JP6853974B2 - Biological substance detector - Google Patents

Description

The present invention relates to a biomaterial detection device using surface plasmon resonance or optical waveguide mode.

Sensors using surface plasmon resonance (SPR) are known as sensors for detecting various substances such as proteins in biological samples, pathogens, metal ions in water, and organic molecules (for example, Non-Patent Documents 1 to 7). reference). This type of sensor is called an SPR sensor, and a Klechman-arranged type sensor is becoming widespread.

The Klechman-arranged SPR sensor emits light from an optical prism, a detection plate composed of a metal layer arranged on the bottom surface side of the optical prism, a light irradiation unit that irradiates the optical prism with the incident light, and the optical prism. It is composed of an optical detection unit that receives the reflected light and detects the intensity of the reflected light.
When the incident light is incident on the optical prism under the condition of total internal reflection, surface plasmon resonance occurs at a certain incident angle θ due to the evanescent wave exuding to the metal layer side. When the surface plasmon resonance occurs, the evanescent wave is absorbed by the surface plasmon, so that the intensity of the reflected light is remarkably reduced near the incident angle θ. Since the conditions under which the surface plasmon resonance appears vary depending on the permittivity on the detection surface of the metal layer opposite to the surface on which the optical prism is arranged, the SPR sensor has a dielectric constant on the detection surface. It is possible to detect the presence of a substance that changes the optics.

Further, an optical waveguide mode sensor is known as a sensor for detecting the presence or the like of a substance that changes the permittivity on the detection surface (see, for example, Patent Documents 1 to 5).
The optical waveguide mode sensor has a structure similar to that of the Kletchman-arranged SPR sensor, and is composed of the optical prism, the detection plate, the light irradiation unit, and the light detection unit. It differs from the SPR sensor in that it is formed of a laminate of a dielectric layer and a metal layer or the like.
When the incident light is incident on the optical prism under the condition of total internal reflection, the incident light propagates in the dielectric layer at a specific incident angle θ, which is also called an optical waveguide mode (leakage mode or leaky mode). ), The optical waveguide mode is excited, and the intensity of the reflected light is remarkably reduced near the incident angle θ. The excitation conditions of the optical waveguide mode change depending on the permittivity on the detection surface of the detection plate opposite to the surface on which the optical prism is arranged.
Therefore, the optical waveguide mode sensor can detect the presence or the like of a substance that changes the dielectric constant on the detection surface, similarly to the SPR sensor.

When a biological substance such as a bacterium, a fungus, or a virus is detected by using the SPR sensor and the optical waveguide mode sensor, a chip for introducing a sample solution suspected of having the biological substance is prepared, and the biological substance and the biological substance are used. The detection surface modified on the surface with a specifically binding antibody or the like is brought into contact with the sample liquid on the chip.
This method enables detection at an earlier stage than the widely used method for detecting a biological substance, which is performed by introducing the biological substance into a solid medium and then observing the culture state using a microscope. In addition, the growth rate and the amount of growth of the biological substance can be detected in a timely manner according to the change in the dielectric constant.

By the way, when the biological substance is infectious or harmful, it is necessary to replace and dispose of the chip after each use from the viewpoint of safety and health.
However, as the chip, a thick glass substrate or a resin substrate is used, and since the waste is heavy and has a large volume, the disposal cost for transportation is high and the energy required for incineration and melting is also large. However, there is a problem that the disposal cost is further increased. Further, there is a case where it is required to perform crushing treatment after sterilization for disposal, and there is a problem that the disposal cost is further increased.

Japanese Patent No. 4581135 Japanese Patent No. 4595072 Japanese Unexamined Patent Publication No. 2007-271596 Japanese Patent No. 5131806 Japanese Patent No. 5424229

W.Knoll, MRS Bulletin 16, pp.29-39 (1991) W.Knoll, Annu.Rev.Phys.Chem.49, pp.569-638 (1998) H.Kano and S.Kawata, Appl.Opt.33, pp.5166-5170 (1994) C.Nylander, B.Liedberg, and T.Lind, Sensor.Actuat.3, pp.79-88 (1982/83) K.Kambhampati, T.A.M.Jakob, J.W.Robertson, M.Cai, J.E.Pemberton, and W.Knoll, Langmuir 17, pp.1169-1175 (2001) O.R.Bolduc, L.S.Live, and J.F.Masson, Talanta 77, pp.1680-1687 (2009) I.Stammler, A.Brecht, and G.Gauglitz, Sensor.Actuat.B54, pp98-105 (1999)

An object of the present invention is to solve the above-mentioned problems in the prior art and to provide a biological substance detection device capable of sensing operation using surface plasmon resonance or optical waveguide mode while using a detection sheet that is easy to dispose of. To do.

