JP3691837B2 - Biochip analyzer - Google Patents

Description

【Technical field】
[0001]
In the present invention, a specimen gene sample is labeled with a fluorescent dye, and this is reacted with a separate gene or gene product arranged on a large number of microspots on a biochip substrate, and the labeled sample is bound thereto. It relates to biochip analyzers that produce, react, and read biochips using a parallel assay method that measures the position of the chip and the degree of binding, especially for gene analysis and quantification of genes or gene products present in samples. Similarly, the present invention relates to a biochip analyzer used for protein analysis and immunology-related analysis.
[Background]
[0002]
In recent years, parallel assay methods in which a large number of samples and specimen samples are reacted in parallel and analyzed individually in the fields of gene analysis, protein analysis, and immunology have come to be used. This analysis method uses a biochip which is a glass plate or a plastic plate or a silicon wafer that is solid-phased on a large number of biological samples and arranged independently. This method has many advantages such as being able to acquire data quickly, saving reagents, and being able to analyze many simultaneously. A parallel gene analysis method using a substrate in which a large number of genes or gene products are plotted on a substrate, particularly called a microarray or gene chip (hereinafter collectively referred to as a biochip), has attracted particular attention and has become widely used. .
The gene analysis using oligonucleotides will be described as an example. This analysis method can also be used for protein analysis and immune-related analysis. Conventionally, the production, reaction, and reading of biochips have been performed separately using dedicated machines.
The problems related to biochip fabrication, reaction, and reading are described below.
Biochip manufacturing methods such as photolithography, jet printing using piezoelectric technology, and mechanical microspot have been developed. Among these, the photolithography method is a technology for producing a biochip using the exposure technology of the semiconductor element manufacturing method. Biochip production by this technique is carried out by selectively irradiating light through a photomask at a predetermined position of a predetermined oligonucleotide on a glass substrate, and the light that has passed through the unmasked region corresponds to four types of phospho The amidite reagent is activated to cause a coupling reaction, and in each coupling step, a base is added one by one in a predetermined region to synthesize and extend the oligonucleotide directly on the substrate. This method uses a photoactivation reaction to directly synthesize a large number of oligonucleotides with different sequences in a microspot on the substrate. Therefore, if oligonucleotide sequence data is available, a biochip can be produced. is there. However, since the above method has a drawback of requiring a large-scale apparatus according to the semiconductor element manufacturing method, the user of the biochip currently purchases and uses a biochip manufactured by a specialized manufacturer.
Non-patent document 1 or non-patent document 2 discloses the synthesis of oligonucleotides utilizing photolithography and a photo-activated coupling reaction.
However, the announced technology required a large-scale device. As an example of a miniaturized biochip manufacturing apparatus in this field, an apparatus using a digital micromirror device (hereinafter referred to as DMD) is proposed in Patent Document 1. The DMD is a reflective spatial light modulation element using a micromirror as an image element. As an example, 1280 × 1024 micromirrors arranged in two dimensions are commercially available. However, for the production of a highly reliable biochip, it is important to eliminate the variation due to the spot position, that is, the uniformity of the in-plane distribution of the exposure light as an optical condition. In order to ensure the optical conditions described above, the apparatus described in Patent Document 1 is not practical because an expensive optical device such as an integrator optical device using a fly-eye lens must be separately installed and used together. .
An exposure method and apparatus using DMD as a spatial light modulation element are proposed in Patent Document 2 and Patent Document 3. The above conventional exposure technique does not function due to the structure of the DMD. That is, as shown in FIG. 4, when the entire DMD is illuminated with convergent light, the light that illuminates the target sample selected by the tilted micromirror does not focus on the plane h parallel to the DMD installation surface. A confocal optical system cannot be constructed. Further, as shown in FIG. 5, even when the DMD is illuminated with parallel light, all the light for illuminating the selected target sample is condensed at one point by the tilted micromirror only with the objective lens. Therefore, the observation point of the sample is illuminated with light diffused from the condensing point. In addition, when the DMD is illuminated with parallel light, the spread of reflected light by the micromirror cannot be ignored.
In addition to the above reasons, illumination is performed with incident light perpendicular to DMD, which must be illuminated by introducing incident light from an oblique direction. Furthermore, an unstable state, that is, a state where the micromirror does not tilt (power OFF state) is used.
In the method and apparatus proposed in Patent Document 1, in an exposure apparatus used as a biochip manufacturing apparatus, measures are taken to eliminate variations due to the position of the exposure surface and to make the in-plane distribution of the exposure surface constant. Absent. Specifically, an expensive optical system using a fly-eye lens or the like is installed outside, and a means for making the illumination light uniform must be provided separately.
Next, a conventional technique related to a biochip reading method and apparatus will be described. Regardless of the biochip manufactured by the photolithographic method, the biochip manufactured by another method is also reacted with the fluorescently labeled test sample, and the fluorescence intensity of the sample bound to the micro spot on the biochip is detected. . A confocal scanning fluorescence detector is used for this detection.
Since the confocal scanning fluorescence detection apparatus has a shallow depth of focus and can exclude fluorescence emitted from other than the observation point out of focus, high-accuracy biochip reading is possible. However, the beam that excites fluorescence must be scanned or moved by moving the sample itself.
In the method of scanning the beam, it is necessary to create a flat detection field using an F-theta lens or the like, and it is difficult to employ an objective lens having a large numerical aperture. On the other hand, the method of scanning the sample has a drawback that the mechanical and optical design is complicated because the flatness corresponding to the shallow depth of focus of the confocal system must be maintained. As scanning means for solving this problem, an apparatus using a DMD as a spatial light modulator is disclosed as a scanning microscope in Patent Document 4, Patent Document 5, and the like.
