1. Field of the Invention
The present invention relates to a target substance capturing device that detects a target substance and a target substance detecting device including the target substance capturing device.
2. Description of the Related Art
As means to detect a target substance such as protein or a cell, or to measure the concentration, biosensors using a photonic crystal are known (for example, Non Patent Literatures 1 and 2). The biosensors described in Non Patent Literature 1 (hereinafter, Prior Art 1) and Non Patent Literature 2 (hereinafter, Prior Art 2) irradiates a photonic crystal substrate on which a gold thin film is formed with light, and measures change of a peak of a wavelength of reflected light reflected at the photonic crystal substrate, thereby to detect the target substance or measure the concentration of the target substance, and the like.
Prior Art 1 (Non Patent Literature 1): “Investigation of Plasmon resonances in metal films with nanohole arrays for biosensing applications”: Takumi Sannomiya, Olivier Scholder, Konstantins Jefimovs, Christian Hafner, and Andreas B. Dahlin, Received 10th December 2010, Revised 1th February 2011
Prior Art 2 (Non Patent Literature 2): “Periodic nanohole arrays with shape-enhanced plasmon resonance”: Antoine Lesuffleur, Hyungsoon Im, Nathan C. Lindquist and Sang-Hyun Oh, Received 16 Apr. 2007; accepted 17 May 2007; published online 13 Jun. 2007
In a case of detecting the target substance using the biosensors, the biosensors are detached to change a liquid to be detected, and when mounting the biosensors again after detached, there is a possibility that a mounting state differs. If the mounting state of the biosensor differs, detection sensitivity of the target substance may be decreased due to the difference in the mounting state.
Further, the photonic crystal has a microstructure. Therefore, it is difficult to finely control the shape, even if a manufacturing process is the same. Therefore, variation occurs in each sensor, and measurement accuracy of the target substance may be decreased.
Further, Prior Art 2 describes that real-time measurement has been performed using the biosensor. In Prior Art 2, the reflected light of the light irradiating the photonic crystal substrate that is exposed to a solution in a flow path is observed in each fixed time. Typically, change of the reflected light of light irradiating the photo crystal substance is fast as the flow speed of the solution is larger. However, if the flow speed of the solution is made large, the amount of the solution that passes through the photonic crystal substrate without having a reaction with the photonic crystal substrate becomes large, and the amount of the solution necessary to reach an equilibrium state becomes large. Therefore, a target substance capturing device that can decrease the amount of the solution necessary to reach the equilibrium state while causing the change of the reflected light of light irradiating the photonic crystal substrate to be fast is desired.
An objective of the present invention is to realize at least one of suppression of decrease in detection sensitivity of a target substance, and providing of a target substance capturing device that can decrease the amount of a solution necessary to reach an equilibrium state while causing change of reflected light of light irradiating a photonic crystal substrate to be fast.
According to an aspect of the invention, a target substance capturing device comprises a supporting member to place and support a metal-film coated structure that captures a target substance, the supporting member including at least two holes opening at portions different from a portion where the metal-film coated structure is placed; a holding member to put the metal-film coated structure in between the holding member and the supporting member, the holding member including an opening portion that overlaps with the holes of the supporting member, and a portion that captures the target substance of the metal-film coated structure placed on the supporting member; and a covering member having transparency and covering the opening portion of the holding member. Accordingly, it is not necessary to detach the metal-film coated structure even if a liquid to be detected is changed. Therefore, a decrease in detection sensitivity of the target substance due to difference in a mounting state of the metal-film coated structure can be suppressed.
According to further aspect of the invention, the holes are two of a supply hole and an discharge hole, the supply hole supplying a liquid containing the target substance to a space surrounded by the covering member, an inner surface of the opening portion, and the supporting member, and the discharge hole discharging the liquid from the space. Accordingly, the liquid can be supplied to the opening portion and can be discharged through the opening portion.
According to further aspect of the invention, a portion of the holding member, the portion being in contact with the metal-film coated structure, is formed of at least silicone. Accordingly, the metal-film coated structure can be easily detached from the holding member.
According to further aspect of the invention, the supporting member is formed of a fluororesin. Accordingly, the metal-film coated structure can be easily detached from the supporting member.
According to further aspect of the invention, the supporting member has transparency. Accordingly, not only the reflected light of the light irradiating the metal-film coated structure but also transmitted light can be observed.
According to further aspect of the invention, the supporting member includes a plurality of claws to engage with the holding member that puts the metal-film coated structure in between the holding member and the supporting member, at a side where the metal-film coated structure is placed. Accordingly, the holding member and the covering member can be easily mounted to the supporting member, and the holding member and the covering member can be easily detached from the supporting member.
According to further aspect of the invention, the covering member is fitted into the opening portion of the holding member. Accordingly, the holding member and the covering member can be easily mounted to the supporting member, and the holding member and the covering member can be easily detached from the supporting member.
According to further aspect of the invention, a target substance detecting device includes the target substance capturing device as described above. This target substance detecting device includes the above-described target substance capturing device. Therefore, a decrease in the detection sensitivity of the target substance can be suppressed.
According to further aspect of the invention, the target substance detecting device includes a liquid sending device supplying the liquid to the space through the hole, and to discharge the liquid from the space through the hole. Accordingly, the liquid can be easily supplied to the opening portion of the holding member, and can be easily discharged through the opening portion.
According to further aspect of the invention, the photo-detection section includes a first spectrometer and a second spectrometer having higher resolution of a wavelength of detectable light than the first spectrometer, and the processing unit obtains the wavelength of an extreme value of the reflected light, using the first spectrometer, and then obtains the wavelength of an extreme value of the reflected light, within a range of the wavelength of an extreme value obtained by the first spectrometer, using the second spectrometer. Accordingly, the wavelength of the extreme value of the reflected light can be promptly and accurately obtained.
According to further aspect of the invention, the wavelength of an extreme value of the reflected light is obtained, by performing fitting of at least one of a detection result of the first spectrometer and a detection result of the second spectrometer, with a function. Accordingly, higher resolution than pixel resolution of the spectrometer can be realized. Therefore, the wavelength of the reflected light in the extreme value can be more accurately obtained.
According to further aspect of the invention, the target substance detecting device includes a cooling unit configured to cool the photo-detection section. Accordingly, a noise due to heat can be decreased in detecting a spectrum of the reflected light.
According to another aspect of the invention, a target substance capturing device includes a supporting member to place and support a metal-film coated structure that captures a target substance; a holding member to put the metal-film coated structure in between the holding member and the supporting member, the holding member including a plurality of opening portions that overlaps with a portion that captures the target substance of the metal-film coated structure; a covering member having transparency and covering the opening portions of the holding member; and holes provided in the supporting member, and two of the holes opening to one of the opening portions, respectively, in a state where the metal-film coated structure is put in between the holding member and the supporting member. This target substance capturing device can introduce the liquid to each opening portion included in the supporting member. Therefore, the metal-film coated structure can be calibrated at the same time as the detection. As a result, the target substance capturing device can realize highly accurate measurement.
In one of the opening portions, a supply hole that supplies a liquid containing the target substance to the opening portion, and an discharge hole that discharges the liquid from the opening portion are provided as the holes. Accordingly, the liquid can be supplied to each opening portion, and can be discharged through the each opening portion.
A portion of the holding member, the portion being in contact with the metal-film coated structure, is formed of at least silicone. Accordingly, the metal-film coated structure can be easily detached from the holding member.
The supporting member is formed of a fluororesin. Accordingly, the metal-film coated structure can be easily detached from the supporting member.