The means for solving the above-mentioned problems are as follows. That is,
<1> An optical prism capable of introducing incident light toward the bottom surface and emitting reflected light of the incident light reflected from the bottom surface side to the outside, and an optical prism on the bottom surface side of the optical prism. A detection layer that is arranged and has a surface opposite to the surface on which the optical prism is arranged as a detection surface and causes a change in the characteristics of the reflected light according to a change in the dielectric constant on the detection surface, and the optical prism. On the other hand, a light irradiation unit capable of irradiating linearly polarized light as the incident light, a light detection unit capable of receiving the reflected light emitted from the optical prism and detecting the characteristics of the reflected light, and a natural A detection sheet in which a solid medium layer capable of culturing biological substances is formed on a base material layer made of either paper or synthetic paper, and the medium layer side is arranged so as to be in contact with the detection surface. And, a biological substance detection device characterized by having.
<2> The water absorption of the base material layer at a contact time of 10 ms measured according to the Bristow method is at most 10 g / m ² , and the surface free energy on the surface of the base material layer on which the medium layer is formed. However, the biological substance detection device according to the above <1>, which has a minimum of 50 mJ / m ^2.
<3> The biological substance detection device according to any one of <1> to <2>, wherein the thickness of the base material layer is at least 200 μm.
<4> The biological substance detection device according to any one of <1> to <3>, wherein the material for forming the medium layer is a medium material containing agar.
<5> The biological substance detection device according to any one of <1> to <4>, wherein the linearly polarized light that can be irradiated by the light irradiation unit is p-polarized light and the detection layer is a metal layer.
<6> The linearly polarized light that can be irradiated by the light irradiation unit is either s-polarized light or p-polarized light, and the detection layer is a laminated body in which a metal layer and a dielectric layer are laminated in this order when viewed from the optical prism side. The biological substance detection device according to any one of <1> to <4>.
<7> Any of the above <5> to <6>, wherein the metal layer is a layer in which the real part has a negative value and the imaginary part has a positive value in the complex permittivity when irradiated with linearly polarized light. The biological substance detection device described in the above.
<8> From the above <1>, which has a support plate that is optically in close contact with the bottom surface of the optical prism, and the detection layer is formed on the surface of the support plate opposite to the surface on which the optical prism is arranged. The biological substance detection device according to any one of <7>.

According to the present invention, there is provided a biological substance detection device capable of solving the above-mentioned problems in the prior art and capable of sensing operation using surface plasmon resonance or optical waveguide mode while using a detection sheet that is easy to dispose of. can do.

It is explanatory drawing for demonstrating the structure of the biological substance detection apparatus which concerns on 1st Embodiment. It is explanatory drawing for demonstrating the structure of the modification of the biological substance detection apparatus which concerns on 1st Embodiment. It is explanatory drawing for demonstrating the structure of the biological substance detection apparatus which concerns on 2nd Embodiment. It is explanatory drawing for demonstrating the structure of the modification of the biological substance detection apparatus which concerns on 2nd Embodiment. It is a figure (1) which shows the measurement result of the reflected light intensity. It is a figure (2) which shows the measurement result of the reflected light intensity. It is a figure which shows the relationship between the water absorption degree and the surface free energy in each sample paper. It is a figure which shows the state of the detection sheet which formed the culture medium layer on the sample paper A. It is a figure which shows the state of the detection sheet which formed the culture medium layer on the sample paper E.

(First Embodiment)
The biological substance detection device according to the first embodiment of the present invention will be described with reference to the drawings. FIG. 1 is an explanatory diagram for explaining the configuration of the biological substance detection device according to the first embodiment.
As shown in FIG. 1, the biological substance detection device 10 has a sensing structure of a Kretschmann-arranged SPR sensor composed of an optical prism 1, a support plate 2, a detection layer 3, a light irradiation unit 6, and a light detection unit 7. In addition, it has a detection sheet 5 into which the subject is introduced.

The optical prism 1 is capable of introducing the incident light incident toward the bottom surface, and is capable of emitting the reflected light of the incident light reflected from the bottom surface side to the outside. In the illustrated example, a triangular prism is used, but the shape of the prism is not particularly limited, and instead, a known optical prism such as a trapezoidal prism, a semi-cylindrical prism, or a hemispherical prism can be used.
Further, the material for forming the optical prism 1 is not particularly limited, and can be appropriately selected from known glass materials such as silica glass.

The support plate 2 is a member that is optically brought into close contact with the bottom surface of the optical prism 1, and is optically brought into close contact with the optical prism 1 using, for example, a known refractive index matching liquid.
The material for forming the support plate 2 is a material that transmits the linearly polarized light in the state of forming the support plate 2, and for example, the same material as the material for forming the optical prism 1 is preferable.

The detection layer 3 is arranged on the bottom surface side of the optical prism 1, and the surface opposite to the surface on which the optical prism 1 is arranged is used as a detection surface, and the characteristics of the reflected light are changed according to the change in the dielectric constant on the detection surface. It is said to be a layer that causes changes in the optics. In this example, it is a single layer of a metal layer made of metal, and is formed on a surface of the support plate 2 opposite to the surface on which the optical prism 1 is arranged.
The metal layer is a layer in which the real part in the complex permittivity when irradiated with the linearly polarized light has a negative value and the imaginary part has a positive value from the viewpoint of expressing the surface plasmon resonance. Is preferable.
The material for forming the metal layer is not particularly limited, and examples thereof include known materials for forming the metal layer applied to the SPR sensor, including at least one of gold, silver, copper, platinum, and aluminum. The forming material is preferable.
The thickness of the metal layer is not particularly limited, and is, for example, about 1 nm to 50 nm.

