JP2009085918A - Method for detecting scattered light - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve the S/N ratio by detecting scattered light at an appropriate acceptance angle in scattered light detection, using total reflection illumination. <P>SOLUTION: In a method for detecting scattered light using the total reflection illumination, the scattered light 20 having directivity is produced by approximately equalizing the size of the wavelength of a measurement light 9 to the size of the diameter of a scattering label microparticle 5 which labels a substance to be detected 2. The scattered light 20, having directivity, is detected from an appropriate direction. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a scattered light detection method using evanescent light.

  Conventionally, a detection method using total reflection illumination has attracted attention in bioassay for detecting proteins, DNA, and the like. This detection method consists of light that oozes out from the interface when the measurement light is totally reflected at the interface of different refractive index, that is, evanescent light, and a detected substance contained in the sample or a label attached to the detected substance. In this method, the presence or amount of the substance to be detected is detected by analyzing optical interactions such as scattering, absorption, and light emission.

  As an example of such a detection method, there is a fluorescence detection method using a fluorescent label.

  The fluorescence detection method has become an indispensable tool for bioresearch, coupled with the enhancement of the performance of photodetectors such as cooled CCDs. Also in materials used for fluorescent labels, fluorescent dyes with high fluorescence quantum yield, particularly in the visible region, such as FITC (fluorescence: 525 nm, fluorescence quantum yield: 0.6) and Cy5 (fluorescence: 680 nm, fluorescence quantum yield). A fluorescent dye exceeding 0.2 which is a practical standard such as a rate of 0.3) has been developed and widely used. Furthermore, high-sensitivity detection that cuts 1 pM (picomolar) has been realized by increasing the fluorescence signal by using the enhancement of the electric field by surface plasmons.

  However, fluorescent dyes have a weak π bond in terms of chemical structure due to the property of absorbing and emitting light, so they are altered by irreversible destruction by strong light and chemical reactions with oxygen and ozone in the atmosphere. There is a problem. This causes a so-called discoloration in which the total amount of fluorescence emitted from the entire fluorescent dye decreases over time, so that the intensity of measurement light cannot be increased beyond a certain level, and there is a limit to increasing sensitivity.

  Therefore, another example of the detection method using total reflection illumination is a scattered light detection method using a scattering label as shown in Patent Document 1 and Patent Document 2.

The scattered light detection method uses elastic scattering of evanescent light by a scattering label such as metal fine particles, and does not have the above-described problem of fading. Furthermore, since the amount of detected light obtained is larger than that of a general fluorescence detection method using a fluorescent dye, it is used as a method used when higher detection sensitivity is required.
Japanese National Patent Publication No. 10-506190 JP-T-8-500667

  However, in the scattered light detection method as described above, there is a problem of noise-like scattered light of evanescent light caused by impurities contained in the sample, unevenness of the sample supply surface, and the like. This is because the noise-like scattered light described above becomes noise with respect to the scattered light by the scattering label that is originally a detection target, so that the S / N ratio is lowered and the quantitativeness of the scattered light detection is lost. is there.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a scattered light detection method with improved S / N ratio and higher quantitativeness.

  In order to achieve the above object, the present inventor has paid attention to the fact that the scattered light has directivity when the wavelength of the measurement light and the diameter of the scattering marker fine particles are approximately the same. It came.

That is, in the scattered light detection method according to the present invention, the measurement light is incident on the detection surface composed of one surface of the dielectric prism substrate so as to satisfy the total reflection condition on the detection surface, and evanescent light is generated on the detection surface. A scattered light detection method for detecting the amount of a substance to be detected by detecting the amount of scattered light generated by scattering of evanescent light to scattering labeled fine particles contained in a sample supplied to a detection surface In
The wavelength λ of the measurement light and the diameter Φ of the scattering marker fine particles satisfy the following formula (1):
Among the scattered light, the scattered light having directivity is detected.
0.25 ≦ Φ / λ (1)

