JP2007292564A - Biosensor chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosensor chip that has very high detection sensitivity, capable of detecting a trace amount of biological substance. <P>SOLUTION: In the biosensor chip that uses a fluorescent method as the principle of detection, a fixing part (A) of a specific bonding substance (X) and a photonic crystal (B) for covering a part of the fixing part (A) are installed. In the biosensor chip that employs a biological molecule detecting method, fluorescence is made to be reflected and amplified between the photonic crystals (B), and the amplified fluorescence is measured by a fluorescence analyzer. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a biosensor chip. In particular, the present invention relates to a biosensor chip with high detection sensitivity that can be used for clinical diagnostic applications.

Today, there are various DNA chips, protein chips, immunoreagents, etc. for basic research such as genetic analysis and protein function elucidation, development of new drugs using the analyzed data, and clinical diagnosis of cancer, hormones, myocardial infarction, etc. Used in various research and medical institutions.
The turbidimetric method, the agglutination method, the surface plasmon method, the fluorescence method, and the luminescence method have been developed as the detection principle of biomolecules, but the fluorescence method and the luminescence method have become mainstream from the viewpoint of detection sensitivity. Both the fluorescence method and the luminescence method can detect even a very small amount of biological material contained in a sample such as blood.

  The fluorescence method is a technique in which a biomolecule on which a fluorescent substance such as a fluorescent dye is immobilized is used as a label, the fluorescent substance is fluorescently emitted by excitation light such as a laser, and a physical quantity is measured from visualization or fluorescence intensity. Although the detection sensitivity is slightly inferior to that of the luminescence method, it is a widely used method because the reagent is highly stable, easy to handle, and the analyzer is relatively inexpensive.

  Although it can be said that the detection sensitivity of the fluorescence method and the luminescence method is very high at present, there is still a strong need for improving the detection sensitivity. This is because if the detection sensitivity is further improved, an extremely small amount of biomolecules that cannot be detected by the conventional method can be detected, and a new diagnostic marker or the like can be expected. In addition, there is an effect that the sample amount is greatly reduced, the measurement time is shortened and the measurement accuracy is improved. In particular, further improvement in detection sensitivity is strongly desired for the fluorescence method having the above-described excellent characteristics.

  As a method for improving detection sensitivity, for example, in an immunoassay method of an antibody using a solid phase, avidin or streptavidin and their biotinylated polymer substance immobilized on a solid phase carrier are immobilized on an antibody in a sample. An antibody immunoassay method (Patent Document 1) characterized by reacting a solid phase to which a derivative is bound, a biotinylated peptide antigen and a labeled peptide antigen simultaneously, and fluorescence for observing an antigen-antibody reaction on the surface of an optical waveguide An immunoassay method comprising: a capturing step of bringing the optical waveguide into contact with a specimen and capturing the measurement target in the specimen on the surface of the optical waveguide; and a fluorescent chromophore on the measurement target captured in the capturing step A binding step of binding a labeled antibody having a concentration, and an excitation light is introduced into the optical waveguide to form a total internal reflection light, and an evanescent wave is emitted on the surface of the optical waveguide. A series of steps consisting of an excitation step for exciting the fluorescent chromophore and an observation step for observing light collected by propagating through the optical waveguide, and is repeated at least twice. There has been proposed a fluorescence immunoassay method characterized by measuring the rate of change of the observed light quantity with respect to the contact time (Patent Document 2).

However, none of these methods is a method for directly enhancing the fluorescence signal, and a significant improvement in detection sensitivity cannot be expected.
JP 9-178747 A JP 2004-205286 A

  That is, an object of the present invention is to provide a biosensor chip with extremely high detection sensitivity that can detect an extremely small amount of biological material that cannot be detected by a conventional method.

The biosensor chip of the present invention is a biosensor chip based on a detection method using a fluorescence method, and covers a fixed part (A) of a specific binding substance (X) and a part of the fixed part (A). It is characterized by installing a crystal (B). A biosensor chip employing a method for detecting a biological material, characterized in that fluorescence is reflected and amplified between photonic crystals (B) and the enhanced fluorescence is measured with a fluorescence analyzer.
That is, the present invention is a biosensor chip having a detection principle based on a fluorescence method, and a biosensor chip provided with a photonic crystal (B) that covers a fixing part (A) of a specific binding substance (X), and This is a method for detecting a biological substance using the biosensor chip.

The biosensor chip of the present invention enhances the fluorescence signal directly by the photonic crystal (B), so it has extremely high detection sensitivity and can detect ultra-small amounts of biomolecules that cannot be detected by conventional methods. To. If a fluorescence analyzer is not used, it is possible to visualize those that could not be qualitatively analyzed, and excellent effects such as the ability to use new diagnostic markers can be expected. It is possible to greatly reduce the sample amount, shorten the measurement time, and improve the measurement accuracy.
In addition, since the fluorescence method is used as a detection principle, there are advantages of the fluorescence method that the reagent is highly stable, easy to handle, and the analyzer is relatively inexpensive.

As the fluorescence method of the present invention, a publicly-known known fluorescence method can be applied, using a biomolecule immobilized with a fluorescent substance as a label, causing the fluorescent substance to emit fluorescence with excitation light, This is a technique for measuring a physical quantity from visualization or fluorescence intensity.
A commercially available fluorescent material can be used as the fluorescent material. For example, fluorescein, rhodamine B isothiocyanate, lysamine-rhodamine B sulfonyl chloride, anilinonaphthalene sulfonic acid, 2-methoxy 2,4-diphenyl 3 (2H) furanone, methylumbelliferone, dansyl chloride, lucifer yellow, erythrosin, pyrene , Fluorescent dyes such as Texas Red, Eosin, Fluoresamine, and Chlorophyll.

