Patent Application
20130230931
The present invention relates to a target substance detecting apparatus that detects a target substance, and a target substance detecting method.
There has been known a biosensor using a photonic crystal as a unit for detecting a target substance such as protein or a cell or for measuring a concentration thereof (e.g., Non Patent Literature 1). In this technique, light is irradiated to a photonic crystal substrate having a metal thin film formed thereon, and the reflected light is observed, whereby a concentration of a target substance, that is a subject to be detected, is measured.
The biosensor described in Non Patent Literature 1 irradiates light to the photonic crystal substrate with a bundle fiber formed by bundling plural optical fibers. The light emitted from the bundle fiber diffuses with a certain angle, so that the intensity of the reflected light reflected on the photonic crystal substrate is extremely low. Therefore, the biosensor described in Non Patent Literature 1 might have deteriorated detection accuracy for the target substance.
The present invention aims to provide a target substance detecting apparatus and a target substance detecting method that can accurately detect a target substance.
According to a first aspect of the present invention, a target substance detecting apparatus includes: a target substance trapping unit configured to include a structure, which has a reflection surface including concave portions and convex portions formed regularly on its surface, covered by a metal film, and trapping a target substance, the structure reflecting light irradiated to the reflection surface;
an optical detecting unit configured to irradiate parallel light to the reflection surface, and to detect reflected light of the parallel light reflected on the reflection surface; and a processing unit configured to obtain a wavelength at an extreme value of the reflected light detected by the optical detecting unit, and to detect at least whether the target substance is present or not based upon a shift of the obtained wavelength at the extreme value.
According to another aspect of the present invention, the target substance trapping unit includes a target substance trapping substance that is fixed on the reflection surface for trapping the target substance.
According to another aspect of the present invention, in the target substance trapping unit, the reflection surface on which a target substance of the same type as the target substance, which is the subject to be detected, is fixed in a constant amount is brought into contact with a mixture of a target substance trapping substance, which specifically reacts with the target substance fixed on the reflection surface, in a known amount, the target substance that is the subject to be detected, and a sample containing the target substance that is the subject to be detected.
According to another aspect of the present invention, the outermost surface of the metal film is gold.
According to another aspect of the present invention, the thickness of the metal film is 30 nm or more and 1000 nm or less.
According to another aspect of the present invention, the optical detecting unit includes: a first optical fiber that guides the light from the light source; a collimator lens that makes the light emitted from the first optical fiber the parallel light; and a second optical fiber that receives the reflected light, and guides the received light to a light-receiving unit.
According to another aspect of the present invention, the first optical fiber and the second optical fiber are integral on an emission side of the first optical fiber and an incident side of the second optical fiber.
According to another aspect of the present invention, the structure is a photonic crystal.
According to a second aspect of the present invention, a target substance detecting apparatus includes: a target substance trapping unit configured to include a photonic crystal, which has a reflection surface including concave portions and convex portions formed regularly on its surface, covered by a metal film, and trapping a target substance, the structure reflecting light irradiated to the reflection surface; an optical detecting unit configured to irradiate parallel light to the reflection surface, and to detect reflected light of the parallel light reflected on the reflection surface; and a processing unit configured to obtain a wavelength at an extreme value of the reflected light detected by the optical detecting unit, and to obtain a concentration of the target substance based upon a shift of the obtained wavelength at the extreme value.
According to another aspect of the present invention, the target substance trapping unit includes a target substance trapping substance that is fixed on the reflection surface for trapping the target substance.
According to another aspect of the present invention, in the target substance trapping unit, the reflection surface on which a target substance of the same type as the target substance, which is the subject to be detected, is fixed in a constant amount is brought into contact with a mixture of a target substance trapping substance, which specifically reacts with the target substance fixed on the reflection surface, in a known amount, the target substance that is the subject to be detected, and a sample containing the target substance that is the subject to be detected.
According to a third aspect of the present invention, a target substance detecting method includes: trapping a target substance on a reflection surface of a structure that has the reflection surface including concave portions and convex portions formed regularly on its surface, covered by a metal film, and reflecting light irradiated to the reflection surface; irradiating parallel light on the reflection surface that traps the target substance; obtaining a wavelength at an extreme value of the reflected light of the parallel light reflected on the reflection surface; and obtaining a concentration of the target substance based upon a shift of the obtained wavelength at the extreme value.