In the scattered light detection method according to the present invention, the scattered light having directivity is
Of the vector indicating the scattering direction of the scattered light and the vector indicating the incident direction of the measurement light with respect to the detection surface, the angle θ formed by the parallel component vector in the direction parallel to the detection surface satisfies the following formula (2): It is desirable that
0 ° ≦ θ ≦ 60 ° (2)

  Here, the “detection surface” is a surface of the dielectric prism substrate to which a sample or the like is supplied and brought into contact, and the measurement light is incident through the dielectric prism substrate so that the total reflection condition is satisfied on this surface. The evanescent light is generated on the surface of this surface.

  In addition, “scattered light having directivity” is scattered light having directivity in the direction indicated by the parallel component vector in the direction parallel to the detection surface of the vectors indicating the incident direction of the measurement light with respect to the detection surface. Details will be described later.

  Further, in the scattered light detection method according to the present invention, among the scattered light having directivity, the scattered light that has entered the dielectric prism substrate side with respect to the detection surface is separated from the reflected light of the measurement light on the detection surface. It is desirable to detect them.

  In the scattered light detection method according to the present invention, the scattering label fine particles are desirably metal fine particles.

  On the other hand, the scattered light detection device according to the present invention is a scattered light detection device used in the scattered light detection method according to claim 1, and has a light source that emits measurement light, a detection surface, and measurement light. Is disposed at a position that satisfies the total reflection condition on the detection surface, and a photodetector disposed at a position that detects scattered light having the directivity of evanescent light by the scattering marker fine particles. It is characterized by comprising.

  In the scattered light detection device according to the present invention, the dielectric prism substrate is configured to transmit scattered light having directivity among scattered light that has entered the dielectric prism substrate side with respect to the detection surface and measurement light on the detection surface. It is desirable to have a separation mechanism that separates the reflected light.

  According to the scattered light detection method of the present invention, in the scattered light detection method using total reflection illumination, the directivity of the scattered light generated when the wavelength of the measurement light and the diameter of the scattering marker fine particle are approximately the same. The scattered light having this directivity is detected as the main detection target. As a result, since scattered light can be detected efficiently, it is possible to improve the quantitativeness of detection.

  Hereinafter, although the best embodiment in the present invention is described using a drawing, the present invention is not limited to this.

Scattered light detection method
In the scattered light detection using total reflection illumination, the present inventor found that when the wavelength of the measurement light and the diameter of the scattering marker fine particles are approximately the same, the scattered light has a different intensity depending on the direction of scattering. I found the directivity.

  FIG. 1 shows the relationship between the light receiving angle θ of the scattered light 20 and the intensity (FIG. 1a) and S / N ratio (FIG. 1b) of the scattered light 20 of the evanescent light 22 by the Au fine particles 5 (diameter Φ: 150 nm, 350 nm). FIG. The S / N ratio is represented by the detection signal ratio of the scattered light 20 to the detection signal (BG signal) when the Au fine particles 5 are not present. As shown in FIG. 2a, the light receiving angle θ is equal to the parallel component vector 9iy in the direction parallel to the detection surface 6s out of the vector 9i indicating the incident direction of the measurement light 9 with respect to the detection surface 6s, and the photodetector 10. Is an angle formed by the normal vector 21. Here, the normal vector 21 includes the parallel component vector 9iy and is included in a plane perpendicular to the detection surface 6s, and has an outward direction (a direction in which no sample is present with respect to the photodetector 10). .

  As can be seen from FIG. 1, in the Au fine particles 5 having both diameters of 150 nm and 350 nm, the intensity of the scattered light 20 increases and the S / N ratio increases as the light receiving angle θ decreases. In particular, the S / N ratio in the case of detection at a light receiving angle θ = 10 ° is the Au fine particle 5 with Φ = 150 nm compared to the detection at the conventional light receiving angle (θ = 90 °, detection from the z direction in the figure). In the case of using the Au fine particles 5 with Φ = 350 nm, it is improved to about 10 times.

