Abstract:
A test piece for use in biological analyses includes a plurality of different known specific binding substances disposed in predetermined positions on a substrate. The specific binding substances are disposed on a plurality of surfaces provided by the substrate and arranged in the direction of thickness of the substrate.

Description:
This is a DIVISIONAL of application Ser. No. 09/572,886 filed May 16, 2000, the disclosure of which is incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to a test piece for use in DNA analysis, immunological analysis, and the like, and a system for reading out image information from the test piece. 
     2. Description of the Related Art 
     Recently, genetic engineering has exhibited rapid progress, and the human genome project for decoding the base sequence of human genomes which amount to 100,000 in number is progressing. 
     Further, enzyme immunoassay, fluorescent antibody technique and the like utilizing antigen-antibody reactions have been used in diagnoses and studies, and studies for searching DNAs which affect genetic diseases are now progressing. In such a situation, a microarray technique is now attracting attention. 
     In the microarray technique, a microarray chip (sometimes called a DNA chip) comprising a plurality of known cDNAs (an example of specific binding substances) coated in a matrix on a substrate such as a membrane filter or a slide glass at a high density (at intervals of not larger than several hundred μm) is used and DNAs (an example of organism-originating substances) taken from cells of a normal person A and labeled with a fluorescent dye a and DNAs taken from cells of a genetic-diseased person B and labeled with a fluorescent dye b are dropped onto the microarray chip by pipettes or the like, thereby hybridizing the DNAs of the specimens with the cDNAs on the microarray chip. Thereafter, exciting light beams which respectively excite the fluorescent dyes a and b are projected onto the cDNAs by causing the exciting light beams to scan the microarray chip and fluorescence emitted from each other of the cDNAs is detected by a photodetector. Then the cDNAs with which the DNAs of each specimen are hybridized are determined on the basis of the result of the detection, and the cDNAs with which the DNAs of the normal person A are hybridized and those with which the DNAs of the diseased person B are hybridized are compared, whereby DNAs expressed or lost by genetic disease can be determined. 
     In the microarray technique, it is necessary to precisely two-dimensionally scan the microarray chip coated with cDNAs at a high density, and there has been proposed a radiation image read-out apparatus with such a precise scanning system. See, for instance, Japanese Unexamined Patent Publication No. 10(1998)-3134. 
     The kinds of cDNAs to be used sometimes amount to several tens of thousands and in such a case, the cDNAs must be coated on a plurality of substrates. However when the number of the microarray chips to be used increases, replacement of microarray chips becomes troublesome. 
     SUMMARY OF THE INVENTION 
     In view of the foregoing observations and description, the primary object of the present invention is to provide a test piece on which an increased number of specific binding substances such as cDNAs can be disposed, and a system for reading out image information from the test piece. 
     In accordance with one aspect of the present invention, there is provided a test piece such as a microarray chip for use in biological analyses comprising a plurality of different known specific binding substances such as cDNAs disposed in predetermined positions on a substrate such as a slide glass, wherein the improvement comprises that 
     the specific binding substances are disposed on a plurality of surfaces provided by the substrate and arranged in the direction of thickness of the substrate. 
     The plurality of surfaces provided by the substrate and arranged in the direction of thickness of the substrate may be opposite sides of the substrate or may be provided by a multi-layered substrate formed by a plurality of substrates which are stacked and bonded together so that the surfaces on which the specific binding substances are disposed are substantially in parallel to each other. 
     It is preferred that the specific binding substances be disposed on the surfaces in positions where the specific binding substances on the respective surfaces do not interfere with each other in the direction of thickness of the substrate, that is, the specific binding substances on the respective surfaces do not overlap with each other in the direction of thickness of the substrate. 
     The substrate may be formed of any material so long as the specific binding substances can be spotted and stably held on the substrate and the substrate is optically transparent to the exciting light and the fluorescence emitted from the specific binding substances upon exposure to the exciting light. For example, the substrate may be a membrane filter or a slide glass. Further the substrate may be subjected to pretreatment so that the specific binding substances are stably held on the substrate. 
