Multiwell plate

Abstract

To stably perform bioluminescence measurement and prevent crosstalk.SOLUTION: A multiwell plate (1) according to the present invention comprises a plate outer wall (10) and a plurality of independent wells arranged in a two-dimensional array, wherein the plurality of wells include: a substrate input well (11) having a wall portion (11w) that is lower in height than the plate outer wall (10); and a sample input well (12) having a wall portion (12w) that is equal in height to the plate outer wall (10), with a notch (12a) being provided in the wall portion (12w).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a multi-well plate. [Background technology]

[0002] Bioluminescence measurement is used as a means of measuring gene expression in cells. Bioluminescence measurement is a measurement method that can analyze gene expression in real time as luminescence intensity while maintaining the viability of the cells. For example, a method is known in which a luminescent substrate is added to a cell sample introduced with a luminescent gene using a multi-well plate.

[0003] In such a measurement method using a multi-well plate, crosstalk between adjacent wells becomes a problem. Crosstalk occurs when strong light leaks into adjacent wells on the top, bottom, left and right, and as a result, the amount of luminescence measured is added up. As a technique to deal with such a problem, for example, Patent Document 1 describes a sample holding member having a filter member fixed to the microplate at a predetermined position through which luminescence from the sample held in the well of the microplate passes. In addition, an example is described in which a microplate formed of a material that is opaque to luminescence from the sample, such as a black material, is used. In addition, Patent Document 2 describes a luminescence reaction measurement method in which a target component in a measurement sample is detected sequentially by luminescence reaction, characterized in that a luminescence stopper is added to a container after measurement to stop the luminescence, and then the luminescence of the next container is measured. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2007-298380 A [Patent Document 2] Japanese Patent Application Publication No. 10-318929 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the techniques described in the above-mentioned patent documents, for example, in Patent Document 1, there is a concern that increasing the opacity of the wells makes it difficult to detect weak light, and that luminescence cannot be detected when measuring a sample with low luminescence intensity. Also, in Patent Document 2, there are many components such as containers, tubes, and pumps required for adding the substrate and luminescence stopping agent, and in order to prevent crosstalk, each well is measured while repeatedly starting and stopping the luminescence, which results in a problem of long measurement time and high cost.

[0006] In addition, when a volatile substance is used as the luminescent substrate, the entire upper surface of the multi-well plate needs to be covered and sealed with a film to prevent diffusion of the substrate. When a commercially available multi-well plate is used for such bioluminescence measurement, the wells containing cell samples are sealed with a film because the height of the well outer wall is the same as that of the multi-well plate outer wall, and the luminescent substrate may not reach the cell sample and may not emit light. On the other hand, if the height of the well wall of the multi-well plate is adjusted by processing such as cutting the top end of the well, the luminescent substrate may reach the cell sample and emit light, but the above-mentioned crosstalk occurs, making it difficult to measure the exact amount of luminescence. It is presumed that the same problem occurs with the microplate of Patent Document 1. In Patent Document 2, the plate cannot be covered with a film because a luminescence stopper is added each time.

[0007] In view of the above problems, an object of one aspect of the present invention is to realize a technique that can stably and efficiently measure bioluminescence and further suppress crosstalk during measurement. [Means for solving the problem]

[0008] In order to solve the above problems, one embodiment of the multiwell plate of the present invention is a multiwell plate used for bioluminescence measurement, which comprises an outer plate wall and a plurality of independent wells arranged in a two-dimensional array, the plurality of wells including a substrate input well having a wall portion of a lower height than the outer plate wall, and a sample input well having a wall portion of the same height as the outer plate wall and with a notch provided in the wall. Effect of the Invention

