The present application claims priority from Japanese application JP 2007-113095 filed on Apr. 23, 2007, the content of which is hereby incorporated by reference into this application.
1. Field of the Invention
The present invention relates to a chemiluminescent detection system, and more specifically, to one in which detection results of luminescence from a plurality of reaction chambers are used to analyze nucleic acid and analyze a base sequence of genes or the like.
2. Background Art
Methods using gel electrophoresis and fluorescence detection are widely used to determine a DNA base sequence. This method makes many copies of a DNA fragment to be subjected to an array analysis first. Fluorescently-labeled nucleic-acid fragments of various lengths are created using a 5′ end of DNA as a starting point. Moreover, fluorescent labels with different wavelengths are added according to the type of 3′ terminal base species of these DNA fragments. The difference in length is identified using gel electrophoresis by a difference of one base and luminescence emitted from each fragment group is detected. The type of DNA terminal base species of the DNA fragment group being measured is known from its luminescence wavelength color. Since DNA passes through the fluorescent detection section from a short fragment group one by one, it is possible to know the type of terminal base species from short DNAs one by one by measuring fluorescent colors. The array is determined in this way. Such a fluorescent DNA sequencer is widely used, and also has taken an active part in a human genome analysis a great deal. According to this method, a technique of increasing the number of genes analyzed and processes per unit using many glass capillaries having an inner diameter of approximately 50 μm and also using a method of terminal detection or the like is disclosed (e.g., see Non-Patent Document 1).
On the other hand, attention is being focused on an array decision method (e.g., see Patent Documents 1 and 2) by phased chemical reaction represented by a pyrosequence from the standpoint of convenience of handling. An outline of this is as follows. A primer is hybridized with a target DNA strand and four types of complementary strand synthesized nucleic acid substrates (dATP, dCTP, dGTP, dTTP) are added to a reactive liquid one type at a time to realize synthesis reaction of complementary strand. Once the synthesis reaction of complementary strand takes place, DNA complementary strands extend and pyrophoric acid (PPi) is generated as a by-product. The pyrophoric acid is converted to ATP by the functioning of coexisting enzyme, reacts under the coexistence of luciferin and luciferase and produces luminescence. It is understood by detecting this light that the added complementary strand synthesized substrates are taken into the DNA strand, and it is possible to know array information on the complementary strand, therefore array information on the target DNA strand.
This method enables high throughput by using a flow cell provided with many reaction chambers and an example where the number of genes analyzed and processes is greatly increased by applying the above described method is reported (e.g., see Non-Patent Document 2). In this application example, a flow cell that has a plurality of micro-reaction chambers on one side is used as a reaction plate. Many units in which target DNA strands are grouped by type and fixed to a sepharose bead of approximately 35 μm in diameter are prepared and approximately 108 DNAs of the same type are fixed to each sepharose bead. After hybridizing the primer with these DNAs, one bead is put in each micro-reaction chamber. Moreover, the reaction chamber is filled with microparticles of 0.8 μm in diameter to which bioluminescent enzyme (luciferase) or the like is fixed. The filling of these beads is done by introducing a solution containing the bead into the flow cell and making it precipitate using a centrifuge. A DNA base sequence analysis is performed by successively introducing four types of complementary strand synthesized nucleic acid substrates (dATP, dCTP, dGTP, dTTP) for extension reaction from the flow cell upstream to realize synthesis reaction of complementary strand, but when the synthesis reaction of complementary strand progresses, pyrophoric acid is generated. The pyrophoric acid is converted to ATP and luciferase reaction takes place and bioluminescence produced in that case is observed. Some of devices that detect chemical luminescence and fluorescence using many such micro-reaction chambers are reported. Examples include a case where an anchor probe is fixed to one end face of a fiber optic plate instead of fixing DNAs to a bead, made to couple with a circular nucleic acid template and realize array determination and a multi-type analysis through bioluminescence (e.g., see Patent Document 2) and a case where the above described fiber optic plate is etched, the central portion of the fiber is removed and a reaction chamber is created, a multi-well titerplate (hereafter, abbreviated as “plate”) is constructed and used as part of the flow cell (e.g., see Non-Patent Document 3). Furthermore, for example, Patent Document 4 discloses a plate provided with a membrane or the like to reduce contamination by diffusion in the horizontal direction of matters generated in individual micro-reaction chambers in this plate or more specifically pyrophoric acid.
These techniques detect luminescence from a reaction layer distributed on a plane by making it form an image in an area sensor using a coupling lens. In this case, since the image formed on the detector may be distorted or the position at which the image is formed may be relatively shifted, detection is generally performed with a plurality of detection pixels associated with one reaction chamber. Moreover, the number of reaction chambers should be set to from a fraction to one tenth of the number of pixels of the detector. Therefore, creating an apparatus provided with many reaction cells requires a very big image pickup element including an area sensor, which cannot help but result in an expensive apparatus.
