JP6900325B2 - Sensor chip and sensing system - Google Patents

Description

The present invention relates to a sensor chip in which a signal emitted from a labeling substance is enhanced by utilizing plasmon resonance of a metal-based particle aggregate, and a sensing system including the sensor chip.

Emission analysis methods that qualitatively or quantitatively detect a substance to be detected labeled with a luminescent substance such as a fluorescent substance by analyzing the luminescence from the luminescent substance are various methods that can realize high-speed analysis. Plays an important role in the field of. For example, by using a luminescence analysis method for a nucleic acid labeled with a luminescent substance, it is possible to determine a base sequence, analyze a gene mutation, measure a gene expression level, and the like at high speed.

However, when the substance to be detected is fixed using a general sensor chip, the intensity of light emission from the luminescent substance is weak, and a high-sensitivity spectroscope may be required for high-sensitivity analysis. Further, when the amount of the substance to be detected is small, highly sensitive analysis becomes more difficult, and a plurality of measurements may be required or the substance may not be detected. The application of a plasmon material composed of metal particles to a light emitting element has been studied for the purpose of solving the problem of such a light emission analysis method and enhancing the light emission of a light emitting substance.

It has been conventionally known that when metal particles are miniaturized to nano size, they exhibit functions that were not seen in the bulk state, and among them, "localized plasmon resonance" is expected to be applied. is there. Plasmon is a coarse and dense wave of free electrons generated by the collective vibration of free electrons in a metal nanostructure.

In recent years, the above-mentioned technical field dealing with plasmons has attracted a great deal of attention as "plasmonics" and active research has been carried out. Such research is on luminescent substances utilizing the localized plasmon resonance phenomenon of metal nanoparticles. Includes those intended to enhance luminescence.

For example, Japanese Patent Application Laid-Open No. 2007-139540 (Patent Document 1), Japanese Patent Application Laid-Open No. 08-271431 (Patent Document 2), and International Publication No. 2005/033333 (Patent Document 3) utilize the localized plasmon resonance phenomenon. A technique for enhancing fluorescence is disclosed. In addition, T. Fukuura and M. Kawasaki, "Long Range Enhancement of Molecular Fluorescence by Closely Packed Submicro-scale Ag Islands", e-Journal of Surface Science and Nanotechnology, 2009, 7, 653 (Non-Patent Document 1) Studies on localized plasmon resonance with silver nanoparticles have been shown.

JP-A-2007-139540 Japanese Unexamined Patent Publication No. 08-271431 International Publication No. 2005/0333335

T. Fukuura and M. Kawasaki, "Long Range Enhancement of Molecular Fluorescence by Closely Packed Submicro-scale Ag Islands", e-Journal of Surface Science and Nanotechnology, 2009, 7, 653

However, the conventional luminescence enhancement using the localized plasmon resonance phenomenon of metal nanoparticles has the following problems.

That is, the factors of the luminescence enhancing action by the metal nanoparticles are 1) the electric field near the particles is enhanced by the generation of localized plasmons in the metal nanoparticles (first factor), and 2) excitation. The energy transfer from the excited molecule (such as a luminescent material molecule) excites the vibrational mode of the free electrons in the metal nanoparticles, resulting in a luminescent induced dipole larger than the luminescent dipole of the excited molecule. Where there are two factors that occur in metal nanoparticles, which increases the luminescence quantum efficiency itself (second factor), the luminescence-induced dipole in the second factor, which is the larger factor, is the metal nano. In order to effectively generate particles, the distance between the metal nanoparticles and the excited molecule is within the range where energy transfer by the Dexter mechanism, which is the direct movement of electrons, does not occur, and the energy transfer of the Felster mechanism is expressed. It is required to be within the range (1 nm to 10 nm). This is because the occurrence of luminescence-induced dipoles is based on Felster's theory of energy transfer (see Non-Patent Document 1 above).

Generally, in the range of 1 nm to 10 nm, the closer the distance between the metal nanoparticles and the excited molecule is, the easier it is for luminescence-induced dipoles to occur, and the more the luminescence enhancing effect is enhanced, while the above distance is increased. As the localized plasmon resonance no longer effectively affects the luminescence enhancing effect, the luminescence enhancing effect gradually weakens, and when the energy transfer of the Felster mechanism is exceeded (generally at a distance of about 10 nm or more), the luminescence enhancing effect is achieved. Could hardly be obtained. Also in the luminescence enhancing methods described in Patent Documents 1 to 3, the distance between the metal nanoparticles effective for obtaining an effective luminescence enhancing effect and the excited molecule is 10 nm or less.

As described above, in the conventional localized plasmon resonance using metal nanoparticles, there is an essential problem that the range of action is limited to an extremely narrow range of 10 nm or less from the surface of the metal nanoparticles. This problem inevitably leads to the problem that almost no enhancing effect is observed in an attempt to enhance the luminescence of the labeling substance by utilizing the localized plasmon resonance by the metal nanoparticles in the sensor chip to improve the sensitivity. ..

That is, in the sensor chip, the substance to be detected that is specifically bound to the trapping substance placed on the sensor chip is detected based on the signal derived from the labeling substance that exists in the vicinity of the specific binding. When the distance from the surface of the metal nanoparticles to the labeling substance in such a configuration is examined, it usually exceeds 10 nm, which is outside the range of action of the localized plasmon resonance described above. This is because it is necessary to form a scaffold for fixing the trapping substance, and it is necessary to consider the length of the trapping substance and the substance to be detected itself. Further, in the configuration in which the distance from the surface of the metal nanoparticles to the labeling substance is shortened, the protection of the surface of the metal nanoparticles becomes insufficient, and the action of the localized plasmon resonance is reduced due to the deterioration of the metal nanoparticles.

Therefore, the present invention includes a sensor chip capable of enhancing a signal derived from a labeling substance by localized plasmon resonance and preventing deterioration of metal nanoparticles that cause localized plasmon resonance, and a sensing system including the sensor chip. The purpose is to provide.

That is, the present invention includes the following.
[1] A sensor chip used to detect a substance to be detected.
With the board
A metal-based particle aggregate layer formed on the substrate and
A protective layer that covers the metallic particle aggregate layer and
A capture layer formed on the protective layer and having a capture substance that specifically binds to the substance to be detected, and a capture layer.
With
The metal-based particle aggregate layer is composed of a particle aggregate in which 30 or more metal-based particles are two-dimensionally arranged apart from each other, and the metal-based particles have an average particle size of 200 to 1600 nm. A sensor chip having an aspect ratio in the range of 55 to 500 nm and an aspect ratio defined by the ratio of the average particle size to the average height in the range of 1 to 8.

[2] The sensor chip according to [1], which detects the substance to be detected by detecting a signal corresponding to a state of specific binding between the trapped substance and the substance to be detected.

[3] The sensor chip according to [2], wherein the signal is a signal derived from a labeling substance existing in the vicinity of a specific bond between the trapping substance and the detected substance.

[4] The labeling substance is a luminescent substance and is a luminescent substance.
The sensor chip according to [3], wherein the signal is a signal derived from absorption or light emission of light by the labeling substance.

[5] The sensor chip according to [3] or [4], wherein the labeling substance is previously bound to the trapping substance or the substance to be detected.

[6] The sensor chip according to any one of [1] to [5], wherein the protective layer has an average thickness of 10 nm or more.

[7] The sensor chip according to any one of [1] to [6], wherein the substance to be detected is a molecule having a major axis of 5 nm or more.

[8] The molecule to be detected has a base and has a base.
The sensor chip according to any one of [1] to [7], wherein the capture molecule has a binding active group capable of binding a base.

[9] The sensor chip according to [4] and
An excitation light source that irradiates the capture layer with excitation light that excites the emission of the labeling substance.
A detector that detects light emission from the labeling substance and
Sensing system with.

According to the present invention, a sensor chip capable of enhancing a signal derived from a labeling substance by localized plasmon resonance and preventing deterioration of metal nanoparticles that cause localized plasmon resonance, and a sensing including the sensor chip. The system can be provided.

It is sectional drawing which shows an example of the sensor chip of this invention. FIG. 2A is a schematic cross-sectional view showing an example of a sensor chip that detects nucleotides, and FIG. 2B is a schematic cross-sectional view showing a state in which a substance to be detected is captured by the sensor chip. It is sectional drawing which shows an example of the sensing system of the emission spectrum of a sensor chip. 3 is an SEM image (10000 times and 50,000 times scale) when the metal particle aggregate layer in the metal particle aggregate layer laminated substrate obtained in Production Example 1 is viewed from directly above. It is an AFM image of the metal-based particle aggregate layer in the metal-based particle aggregate layer laminated substrate obtained in Production Example 1. It is an SEM image (10000 times and 50,000 times scale) when the metal-based particle aggregate layer in the metal-based particle aggregate layer laminated substrate obtained in Production Example 2 is seen from directly above. It is an AFM image of the metal-based particle aggregate layer in the metal-based particle aggregate layer laminated substrate obtained in Production Example 2. FIG. It is a figure which shows the emission spectrum of Example 1b and Comparative Example 1. It is a figure which plotted the conversion value of the light emission intensity of Examples 1a to 1e with respect to the value which added the average thickness of the capture layer of a protective layer.

[Sensor chip configuration]
FIG. 1 is a diagram schematically showing an example of the sensor chip of the present invention. The sensor chip 1 of the present invention is used for detecting a substance to be detected, and is provided on a substrate 10 and a substrate 10, and 30 or more metal-based particles 200 are arranged so as to be separated from each other. A metal-based particle aggregate layer 20 composed of the particle aggregates, a protective layer 30 covering the metal-based particle aggregate layer 20, and a capture layer provided on the protective layer 30 and having a capture substance that specifically binds to the molecule to be detected. 40 and.

