JP2007093355A - Optical sensing method and detection system of target material - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a system capable of detecting a target material highly accurately by a simple optical means based on a structural color change. <P>SOLUTION: This detection method uses a substrate 10; and a structural color evaluation device 20 equipped with a receptor 2 bondable specifically to the target material 1, an additional material 6 bonded to the receptor, which is the additional material for heightening the degree of the structural color change, and a capturing material 3 immobilized on the substrate in the bonded state to the receptor, which is the capturing material having a property capable of liberating the receptor in a prescribed condition wherein the target material exists. The device and a sample are incubated in the condition wherein the receptor and the additional material can be liberated from the device in accordance with existence of the target material in the sample, and the degree of the structural color change based on liberation of the receptor is optically examined. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for detecting a target substance by optical means. More specifically, the present invention relates to a method for detecting a target substance based on a change in structural color, and relates to a detection system that can implement such a detection method.

With the progress of nanotechnology, a method for detecting a target substance based on structural color development, that is, change in structural color, has been proposed. The structural color is a color expressed by reflection of light, etc., and light interference (for example, interference color due to thin film interference or multilayer film interference) due to the fine structure (typically nano-order fine structure) of the object to be processed. , Diffraction (diffraction grating), light scattering, etc. are known. Realization of a rapid and simple target substance detection method based on these structural color changes and a detection system with a simple configuration is expected.
For example, in Patent Document 1, the test solution is obtained by changing the structural color of the gel by bringing the sugar-responsive polymer gel into contact with the sugar-containing solution and observing the degree of the change as a change in the reflection spectrum. A method for detecting the sugar concentration in the medium is described.
Further, Patent Document 2 discloses a substrate having a surface that imparts optical properties, a thin film-like intermediate layer supported by the substrate surface, and a receptor substance (target substance, that is, a test substance and the like) fixed to the intermediate layer. A thin film device having a binding substance) so that the presence or amount of the target substance can be detected from the resulting change in the thin film intermediate layer or optical thickness when the target substance is bound to the receptor substance. Describes a thin film device in which the receptive substance is densely fixed to the thin film intermediate layer.

JP 2004-27195 A JP 2004-45421 A

  However, the method described in Patent Document 1 is a method that uses a special material (sugar-responsive polymer gel), has limited applications, and lacks versatility. In addition, the method described in Patent Document 2 requires that a receptor material (for example, an antibody) be densely attached to a thin film portion formed on a substrate, which can increase the manufacturing cost of the device and can also be used for each device. The amount of retention may vary. This is not preferable because it may cause a decrease in detection accuracy of the target substance.

  The present invention was created to solve the above-described conventional problems in detection methods and detection systems based on structural colors, and provides a general-purpose method capable of detecting a target substance with high accuracy based on structural color changes. The purpose is to do. Another object of the present invention is to provide a detection system (including articles used in the detection method) that can suitably perform such a detection method.

The present invention provides a method for optically detecting a specific target substance contained in a sample by means and processes different from the conventional ones.
That is, one target substance detection method disclosed herein includes (1) a base material, a receptor capable of specifically binding to the target substance, and immobilized on the base material and bound to the receptor. A capture substance in a state that is capable of being released by the receptor under a predetermined condition in which the target substance is present, and an additional substance bound to the receptor and having a degree of structural color change And a surface layer formed by arranging the trapping substance, the receptor and the additional substance so that the structural color can be measured. (2) Conditions for applying the sample to the device and allowing the additional substance and the receptor to be released from the surface layer in accordance with the presence of the target substance in the sample. Ink the device and sample with It encompasses be sorbate, and, a, to investigate the the surface layer of the base material, the additional material and the degree of structural color changes based on the free receptor after incubation (3) above.

In this specification, the “receptor” is a term that refers to a substance that can specifically (selectively) bind to a specific target substance to be detected, and is not limited to a specific form. Substances having a high binding constant in relation to the target substance, for example, antigens involved in specific antigen-antibody reactions (including various fragments in addition to native antigen substances) and antibodies (various other than native antibody substances) Any one of the modifications and the fragments (that is, the other may be a target substance) is a typical example included in the receptor referred to herein.
In addition, the “capturing substance” here means reversible binding with a certain degree of specificity (selectivity) by various binding mechanisms (for example, hydrogen bond, hydrophobic bond) to the selected receptor. The binding force (a typical index includes a binding constant) refers to a substance that does not reach the binding force (binding constant) between the target substance of interest and the receptor. For example, when an antibody against a specific antigen is used as a receptor, a structural analog (so-called analog) having a site that is structurally similar to the epitope of the antigen may be a typical example included in the capture substance here. . For example, with respect to an avidin-biotin complex having a high binding constant, a biotin analog that can form a lower binding constant complex with avidin (for example, dethiobiotin described later) corresponds to the capture substance herein. This is a typical example.
Here, the “additional substance” refers to a substance that can bind to the receptor and can increase the degree of structural color change.
In addition, here, “change in structural color” means a change in structural coloration based on a change in the microstructure of an object that can be measured by various optical means (typically a change in the wavelength of light based on a structural change). ), And it does not matter whether the change can be seen directly.