[0009] According to one aspect of the present invention, bioluminescence measurement can be performed stably and efficiently, and crosstalk during measurement can be suppressed. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a perspective view of a multi-well plate according to an embodiment of the present invention. [Diagram 2] FIG. 2 is a top view of a multiwell plate according to an embodiment of the present invention. [Diagram 3] FIG. 2 is a partial enlarged view of the top surface of a multiwell plate according to an embodiment of the present invention. [Figure 4] 3A and 3B are cross-sectional views of a multiwell plate according to an embodiment of the present invention, where (A) is a cross-sectional view taken along line AA in FIG. 2, and (B) is a cross-sectional view taken along line BB in FIG. [Diagram 5] FIG. 2 is a graph showing the change over time in luminescence value of cyanobacteria in an example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, one embodiment of the present invention will be described in detail with reference to Figures 1 to 4. In this embodiment, a 96-well plate in which a plurality of cylindrical wells are arranged at equal intervals vertically and horizontally will be described as an example, but the present invention is not limited thereto.

[0012] A multiwell plate 1 according to an embodiment of the present invention is used for bioluminescence measurement. FIG. 1 is a perspective view showing an outline of the configuration of a multiwell plate according to an embodiment of the present invention. FIG. 2 is a top view of the multiwell plate 1. As shown in FIGS. 1 and 2, the multiwell plate 1 includes a plate outer wall 10 and a plurality of independent wells arranged in a two-dimensional array. As shown in FIG. 1, the plurality of wells includes a plurality of substrate input wells 11 having a wall portion with a height lower than that of the plate outer wall 10, and a plurality of sample input wells 12 having a wall portion with the same height as the plate outer wall 10 and having a notch 12a in the wall portion. The plurality of wells in the multiwell plate 1 also includes a plurality of normal unprocessed wells 13 whose walls are not processed.

[0013] In this specification, the term "multiple independent wells" refers to a well shape in which there is a gap 14 between each well in the upper part of the plate. Note that in this specification, the "upper part" refers to the upper surface side of the plate.

[0014] As used herein, a "sample" refers to a cell into which a luminescence gene that codes for an enzyme (protein) that emits light in the presence of a substrate, such as luciferase, has been introduced, for example, a cell into which a gene cassette with an arbitrary promoter sequence connected upstream of the luminescence gene has been introduced. When the luminescence gene is expressed in the cell and the corresponding substrate is present, luminescence can occur.

[0015] In the present specification, the term "substrate" refers to a substrate that causes the enzyme to emit luminescence. In the present specification, the substrate is selected to be volatile depending on the measurement method to be adopted.

[0016] <Plate exterior wall> The plate outer wall 10 is the part that forms the outer frame that surrounds the entire outside of the multiwell plate 1. In a normal multiwell plate, the height of the outer frame is such that the upper end of the outer frame is on the same plane as the upper ends of each well. In the case of the plate outer wall 10 of the multiwell plate 1, the upper end of the plate outer wall 10 is also at the same height as the upper ends of each well or at a higher position. Therefore, when the upper surface of the multiwell plate 1 is covered with a film or the like, it is possible to seal the inside of the plate and prevent the volatile substrate from diffusing outside the plate.

[0017] <Sample insertion well> The sample input well 12 will be described in more detail below with reference to Figures 1 and 3. Figure 3 is an enlarged view of part C in Figure 2, showing the sample input well 12 on an enlarged scale.

[0018] As shown in FIGS. 1 and 3, the sample introduction well 12 is provided with a notch 12a. The notch 12a is provided in a part of the wall 12w of the sample introduction well 12, and in this embodiment, is provided at the upper end of the wall 12w. In this embodiment, the position of the notch 12a in the wall 12w is a position where, when the multi-well plate 1 is viewed from above, the light 12b from the center of the sample introduction well 12 passing through the notch 12a does not pass through any well adjacent to the sample introduction well 12. For example, the notch 12a is provided on the wall 12w at a position 30° to 60° clockwise from a virtual reference line L extending from the center of the sample introduction well 12 toward the center of any adjacent well, so that the entire notch 12a fits therein. 3, θ1 and θ2 indicate the angles that one end and the other end of the cutout 12a in the circumferential direction make with the virtual reference line L, respectively, and here, 30°≦θ1<θ2≦60°. In one embodiment, both ends of the cutout 12a in the circumferential direction are provided at positions of 30° and 60°. In other words, in one embodiment, θ1=30°, θ2=60°.

