JP2009058427A - Method and device for identifying surface molecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of selectively identifying molecules (object molecules) existing on a matter surface and can identify a plurality of kinds of object molecules and its positional information with high resolution. <P>SOLUTION: This method of identifying the object molecules existing on the sample surface comprises (A) a step of preparing two or more kinds of particulate groups of different physical characteristics where each particulate is decorated by a molecule specifically binding to different molecule; (B) a step of bringing the particulate into contact with the sample surface, binding the particulate of the particulate group to the object molecule, and fixing it to the sample surface; and (C) a step of scanning the sample surface to which the particulate is fixed using a scanning probe microscope and acquiring information capable of identifying each object molecule. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for identifying molecules present on the surface of a substance and a device for identifying molecules present on the surface of a substance.

The technology for identifying molecules existing on the surface of a substance (hereinafter referred to as “target molecules”) is an important technology for evaluating chemically modified substrates (for example, bichips) and observing the state of cell membrane surfaces. is there. Conventional identification techniques can be broadly classified into the following three.
[1] A method in which a fluorescent molecule is bound to a target molecule, and fluorescence is measured and identified. In this method, target molecules existing on the cell membrane surface can be identified (see Non-Patent Document 1). Moreover, a plurality of types of target molecules can be identified by using fluorescent molecules having different wavelengths (see Non-Patent Document 2).
[2] A method in which fine particles (having a particle size of about 100 nm) are bound to target molecules and identified with an optical microscope. In this method, fine particles modified with a molecule that specifically binds to the target molecule can be bound to the target molecule, the target molecule can be identified, and the movement of the target molecule can be analyzed (see Non-Patent Document 3). .
[3] A method of identifying a target molecule by scanning the surface of a substance with a chemically modified probe using a scanning probe microscope such as an atomic force microscope (AFM). In this method, the sharpened probe is chemically modified with a molecule that specifically interacts with the target molecule, and the target molecule can be identified with a scanning probe microscope. This method has an advantage of high spatial resolution (see Patent Document 1 and Non-Patent Document 4).
International Publication No. 2004/092712 Pamphlet Yu Ohsugi, Kenta Saito, Mamoru Tamura and Masataka Kinjo “Lateral Mobility of Membrane-Binding Proteins in Living Cells Measured by Total Internal Reflection Fluorescence Correlation Spectroscopy”, Biophysical Journal 91, 3456-3464 (2006). Schwille, P., FJ Meyer-Almes, and R. Rigler. 1997. Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophys. J. 72: 1878-1886. A. Kusumi, Y. Sako, and M. Yamamoto. “Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovideo microscopy). Effects of calcium-induced differentiation in cultured epithelial cells.” Biophys. J. 65, 2021 -2040 (1993). Peter Hinterdorfer and Yves F Dufrene, “Detection and localization of single molecular recognition events using atomic force microscopy”, Nature Methods 3, 347-355 (2006).

  However, in the above-described method using the fluorescent molecule [1], since the fluorescent molecule may bind to a molecule existing inside or outside the cell in addition to the cell surface molecule, only the target molecule on the cell surface is detected. Is not easy. Moreover, since an optical microscope is used, the resolution is limited. In the method of identifying using the optical microscope of [2], the resolution is limited due to the diffraction limit of light as in the method of [1]. It is also difficult to identify multiple types of target molecules. In the method using the chemically modified probe of [3], since one kind of molecule is modified to one probe, it is not easy to simultaneously identify many kinds of target molecules.

  Therefore, the present inventor can selectively identify only molecules (target molecules) existing on the surface of a substance with high resolution, and can identify multiple types of target molecules with high resolution including positional information. We considered providing a possible method.

