JP2010175327A - Metal nanoparticle complex and method for manufacturing the same, and biochip and method for manufacturing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new metal nanoparticle complex useful as a biochip material and a method for manufacturing the same, and to provide a biochip using the metal nanoparticle complex and a method for manufacturing the complex. <P>SOLUTION: In the metal nanoparticle complex, a functional molecule is chemically bonded through a bridging linker part to metal nanoparticles whose surface is chemically modified with phospholipid molecules. The biochip has on a substrate, a complex arrangement part on which the metal nanoparticle complex is arranged. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a metal nanoparticle composite and a production method thereof, and a biochip using the metal nanoparticle composite and a production method thereof.

It is known that metal nanoparticles having a nanometer-level size exhibit a unique optical property called localized surface plasmon absorption due to resonance oscillation between the free electrons of the metal nanoparticles and the oscillating electric field of incident light. It has been. Further, it has been found that in a structure in which metal nanoparticles are organized, the local electric field strength between adjacent metal nanoparticles is extremely large (see Non-Patent Document 1).
Recently, research and development of functional materials using these properties of metal nanoparticles has become active, and application in a wide range of fields such as biosensing, electronic materials, solar cells, nonlinear optical materials, plasmonic waveguides, etc. Has been. Especially in the biosensing field, by using metal nanoparticles, it is possible to detect and diagnose biomolecules in a solution easily, in a short time, and at low cost without the need for a large device. Various studies have been made.
One of the reasons is that, when metal nanoparticles are accumulated on the substrate surface with good regularity, the expression of specific physical properties derived from the shape, orientation, structural dimensionality, etc. can be expected.
For example, as described above, when metal nanoparticles are organized, the local electric field strength between adjacent metal nanoparticles becomes very large, so that the spectroscopic measurement of molecules adsorbed in the gaps between the metal nanoparticles can be performed. If it does, the surface enhancement effect which the optical signal intensity of a spectrum will increase extremely will express. This property is an important technical element for achieving high sensitivity.
Moreover, if a rod-shaped thing (metal nanorod) is used as a metal nanoparticle, by adjusting the aspect ratio (ratio of [length in the major axis] / [length in the minor axis (diameter)]) Depending on the target detection molecule, it is possible to impart variability that allows the localized surface plasmon absorption wavelength to be freely changed from visible light to the near infrared region.
By utilizing these characteristics, the target substance to be inspected can be obtained in a short time from simple spectroscopic methods such as absorbed light, reflected light, fluorescence, infrared light, Raman light, and plasmon resonance of metal nanoparticles. Qualitative and quantitative analysis can be performed with high sensitivity.

In recent years, the development of metal nanoparticle thin films in which metal nanoparticles are regularly arranged on a substrate using the properties of metal nanoparticles as described above, and the organized structure formed thereby is precisely controlled has been developed. Has been tried.
By modifying biomolecules such as antibodies and enzymes on the metal nanoparticle thin film, it is considered that a biosensor capable of examining and diagnosing a biomolecular reaction with high sensitivity in a single molecule unit can be provided. Also in the field of nanodevice development, organized metal nanoparticle thin films can be a novel nano-based material that develops new optoelectronic properties. In particular, when metal nanorods are used, the absorption wavelength varies depending on the aspect ratio. Therefore, in biosensing applications, selectivity can be expressed by changing the sensing wavelength according to the desired target molecule. it can. Further, by regularly arranging the metal nanoparticles, the effect of enhancing the light or electric field by the localized surface plasmon coupling is increased, so that a high-sensitivity sensing function at a single molecule level can be provided.

As a specific example of biosensing using metal nanoparticles, for example, when metal nanoparticles on which two types of deoxyribonucleic acid (DNA) are separately immobilized and the target DNA are mixed, one base in the sequence Has been reported to be easily distinguishable from changes in the plasmon absorption characteristics of metal nanoparticles (see Non-Patent Document 2). In addition, even when one kind of DNA-immobilized metal nanoparticles is used, by adding the complementary DNA and a high concentration sodium chloride solution, the ability to form aggregates of metal nanoparticles is intentionally controlled, A method of discriminating a single base mismatch by “non-crosslinking type agglutination reaction” has also been proposed (see Non-Patent Document 3).
In addition, a sugar chain identification method using metal nanoparticles has been devised (see Non-Patent Document 4). In this method, by combining sugars such as lactose with metal nanoparticles coated with polyethylene glycol (PEG) chains, and adding lectins that specifically bind to the sugars, the state of aggregation and dispersion of the metal nanoparticles is changed. In this way, sugar chains are identified. The PEG chain has a role as a dispersion stabilizer that provides dispersion stability in the solution of the metal nanoparticles, and also has a sophisticated role to play a role as an adsorption inhibitor for preventing nonspecific adsorption of the lectin. Particle surface design is made.
Biosensing systems using semiconductor nanoparticles that emit fluorescence have also been developed. For example, biotin is added to the surface of CdS nanoparticles, and streptavidin (fluorescent dye label) that specifically recognizes biotin is added. When CdS is excited, fluorescence resonance energy transfer (Fluorescent resonance energy transfer (FRET)) occurs only to the fluorescent dye that labels the streptavidin adsorbed on the CdS nanoparticle surface, and the reaction between biotin and streptavidin is quantitatively detected. (See Non-Patent Document 5).

On the other hand, biochips are expected to bring huge market value, and competitive research and development at the world level has been attempted together with the above biosensing technologies.
In general, next-generation biochips include (1) high selectivity (improving molecular discrimination), (2) high sensitivity (molecular level diagnosis / detection, signal amplification), and (3) high throughput (separation / purification). Etc.) (4) Advanced technical elements such as cost reduction, weight reduction and downsizing of the diagnostic apparatus are required.
Examples of detection methods used in conventional biochips include electrochemical techniques, ELIZA (Enzyme-linked immunosorbent assay) methods, surface plasmon resonance (SPR) methods, and quartz crystal (quartz crystal microbial). QCM)) method is the mainstream.
Among these, the electrochemical method immobilizes biomolecules such as enzymes, antibodies, and DNA on the electrode surface as a recognition site that specifically recognizes the target substance, and the electrochemical reaction that occurs when the target substance binds to it. Quantification is performed by detecting a typical signal change and signal amplification by a labeling substance such as an enzyme. The ELIZA method is a method for quantitatively detecting a trace amount of a target substance contained in a sample by using an antigen-antibody reaction using an enzyme-labeled or fluorescent-labeled antibody or antigen.
However, all of these detection methods require labeling to biomolecules and are complicated. In addition, since the labeling causes inhibition of the ability to recognize biomolecules, it is difficult to measure the target substance in a trace amount or at high sensitivity.
On the other hand, the SPR method for detecting the shift amount of the plasmon resonance angle when the target substance is adsorbed on the surface of the metal thin film and the QCM method for detecting the change in the frequency are not labeled with enzymes or fluorescent molecules on the biomolecules (non- Label), and has the advantage of being able to detect and quantify the target substance.
However, the SPR method is unsuitable for on-site monitoring of a target substance because a series of detection operations is complicated, such as requiring a large optical measurement system and optimization of measurement parameters in order to improve sensitivity. Further, the QCM method can be easily measured with a simple apparatus, but the noise is large and high-sensitivity measurement cannot be realized.
Furthermore, all of these conventional detection methods are aimed at increasing the signal intensity on the macroscopic surface on which the molecular recognition reaction has been performed to facilitate detection, and are the technologies required for next-generation biochips. It is difficult to satisfy the elements.

Supervised by Satoshi Yamada, “Design and application technology of plasmon nanomaterials”, first edition, 2006, CM Publishing N.L. Rosi et al., “Chemical Review”, Vol. 105, 2005, pp. 1547-1562. K. Sato et al., “Journal of American Chemical Society”, 125, 2003, pp. 8102-8103. H. Otsuka et al., “Journal of American Chemical Society”, Vol. 123, 2001, pages 8226-8230. Y. Nagasaki et al., “Langmuir”, 20, 2004, 6396-6400.

Metal nanoparticles are promising as biochip materials that solve the problems in the detection methods used in conventional biochips. That is, by utilizing the characteristics as described above, the target substance to be inspected can be obtained from simple spectroscopic methods such as absorption light, reflection light, fluorescence, infrared light, Raman light, and plasmon resonance of metal nanoparticles. Therefore, it is possible to provide a biochip capable of performing qualitative and quantitative analysis with high sensitivity in a short time. In addition, if metal nanoparticles are integrated on a substrate at a high density, reaction points on the biochip can also be integrated at a high density, thereby improving throughput and reducing the size of the biochip.
However, metal nanoparticles that have been studied for application to biosensing, etc. have controllability on the substrate (arrangement of metal nanoparticles, dimensionality of array structure, particle spacing at nanometer level, etc.) Is not enough. Therefore, it is difficult to accumulate the metal nanoparticles on the substrate at a high density. In addition, the metal nanoparticles have a problem of low biocompatibility for use in diagnosis / detection of biomolecules such as the surface being coated with a surfactant.
The present invention has been made in view of the above circumstances, and is a novel metal nanoparticle composite useful as a biochip material and a production method thereof, and a biochip using the metal nanoparticle composite and a production method thereof The purpose is to provide.

