide group (—S—S—) to form a so-called mercaptide and chemisorbing and immobilizing a molecule for detection.
[0015]
The metal thin film part is formed on the detection surface of the intermolecular interaction detection surface including at least a step in which the metal thin film part is scattered in an island-like manner at an interval by using any method of vapor deposition or sputtering. It can be formed by a manufacturing method. That is, the metal thin film portion is formed as a metal vapor deposition film or a metal sputter film scattered in an island shape by the manufacturing method. In addition, the board | substrate with which this metal thin film part is formed can also be formed with a stearic acid single crystal board | substrate other organic compound crystal board | substrates which are not couple | bonded with a mercapto group (-SH) or a disulfide group (-S-S-). .
[0016]
The metal thin film portion made of a metal vapor deposition film may be formed by appropriately selecting various known vapor deposition techniques. For example, vapor deposition using an epitaxy phenomenon can also be suitably employed. By using the epitaxy phenomenon, a metal thin film portion (epitaxial film) arranged approximately regularly on the detection surface can be formed. Therefore, there is an advantage that the molecules for detection can be fixed uniformly (regularly) on the detection surface. is there.
[0017]
Here, the main technical terms in the present application are defined. First, “molecular interaction detection surface” in the present application means a surface region on a substrate having a function capable of immobilizing detection molecules.
[0018]
The “detection molecule” is a probe molecule containing a DNA probe or other nucleotide chain, peptide, protein, lipid, etc., immobilized on the detection surface. The nucleotide chain means a polymer of a phosphate ester of a nucleoside in which a purine or pyrimidine base and a sugar are glycosidically bonded, and an oligonucleotide, a polynucleotide containing a DNA probe, a DNA in which a purine nucleotide and a pyrimidine nucleotide are polymerized. (Full length or a fragment thereof), cDNA obtained by reverse transcription (cDNA probe), RNA, polyamide nucleotide derivative (PNA) and the like are widely included.
[0019]
The “target molecule” is a molecule having a property of specifically reacting with the detection molecule. For example, a nucleotide chain having a base sequence complementary to the detection nucleotide chain, and in some cases, labeled with a fluorescent substance or the like.
[0020]
“Intermolecular interaction” refers to hybridization, antigen-antibody reaction, enzyme response reaction and other intermolecular (polymer), which are complementary strand (duplex) formation reactions between nucleotide strands having complementary base sequence structures. , Regardless of the chemical reaction format.
[0021]
The “reaction region” is a sample solution storage region that can mainly provide a field of hybridization or other intermolecular interaction in the liquid phase, and is a space formed on the detection surface formed on the substrate or It is an area.
[0022]
“Steric hindrance” makes it difficult to access the reaction partner molecule due to the presence of bulky substituents near the reaction center in the molecule, the posture of the reaction molecule, and the three-dimensional structure (higher order structure). This means a phenomenon in which a desired intermolecular interaction is less likely to occur.
[0023]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, preferred embodiments of an intermolecular interaction detection surface (hereinafter simply referred to as “detection surface”) according to the present invention will be described with reference to the accompanying drawings. First, FIG. 1 is a perspective view showing a configuration of a detection surface according to the present invention, and FIG. 2 is a sectional view of the detection surface portion. The detection surface denoted by reference numeral 1 is formed on a substrate 2 made of a material such as glass, quartz, or silicon, and constitutes a part of a detection unit for intermolecular interaction.
[0024]
Metal thin film portions 11 made of gold or the like are formed on the detection surface 1 so as to be scattered like islands, and a reaction field for intermolecular interaction is provided above the detection surface 1. Reaction region R is provided (see FIG. 2).
[0025]
The method for forming the metal thin film portion 11 on the detection surface 1 is not particularly limited in the present invention. That is, any method that can form the metal thin film portion 11 so as to be scattered like islands instead of the entire detection surface 1 can be used as appropriate.
[0026]
For example, after a mercapto group or disulfide group pattern is formed on the substrate 1 in advance, metal nanoparticles such as gold and microparticles can be chemically adsorbed on the pattern.
[0027]
Further, as shown in FIG. 3A, a metal X is laminated on the entire detection surface 1 by epitaxy vapor deposition or other vapor deposition or sputtering, and then the metal X is partially deposited by a known etching technique. The metal thin film portions 11 can be distributed like islands on the detection surface 1 (see FIG. 3B). In forming the metal thin film portion 11, the amount of vapor deposition or sputtering may be adjusted, or the substrate 1 may be cooled. A substrate 2 having poor wettability may be used.
