JP5720129B2 - Particle fixing structure, particle analysis apparatus, and analysis method - Google Patents

Description

  The present invention relates to a particle fixing structure for fixing analyte particles, and an analyzer and an analysis method for analyte particles.

  In recent years, a technique has been proposed and created that enables individual analysis and observation of the properties and structure of each particle by regularly and regularly arranging fine particles such as biological samples (eg, cells) on a substrate. Applications in various fields such as medicine, treatment, testing and analysis are expected. In Patent Document 1, a cell suspension is added onto a substrate having micropores (microwells) arranged in an array, and after waiting for the cells to settle in the well, the cells remaining outside the well are removed. A method of fixing cells one by one in each well by repeatedly performing a process of washing away is disclosed. In Patent Document 2, a suspension containing fine particles is introduced into a space between an upper electrode and a lower electrode having an insulator layer in which a large number of through-holes are formed, and an AC voltage is applied between both electrodes. A method of introducing and fixing fine particles into a through-hole by dielectrophoretic force when applied is disclosed. If a substrate on which a large number of fine particles are individually fixed can be produced by a method as described in Patent Documents 1 and 2, for example, the analysis of individual fine particles can be easily and collectively performed by observing fluorescence caused by excitation light irradiation. Can be done. As an example, Patent Document 1 fixes fluorescently labeled lymphocytes one by one in a well and observes changes in fluorescence intensity before and after stimulation with an antigen at a very low frequency in a population. Methods for identifying existing antigen-specific lymphocytes have been proposed.

  Patent Document 3 includes a substrate capable of introducing a hydroxyl group on the surface thereof, a metal-based film provided with a plurality of wells reaching the substrate, and a crosslinkable polymer film disposed on the metal-based film. A biochip substrate is disclosed. However, the substrate of Patent Document 3 is for immobilizing nucleic acids and not for immobilizing fine particles such as cells.

Japanese Patent No. 3723882 JP 2007-296510 A JP 2007-78631 A

  When performing fluorescence detection of a large number of fine particles arranged on a substrate as in Patent Documents 1 and 2, the following problems arise. First, the fluorescence generated by the excitation light irradiation includes autofluorescence generated from the substrate itself, in addition to fluorescence (fluorescence signal) generated from the fluorescently labeled fine particles. This affects as background noise and reduces the detection accuracy of the intensity of the fluorescent signal. Further, depending on the configuration of the measurement apparatus and measurement conditions, in addition to fluorescence, light from the outside such as room light is also detected as background light. Second, in the case where the distance between the fine particles is narrow, a decrease in detection accuracy due to leakage light (crosstalk noise) from adjacent fine particles is also a problem. Thirdly, in order to prevent fluorescence fading due to long-time excitation light irradiation, it is also required to shorten the detection time of fluorescence intensity generated from a fluorescently labeled biological sample.

  Therefore, the present invention has been proposed in view of the conventional situation, and its purpose is to reduce background noise, crosstalk noise, etc., and to perform optical observation of a large number of fine particles with high sensitivity and high accuracy. It is to provide a technology that makes it possible to perform.

  In order to achieve the above object, the present invention adopts the following configuration. That is, the particle-fixing structure according to the present invention (hereinafter also simply referred to as “structure”) uses each of the analyte particles in order to detect light emitted by a substance indicating the presence of a component constituting the analyte particles. A structure for fixing particles having a plurality of holding holes for holding, comprising: a flat substrate; and a holding portion disposed on the substrate and formed with the plurality of holding holes, wherein the holding portion is A light shielding film provided on at least the upper surface side of the holding part, and an insulator film provided between the light shielding film and the substrate, wherein the holding hole is provided on the upper surface side of the holding part. The light-shielding film is located at a position where the light-shielding film is opened, and the insulating film is extended to the substrate.

  According to this configuration, by providing the light shielding film in the holding portion, for example, background noise due to autofluorescence of the insulator film itself or cross noise due to leakage light from the adjacent holding hole, etc. Therefore, it is possible to detect only the light emitted by the observation target substance in each holding hole with high sensitivity and high accuracy. And since it can detect with high sensitivity and high accuracy, the effect of shortening the detection time can be expected. In particular, in the structure for fixing particles according to the present invention, since the light shielding film is provided on the upper surface side of the holding part, the autofluorescence of the insulator film positioned below the light blocking film is prevented from leaking above the structure. It becomes easy. In addition, this structure can block light from the outside that is incident on the structure from above and outside the structure, so that the influence of external light in detecting the analyte particles can be reduced. In addition, since at least one insulator film exists below the light shielding film, the effect of further suppressing the autofluorescence of the insulator film is enhanced depending on the material and film thickness of the light shielding film.

  Here, the structure of the present invention further includes a storage unit that stores the suspension containing the analyte particles on the holding unit, and the holding hole is provided so as to communicate with the storage unit. It is preferable. As a result, the analyte particles can be easily introduced into the holding holes. As a method for introducing the analyte particles into the holding hole, natural sedimentation (gravity) or dielectrophoretic force can be used, but a large number of holdings can be performed in a very short time of about several seconds. Since analyte particles can be introduced into the pores, a method utilizing dielectrophoretic force is more preferable.

  In order to cause the dielectrophoretic force to act on the analyte particles, an alternating electric field that concentrates the electric lines of force on the holding hole may be applied in a state in which the container and the holding hole are filled with the suspension. As a configuration for applying such an alternating electric field, for example, a pair of electrodes respectively disposed at positions corresponding to different holding holes are provided on the surface of the substrate on the holding unit side, The structure of extending from the upper surface of the holding portion to the electrode on the substrate can be employed. In addition, a first electrode disposed at a position corresponding to the holding hole is provided on a surface of the substrate on the holding unit side, and the holding hole is formed on the substrate from the upper surface of the holding unit. It is also possible to adopt a configuration in which the second electrode is provided on the side opposite to the first electrode with the holding portion and the accommodating portion interposed therebetween. In either case, it is possible to introduce the analyte particles in the suspension into the holding hole by applying an AC voltage having a predetermined waveform between the two electrodes.

Here, the light shielding film may be provided on the entire upper portion of the holding portion other than the plurality of holding holes, or the opening portion of the holding hole on the upper surface of the holding portion. It may be provided around. In the former case, the light shielding performance by the above-described light shielding film can be suitably exhibited. Even in the latter case, the upper surface of the holding part is divided into a part covered with a light shielding film and a part where the insulator film is exposed, and the difference in hydrophilicity between the light shielding film and the insulator film Using this, it is possible to suitably detect light related to the subject particle. For example, when the light-shielding film has hydrophilicity and the insulator film has hydrophobicity, after introducing the water-soluble liquid into the holding holes, the holding holes are sealed with the water-insoluble liquid, It is possible to hold the aqueous solution only around the holding hole. As a result, for example, the reactivity with respect to a plurality of analyte particles can be examined by fixing to the holding hole.

  The substrate is preferably made of a translucent material, and the electrode provided on the surface of the substrate on the holding part side is preferably ITO. Thereby, the light emitted from the holding hole can be observed through the substrate from the lower side of the substrate. Any material can be used for the light-shielding film as long as it has a light-shielding property, but a metal film may be used as an example.

  The particle analyzing apparatus according to the present invention includes the above-described structure for fixing particles, a power source for applying an alternating voltage for generating a dielectrophoretic force on the electrode, and after fixing a voltage from the power source, And a detector for detecting light emitted by a substance indicating the presence of a component constituting the analyte particle held in the holding hole of the structure.

  The analyte particle analysis method according to the present invention is issued by a substance that fixes the analyte particle in the holding hole on the particle fixing structure described above and indicates the presence of a component constituting the immobilized analyte particle. It is characterized by detecting light. Here, the substance indicating the presence of the component constituting the analyte particle is a labeling substance described later.

  According to the present invention, it is possible to reduce background noise, crosstalk noise, and the like, and to optically observe a large number of fine particles with high sensitivity and high accuracy.

It is a figure for demonstrating the structure which concerns on 1st Embodiment of this invention. It is sectional drawing of the structure of FIG. It is a figure for demonstrating the structure and particle | grain fixing apparatus which concern on 2nd Embodiment of this invention. It is sectional drawing of the structure of FIG. It is a figure for demonstrating the structure and particle | grain fixing apparatus which concern on 3rd Embodiment of this invention. It is a figure for demonstrating the structure and particle | grain fixing apparatus which concern on 4th Embodiment of this invention. It is sectional drawing of the structure of FIG. It is a 1st figure for demonstrating the fixing method of the particle | grains by dielectrophoresis. It is a 2nd figure for demonstrating the fixing method of the particle | grains by dielectrophoresis. It is a figure for demonstrating the analysis method of particle | grains. It is a figure which shows the example which applied the apparatus of this invention to the detection apparatus of the abnormal cell. FIG. 6 is a first diagram for explaining a method for manufacturing the structure according to the first embodiment. FIG. 10 is a second diagram for illustrating the method for manufacturing the structure according to the first embodiment. FIG. 10 is a first diagram for explaining a method for manufacturing the structure according to the second embodiment. FIG. 10 is a second diagram for explaining the structure manufacturing method according to the second embodiment. FIG. 10 is a third diagram for explaining the structure manufacturing method according to the second embodiment.

  Exemplary embodiments of the present invention will be described in detail below with reference to the drawings. However, the shapes, dimensions, materials, and the like of members described in the following embodiments and corresponding drawings are only specific examples for achieving the object of the present invention, and the scope of the present invention is limited to only those configurations. It is not intended.