The metal-based particle aggregate layer 20 is composed of a particle aggregate in which 30 or more metal-based particles are two-dimensionally arranged apart from each other, and the metal-based particles have an average particle size of 200 to 1600 nm. Within the range, the average height is in the range of 55 to 500 nm, and the aspect ratio defined by the ratio of the average particle size to the average height is in the range of 1 to 8.

In the sensor chip of the present invention, the trapped substance in the trapping layer 40 and the substance to be detected are specifically bonded to capture the substance to be detected on the sensor chip. In detection using a sensor chip, the substance to be detected can be analyzed qualitatively or quantitatively by a signal corresponding to the state of specific binding between the substance to be captured and the substance to be detected.

By using the sensor chip of the present invention, the signal corresponding to the specific bonding state of the trapped substance and the detected substance can be enhanced by the localized plasmon resonance generated by the metal-based particle aggregate layer 20, and the signal is highly sensitive. Detection is possible. The signal corresponding to the specific binding state is, for example, a signal derived from a labeling substance existing in the vicinity of the specific binding between the trapping substance and the detected substance. Further, according to the metal-based particle aggregate layer 20 according to the present invention, the range of action of the localized plasmon resonance can be lengthened, so that the distance from the metal-based particle aggregate layer 20 to the labeling substance is long. Can also obtain the signal enhancing effect. Further, since the action range of the localized plasmon resonance is not limited to a narrow range, the degree of freedom in the configuration of the sensor chip that can obtain the signal enhancing effect by the localized plasmon resonance is increased, and the metal-based aggregate layer 20 is formed by the protective layer 30. It can be a sufficiently protected configuration. With such a configuration, deterioration of the metal-based particle aggregate layer 20 can be prevented, and reduction of the effect of localized plasmon resonance can be suppressed.

Here, the main technical terms used in the present specification will be described.
A "capture substance" is a substance that functions to capture a substance that specifically binds to it (substance to be detected), and is fixed in the capture layer or exists in a free state in the capture layer. Or it may be fixed to the surface of the protective layer. As long as it is a substance having the above functions, any organic substance or inorganic substance can be used as a capturing substance, and examples thereof include biological substances such as nucleosides, nucleotides, nucleic acids, proteins and sugars, viruses, cells and the like. .. Further, a substance having a binding active group that can be bonded to the functional group of the substance to be detected by electrostatic interaction can be used.

The “substance to be detected” is a substance to be detected qualitatively or quantitatively, and is a substance that specifically binds to a trapping substance. All organic and inorganic substances can be detected substances, and for example, biological substances such as nucleosides, nucleotides, nucleic acids, proteins and sugars, viruses, cells and the like can be detected substances.

In the above, the nucleic acid means a polymer (nucleotide chain) of a phosphate ester of nucleoside in which a purine base or a pyrimidine base and a sugar are glycosided, and an oligonucleotide including probe DNA, a polynucleotide, a purimidine nucleotide and a pyrimidine nucleotide are polymerized. Contains DNA (full length or fragment thereof), RNA, polyamide nucleotide derivative (PNA) and the like. The nucleoside is a compound in which a base and a sugar are glycosidic bonded, and the nucleotide is a compound in which phosphoric acid is bonded to the nucleoside, and both the nucleoside and the nucleotide are compounds containing a base.

The "specific bond" broadly means a non-covalent bond between substances, a covalent bond, a chemical bond including a hydrogen bond, and examples thereof include an interaction between protein molecules and an electrostatic interaction between molecules. .. Examples of the substance to be detected that specifically binds to the capture substance include capture of sugar chains by lectins, capture of molecules by inclusion compounds, and the like.

"Sensor chip" means a sensor chip used to detect a substance to be detected by advancing a specific bond between the substance to be detected and the substance to be detected in a reaction region near the capture layer, and the substance to be detected and the substance to be detected. There are no restrictions on the type of. A sensor chip in which the substance to be detected is a substance derived from a living body, a virus, a cell, or the like is also called a biochip. The detection of the substance to be detected in the sensor chip is performed by detecting a signal corresponding to the state of the specific bond between the capture substance and the substance to be detected, and is present in the vicinity of the specific bond between the capture substance and the substance to be detected, for example. This is done by detecting a signal derived from the labeling substance. In the present invention, such a signal is not limited as long as it is a signal that can be enhanced by the localized plasmon resonance generated by the metal-based particle aggregate layer. Examples of such a signal include a signal derived from light absorption or light emission by the light emitting substance when the labeling substance is a light emitting substance. The luminescent substance is a substance that emits light by injecting excitation energy such as excitation light. The principle of light emission in a luminescent substance is not limited, and examples thereof include fluorescence, phosphorescence, and chemiluminescence.

The labeling substance may be one that is preliminarily bound to the trapping substance or the substance to be detected, or may be a labeling substance that specifically binds to the product obtained by the specific binding of the trapping substance and the test substance. Good. In the following, the case where a luminescent substance is mainly used as a labeling substance will be described in detail.

<Metallic particle aggregate layer>
In a preferred embodiment of the sensor chip of the present invention, the metal-based particle aggregate layer has any of the following characteristics.

[I] The metal-based particles constituting the metal-based particle aggregate layer are arranged so that the average distance from the adjacent metal-based particles is within the range of 1 to 150 nm (first embodiment).
[Ii] In the absorption spectrum in the visible light region, the metal-based particle aggregate layer comprises metal-based particles having the same particle size as the average particle size, the same height as the average height, and the same material among the metal-based particles. Compared to the reference metal particle aggregate (X) arranged so that the distances are all within the range of 1 to 2 μm, the maximum wavelength of the peak on the longest wavelength side shifts to the short wavelength side in the range of 30 to 500 nm. (Second embodiment),
[Iii] In the absorption spectrum in the visible light region, the metal-based particle aggregate layer comprises metal-based particles having the same particle size as the average particle size, the same height as the average height, and the same material among the metal-based particles. Compared to the reference metal particle aggregate (Y) arranged so that the distances are all within the range of 1 to 2 μm, the absorbance at the maximum wavelength of the peak on the longest wavelength side is higher than that of the reference metal particle aggregate (Y) with the same number of metal particles. High (third embodiment).

(First Embodiment)
The sensor chip of the present embodiment provided with the metal-based particle aggregate layer having the above-mentioned characteristic [i] is extremely advantageous in the following points.

(1) Since the metal-based particle aggregate layer according to the present embodiment exhibits extremely strong plasmon resonance, a stronger luminescence enhancing effect can be obtained as compared with the case where a conventional plasmon material is used, thereby achieving luminous efficiency. Can be dramatically increased. The strength of the plasmon resonance exhibited by the metal-based particle aggregate layer is not merely the sum of the localized plasmon resonance exhibited by the individual metal-based particles at a specific wavelength, but is stronger than that. That is, when 30 or more metal-based particles having a predetermined shape are densely arranged at the above-mentioned predetermined intervals, the individual metal-based particles interact with each other to develop extremely strong plasmon resonance. It is considered that this was expressed by the interaction between the localized plasmons of the metallic particles.

In general, when the absorption spectrum of a plasmon material is measured by the absorptiometry method, a plasmon resonance peak (hereinafter, also referred to as a plasmon peak) is observed as a peak in the ultraviolet to visible region, and the magnitude of the absorbance value at the maximum wavelength of this plasmon peak is used. , The strength of the plasmon resonance of the plasmon material can be roughly evaluated, but when the absorption spectrum of the metal-based particle aggregate layer according to the present embodiment is measured in a state where it is laminated on a glass substrate, The absorbance of the plasmon peak on the longest wavelength side in the visible light region at the maximum wavelength can be 1 or more, 1.5 or more, and even 2 or so.

The absorption spectrum of the metal-based particle aggregate layer is measured by the absorptiometry in a state of being laminated on a glass substrate. Specifically, the absorption spectrum is on the back surface side of the glass substrate on which the metal-based particle aggregate layer is laminated (the side opposite to the metal-based particle aggregate layer), and is ultraviolet to visible from the direction perpendicular to the substrate surface. A substrate having the same thickness and material as the substrate of the metal-based particle aggregate film laminated substrate and the intensity I of the transmitted light in all directions transmitted to the metal-based particle aggregate layer side by irradiating the incident light in the optical region. , The same incident light as before is irradiated from the direction perpendicular to the surface of the substrate on which the metal particle aggregate film is not laminated, and the intensity I ₀ of the transmitted light in all directions transmitted from the opposite side of the incident surface is integrated. Obtained by measurement using a spherical spectrophotometer. At this time, the absorbance, which is the vertical axis of the absorption spectrum, is calculated by the following equation:
Absorbance = -log ₁₀ (I / I ₀ )
It is represented by.

(2) Since the range of action of plasmon resonance by the metallic particle aggregate layer (the range covered by the plasmon enhancing effect) is significantly extended, a stronger luminous enhancement effect can be obtained as compared with the case of using a conventional plasmon material. It can be obtained, which contributes to a dramatic improvement in luminous efficiency as described above. That is, even if the average thickness of the first layer covering the metal-based particle aggregate is 10 nm or more or is supported in order to sufficiently protect the metal-based particle aggregate layer by the significant extension of this range of action. Even if the substance and the substance to be detected are large, it is possible to enhance the light emission of the labeling substance, which can significantly improve the light emission efficiency of the sensor chip.

It is considered that such an elongation action is also exhibited by the interaction between the localized plasmons of the metal-based particles generated by densely arranging 30 or more metal-based particles having a predetermined shape at predetermined intervals. According to the metal-based particle aggregate layer of the present embodiment, the range of action of plasmon resonance, which was conventionally limited to within the range of Felster distance (about 10 nm or less), can be extended to, for example, about several hundred nm. it can.

