Patent Application
20170260573
The present invention relates to the analysis of a biomolecule, specifically, a spot array substrate for decoding the base sequence of a nucleic acid polymer at high efficiency and high accuracy, a method for producing the same, and a nucleic acid polymer analysis system using the spot array substrate.
Patent Literature 1 describes a method for using a DNA chip to detect the expression of conventionally known genes, and describes that a transparent resin (polycarbonate, polypropylene and cycloolefin-based polymer) having little self-fluorescence is used to detect fluorescent light indicating the presence of a gene. Patent Literature 1 describes that a plurality of concave portions are formed (arrayed) on the substrate surface as the array spot, that the substrate's bottom surface is desirably flat, and that the detection of the target DNA can be performed by immobilizing an oligo DNA on the inner surface of the concave portion, immobilizing a conventionally known probe DNA in advance on the bottom surface of a concave portion, and adding a solution filled with the sample which is the object for analysis.
Patent Literature 2 describes a method for using a microarray to detect the expression of conventionally known genes, and describes that a transparent resin (cycloolefin-based polymer) having little self-fluorescence is used to detect fluorescence indicating the presence of a gene. Patent Literature 2 describes the method for immobilizing an oligo DNA on a substrate as a well-known method in the field, and describes that the presence or absence of expression can be determined by hybridizing with the target DNA on a substrate on which a conventionally known probe DNA is immobilized on an array spot in advance.
Patent Literature 3 describes a method for using a microarray to detect the expression of conventionally known genes, and describes that a functional group can be used in order to immobilize an oligo DNA on a substrate and that a polymer having chemical and thermal stability, low fluorescence and optical stability, preferably a cycloolefin polymer, and preferably Zeonex(R) or Zeonor(R) is used in the substrate.
Patent Literature 4 describes a method for using a microarray to detect the expression of a conventionally known gene, and describes hybridization with the target DNA on a substrate on which a conventionally known probe DNA is immobilized on the array spot in advance, and use of a substrate including a multi-well (namely, the concave portion of the array) cycloolefin polymer when performing fluorescence detection with the substrate.
Patent Literature 5 describes a method for using an Si substrate which forms a highly integrated array of the concave portion having an electrode on the bottom surface by a semiconductor process to analyze a nucleic acid polymer. Patent Literature 5 describes that the base sequence can be decoded by loading the beads which form the replicas of the template DNAs derived from the object for analysis on the surface in advance in the concave portion which is formed as an array on the substrate surface, and detecting the ions emitted from the replica of these template DNAs due to the reaction with a reagent with an electrode provided on the bottom surface of the concave portion.
Patent Literature 1: JP 2009-60859 A
Patent Literature 2: JP 2009-513137 A (US 2007/0099222 A1)
Patent Literature 3: JP 2009-537126 A
Patent Literature 4: JP 2004-500867 (US 2003/0003496 A1)
Patent Literature 5: JP 2010-513869 A
Various methods such as a method for optically measuring and discriminating bases and a method for electrically measuring and discriminating bases have been examined as methods for analyzing nucleic acid polymers such as DNA and RNA. As one method, a fluorescence system DNA sequencer decodes the bases by analyzing the fluorescence generated when reacting a reagent including a fluorescent dye and an enzyme with a DNA. It is anticipated that studying and elucidating the causal relationship between nucleotide sequence information and pathology revealed by such nucleic acid polymer analysis techniques will expand the applications to medical diagnosis. However, presently, methods are mostly used in research applications and the cost of one analysis is also high. It is furthermore desirable to greatly reduce the analysis cost in order for the method to spread widely as a medical diagnostic technology in the near future. Meanwhile, improvements for obtaining a large amount of data from a small number of samples in a high throughput, and improvements for reducing (cost reduction including consumable parts for analysis, reagents, etc.) the analysis cost necessary to obtain the same amount of data are continuously pursued.
It is necessary to improve the data acquisition efficiency in order to increase the reduction of the amount of the sample, the miniaturization of the analysis cell, and the volume of data acquired. Conventionally, a fluorescence system DNA sequencer measures the fluorescence generated by the reaction in a state in which the object for analysis is randomly scattered in a plane, and practical application has started in the form which discriminates the bases from the data analysis of a fluorescence bright spot of random. positions. However, because the analysis was random, the region in which the object for analysis might not be present and the region in which the analysis was not possible due to the overlapping and mixing of the fluorescence from an adjacent object for analysis existed, and the usage efficiency in the region on the substrate of a flow cell where the reaction event occurs was low. As a result, the sample which was the object for analysis which did not contribute to the analysis was generated, and the area of the flow cell for analysis had to be expanded in order to separate from the events of a nearby region to obtain a sufficient amount of data.
An integrated array has been considered as a promising method for solving this problem. It is one general concept for highly efficiently executing the task in the smallest area possible. It is possible to eliminate interference with an adjacent fluorescence event and maximize the volume of data acquired per unit substrate area by maintaining the spot in which the object for analysis can be immobilized on the substrate of the flow cell at a pitch which is a specific interval or more from an adjacent spot and arranging discretely and as closely as possible.
However, in order to form a highly integrated spot array on the substrate of the flow cell, it is necessary to use some fine patterning technology such as a semiconductor process like lithography and dry etching micro-machining or drawing and machining by a beam in the production of the substrate. Needless to say, if the semiconductor process which can form various patterns on an Si wafer is used, it is possible to mass-produce the substrate on which a highly integrated spot array is formed, but a considerable increase of the cost of producing the substrate is inevitable compared to flat substrates which have been used in conventional analysis of random fluorescence. A production technique which will greatly reduce the substrate material cost and the cost of producing the spot array is desired.
This technique is different from a DNA sequencer for decoding an unknown base sequence, but a DNA chip (microarray) may be provided as the technology for determining the presence or absence of a conventionally known base sequence. However, this is limited to the application for determining the presence or absence of the same alignment as a conventionally known probe DNA, thus, the number of array spots placed on the chip is different from the situation in which the DNA sequence for which throughput is sought by requiring nothing more than the number of objects for analysis and acquiring as much data as possible by increasing in parallel the degree of integration of the array as much as possible. Therefore, presently, the degree of integration of DNA chips is not so high, and many are measured with an array spot having large dimensions of up to a level of several 100 to several 10 μm. The spot region is arranged in an array for examining the target DNA which is the object for analysis, and the respective spot regions are subjected to functional group modification, the immobilization of the oligo DNA, the immobilization of the probe DNA, etc. However, since the dimensions are large, the spot region can be produced without patterning at the micronization level (several μm to several 100 nm level) which uses a high cost semiconductor process. Further, if the self-fluorescence of the substrate material that interferes with the fluorescence measurement is sufficiently low, there are few other restrictions regarding changing the substrate material. Therefore, cost reductions such as the application of a resin substrate have been made for DNA chips. Patent Literatures 1 to 4 disclose the formation of DNA chips which use a resin substrate having low self-fluorescence in the DNA chip to form functional groups and a spot such as oligo DNA in an array shape on the surface.