[0019] Here, the "adjacent well" refers to the well closest to the sample input well 12. Therefore, when multiple wells are arranged at equal intervals in the vertical and horizontal directions as in the multi-well plate 1 of this embodiment, there are four adjacent wells for any sample input well 12. Therefore, there are four "positions where the light 12b from the center of the sample input well 12 passing through the notch 12a does not pass through any well adjacent to the sample input well 12" and "positions at 30° to 60° clockwise from the virtual reference line L from the center of the sample input well 12 toward the center of any adjacent well", but the notch 12a may be provided in only one of them, or in two, three, or four of them. In addition, when there are multiple sample input wells 12, the directions in which the notches 12a are provided may all be the same or different.

[0020] The notch 12a is for allowing the volatile substrate to reach the sample input well 12 even when the upper surface of the multiwell plate 1 is covered and sealed with a film or the like. Therefore, as long as the purpose is achieved, there is no limitation on the position. However, by providing the notch 12a at the above position, the amount of light emitted by the sample input well 12 leaking into the adjacent wells can be reduced, which is effective in suppressing the influence of the light 12b on the adjacent wells. If the influence or possibility of crosstalk is small or some crosstalk is acceptable, the position of the notch 12a can be, for example, 25° to 65° or 20° to 70° clockwise from the virtual reference line L instead of 30° to 60°.

[0021] The depth of the notch 12a may be any depth that allows the substrate diffused from the substrate input well 11 to easily reach the sample in the sample input well 12, and may be, for example, in the range of 0.5 to 3 mm from the upper end of the wall, and preferably in the range of 1 to 2 mm from the upper end of the wall. The width of the notch 12a may be any width that allows the substrate diffused from the substrate input well 11 to easily reach the sample in the sample input well 12, and for example, in the case of a well with a circumferential length of 25.1 mm, the length in the circumferential direction is in the range of 0.5 to 3 mm, and preferably in the range of 1 to 2 mm.

[0022] <Substrate injection well> The substrate input wells 11 will be described with reference to Figures 1 and 4. Figure 4(A) is a cross-sectional view taken along line AA in Figure 2, and Figure 4(B) is a cross-sectional view taken along line BB in Figure 2. The arrangement pattern of the various wells in the multiwell plate 1 shown in Figure 2 is the same as that in the multiwell plate 1 shown in Figure 1. Therefore, there are no substrate input wells 11 on line AA in Figure 2, but there are substrate input wells 11 at the third, fifth, seventh, and ninth positions from the left on line BB.

[0023] As shown in FIG. 4B, the height of the wall 11w of the substrate injection well 11 is lower than the plate outer wall 10. Therefore, when the upper surface of the plate is sealed with a film or the like, a gap is generated between the upper part of the substrate injection well 11 and the film, and the substrate can diffuse to the outside of the substrate injection well 11. Therefore, the height of the wall of the substrate injection well 11 may be a height that generates a gap that allows the substrate to sufficiently diffuse from the inside of the substrate injection well 11 through the gap with the film, and is preferably, for example, 0.5 to 9 mm lower than the upper end of the plate outer wall 10. In the example shown in FIG. 4, the height of the plate outer wall 10 is 11 mm, and the wall of the substrate injection well 11 is designed to be 5.5 mm lower than the upper end of the plate outer wall 10.

[0024] On the other hand, as shown in FIG. 4(A) and FIG. 4(B), the wall portion 12w of the sample input well 12 remains at the same height as the outer wall 10 of the plate.

[0025] <Arrangement of each well> The arrangement of each well is not particularly limited and may be appropriately designed according to the purpose. In the multi-well plate 1 of this embodiment, the substrate input wells 11 and the sample input wells 12 are arranged at locations other than the outermost periphery. In other words, unprocessed wells 13 are arranged at the outermost periphery. In order to diffuse the substrate more uniformly within the multi-well plate 1, the substrate input wells 11 are arranged so that they are not adjacent to each other.