The biological substance used for labeling is a target substance, that is, a biological substance that specifically binds to a substance that specifically binds to a specific binding substance (X) described later.
The fluorescent substance can be immobilized on the biological substance by a known method.
As the excitation light, known excitation light can be used, and a xenon lamp and various laser lights can be used. From the viewpoint of improving detection sensitivity, it is preferable to use laser light as excitation light.
The excitation light can be irradiated from various directions as long as the fixing part (A) can be irradiated, but it is preferable to irradiate from the surface covered with the photonic crystal (B). .

As will be described later, since the fluorescence signal is enhanced by reflection amplification by a photonic crystal, excitation is performed by two-photon absorption when the light absorption of the fluorescent substance is close to the fluorescence wavelength so that the excitation light is not reflected. It is preferable. Two-photon absorption is a method that uses two photons for excitation, and it is possible to use long-wavelength excitation light that is not reflected by the photonic crystal (B). In addition to two-photon absorption, a method using excitation light having a wavelength lower than that of the photonic band described later can be used.
A commercially available fluorescence analyzer etc. can be used for the measurement of fluorescence intensity. For example, a high-sensitivity fluorescence analyzer (manufactured by Taiyo Electric Co., Ltd.), a spectrofluorometer (manufactured by Hitachi High-Tech) can be used.

In the present invention, the specific binding substance (X) can be used without limitation as long as it is a substance that specifically binds to a target substance contained in a sample such as blood, for example, an immune protein, an enzyme, a microorganism, a nucleic acid, a low molecular weight organic substance. Examples thereof include compounds, non-immune proteins, immunoglobulin-binding proteins, sugar-binding proteins, sugar chains that recognize sugars, fatty acids or fatty acid esters, or polypeptides or oligopeptides having ligand-binding ability.
Examples of the blood sample include blood (serum, plasma), urine, stool, sputum, cerebrospinal fluid and the like.
The target substance that specifically binds to the specific binding substance (X) contained in the sample is the same as the specific binding substance (X). For example, when (X) is an antibody, the target substance is The antigen recognized by the antibody, for example, when (X) is a sugar chain, the target substance is a lectin, for example, when (X) is a gene, the target substance is a complementary gene.

  Examples of immune proteins include antigens, antibodies and haptens. As the antibody, an antibody against the following antigen can be used, and examples of the antigen include drugs, low molecular hormones, tumor markers, viruses, high molecular hormones, cytokines, various growth factors and the like. Examples of the drug include theophylline, phenytoin, valproic acid and the like. Examples of the low molecular hormone include thyroxine, estrogen, and estradiol. Examples of tumor markers include CEA, AFP, and CA19-9. Examples of viruses include HIV, HCV, HBV and the like. Examples of the polymer hormone include thyroid stimulating hormone, insulin and the like. Examples of cytokines include IL-1, IL-2, and IL-6. Examples of various growth factors include EGF, PDGF, and the like. These antibodies may be monoclonal antibodies or polyclonal antibodies, and may be F (ab ') 2, Fab', Fab which are degradation products of antibodies.

As an enzyme, an oxidoreductase, a hydrolase, an isomerase, a desorption enzyme, a synthetic enzyme, etc. can be used. Specifically, glucose oxidase can be used if the target substance is glucose, and cholesterol oxidase can be used if the target substance is cholesterol. In addition, when targeting pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc., target substances that are metabolized from them, such as acetylcholinesterase, Enzymes such as catecholamine esterase, noradrenaline esterase and dopamine esterase can be used.
The microorganism is not particularly limited, and various microorganisms including Escherichia coli can be used.

  As the nucleic acid, those that hybridize complementarily with the target nucleic acid can be used. As the nucleic acid, either DNA (including cDNA) or RNA can be used. The type of DNA is not particularly limited, and may be any of naturally derived DNA, recombinant DNA prepared by gene recombination technology, or chemically synthesized DNA. Chemical synthesis of DNA (including cDNA) and RNA can be performed using a commercially available synthesizer, or a commercially available DNA probe can be used as it is.

Examples of the low molecular weight organic compound include any compound that can be synthesized by an ordinary organic chemical synthesis method.
The non-immune protein is not particularly limited, and for example, avidin (streptavidin), biotin or a receptor can be used.
As the immunoglobulin-binding protein, for example, protein A or protein G, rheumatoid factor and the like can be used.
Examples of sugar-binding proteins include lectins. Examples of the fatty acid or fatty acid ester include stearic acid, arachidic acid, behenic acid, ethyl stearate, ethyl arachidate, and ethyl behenate.

The specific binding substance (X) is immobilized on the immobilization part (A), and a known method such as a chemical binding method or a physical adsorption method can be used as the immobilization method.
When the specific binding substance (X) is a protein such as an immunity protein or enzyme, the chemical binding method is to introduce a functional group such as an amino group or a sulfhydryl group into the fixing part (A) and perform specific binding. A method of crosslinking a functional group such as an amino group and a sulfhydryl group of the substance (X) with glutaraldehyde, succinaldehyde, m-maleimidobenzoyl-N-hydrosuccinimide ester, o-phenylenebismaleimide, etc. can be used (for example, US No. 4,280,992 and US Pat. No. 3,565,761).
In addition, a method in which a carboxyl group is introduced into the fixing part (A) and bound to the amino group of the specific binding substance (X) through carbodiimide can be mentioned. The case where a polystyrene resin having a carboxyl group on the surface is used as the material of the fixing part (A) will be specifically described. First, after reacting a carboxyl group and carbodiimide at room temperature for about 1 hour, unreacted carbodiimide is sufficiently washed away. Next, after adding a protein such as an AFP antibody and reacting for about 2 hours, the protein can be chemically immobilized by washing away the unreacted protein.