The device used in the detection method of the present invention is a state in which a capture substance having the above-described contents, a receptor for a target substance of interest, and a state of binding to the receptor are formed on a substrate (device) for measuring a structural color. A surface layer on which the additional substances are arranged.
In this method, the additional substance-bound receptor is released from the substrate surface during the incubation by interaction with the target substance contained in the sample. The fine structure of the substrate surface layer changes due to the liberation of the receptor, and the change is optically regarded as a structural color change and evaluated.
Unlike the conventional method described in the above publication, which measures the change in structural color based on the target substance adhering (binding) to the surface of the substrate, this method is different from the target substance in the sample. The corresponding degree of structural color change is realized by the release of the receptor. For this reason, it is hard to be influenced by the size of the target substance itself and the binding form of the target substance to the base material.
Furthermore, since the additional substance is released from the substrate simultaneously with the receptor, the degree of change in the structural color before and after the receptor is released from the substrate can be made relatively large. Therefore, according to the detection method of this configuration, the target target substance can be detected qualitatively or quantitatively with high accuracy and simplicity by structural color measurement.

Preferably, a substance having a molecular size larger than that of the target substance to be detected is used as the receptor provided on the substrate. According to the method of this aspect, even if the target substance has a small molecular size, larger receptors and additional substances can be liberated and the structural color of the device surface can be greatly changed. The target substance can be detected.
Particularly preferably, the receptor used has a chain molecular structure. In the case of a receptor having a chain molecular structure (including a rod-like structure and a spiral structure), the chain molecular structure of the receptor is regularly arranged in a state where it is captured by a capture substance on the substrate surface. A film structure can be formed. In the state where the receptor is released from the capture substance due to the presence of the target substance, a remarkable structural color change can be realized by such a change in the film structure. For this reason, according to the method of this configuration, more accurate target substance detection can be performed by simple structural color measurement.

  Preferably, a spherical substance that can be disposed on the substrate is used as the additional substance. When the additional substance is spherical, even if the additional substance is arranged (aligned) on the substrate in the form of a film, the receptor and the target substance can be easily contacted and bonded via the gap between the spherical substances. it can. For this reason, the detection of the target substance is not inhibited by the presence of the additional substance.

  Preferably, a liquid sample that can contain a target substance of interest is used as the sample, and the incubation is performed in a state where the liquid sample is in contact with the substrate surface layer. According to this aspect, the release of the receptor and the additional substance from the capture substance is promoted, and the structural color change can be easily caused.

  Also preferably, the degree of change in the structural color is determined based on a comparison of reflection spectra in the substrate surface layer measured before and after the incubation. According to this aspect, the change in structural color can be easily measured.

Moreover, this invention provides the system (measurement apparatus) suitable for implementing the detection method disclosed here as another aspect, and the instrument used for comprising this system.
The present invention provides a structural color evaluation device that optically detects a specific target substance contained in a sample.
The device includes a substrate, a receptor capable of specifically binding to the target substance, a capture substance fixed on the substrate and bound to the receptor, wherein the target substance is present. Under such conditions, a capturing substance having a property that the receptor can be released and an additional substance that is bound to the receptor and increases the degree of structural color change are provided. This device is characterized in that a surface layer formed by arranging the capture substance, the additional substance, and the receptor is formed on the substrate so that the structural color can be measured.
As described above, according to the device having such a configuration, the detection method disclosed herein can be suitably implemented.

  Preferably, a substance having a molecular size larger than the target substance to be detected is provided as the receptor. For example, a receptor having a chain molecular structure is provided. The device having such a configuration can optically detect a target substance with high accuracy and simplicity.

  Preferably, the additional substance is a spherical substance that can be disposed on the substrate. The device having such a configuration can easily contact and bind the receptor and the target substance through the gap between the spherical substances regardless of the presence of the additional substance, so that the target substance can be detected with high sensitivity. It can be performed.

The present invention also provides a kit (ie, a combination of several components) used to manufacture a device that optically detects a specific target substance contained in a sample. The kit includes a base material, a receptor that can specifically bind to the target substance, an additional substance that binds to the receptor and increases the degree of structural color change, And a capture substance that binds to the receptor and has a property that the bound receptor can be released under a predetermined condition in which the target substance exists. Further, in this kit, a surface layer is formed on the substrate so that the capturing substances are arranged so that structural color can be measured, and the additional substance and the receptor are formed with the surface layer. Equipped in a form that can be stored independently of the substrate.
According to the kit having such a configuration, the above-described device can be easily manufactured before performing the detection method disclosed herein.

