JP2023072437A - Detection preparation method, detection preparation system, particle group and manufacturing method of particle group - Google Patents

Abstract

To enable detection accuracy of a target substance to be easily improved.SOLUTION: A detection preparation method comprises the steps of: preparing a particle group 20 having a composite particle 23 in which a target substance 11 or a simulated substance 24 simulating the target substance 11 is coupled to a first dielectric particle 21 modified with a substance specifically coupled to the target substance 11 or the simulated substance 24 and a second dielectric particle 22 having a different behavior by the dielectrophoresis from the composite particle 23; applying AC voltage to an electrode for applying an electrical field to a solvent containing the particle group 20; imaging the solvent with an image pick-up device; and searching for a prescribed frequency that can separate by the dielectrophoresis the composite particle 23 from the second dielectric particle 22 on the basis of an image captured by the image pick-up device while changing the frequency of AC voltage. Any one of the composite particle 23 and the second dielectric particle 22 has a mark (fluorescence substance 25) that may visually distinguish both of them in the image.SELECTED DRAWING: Figure 11

Description

The present disclosure relates to a detection preparation method, a detection preparation system, a particle swarm, and a particle swarm preparation method for making preparations before detecting a target substance such as a virus.

Patent Document 1 discloses a method for retaining particles contained in a sample and a method for calibrating means for retaining/detecting particles. This method of holding particles includes the steps of: introducing a sample containing porous particles made of a substance other than a biological substance into a particle holding means having a holding portion capable of holding particles; and holding the part. Moreover, this calibration method includes a step of determining whether the holding step and the detection step are performed normally based on the particle detection result by the detection unit.

JP 2018-146571 A

The present disclosure provides a detection preparation method and the like that easily improve detection accuracy of a target substance.

A detection preparation method according to an aspect of the present disclosure includes a target substance or a mimetic substance that mimics the target substance, and first dielectric particles modified with a substance that specifically binds to the target substance or the mimetic substance. A particle group having bound composite particles and second dielectric particles different in dielectrophoretic behavior from the composite particles is prepared. In the detection preparation method, an alternating voltage is applied to electrodes for applying an electric field to the solvent containing the particle group. In the detection preparation method, an image of the solvent is captured by an imaging device. In the detection preparation method, searching for a predetermined frequency at which the composite particles and the second dielectric particles can be separated by dielectrophoresis based on the image captured by the imaging device while changing the frequency of the AC voltage. do. Either one of the composite particles and the second dielectric particles has a label that allows visual distinction between the two in the image.

A detection preparation system according to one aspect of the present disclosure includes a container, an imaging device, and a search unit. The container comprises composite particles obtained by binding a target substance or a mimetic substance simulating the target substance and first dielectric particles modified with a substance that specifically binds to the target substance or the mimetic substance, and A solvent containing a particle group having second dielectric particles different in behavior by dielectrophoresis from the composite particles is contained. The imaging element images the solvent contained in the container. The search unit detects the composite particles and the second dielectric particles based on the image captured by the imaging device while changing the frequency of the AC voltage applied to the electrodes for applying an electric field to the solvent. Search for a predetermined frequency that can be separated by dielectrophoresis. Either one of the composite particles and the second dielectric particles has a label that allows visual distinction between the two in the image.

A particle group according to an aspect of the present disclosure includes: first dielectric particles modified with a substance that specifically binds to a target substance or a mimetic substance simulating the target substance; Alternatively, the composite particles to which the simulated substance is bound and the second dielectric particles that behave differently by dielectrophoresis are included. Either one of the composite particles and the second dielectric particles has a label that allows visual distinction between the two in the image captured by the imaging device.

A method for producing a particle group according to an aspect of the present disclosure includes mixing dielectric particles modified with a substance that specifically binds to a target substance or a mimetic substance simulating the target substance, and the target substance or the mimetic substance. By doing so, composite particles including first dielectric particles in which the target substance or the simulated substance is bound to the dielectric particles, and second dielectric particles that behave differently from the composite particles by dielectrophoresis, to make. Either one of the composite particles and the second dielectric particles has a label that allows visual distinction between the two in the image captured by the imaging device.

In addition, these general or specific aspects may be realized by a system, apparatus, integrated circuit, computer program, or a recording medium such as a computer-readable CD-ROM. and any combination of recording media.

According to the detection preparation method and the like according to one aspect of the present disclosure, there is an advantage that it is easy to improve the detection accuracy of the target substance.

FIG. 1 is a perspective view showing a schematic configuration of a detection system according to an embodiment. FIG. 2 is a block diagram and cross-sectional view showing a schematic configuration of the detection system according to the embodiment. FIG. 3 is a plan view showing the configuration of the electrode set according to the embodiment. FIG. 4 is a flow chart showing the detection method according to the embodiment. FIG. 5 is a diagram showing a formation process of composite particles in the embodiment. FIG. 6 is a graph showing the set frequency of the AC voltage in the embodiment. FIG. 7A is a schematic diagram showing a particle that has migrated to the first electric field region. FIG. 7B is a schematic diagram showing particles that have migrated to the first and second electric field regions. FIG. 7C is a schematic diagram showing particles that have migrated to the second electric field region. FIG. 8 is a graph showing the crossover frequency for each particle type in the embodiment. FIG. 9 is a block diagram and cross-sectional view showing a schematic configuration of a detection preparation system according to an embodiment. FIG. 10 is a diagram showing an example of particle groups in the embodiment. FIG. 11 is a flow chart showing a detection preparation method according to the embodiment. FIG. 12 is an explanatory diagram of a predetermined frequency search process in the embodiment. FIG. 13 is a schematic diagram showing a state in which the particle group moves to the first electric field region and the second electric field region according to the embodiment. FIG. 14 is a first plan view showing the configuration of the electrode set according to the modification. FIG. 15 is a second plan view showing the configuration of the electrode set according to the modification. FIG. 16 is a diagram showing a formation process of composite particles according to a modification.

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

It should be noted that the embodiments described below are all comprehensive or specific examples. Numerical values, shapes, materials, components, arrangement positions and connection forms of components, steps, order of steps, and the like shown in the following embodiments are examples, and are not intended to limit the scope of the claims.

Also, each figure is not necessarily strictly illustrated. In each figure, substantially the same configuration may be denoted by the same reference numerals, and redundant description may be omitted or simplified.

In the following, terms that indicate the relationship between elements such as parallel and perpendicular, terms that indicate the shape of elements such as rectangular, and numerical ranges do not represent only strict meanings, but are substantially It means that the range equivalent to , for example, a difference of about several percent is also included.

In addition, hereinafter, detecting a target substance includes finding the target substance and confirming the presence of the target substance, as well as measuring the amount (e.g., number or concentration, etc.) of the target substance or its range.

(Embodiment)
A detection preparation method and a detection preparation system according to embodiments are methods and systems for preparing before executing the detection method and before using the detection system. The detection method and detection system are methods and systems for separating composite particles and unbound particles in a liquid by dielectrophoresis (DEP) and detecting a target substance contained in the separated composite particles.

Here, dielectrophoresis is a phenomenon in which force acts on dielectric particles exposed to a non-uniform electric field. This force does not require charging of the particles.

A target substance is a substance to be detected, and is, for example, a molecule such as a pathogenic protein, a virus (coat protein, etc.), or a bacterium (polysaccharide, etc.). A target substance may also be called an analyte or a detection target.

Before describing the detection preparation method and the detection preparation system according to the embodiments, specific embodiments of the detection system and the detection method for realizing the detection of a target substance using dielectrophoresis will be described below with reference to the drawings. explained in detail.