In order to achieve the above object, the present invention employs the following configuration.
[1] A metal nanoparticle composite characterized in that a functional molecule is chemically bonded to a metal nanoparticle whose surface is chemically modified with a phospholipid molecule via a crosslinking linker moiety.
[2] The metal nanoparticle composite according to [1], wherein the metal nanoparticles are metal nanorods.
[3] The functional molecule is at least one functional protein selected from the group consisting of an antibody, an antigen, an enzyme, a membrane protein, a receptor protein, a cytoskeleton, and a motor protein [1] or [2] The metal nanoparticle composite according to 1.
[4] A method for producing a metal nanoparticle composite according to any one of [1] to [3],
A method for producing a metal nanoparticle composite, comprising a step of chemically bonding a metal nanoparticle whose surface is chemically modified with a phospholipid molecule and a functional molecule using a crosslinking linker agent.
[5] The method for producing a metal nanoparticle composite according to [4], wherein the crosslinking linker agent has a succinimidyl group which may have a substituent and a sulfur-containing organic substituent.
[6] A biochip having a composite placement part in which the metal nanoparticle composite according to any one of [1] to [3] is placed on a substrate.
[7] The biochip according to [6], wherein an array structure in which the metal nanoparticle composites are arranged one-dimensionally, two-dimensionally, or three-dimensionally is formed in the composite placement part.
[8] The structure according to [6] or [7], including a plurality of the complex arrangement parts, wherein the types and concentrations of the functional molecules or the types or concentrations of the functional molecules in the plurality of complex arrangement parts are different. Biochip.
[9] A method for producing a biochip according to any one of [6] to [8],
On the substrate, the metal nanoparticles constituting the metal nanoparticle composite are developed using the self-organization ability of phospholipid molecules that chemically modify the surface thereof, and the metal nanoparticle composite is one-dimensional or two-dimensional. A biochip manufacturing method comprising a step of forming a three-dimensional array structure.
[10] The process of forming an array structure in which the metal nanoparticle composites are arranged one-dimensionally or two-dimensionally is repeated a plurality of times to form an array structure in which the metal nanoparticle composites are arrayed in three dimensions [9] ] The manufacturing method of the biochip of description.

  ADVANTAGE OF THE INVENTION According to this invention, the novel metal nanoparticle composite useful as a biochip material, its manufacturing method, the biochip using this metal nanoparticle composite, and its manufacturing method can be provided.

It is a schematic diagram explaining one Embodiment of the metal nanoparticle composite_body | complex of this invention. It is a figure explaining one Embodiment of the manufacturing method of the metal nanoparticle composite_body | complex of this invention. In the biochip of this invention, it is a schematic diagram which shows one Embodiment in case the arrangement | sequence structure where the metal nanoparticle composite_body | complex is arranged in one dimension is formed. In the biochip of this invention, it is a schematic diagram which shows one Embodiment in case the arrangement | sequence structure where the metal nanoparticle composite_body | complex is arranged in two dimensions is formed. In the biochip of this invention, it is a schematic diagram which shows one Embodiment in case the arrangement | sequence structure where the metal nanoparticle composite_body | complex is arranged in two dimensions is formed. In the biochip of this invention, it is a schematic diagram which shows one Embodiment in case the arrangement | sequence structure where the metal nanoparticle composite_body | complex is arranged in three dimensions is formed. In the biochip of this invention, it is a schematic diagram which shows one Embodiment in case the arrangement | sequence structure where the metal nanoparticle composite_body | complex is arranged in three dimensions is formed. In the biochip of this invention, it is a schematic diagram which shows one Embodiment in case the arrangement | sequence structure where the metal nanoparticle composite_body | complex is arranged in three dimensions is formed. It is a schematic diagram which shows one Embodiment in case the biochip of this invention is a microchannel type. It is a schematic diagram which shows one Embodiment in case the biochip of this invention is a multi-array type | mold. The graph which shows the absorption spectrum of each aqueous dispersion of the gold nanorod, the phospholipid modified gold nanorod and the gold nanorod complex produced in Production Example 1, Production Example 2 and Example 1, and the Alexa-labeled anti-IgG antibody stock solution used. It is. The graph which shows the fluorescence spectrum of each aqueous dispersion of the gold nanorod, the phospholipid modified gold nanorod, and the gold nanorod complex produced in Production Example 1, Production Example 2 and Example 1, and the Alexa-labeled anti-IgG antibody stock solution used. It is. It is a graph which shows the result of the flow cytometry measurement about the aqueous dispersion of each of the phospholipid modification gold | metal | money nanorod and gold | metal nanorod composite_body | complex manufactured in manufacture example 2 and Example 1. FIG. 2 is a transmission micrograph and a fluorescence micrograph of a gold nanorod composite produced in Example 1. FIG. 2 is a laser fluorescence micrograph of a gold nanorod composite produced in Example 1. FIG. FIG. 3 is a schematic diagram for explaining a measurement system used when detecting a biomolecular interaction in Test Example 1. In Test Example 1, it is a total reflection fluorescence micrograph at the time of detecting the interaction between biomolecules. In Experiment 2, it is a total reflection fluorescence micrograph at the time of observing the time change of interaction between biomolecules. In Test Example 2, it is a graph showing the number of white bright spots with respect to the reaction time when the time change observation of the interaction between biomolecules is performed. In Test Example 2, it is a graph showing a histogram of average luminance with respect to the number of white bright spots when time change observation of interaction between biomolecules is performed. It is a total reflection fluorescence micrograph which shows the result of Test Example 3 (comparative test).

<Metal nanoparticle composite>
In the metal nanoparticle composite of the present invention, a functional molecule is bonded to a metal nanoparticle whose surface is chemically modified with a phospholipid molecule (hereinafter sometimes referred to as a phospholipid-modified metal nanoparticle) via a crosslinker linker moiety. Are chemically bonded.
Here, the “metal nanoparticles” in the present invention refer to metal particles having a particle diameter in the range of 1 to 999 nm. The chemical modification and chemical bond indicate a bond by a covalent bond, an ionic bond or a hydrogen bond.

[Phospholipid-modified metal nanoparticles]
The phospholipid-modified metal nanoparticles are obtained by chemically modifying the surface of the metal nanoparticles with phospholipid molecules. Since the surface is modified with phospholipid molecules, the phospholipid-modified metal nanoparticles and the metal nanoparticle composites having the phospholipid-modified metal nanoparticles have good dispersion stability in water or organic solvents. is there.

The metal constituting the metal nanoparticle constituting the phospholipid-modified metal nanoparticle is preferably one that can cause localized plasmon resonance. Examples of the metal include gold, silver, copper, and gold, silver, and copper. An alloy containing two or more types can be used. Moreover, you may use together the metal nanoparticle using the metal from which a kind differs.
The metal nanoparticles are preferably rod-shaped, that is, metal nanorods. Metal nanorods exhibit high wavelength selectivity because their absorption characteristics change according to the aspect ratio. Therefore, the measurement wavelength can be selected by changing the shape of the metal nanorods. Furthermore, the polarization characteristic using the difference in absorption wavelength between the long axis surface and the short axis surface of the metal nanorods can be exhibited.
However, the present invention is not limited to this, and the shape of the metal nanoparticles may be substantially spherical. Even when metal nanoparticles other than metal nanorods are used, the same effect as described above is exhibited.
The metal nanorods preferably have an aspect ratio greater than 1. In particular, it is preferable that the gold nanorod has an aspect ratio of 1 to 5 because optical characteristics due to the nanosize effect of the particles are remarkably exhibited.
The metal nanorods preferably have a diameter of several nm to several tens of nm and a length of several nm to several hundred nm.
As the metal nanorods, gold nanorods are particularly preferable because of the excellent effects described above.

The metal nanoparticles can be produced using a known method.
For example, gold nanorods are (1) a method that combines chemical reduction and photoreaction (see, for example, Y. Niidome et al., “Chemical Communications”, 2003, pages 2376-2377). (2) A chemical synthesis method in which a gold nanoseed produced by chemical reduction is grown in a rod shape (for example, TK Sau et al., “Langmuir”, Volume 20, 2004, (See pages 6414-6420).
In particular, the method (1) is preferable because a gold nanorod having a uniform shape can be easily synthesized in a very short time and high reproducibility can be obtained under the same synthesis conditions.

Hereinafter, the method (1) will be described in more detail.
In this method, first, a gold salt solution is chemically reduced.
Examples of the gold salt solution as a starting material include a gold halide aqueous solution and a gold cyanide aqueous solution. The concentration of the gold salt solution is preferably about 1 to 10 mM.
The gold salt solution preferably contains a surfactant such as hexadecyltrimethylammonium bromide. By the addition of the surfactant, the surfactant is adsorbed on the surface of the gold nanorods 11, preventing the gold nanorods from aggregating and stably dispersing in the aqueous solution. The concentration of the surfactant is preferably 20 to 100 mM. When the surfactant concentration is less than 20 mM, the dispersion stability of the gold nanorods 11 is insufficient and tends to aggregate. On the other hand, when the concentration exceeds 100 mM, the viscosity of the resulting solution increases, and there is a possibility that the gold nanorod growth reaction rate decreases, the growth particles become non-uniform, and the handling property decreases.
The gold salt solution preferably contains acetone and / or cyclohexane. Acetone serves as an initiator of the photoreaction to promote the rod growth reaction and can adjust the growth rate of the gold nanorods by light irradiation. Moreover, the yield of gold nanorods having a desired aspect ratio can be changed by appropriately selecting the amount of acetone added. When the gold salt solution contains cyclohexane, the yield of gold nanorods can be increased and the aspect ratio can be made uniform.
The chemical reduction of the gold salt solution can be performed by adding a reducing agent to the gold salt solution.
As the reducing agent, those having a low generation rate of gold clusters (nanoparticle growth nuclei) are preferable, and specifically, ascorbic acid, citric acid and salts thereof are preferable. The reducing agent may be added in an amount sufficient to reduce the gold salt.