The method for forming the detection layer 3 is not particularly limited, and examples thereof include a known vapor deposition method. In this example, the surface of the support plate 2 opposite to the surface on which the optical prism 1 is arranged is arranged. It can be formed by depositing the forming material on top of it.
Further, the detection layer 3 can be used repeatedly by performing cleaning such as autoclave treatment each time it is used, and can also be made disposable by removing it from the optical prism 1 together with the support plate 2.

In the detection sheet 5, a medium layer 5b is formed on the base material layer 5a, and the detection sheet 5 is arranged so that the medium layer 5b side is in contact with the detection surface.

The base material layer 5a is made of either natural paper or synthetic paper.
Therefore, the base material layer 5a is flammable and can be easily incinerated together with the medium layer 5b at the time of disposal. That is, according to the Infectious Waste Guideline (2014) published by the Ministry of the Environment of Japan, if a substance that is infectious or has the possibility of being infectious is handled, it is treated as infectious waste (1) incinerated. It will be disposed of according to one of the following categories: incinerated by equipment (burnable), (2) melted by melting equipment (non-burnable), and (3) crushed after sterilization. However, the detection sheet 5 having the base material layer 5a formed of the paper material is classified into the above (1) with the medium layer 5b formed, and is incinerated at the lowest cost. Is the target of.
Further, when the base material layer 5a is formed of the paper material, the volume and weight of the waste are reduced and the disposal cost in transportation is also reduced as compared with the case where the base material layer 5a is formed of a thick glass substrate or a resin substrate. be able to.
In addition, in this specification, "flammable" means the property that combustion continues when ignited at room temperature and normal pressure.

The medium layer 5b is a solid layer (solid medium) capable of culturing biological substances.
The material for forming the medium layer 5b is not particularly limited as long as such a biological substance can be cultivated, and a known medium material can be used. However, since the solid medium solidified appropriately can be easily obtained, agar is used. A medium material containing the above is preferable.
The medium material is not particularly limited, and examples thereof include known medium materials such as LB medium, cellulose medium, and alginate gel medium.
The thickness of the medium layer 5b is not particularly limited, but is preferably 50 μm or more from the viewpoint of maintaining the water retention state during the culture of the biological substance. The upper limit of the thickness is about 10 mm.
The biological substance is not particularly limited, and examples thereof include bacteria, fungi, and viruses.

Since the base material layer 5a is formed of the paper material and has water absorption, it is useful for forming the medium layer 5b. That is, when the medium layer 5b is formed using the LB medium containing agar, nutrients, salts, etc., the composition obtained by adding water to the LB medium for sterilization is heated to a temperature of about 120 ° C. in an autoclave. Then, the LB medium material is dissolved and dispersed in water to prepare a precursor solution, and then the precursor solution is applied onto the base material layer 5a before the temperature drops to about 60 ° C. at which solidification begins. A medium layer 5b in which the precursor liquid is solidified is formed on the base material layer 5a. At this time, the precursor liquid before the start of solidification partially permeates into the base material layer 5a, so that the medium layer 5b and the base material layer 5a after solidification are stably fixed. .. When the base material layer 5a is made of a material having no water absorption, it becomes difficult to stably fix the base material layer 5a and the medium layer 5b.
Examples of the method of applying the precursor liquid onto the base material layer 5a include a known squeezing method, a die coding method, a coating method similar to screen printing or inkjet printing, and the like.

By the way, the water absorption of paper varies depending on the type, but the base material layer 5a formed of the paper material is defined by the Bristow method (Japan Pulp and Technology Association (Japan Tappi) regulation, pulp and paper test method No. 51: It is preferable that the water absorption at a contact time of 10 millisecond measured according to 2000, "Paper and Paperboard-Liquid Absorption Test Method-Bristow Method") is at most 10 g / m ^2.
That is, when the water absorption degree ^{exceeds 10 g / m 2} , large wrinkles are likely to be generated in the base material layer 5a after drying due to expansion during water absorption and contraction during drying, and at the same time, the medium layer on the base material layer 5a. Large wrinkles are likely to occur in 5b. Therefore, when the surface of the medium layer 5b and the detection surface of the detection layer 3 are brought into contact with each other, air bubbles easily enter the interface, and the detection of the biological substance may be hindered.
Further, the lower limit of the water absorption is preferably at ^{least 1 g / m 2} from the viewpoint of stably fixing the base material layer 5a and the medium layer 5b by the permeation of the precursor liquid.
The contact time is set to 10 milliseconds because it is a sufficient time to determine the suitable water absorption of the base material layer 5a.