[Configuration of detection system]
First, the configuration of the detection system will be described with reference to FIGS. 1 and 2. FIG. FIG. 1 is a perspective view showing a schematic configuration of a detection system according to an embodiment. Also, FIG. 2 is a cross-sectional view showing a schematic configuration of the detection system according to the embodiment. In FIG. 1 , the separator 110 is shown only in outline so that the inside of the separator 110 can be seen by transmitting a portion other than the first substrate 111 . In addition, FIG. 1 is used to explain the relationship between the separator 110 and other components. is not limited to 2 shows a block diagram showing each component of the detection system 100 along with a cross-sectional view of the separator 110 shown in FIG. 1 taken along a direction parallel to the plane of the paper. 1, the thickness of a part of the separator 110 shown in FIG. 2 is omitted.

As shown in FIGS. 1 and 2, the detection system 100 includes a separator 110, a power supply 120, a light source 130, an imaging device 140, and a detection section 150. FIG.

The separator 110 is a container containing the sample 10 that may contain the target substance 11 and has a space 1121 inside. The sample 10 is accommodated in the space 1121 concerned. The separator 110 separates the composite particles 13 and the unbound particles 12 in the liquid (that is, in the external liquid of the sample 10) in the space 1121 by dielectrophoresis. Here, the separator 110 positionally separates the composite particles 13 and the unbound particles 12 . Sample 10 contains unbound particles 12 , and when target substance 11 is contained, further contains composite particles 13 formed by target substance 11 and unbound particles 12 . Also, the sample 10 may be contaminated with contaminants 14 .

The composite particles 13 are composites in which the target substance 11 and dielectric particles 12a (see FIG. 5 described later) modified with a substance having a property of specifically binding to the target substance 11 are bound. That is, in the composite particles 13, the target substance 11 and the dielectric particles 12a are bound via a substance having a property of specifically binding to the target substance 11. As shown in FIG.

Dielectric particles 12a are particles that can be polarized by an applied electric field. Dielectric particles 12a may contain, for example, a fluorescent material. When light having a wavelength that excites the fluorescent substance is emitted from the light source 130, which will be described later, the dielectric particles 12a can be detected by detecting light in the wavelength band of fluorescence emission. Moreover, the dielectric particles 12a are not limited to particles containing a fluorescent material. For example, polystyrene particles containing no fluorescent material, glass particles, or the like may be used as the dielectric particles 12a.

Here, the substance having the property of specifically binding to the target substance 11 is a substance capable of specifically binding to the target substance 11, and is also called a specific binding substance 12b (see FIG. 5 described later). Examples of combinations of specific binding substances 12b for target substances 11 include antibodies for antigens, enzymes for substrates or coenzymes, receptors for hormones, protein A or protein G for antibodies, avidins for biotin, calmodulin for calcium, sugars Examples include lectins, or tag-binding substances such as nickel-nitrilotriacetic acid or glutathione for peptide tags such as 6x histidine or glutathione S transferase.

The unbound particles 12 are dielectric particles 12 a that do not form composite particles 13 . That is, the unbound particles 12 are dielectric particles 12 a that are not bound to the target substance 11 . Unbound particles 12 are also referred to as the free (F) component. On the other hand, the dielectric particles 12a and the specific binding substance 12b contained in the composite particles 13 are also called bind (B) components.

Now, the internal configuration of the separator 110 will be described. As shown in FIG. 2, the separator 110 includes a first substrate 111, spacers 112, and a second substrate 113. As shown in FIG.

The first substrate 111 is, for example, a glass or resin sheet. The first substrate 111 has a top surface that defines the bottom of the space 1121 , and an electrode set 1111 to which an AC voltage is applied from the power supply 120 is formed on the top surface. The electrode set 1111 includes a first electrode 1112 and a second electrode 1113 and can generate a non-uniform electric field (also called an electric field gradient) on the first substrate 111 . That is, 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 with reference to FIG.

A 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 a through-hole sandwiched between the first substrate 111 and the second substrate 113 . As described above, in space 1121 is introduced sample 10 , which may include composite particles 13 and unbound particles 12 . The spacer 112 is an outer wall that surrounds the through hole and has an inner surface that defines the space 1121 . The spacer 112 is made of a material such as resin having high adhesion to the first substrate 111 and the second substrate 113, for example.

The second substrate 113 is a transparent sheet made of glass or resin, for example, and is arranged on the spacer 112 . For example, a polycarbonate substrate can be used as the second substrate 113 . A supply hole 1131 and a discharge hole 1132 connected to the space 1121 are formed in the second substrate 113 so as to pass through 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 . That is, the second substrate 113 is not an essential component. For example, the space 1121 for establishing the separator 110 as a container is formed only by the first substrate 111 and spacers 112 defining the bottom and inner surfaces, respectively.

The power supply 120 is an AC power supply and applies an AC voltage to the electrode sets 1111 of the first substrate 111 . The power supply 120 may be any power supply that can supply AC voltage, and is not limited to a specific power supply. Alternatively, the AC voltage may be supplied from an external power source, in which case power source 120 may not be included in detection system 100 .

The light source 130 irradiates the sample 10 in the space 1121 with the irradiation light 131 . The irradiation light 131 is irradiated into the sample 10 through the transparent second substrate 113 . A detection light 132 corresponding to the irradiation light 131 is generated from the sample 10 , and the dielectric particles 12 a contained in the sample 10 are detected by detecting the detection light 132 . For example, as described above, when a fluorescent substance is contained in the dielectric particles 12a, the fluorescent substance is excited by being irradiated with the excitation light as the irradiation light 131, and the fluorescence emitted from the fluorescent substance is detected as the detection light 132. .

As the light source 130, any known technology can be used without particular limitation. For example, lasers such as semiconductor lasers and gas lasers can be used as the light source 130 . As the wavelength of the irradiation light 131 emitted from the light source 130, a wavelength with which interaction with substances contained in the target substance 11 is small is used. For example, if the target substance 11 is a virus, the irradiation light 131 with a wavelength of 400 nm to 2000 nm is selected. Also, as the wavelength of the irradiation light 131, a wavelength that can be used by a semiconductor laser (for example, 600 nm to 850 nm) may be used.

Note that light source 130 may not be included in detection system 100 . For example, when the size of the dielectric particles 12a is large, observation becomes possible by combining an optical element such as a lens, and it is not necessary to use a light emission phenomenon such as fluorescence emission. That is, the dielectric particles 12a may not contain a fluorescent substance, and in this case, the irradiation light 131 from the light source 130 may not be emitted. In this case, instead of the light source 130, external light emitted from the sun, a fluorescent lamp, or the like can be used to detect the dielectric particles 12a.

The imaging device 140 is a CMOS (Complementary Metal Oxide Semiconductor) image sensor, a CCD (Charge Coupled Device) image sensor, or the like, and receives detection light 132 generated from the sample 10 to generate and output an image. The imaging element 140 is built in, for example, the camera 141 or the like, is arranged horizontally on the board surface of the first substrate 111, and corresponds to the electrode set 1111 via an optical element (not shown) such as a lens included in the camera 141. Take an image of the part to be In this way, the imaging device 140 is used to photograph the composite particles 13 separated from the unbound particles 12 by the separator 110 and detect the target substance 11 contained in the composite particles 13 .

