The present invention relates to a device and a method for detecting a substance to be measured.
There have been increasing needs for a method for detecting a biological substance, such as a virus, a bacterium, or a fungus, that exists in a solution of a biological sample. As a method for detecting a biological substance having a size of several hundreds of nanometers, such as a virus, is known an optical detection method with near-field light (e.g., Patent Literature 1). “Near-field light” refers to light generated, when light entering a low-refractive-index medium from a high-refractive-index medium is totally reflected by the interface, only near to the interface on the side of the low-refractive-index medium, and has the property of being rapidly attenuated as it goes away from the interface.
However it may be difficult to detect bacteria, fungi, or other biological substances by the optical detection method with near-field light because they have a size of several micrometers.
An object of a device and a method for detecting a substance to be measured according to an embodiment of the present disclosure is to conveniently detect a biological substance, such as a bacterium or a fungus.
A device for detecting a substance to be measured according to an embodiment of the present disclosure includes a container that retains a solution containing the substance to be measured and a magnetic labeling substance that binds specifically to the substance to be measured, a flow generating unit (flow generator) that generates a flow in a first direction at least in the solution, a magnetic field generating unit (magnetic field generator) that generates a magnetic field gradient in the solution, and a detection unit (detector) that detects composite particles, based on motion of particles in a predetermined region in the solution, the composite particles being the substance to be measured to which the magnetic labeling substance is bound.
The predetermined region in the solution is preferably separated from an inner wall surface of the container.
The flow generating unit is preferably a light source that radiates spatial light into the container.
The solution may contain another substance that is not the substance to be measured nor the magnetic labeling substance, and the detection unit may detect the composite particles, based on motion of the composite particles and the other substance in the predetermined region in the solution.
The magnetic field generating unit preferably moves the composite particles in a second direction different from the first direction.
The magnetic field generating unit may move the composite particles in a second direction identical to the first direction.
The detection unit preferably detects the composite particles, based on directions of motion of the composite particles and the other substance.
The detection unit preferably detects the composite particles, based on speeds of motion of the composite particles and the other substance.
The flow generating unit may heat the solution to cause convection therein to generate the flow in the first direction at least in part of the solution.
The flow generating unit may rotate the container to generate the flow in the first direction at least in part of the solution.
The flow generating unit may stir the solution to generate the flow in the first direction at least in part of the solution.
The composite particles preferably further include a fluorescent labeling substance, and the detection unit preferably optically detects the fluorescent labeling substance to detect particles to which the fluorescent labeling substance is bound, and detects the composite particles, based on motion of the detected particles.
A method for detecting a substance to be measured according to an embodiment of the present disclosure includes the steps of retaining in a container a solution containing the substance to be measured and a magnetic labeling substance that binds specifically to the substance to be measured, generating a flow in a first direction at least in the solution, generating a magnetic field gradient in the solution, and detecting composite particles, based on motion of particles in a predetermined region in the solution, the composite particles being the substance to be measured to which the magnetic labeling substance is bound.
The detection device and method according to an embodiment of the present disclosure enable conveniently detecting a biological substance, such as a bacterium or a fungus.
Hereinafter, devices and methods for detecting a substance to be measured according to embodiments of the present disclosure will be described with reference to the drawings. However, note that the technical scope of the present invention is not limited to these embodiments and includes the invention described in the claims and equivalents thereof.
First, a device for detecting a substance to be measured according to embodiment 1 of the present disclosure will be described.
The container 1 retains a solution 14 containing a substance 11 to be measured and a magnetic labeling substance 12 that binds specifically to the substance 11 to be measured. The magnetic labeling substance 12 preferably binds to all the substance 11 to be measured in the solution 14 to form composite particles 13. It is not necessary that these substances bind together at the very moment when the substance 11 to be measured and the magnetic labeling substance 12 are injected into the container 1. More specifically, for example, a flow of the solution 14 generated in the container 1 may facilitate a reaction by which the magnetic labeling substance 12 binds to the substance 11 to be measured, thereby generating the composite particles 13. Examples of the substance 11 to be measured include candida, Escherichia coli (E. coli), and CRP (C-reactive protein). Specific examples of the steps for detecting such a substance will be described below.
