Virus detection method using well array, well array and detection device

Abstract

[Problem] To detect a virus quickly and with a high degree of sensitivity. [Solution] A virus detection method for using a well array, in which a plurality of wells are formed such that adjacent wells are defined by lateral walls that rise on a substrate, to detect a virus in a test liquid on the basis of detected light emission from the wells, wherein the well array has a detection region where a well formation surface area, which is the substrate surface area of the region where the wells are formed, is at least 5 mm2, the overall well volume obtained by adding the volumes of the individual wells is at least 17 nL, the depth of the wells is 3.5-40 μm, the opening surface areas of the wells are 18-1,700 μm<sp / > 2, the volumes of the wells are 74-6,000 fL, and the lateral wall thicknesses between adjacent wells are 0.5-15 μm, and the light emission from the wells is detected using a detection unit having an observation field of view fixed at a size including the detection region.

Description

[Technical Field]

[0001] The present invention relates to a virus detection method, a well array, and a detection device using a detection system in which the size of the detection elements is set for high-speed and high-sensitivity virus detection. [Background technology]

[0002] A known protein detection method is the digital ELISA (Enzyme-Linked Immunosorbent Assay) method, in which a group of magnetic beads, which may include magnetic beads in a state where no protein has been captured and magnetic beads in a state where the protein to which a label molecule having an enzyme has been bound, is individually moved and placed into each microwell of a microwell array having many microwells arranged on a substrate using a magnet or gravitational sedimentation, and the number of colors developed in the microwells is digitally counted (see Non-Patent Documents 1 to 5 and Patent Documents 1 to 6). It has also been reported that the digital ELISA method can be used to detect viral proteins (see Non-Patent Document 6).

[0003] A fluorescent substrate is contained in each microwell, and when the contained magnetic beads capture the protein, i.e., when the protein is present in the microwell, color (light emission) occurs due to an enzymatic reaction between the enzyme and the fluorescent substrate, and the presence or absence of the protein is observed as the presence or absence of color in the microwell. At the same time, the number of colored microwells is digitally counted (e.g., colored microwells are counted as "1" and uncolored microwells are counted as "0"), thereby detecting the number of viruses in the sample and achieving highly quantitative protein detection. Furthermore, when this method is used to detect viral proteins as in Non-Patent Document 6, highly quantitative virus detection is achieved.

[0004] However, in the digital ELISA method, the process of moving each magnetic bead into the microwell using the magnet or gravitational sedimentation is itself a labor-intensive and time-consuming process. Furthermore, if the labeled molecule is adsorbed to the magnetic beads without the protein, color will appear in the microwells where the protein is not present. Therefore, pre-washing must be performed to an extent that the capture state of the protein on the magnetic beads is not disrupted, thereby removing unnecessary labeled molecules from the magnetic beads, which requires a significant amount of labor and time for the detection operation. Therefore, the digital ELISA method using magnetic beads has the problem that it requires a significant amount of labor and time for the detection of the protein.

[0005] For this reason, a method of capturing viruses directly in microwells (hereinafter referred to as direct virus capture method) has been proposed as a digital detection method that does not use the magnetic beads (see Patent Document 7, Non-Patent Documents 7 and 8). In this direct virus capture method, detection is performed, for example, according to the procedures shown in Figures 1(a) and 1(b). Figure 1(a) is a diagram (1) showing the detection procedure in the conventional technology, and Figure 1(b) is a diagram (2) showing the detection procedure in the conventional technology.

[0006] In this example of the direct virus capture method, a detection chip 100 is used in which a space 130 is formed between a lower layer 110 and an upper layer 120, as shown in the figure. Side walls 111, the upper surfaces of which are hydrophobic, are erected at predetermined intervals on the lower layer 110. Wells 115a to 115d are configured as extremely small spaces defined by adjacent side walls 111 and the upper surface of the lower layer 110, and the lower layer 110 and side walls 111 configure a well array with the well arrangement of wells 115a to 115d. For such a detection chip 100, first, a hydrophilic solvent 140 containing a virus 161 and a fluorescent chromogenic substrate ((4-methylumbelliferyl)-α-DN-acetylneuraminic acid) 162 that reacts with an enzyme contained in the virus is introduced into the space 130 through an inlet 121 formed in the upper layer 120 (see Figure 1(a)). Next, hydrophobic solvent 150 (a liquid that is difficult to mix with hydrophilic solvent 140) is introduced into space 130 from sample introduction portion 121 formed in upper layer portion 120, and hydrophilic solvent 140 is pushed toward well 115d, while hydrophilic solvent 140 is sealed in wells 115a to 115c (see FIG. 1(b)). In other words, viruses 161 are captured directly in the wells without using the magnetic beads. In the well, the reaction between the virus 161 confined in the minute space and the fluorescent chromogenic substrate 162 progresses over time, producing a chromogenic reaction product 163 (see FIG. 1(b)). Therefore, in wells 115a and 115c where viruses 161 are present, color is developed by reaction product 163, and the presence or absence of viruses 161 can be observed as the presence or absence of color development in wells 115a to 115c. At the same time, the number of wells that have developed color is digitally counted, thereby detecting the number of viruses 161 in the sample.