     The specific binding substances include hormones, tumor markers, enzymes, antibodies, antigens, abzymes, other proteins, nucleic acids, cDNAs, DNAs, RNAs, and the like, and means those which can be specifically bound with an organism-originating substance. The means of the expression “known” differs by the specific binding substance. For example, when the specific binding substance is a nucleic acid, “known” means that the base sequence, the lengths of the bases and the like are known, and when specific binding substance is protein, “known” means that the composition of the amino acid is known. The specific binding substances are disposed by one kind for each position. 
     In accordance with another aspect of the present invention, there is provided a system for reading out image information from the test piece of the present invention comprising 
     a test piece holder portion which holds a test piece of the present invention the specific binding substances on which have been hybridized with an organism-originating substance labeled with fluorescent dye, 
     an exciting light source which emits exciting light for exciting the fluorescent dye, 
     a photoelectric read-out means which photoelectrically reads out fluorescence emitted from the fluorescent dye upon exposure to the exciting light, 
     a scanning means which has an optical head for projecting the exciting light onto the test piece and leading fluorescence, which is emitted from the fluorescent dye and travels through the surface of the test piece onto which the exciting light is projected, to the photoelectric read-out means, and causes the exciting light to scan the test piece, and 
     a controller which controls the exciting light source, the photoelectric read-out means and the scanning means so that fluorescence emitted from the specific binding substances upon exposure to the exciting light is detected for each of the surfaces of the test piece. 
     The test piece holder portion may comprise a table on which the test piece is placed. In this case, the test piece is placed on the table with its one side in contact with the table, and accordingly, it is necessary that the table is transparent to at least the fluorescence. When the test piece holder portion is in the form of a member which supports only the four corners of the test piece, the test piece holder portion need not be transparent. 
     The organism-originating substance may be a wide variety of substances originated from an organism including hormones, tumor markers, enzymes, proteins, antibodies, various substances which can be antigens, nucleic acids, cDNAs, mRNAs and the like. 
     The exciting light is light suitable for exciting the fluorescent dye including a laser beam. 
     As the photoelectric read-out means, a photomultiplier which can detect at a high sensitivity weak light such as fluorescence may be suitably used. However, various known photoelectric read-out means such as a cooled CCD may be used without limited to the photomultiplier. 
     In accordance with the present invention, since the specific binding substances are disposed on a plurality of surfaces provided by the substrate and arranged in the direction of thickness of the substrate, an increased number of specific binding substances can be disposed on one test piece and accordingly, the number of test pieces to be used can be less even if a large number of specific binding substances are used, whereby the frequency at which the test pieces are replaced can be reduced and reading operation can be effectively performed. 
     When the specific binding substances are disposed on opposite sides of a single substrate, the test piece can be manufactured at low cost. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a perspective view of a test piece in accordance with a first embodiment of the present invention, 
     FIG. 2 is a cross-sectional view taken along line I—I in FIG. 1, 
     FIG. 3 is a cross-sectional view of a test piece in accordance with a second embodiment of the present invention, 
     FIG. 4A is a perspective view of an image information read-out system in accordance with a third embodiment of the present invention, 
     FIG. 4B is a schematic side view of the image information read-out system, 
     FIG. 5 is a view for illustrating the operation of the image information read-out system of the third embodiment, 
     FIG. 6A is a perspective view of an image information read-out system in accordance with a fourth embodiment of the present invention, and 
     FIG. 6B is a schematic side view of the image information read-out system. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     In FIGS. 1 and 2, a test piece  1  in accordance with a first embodiment of the present invention comprises a substrate  2  which is a slide glass in this particular embodiment, and a plurality of different cDNAs disposed in a plurality of positions on opposite sides (upper and lower sides) of the substrate  2 . The base sequences of the cDNAs are known and correspond to different DNAs. The kind of each cDNA and the position of each cDNA are predetermined. 