According to a fourth aspect of the present invention, a target substance detecting method includes: fixing a target substance of the same type as a target substance that is a subject to be detected, in a constant amount, on a reflection surface of a structure that has the reflection surface including concave portions and convex portions formed regularly on its surface, covered by a metal film, and reflecting light irradiated to the reflection surface; allowing a mixture of a target substance trapping substance, which specifically reacts with the target substance fixed on the reflection surface, in a known amount, and a sample containing the target substance that is the subject to be detected, to be brought into contact with the reflection surface; irradiating parallel light to the reflection surface with which the mixture is in contact; obtaining a wavelength at an extreme value of the reflected light of the parallel light reflected on the reflection surface; and obtaining a concentration of the target substance based upon a shift of the obtained wavelength at the extreme value.
According to another aspect of the present invention, the metal film is gold, and thickness thereof is 30 nm or more and 1000 nm or less.
According to another aspect of the present invention, the structure is a photonic crystal.
The present invention can provide a target substance detecting apparatus and a target substance detecting method that can accurately detect a target substance.
An embodiment (hereinafter referred to an exemplary embodiment) for embodying the present invention will be described in detail with reference to the drawings.
The photonic crystal biosensor 200 serving as the target substance trapping unit will firstly be described. The photonic crystal biosensor 200 serving as the target substance trapping unit is a structure that has a reflection surface including concave portions and convex portions regularly formed on its front surface, and from which a reflected light is obtained when light (parallel light) with a specific wavelength is irradiated to the reflection surface. The photonic crystal biosensor 200 serving as the target substance trapping unit may include a target substance trapping substance that is fixed on the reflection surface, and that traps the target substance.
The structure from which the reflected light is obtained with a specific wavelength when light is irradiated to the reflection surface on which the concave portions and convex portions are regularly formed is generally called a photonic crystal.
The photonic crystal 100 according to the exemplary embodiment of the present invention includes a reflection surface 112 on which convex portions 111 are regularly formed. The surface on which the convex portions 111 are regularly formed is the reflection surface 112 of the photonic crystal 100. When light is irradiated to the reflection surface 112, light of a specific wavelength dependent upon the shape and material of the photonic crystal 100 is reflected. In the present exemplary embodiment, a diameter D of the columnar convex portion 111 is about 250 nm. The distance C between the centers of the columnar convex portions 111 is about 500 nm. The columnar convex portions 111 are arranged in a triangular lattice. The height H of the columnar convex portion 111 is about 200 nm. It is preferable that the diameter D of the columnar convex portion 111 is 50 nm or more and 1000 nm or less. The distance C between the columnar convex portions 111 is preferably more than 100 nm and not more than 2000 nm. The size of the columnar convex portion 111 is not limited to those described above.
The shape and size of the photonic crystal 100 serving as the structure according to the present exemplary embodiment are not limited to those illustrated in
Examples of usable material of the photonic crystal 100 include an organic material such as a synthetic resin, and an inorganic material such as a metal or ceramic.
Examples of usable synthetic resin include thermoplastic resin such as polyethylene, polypropylene, polymethylpentene, poly cycloolefin, polyamide, polyimide, acryl, polymethacrylic acid ester, polycarbonate, polyacetal, polytetrafluoloethylene, polybutylene terephthalate, polyethylene terephthalate, polyvinyl chloride, polyvinylidene chloride, polystyrene, polyphenylene sulfide, polyether sulphone, or polyether ether ketone; and thermosetting resin such as phenolic resin, urea resin, or epoxy resin.
Examples of usable ceramic include silica, alumina, zirconia, titania, or yttria.
Various alloys such as a ferrous material can be used as the metal. Specifically, stainless steel, titanium, or titanium alloy can preferably be used.
Among various materials described above, poly cycloolefin synthetic resin or silica ceramic is more preferable from the viewpoint of an optical characteristic, processability, resistance to solution containing a target substance (a subject to be a target), adsorption of a target substance trapping substance (specific bonding substance), and resistance to cleaning agent. The poly cycloolefin synthetic resin is the most preferable, since it is excellent in processability.
The photonic crystal 100 is formed by performing microfabrication on the surface of the substrate made of the material described above. Examples of the process include a laser process, thermal nanoimprint, optical nanoimprint, and a combination of a photomask and etching. When the thermoplastic resin such as poly cycloolefin synthetic resin is used as the material, the thermal nanoimprint process is preferable.
In the present exemplary embodiment, as illustrated in
The surface of the photonic crystal 100 is preferably reformed by using 3-triethoxysilylpropylamine (APTES). When the metal film 101 made of Au or Ag is formed on the surface of the photonic crystal 100, the surface of the photonic crystal 100 is preferably reformed by using a carbon chain having a thiol group at one end and a functional group such as an amino group or carboxyl group at the other end, not by using APTES. When the metal film 101 made of a metal other than Au and Ag is formed on the surface of the photonic crystal 100, the surface of the photonic crystal 100 is preferably reformed by using a silane coupling agent having a functional group at one end, such as APTES. Next, one example of a process of forming the photonic crystal 100 by a thermal nanoimprint will be described.