The first of the present invention relates to the following identification method.
[1] A method for identifying target molecules present on the surface of a sample, wherein A) two or more types of fine particle groups having different physical properties, and each of the two or more types of fine particle groups is a different target. Providing a group of microparticles modified with a molecule that specifically binds to the molecule; B) contacting the microparticle with the sample surface to bind to the target molecule and immobilizing on the sample surface; and C) Scanning a sample surface on which microparticles are fixed using a scanning probe microscope to obtain information capable of identifying each of the target molecules.
[2] The method according to [1], wherein the particle size of the particles included in the fine particle group is in the range of 1 nm to 10 μm.
[3] The method according to [1] or [2], wherein the molecule that specifically binds to the molecule is a nucleic acid, peptide, glycopeptide, polysaccharide, or lipid.
[4] The method according to any one of [1] to [3], wherein the physical property of the fine particles is a particle size, and the scanning probe microscope is an atomic force microscope (AFM).
[5] The physical properties of the fine particles are the particle size and the optical properties of the fine particles, and the scanning probe microscope is a near-field optical / atomic force microscope (SNOAM), any of [1] to [4] The method of crab.
[6] Any one of [1] to [5], wherein the change in the position of the target molecule is observed by repeating step C) twice or more for the same part of the sample surface to which the fine particles are bound. The method described in 1.
[7] The method according to any one of [1] to [6], wherein the sample is a substrate on which a target molecule is fixed.
[8] The method according to any one of [1] to [7], wherein the sample is a biochip including a substrate on which DNA or protein is immobilized.
[9] The method according to any one of [1] to [8], wherein the sample is a biological material.
[10] The method according to any one of [1] to [9], wherein the sample is a cell, and the target molecule is a membrane protein, membrane glycoprotein, or lipid.
[11] The method according to [10], wherein the cells are arranged on a substrate.

The second of the present invention relates to the identification device shown below.
[12] An identification device for identifying a target molecule existing on a sample surface, a fine particle providing member for providing fine particles to the sample surface; measuring physical properties of the fine particles fixed on the sample surface; An apparatus comprising: a measuring member that obtains identifiable information; and an output member that outputs previous period information.

  According to the present invention, the position and type of a plurality of types of molecules present on the surface of a substance can be identified with high resolution. Further, according to the present invention, it is possible to observe the behavior over time of a plurality of types of molecules present on the surface of a substance.

1. About the identification method of the surface molecule by this invention By the identification method of this invention, the target molecule which exists in the sample surface can be identified. The sample is not particularly limited as long as it is a substance on which the target molecule to be identified exists.

  The sample is, for example, a solid substrate on which target molecules are fixed. Examples of such fixed substrates include biochips such as DNA chips and protein chips. A DNA chip is a substrate on which oligonucleotides are immobilized on the surface, and a protein chip is a substrate on which proteins are immobilized on the surface. When a DNA chip is used as a sample, an oligonucleotide immobilized on the substrate is a target molecule, and when a protein chip is used as a sample, a protein immobilized on the substrate is a target molecule. The material of the solid substrate is generally a plate shape, a bead shape, or a film shape made of a material such as plastic, glass, or metal, and is usually a plate shape. By using a DNA chip or a protein chip as the sample of the present invention, the quality of these chips can be checked.

  Another example of a sample is a cell. The cell surface is formed by a cell membrane composed mainly of phospholipids. In addition to phospholipids, membrane proteins, membrane glycoproteins, etc. exist in the cell membrane. By the identification method of the present invention, a membrane protein or the like present on the cell membrane surface can be identified as a target molecule.

  Identifying a target molecule means identifying the type of the target molecule; observing the location of the target molecule and its change over time.

  According to the identification method of the present invention, A) two or more kinds of fine particle groups having different physical characteristics, each fine particle of the two or more kinds of fine particle groups is modified with a molecule that specifically binds to a different target molecule. Preparing a group of fine particles; B) bringing the fine particles into contact with the sample surface to bind to the target molecules and immobilizing them on the sample surface; and C) scanning the sample surface with the fine particles immobilized thereon Scanning with a probe microscope to obtain information capable of identifying each target molecule.

  In step A), two or more types of fine particle groups having different physical properties are prepared. Examples of physical properties include the size (particle size) of fine particles, and a combination of particle size and optical properties.

  The fine particles of two or more types of fine particle groups having different physical properties are modified differently for each type. In other words, each type of fine particle is modified with a molecule that specifically binds to the target molecule (hereinafter referred to as “binding molecule”), but the type of the binding molecule is different, and each type is specific to another target molecule. Can be combined. This makes it possible to simultaneously identify two or more types of target molecules.

