JP2020153904A - Sensor substrate manufacturing method, sensor substrate, and detection device - Google Patents

Abstract

To provide a sensor substrate manufacturing method, etc., with which detection accuracy is improved.SOLUTION: Provided is a manufacturing method for a sensor substrate 100 having a metal layer 2 on a substrate 1, in which a localized surface plasmon resonance occurs, comprising: a binding step for binding a first coating molecule 9 onto the metal layer 2; and an immobilization step for binding a molecular recognition substance 10 that specifically binds to a prescribed specimen 20 to the first coating molecule 9 and immobilizing these. In the binding step, the metal layer 2 is irradiated with light of a wavelength that causes a localized surface plasmon resonance to occur, and the first coating molecule 9 is bound to a first region 11 on the metal layer 2 with higher density than in a second region 12 on the metal layer 2, electric field augmentation due to a localized surface plasmon resonance in the first region 11 being larger than electric field augmentation in the second region 12.SELECTED DRAWING: Figure 5

Description

The present disclosure relates to a method for manufacturing a sensor substrate for detecting a substance to be detected in a sample, for example, a pathogen or an allergen, a sensor substrate, and a detection device.

As a method for detecting a trace amount of a substance to be detected, there is a detection method using localized surface plasmon resonance generated on the surface of a metal nanostructure. In the detection method using localized surface plasmon resonance, the sensitivity of the substance to be detected is increased by utilizing the local electric field enhancement of tens to millions of times caused by the localized surface plasmon resonance. Detection methods using localized surface plasmon resonance include, for example, a surface-enhanced Raman scattering method capable of molecular-level analysis using a Raman scattering spectrum enhanced by localized surface plasmon resonance, and surface plasmon enhancement using an antigen-antibody reaction. Fluorescent immunoassay methods (see, for example, Patent Document 1) and the like. In the detection method of Patent Document 1, first, an antibody is immobilized as a molecular recognition functional substance on a sensor unit including a metal layer, and an antibody labeled with a fluorescent substance (hereinafter referred to as a labeled antibody) is bound thereto. The detected substance is bound. Then, by irradiating the sensor unit with excitation light that excites the plasmon in the metal layer, a photoelectric field enhanced by the plasmon (hereinafter referred to as an enhanced electric field) is generated. At this time, the amount of the substance to be detected can be detected by measuring the amount of fluorescence generated from the fluorescent substance of the labeled antibody existing in the enhanced electric field.

Further, Patent Document 2 describes a substrate having a dielectric on its surface, a plurality of metal particles formed on the dielectric, and an organic molecular film formed on the dielectric between the metal particles to which a target molecule is attached. The optical device having the above is disclosed. The gap between the metal particles is set to less than 10 nm in order to strengthen the enhanced electric field on the dielectric, and the inlet side of the gap between the metal particles is widened so that the target molecule can easily reach the organic molecular film on the dielectric. ..

JP-A-2010-0439334 Japanese Unexamined Patent Publication No. 2013-231637 Japanese Unexamined Patent Publication No. 2003-321479

However, the above-mentioned conventional technique may have insufficient detection sensitivity.

Therefore, the present disclosure provides a method for manufacturing a sensor substrate with improved detection sensitivity.

The method for manufacturing a sensor substrate according to one aspect of the present disclosure is a method for manufacturing a sensor substrate having a metal layer on which localized surface plasmon resonance occurs, and a first coating molecule is bonded onto the metal layer. The binding step includes a binding step of binding and immobilizing a molecular recognition functional substance that specifically binds to a predetermined sample to the first coating molecule, and in the binding step, the metal layer is subjected to the above. By irradiating light having a wavelength that causes localized surface plasmon resonance, the first coating molecule is bound to the first region on the metal layer at a density higher than that of the second region on the metal layer. The electric field enhancement due to the localized surface plasmon resonance in the first region is larger than the electric field enhancement in the second region.

The sensor substrate according to one aspect of the present disclosure is a sensor substrate having a metal layer on which localized surface plasmon resonance occurs, which is immobilized on the metal layer and specifically binds to a predetermined sample. The molecular recognition functional substance is provided, and the molecular recognition functional substance is present in the first region on the metal layer at a higher density than the second region on the metal layer, and the localized surface in the first region. The electric field enhancement by plasmon resonance is larger than the electric field enhancement in the second region.

The detection device according to one aspect of the present disclosure includes the sensor substrate, a light irradiation unit that irradiates light having a wavelength that causes localized surface plasmon resonance, and a light detection unit that detects a photoreaction signal from the sensor substrate. Has.

According to the present disclosure, a sensor substrate with improved detection sensitivity can be manufactured.

FIG. 1 is an electron microscope image of a nanostructure in which localized surface plasmon resonance occurs. FIG. 2 is a cross-sectional distribution map showing the distribution of the electric field intensity amplification factor obtained by simulation for the nanostructure in which localized surface plasmon resonance occurs. FIG. 3 is an observation image of observing the emission distribution when a fluorescence immunoassay is performed using a sensor substrate provided with a metal layer in which localized surface plasmon resonance occurs. FIG. 4 is a schematic view of the sensor substrate used for observing the light emission distribution shown in FIG. FIG. 5 is a schematic process diagram showing a method of manufacturing the sensor substrate according to the first embodiment. FIG. 6 is a schematic view showing a detection device using the sensor substrate according to the first embodiment. FIG. 7 is a process schematic diagram showing a method of manufacturing the sensor substrate according to the second embodiment. FIG. 8 is a process schematic diagram showing a method of manufacturing a sensor substrate according to a modified example of the second embodiment.

(Findings that led to this disclosure)
In localized surface plasmon resonance, free electrons on the surface of a metal nanostructure having a shape smaller than the wavelength of the irradiated light resonate with the electromagnetic field of the irradiated light and vibrate, resulting in a locally very strong electric field (hot). Site), that is, electric field enhancement occurs. This electric field enhancement decreases sharply away from the metal surface. When the metal nanostructures are adjacent to each other, the strongest electric field is generated in the gap between the adjacent metal nanostructures, and the smaller the distance between the adjacent metal nanostructures, the larger the electric field.

The prior art described in Patent Document 2 uses an optical device in which metal particles are provided on the surface of a dielectric with narrow gaps, and organic molecules that selectively capture a target are bonded between the metal particles on the dielectric. .. Since the organic molecule that captures the target is on the dielectric, if the gap between the metal particles on the dielectric becomes large, a strong electric field between the metal particles due to localized surface plasmon resonance cannot be obtained. Therefore, in the optical device of Patent Document 2, the gap between the metal particles is set to less than 10 nm. On the other hand, the size of target allergens derived from viruses and organisms, and organic molecules that capture targets such as antibodies is several nm to several hundred nm, and is often larger than 10 nm. Therefore, in Patent Document 2, the width of the metal particles on the dielectric becomes narrower as the height from the surface of the dielectric increases so that the target is inserted into the gap between the metal particles. In the optical device of Patent Document 2, (1) the metal particle shape has the above-mentioned design restrictions, (2) the place where the electric field is enhanced is not stable due to the shape error of the metal particle, and (3). If the width of the gap between the metal particles, which is less than 10 mm, varies, for example, even with an error of 1 nm, the electric field strength changes significantly and the detection performance varies greatly, which is a manufacturing problem.

