JP7217516B2 - Target substance detection device and charge processing particles - Google Patents

Description

INDUSTRIAL APPLICABILITY The present invention is a target substance detection device that detects a target substance by utilizing changes in optical signals when the target substance present in a liquid sample is moved using an electric field, and is used for detecting the target substance. It relates to charge processing particles.

In recent years, methods have been developed for detecting and quantifying minute substances present in a solution, particularly bio-related substances such as DNA, RNA, proteins, viruses, and bacteria. Such methods include, for example, surface plasmon resonance (SPR) immunoassay, total internal reflection illumination fluorescence microscopy (TIRFM), surface plasmon resonance excitation enhanced fluorescence spectroscopy (SPFS) and the like.

The surface plasmon resonance immunoassay method is a technique that combines the specific selectivity of the antigen-antibody reaction with a surface plasmon resonance sensor, which is a highly sensitive refractometer. - Antibody binding can be detected and quantified in real time with high accuracy (see Non-Patent Document 1).

In the total internal reflection fluorescence microscope, the incident light is totally reflected at the interface between the sample and the cover glass or slide glass, and the resulting evanescent field is used as excitation light to perform fluorescence observation with little background light that causes noise. technology (see Patent Document 1). This technique is a technique capable of realizing super-resolution and enables single-molecule observation.

The surface plasmon resonance excitation-enhanced fluorescence spectroscopy uses an optical arrangement called the Kretschmann arrangement, and the total reflection of incident light at the interface between the gold thin film layer on the glass surface in contact with the prism and the liquid sample causes It is characterized by exciting surface plasmon resonance and forming an enhanced electric field on the gold thin film surface. This is a technique for observing fluorescence with little background light by exciting fluorescent molecules existing in the enhanced electric field by using the light enhanced near the surface of the gold thin film by surface plasmon resonance as excitation light to generate strong fluorescence (patented Reference 2).

In addition, as a method of obtaining an enhanced electric field by generating an electric field enhancement by such total reflection of light, there are known methods such as those described in Non-Patent Documents 2 to 8, for example. The present inventors placed a detection plate in which a silicon layer and a _SiO2 layer were laminated in this order on a silica glass substrate on a trapezoidal prism made of silica glass, and detected light under conditions of total reflection on the surface of the detection plate via the prism. Non-Patent Document 2 reported a method of obtaining an enhanced electric field by irradiating .

Non-Patent Document 3 discloses a method of generating an enhanced electric field by generating surface plasmon resonance using the Kretschmann configuration. Non-Patent Document 4 discloses a method of obtaining an enhanced electric field by injecting light into a prism in the Kretschmann configuration using a dove prism to generate surface plasmon resonance. Non-Patent Document 5 and Non-Patent Document 6 disclose a method of obtaining an enhanced electric field using a resonant mirror. In Non-Patent Document 7, a metal layer and a transparent dielectric layer are laminated in this order on a prism to form a structure called a leaky mode sensor, and light is irradiated through the prism to irradiate the surface of the dielectric layer. A method for obtaining an enhanced electric field at is disclosed. In Non-Patent Document 8, a metal layer is formed on a prism, and two types of transparent dielectric layers with different refractive indices are laminated one by one on top of it to generate a stronger enhanced electric field than the leaky mode sensor structure. A method for obtaining is disclosed.

Furthermore, Patent Documents 3 and 4 disclose a method of obtaining an enhanced electric field by giving a channel a prism shape that generates surface plasmon resonance, and generating surface plasmon resonance on the bottom surface or the side surface of the channel.

Also known is a method using magnetic particles as a label in order to facilitate the adsorption or proximity of a target substance to a detection surface and to realize measurement in a short period of time (Patent Documents 5 and 6). In these methods, a combined body of a magnetic label, a photoresponsive labeling substance and a target substance is attracted to a local region by applying a magnetic field, and only a predetermined region including this local region is irradiated with excitation light. Detection is performed while excluding the signal of the photoresponsive label that does not form a conjugate between the target substance and the magnetic label.

Further, as a method for detecting and quantifying the bio-related substance, a method using propagating light as detection light has also been proposed. For example, such methods include fluorescence immunoassay (FIA method), enzyme-linked immunosorbent method (ELISA method), and the like.
In the FIA method, an antibody that specifically binds to a target substance such as a specific bacterium or virus is used to bind a fluorescent dye, and the target substance is detected and quantified by observing the emission of the fluorescent dye with a fluorescence microscope or the like. method.
In the ELISA method, the target substance is immobilized on a detection plate using an antigen-antibody reaction, then an enzyme-labeled antibody is bound, a substrate that develops color with the enzyme is added, and the target substance is detected from the color change. is detected and quantified.
All of these methods are widely used as well-established methods for detecting bio-related substances, but these methods require multiple reaction steps and repeated washing steps, and it takes a lot of time and effort to obtain measurement results. I have a problem that I need. In addition, there is a demand for further improvement in detection sensitivity.

A measurement method using magnetic particles has been proposed as a method for improving the detection sensitivity of the target substance detection using the bio-related substance detection method. For example, a detection method in which a conjugate containing the target substance and the magnetic particles is collected on the bottom side of a liquid sample container and immobilized on the bottom surface of the container by an antigen-antibody reaction between an antibody placed on the bottom surface of the container and the conjugate. is disclosed (see Patent Document 7).
However, in the measurement method using such magnetic particles, although the detection sensitivity can be improved by the concentration effect of collecting the binder at the detection position by the magnetic field, contaminants floating at the detection position of the concentration destination, the liquid A noise signal caused by the contaminants adsorbed on the bottom surface of the sample container, scratches on the bottom surface of the liquid sample container, fluctuations in the light source output of the detection light used for detection, etc., and an optical signal based on the conjugate. can not be distinguished, there is a problem of low detection accuracy. Such a problem becomes even more conspicuous when detecting the minute substances.
In addition, in order to eliminate the noise signal based on the contaminants adsorbed on the bottom surface of the liquid sample container, it is necessary to perform a cleaning process to remove the contaminants for each detection, and the detection efficiency is still low. be.

In order to solve these problems, the present inventor has proposed an external force-assisted sensor, which is a target substance detection device provided with a magnetic field applying section (see Patent Documents 8 to 10). In this proposal, by observing with a photodetector an optical signal based on the combination of the magnetic particles and the target substance that moves along with the application of the magnetic field from the magnetic field applying unit, the target substance floats at the detection position. A noise signal caused by contaminants, the contaminants adsorbed on the bottom surface of the liquid sample container, scratches on the bottom surface of the liquid sample container, fluctuations in the light source output of the detection light used for detection, etc.; to distinguish between optical signals based on
That is, since the noise signal or the like does not move while the conjugate moves due to the application of the magnetic field, it can be determined that the optical signal is caused by the conjugate by observing the movement of the detected optical signal. It is possible to distinguish whether the noise is caused by the noise signal or the like, and by extension, the accuracy and efficiency of detection are improved.