     As shown in FIG. 2, the cDNAs on the upper side of the substrate  2  and those on the lower side of the substrate  2  are positioned not to overlap each other in the direction of thickness of the substrate  2 . Further the upper and lower sides of the substrate  2  have been subjected to surface treatment so that the cDNAs are bonded to the surfaces and accordingly, the cDNAs on the lower side of the substrate  2  cannot peel off the substrate  2 . The thickness of the substrate  2  is about 1 mm, and each of the cDNAs is disposed on the surface of the substrate  2  in a spot of a diameter 30 to 100 μm with the spot intervals of about 300 μm. 
     FIG. 3 shows a test piece in accordance with a second embodiment of the present invention. The test piece shown in FIG. 3 is provided with a substrate formed of a pair of substrate segments  2 A and  2 B which are bonded together with a spacer  3  interposed therebetween. The cDNAs are disposed on the upper surfaces of the respective substrate segments  2 A and  2 B not to overlap each other in the direction of thickness of the substrate segments  2 A and  2 B. The spacer  3  may either be discontinuous or continuous over the entire periphery of the substrate segments  2 A and  2 B. 
     FIGS. 4A and 4B show an image information read-out system in accordance with a third embodiment of the present invention for reading out image information from the test piece  1  shown in FIG.  1 . The image information read-out system comprises a transparent sample table  20  on which the test piece  1 , having applied with an organism-originating substance labeled with a fluorescent dye, is supported at its four corners, a laser  30  which emits a laser beam L in a wavelength band exciting the fluorescent dye, a lens  31  which converges the laser beam L as emitted from the laser  30  into a thin laser beam, a photomultiplier  40  which photoelectrically detects fluorescence K 1  and K 2  emitted from the cDNAs upon exposure to the laser beam L (K 1  represents the fluorescence emitted from the cDNAs on the upper surface of the substrate  2  and K 2  represents the fluorescence emitted from the cDNAs on the lower surface of the substrate  2 ), an optical head  50  which causes the laser beam L to impinge upon the test piece  1  on the sample table  20  and leads the fluorescence K 1  or K 2  to the photomultiplier  40 , a laser beam cut filter  41  disposed on the optical path between the optical head  50  and the photomultiplier  40 , a condenser lens  55  which is disposed between the test piece  1  and the photomultiplier  40  and forms a confocal optical system together with a lens  53 , an aperture plate  56  which has an aperture  56   a  which permits to impinge upon the lens  41  only light from a portion of the test piece  1  on which the laser beam L is converged by the lens  53 , a main scanning system  60  which moves the optical head  50  in the direction of arrow X at a constant speed, a sub-scanning means  80  which moves the laser  30 , the optical head  50 , the condenser lens  55 , the aperture plate  56 , the laser beam cut filter  41  and the photomultiplier  40  in the direction of arrow Y (perpendicular to the direction of arrow X) integrally with each other, a logarithmic amplifier  42  which logarithmically amplifies a detecting signal output from the photomultiplier  40 , and an A/D converter  43  which digitizes the amplified detecting signal. 
     The laser  30  is arranged to emits the laser beam L in the direction of arrow X, and the photomultiplier  40  is arranged to detect the fluorescence K 1  or K 2  impinging thereupon in the direction of arrow X. 
     The optical head  50  comprises a plane mirror  51  which reflects the thin laser beam L, traveling in the direction of arrow X, in a direction perpendicular to the surfaces of the test piece  1 , a mirror  52  which is provided with an aperture  52   a  through which the laser beam L reflected by the plane mirror  51  impinges upon the test piece  1  and reflects the major parts of the fluorescence K 1  or K 2 , emitted downward from the lower surface of the test piece  1 , to impinge upon the photomultiplier  40 , and the lens  53  which collimates the fluorescence K 1  or K 2  which emits downward from the test piece  1  as divergent light. The plane mirror  51 , the mirror  52  with the aperture  52   a  and the lens  53  are integrated into a unit. The lens  53  is movable in the direction of thickness of the test piece  1  or in the direction of arrow z to move a focal point of the lens  53  selectively to the upper surface of the substrate  2  and to the lower surface of the same. When the fluorescence K 1  from the upper surface of the test piece  1  is to be detected, the focal point of the lens  53  is moved to the upper surface of the test piece  1  and when the fluorescence K 2  from the lower surface of the test piece  1  is to be detected, the focal point of the lens  53  is moved to the lower surface of the test piece  1 , whereby the florescence K 1  and K 2  can be collimated to beams of substantially the same diameters. 