<Basic structure of particle fixing structure>
First, the basic structure of a particle fixing structure according to the present invention (hereinafter also simply referred to as “structure”) will be described.

  A structure for fixing particles is a member that facilitates manipulation, observation, analysis, etc. of a large number of sample particles by individually arranging and holding (fixing) a large number of sample particles to be examined. It is. Examples of the analyte particles that can be fixed by the structure of the present invention include biological sample fine particles, inorganic material fine particles (silica, zirconia, nickel oxide, etc.), and organic material fine particles (polystyrene, etc.). Among them, the present invention can be preferably applied to fixation of biological sample-based fine particles represented by cells and virus particles. Therefore, in the following embodiments, biological sample-based fine particles (hereinafter referred to as “biological sample”) are mainly described. Is described as an example.

(First embodiment)
1 and 2 schematically show the structure according to the first embodiment. FIG. 1A is a plan view of a structure, and FIG. 1B is an exploded perspective view for explaining a layer structure of the structure. 2A is a cross-sectional view taken along the line AA of FIG. 1A, and FIG. 2B shows a state in which a biological sample (for example, a cell) is fixed to the structure.

  As shown in FIGS. 1 and 2, the structure 14 includes a flat substrate 15, a holding unit 20 disposed on the substrate 15, and a suspension containing a biological sample introduced onto the holding unit 20. And a spacer 16 for forming a space 45 (also referred to as an accommodating portion). The holding unit 20 includes a stacked body of the insulator film 18 and the light shielding film 19, and the light shielding film 19 is disposed above the insulator film 18. The holding unit 20 has a plurality of holding holes (through holes) 9 formed so as to extend (arrive) to the substrate 15 through the insulator film 18 and the light shielding film 19. Here, the holding hole 9 is a bottomed cylindrical hole having the substrate 15 as a bottom surface, and extends from the upper surface of the substrate 15 in the film thickness direction of the holding portion 20 (a laminated body of the insulator film 18 and the light shielding film 19). Thus, it is formed so as to open to the accommodating portion 45. For convenience of explanation and illustration, only a few holding holes 9 are shown in the drawing, but in an actual structure, thousands to millions or tens of millions of holding holes 9 are provided. .

  According to the above configuration, for example, by introducing a suspension containing a biological sample from the introduction port 24 provided in the spacer 16 into the accommodating portion 45 and allowing the biological sample to settle in the holding hole 9 by gravity, The biological sample 2 can be fixed to each holding hole 9 as shown in FIG. And after performing required operation, such as a label | marker, with respect to the structure 14 to which the biological sample 2 was fixed to each holding hole 9, for example, the excitation light 6 is sent from the upper side (container side) of FIG. Similarly, when the fluorescent light 7 is observed through the substrate 15 from above, the light-shielding film 19 blocks the autofluorescence of the insulator film 18 and the leaked light from the adjacent holding hole 9. Accordingly, optical noise such as background noise and crosstalk noise can be reduced. This makes it possible to detect a weak light signal emitted by a labeling substance (described later) specifically bound to the biological sample 2 with high sensitivity and high accuracy. Further, even when the fluorescence 7 is observed from the lower side of FIG. 2B, the leaked light from the adjacent holding hole 9 is somewhat blocked by the light shielding film 19, so that it can be detected with high sensitivity and high accuracy. It becomes possible.

  Next, another embodiment of the particle fixing structure will be described. The structure according to the first embodiment introduces fine particles into the holding hole by the action of gravity, whereas the structures according to the second to fourth embodiments described below mainly hold the fine particles using the dielectrophoretic force. A configuration for introducing into the hole is adopted.

(Second Embodiment)
3 and 4 schematically show the structures of the structure and the particle fixing device according to the second embodiment. FIG. 3 is an exploded perspective view for explaining the layer structure of the structure, and FIG. 4 is a cross-sectional view of the structure. FIG. 4 shows a cross section of the same portion as the AA line in FIG. 1A (the same applies to the following cross-sectional views). The structure 14 according to the second embodiment includes a comb-like electrode 21 including a pair of electrodes 22 and 23 on the surface of the substrate 15 on the holding unit side. Each of the electrodes 22 and 23 has a plurality of strip electrodes arranged in parallel along the arrangement direction of the holding holes 9, and the strip electrode of one electrode 22 and the strip electrode of the other electrode 23 are staggered. It is arranged to be. As shown in FIG. 4, each strip electrode is provided at a position corresponding to the holding hole 9, and the electrode is exposed at the bottom of each holding hole 9. The electrodes 22 and 23 are connected to the AC power source 4 through the conductive wires 3. In this configuration, the accommodating portion 45 is filled with a suspension containing a biological sample, and an AC voltage is applied between the AC power source 4 and the electrodes 22 and 23 to cause a dielectrophoretic force to act on the biological sample. Can be introduced and fixed into the holding hole 9 where the lines of electric force are concentrated. Details of dielectrophoresis will be described later.

(Third embodiment)
FIG. 5 shows a structure and a particle fixing device according to the third embodiment. The difference from the structure of the second embodiment shown in FIG. 4 is that an upper lid 17 that covers the accommodating portion 45 is provided on the spacer 16. By providing the upper lid 17, the moisture of the suspension containing the biological sample introduced into the storage unit 45 is prevented from evaporating, or the suspension containing the biological sample is stably supplied from the introduction port 24. There is an advantage that it can be supplied within. The reason why the supply of the suspension is stabilized is considered to be because the streamline of the fluid flowing between the upper lid 17 and the accommodating portion 45 of the spacer 16 tends to be laminar parallel to the substrate plane. In the structure of the first embodiment, an upper lid may be provided to obtain the same effect. On the other hand, the configuration without the upper lid as in the first embodiment and the second embodiment can provide the effects of simplification of manufacturing and cost reduction by reducing the number of members, and among the biological samples fixed in the holding holes 9 There is an advantage that an operation such as collecting any of the above by means of a micropipette or the like can be facilitated. Therefore, it is only necessary to select whether or not to provide an upper lid according to the application and purpose. In the embodiment in which the upper lid is not provided or in the embodiment in which the upper lid is detachable, the suspension containing the biological sample is directly introduced from above in the housing portion 45 without providing the introduction port 24 or the like in the spacer 16. Or can be discharged.

(Fourth embodiment)
6 and 7 show a structure and a particle fixing device according to the fourth embodiment. FIG. 6 is an exploded perspective view for explaining the layer structure of the structure, and FIG. 7 is a cross-sectional view of the structure. The structure 14 according to the fourth embodiment has a structure in which a lower electrode substrate 36 and an upper electrode substrate 35 are disposed on the lower side and the upper side of the holding unit 20, respectively. The lower electrode substrate 36 has a configuration in which an electrode layer is disposed on the surface on the holding portion side of the substrate 15 described in the above embodiment (that is, between the substrate 15 and the insulator film 18). The illustration is omitted from 7. The shape of the electrode layer is not limited as long as it is formed so as to be exposed at the bottom of the holding hole 9, but the configuration in which the electrode layer is provided on the entire surface of the substrate as shown in FIG. 6 is preferable from the viewpoint of facilitating the manufacturing process. It is a form. On the other hand, the upper electrode substrate 35 may have a configuration in which an electrode layer is provided on the substrate in the same manner as the lower electrode substrate 36, or a plate-like member made of a conductive material may be used as the upper electrode substrate 35. In this embodiment, the upper electrode substrate 35 also serves as an upper lid that covers the accommodating portion 45. In this configuration, the accommodating portion 45 is filled with a suspension containing a biological sample, and an AC voltage is applied between the upper electrode substrate 35 and the lower electrode substrate 36 from the power source 4, thereby applying a dielectrophoretic force to the biological sample. Thus, the biological sample can be introduced and fixed into the holding hole 9 where the lines of electric force are concentrated.

Also in the structures according to the second to fourth embodiments described above, the light shielding film 19 is provided so as to cover the upper surface of the holding portion 20, that is, the insulator film 18. Similarly, when optically observing a biological sample, optical noise such as background noise and crosstalk noise can be reduced, and weak light signals emitted from the biological sample can be detected with high sensitivity and high accuracy. It becomes possible. In the embodiments described above, the holding portion 20 is formed so as to cover the entire surface of the insulator film 18 with the light shielding film 19.
However, as long as the periphery of the opening of the holding hole 9 is covered, a configuration in which a part of the insulator film 18 is covered may be employed. That is, the arrangement of the light shielding film 19 can be adjusted as appropriate so that the fluorescence emitted from one holding hole 9 is not disturbed by the fluorescence from other holding holes or the autofluorescence of the insulator film 18.

  In addition, the container 45 has a box shape that can be sealed, and the specific gravity of the suspension containing the biological sample is equal to or higher than the specific gravity of the biological sample so that the biological sample floats upward in the suspension. (Even if the specific gravity of the biological sample is large, it is easy to make the specific gravity of the suspension higher than that). The holding part having the holding hole 9 that opens downward above the containing part 45 20 may be provided. This is a structure having a configuration in which the structure of the third or fourth embodiment is turned upside down. However, considering the fact that gravity can also be used for the manipulation of biological samples, and the fractional collection of biological samples after immobilization and the fractional collection of genes eluted from biological samples by extraction, such operations can be performed by aspiration from the top, etc. In order to be able to be implemented by a very simple operation, it is preferable that the holding portion 20 is disposed below the housing portion 45, and the upper lid 17 and the upper electrode substrate 35 of the housing portion 45 are configured to be removable from the spacer 16. It is preferable to keep it.

<Description of components>
Next, each constituent member of the above-described particle fixing structure 14 will be described in detail.