[0026] <Multi-well plate> The material of the multiwell plate 1 may be any material that does not affect the samples and substrates introduced therein and is not affected by the samples and substrates introduced therein, and may be, for example, polystyrene.

[0027] The color of the multiwell plate 1 may be, for example, white, black, or transparent, but is preferably white since it is also applicable to samples with low luminescence intensity.

[0028] The multi-well plate 1 of this embodiment is a 96-well plate in which each well is cylindrical, but the number and shape of the wells can be appropriately set according to the purpose of use. For example, a 24-well plate, a 48-well plate, or a 384-well plate can be used, and the well shape can be a rectangular parallelepiped, etc.

[0029] The method for manufacturing the multiwell plate 1 is not particularly limited, and may involve subjecting a commercially available multiwell plate to the above-mentioned processing, or may involve forming the well structure after the above-mentioned processing is performed during the manufacturing stage of the multiwell plate.

[0030] In the bioluminescence measurement in this embodiment, the expression of the gene of interest is analyzed in real time as luminescence intensity while maintaining the viability of the sample by reacting the sample with a substrate introduced into the multiwell plate 1 to generate bioluminescence. Specifically, a cell sample is introduced into at least one sample introduction well 12 in the multiwell plate 1, a substrate is introduced into at least one substrate introduction well 11, and the top surface of the multiwell plate 1 is sealed with a film or the like. The substrate volatilized from the substrate introduction well 11 diffuses into the gap 14 in the multiwell plate 1 and reaches the cell sample in the sample introduction well 12. The substrate is then taken up into the cell and undergoes an enzymatic reaction with the above-mentioned luminescence gene, thereby observing bioluminescence in the cell sample, and the luminescence intensity can be measured.

[0031] In the multiwell plate according to the present invention, when a substrate and a sample are placed in each well and the top of the plate is sealed, the low height of the substrate-introducing wells 11 creates a gap between the top of the well and the film, allowing the substrate to diffuse outside the well and throughout the sealed multiwell plate 1. Furthermore, the sample-introducing wells 12 have a notch and are the same height as the outer wall of the plate, so that the substrate can reach the sample in the well and crosstalk caused by light leaking into the peripheral wells can be suppressed at the same time. Therefore, by using the multiwell plate according to the present invention, bioluminescence measurement can be performed stably and efficiently.

[0032] <Summary> To summarise the above embodiments, the present invention can also be summarised as follows. (1) A multi-well plate used for bioluminescence measurement, comprising an outer plate wall and a plurality of independent wells arranged in a two-dimensional array, the plurality of wells including a substrate input well having a wall portion at a height lower than the outer plate wall, and a sample input well having a wall portion at the same height as the outer plate wall and a notch provided in the wall. (2) The multi-well plate described in (1), wherein the notch is positioned such that light from the center of the sample input well that passes through the notch does not pass through a well adjacent to the sample input well. (3) The multi-well plate described in (2), wherein the notch is provided on the wall portion at a position 30° to 60° clockwise from an imaginary reference line extending from the center of the sample insertion well to the center of the adjacent well. (4) A multi-well plate described in any one of (1) to (3), wherein the wells on the outermost periphery are neither the substrate input wells nor the sample input wells, and the substrate input wells are not adjacent to each other. (5) The multiwell plate according to any one of (1) to (4), which is a 96-well plate.

[0033] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. EXAMPLES

[0034] An example of luminescence measurement using the multiwell plate of the present invention will be described below.