     The laser beam cut filter  41  is a filter which transmits the fluorescence K 1  and K 2  but does not transmit the laser beam L so that a part of the laser beam L scattered by the test piece  1 , the sample table  20  and the like cannot impinge upon the photomultiplier  40 . 
     Operation of the image information read-out system of this embodiment will be described, hereinbelow. 
     The position of the lens  53  is first adjusted so that the focal point of the lens  53  is on the upper surface of the substrate  2 . Then the main scanning means  60  moves the optical head  50  at a constant high speed in the direction of arrow X. At each moment during movement of the optical head  50 , the laser  30  emits a laser beam L in the direction of arrow X and the lens  31  converges the laser beam L into a thin laser beam. The thin laser beam L enters the optical head  50 . The laser beam L is then reflected upward by the plane mirror  51  and impinges upon a fine area on the upper surface of the test piece  1  through the aperture  52   a  of the mirror  52  and the lens  53 . 
     When an organism-originating substance labeled with fluorescent dye exists in the fine area exposed to the laser beam L, the fluorescent dye is excited by the laser beam L and emits fluorescence K 1 . 
     The fluorescence K 1  spread around the area and the part of the fluorescence K 1  traveling downward from the lower surface of the test piece  1  is collimated by the lens  53  of the optical head  50  into a substantially parallel downward beam and impinges upon the mirror  52 . Though the part of the fluorescence K 1  impinges upon the aperture  52   a  travels further downward through the aperture  52   a  (the diameter of the aperture  52   a  is sufficiently small as compared with the beam diameter), the major part of the fluorescence K 1  is reflected by the mirror  52  to travel in the direction of arrow X and to impinge upon the photomultiplier  40  through the condenser lens  55 , the aperture  56   a  of the aperture plate  56  and the laser bean cut filter  41 . 
     Though a part of the laser beam L impinging upon the test piece  1  is scattered by the test piece  1 , the sample table  20  and the like and travels toward the photomultiplier  40 , it is prevented from impinging upon the photomultiplier  40  by the laser beam cut filter  41 . Further since the test piece  1  and the photomultiplier  40  are optically connected by a confocal optical system, fluorescence from a part of the test piece other than the part exposed to the laser beam L is prevented from impinging upon the photomultiplier  40  and blur of a fluoresce image obtained can be avoided even if the area exposed to the laser beam L is shifted or enlarged. 
     The fluorescence K 1  impinging upon the photomultiplier  40  is photoelectrically detected by the photomultiplier  40  and read out as an electric signal. The electric signal is amplified by the amplifier  42  and is converted to a digital signal by the A/D converter  43 . 
     During these steps, the optical head  50  is kept moved in the direction of arrow X by the main scanning system  60 , and a digital signal is output from the A/D converter  43  for each main scanning position on the test piece  1 . 
     Each time the main scanning along one line is ended, the sub-scanning means  80  slightly moves the laser  30 , the optical head  50 , the laser beam cut filter  41  and the photomultiplier  40  in the direction of arrow Y (sub-scanning) and the main scanning is repeated. The sub-scanning may be effected in parallel to the main scanning. 
     Thus the entire area of the upper surface of the test piece  1  is two-dimensionally scanned by the laser beam L, and image information representing the distribution of the organism-originating substances labeled by the fluorescent dye on the upper surface of the substrate  2  is obtained. 