Preferably, a substance having a larger molecular size than the target substance to be detected is provided as the receptor. Particularly preferably, a receptor having a chain molecular structure is provided. Preferably, a spherical substance that can be disposed on the substrate is provided as an additional substance.
The kit having such a configuration can manufacture a device capable of optically detecting a target substance with high accuracy and simplicity.

  Hereinafter, preferred embodiments of the present invention will be described. It should be noted that technical matters other than the contents particularly mentioned in the present specification and necessary for the implementation of the present invention can be grasped as design matters for those skilled in the art based on the prior art. The present invention can be carried out based on the technical contents disclosed in the present specification and drawings and the common general technical knowledge in the field.

  The detection method of the present invention only needs to be a method for detecting a predetermined target substance (test target substance) by measuring the structural color, regardless of whether it is qualitative or quantitative. The sample to be detected can be in various forms (eg, liquid, vapor, solid, powder, gel), but is preferably a liquid sample. For example, liquid samples taken from humans or other organisms, nature or man-made products (blood or other body fluids, sap, sea water, land water, liquids in food or chemicals, natural or artificial oils, etc.), or A liquid sample prepared by dissolving or dispersing a non-liquid sample in an appropriate solvent can be appropriately diluted or concentrated as necessary. For example, the detection method of the present invention is applicable to harmful substances contained in human or other mammalian body fluids (for example, proteins, nucleic acids, toxins originating from microorganisms such as viruses and bacteria) or harmful substances contained in nature (for example, endocrine secretion). Disturbing substances, that is, various organic chlorine compounds and polymer compounds suspected as environmental hormones, can be detected by simple optical means.

The device used in the detection method of the present invention may be any device that can measure the change in structural color, and the material and shape of the substrate are not particularly limited. Although not limited, when utilizing a structural color change due to thin film interference or multilayer film interference in the substrate surface layer, interference light is easily generated in the substrate surface layer, for example, on a substrate having good light reflectivity, such as a surface. A silicon substrate provided with a silicon oxide film is a suitable example. Or when utilizing the change of the structural color by the diffraction of the light which permeate | transmitted the base material, a base material with good light transmittance, for example, a quartz substrate, is illustrated.
The shape of the surface of the substrate (the site that expresses the structural color to be measured) is not particularly limited as long as it is suitable for the measurement of the structural color, but a flat surface shape is preferable from the viewpoint of spectrum observation. Accordingly, a preferred embodiment of the device disclosed herein is characterized by including a flat substrate (substrate).

  The structural color may be based on any principle. For example, a structural color based on interference of light irradiated and reflected on the film in a thin film or a multilayer film formed on a substrate can be given as a typical example. Alternatively, another typical example is a structural color based on diffraction of light having directionality depending on a wavelength in a group of substances regularly arranged on a substrate. These structural color generation mechanisms and other structural color generation mechanisms (for example, structural colors based on light scattering) are technical common sense and are not particularly necessary for the disclosure of the present invention. Detailed description is omitted.

  The structural color measuring means is not particularly limited. Means capable of accurately detecting structural changes on the substrate are preferred. The methods disclosed herein typically measure structural changes in the substrate (device) surface layer, i.e. structural color changes due to interference and / or diffraction of reflected light. One suitable measuring means in this case is measurement of the reflection spectrum. This kind of reflection spectrum can be easily measured by using a reflectance measuring device equipped with various light sources, spectrometers, photodetectors and the like.

In order to express the structural color, preferably, a surface layer in which the capturing material is arranged on the surface of the substrate is formed using a desired capturing material. For example, it is preferable to form a monomolecular layer made of a trapping substance. For this purpose, it is preferable to employ a capture substance having a chain molecular structure (see Examples described later). The capture substance can be bound (fixed) to the substrate surface by various binding mechanisms (for example, a hydrophobic bond, a hydrogen bond, a covalent bond, and an ionic bond).
For example, based on the Langmuir-Blodgett method (LB method), polymer compounds having relatively long alkyl (or alkenyl or alkynyl) chains are regularly arranged on the surface of the substrate, and van der Waals forces between the chains are used. High density and highly oriented monolayers (ie self-assembled monolayers) can be formed.

