JP2009204484A - Sensing device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sensing device capable of detecting a substance to be detected in a sample with high precision and high reproducibility. <P>SOLUTION: The sensing device has a prism, a metal film arranged on one surface of the prism, a substrate to which a flow channel for supplying the sample on the metal film is formed and which is arranged on one surface of the prism, a light source for emitting light, an incident light optical system for making the light emitted from the light source incident on the prism at an angle totally reflecting the light emitted from the light source by the boundary surface of the prism and the metal film, and a light detection means for detecting the light generated in the vicinity of the metal film. The incident light optical system is constituted so that a plurality of beams of light different in incident angle and substantially equal in intensity are ejected on the boundary surface at respective positions thereof. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a sensing device that detects a substance to be detected using an enhancement field that is generated when light is incident on a detection surface at a predetermined incident angle.

As a method for detecting (or measuring) a target substance with high sensitivity and ease in biomeasurement (measurement of biomolecular reaction), etc., a fluorescent substance that is excited by light of a specific wavelength and emits fluorescence (that is, a substance having fluorescence) ) To detect (or measure) a substance to be detected.
For example, when the substance to be detected is a fluorescent substance, this fluorescence method irradiates the sample to be examined, which is considered to contain the substance to be detected, with excitation light having a specific wavelength, and detects the fluorescence at that time to detect the substance to be detected. It is a method of confirming the existence of.
Further, in the fluorescence method, even when the substance to be detected is not a fluorescent substance, a specific binding substance that specifically binds to the substance to be detected is labeled with a fluorescent substance, and this specific binding substance is bound to the substance to be detected. Thereafter, in the same manner as described above, the presence of the substance to be detected can be confirmed by detecting fluorescence (specifically, the fluorescence of the fluorescent substance that labels the specific binding substance bound to the substance to be detected).

  In addition, as a method of detecting a substance to be detected with higher sensitivity using a fluorescence method, a method using an enhanced electric field by surface plasmon resonance of a metal film to excite the fluorescent substance has been proposed (for example, a patent Document 1, Patent Document 2, and Patent Document 3).

  In any of the methods described in Patent Document 1, Patent Document 2 and Patent Document 3, a metal thin film and a prism (half-column prism, Light is incident on the interface with the triangular glass prism at an angle that satisfies the plasmon resonance condition (plasmon resonance angle) to generate an enhanced electric field in the metal thin film, strongly exciting the substance in the vicinity of the metal thin film, and fluorescence This is a fluorescence detection method utilizing surface plasmon enhanced fluorescence (hereinafter also referred to as “SPF”).

  Here, as described in Patent Document 2, the electric field of the surface plasmon is strongly localized on the metal surface and decays exponentially according to the distance from the metal surface, so that it is adsorbed on the metal surface. Only the immobilized fluorescently labeled antibody (that is, fluorescent substance) can be selectively excited with high probability. Thereby, as described in Patent Document 2, by using the fluorescence detection method using SPF, it is possible to suppress the influence of interfering substances located at a position away from the interface to a minimum, and with high accuracy. It becomes possible to detect a substance to be detected.

  Patent Document 2 and Patent Document 3 provide a rotation mechanism that adjusts the angle of the prism on which the metal film is mounted, and the light emitted from the light source is optimized by adjusting the angle of the prism by this rotation mechanism. It is described that the light is incident on the prism at a plasmon resonance angle.

Moreover, as a method for detecting a substance to be detected using the surface plasmon enhancement effect, there is a method for detecting scattered light other than a method for detecting fluorescence excited by surface plasmon.
In Patent Document 4, a metal film on the surface of a prism, a functional thin film that traps a substance to be detected by an antigen / antibody reaction disposed on the surface of the metal film, and a flow cell that supplies a sample liquid in contact with the functional thin film A surface plasmon sensor is described.
This surface plasmon sensor detects scattered light generated when the electric field of the surface plasmon excited on the metal film using the surface plasmon enhancement effect is disturbed by the target substance present in the functional thin film. The substance to be detected is detected. Thus, the substance to be detected can be detected by a method that detects scattered light instead of fluorescence.

JP 2002-62255 A JP 2001-21565 A JP 2002-257731 A JP-A-10-78390

Here, the plasmon resonance condition of surface plasmon includes the wavelength of illumination light, the incident angle to the metal film, the refractive index and unevenness of the prism, the dielectric constant of the metal film, the thickness and the coarse density, and the sample placed on the metal film. Varies with type, condition, etc. However, in order to detect the maximum intensity with good reproducibility, as described in Patent Documents 2 and 3, a rotation mechanism is provided, the substrate and the prism are rotated, and the optimum angle is detected. There is a problem that the cost increases, and the amount of fluorescence of the fluorescent material on the metal film decreases as the optimum angle is detected.
Further, instead of providing a mechanism for adjusting the incident angle of light, it is conceivable that the temperature adjustment for ensuring (maintaining in a constant state) the physical constants and the positional relationship described above and making the shape of each member the same. However, this is not acceptable for blood diagnostic applications where equipment and chip manufacturing facilities are expensive and demands for cost reduction are particularly severe. In addition, there is a problem that the amount of fluorescence of the fluorescent material on the metal film decreases in the sense that the optimum angle is detected.
These problems were factors that hindered the practical application of sensing devices using plasmons.

  On the other hand, in the fluorescence detection method using SPF as described in Patent Documents 1 to 3 and the detection method described in Patent Document 4 for detecting scattered light generated by disturbing the surface plasmon, Patents 4 As described in Document 1, the light emitted from the light source is collected by a lens or the like to be converted into light having a predetermined angular width and incident on the metal film, so that light having a certain angular width is incident on the metal film. Make it incident. In this way, light of a certain angle width is incident, and the angle adjustment within the convergence angle is not required, thereby reducing the cost.

However, since the light emitted from the light source changes its intensity (that is, has an intensity distribution) depending on the position of the light beam (for example, the distance from the light beam center), the angle at which surface plasmon resonance occurs changes. Thus, there is a problem that the intensity of the electric field generated by the surface plasmon changes.
In addition, since the fluorescence by the fluorescent material changes depending on the intensity of the electric field generated by the surface plasmon even if it is the same fluorescent material, if the intensity of the electric field generated by the surface plasmon changes, the same and the same amount of fluorescent material Even if it exists, since the amount of fluorescence changes and the detection value changes, there is a problem that the detection accuracy is lowered and the reproducibility is lowered. This problem becomes a significant problem when density detection is performed.

In addition, if the substance that binds to the substance to be detected is provided unevenly on the metal film or if the thickness of the metal film is uneven, the substance to be detected adheres unevenly on the metal film. On the other hand, there is a method of irradiating light on a metal film-like region of constant width by setting the focal point of light that is collected by a condenser lens and incident on the metal surface to a position ahead of the metal film. is there.
However, even in this method, when the angle at which surface plasmon resonance occurs is changed as described above, the position of incident light changes at the angle at which surface plasmon resonance occurs. For this reason, there is a problem that the detection value changes due to the thickness of the metal film depending on the irradiation position and unevenness of the detection target substance, the reproducibility is lowered, and the detection accuracy is lowered.
Further, not only when detecting a substance to be detected using an electric field generated by surface plasmons, but also detecting a substance to be detected by using an enhancement field generated when light is incident on a detection surface at a predetermined incident angle. There are similar problems.

  An object of the present invention is to provide a sensing device using a surface plasmon enhancement effect capable of solving the problems based on the above-described conventional technology and detecting a substance to be detected in a sample with high accuracy and high reproducibility. .

  In order to solve the above-described problems, the present invention provides a sensing device that detects a substance to be detected in a sample using an enhancement field generated by making light incident on a detection surface at a predetermined incident angle, and includes a prism. A metal film disposed on one surface of the prism, a substrate disposed on one surface of the prism and provided with a flow path for supplying a sample on the metal film, a light source for emitting light, An incident light optical system for causing the light emitted from the light source to be incident on the prism at an angle at which the light is totally reflected by the boundary surface between the prism and the metal film; and a light detection means for detecting light generated in the vicinity of the metal film; The incident light optical system provides a sensing device characterized in that a plurality of light beams having different incident angles and substantially equal intensities are incident on each position of the boundary surface. .

Here, it is preferable that the incident optical system causes a plurality of light beams having different focal point positions and light beam center inclination angles to enter the boundary surface.
Further, it is preferable that the light source is a light emitting device that emits one light beam, and the incident optical system generates a plurality of light beams having different focal point positions from one light beam emitted from the light source. .
The light source preferably emits coherent light.
The incident optical system includes a selection unit that selects a light beam incident on the boundary surface from the plurality of generated light beams. The selection unit sequentially switches the light beam incident on the boundary surface, and the boundary surface It is preferable not to allow light beams having different focal points to enter simultaneously.
The light source preferably emits incoherent light.