In the case of the cycloolefin polymer, it is preferable that the die DI is heated to about 160° C. (
The target substance trapping substance that traps the target substance will next be described. The target substance is a subject that is to be detected by the target substance detecting apparatus 1, and it may be any one of a polymer such as protein, oligomer, and low-molecular substance. The target substance is not limited to a monoatomic molecule, and it may be a complex made of plural molecules. A bioactive substance present in a living body is used as the target substance, for example, wherein cortisol is preferable. Cortisol is a low-molecular substance having a molecular weight of 362 g/mol. The concentration of cortisol in saliva increases, when a human feels stress. Therefore, the cortisol is focused as a substance for evaluating a degree of stress of a human. When the cortisol is used as the target substance, and its concentration is measured, e.g., when the concentration of the cortisol contained in saliva of a human is measured, the degree of stress can be evaluated.
The target substance trapping substance is a substance that binds to the target substance for trapping the target substance. To bind means not only the chemical binding, but also a binding not based upon the chemical binding, such as the binding with a physical adsorption or van der Waals binding. Preferably, the target substance trapping substance is the one that specifically binds to the target substance for trapping the target substance, and it is preferably an antibody to the target substance serving as an antigen. To specifically react means to selectively form a complex through a reversible or irreversible binding with the target substance. It is not limited to the chemical reaction. A substance, other than the target substance, which specifically reacts with the target substance trapping substance may also be present. Even if a sample includes a substance that reacts with the target substance trapping substance, other than the target substance, and when its affinity is extremely small compared to the target substance, the target substance can be quantized. Examples of usable target substance trapping substance include an antibody to the target substance serving as an antigen, an antibody artificially formed, a molecule made of a substance composing DNA, such as adenine, thymine, guanine, or cytosine, and peptide. When the target substance is cortisol, the target substance trapping substance is preferably a cortisol antibody.
Known methods can be employed for forming the target substance trapping substance. For example, an antibody can be formed by a serum method, hybridoma method, or phage display method. The molecule made of a substance composing the DNA can be prepared by, for example, SELEX (Systematic Evolution of Ligands by Exponential Enrichment). The peptide can be prepared by a phage display method, for example. The target substance trapping substance does not have to be labeled by any enzymes or isotopes. However, it may be labeled by enzymes or isotopes.
In the present exemplary embodiment, the target substance trapping substance is fixed on the reflection surface 112 of the photonic crystal 100. For example, an adsorption is employed as a process of fixing the target substance trapping substance onto the reflection surface 112 of the photonic crystal 100. The adsorption process is as described below, for example. Solution containing the target substance trapping substance is dripped onto the reflection surface 112 of the photonic crystal 100, whereby the target substance trapping substance is adsorbed on the reflection surface 112 for a predetermined time at room temperature, or for a predetermined time as cooled or heated according to need.
The target substance trapping unit allows an antibody (e.g., cortisol antibody) that is bonded only to a specific antigen (e.g., cortisol) to be adsorbed (fixed) beforehand on the surface of the photonic crystal 100. With this process, the photonic crystal biosensor 200 that detects a specific antigen can be formed. This utilizes the optical characteristic of the photonic crystal 100 and various chemical reactions within the body that occur on the surface or in the vicinity of the surface of the photonic crystal 100, e.g., the antigen-antibody reaction in which a specific antigen reacts with only a specific antibody.
The target substance trapping unit may be formed such that a blocking agent (protecting substance) is fixed on the reflection surface 112 of the photonic crystal 100 on which the antibody serving as the target substance trapping substance has already been fixed. The blocking agent is fixed before the target substance is in contact with the target substance trapping unit. The surface of the photonic crystal 100 is generally super-hydrophobic, and it may adsorb impurities other than the antibody serving as the target substance trapping substance by hydrophobic interaction. Since the optical characteristic of the photonic crystal 100 is greatly affected by the surface state, the detection accuracy is enhanced when the impurities are less adsorbed on the surface of the photonic crystal 100.
Therefore, it is preferable that a so-called blocking agent is fixed beforehand on the portion other than the portion where the antibody serving as the target substance trapping substance is adsorbed (fixed) in order to prevent the fixation of the impurities. In order to preliminarily adsorb the blocking agent, the blocking agent is brought into contact with the surface of the photonic crystal 100. Examples of usable blocking agent include skim milk, or bovine serum albumin (BSA). Next, a basic principle in which the photonic biosensor 200 serving as the target substance trapping unit detects the antigen serving as the target substance and its concentration will be described.
As illustrated in
As illustrated in
The type of various biological materials or low-molecular materials, such as protein, which are subjects to be detected, can be changed by changing the type of the combination of antigen and antibody based upon the above-mentioned principle.