(Substrate and electrode)
As a material of the substrate 15, acrylic resin, epoxy resin, synthetic quartz (SiO ₂ ) mainly composed of silicon oxide, ceramics, metal-based material, or the like can be used. Pyrex glass (registered trademark) having silicon oxide and boron oxide as main components and having good workability and a low thermal expansion coefficient can be exemplified as a preferable member. In addition, when a member other than the substrate 15 (for example, the spacer 16) secures the necessary rigidity for the structure, a water-repellent paper, a protein sheet, or the like can be used as the material of the substrate 15.

  The material of the electrode provided on the substrate surface is not particularly limited as long as it has conductivity and is chemically stable. For example, metals such as platinum, gold, copper, and alloys such as stainless steel, transparent conductive materials such as ZnO (zinc oxide), SnO (tin oxide), and ITO (Indium Tin Oxide) are used. it can. When the light emitted from the labeling substance in the holding hole 9 is observed through the electrode substrate, an electrode made of a transparent conductive material is preferably provided on a light-transmitting substrate such as glass. Among these, the ITO electrode is a particularly preferable electrode material in terms of its transparency and film formability.

There are two methods for optically observing the labeling substance in the holding hole 9, “transmission type” and “reflection type”. The transmission type is a method of irradiating light from one side of the bottom of the holding hole 9 and the opening and observing (detecting) the light from the opposite side. This is a method of irradiating light from one side of the opening and observing (detecting) the light from the same side. Any method can be used here, but in the case of the reflection type, both the light irradiation means and the light detection means have to be installed on the same side with respect to the structure body 14, and therefore, compared with the transmission type. There are restrictions on the device configuration. Therefore, the transmission type is more advantageous from the viewpoint of simplifying the device configuration. Further, in the above embodiment, since the light shielding film 19 is disposed on the upper surface of the holding unit 20, it is more preferable to observe the fluorescence from the upper side, that is, the reflection type than the observation from the lower side of the holding unit 20. The effect of reducing optical noise by 19 is great. Therefore, in the structure 14 of the above embodiment, the substrate 15 and the electrode may be a light-transmitting material or a light-transmitting material. Note that, as described above, since the crosstalk noise and the like can be blocked even when the fluorescence is observed as the transmission type, in this case, the substrate 15 and the electrode are preferably made of a translucent material. Although the number of photodetecting means equal to the number of holding holes may be provided, in order to simplify the apparatus configuration and improve maintenance and operability, the number of photodetecting means smaller than the number of holding holes is provided. It is preferable that each holding hole is scanned by driving it with an appropriate actuator. Alternatively, it is also preferable to detect the light of the plurality of holding holes at once by a light detection means such as an image scanner.

(Light shielding film)
The light shielding film 19 is provided on a portion other than the holding hole 9 on the upper surface of the holding unit 20. By arranging in this way, the light emitted from the holding hole is not blocked by the light shielding film, so that it is possible to avoid a decrease in the detection intensity of the light emitted from the labeling substance. It is also possible to observe light through the substrate 15 not only from the opening side of the holding hole 9 but also from the bottom surface side of the holding hole 9. Preferably, as shown in FIGS. 2, 4, 5, 7, etc., the light shielding film 19 may be provided on the entire upper surface of the holding portion 20 except for the holding holes 9. Thereby, it is possible to remove as much as possible the optical noise generated from the part other than the holding hole.

The material of the light shielding film 19 may be any material that has sufficient light shielding properties with respect to light having a wavelength emitted by the labeling substance (for example, light having a wavelength of 380 nm to 780 nm if visible light). Carbon materials, ceramics, resins, etc. can be used. Among them, metals such as chromium (Cr), titanium (Ti), platinum (Pt), niobium (Nb), tantalum (Ta), tungsten (W), aluminum (Al), gold (Au), or metal oxides, etc. This metal material is preferably chromium, which has particularly high light shielding properties and high adhesion to the substrate 15 and the insulator film 18 (C
r) is preferred. The thickness of the light-shielding film can be appropriately set depending on the material, but in the case of a metal film such as chromium, the film thickness is preferably in the range of 50 nm to 10 μm, and a film of 100 nm or more that provides sufficient light-shielding properties. Thickness is particularly preferred. The same applies to other metal films. In the case of a metal film, film formation and hole formation can be performed by general photolithography and etching. In the case of ceramic, for example, processing by spraying or pasting is considered. Moreover, you may affix the resin and film which formed the hole previously by machining to a board | substrate. In addition, if the insulator film 18 is a light-shielding member or a light-shielded member, the insulator film 18 can also serve as a light-shielding film.

(Insulator film)
A part of the holding part 20 is made of an insulating material and the electrode is exposed at the bottom of the holding hole 9 because the electric lines of force are concentrated on the holding hole 9 when an AC voltage is applied to the electrode. This is because the biological sample is moved and fixed in the holding hole. As described above, when only the natural sedimentation (gravity) is used to introduce the analyte particles into the holding hole, an electrode is not required, and therefore an insulator film is not necessary.

  As a material of the insulator film 18, an insulating material, for example, a crosslinkable polymer such as silicone rubber, resist, resin, ceramic, glass, water-repellent paper, or the like can be used. Hereinafter, an insulator film made of a crosslinkable polymer may be referred to as a polymer film. The insulator film is preferably an insulating material having affinity with the analyte particles because the analyte particles are attracted and fixed to the holding holes 9 provided in the insulator film 18. An insulator film having an affinity for analyte particles is a hydrophilic polymer film when the analyte particles are hydrophilic, or a hydrophobic insulator film when the analyte particles are hydrophobic. Is preferred. As a measure of affinity, generally, the contact angle between the droplet formed when a liquid having affinity close to the analyte particles is dropped on the surface of the insulator film and the surface of the insulator film. (The smaller the contact angle, the higher the affinity between the liquid and the surface of the insulator film, and the larger the contact angle, the lower the affinity between the liquid and the surface of the insulator film). Highly hydrophilic polymer films include polyethylene glycol polymers, etc., or glass and titanium oxide, and relatively hydrophobic polymer films include epoxy resin, polystyrene, polyimide, Teflon (registered) Trademark) etc. can be illustrated. In particular, an epoxy resin imparting photosensitivity to ultraviolet rays is preferable, and the SU-8 3000 series (Kayaku Microchem Corporation) capable of producing a structure with a high aspect ratio is more preferable.

  Even when it is unavoidable to use an insulator film having a low affinity for the analyte particles, the affinity between the insulator film and the analyte particles is improved by modifying the surface of the insulator film. be able to. As a method for hydrophilizing a hydrophobic polymer film such as a resin, a known method such as plasma treatment, modification by physical adsorption of protein, or any combination of these methods may be used. Here, the plasma treatment of the polymer film surface means that the polymer film surface is irradiated with an electrically neutral ionized gas (plasma) in which active species such as electrons, ions, and radicals are present. This is a treatment for hydrophilizing the surface of the polymer film by removing organic contaminants on the film surface and changing the chemical bonding state. Here, in the case of modification by physical adsorption of protein, for example, the polymer film is physically adsorbed by immersing the polymer membrane in a protein-containing solution such as BSA (bovine serum albumin) for several minutes to several hours. The membrane surface can be hydrophilized. In addition, as a method for hydrophobizing a hydrophilic polymer film such as a polyethylene glycol diacrylate polymer, a method by chemical modification in which a silane coupling agent is bonded to the hydrophilic polymer surface may be used. A silane coupling agent is a compound composed of an organic substance and silicon. In the molecule, hydrophilic reactive groups (hydroxyl group, carboxyl group, amino group, sulfone group, etc.) and hydrophobic reactive groups (vinyl group, methyl group) Group, ethyl group, propyl group, etc.) having two or more different reactive groups. For this reason, when a hydrophilic polymer film is immersed in a dilute solution of a silane coupling agent, the reactive group showing the hydrophilicity of the silane coupling agent is chemically bonded to the surface of the hydrophilic material, thereby improving the hydrophobicity. Since the reactive group shown covers the surface, the surface of the hydrophobic material can be easily hydrophobicized uniformly.

  In addition, as a hydrophilicity or hydrophobicity evaluation method, the following general methods can be used. That is, by adding pure water to the surface of the polymer film and measuring the contact angle between the droplet formed on the surface of the polymer film and the surface of the polymer film, the hydrophilicity of the surface of the polymer film And the hydrophobicity is evaluated. Since there is no strict definition of hydrophilicity and hydrophobicity, in the present invention, hydrophilicity is defined as the contact angle of 50 ° or less, preferably 40 ° or less, and hydrophobicity is defined as 50 ° or less. It is defined as greater than, preferably greater than 60 °. Furthermore, the measurement of the contact angle uses the θ / 2 method for calculating the contact angle from the angle of the straight line connecting the left and right end points and the vertex of the droplet dropped on the substrate with respect to the solid surface.

(Holding hole)
In order to form the holding hole (through-hole) 9 in the holding unit 20, various methods can be employed depending on the types of the insulator film 18 and the light shielding film 19. For example, in order to form the holding hole 9, a known method such as a method of irradiating a laser or a method of forming the holding part 20 using a mold having a pin for forming the holding hole 9 may be used. it can. In the case of using a photocurable resin or the like, the holding hole 9 may be formed by general photolithography (exposure) and etching (development) using an exposure photomask on which a pattern corresponding to the holding hole 9 is drawn. it can. When forming a holding hole in the light shielding film 19 made of a metal-based material, an insulating film from which a part is selectively removed is used as an etching mask, and a part of the light shielding film is selectively etched by isotropic wet etching. The removal method can be adopted.