Meanwhile, in a DNA sequencer, it will be necessary in the future to continuously acquire unknown sequence data in large quantities and at high throughput, thus, even higher integration is necessary. Therefore, it is necessary to form a spot array at a dimension level of several micrometers to several μm to several 100 nm which is at least one order of magnitude higher than the chip. Presently, the high integration (pitch) of the spot array has a limit due to constraints such as the pitch of the pixels of the photodetector array which detects the fluorescence from the array spot, and thus, the integration remains at the current range of micronization, but it is necessary to acquire unknown sequence data in large quantities and at high throughput, thus, even higher integration will be sought in the future. However, when using a semiconductor process to form a highly integrated array spot at a dimension level of several μm to several 100 nm, a high cost is involved in the production process, thus, for example, the reduction of the total cost of production is insufficient even if a resin substrate is used in order to keep the material cost low. Even though the resin substrate material itself is equivalent to or higher than a material such as quartz and Si in terms of coat and optical performance (low self-fluorescence, etc.), in a DNA sequencer which must improve the data acquisition efficiency by further micronization and higher integration, if a production cost is required in the formation of a highly integrated array, for example, the reduction effect of the final analysis cost will be diminished even if the data acquisition efficiency improves due to higher integration.
However, the application of a highly integrated array has been spreading not only in the analysis of nucleic acid polymers by optical measurement but also in the analysis by electric measurement. Patent Literature 5 discloses the formation of an Si substrate on which a highly integrated array is formed by the semiconductor process in the concave portion having an electrode on the bottom surface, and a method for loading the beads which formed the replica of the template DNA derived from the object for analysis on the surface in advance in each concave portion of the Si substrate surface and electrically measuring and decoding the base sequence. However, in this method, it is necessary to provide a fine electrode for detecting the signals in the concave portion, wiring for connecting thereto, and switches in the substrate to be used, and thus, the substrate is more complicated than the one used in optical measurements. Therefore, the production using the resin substrate having a low material cost is not easy, and all of the substrates must be produced in a high cost semiconductor process.
As stated above, in a DNA sequencer which requires further micronization and higher integration, even with a low cost substrate material, or, even with either optical measurement or electric measurement, as long as a semiconductor process such as lithography or dry etching micro-machining is used to produce all of the highly integrated array substrates, there is a limit to the reduction of the cost of production. It is desired to establish a large-scale production technique without using the semiconductor process in the production of all of the substrates.
The spot array substrate according to the present invention includes a resin substrate having a surface on which an uneven pattern is formed, a plurality of bead sitting positions set in a two-dimensional array within the uneven pattern, and surface-modified beads loaded onto the bead sitting positions of the resin substrate.
As an example, the uneven pattern is a pattern in which the adjacent bead sitting positions are connected with a groove having a width narrower than the dimension of the bead sitting positions.
The surface-modified beads are immobilized onto the bead seating positions of the resin substrate by physical fitting or a chemical bond or both.
The surface-modified beads may be the functional group surface-modified beads or oligo DNA surface-modified beads. Further, the surface-modified beads may be the beads which form in advance the replica of the template DNA derived from the object for analysis on the oligo DNA surface-modified beads.
The surface density of the bead sitting positions, as an example, is preferably 6.6×106/cm2 to 180×106/cm2.
The production method of the spot array substrate according to the present invention includes a step of preparing a resin substrate having a surface on which an uneven pattern is formed and a plurality of bead sitting positions set in a two-dimensional array within the uneven pattern, and a step of loading the surface-modified beads onto the bead sitting positions of the resin substrate.
A centrifugal force or a magnetic force can be used in the step of loading the surface-modified beads onto the bead sitting positions of the resin substrate.
A spot array substrate production kit of the present invention is provided with the resin substrate having a surface on which an uneven pattern is formed, a plurality of bead sitting positions set in a two-dimensional array within the uneven pattern, and surface-modified beads loaded onto the bead sitting positions of the resin substrate. The spot array substrate can be prepared by loading the surface-modified beads such as the functional group surface-modified beads or the oligo DNA surface-modified beads onto the bead sitting positions of the resin substrate. Further, if the beads which form the replica of the template DNA derived from the object for analysis in advance on the beads subjected to surface modification such as the oligo DNA are used as the surface-modified beads, for example, the user of the nucleic acid polymer analysis device can prepare the spot array substrate by loading these beads onto the bead sitting positions after forming the replica of the template DNA on the surface of the oligo DNA surface-modified beads included in the kit by a method such as emulsion PCR.
The nucleic acid polymer analysis device of the present invention is provided with a flow cell in which the aforementioned spot array substrate is incorporated, a reagent supply unit for selectively supplying to the flow cell a plurality of reagents including a reaction reagent including fluorescent dyes which can discriminate the bases of a DNA molecule, a reaction reagent which can cut a dye from dNTP, and a washing reagent for washing inside the flow cell, a temperature control unit for controlling the temperature of the flow cell, a light source for illuminating the excitation light on the spot array substrate, a photodetector array for measuring the fluorescence generated from each bead sitting position of the spot array substrate, and an analyzing device for analyzing a detection signal from the photodetector array.
The analysis method of the nucleic acid polymer according to the present invention includes, as an example, a first step of immobilizing template DNA beads prepared by replicating, on a surface in advance, template DNA derived from a sample which is the object for analysis on the functional group surface-modified beads of the aforementioned spot array substrate, a second step in which the reagent including the fluorescent dye performs an elongation reaction of one base of the template DNA on the template DNA beads immobilized on the functional group surface-modified beads, a third step of measuring optical signals generated from the bead sitting positions of the spot array substrate by illuminating excitation light after the reaction, a fourth step of cleaving the fluorescent dye from the bases in which the optical signals are measured, a fifth step of washing the solution including the cleaved fluorescent dye, and a sixth step of analyzing the optical signals obtained by the third step to determine the base sequence of the template DNA.