[0007] Regarding the size of the wells in the detection chip 100, the distance between adjacent side walls 111 (the distance in the width direction of the wells) is set to be extremely narrow. This is because if the distance is set to be wide, the color of the reaction product 163 will not cover the entire inside of the wells, and the color of wells 115a and 115c will become blurred, making it impossible to distinguish them from well 115b, which does not contain virus 161. As a result, the volume of each of the wells 115a to 115c is set small. Therefore, when virus detection is performed on a test liquid with a low virus concentration, that is, a test liquid in which a small amount of virus 161 is present in a large amount of liquid, the total volume of the wells 115a to 115c falls below the volume of the test liquid necessary to contain one virus 161, and the wells 115a to 115c cannot accommodate a sufficient amount of the test liquid, resulting in a decrease in detection sensitivity.

[0008] In this regard, in the conventional direct virus capture method, the number of wells is increased to prevent the total volume of the wells from being less than the volume of the test solution, i.e., the detection sensitivity is ensured by the number of wells formed. However, the more the number of wells formed, the larger the observation area becomes, and the observation area may exceed the observation field of view in one observation. Therefore, in the conventional direct virus capture method, for example, the detection chip 100 is placed on a moving stage, and the observation field is changed multiple times to perform virus detection. Virus detection by changing the observation field requires a lot of time and effort, such as precise alignment when changing the field and integrating the observation results obtained from multiple observation fields. Therefore, the direct virus capture method of the prior art has the problem that it loses the advantage of not using magnetic beads. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2016 / 6208 [Patent Document 2] Japanese Patent Application Publication No. 2018-91737 [Patent Document 3] International Publication No. 2012 / 121310 [Patent Document 4] Patent No. 3886161 [Patent Document 5] Patent No. 5551798 [Patent Document 6] Patent No. 5363663 [Patent Document 7] Japanese Patent Application Publication No. 2018-38384 [Non-patent literature]

[0010] [Non-Patent Document 1] David M Rissin et al., Nature Biotechnology Vol. 28, No. 6, pp.595 (2010) [Non-patent document 2] Elena Perez-Ruiz et al., Analytica Chimica Acta 1015, pp.74 (2018) [Non-patent document 3] Soo Hyeon Kim et al., Lab on a Chip 12, pp.4986 (2012) [Non-patent document 4] David M Rissin et al., Analytical Chemistry 83, pp.2279 (2011) [Non-Patent Document 5] Stephanie M. Schubert et al., Analytical Chemistry 88, pp.2952 (2016) [Non-patent document 6] Karen Leirs et al., Analytical Chemistry 88, pp.8450 (2016) [Non-Patent Document 7] Yoshihiro Minagawa et al., Lab on a Chip 19, pp.2678 (2019) [Non-patent document 8] Kazuhito V. Tabata et al., Scientific reports 9:1067 (2019) Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to solve the above-mentioned problems in the prior art and to provide a virus detection method, well array, and detection device that are capable of detecting viruses quickly and with high sensitivity.

[0012] The present inventors have conducted extensive research to solve the above problems and have obtained the following findings. First, the inventors considered ensuring detection sensitivity by increasing the depth of the wells, instead of ensuring detection sensitivity by the number of wells formed, thereby eliminating the situation where the total volume of the wells is less than the volume of the test liquid.

[0013] The target detection sensitivity is virus detection in test fluids with a virus concentration of 100 aM (1 virus / 17 nL) or less. This 100 aM virus detection is the sensitivity required to detect the presence or absence of virus in saliva as a screening test for virally infected patients, and is the most desirable detection sensitivity at present for rapid virus testing of a large number of virally infected patients.

[0014] The baseline is observation using a general-purpose observation device equipped with a 4x objective lens and a full HD image sensor. The observation field of view on the detection system side is 5mm from this baseline. 2 When using a 2x objective lens or a high-resolution image sensor, the observation field is set to 5 mm. 2 However, in order to establish a general-purpose detection method that can be used even when a large number of virus-infected patients occur, a setting of 5 mm was adopted. 2 The study will be based on virus detection in the above observation field.

[0015] The well to be examined is represented by the embodiment shown in Figure 2. Figure 2 is an explanatory diagram for explaining the well settings, with the upper side showing the top surface and the lower side showing the front surface. The well 1 shown in FIG. 2 has a depth D, a volume V, an opening area A, and a sidewall thickness T that are to be set.

[0016] (About depth D) If the side wall thickness T is ignored as the lower limit of the depth D, the observation field (5 mm 2 17nL / 5mm) and the virus concentration of the test liquid (1 virus / 17nL or less; the volume of liquid required for detection is 17nL or more), the shallowest depth must be 3.4μm (17nL / 5mm) 2 ). In reality, because of the sidewall thickness T, in order to eliminate the situation where the total volume obtained by accumulating the volumes V of the individual wells 1 is less than the volume of the test liquid, the depth D must be made deeper than 3.4 μm and at least 3.5 μm. On the other hand, if the depth D is too deep, the image sensor will not be able to focus on the entire depth direction, and it will be difficult for the test liquid to fill the entire well 1. In addition, processing the well 1 will be difficult. Therefore, the upper limit of the depth D must be set to 40 μm at most.

