JP2021032757A - Concentration measuring method and concentration measuring device - Google Patents

Abstract

To provide a concentration measuring method with which it is possible to realize improvement on the accuracy of quantifying a detection object substance.SOLUTION: The concentration measuring method includes: arranging a liquid 61 on a substrate 11, the liquid including a composite 6 of a first substance 3 having a magnetic particle 2 fixed thereto, a second substance 5 having a phosphor fixed thereto and a detection object substance 1; irradiating the substrate 11 with excitation light; applying a first magnetic field gradient that attracts the composite 6 in a first direction toward an irradiation area on the substrate 11; applying a second magnetic field gradient that moves the composite 6 in a second direction after stopping the application of the first magnetic field gradient and observing the fluorescence emanated by the phosphor in the composite 6 as the light spot of a two-dimensional image; and counting the number of light spots the positions of which change in the second direction in the two-dimensional image and measuring the concentration of the detection object substance 1. In a fluorescence observation, the first magnetic field gradient is applied again when it is detected that the first light spot the position of which changes in the second direction in the two-dimensional image became extinct; in a concentration measurement, a first light spot is counted as one light spot when it is detected that the first light spot reappeared after the second application of the first magnetic field gradient.SELECTED DRAWING: Figure 5

Description

The present disclosure relates to a concentration measuring method and a concentration measuring device.

In recent years, an optical detection method or the like for detecting a minute target substance (hereinafter, also referred to as a detection target substance) with high sensitivity by using a proximity field has been provided. For example, in Patent Document 1, a complex formed by bonding a substance to be detected, magnetic particles, and fluorescent particles by applying a magnetic field that moves magnetic particles in a direction parallel to the surface of a detection plate on which a proximity field is formed. The concentration of the substance to be detected is quantified by measuring the reduction (including disappearance) or fluctuation (including the movement of the optical signal due to the movement of the complex) of the optical signal.

International Publication No. 2017/187744

However, in Patent Document 1, while the complex is moved in a direction parallel to the surface of the detection plate on which the proximity field is formed, the complex is brought close to each other by convection of a solution containing the complex or Brownian motion of the complex itself. Complexes located in the field may move out of the proximity field and return to the proximity field again. As a result, while the composite was moved in the direction parallel to the surface of the detection plate, the fluorescent particles in the fluorescent composite stopped emitting fluorescence (in other words, the fluorescence disappeared) or disappeared. Later, a phenomenon occurs in which fluorescence is emitted again (in other words, the fluorescence blinks). Therefore, there is a problem that the concentration of the substance to be detected cannot be accurately quantified because the reduction or fluctuation of the optical signal emitted by the complex cannot be accurately measured.

Therefore, the present disclosure provides a method for measuring the concentration of a substance to be detected, which can improve the accuracy of quantification of the substance to be detected.

The concentration measurement method according to one aspect of the present disclosure is a first substance that specifically binds to the detection target substance, that is, a first substance to which magnetic particles are fixed, and a second substance that specifically binds to the detection target substance. A liquid containing a complex formed by binding a second substance, which is a substance to which a phosphor or a scatterer is fixed, and the substance to be detected is arranged on a substrate, and the phosphor generates fluorescence. The substrate is irradiated with excitation light having a wavelength at which the scattering light is generated or a wavelength at which the scattering body generates scattered light, and a first magnetic field gradient that attracts the complex is applied in a first direction toward an irradiation region on the substrate. After stopping the application of the first magnetic field gradient, a second magnetic field gradient that moves the complex in a second direction different from the first direction is applied, and fluorescence generated by the phosphor in the complex or fluorescence or The concentration of the substance to be detected is measured by observing the scattered light generated by the scattering body as a light spot of a two-dimensional image and counting the number of light spots whose position changes in the second direction on the two-dimensional image. Then, in the observation of the fluorescence or the scattered light, when the disappearance of the first light spot whose position changes in the second direction is detected in the two-dimensional image, the first magnetic field gradient is reapplied. In the measurement of the concentration, when the reproduction of the first light spot is detected after the first magnetic field gradient is reapplied, the first light spot is counted as one light spot.

Further, the concentration measuring device according to one aspect of the present disclosure specifically binds to the first substance to which the magnetic particles are fixed, which is the first substance that specifically binds to the detection target substance, and the detection target substance. A holding portion that holds a liquid containing a complex formed by binding a second substance, which is a second substance to which a phosphor or a scatterer is fixed, and the substance to be detected on a substrate, and the fluorescence. A light source that irradiates the substrate with excitation light having a wavelength at which the body emits fluorescence or a wavelength at which the scatterer generates scattered light, and a first direction that attracts the complex toward an irradiation region on the substrate. A first magnetic field application unit that applies one magnetic field gradient, a second magnetic field application unit that applies a second magnetic field gradient that moves the complex in a second direction different from the first direction, and the fluorescence in the composite. Counting the number of observation units that observe the fluorescence generated by the body or the scattered light generated by the scatterer as the light spots of the two-dimensional image and the number of light spots that change position in the second direction on the two-dimensional image. The second magnetic field applying unit is provided with a measuring unit for measuring the concentration of the substance to be detected, and after stopping the application of the first magnetic field gradient, the second magnetic field gradient is applied and the first magnetic field is applied. When the disappearance of the first light spot whose position changes in the second direction is detected in the two-dimensional image, the application unit reapplies the first magnetic field gradient, and the measurement unit reapplies the first magnetic field gradient. When the reproduction of the first light spot is detected after reapplying the magnetic field gradient, the first light spot is counted as one light spot.

It should be noted that these comprehensive or specific embodiments may be realized in a system, method, integrated circuit, computer program or recording medium such as a computer-readable CD-ROM, and the system, method, integrated circuit, computer. It may be realized by any combination of a program and a recording medium.

According to the present disclosure, the quantification accuracy of the substance to be detected can be improved.

FIG. 1 is a diagram showing an example of a complex in the embodiment. FIG. 2 is a schematic configuration diagram showing an example of the concentration measuring device according to the embodiment. FIG. 3 is a diagram showing the distribution of the electromagnetic field on the surface of the substrate and the movement of the complex. FIG. 4 is a graph showing the relationship between the electromagnetic field strength of the substrate surface and the distance from the substrate surface. FIG. 5 is a flowchart showing an example of the operation of the concentration measuring device according to the embodiment. FIG. 6 is a flowchart showing the detailed processing of step S5006 of FIG. FIG. 7 is a diagram schematically showing the process of FIG.

(Knowledge on which this disclosure was based)
In recent years, optical detection methods for detecting or quantifying minute substances to be detected, particularly pathogens such as viruses, have been developed using a proximity field.