The present exemplary embodiment utilizes a phenomenon in which the extreme value of the wavelength of the reflected light is shifted due to the surface plasmon resonance and/or localized surface plasmon resonance upon the irradiation of light to the reflection surface 112 of the photonic crystal 100 having a nanostructure covered by the metal film 101. The present exemplary embodiment detects whether or not there is a target substance trapped on the reflection surface 112 of the photonic crystal 100, and detects the concentration of the target substance.
On the photonic crystal biosensor 200, the antigen 114 is trapped by the antibody 113 serving as the target substance trapping substance fixed on the reflection surface 112 of the photonic crystal 100, whereby the state of the reflection surface 112 is changed, and the change in the reflected light LR is observed. The photonic crystal biosensor 200 outputs an optical physical amount. This physical amount is correlated to the change in the surface state of the reflection surface 112 of the photonic crystal 100, and also correlated to the amount of complex whose antigen 114 is trapped by the antibody 113. Examples of the optical physical amount include the shift amount of the wavelength by which the intensity of the reflected light becomes an extreme value (maximum value or minimum value), an amount of change of the light reflectivity, the shift amount of the wavelength by which the light reflectivity becomes an extreme value (maximum value or minimum value), the intensity of the reflected light, and an amount of change of the extreme value of the intensity of the reflected light. In the present exemplary embodiment, the shift amount of the wavelength by which the intensity or reflectivity of the reflected light becomes the extreme value (maximum value or minimum value) is used.
The optical physical amount is outputted in the manner described below, for example. Light is orthogonally incident on the reflection surface 112 of the photonic crystal 100, and the reflected light is detected. Alternatively, light is incident with an angle to a perpendicular line of the reflection surface 112 of the photonic crystal 100, and the reflected light is detected. The detection of the reflected light can make the target substance detecting apparatus 1 illustrated in
The shape of the opening 240 is not limited to columnar as illustrated. Various shapes can be applied to the opening 240, so long as it has the liquid-droplet holding function. When the opening 240 is columnar, the diameter can be changed according to the type of the combination of the antigen and antibody, a required measurement accuracy, or an optical system of a detector of the reflected light. The diameter of the opening 240 is preferably 0.5 mm to 10 mm. More preferably, the diameter of the opening 240 is about 2 mm to 6 mm, in consideration of the operability and manageability during the rinsing operation and adsorption operation.
The materials for the plate 220 and the lower plate 210 are not particularly limited. Considering the cleanness of the surface, the plate 220 and the lower plate 210 made of stainless steel, poly cycloolefin resin, or silica are preferably used. Another photonic crystal biosensor 200 according to the present invention will next be described.
The plate 220 formed with the opening 240 illustrated in
The plate 220 may be made of a water-repellent material, oil-repellant material, or a material having water-repellent property and oil-repellent property. A surface treatment or coating that exhibits hydrophobic property, hydrophilic property, water-repellent property, or oil-repellent property may be performed to the plate 220. This process can allow the solution to be accurately collected on the concave portion 241.
It is preferable that a fixing member (target-substance trapping unit fixing unit, photonic crystal biosensor fixing unit) for fixing the photonic crystal biosensor 200 on the position determined with respect to the optical detecting unit 300 illustrated in
The photonic crystal biosensor 200 is uniformly formed by thermal nanoimprint. However, if more accurate detection and measurement are expected, it is preferable that the light incident position or the position on which the light is reflected are accurately positioned considering the variation in the optical characteristic of the photonic crystal biosensor 200.
Specifically, the positional relationship between the photonic crystal biosensor 200 and a later-described measurement probe during the measurement after the antigen-antibody reaction and the positional relationship before the antigen-antibody reaction are preferably the same, and the same portion is preferably measured. Accordingly, the distance between the measurement probe and the reflection surface 112 of the photonic crystal biosensor 200 is preferably the same after the antigen-antibody reaction and before the antigen-antibody reaction. The distance is preferably fixed to be 50 to 500 μm. The photonic crystal biosensor 200 includes the plate 220. Therefore, the plate 220 functions as a spacer, whereby the distance between the measurement probe and the reflection surface 112 of the photonic crystal biosensor 200 can be made constant.
It is preferable that the photonic crystal biosensor 200 is marked with a positioning marker that displays the specific position on the reflection surface 112. The positioning marker can be formed by photolithography, sputtering, vapor deposition, lift-off process utilizing these techniques, printing by use of ink, or pattern formation by imprinting.
The marker can be formed on the front surface (on the reflection surface 112) or the back surface (the reverse side of the reflection surface 112) of the photonic crystal biosensor 200, so long as it can read the position. The marker may be formed on the photonic crystal 100 outside the measured portion of the photonic crystal 100. The marker may also be formed on the plate 220 or the lower plate 210 illustrated in FIGS. 6 and 7-1.
