Detection method and detection device

Abstract

This detection method involves: forming a complex by binding a target substance (11) and dielectric particles (12a) modified with a single domain antibody that binds to the target substance (11); separating the complex and unbound particles (12) that are dielectric particles (12a) not forming the complex by means of dielectrophoresis in a liquid; and detecting the target substance (11) included in the separated complex by using an imaging element (140).

Description

[Technical Field]

[0001] The present disclosure relates to a detection method and a detection device for detecting a target substance such as a virus, and to dielectric particles used in the detection method and the detection device. [Background technology]

[0002] Conventionally, optical detection methods have been provided that use near-fields to detect minute target substances with high sensitivity. For example, in Patent Document 1, a target substance is detected by measuring a reduction in an optical signal that occurs when a first magnetic field is applied to move a compound formed by binding a target substance with magnetic particles and fluorescent particles in a direction away from the surface of a detection plate on which the near-field is formed. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2017 / 187744 Summary of the Invention

[0004] However, in Patent Document 1, conjugates formed by nonspecific adsorption, in which magnetic particles and fluorescent particles are bound without the target substance, also move while emitting fluorescence, making it difficult to distinguish them from conjugates containing the target substance. As a result, false positives occur in which the target substance is mistakenly detected by conjugates that do not contain the target substance, reducing detection accuracy.

[0005] Therefore, the present disclosure provides a method for detecting a target substance that can reduce false positives due to non-specific adsorption and improve the accuracy of detecting the target substance.

[0006] A detection method according to one aspect of the present disclosure involves forming a complex by binding a target substance to a dielectric particle modified with a single-domain antibody that binds to the target substance, separating the complex from unbound particles, which are the dielectric particles that have not formed the complex, by dielectrophoresis in a liquid, and detecting the target substance contained in the separated complex using an imaging element.

[0007] A detection device according to one aspect of the present disclosure is a detection device that detects a target substance by dielectrophoresis, and includes: a separator that separates, in a liquid, dielectric particles modified with a single-domain antibody that binds to the target substance, a complex formed by the binding of the target substance and the dielectric particles, and unbound particles, which are the dielectric particles that have not formed the complex, by dielectrophoresis; and an imaging element used to detect the target substance contained in the separated complex.

[0008] A dielectric particle according to one embodiment of the present disclosure is a dielectric particle modified with a single domain antibody that binds to a target substance.

[0009] These comprehensive or specific aspects may be realized as a system, an integrated circuit, a computer program, or a computer-readable recording medium, or as any combination of a method, an apparatus, a system, an integrated circuit, a computer program, and a recording medium. Computer-readable recording media include non-volatile recording media such as CD-ROMs (Compact Disc-Read Only Memory).

[0010] A detection method according to one aspect of the present disclosure can reduce false positives due to non-specific adsorption and improve the detection accuracy of a target substance. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a perspective view showing a schematic configuration of a detection device according to an embodiment. [Figure 2] FIG. 2 is a cross-sectional view showing a schematic configuration of the detection device according to the embodiment. [Figure 3] FIG. 3 is a plan view showing the configuration of the electrode set according to the embodiment. [Figure 4] FIG. 4 is a flowchart showing a detection method according to an embodiment. [Figure 5] FIG. 5 is a diagram showing a process for forming a composite particle according to an embodiment. [Figure 6] FIG. 6 is a graph showing the set frequency of the AC voltage in the embodiment. [Figure 7A] FIG. 7A is a schematic diagram showing a particle moving into a first electric field region. [Figure 7B] FIG. 7B is a schematic diagram showing particles moving into the first electric field region and the second electric field region. [Figure 7C] FIG. 7C is a schematic diagram showing particles that have moved into the second electric field region. [Figure 8] FIG. 8 is a graph showing the crossover frequency for each type of particle according to the embodiment. [Figure 9A] FIG. 9A is a first graph showing the zeta potential of particles according to the embodiment and the comparative example. [Figure 9B] FIG. 9B is a second graph showing the zeta potential of particles according to the embodiment and the comparative example. [Figure 10] FIG. 10 is a first plan view showing the configuration of an electrode set according to a modified example. [Figure 11] FIG. 11 is a second plan view showing the configuration of an electrode set according to a modified example. [Figure 12] FIG. 12 is a diagram showing a process for forming a composite particle according to a modified example. DETAILED DESCRIPTION OF THE INVENTION

[0012] Hereinafter, the embodiments will be specifically described with reference to the drawings.

[0013] The embodiments described below are all comprehensive or specific examples. The numerical values, shapes, materials, components, component placement and connection configurations, steps, and step order shown in the following embodiments are merely examples and are not intended to limit the scope of the claims. The drawings are not necessarily strict illustrations. In the drawings, substantially identical components are designated by the same reference numerals, and duplicated descriptions may be omitted or simplified.

[0014] In the following, terms indicating the relationship between elements, such as parallel and perpendicular, terms indicating the shape of elements, such as rectangular, and numerical ranges do not only indicate the strict meaning, but also include a substantially equivalent range, for example, a difference of a few percent.

[0015] Hereinafter, detecting a target substance includes not only finding the target substance and confirming its presence, but also measuring the amount (for example, number or concentration) or extent of the target substance.

[0016] (Embodiment) In this embodiment, complex particles and unbound particles are separated in a liquid by dielectrophoresis (DEP), and the target substance contained in the separated complex particles is detected.

[0017] Dielectrophoresis is a phenomenon in which forces act on dielectric particles exposed to a non-uniform electric field. This force does not require the particles to be charged.

[0018] A target substance is a substance to be detected, such as a molecule such as a pathogenic protein, a virus (such as an outer coat protein), or a bacterium (such as a polysaccharide). A target substance may also be called an analyte or an object to be detected.

[0019] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of a detection device and a detection method for realizing detection of a target substance using dielectrophoresis will be specifically described with reference to the drawings.

[0020] [Detection device configuration] First, the configuration of the detection device will be described with reference to FIGS. 1 and 2. FIG. 1 is a perspective view showing a schematic configuration of a detection device according to an embodiment. FIG. 2 is a cross-sectional view showing a schematic configuration of a detection device according to an embodiment. In particular, FIG. 1 shows the outline of separator 110 so that the interior of separator 110 can be seen through the view excluding first substrate 111. FIG. 1 is used to explain the relationship between separator 110 and other components, and does not limit the position, orientation, or posture of each component when detection device 100 is used. FIG. 2 is a cross-sectional view of separator 110 shown in FIG. 1 taken along a direction parallel to the paper surface. Note that the thickness of some components of separator 110 shown in FIG. 2 is omitted from FIG. 1.