The analysis method of the nucleic acid polymer according to the present invention includes, as an example, a first step of loading template DNA beads prepared by replicating, on a surface in advance, the template DNA derived from a sample which is the object for analysis in the bead sitting positions of the resin substrate having the aforementioned surface on which an uneven pattern is formed, and a plurality of bead sitting positions set in a two-dimensional array within the uneven pattern, a second step in which the reagent including the fluorescent dye performs an elongation reaction of one base of the template DNA on the template DNA beads loaded onto the bead sitting positions, a third step of measuring the optical signals generated from the bead sitting positions of the spot array substrate by illuminating the excitation light after the reaction, a fourth step of cleaving the fluorescent dye from the bases in which the optical signals are measured, a fifth step of washing the solution including the cleaved fluorescent dye, and a sixth step of analyzing the optical signals obtained in the third step to determine the base sequence of the template DNA.
The analysis method of the nucleic acid polymer according to the present invention includes, as an example, a first step of immobilizing the template DNA derived from a sample which is the object for analysis on the oligo DNA surface-modified beads of the aforementioned spot array substrate, a second step of replicating the template DNA immobilized on the oligo DNA surface-modified beads, a third step in which the reagent including the fluorescent dye performs an elongation reaction of one base of the template DNA on the oligo DNA surface-modified beads, a fourth step of measuring the optical signals generated from the bead sitting positions of the spot array substrate by illuminating the excitation light after the reaction, a fifth step of cleaving the fluorescent dye from the bases in which the optical signals are measured, a sixth step of washing the solution including the cleaved fluorescent dye, and a seventh step of analyzing the optical signals obtained in the fourth step to determine the base sequence of the template DNA.
According to the present invention, a fine structure formation at a dimension level of several μm to several 100 nm is possible, and, the spot array substrate (or a kit for preparing the spot array substrate) made by patterning the material (functional group spot, oligo DNA spot, etc.) of the array spot to an array shape can be mass-produced at a low cost. Further, nucleic acid polymer analysis at a low consumables cost can be realized by using a substrate (or, a kit for preparing a substrate) produced in this manner.
Other problems, configurations, and effects will become apparent from the following description of embodiments.
For the purpose of summarizing the conventional array substrate production technique,
However, in such a production method, it was necessary to use an Si substrate for the semiconductor process in the respective substrate materials, and it was necessary to subject all of the wafers to a treatment by a semiconductor process such as lithography, plasma treatment or dry etching, or vapor deposition. Thus, the reduction of the cost of production was limited. Further, other than the production step illustrated above, there are methods which can form the spot array such as processing by an ion beam and electric beam film deposition, but such a beam drawing production method becomes more costly as the number of spots increases. Thus, it cannot become a low cost mass production method compared to wafer batch processing.
The embodiments of the present invention will be explained in detail below based on the drawings. The configuration and the materials described in the embodiments serve to solely illustrate implementations of the spirit of the present invention, and are not intended to strictly specify materials, dimensions, and other such variables in any way.
A first embodiment describes a production example of the spot array substrate which uses a resin substrate to form a functional group spot array or an oligo DNA spot array.
In the present embodiment, as an example, the Ni convex mold (sub master) 203 shown in
Whether or not the beads entered the bead sitting positions formed by the concave portion was checked by an electron microscope, and the amino modified beads were loaded in 40% of all of the concave portions, but the beads were not loaded in 60% of the concave portions. In order to estimate the cause thereof, the substrate was carefully observed with an optical microscope in a state in which it was immersed in the liquid in which the beads were dispersed, and as a result, a situation was observed in which a variety of large and small air bubbles remained. It can be seen that the air bubbles interfere with the loading of the beads. Therefore, it was assumed that the fact that the air bubbles were likely to remain and the liquid was unlikely to enter the concave portion formed by the convex mold was the cause that made it difficult to fit the beads, and the two air bubble reduction measures of the change in the layout of the mold and the liquid replacement from the alcohol to the aqueous solution were implemented as measures against the above.
The mold shown in
Therefore, lastly, the resin substrate having the improved layout was immersed in the liquid 206 in which the silica surface amino group modified beads were dispersed as shown in
After running the aqueous solution of the reagent for analysis for 12 hours while repeating the temperature control (10 to 70° C.) for controlling the enzyme reaction in order to simulate the use in the subsequent analysis, the beads which entered the bead sitting positions of the intersection point of the grooves were again verified by an electron microscope. As a result, 10% of the beads fell out, and the filling rate decreased to 70% of the total. While the filling rate of the substrate of the present embodiment was slightly low, it was a production method merely for fitting the array spot material on a low-cost molded resin substrate, thus, it was confirmed that the substrate could be produced at a remarkably low cost, and the method was a superior production method.
As an example, in the present embodiment, the bulk material in which the amino group was modified was used as an example of a functional group on the surface of the silica beads as the material of the array spot. Further, an example for loading the beads in the concave portion of the resin substrate by a centrifuge (or a rotary machine similar thereto) has been described. However, it goes without saying that the present embodiment may also be applied to the surface-modified beads in other functional groups, and, the bulk material of the beads is not limited to silica.
The amino group surface-modified magnetic beads 208 containing a magnetic material can be used as, for example, the bulk material of the beads. In this case, as shown in
Further, in the description above, the detailed results such as the loading rate regarding the loading of the beads into the concave portion of the simple spot array layout (bottomed hole) as shown in
While the resin substrates shown in
Note that, the step of loading the beads which are the array spot material, using the centrifugal force and the magnetic force, is the production step of the spot array substrate and is prior preparation operation when seen from the viewpoint of the user of the analysis device. Thus, it is suitable to preferably supply, to a user, the spot array substrate with this step taken by the manufacturer of the spot array substrate. However, a spot array substrate production kit which includes a resin substrate on which an uneven pattern is formed on the surface and in which a plurality of bead sitting positions are set in a two-dimensional array within the uneven pattern, and surface-modified beads such as the functional group surface-modified beads or oligo DNA surface-modified beads which are loaded onto the bead sitting positions of the resin substrate, and a device which uses a centrifuge (or a rotary machine similar thereto) or a magnet to load the magnetic beads are supplied to the user, and the user may implement the loading step of the material of the array spot himself/herself with only a slight difference in the labor and the cost, or the base member itself is produced at a low cost. Thus, the advantage that a low cost analysis can be implemented by the user is not lost.