According to still another aspect of the invention, a target substance detecting device includes the target substance capturing device described above; a photo-detection section provided to each of the opening portions, and irradiating a portion that captures the target substance with parallel light from each of the opening portions, and detecting reflected light of the parallel light reflected at the portion that captures the target substance; and a processing unit configured to obtain a wavelength of an extreme value of the reflected light detected by the photo-detection section, and to detect existence/non-existence of at least the target substance, based on shifting of the obtained wavelength of an extreme value. This target substance detecting device includes the above-described target substance capturing device. Therefore, a decreased in the detection accuracy of the target substance can be suppressed.
The target substance detecting device includes a liquid sending device configured to supply the liquid to the space through the hole, and to discharge the liquid from the space through the hole. Accordingly, the liquid can be easily supplied to each opening portion included in the holding member, and can be easily discharged through the each opening portion.
According to still another aspect of the invention, a target substance capturing device includes a flow path to flow a fluid containing a target substance; and a substrate to capture the target substance, the substrate including a reflection surface that reflects irradiating light. The substrate is arranged in the flow path such that a part of the fluid passes through at least the reflection surface, and the fluid that has passed through the flow path is repeatedly introduced to the flow path.
The target substance capturing device according to the present invention repeatedly introduces a fluid to the reflection surface. Therefore, the solution that has passed through the photonic crystal substrate without having a reaction with the photonic crystal substrate can repeatedly obtain an opportunity to react with the photonic crystal substrate. Therefore, the amount of the solution to reach the equilibrium state is not increased even if the flow speed of the solution is made large. Therefore, the target substance detecting device according to the present invention can decrease the amount of the fluid necessary to reach the equilibrium state while causing change of the reflected light of the light irradiating the photonic crystal substrate to be fast.
The flow path includes a supply port through which the fluid flows in, and a discharge port through which the fluid flows out, and the fluid discharged through the discharge port is introduced to the flow path through the supply port. Accordingly, power that causes the fluid to flow can be installed outside the flow path. Since the flow path is extremely small, assembly of the target substance capturing device becomes easy if the power can be installed outside the flow path. Therefore, the target substance capturing device according to the present invention can be easily assembled, and can decrease the amount of the solution necessary to reach the equilibrium state while causing the change of the reflected light of the light irradiating the photonic crystal substrate to be faster.
The target substance capturing device further includes a container in which a new fluid containing a target substance is stored, wherein the new fluid is introduced to the flow path through the supply port.
The target substance capturing device further includes a plate table; a thin plate configured to overlap on the table in a vertical direction to a surface of the table, and including an opening portion; and a plate cover configured to overlap on the thin plate in a vertical direction to a surface of the table. The flow path is a space surrounded by the table, an inner surface of the opening portion, and the cover. Accordingly, the flow path can be formed to be thin, and a flow speed of the fluid flowing on a plane vertical to the reflection surface can be made large. Accordingly, the target substance can be promptly captured on the reflection surface. Therefore, the target substance capturing device according to the present invention can decrease the amount of the solution necessary to reach the equilibrium state while causing change of the reflected light of the light irradiating the photonic crystal substrate to be faster.
The supply port and the discharge port are through holes provided in the table.
The present invention can realize at least one of suppression of a decrease in detection sensitivity of a target substance, and providing of a target substance capturing device that can decrease the amount of a solution necessary to reach an equilibrium state while causing change of reflected light of light irradiating a photonic crystal substrate to be fast.
Hereinafter, embodiments for implementing the present invention (hereinafter, referred to as embodiments) will be explained in detail based on the drawings.
[Photonic Crystal Biosensor]
In the photonic crystal biosensor 11, the holding device 11H holds the metal-film coated photonic crystal 21. In the holding device 11H, the holding member 23 holds the metal-film coated photonic crystal 21 placed on the supporting member 24, by putting the metal-film coated photonic crystal 21 in between the holding member 23 and the supporting member 24. The covering member 22 covers a surface of the holding member 23 of an opposite side to the supporting member 24. As illustrated in
The supporting member 24 supports the metal-film coated photonic crystal 21 placed thereon. The supporting member 24 includes at least two holes 24HI and 24HE that open to portions different from a portion where the metal-film coated photonic crystal 21 is placed, as illustrated in
The covering member 22 covers the opening portion 23P of the holding member 23, as illustrated in
The liquid such as a solution that contains a target substance capturing material is supplied through the supply hole 24HI to a space 23SP surrounded by the covering member 22, an inner surface of the opening portion 23P, and the supporting member 24, and is held in the space 23SP. The liquid held in the space 23SP is in contact with the portion 21C that captures the target substance of the metal-film coated photonic crystal 21. The liquid held in the space 23SP is held in the space 23SP during detection of the target substance by the target substance detecting device 10 or measurement of the concentration of the target substance. After the detection of the target substance by the target substance detecting device 10 and the like, the liquid held in the space 23SP is discharged through the discharge hole 24HE. To supply the liquid from an outside of the photonic crystal biosensor 11 to the space 23SP, a liquid supply pipe 25 is connected to the photonic crystal biosensor 11. To discharge the liquid to an outside of the photonic crystal biosensor 11 from the space 23SP, a liquid discharge pipe 26 is connected to the photonic crystal biosensor 11. Next, an example of a structure in which the liquid supply pipe 25 and the liquid discharge pipe 26 are connected to the space 23SP will be explained. As the liquid supply pipe 25 and the liquid discharge pipe 26, a tube made of silicon rubber or the like can be used, for example. However, the material is not limited to the silicon rubber tube.
As illustrated in
The photonic crystal biosensor 11 sandwiches the metal-film coated photonic crystal 21 by the supporting member 24 and the holding member 23. The mounting tools 27 and 28 are fastened with bolts 29 illustrated in
In the present embodiment, a portion of the holding member 23, the portion being in contact with the metal-film coated photonic crystal 21, is formed of at least silicone, for example, polydimethylsiloxane (PDMS). The polydimethylsiloxane has high liquid repellency (water repellency), and thus can suppress adhesion of the metal-film coated photonic crystal 21 and the holding member 23. Therefore, when the metal-film coated photonic crystal 21 is replaced, the metal-film coated photonic crystal 21 can be easily detached from the holding member 23. The thickness of the holding member 23 is preferably from 100 μm to 2 mm, both inclusive. With such thickness, fixation of the metal-film coated photonic crystal 21 in between the supporting member 24 and the holding member 23 can be easily handled.
In order to flow the liquid on the metal-film coated photonic crystal 21, a provisional idea of providing a space keeping portion to make a space through which the liquid flows on a sensor surface is considered. The thickness of the space keeping portion depends on a wavelength of plasmon resonance. Therefore, the thickness is limited depending on a wavelength band to be used. If such a structure is employed, the thickness of the space keeping portion requires strict accuracy, and a time and a manufacturing cost are required at the time of manufacturing. The present embodiment does not require the space keeping portion, and thus the manufacturing of the photonic crystal biosensor 11 becomes easy, and the time and the manufacturing cost can be suppressed. Further, with respect to a surface plasmon (SPR) sensor, it is important to fix the sensor and a prism, because the surface plasmon sensor does not function as a sensor if a small gap or bending is formed. However, the metal-film coated photonic crystal 21 of the present embodiment does not require the strict fixation like the SPR sensor.
In the present embodiment, the supporting member 24 is formed of a fluororesin. Although the material of the supporting member 24 is not limited to the fluororesin, the fluororesin has high liquid repellency (water repellency), and thus can suppress adhesion between the metal-film coated photonic crystal 21 and the supporting member 24. Therefore, when the metal-film coated photonic crystal 21 is replaced, the metal-film coated photonic crystal 21 can be easily detached from the holding member 23. The supporting member 24 may have transparency. With such a configuration, transmitted light of light irradiating the metal-film coated photonic crystal 21 can be observed. When the supporting member 24 has transparency, the supporting member 24 is manufactured using glass or a transparent resin, for example. When glass is used for the supporting member 24, the metal-film coated photonic crystal 21 is fixed between the supporting member 24 and the holding member 23, by a joining technology such as self-adsorption between the glass supporting member 24 and the polydimethylsiloxane holding member 23, gluing between the supporting member 24 and the holding member 23, or heat seal of the polydimethylsiloxane holding member 23. Next, the metal-film coated photonic crystal 21 will be explained.