Preferably, the capture substance can be chemically bonded to the substrate surface using a coupling agent (typically a silane coupling agent) or the like. For example, an appropriate surface treatment is performed on the surface of the inorganic oxide of the base material, such as a silicon oxide film formed on the surface of the silicon substrate, and a reactive group (for example, a silanol group (Si-OH), a hydroxyl group, Other hydrophilic groups), for example, a general formula: X—R—Si—Z ₃ (wherein X is a functional group such as an amino group, a vinyl group, an epoxy group, or a methacryloxy group that can bind to an organic material) A suitable silane coupling agent (for example, vinyltriethoxysilane (VTES), 3-glycidoxypropyltriethoxysilane (GPTES), wherein R is an alkyl group, and Z is independently an alkoxyl group or halogen) ), 3-methacryloxypropyltriethoxysilane (MPTES), 3-aminopropyltriethoxysilane (APTES)) The capture agent is an organic substance can be firmly chemically bonded on the inorganic oxide surface. In this case, if the amount of the reactive group is sufficiently present on the surface of the inorganic oxide, a monomolecular layer regularly arranged in one direction is formed by the alkyl chain of the silane coupling agent.

  The receptor used in the detection method of the present invention is appropriately selected according to the content of the target substance to be detected. For example, when the target substance is an antigenic substance contained in the sample (for example, proteins that constitute various toxins or bacterial cells), it can be an antibody or an antibody fragment (for example, a Fab fragment). In addition, when the target substance is a polynucleotide (for example, DNA or RNA possessed by various pathogenic viruses) contained in the sample, at least a part of the polynucleotide (one nucleotide chain in the case of a double strand) The polynucleotide may have a base sequence complementary to the base sequence. In addition to these, when a specific protein is used as a target substance, a substance (ligand) that specifically binds to the protein (that is, exhibits a high binding constant) can be used as a receptor. For example, any of the relationship between various enzymes and their substrates (or analog compounds that antagonize the substrates), various hormones and their receptors, and various nucleic acid-binding proteins and their target nucleic acids is a target substance. And either can be a receptor. A typical example is the relationship between avidin and biotin.

The capture substance used in the detection method of the present invention can reversibly bind to a predetermined receptor under a predetermined condition, while the target substance exists in the presence of the target substance under the condition. It is a substance that can be easily released (can be activated) from the capture substance. The capture substance may be appropriately selected depending on the types and properties of the target substance and the receptor, and is not particularly limited.
This type of capture substance is typically a substance (analog) having a chemical structure similar to the target substance of interest, and the binding constant of the substance with the receptor is greater than the binding constant of the target substance with the receptor. Can be adopted. When a protein such as an enzyme or a hormone is used as a target substance and a coenzyme substrate or receptor for the target substance is used as a receptor, an analog protein having a structure having a lower binding constant than the target substance protein may be used.
For example, as shown in FIG. 1, in a system using biotin 1 as a target substance and avidin 2 as a receptor, a compound similar in structure to biotin 1 and binding of the compound to avidin 2 A substance having a constant lower than the biotin / avidin binding constant, such as dethiobiotin 3 as shown, is one preferred example that can be used as a capture substance in the practice of the present invention.

Alternatively, in a system in which a nucleic acid (DNA or RNA) having a specific base sequence is a target substance and a polynucleotide having a base sequence complementary thereto is a receptor, the homology of the base sequence to the receptor is higher than that of the target nucleic acid. Low polynucleotides (ie, polynucleotides that are less likely to hybridize to the receptor than the target nucleic acid) can be used as capture agents.
The ease with which the receptor is released from the capture substance can be easily determined by appropriately adjusting the incubation conditions. For example, if the sample is a liquid and the receptor and capture substance are proteins and nucleic acids (typically, these bonds are due to van der Waals forces or hydrogen bonds), the salt concentration and processing temperature will vary. By doing so, the degree (rate) of release of the receptor from the capture substance can be adjusted.

  In addition, the additional substance used in the detection method of the present invention is a substance that binds to the receptor in order to increase the degree of change in structural color (typically, the change in wavelength, that is, Δλ is amplified). If it is a substance suitable for this purpose, there will be no restriction | limiting in particular. Molecules or fine particles having a relatively large size that increase the degree of structural color change are preferred. In addition, a stable substance that is not reactive with the target substance (for example, a substance that does not cause nonspecific adsorption with the target substance and the substrate surface) is preferable. In particular, spherical fine particles (or macromolecules) are preferred for the reasons described above. For example, spherical fine particles (preferably having an average diameter of 0.05 μm to 1 μm) made of various polymer substances (polystyrene, silica, latex, etc.) are particularly preferable. Unlike conventional detection methods that employ a structural color change (that is, a structural color change based on adsorption of a target substance on a substrate) by binding such a form of an additional substance to a receptor, the target is a detection target. Even when the molecule (or particle) size of the target substance is small, the presence of the target substance of the small size is increased by the structural color change due to the release of the additional substance-binding receptor that is much larger than the target substance instead. Sensitivity (high accuracy) can be detected.