The material of the fine particles contained in the fine particle group is, for example, metal, resin, ceramic, etc., but is not particularly limited as long as it can be measured with a scanning probe microscope. The particle diameter of the fine particles may be a size within the maximum movable range of the probe from the resolution of the scanning probe microscope. For example, the particle diameter of the fine particles is 1 nm to 10 μm.
The binding molecule is not particularly limited as long as it specifically binds to the target molecule. Examples of binding molecules include nucleic acids, peptides, glycopeptides, polysaccharides, lipids and the like. Here, the modification refers to fixing a binding molecule on the surface of the fine particles. For example, when the material of the fine particles is gold, the binding molecules can be fixed by adding a thiol group to the binding molecules to form a mercaptide bond with a gold atom on the surface of the fine particles. In addition, the binding molecule can be immobilized on the surface of the fine particle by coating the fine particle with a resin containing the binding molecule, or 2) utilizing the strong bond between avidin and biotin.

  In step B), the fine particles are brought into contact with the sample surface, and any of the fine particles is bound to the target molecule and fixed on the sample surface.

  The method of bringing the fine particles into contact with the sample surface may be performed by dropping a solution in which the fine particles are dispersed into the sample, or adding or immersing the sample in the solution. When the sample is immersed in a solvent, a solution in which fine particles are dispersed may be added to the solvent in which the sample is immersed. The solvent of the solution in which the fine particles are dispersed is not particularly limited as long as the binding molecule and the target molecule can exist stably. The solvent is, for example, water. The solution in which the fine particles are dispersed may contain a salt such as sodium chloride, potassium chloride, ammonium chloride, sodium acetate, potassium acetate, or ammonium acetate, or the solution may be a buffer solution.

  When the sample is a solid substrate such as a DNA chip or a protein chip, it is preferable to drop a solution in which fine particles are dispersed on the substrate. If cells are used for the sample, it is preferable to place the cells on the substrate in the culture medium prior to step B). Placing the cells on the substrate includes adhering the cells to the substrate, confining the cells in a hole on the substrate, and preventing the cells from moving. For example, cells can be adhered onto the substrate by seeding cells suspended in a culture solution containing serum on the substrate and culturing the cells for several hours. When using HeLa cells or fibroblasts as a sample, for example, DMEM containing 10% FBS can be used for the culture solution. Further, the substrate on which the cells are arranged is preferably immersed in a solvent. This is because cultured cells are damaged by the surface tension when they are directly exposed to the atmosphere.

  By bringing the microparticles into contact with the sample surface, the binding molecules that modify the microparticles bind to target molecules that exist on the sample surface according to the type. As a result, the fine particles are fixed on the sample surface. Here, the term “bond” refers to forming a complex by any physical adsorption or chemical bond including intermolecular electrostatic bond, hydrophobic bond, ionic bond, and hydrogen bond. Examples of binding include binding of an antigen and an antibody.

  The two or more types of microparticles prepared in step A) may be fixed on the sample surface step by step for each type in step B), or all types of microparticles may be fixed on the sample surface at the same time. Good.

  In step C), the sample surface on which the microparticles are fixed is scanned using a scanning probe microscope to obtain information that can identify the target molecule. That is, the tip of the probe of the scanning probe microscope may be scanned on the sample surface on which the fine particles are fixed.

  Examples of the scanning probe microscope include an atomic force microscope, a near-field optical / atomic force microscope in which an atomic force microscope and a near-field optical microscope are combined, and the like. When the physical property of the fine particles is the particle size, it is preferable to use an atomic force microscope as the scanning probe microscope. An atomic force microscope observes the surface shape of a detection target using a force acting between the detection target and a probe. That is, the surface shape of the detection target is observed by measuring the deflection amount of the cantilever caused by the force acting between the sample and the probe. In the present invention, the measurement mode of the atomic force microscope is preferably a static imaging method (for example, contact mode) or a dynamic imaging method (for example, an amplitude modulation method or a frequency modulation method). When the probe is scanned on the sample surface, the cantilever bends due to repulsive force or attractive force between the probe and the sample. Scanning information can be obtained by acquiring the deflection amount as an electrical signal.