As a method by physical adsorption, for example, when the immobilization part (A) is polystyrene, a carbonate buffer solution (pH 9.0) containing 0.001 to 0.04% (W / V) of a specific binding substance (X) is used. A method of immersing the fixing part (A) for an appropriate time can be used (for example, described in BioSim Biophys Actor, Vol. 251, page 427, 1971).
When the specific binding substance (X) is a nucleic acid, a physical binding method is to impart a positive charge to the fixing part (A) and bind by electrical attraction. This method utilizes the fact that DNA has a negative charge, and is a known method such as a method of coating polylysine on the surface or a method of introducing an amino group by reacting aminosilane with the fixing part (A). Can be used.
As a method for immobilization, a chemical bonding method is preferable from the viewpoint of improving detection sensitivity.

  The immobilization part (A) on which the specific binding substance (X) is immobilized is a part that reacts with the target substance and the specific binding substance (X), and is irradiated with excitation light, and the fluorescence signal is emitted. This is the part that occurs. Specifically, the following operation is performed in the fixing portion (A). First, when a sample containing a target substance is supplied to the fixing part (A), the target substance and the specific binding substance (X) immobilized are reacted, and other substances than the target substance pass through or are washed. Is removed. Next, a fluorescent labeling substance that specifically binds only to the target substance on which the fluorescent substance is immobilized is supplied, and the fluorescent labeling substance is selectively bound only to the location where the target substance exists. Alternatively, the fluorescent labeling substance and the sample may be reacted in advance. By irradiating the bound fluorescent substance with excitation light, a fluorescent signal is generated.

  Therefore, the fixing portion (A) must be configured to perform the above-described operation. Specifically, since the liquid sample is supplied, the fixing portion (A) is preferably present in an analysis cell having a tube, a flow path, a fine container, and the like. Here, the analysis cell means a cell that can be used in a series of analysis operations such as sample supply, reaction between the target substance and specific binding substance (X), and sample discharge. An analysis cell capable of operations such as sample filtration and concentration can also be used.

As the analysis cell, cells having various shapes and sizes can be used. From the viewpoint of coating a photonic crystal (B) described later, a flat cell (C) is preferable. The size of the flat cell (C) is preferably 0.1 to 10 cm on one side and 0.1 to 1000 μm in thickness, more preferably 0.5 to 5 cm on one side and 0.3 to 100 μm in thickness, particularly preferably , One side is 1 to 3 cm, and the thickness is 0.5 to 10 μm.
The internal form of the analysis cell is a form that can accommodate the above-mentioned thin film structure, and from the viewpoint of reducing the amount of sample and improving the reaction efficiency, a micro-channel (D) with a channel diameter of about 1000-1 μm A structure is preferred.
The microchannel (D) can be produced by a nanoimprint method or the like.

The biosensor chip of the present invention has a structure in which a photonic crystal (B) covering the fixing part (A) is installed, that is, the fixing part (A) must be covered with the photonic crystal (B). .
The photonic crystal (B) preferably covers the fixed part (A) widely from the viewpoint of amplification of the fluorescent signal, and may cover only the fixed part (A) of the analysis cell, or capture the fluorescent signal. You may coat | cover all the analysis cells, after ensuring an exit. Covering with a photonic crystal must be performed on a specific part (surface) of the fixed part (A) and a part (surface) opposite to the specific part (surface) across the fixed part (A). Yes, by preparing two or more photonic crystals and sandwiching the analysis cell containing the fixed part (A) or fixed part (A) (Fig. 1), or fixed part (in the photonic crystal (B) A) and the like are listed.

In the latter method, it is necessary to produce a structure such as a flow path in the photonic crystal using a laser processing technique or the like. On the other hand, the former method does not require laser processing or the like, has high productivity, and can be said to be a more preferable method.
As a method of preparing two or more photonic crystals and sandwiching an analysis cell including a fixed part (A) or a fixed part (A), a plate-like or film-like photonic crystal (B) prepared by a method described later Is placed so as to sandwich the plate cell (C), and is heat-bonded or adhered using an adhesive.

This biosensor chip can dramatically improve the detection sensitivity by increasing the signal intensity by reflecting and amplifying the fluorescence signal with the photonic crystal. Here, the photonic crystal is a structure having a periodicity equivalent to the wavelength of light and having a light forbidden band called a photonic band gap.
Therefore, using a photonic crystal having a periodic structure corresponding to the wavelength of the fluorescence signal, the specific part (surface) of the fixed part (A) and the specific part (surface) across the fixed part (A) If the portion (surface) on the opposite side is covered, the fluorescence signal can be reflected, and the conditions of light amplification such as inversion distribution, stimulated emission, and reflection amplification can be achieved.

The enhanced fluorescence signal is taken out from the portion of the fixed part (A) that is not covered with the photonic crystal and measured by a fluorescence analyzer.
By setting a reflecting plate made of a metal such as silver having a high reflectance such as a portion not covered with the photonic crystal, the efficiency of extracting a fluorescent signal can be further increased. In this case, it is important not to cover a part, since the fluorescence signal cannot be taken out if all parts are covered. Specifically, when the analysis cell as shown in FIG. 1 is covered with a photonic crystal from above and below, it is preferable that one of the remaining four surfaces is not provided with a reflector.
The reflection plate can be installed by coating with metal or the like by attaching a film containing metal or the like after the biosensor chip is formed.
Moreover, these reflectors can also be installed on the opposite side of the photonic crystal with the fixed part (A) in between. In this case, a fluorescence signal is amplified between the photonic crystal (B) and the reflector.