Further, as the base material layer 5a, it is preferable that the surface free energy on the surface of the base material layer 5a on which the medium layer 5b is formed is at least 50 mJ / m ^{2 in relation to the water absorption degree.}
That is, when the medium layer 5b is stably fixed on the base material layer 5a, it is necessary to apply the precursor liquid in which the medium material is dissolved and dispersed in water on the base material layer 5a. If the precursor liquid does not get wet and spread on the base material layer 5a, the thickness of the formed medium layer 5b becomes non-uniform, and air bubbles enter the interface between the medium layer 5b and the detection layer 3 to detect the biological substance. It becomes a factor that hinders. Further, if the medium layer 5b is formed with a non-uniform thickness, it also causes uneven culture in the culture of the biological substance in the medium layer 5b. Therefore, it is preferable that the base material layer 5a has an appropriate wettability.
As described above, the wettability can be expressed by using the surface free energy as an index. That is, when the surface free energy is high, the precursor liquid easily wets and spreads uniformly on the surface of the base material layer 5a, and when the surface free energy is low, the precursor liquid is repelled on the surface of the base material layer 5a and is non-uniform. It is easy for lumps and the like to form a thick thickness to occur.
As described above, when the surface free energy on the surface of the base material layer 5a is at least 50 mJ / m ² , the biological substance can be suitably detected as having appropriate wettability. The upper limit of the surface free energy is preferably ^{100 mJ / m 2 at the highest.}

The thickness of the base material layer 5a is not particularly limited, but the purpose is to stably fix the medium layer 5b through permeation of the precursor solution, and to obtain strength for stably supporting the medium layer 5b. Further, for the purpose of suppressing the generation of wrinkles after the formation of the medium layer 5b, the thickness is preferably at least 200 μm.
The base material layer 5a can be handled without touching the medium layer 5b by rolling the medium layer 5b so as to wrap it inside when the detection sheet 5 is discarded. Therefore, the upper limit of the thickness is preferably 500 μm at the thickest from the viewpoint of providing flexibility for easy rolling.

When the detection sheet 5 configured in this way is used, when the SPR sensor and the optical waveguide mode sensor are used, the surface treatment on the detection surface (such as an antigen that specifically binds to the biological substance) that has been conventionally performed is performed. (Formation) is not required, and the setting for detection can be greatly simplified.
That is, since the medium layer 5b is formed as a gel-like solid medium that easily adheres to the detection surface of the detection layer 3, a state or medium in which a sample solution suspected to be present with the biological substance is dropped onto the medium layer 5b. The setting required for detection is completed only by bringing the layer 5b into contact with the sample liquid and bringing the medium layer 5b side of the detection sheet 5 into contact with the detection surface of the detection layer 3 in a state where the biological substance is cultured. be able to.

The light irradiation unit 6 has a laser 6a and a polarizing element 6b that polarizes the light emitted from the laser 6a into linearly polarized light, and can irradiate the optical prism 1 with the linearly polarized light as the incident light. If the optical prism 1 can be irradiated with the linearly polarized light as the incident light, an arbitrary light irradiation unit composed of a known light source, a polarizing element, or the like is selected instead of the configuration of the light irradiation unit 6. May be used. For example, a light source such as a laser or an LED, a collimator that uses the light emitted from the light source as collimating light, a polarizing plate that polarizes the light emitted from the light source into the linear polarization, and the collimeter that emits light emitted from the light source. It can be configured by appropriately selecting from known optical members such as an optical fiber leading to an optical fiber, a condensing lens that condenses light from the light source and causes it to enter the optical prism 1.
The light irradiation unit 6 can be driven in a direction of increasing or decreasing the incident angle by a known driving device (not shown) in order to specify the incident angle θ at which the intensity of the reflected light decreases.
When the detection layer 3 is formed of the metal layer, the linearly polarized light is p-polarized light.

The photodetector 7 receives the reflected light emitted from the optical prism 1 and can detect the characteristics of the reflected light.
The photodetector 7 is not particularly limited, and can be configured by using a known photodetector such as a thermopile sensor, a diode sensor, a CMOS sensor, or a CCD sensor.

In the biological substance detection device 10 configured as described above, when the incident light is introduced into the optical prism 1 so as to satisfy the total reflection condition, the evanescent light exudes from the support plate 2 toward the detection layer 3. The surface plasmon is excited in the vicinity of the detection surface of the detection layer 3.
Here, when the light irradiation unit 6 is driven in a direction of increasing or decreasing the incident angle, surface plasmon resonance in which the evanescent light is absorbed by the surface plasmon is exhibited at a specific incident angle θ, and the intensity of the reflected light is increased. Is significantly reduced. The intensity of the reflected light is detected by the photodetector 7 for each incident angle.
Since the detected incident angle θ changes in shift according to the change in the dielectric constant in the vicinity of the detection surface, the living body existing in the medium layer 5b arranged on the detection surface through the shift change in the incident angle θ. The substance can be detected, and the increase in the biological substance (for example, the growth amount of bacteria and the like, the growth rate, etc.) can be detected in a timely manner based on the analysis of the shift change amount of the incident angle θ.