[0017] (Volume V) Based on this knowledge of the depth D, the present inventors prepared a cubic well 1 with a depth D of 30 μm and performed a preliminary test for virus detection. FIG. 3 shows an electron microscope image of a main part of the well array including well 1 used in the preliminary test. In the preliminary test, a highly concentrated virus-containing solution was diluted to a predetermined concentration as the test solution. The virus was influenza A virus, and (4-methylumbelliferyl)-α-DN-acetylneuraminic acid was used as a fluorescent substrate that reacts with a viral enzyme to produce color.

[0018] In this preliminary test, virus detection was attempted while changing the test conditions regarding the size of well 1, and it was found that detection of the virus failed when the virus concentration in the test liquid contained in one well 1 was less than 1 virus / 6,000 fL. The fluorogenic substrate emits a faint light in the liquid even when the virus is not present. Therefore, unless the virus concentration in the test solution is relatively high, it is impossible to distinguish whether the luminescence in Well 1 is due to the luminescence of the reaction product derived from the fluorogenic substrate that reacted with the virus, or due to the luminescence of the fluorogenic substrate that is not involved in the virus. When a substance that emits light even when not involved in the virus (such as the fluorogenic substrate or aggregation-induced luminescent substance) is used as the luminescence source, the detection limit of the virus concentration is estimated to be 1 virus / 6,000 fL. Therefore, in order to distinguish the luminescence of well 1 in a state in which one virus is trapped from the luminescence in a state in which the virus is not involved, the volume of the specimen liquid contained in well 1 must be 6,000 fL or less. Even when the target specimen liquid has a virus concentration of 100 aM (1 virus / 17 nL), if the volume V of well 1 in a state in which one virus is trapped exceeds 6,000 fL, the virus concentration of the specimen liquid in this well 1 will reach the detection limit of less than 1 virus / 6,000 fL, and it will be impossible to distinguish this well 1 from other wells 1 in which no virus is present. Therefore, the upper limit of the volume V of the well 1 must be set to 6,000 fL.

[0019] On the other hand, the lower limit of the volume V of the well 1 is estimated as follows. The number of pixels in the full HD standard is 1,920 x 1,080. Furthermore, imaging one well 1 requires at least 3 x 3 pixels. Therefore, when the general-purpose observation device is used, the total number of wells 1 formed is limited to approximately 230,000, according to the number of pixels (640×360). Since the amount of liquid required to achieve virus detection at 100 aM is 17 nL, the volume V of one well 1 must be at least 74 fL based on the relationship 17 nL / 230,000 viruses. Therefore, the lower limit of the volume V of the well 1 must be set to 74 fL.

[0020] (Opening area A) The upper limit of the opening area A of the well 1 is automatically determined from the depth D and volume V of the well 1 . That is, when the depth D is at the lower limit of 3.5 μm, if we try to obtain the upper limit of the volume V, 6,000 fL, the opening area A is approximately 1,700 μm 2 This becomes: Therefore, the upper limit of the opening area A is set to 1,700 μm 2 When the opening shape is a square, the upper limit of the length of one side of the opening shape is approximately 41 μm.

[0021] On the other hand, the lower limit of the opening area A of the well 1 is estimated as follows. As mentioned above, the upper limit of the total number of wells 1 formed when using the general-purpose observation device is 230,000. 2 is. Therefore, ignoring the side wall thickness T, 5 mm 2 Based on the relationship of / 230,000 pieces, the minimum per well 1 is 21.7 μm 2 (For example, an opening of 4.7 μm × 4.7 μm) is required. The opening area A ignoring the sidewall thickness T is 21.7 μm 2 If the resolution is less than this, the image of well 1 will be too small for a full HD imaging device to distinguish each well. In reality, there is a sidewall thickness T. If the sidewall thickness T is assumed to be 0.5 μm (the reason for this will be explained later), the opening area A of the well 1 must be at least 18 μm. 2 (For example, an opening of 4.2 μm x 4.2 μm) Therefore, the lower limit of the opening area A is 18 μm 2 It is necessary to do so.

[0022] (Side wall thickness T) The upper limit of the sidewall thickness T must be 15 μm at most, since if it is too thick, the total volume obtained by adding up the volumes V of the individual wells 1 is likely to be less than the volume of the test liquid. Therefore, the upper limit of the sidewall thickness T must be set to 15 μm. On the other hand, as for the lower limit of the sidewall thickness T, if it is too thin, it becomes difficult to process and also becomes fragile. Therefore, the lower limit of the sidewall thickness T must be set to 0.5 μm.

