BIOLOGICAL SUBSTANCE DETECTION METHOD USING WELL ARRAY AND PARTICLES, WELL ARRAY, AND DETECTION DEVICE

Abstract

To detect a biological substance at high speed and high sensitivity by using a well array and particles. The biological substance detection method of the present invention includes a step of preparing a test liquid containing particles at a concentration of at least 5?10.sup.7 particles/mL, a step of allowing the particles to trap the biological substance to form a trapped body, a step of sending the test liquid onto the well array to store a plurality of the particles including the trapped body in the wells while storing the number of the particles to be stored in one well to a minimum average storage number N.sub.min represented by the following formulas (1) and (2), and a step of detecting the color development of the well with an image pickup element.

[00001] $\begin{matrix} [Mathematical Formula 1] & ? \\ N_{\min} = C ? V_{L} ? \frac{P_{image}}{P_{total}} & (1) \end{matrix}$ $\begin{matrix} N_{\min} ? 8 & (2) \end{matrix}$

In the formula (1), C represents a concentration of the particles in the test liquid; V.sub.L represents an amount of the test liquid and is at least 5 ?L; P.sub.total represents the number of pixels of the image pickup element and is at least 300,000 pixels; and P.sub.image represents the number of pixels of a pixel group used for imaging one of the wells in the image pickup element and is at least 9 pixels.

Claims

1. A biological substance detection method using a well array formed by defining a plurality of wells adjacent to each other with a side wall stood on a substrate and particles capable of trapping a biological substance and detecting the biological substance in a test liquid based on color development detection of the wells, comprising: a test liquid preparation step for preparing the test liquid containing the particles at a concentration of at least 5?10.sup.7 particles/mL, a biological substance trapping step for allowing the particles to trap the biological substance to form a trapped body, a particle storing step for sending the test liquid onto the well array to store a plurality of the particles including the trapped body in the wells while storing the number of the particles to be stored into one of the wells to at least a minimum average storage number N.sub.min represented by the following formulas (1) and (2), and a color development detection step for detecting color development of the wells with an image pickup element having at least 300,000 pixels. $\begin{matrix} [Mathematical Formula 1] & ? \\ N_{\min} = C ? V_{L} ? \frac{P_{image}}{P_{total}} & (1) \end{matrix}$ $\begin{matrix} N_{\min} ? 8 & (2) \end{matrix}$ wherein, in the formula (1), C represents the concentration of the particles in the test liquid and is at least 5?10.sup.7 particles/mL; V.sub.L represents the amount of the test liquid and is at least 5 ?L; P.sub.total represents the number of pixels of the image pickup element and is at least 300,000 pixels; and P.sub.image represents the number of pixels of a pixel group used for imaging one of the wells in the image pickup element and is at least 9 pixels (3?3 pixels).

2. The biological substance detection method according to claim 1, wherein the particles are magnetic particles having a diameter of 0.1 ?m to 9 ?m.

3. The biological substance detection method according to claim 1, wherein the test liquid preparation step is to prepare the test liquid while adjusting the concentration of the particles to 5?10.sup.7 particles/mL to 5?10.sup.9 particles/mL.

4. The biological substance detection method according to claim 1, wherein the wells each have a volume of 2 fL to 100 ?L.

5. The biological substance detection method according to claim 1, wherein the color development detection step is carried out by fixing an observation visual field of the image pickup element to have a size including a well formation area which is an area of the substrate in a well formation region.

6. The biological substance detection method according to claim 1, wherein color development of the wells in the color development detection step is due to a reaction between the biological substance of the trapped body and a color producing reagent causing the color development of the wells.

7. A well array for use in the biological substance detection method as claimed in claim 1, which can store a plurality of the particles including the trapped body in one of the wells while storing the number of the particles to at least a minimum average storage number N.sub.min represented by the following formulas (1) and (2): $\begin{matrix} [Mathematical Formula 2] & ? \\ N_{\min} = C ? V_{L} ? \frac{P_{image}}{P_{total}} & (1) \end{matrix}$ $\begin{matrix} N_{\min} ? 8 & (2) \end{matrix}$ wherein, in the formula (1), C represents the concentration of the particles in the test liquid and is at least 5?10.sup.7 particles/mL; V.sub.L represents the amount of the test liquid and is at least 50 ?L; P.sub.total represents the number of pixels of the image pickup element and is at least 300,000 pixels; and P.sub.image represents the number of pixels of a pixel group used for imaging one of the wells in the image pickup element and is at least 9 pixels (3?3 pixels).

8. A detection device, comprising: a detection chip having the well array as claimed in claim 7, and a detection unit having an image pickup element having a pixel number of at least 300,000 pixels.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG. 1 is an explanatory drawing for explaining the setting of wells.

[0055] FIG. 2(a) is an electron microscope image in which Preparation Example 1 of a well array has been imaged.

[0056] FIG. 2(b) is a partially enlarged picture of FIG. 2(a).

[0057] FIG. 3(a) shows an electron microscope image in which Preparation Example 2 of a well array has been imaged.

[0058] FIG. 3(b) is a partially enlarged picture of FIG. 3(a).

[0059] FIG. 4 is a drawing showing an embodiment of a detection device.

