Apparatus with an analysis unit

Abstract

Provided is a device (1) having: a chip (20) including, on a first surface (21) thereof, a metal body capable of excitation of localized surface plasmon resonance; and an analysis unit (30) for scanning the first surface of the chip in at least one dimension with a laser in a state in which the first surface is in contact with a sample (3), and recording, in association with the scanning, scattered light (3) amplified by the first surface. The chip includes a substrate (25), and, on the first surface of the substrate, a first layer (26) provided with a repeating relief structure, and a second layer (27) which is provided via the first layer and which includes a metal body.

Description

[Technical Field]

[0001] The present invention relates to an apparatus having an analysis unit. [Background technology]

[0002] Japanese Patent Application Laid-Open Publication No. 2015-152492 discloses an analytical device capable of obtaining a high enhancement in an enhancement spectrum and detecting and analyzing a target substance with high sensitivity. The analytical device includes an electric field enhancing element including a metal layer, a light-transmitting layer provided on the metal layer and transmitting excitation light, a plurality of metal particles provided on the light-transmitting layer and arranged in a first direction and a second direction intersecting the first direction, a light source that irradiates the electric field enhancing element with at least one of linearly polarized light polarized in the first direction, linearly polarized light polarized in the second direction, and circularly polarized light as the excitation light, and a detector that detects the light emitted from the electric field enhancing element. In this analytical device, localized surface plasmons excited in the metal particles and propagating surface plasmons excited at the interface between the metal layer and the light-transmitting layer interact electromagnetically.

[0003] Japanese Patent Laid-Open Publication No. 2017-181308 discloses a metal nanostructure array and an electric field enhancement device that have a high degree of enhancement of Raman scattering intensity. This device has a plurality of convex nanostructures formed at predetermined intervals on a substrate. For a metal nanostructure array used in an electric field enhancement device such as a substrate for surface-enhanced Raman spectroscopy, the base of each convex nanostructure is formed from metal, and a polycrystalline metal film consisting of shape-anisotropic crystal grains is formed from the same or a different metal so as to cover the base. DISCLOSURE OF THE INVENTION

[0004] One aspect of the present invention is an apparatus having a chip including a metal body capable of exciting localized surface plasmon resonance on a first surface, and an analysis unit that scans the first surface in at least one dimension with a laser while the first surface is in contact with a sample containing an analyte, and records enhanced scattered light from the first surface in association with the scan. The analysis unit may record a spectrum of the scattered light in association with the scan, or may record a fingerprint of the scattered light in association with the scan.

[0005] By scanning the first surface of the chip with a laser in at least one dimension, including at least one of the thickness and surface directions, it is possible to identify hot spots suitable for detecting analytes on the first surface of the chip during scanning, and to obtain enhanced scattered light, typically surface-enhanced Raman scattering (SERS) light in Raman analysis, due to the coincidence of the hot spots suitable for detecting analytes in the sample with the laser spot exciting the scattered light. By recording the SERS light in conjunction with the scan, for example, synchronously, it is possible to identify points on the chip suitable for detecting analytes, thereby increasing the detection sensitivity of the analytes. Furthermore, statistical processing of the SERS light recorded in conjunction with the scan can improve the accuracy and reproducibility of measuring the concentration of analytes contained in the sample. Scanning can be performed by moving the spot, or by switching between multiple laser spots, as in a multifocal type.

[0006] The chip may include a base (substrate), a first layer having a repeated uneven structure on a first surface of the base (substrate), and a second layer including metal objects capable of exciting localized surface plasmon resonance, which are provided through the first layer. By disposing the metal objects capable of exciting localized surface plasmon resonance as the second layer through the first layer having a repeated nano-level uneven structure, it is possible to control the spacing of the metal objects at the nano-level. This increases the likelihood that hot spots (hot sites) suitable for localized surface plasmon excitation, which generates an enhanced electric field that contributes to the measurement of an analyte in a sample, will be present on the first surface of the chip.

[0007] The first layer and / or the second layer may include a structure for capturing microorganisms and / or proteins. When microorganisms or proteins are the target of analysis or detection, capturing the target makes it easier to obtain SERS light resulting from the target. The first layer and the second layer may, individually or collectively, form a microstructure, submicrostructure, nanostructure, or subnanostructure. Therefore, a structure suitable for capturing microorganisms or proteins may be incorporated. For example, the first layer and the second layer may, individually or collectively, have a structure suitable for capturing viruses of several tens to several hundreds of nanometers, a structure suitable for capturing bacteria of several micrometers, or a structure suitable for capturing cells of several to several hundreds of micrometers. The first layer and the second layer may, individually or collectively, have a structure suitable for capturing proteins, including antibodies, of several to several tens of nanometers.

[0008] The chip may include multiple sectors in which at least one of the first layer and the second layer differ in configuration. The chip may also include a third layer containing an affinity ligand attached to at least a portion of the surface of the metal body of the second layer, and the multiple sectors may include sectors in which at least one of the first layer configuration, the second layer configuration, and the affinity ligand differ. The chip may also include a fourth layer overlaid on the second layer or the third layer and including a structure for capturing microorganisms and / or proteins. The fourth layer may be a nanostructure using nanomaterials such as carbon nanotubes. The fourth layer may be a microstructure or nanostructure formed on a different base (substrate) by a method such as nanoimprinting or etching. The fourth layer may include different structures for capturing different microorganisms or proteins, either on a sector-by-sector basis or to separate the sectors.