When this disclosed apparatus is used for reading a biochip, there is a problem that variations occur between reading apparatuses. Since the biochip spot is very small, a shortage of fluorescence emission is inevitable. If this amount of fluorescent light emission is insufficient, the illumination light and detection sensitivity of the apparatus vary depending on the position, and the above problem occurs. That is, it is because the detection is not performed by uniform illumination, but the above two patents do not take measures. In addition, there are dedicated devices for producing and reading biochips, which are currently implemented individually.
[0003]
[Patent Document 1]
Japanese Unexamined Patent Publication No. 2000-40660
[Patent Document 2]
Japanese Patent Laid-Open No. 10-112579
[Patent Document 3]
Special table 2001-500628
[Patent Document 4]
U.S. Pat.No. 5,587,832
[Patent Document 5]
Japanese Patent Laid-Open No. 11-194275
[Non-Patent Document 1]
Foder et al. (1991) Science, Vol.251, P767
[Non-Patent Document 2]
Chee et al. (1996) Science, Vol.274, P610
DISCLOSURE OF THE INVENTION
[Problems to be solved by the invention]
[0004]
The main object of the present invention is to provide a biochip analyzer having biochip production, reaction, and reading functions by solving the problems of biochip analysis using a conventional parallel assay method. .
Another object of the present invention is to provide a biochip analyzer that can produce a biochip with high reproducibility by using a spatial light modulation element having an illumination light correction function for realizing uniform illumination. is there.
Another object of the present invention is to provide a biochip analyzer capable of high-resolution, high-speed scanning and high reliability, eliminating variations due to the position of illumination excitation light and inspection sensitivity when reading a biochip. It is to be.
[Means for Solving the Problems]
[0005]
The biochip analyzer according to claim 1 includes a first optical system composed of a single spatial light modulation element and a planar light source for exposure illumination, and the spatial light modulation element using the planar light source for exposure illumination. The spatial light modulator is formed by an exposure pattern determined from an array layout of spots of nucleotides or peptides or sugar chains arranged on a biochip and a primary structure of nucleotides or peptides or sugar chains on each spot. And selectively exposing a spot position on the biochip substrate where nucleotides, peptides or sugar chains are arranged, causing a photo-activated coupling reaction at a predetermined position with a plurality of types of bases or amino acids or sugars. A biochip preparation means for synthesizing and extending a peptide or a sugar chain is reacted with the prepared biochip and a fluorescently labeled test sample. And a second optical system composed of a fluorescence excitation planar light source and an imaging means for performing fluorescence imaging through the second optical system, wherein the spatial light modulation element is used as the fluorescence excitation planar light source. The imaging device illuminates and selectively illuminates a micro area on the biochip reacted with the test sample by the biochip reaction means, and selectively captures only the fluorescence emitted from the micro area by the imaging means. A biochip reading means for performing confocal scanning, and a spatial light modulation element when a spatial light modulation element is imaged with a planar light source for exposure illumination by temporarily setting a uniform fluorescent plate instead of a sample to be exposed in advance The exposure light distribution measuring means for measuring the in-plane distribution of the exposure light before correction corresponding to each pixel and a uniform fluorescent plate are temporarily installed instead of the biochip in advance, An excitation light distribution measuring means for measuring the in-plane distribution of the excitation light intensity before correction corresponding to each pixel of the spatial light modulation element when the interspace light modulation element is imaged and illuminated, and the exposure light distribution measuring means The measured reciprocal of the intensity of the exposure light before correction corresponding to each pixel is stored in a table, the exposure light correction means for controlling each pixel according to a predetermined exposure pattern with reference to the table at the time of exposure, Illumination light correction means for controlling each pixel by referring to the table when reading and storing the reciprocal of the intensity of excitation light before correction corresponding to each pixel measured by the excitation light distribution measurement means, And a control means for controlling each means.
[0006]
The biochip analyzer according to claim 2 comprises an optical system composed of a single spatial light modulator and a planar light source for exposure illumination, and the spatial light modulator is connected by the planar light source for exposure illumination. The spatial light modulator is controlled on the biochip substrate according to the array layout of the spots of nucleotides, peptides, or sugar chains that are image-illuminated and arranged on the biochip and the primary structure of nucleotides, peptides, or sugar chains on each spot Bios in which the nucleotide, peptide, or sugar chain spot position is selectively exposed to cause a photo-activated coupling reaction at a predetermined position with a plurality of types of bases, amino acids, or sugars, and the nucleotide, peptide, or sugar chain is synthesized and elongated. A spatial light modulation element using a planar light source for exposure illumination, in which a chip preparation means and a uniform fluorescent plate are temporarily installed instead of the sample to be exposed. Exposure light distribution measuring means for measuring an in-plane distribution of exposure light intensity before correction corresponding to each pixel of the spatial light modulation element when imaged illumination is performed, and each pixel measured by the exposure light distribution measuring means Exposure light correction means for storing the reciprocal of the intensity of the exposure light before correction corresponding to the table, and storing each table in accordance with a predetermined exposure pattern with reference to the table during exposure. Features.