  In the said embodiment, although the cylindrical holding hole 9 was illustrated, the shape of the holding hole 9 is not restricted to this. For example, the plane (opening) shape of the holding hole 9 may be an ellipse or a polygon (including a polygon with rounded corners). A shape in which the diameter (width) of the holding hole 9 increases stepwise or continuously from the bottom toward the opening is also preferable.

A configuration in which the plurality of holding holes 9 are regularly arranged in the plane of the holding unit 20 is suitable, and specifically, the plurality of holding holes 9 are preferably arranged in an array. Strictly speaking, the array shape means that the holding holes are two-dimensionally arranged in rows and columns, but in the present invention, the holding holes are only in the row direction (lateral direction) or in the columns. A configuration arranged one-dimensionally at equal intervals only in the direction (longitudinal direction) is also expressed as an array. By arranging the holding holes in an array like this, the electric field generated by the voltage applied between the electrodes is generated almost uniformly in all the holding holes, and the biological sample is similarly applied to all the holding holes. It can be guided and fixed. In addition, when biological samples are regularly arranged in an array, it becomes easy to individually detect and analyze the light signals of each labeling substance, and furthermore, it has a specific property among all the fixed samples. There are also advantages such as the ability to quantitatively evaluate the number and ratio of samples with, and easily identify the position (address) on the substrate of the sample where a specific optical signal was detected. . In addition, after optical detection, in order to make it easy to operate a sample at a specific address on the substrate, the information of the optical signal detected from each holding hole and the position information are stored in association with each other. It is preferable to provide storage means.

(spacer)
The spacers 16 constituting the housing part 45 are for securing a space for holding the suspension of the biological sample. The spacer 16 may be made of, for example, an insulator such as glass, ceramic, or resin, or a conductive material such as metal as long as the upper electrode substrate 35 and the lower electrode substrate 36 are not electrically connected. You may comprise as. For example, the spacer 16 can be molded and bonded at the same time by pouring and curing a rapid-curing type adhesive into a spacer mold disposed on the holding unit 20. Alternatively, the spacer 16 molded separately can be bonded to the holding unit 20 with an adhesive, or can be fused to the holding unit 20 by applying heat and pressure. Alternatively, the spacer 16 can be produced by using a surface adhesive resin such as PDMS (poly-dimethylsiloxane) or a silicon sheet, and this can be bonded to the holding portion 20 by pressure bonding. Although the accommodating part 45 is comprised by the spacer, the shape does not need to be a square as shown in the 1st thru | or 4th embodiment, and various shapes, such as circular, an ellipse, and a rhombus, can be employ | adopted for it. . In addition, the spacer can be provided with inlets and outlets (24, 25) for introducing and discharging the suspension. However, when the suspension is introduced directly from above the housing portion 45, the spacer is introduced. No mouth or outlet is required. Further, the introduction port and the discharge port do not need to be provided in a straight line at positions facing each other as shown in the first to fourth embodiments. In addition, for example, by preparing an insulating box itself and arranging a structure on the inner bottom surface thereof, the accommodating portion 45 can be configured without using a spacer. In this embodiment, for example, a structure including one of a pair of electrodes is disposed on the inner bottom surface of the box, and the other electrode paired on the inner top surface is disposed, or the top surface of the box itself is disposed. The other electrode can also be used.

  In the above embodiment, the biological sample to be provided to the apparatus is provided with the introduction channel provided in the spacer 16 and the introduction port 24 communicating with the channel, the discharge channel discharging the suspension, and the discharge port 25 communicating with the channel. Suspension can be supplied and discharged quickly. The size and shape of the spacer 16 and the space and thickness inside the spacer may be determined in relation to the amount of suspension to be accommodated in the accommodating portion, and are not particularly limited. For example, when the size of the spacer is about 40 mm long × about 40 mm wide, the space inside the spacer may be about 20 mm long × 20 mm wide, and the thickness of the spacer is 0 It may be about 5 to 2.0 mm.

(AC source)
In the particle fixing device shown in the second to fourth embodiments, an AC power supply 4 is connected to the pair of electrodes of the structure 14 via the conductive wire 3. The AC power supply 4 only needs to be able to apply a voltage sufficient to move the biological sample to the holding hole 9 and generate an electric field to be fixed between the electrodes. Specifically, a power source capable of applying an alternating voltage having a peak voltage of about 1 V to 20 V and a waveform of a sine wave, a rectangular wave, a triangular wave, a trapezoidal wave or the like having a frequency of about 100 kHz to 3 MHz can be exemplified. It is particularly preferable that an alternating voltage having a waveform that can be moved and fix only one biological sample in one holding hole is applied between the electrodes. A rectangular wave is preferably used as the AC voltage having such a waveform. Compared to the case where the waveform is a sine wave, a triangular wave, or a trapezoidal wave, the rectangular wave reaches a peak voltage that is set instantaneously, so that it is possible to quickly move the biological sample toward the holding hole. The probability of entering the holding hole so that the above biological samples overlap can be reduced (the probability that only one biological sample can be fixed to one holding hole is increased). A biological sample can be regarded as a capacitor electrically. However, while the peak voltage of the rectangular wave does not change, it is difficult for current to flow through the biological sample captured in the holding hole, and it is difficult to generate electric lines of force. As a result, the dielectrophoretic force is hardly generated in the holding hole to which the biological sample is fixed. Therefore, once a biological sample is fixed in the holding hole, the probability that another biological sample is fixed in the same holding hole is reduced, and instead, another holding in which electric lines of force are generated and dielectrophoretic force is generated. The biological sample is sequentially fixed in the holes (empty holding holes where the biological sample is not fixed).

  In addition, in the apparatus of this invention, it is preferable to employ | adopt the power supply which generate | occur | produces the alternating voltage which does not have a direct-current component. When an alternating voltage having a direct current component is applied, the biological sample moves by receiving a force biased in a specific direction due to the electrostatic force (electrophoretic force) generated by the direct current component, and is difficult to fix to the holding hole by the dielectrophoretic force. Because. In addition, when an AC voltage having a DC component is applied, ions contained in the suspension containing the biological sample generate an electric reaction on the electrode surface and generate heat, thereby causing the biological sample to move due to the dielectrophoretic force. Cannot be controlled, and it is difficult to move and fix the holding hole. The AC voltage having a DC component refers to a voltage whose frequency duty ratio is not 50%, a voltage having an offset, a voltage having an extremely long period (for example, 1 second or more), and the like.

(Retention hole spacing and dimensions)
In the apparatus of the present invention, the waveform of the applied AC voltage is preferably rectangular, so that only one biological sample can be fixed to one holding hole. In addition, it is preferable that the arrangement, size, and shape of the holding holes be an arrangement, size, and shape suitable for fixing only one biological sample to one holding hole. For example, with respect to the arrangement, it is preferable to arrange the holding holes in an array on the surface of the holding unit, but the probability that a plurality of biological samples will be fixed to one holding hole if the interval between adjacent holding holes is too narrow. In addition, since the biological sample suspension in the holding holes on both sides is mixed or crosstalk noise is generated, it does not affect the analysis of the biological sample. Conversely, when the interval between the adjacent holding holes is wide, the biological sample is left between the adjacent holding holes (54 in FIG. 9), and the probability that a holding hole that cannot fix the biological sample is increased. Therefore, the interval and dimensions (diameter and depth) of the holding holes may be set according to the particle size of the biological sample to be fixed. Specifically, the interval between adjacent holding holes is preferably in the range of 0.5 to 20 times the particle size of the biological sample to be fixed. The diameter and depth of the holding hole are preferably in the range of 1 to 5 times the diameter of the biological sample. By doing so, electrostatic force is generated on the electrode surface on the bottom surface of the holding hole and the biological sample, and the biological sample is securely fixed to the holding hole and good observation can be performed.

(Biological sample)
Preferred analyte particles are biological samples such as cells, virus particles, DNA, proteins and the like.
The cell is not particularly limited as long as it is made of a dielectric that can be collected by dielectrophoresis. For example, substances that can be labeled on the surface or inside (specific substances) such as blood, lymph, cerebrospinal fluid, sputum, urine, stool cells (live cells), microorganisms or protozoa present in the body or environment, or cultured cells ) Can be exemplified as a preferable biological sample. More specifically, cancer cells that metastasize remotely through blood or lymph, such as stomach cancer, colon cancer, esophageal cancer, liver cancer, lung cancer, pancreatic cancer, bladder cancer, uterine cancer (epithelial tumor). Examples thereof include cells derived from blood cells such as lymphocytes and leukocytes (lymphoma, leukemia). In addition, cancer cells have a specific protein such as collagenase on the cell surface, and the protein infiltrates and metastasizes by dissolving collagen (extracellular matrix) around the cancer cell. A cancer cell can be illustrated.

Examples of the virus include viruses such as herpes virus, hepatitis virus, HIV virus (human immunodeficiency virus), and ATL virus (adult T cell leukemia virus). As DNA and protein, each has a length or molecular weight of 40 kbp or more, 100,000 Da or more, preferably 100 kbp
As mentioned above, it is preferable that it is 500,000 Da or more. Examples of the biological sample include fine particles obtained by capturing proteins, peptides, DNA, RNA, polysaccharides, lipids, viruses, and the like on carrier particles modified so that these substances can be captured. The carrier particles are not particularly limited as long as they are made of a dielectric. The detection sensitivity is expected to be improved because the protein can be concentrated on the fine particles rather than the detection in a state where various proteins are mixed in the solution. In addition, when a plurality of types of specific fine particles are prepared for each protein or the like, multi-analyte detection is possible. Proteins include various tumor markers such as CEA (carcinoembryonic antigen, digestive system tumor marker such as esophagus, stomach, rectum), CA19-9 (carbohydrate antigen 19-9, digestion of pancreatic / biliary tract cancer, etc.) Tumor markers), PSA (prostate specific antigen), and the like.

