JPH0933427A - Biosensor and concentration measuring device therewith - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve ease of handling of a biosensor wherein an optical system is adopted to form evanescent wave joint and to reduce the cost thereof. SOLUTION: A biosensor 20 is provided with acryl boards 22R and 22L jointed with each other, and a facing board main surface 23 is formed of total reflecting face through vapor-deposition of chromium thin film. An Au thin film 28 is applied to upper end faces 26R and 26L to form an optical reflecting face 29, and a light source unit 30 is built in and fixed in a recessed part 24 for housing a light source unit in a manner that a focus point is set on the face 29. In addition, CCD image sensing elements 27R and 27L are fixed on outgoing-side end faces 25R and 25L. Therefore, a light emitting from the unit 30 is reflected totally on the facing surface 23 and arrives at the face 29 between the faces 23 as a transmitting route, and it is reflected on the face 29, then it is received by the elements 27R and 27L through a transmitting route between the faces 23.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor for measuring a substrate to be measured in a solution to be measured using an optical system, and more specifically to a geometric total reflection on a light reflecting surface provided with a metal thin film. The present invention relates to an optical system that has a light-transmitting light-transmitting medium that reflects light under certain conditions and uses an optical system that forms an evanescent wave coupling between the light-transmitting medium and the metal thin film. Further, the present invention relates to a concentration measuring device that obtains the concentration of a substrate to be measured in a solution to be measured using this biosensor.

[0002]

2. Description of the Related Art Generally, in a biosensor, a substrate to be measured, which is a blood component such as a specific protein or antigen in blood, or a urine component such as glucose or ascorbic acid in urine, has a function of discriminating these substrates. A biological substance that has a biochemical reaction with the substrate is used. Then, various physicochemical displacements associated with the progress of this biochemical reaction are detected by the physicochemical device, and the specification of the measurement target substrate, its concentration, etc. are detected. For example, one that detects the substrate concentration through the electrode reaction of an electrode active substance that is consumed or produced by a biochemical reaction, or one that detects the enthalpy change that occurs with the progress of the biochemical reaction with a thermistor to detect the substrate concentration. These biosensors have been put to practical use since early on.

On the other hand, in recent years, attention has been paid to changes in the dielectric constant with the progress of biochemical reactions, and biosensors have been proposed for measuring a substrate to be measured in a solution to be measured using an optical device. (JP-A-1-138443). In this biosensor, as an optical system, a light-transmitting light-transmitting medium that reflects light under a geometrical total reflection condition is provided on a light-reflecting surface provided with a metal thin film. An optical system that forms an evanescent wave coupling is used. The measuring principle is as follows.

When p-polarized light is applied to a light-transmitting medium of an optical system for forming an evanescent wave coupling on a light-reflecting surface at various incident angles satisfying the condition of total internal reflection, when the incident angle has a certain value, it is peculiar. The phenomenon occurs. That is, when the p-polarized light is applied to the light reflecting surface, an evanescent wave having a wave number having an incident angle θ as a variable is generated on the light transmitting medium side film surface of the metal thin film. Since the metal can be regarded as a solid plasma, a surface plasmon wave as a wave of quantum charge density is generated by the light tunnel effect on the film surface of the metal thin film on the anti-light transmission medium side. The surface plasmon wave is generated as a wave between the metal thin film and the medium in contact with the film surface on the side opposite to the light-transmitting medium as a boundary surface.

When the incident angle θ has a certain value, a surface plasmon resonance phenomenon occurs in which the wave numbers of the evanescent wave and the surface plasmon wave coincide with each other, and the light energy is used as the excitation energy of the surface plasmon wave. Be seen. At this time, in terms of energy, there is a relation that the energy of the light incident on the light reflecting surface is equal to the sum of the energy used to excite the surface plasmon wave and the energy of the reflected light from the reflecting surface. Therefore, by measuring the state of changes in the reflection angle and the energy (light amount), it is possible to determine the presence or absence of the surface plasmon resonance phenomenon, and consequently the incident angle when the phenomenon occurs. On the other hand, there is a correlation between the incident angle when the surface plasmon resonance phenomenon occurs and the refractive index of the medium, and this refractive index can be defined by the dielectric constant of the medium from Maxwell's equation, and the biochemical reaction of biological substances There is a correlation between the progress and the dielectric constant. Therefore, the angle of incidence at that time is determined from the angle of reflection when the amount of reflected light suddenly decreases, and the degree of progress of the biochemical reaction by the biological substance, that is, the substrate concentration is calculated from the above correlations.

By the way, in such a biosensor, the metal thin film is located in the vicinity of the film surface on the side opposite to the light-transmitting medium, specifically, in the evanescent region (about 10) which causes the tunnel effect.
At 0 nm), a biochemical reaction between the substrate and the biological substance needs to occur. Therefore, the patent application publication No. 4-50
As proposed in 1605, it is generally practiced to immobilize a layer on which a biological material is immobilized, a so-called ligand layer, on the above-mentioned film surface of a metal thin film.

As described above, the biosensor for measuring the substrate to be measured in the solution to be measured using the optical system for forming the evanescent wave bond is not affected by the degree of coloring or opacity of the solution to be measured, or Since it has the advantage that it is only necessary to fix a biological substance that causes a biochemical reaction with a substrate on the surface of the metal thin film on the side of the anti-light transmission medium, it is rapidly spreading.

[0008]

However, in the above-mentioned conventional biosensor, when the light is incident in the fan-shaped beam on the light transmitting medium formed by the evanescent wave coupling with the metal thin film, the prism or the hemispherical lens is used. Alternatively, a semi-cylindrical lens or the like is used. These optical devices are expensive because of their high processing accuracy. Further, not only during handling during assembly but also during measurement, it is necessary to take care not to impede the transmission of light by touching a hemispherical lens or the like with a fingerprint, for example, and it is troublesome and inconvenient.

By the way, the measurement accuracy of the substrate concentration depends on the accuracy of the correspondence between the incident angle of the incident light reaching the light reflecting surface and the change in the light amount of the reflected light reflected from the light reflecting surface and emitted from the light transmitting medium. However, in the sensor proposed in the above publication, the light-transmitting medium that forms the evanescent wave coupling with the metal thin film and the prism or the hemispherical lens for making the light incident on the light-transmitting medium in a fan-shaped beam are separate bodies. In addition, there are the following problems regarding the measurement accuracy of the substrate concentration.

When the light transmission medium and the prism or hemispherical lens are separate bodies, an appropriate refraction is provided between the light transmission medium and the prism or the like so as not to adversely affect the transmission of light between the two. It is essential to interpose a refractive index-harmonic fluid having a refractive index, so-called matching oil. Therefore, the fluidity of the matching oil is increased due to an increase in the environmental temperature, etc., and the oil flows between the light transmission medium and the prism or hemispherical lens to enter or exit the prism or the curved surface of the hemispherical lens. In addition, the light transmission is hindered and inadvertent scattering occurs. Moreover, the degree of inhibition and the degree of scattering are not uniform on the light incident side and the light emitting side. Therefore, the accuracy of the correspondence between the changes in the amounts of the incident light and the reflected light is reduced, resulting in a decrease in the measurement accuracy.

Therefore, it is necessary to carefully control the amount of use (intervening amount) of the matching oil so as not to cause the matching oil to flow, and to prevent the ambient temperature from rising or to perform measurement in a constant temperature environment. In other words, it also lacks usability in that it requires such consideration.

However, it is possible to make the prism or hemispherical lens much larger than the light transmitting medium to widen the flat portion of the lens and make it difficult for the flowing matching oil to reach the curved surface of the lens, but it is expensive and complicated to handle. There is no change. Further, the metal thin film may be directly provided on the prism or the hemispherical lens so that the matching oil is not used, but it is still expensive and complicated to handle.

The present invention has been made to solve the above problems, and an object thereof is to improve the usability of the sensor and reduce the cost. It is also an object of the present invention to improve the accuracy of the concentration measurement of the substrate to be measured, which is obtained by using this biosensor.

[0014]

[Means for Solving the Problem and Its Action / Effect] In order to solve the problem, the biosensor of the first invention comprises:
An optical system having a light-transmissive light-transmitting medium that reflects light under a geometric total reflection condition on a light-reflecting surface provided with a metal thin film, and forming an evanescent wave coupling between the light-transmitting medium and the metal thin film. A biosensor for measuring a substrate to be measured in a solution to be measured in contact with the metal thin film,
The optical system is an incident light side optical system that collects and irradiates light from a light source on the light reflecting surface, and receives reflected light that is reflected by the light reflecting surface and is emitted to the outside from the light transmitting medium, An exit side optical system that detects the amount of the reflected light for each reflection angle, a polarization means that p-polarizes the light in the incident light side optical system or the exit side optical system, and a light that is geometrically totally reflected And a translucent substrate for transmitting the light by confining the wave of light between the totally reflective surfaces, and the translucent substrate is provided with the transmissive substrate to reach the light transmissive medium. A light transmission path for the light-emission-side optical system is formed, and a light transmission path for the light-emission-side optical system from the light transmission medium is formed.

In the biosensor of the first invention having the above structure, when transmitting light from the light source in the incident light side optical system to the light transmitting medium forming the evanescent wave coupling with the metal thin film, the light transmitting path is transmitted. It is formed of a light-sensitive substrate. Also, the transmission path of the light when receiving the reflected light reflected by the light reflection surface and emitted from the light transmission medium to the outside by the emission side optical system,
The transparent substrate is used. The translucent substrate transmits the light by confining the wave of the light between the total reflection surfaces while totally reflecting the light on the opposed total reflection surfaces, so that all the light incident on the translucent substrate is transmitted. The light is transmitted to the medium, and further to the light reflection surface, and all the light is subjected to total reflection on the light reflection surface and is then received by the emission side optical system. Then, the light emitted from the light source is p-polarized by the polarization means in the incident light side optical system or the emission side optical system, and the p-polarized light is received in the emission side optical system, and the light amount is detected for each reflection angle.

That is, in the biosensor of the first invention,
In optically performing substrate measurement by utilizing the surface plasmon resonance phenomenon at the light reflecting surface formed by the metal thin film and the light transmitting medium, transmission of light to the light transmitting medium and light transmission from the light transmitting medium are performed. Only a translucent substrate with the total reflection surfaces facing each other is used for transmission. Therefore, an optical device with high processing accuracy such as a prism or a hemispherical lens is not required, and even if the surface of the translucent substrate is touched during assembly, the surface is regarded as a total reflection surface. Therefore, there is no problem in light transmission. As a result, according to the biosensor of the first invention, it is possible to improve both the usability of the sensor and the cost reduction.

Further, even if the matching oil is used between the light transmitting medium and the light transmitting substrate, the problem caused by the flow can be dealt with as follows. First, by appropriately changing the size and shape of the substrate, it is possible to sufficiently separate the joining surface of the light transmissive medium in the translucent substrate and the light incident end surface and the light exit end surface of the light transmissive substrate. Further, an oil sump can be installed at a location where the matching oil flows. Further, a polarizing plate having a larger area than this incident end face can be closely attached to the incident end face. Therefore, from these things, it is possible to make it difficult for the flowing oil to reach the incident end face and the output end face of the translucent substrate.