[0035] <Preparation of luminescent sample> In this example, a recombinant cyanobacterium, Synechocystis sp. PCC 6803, was used as the luminescent sample. aqpz A cyanobacterium (hereafter referred to as P aqpz Two types of cyanobacteria were used: one with no promoter upstream of the luminescence gene (hereafter referred to as "vector cyanobacteria") as a negative control, and the other with no promoter upstream of the luminescence gene (hereafter referred to as "vector cyanobacteria"). The above two types of cyanobacteria recombinants were cultured in liquid and spread on BG11 agar medium containing the nitrogen source NaNO3, and exposed to white light (50 μmol of photons m -2 s-1 ) and cultured at 30°C for 5 days to obtain cyanobacterial colonies.

[0036] <Well plate processing> In this embodiment, a multi-well plate was used that had been processed from a 96-well plate (CulturPlate-96, white, manufactured by PerkinElmer). Specifically, for some wells in the multi-well plate, a notch with a width of 1 to 2 mm was provided at a 45° angle from the adjacent well located above the well to the adjacent well located to the right of the well (hereinafter, this well is also referred to as a "notched well"). In addition, for some other wells, the wall was cut to half its height to lower the height of the wall (hereinafter, this well is also referred to as a "low-wall well"). The arrangement pattern of the notched wells and low-wall wells in the plate that had been processed as described above was the same as that shown in FIG. 1. Hereinafter, the plate that had been processed as described above is also referred to as the "plate of this embodiment."

[0037] Example 1: aqpz Bioluminescence measurement of cyanobacteria and Vector cyanobacteria After the above incubation, P aqpz One colony each of the cyanobacteria and Vector cyanobacteria was punched out into six cut-out wells using a glass tube. In addition, 20 μL of luminescent substrate (1% n-decanal) was added to all bottom-walled wells. After adding the cyanobacteria and luminescent substrate, the top of the 96-well plate was sealed with a gas-impermeable transparent film (TopSeal-A PLUS, manufactured by PerkinElmer). After sealing, the luminescence was measured continuously for 5 days at 30°C and constant light conditions of 3000 lux after 12 hours of dark adaptation using a highly sensitive bioluminescence measuring device (CL96, manufactured by Churitsu Electric Co., Ltd.). The luminescence value was measured for 3 seconds every 30 minutes after a dark period of 180 seconds.

[0038] <Controlled Test> As a control test, a similar 96-well plate (PerkinElmer CulturPlate-96, white) was prepared in which the walls of all wells were cut to half their height (hereinafter referred to as the "all-well low-wall plate"), and a plate without any cutting or wall scraping (hereinafter referred to as the "untreated plate"). aqpz A single colony of cyanobacteria was added to each well (18 wells on low-walled plates, 15 wells on untreated plates) or a total of 20 μL of luminescent substrate (1% n-decanal) was added, and the top of the plate was sealed with film under the same conditions as above, and measurements were performed using a high-sensitivity bioluminescence measuring device.

[0039] Figure 5 shows the change over time in the luminescence intensity of the cyanobacteria due to the processing of the wells. In Figure 5, the vertical axis shows the luminescence intensity per colony, and the horizontal axis shows the actual time after switching to constant light conditions. The data show the average value ± SE of six independent samples. aqpz Please refer to the fact that luminescence measurements were performed on similar cyanobacteria into which AqpZ was introduced and it was confirmed that luminescence was observed (Akai et al., Aquaporin AqpZ Is Involved in Cell Volume Regulation and Sensitivity to Osmotic Stress in Synechocystis sp. Strain PCC 6803, Journal of Bacteriology, 194(24): 6828-6836, 2012).

[0040] When using the wells of a non-treated plate, P aqpz The results of the measurement of blue-green algae (in Fig. 5, "P aqpz The luminescence value of the Vector cyanobacteria (in FIG. 5, "Vector, after improvement (plate of the present invention, n=6)") was equivalent to that of the Vector cyanobacteria (in FIG. 5, "Vector, after improvement (plate of the present invention, n=6)"). In other words, when the wells of the untreated plate were used, it was found that the cells did not emit light. aqpzThe wells containing the algae and luminescent substrate are sealed with a film, so the luminescent substrate is absorbed by the P aqpz It is thought that it did not reach the cyanobacteria.