The flow generating unit 2 generates a flow in a first direction 21 at least in the solution 14. For example, as shown in
In the example shown in
The magnetic field generating unit 3 generates in the solution 14 a magnetic field gradient for moving the composite particles 13 in a second direction 31 different from the first direction 21. The composite particles 13 are moved in the second direction 31 by the resultant of force in the first direction 21 and force caused by the magnetic field gradient. As the magnetic field generating unit 3, for example, a magnet or an electromagnet can be used.
The detection unit 4 includes an imaging unit 44 and a processing unit 45. The imaging unit 44 has the function of taking a picture to capture an image. As the imaging unit 44, for example, an image capturing device, such as a camera or a video camera for capturing still images or moving images, may be used. The processing unit 45 has the function of detecting composite particles from the captured images. As the processing unit 45, for example, a computer including a CPU and a memory can be used. The function of the processing unit 45 detecting composite particles from images captured by the imaging unit 44 is executed by the CPU in the processing unit 45 in accordance with a program prestored in the memory in the processing unit 45. The detection unit 4 detects the composite particles 13, which are the substance 11 to be measured to which the magnetic labeling substance 12 is bound, based on motion of particles in the predetermined detection region 16 in the solution 14. Illumination light 51 radiated from the illumination device 5 is reflected by a mirror 43 to illuminate the solution 14. As the illumination light 51, spatial light can be used. In other words, the illumination device 5 is a light source that radiates spatial light into the container 1. Spatial light (also referred to as “propagating light”) is ordinary light propagating in space rather than localized light, such as near-field light. More specifically, spatial light generally refers to light that does not include near-field light, which is rapidly attenuated at a position several hundreds of nanometers to several micrometers away from its source. In the present description also, it refers to light that does not include near-field light, i.e., light that is not rapidly attenuated at a position several hundreds of nanometers to several micrometers away from the interface between the container and the solution. Since the predetermined region 16 in the present description is a region separated several micrometers or more from the inner wall surface of the container 1, near-field light is not used in the predetermined region 16. Detection light 41 reflected by the composite particles 13 in the solution 14 enters the imaging unit 44 of the detection unit 4.
The magnetic labeling substance 12 binds specifically to the substance 11 to be measured. The solution 14 may contain other substances 17 that are not the substance 11 to be measured nor the magnetic labeling substance 12. The “other substances” are not the substance to be measured and include impurities. The magnetic labeling substance 12 does not bind to the other substances 17. As shown in
The composite particles 13 is simultaneously acted on not only by force in the first direction 21 but also by force in a direction different from the first direction 21 caused by the magnetic field gradient. If only the force in a direction different from the first direction 21 caused by the magnetic field gradient acts on the composite particles 13, the other substances 17, which are not the targets for measurement, also move simultaneously by being pulled by the composite particles 13, which may result in erroneous detection of the number of particles. Thus the detection device according to the embodiment of the present disclosure makes force in two different directions, i.e., in the first direction 21 and a direction different from the first direction 21 act on the composite particles 13, allowing for separating the other substances 17 from the composite particles 13.
The magnetic field generating unit 3 may move the composite particles 13 in a second direction 31, which is identical to the first direction 21. In this case, the detection unit 4 can detect the composite particles 13, based on the speeds of motion of the composite particles 13 and the other substances 17. Even if the second direction 31, the direction of motion of particles caused by the magnetic field gradient, is identical to the first direction 21, the direction of motion of particles caused by the flow generating unit 2, the composite particles 13 including the magnetic labeling substance 12 move faster than the other substances 17, which do not include the magnetic labeling substance 12, due to the magnetic field gradient. Thus the composite particles 13 can be detected from obtained images, based on the fact that their speeds differ. If the second direction 31, the direction of motion of particles caused by the magnetic field gradient, is opposite to the first direction 21, the direction of motion of particles caused by the flow generating unit 2, the speeds and direction of motion of the composite particles 13 including the magnetic labeling substance 12 differ from those of the other substances 17, which do not include the magnetic labeling substance 12, due to the magnetic field gradient. Thus the composite particles 13 can be detected from obtained images.
The following describes a method for detecting a substance to be measured according to embodiment 1 of the present disclosure.