  By scanning the surface of the sample on which the fine particles are fixed with an atomic force microscope, the presence of the fine particles fixed on the sample surface can be confirmed, and the particle size of the fine particles can be confirmed. The presence of the target molecule is confirmed by the presence of the fine particle, and the position of the target molecule on the sample surface is specified by the position of the existing fine particle. And the kind of object molecule | numerator is specified by the particle size of the microparticles | fine-particles which exist.

  Further, as the scanning probe microscope, a near-field optical / atomic force microscope (SNOAM) in which an atomic force microscope and a near-field optical microscope are combined can be used. In the near-field optical / atomic force microscope, a probe applicable to the near-field optical microscope and the atomic force microscope is used. By using a near-field optical / atomic force microscope, the particle size of the fine particles and the optical characteristics of the fine particles can be observed simultaneously. An example of a near-field optical / atomic force microscope is disclosed in Japanese Patent Application Laid-Open No. 2001-194286. When the near-field optical / atomic force microscope is used, the particle size, fine particles and optical properties of the fine particles can be observed simultaneously, so that more types of target molecules can be simultaneously identified.

  Here, the near-field optical microscope is a type of scanning probe microscope that can specify the optical characteristics of the sample surface with high spatial resolution using an evanescent field (light). In the near-field optical microscope, scanning information can be obtained by converting the scattered light intensity of the evanescent field into an electric signal. Since the evanescent field is not limited by the light diffraction limit, the use of a near-field optical microscope has the advantage that the optical properties of the sample can be obtained with a spatial resolution exceeding the light diffraction limit. An example of a near-field optical microscope is disclosed in Japanese Patent Laid-Open No. 4-291310.

  When using cells for the sample, it is preferable to perform scanning while the cells are immersed in the solution. This is because cultured cells are damaged by the surface tension when they are directly exposed to the atmosphere. Examples of the solution in which the cells are immersed include a medium and a TE buffer solution. By immersing the cells in a medium or a buffer solution, it is possible to scan the cell surface while the cells are alive.

  The same part of the sample surface may be repeatedly scanned with the scanning probe microscope over time. By repeatedly scanning over time, it is possible to observe the movement of the fine particles fixed to the target molecule existing on the sample surface. A target molecule (for example, a membrane protein of a cell membrane) existing on the surface of a sample may change (move) over time due to free diffusion or interaction. As the target molecule moves, the fine particles bound to it move, so that the behavior of the target molecule on the sample surface can be understood by observing the movement of the fine particles.

  Information obtained by scanning may be visualized. For example, information obtained by scanning may be displayed on a display or the like as three-dimensional information. The scanning information may be visualized by an arbitrary method.

2. About the surface molecule identification apparatus of the present invention The above-described target molecule identification method can be implemented using, for example, the apparatus shown below. The target molecule identification apparatus of the present invention includes a fine particle providing member that provides fine particles to a sample surface, a measurement member that measures the fine particles fixed on the sample surface and obtains information that can identify the target molecule, and the information Output member.

  The fine particle providing member in the production apparatus of the present invention may be a member that drops the fine particles onto the sample surface or a member that adds the sample to an aqueous solution containing the fine particles. Examples of the fine particle providing unit that drops fine particles onto the sample surface include a fixed nozzle or a disposable tip nozzle.

  The measurement member measures physical properties such as the particle size of the fine particles bonded and fixed to the target molecules on the sample surface and the optical properties of the fine particles, and obtains information capable of identifying the target molecules. Examples of the measurement member include a scanning probe microscope. As an example of the scanning probe microscope, an atomic force microscope or a near-field optical / atomic force microscope in which an atomic force microscope and a near-field optical microscope are combined can be used. By using a near-field optical / atomic force microscope, it is possible to simultaneously obtain information on the particle size of the fine particles and the optical characteristics of the fine particles.