The central portion of each marker is defined as a measurement area A for measuring the reflected light. Specifically, in the photonic crystal biosensor 200 illustrated in
The opening 511 is covered by the sheet 520 after the target substance is placed in a space 512 enclosed by the inner wall of the opening 511 of the perforated cover 510. In this case, the inner wall of the opening 511 of the perforated cover 510, the inner wall of the opening 240 of the photonic crystal biosensor 200, and the reflection surface 112 of the photonic crystal 100 form the liquid-droplet holding portion. The sheet 520 functions as a covering member. The inner wall of the opening 511 means the inner wall of the perforated cover 510, which is the border surface of the perforated cover 510 and the opening 511.
The perforated cover 510 and the sheet 520 can prevent evaporation of the solution dripped into the opening 240 of the photonic crystal biosensor 200, thereby being capable of suppressing the change in the concentration of the solution due to the evaporation during the antigen-antibody reaction. The perforated cover 510 and the sheet 520 also have an effect of preventing foreign matters from externally mixing into the solution.
Since the solution is filled in the space 512 formed by the perforated cover 510, the sheet 520, and the inner wall of the opening 240 of the photonic crystal biosensor 200, the reflected light can more accurately be measured with the solution being filled. In this case, the sheet 520 is preferably made of a transparent material, and more preferably, the sheet 520 is made of a material that absorbs less light of wavelength at the extreme value of the intensity of the reflected light. For example, silica is preferable for the material of the sheet 520, when the reflected light is measured within the range from a visible-light region to an ultraviolet region. The optical detecting unit 300 of the target substance detecting apparatus illustrated in
The optical detecting unit 300 of the target substance detecting apparatus illustrated in
With this structure, the measurement probe 320 can emit the incident light LI irradiated to the reflection surface 112 of the photonic crystal 100 of the photonic crystal biosensor 200 and can allow the reflected light LR from the reflection surface 112 to be incident thereon on almost the same position. The measurement probe 320 is configured as described above, and the light from the measurement probe 320 is made parallel by using the collimator lens 360. Therefore, the optical detecting unit 300 can allow the incident light LI that is the parallel light to be perpendicularly incident on the reflection surface 112. Further, the optical detecting unit 300 can receive the reflected light LR that is perpendicularly reflected on the reflection surface 112. With this structure, the measurement probe 320 can minimize the reduction in the intensity of the reflected light, and can mainly detect the component of 0th order light of the reflected light LR. Accordingly, the correct information of the reflection surface 112 of the photonic crystal 100 can be acquired. As a result, the optical detecting unit 300 including the measurement probe 320 has enhanced detection accuracy of the target substance and the enhanced measurement accuracy of the concentration. The process of detecting the reflected light LR is not limited to the process of using the measurement probe 320. For example, a half mirror may be arranged between the collimator lens 360 and the reflection surface 112, and the reflected light LR may be separated by the half mirror to be guided to the optical detecting apparatus 330 from the second optical fiber 350. The thickness of the metal film 101 formed on the reflection surface 112 of the photonic crystal 100 will next be described.
When the thickness of the metal film 101 is small, a part of the incident light to the photonic crystal 100 might transmit the metal film 101. As a result, an information amount acquired from the reflected light might be reduced, or a lot of unnecessary information might be included in the reflected light from the photonic crystal 100, such as diffraction light or the reflected light from the back surface of the photonic crystal 100. Increasing appropriately the thickness of the metal film 101 can reduce the unnecessary information included in the reflected light from the photonic crystal 100 so as to be capable of enhancing the detection accuracy of the target substance and the measurement accuracy of the concentration. When the thickness of the metal film 101 is appropriately small, a fine pattern can easily be formed on the front surface of the photonic crystal 100, thus preferable. For example, it becomes easy to secure the dimension of the pattern, since the corner of the pattern becomes sharp.