The filling rate of the beads is preferably high in practical application, and it is desirable to form at least 90% of the effective array spot. Therefore, the second embodiment illustrates an example for producing the chip which uses the resin substrate to form a surface-modified spot array such as the functional group spot array and the oligo DNA spot array, and at that time, improving the filling rate of the beads, and implementing measures so that the beads do not fall out during the analysis.
The mold 401 shown in
Note that, in the case where the magnetic beads are loaded not by a centrifuge but by a magnetic force, after using the device having a temperature adjusting function which uses a magnet to load magnetic beads and fitting the beads into the bead sitting positions of the uneven pattern while raising the temperature in the same sense, the application of the magnetic force may be turned off after the temperature is reduced, or the resin substrate may be lowered from the device exerting the magnetic force after the temperature is reduced.
The thermal expansion coefficient of a general resin material is in the range from 5×10−5 and half or larger to 10−4[/° C.], and the cycloolefin-based polymer used in the present embodiment also belongs to this category. Because of orders of higher magnitude compared to, for example, the thermal expansion coefficient of 10−7 of SiO2 which is the main raw material of the silica beads, easy fitting of the beads due to the thermal expansion at raised temperatures and a fixation effect by the tightening of the beads at a lowered temperature can be expected. However, even when the magnetic beads rather than the silica beads are selected, for example, the thermal expansion coefficient of iron and iron oxide is no greater than 5×10−5 and half or smaller, and is also smaller than that of the thermal expansion coefficient of the resin. By the selection of the combination of the resin material of the substrate and the material of the beads, even if details are different such as the optimal temperature conditions, the optimal dimension design of, for example, the concave portion of the uneven pattern and the beads constituting bead sitting positions, namely gap design for easy loading and making it difficult to fall out, and the optimal design of the normal tapered angle of the concave portion, if the thermal expansion coefficient of the resin substrate is higher than that of at least the beads, although the range of the effect becomes different, the improvement of the filling rate of the beads and the reduction of the falling out rate can be expected.
The specific dimensions of the expansion amount, for example, in the case of a combination of the silica beads having a diameter of 1 μm and cycloolefin-based polymers, relate to the detailed dimension design of the concave portion. If the diameter (dimension of upper part of the hole) of the inlet of the concave portion (for example, the hole) for loading the silica beads is designed to be slightly larger than 1 μm, and the diameter (dimension of lower part of the hole) of the bottom surface of the concave portion is designed to be slightly smaller than 1 μm as stated in the first embodiment, the beads become easy to load. With respect to the case of using the difference in the thermal expansion coefficient stated in the second embodiment, when the dimensions in the case of raising the temperature to 90° C. are calculated, the hole of the cycloolefin-based polymer substrate becomes approximately 5 nm larger. Meanwhile, the amount of increase of the diameter of the silica beads is two digits smaller than this, thus, it is considered to hardly change at all. Due to the 5-nm increase of the diameter of the hole, the beads are easily loaded into the hole, and fit snugly to a deeper depth. Then, by cooling the cycloolefin-based polymer, the diameter of the hole contracts by 5 nm, thus, it is expected that the beads are securely clamped after loading, and become difficult to fall out.
Whether or not the beads entered the bead sitting positions set in the intersection point of the groove was checked surrounding the rhombic pattern by an electron microscope in order to verify the effect of the improvement measures of the present embodiment. It was verified that the filling rate improved and the amino group modified beads were loaded in 92% of all the bead sitting positions. It is assumed that the resin expanded more than the beads by increasing the temperature to 90° C., and as a result, the beads became easy to fit and the loading rate of the beads improved.
Next, in the same manner as the first embodiment, after running the aqueous solution of the reagent for analysis for 12 hours while repeating the temperature control (10 to 70° C.) for controlling the enzyme reaction in order to simulate the use in the subsequent analysis, the beads which entered the bead sitting positions were again checked by an electron microscope, and as a result, it was found that the filling rate was maintained at 92% of all. It is considered that when the improvement measures were used, the beads were fit and securely immobilized, and the beads became difficult to fall out. Compared with the first embodiment, an additional temperature adjusting function is necessary in the device which uses a centrifuge or a magnet to load the magnetic beads, but the increase of the cost of production is not large. It is considered that, compared to using an Si wafer for all of the chips and producing by a semiconductor process, the effect of the addition of the temperature adjusting function to the cost increase is remarkably small.
For comparison, the material of the substrate was made from Si, and the evaluation of the loading and the falling out of the beads was performed in the same manner. As stated above, when the material of the substrate was a cycloolefin-based polymer, the dimensions of the concave portion of the bead sitting positions increased due to the temperature increase of the substrate and the beads became easy to fit. However, when Si is the material of the substrate, the thermal expansion coefficient is 10−6 and half or smaller, thus, the dimensional change of the concave portion of the bead sitting positions is small (Si hardly expands compared to resin, even if the temperature increases). Further, the dimensions of the silica beads hardly change. Therefore, it is considered that a sufficient effect to make the beads fit easily and become difficult to fall out cannot be obtained by controlling the temperature.
The procedures of the evaluation are shown in
A semiconductor process (lithography, dry etching, asking) in the same manner as when preparing the mold was used to micromachine the Si wafer, and as shown in
It was checked by an electron microscope whether or not the beads entered the bead sitting positions set at the intersection point of the grooves surrounding the rhombic pattern, and it was verified that the filling rate of the beads decreased compared to when the material of the substrate was a cycloolefin-based polymer and that the amino group modified beads were loaded into 70% of all the bead sitting positions. Further, after running the aqueous solution of the reagent for analysis for 12 hours while repeating the temperature control (10 to 70° C.) for controlling the enzyme reaction in order to simulate the use in the subsequent analysis, the beads which entered the bead sitting positions were again checked by an electron microscope, and as a result, it was found that the filling rate further decreased to 60% of all. Thus, it was found that when the substrate material was made from Si, the effect of the beads becoming easy to fit due to the thermal expansion caused by an increase in the temperature of the substrate, and the effect of the loaded beads being tightened and becoming difficult to fall out due to the decrease of the temperature of the substrate could not be obtained.