Next, in step S102, the flow generating unit 2 generates a flow in the first direction 21 at least in the solution 14. In the example shown in
Next, in step S103, the magnetic field generating unit 3 generates a magnetic field gradient in order to move the composite particles 13 in the second direction 31 different from the first direction 21.
Next, in step S104, the detection unit 4 detects particles moving in the second direction 31. More specifically, the imaging unit 44 of the detection unit 4 captures images of the detection region 16 in the solution 14, and the processing unit 45 executes a process for detecting the composite particles 13 and the other substances 17 (described below), using these captured images. The following describes the “method for detecting the composite particles 13,” which is divided into a “method for adjusting the focus of an image,” a “method for image processing by which the detection unit detects the composite particles from an obtained image,” and a “method for recognizing particles moving in obtained images.”
First, a method by which the detection unit 4 adjusts the focus of an image (the focal depth at capturing the image) will be described in detail.
The imaging unit 44 has the function of adjusting the focus, and can be set so as to have a predetermined focal depth in the detection region 16.
Thus it is preferable that the detection region 16 be set in a region 16b or 16c located a predetermined distance away from the bottom of the container 1, and that the focus f2 or f3 of the imaging unit 44 be set near the center of the corresponding region, as shown in
The following describes a method for image processing by which the detection unit detects the composite particles from an obtained image.
The following describes a method by which the detection unit 4 recognizes particles moving in obtained images.
When fluorescent light is not used, all the substances in the sample solution that scatter light are recorded in the images, and thus if there are particles receiving force caused by the magnetic field gradient other than the composite particles 13, these particles are also recorded in the images. For this reason, it is necessary to separate the particles recorded in the images.
More specifically, a process is necessary for excluding a separate magnetic labeling substance 12 and particles of impurities or other substances to which the magnetic labeling substance 12 is nonspecifically bound, using the speeds and directions of movement vectors. This process will be described below.
First, a separate magnetic labeling substance 12 will be considered. A separate magnetic labeling substance 12 moves faster than a composite particle in the same magnetic field gradient because it does not have an extra load (a counterpart to form a composite particle) that would exist if it were a composite particle. It can be separated by setting a threshold at the known maximum moving speed of the composite particles and excluding target particles having a speed greater than the threshold. Since the moving speed varies depending on the magnitude of the magnetic field gradient, i.e., the place in the detection region, it is necessary to set thresholds for respective places beforehand by calculation or measurement and to store them in the processing unit 45.
The magnetic labeling substance 12 nonspecifically bound to impurities can be separated by setting a threshold at the known minimum speed of the composite particles 13 and excluding target particles having a speed less than the threshold. However, theoretically, if the magnetic labeling substance 12 has properties (the size, molecular weight, and surface state) similar to those of the substance 11 to be measured, the specificity of binding to the substance 11 to be measured needs to be set sufficiently high as necessary.
The detection unit of the detection device according to embodiment 1 of the present disclosure may detect the composite particles, based on the moving speeds of objects moving in the predetermined region of the container. The moving directions and speeds of particles are determined from the database of particle coordinates created as described above. More specifically, when the magnetic field generating unit 3 is disposed at the center of the screen as shown in
The following describes a device for detecting a substance to be measured according to modified example 1 of embodiment 1 of the present disclosure. In the above embodiment is shown the example in which the flow generating unit 2 heats the solution 14 to cause convection therein to generate a flow in the first direction 21 at least in part of the solution 14, but the invention is not limited to this example. More specifically, the flow generating unit 2 may rotate the container 1 to generate a flow in the first direction 21 at least in part of the solution 14.
The following describes a device for detecting a substance to be measured according to modified example 2 of embodiment 1 of the present disclosure. The detection device according to modified example 2 is characterized in that the flow generating unit stirs the solution to generate a flow in a first direction at least in part of the solution.
As shown in
The following describes a method for determination by which the composite particles and the other substances are separated using movement vectors of particles. First, a “rotation process” is performed for removing motion of particles arising from rotation of the container 1. When the rotation rate of the container 1 is known, the obtained images are rotated opposite to the rotating direction of the container 1.
As a more convenient method for determination, changes in the distance from the origin of XY coordinates of the particles can be used in
The following describes a device for detecting a substance to be measured according to modified example 3 of embodiment 1 of the present disclosure. In the above embodiment is shown the example in which the detection unit 4 placed above the container 1 is used to detect the composite particles, but the invention is not limited to this example. The composite particles may be detected from the side surface of the container in parallel with the horizontal direction.