[0009] The analysis unit may include a unit that sequentially scans multiple sectors. The second layer may have metal bodies provided at the tips of the convex portions of the first layer, and may include regions where the spacing between the metal bodies is narrower than the spacing between the convex portions of the first layer. The first layer may include regions where the spacing between the convex portions is different. The second layer may be designed to prioritize the formation of hot spots, the first layer may be designed to prioritize the reflection or transmission of SERS light, or the first layer may be designed for the purpose of dispersing or concentrating the analyte in the sample.

[0010] The analysis unit may include a unit that irradiates the first surface with the laser through the sample. The analysis unit may also include a unit that focuses at least two laser beams onto a common spot on the first surface. This facilitates control of the position of the laser spot that excites scattered light on the first surface of the chip, thereby improving scanning accuracy. Examples of measurement methods using at least two laser beams include coherent anti-Stokes Raman analysis (coherent anti-Stokes Raman scattering, CARS), stimulated Raman analysis (stimulated Raman scattering, SRS), and time-resolved CARS.

[0011] The sample may be solid, gaseous, or liquid, as long as it has a property or shape that allows it to contact the first surface of the chip. The device may be a device or monitor that detects the presence or absence and / or concentration of a component of the sample to be measured. The device may further include a light-transmitting holder that holds or flows a fixed amount of sample, the holder having a light-transmitting chip attached or embedded in its wall. The device may further include a route through which a sample containing wastewater from a living body flows, the route having a chip attached or embedded in its wall.

[0012] Another aspect of the present invention is a chip for electric field enhancement, comprising a substrate and a metal body capable of exciting localized surface plasmon resonance provided on a first surface of the substrate. The chip may include a first layer and a second layer laminated on the substrate. The chip may further include a third layer, and may include multiple sectors in which at least one of these layers has a different configuration.

[0013] Another aspect of the present invention is a method for detecting the presence and / or concentration of an object in a sample using the above-described chip. The method includes scanning a first surface with a laser in at least one dimension while the first surface is in contact with the sample, and recording enhanced scattered light from the first surface in association with the scanning. If the chip includes multiple sectors, the recording may include sequentially scanning the multiple sectors.

[0014] Yet another aspect of the present invention is a program for an apparatus that uses the above-described chip to detect the presence and / or concentration of a target substance in a sample. The program (program product) has instructions for causing the apparatus to scan a first surface with a laser in at least one direction while the first surface is in contact with the sample, and record enhanced scattered light at the first surface in association with the scan. The program (program product) may be provided recorded on a computer-readable recording medium. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a diagram showing an example of a measurement device. [Figure 2] 1A and 1B show enlarged cross-sectional views of some examples of electric field enhancing tips. [Figure 3] FIG. 1 is a diagram showing an example of the surface of an electric field enhancing chip. [Figure 4] 10 is a flowchart showing an example of a measurement method. [Figure 5] 1A and 1B are diagrams showing different examples of devices having chips. [Figure 6] FIG. 10 shows yet another example of a device having a chip. MODE FOR CARRYING OUT THE INVENTION

[0016] 1 shows an example of an apparatus for measuring or detecting an analyte in a sample such as a liquid. This measuring apparatus (detecting apparatus) 1 includes a light-transmitting holder 10 that holds a predetermined amount (quantity) of sample 3 containing an analyte (substance to be analyzed, object to be measured) therein or that flows at a predetermined flow rate, and a circulation system 13 that circulates or supplies sample 3 between a source 12 of sample 3 and holder 10. Measuring apparatus 1 further includes an electric field enhancing chip (detection or measurement chip) 20 attached to or embedded in one wall surface 15 of holder 10, and an analysis unit (spectroscopic analysis unit) 30 arranged to irradiate lasers 31 and 32 onto a surface (front surface, first surface) 21 of chip 20 that contacts sample 3. An example of the spectroscopic analysis module 30 is a Raman analyzer, and in particular, a CARS (Coherent Anti-Stokes Raman Scattering) analyzer, an SRS (Stimulated Raman Scattering) analyzer, a time-resolved CARS analyzer, or the like, which are suitable for trace analysis, may be used.

[0017] The CARS analysis unit 30 in this example includes a laser light source 35, a head module 36 that irradiates (focuses) a common position (region, laser spot, spot) 34 on the surface 21 of the enhancement chip 20 with pump light 31 and Stokes light 32, which are laser light obtained from the laser light source 35 via the sample 3, a scan unit 37 that moves the laser spot 34 formed by the pump light 31 and Stokes light 32 along the surface 21 of the chip 20, a detection module (spectrometer) 38 that detects scattered light (surface-enhanced Raman light, SERS) 33 enhanced on the surface 21 of the enhancement chip 20 from the back surface 22 of the enhancement chip 20, and an analysis control unit 39 that records and analyzes the scattered light 33 detected by the detection module 38 in association with the scan.