[0007]
The biochip analyzer according to claim 6 comprises an optical system composed of a single spatial light modulator and a planar light source for fluorescence excitation, and imaging means for performing fluorescence imaging through the optical system, Only a fluorescent light emitted from the microscopic area by selectively illuminating a microscopic area on the biochip reacted with the test sample by the biochip reaction means by illuminating the spatial light modulator with a planar light source. A biochip reading means for performing confocal scanning to selectively pick up images with the imaging means, and a uniform fluorescent plate in advance instead of the biochip, and the spatial light modulator is imaged by the planar light source for fluorescence excitation Excitation light distribution measuring means for measuring the in-plane distribution of the excitation light intensity before correction corresponding to each pixel of the spatial light modulation element, and corresponding to each pixel measured by the excitation light distribution measuring means. Storing the reciprocal of the intensity of the excitation light before the correction table of to an excitation light correcting means at the time of reading by referring to the table, to control each pixel,
It is characterized by having.
【The invention's effect】
[0008]
According to the present invention, a biochip analyzer having biochip production, reaction, and reading functions uses a spatial light modulation element having an illumination light correction function that realizes uniform illumination. It is possible to produce highly biochips. Further, when reading the biochip, it is possible to eliminate variations due to the position of the illumination excitation light and the inspection sensitivity, perform high-speed scanning with high resolution, and provide high reliability.
BEST MODE FOR CARRYING OUT THE INVENTION
[0009]
Embodiments of the present invention will be described with reference to the accompanying drawings.
[Example 1]
[0010]
The biochip analyzer in the first embodiment can perform a series of steps of biochip production, reaction with a test sample, and reading. In the present embodiment, the illumination for fluorescence excitation at the time of biochip production and the fluorescence excitation illumination at the time of biochip reading are configured by a common optical system, and the exposure light monitor at the time of biochip production and the imaging at the time of biochip reading and the excitation light monitor This is a biochip analyzer comprising a common optical system.
[0011]
In FIG. 1, a biochip 1, an objective lens 2, an imaging lens 3, and a DMD 6 that is a spatial light modulation element are installed in the center of the optical system, and a central optical axis is formed. The objective lens 2 and the imaging lens 3 operate as follows when the biochip 1 is manufactured and read. At the time of production, the biochip 1 is illuminated and exposed, and at the time of reading, the surface of the DMD 6 is imaged on the biochip 1, and further, the fluorescence emitted from the spot of the biochip 1 is imaged on the surface of the DMD 6.
[0012]
The light from the light source 20 enters the fiber optic plate 24 whose exit end is cut obliquely, and a planar light source is formed at the exit end. A real image of the planar light source is formed on the mirror surface of the DMD 6 via the dichroic mirror 26 by the lens function of the lens 25. The exit end of the fiber optic plate 24, the lens 25, and the mirror surface of the DMD 6 satisfy the Scheinproof condition. In the present embodiment, a symptom system in which the lens is tilted so as to satisfy the Scheimpflug condition is employed, but an imaging system in which the lens is shifted can also be employed.
[0013]
The light source 20 is composed of a light source A20, a light source B20, and a light source C20, and can be subjected to two-wavelength fluorescence excitation during reading and exposure during production. A mirror 23 (23A, 23B) is installed to switch the three light sources. When the mirror 23A is introduced at a predetermined position, the light source A20 is used, and when the mirror 23B is introduced at a predetermined position (solid line in the figure), the light source B20 is used. Further, the mirrors 23A and 23B are retracted to the positions of the broken lines in the drawing, and when neither mirror is introduced, the exposure light source C20 is used. The light source of each wavelength of the optical system can be added as necessary.
[0014]
Next, an embodiment of the planar light source will be described. The planar light source can also be constituted by a surface light emitter such as an electroluminescence (EL) element. However, if the light emitting layer is thick, when the sample is imaged and illuminated, the image in the sample thickness direction (Z direction) becomes large, and the resolution in the Z direction does not increase. The thinner the planar light source is, the more advantageous it is to increase the resolution in the Z direction. From the above consideration, a thin film scatterer with a very thin scattering layer illuminated with predetermined light can also be used as a planar light source. For example, Light Shaping Duffuser (POC USA CA. Torrance) is known as a thin film scatterer.
[0015]
In the present invention, a planar light source is imaged and illuminated on a spatial light modulation element, and imaging is performed in correspondence with this pixel. Therefore, one or a plurality of light emission points correspond to one pixel of the spatial light modulation element. If so, a discrete array of point light sources can be used instead of a continuous surface light emitter such as an EL element. Examples of the on-plane array include a two-dimensional array of LEDs and a laser diode, and a two-dimensional array of microlenses and pinholes illuminated by a predetermined method. The on-surface array can be regarded as a fine and sufficient point light source, and is configured by a two-dimensional array of point light sources corresponding to one-to-one or a plurality of one-to-one with respect to the pixels of the spatial light modulator by combining with a reduction optical system. .
[0016]
Similarly, predetermined light is incident from one end of an optical fiber bundle in which a number of optical fibers are bundled, and the light exit end on the opposite side, the light exit end of the fiber optic plate, and the light exit end of the urexite are also flat. Can be used as a light source. For example, in a fiber optic plate in which 3 μ-diameter fibers are bundled, 16 to 25 point light sources that are emission ends of one fiber are allocated to one pixel of the spatial light modulator. In the case of DMD, one pixel of the spatial light modulation element is a 16 μ square micromirror. In the case of a reflective liquid crystal panel, it is a 13.5 μ square liquid crystal element. When the illumination light correction means is provided, it is sufficient that at least one point light source is allocated to any pixel of the spatial light modulator.