The detection device according to modified example 3 of embodiment 1 of the present disclosure can detect the moving composite particles 13 in the detection region 16 in a direction substantially orthogonal to the direction of the magnetic field gradient, allowing for detecting the same composite particle for a longer time than when observing in a direction substantially the same as the direction of the magnetic field gradient.
According to the detection device and method according to embodiment 1, the substance to be measured can be easily detected by detecting the composite particles that are the substance to be measured to which the magnetic labeling substance is bound, as described above.
The following describes a device for detecting a substance to be measured according to embodiment 2 of the present disclosure.
The container 1 retains a solution 14 containing a substance 11 to be measured as well as a magnetic labeling substance 12 and a fluorescent labeling substance 15 that bind specifically to the substance 11 to be measured. The magnetic labeling substance 12 and the fluorescent labeling substance 15 preferably bind to all the substance 11 to be measured in the solution 14 to form composite particles 13e.
It is not necessary that the magnetic labeling substance 12 and the fluorescent labeling substance 15 bind to the substance 11 to be measured at the very moment when they are injected into the container 1. More specifically, for example, a flow of the solution 14 generated in the container 1 may facilitate a reaction by which the magnetic labeling substance 12 and the fluorescent labeling substance 15 bind to the substance 11 to be measured, thereby generating the composite particles 13e.
Illumination light 51 radiated from the illumination device 5 passes through an illumination-side optical filter 52, and is reflected by a mirror 43 to illuminate the solution 14. As the illumination light 51, spatial light can be used. Detection light 41 reflected by the composite particles 13e and substances 17 other than the substance to be measured in the solution 14 enters the detection unit 4 through a detection-side optical filter 42. The illumination-side optical filter 52 passes light having wavelengths such that it illuminates and thereby excites the fluorescent labeling substance 15 so as to emit fluorescent light, but does not pass light having the other wavelengths. The detection-side optical filter 42 passes the fluorescent light emitted from the fluorescent labeling substance 15, but does not pass light having the other wavelengths.
Of fluorescent labeling substances 15, some bind specifically to the substance 11 to be measured, and others do not. The present embodiment describes the case in which a fluorescent labeling substance 15 that binds specifically to the substance 11 to be measured is used.
First, in step S201, the container 1 is made to retain the solution 14 containing the substance 11 to be measured as well as the magnetic labeling substance 12 and the fluorescent labeling substance 15 that bind specifically to the substance 11 to be measured.
Next, in step S202, the flow generating unit 2 generates a flow in the first direction 21 at least in the solution 14. In the example shown in
Next, in step S203, the magnetic field generating unit 3 generates a magnetic field gradient in order to move the composite particles 13e in the second direction 31 different from the first direction 21.
Next, in step S204, the detection unit 4 detects the fluorescent labeling substance 15 to detect particles moving in the second direction 31 to which the fluorescent labeling substance 15 is bound.
A method for separating the composite particles and the other substances will be specifically described, using the defined movement vectors. The following description relates to the case in which fluorescent light is used, but can also be applied similarly to the case in which fluorescent light is not used.
When fluorescent light is used, the other substances to be separated are particles that are not the composite particles 13 and that do not receive force in the second direction 31 caused by the magnetic field gradient. The other substances to be separated are, for example, a separate fluorescent labeling substance 15 that is not bound to any particle and particles of substances, such as impurities, that are not the substance to be measured and to which the fluorescent labeling substance 15 is bound.
In
The composite particles 13 receive the force in the second direction 31 (see
The reason the center of the circle is deviated from the origin is that both the force in the first direction 21 and the force in the second direction 31 act on the composite particles 13. In other words, the resultant of movement vector A and a movement vector caused by the force of the magnetic field gradient is movement vector B.
If the force in the first direction 21 is zero, the center of the circle of movement vectors B′ will agree with the origin, as shown in
By the above technique, the composite particles and the other substances can be separated, using the speeds and directions of movement of particles. The steps thereof are summarized as follows.
