WO2017130839A1 - Puce de capteur et système de détection - Google Patents

Puce de capteur et système de détection Download PDF

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WO2017130839A1
WO2017130839A1 PCT/JP2017/001794 JP2017001794W WO2017130839A1 WO 2017130839 A1 WO2017130839 A1 WO 2017130839A1 JP 2017001794 W JP2017001794 W JP 2017001794W WO 2017130839 A1 WO2017130839 A1 WO 2017130839A1
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metal
substance
particles
sensor chip
average
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PCT/JP2017/001794
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Japanese (ja)
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知浩 福浦
佳代 住田
斉藤　幸一
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住友化学株式会社
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Priority to JP2017564205A priority Critical patent/JP6900325B2/ja
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y20/00—Nanooptics, e.g. quantum optics or photonic crystals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the present invention relates to a sensor chip in which a signal emitted from a labeling substance is enhanced using plasmon resonance of a metal-based particle aggregate and a sensing system including the sensor chip.
  • Luminescence analysis methods for qualitatively or quantitatively detecting a target substance labeled with a luminescent substance such as a fluorescent substance by analyzing luminescence from the luminescent substance are various methods that can realize high-speed analysis. Plays an important role in the field. For example, for a nucleic acid labeled with a luminescent substance, determination of the base sequence, analysis of gene mutation, measurement of gene expression level, etc. can be performed at high speed by using the luminescence analysis method.
  • Plasmon is a free-electron rough wave generated by collective oscillation of free electrons in a metal nanostructure.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2007-139540
  • Patent Document 2 Japanese Patent Application Laid-Open No. 08-271431
  • Patent Document 3 International Publication No. 2005/033335
  • Patent Document 3 utilize the localized plasmon resonance phenomenon.
  • a technique for enhancing fluorescence is disclosed.
  • 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 by silver nanoparticles are shown.
  • JP 2007-139540 A Japanese Patent Application Laid-Open No. 08-271431 International Publication No. 2005/033335
  • the factors of the light emission enhancing action by the metal nanoparticles are 1) the electric field in the vicinity of the particles is enhanced by the occurrence of localized plasmons in the metal nanoparticles (first factor), and 2) the excitation
  • the energy transfer from the excited molecules (such as luminescent material molecules) excites the free electron vibrational mode in the metal nanoparticles, resulting in a luminescent induced dipole that is larger than the luminescent dipole of the excited molecule.
  • the luminescence-induced dipole in the second factor which is a larger factor, is converted into the metal nanoparticle.
  • the distance between the metal nanoparticles and the excited molecules is within a range where energy transfer by the Dexter mechanism, which is direct electron transfer, does not occur. , It is necessary to within the range energy transfer Förster mechanism is expressed (1nm ⁇ 10nm). This is because the occurrence of the luminescence-induced dipole is based on Förster energy transfer theory (see Non-Patent Document 1 above).
  • the distance between the metal nanoparticle and the molecule to be excited is set to 10 nm or less.
  • the detection target substance specifically bound to the capture substance arranged on the sensor chip is detected based on the signal derived from the labeling substance existing in the vicinity of the specific binding.
  • the distance from the surface of the metal nanoparticle to the labeling substance in such a configuration is examined, it usually exceeds 10 nm, which is outside the above-described range of action of localized plasmon resonance. This is because it is necessary to form a scaffold for fixing the capture substance, and it is necessary to consider the length of the capture substance and the substance to be detected itself.
  • the protection of the surface of the metal nanoparticle is insufficient, and the action of localized plasmon resonance is reduced due to the deterioration of the metal nanoparticle.
  • the present invention can enhance a signal derived from a labeling substance by localized plasmon resonance, and prevent a deterioration of metal nanoparticles that cause localized plasmon resonance, and a sensing system including the sensor chip
  • the purpose is to provide.
  • the present invention includes the following.
  • a sensor chip used for detecting a substance to be detected A substrate, A metal-based particle assembly layer formed on the substrate; A protective layer covering the metal-based particle assembly layer; A capture layer formed on the protective layer and having a capture substance that specifically binds to the substance to be detected;
  • the metal-based particle assembly layer is a particle assembly 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 diameter of 200 to 1600 nm.
  • a sensor chip having a range, an average height in a range of 55 to 500 nm, and an aspect ratio defined by a ratio of the average particle diameter to the average height in a range of 1 to 8.
  • the labeling substance is a luminescent substance
  • the sensor chip according to [3] wherein the signal is a signal derived from light absorption or light emission by the labeling substance.
  • the molecule to be detected 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.
  • 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 sensing provided with the sensor chip A system can be provided.
  • FIG. 2A is a schematic cross-sectional view showing an example of a sensor chip for detecting nucleotides
  • FIG. 2B is a schematic cross-sectional view showing a state in which a substance to be detected is captured by the sensor chip.
  • 2 is an AFM image of a metal-based particle assembly layer in a metal-based particle assembly layer laminated substrate obtained in Production Example 1.
  • FIG. 5 is a diagram in which converted values of emission intensity of Examples 1a to 1e are plotted against values obtained by adding the average thicknesses of the capturing layers of the protective layer.
  • FIG. 1 is a diagram schematically showing an example of a sensor chip of the present invention.
  • the sensor chip 1 of the present invention is used to detect a substance to be detected, and is provided on a substrate 10 and 30 or more metal-based particles 200 arranged apart from each other.
  • a metal-based particle assembly layer 20 composed of a particle assembly, a protective layer 30 covering the metal-based particle assembly layer 20, and a capture layer provided on the protective layer 30 and having a capture substance that specifically binds to a molecule to be detected. 40.
  • the metal-based particle assembly layer 20 is composed of a particle assembly 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 diameter 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 diameter to the average height is in the range of 1 to 8.
  • the trapping substance of the trapping layer 40 and the target substance are specifically bound, and the target substance is trapped on the 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 capture substance and the substance to be detected.
  • a signal corresponding to the state of specific binding between the capture substance and the substance to be detected can be enhanced by localized plasmon resonance generated by the metal-based particle assembly layer 20, and high sensitivity. Detection is possible.
  • the signal corresponding to the specific binding state is, for example, a signal derived from a labeling substance that exists in the vicinity of the specific binding between the capture substance and the substance to be detected.
  • the range of action of localized plasmon resonance can be lengthened, so that the distance from the metal-based particle assembly layer 20 to the labeling substance is long. Also, the signal enhancement effect can be obtained.
  • the range of action of localized plasmon resonance is not limited to a narrow range, the degree of freedom in the configuration of a sensor chip that can obtain a signal enhancement effect by localized plasmon resonance is increased, and the metal-based assembly layer 20 is protected by the protective layer 30.
  • the structure can be sufficiently protected. With such a configuration, deterioration of the metal-based particle assembly layer 20 can be prevented, and reduction of the effect of localized plasmon resonance can be suppressed.
  • Capture substance is a substance that functions to capture a substance that specifically binds to this substance (substance to be detected), and is present in a fixed state in the capture layer or in a free state in the capture layer. Or fixed on the surface of the protective layer. Any organic or inorganic substance can be used as the capture substance as long as it has the above-mentioned functions. Examples include biological substances such as nucleosides, nucleotides, nucleic acids, proteins, and sugars, viruses, and cells. . In addition, a substance having a binding active group that can bind to the functional group of the substance to be detected by electrostatic interaction can be used.