  FIG. 2 shows a rough procedure and mechanism of the detection method disclosed here. In the detection method of the present invention, first, as shown in FIG. 2A, at least the structural color measurement of the surface of an appropriate base material (for example, a silicon substrate having a silicon oxide film on the surface) 10 on which structural color evaluation can be performed. A capture substance (in the figure, the above-mentioned dethiobiotin) 3 is bound in a state where it is arranged in a part related to the above. Typically, a capture substance (dethiobiotin) 3 is bonded (fixed) through a spacer (crosslinked molecule) 4 bonded to the surface of the substrate 10 using a silane coupling agent or the like. As a result, a surface layer (preferably a monomolecular layer) made of the capturing substance 3 (preferably oriented in one direction by the spacer 4) is formed on the surface of the base material 10. Such a monomolecular layer exhibits a predetermined spectrum (a spectrum having a maximum value or a minimum value of reflected light intensity in the visible light wavelength region or both).

Next, a receptor 2 that is a receptor 2 for the target substance (in the figure, avidin described above) 2 and is bound with an appropriate additional substance 6 in advance is added to the substrate 10 and incubated under appropriate conditions. By doing so, the additional substance 6-binding type receptor 2 is captured by the layer (preferably a monomolecular layer) made of the capture substance 3 on the substrate 10. Here, when the capture substances 3 are regularly arranged like a monolayer, typically, the structural color evaluation can also be performed for the additional substance 6-binding type receptor 2 captured therein. It can arrange | position on the base-material surface in the state arranged so. Accordingly, the surface of the base material 10 in this state exhibits a predetermined spectrum (a spectrum having the maximum value or the minimum value of the reflected light intensity in the visible light wavelength region, or both).
As shown in the drawing, the method performed for binding the additional substance 6 to the receptor 3 is not particularly limited, and the binding can be performed by a conventionally known method. For example, the additional substance 6 may be directly bonded to the receptor 2 by physical adsorption action, but a spacer (crosslinked molecule) 5 bonded to the surface of the additional substance 6 using a silane coupling agent or the like as shown in the figure. It is preferable to covalently bind the receptor 2 via

  In addition to the addition substance 6 (including the cross-linking molecule 5), the receptor 3 and the capturing substance 4 (including the cross-linking molecule 4), various substances are provided on the base material 10 for various purposes. Can do. For example, it is preferable to coat the substrate surface with a compound such as BSA in order to avoid non-specific adsorption.

  In this way, the device 20 of the present invention is manufactured. The kit provided by the present invention typically includes the substrate 10 bound to the capture substance 3 and the substrate 10 so that such a device can be constructed before the detection method is performed. Stocked in a storable form (for example, a solution containing a suitable salt or a lyophilized powder so that the receptor to which the adduct is bound can maintain a suitable molecular structure (shape)) 2 is included.

Then, a sample that may contain the target substance (the above-mentioned biotin in the figure) 1 is applied to the device 20 having such a configuration, and as shown in FIG. The device 20 and the sample may be incubated (ie, kept in a reaction state) under appropriate conditions that allow the additional substance 6 and the receptor 2 to be released from the surface layer of the material 10. By this incubation (continuation of treatment for a predetermined time), as shown in the figure, the receptor 2 having a high binding constant with the target substance 1 is changed from the capture substance 3 to the additional substance 6 on the substrate surface layer in accordance with the presence of the target substance 1. And is typically bound to the target substance 1 in the sample. As a result, as shown in the figure, the fine structure of the surface of the base material 10 is greatly changed, and the change (that is, the change from the state shown in FIG. 2A to the state shown in FIG. 2B) is a structural color. It can be detected optically as a change.
Such structural color change can be detected by various conventionally known optical techniques. For example, measurement of a reflection spectrum, measurement of an absorption spectrum, and the like can be easily realized by using a commercially available optical instrument (such as a spectrophotometer) (see examples described later).

Accordingly, detection systems that can be provided by the present invention can include various devices that use the structural color evaluation device disclosed herein and that can optically measure changes in the structural color of the device. For example, various light sources, a spectroscope (spectrophotometer), and an analysis device (computer) that analyzes data obtained by the spectroscope can be provided. For example, the structural color change of the device can be optically measured by the apparatus 100 configured as shown in FIG. That is, the apparatus 100 generally includes a light source 102, a spectroscope 104, and an analysis apparatus (PC unit) 101. The light source 102 and the spectroscope 104 are optically connected to the measuring unit 106 by optical fibers 103 and 105, respectively. A measuring container 108 is attached to the measuring unit 106, and a predetermined sample containing a target substance, that is, an object to be processed (specimen) and a device 110 according to the present invention are arranged in the container 108. Then, after incubation for a predetermined time, measurement light output from the light source 102 via the optical fiber 103 is projected onto the surface of the device 110, and the reflected light is received by the spectroscope 104 via the optical fiber 105. Then, the analysis device (PC unit) 101 qualifies or quantifies the target substance based on the measured reflected light spectrum or the like. In addition, since such an apparatus structure itself is the same as that of the existing apparatus, the detailed description beyond this is abbreviate | omitted.
Moreover, such an apparatus 100 can include various incubators (for example, thermostats) that can incubate the structural color evaluation device 110. What is necessary is just to employ | adopt a suitable component according to the property of a target substance and / or a sample for the component of these systems.