[0021] As shown in FIGS. 1 and 2, the detection device 100 includes a separator 110, a power source 120, a light source 130, an image sensor 140, and a detection unit 150.

[0022] The separator 110 is a container that contains the sample 10 containing the target substance 11, and has a space 1121 therein. The sample 10 is contained in the space 1121. The separator 110 separates the complex particles 13 and the unbound particles 12 in the liquid (i.e., in the liquid outside the sample 10) by dielectrophoresis within the space 1121. Here, the separator 110 separates the complex particles 13 and the unbound particles 12 based on their positions. The sample 10 produced by mixing the target substance 11 contains the complex particles 13 in which the target substance 11 and the dielectric particles 12a are bound, and the unbound particles 12. Impurities 14 may be mixed into the sample 10.

[0023] The composite particle 13 is a composite formed by binding a target substance 11 to a dielectric particle 12a (see FIG. 5 described later) modified with a substance that has the property of specifically binding to the target substance 11. That is, in the composite particle 13, the target substance 11 and the dielectric particle 12a are bound via the substance that has the property of specifically binding to the target substance 11.

[0024] The dielectric particles 12a are particles that can be polarized by an applied electric field. The dielectric particles 12a may contain, for example, a fluorescent material. When light having a wavelength that excites the fluorescent material is irradiated from a light source 130 described later, the dielectric particles 12a can be detected by detecting light in the fluorescent emission wavelength band. The dielectric particles 12a are not limited to particles containing a fluorescent material. For example, polystyrene particles, glass particles, etc. that do not contain a fluorescent material may be used as the dielectric particles 12a.

[0025] A substance that has the property of specifically binding to a target substance is a substance that can specifically bind to target substance 11, and is also called specific binding substance 12b (see FIG. 5 described below). As specific binding substance 12b for target substance 11, a single domain antibody, particularly a VHH antibody, for the target substance is used.

[0026] The unbound particles 12 include dielectric particles 12a that do not form complex particles 13. In other words, the unbound particles 12 are dielectric particles 12a modified with specific binding substances 12b, and are not bound to target substances 11. The unbound particles 12 are also called free (F) components. On the other hand, the complex particles 13 include dielectric particles 12a modified with specific binding substances 12b that are bound to target substances 11. The dielectric particles 12a and specific binding substances 12b bound to target substances 11 are also called bound (B) components.

[0027] Here, a description will be given of the internal configuration of separator 110. As shown in Fig. 2, separator 110 includes first substrate 111, spacer 112, and second substrate 113.

[0028] The first substrate 111 is, for example, a glass or resin sheet. The first substrate 111 has an upper surface that defines the bottom of the space 1121, and an electrode set 1111 to which an AC voltage is applied from a power source 120 is formed on the upper surface. The electrode set 1111 includes a first electrode 1112 and a second electrode 1113, and can generate a non-uniform electric field (also referred to as an electric field gradient) on the first substrate 111. In other words, the electrode set 1111 is an example of an electric field gradient generator that generates (or forms) an electric field gradient. Details of the electrode set 1111 will be described later using FIG. 3.

[0029] The spacer 112 is disposed on the first substrate 111. A through-hole corresponding to the shape of the space 1121 is formed in the spacer 112. In other words, the space 1121 is formed by the through-hole sandwiched between the first substrate 111 and the second substrate 113. As described above, the sample 10 containing the composite particles 13 and the unbound particles 12 is introduced into the space 1121. The spacer 112 is an outer wall surrounding the through-hole, and has an inner surface that defines the space 1121. The spacer 112 is made of, for example, a material such as a resin that has high adhesion to the first substrate 111 and the second substrate 113.

[0030] The second substrate 113 is a transparent sheet made of, for example, glass or resin, and is placed on the spacer 112. For example, a polycarbonate substrate can be used as the second substrate 113. The second substrate 113 has a supply hole 1131 and a discharge hole 1132 that are connected to the space 1121 and that penetrate the plate surface. The sample 10 is supplied to the space 1121 through the supply hole 1131 and discharged from the space 1121 through the discharge hole 1132. Note that the separator 110 may be configured without the second substrate 113. In other words, the second substrate 113 is not an essential component. For example, the space 1121 that enables the separator 110 to function as a container is formed by the first substrate 111 and the spacer 112, which respectively define the bottom and inner side surface.

[0031] The power supply 120 is an AC power supply and applies an AC voltage to the electrode set 1111 of the first substrate 111. The power supply 120 may be any power supply that can supply an AC voltage, and is not limited to a specific power supply. The AC voltage may be supplied from an external power supply, in which case the power supply 120 does not need to be included in the detection device 100.

[0032] The light source 130 irradiates the sample 10 in the space 1121 with irradiation light 131. The irradiation light 131 is irradiated into the sample 10 through the transparent second substrate 113. The sample 10 generates detection light 132 in response to the irradiation light 131, and the detection light 132 is detected to detect the dielectric particles 12a contained in the sample 10. For example, as described above, if the dielectric particles 12a contain a fluorescent substance, the fluorescent substance is excited by irradiating it with excitation light as the irradiation light 131, and the fluorescence emitted from the fluorescent substance is detected as detection light 132.

[0033] As the light source 130, any known technology can be used without any particular limitations. For example, a laser such as a semiconductor laser or a gas laser can be used as the light source 130. The wavelength of the irradiated light 131 emitted from the light source 130 is set to a wavelength that has little interaction with the substance contained in the target substance 11. For example, if the target substance 11 is a virus, the irradiated light 131 having a wavelength of 325 nm to 2000 nm is selected. The wavelength of the irradiated light 131 may be a wavelength that can be used by a semiconductor laser (for example, 600 nm to 850 nm).

[0034] The light source 130 does not have to be included in the detection device 100. For example, when the size of the dielectric particles 12a is large, observation can be performed by combining optical elements such as lenses, and a light emission phenomenon such as fluorescence does not have to be used. In other words, the dielectric particles 12a do not have to contain a fluorescent substance, and in this case, the light source 130 does not have to irradiate the irradiated light 131. The sun, a fluorescent lamp, or the like can be used as the light source 130, and the irradiated external light can be used to detect the dielectric particles 12a.