  Accordingly, it can be said that the scattered light 20 of the evanescent light 22 has directivity in the vector 9iy direction (that is, the y direction in FIG. 2A). Here, since the “vector 9 iy direction” represents the traveling direction of the evanescent light 22, it is defined as the “forward direction” in the present invention.

  Furthermore, if the symmetry in the scattering phenomenon is taken into consideration, it is considered that the directivity of the scattered light 20 can be expanded in the direction surrounded by the conical surface C as shown in FIG. Here, the conical surface C in FIG. 2b is a conical surface that opens in the forward direction with a vertex placed on the scattering fine particle 5 and a straight line passing through the vertex and parallel to the vector 9iy.

  Next, the results of examining the relationship between the wavelength of the measurement light and the type and diameter of the scattering label fine particles are shown below.

  3, 4, and 5 show the light reception angle θ and the S of the evanescent light scattered by the Au, Ag, and Cu fine particles (diameter Φ: 150 nm, 350 nm) when the measurement wavelength λ is 405 nm, 532 nm, and 640 nm, respectively. It is a figure which shows the relationship of / N ratio.

  From this, it can be seen that in the case of θ = ˜60 °, the improvement of the S / N ratio with respect to the conventional light receiving angle (θ = 90 °) hardly depends on the type of the scattering label fine particles. On the other hand, when attention is paid to the diameter Φ of the scattering-labeled fine particles, when the wavelength λ is 405 nm, the improvement in the S / N ratio as described above is observed at both the diameters Φ of 150 nm and 350 nm. It can be seen that the degree of improvement decreases with increasing the length of 532 nm and 640 nm. In particular, when the wavelength λ of the measurement light is 640 nm and the diameter Φ of the scattering labeling fine particles is 150 nm, the above improvement in the S / N ratio is not observed. That is, when the value of Φ / λ representing the ratio of the diameter Φ of the scattering label fine particles to the wavelength λ of the measurement light is 0.25 or more, the scattered light of the evanescent light exhibits sufficient directivity, but 0 When it is less than .25, sufficient directivity is not exhibited.

  On the other hand, there is no upper limit for the value of Φ / λ. For example, even when Φ / λ = 100, the scattered light of evanescent light has directivity in the forward direction.

  This does not depend on the type of the scattering label fine particles.

However, the diameter Φ of the scattering labeling fine particles is desirably 1 μm or less, and more desirably 500 nm or less. This is because if the diameter of the scattering labeled fine particles is too large, the volume increases and it becomes physically obstructive, causing secondary scattering (a phenomenon in which the scattered light to be detected is further scattered) and becoming heavy. This is due to the effect that the substance to be detected is easily precipitated and the detected substance cannot be labeled well. On the other hand, it is desirable to use visible light in a band of about 320 to 750 nm, which is generally used, from the viewpoint of ease of handling and cost. Considering the above, as a more desirable condition for obtaining the effect of the present invention, the upper limit of the value of Φ / λ can be about 3.1 (= 1000 nm / 320 nm). It is not limited to this.
3, 4, and 5 suggest that most of the scattered light having the directivity as described above is included in the range represented by the following formula (2). Therefore, in consideration of symmetry, in the present invention, “scattered light having directivity” passes through the conical surface C shown in FIG. 2b in which the range of the angle θ formed by the axis and the bus is expressed by the following equation (2). More desirably, the scattered light is emitted.
0.25 ≦ Φ / λ (1)
0 ° ≦ θ ≦ 60 ° (2)

<First Embodiment>
FIG. 6 is a schematic partial cross-sectional view of the scattered light detection device used when detecting the antigen 2 from the sample 1 containing the antigen 2 as the substance to be detected using the scattered light detection method according to the present embodiment.