However, in the magnetic field applying section of the external force assisted sensor, whether it is configured with a permanent magnet or an electromagnet, in order to generate a magnetic field sufficient to move the combined body, , requires a certain size. In addition, since the electronic parts constituting the photodetector may be adversely affected by the strong magnetic field from the magnetic field applying unit, the distance between the magnetic field applying unit and the photodetector is narrowed by a certain amount or more. I can't.
Therefore, it is difficult to reduce the size of the device in the external force assisted sensor in which the magnetic field applying unit is arranged, and furthermore, the portability is poor, so it is necessary to carry it around for purposes such as performing detection at an outdoor detection site. There is a problem that it is not suitable for use.

JP-A-2002-236258 WO2015/194663 JP 2013-24606 A JP 2010-145408 A JP 2011-33454 A JP-A-2005-77338 JP-A-4-102062 International Publication No. 2017/187744 International Publication No. 2018/100779 International Publication No. 2018/100780

D. R. Shankaran et al. Sensors and Actuators B, Vol. 121 (2007) 158-177 M. Fujimaki et al. Optics Express, Vol. 23 (2015) pp.10925 -10937 C. Nylander et al. Sensors and Actuators, Vol. 3 (1982/83) pp. 79-88 O. R. Bolduc et al. Talanta, Vol. 77 (2009) pp. 1680 - 1687 R. Cush et al. Biosensors and Bioelectronics, Vol. 8 (1993) pp. 347-353 P.E. Buckle et al. Biosensors and Bioelectronics, Vol. 8 (1993) pp. 355-363 R. P. Podgorsek et al. Sensors and Actuators B, Vol. 38-39 (1997) pp. 349-352 S. Hayashi et al. Applied Physics Express, Vol. 8, 022201 (2015)

The present invention solves the above-mentioned problems in the conventional technology, and provides a target substance detection device that is excellent in detection accuracy and efficiency, is small and has excellent portability, and charged processing particles used in the target substance detection device. intended to

Means for solving the above problems are as follows. Namely
<1> A liquid sample containing a target substance and charged particles that form a bond with the target substance is introduced onto the surface and irradiated with light to generate detection light of either propagating light or near-field light. a liquid sample holding part having a liquid sample introduction plate arranged thereon and capable of holding the liquid sample on the surface of the liquid sample introduction plate; a moving electric field applying unit provided with a pair of electrodes disposed on the surface side of the liquid sample introduction plate and configured to move the combined body in the liquid sample between the electrodes by applying a voltage; an optical signal detection unit capable of detecting the movement of the conjugate from a signal change of the optical signal based on the detection light , wherein the moving electric field applying unit is separated from the surface of the liquid sample introduction plate. A target substance detection device comprising a pair of surface electrodes formed by
< 2 > Further, it has an attracting electric field application unit provided on the surface side of the liquid sample introduction plate, and having a pair of electrodes for moving the binding bodies in the liquid sample between the electrodes by applying a voltage and attracting them to the surface. The target substance detection device according to <1 > .
< 3 > An attracting electric field applying section is formed on the surface of the liquid sample introducing plate at a position between a pair of surface electrodes spaced apart from each other and formed on the surface of the liquid sample introducing plate constituting the moving electric field applying section. The above-described < 2 >, comprising a surface electrode, and a cover electrode formed of a transparent electrode material and arranged to face the surface electrode in the vertical direction so as to cover the liquid sample introduced onto the liquid sample introduction plate. target substance detection device .

According to the present invention, a target substance detection device that can solve the above-mentioned problems in the conventional technology, is excellent in detection accuracy and efficiency, is small and has excellent portability, and charged processing particles used in the target substance detection device can be provided.

BRIEF DESCRIPTION OF THE DRAWINGS It is explanatory drawing which shows the outline|summary of the target substance detection apparatus which concerns on embodiment of this invention. 1 is an explanatory diagram (1) showing an outline of a method for preparing a liquid sample 4; FIG. FIG. 2 is an explanatory diagram (2) showing an outline of a method for preparing a liquid sample 4; 3 is an explanatory diagram (3) showing an outline of a method for preparing a liquid sample 4; FIG. FIG. 4 is an explanatory diagram (4) showing an overview of the preparation method of the liquid sample 4; FIG. 5 is an explanatory diagram (5) showing an overview of the preparation method of the liquid sample 4; FIG. 4 is an explanatory diagram showing a main part of a modified example of the target substance detection device 1; Before voltage application to the surface electrodes 3A and 3B, and after 30 seconds, 60 seconds, 90 seconds, 120 seconds, 150 seconds after applying a DC voltage of 2.8 V to the surface electrodes 3A and 3B, and After 180 seconds, images acquired with an exposure time of 10 seconds are superimposed and displayed.

(Target substance detection device)
The target substance detection device of the present invention has a liquid sample holding section, a light irradiation section, a moving electric field application section, and an optical signal detection section, and optionally other sections.

<Liquid sample holder>
The liquid sample holding section is a section in which a liquid sample introduction plate is arranged and a liquid sample is held on the surface of the liquid sample introduction plate.

- liquid sample -
The liquid sample contains a target substance and charged particles that form a conjugate with the target substance.

--Target substance--
The target substance is not particularly limited and can be selected according to the purpose. Examples thereof include DNA, RNA, protein, viruses, bacteria, contaminants and the like.
Specific sample liquids to be detected for the target substance include, for example, solid samples such as blood, saliva, urine, liquid chemicals, environmental water, sewage, beverages, homogenized food solutions, wipes, and powders. Examples thereof include a solution dissolved in a solvent such as water, and a gas phase concentrated liquid in which gases, fine particles, etc. in the gas phase are captured.
Therefore, as a specific example of the liquid sample, one obtained by adding the charged particles and the like to the subject liquid can be mentioned.

--charged particles--
The charged particles combine with the target substance to form the conjugate, and have the effect of moving the target substance along with it based on the electric field applied from the moving electric field applying unit.
The charged particles are not particularly limited as long as they have such an action, and can be appropriately selected according to the purpose. From the viewpoint of ease of use, the following charged particles are preferred.
In addition, the charged particles can be dispersed in a liquid and stored. By preparing and storing the charged particles, the liquid sample can be prepared when the target substance is detected. can be done efficiently.

--- Charged particles ---
In the charged particles, a binding substance capable of binding to the target substance is bound to the first functional group of a part of the particle body surface-modified with a plurality of first functional groups or a derivative group thereof, and A particle in which a charged molecule having a plurality of second functional groups that ionize into cations or anions in a liquid is bound to the first functional group that is not bound to the binding substance or a derived group thereof. .