Preferably, the light source is composed of a plurality of individual light sources that emit light of the same wavelength, and the incident light optical system uses light emitted from one individual light source as one light beam.
Further, it is preferable that the incident light optical system has a single condensing lens, and the individual light sources cause the emitted light to enter the condensing lens at different angles.

Here, it is preferable that the incident light optical system includes a scattering unit that scatters light emitted from the light source.
Moreover, it is preferable that the said scattering part is a scattering plate arrange | positioned on the optical path of the light inject | emitted from the said light source.
Moreover, it is preferable that the said scattering part is the sand printing surface formed in the surface of the said prism.

Further, it is preferable to have a calculation unit that calculates the concentration of the substance to be detected in the sample based on the detection result of the light detection unit.
Further, the substance to be detected is preferably a fluorescent substance or a substance labeled with a fluorescent substance.

According to the present invention, even when the plasmon resonance angle becomes different by causing a plurality of light beams having different incident angles to enter each position on the boundary surface, or when the amount of the fluorescent material changes depending on the position. However, it is possible to make the intensity of the enhanced electric field generated due to the generated surface plasmon uniform. Since the intensity of the surface plasmon can be made uniform in this way, the substance to be detected in the sample can be detected with high reproducibility and high accuracy.
In addition, since detection with high reproducibility can be performed even at different plasmon resonance angles, the allowable range of design error can be increased, and the apparatus cost can be reduced.

  A sensing device according to the present invention will be described in detail based on embodiments shown in the accompanying drawings.

  FIG. 1 is a block diagram showing a schematic configuration of a sensing device 10 which is an embodiment of the sensing device of the present invention. FIG. 2A shows a light source 12 and incident light optics of the sensing device 10 shown in FIG. It is a top view which shows schematic structure of the system | strain 14 and the sample unit 16, and FIG.2 (B) is a BB sectional drawing of FIG. 2 (A).

As shown in FIGS. 1, 2A, and 2B, the sensing device 10 basically includes a light source 12 that emits light of a predetermined wavelength and light emitted from the light source 12 (hereinafter referred to as “excitation light”). The incident light optical system 14 that guides and collects the light and the sample (that is, the measurement target) 82 that contains the substance 84 to be detected are held, and the light condensed by the incident light optical system 14 is incident thereon. The sample unit 16, the light detection means 18 for detecting light emitted from the measurement position of the sample unit 16, and the detection target substance are detected based on the detection result of the light detection means 18 (that is, detected by the light detection means 18). And calculating means 20 for determining the presence / absence and concentration of the detected substance by detecting the detected substance 84 contained in the sample 82 (and measuring).
The sensing device 10 further includes a function generator (hereinafter also referred to as “FG”) 24 that modulates excitation light, and a light source driver 26 that causes a current proportional to the voltage generated by the FG 24 to flow to the light source 12.
Here, the FG 24 is a signal generator that generates a repetitive clock of high and low voltages. The FG 24 sends a signal to the light source driver 26, and the light source driver 26 sends a current proportional to the voltage to the light source 12, so that the light source 12 emits light modulated according to the clock. The clock of the FG 24 is connected to the lock-in amplifier 64, and the lock-in amplifier 64 takes out only the signal synchronized with the clock sent from the FG 24 from the output of the light detection means 18.
Although not shown, each part of the sensing device 10 is supported by a support mechanism in order to fix the mutual positional relationship.

  The light source 12 is a semiconductor laser that emits light of a predetermined wavelength. Note that the light source is not limited to a semiconductor laser, and an LED, a lamp, an SLD, or the like can also be used.

  The incident light optical system 14 includes a collimator lens 30, a polarizing filter 32, a light splitting unit 34, and a condensing lens 36. In the optical path of excitation light, the collimator lens 30, the polarizing filter 32, and the light from the light source 12 side. The dividing unit 34 and the condenser lens 36 are arranged in this order. Therefore, the light emitted from the light source 12 passes through the collimator lens 30, the polarization filter 32, the light splitting unit 34, and the condenser lens 36 in this order, and then enters the sample unit 16.

The collimator lens 30 converts light emitted from the light source 12 and radially diffusing at a predetermined angle into parallel light.
The polarizing filter 32 is a filter that polarizes the transmitted light in the direction of P-polarized light with respect to the reflection surface of the sample unit 16 described later.

Next, the light splitting unit 34 will be described with reference to FIGS. Here, FIG. 3A is an explanatory diagram showing an optical path until light emitted from the light source enters the sample unit, and FIG. 3B until light emitted from the light source enters the sample unit. It is explanatory drawing which shows the breadth of each light beam in this optical path. 3A and 3B show the same surface as the surface shown in FIG.
As shown in FIGS. 3A and 3B, the light splitting unit 34 includes a first half mirror 102a, a second half mirror 102b, a third half mirror 102c, a first cylindrical lens 104a, and a second cylindrical lens 104b. The third cylindrical lens 104c, the first mirror 106a, the second mirror 106b, the third mirror 106c, and the third mirror 106d. The collimator lens 30 converts the light into parallel light and the light polarized by the polarizing filter 32. The light is branched into three light beams (a first light beam 108 a, a second light beam 108 b, and a third light beam 108 c) and is incident on the condenser lens 36.

Here, each of the first half mirror 102a, the second half mirror 102b, and the third half mirror 102c is a beam splitter that transmits half the light as it is, and reflects the half light.
The first cylindrical lens 104 a and the second cylindrical lens 104 b are columnar concave lenses whose axial direction is parallel to the longitudinal direction of the flow path 45 of the sample unit 16 to be described later. The diffused light is diffused at a predetermined angle only on a plane perpendicular to the columnar axis.
The third cylindrical lens 104c is a columnar convex lens whose axial direction is parallel to the longitudinal direction of the flow path 45 of the sample unit 16 to be described later. The third cylindrical lens 104c is a columnar light that is collimated by the collimator lens 30. The light is condensed at a predetermined angle only on a surface perpendicular to the axis of (a surface parallel to the surface shown in FIG. 2B).
The first mirror 106a, the second mirror 106b, the third mirror 106c, and the third mirror 106d are mirror surfaces, and reflect incident light.

Next, the arrangement relationship of each member and the optical path of each light beam will be described.
First, on the straight line connecting the polarizing filter 32 and the condenser lens 36, the first half mirror 102a, the first cylindrical lens 104a, the second half mirror 102b, and the third half mirror 102c are arranged in this order from the polarizing filter 32 side. Has been placed.
Here, the first half mirror 102a is disposed such that the reflection surface is inclined by 45 ° with respect to the optical axis of light emitted from the light source 12 (hereinafter simply referred to as “optical axis L”). That is, in the first half mirror 102a, the reflecting surface is arranged in a direction rotated by 45 ° in the clockwise direction in the drawing with respect to the surface perpendicular to the optical axis L. Further, the second half mirror 102b is arranged such that the reflecting surface is rotated at an angle obtained by subtracting a minute angle from 45 ° with respect to the surface perpendicular to the optical axis L, and rotated counterclockwise in the figure. The mirror 102c is arranged such that the reflecting surface is rotated by a minute angle in the counterclockwise direction in the drawing relative to the first half mirror 102a.

The light transmitted through the polarizing filter 32 is divided into transmitted light and reflected light by the first half mirror 102a, and the transmitted light becomes the first light beam 108a and the reflected light becomes the second light beam 108b.
The first light beam 108a is then diffused at a predetermined angle by the first cylindrical lens 104a (see FIG. 3B), passes through the second half mirror 102b, and further through the third half mirror 102c, and is collected by the condenser lens 34. Focused. In the first light beam 108a, half of the output is split as reflected light by the second half mirror 102b, and half of the output is split as reflected light by the third half mirror 102c. Since these divided lights are unnecessary light, they are emitted outside the apparatus or absorbed by a light absorber.

The first mirror 106a and the second mirror 106b have a first half mirror 102a, a second half mirror 102b, a first mirror 106a, and a second mirror 106b closer to the traveling direction of the second light beam 108b than the first half mirror 102a. It is arranged in the positional relationship that becomes the vertex of the rectangle. That is, the line connecting the first half mirror 102a and the second half mirror 102b is parallel to the line connecting the first mirror 106a and the second mirror 106b, and the first half mirror 102a and the first mirror 106a are connected to each other. The connected lines and the lines connecting the second half mirror 102b and the second mirror 106b are parallel to each other, and the angles formed by the lines connecting the respective parts are arranged at a right angle.
The first mirror 106a is disposed in a direction in which the reflecting surface is rotated by 45 ° in the clockwise direction in the drawing with respect to the surface perpendicular to the optical axis L, and the second mirror 106b has a reflecting surface whose optical axis is the optical axis. It is arranged in a direction rotated 45 ° counterclockwise in the figure with respect to a plane perpendicular to L.
A second cylindrical lens 104b is disposed between the second mirror 106b and the second half mirror 102b.