That is, in the biosensor according to the first aspect of the present invention, when the matching oil is used, even if the matching oil flows for some reason, a simple measure such as an appropriate change in the size and shape of the substrate is taken. Only by collecting the matching oil, it is possible to make it difficult for the matching oil to reach the light incident end surface or the light exit end surface of the translucent substrate. Further, even if the matching oil flows and reaches the incident end face and the emitting end face, the influence thereof can be eliminated. Therefore, according to the biosensor of the first invention, it is possible to avoid a decrease in measurement accuracy due to the matching oil and maintain high measurement accuracy.

The behavior of light in the biosensor of the first invention is as follows: transmission of light up to the light transmitting medium and transmission of light after being totally reflected by the light reflecting surface and emitted from the light transmitting medium. It is the same as the conventional one except that the total reflection is performed on the total reflection surface of the transparent substrate. Therefore, the light that is condensed and emitted from the light source to the light reflecting surface is transmitted through the light transmitting substrate, reaches the light reflecting surface of the light transmitting medium, and is totally reflected by the light reflecting surface. Then, for incident light incident at a specific incident angle, a surface plasmon resonance phenomenon in which the evanescent wave on the light-transmitting medium-side film surface of the metal thin film and the surface plasmon wave on the exposed surface of the metal thin film resonate with their wave numbers matching each other. cause. When this surface plasmon resonance phenomenon occurs, the energy of light is used as the excitation energy of the surface plasmon wave, so the energy of light reflected from the light reflecting surface decreases. In this case, when the exposed surface of the metal thin film comes into contact with the solution to be measured of the substrate to be measured via the ligand layer, the biochemical reaction between this substrate and the biological substance proceeds, and the dielectric constant of the solution to be measured, Since the refractive index changes, the incident angle θ reflects the substrate concentration in the solution to be measured.

Light of various incident angles is incident on the light reflecting surface at one time by condensing and is reflected. Therefore, reflected light totally reflected at various reflecting angles corresponding to each incident angle is reflected from the light reflecting surface. The light emitted from the light transmissive medium to the translucent substrate at one time, and the reflected light of each reflection angle passes through the translucent substrate while being totally reflected at the total reflection surface. At this time, only the reflected light having a reflection angle corresponding to the incident angle θ is emitted from the light transmissive medium as light with a small amount of energy lost to reach the translucent substrate and corresponds to an incident angle other than θ. The reflected light having a reflection angle that is emitted from the light transmitting medium to the light transmissive substrate as light with a high light amount without energy loss. After that, the light is emitted from the transparent substrate to the outside.

Therefore, if the reflected light from the light reflecting surface for each incident angle is received and the amount of the light is detected, the incident angle and the amount of the light can be correlated, so that the incident when the amount of the reflected light is at the lowest level. The angle θ can be obtained. As a result, the correlation between this incident angle θ and the refractive index of the medium, the correlation between the refractive index of the medium and the dielectric constant, the correlation between the progress of the biochemical reaction by the biological substance and the dielectric constant, etc. From this, the substrate concentration can be calculated.

In the structure of the above-mentioned first invention, the light transmitting medium is a part of the light transmitting substrate, and the light reflecting surface is formed by providing the metal thin film on one end surface of the light transmitting substrate. .

In the biosensor having this structure, since the light transmitting medium is used as a part of the light transmitting substrate and a metal thin film is provided on one end surface of the light transmitting substrate to form the light reflecting surface, it is necessary to use matching oil. There is no. Therefore, according to the biosensor having this configuration, it is not necessary to consider the use environment temperature or the like that causes the matching oil to flow, and the usability of the sensor can be improved.

Further, in the above-mentioned first invention,
The incident light side optical system is provided with a light source unit that integrates a light source and a lens that focuses light emitted from the light source on a focal point, and the light source unit and the translucent substrate include the lens. Is focused on the light reflecting surface and tilts the optical axis of the lens with respect to the light reflecting surface,
The light-transmissive substrate is fixedly provided on the other end face side that intersects with the one end face, and the output side optical system is reflected at the light reflection face and reflected to the outside by the reflection light at the light reflection face. A light receiving unit that receives light at each angle at one time and detects the light amount of the reflected light at each reflection angle is provided.

In the biosensor having this structure, the light source unit forming the incident light side optical system is fixed to the translucent substrate at the other end face side intersecting with one end face thereof. At the time of this fixing, the lens of the light source unit focuses on the light reflecting surface, and the optical axis of this lens is tilted with respect to the light reflecting surface. Therefore, on the total reflection surface, a surface plasmon resonance phenomenon occurs due to incident light at a specific incident angle of the condensed light. On the other hand, the light receiving means forming the emission side optical system is configured to receive the reflected light reflected by the light reflecting surface at once for each reflection angle and detect the light amount for each reflection angle. Therefore, it becomes possible to correlate the incident angle and the light amount through the light receiving means without moving the emission side optical system for each reflection angle, and the light receiving means can be fixed.

Therefore, in the biosensor having this configuration,
The relative positional relationship between the light source unit forming the incident light side optical system, the translucent substrate, and the light receiving means forming the emission side optical system has only to be fixed once, and the positional relationship is affected by external vibration and the like. Without being maintained. As a result, according to the biosensor of this configuration, maintenance and inspection are not required after the sensor is completed, so that the usability can be further improved.

Further, in the above-mentioned first invention,
The light source unit was directly fixed to the other end surface of the translucent substrate that intersects the one end surface with an inclination, with a translucent thin plate interposed.

In the biosensor having this structure, when fixing the light source unit to the translucent substrate, the light source unit was directly fixed to the other end surface of the translucent substrate with a translucent thin plate interposed. Therefore, the flowing matching oil is not blocked by the thin plate and reaches the optical path from the light source unit, and the optical path is not blocked by the flowing matching oil. Therefore, the transmission of light from the optical unit to the transparent substrate is performed without any hindrance. As a result, according to the biosensor having this configuration, it is possible to more reliably avoid a decrease in measurement accuracy due to matching oil, and it is possible to maintain high measurement accuracy.

Further, in the above-mentioned first invention,
The polarizing means is a polarizing plate for p-polarizing transmitted light during the transmission, and is interposed between the light source unit and the other end surface of the transparent substrate as the transparent thin plate.

In the biosensor having this structure, the polarizing means is interposed between the light source unit and the other end face of the transparent substrate as a p-polarized thin plate-shaped polarizing plate. The incident of p-polarized light on the optical path and the avoidance of the flow path of the flowing matching oil are avoided. Therefore, according to the biosensor having this configuration, since a member only for shielding the flowing matching oil is not required, it is possible to further reduce the cost by reducing the number of parts.

Further, in the above-mentioned configuration of the first invention,
The light source unit is incorporated into a recess formed on the other end face side of the translucent substrate so as to be inclined with respect to the one end face, and light is incident from the bottom of the recess into the translucent substrate. I decided.

In the biosensor having this structure, the light source unit is incorporated in the recess formed on the other end face side of the transparent substrate so as to be inclined with respect to the one end face, and the light is introduced from the bottom of the recess into the transparent substrate. Incident. Since this recess is inclined with respect to the one end face forming the light reflecting surface, it is difficult for the matching oil flowing from the light reflecting surface to enter the inside of the recess. Therefore, according to the biosensor having this configuration, it is possible to maintain a high measurement accuracy by avoiding a decrease in the measurement accuracy due to the matching oil with a simple configuration in which a recess is formed in the translucent substrate.

Further, in the above-mentioned configuration of the first invention,
The end face of the translucent substrate, which corresponds to the end of the transmission path of the formed optical system of the emission side, is inclined with respect to the facing total reflection surface, and reflects light under a geometric total reflection condition. The total reflection inclined end surface is assumed to be the total reflection inclined end surface, and the facing total reflection surface in the range where the light reflected by the total reflection inclined end surface reaches is a transmission surface that transmits light to the outside only at least in the range, and the emission side The optical system is configured to receive light that has passed through the transmission surface and is emitted to the outside.

In the biosensor having this structure, the behavior of the reflected light reflected by the light reflecting surface on the transparent substrate is as follows. The reflected light is transmitted while being totally reflected by the total reflection surface of the transparent substrate for each reflection angle, and reaches the end of the light transmission path formed by the transparent substrate. The end surface of the translucent substrate, which corresponds to the end of the transmission line, is a total reflection inclined end surface that is inclined with respect to the opposing total reflection surface and that reflects light under geometric total reflection conditions. Therefore, when the reflected light for each reflection angle reaches the total reflection inclined end face, the reflected light is totally reflected by the end face and changes its traveling direction toward the facing total reflection face of the translucent substrate. By the way, since the facing total reflection surfaces in the range where the light reflected by the total reflection inclined end surface reaches are the transmission surfaces that transmit the light to the outside at least within that range, the total reflection inclined end surface is reflected. The reflected light is emitted from the translucent substrate to the outside. Therefore, the reflected light can be received on the total reflection surface side of the translucent substrate, and the light transmitted through the transmission surface and emitted to the outside is received by the emission side optical system.

That is, in the biosensor having this structure, a part of the opposed total reflection surfaces of the translucent substrate is used as the transmissive surface, and the reflected light reflected by the light reflective surface is transmitted from the transmissive surface to the outside of the substrate. Emit. Therefore, according to the biosensor of this configuration, the light receiving means can be installed on the total reflection surface side of the light-transmitting substrate. In addition to facilitating the handling, it is possible to increase the versatility by relaxing restrictions from the installation location of the light receiving means, for example, its shape and size.

Further, in the configuration of the above first invention,
A first translucent substrate and a second translucent substrate, wherein both translucent substrates form the emission side optical system of the first and second translucent substrates. The light transmission path is arranged so that the transmitted light is received by a single emission-side optical system, and one of the first and second light-transmissive substrates is a transparent substrate. When the transmitted light is received by one emission side optical system, the other translucent substrate is provided with a light reception blocking means for blocking the reception of light by the single emission side optical system. Have.

The biosensor having this structure has first and second translucent substrates for transmitting light to and from the light transmissive medium. The light transmitted by each of the substrates is received by a single emission side optical system after being totally reflected by the light reflecting surface. At this time, when light is received by the emission side optical system for one of the first and second light-transmitting substrates, the other light-transmitting substrate is detected by the light-reception blocking unit. The reception of light into the emission side optical system is blocked. Therefore, in the emission side optical system, light is received only by this one transparent substrate, and the amount of light is detected for each reflection angle. Therefore, according to the biosensor of this configuration, the substrate concentration of the solution to be measured corresponding to each of the first and second translucent substrates can be detected with high accuracy by using a single emission-side optical system. .

In this case, in order to arrange the first and second translucent substrates so that the light transmitted through the transmission paths is received by the single emission side optical system, the following may be done. . First, both translucent substrates are arranged such that the principal surfaces of the first and second translucent substrates are bonded to each other, or the principal surfaces of the substrates are opposed to each other with a slight distance therebetween. Then, the single emission side optical system may be arranged so that the light receiving portion of the light is on the extension of the main surfaces of both substrates which are joined or closely opposed to each other.