In view of the above problems, the present inventor has diligently studied a highly sensitive sensor substrate and a method for manufacturing the same, in which the electric field enhancement by localized surface plasmon resonance works efficiently.

FIG. 1 is an electron microscope image (hereinafter referred to as an SEM image) of the nanostructure 45 in which localized surface plasmon resonance occurs at near infrared rays having a wavelength of 780 nm. 1 (a) is a plan view of the nanostructure 45, and FIG. 1 (b) is a cross-sectional view of the nanostructure 45 of FIG. 1 (a) cut along the line I-I. is there. In the nanostructure 45 shown in FIG. 1, a metal layer 62 made of gold is formed by vacuum film formation on a resin substrate 61 in which a large number of nano-sized fine protrusions 60 created by nanoimprint are arranged in six directions. As shown in the cross-sectional view of FIG. 1B, the metal layer 62 has a plurality of columnar convex portions 62a whose tips are slightly rounded in cross-sectional view. The plurality of convex portions 62a are lined up with a narrow gap in between. As shown in the plan view of FIG. 1A, the plurality of convex portions 62a have a substantially hexagonal shape in a plan view and are arranged in a hexagonal close-packed shape. The pitch of the plurality of convex portions 62a is 460 nm, the height of each of the plurality of convex portions 62a is a little over 400 nm, and the gap between the adjacent convex portions 62a is about 10 nm or more and 20 nm or less.

FIG. 2 shows a 3D model of the nanostructure, and a surface localized on the metal layer of the nanostructure on the nanostructure having a metal layer having a plurality of convex portions 70 (the gap between adjacent convex portions is 16 nm). It is a cross-sectional distribution diagram which shows the distribution of the electric field intensity amplification factor obtained by simulation at the time of irradiating light of 780 nm which is a wavelength which causes plasmon resonance (hereinafter, referred to as a plasmon resonance wavelength). The portion having the largest electric field enhancement is the region 71 of the narrow gap portion in the middle of the convex portion 70, and the electric field intensity amplification factor in the region 71 is a little over 300 times. Further, in the vicinity of the inlet where the gap between the two convex portions 70 is widened, that is, in the region 72 near the gap opening between the two convex portions 70, an electric field enhancement having an electric field strength amplification factor of less than 100 times occurs.

FIG. 3 is an observation image of observing the emission distribution when a fluorescence immunoassay is performed using a sensor substrate 200 having a nanostructure in which localized surface plasmon resonance occurs. Specifically, FIG. 3 is a plan view showing a state of light emission of the sensor substrate 200, and a white portion is shown as a light emitting point. Specifically, in FIG. 3, a VHH antibody, which is a small antibody, is bound as a molecular recognition functional substance on a substrate provided with a nanostructure made of a gold metal layer similar to the nanostructure 45 shown in FIG. The results of a sandwich-type fluorescence immunoassay were shown in which a labeled antibody was bound to the nuclear protein of influenza virus as a sample. In the observation of the luminescence distribution, the nanostructure after the assay is irradiated with light having a plasmon resonance wavelength of 780 nm using a STORM (Stochastic Optical Reaction Microscopy) type super-resolution microscope that irradiates the nanostructure with a laser beam having a plasmon resonance wavelength. There is. The white horizontal bar at the lower right shown in FIG. 3 is a 1 μm scale bar, and has a hexagonal mesh-like pitch having the same pitch of 460 nm as the pitch of the convex portion 62a of the SEM image shown in FIG. 1 (a). Light is observed, and it can be seen that the light emitting points are distributed in the vicinity of the gaps between the convex portions.

For comparison, a similar fluorescence immunoassay was performed using a laser beam with a wavelength of 488 nm that does not cause localized surface plasmon resonance in the metal layer and a labeled antibody that is excited at a wavelength of 488 nm and emits fluorescence. FIG. No pattern due to the mesh-like light as shown in is observed, and it is considered that the emission intensity of fluorescence is 1/100 or less of the case where localized surface plasmon resonance occurs. Therefore, the luminescence distribution shown in FIG. 3 does not indicate that the sample and the antibody are unevenly attached, but shows the distribution of fluorescence derived from the electric field enhancement due to the localized surface plasmon resonance. I understand.

Further, in the observation of the light emission distribution using FIG. 3, the position corresponding to the narrow gap portion of the region 71 shown in FIG. 2, which has the strongest electric field intensity, does not shine particularly strongly, and the light emission spreads in the vicinity of the gap portion. From this, it is considered that the electric field enhancing portion near the surface of the metal layer on the gap opening side of the convex portion, which corresponds to the region 72 shown in FIG. 2, mainly contributes to light emission.

FIG. 4 is a schematic view of the sensor substrate 200 used for observing the light emission distribution shown in FIG. As shown in FIG. 4, the molecular recognition functional substance 10 bound to the metal layer 62 via the coating molecule 65 and evenly distributed captures the sample 20 bound to the labeled antibody 21. When irradiated with light having a plasmon resonance wavelength, only the labeled antibody 21a in the vicinity of the gap opening of the convex portion 62a emits strong light.

As described above, the present inventor used a nanostructure in which substantially hexagonal convex portions 62a as shown in FIG. 1 are adjacently arranged in a uniform hexagonal close-packed form with a gap of 10 nm or more and 20 nm or less. We created a sensor board and confirmed that high-sensitivity detection is possible. However, when irradiating light with a plasmon resonance wavelength, the narrower the gap between the adjacent convex portions 62a, the higher the electric field intensity, but when the gap is set to less than 10 nm, the gap between the convex portions 62a due to the variation of the gap. There is also a part where is lost, and the reproducibility of detection cannot be obtained. Therefore, the pitch and height of the convex portions 62a are preferably about half the wavelength of the light used for detection, and it is possible to create a gap between the convex portions 62a having a height exceeding 100 nm with a variation of several nm or less. It is difficult to change the construction method, and the gap between the adjacent convex portions 62a needs to be at least 10 nm or more. On the other hand, when the gap between the adjacent convex portions 62a is 40 nm or less, the effect of enhancing the electric field by the localized surface plasmon resonance is secured at a significant level. Therefore, in order to obtain a sensor substrate capable of high-sensitivity detection, the gap between the convex portions 62a is preferably 10 nm or more and 40 nm or less.