[0028]
Here, the detection surface 1 having the above-described configuration in which the metal thin film portions 11 are distributed in an island-like manner is a single-stranded detection nucleotide molecule D ₁ or disulfide having a mercapto group, schematically illustrated in FIG. The single-stranded detection nucleotide molecule D ₂ having a group can be selectively chemisorbed and immobilized on the surface of the metal thin film portion 11. This fixed state is schematically shown in FIG. In FIG. 5B, the single-stranded target nucleotide molecule T is hybridized with the single-stranded detection nucleotide molecule D ₁ (or D ₂ ) immobilized on the metal thin film portion 11. Thus, a state in which a double strand (complementary strand) is formed is schematically shown.
[0029]
Further, the double-stranded nucleotide molecule D ₃ for detection having a mercapto group or the double-stranded nucleotide molecule for detection D ₄ having a disulfide group schematically shown in FIG. It can be selectively chemisorbed on the surface and immobilized. This fixed state is schematically shown in FIG. The detection nucleotide molecules D ₄ duplexes, and single-stranded denatured even better, or after immobilization using as a detection molecule such as an enzyme response reaction and used as a double-stranded after immobilization Then, it may be used as a detection molecule for detecting a hybridization reaction or the like.
[0030]
The detection molecule D is not limited to a single-stranded or double-stranded nucleotide molecule (D _{1 to} D ₄ ), and is a molecule having a mercapto group or a disulfide group that is chemically adsorbed to the metal thin film portion 11. If it exists, it can be selected regardless of whether it is a polymer or a low molecule.
[0031]
As shown in FIG. 5 and FIG. 6, the detection surface 1 according to the present invention is formed so that the detection molecules D (D _{1 to} D ₄ ) are scattered like islands on the detection surface 1. As a result of being immobilized only on the metal thin film portion 11, the integration density of the detection molecules D on the detection surface 1 is lower than when the detection molecules D are immobilized on the entire detection surface 1.
[0032]
For this reason, the influence of the steric hindrance when the target molecule T added or sent later to the reaction region R approaches the immobilized detection molecule D is small. As a result, the efficiency of hybridization and other intermolecular interaction can be improved. That is, the reaction time can be shortened.
[0033]
When the detection molecule D immobilized on the metal thin film portion 11 of the detection surface 1 is a nucleotide chain having a phosphate ion and negatively charged, a counter electrode (not shown) is arranged in the reaction region R. In addition, a configuration in which a high frequency voltage is applied thereto to form a uniform electric field (an electric field where electric lines of force are not concentrated in part) in the reaction region R between the counter electrodes can be employed. Since the detection molecules D can be elongated by the action of the electric field, it is possible to further eliminate the influence of steric hindrance in addition to the configuration in which the integration density is lowered.
[0034]
The condition of the electric field of the counter electrode is preferably about 1 × 10 ⁶ V / m and about 1 MHz (Masao Washizu and Osamu Kurosawa: “Electrostatic Manipulation of DNA in Microfabricated Structures”, IEEE Transaction on Industrial Application Vol.26, No.26, p.1165-1172 (1990)).
[0035]
The detection surface 1 having the above-described configuration can be provided on a substrate 2 of various sensor chips for detecting a DNA chip (microarray) or an interaction between molecules. It should be noted that the detection of whether or not there is an interaction between the detection molecule D immobilized on the detection surface 1 and the target molecule T is a well-known conventional method such as an optical method, a crystal oscillator principle, or a surface plasmon resonance principle. This method may be performed. A fluorescent intercalator such as POPO-1 or TOTO-3 that specifically binds between the bases of labeled fluorescent dyes or double-stranded nucleotides is irradiated with excitation light, and the resulting fluorescence is detected using a conventional detector. You can also.
[0036]
For example, the reaction region R is excited by irradiating laser light (for example, blue laser light), the magnitude of the fluorescence intensity is detected by a detector (not shown), and the detection molecule D and the target molecule T Determine the state of hybridization between. Finally, the fluorescence intensity for each reaction region R is A / D converted, and the binding reaction ratio can be visualized by being distributed on a computer screen.
[0037]
【The invention's effect】
According to the present invention, the integration density of detection molecules immobilized on the detection surface of the substrate can be kept low, resulting in hybridization between the detection molecule and the target molecule in the reaction region on the detection surface, and the like. The intermolecular interaction of can be made highly efficient.