As described above, the metal-based particle aggregate layer according to the present embodiment uses relatively large metal-based particles in which dipole-type localized plasmons are unlikely to occur in the visible light region by itself. By densely arranging a specific number or more of large metallic particles (although they must have a predetermined shape) at specific intervals, the large metallic particles are included. An extremely large number of surface free electrons can be effectively excited as plasmons, which makes it possible to realize extremely strong plasmon resonance and a remarkable extension of the working range of plasmon resonance.

Further, the sensor chip of the present embodiment has a structure in which a specific number or more of relatively large metal-based particles having a specific shape are arranged two-dimensionally at specific intervals in the metal-based particle aggregate layer. Due to having, the following advantageous effects can be obtained.

(3) In the metal-based particle aggregate layer according to the present embodiment, the maximum wavelength of the plasmon peak is peculiar in the absorption spectrum in the visible light region, depending on the average particle size of the metal-based particles and the average distance between the particles. Since it can exhibit a shift, it is possible to particularly enhance the emission in a specific (desired) wavelength region. Specifically, as the average distance between the particles is kept constant and the average particle size of the metal particles is increased, the maximum wavelength of the plasmon peak on the longest wavelength side in the visible light region shifts to the short wavelength side (blue shift). ). Similarly, as the average particle size of the large metallic particles is kept constant and the average distance between the particles is reduced (when the metallic particles are arranged more densely), the plasmon peak on the longest wavelength side in the visible light region The maximum wavelength shifts to the short wavelength side. This peculiar phenomenon is due to the Mie scattering theory generally accepted for plasmon materials (according to this theory, the maximum wavelength of the plasmon peak shifts to the long wavelength side (red shift) as the particle size increases). It is contrary to that.

The peculiar blue shift as described above also has a structure in which the metal-based particle aggregate layer densely arranges large metal-based particles at specific intervals, and accordingly, the station of the metal-based particles It is considered that this is due to the interaction between the plasmons in the plasmon. The metal-based particle aggregate layer (in a state of being laminated on a glass substrate) according to the present embodiment has an absorption spectrum in a visible light region measured by an absorptiometry according to the shape of the metal-based particles and the distance between the particles. The plasmon peak on the longest wavelength side can exhibit a maximum wavelength in the wavelength region of, for example, 350 to 550 nm. Further, the metal-based particle aggregate layer according to the present embodiment is typically about 30 to 500 nm (compared to the case where the metal-based particles are arranged at a sufficiently long inter-particle distance (for example, 1 μm)). For example, a blue shift of 30-250 nm) can occur.

A sensor chip including such a metal-based particle aggregate layer in which the maximum wavelength of the plasmon peak is blue-shifted is extremely advantageous in the following points, for example. That is, even when a blue luminescent substance having a relatively low luminous efficiency is used as a labeling substance, the luminous efficiency can be enhanced to a sufficient extent.

Next, a specific configuration of the metal-based particle aggregate layer according to the present embodiment will be described.
The metal-based particles constituting the metal-based particle aggregate layer are particularly limited as long as they are made of a material having a plasmon peak in the ultraviolet to visible region in the absorption spectrum measurement by the absorptiometry when the nanoparticles or their aggregates are formed. For example, precious metals such as gold, silver, copper, platinum, and palladium, metals such as aluminum and tantalum; alloys containing the precious metals or metals; metal compounds containing the precious metals or metals (metal oxides, metal salts, etc.) ) Can be mentioned. Among these, precious metals such as gold, silver, copper, platinum, and palladium are preferable, and silver is more preferable because it is inexpensive and has low absorption (the imaginary part of the dielectric function is small at the visible light wavelength).

The average particle size of the metal particles is in the range of 200 to 1600 nm, and in order to effectively obtain the effects of (1) to (3) above, it is preferably 200 to 1200 nm, more preferably 250 to 500 nm, and even more preferably. Is in the range of 300 to 500 nm. What should be noted here is that, for example, a large metal-based particle having an average particle size of 500 nm has almost no enhancing effect by localized plasmon by itself, as described above. On the other hand, in the metal-based particle aggregate layer according to the present embodiment, remarkably strong plasmon resonance and plasmon are formed by densely arranging a predetermined number (30) or more of such large metal-based particles at predetermined intervals. It realizes a remarkable extension of the action range of resonance and the effect of (3) above.

The average particle size of the metal-based particles is defined as the average particle size of 10 particles randomly selected from the SEM observation image from directly above the metal-based particle aggregate (film) in which the metal-based particles are arranged in two dimensions. Randomly draw 5 tangent diameters in the image (however, all straight lines with tangent diameters can pass only inside the particle image, and one of them is the longest straight line that can be drawn only inside the particle. ), It is the average value of the selected 10 particle sizes when the average value is taken as the particle size of each particle. The tangent diameter is defined as a perpendicular line connecting the contour (projected image) of a particle between two parallel lines in contact with it (Nikkan Kogyo Shimbun, "Particle Measurement Technology", 1994, p. 5). ..

More specifically, the method for measuring the average particle size will be described. First, the SEM observation image is measured using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd. Next, the obtained observation image is read in a width of 1280 pixels and a height of 960 pixels using the free image processing software "ImageJ" manufactured by the National Institutes of Health. Next, using the random number generation function "RANDBETWEEN" of the spreadsheet software "excel" manufactured by Microsoft, 10 random numbers (x ₁ , x ₂ , x ₃ , x ₄ , x ₅ , x _{6) from 1 to 1280.} , X ₇ , x ₈ , x ₉ , x ₁₀ ), 1 to 960 to 10 random numbers (y ₁ , y ₂ , y ₃ , y ₄ , y ₅ , y ₆ , y ₇ , y ₈ , y ₉ , y ₁₀ ) are obtained respectively. 10 sets of random number combinations (x ₁ , y ₁ ), (x ₂ , y ₂ ), (x ₃ , y ₃ ), (x ₄ , y ₄ ), (x ₅ , y 1) from each of the obtained 10 random numbers. We obtain y ₅ ), (x ₆ , y ₆ ), (x ₇ , y ₇ ), (x ₈ , y ₈ ), (x ₉ , y ₉ ) and (x ₁₀ , y _{10). Ten sets of coordinate points (x 1} , y ₁ ), (x ₂ , y ₂ ), where the numerical value of the random number generated from 1 to 1280 is the x coordinate and the numerical value of the random number generated from 1 to 960 is the y coordinate. (X ₃ , y ₃ ), (x ₄ , y ₄ ), (x ₅ , y ₅ ), (x ₆ , y ₆ ), (x ₇ , y ₇ ), (x ₈ , y ₈ ), (x _{Obtain 9} , y ₉ ) and (x ₁₀ , y ₁₀ ). Then, the above average value of the tangential diameters is obtained for each of the total of 10 particle images including the coordinate points, and then the average particle size is obtained as the average value of the average values of the 10 tangent diameters. If at least one of the 10 coordinate points, which is a set of 10 random number combinations, is not included in the particle image, or if two or more coordinate points are included in the same particle, this random number combination is discarded. Then, random number generation is repeated until all 10 coordinate points are included in different particle images.

The average height of the metallic particles is in the range of 55 to 500 nm, preferably in the range of 55 to 300 nm, more preferably in the range of 70 to 150 nm in order to effectively obtain the effects (1) to (3) above. Is. The average height of the metal-based particles is 10 when 10 particles are randomly selected in the AFM observation image of the metal-based particle aggregate layer (film) and the heights of these 10 particles are measured. It is the average value of the measured values of.

The aspect ratio of the metallic particles is in the range of 1 to 8, preferably in the range of 2 to 8, and more preferably in the range of 2.5 to 8 in order to effectively obtain the effects of (1) to (3) above. Inside. The aspect ratio of the metallic particles is defined by the ratio of the average particle size to the average height (average particle size / average height). The metallic particles may be spherical, but preferably have a flat shape having an aspect ratio of more than 1.

From the viewpoint of exciting highly effective plasmons, the metal particles preferably have a smooth curved surface, and more preferably have a flat shape having a smooth curved surface. It may contain some fine irregularities (roughness), and in this sense, the metal-based particles may have an irregular shape.

In view of the uniformity of the strength of plasmon resonance in the layer plane of the metal-based particle aggregate, it is preferable that the size variation between the metal-based particles is as small as possible. However, even if there is some variation in the particle size, it is not preferable that the distance between the large particles is large, and it is preferable that the small particles fill the space between the large particles to facilitate the interaction between the large particles.

In the metal-based particle aggregate layer according to the present embodiment, the metal-based particles are arranged so that the average distance between the metal-based particles and the adjacent metal-based particles (hereinafter, also referred to as the average inter-particle distance) is within the range of 1 to 150 nm. Will be done. By arranging the metal-based particles densely in this way, it is possible to realize a remarkably strong plasmon resonance, a remarkably extended range of action of the plasmon resonance, and the effect of (3) above. The average distance is preferably in the range of 1 to 100 nm, more preferably 1 to 50 nm, and further preferably 1 to 20 nm in order to effectively obtain the effects of (1) to (3). If the average interparticle distance is less than 1 nm, electron transfer based on the Dexter mechanism occurs between the particles, which is disadvantageous in terms of deactivation of localized plasmons.

The metal-based particle aggregate layer in which the metal-based particles are arranged apart from each other does not exhibit conductivity as the layer. Specifically, the metal-based particle aggregate layer 20 is subjected to a multimeter [tester (tester (tester)). When a pair of tester probes manufactured by Hulett-Packard Co., Ltd. "E2378A")] are brought into contact with each other at a distance of 10 to 15 mm, and when the range setting is "30 MΩ", the resistance value is 30 MΩ or more under the measurement conditions. "Overload" is displayed. When electrons can be transferred between some or all metallic particles, the plasmon peak loses its sharpness and approaches the absorption spectrum of bulk metal, and high plasmon resonance cannot be obtained. Therefore, it is preferable that the metal-based particles are surely separated from each other and that no conductive substance is interposed between the metal-based particles.