As described above, it was found that a highly sensitive sensor substrate can be produced by shaping the nanostructure that causes localized surface plasmon resonance into an appropriate shape. On the other hand, as shown in FIGS. 2 to 4, the labeled antibody 21 that causes the detected luminescence is limited to the labeled antibody 21 located near the gap on the distal end side of the convex portion 62a, and other than that. The labeled antibody 21 in the region of is not contributing to the detection. Based on these findings, we have diligently studied the configuration of the sensor substrate and its manufacturing method, in which localized surface plasmon resonance acts more efficiently and stably and the detection accuracy is improved. , Disclosed in the following embodiments.

The method for manufacturing a sensor substrate according to one aspect of the present disclosure is a method for manufacturing a sensor substrate having a metal layer on which localized surface plasmon resonance occurs, and a first coating molecule is bonded onto the metal layer. The binding step includes a binding step of binding and immobilizing a molecular recognition functional substance that specifically binds to a predetermined sample to the first coating molecule, and in the binding step, the metal layer is subjected to the above. By irradiating light having a wavelength that causes localized surface plasmon resonance, the first coating molecule is bound to the first region on the metal layer at a density higher than that of the second region on the metal layer. The electric field enhancement due to the localized surface plasmon resonance in the first region is larger than the electric field enhancement in the second region.

As a result, even if the distribution of the region where the electric field is enhanced by the localized surface plasmon resonance varies depending on the shape of the metal layer in which the localized surface plasmon resonance occurs, the localized surface is formed on the metal layer during the manufacturing process. By irradiating light with a wavelength that causes plasmon resonance, the first coating molecule is bound to the first region, which has a larger electric field enhancement than the second region, at a density higher than that of the second region. Since the molecular recognition functional substance is immobilized on the first coated molecule, the molecular recognition functional substance is also immobilized on the first region where the electric field enhancement is large at a higher density than the second region. Therefore, it is possible to manufacture a sensor substrate in which the sample is easily captured and the detection sensitivity is improved in the first region where the electric field enhancement is large.

Further, in the method for manufacturing a sensor substrate according to one aspect of the present disclosure, in the binding step, one end is bonded to the metal layer and the other end is not suitable for the molecular recognition functional substance and the sample. The second step of binding the active second coating molecule to the metal layer and the second bonding of the metal layer to the metal layer by irradiating the metal layer with light having a wavelength that causes the localized surface plasmon resonance. Among the coated molecules, the step of removing the second coated molecule of the first region and the step of binding the first coated molecule to the first region from which the second coated molecule has been removed may be included. Good.

As a result, the molecular recognition functional substance and the second coated molecule that is inactive with respect to the sample are bound to the second region where the electric field enhancement is smaller than that of the first region. Therefore, the molecular recognition functional substance is less likely to be immobilized in the second region, and the capture of the sample in the second region is suppressed. Therefore, it is possible to manufacture a sensor substrate in which the possibility of capturing a sample is high and the detection sensitivity is further improved in the first region where the electric field enhancement is large.

Further, in the method for producing a sensor substrate according to one aspect of the present disclosure, the first coated molecule has a bonding group that binds to the metal layer at one end and binds to the molecule recognition functional substance at the other end. In the bonding step, the binding group of the first coating molecule or a precursor obtained by protecting the functional group with a photodissociable protective group is placed on the metal layer, and the metal layer is subjected to the treatment. The first coated molecule in which the photodissociable protective group of the precursor is dissociated may be bound to the first region by irradiating with light having a wavelength that causes the localized surface plasmon resonance.

As a result, the metal layer is irradiated with light having a wavelength that causes localized surface plasmon resonance, the photodissociative protecting group is dissociated from the precursor, and the first coating molecule is bound to the first region where the electric field enhancement is large. .. Since the object to be protected and the conditions for dissociating the photodissociable protecting group can be changed depending on the type of the photodissociable protecting group, it becomes easy to manufacture a sensor substrate with improved detection sensitivity.

Further, in the method for producing a sensor substrate according to one aspect of the present disclosure, the binding group of the precursor is protected by the photodissociating protecting group, and in the binding step, the solution containing the precursor is used. The first, in which the photodissociative protecting group of the precursor is dissociated by irradiating the metal layer with light having a wavelength that causes localized surface plasmon resonance in contact with the metal layer. The coating molecule may be attached to the first region.

As a result, the first region with large electric field enhancement can be achieved by a simple operation of irradiating the metal layer with light having a wavelength that causes localized surface plasmon resonance while the solution containing the precursor is in contact with the metal layer. The first coating molecule binds to. Therefore, a sensor substrate with improved detection sensitivity can be easily manufactured.

Further, in the method for manufacturing a sensor substrate according to one aspect of the present disclosure, in the bonding step, after the first coating molecule is bonded to the first region, one end is bonded to the metal layer and the other. At the end, the molecular recognition functional substance and a second coated molecule that is inactive with respect to the sample may be bound to the second region.

As a result, the second coated molecule, which is inactive for the molecular recognition functional substance, binds to the second region, which has a smaller electric field enhancement than the first region. Therefore, the molecular recognition functional substance is less likely to be immobilized in the second region, and the capture of the sample in the second region is suppressed. Therefore, it is possible to manufacture a sensor substrate in which the possibility of capturing a sample is high and the detection sensitivity is further improved in the first region where the electric field enhancement is large.

Further, in the method for producing a sensor substrate according to one aspect of the present disclosure, the functional group of the precursor is protected by the photodissociable protecting group, and in the bonding step, the precursor is placed on the metal layer. Even if the photodissociative protecting group is dissociated from the precursor bonded to the first region by irradiating the metal layer with light having a wavelength that causes the localized surface plasmon resonance. Good.

As a result, with the precursor bonded on the metal layer, the metal layer is irradiated with light having a wavelength that causes localized surface plasmon resonance, and the first region has a large electric field enhancement. The coating molecules bind. Therefore, a sensor substrate with improved detection sensitivity can be easily manufactured. In addition, a precursor having a protected functional group that binds to a molecular recognition functional substance binds to the second region, which has a smaller electric field enhancement than the first region. Therefore, the molecular recognition functional substance is less likely to be immobilized in the second region, and the capture of the sample in the second region where the electric field enhancement is small is suppressed. Therefore, it is possible to manufacture a sensor substrate with further improved detection sensitivity.

Further, the sensor substrate according to one aspect of the present disclosure is a sensor substrate having a metal layer on which localized surface plasmon resonance occurs, and is immobilized on the metal layer to be specific to a predetermined sample. The molecular recognition functional substance is provided to be bound, and the molecular recognition functional substance is present in the first region on the metal layer at a higher density than the second region on the metal layer, and the station in the first region. The electric field enhancement due to surface plasmon resonance is larger than the electric field enhancement in the second region.