  The biological sample suspension used for the apparatus may be any suspension that can move the biological sample as described above by dielectrophoresis. For example, an aqueous solution of saccharides such as mannitol, glucose and sucrose and a suspension containing a sample to be analyzed into an aqueous solution containing an electrolyte such as calcium chloride or magnesium chloride or a protein such as BSA (bovine serum albumin) in the aqueous solution. It can be illustrated as a preferred biological sample suspension.

<Dielectrophoresis and particle analysis>
Next, a method for fixing particles using dielectrophoresis and a method for analyzing the fixed particles will be described with reference to FIGS. Here, the structure and the fixing device of the fourth embodiment (FIG. 7) will be described as an example. However, in the case of the structure and the fixing device of other embodiments, the particle fixing and analysis are performed in the same manner. It can be performed.

First, as shown in FIG. 8, a suspension containing a biological sample is injected from the introduction port 24 of the spacer 16, and the inside of the accommodating portion 45 and the holding hole 9 is filled with the suspension. When the AC voltage having the waveform described above is applied from the AC power supply 4, the electric force lines 12 are concentrated in the holding hole 9, and the dielectrophoretic force 26 acts on the biological sample 28. Thereby, the biological sample 28 moves toward the holding hole 9 along the electric lines of force 12, and one biological sample can be introduced and fixed to one holding hole 9. The principle of dielectrophoretic force will be described with reference to FIG. Polarization occurs in the biological sample 28 in a solution placed in an alternating voltage, that is, an alternating electric field, that is, dielectric particles such as cells, and positive and negative charges are induced. Here, as shown in FIG. 8, when a non-uniform electric field (electric field lines 12 shown in FIG. 8) is applied to the holding holes 9 provided in the insulator film 18 on the lower electrode substrate 36, The biological sample 28 is drawn in the direction in which the electric field concentrates (the direction in which the electric lines of force 12 are dense), that is, in the direction of the holding hole 9. This is the dielectrophoretic force 26. In general, the dielectrophoretic force is proportional to the square of the volume of the particle, the difference between the dielectric constant of the particle and the solution, and the magnitude of the non-uniform electric field. For example, when an AC electric field having a frequency of 100 kHz to 3 MHz and 1 × 10 ⁵ to 5 × 10 ⁵ V / m is applied to a particle having a diameter of about 5 to 10 μm, the dielectrophoretic force acts to cause the particle to It is drawn in the direction of concentration. In this case, the biological sample 28 is guided to the holding hole 9 mainly by dielectrophoretic force, gravity, and electrostatic force from the electrode. The number of biological samples in the suspension supplied to the storage unit is not particularly limited, but considering the effective use of the biological sample, the number of holding holes provided in the holding unit of the structure It is preferable that the number is approximately the same.

Next, as shown in FIG. 9, while the alternating voltage is applied, the inside of the holding hole 9 modified with the substance 27 that binds to the biological sample and the biological sample 28 are bonded. If the substance 27 couple | bonded with a biological sample couple | bonds with the biological sample 28, there will be no restriction | limiting in particular. For example, a substance that recognizes a specific substance on the surface of a biological sample, Biocompatible A having a lipid oleyl group that binds to a lipid bilayer of a cell
Examples include nfor for membrane (BAM) and substances that electrostatically bind to biological samples. Examples of the combination of a specific substance on the surface of a biological sample and a molecule that binds to the substance include a receptor-ligand, a sugar chain-lectin, and an antigen-antibody. In consideration of binding to a biological sample in a relatively short time, a substance that electrostatically binds to the surface of the biological sample can be exemplified as a preferable substance that binds to the biological sample. Such substances include polycationic agents such as poly-L-lysine.

In addition, as a substance that binds to a biological sample, a marker that is versatile in various cancer cells (common or not specific among cancer cells), such as EpCAM (CD326, epithelial specific antigen (ESA), or human If a substance that binds to epithelial antigen (HEA) is used, only cancer cells in the blood are fixed and cells other than cancer cells (blood cells, etc.) are not fixed. Cells other than cancer cells can be detached from the holding holes, and only cancer cells can be reliably fixed and detected. As will be described later, in order to detect a specific cancer cell, it is preferable to detect a specific substance present only in the specific cancer cell using a labeling substance. However, for example, when a wide variety of cancer cells are used as a target sample and the purpose is to know whether or not the target sample is present in a biological sample, a holding hole is formed with a substance that binds to a versatile marker. It is only necessary to modify the inside and to detect the presence or absence of cells after washing. In such a case, for example, light may be irradiated from above the holding hole and only whether or not the light irradiated downward from the holding hole arrives, that is, the shadow of the fixed cell may be detected.

  Furthermore, if a substance that specifically recognizes a cell or the like that is not desired to be fixed to the holding hole (for example, an antibody against the cell) is disposed on a light shielding film other than the holding hole (for example, 54 in FIG. 9) It is possible to prevent entry into the hole, that is, target cells (cells that have entered the holding hole) and non-target cells (cells fixed on the light-shielding film outside the holding hole).

  In addition, a substance (for example, an antibody that specifically binds to the common antigen) that binds to a cancer cell's versatile (common) marker (for example, an antigen) is placed in the holding hole, and the dielectrophoretic force After fixing the cancer cells to the retention holes by using, a substance that binds to HER2 (antigen) specifically expressed in breast cancer, lung cancer, etc. among the cancer cells (antibody against HER2 (anti-HER2 antibody)) ) Can be used to estimate the type of fixed cancer cells.

  The method for modifying the holding hole 9 with the substance 27 that binds to the biological sample is not particularly limited as long as the inside of the holding hole 9 and the biological sample 28 can be bonded. For example, by using a self-assembled monolayer (Self-Assembled Monolayer, SAM film) and reacting a substance 27 that binds to a biological sample having an amino group or the like on an electrode made of a metal oxide film or the like on the bottom surface of the holding hole. The inside of the holding hole can be specifically modified. In addition, after capturing the biological sample 28 in the holding hole 9, the inside of the holding hole 9 and the biological sample 28 are coupled by injecting a solution containing a substance 27 that binds to both the biological sample and the inside of the holding hole 9 into the storage unit. It is possible. When binding is performed by injecting a solution containing the substance 27 that binds to the biological sample, a solution in which the biological sample 28 is suspended after the binding reaction, for example, an aqueous solution of saccharides such as mannitol, glucose, sucrose, and the like, calcium chloride or The substance 27 that binds to the biological sample in the container is preferably washed and removed with an aqueous solution containing an electrolyte such as magnesium chloride or a protein such as BSA (bovine serum albumin). Thus, the biological sample 28 captured in the holding hole 9 by the substance 27 that binds to the biological sample is not detached from the holding hole 9 even if an AC voltage is not applied.

  Subsequently, as shown in FIG. 10, a reagent solution necessary for analysis of analyte particles such as a biological sample is injected into the container. The reagent used for the analysis of the analyte particles is not particularly limited as long as a substance present on the surface or inside of the analyte particles can be optically detected.

  A component constituting an analyte particle such as a biological sample, for example, a substance existing on the surface or inside of the analyte particle (hereinafter sometimes referred to as “specific substance”) has a specific property among the analyte particles. It is a substance that exists specifically on the surface or inside of a particle (hereinafter, also referred to as “target sample”) having s. “Specifically present in the target sample” means that it exists in the target sample and does not exist in particles other than the target sample, or exists more in the target sample than particles other than the target sample.

When the analyte particles are cells, specific substances include antigens, receptors, sugar chains, nucleic acids and the like. Examples of the antigen include antigens on tumor cells, major histocompatibility antigens (MHC, HLA in the case of humans), and the like. Examples of antigens on tumor cells include EpCAM. Examples of the receptor include hormone receptor, Fc receptor, virus receptor and the like. Examples of the nucleic acid include DNA and RNA. A protein such as a tumor marker may be used directly as a biological sample, or may be supported on carrier particles as described above. More specifically, for example, if the target sample is various cancer cells, a marker common to various cancer cells may be a specific substance. If the target sample is breast cancer or lung cancer, HER2 (antigen) that is specifically expressed in these cancers may be a specific substance.

  The specific substance as described above can be observed (detected) by labeling with a substance indicating the presence of the specific substance, for example, a substance that specifically binds to the specific substance. For example, an antigen is bound to an antibody against the antigen, a ligand is bound to the receptor, and a lectin is bound to a sugar chain. Therefore, a substance that specifically binds to a specific substance such as these and a substance that emits an optically detectable signal are combined to form a labeling substance. However, for example, when a specific substance binds or reacts only with an arbitrary substance and, as a result, an optically detectable signal is emitted, the arbitrary substance itself can be used as a labeling substance.

  The labeling substance 31 for detecting the specific substance of the present invention is not particularly limited as long as the target sample can be detected. The labeling substance refers to any substance that can indicate the presence of a component constituting the analyte particle. Representative examples of useful detectable labeling substances are substances that can specifically bind to a specific substance such as an antibody, ligand, or lectin as described above based on the properties of fluorescence, phosphorescence, or luminescence. Can be exemplified.