  • “Substance to be detected” is a substance to be subjected to qualitative or quantitative detection, and is a substance that specifically binds to a capture substance. Any organic substance and inorganic substance can be detected substances, and for example, nucleosides, nucleotides, nucleic acids, proteins, saccharides and other biological substances, viruses, cells and the like can be detected substances.
  • nucleic acid means a polymer (nucleotide chain) of a phosphate ester of a nucleoside in which a purine base or pyrimidine base and a sugar are glycosidically linked, and an oligonucleotide, polynucleotide, purine nucleotide and pyrimidine nucleotide containing a probe DNA are polymerized.
  • DNA full length or a fragment thereof
  • RNA polyamide nucleotide derivative (PNA) and the like.
  • a nucleoside is a compound in which a base and a sugar are glycoside-bonded
  • a nucleotide is a compound in which a phosphate is bonded to a nucleoside, and both the nucleoside and the nucleotide are compounds containing a base.
  • Specific binding broadly means chemical bonds including non-covalent bonds, covalent bonds, and hydrogen bonds between substances, and examples include interactions between protein molecules, electrostatic interactions between molecules, and the like.
  • Examples of the substance to be detected and the substance to be specifically bound to the capturing substance include capture of a sugar chain by a lectin and capture of a molecule by an inclusion compound.
  • “Sensor chip” means a sensor chip used to detect a detected substance by causing specific binding between a captured substance and a detected substance in a reaction region in the vicinity of the capturing layer.
  • the type of is not limited.
  • a sensor chip in which a substance to be detected is a biological substance, virus, cell, or the like is also referred to as a biochip.
  • the detection of the detection substance in the sensor chip is performed by detecting a signal corresponding to the specific binding state between the capture substance and the detection substance, for example, in the vicinity of the specific binding between the capture substance and the detection substance. This is done by detecting a signal derived from the labeling substance.
  • the signal is not limited as long as the signal can be enhanced by localized plasmon resonance generated by the metal-based particle assembly layer.
  • a signal when the labeling substance is a luminescent substance, a signal derived from absorption or emission of light by the luminescent substance is exemplified.
  • a luminescent substance is a substance that emits light by injection of excitation energy such as excitation light.
  • the principle of light emission in the luminescent material is not limited, and examples thereof include fluorescence, phosphorescence, and chemiluminescence.
  • the labeling substance may be preliminarily bound to the capture substance or the detection substance, or may be a labeling substance that specifically binds to a product obtained by specific binding of the capture substance and the test substance. Good.
  • a luminescent substance is used as a labeling substance will be described in detail.
  • the metal-based particle assembly layer has any of the following characteristics.
  • the metal-based particles constituting the metal-based particle assembly layer are arranged so that the average distance from the adjacent metal-based particles is within a range of 1 to 150 nm (first embodiment).
  • the metal-based particle assembly layer has a same particle diameter, the same height as the average height, and the same material in the absorption spectrum in the visible light region. Compared to the reference metal particle aggregate (X) arranged so that the distances are all in the range of 1 to 2 ⁇ m, the maximum wavelength of the peak on the longest wavelength side is shifted to the short wavelength side in the range of 30 to 500 nm.
  • the metal-based particle assembly layer has a same particle diameter as the average particle diameter, the same height as the average height, and the same material in the absorption spectrum in the visible light region. Compared with the same number of metal particles compared to the reference metal particle aggregate (Y) arranged so that the distances are all within the range of 1 to 2 ⁇ m, the absorbance at the maximum wavelength of the peak on the longest wavelength side is larger. High (third embodiment).
  • the metal-based particle assembly layer according to the present embodiment exhibits extremely strong plasmon resonance, a stronger light emission enhancement effect can be obtained as compared with the case where a conventional plasmon material is used. Can be dramatically improved.
  • the intensity of the plasmon resonance exhibited by the metal-based particle assembly layer is not a mere sum of the localized plasmon resonances exhibited by individual metal-based particles at a specific wavelength, but is more than that. That is, when 30 or more metal particles having a predetermined shape are densely arranged at the predetermined interval, individual metal particles interact with each other, and extremely strong plasmon resonance is expressed. This is considered to be expressed by the interaction between the localized plasmons of the metal-based particles.
  • a plasmon resonance peak (hereinafter also referred to as a plasmon peak) is observed as a peak in the ultraviolet to visible region.
  • the strength of the plasmon resonance of the plasmon material can be roughly evaluated, but the metal-based particle assembly layer according to the present embodiment is measured when the absorption spectrum is measured in a state of being 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 can be 1 or more, further 1.5 or more, and even about 2.
  • the absorption spectrum of the metal-based particle assembly layer is measured in a state of being laminated on a glass substrate by absorptiometry. Specifically, the absorption spectrum is from the back side of the glass substrate on which the metal-based particle assembly layer is laminated (the side opposite to the metal-based particle assembly 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 particle assembly film laminated substrate, and the intensity I of transmitted light in all directions irradiated with incident light in the light region and transmitted to the metal particle assembly layer side; Irradiating the same incident light from the direction perpendicular to the surface of the substrate on which the metal-based particle assembly film is not laminated, and integrating the transmitted light intensity I 0 in all directions transmitted from the opposite side of the incident surface. It is obtained by measuring using a sphere spectrophotometer. At this time, the absorbance, which is the vertical axis of the absorption spectrum, has the following formula: Absorbance ⁇ log 10 (I / I 0 ) It is represented by
  • Such an elongation action is also considered to be manifested by the interaction between localized plasmons of metal-based particles produced by densely arranging 30 or more predetermined-shaped metal particles at a predetermined interval.
  • the metal-based particle assembly layer of the present embodiment it is possible to extend the plasmon resonance operating range, which has been generally limited to within the range of the Forster distance (about 10 nm or less) to about several hundred nm, for example. it can.
  • the metal-based particle assembly layer according to the present embodiment does not use dipole-type localized plasmons alone, although relatively large metal-based particles are used in the visible light region.
  • a large number of large metal-based particles (although it is necessary to have a predetermined shape) are densely arranged at specific intervals so that the large-sized metal particles are included.
  • An extremely large number of surface free electrons can be effectively excited as plasmons, and it is possible to realize extremely strong plasmon resonance and a significant extension of the range of action of plasmon resonance.
  • the sensor chip of the present embodiment has a structure in which a specific number or more of relatively large metal particles having a specific shape of the metal particle aggregate layer are two-dimensionally spaced apart at a specific interval. Due to the possession, the following advantageous effects can be obtained.
  • the maximum wavelength of the plasmon peak is unique depending on the average particle diameter of the metal-based particles and the average distance between the particles. Since it can show a shift, it is possible to particularly enhance the light emission in a specific (desired) wavelength region. Specifically, 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) as the average particle size of the metal-based particles is increased with a constant average distance between the particles.
  • the plasmon peak on the longest wavelength side in the visible light region is reduced.
