Optical Detection Method and Optical Detection Device Using Droplets

Abstract

To provide an optical detection method and the like capable of optical detection of target substances with high throughput and high accuracy, using a detection device capable of being configured simply, compactly and inexpensively.SOLUTION: An inventive optical detection method includes: a droplet holding step of causing a square-shaped droplet holding part 2, which has a depth of at least 3.5 times the diameter of droplets D1 and D2 and opening area of at least 100 times the cross-sectional area of the droplets, to hold a water-in-oil emulsion 2, in which a large number of droplets D1 and D2 encapsulating a sample liquid that may contain the target substance and a luminescence / coloring agent that reveals the presence of the target substance by luminescence / coloration are dispersed in the oil, and a refractive index difference between a water phase constituting the droplets D1 and D2 and an oil phase constituting the oil is less than 0.01; and a luminescence / color detection step of detecting the presence or absence of light emission and color development of droplets D1 and D2 with a detection unit that is placed on either an opening surface or the bottom surface of a droplet holding part 1 that holds the droplets D1 and D2.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to an optical detection method and an optical detection apparatus for improving throughput in the detection of a target substance using droplets by an array method.

Background Art

[0002] There is known a droplet detection method in which a test solution that may contain a target substance is divided into small-volume droplets in oil, and the target substance is detected through the light emission / color development of the droplets based on the reaction between the target substance and a light-emitting / color-developing agent. As implementation methods of the droplet detection method, two implementation methods, a flow method and an array method, have been proposed.

[0003] In the flow method, an oil-in-water droplet emulsion in which droplets of the test solution are dispersed in the oil with the test solution as an aqueous phase is prepared, and this oil-in-water droplet emulsion is fed into a flow cell. Laser light is irradiated onto the droplets flowing through the flow cell, and the light emission / color development state of the droplets to be detected is detected by a detector (see Patent Document 1 and Non-Patent Documents 1 to 2). For example, when the light-emitting / color-developing agent causes fluorescence emission in the droplets in response to the presence of the target substance, the presence or absence of fluorescence emission of each of the droplets flowing through the flow cell is sequentially measured (for example, digital measurement with the droplets that fluoresce being "1" and the droplets that do not fluoresce being "0"), and the target substance in the test solution is detected. When a known flow cytometer is used as the droplet detection device, a water-oil-water emulsion further dispersed in an aqueous phase may be used after forming the oil-in-water droplet emulsion (see Non-Patent Documents 3 and 4).

[0004] However, in the flow method, a flow path, a pump, etc. for flowing the droplets through the flow cell are required, the detection device becomes complicated and large-sized, and it becomes expensive. In addition, since measurement is performed on the droplets flowing through the flow cell, the time allotted for measuring one droplet is short, and a high-intensity laser light source and a highly sensitive detector are required. Also from this point, the detection device becomes expensive. In addition, in order to increase the throughput of the detection of the droplets, a plurality of the flow cells are arranged in parallel with their liquid feeding directions aligned, and the droplets flowing through these plurality of flow cells are simultaneously measured. In this case, the same number of detectors as the number of the flow cells arranged in parallel are arranged to perform measurement for each flow cell, or an image sensor having an observation field of view in a region straddling all of the flow cells arranged in parallel is arranged as the detector to perform measurement for the entire plurality of flow cells. In either case, this becomes a factor for the high cost of the detection device. That is, in the former case, the cost increases as the number of detectors increases, and in the latter case, the cost is high because it is necessary to use a high-performance camera with high speed and high definition for the image sensor. That is, in the flow method, there is a problem that the detection device becomes complicated, large-sized, and expensive, and when trying to increase the throughput of the detection device, this problem becomes even more significant.

[0005] In addition, in the flow method, while the droplets flowing on the front side with respect to the detector can be detected with high sensitivity, there is a problem that it is difficult to detect the droplets flowing on the back side. That is, when detecting the optical signal of the droplets by the photodetector, if two or more of the droplets simultaneously flow into the measurement point, the optical signal based on the droplets flowing on the back side is blocked by the light scattering at the interface between the droplets flowing on the front side and the oil. In addition, when the flow cells are arranged in parallel and laser light is irradiated through them to simultaneously detect the droplets flowing through the flow cell on the near side and the flow cell on the far side, there is a problem that the laser light is affected by light scattering and refraction in the flow cell on the near side, making it difficult to detect the droplets flowing through the flow cell on the far side. Regarding this problem of parallel arrangement, Patent Document 1 proposes suppressing the light scattering and the like by adjusting the refractive index of the droplets to be close to the refractive index of the oil to prepare the water-in-oil emulsion. However, in the flow method, due to the detection principle that the detection timing of the droplets is an instant in the flow, ultimately, regarding the detection of the overlapping droplets in the near and far sides within one flow cell, a signal change over time cannot be expected. Consequently, the light signal of the droplets flowing through the far side within one flow cell cannot be detected in a form hidden by the light emission and color development of the droplets flowing through the near side, resulting in detection leakage. To prevent such detection leakage, in order to eliminate the overlap between the droplets flowing through the near side and the droplets flowing through the far side, it is necessary to arrange the flow cells in a single row and send the droplets in a line to the flow cells. However, in this case, the detection throughput is lower than that of the detection device with the flow cells arranged in parallel.