[0035] The imaging element 140 is a CMOS image sensor, a CCD image sensor, or the like, and generates and outputs an image by receiving detection light 132 generated from the sample 10. The imaging element 140 is, for example, built into a camera 141 or the like and arranged horizontally on the surface of the first substrate 111, and images a location corresponding to the electrode set 1111 via an optical element (not shown), such as a lens, included in the camera 141. In this way, the imaging element 140 is used to capture an image of the complex particles 13 separated from the unbound particles 12 by the separator 110, and to detect the target substance 11 contained in the complex particles 13.

[0036] In an example in which the dielectric particles 12a contain a fluorescent substance, the imaging element 140 captures an image of fluorescence emitted from the fluorescent substance contained in the dielectric particles 12a. The detection device 100 may include a photodetector instead of the imaging element 140. In this case, the photodetector may detect detection light 132, such as fluorescence, from a region on the first substrate 111 where the composite particles 13 separated by dielectrophoresis are collected. When a photodetector is used instead of the imaging element 140 in this way, analysis by the detection unit 150, which will be described later, is not necessary. Therefore, the detection device 100 can be realized without including the detection unit 150.

[0037] The detection device 100 may include an optical lens and / or an optical filter between the light source 130 and the separator 110 and / or between the separator 110 and the image sensor 140. For example, a long-pass filter that can block the irradiation light 131 from the light source 130 and pass the detection light 132 may be installed between the separator 110 and the image sensor 140.

[0038] The detection unit 150 acquires the image output by the image sensor 140, and detects the dielectric particles 12a contained in the sample 10 based on the image. In particular, the detection device 100 in this embodiment can count the composite particles 13 and the unbound particles 12 individually. That is, it is possible to distinguish and detect the dielectric particles 12a that form the composite particles 13 from the dielectric particles 12a contained in the unbound particles 12. Therefore, by detecting the dielectric particles 12a based on the image, the detection unit 150 detects the composite particles 13 in the sample 10.

[0039] For example, the detection unit 150 uses a control image captured in advance that does not contain dielectric particles 12a and compares the acquired image with the control image to detect bright spots with different brightness values. Specifically, when detecting luminescence as the detection light 132, points with high brightness values ​​in the acquired image relative to the control image are detected as bright spots, and when detecting transmitted light, scattered light, etc. as the detection light 132, points with low brightness values ​​in the acquired image relative to the control image are detected as bright spots. In this way, the detection unit 150 obtains the detection result of the composite particles 13 in the sample 10.

[0040] The detection unit 150 is realized by, for example, executing a program for the image analysis using a circuit such as a processor and a storage device such as a memory, but may also be realized by a dedicated circuit. The detection unit 150 is, for example, built into a computer.

[0041] [Shape and arrangement of electrode set on first substrate] Next, the shape and arrangement of the electrode set 1111 on the first substrate 111 will be described with reference to Fig. 3. Fig. 3 is a plan view showing the configuration of the electrode set according to the embodiment. Fig. 3 shows the configuration of the electrode set 1111 when viewed from the imaging element 140 side. For simplification, Fig. 3 shows a schematic diagram showing a portion of the electrode set 1111.

[0042] As described above, the electrode set 1111 includes a first electrode 1112 and a second electrode 1113 disposed on the first substrate 111. Each of the first electrode 1112 and the second electrode 1113 is electrically connected to the power source 120.

[0043] The first electrode 1112 includes a first base 1112a extending in a first direction (the left-right direction on the paper in FIG. 3 ) and two first protrusions 1112b protruding from the first base 1112a in a second direction (the up-down direction on the paper in FIG. 3 ) intersecting the first direction. A first recess 1112c is formed between the two first protrusions 1112b. The two first protrusions 1112b are disposed opposite the second electrode 1113. In other words, the first electrode 1112 has first protrusions 1112b protruding from the first base 1112a in a direction that intersects the first direction and is convex toward the second electrode 1113. The first protrusions 1112b face a second recess 1113c of the second electrode 1113. The length in the first direction and the length in the second direction of each of the two first protrusions 1112b and the first recess 1112c are, for example, about 5 micrometers. Note that the sizes of the two first protrusions 1112b and the first recess 1112c are not limited to this.

[0044] The shape and size of the second electrode 1113 are substantially the same as those of the first electrode 1112. That is, the second electrode 1113 also includes a second base 1113a extending in a first direction (the horizontal direction on the paper in FIG. 3 ) and two second protrusions 1113b protruding from the second base 1113a in a second direction (the vertical direction on the paper in FIG. 3 ) intersecting the first direction. A second recess 1113c is formed between the two second protrusions 1113b. The two second protrusions 1113b are disposed opposite the first electrode 1112. That is, the second electrode 1113 has a second protrusion 1113b protruding from the second base 1113a in a direction intersecting the first direction and protruding toward the first electrode. The second protrusion 1113b faces the first recess 1112c of the first electrode 1112.

[0045] By applying an AC voltage between the first electrode 1112 and the second electrode 1113, a non-uniform electric field is generated on the first substrate 111. The AC voltage applied to the first electrode 1112 and the AC voltage applied to the second electrode 1113 may have the same voltage waveform but may have a phase difference. The phase difference of the applied AC voltages may be, for example, 180 degrees.

[0046] The position of the electrode set 1111 is not limited to on the first substrate 111. The electrode set 1111 may be disposed in the vicinity of the sample 10 in the space 1121. Here, the vicinity of the sample 10 means a range in which an electric field can be generated in the sample 10 by an AC voltage applied to the electrode set 1111. In other words, the electrode set 1111 may be in direct contact with the sample 10 in the space 1121, or may form an electric field in a region including the sample 10 from outside the space 1121.

[0047] [Distribution of electric field strength on the first substrate] Here, the electric field intensity distribution of the non-uniform electric field generated on the first substrate 111 will be described with reference to FIG.

[0048] 3, the non-uniform electric field forms a first electric field region A with a relatively high electric field strength and a second electric field region B with a relatively low electric field strength on the first substrate 111. The first electric field region A is a region with a higher electric field strength than the second electric field region B, and is the region between the opposing first convex portion 1112b and second convex portion 1113b. More specifically, the first electric field region A is formed at a position where the ends of the first convex portion 1112b and the second convex portion 1113b in the first direction face each other.