Above, in the method for producing the surface-modified spot array in which a resin substrate uses a mold to form an uneven pattern on the surface and in which a resin substrate with a plurality of bead sitting positions set in a two-dimensional array within the uneven pattern is used, it was shown that not only the material cost and the cost of production were cheaper but the property that the resin has a high thermal expansion coefficient was actively utilized to improve the loading rate of the beads by the temperature adjusting function and reduce the falling out ratio of the beads, namely, the yield of the array spot production could be improved.
Note that, in the same manner as the first embodiment, it is desirable that the spot array substrate is supplied to the user as a finished product by the manufacturer. However, a device including the spot array substrate production kit which includes the molded substrate and the beads which are the material of the array spot and a centrifuge having a temperature adjusting function (or a rotary machine similar thereto) or a temperature adjusting function which uses a magnet to load the magnetic beads is supplied to the user, and the user may implement the loading step of the material of the array spot himself/herself and produce the base member itself at a low cost. Thus, the advantage that a low cost analysis can be implemented by the user is not lost.
Next, the third embodiment used the resin substrate to produce a chip which formed the functional group spot array or an oligo DNA spot array, and especially at that time, the effect of the chemical bond at a fixed location of the beads and the effect of narrowing the particle size distribution of the beads were examined as other measures for raising the filling rate of the beads and preventing the beds from falling out during the analysis.
In the present embodiment, while there is an increase in the number of steps, the following four steps were added to form the chemical bond after producing the resin substrate with a plurality of bead sitting positions set in a two-dimensional array within the surface uneven pattern by the formation steps shown in the second embodiment.
First, as shown in
After adding these 4 steps, finally, as the evaluation of the filling rate of the beads, after the cycloolefin-based polymer substrate 511 of the rhombic pattern in which only the inner surface of the groove modified by the epoxy group was immersed in isopropyl alcohol once in the same manner as the case of
Note that, when the magnetic beads are loaded using a magnet and not a centrifuge, it suffices that the device having a temperature adjusting function for loading the magnetic beads using a magnet is used in the same manner, and after the beads are fit upon raising the temperature, the temperature is reduced and the application of the magnetic force is turned off, or, the temperature is reduced and the loaded beads are removed from the device influencing the magnetic force.
In the evaluation, therefore, it was estimated that a chemical bond would form by the amino group of the amino group modified beads which is an example of the spot array material and the epoxy group on the inside of the groove of the substrate, and it was anticipated that the filling rate of the beads would furthermore increase. In order to verify the effect of the chemical bond, whether or not the beads entered the bead sitting positions set in the intersection point of the groove surrounding the rhombic pattern was checked by an electron microscope. It was verified that the filling rate further improved, and the amino modified beads were loaded into 96% of the bead sitting positions among the entirety. It is assumed that the filling rate of the beads improved by the addition of an immobilization effect due to the chemical bond.
Next, as in the first and second embodiments, after repeating the temperature control (10 to 70° C.) for controlling the enzyme reaction while running the aqueous solution of the reagent for analysis for 12 hours in order to simulate the use in the subsequent analysis, the beads which entered the bead sitting positions were again verified by an electron microscope, and as a result, the filling rate decreased slightly, and was 94% of all of them. While a reduction of 2% was observed, a high filling rate of the beads and a high bead residual ratio (=the filling rate−the falling out rate) were obtained by addition of the immobilization effect due to the chemical bond, compared to the case without the immobilization effect. Note that, other than the epoxysilane film formed by the epoxy-based silane coupling agent, for example, an isocyanate silane film and the like formed by an isocyanate-based silane coupling agent can be used as the material which can form a chemical bond with the amino group modified beads. An examination was conducted on a trial basis in the same manner, and as a result, the beads having a filling rate of 95% which is almost the same as the case of the epoxysilane film could be obtained, and, a bead residual ratio of 94% could be obtained after a 12-hour run which simulated the analysis.
Even in the case where either functional group is used, it can be interpreted that the chemical bond increases the ability to maintain the beads in the bead sitting positions on the substrate. However, when compared with the first and second embodiments, there is an increase of the 4 steps of
Note that, also in the method which uses the effect by the chemical bond, it is desirable that the spot array substrate is supplied to the user as a finished product by the manufacturer, in the same manner as the First and Second Embodiments. Certainly, it is possible to be in a form in which the user implements the fitting step of the array spot material, and in this case, the base member itself is produced at a low cost, thus, the advantage that the analysis can be implemented at a low cost is not lost. However, in the case of the present embodiment, when supplying the set of the molded substrate, the beads which become the array spot material, and the device having a temperature adjusting function which uses the centrifuge having the temperature adjusting function and the magnet to load the magnetic beads, from the viewpoint of the protection of the functional group (in the present embodiment, the epoxy group, or, the isocyanate group) in a positon in which the beads of the formed substrate are immobilized, it is safer to supply to the user with a cover of the resist resin attached which protects the functional group on the inside of the groove. In this case, the user must first remove the resist resin with an organic solvent (acetone, etc.), and this increases the work steps and increases the complexity for the user compared to the First and Second Embodiments.
However, a specific problem was discovered when the chemical bond in the present embodiment was used. As stated above, with respect to the bead sitting positions set in the intersection point of the groove surrounding the rhombic pattern, while the results that the loading rate of the amino group modified beads was 96% and that the bead residual ratio after simulating the analysis with a 12-hour run was 94% were good, and the nonspecific adsorption of the amino group modified beads was found here and there on the upper surface of the rhombic pattern irrespective of the bead sitting positions, namely, on the surface in which the cycloolefin-based polymer was exposed by removing the film forming the functional group by dry etching. In the first and second embodiments, the surface of the cycloolefin-based polymer substrate with an uneven pattern formed on the surface by injection molding and thermal deformation (thermal imprint) maintains the hydrophobicity, and nonspecific adsorption was not a problem. However, the surface of the substrate of the cycloolefin-based polymer of the present embodiment is the surface of the cycloolefin-based polymer which was exposed again after forming another film thereon and removing the film by O2 plasma etching. Therefore, it was assumed that, when the cycloolefin-based polymer was exposed by, for example, dry etching, the surface of the cycloolefin-based polymer was a state of the surface functional group which was different from the usual surface of the cycloolefin-based polymer such as 0 which was bound to the C atom on the surface of the cycloolefin polymer to form, for example, an —OH group bonded to C on the surface due to the O2 plasma used in dry etching.
