Abstract:
Identically shaped spots can be formed sequentially and stably by a spotting pin comprising a bar-like plunger  20 . Four projections each formed in the shape of a top portion of a quadrangular pyramid are formed on the head of the plunger  20 . The apexes  21  of the quadrangular pyramids constituting the projections are located inside a virtual plane extending from the peripheral wall of the plunger.

Description:
BACKGROUND OF THE INVENTION  
         [0001]    1. Technical Field  
           [0002]    The present invention relates to a spotting pin for spotting solutions containing biomolecules on a support in the process of manufacturing biochips.  
           [0003]    2. Background Art  
           [0004]    Biochips are conventionally manufactured by spotting solutions containing biopolymers, such as multiple kinds of DNA, RNA, proteins, or oligonucleotides, on a support, such as a slide glass or a nylon membrane. In such a manufacturing process, spotting pins are used. Various kinds of spotting pins have been developed, including a split-type spotting pin capable of sequential spotting operations based on capillary action, such as that observed in a fountain pen tip. Another example is a solid-type spotting pin in which a spotting solution is caused to adhere to the pin tip before each stamping. The solid-type spotting pin is advantageous in that it is easy to wash and that it wastes less spotting solution. However, it has been difficult to sequentially create uniform spots and produce large quantities of biochips with uniform quality using the solid-type spotting pin. This problem is being overcome in recent years, as evidenced by JP Patent Publication (Kokai) No. 2000-15272 A1. This describes a spotting pin with a pin head on which a cross-shaped groove is formed in order to increase the amount of spotting solution that can adhere to the pin head.  
           [0005]    [0005]FIG. 12 is a perspective view of the tip of a conventional flat-cut pin that is cut in parallel to the contact surface. The flat-cut pin is used when spotting a solution on a water-absorbing support, such as a nylon membrane. It has the problem that it cannot create spots with circular edges and a stable shape if the spotting speed is high. FIG. 13 is a perspective view of the head of a conventional spotting pin (V-cut pin) in which two V-shaped grooves are formed in the shape of a cross on the head of the body, which is substantially cylindrical in shape, with each V having a wedge angle of 90°. The V-cut pin has four projections  131  to  134 . The projections are substantially triangular-pyramidal in shape, each having an apex located on the external surface of a substantially cylindrical body  130 . The V-cut pin forms a spot that tends look like a square, as shown in FIG. 14, and its shape is unstable. If identically shaped spots cannot be obtained, the reproducibility or analysis of an experiment utilizing a biochip may suffer.  
           [0006]    Furthermore, there is a need for a technique that enables spots to be formed at high densities, because multiple kinds of biomolecules being spotted in a narrower area would not only allow large quantities of gene expression to be analyzed at once, for example, but would also help reduce the amount of samples used. For this purpose, a spotting pin is required that is capable of stamping small identically shaped spots in a stable manner.  
         SUMMARY OF THE INVENTION  
         [0007]    It is therefore an object of the invention to provide a spotting pin capable of forming spots with desired diameters in a sequential and stable manner on a biochip support, such as a slide glass or a nylon membrane.  
           [0008]    The above object is achieved by a spotting pin according to the invention in which the head of the pin in which a solution is held is shaped in an advantageous manner.  
           [0009]    In one aspect, the invention provides a spotting pin comprising a bar-like body and four projections formed on the head of the body, each of the projections formed in the shape of a top portion of a quadrangular pyramid. The apexes of the quadrangular pyramids constituting the projections are located inside a virtual plane extended from the peripheral wall of the body. This spotting pin makes it possible to spot a spotting solution containing biomolecules on a support, such as a slide glass or a nylon membrane, sequentially and stably, and to obtain spots of identical shape.  
           [0010]    The tip of each quadrangular pyramid constituting a projection is preferably cut to be in a plane perpendicular to the central axis of the body. By thus cutting the tip, the possibility of the spotting pin and/or the support being damaged when the former comes into contact with the latter can be reduced.  
           [0011]    Of a plurality of wall surfaces possessed by two adjacent projections, the two wall surfaces located toward the center of the body are in a first common plane, while the two wall surfaces located farther from the center of the body are in a second common plane.  
           [0012]    The first common plane preferably intersects a plane perpendicular to the central axis of the body at an angle of between 30° and 60°. The second common plane preferably intersects the plane perpendicular to the central axis of the body at an angle of between 30° and 60°. The wedge angle formed by the first and second common planes is preferably in the range of 60° and 120°. The distance between the apexes of quadrangular pyramids constituting adjacent projections may be in the range of 50 to 250 μm.  
           [0013]    The biomolecules that can be spotted by the spotting pin of the invention are not limited to DNA. The inventive spotting pin can be used in spotting any kind of biomolecule, such as RNA, proteins, or mixtures thereof, on a support such as a slide glass or a nylon membrane. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0014]    FIGS.  1 ( a ) and  1 ( b ) show an overall structure of the spotting pin according to the invention.  
         [0015]    [0015]FIG. 2 shows a perspective view illustrating the shape of the head of a W-cut pin according to the invention.  