For example, in the optical detection method described in Patent Document 1, first, two types of antibodies that specifically bind to a substance to be detected (for example, an antigen such as a virus) are prepared. One antibody is immobilized on magnetic particles having magnetism, and the other antibody is labeled with labeled particles (scattered particles or fluorescent particles). These two types of antibodies are mixed with a test solution containing a substance to be detected to prepare a mixed solution. The antigen-antibody reaction proceeds in this mixed solution, and the two types of antibodies are specifically bound (sandwiched) with the substance to be detected sandwiched between them, and an antigen-antibody complex of magnetic particles-substance to be detected-labeled particles (hereinafter, referred to as (Simply called a complex) is formed. The mixed solution is retained on the surface of the detection plate, which generates a near field on the surface when irradiated with light from the back surface. A magnetic field gradient is applied in the direction perpendicular to the detection plate to attract the complex in the mixed solution near the surface of the detection plate. Here, when the complex is irradiated with a near field and a two-dimensional image of the mixed solution is acquired from the surface side, the complex appears on the two-dimensional image as a light spot that emits an optical signal (scattered light or fluorescence). Then, when the magnetic field gradient is applied in the direction parallel to the surface of the detection plate, the light spot on the two-dimensional image moves in the direction of the magnetic field gradient. The concentration of the substance to be detected is quantified by counting the moving light spots (moving light spots).

However, the above-mentioned method has the following problems.

While moving the complex in a direction parallel to the surface of the substrate, the complex located in the proximity field moves out of the proximity field due to convection of the mixed solution containing the complex, Brownian motion of the complex itself, etc. In other words, it may leave the proximity field area) and return to the proximity field again (in other words, invade the proximity field area). As a result, while the complex is moved in the direction parallel to the surface of the detection plate, the fluorescent particles in the fluorescent complex do not fluoresce (that is, the fluorescence disappears) and fluoresce again (that is, the fluorescence disappears). That is, the phenomenon that the fluorescence blinks) occurs. Due to this phenomenon, a blinking moving light spot appears while moving.

Here, when one blinking moving light spot is counted as a plurality of moving light spots, it counts more than the original moving light spot, so that the concentration of the substance to be detected is calculated to be higher than the actual concentration. In order to avoid this, unless the moving light spots with a short period until extinction are counted, even if the moving light spots fluoresce again after disappearing, they are not counted as the moving light spots, so that the concentration of the substance to be detected is , Calculated lower than the actual concentration.

Therefore, in the above-mentioned method, since the moving light spot of the complex cannot be accurately counted, there is a problem that the quantification accuracy of the substance to be detected is lowered.

(Summary of this disclosure)
Therefore, in the concentration measurement method according to one aspect of the present disclosure, the first substance that specifically binds to the detection target substance and the magnetic particles are fixed is specifically bound to the detection target substance. A liquid containing a complex formed by binding the second substance, which is a second substance to which a phosphor or a scatterer is fixed, and the detection target substance is placed on a substrate, and the phosphor fluoresces. The substrate is irradiated with excitation light having a wavelength at which the scattering light is generated or a wavelength at which the scattering body generates scattered light, and a first magnetic field gradient that attracts the complex is applied in the first direction toward the irradiation region on the substrate. Then, after stopping the application of the first magnetic field gradient, a second magnetic field gradient that moves the complex in a second direction different from the first direction is applied, and the phosphor in the complex is generated. The concentration of the substance to be detected is detected by observing the scattered light generated by fluorescence or the scattering body as a light spot of a two-dimensional image and counting the number of light spots whose position changes in the second direction on the two-dimensional image. When the disappearance of the first light spot whose position changes in the second direction is detected in the two-dimensional image in the observation of the fluorescence or the scattered light, the first magnetic field gradient is reapplied. Then, in the measurement of the concentration, when the reproduction of the first light spot is detected after the first magnetic field gradient is reapplied, the first light spot is counted as one light spot.

As a result, while the complex located in the irradiation area on the substrate is attracted and moves in the second direction, the complex moves out of the irradiation area due to convection of the liquid or Brownian motion of the complex itself. Also, the complex can be pulled back into the illuminated area by reapplying the first magnetic field gradient. Along with this, the disappeared and reproduced moving light points are counted as one moving light point, so that the possibility of overlapping counting of the moving light points is reduced. Therefore, according to the concentration measurement method according to one aspect of the present disclosure, the counting accuracy of the complex is improved, so that the quantification accuracy of the substance to be detected is improved.

For example, in the concentration measurement method according to one aspect of the present disclosure, when the reproduction of the first light spot is not detected after reapplying the first magnetic field gradient in the concentration measurement, the first light spot is used. You don't have to count.

As a result, the moving light spots corresponding to the labeled particles that move regardless of the magnetic field gradient are not counted, so that the moving light points corresponding to the complex can be accurately counted. For example, labeled particles (phosphor or scatterer) in the vicinity of magnetic particles (pointing to magnetic particles that are unbonded to the substance to be detected) or composites that are attracted in the second direction by the second magnetic field gradient move. Can reduce the likelihood that labeled particles will be counted as a complex if they are affected by a slight flow of liquid. Therefore, according to the concentration measurement method according to one aspect of the present disclosure, the counting accuracy of the complex is improved, so that the quantification accuracy of the substance to be detected is improved.

For example, in the concentration measurement method according to one aspect of the present disclosure, when the disappearance of the first light spot is detected in the observation of the fluorescence or the scattered light, the application of the second magnetic field gradient is stopped. The first magnetic field gradient may be reapplied.

Thereby, when the light spot is reproduced at the position where the first light spot disappears (including the position in the vicinity thereof), the reproduced light spot can be counted as the first light spot. Therefore, it becomes easy to determine whether or not the light spots are the same, and the processing amount is reduced. Therefore, according to the concentration measuring method according to one aspect of the present disclosure, the processing efficiency is improved.

For example, in the concentration measurement method according to one aspect of the present disclosure, the substrate may form a near field of the excitation light on the surface of the substrate by being irradiated with the excitation light.

This reduces the detection (false positive) of the light (fluorescent or scattered light) emitted by the labeled particles (pointing to the labeled particles unbonded to the substance to be detected) as the light emitted by the labeled particles in the complex. be able to. Since the proximity field is formed near the surface of the substrate (for example, several hundred nm to 1 μm), only the labeled particles located near the surface of the substrate are excited by the excitation light among the liquids arranged on the substrate. .. However, since the labeled particles are not attracted in the second direction even when the second magnetic field gradient is applied, they are basically detected as light spots that do not move, unlike the complex that is attracted and moves in the second direction. Therefore, according to the concentration measurement method according to one aspect of the present disclosure, false positives can be reduced, and the quantification accuracy of the substance to be detected is improved.