As a result, since the molecular recognition functional substance is present in the first region where the electric field enhancement is larger than that in the second region at a density higher than that in the second region, it becomes easy to capture the sample in the first region. Therefore, the detection sensitivity of the sensor substrate can be improved.

Further, in the sensor substrate according to one aspect of the present disclosure, one end is bound to the metal layer, the other end is a first coated molecule that is bound to the molecular recognition functional substance, and one end is the metal layer. The other end comprises the molecular recognition functional substance and a second coated molecule that is inactive with respect to the sample, and the first coated molecule has a higher density in the first region than in the second region. The second coated molecule is bound to the second region at a higher density than that of the first region, and the molecular recognition functional substance is bound to the first coated molecule. May be good.

As a result, in the second region where the electric field enhancement is smaller than that in the first region, the molecular recognition functional substance and the second coated molecule which is inactive for the sample are present at a higher density than the first region. Specimen capture is suppressed. Further, in the first region where the electric field enhancement is large, the first coated molecule bound to the molecular recognition functional substance is bound at a higher density than the second region. Therefore, there is a high possibility that the sample will be captured in the first region, and the detection sensitivity of the sensor substrate can be further improved.

Further, in the sensor substrate according to one aspect of the present disclosure, the molecular recognition functional substance is an antibody, and the metal layer is made of gold, silver, or an alloy containing gold or silver as a main component, and the first coating The molecule has a thiol group at one end, a functional group that binds to the molecular recognition functional substance at the other end, a repeating sequence of hydrophilic groups that connect to the functional group, and is on the metal layer. The second coated molecule has a thiol group at one end and a hydroxyl group at the other end, and has a repeating sequence of hydrophilic groups connected to the hydroxyl group. It may have and form a self-assembled monomolecular film on the metal layer.

As a result, the metal layer and the first coating molecule, and the metal layer and the second coating molecule are easily bonded, and a self-assembled monolayer is formed on the metal layer. Therefore, since the first-coated molecule and the second-coated molecule exist as a self-assembled monolayer on the metal layer, non-specific adsorption of the sample to the metal layer is suppressed. Therefore, the detection sensitivity of the sensor substrate can be further improved.

Further, the detection device according to one aspect of the present disclosure includes the sensor substrate, a light irradiation unit that irradiates light having a wavelength that causes localized surface plasmon resonance, and light that detects a photoreaction signal from the sensor substrate. It has a detection unit.

As a result, since the sensor substrate with improved detection sensitivity is used, the detection accuracy can be improved.

Hereinafter, embodiments will be specifically described with reference to the drawings.

It should be noted that all of the embodiments described below show comprehensive or specific examples. The numerical values, shapes, materials, components, arrangement positions and connection forms of the components, processes, order of processes, etc. shown in the following embodiments are examples, and do not intend to limit the scope of claims. Further, among the components in the following embodiments, the components not described in the independent claims indicating the highest level concept are described as arbitrary components.

Moreover, each figure is not necessarily exactly illustrated. In each figure, substantially the same configuration is designated by the same reference numerals, and duplicate description is omitted or simplified.

(Embodiment 1)
First, the method of manufacturing the sensor substrate according to the first embodiment will be described with reference to FIG. FIG. 5 is a process schematic diagram showing a method of manufacturing the sensor substrate 100 according to the first embodiment. The sensor substrate 100 is a sensor substrate having a metal layer 2 in which localized surface plasmon resonance occurs, and includes a molecular recognition functional substance 10 that is immobilized above the metal layer 2 and specifically binds to a predetermined sample.

In the method for manufacturing the sensor substrate 100 according to the first embodiment, the nanostructure 40 in which the metal layer 2 in which the localized surface plasmon resonance occurs is formed on the substrate 1 is used.

The metal layer 2 is composed of a metal such as gold, silver, and aluminum, and may be composed of gold, silver, or an alloy containing gold or silver as a main component. The metal layer 2 has a plurality of convex portions 2a. The plurality of convex portions 2a are arranged adjacent to each other with the gap 3 interposed therebetween. In FIG. 5, only two convex portions 2a out of the plurality of convex portions 2a are shown, and the other convex portions are omitted, but the number of convex portions 2a is not particularly limited. .. Specifically, for example, as shown in the SEM image of FIG. 1, a nanostructure in which a large number of convex portions made of gold are densely packed in six directions is preferable. The plasmon resonance wavelength varies depending on the type of metal used for the metal layer and the shape of the nanostructure 40, and can be designed by simulation or the like. The pitch of the convex portion 2a in the case of the periodic structure is about ½ of the plasmon resonance wavelength. Further, as described above, the gap 3 between the adjacent convex portions 2a is preferably 10 nm or more and 40 nm or less. For example, in the nanostructure having the shape shown in FIG. 1, when the pitch of the convex portion is 460 nm and the height is about 400 nm, the plasmon resonance wavelength is about 780 nm. Further, even in the same nanostructure, the plasmon resonance wavelength slightly shifts depending on the refractive index of the medium in contact with the metal and the incident angle of light. Therefore, the absorption spectrum of the nanostructure is measured to measure the wavelength and incident angle of light. Should be adjusted as appropriate.

After washing such a nanostructure 40 with a detergent, oxygen plasma, or the like, as shown in FIG. 5A, the nanostructure 40 is placed in an aqueous solution 4 containing a second coating molecule 5, and the aqueous solution 4 is placed in a metal layer. 2 is contacted at room temperature to 40 ° C. for a certain period of time (for example, 16 hours at room temperature or 1 hour at 40 ° C.).

The second coated molecule 5 is a molecule in which one end is bound to the metal layer and the other end is inactive with respect to the molecular recognition functional substance and the sample. Specifically, the second coated molecule 5 has a bonding group such as a thiol group or a phosphonic acid group that binds to a metal at one end, and a hydroxyl group that is inactive with respect to a molecular recognition functional substance at the other end. It is preferable that the molecule has a terminal group of the above and forms a self-assembled monolayer (hereinafter referred to as SAM film). Further, in the second coated molecule 5, it is preferable that the repeating sequence of the hydrophilic group, which is inactive for other proteins including the sample to be detected, is connected to the terminal group. Examples of the repeating sequence of the hydrophilic group include a repeating sequence of an ethylene glycol group, a phospholipid group, and the like. The second coating molecule 5 may be, for example, Hydroxy-EG _3- undecanetic (EG is an ethylene glycol group).

By bringing the aqueous solution 4 into contact with the metal layer 2, as shown in FIG. 5B, the second coating molecule 5 binds to the metal layer 2 to form a SAM film containing the second coating molecule 5. ..