As the biological substance detection device 10, unlike the biological substance detection device 10'shown in FIG. 2, the support plate 2 which is separate from the optical prism 1 is not provided, and the detection layer 3 is directly on the bottom surface of the optical prism 1. May be formed. Note that FIG. 2 is an explanatory diagram for explaining the configuration of a modified example of the biological substance detection device according to the first embodiment.

(Second Embodiment)
Next, the second embodiment of the present invention will be described with reference to the drawings. FIG. 3 is an explanatory diagram for explaining the configuration of the biological substance detection device according to the second embodiment.
As shown in FIG. 3, the biological substance detection device 20 is composed of an optical prism 21, a support plate 22, a metal layer 23, a dielectric layer 24, a detection sheet 25, a light irradiation unit 26, and a light detection unit 27.

The biological substance detection device 10 according to the first embodiment is configured by using the Kletchman-arranged SPR sensor. On the other hand, the biological substance detection device 20 according to the second embodiment is configured by using an optical waveguide mode sensor.
The optical waveguide mode sensor can be configured by using the same optical system as the SPR sensor, and includes an optical prism 21, a support plate 22, a metal layer 23, a detection sheet 25 (base material layer 25a and a medium layer 25b), and the like. The light irradiation unit 26 (laser 26a, polarizing element 26b, etc.) and the light detection unit 27 include an optical prism 1, a support plate 2, a metal layer 3, and a detection sheet 5 in the biological substance detection device 10 according to the first embodiment. Items common to the light irradiation unit 6 and the light detection unit 7 can be applied.
Hereinafter, matters different from the biological substance detection device 10 according to the first embodiment will be described.

In the biological substance detection device 20 according to the second embodiment, instead of the detection layer 3, the detection layer is composed of a laminated body in which the metal layer 23 and the dielectric layer 24 are laminated in this order when viewed from the optical prism 21 side. Is arranged, and the surface of the dielectric layer 24 that comes into contact with the medium layer 25b is defined as the detection surface.

The material for forming the metal layer 23 is not particularly limited, and examples thereof include known metal materials applied to the optical waveguide mode sensor. However, a forming material containing at least one of gold, silver, copper, platinum, and aluminum may be used. Preferably, gold is particularly preferable in consideration of a cleaning treatment such as high-pressure steam sterilization at the time of reuse.
The thickness of the metal layer 23 is not particularly limited, and is, for example, about 1 nm to 50 nm.
Further, the metal layer 23 is preferably a layer in which the real part has a negative value and the imaginary part has a positive value in the complex dielectric constant when irradiated with the linearly polarized light.
The method for forming the metal layer 23 is not particularly limited, and examples thereof include a known vapor deposition method. In this example, the surface of the support plate 22 opposite to the surface on which the optical prism 21 is arranged is arranged. It can be formed by depositing the forming material on top of it.

The material for forming the dielectric layer 24 is not particularly limited, and a known material for forming the dielectric layer applied to the optical waveguide mode can be applied. For example, a resin material such as an acrylic resin, silica, or alumina can be applied. , Metal oxides such as zinc oxide, and metal nitrides such as silicon nitride and aluminum nitride. Considering the cleaning treatment by high-pressure steam sterilization at the time of reuse, the metal oxides are preferable, and the silica is particularly preferable. Especially preferable.
The thickness of the dielectric layer 24 is not particularly limited, but is, for example, about 1 nm to 1 μm.

The method for forming the dielectric layer 24 is not particularly limited, and known physical and chemical forming methods can be applied depending on the forming material.
Further, when the detection layer is cleaned by high-pressure steam sterilization or the like and reused, a bonding layer formed of chromium, titanium, or the like is formed between the metal layer 23 and the dielectric layer 24 in order to avoid peeling. (Not shown) may be formed with a thickness of about 1 nm.
Further, the dielectric layer 24 may be formed as a single layer alone, or may be formed as a laminate of two or more layers using the forming material.

When the detection layer is formed of the metal layer 23 and the dielectric layer 24, it can be used repeatedly by performing cleaning such as autoclave treatment each time it is used, and it can be removed from the optical prism 21 together with the support plate 22. , Can be disposable.
When the detection layer is formed of the metal layer 23 and the dielectric layer 24, either p-polarized light or s-polarized light can be used as the linearly polarized light used as the incident light.

In the biological substance detection device 20 configured as described above, when the incident light is incident on the optical prism 21 under the condition of total internal reflection, the incident light propagates in the dielectric layer 24 at a specific incident angle θ. The optical waveguide mode is excited in combination with the optical waveguide mode, and the intensity of the reflected light is remarkably reduced near the incident angle θ. The excitation conditions of the optical waveguide mode change depending on the permittivity on the detection surface.
Since the incident angle θ shifts according to the change in the dielectric constant in the vicinity of the detection surface, the biological substance existing in the medium layer 25b arranged on the detection surface is detected through the shift change of the incident angle θ. In addition, based on the analysis of the shift change amount of the incident angle θ, the increase in the biological substance (for example, the growth amount of bacteria and the like, the growth rate, etc.) can be detected in a timely manner.