It should be noted that one of the first and second translucent substrates is associated with a ligand layer on which a biological substance having an activity on a substrate is immobilized, and the other is immobilized on the substrate with a deactivated biological substance. If the ligand layers are made to correspond to each other, the substrate concentration of the solution to be measured can be detected with higher accuracy only by using a single emission side optical system.

Further, in the above-mentioned first invention,
The light reception blocking means includes a first light source for guiding light to a light transmission path of the incident light side optical system formed by the first light transmissive substrate, and the second light transmissive substrate. A second light source for guiding light to the formed light transmission path of the incident light side optical system, and when one of the first and second light sources is in a lighting state, the other light source. And a lighting means for controlling lighting of both light sources.

In the biosensor having this structure, the first, second
When one of the light sources is in a lighting state, the lighting means controls the other light source to be in a non-lighting state, so that only the light of either one of the light sources is reflected on the light reflecting surface by one of the translucent substrates. Not transmitted. Therefore, light is received by the emission side optical system only for any one of the transparent substrates. Therefore, even with the biosensor having this configuration, the substrate concentration of the solution to be measured corresponding to each of the first and second translucent substrates can be detected with high accuracy only by using a single emission-side optical system.

Further, in the configuration of the above first invention,
The light-reception blocking means is provided on at least one of an entrance-side end surface and an exit-side end surface of the light transmission path in the first light-transmissive substrate, and shields and transmits light to the light transmission path. And a second shutter that is provided on at least one of an end face and an end face of the light transmission path of the second light-transmissive substrate and shields and transmits light to the light transmission path. And the first and second
When one of the two shutters is in the transmissive state, the other shutter is closed to control both shutters.

In the biosensor having this structure, the first and second
When one of the shutters is in the transmissive state, the other shutter is controlled by the shutter control means to be in the shielded state. It does not reach the emission side optical system after being reflected at. Therefore, light is received by the emission side optical system only for any one of the transparent substrates. Therefore, even with the biosensor having this configuration, the substrate concentration of the solution to be measured corresponding to each of the first and second translucent substrates can be detected with high accuracy only by using a single emission-side optical system.

In this case, the first and second shutters have various structures such as a so-called mechanical shutter that opens and closes the shield plate, a so-called liquid crystal shutter that shields and transmits light depending on the molecular arrangement of liquid crystals. Can be taken.

Further, in order to solve such a problem, the concentration measuring device of the second invention is a concentration measuring device for obtaining the concentration of the substrate to be measured in the solution to be measured, and the concentration measuring device according to any one of claims 1 to 10. Of the biosensor and the emission side optical system of the biosensor, the amount of reflected light for each reflection angle detected by the emission side optical system is used as an input signal, and the input signal is subjected to predetermined filtering to produce noise. Filter means that uses the signal from which the signal has been removed as an output signal, and computing means that computes the concentration of the substrate to be measured based on the output signal from the filter means.

In the concentration measuring apparatus according to the second aspect of the present invention, the amount of reflected light for each reflection angle detected by the optical system on the emission side of the biosensor is output as an output signal after noise is removed through a predetermined filtering process by the filter means. . Therefore, the correlation between the incident angle and the light amount thereof can be obtained with high accuracy, and the incident angle θ when the light amount of the reflected light is at the lowest level can be accurately determined. The noise in this case includes noise caused by the physical or chemical characteristics of the optical system or the transparent substrate on the incident light side or the outgoing light side, and the degree of the noise depends on the optical system or the transparent substrate. It depends on individual differences such as. By performing the concentration calculation based on this highly accurate output signal, the calculation precision of the concentration of the measurement target substrate by the calculation means is increased. As a result, the second
According to the concentration measuring device of the invention described above, it is possible to improve the calculation accuracy of the incident angle θ when the light amount of the reflected light is at the minimum level, and improve the calculation accuracy of the concentration of the measurement target substrate.

In the concentration calculation, as in the conventional case, the correlation between the incident angle and the refractive index of the medium, the relationship between the refractive index of the medium and the dielectric constant, the progress of the biochemical reaction by the biological substance and the dielectric constant are calculated. The correlation and the like are also taken into consideration.

[0048]

BEST MODE FOR CARRYING OUT THE INVENTION Next, an embodiment of the biosensor according to the present invention will be described based on its examples. FIG. 1 is a front view, a plan view and a right side view of a biosensor 20 of the first embodiment.

As shown in the figure, the biosensor 20 is formed into a rectangular parallelepiped approximately the size of a business card, and is formed by joining two acrylic substrates 22R and 22L at the substrate main surface 23. As shown in FIG. 1 and FIG. 2 which is a schematic exploded perspective view, the acrylic substrates 22R and 22L have the same external shape, and are symmetrical in forming the light source unit housing recess 24 having a hemispherical cross section. . Each acrylic substrate 2
2R, 22L front and back substrate main surface 23, well-known evaporation,
A chromium thin film is formed over the entire surface by a sputtering method or the like, and no chromium thin film is formed on the peripheral end faces of each acrylic substrate 22R, 22L.
In this case, the light source unit housing recess 24 in each of the acrylic substrates 22R and 22L has no chrome thin film formed on at least the bottom surface thereof.

Therefore, each acrylic substrate 22R, 22L
Makes the front and back substrate main surfaces 23 face each other as a total reflection surface that reflects light under a geometrical total reflection condition. Therefore, each of the acrylic substrates 22R and 22L transmits the light incident from the peripheral end surface or the bottom surface of the light source unit housing recess 24 by confining the wave of the light between the front and back substrate main surfaces 23.

The light source unit accommodating recesses 24 of the acrylic substrates 22R and 22L are each formed as a columnar recess having a hemispherical cross section, and the upper end surface 26 of the upper end of each acrylic substrate is formed.
It is formed so as to be inclined at a predetermined angle with respect to R and 26L. Further, the respective acrylic substrates 22R and 22L are joined so that their upper end surfaces 26R and 26L form the same plane. Therefore, although the light source unit housing recess 24 has a hemispherical cross section in each of the acrylic substrates 22R and 22L alone, when both acrylic substrates are joined, a cylindrical recess is formed and its central axis forms the same plane. Upper surface 26R, 2
It will be inclined with respect to 6L. Then, a light source unit 30 described later is housed in the light source unit housing recess 24.

As described above, gold thin films (Au thin films) 28 having a film thickness of 50 nm are formed on both upper end surfaces 26R and 26L, which have the same plane, by vapor deposition over both end surfaces. That is, the upper end surfaces 26R and 26L become the light reflection surfaces 29 of the total reflection surfaces that reflect the light under the geometric total reflection condition over the vapor deposition range of the Au thin film 28. Then, the acrylic substrate 22
An evanescent wave coupling is formed on the light reflecting surface 29 by the R and 22L and the Au thin film 28. Further, on the exposed film surface (hereinafter, simply referred to as surface) of the Au thin film 28, a ligand layer (not shown) on which a biological substance having a function of discriminating against a measurement target substrate and causing a biochemical reaction with the substrate is immobilized. Are formed.

In this case, the Au thin film 28 of one acrylic substrate, for example, the acrylic substrate 22L on the front side in FIG. 1 is used.
Has a ligand layer on which an active biological substance is immobilized, and the Au thin film 2 on the other acrylic substrate 22R.
8 has a ligand layer formed by immobilizing deactivated biological material. That is, the front acrylic substrate 22L is used as the substrate measuring sensor unit, and the other acrylic substrate 22R is used as the correcting sensor unit. That is, the acrylic substrate 22R and the acrylic substrate 22L are used for measuring the substrate and for correcting the substrate, and form a set of measurement systems. The inactivation of biological material is caused by strong acid or strong alkali, or irradiation with electron beams such as ultraviolet rays or ultrasonic treatment, 70 ° C.
It is carried out by deactivation treatment such as heat treatment to a certain degree.

As shown in the sectional view of FIG. 3, the light source unit 30 emits light of a single wavelength such as a p polarizing plate 32 at one end and an LED (light emitting diode), an LD (semiconductor laser) at the other end. A light source 34 is provided. In addition, in the outer cylinder 36,
A lens 38 including a pair of curved glass 37a and 37b having a columnar curved surface is fixedly provided via a lens holder sleeve 39. Therefore, the light source unit 30 condenses the light p-polarized by the p-polarizer 32 (p-polarized light) to the focal point f by the lens 38 and irradiates the light from the p-polarizer 32 side to the outside. Since the curved surfaces of the curved glasses 37a and 37b are columnar curved surfaces, the lens 38 collects the p-polarized light linearly along a line orthogonal to the paper surface at the focal point f.

The light source unit 30 is positioned forward and backward in the light source unit accommodation recess 24 so that the linear focus f is included in the light reflecting surface 29 of the upper end surfaces 26R and 26L of the acrylic substrates 22R and 22L. It is adjusted and assembled and fixed in the recess. Therefore, the light emitted from the light source 34 of the light source unit 30 is emitted from the p polarizing plate 3
The light is p-polarized by 2 and enters the acrylic substrates 22R and 22L from the bottom surface of the light source unit housing recess 24. And
The p-polarized light is totally reflected by the principal surface 23 of the acrylic substrates 22R and 22L, which are the facing total reflection surfaces of the acrylic substrates 22R and 22L.
It is transmitted in 22L. In other words, the p-polarized light radially travels through the acrylic substrates 22R and 22L so as to be focused on the line-shaped focus f included in the light reflecting surface 29, and the light is reflected by the upper end surfaces 26R and 26L of the acrylic substrates 22R and 22L. To surface 29. Therefore, the light reflecting surface 29 has p
The light, which is p-polarized by the polarizing plate 32 and is a wave in the incident plane, has a predetermined range of incident angles (θ1 to θ2) determined by the focal length F of the lens 38, the aperture length D, the formation angle of the light source unit housing recess 24, and the like. ) To reach.

In addition, the reflected light totally reflected by the light reflecting surface 29 is emitted from the acrylic substrates 22R and 22L, and the output side end faces 25R and 25L of the acrylic substrates 22R and 22L detect the amount of received light and generate electricity. CCD to convert to signal
The image pickup devices 27R and 27L are fixed in close contact with each other. Therefore, the p-polarized light that has entered the acrylic substrates 22R and 22L from the bottom surface of the light source unit housing recess 24 reaches the light reflection surface 29 and from the light reflection surface 29 to the emission side end surfaces 25R and 25L. Each acrylic substrate 22
The light-emitting side end faces 25R and 25L are reached using the space between the R and 22L facing main substrate surfaces 23 as a transmission path. The state of transmission of the p-polarized light during this period will be described by taking as an example the steps up to the emission side end faces 25R and 25L. As shown in FIG. 4, the p-polarized light is totally reflected by the main surface 23 of the substrate, which is a facing total reflection surface. At the same time, the wave is confined and transmitted between the main surfaces 23 of the substrate.

Then, the CCD image pickup devices 27R, 27
The amount of light is detected by L. Output side end face 25R,
Like the incident light on the light reflecting surface 29, the light reaching 25 L is a wave having an amplitude within the incident surface, and the CCD image pickup device 27
In R and 27L, the light amount for each reflection angle, that is, the light amount for each incident angle (θ1 to θ2) in the above range is detected. This CC
If the D image pickup devices 27R and 27L are area sensors (two-dimensional matrix type sensors), it is not necessary to provide a plurality of image pickup devices.