[Metal-Film Coated Photonic Crystal]
First, the photonic crystal 65 will be explained. The photonic crystal is a structure that has a reflection surface having a surface where recessed portions having a predetermined depth or protruding portions having a predetermined height are periodically formed, and can obtain reflected light when the reflection surface is irradiated with light having a specific wavelength (parallel light). The structure that can obtain the reflected light having a specific wavelength when the reflection surface having a surface where recessed portions or protruding portions are periodically formed is irradiated with the light is typically called a photonic crystal.
The photonic crystal is a structure having a grid structure with a subwavelength interval. Then, when a surface of the structure (hereinafter, referred to as reflection surface) is irradiated with light having a wide region wavelength, the photonic crystal reflects or transmits light in a specific wavelength band depending on a surface state of the photonic crystal. The surface state of the photonic crystal depends on the shape or the material of the photonic crystal, for example. By reading change of the reflected light or the transmitted light, change of the surface state of the photonic crystal can be quantified. Examples of the change of the surface state of the photonic crystal include absorption of a substance to the surface, and structure change. When the photonic crystal having a surface on which a metal thin film is formed is irradiated with light, an extreme value (a maximum value of a minimum value) appears in reflectance of light or transmittance of light. The extreme value of the reflectance or the transmittance depends on a type of the metal, a film thickness of the metal, and the shape of the surface of the photonic crystal. By reading the reflectance of light or the transmittance of light, the change of the surface state of the photonic crystal can be quantified. The metal thin film will be explained below. To quantify the change of the surface state of the photonic crystal from the change of the reflected light or the transmitted light, the following method can be used. For example, an amount of change of the reflectance or the transmittance in the extreme value (a maximum value of a minimum value), or a shift amount of a wavelength with which the reflectance or the transmittance becomes the extreme value is obtained. Note that, when there is a plurality of extreme values of the reflectance and the transmittance, an arbitrary extreme value is focused. Then, the amount of change of the focused extreme value or the shift amount of the wavelength with which the focused extreme value is obtained is obtained, so that the change of the surface state of the photonic crystal can be quantified.
As illustrated in
The shape and the dimensions of the photonic crystal 65 are not limited to the shape illustrated in
As the synthetic resin, a thermoplastic resin such as polyethylene, polypropylene, polymethylpentene, polycycloolefin, polyamide, polyimide, acryl, polymethacrylic acid ester, polycarbonate, polyacetal, polytetrafluoroethylene, polybutylene terephthalate, polyethylene terephthalate, polyvinyl chloride, polyvinylidene chloride, polystyrene, polyphenylene sulfide, polyether sulfone, or polyetheretherketone, or a thermosetting resin such as a phenol resin, a urea resin, or an epoxy resin, can be used.
As the ceramic, a ceramic such as silica, alumina, zirconia, titania, or yttria can be preferably used. As the metal, various alloys including a steel material can be used. To be specific, stainless steel, titanium or a titanium alloy can be preferably used.
Among the above-described various materials, a polycycloolefin-based synthetic resin or a silica-based ceramic is more preferable, in light of optical characteristics, processability, tolerance to a solution containing a target substance (a substance to be targeted), absorbability of the target substance capturing material (specific bonding substance), and tolerance to a washing agent. Between the polycycloolefin-based synthetic resin and the silica-based ceramic, the polycycloolefin-based synthetic resin is most preferable because of excellent processability.
The photonic crystal 65 is manufactured by application of fine processing to a surface of a substrate made of the above-described material. As a processing method, laser processing, heat nanoimprint, optical nanoimprint, or a combination of a photo mask and etching can be used. Especially, when the thermoplastic resin such as the polycycloolefin-based synthetic resin is used as the material, a method by the heat nanoimprint is preferable.
Next, the metal film 66 will be explained. In the present embodiment, as illustrated in
When the film thickness of the metal film 66 is small, a part of incident light on the photonic crystal 65 may transmit the metal film 66. As a result, there is a possibility that an amount of information obtained from reflected light from the photonic crystal 65 decreases, and that unnecessary information, such as diffracted light or reflected light from a back surface of the photonic crystal 65 is included in the reflected light. By appropriately making the film thickness of the metal film 66 large, the unnecessary information included in the reflected light from the photonic crystal 65 can be decreased, and detection accuracy of the target substance and measurement accuracy of the concentration can be improved. Further, suitably small film thickness of the metal film 66 is preferable in easily manufacturing a detailed pattern shape on the surface 67 of the photonic crystal 65. For example, a corner of the pattern becomes sharp, and the dimension of the pattern can be easily secured. Based on the above perspective, the film thickness of the metal film 66 is preferably from 30 nm to 1000 nm, both inclusive, and is more preferably from 150 nm to 500 nm, both inclusive, and is still more preferably from 200 nm to 400 nm, both inclusive, in the present embodiment. This is because change of the reflectance to the wavelength becomes nearly similar when the film thickness of the metal film 66 exceeds 200 nm.
The metal film 66 can be formed on the reflection surface 69 of the photonic crystal 65 by means of sputtering, a deposition apparatus, or the like. It is preferable to form an outermost surface of the metal film 66 with Au. When Ag, Pt, or Al is used as the metal film 66, the wavelength of the reflected light in each extreme values becomes 1.5 times that of the case where Au is used as the metal film 66. As explained above, sensitivity of Ag, Pt, and Al is 1.5 times that of Au. Since Ag is easily oxidized, it is preferable to form an oxide thin film of Au or SiO2, which is less easily oxidized, after forming Ag on the reflection surface 69 of the photonic crystal 65. In this case, a film of Au having the thickness of 5 nm can be formed on a surface of a film of Ag having the thickness of 200 nm. When the film of Au having the thickness of 5 nm is formed on the film of Ag having the thickness of 200 nm, the sensitivity becomes 1.5 times that of a film of Au having the thickness of 200 nm. Further, no change of the sensitivity is seen between existence and non-existence of the film of Au of 5 nm. Al is also easily oxidized similarly to Ag, and thus it is preferable to form an oxide thin film of Au or SiO2, which is less easily oxidized, after forming a film of Al on the surface 67 of the photonic crystal 65. In a case of Pt, it is also preferable to form the oxide thin film of Au or SiO2 because of modification with an antibody or the like.
Further, it is preferable to reform the reflection surface 69 of the photonic crystal 65, using 3-triethoxysilylpropylamine (APTES) or the like. When the metal film 66 of Au or Ag is formed on the reflection surface 69 of the photonic crystal 65, it is preferable to reform the reflection surface 69 of the photonic crystal 65, using a carbon chain having a thiol group in one end, and a functional group such as an amino group or a carboxyl group in the other end, instead of APTES. When the metal film 66 of other than Au or Ag is formed on the reflection surface 69 of the photonic crystal 65, it is preferable to reform the reflection surface 69 of the photonic crystal 65, using a silane-based coupling agent having a functional group in one end, for example, APTES.