After the chemical reduction, a photoreaction is performed. The photoreaction is a reaction in which gold nanorods are obtained by irradiating a gold salt solution to which a reducing agent is added with light such as ultraviolet rays.
Here, in light irradiation, a substance that induces a phenomenon in which the growth reaction of gold fine particles is biased toward a specific crystal plane, that is, a substance that promotes growth in the long axis direction of the gold nanorod 11 (hereinafter referred to as a growth promoting substance). It is preferable to add. As the growth promoting substance, silver salts such as silver nitrate, silver chloride and silver bromide are preferable. A gold nanorod having a long major axis can be obtained as the concentration of the silver salt added increases, but the addition of an excess silver salt tends to lower the reaction rate. From this, it is preferable that the addition concentration of silver salt is about 1 μM to 1 mM.
In the photoreaction, the amount and shape of the gold nanorods can be controlled by appropriately selecting the light irradiation time, light irradiation intensity, and irradiation light wavelength. The wavelength of irradiation light is effective for gold nanorod growth, and is preferably 300 nm or less, more preferably 254 nm. As the light source, for example, a low-pressure mercury lamp, a high-pressure mercury lamp, an ultrahigh-pressure mercury lamp, a xenon lamp, or the like can be used. Furthermore, a band pass filter that selectively transmits only light in a desired wavelength band (for example, 254 nm) may be disposed between the light source and the sample. If a bandpass filter is disposed between the light source and the sample and irradiated with light, the gold nanorod growth reaction can be further promoted.

Purification may be performed after the photoreaction. When a surfactant is contained in the gold salt solution, the aqueous solution containing gold nanorods obtained through the above steps contains an excess of surfactant, and the subsequent formation of phospholipid-modified gold nanorods. May interfere. Therefore, removal of excess surfactant by purification can prevent formation inhibition of phospholipid-modified gold nanorods.
As purification, it is preferable to repeat the operations of centrifugation, supernatant removal, and pure water washing. Furthermore, the centrifugation operation is preferably performed under conditions of 5 ° C. or more and less than 15 ° C., 5000 rpm or more per minute, and 10 minutes or more. Under such conditions, the amount of gold nanorods suspended in the supernatant solution after centrifugation is suppressed, and the amount of gold nanorods recovered can be increased. However, when this purification process is repeated three times or more, the surfactant on the gold nanorod surface is completely peeled off, and the gold nanorod tends to be unable to be stably dispersed in the aqueous solution.

[Production of phospholipid-modified metal nanoparticles]
Phospholipid-modified metal nanoparticles can be obtained by the method described in JP-A No. 2006-179395, H. Nakashima et al., “Langmuir”, Vol. 24, 2008, pages 5654-5658. It can produce with reference to. For example, it can be produced by mixing and stirring an aqueous solution containing gold nanorods prepared by the above method (1) and a dispersion in which phospholipid molecules are dispersed in water or an organic solvent at an arbitrary concentration.
When gold nanorods are prepared by the above method (1), a cationic surfactant (such as hexadecyltrimethylammonium bromide) added during preparation is electrostatically coordinated on the surface of the gold nanorods. By adding a phospholipid molecule having a higher chemical binding force with gold, a ligand exchange reaction on the surface occurs. For this reason, the gold nanorod surface can be easily chemically modified with phospholipid molecules.
When phospholipid molecules are dispersed in an organic solvent, it is preferable to use a polar organic solvent that is immiscible with water, for example, a halogen-based solvent such as chloroform or methylene chloride. Phospholipid molecules and phospholipid-modified gold nanorods 13 are easily dispersed in this type of organic solvent.

The phospholipid molecule used for the production of phospholipid-modified metal nanoparticles has a reactive group capable of reacting with the metal constituting the metal nanoparticle as a substituent at the end because the surface of the metal nanoparticle can be easily chemically modified. Those are preferred.
Here, in the present specification and claims, “can react” means that a chemical bond can be formed.
As the reactive group, those used as a reactive group in a compound conventionally used for surface treatment of the metal can be used depending on the metal constituting the metal nanoparticle.
For example, when the metal nanoparticle is a gold nanoparticle, a sulfur-containing organic substituent is preferable because of its high chemical bond strength with gold. The sulfur-containing organic substituent is not particularly limited as long as it can react with gold to form an Au—S bond, but it is excellent in reactivity, and therefore an organic group containing a mercapto group (—SH). , At least one selected from the group consisting of a thioacetyl group, a sulfide group and a disulfide group is preferable. Among these, an organic group containing a mercapto group is preferable, and a mercaptoalkyl group is particularly preferable.
As the phospholipid molecule, a phospholipid molecule represented by the following general formula (12-1) is particularly preferable. In the phospholipid molecule represented by formula (12-1), terminal -SH reacts with gold to form an Au-S bond.

[Wherein R ^s is a sulfur-containing organic substituent, R ¹ and R ² are each independently a linear aliphatic hydrocarbon group, and X ¹ is an alkali metal or a hydrogen atom. ]

In the formula (12-1), examples of the sulfur-containing organic substituent in R ^s include the same ones as described above. The R ^s, preferably an organic group containing a mercapto group, more preferably a mercaptoalkyl group, linear mercaptoalkyl group. Examples of the mercaptoalkyl group include HS— (CH ₂ ) _r — [wherein r is an integer of 1 to 20. ].
Aliphatic hydrocarbon radical straight in R ^1, R ^2, which may be saturated, may be unsaturated, saturated aliphatic hydrocarbon group, i.e., the alkyl group. Although carbon number of this aliphatic hydrocarbon group is not specifically limited, Usually, 8-24 are preferable and 14-18 are more preferable. By adjusting the length of the carbon chain of the aliphatic hydrocarbon group, when the metal nanoparticle composite of the present invention is arranged to form an array structure, the distance between adjacent metal nanoparticle composites is adjusted. The longer the carbon chain, the wider the spacing between adjacent metal nanoparticle composites. For example, when the carbon chain has an arbitrary length of 8 to 24 carbon atoms, the distance between the metal nanoparticle composites in the array structure can be adjusted to about 1 to 5 nm.
Examples of the alkali metal in X ¹ include sodium and potassium.
Specific examples of the phospholipid molecule represented by the formula (12-1) include a sodium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol represented by the following chemical formula. .

[Functional molecules]
In the present invention, as the functional molecule, a specific binding substance that causes some kind of interaction (for example, formation of a chemical bond, adsorption, etc.) specifically with the functional molecule may exist, that is, a sample serving as a specimen. It can specifically bind to or adsorb target substances and candidate target substances (for example, biomolecules such as proteins, peptides, and low molecular compounds derived from living bodies) and can be immobilized on phospholipid-modified gold nanoparticles. Any molecule can be used.
As the functional molecule, a functional protein is preferable from the viewpoint that a biochip utilizing a specific binding ability between biomolecules can be developed. The functional protein is preferably at least one selected from the group consisting of an antibody, an antigen, an enzyme, a membrane protein, a receptor protein, a cytoskeleton, and a motor protein.
However, in the present invention, the functional molecule is not limited to a functional protein, and as described above, a specific binding substance that specifically binds can be present and can be immobilized on phospholipid-modified metal nanoparticles. All molecules are available. Examples of functional molecules other than proteins include sugar chain compounds, DNA, and ribonucleic acid (RNA).

[Crosslinked linker]
The crosslinked linker portion is a portion derived from a crosslinked linker agent, and is formed by reacting the crosslinked linker agent with phospholipid-modified metal nanoparticles and a functional molecule.
As the crosslinking linker agent, a group capable of reacting with a functional molecule (hereinafter referred to as a reactive group (P)) and a group capable of reacting with a phospholipid-modified metal nanoparticle (hereinafter referred to as a reactive group (Q)). The thing which has these is used.

The reactive group (P) may be appropriately set according to the structure of the functional molecule to be chemically bonded. For example, when a functional protein is used as the functional molecule, those that have been used as reactive groups in compounds conventionally used for chemical modification of proteins can be used.
In the present invention, when a functional protein is used as the functional molecule, the reactive group (P) is preferably a succinimidyl group. The succinimidyl group forms a covalent bond by efficiently coupling with an amino group in the protein structure, particularly an amino group at the N-terminus.
The succinimidyl group, a sulfonic acid (salt) group (-SO 3 _X ^{2 _[X} 2 is an alkali metal or a hydrogen atom.]) May have a substituent such as.
As the reactive group (P), a succinimidyl group which may have a sulfonic acid (salt) group is particularly preferable.