[0006] On the other hand, in the array method, the water-in-oil emulsion is introduced into the droplet holding portion, and the presence or absence of light emission and color development of the droplets held in the droplet holding portion is statically detected by the detection device having the light source and the detector (see Non-Patent Documents 5 to 8). According to the array method, various members for liquid feeding such as the pump in the detection device used in the flow method are unnecessary, and there are no restrictions on using a high-intensity laser light source as the light source, a high-sensitivity detector, or the high-performance camera as the detector. Therefore, the detection device can be configured simply, compactly, and inexpensively.

[0007] Incidentally, the light emission / color development detection of the droplets in the array method is performed on the droplets located on any one of the liquid surface side, the liquid bottom side, and the side surface side of the water-in-oil emulsion held in the droplet holding part, that is, the droplets on the front side as viewed from the detector. This is because the light signal of the droplets located on the back side as viewed from the detector cannot be detected due to the influence of light scattering at the interface between the droplets located on the front side and the oil. This problem becomes more prominent as the droplet holding part holds the water-in-oil emulsion with a depth, width, that is, a three-dimensional depth larger than the diameter of the droplets, because the number of droplets with detection omission increases. On the other hand, in order to avoid detection omission, if the two-dimensional area, that is, the area of the droplet holding part is set large, this area exceeds the size of the observation field of the detector, and it becomes necessary to move the observation field. Since it takes a lot of time to move the observation field even when using an automatic moving stage, the detection throughput decreases. That is, the array method has a problem that it is restricted by the two-dimensional arrangement number of the droplets and cannot obtain a large throughput.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0010] An object of the present invention is to solve the above problems in the prior art and provide an optical detection method and an optical detection device capable of optically detecting a target substance with high throughput and high precision using a detection device that can be configured simply, in a small size, and at low cost.

[0011] To solve the above problems, the inventors of the present invention, in the array method, rather than having the water-in-oil emulsion held in the droplet holding part with a depth, width, or three-dimensional depth larger than the diameter of the droplets, hold the water-in-oil emulsion in a way that it has a depth at least 3.5 times as deep as the diameter of the droplets and an opening area at least 100 times as small as the cross-sectional area of the droplets, thereby improving the detection throughput. At the same time, the problem that the droplets located on the back side as viewed from the detector are subject to the influence of light scattering at the interface between the droplets located on the front side and the oil and result in detection omission is solved by refractive index matching that brings the refractive index of the droplets closer to the refractive index of the oil. That is, instead of moving the observation field of view of the detector, by expanding the observation region of the detector in the depth direction and width direction of the droplet holding part, both high throughput and high-precision detection are achieved simultaneously. Further, in the array method, performing the refractive index matching also solves the problem of detection omission due to the instantaneous overlap of the droplets in the flow method. That is, in the detection of the droplets by the array method, by tracking the signal change of the droplets over time, each of the droplets that overlap between the front and the back can be detected, and the time required for tracking is also very short.

Means for Solving the Problems

[0012] The present invention is based on the above findings, and the means for solving the above problems are as follows. That is, <1> A test liquid that may contain a target substance and a luminescent / color-developing agent that makes the presence of the target substance manifest by luminescence / color development are encapsulated, and a large number of droplets with a diameter of 1 μm to 100 μm are dispersed in oil. A water-in-oil emulsion in which the refractive index difference between the aqueous phase constituting the droplets and the oil phase constituting the oil is less than 0.01 is placed in a rising droplet holding part having a depth at least 3.5 times as deep as the diameter of the droplets and an opening area at least 100 times as small as the cross-sectional area of the droplets. On the other hand, by stacking the layers of the droplets arranged in layers, the droplets are arranged in a three-dimensional stateA droplet holding step for holding droplets, and a light emission / color development detection step for detecting the light emission / color development of the droplets by a detection unit disposed on either the opening surface or the bottom surface of the droplet holding unit that holds the droplets. The light emission / color development detection step is a step of counting the number of droplets in the light emission / color development state and detecting the target substance by the detection unit configured to be movable in the focal position and having an image sensor. After the first imaging of capturing an image at different focal positions in the foreground and the background as viewed from the detection unit, a second imaging is performed to recapture the image in the same imaging sequence and at the same focal position as the first imaging. An optical detection method characterized by this. <2> An oil-in-water droplet emulsion in which a test liquid that may contain a target substance and a light emission / color development agent that makes the presence of the target substance manifest by light emission / color development are encapsulated, and a large number of droplets having a diameter of 1 μm to 100 μm are dispersed in oil, and the refractive index difference between the aqueous phase constituting the droplets and the oil phase constituting the oil is less than 0.01. The shortest distance between one side surface and the other side surface facing the one side surface is at least 3.5 times the length of the diameter of the droplets, and the area of the inner wall surface of each of the one side surface and the other side surface has an area of at least 100 times the cross-sectional area of the droplets. In a rising droplet holding unit On the other hand, by stacking the layers of the droplets arranged in layers, the droplets are arranged in a three-dimensional state A droplet holding step for holding droplets, and a light emission / color development detection step for detecting the light emission / color development of the droplets by a detection unit disposed on the one side surface side of the droplet holding unit that holds the droplets. The light emission / color development detection step is a step of counting the number of droplets in the light emission / color development state and detecting the target substance by the detection unit configured to be movable in the focal position and having an image sensor. After the first imaging of capturing an image at different focal positions in the foreground and the background as viewed from the detection unit, a second imaging is performed to recapture the image in the same imaging sequence and at the same focal position as the first imaging. An optical detection method characterized by this. <3> The optical detection method according to any one of <1> to <2>, wherein the focal position of the detection unit is controlled by a focal position electric control unit having either a liquid lens or an electro-optical crystal lens.