From this viewpoint, the thickness of the metal film 101 is preferably set to be 30 nm or more and 1000 nm or less, more preferably 150 nm or more and 500 nm or less in the present exemplary embodiment. From the result in
It is found from
The reflectivity is calculated from the intensity of the reflected light. Therefore, the reflectivity is greatly affected by the magnitude of the intensity of the reflected light, and this influence appears as noise. The intensity of the reflected light of the optical detecting unit 300 that irradiates the parallel light is larger. Therefore, as illustrated in
In the present exemplary embodiment, a material layer 252 with a thickness of about 2 mm is formed on the reflection surface 112 of the photonic crystal 100, and the material layer 252 is protected by a cover glass 250 having a thickness of about 0.2 mm, as illustrated in
Au, Ag, Pt, and Al were evaluated as metals used for the metal film 101 on the surface of the photonic crystal 100 by using the method of evaluating the sensitivity of the sensor. As a result, when Ag, Pt, and Al were used for the metal film 101 on the surface of the photonic crystal 100, the wavelengths λa, λb, and λc of the reflected light at the respective extreme values Pa, Pb, and Pc were 1.5 times as large as those in the case where Au was used for the metal film 101. As described above, Ag, Pt, and Al have sensitivity 1.5 times larger than Au. It is preferable that an oxide thin film made of Au or SiO2, which is difficult to be oxidized, is formed after the Ag is formed on the surface of the photonic crystal 100, since Ag is easy to be oxidized. In the present exemplary embodiment, Au film having a thickness of 5 nm was formed on the surface of the Ag film having a thickness of 200 nm. The sensitivity was obtained in this test. In the example in which the Au film with a thickness of 5 nm was formed on the surface of the Ag film with a thickness of 200 nm, the sensitivity was 1.5 times as large as the case where the Au film with a thickness of 200 nm was used. The change in the sensitivity was not observed depending upon the presence of the Au film with a thickness of 5 nm. It is preferable that an oxide thin film made of Au or SiO2, which is difficult to be oxidized, is also formed after the Al is formed on the surface of the photonic crystal 100, since Al is easy to be oxidized like Ag. It is also preferable that the oxide thin film made of Au or SiO2 is formed for the Pt in order to modify with the antibody.
The processing unit 600 in the target substance detecting apparatus 1 illustrated in
Firstly, the reflected light, e.g., the spectrum of the intensity of the reflected light, from the reflection surface 112 upon the irradiation of light to the reflection surface 112 is measured for the photonic crystal 100 by using a detector having a predetermined optical measurement system, i.e., the target substance detecting apparatus 1 (step S101). In this case, cortisol serving as the target substance is not trapped on the reflection surface 112. The metal film 101 is formed on the reflection surface 112. The wavelength of light irradiated to the reflection surface 112 is, for example, 300 nm or more and 900 nm or less.
Subsequently, cortisol antibody solution (cortisol antibody concentration 1 μg/ml to 50 μg/ml) is dripped onto the reflection surface 112 that is the surface of the photonic crystal 100. The photonic crystal 100 is left to stand for a predetermined time, or according to need, is left to stand for a predetermined time at predetermined temperature, in order to allow the cortisol antibody to be adsorbed on the reflection surface 112 that is the surface of the photonic crystal 100 (step S102).
Subsequently, phosphate buffered saline (PBS) is dripped on the reflection surface 112 that is the surface of the photonic crystal 100. Thereafter, a rising process for removing the PBS with centrifugal force is performed plural times (step S103).
Subsequently, skim milk is dripped on the reflection surface 112 of the photonic crystal 100 as the blocking agent 115. The photonic crystal 100 is left to stand for a predetermined time, or according to need, is left to stand for a predetermined time at predetermined temperature, in order to allow the skim milk to be adsorbed on the non-adsorption portion of the cortisol antibody on the reflection surface 112 of the photonic crystal 100 (step S104). Thereafter, a rising process is performed plural times by using phosphate buffered saline as described above (step S105). With the above-mentioned processes, a predetermined process is executed on the reflection surface 112 of the photonic crystal 100, whereby the photonic crystal biosensor 200 is formed.
Subsequently, saliva serving as solution containing cortisol is prepared. The preparation such as the sampling of saliva or removal of impurities is executed by using a commercially available saliva sampling kit. The preparation of saliva may be carried out on any timing before the saliva is dripped on the photonic crystal biosensor 200. For example, the preparation of saliva may be carried out before the photonic crystal biosensor 200 is formed, may be carried out simultaneous with the formation of the photonic crystal biosensor 200, or may be carried out after the intensity of the reflected light is measured.
Subsequently, the saliva to which the sampling and preparation have already been executed in an amount of 10 μL to 50 μL is dripped onto the photonic crystal biosensor 200 (step S106). The photonic crystal biosensor 200 is left to stand for a predetermined time, or according to need, is left to stand for a predetermined time at predetermined temperature, in order to perform the antigen-antibody reaction (step S107), and the rinsing process same as that described above is carried out plural times by using phosphate buffered saline (step S108).
Subsequently, light is irradiated to the reflection surface 112 of the photonic crystal biosensor 200 on which the cortisol has been adsorbed, by using the detector having the predetermined optical system, i.e., the target substance detecting apparatus 1, as in the process described above. The irradiated light is the same as the light irradiated to the reflection surface 112 at step S101. The target substance detecting apparatus 1 measures the reflected light from the reflection surface 112, e.g., the spectrum of the intensity of the reflected light (step S109).