1) Successively determine vectors of movement of the particles at a certain time and after the elapse of a certain time period, as shown in
2) Judge particles whose movement vectors are concentrated near the center as shown in
3) Judge particles whose movement vectors are in a circle centered at the final point of movement vector A as shown in
4) Determine that candidates for the composite particles having movement vectors whose magnitudes vary with the passage of time and whose directions are unchanged are the composite particles 13, and count the number of particles of the candidates.
The number of composite particles can be counted from successively obtained images by the processing unit 45 executing the above process.
The time intervals at which the movement vectors are determined can be adjusted depending on the moving speeds of the particles and the frame rate of image capturing by a camera or other devices.
According to the detection device and method according to embodiment 2 of the present disclosure, particles smaller than the composite particles 13 in embodiment 1 can be detected by detecting particles to which the fluorescent labeling substance 15 is bound.
When a fluorescent labeling substance 15 that does not bind specifically to the substance 11 to be measured is used, the fluorescent labeling substance 15 may bind to the other substances 17. Even if the fluorescent labeling substance 15 is bound to the other substances 17, the composite particles can be distinguished from the other substances 17 to which the fluorescent labeling substance 15 is bound, and detected, based on the difference in motion. Further, there may be a fluorescent labeling substance 15 that is not bound to particles of the substance 11 to be measured, in the container 1. Even if there is a fluorescent labeling substance 15 that is not bound to particles of the substance 11 to be measured, the detection unit 4 can distinguish the composite particles from such a fluorescent labeling substance 15, and detect them, based on the difference in motion.
In the detection device and method according to embodiment 2, the region in the detection region 16 irradiated by the illumination device 5 with the illumination light 51 is preferably set so as to avoid the region of the magnetic field generating unit 3.
The following describes a method for image processing by which the detection unit detects the composite particles from an obtained image.
The following describes two specific examples of the detection method performed by the detection device according to embodiment 2 of the present disclosure. The first example is a detection method with a fluorescent labeling substance.
First, as shown in
Next, as shown in
The second example is a detection method in which fluorescent staining is performed.
First, as shown in
Next, as shown in
The values shown in the above are merely examples, and the invention is not limited thereto.
The following describes a device for detecting a substance to be measured according to embodiment 3 of the present disclosure.
The detection device 105 according to embodiment 3 of the present disclosure is characterized by using a portable device 200, such as a smartphone, to detect the substance to be detected. The container 1, the magnetic field generating unit 3, and the illumination device 5 are housed in a measurement housing 100. The measurement housing 100 is composed of an upper housing 100a and a lower housing 100b. The illumination device 5 is housed in the lower housing 100b. The container 1 is placed on the upper surface of the lower housing 100b. The magnetic field generating unit 3 is disposed on the side surface of the container 1. The portable device 200 is placed on the upper surface of the upper housing 100a, which has an opening 201 so that detection light 41 can enter the detection unit 4, which is, for example, a camera of the portable device 200. The illumination device 5 irradiates the container 1 with illumination light 51 from below, and the detection light 41 enters the detection unit 4 of the portable device 200. Since the container 1 is heated by the illumination light 51, the illumination device 5 serves as the flow generating unit 2.
The measurement principle of the detection device 105 according to embodiment 3 of the present disclosure is the same as that of the detection device 101 according to embodiment 1. Images captured by the detection unit 4 of the portable device 200 can be displayed in an image display area 200b in a display 200a of the portable device 200. Data analyzed from the obtained images, such as the number and moving speeds of particles of the substance to be measured, can be displayed in a data display area 200c in the display 200a. The substance to be measured can be more conveniently detected by detection of images and execution of image processing with a portable device, as in the embodiment of the present disclosure.
The following describes examples of the container used in the detection devices according to embodiments 1 to 3. The flat-bottomed container has mainly been described as an example of the container used in the above embodiments, but the container is not limited to this example. More specifically, the container 1 may have a curved bottom, as in
The shapes shown in
As shown in
The following describes specific examples of the steps of detection, taking candida, E. coli, and CRP (C-reactive protein) as examples of the substance 11 to be measured.
An example in which candida is detected without using a fluorescent labeling substance will be described. The size of candida, which is a fungus, is approximately 5 to 10 [μm]. Candida is an indigenous fungus inhabiting, for example, the saliva, body surface, and digestive tract of humans. As shown in
Candida albicans antibody labeled with biotin is obtained by combining Anti-Candida albicans, Mouse (B341M)_IgG available from GeneTex Inc., with EZ-Link NHS-LC-Biotin available from Thermo Fisher Inc. As the magnetic labeling substance 12 labeled with avidin was used Dynabeads M-280 Streptavidinis available from Invitrogen Corp.