In an example where the dielectric particles 12a contain a fluorescent substance, the imaging device 140 captures fluorescence emitted from the fluorescent substance contained in the dielectric particles 12a. Note that the detection system 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 gather. Note that when a photodetector is used instead of the imaging device 140 in this way, the detection unit 150 may detect the target substance 11 bound to the dielectric particles 12a based on the intensity of the detection light 132 .

Note that detection system 100 may include an optical lens or an optical filter between light source 130 and separator 110 or between separator 110 and imaging device 140 . For example, a long-pass filter that can block the illumination light 131 from the light source 130 and pass the detection light 132 may be placed between the separator 110 and the imaging device 140 .

The detection unit 150 acquires an image output by the imaging element 140, and detects the dielectric particles 12a contained in the sample 10 based on the image. In particular, the detection system 100 according to the embodiment can count each of the complex particles 13 and the unbound particles 12 individually. That is, the dielectric particles 12a forming the composite particles 13 and the dielectric particles 12a contained in the unbonded particles 12 can be detected separately. Therefore, by detecting the dielectric particles 12 a based on the image, the detection unit 150 detects the target substance 11 contained in the composite particles 13 in the sample 10 .

For example, the detection unit 150 detects bright spots having different brightness values by comparing the obtained image and the control image using a control image that does not contain the dielectric particles 12a captured in advance. Specifically, when luminescence is detected as the detection light 132, a point with a high luminance value in an image obtained with respect to the control image is set as a bright point, and transmitted light, scattered light, or the like is detected as the detection light 132. , a point with a low luminance value in the image acquired with respect to the control image may be detected as a bright point. Thus, the detection unit 150 obtains the detection result of the composite particles 13 in the sample 10 .

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

[Shape and Arrangement of Electrode Set]
Next, the shape and arrangement of the electrode set 1111 on the first substrate 111 will be described with reference to FIG. FIG. 3 is a plan view showing the configuration of the electrode set 1111 according to the embodiment. FIG. 3 shows the configuration of the electrode set 1111 when viewed from the imaging device 140 side. For simplification, FIG. 3 shows a schematic configuration diagram showing a part of the electrode set 1111. As shown in FIG.

As explained above, the electrode set 1111 has 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 .

The first electrode 1112 has a first base portion 1112a extending in a first direction (horizontal direction in FIG. 3) and protruding from the first base portion 1112a in a second direction (vertical direction in FIG. 3) intersecting with the first direction. and two first protrusions 1112b. A first recess 1112c is formed between the two first protrusions 1112b. The two first protrusions 1112b are arranged to face the second electrode 1113 (particularly, a second protrusion 1113b to be described later). The length in the first direction and the length in the second direction of each of the two first protrusions 1112b and the first recesses 1112c are both, for example, approximately 5 micrometers. Note that the sizes of the two first protrusions 1112b and the first recesses 1112c are not limited to this.

The shape and size of the second electrode 1113 are substantially the same as the shape and size of the first electrode 1112 . That is, the second electrode 1113 also has a second base portion 1113a extending in the first direction (horizontal direction on the paper surface in FIG. 3) and a second base portion 1113a extending in a second direction (vertical direction on the paper surface in FIG. 3) intersecting the first direction. and two projecting second protrusions 1113b. A second recess 1113c is formed between the two second protrusions 1113b. The two second protrusions 1113b are arranged to face the first electrode 1112 (in particular, the first protrusion 1112b).

A non-uniform electric field is generated on the first substrate 111 by applying an AC voltage to the first electrode 1112 and the second electrode 1113 . The AC voltage applied to the first electrode 1112 and the AC voltage applied to the second electrode 1113 may be substantially the same, or may have a phase difference. For example, 180 degrees can be used as the phase difference of the applied AC voltage.

Note that the position of the electrode set 1111 is not limited to the first substrate 111 . The electrode set 1111 may be arranged near 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 within the sample 10 by the AC voltage applied to the electrode set 1111 . That is, the electrode set 1111 may be in direct contact with the sample 10 within the space 1121 and may form an electric field in the region containing the sample 10 from outside the space 1121 .

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

As shown in FIG. 3, the uneven 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 . be done. The first electric field region A is a region having an electric field strength higher than that of the second electric field region B, and is a region between the first convex portion 1112b and the second convex portion 1113b facing each other.

The electric field strength depends on the inter-electrode distance of the pair of electrodes that generate the electric field. The electric field strength decreases as the distance between the electrodes increases, and increases as the distance between the electrodes decreases. The position where the ends of the first projection 1112b and the second projection 1113b face each other in the first direction is the position where the distance between the first electrode 1112 and the second electrode 1113 is the shortest in the electrode set 1111. , the electric field strength is the highest. The first electric field area A is an area of a predetermined range including the position where the distance between the first electrode 1112 and the second electrode 1113 is the shortest.

In addition, the second electric field region B is a region having an electric field strength lower than that of the first electric field region A, and is between the first convex portion 1112b and the second concave portion 1113c facing each other, or between the first concave portion 1112c and the second concave portion 1112c facing each other. It is formed in the region between the two convex portions 1113b. This region is the position where the distance between the first electrode 1112 and the second electrode 1113 is the longest, and in particular, the closer to the first recess 1112c or the second recess 1113c, the lower the electric field strength. 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 intensity is particularly low.

[Detection method using detection system]
A target substance detection method using the detection system 100 configured as described above will be described with reference to FIGS. 4 to 6. FIG. FIG. 4 is a flow chart showing the detection method according to the embodiment.

First, in the detection method, the target substance 11 and the dielectric particles 12a modified with the specific binding substance 12b are bound to form the composite particles 13 (S110). Here, the formation process of composite particles 13 will be described with reference to FIG. FIG. 5 is a diagram showing a formation process of composite particles 13 in the embodiment.

First, as shown in (a) of FIG. 5 , unbound particles 12 are introduced into a sample 10 containing a target substance 11 . Note that the sample 10 may contain contaminants 14 in addition to the target substance 11 . The contaminants 14 may be various particles depending on their origin. The contaminants 14 are, for example, dust, viruses, cells, or broken pieces of the detection system such as part of the separator 110 that are collected at the same time as the target substance 11 is collected. In either case, contaminants 14 preferably do not bind to specific binding substance 12b.

That is, the specific binding substance 12b is selected from substances having such high binding specificity that it does not bind to the contaminants 14 . In other words, when detecting the target substance 11 in the sample 10 expected to be contaminated with the contaminants 14, the specific binding substance 12b having sufficient binding specificity is selected. Here, an example of using antibody-modified dielectric particles 12a modified with an antibody having relatively high binding specificity to the target substance 11 as the specific binding substance 12b will be described.

An antibody is an example of a substance that has the property of specifically binding to the target substance 11 . Here, an anti-target substance VHH (Variable domain of Heavy chain of Heavy chain antibody) antibody is employed as the antibody, but it is not limited to this. In addition to VHH antibodies, other single domain antibodies capable of binding to the target substance 11 are used as antibodies. Single domain antibodies include the above-mentioned VHH antibodies, epitope recognition site-containing single domain peptide chains of immunoglobulin variable regions, recombinantly expressed using hosts such as E. coli, and broadly defined single domain antibodies. , a target recognition molecule with a molecular weight of about 7-20 kDa, consisting of a polypeptide forming a helix-loop-helix. Such target recognition molecules are known as so-called microantibodies. The sizes (diameters) of the target substance 11, the dielectric particles 12a and the VHH antibody are approximately 100 nm, approximately 300 nm and approximately 5 nm, respectively.

After the sample 10 shown in FIG. 5(a) is allowed to stand at a predetermined temperature for a predetermined time, the target substance 11 and the unbound particles 12 are bound by an antigen-antibody reaction as shown in FIG. 5(b). Composite particles 13 are thus formed. At this time, the size of the composite particles 13 is about 700 nanometers.