         [0016]    [0016]FIG. 3 shows a top view of the head of the W-cut pin according to the invention.  
         [0017]    [0017]FIG. 4 shows a side view of the W-cut pin as seen from the direction of an arrow A.  
         [0018]    [0018]FIG. 5 shows a spot formed with the W-cut pin according to the invention.  
         [0019]    FIGS.  6 ( a ) and  6 ( b ) show states of DNA solution held by spotting pins when the head of each spotting pin is in contact with a biochip support.  
         [0020]    [0020]FIG. 7 shows a perspective view illustrating the shape of the head of a W flat-cut pin according to the invention.  
         [0021]    [0021]FIG. 8 shows a top view of the head of a W flat-cut pin according to the invention.  
         [0022]    [0022]FIG. 9 shows a side view of the W flat-cut pin as seen from the direction of an arrow B.  
         [0023]    [0023]FIG. 10 shows a photograph of the head of the W flat-cut pin according to the invention.  
         [0024]    [0024]FIG. 11 shows an example of spotting equipment.  
         [0025]    [0025]FIG. 12 shows a perspective view of a conventional flat-cut pin whose head is cut in parallel with the contact plane.  
         [0026]    [0026]FIG. 13 shows a perspective view of the head of a conventional spotting pin (V-cut pin).  
         [0027]    [0027]FIG. 14 shows a spot formed with the V-cut pin. 
     
    
     DESCRIPTION OF THE INVENTION  
       [0028]    The invention will be hereafter described by way of embodiments with reference made to the drawings.  
         [0029]    FIGS.  1 ( a ) and  1 ( b ) show the overall structure of a spotting pin  10  according to the invention. FIG. 1( a ) is a side view, and FIG. 1( b ) is a cross-sectional view. The spotting pin  10  comprises a rod-shaped plunger  11 , to the head of which a solution is attached, and which is stamped onto a biochip support. It also comprises a cylindrical barrel  12  with a closed bottom. into which a rear end of the plunger  11  is inserted, and a spring  13  disposed inside the barrel  12  for pushing the rear end of the plunger  11 . A spot is formed on the support by bringing the head of the plunger  11 , to which a spotting solution has been attached, into contact with the support, and then pushing the barrel  12  toward the support against the force of the spring  13 .  
         [0030]    By forming the plunger  11  with austenitic stainless steel, increased strength as well as acid and chemical resistance can be obtained. By diamond-coating the inside of the barrel  12  and minimizing the force of the spring  13 , smooth movement of the plunger can be obtained, and the pressure with which the slide glass or nylon membrane is stamped for spotting can be minimized. This helps prevent the deterioration of the spotting pin, making it possible to form identically shaped spots in a sequential and stable manner.  
         [0031]    Referring to FIGS. 2, 3, and  4 , the shape of an example of the spotting pin according to the invention (to be hereafter referred to as. a W-cut pin) will be described. FIG. 2 shows a perspective view illustrating the head of the W-cut pin. FIG. 3 shows a top view of the head of the pin. FIG. 4 shows a side view of the pin as seen from an arrow A shown in FIGS. 2 and 3. The W-cut pin includes four independent quadrangular-pyramidal projections. They are formed by first cutting four independent triangular-pyramidal projections by cutting two V-shaped grooves across each other on the head of the substantially cylindrical plunger  20 . Then, the outside of the four triangular-pyramidal projections is diagonally cut in a plane inclined with respect to the V-shaped grooves. The four quadrangular-pyramidal projections formed on the head of the plunger  20  are thus divided from one another by the two V-shaped grooves. Each projection includes an apex  21 , two planes  22  and  23  forming two inclined surfaces of the two V-shaped grooves perpendicular to one another, a plane  24  slicing diagonally one V-shaped groove  31 , and a plane  25  slicing diagonally the other V-shaped groove  32 .  
         [0032]    Two adjacent projections, such as the upper-right projection (a first projection) and the upper-left projection (a second projection) in FIG. 3, will be considered. A wall surface  23  of the first projection and a wall surface  43  of the second projection form one of the two walls of the same V-shaped groove  31  and are therefore coplanar. A wall surface  25  on the periphery of the first projection and a wall surface  45  on the periphery of the second projection are cut diagonally with respect to the V-shaped groove  32  and are therefore coplanar. The individual wall surfaces of the other two adjacent projections also have similar relationships to the wall surfaces  23  and  25  of the first projection and the wall surfaces  43  and  45  of the second projection.  
         [0033]    A plane formed by the wall surfaces  23  and  43  intersects a plane perpendicular to the central axis of the plunger  20  with an angle  01 . In the present embodiment, the angle θ 1  is about 45°. The angle should preferably be in the range of 30° to 60°. When θ 1  is less than 30°, a sufficient amount of solution cannot be attached to the pin head. When greater than 60°, the solution attached to the pin head cannot be stamped onto the support in a proper manner.  
         [0034]    A plane formed by the wall surfaces  25  and  45  intersects a plane perpendicular to the central axis of the plunger  20  with an angle θ 2 . In the present embodiment, the angle θ 2  is about 45°. The angle should preferably be within the range of 30° to 60°. If the angle is less than 30° or more than 60°, circular spots cannot be stably obtained.  