  • This unique 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 longer wavelength side (red shift) as the particle size increases). It is contrary.
  • the peculiar blue shift as described above also has a structure in which the metal-based particle aggregate layer has large metal particles densely arranged at specific intervals. This is thought to be due to the interaction between plasmons.
  • the metal particle assembly layer according to the present embodiment (the state laminated on the glass substrate)
  • the plasmon peak on the longest wavelength side can exhibit a maximum wavelength in a wavelength region of 350 to 550 nm, for example.
  • the metal-based particle assembly layer according to the present embodiment typically has a thickness of about 30 to 500 nm as compared with the case where the metal-based particles are arranged with a sufficiently long inter-particle distance (for example, 1 ⁇ m) ( For example, a blue shift of 30 to 250 nm may occur.
  • Such a sensor chip including a metal-based particle assembly 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 light-emitting substance having a relatively low light emission efficiency is used as a labeling substance, the light emission efficiency can be enhanced to a sufficient level.
  • the metal-based particles constituting the metal-based particle assembly layer are not 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 absorptiometry when it is a nanoparticle or an aggregate thereof.
  • noble metals such as gold, silver, copper, platinum and palladium, metals such as aluminum and tantalum; the noble metals or alloys containing metals; metal compounds including the noble metals or metals (metal oxides, metal salts, etc. ).
  • noble metals such as gold, silver, copper, platinum, and palladium are preferable, and silver is more preferable because it is inexpensive and has low absorption (small imaginary part of dielectric function at visible light wavelength).
  • the average particle diameter of the metal-based particles is in the range of 200 to 1600 nm.
  • the metal-based particle assembly layer according to the present embodiment has a remarkably strong plasmon resonance and plasmon resonance by arranging a predetermined number (30) or more of such large metal-based particles densely at a predetermined interval. The remarkable extension of the action range of resonance and the effect (3) are realized.
  • the average particle size of the metal-based particles means that 10 particles are randomly selected in the SEM observation image from directly above the metal-based particle aggregate (film) in which the metal-based particles are two-dimensionally arranged. Randomly draw 5 tangential diameters in the image (however, any straight line with a tangential diameter can only pass through the interior of the particle image, one of which is the straightest line that can be drawn the longest only through the interior of the particle) And the average value of the 10 selected particle sizes when the average value is the particle size of each particle.
  • the tangent diameter is defined as a perpendicular line connecting the interval (projection image) of a particle between two parallel lines in contact with it (Nikkan Kogyo Shimbun, “Particle Measurement Technology”, 1994, page 5). .
  • the measurement method of the average particle diameter will be described more specifically.
  • the SEM observation image is measured using a scanning electron microscope “JSM-5500” manufactured by JEOL Ltd.
  • the obtained observation image is read at 1280 pixels by 960 pixels by using free image processing software “ImageJ” manufactured by the National Institutes of Health.
  • the average height of the metal-based particles is in the range of 55 to 500 nm, and in order to effectively obtain the effects (1) to (3), preferably in the range of 55 to 300 nm, more preferably 70 to 150 nm. It is.
  • the average height of the metal-based particles is 10 particles when 10 particles are selected at random in the AFM observation image of the metal-based particle assembly layer (film) and the heights of these 10 particles are measured. Is the average value of the measured values.
  • the aspect ratio of the metal-based particles is in the range of 1 to 8, and preferably 2 to 8, more preferably 2.5 to 8 in order to effectively obtain the effects (1) to (3). Is within.
  • the aspect ratio of the metal-based particles is defined by the ratio of the average particle diameter to the average height (average particle diameter / average height).
  • the metallic particles may be spherical, but preferably have a flat shape with an aspect ratio exceeding 1.
  • the metal particles preferably have a smooth curved surface, and more preferably have a flat shape with a smooth curved surface. Some minute irregularities (roughness) may be included, and in this sense, the metal-based particles may be indefinite.
  • the size variation between the metal-based particles is as small as possible.
  • the distance between the large particles is increased, and it is preferable that the interaction between the large particles is facilitated by filling the space between the small particles.
  • the metal-based particles are arranged so that the average distance between the adjacent metal-based particles (hereinafter also referred to as the average interparticle distance) is in the range of 1 to 150 nm. Is done.
  • the average distance is preferably in the range of 1 to 100 nm, more preferably 1 to 50 nm, and still more preferably 1 to 20 nm in order to effectively obtain the effects (1) to (3).
  • the average interparticle distance is less than 1 nm, electron transfer based on the Dexter mechanism occurs between particles, which is disadvantageous in terms of deactivation of localized plasmons.
  • the metal-based particle assembly layer in which the metal-based particles are arranged apart from each other does not exhibit conductivity as the layer.
  • the metal-based particle assembly layer 20 includes a multimeter [tester ( When a pair of tester probes (“E2378A” manufactured by Hewlett-Packard Co., Ltd.) are brought into contact with each other at a distance of 10 to 15 mm and the range setting is “30 M ⁇ ”, the resistance value is 30 M ⁇ or more under the measurement conditions. “Overload” is displayed. When electrons can be exchanged between some or all of the metal-based particles, the plasmon peak loses sharpness, approaches the absorption spectrum of the bulk metal, and high plasmon resonance cannot be obtained. Therefore, it is preferable that the metal-based particles are reliably separated from each other, and no conductive substance is interposed between the metal-based particles.
  • the average interparticle distance is 30 particles randomly selected, and for each selected particle, It is the average value of the interparticle distances of these 30 particles when the interparticle distance between adjacent particles is obtained.
  • the inter-particle distance between adjacent particles is a value obtained by measuring the distances between all adjacent particles (the distance between the surfaces) and averaging them.
  • the SEM observation image is measured using a scanning electron microscope “JSM-5500” manufactured by JEOL Ltd.
  • the obtained observation image is read at 1280 pixels by 960 pixels by using free image processing software “ImageJ” manufactured by the National Institutes of Health.
  • a random number generation function “RANDBETWEEN” of spreadsheet software “excel” manufactured by Microsoft Corporation is used, and 1 to 1280 to 30 random numbers (x 1 to x 30 ), 1 to 960 to 30 random numbers (y 1 to y 30 ) are obtained respectively.
  • (X 30 , y 30 ) is obtained from 30 random number combinations (x 1 , y 1 ) from the obtained 30 random numbers.
  • 30 sets of coordinate points (x 1 , y 1 ) to (x 30 , y 30 ) are set with the x-coordinate of the random number generated from 1 to 1280 and the y-coordinate of the random number generated from 1 to 960. obtain. Then, for each of a total of 30 particle images including the coordinate point, an interparticle distance between the particle and an adjacent particle is obtained, and then an average particle is obtained as an average value of the interparticle distance between the 30 adjacent particles. Get the distance between. If at least one of the 30 coordinate points that are 30 random number combinations is not included in the particle image, or if two or more coordinate points are included in the same particle, the 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 particles contained in the metal particle aggregate layer is 30 or more, preferably 50 or more.
  • the number of metal-based particles contained in the metal-based particle aggregate can be, for example, 300 or more, and further 17500 or more.
