Abstract:
A microarray chip manufacturing apparatus includes a plurality of picking pins which pick specific binding substances linearly or two-dimensionally arranged at first spaces and spot the specific binding substances onto a flat chip at second spaces narrower than the first spaces. The picking pins are movable so that the spaces among the picking pins can be switched between the first spaces and the second spaces, and a space switching mechanism switches the spaces among the picking means to the first spaces when the picking means pick the specific binding substances and to the second spaces when the picking means spot the specific binding substances onto the chip.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to an apparatus for manufacturing microarray chip for use in DNA analysis, immunological analysis, and the like, and more particularly to an improvement in the structure for spotting specific binding substances onto a chip. 
     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 DNA chip) comprising a plurality of known cDNAs (an example of specific binding substances) spotted in a matrix on a chip such as of a membrane filter or a slide glass at a high density (at intervals of not larger than several hundred μm) as shown in FIG. 5 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 of the cDNAS is detected by a photodetector. Then the cDNAs with which the DNAs of each specimen are hybritized 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 the genetic disease can be determined. This information contributes to gene therapy. 
     The number of specific binding substances such as cDNAs have been spotted on a chip such as of a membrane filter or a slide glass at a high density by means of a microarray chip manufacturing apparatus(a spotter or an arrayer) as shown in FIG.  3 . 
     In the microarray chip manufacturing apparatus shown in FIG. 3, there is used a microtiter plate  10  having a number of specific binding substance holes  10   a  which are two-dimensionally arranged at pitches P larger than pitches at which the specific binding substances  11  are to be spotted onto a chip  20 . The specific binding substance holes  10   a  are respectively filled with the specific binding substances  11 . The microarray chip manufacturing apparatus comprises a spot head  30  provided with a plurality of, e.g., two×two, specific binding substance picking pins  31  which are arranged at the same pitches as the pitches P at which the specific binding substance holes  10   a  of the microtiter plate  10  are arranged, and a means for moving the spot head  30 . 
     The microtiter plate  30  is about 8 cm×12 cm in size and the number of the specific binding substance holes  10   a  on the microtiter plate  30  is generally in the range of 8×12 (=96) to 32×48 (=1536). To the contrast, the chip  20  on which the specific binding substances  11  are spotted is about 2 cm×2 cm in size operation of a microarray chip manufacturing apparatus which spots  96  specific binding substances  11  onto a chip  20  by the use of a microtiter plate  10  having 8×12 specific binding substance holes  10   a  will be described, hereinbelow. 
     First the spot head  30  is moved to a position where the four picking pins  31  are aligned with the upper left four (2×2) specific binding substance holes  10   a  and then moved downward so that the picking pins  31  are inserted into the respective specific binding substance holes  10   a  and the specific binding substances  11  therein adheres to the picking pins  31 . Thereafter, the spot head  30  is moved upward and to the chip  20  and then moved downward so that the picking pins  31  are brought into contact with the chip  20 , whereby the specific binding substances  11  on the respective picking pins  31  are spotted onto the chip  20  as shown in FIG.  4 A. In FIG.  4 A and the following FIGS. 4B to  4 D, the small circles indicated at × show the specific binding substance holes  10   a  into which the picking pins  31  are inserted at that time. When a plurality of microarray chips are to be manufactured, the same procedure is repeated the like number of times. 
     Then the specific binding substances  10   a  in the four holes  10   a  on the right side of the holes  10   a  the specific binding substances  11  in which are initially spotted (i.e., third and fourth holes  10   a  in the uppermost line as numbered from the left and third and fourth holes  10   a  in the second uppermost line as numbered from the left) are picked. At this time, since the specific binding substances  11  in the respective holes  10   a  are generally different from each other, it is necessary to clean the pins  31  by ultrasonic cleaning and to dry the same prior to picking the next specific binding substances  11  in order to prevent the preceding specific binding substances  11  from mingling with the next specific binding substances  11 . After cleaning and drying the pins  31 , the pins  31  are inserted into the next four holes  31  and the spot head  30  are moved so that the specific binding substances  11  in the holes  31  are spotted onto the chip  20  on the right side of the preceding spots at distances therefrom smaller than the distance (pitch) P between the pins  31  (e.g., P/6) as shown in FIG.  4 B. 