The metal-film coated photonic crystal 21 is obtained such that the reflection surface 69 of the photonic crystal 65 is coated with the metal film 66. Therefore, recessed portions (non-flat portions) 68B of the metal-film coated photonic crystal 21 are periodically formed in the reflection surface 69, corresponding to the recessed portions 68A of the photonic crystal 65. The recessed portions 68B are arranged in a triangular grid manner, similarly to the recessed portions 68A. Further, although depending on the thickness of the metal film 66, a diameter Db of the recessed portion 68B is preferably from 50 nm to 1000 nm, both inclusive, and is more preferable from 100 nm to 500 nm, both inclusive. Further, a distance C2 between centers of the recessed portions 68B is preferably from 100 nm to 2000 nm, both inclusive, and is more preferably from 200 nm to 1000 nm, both inclusive, similarly to the distance C1 between centers of the recessed portions 68A. Further, an aspect ratio (H2/Db) of the recessed portion 68B, where the depth of the recessed portion 68B is H2, is preferably from 0.1 to 10, both inclusive, and is more preferably from 0.5 to 5.0, both inclusive. Note that dimensions of the recessed portion 68B are not limited to the above-described dimensions.
tan θ=L3/L2 (1)
0≦tan θ≦1.0 (2)
When a surface of a metal provided with a structure in which recessed holes are periodically arranged is irradiated with light, a peak is observed in a wavelength spectrum of reflected light. A wavelength (peak wavelength) with which the reflectance to the wavelength of the reflected light is maximized can be typically obtained by the following expression (3). In the expression (3), λpeak is the peak wavelength, a0 is a period of the hole, i and j are an order of diffraction, ∈m is a dielectric constant of the metal, and ∈d is a dielectric constant of an environment.
According to the expression (3), the peak wavelength can be obtained when a period of arranging the recessed portion 68B is given. When the spectrum of the peak wavelength is observed, the position of the peak wavelength can be more easily identified if a width of the spectrum of the peak wavelength is smaller. Therefore, if the period of arranging the recessed portion 68B is clearly given, the width of the spectrum of the peak wavelength becomes small, and the position of the peak wavelength can be easily identified.
The metal-film coated photonic crystal 21 has a periodic structure in which the recessed portions 68B are periodically formed on the reflection surface 69. The wall surface 68a of the recessed portion 68B is formed on the reflection surface 69 to satisfy the expressions (1) and (2), so that the width of the form of the wavelength spectrum of the reflected light becomes narrow, and the peak wavelength of the reflected light can be easily identified. Accordingly, the target substance can be accurately detected. As a result, the sensor sensitivity of the photonic crystal biosensor 11 can be improved. Note that the width of the form of the wavelength spectrum of the reflected light is a half-value width or the like.
It is preferable to form the recessed portion 68B to satisfy the following expression (2)′. The wall surface 68a of the recessed portion 68B is formed to satisfy the above-described expression (1) and the following expression (2)′, so that the form of the wavelength spectrum of the reflected light becomes narrower, and the peak wavelength of the reflected light can be more easily identified. As a result, the target substance can be more accurately detected.
0≦tan θ≦0.7 (2)′
From the expressions (2) and (2)′, the angle θ is 0 degrees or more. When the angle θ=0 degrees, a connection portion K of the surface 67 of the metal-film coated photonic crystal 21, and the wall surface 68a of the recessed portion 68B have an angle of appropriately 90 degrees. When the connection portion K has the angle of approximately 90 degrees, control of the shape of the metal-film coated photonic crystal 21, especially, control of the shape of the recessed portion 68B becomes difficult. That is, it becomes difficult to obtain an expected shape of the recessed portion 68B. By satisfying tan θ>0, that is, by causing the angle θ to be larger than 0, the expected shape of the recessed portion 68B can be easily obtained, and thus it is preferable. Further, the metal-film coated photonic crystal 21 is washed with relatively high-pressure water. If the angle of the connection portion K becomes approximately 90 degrees, a corner is apt to be removed. As a result, there is a possibility that the recessed portion 68B does not have the expected shape. By satisfying tan θ>0, that is, by causing the angle θ to become larger than 0, the possibility of removal of the corner of the connection portion K decreases. Therefore, the recessed portion 68B has the expected shape after the washing, and thus it is preferable. Further, by satisfying tan θ>0, that is, by causing the angle θ to become larger than 0, water can easily enter the recessed portion 68B, and thus the target substance can be reliably captured in the recessed portion 68B.
[Method of Manufacturing Photonic Crystal]
When the resin P is a cycloolefin-based polymer, the die DI is heated to about 160° C. and is pressed with pressure of about 12 MPa for a predetermined time. It is preferable to release the die when the surface temperature of the die DI becomes about 60° C. After the photonic crystal 65 is manufactured, the metal film 66 is formed on a surface, which was in contact with the die DI, by means of sputtering or a deposition apparatus, as illustrated in
[Target Substance Capturing Material]
Next, the target substance capturing material that captures the target substance will be explained. The target substance is an object to be detected by the target substance detecting device 10, and may be any of a macromolecule such as a protein, an oligomer, or a low molecule. The target substance is not limited to a single molecule, and may be a complex made of a plurality of molecules. Examples of the target substance include a pollutant in the atmosphere, a toxic substance in water, and a biomarker in a human body. Among them, cortisol is preferable. Cortisol is a low-molecular substance having a molecular weight of 362 g/mol. The cortisol concentration in saliva increases when a human feels stress. Therefore, cortisol attracts attention as a substance with which the degree of stress felt by the human is evaluated. If the cortisol concentration contained in saliva of a human is measured using the cortisol as the target substance, the degree of stress can be evaluated. By evaluating the degree of stress, it can be determined whether a person to be measured is in a level of stress state leading to a mental disease such as depression.
The target substance capturing material is a material to bond with the target substance to capture the target substance. Here, bonding may be non-chemical bond, such as bonding by physisorption or Van der Waals force, in addition to the chemical bond. Preferably, the target substance capturing material specifically reacts with the target substance, and captures the target substance, and is preferable to be an antibody having the target substance as an antigen. The specific reaction means selective combination with the target substance in a reversible or irreversible manner to form a complex, and is not limited to a chemical reaction. Further, a substance to specifically react with the target substance capturing material may exist other than the target substance. Even if there is a substance to react with the target substance capturing material in a sample, other than the target substance, the target substance can be quantified when affinity of the substance is extremely smaller than that of the target substance. As the target substance capturing material, an antibody having the target substance as an antigen, an artificially manufactured antibody, a molecule configured from a substance that configures DNA such as adenine, thymine, guanine and cytosine, a peptide, or the like can be used. When the target substance is cortisol, the target substance capturing material is preferably a cortisol antibody.
To manufacture the target substance capturing material, a known method can be employed. For example, the antibody can be manufactured by a serum test, a hybridoma method, or a phage display method. The molecule configured from a substance that configures DNA can be manufactured by systematic evolution of ligands by exponential enrichment (SELEX method), for example. The peptide can be manufactured by a phage display method, for example. The target substance capturing material is not necessarily labeled with some sort of enzyme or isotope. However, the target substance capturing material may be labeled with enzyme or isotope.
In the present embodiment, the target substance capturing material is fixed to the reflection surface 69 of the metal-film coated photonic crystal 21 illustrated in
The photonic crystal biosensor 11 allows an antibody (for example, a cortisol antibody), which is bonded only with a specific antigen (for example, cortisol), to be absorbed by (fixed to) the surface of the reflection surface 69 of the metal-film coated photonic crystal 21 in advance. Accordingly, the photonic crystal biosensor 11 can detect the specific antigen. This uses various optical characteristics of the photonic crystal 65, and biological reaction/chemical reaction occurring on the surface or in the vicinity of the surface of the photonic crystal 65, for example, an antigen/antibody reaction in which the specific antigen reacts only with the specific antibody.
The photonic crystal biosensor 11 may be constituted such that a blocking agent (protecting substance) is fixed on the reflection surface 69 to which the antibody as the target substance capturing material is fixed. The blocking agent is fixed before the target substance is brought to come in contact with the photonic crystal biosensor 11. The surface of the reflection surface 69 of the photonic crystal 65 is typically super-hydrophobic. Therefore, impurities other than the antibody as the target substance capturing material may be absorbed by the reflection surface 69 due to hydrophobic interaction. In addition, the optical characteristics of the photonic crystal 65 are substantially influenced by the surface state. Therefore, it is preferable that the impurities are not absorbed by the reflection surface 69 of the photonic crystal 65. The fixation of the blocking agent to the reflection surface 69 of the photonic crystal 65 improves the detection accuracy of the reflected light.