Advantages of the Invention

[0013] According to the present invention, it is possible to solve the above problems in the prior art, and to provide an optical detection method and an optical detection device capable of optically detecting a target substance with high throughput and high precision by using a detection device that can be configured simply, in a small size, and at low cost.

Brief Description of the Drawings

[0014]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5(a)

Figure 5(b)

Figure 5(c)

Figure 6(a)

Figure 6(b)

Embodiments for Carrying Out the Invention

[0015] (Optical Detection Method) The optical detection method of the present invention includes a droplet holding step and a light-emitting / color-developing detection step.

[0016] <Droplet holding step> The droplet holding step is a step of holding a water-in-oil (W / O) emulsion in which the refractive index difference between the aqueous phase constituting the droplet and the oil phase constituting the oil is less than 0.01 in a droplet holding section.

[0017] -Water-in-oil emulsion- The water-in-oil emulsion is prepared by dispersing a large number of the droplets, in which a test liquid that may contain a target substance and a luminescent / coloring agent that makes the presence of the target substance manifest by luminescence / color development are encapsulated, in the oil. In this specification, "luminescence / color development" means that at least one of the state changes such as luminescence by fluorescence or chemiluminescence and color development by coloring or discoloration occurs before and after detection, and "luminescent / coloring agent" means an agent that causes the state change in the droplet.

[0018] The target substance is not particularly limited, and examples thereof include DNA, RNA, proteins, viruses, bacteria, etc. that are detection targets in known biological substance detection methods such as ELISA, immunoassay, and PCR methods. The test liquid is not particularly limited, and examples thereof include blood, saliva, urine, environmental water, etc.

[0019] The luminescent / coloring agent is not particularly limited, and examples thereof include luminescent / coloring agents used in known biological substance detection methods such as ELISA, immunoassay, and PCR methods. For example, a luminescent / coloring substance that adsorbs to the target substance and causes luminescence / color development in the droplet, a reagent that generates a coloring substance by an enzymatic reaction with a protein in the target substance, a reagent that generates a fluorescent substance by an enzymatic reaction with a protein in the target substance, a reagent that generates a chemiluminescent substance by an enzymatic reaction with a protein in the target substance, a labeling substance having a recognition site that specifically recognizes the target substance, etc.

[0020] The luminescent / colorimetric substance that adsorbs to the target substance and causes luminescence / color development in the droplet is not particularly limited, and examples include aggregation-induced emission (AIE) substances. Examples of the aggregation-induced emission substance include compounds that exhibit the AIE effect described in JP-A-2010-112777.

[0021] Examples of the reagent that generates a colorimetric substance by an enzymatic reaction with the protein in the target substance include 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside that colors the protein as β-galactosidase.

[0022] Examples of the reagent that generates a fluorescent substance by an enzymatic reaction with the protein in the pre-target substance include derivatives containing 4-methylumbelliferone such as (4-methylumbelliferyl)-α-D-N-acetylneuraminic acid that reacts with neuraminidase in influenza virus to generate 4-methylumbelliferone, which is a fluorescent substance, derivatives containing fluorescein, derivatives containing resorufin, and derivatives containing rhodamine.

[0023] Examples of the reagent that generates a chemiluminescent substance by an enzymatic reaction with the protein in the target substance include luciferin that emits light when the protein is luciferase.

[0024] Examples of the labeling substance include enzyme labels and fluorescent dye labels obtained by labeling an antibody that recognizes the target substance with an enzyme or a fluorescent dye. When using the enzyme label, it is used in combination with a reagent that generates a colorimetric substance by an enzymatic reaction with the enzyme label, a reagent that generates a fluorescent substance by an enzymatic reaction with the enzyme label, a reagent that generates a chemiluminescent substance by an enzymatic reaction with the enzyme label, etc., to act as the luminescent / coloring agent.

[0025] The droplets are formed in the hydrophilic aqueous phase. For example, a water dispersion of the test solution and the luminescent / color-developing agent, or a dispersion of the test solution and the luminescent / color-developing agent in an aqueous buffer used in known biological substance detection methods such as ELISA, immunoassay, and PCR methods is prepared as a droplet source.

[0026] The oil is formed in the hydrophobic oil phase and is prepared, for example, from fluorinated oil, silicone oil, hydrocarbon oil, and mixed oils thereof.

