JP5241274B2 - Detection method of detected substance - Google Patents

Description

The present invention relates to a detection method for a substance to be detected, which detects a substance to be detected by using an enhancement field generated by making light incident on a detection surface at a predetermined incident angle.

As a method for detecting (or measuring) a substance to be detected 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 for 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, Patent Documents). 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. Only the immobilized fluorescently labeled antibody (that is, the 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 is also 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.
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.
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 method for detecting a substance to be detected, which can solve the problems based on the above prior art and can detect the substance to be detected in a sample with high accuracy and high reproducibility.

In order to solve the above problems, the present invention is a method for detecting a substance to be detected in a sample by using an enhancement field generated by making light incident on a detection surface at a predetermined incident angle, Is a predetermined wavelength that causes the prism to generate an enhancement field on the metal film regardless of the sample at an angle at which the metal film to be supplied is totally reflected at the boundary surface between the prism and the metal film disposed on one surface. The present invention provides a method for detecting a substance to be detected, in which light having a spectral distribution whose maximum spectrum intensity is 1.1 times or less of the minimum intensity is incident to detect light generated in the vicinity of the metal film .

Here, it is preferable that the light having a spectral distribution incident on the prism is generated by using a spectrum adjusting unit.
The predetermined wavelength region preferably has a wavelength width of 15 nm or more.
The light source is preferably a light source that emits white light.
The spectrum adjusting means is preferably a filter having a different transmittance for each wavelength.

  Further, light having a spectral distribution incident on the prism is emitted from a light source, and the light emitted from the light source is collected from a light collecting lens that collects the light emitted from the light source and the light emitted from the light source. Is preferably incident on the prism at an angle that causes total reflection at the boundary surface between the prism and the metal film.

  Here, the light source has a plurality of individual light sources that emit light having different wavelengths, and combines the light emitted from the individual light sources, and the maximum intensity of the spectrum in a predetermined wavelength region is 1 with the lowest intensity. It is preferable to emit light having a spectral distribution that is less than 1 time.

Further, based on the detection result of the optical discovery, it is preferable to have a calculating means for calculating the concentration of the substance to be detected in said sample.
Further, the substance to be detected is preferably a fluorescent substance or a substance labeled with a fluorescent substance.
Furthermore, it is preferable that the maximum intensity of the spectrum in a predetermined wavelength region of the light having the spectrum distribution is 1.03 times or less of the minimum intensity.

According to the present invention, light having a predetermined wavelength width is emitted from a light source, and light having a small difference between the maximum intensity and the minimum intensity at a predetermined wavelength width is made light that is incident on the prism by the spectrum adjustment unit. Even when the plasmon resonance angle at which the surface plasmon enhancement effect occurs in each case changes in each measurement, that is, when the wavelength at which the predetermined plasmon resonance angle changes every measurement, the enhanced electric field generated due to the surface plasmon The strength can be made uniform. Since the intensity of the enhanced electric field generated due to the surface plasmon can be made uniform as described above, the detection target substance in the sample can be detected with high reproducibility and high accuracy.
In addition, when the plasmon resonance angle at which the surface plasmon enhancement effect occurs at an arbitrary wavelength changes from measurement to measurement, that is, when the wavelength at which the predetermined plasmon resonance angle changes, the design can be performed with high reproducibility. The allowable range of error can be increased, and the apparatus cost can be reduced.

A method for detecting a substance to be detected according to the present invention will be described in detail based on an embodiment shown in the accompanying drawings.

FIG. 1 is a block diagram showing a schematic configuration of a sensing device 10 that is an embodiment of a sensing device that implements the method for detecting a substance to be detected of the present invention, and FIG. 2 (A) shows the sensing shown in FIG. It is a top view which shows schematic structure of the light source 12, the incident light optical system 14, and the sample unit 16 of the apparatus 10, 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 having a predetermined wavelength width, and light emitted from the light source 12 (hereinafter “excitation light”). The incident light optical system 14 that guides and collects the light, the spectrum adjustment means 15 that adjusts the spectrum of the light emitted from the light source 12, and the sample (that is, the measurement target) 82 that contains the substance 84 to be detected. , The sample unit 16 into which the light condensed by the incident light optical system 14 is incident, the light detection means 18 for detecting the light emitted from the measurement position of the sample unit 16, and the detection by the light detection means 18 Based on the result, the detection substance is detected (that is, the signal detected by the light detection means 18 is digitized to determine the presence / absence and concentration of the detection substance) and contained in the sample 82 Detected substance 4 detects (and measures).
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 light emitting device that emits light having a predetermined wavelength width. In the present embodiment, an LED (EBR3368S manufactured by Stanley) that emits light having an intensity distribution with a peak of 660 nm is used.