Therefore, it is preferable to fix the blocking agent in advance so that the impurities and the like are not fixed to a portion other than the portion where the antibody as the target substance capturing material is absorbed by (fixed to) the reflection surface 69 of the photonic crystal 65. To absorb the blocking agent in advance, the blocking agent is brought to come in contact with the surface of the photonic crystal 65. As the blocking agent, skim milk, bovine serum albumin (BSA), or the like can be used.
As illustrated in
Next, the photo-detection section 12 illustrated in
In the photonic crystal biosensor 11, when the antigen 76 is captured by the antibody 74 fixed to the reflection surface 69, the state of the reflection surface 69 is changed, and the reflected light LR is changed. The photonic crystal biosensor 11 outputs an optical physical amount. The physical amount correlates with the change of the surface state in the reflection surface 69 of the metal-film coated photonic crystal 21, and correlates with the amount of the complex 77 that is formed such that the antigen 76 is captured by the antibody 74 fixed to the reflection surface 69. The optical physical amount is, for example, the shift amount of the wavelength with which the intensity of the reflected light LR becomes the extreme value, the amount of change of the reflectance of light, the shift amount of the wavelength with which the reflectance of light becomes the extreme value, the amount of change of the intensity of the reflected light LR or of the extreme value of the intensity of the reflected light LR, and the like. In the present embodiment, the shift amount of the wavelength with which the intensity of the reflected light LR or the reflectance of light becomes the extreme value is used.
To output the optical physical amount, the following processes are performed, for example. Light is vertically incident on the reflection surface 69 of the metal-film coated photonic crystal 21, and the reflected light LR is detected. The light can be incident on the reflection surface 69 of the metal-film coated photonic crystal 21 with an angle with respect to a perpendicular line of the reflection surface 69 of the metal-film coated photonic crystal 21, and the reflected light LR can be detected. By detection of the reflected light LR, the target substance detecting device 10 illustrated in
[Photo-Detection Section]
Next, the photo-detection section 12 illustrated in
A control device connected with the light source 51, the photo-detection device 53, and the like, and which controls the light source 51 and processes a signal from the photo-detection device 53 may be provided, as needed.
As illustrated in
Since the measuring probe 52 has such a structure, the measuring probe 52 can emit the incident light LI irradiating the reflection surface 69 of the photonic crystal 65, and receive the reflected light LR from the reflection surface 69, respectively, to and from an approximately the same position. The measuring probe 52 is caused to have the above-described structure, and the light from the measuring probe 52 is caused to be the parallel light using the collimating lens 56, so that the photo-detection section 12 allows the incident light LI of the parallel light to be vertically incident on the reflection surface 69. Further, the photo-detection section 12 can receive the reflected light LR vertically reflected on the reflection surface 69. Accordingly, the measuring probe 52 can minimize a decrease in the reflected light intensity, and can mainly detect 0-order light component of the reflected light LR. As a result, the processing unit 13 can obtain accurate information of the reflection surface 69 of the metal-film coated photonic crystal 21. Therefore, the detection accuracy of the target substance and the measurement accuracy of the concentration are improved. A technique of detecting the reflected light LR is not limited to the above-described measuring probe 52. For example, a half mirror is arranged between the collimating lens 56 and the reflection surface 69, and the reflected light LR is divided by the half mirror, and guided through the second optical fiber 55 to the photo-detection device 53. The collimating lens 56 may include an antireflection film. Accordingly, an influence of the reflected light from the collimating lens 56 is decreased. Therefore, a noise generated at the time of measurement can be decreased.
The photo-detection device 53 illustrated in FIG. 1 includes a spectrometer that detects a light spectrum of the reflected light LR. As the spectrometer, there are a monochromator and a multichannel spectrometer. In the present embodiment, the multichannel spectrometer is employed from the viewpoint of fast detection speed. The multichannel spectrometer is a device that disperses the incident light into a plurality of different wavelength regions, using a prism, grating, and the like, and detects a spectrum with photo-detection elements arrayed in an array manner. The multichannel spectrometer can obtain measurement result for each pixel of the photo-detection elements arrayed in an array manner, at a pitch of a specific wavelength width. A product of division of a measurement range of one spectrometer by the number of pixels is called pixel resolution. The pixel resolution is resolution of the wavelength of the light which the spectrometer can detect.
In the spectrometer in which the photo-detection elements are arrayed in an array manner, as a method of reading a signal from the photo-detection elements arrayed in an array manner, there are a charge coupled device (CCD) system, and a complementary metal oxide semiconductor (CMOS) system. In the present embodiment, either system may be employed. As the photo-detection elements, photodiodes, avalanche photodiodes, photomultiplier tubes, or the like may be arrayed in an arrayed manner.
The wavelengths λ1, λ2, λ3, λ4, and λ5 become large by 1 μm in this order, and the wavelengths λ11, λ12, λ13, λ14, and λ15 become large by 0.1 μm in this order. Therefore, pixel resolution P1 of the spectrometer 53SA is 1 nm, and pixel resolution P2 of the spectrometer 53SB is 0.1 nm. When the number pixels (pixel number) is the same between the spectrometer 53SA and the spectrometer 53SB (the pixel number is five in the example of
In the example illustrated in
In step S3, the processing unit 13 performs peak fitting, using (2×n+1) detection results. In the peak fitting, the above-described arbitrary function is used. The processing proceeds to step S4, and the processing unit 13 calculates a residual between a curved line obtained by the peak fitting, and the detection result of the photo-detection device 53, and when the calculated residual is smaller than a set value determined in advance (Yes in step S4), the processing proceeds to step S5. In step S5, the processing unit 13 estimates the peak position from the fitting function, that is the function used in the peak fitting. In step S4, when the calculated residual is equal to or more than the set value determined in advance (No in step S4), the processing proceeds to step S6. In step S6, the processing unit 13 changes at least one of the fitting function or an initial parameter, and executes steps S3 and S4. The n-order function (n is an integer of two or more), the Lorentz function, the Gaussian function, the Voigt function, and the beta function, which have been exemplarily explained as the fitting function, are on a par. A function closest to the detection result group of an object to be subjected to the peak fitting, that is, a function having a shape with the smallest residual is the function most suitable for the case.
[Liquid Handling Section]
Next, the liquid handling section 14 illustrated in
A liquid handling section 14b illustrated in
[Modification of Photonic Crystal Biosensor]
The present embodiment and its modifications guide the liquid to the opening portion 23P included in the holding member 23 of the photonic crystal biosensor 11 or the like. Accordingly, the liquid in the opening portion 23P can be replaced in a state where the metal-film coated photonic crystal 21 is sandwiched between the supporting member 24 and the holding member 23. As a result, a measurement noise due to an error in mounting the metal-film coated photonic crystal 21 decreases. As a result, the detection sensitivity of the target substance can be improved. The configurations of the present embodiment and its modifications can be appropriately applied or combined in embodiments below.