For example, in the concentration measurement method according to one aspect of the present disclosure, the two-dimensional image includes a first image, a second image, and a third image obtained with the passage of time, and in the observation of the fluorescence or the scattered light. Further, the motion vector of the first light spot is derived from the first image and the second image, and the position of the first light spot in the third image is predicted by using the derived motion vector. The disappearance of the first light spot may be detected based on the presence or absence of the light spot at the position of the first light spot predicted in the third image.

As a result, the moving first light spot can be identified over time, so that the possibility of erroneously counting as another moving light spot can be reduced. Therefore, the measurement accuracy of the moving light point is improved. Further, since the disappearance of the first light spot is detected by using the predicted image and the actually captured image, the concentration measurement of the substance to be detected can be continuously performed. Therefore, the concentration of the substance to be detected can be efficiently measured. Therefore, according to the concentration measurement method according to one aspect of the present disclosure, the quantification accuracy and quantification efficiency of the substance to be detected are improved.

Further, the concentration measuring device according to one aspect of the present disclosure specifically binds to the first substance to which the magnetic particles are fixed, which is the first substance that specifically binds to the detection target substance, and to the detection target substance. A holding portion that holds a liquid containing a complex formed by binding a second substance, which is a second substance to which a phosphor or a scatterer is fixed, and the detection target substance on a substrate, and the fluorescence. A light source that irradiates the substrate with excitation light having a wavelength at which the body generates fluorescence or a wavelength at which the scatterer generates scattered light, and a first direction that attracts the complex toward an irradiation region on the substrate. A first magnetic field application unit that applies one magnetic field gradient, a second magnetic field application unit that applies a second magnetic field gradient that moves the complex in a second direction different from the first direction, and the fluorescence in the composite. The detection is performed by observing the fluorescence generated by the body or the scattered light generated by the scatterer as the light spots of the two-dimensional image and counting the number of light spots whose position changes in the second direction on the two-dimensional image. A measuring unit for measuring the concentration of the target substance is provided, the second magnetic field applying unit applies the second magnetic field gradient after stopping the application of the first magnetic field gradient, and the first magnetic field applying unit applies the second magnetic field gradient. When the disappearance of the first light spot whose position changes in the second direction is detected in the two-dimensional image, the first magnetic field gradient is reapplied, and the measuring unit applies the first magnetic field gradient. When the reproduction of the first light spot is detected after reapplication, the first light spot is counted as one light spot.

As a result, while the complex located in the irradiation area on the substrate is attracted and moves in the second direction, the complex moves out of the irradiation area due to convection of the liquid or Brownian motion of the complex itself. Also, the complex can be pulled back into the illuminated area by reapplying the first magnetic field gradient. Along with this, the disappeared and reproduced moving light points are counted as one moving light point, so that the possibility of overlapping counting of the moving light points is reduced. Since the moving light points are counted as one moving light point, the possibility of duplicate measurement is reduced. Therefore, according to the concentration measuring device according to the present embodiment, the counting accuracy of the complex is improved, so that the quantification accuracy of the substance to be detected is improved.

It should be noted that these comprehensive or specific embodiments may be realized in a system, method, integrated circuit, computer program or recording medium such as a computer-readable CD-ROM, and the system, method, integrated circuit, computer. It may be realized by any combination of a program and a recording medium.

Hereinafter, embodiments will be specifically described with reference to the drawings. It should be noted that all of the embodiments described below show comprehensive or specific examples. Numerical values, shapes, materials, components, arrangement positions and connection forms of components, steps, order of steps, etc. shown in the following embodiments are examples, and are not intended to limit the present disclosure. Further, among the components in the following embodiments, the components not described in the independent claims indicating the highest level concept are described as arbitrary components.

Moreover, each figure is not necessarily exactly illustrated. In each figure, substantially the same configuration is designated by the same reference numerals, and duplicate description may be omitted or simplified.

In addition, in the following, terms indicating relationships between elements such as parallel and vertical, terms indicating the shape of elements such as a cylindrical shape, and numerical ranges do not only represent strict meanings but are substantial. Including measuring a range equivalent to, for example, the amount (eg, number or concentration, etc.) of the substance to be detected or the range thereof.

Further, in each figure, the X-axis direction, the Y-axis direction, and the Z-axis direction, which are orthogonal to each other, will be described as appropriate. In particular, the positive side in the Z-axis direction may be described as the upper side, and the negative side may be described as the lower side.

(Embodiment)
Hereinafter, embodiments will be described with reference to FIGS. 1 to 7.

[Complex]
First, the complex will be described with reference to FIG. FIG. 1 is a diagram showing an example of the complex 6 in the embodiment.

The complex 6 is formed by binding the first substance 3 to which the magnetic particles 2 are fixed, the second substance 5 to which the labeled particles 4 are fixed, and the detection target substance 1. More specifically, the first substance 3 to which the magnetic particles 2 are fixed and the second substance 5 to which the phosphor is fixed are bound to different sites of the detection target substance 1 and sandwich the detection target substance 1. By binding (ie, sandwich binding), the complex 6 is formed.

The substance to be detected 1 is a molecule to be detected, for example, a protein, a lipid, a sugar, a nucleic acid, or the like.

The first substance 3 has a property of specifically binding to the substance to be detected 1, and the magnetic particles 2 are fixed. The combination of the first substance 3 with respect to the substance 1 to be detected includes, for example, an antibody against an antigen, an enzyme against a substrate or a coenzyme, a receptacle against a hormone, a protein A or protein G against an antibody, avidins against biotin, calmodulin against calcium, and a lectin against sugar. And so on.

The second substance 5 has a property of specifically binding to the substance to be detected 1, and the labeled particles 4 are fixed. Since the combination of the second substance 5 with respect to the detection target substance 1 is the same as that of the first substance 3, the description thereof is omitted here. The first substance 3 and the second substance 5 may be the same type of substance or may be different types of substances. When the first substance 3 and the second substance 5 are substances of the same type, these substances are, for example, antibodies that specifically bind to the substance to be detected 1 (for example, a virus). At this time, each of these substances may be bound to a different binding site of the substance to be detected 1. That is, these substances should be less competitive.

The magnetic particle 2 is a particle having paramagnetism or ferromagnetism. Paramagnetism means magnetism that has no magnetization when there is no external magnetic field and is weakly magnetized in the direction of the magnetic field gradient when a magnetic field gradient is applied. Ferromagnetism means magnetism that can have spontaneous magnetization without an external magnetic field. That is, the magnetic particles 2 move in the direction of the magnetic field gradient by applying the magnetic field gradient. The magnetic particles 2 may have a shell structure. For example, the magnetic particles 2 may have paramagnetic or ferromagnetic particles as the inner core, and the inner core may be coated with a resin, glass, a noble metal, or the like. This can protect the inner core from oxidation.