According to the method disclosed herein, a change in structural color in a device (base material surface layer) based on the presence of a target substance in a sample can be easily detected by simple optical means. That is, according to the detection method disclosed herein, the target substance contained in the sample can be easily and qualitatively detected by examining the degree of change in the structural color.
In addition, for example, by examining the degree of structural color change in each standard sample using several standard samples containing known amounts of target substances different from each other in advance, by creating a calibration curve from those data, The target substance in the sample to be measured can be quantitatively measured.

  In the method disclosed herein, the detection sensitivity of the target substance can be improved by increasing the molecular structure of the receptor. That is, the larger the size of the receptor that is released under a predetermined condition, the greater the change in the surface structure of the base material that is involved in the structural color development as compared to before the release of the receptor. In other words, even if a small amount of the receptor is liberated, the degree of structural color change (for example, the rate of change of the reflection spectrum) increases, and this can be easily detected by optical means. For example, by modifying a receptor molecule such as avidin described above with a chain molecule (eg, a long chain fatty acid or an aliphatic compound such as paraffinic, olefinic and acetylenic hydrocarbons) on the substrate. A modified receptor having a large molecular size can be obtained while maintaining a regular arrangement. The method for modifying the receptor is not particularly limited because it varies depending on the properties of the receptor used (core part involved in binding to the target substance). Various conventionally known modification techniques may be employed depending on the type of receptor. For example, protein receptors can be modified (coupled) with long chain fatty acids by conventional techniques.

  The present invention will be described in more detail with reference to the following examples. However, the present invention is not intended to be limited to those shown in the examples.

<Production of structural color plate>
In this example, a biotin detection device based on a structural color change was manufactured using a structural color plate composed of silicon and a silicon oxide surface film as a base material.
That is, a silicon wafer (plane orientation (100), n-type, thickness 0.5 mm, single-side polished, Niraco Co., Ltd. product) is cut into an appropriate size by glass cutting and immersed in acetone and nitric acid for 24 hours for cleaning. did. Subsequently, by performing annealing for 3 hours at a constant temperature (950 to 1120 ° C.) using an electric furnace, the thickness of the oxide layer on the silicon surface is increased, and silanol groups (reactive hydroxyl groups: —OH) are formed on the surface. A plate 30 was obtained (FIG. 4A).
Next, the plate was immersed in an ethanol solution of 1% by mass of 3-glycidoxypropyltriethoxysilane (GPTES) at room temperature for 24 hours. Thereby, as shown in FIG. 4B, a silane coupling agent (GPTES) was fixed to the surface of the plate 30 and an epoxy group was introduced to the surface of the plate.
Next, the plate was immersed in a PBS solution (pH 7.4) of 0.05% by mass bovine serum albumin (BSA) for 24 hours at room temperature. As a result, as shown in FIG. 4C, the epoxy group and the amino group of BSA reacted and bonded, and BSA molecules were introduced into the cross-linked molecules on the plate 30 surface. In addition, BSA molecules were immobilized (coated) on the blank area on the surface of the plate 30.

<Binding of dethiobiotin to structural color plate>
Next, dethiobiotin hydrazide (DSB-H), which is an analog of biotin, was bound to the terminal carboxyl group of BSA at the tip of the spacer formed on the surface of the plate 30.
That is, 2.9 mM DSB-H, 5 vol% DMSO (dimethyl sulfoxide), 6.2 mM EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide), and 0.1 M MES (4-morpholineethanesulfonic). The plate was immersed in an aqueous solution (pH 5.5) containing acid) at 0 ° C. for 1 hour and then at room temperature for 50 hours. As a result, as shown in FIG. 4D, a plate 30 is obtained in which DSB-H, which is a capture substance according to the present example, is bound to the surface of the plate 30 via a long-chain spacer containing BSA. It was.