Therefore, after the O2 dry etching step of
The bead loading test and the falling out test which used the temperature adjusting function were performed to this substrate in the same manner, and the good results such as the loading rate of the amino group modified beads being 96% and the bead residual ratio after simulating the analysis with a 12-hour run being 94% were unchanged. Further, nonspecific adsorption was not observed in the range observed by the electron microscope. It is assumed that even in the same resin substrate, if surface oxidation and the like occurs due to a plasma treatment, etc., the surface state will change, and in this case, it was found that the surface having similar characteristics as the original resin material (hydrophobic plastic) surface could be formed by performing a fluorocarbon-based, etc., hydrophobic surface treatment, and could be used in the prevention of nonspecific adsorption.
However, apart from the nonspecific adsorption on the surface (if a rhombic pattern, the rhombus-shaped upper surface) exposed to O2 plasma due to the dry etching of the cycloolefin-based substrate, the beads having a small particle size were discovered immobilized in a position which was not an intersection point within the groove surrounding the rhombic pattern, although it was not frequent. As shown in
Taking the variation in the particle diameter of the beads into consideration, it is also possible to avoid the above problem by changing the groove width to be more narrow, but other than that, by slightly changing the production method shown in
Therefore, further simplification of the steps for forming the epoxy group or the isocyanate group for the chemical bond was examined as another examination of the method utilizing the effect of the chemical bond. This is not a complicated method such as removing an epoxy group or an isocyanate group of the unnecessary portion (the upper surface of the rhombic pattern) after performing gas-phase CVD or the resist resin coating (or, the loading of the silica beads as the protective mask) as shown in
The substrate prepared as such was evaluated, and as a result, it was found that 95% of the amino beads could be loaded in almost the same manner as the substrate prepared by the production method of
Other than forming a film with the liquid silane coupling agent by the gas-phase of the vacuum device, a method which can modify the inside of the groove by the capillary phenomenon in the liquid state could be confirmed by a manual operation. If the adhesion with the flat plate and the extrusion at the portion in which the drop is dripped is properly prevented, the amino bead sitting positions can be chemically modified by the treatment with a silane coupling agent in a liquid state, and the filling rate of the amino beads the filling rate can be improved.
Changing the viewpoint slightly, lastly, the effect of the narrowing of the particle size distribution of the beads was examined as another measure for further improving the filling rate of the beads and the residual ratio of the array spot material. First, it is assumed that the reason why the filling rate of the beads was improved by the method which uses the effect of the chemical bond stated above and the reason why the residual ratio of the beads decreased by only 2% after being used to simulate the analysis are that, it was deduced that due to the extent of the particle size distribution of the beads, the beads which were considerably smaller than the average particle diameter were not physically fitted tightly but were immobilized only by chemical bonding, and the beads fell out while used to simulate the analysis, thus, it was essential to properly arrange the particle size of the beads to narrow the distribution. Further, it is anticipated that the beads which are immobilized in a position which is not the intersection point within the groove can be eliminated by the narrowing of the particle size distribution of the beads. Following this consideration, the same treatment as in
To verify the effect of the narrowing of the particle size distribution of the beads, whether or not the beads entered the bead sitting positions set in the intersection point of the groove surrounding the rhombic pattern was checked by an electron microscope, and it was verified that the filling rate improved compared to the case of CV10% shown in the second embodiment, and the amino group modified beads were loaded into 95% of the bead sitting positions among the entirety. Further, the bead residual ratio after simulating the analysis with a 12-hour run remained 95%, and in addition, nonspecific adsorption was not observed at all in the range observed by the electron microscope. It is considered that as a result of changing from CV10% to CV3%, the ratio of the beads that are too large to be loaded for being too large compared to the average and the beads which can be loaded, but immediately fall out for being too small compared to the average decreased to improve the dimensional compatibility with the bead sitting positions of the resin substrate, and the filling rate of the beads and the residual ratio became large.
In the third embodiment, in order to further improve the results of the 92% filling rate of the beads obtained by the second embodiment and the bead residual ratio of 92% after the analysis simulation, a bead filling rate of 96% and a bead residual ratio of 94% were obtained by the combined use of chemical bonding at the bead sitting positions and a hydrophobic treatment for preventing non-specific adsorption. However, when the particle size distribution of the beads was reduced from CV10% to CV3%, the filling rate of the beads became 95%, and the bead residual ratio became 95%. Only the beads having a narrow particle size distribution were selected during the production process, and have a higher cost than the beads having a wide particle size distribution, but it is considered that, at least, due to the production process for subjecting each mass production substrate to film forming, etching and the like to perform a surface-modified treatment for the chemical bond and a non-specific adsorption preventive treatment, the increase of the cost is low, and the production process is also remarkably simple.
Examples regarding several methods which form the array spot at a formation rate of 90% or more were shown above, and the relative merits such as the cost of production were examined. Therefore, there are several implementation methods, but the following Fourth and Fifth Embodiments show examples, among the methods, using “the production steps as the second embodiment, but a substrate having an array spot formation rate of 95% produced using the CV3% beads” illustrated at the end of the third embodiment to perform the analysis of a nucleic acid polymer, and a decoding of the base sequence.
The fourth embodiment illustrates an example of the analysis which used the flow cell which incorporated the spot array substrate.
Note that, as stated in the First to Third Embodiments, it is possible that the resin substrate (for example, the form arranged in the flow cell) in which the bead sitting positions of the two-dimensional array were set with the uneven pattern formed on the surface and the beads (for example, the liquid in which the beads were dispersed) surface-modified with a functional group (in the present embodiment, an amino group) are supplied separately to the user, and the user fits the beads in the bead sitting positions of the substrate to form the functional group spot array substrate and uses this substrate.
The temperature control of the flow cell 701 is possible by the temperature adjusting function 712. Further, the reaction reagent unit 702 which supplies several reaction reagents is installed and held at a low temperature where the enzyme reaction does not progress within the reagent, and is connected to the flow cell 701 with piping tubes. In the present embodiment, four types of reagents 703 to 706 can be supplied by the reaction reagent unit 702. The reagent 703 is introduced to the bead surface on which the template DNA having an unknown sequence in at least the portion which is the object for analysis modified by the oligo DNA (known sequence) in advance, and includes the beads (beads 715 which form the replica of the template DNA on the surface by emulsion PCR) amplified by emulsion PCR. The reagent 704 includes dATP-fluorescent dye 1, dGTP-fluorescent dye 2, dCTP-fluorescent dye 3, d-TTP-fluorescent dye 4, a known sequence (oligo DNA) used in the bead-side fixed terminal of the beads for emulsion PCR and a complementary primer, and the elongation reaction enzyme. The reagent 705 is a dye bond cleaving reagent which cleaves the bond between the four types of dNTP and the fluorescent dye. The reagent 706 is an electrolyte solution.