The particle body is not particularly limited, and known particles can be mentioned. However, from the viewpoint of mobility accompanying the application of an electric field, it is formed mainly of any one of polystyrene, silica, dextran, polyvinyl alcohol and polyester. It is preferable that the particles are spherical particles.
The diameter of the spherical particles is not particularly limited, but is preferably 50 nm to 10 μm when detecting by propagating light, and preferably 50 nm to 2 μm when detecting by near-field light.
Further, the particle main body surface-modified with a plurality of the first functional groups is used in various applications, and can be appropriately selected from known ones and used.

The first functional group is not particularly limited, but a carboxy group (COOH), an amino group (NH ₂ ), and a sulfhydryl group (SH) are used from the viewpoint of stably binding the binding substance and the charged molecule. preferable.
The derivatizing group of the first functional group includes a group derivatized to bind the binding substance and the charged molecule to the first functional group.
For example, when the first functional group is a carboxy group (COOH), the formed active ester (NHS ester The carboxy group residue (CO) in the amide bond (CONH) formed by dehydration condensation of the binding substance having an amino group in the body) and the charged molecule, or the binding substance and the The above-mentioned carboxy group residue (COO) when a charged molecule is ester-bonded (COOR) corresponds to the above-mentioned derivative group.

The binding substance is not particularly limited as long as it binds to the first functional group or its derivative group at one part and binds to the target substance at the other part. Substances that give known antigen-antibody reaction, binding by aptamer, DNA hybridization, biotin-avidin binding, chelate binding, etc. can be used. Specific examples include antibodies, aptamers, DNA probes, RNA probes, peptides, sugar chains, protein A, protein G, avidin and its derivatives (such as neutravidin), streptavidin, biotin and its derivatives.

The charged molecule is preferably a molecule having, in one molecule, a plurality of second functional groups that bind to the first functional group or its derivative group and ionize into cations or anions in a liquid. is necessary. Having a plurality of the second functional groups in one molecule increases the electric charge and improves the mobility associated with the application of an electric field.
Now, taking as an example the polystyrene beads surface-modified with the carboxy group when the carboxy group is selected as the first functional group, the polystyrene beads are dispersed in a neutral liquid. , the carboxyl group is negatively charged by anionization, but when a part of the carboxyl group is bound to the binding substance by an amine coupling reaction or the like, the binding substance and the unbound negative charge on the surface of the polystyrene bead become negative. The number of ionized carboxyl groups is reduced and the charge is reduced. As a result, it is difficult to obtain the electric charge necessary for movement upon application of an electric field.
In fact, in experiments using the polystyrene beads surface-modified with the carboxy group, when only IgG antibody molecules are bound as the conjugate, there are cases where the movement of the polystyrene beads in the electric field cannot be confirmed, In the case performed under the same conditions except that glutamic acid as the charged molecule was bound in addition to the IgG antibody molecule, movement of the polystyrene beads in the electric field was confirmed.

The second functional group is not particularly limited, and when it is ionized into cations in a liquid, examples thereof include an amino group, an imino group, and an imidazole group, and when it is ionized into anions in a liquid. includes, for example, a carboxy group, a sulfo group, a phosphate group, a thiol group, a hydroxy group, and the like.

Suitable compounds satisfying the above charged molecule requirements include glutamic acid and aspartic acid having two carboxy groups in one molecule, and arginine having two amino groups in one molecule.

Further, the binding of the binding substance and the charged molecule to the first functional group or derivative thereof may be a direct binding or an indirect binding. Interposed between the first functional group or a derivative thereof and the binding substance and the charged molecule can be a conjugate molecule that mediates these bonds.
The conjugating molecule is not particularly limited and can be appropriately selected depending on the purpose. Examples include protein A, protein G, avidin and its derivatives (neutravidin etc.), streptavidin, biotin and its derivatives. be done.

The charged particles, including the charged particles, need to have a moving speed in the liquid at which movement in response to application of an electric field can be determined by signal detection by the optical signal detection unit.
The moving speed in the liquid is such that when the surface of the liquid sample introduction plate is observed by the optical signal detection unit, the light spot of the conjugate containing the charged particles continuously moves by one pixel or more within a practical observation time. or a speed exceeding the average amount of random movement due to Brownian motion, whichever is greater.
Here, the practical observation time is 20 minutes or less in on-site measurement, preferably 5 minutes or less.
In addition, it may not be possible to distinguish between the light spot movement due to the electric field application and the light spot movement due to the Brownian motion with only the two observation images before and after the electric field application. should be obtained at least three times in succession. Therefore, the time taken to acquire one image is 400 seconds or less, preferably 100 seconds or less.
The size of one pixel of the observed image varies depending on the element of the image sensor, but generally ranges from 1 μm to 10 μm. Therefore, it is preferable that the moving amount of the combined body in the liquid is 10 μm or more.
Therefore, the speed at which the movement of the light spot of the conjugate by one pixel or more can be continuously confirmed within the practical observation time is 0.025 μm/s or more, preferably 0.1 μm/s or more. .
On the other hand, the average amount of movement Δx of random movement due to Brownian motion is obtained by the following formula (1) for spherical particles.

ただし、前記式（１）中、ｔは、観察時間を示し、ｋ_Ｂは、ボルツマン定数を示し、Ｔは、絶対温度を示し、ηは、溶媒の粘度を示し、ｄは、粒子径を示す。
前記式（１）に示されるように平均移動量Δｘは、前記荷電粒子の粒子径が小さくなるほど大きくなる。ここでは、直径１００ｎｍの前記荷電粒子を基準粒子として考え、温度を２０℃、溶媒を水として計算を行うと、観察時間１００秒において、平均移動量Δｘが２９μｍとなり、観察時間４００秒において平均移動量Δｘが５８μｍとなる。
したがって、ブラウン運動によるランダム移動の平均移動量を上回る速度は、０．３μｍ／ｓ以上となる。
よって、前記実用的な観察時間内で前記荷電粒子を含む前記結合体の光点の１ピクセル以上の移動が連続して確認できる速度、あるいは、ブラウン運動によるランダム移動の平均移動量を上回る速度のいずれか大きい方とする前記移動速度は、０．３μｍ／ｓ以上である。

However, in the formula (1), t indicates the observation time, k _B indicates the Boltzmann constant, T indicates the absolute temperature, η indicates the viscosity of the solvent, and d indicates the particle size. .
As shown in the formula (1), the average moving amount Δx increases as the particle diameter of the charged particles decreases. Here, when the charged particles with a diameter of 100 nm are considered as the reference particles, the temperature is 20° C., and the solvent is water, the average movement amount Δx is 29 μm at the observation time of 100 seconds, and the average movement amount is 29 μm at the observation time of 400 seconds. The amount Δx becomes 58 μm.
Therefore, the speed exceeding the average amount of random movement due to Brownian motion is 0.3 μm/s or more.
Therefore, the speed at which the movement of one or more pixels of the light spot of the conjugate containing the charged particles can be continuously confirmed within the practical observation time, or the speed exceeding the average amount of random movement due to Brownian motion. The moving speed, whichever is greater, is 0.3 μm/s or more.