The second light beam 108b travels from the first half mirror 102a toward the first mirror 106a, is reflected by the first mirror 106a, is reflected by the second mirror 106b, and is diffused by the second cylindrical lens 104b (FIG. 3). (See (B)), the light is reflected by the second half mirror 102 b, passes through the third half mirror 102 c, and is collected by the condenser lens 34. Here, the reflection surface of the second half mirror 102b is arranged in a direction rotated by a minute angle clockwise from 45 ° with respect to the traveling direction of the second light beam 108b immediately before entering the second half mirror 102b. Therefore, when the second half mirror 102b is centered, the optical axis of the second light beam 108b is inclined by a predetermined angle clockwise from the optical axis of the first light beam 108 by a predetermined angle.
In the second light beam 108b, half of the output is split as transmitted light by the second half mirror 102b, and half of the output is split as reflected light by the third half mirror 102c.
The transmitted light divided by the second half mirror 102b becomes a third light beam 108c. Moreover, since the reflected light divided | segmented by the 3rd half mirror 102c is unnecessary light, it is inject | emitted out of an apparatus or it is made to absorb with a light absorber.

The third mirror 106c and the fourth mirror 106d are arranged so that the second half mirror 102b, the third half mirror 102c, the third mirror 106c, and the fourth mirror 106d are closer to the traveling direction of the third light beam 108c than the second half mirror 102b. It is arranged in the positional relationship that becomes the vertex of the rectangle. That is, the line connecting the second half mirror 102b and the third half mirror 102c and the line connecting the third mirror 106c and the fourth mirror 106d are parallel, and the second half mirror 102b and the third mirror 106c are connected to each other. The connected lines and the lines connecting the third half mirror 102c and the fourth mirror 106d are parallel to each other, and the angles formed by the lines connecting the respective parts are arranged at a right angle.
The third mirror 106c is arranged in a direction in which the reflecting surface is rotated by 45 ° counterclockwise in the drawing with respect to the surface perpendicular to the optical axis L, and the fourth mirror 106d has a reflecting surface that is light. It is arranged in a direction rotated by 45 ° in the clockwise direction in the drawing with respect to a plane perpendicular to the axis L.
A second cylindrical lens 104b is disposed between the second mirror 106b and the second half mirror 102b.

The third light beam 108c travels from the second half mirror 102b in the direction of the third mirror 106c, is reflected by the third mirror 106c, is reflected by the fourth mirror 106d, and is collected by the third cylindrical lens 104b (see FIG. 3 (B)), the light is reflected by the third half mirror 102c and condensed by the condenser lens 34. Here, the reflecting surface of the third half mirror 102c is arranged in a direction rotated by a minute angle counterclockwise from 45 ° with respect to the traveling direction of the third light beam 108c immediately before entering the third half mirror 102c. Therefore, when the third half mirror 102 c is the center, the optical axis of the third light beam 108 c is inclined by a predetermined angle counterclockwise by a predetermined angle from the optical axis of the first light beam 108.
The third light beam 108c is also split by the third half mirror 102c with half of the output as transmitted light.
Since the transmitted light divided by the third half mirror 102c is unnecessary light, it is emitted outside the apparatus or absorbed by a light absorber.

  All of the light components of the first light beam 108a, the second light beam 108b, and the third light beam 108c are emitted from the light source 12, and then once each to the first half mirror 102a, the second half mirror 102b, and the third half mirror 102c. In order to transmit, the intensity | strength of light will become 1/8 of the time of being inject | emitted from the light source 12, all. The third light beam 108c is diffused as a part of the second light beam 108b by the second cylindrical lens 104b, and then condensed by the third cylindrical lens 104c. Thus, by condensing with the third cylindrical lens 104c, the third light beam 108c has an optical path length longer than that of the second light beam 108b, but is incident on the condensing lens at the same diffusion angle as the second light beam 108b. Can do.

  The light splitting unit 34 is configured as described above, and the light transmitted through the polarization filter 32 is different from each other in the angle of the optical axis and the first light beam 108a, the second light beam 108b, and the third light beam having different focal points. The light beam 108 c is divided and incident on the condenser lens 36.

Returning to FIG. 1 and FIGS. 2A and 2B, the other parts will be described.
As shown in FIGS. 2A and 2B, the condenser lens 36 is a columnar lens whose axial direction is parallel to the longitudinal direction of the flow path of the sample unit, which will be described later. The two light beams 108b and the third light beam 108c are condensed only on a surface perpendicular to the columnar axis (a surface parallel to the surface shown in FIG. 2B).

  Next, the sample unit 16 includes a prism 38, a metal film 40, a substrate 42, and a transparent cover 44, and a sample 82 containing a substance 84 to be detected on the metal film 40 formed on one surface of the prism 38. Is placed.

The prism 38 is a prism having a substantially triangular prism shape whose cross section is an isosceles triangle (precisely, a hexagonal prism shape obtained by cutting each vertex portion of the isosceles triangle perpendicularly or parallel to the bottom surface of the isosceles triangle). Are arranged on the optical path of light (that is, the first light beam 108a, the second light beam 108b, and the third light beam 108c) emitted from the light beam and collected by the incident light optical system 14.
The prism 38 is arranged in such a direction that the light condensed by the incident light optical system 14 enters from a surface constituted by one of the two oblique sides of the isosceles triangle among the three side surfaces.
The prism 38 can be formed of a known transparent resin or optical glass. For example, ZEONEX (registered trademark) 330R (refractive index 1.50) manufactured by Nippon Zeon Co., Ltd. can be used as a material. In addition, the prism 38 is preferably formed of a resin rather than optical glass because the cost can be further reduced. For example, the amorphous polyolefin containing polymethyl methacrylate (PMMA), polycarbonate (PC), and cycloolefin is used. It is preferably formed of a resin such as (APO).
The prism 38 has such a configuration, and the light collected by the incident light optical system 14 is incident from a surface formed by one of the two oblique sides of the isosceles triangle, and the isosceles triangle The light is reflected from the surface formed by the base and emitted from the surface formed by the other of the two oblique sides of the isosceles triangle.

The metal film 40 is a metal thin film formed on a part of a surface formed by the base of the isosceles triangle of the prism 38 (specifically, a region including a region irradiated with light incident on the prism 38). is there.
Here, as a material used for the metal film 40, metals such as Au, Ag, Cu, Pt, Ni, and Al can be used. In addition, when using a liquid as a sample, in order to suppress reaction with a liquid, it is preferable to use Au and Pt.
Various methods can be used for forming the metal film 40. For example, the metal film 40 can be formed on the prism 38 by sputtering, vapor deposition, plating, pasting, or the like.
Here, FIG. 4 is an enlarged schematic view showing a part of the metal film 40 of the sample unit 16 shown in FIGS. 2A and 2B in an enlarged manner.
As shown in FIG. 4, a primary antibody 80, which is a specific binding substance that specifically binds to the detection target substance 84, is immobilized on the surface of the metal film 40.

The substrate 42 is a plate-like member arranged on the surface formed by the bases of the isosceles triangles of the prism 38, and the flow path 45 for supplying the sample 82 to the metal film 40 is formed.
The flow path 45 is formed at a linear line portion 46 formed across the metal film 40 and at one end of the line portion 46, and a starting end serving as a liquid reservoir to which a sample 82 is supplied during measurement. A portion 47 and a terminal portion 48 that is formed at the other end of the linear portion 46 and serves as a reservoir for the sample 82 that has been supplied to the starting end portion 47 and passed through the linear portion 46 to arrive.
Further, a secondary antibody placement region 49 on which a secondary antibody 88 labeled with a fluorescent material 86 is placed is provided on the linear portion 46 closer to the start end portion 47 than the metal film 40.
Here, the secondary antibody 88 is a specific binding substance that specifically binds to the substance 84 to be detected.

The transparent cover 44 is a transparent plate-like member bonded to the surface of the substrate 42 opposite to the surface in contact with the prism 38. The transparent cover 44 seals the flow path 45 formed in the substrate 42 by closing the surface of the substrate 42 opposite to the surface in contact with the prism 38.
The transparent cover 44 has openings formed in a portion corresponding to the start end portion 47 of the flow channel 45 and a portion corresponding to the end portion 48 of the flow channel 45. In addition, the transparent cover 44 may be provided with an openable / closable lid at an opening formed at a position corresponding to the start end portion 47 (and also the end portion 48).
The sample unit 16 is basically configured as described above. Here, the prism 38, the metal film 40, and the substrate 42 are preferably formed integrally.