In general, when detecting a target substance 11 whose existing amount is unknown, an excess amount of unbound particles 12 is added to the estimated amount of the target substance 11, and the complex particles 13 are added to almost all the target substances 11. form. Since the amount of the composite particles 13 correlates with the amount of the target substance 11 , the target substance 11 can be detected indirectly by detecting the composite particles 13 . Here, by adding an excessive amount of unbound particles 12, as shown in FIG. .

Note that the configuration of the composite particles 13 shown in FIG. 5(b) is an example, and is not limited to this. For example, the number of dielectric particles 12a included in composite particle 13 may be one, or may be three or more. Further, for example, the number of target substances 11 contained in the composite particle 13 may be two or more.

Returning to the description of the flowchart of FIG. 4, next, in the detection method, the complex particles 13 and the unbound particles 12 are separated by dielectrophoresis in a liquid (external liquid of the sample 10) (S120). Specifically, the power supply 120 is operated to apply an alternating voltage to the electrode set 1111 to generate a non-uniform electric field within the sample 10 on the first substrate 111 . As a result, dielectrophoresis acts on the composite particles 13 and the unbound particles 12, and each of the composite particles 13 and the unbound particles 12 moves. The contaminants 14 are also separated in the same manner, but in this separation, it is necessary to separate the substance to be detected from the others. That is, the composite particles 13 are separated from the unbound particles 12 and the contaminants 14, and the unbound particles 12 and the contaminants 14 need not be separated. The aggregated unbound particles 12 are decomposed into a plurality of independent unbound particles 12 by dielectrophoresis.

In order to separate the complex particles 13 and the unbound particles 12 by dielectrophoresis as described above, the frequency of the AC voltage applied to the electrode set 1111 is set to a predetermined frequency. Dielectrophoresis in different directions can be applied to the composite particles 13, the unbound particles 12, and the contaminants 14 by applying an AC voltage of a predetermined frequency. For example, a predetermined frequency at which negative dielectrophoresis (nDEP) acts on the composite particles 13 and positive dielectrophoresis (pDEP) acts on the unbound particles 12 and the contaminants 14 is the frequency of the AC voltage. Assume it is set. In this case, the composite particles 13 move to the second electric field region B where the electric field strength is relatively low, and the unbound particles 12 and the contaminants 14 move to the first electric field region A where the electric field strength is relatively high. . As a result, the composite particles 13 are positionally separated from the unbound particles 12 and contaminants 14 .

Here, the predetermined frequency of the AC voltage will be explained with reference to FIG. FIG. 6 is a graph showing the frequency of AC voltage in the embodiment. In the graph shown in FIG. 6 , the vertical axis represents the Real-part of Clausius-Mossotti factor and the horizontal axis represents the frequency of the AC voltage applied to the electrode set 1111 .

If the real part of the Clausius-Mossotti coefficient is positive, the particles will experience positive dielectrophoresis and move to areas of higher electric field strength. Conversely, if the real part of the Clausius-Mossotti coefficient is negative, the particles will experience negative dielectrophoresis and migrate to regions of lower electric field strength.

As shown in FIG. 6, the real part of the Clausius-Mossotti coefficient depends on particle size and frequency. When the frequency of the alternating voltage is "F", the real part of the Clausius-Mossotti coefficient is negative for particles of 700 nm, corresponding to the size of composite particles 13, and for particles of 300 nm, corresponding to unbonded particles 12, The real part of the Clausius-Mossotti coefficient is positive. Therefore, by setting the frequency "F" as a predetermined frequency of the AC voltage, negative dielectrophoresis can be applied to the composite particles 13 and positive dielectrophoresis can be applied to the unbound particles 12. can.

Returning to the description of the flowchart of FIG. 4, finally, in the detection method, the target substance 11 contained in the separated composite particles 13 is detected (S130). For example, the imaging device 140 images the second electric field region B and outputs an image including the composite particles 13 . The detection unit 150 performs appropriate image analysis processing on the output image to detect the composite particles 13 . As described above, the detection method can detect the target substance 11 contained in the composite particle 13 .

Setting of the predetermined frequency described above will be described in more detail below with reference to FIGS. 7A to 8. FIG. 7A is a schematic diagram showing a particle that has migrated to the first electric field region A. FIG. Also, FIG. 7B is a schematic diagram showing particles that have migrated to the first electric field region A and the second electric field region B. As shown in FIG. Also, FIG. 7C is a schematic diagram showing particles that have migrated to the second electric field region B. As shown in FIG. 7A to 7C show plan views of the electrode set 1111 viewed from the same direction as in FIG. The particles 15 here are general particles that move under the action of dielectrophoresis, and can correspond to any of the composite particles 13, unbound particles 12, and contaminants 14 described above.

As shown in FIG. 7A, when an alternating voltage is applied to the electrode set 1111 at a frequency at which the real part of the Clausius-Mossotti coefficient of the particles 15 becomes positive, positive dielectrophoresis causes the particles 15 to move to the first electric field Move to area A.

In addition, as shown in FIG. 7B, when an AC voltage having a frequency at which the real part of the Clausius-Mossotti coefficient of the particle 15 is near 0 is applied to the electrode set 1111, the particle 15 is moved to the first Move to the electric field region A and the second electric field region B. This occurs because each of the plurality of particles 15 exhibits slightly different properties, resulting in a state in which the particles 15 moving by positive dielectrophoresis and the particles 15 moving by negative dielectrophoresis are mixed.

In addition, as shown in FIG. 7C, when an alternating voltage with a frequency at which the real part of the Clausius-Mossotti coefficient of the particles 15 becomes a negative value is applied to the electrode set 1111, the particles 15 move to the second 2 Move to the electric field region B. Thus, the particles 15 reverse the direction of dielectrophoresis exerted from positive dielectrophoresis to negative dielectrophoresis for each frequency of the alternating voltage applied to the electrode set 1111 . The frequency of the alternating voltage at which the direction of dielectrophoresis is reversed (hereinafter also referred to as crossover frequency) varies depending on the type of particles 15 . When the state shown in FIG. 7B continues over a wide frequency band (in other words, the crossover frequency has a bandwidth), the minimum frequency within this bandwidth that causes the state shown in FIG. 7B is defined as the crossover frequency.

FIG. 8 is a graph showing the crossover frequency for each type of particles 15 in the embodiment. In the graph shown in FIG. 8 , the vertical axis indicates the positive/negative of the real part of the Clausius-Mossotti coefficient, and the horizontal axis indicates the frequency of the AC voltage applied to the electrode set 1111 . In the graph shown in FIG. 8, the vertical axis indicates only the positive or negative value of the real part of the Clausius-Mossotti coefficient. only one of Further, in the graph shown in FIG. 8, (i) dielectric particles alone, (ii) VHH antibody-modified dielectric particles, and (iii) VHH antibody-modified dielectric particles + target substance (that is, composite particles) are shown. shows the results.

As shown in FIG. 8, for all particles (i) to (iii), crossover frequencies were confirmed within the frequency range of 100 kHz to 500 kHz, and as the frequency increased, positive dielectrophoresis shifted to negative dielectrophoresis. It turns out that it is reversed. Also, the crossover frequency differs for each type of particle.