  Examples of the reactive group (Q) are the same as those mentioned as the reactive group capable of reacting with the metal constituting the metal nanoparticle in the description of the phospholipid molecule. A sulfur-containing organic substituent is preferable because of its high chemical bond strength with gold, and particularly preferably at least one selected from the group consisting of an organic group containing a mercapto group, a thioacetyl group, a sulfide group, and a disulfide group. . Among these, an organic group or a disulfide group containing a mercapto group is preferable, and a mercaptoalkyl group or a disulfide group is more preferable.

  In the present invention, as the crosslinking linker agent, those having a succinimidyl group which may have a substituent and a sulfur-containing organic substituent are preferable, and a succinimidyl group which may have a sulfonic acid (salt) group. And those having a sulfur-containing organic substituent are more preferred. Examples of such a crosslinking linker agent include compounds represented by the following general formula (14-1).

[Wherein, X ² is an alkali metal or a hydrogen atom, m is 1 or 0, R ³ is a hydrogen atom or —S—R ⁵ (R ⁵ is a monovalent organic group); R ⁴ is a divalent organic group. ]

Examples of the alkali metal in X ² include sodium and potassium, and sodium is preferable.
Examples of the divalent organic group for R ⁴ include an alkylene group. The alkylene group is preferably linear. The number of carbon atoms of the alkylene group is preferably 1 to 12 in consideration of solubility in water. In the alkylene group, a divalent group containing a hetero atom such as —NH—C (═O) — may be interposed in the carbon chain.
The monovalent organic group represented by R ^5, is not particularly limited, the terminal preferably has a nitrogen-containing cyclic group. Examples of the nitrogen-containing cyclic group include the succinimidyl group and 2-pyridyl group.

Specific examples of the compound represented by the formula (14-1) include, for example, 3,3′-dithiobis (sulfosuccinimidylpropionate) (the following chemical formula (14a), hereinafter referred to as DTSSP), dithiobis. (Succinimidyl propionate) (the following chemical formula (14b), hereinafter referred to as DSP), sulfosuccinimidyl 6- (3 ′-[2-pyridyldithio] propionamide) hexanoate (the following chemical formula (14c) , Hereinafter referred to as Sulfo-LC-SPDP), succinimidyl 6- (3- [2-pyridyldithio] propionamide) hexanoate (the following chemical formula (14d), hereinafter referred to as LC-SPDP), N-succinimidyl 3- ( 2-pyridyldithio) propionate (the following chemical formula (14e), hereinafter referred to as SPDP), 1 - mercaptoundecanoic acid (11-MUA) and N- hydroxysuccinimide the reaction product of an imide (NHS) (. The following chemical formula (14f), hereinafter referred to MUA-NHS), and the like. MUA-NHS can be synthesized, for example, by coupling reaction of 11-MUA and NHS in the presence of 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide (EDAC) catalyst.
Among these, when DTSSP, DSP, Sulfo-LC-SPDP, LC-SPDP or SPDP is used as a crosslinking linker agent, dithiothreitol (DTT), which is a sulfur-containing reducing agent and also known as Cleland reagent, is optionally used. It is preferred to use and reduce the -S-S- moiety to -SH. Thereby, it can be chemically bonded to the gold surface more efficiently. DTT is also referred to as threo-1,4-dimercapto-2,3-butanediol, and is represented by the chemical formula: HS—CH ₂ —CH (OH) —CH (OH) —CH ₂ —SH.
As the crosslinking linker agent, DTSSP is preferable in consideration of solubility in water and reaction efficiency.

Hereinafter, 1st embodiment of the metal nanoparticle composite_body | complex of this invention is described using drawing. In the present embodiment, the metal nanoparticles constituting the metal nanoparticle composite are gold nanorods, and the functional molecule is an antibody.
FIG. 1A shows a schematic diagram of a metal nanoparticle composite (gold nanorod composite 10) of the present embodiment.
The gold nanorod composite 10 is formed by chemically bonding an antibody 15 to a phospholipid-modified gold nanorod 13 obtained by chemically modifying the surface of a gold nanorod 11 with a phospholipid molecule 12 via a cross-linking linker portion 14.
The phospholipid molecule 12 includes a hydrophilic portion (larger circle in the figure), two linear hydrophobic portions bonded to the hydrophilic portion, and a connecting portion that connects the hydrophilic portion and the surface of the gold nanorod 11. (Smaller circle in the figure). FIG. 1 (b) shows the structure of the phospholipid molecule 12 formed when the sodium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol mentioned above is used.
The antibody 15 has a Y-shaped structure composed of two heavy chains having a bent structure and two light chains bonded to each heavy chain. One end of the cross-linking linker portion 14 is bonded to one of the two ends on the side where the surface is present.

[Method for producing metal nanoparticle composite]
The metal nanoparticle composite can be produced, for example, by a production method including a step of chemically bonding a phospholipid-modified metal nanoparticle and a functional molecule using a crosslinking linker agent.
The said process can be implemented by the procedure of the following (A) or (B), for example.
(A): A functional molecule and a crosslinking linker agent are reacted, and then reacted with phospholipid-modified metal nanoparticles.
(B): A phospholipid-modified metal nanoparticle and a cross-linking linker agent are reacted, and then reacted with a functional molecule.

A method for producing a metal nanoparticle composite according to the procedure (A) will be described with reference to FIG. In the drawings described below, components corresponding to those shown in FIG. 1 are given the same reference numerals, and detailed descriptions thereof are omitted.
In this example, first, a solution containing the antibody 15 ′ and the cross-linking linker agent 14 ′ at an arbitrary concentration (such as a phosphate buffer solution) is mixed and incubated at room temperature for several hours. Thereby, the reaction is completed, and one end of the cross-linking linker agent 14 ′ is bonded to one end of the antibody 15 ′.
An arbitrary concentration of phospholipid-modified gold nanorod 13 solution is added thereto. Then, the other end of the crosslinking linker agent 14 ′ bonded to the end of the antibody 15 ′ reacts with the phospholipid-modified gold nanorod 13 to form the gold nanorod complex 10.

  After the above reaction, further purification may be performed. For example, unreacted substances and impurities other than the target substance can be removed by repeatedly performing operations such as centrifugation, supernatant removal, redispersion in a buffer solution, and pure water washing.

<Biochip>
The biochip of the present invention has a composite arrangement part in which the metal nanoparticle composite of the present invention is arranged on a substrate.
Various substrates such as silicon, glass, quartz, sapphire, mica, ITO, gold, platinum, copper, silicon nitride, indium phosphide, etc .; organic materials such as polystyrene, polyethylene terephthalate, polycarbonate, polydimethylsiloxane, etc. A solid substrate made of any material can be used without any limitation. In addition, a surface of an inorganic substrate such as a glass substrate that has been subjected to a surface treatment such as a water-repellent polymer coat can be used.
When the biochip is used, when an inverted microscope is used for detection, a sample is disposed on the upper part of the substrate and detection is performed from the lower part of the substrate.
Moreover, it is preferable that a board | substrate is a material excellent in workability.
Further, the substrate preferably has a nanometer-scale flatness in order to precisely orient the gold nanorod composite 10 having a diameter of about 10 nm on the substrate surface.
Although the thickness of a board | substrate is not specifically limited, Usually, it is about 0.1-20 mm.

A method for forming the composite arrangement part on the substrate is not particularly limited, and a known method can be applied.
In the present invention, a method of developing the phospholipid-modified metal nanoparticles on a substrate using the self-assembly ability of phospholipid molecules is preferably used.
At this time, the phospholipid-modified metal nanoparticles developed on the substrate may be in a state in which the functional molecules are chemically bonded or in a state in which they are not chemically bonded. That is, as in the following method (1), phospholipid-modified metal nanoparticles having functional molecules chemically bonded thereto, that is, the metal nanoparticle composite of the present invention may be developed on a substrate. After the phospholipid-modified metal nanoparticles with no functional molecules chemically bonded thereto are spread on the substrate or simultaneously with the development, the functional molecules are chemically bonded to the phospholipid-modified metal nanoparticles. And may be a metal nanoparticle composite.
Method (1): A method comprising a step of spreading the metal nanoparticle complex on a substrate by utilizing the self-organization ability of the phospholipid molecule.
Method (2): a step (2-1) in which a phospholipid-modified metal nanoparticle is developed on a substrate using the self-organization ability of the phospholipid molecule;
A step (2-2) of chemically bonding the phospholipid-modified metal nanoparticles and the functional molecule with a crosslinking linker agent.
In the method (2), the step (2-1) and the step (2-2) may be performed at the same time, or after the step (2-1) is performed, the step (2-2) may be performed.