Here, the concentration measuring device 45 for measuring the substrate concentration using the biosensor 20 described above will be explained. The concentration measuring device 45 is the same as the biosensor 20 described above.
And an electronic control unit 40 connected to the CCD image pickup devices 27R and 27L. This electronic control unit 40 is
As shown in FIG. 5, a well-known CPU, ROM, RAM, and a logical operation circuit 40a configured by connecting I / O ports to each other via a common bus, and a low-pass filter 40b configured by various electronic devices ( For example, FIR
Type (finite-duration impulse-response) digital low-pass filter). This electronic control unit 40
Is an electric signal from the CCD image pickup devices 27R and 27L, that is, an amount of reflected light for each reflection angle is input to the low-pass filter 40b, and an output signal from which noise superimposed as a high-frequency component is removed is output from the low-pass filter 40b.
hand it over to a. Then, in this logical operation circuit 40a,
From the distribution of the light quantity for each reflection angle, the reflection angle when the light quantity of the reflected light is at the lowest level, that is, the incident angle when the light quantity is at the lowest level is calculated. Note that this calculation is performed by each CCD image sensor 27.
It is performed separately based on the signals from R and 27L. In addition, the electronic control unit 40 controls the light source 34 of the light source unit 30.
Is turned on and the measurement of the substrate concentration is started in response to a pressing operation of a measurement start switch (not shown). Further, the electronic control unit 40 includes the monitor 100 and the printer 10.
It is connected to 2 and monitors the measurement result of the substrate concentration 10
It is displayed on 0 or is printed on the printer 102.

When measuring the substrate concentration by the biosensor 20 described above, the respective acrylic substrates 22R, 22R
The Au thin film 28 on the upper surfaces 26R and 26L of 2L is
Each is immersed in the solution to be measured. Then, in the acrylic substrate 22L in which the ligand layer on which the active biological substance is immobilized is formed on the surface of the Au thin film 28, the biochemical reaction between the substrate and the biological substance proceeds on the surface of the Au thin film 28, and the solution to be measured is The dielectric constant of, and consequently its refractive index changes. However, on the other acrylic substrate 22R, since the biological substance is inactivated, the biochemical reaction with the measurement target substrate does not proceed, and the dielectric constant of the solution to be measured on the surface of the Au thin film 28, by extension, Its refractive index does not change.

Next, the substrate concentration measuring routine executed by the biosensor of the first embodiment having the above-mentioned structure will be described with reference to the flowchart of FIG. FIG. 6 is a flowchart showing the processing of the routine, which is started after the power is turned on.

When the power is turned on, it is determined whether or not the light source 34, which is controlled to be lit by a light source lighting routine (not shown), is in a stable lighting state, for example, based on the elapsed time from the start of lighting (step S100), Wait until a positive decision is made. If it is determined that the light source 34 is stably turned on, it is determined that the measurement of the substrate concentration can be started, and it is determined whether or not the measurement start switch has been pressed (step S110).
Wait until the switch is turned on.

In the measurement of the substrate concentration, that is, before the measurement start switch is turned on, the acrylic substrate 22R and
Immersion of the solution to be measured of the Au thin film 28 in 2 L is performed. When this work is completed, the measurement start switch is turned on.

When the operator turns on the measurement start switch, the CCD image pickup device 27 receives the signal.
Reading of sensor outputs from R and 27L is started and the values are stored (step S130). Then, the elapsed time from the start of reading is measured and it is determined whether or not a predetermined time has elapsed (for example, 3 to 10 minutes) (step S1
40) Continue reading and storing the sensor output until a positive determination is made. During the reading and storage of the sensor output, on the surface of the Au thin film 28 on each of the acrylic substrates 22R and 22L, the dielectric constant of the solution to be measured changes as described below depending on the presence or absence of the activity of the biological material, and after the change, The value stabilizes, and this state is detected by the CCD image pickup devices 27R and 27L.

On the surface of the Au thin film 28 of the acrylic substrate 22L on the side where the active biological substance is fixed, the biochemical reaction between this biological substance and the measurement target substrate proceeds to the extent defined by the substrate concentration. , The dielectric constant of the measured solution,
Furthermore, its refractive index changes as the biochemical reaction progresses and stabilizes at a value defined by the substrate concentration. At this time, the change in the dielectric constant of the solution to be measured, and by extension, its refractive index,
It is observed as a phenomenon of energy of reflected light when the surface plasmon resonance phenomenon occurs due to the evanescent wave coupling formed by the acrylic substrate 22L and the Au thin film 28 of the light reflecting surface 29.

That is, the p-polarized light emitted from the light source 34 is incident on the acrylic substrate 22L from the bottom surface of the light source unit accommodating recess 24, and evanescent wave coupling is formed at the incident angle in the above range (θ1 to θ2). To the light reflection surface 29. At this time, an angle (θS
The p-polarized light incident at the incident angle of 1) causes the evanescent wave on the light-reflecting surface 29 side of the Au thin film 28 and the surface plasmon wave on the measured solution side of the Au thin film 28 to resonate by matching their wave numbers. Causes the surface plasmon resonance phenomenon. When this surface plasmon resonance phenomenon occurs, the energy of light having an incident angle of θS1 is used as the excitation energy of the surface plasmon wave, and the energy of reflected light having a reflection angle of θS1 from the light reflecting surface 29 decreases.

Therefore, as shown in FIG. 7, from the CCD image pickup device 27L which receives the reflected light from the light reflecting surface for each incident angle (θ1 to θ2) on the acrylic substrate 22L at the reflection angle of θ1 to θ2. In addition, the correlation between each incident angle of the incident angles (θ1 to θ2) and its light quantity is obtained (FIG. 7).
(A)).

On the other hand, on the surface of the Au thin film 28 of the other acrylic substrate 22R on which the deactivated biological material is fixed, since the biochemical reaction with the substrate to be measured does not proceed, the dielectric constant of the solution to be measured and the Moreover, its refractive index is constant as it is at the initial value. However, the surface plasmon resonance phenomenon occurs due to the p-polarized light incident at an incident angle of a certain angle (θS0), and from the CCD image pickup device 27R corresponding to this acrylic substrate 22R, as shown in FIG. 7, the incident angles (θ1 to θ2 The correlation between each incident angle and the amount of light is obtained (Fig. 7).
(B)), when the incident angle is θ S0, the energy of reflected light decreases.

Next, in step S140, both C
Based on the correlation between the reflection angle and the light amount obtained from the CD image pickup devices 27R and 27L, the respective acrylic substrates 22
For the solutions to be measured corresponding to R and 22L, the incident angles (θS1, θS0) when the amount of reflected light is at the lowest level are obtained (step S150). Then, this incident angle (θS1, θ
S0), the correlation between the incident angle at which the amount of light reaches the lowest level and the refractive index of the medium (solution to be measured), the relationship between the refractive index of the medium and the dielectric constant, the progress of the biochemical reaction by the biological material and the dielectric constant The substrate concentration is calculated from the correlation with (step S
160). It should be noted that the correlation between the incident angle at which the amount of light is at the lowest level and the refractive index of the medium (solution to be measured), the relationship between the refractive index of the medium and the dielectric constant, the progress of the biochemical reaction by the biological substance, and the dielectric constant The correlation and the like are stored in advance in the ROM of the logical operation circuit 40a in the electronic control unit 40.

Here, how the incident angles (θS1, θS0) are calculated in step S150 will be described in detail. The CCD image pickup devices 27L and 27R are the light source unit 3
In addition to 0, the light quantity distribution of the reflected light on which the noise of the high frequency component caused by the physical or chemical characteristics of the acrylic substrates 22R and 22L is superimposed is input, and the distribution is output to the low-pass filter 40b as an electric signal (FIG. 8). (A)). This noise is generated at the location of the same pixel number of the CCD image pickup device even if the measurement is repeated. The signal on which the noise is superimposed is filtered by the low-pass filter 40b of the electronic control unit 40 into the signal from which the noise of the high frequency component is removed, and is input to the logical operation circuit 40a (FIG. 8).
(B)). Then, the filtered signal (the signal whose reflectance corresponds to the pixel number) is converted into the pixel number by the variable x,
The incident angle (θS1, θS0) is calculated by performing a well-known nth-order regression analysis using a regression equation in which the reflectance is the variable y. FIG. 9 shows the result of the quadratic regression analysis using the quadratic regression curve in an enlarged manner in the vicinity of the lowest light level. The incident angle (θS1, θS0) is finally calculated by this secondary regression analysis. Since θS1 and θS0 are calculated based on the signals obtained by filtering the electric signals from the respective CCD image pickup devices, they are naturally calculated as different values.

Thereafter, the calculated substrate concentration is monitored 100
(See FIG. 5) or the printer 10 together with the display.
2 (see FIG. 5) and print out (step S
170) and terminates this routine once. After that, when the measurement start switch is pressed through preparations such as immersion of the Au thin film 28 in a new solution to be measured, the above-described processing is repeated to calculate the substrate concentration.

If the substrate to be measured is an antigen, one acrylic substrate 2 is used in the biosensor 20 of the first embodiment described above.
The surface of the 2 L Au thin film 28 is provided with a ligand layer on which an antibody against this antigen is fixed, and the other acrylic substrate 22R.
The antigen concentration can be measured by fixing the antibody inactivated by heat on the surface of the Au thin film 28.

As described above, in the biosensor 20 of the first embodiment, the transmission of light from the light source unit 30 to the light reflecting surface 29 and the CCD image pickup device 27 from the light reflecting surface 29.
Main surface 2 of the substrate, which is a total reflection surface, for transmitting light to R and 27L
The acrylic substrates 22R and 22L facing each other are only used, and an optical device such as a hemispherical lens with high processing accuracy is not required. Further, in this biosensor 20, the acrylic substrates 22R and 22L are provided with A on the upper end surfaces 26R and 26L.
The u thin film 28 was provided and the light reflecting surface 29 was formed on the upper end face, and the acrylic substrates 22R and 22L and the Au thin film 28 formed evanescent wave coupling. That is, in the biosensor 20 of the first embodiment, the light transmitting medium forming the light reflecting surface 29 is covered by the acrylic substrates 22R and 22L, and a separate light transmitting medium for forming the light reflecting surface 29 is not required. Therefore, it is not necessary to use the matching oil, and it is not necessary to consider the amount of the matching oil or the temperature of the environment in which it is used. Moreover, inexpensive acrylic substrates 22R and 22L in which chromium thin films are simply formed on the front and back substrate main surfaces are used,
Even if a fingerprint or the like is attached to the main surface of the substrate by touching it, light transmission on the acrylic substrate is not hindered at all. Therefore, according to the biosensor 20 of the first embodiment, the usability of the sensor can be improved and the cost can be reduced.