By the way, the movement speed of the charged particles in the liquid increases as the strength of the electric field increases. On the other hand, if the voltage applied to the constituent electrodes of the moving electric field applying section is too high, the electrolysis of the liquid in the liquid sample proceeds and bubbles are generated, which may hinder observation of the movement of the combined body. . In addition, a high applied voltage is not suitable because it requires a large power supply unit, and from the viewpoint of providing the target substance detection device that is compact and excellent in portability, it is preferable that it can be driven by a battery.
The voltage of lithium ion polymer secondary batteries, which are widely used in mobile phones, laptop computers, etc., is 3.7V, and the voltage of coin-type lithium batteries, which are widely used in small devices, is 3.0V.
Therefore, a suitable voltage to be applied to the moving electric field applying section selected from the driving sources of the moving electric field applying section is 3.0 V or less.
On the other hand, as the lower limit of the applied voltage, it is necessary to apply a voltage exceeding the voltage required to cause the electrolytic reaction, since the electrode must cause an electrolytic reaction in order to cause electrophoresis. The type of electrolytic reaction that occurs varies depending on the constituents of the liquid sample. Considering water as a representative electrolyzed reactant, a voltage exceeding 1.23 V is applied to cause the electrolytic reaction. (See reference 1 below). Therefore, the lower limit of the applied voltage is about 1.3V.
From the above, when a voltage within the range of 1.3 V to 3.0 V is applied to the pair of electrodes constituting the moving electric field applying unit, the charged particles have a thickness of 0.3 μm at any voltage value. /s or more is preferable.
As the charged particles having the charged molecules bonded to the surface of the particle body, the polystyrene beads used in the examples described later were found to have a voltage of 2.8 V from the results of observation of the moving light point of the conjugate. confirmed that it moves a distance of 280 μm in 180 seconds, and the moving speed in the liquid is 1.6 μm/s, which greatly exceeds 0.3 μm/s.
In this specification, the term "moving speed in liquid" indicates the speed at which a substance moves between a pair of electrodes by electrophoresis when a solution containing 90% by volume or more of water is used as the liquid.
Reference 1: Watanabe et al., Basic Science Course Electrochemistry, Maruzen (2001)

--labeling substance--
The target substance is detected by the optical signal detector through the movement of the optical signal emitted from the conjugate.
When the target substance does not emit an optical signal such as fluorescence, the target substance is bound to a photoresponsive labeling substance for optical signal detection by the optical signal detection unit, and the target substance is bound to the target substance based on the movement of the binding substance. Detect signal changes in the optical signal.

The labeling substance is a substance that binds to the conjugate via the target substance and emits an optical signal upon receiving light resulting from light irradiation from the light irradiation unit.
The labeling substance is not particularly limited as long as it has such properties, and can be appropriately selected according to the purpose. Examples include known fluorescent substances such as fluorescent dyes and quantum dots. .

The method for binding the target substance and the labeling substance is not particularly limited and can be appropriately selected according to the purpose. - Known binding methods such as avidin binding, chelate binding, and amide binding can be used.
When a dye is used as the labeling substance, staining the target substance with the dye is also effective as a method of binding the target substance and the labeling substance.
Examples of the method of binding by physical adsorption include a method of binding the target substance and the labeling substance using electrostatic binding force such as hydrogen bonding.
Among these binding methods, the antigen-antibody reaction, the aptamer binding, the DNA hybridization, the biotin-avidin binding, the chelate binding, and the like are binding methods in order to prevent the labeling substance from binding to the contaminants. Preferably, the method specifically binds the target substance and the labeling substance.

－Liquid sample introduction plate－
The liquid sample introduction plate is a member on which the liquid sample is introduced onto the surface and which is irradiated with light to generate detection light of either the propagating light or the near-field light.
As the liquid sample introduction plate, the liquid sample is introduced onto the front side, and the transmitted light of the light irradiated from the back side or the front side is used as the propagating light, and the side opposite to the side irradiated with the light is used. a light-transmitting plate capable of propagating to the surface, a reflecting plate capable of propagating above the surface as the propagating light, the reflected light of the light irradiated from the surface side while the liquid sample is introduced onto the surface, and the liquid An introduction plate through which the sample is introduced onto the surface, and the near-field light is generated on the surface by light irradiated onto the surface under conditions of total internal reflection while the liquid sample is introduced onto the surface. It is formed by any of the possible sensing plates.
The propagating light is generally defined as light that does not include near-field light that exhibits abrupt attenuation at a distance of several hundred nm to several μm from the source. It means that it does not contain near-field light, and means light that does not exhibit rapid attenuation at a distance of several hundred nm to several μm from the surface of the liquid sample introduction plate. Further, the near-field light means light that exhibits abrupt attenuation at a position separated from the surface of the liquid sample introduction plate by a distance of several hundred nm to several μm.

The light-transmitting plate is not particularly limited and can be appropriately selected according to the purpose. can be used.
The reflecting plate is not particularly limited and can be appropriately selected according to the purpose. A reflector can be used.
The introduction plate is not particularly limited and can be appropriately selected according to the purpose. A shaped member can be used.
Further, the detection plate is not particularly limited and can be appropriately selected according to the purpose, and a known detection plate such as a detection plate used for a known surface plasmon resonance sensor or a known optical waveguide mode sensor is used. be able to.

The liquid sample introduction plate is not particularly limited and can be appropriately selected according to the purpose, but the surface is preferably treated with an adsorption inhibitor that suppresses adsorption of the conjugate. When such a surface treatment is applied, the conjugate is suppressed from being adsorbed to the surface of the liquid sample introduction plate, and the movement of the conjugate due to the electric field can be assisted.
The adsorption inhibitor is not particularly limited, and can be appropriately selected from known adsorption inhibitors according to the type of substance constituting the conjugate.
For example, as the surface treatment method, when the target substance is the protein, a known blocking method for suppressing adsorption of the protein can be selected. The blocking method is not particularly limited, and examples thereof include a method using polyethylene glycol, a method using ethanolamine, and a method using skim milk.