[0017]
The imaging system includes an imaging lens 27, a filter 28 (28A, 28B, 28C), and a CCD 29, and captures a fluorescent real image of the biochip 1. The filter 28 is installed so as to be selectable between the imaging lens 27 and the CCD 29, and transmits a wavelength corresponding to the fluorescent dye. For example, band pass filters having center wavelengths of 520 nm, 570 nm, and 670 nm are installed in the FITC, Cy3, and Cy5 fluorescent dyes, respectively. The DMD 6, the imaging lens 27, and the CCD 29 satisfy the Scheimpflug condition. This imaging system is also used for uniformizing excitation light or exposure light. Similarly, it is also used as a monitor in measuring the in-plane distribution of light.
[0018]
Computer system 3 is software for offline control, such as DMD6 control, chemical solution management, temperature management, CCD29 control, illumination light correction that makes exposure light and excitation light uniform, which is necessary for analysis of biochip 1 for parallel assay. It comprises a computer 30, a display device 31, a keyboard 32, and a pointing device 33 that are equipped with analysis software and user interface software for the biochip 1 to be used and are connected to a network such as the Internet.
[0019]
The constant temperature chemical supply system 4 includes a flow cell 41 in which the worn biochip 1 forms an upper lid and maintains a set temperature, a reaction reagent container 42 in which an electromagnetic valve 44 controlled from the computer 30 is installed, a cleaning liquid container 43, A test sample introduction means is provided, and a chemical solution and a cleaning solution are supplied in synchronization with each step of the photo-activated coupling reaction at the time of biochip production. The flow cell 41 is used not only for the production of the biochip 1 but also for the reaction with the test sample and the reading. The above reaction is particularly called hybridization when it is used for gene analysis.
[0020]
After the production of the biochip 1 is completed, a test sample is introduced into the flow cell 41 from the introduction port 45 and reacted. Reading is performed with the biochip 1 in a wet state or a dry state. Reading in the wet state is characterized by a high fluorescence yield, and is performed by exposing the biochip 1 to a liquid. Reading in the dry state is performed by introducing clean dry nitrogen gas from the inlet 45 and drying the biochip 1. The temperature of the flow cell 41 is configured to be able to be set to a predetermined value, and the influence of the temperature change when the biochip 1 is read is examined. In addition, an adjustment screw (not shown) for tilting the entire apparatus by 10 to 20 degrees is used. This is for expelling bubbles generated in the flow cell and analyzing the biochip 1 stably.
[0021]
According to the present embodiment, all the analysis operations of the biochip 1 can be performed without attaching or detaching the biochip 1 from the flow cell 41, so that the efficiency can be improved and the contamination is small.
The DMD 6 employed as the spatial light modulation element is a minute light deflecting element and has a structure in which a large number of minute micromirrors are arranged. Individual micromirrors can be individually tilted at two predetermined angles by digital control means. That is, each micromirror has two stable states, and has a function of switching light introduced from the outside to optical paths in two directions. DMD6 has several types of micromirrors such as 800 × 600, 1024 × 768, 1280 × 1024, 1920 × 1080, and two types of tilt angles are available: ± 10 degrees and ± 12 degrees. Any of them can be applied. By controlling DMD6, pattern exposure (during production) and confocal scanning fluorescence detection (during reading) were realized.
[0022]
DMD6 is used for both biochip fabrication and biochip reading. At the time of production, the light source unit 20 is switched to the exposure light source C. The light from the exposure light source C enters the fiber optic plate 24, and a planar light source formed at the exit end illuminates the DMD 6. The imaging illumination light is reflected by the DMD 6 micromirror tilted in accordance with the exposure pattern. The reflected light forms an image through the imaging lens 3 and the objective lens 2 and a predetermined spot position on the biochip 1 is exposed.
At the time of reading, the exposure light source C used for production is switched to the fluorescence excitation light source A or B having a different wavelength, and the mirror 23A or 23B is inserted in the optical path. The light from the light source A or B enters the fiber optic plate 24, and a planar light source formed at the exit end is imaged on the DMD 6 to become a second light source. A microscopic region of the second light source is cut out by the tilted micromirror, and reduced image is formed on the biochip by the optical path formed by the imaging lens 3 and the objective lens 2. Fluorescence emitted from this imaging point travels backward along the optical path and forms an image on the micromirror. The image on the micromirror is selected only for fluorescence by the dichroic mirror 26, imaged again by the imaging lens 27, and imaged by the CCD 29. At this time, the S / N is increased by excluding the excitation light mixed by the fluorescent filter 28 (28A, 28B, 28C).
[0023]
In this embodiment, a reduction optical system including the objective lens 2 and the imaging lens 3 is used, but the present invention is not limited to this. As the optical system, an equal-magnification optical system or an enlargement optical system is selected according to the size of the biochip 1.
[0024]
The uniformization of exposure light during the production of a biochip will be described. A uniform fluorescent plate is installed on the flow cell 41, the fluorescence intensity corresponding to each micromirror is monitored by the CCD 15, and the intensity distribution of the exposure light before being uniformed is measured. Next, an exposure light correction table is created by calculating the reciprocal of the fluorescence intensity for each micromirror so that the exposure light intensity at the position corresponding to each micromirror has a constant value. The following two methods use an exposure light correction table.