The mixed solution 14 reacts in the container 1 where convection occurs, forming composite particles 13 composed of candida, Candida albicans antibody, and the magnetic labeling substance 12. The flow generating unit 2 (e.g., convection, movement or rotation of the container, a flow cell, or gravity), which generates a flow in the first direction 21 in the solution 14 may be a means for generating the convection. For example, illumination light 51 from the illumination device 5 causes convection, generating a flow in the first direction 21.
When an external magnetic field is applied to the container 1, the magnetic labeling substance 12 exhibits characteristic motion. More specifically, the composite particles 13 including candida, the substance 11 to be measured, and the magnetic labeling substance 12 exhibit characteristic motion. The magnetic field generating unit 3 (e.g., a magnet, an electromagnet, or a magnetic film), which generates a magnetic field gradient in the detection region 16, may be used as a means for generating the external magnetic field.
It is irradiated by the illumination device 5 with spatial light (either transmitted light or epi-illumination light will do) as the illumination light 51, and detection light 41 reflected by the composite particles 13 is observed by the detection unit 4 at magnification of 50 to 1000. Then, the shapes and behavior of the composite particles 13, the magnetic labeling substance 12, and other substances can be seen. The composite particles 13 including candida can be distinguished by the shape specific to candida (yeast-like or mycelioid shape), the shape of the composite particles 13, and the characteristic motion in the second direction 31 different from the first direction 21 caused by the external magnetic field. Quantitative detection of candida, the substance 11 to be measured, could be achieved by obtaining two-dimensional images, using a means for optical detection (e.g., an image sensor) as the detection unit 4, and further analyzing the images.
The following describes the magnetic labeling substance used in the detection devices and methods according to the examples of the present disclosure. The magnetic labeling substance 12 has a structure of magnetic beads used for biomedical application, and as the magnetic substance, spinel ferrite is generally used. The size of the magnetic labeling substance 12 varies from nanometers to micrometers. A nano-sized substance has a larger surface area and diffuses wider in the solution on average by the Brownian movement, resulting in a high reactivity with the substance to be measured. However, since its particle size is small, magnetic force is weak. As the magnetic labeling substance 12, one having a size of 10 [nm] to 10 [μm] can be used.
An example in which E. coli is detected without using a fluorescent labeling substance will be described. E. coli, which is a bacterium, has a minor axis of 0.4 to 0.7 [μm] and a major axis of 2.0 to 4.0 [μm]. It is one of major species of bacteria existing in the environment. As shown in
The mixed solution 14 reacts in the container 1 where convection occurs, forming composite particles 13 composed of E. coli, an anti-E. coli antibody, and the magnetic labeling substance 12. The flow generating unit 2 (e.g., convection, movement or rotation of the container, a flow cell, or gravity), which generates a flow in the first direction 21 in the solution 14 may be a means for generating the convection. For example, illumination light 51 from the illumination device 5 causes convection, generating a flow in the first direction 21. After that, a magnetic field is applied by the magnetic field generating unit 3 to move the composite particles 13 in the second direction 31, and the composite particles 13 are detected with the illumination light 51, which is spatial light. These steps are similar to those in the case of candida described above, and thus description thereof is omitted.
An example in which candida is detected using a fluorescent labeling substance will be described. As shown in
Candida albicans antibody labeled with biotin is obtained by combining Anti-Candida albicans, Mouse (B341M)_IgG available from GeneTex Inc., with EZ-Link NHS-LC-Biotin available from Thermo Fisher Inc. As the magnetic labeling substance 12 labeled with avidin was used Dynabeads M-280 Streptavidinis available from Invitrogen Corp. Moreover, a method with a fluorescent labeling substance or a method of applying fluorescence resonance energy transfer may be used as a means for fluorescent labeling.
The antibody may be β1,3-glucan antibody or others that react specifically to a fungus, besides Candida albicans antibody.