The structure of the liquid sample holding part is not particularly limited, and can be appropriately selected according to the purpose. may be sandwiched between the plate-like translucent member and the liquid sample introduction plate, and the liquid layer of the liquid sample may be held on the surface of the liquid sample introduction plate.
Further, the liquid sample holding portion may be configured by a square-shaped liquid cell whose bottom surface is formed by the liquid sample introduction plate.
In addition, as the liquid sample holding portion, the region on the surface of one liquid sample introduction plate may be divided into a plurality of channels to form a multi-channel.

<Light irradiation part>
The light irradiation section is a section that irradiates the liquid sample introduction plate with the light to generate the detection light.
The light irradiation section is formed by any one of a rear surface side light irradiation section, a front side light irradiation section, a side surface side light irradiation section, and a total reflection light irradiation section.

The rear surface side light irradiation section can irradiate the light from the rear surface of the liquid sample introduction plate when the liquid sample introduction plate is formed of the transparent plate.
The configuration of the backside light irradiation section is not particularly limited and can be appropriately selected according to the purpose. For example, it can be configured in the same manner as a known light irradiation section used in a known transmission microscope. .

The surface-side light irradiation section can irradiate the light from the surface side of the liquid sample introduction plate when the liquid sample introduction plate is formed of either the light-transmitting plate or the reflection plate.
The configuration of the surface-side light irradiation unit is not particularly limited and can be appropriately selected according to the purpose. In addition, when it is formed of the light-transmitting plate, it can be configured in the same manner as a known light irradiation section used in a known transmission microscope.

The side-side light irradiator is configured to apply light to the liquid sample held on the liquid sample introduction plate from the side surface of the liquid sample introduction plate when the liquid sample introduction plate is formed by the introduction plate. The light can be applied in a direction parallel to the in-plane direction of the surface of the sample introduction plate.
The configuration of the side light irradiation section is not particularly limited and can be appropriately selected according to the purpose. For example, it can be configured in the same manner as a known light irradiation section.

The total reflection light irradiation section can irradiate the surface with the light under total reflection conditions when the liquid sample introduction plate is formed of the detection plate. Regarding the total reflection on the surface, there is no particular limitation on the incident direction of the light as long as the conditions for total reflection on the surface of the detection plate can be satisfied. For example, by forming the surface of the detection plate into a waveguide structure, a grating formed on the front surface side, the back surface side, or the side surface side of the detection plate, or arranged on the front surface side, the back surface side, or the side surface side of the detection plate. The light can be introduced into the waveguide structure from the surface side through the prism, and the total reflection condition on the detection plate surface can be satisfied by utilizing the total reflection within the waveguide structure. Also, the prism may be formed as a partial structure of the detection plate.
The configuration of the total reflection light irradiation unit is not particularly limited, and can be appropriately selected according to the purpose. can be configured to

The light sources in the back side light irradiation section, the front side light irradiation section, the side side light irradiation section, and the total reflection light irradiation section are not particularly limited, and can be appropriately selected according to the purpose. Light-emitting devices such as known lamps, LED devices, and laser light irradiation devices can be used.
Further, as the back side light irradiation section, the front side light irradiation section and the total reflection light irradiation section, optical elements other than the light source are not particularly limited, and known optical microscopes, known surface plasmon resonance sensors, A known optical element used in a known optical waveguide mode sensor can be appropriately adopted and constructed according to the purpose.

<Moving electric field applying unit>
The moving electric field application unit is provided on the surface side of the liquid sample introduction plate, and includes a pair of electrodes for moving the combined body in the liquid sample between the electrodes by application of a voltage. That is, the moving electric field applying section has an effect of electrophoresis of the conjugate between the pair of electrodes by an electric field generated by voltage application.
By arranging such a moving electric field application unit, it is possible to reduce the size and improve the portability of the device as compared with the conventional example in which a magnetic field is used as an external force for moving the combined body.

The moving electric field application unit is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include known electrodes and a power source for applying voltage to the electrodes.
Specifically, depending on whether the charged particles are positively or negatively charged, a voltage is applied to the pair of electrodes to move the target substance bound to the charged particles in the negative or positive direction by electrophoresis. .
The direction in which the combined body is moved is either a direction having a vector component in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate or a direction having a vector component in a direction away from the liquid sample introduction plate. direction.
When the conjugate is moved in a direction parallel to the in-plane direction of the surface of the liquid sample introduction plate, for example, the shift of light points over time and the light spot in the two-dimensional image in the process before and after the movement of the conjugate. The target substance can be detected as the disappearance of (move out of the observation field of view). Further, when the conjugate is moved in a direction away from the liquid sample introduction plate, for example, changes in the size of the light spots over time and disappearance of the light spots ( The target substance can be detected by moving out of the observation field of view).

The moving electric field application unit is not particularly limited, but from the viewpoint of miniaturization and improvement of portability of the device, a pair of surface electrodes formed on the surface of the liquid sample introduction plate with a space therebetween; a surface electrode formed on the surface of the liquid sample introduction plate; and a cover electrode formed of a transparent electrode material and arranged to face the surface and cover the liquid sample introduced onto the liquid sample introduction plate. It is preferable that at least one of the pair of configured electrodes is used.
When configured as the surface electrode, the liquid sample introduction plate and the moving electric field applying section can be integrated, and an increase in the size of the apparatus can be suppressed. Further, when configured as the cover electrode, it can be arranged in place of the cover glass, and an increase in the size of the device can be suppressed.
Materials for forming the respective surface electrodes are not particularly limited, and known electrode materials can be used. Examples of methods for forming the surface electrodes on the liquid sample introduction plate include a sputtering method, a vacuum vapor deposition method, a chemical vapor deposition method, a sol-gel method, and a coating method.
The transparent electrode material is not particularly limited, and known materials such as indium tin oxide (ITO), tin dioxide, and IGZO can be used.
The cover electrode may be formed of the transparent electrode material itself, or may be formed by forming an electrode layer of the transparent electrode material on one surface of the cover glass.

<Optical signal detector>
The optical signal detection section can detect movement of the conjugate from a signal change of an optical signal based on the detection light.

The optical signal detection unit is not particularly limited and can be appropriately selected depending on the purpose. Can be configured.
Further, the optical signal detection section is not particularly limited, but is preferably capable of acquiring a two-dimensional image of the state of the detection area on the surface of the liquid sample introduction plate. If the two-dimensional image can be obtained, positional information and size information of the optical signal in the two-dimensional image appearing as a light spot or a dark spot can be easily obtained, and the two-dimensional image before and after the movement of the conjugate can be easily obtained. By comparing the images, it is possible to determine whether the optical signal is information related to the conjugate, or whether the conjugate is affected by a scratch on the surface of the liquid sample introduction plate, the contaminants, fluctuations in light source output, or the like. It is possible to clearly distinguish whether the information is irrelevant or not. In order to enable acquisition of such a two-dimensional image, an imaging device may be selected as the optical signal detection section.
The imaging device is not particularly limited and can be appropriately selected according to the purpose. For example, a known image sensor such as a CCD image sensor or a CMOS image sensor can be used.
As a method for detecting the optical signal by the optical signal detection section, the area outside the imaging range of the optical signal detection section and the generation area of the near-field light (several hundred nm from the surface of the liquid sample introduction plate) In order to prevent detection failure of the conjugate existing outside (area up to several μm above), there is a method in which the conjugate is once placed on or near the surface of the liquid sample introduction plate and then detected. preferable.
Detecting the target substance includes detection of the presence or absence of the target substance, detection of the abundance of the target substance (quantitative measurement), real-time observation of the presence of the target substance, and the like.