  The incident light optical system 14 includes a collimator lens 30, a cylindrical lens 32, and a polarization filter 34. The collimator lens 30, the cylindrical lens 32, and the polarization filter 34 are arranged in this order from the light source 12 side in the optical path of the excitation light. Has been. Therefore, the light emitted from the light source 12 passes through the collimator lens 30, the cylindrical lens 32, and the polarization filter 34 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.
As shown in FIGS. 2A and 2B, the cylindrical lens 32 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 collected light is condensed only on a plane perpendicular to the columnar axis (a plane parallel to the plane shown in FIG. 2B).
The polarizing filter 34 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 spectrum adjusting unit 15 is a filter having a different transmittance for each wavelength region, and is disposed on the optical path of the excitation light between the cylindrical lens 32 and the polarizing filter 34 of the incident light optical system 14. The spectrum adjusting unit 15 adjusts the spectral distribution of the light emitted from the light source 12.
Here, FIG. 3A is a graph showing the relationship between the transmittance of the spectrum adjusting unit 15 and the light emitted from the light source, and FIG. 3B is a graph of the light that has passed through the spectrum adjusting unit 15. It is a graph which shows spectrum distribution and spectrum distribution of the light inject | emitted from the light source. Here, in FIG. 3A, the horizontal axis represents wavelength [nm], and the vertical axis represents relative intensity and transmittance. In FIG. 3B, the horizontal axis represents wavelength [nm] and the vertical axis represents relative intensity.

As shown in FIG. 3A, the spectrum adjusting unit 15 is a filter having a transmittance distribution that is different in transmittance for each wavelength and inversely proportional to the spectrum distribution (intensity distribution) of light emitted from the light source. That is, the spectrum adjusting unit 15 has a low transmittance in a wavelength region where the intensity of light emitted from the light source 12 is high, and the transmittance increases as the wavelength region in which the intensity of light emitted from the light source 12 is low. Distribution.
The spectrum adjusting unit 15 has a transmittance distribution as described above, and converts light emitted from a light source, which is light having a spectral distribution having different intensities for each wavelength as indicated by a dotted line in FIG. By transmitting the light with different transmittances, the light in the constant wavelength region as shown by the solid line in FIG.

  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 the light emitted from 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.
Note that, as in the present embodiment, when an LED that emits light with an intensity distribution having a peak at 660 nm is used as the light source 12, the excitation wavelength is 660 nm, the fluorescence wavelength is 690 nm, and the particle diameter is Φ = Use fluorescent beads having 210 nm and COOH groups (Bangs Laboratories, FC02F / 7040) or fluorescent elements having a maximum excitation wavelength of 650 nm and a fluorescence wavelength of 668 nm (Molecular Probes, Alexa647). Is preferred.

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.

  Here, the light source 12, the incident light optical system 14, and the sample unit 16 totally reflect the light incident on the prism 38 from the incident light optical system 14 at the boundary surface between the prism 38 and the metal film 40, and the other of the prisms. They are arranged in a positional relationship for injection from the surface.

  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. The excitation light is emitted from the light source 12 and then passes through the incident light optical system 14 and the spectrum adjusting means 15. Specifically, the excitation light is converted into parallel light by the collimator lens 30, and then collected in only one direction by the cylindrical lens 32, and the intensity of a certain wavelength width is substantially uniformed by the spectrum adjusting unit 15, Polarized by the filter 34.
The light that has passed through the incident light optical system 14 and the spectrum adjusting unit 15 is incident on the prism 38 and reaches the boundary surface between the prism 38 and the metal film 40 as light having a predetermined angular width. Are totally reflected at the boundary surface of the light and emitted from the prism 38. The cylindrical lens 32 collects light so that a position beyond a certain distance beyond the boundary surface between the prism 38 and the metal film 40 becomes a focal point.
In addition, the parallel light generated by the collimator lens 30 is condensed in only one direction by the cylindrical lens 32, so that the direction parallel to the extending direction of the linear portion 46 at the boundary surface between the prism 38 and the metal film 40 is obtained. Can be made incident at the same angle.