[0025]
The first method controls the exposure time for each micromirror. When producing a biochip, the exposure light correction table is referred to, and the exposure time for each micromirror is variably controlled to shorten or extend. In the second method, the exposure time is kept constant for all micromirrors, but intensity modulation is performed by applying binary pulse width modulation (BPWM) to each micromirror. At the time of manufacture, the exposure light correction table is referred to, and intensity correction is performed on the reflected light of each micromirror, so that uniform exposure light can be obtained as a whole. This exposure light correction method is used not only at the time of manufacturing a biochip but also at the time of reading described later.
[0026]
An illumination method for forming a confocal optical system at the time of reading a biochip will be described. Light from the light source passes through the fiber optical plate 24, and a planar light source is generated on the exit end face thereof. When this planar light source is imaged on the surface of DMD 6, a real image of the planar light source is generated on the surface of DMD 6. By tilting one micromirror selected on the DMD 6, the extracted microscopic area (16 μ square) of the planar light source is illuminated on the spot of the biochip 1 through the objective lens 2. That is, the planar light source imaged on the selected micromirror is reduced and imaged through the objective lens 2 on the minute area of the specified reading surface of the biochip 1 to illuminate the minute area. Only the fluorescence emitted from this minute region is imaged through the aforementioned micromirror. When one tilting micromirror is sequentially switched and scanned on the DMD 6, the biochip 1 is scanned by confocal fluorescence.
[0027]
In the above-described confocal optical system illumination method, one micromirror is scanned, but the present invention is not limited to this. As another method, if the micromirrors are sufficiently separated on the DMD 6 to maintain a spatial light modulation function for removing defocused light and stray light, a plurality of micromirrors can be simultaneously translated and scanned. For example, high speed can be realized by scanning 2000 micromirrors in parallel.
[0028]
The confocal fluorescence scanning means and the excitation light correcting means are realized simultaneously by a single spatial light modulation element. The intensity distribution of the illumination light at the time of reading is adjusted by a (uniform) uniform fluorescent screen. The fluorescence intensity before homogenization is measured, and an excitation light correction table is created. As many excitation light correction tables as the number of illumination light sources are provided. These excitation light correction tables are referred to according to the light source used at the time of reading, and variations in illumination light and detection sensitivity depending on the position are corrected by the first or second exposure light correction method.
[0029]
The above correction function is realized by a computer and software installed therein.
[Example 2]
[0030]
A second embodiment of the present invention will be described. The second embodiment is a biochip analyzer using a reflective liquid crystal panel called LCOS (Liquid Crystalon Silicon) as a spatial light modulator.
[0031]
The reflection type liquid crystal panel is a liquid crystal cell in which individual pixels are minute, and the polarization direction of light reflected by an applied voltage (analog amount) is controlled. Combining a reflective liquid crystal panel and PBS (Polarized Beam Splitter) can be used for spatial light modulation and intensity modulation. The reflective liquid crystal panel has, for example, 1365 × 1024 liquid crystal cells each having a size of 13.5 μ × 13.5 μ integrated. In addition, there are reflective liquid crystal panels having various liquid crystal cell sizes and integration numbers. The present invention can also use a transmissive liquid crystal. The transmissive LCD panel is partly covered by switching elements such as TFTs, so the effective pixel area does not cover the entire surface, and the entire screen cannot be controlled smoothly. .
[0032]
FIG. 2 shows the optical system of the second embodiment, in which the computer system and the constant temperature chemical supply system shown in FIG. 1 are omitted. An exposure light source 20C used at the time of manufacturing the biochip and an excitation light source 20A used at the time of reading the biochip are installed. These light sources are configured to be switched by introducing a mirror 23C into the optical path. That is, when the biochip is manufactured, the mirror 23C is removed from the optical path and the exposure light source 20C is used. When the biochip is read, the mirror 23C is introduced into the optical path and the excitation light source 20A is used. Light from any of the light sources is introduced into the fiber optic plate 24, and a planar light source is generated at the exit end. An imaging lens 25 that forms an image of this planar light source on the reflective liquid crystal panel 60, a dichroic mirror 26 that transmits fluorescence and reflects excitation light and exposure light, and a PBS 61 that branches light depending on the polarization angle are installed. An imaging lens 3 and an objective lens 2 are installed between the biochip 1 and the PBS 61. The surface of the reflective liquid crystal panel 60 is imaged on the surface of the biochip 1 via the PBS 61. The CCD 29 is used for capturing a fluorescent image or monitoring illumination light. The surface of the reflective liquid crystal panel 60 is imaged on the CCD 29 by the imaging lens 27.
[0033]
In the above optical system, the polarization angle of the reflected light from the liquid crystal cell is controlled by an analog voltage applied to the liquid crystal cell. The amount of light is controlled by the branching action of the PBS 61 light transmitted or reflected by the controlled polarization angle of the light. Specifically, when no voltage is applied to the liquid crystal cell, light from the light source passes through the PBS 61 and illuminates the biochip. When a predetermined maximum voltage is applied to the liquid crystal cell, the light from the light source is reflected by the PBS 61 and does not illuminate the biochip. When an intermediate voltage equal to or lower than the maximum voltage is applied to the liquid crystal cell, the biochip is illuminated with a light amount inversely proportional to the applied voltage.
[0034]
In this embodiment, an analog RGB image output terminal of a computer and a liquid crystal panel control board (not shown) are connected. The spatial light modulation function is realized by controlling the voltage of each liquid crystal cell described above. The spatial light modulation function using a liquid crystal cell is a mask function that generates an exposure pattern during biochip fabrication, a confocal fluorescence scanning function during biochip reading, or illumination that is used both during biochip fabrication and reading. Used as a light correction function. Since these functions are the same as those in the first embodiment, the description thereof is omitted.