Further, the optical signal detection unit is not particularly limited, but when the liquid sample contains charged contaminants, it is used to distinguish between the contaminant-derived optical signal and the conjugate-derived optical signal. , an optical filter that removes the optical signal originating from the contaminants can be preferably employed.

<Other Sections>
The other parts are not particularly limited and can be appropriately selected depending on the purpose. Any part used for an optical waveguide mode sensor or the like can be mentioned.

－Attracting electric field application part－
The attracting electric field application unit is provided on the surface side of the liquid sample introduction plate, and includes a pair of electrodes for moving the combined body in the liquid sample between the electrodes and attracting it to the surface by applying a voltage. In other words, the attracting electric field applying section has a function of causing electrophoresis of the conjugate between the pair of electrodes by an electric field generated by applying a voltage and attracting the conjugate to the surface.
In the optical signal detection section, the vicinity of the surface of the liquid sample introduction plate is used as a detection area. Therefore, in the case where the attracting electric field application section is not provided, the combined body in the liquid sample is detected by the liquid sample introduction plate. Although it is necessary to wait until the gravitational sedimentation to the vicinity of the surface before executing the detection operation, when the attracting electric field applying unit is arranged, it is not necessary to wait for the gravitational sedimentation, and the detection operation can be executed efficiently.

The attracting electric field applying unit is not particularly limited, and can be configured in the same manner as the moving electric field applying unit. a surface electrode formed on the surface of the liquid sample introduction plate at a position between the pair of surface electrodes spaced apart from each other on the surface of the sample introduction plate; and a surface electrode formed of a transparent electrode material and perpendicular to the surface electrode. It is preferable to provide a cover electrode arranged so as to cover the liquid sample introduced onto the liquid sample introduction plate so as to face each other in the direction.
The cover electrode has two functions of attracting the combined body to the surface side of the liquid sample introduction plate and moving it away from the surface side by switching the level of potential between the pair of electrodes. In addition, as a counter electrode in the pair of electrodes constituting the moving electric field applying section and the attracting electric field applying section, both functions of these sections can be provided.

The configuration of each part such as the liquid sample holding part, the light irradiation part, the optical signal detection part, etc. in the target substance detection device is described in International Publication No. 2017/187744 concerning an external force assisted sensor using an external force (magnetic field). No. 2018/100779, International Publication No. 2018/100780.

(Electrically charged particles)
In the charge-treated particles of the present invention, a binding substance capable of binding to the target substance is bound to the first functional group of a part of the particle body surface-modified with a plurality of first functional groups or a derivative group thereof, In addition, a charged molecule having a plurality of second functional groups in one molecule that ionize into cations or anions in a liquid is bound to the first functional group that is not bound to the binding substance or a derived group thereof. characterized by
The charged particles are used as the charged particles for the target substance detection device of the present invention, and the items described as the charged particles in the description of the target substance detection device can be applied. , redundant explanations are omitted.

Hereinafter, embodiments of the present invention will be described more specifically with reference to the drawings.

FIG. 1 shows an outline of a target substance detection device according to an embodiment of the present invention. FIG. 1 is an explanatory diagram showing the outline of the target substance detection device according to the embodiment of the present invention.
As shown in FIG. 1, the target substance detection device 1 includes a liquid sample introduction plate 2, a light irradiation section composed of a light source 6 and an optical prism 8, and a pair of surface electrodes 3A as constituent electrodes of the moving electric field application section. , 3B and an optical signal detection unit 7 (imaging device). Note that the imaging device is composed of, for example, a known CCD image sensor or the like, and is capable of acquiring a two-dimensional image.

The liquid sample introduction plate 2 receives a liquid sample 4 introduced onto the surface and is irradiated with light L irradiated on the surface under total reflection conditions, and is capable of generating near-field light above the surface. Formed in a board. Further, the liquid sample introduction plate 2 itself constitutes the liquid sample holding portion, and after the liquid sample 4 is introduced onto the surface, the liquid sample can be collected by disposing a cover glass 5 so as to cover the liquid sample 4 . Hold 4.
The light irradiation section is configured as a total reflection light irradiation section that can irradiate the surface of the liquid sample introduction plate 2 with the light L irradiated from the light source 6 via the optical prism 8 under total reflection conditions. .
The surface electrodes 3A and 3B are spaced apart from each other on the side surface of the liquid sample introduction plate 2. In the optical signal detection section 7, a detection area including the light irradiation area between the surface electrodes 3A and 3B is set. be done.

A target substance detection method using the target substance detection device 1 will be described.
First, a method for preparing a liquid sample 4 containing the charged particles as the charged particles used in the target substance detection device 1 will be described with reference to FIGS. 2(a) to 2(e). FIG. 2(a) is explanatory diagrams (1) to (5) showing an overview of the preparation method of the liquid sample 4. FIG.

First, as shown in FIG. 2(a), surface-modified particles are prepared in which the surface of a particle body P such as polystyrene beads is modified with a plurality of _first functional groups R1. In this example, the first functional group _R1 is assumed to be ionized (anionized) in liquid.
Next, as shown in FIG. 2(b), the surface-modified particles are reacted with an antibody Ig that specifically binds to an antigen (the target substance) as the binding substance in a liquid, and the surface-modified particles are Antibody Ig is bound to said derivative group of one first functional group R ₁ of .
Next, as shown in FIG. 2(c), the charged molecule having two second functional groups _R2 in one molecule that are ionized (anionized) in the liquid is added, and the other of the surface-modified particles is added. The charged molecule is bound to the derivative group of the _first functional group R1 to obtain the charged particles. At this time, the charge-treated particles have more ionized functional groups than the surface-modified particles shown in FIG.
Next, as shown in FIG. 2(d), the specimen solution containing the antigen Ag (the target substance) is added, and the antigen-antibody reaction between the antibody Ig and the antigen Ag causes the antigen Ag (the target substance) and the The conjugate is obtained by binding the charged particles.
Here, when the antigen Ag (the target substance) receives the near-field light and does not emit an optical signal such as fluorescence, as shown in FIG. to obtain the conjugate in which the charged particles, the antigen Ag (the target substance) and the labeling substance O are bound.
As described above, the liquid sample 4 is prepared.