The development of metal nanoparticle composites or phospholipid-modified metal nanoparticles using the self-organization ability of phospholipid molecules can be achieved by, for example, dispersing the metal nanoparticle composite or phospholipid-modified metal nanoparticles in a dispersion medium such as water. And the dispersion is placed on a substrate and dried to remove the dispersion medium. When the complex arrangement part is formed in this way, in the complex arrangement part, the phospholipid-modified metal nanoparticles are linked by the self-assembly (intermolecular interaction) of the phospholipid molecules in the drying process. , Integrate.
The method for arranging the dispersion on the substrate is not particularly limited, and a known method such as a casting method, a spin coating method, a Langmuir-Blodget (LB) method, or the like can be used.
At this time, by adjusting the following requirements (a) to (e) and the like, the integrated structure (array direction and structural dimensionality) of the phospholipid-modified metal nanoparticles on the substrate can be precisely controlled.
(A) Surface characteristics (hydrophilicity, hydrophobicity) of the substrate.
(B) Chemical affinity and surface tension of the dispersion medium with respect to the substrate.
(C) A method of developing the dispersion liquid on the substrate (developing method, number of times of development, direction of development, etc.).
(D) Drying conditions of the dispersion (drying temperature, drying speed, etc.).
(E) Structure of phospholipid molecule

For example, on the surface of a hydrophilic substrate such as a cover glass, the metal nanoparticle complex or phospholipid-modified metal nanoparticle expands along the surface due to the self-organization ability of phospholipid molecules, and gold nanorods do not overlap each other. A single layer array structure is formed.
In addition, the interparticle spacing of the metal nanoparticle complex or phospholipid-modified metal nanoparticle to be arranged can be easily controlled according to the hydrocarbon chain length of the phospholipid molecule modified on the surface of the metal nanoparticle.
The distance between the metal nanoparticle composites in the array structure is preferably adjusted to 1 to 5 nm.

In addition, when the dispersion is developed using a spin coating method, the metal nanoparticle composite or the phospholipid-modified metal nanoparticles are disposed on the substrate at a relatively low density.
In this case, an array structure in which the metal nanoparticle composites are arranged one-dimensionally can be formed. Here, the “one-dimensional array structure” refers to an array structure in which metal nanoparticle composites are integrated and arrayed in one layer and one row on a substrate.
FIG. 3 shows an embodiment in which an array structure in which metal nanoparticle composites are arranged one-dimensionally is formed in the biochip of the present invention.
In the biochip 31 of the present embodiment, a plurality of phospholipid-modified gold nanorods 13 are arranged in a row in a partial region on the substrate 21 in parallel with the surface of the substrate 21 and in contact with the major axis surface. A one-dimensional array structure is formed, and in other regions, a one-dimensional array structure is formed in which a plurality of phospholipid-modified gold nanorods 13 are arranged in a row in parallel with the surface of the substrate 21 and in contact with each minor axis surface. Has been. In each one-dimensional array structure, the antibody 15 is bound to a part or all of the phospholipid-modified gold nanorods 13 via a cross-linking linker part (not shown) to form the gold nanorod complex 10.
Thus, in the case of forming a one-dimensional array structure in which either the long-axis surface or the short-axis surface of the phospholipid-modified gold nanorod 13 is in contact with each other, either the long-axis surface or the short-axis surface of the gold nanorod 11 is selected. By selectively introducing the phospholipid molecule 12 into this, the connecting direction can be changed to either the vertical or horizontal direction.

In addition, when the dispersion is developed using the cast method or the LB method, the metal nanoparticle composite or the phospholipid-modified metal nanoparticles are disposed on the substrate at a relatively high density.
In this case, an array structure in which metal nanoparticle composites are two-dimensionally arranged can be formed. Here, “a two-dimensional arrangement structure” indicates an arrangement structure that is integrated and arranged in two layers or more on a substrate.
4 to 5 show an embodiment in which an array structure in which metal nanoparticle composites are two-dimensionally arranged is formed in the biochip of the present invention.
In the biochip 32 shown in FIG. 4, on the substrate 21, a plurality of phospholipid-modified gold nanorods 13 are each in parallel with the surface of the substrate 21, one layer, 6 rows in the X direction × 4 rows in the Y direction. An arrayed two-dimensional horizontal array structure is formed. In the two-dimensional lateral arrangement structure, the antibody 15 is bonded to a part or all of the phospholipid-modified gold nanorods 13 via a crosslinker linker (not shown) to form the gold nanorod complex 10.
In the biochip 33 shown in FIG. 5, a two-dimensional longitudinal array in which a plurality of phospholipid-modified gold nanorods 13 are stacked on a substrate 21 so as to be perpendicular to the surface of the substrate 21 and in contact with one layer and a long axis surface. A structure is formed. In the two-dimensional longitudinal array structure, the antibody 15 is bound to a part or all of the phospholipid-modified gold nanorods 13 via a cross-linking linker part (not shown) to form the gold nanorod complex 10.
As shown in FIGS. 4 to 5, the above-described requirements (a) and (b) mainly affect the alignment structure in which the metal nanorod composite 10 is aligned perpendicularly or parallel to the substrate 21. By precisely controlling these requirements, a desired alignment structure can be selectively produced. For example, by using a micro-channel or a nano-channel and depositing the dispersion liquid from a certain direction, the metal nanorod composites 10 are aligned in parallel to the substrate 21 in a certain direction. A structure can be formed.

Further, by repeating the step of forming the one-dimensional or two-dimensional array structure, it is possible to form an array structure in which the metal nanoparticle composites are arrayed in three dimensions. Here, “a three-dimensional array structure” indicates an array structure that is integrated and arranged in two or more layers on a substrate.
FIGS. 6 to 8 show an embodiment in which an array structure in which metal nanoparticle composites are arranged three-dimensionally is formed in the biochip of the present invention.
In the biochip 34 shown in FIG. 6, a two-dimensional lateral arrangement structure in which a plurality of phospholipid-modified gold nanorods 13 are arranged in one layer and 5 × 4 rows on the substrate 21 in parallel with the surface of the substrate 21. However, all have a three-dimensional array structure in which four layers are stacked in the same array direction. In the three-dimensional lateral arrangement structure, the antibody 15 is bound to a part or all of the phospholipid-modified gold nanorods 13 via a cross-linking linker part (not shown) to form the gold nanorod complex 10.
In the biochip 35 shown in FIG. 7, a two-dimensional vertical array structure in which a plurality of phospholipid-modified gold nanorods 13 are arranged in 6 × 8 rows on the substrate 21 perpendicular to the surface of the substrate 21, respectively. A stacked three-dimensional array structure is formed. In the three-dimensional lateral arrangement structure, the antibody 15 is bound to a part or all of the phospholipid-modified gold nanorods 13 via a cross-linking linker part (not shown) to form the gold nanorod complex 10.
In the biochip 36 shown in FIG. 8, a two-dimensional lateral arrangement structure in which a plurality of phospholipid-modified gold nanorods 13 are arranged on the substrate 21 in parallel with the surface of the substrate 21 and in contact with the minor axis surface, A three-dimensional array structure (three-dimensional raft structure) is formed by alternately stacking six layers of one-dimensional or two-dimensional array structures arranged in contact with the major axis surface. In the three-dimensional lateral arrangement structure, the antibody 15 is bound to a part or all of the phospholipid-modified gold nanorods 13 via a cross-linking linker part (not shown) to form the gold nanorod complex 10.
At this time, in order to control the alignment structure more precisely, the alignment structure can be controlled more precisely by using microchannels or nanochannels and flowing and depositing the dispersion from a certain direction. it can. For example, the first layer is formed by flowing the dispersion in a certain direction, and then the second layer is formed by changing the flow direction of the dispersion by 90 degrees, and this is repeated further, as shown in FIG. Thus, it is possible to construct a three-dimensional raft structure in which columns and rows are alternately changed for each layer.

  Since phospholipids are very fluid molecules, after placing the metal nanoparticle composite in the composite placement portion as described above, heat treatment is performed to such an extent that the functional molecules are not destroyed. It is also possible to rearrange the complex.

As described above, in the biochip in which the array structure in which the metal nanoparticle composite is arranged one-dimensionally, two-dimensionally or three-dimensionally is formed in the composite placement portion, the orientation structure of the metal nanoparticles is precisely controlled. Therefore, a local strong electric field appears between adjacent metal nanoparticles due to the surface plasmon coupling phenomenon. The spectroscopic signals of the analyte molecules arranged between the gold nanoparticles, for example, optical signals such as Raman spectroscopy, fluorescence spectroscopy, infrared spectroscopy, reflected light, near-field light, and surface plasmon spectroscopy are dramatically amplified and observed. For this reason, if such a biochip is used, high-sensitivity observation on a single molecule scale becomes possible.
In conventional methods, when detecting sample molecules themselves or detecting dye molecules in biological tissues labeled with fluorescent dyes, the signal intensity per molecule is weak and observation is unstable due to quenching action. Thus, one detection point could not be reduced so much.
However, in the present invention, high sensitivity at the single molecule level can be expected, so it is possible to track the dynamic behavior of single biomolecules in tissues and to detect and quantify biomolecular interactions per single molecule. It becomes.
Since the optical signal enhancement effect by plasmon coupling as described above can be obtained, the biochip of the present invention utilizes the enhancement effect to detect and detect the target molecule by detecting the enhanced optical signal. -It is preferably used as a light-enhanced detection type biochip for quantitative determination. Thereby, diagnosis, detection, quantification, etc. of the target molecule can be carried out with high sensitivity at the single molecule level.
In the biochip of the present invention, in consideration of the optical signal enhancement effect by plasmon coupling as described above, the interval between the metal nanoparticle composites in the array structure is adjusted to 1 to 5 nm. Preferably it is. As described above, the interparticle spacing can be easily controlled according to the hydrocarbon chain length of the phospholipid molecule modified on the surface of the metal nanoparticle.