[0049] The electric field strength depends on the distance between the electrodes that generate the electric field. The longer the distance between the electrodes, the lower the electric field strength, and the shorter the distance between the electrodes, the higher the electric field strength. The position where the ends of the first convex portion 1112b and the second convex portion 1113b in the first direction face each other is the position in the electrode set 1111 where the distance between the first electrode 1112 and the second electrode 1113 is the shortest, and the electric field strength is the highest. The first electric field region A is a region of a predetermined range that includes such a position where the distance between the first electrode 1112 and the second electrode 1113 is the shortest.

[0050] The second electric field region B is a region having a lower electric field strength than the first electric field region A, and is formed in the region between the opposing first convex portion 1112b and second concave portion 1113c, or between the opposing first concave portion 1112c and second convex portion 1113b. This region is the position where the distance between the first electrode 1112 and the second electrode is longest, and the electric field strength is particularly low closer to the first concave portion 1112c or the second concave portion 1113c. The second electric field region B is a region including the bottoms of the first concave portion 1112c and the second concave portion 1113c, where the electric field strength is particularly low.

[0051] [Detection method using a detection device] A method for detecting a target substance using the detecting device 100 configured as above will be described with reference to Figures 4 to 6. Figure 4 is a flowchart showing the detection method according to the embodiment.

[0052] First, a target substance 11 is bound to a dielectric particle 12a modified with a specific binding substance 12b to form a composite particle 13 (S110). The process of forming the composite particle 13 will now be described with reference to Fig. 5. Fig. 5 is a diagram showing the process of forming a composite particle in an embodiment.

[0053] As shown in FIG. 5(a), a target substance 11 and unbound particles 12 are mixed to produce a sample 10.

[0054] In the present disclosure, a single-domain antibody, particularly a VHH antibody, specific to the target substance 11 is used as the substance 12b that specifically binds to the target substance 11. An antibody is an example of a substance that has the property of specifically binding to the target substance 11. In addition to the VHH antibody, other single-domain antibodies capable of binding to the target substance 11 may be used. Single-domain antibodies include the above-mentioned VHH antibody, recombinants obtained by recombinantly expressing a single-domain peptide chain of an immunoglobulin variable region containing an epitope recognition site using a host such as Escherichia coli, and single-domain antibodies in the broad sense, such as target recognition molecules with a molecular weight of approximately 7 to 20 kDa that consist of a polypeptide that forms a helix-loop-helix. Such target recognition molecules are known as so-called microantibodies. The sizes of the target substance 11, the dielectric particles 12a, and the VHH antibody are approximately 100 nanometers, approximately 300 nanometers, and approximately 5 nanometers, respectively. Note that in the present disclosure, antibodies other than single-domain antibodies, such as IgG antibodies, are not used as the substance that specifically binds to the target substance.

[0055] After the sample 10 shown in Fig. 5(a) is allowed to stand in a liquid at a predetermined temperature for a predetermined time, the target substance 11 and the unbound particles 12 are bound to each other by an antigen-antibody reaction to form complex particles 13, as shown in Fig. 5(b). At this time, the size of the complex particles 13 is approximately 700 nanometers.

[0056] Generally, when detecting a target substance 11 whose amount is unknown, an excess amount of unbound particles 12 is introduced to form complex particles 13 for almost all of the target substance 11. Since the amount of complex particles 13 correlates with the amount of target substance 11, the target substance 11 can be indirectly detected by detecting the complex particles 13. Here, by introducing an excess amount of unbound particles 12, the unbound particles 12 that have not bound to the target substance 11 remain alone or in an aggregated state, as shown in FIG. 5(b).

[0057] 5(b) is an example and is not limiting. For example, the number of dielectric particles 12a contained in the composite particle 13 may be one, or may be three or more. For example, the number of target substances 11 contained in the composite particle 13 may be two or more.

[0058] Returning to the explanation of the flowchart in FIG. 4, next, the complex particles 13 and the unbound particles 12 are separated by dielectrophoresis in a liquid (external liquid of the sample 10) (S120). Specifically, an AC voltage is applied to the electrode set 1111, generating a non-uniform electric field in the sample 10 on the first substrate 111. This causes dielectrophoresis to act on the complex particles 13 and the unbound particles 12, causing each of the complex particles 13 and the unbound particles 12 to move. Impurities 14 are also separated in the same manner, but this separation requires separating the specimen from others. In other words, the complex particles 13 are separated from the unbound particles 12 and the impurities 14. It is not necessary to separate the unbound particles 12 from the impurities 14. Note that the aggregated unbound particles 12 are decomposed into a plurality of individual unbound particles 12 by dielectrophoresis.

[0059] For this purpose, the frequency of the AC voltage applied to the electrode set 1111 is set to a predetermined frequency. Application of an AC voltage of a predetermined frequency can cause dielectrophoresis in different directions to occur on the complex particles 13, the unbound particles 12, and the impurities 14. For example, if the frequency of the AC voltage is set to a predetermined frequency at which negative dielectrophoresis (nDEP) occurs on the complex particles 13 and positive dielectrophoresis (pDEP) occurs on the unbound particles 12 and the impurities 14, the complex particles 13 will move to the second electric field region B, where the electric field strength is relatively low, and the unbound particles 12 and the impurities 14 will move to the first electric field region A, where the electric field strength is relatively high. As a result, the complex particles 13 will be separated from the unbound particles 12 and the impurities 14 in terms of their positions.

[0060] Here, the predetermined frequency of the AC voltage will be described with reference to Fig. 6. Fig. 6 is a graph showing the set frequency of the AC voltage in the embodiment. In the graph of Fig. 5, the vertical axis represents the real part of the Clausius-Mossotti factor, and the horizontal axis represents the frequency of the AC voltage applied between the electrode set 1111.

[0061] If the real part of the Clausius-Mossotti coefficient is positive, the particle experiences positive dielectrophoresis, which causes it to move towards regions of higher electric field strength. Conversely, if the real part of the Clausius-Mossotti coefficient is negative, the particle experiences negative dielectrophoresis, which causes it to move towards regions of lower electric field strength.

[0062] As shown in Figure 6, the real part of the Clausius-Mossotti coefficient depends on the particle size and frequency. At frequency F, the real part of the Clausius-Mossotti coefficient is negative for particles of 700 nanometers, which corresponds to the size of composite particle 13, and positive for particles of 300 nanometers, which corresponds to unbound particle 12. Therefore, by setting frequency F as a predetermined frequency of the AC voltage, negative dielectrophoresis can be applied to composite particle 13 and positive dielectrophoresis can be applied to unbound particle 12.