On the other hand, a recessed part is made by etching an optical fiber and the recessed part can be used as a reaction chamber, too. In this case, there is also an attempt to couple an end of the fiber opposite to the reaction chamber provided at the end of the fiber with pixels of the area sensor. However, it is necessary to create an image pickup element and a reaction chamber as a single unit in this case, and there are disadvantages that this is not user-friendly and it is hard to control the temperature of the reaction chamber. Enzyme reaction is preferably realized at 35° C. or more, and this is because the image pickup element is preferably cooled for use because noise increases as the temperature of the image pickup element rises. Furthermore, since it is convenient that the reaction cell be made easily detachable because the reaction cell needs to be cleaned or disposed of according to circumstances. Furthermore, since an array of optical fibers is not completely regular, it is impossible to provide a one-to-one correspondence with the completely regularly arranged pixels over a wide area.
Moreover, Patent Document 4 also describes that fluorescence from a plurality of nucleic acids fixed to a nucleic acid chip is measured in one-to-one correspondence therewith.
However, there is no description on how the chip to which nucleic acids are fixed and the image pickup element are spatially (accurately) arranged, but in a system in which an inverted image is formed on a detection element through normal lens coupling, micro distortion of the image often has a considerable influence on the measuring result.
A pyrosequence analysis technique using a flow through detector made up of a plurality of micro-reaction chambers arranged in parallel has a short reading base length, but can achieve high throughput by increasing the number of reaction chambers compared with conventional gel electrophoresis. Under present circumstances in which array databases of various living things including a human genome array database are being put in order, if an array of many DNA fragments can be determined no matter how short the array may be, the influence on medical treatment and other fields is significant.
On the other hand, the number of feasible micro-reaction chambers is limited by the number of pixels of a semiconductor image pickup element in the case of chemiluminescent detection. The above described conventional technique performs detection with nine pixels associated with one reaction chamber and requires dummy pixels (pixels which do not need intensity of light received in the pixels as data) to further reduce crosstalk of signals. Moreover, the conventional technique can only use a reaction chambers corresponding in number to one tenth of pixels of the image pickup element (=solid-state element with many pixels formed on the same substrate). Actually, according to Non-Patent Document 2, the pixel size of a CCD that is an image pickup element is 15×15 μm and 480 micro-reaction chambers per square millimeter are measured using approximately 4500 pixels per square milimeter. That is, the number of micro-reaction chambers realized is approximately 1/10 of the number of pixels.
It is certainly possible to increase the number of micro-reaction chambers that can be measured by increasing the number of pixels of the image pickup element, but as described above, the image pickup element requires a large area, and not only the image pickup element becomes expensive but also the optical system to guide light to the image pickup element generally becomes expensive.
In this respect, it is understandable that throughput can be improved ten times if the number of pixels and the number of micro-reaction chambers are made equal.
However, to achieve this, the pixels and the reaction chambers need to be detected associated with each other and even if an image of a reaction chamber is formed in the area sensor by means of lens coupling as in the case of the conventional technique, this actually does not go well due to micro distortion of the lens (the detection accuracy is not good). Moreover, because the number of pixels of the image pickup element matches the number of reaction chambers, focus adjustment and alignment are essentially difficult even using a lens system with no distortion. In other words, it is difficult to make high throughput compatible with high detection accuracy.
Furthermore, as for the system shown in the conventional example in which a reaction chambers is constructed by etching one end of an optical fiber and causing the other end to closely contact the pixels of a detection element, it is not suitable for a system made detachable for replacement or cleaning of the reaction chamber. When the reaction chamber is constructed by etching one end of the optical fiber in this way, the position of the reaction chamber is determined by the position of the optical fiber, but the position of the optical fiber is not completely regular and it is difficult to make the position of the optical fiber correspond one-to-one with the pixels on the completely regularly arranged pixels on the image pickup element. Therefore, the development of new techniques which are different from these conventional examples is demanded.
Moreover, when the number of micro-reaction chambers is increased so as to substantially match the number of pixels on the image pickup element, positioning of the plate becomes important. That is, an apparatus that executes a pyrosequence analysis is provided with a plate wherein different arrays of nucleic acid of many types to be analyzed are fixed at different positions, an image pickup element for receiving light from the micro-reaction chambers thereon and an optical system for guiding luminescence from the plate to the image pickup element, but in this case, the image pickup and the relative position with respect to the optical system must be adjusted every time the plate is replaced. In this case, it is not possible to know beforehand at which part of the plate luminescence occurs and since the micro-reaction chambers on the plate are smaller than the resolution of the image pickup element, there is also a problem that focus, inclination and in-plane displacement of pixels cannot be adjusted so that the light from the micro-reaction chambers is condensed only on a specific pixel.