The biological substance detection device 20 is not provided with a support plate 22 that is separate from the optical prism 21 as in the biological substance detection device 20'shown in FIG. 4, and the detection layer is directly on the bottom surface of the optical prism 21. (Metal layer 23, dielectric layer 24) may be formed. Note that FIG. 4 is an explanatory diagram for explaining the configuration of a modified example of the biological substance detection device according to the second embodiment.

(Other variants)
In each of the biological substance detection devices according to the first and second embodiments, the analysis is performed by driving the light irradiation unit and measuring the shift change of the incident angle θ in which the intensity of the reflected light decreases. When driving the irradiation unit, the optical system becomes large. Therefore, a wavelength dispersion type optical system in which the light irradiation unit is fixed for analysis may be adopted.
For example, in the light irradiation unit 6 according to the first embodiment, an optical system that irradiates the optical prism 1 with incident light in a wide wavelength band may be adopted by using a white light source instead of the laser 6a.
In this case, the light detection unit 7 detects the wavelength spectrum of the reflected light, and it is observed that the intensity of the reflected light is remarkably reduced at a specific wavelength in the wavelength spectrum.
The curved shape in the wavelength spectrum in which the intensity of the reflected light is remarkably reduced at the specific wavelength is called a dip, and in the biological substance detection device composed of the wavelength dispersive optical system, the dip position is detected. The biological substance is detected by utilizing the shift change to the short wavelength side or the long wavelength side according to the change in the dielectric constant on the surface.

In addition, although various explanations have been given by taking each biological substance detection device according to the first and second embodiments as an example, in addition to the matters described, the known Kretschmann arrangement type has been described for configurations other than the configuration of the detection sheet. It can be configured based on the matters applicable to the SPR sensor and the optical waveguide mode sensor. On the flip side of this, the biological substance detection device of the present invention can be configured by using the existing SPR sensor and the optical waveguide mode sensor as they are, and has a cost advantage that it does not require the introduction of a new sensor. Have.

Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples.

(Example 1)
The biological substance detection device according to Example 1 was manufactured with the configuration shown in FIG. Specifically, it was manufactured as follows.

In the biological substance detection device according to the first embodiment, light emitted from the laser having a wavelength of 632.8 nm is introduced into the polarizing plate, polarized as p-polarized light, and then incident on the optical prism. In addition, the optical system was set so that the thermopile sensor receives the reflected light emitted from the optical prism.
As the optical prism, a glass optical prism (triangular prism, apex angle 90 °) having a refractive index of 1.73 with respect to light having a wavelength of 632.8 nm was used.
Gold was deposited as a detection layer on a 25 mm square support plate (refractive index 1.73) made of the same material as the optical prism to a thickness of 47 nm. The surface of the support plate opposite to the surface on which the detection layer was vapor-deposited was brought into close contact with the bottom surface of the optical prism via a refractive index matching liquid.
The detection sheet is prepared by forming a medium layer formed of an LB agar medium material on the base layer using a commercially available glossy paper for inkjet printing (Crispia, manufactured by Epson) cut into 25 mm squares as a base layer. did. The medium layer is formed by heating and sterilizing a composition obtained by adding water to the LB medium to a temperature of about 120 ° C. in an autoclave to prepare a precursor solution in which the LB medium is dissolved and dispersed in water. Before the temperature drops to about 60 ° C. at which solidification begins, the precursor liquid is applied onto the substrate layer by a squeezing method, and then cooled to 37 ° C. to solidify the precursor liquid. It was.
Finally, the surface of the detection layer opposite to the surface on which the optical prism is arranged is set as the detection surface, and the detection surface is brought into close contact with the medium layer of the detection sheet to manufacture the biological substance detection device according to Example 1. did.

For the biological substance detection device according to Example 1, the incident angle of the incident light with respect to the optical prism was changed from 50 ° to 75 °, and the reflected light intensity at each incident angle was measured. The measurement result of the reflected light intensity is shown in FIG.
As shown in FIG. 5, a decrease in the reflected light intensity is confirmed at around 63.5 °. As described above, in the biological substance detection device according to the first embodiment, it is possible to confirm the decrease in the reflected light intensity due to the surface plasmon resonance.