[0023] From the above investigations, the present inventors have obtained knowledge about a virus detection method, well array, and detection device for performing high-speed virus detection at 100 aM using the general-purpose observation device. [Means for solving the problem]

[0024] The present invention is based on the above findings, and provides the following means for solving the above problems. <1> A virus detection method for detecting viruses in a test liquid based on luminescence detection of a well array formed by dividing adjacent wells with side walls erected on a substrate, wherein the well array has a well formation area of ​​5 mm, which is the area of ​​the substrate in the region where the wells are formed. 2 The total volume of the wells obtained by integrating the volumes of the individual wells is 17 nL or more, the depth of the wells is 3.5 μm to 40 μm, and the opening area of ​​the wells is 18 μm 2 ~1,700μm 2 The wells have a volume of 74 fL to 6,000 fL, and the thickness of the sidewall between adjacent wells is 0.5 μm to 15 μm. The luminescence detection of the wells is carried out by a detection unit having a fixed observation field of view that is large enough to include the test region. The upper surface of the side wall is subjected to hydrophobic treatment. A virus detection method characterized by: <2> The observation field of the detection unit is set to the same size as the test area. <1> The virus detection method described in <3> The above method involves adsorbing a luminescent substance onto the surface of the virus to make the wells luminous. <1> from <2> 1. A virus detection method according to any one of the preceding claims. <4> The above-mentioned luminescent material is an aggregation-induced luminescent material. <3> The virus detection method described in <5> The wells are illuminated using a reagent that generates a fluorescent substance through an enzyme reaction with a protein contained in the virus. <1> from <2> 1. A virus detection method according to any one of the preceding claims. <6> The wells are illuminated using a reagent that generates a chemiluminescent substance through an enzyme reaction with a protein contained in the virus. <1> from <2> 1. A virus detection method according to any one of the preceding claims. <7> A well array is formed by defining adjacent wells with side walls erected on a substrate, and the well formation area, which is the area of ​​the substrate in the region where the wells are formed, is 5 mm 2The total volume of the wells obtained by integrating the volumes of the individual wells is 17 nL or more, the depth of the wells is 3.5 μm to 40 μm, and the opening area of ​​the wells is 18 μm 2 ~1,700μm 2 The well volume is 74 fL to 6,000 fL, and the thickness of the side wall between adjacent wells is 0.5 μm to 15 μm. The upper surface of the side wall is subjected to hydrophobic treatment. A well array characterized by: <8> The aforementioned <7> A detection device comprising: a detection chip on which the well array described in claim 1 is arranged; and a detection unit capable of fixing an observation field of view to a size that includes the test area in the well array. <9> The observation field of the detector is set to the same size as the test area. <8> The detection device according to claim 1. [Effects of the Invention]

[0025] According to the present invention, it is possible to solve the above-mentioned problems in the prior art and to provide a virus detection method, well array, and detection device that are capable of detecting viruses quickly and with high sensitivity. [Brief explanation of the drawings]

[0026] [Figure 1(a)] FIG. 1 is a diagram (1) showing a detection procedure in the prior art. [Figure 1(b)] FIG. 2 is a diagram showing the detection procedure in the prior art. [Figure 2] FIG. 10 is an explanatory diagram for explaining the setting of wells. [Figure 3] FIG. 1 shows an electron microscope image of a main part of a well array including well 1 used in a preliminary test. [Figure 4] FIG. 1 shows an electron microscope image of an example of well array fabrication. [Figure 5] 1 illustrates an example embodiment of a detection device. [Figure 6] FIG. 1 is a diagram showing a fluorescent image of a sample containing a virus in an example. [Figure 7]FIG. 10 is a diagram showing a fluorescent image of a virus-free specimen in an example. DETAILED DESCRIPTION OF THE INVENTION

[0027] (Well Array) An example of an embodiment of a well array according to the present invention is shown in Figure 4. Figure 4 is an electron microscope image of an example of the well array fabrication. As shown in this electron microscope image, the well array has a structure in which a plurality of wells are formed by defining adjacent wells with side walls erected on the substrate.

[0028] The material for forming the well array is not particularly limited and can be appropriately selected depending on the purpose. Examples include glass materials, semiconductor materials, and resin materials. Furthermore, the method for forming the well array is not particularly limited and can be selected appropriately depending on the purpose. Examples include known methods such as forming the well array by etching after pattern drawing by lithography, and forming the well array by injection molding or imprinting using a mold having the shape of the well. For example, when the well array is formed using lithography and reactive ion etching with silicon as the forming material, the processing limit is about 0.1 μm, and the well array can be formed with high precision. The upper surface of the sidewall is preferably subjected to a hydrophobic treatment so as to facilitate the placement of the test liquid in the well. The method for the hydrophobic treatment is not particularly limited, and examples thereof include known methods such as coating a hydrophobic polymer (e.g., photoresist) and forming a hydrophobic molecular layer (e.g., dimethyldichlorosilane). The well array shown in FIG. 4 was fabricated by lithography and reactive ion etching using a silicon substrate as the forming material.

[0029] The well array is characterized by the conditions of the wells formed by the above-described forming method, which will be explained below with reference to FIG.