[0041] On the other hand, when the plate of this embodiment is used, P aqpz The luminescence from the nucleus was detected. aqpz After improvement (plate of the present invention, n=6)). In the plate of this example, the luminescent substrate was P aqpz It was determined that the light had diffused to the cyanobacteria and emitted light.

[0042] Example 2: Confirmation of Crosstalk Occurrence The occurrence of crosstalk depending on whether or not the well plate was processed was compared using the method described below.

[0043] First, in addition to the unprocessed plate, all-well low-wall plate, and plate of this embodiment, a 96-well plate (PerkinElmer CulturPlate-96, white) was prepared in which all wells had a notch of the same width on the left and right sides as the plate of this embodiment (hereinafter, also referred to as "well left and right notch plate"). For the wells in each plate, the coordinates A to H were assigned to the rows and 1 to 12 to the columns, and the 4 rows x 3 columns of B2 to B4, C2 to C4, D2 to D4, and E2 to E4 sections were used.

[0044] The luminescence source used was the luminescence reagent contained in Lucipack Pen-AQUA (Kikkoman Biochemifa Corporation). Specifically, the entire amount of luminescence reagent in one Lucipack Pen-AQUA kit was taken out and dissolved in 10 mL of water to prepare a solution, and 100 μL of the solution was added to the C3 well of each plate. The wells were made to emit light by adding 10 μL of 0.25 μM ATP solution, and the luminescence values ​​immediately after the addition of the ATP solution were compared between each plate. A high-sensitivity bioluminescence measuring device (CL96, manufactured by Churitsu Electric Co., Ltd.) was used to measure the luminescence value, and the ATP solution was added immediately before the measurement.

[0045] Table 1 shows the average value of three measurements. In the unprocessed plate, the luminescence values ​​in all wells not containing a luminescent sample, except for coordinate C3, were 207 to 593 cps. On the other hand, in the plate of this example, the luminescence values ​​in wells other than coordinate C3 were 278 to 641 cps. In other words, in the plate of this example, it was recognized that crosstalk to wells other than coordinate C3 was suppressed to the same level as in the unprocessed plate.

[0046] In contrast, in the all-well low-wall plate, the luminescence values ​​in the wells adjacent to the coordinate C3 (B3, C2, C4, D3) were 671 to 1568 cps, and an increase in the luminescence value was observed. In addition, in the all-well left-right notch plate, the luminescence values ​​in the wells adjacent to the coordinate C3 (C2, C4) were 1080 and 1222 cps, and an increase in the luminescence value was observed. In other words, when there is a space such as a notch in the position where the light emitted from the center of the well C3 passes through the adjacent wells, crosstalk was observed to occur in the adjacent wells. [Table 1] [Industrial Applicability]

[0047] The present invention can be used to measure gene expression in cells. [Explanation of symbols]

[0048] 1 multi-well plate 10 Plate exterior wall 11 Substrate injection well 11w Substrate injection well wall 12 Sample input wells 12a Notch 12w Sample well wall 13 Raw Wells 14 void

Claims

1. Plate outer wall; a plurality of independent wells arranged in a two-dimensional array; A multi-well plate used for bioluminescence measurement, wherein the multiple wells include a substrate input well having a wall portion at a height lower than the outer wall of the plate, and a sample input well having a wall portion at the same height as the outer wall of the plate and a notch provided in the wall.

2. 2. The multi-well plate of claim 1, wherein the notch is positioned such that light from the center of the sample input well that passes through the notch does not pass through a well adjacent to the sample input well.

3. The multi-well plate of claim 2, wherein the notch is provided on the wall portion at a position 30° to 60° clockwise from a virtual reference line extending from the center of the sample insertion well to the center of any adjacent well.

4. The outermost wells are neither the substrate input wells nor the sample input wells, The multi-well plate of claim 1 , wherein the substrate input wells are not adjacent to each other.

5. The multi-well plate according to any one of claims 1 to 4, which is a 96-well plate.

Images