The present invention has been implemented in view of such circumstances and is intended to provide a pyrosequence analysis technique that can achieve high throughput at low cost and realize fluorescence detection with high accuracy.
In order to solve the above described problems, the chemiluminescent detection system according to the present invention uses optical means capable of forming images in a one-to-one correspondence of pixels with reaction chambers. Use of such optical means together with position control of the reaction chambers makes it possible to form one-to-one correspondence without distortion on the image pickup element. Furthermore, by providing a spatially separated lens system between the plate provided with the reaction chambers and the image pickup element, they are enabled to operate at mutually different temperatures. As such a lens system, for example, a rod lens array capable of forming an erecting image (normal image) or a micro lens array and optical fiber bundles can be used. The plate on which a plurality of micro-reaction chambers are regularly arranged based on a design, the image pickup element and the optical system are arranged at predetermined positions so that light from all the micro-reaction chambers can be detected by individual and corresponding pixels on the image pickup element. Especially, the reaction chambers, lens system, optical system, and image pickup element are arranged taking into account the temperature expansion coefficient of the plate in which the reaction chambers are formed so as to match the interval between the centers of images of the micro-reaction chambers on the image pickup element with the interval between the centers of pixels of the image pickup element when adjusted to an optimum temperature for chemical reaction produced in the reaction chambers.
That is, the chemiluminescent detection system according to the present invention is a chemiluminescent detection system that detects light from a plurality of reaction chambers and includes a flow cell that has a plate on which a plurality of reaction chambers are one-dimensionally or two-dimensionally arranged, optical detection means having a plurality of pixels, the interval of pixels of the optical detection means substantially matching the interval of the reaction chambers on the plate, and an optical system for forming images of the plurality of reaction chambers in the optical detection means. Luminescence of each of the plurality of reaction chambers is detected in a one-to-one correspondence with different pixels in the optical detection means. Such a function may also be provided that DNA samples are fixed to beads and stored in the plurality of reaction chambers individually and synthesis reaction of complementary strand is executed in such a condition to enable luminescence reaction to be produced continuously.
Furthermore, the present chemiluminescent detection system includes means for adjusting, after the plate provided with the reaction chambers is arranged, relative positions of the lens system, image pickup element and optical system. This adjusting means is constructed of an illuminant, reflector or light transmitter on the plate for making images correspond one-to-one with pixels on the image pickup element of the reaction chamber and intended to adjust the position and angle of the plate based on the detection result of light.
To measure luminescence from the reaction chambers most efficiently at pixels in a one-to-one correspondence with the reaction chambers, the luminescence area of the reaction chamber (size of the reaction chamber) is configured so as to be smaller than the pixel area. Moreover, in order to measure luminescence from the reaction chambers most efficiently at pixels in a one-to-one correspondence with the reaction chambers, a reflection coating may be formed in the inner wall of the reaction chambers so as to improve the optical radiation efficiency of upward luminescence and measure light from the top surface of the plate efficiently.
Moreover, it is also possible to arrange two-dimensionally arrayed reaction chambers using a line sensor made up of one-dimensionally arrayed pixels in such a way that the reaction chambers arrayed in a direction parallel to the array direction of the pixels of the line sensor are made to correspond one-to-one with the pixels and the plate is relatively moved with respect to the image pickup element so as to enable chemical luminescence from the two-dimensionally arrayed reaction chambers to be measured.
According to the present invention, it is possible to realize a chemiluminescent detection system capable of performing fluorescence detection for a pyrosequence analysis at low cost and with high throughput and high accuracy.
Further features of the present invention will be made clear with preferred embodiments for implementing the present invention and attached drawings which will be described below.
Hereafter, embodiments of the present invention will be explained with reference to the attached drawings. However, it should be noted that the embodiments are merely examples for implementing the present invention and are not intended to limit the present invention. Moreover, components common among drawings will be assigned the same reference numerals.
In
Furthermore, the chemiluminescent detection system 1 is constructed of reagent chambers 106 to 109 that contain four types of nucleic acid substrate (dATP, dGT, dCTP, dTTP) to dispense a reagent into the flow cell one by one as a system that sends a solution of the reagent to the micro-reaction chambers, a cleaning reagent chamber 110 that contains a cleaning reagent for cleaning the inside of the flow cell after measurement of extension reaction, a conditioning reagent chamber 111 that contains a conditioning reagent to flush the remaining cleaning reagent component in the cell after cleaning, an injection section for selectively injecting the reagents into the flow cell (selection valve 112 and pump 113 to handle the reagents) and a waste fluid bottle 114 or the like. The chemiluminescent detection system 1 is further provided with a Peltier element 120 for setting the temperature of a reagent solution in the flow cell to an optimum temperature for a pyrosequence, a thermistor as a temperature sensor, and a temperature controller 122 for controlling an electric current to be flown into the Peltier element from the temperature measured with the thermistor. Moreover, the system is cooled down to −20° C. to decrease noise by a dark current of the image pickup (CCD) element 103. This cooling temperature is determined according to the intensity of chemical luminescence and the intensity of background luminescence which does not derive from an extension of target DNA, but is generally set to a room temperature or below. On the other hand, the temperature of the plate controlled by the Peltier element 120 is set to an optimum temperature for chemical luminescence, 40° C. here. This temperature also varies depending on enzyme used, but is generally set to the room temperature or above.