Further, isopropyl alcohol 707 and the electrolyte solution 708 which is an equivalent of the reagent 706 were separately prepared. These solutions are injected into the flow cell by a pipette. Isopropyl alcohol 707 is used in the solution replacement within the flow cell, and another alcohol such as ethyl alcohol may be used.
A light source 709 for generating the excitation light for exciting each type of fluorescent dye contained in the reagent 704, an optical system 710 for measuring the fluorescence from the fluorescent dye, and a photodetector array 711 are arranged above the flow cell 701. The illumination of the excitation light and the fluorescence detection are possible even if with a non-transparent substrate, and, if a transparent thin substrate is used, the excitation and the detection are possible even from below the substrate, thus, in the present embodiment, the vertical arrangement of the optical system and the detector is not specifically limited. If the temperature adjustment, photoexcitation, and fluorescence observation can be performed, the optical system can be arranged either above or below the flow cell. Further, the optical signals received by the photodetector array 711 are converted to electric signals and transferred to the analyzing device 713. The analyzing device 713 performs analyses such as the discrimination of the bases from the transferred signals and the connecting of fragment data, and decodes the base sequence of the nucleic acid polymer.
It is well known that, as the fluorescent dye which can be used in the fluorescent dyes 1 to 4 contained in the reagent 704, Alexa488, Cy3, Cy5, Cy5.5, Alexa555, Alexa647, Alexa680, dN6G (dichloro-rhodamine 6G), dN110, dTAMNA (dichloro-carboxyteetramethyl-rhodamine), dROX (dichloro-carboxy-X-rhodamine), etc., and various fluorescent dyes are commercially available, and a suitable dye can be selected so that the wavelengths do not partially overlap. Further, it is also well known that selecting at least one type or more suitable wavelengths as the wavelengths of a light source 709 for efficiently exciting the fluorescent dye by the selection of these dyes.
In the present embodiment, first, isopropyl alcohol 707 was injected through the inlet of the flow cell 701 by a manual operation using a pipette and after verifying that there were no air bubbles, the isopropyl alcohol 707 was replaced with the electrolyte solution 708 by a manual operation using a pipette. The operation for replacing from alcohol to the electrolyte solution 708 may also be performed by automatic solution sending, and, in some cases, it is also possible to use the method, etc., for substituting from an alcohol to an electrolyte solution 708 on the flow cell maker side and fill the liquid in advance to supply to the user. In any event, next, the flow cell 701 was set in the nucleic acid polymer analysis device with the electrolyte solution 708 filling the cell and the air bubbles removed.
Next, the reagent 703 containing the beads 715 which formed the replica of the template DNA on the surface was automatically injected into the flow cell 701 by a solution sending unit, the solution sending was stopped once in this state, the reagent 703 was set to the optimal temperature, left standing for three hours, and immobilized on the amino group-modified spot array. The spot array material (amino group surface-modified beads 714) of which 95% of all of the bead sitting positions were surface-modified with the amino group during production was embedded in the cycloolefin substrate incorporated in the flow cell 701, but the beads 715 which formed the replica of the template DNA by emulsion PCR could be immobilized on the surface in 92% of the spots among this 95%. 3% of the spots were not immobilized. Then, a suitable temperature was set, the reagent 704 was supplied to the flow cell, hybridized with a primer, and the elongation reaction was conducted with an enzyme. At this time, since the dNTP within the reagent was terminated by the fluorescent dye, after conducting the elongation reaction on the first base type of the template DNA, the reaction was stopped. Then, the excitation light was illuminatedby the light source 709, and the fluorescence emitted at this time was measured by the optical system 710 and the photodetector array 711.
Note that, in the present embodiment, it is necessary that one bead which replicates the template DNA by emulsion PCR in advance is the bead in which one type of template DNA is replicated. In emulsion PCR, with the exception that one type of template DNA is replicated on the beads, in terms of probability, there are cases where the beads in which the template DNA could not be immobilized and when two different types of template DNA were replicated on one bead exist as defective beads. In these cases, the signals of the bases cannot be obtained, or, two types of signals are simultaneously obtained, and thus, cannot be used as data. There is another method regarding the method and the like for the adjustment of the concentration of the aqueous solution for decreasing the defective beads in which two types of template DNA were replicated, and selecting the beads in which the template DNA was immobilized beads and in which the template DNA was not immobilized, thus, the validity of the data obtained from the array spot can be improved. However, this is a well-known technology in emulsion PCR, and the presence or absence of the use of these technologies is not specifically limited in the present embodiment.
The present embodiment shows an example in which the template DNA was amplified in advance on the surface of the beads by emulsion PCR, and the template DNA immobilized on the array spot on which the beads were surface-modified by an amino group was analyzed by fluorescence measurement. However, it is also possible to use a kit of the beads with the resin substrate in the analysis of the beads which amplified the template DNA on the surface by emulsion PCR. For example, the resin substrate (without the loading of the functional group surface-modified beads, for example, the form arranged in the flow cell) in which the bead sitting positions of the two-dimensional array are set due to the uneven pattern formed on the surface, and the kit of the oligo DNA surface-modified beads (for example, the liquid in which the beads are dispersed) are separately supplied to the user, the oligo DNA surface-modified beads of the kit are used to amplify the template DNA which is the object for analysis on the beads in advance by emulsion PCR. Without supplying the template DNA beads containing the reagent 703 to the flow cell, a centrifugal force and the like is utilized to form the spot array substance by fitting into the bead sitting positions of the substrate, and this may be used to perform the analyses. In this case, it is also necessary for the user to perform the loading step of the beads himself/herself, but the base member itself is produced at a low cost, thus, the advantage that the user can perform a low cost analysis does not change.
The Fifth Embodiment illustrates another example of the analysis which uses the flow cell which incorporates the spot array substrate.
As stated in the first to fourth embodiments, it is possible that the resin substrate in which the bead sitting positions of the two-dimensional array were set with the uneven pattern formed on the surface and the beads surface-modified with an oligo DNA are supplied separately to the user, and the user fits the beads in the bead sitting positions of the substrate to form the functional group spot array substrate and uses this substrate.