The wavelength at the extreme value of the intensity of the reflected light from the photonic crystal biosensor 200 changes due to the influence from the antigen-antibody reaction on the reflection surface 112 or in the vicinity of the reflection surface 112. Therefore, the cortisol in the saliva can be detected from the difference, before and after the reaction, in the wavelength at the extreme value of the intensity of the reflected light, i.e., the wavelength shift amount Δλ. The concentration of the cortisol in the saliva can be detected from the wavelength shift amount Δλ.
Accordingly, the processing unit 600 in the target substance detecting apparatus 1 obtains the shift (wavelength shift amount Δλ) of the wavelength λ2 at the extreme value (minimum value) of the intensity (or reflectivity) of the reflected light measured at step S109 (step S110). The wavelength shift amount Δλ is the difference λ2−λ1 between the wavelength λ2 after the target substance is trapped on the reflection surface 112 and the wavelength λ1 corresponding to the extreme value (minimum value) of the intensity (or reflectivity) of the reflected light when the target substance is not trapped on the reflection surface 112.
The processing unit 600 determines that the saliva includes cortisol when the wavelength shift amount Δλ is not less than a predetermined amount (step S111). The processing unit 600 determines the concentration of the cortisol by using a relational expression between the wavelength shift amount Δλ and the concentration of the cortisol based upon the wavelength shift amount Δλ (step S111). The relational expression is obtained beforehand, and stored in the storage unit of the processing unit 600.
In the above-mentioned example, the wavelength shift amount Δλ is obtained by using the wavelength at the extreme value of the intensity of the reflected light on the reflection surface 112 on which the target substance is not trapped, but the invention is not limited thereto. For example, the wavelength shift amount Δλ may be obtained by using the wavelength at the extreme value of the intensity of the reflected light from the reflection surface 112 to which the rinsing process (step S103 or step S105) has already been performed. When there are plural extreme values at step S101 and step S109, the target extreme value is selected as needed. The wavelengths λ1 and λ2 are obtained for the selected extreme value.
The target substance detecting apparatus 1 can detect the target substance (in this example, cortisol) from at least solution. The target substance detecting apparatus 1 can also obtain the concentration of the target substance in the solution by using the relationship between the wavelength shift amount Δλ and the concentration of the target substance. In the present exemplary embodiment, light that is the parallel light is perpendicularly irradiated to the reflection surface 112 of the photonic crystal biosensor 200. Simultaneous with the irradiation, the target substance detecting apparatus 1 receives the reflected light perpendicularly reflected from the reflection surface 112, thereby detecting the target substance or obtaining the concentration. With this configuration, the detection accuracy of the target substance and the measurement accuracy of the concentration can be enhanced. As illustrated in
A second embodiment is different from the first embodiment in that a target substance in a constant amount is fixed on a reflection surface of a structure, and the reflection surface on which the target substance is fixed is brought into contact with a mixture of a target substance trapping substance, which specifically reacts with the target substance, in a known amount, the target substance that is the subject to be detected, and a sample. The second embodiment is the same as the first embodiment in that the target substance detecting apparatus detects the target substance and obtains the concentration of the target substance based upon the wavelength shift at the extreme value of the intensity or reflectivity of the reflected light.
An antibody (secondary antibody) to an antibody is used as a complex binding substance, and it is reacted with a complex 116 (see
In this case, after the complex (first complex) 116 is formed on a reflection surface 112 of the photonic crystal 100, the complex binding substance (e.g., secondary antibody) that specifically reacts with the first complex 116 is brought into contact with the reflection surface 112 of the photonic crystal 100 in an amount exceeding the amount of the first complex 116. Then, the first complex 116 is completely converted into the second complex. Thereafter, the photonic crystal biosensor 200 outputs a physical amount (in the present exemplary embodiment, the wavelength at the extreme value of the intensity or reflectivity of the reflected light) correlated to the amount of the second complex. With this process, the second complex is detected and quantized. The amount of the second complex is the same as the amount of the first complex 116. Therefore, the first complex 116 can be quantized.
As illustrated in
The antigen 114 serving as the target substance is fixed on the reflection surface 112 of the photonic crystal 100. Since the photonic crystal 100 has the metal film 101 formed on its surface, it is preferable that the reflection surface 112 that is the surface of the metal film 101 is reformed by using a carbon chain having a thiol group at one end and a functional group such as an amino group or carboxyl group at the other end. When the metal film 101 made of a metal other than Au or Ag is formed on the surface of the photonic crystal 100, it is preferable that the reflection surface 112 of the photonic crystal 100 is reformed by using silane coupling agent having a functional group at one end, such as APTES. Examples of the process of fixing the antigen 114 on the reflection surface 112 of the photonic crystal 100 include a chemical binding process and physical binding process such as a covalent binding, chemical adsorption, or physical adsorption. These processes can appropriately be selected according to the property of the antigen 114. For example, when the adsorption is selected as the process of fixing the antigen, the process described below is executed. For example, solution containing the antigen 114 is dripped on the reflection surface 112 of the photonic crystal 100, whereby the antigen 114 is adsorbed on the reflection surface 112 for a predetermined time at room temperature, or according to need, for a predetermined time as cooled or heated.