Examples of the structure of the photonic crystal include a hexagonal close-packed structure, a cubic close-packed structure, a body-centered cubic packed structure, a log pile structure, and a diamond structure.
Among these structures, a hexagonal close-packed structure and a cubic close-packed structure in which fine particles can be used as a structural unit from the viewpoint of productivity and the like are preferable.

The fine particles have a true spherical shape and a number average particle diameter of preferably 100 to 1000 nm, more preferably 150 to 800 nm, and particularly preferably 200 to 700 nm. When the average particle diameter is within this range, the fluorescent signal can be efficiently reflected and amplified.
The particle size distribution of the fine particles is preferably sharp, and specifically, the coefficient of variation is preferably 10% or less, more preferably 7% or less, and particularly preferably 5% or less. When the particle size distribution is within this range, a photonic crystal with high amplification efficiency can be produced. Here, the variation coefficient means a percentage of a value obtained by dividing the standard deviation by the average value. It is possible to produce a photonic crystal suitable for a highly sensitive biosensor chip, such as irregular particles or a broad particle size distribution, where there are defects in which particles are not accumulated in the photonic crystal. Can not.

  The composition of the fine particles is preferably a polymer or metal oxide mainly composed of styrene and methyl methacrylate, and the fine particles having these compositions can be synthesized by a known method. For example, when styrene is the main component, an emulsion polymerization method, a suspension polymerization method, a soap-free emulsion polymerization method, and the like can be mentioned. In the case of silica, a sol-gel method and the like can be mentioned. Many fine particles having such a composition and having the above-mentioned preferable average particle diameter and particle size distribution are commercially available, and those commercially available products can also be used.

As a photonic crystal production method, a micromanipulator or the like is used to collect the structural units one by one, or microparticles are used as the structural unit, which are collected by gravity, centrifugal force, capillary force, liquid bridging force, etc. The method of doing is mentioned.
As the former method, a structural unit, specifically, a columnar body or a plate-like body made of a metal compound or a synthetic polymer prepared by a known fine processing technique (lithography and etching, laser processing, etc.) is confirmed with an electron microscope or the like. One way is to collect them one by one. According to this method, it is possible to produce a log pile structure capable of easily obtaining a photonic band gap.

On the other hand, the latter method is preferable to the former method because it is not necessary to perform accumulation while confirming with an electron microscope or the like, and mass production can be performed very easily if the accumulation driving force is appropriately selected. According to the latter method, the photonic crystal has a hexagonal close-packed structure and / or a cubic close-packed structure. As will be described later, when a template having a specific shape is used, a cubic close-packed structure is selectively obtained.
Here, the cubic close-packed structure and the hexagonal close-packed structure are both close-packed structures, and the filling rate is 74% by volume.

The cubic close-packed structure is also called a face-centered cubic lattice structure, and has a structure in which particles are positioned at the vertices of the regular tetragonal unit cell and the center of each surface, and the close-packed surfaces are stacked in the order ABCCABBC.
In the hexagonal close-packed structure, the unit cell is represented by a regular hexagonal column, and there are three particles at each corner and center of the top and bottom surfaces of this regular hexagonal column and at a height of 1/2 inside the hexagonal column. The atom located in the center of the bottom is in contact with the 6 particles at the corner of the bottom and 3 particles above and below (12 particles in total), and has the structure in which the most dense surfaces are stacked in the order of ABABAB.
In addition, a method that does not use a structural unit, that is, a method that directly processes a resin material or the like that does not have a certain form with a laser can also be mentioned as a method for producing a photonic crystal. As a specific method, there is a method of obtaining a periodic structure by selectively curing only a portion irradiated with a laser by irradiating a photosensitive resist with a short pulse laser (a method described in JP-A-2004-126312). Can be mentioned.

A template substrate (Q) is required in the method of using fine particles and accumulating by gravity, centrifugal force, capillary force, liquid bridging force and the like. As the substrate (Q) having a template function, various substrates can be used as long as they can regularly collect fine particles, and a flat substrate without irregularities, a substrate with holes, and grooves are formed. Substrate etc. can be used. Since the shape and size of the resulting photonic crystal (B) are determined by the shape and size of these substrates, a substrate with a uniform hole and groove size is required, and a substrate with holes formed It is preferable to use a substrate on which grooves are formed. By using these substrates (Q), a photonic crystal having very few defects and high regularity can be produced, and the sensitivity of the biosensor chip is further improved.
As a material constituting the substrate (Q), it is possible to use a material made of an inorganic compound such as silicon, silica, titanium oxide or a material made of a synthetic polymer such as an acrylic resin, an epoxy resin, a polyimide resin or a photosensitive resin. it can.

An example of the substrate in which the hole is formed is a substrate as shown in FIG. The shape of the hole shown in Fig. 2 is an inverted pyramid type that forms an angle of 54.7 ° with respect to the surface of the substrate. The substrate with such a hole with a uniform size uses light or an electron beam. It can be produced by a known fine processing technique such as lithography and etching.
For example, the inverted pyramid type substrate shown in FIG. 2 can be produced by electron beam lithography and anisotropic etching. Specifically, a resist film coated on a silicon substrate is patterned by electron beam (electron beam lithography), and then 100 surfaces are selectively dissolved (anisotropic etching) with an aqueous potassium hydroxide solution. it can. By using a substrate having an inverted pyramid type hole and collecting fine particles in order from the tip of the pyramid, a periodic structure consisting only of a cubic close-packed structure can be produced.
If this method is used, not only inverted pyramid type holes, but also V-shaped cross-sections with high aspect ratio grooves can be accurately produced. Columnar holes and the like can also be produced.