[0030] <Well formation area> The well array has a well formation area of ​​5 mm, which is the area of ​​the substrate in the region where wells 1 are formed in the test region. 2 The well formation area refers to the area on the substrate where a plurality of wells 1 are formed as a group of wells 1 to be observed, and is different from the area for forming one well 1. The well formation area is at least 5 mm 2 The reason for this is that it is based on the observation field of view when the general-purpose observation device, which is configured to have the 4x objective lens and the full HD standard image sensor, is used as the detection unit. That is, the wider the area observed at one time, the more wells can be observed at one time, resulting in higher virus detection sensitivity. 2 If the distance is less than 5 mm, multiple images will be required to perform highly sensitive detection, making the detection process complicated. 2 5mm in the observation field of view 2 The setting is based on the fact that it is possible to observe the emitting and non-emitting states of each well 1 formed in the well forming area.

[0031] However, the detection unit may be configured as a wide-field observation device, in which case the observation field can be set larger while maintaining the required visibility. For example, using a full HD (1,920 x 1,080 pixel) image sensor, the observation field is set to 20 mm using the 2x objective lens. 2 This means that more sensitive virus detection is possible with a single observation. Furthermore, using a 4K image sensor (3,840 x 2,160 pixels) or an 8K image sensor (7,680 x 4,320 pixels) makes it possible to capture an even larger area at the same resolution, resulting in higher sensitivity. In this sense, the upper limit of the observation field is set to 320 mm, based on the imaging limit of known image sensors. 2 At the same time, from the viewpoint of performing high-sensitivity detection by making full use of the observation field, the upper limit of the well formation area is set to 320 mm, which is the same as the size of the observation field. 2 However, when considering ease of use and manufacturing costs, the well formation area is set to 100 mm 2 It is more preferable to set the upper limit at about (=1 cm x 1 cm).

[0032] <Total volume of wells> The well array has a total volume of wells 1 of 17 nL or more, which is the sum of the volumes of the individual wells 1 in the test region. The reason why the total volume is at least 17 nL is to obtain a detection sensitivity that enables virus detection in the test liquid with a virus concentration of 100 aM (1 virus / 17 nL) or less using the general-purpose observation device.With such detection sensitivity, the presence or absence of a virus can be detected in saliva as a screening test for virally infected patients. The total volume can be set depending on the desired virus concentration, with the upper limit being approximately 4,000 nL, taking into account the upper limit of the well formation area and the limit of the depth D of well 1 described below.

[0033] <Well depth> The depth D of the well 1 is set to 3.5 μm to 40 μm. If the depth D is less than 3.5 μm, it is not possible to obtain the detection sensitivity required to detect viruses in the test liquid having a virus concentration of 100 aM (1 virus / 17 nL) or less using the general-purpose observation device. Furthermore, if the depth D exceeds 40 μm, the focus of the imaging element will not be adjusted throughout the depth direction. Furthermore, it will be difficult for the test liquid to fill the entire well 1. In addition, processing the well 1 will be difficult.

[0034] <Well opening area> The opening area A of well 1 is 18 μm 2 ~1,700μm 2The opening area A is 18 μm 2 If the opening area A is less than 1,700 μm, when the general-purpose observation device is used, the image of the well 1 becomes too small to distinguish each well. 2 If the volume V of well 1 exceeds this value, it will be the volume at which the detection limit is reached when the light source is a substance that emits light in a state unrelated to the virus, and it will be impossible to distinguish between the luminescence of well 1 in which the virus is present and the luminescence of well 1 in which the virus is not present.

[0035] The opening area A of the well 1 is at least 18 μm 2 However, this setting is based on an estimate when imaging one well 1 with three pixels. To improve visibility, when imaging one well 1 with four pixels, the lower limit of the opening area A of well 1 is set to 25 μm. 2 is set to 2, the opening shape of the well 1 is square, but the opening shape is not particularly limited and may be a regular polygon such as an equilateral triangle or a regular hexagon (honeycomb structure), a rectangle, a circle, or an ellipse. Furthermore, the well 1 is not particularly limited as long as it is columnar (box-shaped).

[0036] <Well volume> The volume V of the well 1 is set to 74 fL to 6,000 fL. If the volume V is less than 74 fL, it is impossible to obtain the detection sensitivity necessary to detect viruses in the test solution having a virus concentration of 100 aM or less using the general-purpose observation device. Furthermore, if the volume V exceeds 6,000 fL, this volume reaches the detection limit when a substance that emits light in a state unrelated to viruses is used as the light source, and it is impossible to distinguish between the luminescence of wells 1 in which viruses are present and the luminescence of wells 1 in which viruses are not present. It is possible to obtain a clearer well image by imaging one well 1 with 4 × 4 pixels or more. In this case, the total number of wells 1 formed when using the general-purpose observation device is limited to approximately 130,000, based on the number of pixels (480 × 270). In this case, the volume V of one well 1 must be at least 130 fL based on the relationship of 17 nL / 130,000 cells. Therefore, the lower limit of the volume V of the well 1 is more preferably 130 fL.