[0063] Returning to the explanation of the flowchart in Fig. 4, finally, target substance 11 contained in separated complex particle 13 is detected (S130). For example, image capture element 140 captures an image of second electric field region B and outputs an image including complex particle 13. Detection unit 150 performs image analysis on the output image to detect complex particle 13. In this way, target substance 11 contained in complex particle 13 is detected.

[0064] The setting of the predetermined frequency described above will be explained in more detail below with reference to FIGS. 7A to 9B. FIG. 7A is a schematic diagram showing particles that have moved to the first electric field region. FIG. 7B is a schematic diagram showing particles that have moved to the first electric field region and the second electric field region. FIG. 7C is a schematic diagram showing particles that have moved to the second electric field region. FIGS. 7A to 7C show plan views of an electrode set 1111 viewed from the same direction as FIG. 3 and particles 15 that are dielectrophoretically migrated in the electrode set 1111. The particles 15 here are general particles that move due to the action of dielectrophoresis, and may correspond to any of the above-mentioned complex particles 13, unbound particles 12, and impurities 14.

[0065] As shown in Figure 7A, when an AC voltage of a frequency at which the real part of the Clausius-Mossotti coefficient of particle 15 becomes a positive value is applied between electrode set 1111, particle 15 moves to first electric field region A by positive dielectrophoresis.

[0066] 7B, when an AC voltage having a frequency at which the real part of the Clausius-Mossotti coefficient of the particles 15 is near 0 is applied between the electrode set 1111, the particles 15 move by positive dielectrophoresis to the first electric field region A and the second electric field region B. This occurs because each of the multiple particles 15 exhibits slightly different properties, resulting in a mixture of particles 15 moving by positive dielectrophoresis and particles 15 moving by negative dielectrophoresis.

[0067] As shown in Figure 7C, when an AC voltage having a frequency at which the real part of the Clausius-Mossotti coefficient of particle 15 is negative is applied between electrode set 1111, particle 15 moves to second electric field region B due to negative dielectrophoresis. Thus, the direction of dielectrophoresis of particle 15 reverses from positive to negative dielectrophoresis with each frequency of the AC voltage applied between the electrode sets. The frequency of the AC voltage at which this dielectrophoresis direction reversal (hereinafter referred to as the crossover frequency) varies depending on the type of particle 15. Note that when the state of Figure 7B continues over a wide frequency band (i.e., there is a frequency bandwidth), the lowest frequency at which the state of Figure 7B occurs is defined as the crossover frequency.

[0068] FIG. 8 is a graph showing the crossover frequency for each type of particle in the embodiment. In FIG. 8, the vertical axis shows the positive or negative sign of the real part of the Clausius-Mossotti coefficient, and the horizontal axis shows the frequency of the AC voltage applied between the electrode set 1111. The vertical axis of FIG. 8 shows either +1 or −1, which is obtained by dividing the value of the real part of the Clausius-Mossotti coefficient by the absolute value of that value, to indicate the positive or negative sign of the real part of the Clausius-Mossotti coefficient. The graph in FIG. 8 shows the results for (i) dielectric particles alone, (ii) VHH antibody-modified dielectric particles and target substance, (iii) VHH antibody-modified dielectric particles alone, and (iv) IgG antibody-modified dielectric particles alone.

[0069] As shown in Figure 8, the crossover frequency was confirmed within the frequency range of 100 kHz to 500 kHz for all particles 15, and it was found that as the frequency increased, the dielectrophoresis reversed from positive to negative. The crossover frequency differed for each type of particle 15.

[0070] Although not shown, the crossover frequency between IgG antibody-modified dielectric particles and the target substance could not be confirmed within the frequency range of 100 kHz to 500 kHz, and the real part of the Clausius-Mossotti coefficient always showed a negative value. In other words, the crossover frequency between IgG antibody-modified dielectric particles and the target substance is thought to exist in a frequency range below 100 kHz. However, as mentioned above, the frequency of the AC voltage at which the dielectrophoretic direction reverses often has a bandwidth.

[0071] (iv) In order to separate the IgG antibody-modified dielectric particles alone from the IgG antibody-modified dielectric particles plus the target substance, it is necessary to set a predetermined frequency at which one moves by positive dielectrophoresis to the first electric field region A and the other moves by negative dielectrophoresis to the second electric field region B. However, since the crossover frequencies of the (iv) IgG antibody-modified dielectric particles alone and the IgG antibody-modified dielectric particles plus the target substance are substantially the same, the particles 15 exhibit the same dielectrophoretic behavior regardless of the frequency of the AC voltage applied.

[0072] In contrast, because there is a 300 kHz difference between the crossover frequency of (ii) VHH antibody-modified dielectric particles + target substance and the crossover frequency of (iii) VHH antibody-modified dielectric particles alone, it is possible to set a predetermined frequency at which one moves to the first electric field region A by positive dielectrophoresis and the other moves to the second electric field region B by negative dielectrophoresis. In other words, to separate (ii) VHH antibody-modified dielectric particles + target substance from (iii) VHH antibody-modified dielectric particles alone, an AC voltage with a frequency of 100 kHz to 400 kHz needs only to be applied. Furthermore, even when the frequency has a bandwidth, the predetermined frequency can be selected from a frequency range of 150 kHz to 350 kHz, and to further improve separation performance, the predetermined frequency can also be selected from a frequency range of 200 kHz to 300 kHz.

[0073] Thus, in separation of two types of particles 15 by dielectrophoresis, an AC voltage of a predetermined frequency that is greater than the first frequency and less than the second frequency is applied to the electrode set 1111. When an AC voltage of the first frequency is applied, both positive and negative dielectrophoresis act on one of the two types of particles 15, and when an AC voltage of the second frequency is used, both positive and negative dielectrophoresis act on the other of the two types of particles 15. In other words, a predetermined frequency is set between the crossover frequency of one of the particles 15 and the crossover frequency of the other of the particles 15.