In addition, the resist film pattern itself produced by electron beam lithography or the like on a flat substrate made of an inorganic compound or the like can be used as the substrate (Q), or a mold substrate can be produced by microfabrication technology, and the heat softening point can be obtained. A resin substrate produced by a method (nanoimprint method) of transferring a pressing pattern to a thermoplastic resin or the like heated as described above can also be used.
As the size (μm) of one hole, the diameter is preferably 1 to 1000, more preferably 5 to 500, and particularly preferably 10 to 100.

Examples of the substrate on which the groove is formed include a substrate as shown in FIG. 3 and can be manufactured by the same fine processing technique. Here, the groove means a linear shape having a higher aspect ratio (major axis / minor axis, where the minor axis is the diameter of the cross section of the substrate surface) than the hole. Specifically, the aspect ratio is 10 times or more. (A hole whose aspect ratio is less than 10 times is used as a hole).
Examples of the cross-sectional shape of the groove include a quadrangle and a V-shape as shown in FIG.
As the size (μm) of one groove, the minor axis is preferably 1 to 1000 and the major axis is preferably 10 to 50,000, more preferably the minor axis is 5 to 500, and the major axis is 50 to 20000, particularly preferably short. The diameter is 10-100 and the major axis is 100-10000.
As these grooves, those in which both ends of the groove are cut out, that is, those in which both ends of the groove are present at the end of the substrate, those in which only one end is cut out, or those in which both ends are not cut out are used. can do.

As a method for accumulating fine particles on these substrates (Q) by gravity, centrifugal force, capillary force, liquid cross-linking force, etc., known methods can be used.
The method of collecting fine particles by gravity is also referred to as a natural sedimentation method, and is a method of collecting fine particles by dropping a slurry of fine particles on a substrate (Q) and using gravity as an integrated driving force. Here, the integrated driving force means a force that acts to accumulate fine particles on the substrate (Q).
The fine particle accumulation method by liquid cross-linking force (liquid cross-linking force method) is a method in which fine particle slurry is dropped onto a substrate (Q), and the fine particles are accumulated using the liquid cross-linking force generated by evaporation of the solvent as an integrated driving force.

  The integration method using centrifugal force increases the production efficiency of the photonic crystal by using centrifugal force as the integrated driving force, and further improves the uniformity of periodic structural units that are difficult to produce by conventional methods. This is a method capable of producing a small photonic crystal. For example, when a centrifugal force of 10 G is applied, the accumulation speed becomes 10 times. In addition, when a strong centrifugal force of about 5000 G is applied, small particles with a particle size of about 300 nm can be easily collected, and a photonic crystal with a small structural unit suitable for a biosensor chip can be produced. . Further, as a method for improving the uniformity of the photonic crystal, it is preferable not to use the liquid crosslinking force, which makes it difficult to optimize the accumulation conditions. A specific operation includes not evaporating the solvent during the accumulation process.

Centrifugal force is an inertial force in the direction from the center of centrifugal force to the outside, and is given by the following equation.
F = mrω ²
Here, m is the mass of the true spherical particle (P), r is the distance from the center of the centrifugal field to the true spherical particle (P), and ω is the angular velocity (number of revolutions × 2π). To be precise, r is the distance from the center of the centrifuge field (centrifugal center) to the spherical particle (P), but since the particles always move, the distance from the center of the centrifuge to the substrate (Q) for convenience. Treat as.
The strength of the centrifugal force to be used varies depending on the particle diameter and the specific gravity of the particles, but it is better from the viewpoint of improving the production speed. However, in order to produce a highly regular and uniform aggregate of spherical particles (P), not only the strength of the centrifugal force but also parameters such as processing time, particle slurry concentration, and solvent evaporation rate are optimal. It is necessary to make it.

For example, a polystyrene particle having a number average particle diameter of 500 nm as a fine particle, and a substrate (Q) having a square hole diameter of 10 μm in diameter and having a depth of 12 μm and an inverted pyramid type hole shown in FIG. The preferable range of these parameters when using is as follows from the viewpoints of production efficiency and uniformity of the resulting photonic crystal (B).
The strength of the centrifugal force is preferably 10 to 10,000 G, more preferably 100 to 8000 G, and particularly preferably 500 to 5000 G.
The treatment time varies depending on the strength of the centrifugal force, but is preferably 5 to 3600 minutes, more preferably 10 to 1000 minutes, and particularly preferably 15 to 120 minutes.
The concentration of the fine particle slurry varies depending on the amount of the particle slurry, but is preferably 0.01 to 20% by weight, more preferably 0.02 to 15% by weight, and particularly preferably 0.05 to 10% by weight. The method for preparing the fine particle slurry can be adjusted by dispersing the fine particles in water using a surfactant or an ultrasonic disperser. The fine particles are synthesized from the raw material monomers by soap-free emulsion polymerization in water. Therefore, it is preferable to use it as a fine particle slurry as it is.
The evaporation of the solvent is preferably not performed during the accumulation process as described above.

As a specific manufacturing method, holes or grooves formed in the substrate (Q) are filled with a slurry of fine particles, and centrifugal force is applied perpendicularly to the surface of the substrate (Q) to accumulate the fine particles in the holes or grooves. It is a method to do.
The reason why the fine particles are not used as they are but as a slurry dispersed in a solvent is to give the particles mobility. When there is no solvent or when the solvent ceases to evaporate, adhesion between the particles or between the particles and the substrate occurs immediately, and the mobility of the particles is lost, so a highly uniform photonic crystal (B) can be produced. Can not.