  As shown in the figure, this scattered light detection device includes a light source 8 that emits measurement light 9, and a material that transmits the measurement light 9 that is disposed at a position that satisfies the total reflection condition on the detection surface 6s. The dielectric prism substrate 6, the primary antibody 3 fixed on the detection surface 6 s, the sample holder 7 that holds the sample 1 so that the sample 1 is in contact with the detection surface 6 s, and the sample with respect to the detection surface 6 s And a photodetector 10 arranged at a position where the scattered light 20 having directivity can be efficiently detected from a substantially forward direction on the side to which the light is supplied. In the figure, the antigen 2 contained in the sample 1, the scattering-labeled fine particles 5 having a diameter approximately the same as the wavelength of the measuring light 9, and the specific properties of the scattering-labeled fine particles 5 are imparted. A secondary antibody 4 is also shown.

  The measuring light 9 may be, for example, a single wavelength light obtained from a laser light source or the like, or a broad light obtained from a white light source or the like, and is not particularly limited. However, from the viewpoint of increasing the directivity of the scattered light, the wavelength λ is appropriately selected according to the detection conditions. In particular, the diameter Φ of the scattering-labeled fine particles 5 that label the substance 2 to be detected and the above formula (1) Satisfy the relationship. Furthermore, for the reason described above, it is desirable to use visible light in a band of about 320 to 750 nm that is generally used.

  The light source 8 may be, for example, a laser light source and is not particularly limited, but can be appropriately selected according to detection conditions. Further, if necessary, the light source 8 is provided with a mirror, a lens, or the like for guiding the measurement light 9 so that the measurement light 9 is incident through the dielectric prism substrate 6 so as to satisfy the total reflection condition on the detection surface 6s. It is desirable to combine light guide systems and the like as appropriate.

  The dielectric prism substrate 6 is made of a transparent material such as resin or glass. When a resin substrate is used, there is an advantage that the substrate is inexpensive, and when a glass substrate is used, scattering due to impurities and surface irregularities (this scattering becomes noise for the detection signal) is small. Since there is an advantage, it is desirable to select appropriately according to the application and detection conditions. When the dielectric prism substrate 6 is formed from a resin, polymethyl methacrylate (PMMA), polycarbonate (PC), amorphous polyolefin (APO) containing cycloolefin, or the like can be used.

  The primary antibody 3 and the secondary antibody 4 are antibodies against the antigen 2. The antibody to be used can be appropriately selected according to detection conditions (particularly, antigen 2). As a method for immobilizing the primary antibody 3, for example, in the case of this embodiment, the detection surface 6s can be subjected to a silane coupling treatment by a method comprising the following steps (1) to (3).

(1) Introduction of amino group by silane coupling agent (APS) 18 ml of EtOH, 1.98 ml of MilliQ water, 20 ul of 1N HCl are put into a test bottle with a screw cap, and after mixing, warm at 60 ° C. in an incubator. Then, add 24.3 mg of APS to the test bottle and stir well. Then, a cuvette for holding the liquid is provided on the prism, and 10 ml of the above APS mixed solution is dispensed therein. With the liquid in the cuvette, the prism is placed in an incubator at 60 ° C. and allowed to react for 12 minutes. Thereafter, stirring and washing in the cuvette is performed 5 times with EtOH / MilliQ water (volume ratio 9: 1). Then, all the liquid in the cuvette is taken out, placed in an incubator at 90 ° C., and heated for 180 minutes.

APS: 3-aminopropyltrimethoxysilane
(2) Modification with divinyl sulfone Add 10 ml of 4 wt% divinylsulfone solution and 30 ml of MilliQ water to a test tube with a screw cap, and mix well to prepare a 1% divinylsulfone aqueous solution. And the said aqueous solution is dispensed to the cuvette on a prism, and it makes it react for 60 minutes at room temperature. After that, stirring and washing in the cuvette is performed 5 times with MilliQ water.

(3) Immobilization of primary antibody 3 A diluted solution of primary antibody 3 is dispensed into a cuvette and allowed to air dry.