  The light source 12, the incident light optical system 14, and the sample unit 16 cause the light incident on the prism 38 from the incident light optical system 14 to be totally reflected at the boundary surface between the prism 38 and the metal film 40, and thereby the other surface of the prism. It is arranged in a positional relationship for injection.

  The light detection means 18 includes a detection light optical system 50, a photodiode (hereinafter referred to as “PD”) 52, and a photodiode amplifier (hereinafter referred to as “PD amplifier”) 54, and the metal film of the sample unit 16. The light on 40 (that is, the light emitted from the sample 82 on the metal film 40) is detected.

  The detection light optical system 50 includes a first lens 56, a cut filter 58, a second lens 60, and a support portion 62 that supports them, and is on the metal film 40 (more precisely, in the vicinity of the metal film 40). ) Is collected and made incident on the PD 52. The detection light optical system 50 is arranged on the optical path of the light emitted from the metal film 40 in the order of the first lens 56, the cut filter 58, and the second lens 60 in order from the metal film 40 side. Has been.

The first lens 56 is a collimator lens, and is disposed so as to face the metal film 40. The first lens 56 emits light on the metal film 40 and changes the light reaching the first lens 56 into parallel light.
The cut filter 58 is a filter having a characteristic of selectively cutting light having the same wavelength as the excitation light and passing light having a wavelength different from that of the excitation light (for example, fluorescence caused by the fluorescent material 86). Of the light that has been converted into parallel light by the lens 56, only light having a wavelength different from that of the excitation light is allowed to pass.
The second lens 60 is a condensing lens, condenses the light transmitted through the cut filter 58, and makes it incident on the PD 52.
The support portion 62 is a holding member that integrally holds the first lens 56, the cut filter 58, and the second lens 60 while being spaced apart from each other by a predetermined distance.

The PD 52 is a photodetector that converts received light into an electrical signal, and condenses the incident light that is collected by the second lens 60 into an electrical signal. The PD 52 sends the converted electrical signal to the PD amplifier 54 as a detection signal.
The PD amplifier 54 is an amplifier that amplifies the detection signal, amplifies the detection signal sent from the PD 52, and sends it to the calculation means 20.

  The calculation means 20 includes a lock-in amplifier 64 and a PC (that is, a calculation unit) 66, and calculates the mass, concentration, etc. of the detection target from the detection signal.

  The lock-in amplifier 64 is an amplifier that amplifies a frequency component equal to the reference signal in the detection signal, and amplifies a signal component synchronized with the reference signal sent from the FG 24 among the detection signals amplified by the PD amplifier 54. . The detection signal amplified by the lock-in amplifier 64 is sent (output) to the PC 66.

The PC 66 converts the detection signal supplied from the lock-in amplifier 64 into a digital signal, and detects the concentration of the substance to be detected in the sample based on the converted signal. Here, the concentration of the substance to be detected in the sample can be calculated from the relationship between the number of substances to be detected and the amount of liquid. The number of substances to be detected can be calculated by calculating the relationship between the intensity of the detection signal and the number of substances to be detected using a known substance to be detected and creating a calibration curve. It should be noted that the concentration can be easily and accurately calculated by setting the amount of the sample liquid supplied to the flow path 45 of the substrate 42 of the sample unit 16 to be a constant amount (or by designing it to be a constant amount). it can.
The sensing device 10 is basically configured as described above.

  Hereinafter, the present invention will be described in more detail by describing the operation of the sensing device 10. 5A to 5C are explanatory views showing the flow of the sample 82 in the sample unit 16, respectively. FIG. 6 is an enlarged view showing a part of the metal film 40 reached by the sample 82. FIG. It is a schematic diagram.

First, as shown in FIG. 5A, a sample 82 containing a substance to be detected 84 is dropped onto the start end 47 of the flow path 45 of the substrate 42 of the sample unit 16.
The sample 82 dropped on the start end 47 moves in the tube formed by the linear portion 46 and the glass cover 44 toward the end portion 48 due to the capillary shape.

  The sample 82 moving along the linear portion 46 from the start end portion 47 toward the end portion 48 reaches the secondary antibody placement region 49 of the linear portion 46 as shown in FIG. When the sample 82 reaches the secondary antibody placement region 49, an antigen-antibody reaction occurs between the target substance 84 contained in the sample 82 and the secondary antibody 88 placed in the secondary antibody placement region 49. The detected substance 84 and the secondary antibody 88 bind to each other. Further, since the secondary antibody 88 is labeled with the fluorescent material 86, the detection target substance 84 bound to the secondary antibody 88 is in a state of being labeled with the fluorescent material 86.

  The sample 82 that has passed through the secondary antibody placement region 49 further moves to the end portion 48 side through the linear portion and reaches the metal film 40. When the sample 82 reaches the metal film 40, an antigen-antibody reaction occurs between the detection target substance 84 contained in the sample 82 and the primary antibody 80 immobilized on the metal film 40, as shown in FIG. The detected substance 84 is captured by the primary antibody 80. Here, since the detection target substance 84 captured by the primary antibody 80 is in a state of being labeled with the fluorescent substance 86 in the secondary antibody placement region 49, the primary antibody 80 that has captured the detection target substance 84 is the fluorescent substance. It becomes a state labeled at 86. That is, the substance to be detected 84 is sandwiched between the primary antibody 80 and the secondary antibody 88.

The sample 82 that has passed through the metal film 40 moves to the end portion 48. In addition, the detected substance 84 not captured by the primary antibody 80, the secondary antibody 88 not bound to the detected substance 84, and the fluorescent substance 86 also move to the terminal portion 48 together with the sample 82.
As a result, as shown in FIG. 5C, the detection target substance 84 bound to the secondary antibody 88 on the metal film 40, labeled with the fluorescent substance 86, and captured by the primary antibody 80 remains. Become.

As described above, when only the secondary antibody 88 labeled with the fluorescent material 86, the detected substance 84, and the immobilized primary antibody 80 remain on the metal film 40, the metal film 40 is irradiated with excitation light. To do.
Specifically, excitation light is emitted from the light source 12 based on the current flowing from the light source driver 26 based on the intensity modulation signal determined by the FG 24. After the excitation light is emitted from the light source 12, a part of the excitation light is blocked by the light shielding plate 36 by the incident light optical system 14, is converted into parallel light by the collimator lens 30, and is then polarized by the polarization filter 32. The light splitting unit 34 makes the first light beam 108 a, the second light beam 108 b, and the third light beam 108 b, and then condenses only in one direction by the condenser lens 36.
The first light beam 108a, the second light beam 108b, and the third light beam 108b collected by the condenser lens 36 are incident on the prism 38 and reach the boundary surface between the prism 38 and the metal film 40 as light having a predetermined angular width. Then, the light is totally reflected at the boundary surface between the prism 38 and the metal film 40 and is emitted from the prism 38. The condensing lens 36 condenses the condensing points of the first light beam 108a, the second light beam 108b, and the third light beam 108b so that they are positioned beyond the boundary surface between the prism 38 and the metal film 40 by a certain distance. .
Further, by collecting light in only one direction by the condensing lens 36, light having the same angle is incident in a direction parallel to the extending direction of the linear portion 46 at the boundary surface between the prism 38 and the metal film 40. be able to.

The excitation light (that is, the first light beam 108a, the second light beam 108b, and the third light beam 108b) incident on the prism 38 is totally reflected on the boundary surface between the prism 38 and the metal film 40, so that the flow path of the metal film 40 is obtained. An evanescent wave oozes out on the 45 side surface (the surface opposite to the prism 38 side), and surface plasmons are excited in the metal film 40 by the evanescent wave. This surface plasmon causes an electric field distribution on the surface of the metal film 40, and an electric field enhancement region is formed.
At this time, the excitation light incident at a predetermined angle width is generated by the incident excitation light having a predetermined angle (specifically, an angle satisfying the plasmon resonance condition) on the boundary surface between the prism 38 and the metal film 40. The evanescent wave and the surface plasmon resonate to generate surface plasmon resonance (plasmon enhancement effect). Thus, a stronger electric field enhancement is formed in a region where surface plasmon resonance (plasmon enhancement effect) occurs. Here, the plasmon resonance condition is a condition in which the wave number vector of the evanescent wave generated by the incident light is equal to the fraction of the surface plasmon, and the wave number matching is established. It is determined based on various conditions such as the state, the thickness and density of the metal film, the wavelength of excitation light, and the incident angle. In the present invention, the plasmon resonance angle and the incident angle of the excitation light (that is, each light beam) are angles formed with a line perpendicular to the metal surface.