[0060] FIG. 5(a) is an explanatory drawing (1) for explaining the detection manner of a biological substance.

[0061] FIG. 5(b) is an explanatory drawing (2) for explaining the detection manner of a biological substance.

[0062] FIG. 5(c) is an explanatory drawing (3) for explaining the detection manner of a biological substance.

[0063] FIG. 6 is a graph showing the results of Example of the biological substance detection according to the present invention.

MODE FOR CARRYING OUT THE INVENTION

[Biological Substance Detection Method]

[0064] The biological substance detection method of the present invention is to use a well array formed by defining a plurality of wells adjacent to each other with a side wall stood on a substrate and particles capable of trapping a biological substance, and to detect the biological substance in a test liquid based on the detection of color development of the wells, which includes a test liquid preparation step, a biological substance trapping step, a particle storing step, and a color development detection step.

[0065] The biological substance is not particularly limited and examples include DNA, RNA, proteins, viruses, and bacteria to be detected by a known biological substance detection method such as an ELISA method and an immunoassay method. In particular, the examples include biological substances having a diameter of 1 nm to 500 nm such as protein (having a diameter of about 5 nm) and pathogenic viruses such as an influenza virus and a corona virus (having a diameter of about 100 nm).

[0066] A liquid sample containing the biological substance is not particularly limited and examples include blood, saliva, urea, and environmental water.

[0067] The test liquid preparation step is to prepare the test liquid by setting the concentration of the particles to at least 5?10.sup.7 particles/mL.

[0068] For example, for the detection of a virus contained in the saliva, the test liquid is prepared by adding the particles to the saliva.

[0069] A method of setting a specific concentration of the particles in the test liquid is not particularly limited and examples include a method of diluting a known particle-containing liquid containing the particles at a high concentration with a known buffer solution.

[0070] The particles are not particularly limited and examples include known plastic particles, metal particles, ceramic particles, and magnetic particles. The magnetic particles are preferred, because they can be stored in the wells more easily using a magnet, in comparison with the plastic particles, metal particles, and ceramic particles that precipitate in the wells by their own weight.

[0071] The particle size of the particles is not particularly limited and is preferably 0.1 ?m to 9 ?m and more preferably 0.2 ?m to 5 ?m.

[0072] As the particles, spherical ones and also those having a plurality of particle sizes such as oval ones can be used. When the particles are oval, the maximum diameter corresponds to the particle size described above in the particle size range.

[0073] The particle size of the particles will hereinafter be described in detail.

[0074] When the particle size is too small, even if the magnetic particles are used, they have low responsiveness to magnetic force so that it sometimes takes a lot of time to perform an operation of attracting the particles by a magnet and storing them in the well.

[0075] The lower limit of the particles size is therefore preferably 0.1 ?m or more and more preferably 0.2 ?m or more.

[0076] On the other hand, using the particles having a larger particle size can shorten the reaction time with the biological substance, but too large particles require a large observation visual field and may prolong the detection time during color development detection of the wells, which will hereinafter be described in detail.

[0077] The depth of the well is preferably 40 ?m at most. The well deeper than this depth is likely to cause such inconveniences that the image pickup element loses its focus in the whole depth direction of the well, it becomes difficult to process the well, and the test liquid cannot easily be stored in the well.

[0078] When preparing 5 ?L of the test liquid having the particle concentration of 5?10.sup.7 particles/mL, the total number of the particles contained in the test liquid is 2.5?10.sup.5 particles.

[0079] The particles even in the spherical form are not closest-packed in the wells so that, similarly to another form, they are assumed to be packed in a cubic lattice form.

[0080] In this assumption, when the particles have a particle size of 1 ?m, the total volume (capacity) of the well array obtained by integrating the volumes of all the wells is required to be 2.5?10.sup.5 ?m.sup.3 (1 ?m?1 ?m?1 ?m?2.5?10.sup.5 particles). Similarly, when the particles have a particle size of 5 ?m, the total volume is required to be 3.13?10.sup.7 ?m.sup.3, and when the particles have a particle size of 6 ?m, the total volume is required to be 5.4?10.sup.7 ?m.sup.3.

[0081] Supposing that the well has a depth of 40 ?m or less, a total opening area of the well array obtained by integrating the opening area of all the wells (an area obtained by integrating the formation area of all the wells on the substrate of the well array) is required to be 0.00625 mm.sup.2 or more (2.5?10.sup.5 ?m.sup.3/40 ?m or less) when the particles have a particle size of 1 ?m. Similarly, the total opening area is required to be 0.169 mm.sup.2 or more when the particles have a particle size of 3 ?m; 0.4 mm.sup.2 or more when the particles have a particle size of 4 ?m; 0.781 mm.sup.2 or more when the particles have a particle size of 5 ?m; 1.35 mm.sup.2 or more when the particles have a particle size of 6 ?m; 2.14 mm.sup.2 or more when the particles have a particle size of 7 ?m; 3.2 mm.sup.2 or more when the particles have a particle size of 8 ?m; and 4.56 mm.sup.2 or more when the particles have a particle size of 9 ?m; and 6.25 mm.sup.2 or more when the particles have a particle size of 10 ?m.