The average inter-particle distance is an SEM observation image from directly above the metal-based particle aggregate layer in which the metal-based particles are two-dimensionally arranged. It is an average value of the interparticle distances of these 30 particles when the interparticle distances with adjacent particles are obtained. The inter-particle distance between adjacent particles is a value obtained by measuring the distances between all adjacent particles (distances between surfaces) and averaging them.

More specifically, the method for measuring the average interparticle distance will be described. First, the SEM observation image is measured using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd. Next, the obtained observation image is read in a width of 1280 pixels and a height of 960 pixels using the free image processing software "ImageJ" manufactured by the National Institutes of Health. Next, using the random number generation function "RANDBETWEEN" of the spreadsheet software "excel" manufactured by Microsoft, 1 to 1280 to 30 random numbers (x _{1 to x 30} ) and 1 to 960 to 30 random numbers (y). _{1 to} y ₃₀ ) are obtained respectively. From each of the obtained 30 random numbers, 30 sets of random number combinations (x ₁ , y ₁ ) are obtained (x ₃₀ , y _{30). 30 sets of coordinate points (x 1} , y ₁ ) to (x ₃₀ , y ₃₀ ) are set with the numerical value of the random number generated from 1 to 1280 as the x coordinate and the numerical value of the random number generated from 1 to 960 as the y coordinate. obtain. Then, for each of the total of 30 particle images including the coordinate points, the interparticle distance between the particle and the adjacent particle is obtained, and then the average particle is used as the average value of the interparticle distance between the 30 adjacent particles. Get the distance. If at least one of the 30 coordinate points, which is a combination of 30 random numbers, is not included in the particle image, or if two or more coordinate points are included in the same particle, this random number combination is discarded. Then, random number generation is repeated until all 30 coordinate points are included in different particle images.

The number of metal-based particles contained in the metal-based particle aggregate layer is 30 or more, preferably 50 or more. By forming an aggregate containing 30 or more metal-based particles, extremely strong plasmon resonance and extension of the action range of plasmon resonance are exhibited by the interaction between the localized plasmons of the metal-based particles.

In light of the general element area of the sensor chip, the number of metal-based particles contained in the metal-based particle aggregate can be, for example, 300 or more, or even 17,500 or more.

The number density of the metal-based particles in the metal-based particle aggregate layer ^{is preferably 7 particles / μm 2} or more, and more preferably 15 ^{particles / μm 2} or more.

In the metal-based particle aggregate layer, the metal-based particles are insulated from each other, in other words, they are non-conductive with respect to adjacent metal-based particles (non-conductive as a metal-based particle aggregate layer). Is preferable. When electrons can be transferred between some or all metallic particles, the plasmon peak loses its sharpness and approaches the absorption spectrum of bulk metal, and high plasmon resonance cannot be obtained. Therefore, it is preferable that the metal-based particles are surely separated from each other and that no conductive substance is interposed between the metal-based particles.

(Second Embodiment)
In the absorption spectrum in the visible light region, the sensor chip of the present embodiment has a peak maximum wavelength in the range of 30 to 500 nm on the longest wavelength side as compared with the reference metal particle aggregate (X) on the short wavelength side. It includes a metal-based particle aggregate layer (having the characteristics of [ii] above) that is shifted to. The sensor chip of the present embodiment provided with the metal-based particle aggregate layer having such characteristics is extremely advantageous in the following points.

(I) In the metal-based particle aggregate layer according to the present embodiment, since the maximum wavelength of the plasmon peak on the longest wavelength side exists in a specific wavelength region in the absorption spectrum in the visible light region, it is specific (desired). It is possible to particularly enhance the light emission in the wavelength region. Specifically, in the metal-based particle aggregate layer according to the present embodiment, when the absorption spectrum is measured, the maximum wavelength of the plasmon peak is higher than the maximum wavelength of the reference metal-based particle aggregate (X) described later. , 30 to 500 nm (for example, 30 to 250 nm) shifts to the short wavelength side (blue shift), and typically, the maximum wavelength of the plasmon peak is in the range of 350 to 550 nm.

The blue shift has a structure in which a metal-based particle aggregate layer has a structure in which a specific number or more of large metal-based particles having a specific shape are arranged two-dimensionally separated from each other. It is considered that this is due to the interaction between the localized plasmons of.

The metal-based particle aggregate layer according to the present embodiment having a plasmon peak in the blue or near wavelength region is extremely useful for enhancing the light emission of a sensor chip that detects a substance to be detected having a luminescent substance in the blue or near wavelength region. Therefore, in the sensor chip provided with such a metal-based particle aggregate layer, the luminous efficiency can be sufficiently enhanced even when a blue light emitting material having a relatively low luminous efficiency is used.

Here, when comparing the maximum wavelength of the peak on the longest wavelength side and the absorbance at the maximum wavelength between a certain metal-based particle aggregate and the reference metal-based particle aggregate (X), both are microscopically measured. (Using a Nikon "OPTIPHOT-88" and a spectrophotometer ("MCPD-3000" manufactured by Otsuka Electronics Co., Ltd.), the absorbance spectrum is measured by narrowing the measurement field.

The reference metal-based particle aggregate (X) is a metal-based particle A having an average particle size, the same particle size, height, and the same material as the average height of the metal-based particle aggregate layer to be measured by the absorption spectrum. It is a metal-based particle aggregate arranged so that the distances between the metal-based particles are all within the range of 1 to 2 μm, and the absorption spectrum can be measured using the above-mentioned microscope in a state of being laminated on a glass substrate. It has the size of.

The absorption spectrum waveform of the reference metal-based particle aggregate (X) is the particle size and height of the metal-based particle A, the dielectric function of the material of the metal-based particle A, and the dielectric function of the medium (for example, air) around the metal-based particle A. , It is also possible to theoretically calculate by the 3D-FDTD method using the dielectric function of a substrate (for example, a glass substrate).

Further, the sensor chip of the present embodiment has a structure in which a specific number or more of relatively large metal-based particles having a specific shape are arranged two-dimensionally apart from each other in the metal-based particle aggregate layer. As a result, (II) the metallic particle aggregate layer can exhibit extremely strong plasmon resonance, so that a stronger luminescence enhancing effect can be obtained as compared with the case where a conventional plasmon material is used. It is possible to dramatically increase the light emission efficiency (similar to the effect (1) of the first embodiment above), and (III) the range of action of plasmon resonance by the metal-based particle aggregate layer (enhancement effect by plasmon). Since the range) can be significantly extended, a stronger luminescence enhancing effect can be obtained as compared with the case where a conventional plasmon material is used, and similarly, the luminescence efficiency can be dramatically increased (the above-mentioned first). The effect of the first embodiment (similar to (2)), and the like can be achieved. The metal-based particle aggregate layer according to the present embodiment has an absorbance of 1 or more at the maximum wavelength of the plasmon peak on the longest wavelength side in the visible light region when the absorption spectrum is measured in a state where the metal-based particle aggregate layer is laminated on a glass substrate. It can be 1.5 or more, and even 2 or more.

Next, a specific configuration of the metal-based particle aggregate layer according to the present embodiment will be described. The specific configuration of the metal-based particle aggregate layer according to the present embodiment is the specific configuration of the metal-based particle aggregate layer according to the first embodiment (material of metal-based particles, average particle size, average height, aspect). The ratio, the average inter-particle distance, the number of metal-based particles, the non-conductive nature of the metal-based particle aggregate layer, etc.) can be basically the same. Definitions of terms such as average particle size, average height, aspect ratio, and average interparticle distance are also the same as in the first embodiment.

The average particle size of the metal-based particles is in the range of 200 to 1600 nm, and in order to effectively obtain the effects of (I) to (III) above, it is preferably 200 to 1200 nm, more preferably 250 to 500 nm, and even more preferably. Is in the range of 300 to 500 nm. In the metal-based particle aggregate layer according to the present embodiment, remarkably strong plasmon resonance and plasmon resonance are formed by forming an aggregate in which a predetermined number (30) or more of such large metal-based particles are two-dimensionally arranged. It is possible to realize a remarkable extension of the working range of. Further, in order to exhibit the above-mentioned characteristic [ii] (shift of plasmon peak to the short wavelength side), it is essential that the metal-based particles have a large average particle size of 200 nm or more, preferably 250 nm or more. Is.

In the metal-based particle aggregate layer according to the present embodiment, the maximum wavelength of the plasmon peak on the longest wavelength side in the visible light region depends on the average particle size of the metal-based particles. That is, when the average particle size of the metal-based particles exceeds a certain value, the maximum wavelength of the plasmon peak shifts (blue shifts) to the short wavelength side.

The average height of the metallic particles is in the range of 55 to 500 nm, preferably in the range of 55 to 300 nm, more preferably in the range of 70 to 150 nm in order to effectively obtain the effects (I) to (III) above. Is. The aspect ratio of the metallic particles is in the range of 1 to 8, preferably in the range of 2 to 8, and more preferably in the range of 2.5 to 8 in order to effectively obtain the effects (I) to (III) above. Inside. The metallic particles may be spherical, but preferably have a flat shape having an aspect ratio of more than 1.