     After these procedure (including picking, spotting, cleaning and drying) is repeated six times and the specific binding substances  11  in the upper right four holes  10   a  (i.e., first and second holes  10   a  in the uppermost line as numbered from the right and first and second holes  10   a  in the second uppermost line as numbered from the right) are spotted onto the chip  20 , twenty-four specific binding substances  11  are spotted onto the chip  20  in two lines as shown in FIG.  4 C. 
     Thereafter, the specific binding substances  11  in the holes  10   a  on the third and fourth lines are spotted onto the chip  20  on the lower side of the preceding spots at distances therefrom smaller than the distance (pitch) P between the pins  31  (e.g., P/4) as shown in FIG. 4 c.    
     The procedure described above is repeated until the specific binding substances  11  in all the ninety-six holes  10   a  are spotted onto the chip  20  as shown in FIG.  4 D. 
     In the conventional microarray chip manufacturing apparatus, the picking pins must be cleaned and dried each time they spot four specific binding substances. This means that cleaning and drying must be repeated twenty-four times four a chip  20  bearing thereon ninety-six (96) specific binding substances  11  (96/4=24) and three hundred and eighty-four (384) times for a chip  20  bearing thereon one thousand five hundred and thirty-six (1536) specific binding substances  11  (1536/4=384). The operation of cleaning and drying the picking pins requires a long time and deteriorates the efficiency of the spotting. 
     Further, the preceding specific binding substances  11  adhering to the picking pins cannot be perfectly cleaned, which gives rise to a problem that there is fear that the next specific binding substances  11  can be contaminated by the preceding specific binding substances. 
     SUMMARY OF THE INVENTION 
     In view of the foregoing observations and description, the primary object of the present invention is to provide a microarray chip manufacturing apparatus which can spot specific binding substances onto a chip in a shorter time. 
     The microarray chip manufacturing apparatus in accordance with the present invention is characterized in that the spaces among the picking means (e.g., picking pins, picking nozzles or the like) are changed when the picking means spot specific binding substances from those when the picking means pick the specific binding substances. 
     That is, in accordance with the present invention, there is provided a microarray chip manufacturing apparatus comprising a plurality of picking means which pick specific binding substances linearly or two-dimensionally arranged at first spaces and spot the specific binding substances onto a flat chip at second spaces narrower than the first spaces, wherein the improvement comprises 
     that the picking means are movable so that the spaces among the picking means can be switched between said first spaces and said second spaces, and a space switching means switches the spaces among the picking means to said first spaces when the picking means pick the specific binding substances and to said second spaces when the picking means spot the specific binding substances onto the chip. 
     The specific binding substances may be, for instance, organism-originating substances such as cDNAs, and may be held in holes formed on a microtiter plate arranged linearly or two-dimensionally. 
     When the specific binding substances are held on a microtiter plate, the first spaces may be, for instance, about 1 mm to 10 mm and the second spaces may be, for instance, about 100 μm to 500 μm. 
     The flat chip is a substrate such as of a membrane filter or a slide glass which is spotted with the specific binding substances to form a microarray chip. 
     The space switching means may be of any structure so long as it can switch the spaces among the picking means between said first spaces and said second spaces. For example, the space switching means may comprise bellows connecting the picking means and a means for linearly or two-dimensionally expanding and contracting the bellows. 
     It is preferred that the picking means be provided in the same number as the number of the specific binding substances so that all the specific binding substances can be spotted onto the chip in one action. 
     In the microarray chip manufacturing apparatus of the resent invention, by switching the spaces among the picking means to the first spaces, equal to the spaces at which the specific binding substances are arranged, when the picking means pick the specific binding substances and to the second spaces, at which the specific binding substances are to be spotted onto the chip, when the picking means spot the specific binding substances onto the chip, a larger number of specific binding substances can be spotted at one time, whereby the time required to spot the specific binding substances can be shortened and at the same time, contamination of the specific binding substances with those spotted previously due to repeated use of the same picking means can be suppressed. 