[0018] The analysis control unit (control unit) 39 may be implemented as software (program, program product) 41 in a PC 40 equipped with computer resources including a CPU and memory. The program 41 may be provided by being recorded on an appropriate type of computer-readable memory (recording medium). The analysis control unit 39 may record the SERS light detected (measured) by the detection module 38 as a spectrum containing the SERS light, may record characteristic components (fingerprints) extracted from the spectrum, or may record information compressed in an appropriate manner, such as a difference from a base waveform.

[0019] The fluid sample 3 containing a liquid (including aqueous solutions and other solutions) or gas to be measured by the measuring device 1 may be any fluid, such as a fluid used in a manufacturing process, a fluid discarded during a manufacturing process, or a liquid containing a substance (analyte) to be measured, such as air, river water, wastewater, blood, serum, body fluid, culture medium, or amplification solution. The sample 3 may be a solidified substance containing a chip 20, or a solid with a chip 20 attached to part of its surface. The sample 3 may be any substance or shape that can contact or adhere to the surface (first surface) 21 of the chip 20.

[0020] The sample 3 may be one excreted from a living body. An example of the sample 3 may be a urine sample. The sample 3 may be dialysis effluent. The sample 3 may be exhaled air (exhaled gas). The measuring device 1 can detect trace components contained in these fluid samples 3 using surface-enhanced Raman analysis. A pulsed laser may be used as the laser light source 35. For example, a pulsed laser of several kHz or around 5 kHz can be used. Spectra that are enhanced and detected by this method include luminescence, fluorescence, Raman scattered light, and nonlinear Raman.

[0021] 2 shows an enlarged cross section of the structure of a chip 20 for electric field enhancement. The chip 20 includes a substrate 25 and a first layer 26 provided on a first surface (front surface) 21 of the substrate 25 that contacts a sample 3 containing an analyte. The first layer 26 includes a repeated uneven structure 26a. The chip 20 further includes a second layer 27 that is provided on top of the first layer 26 and includes metal bodies 27a capable of exciting localized surface plasmon resonance. An example of the substrate 25 is a glass plate, but it may be made of other materials such as a silicon substrate.

[0022] The chip 20 may be configured such that the surface 21 of the substrate (base, base) 25 is irradiated with pump light 31 and Stokes light 32 via the sample 3, and SERS light 33 emitted forward relative to these lights is detected from the back surface 22. In this case, it is desirable that the substrate 25 has sufficient transparency to electromagnetic waves (light) in the wavelength range of the SERS light 33, for example, 600-800 nm. The substrate 25 does not have to have transparency to electromagnetic waves (light) in the wavelength range of the pump light 31 and Stokes light 32, for example, 1000-1300 nm. The chip 20 may be configured such that the surface 21 of the substrate 25 is irradiated with pump light 31 and Stokes light 32 via the sample 3, and SERS light 33 emitted backward relative to these lights is detected. The chip 20 may be irradiated with pump light 31 and Stokes light 32 through the back surface 22 of the substrate 25. In this case, it is desirable that the substrate 25 has sufficient transparency to electromagnetic waves (light) in the wavelength range of the pump light 31 and Stokes light 32.

[0023] The fine relief structure 26a of the first layer 26 can be formed using a resist or other highly heat-resistant resin (such as polyether sulfone) by a method suitable for manufacturing nanostructures, such as nanoimprinting or etching. The relief structure 26a may be a microstructure, submicrostructure, nanostructure, or subnanostructure. For example, when a nanostructure is formed, the thickness of the first layer 26, i.e., the height 26h of the relief structure 26a, may be 10-1000 nm or 100-300 nm. Furthermore, the pitch or spacing 26p of the relief structure 26a may be 1-1000 nm or 50-300 nm.

[0024] The metal bodies 27a of the second layer 27 are supported by the concave-convex structure 26a of the first layer 26 and are spaced apart from the substrate 25 by a distance 26h of several tens to several hundreds of nanometers by the concave-convex structure 26a, or are provided in a floating state. The metal bodies 27a, which serve as electric field enhancing elements capable of exciting localized surface plasmon resonance, may include metals such as gold, silver, copper, aluminum, platinum, chromium, nickel, palladium, tungsten, rhodium, and tellurium, or alloys containing any of these metals. The metal bodies 27a may also be semimetals such as silicon, polysilicon, gallium, and arsenic, mixtures containing any of these metals, or alloys of any of the semimetals with any of the above metals.

[0025] Each metal body 27a supported by the concave-convex structure 26a may have a shape extending from the concave-convex structure 26a, such as a rod, or may have a shape having a predetermined volume in a direction perpendicular to the concave-convex structure 26a, such as a sphere or ellipsoid. The diameter of the apex of the metal body 27a having a volume, such as a sphere or ellipsoid, may be 50 nm or less. A gap 27p may be provided between adjacent metal bodies 27a. A gap 27p suitable as a hot spot may be several nanometers, for example, 10 nm or less.