[0037] <Side wall thickness> The sidewall thickness T, which is the thickness of the sidewall between adjacent wells 1, is set to 0.5 μm to 15 μm. If the sidewall thickness T is less than 0.5 μm, it will be difficult to process and will be prone to breakage. If the sidewall thickness T exceeds 15 μm, the total volume will likely be less than the volume of the test liquid. Note that if the spacing between adjacent wells 1 is not constant, such as when the wells 1 are formed in a circular or elliptical shape, the sidewall thickness T is determined by the thickness of the thinnest part of the spacing between adjacent wells 1.

[0038] The well array configured as described above can simultaneously satisfy various settings for well 1 in the test area, thereby achieving detection sensitivity that enables virus detection in the test solution with a virus concentration of 100 aM or less using the general-purpose observation device. It can also be used for virus detection using the direct virus capture method, which does not use magnetic beads, and enables virus detection in a short period of time. Additionally, virus detection within one minute can be achieved under suitable conditions.

[0039] <Test area> In addition to the wells that contain the test liquid and perform luminescence detection, the well array may also be formed with optional wells that are not subject to luminescence detection, i.e., that are located outside the observation field of the detection unit. For example, it is preferable that the area of ​​the observation field in the detection unit, to which the observation field is fixed, and the area of ​​the formation region of the wells that contain the test liquid and perform luminescence detection are of equal size, but from the viewpoint of allowing room for observation operations when finely aligning the observation field with the formation region of the wells to be observed, a "waste area" outside the observation field may be provided in the well array, and the optional wells may be formed in this area. In other words, when the arbitrary wells are not present, attempting to align the observation field with the well formation region may require time due to the minute size of the wells, whereas when the arbitrary wells are present, alignment can be easily achieved by utilizing the visibility of the outer contour recognized by the outermost arbitrary well. In other words, when the arbitrary wells are present, alignment between the observation field and the well formation region can be achieved simply by determining the boundary on the outer contour and setting the observation field inside it. Note that the arbitrary wells may be formed to be the same size as the wells that will contain the test liquid and perform luminescence detection, or they may be formed as alignment adjustment wells that are different in size from the wells that will contain the test liquid and perform luminescence detection. In this specification, the well formation region to be observed is referred to as the "test region" to distinguish it from the "waste space" region that is not the target of observation. In this sense, it should be noted that the terms "well formation area," "total well volume," "well depth," "well opening area," "well volume," and "sidewall thickness" mean the "well formation area," "total well volume," "well depth," "well opening area," "well volume," and "sidewall thickness" in the "test region," respectively.

[0040] (Detection device) The detection device of the present invention comprises a detection chip on which the well array of the present invention is arranged, and a detection unit. An example of an embodiment of the detection device is shown in FIG. As shown in FIG. 5, the detection device 10 has a detection chip 2 on which a well array 1′ in which a plurality of the above-described wells 1 are formed is arranged, and a detection unit 3.

[0041] The detection chip 2 is not particularly limited except that it has wells 1 and well array 1' with the above-described characteristics, and can be fabricated in accordance with known configurations, for example, it can be fabricated in accordance with the configuration of detection chip 100 (see FIGS. 1(a) and 1(b)) in which space 130 is formed between lower layer 110 and upper layer 120. In addition, for example, it can be configured such that the top of well array 1' is sealed with transparent resin or silicone rubber, or a hydrophobic solvent is dropped onto the top of well array 1' to seal the top of well 1, and a cover glass is placed on top of it.

[0042] The detection unit 3 is capable of fixing the observation field to a size that includes the test region in the well array 1'. The detector 3 is configured in accordance with the configuration of a detector employed in a known microscope, and may be configured, for example, as the general-purpose observation device configured with the 4x objective lens and the full HD image sensor. Alternatively, instead of the general-purpose observation device, it may be configured as the wide-field observation device configured with the 2x objective lens and a 4K image sensor, 8K image sensor, or the like. The observation field of the detection unit 3 may be any size that includes the test area, but is preferably the same size as the test area. In this way, when the observation field and the detection area are set to have the same size, the observation field can be used without waste and high-sensitivity detection can be performed. When detecting fluorescence as the emission from well 1, the detection device is configured with a light irradiation unit for irradiating light to excite fluorescence. This light irradiation unit is configured in accordance with the configuration of a known light irradiation unit for fluorescence excitation, and is configured, for example, with a light source for fluorescence excitation selected according to the excitation wavelength of the substance that causes fluorescence emission in well 1. Known light sources such as lasers, LEDs, and lamps can be used as the light source, and may be configured in combination with lenses and optical filters as necessary.

[0043] The detection device configured as described above can simultaneously satisfy the various settings of the well array, thereby achieving detection sensitivity that enables virus detection in the test solution with a virus concentration of 100 aM (1 virus / 17 nL) or less using the general-purpose observation device. Furthermore, the device can be used for virus detection using the direct virus capture method, which does not use magnetic beads, and enables virus detection in a short period of time. Additionally, virus detection within one minute can be achieved under suitable conditions.

[0044] (Virus detection method) The virus detection method of the present invention is a method of detecting viruses in the test liquid based on luminescence detection of the wells using the well array of the present invention, and the luminescence detection of the wells is carried out in a detection unit in which the observation field is fixed to a size that includes the test area.