[0038]
Control of the integration density of detection molecules on the detection surface can be achieved by shortening the reaction time, but control by reaction time requires strict control of reaction temperature and reaction environment. For this reason, it is easier to control the interaction between the detection surfaces on which the detection molecules are immobilized. In the present invention, since the distribution of detection molecules can be controlled by the area and formation density of the island-like pattern of the metal thin film portion on the detection surface, the detection molecules can be immobilized on the detection surface at an arbitrary integration density. .
[0039]
The present invention can reliably achieve improvement in efficiency of hybridization and other intermolecular interaction, shortening of reaction time, prevention of false positive or false negative on the detection surface of a sensor chip such as a DNA chip.
[0040]
The present invention efficiently carries out detection of intermolecular interactions including detection of hybridization and antigen-antibody reaction, which are essential in gene mutation analysis, SNPs (single nucleotide polymorphism) analysis, gene expression frequency analysis, etc. Therefore, it has the technical significance of providing it to drug discovery, clinical diagnosis, pharmacogenomics, forensic medicine and other related industries.
[Brief description of the drawings]
FIG. 1 is a perspective view showing a configuration of a detection surface (1) according to the present invention. FIG. 2 is a sectional view of the detection surface (1). FIG. 3 (A) Metal X is deposited on the entire detection surface 1. FIG. 4B is a view showing a state where the metal X is sputtered and laminated. FIG. 4A is a view showing a state after the metal X is partially peeled off by a known etching technique. FIG. 4A is for detecting a single strand having a mercapto group. duplexes with two detecting nucleotide molecule D ₃ or a disulfide group of chains having a schematically shown FIG. (B) a mercapto group of detecting nucleotide molecule D ₂ of the single strand having a nucleotide molecule D ₁ or disulfide group FIG. 5 is a diagram schematically showing a detection nucleotide molecule D ₄ of FIG. 5A. (A) A single-stranded detection nucleotide molecule (D ₁ ) is formed to be scattered like islands on the detection surface (1). Fixed to the thin metal film part (11) Against diagram showing how (B) the detecting nucleotide molecule single-stranded immobilized (D ₁ or D _2), the target nucleotide molecule T of the single-stranded by hybridization duplexes (complementary The figure which shows a mode that the chain | strand) is formed.
FIG. 6 shows a state in which double-stranded detection nucleotide molecules (D ₂ ) are immobilized on metal thin film portions (11) formed to be island-like scattered on detection surface (1). FIG. 7 is a diagram (cross-sectional view) schematically showing a configuration of a conventional detection surface.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Detection surface 2 Substrate 11 Metal thin film part D Molecule for detection R Reaction area T Target molecule

Claims (6)

Detection surface formed on a substrate facing a reaction region that provides a field for intermolecular interaction between a detection molecule having a mercapto group (-SH) or a disulfide group (-SS-) and a target molecule Because
The detection surface is formed of an organic compound film that does not bind to a mercapto group (—SH) or a disulfide group (—S—S—),
A metal thin film portion formed of a gold thin film is provided on the detection surface at an interval at which steric hindrance in the intermolecular interaction does not occur , and the detection is performed on the metal thin film portion via the mercapto group or disulfide group. Intermolecular interaction detection surface on which molecules are immobilized.   The intermolecular interaction detection surface according to claim 1, wherein the metal thin film portion is formed of a metal vapor deposition film or a metal sputter film. The intermolecular interaction detection surface according to claim 1 or 2, wherein the detection molecule is a single-stranded or double-stranded nucleotide molecule. The intermolecular interaction detection surface according to any one of claims 1 to 3, wherein the intermolecular interaction is hybridization. An intermolecular interaction detection substrate comprising at least the intermolecular interaction detection surface according to any one of claims 1 to 4 . A detection surface formed on a substrate facing a reaction region that provides a field of intermolecular interaction between a detection molecule and a target molecule is a mercapto group (-SH) or a disulfide group (-S-S-). Formed from an organic compound film that does not bond with
Using either deposition or sputtering techniques, metal thin film portions formed from a gold thin film are scattered on the detection surface in an island-like manner at intervals that do not cause steric hindrance in the intermolecular interaction. A method for producing a surface for detecting an interaction between molecules, which comprises at least a step of causing the reaction to occur.

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