[0082] Assuming that the color development detection of the wells is performed at the detection unit comprised of the general-purpose observation device, an observation visual field is about 5 mm.sup.2 when a 4? objective lens is used in the general-purpose observation device and it is about 0.8 mm.sup.2 when a 10? objective lens is used. As the observation visual field is smaller, the visibility of the wells is higher and the color development detection is easier.

[0083] When the total opening area of the well array exceeds the observation visual field, the area exceeding the observation visual field is required to be observed by moving the observation visual field. It therefore takes time for detecting the color development of the wells.

[0084] Consequently, the upper limit of the particle size is preferably 9 ?m or less under the conditions where the total opening area does not exceed the observation visual field at the observation visual field of 5 mm.sup.2 and it is more preferably 5 ?m or less under the conditions where the total opening area does not exceed the observation visual field at the observation visual field of 0.8 mm.sup.2.

[0085] The lower limit of the concentration of the particles in the test liquid may be 5?10.sup.7 particles/mL or more and is more preferably 1?10.sup.8 particles/mL or more because with an increase in the concentration, the reaction time with the biological substance can be shortened.

[0086] When the concentration is too high, it may be over performance for shortening of the reaction time. In addition, it may increase the observation visual field and prolong the detection time during the color development detection of the wells. The following is the details thereof.

[0087] When the particles have a particle size of 1 ?m on the same assumption as when finding the preferable particle size (the amount of the test liquid is 5 ?L and the depth of the well is 40 ?m or less), the total volume is 5?10.sup.6 ?m.sup.3 and the total opening area is 0.125 mm.sup.2 or more at the concentration of 1?10.sup.9 particles/mL; the total volume is 1?10.sup.7 ?m.sup.3 and the total opening area is 0.25 mm.sup.2 or more at the concentration of 2?10.sup.9 particles/mL; the total volume is 2?10.sup.7 ?m.sup.3 and the total opening area is 0.50 mm.sup.2 or more at the concentration of 4?10.sup.9 particles/mL; the total volume is 4?10.sup.7 ?m.sup.3 and the total opening area is 1.0 mm.sup.2 or more at the concentration of 8?10.sup.9 particles/mL; the total volume is 8?10.sup.7 ?m.sup.3 and the total opening area is 2.0 mm.sup.2 or more at the concentration of 1.6?10.sup.10 particles/mL; and the total volume is 2.0?10.sup.8 ?m.sup.3 and the total opening area is 5.0 mm.sup.2 or more at the concentration of 4.0?10.sup.10 particles/mL. The visual field to be observed in practice has an area obtained by adding, to the total opening area, the thickness of the side wall, that is, the area of the side wall portion so that at the concentration of 4.0?10.sup.10 particles/mL, an observation visual field more than 5.0 mm.sup.2 becomes necessary in practice. This means that at the concentration more than 4.0?10.sup.10 particles/mL, the total opening area is larger relative to the observation visual field (5 mm.sup.2).

[0088] Thus, an excessively high concentration may be a limitation for setting the total opening area of the well array to be the observation visual field (5 mm.sup.2) or less.

[0089] This limitation is relaxed as the particle size of the particles is smaller (for example, 0.1 ?m), but an unnecessary increase in the number of the particles is wasteful.

[0090] Accordingly, the upper limit of the concentration is preferably 4?10.sup.10 particles/mL or less based on the balance between the detection time and the reaction time, and is more preferably 5?10.sup.9 particles/mL or less in consideration of the thickness of the side wall.

[0091] The biological substance trapping step is to allow the particles to trap the biological substance to form a trapped body. The trapped body is formed by binding the biological substance to the particles.

[0092] A mode of allowing the particles to trap the biological substance is not particularly limited and can be selected as needed depending on the purpose. Examples thereof include an antigen-antibody reaction, DNA hybridization, biotin-avidin binding, and amino binding. For example, in the antigen-antibody reaction, the particles with a large number of antibodies, which specifically bind to the biological substance used as an antigen, bound to their surfaces are used.

[0093] As the particles capable of trapping the biological substance, commercially available ones may be used, or they may be formed by a known method.

[0094] In the test liquid containing the particles at a low concentration, contact opportunities between the biological substance and the particles are limited and the reaction time is longer. The test liquid preparation step is therefore performed as a preliminary step of the biological substance trapping step to obtain the test liquid having an increased particle concentration.

[0095] Examples of a method of performing the biological substance trapping step include a method of stirring the test liquid to bring the particles into contact with the biological substance.

[0096] The particle storing step is to send the test liquid onto the well array to store, in one of the wells, a plurality of the particles including the trapped body while storing the number of the particles to at least a minimum average storage number N.sub.min represented by the following formulas (1) and (2).

[00005] $\begin{matrix} [Mathematical Formula 4] & ? \\ N_{\min} = C ? V_{L} ? \frac{P_{image}}{P_{total}} & (1) \end{matrix}$ $\begin{matrix} N_{\min} ? 8 & (2) \end{matrix}$ [0097] wherein, in the formula (1), C represents the concentration of the particles in the test liquid and is at least 5?10.sup.7 particles/mL; V.sub.L represents an amount of the test liquid and is at least 5 ?L; P.sub.total represents the number of pixels of the image pickup element and is at least 300,000 pixels; and P.sub.image represents the number of pixels of a pixel group used for imaging one of the wells in the image pickup element and is at least 9 pixels (3?3 pixels).