From the viewpoint of exciting highly effective plasmons, the metal particles preferably have a smooth curved surface, and more preferably have a flat shape having a smooth curved surface. It may contain some fine irregularities (roughness), and in this sense, the metal-based particles may have an irregular shape. Further, in view of the uniformity of the strength of plasmon resonance in the surface of the metal-based particle aggregate layer, it is preferable that the size variation between the metal-based particles is as small as possible. However, as described above, even if there is some variation in the particle size, it is not preferable that the distance between the large particles is large, and the small particles fill the space between them to facilitate the interaction between the large particles. Is preferable.

In the metal-based particle aggregate layer according to the present embodiment, the metal-based particles are preferably arranged so that the average distance (average inter-particle distance) from the adjacent metal-based particles is within the range of 1 to 150 nm. .. It is more preferably in the range of 1 to 100 nm, further preferably 1 to 50 nm, and particularly preferably in the range of 1 to 20 nm. By arranging the metal-based particles densely in this way, the interaction between the localized plasmons of the metal-based particles is effectively generated, and the above-mentioned effects (I) to (III) are likely to be exhibited. Since the maximum wavelength of the plasmon peak depends on the average interparticle distance of the metal-based particles, the degree of blue shift of the plasmon peak on the longest wavelength side and the maximum wavelength of the plasmon peak can be controlled by adjusting the average interparticle distance. It is possible to do. If the average interparticle distance is less than 1 nm, electron transfer based on the Dexter mechanism occurs between the particles, which is disadvantageous in terms of deactivation of localized plasmons.

As another means other than the above for expressing the above-mentioned characteristic of [ii] (shift of plasmon peak to the short wavelength side), for example, a dielectric substance having a dielectric constant different from that of air between metal-based particles (described later). As described above, a method of interposing (preferably a non-conductive substance) can be mentioned.

The number of metal-based particles contained in the metal-based particle aggregate layer is 30 or more, preferably 50 or more. By forming an aggregate containing 30 or more metallic particles, the interaction between the localized plasmons of the metallic particles is effectively generated, and the above-mentioned characteristics [ii] and the above-mentioned effects (I) to (III) are obtained. Can be expressed.

In light of the general element area of the sensor chip, the number of metal-based particles contained in the metal-based particle aggregate can be, for example, 300 or more, or even 17,500 or more.

The number density of the metal-based particles in the metal-based particle aggregate layer ^{is preferably 7 particles / μm 2} or more, and more preferably 15 ^{particles / μm 2} or more.

Also in the metal-based particle aggregate layer of the present embodiment, as in the first embodiment, the metal-based particles are insulated from each other, in other words, they are non-conductive (metal) with respect to adjacent metal-based particles. It is preferable that the system particle aggregate layer is non-conductive).

(Third Embodiment)
In the absorption spectrum in the visible light region, the sensor chip of the present embodiment has a peak at the maximum wavelength on the longest wavelength side in comparison with the same number of metal particles as the reference metal particle aggregate (Y). It includes a metal-based particle aggregate layer having high absorbance (having the characteristics of [iii] described above). The sensor chip of the present embodiment provided with the metal-based particle aggregate layer having such characteristics is extremely advantageous in the following points.

(A) In the metal-based particle aggregate layer according to the present embodiment, the absorbance at the maximum wavelength of the peak on the longest wavelength side in the visible light region, which is the plasmon peak, is such that the metal-based particles do not interact with each other. Larger than the above-referenced metal-based particle aggregate (Y), which can be regarded as simply an aggregate, and therefore exhibiting extremely strong plasmon resonance, a stronger luminescence enhancement as compared with the case of using a conventional plasmon material. The effect can be obtained, and thus the light emission efficiency can be dramatically increased. It is considered that such strong plasmon resonance is expressed by the interaction between the localized plasmons of the metallic particles.

As described above, the strength of the plasmon resonance of the plasmon material can be roughly evaluated from the magnitude of the absorbance value at the maximum wavelength of the plasmon peak. When the absorbance spectrum was measured with this laminated on a glass substrate, the absorbance at the maximum wavelength of the plasmon peak on the longest wavelength side in the visible light region was 1 or more, 1.5 or more, and even 2 Can be a degree.

As described above, when comparing the maximum wavelength of the peak on the longest wavelength side and the absorbance at the maximum wavelength between a certain metal-based particle aggregate and the reference metal-based particle aggregate (Y), both are used. , A microscope (“OPTIPHOT-88” manufactured by Nikon Co., Ltd. and a spectrophotometer (“MCPD-3000” manufactured by Otsuka Electronics Co., Ltd.)) is used to narrow the measurement field and measure the absorbance spectrum.

The reference metal-based particle aggregate (Y) is a metal-based particle B having an average particle size, the same particle size, height, and the same material as the average height of the metal-based particle aggregate layer to be measured for absorption spectrum. It is a metal-based particle aggregate arranged so that the distances between the metal-based particles are all within the range of 1 to 2 μm, and the absorption spectrum can be measured using the above-mentioned microscope in a state of being laminated on a glass substrate. It has the size of.

When comparing the absorbance at the maximum wavelength of the peak on the longest wavelength side between the metal-based particle aggregate layer to be measured for absorption spectrum measurement and the reference metal-based particle aggregate (Y), it is described below. As described above, the absorption spectrum of the reference metal-based particle aggregate (Y) converted so that the number of metal-based particles is the same is obtained, and the absorbance at the maximum wavelength of the peak on the longest wavelength side in the absorption spectrum is compared. To do. Specifically, the absorbance spectra of the metal-based particle aggregate and the reference metal-based particle aggregate (Y) are obtained, and the absorbance at the maximum wavelength of the peak on the longest wavelength side in each absorption spectrum is determined by the respective coverage. Calculate the value divided by (the coverage of the substrate surface with metal particles) and compare them.

Further, the sensor chip of the present embodiment has a structure in which a specific number or more of relatively large metal-based particles having a specific shape are arranged two-dimensionally apart from each other in the metal-based particle aggregate layer. As a result, (B) the range of action of the plasmon resonance by the metallic particle aggregate layer (the range of the enhancement effect by the plasmon) can be significantly extended, so that the range of action of the plasmon resonance is more than that of the case of using the conventional plasmon material. A strong luminescence enhancing effect can be obtained, whereby the luminescence efficiency can be dramatically increased (similar to the effect (2) of the first embodiment above), and (C) a metal-based particle aggregate layer. Since the maximum wavelength of the plasmon peak of the above can exhibit a peculiar shift, it is possible to enhance the light emission in a specific (desired) wavelength region (similar to the effect (3) of the first embodiment), etc. Can produce the effect of.

The metal-based particle aggregate layer (in a state of being laminated on a glass substrate) of the present embodiment has an absorption spectrum in a visible light region measured by an absorptiometry according to the shape of the metal-based particles and the distance between the particles. The plasmon peak on the longest wavelength side may exhibit a maximum wavelength in the wavelength region of, for example, 350 to 550 nm. Further, the metal-based particle aggregate layer of the present embodiment is typically about 30 to 500 nm (for example,) as compared with the case where the metal-based particles are arranged at a sufficiently long inter-particle distance (for example, 1 μm). A blue shift of 30-250 nm) can occur.

Next, a specific configuration of the metal-based particle aggregate layer according to the present embodiment will be described. The specific configuration of the metal-based particle aggregate layer according to the present embodiment is the specific configuration of the metal-based particle aggregate layer according to the first embodiment (material of metal-based particles, average particle size, average height, aspect). The ratio, the average inter-particle distance, the number of metal-based particles, the non-conductive nature of the metal-based particle aggregate layer, etc.) can be basically the same. Definitions of terms such as average particle size, average height, aspect ratio, and average interparticle distance are also the same as in the first embodiment.

The average particle size of the metal-based particles is in the range of 200 to 1600 nm, and the above-mentioned feature of [iii] (the absorbance at the maximum wavelength of the plasmon peak on the longest wavelength side is higher than that of the reference metal-based particle aggregate (Y). In order to effectively obtain the effects of (A) to (C) above, it is preferably in the range of 200 to 1200 nm, more preferably 250 to 500 nm, and further preferably 300 to 500 nm. .. In this way, it is important to use relatively large metal particles, and by forming an aggregate in which a predetermined number (30) or more of large metal particles are arranged in two dimensions, extremely strong plasmon resonance occurs. Furthermore, it is possible to significantly extend the range of action of plasmon resonance and to shift the plasmon peak to the short wavelength side.

The average height of the metallic particles is in the range of 55 to 500 nm, and in order to effectively obtain the characteristics of the above [iii] and the effects of the above (A) to (C), it is preferably 55 to 300 nm. More preferably, it is in the range of 70 to 150 nm. The aspect ratio of the metallic particles is in the range of 1 to 8, and in order to effectively obtain the characteristics of the above [iii] and the effects of the above (A) to (C), preferably 2 to 8 or more. It is preferably in the range of 2.5 to 8. The metallic particles may be spherical, but preferably have a flat shape having an aspect ratio of more than 1.

From the viewpoint of exciting highly effective plasmons, the metal particles preferably have a smooth curved surface, and more preferably have a flat shape having a smooth curved surface. It may contain some fine irregularities (roughness), and in this sense, the metal-based particles may have an irregular shape.

Since the above-mentioned characteristics of [iii] can be effectively obtained, the size and shape (average particle size, average height, aspect ratio) of the metal-based particles constituting the metal-based particle aggregate layer are as uniform as possible. It is preferable to have. That is, by making the size and shape of the metal-based particles uniform, the plasmon peak is sharpened, and accordingly, the absorbance of the plasmon peak on the longest wavelength side is higher than that of the reference metal-based particle aggregate (Y). It tends to be expensive. Reducing the variation in size and shape between metallic particles is also advantageous from the viewpoint of uniformity of plasmon resonance intensity in the surface of the metallic particle aggregate layer. However, as described above, even if there is some variation in the particle size, it is not preferable that the distance between the large particles is large, and the small particles fill the space between them to facilitate the interaction between the large particles. Is preferable.