Next, the structure of the flow cell 101 will be explained with reference to
The shape of the micro-reaction chambers 201 is preferably columnar, for example. The shape is determined depending on the material and the manufacturing method of the substrate. Various plates can be used such as a plate manufactured through cutting using a stainless steel material as a substrate, a plate manufactured through a mask and wet etching using a silicon wafer, a plate manufactured through a bluster process with particles using glass such as slide glass and a plate manufactured through injection molding of a metal mold using polycarbonate, polypropylene, polyethylene or the like. However, these are by no means intended to limit the material and manufacturing method of the micro-reaction layer.
Furthermore, for example, the flow cell 101 used includes 4096×4096 micro-reaction chambers formed at intervals of 15 μm in a square area, 6.144 cm on a side on the plate 202. When the plate 202 is formed using glass, for example, it must be created by taking into account thermal expansion due to the difference in the temperature when the micro-reaction chambers are formed and the temperature (40° C.) when the plate is installed and chemical luminescence is measured. That is, the temperature of the micro-reaction chambers is set to 20° C. and 4096×4096 micro-reaction chambers are formed in an area smaller by 9.8 μm than 6.144 cm. Furthermore, when polycarbonate is used, molding is performed at 200° C. using a metal mold, and the square area in which the micro-reaction chambers are arranged on the metal mold is manufactured to be greater by 368.6 μm than 6.144 cm and used as the metal mold. Thus, by manufacturing the system with thermal expansion and contraction coefficients of the plate taken into consideration, it is possible to reliably realize the one-to-one correspondence between the micro-reaction chambers and pixels at the time of detection.
Next, the image pickup element 103 will be explained. The image pickup element 103 may be any area sensor which is a two-dimensional image pickup element or any line sensor which is a one-dimensional image pickup element as long as it is a light receiving element provided with many pixels. However, it is effective to use either a CCD (Charge Coupled Device) with low transfer noise of data or a CMOS sensor whose manufacturing cost is low. In this case, the image pickup element may be preferably cooled electronically to reduce dark current noise. Actually, measurement is performed using the CCD element at an element temperature of −20° C. or less.
Furthermore, when the pixel size increases, not only the element cost increases, but also it is not possible to produce the image pickup element with many pixels from the standpoint of manufacturing yield. Therefore, the pixel size is preferably set to 15 μm in both cases of CCD and CMOS. Furthermore, the array format of pixels is generally tetragonal lattice or rectangular lattice, but the array format may also be hexagonal lattice or a honeycomb structure combining an octagon and square. In this case, the micro-reaction chambers also need to be arrayed in the same manner. This embodiment adopts tetragonal lattice.
Since the arrangement cycle of the pixels of the image pickup element 103 should match the arrangement cycle of the micro-reaction chambers 201, the micro-reaction chambers 201 are preferably made smaller than the pixels of the image pickup element 103. However, when the size of the micro-reaction chambers 201 is reduced and the spacing between the micro-reaction chambers 201 is reduced, Ppi or ATP that becomes the substrate of chemical luminescence diffuses within an exposure time, making it difficult to make a distinction from luminescence of the adjoining micro-reaction chambers 201. Therefore, the spacing between the micro-reaction chambers 201 and the pixel size determined therefrom should be longer than the length determined from the diffusion distance. This length is approximately 1 μm. On the other hand, when the pixel size is made greater than a predetermined size, the size of the whole image pickup element made up of a CCD or CMOS sensor manufactured on a semiconductor substrate increases, which becomes unrealistic not only from the standpoint of cost but also from the standpoint of manufacturing yield. The pixel size which constitutes a limit within which the size of the whole image pickup element can be increased is approximately 30 μm. Therefore, when the semiconductor image pickup element such as CCD or CMOS sensor is used, the pixel size should preferably be set to 1 μm to 30 μm.
In the case of a flat panel display whose element is formed on a glass substrate, an increase of the pixel size is not led to any cost increase, but there is a limitation to cooling thereof. Therefore, it is preferable to set 30 μm to 150 μm as the pixel size.