The labeled particle 4 is a particle containing a substance (labeled substance) that makes it possible to distinguish the substance 1 to be detected from another substance. The labeled particle 4 is, for example, a phosphor or a scatterer.

The phosphor radiates fluorescence by irradiation with excitation light having a predetermined wavelength. The phosphor may be, for example, an organic molecule or a quantum dot, and is a fluorescence composed of an organic fluorescent molecule, an inorganic phosphor, a resin (for example, polystyrene or acrylic) or glass incorporating the quantum dot or the like. It may be a particle. The particle size of the fluorescent particles is from several tens of nm to several hundreds of nm. For fluorescent particles, the fading of fluorescence is reduced by impregnating the resin or glass with a fluorescent deactivation inhibitor. Further, the fluorescent particles are subjected to various surface modifications such as an amino group and a carboxy group to improve the bondability with the detection target substance 1. Further, the fluorescent particles contain resin or glass to improve the dispersibility in water.

The scatterer emits scattered light by irradiating light having a predetermined wavelength. The scatterer is composed of, for example, a material that is not easily affected by the magnetic field gradient even if a magnetic field gradient is applied. Such materials include, for example, non-magnetic metals such as gold, silver, aluminum, manganese, chromium, platinum, copper, zinc, lead, or manganese, alloys of these metals, metal oxides or metal nitrides. It may be a resin such as polycarbonate, polyethylene, polypropylene, vinyl chloride, epoxy resin, or ABS resin. Further, the scatterer may be, for example, a particle having a rectangular cross-sectional shape, an ellipse close to a rectangle, a sine shape, or a trapezoidal shape, and the surface of the particle may have irregularities. The particle size of the scatterer has a major axis (for example, the diameter of the circumscribed circle) and may be 30 nm or more, 50 nm or less, 300 nm or less, or 200 nm.

In FIG. 1, a plurality of first substances 3 are fixed to the magnetic particles 2, but the present invention is not limited to this. For example, only one first substance 3 may be fixed to the magnetic particles 2. Further, in FIG. 1, a plurality of second substances 5 are fixed to the labeled particles 4, but the present invention is not limited to this. For example, only one second substance 5 may be immobilized on the labeled particles 4.

The configuration of the complex 6 shown in FIG. 1 is an example, and is not limited thereto. For example, the number of magnetic particles 2 contained in the composite 6 may be one or three or more. Further, for example, the number of the detection target substances 1 contained in the complex 6 may be two or more.

[Overview of concentration measuring device]
Subsequently, the concentration measuring device according to the present embodiment will be described with reference to FIG. FIG. 2 is a schematic configuration diagram showing an example of the concentration measuring device 100 according to the embodiment. In FIG. 2, the holding portion 10 shows a cross section cut on a plane (XZ plane) orthogonal to the main plane (XY plane) of the substrate 11.

The concentration measuring device 100 optically measures the concentration of a trace amount of the substance 1 to be detected in the liquid 61. More specifically, the concentration measuring device 100 collects the complex 6 in the liquid 61 in the irradiation region on the substrate 11 by the first magnetic field gradient, and in the direction parallel to the surface 11a of the substrate 11 by the second magnetic field gradient. Move the complex 6. Then, the concentration measuring device 100 observes the fluorescence emitted by the phosphor in the moving complex 6 or the scattered light emitted by the scatterer as a moving light point of the two-dimensional image. At this time, the moving light spot in the two-dimensional image may blink. This phenomenon will be described with reference to FIGS. 3 and 4.

FIG. 3 is a diagram showing the distribution of the electromagnetic field (so-called proximity field) on the surface 11a of the substrate 11 and the movement of the complex 6. FIG. 4 is a graph showing the relationship between the electromagnetic field strength of the surface 11a of the substrate 11 and the distance from the surface 11a of the substrate 11. In the present embodiment, the region in which the proximity field is formed by the irradiation of the excitation light 21 is referred to as an irradiation region.

As shown in FIGS. 3 and 4, the proximity field is formed at a distance of 0.75 μm from the surface 11a of the substrate 11. The proximity field is formed by total reflection of the excitation light 21 emitted from the back surface 11b of the substrate 11 at the interface between the substrate 11 and the liquid 61.

When the complex 6 is collected in the vicinity of the surface 11a (for example, within 0.5 μm) of the substrate 11 by the first magnetic field gradient, the phosphor or the scatterer in the complex 6 is excited in the proximity field. However, while moving the composite 6 in a direction parallel to the surface of the substrate 11, the composite 6 moves away from the irradiation region on the surface of the substrate 11 due to convection of the liquid 61 or Brownian motion of the composite 6 itself. May move to and out of proximity. At this time, the moving light spots in the two-dimensional image disappear. Further, as shown in FIG. 3, the complex 6 that has gone out of the proximity field may return to the proximity field due to convection of the liquid 61 or Brownian motion of the complex 6 itself. At this time, the disappeared moving light spots in the two-dimensional image are reproduced.

When the density measuring device 100 according to the present embodiment detects that the moving light spot in the two-dimensional image has disappeared, it applies a first magnetic field gradient that attracts the complex 6 toward the irradiation region. Then, when the density measuring device 100 detects that the moving light spots in the two-dimensional image have reappeared, the disappearing and reproduced moving light spots are counted as one light spot. As a result, it is possible to reduce the duplication of counting one blinking moving light spot as two or more moving light spots.

[Concentration measuring device configuration]
As shown in FIG. 2, the concentration measuring device 100 includes a holding unit 10, a light source 20, a first magnetic field applying unit 31, a second magnetic field applying unit 32, and a measuring unit 50. Hereinafter, each component of the concentration measuring device 100 will be described in order.

The holding portion 10 puts the complex 6, the first substance 3 on which the magnetic particles 2 are fixed, and the second substance 5 on which the labeled particles 4 (fluorescent or scatterer) are fixed, and the liquid 61 on the substrate 11. Hold. The liquid 61 may be prepared in advance or may be prepared in the space 15 of the holding portion 10. In the present embodiment, the holding portion 10 includes a substrate 11 and a prism 12 capable of forming a close field by irradiation with excitation light. Specifically, the substrate 11 is arranged on the front surface of the prism 12, and the back surface 11b of the substrate 11 is optically bonded to the surface of the prism 12. As a result, the substrate 11 functions as a substrate capable of forming a close field on the surface 11a.