     Thereafter the optical head  50  is returned to the initial position by the main scanning means  60  and the sub-scanning means  80 . Then the lens  53  is moved downward by d 0  (FIG. 5) so that the focal point of the lens  53  is on the lower surface of the substrate  2 . Then the fluorescence K 2  emitted from the lower surface of the test piece  1  is detected and converted to a digital signal in the same manner as described above and image information representing the distribution of the organism-originating substances labeled by the fluorescent dye on the lower surface of the substrate  2  is obtained. 
     The image information representing the distribution of the organism-originating substances labeled by the fluorescent dye on the upper and lower surfaces of the substrate  2  is displayed on a monitor (not shown). 
     Thus in the image information read-out system of this embodiment, image information can be read out from opposite sides of the test piece  1  of the first embodiment of the present invention, where cDNAs are disposed on opposite sides of the substrate  2 . 
     The image information read-out system of this embodiment can be used to read out image information from the test piece of the second embodiment of the present invention shown in FIG.  3 . In this case, the lens  53  is moved so that the focal point of the lens  53  is selectively moved to the upper surface of the substrate segment  2 A and that of the substrate segment  2 B. 
     Though, in the embodiment described above, the fluorescence K 1  emitted from the cDNAs on the upper surface of the substrate  2  and the fluorescence K 2  emitted from the cDNAs on the lower surface of the substrate  2  are separately detected by moving the focal point of the lens  53  forming a confocal optical system, it is possible to separately detect the fluorescence K 1  and the fluorescence K 2  by moving the aperture plate  56  along the optical axis with the lens  53  kept stationary. That is, when the laser beam L is projected onto the test piece  1  with the focal point of the lens  53  set at the middle between the upper and lower surfaces of the substrate  2 , the fluorescence K 1  is emitted from the cDNAs on the upper surface of the substrate  2  and the fluorescence K 2  is emitted from the cDNAs on the lower surface of the substrate  2 . Depending on the position of the aperture plate  56  along the optical axis, only one of the fluorescence K 1  and the fluorescence K 2  can pass through the aperture  56   a  in the aperture plate  56 . 
     FIGS. 6A and 6B show an image information read-out system in accordance with a fourth embodiment of the present invention for reading out image information from the test piece  1  shown in FIG.  1 . The image information read-out system of this embodiment comprises a sample table  20 , a first laser  30 A, a first lens  31 A, a first photomultiplier  40 A, an optical head  50 , a first laser beam cut filter  41 A, a first condenser lens  55 A, a first aperture plate  56 A, a main scanning system  60 , a sub-scanning means  80 , a first logarithmic amplifier  42 A, and a first A/D converter  43 A, which are basically the same as the sample table  20 , the laser  30 , the lens  31 , the photomultiplier  40 , the optical head  50 , the laser beam cut filter  41 , the condenser lens  55 , the aperture plate  56 , the main scanning system  60 , the sub-scanning means  80 , the logarithmic amplifier  42 , and the A/D converter  43  employed in the third embodiment. The image information read-out system of this embodiment further comprises a second laser  30 B, a second lens  31 B, a second condenser lens  55 B, a second aperture plate  56 B, a second laser beam cut filter  41 B, a second photomultiplier  40 B, a second logarithmic amplifier  42 B, a second A/D converter  43 B, a polarization beam splitter  62  which transmits the laser beam L 1  emitted from the first laser  30 A and reflects the laser beam L 2  emitted from the second laser  30 B, and a half-silvered mirror  63  which transmits a part of the fluorescence K 1  and the fluorescence K 2  to impinge upon the first photomultiplier  40 A, and reflects the other part of the fluorescence K 1  and the fluorescence K 2  to impinge upon the second photomultiplier  40 B. 
     The first and second lenses  31 A and  31 B are identical to each other. The first and second lasers  30 A and  30 B are basically identical to each other except that the laser beam L 1  emitted from the first laser  30 A is polarized in the vertical direction as seen in FIG.  6 B and the laser beam L 2  emitted from the second laser  30 B is polarized in a direction perpendicular to the surface of the paper on which FIG. 6B is drawn. With this arrangement, the laser beam L 1  transmits the polarization beam splitter  62  and the laser beam L 2  is reflected by the same. 