  Examples of the labeling substance include a labeling substance itself, for example, a substance that emits fluorescence or phosphorescence, or a substance that emits light, such as a fluorescent dye, bound to a substance that can specifically bind to a specific substance. In addition, the labeling substance is a reaction that emits light, for example, a reaction that generates a substance that emits fluorescence or phosphorescence, or a substance that catalyzes a luminescence reaction bound to a substance that can specifically bind to a specific substance. Also good. For example, examples of fluorescent dyes include FITC (fluorescein isocyanate), PE (phycoerythrin), rhodamine and the like. Examples of substances that catalyze the reaction include peroxidase, β-galactosidase, alkaline phosphatase, and luciferase. For example, when a specific substance reacts only with an arbitrary substance and, as a result of the reaction, an optically detectable signal is emitted, the arbitrary substance itself may be used as a labeling substance.

  In addition, the use of a labeling substance in which a quencher that inhibits fluorescence, phosphorescence, or luminescence is combined with a substance that specifically binds to a specific substance can be exemplified. In this case, if the biological sample is stained with a fluorescent, phosphorescent or luminescent substance and further reacted with the labeling substance, the fluorescent, phosphorescent or phosphorous from the holding hole to which the biological sample having the specific substance is fixed. Since the emission is decreased by the quencher, this decrease may be detected. In addition, it can be exemplified that the labeling substance is used in combination with a substance that binds a versatile (common) marker to a whole cell in a biological sample and a substance that fluoresces, phosphorescents, or emits light.

  As described above, the labeling substance is a substance in which a substance that specifically binds to the specific substance and a substance that emits an optically detectable signal are bound to each other, even if the labeling substance itself binds or reacts with the specific substance. There may be. When a substance that emits an optically detectable signal is bound to a substance that specifically binds to a specific substance, the both may be directly bound by a known chemical method or the like. You may couple | bond indirectly via the substance couple | bonded with respect to the substance to couple | bond. For example, when the substance that specifically binds to a specific substance is an antibody, the antibody against the animal immunoglobulin used for the preparation of the antibody or the substance that generates a signal with protein A or protein G may be bound. . Further, for example, a substance that specifically binds to a specific substance and biotin are bound, and a substance that generates a signal is bound to avidin or streptavidin. In this case, avidin or streptavidin bound to a substance that generates a signal binds to biotin bound to a substance specifically bound to the specific substance, and as a result, (specific substance)-(specific to the specific substance). The specific substance is indirectly labeled by forming a complex of (substance to be bound)-(biotin)-(avidin or streptavidin)-(substance that generates a signal). Such indirect cases are also included in the specific substance referred to in the present invention.

  In the case where the specific substance is a nucleic acid, a substance that binds directly or indirectly to (or can bind to) a substance that generates a signal and that uses a probe that binds to the nucleic acid or generates a signal The nucleic acid can be detected by amplification of the nucleic acid using a primer that is directly or indirectly bound to (or capable of binding to). For amplification of nucleic acid, a PCR method, a LAMP method, an RT-PCR method, a NASBA method, a TMA method, or a TRC method, which are known per se, can be used. The TaqMan method uses an oligonucleotide having a 5 ′ end modified with a fluorescent substance and a 3 ′ end modified with a quencher substance as a probe. Since this probe has a quencher substance nearby together with a fluorescent substance, the generation of fluorescence is suppressed. When the TaqMan probe hybridized to the target nucleic acid is degraded by the 5 ′ → 3 ′ exonuclease activity of Taq DNA polymerase during the PCR extension step, the fluorescent dye is released from the probe and suppressed by the quencher. Is released and fluorescence is generated. Examples of the nucleic acid include DNA or RNA. When amplifying RNA, a reverse transcription reaction may be performed using RNA as a template, and the generated cDNA may be amplified by a PCR method (RT-PCR). Further, the substance that generates a signal may be a combination of quenchers that inhibit fluorescence, phosphorescence, or luminescence, as described above.

  As described above, a primer that binds to a nucleic acid generated by an amplification reaction is also included in the substance that specifically binds to a specific substance in the present invention.

  When detecting cells as target samples by PCR, heating and cooling in the PCR reaction may be performed by placing a particle fixing structure on a heat block of a thermal cycler. A heat block may be provided and performed on the heat block.

  The light emitted by the labeling substance, that is, the light emitted from the labeling substance or the light emitted by the reaction catalyzed by the labeling substance indicates the presence of the component constituting the analyte particle, that is, the specific substance.

  FIG. 11 shows an application example of the biological sample analyzer of the present invention. For example, a suspension in which the presence of abnormal cells 32 such as cancer is suspected is applied to a biological sample analyzer, and the cells are captured in the holding holes. On the other hand, an abnormal cell is detected by introducing a labeling substance for detecting a specific substance on the surface or inside the abnormal cell 32 that is the object of the detection. Furthermore, abnormal cells such as cancer detected by the fluorescence microscope 33 are collected with a micropipette as the biological sample collecting means 34, and a detailed biological sample can be analyzed.

  In the above description, the micropipette has been described as the biological sample collection means. However, the biological sample collection means is not particularly limited as long as the biological sample can be collected. In addition to the micropipette, the biological sample can be precisely collected using electroosmotic flow. Biological sample collecting means that can be collected can be used.

  As another application example of the biological sample analyzer of the present invention, a crushing means for crushing the biological sample may be provided. The crushing means is not particularly limited as long as it can crush the biological sample and elute the nucleic acid contained in the biological sample to the outside of the biological sample. For example, a means for applying a voltage to a biological sample, which includes a pair of electrodes and a power source for applying a DC voltage or an AC voltage having a low frequency of about 1 Hertz to the electrodes, is fixed to the holding hole of the holding unit. be able to. If the biological sample is a cell that does not have a cell wall, the cell can be disrupted by applying a voltage of about 1 volt to the cell membrane. In addition, as a pair of electrode in the said means, the electrode for making a biological sample act on a dielectrophoretic force and making it move to a holding | maintenance part can be combined. Moreover, for example, a heating means for heating the biological sample fixed in the holding hole of the holding unit can be exemplified. A biological sample can be crushed by placing it at a temperature of 90 ° C. for several minutes. Moreover, for example, an ultrasonic wave generation unit that vibrates a biological sample fixed in the holding hole of the holding unit can be exemplified. A biological sample can be crushed by applying a vibration of 20 to 40 kilohertz continuously or intermittently with an output of 100 to 200 watts. The illustrated means for applying a voltage, heating means, and ultrasonic wave generation means can be used only alternatively. For example, both the heating means and the ultrasonic wave generation means can be used.

  When detecting the nucleic acid eluted from the crushed biological sample after amplification by PCR method, first, after fixing the biological sample in the holding hole of the holding unit, for example, a primer, an enzyme, A reaction solution containing an enzyme substrate or the like is provided, and a solution (a solution used for suspending biological cells) present in a holding hole communicating with the housing portion is replaced with the reaction solution. In the PCR reaction, the temperature of the gene eluted from the biological sample fixed in the holding hole is raised to about 90 ° C. At that time, heat convection occurs inside the holding hole, and the gene derived from the biological sample in the holding hole moves outside the holding hole. Since it may diffuse and contaminate the biological sample suspension in the adjacent holding hole, silicon oil or the like is dropped into the holding hole when the solution replacement is completed, and the temperature response in the PCR reaction solution It is preferable to suppress the thermal convection by adding a polymer. If a biological sample is suspended in a reaction solution for PCR reaction and moved to a holding hole by dielectrophoresis, an overcurrent will occur if voltage is applied for various electrolytes contained in the reaction solution for PCR reaction. Since the heat convection due to heat generation occurs and it is difficult to move and fix the biological sample to the holding portion, it is preferable to perform such solution replacement. After sealing, nucleic acid is detected by crushing as described above.

  In the configuration shown in FIG. 11, the light shielding film 19 covers the entire surface of the insulator film 18, but instead the insulator film 18 as long as the light shielding film 19 covers the periphery of the opening of the holding hole 9. You may employ | adopt the structure which covers a part of. In such a configuration, the upper surface of the holding portion 20 is divided into a portion covered with a light shielding film and a portion where the insulator film is exposed. Therefore, it is possible to suitably detect fluorescence related to the biological sample by utilizing the difference in hydrophilicity between the light shielding film 19 and the insulator film 18. For example, when the light shielding film 19 is a metal film and has hydrophilicity, and the insulator film 18 is a polymer film and has hydrophobicity, after introducing a water-soluble liquid into the holding hole 9 By sealing the holding holes 9 with a water-insoluble liquid, the aqueous solution can be held only around each holding hole. As a result, it is possible to examine the reactivity with respect to a plurality of biological samples by fixing to the holding hole 9.

<Effect>
The structure according to the above-described embodiment of the present invention, and the fixing device and the analysis device using the structure have the following effects.

  The structure of the present embodiment and the fixing device using the structure can quickly fix one particle to each holding hole provided in the holding unit. In addition, since the plurality of holding holes are arranged in an array, a plurality of particles can be separated one by one and fixed in an array, which makes it easy to analyze individual particles collectively. . Furthermore, by providing a light-shielding film on the holding part, it is possible to reduce light noise such as background noise and crosstalk noise, and only light emitted by the observation target substance in the holding hole is highly sensitive and accurate. Can be detected. And since it is possible to detect light with high sensitivity and high accuracy, it is possible to shorten the detection time.

<Example>
Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to the following examples.

Example 1
In Example 1, the structure and fixing device shown in FIG. 7 are used.