Here, the solvent is not particularly limited in terms of the structure of the invention as long as the fine particles can be dispersed without being dissolved, and organic solvents such as water, alcohol, acetone and toluene can be used. From the viewpoint of a clean process or the like, it is preferable to use water, a lower alcohol or a mixed solvent thereof as a solvent.
Since the Brownian motion of the particles promotes regular accumulation, the method of adding more mobility to the particles such as periodically increasing / decreasing the strength of the centrifugal force, increasing the temperature, applying some vibration, etc. It is effective as a method for producing a high photonic crystal (B).
Applying a centrifugal force perpendicular to the surface of the substrate (Q) means that if the microparticles are a substrate with the surface of the substrate (Q) and holes or grooves formed, the force perpendicular to the bottom of the holes or grooves Means to be accumulated. Specifically, as the centrifugal force increases, the angle of the centrifuge tube on which the substrate (Q) is installed changes, using a so-called swing rotor type centrifuge (Fig. 4) or strong centrifugal force. In this case, a method using a centrifuge capable of installing the substrate (Q) perpendicular to the direction in which the centrifugal force is applied may be used. In the latter method, the case where the centrifugal force is strong means a case where a centrifugal force of about 500 G or more is applied. This is because the influence of gravity can be ignored when the centrifugal force increases.

  As a centrifuge that can be used in these methods, many commercially available centrifuges can be used. For example, cooling high-speed centrifuges H2600, H923, H2000B, small table centrifuges H11NA, H19FMR (manufactured by Kokusan), high-speed cooling centrifuges GRX250, SRX201 (manufactured by Tommy Seiko Co., Ltd.) and the like can be used. As the rotor (centrifuge tube) that can be used, RF109L, RF127 (for H19FMR) and SH1 (for H2600) can be used in the former method, and DWH1 (for H2600) is used in the latter method. it can.

The photonic crystal (B) can also be produced by applying a centrifugal force parallel to the surface of the substrate (Q) rather than perpendicularly.
In this method, if necessary, an upper cover is installed in the groove formed in the substrate (Q), a liquid amount adjusting unit is provided on one end of the groove located outside the centrifugal field or on the upper cover just above the one end, and the groove contains fine particles. In this method, after the slurry is filled, fine particles are accumulated in the groove while applying a centrifugal force in a direction parallel to the surface of the substrate (Q) and adjusting the liquid amount adjusting unit as necessary. A conceptual diagram of an apparatus used in this method is shown in FIG.

The top cover is a cover that is installed on the top of the groove formed on the substrate (Q) or the entire surface of the substrate (Q) for the purpose of preventing the discharge of the slurry of fine particles. Is preferred.
The liquid volume control unit installed on one end of the groove located outside the centrifuge field or on the top cover just above one end is a filter or valve that discharges only the solvent and does not allow the passage of fine particles. It is preferable that it can be adjusted. After the fine particles are collected, the solvent may be discharged by opening the closed valve, or the solvent may be discharged in the process of collecting the fine particles using a filter or an appropriately opened valve. However, from the viewpoint of improving the uniformity of the photonic crystal, it is preferable that the solvent is not completely discharged until the accumulation of the fine particles is completed.
Applying centrifugal force in a direction parallel to the surface of the substrate (Q) after filling the groove with fine particle slurry is perpendicular to the cross section of one end of the groove located outside the centrifuge field formed on the substrate. Means applying centrifugal force. Therefore, the fine particles are sequentially accumulated from one end of the groove located outside the centrifugal field.

In the above-described method, a photonic crystal (B) is produced by filling a slurry of fine particles from one end inside the centrifugal field with centrifugal force applied in a direction parallel to the surface of the substrate (Q). You can also. According to this method, the production efficiency is further increased and it is extremely effective as a continuous production method.
A conceptual diagram of an apparatus used in this method is shown in FIG. There is a recess (WP) for dripping a fine particle slurry in the center of the centrifuge and temporarily storing it, and a substrate (Q) is placed around it. The substrate mounting portion (QS) is preferably designed and installed such that the wall of the recess is removed, and the bottom of the groove formed in the substrate (Q) and the bottom of the WP are the same height. In the configuration of the present invention, the number of substrate mounting portions (QS) is not limited, and the productivity is further improved by receiving a large number.
When the upper cover is installed, a fine particle slurry supply unit (WI) is provided at the centrifugal center, and the slurry can be dropped onto the centrifugal center from here.

A commercially available spin coater or the like can be used as a centrifuge that can be used in a method of applying a centrifugal force parallel to the surface of the substrate (Q). For example, 1H series (manufactured by Mikasa), ACT series, ASS series (manufactured by Active), K359 series (manufactured by Kyowa Riken) and the like can be mentioned.
As the rotation speed of the spin coater, a preferable rotation speed varies depending on the rotation radius or the like, but it is preferable to adjust so that the above-described strong centrifugal force is applied.

An accumulation method using capillary force is a method of accumulating true spherical particles using a phenomenon (capillary phenomenon) in which a solvent in a thin tube ascends in the tube as an accumulation driving force.
The height at which the solvent rises depends on the surface tension of the solvent, the ease of wetting of the tube, the density of the solvent, and the like, and can be calculated by the following equation.
h = 2Tcosθ / pgr
Where T is the surface tension (N / m), θ is the contact angle, p is the density of the solvent (kg / m ³ ), and g is the acceleration of gravity (m / s2)
, R is the radius (m) of the tube.
The fine particles dispersed in the solvent rise together with the rise of the solvent and accumulate in order from the top of the tube. Accumulation of fine particles is finally performed by the liquid crosslinking force generated by the evaporation of the solvent, but unlike the normal liquid crosslinking force, the force works only in the vertical direction. Since the evaporation of the solvent occurs in order from the top of the tube, the force does not work in all directions like the normal liquid bridging force. In this respect, it is distinguished from the liquid crosslinking force, and it is a method that makes it possible to produce a highly uniform photonic crystal (B) that is very easy to control the accumulation conditions.