The near field is a layer of light (evanescent light) that exudes to the medium on the low refractive index side when the light incident on the medium on the high refractive index side at a critical angle or higher is totally reflected at the interface. Here, the light 21 emitted from the back surface 11b of the substrate 11 is totally reflected at the interface between the substrate 11 and the liquid 61, so that the liquid 61 (for example, glass) having a refractive index smaller than that of the substrate 11 (for example, glass) is totally reflected at the interface. For example, the light 21 exudes to the water) side, and a near field of the light 21 is formed. In the present embodiment, the proximity field is formed in the vicinity of the surface 11a of the substrate 11.

The holding portion 10 further includes a transparent cover glass 13 that covers the liquid 61. The liquid 61 is held between the substrate 11 and the cover glass 13. The holding portion 10 may include a spacer 14 that surrounds the liquid 61. The spacer 14 is arranged on the substrate 11 and extends from the substrate 11 toward the cover glass 13.

The light source 20 irradiates the substrate 11 with excitation light 21 having a wavelength at which the phosphor emits fluorescence or a wavelength at which the scatterer generates scattered light. In the present embodiment, the light source 20 irradiates the back surface 11b of the substrate 11 with the excitation light 21 via the prism 12. As the light source 20, known techniques can be used without particular limitation. For example, a laser such as a semiconductor laser or a gas laser can be used as the light source 20. The wavelength of the excitation light emitted from the light source 20 may be a wavelength that has a small interaction with the substance contained in the detection target substance 1 such as a virus. For example, the wavelength of the excitation light 21 may be in the range of 400 nm or more and 2000 nm or less, or may be a wavelength having a small interaction with the constituent substances of water or virus. Further, the wavelength of the excitation light 21 may be a wavelength in the range of 600 nm or more and 850 nm or less, which is a wavelength that can be used by the semiconductor laser.

As described above, in the present embodiment, the excitation light 21 is totally reflected at the interface between the liquid 61 and the substrate 11. As a result, a proximity field is formed on the surface 11a of the substrate 11. The proximity field is formed only in the region near the surface 11a, and rapidly attenuates as the distance from the surface 11a of the substrate 11 increases. In many cases, the distance covered by the near field is about the wavelength of the excitation light 21. Therefore, the proximity field of the excitation light 21 is applied only to the liquid 61 existing in the region near the surface 11a of the substrate 11. That is, in the present embodiment, the region near the surface 11a of the substrate 11 corresponds to the irradiation region to which the excitation light 21 is irradiated.

The configuration of the substrate 11 is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the substrate 11 may be composed of a single layer or a laminated body for the purpose of enhancing the electric field.

The first magnetic field application unit 31 applies the first magnetic field gradient in the first direction (Z-axis minus direction in FIG. 2) toward the irradiation region on the substrate 11. Due to this first magnetic field gradient, the magnetic particles 2 in the liquid 61 move in the first direction. As a result, the complex 6 and the first substance 3 existing in the liquid 61 are collected in the irradiation region of the excitation light. The details of the operation of the first magnetic field application unit 31 will be described later.

The second magnetic field application unit 32 applies a second gradient to the liquid 61 differently from the first direction. A second magnetic field gradient is applied into the liquid 61 to move the complex 6 in a second direction (X-axis minus direction in FIG. 2) different from the first direction. Due to this second magnetic field gradient, the magnetic particles 2 in the liquid 61 move in the second direction. As a result, the complex 6 and the first substance 3 existing in the liquid 61 move in the light irradiation region in the second direction. The details of the operation of the second magnetic field application unit 32 will be described later.

An electromagnet, a permanent magnet, or the like can be used as the first magnetic field application unit 31 and the second magnetic field application unit 32. When an electromagnet is used, the control unit (not shown) switches between application and non-application of the magnetic field gradient by controlling the supply of current to each of the first magnetic field application unit 31 and the second magnetic field application unit 32. Can be done. When a permanent magnet is used, the control unit can switch between application and non-application of the magnetic field gradient by moving each of the first magnetic field application unit 31 and the second magnetic field application unit 32.

The measuring unit 50 observes the fluorescence generated by the phosphor in the composite 6 or the scattered light generated by the scatterer as the light spot of the two-dimensional image, and the position of the light spot that changes its position in the second direction on the two-dimensional image. The concentration of the detection target substance 1 is measured by counting the number. The measuring unit 50 includes a light detecting unit 40 and a processing unit 45.

The light detection unit 40 receives the fluorescence emitted from the phosphor or the scattered light emitted from the scatterer in the irradiation region of the excitation light via the optical system, and outputs an electric signal according to the intensity of the received light. .. The photodetector 40 includes a light-collecting member 41 having a light-collecting property, a filter 42 that selectively transmits light of a part of wavelengths, and an image sensor 43.

The condensing member 41 is, for example, a refraction type lens, and condenses the received light on the image sensor 43. The condensing member 41 may be composed of a single lens or may be composed of a plurality of lenses.

The filter 42 has, for example, an optical property of transmitting only the main wavelength component of the fluorescence or scattered light emitted by the complex 6 and absorbing or reflecting the others.

The image sensor 43 is, for example, a two-dimensional image sensor, which forms an image of the light collected by the condensing member 41 on a light receiving surface, photoelectrically converts the light and darkness of the image into the amount of electric charge, and reads it out. To convert it into an electric signal.

The processing unit 45 detects the substance 1 to be detected based on the amount of change in the output signal of the photodetector 40 (that is, the amount of change in the intensity of fluorescence or scattered light). For example, the processing unit 45 converts the electric signal from the image sensor 43 into two-dimensional image data. The two-dimensional image includes a first image, a second image, and a third image obtained over time. The processing unit 45 observes the fluorescence generated by the phosphor in the composite 6 or the scattered light generated by the scatterer as a light spot of the two-dimensional image. At this time, in the two-dimensional image, the fluorescence emitted by the phosphor fixed to the second substance 5 or the scattered light emitted by the scatterer is also observed as a light spot. The processing unit 45 indicates a light spot that changes its position in the second direction on the two-dimensional image and indicates fluorescence emitted by the phosphor in the composite 6 or scattered light generated by the scatterer (that is, the first light spot). Identify as. In the observation of fluorescence or scattered light, the processing unit 45 further derives a motion vector of the first light spot from the first image and the second image, and uses the derived motion vector to obtain the first light spot in the third image. Is predicted, and the disappearance of the first light spot is detected based on the presence or absence of the light spot at the position of the first light spot predicted in the third image. At this time, the first magnetic field application unit 31 reapplies the first magnetic field gradient. When the reproduction of the first light spot is detected after the first magnetic field gradient is reapplied, the measuring unit 50 counts the first light spot as one light spot. In the density measurement, the measuring unit 50 does not count the first light spot if the reproduction of the first light spot is not detected after the first magnetic field gradient is reapplied. The details of these operations will be described later.