Since the excitation light is totally reflected at the boundary surface between the prism 38 and the metal film 40, an evanescent wave oozes out on the surface on the flow channel 45 side of the metal film 40 (surface opposite to the prism 38 side), 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 of the sample, the thickness and density of the metal film, the wavelength of the excitation light, and the incident angle. In the present invention, the plasmon resonance angle and the incident angle of the excitation light 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, light having a predetermined wavelength width is emitted from the light source 12, and the spectrum adjusting unit 15 causes the intensity of each wavelength in the predetermined wavelength region to be substantially uniform (that is, a spectrum having a small difference between the maximum intensity and the minimum intensity). Distribution), even when the plasmon resonance angle is deviated, an enhanced electric field with a small intensity difference can be generated.

  Hereinafter, the relationship between the wavelength and the plasmon resonance angle will be described in more detail with reference to FIG. Here, FIG. 7 is a graph showing the relationship between the incident angle of single-wavelength excitation light and the reflectance at the boundary surface, where the horizontal axis is the incident angle [°] and the vertical axis is the reflectance. Here, the reflectance is the ratio of the intensity of light reflected at the boundary surface between the prism 38 and the metal film 40 to the intensity of light incident on the boundary surface between the prism 38 and the metal film 40. Further, the light that is not reflected at the interface between the prism 38 and the metal film 40 is converted into surface plasmons. Therefore, the lower the reflectance (the smaller the amount of light reflected at the boundary surface), the stronger the surface plasmon is generated, and the stronger electric field is formed.

  In FIG. 7, a metal film is formed of gold with a thickness of 50 nm, water is used as a sample, a prism with a refractive index n = 1.60 is used as a prism, and light with a wavelength λ = 656 nm is used as excitation light. The measurement example 1, the metal film, the sample, and the excitation light used are the same as those in the measurement example 1. The measurement example 2 uses a prism having a refractive index n = 1.65 as the prism. 1 shows a measurement result of Measurement Example 3 in which a prism having a refractive index n = 1.65 is used as the prism, and light having a wavelength λ = 701 nm is used as excitation light.

As shown in Measurement Example 1 and Measurement Example 2, when the sample unit (in the measurement example, the refractive index of the prism) is different, the angle with the highest conversion efficiency to the surface plasmon, that is, the resonance angle changes. Therefore, even when excitation light having the same intensity is incident at the same angle, the amount of surface plasmon generated varies depending on the sample unit. Specifically, when light is incident at an incident angle of 63 degrees, the reflectance in the measurement example 2 is increased by 10% in the measurement example 2 than in the measurement example 1, so that the intensity of the surface plasmon is decreased. .
Next, as shown in Measurement Example 1 and Measurement Example 3, even when the sample units are different, the same reflectance distribution can be obtained by adjusting the wavelength of the excitation light. That is, by changing the wavelength of the emitted light without changing the incident angle, the enhanced electric field intensity caused by the surface plasmon can be made the same.
In other words, since the plasmon resonance angle also changes depending on the wavelength, even if the plasmon resonance angle at an arbitrary wavelength is changed using different sample units, the same incident angle can be obtained by changing the wavelength of the excitation light. Only an amount can generate an enhanced electric field due to surface plasmon resonance.

  Here, the sensing device 10 makes light of a wavelength range of a certain width incident with substantially uniform intensity, so that even if the plasmon resonance angle at an arbitrary wavelength of the sample unit differs, The wavelength of the same reflectance distribution can be included. That is, the sensing device 10 can generate a surface plasmon enhancement effect because light of a predetermined wavelength width is incident even when the wavelength at which the incident angle of light becomes the plasmon resonance angle is different for each sample unit. . In addition, by making light of a predetermined wavelength width incident, the wavelengths before and after the wavelength that efficiently generates the surface plasmon enhancement effect can also be incident on each sample unit. Thus, the intensity of the enhanced electric field generated can be made substantially the same.