The biochip of the present invention preferably has a plurality of complex arrangement portions. In addition, it is preferable that metal nanoparticle complexes having different types and concentrations of functional molecules or different types or concentrations of functional molecules are arranged in the plurality of complex arrangement portions, respectively.
Usually, when diagnosing / detecting a biomolecule, a sample contains many analyte molecules. Therefore, the biochip is required to have a device structure in order to simultaneously determine and quantify the molecules of the multiple samples in a short time in a molecular selective manner. That is, it is desirable to provide a biochip with a high-throughput screening function for biomolecules as specimens.
In the present invention, a plurality of complex arrangement portions are provided, and the types and concentrations of functional molecules or metal nanoparticle complexes having different types or concentrations of functional molecules are arranged in the plurality of complex arrangement portions, respectively. By doing so, such a high-throughput screening function can be provided.

Examples of the element structure of such a biochip include a microchannel type and a multi-array type.
In the micro-channel type element structure, in addition to the composite placement portion, a sample placement portion for placing a sample, and a micro-flow passage connecting the sample placement portion and the composite placement portion are provided on the substrate. In addition, a reagent arrangement section for arranging the reagent, wiring, and the like are provided as necessary.
FIG. 9 shows an embodiment in which the biochip of the present invention has a micro-channel device structure. In the biochip 37 of the present embodiment, three sample placement units 22, one reagent placement unit 23, and one detection unit 24 (drain) are provided on the substrate 21. In addition, composite arrangement portions 25a to 25c in which gold nanorod complexes formed by chemically bonding different types of antibodies a to c to the phospholipid-modified gold nanorods 13 are provided.
In addition, each sample placement unit 22, reagent placement unit 23, and complex placement unit 25 are connected to each other by a micro flow channel 26. Here, the “micro channel” indicates a channel having a channel width of micrometer size. The channel width is preferably about 1 to 100 μm. The microchannel 26 can be easily manufactured by a known method using a material such as polydimethylsiloxane (PDMS).
A wiring 27 is provided on the substrate 21 across the microchannel 26. By connecting a voltage application device via the wiring 27, it is possible to apply a voltage to the sample flowing through the microchannel 26 and cause a reaction thereby.
Diagnosis / detection using the microchannel biochip 37 can be performed, for example, by the following procedure. First, different samples A to C are arranged on the three sample placement portions 22, respectively. Then, each sample flows into the microchannel and is mixed. A voltage is arbitrarily applied to the mixed sample, a reagent is mixed, etc., and only the reaction product (with impurities removed) in the mixed sample is separated and allowed to flow into the complex arrangement portions 25a to 25c. . At this time, for example, when a target molecule that reacts with the antibody a is present in the sample, the target molecule is bound to the gold nanorod complex in the complex arrangement portion 25a. For this reason, when the spectroscopic measurement of each of the composite placement portions 25a to 25c is performed using an optical probe or the like, an increase in light intensity due to the surface enhancement effect is observed only in the composite placement portion 25a.
By using such a microchannel biochip, it is possible to identify, quantify, and diagnose target molecules contained in a sample in a molecular selective manner with high sensitivity.

In the multi-array type element structure, a plurality of independent complex arrangement portions are provided on a substrate.
FIG. 10 shows an embodiment in which the biochip of the present invention has a multi-array element structure. In the biochip 38 of the present embodiment, a gold nanorod composite in which different types of antibodies a to e are chemically bonded to the phospholipid-modified gold nanorods 13 on the substrate 21 in 8 rows in the short side direction × 3 rows in the long side direction. The dot-shaped composite body arrangement | positioning parts 25a-25e by which the body was arrange | positioned are formed. Moreover, in each complex arrangement | positioning part 25a-25e, the arrangement | positioning number of each gold nanorod complex, ie, the density | concentration of the antibody in each complex arrangement | positioning part, is changed in steps in the short side direction.
Such a multi-array biochip 38 can be produced, for example, by spotting a dispersion liquid having metal nanorod complexes, to which different antibody species are bound, at different antibody concentrations, on a substrate.
The substrate used here is not particularly limited, and can be appropriately selected from substrates of various materials as described above, for example, a cover glass, a slide glass, a water-repellent polymer-coated glass, a silicon substrate, and the like.
The spotting method is not particularly limited, and the known methods mentioned above as the method of arranging the dispersion on the substrate can be used. For example, as a spotting method using a casting method, a method using a solution discharge dispenser, a capillary method (a method in which the dispersion is pressed onto a substrate and applied by a capillary effect), an ink jet method, and the like can be given.
In the diagnosis / detection using the multi-array biochip 38, for example, a target sample is arranged in each sample arrangement unit, and the spectral arrangement using the optical probe is performed on each complex arrangement unit in the same manner as described above. Can be implemented. For example, when the sample includes a fluorescently labeled secondary antibody against the antibody a, the secondary antibody binds to the gold nanorod complex in which the antibody a is chemically bonded to the phospholipid-modified gold nanorod 13. In the complex arrangement part 25a where is arranged, an increase in the light intensity of the fluorescence spectrum due to the surface enhancement effect is observed.
By using such a multi-array biochip, it becomes possible to simultaneously detect and detect a plurality of target molecules contained in a sample with high efficiency and high sensitivity.

EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited to these Examples at all.
<Production Example 1 (Production of gold nanorods)>
100 μL of acetone (65 μL), cyclohexane (45 μL) and silver nitrate (10 mM aqueous solution) as a growth promoting substance in 3 mL of an aqueous solution in which gold chloride (III) acid (2 mM) and hexadecyltrimethylammonium bromide (80 mM) were dissolved. It was dripped. Furthermore, 200 microliters of ascorbic acid (40 mM aqueous solution) which is a reducing agent of chloroauric acid was added, and it stirred for 10 minutes at room temperature, and obtained the transparent gold salt solution.
Next, this gold salt solution was transferred to a 1 cm quartz cell and irradiated with light for 10 minutes using a UV light source (UV LIGHT SOURCE, HOYA-SCHOTT, HLS 100UM) while stirring. At that time, light in the visible region was cut using an ultraviolet transmission filter (Sigma light machine, UTVAF-50S-33U), and only a specific wavelength near 254 nm was irradiated. As a result, an aqueous dispersion in which gold nanorods were dispersed was obtained. The gold nanorods had a rod diameter of about 10 nm and a rod length of about 40 nm (average aspect ratio of 3.9).
The gold nanorods were stably dispersed in water without aggregation even after several days. This is presumably because the surface is coated with a surfactant, hexadecyltrimethylammonium bromide.

<Production Example 2 (Production of Phospholipid-Modified Gold Nanorod)>
Phospholipid molecules were dispersed by adding pure water to 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (sodium salt) and subjecting to ultrasonic treatment (room temperature, 10 to 15 minutes). An aqueous dispersion (phospholipid molecule concentration 1 mM) was prepared. 100 μL of this aqueous dispersion was dropped into 100 μL of a gold nanorod-containing liquid and stirred at room temperature for 10 to 15 minutes to obtain an aqueous dispersion of phospholipid-modified gold nanorods.
The phospholipid-modified gold nanorods were stably dispersed in water without aggregation even after several days. This is presumed to be because the surface is coated with phospholipid molecules.
In addition, the gold nanorod-containing liquid used here was previously centrifuged (10000 rpm) in order to increase the efficiency of the complexing reaction by exposing the gold nanorod aqueous dispersion obtained in Production Example 1 as much as possible. 15 minutes, 10 ° C.), removal of the supernatant, and redispersion in pure water were repeated twice to remove and purify excess surfactant.

The following evaluation was performed for each of the aqueous dispersion of gold nanorods obtained in Production Example 1 and the aqueous dispersion of phospholipid-modified gold nanorods obtained in Production Example 2.
[Absorption spectrum measurement]
The absorption spectrum (wavelength 300 to 900 nm) of each aqueous solution was measured. The results are shown in FIG.
As shown in FIG. 11, the gold nanorods showed two absorption maxima at 690.5 nm and 522.0 nm. They are peaks corresponding to localized surface plasmon absorption derived from the major axis and minor axis of the rod, respectively.
On the other hand, in the phospholipid-modified gold nanorods, peaks corresponding to the major axis and minor axis of the rod are observed at 694.0 nm and 529.5 nm, respectively, and each peak is slightly shifted to the longer wavelength side compared to the gold nanorods. Was. Moreover, the peak was broadened, and this result suggested that aggregates were formed in water. That is, it is presumed that the aqueous dispersion of phospholipid-modified gold nanorods contains aggregated particles in which adjacent phospholipid-modified gold nanorods aggregate due to the interaction of phospholipid molecules.
In addition, the light absorbency shown in the graph of FIG. 11 is the value which normalized the light absorbency measured about each aqueous solution with the peak top of a long wavelength side.

[Dynamic light scattering measurement]
Moreover, about each water dispersion liquid, the dynamic light-scattering measurement was performed and the particle size distribution of each gold nanorod and each phospholipid modification gold nanorod in each aqueous solution was observed.
As a result, the average particle size of the gold nanorods was 78.4 nm, and the average particle size of the phospholipid-modified gold nanorods was 173.0 nm.
In the particle size distribution measurement by dynamic light scattering measurement, there is a tendency to show an average particle size value slightly larger than the actual particle size. However, in view of this point, the average particle size of the phospholipid-modified gold nanorod is It was significantly larger than the nanorods, suggesting that phospholipid-modified gold nanorods may form aggregated particles in water.