  • the number density of the metal-based particles in the metal-based particle assembly layer is preferably 7 / [mu] m 2 or more, more preferably 15 / [mu] m 2 or more.
  • the metal-based particles are insulated from each other, in other words, non-conductive (non-conductive as a metal-based particle assembly layer) between adjacent metal-based particles. Is preferred. When electrons can be exchanged between some or all of the metal-based particles, the plasmon peak loses sharpness, approaches the absorption spectrum of the bulk metal, and high plasmon resonance cannot be obtained. Therefore, it is preferable that the metal-based particles are reliably separated from each other and no conductive substance is interposed between the metal-based particles.
  • the sensor chip of the present embodiment has a peak maximum wavelength on the shortest wavelength side in the range of 30 to 500 nm compared to the reference metal-based particle aggregate (X) in the absorption spectrum in the visible light region. And a metal-based particle assembly layer (having the above-mentioned feature [ii]).
  • the sensor chip according to the present embodiment including the metal-based particle assembly layer having such characteristics is extremely advantageous in the following points.
  • the maximal wavelength of the plasmon peak on the longest wavelength side exists in a specific wavelength region.
  • the light emission in the wavelength region can be particularly enhanced.
  • the maximum wavelength of the plasmon peak is larger than the maximum wavelength of the reference metal-based particle assembly (X) described later. , Shifted to the short wavelength side (blue shift) in the range of 30 to 500 nm (for example, in the range of 30 to 250 nm).
  • 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 specific number or more of large metal particles having a specific shape are arranged two-dimensionally apart from the metal particle aggregate layer. This is thought to be due to the interaction between the localized plasmons of.
  • the metal-based particle assembly layer according to the present embodiment having a plasmon peak in blue or in the vicinity wavelength region thereof is extremely useful for enhancing the emission of a sensor chip that detects a target substance having a light emitting substance in blue or in the vicinity wavelength region.
  • the light emission efficiency can be increased to a sufficient level.
  • the absorption spectrum is measured by narrowing the measurement field of view.
  • the reference metal-based particle aggregate (X) is an average particle diameter of the metal-based particle aggregate layer to be subjected to absorption spectrum measurement, a metal particle A having the same particle size, height and the same material as the average height, An aggregate of metal particles arranged so that the distance between the metal particles is all within the range of 1 to 2 ⁇ m, and is capable of measuring an absorption spectrum using the above microscope in a state of being laminated on a glass substrate. It has the magnitude
  • the absorption spectrum waveform of the reference metal-based particle aggregate (X) includes 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 the substrate (for example, a glass substrate).
  • the sensor chip of the present embodiment has a structure in which a specific number or more of relatively large metal particles having a specific shape of the metal particle aggregate layer are two-dimensionally spaced apart.
  • the metal-based particle assembly layer can exhibit extremely strong plasmon resonance, so that a stronger emission enhancement effect can be obtained compared to the case of using a conventional plasmon material.
  • the luminous efficiency can be dramatically increased (similar to the effect (1) of the first embodiment), and (III) the range of plasmon resonance by the metal-based particle assembly layer (the enhancement effect of the plasmon) Range) can be remarkably extended, so that a stronger light emission enhancement effect can be obtained compared to the case of using a conventional plasmon material, and the light emission efficiency can be dramatically increased as well. It becomes (similar to the effect of the first embodiment (2)), may provide an advantage, such as.
  • the metal-based particle assembly layer according to the present embodiment has an absorbance at the maximum wavelength of the plasmon peak at the longest wavelength side in the visible light region when the absorption spectrum is measured in a state where it is laminated on the glass substrate. Further, it can be 1.5 or more, and even more preferably about 2.
  • the specific configuration of the metal-based particle assembly layer according to this embodiment is the specific configuration of the metal-based particle assembly layer according to the first embodiment (material of metal-based particles, average particle diameter, average height, aspect ratio). Ratio, average interparticle distance, number of metal-based particles, non-conductivity of the metal-based particle assembly layer, etc.). Definitions of terms such as average particle diameter, average height, aspect ratio, and average interparticle distance are the same as those in the first embodiment.
  • the average particle diameter of the metal-based particles is in the range of 200 to 1600 nm.
  • the average particle diameter of the metal-based particles is in the range of 200 to 1600 nm.
  • extremely strong plasmon resonance and plasmon resonance are obtained by forming an aggregate in which a predetermined number (30) or more of such large metal-based particles are two-dimensionally arranged.
  • the metal-based particles must have a large average particle size of 200 nm or more, preferably 250 nm or more. It is.
  • the maximum wavelength of the plasmon peak on the longest wavelength side in the visible light region depends on the average particle diameter of the metal-based particles. That is, when the average particle diameter of the metal-based particles exceeds a certain value, the maximum wavelength of the plasmon peak shifts (blue shift) to the short wavelength side.
  • the average height of the metal-based particles is in the range of 55 to 500 nm. In order to effectively obtain the effects (I) to (III), the average height is preferably in the range of 55 to 300 nm, more preferably in the range of 70 to 150 nm. It is.
  • the aspect ratio of the metal-based particles is in the range of 1 to 8, and preferably 2 to 8, more preferably 2.5 to 8 in order to effectively obtain the effects (I) to (III). Is within.
  • the metallic particles may be spherical, but preferably have a flat shape with an aspect ratio exceeding 1.
  • the metal particles preferably have a smooth curved surface, and more preferably have a flat shape with a smooth curved surface. Some minute irregularities (roughness) may be included, and in this sense, the metal-based particles may be indefinite.
  • 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 increases, and the interaction between the large particles is facilitated by filling the space between the small particles. It is preferable.
  • the metal-based particles are preferably arranged so that the average distance between the adjacent metal-based particles (average interparticle distance) is in the range of 1 to 150 nm. . More preferably, it is in the range of 1 to 100 nm, still more preferably 1 to 50 nm, and particularly preferably 1 to 20 nm.
  • the degree of blue shift of the plasmon peak on the longest wavelength side and the maximum wavelength of the plasmon peak are controlled by adjusting the average interparticle distance. Is possible.
  • the average interparticle distance is less than 1 nm, electron transfer based on the Dexter mechanism occurs between particles, which is disadvantageous in terms of deactivation of localized plasmons.
  • the number of metal particles contained in the metal particle aggregate layer is 30 or more, preferably 50 or more.
  • the number of metal-based particles contained in the metal-based particle aggregate can be, for example, 300 or more, and further 17500 or more.
  • the number density of the metal particles in the metal particle assembly layer is preferably 7 particles / ⁇ m 2 or more, and more preferably 15 particles / ⁇ m 2 or more.
  • the metal-based particles are insulated from each other, in other words, non-conductive (metal) between adjacent metal-based particles.
  • the non-conductive as the system particle aggregate layer is preferable.
  • the sensor chip according to the present embodiment has a peak at the maximum wavelength on the longest wavelength side in comparison with the same number of metal particles compared to the reference metal particle aggregate (Y).
  • a metal-based particle assembly layer having a high absorbance (having the characteristics of [iii] above) is provided.
  • the sensor chip according to the present embodiment including the metal-based particle assembly layer having such characteristics is extremely advantageous in the following points.