In the metal-based particle aggregate layer according to the present embodiment, the metal-based particles are preferably arranged so that the average distance (average inter-particle distance) from the adjacent metal-based particles is within the range of 1 to 150 nm. .. It is more preferably in the range of 1 to 100 nm, further preferably 1 to 50 nm, and particularly preferably in the range of 1 to 20 nm. By arranging the metal-based particles densely in this way, the interaction between the localized plasmons of the metal-based particles is effectively generated, and the above-mentioned characteristics [iii] and further the above-mentioned effects (A) to (C) are obtained. Can be effectively expressed. If the average interparticle distance is less than 1 nm, electron transfer based on the Dexter mechanism occurs between the particles, which is disadvantageous in terms of deactivation of localized plasmons.

The number of metal-based particles contained in the metal-based particle aggregate layer is 30 or more, preferably 50 or more. By forming an aggregate containing 30 or more metal-based particles, the interaction between the localized plasmons of the metal-based particles is effectively generated, and the above-mentioned characteristics of [iii], as well as the above-mentioned (A) to (C). The effect of can be effectively expressed.

In light of the general element area of the sensor chip, the number of metal-based particles contained in the metal-based particle aggregate can be, for example, 300 or more, or even 17,500 or more.

The number density of the metal-based particles in the metal-based particle aggregate layer ^{is preferably 7 particles / μm 2} or more, and more preferably 15 ^{particles / μm 2} or more.

Also in the metal-based particle aggregate layer of the present embodiment, as in the first embodiment, the metal-based particles are insulated from each other, in other words, they are non-conductive (metal) with respect to adjacent metal-based particles. It is preferable that the system particle aggregate layer is non-conductive).

As described above, the metal-based particle aggregate layer according to the present embodiment having the above-mentioned characteristics of [iii] has the metal species, size, shape, average distance between the metal-based particles, and the like of the metal-based particles constituting the layer. It can be obtained by control.

The metal-based particle aggregate layer included in the sensor chip of the present invention preferably has any one of the above-mentioned features [i] to [iii], and has any two or more features of [i] to [iii]. It is more preferable to have all the characteristics of [i] to [iii].

<Manufacturing method of metallic particle aggregate layer>
The metal-based particle aggregate layer according to the present invention including the metal-based particle aggregate layer according to the first to third embodiments can be produced by the following method.

(1) A bottom-up method in which metallic particles are grown from minute seeds on a substrate.
(2) A method of coating metal particles having a predetermined shape with a protective layer made of an amphipathic material having a predetermined thickness, and then forming a film on a substrate by an LB (Langmuir-Blodget) film method.
(3) In addition, a method of post-treating a thin film produced by vapor deposition or sputtering, a resist process, an etching process, a casting method using a dispersion liquid in which metal-based particles are dispersed, and the like.

In the above method (1), it is important to include a step of growing metal-based particles at an extremely low speed (hereinafter, also referred to as a particle growth step) on a substrate adjusted to a predetermined temperature. According to the production method including such a particle growth step, 30 or more metal-based particles are two-dimensionally arranged apart from each other, and the metal-based particles have a shape within a predetermined range (average particle size 200 to 200 to). A layer (thin film) of a metal-based particle aggregate having an average height of 1600 nm, an average height of 55 to 500 nm, and an aspect ratio of 1 to 8), more preferably an average particle distance (1 to 150 nm) within a predetermined range can be obtained with good control. it can.

In the particle growth step, the rate of growing metal-based particles on the substrate is preferably less than 1 nm / min in average height growth rate, and more preferably 0.5 nm / min or less. The average height growth rate referred to here can also be called the average deposition rate or the average thickness growth rate of metal-based particles, and the following formula:
Average height of metallic particles / Growth time of metallic particles (supply time of metallic materials)
Defined in. The definition of "average height of metallic particles" is as described above.

The temperature of the substrate in the particle growth step is preferably in the range of 100 to 450 ° C., more preferably 200 to 450 ° C., further preferably 250 to 350 ° C., particularly preferably 300 ° C. or its vicinity (about 300 ° C. ± 10 ° C.). ).

In the manufacturing method including a particle growth step of growing metallic particles at an average height growth rate of less than 1 nm / min on a substrate whose temperature is adjusted in the range of 100 to 450 ° C., the particles were supplied in the early stage of particle growth. A plurality of island-like structures made of metal-based materials are formed, and these island-like structures grow large with the supply of further metal-based materials and coalesce with the surrounding island-like structures, and as a result, Although the individual metal-based particles are completely separated from each other, a metal-based particle aggregate layer in which particles having a relatively large average particle size are densely arranged is formed. Therefore, a metal particle aggregate layer composed of metal particles controlled to have a shape (average particle size, average height and aspect ratio) within a predetermined range, and more preferably an average particle distance within a predetermined range. It becomes possible to manufacture.

In addition, the average particle size, average height, and aspect of the metal particles grown on the substrate are adjusted by adjusting the average height growth rate, the substrate temperature, and / or the growth time of the metal particles (supply time of the metal material). It is also possible to control the ratio and / or average interparticle distance within a predetermined range.

Further, according to the manufacturing method including the particle growth step, various conditions other than the substrate temperature and the average height growth rate in the particle growth step can be selected relatively freely, so that the desired size can be obtained on the desired size substrate. There is also an advantage that the metal-based particle aggregate layer can be efficiently formed.

When the average height growth rate is 1 nm / min or more, or when the substrate temperature is less than 100 ° C or more than 450 ° C, the island-like structure and the surrounding island-like structure and the continuum are formed before the island-like structure grows large. It is not possible to obtain a metal-based aggregate composed of metal-based particles having a large particle size that are formed and completely separated from each other, or it is not possible to obtain a metal-based aggregate composed of metal-based particles having a desired shape. (For example, the average height, the average interparticle distance, and the aspect ratio are out of the desired range).

The pressure for growing metallic particles (pressure in the apparatus chamber) is not particularly limited as long as the pressure allows the particles to grow, but is usually less than atmospheric pressure. The lower limit of the pressure is not particularly limited, but it is preferably 6 Pa or more, more preferably 10 Pa or more, and further preferably 30 Pa or more because the average height growth rate can be easily adjusted within the above range.

The specific method for growing the metal-based particles on the substrate is not particularly limited as long as the particles can grow at an average height growth rate of less than 1 nm / min, and examples thereof include a sputtering method and a vapor deposition method such as vacuum deposition. it can. Among the sputtering methods, the direct current (DC) sputtering method is used because the metal-based particle aggregate layer can be grown relatively easily and the average height growth rate of less than 1 nm / min can be easily maintained. Is preferable. The sputtering method is not particularly limited, and a DC argon ion sputtering method in which an ion gun or an argon ion generated by plasma discharge is accelerated by an electric field to irradiate the target can be used. Other conditions such as the current value, the voltage value, and the distance between the substrate and the target in the sputtering method are appropriately adjusted so that the particles grow at an average height growth rate of less than 1 nm / min.

In order to obtain a metal-based particle aggregate layer composed of metal-based particles having a shape within a predetermined range (average particle size, average height and aspect ratio), and more preferably an average particle distance within a predetermined range, with good control. In addition to setting the average height growth rate to less than 1 nm / min in the particle growth step, it is preferable that the average particle size growth rate is less than 5 nm, but the average height growth rate is less than 1 nm / min. In this case, the average particle size growth rate is usually less than 5 nm. The average particle size growth rate is more preferably 1 nm / min or less. The average particle size growth rate is the following formula:
Average particle size of metal particles / Growth time of metal particles (supply time of metal materials)
Defined in. The definition of "average particle size of metallic particles" is as described above.

The growth time of the metal-based particles (supply time of the metal-based material) in the particle growth step is at least the shape of the metal-based particles supported on the substrate within a predetermined range, and more preferably the average inter-particle distance within a predetermined range. It is the time to reach and less than the time to start deviating from the shape and average interparticle distance within the predetermined range. For example, even if the particles are grown at the average height growth rate and the substrate temperature within the above predetermined range, if the growth time is extremely long, the amount of the metal-based material carried becomes too large and the particles are separated from each other. It does not become an aggregate of the arranged metal-based particles but becomes a continuous film, or the average particle size and the average height of the metal-based particles become too large.

Therefore, it is necessary to set the growth time of the metallic particles to an appropriate time (stop the particle growth process at an appropriate time), and such a time setting can be obtained, for example, by conducting a preliminary experiment in advance. This can be performed based on the relationship between the average height growth rate and the substrate temperature obtained, the shape of the metal-based particles in the obtained metal-based particle aggregate, and the average distance between the particles. Alternatively, the time until the thin film made of the metal-based material grown on the substrate exhibits conductivity (that is, the time when the thin film becomes a continuous film instead of the metal-based particle aggregate film) is determined in advance by a preliminary experiment. The particle growth process may be stopped by the time this time is reached.

The surface of the substrate on which the metal-based particles are grown is preferably as smooth as possible, and more preferably smooth at the atomic level. The smoother the surface of the substrate, the easier it is for the growing metal particles to coalesce and grow with other surrounding metal particles due to the thermal energy received from the substrate, resulting in a film of larger size metal particles. It tends to be easy to obtain.

The substrate on which the metal particles are grown can be used as it is as the substrate of the sensor chip. That is, a substrate (metal-based particle aggregate layer laminated substrate) on which the metal-based particle aggregate layer is laminated and supported, which is produced by the above method, can be used as a constituent member of the sensor chip.