At this time, if the fluorescent material 86 is present in the area where the evanescent wave is exuded, it is excited to generate fluorescence. In addition, this fluorescence is enhanced by the effect of electric field enhancement by surface plasmons existing in a region substantially equivalent to the region where the evanescent wave oozes out, particularly by the effect of electric field enhancement enhanced by surface plasmon resonance.
It should be noted that the fluorescent material outside the area where the evanescent wave exudes is not excited and thus does not generate fluorescence.
In this way, the fluorescence of the fluorescent substance 86 that labels the detection target substance 84 fixed on the metal film 40 is excited and enhanced.
The light emitted from the fluorescent material 86 enters the first lens 56 of the light detection means 18, passes through the cut filter 58, is collected by the second lens 60, enters the PD 52, and is converted into an electrical signal. In addition, since the light having the same wavelength as the excitation light among the light incident on the first lens 56 cannot pass through the cut filter 58, the excitation light component does not reach the PD 52.

The electric signal generated by the PD 52 is amplified by the PD amplifier 54 as a detection signal, and the signal component synchronized with the reference signal is amplified by the lock-in amplifier 64. As a result, the light generated due to the excitation light can be amplified, so that other noise components (for example, fluorescent light in the room, sensor light in the apparatus, etc., enter the PD 52 from other than the detection light optical system 50. And the light emitted from the fluorescent material 86 can be reliably identified.
The detection signal amplified by the lock-in amplifier 64 is sent to the PC 66.
The PC 66 A / D-converts the signal and detects the concentration of the detected substance 84 in the sample 82 from the calculation result of the detected substance 84 based on the calibration curve stored in advance.
The sensing device 10 detects the concentration of the detection target substance 84 in the sample 82 as described above.

According to the sensing device 10, the boundary between the prism 38 and the metal film 40 is made to be incident by entering three light beams having different focal points, that is, the first light beam 108 a, the second light beam 108 b, and the third light beam 108 c. Three light beams having different angles can be incident on each position of the surface.
As a result, the intensity of the enhanced electric field generated at each position (more precisely, the enhanced electric field generated due to the surface plasmon) can be prevented from changing due to the plasmon resonance angle, and the metal film can be used regardless of the plasmon resonance angle. The intensity of the enhanced electric field generated above can be made uniform.

More specifically, when light having a difference in intensity such as a normal distribution is used as excitation light, if the plasmon resonance angle is different, the intensity of the excitation light greatly changes depending on the angle.
Here, FIGS. 7A and 7B are schematic diagrams showing the relationship between the intensity of the conventional excitation light and the generation conditions of surface plasmon resonance, respectively.
FIG. 7A shows the intensity distribution 90 of the excitation light, the prism 38, and the metal when the excitation light is light having a normal distribution intensity distribution and the light near the center of the light flux of the optical excitation light coincides with the plasmon resonance angle θ1. An intensity distribution 92 of the light after being totally reflected at the boundary surface with the film 40 is shown. FIG. 7B shows that the excitation light is a light having a normal distribution intensity distribution and is separated from the center of the luminous flux of the excitation light. An intensity distribution 90 ′ of excitation light when the light at the position coincides with the plasmon resonance angle θ2 and an intensity distribution 92 ′ of light after being totally reflected at the boundary surface between the prism 38 and the metal film 40 are shown.
As shown in FIG. 7A, when surface plasmon resonance occurs with light incident at a plasmon resonance angle θ1, light having an intensity near the apex of the intensity distribution 90 (light having an intensity indicated by a circle in FIG. 7A). ) Becomes light converted to surface plasmon resonance. In addition, as shown in FIG. 7B, when surface plasmon resonance is generated by light incident at a plasmon resonance angle θ2, light having an intensity near the middle of the intensity distribution 90 ′ (in the middle circle in FIG. 7B). Light having the intensity shown) is converted into surface plasmon resonance. For this reason, the intensity of light that contributes to surface plasmon resonance has a significantly different value. Further, as shown in the intensity distribution 92 and the intensity distribution 92 ′, the intensity distribution of the reflected light is greatly reduced in the intensity distribution 92 than in the intensity distribution 92 ′. That is, the energy used for the surface plasmon resonance is larger in the plasmon resonance angle θ1 than in the case of the plasmon resonance angle θ2.
Thus, when the intensity of light converted to surface plasmon resonance is greatly different, the intensity of the enhanced electric field generated on the metal film is also greatly different. Specifically, the strength of the enhanced electric field is larger in the case shown in FIG. 7A than in the case shown in FIG.
Further, since the position where the surface plasmon resonance occurs (the position of the incident light at the plasmon resonance angle) is different, unevenness of the primary antibody 80 on the surface of the metal film, unevenness of adhesion of the detected substance 84 to the primary antibody 80, etc. If so, even if the intensity of the enhanced electric field generated on the metal film is the same, the amount of fluorescence changes depending on the position of the metal film.

Next, FIG. 8A is an explanatory diagram showing the relationship between the first light beam 108a and the metal film 40 (more precisely, the boundary surface between the prism 38 and the metal film 40), and FIG. FIG. 8 is an explanatory diagram showing a relationship between the second light beam 108b and the metal film 40, and FIG. 8C is an explanatory diagram showing a relationship between the third light beam 108c and the metal film 40. FIG. 9 is a graph showing the relationship between the incident angle of each light beam and the amount of light. In FIG. 9, the horizontal axis is the incident angle [°] to the boundary surface, and the vertical axis is the relative light quantity of each light beam.
As shown in FIG. 8A, the sensing device 10 causes the first light beam 108a to enter the metal film 40 so that the center of the light beam is at the position of the origin 0, and as shown in FIG. The second light beam 108b is incident on the metal film 40 so that the angle formed with the metal film 40 is larger than that of the first light beam 108a and the center of the light beam is at the position of the origin 0, and FIG. ), The third light beam 108c is incident on the metal film 40 so that the angle formed with the metal film 40 is smaller than that of the first light beam 108a and the center of the light beam is at the origin 0 position. Let
In this way, by making the first light beam 108a, the second light beam 108b, and the third light beam 108c incident from three directions having different angles, the intensity of light incident on the metal film 80 is set to a predetermined angle as shown in FIG. The range can be made substantially constant.
Thus, by making the light intensity uniform at the incident angle, the intensity of the enhanced electric field generated on the metal film can be made substantially constant regardless of the plasmon resonance angle.

Here, FIG. 10 (A) and FIG. 10 (B) are graphs showing the relationship between each light flux and the intensity of fluorescence generated in the sample unit, respectively. 10A and 10B show the case where the amount of the fluorescent material is the same.
For example, when the plasmon resonance angle is close to the incident angle at the center of the first light beam 108a at the time of measurement, the intensity of the enhanced electric field generated by entering the first light beam 108a is increased, and the second light beam 108b and the third light beam are transmitted. The intensity of the enhanced electric field generated by the incident becomes low. Therefore, as shown in FIG. 10A, the intensity of the fluorescence (of the fluorescent substance that labels the substance to be detected) caused by the first light flux 108a increases, and the fluorescence caused by the second light flux 108b and the third light flux 108c. The strength of is reduced.
On the other hand, when the plasmon resonance angle is lower, such as when measuring a sample unit having a lower refractive index of the sample than the measurement shown in FIG. 10 (A), the plasmon resonance angle is measured as shown in FIG. 10 (A). In this case, the incident angle at the center of the first light beam 108a is far from the incident angle at the center of the second light beam 108b. Therefore, the intensity of the fluorescence caused by the second light flux 108b is increased, and the intensity of the fluorescence caused by the first light flux 108a and the third light flux 108c is reduced.
As described above, the intensity of the fluorescence caused by one light beam changes depending on the plasmon resonance angle at the time of measurement, but if the intensity of the fluorescence caused by any one of the three light beams decreases, Since the intensity of the luminous flux increases, the intensity of the enhanced electric field can be made substantially constant regardless of the plasmon resonance angle, and when the amount of the fluorescent material is the same, the intensity of the fluorescence can be made the same, Highly reproducible and highly accurate measurement can be performed.