Next, as shown in FIG. 5 (c), when the metal layer 2 is irradiated with monochromatic light 6 such as laser light having a plasmon resonance wavelength, an electric field enhancement is generated in the first region 11 near the gap portion of the convex portion 2a. Occurs. The bond between the metal layer 2 and the second coated molecule 5, for example, the gold-sulfur bond between gold and the thiol group of the second coated molecule 5, is 5 W / cm ² visible or near infrared laser in a nitrogen atmosphere. It is known that the bond is cleaved by the oxidation of the thiol group by irradiation. Therefore, by irradiating a laser beam having a plasmon resonance wavelength with an intensity that breaks the bond between the metal layer 2 and the second coating molecule 5 due to the enhancement of the electric field by the localized surface plasmon resonance, the metal layer 2 is bonded to the first region 11. The bond between the second coated molecule 5 and the metal layer 2 is selectively cleaved. As a result, the second coated molecule 5 bound to the first region 11 on the metal layer 2 is removed, and as shown in FIG. 5D, the first region 11 whose electric field is enhanced by the localized surface plasmon resonance. The second coated molecule 5 of the above is removed, and the second coated molecule 5a bound to the second region 12 whose electric field enhancement is smaller than that of the first region 11 remains undamaged. That is, since the electric field enhancement by the localized surface plasmon resonance of the first region 11 is larger than the electric field enhancement of the second region 12, the second coating molecule 5 bound to the first region 11 on the metal layer 2 is removed. The second coated molecule 5a bound to the second region 12 on the metal layer 2 remains.

For example, in the nanostructure shown in FIG. 1, the electric field intensity amplification factor is a little less than 100 times, so that the nanostructure is immediately washed with water after being irradiated with a laser beam of 50 to 100 mW / cm ² at 780 nm, which is a plasmon resonance wavelength, for 1 minute. As a result, the second coating molecule in the first region is selectively removed. Even if the shape or material of the nanostructure is different, selective removal is possible by irradiating light with a plasmon resonance wavelength at an intensity that takes into consideration the electric field intensity amplification factor. When silver or aluminum is used as the metal layer, the plasmon resonance wavelength can be shortened to the ultraviolet region. In this case, by irradiating the second coating molecule on the metal layer with a laser beam having a wavelength in the ultraviolet region in water, the metal-sulfur bond is broken and the second coating molecule in the first region is selectively removed. ..

Next, as shown in FIG. 5 (e), the metal layer 2 is filled with an aqueous solution 8 such as a phosphoric acid buffer solution containing the first coating molecule 9, and the aqueous solution 8 is applied to the metal layer 2 at room temperature to 40 ° C. Contact for a period of time (eg, 16 hours at room temperature or 1 hour at 40 ° C.).

The first coated molecule 9 is a molecule in which one end is bound to a metal layer and the other end is bound to a molecular recognition functional substance. Specifically, the first coated molecule 9 has a binding group that binds to a metal such as a thiol group or a phosphonic acid group at one end, and a carboxy group that binds to a molecular recognition functional substance such as an antibody at the other end. , Amino group, or a molecule having a functional group such as an epoxy group and forming a SAM film. The first coating molecule 9 may be, for example, Carboxy-EG _6- undequanethial. For example, a carboxy group is generally used as a functional group that binds to an amino group of a protein, and a protein is passed through an NHS ester obtained by reacting an N-Hydroxy succiniumide (hereinafter referred to as NHS) with a carboxy group. Reacts with and binds to the amino group of. If an unreacted carboxy group remains, it is ionized in water to cause non-specific adsorption. Therefore, as the first coating molecule 9, Carboxy-EG _6- undekanethial and the second coating molecule 5 are used in the aqueous solution 8. The density of the carboxy group may be adjusted by mixing with Hydroxy-EG _3- undekanethyl to adjust the density of the first coating molecule 9 bound to the first region 11.

By bringing the aqueous solution 8 into contact with the metal layer 2 for a certain period of time, as shown in FIG. 5 (f), the first coated molecule is formed in the first region 11 on the metal layer 2 from which the second coated molecule 5 has been removed. 9 binds to form a SAM film containing the first coated molecule 9. That is, the first coating molecule 9 binds to the first region 11a, which has a larger electric field enhancement due to localized surface plasmon resonance than the second region 12, at a density higher than that of the second region 12.

By binding the functional group of the first coated molecule 9 and the molecular recognition functional substance 10 such as an antibody by a chemical reaction such as the method via the NHS ester described above, the molecule is shown in FIG. 5 (g). The sensor substrate 100 according to the first embodiment, wherein the first coated molecule 9 to which the recognition functional substance 10 is bound is selectively bound to the first region 11a near the gap opening of the adjacent convex portion 2a on the metal layer 2. Is created. That is, the sensor substrate 100 in which the molecular recognition functional substance 10 exists in the first region 11a at a higher density than that in the second region is created.

When the gap between the adjacent convex portions 2a is, for example, 10 nm or more and 20 nm or less, the size of the molecular recognition functional substance 10 is often larger than the gap between the adjacent convex portions 2a. Therefore, the molecular recognition functional substance 10 cannot enter the gap between the adjacent convex portions 2a, and therefore, as shown in FIG. 5 (g), the first region 11b of the convex portion 2a close to the substrate 1 is bonded. The molecular recognition functional substance 10 is not bound to the coated molecule 9. When the size of the molecular recognition functional substance 10 is smaller than the size of the gap between the adjacent convex portions 2a, the molecular recognition functional substance 10 is also bound to the first coated molecule 9 bound to the first region 11b. You may be.

As described above, the method for manufacturing the sensor substrate 100 according to the first embodiment utilizes the electric field enhancement by the localized surface plasmon resonance to generate the electric field enhancement in the vicinity of the gap (first region) of the adjacent convex portion 2a. The bonding state between the metal layer 2 of 11) and the coating molecule on the metal layer 2 is locally and selectively changed. As a result, even if the height of the nanostructure, the width and shape of the gaps vary during manufacturing, and the distribution of the electric field enhancement of the localized surface plasmon resonance varies, it is selective and local to the first region 11 where the electric field enhancement is large. There is a remarkable effect that the molecular recognition functional substance 10 can be immobilized.

In the sensor substrate 100 according to the first embodiment manufactured in this manner, the molecular recognition functional substance 10 has an electric field enhancement on the metal layer 2 due to localized surface plasmon resonance, which is larger than that of the second region 12. Is unevenly distributed in. As a result, the sample is captured in the first region 11 where the electric field enhancement due to the localized surface plasmon resonance occurs on the tip side of the convex portion 2a to which the sample molecule of the polymer such as protein can approach, so that the sample is detected. Sensitivity is improved. Therefore, it is possible to realize a highly sensitive sensor substrate capable of stably exerting the effect of strong electric field enhancement caused by localized surface plasmon resonance.