[0026] As shown in Fig. 2(a), the metal bodies 27a may be configured such that the bulging tops 26b of the concave-convex structure 26a are covered with a thin metal film 27b having a thickness of about several nanometers. As shown in Fig. 2(b), the metal bodies 27a may be configured such that metal nanostructures 27c are supported by the convex portions of the concave-convex structure 26a. The structure of the metal bodies 27a, which have a predetermined volume and can control the gaps 27p between adjacent metal bodies 27a, is not limited to shapes formed by curved surfaces such as spheres or ellipsoids, but may also be polyhedrons or shapes with acute or obtuse corners or steps.

[0027] The first layer 26 and the second layer 27 of the chip 20 may be integrally formed using the same metal material. The first layer 26 and the second layer 27 of the chip 20 may also be formed using different materials. By separately forming these layers 26 and 27, the first layer 26 can be structured to be manufactured stably and precisely as a nanostructure, and the second layer 27 can utilize the stably manufactured nanostructure of the first layer 26. This allows for the selection of a metal 27a that is more suitable for hot spots than structural stability. This allows for the provision of a chip 20 that includes metal 27a that is suitable for hot spots as a whole and has a stable metal nanostructure. This allows for the provision of a chip 20 that allows for more efficient and uniform formation of electric field-enhanced hot sites using metal nanostructures.

[0028] The chip 20 may include an area where the spacing 27p between the metal bodies 27a in the second layer 27 is narrower than the spacing 26p between the convex portions in the first layer 26. The second layer 27 can have metal bodies 27a suitable for electric field enhancement arranged at a narrow spacing (gap) 27p of about nanometers, which is suitable for forming hot spots. Meanwhile, the first layer 26 can have structures 26a made of a highly transparent material arranged at a relatively wide spacing (gap) 26p so as to guide the scattered light 33 enhanced by the second layer 27 to the back side 22 of the substrate 25. This provides a structure suitable for a chip 20 in which the surface 21 of the substrate 25 is irradiated with pump light 31 and Stokes light 32 via the sample 3, and SERS light 33 emitted forward relative to these light beams is detected through the back side 22 of the substrate 25. This configuration is also expected to suppress transmission of the pump light 31 and Stokes light 32 irradiated on the surface 21 to the back side 22.

[0029] As shown in FIG. 2(c), the chip 20 may further include a third layer 28 containing affinity ligands 28a attached to at least a portion of the surface of the metal structures 27a of the second layer 27. The affinity ligands 28a are ligands containing capture molecules, which may include any molecule capable of binding to multiple target analytes. Examples of capture molecules include, but are not limited to, antibodies, antibody fragments, recombinant antibodies, single-chain antibodies, receptor proteins, binding proteins, enzymes, inhibitor proteins, lectins, cell adhesion proteins, oligonucleotides, polynucleotides, nucleic acids, and aptamers. The affinity ligands 28a can increase the concentration of analytes near the nanostructured metal structures 27a suitable for electric field enhancement, further improving the detection sensitivity of the analytes.

[0030] The analyte (detection target) to be analyzed (detected, measured) using these chips 20 refers to any atom, chemical substance, molecule, compound, composition, microorganism, or aggregate to be detected and / or identified, including, but not limited to, amino acids, peptides, polypeptides, proteins, glycoproteins, lipoproteins, nucleosides, nucleotides, oligonucleotides, nucleic acids, sugars, carbohydrates, oligosaccharides, polysaccharides, fatty acids, lipids, hormones, metabolites, cytokines, chemokines, receptors, neurotransmitters, antigens, allergens, antibodies, substrates, metabolites, cofactors, inhibitors, drugs, pharmaceuticals, nutrients, prions, toxins, poisons, explosives, pesticides, chemical warfare agents, biological hazards, radioisotopes, vitamins, heterocyclic aromatic compounds, carcinogens, mutagens, narcotics, amphetamines, barbiturates, hallucinogens, waste products, and / or pollutants. Microorganisms include, but are not limited to, viruses, bacteria, and cells.

[0031] The first layer 26 and the second layer 27 may, either individually or in combination, form a microstructure, a submicrostructure, a nanostructure, or a subnanostructure. Therefore, these layers 26 and 27 can further improve the detection efficiency of the chip 20 by introducing a structure that physically captures the target substance. For example, the first layer 26 and the second layer 27 may, either individually or in combination, have a structure suitable for capturing viruses of several tens to several hundreds of nanometers, a structure suitable for capturing bacteria of several micrometers, or a structure suitable for capturing cells of several to several hundreds of micrometers. The first layer 26 and the second layer 27 may, either individually or in combination, have a structure suitable for capturing proteins, including antibodies, of several to several tens of nanometers. The first layer 26 and the second layer 27 may, either individually or in combination, include a structure suitable for capturing specific molecules, such as genes (DNA).

[0032] 3A and 3B schematically show the configuration of the surface 21 of a chip 20 for electric field enhancement. As shown in Fig. 3A, the chip 20 may include multiple sectors (areas, regions) 29 in which at least one of the configurations of the first layer 26 and the second layer 27 differ. When the chip 20 further includes a third layer 28 containing affinity ligands 28a, the chip 20 may include multiple sectors 29 in which at least one of the configurations of the first layer 26, the configuration of the second layer 27, and the affinity ligands 28a differ.