Here, since there is a difference of 300 kHz between (ii) the crossover frequency of the VHH antibody-modified dielectric particles and (iii) the crossover frequency of the VHH antibody-modified dielectric particles + target substance, one of the crossover frequencies is positive dielectrophoresis in the first electric field. It is possible to set a predetermined frequency at which one migrates to region A and the other to the second electric field region B due to negative dielectrophoresis. That is, in order to separate (ii) the VHH antibody-modified dielectric particles and (iii) the VHH antibody-modified dielectric particles + target substance, an AC voltage with a frequency of 100 kHz to 400 kHz may be applied to the electrode set 1111. . Furthermore, even when the crossover frequency has a bandwidth, it is sufficient to select a predetermined frequency from the frequency range of 150 kHz to 350 kHz. can also be selected.

Thus, in the separation of the two types of particles 15 by dielectrophoresis, the first crossover frequency, which is the crossover frequency of one of the two types of particles 15, is greater than the crossover frequency of the other particle 15. An alternating voltage having a predetermined frequency less than the second crossover frequency is applied to the electrode set 1111 . That is, a frequency between the first crossover frequency of one particle 15 and the second crossover frequency of the other particle 15 is selected as the predetermined frequency.

As described above, the predetermined frequency of the AC voltage applied to the electrode set 1111 is appropriately set in consideration of the crossover frequency. At this time, it is necessary that the two or more types of particles 15 to be separated have mutually different crossover frequencies.

[Detection preparation method and detection preparation system]
As described above, when detecting the target substance 11 in the detection method and detection system 100 , an alternating voltage of a predetermined frequency is applied to the electrode set 1111 . As long as the same target substance 11 is detected, there is basically no need to change this predetermined frequency once it is set. However, in the following situations, it may be necessary to calibrate the predetermined frequency.

That is, the dielectric particles 12a, the electrode set 1111, and the like used in the detection method and detection system 100 are consumables because they may become contaminated during detection. Therefore, it is necessary to replace the consumables such as the dielectric particles 12a and the electrode set 1111 with new ones. When the consumables are replaced with new ones in this way, the behavior of the particles 15 due to dielectrophoresis changes not a little if the predetermined frequency before the replacement is used, and the particles 15, particularly the composite particles 13, are separated with high accuracy. In other words, the detection accuracy of the target substance 11 may be lowered.

Therefore, for example, every time consumables are replaced with new ones, the predetermined frequency of the AC voltage used in the detection method and detection system 100 is calibrated by the detection preparation method and detection preparation system 200 (see FIG. 9) described below. Therefore, it is possible to improve the detection accuracy of the target substance 11 .

First, the configuration of the detection preparation system 200 according to the embodiment will be described with reference to FIG. FIG. 9 is a block diagram and cross-sectional view showing a schematic configuration of the detection preparation system 200 according to the embodiment. The detection preparation system 200 includes a container 210, a power supply 220, an excitation light source 230, an imaging element 240, and a search section 250, as shown in FIG. In an embodiment, detection preparation system 200 further comprises filter 241 and mirror 242 . Note that the detection preparation system 200 does not have to include the filter 241 and the mirror 242 . Although not shown in FIG. 9, if the amount of sunlight or outside light is not sufficient when observing an image captured by the imaging element 240, the detection preparation system 200 may further include a transmission light source. .

Here, the container 210, the power supply 220, and the imaging element 240 in the detection preparation system 200 have the same configurations as the separator 110, the power supply 120, and the imaging element 140 in the detection system 100, respectively. Therefore, descriptions of the container 210, the power source 220, and the imaging element 240 are omitted here.

However, in the detection preparation system 200 , the container 210 accommodates the particle group 20 as shown in FIG. 10 instead of the sample 10 . FIG. 10 is a diagram showing an example of particle group 20 in the embodiment. The particle group 20 includes composite particles 23 and second dielectric particles 22 . The composite particles 23 are obtained by binding the target substance 11 or the mimic substance 24 simulating the target substance 11 and the first dielectric particles 21 modified with a substance that specifically binds to the target substance 11 or the mimic substance 24. particles. The second dielectric particles 22 are unbound particles that are not bound to either the target substance 11 or the mimetic substance 24, and are particles that behave differently from the composite particles 23 by dielectrophoresis. That is, the composite particles 23 and the second dielectric particles 22 can be separated by applying appropriate dielectrophoresis. The first dielectric particles 21 and the second dielectric particles 22 may be the same dielectric particles or different dielectric particles.

Here, the simulant substance 24 is a substance different from the target substance 11, but when it binds to the first dielectric particles 21, the same dielectrophoresis effect as when the target substance 11 binds to the first dielectric particles 21 is achieved. It is a substance that behaves according to That is, the composite particles 23 exhibit the same dielectrophoretic behavior regardless of whether the target substance 11 is bound or the mimetic substance 24 is bound. Here, "same behavior" means that the behavior is completely the same, and that the behavior is slightly different but within an allowable error range.

In addition, one of the composite particles 23 and the second dielectric particles 22 has a label that can visually distinguish the two in the image captured by the imaging device 240 . Here, "both can be visually distinguished" means that when both the composite particles 23 and the second dielectric particles 22 are observed, the composite particles 23 and the second dielectric particles 22 can be distinguished. means that it is possible Since both the composite particles 23 and the second dielectric particles 22 have nano-order particle sizes, the "observation" referred to here is not observation with the naked eye, but the composite particles 23 and the second dielectric particles. 22 are both magnified to the extent that they can be observed with the naked eye. In addition, the term “visually distinguishable between the two in the image” used herein means that an appropriate image analysis process is performed on an image obtained by capturing both the composite particles 23 and the second dielectric particles 22. , means that the composite particles 23 and the second dielectric particles 22 can be distinguished from each other by, for example, the shading of pixels.

In embodiments, the label includes a fluorescent substance 25 . In the example shown in FIG. 10, the second dielectric particles 22 have fluorescent material 25 embedded therein. Thereby, the second dielectric particles 22 have the fluorescent substance 25 as a label. The fluorescent substance 25 may be immobilized on the second dielectric particles 22 by modifying the surface of the second dielectric particles 22 by chemical bonding. First dielectric particle 21 does not have fluorescent substance 25 as a label, and therefore composite particle 23 does not have fluorescent substance 25 as a label either.

Here, the fluorescent substance 25 emits fluorescent light upon receiving predetermined light 231 from an excitation light source 230, which will be described later. In other words, the second dielectric particles 22 containing the fluorescent material 25 emit fluorescent light when the fluorescent material 25 is excited by receiving the predetermined light 231 from the excitation light source 230 . On the other hand, the composite particles 23 that do not contain the fluorescent substance 25 do not emit fluorescence even if they receive the predetermined light 231 from the excitation light source 230 . Therefore, in the image captured by the imaging element 240, the complex particles 23 and the second dielectric particles 22 can be visually distinguished from each other by the presence or absence of fluorescence emission.

The excitation light source 230 is a light source that emits predetermined light 231 having a wavelength (for example, five hundred and several tens of nanometers) that excites the fluorescent material 25 . In an embodiment, excitation light source 230 is the same as light source 130 in detection system 100 . That is, the excitation light source 230 may also be used as the light source 130 .

Here, the predetermined light 231 irradiated to the particle group 20 from the excitation light source 230 is the same as the irradiation light 131 irradiated to the sample 10 from the light source 130, but depending on the predetermined light 231 from the particle group 20 The detected light 232 is different from the detected light 132 corresponding to the irradiation light 131 from the sample 10 . That is, in the detection light 132 from the sample 10, when the dielectric particles 12a contain a fluorescent substance, fluorescence emission from all the dielectric particles 12a regardless of the types of the composite particles 13 and the unbound particles 12 will be included. On the other hand, in the detection light 232 from the particle group 20, since the composite particles 23 do not emit fluorescence, fluorescence emitted only from the second dielectric particles 22 is included.