  The sample holder 7 is particularly limited as long as it can hold the sample 1 so as to be in contact with the detection surface 6s and does not interfere with detection of scattered light scattered from the label contained in the sample 1. is not. That is, in the present embodiment, since the scattered light is detected from the forward direction in which the directivity is shown, for example, as shown in FIG. 6, at least the forward direction portion where the scattered light is detected is a material that transmits the light. The one consisting of is desirable. The other parts are preferably made of a material that does not transmit light. This is from the viewpoint of blocking unintentional external light.

  The photodetector 10 quantitatively detects scattered light scattered by the label contained in the sample 1, and for example, LAS-1000 plus (trade name) manufactured by FUJIFILM Corporation can be suitably used. However, the present invention is not limited to this, and can be appropriately selected according to detection conditions, and a CCD, PD (photodiode), photomultiplier tube, c-MOS, or the like can be used. Further, in the present embodiment, the photodetector 10 is disposed at a position where the scattered light 20 having directivity is efficiently detected from a substantially forward direction on the side to which the sample is supplied with respect to the detection surface 6s. It is desirable. In particular, it is more desirable that the outward normal vector of the photodetector 10 is included in a plane including the parallel component vector 9iy in FIG. 2 and perpendicular to the detection surface 6s. Furthermore, it is desirable that the angle formed by the outward normal vector and the detection surface 6s is within the range expressed by the above equation (2).

  Then, as shown in FIG. 7, in the scattered light detection method according to the present embodiment, the primary antibody 3 is fixed to the detection surface 6s, which is one surface of the dielectric prism substrate 6, and the measurement light 9 is completely applied to the detection surface 6s. Incidence is made so as to satisfy the reflection condition, evanescent light 22 is generated on the surface of the detection surface 6 s, and a BG signal due to unevenness on the detection surface 6 s is detected by the photodetector 10, and then the sample 1 containing the antigen 2 (FIG. 7a), the antigen 2 is bound to the primary antibody 3 (FIG. 7b), and then the secondary antibody 4 that is labeled with the scattering-labeled microparticles 5 and specifically binds to the antigen 2 is flowed (FIG. 7c). ), Bound to the antigen 2 bound to the primary antibody 3, and fixed to the detection surface 6s by the so-called sandwich method (FIG. 7d). Thereafter, the evanescent light 22 is generated in the same manner to detect the detection surface. Fixed to 6s According to scattering labeled microparticles 5, it is characterized in that the scattered light 20 having a directivity of the evanescent light 22 is detected by the photodetector 10 (FIG. 7e).

  The kind of the scattering labeling fine particles 5 is appropriately selected depending on the detection conditions without any particular limitation, but is preferably a metal fine particle from the viewpoint of scattering properties and diameter control, and more preferably a group consisting of Au, Ag, Cu, and Pt. It is more desirable that the fine metal particles be composed of at least one or more kinds of metals selected. On the other hand, the diameter Φ satisfies the wavelength λ of the measuring light 9 and the above formula (1). Furthermore, for the reasons described above, it is desirable that the thickness be 1 μm or less, and more desirably 500 nm or less.

Hereinafter, the operation of this embodiment will be described with reference to FIG.
A primary antibody 3 that specifically binds to the antigen 2 is immobilized on the detection surface 6s. The BG signal is detected using the state at this time as the background. Thereafter, the sample 1 is supplied into the sample holder 7, and the antigen 2 in the sample 1 is bound to the primary antibody 3 and fixed. Then, the secondary antibody 4 that is labeled with the scattering labeling fine particles 5 and specifically binds to the antigen 2 is flowed, and the secondary antibody 4 binds to the antigen 2 to fix the scattering labeling fine particles 5 to the detection surface 6s. Is done. Thereafter, measurement light 9 emitted from the light source 8 is incident through the dielectric prism substrate 6 so as to satisfy the total reflection condition on the detection surface 6s. At this time, evanescent light 22 is generated on the surface of the detection surface 6s. Then, the evanescent light 22 is illuminated on the scattering marker fine particles 5 fixed to the detection surface 6s. By subtracting the BG signal from the detection signal at this time, the scattered light 20 of the evanescent light 22 scattered by the scattering labeling fine particles 5 can be analyzed, and the antigen 2 can be detected.