[Operation of concentration measuring device]
The operation of the concentration measuring device 100 configured as described above will be described with reference to FIG. FIG. 5 is a flowchart showing an example of the operation of the concentration measuring device 100 according to the embodiment. Here, the description will be given with reference to FIG.

First, in the introduction section (not shown), the liquid 61 prepared in advance is arranged on the substrate 11 (S5001). For example, the liquid 61 may be introduced into the space 15 through an introduction hole (not shown) provided between the cover glass 13 and the spacer 14. The space 15 is a space in which the liquid 61 can be sealed. As a result, the holding unit 10 holds the liquid 61 on the substrate 11. The liquid 61 is prepared by preparing a solution containing the substance 1 to be detected, a solution containing the first substance 3 on which the magnetic particles 2 are fixed, and a second substance on which the labeled particles 4 (here, a phosphor) are fixed. This is done by mixing the solutions containing 5 in no particular order.

Next, the light source 20 irradiates the substrate 11 with the excitation light 21 (S5002). As a result, a part of the liquid 61 is irradiated with the excitation light 21. In the present embodiment, the light source 20 irradiates the back surface 11b of the substrate 11 with the excitation light 21 via the prism 12. As a result, a proximity field is formed on the surface 11a of the substrate 11. Here, the region where the proximity field is formed is referred to as an irradiation region. The irradiation of the excitation light 21 is continuously performed until the concentration measurement process is completed.

The first magnetic field application unit 31 applies the first magnetic field gradient to the liquid 61 in the first direction (downward in FIG. 2) (S5003). Due to this first magnetic field gradient, the magnetic particles 2 in the liquid 61 move in the first direction. As a result, the complex 6 and the first substance 3 existing in the liquid 61 are collected in the irradiation region of the excitation light. The application of the first magnetic field gradient is performed for a predetermined period. The predetermined period is a period in which the complex 6 dispersed in the liquid 61 has a sufficient length to move into the irradiation region. The length of the predetermined period is set according to the degree of dispersibility and magnetism of the particles in the liquid 61 and the strength of the first magnetic field gradient.

Next, the measuring unit 50 starts detecting fluorescence (not shown). Subsequently, the first magnetic field application unit 31 stops the application of the first magnetic field gradient (S5004).

Next, the second magnetic field application unit 32 applies the second magnetic field gradient to the liquid 61 in a second direction (leftward in FIG. 2) different from the first direction (S5005). As a result, the complex 6 in the liquid 61 moves in the second direction. As a result, the complex 6 moves in the second direction in the irradiation region of the excitation light 21.

Next, the measuring unit 50 observes the fluorescence emitted by the phosphor in the composite 6 as the light spot of the two-dimensional image, and counts the light spots whose position changes in the second direction on the two-dimensional image (S5006). The measuring unit 50 ends the fluorescence detection (not shown).

Subsequently, the detailed processing of step S5006 will be described with reference to FIGS. 6 and 7. FIG. 6 is a flowchart showing the detailed processing of step S5006 of FIG. FIG. 7 is a diagram schematically showing the process of FIG.

First, the photodetector 40 captures the first image and the second image (S6001). As shown in FIGS. 7A and 7B, the first image is a two-dimensional image captured before the second image. The light detection unit 40 outputs each of the first image and the second image to the processing unit 45.

Next, the processing unit 45 acquires the first image and the second image output from the light detection unit 40, and detects the light spot in the first image and the light spot in the second image (FIG. 6). Not shown). The processing unit 45 detects light spots (for example, first light spot 7, second light spot 8, and third light spot 9) in the first image and the second image (see FIG. 7). Subsequently, the processing unit 45 extracts a light spot (moving light spot) whose position changes in the second direction on the first image and the second image. In this example, the moving light spots are the first light spot 7 and the second light spot 8. FIG. 6 describes the process related to the first light spot 7.

The processing unit 45 derives the motion vector 7a of the first light spot 7 (see (c) of FIG. 7) from the first image and the second image (S6002). A motion vector 8a is derived for the second light spot 8.

Next, the processing unit 45 uses the derived motion vector 7a to predict the position of the first light spot 7 in the third image captured after a predetermined time (S6003).

Next, the light detection unit 40 captures a third image (S6004) and outputs the third image to the processing unit 45.

When the third image is acquired, the processing unit 45 determines whether or not a light spot is detected at the predicted position of the first light spot 7 in the third image (S6005). When the processing unit 45 determines that a light spot is detected at the predicted position of the first light spot 7 in the third image (Yes in S6005), the light whose position changes in the second direction on the two-dimensional image. Count the points (S6006).

On the other hand, when the processing unit 45 determines that the light spot is not detected at the predicted position of the first light spot 7 in the third image (No in S6005), that is, the disappearance of the first light spot 7 is detected. If so, the first magnetic field application unit 31 reapplies the first magnetic field gradient (S6008) after the application of the second magnetic field gradient is stopped (S6007).

Next, the processing unit 45 determines whether or not the extinguished first light spot 7 is reproduced (S6009). When the processing unit 45 determines that the light spot is detected at the position where the first light spot 7 disappears in the third image, that is, when it determines that the disappeared first light spot 7 is reproduced (Yes in S6009). ), The reproduced light spots are included in the light spots whose position changes in the second direction on the two-dimensional image and counted (S6010).

On the other hand, when the processing unit 45 determines that the light spot is not detected at the position where the first light spot 7 disappears in the third image, that is, when it determines that the disappeared first light spot 7 is not reproduced ( No in S6009), the light spots whose position changes in the second direction on the two-dimensional image are counted without including the extinguished light spots (S6011).

The order of the steps shown in FIGS. 5 and 6 is an example and is not limited thereto. For example, the formation of the proximity field in step S5002 of FIG. 5 may be started after the application of the first magnetic field gradient in step S5003 or before the placement of the liquid 61 on the substrate 11 in step S5001. Good. Further, the stop of the application of the second magnetic field gradient in step S6007 of FIG. 6 may be executed after determining whether or not the extinguished first light spot 7 in step S6009 is reproduced. In this case, it is determined whether or not the first light spot 7 is reproduced at the position predicted by using the motion vector 7a.