A method of detecting the target substance by the target substance detection device 1 will be described with reference to FIG. 1 again.
First, the prepared liquid sample 4 is introduced onto the surface of the liquid sample introduction plate 2 and held.
After waiting for the combined body floating in the liquid layer of the liquid sample 4 to settle near the surface of the liquid sample introduction plate 2 by gravity, the light L emitted from the light source 6 is directed through the optical prism 8 to the liquid sample introduction plate. 2 is irradiated under total reflection conditions, and the optical signal detector 7 acquires an optical signal S based on the near-field light on the surface.

Next, a voltage is applied to the surface electrodes 3A and 3B to move the combined body. In this example, the surface electrode 3A is used as a negative electrode and the surface electrode 3B is used as a positive electrode, and the negatively charged particles are caused to electrophoretically move from the surface electrode 3A side to the surface electrode 3B side.
Next, the optical signal detector 7 acquires the optical signal on the surface of the liquid sample introduction plate 2 after the conjugate is moved while maintaining the detection area (observation field). In addition, it is preferable that the moving state of the conjugate is grasped as a plurality of states by separating a series of movements with time. In other words, it is preferable to capture a series of movements of the conjugate from the start of movement to the end of movement by dividing them into a plurality of parts, and acquire a plurality of optical signals at each time point. Such an optical signal acquisition method makes it easy to distinguish between random movement due to Brownian motion and movement due to an electric field.

In the target substance detection device 1 configured as described above, the optical signal based on the movement of the conjugate is detected by the scratches on the surface of the liquid sample introduction plate 2, contaminants adsorbed to or present on the surface of the liquid sample introduction plate 2, The target substance can be detected with high precision because it can be detected by clearly distinguishing it from noise signals that do not cause movement such as fluctuations in light source output. In addition, even if the contaminants are adsorbed on the surface of the liquid sample introduction plate 2, the detection can be performed ignoring the presence thereof, so the liquid sample introduction plate 2 must be washed for each detection. efficient detection.
Furthermore, compared to the conventional external force assisted sensor in which the magnetic field application unit is arranged for the purpose of using the magnetic field as the external force, the target substance detection device 1 in which the surface electrodes 3A and 3B that use the electric field as the external force are arranged It is possible to suppress an increase in the size of the device, and to provide a small device with excellent portability.

Next, a modification of the target substance detection device 1 will be described with reference to FIG. In addition, FIG. 3 is an explanatory diagram showing a main part of a modification of the target substance detection device 1. As shown in FIG.
As shown in FIG. 3, in this modification, surface electrodes 13A, 13B, and 13C are arranged on the surface of the liquid sample introduction plate 2 in place of the surface electrodes 3A and 3B, and a cover glass 5 is replaced with a cover. An electrode 19 is arranged.
In this modification, the area between the surface electrodes 13A and 13C or the area between the surface electrodes 13C and 13B is used as the optical signal detection area by the optical signal detection unit 7, and the surface electrodes 13A and 13B are Like the surface electrodes 3A and 3B, they act as the moving electric field applying section.
The surface electrode 13C and the cover electrode 19 act as the attracting electric field applying section that attracts the combined body floating in the liquid layer of the liquid sample 4 to the surface side of the liquid sample introduction plate 2 by applying voltage.
By switching the polarity of the applied voltage, the cover electrode 19 serves as a counter electrode for the pair of electrodes that constitute the moving electric field applying section and the attracting electric field applying section, thereby exhibiting both functions of these sections. can be made That is, the cover electrode 19 and the surface electrode A (or the surface electrode B) can constitute the pair of electrodes in the moving electric field application section, and the cover electrode 19 and the surface electrode 13C can constitute the pulling electric field application section. A pair of electrodes in can be configured.
According to the modified example having such an attracting electric field applying section, it is not necessary to wait for the combined bodies floating in the liquid layer of the liquid sample 4 to gravitationally settle near the surface of the liquid sample introduction plate 2. The target substance can be efficiently detected.

A target substance detection device according to an example was produced according to the configuration of the target substance detection device 1 shown in FIG. In the following, for convenience of explanation, each constituent part of the target substance detection device according to the first embodiment will be explained using the same reference numerals as those used in the explanation of the target substance detection device 1 .
Specifically, a 0.75 mm thick SiO ₂ substrate was used as the liquid sample introduction plate 2 . As the light source 6, a semiconductor laser (Thorlabs, Model No. CPS635) with a central wavelength of 635 nm was used. An optical prism 8 made of SiO ₂ glass is placed in close optical contact with the rear surface of the liquid sample introduction plate, and light from the light source 6 is made incident on the surface of the liquid sample introduction plate 2 at an incident angle of 67°. It was configured.
Further, as the moving electric field application unit, indium tin oxide electrodes with a thickness of 300 nm are formed as the surface electrodes 3A and 3B at a distance of 5 mm, and the surface electrodes 3A and 3B are connected via a fine copper wire fixed on each electrode. was connected to a function generator (NF Circuit Design Block Co., Model No. WF1974) to apply a DC voltage.
Further, in order to prevent adsorption of the conjugate to the liquid sample introduction plate 2 and the surface electrodes 3A and 3B, a monomolecular film of methoxytriethylene glycol-triethoxysilane was formed on the surfaces of these members as an adsorption prevention layer.
As the optical signal detector 7, an optical microscope equipped with a 5x objective lens and a cooled CCD camera (Model No. BU-59LIR, Bitlan) is used. It is configured to acquire images. In addition, the optical signal of the scattered light generated by the scattering of the light by the near field by the charged particles and contaminants adhering to the surface of the liquid sample introduction plate 2 is excluded, and the light signal from the fluorescent dye in the labeling substance is excluded. In order to acquire only the optical signal, a bandpass filter with a transmission wavelength of 680 nm (Thorlabs, Model No. FB680-10) was placed in the optical path between the objective lens and the CCD camera. That is, as the optical signal detector 7, a CCD camera equipped with the 5-fold objective lens and the band-pass filter with a transmission wavelength of 680 nm is used.