  Here, in the above example, it is the scattering labeled fine particles 5 that are actually confirmed by the scattered light detection, but basically, in the above process, the antigen 2 is bound to the scattering labeled fine particles 5. The presence of the antigen 2 is indirectly confirmed by confirming the presence of the scattering-labeled fine particles 5.

  In the scattered light detection method according to the present embodiment, since the wavelength λ of the measurement light 9 and the diameter Φ of the scattering label fine particles 5 satisfy the above formula (1), the evanescent light 22 generated by the scattering label fine particles 5 The scattered light 20 has directivity with respect to the forward direction. And by receiving the scattered light 20 having the directivity from the front direction, it is possible to detect the scattered light 20 more efficiently than when detecting from the conventional light receiving direction (upward in FIG. 6). , The S / N ratio can be improved.

  The evanescent light 22 as described above reaches only a region of about several hundred nm from the detection surface 6s. Due to this characteristic, the primary antibody 3 is used to aggregate the pair of the scattering-labeled fine particles 5 and the antigens 2 onto the detection surface 6s, so that the impurities 90 and the floating scattering-labeled fine particles that remain in the sample 1 unintentionally. Since the influence such as scattering from 5 ′ can be greatly reduced, the S / N ratio can be further improved.

  Furthermore, by using the primary antibody 3 to aggregate the pairs of the scattering labeled fine particles 5 and the antigens 2 on the detection surface 6s, the amount of the scattering labeled fine particles 5 illuminated by the evanescent light 22 can be increased. Thereby, a lot of scattered light 20 can be generated, and as a result, scattered light detection with a higher S / N ratio becomes possible.

  Further, since the scattered light 20 having directivity can be detected and the S / N ratio can be improved, an expensive dielectric prism substrate having no irregularities on the surface is not necessarily required, and thus the scattered light can be detected at a low cost. Can be performed.

  As described above, in the present embodiment, scattered light can be detected efficiently, so that the quantitativeness of detection can be improved and the cost can be reduced.

<Second Embodiment>
As shown in FIG. 8, the scattered light detection device used in the present embodiment has a directivity on the dielectric prism substrate 6 of the device used in the first embodiment shown in FIG. 6. A separation mechanism 23 is provided for separating the scattered light 20 ′ entering the dielectric prism substrate 6 with respect to the detection surface 6 s and the reflected light 9 r of the measurement light 9 on the detection surface 6 s. The photodetector 10 is arranged so as to detect the scattered light 20 ′ separated by the separation mechanism 23. Other configurations are the same as those in the case of the first embodiment, and description of elements equivalent to those in the first embodiment shown in FIG. 6 is omitted unless particularly necessary.

  The scattered light detection method according to the present embodiment is the same as that in the first embodiment until the evanescent light 22 is generated after the scattering labeled fine particles 5 are fixed to the detection surface 6s. Of the generated scattered light 20 having directivity of the evanescent light 22, the separation mechanism 23 separates the reflected light 9 r of the measurement light 9 on the detection surface 6 s from the detection surface 6 s toward the dielectric prism substrate 6. The scattered light 20 ′ that has entered is detected by the photodetector 10.

  Also in this embodiment, the same effect as that of the first embodiment can be obtained.

  Furthermore, in the present embodiment, the scattered light 20 ′ that has entered the dielectric prism substrate 6 side with respect to the detection surface 6 s is detected from the scattered light 20 having the directivity of the evanescent light 22. Thereby, detection is performed using a sample that does not easily transmit light such as blood, which is useful when it is difficult to detect the scattered light 20 from the sample 1 side with respect to the detection surface 6s.