  • the absorbance at the maximum wavelength of the peak on the longest wavelength side in the visible light region that is the plasmon peak is such that the metal-based particles have no interparticle interaction. Since it is larger than the above reference metal-based particle aggregate (Y), which can be regarded simply as an aggregate, and therefore exhibits extremely strong plasmon resonance, it has a stronger emission enhancement compared to the case of using a conventional plasmon material. An effect can be obtained, and thereby the luminous efficiency can be dramatically increased. Such strong plasmon resonance is considered to be expressed by the interaction between localized plasmons of metal-based particles.
  • the metal-based particle assembly layer according to this embodiment is When the absorption spectrum is measured in a state where this is laminated on the glass substrate, the absorbance at the maximum wavelength of the plasmon peak on the longest wavelength side in the visible light region is 1 or more, further 1.5 or more, and even 2 Can be about.
  • the reference metal-based particle aggregate (Y) has an average particle diameter, a metal particle B having the same particle size, height and the same material as that of the average particle diameter of the metal-based particle aggregate layer to be subjected to absorption spectrum measurement.
  • the absorption spectrum of the reference metal-based particle aggregate (Y) converted so as to have the same number of metal-based particles is obtained, and the absorbance at the maximum wavelength of the peak on the longest wavelength side in the absorption spectrum is compared.
  • the absorption spectrum of each of the metal-based particle assembly and the reference metal-based particle assembly (Y) is obtained, and the absorbance at the maximum wavelength of the peak on the longest wavelength side in each of the absorption spectra is expressed as the coverage ratio. The value divided by (the coverage of the substrate surface with the metal particles) is calculated and compared.
  • the sensor chip of the present embodiment has a structure in which a specific number or more of relatively large metal particles having a specific shape of the metal particle aggregate layer are two-dimensionally spaced apart.
  • the range of action of plasmon resonance by the metal-based particle aggregate layer (B) can be significantly extended, and compared with the case where a conventional plasmon material is used.
  • a strong light emission enhancement effect can be obtained, thereby making it possible to dramatically increase the light emission efficiency (similar to the effect (2) of the first embodiment), and (C) the metal-based particle assembly layer Since the maximal wavelength of the plasmon peak can exhibit a peculiar shift, it is possible to enhance light emission in a specific (desired) wavelength region (similar to the effect (3) of the first embodiment), etc. Effect of It can be.
  • the metal-based particle assembly layer of the present embodiment (the state laminated on the glass substrate) is measured 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 a wavelength region of 350 to 550 nm, for example.
  • the metal-based particle assembly layer of the present embodiment typically has a thickness of about 30 to 500 nm (for example, compared to the case where the metal-based particles are arranged with a sufficiently long inter-particle distance (for example, 1 ⁇ m). 30-250 nm) can be produced.
  • the specific configuration of the metal-based particle assembly layer according to this embodiment is the specific configuration of the metal-based particle assembly layer according to the first embodiment (material of metal-based particles, average particle diameter, average height, aspect ratio). Ratio, average interparticle distance, number of metal-based particles, non-conductivity of the metal-based particle assembly layer, etc.). Definitions of terms such as average particle diameter, average height, aspect ratio, and average interparticle distance are the same as those in the first embodiment.
  • the average particle diameter of the metal-based particles is in the range of 200 to 1600 nm, and the characteristics of [iii] above (the absorbance at the maximum wavelength of the plasmon peak on the longest wavelength side is that of the reference metal-based particle aggregate (Y)).
  • it is preferably in the range of 200 to 1200 nm, more preferably 250 to 500 nm, and still more preferably 300 to 500 nm. .
  • 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 two-dimensionally arranged, extremely strong plasmon resonance is achieved.
  • the average height of the metal-based particles is in the range of 55 to 500 nm, and in order to effectively obtain the characteristics of [iii] and further the effects (A) to (C), preferably 55 to 300 nm, More preferably, it is in the range of 70 to 150 nm.
  • the aspect ratio of the metal-based particles is in the range of 1 to 8, and preferably 2 to 8, in order to effectively obtain the characteristics of [iii] and further the effects (A) to (C). Preferably it is in the range of 2.5-8.
  • the metallic particles may be spherical, but preferably have a flat shape with an aspect ratio exceeding 1.
  • the metal particles preferably have a smooth curved surface, and more preferably have a flat shape with a smooth curved surface. Some minute irregularities (roughness) may be included, and in this sense, the metal-based particles may be indefinite.
  • the metal particles constituting the metal particle assembly layer are as uniform in size and shape (average particle diameter, average height, aspect ratio) as possible.
  • the plasmon peak is sharpened. 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 higher.
  • the reduction in the size and shape variation between the metal-based particles is advantageous also from the viewpoint of uniformity of the intensity of plasmon resonance in the plane of the metal-based particle assembly layer.
  • the distance between the large particles increases, and the interaction between the large particles is facilitated by filling the space between the small particles. It is preferable.
  • the metal-based particles are preferably arranged so that the average distance between the adjacent metal-based particles (average interparticle distance) is in the range of 1 to 150 nm. . More preferably, it is in the range of 1 to 100 nm, still more preferably 1 to 50 nm, and particularly preferably 1 to 20 nm.
  • the average interparticle distance is less than 1 nm, electron transfer based on the Dexter mechanism occurs between particles, which is disadvantageous in terms of deactivation of localized plasmons.
  • the number of metal particles contained in the metal particle aggregate layer is 30 or more, preferably 50 or more.
  • the number of metal-based particles contained in the metal-based particle aggregate can be, for example, 300 or more, and further 17500 or more.
  • the number density of the metal particles in the metal particle assembly layer is preferably 7 particles / ⁇ m 2 or more, and more preferably 15 particles / ⁇ m 2 or more.
  • the metal-based particles are insulated from each other, in other words, non-conductive (metal) between adjacent metal-based particles.
  • the non-conductive as the system particle aggregate layer is preferable.
  • the metal-based particle assembly layer according to the present embodiment having the above feature [iii] includes the metal species, size, shape, average distance between the metal-based particles, and the like of the metal-based particles constituting the metal-based particle assembly layer. It can be obtained by control.
  • the metal-based particle assembly layer provided in the sensor chip of the present invention preferably has any one of the above features [i] to [iii], and any two or more features of [i] to [iii]. It is more preferable to have all of the features [i] to [iii].
  • the metal-based particle assembly layer according to the present invention including the metal-based particle assembly layer according to the first to third embodiments can be produced by the following method.
  • a bottom-up method for growing metal-based particles from a small seed on a substrate (2) A method in which a metal particle having a predetermined shape is coated with a protective layer made of an amphiphilic material having a predetermined thickness and then formed into a film on a substrate by an LB (Langmuir Broadgett) film method, (3) In addition, a method of post-processing 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 particles are dispersed, and the like.
  • a step of growing metal-based particles (hereinafter also referred to as a particle growth step) on a substrate adjusted to a predetermined temperature at a very low speed.
  • a particle growth step a step of growing metal-based particles (hereinafter also referred to as a particle growth step) on a substrate adjusted to a predetermined temperature at a very low speed.