As the substrate constituting the lower electrode substrate 36, a glass substrate having a length of 78 mm, a width of 56 mm, and a thickness of 1 mm is used. The spacer 16 is 20 mm long × 20 mm wide × 1.5 mm thick on the holding part.
It is made of a silicon sheet so as to form a housing portion. In addition, the spacer 16 is provided with an inlet 24 and an outlet 25 for introducing and discharging a suspension containing a biological sample.

  The holding portion having a plurality of holding holes 9 is integrally formed on the lower electrode substrate by the photolithography and etching methods shown in FIGS. 12A and 12B. The resist 40 is applied to the ITO film-forming surface of the glass substrate 30 on which the ITO 37 is formed using a spin coater so as to have a film thickness of 5 μm, naturally dried for 1 minute, and then pre-baked (95 ° C., 3 minutes) using a hot plate. )I do. An epoxy negative resist is used for the resist 40. Subsequently, a fine hole pattern having a diameter of 8.5 μm arranged in an array of 600 × 600 horizontal is drawn in an area of 30 mm × 30 mm, with the holding hole and the holding hole having a vertical and horizontal interval of 50 μm. The resist 40 is exposed 42 with a UV exposure machine using the exposure photomask 41 and developed with a developer 43. The exposure time and the development time are adjusted so that the depth of the holding hole is 5 μm, which is equal to the film thickness of the resist 40. Thereafter, post-baking (180 ° C., 30 minutes) is performed using a hot plate to harden the resist structure.

  Next, a Cr film 38 having a film thickness of 100 nm is formed on the resist 40 structure having a plurality of holding holes by sputtering. Subsequently, a resist 46 is applied on the formed Cr film 38 to a thickness of 1 μm from the surface of the lower resist film by using a spin coater, naturally dried for 1 minute, and then pre-baked (95 3 minutes). A positive type resist 46 is used. The above is the description of the process shown in FIG. 12A, and the continuation is shown in FIG. 12B.

After that, as shown in FIG. 12B, the diameter φ8.5 μm arranged in an array of 600 × 600 horizontal in an area of 30 mm × 30 mm in the vertical and horizontal intervals between the holding holes and the holding holes of 50 μm. Using the exposure photomask 55 on which the fine hole pattern is drawn, the fine hole pattern is superimposed on the lower holding hole, the resist 46 is exposed 42 with a UV exposure machine, and developed with a developer 47. The exposure time and the development time are adjusted so that the depth of the holding hole is 6 μm, which is equal to the total thickness of the lower and upper resist layers. After the development, the exposed Cr film is peeled off with a 30% diammonium cerium nitrate solution 49 so that the ITO 37 is exposed on the bottom surface of the holding hole. Finally, the resist 46 is peeled off by the remover 56 so that the Cr film 38 which is a metal film is exposed. Thereafter, post baking (180 ° C., 30 minutes) is performed using a hot plate, the resist is solidified, and a light-shielding polymer film in which a metal film is disposed on the polymer film in which the holding holes are formed is integrated. The lower electrode substrate 36 is manufactured.

  Next, the spacer 16 is laminated and pressure-bonded on the lower electrode substrate 36 integrated with the light-shielding polymer film as shown in FIGS. The surface of the silicon sheet is sticky, and the parts are brought into close contact with each other by pressure bonding, and the suspension containing the biological sample can be put into the spacer without leakage. Since the area formed by hollowing out the spacer is 20 mm long × 20 mm wide, the number of holding holes existing in the accommodating portion is about 160,000. An upper electrode substrate 35 is disposed on the spacer 16 on the spacer 16, and a power source (signal generator) 4 is connected to each of the upper electrode substrate 35 and the lower electrode substrate 36 through the conductive wire 3.

As a biological sample, mouse spleen cells (particle size: about 6 μm) are suspended in a 300 mM mannitol aqueous solution, and a cell suspension is prepared to a density of 2.7 × 10 ⁵ cells / mL. .

  Subsequently, 600 μL of the cell suspension is injected from the introduction port 24 of the spacer 16 using a syringe (number of introduced cells: about 160,000), and a rectangular wave AC voltage having a voltage of 20 Vpp and a frequency of 3 MHz is applied between the electrodes by a signal generator. Apply to. Thus, one cell can be fixed to each of the plurality of holding holes formed in an array in an extremely short time of about 2 to 3 seconds. “Fix” means that a cell has entered the holding hole, and the same definition applies in the following comparative examples. At this time, the biological sample fixation rate in which approximately one cell enters one holding hole is approximately 90%. The biological sample fixation rate means that 225 holding holes (15 vertical x 15 horizontal) can be seen in the field of view of the microscope, and one biological sample is contained when the biological sample is introduced and fixed. It is defined as the number of holes divided by 225. The calculation method of the biological sample fixation rate is the same in the following examples and comparative examples.

Next, 600 μL of 2.5 × 10 ⁻⁴ % poly-L-lysine is injected into the container, and after standing for 3 minutes, voltage application is stopped. Next, by injecting a phosphate buffer solution (pH 7.2) to wash the poly-L-lysine in the housing part, cells can be electrostatically bound into the holding hole.

  Subsequently, B cells in the mouse spleen cell population are detected. A specific substance that is a target for detecting B cells is a CD19 molecule present on the surface of the B cells. The CD19 molecule is a B cell surface receptor and is found in cells throughout the differentiation of B lineage cells that differentiate from stem cell stages to eventually plasma cells, which include pre-B cells, B cells (Including naive B cells, antigen-stimulated B cells, memory B cells, plasma cells, and B lymphocytes) and follicular dendritic cells.

Next, a PE-labeled CD19 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), which is a labeling substance, is fed to a 600 μL container, and B cells are labeled by antigen-antibody reaction (4 ° C., 10 minutes). Wash with acid buffer and perform B cell detection. The labeled B cells are observed with a CCD camera through a fluorescence microscope (U-RFL-T / IX71, Olympus, Japan). Thereby, compared with the fluorescence microscope image of the cell before labeling, after labeling, only the fluorescence intensity on the surface of the B cell is increased, and the B cell can be detected.

(Example 2)
In Example 2, the structure and the fixing device shown in FIG. 4 are used.

As the substrate, a glass substrate 30 of 70 mm length × 40 mm width × 1 mm thickness is used. The spacer 16 is used by hollowing out the center of a silicon sheet having a length of 40 mm, a width of 40 mm, and a thickness of 1.5 mm into a length of 20 mm and a width of 20 mm. Moreover, as shown in FIG. 3, the inlet 24 and the outlet 25 for introducing and discharging the suspension contained in the biological sample are provided.

  The holding part having the plurality of through holes 9 and the comb-like electrode 21 are integrally formed on the member by the photolithography and etching methods shown in FIGS. 13A to 13C. As shown in FIG. 13A, an ITO 37 film having a thickness of 100 nm is formed on one surface of the glass substrate 30 by sputtering. Next, a resist 46 is applied on the formed ITO 37 to a film thickness of 1 μm using a spin coater, naturally dried for 1 minute, and then pre-baked (105 ° C., 15 minutes) using a hot plate. A positive type resist 46 is used.

  Next, using an exposure photomask 39 in which a comb-like electrode pattern in which an electrode a having a width of 10 μm and an electrode b having a width of 10 μm are formed at an interval of 50 μm in an area of 30 mm in length × 30 mm in width is used with a UV exposure machine. The resist 46 is exposed 42 and developed with a developer 47. The exposure time and development time are adjusted so that the film thickness peeled off by development is 1 μm, which is equal to the film thickness of the resist, so that the ITO is exposed on the bottom surface of the through hole. After the development, the exposed ITO film is peeled off by an ITO etching solution (ITO-Etchant, Wako Pure Chemical Industries) 48. Finally, the resist is peeled off by the remover 56 to form the comb-like electrode 21.

  Next, a process is demonstrated based on FIG. 13B. A resist 40 is applied on the substrate on which the comb-like electrode 21 manufactured as described above is disposed using a spin coater so as to have a film thickness of 5 μm. After naturally drying for 1 minute, prebaking is performed using a hot plate. (95 ° C., 3 minutes). An epoxy negative resist is used for the resist 40. Subsequently, in the area of 30 mm in length × 30 mm in width, a fine hole pattern with a diameter of 8.5 μm arranged in an array of 600 in length and 600 in width with the vertical and horizontal intervals between the through holes being 50 μm is drawn. Using the exposure photomask 41, the fine hole pattern is superimposed on the comb-shaped electrode, the resist 40 is exposed 42 with a UV exposure machine, and developed with a developer 43. The exposure time and the development time are adjusted so that the depth of the through hole is 5 μm, which is equal to the film thickness of the resist 40. Thereafter, post-baking (180 ° C., 30 minutes) is performed using a hot plate to harden the resist structure.

  Next, a Cr film 38 having a thickness of 100 nm is formed on the resist structure having a plurality of through holes by sputtering. Subsequently, a resist 46 is applied on the formed Cr film 38 to a thickness of 1 μm from the surface of the lower resist film by using a spin coater, naturally dried for 1 minute, and then pre-baked (95 3 minutes). Hereinafter, the process will be described with reference to FIG. 13C. After that, in the area of 30 mm in length and 30 mm in width, a fine hole pattern having a diameter of 8.5 μm arranged in an array of 600 vertical by 600 horizontal, with the vertical and horizontal intervals between the through holes being 50 μm, was drawn. Using the photomask 55 for exposure, the fine hole pattern is superimposed on the lower through hole, the resist 46 is exposed 42 with a UV exposure machine, and developed with a developer 47. The exposure time and the development time are adjusted so that the depth of the through-hole is 10 μm, which is equal to the total thickness of the lower layer and upper layer resists, and after development, the Cr film exposed by 30% diammonium cerium nitrate solution 49 is used. The ITO 37 is peeled off so that the ITO 37 is exposed on the bottom surface of the through hole. Finally, the resist 46 is peeled off by the remover 56 so that the Cr film 38 which is a metal film is exposed. Thereafter, post baking (180 ° C., 30 minutes) is performed using a hot plate, the resist is solidified, and a light-shielding polymer film in which a metal film is disposed on a polymer film in which a through hole is formed is integrated. The comb-shaped lower electrode substrate 21 is manufactured.