Further, in the second region 12 near the center of the plurality of convex portions 2a on the distal end side of the sensor substrate 100, the metal layer 2 is a SAM film containing the molecular recognition functional substance 10 and the second coated molecule 5a inactive with respect to the sample. Is covered. That is, the second coated molecule 5a is bound to the second region 12 at a higher density than that of the first region 11. On the other hand, the first coated molecule 9 is bound to the first region 11a at a higher density than that of the second region 12. As a result, in the second region 12, where the electric field enhancement is smaller than that of the first region 11, it becomes difficult to adsorb the molecular recognition functional substance 10 and samples such as other proteins. Therefore, the molecular recognition functional substance 10 is placed in the vicinity of the gap opening. It can be easily unevenly distributed in one region 11 and non-specific adsorption can be reduced.

The gap between the adjacent convex portions 2a is preferably 10 nm or more from the viewpoint of reducing the influence of variations in manufacturing, and is preferably 40 nm or less from the viewpoint of ensuring the enhancement of the electric field.

Next, a method of detecting a sample such as a pathogen and an example of a detection device will be described using the sensor substrate 100 according to the first embodiment manufactured in this manner. FIG. 6 is a schematic view showing a detection device 300 using the sensor substrate 100 according to the first embodiment. The detection device 300 includes a sensor substrate 100, a light irradiation unit 30, a beam splitter 32, and a light detection unit 33.

First, as shown in FIG. 6A, the sensor substrate 100 manufactured in the schematic process diagram shown in FIG. 5 is placed in a well (not shown), and a labeled antibody labeled with a phosphoric acid in advance is provided. A test solution 19 obtained by mixing 21 and a sample 20 which is a substance to be detected such as a virus in a phosphate buffer solution (hereinafter referred to as PBS) is put into a well, and the test solution 19 is brought into contact with the sensor substrate 100. A part of the sample 20 binds to the labeled antibody 21 in the test solution 19 to form composite particles 22. The composite particle 22 containing the sample 20 approaches the surface of the metal layer 2 of the sensor substrate 100 by Brownian motion, and does not bind to the second coated molecule 5a that is not bound to the molecular recognition functional substance 10, but the first coating. It binds to the molecular recognition functional substance 10 bound to the molecule 9. By washing with PBS after leaving it for a sufficient time, the labeled antibody 21 and the sample 20 which are not bound to the molecular recognition functional substance 10 are washed away, and as shown in FIG. 6 (b), the metal is washed away. The sample 20 of the composite particle 22 is bound to the molecular recognition functional substance 10 which is unevenly distributed in the first region 11a on the layer 2.

Then, when the sensor substrate 100 is irradiated with the monochromatic light 31 via the beam splitter 32 by the light irradiation unit 30 such as a semiconductor laser that emits light having a plasmon resonance wavelength, the composite particles captured in the first region 11 where the electric field enhancement is large. The labeled antibody 21 of 22 emits strong fluorescence, and the sample 20 can be detected with high sensitivity by detecting the fluorescence with a photodetector 33 such as a photosensor that detects this fluorescence as a photoreaction signal.

As the light irradiation unit 30, known techniques can be used without particular limitation, and for example, a laser light source such as a semiconductor laser or a gas laser can be used. As the light irradiation unit 30, a light source that irradiates excitation light having a wavelength that has a small interaction with the sample 20 may be used.

As the light detection unit 33, a known technique can be used without particular limitation as long as it can disperse light in a specific wavelength band and can detect it. For example, it transmits light in a specific wavelength band in order to disperse light. It is possible to use an interference filter, a Zellny turner type spectroscope that disperses using a diffraction lattice, an Echel type spectroscope, and the like.

Using the nanostructure shown in FIG. 1, the first coated molecule, the second coated molecule, and the molecular recognition functional substance described above, the sensor substrate according to the first embodiment is manufactured by the manufacturing method shown in FIG. , A sandwich-type fluorescent immunoassay using a labeled antibody and a sample (influenza virus nuclear protein) was performed. As shown in FIG. 4, the molecular recognition functional substance was evenly distributed on the metal layer, and the conventional method was used. Compared with the case where the same sandwich type fluorescence immunoassay was performed on the manufactured sensor substrate, it was possible to obtain an optical signal intensity several tens of times higher. Further, in the sensor substrate according to the first embodiment, the noise due to non-specific adsorption generated in the test solution containing no sample is reduced to less than a fraction of that of the sensor substrate manufactured by the conventional method, and is 100. It was confirmed that it was possible to achieve more than double the sensitivity.

In this embodiment, an example of detection by the fluorescence immunoassay method has been described, but the same effect can be obtained if the method is not limited to this and the detection is performed by localized surface plasmon resonance such as the surface plasmon enhancement Raman method. is there. The case of an antibody has been described as an example of a molecular recognition functional substance, but the case is not limited to this, and proteins such as protein A / G, surface-modified nanoparticles, and the like can also be used.

(Embodiment 2)
Next, a method of manufacturing the sensor substrate according to the second embodiment will be described with reference to FIG. 7. In the following description of the second embodiment, the differences from the first embodiment will be mainly described, and the description of the common points will be omitted or simplified. In the method for manufacturing a sensor substrate according to the second embodiment, the localized surface plasmon resonance is used to apply the first coated molecule to the first region where the electric field is enhanced before the second coated molecule is bonded onto the metal layer. Combine locally.

FIG. 7 is a process schematic diagram of a method for manufacturing the sensor substrate 110 according to the second embodiment. In the method for manufacturing the sensor substrate 110 according to the second embodiment, a metal layer 2 having a structure in which a plurality of convex portions 2a are arranged adjacent to each other with a gap thereof is formed on the substrate 1 as in the first embodiment. The nanostructure 40 is used. As shown in FIG. 7A, the nanostructure 40 is immersed in the reaction solution 50 containing the precursor of the first coating molecule 9, and is irradiated with monochromatic light 51 having a plasmon resonance wavelength. The reaction solution 50 is a solution in which the precursor of the first coating molecule 9 is dissolved in a solvent such as water or ethanol. Specifically, the precursor contained in the reaction solution 50 is the first coated molecule 9 having a linking group that binds to the metal layer at one end and a functional group that binds to the molecular recognition functional substance at the other end. , A molecule whose binding group is protected by a photodissociative protective group. The first coated molecule 9 is a molecule similar to that of the first embodiment, and may be, for example, Carboxy-EG _6- undekanethial. Then, the thiol group, which is a binding group, is bonded to a photodissociative protecting group, for example, a nitrobenzyl group or a coumarin derivative group to synthesize a so-called caged compound, and a precursor of the first coating molecule 9 is obtained. A method for synthesizing a caged compound is described in, for example, Japanese Patent Application Laid-Open No. 2003-321479 (Patent Document 3), and a photodissociative protecting group-introducing agent used in the reaction is sold by Tokyo Chemical Industry Co., Ltd. and the like. ing.