[0027] The refractive index difference between the aqueous phase and the oil phase is less than 0.01. That is, the aqueous phase and the oil phase are refractive index-matched to have substantially the same refractive index, and light scattering at the interface between the droplet and the surrounding oil is suppressed. The refractive index of the aqueous phase constituting the droplet is often close to the refractive index of water and is approximately 1.33. Therefore, when the refractive index of the oil phase is less than 1.33, a component with a high refractive index is added to prepare the oil phase, and the refractive index of the oil phase is matched with the refractive index of the aqueous phase. Examples of such a component include commercially available fluorinated oils with a refractive index of about 1.36. When the refractive index of the oil phase exceeds 1.33, a component with a low refractive index is added to prepare the oil phase, and the refractive index of the oil phase is matched with the refractive index of the aqueous phase. Examples of such a component include commercially available fluorinated oils with a refractive index of about 1.30. Also, the refractive index of the aqueous phase constituting the droplet may be made close to the refractive index of the oil phase. Examples of components for adjusting the refractive index of the aqueous phase include a 40% by mass aqueous sucrose solution with a refractive index of about 1.40. These components for adjusting the refractive index are appropriately selected from commercially available products according to the refractive indices of the aqueous phase and the oil phase. Note that the water-in-oil emulsion may contain a known surfactant (emulsifier) for preventing unintended aggregation or binding of the droplets.

[0028] The method for preparing the water-in-oil droplet emulsion is not particularly limited. For example, the fluid constituting the aqueous phase and the fluid constituting the oil phase can be added to an arbitrary container and emulsified by known stirring treatment, shaking treatment, or ultrasonic treatment, and then introduced into the droplet holding portion by pipetting or the like. Alternatively, the fluid constituting the aqueous phase and the fluid constituting the oil phase can be directly added to the droplet holding portion, and the droplet holding portion can be emulsified by performing known stirring treatment, shaking treatment, or ultrasonic treatment. Further, for example, an emulsion preparation method using a known microchannel as disclosed in Non-Patent Documents 1 to 4, 7, and 8 may be used.

[0029] The droplets are spherical, and their diameter is not particularly limited, but is preferably 1 μm to 100 μm. If the diameter is less than 1 μm, the number of molecules of the luminescent / color-developing agent contained in the droplets may be small, making it difficult to detect the optical signal. On the other hand, if the diameter exceeds 100 μm, the background signal caused by the autofluorescence component in the test solution, the unreacted luminescent / color-developing agent, etc. with respect to the optical signal generated by the reaction between the target substance and the luminescent / color-developing agent may increase, making it difficult to detect the droplets in which the reaction has occurred. The diameter of the droplets can be controlled, for example, by adjusting the energy density applied to the droplets in known stirring treatment, shaking treatment, or ultrasonic treatment. Also, in an emulsion preparation method using a known microchannel, it can be controlled by adjusting the width, height, flow rate, etc. of the droplet formation channel. Note that the diameter of the droplets can be grasped as the average value of a plurality of the droplets. For example, it is grasped as the average value of 10 droplets arbitrarily selected from a plurality of the droplets held in the droplet holding portion.

[0030] The droplet holding portion is configured as a rising member having a depth that is at least 3.5 times the diameter of the droplets and an opening area that is at least 100 times the cross-sectional area of the droplets. That is, a plurality of the droplets can be introduced in the depth direction and width direction of the droplet holding portion, and the droplets are held in the droplet holding portion in a three-dimensional arrangement. Note that the cross-sectional area of the droplet corresponds to the maximum cross-sectional area of this droplet.

[0031] The depth of the droplet holding portion may be at least 3.5 times shallower than the diameter of the droplet. However, the greater the depth, the more the number of droplets included in the observation field of view can be increased. Therefore, it is preferably 10 times or more, more preferably 50 times or more, relative to the diameter of the droplet. On the other hand, if the depth is too deep, the later-described focus position moving mechanism may be enlarged. Therefore, as the upper limit of the depth, it is preferably 100 mm or less, which is within the moving range of a general focus position moving mechanism.

[0032] The opening area of the droplet holding portion may be at least 100 times smaller than the cross-sectional area of the droplet. However, the larger the opening area, the more the number of droplets included in the observation field of view can be increased. Therefore, it is preferably 1,000 times or more, more preferably 10,000 times or more, relative to the cross-sectional area of the droplet. It is preferable that it is. On the other hand, if the opening area is too large, the number of droplets outside the observation field of view of the later-described image sensor may increase. Therefore, as the upper limit of the opening area, it is less than 2 times the observation field of view when the lens magnification is 1 with a 1 / 1.2-inch full HD image sensor, which is 100 mm 2 or less is preferable. However, the droplet holding portion may be configured as a flat-bottom multi-cell formed in a plurality on a known array substrate. In this case, the upper limit of the opening area can be set in consideration of the relationship between the total opening area obtained by integrating the individual opening areas of the multi-cells and the size of the observation field of view.

[0033] Incidentally, the preferable ranges of the depth and the opening area are applicable when detection is performed from either the opening surface or the bottom surface side of the droplet holding portion in the light emission / color development detection step described later. When detection is performed from the side surface side of the droplet holding portion, the depth is read as the shortest distance between one side surface and the other side surface facing the one side surface in the droplet holding portion, and the opening area is read as the area of the inner wall surfaces of the one side surface and the other side surface, respectively, and then applied. However, regarding the upper limit of the area of the inner wall surfaces of the one side surface and the other side surface, unlike the upper limit of the opening area (100 mm 2 or less), it can be set to 1,000 mm 2 or less. This is because it is also possible to use the droplet holding portion filled with the water-in-oil emulsion to a depth of about 1 / 10 without filling the entire droplet holding portion. If the area of the inner wall surfaces of the one side surface and the other side surface exceeds 1,000 mm 2 , an increase in the size of the apparatus is a concern. As a specific configuration of the liquid holding portion when detection is performed from the side surface side, the shortest distance between the one side surface and the other side surface facing the one side surface is at least 3.5 times the diameter of the droplet, and the area of the inner wall surfaces of the one side surface and the other side surface is at least 100 times the cross-sectional area of the droplet. More specifically, a configuration in which the above reading is applied for matters other than the upper limit of the area of the inner wall surfaces of the one side surface and the other side surface can be mentioned.