[0098] As examples of a larger number of pixels of the image pickup element, there are 2,073,600 pixels (1,920?1,080 pixels) for a full HD image pickup element, 8,294,400 pixels (3,840?2,160 pixels) for a 4K image pickup element, 33,177,600 pixels (7,680?4,320 pixels) for an 8K image pickup element, and about 61,000,000 pixels for a full-size CMOS image sensor.

[0099] The pixel group means a pixel group which consists of pixels juxtaposed in a first direction and pixels juxtaposed in a second direction orthogonal to the first direction and in which these pixels are arranged in matrix form. In a pixel group arranged in a matrix of 3 rows and 3 columns, that is, a group of 3?3 pixels (9 pixels), one pixel at the center is used for the color development detection of the wells. The pixel group may be comprised of 4?4 pixels (16 pixels) or the like and in this case, the number of pixels used for the color development detection of the wells can be increased and this leads to improvement in visibility.

[0100] The term average in the minimum average storage number N.sub.min is for taking into account the variation during storing the particles in the well and the individual wells themselves are set to be able to contain at least N.sub.min particles. Whether or not at least N.sub.min particles are stored in the well is confirmed by average value in consideration of the variation. For example, it is confirmed from average value obtained by counting the total number of the particles stored in the wells in the arbitrarily selected group of the wells (for example, four wells arranged in two rows and two columns and adjacent to each other) from an electron microscope image or optical microscope image and then dividing the total number by the number of the wells in the group of the wells to be counted.

-Well Array-

[0101] The well array is formed by defining a plurality of the wells adjacent to each other by the side wall stood on a substrate.

[0102] The well array is comprised so that one of the wells can contain a plurality of the particles including the trapped body with at least the minimum average storage number N.sub.min represented by the aforesaid formulas (1) and (2).

[0103] The total number of the wells to be formed in the well array is not particularly limited but ideally it is equal to an observation limit P.sub.total/P.sub.image. The observation limit P.sub.total/P.sub.image will next be described specifically. Supposing that the P.sub.total is 640?480 pixels (=307,200 pixels) of the VGA image pickup element and the P.sub.image is 3?3 pixels (=9 pixels), the observation limit P.sub.total/P.sub.image is about 34,080 wells (213?160 wells). If the pixel number of the VGA image pickup element is more plainly 300,000 pixels, the observation limit is 33,333 wells.

[0104] A preferable lower limit of the total number of the wells will next be examined.

[0105] The premise is that even if the total number of the wells is less than P.sub.total/P.sub.image, the volume of one of the wells may be set so that the storage number of the particles may exceed the minimum average storage number N.sub.min.

[0106] This means that the assumption that the total number of the wells is set at about 33,333 (300,000 pixels/9 pixels) is a baseline for setting the minimum average storage number at 8 on average. The total number of the wells can arbitrarily be reduced from the aforesaid assumption by making the storage number larger than the average value of 8 through volume setting of one of the wells.

[0107] However, the actual volume of the well has an upper limit because of the reason described later and the preferable maximum volume of the well is 100 ?L (=0.1?10.sup.6 ?m.sup.3).

[0108] On the other hand, when the preferably usable particles having a particle size of 1 ?m are used, the total volume of the well array is required to be 2.5?10.sup.5 ?m.sup.3 or more to store, in the wells, all of 2.5?10.sup.5 particles (the total number of particles when 5 ?L of the test liquid having a particle concentration of 5?10.sup.7 particles/mL is prepared).

[0109] Therefore, the lower limit of the total number of the wells could be preferably 3 (2.5?10.sup.5 ?m.sup.3/(0.1?10.sup.6 ?m.sup.3)) or more.

[0110] In an actual detection case, on the other hand, even when a detection object is the test liquid not containing the biological substance, a certain number of color-developed wells (false-positive wells) are detected due to a detection reagent being unwashed or the like. The present inventors repeated a preliminary test using enzymes, including ?-galactosidase, which cause color development of a well 1, and confirmed the occurrence of about tens of the false-positive wells at most.

[0111] When carrying out detection of the test liquid containing the biological substance, therefore, in order to detect the number of color-developed wells as a significant signal derived from the biological substance, it is required to take into account the variation and set the number of the wells so that color-developed wells as significantly more than the false-positive wells (for example, ?+3.3? wells or more in which ? represents an average number of the false-positive wells and ? represents a standard deviation) can be detected. From this viewpoint, the lower limit of the total number of the wells is preferably 100 or more.

[0112] By the way, in the biological substance detection method of the present invention, quantitativity according to the Poisson distribution is secured in terms of the statistical probability insofar as the total number of the wells exceeds the number of the biological substance contained in the test liquid.

[0113] In addition, with an increase in the total number of the wells used for detection, a dynamic range in digital counting of the number of color-developed wells expands, which is remarkably advantageous in performing high-speed and high-sensitivity quantitative detection of the biological substance.