The amount of the antigen 114 fixed on the photonic crystal 100 is constant. With this process, when the antigen 114 to be fixed and the antibody 113 serving as the target substance trapping substance form the complex 116, the photonic crystal biosensor 200 can output the physical amount correlated to the amount of the formed complex 116. The constant amount of the antigen 114 to be fixed may appropriately be changed. For example, the amount can be set to be an optimum amount depending upon the range of the amount of the antigen 114 contained in a sample S.
The photonic crystal 100 may be formed such that a blocking agent (protecting substance) 115 is fixed on the reflection surface 112 on which the antigen 114 is fixed as illustrated in
Therefore, it is preferable that the so-called blocking agent 115 is preliminarily fixed on the portion other than the portion where the antigen 114 is adsorbed (fixed) in order to prevent the impurities from being fixed. In order to adsorb the blocking agent 115 beforehand, the blocking agent 115 is brought into contact with the surface of the photonic crystal 100. Examples of usable blocking agent 115 include skim milk, or bovine serum albumin (BSA).
The detection and quantization of the antigen 114 are executed based upon the optical physical amount outputted from the photonic crystal biosensor 200, e.g., based upon the shift amount of the wavelength by which the light intensity becomes the extreme value, or the shift amount of the wavelength by which the light reflectivity becomes the extreme value. The detection and quantization (e.g., the determination of concentration) of the antigen 114 executed based upon the shift amount of the wavelength by which the light reflectivity becomes the extreme value will be carried out as described below.
As illustrated in
Subsequently, a mixture M of the sample S and the antibody 113 in a known amount illustrated in
When the second complex is formed on the reflection surface 112, light is irradiated to the reflection surface on which the second complex has already been formed. The wavelength by which the intensity (or reflectivity) of the reflected light in this case becomes the extreme value (in this example, the minimum value) is defined as λ2. If there are plural extreme values, the target extreme value is selected as needed. The wavelengths λ1 and λ2 are obtained for the selected extreme value.
The wavelength shift amount Δλ of the wavelength by which the light reflectivity becomes the extreme value is λ2−λ1. The wavelength shift amount Δλ changes according to the change in the surface state of the reflection surface 112 of the photonic crystal 100. The antigen 114 is detected and quantized based upon Δλ. The quantization (in this example, the determination of the concentration of the antigen 114) of the antigen 114 will next be described.
It is supposed that the amount of the binding portion of the antigen 114 contained in the sample S is defined as X, and the known amount of the antibody 113 is defined as C. In this case, X<C is established. In the mixture M, the antigen 114 and the antibody 113 cause the antigen-antibody reaction to form the complex 116. Since X<C, the amount of the antibody 113 in the mixture M becomes C−X. When the mixture M is brought into contact with the reflection surface 112 on which the antigen 114 in a constant amount is fixed, the antibody 113 in the mixture M causes the antigen-antibody reaction with the antigen 113 on the reflection surface 112, whereby the complex 116 is formed. The amount of the antigen 114 fixed on the reflection surface 112 is not less than C−X of the amount of the antibody 113 in the mixture M.
When all antibodies in the mixture M cause the antigen-antibody reaction with the antigens 114 on the reflection surface 112, the amount of the complex 116 becomes C−X. The wavelength shift amount Δλ obtained from the wavelengths λ1 and λ2 that are measured after and before the mixture M is brought into contact with the reflection surface 112 corresponds to the amount of the complex 116 fixed on the reflection surface 112. Accordingly, Δλ=k×(C−X) is established, wherein k is a constant for converting the wavelength shift amount Δλ into the amount of the complex 116. The relationship between the amount of the complex 116 fixed on the reflection surface 112 and the wavelength shift amount Δλ is determined beforehand. The amount X of the antigen 114 can be obtained from C−Δλ/k according to the above-mentioned relational expression. The concentration of the antigen 114 can be obtained based upon the amount X of the antigen 114.
The exemplary first and second embodiments have been described above. However, the exemplary first and second embodiments are not limited to those described above. The components in the above-mentioned first and second embodiments include those that can easily be arrived by a person skilled in the art, those substantially equal, and equivalents.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2012/055245 | 3/1/2012 | WO | 00 | 8/28/2012 |