     The distance d 2  between the beam radiating end of the second laser  30 B and the second lens  31 B is set larger than the distance d 1  between the beam radiating end of the first laser  30 A and the first lens  31 A so that the diameter of the laser beam L 1  on the upper surface of the substrate  2  becomes equal to the diameter of the laser beam L 2  on the lower surface of the substrate  2 . 
     Further, the distance D 2  between the second condenser lens  55 B and the second aperture plate  56 B is set larger than the distance D 1  between the first condenser lens  55 A and the first aperture plate  56 A. In the fourth embodiment, the leaser beams L 1  and L 2  are simultaneously emitted from the first and second lasers  30 A and  30 B, and the fluorescence K 1  and the fluorescence K 2  emitted respectively from the upper and lower surfaces of the test piece  1  are simultaneously detected. The fluorescence K 1  and the fluorescence K 2  emitted respectively from the upper and lower surfaces of the test piece  1  simultaneously travel in the direction of arrow X. The fluorescence K 1  and the fluorescence K 2  emitted respectively from the upper and lower surfaces of the test piece  1  are separated by the half-silvered mirror  63  to parts which respectively travel to the first and second photomultipliers  40 A and  40 B. Either of the parts includes both the fluorescence K 1  and the fluorescence K 2 , and accordingly, the distance D 2  between the second condenser lens  55 B and the second aperture plate  56 B is set larger than the distance D 1  between the first condenser lens  55 A and the first aperture plate  56  so that only the fluorescence K 1  can pass through the aperture of the first aperture plate  56 A and only the fluorescence K 2  can pass through the aperture of the second aperture plate  56 B, whereby the fluorescence K 1  and the fluorescence K 2  are separately detected by the photomultipliers  40 A and  40 B, respectively. 
     Operation of the image information read-out system of this embodiment will be described, hereinbelow. 
     When the laser beams L 1  and L 2  are projected onto the upper and lower surfaces of the test piece  1 , fluorescence K 1  and fluorescence K 2  are emitted from the upper and lower surfaces of the test piece  1 , respectively, and simultaneously travel in the direction of arrow X as a light bundle. The light bundle is divided into two light bundles by the half-silvered mirror  63 , one traveling toward the first photomultiplier  40 A and the other traveling toward the second photomultiplier  40 B. The fluorescence K 1  included in said one light bundle impinges upon the first photomultiplier  40 A through the first condenser lens  55 A, the first aperture plate  56 A and the first laser beam cut filter  41 A and is detected by the first photomultiplier  40 A, whereas the fluorescence K 2  included in said the other light bundle impinges upon the second photomultiplier  40 B through the second condenser lens  55 B, the second aperture plate  56 B and the second laser beam cut filter  41 B and is detected by the second photomultiplier  40 B. 
     The fluorescence K 1  and the fluorescence K 2  are photoelectrically converted to electric signals respectively by the first and second photomultipliers  40 A and  40 B, and the electric signals are amplified by the first and second amplifiers  42 A and  42 B and then digitized by the first and second A/D converters  43 A and  43 B. Then visible images are displayed on a monitor (not shown) on the basis of the digitized electric signals. 
     Thus also in the image information read-out system of this embodiment, image information can be read out from opposite sides of the test piece  1  of the first embodiment of the present invention, where cDNAs are disposed on opposite sides of the substrate  2 . 
     The image information read-out system of this embodiment can be used to read out image information from the test piece of the second embodiment of the present invention shown in FIG.  3 . In this case, positions of the first and second lenses  31 A and  31 B and the first and second aperture plates  56 A and  56 B are adjusted to read out image information from the substrate segments  2 A and  2 B. 
     Though, in the embodiments described above, the cDNAs are disposed not to overlap each other in the direction of thickness of the substrate  2  or the substrate segments  2 A and  2 B, they may overlap each other in the direction of thickness of the substrate  2  or the substrate segments  2 A and  2 B when the image information read-out system comprises a confocal optical system.