In the biosensor 20, the light source unit 30 for irradiating the light reflecting surface 29 with the light from the light source 34.
Are fixed in the light source unit housing recesses 24 of the acrylic substrates 22R and 22L, and the CCD image pickup devices 27R and 27L that receive the light totally reflected by the light reflecting surface 29 are also output side end faces 25R of the acrylic substrates 22R and 22L. , Fixed at 25 L. Therefore, in the biosensor 20, the relative positional relationship between the light source unit 30, the acrylic substrates 22R and 22L, and the CCD image pickup devices 27R and 27L need only be temporarily fixed, and the positional relationship is affected by external vibration or the like. Can be maintained without being done. Therefore, according to the biosensor 20 of the first embodiment, since maintenance and inspection are not required after the sensor is completed, the usability thereof can be further improved.

Further, the biosensor 20 is composed of acrylic substrates 22R and 22L, a light source unit 30 and a C.
The CD image pickup devices 27R and 27L are merely formed, and the chromium thin film is merely formed on the substrate main surfaces of the acrylic substrates 22R and 22L. Therefore, according to the biosensor 20, the mass productivity of the sensor can be improved and the cost can be further reduced.

In the biosensor 20, the acrylic substrate 22L on the side on which the active biological material is fixed and the acrylic substrate 22R on the side on which the inactivated biological material is fixed are used together, and the acrylic substrates 22R and 22L are used. The reflected light from the light reflecting surface 29 of the corresponding Au thin film 28 portion is received. Therefore, according to the biosensor 20 of the first embodiment,
Due to the relationship between the incident angle and the amount of light on the side where the deactivated biological material is fixed, the measurement error caused by factors other than biochemical reactions (temperature of the solution, dielectric constant, etc.) is eliminated to improve the measurement accuracy. be able to.

In the biosensor 20 described above, the output signal from which the noise of the high frequency component has been removed by the low pass filter 40b is subjected to regression analysis to calculate the incident angle (θS1, θS0) at which the amount of reflected light is at the lowest level. . Therefore, noise that cannot be completely removed by the low-pass filter 40b is also eliminated, so that the measurement accuracy of the substrate concentration can be further improved through the improvement of the calculation accuracy of the incident angle (θS1, θS0). Moreover, since the low-pass filter 40b is configured by the FIR type digital low-pass filter, the effective number of the reflected light amount (reflection intensity) is increased to increase the apparent resolution, and the calculation accuracy of the incident angles (θS1, θS0) is further increased. Was able to increase.

Here, a modification of the first embodiment will be described. In this modification, the main configuration is the same as that of the biosensor 20 of the first embodiment described above, and the configuration is different in the following points. In the following description, in order to avoid duplication of description, the same members as those in the first embodiment will be designated by the same reference numerals and the description thereof will be omitted.

In the biosensor 21 of the modified example of the first embodiment, as shown in FIG. 10, the emitting side end faces 25R, 25L are provided.
Was formed as follows. That is, the exit side end surface 2
5R and 25L are lenses 38 in the light source unit 30.
The light along the optical axis is reflected by the light reflecting surface 29 so as to be orthogonal to the traveling path. According to the biosensor 21 of this modified example, in the CCD image pickup devices 27R and 27L, the light amount for each incident angle (θ1 to θ2) is regarded as the light having almost the same optical path length after being reflected by the light reflecting surface 29. Can be measured.

Next, the second embodiment will be described. The biosensor 20 according to the second embodiment is also the same as the biosensor 20 according to the first embodiment described above, except for the following points.

The biosensor 50 of the second embodiment is shown in FIG.
As shown in, the plate seat surface 42 for mounting the sample plate, which is partitioned by the biaxial groove 41, is provided substantially at the center of the upper end surfaces 26R, 26L of the acrylic substrates 22R, 22L. The plate seat surface 42 is lowered one step by the upper end surfaces 26R and 26L, and the step is taken into consideration in consideration of the thickness of the acrylic resin sample plate 44 placed with the matching oil 43 interposed on the seat surface. Have been set. That is, when the sample plate 44 is placed on the plate seat surface 42 with the matching oil 43 interposed, the p-polarized light from the light source unit 30 is the upper surface of the sample plate 44, that is, the light formed by vapor deposition of the Au thin film 28. The plate seat surface 42 is formed with a step so that it is condensed on the reflecting surface 29. Matching oil 4
No. 3 is an oil whose refractive index is about the same as that of acrylic resin.

Similar to the first embodiment, the surface of the Au thin film 28 above the acrylic substrate 22L is the surface of the Au thin film 28 where the ligand layer on which the antibody against the antigen is immobilized is above the acrylic substrate 22R. Are each provided with a ligand layer on which the inactivated antibody is immobilized.

Therefore, the biosensor 5 of the second embodiment is
According to 0, by exchanging the sample plate 44 on which the ligand layer corresponding to the substrate to be measured is formed, it is possible to use for various kinds of substrate measurement as in the conventional sensor.

Further, even if the matching oil 43 flows due to an increase in environmental temperature, the biosensor 50 can temporarily store the flowing matching oil 43 in the groove 41. Further, in the biosensor 50, since the plate seat surface 42 is separated from the emission side end surfaces 25R, 25L at approximately the center of the upper end surfaces 26R, 26L, the flowing matching oil 43 does not reach the emission side end surfaces 25R, 25L. Few. Moreover, the light source unit 30 has the upper end surface 2
Light source unit storage recess 2 inclined with respect to 6R and 26L
As a result, the flowing matching oil 43 rarely enters the light source unit housing recess 24. Therefore, the flowing matching oil 43
The light transmission is not hindered by this.

Therefore, the biosensor 50 of the second embodiment is
According to this, it is possible to achieve an improvement in versatility with respect to the measurement substrate and avoidance of a decrease in measurement accuracy due to the matching oil 43 with a simple configuration.

Next, a third embodiment will be described. In the third embodiment, the biosensor 2 of the first embodiment described above is used.
0, acrylic substrates 22R and 22L, and light source unit 3
0, the CCD image pickup devices 27R and 27L are used, and the configuration is the same, but the configurations are different in the following points.

In the biosensor 60 of the third embodiment, as shown in FIG.
As shown in FIG. 2, the acrylic substrates 22R and 22L each have an emission-side inclined end surface 6 in which the end surface on the side where the reflected light totally reflected by the light reflection surface 29 reaches is inclined with respect to the opposing substrate main surface 23.
1R and 61L. In this case, as shown in FIG.
The inclination angles of the outgoing side inclined end faces 61R and 61L with respect to the substrate main surface 23 of the acrylic substrates 22R and 22L are 45 °. A thin film of chrome is vapor-deposited on the surfaces of the emission side inclined end faces 61R and 61L.
R and 61L are total reflection surfaces that reflect light under geometric total reflection conditions.

The main surface 23 of the substrate, which is a total reflection surface over the entire surface by the chromium thin film, is the emitting side inclined end surface 61.
In a range facing R and 61L, specifically, a range of width T × height H shown by diagonal lines in FIG.
It is set to 3A. That is, the chromium thin film is not formed on the main surface 23 of the substrate over the range of the light transmitting surface 23A. The CCD image pickup devices 27R and 27L are fixed to the light transmitting surface 23A. In addition, as described above, the main surface of the substrate 23 that does not require the chromium thin film only in a partial range
To achieve this, a chromium thin film may be formed on the main surface 23 of the substrate while masking the area. In addition, the light transmitting surface 2
The width T of 3A is equal to that of the acrylic substrates 22R and 22L because the emission side inclined end faces 61R and 61L are inclined at 45 °.
Equal to the thickness of.

Therefore, the light totally reflected by the light reflecting surface 29 (p
The polarized light is reflected by the emission-side inclined end faces 61R and 61L and travels toward the light-transmissive face 23A, as shown in FIG. 14, which shows how light is transmitted near the emission-side inclined end faces 61R and 61L. The light is transmitted to the outside from the light transmitting surface 23A of the acrylic substrates 22R and 22L. Then, the light amount of the light emitted to the outside is detected as the light amount for each incident angle (θ1 to θ2) on the light reflecting surface 29 by the CCD image pickup devices 27R and 27L on the surface of the light transmitting surface 23A.

Biosensor 6 of the third embodiment described above
In the case of 0, the CCD image pickup devices 27R and 27L for detecting the light amount for measuring the substrate may be provided on the light transmitting surface 23A in the substrate main surface 23 of the acrylic substrates 22R and 22L.
Therefore, according to this biosensor 60, since the mounting portion is wide, the assembling work and the handling of the CCD image pickup devices 27R and 27L can be simplified, and the shape and size of the CCD image pickup devices 27R and 27L used. The versatility can be enhanced by relaxing restrictions such as size.

Here, a first modification of the third embodiment will be described. In this modification, the following configuration is added to the biosensor 60 of the third embodiment described above. That is, FIG.
As shown in, the acrylic substrate 22R, the acrylic substrate 22
A cylinder lens 62 made of acrylic resin was fixed to the light transmitting surface 23A of L. Then, as shown in FIG.
The image pickup devices 27R and 27L are arranged so as to face the cylinder lens 62 so that the cylinder lens 62 focuses on the light receiving portion. Therefore, according to the biosensor 60 of this modified example, it is possible to collect the light emitted from the light transmitting surface 23A of the acrylic substrates 22R and 22L and obtain the light amount thereof, and thus, for each incident angle (θ1 to θ2). It is possible to increase the amount of measuring light and improve the detection sensitivity.

Next, a second modification of the third embodiment will be described. In this modification, the biosensor 60 of the third embodiment described above is used as the biosensor 2 of the modification of the first embodiment.
It was constructed by modifying 1 as follows. That is, in the biosensor 64 of this modified example, FIG.
As shown in FIG. 18 of the sectional view taken along the line 8-18, the emission side inclined end surfaces 61R and 61L are orthogonal to the locus along which the light along the optical axis of the lens 38 in the light source unit 30 is reflected by the light reflection surface 29 and travels. Thus, the corner end faces are formed so as to be inclined at 45 ° with respect to the substrate main surface 23 of the acrylic substrates 22R and 22L. Then, similarly to the third embodiment, the emission side inclined end surfaces 61R and 61L are made into a total reflection surface through a thin film deposition of chromium on the surface.

Further, as shown by the slanting lines in FIG. 17, the area facing the outgoing side inclined end surfaces 61R and 61L is the light transmitting surface 23A, and the light transmitting surface 23A has CCs.
The D image pickup devices 27R and 27L are fixed. Therefore,
According to the biosensor 64 of this modified example, in addition to simplifying the handling by fixing the CCD image pickup devices 27R and 27L to the substrate main surface 23, the optical path length after being reflected by the light reflecting surface 29 is almost the same. As light, the amount of light for each incident angle (θ1 to θ2) can be measured.

Next, a fourth embodiment will be described. In the fourth embodiment, the biosensor, the acrylic substrates 22R and 22L, and the light source units 30 and C of the above-described embodiments are used.
The configuration is the same in that the CD image pickup devices 27R and 27L are used, and the configuration is different in the following points.

In the biosensor 70 of the fourth embodiment, as shown in FIG.
As shown in FIG. 9, the light source unit 30 is fixedly provided as follows. That is, the incident-side end surface 7 that intersects the upper end surfaces 26R and 26L of the acrylic substrates 22R and 22L at an angle.
1R and 71L are light-transmitting end faces, and the light source unit 30 is directly fixed to the incident-side end faces 71R and 71L with an acrylic light-transmitting thin plate 72 interposed. The incident side end surfaces 71R and 71L are the upper end surfaces 26R and 26L.
When the acrylic substrates 22R and 22L are joined so as to be on the same plane, they are also on the same plane.