The supporting member 24C on which the metal-film coated photonic crystal 21 is placed includes a plurality of holes 24I1, 24I2, 24I3, 24E1, 24E2, and 24E3. The opening portions 23P1, 23P2, and 23P3 also overlap with the holes 24I1, 24I2, 24I3, 24E1, 24E2, and 24E3 of the supporting member 24C. Two of the holes 24I1, 24I2, 24I3, 24E1, 24E2, and 24E3 are open to one of the plurality of opening portions 23P1, 23P2, and 23P3, respectively, in a state where the metal-film coated photonic crystal 21 is put in between the holding member 23C and the supporting member 24C. To be specific, the holes 24I1 and 24E1 open to the opening portion 23P1, the holes 24I2 and 24E2 open to the opening portion 23P2, and the holes 24I3 and 24E3 open to the opening portion 23P3. The hole 24I1 supplies a liquid such as a solution that contains a target substance capturing material to the opening portion 23P1, the hole 2412 supplies the solution to the opening portion 23P2, and the hole 24I3 supplies the solution to the opening portion 23P3. The hole 24E1 discharges the liquid such as a solution that contains a target substance capturing material from the opening portion 23P1, the hole 24E2 discharges the liquid from the opening portion 23P2, and the hole 24E3 discharges the solution from the opening portion 23P3. Hereinafter, the holes 24I1, 24I2, and 24I3 are appropriately called liquid supply holes 24I1, 24I2, and 24I3, and the holes 24E1, 24E2, and 24E3 are called liquid discharge holes 24E1, 24E2, and 24E3. With such a configuration, the photonic crystal biosensor 11C can introduce the liquid to each of the opening portions 23P1, 23P2, and 23P3, and thus can evaluate different types of liquid with one metal-film coated photonic crystal 21.
In the introduction of the liquid to each of the opening portions 23P1, 23P2, and 23P3, the liquid discharge holes 24E1, 24E2, and 24E3 may be connected with an inlet of a pump, and the liquid may be introduced to the opening portions 23P1, 23P2, and 23P3, using negative pressure. In this case, the pumps may be provided corresponding to the respective opening portions 23P1, 23P2, and 23P3, or one pump may supply the liquid to the opening portions 23P1, 23P2, and 23P3, and discharges the liquid from the opening portions 23P1, 23P2, and 23P3. Further, the liquid supply holes 24I1, 24I2, and 24I3 may be connected with outlets of pumps, and the liquid may be introduced to the opening portions 23P1, 23P2, and 23P3, using positive pressure. In this case, the pumps are provided corresponding to the respective opening portions 23P1, 23P2, and 23P3.
It is difficult to finely control the shape of the metal-film coated photonic crystal 21 even if the metal-film coated photonic crystal 21 is manufactured by the same manufacturing process because the metal-film coated photonic crystal 21 has a microstructure. Therefore, variation occurs in each metal-film coated photonic crystal 21. The photonic crystal biosensor 11C can introduce the liquid to each of the opening portions 23P1, 23P2, and 23P3, and can calibrate the metal-film coated photonic crystal 21 at the same time of inspection. As a result, the photonic crystal biosensor 11C can realize highly accurate measurement. For example, a solution to be inspected and a standard solution having known characteristics (for example, the concentration and the like) are introduced to the portion 21C that captures the target substance of the metal-film coated photonic crystal 21 at the same time. The concentration of the standard solution is known in advance. Therefore, a calibration curve is obtained from a detection result of the standard solution and a detection result of the solution to be inspected, so that calibration of the metal-film coated photonic crystal 21 can be performed at the same time of the inspection. As a result, the photonic crystal biosensor 11C can highly accurately measure the concentration and the like of the target substance contained in the solution to be inspected. Further, a volume of a space surrounded by the opening portions 23P1, 23P2, and 23P3, the supporting member 24C, and the covering member 22 is smaller than a volume of a space surrounded by the opening portion 23P, the supporting member 24, and the covering member 22 in the first embodiment. Therefore, the amount of the liquid to be supplied to the opening portions 23P1, 23P2, and 23P3 can be small. Therefore, the present embodiment is especially preferable when an expensive liquid is used.
When the holding member 23C has two or more opening portions, the photonic crystal biosensor 11C can calibrate the metal-film coated photonic crystal 21. Therefore, it is preferable that the holding member 23C includes two or more opening portions. Further, to more accurately calibrate the metal-film coated photonic crystal 21, it is preferable to introduce a plurality of standard solutions, in addition to the solution to be inspected. Therefore, it is more preferable that the holding member 23C has three or more opening portions.
The photo-detection unit 50 stores the plurality of measuring probes 52C in a casing 43, as illustrated in
As illustrated in
The collimating lens 56C is a spherical lens. As illustrated in
As explained above, the present embodiment can introduce the liquid to each of the opening portions 23P1, 23P2, and 23P3 included in the photonic crystal biosensor 11C. Therefore, the metal-film coated photonic crystal 21 can be calibrated at the same time of an inspection. As a result, the present embodiment can realize highly accurate measurement. Further, the volume of a space surrounded by the opening portions 23P1, 23P2, and 23P3, the supporting member 24C, and the covering member 22 is small. Therefore, the amount of the liquid to be supplied to the opening portions 23P1, 23P2, and 23P3 can be small.
(Photonic Crystal Biosensor)
First, a photonic crystal biosensor 11c will be explained. The photonic crystal biosensor 11c includes a metal-film coated photonic crystal 21, a table 83, a thin plate 84, and a cover 82. In the third embodiment, the photonic crystal biosensor 11c has a structure in which the metal-film coated photonic crystal 21 is arranged in a flow path 84f formed by the table 83, the thin plate 84, and the cover 82. The metal-film coated photonic crystal 21 is similar to that in the first embodiment, and thus description is omitted.
(Method of Manufacturing Photonic Crystal Biosensor)
Next, an example of manufacturing of the photonic crystal biosensor 11c illustrated in
The photonic crystal biosensor 11c includes a supply pipe 96 and a discharge pipe 97. The solution is supplied through the supply pipe 96 to the flow path 84f. The solution is discharged through the discharge pipe 97 from the flow path 84f.
Materials of the table 83 and the cover 82 are not especially limited. However, it is preferable to use stainless steel, a poly cycloolefin-based polymer resin, or silica, in light of cleanliness of the cover 82 and the surface of the table 83.
One of the two through holes 83h is a supply port that allows the solution to flow into the flow path 84f. The other of the two through holes 83h is a discharge port that allows the solution to be discharged from the flow path 84f. A supply pipe 96 including a connector 79 on a tip is connected with one of the two through holes 83h. A discharge pipe 97 including a connector 79 on a tip is connected with the other of the two through holes 83h. The solution flows in the flow path 84f through the supply pipe 96, and flows out from the flow path 84f through the discharge pipe 97. Further, the connectors 79 block the two through holes 83h. Therefore, the connectors 79 decrease a possibility that the solution leaks from the flow path 84f. Note that the through holes 83h, the supply pipe 96, and the discharge pipe 97 may not be provided. Even when the through holes 83h, the supply pipe 96, and the discharge pipe 97 are not provided, the solution circulates in the flow path 84f as long as the flow path 84f is formed in an annular manner. Further, three or more through holes 83h may be provided.
The photonic crystal biosensor 11c is uniformly manufactured by heat nanoimprint or the like. To cause the target substance detecting device 10c to be able to more accurately detect the reflected light, it is preferable to accurately position an incident part and a reflection part of the light irradiating the photonic crystal biosensor 11c.
That is, it is preferable that a positional relationship between the photonic crystal biosensor 11c and a measuring probe explained below at the time of measurement is the same before and after an antigen/antibody reaction, and that the same portion is measured. Therefore, it is preferable that a distance between the measuring probe and a reflection surface 69 of the photonic crystal biosensor 11c is the same before and after the antigen/antibody reaction, and it is preferable to fix the distance from 50 μm to 500 μm. The photonic crystal biosensor 11c includes the cover 82, so that the cover 82 functions as a spacer, and can keep the distance between the measuring probe and the reflection surface 69 of the photonic crystal biosensor 11c constant.