The excitation light source 230 may be used only in the detection preparation method and detection preparation system 200, and is preferably detachable so that it can be used when not used in the detection method and detection system 100. FIG. Further, the excitation light source 230 may be a light source composed of a solid light emitting device such as an LED (Light Emitting Diode), in addition to a laser such as a semiconductor laser and a gas laser.

The filter 241 is provided between the container 210 and the imaging device 240 and blocks the predetermined light 231 emitted by the excitation light source 230 . Specifically, the filter 241 is a long-pass filter that blocks the predetermined light 231 and allows the detection light 232 to pass.

The mirror 242 is provided between the container 210 and the filter 241 and is a dichroic mirror that reflects the scattered light and allows the predetermined light 231 and the detection light 232 to pass through.

The searching unit 250 detects the composite particles 23 and the second dielectric particles 22 by dielectrophoresis based on the image captured by the imaging device 240 while changing the frequency of the AC voltage applied to the electrodes (electrode set 1111). Search for separable predetermined frequencies. The search process for the predetermined frequency by the search unit 250 will be described in detail later in [Detection preparation method using detection preparation system].

The searching unit 250 is realized by executing a program for searching for the predetermined frequency using a circuit such as a processor and a storage device such as a memory, for example, but is realized by a dedicated circuit. good too. The search unit 250 is built in, for example, a computer.

[Detection preparation method using detection preparation system]
An example of the detection preparation method (operation of the detection preparation system 200) according to the embodiment will be described below with reference to FIG. FIG. 11 is a flow chart showing a detection preparation method according to the embodiment. First, the particle group 20 is produced by steps S210 to S230 described below. That is, by modifying the dielectric particles with the VHH antibody and the fluorescent substance 25, the dielectric particles are provided with the fluorescent substance 25 as a label (S210). The dielectric particles to which this fluorescent substance 25 is applied are the second dielectric particles 22 . As in the example shown in FIG. 10, the fluorescent substance 25 may be applied to the dielectric particles by embedding the fluorescent substance 25 inside the dielectric particles.

Next, by modifying the VHH antibody to a dielectric particle different from the dielectric particles imparting the fluorescent substance 25 with the VHH antibody, and mixing the VHH antibody-modified dielectric particles with the target substance 11 or the mimic substance 24, the composite Body particles 23 are produced (S220). The dielectric particles in this composite particle 23 are the first dielectric particles 21 . Then, the particle group 20 is produced by mixing the second dielectric particles 22 produced in step S210 and the composite particles 23 produced in step S220 (S230).

Next, the solvent containing the particle group 20 is accommodated in the space 1121 of the container 210, and the power source 220 is operated to apply an AC voltage to the electrode set 1111 (S240). As a result, a nonuniform electric field is generated in the solvent on the first substrate 111, dielectrophoresis acts on the composite particles 23 and the second dielectric particles 22 included in the particle group 20, and the composite particles 23 and Each second dielectric particle 22 moves. An image including the particle group 20 is output by capturing an image of the solvent with the image sensor 240 while applying an AC voltage to the electrode set 1111 (S240).

Then, the search unit 250 searches for a predetermined frequency based on the image captured by the imaging element 240 while changing the frequency of the AC voltage (S260). A search process for a predetermined frequency by the search unit 250 will be described below with reference to FIGS. 12 and 13. FIG. FIG. 12 is an explanatory diagram of a predetermined frequency search process in the embodiment. FIG. 13 is a schematic diagram showing a state in which the particle group 20 has moved to the first electric field region A and the second electric field region B according to the embodiment.

Here, the composite particles 23 do not have a label (here, the fluorescent substance 25), whereas the second dielectric particles 22 have a label. Therefore, in the image captured by the imaging device 240, the composite particles 23 are displayed as black dots, while the second dielectric particles 22 that emit fluorescence due to the predetermined light 231 from the excitation light source 230 are displayed as bright dots. be done. Therefore, the search unit 250 performs appropriate image analysis processing on the image captured by the imaging element 240 to detect black points (composite particles 23) and bright points (second dielectric particles 22) in the image, respectively. It is possible to count.

The search unit 250 detects the crossover frequency of the second dielectric particles 22 and the crossover frequency of the composite particles 23 while changing the frequency of the AC voltage applied to the electrode set 1111 from a relatively high frequency to a low frequency. Explore. In the graph shown in FIG. 12 , the vertical axis represents the real part of the Clausius-Mossotti coefficient and the horizontal axis represents the frequency of the AC voltage applied to the electrode set 1111 . In FIG. 12, curve L1 represents the dielectrophoretic properties acting on the composite particles 23, and curve L2 represents the dielectrophoretic properties acting on the second dielectric particles 22. As shown in FIG.

The searching unit 250 first searches for the second crossover frequency f2, which is the frequency of the AC voltage at which positive dielectrophoresis and negative dielectrophoresis acting on the second dielectric particles 22 are switched. Specifically, the search unit 250 determines that the ratio of the number of the second dielectric particles 22 in the second electric field region B to the total number of the second dielectric particles 22 exceeds a threshold value (for example, 80% or more). Therefore, the frequency at which the ratio of the number of the second dielectric particles 22 in the first electric field region A to the total number of the second dielectric particles 22 exceeds the threshold value is searched for as the second crossover frequency f2.

Next, the searching unit 250 searches for the first crossover frequency f1, which is the frequency of the AC voltage at which positive dielectrophoresis and negative dielectrophoresis acting on the composite particles 23 are switched. Specifically, the searching unit 250 shifts from a state in which the ratio of the number of the composite particles 23 in the second electric field region B to the total number of the composite particles 23 exceeds the threshold to the total number of the composite particles 23 The frequency of the AC voltage at which the ratio of the number of composite particles 23 in the first electric field region A switches to a state exceeding the threshold value is searched for as the first crossover frequency f1.

The searching unit 250 then searches for frequencies between the first crossover frequency f1 and the second crossover frequency f2 as predetermined frequencies. In the example shown in FIG. 12, the searching unit 250 searches for the average value of the first crossover frequency f1 and the second crossover frequency f2 as the predetermined frequency f0. This predetermined frequency f0 is used in the detection method and detection system 100 described above.

FIG. 13 shows an example of an image of the solvent captured by the imaging device 240 in a state in which an AC voltage with a predetermined frequency f0 is applied to the electrode set 1111. FIG. As shown in FIG. 13, in this state, the composite particles 23 move to the second electric field region B due to the action of negative dielectrophoresis. On the other hand, the second dielectric particles 22 move to the first electric field region A due to the action of positive dielectrophoresis.

In FIG. 13, even if positive dielectrophoresis is acting, there are second dielectric particles 22 that cannot move to the first electric field region A and stay in the second electric field region B. Particles may behave differently than dielectrophoresis works. Even when such irregular particles are present, the search unit 250 compares the ratio of the number of particles exhibiting correct behavior by dielectrophoresis as described above with the threshold, thereby determining the crossover frequency of the particles. It is possible to explore.

[Effects, etc.]
As described above, the detection preparation method according to the embodiment includes the target substance 11 or the mimetic substance 24 that simulates the target substance 11, and the first Particle groups 20 having composite particles 23 bonded to dielectric particles 21 and second dielectric particles 22 having dielectrophoretic behavior different from the composite particles 23 are prepared, and an electric field is applied to a solvent containing the particle groups 20. An alternating voltage is applied to the electrode (electrode set 1111) for application, the solvent is imaged by the imaging element 240, and the composite particles 23 and This is a method of searching for a predetermined frequency that can be separated from the second dielectric particles 22 by dielectrophoresis. Either one of the composite particles 23 and the second dielectric particles 22 has a label that can visually distinguish the two in the image.