<Binding of avidin to DSB-H on the surface of the structural color plate>
Avidin, which is a receptor according to this example, was bonded to the surface of the plate (base material for structural color evaluation) 30 obtained as described above. That is, a phosphate buffer (pH = 7.4) containing 1 × 10 ⁻⁵ M avidin was prepared. The plate 30 was immersed in this buffer at room temperature for 24 hours. Thereafter, the plate 30 was lifted from the buffer and washed with ultrapure water. As a result, as shown in FIG. 5A, a plate 30 was obtained in which avidin 32 was bound to DSB-H (reference numeral 33) on the surface of the plate 30.

<Binding of additional substances to avidin on the surface of the structural color plate>
Next, on the surface of the plate (structural color evaluation base material) 30 obtained as described above, polystyrene (PS) fine particles (average particle diameter of 100 nm, refractive index of 1.59) as an additional substance according to this example. : Duke Scientific Corporation product).
That is, in a PBS-M solution (containing 1 mg / mL nonionic surfactant (n-Octanoyl-N-methylglucamide: trade name MEGA-8)) containing 1.3 mg / mL PS fine particles. The plate 30 was immersed for 1 hour at room temperature. Thereafter, the plate 30 was lifted from the buffer and washed with ultrapure water. As a result, as shown in FIG. 5B, a plate 30 was obtained in which the PS fine particles 36 bonded to the avidin 32 on the surface of the plate 30 were regularly arranged as viewed from the cross section. The surface layer of the obtained plate 30 was observed with an atomic force microscope (AFM). The result (AFM image) is shown in FIG. As is clear from this image, PS fine particles were densely arranged on the surface of the obtained plate (device for structural color evaluation).

<Avidin release by adding biotin>
A phosphate buffer (pH = 7.4) containing 4.1 × 10 ⁻³ M biotin (product of Tokyo Chemical Industry Co., Ltd.) as a target substance was prepared. The plate 30 obtained above was immersed in this buffer at room temperature for 20 hours for incubation. Thereafter, the plate 30 was lifted from the buffer and washed with ultrapure water. In order to prevent denaturation of avidin, this operation was performed without always drying the plate.

<Measurement of reflection spectrum>
Visible light reflection spectrum measurement using a commercially available UV-visible spectrophotometer (JASCO Corporation product, model: V-550) and an absolute reflectance measuring device (JASCO Corporation product, model: ARS-474) Thus, the performance of the device according to this example was evaluated. As a comparative control, the reflection spectrum was measured in the same manner for the plate before being immersed in the biotin-containing buffer. Specifically, the plate (device) was fixed to an absolute reflectance measurement holder at an incident angle of 10 ° and measured at room temperature. By dividing the spectrum of the untreated silicon from the spectrum obtained by this measurement, a spectrum caused only by the structural color was obtained.
FIG. 7 is a spectrum diagram showing the result. The horizontal axis represents wavelength (wavelength: nm), and the vertical axis represents reflectivity (reflectivity:%).
As is apparent from the spectrum diagram, before immersion in the biotin-containing buffer (without
After immersion in a biotin-containing buffer (see curve plotted as biotin) compared to the biotin plotted curve), the receptor avidin with PS microparticles is released and the structure of the plate surface layer changes. The change could be detected as a change in structural color (typically based on a change in optical interference). That is, a wavelength shift (color change) in the visible light reflection spectrum was confirmed. Specifically, a wavelength shift (Δλ) of approximately −40 nm was confirmed by incubation with a biotin-containing sample. An AFM image that supports this is shown in FIG. As is clear from this image, it was confirmed that a large number of PS fine particles were released on the plate surface after being immersed in the biotin-containing buffer. Further, as shown in FIG. 9, this color change is at a level that can be confirmed with the naked eye.

Next, the biotin concentration contained in the phosphate buffer (pH = 7.4) was changed to 0M, 2.0 × 10 ⁻⁴ M, 4.1 × 10 ⁻⁴ M, 2.0 × 10 ⁻³ M and The visible light reflection spectrum measurement was performed by performing the same process while changing to 4.1 × 10 ⁻³ M. The results are shown in FIG. The horizontal axis is the biotin concentration (M), and the vertical axis is the minimum wavelength shift (Δλmin / nm). As is clear from the wavelength shift (color change) with respect to each biotin concentration plotted in this figure, in the device according to this example, the concentration of biotin (target substance) is as low as 2.0 × 10 ⁻⁴ M. Even in this case, a large value of wavelength shift (-15 to -20) was observed.

  Specific examples of the present invention have been described in detail above, but these are merely examples and do not limit the scope of the claims. The technology described in the claims includes various modifications and changes of the specific examples illustrated above.