Normal mouse IgG was selected as the target substance.
Further, as the charged particles (the charged particles), an anti-mouse IgG-donkey antibody as the binding substance for polystyrene beads with a diameter of 100 nm surface-modified with a carboxyl group as the first functional group, As the charged molecule, a molecule bound with glutamic acid having two carboxy groups in one molecule was used.
The charged particles were prepared as follows.
A Polylink Protein Coupling Kit (manufactured by Polyscience, containing Polylink Coupling Buffer as a buffer solution and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as an activating agent) is used as a preparation reagent. .
First, a dispersion liquid obtained by dispersing 12.5 mg of the polystyrene beads in 170 μL of the buffer solution was mixed with 10 μL of an EDAC solution having a concentration of 200 mg/mL prepared by adding the EDAC to the buffer solution to prepare a mixed solution. , the mixture was allowed to react for 15 minutes to convert the carboxy groups on the surface of the polystyrene beads to activity-inducing groups.
Next, the concentrate of the polystyrene beads obtained from the mixed solution was redispersed in another 170 μL of the buffer solution by washing the beads after removing the supernatant by centrifugation to prepare a redispersed solution.
Next, 10 μL of the anti-mouse IgG solution having a concentration of 5 μg/mL prepared by adding the anti-mouse IgG dispersion (manufactured by Jackson ImmunoResearch Laboratories) to the buffer solution is mixed with the redispersion solution for 30 minutes. The anti-mouse IgG was bound to the activity-inducing group by reacting at room temperature to prepare a primary treatment solution for the polystyrene beads.
Next, the concentration of 500 ng/mL prepared by adding glutamic acid (manufactured by Fuji Film Wako Pure Chemical Co., Ltd.) to phosphate buffered saline (manufactured by Fuji Film Wako Pure Chemical Co., Ltd.) is added to the primary treatment solution of the polystyrene beads. and allowed to react at room temperature for 24 hours to bind the glutamic acid to the activity-inducing groups that have not bound to the anti-mouse IgG, thereby preparing a secondary treatment solution for the polystyrene beads.
After that, the beads are washed three times with respect to the secondary treatment solution of the polystyrene beads, and one group out of the groups of the carboxy groups on the surface of the polystyrene beads binds to the anti-mouse IgG, and the other group binds to the anti-mouse IgG. The charge-treated particles were prepared as surface-treated particles of the polystyrene beads in which the groups were bound to the glutamic acid.
As the labeling substance, particles obtained by binding an anti-mouse IgG-rabbit antibody (manufactured by Jackson ImmunoResearch Laboratories) to polymer beads (manufactured by Tamagawa Seiki Co., Ltd.) containing a fluorescent dye Cyanine 5 with a diameter of 400 nm were used.
The target substance, the charged particles, and the labeling substance were all dispersed in a phosphate buffered saline, and the pH of the dispersion was adjusted to 6.7.
The liquid sample 4 was prepared by mixing the target substance dispersion with the charged particle dispersion and the labeling substance dispersion.

Detection of the target substance was carried out as follows.
First, after introducing 100 μL of the liquid sample 4 onto the liquid sample introduction plate 2 , the cover glass 5 was arranged to hold the liquid sample 4 on the liquid sample introduction plate 2 .
Next, light L was irradiated from the light source 6 to form a near-field on the surface of the liquid sample introduction plate 2 .
Next, a voltage was applied to the surface electrodes 3A and 3B, and the state of movement of the conjugate was detected by the optical signal detector 7. FIG.

FIG. 4 shows the detection results. In addition, FIG. 4 shows the results before voltage application to the surface electrodes 3A and 3B, and 30 seconds, 60 seconds, 90 seconds, and 120 seconds after applying a DC voltage of 2.8 V to the surface electrodes 3A and 3B. After 150 seconds and 180 seconds, images obtained with an exposure time of 10 seconds are superimposed and displayed.
In addition, the field of view in FIG. 4 is 0.9 mm × 0.9 mm, "1" in the figure is the light spot before voltage application, Point, "3" is the light spot 60 seconds after applying the DC voltage, "4" is the light spot 90 seconds after applying the DC voltage, "5" is the light spot after applying the DC voltage. 120 seconds after applying the DC voltage, "6" indicates the light spot after 150 seconds, and "7" indicates the light spot after 180 seconds after applying the DC voltage. .
The detection area of the image shown in FIG. 4 is the area between the surface electrode 3A and the surface electrode 3B in the vicinity of the surface of the liquid sample introduction plate 2, and the surface electrode 3A is arranged as a negative electrode in the left direction in the drawing. , and a surface electrode 3B as a positive electrode is arranged in the right direction.

As indicated by light spots "1" to "7" in FIG. 4, it is confirmed that the light spots in the field of view move toward the surface electrode 3B (positive electrode) over time.
Here, the moving light spot is the movement of the combined body composed of the target substance, the charged particles and the labeling substance on the surface of the liquid sample introduction plate 2 due to the voltage applied to the surface electrodes 3A and 3B. It was detected by
In this way, by observing the movement of the optical signal due to voltage application to the surface electrodes 3A and 3B, the optical signal based on the movement of the conjugate can be detected by the scratches on the surface of the liquid sample introduction plate 2 and the surface. It can be detected by clearly distinguishing it from noise signals (non-moving light spots in FIG. 4) such as foreign substances adsorbed or present on the surface and fluctuations in light source output.

1 target substance detection device 2 liquid sample introduction plate 3A, 3B, 13A, 13B, 13C surface electrode 4 liquid sample 5 cover glass 6 light source 7 optical signal detector 8 optical prism 19 cover electrode P particle body R ₁ first functional group R2 _second functional group Ig antibody Ag antigen

Claims (3)

A liquid sample introduction in which a liquid sample containing a target substance and charged particles that form a bond with the target substance is introduced onto the surface and irradiated with light to generate detection light of either propagating light or near-field light. a liquid sample holding part provided with a plate and capable of holding the liquid sample on the surface of the liquid sample introduction plate;
a light irradiation unit that irradiates the liquid sample introduction plate with the light to generate the detection light;
a moving electric field applying unit including a pair of electrodes disposed on the surface side of the liquid sample introduction plate and configured to move the combined body in the liquid sample between the electrodes by applying a voltage;
an optical signal detection unit capable of detecting movement of the conjugate from a signal change of an optical signal based on the detection light;
has
The target substance detection device , wherein the moving electric field applying section is composed of a pair of surface electrodes spaced apart from each other on the surface of the liquid sample introduction plate . 1. Further comprising an attracting electric field applying unit provided on the surface side of the liquid sample introduction plate and having a pair of electrodes for moving the combined bodies in the liquid sample between the electrodes by applying a voltage and attracting them to the surface. The target substance detection device according to . a surface electrode formed on the surface of the liquid sample introduction plate at a position between a pair of surface electrodes spaced apart from each other on the surface of the liquid sample introduction plate, wherein the attracting electric field application unit constitutes the moving electric field application unit; 3. The target substance detection according to claim 2, further comprising: a cover electrode formed of a transparent electrode material and arranged to face the surface electrode in the vertical direction so as to cover the liquid sample introduced onto the liquid sample introduction plate. Device.

Images