[0033] As shown in FIG. 3(b), the chip 20 may further include regions or sectors 29 in which the spacing (gap, pitch) 26p between the protrusions of the concave-convex structure 26a of the first layer 26 is different, resulting in different spacing (gap, pitch) 27p between the metal bodies 27a. The spacing 27p between the metal bodies 27a and the spacing 26p between the concave-convex structure 26a may differ depending on whether the direction prioritizes the formation of hot spots or the direction prioritizes the effect on the transmission of scattered light 33. The spacings 26p and 27p may differ relative to the flow direction of the sample 3, may differ depending on the adhesion of the analyte to be measured in each sector 29, or may differ depending on the concentration and detection sensitivity of the analyte. The spacings 26p and 27p may differ depending on the sector 29, taking into account manufacturing tolerances of the chip 20 or various other factors, and different regions may be included within each sector 29.

[0034] The enhancement of the electric field due to surface plasmons is typically utilized in surface-enhanced Raman scattering (SES). Surface-enhanced Raman scattering can be caused by not only the electromagnetic effect of surface plasmons but also chemical effects related to the chemical adsorption of molecules. In this case, the enhancement is thought to be due to the resonant Raman effect mediated by the charge transfer state between the chemically adsorbed molecules and the metal. The surface structure of the metal that generates surface plasmons is thought to be the most important factor determining the enhancement. However, the surface electric field varies depending on the metal structure, and in the case of metal particles, it depends on their size and shape. Therefore, the enhancement of Raman scattering can vary depending on various parameters such as the shape and size of the metal nanostructure, the localization of the electromagnetic field, the adsorption of the target substance, and the laser focus intensity. It is also related to the manufacturing tolerances of the nanostructures formed on the surface 21 of the chip 20.

[0035] For this reason, in this measurement device 1, multiple sectors 29 with different conditions for electric field enhancement by surface plasmons are prepared on the surface 21 of the chip 20, and the analysis unit 30 scans these sectors 29 with a laser spot 34 formed by pump light 31 and Stokes light 32. By providing multiple sectors 29 with different conditions for electric field enhancement by surface plasmons for each sector 29, surface-enhanced Raman scattering (SERS) light 33 related to or specific to each of the target substances can be obtained with a high probability for each sector 29. The analysis unit 30 (control unit 39) records the SERS light 33 in conjunction with the scanning of the surface 21 of the chip 20, for example, in synchronization with the scanned position or timing, thereby making it possible to identify the timing or position of a hot spot suitable for detecting each target substance contained in the sample 3 for each sector 29, and further, for each position of the spot 34 formed in the sector 29.

[0036] Therefore, the measuring device 1 can detect the target substance contained in the sample 3 with high accuracy and high reproducibility by using a chip 20 equipped with multiple sectors 29. The analysis unit 30 (control unit 39) may also have a function for statistically processing the SERS light 33 recorded in association with the scan. This makes it possible to improve the measurement accuracy of the concentration of the target substance contained in the sample 3, and also improve reproducibility. The measuring device 1 may also have a cell 10 that can control the volume or flow rate of the sample 3 during measurement, thereby improving quantitative measurement accuracy.

[0037] The scanning direction may be controlled by the analysis control unit 39 of the analysis unit 30 via the scan unit 37. The scanning direction may be one-dimensional or two-dimensional along the arrangement of the sectors 29 of the chip 20. Furthermore, the state between the metal bodies 27a of the second layer 27, the shape of the metal bodies 27a, and the adhesion state of the substance to be measured may vary depending on the thickness direction of the chip 20. Therefore, the scanning direction may be set in the thickness direction of the chip 20, and the analysis unit 30 may have a function for scanning in three dimensions.

[0038] The method of moving (scanning) the laser spot 34 by the scan unit 37 may involve physically moving the head 36, or may involve controlling the optical path using a polygon mirror or a micromirror device. The analysis unit 30 may also employ a multi-focus type head 36 to switch between multiple laser spots for scanning.

[0039] The SERS light 33 obtained on the surface 21 of the chip 20 may be detected as reflected light (backscattered light) from the surface 21 of the chip 20. In the device 1 of this example, the substrate 25 of the chip 20 is made transparent to the SERS light 33, so that the SERS light 33 transmitted through the chip 20, i.e., forward scattered light, can be detected by a spectrometer (photodetector) 38. By detecting the transmitted SERS light 33, it is possible to suppress a decrease in intensity due to the SERS light 33 propagating through the sample 3 and the generation of noise due to interaction with the sample 3, thereby enabling more accurate detection of the substance to be measured.

[0040] As described above, this device 1 enables highly sensitive and quantitative measurement by enhancing signal intensity through directional surface plasmon polaritons using a laser. Conventional surface plasmon polariton techniques detect only reflected light due to omnidirectional scattering. This technique utilizes uniform or sector-level uniform nanostructures formed on a transparent substrate 25 as the electric field enhancement device (chip) 20. By optimizing the directionality and adjusting the focal spot size of the irradiation, the excitation light is efficiently utilized, and the enhanced signal can be efficiently detected as both reflected and transmitted light. Furthermore, by using a chip 20 equipped with a transparent substrate 25, this technique allows SERS light 33 generated on the surface 21 to be detected by a detector 38 via a fixed distance from the back surface 22, without passing through the solvent (sample) 3. This enhances the sensitivity and reproducibility of the SERS light 33 obtained during measurement. This enables reproducible and quantitative spectroscopic measurements.