<Example 1 (production of gold nanorod composite)>
(1-1: Modification of antibody ends with a crosslinking linker)
Dispense 50 μL of a stock solution (Alexa Floor 488 rabbit anti-mouse IgG (H + L), 2.0 mg / mL) of anti-IgG antibody labeled with fluorescent dye Alexa (Alexa-labeled anti-IgG antibody), and 150 μL of phosphoric acid A solution with a protein concentration of 0.1 mg / 200 μL was prepared by dissolving in a buffer solution (PBS buffer, 0.1 M, 0.15 M sodium chloride, pH 7.2).
Further, DTSSP (3,3′-dithiobis (sulfosuccinimidylpropionate)) as a crosslinking linker agent was dissolved in pure water to prepare a 10.0 mM solution.
After mixing these two solutions, they were gently shaken and incubated at room temperature (23 ° C.) for 3 hours. As a result, a solution containing an anti-IgG antibody whose end was modified with a crosslinking linker agent (crosslinking linker-modified anti-IgG antibody) was obtained as a reaction product.

(1-2: Complexation)
After mixing 50 μL of the gold nanorod aqueous dispersion obtained in Production Example 2 and 50 μL of the solution obtained in (1-1) above, the mixture was gently shaken and incubated overnight at room temperature. Thereafter, centrifugation (10,000 rotations / minute, 15 minutes, 10 ° C.), supernatant removal, and redispersion in phosphate buffer are repeated several times to purify the unreacted anti-IgG antibody and impurities. To obtain an aqueous dispersion of the gold nanorod composite.

The following evaluation was performed about the aqueous dispersion liquid of the obtained gold nanorod composite.
[Absorption spectrum measurement]
The absorption spectrum (wavelength 300 to 900 nm) of the aqueous dispersion was measured. The results are shown in FIG.
As shown in FIG. 11, in the gold nanorod composite, absorption peaks derived from localized surface plasmons were observed at 701.5 nm and 530.0 nm. The shape of the peak was the same as that of the phospholipid-modified gold nanorod obtained in Production Example 2.
In addition, the light absorbency shown in the graph of FIG. 11 is the value which normalized the light absorbency measured about each aqueous solution of a gold | metal | money nanorod, a phospholipid modification gold | metal | money nanorod, and a gold | metal | money nanorod complex with the peak top on the long wavelength side.
In addition, the Alexa-labeled anti-IgG antibody stock solution used for the production of the gold nanorod composite had an absorption peak at 494.5 nm, but the aqueous dispersion of the gold nanorod composite was clearly shown at 494.5 nm. No absorption peak was confirmed. This is probably because the concentration of Alexa-labeled anti-IgG antibody surface-modified to gold nanorods is low, which may be hidden in the absorbance of gold nanorods and cannot be clearly identified as a peak. .

[Fluorescence intensity measurement]
The fluorescence spectrum (wavelength 450 to 700 nm) of the aqueous dispersion was measured. Further, as a reference, the same measurement was carried out for the gold nanorod aqueous dispersion, the phospholipid-modified gold nanorod aqueous dispersion, and the Alexa-labeled anti-IgG antibody stock solution. The results are shown in FIG.
As shown in FIG. 12, in the gold nanorod complex, a fluorescence peak having a maximum at 516.0 nm was observed, and it was confirmed that the Alexa-labeled anti-IgG antibody was coupled to the phospholipid-modified gold nanorod.
On the other hand, gold nanorods and phospholipid-modified gold nanorods did not exhibit fluorescence properties, and the Alexa-labeled anti-IgG antibody itself had a fluorescence peak at 513 nm.
The fluorescence intensity shown in the graph of FIG. 12 is a value obtained by standardizing the fluorescence intensity measured for the gold nanorod complex aqueous dispersion and the stock solution of Alexa-labeled anti-IgG antibody at the peak top.

[Flow cytometry measurement]
Flow cytometry measurement of the aqueous dispersion was performed. For reference, the same measurement was performed on an aqueous dispersion of phospholipid-modified gold nanorods. In flow cytometry, the particle size distribution of single particles in the sample flowing in the liquid is observed from the forward scattering intensity indicated on the X axis, and the fluorescent molecules in each single particle are determined from the fluorescence intensity indicated on the Y axis. The presence or absence of a component is observed.
The measurement results of the phospholipid-modified gold nanorods are shown in FIGS. 13 (a) to (b), and the measurement results of the gold nanorod composite are shown in FIGS. 13 (c) to (d).
From these results, in the phospholipid-modified gold nanorods, no fluorescent component was observed for each particle, but in the gold nanorod complex, as the particle size slightly increased, nanoparticles having a fluorescent component were observed, It was confirmed that Alexa-labeled anti-IgG antibody was coupled to phospholipid-modified gold nanorods.

[Microscopic observation]
FIG. 14 shows a transmission micrograph of the gold nanorod composite (FIG. 14A, observation: under white light, using a 10 × objective lens) and a fluorescence micrograph (FIG. 14B, excitation: mercury lamp, observation: green) Using a fluorescent filter in the region, using a 10 × objective lens). FIG. 15 shows a laser fluorescence micrograph of a gold nanorod composite (excitation: 488 nm argon laser, observation: using a fluorescent filter for 505-525 nm region observation, FIG. 15 (a) using a 10 × objective lens, FIG. (B) shows a partially enlarged image thereof).
In each observation, 5 μL of the gold nanorod composite aqueous dispersion was dropped on a slide glass and dried, and the observation was performed using the sample as a sample.
In the transmission microscope image of FIG. 14 (a), a gold nanorod composite dried in a stripe shape is observed, and in the fluorescence microscopic image of FIG. 14 (b), the dry stripe of the gold nanorod composite confirmed by the transmission microscope image. A green fluorescent image was observed along the line. This also demonstrated the coupling between Alexa-labeled anti-IgG antibody and phospholipid-modified gold nanorods. Further, in FIG. 14B, since no fluorescent component is seen in the region other than the dry stripes of the gold nanorod complex, the unreacted Alexa-labeled anti-IgG antibody is not mixed in the aqueous dispersion and purified. It was confirmed that it was almost removed during the process.
Similarly, also in the laser fluorescence microscope image of FIG. 15A, the state of the gold nanorod composite having a fluorescent component was observed more clearly. In the partially enlarged image of FIG. 15 (b), large dot-like particles were observed, suggesting the presence of aggregating components of the gold nanorod composite.

<Example 2 (Manufacture of biochip)>
5 μL of the phospholipid-modified gold nanorod aqueous dispersion obtained in Production Example 2 was dropped on a cover glass, dried at room temperature and in the atmosphere, and the solvent was removed. Subsequently, 60 to 100 μL of a phosphate buffer solution was dropped at the position where the phospholipid-modified gold nanorod aqueous solution was dropped, and then 5 μL of an IgG (mouse-IgG, primary antibody) solution whose end was modified with a crosslinking linker was added thereto. Added and incubated at room temperature for 30 minutes to 1 hour. By doing so, a biochip in which a gold nanorod complex in which IgG was chemically bonded to a phospholipid-modified gold nanorod was arranged on the surface was produced. Thereafter, in order to remove the unreacted IgG on the biochip surface, a washing operation was performed several times using a phosphate buffer.
In addition, the IgG (mouse-IgG, primary antibody) solution whose end was modified with a crosslinking linker used here was IgG (mouse-IgG) instead of Alexa-labeled anti-IgG antibody in (1-1) of Example 1. It was prepared in the same manner except that IgG, a primary antibody) was used.
On the surface of a hydrophilic substrate such as a cover glass, as described above, the phospholipid-modified gold nanorods expand along the surface due to the self-organization ability of the phospholipid molecules, and the gold nanorods do not overlap with each other. Form a structure. Therefore, phospholipid-modified gold nanorods can be two-dimensionally and densely integrated by performing the above treatment.

<Example 3 (Production of multi-array biochip)>
A gold nanorod complex in which an anti-IgG antibody is coupled to a phospholipid-modified gold nanorod in the same manner as in Example 1 except that an anti-IgG antibody (secondary antibody) solution is used instead of the Alexa-labeled anti-IgG antibody stock solution. An aqueous dispersion was obtained.
10 nL of this aqueous dispersion is discharged onto a substrate (material: slide glass or silicon, long side 75 mm × short side 25 mm) at intervals of 1 mm in length and width using a solution discharge dispenser, and dried at room temperature and in the atmosphere. gave. At this time, the aqueous dispersion was discharged in a total of 50 spots, 5 spots in the long side direction and 10 spots in the short side direction. Each dot after drying had a diameter of about 200 μm. In addition, adjacent dots did not overlap and were arranged in a multi-array with good position controllability.
Separately, an aqueous dispersion of 4 types of gold nanorod complexes was used in the same manner as above except that the anti-IgG antibody was changed to 4 types of heterologous antibodies (anti-IgA antibody, anti-IgD antibody, anti-IgE antibody, anti-IgM antibody). Got. Each of these aqueous dispersions was ejected onto the substrate in 50 spots in the same manner as described above, and dried at room temperature and in the atmosphere.
As a result, a multi-array biochip having a total of 250 spots each having 50 spots each having 5 types of antibodies immobilized thereon was obtained.