It is explanatory drawing which shows typically the measurement principle of the detection method of this invention. It is explanatory drawing which shows typically the microscopic state of the device for structural color evaluation used for the detection method of this invention, (A) is the state by which the capture | acquisition substance, the receptor, and the additional substance were arrange | positioned on the base-material surface, ( B) shows a state in which the receptor and the additional substance are released from the capture substance due to the presence of the target substance. 1 is a block diagram schematically showing a preferred embodiment of an optical detection apparatus for a target substance. It is a flowchart which illustrates typically the manufacture procedure of the device for structural color evaluation which concerns on one Example, (A) shows the state which introduce | transduced the silanol group into the base-material surface, (B) is a silane coupling agent. The cross-linked molecule (spacer) is shown immobilized on the surface of the substrate, (C) shows the state where the BSA molecule is introduced at the tip of the cross-linked molecule, and (D) uses the carboxyl group on the surface of the BSA molecule. This shows a state in which dethiobiotin hydrazide is bound (fixed). FIG. 5 is a flowchart schematically illustrating the manufacturing procedure of the structural color evaluation device following FIG. 4, wherein (A) shows a state in which avidin is bound to dethiobiotin hydrazide immobilized on a base material, and (B) shows the relevant step. It shows a state where spherical polystyrene fine particles as an additional substance are bound to avidin. It is the image (AFM image) obtained by atomic force microscope (AFM) observation of the surface of the device for structural color evaluation manufactured in one Example. It is a diagram of the reflection spectrum which shows the detection result obtained in one Example, a horizontal axis shows a wavelength (nm) and a vertical axis | shaft shows a reflectance (%). It is the image (AFM image) obtained by atomic force microscope (AFM) observation of the surface of the device after immersion in the biotin solution used in one Example. It is a color photograph which shows the color change of the device (base material) surface visually recognized in one Example. It is a diagram which shows the relationship between a biotin density | concentration and wavelength shift when using the device for structural color evaluation manufactured in one Example, a horizontal axis is biotin density | concentration (M), and a vertical axis | shaft is minimum wavelength shift ((DELTA) lambdamin / nm). ).

Explanation of symbols

1 Target substance (biotin)
2,32 receptor (avidin)
3,33 capture substance (dethiobiotin analog)
4,5 Spacer (crosslinking molecule)
6 Additional substances 36 Additional substances (polystyrene fine particles)
DESCRIPTION OF SYMBOLS 10,30 Base material 20,110 Structural color evaluation device 100 Optical detection apparatus

Claims (9)

A method for detecting a specific target substance contained in a sample comprising:
A substrate;
A receptor capable of specifically binding to the target substance;
A capturing substance fixed on the substrate and bound to the receptor, and having a property that the receptor can be released under a predetermined condition in which the target substance exists;
An additional substance bound to the receptor and for increasing the degree of structural color change,
A structural color evaluation device is provided, wherein a surface layer is formed on the substrate so that the capturing substance, the receptor and the additional substance are arranged so that the structural color can be measured. thing;
Applying a sample to the device and incubating the device and the sample under conditions that allow the additional substance and receptor to be released from the surface layer in response to the presence of a target substance in the sample; and after the incubation Examining the degree of structural color change based on the liberation of the additional substance and the receptor on the surface layer of the base material;
A method for detecting a target substance.   The method according to claim 1, wherein a substance having a molecular size larger than a target substance to be detected is used as the receptor.   The method according to claim 1 or 2, wherein a spherical substance that can be disposed on the substrate is used as the additional substance.   The method according to any one of claims 1 to 3, wherein a liquid sample is used as the sample, and the incubation is performed in a state where the liquid sample is in contact with the substrate surface layer.   The method according to claim 1, wherein the degree of change in the structural color is determined based on a comparison of reflection spectra in the substrate surface layer measured before and after the incubation. A device for optically detecting a specific target substance contained in a sample,
A substrate;
A receptor capable of specifically binding to the target substance;
A capturing substance fixed on the substrate and bound to the receptor, and having a property that the receptor can be released under a predetermined condition in which the target substance exists;
An additional substance bound to the receptor and for increasing the degree of structural color change,
A structural color evaluation device, wherein a surface layer is formed on the substrate so that the capturing substance, the additional substance, and the receptor are arranged so that the structural color can be measured.   The device according to claim 6, comprising a substance having a molecular size larger than a target substance to be detected as the receptor.   The device according to claim 6 or 7, wherein the additional substance is a spherical substance that can be disposed on the substrate. A kit used to manufacture a device for optically detecting a specific target substance contained in a sample,
A substrate;
A receptor capable of specifically binding to the target substance;
An additional substance bound to the receptor for increasing the degree of structural color change;
A capturing substance fixed on the substrate, which binds to the receptor and has a property that allows the bound receptor to be released under predetermined conditions in which the target substance exists;
With
Here, a surface layer is formed on the base material by arranging the capture substances so that the structural color can be measured, and the additional substance and the receptor are the base material on which the surface layer is formed. A kit equipped in an independently storable form.