Next, the detection sheet in the biological substance detection device according to Example 1 was once removed from the detection layer, and E.I. E. with the introduction of Coli. The Coli suspension was added dropwise, and the detection sheet was brought into close contact with the detection layer again. In addition, E. As the Coli suspension, E. cerevisiae cultured in another LB medium solution. The one prepared by diluting Coli to a concentration of 1 / 10,000 was used.
In this state, the incident angle of the incident light with respect to the optical prism was changed from 50 ° to 75 ° to the biological substance detection device according to Example 1, and the reflected light intensity at each incident angle was measured.
Further, the same measurement was performed by allowing the detection sheet once removed from the detection layer to stand at a temperature of 37 ° C. for a certain period of time, and then attaching it to the detection layer again. The standing time was determined by E.I. The time was set to 1 hour after the dropping of the Coli suspension and 4 hours after the dropping. The measurement result of the reflected light intensity in each state is shown in FIG.
As shown in FIG. 6, E.I. Immediately after dropping the Coli suspension (0 h), a decrease in the reflected light intensity was confirmed at around 63.6 °, and E.I. A shift change from the incident angle (63.5 °) is confirmed, in which a decrease in the reflected light intensity is confirmed when the measurement is performed without dropping the Coli suspension.
Therefore, the biological substance detection device according to the first embodiment can detect the biological substance in the medium layer through the shift change.

Further, as shown in FIG. 6, E.I. Although the shift change was not clearly confirmed 1 hour after the dropping of the Coli suspension, a decrease in the reflected light intensity was confirmed at around 63.7 ° after 4 hours. It is possible to clearly confirm the shift change from the incident angle (63.6 °) at which the decrease in the reflected light intensity is confirmed immediately after the Coli suspension is dropped. Such shift changes are caused by E. coli in the medium layer. It means that Colli has grown or proliferated.
Therefore, the biological substance detecting apparatus according to the first embodiment can detect the increase in the biological substance in a timely manner by analyzing the time for causing the shift change, the amount of the shift change, and the like.

(Water absorption test)
Next, in order to confirm the fixed state of the base material layer and the medium layer and the state of the shape change of the detection sheet due to the water absorption of the paper material forming the base material layer, the base material layer As a water absorption test, a measurement test of water absorption and surface free energy was performed.
The water absorption degree is determined according to the Bristow method (Japan Pulp and Paper Technology Association (Japan Tappi) regulation, Pulp and Paper Test Method No. 51: 2000, "Paper and Paperboard-Liquid Absorption Test Method-Bristow Method"). It was measured as the water absorption in millisecond.
Further, the surface free energy was calculated as follows as the surface free energy on the surface of the base material layer on which the medium layer is formed. That is, the static contact angle of the water and diiodomethane dropped on the surface of the base material layer by the dropping method using water and diiodomethane is measured using a contact angle measuring device (DM-500, manufactured by Kyowa Interface Science Co., Ltd.). Then, based on the obtained measured values, the surface free energy value was calculated by the Owns-Wendt method. This calculation was performed using the surface free energy analysis tool of the multifunctional integrated analysis software (FAMAS) attached to the contact angle measuring device.
Further, in these water absorption tests, the paper (manufactured by Epson, Crispia) used as the base material layer in the biological substance detection device according to Example 1 was used as sample paper A, and the paper manufactured by Pictorico, Pictorico Pro was used as sample paper B. These sample papers A to E are made by Nippon Paper Pavilia, Oper MDP as sample paper C, Oji Paper, mirror coated platinum as sample paper D, and Mitsubishi Paper Mills, matte coated paper as sample paper E. Went to.
The test results are shown in FIG. 7 and Table 1 below. Note that FIG. 7 is a diagram showing the relationship between the water absorption degree and the surface free energy of each sample paper.

As shown in FIGS. 7 and 1, the sample papers A and B have a water absorption of 10 g / m ² or less and a surface free energy of 50 m J / m ² or more. Therefore, when the medium layer is formed on the sample papers A and B, the sample papers A and B are less susceptible to the influence of the decrease in strength due to excessive water absorption and the generation of wrinkles due to drying according to the degree of water absorption. As a result, it is possible to prevent the smoothness of the medium layer from being impaired. Further, according to the surface free energy, the medium material (dispersion liquid) forming the medium layer on the sample papers A and B is appropriately wetted and spread to stably fix the medium layer with a uniform thickness. be able to.
Further, the sample papers C and D have the water absorption degree of 10 g / m ² or less, but the surface free energy is less than ^{50 m J / m 2.} Therefore, when the medium layer is formed on the sample papers C and D, the medium material (dispersion liquid) is easily repelled according to the surface free energy of the surface of the sample papers C and D (water repellent), and the medium material becomes. The thickness of the medium layer tends to be uneven due to lumps and the like.
Further, the sample paper E has the surface free energy of 50 mJ / m ² or more, but the water absorption degree of ^{the sample paper E exceeds 10 g / m 2} of the water absorption degree. Therefore, when the medium layer is formed on the sample paper E, the sample paper E is susceptible to a decrease in strength due to excessive water absorption according to the water absorption degree, wrinkles due to drying, and the like. The smoothness of the medium layer is easily impaired.

Actually, using the sample papers A and E as the base material layer, the medium layer was formed as follows, and the situation was confirmed.
The medium layer was formed on the sample papers A and E in the same manner as the method for forming the medium layer on the base material layer in Example 1. That is, the composition obtained by adding water to the LB medium is heated and sterilized in an autoclave to a temperature of about 120 ° C. to obtain a precursor solution in which the LB medium is dissolved and dispersed in water, and then solidification begins. Before the temperature drops to about 60 ° C., the precursor liquid is applied onto the substrate layer by a squeezing method, and then cooled to 37 ° C. to solidify the precursor liquid on sample papers A and E. The medium layer was formed in.
FIG. 8 shows the state of the detection sheet in which the medium layer is formed on the sample paper A. Further, FIG. 9 shows the state of the detection sheet in which the medium layer is formed on the sample paper E.