The spacers 16 are stacked on the comb-like lower electrode substrate 21 thus manufactured as shown in FIG. The surface of the silicon sheet is sticky, and the parts are brought into close contact with each other by pressure bonding, and the suspension containing the biological sample can be put into the spacer 16 without leakage. Since the area in which the spacer is hollowed is 20 mm long × 20 mm wide, the number of through holes existing in the accommodating portion is about 160,000. A power source (signal generator) 4 for applying a voltage between the electrodes is connected by a lead wire.

As a biological sample, mouse spleen cells (particle size: about 6 μm) are suspended in a 300 mM mannitol aqueous solution, and a cell suspension is prepared to a density of 2.7 × 10 ⁵ cells / mL. .

Subsequently, 600 μL of the cell suspension is injected from the introduction port 24 of the spacer 16 using a syringe (number of introduced cells: about 160,000), and a rectangular wave AC voltage having a voltage of 20 Vpp and a frequency of 3 MHz is applied between the electrodes by a signal generator. Apply to. Thus, one cell can be fixed to each of the plurality of holding holes formed in an array in an extremely short time of about 2 to 3 seconds. Next, 600 μL of 2.5 × 10 ⁻⁴ % concentration of poly-L-lysine is injected into the container, and after standing for 3 minutes, voltage application is stopped. Next, by injecting a phosphate buffer solution (pH 7.2) to wash the poly-L-lysine in the housing part, cells can be electrostatically bound into the holding hole.

  Subsequently, B cells in the mouse spleen cell population are detected. A specific substance that is a target for detecting B cells is a CD19 molecule present on the surface of the B cells. The CD19 molecule is a B cell surface receptor and is found in cells throughout the differentiation of B lineage cells that differentiate from stem cell stages to eventually plasma cells, which include pre-B cells, B cells (Including naive B cells, antigen-stimulated B cells, memory B cells, plasma cells, and B lymphocytes) and follicular dendritic cells.

Next, a PE-labeled CD19 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), which is a labeling substance, is fed to a 600 μL container, and B cells are labeled by antigen-antibody reaction (4 ° C., 10 minutes). Wash with acid buffer and detect B cells. The labeled B cells are observed with a CCD camera through a fluorescence microscope (U-RFL-T / IX71, Olympus, Japan). Thereby, compared with the fluorescence microscope image of the cell before labeling, after labeling, only the fluorescence intensity on the surface of the B cell is increased, and the B cell can be detected.

  Biological sample collecting means is installed in the fixing device. As the biological sample collecting means, a pipette capable of accurately collecting a biological sample using an electroosmotic flow is used. Thereby, the B cell detected with the fluorescence labeled antibody can be collected while observing with a microscope.

(Example 3)
Using the same structure and fixing device as in Example 1, the detection accuracy of one labeled cell fixed to one holding hole can be confirmed as follows.

First, a part of mouse spleen cells is stained with CellTracker Green CMFDA (Invitrogen). The sample to be detected is the CellTracker Gr.
3 cells stained with een CMFDA and unstained mouse spleen cells (particle size of about 6 μm)
: Using the sample mixed in 7, prepare a cell suspension to a density of 2.7 × 10 ⁵ cells / mL.

Subsequently, 600 μL of the cell suspension is injected from the introduction port 24 of the spacer 16 using a syringe (number of introduced cells: about 160,000), and a rectangular wave AC voltage having a voltage of 20 Vpp and a frequency of 3 MHz is applied between the electrodes by a signal generator. And one cell is fixed to each of the plurality of holding holes. Next, 600 μL of 2.5 × 10 ⁻⁴ % poly-L-lysine is injected into the container, and after standing for 3 minutes, voltage application is stopped. Next, a phosphate buffer solution (pH 7.2) is injected to wash the poly-L-lysine in the container, thereby electrostatically binding the cells into the holding holes.

Cells stained with CellTracker Green CMFDA are observed with a CCD camera through a fluorescence microscope (U-RFL-T / IX71, Olympus, Japan).
Thereby, the fluorescence of the labeled cell can be detected from the plurality of spleen cells fixed in the holding hole.

(Comparative example)
For comparison, the following operation was performed using a structure without a light-shielding film. First, 600 μL of the spleen cell suspension (number of cells: about 160,000) was injected from the introduction port of the spacer using a syringe, and a rectangular wave AC voltage having a voltage of 20 Vpp and a frequency of 3 MHz was applied between the electrodes by a signal generator. However, one cell could be fixed to each of the plurality of holding holes in an extremely short time of about 2 to 3 seconds.

Next, 600 μL of 2.5 × 10 ⁻⁴ % concentration of poly-L-lysine was injected into the container, and after standing for 3 minutes, voltage application was stopped. Next, by injecting a phosphate buffer solution (pH 7.2) and washing the poly-L-lysine in the container, cells could be electrostatically bound into the through-hole.

Subsequently, B cells in the mouse spleen cell population were detected. As in Example 1, a specific substance as a target for detecting B cells is a CD19 molecule present on the surface of the B cell, and a B cell by PE label-CD19 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) as a labeling substance (4 ° C., 10 minutes). Labeled B cells were transferred to the CCD through a fluorescence microscope (U-RFL-T / IX71, Olympus, Japan).
Observed with a camera. As a result, optical noise due to fluorescence scattering from the insulator film (polymer film) around the holding hole was large, and the fluorescence on the surface of the B cell fixed in the holding hole could not be detected with high accuracy.

2: Biological sample 3: Conductive wire 4: AC power supply 9: Holding hole 12: Line of electric force 14: Particle fixing structure 15: Substrate 16: Spacer 17: Top lid 18: Insulator film 19: Light shielding film 20: Holding part 21: Comb electrode 22: Electrode 23: Electrode 24: Inlet 25: Discharge 26: Dielectrophoretic force 27: Substance binding to biological sample 28: Biological sample 30: Glass substrate 31: Labeling substance 32: Abnormal cell 33: Fluorescence microscope 34: Biological sample collecting means 35: Upper electrode substrate 36: Lower electrode substrate 37: ITO
38: Cr film 39: Photomask for exposure (comb pattern)
40: Resist (negative type)
41: Photomask for exposure (fine hole pattern: for negative resist)
42: Exposure 43: Developer (negative type)
44: Lower electrode substrate integrated with light-shielding polymer film 45: Accommodating portion (space)
46: Resist (positive type)
47: Developer (positive type)
48: ITO etching solution 49: Ammonium cerium nitrate solution 54: Between holding holes (light-shielding film)
55: Photomask for exposure (fine hole pattern: for positive resist)
56: Remover

Claims (9)

A structure for fixing particles, having a plurality of holding holes for holding the analyte particles in order to detect light emitted by a substance indicating the presence of a component constituting the analyte particles,
A flat substrate;
A holding portion disposed on the substrate and formed with the plurality of holding holes,
The holding part includes at least a light shielding film provided on an upper surface side of the holding part, and an insulator film provided between the light shielding film and the substrate, and the holding hole includes the holding part. the opening in the light shielding film, and extend to the substrate via the insulating film located on the surface side up,
The light shielding film is made of a hydrophilic material, the insulator film is made of a hydrophobic material,
The light shielding film is provided around the opening portion of the holding hole in the upper surface of the holding portion.
Particle fixing structure. A storage unit disposed on the holding unit and storing a suspension containing the analyte particles;
The holding hole communicates with the accommodating portion;
The particle fixing structure according to claim 1 . A pair of electrodes disposed at positions corresponding to different holding holes are provided on the surface of the substrate on the holding portion side,
The holding hole extends from the upper surface of the holding part to the electrode on the substrate.
The structure for particle | grain fixation of Claim 1 or Claim 2 . A first electrode disposed at a position corresponding to the holding hole is provided on the surface of the substrate on the holding unit side, and the holding hole is formed on the substrate from the upper surface of the holding unit. Extending to the electrode,
A second electrode is provided on a side opposite to the first electrode across the holding portion and the accommodating portion;
The structure for fixing particles according to claim 2 . The substrate is made of a translucent material, and the electrode provided on the surface of the substrate on the holding portion side is I
Is TO,
The structure for particle | grain fixation of Claim 3 or Claim 4 . The light shielding film is a metal film,
The structure for particle | grain fixation as described in any one of Claims 1-5 . The structure for fixing particles according to claim 3 or 4 ,
A power source for applying an alternating voltage for generating a dielectrophoretic force on the electrode;
A detection unit for detecting light emitted by a substance indicating the presence of a component constituting the analyte particle held in the holding hole of the particle fixing structure after voltage application from the power source;
A particle analyzer. A specimen particle is fixed and fixed to a holding hole on the particle fixing structure according to any one of claims 1 to 6 or the particle fixing structure of the particle analyzer according to claim 7. A method for analyzing analyte particles, comprising: detecting light emitted by a substance indicating the presence of a component constituting the analyte particles. The analysis method according to claim 8 , wherein the substance indicating the presence of a component constituting the analyte particle is a labeling substance that specifically binds to the component.

Images