[0041] In particular, when CARS is used as the excitation light, 6 ~10 7 The surface plasmon polariton is expected to increase the intensity by 10 times. 2 ~10 12 The light to be measured is not limited to CARS, but may be stimulated Raman spectroscopy (SRS), coherent Stokes Raman spectroscopy (CSRS), or the like.

[0042] If long-term laser irradiation is required, a highly heat-resistant resin (such as polyether sulfone) may be used for the resist forming the first layer 26. If durability is required, the metal surface may be coated (with silica glass, for example). The appropriate wavelength range may be adjusted by modifying the nanostructure or the thickness of the silica glass layer on the metal surface. Since the chip 20, including the substrate 25, also serves as the source of CARS or Raman light, it is desirable for the substrate 25 to be made of a material whose output spectrum does not overlap with the spectrum of the object being measured (fingerprint region). Since the substrate 25 occupies the majority of the chip 20's volume, it is particularly desirable for the material to not overlap with the fingerprint region; a glass substrate is more effective than a silicon substrate.

[0043] 4 is a flowchart showing an example of a measurement method using the chip 20 in the measurement device 1. This measurement method may be provided by the program 41 as a control method for the measurement device 1. When measurement of the sample 3 starts in step 81, in step 82 the analysis control unit 39 uses the scan unit 37 to scan multiple sectors 29 provided on the first surface 21 of the chip 20 in a predetermined pattern with laser light (pump light 31 and Stokes light 32). At the same time, in step 83 the analysis control unit 39 uses the detection module 38 to measure enhanced scattered light (SERS light) 33 in each sector 29 of the first surface 21 and record it in association with the scan.

[0044] In step 82, if the chip 20 has a plurality of sectors 29 arranged one-dimensionally, for example, in a row, the scanning unit 37 may move the pump beam 31 and the Stokes beam 32 continuously in one dimension at a constant speed. The scanning unit 37 may move the pump beam 31 and the Stokes beam 32 intermittently so that spots 34 are formed continuously for a predetermined time at the center or a predetermined position of each sector 29. If the chip 20 has a plurality of sectors 29 arranged two-dimensionally, the scanning unit 37 may move the pump beam 31 and the Stokes beam 32 continuously or intermittently in two dimensions at a constant speed, or may move them continuously or intermittently in three dimensions.

[0045] In step 83, the analysis control unit 39 may record the spectrum or intensity of a predetermined wavelength of the SERS light 33 obtained by the detection module 38 in association with the scan, for example, in association with the position or identification information of the sector 29 from which the SERS light 33 was obtained. The detection result of the SERS light 33 may also be recorded in association with the more precise position of the spot 34 moving within the sector 29.

[0046] After the scanning of the sectors 29 on the surface 21 of the chip 20 is completed in step 84, the analysis control unit 39 analyzes the detection results of the SERS light 33 obtained by the scanning in step 85. All sectors 29 on the surface 21 of the chip 20 may be scanned, or a limited number of sectors 29 predetermined by prior or preliminary measurements may be scanned before proceeding to the analysis process. The analysis control unit 39 may select and analyze SERS light 33 with an intensity or spectrum suitable for quantitative or qualitative measurement of the substance to be measured from the scanned results. The control unit 39 may statistically process the scanned results to determine the presence and / or concentration of the substance to be measured.

[0047] In step 86, the analysis control unit 39 outputs the analysis results to a host application. For example, if the measurement device 1 is a urine monitoring device or part of it, the measurement device 1 can use the chip 20 to simultaneously detect multiple components in urine, including uric acid, urea, and creatinine, which are trace components, with high sensitivity. Therefore, the monitoring device application can provide health management advice to the monitored user based on the urine analysis results of the measurement device 1, for example.

[0048] FIG. 5 shows another example of an apparatus for measuring an analyte in a sample using a chip 20. This apparatus 60 includes a route (pipe) 11 through which a sample 3 containing wastewater 66 from a living body 65, such as a human body, flows. A chip 20 is attached to a portion of the wall 15 of the route (pipe) so that its first surface 21 contacts the sample 3. The apparatus 60 also includes a monitoring device 50 that irradiates the chip 20 with laser light to measure the presence and / or concentration of an analyte (substance to be measured) contained in the sample 3. The monitoring device 50 has an analysis unit 30 that irradiates (focuses) the chip 20 with pump light 31 and Stokes light 32 and acquires scattered light (surface-enhanced Raman light, SERS) 33 enhanced by the surface 21 of the chip 20. The basic configuration of the analysis unit (spectroscopic analysis unit) 30 is the same as that of the analysis unit 30 included in the measurement apparatus 1 described above.