[0034] <Light Emission / Color Development Detection Step> The light emission / color development detection step is a step of detecting the light emission / color development of the droplet by a detection unit disposed opposite to any one of the opening surface, the bottom surface, and the side surface of the droplet holding portion holding the droplet. As this light emission / color development detection step, it is preferable that the detection unit whose focal position is movable detects the light emission / color development of the droplet at different focal positions. That is, when detection is performed with the focal position fixed, among the droplets present at different positions in the droplet holding unit, those present at positions far from the focal position are out of focus of the detection unit and cannot be detected, and the setting of the droplet holding unit that arranges the droplets in a three-dimensional manner may not be utilized. In addition, when the detection unit is arranged on either the opening surface or the bottom surface side of the droplet holding unit, the detection unit is configured such that the focal position is movable in a direction parallel to the depth direction of the droplet holding unit (hereinafter, may be simply referred to as the "depth direction"), and when the detection unit is arranged on the side surface side of the droplet holding unit, the detection unit is configured such that the focal position is movable in a direction parallel to the direction from one side surface to the other side surface (hereinafter, may be simply referred to as the "width direction").

[0035] -Detection unit- The detection unit is configured to include a photodetector that detects the light emission / color development of the droplet and various optical elements for causing the photodetector to detect the light emission / color development of the droplet.

[0036] There is no particular limitation on the optical element. In addition to mirrors, objective lenses, etc. used in known microscopes, as a mechanism for moving the focal position, a focal position electric control unit having either a liquid lens or an electro-optic crystal lens can be particularly preferably cited. That is, from the viewpoint of obtaining high throughput by moving the focal position instead of moving the observation field of view, it is preferable to use the focal position electric control unit capable of switching the focal position of the detection unit in the order of microseconds to milliseconds.

[0037] Although there are no particular restrictions on the photodetector, an image sensor can be preferably cited. If the number of droplets in the light-emitting / color-developing state is counted on the image captured by the detection unit configured to include the image sensor to detect the target substance, the number of droplets can be automatically digitally counted in the image processing, and high throughput can be obtained. In addition, since the temporal change of the optical signal based on the light emission / color development of the droplet can be tracked, each of the droplets overlapping in the foreground and the background as viewed from the detection unit can be detected. That is, since the light emission / color development of each of the droplets can occur with a time difference according to the number of the target substances present in the droplets and the reaction state between the target substance and the light-emitting / color-developing agent, for example, by taking the difference value of the optical signal intensity (luminance value or optical density value) in the time-lapse image, each light emission / color development can be detected when the light emission / color development of the droplets overlapping in the foreground and the background has a time difference. In addition, if information on the temporal change of the optical signal is obtained by detecting the state of the optical signal before the droplet emits light / develops color and the state of the optical signal after the droplet emits light / develops color, respectively, it is also possible to distinguish between the noise signal resulting from signal fluctuations or impurity contamination that do not change over time and the optical signal based on the light emission / color development of the droplet. Thereby, the influence of the noise signal can be reduced, and the optical signal based on the light emission / color development of the droplet can be detected with higher accuracy. There are no particular restrictions on the image sensor, and known CCD image sensors and CMOS image sensors can be used.

[0038] By the way, in the conventional array method, due to light scattering at the interface between the aqueous phase and the oil phase, the droplets arranged in the depth direction or the width direction are not detected. Therefore, the detection throughput is determined by the number of droplets two-dimensionally arranged within one observation field of view. From the resolution of a general image sensor (for example, an image sensor having the number of pixels of full HD), the number of droplets in one observation field is about 10,000 to 100,000. Also, when moving the observation field of view to detect more droplets, it takes more than 1 second without using an expensive electric stage. Therefore, when using a general electric stage, the throughput achievable by the array method is, at most, about 100,000 per second. However, in the present invention, even when using the same image sensor, since the droplets are three-dimensionally arranged within one observation field of view, each time the number of layers of the droplets stacked in the depth direction or the width direction of the droplet holding portion increases to two or three layers, the number of droplets in one observation field can be increased in proportion to the number of layers. Even considering the moving time when moving the focal position, a throughput far exceeding about 100,000 per second can be achieved. For example, when observing droplets with a diameter of 20 μm using a 2-fold objective lens and a 1 / 1.2-inch image sensor having the number of pixels of full HD, the number of droplets that can be detected in one observation field is about 50,000. When performing this detection by switching the focal position, if the detection speed (imaging speed) is set to a video rate of 30 detections (30 images) / s, a throughput of about 1.5 million per second is achieved, which is excellent in achieving a throughput of 1 million per second, corresponding to 10 times the maximum throughput (100,000 per second) achievable by the array method. This detection speed (imaging speed) can be sufficiently achieved using a focal position electric control unit or the like capable of switching the focal position at a response speed on the order of microseconds to milliseconds. Also, when taking the difference value of the optical signal intensity in the time-lapse image for the purpose of tracking the change over time of the droplets overlapping in the foreground and the background as seen from the detection unit, even if the video rate is halved considering the time required for re-imaging, for example, the time required for two images, a throughput of 750,000 per second can be achieved.