[Example 3]
[0035]
A third embodiment of the present invention will be described. The third embodiment is a biochip analyzer that enables two-wavelength analysis without mechanical switching.
In reading a single biochip, it may be necessary to read two wavelengths of fluorescence. For example, the RNA of the lesion site and the RNA of the normal site of the same organ are extracted, and the cDNA obtained by reverse transcription of the RNA of each site is labeled with two types of fluorescent dyes having different wavelengths, such as Cy3 and Cy5, and then mixed. For example, analysis is performed by reacting to a single biochip and comparing the respective ratios.
[0036]
FIG. 3 shows the optical system of the third embodiment, in which the computer system and the constant temperature chemical supply system are omitted. The present embodiment is a biochip analyzer capable of reading two wavelengths, in which two sets of the fluorescence excitation illumination optical system and the imaging optical system of the first embodiment are arranged independently for each wavelength.
[0037]
At the center of the optical system, a biochip 1, an objective lens 2, an imaging lens 3, and a DMD 6 that is a spatial light modulation element are installed to form a central optical axis. The objective lens 2 and the imaging lens 3 illuminate and expose the biochip 1 when producing the biochip. Further, at the time of reading, the surface of DMD 4 is imaged on biochip 1 to excite a predetermined spot, and the fluorescence emitted from the excited spot is imaged on the surface of DMD 4.
[0038]
A optical system (left) and B optical system (right) having different fluorescence detection wavelengths are installed symmetrically with respect to the central optical axis. The A optical system constitutes an exposure light illumination system using a light source C in addition to a fluorescence excitation illumination system and an imaging system having a specific wavelength by the light source A. The B optical system constitutes a fluorescence excitation illumination system and an imaging system having a wavelength different from that of the A optical system. Both optical systems are composed of different optical elements for the respective wavelengths of the light sources A and B for fluorescence excitation, the light source C for exposure illumination, the dichroic mirrors 26A and 26B, and the bandpass filters 28A and 28B. Other elements are the same. Here, only the A optical system will be described.
[0039]
The A optical system includes a light source A having a switchable specific wavelength and a light source C for exposure light, makes light from the switched light source incident on the fiber optical plate 24, and illuminates an image of the planar light source generated at the exit end. The lens 25 forms an image on the surface of the DMD 6. Between the illumination lens 25 and the DMD 6, a dichroic mirror 26A for reflecting excitation light or exposure light and transmitting fluorescence is installed.
[0040]
At the time of producing the biochip, the biochip is produced by switching to the light source C as in the first embodiment. At the time of biochip reading, the biochip 1 is read by the excitation light having a specific wavelength of the switched light source A as in the first embodiment.
With the above configuration, the biochip 1 is scanned by confocal fluorescence, and the fluorescence information of the biochip 1 can be read.
[0041]
The biochip reading using both the A optical system and the B optical system will be described based on a specific example. For example, when the A optical system performs reading using Cy3 as a fluorescent dye, and the B optical system performs reading using Cy5, light sources A and B for fluorescence excitation, dichroic mirrors 26A and 26B, and a bandpass filter 28A 28B is installed with a suitable fluorescence wavelength of 570 nm and 670 nm. Before measurement, a uniform Cy3 fluorescent plate is installed instead of a biochip, and a correction table for the A optical system is created. Similarly, a correction table for the Cy5 B optical system is created.
The biochip 1 is installed, first, the fluorescence information of Cy3 is read by the A optical system, and then the fluorescence information of Cy5 is read by switching to the B optical system.
[0042]
According to the present embodiment, mechanical switching of the light source and the filter is unnecessary, and fluorescence of two wavelengths can be sequentially measured only by turning on the light source and switching of the CCD to be used.
[0043]
Another embodiment of the present invention will be described. In this embodiment, a biochip is produced, reacted, and read using the biochip analyzer of each of the above examples, and data necessary for producing the biochip is provided to the client and read from the client. The present invention provides a biochip analysis online system in which a biochip information center that performs biochip analysis requested by a client based on data is connected to a computer network.
[0044]
This system provides information consisting of a set of oligonucleotide sequence data, a set of sequence data of protein amino acids, and the primary structure of nucleotides, peptides, or sugar chains via a computer network, and collects reading results from clients. The analysis result is returned to the client again. The online system charges for providing information necessary for biochip production and for analysis based on the read data.
[0045]
In FIG. 6, the biochip analyzer of the present invention is installed on the client side. The Biochip Information Center is equipped with a system body that contains an analysis program and a database that stores various biochip production data and biochip analysis data. The computer system 3 of the biochip analyzer and the system main body of the biochip information center are connected by a computer network. There are multiple clients but only one is shown.
[0046]
The system main body incorporates a biochip production processing program and a biochip analysis processing program, and a biochip production database and analysis database are installed. The biochip production database contains a set of base sequences and amino acid sequences for gene-related analysis and protein analysis-related data required for biochip production, and a predetermined data structure for the spot layout on the biochip. Accumulated. The biochip analysis database stores classification information of known genes and proteins that are referred to when performing analysis.
[0047]
The processing flow of this system will be described with reference to FIG. After the preparation for biochip production is completed, the client connects the biochip analyzer to the biochip information center, communicates with the biochip production processing program through the network, and requests a list of biochips. A biochip according to the analysis item is selectively ordered, such as a biochip displayed in a list, such as a biochip for analyzing environmental hormone responsive genes. The biochip information center calculates a charge based on the biochip received from the client and the analysis item. Thereafter, the biochip production data including the set of base sequence data of each spot of the selected biochip and the array layout of the spots on the biochip is transmitted, and the charge is notified. Upon receiving the biochip production data, the biochip analyzer performs biochip production.