[Effects, etc.]
As described above, according to the present embodiment, while the complex 6 is moved in the direction parallel to the surface 11a of the substrate 11, the convection of the liquid 61 containing the complex 6 and the Brownian motion of the complex 6 itself are performed. As a result, the complex 6 located in the proximity field moves out of the proximity field (that is, leaves the proximity field area) and returns to the proximity field again (that is, invades the proximity field area). As a result, the disappearance of fluorescence from the complex 6 is reduced. As a result, the measurement accuracy of the substance 1 to be detected is improved. Further, in the present embodiment, when it is detected that the composite 6 has gone out of the proximity field (disappearance of the moving light spot), a magnetic field gradient is applied in the direction perpendicular to the surface 11a of the substrate 11 (downward). Therefore, it is a temporary application. On the other hand, when a magnetic field gradient is constantly applied in a direction perpendicular to the surface 11a of the substrate 11, the composite 6 is easily attracted to the surface 11a of the substrate 11, and the composite 6 moves in a direction parallel to the surface 11a of the substrate 11. Cannot measure accurately. According to the present embodiment, since the downward magnetic field gradient is temporarily applied when necessary, the possibility that the composite 6 is adsorbed on the surface 11a of the substrate 11 can be reduced.

In the concentration measurement method according to the present embodiment, the first substance 3 which is the first substance 3 which specifically binds to the detection target substance 1 and the magnetic particles 2 are fixed is specifically bound to the detection target substance 1. A liquid 61 containing a complex 6 formed by binding the second substance 5 to be detected and the second substance 5 to which the labeled particles 4 (for example, a phosphor or a scatterer) are fixed and the detection target substance 1. Is arranged on the substrate 11, and the substrate 11 is irradiated with excitation light having a wavelength at which the phosphor emits fluorescence or a wavelength at which the scatterer generates scattered light, and is compounded in the first direction toward the irradiation region on the substrate 11. A first magnetic field gradient that attracts the body 6 is applied, the application of the first magnetic field gradient is stopped, and then a second magnetic field gradient that moves the complex 6 in a second direction different from the first direction is applied, and the complex Detected by observing the fluorescence generated by the phosphor in 6 or the scattered light generated by the scatterer as the light spots of the two-dimensional image and counting the number of light spots whose position changes in the second direction on the two-dimensional image. When the concentration of the target substance 1 is measured and the disappearance of the first light spot whose position changes in the second direction is detected in the two-dimensional image in the observation of fluorescence or scattered light, the first magnetic field gradient is reapplied. However, in the measurement of the density, when the reproduction of the first light spot is detected after the first magnetic field gradient is reapplied, the first light spot is counted as one light spot.

As a result, while the complex 6 located in the irradiation region on the substrate 11 is attracted and moves in the second direction, the complex 6 becomes the irradiation region due to the convection of the liquid 61 or the Brownian motion of the complex 6 itself. Even if it moves to the outside, the complex 6 can be pulled back to the illumination region by reapplying the first magnetic field gradient. Along with this, the disappeared and reproduced moving light points are counted as one moving light point, so that the possibility of overlapping counting of the moving light points is reduced. Since the moving light points are counted as one moving light point, the possibility of duplicate measurement is reduced. Therefore, according to the concentration measurement method according to the present embodiment, the counting accuracy of the complex 6 is improved, so that the quantification accuracy of the substance 1 to be detected is improved.

For example, in the concentration measurement method according to the present embodiment, when the reproduction of the first light spot is not detected after reapplying the first magnetic field gradient in the concentration measurement, the first light spot is not counted. Good.

As a result, the moving light spots corresponding to the labeled particles 4 that move regardless of the magnetic field gradient are not counted, so that the moving light points corresponding to the composite 6 can be accurately counted. For example, the magnetic particles 2 (pointing to the magnetic particles 2 unbonded to the substance to be detected 1) or the composite 6 are attracted in the second direction by the second magnetic field gradient and move to the labeled particles 4 (fluorescence) in the vicinity thereof. It is possible to reduce the possibility that the labeled particles 4 will be counted as the complex 6 when the body or scatterer) is affected by the slight flow of the liquid 61. Therefore, according to the concentration measurement method according to the present embodiment, the counting accuracy of the complex 6 is improved, so that the quantification accuracy of the substance 1 to be detected is improved.

For example, in the concentration measurement method according to the present embodiment, when the disappearance of the first light spot is detected in the observation of fluorescence or scattered light, the application of the second magnetic field gradient is stopped and then the first magnetic field gradient is applied. It may be reapplied.

Thereby, when the light spot is reproduced at the position where the first light spot disappears (including the position in the vicinity thereof), the reproduced light spot can be counted as the first light spot. Therefore, it becomes easy to determine whether or not the light spots are the same, and the processing amount is reduced. Therefore, according to the concentration measurement method according to the present embodiment, the processing efficiency is improved.

For example, in the concentration measurement method according to the present embodiment, the substrate 11 may form a close field of the excitation light on the surface of the substrate 11 by being irradiated with the excitation light.

As a result, the light (fluorescence or scattered light) emitted by the labeled particles 4 (pointing to the labeled particles 4 unbonded to the detection target substance 1) is detected as the light emitted by the labeled particles 4 in the complex 6 (pseudo). Positive) can be reduced. Since the proximity field is formed near the surface of the substrate 11 (for example, several hundred nm to 1 μm), only the labeled particles 4 located near the surface of the substrate 11 are excited among the liquids 61 arranged on the substrate 11. Excited by light. However, since the labeled particles 4 are not attracted in the second direction even when the second magnetic field gradient is applied, they are basically detected as light spots that do not move, unlike the complex 6 that is attracted and moves in the second direction. Therefore, according to the concentration measurement method according to the present embodiment, false positives can be reduced, and the quantification accuracy of the substance 1 to be detected is improved.

For example, in the density measurement method according to the present embodiment, the two-dimensional image includes the first image, the second image, and the third image obtained with the passage of time, and in the observation of fluorescence or scattered light, the second image is further increased. The motion vector of the first light spot is derived from the first image and the second image, the position of the first light spot in the third image is predicted using the derived motion vector, and the predicted first light in the third image. The disappearance of the first light spot may be detected based on the presence or absence of the light spot at the position of the spot.

As a result, the moving first light spot can be identified over time, so that the possibility of erroneously counting as another moving light spot can be reduced. Therefore, the measurement accuracy of the moving light point is improved. Further, since the disappearance of the first light spot is detected by using the predicted image and the actually captured image, the concentration measurement of the substance 1 to be detected can be continuously performed. Therefore, the concentration of the substance 1 to be detected can be efficiently measured. Therefore, according to the concentration measurement method according to the present embodiment, the quantification accuracy and quantification efficiency of the substance 1 to be detected are improved.