         [0035]    In the present embodiment, a wedge angle θ 3  formed by a first common plane and a second common plane is about 90°. The angle θ 3  should preferably be in the range of 60° to 120°. If the angle is less than 60°, durability may be compromised. If the angle is greater than 120°, relatively circular spots may not be obtained.  
         [0036]    In the present embodiment, the distance between the apexes of quadrangular pyramids constituting adjacent projections is 100 μm. The distance may be appropriately set within the range of 50 to 250 μm, depending on the desired size of spots.  
         [0037]    When a DNA solution was spotted using the above-described W-cut pin, circular spot were obtained in a sequential and stable manner, as shown in FIG. 5. As the tip angle of the projections is obtuse as compared with a V-cut pin, the spotting pin is less likely to break. Further, the W-cut pin does not have to be as narrow toward its head as the V-cut pin, so that its machining is easier.  
         [0038]    A spot formed with a V-cut pin is likely to be deformed into a rectangle whereas a spot formed with the W-cut pin is circular. This is believed to result from the following reasons. FIGS.  6 ( a ) and  6 ( b ) schematically show the states of DNA solution held by the spotting pin when its head is in contact with a biochip support  61 . FIG. 6( a ) shows the W-cut pin of the invention. FIG. 6( b ) shows a conventional V-cut pin  130 .  
         [0039]    As shown in FIG. 6( b ), the conventional V-cut pin  130  spots the DNA solution  66  on the support  61  while holding the solution inside the four projections  65  at its pin head. As a result, the spot shape tends to extend from the four apexes of the projections  65 , creating a rectangular spot as shown in FIG. 14. On the other hand, in the W-cut pin according to the invention, the DNA solution  64  exists outside as well as inside the four projections  63  of the W-cut pin  20 , as shown in FIG. 6( a ). Thus, the W-cut pin  20  can create a relatively circular spot on the support  61 , as shown in FIG. 5.  
         [0040]    Now referring to FIGS.  7  to  10 , another embodiment (“W flat-cut pin”) of the spotting pin according to the invention will be described. The W flat-cut pin is a modification of the W-cut pin in which the head is additionally cut in parallel to the contact plane. FIG. 7 shows a perspective view of the head of the W flat-cut pin. FIG. 8 shows a top view of the pin head. FIG. 9 shows a side view as seen from the direction of an arrow B. FIG. 10 shows a photograph of the head of the W flat-cut pin. In FIGS.  7  to  9 , parts similar to those shown in FIGS.  2  to  4  are designated by similar numerals to avoid redundancy.  
         [0041]    In the W-cut pin, each apex of the quadrangular pyramids constituting the four projections formed on the pin head is sharply pointed. In the W flat-cut pin, on the other hand, the apex of each projection is shaved and blunted. Thus, because projection tips  81  of the W flat-cut pin are parallel to the contact surface (i.e., a plane perpendicular to the central axis of the plunger), damage to the projections can be prevented when the pin head comes into contact with the support. Further, the W flat-cut pin can be used in forming spots on a film-like support such as a nylon membrane, which may cause problems when the pin head is sharply pointed.  
         [0042]    [0042]FIG. 11 shows an example of spotting equipment for manufacturing a biochip by spotting a DNA solution or the like on a support using the spotting pin. The spotting equipment comprises a pin head  112  on the underside of which spotting pins  111  are mounted, an X-motor  113 X for driving the pin head  112  in an X-axis direction, a Z-motor  113 Z for driving the pin head in a Z-axis direction, a base  114 , and a Y-motor  113 Y for driving the base  114  in a Y-direction. On the base  114  is mounted a stage  116  carrying a plurality of biochip supports  115  such as, for example, slide glasses or nylon membranes. A microplate  118  containing solutions of biomolecules such as multiple kinds of DNA, for example, is also mounted on the base  114 . As the spotting pins  111 , the type of spotting pin according to the invention as described above is used.  
         [0043]    The X- and Z-direction positions of the pin head  112  are accurately controlled by the X- and Z-motors  113 X and  113 Z, respectively. The Y-direction position of the base  114  is accurately controlled by the Y-motor  113 Y. As a result, equal amounts of the multiple kinds of biomolecule solutions can be sequentially spotted on a plurality of supports  115 . When a separate kind of biomolecule solution contained in the microplate  118  is to be sequentially spotted using the same spotting pins, the spotting pins are washed in a pin washing apparatus  119  prior to charging the next biomolecule Solution into them in order to prevent contamination of the solution. The washing is carried out using a combination of ultrasonic washing and vacuum drying. Specifically, the pins are once vacuum-dried after use, washed with ultrasonic, and then vacuum-dried once again. This prevents contamination of the solution and makes it possible to sequentially spot multiple kinds of biomolecule solutions on the support  115 .  
         [0044]    Thus, in accordance with the invention, spotting solutions containing biomolecules, such as multiple kinds of DNA, RNA, or proteins, can be spotted on a support such as a slide glass or a nylon membrane sequentially and stably, and spots with a desired shape can be obtained.