The mixed solution 14 reacts in the container 1 where convection occurs, forming composite particles 13e composed of fluorescent candida, Candida albicans antibody, and the magnetic labeling substance 12. The flow generating unit 2 (e.g., convection, movement or rotation of the container, a flow cell, or gravity), which generates a flow in the first direction 21 in the solution 14 may be a means for generating the convection. For example, illumination light 51 from the illumination device 5 causes convection, generating a flow in the first direction 21.
When an external magnetic field is applied to the container 1, the magnetic labeling substance 12 exhibits characteristic motion. More specifically, the composite particles 13e including candida, which is the substance 11 to be measured, the magnetic labeling substance 12, and the fluorescent labeling substance 15 exhibit characteristic motion in the second direction 31 different from the first direction 21. The magnetic field generating unit 3 (e.g., a magnet, an electromagnet, or a magnetic film), which generates a magnetic field gradient in the detection region 16, may be used as a means for generating the external magnetic field.
It is irradiated by the illumination device 5 with spatial light having an excitation wavelength of the fluorescent labeling substance (either transmitted light or epi-illumination light will do) as the illumination light 51, and fluorescent detection light 41 reflected by the composite particles 13e is observed by the detection unit 4 at magnification of 50 to 1000. Then, the composite particles 13e including the fluorescent labeling substance 15 and an unreacted fluorescent labeling substance 15 can be observed as light spots. Additionally, the composite particles 13e including the magnetic labeling substance 12 can be distinguished by the characteristic motion in the second direction 31 different from the first direction 21 caused by the external magnetic field. Quantitative detection of candida, the substance 11 to be measured, could be achieved by obtaining two-dimensional images, using a means for optical detection (e.g., an image sensor) as the detection unit 4, and further analyzing the images. Combining a light source of a fluorescence wavelength with other wavelengths, such as white light, allows for obtaining information on the shapes of cells and the background together with information on fluorescent light and motion, which is effective in detecting a complex sample solution.
The following describes the fluorescent labeling substance used in the detection devices and methods according to the examples of the present disclosure. As the fluorescent labeling substance, one having a size of 10 [nm] to 10 [μm] can be used. It is expected that a fluorescent labeling substance 15 labeled with a fluorochrome, such as fluorescein (FITC), has high reactivity, because it has a smaller size than the magnetic labeling substance 12. For this reason, if the fluorescent labeling substance 15 and the magnetic labeling substance 12 are simultaneously added to the solution 14 to start a composite reaction, the fluorescent labeling substance 15 will react faster, which may reduce the magnetic labeling substance 12 that binds to the surface of the substance 11 to be measured.
To prevent this, it would be desirable to add the magnetic labeling substance 12 first to make it react, and then add the fluorescent labeling substance 15. In other words, it is expected that the smaller fluorescent labeling substance 15 can enter space between particles of the magnetic labeling substance 12 that is bound to the substance 11 to be measured. To larger particles, the magnetic labeling substance 12 acts as a three-dimensional barrier. In other words, imbalance of reactions can be prevented by changing the order of reactions according to the size of particles.
An example in which E. coli is detected using a fluorescent labeling substance will be described. As shown in
The mixed solution 14 reacts in the container 1 where convection occurs, forming composite particles 13e composed of the fluorescent labeling substance 15, E. coli, and the magnetic labeling substance 12. The flow generating unit 2 (e.g., convection, movement or rotation of the container, a flow cell, or gravity), which generates a flow in the first direction 21 in the solution 14 may be a means for generating the convection. For example, illumination light 51 from the illumination device 5 causes convection, generating a flow in the first direction 21. After that, a magnetic field is applied by the magnetic field generating unit 3 to move the composite particles 13e in the second direction 31, particles to which the fluorescent labeling substance 15 is bound are detected with the illumination light 51, which is spatial light, and the composite particles 13e are detected, based on motion of the detected particles. These steps are similar to those in the case of candida described above, and thus description thereof is omitted.
An example in which CRP is detected using a fluorescent labeling substance will be described. As shown in
The above detection devices and methods according to the examples of the present disclosure enable detection of micron-sized bacteria, fungi, and other substances in a solution.
Number | Date | Country | Kind |
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2019-053626 | Mar 2019 | JP | national |
Filing Document | Filing Date | Country | Kind |
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PCT/JP2020/011825 | 3/17/2020 | WO | 00 |