When fixing the light source unit 30, as shown in FIG. 20, the light from the light source unit 30 is condensed and focused on the light reflecting surface 29 formed by providing the Au thin film 28 on the upper end surfaces 26R and 26L. The light source unit 30 is fixed to the incident-side end faces 71R and 71L so as to connect with each other. In this case, the light source unit 30 and the acrylic substrates 22R and 22L can be fixed using a holder (not shown).

Further, in this biosensor 70, as shown in FIG.
As shown in FIG. 1, one lumirror sheet 73 is sandwiched between the acrylic substrate 22R and the acrylic substrate 22L in a sandwich shape to join the two acrylic substrates, and the other two lumirror sheets 73 are attached to the other acrylic substrate main surface. 23, and the sheet was brought into close contact with the main surface 23 of each substrate. The lumirror sheet 73 is a light-impermeable sheet having a refractive index greatly different from that of acrylic, and the light-transmissive surface 23A facing the outgoing-side inclined end surfaces 61R and 61L.
The shape is such that it conforms to the main surface 23 of the substrate except the area. Therefore, even in the biosensor 70, the substrate main surface 23 in the range excluding the light transmitting surface 23A facing the emitting side inclined end surfaces 61R and 61L is a total reflection surface. That is, in the fourth embodiment, instead of the vapor deposition of the chromium thin film, the lumirror sheet 73 is used to make the opposing substrate main surface 23 the total reflection surface.

According to the biosensor 70 having the above structure, the following effects can be obtained in addition to the same effects as those of the biosensor 20 and the like of the first embodiment, such as improvement of usability and measurement accuracy. .

The light source unit 30 is connected to the acrylic substrate 22R,
Both acrylic substrates can be thinned because they do not need to be accommodated in 22L. Moreover, even when the light source unit 30 irradiates light to the outside of the substrate by thinning the substrate, the light is not blocked by the light-impermeable lumirror sheet 73 and does not enter the inside of the substrate. There is no problem. Therefore, according to the biosensor 70,
The acrylic substrates 22R and 22L can be made to have a thickness of 5 mm or less, for example, about 2 mm.
The weight can be reduced. Moreover, due to this reduction in size and weight, handling during transportation becomes easier.

In addition, even if the biosensor 70 is used so that the sample plate 44 is placed on the upper end surfaces 26R and 26L with the matching oil 43 interposed as described in the biosensor 50 of the second embodiment, the flow can be reduced. The matching oil 43 is not blocked by the translucent thin plate 72 and does not enter the optical path of light. Therefore, according to the biosensor 70 of the fourth embodiment, it is possible to maintain high measurement accuracy of the measurement target substrate even when the matching oil 43 is used.

In addition to this, the biosensor 20A of the following modified example can be used. As shown in FIG. 22, in the biosensor 20A of this modified example, the emission side end faces 25R and 25L of the biosensor 20 of the first embodiment are connected to the substrate main surface 23.
Similarly, a chromium thin film is vapor-deposited to form a total reflection surface, and the bottom surfaces 75R and 75L of the acrylic substrates 22R and 22L are used as light transmission surfaces. Then, CCD image pickup devices 27R and 27L are fixedly provided on the bottom surfaces 75R and 75L. In the biosensor 20A of this modified example, the reflected light reflected by the light reflecting surface 29 is further reflected by the emission side end faces 25R, 25L, and the light amount of the reflected light is measured at the bottom surfaces 75R, 75L for each reflection angle. Therefore, the acrylic substrate 22R, 2 is provided with an optical path having a length suitable for the measurement resolution of the CCD image pickup device 27R, 27L.
Since it is secured by reflection at 2 L, the biosensor 20
According to A, the miniaturization of the sensor can be further promoted.

Further, in each of the above embodiments, the case where one substrate concentration is measured using the two acrylic substrates 22R and 22L has been described, but it can be modified as follows. This modification will be described by taking the case where the biosensor 60 of the third embodiment is modified as an example. In the biosensor 80 of this modification, as shown in FIG. 23, both acrylic substrates 22R and 22R of the biosensor 60 of the third embodiment.
N sheets of L were used (four sheets are shown in the figure). The acrylic substrates 22L1, 22L2, 22L3, 22L4 and the acrylic substrates 22R1, 22R2, 22R3, 22R4 are
The heights are the same and the lengths are made longer in this order.
Each of these acrylic substrates is superposed and bonded as shown in the figure.

Further, on the upper surface of each acrylic substrate thus bonded, A in the biosensor 60 of the third embodiment is formed.
An Au thin film 81 corresponding to the u thin film 28 is formed by vapor deposition on each acrylic substrate, and the lower surface of the Au thin film 81 serves as a light reflecting surface. Then, each acrylic substrate has a light source unit housing recess cutout 82 that replaces the light source unit housing recess 24.
Is provided. Then, in the light source unit housing recess notch 82, a line-shaped optical unit (not shown) that emits light in a line shape is incorporated and fixed. The line-shaped optical unit is, of course, incorporated and fixed so that the light-reflecting surface is focused.

In this biosensor 80, the acrylic substrate 22L1 and the acrylic substrate 22R1 are paired, the acrylic substrate 22L1 is the acrylic substrate on the side on which the active biological material is fixed, and the acrylic substrate 22R1 is the inactivated biological body. The acrylic substrate on which the substance is fixed is used. The same applies to the acrylic substrate 22L2 and acrylic substrate 22R2, and the acrylic substrate 22Li and acrylic substrate 22Ri. Therefore, in the biosensor 80, one of the pair is for measuring the substrate and the other is for the sensor part for correcting the pair, so that the concentrations of N kinds of substrates can be simultaneously measured.
Moreover, such a sensor capable of measuring various kinds of substrate concentrations can be downsized. The combination of glass substrates forming a pair is arbitrary.

Next, a fifth embodiment will be described. In the fifth embodiment, the biosensor and the acrylic substrates 22R and 22L of the above-described respective embodiments are used and the light source unit 3 is used.
The configuration is the same in that it has 0, and the configuration is different in the following points.

In the biosensor 85 of the fifth embodiment,
As shown in FIG. 24, the acrylic substrates 22R and 22L have incident side end faces 71R and 71L that are inclined similarly to the biosensor 70 of the fourth embodiment described above, and these are used as incident faces of light from the light source. To do. The biosensor 85 includes acrylic substrates 22R and 22L by bonding the substrate main surfaces 23 to each other, and incident side end faces 71R and 7R of both acrylic substrates.
A non-translucent partition plate 86 is provided upright on the 1L side. Therefore, the partition plate 86 allows the acrylic substrate 22
Light enters the R and 22L independently from the incident-side end faces 71R and 71L. In this case, the partition plate 86 may be sandwiched between the two acrylic substrates 22R and 22L.

In addition, the biosensor 85 includes a light source unit 30R and a light source unit 30R for each acrylic substrate 22R and 22L.
30L, each light source unit to acrylic substrate 22
Light is incident on R and 22L. In the biosensor 85, the light emitted from one light source unit to one acrylic substrate does not enter the other acrylic substrate by the partition plate 86. However, both light source units are arranged so that the light condensed and emitted from the light source unit (see FIG. 3) reaches the incident side end surface of the corresponding acrylic substrate, but does not reach the incident side end surface of the adjacent acrylic substrate. If arranged, the light can be incident on the acrylic substrates 22R and 22L from each light source unit without using the partition plate 86. Further, as in the biosensor 70 shown in FIG. 19, each light source unit can be fixed to the incident side end surface of each acrylic substrate, and in this case also, the partition plate 86.
Becomes unnecessary.

The light source units 30R and 30L emit p-polarized light on the light reflecting surface 29 formed of the Au thin film 28 formed by vapor deposition on the upper end surfaces 26R and 26L of the acrylic substrates 22R and 22L, similarly to the sensor described above. It is installed so that it collects and irradiates.

Further, the biosensor 85 has a single CCD image pickup device 27, which is different from the above-mentioned sensors. The CCD image pickup device 27 is arranged so as to be an extension of the substrate main surface 23 of the acrylic substrates 22R and 22L joined as described above, and the emission side end faces 25R and 25.
It is provided facing L. Therefore, as shown in FIG. 25, which is a schematic plan view of the biosensor 85, the light emitted from the light source units 30R and 30L (p
(Polarized light) is individually transmitted between the corresponding acrylic substrates 22R and 22L between the substrate main surfaces 23 to generate a light reflection surface 29.
Leading to. After being totally reflected by the light reflecting surface 29, the p-polarized light is continuously transmitted as reflected light between the substrate main surfaces 23 and is emitted to the outside from the acrylic substrates 22R and 22L via the emission side end faces 25R and 25L. . The reflected light (p-polarized light) emitted from each of the acrylic substrates 22R and 22L is received by the CCD image pickup device 27 arranged as described above.

The light source units 30R and 30L are respectively connected to the electronic control unit 40, and are turned on by a control signal from the electronic control unit 40. In the present embodiment, the electronic control unit 40 controls the light source unit 30 so that when one of the light source units 30R and 30L is in the on state, the other light source unit is in the off state.
Lighting control of R and 30L is performed.

Also in the biosensor 85, the acrylic substrate 22L is made of the Au thin film 2 similarly to the above-mentioned sensors.
8 is used as a substrate measuring sensor part in which a ligand layer having an active biological substance immobilized thereon is formed, and the other acrylic substrate 22R has a ligand layer having an inactivated biological substance immobilized on the Au thin film 28. The formed correction sensor unit is used.

In the concentration measuring device 45 using the biosensor 85 of the fifth embodiment described above, the substrate concentration is measured as follows. The substrate concentration measuring routine executed by the concentration measuring apparatus of the fifth embodiment for measuring the substrate concentration is almost the same as the substrate concentration measuring routine shown in FIG. Description will be made with reference to the flow charts as appropriate.

In the concentration measuring apparatus of the fifth embodiment, first, the light source unit 30L on the acrylic substrate 22L side, which is the substrate measuring sensor section, is lit to achieve stable lighting, and during this period, the acryl sensor section for correction is used. The light source unit 30R on the side of the substrate 22R is kept turned off. This process
This corresponds to step S100 shown in FIG. After the stable lighting of the light source unit 30L, the processing from step S110 to step S150 in FIG. 6 is performed. That is, only for the acrylic substrate 22L that is the substrate measuring sensor portion, irradiation of light (p-polarized light) from the light source unit 30L, transmission of light between the substrate main surfaces 23 of the acrylic substrate 22L, and the light reflecting surface 2 are performed.
Total reflection at 9 and reception of reflected light at the CCD image sensor 27 are performed. As a result, the light amount for each incident angle (θ1 to θ2) is measured by the substrate measuring sensor unit, and the reflection angle θS1 at which the energy of the light is minimum is obtained (FIG. 7).
(A)).