<Test Example 1 (Detection of interaction between biomolecules using a biochip)>
In the measurement system using the total reflection fluorescence microscope (TIRFM) schematically shown in FIG. 16 using the biochip produced in Example 2, biomolecular interaction between antibodies was detected by the following procedure. .
On the biochip 61, 60-100 μL of a phosphate buffer was dropped to form droplets 62. 5 μL of Alexa-labeled anti-IgG antibody stock solution (same as that used in Example 1) was dropped into the droplet 62. Droplets 62 after 0 and 30 minutes after the dropping were observed with TIRFM (excitation: 488 nm argon laser, objective lens: 150 times).
In the measurement system shown in FIG. 16, a dichroic mirror 63 is used as a reflecting mirror, and excitation light is incident on a place off the center of the objective lens 64 on the microscope side by epi-illumination by an oil immersion objective lens. Total reflection is generated at the boundary surface of the chip 61. Then, an evanescent field is formed on this boundary surface, and excitation light is irradiated only in a predetermined thickness range on the droplet 62 side. Therefore, the fluorescence generated within the range irradiated with the excitation light can be measured. In this example, fluorescence in a region (nanointerface) within 100 nm from the interface with the biochip 61 in the droplet 62 was measured.

The results are shown in FIG. FIGS. 17A and 17C show TIRFM photographs after 0 minutes and 30 minutes, respectively. In each photograph, the black part is the cover glass surface of the biochip 61, the gray part is a gold nanorod complex to which IgG (primary antibody) is bound, and the white bright spot part is an Alexa-labeled anti-IgG antibody (secondary antibody). Indicates. FIGS. 17B and 17D are images obtained by setting only a threshold value for contrast in an imaging image obtained after 0 minutes and 30 minutes and extracting only a white bright spot portion.
As shown in FIGS. 17 (a) and (b), no white bright spot is confirmed at the start of the reaction, but as shown in FIGS. 17 (c) and 17 (d), after 30 minutes from the start of the reaction, the biochip Above, many white bright spots derived from the anti-Alexa labeled IgG antibody were observed. Considering the size of the white bright spot, the uniformity of the diameter, and the resolution of the measurement system, each white bright spot has a single molecular unit Alexa-labeled anti-IgG antibody on the surface of the biochip 61. It seems to indicate that it is selectively adsorbed only on the gold nanorod composite.
From this result, it was shown that the detection of the reaction between biomolecules can be performed position-selectively and with high sensitivity at the single molecule level by using the biochip.

<Test Example 2 (observation of time-dependent change in interaction between biomolecules)>
In Test Example 1, the droplet 62 after 15 minutes, 45 minutes, 75 minutes, and 90 minutes after dropping the stock solution of Alexa-labeled anti-IgG antibody was observed with TIRFM as in Test Example 1. As a result, the binding reaction of the Alexa-labeled anti-IgG antibody to the surface of the biochip 61 was observed over time.
FIGS. 18A to 18D are images obtained by extracting only a white bright spot portion by setting a threshold value in contrast in imaging images obtained after 15 minutes, 45 minutes, 75 minutes, and 90 minutes, respectively. . The number of white bright spots is also shown in each figure. As shown in these results, it was confirmed that the number of white bright spots increased as time passed from the start of the reaction.
Further, FIG. 19 shows a graph in which the change in the number of white bright spots with respect to the reaction time is plotted from the number of white bright spots measured at this time. From this graph, it is clear that when the biochip is used, the change in the number of secondary antibodies adsorbed on the biochip surface with time can be quantitatively analyzed.
FIG. 20 is a graph of the histogram of the average luminance for each white luminescent spot (the luminance was analyzed by image analysis software “MetaMorph” mounted on the Olympus TIRFM apparatus) for each reaction time. The figure is shown. As shown in the graph, each white bright spot showed almost stable luminance even when the reaction time passed.
From these results, the biochip of the present invention and the measurement system using the same can perform non-contact detection, diagnosis, quantification, and the like. It was shown that high-sensitivity measurement can be performed stably for a long time.

<Test Example 3 (Comparative Test)>
5 μL of an aqueous dispersion of only the phospholipid-modified gold nanorods (without antibody application) obtained in Production Example 2 was dropped on a cover glass, and dried at room temperature and in the atmosphere to remove the solvent.
On the surface of the cover glass, 100 μL of a phosphate buffer is dropped to form a droplet, and 5 μL of Alexa-labeled anti-IgG antibody stock solution (same as that used in Example 1) is dropped into the droplet. did. In the same manner as in Test Example 1, the droplet 30 minutes after the dropping was observed with TIRFM. As a result, as shown in FIG. 21A, no white bright spot was observed at the nanointerface even 30 minutes after the start of the reaction.
From this result, it was confirmed that Alexa-labeled anti-IgG antibody 66 could not be detected with high sensitivity when phospholipid-modified gold nanorods did not form a complex with the antibody. This is probably because the Alexa-labeled anti-IgG antibody 66 does not bind to the phospholipid-modified gold nanorod 13 as shown in FIG.

The metal nanoparticle composite of the present invention has high biocompatibility because the surface of the metal nanoparticle is chemically modified with phospholipid molecules, and also has high controllability of arrangement on the substrate, and high density on the substrate. Therefore, it is useful as a biochip. The biochip can be used as a biomolecule diagnosis / test chip.
In addition, the metal nanoparticle composite of the present invention is a stable nanoparticle and has excellent durability, and for example, fading after laser irradiation is suppressed. Therefore, the dynamics of the target biomolecule on the biochip can be observed stably for a long time.
In addition, according to the present invention, it is possible to provide a biochip on which a functional protein such as an antibody, an enzyme, or a membrane protein is immobilized as a molecular recognition unit. By using the biochip, biological reactions such as antibody reaction (antibody-antibody reaction, antigen-antibody reaction), enzyme reaction and the like can be observed.
Further, in the present invention, a biochip provided with a high-sensitivity biosensing function having a single-molecule level resolution can be provided by utilizing the enhancement effect of the photoelectron signal, which is a characteristic unique to metal nanoparticles.
Moreover, in the biochip of the present invention, not only the functional protein but also DNA and cells can be immobilized.
Therefore, it is possible to combine the role of a sensor that detects interactions between various biomolecules and cellular metabolites, and the function as a high-throughput screening element that enables quantitative analysis of biomolecules.
Furthermore, since the metal nanoparticle composite of the present invention has high biocompatibility, it can be introduced into living tissues such as cells. Therefore, it can utilize as a cell labeling material (biomarker) which can stain | stain a specific cell nondestructively. Moreover, since it is excellent in durability as described above, the intracellular dynamics of the target biomolecule can be stably observed for a long time. Therefore, highly sensitive bioimaging analysis becomes possible by using the metal nanoparticle composite of the present invention as a biomarker.

  DESCRIPTION OF SYMBOLS 10 ... Gold nanorod complex, 12 ... Phospholipid molecule, 13 ... Phospholipid modification gold nanorod, 14 ... Crosslinking linker part, 15 ... Antibody, 21 ... Substrate, 22 ... Sample arrangement part, 23 ... Reagent arrangement part, 24 ... Detection Part, 25a to 25e ... complex arrangement part, 26 ... micro flow path, 27 ... wiring, 31-38 ... biochip, 61 ... biochip, 62 ... droplet, 63 ... dichroic mirror, 64 ... objective lens, 65 ... Immersion oil, 66 ... Alexa-labeled anti-IgG antibody

Claims (10)

  A metal nanoparticle composite, wherein a functional molecule is chemically bonded to a metal nanoparticle whose surface is chemically modified with a phospholipid molecule via a cross-linking linker moiety.   The metal nanoparticle composite according to claim 1, wherein the metal nanoparticles are metal nanorods.   3. The metal nano of claim 1, wherein the functional molecule is at least one functional protein selected from the group consisting of an antibody, an antigen, an enzyme, a membrane protein, a receptor protein, a cytoskeleton, and a motor protein. Particle complex. A method for producing a metal nanoparticle composite according to any one of claims 1 to 3,
A method for producing a metal nanoparticle composite, comprising a step of chemically bonding a metal nanoparticle whose surface is chemically modified with a phospholipid molecule and a functional molecule using a crosslinking linker agent.   The manufacturing method of the metal nanoparticle composite_body | complex of Claim 4 in which the said crosslinking linker agent has the succinimidyl group which may have a substituent, and a sulfur-containing organic substituent.   The biochip which has a composite_body | complex arrangement | positioning part by which the metal nanoparticle composite_body | complex as described in any one of Claims 1-3 is arrange | positioned on a board | substrate.   The biochip according to claim 6, wherein an array structure in which the metal nanoparticle composites are arranged one-dimensionally, two-dimensionally or three-dimensionally is formed in the complex arrangement part.   The biochip according to claim 6 or 7, wherein the biochip has a plurality of the complex arrangement parts, and the types and concentrations of the functional molecules or the types or concentrations of the functional molecules in the plurality of complex arrangement parts are different. It is the manufacturing method of the biochip as described in any one of Claims 6-8,
On the substrate, the metal nanoparticles constituting the metal nanoparticle composite are developed using the self-organization ability of phospholipid molecules that chemically modify the surface thereof, and the metal nanoparticle composite is one-dimensional or two-dimensional. A biochip manufacturing method comprising a step of forming a three-dimensional array structure.   The metal nanoparticle composite is formed in an array structure in which the metal nanoparticle composite is arranged in three dimensions by repeating the step of forming an array structure in which the metal nanoparticle composite is arrayed in one or two dimensions a plurality of times. Biochip manufacturing method.