Thus, regardless of the sample unit (regardless of the resonance angle of the surface plasmon at a certain wavelength), the intensity of the enhanced electric field generated due to the surface plasmon generated on the metal film can be made substantially uniform, The intensity of the enhanced electric field that enhances the fluorescence of the fluorescent substance can be made constant. Therefore, even when measurement is performed with sample units having different plasmon resonance angles at arbitrary wavelengths (measurement under different conditions), 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, but according to the sensing device of the present invention, light of a certain wavelength width can be incident with no difference in intensity. Thus, even when the plasmon resonance angle is changed using different samples and substances to be detected, it can be detected by the same apparatus 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.
As described above, 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 due to the surface plasmon 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, by allowing highly reproducible measurement with sample units having different plasmon resonance angles at arbitrary wavelengths, the tolerance of the sample unit can be increased, so that the sample unit can be manufactured at low cost. It becomes possible.

  In addition, since it is possible to accurately detect (or measure) the amount and concentration of a substance to be detected with a simple configuration of providing a spectrum adjusting means (a filter in this embodiment), rather than using a rotating mechanism or the like. The apparatus can be made inexpensive.

  Here, in this embodiment, the spectrum adjusting means is arranged between the cylindrical lens and the polarizing filter. However, the arrangement position of the spectrum adjusting means is particularly on the optical path of the excitation light between the light source and the sample unit. It is not limited, The light source side may be sufficient as a collimator lens, The prism side rather than a polarizing filter may be sufficient between a collimator lens and a cylindrical lens.

  In addition, since the substance to be detected can be detected and measured more accurately, the spectrum adjusting means can adjust the spectrum distribution in the predetermined wavelength region to be substantially uniform as in the above embodiment. Most preferably, the intensity of the surface plasmon can be made substantially uniform by setting the spectrum distribution so that the maximum intensity of the spectrum is 1.1 times or less in the predetermined wavelength range. In addition, since the intensity of the enhanced electric field generated due to the surface plasmon can be made more uniform, the spectrum adjusting means has a spectrum in which the maximum intensity of the spectrum is 1.03 times or less in the predetermined wavelength range. A distribution is preferable.

In the above embodiment, an LED light source that emits light in a certain wavelength region is used as the light source. However, the present invention is not limited to this, and a light source that emits light in a certain wavelength region is more specific. The light source may be any light source that emits light in a wavelength range including a predetermined wavelength range that generates a plasmon enhancement effect on the metal film regardless of the sample, and various light sources can be used.
Here, the predetermined wavelength range that generates the plasmon enhancement effect on the metal film regardless of the sample is the combination of the assumed sample unit and sample, and the incident angle of the light of the sensing device is within the wavelength range. It is a wavelength region set so as to include a wavelength that becomes a plasmon resonance angle.
Here, the predetermined wavelength region preferably has a wavelength width of 15 nm or more. By setting the wavelength width within the above range, an electric field having a uniform intensity can be formed even when the plasmon resonance angle is shifted.
Moreover, as a light source, the light of the spectrum distribution as shown in FIG. 8, ie, the white light source (for example, white LED) which inject | emits white light, can be used suitably. In addition, the spectrum distribution of the white light source shown in FIG. 8 is a spectrum distribution in the case of 25 degreeC.
As shown in FIG. 8, since the white light source has a broad spectrum of light, white light is used as the light source and the intensity is uniformed by the spectrum adjusting means, so that more types of sample units and samples can be obtained. In addition, the tolerance can be made wider.

Here, in the sensing device 10, a light source that emits light in a certain wavelength range is used as the light source, and the spectrum intensity of the light emitted from the light source is adjusted by the spectrum adjusting unit, but the present invention is not limited to this. .
For example, a spectrum adjusting unit may be combined with a light source to emit light having a substantially uniform spectrum intensity in a predetermined wavelength range.
Specifically, a plurality of LEDs that emit light having different wavelengths are used as a light source, and the light emitted from each LED is combined so that the spectrum intensity in a predetermined wavelength region is substantially uniform. May be injected.
In addition, by adjusting the intensity of each wavelength of the light emitted from each LED with a high-pass filter, a low-pass filter, a band-pass filter, or the like, the intensity at each wavelength can be adjusted even when they are combined.

Here, since the fluorescence excitation efficiency of the fluorescent material changes depending on the wavelength, the spectrum adjusting means preferably adjusts the spectrum of the excitation light based on the excitation spectrum and the excitation light rate of the fluorescent material.
Further, in the surface plasmon resonance, in addition to the change of the resonance angle depending on the wavelength of the excitation light, the enhancement of the electric field also changes for each wavelength. In general, the enhancement increases as the excitation light has a longer wavelength. Therefore, the spectrum adjusting means preferably adjusts the spectrum of the excitation light based on the relationship between the wavelength of the excitation light and the maximum enhancement of the electric field generated by the surface plasmon resonance generated by the wavelength. For example, it is preferable that the intensity decreases as the wavelength of the excitation light increases. Thereby, the substance to be detected can be detected with higher accuracy.