  In all the above embodiments, the antigen-antibody reaction has been described. However, the present invention is not limited to this, and the subject of the present invention can be achieved by using a reaction utilizing other specific binding properties. Can be solved.

The figure which shows the relationship between the light reception angle of scattered light, the intensity | strength (FIG. 1a) of the scattered light of the evanescent light by Au fine particle (diameter (PHI): 150nm, 350nm), and S / N ratio (FIG. 1b). Conceptual diagram for defining scattered light having directivity in the present invention The figure which shows the relationship between light reception angle (theta) and S / N ratio of the scattered light of the evanescent light by Au, Ag, and Cu microparticles (diameter: 150 nm, 350 nm) in case a measurement wavelength is 405 nm. The figure which shows the relationship between receiving angle (theta) and S / N ratio of the scattered light of the evanescent light by Au, Ag, and Cu microparticles (diameter: 150 nm, 350 nm) in case a measurement wavelength is 532 nm. The figure which shows the relationship between light reception angle (theta) and the S / N ratio of the scattered light of the evanescent light by Au, Ag, and Cu microparticles (diameter: 150 nm, 350 nm) in case a measurement wavelength is 640 nm. The fragmentary sectional view which shows roughly the scattered light detection apparatus in 1st Embodiment The fragmentary sectional view which shows roughly the scattered light detection method in 1st Embodiment The fragmentary sectional view which shows roughly the scattered light detection apparatus in 2nd Embodiment

Explanation of symbols

1 Sample 2 Antigen 3 Specific binding substance (primary antibody)
4 Secondary antibody 5 Scattering labeled fine particle 5 'Floating scattering labeled fine particle 6 Dielectric prism substrate 6a Detection surface 7 Sample holding part 8 Light source 9 Measurement light 10 Photodetector 20 Scattered light 21 Normal vector 22 of the light detector Evanescent Light 23 Scattered light separation mechanism 90 Impurity in sample

Claims (6)

A measurement light is incident on a detection surface consisting of one surface of a dielectric prism substrate so as to satisfy the total reflection condition on the detection surface, evanescent light is generated on the detection surface, and the sample supplied to the detection surface In the scattered light detection method for detecting the abundance of the substance to be detected by detecting the amount of scattered light generated by scattering the evanescent light to the scattering label fine particles contained therein,
The wavelength λ of the measurement light and the diameter Φ of the scattering marker fine particles satisfy the following formula (1):
A scattered light detection method, comprising: detecting the scattered light having directivity among the scattered light.
0.25 ≦ Φ / λ (1) The scattered light having the directivity is
Of the vector indicating the scattering direction of the scattered light and the vector indicating the incident direction of the measurement light with respect to the detection surface, an angle θ formed by a parallel component vector in a direction parallel to the detection surface is expressed by the following equation (2): The scattered light detection method according to claim 1, wherein:
0 ° ≦ θ ≦ 60 ° (2)   Out of the scattered light having the directivity, the scattered light that has entered the dielectric prism substrate side with respect to the detection surface and the reflected light of the measurement light on the detection surface are separately detected. The scattered light detection method according to claim 1, wherein:   The scattered light detection method according to claim 1, wherein the scattering marker fine particles are metal fine particles. A scattered light detection device used in the scattered light detection method according to claim 1,
A light source that emits the measurement light;
The dielectric prism substrate having the detection surface and disposed at a position where the measurement light satisfies a total reflection condition on the detection surface;
A scattered light detection apparatus comprising: a photodetector arranged at a position to detect the scattered light having the directivity of the evanescent light by the scattering label fine particles.   Of the scattered light having the directivity, the dielectric prism substrate enters the dielectric prism substrate side with respect to the detection surface, and the reflected light of the measurement light on the detection surface The scattered light detection apparatus according to claim 5, further comprising a separation mechanism that separates the light from the light source.