Further, the concentration measuring device according to the present embodiment is the first substance 3 that specifically binds to the detection target substance 1 and has the magnetic particles 2 fixed thereto, and is specific to the detection target substance 1. Liquid 61 containing the complex 6 formed by binding the second substance 5 which is the second substance 5 to be bound to the substance 5 and the labeled particles 4 (fluorescent substance or scatterer) are fixed and the substance 1 to be detected. A holding unit 10 that holds the substrate 11 on the substrate 11, a light source 20 that irradiates the substrate 11 with excitation light having a wavelength at which the phosphor emits fluorescence or a wavelength at which the scatterer generates scattered light, and an irradiation region on the substrate 11. A first magnetic field application unit 31 that applies a first magnetic field gradient that attracts the complex 6 in the first direction toward the first direction, and a second magnetic field gradient that moves the complex 6 in a second direction different from the first direction. The light that changes its position in the second direction on the two-dimensional image by observing the fluorescence applied by the two magnetic field application unit 32 and the fluorescence generated by the phosphor in the composite 6 or the scattered light generated by the scatterer as the light spots of the two-dimensional image. A measurement unit 50 for measuring the concentration of the substance 1 to be detected by counting the number of points is provided, and the second magnetic field application unit 32 applies the second magnetic field gradient after stopping the application of the first magnetic field gradient. Then, when the disappearance of the first light spot whose position changes in the second direction is detected in the two-dimensional image, the first magnetic field application unit 31 reapplies the first magnetic field gradient, and the measurement unit 50 receives the measurement unit 50. When the reproduction of the first light spot is detected after reapplying the first magnetic field gradient, the first light spot is counted as one light spot.

As a result, while the complex 6 located in the irradiation region on the substrate 11 is attracted and moves in the second direction, the complex 6 becomes the irradiation region due to the convection of the liquid 61 or the Brownian motion of the complex 6 itself. Even if it moves to the outside, the complex 6 can be pulled back to the illumination region by reapplying the first magnetic field gradient. Along with this, the disappeared and reproduced moving light points are counted as one moving light point, so that the possibility of overlapping counting of the moving light points is reduced. Since the moving light points are counted as one moving light point, the possibility of duplicate measurement is reduced. Therefore, according to the concentration measuring device according to the present embodiment, the counting accuracy of the complex 6 is improved, so that the quantification accuracy of the substance 1 to be detected is improved.

(Other embodiments)
Although the concentration measuring device and the concentration measuring method according to one or more aspects of the present disclosure have been described above based on the embodiment, the present disclosure is not limited to this embodiment. As long as it does not deviate from the gist of the present disclosure, one or more of the present embodiments may be modified by those skilled in the art, or may be constructed by combining components in different embodiments. It may be included within the scope of the embodiment.

Further, one aspect of the present disclosure may be a computer program that causes a computer to execute each characteristic step included in the concentration measurement method. Further, one aspect of the present disclosure may be a non-temporary recording medium that can be read by a computer on which such a computer program is recorded.

The present disclosure is used for a sensor device that detects a substance to be detected easily, at high speed, and with high accuracy.

1 Substance to be detected 2 Magnetic particles 3 1st substance 4 Labeled particles 5 2nd substance 6 Complex 7 1st light spot 7a Motion vector 8 2nd light spot 8a Motion vector 9 3rd light spot 10 Holding part 11 Substrate 11a Surface 11b Back side 12 Prism 13 Cover glass 14 Spacer 15 Space 20 Light source 21 Excitation light 31 First magnetic field application part 32 Second magnetic field application part 40 Light detection part 41 Condensing member 42 Filter 43 Image sensor 45 Processing part 50 Measuring part 61 Liquid 100 Concentration Measuring device

Claims (6)

The first substance that specifically binds to the substance to be detected and the magnetic particles are fixed, and the second substance that specifically binds to the substance to be detected and the phosphor or scatterer is fixed. A liquid containing a complex formed by binding the second substance and the detection target is placed on the substrate.
The substrate is irradiated with excitation light having a wavelength at which the phosphor emits fluorescence or a wavelength at which the scatterer generates scattered light.
A first magnetic field gradient that attracts the complex is applied in the first direction towards the irradiation region on the substrate.
After stopping the application of the first magnetic field gradient, a second magnetic field gradient that moves the complex in a second direction different from the first direction is applied, and fluorescence generated by the phosphor in the complex or fluorescence or The concentration of the detection object is measured by observing the scattered light generated by the scatterer as a light spot of a two-dimensional image and counting the number of light spots whose position changes in the second direction on the two-dimensional image. And
In the observation of the fluorescence or the scattered light,
When the disappearance of the first light spot whose position changes in the second direction is detected in the two-dimensional image, the first magnetic field gradient is reapplied.
In the measurement of the concentration,
When the reproduction of the first light spot is detected after reapplying the first magnetic field gradient, the first light spot is counted as one light spot.
Concentration measurement method. In the measurement of the concentration,
If the reproduction of the first light spot is not detected after reapplying the first magnetic field gradient, the first light spot is not counted.
The concentration measuring method according to claim 1. In the observation of the fluorescence or the scattered light,
When the disappearance of the first light spot is detected, the application of the second magnetic field gradient is stopped and then the first magnetic field gradient is reapplied.
The concentration measuring method according to claim 1 or 2. When the substrate is irradiated with the excitation light, a near field of the excitation light is formed on the surface of the substrate.
The concentration measuring method according to any one of claims 1 to 3. The two-dimensional image includes a first image, a second image, and a third image obtained with the passage of time.
In the observation of the fluorescence or the scattered light, further
The motion vector of the first light spot is derived from the first image and the second image, and the motion vector is derived.
Using the derived motion vector, the position of the first light spot in the third image is predicted.
The disappearance of the first light spot is detected based on the presence or absence of the light spot at the position of the first light spot predicted in the third image.
The concentration measuring method according to any one of claims 1 to 4. The first substance that specifically binds to the detection target and the magnetic particles are fixed, and the second substance that specifically binds to the detection target and the phosphor or scatterer is fixed. A holding portion for holding the liquid containing the complex formed by binding the second substance and the detection target on the substrate, and
A light source that irradiates the substrate with excitation light having a wavelength at which the phosphor emits fluorescence or a wavelength at which the scatterer generates scattered light.
A first magnetic field application unit that applies a first magnetic field gradient that attracts the complex in the first direction toward the irradiation region on the substrate.
A second magnetic field application unit that applies a second magnetic field gradient that moves the complex in a second direction different from the first direction,
The number of light spots in which the fluorescence generated by the phosphor or the scattered light generated by the scatterer in the complex is observed as the light spots of the two-dimensional image and the position changes in the second direction on the two-dimensional image. And a measuring unit that measures the concentration of the substance to be detected by counting
With
The second magnetic field application unit applies the second magnetic field gradient after stopping the application of the first magnetic field gradient.
When the disappearance of the first light spot whose position changes in the second direction is detected in the two-dimensional image, the first magnetic field application unit reapplies the first magnetic field gradient.
When the reproduction of the first light spot is detected after the first magnetic field gradient is reapplied, the measuring unit counts the first light spot as one light spot.
Concentration measuring device.