<Board>
The substrate 10 may be made of any material, but particularly when the metal-based particle aggregate layer is directly laminated on the substrate 10, it is non-conductive from the viewpoint of ensuring the non-conductive property of the metal-based particle aggregate layer. It is preferable to use a conductive substrate. As the non-conductive substrate, glass, various inorganic insulating materials (SiO ₂ , ZrO ₂ , mica, etc.), and various plastic materials can be used.

<Protective layer>
As shown in FIG. 1, the sensor chip of the present invention has a protective layer 30 that covers the surface of each metal-based particle 200 constituting the metal-based particle aggregate layer and protects the metal-based particle 200. The protective layer 30 is preferably insulating. Due to the insulating property, the non-conductive property (non-conductive property between the metal-based particles) of the above-mentioned metal-based particle aggregate layer can be ensured, and the metal-based particle aggregate layer 20 and other layers adjacent thereto can be secured. Electrical insulation between them can be achieved. If a current flows through the metal-based particle aggregate layer 20, there is a possibility that the effect of enhancing light emission due to plasmon resonance cannot be sufficiently obtained. Further, the protective layer 30 covering the metal-based particles can prevent the metal-based particles 200 from coming into direct contact with a layer other than the protective layer 30 or the external environment, and can prevent the metal-based particles 200 from deteriorating.

The material constituting the protective layer 30 is preferably a material having good insulating properties, and for example, spin-on glass (SOG; a material containing, for example, an organic siloxane material), SiO ₂ or Si ₃ N ₄ or the like is used. Can be done. The thickness of the protective layer 30 is not particularly limited as long as it can prevent the metal particles 200 from coming into direct contact with a layer other than the protective layer 30 or the external environment. Since it is preferable that the distance between the labeling substance existing in the vicinity of the specific bond of the substance and the metal-based particle aggregate layer 200 is within a predetermined range, the thinner the range is, the better as long as the desired protection is ensured. The thickness of the protective layer 30 is preferably, for example, 10 to 150 nm, more preferably 20 to 150 nm, and even more preferably 20 to 90 nm. In the present specification, the thickness of the protective layer 30 is a value obtained by subtracting the average height of the metal-based particle aggregate layer 20 from the average thickness from the upper surface of the substrate 10 to the upper surface of the protective layer 30. It is preferably 10 nm or more from the viewpoint of ensuring sufficient protection, and from the viewpoint of ensuring that the effect of enhancing light emission by localized plasmon resonance can be sufficiently obtained for the labeling substance that will be present above the sensor chip. It is preferably 150 nm or less.

According to the present invention, the metal-based particle aggregate layer 20 is provided with a metal-based particle aggregate layer that exhibits strong plasmon resonance and has a significantly extended range of action of plasmon resonance (range of enhancement effect by plasmon). Even when the distance from the upper surface to the labeling substance that will be above the sensor chip is, for example, 15 nm or more, further 25 nm or more, or even more, it is possible to enhance the light emission of the labeling substance, for example. Is. The distance from the upper surface of the metal-based particle aggregate layer 20 to the labeling substance is preferably 170 nm or less, more preferably 150 nm or less.

Therefore, although the distance from the upper surface of the metal-based particle aggregate layer 20 to the labeling substance may be increased by the size of the substance to be detected, in the present invention, the substance to be detected having a major axis of 5 nm or more and further having a major axis of 10 nm or more. It is also possible to enhance the signal derived from the labeling substance in the detection of. The substance to be detected preferably has a major axis of 1 μm or less. When the major axis of the substance to be detected exceeds 100 nm, it is preferable to adjust so that the substance to be detected is captured so that the labeling substance is located within the action range of plasmon resonance. When the substance to be detected is a chain compound such as nucleic acid, the major axis here means the chain length.

According to the present invention, since the detection sensitivity can be enhanced, high sensitivity is obtained regardless of whether a luminescent substance having low luminous efficiency is used as a labeling substance or a small amount of the substance to be detected is used. It becomes possible to detect with.

Further, according to the sensor chip of the present invention, even when a conventional blue (or its neighboring wavelength region) light emitting substance having a relatively low luminous efficiency is used, a metal in which the maximum wavelength of the plasmon peak is shifted to the short wavelength side. By providing the system particle aggregate layer, the luminous efficiency can be enhanced.

The maximum wavelength of the plasmon peak of the metal-based particle aggregate layer preferably matches or is close to the emission wavelength of the luminescent substance used as the labeling substance. Thereby, the luminescence enhancing effect by the plasmon resonance can be enhanced more effectively. The maximum wavelength of the plasmon peak of the metal-based particle aggregate layer can be controlled by adjusting the metal type, average particle size, average height, aspect ratio and / or average interparticle distance of the metal-based particles constituting the plasmon peak.

<Capture layer>
As shown in FIG. 1, the trapping layer 40 has a trapping substance and is formed on the protective layer 30. The trapping substance may be fixedly present in the trapping layer 40, may be present in a free state in the trapping layer, or may be fixedly present on the surface of the protective layer 30. A treatment for inducing a binding active group capable of specifically binding to the substance to be detected may be performed on the surface of the trapping layer 40, and such binding active group may be used as the capturing substance.

[Structure example of sensor chip that detects nucleotides]
FIG. 2A is a schematic cross-sectional view showing an example of a sensor chip that detects nucleotides. FIG. 2B is a schematic cross section showing a state in which the substance to be detected 51 is captured by the capture layer 41 in the sensor chip 110 of this configuration example.

The substance to be detected 51 is a nucleotide to which a fluorescent substance is bound. The material of the capture layer 41 is not limited, and organic substances, inorganic substances, oxides thereof and the like can be used. For example, indium tin oxide (ITO), SiO ₂ , Si ₃ N ₄ , TiO ₂ , Ta ₂ It can be formed of O ₅ , Al ₂ O _{3, etc.} The surface of the capture layer 41 is subjected to a treatment for inducing a binding active group capable of specifically binding to a base contained in a nucleotide as a substance to be detected. Such a binding active group functions as a capture substance. Examples of such a binding active group include a carboxyl group and a hydroxyl group that electrostatically interact with a base.

In this configuration example, the thicknesses of the protective layer 30 and the capture layer 41 are determined so that the fluorescent substance located near the surface of the sensor chip falls within the action range of the localized plasmon resonance generated by the metal-based particle layer. Is preferable. The average thickness of the capture layer 41 can be, for example, 5 to 50 nm. The average thickness of the protective layer 30 can be, for example, 15 to 145 nm. The total of the average thickness of the protective layer 30 and the average thickness of the capture layer 41 is preferably 20 to 150 nm, more preferably 20 to 150 nm, and even more preferably 20 to 90 nm.

[Detection method for substances to be detected]
An example of a method for detecting a substance to be detected using the sensor chip of the present invention will be described. In the following description, a case of detection using a fluorescent substance bound to the substance to be detected will be described as an example based on FIGS. 2 (a) and 2 (b).

On the surface of the protective layer 30 of the sensor chip 110, a sample solution containing the substance to be detected 51 labeled with a fluorescent substance is dropped onto the surface of the capture layer 41. After that, the sensor chip 110 left for a predetermined time is analyzed using, for example, a sensing system described below to detect the substance to be detected.

[Sensing system]
FIG. 3 is a schematic cross-sectional view showing an example of the sensing system of the present invention. In the sensing system, the capture layer 41 side of the sensor chip 110 is irradiated with excitation light 3 from a direction perpendicular to the surface of the capture layer 41 to emit a luminescent substance which is a labeling substance existing in the vicinity of the surface of the sensor chip. Make it emit light. The light emitted from the excitation light source 4 is focused by the lens 5 to obtain excitation light 3, which is then irradiated. After that, the light emission 6 from the sensor chip 110 radiated in the direction of 40 ° with respect to the optical axis of the excitation light 3 is focused by the lens 7 and separated through a wavelength cut filter 8 that cuts the light having the wavelength of the excitation light. It is detected by the measuring instrument 9. As the detector, in addition to the spectroscopic measuring instrument 9, an epi-fluorescence microscope, a total internal reflection fluorescence microscope, a scanning near-field fluorescence microscope, or the like can be used.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Preparation of metal-based particle aggregate layer laminated substrate]
<Manufacturing example 1>
Using a DC magnetron sputtering device, silver particles are grown extremely slowly on a soda glass substrate under the following conditions, and a thin film of metal-based particle aggregates is formed on the entire surface of the substrate to form metal-based particle aggregates. A layered laminated substrate was obtained.

Gas used: Argon,
Chamber pressure (sputter gas pressure): 10 Pa,
Distance between board and target: 100 mm,
Sputtering power: 4W,
Average particle size growth rate (average particle size / sputtering time): 0.9 nm / min,
Average height growth rate (= average deposition rate = average height / spatter time): 0.25 nm / min,
Substrate temperature: 300 ° C,
Board size and shape: A square with a side of 5 cm.

FIG. 4 is an SEM image of the obtained metal-based particle aggregate layer laminated substrate when the metal-based particle aggregate layer is viewed from directly above. FIG. 4 (a) is a magnified image of 10000 times scale, and FIG. 4 (b) is a magnified image of 50,000 times scale. Further, FIG. 5 is an AFM image showing the metal-based particle aggregate layer in the obtained metal-based particle aggregate layer laminated substrate. "VN-8010" manufactured by KEYENCE CORPORATION was used for AFM image photographing (the same applies hereinafter). The size of the image shown in FIG. 5 is 5 μm × 5 μm.