For example, when Carboxy-EG _6- undekanethyl is used as the first coating molecule 9, when the first coating molecule 9 is reacted with 2-nitrobenzyl bromide, which is a photodissociable protecting group-introducing agent, the following reaction formula ( By the reaction of 1), the bromine of 2-nitrobenzyl bromide is eliminated and combined with the sulfur of Carboxy-EG _6- undekanethyl to obtain the precursor shown on the right side of the following reaction formula (1). As described above, since the precursor has a structure in which the binding group of the first coating molecule 9 is protected, it is less likely to react with the metal layer than the first coating molecule 9 before protection, and is inactive with respect to the metal layer. It is a molecule.

HOOC-EG _6- (CH ₂ ) _11- SH + Br-CH _2- C ₆ H ₄ NO ₂
→ HOOC-EG _6- (CH ₂ ) _11- S-CH _2- C ₆ H ₄ NO ₂ (1)
HOOC-EG _6- (CH ₂ ) _11- S-CH _2- C ₆ H ₄ NO ₂
→ HOOC-EG _6- (CH ₂ ) _11- SH + OHC-C ₆ H ₄ NO (2)

When the obtained precursor is irradiated with light having a wavelength in the ultraviolet to near-ultraviolet region, the nitrobenzyl group is dissociated as shown in the above reaction formula (2), and Carboxy-EG ₆ which is the first coating molecule 9 is dissociated. -Benzyl is generated and can be bonded to metals such as gold.

Normally, the reaction represented by the reaction formula (2) is caused by irradiation with light having a wavelength in the ultraviolet to near-ultraviolet region, but when the light intensity is strong, it is caused by a two-photon reaction in the visible to near-infrared region. It also occurs when irradiated with light of a wavelength. As shown in FIG. 7A, when monochromatic light 51 having a plasmon resonance wavelength is irradiated, an electric field enhancement occurs in the first region 11 near the gap portion of the convex portion 2a. Therefore, when the light having a wavelength in the visible light region to the near infrared region is irradiated, the reaction of the reaction formula (2) proceeds in the first region 11 where the electric field enhancement is large, and the photodissociative protecting group is dissociated from the precursor. Then, in the first region 11, the first coating molecule 9, Carboxy-EG _6- underkanethial, is generated.

Then, as shown in FIG. 7 (b), the first coating molecule 9 is bound to the first region 11 on the metal layer 2, and a SAM film containing the first coating molecule 9 having a functional group at the end is formed. Will be done. When the photodissociative protecting group is dissociated from the precursor and the first coating molecule 9 is bonded to the first region 11 on the metal layer 2, laser light of 300 mW / cm ² or more and 1 W / cm ² or less is applied to the metal layer 2. It is recommended to irradiate for about 10 minutes.

Next, as shown in FIG. 7 (c), a second coated molecule 5 having one end bonded to the metal layer 2 and the other end inactive with respect to the molecular recognition functional substance 10, for example, is carried out. An aqueous solution 53 containing the same Hydroxy-EG _6- underkanethyl as in Form 1 of No. 1 is brought into contact with the metal layer 2 for a certain period of time. When the aqueous solution 53 is brought into contact with the metal layer 2 for a certain period of time, as shown in FIG. 7D, the first coating molecule 9 is bound because the electric field enhancement due to the plasmon resonance wavelength is smaller than that of the first region 11. The second coated molecule 5a is bound to the second region 12 which is not covered, and a SAM film containing the second coated molecule 5a is formed.

Then, by binding a functional group such as a carboxy group of the first coated molecule 9 and a molecular recognition functional substance 10 such as an antibody by a chemical reaction such as the method via the NHS ester described above, (e) of FIG. 7 is obtained. As shown, the sensor substrate 110 according to the second embodiment, wherein the molecular recognition functional substance 10 is unevenly distributed in the first region 11a near the gap opening of the adjacent convex portion 2a, which has a larger electric field enhancement than the second region 12. Is manufactured.

In the sensor substrate 110 produced by the above manufacturing method, the molecular recognition functional substance 10 is locally bound to the first region 11a in which the electric field enhancement due to the localized surface plasmon resonance is large, as in the first embodiment. Therefore, highly sensitive detection is possible.

(Modification example)
Next, a method of manufacturing the sensor substrate according to the modified example of the second embodiment will be described with reference to FIG. The method for manufacturing the sensor substrate according to the modified example of the second embodiment uses a precursor in which the functional group of the first coating molecule is protected by a photodispersible protecting group.

FIG. 8 is a process schematic diagram of a method of manufacturing the sensor substrate 120 according to the modified example of the second embodiment. In the method for manufacturing the sensor substrate 120 according to the modified example of the second embodiment, a metal layer 2 having a structure in which a plurality of convex portions 2a are arranged adjacent to each other with a gap thereof is provided on the substrate 1 as in the first embodiment. The formed nanostructure 40 is used.

As shown in FIG. 8A, the aqueous solution 55 is placed in the aqueous solution 55 containing the precursor 56, and the aqueous solution 4 is brought into contact with the metal layer 2 at room temperature overnight.

The precursor 56 according to the modified example of the second embodiment is a precursor of the first coated molecule 9 in which the functional group of the first coated molecule 9 is protected by a photodispersible protecting group. By changing the type of the photodissociative protecting group-introducing agent from the precursor according to the second embodiment, not the binding group of the first coating molecule 9 but the functional group is protected. For example, when Carboxy-EG _6- undekanethiol is used as the first coated molecule 9, the functional group is formed by reacting the first coated molecule 9 with (2-nitrophenyl) diazomethane as a photodissociative protecting group introducer. A precursor 56 having a protected carboxy group is obtained. As described above, since the precursor 56 has a structure in which the functional group of the first coated molecule 9 is protected, it becomes a molecular recognition functional substance that is less likely to bind to the molecular recognition functional substance than the first coated molecule 9 before protection. On the other hand, it is an inactive molecule.

By bringing the aqueous solution 55 into contact with the metal layer 2 for a certain period of time, the precursor 56 is uniformly bonded onto the metal layer 2 as shown in FIG. 8 (b), and a SAM film containing the precursor 56 is formed. To.

As shown in FIG. 8 (c), when monochromatic light 57 having a plasmon resonance wavelength is irradiated, an electric field enhancement occurs in the first region 11, and a photodissociative protecting group is dissociated from the precursor 56. As a result, as shown in FIG. 8D, the first coated molecule 9 is generated in the first region 11, and the SAM film containing the first coated molecule 9 is formed. A SAM film containing a functional group-protected precursor 56a remains in the second region 12, which has a smaller electric field enhancement than the first region 11. As shown in FIG. 8 (e), by binding a molecular recognition functional substance 10 such as an antibody to the carboxy group of the first coating molecule 9, the first region 11a near the gap opening of the adjacent convex portion 2a A molecular recognition functional substance 10 such as an antibody can be bound to. However, in this case, the photodissociative protecting group does not dissociate because the precursor 56a having the functional group protected by the photodissociative protecting group remains in the second region 12 where the electric field enhancement is smaller than that of the first region 11a. As described above, it is necessary to protect the sensor substrate 120 with an ultraviolet cut filter or the like.