[0114] In order to add the quantitative detection of the biological substance to the object, how to set the lower limit of the total number of the wells after satisfying the aforesaid requirement for the lower limit depends on the number of the biological substance contained in the test liquid, that is, the intended use to which the biological substance detection method of the present invention is applied. For example, empirically, for use when the test liquid assumed to contain a small number of the biological substance is to be detected, the total number of the wells is set small (for example, 1,000 wells) so as to satisfy the setting of the dynamic range based on the number of the biological substance. On the contrary, for use when the test liquid assumed to contain a large number of the biological substance is to be detected, the total number of the wells is set large (for example 10,000 wells).

[0115] Regarding the upper limit of the total number of the wells, if the total number of the wells exceeds P.sub.total/P.sub.image, with the entirety of the well array being observed, the color development of the well cannot be detected due to deteriorated visibility. Also, if the observation visual field is divided to partially observe the well array, it takes time to detect the color development of the well because the observation should be carried out by transferring the observation visual field with an extra time.

[0116] In the well array, a well formation area which is an area of the substrate in the well formation region is preferably at least 0.8 mm.sup.2. The well formation area indicates an area on the substrate on which a plurality of the wells are formed as a group of the wells to be observed and it is different from an area for the formation of one of the wells.

[0117] When the well formation area is 0.8 mm.sup.2 and color development of the well is detected with the detection unit comprised of the general-purpose observation device having the 10? objective lens, observation can be carried out without transferring the observation visual field because the observation visual field in the detection unit is about 0.8 mm.sup.2.

[0118] When the well formation area is 5 mm.sup.2 and color development of the well is detected with the detection unit comprised of the general-purpose observation device having the 4? objective lens, observation can be carried out without transferring the observation visual field because the observation visual field in the detection unit is about 5 mm.sup.2.

[0119] The constitution example of the well will next be described referring to FIG. 1. It is an explanatory drawing for explaining the setting of the well.

[0120] The depth D of the well 1 is not particularly limited and it is preferably 2 ?m to 40 ?m. The well 1 is designed for storing the particles therein so that the depth D is required to be larger than the particle size of the particles. The lower limit of the particle size of the usable particles is 0.1 ?m so that if the depth D is less than 0.1 ?m, even the particles having a minimum size are difficult to be stored in the well 1. The particles can be stably stored in the well having a deeper depth D so that even if particles having a small particle size are used, the depth is preferably set at 2 ?m or more. On the other hand, the well having a depth D of more than 40 ?m is likely to cause such inconveniences that the image pickup element loses its focus in the whole depth direction of the well, it becomes difficult to process the well, and the test liquid cannot easily be stored in the well.

[0121] The volume V of the well 1 is set on the baseline that the minimum average storage number N.sub.min of the particles are stored in the well.

[0122] Now consider that there are stored in the well 8 particles, that is, the minimum average storage number N.sub.min when the detection unit is comprised of the image pickup element having 300,000 pixels.

[0123] Supposing that the particles have a particle size of 0.1 ?m, and the well 1 is not closest-packed with the particles and is designed with a margin to pack them in a cubic lattice form, the volume occupied by one of the particles in the well 1 is 0.001 fL. The volume occupied by 8 particles in the well is therefore 0.008 fL. The well 1 is therefore required to have a volume of at least 0.008 fL or more.

[0124] Given the visibility of the well 1 and the optimum value of the depth D, the length of one side of the well 1 smaller than 1 ?m may deteriorate the visibility even in a high magnification microscopic observation system so that the length of one side is preferably 1 ?m or more. As described above, the depth D is preferably 2 ?m or more and therefore the lowest volume of the well satisfying such a size is 2 fL.

[0125] The lower limit of the volume V of the well 1 is therefore preferably 2 fL or more.

[0126] On the other hand, the upper limit of the volume V of the well 1 can be explained as follows.

[0127] When the volume V of the well 1 is excessively large, the concentration of a substance causing color development of the well 1 becomes lower in the well 1, causing deterioration in the visibility of the color development of the well 1. In other words, even when the biological-substance trapped body is stored in the well 1, the color development due to the biological substance in this well 1 cannot be distinguished from another well 1 storing only the particles which have not trapped the biological substance, making it difficult to detect the biological substance through color development or non-color development of the well 1.

[0128] On this point, according to a preliminary test performed by the present inventors with enzymes such as ?-galactosidase that cause color development of the well 1, when there is one molecule of the enzymes in the well 1 filled with the test liquid and having a volume V of 10 ?L, a color developed well 1 containing the enzyme can be distinguished from another well 1 not containing the enzyme and being in a non-color developed state.

[0129] About 100 molecules of the aforesaid enzyme can generally be attached to one virus via a substance, such as an antibody, that specifically adsorbs to a virus so that a detection limit between color development and non-color development of the well 1 can be estimated at 1,000 ?L. If the volume of the well is set at 100 ?L with a margin in the detection limit, color development and non-color development of the well 1 can easily be distinguished from each other.

[0130] Thus, the upper limit of the volume V of the well 1 is preferably 100 ?L or less.