Substrate (Q) that can be used in this method is a substrate in which a groove is formed and a substrate with no unevenness is provided on both ends of the groove, or a minute between two substrates. The thing etc. which have space can be used.
A specific method is to immerse one end of these substrates in a slurry of fine particles, but there is also an improved method for the purpose of improving production efficiency, such as a method of sucking from one end of the upper portion not immersed. Furthermore, by optimizing the integration conditions such as adjusting the hydrophilicity / hydrophobicity of the substrate surface, a photonic crystal (B) with higher uniformity can be produced.

  As a method of using fine particles and accumulating by gravity, centrifugal force, capillary force, liquid cross-linking force, etc., as described above, known methods can be used. From the viewpoint of producing a defect-free photonic crystal (B) suitable for a chip, it is preferable to use centrifugal force or capillary force as the integrated driving force.

The photonic crystal (B) produced by these methods is preferably used for a biosensor chip in a state of being filled in the template substrate (Q). Accordingly, the substrate (Q) is preferably made of a resin or glass that transmits excitation light.
Moreover, as a shape of a photonic crystal (B), it is preferable that it is flat form or a film form from a viewpoint etc. which coat | cover a fixed part (A) or an analysis cell.

[Example]
EXAMPLES Next, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the examples without departing from the gist of the present invention. Unless otherwise specified, “part” means “part by weight” and “%” means “% by weight”.

<Production of substrate (Q)>
Appearance; see Figure 2 Hole shape; inverted pyramid hole size; one side; 10μm square (cross section), depth; 12μm, pyramid angle; 54 °
Substrate (Q) size: 1cm square
After forming an oxide film by heating a silicon substrate (substrate (Q)) with a side of 1 cm at 1000 ° C for 1.5 hours in the atmosphere, piranha solution (sulfuric acid and 30% hydrogen peroxide water at a volume ratio of 1: 9) The organic matter was completely removed by washing with a mixture.

After drying, a resist was applied by a spin coater to form a resist film having a thickness of about 400 nm on a silicon substrate (prebaked at 180 ° C. for 3 minutes). As the resist, α-methylstyrene-α-chloroacrylate copolymer (ZEP520 (positive resist), manufactured by Nippon Zeon Co., Ltd.) was used.
A pattern was drawn on the resist film with an electron beam drawing apparatus so as to correspond to the size of the hole and the like, and then immersed in a developer (ZEP-RD, manufactured by Nippon Zeon Co., Ltd.) to prepare a mask pattern.
An aqueous solution consisting of potassium hydroxide (30%) and isopropyl alcohol (12%) after being immersed in BHF (a mixture of ammonium fluoride and hydrofluoric acid at a volume ratio of 1: 9) for several minutes to remove only the oxide film. For 1 hour to perform anisotropic etching. Finally, the oxide film was removed with BHF to produce a template substrate for a nanoimprint substrate.
Using this template substrate, a nanoimprint substrate made of nickel was obtained. A polystyrene thin film having a thickness of 20 μm was prepared by spin coating, and an inverted pyramid type hole was formed on one surface by an imprint substrate, and then cut into 1 cm square to obtain a substrate (Q). A wall surface having a height of about 8 μm was formed at the edge of the substrate (Q).

<Example 1>
To the substrate (Q), 0.1 g of a fine particle slurry (Molytex 3269A) having an average particle size of 270 nm and a Cv value of 1.8% is dropped, and using a centrifuge (H30R, manufactured by Kokusan) at 4000G for 10 minutes. Fine particles were accumulated by applying a centrifugal force perpendicular to the surface of the substrate (Q). In the centrifuge, the centrifuge tube shown in FIG. 7 was installed on the swing rotor (RF121) so that a centrifugal force was applied perpendicularly to the surface of the substrate (Q). After centrifugation, the substrate (Q) was removed from the centrifuge tube, excess slurry was removed, and a photonic crystal (B-1) was obtained. The appearance of the photonic crystal (B-1) was a film filled in the substrate (Q), and a single crystal structure composed of a cubic close-packed structure was formed on the entire substrate (Q).

Two identical photonic crystal (B-1) films were prepared, and spin coating was applied to the back of one of them, and a carboxyl group-containing polymethyl methacrylate thin film with a thickness of 1 μm was formed. Through nanoimprinting, the channel diameter was 500 μm and 1 μm A microchannel in which circular pillars having a diameter were formed at intervals of 2 μm was produced. The thin film phase having the microchannels becomes a flat cell.
The other photonic crystal film was placed so as to sandwich the flat cell, and heat-bonded by heat treatment at 80 ° C. for 10 seconds.
1 μl of 0.2% by weight 1-ethyl-3dimethylaminopropylcarbodiimide aqueous solution is added to the microchannel and allowed to react at room temperature for 1 hour. Thoroughly wash away unreacted 1-ethyl-3dimethylaminopropylcarbodiimide with phosphate buffer, add 1 μl of phosphate buffer containing 0.2% by weight of AFP antibody, and react at room temperature for 2 hours. The AFP antibody was immobilized on the wall surface of the microchannel and the surface of the biller via carbodiimide. Unreacted AFP antibody was sufficiently washed and removed with a phosphate buffer, and then the microchannel was filled with the phosphate buffer to obtain a biosensor chip-1. The appearance of biosensor chip-1 is as shown in Figure 1. The fixed part (A) of the AFP antibody (specific binding substance (X)) is the entire microchannel and is completely covered in the vertical direction by two photonic crystal films.