[0074] As described above, the frequency of the AC voltage applied between the electrode set 1111 is appropriately set in consideration of the crossover frequency. In this case, the crossover frequencies must be different between the two or more types of particles 15 to be separated. Here, attention is focused on the zeta potential of the particles 15 as a property of the particles 15 themselves that affects the crossover frequency of the particles 15. FIG. 9A is a first graph showing the zeta potential of particles according to the embodiment and a comparative example. FIG. 9B is a second graph showing the zeta potential of particles according to the embodiment and a comparative example.

[0075] 9A and 9B, the zeta potentials of the VHH antibody-modified dielectric particles according to the embodiment and the IgG antibody-modified dielectric particles according to the comparative example are shown by open circles and open squares, respectively. Note that "Bare" on the horizontal axis of the graph indicates the dielectric particles alone, "+Antibody" indicates the antibody-modified dielectric particles alone in which the respective antibodies are modified on the dielectric particles, and "+Antigen" indicates the composite particles in which the antibody-modified dielectric particles are bound to the virus used as the antigen. FIG. 9A shows the zeta potential of each particle when the size of the dielectric particles is 1000 nanometers. FIG. 9B shows the zeta potential of each particle when the size of the dielectric particles is 300 nanometers.

[0076] The zeta potential is calculated from the mobility of the dielectric particles based on the detection of the movement of the dielectric particles using electrophoretic light scattering measurement (so-called laser Doppler method) based on the change in the scattering intensity of laser light irradiated onto the particles.

[0077] 9A, in an example using 1000-nanometer dielectric particles, when in the form of composite particles, the VHH antibody-modified dielectric particles according to the embodiments and the IgG antibody-modified dielectric particles both exhibit equivalent zeta potentials (VHH: -26.4 mV, IgG: -24.1 mV). On the other hand, when in the form of antibody-modified dielectric particles alone, the VHH antibody-modified dielectric particles according to the embodiments and the IgG antibody-modified dielectric particles exhibit different zeta potentials (VHH: -44.2 V, IgG: -36.8 mV). As a result, the zeta potential of the VHH antibody-modified dielectric particles changes more significantly upon virus binding than that of the IgG antibody-modified dielectric particles.

[0078] For example, as shown in the figure, the ratio of the zeta potential of the IgG antibody-modified dielectric particles (i.e., unbound particles) to the complex particles is 1:0.65, whereas the ratio of the zeta potential of the VHH antibody-modified dielectric particles (i.e., unbound particles) to the complex particles is 1:0.59.

[0079] As shown in Figure 9B, a similar trend was observed in an example using 300-nanometer dielectric particles. Specifically, when in the form of composite particles, the VHH antibody-modified dielectric particles according to the embodiment and the IgG antibody-modified dielectric particles both exhibit equivalent zeta potentials (VHH: -21.4 mV, IgG: -21.9 mV). On the other hand, when in the form of antibody-modified dielectric particles alone, the VHH antibody-modified dielectric particles according to the embodiment and the IgG antibody-modified dielectric particles exhibit different zeta potentials (VHH: -52.9 V, IgG: -34.6 mV). This means that the zeta potential of the VHH antibody-modified dielectric particles changes more significantly upon virus binding than that of the IgG antibody-modified dielectric particles.

[0080] For example, as shown in the figure, the ratio of the zeta potential of the IgG antibody-modified complex particles to that of the antibody-modified dielectric particles (i.e., unbound particles) is 1:0.63, whereas the ratio of the zeta potential of the VHH antibody-modified complex particles to that of the antibody-modified dielectric particles (i.e., unbound particles) is 1:0.40.

[0081] To create a sufficient difference in crossover frequency to cause dielectrophoresis in different directions on the two types of particles, the ratio of the zeta potential of the complex particles to that of the unbound particles should be less than 0.65 and less than 0.63. The ratio of the zeta potential of the complex particles to that of the unbound particles should be 0.40 or greater and 0.59 or greater. In other words, the ratio of the zeta potential of the complex particles to that of the unbound particles should be in the range of 0.40 or greater and 0.62 or less.

[0082] [Effects, etc.] As described above, the detection method according to this embodiment forms complex particles 13 by binding a target substance 11 to dielectric particles 12a modified with a single domain antibody (e.g., a VHH antibody) that specifically binds to the target substance 11, separates the complex particles 13 from unbound particles 12, which are dielectric particles 12a that have not formed complex particles 13, by dielectrophoresis in a liquid such as the external liquid of the sample 10, and detects the target substance 11 contained in the separated complex particles 13 using an imaging element 140.

[0083] In this detection method, a target substance 11 is captured as an antigen of a single-domain antibody, and the target substance 11 and dielectric particles 12a form complex particles 13 via the single-domain antibody. The target substance 11 is bound to dielectric particles 12a and is moved by dielectrophoresis. At this time, by separating unbound particles 12 including unbound dielectric particles 12a by dielectrophoresis, the complex particles 13 can be selectively detected, and the target substance 11 correlated with the detected complex particles 13 can be detected.

[0084] Here, magnetic particles bound to fluorescent particles without a target substance and magnetic particles bound to fluorescent particles via a target substance behave in the same way when a magnetic field is applied, making it difficult to selectively detect particles bound to fluorescent particles via a target substance. Therefore, this detection method is subject to a large influence of false positives, resulting in reduced detection accuracy. On the other hand, the detection method of the present embodiment allows for selective detection of composite particles 13 containing target substance 11, because unbound particles 12 not containing target substance 11 and composite particles 13 containing target substance 11 behave differently. Therefore, false positives due to nonspecific adsorption can be reduced, and the detection accuracy of target substance 11 can be improved.

[0085] For example, in separating the composite particles 13 and the unbound particles 12, application of an AC voltage of a predetermined frequency may cause one of positive dielectrophoresis and negative dielectrophoresis to act on the composite particles 13, and the other of positive dielectrophoresis and negative dielectrophoresis to act on the unbound particles 12.

[0086] According to this, by applying positive dielectrophoresis to one of the complex particles 13 and the unbound particles 12 and applying negative dielectrophoresis to the other, the complex particles 13 and the unbound particles 12 can be separated, and the target substance 11 contained in the separated complex particles 13 can be detected. By moving each particle in the opposite direction by positive dielectrophoresis and negative dielectrophoresis, the complex particles 13 and the unbound particles 12 can be separated. Therefore, it becomes possible to selectively detect the complex particles 13, reducing false positives due to nonspecific adsorption and improving the detection accuracy of the target substance 11.