[0039] In the light emission / color development detection step, since there is no time constraint due to the detection timing like the flow method, it is naturally possible to consider the detection of the change over time of the droplets where the front and the back overlap as seen from the detection unit. When re-imaging the change over time of the droplets using the image sensor (imaging multiple times at the same focal position), for example, for the droplets stacked in the depth direction and the width direction within the droplet holding unit, after imaging them once in the stacking order, it is conceivable to image them again in the stacking order without changing the imaging order. By performing such re-imaging, it is possible to increase the time interval between the first imaging and the second imaging while maintaining the throughput, thereby increasing the change over time of the droplets and efficiently detecting the droplets where the front and the back overlap as seen from the detection unit.

[0040] An example of an embodiment of the optical detection method of the present invention will be described while referring to the drawings. FIG. 1 is an explanatory diagram for explaining the holding state of the droplets in the droplet holding unit, and FIG. 2 is an explanatory diagram for explaining the droplets in the light emission / color development state.

[0041] As shown in FIGS. 1 and 2, in the optical detection method, in the droplet holding unit 1, an oil-in-water emulsion 2 in which a large number of droplets D1 and D2 are dispersed, where the droplet D1 encapsulates the test liquid containing the target substance T and the light emission / color development agent R that makes the presence of the target substance T apparent by light emission / color development, and the droplet D2 that does not contain the target substance T, is held (the droplet holding step). Among the droplets D1 and D2, only the droplet D1 containing the target substance T emits light / develops color due to the adsorption or reaction of the light emission / color development agent R with respect to the target substance T. The droplet holding unit 1, in the illustrated example, has a depth that is more than four times and an opening area that is more than ten times the diameter of the droplets D1 and D2, and the droplets D1 and D2 are held in a three-dimensional arrangement within the droplet holding unit 1. Also, the arrangement of the droplets D1 and D2 can be regarded as a layered arrangement arranged at arbitrary intervals in the horizontal direction, and can be regarded as layers L1 to L4 in a state of being stacked from the bottom side towards the opening within the droplet holding unit 1.

[0042] When performing the refractive index matching such that the refractive index difference between the aqueous phase constituting the droplets D1 and D2 and the oil phase constituting the oil in the water-in-oil droplet emulsion 2 is less than 0.01, when detecting the light emission and color development of the droplets D1 and D2 in the droplet holding part 1, for example, from above the droplet holding part 1 (light emission and color development detection step), it is possible to detect the light emission and color development of the droplet D1 not only in the front layer L4 but also in the layers L3 to L1 arranged deeper than this. This is because due to the effect of suppressing scattered light based on the refractive index matching, the non-light-emitting and non-color-developing droplet D2 becomes transparently visible, and the light emission and color development of the droplet D1 in the deeper layer can be detected. In addition, even when detecting from the side surface of the droplet holding part 1, the water-in-oil droplet emulsion 2 can be regarded as constituting a stack of layers of a plurality of droplets D1 and D2 with the width direction as the stacking direction, and it is certain that the light emission and color development of the droplet D1 can be detected in both the front layer and the deeper layer of these layers.

[0043] An example of an optical detection device is shown in FIG. 3. Note that FIG. 3 is an explanatory diagram for explaining an optical detection device configured according to an upright microscope. As shown in FIG. 3, the optical detection device 10 includes a droplet holding part 1, a light source 11 for illumination light, a half mirror 12, a focus position control part 13, and an image sensor 14.

[0044] In the optical detection device 10 configured in this way, the inside of the droplet holding part 1 is illuminated by the illumination light irradiated from the light source 11 through the half mirror 12, and the image of the light emission and color development of the droplet D1 can be captured by the image sensor 14 arranged on the opening surface side of the droplet holding part 1. Further, by moving the focus position by the focus position control part 13, the focus of the image sensor 14 can be aligned with the respective stacking heights of the layers L1 to L4, for example, and the respective light emissions and color developments of the droplets D1 included in the layers L1 to L4 can be detected with good visibility. Note that the focal position by the focal position control unit 13 can be set in advance according to the depth of the droplet holding unit 1, such as the bottom side position, the intermediate position in the depth direction, and the opening side position, regardless of the layers L1 to L4. Further, when an image sensor 14 is arranged on the side surface side of the liquid holding unit 1 (not shown), the setting can be changed to the setting in the width direction of the liquid holding unit 1 according to the setting in the depth direction in the illustrated example. Also, when the light emission / color development of the droplet D1 is fluorescence emission, the light source 11 may have a light source that irradiates the droplet D1 with excitation light that causes fluorescence emission, or an excitation light source may be arranged separately from the light source 11. Also, when detecting the light emission / color development of the droplet D1, for stable holding, the opening portion of the droplet holding unit 1 may be covered with a cover glass or the like.