  • 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 diameter of 200 to 1600 nm, average height of 55 to 500 nm and aspect ratio of 1 to 8), more preferably a metal particle aggregate layer (thin film) having an average interparticle distance (1 to 150 nm) within a predetermined range can be obtained with good control. it can.
  • the rate at which the metal-based particles are grown on the substrate is preferably less than 1 nm / min, more preferably 0.5 nm / min or less in terms of average height growth rate.
  • the average height growth rate here can also be referred to as an average deposition rate or an average thickness growth rate of metal-based particles.
  • Average height of metal particles / Metal particle growth time (metal material supply time) Defined by The definition of “average height of metal particles” is as described above.
  • the temperature of the substrate in the grain 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 in the vicinity thereof (about 300 ° C. ⁇ 10 ° C. ).
  • a production method including a particle growth step of growing metal-based particles at an average height growth rate of less than 1 nm / min on a substrate whose temperature is adjusted within a range of 100 to 450 ° C., it is supplied at the initial stage of particle growth.
  • a plurality of island-shaped structures made of metal-based materials are formed, and these island-shaped structures grow together with the supply of further metal-based materials, and merge with surrounding island-shaped structures.
  • a metal particle aggregate layer in which particles having a relatively large average particle diameter are densely arranged is formed. Therefore, a metal-based particle assembly layer comprising metal-based particles controlled to have a shape within a predetermined range (average particle diameter, average height, and aspect ratio), and more preferably with an average interparticle distance within a predetermined range. It can be manufactured.
  • the average height growth rate, substrate temperature, and / or metal-based particle growth time (metal-based material supply time)
  • the average particle diameter, average height, and aspect of the metal-based particles grown on the substrate It is also possible to control the ratio and / or the average interparticle distance within a predetermined range.
  • conditions other than the substrate temperature and the average height growth rate in the particle growth step can be selected relatively freely.
  • the metal-based particle assembly layer can be formed efficiently.
  • the average height growth rate is 1 nm / min or more, or when the substrate temperature is lower than 100 ° C. or higher than 450 ° C., the surrounding island-like structure and the continuum are formed before the island-like structure grows greatly. It is not possible to obtain a metal-based aggregate composed of large-sized metal particles that are formed and completely separated from each other, or a metal-based aggregate composed of metal-based particles having a desired shape cannot be obtained. (For example, the average height, average interparticle distance, and aspect ratio deviate from the desired ranges).
  • the pressure at the time of growing metal-based particles is not particularly limited as long as it is a pressure capable of particle growth, but is usually less than atmospheric pressure.
  • the lower limit of the pressure is not particularly limited, but 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.
  • a specific method for growing metal-based particles on the substrate is not particularly limited as long as the particles can be grown at an average height growth rate of less than 1 nm / min. it can.
  • a metal-based particle assembly layer can be grown relatively easily, and an average height growth rate of less than 1 nm / min can be easily maintained. Therefore, direct current (DC) sputtering is used. It is preferable.
  • the sputtering method is not particularly limited, and a direct current argon ion sputtering method in which argon ions generated by an ion gun or plasma discharge are accelerated by an electric field and irradiated onto a target can be used. Other conditions such as a current value, a voltage value, and a substrate-target distance in the sputtering method are appropriately adjusted so that particle growth is performed at an average height growth rate of less than 1 nm / min.
  • a metal-based particle aggregate layer composed of metal-based particles having a shape within a predetermined range (average particle diameter, average height, and aspect ratio)
  • the average particle size growth rate is preferably set to less than 5 nm, but the average height growth rate is less than 1 nm / min. In general, the average particle size growth rate is less than 5 nm.
  • the average particle size growth rate is more preferably 1 nm / min or less.
  • the average grain size growth rate is the following formula: Average particle size of metal particles / Metal particle growth time (metal material supply time) Defined by The definition of “average particle diameter of metal-based particles” is as described above.
  • the growth time of metal-based particles in the particle growth step is at least a shape in which the metal-based particles supported on the substrate are in a predetermined range, and more preferably an average interparticle distance within the predetermined range. It is the time to reach and less than the time within which the shape within the predetermined range and the average interparticle distance start to deviate. For example, even if particle growth is performed at an average height growth rate and a substrate temperature within the above predetermined range, if the growth time is extremely long, the amount of the metal-based material loaded becomes too large and separated from each other. It does not become an aggregate of arranged metal-based particles, but becomes a continuous film, or the average particle diameter and average height of the metal-based particles become too large.
  • the particle growth process is stopped at an appropriate time.
  • a time setting can be obtained, for example, by conducting a preliminary experiment in advance.
  • the average height growth rate and the substrate temperature, and the relationship between the shape of the metal-based particles and the average distance between the particles in the obtained metal-based particle aggregate can be performed.
  • the time until the thin film made of the metal-based material grown on the substrate exhibits conductivity is obtained in advance by a preliminary experiment.
  • the particle growth process may be stopped until this time is reached.
  • the substrate surface 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 substrate surface the easier it is for the growing metal-based particles to coalesce with other neighboring metal-based particles due to the thermal energy received from the substrate, so a film made of larger-sized metal-based particles is formed. It tends to be easily obtained.
  • the substrate on which the metal particles are grown can be used as it is as a sensor chip substrate. That is, a substrate (metal particle assembly layer laminated substrate) on which a metal particle assembly layer is laminated and supported by the above-described method can be used as a component of a sensor chip.
  • the substrate 10 may be made of any material, but in particular, when the metal-based particle assembly layer is directly laminated on the substrate 10, from the viewpoint of ensuring the non-conductivity of the metal-based particle assembly layer, It is preferable to use a conductive substrate.
  • 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.
  • 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 assembly layer and protects the metal-based particle 200.
  • the protective layer 30 is preferably insulating. By being insulative, the above-described non-conductivity of the metal-based particle assembly layer (non-conductivity between metal-based particles) can be ensured, and the metal-based particle assembly layer 20 and other layers adjacent thereto can be secured. Can be electrically insulated. If a current flows through the metal-based particle assembly layer 20, the light emission enhancement effect by plasmon resonance may not be sufficiently obtained. Further, the protective layer 30 covering the metallic particles can prevent the metallic particles 200 from coming into direct contact with a layer other than the protective layer 30 or the external environment, and can prevent the metallic particles 200 from being deteriorated.
  • the material constituting the protective layer 30 is preferably a material having good insulating properties.
  • a material having good insulating properties for example, in addition to spin-on glass (SOG; for example, containing an organic siloxane material), SiO 2 , Si 3 N 4, or the like is used. Can do.
  • the thickness of the protective layer 30 is not particularly limited as long as the metal-based particles 200 can prevent direct contact with a layer other than the protective layer 30 or the external environment. Since the distance between the labeling substance existing in the vicinity of the specific binding of the substance and the metal-based particle assembly layer 200 is preferably within a predetermined range, it is better that the distance is as thin as 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 further preferably 20 to 90 nm.
  • the thickness of the protective layer 30 is a value obtained by subtracting the average height of the metal-based particle assembly layer 20 from the average thickness from the upper surface of the substrate 10 to the upper surface of the protective layer 30.