[0045] As the detection section, the same matters as those of the detection section 3 (see FIG. 5) described in the detection device of the present invention can be applied. The observation field of the detection unit may be any size that includes the test area, but is preferably the same size as the test area. When the observation field and the detection area are set to have the same size, the observation field can be used without waste to perform highly sensitive detection.

[0046] There are no particular limitations on the viruses to be detected, and examples include viruses that can be detected by known virus detection methods such as ELISA and immunoassay, such as influenza virus, norovirus, and coronavirus.

[0047] The method for detecting luminescence from the well is not particularly limited, and examples thereof include luminescence detection methods that use adsorption reactions, enzymatic reactions, or chemical reactions that emit light in the presence of viruses. For example, methods include a method in which a luminescent substance is adsorbed onto the surface of the virus to cause the well to emit light, a method in which the well emits light using a reagent that generates a fluorescent substance through an enzymatic reaction with a protein contained in the virus, and a method in which the well emits light using a reagent that generates a chemiluminescent substance through an enzymatic reaction with a protein contained in the virus.

[0048] The luminescent substance is not particularly limited and can be appropriately selected depending on the type of virus, etc. For example, an aggregation-induced luminescence (AIE) substance is preferred. Examples of the aggregation-induced luminescent substance include compounds that exhibit the AIE effect described in JP-A-2010-112777.

[0049] The reagent that generates a fluorescent substance through an enzymatic reaction with a protein in the virus is not particularly limited and can be selected appropriately depending on the type of virus, etc., and examples include derivatives containing 4-methylumbelliferone, such as (4-methylumbelliferyl)-α-DN-acetylneuraminic acid, which generates the fluorescent substance 4-methylumbelliferone through an enzymatic reaction with neuramidase in influenza virus, derivatives containing fluorescein, derivatives containing resorufin, and derivatives containing rhodamine.

[0050] The reagent that generates a chemiluminescent substance through an enzymatic reaction with a protein in the virus is not particularly limited and can be appropriately selected depending on the type of virus, etc., and examples thereof include known artificial luciferins.

[0051] The luminescence detection of the well is carried out by placing the test liquid containing the virus and a substance (reagent) that causes the well to luminesce in the well, and then detecting the luminescence of the well with the detection unit. There are no particular limitations on the method for containing the test liquid in the well. When using a detection chip having a configuration similar to that of detection chip 100 (see Figures 1(a) and (b)), in which space 130 is formed between lower layer 110 and upper layer 120, the test liquid can be contained in the well by preparing the test liquid as a hydrophilic solvent and introducing it into space 130, and then introducing a hydrophobic solvent into space 130 to seal the test liquid in the well. Other methods include preparing the test liquid as a hydrophilic solvent and dropping it onto the well array, then pressing a transparent resin or silicone rubber onto the well array to seal it and contain the test liquid in the wells, and preparing the test liquid as a hydrophilic solvent and dropping it onto the well array, then replacing the hydrophilic solvent on the surface with a hydrophobic solvent, and then placing a cover glass on top to contain the test liquid in the wells.

[0052] According to the virus detection method implemented as described above, by simultaneously satisfying the various settings of the well array, the general-purpose observation device can be used to obtain detection sensitivity that enables virus detection in the test solution with a virus concentration of 100 aM (1 virus / 17 nL) or less. Furthermore, this method can be used for virus detection by the direct virus capture method that does not use magnetic beads, allowing viruses to be detected in a short period of time. Additionally, under suitable conditions, virus detection within one minute can be achieved. [Example]

[0053] In order to confirm the effectiveness of the virus detection method according to the present invention, the following detection test was carried out using influenza viruses as the detection target.

[0054] In the detection test, the well array had an opening shape of a square with one side of 7 μm, a depth D of 10 μm, and an opening area of ​​49 μm 2 The wells had a volume of 490 fL and a sidewall thickness T of 3 μm, and were formed on a silicon substrate in a total of 50,451 wells (251 x 201). 2The wells had a total volume of 25 nL and were fabricated by a known shape processing method using photolithography and dry etching. Furthermore, as a reagent for detecting the influenza virus, a detection reagent was used in which a peptide molecule that binds to the influenza virus is bound to tetraphenylsilole, an aggregation-inducing luminescent substance (synthesized with reference to H Shi et al., Journal of the American Chemical Society 134, pp. 9569 (2012)). A 2,048 x 2,048 pixel (4,194,304 pixel) CMOS camera (Hamamatsu Photonics, ORCA-Flash 4.0 V3) was used to observe the well array. The observation field of view when observed with a 4x objective lens was 3.25 mm x 3.25 mm, allowing the entire well array to be observed.