Further, the photonic crystal biosensor 11c may be marked with a positioning marker that displays a specific position on the reflection surface 69. The marker may be provided by photolithography, sputtering, deposition, or a liftoff process using the aforementioned methods, printing with an ink, pattern formation by imprint, or the like. The marker may be attached to either a surface (the reflection surface 69 side) or a back surface (an opposite side to the reflection surface 69) of the photonic crystal biosensor 11c as long as the position of the marker can be read. Further, the marker may be attached to a photonic crystal 65 itself, except a measuring portion of the photonic crystal 65. Further, the marker may be attached to the cover 82 or the table 83.
(Method of Circulating Solution)
Next, in step S12, the valve 94 is switched and the solution is circulated. When the passage 94b is filled with the solution, the control unit 13c switches the valve 94. Accordingly, as illustrated in
Next, in step S13, the end portion 95e of the supply pipe 95 is pulled up from the new solution 93 stored in the container 92, after termination of measurement of reflected light, and the valve 94 is switched. When the valve 94 is switched, the discharge pipe 97 is connected with the discharge pipe 98 through the passage 94b. Accordingly, the solution inside the flow path 84f, the supply pipe 96, and the discharge pipe 97 is discharged from an end portion 98e of the discharge pipe 98. Further, a method of circulating the solution may not be the above-described method.
In the photonic crystal biosensor 11c according to the third embodiment, the solution that has passed through the space 21u above the reflection surface 69 is repeatedly introduced to the space 21u above the reflection surface 69. Accordingly, the solution that has passed through the space 21u above the reflection surface 69 without having a reaction with the metal-film coated photonic crystal 21 can repeatedly have an opportunity to react with the metal-film coated photonic crystal 21. Therefore, even if a flow speed of the solution is made large, the amount of the solution necessary to reach an equilibrium state is not increased. Therefore, the photonic crystal biosensor 11c according to the third embodiment can decrease the amount necessary to reach the equilibrium state, while making change of the reflected light of the light irradiating the metal-film coated photonic crystal 21 fast.
Further, in the photonic crystal biosensor 11c according to the third embodiment, the solution is supplied through the supply pipe 96 to the flow path 84f. The solution is discharged from the flow path 84f through the discharge pipe 97. Accordingly, the pump 91 for moving the solution can be installed outside the flow path 84f. Since the flow path 84f is very small, when the pump 91 can be installed outside the flow path 84f, assembly of the photonic crystal biosensor 11c becomes easy. Therefore, the photonic crystal biosensor 11c according to the third embodiment can be easily assembled, and can decrease the amount of the solution necessary to reach the equilibrium state, while making the change of the reflected light of the light irradiating the metal-film coated photonic crystal 21 faster.
Further, the photonic crystal biosensor 11c according to the third embodiment includes the flow path 84f formed by being surrounded by the table 83, the inner walls of the thin plate 84 facing opening portion 84h, and the cover 82. Accordingly, the flow path 84f can be formed to be thin, and thus the flow speed of the solution that passes through the space 21u above the reflection surface 69 can be made large. Accordingly, the target substance can be promptly captured on the reflection surface 69. Therefore, the photonic crystal biosensor 11c according to the third embodiment can decrease the amount of the solution necessary to reach the equilibrium state, while making the change of the reflected light of the light irradiating the metal-film coated photonic crystal 21 faster.
Next, an experiment result using the target substance detecting device illustrated in
Further, a relationship between the flow speed of the solution and a cross section shape of the flow path 84f is preferably a relationship where the Reynolds number becomes from 0.01 to 2000, both inclusive. When the Reynolds number is 2000 or less, a turbulent flow component is less likely to occur, and thus a possibility of occurrence of a noise in the measurement result of the reflected light becomes low. Further, when the Reynolds number is 2000 or less, large pressure is less likely to be applied to the flow path 84f, and thus a possibility of leakage of the solution from the flow path 84f becomes low. Further, the relationship between the flow speed of the solution and the cross section shape of the flow path 84f is preferably a relationship where the Reynolds number becomes from 0.01 to 1000, both inclusive. When the Reynolds number is 1000 or less, a stable laminar flow is more likely to occur, and thus a possibility of occurrence of a noise in the measurement result of the reflected light becomes lower.
(Control Unit 13c)
Next, the control unit 13c illustrated in
(Method of Detecting Target Substance)
Next, a method of detecting the target substance (target substance detection method) using a target substance detecting device 10 illustrated in
Next, in step S102, phosphate buffered saline (PBS) is brought to come in contact with the reflection surface 69 of the metal-film coated photonic crystal 21. Following that, rinsing processing that performs removal using centrifugal force or the like is performed several times.
Next, in step S103, skim milk, as a blocking agent 75, is brought to come in contact with the reflection surface 69 of the metal-film coated photonic crystal 21. The reflection surface 69 of the metal-film coated photonic crystal 21 is exposed to the skim milk for a predetermined time, or at a predetermined temperature for a predetermined time, as needed. In this way, the skim milk is absorbed in a non-absorption portion of the cortisol antibody on the reflection surface 69 of the metal-film coated photonic crystal 21.
Following that, in step S104, rinsing processing with the phosphate buffered saline is performed several times, similarly to the rinsing processing (step S102). With the above operation, predetermined processing is applied on the reflection surface 69 of the metal-film coated photonic crystal 21, and the photonic crystal biosensor 11c is formed.
Next, in step S105, the photo-detection section 12 detects reflected light LR from the reflection surface 69 when the reflection surface 69 of the photonic crystal 65 is irradiated with light, and the control unit 13c measures the reflected light LR. The control unit 13c measures a spectrum of reflected light intensity of the reflected light LR. The wavelength of the light (incident light LI) irradiating the reflection surface 69 is, for example, from 300 nm to 2000 nm, both inclusive.
Next, in step S106, first, saliva as a solution containing cortisol is prepared. Sampling of the saliva and pretreatment such as removal of impurities are performed using a commercially available saliva collecting kit. The preparation of the saliva can be performed at any time before the saliva is brought to come in contact with the photonic crystal biosensor 11c. For example, the preparation of the saliva may be performed before the formation of the photonic crystal biosensor 11c, may be performed in parallel with the formation of the photonic crystal biosensor 11c, or may be performed after the measurement of the reflected light intensity. 10 μL to 50 μL of the saliva subjected to the sampling and the pretreatment is brought to come in contact with the photonic crystal biosensor 11c.
Next, in step S107, the reflection surface 69 of the metal-film coated photonic crystal 21 is exposed to the solution containing cortisol, for a predetermined time, or at a predetermined temperature for a predetermine time, as needed. In this way, the antigen/antibody reaction is performed. The antigen/antibody reaction of step 107 is performed while the solution is circulated in step S2 of
Following that, in step S108, rinsing processing is performed with the phosphate buffered saline several times, similarly to the rinsing processing (step S104).
Next, in step S109, the reflection surface 69 of the metal-film coated photonic crystal 21 is irradiated with light, using the target substance detecting device 10c. The light irradiating the reflection surface 69 at this time is the same as the light irradiating the reflection surface 69 in step S15. Then, the target substance detecting device 10c measures the spectrum of the reflected light intensity of the reflected light LR from the reflection surface 69.
The wavelength in the extreme value of the reflected light intensity of the photonic crystal biosensor 11c is changed, by being subject to the antigen/antibody reaction on the reflection surface 69 or in the vicinity of the reflection surface 69. Therefore, cortisol in the saliva can be detected from a difference between the wavelengths in the extreme value of the reflected light intensity before and after the reaction, that is, the wavelength shift amount. Further, the concentration of cortisol in the saliva can be obtained from the wavelength shift amount.
In step S110, the control unit 13c obtains shifting (wavelength shift amount) of the wavelength in the extreme value (minimum value) of the reflected light intensity (or the reflectance) measured in step S109. The wavelength shift amount is, for example, a difference λ2−λ1 between the wavelength λ2 after the target substance is captured on the reflection surface 69, and the wavelength λ1 corresponding to the extreme value (minimum value) of the reflected light intensity (or the reflectance) of when the target substance is not captured on the reflection surface 69.