As shown in FIG. 8, in the detection sheet in which the medium layer is formed on the sample paper A, no significant change in shape is observed in the sample paper A, and the surface of the medium layer is brought into close contact with the detection surface of the detection layer. It has a flat shape that is easy to use.
On the other hand, as shown in FIG. 9, in the detection sheet in which the medium layer is formed on the sample paper E, a plurality of large wrinkles are generated on the sample paper E, and at the same time, the shape of the sample paper E is also formed on the surface of the medium layer. Multiple large wrinkles formed following the change are confirmed. Therefore, when the detection sheet in which the medium layer is formed on the sample paper E is used, it is assumed that air bubbles easily enter the interface between the surface of the medium layer and the detection surface when the detection sheet is in close contact with the detection surface. If air bubbles or the like enter the interface, the detection of the biological substance is hindered, so that it is assumed that a load is applied to the setting when the detection sheet is attached to the detection layer.

When the water absorption degree ^{exceeds 10 g / m 2} , large wrinkles are likely to be generated in the base material layer after drying due to expansion during water absorption and contraction during drying, and at the same time, the medium layer on the base material layer is likely to have large wrinkles. It is considered that large wrinkles are likely to occur.
^{Therefore, as the base material layer, the water absorption is preferably 10 g / m 2} or less according to the measurement results of the water absorption tests of the sample papers A and B, and a paper material having such a water absorption is used. It is considered preferable to be formed.
At the same time, as the base material layer, it is considered preferable that the surface free energy on the surface forming the medium layer is at least 50 mJ / m ^2.
That is, although it is not shown because it is difficult to obtain a photograph for comparison, when the medium layer was formed in the same manner using the sample papers A to D as the base material layer and the situation was confirmed, the sample papers A and A, Compared with B, in the sample papers C and D, the medium material (dispersion liquid) was easily repelled, and the thickness of the medium layer was likely to be non-uniform.
On the other hand, in the sample papers A and B, the medium material (dispersion liquid) easily wets and spreads appropriately, and the thickness of the medium layer can be formed uniformly (see, for example, FIG. 8).

1,21 Optical prism 2,22 Support plate 3 Detection layer 5,25 Detection sheet 5a, 25a Base material layer 5b, 25b Medium layer 6,26 Light irradiation unit 6a, 26a Laser 6b, 26b Polarizing element 7,27 Light detection unit 10, 10'20, 20'Biological substance detector 23 Metal layer 24 Dielectric layer

Claims (8)

An optical prism capable of introducing incident light toward the bottom surface and emitting reflected light of the incident light reflected from the bottom surface side to the outside.
It is arranged on the bottom surface side of the optical prism, and the surface opposite to the surface on which the optical prism is arranged is used as a detection surface, and the characteristics of the reflected light are changed according to the change in the dielectric constant on the detection surface. With the detection layer,
A light irradiation unit capable of irradiating the optical prism with linearly polarized light as the incident light,
A photodetector that receives the reflected light emitted from the optical prism and can detect the characteristics of the reflected light.
Detection in which a solid medium layer capable of culturing biological substances is formed on a base material layer made of either natural paper or synthetic paper, and the medium layer side is arranged so as to be in contact with the detection surface. Sheet and
A biological substance detection device characterized by having. The water absorption of the base material layer at a contact time of 10 ms measured according to the Bristow method is at most 10 g / m ² , and the surface free energy on the surface of the base material layer on which the medium layer is formed is low. The biological substance detection device according to claim 1, both of which are 50 mJ / m ^2. The biological substance detection device according to any one of claims 1 to 2, wherein the thickness of the base material layer is at least 200 μm. The biological substance detection device according to any one of claims 1 to 3, wherein the material for forming the medium layer is a medium material containing agar. The biological substance detection device according to any one of claims 1 to 4, wherein the linearly polarized light that can be irradiated by the light irradiation unit is p-polarized light, and the detection layer is a metal layer. The linearly polarized light that can be irradiated by the light irradiation unit is either s-polarized light or p-polarized light, and the detection layer is composed of a laminated body in which a metal layer and a dielectric layer are laminated in this order when viewed from the optical prism side. The biological substance detection device according to any one of claims 1 to 4. The biological material according to any one of claims 5 to 6, wherein the metal layer is a layer in which the real part has a negative value and the imaginary part has a positive value in the complex permittivity when irradiated with linearly polarized light. Detection device. Any of claims 1 to 7, which has a support plate that is optically in close contact with the bottom surface of the optical prism, and the detection layer is formed on a surface of the support plate opposite to the surface on which the optical prism is arranged. The biological substance detection device according to the above.

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