After that, the light source unit 30L, which has been turned on up to that point, is turned off, and the light source unit 30R on the acrylic substrate 22R side, which is the correction sensor section, is turned on to achieve stable lighting. That is, the light source unit to be turned on stably is changed. Then, after that, only the acrylic substrate 22R, which is the correction sensor unit, is subjected to the processing from Step S110 to Step S150 in FIG. 6, and the reflection angle θS0 at which the light energy is minimum in the correction sensor unit is obtained. (See FIG. 7B). Next, the process from step S160 onward in FIG. 6 is performed to calculate the substrate concentration.

As described above, also in the biosensor 85 of the fifth embodiment, the reflection angles (θS1, θS) at which the energy of light becomes the minimum depending on the presence or absence of the activity on the measurement substrate.
0) can be calculated, and the measurement accuracy can be improved by eliminating the measurement error caused by factors other than the biochemical reaction (temperature of the solution, dielectric constant, etc.). Moreover, according to the biosensor 85, a single C
It can be realized only by using the CD image pickup device 27. Of course, the biosensor 85 can obtain the effects (improvement in usability, cost reduction, etc.) exhibited by the above-described sensors.

Next, the sixth embodiment will be described. In the sixth embodiment, the biosensor 85 of the fifth embodiment described above is used.
And a point using acrylic substrates 22R and 22L or a single C
The configuration is the same in that the CD image pickup device 27 is used, and the configuration is different in the following points.

As shown in FIG. 26, the biosensor 87 of the sixth embodiment is installed on the incident side end faces 71R and 71L of the acrylic substrates 22R and 22L so as to cover the end faces, and the light to the end faces is detected. And liquid crystal shutters 88R and 88L for switching between blocking and transmitting. These liquid crystal shutters 88R and 88L are well-known TN type (twisted nematic type) liquid crystal shutters, and change the arrangement of each liquid crystal by the application of a voltage from the electronic control unit 40 to transmit / shield light. Therefore, this liquid crystal shutter 88
By R and 88L, light is independently incident on the acrylic substrates 22R and 22L from the incident-side end faces 71R and 71L. In this embodiment, the electronic control unit 40 energizes the liquid crystal shutters 88R and 88L so that when one of the liquid crystal shutters 88R and 88L is in the transmissive state, the other liquid crystal shutter is in the closed state. Controlled. Of course, the liquid crystal shutters 88R and 88L may be provided on the emission side end faces 25R and 25L of the respective substrates.

Further, unlike the biosensor 85, the biosensor 87 is provided with only a single light source unit 30, and the light from this light source unit is used as liquid crystal shutters 88R, 88L.
Then, it is incident on the acrylic substrates 22R and 22L. It should be noted that this single light source unit 30 is installed so as to collect and irradiate the p-polarized light on the light reflecting surface 29, similarly to the above-mentioned sensor.

In the concentration measuring device 45 using the biosensor 87 of the sixth embodiment described above, the individual processes of the substrate concentration measuring routine shown in FIG. 6 are executed as follows to measure the substrate concentration. . That is, first, the liquid crystal shutter 88 on the side of the acrylic substrate 22L which is the substrate measuring sensor unit.
L is controlled to be in a light transmitting state, and the liquid crystal shutter 88R on the side of the acrylic substrate 22R which is the correction sensor unit is left in a light shielding state. Specifically, the liquid crystal shutter 88R is energized only, and the liquid crystal shutter 88L is not energized. After that, step S10 of FIG.
The processing from 0 to step S150 is performed. That is, only for the acrylic substrate 22L that is the substrate measuring sensor portion, irradiation of light (p-polarized light) from the light source unit 30, transmission of light between the substrate main surfaces 23 of the acrylic substrate 22L, and total reflection at the light reflecting surface 29 are performed. And the reflected light is received by the CCD image pickup device 27. As a result, in the substrate measurement sensor unit,
The amount of light for each incident angle (θ1 to θ2) is measured, and the reflection angle θS1 at which the energy of the light therein is the minimum is obtained (see FIG. 7A).

After that, energization of the liquid crystal shutter 88L which has been in the transmission state until then is started to keep the shutter in the closed state, and at the same time, the liquid crystal shutter 88R on the side of the acrylic substrate 22R which is the correction sensor portion is applied. Stop energizing and leave it in the transparent state. That is, the shutter in the transparent state is changed. Next, in this state, only for the acrylic substrate 22R which is the correction sensor unit, the step S of FIG.
By performing the processing from 100 to step S150, the reflection angle θS0 that minimizes the light energy is obtained (FIG. 7).
(B)). After that, the process after step S160 in FIG. 6 is performed to calculate the substrate concentration.

As described above, it goes without saying that the biosensor 87 of the sixth embodiment can also obtain the same effect as that of the biosensor 85 of the fifth embodiment.

Although some embodiments of the present invention have been described above, the present invention is not limited to such embodiments and can be carried out in various modes without departing from the scope of the present invention. Of course.

For example, in the above-mentioned embodiment, the light source unit 30 has the p-polarizing plate 32 on its emission side, but the present invention is not limited to this. More specifically, the light source unit 30 may not have the p polarizing plate 32, and the p polarizing plate 32 may be attached to the light receiving surfaces of the CCD image pickup devices 27R and 27L. In addition, the translucent thin plate 72 itself in the biosensor 70 is replaced by the p polarizing plate 32.
The cost can be reduced by reducing the number of parts.

The above embodiments have been described with reference to the case where the surface of the Au thin film 28 (exposed film surface) is provided with a ligand layer on which a biological substance is fixed. The light reflecting surface is formed on the surface of the Au thin film 28. Evanescent region from 29 (about 100
It is also possible to form a monomolecular film of a carboxylic acid alcohol or an organic substance, or a thin film of an inorganic substance such as silicon nitride with a very thin film thickness (2 to 3 nm) that does not exceed 10 nm. Even if a monomolecular film or the like is provided in this way, the surface plasmon resonance phenomenon occurs, so that there is no effect on substrate measurement. When the monomolecular film or the like is provided in this manner, the substrate is not attached to the surface of the Au thin film 28, so that the measurement accuracy can be improved without being affected by the residual substrate at the time of the previous measurement. it can.

Further, instead of the acrylic substrates 22R and 22L, other than the glass substrate, various resin substrates having a light transmitting property can be used. Further, in each of the above examples, the substrate measurement sensor unit using the active biological substance and the correction sensor unit using the inactivated biological substance are used in combination, but the biosensor having only the substrate measurement sensor unit is used. It can also be a sensor.

That is, as shown in FIG. 27, a biosensor 90 having one acrylic substrate 22 can be used. This biosensor 90 also has the Au thin film 28, the light reflecting surface 29, the light source unit 30, the CCD image pickup device 27, and the like, and its main configuration is the same as that of the above-described several biosensors. However, as shown in the drawing, the biosensors described above are characterized in that one acrylic substrate 22 having a chromium thin film formed on the entire surface of the front and back substrate main surfaces 23 by well-known evaporation, sputtering, etc. is used. And the configuration is different. Even with the acrylic substrate 22 in this biosensor 90, as shown in FIG. 28, the substrate main surfaces 23 on the front and back sides are opposed to each other as a total reflection surface that reflects light under a geometrical total reflection condition, and they are installed on the peripheral end faces. The light incident from the light source unit 30 is confined between the front and back substrate main surfaces 23 and transmitted, and the light reflected by the light reflecting surface 29 is CC
The data is confined and transmitted between the front and back substrate main surfaces 23 up to the D image sensor 27.

According to the so-called 1-channel type biosensor 90 described above, the sensor can be constructed with one acrylic substrate 22, so that the cost can be further reduced by further reducing the number of parts. Further, since the light source can be directly attached to the end face of the plate-shaped acrylic substrate 22, the alignment (adjustment of the optical system) can be simplified. Note that the 1-channel type biosensor 90 is more susceptible to the drift due to temperature than the 2-channel type using the two acrylic substrates described above, but the influence on the measurement accuracy if the measurement is performed for a short time. There are few, and there is no problem in measuring the substrate concentration.

In addition, the two-channel type sensors (biosensor 20, biosensor 50, biosensor 85, etc.) using two acrylic substrates, one acrylic substrate 22 and the other acrylic substrate 22, The Au thin film 2
The biomaterial immobilized on the ligand layer formed in 8 may be different. With such a configuration, it is possible to simultaneously measure the concentrations of different substrates in the solution to be measured that are in contact with the ligand layer with high accuracy.

Further, after the filter processing by the low-pass filter 40b, the regression analysis is performed to determine the incident angles (θS1, θS
Although 0) is calculated, the incident angle (θS1, θS0) may be calculated based on the signal after being filtered by the low-pass filter 40b. Even in this case, the incident angle (θS1,
By calculating θS0), the measurement accuracy of the substrate concentration can be improved.

Here, a modified example of the biosensor of the above embodiment will be described with reference to the drawings. As shown in FIG. 29, the biosensor 85 of the fifth embodiment may be a biosensor 85A in which the emission side end faces 25R and 25L of the acrylic substrates 22R and 22L have lens curved surfaces of convex lenses. In addition, the biosensor 85
As shown in FIG. 30, the output side end faces 25R, 25
It is also possible to use the biosensor 85B in which L is an inclined surface inclined with respect to the substrate main surface 23.

Biosensors 85A, 8 of these modified examples
In 5B, the light (reflected light) at the exit side end faces 25R and 25L
Refraction of light changes the traveling path of light toward the CCD image pickup device 27. Therefore, according to these modifications, CC
The measurement sensitivity can be improved by increasing the amount of light received by the D image sensor 27. It goes without saying that such a modification can be performed on the biosensor 87 of the sixth embodiment.

Furthermore, the biosensor 85 of the fifth embodiment is
It can be modified as shown in FIGS. 31 and 32. The biosensor 85C of the modified example shown in FIG. 31 includes a cylinder lens 91 in front of the CCD image pickup device 27. In the biosensor 85C, light is condensed by the cylinder lens 91 in the light receiving area of the CCD image pickup device 27. Therefore, according to the biosensor 85C, the CCD image pickup device 2
The measurement sensitivity can be improved by increasing the amount of received light at 7. In addition, the biosensor 8 of the modification shown in FIG.
In 5D, a translucent block 92 is interposed between the acrylic substrates 22R and 22L and the CCD image pickup device 27 to integrate the acrylic substrate and the CCD image pickup device. Therefore,
According to this biosensor 85D, the acrylic substrate 22
Positioning of the CCD image pickup device 27 with respect to the R and 22L is easy, and the positional relationship can be maintained after the assembling, which improves usability. In this case, it is preferable that the block 92 is made of the same acrylic resin as the acrylic substrates 22R and 22L, because light is not emitted to the outside due to careless refraction of light.

Further, the biosensor 85D can be modified as shown in FIG. This biosensor 85E
Is an acrylic substrate 22R, 22L and a CCD image pickup device 27.
And a light-transmissive block 93 is provided between and, and the outer facing surfaces 93a and 93b of the block are tapered surfaces. Moreover, a chromium thin film is vapor-deposited over the entire outer facing surfaces 93a and 93b, and the biosensor 85E uses the outer facing surfaces 93a and 93b as reflecting surfaces in the block. Therefore, according to the biosensor 85E, in addition to simplifying the positioning of the CCD image pickup device 27, the traveling path of light is changed by the reflection on the outer facing surfaces 93a and 93b to increase the amount of light received by the CCD image pickup device 27. ,
This can improve the measurement sensitivity. In addition,
Needless to say, the modifications shown in FIGS. 31 to 33 can be applied to the biosensor 87 of the sixth embodiment.