  • the thickness is preferably 10 nm or more. From the viewpoint of obtaining a sufficient emission enhancement effect by localized plasmon resonance for a labeling substance that is to be present above the sensor chip. It is preferable that it is 150 nm or less.
  • the metal-based particle assembly layer 20 is provided with the metal-based particle assembly layer that exhibits strong plasmon resonance and in which the action range of plasmon resonance (range in which the enhancement effect by plasmons extends) is significantly extended. Even if the distance from the upper surface to the labeling substance to be present above the sensor chip is, for example, 15 nm or more, further 25 nm or more, and even more than that, it is possible to enhance the emission of the labeling substance, for example It is. In addition, the distance from the upper surface of the metal-based particle assembly layer 20 to the labeling substance is preferably 170 nm or less, and more preferably 150 nm or less.
  • the distance from the upper surface of the metal-based particle assembly 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, a major axis of 10 nm or more.
  • the signal derived from the labeling substance can be enhanced.
  • the substance to be detected preferably has a major axis of 1 ⁇ m 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 plasmon resonance operating range.
  • the major axis here means the chain length.
  • the detection sensitivity can be enhanced, it is possible to increase the sensitivity even when a luminescent substance having a low luminous efficiency is used as a labeling substance or when the amount of the detected substance is small. Can be detected.
  • the sensor chip of the present invention even when a conventional blue (or a wavelength region in the vicinity thereof) light emitting material having a relatively low luminous efficiency is used, the maximum wavelength of the plasmon peak is shifted to the short wavelength side.
  • the luminous efficiency can be enhanced.
  • the maximum wavelength of the plasmon peak of the metal-based particle assembly layer is preferably equal to or close to the emission wavelength of the luminescent substance used as the labeling substance. Thereby, the light emission enhancement effect by plasmon resonance can be heightened more effectively.
  • the maximum wavelength of the plasmon peak of the metal-based particle assembly layer can be controlled by adjusting the metal species, average particle diameter, average height, aspect ratio, and / or average interparticle distance of the metal-based particles constituting the metal-based particle assembly layer.
  • the trapping layer 40 has a trapping substance and is formed on the protective layer 30.
  • the trapping substance exists in a fixed state in the trapping layer 40, exists in a free state in the trapping layer, or exists in a fixed state 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 capture layer 40, and the binding active group may be used as the capture substance.
  • FIG. 2A is a schematic cross-sectional view showing an example of a sensor chip that detects nucleotides.
  • FIG. 2B is a schematic cross-sectional view showing a state in which the detection target substance 51 is captured by the capture layer 41 in the sensor chip 110 of this configuration example.
  • the detected substance 51 is a nucleotide to which a fluorescent substance is bound.
  • the material of the acquisition layer 41 is not limited, and organic substances, inorganic substances, oxides thereof, and the like can be used.
  • ITO indium tin oxide
  • SiO 2 , Si 3 N 4 , TiO 2 , Ta 2 can be used. It can be formed of O 5 , Al 2 O 3 or the like.
  • the surface of the capture layer 41 is subjected to a treatment for inducing a binding active group that can specifically bind to a base of a nucleotide that is a detection target substance.
  • 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 interact electrostatically with a base.
  • the thicknesses of the protective layer 30 and the capturing layer 41 are determined so that the fluorescent material located in the vicinity of the surface of the sensor chip falls within the range of localized plasmon resonance generated by the metal-based particle layer. Is preferred.
  • the average thickness of the trapping layer 41 can be set to, for example, 5 to 50 nm.
  • the average thickness of the protective layer 30 can be set to, for example, 15 to 145 nm.
  • the sum of the average thickness of the protective layer 30 and the average thickness of the trapping layer 41 is preferably 20 to 150 nm, more preferably 20 to 150 nm, and even more preferably 20 to 90 nm.
  • Detection method of detected substance 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 where detection is performed using a fluorescent substance bound to a detection target substance will be described as an example based on FIGS. 2 (a) and 2 (b).
  • a sample solution containing the target substance 51 labeled with a fluorescent substance is dropped on the surface of the capturing layer 41 on the surface of the protective layer 30 of the sensor chip 110. Thereafter, the sensor chip 110 that has been left for a predetermined time is analyzed using, for example, a sensing system described below to detect a substance to be detected.
  • FIG. 3 is a schematic cross-sectional view showing an example of the sensing system of the present invention.
  • a luminescent substance which is a labeling substance present near the surface of the sensor chip, is irradiated by irradiating the excitation light 3 from the direction perpendicular to the surface of the capture layer 41 on the capture layer 41 side of the sensor chip 110. Make it emit light.
  • the light emitted from the excitation light source 4 is condensed by the lens 5 to be the excitation light 3, which is irradiated.
  • 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 collected by the lens 7 and then dispersed through the wavelength cut filter 8 that cuts the light having the wavelength of the excitation light. It is detected by the measuring device 9.
  • the detector besides the spectrophotometer 9, an epifluorescence microscope, a total reflection illumination fluorescence microscope, a scanning near-field light microscope, and the like can be used.
  • FIG. 4 is an SEM image when the metal-based particle assembly layer in the obtained metal-based particle assembly layer laminated substrate is viewed from directly above.
  • FIG. 4A is an enlarged image on a 10000 times scale
  • FIG. 4B is an enlarged image on a 50000 times scale.
  • FIG. 5 is an AFM image showing the metal-based particle assembly layer in the obtained metal-based particle assembly layer laminated substrate.
  • VN-8010 manufactured by Keyence Corporation was used (the same applies hereinafter).
  • the size of the image shown in FIG. 5 is 5 ⁇ m ⁇ 5 ⁇ m.
  • the average particle diameter based on the above definition of the silver particles constituting the metal-based particle assembly layer of this production example was determined to be 335 nm, and the average interparticle distance was 16.7 nm.
  • the average height was determined to be 96.2 nm. From these, the aspect ratio (average particle diameter / 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. Furthermore, it can be seen from the SEM image that the metal-based particle assembly layer of this production example has about 6.25 ⁇ 10 10 (about 25 particles / ⁇ m 2 ) silver particles.
  • the conductivity was confirmed by connecting a tester [Multimeter (“E2378A” manufactured by Hewlett-Packard Company)] to the surface of the metal-based particle assembly layer in the obtained metal-based particle assembly layer laminated substrate. It was confirmed that it does not have.
  • a silver nanoparticle aqueous dispersion (manufactured by Mitsubishi Paper Industries, Ltd., silver nanoparticle concentration: 25% by weight) was diluted with pure water so that the silver nanoparticle concentration was 2% by weight.
  • 1% by volume of a surfactant was added to the silver nanoparticle aqueous dispersion and stirred well, and then 80% by volume of acetone was added to the obtained silver nanoparticle aqueous dispersion at room temperature. was sufficiently stirred to prepare a silver nanoparticle coating solution.
  • the silver nanoparticle coating solution was spin-coated at 1000 rpm on a 1 mm-thick soda glass substrate whose surface was wiped with acetone, and then left for 1 minute in the atmosphere, and then in an electric furnace at 550 ° C. for 40 seconds. Baked.