[0055] In detail, the detection method was as follows: first, 30 μL of the specimen solution containing the influenza virus was mixed with 30 μL of a 0.4 μM aggregation-inducing luminescent substance solution, and 50 μL of the mixture was then dispensed and introduced into the well array. Thereafter, the well array was sealed with fluorine oil, a cover glass was placed over it, and the wells were observed under a fluorescence microscope (Olympus, BXFM) using the 4x objective lens. The binding of the influenza viruses to the aggregation-induced luminescent substance resulted in enhanced fluorescence of the aggregation-induced luminescent substance, and in fact, the wells containing the influenza viruses were observed to be brighter than the wells not containing the influenza viruses. To determine the luminescent wells containing the influenza virus, the luminance values ​​of each well were normalized to the average luminance value of a 200 x 200 pixel area outside the well array where the well array was not formed (normalized luminance value), and this value was used for analysis. The threshold for determining the luminescent wells was set to μ + 3.3σ, where μ is the average value of the normalized luminance measured in the influenza virus-free samples and σ is the standard deviation. Wells exceeding the threshold were counted as luminescent wells, and the value was used as a measurement value corresponding to the influenza virus concentration.

[0056] As a result of this test, the final concentration was 1 × 10 5 An example of a fluorescence image taken of a sample containing 100 copies / mL of influenza virus is shown in FIG. 6, and an example of a fluorescence image taken of the blank sample is shown in FIG. Comparing Figures 6 and 7, it can be seen that in samples containing influenza virus, some wells are brighter (whiter) than the surrounding wells, while in samples not containing influenza virus, most of the wells are of equal brightness. The number of luminescent wells counted for the entire well array was 61 for the sample containing the influenza virus, and 8 for the sample not containing the influenza virus, indicating that a significant distinction was made and that the influenza virus could be detected. Furthermore, if the number of luminescent wells is at least 20, a sample can be significantly distinguished from a sample not containing the influenza virus. Furthermore, since the number of luminescent wells is considered to be proportional to the virus concentration, a final concentration of approximately 3 × 10 4 Virus detection is possible for samples containing up to 100 copies / mL of the influenza virus. Final concentration (3×10 4 The concentration of influenza virus (100 copies / mL) corresponds to 50 aM. It has also been confirmed that the binding reaction between the influenza virus and the agglutination-induced luminescent substance is completed within 10 seconds, and even when time for sample introduction into the well array and fluorescence observation is added, the entire measurement process can be completed within 1 minute. In other words, the present invention enables high-speed and highly sensitive virus detection. [Explanation of symbols]

[0057] 1 well 1' well array 2. Detection chip 3. Detection unit 10. Detection Device L Light 100 detection chips 110 Lower part 111 Side wall 115a~115b Well 120 Upper Management 121 entrance 130 Space 140 Hydrophilic solvent 150 Hydrophobic Solvents 161 Virus 162 Fluorogenic Substrates 163 Reaction Products

Claims

1. A virus detection method for detecting viruses in a test liquid based on luminescence detection of a plurality of wells, the method comprising: using a well array formed by dividing adjacent wells with side walls erected on a substrate; The well array has a well formation area of ​​5 mm, which is the area of ​​the substrate in which the wells are formed. ２ The total volume of the wells obtained by integrating the volumes of the individual wells is 17 nL or more, the depth of the wells is 3.5 μm to 40 μm, and the opening area of ​​the wells is 18 μm. ２ ~1,700μm ２ the well has a volume of 74 fL to 6,000 fL, and the thickness of the side wall between adjacent wells is 0.5 μm to 15 μm; The luminescence detection of the well is carried out in a detection unit in which an observation field is fixed to a size including the test region; A virus detection method characterized in that the upper surface of the side wall is subjected to a hydrophobic treatment.

2. 2. The virus detection method according to claim 1, wherein the observation field of the detection unit is set to a size equivalent to that of the test area.

3. 3. The virus detection method according to claim 1, wherein a luminescent substance is adsorbed onto the surface of the virus to cause the well to emit light.

4. 4. The virus detection method according to claim 3, wherein the luminescent substance is an aggregation-induced luminescent substance.

5. 3. The virus detection method according to claim 1, wherein the wells are made to emit light using a reagent that generates a fluorescent substance upon an enzymatic reaction with a protein contained in the virus.

6. 3. The virus detection method according to claim 1, wherein the wells are made to emit light using a reagent that generates a chemiluminescent substance through an enzymatic reaction with a protein contained in the virus.

7. A well array formed by defining adjacent wells with side walls erected on a substrate, The well formation area, which is the area of ​​the substrate in which the well is formed, is 5 mm ２ The total volume of the wells obtained by integrating the volumes of the individual wells is 17 nL or more, the depth of the wells is 3.5 μm to 40 μm, and the opening area of ​​the wells is 18 μm. ２ ~1,700μm ２ the well has a volume of 74 fL to 6,000 fL, and the thickness of the side wall between adjacent wells is 0.5 μm to 15 μm; A well array characterized in that the upper surface of the side wall is subjected to a hydrophobic treatment.

8. A detection chip on which the well array according to claim 7 is arranged; a detection unit capable of fixing an observation field of view to a size including a test region in the well array; A detection device comprising:

9. 9. The detection device according to claim 8, wherein the observation field of the detection unit is set to a size equivalent to that of the detection region.

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