     Further, when the picking means are provided in the same number as the number of the specific binding substances, all the specific binding substances can be spotted onto the chip at one time, whereby the time required to spot the specific binding substances can be further shortened and at the same time, contamination of the specific binding substances with those spotted previously, which can occur when the same picking means are repeatedly used, can be prevented. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a perspective view of a microarray chip manufacturing apparatus in accordance with a first embodiment of the present invention, 
     FIG. 2 is a perspective view showing in detail the space switching means employed in the microarray chip manufacturing apparatus shown in FIG. 1, 
     FIG. 3 is a perspective view showing a microarray chip manufacturing apparatus in accordance with the prior art, 
     FIGS. 4A to  4 D are views for illustrating operation of the conventional microarray chip manufacturing apparatus, 
     FIG. 5 is a perspective view showing a microarray chip, and 
     FIG. 6 is a perspective view showing a modification of the microarray chip manufacturing apparatus shown in FIG. 1 where the space switching means comprises bellows. 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     In FIG. 1, a microarray chip manufacturing apparatus in accordance with an embodiment of the present invention comprises a spot head  30  on which a plurality of picking pins  31  which pick specific binding substances  11  held in specific binding substance holes  10   a  on a microtiter plate  10  and spot the specific binding substances  11  onto a slide glass  20  are supported, and a moving means  40  for moving the spot head  30  between the microtiter plate  10  and the slide glass  20 . The specific binding substance holes  10   a  are ninety-six in number and are two-dimensionally arranged on the microtiter plate  10  at spaces of P, eight in the longitudinal direction of plate  10  and twelve in the transverse direction of the same (8×12). 
     The picking pins  31  are the same as the holes  10   a  in number (8×12=96) and the spaces among the pins  31  are variable. The spaces among the pins  31  are variable between P and P/6 in the transverse direction of the plate  10  and between P and P/4 in the longitudinal direction of the same. 
     The spot head  30  is provided with a space switching means  33  which switches the spaces among the pins  31 . The space switching means  33  switches the spaces among the pins  31  to P in both the longitudinal and transverse directions of the plate  10  when the pin  31  pick the specific binding substances  11  from the plate  10  and to P/6 and P/4 respectively in the transverse and longitudinal directions of the plate  10  when the pins  31  spot the specific binding substances  11  onto the slide glass  20 . 
     FIG. 2 shows the space switching means  33 . As shown in FIG. 2, the space switching means  33  is like a mechanism for opening and shutting a window shade. In FIG. 2, some of the pins  31  (indicated at  31   a ,  31   b ,  31   c  . . . in FIG. 2) and some elements are eliminated for the purpose of simplicity. 
     In FIG. 2, pins  31   a ,  31   b  and  31   c  on a transverse line are respectively fixed to sliders  33   d ,  33   e  and  33   f  which are mounted on a support shaft  33   a  in the transverse direction to be slidable along the shaft  33   a . A threaded rod  33   b  extends through the sliders  33   d ,  33   e  and  33   f  in parallel to the support shaft  33   a . A guide slider  33   c  is mounted on the support shaft  33   a  at the right side end portion thereof to be slidable along the support shaft  33   a . The guide slider  33   c  has a threaded hole in mesh with the threaded rod  33   b  so that the guide slider  33   c  is moved along the support shaft  33   a  in response to revolution of the threaded rod  33   b . The guide slider  33   c  and the slider  33   d  are connected by a chain  33   g , the sliders  33   d  and  33   e  are connected by a chain  33   h  and the sliders  33   e  and  33   f  are connected by a chain  33   i.    
     Support shafts  33   j  and  33   k  similar to the support shaft  33   a  are provided and the support shafts  33   a ,  33   j  and  33   k  are supported by guide rails  34   a  and  34   b  at their opposite ends to be movable in the direction perpendicular to the longitudinal direction thereof. The support shafts  33   a ,  33   j  and  33   k  are moved along the guide rails  34   a  and  34   b  in parallel to each other by an electric motor not shown. 
     When the chains  33   g ,  33   h  and  33   i  are tensioned straight, the spaces between the pins  31   a  and  31   b  and  31   b  and  31   c  are P, and the spaces between the support shafts  33   a  and  33   j  and  33   j  and  33   k  are also P. 