In step S111, the control unit 13c determines that cortisol exists in the saliva, when there is a predetermined amount or more of the wavelength shift amount. Further, the control unit 13c determines the concentration of cortisol, using a relational expression between the wavelength shift amount and the concentration of cortisol, based on the wavelength shift amount. At this time, the relational expression is obtained in advance, and is stored in a storage unit of the control unit 13c.
In the above-described example, the wavelength shift amount is obtained using the wavelength of the extreme value of the reflected light intensity on the reflection surface 69 in a state where the target substance is not captured. However, an embodiment is not limited to the example. Further, in steps S15 and S19, when there is a plurality of extreme values, an extreme value to be focused is appropriately selected. Then, the wavelength λ1 and the wavelength λ2 are obtained about the selected extreme value.
Note that, in the third embodiment, in the metal-film coated photonic crystal 21, the antibody 74 is fixed to the reflection surface 69. However, an embodiment is not limited to the example, and the metal-film coated photonic crystal 21 may be used where the antibody 74 is not fixed to the reflection surface 69.
A target substance detecting device including a target substance capturing device according to a fourth embodiment will be explained. A target substance capturing device according to the fourth embodiment is similar to the third embodiment, except that a substance to be fixed to a reflection surface 69 of a metal-film coated photonic crystal 21 is an antigen (target substance) 76, and an antibody 74 is absorbed by the antigen 76, and thus overlapping description is omitted.
First, as illustrated in
The amount of the antigen 76 fixed to the metal-film coated photonic crystal 21 is constant. Therefore, when the antibody 74 is absorbed to the antigen 76 fixed to the metal-film coated photonic crystal 21 and a complex 77 (see
Following that, as illustrated in
Next, the reflection surface 69 of the photonic crystal 65 is irradiated with the light (incident light) LI of from 300 nm to 900 nm, both inclusive, in parallel light, and such that the optical axis is perpendicular to the reflection surface 69. A wavelength with which the intensity or the reflectance of the reflected light LR of this time becomes the extreme value (the minimum value in this example) is λ1.
Next, as illustrated in
The wavelength shift amount of the wavelength with which the reflectance of light becomes the extreme value is λ2−λ1. The wavelength shift amount is changed according to change of a surface state on the reflection surface 69 of the metal-film coated photonic crystal 21. Detection and quantification of the antigen 76 are performed based on the wavelength shift amount. The photonic crystal biosensor 11c outputs an optical physical amount. This physical amount correlates with the change of the surface state on the reflection surface 69, and correlates with the amount of the complex 77 formed by the antigen 76 and the antibody 74 fixed to the reflection surface 69.
In the fourth embodiment, cortisol as the antigen 76 is fixed to the metal-film coated photonic crystal 21, and the anti-cortisol as the antibody 74 is brought to react with cortisol. The change of the surface state of the metal-film coated photonic crystal 21 becomes large, and the sensitivity of the photonic crystal biosensor 11c is improved, in the case such as the fourth embodiment in which the anti-cortisol antibody is brought to react with cortisol after cortisol is fixed to the reflection surface 69 of the metal-film coated photonic crystal 21, compared with the case such as the third embodiment in which the antigen 76 is brought to react with the antibody 74 after the antibody 74 is fixed to the reflection surface 69 of the metal-film coated photonic crystal 21.
Next, a method of measuring the concentration of the antigen 76 will be explained. An amount of a combining site of the antigen 76 contained in the sample S is X, and the known amount of the antibody 74 in the mixture M is C. With regard to the relationship between X and C, X is made smaller than C (X<C). In the mixture M, the antigen 76 and the antibody 74 have an antigen/antibody reaction, and the complex 77 is formed. Since X is smaller than C (X<C), the amount of the antibody 74 in the mixture M becomes C−X. Then, the mixture M is brought to come in contact with the reflection surface 69 to which the constant amount of the antigen 76 is fixed, the antibody 74 in the mixture M have the antigen/antibody reaction with the antigen 76 of the reflection surface 69, and the complex 77 is formed. The amount of the antigen 76 fixed to the reflection surface 69 is equal to or more than the amount C−X of the antibody 74 in the mixture M.
When all of the antibodies 74 in the mixture M have the antigen/antibody reaction with the antigen 76 of the reflection surface 69, the amount of the complex 77 becomes C−X. A wavelength shift amount Δλ obtained from the wavelengths λ1 and λ2 measured before and after the mixture M is brought to come in contact with the reflection surface 69 corresponds to the amount of the complex 77 fixed to the reflection surface 69. Therefore, Δλ=k×(C−X) is satisfied. k is a constant for converting the wavelength shift amount Δλ into the amount of the complex 77. The relationship between the amount of the complex 77 fixed to the reflection surface 69 and the wavelength shift amount Δλ is obtained in advance. From the above relational expression, the amount X of the antigen 76 can be obtained by C−Δλ/k. The concentration of the antigen 76 can be obtained based on the amount X of the antigen 76.
Further, in the fourth embodiment, the photonic crystal biosensor 11c may cause a secondary antibody which specially reacts with the complex 77, to react with the complex 77 fixed to the reflection surface 69 of the metal-film coated photonic crystal 21. The secondary antibody functions as a complex binding substance. An excessive amount of the secondary antibody than that of the first complex (complex) 77 is brought to come in contact with the reflection surface 69 of the metal-film coated photonic crystal 21. Then, the secondary antibody is attached to all of the complexes 77 to obtain a second complex. Accordingly, the change of the surface state of the metal-film coated photonic crystal 21 becomes larger. As a result, the sensitivity of the photonic crystal biosensor 11c is further increased. The secondary antibody can be used as it is, or may be used by adding another substance. The change of the surface state of the metal-film coated photonic crystal 21 becomes larger as the secondary antibody is larger. Therefore, after another substance is added to the secondary antibody, the secondary antibody is brought to react with the complex 77, so that the sensitivity of the photonic crystal biosensor 11c is further increased.
When the second complex is formed on the reflection surface 69, the reflection surface 69 of after the second complex is formed is irradiated with light. A wavelength with which the reflected light intensity or the reflectance obtained as a result becomes the extreme value (the minimum value in this example) is λ2. When there is a plurality of extreme values, an extreme value to be focused is appropriately selected. The wavelength λ1 and the wavelength λ2 are obtained about the selected arbitrary extreme value. The photonic crystal biosensor 11c outputs an optical physical amount. This physical amount correlates with the change of the surface state on the reflection surface 69, and correlates with the amount of the second complex fixed to the reflection surface 69. Then, the second complex is detected and quantified. The amount of the second complex is the same as the amount of the complex 77. Therefore, the complex 77 can be quantified.
Configuration elements of the above-described first to third embodiments include those that can be easily assumed by persons skilled in the art, those that are substantially identical, and those in a scope of so-called equivalents. Further, the above-described configuration elements can be appropriately combined. Further, various omissions, replacements, and changes of the configuration elements can be performed without departing from the gist of the present embodiments.
Number | Date | Country | Kind |
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2013-095947 | Apr 2013 | JP | national |
2013-095953 | Apr 2013 | JP | national |
2013-095974 | Apr 2013 | JP | national |
2014-025826 | Feb 2014 | JP | national |
This application is a National Stage of International Application No. PCT/JP2014/061899, filed Apr. 28, 2014, claiming priority based on Japanese Patent Application Nos. 2013-095947, 2013-095953, 2013-095974, filed Apr. 30, 2013, and 2014-025826, filed Feb. 13, 2014, the contents of all of which are incorporated herein by reference in their entirety.
Filing Document | Filing Date | Country | Kind |
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PCT/JP2014/061899 | 4/28/2014 | WO | 00 |