[0087] For example, the predetermined frequency may be greater than the first frequency and less than the second frequency, and when an AC voltage of the first frequency is used instead of the predetermined frequency, the generated electric field gradient may cause both positive and negative dielectrophoresis to act on the composite particles 13, and when an AC voltage of the second frequency is used instead of the predetermined frequency, the generated electric field gradient may cause both positive and negative dielectrophoresis to act on the unbound particles 12.

[0088] According to this, by applying an AC voltage of a predetermined frequency between a first frequency, which is the crossover frequency of the complex particles 13, and a second frequency, which is the crossover frequency of the unbound particles 12, the complex particles 13 and the unbound particles 12 can be separated. An AC voltage of an appropriate frequency can be applied, making it possible to selectively detect the complex particles 13. Therefore, false positives due to nonspecific adsorption can be reduced, and the detection accuracy of the target substance 11 can be improved.

[0089] For example, the ratio of the zeta potential of the composite particles 13 to the zeta potential of the unbound particles 12 may be 1.0 or less, or may be 0.40 or more and 0.62 or less.

[0090] This allows for the selection of a particle configuration that allows separation of the composite particles 13 and the unbound particles 12 by evaluating the zeta potential that affects the crossover frequency of the composite particles 13 and the unbound particles 12. In particular, whether an appropriate detection system can be constructed can be evaluated by whether the ratio of the zeta potential of the composite particles 13 to the unbound particles 12 is 1.0 or less, more preferably in the range of 0.40 to 0.62. This improves the ease of application of the detection method.

[0091] For example, the dielectric particles 12a may contain a fluorescent substance, and in detecting the target substance 11, the separated complex particles 13 may be irradiated with excitation light, and the fluorescence emitted by the fluorescent substance contained in the complex particles 13 may be imaged by the imaging element 140, thereby detecting the target substance 11 contained in the complex particles 13.

[0092] This allows the dielectric particles 12a to be detected with high sensitivity by utilizing the decrease in fluorescence emission. For example, even when the particle size of the dielectric particles 12a cannot be increased due to limitations such as the crossover frequency and zeta potential, the dielectric particles 12a can be detected with high sensitivity. In other words, the improved detection sensitivity allows a wider range of choices for the dielectric particles 12a, and the detection method of this embodiment can be applied to a wider variety of detection systems.

[0093] The detection device 100 of this embodiment is a detection device 100 that detects a target substance 11 by dielectrophoresis, and is equipped with a separator 110 that separates, by dielectrophoresis, dielectric particles 12a modified with a single domain antibody (e.g., a VHH antibody) that specifically binds to the target substance 11, complex particles 13 formed by the binding of the target substance 11 and the dielectric particles 12a, and unbound particles 12, which are dielectric particles 12a that have not formed complex particles 13, in a liquid such as the external liquid of the sample 10, and an imaging element 140 used to detect the target substance 11 contained in the separated complex particles 13.

[0094] Such a detection device 100 can constitute a detection device 100 that realizes the above-described detection method.

[0095] For example, the separator 110 has an electrode set 1111 including a first electrode 1112 and a second electrode 1113 spaced apart from each other, the first electrode 1112 having a first base 1112a extending in a predetermined direction and one or more first protrusions 1112b protruding from the first base 1112a in a direction intersecting the predetermined direction and protruding toward the second electrode 1113, and the second electrode 1113 having a second base 1113a extending in the predetermined direction and one or more first protrusions 1112b protruding from the second base 1113a in a direction intersecting the predetermined direction and protruding toward the first electrode 1112. The electrode set 1111 has one or more protruding second convex portions 1113b, and when an AC voltage is applied to the electrode set 1111, the first convex portion 1112b and the second convex portion 1113b form a first electric field region A including the position where the distance between the first electrode 1112 and the second electrode 1113 is shortest, and the first convex portion 1112b and the second convex portion 1113b form a second electric field region B including the position where the distance between the first electrode 1112 and the second electrode 1113 is longest, and the second electric field region B may have a lower electric field strength than the first electric field region A.

[0096] This allows a gradient in electric field strength to be formed in the electric field depending on the shape of the electrode set 1111. In this way, a non-uniform electric field can be generated by designing the electrode set 1111, so that an electric field suitable for the detection system can be selectively used. This allows the detection device 100 to be applied in a wider range of applications.

[0097] The dielectric particles 12 a according to this embodiment are modified with a single domain antibody that specifically binds to the target substance 11 .

[0098] Such dielectric particles 12a are useful in a detection device 100 or a detection method for detecting a target substance 11 by dielectrophoresis.

[0099] (Variation) Although the detection device and detection method according to one or more aspects of the present disclosure have been described based on the embodiments, the present disclosure is not limited to these embodiments. As long as they do not deviate from the spirit of the present disclosure, various modifications conceivable by those skilled in the art to the present embodiments may also be included within the scope of one or more aspects of the present disclosure.

[0100] For example, in the above embodiment, the electrode set 1111 on the first substrate 111 is illustrated in FIG. 3, but the shape and arrangement of the electrode set are not limited to this. FIG. 10 is a first plan view showing the configuration of an electrode set according to a modified example. For example, an electrode set 2111 may be installed on the first substrate 111 as shown in FIG. 10. In the electrode set 2111 of FIG. 6, the first convex portion 1112b of the first electrode 1112 and the second convex portion 1113b of the second electrode 1113 face each other in the second direction (the up-down direction on the paper in FIG. 10). Even with this electrode set 2111, a non-uniform electric field can be generated by applying an AC voltage.

[0101] The number of electrodes included in an electrode set is not limited to two, and may be three or more. FIG. 11 is a second plan view showing the configuration of an electrode set according to a modified example. For example, as shown in FIG. 11, an electrode set 3111 may be installed on a first substrate 111. The electrode set 3111 in FIG. 11 includes three or more electrodes, and a phase difference is provided between the AC voltages applied to adjacent electrodes. The electrode set 3111 is sometimes called a castellated electrode.

[0102] In the above embodiment, the composite particle 13 is illustrated in Fig. 5, but the configuration of the composite particle is not limited to this. Fig. 12 is a diagram showing a process for forming a composite particle according to a modified example. For example, in the above embodiment, an example in which the dielectric particle 12a contains a fluorescent substance has been described, but as shown in Fig. 12, the dielectric particle 21a and the fluorescent particle 22a may be separate particles.