[0048]
The prepared biochip is manually or in response to a biochip completion trigger signal, the test sample is automatically introduced from the test sample introduction port, and a reaction such as hybridization is performed. The biochip reading is automatically started upon receiving a biochip reaction end trigger signal, and the read data is stored in the memory. The client requests the analysis by designating the biochip to the biochip information center. The biochip information center starts a processing program corresponding to biochip analysis in response to a request from the client, and transmits an analysis item list to the client. The analysis item list includes various processing and analysis items such as visualization processing, cluster analysis, structural hierarchy analysis, and polymorphism analysis. The client selects and orders a predetermined item from the displayed analysis item list, and transmits biochip reading data. The biochip information center receives an analysis item for a designated biochip, performs billing calculation, receives biochip reading data, performs processing and analysis based on the selected analysis item, and transmits the result. The client receives the analysis result and the billing information, displays the confirmation information, confirms the content, and stores it in the memory.
[0049]
This system does not limit the biochip analyzer of the present invention to a limited client. The biochip analyzer of the present invention, the biochip reader, or other existing biochip production or read-only machines compatible with the network can also be used. In addition, this system is connected to the network of the biochip analyzer of the present invention and used in various forms such as analysis from reading of biochips produced by other devices, or analysis from biochip read data prepared in advance. Can do. Furthermore, using this system, it is possible to read biochip read data recorded on a recording medium such as a CD or DVD and request analysis from the biochip information center.
[0050]
According to this system, immediately after communication with the biochip production support processing program on the client side, the biochip production processing, reaction, reading, A series of analyzes using the biochip for analysis is performed fully automatically. This fully automatic operation is performed without operator intervention during the series of analysis operations.
[0051]
Other embodiments of the invention
[0052]
The present invention is not limited to the biochip analyzer of the above embodiment, but can also be applied to an apparatus configured by any one or a combination of biochip production, reaction, and reading functions.
In addition to biochips, the present invention can be applied to the measurement of fluorescence intensity distribution when the conventional Southern blot and Northern blot methods are performed by labeling / detection with a fluorescent dye instead of labeling / detection with a radioisotope. The Southern blot and Northern blot methods described above are analysis methods using hybridization. Examples of the use include measurement of fluorescence intensity distribution of, for example, nylon, nitrocellulose film, and gel film. Further, it can be used not only for the measurement of fluorescence intensity distribution but also for the purpose of measuring the intensity distribution of chemiluminescence in experiments in which labeling is performed with a chemiluminescent substance that does not require excitation light. In this case, in order to correct the illumination light and the detection sensitivity, a sample that uniformly chemiluminescence is used instead of the uniform fluorescent plate used in this embodiment.
[0053]
Industrial applicability
[0054]
(1) Biochip analyzer
The spatial light modulation element can be realized by control from software without using a photomask conventionally used in photolithography.
Unlike the conventional method of uniform illumination using hardware such as a fly-eye lens for exposure light correction, the present invention corrects exposure light for each device, so that variations between devices can be eliminated. It is possible to flexibly cope with switching and replacement of light sources. Further, since the exposure light correction means can be combined with the spatial light modulation element that generates the exposure pattern, it contributes to downsizing and cost reduction of the apparatus.
The reading of the biochip can be realized by controlling the conventional confocal scanning function with software using a spatial light modulator. In addition, the illumination light intensity is adjusted for each spatial light modulation element to improve the in-plane distribution of the illumination light, and variations in illumination light and detection sensitivity due to position are corrected.
[0055]
(2) Biochip online analysis system
The biochip analyzer on the client side and the system body of the biochip information center are connected by a computer network. The biochip analyzer receives the set information of the biochip sequence data and the primary structure data ordered online from the biochip information center. The biochip analyzer performs the steps of biochip production, reaction, and reading based on the above set information, and the biochip reading data is collected by the biochip information center and is analyzed according to the analysis items specified by the client. Perform biochip analysis. The analysis data is transmitted to the client. As a result, the client can analyze the test sample on hand.
[Brief description of the drawings]
[0056]
FIG. 1 is a system configuration diagram of an optical system and a control system of a biochip analyzer using a DMD according to a first embodiment of the present invention.
FIG. 2 is a system configuration diagram of an optical system and a control system of a biochip analyzer using a reflective liquid crystal panel according to a second embodiment of the present invention.
FIG. 3 is a system configuration diagram of an optical system of a biochip analyzer capable of analyzing with two wavelengths according to a third embodiment of the present invention.
FIG. 4 is a diagram showing a focal position when a DMD is illuminated with focused light.
FIG. 5 is a diagram showing a focal position when a DMD is illuminated with parallel light.
FIG. 6 is a conceptual diagram of an online analysis system and a flow chart from biochip production to analysis.