As mentioned above, although the detection method of the to-be-detected substance based on this invention and the sensing apparatus which implements this were demonstrated in detail, this invention is not limited to the above embodiment, The range which does not deviate from the summary of this invention In the above, various improvements and changes may be made.

  For example, in each of the above-described embodiments, a collimator lens and a cylindrical lens serving as a condensing lens are provided as the incident light optical system. However, the present invention is not limited to this, and only the condensing lens may be provided, and the light emitted from the light source may be condensed by the condensing lens without being converted into parallel light.

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, a polarizing filter is not necessarily provided. When a light source that emits prepolarized excitation light, for example, a laser light source (more precisely, a laser light source that emits light in the optical wavelength range) is used as a light source, Since the light emitted from the light is polarized light, the 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.

In the above-described embodiments, the fluorescence of the fluorescent substance enhanced by the enhanced electric field generated due to the surface plasmon is detected in a state where the target substance is bound to the secondary antibody labeled with the fluorescent substance. However, although the detection target substance is detected, the method of labeling the detection target substance with a fluorescent substance is not particularly limited. For example, when the detection target substance itself 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) is a graph which shows the relationship between the transmittance | permeability of a spectrum adjustment means, and the light inject | emitted from a light source, (B) is the spectrum distribution of the light which passed the spectrum adjustment means, and was inject | emitted from the light source. It is a graph which shows spectrum distribution of light. 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. It is a graph which shows the relationship between the incident angle of the excitation light of a single wavelength, and the reflectance in a boundary surface. It is a graph which shows the spectrum distribution of another example of the light source which can be used for the sensing apparatus of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Sensing apparatus 12 Light source 14 Incident light optical system 15 Spectrum adjustment means 16 Sample unit 18 Light detection means 20 Calculation means 24 Function generator (FG)
26 Light source driver 30 Collimator lens 32 Cylindrical lens 34 Polarizing filter 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

Claims (10)

A method for detecting 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,
The angle at which the metal film to which the sample is supplied is totally reflected at the interface between the prism disposed on one surface and the metal film, and causing the prism to generate an enhancement field on the metal film regardless of the sample. A method for detecting a substance to be detected, wherein light having a spectral distribution in which the maximum intensity of the spectrum in the wavelength region is 1.1 times or less of the minimum intensity is incident to detect light generated in the vicinity of the metal film. The method for detecting a substance to be detected according to claim 1, wherein the predetermined wavelength region has a wavelength width of 15 nm or more . 3. The method for detecting a substance to be detected according to claim 1 , wherein the light having a spectral distribution incident on the prism is generated by using spectral adjustment means to emit light emitted from a light source . The method for detecting a substance to be detected according to claim 3, wherein the light source is a light source that emits white light. The method for detecting a substance to be detected according to claim 3 or 4 , wherein the spectrum adjusting means is a filter having different transmittance for each wavelength. Light having a spectral distribution incident on the prism is emitted from a light source,
The prism and the metal film using a condenser lens that condenses the light emitted from the light source, and a polarizing filter that polarizes the light emitted from the light source in a P-polarization direction. The method for detecting a substance to be detected according to any one of claims 3 to 5, wherein the light is incident on the prism at an angle at which the light is totally reflected at the boundary surface. The light source includes a plurality of individual light sources that emit light having different wavelengths, and combines the light emitted from the individual light sources so that the maximum intensity of the spectrum in a predetermined wavelength region is 1.1 times the minimum intensity. The method for detecting a substance to be detected according to any one of claims 3 to 6, wherein light having a spectral distribution as follows is emitted.   The detection method of the to-be-detected substance in any one of Claims 1-7 which has a calculation means to calculate the density | concentration of the to-be-detected substance in the said sample based on the detection result of the said light detection.   The method for detecting a substance to be detected according to claim 1, wherein the substance to be detected is a substance having fluorescence or a substance labeled with a substance having fluorescence.   The method for detecting a substance to be detected according to claim 1, wherein the maximum intensity of the spectrum in a predetermined wavelength region of the light having the spectrum distribution is 1.03 times or less of the minimum intensity.

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