Further, in the above embodiments, when the main surface of the substrate is the total reflection surface, it is not necessary to limit the formation of the chromium thin film or the like. In other words, when a light-transmitting medium formed of acrylic or the like has its main surface in contact with a medium having a significantly different refractive index (for example, air), even if a chromium thin film or the like is not formed on the surface. This is because it is possible to cause total reflection in the main surface of the substrate sufficiently geometrically. Specifically, in the above-described embodiments, in the case of a two-channel type biosensor 20, 21, 50 or the like using two acrylic substrates, a member is provided with a gap between the acrylic substrates or a member having a significantly different refractive index. Since it is not necessary to form a chromium thin film by interposing, the cost can be reduced by reducing the number of steps. Further, in the case of the 1-channel type biosensor 90, it is not necessary to form a chromium thin film unless the acrylic substrate is in contact with a medium whose refractive index is substantially the same, and the cost can be reduced by reducing the number of steps. You can

[Brief description of drawings]

FIG. 1 is a schematic configuration diagram showing a biosensor 20 of a first embodiment from the front, plane and right side.

FIG. 2 is a schematic exploded perspective view of a main part of a biosensor 20.

FIG. 3 is an enlarged cross-sectional view of a light source unit 30 used in the biosensor 20.

FIG. 4 is an end face 25R on the emission side of the biosensor 20,
The schematic diagram explaining the mode of light transmission in 25L.

FIG. 5 is a block diagram showing a schematic configuration of an electronic control unit 40.

FIG. 6 is a flowchart showing the processing of a substrate concentration measurement routine.

FIG. 7 shows C during the processing in the substrate concentration measurement routine.
The graph which shows the correlation of the incident angle obtained from CD image pick-up element 27R, 27L, and its light quantity.

FIG. 8 is an explanatory diagram for explaining how the incident angles (θS1, θS0) are calculated in step S150 of the substrate concentration measurement routine.

FIG. 9 is an explanatory diagram for explaining how to calculate incident angles (θS1, θS0).

FIG. 10 is a biosensor 2 according to a modification of the first embodiment.
1 is a schematic configuration diagram showing 1 from the front, the plane, and the right side.

FIG. 11 is a front view of the biosensor 50 of the second embodiment,
The schematic block diagram shown from a plane and a right side.

FIG. 12 is a front view of the biosensor 60 of the third embodiment,
The schematic block diagram shown from a plane and a right side.

FIG. 13 is a schematic exploded perspective view of a main part of the biosensor 60.

FIG. 14 is an emission-side inclined end surface 6 of the biosensor 60.
The schematic diagram explaining the mode of light transmission in 1R and 61L vicinity.

FIG. 15 is a schematic exploded perspective view of a main part of a biosensor according to a first modified example of the biosensor 60.

FIG. 16 is an emission-side inclined end surface 61R in this modification,
The schematic diagram explaining the mode of the light transmission in the 61L vicinity.

FIG. 17 is a schematic configuration diagram showing a biosensor 64 in a second modified example of the biosensor 60 from the front, the plane, and the right side.

18 is a sectional view taken along line 18-18 of FIG.

FIG. 19 is a schematic perspective view of a biosensor 70 according to a fourth embodiment.

20 is a schematic diagram showing how light is reflected by a light reflecting surface 29 of the biosensor 70. FIG.

FIG. 21 is an exploded perspective view of the biosensor 70.

FIG. 22 is a schematic configuration diagram showing a biosensor 20A of a modified example from the front, the plane, and the right side.

FIG. 23 is a schematic perspective view of a biosensor 80 of a modified example.

FIG. 24 is a schematic perspective view of a biosensor 85 according to a fifth embodiment.

FIG. 25 is a schematic plan view of this biosensor 85.

FIG. 26 is a schematic perspective view of the biosensor 87 of the sixth embodiment.

FIG. 27 is a schematic configuration diagram showing a biosensor 90 of a modified example from the front, the plane, and the right side.

FIG. 28 is a schematic diagram illustrating how light is transmitted in the biosensor 90.

FIG. 29 is a schematic plan view of a biosensor 85A which is a modification of the biosensor 85 of the fifth embodiment.

FIG. 30 is a schematic plan view of a biosensor 85B which is another modification.

FIG. 31 is a schematic plan view of a biosensor 85C which is another modification.

FIG. 32 is a schematic plan view of a biosensor 85D which is another modification.

FIG. 33 is a schematic plan view of a biosensor 85E which is another modification.

[Explanation of symbols]

 20 ... Biosensor 20A ... Biosensor 21 ... Biosensor 22 ... Acrylic substrate 22L ... Acrylic substrate 22L1-22L4, 22Li ... Acrylic substrate 22R ... Acrylic substrate 22R1-22R4, 22Ri ... Acrylic substrate 23 ... Substrate main surface 23A ... Light transmitting surface 24 ... Recesses for storing light source unit 25R, 25L ... Emission side end face 26R, 26L ... Upper end face 27 ... CCD image pickup device 27R, 27L ... CCD image pickup device 28 ... Au thin film 29 ... Light reflecting surface 30 ... Light source unit 30R, 30L ... Light source Unit 32 ... p polarizing plate 34 ... Light source 38 ... Lens 40 ... Electronic control device 40a ... Logical operation circuit 40b ... Low pass filter 45 ... Concentration measuring device 50 ... Biosensor 60, 70, 80, 85 ... Biosensor 85A, 85B 85C, 85D , 85E ... Biosensor 87, 9 Biosensor 61R, 61L ... Inclined end face 62 on exit side Cylinder lens 64 ... Biosensor 71R, 71L Incident end face 72 ... Translucent thin plate 73 ... Lumirror sheet 75R, 75L ... Bottom 81 ... Au thin film 82 ... Light source unit storage Recess notch 86 ... Partition plate 88R, 88L ... Liquid crystal shutter

Claims (11)

[Claims] 1. A light-reflecting surface provided with a metal thin film, comprising a light-transmitting light-transmitting medium that reflects light under geometric total reflection conditions, and the light-transmitting medium and the metal thin film couple evanescent waves. A biosensor for measuring a substrate to be measured in a solution to be measured, which is in contact with the metal thin film, by using an optical system for forming an optical system, wherein the optical system collects light from a light source on the light reflecting surface. An incident light side optical system that irradiates the light, and an output side optical system that receives the reflected light that is reflected by the light reflection surface and is emitted to the outside from the light transmission medium, and that detects the amount of the reflected light for each reflection angle. A polarizing means for p-polarizing light in the incident light side optical system or the exit side optical system and a total reflection surface for reflecting light under a geometric total reflection condition are opposed to each other, and a wave motion of light is generated between the total reflection surfaces. And a translucent substrate for confining light and transmitting light. The flexible substrate forms a light transmission path of the incident light side optical system up to the light transmission medium and a light transmission path of the emission side optical system from the light transmission medium. And biosensor. 2. The biosensor according to claim 1, wherein the light transmissive medium is a part of the translucent substrate,
The biosensor in which the light reflection surface is formed by providing the metal thin film on one end surface of the translucent substrate. 3. The biosensor according to claim 1 or 2, wherein the incident light side optical system is an integrated light source and a lens that condenses light emitted from the light source into a focal point. A light source unit and the translucent substrate, the lens is focused on the light reflecting surface, and the optical axis of the lens is tilted with respect to the light reflecting surface. The substrate is fixedly provided on the other end face side that intersects with the one end face of the substrate, and the emission side optical system reflects the reflected light that is reflected by the light reflection face and is emitted to the outside once for each reflection angle on the light reflection face. A biosensor including a light-receiving unit that receives light on the substrate and detects the amount of the reflected light for each reflection angle. 4. The biosensor according to claim 3, wherein the light source unit has a light-transmissive thin plate interposed on the other end surface that intersects the one end surface of the light-transmissive substrate at an angle. A biosensor that is directly fixed. 5. The biosensor according to claim 4, wherein the polarization means is a polarizing plate that p-polarizes transmitted light during the transmission, and the light source unit and the light-transmitting thin plate serve as the light-transmitting thin plate. A biosensor interposed between the transparent substrate and the other end face. 6. The biosensor according to claim 3, wherein the light source unit is incorporated in a recess formed on the other end surface side of the translucent substrate so as to be inclined with respect to the one end surface. A biosensor in which light is incident from the bottom of the recess into the transparent substrate. 7. The biosensor according to claim 1 or 2, wherein an end surface of the translucent substrate, which corresponds to an end of a light transmission path of the formed emission-side optical system, has the entire facing surface. The total reflection inclined end face that is inclined with respect to the reflection face and reflects light under a geometrical total reflection condition is used. The facing total reflection face in the range where the light reflected by the total reflection inclined end face reaches is A biosensor having a transmission surface that transmits light to the outside only at least in the range, and the emission-side optical system receives light that is transmitted through the transmission surface and emitted to the outside. 8. The biosensor according to claim 1, further comprising a first transparent substrate and a second transparent substrate, wherein the first and second transparent substrates are provided. A light-transmitting substrate, the light-transmitting substrate being disposed so that the light transmitted through the light transmission path of the emission-side optical system formed by the light-transmitting substrate is received by a single emission-side optical system; Regarding one of the first and second translucent substrates, when the transmitted light is received by the single emission side optical system, the other translucent substrate is A biosensor having a light-reception blocking unit that blocks light from being received by the single emission-side optical system. 9. The biosensor according to claim 8, wherein the light reception blocking unit guides light to a light transmission path of the incident light side optical system formed by the first light transmissive substrate. A first light source, a second light source for guiding light to a light transmission path of the incident light side optical system formed by the second light transmissive substrate, and the first and second light sources. When one of the light sources is in a lighting state, the other light source is turned off, and a lighting means for controlling lighting of both light sources is provided. 10. The biosensor according to claim 8, wherein the light-reception blocking means is provided on at least one of an inlet-side end surface and an outlet-side end surface of the light transmission path of the first translucent substrate. A first shutter that shields and transmits light to and from the light transmission path; and a second shutter provided on at least one of an end surface and an exit end surface of the light transmission path of the second transparent substrate. When one of the first and second shutters is in the transmissive state, the second shutter that shields and transmits the light to and from the light transmission path, and the other shutter is shielded so that both shutters are closed. A biosensor having a shutter control means for controlling. 11. A concentration measuring device for determining the concentration of a substrate to be measured in a solution to be measured, which is connected to the biosensor according to any one of claims 1 to 10 and the emission side optical system of the biosensor. Filter means for using, as an input signal, the reflected light amount for each of the reflection angles detected by the emission side optical system, and a signal obtained by subjecting the input signal to predetermined filtering to eliminate noise as an output signal; And a calculation unit that calculates the concentration of the substrate to be measured based on an output signal from the concentration measurement apparatus.