Claims (15)

A single spatial light modulator;
A spot of nucleotides, peptides, or sugar chains that is provided with a first optical system composed of a planar light source for exposure illumination, illuminates the spatial light modulation element with the planar light source for exposure illumination, and is arranged on a biochip. The spatial position of the spot on the biochip substrate is determined by controlling the spatial light modulation element according to an exposure pattern determined from the sequence layout of the nucleotide and the primary structure of the nucleotide, peptide or sugar chain on each spot. A biochip production means for selectively exposing, causing a photo-activated coupling reaction at a predetermined position of a plurality of types of bases or amino acids or sugars, and performing synthetic elongation of nucleotides or peptides or sugar chains;
Biochip reaction means for reacting the prepared biochip with a fluorescently labeled test sample;
A second optical system composed of a planar light source for fluorescence excitation, and an imaging means for performing fluorescence imaging through the second optical system, image-illuminating the planar light source for fluorescence excitation on the spatial light modulator, Bio for performing confocal scanning in which a micro area on a bio chip reacted with a test sample by a bio chip reaction means is selectively illuminated and only the fluorescence emitted from the micro area is selectively imaged by the imaging means. Chip reading means;
Preliminary exposure light corresponding to each pixel of the spatial light modulation element when a uniform fluorescent plate is temporarily set in place of the sample to be exposed and the spatial light modulation element is imaged and illuminated by the planar light source for exposure illumination. Exposure light distribution measuring means for measuring the in-plane distribution of
Preliminary excitation light intensity corresponding to each pixel of the spatial light modulator when a uniform fluorescent plate is temporarily placed instead of a biochip and the spatial light modulator is imaged and illuminated by the planar light source for fluorescence excitation Excitation light distribution measuring means for measuring the in-plane distribution of
The reciprocal of the intensity of exposure light before correction corresponding to each pixel measured by the exposure light distribution measuring means is stored in a table, and each pixel is controlled according to a predetermined exposure pattern by referring to the table during exposure. Exposure light correction means;
Illumination light correction means for controlling each pixel by reading the table and storing the reciprocal of the intensity of the excitation light before correction corresponding to each pixel measured by the excitation light distribution measurement means. ,
Control means for controlling each means;
A biochip analyzer characterized by comprising: A single spatial light modulator;
An array of nucleotides, peptides, or sugar chain spots that are provided with an optical system composed of a planar light source for exposure illumination, image-illuminate the spatial light modulator with the planar light source for exposure illumination, and are arranged on a biochip. The spatial light modulator is controlled by the layout and the primary structure of nucleotides, peptides or sugar chains on each spot to selectively expose the spot positions of nucleotides, peptides or sugar chains on the biochip substrate, and a plurality of types of bases Alternatively, a biochip production means for causing a photo-activated coupling reaction at a predetermined position of an amino acid or a sugar and performing a synthetic elongation of a nucleotide, a peptide or a sugar chain,
Preliminary exposure light corresponding to each pixel of the spatial light modulation element when a uniform fluorescent plate is temporarily set in place of the sample to be exposed and the spatial light modulation element is imaged and illuminated by the planar light source for exposure illumination. Exposure light distribution measuring means for measuring the in-plane distribution of intensity;
The reciprocal of the intensity of exposure light before correction corresponding to each pixel measured by the exposure light distribution measuring means is stored in a table, and each pixel is controlled according to a predetermined exposure pattern by referring to the table during exposure. Exposure light correction means;
A biochip analyzer characterized by comprising: 3. The biochip analyzer according to claim 2, wherein the planar light source is constituted by a fiber optic plate. 3. The biochip analyzer according to claim 2, wherein the planar light source is composed of urexite. 3. The biochip analyzer according to claim 2, wherein the planar light source is constituted by a thin film scatterer. A single spatial light modulator;
An optical system comprising a planar light source for fluorescence excitation, and an imaging means for performing fluorescence imaging through the optical system, and image-illuminating the planar light source for fluorescence excitation onto the spatial light modulator, and the biochip reaction means A biochip reading means for performing confocal scanning for selectively illuminating only a fluorescent region emitted from the microscopic area by selectively illuminating a microscopic area on the biochip reacted with the test sample by ,
Preliminary excitation light intensity corresponding to each pixel of the spatial light modulator when a uniform fluorescent plate is temporarily placed instead of a biochip and the spatial light modulator is imaged and illuminated by the planar light source for fluorescence excitation Excitation light distribution measuring means for measuring the in-plane distribution of
Excitation light correction means for controlling each pixel by reading and storing the reciprocal of the intensity of excitation light before correction corresponding to each pixel measured by the excitation light distribution measurement means, and referring to the table when reading ,
A biochip analyzer characterized by comprising: The biochip analyzer according to any one of claims 1, 2, and 6, wherein the spatial light modulator is a DMD. 7. The biochip analyzer according to claim 1, wherein the spatial light modulator is a reflective liquid crystal panel. 3. The flow cell according to claim 1 or 2, wherein a biochip is placed and manufactured, reacted, and read under temperature control of the biochip, and a chemical solution for synthesizing and extending nucleotides, peptides, or sugar chains on the biochip. And means for supplying the device. 3. The biochip analyzer according to claim 1, wherein the exposure light correction means controls the exposure pattern according to an exposure time required for each pixel of the spatial light modulator. 3. The biochip analyzer according to claim 1 or 2, wherein the exposure light correction means controls the exposure pattern for each pixel of the spatial light modulator by the intensity of the exposure light. 7. The biochip analyzer according to claim 1, wherein the excitation light correction means controls the excitation time for each pixel of the spatial light modulator. 7. The biochip analyzer according to claim 1, wherein the excitation light correcting means controls the intensity of the excitation light for each pixel of the spatial light modulator. 8. The biochip analyzer according to claim 7, wherein the DMD has a plurality of micromirrors simultaneously translated. 2. The biochip analyzer according to claim 1, wherein two sets of biochip reading systems having different fluorescence wavelengths are arranged independently.

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