As described above, this embodiment uses the rod lens array 127 as the optical system. With the rod lens array 127, one lens is realized by increasing the refractive index in the central portion of the columnar glass compared with that of the surrounding portion. An array is formed by one-dimensionally or two-dimensionally arranging these columns in such a way the columns erect in a direction perpendicular to the surface of image. According to this embodiment, the diameter of the columnar lens making up the rod lens array is, for example, 1.115 mm and the length thereof is 8.42 mm. Furthermore, the array is constructed by arranging 60×60 (3600) such columnar lenses in consideration of peripheral effects, too.
The images of the micro-reaction chambers are designed to be formed in the image pickup element when the distance between the rod lens array 127 and micro-reaction chambers 201 matches the distance between the image pickup element 103 and rod lens array 127. This distance is generally several millimeters. The rod lens array 127 used here has this distance of 4.2 mm. In this way, by obtaining images with a certain distance kept between the plate 202 and rod lens array 127, it is possible to provide the channel for a reagent 209 between the micro-reaction chamber 201 and rod lens array 127 as shown in
To make the micro-reaction chambers 201 correspond one-to-one with the pixels of the image pickup element 103, the positions of the plate 202, the optical system (rod lens 127) and the image pickup element need to be adjusted with high accuracy. The configuration and method for realizing this adjustment will be explained below.
As shown in
In this embodiment, alignment between the flow cell 101 and image pickup element 103 is performed when the system is manufactured. As described above, the flow cell 101 and image pickup element 103 are enabled to be mechanically aligned through the convex parts 125 and concave parts 124 during operation. In a word, a dummy plate is prepared in which pinholes of approximately 1 μm in diameter are perforated at positions corresponding to several micro-reaction chambers 201 when the chemiluminescent detection system 1 is manufactured. By fixing the dummy plate to the lens holder 126 and irradiating the dummy plate with light from the back, light-emitting points can be arranged at positions corresponding to the micro-reaction chambers 201. The image pickup element 103 is aligned to allow these light-emitting points to be measured by the corresponding pixels and the image pickup element 103 is fixed. Furthermore, the temperature of this plate 202 and the temperature of the CCD or the like are set to the above described operating temperatures and adjustment is performed. Furthermore, the lens holder 126 is preferably made of glass (quartz) so as not be distorted by variations in the ambient temperature.
Suppose the diameter of the micro-reaction chambers 201 is 12 μm, depth is 12 μm and the spacing between the micro-reaction chambers 201 is 3 μm. In this case, the accuracy of alignment of the image pickup element 103 with respect to the plate 202 is preferably half or less than the above described spacing, that is, 1.5 μm or less.
This embodiment uses a rod lens 127 whose F value is 1 at a 1× magnification, and generally, when a lens system having a fixed F value is used for image formation, the magnification for guiding light from a micro-reaction chamber to a pixel most efficiently is 1×. This is shown in
A two-dimensional (area) sensor is used as the image pickup element here, but a one-dimensional (line) sensor may also be used.
The optical fiber bundle 123 is made up of many optical fibers having a minimal diameter bundled together and fixed and causes images of luminescence from micro-reaction chambers 201 in the vicinity of one end face to be formed in the vicinity of the other end face. As in the case of the rod lens array 127, the optical fiber bundle can make all the micro-reaction chambers 201 correspond one-to-one with pixels of an image pickup element 103 if only aligned adequately without any distortion of images even in the periphery. However, in the case of the optical fiber bundle 123, resolution is limited depending on the diameter of the bundled optical fiber.
Furthermore, in order to directly take in light from the micro-reaction chambers 201 using the optical fiber bundle 123 in the positional relationship between a plate 202 of a flow cell 101 and the image pickup element 103 as shown in
This micro lens array 1501 corresponds one-to-on with the micro-reaction chambers 201, is fixed to the plate 202 and also corresponds one-to-on with the pixels. Moreover, the focal plane of the optical fiber bundle 123 is designed and arranged so that the image of a rear principal point of the micro lens array 1501 (principal point on the image side) is formed in the image pickup element 103. The diameter w of the micro-reaction chamber 201 is set to 10 μm, depth d to 10 μm, channel height h to 5 μm, and distance s between the front principal point of the micro-array lens (principal point on the object side) and the surface of the lens on the channel side to 10 μm. Moreover, the diameter R of one lens making up the micro lens array 1501 is set to 15 μm and these lenses of the micro lens array 1501 are arranged on a tetragonal lattice at intervals of 15 μm in a one-to-one correspondence with the pixels of the image pickup element. Furthermore, the micro lens used has a focal length f of 20 μm. f is determined according to s+h+d/2. By making the focal length f as small as possible under the condition of R>w, it is possible to receive bioluminescence generated in the micro-reaction chambers more efficiently than when it is condensed using an ordinary camera lens.
A camera lens may be used instead of the optical fiber bundle 123, but there is a problem with distortion of images in this case. Therefore, it can be said to be preferable to use the optical fiber bundle 123.