From the SEM image shown in FIG. 4, the average particle size of the silver particles constituting the metal-based particle aggregate layer of this production example based on the above definition was determined to be 335 nm, and the average interparticle distance was determined to be 16.7 nm. Further, from the AFM image shown in FIG. 5, the average height was determined to be 96.2 nm. From these, the aspect ratio (average particle size / average height) of the silver particles is calculated to be 3.48, and it can be seen from the acquired image that the silver particles have a flat shape. Further, from the SEM image, it can be seen that the metal-based particle aggregate layer of this production example has about 6.25 × 10 ¹⁰ (about 25 / μm ^{2) silver particles.}

Further, when a tester [multimeter (“E2378A” manufactured by Hewlett-Packard Co., Ltd.]] was connected to the surface of the metal-based particle aggregate layer in the obtained metal-based particle aggregate layer laminated substrate and the conductivity was confirmed, the conductivity was confirmed. It was confirmed that it did not have.

<Manufacturing example 2>
An aqueous dispersion of silver nanoparticles (manufactured by Mitsubishi Paper Co., Ltd., concentration of silver nanoparticles: 25% by weight) was diluted with pure water so that the concentration of silver nanoparticles was 2% by weight. Next, 1% by volume of a surfactant was added to the aqueous dispersion of silver nanoparticles and stirred well, and then 80% by volume of acetone was added to the obtained aqueous dispersion of silver nanoparticles to keep the temperature at room temperature. To prepare a silver nanoparticle coating solution.

Next, the silver nanoparticle coating solution was spin-coated on a 1 mm thick soda glass substrate whose surface was wiped with acetone at 1000 rpm, left as it was in the air for 1 minute, and then in an electric furnace at 550 ° C. for 40 seconds. It was fired. Then, the silver nanoparticle coating solution was spin-coated on the formed silver nanoparticle layer again at 1000 rpm, left as it was in the air for 1 minute, and then fired in an electric furnace at 550 ° C. for 40 seconds. , A metal-based particle aggregate layer laminated substrate was obtained.

FIG. 6 is an SEM image of the obtained metal-based particle aggregate layer laminated substrate when the metal-based particle aggregate layer is viewed from directly above. FIG. 6A is a 10000x scale magnified image, and FIG. 6B is a 50000x scale magnified image. Further, FIG. 7 is an AFM image showing the metal-based particle aggregate layer in the obtained metal-based particle aggregate layer laminated substrate. The size of the image shown in FIG. 7 is 5 μm × 5 μm.

From the SEM image shown in FIG. 6, the average particle size of the silver particles constituting the metal-based particle aggregate layer of this production example based on the above definition was determined to be 293 nm, and the average particle distance was 107.8 nm. Further, from the AFM image shown in FIG. 7, the average height was determined to be 93.0 nm. From these, the aspect ratio (average particle size / average height) of the silver particles is calculated to be 3.15, and it can be seen from the acquired image that the silver particles have a flat shape. Further, from the SEM image, it can be seen that the metal-based particle aggregate layer of this production example has about 3.13 × 10 ¹⁰ silver particles (about 12.5 ^{particles / μm 2).}

Further, when a tester [multimeter (“E2378A” manufactured by Hewlett-Packard Co., Ltd.]] was connected to the surface of the metal-based particle aggregate layer in the obtained metal-based particle aggregate layer laminated substrate and the conductivity was confirmed, the conductivity was confirmed. It was confirmed that it did not have.

[Example 1: A sensor chip using a nucleotide labeled with a fluorescent substance as a substance to be detected]
(Manufacturing of sensor chip)
The sensor chip shown in FIG. 2A was produced according to the following method. By growing silver particles under the same conditions as in Production Example 1, the metal-based particle aggregate layer 20 described in Production Example 1 was formed on a substrate 10 which is a soda glass substrate having a thickness of 0.5 mm. Immediately thereafter, a spin-on-glass (SOG) solution was spin-coated on the metal-based particle aggregate layer, and a protective layer 30 having a predetermined thickness was laminated. As the SOG solution, an organic SOG material "OCD T-7 5500T" manufactured by Tokyo Ohka Kogyo Co., Ltd. diluted with ethanol was used. Next, an ITO layer (thickness 10 nm) was laminated on the protective layer 30 as a trapping layer 41 by a sputtering method. In this way, five sensor chips (Examples 1a, 1b, 1c, 1d, 1e) having different thicknesses of the protective layer 30 were produced. The thicknesses of the protective layer 30 of Examples 1a, 1b, 1c, 1d, and 1e were 10 nm, 30 nm, 50 nm, 80 nm, and 150 nm, respectively.

[Comparative Example 1: Sensor chip using nucleotides labeled with a fluorescent substance as the substance to be detected]
A sensor chip was produced in the same manner as in Example 1 except that the metal particle aggregate layer was not formed and the thickness of the protective layer 30 was set to 150 nm.

[Evaluation of luminescence enhancing effect of Example 1 and Comparative Example 1]
A solution containing cytidine triphosphate (CTP) to which the fluorescent substance Cy3 was bound was spin-coated on the surfaces of the sensor chips of Example 1b and Comparative Example 1 at a rotation speed of 2000 rpm, and then dried. The solution containing cytidine triphosphate (CTP) to which the fluorescent substance Cy3 was bound was prepared as follows. 200 μL of ethanol was added to a fluorescently labeled nucleonucleotide manufactured by PerkinElmer (product name: CTP analogs Cyanine 3-CTP, product code: NEL580001EA) and stirred. This solution was measured in 100 μL and diluted with 800 μL of ethanol to obtain a solution containing cytidine triphosphate (CTP) to which the fluorescent substance Cy3 was bound. By this step, Cy3-CTP was captured on the surface of the capture layer 40. Then, in the sensing system shown in FIG. 3, the emission spectrum was measured using a fluorescence spectrophotometer (trade name: FP-6500, manufactured by Nippon Spectroscopy Co., Ltd.) as the spectrophotometer 9. The wavelength of the excitation light source was 532 nm.

FIG. 8 shows emission spectra measured using the sensor chips of Example 1b and Comparative Example 1. The emission intensity represented by the integrated value of the emission spectrum observed by the sensor chip of Example 1b (wavelength 545 nm to wavelength 755 nm) is the integrated value of the emission spectrum observed by the sensor chip of Comparative Example 1 (wavelength from wavelength 545 nm to wavelength). It was confirmed that the emission intensity was 10.1 times that represented by 755 nm).

The emission spectra of Examples 1a, 1c, 1d, and 1e were measured in the same manner. With respect to the obtained emission spectrum, a value obtained by converting the emission intensity represented by the integrated value of the emission spectrum (wavelength 545 nm to wavelength 755 nm) into a value when the emission intensity of Comparative Example 1 was set to 1 was calculated. FIG. 9 is a diagram in which the above-mentioned conversion value of the emission intensity is plotted against a value obtained by adding the average thickness of the protective layer and the average thickness of the capture layer. From FIG. 9, when the average thickness of the capture layer 41 is 10 nm, the average thickness of the protective layer is 10 to 150 nm (20 to 160 nmn when the average thickness of the protective layer is added to the average thickness of the capture layer). It was found that the luminescence enhancing effect was obtained in the range, and the highest luminescence enhancing effect was obtained when the average thickness of the protective layer was 30 nm. From these results, assuming that the fluorescent substance, which is a labeling substance, is present almost on the surface of the capture layer, it emits light when the distance from the surface of the metal-based particle aggregate layer to the labeling substance is 20 to 160 nm. It can be seen that the enhancing effect can be obtained. In this evaluation, cytidine triphosphate (CTP) to which the fluorescent substance Cy3 is bound is used as the substance to be detected, and the fluorescent substance Cy3 is almost on the surface of the capture layer even considering that the molecular weight of CTP is 483. Assuming that it exists in, it is considered that there is no problem.

1,110 Sensor chip, 3 excitation light, 4 excitation light source, 5,7 lens, 6 emission from labeled material, 8 wavelength cut filter, 9 spectrophotometer, 10 substrate, 20 metal particle aggregate layer, 30 protective layer , 40, 41 capture layer, 51 substances to be detected, 200 metal particles.

Claims (10)

A sensor chip used to detect a substance to be detected.
With the board
A metal-based particle aggregate layer formed on the substrate and
A protective layer that covers the metallic particle aggregate layer and
A capture layer formed on the protective layer and having a capture substance that specifically binds to the substance to be detected, and a capture layer.
With
The metal-based particle aggregate layer is composed of a particle aggregate in which 30 or more metal-based particles are two-dimensionally arranged apart from each other, and the metal-based particles have an average particle size of 200 to 1600 nm. A sensor chip having an aspect ratio in the range of 55 to 500 nm and an aspect ratio defined by the ratio of the average particle size to the average height in the range of 1 to 8. The sensor chip according to claim 1, wherein the total value of the average thickness of the protective layer and the average thickness of the capture layer is 20 nm to 90 nm. The sensor chip according to claim 1 or 2 , wherein the detected substance is detected by detecting a signal corresponding to a state of specific binding between the captured substance and the detected substance. The sensor chip according to claim 3 , wherein the signal is a signal derived from a labeling substance existing in the vicinity of a specific bond between the trapping substance and the substance to be detected. The labeling substance is a luminescent substance and is
The sensor chip according to claim 4 , wherein the signal is a signal derived from absorption or light emission of light by the labeling substance. The sensor chip according to claim 4 or 5 , wherein the labeling substance is previously bound to the trapping substance or the substance to be detected. The sensor chip according to any one of claims 1 to 6 , wherein the protective layer has an average thickness of 10 nm or more. The sensor chip according to any one of claims 1 to 7 , wherein the substance to be detected is a molecule having a major axis of 5 nm or more. The substance to be detected has a base and has a base.
The sensor chip according to any one of claims 1 to 8 , wherein the capture substance has a binding active group capable of binding a base. The sensor chip according to claim 5 and
An excitation light source that irradiates the capture layer with excitation light that excites the emission of the labeling substance.
A detector that detects light emission from the labeling substance and
Sensing system with.

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