In addition, even when the plasmon resonance angle is shifted, the intensity of the enhanced electric field at each level is made uniform by causing three light beams having different incident angles to enter the boundary surface so that the centers of the light beams are at the same position. Can do. That is, even when the plasmon resonance angle changes, as in the case of the entire fluorescence amount shown in FIG. 10, the enhancement generated due to the light beam of one of the three light beams incident on each position. When the strength of the electric field (more precisely, the enhanced electric field generated due to the surface plasmon generated due to the light beam) decreases, the strength of the enhanced electric field generated due to the light beam of the other light flux increases. In addition, the intensity distribution of each light beam is the same, and the light beam is incident on the boundary surface so that the center of the light beam is at the same position. The intensity of the incident light beam is substantially constant. Thereby, the intensity of the enhanced electric field generated at each position can be made constant regardless of the plasmon resonance angle.
Thereby, since it can detect on fixed conditions, even when there is a detection nonuniformity by the nonuniformity of the primary antibody of a sample unit, etc., a highly reproducible and highly accurate measurement can be performed.

As described above, the intensity of the enhanced electric field generated on the metal film (more precisely, the enhanced electric field generated due to the surface plasmon) can be made substantially uniform regardless of the plasmon resonance angle. The intensity of the enhanced electric field that enhances the fluorescence of the substance can be made constant. Therefore, even when measurement is performed with sample units having different plasmon resonance angles, the intensity of the detection signal with respect to the amount and concentration of the substance to be detected is constant.
Thereby, measurement with high reproducibility can be performed, and the amount and concentration of the substance to be detected can be accurately detected (or measured).
Further, since it is not necessary to detect the plasmon resonance angle, it can be detected in a short time. Moreover, since the fluorescent material is not excited before the measurement for setting the conditions, it is possible to prevent the fluorescence intensity of the fluorescent material from being lowered.

The plasmon resonance angle also varies depending on the sample placed on the metal film and the substance to be detected. However, according to the sensing device of the present invention, the plasmon resonance angle is changed using a different sample and substance to be detected. In some cases (for example, several to ten degrees), it is possible to detect with the same device without adjusting the angle.
For example, a single sensing device can detect a substance to be detected in urine using urine as a sample, or can detect a substance to be detected in blood using blood as a sample.
More specifically, when the sample unit is manufactured with basically the same configuration (the prism is manufactured using PMMA (polymethyl methacrylate)), the whole blood and urine are sampled due to individual differences and physical condition differences. The unit refractive index varies from 1.335 to 1.36.
Here, for example, when the wavelength of the excitation light is 657 nm, when the refractive index is 1.335, the optimum plasmon resonance angle (incident angle at which the enhancement is highest) is 73.3 °, and the refractive index is 1. In the case of .36, the optimum plasmon resonance angle is 77.67 °, and the optimum plasmon resonance angle changes by about 4.3 °.
According to the present invention, as described above, the intensity of the enhanced electric field generated on the metal film can be made substantially uniform regardless of the plasmon resonance angle, so even when the plasmon resonance angle changes by several degrees in this way. Can be measured under the same conditions.
Thus, according to the present invention, the number of objects to be detected can be increased. Moreover, since the intensity of the enhanced electric field generated on the metal film can be made constant irrespective of the plasmon resonance angle, it is possible to prevent the detection accuracy from being varied depending on the detection substance.

  In addition, since it is possible to perform highly reproducible measurement with sample units having different plasmon resonance angles, the tolerance of the sample unit can be increased, so that the sample unit can be manufactured at low cost.

Here, when a laser light source (that is, a light source that emits coherent light) is used as the light source 12 as in the above-described embodiment, a light beam incident on the boundary surface of the sample unit is selected (that is, the light beam is switched). It is preferable to provide a light beam selection means.
Here, as the light beam selection means, a configuration in which a shutter is arranged on the optical path of each light beam of the light splitting unit is exemplified.

It is preferable to sequentially switch the light beam incident on the boundary surface to only the first light beam 108a, only the second light beam 108b, and only the third light beam 108c by the light beam selecting means. More specifically, when the first light beam 108a is incident on the boundary surface, the second light beam 108b and the third light beam 108c are shielded by the corresponding shutter, and the second light beam 108b is incident on the boundary surface. At times, the first light beam 108a and the third light beam 108c are shielded by the corresponding shutter, and when the third light beam 108c is incident on the boundary surface, the first light beam 108a and the second light beam 108b are shielded by the corresponding shutter. To do.
In this way, by sequentially switching the light beams incident on the boundary surface, the fluorescence intensity caused by the surface plasmon generated by each light beam can be detected for each light beam. In addition, it is possible to prevent light interference by preventing two light beams from being simultaneously incident on the same position.

Further, as described above, it is also preferable that the light source emits incoherent light (for example, an LED or a lamp).
By using a light source that emits incoherent light, a plurality of light beams can be simultaneously incident on the boundary surface without causing light interference. Thereby, it is not necessary to provide light selection means, and measurement can be performed stably in a short time.
In addition, when a light source that emits light having a certain degree of coherence, such as an SLD, is used as the light source, it is made incident for each light beam, or a plurality of light beams are incident simultaneously, depending on the application and required detection accuracy. You just have to choose.

Here, in the sensing device 10, a plurality of light beams are generated by dividing one light beam emitted from the light source by the incident light optical system, but the present invention is not limited to this.
Hereinafter, another embodiment of the sensing device of the present invention will be described with reference to FIG. Here, FIG. 11 is a block diagram showing a schematic configuration of a sensing device 200 as another embodiment of the sensing device of the present invention, and FIG. 12 shows the light source 202 and incident light of the sensing device 200 shown in FIG. 2 is a cross-sectional view of an optical system 204 and a sample unit 16. FIG.

  Here, since the sensing device 200 is the same as the sensing device 10 shown in FIG. 1 except for the construction of the light source 202 and the incident light optical system 204, the same components are denoted by the same reference numerals. Detailed description thereof will be omitted, and points unique to the sensing device 200 will be mainly described below.

  As shown in FIGS. 11 and 12, the sensing device 200 basically includes a light source 202 that emits light of a predetermined wavelength, an incident light optical system 204, a sample unit 16, a light detection means 18, and a calculation means. 20. In addition, the sensing device 100 further includes an FG 24 and a light source driver 26.

The light source 202 includes a first individual light source 210 a that emits a first light beam 220 a, a second individual light source 210 b that emits a second light beam 220 b, a third individual light source 210 c that emits a third light beam 220 c, and the light source driver 26. And a distribution unit 212 for flowing the flowing current to the first individual light source 210a, the second individual light source 210b, and the third individual light source 210c (hereinafter collectively referred to as “each individual light source”).
Each individual light source is a semiconductor laser that emits light of a predetermined wavelength. The first individual light source 210a, the second individual light source 210b, and the third individual light source 210c emit light having the same wavelength.
Here, the first light beam 220a is light parallel to the axis of the condenser lens 36 (a line connecting the focal point and the lens center), and the second light beam 220b is a predetermined angle (FIG. 12) with respect to the first light beam 220a. The third light beam 220c is inclined at a predetermined angle (a predetermined angle in the counterclockwise direction) with respect to the first light beam, and the third light beam 220c is opposite to the second light beam 220b with respect to the first light beam 220a. The light is inclined by a predetermined angle in the clockwise direction with respect to the first light flux.
In addition, the distribution unit 212 is connected to the light source driver 26 and each individual light source, and flows the current that is supplied from the light source driver 26 to each individual light source.

The incident light optical system 204 includes a first collimator lens 214a, a second collimator lens 214b, a third collimator lens 214c, a polarizing filter 32, and a condensing lens 36 disposed on the optical path of the excitation light. The first light beam 220a, the second light beam 220b, and the third light beam 220c emitted from the individual light sources are respectively polarized, condensed, and incident on a predetermined position of the sample unit 16 at a predetermined angle.
Here, in the incident light optical system 204, a first collimator lens 214a, a second collimator lens 214b, and a third collimator lens 214c are arranged corresponding to each individual light source. Two polarizing films 34 and one condenser lens 36 are arranged.

The first collimator lens 214a is disposed between the first individual light source 210a and the polarizing filter 32, and uses the first light beam 220a emitted from the first individual light source 210a as parallel light.
The second collimator lens 214b is disposed between the second individual light source 210b and the polarization filter 32, and makes the second light beam 220b emitted from the second individual light source 210b parallel light.
The third collimator lens 214c is disposed between the third individual light source 210c and the polarization filter 32, and uses the third light beam 220c emitted from the third individual light source 210c as parallel light.

  Here, the polarizing filter 32 and the condensing lens 36 have the same configuration as the polarizing filter 32 and the condensing lens 36 of the sensing device 10 shown in FIG.