As described above, in the second embodiment and the production method according to the second embodiment, a precursor in which the binding group or functional group of the first coating molecule is protected by a photodispersible protecting group is arranged on the metal layer to form a metal. The layer is irradiated with light of a wavelength that causes localized surface plasmon resonance, and the first coated molecule in which the photodissociable protecting group of the precursor is dissociated is bound to the first region. As a result, the manufactured sensor substrate can be detected with high sensitivity as in the first embodiment. Further, since the object to be protected and the conditions for dissociating the photodissociable protecting group can be changed depending on the type of the photodissociable protecting group, the sensor substrate can be easily manufactured.

(Other embodiments)
The method for manufacturing the sensor substrate, the sensor substrate, and the detection device according to the present disclosure have been described above based on the embodiments, but the present disclosure is not limited to these embodiments. As long as the gist of the present disclosure is not deviated, various modifications that can be conceived by those skilled in the art are applied to the embodiment, and other forms constructed by combining some components in the embodiment are also included in the scope of the present disclosure. included.

In addition, each of the above embodiments can be changed, replaced, added, omitted, etc. within the scope of claims or the equivalent scope thereof.

For example, in the above embodiment, in the production method shown in FIG. 7, the second coating molecule is bound to the second region, but the present invention is not limited to this. For example, in the state shown in FIG. 7 (b) in which the second coated molecule is not bound on the metal layer, the molecular recognition functional substance is bound to the first coated molecule, and the second coated molecule is formed in the second region. Uncoupled sensor substrates may be manufactured.

The sensor substrate according to the present disclosure can be used as a sensor and a detection device for detecting pathogens such as viruses and allergens such as pollen and food allergies.

1 Substrate 2, 62 Metal layer 2a, 62a, 70 Convex part 3 Gap 4, 8, 53, 55 Aqueous solution 5, 5a Second coating molecule 6, 31, 51, 57 Monochromatic light 9 First coating molecule 10 Molecular recognition functional substance 11, 11a, 11b 1st region 12 2nd region 19 Test solution 20 Specimen 21, 21a Labeled antibody 22 Composite particle 30 Light irradiation unit 32 Beam splitter 33 Light detection unit 40, 45 Nanostructure 50 Reaction solution 56, 56a precursor Body 60 Fine protrusions 61 Resin substrate 65 Coating molecules 71, 72 Regions 100, 110, 120, 200 Sensor substrate 300 Detection device

Claims (10)

A method for manufacturing a sensor substrate having a metal layer on which localized surface plasmon resonance occurs on the substrate.
A bonding step of binding the first coating molecule onto the metal layer,
It includes an immobilization step of binding and immobilizing a molecular recognition functional substance that specifically binds to a predetermined sample to the first coated molecule.
In the bonding step, the metal layer is irradiated with light having a wavelength that causes the localized surface plasmon resonance, so that the first region on the metal layer has a higher density than the second region on the metal layer. , The first coating molecule is bound to
The electric field enhancement due to the localized surface plasmon resonance in the first region is larger than the electric field enhancement in the second region.
Manufacturing method of sensor board. The joining step is
One end is bound to the metal layer, and the other end is a step of binding the molecular recognition functional substance and a second coating molecule that is inactive to the sample to the metal layer.
By irradiating the metal layer with light having a wavelength that causes the localized surface plasmon resonance, the second coating molecule in the first region among the second coating molecules bonded to the metal layer is removed. And the process to do
Includes a step of binding the first coated molecule to the first region from which the second coated molecule has been removed.
The method for manufacturing a sensor substrate according to claim 1. The first coated molecule has a binding group that binds to the metal layer at one end and a functional group that binds to the molecular recognition functional substance at the other end.
In the binding step, a precursor in which the binding group or the functional group of the first coating molecule is protected with a photodissociative protecting group is placed on the metal layer, and the localized surface plasmon resonance is applied to the metal layer. By irradiating with light of a wavelength to be generated, the first coating molecule in which the photodispersible protecting group of the precursor is dissociated is bound to the first region.
The method for manufacturing a sensor substrate according to claim 1. The binding group of the precursor is protected by the photodissociative protecting group.
In the bonding step, the metal layer is irradiated with light having a wavelength that causes the localized surface plasmon resonance in a state where the solution containing the precursor is in contact with the metal layer. The first coating molecule formed by dissociating the photodissociable protective group is bound to the first region.
The method for manufacturing a sensor substrate according to claim 3. In the binding step, after the first coated molecule is bound to the first region, one end binds to the metal layer and the other end is inactive to the molecular recognition functional substance and the sample. The second coated molecule is attached to the second region.
The method for manufacturing a sensor substrate according to claim 4. The functional group of the precursor is protected by the photodissociative protecting group.
In the binding step, the precursor is bound to the first region by binding the precursor onto the metal layer and then irradiating the metal layer with light having a wavelength that causes the localized surface plasmon resonance. Dissociate the photodissociative protecting group from the body,
The method for manufacturing a sensor substrate according to claim 3. A sensor substrate having a metal layer on which localized surface plasmon resonance occurs.
It is provided with a molecular recognition functional substance that is immobilized on the metal layer and specifically binds to a predetermined sample.
The molecular recognition functional substance is present in the first region on the metal layer at a higher density than the second region on the metal layer.
The electric field enhancement due to the localized surface plasmon resonance in the first region is larger than the electric field enhancement in the second region.
Sensor board. One end is bound to the metal layer, and the other end is a first coated molecule that binds to the molecular recognition functional substance.
One end is bound to the metal layer and the other end comprises the molecular recognition functional material and a second coated molecule that is inert to the sample.
The first coated molecule is bound to the first region at a higher density than the second region.
The second coated molecule is bound to the second region at a higher density than the first region.
The molecular recognition functional substance is bound to the first coated molecule.
The sensor substrate according to claim 7. The molecular recognition functional substance is an antibody.
The metal layer is made of gold, silver, or an alloy containing gold or silver as a main component.
The first coated molecule has a thiol group at one end, a functional group that binds to the molecular recognition functional substance at the other end, and a repeating sequence of hydrophilic groups that connect to the functional group. A self-assembled monolayer is formed on the metal layer,
The second coated molecule has a thiol group at one end, a hydroxyl group at the other end, a repeating sequence of hydrophilic groups connected to the hydroxyl group, and is a self-assembled monolayer on the metal layer. Forming a film,
The sensor substrate according to claim 8. The sensor substrate according to any one of claims 7 to 9,
A light irradiation unit that irradiates light having a wavelength that causes the localized surface plasmon resonance,
It has a photodetector that detects a photoreaction signal from the sensor substrate.
Detection device.