[0131] The side wall thickness T, which is a thickness of the side wall between the wells 1 adjacent to each other, is not particularly limited and it is preferably 0.5 ?m to 15 ?m. The side wall thickness T less than 0.5 ?m makes processing of a well difficult and in addition such a well is easy to break. When the side wall thickness T is more than 15 ?m, on the other hand, the well formation area in the well array becomes uselessly wide and a large limitation is likely to be imposed on the setting for the detection of the biological substance without transferring the observation visual field. When the distance between the wells 1 adjacent to each other is not uniform as in the case where the well 1 has a round or oblong shape, the side wall thickness T is determined by the thickness of the thinnest portion between the wells 1 adjacent to each other.

[0132] Although no limitation is imposed on the opening area A of the well 1, it is preferably 1 ?m.sup.2 to 50,000 ?m.sup.2 judging from the visibility and the relation (V/D) between depth D and volume V.

[0133] In an embodiment of the well 1 shown in FIG. 1, the opening shape is square, but the opening shape is not particularly limited. It may be a regular polygon such as a regular triangle and a regular hexagon (a honeycomb structure), a rectangle, a circle, or an ellipse. Also, the well 1 is not particularly limited insofar as it has a columnar (square cup) shape.

[0134] A material for forming the well array is not particularly limited and can be selected as needed depending on the purpose. Examples include known glass materials, semiconductor materials, and resin materials.

[0135] A method of forming the well array is also not particularly limited and can be selected as needed depending on the purpose. Examples include known methods such as a method of pattern drawing by lithography and then etching to form the well array, and a method of forming the well array by injection molding or imprint with a mold having a shape of the well.

[0136] For example, the processing limit of a method of forming the well array by lithography and reactive ion etching with silicon as a formation material is about 0.1 nm and thus the well array can be formed with high resolution.

[0137] Preparation Example 1 of the well array is shown in FIGS. 2(a) and (b). FIG. 2(a) shows an electron microscope image in which Preparation Example 1 of the well array has been imaged and FIG. 2(b) is a partially enlarged photograph of FIG. 2(a).

[0138] Also, Preparation Example 2 of the well array is shown in FIGS. 3(a) and (b). FIG. 3(a) shows an electron microscope image in which Preparation Example 2 of the well array has been imaged and FIG. 3(b) is a partially enlarged photograph of FIG. 3(a).

[0139] Preparation Examples 1 and 2 are each obtained by lithography and reactive ion etching with silicon as a formation material.

[0140] The well array of Preparation Example 1 relates to a preparation example of a shallow type in which a well 1 is formed while setting the opening shape to a square with 10 ?m on each side, a depth D to 3 ?m, and a side wall thickness T to 3 ?m.

[0141] The well array of Preparation Example 2 relates to a preparation example of a deep type in which a well 1 is formed while setting the opening shape to a square with 10 ?m on each side, a depth D to 15 ?m, and a side wall thickness T to 3 ?m.

[0142] The well array of Preparation Example 1 is suitably used for the particles having a small particle size and the well array of Preparation Example 2 is preferably used for the particles having a large particle size.

[0143] The color development detection step is to detect color development of the well by using a commercially available image pickup element.

[0144] In the color development detection step, a generally available image pickup element may be used without a particular problem. An image pickup element having at least 300,000 pixels may be used.

[0145] The aforesaid image pickup element constitutes a detection unit based on a known microscope or the like and used for the detection of color development of the well. In the color development detection of the well with the observation visual field of 5 mm.sup.2, for example, the detection unit may be comprised of a 4? objective lens, while in the color development detection of the well with the observation visual field of 0.8 mm.sup.2, for example, the detection unit may be comprised of a 10? objective lens.

[0146] Although the color development of the well is not particularly limited, it preferably occurs, after the previous particle storing step, due to a reaction between the biological substance in the trapped body and a color producing reagent causing color development of the well.

[0147] The color producing reagent is not particularly limited and examples include a color producing reagent used in a known biological substance detection method such as an ELISA method and an immunoassay method. Specific examples include a color development substance that adsorbs to the biological substance and causes color development of the well, a reagent that forms a fluorescent substance by an enzyme reaction with a protein in the biological substance, a reagent that forms a chemiluminescent substance by an enzyme reaction with a protein in the biological substance, and a labeled substance having a recognition site that specifically recognizes the biological substance.

[0148] The color producing reagent may be added to the well before and after the particle storing step or may be added during preparation of the test liquid in the test liquid preparation step.

[0149] The term color development as used in this specification means a state in which light different in at least one of spectrum and intensity between the well containing the biological substance and the well not containing the biological substance can be detected. The concept color development includes, in addition to a change in coloration from the well not containing the biological substance to the well containing the biological substance, at least one of a change in emission spectrum and a change in emission intensity from the well not containing the biological substance to the well containing the biological substance. The term color development as used in this specification may be replaced with color development or light emission.

[0150] The color development substance that adsorbs to the biological substance to cause color development of the well is not particularly limited and it is for example an aggregation induced emission (AIE) substance. Examples of the aggregation induced emission substance include compounds producing an AIE effect as described in Japanese Patent Laid-Open No. 2010-112777.