The light source 202 and the incident light optical system 204 of the sensing device 200 are configured as described above, and the first light beam 220a emitted from the first light source 210a is converted into parallel light by the first collimator lens 214a, and then the polarization filter. After being polarized by 32 and condensed by the condenser lens 36, it is incident on the sample unit 16. Similarly, the second light beam 220b emitted from the second light source 210b is also converted into parallel light by the second collimator lens 214b, then polarized by the polarizing filter 32, condensed by the condenser lens 36, and then sampled. The third light beam 220c incident on the unit 16 and emitted from the third light source 210c is also converted into parallel light by the third collimator lens 214c, then polarized by the polarization filter 32, and condensed by the condenser lens 36. , Is incident on the sample unit 16.
The light source 202 and the incident light optical system 204 adjust the arrangement position of each individual light source, the mutual angle, the vertex of the condenser lens, etc., and the first light beam 220a, the second light beam 220b, and the third light beam at the boundary surface. The light is incident so that the center of 220c is at the same position.

  In addition, the first light beam 220a, the second light beam 220b, and the third light beam 220c are emitted at different angles, are converted into parallel light, and are incident on the condenser lens 36 at different angles. The positions are different from each other. Also, the incident angles to the boundary surface at the center of the light flux are different from each other.

  As in the sensing device 200, a plurality of individual light sources are provided as light sources, and the light emitted from each individual light source is a light beam having a different focal point and a different tilt angle at the center of the light beam. 10 can be obtained.

Note that when a plurality of individual light sources are used as in the sensing device 200, light interference does not basically occur regardless of the coherence of light emitted from the individual light sources. Even if the light fluxes made incident on the boundary surface at the same time, it is possible to make light with a constant intensity incident.
Further, even when a plurality of individual light sources are used as in the sensing device 200, the individual light sources that allow current to flow are selected by the distribution unit 212, the individual light sources that emit light are sequentially switched, and the light flux that is incident on the boundary surface is sequentially switched. You may do it.

  Here, in the sensing device 10 and the sensing device 200, a plurality of light beams having different focal point positions and light beam center tilt angles are incident on the boundary surface. A plurality of different light beams may be incident. For example, a scattering unit may be provided in the incident light optical system, and light emitted from the light source may be scattered by the scattering unit and then incident on the boundary surface.

FIG. 13 is a block diagram showing a schematic configuration of another embodiment of the sensing device of the present invention. Here, the sensing device 300 is basically the same as the sensing device 10 shown in FIG. 1 except for the configuration of the light source 302, the incident light optical system 304, and the sample unit 306. Are denoted by the same reference numerals, detailed description thereof will be omitted, and points unique to the sensing device 300 will be described below with emphasis.
A sensing device 300 shown in FIG. 13 basically includes a light source 302 that emits light of a predetermined wavelength, an incident light optical system 304, and a sample unit 306. Although not shown, the sensing device 300 includes a light detection unit, a calculation unit, an FG, and a light source driver, like the sensing device 10.

  The light source 302 is an LED that emits light of a predetermined wavelength. As the light source 302, the above-described various light sources such as a laser light source, an SLD, and a lamp can be used.

The incident light optical system 304 has a scattering plate 310 and makes the light emitted from the light source 302 enter the sample unit 306.
The scatter plate (also referred to as a diffuser plate) 310 is a scatter plate that makes light emitted from the light source 302 non-directional light and scatters uniformly within a predetermined angular width, and is emitted from the light source 302. Is scattered and ejected. Here, as the scattering plate 310, a scattering plate made of opal glass can be used.

The sample unit 306 includes a prism 312, a metal film 40, a substrate 42, and a transparent cover 44. Here, since the metal film 40, the substrate 42, and the transparent cover 44 have basically the same configuration as each part of the sample unit 16, detailed description thereof will be omitted. The substrate 42 of the sample unit 306 is different in that a part of the substrate 42 protrudes on the metal film 40, but the basic function is the same as that of the sample unit 16.
The prism 312 is a prism having a rectangular cross section, and is disposed on the optical path of light emitted from the light source 302 and passed through the incident light optical system 304.
The prism 312 is a rectangular parallelepiped prism, and one surface is in contact with the metal film 40 and the substrate 42. The prism 312 is arranged in such a direction that light that has passed through the incident light optical system 304 enters from a surface that is orthogonal to the surface in contact with the metal film 40 and the substrate 42. The prism 312 can be formed of the same material as the prism 38 described above.
The prism 312 has such a configuration, and the light transmitted through the incident light optical system 310 is incident from a surface orthogonal to the surface in contact with the metal film 40 and is reflected by the surface in contact with the metal film 40. The light transmitted through the incident light optical system 310 is emitted from the surface opposite to the surface on which the light is incident.

The sensing device 300 is configured as described above. After the light emitted from the light source 302 with a predetermined angle width is scattered by the scattering plate 310 of the incident light optical system 304, the sensing device 300 and the prism 312 of the sample unit 306 The light is incident on the boundary surface with the metal film 40.
As a result, light scattered at various angles by the scattering plate 310 (that is, a plurality of light beams having different incident angles on the boundary surface) can be incident on each value of the boundary surface.
Specifically, as shown in FIG. 13, the light emitted from the light source 302 is emitted and enters the scattering plate 310 as light having a predetermined width. The light having a predetermined width incident on the scattering plate 310 is uniformly scattered at any angle with a predetermined angle width at each position incident on the scattering plate 310, and the scattered light beams enter the boundary surface. At this time, since the light beams scattered at each position of the scattering plate 310 are incident on the boundary surface, a plurality of light beams scattered at different positions on the scattering plate 310 are incident on each position of the boundary surface. Since the light beams scattered at different positions on the scattering plate 310 have different optical paths, the incident angles on the boundary surfaces are different from each other. From the above, light rays scattered at various angles by the scattering plate 310 (that is, a plurality of light rays having different incident angles on the boundary surface) are incident on each value of the boundary surface.

As described above, the sensing device 300 also causes the plasmon resonance by causing light beams scattered at various angles by the scattering plate 310 (that is, a plurality of light beams having different incident angles to the boundary surface) to enter the respective values of the boundary surface. Regardless of the angle, an enhanced electric field having substantially the same intensity can be formed, and the same effect as the sensing device 10 can be obtained.
In addition, since the sensing device 300 only needs to provide a scattering plate in the incident light optical system, the device configuration can be simplified.

In the sensing device 300, the light emitted from the light source is scattered using a scattering plate in the incident light optical system, but the present invention is not limited to this.
Here, FIG. 14 is a block diagram showing a schematic configuration of another embodiment of the sensing device of the present invention.
Here, since the sensing device 350 shown in FIG. 14 is basically the same as the sensing device 300 shown in FIG. 13 except for the configuration of the incident light optical system 354 and the sample unit 356, the same configuration is used. Elements will be denoted by the same reference numerals, detailed description thereof will be omitted, and points specific to the sensing device 350 will be mainly described below.
A sensing device 350 shown in FIG. 14 basically includes a light source 302 that emits light of a predetermined wavelength, an incident light optical system 354, and a sample unit 356. Although not shown, the sensing device 350 includes a light detection unit, a calculation unit, an FG, and a light source driver, like the sensing device 300.
Here, the sample unit 356 has the same configuration as that of the sample unit 306 except that a sand printing surface 362 of the incident light optical system 354 described later is formed on the surface on which light emitted from the light source 302 of the prism 364 is incident. It is the same composition as.

The incident light optical system 354 includes a collimator lens 360 and a sand printing surface 362 formed on the prism 362 of the sample unit 356, and makes the light emitted from the light source 302 enter the sample unit 306.
The collimator lens 360 converts the light emitted from the light source 302 and radially diffusing at a predetermined angle into parallel light.
The sandprint surface 362 is a scattering portion that is emitted from the light source 302 and is converted into parallel light by the collimator lens 360 and has non-directional light, and is uniformly scattered within a predetermined angular width. The light source 302 of the prism 364 It is formed on the surface on which the light emitted from is incident. Here, the sand-printed surface 362 only needs to have a shape that scatters light, and may have various shapes such as forming fine irregularities. In addition, the method for forming the sand printing surface 362 on the prism 364 is not particularly limited. For example, a method for forming irregularities by mechanical processing such as a file or a method for forming irregularities by chemical processing such as etching. Can be used.

The sensing device 350 is configured as described above. Light emitted from the light source 302 with a predetermined angular width is emitted as parallel light by the collimator lens 360, and is incident on the prism 364 from the surface on which the sand printing surface 362 is formed. Let Accordingly, the parallel light is scattered by the sand printing surface 362 and converted into scattered light, and then enters the prism 364 and enters the boundary surface between the prism 312 and the metal film 40 of the sample unit 306.
As a result, light scattered at various angles by the sand-printed surface 362 (that is, a plurality of light rays having different incident angles on the boundary surface) can be incident on each value of the boundary surface.
As described above, even when the prism is provided with a sand-printed surface instead of the scattering plate, a plurality of light beams having different incident angles can be made incident on each value of the boundary surface, similarly to the sensing device, and the plasmon resonance angle. Regardless, it is possible to form an enhanced electric field having substantially the same intensity, and the same effect as that of the sensing device 10 can be obtained.