According to this, in the image captured by the imaging device 240, the behavior of each of the composite particles 23 and the second dielectric particles 22 that differ from each other due to dielectrophoresis can be visually distinguished. It is easy to search for a given frequency. As a result, there is an advantage that the predetermined frequency used in the detection method and detection system 100 can be calibrated with high accuracy, and the detection accuracy of the target substance can be easily improved.

In addition, in the detection preparation method according to the embodiment, even if the target substance 11, such as a virus, which can be collected in a relatively small amount, is not used, a predetermined frequency can be searched by using the simulated substance 24. , the target substance 11 used in the detection method and detection system 100 can be easily secured. Furthermore, in the detection preparation method according to the embodiment, if a substance harmless to the human body, such as protein, is used as the simulant substance 24, the process of searching for a predetermined frequency is compared with the case where the target substance 11 such as a virus is used. There is also the advantage that the safety of the system can be improved and the cost can be reduced.

Moreover, in the detection preparation method according to the embodiment, the label includes the fluorescent substance 25 .

According to this, there is an advantage that the particles containing the fluorescent substance 25 and the particles not containing the fluorescent substance 25 can be easily visually distinguished from each other by the presence or absence of fluorescence emission.

Further, in the detection preparation method according to the embodiment, when the solvent is imaged by the imaging element 240, the excitation light source 230 that emits the predetermined light 231 having a wavelength that excites the fluorescent substance 25 emits a predetermined amount of light to the solvent. Light 231 is applied.

According to this, since the particles containing the fluorescent substance 25 can be caused to emit fluorescent light, there is an advantage that the fluorescent light emission of the particles containing the fluorescent substance 25 can be easily imaged by the imaging element 240 .

Moreover, in the detection preparation method according to the embodiment, the imaging device 240 images the solvent through the filter 241 that blocks the predetermined light 231 .

According to this, by blocking the predetermined light 231, there is an advantage that it becomes easier for the imaging element 240 to capture the fluorescence emission of the particles containing the fluorescent substance 25. FIG.

Further, in the detection preparation method according to the embodiment, the first crossover frequency f1, which is the frequency of the alternating voltage at which positive dielectrophoresis and negative dielectrophoresis acting on the composite particles 23 are switched, and the second dielectric A frequency between the second crossover frequency f2, which is the frequency of the AC voltage at which positive dielectrophoresis and negative dielectrophoresis acting on the particles 22 are switched, is searched for as the predetermined frequency f0.

According to this, by searching for the crossover frequency of each of the composite particles 23 and the second dielectric particles 22, which can be visually distinguished by the presence or absence of the label, the predetermined frequency f0 can be easily searched. There is an advantage.

Moreover, the detection preparation system 200 according to the embodiment includes a container 210 , an imaging element 240 and a searching section 250 . The container 210 is a complex in which the target substance 11 or the mimetic substance 24 simulating the target substance 11 and the first dielectric particles 21 modified with a substance that specifically binds to the target substance 11 or the mimetic substance 24 are combined. A solvent containing a particle group 20 having particles 23 and second dielectric particles 22 that behave differently from the composite particles 23 by dielectrophoresis is contained. The imaging element 240 images the solvent contained in the container 210 . The searching unit 250 detects the composite particles 23 and the second dielectric based on the image captured by the imaging element 240 while changing the frequency of the AC voltage applied to the electrodes (electrode set 1111) for applying an electric field to the solvent. A predetermined frequency that can be separated from the particles 22 by dielectrophoresis is sought. Either one of the composite particles 23 and the second dielectric particles 22 has a label that can visually distinguish the two in the image.

According to this, the same effect as the detection preparation method according to the embodiment can be obtained.

Moreover, in the detection preparation system 200 according to the embodiment, the label contains the fluorescent substance 25 . The detection preparation system 200 further includes an excitation light source 230 that irradiates the solvent with a predetermined light 231 having a wavelength that excites the fluorescent substance 25 .

According to this, since the particles containing the fluorescent substance 25 can be caused to emit fluorescent light, there is an advantage that the fluorescent light emission of the particles containing the fluorescent substance 25 can be easily imaged by the imaging element 240 .

Moreover, the particle group 20 according to the embodiment includes first dielectric particles 21 and second dielectric particles 22 . The first dielectric particles 21 are modified with a substance that specifically binds to the target substance 11 or a mimetic substance 24 simulating the target substance 11 . The behavior of the second dielectric particles 22 by dielectrophoresis differs from that of the composite particles 23 in which the target substance 11 or the simulated substance 24 is bound to the first dielectric particles 21 . Either one of the composite particles 23 and the second dielectric particles 22 has a label that allows the two to be visually distinguished in the image captured by the imaging element 240 .

According to this, by executing the detection preparation method using the particle group 20, the same effect as the detection preparation method according to the embodiment can be obtained. In this particle group 20, the first dielectric particles 21 are not bound to the target substance 11 or the simulated substance 24, but are bound to the target substance 11 or the simulated substance 24 to form the composite particles 23. It may be in a state where

In addition, in particle group 20 according to the embodiment, the label includes fluorescent substance 25 .

According to this, there is an advantage that the particles containing the fluorescent substance 25 and the particles not containing the fluorescent substance 25 can be easily visually distinguished from each other by the presence or absence of fluorescence emission.

In addition, the method for producing the particle group 20 according to the embodiment includes dielectric particles modified with a substance that specifically binds to the target substance 11 or the mimetic substance 24 simulating the target substance 11, and the target substance 11 or the mimetic substance 24. By mixing the composite particles 23 including the first dielectric particles 21 in which the target substance 11 or the mimetic substance 24 is bound to the dielectric particles, and the composite particles 23, the behavior by dielectrophoresis is different from the second dielectric a method of making a body particle 22; Either one of the composite particles 23 and the second dielectric particles 22 has a label that allows the two to be visually distinguished in the image captured by the imaging element 240 .

According to this, by executing the detection preparation method using the produced particle group 20, the same effect as the detection preparation method according to the embodiment can be obtained.

In addition, in the method for producing the particle group 20 according to the embodiment, the fluorescent substance 25 is immobilized on either the first dielectric particles 21 or the second dielectric particles 22 by chemical bonding, or A label is imparted by embedding a fluorescent substance 25 inside the particle.

According to this, there is an advantage that the particles containing the fluorescent substance 25 and the particles not containing the fluorescent substance 25 can be easily visually distinguished from each other by the presence or absence of fluorescence emission.

(Modification)
Although the detection preparation system and the detection preparation method according to one or more aspects of the present disclosure have been described above based on the embodiments, the present disclosure is not limited to these embodiments. Various modifications conceived by those skilled in the art may be included within the scope of one or more aspects of the present disclosure as long as they do not depart from the spirit of the present disclosure.

For example, in the above embodiments, 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. 14 is a first plan view showing the configuration of an electrode set 2111 according to a modification. For example, an electrode set 2111 may be placed on the first substrate 111, as shown in FIG. In this electrode set 2111, 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 (vertical direction in FIG. 14). Even such an electrode set 2111 can generate a non-uniform electric field when an AC voltage is applied.