Furthermore, as an optical system, an optical fiber bundle that bundles optical fibers having a diameter of 3 μm is used.
The optical fiber bundle 123 and the image pickup element 103 are fixed. On the other hand, the flow cell 101 can be detached from the chemiluminescent detection system 2. In this case, the positions of the optical fiber bundle 123 and the image pickup element 103 are adjusted and fixed appropriately according to the following procedure. That is, concave parts 124 are formed as a plurality of match marks in the optical fiber bundle 123. Convex parts 125 (engagement pins) are attached to the plate 202 as fixed match marks. With the plate 202 attached, the micro-reaction chambers and the image pickup element 103 are aligned so as to face each other, and then the image pickup element 103 is fixed to the optical fiber bundle 123. Moreover, the focal plane of the optical fiber bundle 123 is defined by a spacer 128. By engaging the engagement pins 125 fixed to the plate 202 with the concave parts 124 in the optical fiber bundle 123, the micro-reaction chambers in the plate 202 and the pixels of the image pickup element 103 can be aligned in a one-to-one correspondence. In this case, a pyrosequence may be conducted in the flow cell 101 to align with the image pickup element 103.
The alignment between the flow cell 101 and the image pickup element 103 may also be performed when the system is manufactured. As described above, in operation, the system is designed to be able to realize mechanical alignment using the convex parts 125 and concave parts 124. In a word, a dummy plate is prepared with pinholes of approximately 1 μm in diameter perforated at positions corresponding to several micro-reaction chambers 201 when the chemiluminescent detection system 1 is manufactured. By fixing the dummy plate and irradiating the dummy plate with light from the back, light-emitting points can be arranged at positions corresponding to the micro-reaction chambers 201. The image pickup element 103 is aligned to allow these light-emitting points to be measured by the corresponding pixels and the image pickup element 103 is fixed. Furthermore, the temperature of this plate 202 and the temperature of the CCD or the like are set to the above described operating temperatures and adjustment is performed.
This embodiment uses an optical fiber bundle to form images of chemical luminescence on the plate in a CCD. This is because the optical fiber bundle reduces distortion of the image and reduces the possibility that spacing between images of the micro-reaction chambers may be widened or narrowed in the central and peripheral parts of the images.
A two-dimensional (area) sensor is used as the image pickup element, but a one-dimensional (line) sensor may also be used.
Because the structure of the flow cell and the features of the image pickup element are similar to those of the first embodiment, explanations thereof will be omitted.
In the above described first and second embodiments, alignment of the flow cell 101 is performed during manufacturing so as to eliminate the necessity for alignment when the flow cells 101 is replaced (during operation). However, when the work accuracy of the flow cell 101 is not enough, alignment is needed every time the flow cell 101 is replaced. Therefore, the third embodiment provides a chemiluminescent detection system having a configuration that allows alignment of the flow cell 101 in operation of the system.
As in the case of the first embodiment, the flow cell 101 has the structure shown in
The image pickup element 103 may be any area sensor which is a two-dimensional image pickup element or any line sensor which is a one-dimensional image pickup element as long as it is a light receiving element provided with many pixels. For example, it is effective to use either a CCD (Charge Coupled Device) with low transfer noise of data or a CMOS sensor whose manufacturing cost is low. In this case, the image pickup element needs to be electronically cooled to reduce dark current noise. Actually, measurement is performed using the CCD element at an element temperature of −20° C. or less.
Furthermore, when the pixel size increases, not only the element cost increases, but also it is not possible to produce the image pickup element 103 with many pixels from the standpoint of manufacturing yield. Therefore, the pixel size is preferably set to 20 μm or less in both cases of CCD and CMOS. Furthermore, the pixel array format is generally tetragonal lattice or rectangular lattice, but the pixel array format may also be hexagonal lattice or a honeycomb structure combining an octagon and a square. In this case, the micro-reaction chambers also need to be arrayed in the same manner. This embodiment adopts tetragonal lattice.
The shape of the micro-reaction chamber 201 is preferably columnar, for example. The shape is determined depending on the material and the manufacturing method of the substrate. Various plates can be used such as a plate manufactured through cutting using a stainless steel material as a substrate, a plate manufactured through a mask and wet etching using a silicon wafer, a plate manufactured through a bluster process by means of particles using glass such as slide glass and a plate manufactured through injection molding of a metal mold using polycarbonate, the polypropylene and polyethylene or the like. However, these are by no means intended to limit the material and manufacturing method of the micro-reaction layers.