[0049] In this example, the analysis unit 30 is a type that detects SERS light (backward CARS, Epi-CARS) 33 output opposite the pump light 31 and Stokes light 32 irradiated onto the chip 20, and the head module 36 also functions as a module that collects the SERS light 33 generated on the surface 21 of the chip 20.

[0050] An example of the effluent 66 from the living body 65 is the aforementioned urine. Another example of the effluent 66 is the effluent (dialysis fluid effluent) discharged from the human body via the blood purifier (dialyzer) of the dialysis device. The effluent monitor 50 detects the concentration of a predetermined substance (e.g., the concentration of a substance such as urea or uric acid) in the effluent 66, and can accurately determine the end of the dialysis treatment.

[0051] These devices and methods are suitable for applications in which a liquid sample 3 is measured, and instead of flowing or storing the fluid in a cell 10, the chip 20 may be inserted into a flow system (line) 11 for measurement. These devices and methods enable the simultaneous detection of multiple components, including uric acid, urea, and creatinine, in aqueous solutions (such as body fluids, dialysis fluids, wastewater, food components, and other aqueous solution components) with high sensitivity. Calibration for the substance to be measured may be performed to allow for error in the measurement.

[0052] FIG. 6 shows an example of a virus testing device using a chip 20. This device 70 is a platform that identifies viruses contained in a liquid (test liquid, sample) 3 containing a specimen collected from a human body or the like, placed in a light-transmitting container 16 with a chip 20 attached to a portion of its wall 15. The sample 3 may be a liquid in which the specimen has been cultured. The chip 20 includes a fourth layer 24 including nanostructures for capturing viruses, in addition to the first layer 26, second layer 27, and third layer 28 described above, on a base 25. The chip 20 of this example includes a second base (substrate) 23 that is sufficiently transparent to the pump light 31 and the Stokes light 32, and a fourth layer 24 formed on the second base 23 and including a structure such as concaves and convexes of several to several hundred nanometers or several tens of nanometers that is suitable for capturing viruses. The first base 25 and the second base 23 are stacked so as to sandwich these layers. The fourth layer 24 may include a nanostructure using a nanomaterial such as a carbon nanotube, and may be configured to be laminated to the third layer 28 or the second layer 27 without using the second substrate 23.

[0053] In the virus detection device (virus detection platform) 70, viruses contained in the sample 3 are captured by the fourth layer 24 of the chip 20, and by irradiating (focusing) the chip 20 with pump light 31 and Stokes light 32, CARS light (scattered light) related to the structure or components that make up the virus can be detected as scattered light (surface-enhanced Raman light, SERS) 33 enhanced on the surface (first surface) 21 sandwiched between the two substrates 25 and 23 of the chip 20. In the virus detection device 70, the presence or concentration of viruses can be optically detected. Therefore, the presence or concentration of viruses can be detected (measured) with high accuracy without the viruses being destroyed or the components that make up the viruses being mixed together during detection.

[0054] The virus detection device 70 includes an analysis unit (spectroscopic analysis unit) 30, and its basic configuration is the same as that of the analysis unit 30 included in the measurement device 1 described above. The virus detection device 70 can acquire SERS light 33 indicative of one or more previously identified viruses or unknown viruses, and can identify the presence and concentration of known viruses in real time by comparing their fingerprints. Furthermore, for unknown viruses, the type and danger of the virus can be determined more quickly by comparing the obtained fingerprint with the fingerprints of known viruses. This virus detection device 70 can be configured simply and compactly, and can be provided as an easily portable device.

[0055] The chip 20 may include a structure that facilitates obtaining SERS light 33 containing a fingerprint for identifying a virus in units of sectors (regions, segments) 29, or a structure that facilitates capturing the target virus in the fourth layer 24. The virus-capturing structure may be provided in the first layer 26 and / or the second layer 27. By forming the first layer 26 and the second layer 27, which are primarily intended to provide a configuration suitable for hotspots, and the fourth layer 24, which is primarily intended to capture viruses, as different layers, a chip 20 with a structure more suited to each purpose can be provided. Furthermore, by providing a structure that physically captures the target virus together with or separately from the affinity ligand 28a, target detection can be performed more quickly and accurately.

[0056] Instead of these configurations for capturing viruses, it is also possible to provide a cell analysis platform with a configuration for capturing specific cells, a bacterial analysis platform with a configuration for capturing specific bacteria, an antibody detection platform with a configuration for capturing specific proteins including antibodies, and a DNA analysis platform for capturing and analyzing genes.

[0057] Furthermore, while particular embodiments of the present invention have been described above, various other embodiments and modifications may be devised by those skilled in the art without departing from the scope and spirit of the present invention, and such other embodiments and modifications are within the scope of the following claims, which define the present invention.