  Although the sensing device according to the present invention has been described in detail above, the present invention is not limited to the above embodiment, and various improvements and modifications may be made without departing from the gist of the present invention. Good.

  For example, in each of the sensing device 10 and the sensing device 200, three light beams are incident on the boundary surface of the sample unit. However, the number of incident light beams is not particularly limited, and two or four or more light beams are incident. But you can. By increasing the number of incident light, more accurate detection can be performed.

Further, the intensity of each light beam emitted from the light source and incident on the boundary surface of the sample unit (the distance from the center of the light beam) is made uniform (specifically, the intensity distribution of the light incident on the prism) It is also preferable to provide a light intensity distribution adjusting means so as to reduce the difference between the maximum intensity and the minimum intensity. Here, the light intensity distribution adjusting means is arranged on the optical path of the light, and blocks light having a certain intensity or less from the light emitted from the light source (that is, only the light having a uniform intensity at the center of the light source). Examples include a light-shielding plate that transmits light and an inverse Gaussian filter in which the transmittance is changed in accordance with the intensity.
By providing the light intensity distribution adjusting means as described above, the intensity of light incident on each sample value of each light beam can be made uniform, and the surface plasmon intensity at each value can be made more uniform. As a result, even when there is uneven distribution of the fluorescent material depending on the position of the sample unit, the fluorescence as a whole can be measured more accurately, so that detection with higher reproducibility can be performed.

In the sensing device 10, a cylindrical lens or a condensing lens is used for the incident optical system to collect the light emitted from the light source. However, the present invention is not limited to this, and the light emitted from the light source at a predetermined radiation angle is collected. You may make it inject into the interface of a prism and a metal film, without condensing.
In addition, it is not always necessary to provide a polarizing filter. In particular, when a laser light source is used as a light source, the light emitted from the light source is polarized light, and thus a polarizing film may not be provided.

  In the above-described embodiments, the number or concentration of the detection target substance contained in the sample is detected. However, the present invention is not limited to this, and whether or not the detection target substance is contained in the sample (that is, Whether or not there is a substance to be detected in the sample) may be detected.

Further, in the above-described embodiments, the fluorescence of the fluorescent substance enhanced by the enhanced electric field generated on the metal film is detected in a state where the substance to be detected is bound to the secondary antibody labeled with the fluorescent substance, Although the substance to be detected is detected, the method for labeling the substance to be detected with a fluorescent substance is not particularly limited. For example, when the substance to be detected is a fluorescent substance, it is not necessary to provide a secondary antibody.
In addition, the sensing device of the present invention is a method for detecting scattered light (Raman scattered light) generated when surface plasmon is generated in a state of being attached (or arranged in the vicinity) to a substance to be detected on a metal film. It can also be used for sensing devices.

Further, in the above-described embodiments, an enhanced electric field is formed by generating evanescent waves and surface plasmons on the surface of the metal film and further generating surface plasmon resonance, but the present invention is not limited to this. In other words, the present invention can be used in various systems in which the enhancement intensity changes depending on the incident angle of light on the surface where the enhanced electric field is formed (that is, the enhanced field changes only when light is incident at a predetermined incident angle). For example, it can be used for a method of forming an enhanced electric field by laminating a gold film and a SiO ₂ film having a thickness of about 1 μm on a prism and resonating light incident at a predetermined angle in the SiO ₂ film. .

It is a block diagram which shows schematic structure of one Embodiment of the sensing apparatus of this invention. (A) is a top view which shows schematic structure of the light source of the sensing apparatus shown in FIG. 1, an incident light optical system, and a sample unit, (B) is a BB sectional drawing of (A). (A) And (B) is explanatory drawing which shows the optical path until the light inject | emitted from a light source injects into a sample unit. It is an expansion schematic diagram which expands and shows a part of metal film of the sample unit shown to FIG. 2 (A) and (B). (A)-(C) is explanatory drawing which shows the flow of the sample in a sample unit, respectively. It is an expansion schematic diagram which expands and shows a part of metal film which the sample reached | attained. (A) And (B) is a schematic diagram which shows the relationship between the intensity | strength of the conventional excitation light, and the generation conditions of surface plasmon resonance, respectively. (A) is explanatory drawing which shows the relationship between a 1st light beam and a metal film, (B) is explanatory drawing which shows the relationship between a 2nd light beam and a metal film, (C) is a 3rd light beam. It is explanatory drawing which shows the relationship between a metal film. It is a graph which shows the relationship between the incident angle and light quantity of each light beam. (A) And (B) is a graph which shows the relationship between each light beam and the intensity | strength of the fluorescence which generate | occur | produces in a sample unit, respectively. It is a block diagram which shows schematic structure of other embodiment of the sensing apparatus of this invention. It is sectional drawing of the light source of the sensing apparatus shown in FIG. 11, an incident light optical system, and a sample unit. It is a block diagram which shows schematic structure of other embodiment of the sensing apparatus of this invention. It is a block diagram which shows schematic structure of other embodiment of the sensing apparatus of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Sensing apparatus 12 Light source 14 Incident light optical system 16 Sample unit 18 Light detection means 20 Calculation means 24 Function generator (FG)
26 Light source driver 30 Collimator lens 32 Polarizing filter 34 Light splitting portion 36 Condensing lens 38 Prism 40 Metal film 42 Substrate 44 Transparent cover 45 Flow path 46 Linear portion 47 Start end portion 48 End portion 49 Secondary antibody placement region 50 Detection light Optical system 52 Photodiode (PD)
54 Photodiode amplifier (PD amplifier)
56 First lens 58 Cut filter 60 Second lens 62 Support section 64 Lock-in amplifier 66 PC
80 Primary antibody 82 Sample 84 Detected substance 86 Fluorescent substance 88 Secondary antibody 102a, 102b, 102c (first, second, third) half mirror 104a, 104b, 104c (first, second, third) cylindrical lens 106a, 106b, 106c, 106d (first, second, third, fourth) mirrors 108a, 220a first light beam 108b, 220b second light beam 108c, 220c third light beam 210a, 210b, 210c (first, second 3) Individual light source 212 Distributor 214a, 214b, 214c (first, second, third) collimator lens

Claims (13)

A sensing device that detects a substance to be detected in a sample using an enhancement field generated by making light incident on a detection surface at a predetermined incident angle,
Prism,
A metal film disposed on one surface of the prism;
A substrate disposed on one surface of the prism and having a flow path for supplying a sample on the metal film;
A light source that emits light;
An incident light optical system for causing the light emitted from the light source to be incident on the prism at an angle at which the light is totally reflected at a boundary surface between the prism and the metal film;
Photodetection means for detecting light generated in the vicinity of the metal film,
The incident light optical system causes a plurality of light beams having different incident angles and substantially equal intensities to enter each position of the boundary surface.   The sensing apparatus according to claim 1, wherein the incident optical system causes a plurality of light beams having different focal point positions and light beam center inclination angles to be incident on the boundary surface. The light source is a light emitting device that emits one light beam,
The sensing apparatus according to claim 1, wherein the incident optical system generates a plurality of light beams having different focal point positions from one light beam emitted from the light source.   The sensing device according to claim 3, wherein the light source emits coherent light. The incident optical system has selection means for selecting a light beam to be incident on the boundary surface from a plurality of generated light beams,
5. The sensing device according to claim 4, wherein the selection unit sequentially switches light beams incident on the boundary surface, and does not simultaneously input light beams having different focal points to the boundary surface.   The sensing device according to claim 3, wherein the light source emits incoherent light. The light source is composed of a plurality of individual light sources that emit light of the same wavelength,
The sensing apparatus according to claim 3, wherein the incident light optical system uses light emitted from one individual light source as one light beam. The incident light optical system has one condenser lens,
The sensing device according to claim 7, wherein the individual light sources cause the emitted light to enter the condenser lens at different angles.   The sensing apparatus according to claim 1, wherein the incident light optical system includes a scattering unit that scatters light emitted from a light source.   The sensing device according to claim 9, wherein the scattering unit is a scattering plate arranged on an optical path of light emitted from the light source.   The sensing device according to claim 9, wherein the scattering portion is a sand printing surface formed on a surface of the prism.   The sensing device according to claim 1, further comprising a calculation unit that calculates a concentration of the substance to be detected in the sample based on a detection result of the light detection unit.   The sensing device according to claim 1, wherein the substance to be detected is a fluorescent substance or a substance labeled with a fluorescent substance.

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