From a state in which the flow cell 101 has been mechanically located at an appropriate position to some degree, X, Y, Z, η, φ and θ will be adjusted according to the flow chart in
A more specific adjustment procedure is performed according to
The light sources for alignment 90 are lit (process 2). Next, the flow cell 101 is moved in the x- and y-axis directions by driving the alignment mechanism 105 (driving part) to cause Q1 to align with P1 (process 3). Next, the flow cell 101 is moved in the z-axis direction to maximize the contrast of Q1 (process 4). Since the position of the flow cell 101 shifts in the x- and y-axis directions when the flow cell 101 is moved in the z-axis direction, process 3 is executed again (process 5). In a word, the following operation is executed in processes 3 to 5. The contrast function is moved in such XYZ directions that the position of the flow cell is indicated by coordinates in
In this adjustment operation, η and φ can be adjusted preferentially according to the shape of arrangement of Qi at four points (processes 6 and 8). The process of adjusting in a combination of X, Y, Z and θ precedes over the process of adjusting η or φ which would originally be executed if this process did not exist, and a true maximum value cannot be reached and there is a possibility that alignment and focus adjustment may be finished insufficiently. This process of judging whether or not to preferentially execute angle adjustment from the shape of arrangement of image Qi on the CCD plane of a plurality of light-emitting points is a process indispensable to ensure that a true maximum value of the contrast function is reached and adjustment can be completed correctly.
After process 6, processes 4 to 6 are repeated until the contrast of Q3 and Q4 are no longer improved (process 7). Moreover, after process 8, processes 4, 5 and 8 are repeated until the contrast of Q2 and Q4 are no longer improved (process 9).
Next, Q2 is brought closer to P2 by moving θ by dθ (process 10). Next, processes 6 to 9 are executed (process 11). Furthermore, processes 10 and 11 are repeatedly executed so that Q2 matches P2 (process 12). If Q3 does not match P3 and Q4 does not match P4, dη=dη/2, dφ=dφ/2 and dθ=dθ/2 are set again and processes 3 to 12 are executed again (process 13).
When the contrast values of Q1 to Q4 are finally obtained, the values are saved in a memory (process 14). Moreover, dη=dη/2, dφ=dφ/2 and dθ=dθ/2 are set again, processes 3 to 12 are executed again and the contrast values of Q1 to Q4 are saved in the memory (process 15). Finally, if the difference in the contrast of Q1 to Q4 before and after process 15 is greater than a value set beforehand, dη=dη/2, dφ=dφ/2 and dθ=dθ/2 are set again and processes 3 to 12 are executed again. Otherwise, the adjustment processing is finished (process 16).
In the adjustment processing shown in the flow chart of
In the above described alignment processing, luminescence intensities at the plurality of light-emitting points 90 should substantially match and should not change timewise. To realize such luminescence, there are various methods like (i) a method of introducing a light-emitting device into the multi-well titerplate, (ii) a method of arranging the lighting opposite to the image pickup element with respect to the multi-well titerplate to enable the light from this lighting to be observed only at the part corresponding to the light-emitting point of the multi-well titerplate, (iii) a method of arranging the lighting on the same side as the image pickup element with respect to the multi-well titerplate and arranging a reflector at the position corresponding to the light-emitting point to enable the light to enter the image pickup element to enable illumination light to enter the image pickup element more effectively than the other areas on the multi-well titerplate. Hereinafter, more specific methods will be explained.
First, the cross section of the multi-well titerplate 71 in the case (i) is shown (see
Next, the cross section of the multi-well titerplate 75 in the case (ii) is shown (see
Finally, the cross section of the multi-well titerplate 80 in the case (iii) will be shown (see
The reflector may be made of a glass material, metal or resin material, and the shape may also be a column or rectangular parallelepiped instead of a spherical shape. In order to improve the contrast at points other than the light-emitting points, the plate 80 was prepared using a resin material containing black pigment so as to suppress reflections of light at any points other than the light-emitting points on the surface of the plate. Antireflection treatment may also be performed for the same purpose. Furthermore, to improve the contrast, it is also possible to mix micro semiconductor particles (semiconductor particles of ZnSe of several nm to several tens of nm called “Quantum Dot”), use a laser as the light source and use fluorescence from the beads as the light-emitting points. Semiconductor particles do not produce color fading and are suitable for adjustment by irradiating laser light for a long time. In this case, a light source having a wavelength shorter than the wavelength of luminescence is used for laser excitation and a band stop filter for cutting this wavelength is used between the image pickup element and the plate 80.
Next, the arrangement in the system will be explained about the above described three light-emitting schemes. The system configuration of this embodiment is shown in
On the other hand,
As shown in
Furthermore, in
Various lighting methods are possible if the above described requirements of symmetry are satisfied. If the light-emitting diodes that do not satisfy these symmetrical requirements are arranged, the same intensity of luminescence from the reflection point can no longer be obtained, a great difference is produced in the contrast corresponding to Qi and the accuracy of adjustment of η and θ deteriorates. Deterioration of accuracy may also occur when the contrast remains in a maximum value in the contrast maximization process and may also lead to drastic deterioration of accuracy of alignment depending on the operating conditions.