The temperature control of the flow cell 901 is possible by a temperature adjusting function 912. Further, a reaction reagent unit 902 for supplying several reaction reagents is installed and held at a low temperature where the enzyme reaction does not progress within the reagent, and is connected to the flow cell 901 with piping tubes. In the present embodiment, five types of reagents 903 to 906 and 914 can be supplied by the reaction reagent unit 902. The reagent 903 contains the material (template DNA 916 bound with complementary sequence) which binds the template DNA having an unknown sequence in at least the portion which is the object for analysis to the oligo DNA (known sequence) on the array spot to the complementary sequence. The reagent 914 includes dATP, dGTP, dCTP, dTTP, and an enzyme, and is a reagent for replicating on the array spot. The reagent 904 includes dATP-fluorescent dye 1, dGTP-fluorescent dye 2, dCTP-fluorescent dye 3, dTTP-fluorescent dye 4, a primer of a sequence complementary with the oligo DNA (known sequence) on the array spot, and the elongation reaction enzyme. The reagent 905 is a dye bond cleaving reagent for cleaving the bond between the four types of dNTP and the fluorescent dye. The reagent 906 is an electrolyte solution. Further, in the same manner as the fourth embodiment, isopropyl alcohol 907 and an electrolyte solution 908 which is the equivalent of the reagent 906 were separately prepared. Isopropyl alcohol 907 is used in the solution replacement within the flow cell 901, and another alcohol such as ethyl alcohol may be used.
A light source 909 for exciting each type of fluorescent dye contained in the reagent 904, an optical system 910 for measuring the fluorescence from the various dyes, and a photodetector array 911 are arranged above the flow cell 901. The optical signals received by the photodetector array 911 are converted to electric signals to be transferred to the analyzing device 913 for analyzing the fluorescence. The analyzing device 913 performs analyses such as the discrimination of the bases from the transferred signals and the connecting of fragment data, and decodes the base sequence of the nucleic acid polymer. Note that, in the same manner as the fourth embodiment, various selections of the fluorescent dyes 1 to 4 are contained in the reagent 904, and thus, can be used to select a suitable dye. Further, a suitable wavelength from the light source 909 for efficiently exciting these fluorescent dyes can be selected by the selection of these dyes.
First, isopropyl alcohol 907 was injected through the inlet of the flow cell 901 by a manual operation using a pipette and after verifying that there were no air bubbles, the isopropyl alcohol 907 was replaced with the electrolyte solution 908 by a manual operation using a pipette. Next, the flow cell 901 was set in the nucleic acid polymer analysis device with the electrolyte solution 908 filled in the cell and the air bubbles removed.
Next, the reagent 903 containing the template DNA 916 bound with complementary sequence was automatically injected into the flow cell 901 by a solution sending unit, the solution sending was stopped once in this state, th reagent 903 was set to the optimal temperature, left standing for 10 minutes, and immobilized on the oligo DNA-modified spot array. Since the material of the substrate was hydrophobic, no evidence that the template DNA 916 bound with complementary sequence was adsorbed other than in the spot array was observed, and the nonspecific adsorption was not a problem. Then, a suitable temperature was set, the reagent 914 for replicating on the array spot was supplied to the flow cell, the solution sending was stopped once in this state, the optimal temperature was set to repeat the elongation reaction and the denaturing, and the template DNA was replicated on the array spot. 95% of the oligo DNA-modified spot array material (oligo DNA surface-modified beads 915) during production was embedded in the cycloolefin substrate incorporated into the flow cell 901. Among them, the template DNA 916 bound with complementary sequence could be immobilized in a certain proportion of spots determined by probability theory. In this case, the concentration of the template DNA within the reagent 903 is different. Then, a suitable temperature was set, the reagent 904 was supplied to the flow cell, hybridized with a primer, and the elongation reaction was conducted with an enzyme. At this time, since the dNTP within the reagent was terminated by the fluorescent dye, after conducting the elongation reaction on the first base type of the template DNA, the reaction was stopped. Then, the excitation light was illuminated by the light source 909, and at this time, the emitted fluorescence was measured by the optical system 910 and the photodetector array 911.
Note that, in the present embodiment, it is necessary that the replica of the template DNA immobilized on the one array spot which was modified by the oligo DNA is the replica of one type of template DNA. In the replica on the array spot which was modified by the oligo DNA, with the exception that the template DNA replicated on one spot is one type, in terms of probability, there are cases where the spot in which the template DNA could not be immobilized and where two different types of template DNA were replicated on one spot exist as defective beads. In these cases, the signals of the bases cannot be obtained, or, two types of signals are simultaneously obtained, and thus, cannot be used as data. Another method regarding the method and the like for the adjustment of the concentration of the aqueous solution for decreasing the defective spots in which two types of template DNA were replicated and for immobilizing the template DNA to the spot in which the template DNA could not be immobilized has been proposed, and thus, the validity of the data obtained from the array spot can be improved. For example, the method described in US 2012/0156728 A1, etc., can be used as the protocol for the replica on the substrate of the template DNA. The present embodiment is not limited to, specifically, the details of the replica technology on these substrates, and the details of the protocol are not specifically shown.
Note that, in the fourth and fifth embodiments, the flow cell which incorporated the spot array substrate by the present invention is used to perform the replica on the beads by emulsion PCR and the replica on the array spot of the substrate, and decode the DNA containing the partially unknown base sequence which is the object for analysis, but it goes without saying that the flow cell which incorporates the spot array substrate of the present invention can be used in the decoding not only DNA but also other nucleic acid polymers such as RNA.
Further, in the drawings corresponding to the explanation of the present invention, a mark of a heat filament and a mark of a convex lens are used symbolically in the temperature adjusting function and the optical system. However, the temperature adjusting function also includes functions for cooling such as air cooling, water cooling, and a cooling element, and can be controlled to a fixed temperature by a general temperature control method such as PID control, and the optical system has a light separating function such as a spectrometer and a color filter other than a convex lens, thus, it goes without saying that the color of the fluorescence can be distinguished.
Note that, the present invention is not limited to the above-described embodiments but includes various modifications. The above-described embodiments are explained in detail for better understanding of this invention and are not limited to those including all the configurations described above. A part of the configuration of one embodiment may be replaced with that of another embodiment; and the configuration of one embodiment may be incorporated to the configuration of another embodiment. A part of the configuration of each embodiment may be added, deleted, or replaced by that of a different configuration.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2014-240434 | Nov 2014 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2015/078479 | 10/7/2015 | WO | 00 |