[0045] In the optical detection method using the optical detection device 10 configured as described above, the oil-in-water emulsion 2 is held in the droplet holding unit 1 to have a depth, width, or three-dimensional depth larger than the diameters of the droplets D1 and D2, so that the number of the droplets in one observation field can be increased. At the same time, the problem that the droplet D1 located on the back side as viewed from the image sensor 14 is undetected due to the influence of light scattering at the interface between the droplet D2 located on the front side and the oil is solved by the refractive index matching. That is, instead of moving the observation field of the detector, by expanding the detection area of the image sensor in the depth direction or the width direction of the droplet holding unit 1, both high throughput and high-precision detection are realized simultaneously. Further, the problem that the droplets D1 overlap with each other at the front and the back in the flow method is solved by detecting the light emission / color development of the droplets D1 over time. That is, by adopting the static detection (detection for a stationary fluid) of the droplets in the array method, it is possible to track the change over time of the optical signal based on the light emission / color development of the droplets, and to detect the droplets D1 that overlap with each other at the front and the back. Further, as the optical detection device 10, it can be manufactured simply, in a small size, and at low cost according to a known microscope configuration.

[0046] Another example of the optical detection device is shown in FIG. 4. Note that FIG. 4 is an explanatory diagram for explaining an optical detection device configured according to an inverted microscope. This optical detection device 20 is obtained by changing the upright microscope configuration in the optical detection device 10 to an inverted microscope configuration. Otherwise, it is the same as the optical detection device 10, and the light source 21, the half mirror 22, the focus position control unit 23, and the image sensor 24 can be configured in the same manner as the light source 11, the half mirror 12, the focus position control unit 13, and the image sensor 14, respectively. When the microscope configuration of the optical detection device 20 is used, the droplet holding unit 1 is made of a known transparent member such as a transparent resin.

[0047] (Optical Detection Device) The optical device of the present invention is used in the optical detection method of the present invention and is configured to include a droplet holding unit and a detection unit.

[0048] <Droplet Holding Unit> As one aspect, the droplet holding unit is configured as a rising member (a square columnar cell) having a depth of at least 3.5 times the diameter of the droplet and an opening area of at least 100 times the cross-sectional area of the droplet. Also, as another aspect, the droplet holding unit is configured as a rising member (a square columnar cell) in which the shortest distance between one side surface and another side surface facing the one side surface is at least 3.5 times the diameter of the droplet, and the area of each inner wall on the one side surface and the another side surface is at least 100 times the cross-sectional area of the droplet. Regarding the specific configuration of the droplet holding unit, the matters described in the explanation of the optical detection method can be applied, and duplicate explanations are omitted.

[0049] <Detection Unit> The detection unit is configured as a unit that is disposed opposite to any one of the opening surface, the bottom surface, and the side surface of the droplet holding unit and can detect the light emission and color development of the droplet held in the droplet holding unit. The detection unit is not particularly limited, but it is preferably configured to be able to move the focal position. Further, as this focal position, it is particularly preferable that it is controlled by a focal position electric control unit having either a liquid lens or an electro-optical crystal lens. Further, the detection unit preferably includes an image sensor. Regarding the specific configuration of the detection unit, the matters described for the optical detection method can be applied, and duplicate explanations are omitted.

Example

[0050] (Example) In order to pseudo-reproduce an actual detection situation in which a small number of the droplets that emit light / change color due to the presence of the target substance occur among a large number of droplet groups, as something that pseudo-reproduces the droplets in which the target substance is absent, 10 mM HEPES buffer (colorless sample) was prepared, and as something that pseudo-reproduces the droplets in which the target substance is present, 10 mM HEPES buffer (red sample) added with 4 mg / mL eosin was prepared. The refractive index of the colorless sample and the refractive index of the red sample (refractive index of the aqueous phase) measured using a refractometer (LLG-uniREFRACTO 2) were both 1.334.

[0051] Also, a refractive index adjusting liquid (Cargille Laboratories) was added to the oil for generating water-in-oil droplets (Automated Droplet Genetator oil for Probes, BioRad) to adjust the refractive index, and the oil constituting the oil phase was prepared. The refractive index of the oil phase measured using the refractometer was 1.333. The refractive indices of the aqueous phase and the oil phase were almost the same, and refractive index matching was achieved.

[0052] Next, the colorless sample and the oil were added in equal amounts to a 1.5 mL microtube, and then shaken with a vortex mixer for 30 seconds to prepare a colorless sample emulsion in which the droplets (colorless droplets) of the colorless sample were dispersed in the oil. Next, the red sample and the oil were added to another microtube in equal amounts, and then shaken with a vortex mixer for 30 seconds to prepare a red sample emulsion in which the droplets (red droplets) of the red sample were dispersed in the oil.

[0053] Next, 100 parts by volume of the colorless sample emulsion and 1 part by volume of the red sample emulsion were mixed at a volume ratio, and then introduced into a flat-bottomed well as the droplet holding part. The droplets were stacked and held in the depth direction of the well, and the stacking height of the droplets, which is the distance between the bottom position of the droplet located on the bottommost side of the well and the top position of the droplet located on the most open side of the well, was 3 mm. As described above, as a detection test according to the example, the actual detection situation in which a small number of droplets that emit light and change color due to the presence of the target substance occur among a large number of the droplet groups was pseudo-reproduced. Whether the red droplets that pseudo-reproduce the droplets in which the target substance is present can be detected is a matter of concern.