  The output member outputs information obtained by scanning. A method for outputting information is, for example, visualizing fine particles bound to target molecules on the sample surface.

  Hereinafter, the present invention will be described using examples. However, the scope of the present invention is not construed as being limited to the following examples.

[Preparation of substrate]
Concanavalin A (hereinafter referred to as “ConA”), which is a kind of lectin that is a sugar-binding protein, was dissolved in a phosphate buffer (10 mM pH 7.4) to obtain a 10 mM ConA solution. Wheat germ agglutinin (hereinafter referred to as “WGA”), which is a lectin different from ConA, was dissolved in a similar phosphate buffer to obtain a 10 mM WGA solution.
The surface of the glass substrate (1.5 mm × 1.5 mm) was divided into right and left, and both were coated with different lectins. That is, one of the left and right was dipped in a ConA solution and dried, the other was dipped in a WGA solution and dried, and each was coated to prepare a substrate 1 on which a lectin was fixed. ConA (reference numeral 2) and WGA (reference numeral 3) are fixed to the manufactured substrate 1 (see FIG. 1A).

[Preparation of fine particles]
Colloid solution (Au wt% 0.0044 wt% Tanaka Kikinzoku Kogyo Co., Ltd.) 500 μl containing gold fine particles (particle size 5 nm) and α-linked mannose lipid (2- (2- (2- (2- (2- (2- (2- (8-thio-octyloxy) ethyoxy) ethyoxy) ethyoxy) ethy-α-D-mannanoside) and a mixed solution of water (10 μg / μl) were mixed and stirred at room temperature for 1 hour. Thereafter, the mixture was purified by a gel filtration column to obtain gold fine particles modified with α-bonded mannose. α-binding mannose specifically binds to ConA2. The obtained gold fine particles modified with α-bonded mannose were dispersed in water to obtain an aqueous solution (hereinafter referred to as “aqueous solution A (symbol 8)”) having a gold fine particle concentration of 0.0044 wt%. Gold fine particles (reference numeral 4) having a particle diameter of 5 nm are modified with α-bonded mannose (reference numeral 5) (see FIG. 1B). The α-linked mannose lipid was prepared with reference to K. Niikura et al. “Accumulation of O-GlcNAc-displaying CdTe quantum dots in cells in the presence of ATP”, ChemBioChem, 8, 379-384.
Colloidal solution containing gold fine particles (particle size 50 nm) (citric acid type Au wt% 0.0066 wt% Tanaka Kikinzoku Kogyo Co., Ltd.) 500 μl and N-acetylglucosamine (GlcNAc) lipid (2- (2- (2- (2 -(2- (2- (8-thio-octyloxy) ethyoxy) ethyoxy) ethyoxy) ethyoxy-2-acetamide-2-deoxy-β-D-glucopyroxide) and water (10 μg / μl) ) And stirred at room temperature for 1 hour. Thereafter, the mixed solution was purified by a gel filtration column to obtain gold fine particles modified with N-acetylglucosamine. N-acetylglucosamine specifically binds to WGA. The obtained gold fine particles modified with N-acetylglucosamine were dispersed in water to obtain an aqueous solution (hereinafter referred to as “aqueous solution B (reference numeral 9)”) having a gold fine particle concentration of 0.0066 wt%. Gold fine particles (reference numeral 6) having a particle size of 50 nm are modified with N-acetylglucosamine (reference numeral 7) (see FIG. 1B). N-acetylglucosamine lipid was prepared with reference to K. Niikura et al. “Accumulation of O-GlcNAc-displaying CdTe quantum dots in cells in the presence of ATP”, ChemBioChem, 8, 379-384.

[Provide fine particles to the substrate]
100 μl of each of the aqueous solution A and the aqueous solution B was dropped on the prepared substrate 1 and incubated at room temperature for 15 minutes. Thereafter, the substrate 1 was washed with a buffer solution (not containing gold fine particles) and dried. By this treatment, gold fine particles having a particle diameter of 5 nm (reference numeral 4) and gold fine particles having a particle diameter of 50 nm (reference numeral 6) are attached to the substrate 1 (see FIG. 1C).