The condition necessary for the optical system in this embodiment is to efficiently guide the light emitted from the micro-reaction chambers 201 only to specific pixels and not to guide the light to other pixels. The most common method of achieving this is preferably one that efficiently forms an image of luminescence from the micro-reaction chambers in the image pickup element 103 using the lens system 104 having a small F value as shown in
However, if a combination lens such as a camera lens is used for optical system 104, distortion of the image occurs and it is not possible to make the positions of all the micro-reaction chambers in the plate align with the positions of all the pixels in a simple configuration.
Therefore, a rod lens array or micro lens array and optical fiber bundle may also be used in the third embodiment as well as the first and second embodiments.
In this case, adjustment of focus and the adjustment of angles φ and η become unnecessary. However, adjustment of X, Y and θ in the plane is performed using the above described process. Moreover, it is also possible to measure one flow cell using a plurality of image pickup elements using a plurality of these optical fiber bundles or a branched optical fiber bundle.
As a modification example, a fiber optic plate may be used for the multi-well titerplate 202 and light from the micro-reaction chambers 201 may be measured from the back of the plate 202. Light is measured from the back of the plate in the case of Non-Patent Document 2, but the micro-reaction chambers do not correspond one-to-one with pixels of the image pickup element.
However, even when light is measured from the back of the plate 202, the pixels and reaction chambers 201 can be made to correspond one-to-one with each other only through in-plane displacement and rotation by θ of the multi-well titerplate 202 with respect to the image pickup element 103 if the configuration of this embodiment (alignment mechanism 105 and alignment operation (
Moreover, a grating or a prism may be inserted into the lens system 104 to change detecting pixels depending on the light-emitting wavelength. In this case, when the light-emitting wavelengths of dATP, dTTP, dCTP and dTTP are different, this embodiment makes it possible to identify which base emits light at the position of a pixel that detects luminescence. In a word, fluorescent materials of different wavelengths for four types of dNTP are introduced, a reagent is put at a time to identify which base has extended by wavelength. The type of dNTP is identified in correspondence with a pixel which varies from one wavelength to another. However, when wavelength resolution does not take place, in the same way that one reaction chamber is made to correspond to one pixel, even if the reaction chamber is the same, if the wavelength is different, the wavelength should be made measurable by a different pixel to avoid crosstalk in this modification example. This enables a base array to be determined with high throughput and high accuracy. Describing more specifically, when one type of dNTP is put to cause extension reaction, the type of base is judged by whether or not extension reaction takes place, and on the other hand, in this modification example, only one base is allowed to extend and the type of base is made distinguishable by wavelength. This eliminates any such step that extension does occur even when a reagent is put, and therefore the analysis time is reduced by half (when the reagent is deposited one type at a time, if the array is random, extension reaction occurs at a probability of approximately 50%). Thus, the number of times the reagent is deposited is reduced by half, the analysis time is reduced by half and throughput improves two-fold.
The third embodiment has shown an example where alignment is realized in such a way that one light-emitting point 90 is made to correspond to one pixel. This embodiment shows an example where alignment is realized by making one light-emitting point correspond to four pixels.
Here, when correctly adjusted, the value of this contrast function diverges, and so a reciprocal is taken and the reciprocal is adjusted so as to approximate to minimization (that is, 0). Therefore, when the processing shown in the flow chart of
In this way, the contrast function that can be defined by arranging the light-emitting points at the boundary points displays an excellent characteristic as will be explained below. For a comparison,
Similarly, the values of Formula (2) are plotted in
A fifth embodiment shows an example where the number of light-emitting points is increased instead of four.
Furthermore, in these examples, the accuracy of a contrast function value is improved and accuracy of alignment is improved by arranging points at which luminescence is further reduced compared to the periphery (circles painted in black) at positions of some micro-reaction chambers 201 around the light-emitting points. The black painted parts in
In addition, it is more effective to define a contrast function by grouping instead of individually defining light-emitting points for alignment.
The chemiluminescent detection system of each embodiment detects chemical luminescence from reaction chambers using a nucleic acid analysis, phased synthesis of complementary strands in particular. A gene array analysis is executed using the detection result.
According to the chemiluminescent detection system of each embodiment, a plurality of reaction chambers and pixels of the image-pickup element correspond one-to-one with each other and the detected image is free of distortion. Therefore, the number of reaction chambers defined can be increased to the utmost and an analysis throughput can be analyzed precisely. Moreover, because the number of DNA samples which can be analyzed at a time can be increased to the number of pixels of a detection element, and it is thereby possible to manufacture the system at low cost, too.
Number | Date | Country | Kind |
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2007-113095 | Apr 2007 | JP | national |