[0151] Examples of the reagent that causes an enzyme reaction with a protein in the biological substance and thereby produces a fluorescent substance include 4-methylumbelliferone-containing derivatives such as (4-methylumbelliferyl)-?-D-N-acetylneuraminic acid that enzymatically reacts with neuraminidase in an influenza virus and thereby produces 4-methylumbelliferone which is a fluorescent substance, fluorescein-containing derivatives, resorufin-containing derivatives, and rhodamine-containing derivatives.

[0152] Examples of the reagent that produces a chemiluminescent substance by an enzyme reaction with a protein include luciferin that causes the protein to emit light as luciferase.

[0153] Examples of the labeled substance include a labeled enzyme or a labeled fluorescent dye in which an antibody recognizing the biological substance is labeled by an enzyme or a fluorescent dye.

[0154] An example of a method of performing the biological substance detection method using the magnetic particles as the particles will be described with reference to drawings.

[0155] FIG. 4 shows an embodiment of a detection device to be used for the biological substance detection method. FIGS. 5(a) to (c) show detection manners of the biological substance.

[0156] As shown in FIG. 4, a detection device 10 is comprised of a detection chip 2 in which a well array 1 having a plurality of the wells 1 is placed, a detection unit 4, and a magnetic field application unit 3. The detection chip 2 is not particularly limited and it has a constitution similar to that of a known detection chip used for biological substance observation. The magnetic field application unit 3 is not particularly limited and it is comprised of a permanent magnet, an electromagnet or the like.

[0157] First, a test liquid 5 is prepared so as to have a concentration of magnetic particles 6 of at least 5?10.sup.7 particles/mL and then the test liquid 5 is stirred. For example, after the magnetic particles 6 trap the biological substance to form the trapped body, the test liquid 5 is sent onto the well array 1 (refer to FIG. 5(a)). The number of the magnetic particles 6 shown in the drawings is simplified so as not to complicate the drawings.

[0158] Next, after the test liquid is sent, the magnetic particles 6 are attracted into the well 1 by a magnetic field applied from the magnetic field application unit 3 and the plurality of the magnetic particles 6 including the trapped body are stored in the well 1 (refer to FIG. 5(b)). At this time, the excess test liquid 5 is sent outside of the well array 1. When the magnetic particles 6 are replaced by the plastic particles, metal particles, or ceramic particles, the plastic particles and others are precipitated by their own weight and stored in the well 1.

[0159] Next, the color producing reagent is added in the well 1 and the well 1 is covered with a transparent glass plate 7 to prevent the magnetic particle 6 and the color producing reagent from dissipating from the well 1 (refer to FIG. 5(c)). Alternatively, a hydrophobic solvent may be dropped on the upper portion of the well 1 to seal the upper portion of the well 1 and then the transparent glass plate 7 may be placed thereon. The color producing reagent may be added to the test liquid 5 in advance just before the test liquid 5 is sent onto the well array 1.

[0160] When a reaction between the biological substance trapped in the trapped body and the color producing reagent proceeds, color development due to this reaction occurs. This color development is detected at the detection unit 4 as color development L of the well 1 (refer to FIG. 4).

[0161] When the color producing reagent produces fluorescence in the well 1, the aforesaid reaction is followed by emission of an excited light from an arbitrary light source to cause fluorescence and this color development is detected at the detection unit 4 as color development L of the well 1.

[0162] According to the biological substance detection method described above, all the particles are stored in the well to keep a detection sensitivity, and, in addition, by using the test liquid prepared to have a high particle concentration, a reaction time necessary for trapping the biological substance is reduced and thereby high-speed detection of the biological substance can be achieved.

(Well Array)

[0163] The well array of the present invention is used for the biological substance detection method of the present invention and it can store, in one of the wells, a plurality of the particles including the trapped body while storing the number of the particles to at least the minimum average storage number N.sub.min represented by the following formulas (1) and (2).

[0164] According to the well array, all the particles are stored in the well to keep a detection sensitivity, and, in addition, by using the test liquid prepared to have a high particle concentration, a reaction time necessary for trapping the biological substance is reduced and thereby high-speed detection of the biological substance can be achieved.

[00006] $\begin{matrix} [Mathematical Formula 5] & ? \\ N_{\min} = C ? V_{L} ? \frac{P_{image}}{P_{total}} & (1) \end{matrix}$ $\begin{matrix} N_{\min} ? 8 & (2) \end{matrix}$ [0165] wherein, in the formula (1), C represents the concentration of the particles in the test liquid and it is at least 5?10.sup.7 particles/mL; V.sub.L represents an amount of the test liquid and is at least 5 ?L; P.sub.total represents the number of pixels of the image pickup element and is at least 300,000 pixels; and P.sub.image represents the number of pixels of a pixel group used for imaging one of the wells in the image pickup element and is at least 9 pixels (3?3 pixels).

[0166] The well array can be formed by applying the above matters described in the biological substance detection method and an overlapping description is omitted.

(Detection Device)

[0167] The detection device of the present invention has the detection chip on which the well array of the present invention is placed and the detection unit having the image pickup element with at least 300,000 pixels.

[0168] According to the detection device, all the particles are stored in the well to keep a detection sensitivity, and, in addition, by using the test liquid prepared to have a high particle concentration, a reaction time necessary for trapping the biological substance is reduced and thereby high-speed detection of the biological substance can be achieved.