[0103] As shown in (a) of Figure 12, a target substance 11, unbound particles 21, and antibody-modified fluorescent particles 22 are mixed to produce a sample 10. The unbound particles 21 are dielectric particles 21a of 500 to 1000 nanometers modified with antibodies 21b of approximately 5 nanometers. The antibody-modified fluorescent particles 22 are fluorescent particles 22a of approximately 300 nanometers modified with antibodies 22b of approximately 5 nanometers. Note that the sizes of the particles and antibodies are not limited to those mentioned above.

[0104] The dielectric particles 21a may be, but are not limited to, polystyrene particles. The antibodies 21b and 22b may be, but are not limited to, VHH antibodies. The antibodies 21b and 22b may be different.

[0105] When the sample 10 shown in Fig. 12(a) is left standing at a predetermined temperature for a predetermined time, the target substance 11 binds to the unbound particles 21 and the antibody-modified fluorescent particles 22 through an antigen-antibody reaction, forming complex particles 23, as shown in Fig. 12(b). At this time, the size of the complex particles 23 is 900 to 1400 nanometers. The unbound particles 12 that have not bound to the target substance 11 remain alone or in an aggregated state.

[0106] In this way, by making the dielectric particles 21a larger than the fluorescent particles 22a, the difference in size between the complex particles 23 and the unbound particles 21 can be increased, and separation of the complex particles 23 and the unbound particles 21 by dielectrophoresis can be more reliably performed.

[0107] The frequency of the AC voltage for separating particles 15 based on the zeta potential varies depending on the size of the particles 15, and may therefore be outside the range of the zeta potential described in the above embodiment. The zeta potential may be used as a guide for selecting particles 15 that can be separated using the detection device 100, and the crossover frequency of each particle 15 may be measured to determine the particles 15 to be used for detecting the target substance 11. [Industrial Applicability]

[0108] The device can be used as a detection device for detecting target substances such as influenza viruses. [Explanation of symbols]

[0109] 10 samples 11 Target substance 12, 21 Unbound particles 12a, 21a Dielectric particles 12b Specific binding substances 13, 23 Composite particles 14 Contaminants 15 particles 21b, 22b antibody 22 Antibody-modified fluorescent particles 22a Fluorescent particles 100 Detection device 110 Separator 111 First board 112 Spacer 113 Second board 120 Power supply 130 light source 131 Irradiation Light 132 Detection light 140 image sensor 141 Camera 150 Detector 1111, 2111, 3111 electrode sets 1112 1st electrode 1112a 1st base 1112b First convex part 1112c First recess 1113 2nd electrode 1113a 2nd base 1113b Second convex part 1113c Second recess 1121 Space 1131 Supply hole 1132 Discharge hole

Claims

1. forming a complex by binding a target substance to a dielectric particle modified with a single domain antibody that binds to the target substance; separating the complexes from unbound particles, which are the dielectric particles that have not formed the complexes, by dielectrophoresis in a liquid; The target substance contained in the separated complex is detected using an imaging element. Detection method.

2. In the separation of the complex and the unbound particles, one of positive dielectrophoresis and negative dielectrophoresis acts on the complex by applying an AC voltage of a predetermined frequency, and the other of positive dielectrophoresis and negative dielectrophoresis acts on the unbound particles. The detection method according to claim 1 .

3. the predetermined frequency is greater than the first frequency and less than the second frequency, when an AC voltage of the first frequency is used instead of the predetermined frequency, both positive dielectrophoresis and negative dielectrophoresis act on the complex due to the generated electric field gradient; When the AC voltage of the second frequency is used instead of the predetermined frequency, both positive and negative dielectrophoresis act on the unbound particles due to the generated electric field gradient. The detection method according to claim 2 .

4. The ratio of the zeta potential of the complex to the zeta potential of the unbound particles is 1.0 or less. The detection method according to any one of claims 1 to 3.

5. the dielectric particles contain a fluorescent material; In detecting the target substance, the separated complex is irradiated with excitation light, and the fluorescence emitted by the fluorescent substance contained in the complex is imaged by the image sensor, thereby detecting the target substance contained in the complex. The detection method according to any one of claims 1 to 4.

6. A detection device that detects a target substance by dielectrophoresis, dielectric particles modified with a single domain antibody that binds to the target substance; a separator that separates complexes formed by binding between the target substance and the dielectric particles and unbound particles, which are the dielectric particles that have not formed any complexes, by dielectrophoresis in a liquid; an imaging element used to detect the target substance contained in the separated complex. Detection device.

7. the separator has an electrode set including a first electrode and a second electrode spaced apart from each other; the first electrode has a first base portion extending in a predetermined direction and one or more first protrusions protruding from the first base portion in a direction intersecting the predetermined direction and protruding toward the second electrode; the second electrode has a second base portion extending in the predetermined direction and one or more second protrusions protruding from the second base portion in a direction intersecting the predetermined direction and protruding toward the first electrode, When an AC voltage is applied to the electrode set, a first electric field region including a position where the distance between the first electrode and the second electrode is shortest is formed by the first convex portion and the second convex portion, and a second electric field region including a position where the distance between the first electrode and the second electrode is longest is formed by the first convex portion and the second convex portion, The second electric field region has a lower electric field strength than the first electric field region. The detection device according to claim 6.

8. separating the complexes and unbound particles by dielectrophoresis in a liquid; A detection method for detecting a target substance contained in the separated complex using an imaging element, comprising: the complex is formed by binding the target substance to a dielectric particle modified with a single domain antibody that binds to the target substance, The unbound particles are the dielectric particles that have not formed the complex. Detection method.

9. mixing a plurality of target substances with a plurality of dielectric particles modified with a plurality of single domain antibodies, thereby generating a liquid containing a plurality of first complexes bound to a plurality of first target substances and a plurality of first dielectric particles, and a plurality of second dielectric particles not bound to the plurality of target substances, each of the plurality of dielectric particles being modified with one or more single domain antibodies included in the plurality of single domain antibodies; generating an AC voltage between a first electrode having a concave-convex shape when viewed in a plane and a second electrode having a concave-convex shape when viewed in a plane, thereby generating a non-uniform electric field between the first electrode and the second electrode, and the non-uniform electric field passing through the liquid mixture separates the plurality of first composites and the plurality of second dielectric particles; detecting the separated first complexes using an imaging element; the plurality of target substances includes a first target substance; The plurality of dielectric particles includes a plurality of first dielectric particles and a plurality of second dielectric particles. Detection method.

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