     When the threaded rod  33   b  is revolved by an electric motor (not shown) in one direction, the guide slider  33   c  is moved leftward and pushes slider  33   d  leftward so that the slider  33   d  pushes leftward the slider  33   e  to bring the slider  33   e  in contact with the slider  33   f . Finally, the sliders  33   c ,  33   d ,  33   e  and  33   f  are held in contact with each other side by side. In this state, the spaces between the pins  31   a  and  31   b  and between the pins  31   b  and  31   c  are equal to P/6 and the chains  33   g ,  33   h  and  33   i  hang down. The spaces between the pins supported by the support shafts  33   j  and  33   k  are made equal to P/6 in the same manner. 
     Further the spaces between the pins in the longitudinal direction are made equal to P/4 by moving the support shafts  33   j  and  33   k  toward the support shaft  33   a  along the guide shafts  34   a  and  34   b  by the electric motor (not shown). 
     When the electric motor is reversed to move the support shafts  33   j  and  33   k  away from the support shaft  33   a , the spaces between the pins in the longitudinal direction are returned to P. Further, when the threaded rod  33   b  is reversed, the guide slider  33   c  is moved rightward away from the slider  33   d , and when the chain  33   g  is tensioned straight (the space between the guide slider  33   c  and the slider  33   d  becomes P), the slider  33   d  is pulled rightward. In this manner, when all the chains  33   g ,  33   h  and  33   i  are tensioned straight, the guide slide  33   c  is stopped, whereby the spaces between the pins in the transverse direction are returned to P. 
     Though, in this embodiment, the adjacent pins are connected by chains, the adjacent pins may be connected by bellows  33   z  as shown in FIG. 6, or by X-shaped movable links. 
     Operation of the microarray chip manufacturing apparatus of the embodiment will be described, hereinbelow. 
     After the spaces P among the pins  31  are held at P by the space switching means  33 , the moving means  40  moves the spot head  30  to above the microtiter plate  10  so that the pins  31  on the head  30  are respectively aligned with the specific binding substance holes  10   a  of the plate  10 . 
     Then the moving means  40  moves downward the head  30  so that the pins  31  are inserted into the holes  10   a , whereby the specific binding substances  11  in the holes  10   a  adhere to the pins  31 . Then the moving means  40  moves upward the head  30  and moves the head  30  to above the slide glass  20 . 
     Then the space switching means  33  switches the spaces among the pins  31  in the longitudinal direction to P/4 and the spaces among the pins  31  in the transverse direction to P/6. In this state, all the pins  31  on the head  30  are held in the area of the slide glass  20 , which is 2 cm×2 cm in size. 
     Then the moving means  40  moves downward the head  30  so that the pins  31  are brought into contact with the surface of the slide glass  20  and the specific binding substances  11  on the pins  31  are spotted onto the slide glass  20 . After the specific binding substances  11  are spotted onto the slide glass  20 , the moving means  40  moves upward the head  30  away from the slide glass  20 . 
     As can be understood from the description above, in the microarray chip manufacturing apparatus of this embodiment, by switching the spaces among the picking pins  31  to the spaces equal to the spaces P, at which the specific binding substances  11  are arranged, when the picking pins  31  pick the specific binding substances  11  and to the spaces at which the specific binding substances  11  are to be spotted onto the slide glass  20  when the picking pins  31  spot the specific binding substances  11  onto the slide glass  20  (to P/4 in the longitudinal direction and to P/6 in the transverse direction), a larger number of specific binding substances  11  can be spotted at one time, whereby the time required to spot the specific binding substances can be shortened. 
     Further, in the apparatus in accordance with this particular embodiment, since the picking pins  31  are provided in the same number as the number of the specific binding substances  11 , all the specific binding substances  11  can be spotted onto the slide glass  20  at one time, whereby contamination of the specific binding substances  11  with those spotted previously, which can occur when the same picking pins  31  are repeatedly used without perfectly cleaning the pins  31 , can be prevented. 
     In addition, all of the contents of japanese patent application no. 11(1999)-279090 are incorporated into this specification by reference.