[0169] The detection device can be formed by applying the above matters described in the biological substance detection method and an overlapping description is omitted.

EXAMPLES

[0170] In order to confirm the validity of the biological substance detection method of the present invention, the following detection test was conducted with an influenza virus (biological substance) as a detection target.

[0171] In the detection test, a well array was used in which 155?155 wells, total 24,025 wells, each well having a square opening shape with 10 ?m on each side, a depth D of 5 ?m, and a volume of 500 fL, were formed with a side wall thickness T of 3 ?m on a silicon substrate. The well array was prepared by a known shape processing method using photolithography and dry etching.

[0172] As a particle for trapping influenza viruses, 1 ?m diameter magnetic particles having a solid-phased anti-hemagglutinin antibody were used.

[0173] As a reagent for detecting the influenza viruses, (4-methylumbelliferyl)-?-D-N-acetylneuraminic acid (MUNANA, (produced by Toronto Research Chemicals, M334200)), a reagent for producing a fluorescent substance by an enzyme reaction, was used.

[0174] As a camera for observing the well array, a CMOS camera with 2,048?2,048 pixels (4,194,304 pixels, P.sub.total) (ORCA-Flash4.0 V3, manufactured by Hamamatsu Photonics K.K.) was used.

[0175] The following is the details of the detection method. First, 15 ?L of a specimen liquid containing the influenza viruses was mixed with 15 ?L of a magnetic particle liquid containing the magnetic particles at a concentration of 5?10.sup.8 particles/mL, and the resulting mixture was left to stand at room temperature for 10 minutes to cause reaction therebetween so as to bind the influenza viruses to the magnetic particles.

[0176] Then, 30 ?L of a MUNANA solution having a concentration of 1 mM was added and mixed with the reaction product. A 50 ?L portion of the resulting mixture was collected and introduced into the well array. The concentration (C) of the magnetic particles in the mixture (the test liquid, V.sub.L=50 ?L) collected and introduced into the well array was 1.25?10.sup.8 particles/mL.

[0177] Then, the magnetic particles were drawn into the well array by using a magnet, and the well array was sealed with a fluorine oil and thereafter covered with a cover glass. After that, the well array was observed with a fluorescence microscope (BXFM, manufactured by OLYMPUS CORPORATION).

[0178] The observation was performed under the conditions where a 4? objective lens was used and the wells were each observed by 64 pixels (8?8 pixels, P.sub.image) of the CMOS camera.

[0179] In the well array, a fluorescent substance 4-methylumbelliferone was formed by the enzyme reaction between the influenza viruses and the MUNANA introduced into the wells. In fact, an increase of luminescence due to the enzyme reaction was observed in the wells containing the influenza viruses and the wells were detected as luminescent wells.

[0180] The number of the luminescent wells for 20 minutes of an enzyme reaction time was counted and it was normalized with the number of the wells used for the observation. The value thus obtained was used as a measurement value corresponding to the concentration of the influenza viruses.

[0181] The results of the present test are shown in FIG. 6.

[0182] As shown in FIG. 6, the measurement value of the number of the luminescent wells correlates to the concentration of the influenza viruses and an increasing tendency with an increase in the concentration of the influenza viruses is observed. This means that the presence of the influenza viruses and its concentration can be detected from the number of the luminescent wells.

[0183] It is to be noted that the lower detection limit determined by ?+3.3? (shown by a dotted line in FIG. 6) wherein R is an average number of the luminescent wells measured using a sample not containing the influenza viruses and ? is a standard deviation is 1?10.sup.2 copies/mL.

[0184] The trapping time by the magnetic particles was 10 minutes and the measurement time including the enzyme reaction time (20 minutes) was about 30 minutes.

[0185] The lower detection limit is remarkably small and the measurement time is remarkably short so that this method has performance exceeding that of the PCR method widely used now as a high-sensitivity virus detection method.

[0186] In the present test, the value N.sub.min determined from the formula (1) is about 95 and the average storage number of the magnetic particles in the wells is almost 260, which satisfy the condition of N.sub.min specified by the formula (2). In short, according to the present invention, high-speed and high-sensitivity biological substance detection is realized.

DESCRIPTION OF REFERENCE NUMERALS

[0187] 1: well [0188] 1: well array [0189] 2: detection chip [0190] 3: magnetic field application unit [0191] 4: detection unit [0192] 5: test liquid [0193] 6: magnetic particles [0194] 7: transparent glass plate [0195] 10: detection device [0196] L: color development

BIOLOGICAL SUBSTANCE DETECTION METHOD USING WELL ARRAY AND PARTICLES, WELL ARRAY, AND DETECTION DEVICE

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G01N21/6452

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B01L2200/0652

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G01N2469/10

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B01L2300/0829

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B01L3/5085

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B01L2200/16

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G01N33/18

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G01N33/487

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G01N21/78

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G01N33/493

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G01N33/49

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G01N33/56983

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G01N2021/1765

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G01N33/54386

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G01N2201/04

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B01L2300/0654

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C12M23/12

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G01N15/0612

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G01N33/54326

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G01N33/569

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B01L3/00

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Abstract

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Description