[Scanning with a scanning microscope]
The surface of the substrate 1 to which the gold fine particles (reference numerals 4 and 6) were attached was scanned with an atomic force microscope (AFM) probe (see FIG. 1D). As an atomic force microscope apparatus, MFP-3D (Asylum Research) was used. As the cantilever 10, OMCL-AC160 (Olympus) was used. The scanning method employs a tapping mode in which the cantilever 10 is vibrated at its resonance frequency, and feedback is applied with a change in vibration amplitude for imaging. The imaging frequency was 1-2 Hz.

  The surface shape was imaged by information obtained by scanning. The result of imaging is shown in FIG.

  FIG. 2A shows a state where a region (1 μm × 1 μm) coated with ConA is imaged. FIG. 2C shows a state where a region (1 μm × 1 μm) coated with WGA is imaged. On the other hand, FIG. 2B shows a state in which a boundary region (1 μm × 1 μm) between the region coated with ConA and the region coated with WGA is imaged.

As shown in FIGS. 2A to 2C, gold fine particles of about 5 nm are observed in the region coated with ConA; gold fine particles of about 50 nm are observed in the region coated with WGA; A clear boundary was observed in the boundary region between the coated region and the region coated with WGA.
Thus, it was shown that the target molecule can be identified and the spatial distribution can be measured with a resolution smaller than the particle size of the fine particles (50 nm in this example).

  According to the present invention, “position” and “type” of a plurality of types of molecules existing on the surface of a substance can be identified. Inspecting biochip quality by examining the distribution of nucleic acids and proteins bound to biochips, etc., and identifying biological proteins by identifying membrane proteins present in biological membranes (cell membranes) Can be obtained.

It is a schematic diagram which shows the flow of the identification method of this invention. It is the photograph of the information obtained in the Example.

Explanation of symbols

1 Substrate 2 Concanavalin A (ConA)
3 Wheat malt condensate (WGA)
4 Gold fine particles with a particle size of 5 nm 5 α-bonded mannose 6 Gold fine particles with a particle size of 50 nm 7 N-acetylglucosamine 8 Aqueous solution A
9 Aqueous solution B
10 Cantilever

Claims (12)

A method for identifying target molecules present on a sample surface,
A) Prepare two or more types of fine particle groups having different physical characteristics, and each of the two or more types of fine particle groups is modified with a molecule that specifically binds to a different target molecule. Step;
B) contacting the microparticles with the sample surface to bind to the target molecule and immobilizing it on the sample surface; and C) scanning the sample surface with the microparticles immobilized using a scanning probe microscope, Obtaining information capable of identifying each target molecule.   The method according to claim 1, wherein a particle diameter of the fine particles contained in the fine particle group is in a range of 1 nm to 10 μm.   The method according to claim 1, wherein the molecule that specifically binds to the target molecule is a nucleic acid, a peptide, a glycopeptide, a polysaccharide, or a lipid.   The method of claim 1, wherein the physical property of the microparticle is a particle size and the scanning probe microscope is an atomic force microscope (AFM).   The method of claim 1, wherein the physical properties of the microparticles are particle size and optical properties of the microparticles, and the scanning probe microscope is a near-field optical / atomic force microscope (SNOAM).   The method according to claim 1, wherein the change in the position of the target molecule is observed by repeating the step C) twice or more for the same part of the sample surface on which the fine particles are fixed.   The method according to claim 1, wherein the sample is a substrate on which a target molecule is immobilized.   The method according to claim 7, wherein the sample is a biochip including a substrate on which DNA or protein is immobilized.   The method of claim 1, wherein the sample is a biological material.   The method according to claim 9, wherein the sample is a cell, and the target molecule is a membrane protein, a membrane glycoprotein, or a lipid.   The method of claim 10, wherein the cells are disposed on a substrate. An identification device for identifying a target molecule present on a sample surface,
A fine particle providing member for providing fine particles to the surface of the sample;
An apparatus comprising: a measurement member that measures physical properties of the fine particles fixed on the sample surface and obtains information capable of identifying the target molecule; and an output member that outputs the previous period information.