Claims

1. An apparatus for detecting the presence and / or concentration of a target substance in a fluid sample, comprising: a chip including a metal body capable of exciting localized surface plasmon resonance on a first surface; an analysis unit that scans the first surface of the chip with a laser in at least one dimension while the first surface is in contact with the sample, and records enhanced scattered light at the first surface in association with the scan; The chip comprises a substrate and a first layer having a repeated uneven structure provided on the first surface of the substrate; a second layer including the metal bodies provided via the first layer, the second layer being configured such that the state between the metal bodies, the shape of the metal bodies, and the adhesion state of the measurement target substance contained in the sample change depending on the thickness direction of the chip due to affinity ligands attached to at least a part of the surface of the metal bodies, The analysis unit includes a unit that focuses at least two laser beams onto a common spot on the first surface, including in the thickness direction, and scans the second layer, including in the thickness direction, through the first layer of the chip, in which the uneven structure is repeatedly provided, and the second layer is configured so that the adhesion status of the substance to be measured changes depending on the thickness direction, and acquires the scattered light excited by the at least two laser beams from a hot spot scanned in the thickness direction of the chip.

2. In claim 1, The device, wherein the first layer and / or the second layer comprises a structure that captures microorganisms and / or proteins.

3. In claim 1 or 2, The device, wherein the chip includes a plurality of sectors having different configurations of the first layer and / or the second layer.

4. In any one of claims 1 to 3, The chip comprises a plurality of sectors in which the affinity ligands are different and attached to at least a portion of the surface of the metal body of the second layer.

5. In claim 3 or 4, The device wherein the chip includes a fourth layer comprising a structure that captures microorganisms and / or proteins.

6. In any one of claims 3 to 5, The apparatus, wherein the analyzing unit includes a unit that sequentially scans the plurality of sectors.

7. In any one of claims 1 to 6, The second layer has a metal body provided at each tip of a protrusion of the first layer, and includes an area where the distance between the metal bodies is narrower than the distance between the protrusions of the first layer.

8. In claim 7, The device, wherein the first layer includes regions where the intervals between the protrusions vary.

9. In any one of claims 1 to 8, The analysis unit includes a unit for irradiating the laser onto the first surface through the sample.

10. In any one of claims 1 to 9, The scattered light comprises coherent anti-Stokes Raman scattering (CARS) light, stimulated Raman scattering (SRS) light, or coherent Stokes Raman scattering (CSRS) light.

11. In any one of claims 1 to 10, The device further comprises a holder for holding or flowing a fixed quantity of said sample therein, said holder having said chip attached or embedded in a wall thereof.

12. In any one of claims 1 to 10, The device further comprises a route through which the sample containing wastewater from a living body flows, the route having the chip attached or embedded in a wall surface.

13. A method for detecting the presence and / or concentration of a target substance in a fluid sample using a chip, comprising: The chip includes a substrate, a first layer having a repeated uneven structure on a first surface of the substrate, and a second layer provided via the first layer and including metal bodies capable of exciting localized surface plasmon resonance, the second layer being configured such that the state between the metal bodies, the shape of the metal bodies, and an affinity ligand attached to at least a portion of the surface of the metal bodies cause an attachment state of the measurement target substance contained in the sample to change in the thickness direction of the chip; the detecting includes scanning the first surface in at least one dimension with a laser while the first surface is in contact with the sample, and recording enhanced scattered light at the first surface in association with the scanning; The recording step includes focusing at least two laser beams onto a common spot on the first surface, including in the thickness direction, and scanning the second layer, including in the thickness direction, through the first layer of the chip, in which the uneven structure is repeatedly provided, and the second layer is configured so that the adhesion status of the substance to be measured changes depending on the thickness direction, and acquiring the scattered light excited by the at least two laser beams from a hot spot scanned in the thickness direction of the chip.

14. In claim 13, the chip includes a plurality of sectors in which at least one of the first layer configuration and the second layer configuration is different; The method, wherein the recording includes scanning the plurality of sectors in sequence.

15. In claim 13, the chip includes a plurality of sectors that are different in at least one of the configuration of the first layer, the configuration of the second layer, and the affinity ligand; The method, wherein the recording includes scanning the plurality of sectors in sequence.

16. A program for an apparatus that detects the presence and / or concentration of a target substance in a fluid sample using a chip, comprising: The chip includes a substrate, a first layer having a repeated uneven structure on a first surface of the substrate, and a second layer provided via the first layer and including metal bodies capable of exciting localized surface plasmon resonance, the second layer being configured such that the state between the metal bodies, the shape of the metal bodies, and an affinity ligand attached to at least a portion of the surface of the metal bodies cause an attachment state of the measurement target substance contained in the sample to change in the thickness direction of the chip; The program is the apparatus having instructions for scanning the first surface in at least one dimension with a laser while the first surface is in contact with the sample, and recording enhanced scattered light at the first surface in association with the scanning; The recording step includes focusing at least two laser beams onto a common spot on the first surface, including in the thickness direction, scanning the second layer, including in the thickness direction, through the first layer of the chip, in which the uneven structure is repeatedly provided, and the second layer is configured so that the adhesion status of the substance to be measured changes depending on the thickness direction, and acquiring the scattered light excited by the at least two laser beams from a hot spot scanned in the thickness direction of the chip.

17. In any one of claims 1 to 12, The scattered light acquiring unit scans in three dimensions with the at least two laser beams.

18. In any one of claims 13 to 15, The method, wherein acquiring the scattered light includes scanning in three dimensions with the at least two laser beams.

19. In claim 16, The program, wherein acquiring the scattered light includes scanning in three-dimensional directions with the at least two laser beams.

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