[0054] The well was set in an inverted microscope (the detection unit on the bottom side of the well), and using a 4x objective lens, the results of observing the droplets while moving the focal position in a direction parallel to the depth direction of the well are shown in FIGS. 5(a) to 5(c). Note that FIG. 5(a) shows a microscope image obtained by focusing on the bottom side position (the lower layer of the droplets) of the well in the detection test according to the example, FIG. 5(b) shows a microscope image obtained by focusing on the middle position in the depth direction of the well (the middle layer of the droplets) in the detection test according to the example, and FIG. 5(c) shows a microscope image obtained by focusing on the opening side position (the upper layer of the droplets) of the well in the detection test according to the example. As shown in these figures, light scattering at the interface between the droplets and the surrounding oil is suppressed by the refractive index matching, and the red droplets can be detected in all layers from the upper layer to the lower layer of the droplets. Note that the dark black dots appearing in FIGS. 5(a) to 5(c) are unintended bubbles.

[0055] (Comparative Example) Next, a 10 mM HEPES buffer containing 40% by mass of sucrose (colorless reference sample) was used instead of the colorless sample, and a 10 mM HEPES buffer containing 4 mg / mL of food red added with 40% by mass of sucrose (red reference sample) was used instead of the red sample. A detection test according to the comparative example was conducted in the same manner as the detection test according to the example, except for the above. The refractive indices of both the colorless reference sample and the red reference sample (refractive index of the aqueous phase) are 1.40, and the refractive index matching with the oil phase (refractive index: 1.333) is not achieved.

[0056] In the detection test according to the comparative example, a microscopic image obtained by focusing on the bottom side position of the well (lower layer of the droplet) is shown in Fig. 6(a). Also, in the detection test according to the comparative example, a microscopic image obtained by focusing on the middle position in the depth direction of the well (middle layer of the droplet) is shown in Fig. 6(b). As shown in these figures, the red droplet can be distinguished in the lower layer of the droplet (the bottommost side of the well), but in the middle layer of the droplet, the image of the droplet becomes unclear due to light scattering, and the red droplet cannot be distinguished. From the above results, the effectiveness of the refractive index matching in the case of arranging the droplets three-dimensionally with respect to the liquid sample holding portion to improve the detection throughput is confirmed.

Explanation of Signs

[0057] 1 Droplet holding portion D1 Droplet in non-emitting and non-coloring state D2 Droplet in emitting and coloring state T Target substance R Emitting and coloring agent 10, 20 Optical detection device 11, 21 Light source 12, 22 Half mirror 13, 23 Focus position control unit 14, 24 Image sensor

Claims

1. A liquid droplet holding step of enclosing a test liquid that may contain a target substance and a luminescent / color-developing agent that makes the presence of the target substance apparent by luminescence / color development, dispersing a large number of liquid droplets having a diameter of 1 μm to 100 μm in oil, and having a refractive index difference between the aqueous phase constituting the liquid droplets and the oil phase constituting the oil, being less than 0.01, and laminating layers of the liquid droplets arranged in layers with respect to a rising liquid droplet holding portion having a depth of at least 3.5 times the diameter of the liquid droplets and an opening area of at least 100 times the cross-sectional area of the liquid droplets, so that the liquid droplets are held in a three-dimensionally arranged state; A luminescence / color development detection step of detecting the luminescence / color development of the liquid droplets by a detection unit arranged on either the opening surface or the bottom surface of the liquid droplet holding portion holding the liquid droplets; comprising; The luminescence / color development detection step is a step of counting the number of the liquid droplets in a luminescence / color development state and detecting the target substance by the detection unit configured to have an image sensor and be capable of moving the focal position, and after the first imaging of capturing an image at different focal positions in the front and the back as viewed from the detection unit, a second imaging of re-capturing the image in the same imaging order and at the same focal position as the first imaging is performed. An optical detection method characterized by this.

2. A liquid droplet holding step of enclosing a test liquid that may contain a target substance and a luminescent / color-developing agent that makes the presence of the target substance apparent by luminescence / color development, dispersing a large number of liquid droplets having a diameter of 1 μm to 100 μm in oil, and having a refractive index difference between the aqueous phase constituting the liquid droplets and the oil phase constituting the oil, being less than 0.01, and laminating layers of the liquid droplets arranged in layers with respect to a rising liquid droplet holding portion having a shortest distance between one side surface and another side surface facing the one side surface of at least 3.5 times the diameter of the liquid droplets, and each inner wall area on the one side surface and the other side surface having an area of at least 100 times the cross-sectional area of the liquid droplets, so that the liquid droplets are held in a three-dimensionally arranged state; A luminescence / color development detection step of detecting the luminescence / color development of the liquid droplets by a detection unit arranged on the one side surface side of the liquid droplet holding portion holding the liquid droplets; comprising; The light emission / color development detection step is a step of counting the number of droplets in the light emission / color development state by the detection unit configured to have an image sensor and be capable of moving the focal position, and detecting the target substance. After the first imaging of capturing an image at different focal positions, the front and the back as seen from the detection unit, a second imaging is performed to re-capture the image in the same imaging order and at the same focal position as the first imaging. An optical detection method characterized by this.

3. The optical detection method according to any one of claims 1 to 2, wherein the focal position of the detection unit is controlled by a focal position electric control unit having either a liquid lens or an electro-optical crystal lens.

Images