<Example 2>
Biosensor chip-2 was produced in the same manner except that one photonic crystal film of two photonic crystal (B-1) films was changed to a mirror-treated silicon substrate.

<Example 3>
The blood collected from three volunteers (b1, b2, b3) was centrifuged at 10,000 rpm for 15 minutes in a centrifuge to collect supernatant plasma, which was sample (b1), sample (b2), sample ( b3).
Add 100 μl of phosphate buffer (AFP antigen phosphate buffer) with AFP antibody concentration adjusted to 20.0 pg / ml to 100 μl of sample (b1-3) and mix to test samples (AFPb1), (AFPb2), (AFPb3 ) Was produced.

The following analysis operation was performed using Biosensor Chip-1.
After injecting 0.5 μL of the test sample (AFPb1) into the microchannel, 1 μL of phosphate buffer was injected and washed. Next, labeling was performed by injecting a labeled antibody solution in which rhodamine B isothiocyanate was immobilized.
From the surface of the photonic crystal film, YAG laser was irradiated to excite rhodamine B isothiocyanate, and the fluorescence signal was measured with a photomultiplier tube (R2228, manufactured by Hamamatsu Photonics).
The measured value was 15000 to 15500 counts / second (measured at n = 3) in any of the test samples.
The concentration of AFP antigen phosphate buffer was decreased, and the lower limit of detection (pg / ml) was determined under the same conditions as described above. The detection lower limit concentration was a concentration at which the fluorescence signal could not be measured, and was a concentration that coincided with the blank signal (when only the labeled antibody solution was injected).
As a result, the detection lower limit concentration (pg / ml) of Biosensor Chip-1 was 0.0005 (0.5 fg / mL).

<Example 4>
Using Biosensor Chip-2, the lower detection limit concentration (pg / ml) was determined in the same manner as in Example 3. As a result, the detection lower limit concentration (pg / ml) of Biosensor Chip-1 was 0.0003 (0.3 fg / mL).

<Comparative Example 1>
A biosensor chip was produced in the same manner as in Example 1 except that only one photonic crystal film of biosensor chip-1 was not used. The lower detection limit concentration (pg / ml) was determined in the same manner as in Example 3. As a result, the detection lower limit concentration (pg / ml) of this biosensor chip was 10.
A normal microchannel biosensor chip that does not have one photonic crystal film and cannot exhibit the reflection amplification function has a detection limit concentration (pg / ml) of 10. On the other hand, the sandwich structure biosensor chip-1 sandwiched between two photonic crystals with a fixed part (A) has extremely high detection sensitivity, and the detection lower limit concentration (pg / ml) is 0.0005 (0.5 fg / mL). there were. Therefore, it was illuminated that the reflection amplification effect by the photonic crystal was functioning sufficiently. The same effect was also obtained with Biosensor Chip-2 using a silicon reflector instead of one photonic crystal film.

  The biosensor chip of the present invention has extremely high detection sensitivity and can be used for various applications. It is particularly suitable for research use such as protein analysis and gene analysis, and clinical test use such as tumor test and virus test.

Appearance of biosensor chip. Sandwich structure in which a flat cell (C) with a microchannel (D) is sandwiched between two flat photonic crystals (B) Substrate with holes (inverted pyramid type substrate) Substrate with groove (V-shaped groove) Centrifugal tube for swing rotor Conceptual diagram Conceptual diagram

Explanation of symbols

PC: Photonic crystal AC: Analysis cell Q: Substrate (template)
C: Upper cover SQ: Spin coater substrate DI: Groove PIN: Fixing screw VA: Liquid amount adjusting unit (regulating valve)
QS: substrate mounting part WI: particle slurry supply part WP: particle slurry reservoir CW: solvent evaporation prevention cover OR: O-ring

Claims (10)

A biosensor chip that uses a fluorescence method as a detection principle, and is provided with a photonic crystal (B) that covers a fixed part (A) of a specific binding substance (X). The biosensor chip according to claim 1, wherein the fixing part (A) of the specific binding substance (X) is present in the flat cell (C). 3. The biosensor chip according to claim 2, which has a sandwich structure in which the flat cell (C) is sandwiched between two flat photonic crystals (B). The biosensor chip according to claim 2 or 3, wherein the flat cell (C) has a microchannel (D). 5. The biosensor chip according to claim 4, wherein the fixing part (A) of the specific binding substance (X) is present in the channel of the microchannel (D). 6. The structure according to claim 1, wherein a part of the fixed part (A) of the specific binding substance (X) is covered with a photonic crystal (B) and a part of the fixed part (A) is covered with a reflector. The biosensor chip according to any one of the above. The biosensor chip according to any one of claims 1 to 6, wherein the photonic crystal (B) comprises an aggregate of fine particles. The biosensor chip according to any one of claims 1 to 7, wherein the specific binding substance (X) is an immune protein. A fluorescence method for detecting a biological material by irradiating excitation light to the biosensor chip according to any one of claims 1 to 8, wherein a fluorescence signal is reflected and amplified between photonic crystals (B), A method for detecting a biological substance, comprising measuring an enhanced fluorescence signal with a fluorescence analyzer. 10. The detection method according to claim 9, wherein the fluorescence signal is reflected and amplified between the photonic crystal (B) and the reflector.

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