  • the above-mentioned silver nanoparticle coating solution is again spin-coated at 1000 rpm on the formed silver nanoparticle layer, and then left in the air for 1 minute, and then baked in an electric furnace at 550 ° C. for 40 seconds.
  • a metal-based particle assembly layer laminated substrate was obtained.
  • FIG. 6 is an SEM image when the metal-based particle assembly layer in the obtained metal-based particle assembly layer laminated substrate is viewed from directly above.
  • FIG. 6A is an enlarged image on a 10000 times scale
  • FIG. 6B is an enlarged image on a 50000 times scale.
  • FIG. 7 is an AFM image showing the metal-based particle assembly layer in the obtained metal-based particle assembly layer laminated substrate. The size of the image shown in FIG. 7 is 5 ⁇ m ⁇ 5 ⁇ m.
  • the average particle diameter based on the above definition of the silver particles constituting the metal-based particle assembly layer of this production example was determined to be 293 nm and the average interparticle distance was 107.8 nm. Further, the average height was determined to be 93.0 nm from the AFM image shown in FIG. From these, the aspect ratio (average particle diameter / 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. Furthermore, it can be seen from the SEM image that the metal-based particle assembly layer of this production example has about 3.13 ⁇ 10 10 (about 12.5 particles / ⁇ m 2 ) silver particles.
  • the conductivity was confirmed by connecting a tester [Multimeter (“E2378A” manufactured by Hewlett-Packard Company)] to the surface of the metal-based particle assembly layer in the obtained metal-based particle assembly layer laminated substrate. It was confirmed that it does not have.
  • Example 1 Sensor chip using a nucleotide labeled with a fluorescent substance as a substance to be detected
  • the sensor chip shown in FIG. 2A was manufactured according to the following method.
  • metal-based particle assembly layer 20 described in Production Example 1 was formed on substrate 10 which is a soda glass substrate having a thickness of 0.5 mm.
  • a spin-on-glass (SOG) solution was spin-coated on the metal-based particle assembly layer, and a protective layer 30 having a predetermined thickness was laminated.
  • SOG solution an organic SOG material “OCD T-7 5500T” manufactured by Tokyo Ohka Kogyo Co., Ltd. diluted with ethanol was used.
  • an ITO layer (thickness 10 nm) was laminated on the protective layer 30 as the capture layer 41 by sputtering.
  • five sensor chips (Examples 1a, 1b, 1c, 1d, and 1e) having different thicknesses of the protective layer 30 were produced.
  • the thickness of the protective layer 30 in Examples 1a, 1b, 1c, 1d, and 1e was 10 nm, 30 nm, 50 nm, 80 nm, and 150 nm, respectively.
  • Example 1 Sensor chip using a nucleotide labeled with a fluorescent substance as a substance to be detected
  • a sensor chip was produced in the same manner as in Example 1 except that the metal-based particle assembly layer was not formed and the thickness of the protective layer 30 was 150 nm.
  • a solution containing cytidine triphosphate (CTP) to which the fluorescent substance Cy3 was bound was obtained.
  • Cy3-CTP was captured on the surface of the capture layer 40.
  • the emission spectrum was measured using a fluorescence spectrophotometer (trade name: FP-6500, manufactured by JASCO Corporation) as the spectrometer 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 (wavelength 545 nm to wavelength 755 nm) of the emission spectrum observed with the sensor chip of Example 1b is the integrated value of the emission spectrum observed with the sensor chip of Comparative Example 1 (wavelength 545 nm to wavelength It was confirmed to show 10.1 times the emission intensity represented by 755 nm).
  • the emission spectra were similarly measured for Examples 1a, 1c, 1d, and 1e.
  • a value obtained by converting the emission intensity represented by the integrated value of the emission spectrum (wavelength 545 nm to wavelength 755 nm) to a value when the emission intensity of Comparative Example 1 was set to 1 was calculated.
  • FIG. 9 is a diagram in which the converted value of the emission intensity is plotted against the value obtained by adding the average thickness of the protective layer and the average thickness of the trapping layer. From FIG. 9, when the average thickness of the trapping 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 protective layer).
  • 1,110 sensor chip 3 excitation light, 4 excitation light source, 5, 7 lens, 6 emission from labeling substance, 8 wavelength cut filter, 9 spectrometer, 10 substrate, 20 metal particle aggregate layer, 30 protective layer 40, 41 trapping layer, 51 substance to be detected, 200 metal particles.

Abstract

L'invention concerne une puce de capteur (1) qui est utilisée pour détecter une substance à détecter et qui comprend : un substrat (10) ; une couche d'agrégat de particules à base de métal (20) formée sur le substrat (10) ; une couche protectrice (30) recouvrant la couche d'agrégat de particules à base de métal (20) ; et une couche de piégeage (40) formée sur la couche protectrice (30) et comprenant une substance de piégeage qui se lie spécifiquement à la substance à détecter. La couche d'agrégat de particules à base de métal (20) comprend un agrégat de particules formé par 30 particules métalliques (200) ou plus disposées de manière bidimensionnelle de façon à être mutuellement séparées. Les particules à base de métal (200) ont un diamètre moyen de particule dans la plage de 200 à 1600 nm, une hauteur moyenne dans la plage de 55 à 500 nm, et un rapport d'aspect, qui est défini par un rapport du diamètre moyen de particule sur la hauteur moyenne, compris dans la plage 1 à 8.
PCT/JP2017/001794 2016-01-27 2017-01-19 Puce de capteur et système de détection WO2017130839A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
EP4047353A4 (fr) * 2020-03-25 2023-11-22 Sumitomo Chemical Company Limited Agrégat de particules métalliques, stratifié, et dispositif de détection

Citations (4)

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Publication number Priority date Publication date Assignee Title
JP2008502332A (ja) * 2004-06-18 2008-01-31 ノバルティス・フォルシュングスシュティフトゥング・ツヴァイクニーダーラッスング・フリードリッヒ・ミーシェー・インスティトゥート・フォー・バイオメディカル・リサーチ メチル化核酸の分析
JP2012132804A (ja) * 2010-12-22 2012-07-12 Kyoto Univ 光増強素子
JP2013177665A (ja) * 2011-03-31 2013-09-09 Sumitomo Chemical Co Ltd 金属系粒子集合体
JP2014228322A (ja) * 2013-05-20 2014-12-08 ウシオ電機株式会社 センサ、検査方法

Patent Citations (4)

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JP2008502332A (ja) * 2004-06-18 2008-01-31 ノバルティス・フォルシュングスシュティフトゥング・ツヴァイクニーダーラッスング・フリードリッヒ・ミーシェー・インスティトゥート・フォー・バイオメディカル・リサーチ メチル化核酸の分析
JP2012132804A (ja) * 2010-12-22 2012-07-12 Kyoto Univ 光増強素子
JP2013177665A (ja) * 2011-03-31 2013-09-09 Sumitomo Chemical Co Ltd 金属系粒子集合体
JP2014228322A (ja) * 2013-05-20 2014-12-08 ウシオ電機株式会社 センサ、検査方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4047353A4 (fr) * 2020-03-25 2023-11-22 Sumitomo Chemical Company Limited Agrégat de particules métalliques, stratifié, et dispositif de détection

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