Also, the number of electrodes included in the electrode set is not limited to two, and may be three or more. FIG. 15 is a second plan view showing the configuration of the electrode set 3111 according to the modification. For example, an electrode set 3111 may be placed on the first substrate 111, as shown in FIG. This electrode set 3111 includes three or more electrodes, and alternating voltages applied to adjacent electrodes are provided with a phase difference. Electrode set 3111 may be referred to as Castellated electrodes.

Further, although the composite particle 13 is illustrated in FIG. 5 in the above embodiment, the structure of the composite particle is not limited to this. FIG. 16 is a diagram showing a formation process of composite particles 28 according to a modification. For example, in the above embodiment, an example in which the second dielectric particles 22 contain the fluorescent substance 25 has been described, but as shown in FIG. may be

As shown in (a) of FIG. 16 , antibody-modified dielectric particles 26 and antibody-modified fluorescent particles 27 are introduced into a sample containing target substance 11 or mimetic substance 24 . Antibody-modified dielectric particles 26 are obtained by modifying dielectric particles 26a of 500 nm to 1000 nm with antibodies 26b of about 5 nm. Antibody-modified fluorescent particles 27 are obtained by modifying fluorescent particles (fluorescent substance) 27a of about 300 nm with antibodies 27b of about 5 nm. Note that the size of each particle and antibody is not limited to the size described above.

Polystyrene particles can be used as the dielectric particles 26a, but are not limited to this. In addition, as the antibodies 26b and 27b, VHH antibodies can be used, but they are not limited to this. Also, the antibody 26b and the antibody 27b may be different.

When the sample shown in (a) of FIG. 16 is allowed to stand at a predetermined temperature for a predetermined period of time, as shown in (b) of FIG. Composite particles 28 are formed by combining particles 26 and antibody-modified fluorescent particles 27 . At this time, the size of the composite particles 28 is 900 nm to 1400 nm. The antibody-modified dielectric particles 26 that have not bound to the target substance 11 or mimetic substance 24 remain alone or in an aggregated state. In this case, the antibody-modified dielectric particles 26 contained in the composite particles 28 correspond to the first dielectric particles having fluorescent particles (fluorescent substances) 27a as labels, and the antibody-modified dielectric particles 26 that are unbound particles corresponds to the second dielectric particles.

Thus, by making the dielectric particles 26a larger than the fluorescent particles 27a, the difference between the size of the composite particles 28 and the size of the antibody-modified dielectric particles 26, which are unbound particles, can be increased. Separation of the composite particles 28 and the antibody-modified dielectric particles 26 by electrophoresis can be performed more reliably.

In the embodiment, one of the composite particles 23 and the second dielectric particles 22 contains the fluorescent substance 25 as a visually distinguishable label in the image captured by the imaging device 240, but this is not the only option. can't For example, the label may be that the color of one of the particles is different from that of the other particles, or that the shape of one of the particles is different from that of the other particle.

INDUSTRIAL APPLICABILITY The present disclosure can be used as a detection preparation method or the like for making preparations before detecting a target substance such as an influenza virus.

10 sample 11 target substance 12 unbound particles 12a, 26a dielectric particles 12b specific binding substances 13, 23, 28 composite particles 14 contaminants 15 particles 20 particle group 21 first dielectric particles 22 second dielectric particles 21b, 22b, 26b, 27b antibody 24 mimetic substance 25 fluorescent substance 26 antibody-modified dielectric particle 27 antibody-modified fluorescent particle 27a fluorescent particle 100 detection system 110 separator 111 first substrate 112 spacer 113 second substrate 120 power source 130 light source 131 irradiation light 132 Detection light 140 Imaging device 141 Camera 150 Detecting unit 200 Detection preparation system 210 Container 220 Power supply 230 Excitation light source 231 Predetermined light 232 Detection light 240 Imaging device 241 Filter 242 Mirror 250 Searching unit 1111, 2111, 3111 Electrode set 1112 First electrode 1112a First base 1112b First protrusion 1112c First recess 1113 Second electrode 1113a Second base 1113b Second protrusion 1113c Second recess 1121 Space 1131 Supply hole 1132 Discharge hole A First electric field region B Second electric field region

Claims (11)

A composite particle in which a target substance or a mimetic substance simulating the target substance is bound to first dielectric particles modified with a substance that specifically binds to the target substance or the mimetic substance, and the composite particle Prepare a particle group having second dielectric particles that behave differently from dielectrophoresis,
applying an alternating voltage to an electrode for applying an electric field to the solvent containing the particle group;
Imaging the solvent with an imaging device,
While changing the frequency of the AC voltage, searching for a predetermined frequency at which the composite particles and the second dielectric particles can be separated by dielectrophoresis based on the image captured by the imaging device;
Either one of the composite particles and the second dielectric particles has a label that allows the two to be visually distinguished in the image,
Detection preparation method. the label comprises a fluorescent substance;
The detection preparation method according to claim 1 . When an image of the solvent is captured by the imaging device, the solvent is irradiated with the predetermined light from an excitation light source that emits predetermined light having a wavelength that excites the fluorescent substance.
The detection preparation method according to claim 2. The imaging element images the solvent through a filter that blocks the predetermined light.
The detection preparation method according to claim 3. A first crossover frequency, which is the frequency of the alternating voltage at which positive dielectrophoresis and negative dielectrophoresis acting on the composite particles are switched, and the positive dielectrophoresis acting on the second dielectric particles and a second crossover frequency, which is the frequency of the alternating voltage at which the negative dielectrophoresis switches, as the predetermined frequency.
The detection preparation method according to any one of claims 1 to 4. A composite particle in which a target substance or a mimetic substance simulating the target substance is bound to first dielectric particles modified with a substance that specifically binds to the target substance or the mimetic substance, and the composite particle a container containing a solvent containing a particle group having second dielectric particles that behave differently from dielectrophoretic behavior;
an imaging device that images the solvent contained in the container;
The composite particles and the second dielectric particles can be separated by dielectrophoresis based on the image captured by the imaging element while changing the frequency of the alternating voltage applied to the electrode for applying an electric field to the solvent. and a search unit that searches for a predetermined frequency,
Either one of the composite particles and the second dielectric particles has a label that allows the two to be visually distinguished in the image,
Detection readiness system. the label comprises a fluorescent substance,
further comprising an excitation light source for irradiating the solvent with a predetermined light having a wavelength that excites the fluorescent substance;
7. A detection preparation system according to claim 6. first dielectric particles modified with a substance that specifically binds to a target substance or a mimetic substance simulating the target substance;
Composite particles in which the target substance or the mimetic substance is bound to the first dielectric particles and second dielectric particles that behave differently by dielectrophoresis,
Either one of the composite particles and the second dielectric particles has a label that allows the two to be visually distinguished in an image captured by an imaging device.
particle group. the label comprises a fluorescent substance;
The particle group according to claim 8. Dielectric particles modified with a substance that specifically binds to a target substance or a mimetic substance simulating the target substance are mixed with the target substance or the mimetic substance, so that the target substance or the mimetic substance is mixed with the dielectric particles. Preparing composite particles containing first dielectric particles bound with a simulated substance and second dielectric particles different in behavior due to dielectrophoresis from the composite particles,
Either one of the composite particles and the second dielectric particles has a label that allows the two to be visually distinguished in an image captured by an imaging device.
A method for producing a group of particles. With respect to either one of the first dielectric particles and the second dielectric particles, the label is attached by immobilizing a fluorescent substance on the particles by chemical bonding or by embedding the fluorescent substance inside the particles. Give,
A method for producing a particle group according to claim 10 .

Images